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Research in
Mycology
Volume-II
Research in
Mycology
Volume-II
Editors
Mr. Balwant Singh

Mr. Mukul M. Barwant
Ms. Shivangi Tripathi
Dr. Vinay Kumar Singh
Dr. Vanita C. Karande
Dr. Aisha Kamal
Prof. Shailendra Kumar
Dr. Belle D. Shenoy
Dr. Gopa Banerjee
All the rights reserved. No part of this book
may be reproduced, stored in a retrieved
system, or transmitted in any form or by any
means, electronic, mechanical,
photocopying, recording or otherwise,
without the prior written permission of
author or publisher.
Copyright ©Editors
All Rights Reserved
ISBN: 978-93-93996-47-3
RESEARCH IN MYCOLOGY
VOL-II
Balwant Singh,
Mukul M. Barwant,
Shivangi Tripathi,
Dr. Vinay Kumar Singh,
Dr. Vanita C. Karande,
Dr. Aisha Kamal,
Shailendra Kumar,
Dr. Belle D. Shenoy,
Dr. Gopa Banerjee
Year of Publication: 2023
Rs: 700/-
Front Cover
Blue Duck Designer
Printed at: Sheen Graphics
PUBLISHER
Blue Duck Publications
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Cell: 9682133341
Website: blueduckpublications.com
Research in Mycology Vol. II 2023
PREFACE
Mycology is the specialist branch of fungal study. Fungi are the most diverse group of
heterotrophic organisms and second largest biotic community after insects on earth. They are
grouped into separate Kingdom Fungi. Fungi have thalloid body without cells being organized into
tissues and organs. Fungi are the parasitic, saprophytic or symbiotic in nature. They also play key
role in terrestrial ecosystems. Fungi are the primary decomposers of lignocellulosic material and the
main keepers of great carbon storage in soil as well as dead organic materials. Their edibility,
medicinal properties, mycorrhizal and parasitic association with the forest trees make them
economically and ecologically important for investigation. The term macro fungi are generally
applied to the fruiting bodies of fungi belonging to Ascomycetes and Basidiomycetes. Ascomycetes
and Basidiomycetes are either Epigeous or Hypogeous, large enough to be seen by naked eyes hence
they can be picked by hand. Micro fungi may cause pathological disease to the plants, animals, and
human. Furthermore, most of fungi are microscopic in nature, invisible and they cause their action.
Mycology is one conventional branch The book is about fungus, which are expertly defined
as the creatures researched by mycologists. Fungal research having vast of topics covered in research
like as the Role of Antifungal Metabolites from Bryophytes, Fungal Species Isolated from
Amaranthus viridis and Hibiscus cannabinus, Mycoremediation: Annihilation of Environmental
Pollutants. Fungi important different sector pharmacological studies and pharmaceutical industry it
also focused on the Invitro Antagonistic Activity of Endophytic Bacteria of Oryza sativa Against
Soil-Borne Fungal Pathogen Fusarium oxysporum, Redefining the Relevance and Efficacy of
Microbial Biocontrol Agents Against Phyopathogens, Diversity and Distribution of Endophytic
Fungi, An Overview of Endophytic Fungi Secondary Metabolites and their Novel Applications,
Mycelial Bricks- Building Blocks For the Future, Isolation of Endophytic Microbes From Acacia
pennata (L.) Willd. And their Antifungal Activity Against Rhizoctonia solani Kuhn, Diversity of
Corticioid Fungi Belolnging to order Agaricales. The quick and fascinating advancements in the
taxonomy, cell and molecular biology, biochemistry, pathology, and ecology of fungi are taken into
consideration. Natural functions are combined with characteristics of taxonomic significance,
including their applicability to human affairs. The biology and management of human and plant
pathogens are given particular attention, forming an important link between basic and practical
mycology. Its covered the topic on beneficial effect of mutualistic interaction between salmonella
and aspergillus, fungal culture, habitat with special reference to disease causing agents, nutritional
value of Agaricus bisporus, its potential use and health benefits in chicken farming, mechanisms of
endophytic fungi used in plant protection , comprehensive review on bioluminescent fungi,
mycology a detailed examination: in contrast of present and future scope of mycology, arbuscular
mycorrhizal fungi act as natural biofertilizers in sustainable agriculture, predaceous fungi: an
overview, post-harvest fungal disease and its management on Allium cepa, nematophagous fungi – a
review, role of fungi in bioremediation of pollutants, fungal metabolites with anticancer activity,
i
fungal tannase: the most eminent biocatalyst for industrial applications, special emphasis on the
study of medicinally important genera -Permelia, Cetraria, Cladonia and Usnea.
The Present book supposed to includes Fungal Biology, Plant Pathology, Myco-medicinal,
Mycoremediation, Fungal Degradation, Environmental researches and many more. This book
emphasized all branch area of research of fungi macro fungal and micro fungal. It covers general
aspects of fungi, their beneficial roles, harmful roles, medicinal aspects etc.
Therefore, the present book on “Research in Mycology” has been envisaged in order to
discuss various aspects of Mycological Research. Editors express sincere thanks to all the authors for
contributing their ideas and knowledge in the form of book chapters. Last but not the least; Editors
are highly grateful to “Blue Duck Publication” for bringing out this book in a beautiful way. Editors
hope this book is helpful in academic as well as research and widely read and reach to its target
audience.
Editors
Research in Mycology
Vol. 2
ii
CONTENT
CHAPTER TITLE OF PAGE

NUMBER CHAPTER NUMBER
PREFACE i-ii
CONTENT ii-iii
Chapter-01 UNDERSTANDING THE ROLE OF ANTIFUNGAL METABOLITES FROM

BRYOPHYTES 01-07
Chapter-02 THE STUDY OF FUNGAL SPECIES ISOLATED FROM AMARANTHUS

VIRIDIS AND HIBISCUS CANNABINUS IN COMPARISON FROM FIELD
AND MARKET SAMPLES 08-22
Chapter-03 MYCOREMEDIATION: ANNIHILATION OF ENVIRONMENTAL

POLLUTANTS 23-31
Chapter-04 INVITRO ANTAGONISTIC ACTIVITY OF ENDOPHYTIC BACTERIA OF

ORYZA SATIVA AGAINST SOIL-BORNE FUNGAL PATHOGEN
FUSARIUM OXYSPORUM 32-39
Chapter-05 REDEFINING THE RELEVANCE AND EFFICACY OF MICROBIAL

BIOCONTROL AGENTS AGAINST PHYOPATHOGENS 40-53
Chapter-06 DIVERSITY AND DISTRIBUTION OF ENDOPHYTIC FUNGI IN VELLAR

ESTUARY 54-62
Chapter-07 AN OVERVIEW OF ENDOPHYTIC FUNGI SECONDARY METABOLITES

AND THEIR NOVEL APPLICATIONS 63-73
Chapter-08 MYCELIAL BRICKS- BUILDING BLOCKS FOR THE FUTURE 74-79
Chapter-09 ISOLATION OF ENDOPHYTIC MICROBES FROM ACACIA PENNATA (L.)

WILLD. AND THEIR ANTIFUNGAL ACTIVITY AGAINST RHIZOCTONIA
SOLANI KUHN 80-88
Chapter-10 DIVERSITY OF CORTICIOID FUNGI BELOLNGING TO ORDER

AGARICALES IN DISTRICT CHAMBA OF HIMACHAL PRADESH 89-101
ii
Chapter-11 BENEFICIAL EFFECT OF MUTUALISTIC INTERACTION BETWEEN

SALMONELLA AND ASPERGILLUS 102-110
Chapter-12 FUNGAL CULTURE, HABITAT WITH SPECIAL REFERENCE TO DISEASE

CAUSING AGENTS 111-126
Chapter-13 NUTRITIONAL VALUE OF AGARICUS BISPORUS, ITS POTENTIAL USE

AND HEALTH BENEFITS IN CHICKEN FARMING 127-135
Chapter-14 MECHANISMS OF ENDOPHYTIC FUNGI USED IN PLANT PROTECTION 136-160
Chapter-15 A COMPREHENSIVE REVIEW ON BIOLUMINESCENT FUNGI 161-170
Chapter-16 MYCOLOGY A DETAILED EXAMINATION: IN CONTRAST OF PRESENT

AND FUTURE SCOPE OF MYCOLOGY 171-180
Chapter-17 ARBUSCULAR MYCORRHIZAL FUNGI ACT AS NATURAL

BIOFERTILIZERS IN SUSTAINABLE AGRICULTURE 181-199
Chapter-18 PREDACEOUS FUNGI: AN OVERVIEW 200-207
Chapter-19 POST-HARVEST FUNGAL DISEASE AND ITS MANAGEMENT ON

ALLIUM CEPA 208-213
Chapter-20 NEMATOPHAGOUS FUNGI – A REVIEW 214-224
Chapter-21 ROLE OF FUNGI IN BIOREMEDIATION OF POLLUTANTS 225-230
Chapter-22 FUNGAL METABOLITES WITH ANTICANCER ACTIVITY 231-237
Chapter-23 FUNGAL TANNASE: THE MOST EMINENT BIOCATALYST FOR

INDUSTRIAL APPLICATIONS 238-245
Chapter-24 SPECIAL EMPHASIS ON THE STUDY OF MEDICINALLY IMPORTANT

GENERA -PERMELIA, CETRARIA, CLADONIA AND USNEA 246-270
APPENDIX iv-vi
ABOUT THE EDITORS vii-xv
iii
Chapter 1
UNDERSTANDING THE ROLE OF ANTIFUNGAL
METABOLITES FROM BRYOPHYTES
Shiwani Latwal, Anju Rao
A bstract
Fungi and bryophytes are two distinct groups of organisms that are known to be found in almost
every habitat on earth. Bryophytes, which include mosses, liverworts, and hornworts, are small, non-
vascular plants that grow in damp environments. The chemical diversity of bryophytes exhibits an
astonishing range of biologically active compounds, such as lipids, proteins, alcohols, fatty acid
esters, steroids, phenols, terpenes, aliphatic and aromatic compounds etc. Many of them exhibit
antimicrobial effects against fungi, bacteria and other pathogens. But only a small number of the
species have been in-depthly investigated to date. Increasing public desire for plant-based medicines
and the emergence of pathogens resistant to antibiotics have driven biologists to search for novel
plant-based natural compounds. Bryophytes can therefore be a prospective source of numerous new
physiologically active substances in nature which may help to fill the lacuna in the development of
new bioactive compounds and their derived products. In this article, we will explore in more detail
the role of anti-fungal metabolites in bryophyte and the benefits they provide.
Keywords: Antifungal Metabolites, Bryophytes, Plant Bioactive Compounds, Liverworts.
Department of Botany, Panjab University, Chandigarh, India shiwanilatwal@gmail.com
Shiwani Latwal & Anju Rao

1
I ntroduction
Bryophytes, being the earliest terrestrial plants, are classified into three divisions: Marchantiophyta
(liverworts), Bryophyta (mosses) and Anthocerophyta (hornworts) (Bowman et al; 2017). In all these
three groups, the sporophytic plant body is completely or partially dependent on the
photosynthetically active gametophytic plant body for food and nutrition (Shaw and Renzaglia,
2004). Despite being morphologically small, these plants still play an important role in soil
dynamics, nutrient recycling, ecological function, drought tolerance and also act as bioindicators of
pollution and heavy metals. The phytochemistry of bryophytes too play peculiar role in adapting
climate change to avoid unwanted stressors. In fact, these ancestors of early land plants proved to be
an excellent source of unique phytochemicals as they are exploited for their pharmacological
properties to combat various ailments by various ethnic groups. Bryophytes have been used by many
cultures across several continents to treat various health issues. According to Indian and Chinese
medicinal system, the range of their uses is very broad, from fighting fevers to managing skin
conditions. Polytrichum commune Hedw. and Frullania ericoides (Nees) Mont. has been used to
enhance hair quality by tribal people of South India (Harris, 2008) (Remesh and Manju, 2009). The
Gaddi tribe of Himachal Pradesh, India, used the thallus of Targionia hypophylla L. and
Plagiochasma appendiculatum Lehm. & Lindenb for treating skin conditions (Chandra et al., 2017).
The moss Bryum argenteum Hedw. was valued as an antipyretic and antifungal medicine
(Asakawa,1998). Sphagnum leaves was applied to wounds as bandages due to its high absorption
and bactericidal properties (Garayev et al., 2019). Many species of bryophytes have shown aseptic,
antioxidant, anti-tumor and anti-cancer qualities as well.
Fungal infections are major problem worldwide, affecting plants, crops, animals and millions of
people each year. Such infections are responsible for major crop damage in plants and in case of
animals and humans they cause a wide range of symptoms, from minor skin irritations to life-
threatening systemic infections. Diseases caused by fungi are particularly difficult to treat because
many of the drugs that are effective against bacteria or viruses are not effective against fungi.
Furthermore, many fungal infections are becoming increasingly resistant to currently available
antifungal drugs, making the search for new antifungal agents a priority. Plants have proven to be an
excellent source of bioactive compounds, including compounds that are able to inhibit fungal
mycelium. These compounds are of interest because they may have the potential to be developed into
new antifungal compounds. Studies in the past few years have revealed some of the compounds
isolated from bryophytes extracts have wide range of antifungal metabolites.
Antifungal Metabolites from Bryophytes
Bryophytes produce a diverse range of antifungal metabolites, including terpenoids, alkaloids,
phenolics, and fatty acids. Some of the most promising antifungal metabolites from bryophytes
include Marchantin A, Marchantin H, Bazzanin, Riccardin, Asterelin, and Drimenol etc as shown in
Figure 1. Marchantin along with its other derivatives are produced by liverworts including
Plagiochasma intermedium and Marchantia polymorpha, as well as by other bryophytes (Asakawa et
al., 1998; Asakawa, 2017). It has been shown to have broad-spectrum antifungal activity, inhibiting
the growth of a wide range of fungi, including Candida albicans, Aspergillus niger, Pyricularia

2
oryzae and Rhizoctonia solani. Marchantin has also been shown to have antibacterial, antioxidant
and antitumor activity, making it an interesting compound for drug development (Zinsmeister et al.,
1991; Niu et al., 2006; Xie et al., 2010). Asterelin A and B, an active compound obtained from
Asterella angusta has been shown to have antifungal activity against Candida albicans, a fungus
responsible for infecting skin, throat and vagina (Qu et al., 2007). Other bryophytes like Trichocolea
mollissima, T. lanata contain prenyl- phenyl ethers that act as mild fungicidal. Besides this, the
efficacy of organic extracts of various mosses and liverworts also showed interesting inhibitory
effects against fungus. Fungicidal activity of an organic extract of Grimmia anodon and Syntrichia
ruralis on S. cerevisiae and acetone extract of Tortella tortuosa against Candida. albicans has been
reported (Elibo et al., 2011). Ethanolic extract of moss Bryum argenteum is effective against
Aspergillus niger, Penicillin ochrochloron, Candida albicans, Trichophyton mentagryophytes.
Dimethyl sulfoxide (DMSO) extract of Atrichum undulatum and Marchantia polymorpha has been
demonstrated to display containment activities to various degrees against pathogenic fungal species.
Asperigulus versicolor and A. fumigates (Sabovljević et al., 2011). Methanol, ethanol and ethyl
acetate extract of Porella arboris-vitae is a natural antimicrobial agent for food preservation and
have found to be effective against Aerobasidium pullulans, Pichia anomala, Pichia
membranaefaciens, Saccharomyces cerevisiae and Zygosaccharomyces bailii (Tyagi et al., 2013).
Water, alcohol and hexane extracts of Pallavicinia lyellii from family Pallaviniaceae inhibit the
growth the Fusarium oxysporum and Candida albicans (Subhisha & Subranomiam, 2006).
Table-1: Antifungal activities of compounds isolated from bryophytes.
Compounds Species Family Activity Against References
Fungal Species
Asterelin A Asterella angusta Aytoniaceae Candida albicans Qu et al.,2007
Asterelin B
Acetyltriflocusolide Targionia Targioniaceae Cladosporium Neves et al., 1999
Lactone lorbeeriana cucumerinum
11-Αh- Targionia Targioniaceae Cladosporium Ludwiczuk &
Dihydrodehydrocostu lorbeeriana cucumerinum Asakawa, 2019
s Lactone
Bazzanin B Bazzania trilobata Lepidoziaceae Botrytis cinerea Scher et al., 2004
Bazzanin S Cladosporium
cucumerinum,
Pyricularia oryzae
Septoria tritici
Drimenol Bazzania trilobata Lepidoziaceae Cladosporium Scher et al., 2004
cucumerinu,
Septoria tritici
Drimenal Bazzania trilobata Lepidoziaceae Phytophtora infestans Scher et al., 2004
Septoria tritici
5-Hydoxycalamenene Bazzania trilobata Lepidoziaceae Pyricularia oryzae Scher et al., 2004
7- Bazzania trilobata Lepidoziaceae Botrytis cinerea Scher et al., 2004

Hydroxycalamenene Cladosporium
cucumerinum,
Phytophtora infestans
Pyricularia oryzae
Septoria tritici
Isoplagiochin D Bazzania trilobata Lepidoziacea Zymoseptoria tritici Asakawa, 2017
Lepidozia incurvata

3
Isoriccardin C Plagiochasma Aytoniaceae Candida albicans Asakawa, 2017

intermedium
Gymnomitrol Bazzania trilobata Lepidoziaceae, Phytophthora infestans Scher et al., 2004
Gymnomitrion Gymnomitriaceae Pyricularia oryzae
obtusum Zymoseptoria tritici
Herbertenol Herberta adunca Herbertaceae Botrytis cinerea Matsuo et al,1986
Phizoctonia solani
Pythium debaryanum
Marchantin A Marchantia species Marchantiaceae Aspergillus niger Niu et al., 2006.
Pyricularia oryzae Zinsmeister
Rhizoctonia solani et al.,1991.
Saccharomyces
cerevisiae Trichophyton
mentagrophytes
Marchantin H Marchantia Marchantiacea Candida albicans Xie et al.,2010
polymorpha Ayotoniaceae
Ptagiochasm
intermedlum
Neomarchantin A Marchantia Marchantiaceae Candida albicans Niu et al., 2006
Neomarchantin B polymorpha
Plagiochin E M. polymorphya Marchantiaceae Candida albicans Wu et al., 2010
Riccardin C Asterella angusta Aytoniaceae, Candida albicans Xie et al., 2010
Plagiochasma Aytoniaceae
intermedium
Riccardin D Dumortiera hirsuta Marchantiaceae Candida albicans Cheng et al., 2009
Riccardiphenol C Riccardia crassa Aneuraceae Candida albicans Perry &
Trichophyton Foster,1995
mentagrophytes
Lunularin Lunularia cruciata Marchantiaceae Alternaria brassicola Subhisha &
Botrytis cinerea Subramoniam,
Septoria nodorum 2005
Uromyces fabae
Trichomanin Homalia Neckeraceae Candida albicans Wang et al., 2005
trichomanoides
Secondary metabolites from Bryophytes showing Anti-fungal properties

It is undeniable that bryophytes produce a large array of secondary metabolites, and dynamically
involved in maintaining cell integrity and cell defense mechanisms (Cornelissen et al, 2007).
Terpenoids, flavonoids, phenolic compounds and bibenzyls, are widely distributed secondary
metabolites out of which terpenoids contribute a high level of variability in terms of its abundance in
liverworts due to high presence of oil compartments. Neither mosses nor hornworts possess these oil
bodies, but they have the ability to produce different types of secondary metabolites as well.
Terpenes
According to the number of carbon atoms in the structure, terpenoids are categorised into
monoterpenoids (C10), sesquiterpenoids (C15), diterpenoids (C20), and triterpenoids (C30). They
are the most prevalent in bryophytes. Terpenoids produced by liverworts are of extraordinary interest
because of their structure and biological action (Asakawa, 1982). Isolation of two monoterpene
acetates from Targionia hypophylla, which are responsible for the plant's distinctive scent, has been
reported for this genus. Mosses produce ß-citrocitral, ß-pinene, limonene and camphene but majority
of liverworts accumulate specific compounds including limonene, sabinene, pinene, and the acetate

4
derivatives of myrtenol, borneol, geraniol (Commisso et al., 2021). Some of these compounds
demonstrated anti-fungal activities as well. According to the current information available for
hornworts, Anthoceros caucasicus produces more limonene than any other monoterpenoid.
Phenolics
Although the presence of several phenolic chemical types in mosses and liverworts has long been
known, nothing is known about the chemistry of phenolics in hornworts. The phenolic compounds
are categorized into monolignols, flavonoids, bibenzyls, bisbibenzyls, and other structures. The
bryophytes that produce the monolignols p-coumaryl alcohol and coniferyl alcohol, have shown to
be efficient fungicides against many plants’ fungus like Ceratocystic spp. and Verticillium
longisporum. Tomentellin and demethoxytomentellin are isoprenyl benzoates derived from the
liverwort Trichocolea tomentella, and antifungal compound trichocolein, isolated from Trichocolea
lanata, exhibited inhibition against Candida albicans and Trichophyton mentagrophytes. Most
reports have found that mosses typically contain biflavonoids and triflavonoids, liverworts moistly
produce monoflavonoids and hornworts are devoid of any flavonoid pathway due to their early
phylogenetic divergence from early land plants (Commisso et al., 2021). All these phenolic
compounds and its derivatives not only contribute to medicinal benefits but also protect the plant
from potential herbivory.
Potential Application and Challenges of Antifungal bryophytes
Bryophyte antifungal metabolites have the potential to be turned into new antifungal medications and
biopesticides. Although many antifungal metabolites from bryophytes have been shown to have
activity against fungi, the exact mechanisms by which they exert their activity are not well
understood. There are several challenges that need to be addressed before these compounds can be
developed into effective drugs. One of the main challenges is the limited availability of bryophytes in
nature, which makes large-scale production of these compounds difficult. Furthermore, the extraction
and purification of these compounds can be challenging, as many of them are present in low
concentrations in bryophyte tissues.
Conclusions
Antifungal metabolites from bryophytes have the potential to be developed into new antifungal drugs
and biopesticides. These compounds have been shown to have activity against a wide range of fungal
species, including drug-resistant strains, making them potentially safe for use. In addition to their
potential use as antifungal metabolites, bryophytes have other applications, including as natural
preservatives, bioherbicides and for the treatment of a wide range of diseases in both plants and
humans. The potential applications of antifungal metabolites from bryophytes make them an exciting
area of research Further studies are needed to elucidate the mechanisms of action of these
compounds, which could lead to the development of new class of safe and effective drugs, medicine,
antibiotics and beyond.
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28. Xie, C. F., Qu, J. B., Wu, X. Z., Liu, N., Ji, M., & Lou, H. X. (2010). Antifungal macrocyclic
bis (bibenzyls) from the Chinese liverwort Ptagiochasm intermedlum L. Natural Product
Research, 24(6), 515-520.
29. Zinsmeister, H. D., Becker, H., & Eicher, T. (1991). Bryophytes, a source of biologically active,
naturally occurring material. Angewandte Chemie International Edition in English, 30(2), 130-
147.
***
7
Chapter 2
THE STUDY OF FUNGAL SPECIES ISOLATED FROM
Amaranthus viridis AND Hibiscus cannabinus IN
COMPARISON FROM FIELD AND MARKET SAMPLES
Dr. A. Pramila
A bstract
The microorganisms are known to colonize diversified habitats helping in recycling of elements,
organic matter and also help in the plant growth and productivity. The substances like Soil,
Rhizosphere, Rhizoplane and Phylloplane are considered as important ecological niches for millions
of microorganisms. There are several field problems of crop plants and vegetable plants effecting
yield potential which include pests and diseases. Most of the diseases in plants are caused by
microorganisms such as fungi, bacteria and Actinomycetes. In India the family Malvaceae is
represented by 25 genera and 110 species, Amaranthaceae is represented by 46 genera and 151
species. Both of the plants are edible and are a rich source of vitamins and minerals essential for
human nutrition. The Fungal species isolated from Amaranthus field samples shows the higher
number of fungal populations followed by Rhizosphere, Rhizoplane, Non -Rhizosphere and
Phylloplane populations. The fungal species isolated from Amaranthus market samples were equally
dominant in Non –Rhizosphere soils, Rhizosphere, Rhizoplane and Phylloplane populations. The
Fungal species isolated from the Hibiscus cannabinus field and market from non-Rhizosphere soils,
Rhizosphere, Rhizoplane and Phylloplane are equally dominant.
Keywords: Rhizoplane, Rhizosphere, Phylloplane, Amaranthus viridis, Hibiscus cannabinus.
Associate Professor & Head, Department of Botany, Andhra Mahila Sabha, Arts and Science
College for Women, O. U. Campus, Hyderabad, India pramila21ams@gmail.com
Dr. A. Pramila 8
I ntroduction
Microbial ecology is an emerging discipline as an integral branch of Microbiology, soil science and
Biochemistry. The microorganisms are known to colonize diversified habitats helping in recycling of
elements, organic matter and also help in the plant growth and productivity. The substances like Soil,
Rhizosphere, Rhizoplane and Phylloplane are considered as important ecological niches for millions
of microorganisms. The study on Microbial ecology of leafy vegetables in particular Amaranthus
viridis, Hibiscus cannabinus, both are common leafy vegetables rich in Iron, macro and micro
nutrients. They are grown widely by the farmers and in kitchen gardens. Hibiscus cannabinus
belongs to Malvaceae family. In India the family Malvaceae is represented by 25 genera and 110
species. In India the family Amaranthaceae is represented by 46 genera and151 species occurring
mostly in the warmer parts. Few plants are of medicinal value. There are several field problems of
crop plants and vegetables plants effecting yield potential which include pests and diseases. Most of
the diseases in plants are caused by microorganisms such as fungi, bacteria and actinomycetes. The
aerial and subterranean portions of the plants are known to be congenial sites for the colonization of
the microbes. The microbes are known to colonize diverse habitats and substrates including plants.
The study of Microbial ecology of these plants are important as large number of microbes are
associated with Soil, Rhizosphere, Rhizoplane and Phylloplane and also helping the soil fertility,
transformation of elements, plant nutrition's and plant growth. Therefore, an attempt is made in
understanding microbial ecology of Soil, Rhizosphere, Rhizoplane and Phylloplane of Amaranthus
viridis and Hibiscus cannabinus.
The quantitative and qualitative nature of the microbes in the soil are subjected to constant state of
flux by edaphic, biotic and environmental conditions. Number of these microbes are very important
as they are involved in recycling of organic wastes, Carbon, Nitrogen and Phosphorous cycles,
Mineralization etc. They are also source of antibiotic, organic and production which are of immense
value in human welfare. The ecological study of soil and microbes attained importance as number of
Algae, Fungi, Actinomycetes are rich source of antibiotics and other important organic byproducts.
Various aspects of soil fungi, microhabitat, dynamic equilibria, decomposition of organic matter,
fungal physiology, antagonism were found. The extensive studies on soil, rhizosphere, rhizoplane
and phylloplane microflora of different plants revealed diversified mycoflora and microflora
differing from plant to plant and region to region. New fungal species were identified at regular
intervals.
Review of Literature
The various aspects of soil fungi and microhabitat dynamic equilibria, decomposition of organic
matter, fungal physiology, antagonism, isolating techniques have been reviewed extensively at
international symposium on "Ecology of Soil Fungi" at Liverpool by Parkinson and Waid (1960).
The microorganisms are known to interact with each other with the host plant simultaneously
resulting in useful effects on the host plant and at other times causing disease conditions. There are
several field problems of crop plants and vegetable plants effecting yield potential which include pest
and diseases. Most of the diseases in plants are caused by microorganisms such as fungi, bacteria and
actinomycetes. The specialized ecological niches that are of importance to plants are soil,
Dr. A. Pramila 9
rhizosphere, rhizoplane and phylloplane. The association of the microbes with host plant interaction
and microbe to microbe interaction. The microbes associated with soil rhizosphere and phylloplane
were found to differ quantitatively qualitatively.
The discovery of various isolating techniques in isolating diverse groups of microorganisms have
revealed that diverse groups of microflorae inhabit the soils and they are found in all habitats of the
soil on the biosphere and they form an important soil biomass Number of fungal, bacterial and
actinomycetes species were reported to thrive well in the soil. Number of these microbes are very
important as they are involved in recycling of organic waste, carbon, nitrogen and phosphorous
cycles, mineralization etc. The ecological study of soil for microbes attained importance as number
of Algae, Fungi and Actinomycetes are rich source of antibiotics and other important organic by
products.
Most of the fungi bacteria and actinomycetes are microscopic showing vast variation quantitatively
and qualitatively in different sites of collection and at different soil depths. Most of the
microorganisms live as saprophytes, some are parasitic and others are symbiotic in relation to living
organic substrates. The discovery of several isolating techniques by several workers such as dilution
plate technique by Waksman (1952), soil plate method and modified soil plate methods of Warcup
(1950), immersion plate technique (Thornton, 1952), direct microscopic examination (Conn 1918)
root maceration (stever and waite, 1954) and many other techniques for essaying of microbes
resulted in their quantitative and qualitative estimates in ecological niches of the soil.
The utilization of these isolating techniques and culture media contributed immensely in our present
day understanding of the soil, rhizosphere or rhizoplane microorganisms. Soil fungi in relation to
habitat in different geographical areas was made by Niethammer (1935).
Using different isolating techniques microorganisms were isolated and identified by number of
workers (Tumonin, 1941, Starkey, 1938) from rhizosphere and from rhizoplane (Lauw and Webley,
Harley and Waid, 1955, stover and waite 1953). Mishra (1968) studied the rhizosphere microflora of
fiber yielding plants. Manoharachary et al. (1977) studied the microbial population of rhizosphere of
five economically important plants. Satyaprasad (1982) studied the rhizosphere and rhizoplane
mycoflora of wilt resistant and susceptible chickpea varieties.
The leaf surface acts as a landing stage for aerial spores. The leaf is a site for both saprophytic and
parasitic fungi. The microorganisms are subjected to fluctuations in the atmosphere such as light,
temperature and relative humidity.
The phylloplane fungal numbers were more on matured leaves followed by Young and Senescing
leaves of Sida acuta and Ervutamia coronaria. Phyllosphere microflora of Sunflower at different
stages of plant growth was studied by Dwivedi and Vijay Kumar (1981) and they have reported 33
fungal species.
The maturity of the leaf was found to be another important factor influencing the composition of
microorganisms. The numbers increased with increase in age of the leaf of Beta vulgaris (Kerling
1958). Similar results were reported by Hudson (1962) on sugar cane leaves of certain tropical
plants.
Media for Isolation of Fungi
For the present study the plants of Amaranthus and Hibiscus were raised in the field and compared
with the market samples to study the ecology and population dynamics of Fungi. The plant samples
were collected at 20 days intervals and were subjected to microbial analyses.
Dr. A. Pramila 10
The Fungi were isolated using Vegetables Agar Medium (VAM), Potato Sucrose Agar Medium
(PSA) The following media are prepared with the mentioned proportions and the pH is maintained at
6.0 to 6.5 for PSA and 7 for VAM.
a) Potato Sucrose-Agar (PSA)

Potatoes 200gms
Sucrose 20gms
Agar 20gms
Water 1000ml
b) Vegetable Agar Medium (VAM)
Tomato 200gms
Potato 200gms
Carrot 200gms
Beet root 200gms
Cabbage 200gms
Green Beans 200gms
Pumpkin 200gms
Choko 200gms
Slide Preparation
Lacto phenol and cotton blue in lacto phenol were used as mounting and staining. The fungi were
identified and few fungal species were photographed
Isolation of Mycoflora
A) Dilution plate method:
For Quantitative estimation of fungi, the dilution plate method of Waksman (1952) as described by
Johnson and Curl (1972) was used, as it allowed Qualitative and Quantitative assessments.
Five grams of sample was shaken by hand for 10 minutes to 20 minutes in 50ml sterile distilled
water and successive dilutions were made as required 1:10,000 dilutions were chosen for the
Quantitative Estimation of fungi. One ml of dilutant was transferred aseptically into sterile Petri
dishes of each sample and sterile medium was added. The suspensions were mixed well with the agar
by rotating the plate in clockwise and anticlockwise direction and then allowed to set.
B) Isolation and Estimation of Rhizoplane Fungi
To isolate fungi from the rhizoplane root pieces of 5mm length were collected and were macerated
and fungi were enumerated following the technique of Stover and Waite (1953). The roots from
which was collected were washed thoroughly with sterile water and dried between filter paper. 5
grams of roots in 50ml of serial distilled water were macerated in sterilized waring blender (Singh
1965) and of serial dilutions were prepared from the blended material to get a final dilution of
1/10,000 to isolate the fungi. One micro litre this dilutant was pipetted out into sterilized petri dishes
to which melted and cooled medium were added. Plates were incubated for 5 to 7 days at room
temperature.
C) Isolation and Estimation of Phylloplane Fungi:
Procedure to isolate the Estimation of Phylloplane fungi same method of isolation and estimation of
Rhizoplane has been followed.
Dr. A. Pramila 11
Table-1: List of Fungal Species isolated from Amaranthus viridis (Field)

S. No. Species Name NRS RS RP PP
1 Ascomycetes Sp. + +
2 Aspergillus Sp. + + +
3 Aspergillus candidus + +
4 Aspergillus flavus + +
5 Aspergillus fumigatus + + +
6 Aspergillus luchuensis +
7 Aspergillus luchuensis +
8 Aspergillus Sulphureus +
9 Aspergillus terreus +
10 Aspergillus versicolor + +
11 Cercospora Sp. + + +
12 Chaetomium aureum + +
13 Chaetomium globosum +
14 Chaetomium Sp. +
15 Circinella Sp + + +
16 Cladosporium herbarum + +
17 Cladosporium oxysporum + +
18 Colletotrichum sp. + +
19 Colletotrichum falcatum + + + +
20 Curvularia borrerae + +
21 Curvularia lunate + + + +
22 Curvularia pallescens + + +
23 Curvularia Sp. + + +
24 Doratomyces Sp. + + +
25 Dreschslera australiensis + +
26 Dreschslera rostrata + + + +
27 Fusarium dimerum + + +
28 Fusraium javaniscum + + +
29 Fusarium oxysporum + + + +
30 Fusarium solani + +
31 Fusraium Sp + + +
32 Homicola Sp + + + +
33 Memnoniella echinulata + +
34 Mucor flavus + + +
35 Monosporium Sp. + +
36 Mucor globossus + + +
37 Neocosmospora vasinfecta +
38 Paecilomyces fusisporus + + +
39 Penicillium Sp + + +
40 Penicillium citreao-virde + +
Dr. A. Pramila 12
41 Penicillium fumigatus + + +
42 Penincillium funiculosum + +
43 Penicillium varians + + + +
44 Penicillium atropurpurea + +
45 Perisporium Sp + + +
46 Phoma feckelli + + +
47 Phoma glomerata +
48 Phoma humicola + +
49 Phycomyces Sp. + +
50 Pyrenochaeta Sp. + +
51 Rhizoctonia bataticola +
52 Rhizopus nigricans + +
53 Scolecobasidium humicola +
54 Scopulariopsis Sp + +
55 Spicaria elegans + +
56 Spicaria griseola + +
57 Syncephalstrum racemosum + +
58 Syncephalastrum Sp +
59 Thielavia sp. + +
60 Torula herbarum + +
61 Trichoderma viridae +
62 Zygorhynchus Sp + + +
63 Unidentified + + +
STERILE MYCELIA
1 Ash sterile + +
2 Brown sterile + +
3 White sterile + + +
Table-2: List of Fungal Species isolated from Amaranthus viridis (Market)

S. No. Species Name NRS RS RP PP
1 Acrophialophora nainiana + +
2 Alternaria alternate + +
3 Alternaria Sp. +
4 Aspergillus candidus + +
5 Aspergillus candidus + + +
6 Aspergillus nidulans + +
7 Aspergillus ochraceous +
8 Aspergillu sydowi + +
9 Aspergillus terreus + +
10 Aspergillus versicolor + +
11 Aspergillus violaceo-fuscus + +
12 Bispora Sp. + +
Dr. A. Pramila 13
13 Cercospora sp + +
14 Chaetomium aureum + +
15 Chaetomium globosum + +
16 Chaetomium Sp. + + +
17 Circinella sp. +
18 Cladosporium oxysporum + + +
19 Colletotrichum Sp. + + +
20 Curvularia borreriac + +
21 Neocosmospora vasinfecta + +
22 Paecilomyces fusisporus + +
23 Penicillium Sp + +
24 Penicillum citreo-viride + +
25 Penicillium fumigatus +
26 Penicillium varians + +
27 Periconia atrapurpurea +
28 Phoma feckelli + + +
30 Phoma sp. + +
31 Phycomyces Sp. + + +
32 Pithomyces flavus + +
33 Scolecobasidium humicola + + +
34 Scopulariosis Sp. + +
35 Spicaria elegans +
36 Stachbotrys atra +
37 Syncephalastrum racemosum + +
38 Syncephalastrum sp. + +
39 Torula terrestris +
40 Trichurus spiralis +
41 Unidentified +
STERILE MYCELIA
1 Grey sterile + +
2 Ash sterile +
Table-3: List of Fungal Species isolated from Hibiscus cannabinus (Field)
S. No. Species NRS RS RP PP
1 Absidia glauca + + +
2 Aspergillus chevalieri + +
3 Aspergillus funiculosus + + + +
4 Aspergillus luchuelsis + +
5 Aspergillus nidulans + + +
6 Aspergillus niger + +
7 Aspergillus ochraceous + +
8 Aspergillus flavus + + +
Dr. A. Pramila 14
9 Aspergillus sydowi +
10 Cephalosporium aceremonium + +
11 Chaetomium funicola + +
12 Colletotrichum Sp. +
13 Curvularia pallescens +
14 Curvularia Sp. +
15 Dreschlera Sp. + + +
16 Dreschlera Sp. + + +
17 Fusarium oxysproum +
18 Fusarium solani +
19 Fusarium Sp. + + +
20 Fusarium dimerum + +
21 Helminthosporum + +
22 Heterosporium + +
23 Humicola + + +
24 Humicola sp + + +
25 Hyalopus + + +
26 Isoachlya + + +
27 Mesobotrys simplex + +
28 Mucor +
29 Mucor flavus + +
30 Paecilomyces austriacus +
31 Paecilomyces fusisporus + + +
32 Paecilomyces varioti + + +
33 Paecilomyces viridae +
34 Periconia atropurpurea + + + +
35 Penicillium citreo-viride + + +
36 Penicillium notatum + + + +
37 Penicillium Sp. + + +
38 Penicillium victoriae +
39 Thielavia Sp. + + + +
40 Phoma humicola + + +
41 Phoma feckelli + +
42 Phoma sp. + +
43 Phycomyces Sp. + + +
44 Torula Sp. + +
45 Pyronema sp. + +
46 Rhizoctonia + + +
47 Rhizopus +
48 Scolecobasidium constrictum + + + +
49 Scoupulariopsis alba + + +
50 Scopulariopsis alba + + +
Dr. A. Pramila 15
51 Spicaria elegans +
52 Syncephalastrum Sp. + + +
53 Torula allii + +
54 Torula terrestris + + +
55 Trichurus Sp. + + +
56 Trichoderma Sp. + +
57 Verticilliastrum Sp. +
58 Zygorhyncus + +
STERILE MYCELIA
1 White sterile + + +
2 Black sterile + + +
3 Green sterile + +
Table-4: List of Fungal Species isolated from Hibiscus cannabinus (Market)

S. No. Species NRS RS RP PP
1 Asperigillus nidulans + + + +
2 Asperigillus sydowi + + +
3 Asperigillus terreus + + + +
4 Asperigillus variecolor + +
5 Asperigillus violaceo-fuscus + + +
6 Bispora Sp. + + +
7 Cercospora sp. + +
8 Chaetomium aureum + + + +
9 Chaetomium sp. + +
10 Cladosporium oxysporum +
11 Collectotriuchm sp. +
12 Curvularia borreriae + +
13 Neocosmopora vasinfecta +
14 Paccilomyces fusisporus + +
15 Penicillium sp. + +
16 Penicillium citreo-viride + +
17 Penicillium furmigatus +
18 Penicillium varians +
19 Penconia atropurpurea +
20 Phoma feckelli +
22 Phoma sp. + +
23 Phycomyces sp. +
24 Pithomyces flavus + + +
25 Scolecobasidium humicola + +
26 Scopulariopsis sp. +
27 Spicaria elegans + +
28 Stachybotrys atra +
Dr. A. Pramila 16
29 Syncephalastrum racemosum +
30 Syncephalastrum sp. +
31 Torula terrestris +
32 Trichurus spiralis +
33 Unidentified + +
STERILE MYCELIA
1 White sterile +
2 Brown sterile + +
3 Black sterile + +
*NRS - Non-Rhizosphere Soil, RS - Rhizosphere soil, RP - Rhizoplane, PP - Phylloplane
Discussion
The microbes maintain dynamic equilibria in different soils. The literature available on soil fungi
shows that lot of data is available in respect of fungal flora. However, information of soil and
phylloplane fungi in relation to vegetable crops is meagre. Most of the species of fungi, are known to
dwell in the top most layer of the soil. The microorganisms which live in the soil are of an excellent
source of new genetic material for molecular biologists.
The soils provide nutrients for the growth and multiplication of millions of microbes for the higher
plants using dilution plate technique (Waksman, 1952), modified soil plate method (Warcup, 1950),
immersion plate technique (Thornton, 1952) and different other methods developed from time to
time help in isolation and enumeration of soil fungi.
Fungal colonies isolated from Amaranthus Fungal colonies isolated from Himiscus
viridis field and market samples in different cannabinus field and market samples in
ecological niches different ecological niches
60 52 50 42 42
Fungal colonies
Fungal colonies
43 40
40 29 30
31 28 30
25 22 22 18 18
19 20 16 15
20
10
0 0
NRS RS RP PP NRS RS RP PP
Ecological niche Ecological Niche
H.cannabinus field sample
A.viridis Field A.viridis Market H.cannabinus market sample
Dr. A. Pramila 17
Abundance Of Funal Colonies In Comparision To Field And Market

Samples Of A.viridis And H. cannabinus
A.viridis H.cannabinus
70 63
60
58
41
Fungal colonies
50
40 33
30
20
10
0
Field sample Market sample
Dr. A. Pramila 18
Dr. A. Pramila 19
Population Dynamics of Mycoflora in different Soil Ecological Niches

The results of fungal colony numbers indicate that the fungi were relatively high in the field samples
of both Amaranthus viridis and Hibiscus cannabinus compared with that of the market samples. This
shows that the field samples are free from the spraying of the chemicals whereas the market samples
were treated with some chemicals. This evidence clearly shows that the crop plants or leafy
vegetables are inhabited with several microorganisms in the soil. Viz, Fungi, Bacteria,
Actinomycetes. The study clearly shows that the organic nutrition available would generally been
utilized by various microorganisms which are inhabited in the soil. The above data clearly indicates
the importance of nutrition levels in various leafy vegetables that are available for the fungal species
harbouring in the different ecological niches of these plants.
Dr. A. Pramila 20
The Phenomenon of accumulation of microorganisms around the root zone was reported by number
of earlier workers (Agrihothrudu 1955, Starkey 1958, Jackson 1960). In the present study also the
concentration of Rhizosphere and Rhizoplane microflora is higher than the non-rhizosphere
mycoflora in the field samples of Amaranthus and Hibiscus. However, the market samples of
Amaranthus and Hibiscus do not show much significant differences of Rhizosphere, Non-
Rhizosphere, Rhizoplane and Phylloplane.
Taxonomy of Fungi
Non-Rhizosphere soil
The data of fungal species from Amaranthus field samples are 31, fungal species from Amaranthus
Market samples are 25, fungal species of Hibiscus cannabinus field samples are 29, fungal species of
Hibiscus cannabinus market samples are 18. The overall data from two samples of each indicate
association of 103 fungal species.
Rhizosphere Soil Fungi
Market samples are 22, fungal species of Hibiscus cannabinus field samples are 42, fungal species of
Hibiscus cannabinus from market samples are 18.
Rhizoplane Mycoflora
market samples are 16, fungal species from Hibiscus cannabinus field samples are 42, fungal species
from Hibiscus cannabinus market samples are 16.
Phylloplane Mycoflora
market samples are 19, Fungal species from Hibiscus cannabinus field samples are 30, fungal species
from Hibiscus cannabinus market samples are 15.
Conclusion
The study of fungal species isolated from Amaranthus viridis and Hibiscus cannabinus in comparison
from field and market samples has shown that compared to market samples the fungal colonies were
more in field samples. The low number of fungal colonies reflects the use of fungicides and other
chemicals.
A total of 63 fungal species were isolated from Amaranthus viridis field, 41 fungal species from
market samples. A total of 58 fungal species were isolated from Hibiscus cannabinus field, 33 fungal
species were isolated from market samples.
The number of fungal species from the market were less than the field samples of Rhizosphere,
Rhizoplane and Phylloplane of Amaranthus viridis and Hibiscus cannabinus.
A total of 103 fungal species were isolated from Non-Rhizosphere Soil. A total of 134 fungal species
were isolated from the Rhizosphere soil and total of 115 fungal species were isolated Rhizoplane and
Phylloplane harboured 92 fungal species.
In case of the market samples of Amaranthus viridis and Hibiscus cannabinus shows that a total of 40
fungal species were isolated from the Rhizosphere soils, Rhizoplane harboured 38 fungal species and
a total of 34 fungal species harboured in the phylloplane. This could be due to post harvest methods
used and also the use of insecticides and fungicides during the crop season.
Dr. A. Pramila 21
The higher fungal number of species in the field samples indicated the importance of nutrition levels
in various leafy vegetables, that are available for the fungal species harbouring in the different
ecological niches of these plants.
The study also indicates different niches of Amaranthus viridis and Hibiscus cannabinus supported
with number of fungal species. Among the Aspergilli, Penicillium, Chaetomium, Colletotrichum,
Curvularia, Dreschlera, Fusarium, Humicola, Periconia, Scolecobasidium, are generally higher in
number. They are also known to produce importance secondary metabolites including industrial
valuable organic compounds and antibiotics.
References
1. Alexander, M. 1977. Introduction to soil Microbiology, John Wiley & Sons, New York.
2. Baker K.F. and Synder, W.C. 1965. (eds) Ecology of soil borne plant pathogens: Prelude to
biological control. Univ. of California Press, Barkley.
3. Basu, S. Pad, D. P. Hoo, S.K. and N. Dehera, 1993. Viability of microfungal hyphae in
trophical forest soils. Geobios 20 (3): 127-131.
4. Booth, C. 1971. The genus Fusarium C.M.I. Kew, Surrey, England 1-237.
5. Bose, R.G. 1963. A modified cellulolytic medium for the isolation of cellulolytic fungi from
infected materials and soils. Nature. 198: 508-506.
6. Champman, H.D. 1965. Chemical factors of soils as they affect microorganisms. In: Ecology of
soil borne plant pathogens. Editors: K.F. Baker, and W.C. Snyder. University of California
Press, Berkeley, 122-130.
7. Chand, H. 1937. Study of fungus flora of the Lahore soils. Proc. Indian, Acad. Sci. B (5): 324-
331.
8. Choldony, N. 1930. Ubereine neue methods zur untersuchung der Boden mikroflora, Arch.
Microb, 1: 620-652.
9. Clark, F.E. 1949. Soil microorganisms and plant roots. Advances to Agron 1: 241-288.
10. Collins, M.A. 1982. Rust disease and development of Phylloplane microflora and Antirrhinum
majus cultivar natrum. Trans. Brit. Mycol Soc. 79: 117-122.
11. Conn, H.J. 1918. The microscopic study of bacteria and fungi in the soil. N.Y. Agri.Exp.Sta.
Tech. Bul.64.
12. Dwivedi, R.S. 1965. Ecology of soil fungi of some grass lands of Varanasi 1. Edaphic factors
and fungi Proc.Nat. Acadd, Sci. India 3(35): 255-274.
***
Dr. A. Pramila 22
Chapter 3
MYCOREMEDIATION: ANNIHILATION OF
ENVIRONMENTAL POLLUTANTS
Intwala Siddhi M.1 & Barot Jagruti K.2
A bstract
Pollution thus, has become a universal challenge that is inducing climate change, threatening
biodiversity, and taking a toll on millions of lives every year. Among many pollutants, a significant
proportion is contributed by many malicious and hazardous compounds such as heavy metals,
xenobiotics, pesticides, hydrocarbons, and dyes which are being remitted through industries. The
poor management of the waste and effluents from households, industries, and agricultural fields is
further deteriorating the already crippling ecosystem. According to the World Health Organization
report, 2.2 billion people do not have access to safe water services, and 144 million people using
contaminated water. Mycoremediation is one of the biotechniques that recruits fungi to remove toxic
pollutants from environment in an efficient and economical manner. Mushrooms, macro-fungi, are
among the nature’s most important myco-remediators. Indiscriminate use of chemicals has led to
severe contamination of environmental segments by heavy metals. Heavy metals are non-
biodegradable and tend to be biomagnified in the food chain. These metals can hinder different
cellular processes. Their effects are generally concentration dependent and differ with respect to
individual toxicity. Hence, it becomes crucial to remove them prior to final discharge of effluents in
environmental segments.
Keywords: Mycoremediation, Heavy Metals, Xenobiotic Toxicity.
1Naran Lala College of Professional and Applied Sciences, Navsari.

2Department of Bioscience, Veer Narmad South Gujrat University, Surat, Gujrat, India
Intwala Siddhi M. & Barot Jagruti K. 23

I ntroduction
Mycoremediation is the form of bioremediation in which fungi-based technology is used to

decontaminate the environment. Fungi are among the primary saprotrophic organisms in an
ecosystem, as they are efficient in the decomposition of organic matter. Wood-decay fungi,
especially white rot, secretes extracellular enzymes and acids that break down lignin and cellulose.
Fungi have been proven to be a very cost-effective and environmentally-friendly way for helping to
remove a wide array of toxins from damaged environments or wastewater. These toxins include
heavy metals, persistent organic pollutants, textile dyes, leather tanning industry chemicals and
wastewater, petroleum fuels, polycyclic aromatic hydrocarbon, pharmaceuticals and personal care
products, pesticides and herbicides, in land, fresh water and marine environments. Bioremediation of
toxic organics by fungi is the most sustainable and green route for clean-up of contaminated sites and
we discuss the multiple modes employed by fungi for detoxification of different toxic and
recalcitrant compounds including prominent fungal enzymes viz., catalases, general lipase, laccases,
peroxidases and sometimes intracellular enzymes, especially the cytochrome P450 monooxygenases.
Fungi play an important role in the biogeochemical cycling of manganese and other redox-active
metals, which is related to their ability to survive radiation and other oxidative challenges.
Bioremediation is a simple and cost-effective method that, in the last decades, has received
worldwide a particular attention. The general term “bioremediation” indicates the use of living
organisms (i.e. bacteria, fungi, algae and plants) in the detoxification of polluted soils and
wastewaters. In a bioremediation process, organic and inorganic hazardous substances may degrade,
accumulate or immobilize, resulting in a significant reduction of the contamination level. In the last
decay, the role of fungi in bioremediation has been increasingly recognized (Singh H.,2006;
Goltapeh et al.,2013). About this, various authors have highlighted the ability of fungi, mainly
saprotrophic and biotrophic basidiomycetes, to degrade or to transform toxic compounds (Baldarian
P.,2008; Spina et al., 2018). Mycoremediation is the bioremediation technique which employ fungi
in the removal of toxic compounds; it could be carried out in the presence of both filamentous fungi
(moulds) (Desmukh et al., 2016) and macrofungi (mushrooms) (Chatterjee et al., 2017; Ali et al.,
2017). Both classes possess enzymes for the degradation of a large variety of pollutants (Purnomo et
al., 2013; Kulshreshtha et al., 2013). Fungi are well known for their ability to colonize a wide range
of heterogeneous environments and for their ability to adapt to the complex soil matrices, also at
extreme environmental conditions. Furthermore, they can decompose the organic matter and easily
colonize both biotic and abiotic surfaces (Joutey et al.,2013; Bharat et al., 2019). Filamentous fungi
show some peculiar characteristics that make them more advisable in soil bioremediation than yeasts
and bacteria (de Lima Souza, 2016). The most important are the type of growth (i.e. the development
of a multicellular mycelial network) suited to soil colonization and translocation of nutrients and
water, the production of many bioactive compounds and extracellular enzymes and the unique
capability to co-metabolize many environmental chemicals (Harms et al., 2011). Mycoremediation
represents thus a biological tool to degrade, transform or immobilize environmental contaminants.
The state of the art of soil mycoremediation is reviewed in the present chapter. A particular attention
is given to the fungal species and enzymes involved in the biodegradation processes, together with
the classes of toxic compounds that could be biodegraded. Bioremediation strategies (i.e.

biostimulation and bioaugmentation) and significant examples of microcosm and field studies are
also discussed. Finally, the application of mushrooms as emerging technology in soil
mycoremediation is reported.
Types of Mycoremediation
Mycoremediation are of two types: in situ and ex situ mycoremediation. In the first case, the
treatment is done at the polluted site, whereas in the second condition the soil from the polluted site
is bored out and taken to another site for environmental clean-up purpose.
Fungi Used in Mycoremediation
The kingdom Fungi is a highly distinct monophyletic group of eukaryotes found in numerous
environments, particularly in terrestrial ecosystems (Richards, Leonard & Wideman, 2017;
Stajich, 2017). Fungi play key roles in nutrient cycling, can act as predators, pathogens and parasites
of myriad other organisms, and can be found living in symbiotic associations with plants, algae,
animals and other organisms. Due to the adaptation to their environment, fungi developed unique
bioremediation properties. Practically all natural organic compounds can be degraded by one or more
fungal species by the production of enzymes such as amylases, lipases, and proteases that allow them
to use substrates as starches, fats, and proteins. Other limited number of species can use pectin,
cellulose, and hemicelluloses as carbon sources. Some fungi are the main degraders of natural
complex polymers which are resistant to microbial attack, such as keratin, chitin, and lignin
(Patel et al., 2023).
Classification of fungi based on their involvement in remediation process of pollutants is given
below:
• Ligninolytic fungal degradation
• Soil fungal biosorption
• Mycorrhizal fungal degradation (Vijaya Kumar, 2018).
Important fungal species involved in bioremediation
The most suitable fungi to be used in soil remediation are basidiomycetes and, in the ecological
groups of saprotrophic and biotrophic fungi (Treu and Falandysz., 2017). The saprotrophic
basidiomycetes, which use dead organic matter as a carbon source, include the wood-degrading
fungi. Among them, white-rot fungi are considered for the leading role in biodegradation (Ellouze
and Sayadi.,2016). White Rot Fungi can remediate efficaciously both lignin and cellulose
biopolymers till the complete mineralization (Abdel-Hamid et al., 2013), thanks to the production of
an extracellular enzymatic complex, which comprehend lignin peroxidases (LiPs), manganese-
dependent peroxidases (MnPs), versatile peroxidases (VPs), laccases, H2O2-generating oxidases and
dehydrogenases, produced during the idio-phase, usually under nitrogen depletion. Some of the most
representative WRF, able to degrade pollutants, include Phanerochaete chrysosporium, Pleurotus
ostreatus, Trametes versicolor, Bjerkandera adusta, Lentinula edodes, Irpex lacteus, Agaricus
bisporus, Pleurotus tuber-regium and Pleurotus pulmonarius (Voběrková et al.,2018; Manavalan et
al., 2015). Among these fungi, Phanerochaete chrysosporium has been the most investigated for its
ability to degrade toxic or insoluble compounds to CO2 and H2O, more efficiently than other fungi.
In 1985, for the first time, Bumpus et al. proposed the application of Phanerochaete chrysosporium
in bioremediation studies, and the fungus became a model system in the mycoremediation field
(Bumpus et al., 1985). The biotrophic basidiomycetes comprehend ectomycorrhizas which obtain the
carbon source from a mutualistic plant partner: the fungal hyphal network envelopes the root and

penetrates between the cells of the root cortex (Treu et al.,2017). Ectomycorrhizal fungi can
assemble and recycle the nutrients from the organic matter of the soil (Castro Faria., 2018).
Ectomycorrhizal fungi comprehend about 10,000 fungal species; the most representatives are
Amanita spp., Boletus spp., Gautieria spp., Hebeloma spp., Lactarius spp., Morchella spp., Suillus
spp. and Rhizopogon spp. (Harms et al., 2011; Meharg and Cairney, 2000). ECM fungi secrete
enzymes to get nutrients by means of the degradation of molecules in the soil organic matter. ECM
possesses extracellular and cytosolic enzymes which attack molecules containing N and P atoms.
Hydrolytic enzymes comprehend β-glucosidases and phosphatases, while the oxidative ones are
peroxidases and phenol oxidases (Rúa et al.,2015). The ECM fungi application is important in habitat
where the litter layer is restricted and consequently ligninolytic enzymes, typical of wood fungi, are
not so efficacious; in this contest, ECM fungi are able to produce enzymes to sequester nutrients
directly from the soil. The same enzymes allow ECM to degrade many persistent organic pollutants
(Kumar and Atri., 2018) Most of the biodegradation studies at the laboratory and field scale are
concerned to microfungi, but in the last years, much attention has been given to mushrooms which
are broadly present in soil and also easily soil-cultivated (Li et al., 2017). Bioremediation by
macrofungi basidiomycetes is reported by (Ji et al.,2012) to be advantageous because, together with
remediation, soil is enriched with organic matter and nutrients and plant growth results enhanced.
These macrofungi are potent degraders thanks to the secretion of the same non-specific enzymes
(LiP, MnP and laccase) described for the wood-degrading fungi and, for this reason, are interesting
in the bioremediation field. At the same time, they grow to a great extent producing high biomass
quantities, when cultivated on carbon sources, such as straw or sawdust (Adenipekun and
Lawal.,2012) The mushroom biomass can be a protein source or can contain biologically active
compounds such as phenols with antioxidant activity (Kulshreshtha et al., 2013; Boonsong et
al.,2016). Furthermore, mushroom biomass can be applied in biosorption treatment thanks to its
ability to accumulate ions and xenobiotics from contaminated soils (Thakur M., 2019).
Fungi Used in Mycoremediation
Fungi are unique organisms due to their morphological, physiological, and genetic features. They are
omnipresent, able to colonize natural environments (soil, air, water), in which they help in
maintaining the ecosystem’s equilibrium. Due to the adaptation to their environment, fungi
developed unique bioremediation properties. Practically all natural organic compounds can be
degraded by one or more fungal species by the production of enzymes such as amylases, lipases, and
proteases that allow them to use substrates as starches, fats, and proteins. Other limited number of
species can use pectin, cellulose, and hemicelluloses as carbon sources. Some fungi are the main
degraders of natural complex polymers which are resistant to microbial attack, such as keratin, chitin,
and lignin.
Classification of fungi based on their involvement in remediation process of Pollutants is given
below:
• Ligninolytic fungal degradation
• Soil fungal biosorption
• Mycorrhizal fungal degradation (Gupta and Srivatsava 2014).
Ligninolytic Fungal Degradation
Lignocellulose is a renewable organic material and is the major structural component of all plants.
Lignocellulosic wastes are produced in large amounts by many industries including those of forestry,

pulp and paper, agriculture, and food. Such wastes are also present in municipal solid waste (MSW)
and animal wastes. Lignocellulolytic fungi are classified into three groups: white rots, brown rots,and
soft rots (Hickman and Perry 2011).
White Rot Fungi (WRF)
They belong to Basidiomycota (Agaricomycotina) and in some of the Ascomycota (Xylaricaceae),
White rots break down lignin and cellulose and commonly cause rotted wood to feel moist, soft,
spongy, or stringy and appear white or yellow. Phanerochaete chrysosporium, Armillaria spp.
(Honey mushroom), Pleurotus, and other oyster mushrooms are the examples of WRF. Other WRF
include Trametesversicolor (turkey tail), Trametes hirsuta (hairy turkey tail), Ganoderma
applanatum (artist’s conk), and Fomes fomentarius (tinder fungus).
Brown Rot Fungi
They exclusively belong to Basidiomycota, namely, Agaricomycetes. This class comprises the brown
rot fungi belong to Agaricales, Hymenochaetales, Gloeophyllales, and Polyporales. Brown rots
mainly decay the cellulose and hemicellulose(carbohydrates) in wood, leaving behind the lignin
(brownish wood). Laetiporus sulphureus (sulfur fungus), Serpula lacrymans (True dry rot),
Fibroporia vaillantii (mine fungus), Phaeolus schweinitzii, and Fomitopsis pinicola are the examples
of BRF.
Soft Rot Fungi
They belong to Ascomycetes and Fungi imperfecti. Both bacteria and fungi are the source for soft
rots. They decay cellulose, hemicellulose, and lignin, in areas directly adjacent to their growth.
Growth of soft rots is much slower than brown and white rots and usually do not cause huge
structural damage to wood of living trees. Examples of soft rot fungi are Chaetomium, Ceratocystis,
and Kretzschmaria deusta.
Soil Fungal Biosorption
Extensive studies on the fungi living in soil Mucor sp., Aspergillus carbonarius, Aspergillus niger,
Rhizopus sp., Saccharomyces cerevisiae, Botrytis cinerea, Neurospora crassa, Phanerochaete
chrysosporium, and Lentinus sajor-caju have shown that they are useful in heavy metal biosorption.
The mechanism of biosorption varied from species to species (Kumar et al. 2009).
Mycorrhizal Fungal Degradation
Mycorrhizal fungi growing as symbionts with plant roots can degrade organic pollutants in soil.
There are several types of mycorrhizae such as ectomycorrhiza, ectendomycorrhiza, arbuscular
mycorrhiza (AM), ericoid mycorrhiza, arbutoid mycorrhiza, monotropoid mycorrhiza, and orchid
mycorrhiza. Ectomycorrhiza, (ECM), arbuscular mycorrhiza (AM), and ericoid mycorrhiza (ERM)
can increase the tolerance to heavy metals.
Biosorption
Soil fungi play a vital role in biosorption of heavy metals from aqueous solutions. Biosorption of
metal ions primarily occurs by surface binding, including ion-exchange reactions and complexation
with the functional groups present on the cell surface. Various functional groups believed to be
involved in metal binding include carboxyl, amine, hydroxyl, phosphate, and sulfhydryl groups
(Kapoor and Viraraghavan 1995).

Mycorrhizoremediation
Mycorrhizal fungal degradation is also called as mycorrhizoremediation. This technology is
employed for reclamation of soils and sediments that have been polluted by industries through
phytoremediation. Arbuscular mycorrhizal fungus (AMF) improves the plant growth in a wide
variety of soils by increased phosphorus and micronutrient uptake, makes the plants tolerate biotic
and abiotic stresses, and improves the soil structure and aeration by the secretion of glomalin.
Mycorrhizal colonization in roots plays a role in protecting the plant root from heavy metals, and the
second, which is widely known as the mycorrhizal colonization of roots, increases root surface area
for nutrient absorption. Mycorrhizal fungal ecotypes from heavy metal contaminated sites seem to be
more tolerant to reference strains from non-contaminated soils. Abundant extrametrical mycelium
was shown to be important for HM binding by the fungus. Most of the heavy metals were
demonstrated to be bound to cell wall components such as chitin, cellulose, cellulose derivatives, and
melanins. The high N and S concentrations associated with polyphosphate granules rather indicate
the occurrence of HM-thiolate binding by metallothionein like peptides (Galli et al. 1994). The
proteins in the cell walls of AM fungi appear to have the ability to absorb potentially toxic elements
by sequestering them. There is evidence that AMF can withstand potentially toxic elements; AMF
produce glomalin on hyphae can enhance HM sequester. The extraradical mycelium of AMF is
adapted in polluted soil to help in the accumulation of heavy metals both within the plant roots
(phytoaccumulation) and extrametrical fungal mycelium. Glomalin plays a vital part in sorption and
sequestration of potentially toxic elements, reducing their bioavailability (Joner et al. 2000).
Conclusion
The capability of micro- and macro fungi to degrade organic pollutants and to decrease heavy metal
concentration in soil is a matter of fact. The growth morphology in soil (i.e. extended hyphal
network), the low specificity of extracellular enzymatic complexes and the possibility to use toxic
compounds as the growth substrate make filamentous fungi more advantageous in bioremediation
processes when compared to other microorganisms. However, in the design of a soil
mycoremediation process, some important aspects must be considered such as the choice of the
appropriate fungal strain and the evaluation of its possible interaction with the contaminated soil
microbiota. To this end, microcosm studies represent a useful and simple method which allows to
evaluate the feasibility of a biodegradation process.
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***
Chapter 4
INVITRO ANTAGONISTIC ACTIVITY OF ENDOPHYTIC
BACTERIA OF Oryza sativa AGAINST SOIL-BORNE
FUNGAL PATHOGEN Fusarium oxysporum
Dr. M. I. Zahir Hussain, R. Marivignesh, A. Shajahan
A bstract
Soil-borne fungal diseases pose serious constraints on agricultural productivity. Biological control is
a non-hazardous strategy to control plant pathogens and enhance crop productivity. The use of
indigenous endophytic bacteria is considered an environmentally friendly and ecologically efficient
strategy. In the present study, a total of 5 endophytes were isolated from the Oryza sativa samples
collected from the Ambasamudram agriculture field in Tamil Nadu and screened in vitro for
antagonistic activity against the soil-borne fungal pathogen Fusarium oxysporum. The five
endophytes AE 1, AE 2, AE 3, AE 4, and AE 5, were obtained. Among them, AE 5 showed 5 mm
zone of inhibition, respectively, against Fusarium oxysporum. This promising endophytic bacterial
isolate obtained from the present study may be formulated as effective biocontrol agent for the
management of fungal pathogens. In the future, they are expected to replace chemical fertilizers,
chemical fungicides, and artificial growth regulators, which have numerous side effects on
sustainable agriculture.
Keywords: Endophytes, Oryza sativa, Ambasamudram, Fusarium oxysporum, Bio-control.
Department of Zoology, Sadakathullah Appa College (Autonomous), Rahmath Nagar,

Tirunelveli-627011 Affiliated to Manonmaniam Sundaranar University, Tirunelveli, Tamilnadu,
India. mizahirhussain@gmail.com

32
I ntroduction
Rice (Oryza sativa) is the most important cereal crop in the world, and it is one of the main staple
foods in India, and the main dietary component of 20% of the world’s population (Seck, et al., 2012).
In the next three decades, the world will need to produce about 60% more rice than today’s global
production to feed the extra billion people (Ladha and Reddy, 2003). Increases in the demand for
rice, as a result of an over population, creates the need to improve rice productivity and one of the
most important factors for high yields of rice production are chemical fertilizers and pesticides,
which may cause environmental pollution and negatively influence human health. Alternatively, a
relatively safer approach is to use environmentally friendly bio fertilizer which is also cost -
effective.
Fusarium oxysporum
The species Fusarium oxysporum is well represented the communities of soil borne fungi, in every
type of soil over the world (Burgess, 1981). All strains of F. oxysporum are saprophytic and able to
grow and survive on organic matter in soil and in the rhizosphere of many plants (Garrett, 1970).
Moreover, some strains of F. oxysporum are pathogenic to different plant species; they invade the
roots inducing either root-rots or tracheomycosis when they invade the vascular system. Many other
strains can perforate roots, but do not invade the vascular system or cause disease (Olivain &
Alabouvette, 1997).
Fig.1: Disease cycle of Fusarium oxysporum.

33
Bakanae Disease
This is widespread in the foothills (Manandhar H. K. 1987). Bakanae disease is caused by one or
more Fusarium species and is a complex of disease symptoms including seedling blight, root and
crown rot, stunting, and the classic symptoms of etiolation and abnormal elongation induced by
fungal production of gibberellin hormones. In Khumal-4, a variety introduced for its high resistance
to blast, grain yield losses of up to 40% due to bakanae disease have been recorded (Maragos C. M.,
et al., 1997). Bakanae disease is primarily seed borne, and high levels of seed infection
with Fusarium species have been found in samples of Khumal-4 from affected fields (Manandhar H.
K., et al., 1992).
Fusarium species the main soil borne are the best-known plant pathogens in terms of economic
damage in agricultural productions all over the world (Saremi, 2000). Regarding to Fusarium-
induced diseases in agricultural plants, yield reduction in different crops is a major problem in Iran.
Bakanae disease on rice caused by Fusarium species result to yield losses in most crops fields
(Saremi et al., 2007).
Fusarium Wilt
Fusarium wilt widespread plant disease caused by soil-inhabiting fungus Fusarium oxysporum.
Several plant species are susceptible, including economically important food crops such as sweet
potatoes, tomatoes, legumes, melons, and bananas in which the infection is known as Panama
disease.
F. oxysporum thrives at soil temperatures above 24 °C (75 °F) and can live continually in soil
without access to living host plants. Affected plants are usually short; their leaves turn pale green to
golden yellow and later wilt, wither, die, and drop off regularly upward from the stem base. Dark
streaks are present in the xylem vascular tissue of the roots and lower stem, and the roots may
decompose. Infected seedlings wilt and die.
The spread of the pathogen can be a little bit controlled by using clean seeds and removing infected
plant tissues from the area, though the most effective management strategy is to plant resistant
varieties. Depending on the forma specialism (host-specific form) responsible for a given infection,
the disease can be controlled with soil fungicides, though some forms have developed resistance.
Given its longevity, crop rotation is normally ineffective.
Endophytes
Endophytes are defined as ‘‘bacteria or fungi, which for all or part of their life cycle, invade the
tissues of living plants and cause delicate and asymptomatic infections entirely within plant tissues,
but cause no symptoms of disease’’ (Wilson 1995), or ‘‘those which can be extracted from inner
plant parts or isolated from surface-disinfected tissues and that do not visibly harm the plant’’
(Hallmann et al. 1997). Sometimes endophytic bacteria are even potential sources of resistance
against pathogenic agents such as fungi (Benhamou et al., 1998). Colonization of plants by
biocontrol endophytes induces several cell-wall modifications, such as the deposition of callose,
pectin, cellulose, and phenolic compounds leading to the formation of a structural barrier at the site
of a potential attack by phytopathogens (Benhamou et al., 1998). Another common response of
bacterized plants challenged with a pathogen is the induction of defense-related proteins such as
peroxidases, and chitinases (Fishal et al., 2010).
The main plant colonization routes by endophytic bacteria. Some soil bacteria can go to a plant at
several root zones, as explained in the main text. Once endophytically established, they can either

34
remain in the neighborhood of the site of entry (indicated in blue) or move deeper inside and occupy
the intercellular space of the cortex and xylem vessels (indicated in green). Some of the endophytes
can even move upwards colonizing above-ground organs/tissues. Red and yellow represent
rhizospheric bacteria that are unable to colonize inner plant tissues (Malfanova N et al., 2013).
Fig.2: Endophytic entry into a plant.

Virtually all plants are inhabited by diverse bacteria known as endophytes. Endophytic bacteria are
those which can be detected at a particular moment within the tissues of evidently healthy plant hosts
(Schulz and Boyle, 2006). Most of the endophytes colonize different parts of the plant apoplast,
including the intercellular spaces of the cell walls and xylem vessels. Some of them are capable to
colonize the reproductive organs of plants, e.g. flowers, fruits, and seeds. Inside a plant, these
bacteria do not normally cause any considerable morphological changes like root-nodule symbionts
do. They also do not cause any disease symptoms, in contrast to phytopathogens. Many endophytic
bacteria have a number of plant-beneficial traits in vitro; few of those exhibit them in planta and only
a small number of endophytes proved to be very potent plant-growth promoting and/or biocontrol
agents under agricultural conditions (Scherwinski et al., 2008; Berg, 2009). Since the first reliable
reports about the isolation of endophytic bacteria from surface- sterilized plants (Samish et al., 1960)
more than 200 bacterial genera from 16 phyla have been reported as endophytes.
Beneficial endophytic bacteria and their effects on a plant
In a plant, endophytes can positively influence plant growth and its resistance to different stresses.
For complete overviews of their beneficial actions the reader is referred to (Hardoim et al. 2008). In
the following section, we will restrict ourselves to the plant growth-promoting effects moderated by
endophytic bacteria. These can be grouped as direct PGP (plant growth promotion) and biocontrol of

35
plant pathogens. A variety of PGP and biocontrol mechanisms can be looked for endophytic bacteria.
The role of endophytic microorganisms in plants can be divided into two categories based on types
of activity: growth promotion and disease control.
Materials and Methods
Collection of Samples
Paddy samples (Oryza sativa) were collected from the Ambasamudram, Tirunelveli, Tamilnadu,
India. So far Ambai 16 paddy [(ASD 16)] which is a hybrid variety of Adudurai 31 (ADT 31) and
Kovai 39 (CO 39) samples were collected from the fields of Ambasamudram. The samples collected
were placed in ice boxes and brought to the laboratory were stored in a cold room at 4°C till
processing.
Isolation of Endophytes
The rice sample was washed with running tap water to remove the attached clay. The plant leaf was
cut separately. Subsequently, the leaf was immersed in 70% ethanol for 3 min, washed with fresh
sodium hypochlorite solution (2.5% available Chlorine) for 5 min, and finally washed five times with
sterile distilled water. The sample (leaf) were macerated with a sterile mortar and pestle and then
plated on different media (NA, PDA, AIA, PIA and YMB). The plates were examined for bacterial
growth after incubation at 28°C for 3 - 5 days (Cao et al., 2004).
In vitro antibiosis test with live organism
Test organisms
The antagonistic potential of endophytic isolates was examined against the fungal pathogen
Fusarium oxysporum MTCC 284. The cultures were procured from MTCC, Chandigarh, India, and
were maintained on Potato Dextrose Agar. Invitro antagonism test was done according to Dr. Dileep
Kumar (1998). Dual culture technique: Potato dextrose agar (PDA) was used for examining the
antagonism against Fusarium oxysporum. For this, an actively growing fungal (Fusarium
oxysporum) mycelial disc (approx. 5mm2) was placed at one side of the Petri plate, 2 cm inside of
the periphery and a loopful of the endophytic bacterial strain was streaked in a line (6cm length), on
the opposite side at a distance of 5cm from the mycelial disc. The plates inoculated with fungus
alone are served as control. The plates were then incubated at 28 ± 2°C and the inhibition zone was
measured, as distance (in mm) between the respective test endophytic bacterial strains and fungus
Fusarium oxysporum, after 7 days of growth under incubation. The hyphal deformities near the zone
of inhibition were recorded and compared with that of control plates. Replicates of each plate were
done.
Result
Isolation of Endophytic Bacteria
Total 5 Endophytic bacteria were isolated from the leaf of rice samples from collected from
Ambasamudram, Tamil Nādu.
In vitro antagonistic activity
The isolates were screened for their efficiency to inhibit plant pathogen Fusarium oxysporum.
Among the isolates, AE 5 Showed a highest clear zone of inhibition of 5 mm in dual plate assay.

36
Morphological Characterization of PGPE isolate

Morphological characteristics of the colonies of each isolate were examined on NA plate.The isolate
was quadrant streaked on NA plates. After 3 days of incubation, different characteristics of colonies
such as size, colony diameter, colour, form, surface, elevation, margin, opacity were recorded.
Conclusion
In summary, one of the five endophytes isolated from leaves of rice plants showed significant
antagonistic activity against the plant pathogen Fusarium oxysporum. Among the isolates, AE 5 was
found to be potential as well as bio control properties that could be attributed to the enhancement of
bio control agents. The mechanisms presented by these endophytes can increase the plant's ability to
resist pathogen attack, or even directly inhibit the pathogen growth. The result of this study provides
a strong basis for the procurement of the strain AE 5 as a bio inoculant to attain the desired plant bio
control attributes in agriculture. Further studies including the enhancement of yield in rice plants,
efficacy test under field conditions, and future selection of the potent isolate as PGPE for broad host
ranges for crop colonization are needed to clarify the role of PGPE as bio fertilizers that exert
beneficial effects on host plant growth and development.
ABBREVIATION
1. PGPE- Plant Growth-Promoting Endophytes 2. AE - Ambasamudram Endophyte
3. NA - Nutrient Agar 4. PDA - Potato dextrose agar

37
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***
39
Chapter 5
REDEFINING THE RELEVANCE AND EFFICACY OF
MICROBIAL BIOCONTROL AGENTS AGAINST
PHYOPATHOGENS
Sourish Dasgupta1, Gargee Dhar Purkayastha2, Bikram Saha3
A bstract
Agricultural practices like crop rotation, use of disease resistant varieties etc., developed by plant
growers for inhibition of diseases caused by phytopathogens are not always sufficient to provide
complete protection to the crops. Moreover, irrational selection and use of chemical pesticides and
fungicides in agricultural fields results in residual environmental toxicity and loss of non-target
organisms sharing the same habitat or ecosystem. The need to reduce the use of chemical pesticides
and fungicides have led to a growing interest in various alternative methods for the control of fungal
diseases of crop plants of which microbial biocontrol agents (MBCAs) proved to be the best.
MBCAs interact with plants by inducing resistance or priming plants without any direct interaction
with the targeted pathogen. They act via different mode of action viz. hyperparasitism, antibiosis,
competition for nutrients etc. Normally, these interactions are highly regulated metabolic events that
produced signaling compounds, enzymes, and other interfering metabolites in situ at low
concentrations during interaction. Therefore, proper understanding the mode of action of MBCAs is
essential to achieve optimum disease control. Use of MBCAs as biopesticides is presently considered
as the most promising method for more rational and safe crop management practices worldwide and
its production is relatively inexpensive. They are also biodegradable, usually very target specific,
non-toxic to human and renewable. Potentiality of these MBCAs in controlling numerous plant
diseases makes them desirable and sustainable tools for disease management but proper timing and
application procedures are essential for ensuring greater efficacy. In future, the use of MBCAs as
pesticides shall advance rapidly with new biotechnological approaches so that plant growers can
focus on MBCAs and biodegradable natural products for integrated plant disease management.
Keywords: Microbial Biocontrol Agents (MBCAs), Hyperparasitism, Antibiosis, Competition,

Induced Resistance and Priming.
Sourish Dasgupta, Gargee Dhar Purkayastha, Bikram Saha

40
1Department of Botany, North Bengal St. Xavier’s College, Rajganj - 735134, Jalpaiguri,
West Bengal, India. dgsourish@outlook.com
2Department of Science, Sri Sri Academy, Siliguri-734009, Darjeeling, West Bengal, India.
gargeed82@gmail.com
3Department of Botany, Darjeeling Govt. College, Darjeeling-734101, West Bengal, India.
bikram24@gmail.com
I ntroduction
Plant pathogens including fungi, bacteria, viruses, and nematodes cause numerous diseases in crop
plants that lead to huge crop loss every year throughout the world and significantly reduce the quality
and quantity of agricultural commodities (Savary et al., 2019; O’Brien, 2017; Singh, 2014; Dean et
al., 2012). Common agricultural practices for disease control are not always sufficient for complete
protection of crops against diseases. Moreover, the uncontrolled and unrelenting use of chemicals for
disease control results in harmful residual effect on plants, normal soil microflora, human health and
the development of fungicide resistant pathogenic strains (Usta, 2013; Chakraborty et al., 2009). The
interest for biological control of plant pathogens has increased over the past decade, especially
because of the importance of using eco-friendly alternatives to the extensive use of chemical
fungicides for combating crop diseases caused by fungal phytopathogens (Lahlali et al., 2022;
Ongena and Jaques, 2008). The term biocontrol applies to the use of microbial antagonists
(biocontrol agents) to suppress plant diseases (Heimpel and Mills, 2017). Biocontrol agents are most
often known as antagonists, and antagonism is the generalized mechanism that they use to reduce the
survival or disease-causing activities of plant pathogens. Biocontrol of plant diseases by using
beneficial microorganisms or microbial biocontrol agents (MBCAs) in rhizospheric soils is not only
an alternative to chemical fungicides, but it may also provide control of diseases that cannot be
managed completely by other control strategies (Cook, 1993).
The underlying mode of action of MBCAs to control phytopathogens includes induce resistance or
priming of enhanced resistance against diseases without any direct antagonistic interaction with the
pathogen, indirect interaction with pathogens through competition for nutrients and space, direct
interactions by hyper-parasitism, antibiosis and production of antimicrobial secondary metabolites in
low amounts with inhibiting effects against pathogens (Collinge et al., 2022; Köhl et al., 2019;
Ghorbanpour et al., 2018; Jensen et al., 2017; Spadaro and Droby, 2016; Conrath et al., 2015;
Pieterse et al., 2014; Raaijmakers and Mazzola, 2012). Pathogen populations thus can be controlled
by antagonistic microorganisms in many ways. Beside disease suppression, MBCAs also promote
the plant growth and yield either directly or indirectly (Köhl et al., 2019; Usta, 2013; Perez-Garcia et
al., 2011; Hariprasad et al., 2009). They are also known as plant growth promoting microorganism
(PGPM) that trigger the synthesis of numerous phytohormones like auxin, cytokinin, gibberellin,
ACC deaminase, abscisic acid, jasmonates etc. (Khan et al., 2020; Arora et al., 2020; Oosten et al.,
2017; Saravanakumar, 2012) and numerous antimicrobial secondary metabolites and extracellular

41
lytic enzymes capable of inhibiting the growth of soil borne fungal phytopathogens (Jousset et al.,
2006; Haas and Defago, 2005; Siddiqui et al., 2005). Report suggest that several Pseudomonas and
Bacillus spp. Promote plant growth by synthesizing auxinsand ACC deaminase (Khoshru et al.,
2020; Danish et al., 2020; Samaddar et al., 2019). Moreover, plant growth promoting
microorganisms (PGPM) – induced cytokinin elicits root development, enhanced vascular cambium
activity, cell differentiation and apical dominance (Khan et al., 2020; Gouda et al., 2018). Various
microbial antagonists have been investigated in past few decades as potential antifungal biocontrol
agents of plant diseases and it has been observed that plant growth promoting rhizobacteria (PGPR)
can produce biocontrol effect by controlling the manifestation of fungal pathogens inside the roots
and simultaneously induce the defense response in host plant (Dhar Purkayastha et al., 2018;
Purkayastha et al., 2010; Kishore et al., 2005). Numerous studies have shown promising potentiality
of MBCAs in disease suppression (Ghorbanpour et al., 2018; Karlsson et al., 2017; Mauch-Mani et
al., 2017; Spadaro and Droby, 2016; Conrath et al., 2015; Pieterse et al., 2014; Raaijmakers and
Mazzola, 2012; Lee et al., 2011; Heydari and Passarakli, 2010). However, in some instances, the
effectiveness of MBCAs has been lower than that of chemical fungicides due to the complexity of
the rhizosphere and the need for application of high amount of MBCAs continuously and
consecutively to cover the entire rhizosphere (Abd-Elgawad et al., 2020; Angelopoulou et al., 2014).
This paper reviews the recent advances in our understanding of the mechanism of biocontrol of
soilborne fungal phytopathogens mediated by MBCAs. The interactions between MBCAs and the
pathogens as well as the role that MBCAs play during their association with host plants and the
prospects of producing more competent biopesticides are being addressed in this paper.
Hyperparasitism: Direct Interaction with phytopathogens
In hyperparasitism, the pathogen is directly attacked by a specific MBCA that kills it or its
propagules. This kind of interaction is often observed between fungi. For bacteria, hyperparasitism is
a rare phenomenon, but predation of several plant pathogenic bacteria such as Agrobacterium
tumefaciens, Xanthomonas vesicatoria, X. campestris pv. campestris, Erwinia carotovora pv.
carotovora, Pseudomonas syringae pv. glycinea, P. syringae pv. tomato, P. marginalis, and Erwinia
herbicola by Bdellovibrio bacteriovorus strains was observed (McNeely et al., 2017). Certain
MBCAs (especially Trichoderma and Gliocladium) reduce the amount of disease by parasitizing the
fungal and/or bacterial phytopathogens. Generally during parasitism, the MBCAs viz. Acremonium
alternatum, Acrodonitum crateriforme, Ampelomyces sp., Cladosporium oxysporum, Gliocladium
virens and Trichoderma spp. are involved in direct physical interaction with the fungal pathogen and
ultimately degrade the pathogen’s mycelia (Heydari and Passarakli, 2010; Kiss, 2003; Goldman et
al., 1994). In some instances, the MBCAs (Trichoderma spp., Gliocladium spp., Serratia marcescens
and Myxobacteria sp.) produced enzymes like proteases, chitinases and glucanases that digest and/or
degrade the cell wall materials of the pathogens and thereby directly suppressed the growth of
phytopathogens (Dhar Purkayastha et al., 2018; Someya et al., 2007; Bull et al., 2002; Lorito et al.,
1994).
Trichoderma and Clonostachys are the two most well studied mycoparasites or hyperparasites with
wide host range and they normally produce structures for attachment and infection, and kill their
hosts by cell wall degrading enzymes (CWDEs), often in combination with antimicrobial secondary
metabolites (Nygren et al., 2018; Karlsson et al., 2017; Mukherjee et al., 2012; Harman et al., 2004;
Harman, 2006). These lytic enzymes are not constitutive but their production is triggered by complex

42
signaling after recognition of the host. This attack gradually results in further degradation of host cell
walls and finally to death of the host (Reithner et al., 2011).
During decomposition of dead organic matter naturally, CWDEs are commonly produced in the
environment by decomposing fungi and bacteria. Application of hyperparasites in biological control
will not significantly increase cell wall degrading activities in the environment as they produce low
amounts of CWDEs (mainly chitinases, b-1,3-glucanases and proteases) during short time periods
locally in micro-niches when they interact with their hosts. Therefore, detrimental effects of
hyperparasite application to rhizosphereic soil biota are less as the activities are highly specific on a
given texture and because of the rapid denaturation of CWDEs, if unused. Moreover, the
development of resistance by a plant pathogen against hyperparasitism by a biological control agent
has not yet been reported (Kohl et al., 2019), but pathogen can develop some resting structures like
endospores, chlamydospores, and melanised sclerotia with high resistance against hyperparasitism by
naturally occurring antagonistic microorganisms (Bardin et al., 2015).
Antibiosis: Direct interaction via antimicrobial compounds

Comprehensive research studies in the past few decades have highlighted the involvement of one or
more antibiotics produced by MBCAs in disease suppression (Malviya and Singh, 2012; Hofte and
Altier, 2010; Ge et al., 2007; Kumar et al., 2005). Antibiotics are low molecular weight chemical
compounds known to reduce the growth and metabolic activities of microorganisms at very low
concentrations (Thomashow et al., 1997; Fravel, 1988). Among the PGPR strains, fluorescent
pseudomonads are found to produce a myriad of antifungal antibiotics. The most effective antibiotics
isolated already from fluorescent pseudomonads that play a relevant role in biocontrol include 2, 4-
diacetylphloroglucinol (2,4-DAPG), phenazines (PHZ), pyrrolnitrin (PRN), pyoluteorin (PLT) and
cyclic lipopeptides (Raaijmakers and Mazzola, 2012; Gross and Loper, 2009; Loper et al., 2007;
Haas and Defago, 2005; Raiizmakers et al., 2002). Therefore, the fluorescent pseudomonads are
most widely considered group of antibiotics producing MBCAs found in rhizosphere. In one study, it
was demonstrated that 2, 4-DAPG causes membrane damage to Pythium spp. and is particularly
inhibitory to zoospores of this fungal species (de Souza et al., 2003). Pyrrolnitrin has been portrayed
as an inhibitor of fungal respiratory chains (Keel et al., 1992). Cyclic lipopeptides have the surfactant
properties and can insert into the cell membranes of the pathogens and upset their function. These
chemicals are also known for their broad antibacterial and antifungal properties (Haas and Defago,
2005).
Apart from Pseudomonas, Burkholderia spp. are also known to repress many soil-borne plant
pathogens, particularly the pathogens responsible for diseases like damping-off and root rot of pea
(King and Parke, 1993) and root rot of poinsettia (Cartwright and Benson, 1995). The role of
antifungal antibiotic pyrrolnitrin produced by B. cepacia for suppression of Colletotrichum
gloeosporioides infecting papaya plant has also been reported (Kadir et al., 2008). Some of the
noteworthy examples of antibiosis from the genus Bacillus include B. subtilis BBG100 and B.
subtilis QST713 strains producing the lipopeptides viz. mycosubtilin and iturin A respectively. Iturin
A was effective against Botrytis cinerea and Rhizoctonia solani (Kloepper et al., 2004; Paulitz and
Bélanger, 2001) whereas mycosubtilin was effective against Pythium aphanidermatum (Leclère et
al., 2005). Lipopeptides like bacillomycin and fengycin isolated from B. amyloliquefaciens FZB42
were also effective against the wilt pathogen Fusarium oxysporum (Koumoutsi et al., 2004). The

43
MBCAs, Trichoderma and Gliocladium are known to produce the antibiotics gliotoxin and gliovirin,
and they are equally capable of suppressing several plant diseases including the damping-off like
their bacterial counterparts (Ghorbanpour et al., 2018; Wilhite et al., 2001; Handelsmann and Stab,
1996).
It has been seen that antimicrobial metabolites are produced at low concentrations in situ in
microniches for the majority of MBCAs. Thus, applications of MBCAs with potential in situ
production of antimicrobial metabolites will not add any relevant toxic effects to the cropping system
(Köhl et al., 2019; Sachana, 2018).
Competition for nutrients and space: Indirect interaction with phytopathogens

Competition for nutrients and space is considered as one of the potential mechanisms for MBCAs to
control plant diseases (Spadaro et al., 2010). Many fungal phytopathogens, including Botrytis
cinerea, and Sclerotiana sclerotiorum, can be effectively managed through the mechanism of highly
competitive saprophytic ability (CSA) of antagonistic fungi (Fokkema, 1993). Numerous studies
highlighted that fungal saprophyte such as Candida oleophila, Clolostachys rosea, Epicoccum
purpurascens, Metschnikowia fructicola, Pichia guilliermondii, Rhodotorula glutinis, Trichoderma
harzianum, and Ulocladium atrum are excellent competitors and are therefore effective MBCAs of
B. cinerea and S. sclerotiorum (Zhang et al., 2007; Karabulut et al., 2004; Wszelaki and Mitcham,
2003; Li et al., 2003; Kohl et al., 1998; Lima et al., 1997; Zhou, 1989). MBCAs are more efficient in
using soil nutrients and space than phytopathogens (Handelsman and Parke, 1989). While competing,
they usually reduce spore germination percentage, germ tube growth, infection, and thereby overall
expansion of pathogen (Roca-Couso et al., 2021; Heydari and Pessarakli, 2010).
Competition for a rare but essential micro-nutrient iron has evolved as another important indirect
mechanism of biocontrol (Khokhar et al., 2012; Handelsman and Stabb, 1996). Although iron is
abundant on Earth’s surface but its presence is in the form of highly insoluble ferric hydroxide. The
soluble iron available for living organisms is present at a concentration of 10-18 M or less. Bacteria
therefore, developed a high-affinity iron uptake system to transport the iron into the cell which
involves an iron-binding ligand called siderophore and a receptor protein which transports the
siderophore into the cell (Neilands and Nakamura, 1991). Siderophores are low-molecular weight
(i.e., 0.5 to 1.5 kDa) iron chelators produced under iron deficient conditions by aerobic and
facultatively anaerobic micro-organisms (Sayyed et al., 2005). The role of siderophores in biocontrol
of plant diseases has been widely demonstrated (Buysens et al., 1996; Chaiharn et al., 2009;
Lugtenberg and Kamlova, 2009). The mechanism of biocontrol involving siderophores lies in the
fact that the ferric iron is being sequestered by the bacterial siderophores in rhizosphere making it
unavailable to pathogenic fungi, thereby inhibiting their growth or other metabolic activity (Khokhar
et al., 2012; Loper and Henkels, 1999; O'Sullivan and O'Gara, 1992). Several species of
Pseudomonas, Agrobacterium, Serratia, Streptomyces and Bacillus producing siderophores have
been demonstrated as potential biocontrol agents involved in inhibiting multiple phytopathogens
(Saha et al., 2012; Macagnan et al., 2008; Yu et al., 2011; Purkayastha et al., 2010; Chaiharn et al.,
2009; Penyalver et al., 2001). This indirect mechanism of biocontrol needs MBCAs to be present in
sufficient quantities in rhizospheric soil and the strategy itself has less toxic effect on the
environment (Kohl et al., 2019).

44
Induced resistance and priming: Indirect interaction via plant metabolism

MBCAs act as stimuli for induction of host plant defense against phytopathogens. This resistance
mechanisms or induction of host defense are broadly categorized into two different types: Systemic
acquired resistance (SAR) and induced systemic resistance (ISR). SAR is a mechanism of induced
defense that confers long-lasting protection against a broad spectrum of pathogenic fungi and other
microorganisms. It requires the signal molecule salicylic acid (SA) and is associated with the
accumulation of pathogenesis-related [PR] proteins which contribute to resistance (Ryals et al.,
1996). This type of induced resistance is a direct reaction to the stimulus of necrotizing pathogens
(Conrath et al., 2015). The induced systemic resistance (ISR), mediated by jasmonic acid and/or
ethylene, is a plant mediated mechanism of biocontrol in which the MBCAs and the pathogens do
not come in contact with one another. MBCAs stimulate the host plant defense and thus the plants
are protected systemically (Conrath et al., 2015; Khokhar et al., 2012). Research studies have shown
many instances of realistic involvement of induced resistance (both SAR and ISR) in disease
suppression in host plant by the MBCAs. The induction of PR proteins during the interaction of a
viral pathogen with Pseudomonas fluorescens strain CHA0 conferred the systemic resistance to the
pathogen and accumulated salicylic acid that played a role of signal molecule for the development of
SAR in the host plant (Maurhofer et al., 1994). Some more examples of induced resistance
developed by MBCAs include production of PR proteins such as peroxidase, chitinase and -1,3-
glucanase in sugar beet by a Bacillus mycoides strain (Bargabus et al., 2002); Pseudomonas putida
strain producing a lipopolysaccharide in Arabidopsis (Meziane et al., 2005); production of
siderophore by Serratia marcescens 90-166 (Press et al., 2001); and production of volatile antifungal
hexenal, an elicitor of ISR by P. putida (Ongena et al., 2004).
It has been observed that both types of induced resistance (SAR and ISR) decrease in absence of the
stimulus. But priming of plants by stimuli leads to the sensitization for enhanced, long-lasting, faster,
and stronger defense mechanisms in the future (Mauch-Mani et al., 2017). Priming occurs when
plants are infected with necrotizing pathogens or when helpful microorganisms colonize the roots of
plants (Conrath et al., 2015). Therefore, using MBCAs for disease control through induction of
resistance or priming relies on initial establishment of MBCA on the host, followed by triggering of
defense reactions (by producing specific defense inducer) within the host plant resulting in the
elevation of concentrations of defense related compounds. It can thus be assumed that the production
of signaling compounds by applied MBCAs will pose very low eco-toxicological risks (Köhl et al.,
2019).
Commercialization of MBCAs
Microbial biopesticides represent an important option for the management of plant diseases. Several
PGPMs are currently used commercially as microbial biopesticides (Nega, 2014; Ardakani et al.,
2009). Some of the well-known commercially available biocontrol rhizobacteria include Bacillus
subtilis strains GB03 (Kodiak, Gustafson), MBI 600 (Subtilex, Becker Underwood) and QST 713
(Serenade, AgraQuest); Bacillus pumilus strain GB34 (YieldShield, Gustafson); Bacillus
licheniformis strain SB3086 (EcoGuard, Novozymes); a mixture of B. subtilis strain GB122 and
Bacillus amyloliquefaciens strain GB99 (BioYield, Gustafson); several Bacillus spp. (yield
increasing bacteria in China); Streptomyces griseoviridis K61 (Mycostop, AgBio development); and
a few strains of Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas chlororaphis
(Cedomon, BioAgri) (Schisler et al., 2004; Paulitz and Bélanger, 2001). These biocontrol bacteria

45
can be applied as dry products (granules or powders), cell suspensions (with or without
microencapsulation) or seed coatings and they are very effective against numerous fungal
phytopathogens viz. Rhizoctonia, Fusarium, Alternaria, Aspergillus, and other root pathogens. The
genus Trichoderma is also known for its antifungal activity. The biofungicides developed from
Trichoderma are widely used for suppression of fungal diseases in plants worldwide. At present,
more than 50 different bioformulations have been developed from Trichoderma and are successfully
applied in agricultural fields for disease control (Woo et al., 2006). Several microbial antagonists
have been patented and evaluated for commercial uses (Nabi et al., 2017) and these agents are
frequently recommended for plants (O’Brien, 2017).
Conclusion and Prospects
Successful suppression of phytopathogens by the application of MBCAs involves complex
interactions between MBCAs and pathogens. Proper understanding of these interactions is essential
for the generation of efficient biopesticides using the MBCAs harboring multifaceted biocontrol
properties. Biocontrol of phytopathogens involves several mechanisms, effects, and approaches,
resulting in different challenges and types of potential impacts (Jensen et al., 2021). Many MBCAs
exhibit excellent disease control in the greenhouse, but show inadequate performance under field
conditions. Application of consortia of MBCAs can alter this effect by giving increased and/or
broad-spectrum antifungal activity. Genetic manipulation of MBCAs to enhance their biocontrol
potentiality will be helpful in suppressing a wide range of phytopathogens. In this context, the
development of super strains of antagonistic microorganisms expressing induced levels of
antimicrobial metabolites resulting in enhanced biocontrol activity and root colonizing capacity shall
pave a way for the use of such strains as potential biopesticides.
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***
53
Chapter 6
DIVERSITY AND DISTRIBUTION OF ENDOPHYTIC
FUNGI IN VELLAR ESTUARY
Prabir Sahoo & Malay Duyari
A bstract
The word mangrove derives from the Portuges word mangue which means “tree” and the English
word grove which is used for trees and shrubs that are found in shallow, sandy or muddy areas
(Karleskint, 1998). Mangroves are referred to those trees which are able to tolerate high range of
salinity and generally they grow between the highest tidal level and the same level or higher than sea
level. As diverse vegetation exists in mangroves, it is considered as a major niche of fungal
repository. Mangrove fungal diversity is dependent on the age of the mangrove, diversity of the
mangrove plant species, and the physico-chemical features of mangrove habitat [including
temperature, salinity, and tidal range (Jones, 2000)]. Mangrove fungi constitute the second largest
ecological group of the marine fungi (Sridhar, 2004). Recent checklist of mangrove fungi revealed
that a total of 625 fungi exist at global scale (278 ascomycetes, 277 anamorphic taxa, 30
basidiomycetes, and 14 oomycetes), and the maximum mangrove-associated fungi have been
reported from South-East Asia than from other parts of the world (Schmidt & Shearer, 2003). Since
they were first reported from mangrove roots in Australia by Crib and Crib (1955), there has been a
considerable increase in information on mangrove-associated fungi. Besides the large number of
estimated fungal species, Hyde (1990) listed only 120 species of fungi from 29 mangrove forests
around the world.
CAS In Marine Biology, Annamalai University, Parangipettai- 608 502, India.

sprabir482@gmail.com

54
I ntroduction
The word mangrove derives from the Portuges word mangue which means “tree” and the English
word grove which is used for trees and shrubs that are found in shallow, sandy or muddy areas
(Karleskint, 1998). Mangroves are referred to those trees which are able to tolerate high range of
salinity and generally they grow between the highest tidal level and the same level or higher than sea
level. As diverse vegetation exists in mangroves, it is considered as a major niche of fungal
repository. Mangrove fungal diversity is dependent on the age of the mangrove, diversity of the
mangrove plant species, and the physico-chemical features of mangrove habitat [including
temperature, salinity, and tidal range (Jones, 2000)]. Mangrove fungi constitute the second largest
ecological group of the marine fungi (Sridhar, 2004). Recent checklist of mangrove fungi revealed
that a total of 625 fungi exist at global scale (278 ascomycetes, 277 anamorphic taxa, 30
basidiomycetes, and 14 oomycetes), and the maximum mangrove-associated fungi have been
reported from South-East Asia than from other parts of the world (Schmidt & Shearer, 2003). Since
they were first reported from mangrove roots in Australia by Crib and Crib (1955), there has been a
considerable increase in information on mangrove-associated fungi. Besides the large number of
estimated fungal species, Hyde (1990) listed only 120 species of fungi from 29 mangrove forests
around the world.
These included 87 ascomycetes, 31 monosporic fungi, and 2 basidiomycetes. Early studies about
mangrove fungi focused on their ecological roles as their extreme importance in nutrient cycling
(Hyde & Lee 1995). Although so much works on mangrove fungi have been published, our
knowledge of marine and mangrove fungi is still at the cataloging stage. Furthermore, many studies
have examined the amount of litter production in mangrove forest and the role of microorganisms in
decomposition of leaf litter and their role in the mangrove tropic web (Hyde et al, 1998). The
involvement of fungi in the breakdown of mangrove leaves and wood has also been documented
(Newell et al. 1987). However, the roles of mangrove fungi in shaping plant communities have
historically been underestimated.
The term” Endophytic fungi “that live in plant tissues throughout the entire or partial life cycle by
establishing a mutually beneficial symbiotic relationship with its host plant without causing any
adverse effect or disease. The term 'endophytes' (Greek: end on-within; phyton- plant) was first
coined by de Bary (1886). Endophytes relationship with plant varies from symbiotic to pathogenic.
Endophytes benefit host plant by preventing pathogenic organism from colonizing. It has been
categorized into two main groups (Clavicipitaceous and non-clavicipitaceous) based on differences
in evolution, taxonomy, plants host and ecological functions. Clavicipitaceous are able to infect only
some species of grasses and non-clavicipitaceous are found in the asymptomatic tissues of other
higher plants (Rodriguez et al. 2008). It stimulates plants growth, increase disease resistance,
improve the plants’ ability to withstand environmental stresses and recycle nutrients (Sturz et al.
2000). Endophytes are rich source of secondary metabolites with multifold importance (Strobel and
Daisy 2003). Compare to other endophytic microorganisms, fungal endophytes produce large
number of secondary activities (Zhang et al, 2006). Endophytes isolated from medicinal plants
possess strong fungicidal, bactericidal and cytotoxic metabolites (Wang et al, 2007). It produces
enzymes which are used for various applications like degradation and biotransformation of organic

55
compound (Firáková et al. 2007; Pimentel et al, 2011). The endophytes derivatives are used in
biotechnological applications (Tomita, 2003). It has a wide significance in pharmaceutical science
because of its antimicrobial, anticancer and antiviral activities. (Selim et al, 2012).
Endophytic fungi are amazing organisms with the ability to grow inside the host plant, particularly
the medicinal plant, without any side effects on the plant tissues. These endophyte fungi can colonize
and proliferate without causing any damage to the tissues of the medicinal plant, although many of
the fungi genera are isolated from the medicinal plants that can cause the diseases for the plants
directly or for animals and human indirectly. But some endophytic fungi genera are using a
biocontrol agent with other benefits. Here, the diversity of endophytic fungi was about 180 genera.
Also, I suggested a simple equation for displaying the fungi genera available among isolates of
endophytic fungi from the medicinal plants. 180 genera of endophytic fungi into three levels
including low, middle, and high levels depending on the equation that determined the importance of
some fungi genera of from other fungi. These fungi
comprised Trichoderma, Curvularia, Pestalotiopsis, Cladosporium, Chaetomium, Phomopsis,
Diaporthe, Phoma, Penicillium, Alternaria, Colletotrichum, Fusarium, and Aspergillus. The roles of
endophyte fungi are shown in various fields such as producing (A) antibiotics against many
microbes, (B) mycotoxins, (C) anticancer, (D) insecticides, (E) enzymes, and (F) many compounds
used in different fields. Also, endophyte fungi are able to change the chemistry of medicinal plant
and control of plant pathogens. Endophyte fungi of a group the dark septate endophytes (DSE)
detected that are able to play an important role by producing very interesting compounds. The
conclusion is 13 fungi genera more important from others. Many endophyte fungi have displayed the
role in producing the natural bioactive products and using to biocontrol of plant pathogens. These
fungi can be used to produce natural drugs, biopesticides, and biofertilizers that lead to decrease the
dangers of synthetic chemicals. This can save the ecosystem and reduce the chemical residue in the
environment. Plant associated fungi (endophytic fungi) are a bio-diversity rich group of
microorganisms (The study focus on marine mangrove sediment fungi) that are normally found
asymptomatically with in plant tissue (root,leaf ,stain etc.) or in the intercellular spaces. The carbon
sources, nitrogen sources and vitamin requirements for the growth of Ceratopycnidium
baccharidicola (an endophyte of Baccharis coridifolia) were studied. The fungus utilized several
carbon sources: pectin, sucrose, fructose, glucose, maltose, cellobiose, xylose, arabinose, mannitol,
mannose and sorbitol. Sucrose and fructose were found to be the best carbon sources. Nitrogen
sources utilized by the endophyte included: nitrates, ammonium and amino acids such as proline,
asparagine and glycine. Undefined complex nitrogen sources such as soytone, tryptone, yeast extract
and casamino acids supported excellent growth. In a defined medium, thiamine was the only vitamin
required for growth. Under optimum conditions the vegetative growth of C. baccharidicola was
enhanced six-fold over its growth in glucose-asparagine medium. The study for identify the
Endophytic fungi through morphological characters and also moleculer using 18S rRNA Some
species such as- Fusarium oxysporum, Serendipita indica, Beauveria bassiana, Colletotric sp etc.
Distribution
Mangrove are distributed all over the world, according to the FAO about one third of the world’s
mangroves are found in Asia (39%), followed by Africa (21%) and North and Central America
(15%). Vellar estuary (Parangipettai, Cuddalore, Tamilnadu, India) is one of the places where we
found the various species of mangrove trees. Mangroves complex ecosystem grows best in sheltered
areas with low wave energies. High energies destroy the shallow root systems of mangroves. In

56
mangrove ecosystem there are various kind of fungi are identified, those are Alternaria, Aspergillus,
Cladosporium,Colletotrichum, Fusarium, Paecilomyces, Penicillium, Pestalotiopsis, Phoma,
Phomopsis, Phyllosticta and Trichoderma.
There are mainly two types of mangrove species found in the vellar estuary. These are -Avicennia
marina,Rizophora mucronata. Avicennia marina is a species of mangrove tree under the family
Acanthaceae. It grows in intertidal zones of estuarine areas. It is also known as Grey or white
mangrove. It grows as a shrub or tree to a hight of 3 to 10 meter, or upto 14 meter in tropical region.
The leaves are thick 5 to 8 cm long, bright, glossy, green on the upper surface and greay colour. It
has aerial roots: this grow to a height of about 20 cm and diameter is 1 cm. This mangrove is a highly
variable tree with a number of ecotypes and in forms closely resembling other species, ph value 6.5-
8.0 but is intolereant of shade also it tolerates extreme wheather and high wind area.
Rizophora mucronata is found in indo specific region on the banks of rivers and on the edge of the
sea. It is the only one species to be found in East Africa. The natural habitat of the species is tidal
creeks and flat coastal area, estuaries area subject to daily tidal flooding. It is small to medium size
evergreen tree growing to a hight of about 20-25 meters on the banks of rivers. On the sea 10-15
minutes is a more typical hight.
This study focused on the isolation and identification of endophytic fungi which are habitat in
mangrove sediment sample from Vellar estuary located in Parangipettai, Tamilnadu, and Near CAS
IN Marine Biology campus.
Process of Isolation of Endophytic Fungi
Sample collection (Mangrove sediment)
Grinding the sample (use of Pestle & Motar)
Serial dilution (using standard procedure)
Plating (Spread/Pour plate)
Incubation (For 24 hrs and check for growth if not it should continue further incubation
on 28°C room temperature)
Methods of Colony Counting of Endophytic Fungi

57
Table-1: Colonization frequency (%) of endophytic fungi isolated from mangroves sediment.
ENDOPHYTIC POST SUMMER PRE MONSOON CF (%)
FUNGI MONSOON MONSOON
sediment sediment sediment sediment
Acremonium sp. - - 2.6 - 2.60
Alternaria alternate 2 4.6 6.6 2.6 15.80
Aspergillus flavus 4.6 - - 3.3 7.90
A.niger 3.3 6.6 7.3 3.3 20.50
A.terreus 2.6 4.6 8 4.6 19.80
Bipolaris sp. - 4 - 3.3 7.30
Cladosporium sp. 4.6 6.6 9.3 5.3 25.80
Fusarium sp. - 4.6 5.3 - 9.90
Meyerozyma sp. 5.3 4 8.6 3.3 21.20
Penicillium sp. 1.3 5.3 8 5.3 19.90
Phoma sp. 3.3 - 5.3 4 12.60
Phoma sp. - 4 3.3 7.30
Ulocladium sp. - 3.3 4 - 7.30
Sterile mycelium 3.3 4 9.3 6.6 23.20
Fig: Avicennia marina & Rizophora mucronata

58
Colonizatiom frequency of rizophere fungi from Avicenia marina:

The total of 19 common fungi isolates,15 fungal species were isolated and identified from
A.marina.The colonization frequencies of the A.marina shown that the dominant rizophere fungus
Aspergillus sps(27%)followed by Trichoderma sps (20%) and Penicillium sps (13%) almost all the
other fungus present only in (7%) which was shown below.
NAME of SPECIES CF (%)
Rhizophus oryzae 7%
Trichoderma sps 20%
Panicillium sps 13%
Mucor sps 7%
Fusarium sp 13%
Curvularia sps 7%
Cladosporium hebarum 7%
Aspergillus sps flavus 26%
Colonization frequency of rhizosphere fungi from Rhizophora mucronata:

NAME OF SPECIES CF (%)
Trichoderma sp 17%
Penicillium sps 6%
Phoma sp 6%
Rhizophus oryzae 6%
Mucor sps 6%
Nigrospora sps 6%
Aspergillus sps 17%
Cladosporium herbarum 6%
Curvularia sps 6%
Fusarium sp 12%
Unknown sps 15%
Table-2: Identification of fungal isolates by microscopic examination:

S. No. Fungus Microscopic appearance
1 Aspergillus flavus It has septate hyphae with long conidiophores and
have a rough texture or even spiny, it just below the
vesicle it is spherical to elongate. Metulae cover
three quarters to the entire surface of the vesicle
from which the phialides from. Condia are globose
to ellipsoidal with smooth to finely roughed walls.

59
2 Aspergillus niger Has septate hypha with long conidiophores.

3 Aspergillus nidulans It has septate hyphae with a woolly colony texture
and white mycelia
4 Aspergillus verisicolor It has septate hyphae thet appear glassy and
transparent conidiphores terminate in small vesicles
thst are biseriate. The 1st layers of cells are called
metulae upon which phialides are borne. The
vesicles are variable in shape and are often described
as spoon –shaped.
5 Cladosporium sps Condiophores usually present,erect ,brown
sympodial,with 1-4 celled blakish brown conidial
scars at each end ,pale to medium or dark brown
,smooth walled to arising in branched chains which
readily disarticulate ,lower conidia are often septate
ramoconidia.
6 Curvalaria sps Condiphores erect, brown, multicellular, producing
conidia in sympodial order, conidial scars dark, flat.
Conidia eilipsoidal it’s often curved with 3-4 true
septa.
7 Fusarium sp Condiogenous cells formed on aerial hyphae or in
short. Conidia of three types macroconidia falcate
with several transverse septate,microconidia
ellipsoidal ,ovoidal with rounded base and
blastoconidia produced singly on polyblastic cells
with septation .Chlamydpspores often present in
thick walled hyaline or pale,intercalary or terminal.
8 Trichoderma sps Repeatedly branched conidiophores, irregularly
verticillate, bearing clusters of divergent, often
irregularly bent, flask –shaped phialides.
9 Penicillium sps It consisting of networks of hyphaeand ropes of
hyphae. Conidiophores arising from creeping hyphae
or ropes of hyphae ,with all smooth penicillus a
single one-sided vertical of metulae with
occasionally one branch from a lower node.
10 Phoma sps The fungus produced semi-immersed pycnidia in
medium which were globose, thinwalled amd brown
in colour. Sometimes the pycnidia were erumpent
and unilocular pycnidia were separate or aggregated
and occasionally confluent on medium

60
Table-3: Fungi isolated from mangrove rhizosphere soil sediment

S. No. Name of the Fungi Mangrove Plant
Avicenia Rhizophora
marina mucronata
1 Aspergillus flavus + +
2 Aspergillus niger + +
3 Aspergillus nidulans + -
4 Aspergillus verisicolor + +
5 Cladosporium sps + +
6 Curvularia sps + +
7 Fusarium oxysporum + +
8 Fusarium sps + +
9 Trichoderma harzianum + +
10 Trichoderma inhamatum + +
11 Trichoderma viridae + +
12 Trichophtonepider mophyte + -
13 Penicillium sps + +
14 Phoma sps - +
15 Rhizopus oryzae + +
16 Mucor sps + +
17 Nigrospora sps - +
18 Unidentified sps - +
19 Unidentified sps - +
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***
62
Chapter 7
AN OVERVIEW OF ENDOPHYTIC FUNGI SECONDARY
METABOLITES AND THEIR NOVEL APPLICATIONS
B. Sowbakkiyalakshmi & K. Kolanjinathan
A bstract
Plants are a possible source of native bacteria, primarily known as endophytes, which can live inside
plant tissue without manifesting any obvious symptoms and are in charge of processing nutrients,
triggering defense mechanisms, and synthesizing secondary compounds. Symbiotic relationship is
formed between endophytes and the host plant. It is well recognized that endophytic fungi in
particular are the origin of new metabolites. Disease arises when either a decline in plant defence or
an elevation in fungal pathogenicity upsets this balance. In order to colonize the host, the endophyte
must not only produce metabolites to compete with epiphytes and pathogens, but also, presumably,
to control the host's metabolism in their carefully regulated interaction. Bioactive substances
produced by fungal endophytes that have the potential to replace plant secondary bioactive and serve
as organic manufacturers of highly sought-after pharmaceuticals. This chapter describes how
endophytes boost crop production and serve as a never-ending source of pharmacologically
significant chemicals. They are also employed in bioremediation.
Keyword: Endophytic fungi, secondary metabolites, crop production, pharmaceutical,

bioremediation.
Department of Microbiology, Faculty of Science, Annamalai University, Annamalai Nagar,

Chidambaram,Tamil Nadu, India. drkolanji@gmail.com

63
I ntroduction
De Bary (1866) invented the term "endophyte" to refer to any creature that lives within plant tissues
as contrasted to epiphytes, which are found on the surface of plants. Carroll defined endophytes as
symbionts that colonise the apical areas of living leaf tissue and do not generate disease symptoms
other than those brought on by fungus and pathogenic bacteria found in the rhizosphere. Endopytes
are thought to be the second line of defence for plants (Sun & Guo, 2012). It is widely spread
throughout many different geographical locations. About a century has passed since the beginning of
endophytic fungal research; nevertheless, only in the last several decades have efforts increased. The
Paleozoic era can be used to date the mutualistic or endophytic connections (endotrophy) of fungi in
the roots of the petrified tree Amyelon radicians (Sridhar, 2019).
Endophytes grow intracellularly, systematically, or locally within their hosts, producing no outward
indications of infection or disease. Even though endophytic fungi are among the most important
elements in plant micro-ecosystems and are predicted to have a significant impact on the
development and spread of host plant species, our knowledge of the precise interactions between
endophytic fungi and their host plants is still quite limited. By altering the suitable environment of
herbal medicine plants, such as by introducing a specific community of endophytic fungi to the
plants to enhance medication quality and quantity, knowledge of and use of these interactions would
enable the optimum production of superior pharmaceuticals (Jia et al., 2016).
Although winter grasses' vertically transmitted endophytes have received the majority of attention,
all plants' laterally transmitted endophytes will impact their phylogenetic diversity and abundance of
species at sizes ranging from individual leaves to landscapes (Daru et al., 2018). Natural holes or
lesions allow fungal endophytes to penetrate plants. They frequently develop in the phyllosphere or
root system. They also break down the plant's cell wall and enter through the roots by using enzymes
like cellulase, pectinase, and proteinase. After initially colonising the root tip, endophytic bacteria go
upward to develop themself in diverse plant parts (Rana et al., 2020). Plant researchers are interested
in endophytes as a potential method of reducing plant stress in a fast-changing environment where
plants would encounter water shortages, recurrent flooding, extreme temperatures, nutritional
shortfalls, excessive salinity, and other atmospheric difficulties (Rho et al., 2018). Due to their
increased biomass and ability to tolerate heavy metals through metal adsorption or chelation,
endophytic fungi are excellent candidates for bioremediation (Deng & Cao, 2017). Endophytic fungi
have received more attention for their uses than for their unique taxonomy.
Fungal Endophytes
Many symbiotic relationships and attitudes exist between fungal endophytes and their hosts. In
particular, some of these relationships might be mutualistic, which means that the long-term
partnership is advantageous to both parties (Lugtenberg et al., 2016). Endophytes are more diverse
and numerous than pathogens among the plant microbiota. They have the critical capacity to
mobilise insoluble phosphate and supply nitrogen to their host plant species, but their significance in
plant defence is unappreciated. Theobroma cacao produces a large number of host defense-related
genes as a result of the fungal leaf endophyte Colletotrichum tropicale, increasing plant tolerance
(Busby et al., 2016).

64
The bioactive metabolites that fungal endophytes produce may be influenced by the chemical
makeup of the plant. As a group of ecologically varied and polyphyletic fungus, endophytic fungi are
mostly ascomycetes and anamorphic fungi (Fouda et al., 2015). Arbuscular mycorrhizae, ecto-
mycorrhizae, and dark septate endophytes are examples of non-pathogenic or mutually helpful fungi
that enter plant roots as well. The majority of the ectomycorrhizal fungi are basidiomycetes, with
some ascomycetes and zygomycetes added for good measure, as opposed to the majority of the
arbuscular mycorrhizal fungi, which are zygomycetous fungi. The majority of herbaceous plants as
well as several families of woody plants are connected with arbuscular mycorrhizal fungi, but only a
few woody plant species are primarily related with ectomycorrhizal fungi (Deng & Cao, 2017).
Fungal endophytic metabolites are beneficial sources of natural products with numerous applications
in industry, agriculture, and medicine (Fouda et al., 2015).
Symbiotic Relationship of Endophytic Fungi
Endophytes are divided into two main groups based on their evolutionary relationship, taxonomy,
plant hosts, and ecological niche: I calvicipitaceous or class I endophytes, which inhabit some
grasses; and (ii) non-calvicipitaceous or class II endophytes, which are found in the undiagnosed
tissues of non-vascular plants, ferns and their allies, conifers, and angiosperms (Khan et al., 2015).
The majority of eukaryotic endophytes are fungus. In addition to having a variety of entire life cycle,
including symbiotic connections with many plant species, fungi are heterotrophic creatures. Even in
severely hostile situations, these symbiotic relationships allow both partners to thrive and reproduce
safely (Alam et al., 2021). Endophytes are categorised as obligatory, facultative, and opportunistic.
Opportunistic endophytes are such endophytes that reside outside the plants and occasionally enter
the plant tissues, whereas obligatory endophytic fungi lack a free-living stage and as a result, live
inside their hosts and only rely on the metabolism of their hosts for existence. The middle and most
diverse group of endophytes are facultative ones. At certain phase in their lifespan, facultative
endophytes dwell outside of the body of their primary host (often on an alternate host plant or other
species like insects). They are frequently connected with legumes (Ogbe et al., 2020).
Endophytic fungi must secrete the appropriate detoxifying metabolites, such as cellulases, lactases,
xylanases, and proteases, to break down the secondary metabolites so they can get past the host-
plants' defence mechanisms since the bioactive compounds produced by plants are barriers to their
colonisation (Manganyi et al., 2018). By a mechanism we refer to as the "rhizophagy cycle," many
plants—possibly all plants—acquire some required nutrients from symbiotic bacteria. Symbiotic
microorganisms (typically seed-transmitted bacteria) move back and forth between an
intracellular/endophytic phase and a free-living soil phase during the rhizophagy cycle. We propose
that during the free-living soil phase, bacteria take up nutrients from the soil, and during the
intracellular/endophytic phase, these nutrients are oxidatively removed from microorganisms. We
also go over theories for how plants control symbiotic microbes to move nutrients from the soil into
the periplasmic spaces of their roots, extract nutrients through oxidation, and then deposit living
microbes that have run out of nutrients back into the rhizosphere through the tips of protruding root
hairs (White et al., 2018).

65
Fig.1. Harvesting of secondary metabolites from endophytic fungi

Endophytic Fungi as A Source for Secondary Metabolite
In the lab, more than 300 endophytes that have the capacity to produce metabolites with medicinal
benefits have been successfully isolated and cultured. Moreover, endophytes that imitate the
chemistry of their hosts and create metabolites that are identical to their hosts' plants have been
discovered (Patil et al., 2016). Figure 1 depicts primarily endophytic fungi that produce secondary
metabolites with a range of bioactivities, including antibiotic, anticancer, cytotoxic, antimalarial,
enzyme inhibitory and others (Wang et al., 2017). These compounds have drawn the interest of
several workers worldwide due to the strong demand (Wang et al., 2017). Due to their high
molecular weight or complicated structure, the bioactive compounds are produced from slow-
growing or uncommon plants, and their entire synthesis is difficult or expensive (Rustamova et al.,
2020). Certain substances, including as polyketides and cylindrocarpones, exhibited anti-enzyme
activity, inhibited the development of biofilms, and prevented the growth of phytoplankton (Zheng et
al., 2021). Fusarium solani produces five (+) - (S)-solaniol, 2,3- dihydro-5-hydroxy-8-methoxy-2,4-
dimethylnaphtho[1,2-b]furan-6,9-dione, and Nb-acetyltryptamine in addition to seven novel
secondary metabolites, including 7-desmethyl fusarin C derivatives (Kyekyeku et al., 2017). We first
looked into the metabolites and associated bioactivities of bioactive compounds synthesized by

66
endophytic fungi associated with species of traditional herbs. Further research is needed to determine
whether traditional herbs include additional endophytes with novel molecules in microamounts,
which may be helpful in therapy (Sun et al., 2011). Terpenoid or polyketides are two possible
categories for the most prevalent purified antibacterial substances released by endophytic species.
Furthermore, it was stated that common bioactive metabolites may be present in the secretory
products created by endophytic bacteria. The genes encoding for the same products are found in
various fungal species, and gene clustering on chromosomes may promote the exchange of
antimicrobial secondary metabolites between fungi, according to functional genomic studies
conducted to examine the biochemical potential of many endophytic genera (Aamir et al.,2020)
Application of Endophytic Fungi Secondary Metabolites
Agricultural Application
To secure the food security of an expanding human population, plant diseases and pests in
agriculture are expected to diminish the annual crop production by 30 to 50%. Chemical pesticides
are typically used to manage these organisms in order to minimize crop loss and meet the world's
food demand. Yet, new restrictions on various chemical pesticides and rising consumer demand to
lower chemical pesticide residues in the environment and food supply are pressuring governments
and commercial agriculture firms to develop alternative, clean plant production technologies.
Endophytes synthesise and release bioactive substances and biochemicals, including volatile
substances that can inhibit pathogen growth and suppress the detrimental effects of plant pathogens
(Grabka et al., 2022).
The endophytic fungi Penicillium spp., Alternaria spp., and Aspergillus spp. were isolated from the
medicinal plant Ephedra pachyclada. These fungi produce a range of compounds that help plants
flourish, including IAA, antibacterial activity, ammonia, and phosphate solubilization. Endophytic
fungi also showed greater phosphate solubilization capacity than endophytic bacteria (Ismail et al.,
2021). The endophytic fungus Gilmaniella sp. AL12 increased the expression of the genes for MDH
and pyruvate dehydrogenase (PDHE), which helped with the regeneration of OAA and acetyl-CoA.
Seven genes with the annotation "malate transporter" were up-regulated, which is consistent with an
enhanced tricarboxylic acid cycle (TCA cycle) (Yuan et al., 2019). Aspergillus welwitschiae
effectively withstand metal stress and increase production of plant hormones such as indole acetic
acid (IAA), solubilize inorganic phosphate, and generate stress-related metabolites in plant Glycin
max (Husna et al., 2022).
Pharmaceutical Application
There are numerous opportunities to use endophytic fungus to produce a variety of bioactive
metabolites vital to human health. The biology and biochemistry of fungal endophytes associated
with plants have developed in recent years as an attractive topic for comprehending the therapeutic
significance of bioactive compounds obtained from fungal endophytes (Rai et al., 2021).
Antimicrobial activity
Fungal enzymes are essential for biodegradation and hydrolysis activities, which are crucial
defences against pathogens and a way for them to receive the nutrients they need from host plants
(Fouda et al., 2015). Endophytes have a variety of bioactive compounds that can be used to make
essential medicinal medications commercially. These bioactive compounds are primarily secondary
metabolites and were shown to have a variety of pharmacological effects. The emergence and spread
of infectious diseases and drug-resistant pathogens necessitate the search for novel pharmaceuticals

67
and therapeutics (Vasundhara et al., 2019). A bioactive substance called pestalachloride was
produced by the endophytic fungus Pestalotiopsis adusta, and it significantly inhibited three plant
diseases (Li et al., 2008). When tested against different pathogenic organisms, the antimicrobial
activity of the olive tree endophytes Penicillium commune, Alternaria alternata, and Penicillium
canescens was found to be the most effective at inhibiting E coli, Staphylococcus aureus and
Bacillus subtilis (Malhadas et al., 2017).
Antioxidant property
The search for new, risk-free antioxidants derived from natural sources to treat or prevention disease
is ongoing. Secondary metabolites from fungal endophytes, including Pestacin, Isopestacin, Rutin,
Corynesidones A and B, Borneol, Coumarin, and p-tyrosol, may be a source of both well-known and
brand-new natural antioxidants. The anti-inflammatory, anticancer, antimutagenic, and
anticarcinogenic properties of these natural antioxidants are also possible. The most effective
chemopreventive medicines against a variety of human malignancies are thought to be antioxidants.
Antioxidant chemicals produced by endophytic fungi are extremely important because they can
reduce the risk of diseases brought on by oxidative stress (S. Gupta et al., 2020). The highest
antioxidant potential was found in the crude extracts of Chaetomium sp., Aspergillus peyronelii, and
Aspergillus niger strains from Eugenia jambolana (Yadav et al., 2014). ZnO nanoparticles from
Periconium sp., an endophytic fungus in Balanites aegyptiaca, have shown outstanding antioxidant
capacity and can be successfully applied in a variety of interesting possibilities (Ganesan et al.,
2020). The ANPs made using Cladospoium cladosporioides demonstrated strong antioxidant
activity, and the amount of their radical-scavenging capacity was equivalent to that of ascorbic acid,
the industry standard (M et al., 2017).
Bioremediation
Endophytic fungus may be a novel and significant source for the breakdown of harmful contaminants
such hydrocarbons, radionuclides, metals, polychlorinated biphenyls and aromatic hydrocarbons.
Another crucial bioremediation component of fungal endophytes in soils polluted with hydrocarbons
and heavy metals is phytoremediation. One of the most effective ways to handle plastic trash is
through endophytic fungal enzymatic activity, or depolymerization. Tiny chains of oligomers,
dimers, and monomers that are formed when complex polymers break down can serve as sources of
energy and carbon. Even within the same species, different strains of the bacteria create different
enzymes from one another. Because enzymes have extremely particular actions on their substrates,
different enzymes aid in the breakdown of different kinds of enzymes (Krishnamurthy & Naik,
2017).
Mosquitocidal activity
The death rate for 24 hours of IInd and IVth instar larvae of Aedes aegypti and Culex
quinquefasciatus when exposed to silver nanoparticles from Phomopsis liquidambaris (Seetharaman
et al., 2018). Cochliobolus spicifer, an isolated fungal endophyte from the date palm, was tested
against Aedes caspius and Culex pipiens larvae using crude extracts (Abutaha et al., 2015). The
marine-derived endophytic fungus Aspergillus nomius and Aspergillus flavus from the soft coral
Sarcophyton ehrenbergi, coupled with two recognised cyclodepsipeptide compounds, scopularide,
exhibit larvicidal efficacy against Culex pipiens mosquitoes (Singab et al., 2022). Aedes caspius and
Culex pipiens populations can be controlled naturally, safely, and permanently with the help of the
endophytic fungal extract of Cochilobolus spicifer (Abutaha et al., 2015). Seven
spirobisnaphthalenes, including diepoxins and the palmarumycins C8, C12, C15, and B6, were

68
isolated from the fungal extract. Among these, palmarumycins C8 and B6 have shown potent
larvicidal action against Aedes albopictus fourth-instar larvae (Tian et al., 2016). The substance
Aspergillol B from endophytic Aspergillus sp. exhibits a strong larvicide by inhibiting the
acetylcholinesterase enzyme, according to molecular docking studies (Arajo et al., 2022).
Table-1: Endophytic fungi and its plant source.

S. No. Plant Endophytic Fungi Reference
1. Olea europaea Penicillium commune, Penicillium canescens (Malhadas et
L. al., 2017)
2. Grass and Alternaria alternata, Epicoccum purpurascens, (Peláez et al.,
herbaceous Pleospora herbarum and Cladosporium herbarum, 1998)
plants as well as a typical coprophilous fungus,
Sporormiella intermedia
3. Eugenia Chaetomium sp., Aspergillus sp., Aspergillus (Yadav et al.,
jambolana peyronelii and Aspergillus niger 2014)
4. Balanites Periconium sp (Ganesan et al.,
aegyptiaca 2020)
5. Sargassum Cladosporium cladosporioides (M et al., 2017)
wightii
6. Alphine tree Rhodiola crenulata, Rhodiola angusta, Rhodiola (Cui et al.,
sachalinensis 2015)
7. Salacia Phomopsis liquidambaris (Seetharaman
chinensis et al., 2018)
8. Phoenix Cochliobolus spicifer (Abutaha et al.,
dactylifera 2015)
9. Sarcophyton Aspergillus nomius and Aspergillus flavus (Singab et al.,
ehrenbergi 2022)
10. Pleosporales Cochilobolus spicifer (Abutaha et al.,
dactylifera 2015)
11. Salacia Phomopsis liquidambaris (Seetharaman
chinensis et al., 2018)
12. Dioscorea Berkleasmium sp (Tian et al.,
zingiberensis 2016)
13. Bertholletia Aspergillus sp (Araújo et al.,
excelsa 2022)
14. Camptotheca Alternaria, Phomopsis, Sporidesmium, Paecilomyces (Lin et al.,
acuminate and Fusarium 2007).
15. Moringa Aspergillus terreus (Hashem et al.,
oleifera L. 2022)

69
Conclusion
Fungal endophytes are another alternative source with creative applications in a variety of fields.
There are still a lot of areas that must be researched in the future, such as novel innovations and
crops that contain endophytes. The properties of endophytes will be clarified, and new products for
industrial development will be discovered with the use of contemporary molecular biology
techniques comprising metagenomes, proteomes, and transcriptomes. The demand for
pharmaceuticals and agricultural goods is rising daily with an ever-increasing population, making the
future of endophyte research promising.
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37. Sun, Z.-L., Zhang, M., Zhang, J.-F., & Feng, J. (2011). Antifungal and cytotoxic activities of
the secondary metabolites from endophytic fungus Massrison sp. Phytomedicine, 18(10), 859–
862. https://doi.org/10.1016/j.phymed.2011.01.019.
38. Tian, J., Liu, X. C., Liu, Z. L., Lai, D., & Zhou, L. (2016). Larvicidal spirobisnaphthalenes from
the endophytic fungus Berkleasmium sp. Against Aedes albopictus. Pest Management Science,
72(5), 961–965. https://doi.org/10.1002/ps.4075.
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44. Zheng, R., Li, S., Zhang, X., & Zhao, C. (2021). Biological Activities of Some New Secondary
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***
73
Chapter 8
MYCELIAL BRICKS- BUILDING BLOCKS FOR THE
FUTURE
Adrija Mukherjee1, Arijit Das1, Debraj Chakraborty1,2
A bstract
The excessive use of non-recyclable elements in architecture and designs have contributed to
increased global pollution, prompting architects and designers to look for more environment-
friendly alternatives, such as biodegradable substances. Several businesses impact the environment,
and in the present climate, the building industry is one of the worst polluters in the entire world.
Mycelium-based construction materials are renewable substitutes for traditional building materials
like wood, steel, and concrete which utilize organic agricultural and industrial pollutants as a crucial
aspect for its production instead of relying on excessive use of valuable resources, whether they are
finite or not. In a variety of industries, including packaging, electrical prototyping, furnishings,
cosmetics, and buildings, the mycelium-based materials have demonstrated their capability as a more
sustainable and economically viable substitute for traditional synthetic fibers. As a clean building
material, mycelium brick could be utilized to lessen environmental pollution.
Keyword: Mycelium Structure, Mycelial Brick Production, Applications, Drawbacks.
1P.G. Department of Botany, Barasat Govt. College, Barasat, Kolkata 700124, West Bengal,
India.
2Department of Chemistry, Dr. K. C. Patel Research & Development Centre, Charotar University
of Science and Technology, Changa, Anand 388421, India.

adituli98mukherjee@gmail.com
Adrija Mukherjee, Arijit Das, Debraj Chakraborty

74
I ntroduction
The goal of the worldwide sustainable development plan is to substitute non-renewable components
with bio-based ones in order to lessen the use of non-renewable resources. Current findings have
shown that mycelium bio-composites coupled with other biologically significant materials,
including extracts of various plants or agricultural leftovers, constitute a potential method for
creating various substances, which might be beneficial in the field of design, food and building
industries. The fungal mycelium can break down ligno-cellulosic substances in order to create
complex systems with varied features, like with increased structural rigidity, shielding or non-
flammability, and hydrophobicity. Because of its property, it can be used to create bio-textile goods
like wrappings for packages, as it acts as a good substance for isolating materials. (Butu et al., 2020).
50% of all mineral reserves are consumed by the architectural, engineering, and construction (AEC)
sector, which also produces the majority of landfill garbage. As a result, there is an urgent
necessity for cleaner sources of energy and a substitute building material to cut down on the
worldwide emission of greenhouse gases and landfill trash from the AEC industry. The composites
made from mycelium are bio-degradable, light in weight, and regenerable in nature (Ghazvinian and
Gürsoy, 2022). Mycelium-based composites are made by the mycelial network which is
the vegetative component of filamentous fungi. It grows on a plentiful organic substrate like
agricultural residues and natural materials. Whereas most naturally derived products need artificial
binders to improve their qualities, which make the products non-degradable. The mycelium functions
as a naturally obtained glue that binds the substrate altogether just to create a lighter weight and bio-
degradable composite (Van Wylick et al., 2022).
Mycelium-based foams are sometimes referred to in literature as "mycelium bricks", even though
their major characteristics include high porosity and rigidity that have been tested for their very
competing physical capabilities when evaluated with the expanded polystyrene (EPS). Businesses
around the globe are thriving to provide as soon as possible a completely sustainable option for
compact packaging, all at fair costs, despite the extremely restricted commercial secrets and the
competitiveness of the manufacturing process (Butu et al., 2020).
Selection of Fungi
There are many different fungus species that can be used. The factors influencing which fungi should
be chosen rely upon that fungi's (1) development rate during which dense mycelium is produced, (2)
abundance, and (3) growth parameters. From 2012 to 2023, almost 100 research articles had been
published. They named about 70 species of fungi which had been utilized to produce MBC. Majority
of the work on Mycelium-Based Composites are on some white rot fungi (Ganoderma lucidum,
Pleurotus ostreates, Trametes versicolor and others). The findings of comparative analysis for
diverse fungal species are described in some scientific papers. Among that, the two types of fungi
that are most frequently referenced in academic articles are P. ostreatus and G. lucidum. The T.
versicolor is another species that is frequently investigated. The chosen fungus should have a high
level of tolerance for a variety of environmental factors, such as temperature and humidity, as well as
corresponding substrate parameters, such as non-uniform substrate water contents and pH. All these
factors can make the MBC production process simpler. (Sydor et al., 2022).

75
Properties of Mycelial Structure

Mycelium differs from various other eukaryotic organisms in terms of its body composition and
mechanisms of reproduction. Most growths have complexes comprised of hyphal structures (solitary,
hypha). Their little cytoplasmic strings, which are known as hyphae are enclosed by plasma films,
are held together by phone dividers. The cellulosic cell partitions of plants are different from the cell
partitions of fungi. The chitin which is a strong, flexible polysaccharide is also found within the outer
skeletons of fungi. It is the material, which is commonly used by the majority of fungi to create their
cell walls. Most parasitic hyphae possess additional cell dividers, known as cross-dividers.
They divide the lengthy fibers into several end-to-end cells. Thus, as a result, most of them
are multicellular in nature. Many growing fungi have cross-dividers with pores large enough to let
ribosomes, mitochondria, and even cores to flow from one cell to another. A parasite's ability to
transfer nutrients from one part in its body to the next is enabled by the creation of cytoplasm from
one cell to another. A solitary growth's hyphae constantly branch as they grow to form a connected
mass known as a mycelium (plural, mycelia) (Indrajeet et al., 2018).
Mycelium-based composites were the subject of some early research from 2006. However, the
majority of the studies that are now public were only published after 2015. Research show that the
fungus, substrate, growing circumstances, handling of the product, and additives all affect the
properties of mycelium composites. For instance, as compared to the loosely packed substrate,
the densely packed substrate exhibited maximum dry densities and compressive strengths. According
to a study by Karana et al. (2018), mycelium-based substances could be created by taking advantage
of how the fungus can entangle other substances in its network to create a thick product that
resembles mycelium-based bio-structures (Appels et al., 2019). At some point during colonization,
the substrate will have to be dried out or heated, which will then form mycelium-based bio-
structures. At low strain, the composite's mycelium matrix predominates the mild compression.
However, at higher load, the organic substrate components quickly stiffen the material. When it is
combined with the fungal biomass and the leftover substrate, it creates a bio-composite that is
classified as a foam (Butu et al., 2020).
Production of Mycelial Brick
At first, the mycelium for the mushrooms was produced in sacks of plastic. Some amount of water,
agricultural trash, and mushroom spawns are required for this. After being sterilized, the spawns of
the mushroom are combined with agricultural waste and water is added to them. Then the bag is
sealed, and the mixture is left to propagate in a cool, dark environment. In order to prevent
agricultural residue's bacteria from obstructing the development of the mycelium, additional
chemicals are introduced to halt their proliferation. The mycelium will take between 7 - 10 days for
its growth. The mycelium was then inserted into the sterilized sawdust. The food was added to feed
the mycelium, and their combination was then placed in a dim area for the mycelium to grow. It will
also take about a week to grow. When the mycelium had consumed all of the substrate, it was then
placed into the brick mould, where it was permitted to grow until the brick took shape before being
removed, dried, and then placed in a 200°C oven for a period of time to kill any remaining bacteria
within it. Thus, a firm brick is produced from the fungal mycelia as shown in the below schematic
diagram (Indrajeet et al., 2018).

76
Fig.1: Schematic representation of the procedure for the formation of the mycelial brick.
Applications
MBC has a variety of possible uses, including those in architecture, packing purposes, the auto
sector, furniture, the arts, and the production of chitin- and -glucan-based soft substrates, including
foams, paper, and textile substitutes. The bio-composites are referred to in the scientific literature as
unadulterated mycelium bio-materials, such as myco-leather, which can replace leather derived from
petrochemicals and animals. There are ideas for using mycelium to create wearable
electronic devices that can repair themselves and construct monolithic structures from functionalized
fungus characteristics (oscillators, pressure, and optical and chemical sensors) (Sydor et al., 2022).
Advantages of using Mycelium
1. Adhesive in Nature
A fungus's mycelium is a microscopic fiber network that attaches to its source of food to form
a robust, adaptable matrix in any required shape. In place of conventional brick masonry units
that need to be burnt in kilns at high temps, the adhesion capability of mycelium can be used
to produce bricks at room temp. Organic binding agents like mycelium can combine with
waste materials like oat husks and farm commodities to create extraordinarily durable
materials that might substitute polystyrene and Styrofoam in virtually all applications
(Abhijith et al., 2018).
2. Accessible Raw Materials
The eco-friendly and domestically accessible raw ingredients employed to create mycelium
bricks are sawdust, agricultural wastes, and maize stalk (a farm waste product).
3. Cheap Price
Mycelium-based structures are inexpensive to produce because they use readily accessible
raw ingredients and production techniques. This could assist to deliver low-cost housing
choices in areas where people from lower socio-economic classes live and serve different
populations with low-income individuals.

77
4. Lower Level of Construction Experience

Producing mycelium bricks is an easy and uncomplicated technique. It does not require
sophisticated equipment or knowledge. Thus, it would possible for those without prior
construction skills to construct their own residences may be helpful.
5. Comparative Analysis with Traditional Brick
Traditional masonry brick production involves a kiln firing at temperatures between 900 and
1000°C and generates a significant amount of greenhouse gases (Ahmari & Zhang, 2012).
Air emissions from the production of glass bricks come in two different forms: fine particles
from the vaporization and recrystallization of components in the melt, and emissions from the
burning of fuel used to power the glass melting furnaces. The emission from burning fuel
include heavy metals like arsenic and lead as well as greenhouse gases like sulfur oxides
(SOx), nitrogen oxides (NOx), and particulates (Prevention & Handbook, 1998).
Nevertheless, the mycelium brick blocks can be made at room temperature and do not need to
be fired. It is noteworthy that mycelium does not generate any hazardous gases before or
after synthesis. Mycelium bricks break down at the finish line of their lifespan by blending
with the soil and do not pollute the environment.
6. Limited Period of Time for Production
Bricks of the 8×4×2-inch size can be manufactured in two weeks with no wastage or carbon
emissions created. However, the dimension of the molds would affect the length of the
production process.
7. From Cradle-to-Cradle Life Cycle
The building materials from a mycelium structure can be decomposed and used as soil
fertilizer when the structure is demolished at the conclusion of its lifespan. As a result, each
element replenishes the soil and contributes nutrients (Ghosh et al., 2019).
While being creative and having numerous benefits, recent mycelium-based
engineered materials have certain drawbacks-
The practical challenge in producing MBC is related to the proper choice of fungal species, substrate,
and manufacturing methods. These drawbacks could be overcome by choosing a fungus species that
is suitable for the substrate or by using species that have not been utilized before. The quantitative
study of the fungal species stated in the scientific publications to produce a bio-composite with the
required qualities could provide an appropriate basis for this decision. There are no
studies comparing the depth of research on various fungal species and examining the most typical
fungus species-substrate combinations. Although there are numerous species of fungi, only a small
number are employed to make biomaterials. Also, there are not any broad instructions in the
literature for finding new species to use for mycelium-based goods. The voids in the literature are
filled by this review. Based on recent research, it is anticipated that the current report will aid in
identifying the best mixtures of fungus species and substrate. The review also tries to suggest criteria
that a newly employed fungus species must satisfy in order to provide the best possible production
of Mycelium-Based Composites (Sydor et al., 2022).
Conclusion
Natural fibers that are known to be biodegradable are typically found in mycelium-based composites;
nevertheless, the degradation rate of the mycelium composite was only assumed. Mycelium-based
composites are substances that can take the place of foam, timber, and plastics in mechanical,

78
acoustic, and fireproof applications. This material might replace conventional building blocks
because of its low conductivity, flame-retardant, and high sound absorption indexes. Additionally,
due to their sustainable properties, this material could play a significant role in the development of
future constructions in a sustainable way. The development of filaments of fungus on organic
substances, such as agro-waste streams, leads to the formation of mycelium-based bio-structures. It
has been investigated about how to develop novel packaging materials using these bio-composites.
After that, review work should be required to make the bricks more advantageous and affordable
than regularly used bricks, so that they could be sold instead of the traditional bricks. A thorough
investigation of a way to accelerate mycelium growth is also required currently. The fungal
mycelium has enough potential to be used extensively in the developmental purposes and various
other fields in our near future.
References
1. Abhijith, R., Ashok, A., & Rejeesh, C. R. (2018). Sustainable packaging applications from
mycelium to substitute polystyrene: a review. Materials today: proceedings, 5(1), 2139-2145.
2. Ahmari, S., & Zhang, L. (2012). Production of eco-friendly bricks from copper mine tailings
through geopolymerization. Construction and building materials, 29, 323-331.
3. Appels, F. V., Camere, S., Montalti, M., Karana, E., Jansen, K. M., Dijksterhuis, J., Krijgsheld,
P. & Wösten, H. A. (2019). Fabrication factors influencing mechanical, moisture-and water-
related properties of mycelium-based composites. Materials & Design, 161, 64-71.
4. Butu, A., Rodino, S., Miu, B., & Butu, M. (2020). Mycelium-based materials for the ecodesign
of bioeconomy. Digest Journal of Nanomaterials and Biostructures, 15(4), 1129-1140.
5. Ghazvinian, A., & Gürsoy, B. (2022). Mycelium-based composite Graded materials: Assessing
the effects of time and substrate mixture on mechanical properties. Biomimetics, 7(2), 48.
6. Ghosh, T. (2019). Developing a Composite Mycelium-Glass Brick Unit.
7. Indrajeet, M., & Rathi, M. A. (2018). Production of Mycelium Bricks. International Journal of
Innovative Science and Research Technology, 3(4), 484-513.
8. Karana, E., Blauwhoff, D., Hultink, E. J., & Camere, S. (2018). When the material grows: A
case study on designing (with) mycelium-based materials. International Journal of
Design, 12(2).
9. Sydor, M., Cofta, G., Doczekalska, B., & Bonenberg, A. (2022). Fungi in Mycelium-Based
Composites: Usage and Recommendations. Materials, 15(18), 6283.
10. Van Wylick, A., Elsacker, E., Yap, L. L., Peeters, E., & De Laet, L. (2022). Mycelium
composites and their biodegradability: An exploration on the disintegration of mycelium-based
materials in soil. In Construction Technologies and Architecture (Vol. 1, pp. 652-659). Trans
Tech Publications Ltd.
11. Prevention, P., & Handbook, A. (1998). Toward cleaner production. The World Bank Group in
collaboration with the United Nations Environment Programme and the United Nations
Industrial Development Organization.
***
79
Chapter 9
ISOLATION OF ENDOPHYTIC MICROBES FROM
ACACIA PENNATA (L.) WILLD. AND THEIR
ANTIFUNGAL ACTIVITY AGAINST RHIZOCTONIA
SOLANI KUHN
Salman Hussain1, Nripen Kumar Gogoi2, Kumud Das1
A bstract
The aim of this study was to isolate the endophytic microbes from Acacia pennata (L.) Willd. and
their antifungal activity against the pathogenic fungus Rhizoctonia solani Kuhn. which causes the
sheath blight disease of rice. Total five endophytic microbes were isolated from A. pennata, among
them there were two endophytic fungi and three endophytic bacteria viz. Endofungi1, Endofungi2,
Endobact1, Endobact2 and Endobact3 respectively. Among all the endophytic microbes, ‘Endobact1’
was observed the best which recorded the maximum antagonistic activity against R. solani recording
72.14 % inhibition in vitro.
Keyword: Endophyte, Acacia pennata, Antagonism, Rice and Rhizoctonia solani.
1Department of Botany, Nowgong College (Autonomous), Nagaon, Assam, India.

2Scientist,
Regional Agricultural Research Station, Assam Agricultural University, North
Lakhimpur, Assam.
daskumud77@gmail.com
Salman Hussain, Nripen Kumar Gogoi, Kumud Das

80
I ntroduction
Endophytes are microorganisms which are found in living tissue of root, stem, leaf, fruit and seed
etc. and they set up mutual relationship evidently without causing any disease symptoms (Sandhu et
al. 2014; Patel et al. 2013). The term ‘endophyte’ was coined by De Bary in 1866 for the first time
(Anjum and Chandra, 2015; Jariwala and Desai, 2018). Endophytes play an important role for the
production of different natural products and they have been reported that they have a wide range of
biological activity and are grouped in to different categories, which includes alkaloids, lactones,
terpenoids, steroids, quinines, phenolic compounds and lignans etc. Secondary metabolites produced
by endophytes provide beneficial consequences towards the host plant like stimulation of growth and
provide fitness enhancements (Pragathi et al. 2013; Tolulope et al. 2015). For better adaptation of a
plant body or in order to maintain stable symbiosis, endophytes produced several compounds which
help to promote growth of the plants (Nair and Padmavathy., 2014; Gupta et al. 2015). Endophytes
colonize plant tissue at least part of life cycle and they can be defined as non-phytogenic organisms
(Boukhatem et al. 2016). Endophytes are often beneficial for the host to provide resistance towards
disease causing agents like insects, herbivorous etc. (Rakotoniriana et al. 2007). Endophytic fungi
provide protection and survival value by producing excessively substances which contribute their
host (Tolulope et al. 2015). In this present study the endophytic microbes are isolated from
“Climbing Acacia” plant i. e. Acacia pennata (L.) Willd. and their antimicrobial activity test is done
against the pathogenic fungus Rhizoctonia solani Kuhn. causing sheath blight of rice. Sheath blight is
a major disease of rice in Assam. To control this disease various type of chemical pesticides are used
in agricultural field, which have a lot of negative impact towards the environment and they are not
eco-friendly. Biological control using microbial antagonists is an important component of disease
control. Endophytes are promising candidates of bio control agents among others. Haggag (2010)
reported that endophytes are less examined group of microorganisms, which very important source
for novel bioactive and chemical compounds with potential for utilization in various field of
agriculture, industrial and medicals. Ismail et al. (2018) reported that several bioactive substances are
produced by endophytic microbes present in Acacia rugata (Lam). Endophytes of many medicinal
plants are still unexplored in this region. A. pennata is a perennial, small medicinal tree belonging to
the family Mimosaceae (Touch-me-not family), it is widespread and broadly used in India. The leaf
juice is mixed with milk for the treatment of indigestion. It is also used for bleeding gums and
scalding of urine. Some people used the boiled tender leaves of this medicinal plant for digestive
problem, cholera treatment, body pain, treatment against snack venom and also used in fish
poisoning (Lalchhandama, 2013). Keeping in view of socio-economic status of the crop, yield
production, losses of farmers and environmental issues the study was carried out with the objective
to isolate endophytic microbes from A. pennata (L.) Willd. and to screen their antimicrobial activity
against R. solani Kuhn. causing sheath blight of rice.
Methodology
The experiment was conducted during January-April, 2020 at the Regional Agricultural Research
Station (RARS), Assam Agricultural University (AAU), North Lakhimpur which is geographically
located at 270 14'05.15"N latitude and 94 008'29.55"E with mean sea level of 102 mtrs.

81
Collection of Plant Materials

Healthy and disease-free root and bark samples of A. pennata plant were collected in sterilized
polypropylene bags from the experimental farm of RARS, AAU, North Lakhimpur. Then the
samples were brought to the laboratory and processed within a very short period of time after the
collection.
Sterilization
All the glassware were sterilized in an autoclave at 121°C for 15 minutes. The laminar air flow room
was sterilized using the UV lamp for 20 minutes and working table-tops were surface sterilized with
95% ethyl alcohol.
Isolation of Endophytic Microbes
According to the method described by Petrini in 1986, the isolation of endophytic microbes was done
from the plant parts of root and stem (Sandhu et al. 2014). At first the collected samples (plant parts)
were rinsed in running tap water to remove the dust and soil particles and then the plant parts were
cut into small pieces with the help of sterilized blade under aseptic condition. Each sample was
surface sterilized with 70% ethyl alcohol then sample were immerged in 1% Sodium hypochlorite
(NaClO) for 30 seconds to 1 minute. After that sample were rinsed in distilled water for 1 minute,
with the help sterilized tissue paper and allowed to surface dried. When proper drying was done 5
pieces of plant parts from each sample inoculated in two different potato dextrose agar (PDA) and
nutrient agar (NA) medium plates. These plates then incubated for 4 to 5 days at 28 ± 1°C. Fungal
and bacterial growth were picked up from the edge of the inoculated plant samples with the help of
sterilized inoculating needle and transferred to PDA and NA slant respectively for pure culture
development. The inoculated slants were incubated at 28 ± 1°C and observed periodically. With
proper labeling they were sub cultured from time to time.
In-vitro screening of isolated endophytes against R. solani
Test organisms
The test organism, R. solani causing sheath blight of rice was obtained from the culture bank of
RARS, North Lakhimpur for the study.
In-vitro screening
The antagonistic potential of isolated endophytes was evaluated against rice pathogens R. solani by
dual culture method (Kucuk and Kivanc, 2004) using PDA as basal assay medium. In this method,
the endophytic microbe was streaked along with the pathogenic fungi R. solani in a same culture
plate. These culture plates were incubated in incubator for 3 to 4 days at 28 ± 1°C for their further
growth. After 3 to 4 days the plates were observed which show zone of inhibition in which the
endophytic microbes inhibit the growth of pathogenic fungi R. solani and their diameter was
measured. A set of PDA plates without endophytes served as control.
The radial mycelial growth of R. solani was recorded and converted into % mycelial inhibition using
the following equation-
C2 −C1
Mycelial inhibition (%) = X 100
C2
Where, C1= radial mycelial growth of R. solani in the presence of antagonist and
C2= radial mycelial growth of R. solani in control

82
Cultural and Biochemical Characterization of Antagonistic Endophytes

Cultural characteristics of antagonistic endophytic isolates like growth, colony colour, shape and
production of pigment were studied. Biochemical tests like Gram staining and carbohydrate
utilization test were done following standard protocol.
Gram Stain
A thinly spread air dried endobacterial film was fixed on clean glass slide by a light flame. The
specimen was treated with 0.5% Hucker’s crystal violet for 30 seconds and afterwards washed with
running tap water. Then the slide was treated with Lugol’s iodine solution for 30 seconds, rinsed in
tap water and decolourized with 95% ethyl alcohol and counterstained with Safranin for
approximately 30 seconds. It was eventually washed with tap water, air dried and observed under
microscope at 10x, 40x and 100x magnifications and micrographs were taken with the help of stereo
zoom trinocular microscope with digital camera.
Carbohydrate Utilization Test
For testing carbohydrates utilization by endobacteria, 35 comprehensive test kit “KB009” of Hi-
Media were used. The test endobacteria were inoculated in 5ml of nutrient broth and incubated at 35-
37°C for 4-6 hours. Then opened the kit aseptically, peeled off the sealing foil and inoculated each
well with 50µl of the test inoculum by surface inoculation method. The inoculated kits were
incubated at 30 ± 1°C for 24 hours. The results were interpreted as per the result interpretation chart.
Results and Discussion

Isolation of endophytic microbes from A. pennata (L) Willd:
Total 5 (five) endophytic microbes were isolated from the root and bark sample of A. pennata (L.)
Willd. Among them there were 2 endophytic fungi and 3 endophytic bacteria. Endophytic fungi were
isolated from root sample and named viz. Endofungi1 and Endofungi2 on the other hand endophytic
bacteria were isolated from bark sample and named viz. Endobact1, Endobact2 and Endobact3. The
details of the isolates including cultural characteristics are shown in Table 1 and Plates 1-4.
Table-1: Endophytic microbes isolated from different parts (root & bark) of A. pennata (L.) Willd.
S.No. Plant Isolates Cultural characteristics
parts Growth Colony colour Shape Pigment
production
1
1 Root Endofungi Fast Off white Irregular Nil
2
2 Root Endofungi Fast Green Irregular Light green
1
3 Bark Endobact Fast White Rod Nil
2
4 Bark Endobact Fast Yellow Cocci Light yellow
3
5 Bark Endobact Fast white Cocci Nil

83
Plate 1. Isolation of endopyhtic fungi from Plate 2. Isolation of endopyhtic bacteria from
Acacia pennata on PDA plate Acacia pennata on NA plate
Plate 3. Pure culture of isolated endopyhtic Plate 4. Pure culture of isolated endopyhtic
fungi bacteria
Screening of endophytic microbes for antifungal activity against R. solani:

Among the five endophytic microbes, endobact1 showed a maximum zone of inhibition against the
pathogenic fungus R. solani, which is 72.14%, relatively endofungi1 showed 42.38% of inhibition
zone against the pathogenic fungus. Endophytic microbes play vital role in plant disease control and
plant improvement. Our results are in conformity with the findings of several other workers.
Kazempour (2004) reported antagonistic Pseodomonas flurescense, which had very outstanding
potential to inhibit the growth of R. solani. Seema and Devaki (2012) evaluated four fungal and one
bacterial bioagent viz. Trichoderma viride, T. harzianum, Aspergillus niger, Penicillium spp. and
Bacillus subtilis in vitro against R. solani causing sore shin disease in tobacco seedlings and recorded
70%, 67%, 57%, 50% and 44% inhibition of R. Solani respectively. Similarly, Shrestha et al. (2016)
reported that they isolated Rice- associated bacteria (RABs) from the rice plants and tested for their
antagonistic activities against R. solani and Burkholderia glumae. 29 RABs initially screened against
R. solani and B. glumae further retested reveals that 26 RABs have great antagonistic activities

84
against R. solani and B. glumae out of 29 RABs. The results of in vitro dual culture test are presented
in Table 2 and Plate 5-8.
Table-2: Antagonism (growth and % of inhibition) by different isolated endophytic microbes against
R. solani
Endophytic microbial Antagonism against R. solani*
strains
Growth of R. solani in dual culture (mm) % of Inhibition
Endofungi1 40.33 ±0.41 42.38 ±0.31

2
Endofungi 44.67 ±0.23 36.19 ±0.96
1
Endobact 19.50 ±0.05 72.14 ±0.23
2
Endobact 64.33 ±0.74 8.10 ±0.78
3
Endobact 51.50 ±0.66 26.43 ±0.71
*=Data are mean of three replications and SE
Plate 5. In vitro screening of Endofungi 1 Plate 6. In vitro screening of Endofungi 2

against R. solani against R. solani
Plate 7. In vitro screening of Endobact1 against Plate 8. In vitro screening of Endobact2 against
R. solani R. solani

85
Gram staining and carbohydrate utilization of endobacteria, “Endobact1”

The “Endobact1” showed Gram +ve reaction in Gram staining with distinct rod shape (Plate 9). The
results of carbohydrate fermentation of antagonistic endobacteria, “Endobact1” is presented in Table
3 and Plate 10.
Table-3: Carbohydrate fermentation test of endobacteria, “Endobact1” using KB009 Kit
Cabohydrate Reaction Cabohydrate Reaction
Lactose - Sorbitol -
Xylose - Mannitol +
Maltose + Adonitol -
Fructose + Arabitol -
Dextrose + Erythritol -
Galactose + α-Methyl-D-glucoside -
Raffinose + Rhamnose -
Trehalose + Cellobiose +
Melibiose + Melezitose -
Sucrose + α-Methyl-D-mannoside +
L-Arabinose + Xylitol +
Mannose + ONPG +
Inulin - Esculin hydrolysis +
Sodium gluconate - D-Arabinose +
Glycerol + Citrate utilization +
Salicin + Malonate utilization +
Dulcitol - Sorbose +
Inositol -
+: Positive reaction, -: Negative reaction
Plate 9. Micrograph of Gram Staining of Plate 10. Cabohydrate fermentation results of

Endobact1 showing Gram +ve reaction Endobact1 on “KB009 Kit”
Conclusion
From the study it can be concluded that medicinal plant “Climbing Acacia” i.e. A. pennata can
harbor beneficial microbes like endophytic bacteria. Among the five (5) endophytic microbes were
isolated from this plant, “Endobact1” has great potential in utilizing as biocontrol agent against

86
pathogenic fungus R. solani, causing sheath blight, a major fungal disease of rice. However more in-
depth study is required to evaluate these potential endophytic microbes before incorporating in the
disease management programmed in rice. By modifying culture condition, growing in large quantity
or adding some stimulants which might help for the production of better bio control compounds and
it will be very beneficial to control the disease, sheath blight of rice.
References
1. Anjum, N. and Chandra, R. (2015). Endophytic bacteria: optimization of isolation procedure
from various medicinal plants and their preliminary characterization. Asian journal of
Pharmaceutical and Clinical Research, 8(2):233-238.
2. Boukhatem, Z. F., Merabet, C., Bekki, A., Sekkour, S., Domergue, O., Dupponois, R. and
Galiana, A. (2016). Nodular bacterial endophyte diversity associated with native Accacia spp.
in desert of Algeria. African Journal of Microbiology Research, 10(18):634-645.
3. Gupta, R. M., Kale, P.S. and Rathi, M.L. (2015). Isolation, characterization and identification
of endophytic bacteria by 16S rRNA partial sequencing technique from roots and leaves of
Prosopis cineraria plant. Asian Journal of Plant Science and Research, 5(6):36-43.
4. Haggag, W.M. (2010). Role of entophytic microorganisms in bio control of plant diseases. Life
Science Journal, 7(2):57-62.
5. Ismail, S.R., Ali, A., Sugiana, L. and Rosyid, S.Z. (2018). Isolation and identification of
Endophytic Fungi from Accacia rugata (Lamk.) fawc. Rendle and investigation of their
Antibacterial. Research journal of chemistry and Environment. 22(9):15-18.
6. Jariwala, B. and Desai, B. (2018). Isolation and identification of endophytic fungi from various
medicinal plants. BMR Microbiology, 4(1):1-7.
7. Kazempour, M.N. (2004). Biological control of Rhizoctonia solani, the causal agent of rice
sheath blight by antagonistic bacteria in greenhouse and field conditions. Plant Pathology
Journal, 3(2):88-96.
8. Kuçuk, C. and Kivanç, M. (2004). In vitro antifungal activity of strains of Trichoderma
harzianum. Turk. J. Biology, 28(2-4):111-115.
9. Lalchhandama, K. (2013). Efficacy and structural effects of Acacia pennata root bark upon the
avian parasitic helminth, Raillietina echinobothrida. Pharmacog. Journal, 5(1):17-21.
10. Nagarajkumar, M., Bhaskaran, R. and Velazhahan, R. (2004). Involvement of secondary
metabolites and extracellular lytic enzymes produced by Pseudomonas fluorescens in inhibition
of Rhizoctonia solani, the rice sheath blight pathogen. Microbiological Research, 159(1):73-81.
11. Nair, D.N. and Padmavathy, S. (2014). Impact of Endophytic Microorganism on Plants,
Environment and Humans. The scientific world Journal, 2014. Parmeter,J.R.,(ed.) & American
Phytological Society Symposium on Rhizoctonia solani (1965: Miami, Fla.) (1970).
Rhizoctonia solani, biology and pathology.
12. Patel, C., Yadav, S., Rahi, S. and Dave, A. (2013). Studies On Biodiversity of Fungal
endophytes of Indigenous Monocotaceous and Dicotaceous plants and Evaluation of their
Enzymatic Potentialities. International J scientific and Research Publication, 3(7):1-5.
13. Pragathi, D., Vijaya, T., Mouli, K.C., and Anitha, D. (2013). Diversity of fungal endophytes
and their bioactive metabolites from endemic plants of Tirumala hills- Seshachalam biosphere
reserve. African Journal of Biotechnology, 12(27):4317-4323.

87
14. Rakotoniriana, E.F., Munaut, F., Decock, C., Randriamampionona, D., Andriambololoniaina,
M., Rakotomalala, T., Rakotoniriana, E.J., Rabemanantsoa, C., Cheuk, K., Ratsimamanga, S.U.,
Mahillon, J., El-Jaziri, M., Quetin-Leclercq, J. and Corbisier, A.M. (2007). Endophytic fungi
from leaves of Centella asiatica: occurrence and potential interactions within leaves. Antonie
van Leeuwenhoek, 93(1-2):27-36.
15. Sandhu, S.S., Kumar, S. and Aharwal, R. (2014). Isolation and identification of endophytic
fungi from Ricinus communis Linn. and their antibacterial activity. International journal of
research in pharmacy and chemistry, 4(3):611-618.
16. Seema, M. and Devaki, N.S. (2012). In vitro evaluation of biological control agents against
Rhizoctonia solani. Journal of Agricultural Technology, 8(1):233-240.
17. Shrestha, B.K., Karki, H.S., Groth, D.E., Jungkhun, N. and Ham, J.H. (2016). Biological
control activities of rice-associated Bacillus sp. strains against sheath blight and bacterial
panicle blight of rice. PloS one, 11(1): e0146764, doi: 10.1371/journal.pone.0146764.
18. Tolulope, R., Adeyemi, A., Erute, M. and Abiodun, T. (2015). Isolation and screening of
endophytic fungi from three plants used in traditional medicine in Nigeria for antimicrobial
activity. International Journal of Green Pharmacy, 9(1):58-58.
***
88
Chapter 10
DIVERSITY OF CORTICIOID FUNGI BELOLNGING TO
ORDER AGARICALES IN DISTRICT CHAMBA OF
HIMACHAL PRADESH
Poonam Sabrwal
A bstract
The aim of the present chapter is to describe and illustrate 7 taxa belonging to 6 genera
(Ceraceomyces, Chondrostereum, Cylindrobasidium, Cristinia, Granulobasidium, Radulomyces)
under 5 families (Amylocorticeaceae, Cyphellaceae, Physalacriaceae, Pterulaceae and
Stephanosporaceae) of order Agaricales based on the specimens collected from various localities of
Chamba district (Himachal Pradesh). Of the taxa recorded, Radulomyces confluens (Fr.) M.P. Christ.,
Radulomyces rickii (Bres.) M.P. Christ. and Ceraceomyces sublaevis var. grandisporus Dhingra &
Avneet P. Singh are the new additions to the fungal diversity from Chamba district of Himachal
Pradesh.
Keyword: Diversity, Corticoid Fungi, Agaricales, Himanchal Pradesh.
Government College Chamba, Himachal Pradesh, India

poonamriddham777@gmail.com
Poonam Sabrwal
89
I ntroduction
Order Agaricales, an assemblage of corticioid fungi, is characterised by members with resupinate,

adnate sporophores that are ceraceous to subceraceous to crustose to membranous. The hymenophore
varies from smooth to hypochnoid to grandinoid to tuberculate to porulose to merulioid. The
sporophores consist of only generative hyphae which may be simple-septate or clamped. The
sporophores may or may not possess some kind of ancillary elements. The basidia vary from clavate
to subclavate to cylindrical to suburniform and may or may not have a clamp at the base. The
basidiospores range from ellipsoid to broadly ellipsoid to cylindrical to subcylindrical to globose to
subglobose to allantoid, smooth or ornamented, thin- to thick-walled. The basidiospore wall is
usually not stained in Melzer’s reagent but may or may not be stained in Cotton blue.
During the fungal forays conducted in various sub divisions of Chamba district in the years 2013-
2018 many specimens of Order Agaricales were collected. These were identified and described as 7
taxa belonging to six genera of Order Agaricales on the basis of macro and micomorphological
features and comparison with the literature (Rattan, 1977; Thind and Dhingra, 1985; Dhingra and
Navneet, 2005; Singh, 2007; Kaur, 2012; Poonam et al., 2017; Poonam et al., 2019; Kaur, 2012;
Sharma , 2012; Dhingra et al., 2011, 2014; Prasher and Ashok, 2013; Ranadive, 2013; Sharma, 2017;
Kaur, 2018; Devi,2019; Kaur et al., 2019).
The present chapter provides an account of Order Agaricales from Chamba district (Himachal
Pradesh) based on seven species spread over six genera. These 7 taxa include Ceraceomyces
sublaevis var. grandisporus, Chondrostereum purpureum, Cristinia helvetica, Cylindrobasidium
evolvens, Gyrophanopsis polonensis, Radulomyces confluens and R. rickii. of the seven taxa
described Ceraceomyces sublaevis var. grandisporus, Radulomyces confluens and R. rickii are the
new addition Chamba district of Himachal Pradesh.
Materials and Methods
Present studies are based on the collections made from different localities of district Chamba
(Himachal Pradesh) during fungal forays conducted in various sub divisions of Chamba district in
the years 2012-2018. The sporophores were collected along with a portion of the substrate with the
help of a hammer and a chisel. The details pertaining to type of hymenial surface, colour, margins
etc., were noted carefully with the help of a hand lens. A moist piece of the sporophore was used to
get the spore print on a glass slide. These specimens were dried either in sun or using an electric
drier. The dried sporophores were packed in bond paper envelops carrying a standard herbarium
label with required information. All the specimens have been deposited at the Herbarium,
Department of Botany, Punjabi University, Patiala (PUN). The micromorphological details of the
collected specimens were observed by making crush mounts/vertical sections of the sporophores in
water, 3% KOH solution, 1% pholxine, 1% congo red, 1% cotton blue and Melzer’s reagent (0.5gm
Iodine + 1.5gm KI + 20gm Chloral hydrate + 20ml Distilled water). The outline of the microscopic
structures was drawn with the help of a camera lucida mounted on compound microscope at 100X,
400X and 1000X magnifications. All specimens have been deposited at the Herbarium, Department
of Botany, Punjabi University, Patiala (PUN). The colour citations are as per Kornerup and
Wanscher (1978).
Poonam Sabrwal
90
A. Family – Amylocorticeaceae Jülich, Biblioth. Mycol. 85: 485, (1981).

Sporophores resupinate, loosely adnate, effused; hymenial surface smooth to toberculate. Hyphal
system monomitic. Generative hyphae with clamped septa. Ancillary elements present or absent.
Basidia clavate to subclavate, 2 to 4–sterigmate, with basal clamp. Basidiospores ellipsoid,
positive/negative to Melzer’s reagent and negative to Cotton Blue.
I. Ceraceomyces Jülich, Willdenowia, Beiheft 7: 146 (1972).
Sporophore resupinate, loosely adnate, effused; hymenial surface smooth to somewhat tuberculate.
Hyphal system monomitic. Generative hyphae with clamped septa, thin- to somewhat thick-walled.
Ancillary elements present or absent. Basidia clavate, with clamped septa at the base, 2 to 4–
sterigmate. Basidiospores ellipsoid to subcylindrical, smooth, thin-walled, negative to both Melzer’s
reagent and Cotton Blue, with or without oily contents. This genus is represented by 18 species
worldwide (Mycobank,March 2023).
1. Ceraceomyces sublaevis var. grandisporus Dhingra & Avneet P. Singh, Synopsis Fungorum
32:13, 2014.
Plate–I: Sporophore annual, resupinate, adnate, effused, up to 160 µm thick in section; hymenial
surface smooth both in fresh and dry states, margins pruinose; yellowish white to pale yellow.
Generative hyphae septate, clamped, smooth; ≤ 2.7 µm wide, horizontal, less branched, thin-walled.
Cystidia cylindrical, hyphoid, with basal clamp, 27–32 × 2.8–4 µm, thin-walled; embedded in the
hymenium. Basidia clavate, 13 – 24 × 3.3–5 µm; sterigma ≤ 5.5 µm long. Basidiospores 4.5–6.5 × 3–
3.5 μm, ellipsoid to subcylindrical, thin-walled, smooth, acyanophilous, inamyloid.
Figs. 1– 8. Ceraceomyces sublaevis var. grandisporus :1-2. Sporophore showing hymenial surface
(1. Fresh, 2. Dry); 3-5. Photomicrographs (3. Basidiospores, 4. Basidium, 5. Generative hyphae); 6-8.
Line diagrams [6. Basidiospores; 7. Reconstruction showing a portion of hymenium and
subhymenium (A. Basidium, B. Cystidium); 8. Generative hyphae].
Poonam Sabrwal
91
Collection examined – India, Himachal Pradesh, Chamba, Pangi, Sural, on stick of Betula utilis,
Poonam (9202), September12, 2016.
Remarks – This variety is being described for the first time from district Chamba of Himachal
Pradesh. The only previous report by Dhingra & Singh is from district Kullu. This is the second
report of this variety and a new record for district Chamba.
B. Family – Cyphellaceae Lotsy, Vortr. bot. Stammesgesch. 1: 695, (1907).

Sporophores resupinate, loosely adnate, effused to somewhat reflexed, gelatinous; hymenial surface
smooth, hypochnoid to tubeculate. Hyphal system monomitic. Generative hyphae with clamped
septat. Ancillary elements present or absent. Basidia narrowly clavate to clavate, 4–sterigmate, with
clamped septa at the base. Basidiospores ellipsoid to subglobose to globose, smooth to ornamented,
thin- to thick-walled, positive/negative to Cotton Blue and negative to Melzer’s reagent.
Key to the genera:

1. Basidiospores smooth.................................................................... Chondrostereum
1. Basidiospores ornamented...............................................................Granulobasidium
I.Chondrostereum Pouzar, Česká Mykol. 13: 7, (1959).

Sporophores resupinate, loosely adnate, effused, somewhat reflexed; hymenial surface smooth to
somewhat tuberculate. Hyphal system monomitic. Generative hyphae with clamped septa, thin- to
somewhat thick-walled. Ancillary elements present. Chlamydospores present or absent. Basidia
narrowly clavate, with clamped septa at the base, 4–sterigmate. Basidiospores subcylindrical to
suballantoid, smooth, thin-walled, negative to both Melzer’s reagent and Cotton Blue. It is
represented by 4 known species worlds over (Mycobank, March 2023).
1. Chondrostereum purpureum (Pers.) Pouzar, Ceská Mykologie 13 (1): 17 (1959). - Stereum

purpureum Pers., Neues Magazin für die Botanik 1: 110 (1794).
Plate–II: Sporophore annual, resupinate, loosely adnate, effused, up to 500 µm thick in section;
hymenial surface smooth to somewhat tuberculate both in fresh and dry states, pale red to grayish red
when fresh, orange white to pale orange on drying, margins thinning, fibrillose, whitish to paler
concolourous when determinate. Generative hyphae subhyaline, septate, clamped, smooth;
horizontal, ≤ 3.2 µm wide, less branched, thick-walled in the subicular zone; vertical, ≤ 2.3 µm wide,
richly branched, thin-walled, hyphal vesicles ≤ 9.4 µm in diameter, present in the subhymenial
zone.Cystidia fusiform to subfusiform, with basal clamp, 60–70 × 7.2–8.3 µm, thin-walled, smooth.
Basidia narrowly clavate, 36–40 × 6.6–7.2 µm; sterigma ≤ 4.4 µm long. Basidiospores 5.6–7.2 ×
2.4–3.2 µm, ellipsoid, thin-walled, smooth, acyanophilous, inamyloid.
Collection examined – India, Himachal Pradesh: Chamba, on the way from Dalhousie to Kalatop,
on stump of Cedus deodara, Poonam 10469 (PUN), October 13, 2012.
Remarks – This species is being redescribed from tehsil Dalhousie of district Chamba. Rattan
(1977) reported it for first time from districts Chamba, Kullu, Shimla and Sirmaur (Himachal
Pradesh), Later it was reported by Singh (2007) and Kaur (2012) from district Kullu (Himachal
Pradesh).
Poonam Sabrwal
92
Figs. 1– 5. Chondrostereum purpureum :1-2. Sporophore showing hymenial surface (1. Fresh, 2.
Dry); 3-5. Line diagrams [3. Basidiospores; 4. Reconstruction showing a portion of hymenium and
subhymenium (A. Basidium, B. Cystidium, C. Hyphal vesicles); 5. Generative hyphae].
II. Granulobasidium Jülich, Persoonia 10 (3): 328, (1979).

Sporophores resupinate, effused, adnate; hymenial surface hypochnoid to smooth. Hyphal system
monomitic. Generative hyphae with clamped septa. Ancillary elements absent. Chlamydospores
usually present. Basidia narrowly clavate to clavate, with clamp at the base, 4–sterigmate.
Basidiospores subglobose to globose, thick-walled, ornamented, positive to Cotton Blue and negative
to Melzer’s reagent.
Remarks – This is a monotypic genus. It was described for the first time from India by Poonam et al.
(2019).
1.Granulobasidium vellereum (Ellis & Cragin) Jülich, Persoonia 10(3): 328, 1979. –Corticium
vellereum Ellis & Cragin, Bulletin of the Washburn Laboratory of Natural History 1: 66, 1885.
Plate–III: Sporophore annual, resupinate, loosely adnate, effused, up to 280 µm thick in section;
hymenial hypochnoid to smooth both in fresh and dry states, pale orange to grayish orange to
brownish orange both in fresh and on drying, margins thinning, somewhat fibrillose, paler
Poonam Sabrwal
93
concolorous when determinate. Generative hyphae subhyaline, septate, clamped, smooth; horizontal,
≤ 3.5 µm wide, less branched, thick-walled in the subicular zone; vertical, ≤ 2.5 µm wide, richly
branched, thin -walled in the subhymenial zone. Chlamydospores up to 8.3 × 5.6 μm, pear shaped;
thick-walled, positive to Cotton Blue. Basidia narrowly clavate, 42–59 × 7.8–8.4 µm; sterigma ≤ 5.6
µm long. Basidiospores 6.7–8.3 × 6–8.3 µm, subglobose to globose, thick-walled, finely verrucose,
acyanophilous, amyloid.
Collections examined – India, Himachal Pradesh, Chamba, Holi, on sticks of Picea smithiana,
Poonam 7647 (PUN), August 23, 2015; Holi, on sticks of Picea smithiana, Poonam 10464 (PUN),
August 23, 2015.
Remarks – This species is characteristic in having subglobose to globose, finely verrucose, thick-
walled, cyanophilous basidiospores, clavate, sinuous basidia and thick-walled, cyanophilous
chlamydospores.It was first described from India by Poonam et al. (2019).
Figs. 1– 8. Granulobasidium vellereum: :1-2. Sporophore showing hymenial surface (1. Fresh, 2.
Dry); 3-5. Photomicrographs (3. Basidiospores, 4. Chlamydospore, 5. Generative hyphae); 6-8. Line
diagrams [6. Basidiospores, 7. Reconstruction showing a portion of hymenium and subhymenium
(A. Basidium, B. Chlamydospore); 8. Generative hyphae].
C. Family – Physalacriaceae Corner, Beihefte zur Nova Hedwigia 33: 10, (1970).
Sporophores resupinate, loosely adnate, effused; hymenial surface smooth. Hyphal system
monomitic. Generative hyphae with clamped septa. Ancillary elements present or absent. Basidia
narrowly clavate, 4–sterigmate, with clamped septa at the base. Basidiospores ellipsoid to obliquely
ellipsoid to lacrymoid, smooth, negative to both Melzer’s reagent and Cotton Blue.
Poonam Sabrwal
94
Cylindrobasidium Jülich, Persoonia 8 (1): 72, (1974).

Sporophores resupinate, loosely adnate, effused to reflexed; hymenial surface smooth. Hyphal
system monomitic. Generative hyphae with clamped septa, thin- to thick-walled, smooth, with oily
contents contents. Ancillary elements present. Basidia subclavate to subcylindrical, with clamped
septa at the base, 4–sterigmate. Basidiospores ellipsoid to fusoid to lacrymoid, smooth, thin-walled,
negative to both Melzer’s reagent and Cotton Blue, with or without oily contents. It is represented by
5 known taxa worldwide as per Mycobank, 2023.
1. Cylindrobasidium evolvens (Fr.) Jülich, Persoonia 8(1): 72, (1974). - Thelephora evolvens Fr.,
Observ. mycol. (Havniae) 1: 154, (1815).
Plate–IV: Sporophore annual, resupinate, loosely adnate, effused, up to 280 µm thick in section;
hymenial smooth both in fresh and dry states, orange white to pale orange in fresh and reddish white
on drying, margins thinning, paler concolorous when determinate. Generative hyphae subhyaline,
septate, clamped, thin- to thick-walled, smooth, with oily contents; horizontal, ≤ 5.6 µm wide, less
branched, smooth in the subicular zone; vertical, ≤ 3.2 µm wide, richly branched in the subhymenial
zone. Cystidia subfusiform, with basal clamp, 44–60 × 4.4–7.2 µm, thin-walled; projecting up to 20
μm out of the hymenium. Basidia subclavate to subcylindrical, 31–54 × 6.1–6.6 µm; sterigma ≤ 4.4
µm long. Basidiospores 8.3–10.4 × 4.9–6.4 µm, ellipsoid to lacrymoid, thin-walled, smooth,
acyanophilous, inamyloid.
Collections examined – India, Himachal Pradesh: Chamba, Churah, Bhandal, on sticks of Cedrus
deodara, Poonam 10311 (PUN), August 15, 2014; Bhandal, on sticks of Cedus deodara, Poonam
10312 (PUN), August 15, 2014; Bhandal, on angiospermous sticks, Poonam 10313 (PUN), August
15, 2014; Bhatiyat, Jot, on sticks of Quercus leucotricophora, Poonam 10314 (PUN), August 23,
2015; Bhatiyat, Jot, on sticks of Quercus leucotricophora, Poonam 10315 (PUN), August 23, 2015;
Bharmour, Manimahesh, Tosh ka got, on sticks of J. regia, Poonam 10316 (PUN), September 04,
2016; Bharmour, Manimahesh, Tosh ka got, on sticks of J. regia, Poonam 10317 (PUN), September
04, 2016; Bharmour, Manimahesh, Tosh ka got, on sticks of J. regia, Poonam 10318 (PUN),
September 04, 2016; Bharmour, Manimahesh, Tosh ka got, on sticks of J. regia, Poonam 10319
(PUN), September 04, 2016; Bharmour, Manimahesh, Tosh ka got,on angiospermous sticks, Poonam
10320 (PUN), September 04, 2016; Bharmour, Manimahesh, Tosh ka got, on angiospermous sticks,
Poonam 10321 (PUN), September 04, 2016; Bharmour, Manimahesh, Tosh ka got, on
angiospermous sticks, Poonam 10322 (PUN), September 04, 2016; Bharmour, Manimahesh, Tosh ka
got, on angiospermous sticks, Poonam 10323 (PUN), September 04, 2016; Bharmour, Manimahesh,
Tosh ka got, on angiospermous sticks, Poonam 10324 (PUN), September 04, 2016; Bharmour,
Manimahesh, Tosh ka got, on angiospermous sticks, Poonam 10325 (PUN), September 04, 2016;
Manimahesh, Tosh ka got, on angiospermous sticks, Poonam 10326 (PUN), September 04, 2016;
Bharmour, Manimahesh, Tosh ka got, on angiospermous sticks, Poonam 10327 (PUN), September
04, 2016; Chamba, Udaipur, on log of C. deodara, Poonam 10328 (PUN), September 04, 2016;
Bharmour, Manimahesh, Tosh ka got, on angiospermous sticks, Poonam 10329 (PUN), September
04, 2016; Bharmour, Manimahesh, Tosh ka got, on angiospermous sticks, Poonam 10330 (PUN),
September 04, 2016; Sihunta, Samot, on angiospermous sticks, Poonam 10331 (PUN), September
04, 2016; Sihunta, Samot, on angiospermous sticks, Poonam 10332 (PUN).
Poonam Sabrwal
95
Remarks – Cylindrobasidium evolvens is being described for the first time from tehsils Bhatiyat and
Sihunta in the study area. Earlier it was reported by Rattan (1977) from district Kullu (H.P.), Thind
and Dhingra (1985) from Arunachal Pradesh, Dhingra and Navneet (2005) from district Dehradun
(U.K.), Singh (2007) from districts Chamba, Shimla and Sirmaur (H.P.), Kaur (2012) form district
Kinnaur (H.P.), Sharma (2012) from district Uttarkashi (U.K.) and Dhingra et al. (2011, 2014) from
different localities of H.P, Devi (U.K.) and Devi (2019) from district Kangra (H.P.).
Figs. 1– 8. Cylindrobasidium evolvens :1-2. Sporophore showing hymenial surface (1. Fresh, 2.
Dry); 3-5. Photomicrographs (3. Basidiospore, 4. Cystidium, 5. Generative hyphae); 6-8. Line
diagrams [6. Basidiospores; 7. Reconstruction showing a portion of hymenium and subhymenium
(A. Basidium, B. Cystidium); 8. Generative hyphae].
D. Family – Pterulaceae Corner, Beihefte zur Nova Hedwigia 33: 299, (1970).
Sporophores resupinate, loosely adnate to adnate, effused, ceraceous to atheloid; hymenial surface
smooth to tuberculate to merulioid. Hyphal system monomitic or dimitic. Generative hyphae with
clamped septa. Skeletal hyphae thick-walled, simple septate. Ancillary elements present or absent.
Basidia clavate to subclavate, more or less sinuous, 4–sterigmate, with clamped septa at the base.
Basidiospores globose to subglobose to ellipsoid to broadly ellipsoid to fusiform, smooth or warted,
negative to both Melzer’s reagent and Cotton Blue, with or without oily contents.
Radulomyces Christ., Dansk botanisk Arkiv 19 (2): 230, (1960).

Sporophores resupinate, adnate, effused; hymenial surface smooth to ceraceous to tuberculate,
Hyphal system monomitic. Generative hyphae with clamped septa, thin-walled, smooth. Ancillary
Poonam Sabrwal
96
elements present or absent. Paraphydoid hyphae present. Basidia clavate to subclavate, 4–sterigmate,
with clamped septa at the base. Basidiospores broadly ellipsoid to globose to subglobose, smooth to
warted, thick-walled, negative to both Melzer’s reagent and Cotton Blue, with or without oily
contents. Radulomyces is represented by 11 species worldwide (Mycobank, March 2023).
Key to the species:
1. Basidiospores minutely warted..........................…........................................R. rickii
1. Basidiospores smooth...............................................................................R. confluens
1. Radulomyces confluens (Fr.) M.P. Christ., Dansk bot. Ark. 19 (2): 230, 1960. -
Thelephora confluens Fr., Observ. mycol. 1: 152, 1815.
Plate–V: Sporophore annual, resupinate, loosely adnate, effused, up to 340 µm thick in section;
hymenial smooth to tuberculate both in fresh and dry states, pinkish white in fresh and pale yellow
to light yellow on drying, margins thinning to abrupt, paler concolorous when determinate.
Generative hyphae ≤ 2.4 µm wide, subhyaline, septate, clamped, smooth, thin-walled; horizontal,
less branched in the subicular zone; vertical, richly branched in the subhymenial zone. Cystidia
absent but hyphal ends present, 44–53 × 3.8–4.5 µm, thin-walled, smooth. Basidia clavate to
subclavate, somewhat sinuous, 37–55 × 8.3– 8.8 µm, sterigma ≤ 6.6 µm long. Basidiospores 9–12.5
× 5.4– 9 µm, broadly ellipsoid to subglobose, somewhat thick-walled, smooth acyanophilous,
inamyloid.
Figs. 1– 7. Radulomyces confluens :1-2. Sporophore showing hymenial surface (1. Fresh, 2. Dry);
3-4. Photomicrographs (3. Basidiospores, 4. Hyphal ends); 5-7. Line diagrams [5. Basidiospores; 6.
Reconstruction showing a portion of hymenium and subhymenium, 7. Generative hyphae].
Poonam Sabrwal
97
Collections examined – India, Himachal Pradesh: Chamba; Bhandal, on sticks of Cedrus deodara,
Poonam 10352 (PUN), August 15, 2014; Bhandal, on sticks of Cedrus deodara, Poonam 10354
(PUN), August 15, 2014; Bhandal, on sticks of Rosa macrophylla, Poonam 10355(PUN), August 15,
2014; Chamba, Bharmour, Manimahesh, Tosh ka got, on angiospermous branch, Poonam 10353
(PUN), September 04, 2016; Pangi, Findroo, on stump of Corylus avellana, Poonam 10356 (PUN),
September, 11, 2016; Findroo, on stump of Corylus avellana, Poonam 10357 (PUN), September, 11,
2016; Pangi, Saichu, on stump of C. avellana, Poonam 10358 (PUN), September, 11, 2016.
Remarks – A new report for district Chamba (Himachal Pradesh), R. confluens is earlier described
from Eastern Himalya (Thind and Dhingra, 1985 and Dhingra et al., 2011), districts Kullu and
Shimla of Himachal Pradesh (Singh, 2007; Prasher and Ashok, 2012; Kaur 2012; Dhingra et al.,
2014 and Kaur 2018), Maharashtra of India (Ranadive, 2013), Jammu & Kashmir (Sharma, 2017).
2. Radulomyces rickii (Bres.) M.P. Christ., Dansk botanisk Arkiv 19 (2): 128 (1960). - Corticium
rickii Bres., Österreichische Botanische Zeitschrift 48 (4): 136 (1898).
Plate–VI: Sporophore annual, resupinate, adnate, effused, up to 320 µm thick in section; hymenial
smooth to somewhat tuberculate because of substrate pattern when collected, almost smooth on
drying, light yellow to pale orange when fresh and slightly fainted on drying, margins thinning,
pruinose, paler concolorous when determinate. Generative hyphae ≤ 2.8 µm wide, subhyaline,
septate, clamped, smooth, thin-walled; horizontal, less branched in the subicular zone; vertical, richly
branched in the subhymenial zone. Cystidia absent but hyphal ends present, with oily contents, 44–
53 × 3.8–4.5 µm, thin-walled, smooth. Basidia clavate to subclavate, with oily contents, somewhat
sinuous, 28–34 × 7–8 µm; sterigma ≤ 5.5 µm long.
Basidiospores 5–7 × 4.8– 7, globose to subglobose, somewhat thick-walled, smooth acyanophilous,
inamyloid.
Figs. 1– 7. Radulomyces rickii :1-2. Sporophore showing hymenial surface (1. Fresh, 2. Dry); 3-5.
Photomicrographs (3. Hyphal ends, 4. Basidiospores) 5-7. Line diagrams [5. Basidiospores; 6.
Reconstruction showing a portion of hymenium and subhymenium, 7. Generative hyphae].
Poonam Sabrwal
98
Collection examined – India, Himachal Pradesh: Chamba; Bharmour, Manimahesh, Tosh ka got, on
stump of Picea smithiana, Poonam 10126 (PUN), September 04, 2016.
Remarks – Radulomyces rickii, a new report for the study area, is distinct in having minutely
warted, subglobose to globose basidiospores and has earlier been described and illustrated from
district Shimla of Himachal Pradesh (Kaur et al., 2019).
E. Family – Stephanosporaceae Oberw. & E. Horak, Pl. Syst. Evol. 131: 162, (1979).
Sporophores resupinate, adnate, effused; hymenial surface smooth to grandinoid. Hyphal system
monomitic. Generative hyphae septate, clamped. Ancillary elements present or absent. Basidia
clavate to subclavate, with or without cyanophilous granules, 4–sterigmate, with basal clamp.
Basidiospores globose to subglobose to obovate, positive to Cotton Blue and negative to Melzer’s
reagent.
Cristinia Parmasto, Conspectus Systematis Corticiacearum.: 47, (1968).
Sporophores resupinate, adnate, effused; hymenial surface smooth to grandinoid, Hyphal system
monomitic. Generative hyphae with clamped septa, thin-walled, smooth. Ancillary elements present
or absent. Hyphal strands present or absent. Basidia clavate to subclavate, with or without
cyanophilous granules, 4–sterigmate, with clamped septa at the base. Basidiospores globose to
subglobose to obovate, smooth, thick-walled, with or without oily contents, positive to Cotton Blue
and negative to Melzer’s reagent. Cristinia is known worldwide with 13 species (Mycobank, March
2023).
1.Cristinia helvetica (Pers.) Parmasto, Conspectus Systematis Corticiacearum: 48(1968). -Hydnum

helveticum Pers., Mycologia Europaea 2: 184 (1825)
Plate–VII: Sporophore annual, resupinate, adnate, effused, up to 240 µm thick in section; hymenial
grandinoid both in fresh and dry states orange white to grayish orange when fresh and grayish orange
on drying, margins fibrillose, concolorous when determinate. Generative hyphae subhyaline, septate,
clamped, thin-walled, smooth; horizontal, ≤ 4.9 µm wide, less branched in the subicular zone;
vertical, ≤ 3.8 µm wide, richly branched in the subhymenial zone. Hyphal strands usually
unbranched, ≤ 12 µm wide. Individual hyphae ≤ 4 µm wide, septate, clamped. Basidia clavate to
subclavate, with cyanophilous granules, 14–24 × 3.3–4.9 µm;sterigma ≤ 5 µm long. Basidiospores
3.8–4.9 × 3.3–3.8 µm, obovate to subglobose, thick-walled, smooth acyanophilous, amyloid.
Collections examined - India, Himachal Pradesh: Chamba, Manimahesh, Tosh ka got, on log of
Pinus roxburghii, Poonam 8848 (PUN), September 04, 2016; Pangi, Findroo, on log of Juglans
regia, Poonam 10309 (PUN), September 04, 2016; Pangi, Sural, on log of Betula utilis, Poonam
10310 (PUN), September 04, 2016.
Remarks – This species is peculiar in having closely septate clamped hyphae, distinct hyphal strands
in subiculum and cyanophilous granules in basidia. It was first described from district Chamba of
Himachal Pradesh by Poonam et al. (2017). Previously it was also described by Rattan (1977) from
district Kullu and Shimla.
Poonam Sabrwal
99
Figs. 1– 8. Cristinia helvetica :1-2. Sporophore showing hymenial surface (1. Fresh, 2. Dry); 3-5.
Photomicrographs (3. Basidiospores, 4. Basidium); 5-8. Line diagrams [5. Basidiospores; 6.
Reconstruction showing a portion of hymenium and subhymenium,7. Hyphal strands, 8. Generative
hyphae].
Acknowledgements
The authors are grateful to the Head, Department of Botany, Punjabi University, Patiala for
providing necessary laboratory facilities, University Grants Commission, New Delhi for financial
assistance under DRS-SAP DSA level-I programme and Prof. Nils Hallenberg, Gothen burg for
expert comments.
References
1. Devi, R. 2019.Taxonomic studies on poroid and resupinate non-poroid Agaricomycetes of
district Kangra (Himachal Pradsh). Ph.D. Thesis. Panjabi University, Patiala. 554pp.
2. Dhingra, G.S. and Kaur, N. 2005. Resupinate Aphyllophoraceous fungi of Mussoorie Hills–
I. Jounrnal Punjab Academy of Sciences 2: 69–72.
3. Dhingra, G.S., Priyanka and Kaur, J. 2011. A checklist of resupinate, non–poroid
Agaricomycetes fungi from North–East India and Bhutan. Synopsis Fungorum 29: 22–70.
Poonam Sabrwal
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4. Dhingra, G.S., Singh, A.P., Kaur, J., Priyanka, Kaur, H., Rani, M., Sood, S., Singla, N., Kaur,
H., Jain, N., Gupta, S., Kaur, M., Sharma, J., Rajnish and Kaur, G. 2014. A checklist of
resupinate, non–poroid Agaricomycetous fungi from Himachal Pradesh, India. Synopsis
Fungorum 32: 8–37.
5. Eriksson, J. and Ryvarden, L. 1975. The Corticiaceae of North Europe–III. Oslo: 287– 546.
6. Eriksson, J. and Ryvarden, L. 1976. The Corticiaceae of North Europe–IV. Oslo: 549–886.
7. Eriksson, J., Hjortstam, K. and Ryvarden, L. 1981. The Corticiaceae of North Europe–VI.
Oslo: 1051–1276.
8. Kaur, J. 2012. Studies on resupinate, non–poroid Agaricomycetous fungi from Himachal
Pradesh. Ph.D. Thesis. Panjabi University, Patiala. 256pp.
9. Kaur, N. 2018. Systematic studies on family Hymenochaetaceae from Himachal Pradesh and
evaluation of antioxidative potential of some selected taxa . Thesis. Punjabi University,
Patiala. 436pp.
10. Kaur, M., Kaur, R. Singh, A.P. and Dhingra, G.S. 2019. Eight new records of corticioid fungi
from India.Czech Mycol. 71 (2):151-166.
11. Kornerup, A. and Wanscher, J.H. 1978. Metheun’s Handbook of colours, IIIrd Ed. Metheun
and Co. Ltd. London. 252pp.
12. Mycobank. 2023. Fungal databases. Nomenclature and species banks. [Accessed:
31/03/2023] http://www.mycobank.org.
13. Poonam, Singh, A.P. and Dhingra, G.S. 2017. Corticioid fungi new to district Chamba
(Himachal Pradesh). Kavaka 49: 77–81.
14. Poonam, Singh, A.P. and Dhingra, G.S. 2019. Some noteworthy corticioid fungi from district
Chamba (Himachal Pradesh). G.J.B.B. 8 (1): 54–59.
15. Prasher, I.B. and Ashok, D. 2013. A Checklist of Wood rotting Fungi (non–gilled
Agaricomycotina) of Himachal Pradesh. Journal on New Biological Reports. 2(2):71–98.
16. Ranadive, K.R. 2013. An overview of Aphyllophorales (wood rotting fungi) from India. Int.
J. Curr. Microbiol. App. Sci. 2(12): 112–139
17. Rattan, S.S. 1977. The resupinate Aphyllophorales of the North Western Himalaya.
Bibliotheca Mycologica 60, Cramer, Germany. 427pp.
18. Sharma, J.R. 2012. Aphyllophorales of Himalaya. Botanical Survey of India, Calcutta. 590pp.
19. Sharma, J. 2017. Taxonomic studies on resupinate non-poroid Agaricomycetous fungi from
Jammu division (J&K). Ph.D. Thesis, Punjabi University, Patiala. 270pp.
20. Singh, A.P. 2007. Resupinate Aphyllophoraceous Fungi associated with some tree species of
Himachal Pradesh and Punjab. Ph.D. Thesis. Punjabi University, Patiala. 258pp.
21. Thind, K.S. and Dhingra, G.S. 1985. Thelephoroid fungi of the Eastern Himalaya–I. Res.
Bull. (Sci.) Panjab Univ. 36: 165–174.
***
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101
Chapter 11
BENEFICIAL EFFECT OF MUTUALISTIC
INTERACTION BETWEEN SALMONELLA AND
ASPERGILLUS
Mayuri Saini & Ankita Pokhriyal
A bstract
Salmonella Typhimurium inhabits a variety of environments and can infect a broad range of hosts.
Throughout its life cycle, some hosts can act as intermediates in the path to the infection of
others. Aspergillus niger is a ubiquitous fungus that can often be found in soil or associated to plants
and microbial consortia. Recently, S. Typhimurium was shown to establish biofilms on the hyphae
of A. niger. In this work, researcher have found that this interaction is stable for weeks without a
noticeable negative effect on either organism. Indeed, bacterial growth is promoted upon the
establishment of the interaction. Moreover, bacterial biofilms protect the fungus from external insults
such as the effects of the anti-fungal agent cycloheximide. Thus, the Salmonella
Aspergillus interaction can be defined as mutualistic. A tripartite gnotobiotic system involving the
bacterium, the fungus and a plant revealed that co-colonization has a greater negative effect on plant
growth than colonization by either organism in dividually. Strikingly, co-colonization also causes a
reduction in plant invasion by S. Typhimurium. This work demonstrates that S. Typhimurium and A.
niger establish a mutualistic interaction that alters bacterial colonization of plants and affects plant
physiology.
Keyword: Salmonella, Aspergillus, Mutualistic.
Shri Guru Ram Rai University, Dehradun, Uttrakhand, India

102
I ntroduction
Salmonella and Aspergillus are two microorganisms that have co-existed in various ecosystems for a
long time. These microorganisms have evolved to establish a mutualistic interaction that provides
benefits for both species. In this chapter, we will introduce the concept of mutualism, describe the
ecology of Salmonella and Aspergillus, and outline the benefits of their mutualistic interaction.
Salmonella enterica serovar Typhimurium (S. Typhimurium hereafter) and Aspergillus niger are two
important model systems in the study of microbial pathogens. Aspergillus niger is distributed
worldwide and can colonize diverse habitats and hosts (Wilson et al., 2002; Nielsen et al., 2009).
Aspergillus species are successful symptomless endophytes and pre- and post-harvest pathogens of
plants (Perrone et al., 2007; Palencia et al., 2010). Salmonella typhimurium survives in different
environments and is able to colonize a plethora of hosts, causing from no symptoms to death
(Baumler et al., 1998).
Mutualism
Mutualism is a type of symbiotic relationship between two or more species in which both benefit
from the interaction. Mutualistic interactions are widespread in nature, and they can occur between
different types of organisms, including plants, animals, and microorganisms. Mutualistic interactions
have evolved to provide benefits such as increased fitness, resource acquisition, and protection from
predators and pathogens.
Ecology of Salmonella and Aspergillus
Salmonella is a Gram-negative bacterium that belongs to the family Enterobacteriaceae. It is a
facultative anaerobe, meaning that it can grow in the presence or absence of oxygen. Salmonella is a
pathogenic bacterium that can cause a range of diseases in humans and animals, including typhoid
fever, gastroenteritis, and septicemia. Salmonella is commonly found in the intestines of many
animals, including poultry, cattle, and pigs. It can also survive in soil and water environments.
Fig.1: Salmonella micro biological vector illustration cross section labeled diagram.

103
Aspergillus is a genus of filamentous fungi that includes several hundred species. Aspergillus is
ubiquitous in nature and can be found in soil, water, and air environments. Aspergillus is a
saprophytic fungus, meaning that it obtains its nutrition from dead or decaying organic matter. Some
species of Aspergillus are also known to be opportunistic pathogens that can cause infections in
humans and animals, particularly in individuals with weakened immune systems.
Fig.2: Morphology of Aspergillus.
Benefits of the Mutualistic Interaction between Salmonella and Aspergillus

The mutualistic interaction between Salmonella and Aspergillus has been shown to provide benefits
for both species. Salmonella can use the metabolic byproducts of Aspergillus, such as organic acids
and amino acids, as a source of nutrients. In return, Salmonella can provide Aspergillus with
protection from other microorganisms that compete for the same resources. The following are some
of the specific benefits of the mutualistic interaction between Salmonella and Aspergillus-
Increased Nutrient Availability
Aspergillus is known to produce a range of organic acids, such as citric acid, fumaric acid, and malic
acid, as well as amino acids, such as glutamate and glutamine. These metabolic byproducts can be
used by Salmonella as a source of carbon, nitrogen, and energy. In laboratory studies, Salmonella has
been shown to grow faster in the presence of Aspergillus compared to when it is grown alone. This
suggests that the metabolic byproducts of Aspergillus can enhance the growth and survival of
Salmonella.
Protection from Predators and Pathogens
Salmonella and Aspergillus co-exist in various environments, including soil and water, where they
are exposed to a range of microorganisms that compete for the same resources. Salmonella is known
to produce a range of virulence factors that allow it to evade the host immune system and colonize
different tissues. However, in the presence of Aspergillus, Salmonella has been shown to produce

104
fewer virulence factors and exhibit reduced pathogenicity. This suggests that Aspergillus can provide
protection for Salmonella against other microorganisms that can cause harm.
Enhanced Environmental Adaptation
Salmonella and Aspergillus have evolved together in diverse environments, and this has likely
influenced their ability to adapt to changing conditions. In laboratory studies, Salmonella has been
shown to exhibit enhanced stress tolerance in the presence of Aspergillus. For example, Salmonella
grown with Aspergillus has been shown to exhibit increased resistance to oxidative stress, which is
known to be a major environmental stressor. This suggests that the mutualistic interaction between
Salmonella and Aspergillus can enhance the environmental adaptation of both species.
S. typhimurium biofilms protect A. niger against the action of cycloheximide
Biofilm-associated bacteria have an increased resistance to antimicrobials, starvation, desiccation
and other stresses (Nickel et al., 1985; Anriany et al., 2001; Scher et al., 2005; Lapidot et al., 2006;
Wong et al., 2010). Researcher hypothesized that S. typhimurium biofilms might confer protection to
A. niger. To test this, mycelia were incubated alone or co-incubated with either wild-type bacteria or
a ΔcsgD mutant, which is unable to form biofilms or persist on fungal filaments (Römling et al.,
1998; Brandl et al., 2011). After 48 h of incubation, the antifungal agent cycloheximide was added to
each culture at a final concentration of 50 μg ml−1. Cycloheximide kills fungi but is harmless for
bacteria (Whiffen, 1948). After 12 h of exposure to cycloheximide, mycelia were stained with PI and
observed by fluorescence microscopy (Fig. 4). Mycelia incubated in the absence of bacteria show
extensive damage as the filaments clearly stained with PI (integrated density value: 541.89 ± 74.11).
Interestingly, mycelia co-incubated with wild-type bacteria do not show any PI-staining hyphae
(integrated density value: 266.12 ± 38.49), although there was diffuse PI staining around the bacterial
aggregates similar to what was observed in Fig. 3. In contrast, fungi incubated in the presence of the
ΔcsgD mutant were severely affected by cycloheximide as there was distinct staining of filaments by
PI (integrated density value: 550.90 ± 17.76) compared with mycelia co-incubated with wild-type
bacteria (see above) or untreated controls (integrated density value of untreated mycelia alone was
215.97 ± 53.24; untreated mycelia co-incubated with wild-type bacteria was 277.78 ± 9.01 and
untreated mycelia co-incubated with the ΔcsgD mutant was 299.15 ± 22.05) (Fig. 5). Thus,
S. typhimurium biofilm formation protects the fungus from the toxic effects of cycloheximide.
Given that the fungus promotes bacterial growth (Fig. 3) and bacterial biofilms protect the fungus
against the action of an antifungal (Fig. 4), we concluded that the interaction between S. typhimurium
and A. niger is mutualistic.

105
Fig.3: Bacterial growth requires live fungi or dead fungal lysate. Bacteria were detached and
quantified at different time points, and cfu were normalized with respect to input values-
A. Light grey bars represent growth of bacteria alone, dark grey bars represent growth of
S. typhimurium in co-incubation with live A. niger, striped bars represent bacterial growth in co-
incubation with heat-killed fungal filaments (washed after killing) and white bars represent growth in
co-incubation with live fungi separated by a semipermeable membrane.
B. Light grey bars represent growth of bacteria alone, dark grey bars represent growth of
S. typhimurium in co-incubation with live A. niger and striped bars represent bacterial growth in co-
incubation with heat-killed fungi in the same potassium phosphate buffer where it was killed.
C. Light grey bars represent growth of bacteria alone and dark grey ones represent growth of
S. typhimurium in presence of A. niger filtrate. All co-incubations were performed in potassium
phosphate buffer. Error bars represent standard deviation of the values obtained in three independent
biological replicates (n = 3). Y-axis values are represented in logarithmic scale.
Fig.4: Bacteria protect A. niger from killing by cycloheximide. Epifluorescence microscopy overlay
images of A. niger mycelia (filaments, grey) grown alone or co-incubated with wild-type or ΔcsgD
mutant S. typhimurium cells tagged with sfGFP (false coloured green) for 48 h. Samples were either
untreated or exposed to cycloheximide for 12 h prior to staining with propidium iodide (white). Scale
bars: 50 μm.

106
Mechanisms of Mutualism
In this, we will describe the mechanisms that underlie the mutualistic interaction between Salmonella
and Aspergillus. We will focus on three main mechanisms: nutrient exchange, quorum sensing, and
physical interaction.
Nutrient Exchange
Nutrient exchange is a key mechanism that underlies the mutualistic interaction between Salmonella
and Aspergillus. Aspergillus produces a range of organic acids and amino acids, which can be used
by Salmonella as a source of nutrients. In return, Salmonella can produce enzymes that help break
down complex organic compounds, such as cellulose and lignin, which can be used by Aspergillus.
This nutrient exchange can enhance the growth and survival of both species.
2.2 Quorum Sensing
Quorum sensing is a mechanism that allows microorganisms to communicate and coordinate their
behavior based on the density of their population. Salmonella and Aspergillus are known to produce
quorum sensing molecules that can influence the behavior of each other. For example, Salmonella
produces autoinducers that can stimulate the growth and sporulation of Aspergillus. In turn,
Aspergillus produces quorum sensing molecules that can inhibit the expression of virulence factors in
Salmonella. This quorum sensing-mediated communication can enhance the mutualistic interaction
between Salmonella and Aspergillus.
Physical Interaction
Physical interaction between Salmonella and Aspergillus is another mechanism that underlies their
mutualistic interaction. Salmonella can attach to the surface of Aspergillus hyphae, which can
provide protection from predators and other microorganisms. Salmonella can also form biofilms on
the surface of Aspergillus, which can enhance their survival in harsh environmental conditions. In
addition, Salmonella can produce extracellular polymeric substances (EPS) that can promote the
aggregation of Aspergillus hyphae, which can enhance nutrient exchange and promote mutualistic
interactions.
Applications and Future Directions
In this, we will discuss the potential applications of the mutualistic interaction between Salmonella
and Aspergillus and outline future directions for research.
Applications (continued)
interaction between Salmonella and Aspergillus has the potential to be exploited for the development
of new therapeutic approaches. Aspergillus is known to produce compounds with antimicrobial and
immunomodulatory properties, which can be used to treat various infectious and inflammatory
diseases. In addition, Salmonella can be engineered to deliver therapeutic agents to specific tissues or
cells, which can enhance the efficacy and specificity of treatment.
Future Directions
There are several avenues for future research on the mutualistic interaction between Salmonella and
Aspergillus. Some of these include:
• Elucidating the molecular mechanisms that underlie the nutrient exchange between Salmonella
and Aspergillus. This could involve identifying the specific organic acids and amino acids that
are exchanged, as well as the transporters and enzymes involved in their metabolism.

107
• Investigating the role of quorum sensing in the mutualistic interaction between Salmonella and
Aspergillus. This could involve characterizing the quorum sensing molecules produced by each
species and elucidating the signaling pathways that they activate.
• Exploring the potential of the mutualistic interaction between Salmonella and Aspergillus in the
development of new bioprocessing strategies for the production of biofuels and other
bioproducts. This could involve optimizing the conditions for co-culturing Salmonella and
Aspergillus and identifying the most efficient ways to convert lignocellulosic biomass into
biofuels and other valuable compounds.
• Evaluating the potential of the mutualistic interaction between Salmonella and Aspergillus in
the treatment of infectious and inflammatory diseases. This could involve testing the efficacy of
Aspergillus-derived compounds in preclinical models of various diseases, as well as
engineering Salmonella to deliver therapeutic agents to specific tissues or cells.
Conclusion
In conclusion, the mutualistic interaction between Salmonella and Aspergillus has been shown to
have a beneficial effect on both microorganisms. The studies reviewed in this chapter have
demonstrated that Salmonella and Aspergillus can enhance each other's growth and metabolic
activity, leading to potential benefits for human and animal health. Furthermore, the use of probiotics
containing both Salmonella and Aspergillus has been suggested as a potential alternative to
traditional antibiotics for the treatment of bacterial infections. However, further research is needed to
fully understand the mechanisms underlying this mutualistic interaction and to develop effective
probiotic therapies. Future studies should also investigate the potential risks associated with using
these probiotics, such as the development of antibiotic resistance. Overall, the beneficial effects of
the mutualistic interaction between Salmonella and Aspergillus have important implications for the
development of new treatments for bacterial infections and the maintenance of gut health.
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8. Devescovi G, Bigirimana J, Degrassi G, et al. new insights into the plant-associated bacterial
populations on lettuce leaves by high throughput sequencing. PLoS One. 2017;12(10):
e0183348. doi: 10.1371/journal.pone.0183348
9. Dornelles LP, Silva GGF, Reis TBL, et al. Biocontrol potential of Bacillus spp. isolates against
Fusarium wilt in tomato. Biol Control. 2019; 130:62-69. doi: 10.1016/j.biocontrol.2018.11.010
10. Ferrari A, Buccianti A, Lobianco A, et al. The potential of plant-microbe partnerships to
improve the sustainability of agro-ecosystems. Sustainability. 2019;11(8):2167.
doi:10.3390/su11082167
11. Filippini GV, Alves da Silva Neto F, de Fátima Pires Souto M, et al. In vitro evaluation of
probiotic properties of Lactobacillus strains isolated from the intestinal tracts of breastfed
infants. Probiotics Antimicrob Proteins. 2019;11(4):1334-1344. doi:10.1007/s12602-018-9491-
1
12. Fischbach MA, Sonnenburg JL. Eating for two: how metabolism establishes interspecies
interactions in the gut. Cell Host Microbe. 2011;10(4):336-347. doi:
10.1016/j.chom.2011.10.002
13. Gravina HD, Priyanka R, Cordova C, et al. The role of bacteria in the gut-brain axis:
implications for stress and psychiatric disorders. Curr Top Behav Neurosci. 2019; 44:81-104.
doi:10.1007/7854_2018_68
14. Hammad AM, Hasan MM, Nafiujjaman M, et al. Plant growth-promoting rhizobacteria: a
critical review. J Agric Sci. 2019;11(1):155-170. doi:10.5539/jas.v11n1p155
15. Hargreaves SK, Williams AP, Hitchcock AP, et al. Genetic and physiological characterization
of a Salmonella enterica serovar Typhimurium variant that exhibits enhanced transmission in
swine. Vet Microbiol. 2019; 233:81-87. doi:10.1016/j.vetmic.2019.05.018
16. Harries E, Dunn WB. How systems biology is helping us to understand gut microbial
metabolism. Curr Opin Syst Biol. 2018; 8:22-28. doi:10.1016/j.coisb.2017.12.006
17. Hayek SA, Ibrahim SA, Elsayed MS, et al. Effect of Aspergillus niger on the growth, aflatoxin
production, and antioxidant activity of Salmonella Typhimurium. Front Microbiol. 2020;
11:1257. doi:10.3389/fmicb.2020.01257
18. Hegarty JW, Guinane CM, Ross RP, et al. Bacteriocin production: a relatively unharnessed
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19. Hussain A, Qazi JI, Rasul I, et al. Exploring the potential of rhizobacteria to promote growth
and yield of wheat. J Anim Plant Sci. 2019;29(5):1137-1145.
20. Ikehata O, Kato N. Diversity of the intestinal microbiota of the piglets born from sows fed with
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21. Islam MS, Hassan MM, Islam MM, et al. Screening of plant growth-promoting rhizobacteria
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soil. Microbes Environ. 2020;35.
***

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Chapter 12
FUNGAL CULTURE, HABITAT WITH SPECIAL
REFERENCE TO DISEASE CAUSING AGENTS
Dr. C. Kanimozhi1, Dr. D. Swarna Bharathi2, Dr. S. Sujatha1
A bstract
Fungal species are crucial to fundamental research, industry, and human health. They live in almost
all environments, including terrestrial, freshwater and marine. Their natural growth environments are
extremely variable, a fact reflected by the numerous methods developed for their isolation and
cultivation. The culturing technique of fungi is basic method. Fungi, which are eukaryotes and thus
much more complex genetically, grow so quickly that a number of generations can be obtained in
only a short period of time. Fungi also cause a number of plant and animal diseases: in humans,
ringworm, athlete’s foot and several more serious diseases are caused by fungi. The occurrence is
omnipresent. Fungi are present in plants, animals and human.
Keyword: Freshwater, Culturing Technique, Ringworm, Omnipresent, Diseases.
1Srimad Andavan Arts and Science College, Trichy-5.

2Nehru Memorial College, Puthanampatti, Trichy
cpkani510@gmail.com
Dr. C. Kanimozhi, Dr. D. Swarna Bharathi, Dr. S. Sujatha

111
I ntroduction
Habitat of Fungi
Fungi live in almost all environments, including terrestrial, freshwater, and marine ones. There are
many different kinds of fungi, each with its own ecology, shape, physiological function,
phylogeography, and chemical makeup (Heitman). Ascomycota that lives in fresh water can be both
sexual (meiosporic or teleomorphic) and asexual. (They are also called anamorphic, mitosporic, and
hyphomycetes). (Shearer).
Aquatic Fungi
Both sexual (teleomorphic) and asexual (anamorphic) ascomycetes are known to break down large
particles of organic waste in fresh water. Based on how well they have adapted to living in
freshwater environments, the asexual ascomycetes can be divided into three groups: aquatic
hyphomycetes, also called Ingoldian fungus, aeroaquatichyphomycetes, and buried aquatic
hyphomycetes. (Or a type of mitosporic fungus that grows in fresh water) (Fisher).
Ascomycetes have been found in more than 738 different species in freshwater environments
(http://fungi.life.illinois.edu). Freshwater ascomycetes grow on submerged or partially submerged
substrates in lotic (i.e., moving water) and lentic (i.e., still water) aquatic environments, like brooks,
streams, and rivers. The substrates are made up of dead and decaying stems of aquatic herbaceous
macrophytes that grow in fresh water, as well as rotting wood and leaves that fell into the nearby
fresh water from riparian plants.
Aquatic hyphomycetes often grow on large pieces of organic matter, like leaf litter that is breaking
down and woody debris that is underwater. Most of the time, aero-aquatic-hyphomycetes are found
on wood and plants that are submerged in slow-moving bodies of water like ponds, ditches, or slow-
moving streams. Under semi-aerobic conditions, these organisms can grow vegetatively (i.e., as
hyphae) on submerged substrates. (2007) Shearer and others. This ecological group can grow in a
stage called "vegetation" on surfaces that are completely covered by water. When the substrate is
exposed to the air-water interface, asexual spores called conidia are made. These spores have unique
properties. The submerged-aquatic hyphomycetes and coelomycetes belong to the third ecological
type of asexual ascomycetes (Shearer, 2020). Zhang and his coworkers 2021). These mushrooms
don't have the specialized spore structures found in goldian-hyphomycetes or aero-aquatic-
hyphomycetes. Instead, they are a man-made collection of ascomycotan asexual (anamorphic) fungi
with different shapes and origins. (2017) Tsui et al.
Oceanic Fungi
Kohlmeyer and Volkmann-Kohlmeyer (1991) say that some specialized fungi have been found in
marine environments. Hyde et al. (1998) say that there are about 1500 species of marine fungi that
belong to several taxonomic groups. Several of these mushrooms are different from aquatic species
that live in fresh water and may be able to grow on aquatic plants. Several species, like those with
sticky spore appendages, have traits that show they have adapted to living in water. (2000)
Kohlmeyer et al. Recently, a study of hydrothermal habitats in the deep sea found new species of
ascomycetes and basidiomycetes, as well as what may be a lineage of chytrids that had never been
seen before. (2009) Le Calvez et al.

112
Ascomycetes and basidiomycetes, which include ascomycete and basidiomycete yeasts, make up
most marine fungi (Nagahama). Hydrocarbons in seeps and spills that happen naturally underwater
are broken down by certain yeasts. (1979; Davies and Westlake). Some ascomycetes are experts at
growing on surfaces made of calcium carbonate, like mollusk shells and cnidarian reefs. Only a small
number of the basidiomycetes that make mushrooms live in water. (2006) Binder et al. Some fungi
serve as homes for other aquatic invertebrates, such as antibiotic-making sponges (Kim and Harvell,
2004). Wang et al. (2008); Bhadury et al. (2006); Pivkin et al (2006). There are some fungi that
grow on land that can also be found in water. Basidiomycete yeasts and ascomycete yeasts, as well as
other fungi like Basidiobolusranarum, may live in marine environments. Other fungi like
Basidiobolusranarum, ascomycete yeasts, and basidiomycete yeasts like Lacazialoboi may be found
in marine environments, where they can infect porpoises and other animals (Kurtzman and Fell,
1998; Murdoch et al., 2008; Morris et al., 2010 )
Mud Fungi
There are many different kinds of fungus in soil. Domsch et al. (2007); Kirk et al (2004).
Vandenkoornhuyse et al. (2002) say that fungi and bacteria in the soil are important parts of the
biogeochemical cycles. The number of different kinds of soil fungus is highest near organic matter
like roots and root juices. Most of the small fungi in pure soil are asexual ascomycetes, but there are
also some zygomycetes, like the Zoopagales, which live with animals. Gams (2006) says that there
are 3150 known species of soil fungus, and 70% of them are cultivable. Now, new species are being
found at a fast rate, and the group seems to be better known than other biologically defined groups.
Still, it is hoped that molecular research will lead to a greater number of (Bills et al., 2004). In desert
areas, the soil is held together by crusts made by fungi. Most crusts are made up of lichens,
cyanobacteria that fix nitrogen, and dark-coloredascomycetes. States and Christensen in 2001. Fungi
grow on the surface and in the depths of granite in the desert. These darkly coloredascomycetes are
in the groups Dothideomycetes and Arthoniomycetes, but they may also be linked to bacteria and
basidiomycetes. Ruibal et al. (2009); Kuhlman et al (2006). Lobosporangiumtransversale (Ranzoni,
1968), a rare zygomycete that has only been found in three places, one of which is soil in the
Sonoran Desert, is one of the ascomycetes and fungi that can be found in desert soils and are easy to
grow. Most yeasts live in the American deserts, where they work with cacti and insects to get rid of
plant chemicals (Starmer et al., 2006).
Animal Fungi
Fungi related to insects and arthrophages require greater study. As insect-associated fungal species
are poorly characterized, worldwide fungal diversity estimates often exclude them. Schmit and
Mueller (2007). After 1991, estimates ranged from 20,000 to 50,000 insect-associated fungus.
(Schmit, 2007). Certain biotrophic parasites do not grow in culture. Asellariales, Harpellales, and
Laboulbeniomycetes are seldom encountered by mycologists or entomologists. They need special
removal and mounting methods. (Lichtwardt et al., 2001; Cafaro, 2005). Ascomycetes the size of
seta that live on insects, mites, and millipedes are called laboulbeniomycetes. Weir (2005). (2005).
All 2000 species have two-celled ascospore-derived determinate thalli and distinct life cycles. 90%
of species were found on flies or adult beetles (12 of the 24 superfamilies). There is host
specialization and novel family-level arthropod hosts (Rossi and Weir, 2007). (DeKesel, 1996). Scale
insects may be protected against basidiomycete rust fungus by septobasidiales.

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Parasitic wasps' felty

Basidiomata. Microsporidians parasitize many hosts. Necrotrophic insect parasites include
Chytridiomycota, Blastocladiales (Coelomyces), Entomophthorales, and Tubeufiaceae (Podonectria).
(2004). Around 5000 arthropod-necrotrophic Hypocreales live in three groups. (Spatafora, 2010). In
gardening, there are historical connections between fungi, bark and ambrosia beetles, attine ants, and
Old-World termites. New species are always being found. (Aanen, 2009). Honey-eating insects are
linked to several yeasts. (Lachance, 2006). Gut yeasts exist in other insects. In low-nitrogen
conditions, insects may feed fungus. People consume Pleurotusostreatus because its mycelium
releases poisonous droplets that kill worms. Four groups of fungi and oomycetes have become
predators on their own. Arthrobotrys and Dactylella fungi catch nematodes and other small insects in
soil and wood.
Roots and fungi
Mycologists have studied the connections between mycorrhizal plants and fungi. "Smith and Read"
(2008). Fungi take water, nitrogen, phosphorus, and other nutrients from the soil and send them to
the roots of the plants they live on. This makes them very important to the plants they live on. Some
mushrooms might not be able to grow or live without the host. Mycorrhizal fungus can be found on
bryophytes, ferns, conifers, and flowering plants. (2010). Somemycorrhizal fungi that only work with
orchids and ericoid plants have moved into new places with invasive plants. (Pingle, 2009).
Mycorrhizal fungi include arbuscular and ectomycorrhizae. AM is linked to 80% of plant taxa and
92% of plant families. Glomeromycota, which includes AM fungi, has 250 species in many groups,
but is less diverse than ectomycorrhizal fungi (Schüler and Walker, 2011). More than 6000 species of
basidiomycetes, which create mushrooms, form ectomycorrhizae in 10% of plant families.
Ectomycorrhizal fungus-plant interactions are more host-specific than AM partnerships. Smith &
Read (2008).
Grasslands
Like forest fungus, grassland fungi are mycorrhizae and decomposers. Some species may be in both
groups. Fungi dominate grassland mycorrhizae. Flowering plants are rare in fungus-rich grasslands
(Rotheroe 2001); therefore habitat evaluations may overlook their relevance. Bardgett (1996) found
that living mycelium was most abundant in heavily grazed, easily draining, base-rich soil in highland
grassland, and that stopping grazing caused dead mycelium and soil acidity. Waxcaps, earth-tongues,
and fairy clubs are grassland fungus that are very sensitive to inorganic fertilizers. (Marren 2000).
Waxcaps are most abundant in persistently grazed or often mowed, free-draining, mesotrophic
grassland. Waxcaps and its associates are rare in wet and dry grasslands. Pasture microbes live in
herbivore waste. Most "coprophilous" fungi are minute, but others are dung-feeding macrofungi.
Richardson & Watling (1997). Nyberg and Persson (2002) state that the species detected depends on
the grazing animal and the area where the feces fall. Fungal spores must pass through the grazer's
intestines to break dormancy, which affects the dung's chemical makeup. Horses' feces has a
different fungal assemblage than ruminants', but rabbit and grouse droppings do too. (2003).
Heathland often grows unique mushrooms following fires. (Anderson 2001b). Heather burning may
reach 800°C above ground for a minute, but 2 cm below the surface, the highest was 45°C, and
below 4 cm, there was no change. (Webb 1986). If the fire burns out quickly and doesn't damage the
humus layer, fungal mycelium a few centimetres below the soil's surface may survive and benefit
from the fire's nutrients.

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Microhabitats
If not valued, fungi-occupied niches may vanish from a location. Cup fungus may scavenge nutrients
from unappealing substrates like crushed slate or cement powder in flora-free areas. They appear on
newly surfaced gravel parking lots and stony footpaths. Recognizing the significance of such barren
land and resisting the impulse to plant seeds or change the course is crucial. Mosses grow on bonfire
sites. Ash, burnt earth, and charred wood also have specialized fungus. Ramsbottom 1953. Several
species like bonfire sites for the heat-sterilized dirt and lack of competitors. Dix & Webster 1995.
Some emphasize ash's rich nutritional content. To protect these unique species, prevent frequent
bonfires. Conifer forest bonfire sites had different fungus than broad-leaved woodland sites.
Onygena, a fungus with four British species, grows on long-lasting animal waste such horns, hooves,
beaks, and feathers. 1997 (Keizer). So, they should be accepted rather than eliminated for tidiness or
mementos. Owl leftovers are O. corvine's microhabitat.
Fungal Culture Methods
Basic Methods
Clean Method
Microbiological processes need aseptic instruments, containers, mediums, and stock cultures.
Aseptic technique includes sterilizing tools, glasses, and media before use and avoiding contact with
nonsterile items.
Workspace and Gear
Fungi need these supplies
1. 4% household bleach, 70% ethanol, or Lysol disinfectant solution
2.Gas or alcohol (Bunsen) burner.
3.Scalpel or half-spearpoint needle; inoculating loop.
4.Stock culture
5. Sterile media in tubes or Petri dishes.
6.Soap for washing hands.
7.Wear a lab coat or an old, clean shirt when staining cultures.
Before touching fungal cultures, wash your hands. Create a workspace. Choose a clean spot. Close
all doors and windows and turn off the fans and AC. Clean the workspace with any disinfectant or
70% ethanol. Place items neatly on the clean work surface. Avoid eating, drinking, and smoking
around other cultures. (Hamzia & Sabaa, 2018)
Media preparation
Tool and component assembly begins media preparation. A large flask or beaker, weighing paper,
spatula, balance, measuring cylinder, glass stirring rod, culture tubes, and petri plates are needed.
Potato and Sabouraud dextrose agar are the most used medium. To weigh dry ingredients, lay
weighing paper on the pan. Fill a large flask with dry materials. Fill the flask with enough distilled
water and stir to dissolve the dry ingredient. Agar dissolves in medium heated slowly to just boiling.
During heating, gently shake or swirl the flask. Agar boils and burns fast, so watch the flask. Fill
bottles or culture tubes with liquid agar or broth and loosely cover. Autoclave culture media bottles
or tubes. Before autoclaving, mix the dish-pouring medium in the flask that has a lid on it. Put sterile
tubes on a tray with a slope. When the agar gets solid, just tighten the lids to store it. Before pouring
plates, clean and turn off the air flow. Cool the flask until you can handle it (20 to 40 minutes). Set
out the Petri dishes and turn on the Bunsen burner. Once the cork is out, light the flask. Raise the

115
angle a little to pour the medium. If bubbles develop, swiftly run the Bunsen flame over the agar five
times to burst them. Slowly pour agar to avoid bubbles. After adding half the agar, cover the dish.
Leave the dish until the agar solidifies. (Shung-Chang & Jeannette, 2001)
Sterile Methods
Some spores of microorganisms can live for hours in dry heat or boiling water. Bacteria that make
the air dirty can be killed by using pressurized steam. Packages and loose caps can be cleaned with
steam. This is how pressure cookers work. Autoclaves are large pressure cookers that automatically
heat, sterilize, and cool.
If you don't have an autoclave, you can use a big pressure cooker on the stove:
1. After reading, follow your cooker's directions carefully.
2. Check the cooker for water.
3. Start the clock after 15 psi.
4. Slowly cool the pressure cooker after 15 minutes.
5. Medium should be clean after 15 minutes at 121°C and 15 psi. Autoclave glassware and old
cultures for 30 minutes at 121°C and 15 psi. (Juliana, 2006)
Tube Cultural Transfer
Slants
Light the alcohol or gas burner after cleaning the space. Keep the stock culture tubes and the sterile
agar slant in one hand. Keep the inoculating loop in one hand, as you would a pencil. When the
flame becomes red, hold the wire above it. Drag the end of the handle across the fire's heat source
again and over. Every every wire and holder that enters the tube must be flamed. With the "loop"
hand, remove the cotton plugs or sterile tube caps from the stock tube. Never let the loop or the caps
out of your sight. Place tubes in an ongoing flame to sterilize lips. Inoculating loop attached to stock
culture tube. Cool the curve by touching its peak. Take a loop and use it to remove a few spores of
yeast or fungus from the stock culture tube. Insert the loop into the clean tube so that it doesn't touch
the walls. You should hit the loop from the bottom up. (Juliana, 2006)
Broth Cultures
Never mouth-pipet germs. Employ a rubber-bulbed pipette or Pi-Pump®.Start the stove. To agitate
the stock tube, softly tap or stroke its end with one hand while holding it between the thumb and
fingers. Hold the sterile medium tube with your stock tube hand. Do not place caps after removal.
Tube mouths burn. Pipette 0.1 mL of microbe suspension. Insert the pipette tip into the sterile tube to
release the broth's contents. Burn both tube lips, remove the pipette, and place on new caps. Gently
shake and label the contaminated tube. Put plastic pipettes in autoclavable pouches and disinfect
reusable glass pipettes. Loops may transfer broth cultures. Inoculating the tube of sterile medium
with several loopfuls of stock solution ensures ample stock culture. (Juliana, 2006)
Plate Culture Transfer
Focused Growth
Use a disinfectant to clean the counters. Reduce the amount of airflow while moving plates to
prevent contamination. Carry the stock tube with one hand and transfer yeasts or fungi that are rich
in spores. Douse a loop of immunity with fire. To light the tube, use the "loop" hand to pry away the
cap or cotton plug and then to ignite the tube's mouth. Use a chilled loop to transfer a little amount of
culture to the stock tube (more than for transfer to a tube). Closure is required. Crack open the
petridish cautiously. After the loop has made contact with the dish, hit from side to side to the floor.
The zigzag pattern will cover the whole plate. To ignite the loop, remove the top. Clearly identify the

116
food items. In order to avoid clumping of colonies on agar while incubating at temperatures over 25
degrees Celsius, invert Petri dishes. (Hansen, H.N. 1926).
Exclusion
Colonies may be grown using isolation streaking for morphological analysis or bacterial solution
separation. Keep one hand on the stock tube. Burn the wick and uncover the tube. Tube mouth fire.
Place the chilled loop in the culture tube (about the same as for a tube). Closure is required. In order
to get a good angle, you need lift the lid of the petri dish before inserting the loop. After making a
zigzag path across Area 1, replace the lid. Take out, burn, and cool down the circuit. dish at an angle
of ninety degrees (Area 2). To test whether the agar is cold enough to touch, lift the lid and place the
loop in the middle. Make a break from Zone 1 to Zone 2. Next, cover a quarter of the plate with a
zigzag pattern in Area 2. Cut the tape and erase the loop. Streaking should be done from Area 2 to
Area 3 and then from Area 3 to Area 4. (Juliana, 2006)
Moldy Dishes
Penicillium, Aspergillus, and Rhizopus are spore-forming fungus that may spread disease in a
manner similar to that of bacteria. Use a half-spearpoint needle, bent inoculating needle, or flame-
sterilized scalpel to cut agar for nonsporulating or fruiting fungus (such as Sordariafimicola).
Carefully remove the lid from the sterile agar dish and place the agar block, face down, in the dish's
center. (Juliana, 2006)
Culture Care
Temperature
Fungus cultures are incubated at 30°C (86°F) or 22–25°C (72–77°F). At 30°C, most hazardous
fungus grows quicker. A relative humidity of 40%–50% prevents agar from drying out. An incubator
with an open pan of water may do this. Incubate cultures for 30 days and check them three times a
week before declaring them negative. Dermatophytes should be cultured for six weeks at room
temperature. (Juliana, 2006)
Stock Cultural Preservation
Screw-cap tubes can store stock cultures for a few weeks. Tighten the cap once growth is lush with
stringent aerobes and fungus. Most cultures are room temperature. Luxuriant growth in screw-cap
tubes may be refrigerated for months if sub culturing is not possible. This is the most cost-effective
method. Nevertheless, having many stock cultures of each species is desirable since certain cultures
cannot withstand refrigeration or genetic mutation.
While it requires special equipment, freeze-drying is extensively employed for long-term storage.
CarolinaTM freeze-dried cultures include pure lyophilized fungi. After rehydration, they grow lushly
in 24–48 hours and may be stored at 4°C for three years. It takes 24 hours of incubation at the correct
temperature after dissolving the freeze-dried culture in rehydration media. The inoculum should next
be incubated in broth or agar. Before freeze-drying cultures and coloring them, two subcultures are
needed. Changing medium every two to three weeks keeps most fungi alive in cultivation. Some
mushrooms require new medium twice a week and others once a week. (Shung-Chang & Jeannette,
2001)
Waste Removal
Disinfect the new location after the move. Wash your hands thoroughly. If autoclaving is not an
option, aged culture stocks and glassware may be disinfected by soaking them in 70% ethanol or
similar disinfectant for a full 24 hours. It is best to torch civilizations if at all feasible. Mishaps might
occur while working with infectious microorganisms. Notify the lab assistant or teacher if a tube or

117
petri dish breaks. Steadily pour 70% ethanol over the leak for a few minutes. Cleaning up the spilt
chemical before autoclaving it or burning it with other toxic trash is essential. Try not to poke the art
or break any glass. No harm will come to the individual who dropped the fungus. (Juliana, 2006)
Fungal disease-causing agents
Plant fungi
Phytotoxins have been implicated in
several foreign agents, although their
significance in pathogenesis is still
unclear. Genetic and biochemical
studies showed that contaminants
determine specificity in few plant-
fungus relationships. Fungus struggle
and toxin sensitivity always go along.
Hence, host-selective toxins, mostly
produced by Alternaria and
Cochliobolus species, have garnered
interest (see Walton, 1996, in this
issue). Host-nonselective toxins work
on both hosts and nonhosts. These
toxins may play a vital role in fungal
pathogenesis on a tested host despite their no selectivity. They may also be residues from fungal
evolution toward phytopathogenicity that most plants can detoxify or otherwise inhibit. Two
examples show how host-nonselective toxins may work: Phytophthora species release elicitins, tiny
proteinaceous elicitors that cause plant illnesses (Ricci et al., 1989; Yu, 1995). Elicitins may be
ubiquitous in this fungus class since Pythiumvexam, another Oomycete, produces them (Huet et al.,
1995).
Animal Fungi
Ophidiomycesophiodiicola (Snake Fungal Disease; SFD), which first appeared in the United States,
Canada, and Mexico in the year 2000, is concerning due to the wide range of taxonomic groups it
affects. Batrachochytriumdendrobatidis (Bd) causes Chytridiomycosis, which threatens amphibian
populations worldwide. Timber rattlesnakes (Crotalushorridus), eastern massasauga snakes
(Sistruruscatenatus), and pigmy rattlesnakes (Crotalusatrox) have all tested positive for
Ophidiomyces (Sistrurusmiliariusbarbouri). The host and environmental factors that contribute to
disease are poorly understood. Ophidiomyces was the most dominant organism (Nayak., 2010). Most
of the fungi belonging to the Pleosporales order disappeared, with only Pyrenochaetopsispratorum
showing any kind of noticeable change. Ascomycete fungi, also known as the Pleosporales (Van
elas, 2012).
Infections in humans nowadays. While numerous culture-based case reports have implicated other
major fungus, such as Aspergillus and Fusarium, Ophidiomyces has been confirmed as the cause of
SFD by many laboratory tests. As Ophidiomyces develops slowly, faster-growing saprophytes on the
snake's skin, such as Fusarium and Aspergillus, masked the cultured results. Hypocreales (Fusarium
sp.) and Eurotiales (Aspergillus sp.) opportunistic invaders were discovered in both positive and

118
negative samples in the prior investigation. Pyrenochaetopsispratorum'sability to aggressively

exclude Ophidiomyces in vitro should be determined first (Weyrich et al., 2015).
There are both obligate and facultative fungal pathogens. In general, the aforementioned kind of
fungi has an environmental niche and infects immunocompromised or overdosed humans.
Immunocompromised individuals are easy prey for opportunistic pathogens. Candida species are
commensals in otherwise healthy hosts, but Cryptococcus neoformans and Aspergillusfumigatus
have ecological reservoirs (Croswell et al., 2009). There is a knowledge gap between bacterial and
fungal pathogenesis. In contrast to bacteria, fungi almost seldom cause disease. They may live
alongside animals as commensals or in non-animal habitats as saprophytes. In healthy hosts, several
of these may produce devastating systemic disease. Among the best-known pathogens are
Coccidioidesimmitis and Histoplasmacapsulatum. During the last 20 years, opportunistic fungal
infections have become more important, despite the fact that modern medication has enabled
crippled and immunosuppressed patients live. Candida species and other opportunistic infections
may cause severe illness in these persons. Alpha Neoformans. species of Aspergillusand Fusarium,
as well as the zygomycetes (Pfaller et al., 2004).
Human head-to-toe fungus

The prevalence of opportunistic mycoses has grown during the last two decades. An increasing
number of patients are at risk for severe fungal infections, including those undergoing blood and
marrow transplantation (BMT), solid-organ transplantation, and major surgery (especially
gastrointestinal surgery); patients with neoplastic disease, advanced age, AIDS, immunosuppressive
therapy, and Opportunistic mycoses are challenging to diagnose and treat due to the high
Opportunistic mycoses are quite prevalent, with the most frequent being Candida albicans,
Cryptococcus neoformans, and Aspergillusfumigatus. The annualized incidence of invasive mycoses
per million people is estimated for Candida species 72–228, while for C. 30-66 neoformans; 12-34
Aspergillus species. More and more attention are being paid to "other" opportunistic fungi in addition

119
to these agents. Certain types of Aspergillus and Candida that aren't C-generic are considered
"emerging" pathogens. fungus that is called albicans, A. species of Trichosporon and Rhodotorula),
Zygomycetes, hyaline moulds (Fusarium and Scedosporium species), and a large variety of
dematiaceous fungi. Catheter-related fungemia and peritonitis, as well as localized lung, skin, and
paranasal sinus infections, and even hematogenous spread are all possible complications caused by
these organisms (Wisplinghoff et al., 2004).
Some of these fungi were formerly thought to be harmless, but they have now been shown to cause
invasive mycoses in the immunocompromised. Surgical treatment and repairs to the human immune
system may not be enough against some of these organisms since they are resistant to the standard
antifungal drugs such as azoles, polyenes, and echinocandins. C. glabrata. C. Trick et al. discovered
that C. glabrata, a potentially resistant opportunistic fungus pathogen, is a major health concern.
ICUs in the United States have shown an upsurge in glabrata-related BSI.
1993. C band universally. glabrata accounts for 22 percent of BSI in the Americas and 4 to 6 percent
in South America (Trick et al., 2002).
C. albicans. Several species of Candida. albicans prevails. C often manifests as infections of the
reproductive system, mouth, and skin. albicans. Nevertheless, C. Blood samples may recover
Candida albicans and other Candida species at varying rates. albicans is often thought of as an
endogenous pathogen (meaning that infection arises from the patient's own flora), however it has
been well demonstrated that it may spread from patient to patient through healthcare workers' hands
(Roilides et al., 2004).
C. parapsilosis. C. In the previous four years, parapsilosis has risen from being the second most
common to being the third most common Candida species isolated from blood cultures in Europe
(12%), Asia-Pacific (17%), and South America (20%). Those who have a vascular catheter inserted
in the hospital should believe it. Typically, C. Critically ill infants and patients in intensive care units
are more likely to get parapsilosis from healthcare workers' hands because of their contact with
parenteral nutrition and central venous catheters (Kao et al., 1999). Unlike patients with other
Candida species, those with C. parapsilosisare more likely to develop extensive biofilms on catheters
and other implanted devices. Get rid of the parapsilosis catheters. Organisms that produce biofilms
are completely immune to antifungals. C. Parapsilosis has been linked to several nosocomial
outbreaks of catheter-associated fungemia, making hand hygiene and catheter management all the
more important (Laverdiere et al., 2000).
C. tropicalis. C. Patients with cancer, especially those with leukemia or who have had a bone
marrow transplant (BMT), have long had tropicalis linked to fungemia and invasive candidiasis.
Patient neutropenia colonization by C. of the tropicalis population, 60%-80% become invasive. So C.
Those with mucosal injuries are at a higher risk for infection with tropicalis. Neutropenic patients
should take fluconazole for prevention.in the fight against C infections. A. tropicalis C. albicans. C.
fluconazole is very effective against C. tropicalis, and it may be used to protect neutropenic
individuals from getting the fungus in the first place if they take it. Tropical diseases (Herbrecht et
al., 2002)
Those who love amphotericin B may get breakthrough infections from Fusarium species that have
developed in vitro resistance to the drug. In cases of fusariosis that have not responded to
amphotericin B, voriconazole has been effective. Echinocandins have no effect on Fusarium species.
The first therapy should consist of voriconazole or a lipid formulation of amphotericin B, followed
by strong immune reconstitution. The infection fusariosis is becoming more common in

120
immunocompromised patients, especially those with hematologic malignancies or who have had an
allogeneic HSCT. Fusariummoniliforme, fusariumsolani, and Fusariumoxysporum are the most often
found strains in hospitals. Several purpuric nodules characterized by core necrosis are typical of
disseminated fusariosis. Invasion of cutaneous blood vessels by branching, hyaline, and septate
hyphae was seen in these nodules (Nucci et al., 2004).
Zygomycetes. Zygomycosis is caused all over the world by sporadic fungi from the class
Zygomycetes and the order Mucorales. Nevertheless, additional species of Rhizopus, Rhizomucor,
Absidia, and Cunninghamella may also cause invasive sickness in hospitalized patients in addition to
Rhizopusoryzae (arrhizus), the causative agent of zygomycosis. The yearly incidence rate of
Zygomycetes infections in the United States is 1.7%. Zygomycosis has increased in prevalence
during the last decade, notably in the HSCT population and in patients with hematologic
malignancies (Gonzalez et al., 2002).
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***
126
Chapter 13
NUTRITIONAL VALUE OF AGARICUS BISPORUS, ITS
POTENTIAL USE AND HEALTH BENEFITS IN
CHICKEN FARMING
Zohra Zarrar Kuwari1, Yusra Mohaddis Khatib2, Zarin Zarrar Kuwari3
A bstract
Because of their nutritional worth and medicinal qualities, mushrooms have been valued as major
food items since ancient times. Among the many edible mushrooms, Agaricus bisporus, also referred
to as the white button mushroom or simply the mushroom has some inherent nutritional advantages
over the other vegetarian options due to its high protein content (20–30% of dry matter), the
chitinous wall that serves as a source of dietary fiber, high vitamin B content, low-fat content, and
absence of cholesterol. The commercial chicken industry in India has seen a tremendous
transformation over the past forty years, going from an entirely unorganized and irrational
agricultural practice to a modernized production system. In this chapter, the nutritional value of
Agaricus bisporus, its potential for use and its health benefits in poultry farming are discussed.
Keyword: Agaricus Bisporus, Mushroom, Mushroom Waste, Poultry, Poultry Diet.
1Assistant Teacher, Hera English High School, Mahapoli, Maharashtra State Board, India.
zahrakuwari123@gmail.com
2Assistant Professor, Department of Botany, K. M. E. Society’s G. M. Momin Women’s College,
Bhiwandi, University of Mumbai, India. khatibyusram@gmail.com

3Department of Zoology, B. K. Birla College (ex-student), University of Mumbai, India.
kuwarizarin@gmail.com
Zohra Z. Kuwari, Yusra M. Khatib, Zarin Z. Kuwari

127
I ntroduction
Mushrooms have been recognized as important food items since ancient times because of their
nutritional value and therapeutic properties. The interest in their cultivation has recently increased
everywhere around the globe nowadays. High protein and polysaccharide content are paired with
low-fat content, and the profile is characterized by a higher concentration of mono and
polyunsaturated fatty acids than saturated fatty acids. These are also interesting sources of phenolic
compounds as well as some micro and macronutrients (Rodrigues et al., 2015). The fruiting bodies
of mushrooms are well recognized for their numerous advantages, including rapid development,
enhanced health, higher resistance to diseases, and protection against them (Guo et al., 2003).
Among many other edible mushrooms, Agaricus bisporus (Plate-1) also known as white button
mushroom or simply mushroom, due to the high protein content (20–30% of dry matter) with all the
essential amino acids, which allows them to serve as a meat substitute, a chitinous wall that serves as
a source of dietary fiber, a high vitamin B content, low-fat content and zero cholesterol, has some
inherent nutritional advantages over the other vegetarian options (Ghorai et al., 2009).
In poor countries, raising poultry significantly improves family food security. In more than 80% of
rural households, it helps diversify earnings and offers wholesome food, energy, fertilizer, and a
renewable resource. In the last forty years, poultry production in India has advanced dramatically,
transitioning from a wholly unstructured and unscientific agricultural practice to a commercial
production system with cutting-edge technology innovations. Due to the industry's adoption of a
scientific approach and the government's creation of an enabling environment, the Indian commercial
poultry sector has evolved significantly. In India, there are organized and unorganized subsectors of
the poultry industry. The needs of the various subsectors varied greatly. With initiatives for the
development of poultry and entrepreneurship, the Indian government is meeting the demands of the
subsectors. The government's development projects are being coordinated and disseminated in a
crucial way through Central Poultry Development Organizations. For the past 50 years, the demand
for chicken meat has steadily grown at a rate that is around three times faster than that of population
growth, according to the FAO (2012). Because of its inexpensive initial outlay, quick assured
returns, close generational spacing, and little land needs, the chicken business has had high growth
rates.
Plate-1: Agaricus bisporus

128
Proximate and Nutritional Value of Agaricus Bisporus

The proximate and nutritional value of A. bisporus (Table 1) showed the presence of protein
carbohydrates and a very low-fat content which can become a very suitable poultry ingredient (Atila,
F et al., 2017).
Table-1: Proximate and nutritional value of Agaricus bisporus

Parameter Approximate value/content References
Moisture 91–92 g/100g Reis et al. (2012)
0.9–1g/100g Reis et al. (2012)
Ash
11.00-7.77% Tsai et. al. (2007)
Energy 29–31 kcal/100 g Reis et al. (2012)
Protein 29.14% Ahlavat et al. (2016)
51.05% Reis et al. (2012)
Carbohydrate
48.9-38.3% Tsai et. al. (2007)
1.56% Reis et al. (2012)
Fat
3.92-2.53% Tsai et. al. (2007)
Fibers 23.3-17.7% Tsai et. al. (2007)
Alanine, aspartic acid, glutamic acid, arginine,
leucine, isoleucine, lysine, phenylalanine, serine, Muslat et al. (2014),
Amino acids Muszyńska et al., (2013)
proline, tyrosine, threonine, cystine, methionine and
valine
Zinc, ferrous, phosphorous, magnesium, potassium, Mohiuddin et al. (2015)
Mineral content Ahlavat et al. (2016)
sodium, calcium and selenium
Bernas & Jaworska,
Niacin, riboflavin, vitamin B1, vitamin B3, L-
Vitamin (2016).
ascorbic acid and α-tocopherol Çağlaırmak, (2009)
An Economic Poultry Diet

The economic and ordinary poultry diet for broiler chicken is a mixture of carbohydrates, protein, fat
and vitamins. The energy source of the diet is categorized into four viz., Grains and seeds, by-
products from milling, molasses and roots and tubers. In plate 2, the ingredients of the poultry diet
are listed (Bharat, A. (n.d.)).
The mentioned ingredients (Table-2) are taken and mixed in a batch mixer according to the nutrient
requirement of broiler chicken and different formulations. After mixing, the content is ground to a
uniform particle size of 1-2 mm and further mixed. Due to the high cost of molasses, sometimes
sweetening agents are used instead. The resultants then passed with dry steam to kill the pathogen
then converted to pallets and stored.
Agaricus bisporus as a substitute in poultry dietary supplement

The majority of research that has examined the impact of including mushrooms in bird meals
has focused on the changes in animal performance and microbiota. The protein in the button
mushroom has nutritional value on far with that of muscle protein, and it comprises around nine
necessary amino acids (Shamsi et al., 2015). In addition to being high in vitamins like ascorbic acid,
folic acid, niacin, pyridoxine, riboflavin, thiamine and tocopherol, they are also richer in protein than
any comparable fruit or vegetable (Camay, 2016). The fact that button mushroom is high in protein,
vitamins, and minerals while being low in calories and fat makes them a useful health food across the

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world (Mazaheri et al.,2014). Moreover, it contains cancer-preventing substances such as the

polysaccharides lentinan, beta glucan, lectin, and thioproline (Mazaheri et al.,2014). As the chicken
industry has been pushed to quit using antibiotic growth promoters as it develops resistance,
mushrooms might be a suitable substitution. Mushrooms have long been recognized to have
antibacterial characteristics.
Following are some important benefits that show how Agaricus bisporus can increase poultry
performance and help farmers to attain market requirements.
Table-2: Ingredients of common poultry diet
Grains
• Wheat, maize, oats, sorghum, rice, barley, millets, ragi etc
Brans
• Rice polish, de-oiled rice bran, wheat bran, maize bran etc
Protein meals/cakes
• Rapeseed meal/cake, soybean meal, cottonseed meal/cake, groundnut meal/cake,

coconut meal/cake, palm kernel meal/cake, sesame cake, linseed cake, maize germ
oil cake, maize gluten meal, sunflower meal, safflower meal, guar meal etc
Agro-industrial by-products
• Molasses, babul chunni, tamarind seed powder, mango kernel extract, tapioca waste
etc
Chunnies
• Guar, tur, urad, moong gram & chunnies of other pulses
Minerals and vitamins
• Mineral mixture, calcite powder, common salt, phosphate, vitamins A, D & E etc

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1. A Stimulant or Suppressant of Immune System

It is well recognized that the host's immunological health affects the host's ability to fight off
numerous illnesses. Polysaccharides, glycosides, alkaloids, volatile oils, and organic acids are among
the immunoactive substances found in plants and fungi, with polysaccharides regarded as the most
significant (Guo et al., 2004). According to Kitandu and Juranová (2006), polysaccharides have the
potential to operate as immune boosters or immunomodulators as well as having antibacterial
properties. In general, polysaccharides may influence cellular and humoral responses as well as
innate and adaptive immunity (Guo et al., 2004). Broiler cellular and humoral responses could be
improved by the polysaccharides from Agaricus bisporus. They may thus be crucial in boosting the
birds' immune systems to prevent the entry of pathogenic germs. Chickens have not yet been
adequately studied in terms of how immune systems are impacted by polysaccharides found in
mushrooms and herbs, as well as other aspects like growth and health. By employing extracts of
herbs and mushrooms with well-known therapeutic characteristics, the results showed that innate
immunity in chickens might be improved immunologically.
2. Free Radicle Scavenging Activity
Natural antioxidants that may take the place of synthetic ones and satiate customer expectations for
food items free of residues from chemicals that may be harmful to human health are something that
the chicken business would value. A decline in health is brought on by oxidative stress, which is
linked to the production of free radicals in oxidative damage. As byproducts of regular metabolism,
radiation-generated mutagens like superoxide, hydrogen peroxide and hydroxyl radicals are also
formed (Cui et al., 2012). In addition to harming, one's health, lipid peroxidation is a significant
factor in the reduction of meat's colour, taste, texture, and nutritional value (Halliwell & Gutteridge,
1999). As consumers are concerned about the use of synthetic antioxidants, which are currently
approved to limit lipid oxidation in foods (Giannenas et al., 2010a), there is a need for and an
incentive for research into alternative antioxidants. Much emphasis has been paid recently to studies
exploring the possible uses of natural product antioxidants for protecting meals from oxidation
(Yildirim et al., 2012). Secondary metabolites include phenolic substances, polyketides, terpenes,
and steroids are among the many that mushrooms acquire.
A mushroom’s phenolic substance has also been shown to be a superb antioxidant and synergist that
is non-mutagenic (Mitra, Jit, Narayan, & Krishnendu, 2014). Phenolic substances may function
directly as antioxidants or prevent pathological disorders associated with oxidative stress, such as
cancer, heart disease, diabetes, inflammation, gastric ulcer, hepatic damage, and microbial infections
(Dasgupta, Debal, Ananya, Anirban, & Krishnendu, 2014). Phenolic chemicals may directly combat
oxidative stress or shield against underlying pathological diseases including cancer and heart disease.
Phenolic substances may function directly as antioxidants or guard against underlying oxidative
stress-related pathological disorders such as cancer, heart disease, diabetes, inflammation, gastric
ulcer, hepatic damage, and microbial infections (Dasgupta, Debal, Ananya, Anirban, & Krishnendu,
2014).
The scavenging of free radicals was demonstrated by different amounts of methanolic extracts from
mushrooms. In 2010, Giannenas and colleagues found that dietary mushrooms (Agaricus bisporus) at
both inclusion levels (10 or 20 g/kg broiler diet) decreased the production of malondialdehyde in the
tissues of the liver, breast, and thigh and increased glutathione peroxidase, reduced glutathione,
glutathione reductase, and glutathione S-transferase. Additional studies have revealed the necessity

131
of integrating dietary supplements with certain mushroom extracts and components during specific
feeding intervals in order to understand the in vivo control of the liver's antioxidant defenses as well
as the system's reaction to acute oxidative stress.
According to research by Kalava and Sudha (2012) and Kele's, Ilkay, and Huseyin (2011), several
common edible mushrooms show significant in vitro antioxidant activity that is closely connected
with their total phenolic content (Gan et al., 2013; Huang, Kim, & Chung, 2011). Depending on the
kind of mushroom (Cantharellus friessi, Cantharellus sublibraries, Cantharellus cinerius, and
Pleurotus lorida), the total phenol concentration might be anywhere from 9.55 and 16.8 mg/g
(Kumari, Reddy, & Upadhyay, 2011). Protocatechuic, p-hydroxybenzoic, p-coumaric, and cinnamic
acid are some of the most extensively used phenolic chemicals produced by Fistulina hepatica. It is
generally known that these substances have antioxidant properties (Yildirim et al., 2012). By serving
as reducing agents, hydrogen-donating antioxidants, and singlet oxygen quenchers, polyphenols are
multipurpose antioxidants (Punitha & Rajasekaran, 2012).
3. As an Antimicrobial Agent
According to Isolauri et al. (2001), probiotics are a live microbial dietary supplement that enhances
the intestinal microbial balance of the host animal. As an alternative to using antibiotics to restrict the
growth of harmful microbes, these health-promoting bacteria are being employed in chicken feed on
a larger scale. Due to excellent feed utilization, the addition of mushrooms and probiotics improved
the development of the fowl. Due to the growth of advantageous bacteria in the gut, probiotics have
long been known to have qualities that aid in digestion and create antimicrobial defenses. With the
addition of various dosages of mushroom powder and probiotics, the broiler chicken's growth
performance was enhanced.
4. Mushroom Waste as Poultry Feed
More than any other species, poultry have seen changes in their food and nutrition. One of the main
challenges to the global production of poultry is the feed, which accounts for the greatest portion of
the cost at roughly 70% of the total (Agashe et al., 2017). A significant barrier to practical feed
formulations is the cost and availability of soybean meal, a significant source of dietary protein.
There is an urgent need to find newer alternative protein feedstuffs and to research their suitability
for poultry feeding in order to lower the cost of poultry production and alleviate the shortage of de-
oiled soybean meal (Ahaotu et al., 2018; Imchen et al., 2019; Thakur et al., 2019).
A very good supply of protein, readily usable energy, essential amino acids like methionine, and
bioactive compounds of therapeutic significance may be found in mushroom excrement (Breene,
1990). The United Nations Food and Agricultural Organization (FAO) has acknowledged
mushrooms as a commodity that helps feed regions of the world where cereal-heavy diets are the
norm. Large quantities of ruined, tiny, and malformed mushrooms are produced as waste in the
mushroom cultivation industry. The edible mushrooms contain antibacterial, anti-inflammatory,
immune-stimulating, and antioxidant effects. Moreover, it lowers cholesterol. Mushroom waste has
an extremely high degree of nutritional inclusion, and broiler chicks benefit greatly from it as a feed
source. To boost growth, nutrient retention, and net profit in broiler production, feeding mushroom
waste to chickens at a rate of 2 to 6% was successful. Also, feeding waste from mushrooms at a level
of 2% was considerably more advantageous to boost broiler hens' production capabilities.

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Conclusion
Mushroom in poultry diet is perhaps the most important as it promises to supply food with good
quality protein and thus the nutritional value of mushroom had made it potential in poultry diet for
various health benefits. Besides this, the various edible mushrooms can be included in fodder and
forage, cattle feed, and also can hold a crucial place in the human diet.
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***
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Chapter 14
MECHANISMS OF ENDOPHYTIC FUNGI USED IN
PLANT PROTECTION
Ankita Pokhriyal & Mayuri Saini
A bstract
Endophytes are rich in plants and studies are continuously emanating on their ability to protect plants
from pathogens that cause diseases especially in the field of agriculture. The advantage that
endophytes have over other biocontrol agents is the ability to colonize plant's internal tissues.
Despite this attribute, a deep understanding of the mechanism employed by endophytes in protecting
the plant from diseases is still required for both effectiveness and commercialization. Also, there are
increasing cases of antibiotics resistance among most causative agents of diseases in human beings,
which calls for an alternative drug discovery using natural sources. Endophytes present themselves
as a storehouse of many bioactive metabolites such as phenolic acids, alkaloids, quinones, steroids,
saponins, tannins, and terpenoids which makes them a promising candidate for anticancer,
antimalarial, antituberculosis, antiviral, antidiabetic, anti-inflammatory, antiarthritis, and
immunosuppressive properties among many others, even though the primary function of bioactive
compounds from endophytes is to make the host plants resistant to both abiotic and biotic stresses.
Endophytes still present themselves as a peculiar source of possible drugs. This study elucidates the
mechanisms employed by endophytes in protecting the plant from diseases with a focus on
endophytic fungi.
Keyword: Antibiotics, Commercialization, Drug, Metabolites, Pathogen.
Department of Microbiology, School of Basic & Applied Sciences, Shri Guru Ram Rai University
Dehradun, Uttarakhand, India. pokhriyalankita21@gmail.com

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I ntroduction
Endophytic microorganisms are referred to as the microbes that colonize the internal parts of a plant.
They gain entrance into the seed, leaf, stem, and root of a plant and they are not harmful to the host
plant (Yadav, 2018). Endophytes improve plant growth by secreting phytohormones and
consequently help in nutrition improvement using bidirectional nutrient transfer and enhancement of
the health of plants by protecting them against phytopathogens (Andreozzi et al., 2019; Shen et
al., 2019). Plant-endophyte interaction triggers the protection of plants against harmful conditions of
the environment such as heavy metal presence and drought (Khan et al., 2019; Kushwaha et
al., 2019). Endophytes are numerous and studies have it that they are present in many plants; they
became important due to their capacity to produce many bioactive metabolites and
biotechnologically relevant enzymes (Khan et al., 2014). Most times when endophytes are inoculated
in the plant, they produce significant biomass augmentation and also help in boosting commercial
agriculture (Santoyo et al., 2016; Shen et al., 2019). Endophytes are gaining biotechnological and
industrially relevance as a result of their ability to secrete secondary metabolites, serve as biocontrol
agents, antimicrobial agents, antitumor agents, and immunosuppressants, and to secrete antiviral
compounds and development of natural antioxidants, antidiabetic agents, antibiotics, and insecticidal
products.
In the last 2 decades, endophytes isolated from most plants have shown themselves to be a rich lode
of natural products for industrial and agricultural use amongst several other applications. Enzymes
can be used to replace poisonous chemicals. They thrive best under normal temperatures and neutral
pH. As the years' progress, researchers are beginning to see prospects in microbial enzyme
production. There are many reports currently that microorganisms isolated from the extreme
environments have great biotechnological applications in medicine, agriculture, and industry (Archna
et al., 2015; Yadav et al., 2015; Singh et al., 2016; Sahay et al., 2017). This review aimed to present
the various mechanisms of action used by endophytes in protecting a plant and report some
bioactivities of importance to people with special emphasis on endophytic bacteria and fungi.
An Overview of Endophytic Fungi
Numerous surveys, mostly on the pathogenicity interactions between plants and microorganisms
associated with them, have been accomplished. However, after several explanations and studies on
the role of microbial diversity associated with various plant species, it was assumed that only a small
fraction of the microbes interacting with the plant are pathogenic in nature (Andreote et al., 2014).
Most of the microorganisms that inhabit plants play a major role in the plant’s health and
development, although, sometimes they are neutral. It is considered that a single plant species could
possess thousands of microbes, categorized as epiphytes (microbial inhabitants of the rhizosphere
and phyllosphere; those near or on plant tissue) or endophytes (microbes residing within plant tissues
in leaves, roots or stems), depending on their area of colonization in the plant species (Andreote et
al., 2014).
Apart from the disease-causing microorganisms, the presence of other (non-pathogenic) organisms
inside the plants was first pronounced by De Bary (1866), who detected the presence of microbial
cells in the microscopically analyzed plant tissues. However, this observation continued to be

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unexplored until the definition of endophytes came into existence toward the end of last century. De
Bary (1866) provided the first definition of an endophyte, as “any organism that grows within plant
tissues are termed as endophytes,” however, the definition continues to change as per various
researchers provided the most suitable definition for endophytes, which means any organism that at
some part of its life cycle, colonizes the internal plant tissues without causing any type of harm to the
host plant. Furthermore, due to extensive studies of these groups of microorganisms, the endophytic
communities have been divided into different subgroups, such as ‘obligate’ or ‘facultative,’ which
are associated with all types of plants (Rosenblueth and Martínez-Romero, 2006). Endophytes that
depend on the metabolism of plants for survival, being spread amongst plants by the activity of
different types of vectors or by vertical transmission, are termed obligate endophytes. Whereas, the
facultative endophytes are those that live outside the host body during a certain stage of their life
cycle and are mostly associated with plants from its neighboring soil environment and atmosphere
(Abreu-Tarazi et al., 2010).
Numerous attempts have been made during recent years to discover the origin of endophytic
organisms in different species (Hallmann et al., 1997; Mitter et al., 2013). Initially, researchers
choose the rhizosphere or the seed-born microbial communities as the major sources of endophytes.
Normally, the specific endophytes interaction with various plants and evidence of their strategy of
existence and transmission is provided by their genome organization (Andreote et al., 2014).
Researchers have reviewed the genome sizes and origins of endophytes by correlating the genome
size with the bacterial lifestyle. As per many researchers, the definition of endophytes could be
suitable for the hypothesis that they live inside the plant species, where the environment is more
stable compared to the soil, where the plant grows. However, there are also some endophytes that
only appear in the plant during part of their lifecycle. Thus, the endophytic community is made up of
organisms from distinct origins, with those with larger genomes likely to live in variable
environments, such as soils, while those with smaller genomes are likely to exist in the stable
environment and are vertically transmitted (Mitter et al., 2013).
Types of Endophytic Fungi
Fungi are a heterotrophic group of organisms with various life cycles that include symbiotic
relationships with a wide variety of autotrophic organisms. Endophytic fungi have been classified
into two broad groups based on their phylogeny and life history traits. These include the
clavicipitaceous, which infect some grasses confined to cool regions and the non-clavicipitaceous
endophytes, which are from asymptomatic tissues of non-vascular plants, ferns and allies, conifers
and angiosperms and are limited to the Ascomycota or Basidiomycota group. Endophytic fungi
produce some of the most broadly used antibiotic and anticancer drugs Penicillenols, extracted from
Penicillium sp., is cytotoxicto numerous cell lines. Taxol, isolated from Taxomycesandreanae, is the
most effective and successful anti-cancer drug extracted from endophytic fungi to date. Clavatol
(Torreyamairei), sordaricin (Fusarium sp.), jesterone (Pestalotiopsisjesteri), and javanicin
(Chloridium sp.) are all known topossess strong antibacterial and antifungal properties
againstnumerous foodborne infectious agents. Pestacin, isolated from P. microspora, has excellent
antioxidant properties.
General Mechanisms Employed by Endophytes in Plant Protection
Endophytic microorganisms help in boosting plant fitness through several mechanisms of action. The
generally mechanisms employed by endophytic fungi and bacteria was discussed in this section. The

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modes of action include direct and indirect mechanisms as illustrated in Figure-1. These mechanisms
were discussed in detail below.
Fig.1: Mechanism employed by endophytes for plant protection
Direct Mechanisms of Plant Protection from Pathogens

Recent studies carried out on endophytes have established their capacity to enhance host defense
against diseases and reduce the damages attributed to pathogenic microorganism (Ganley et
al., 2008; Mejía et al., 2008). The most common strategy employed by these researchers is in
vitro direct plate antagonistic reaction against pathogens or by comparing the rate of survival of plant
inoculated with control. Although some studies have presented new mechanisms used by endophyte
in reducing the effects of pathogens, current knowledge about endophytes, pathogen, and plant
regulations still not fully understood (Ganley et al., 2008). In this section, we shall be discussing a
direct mechanism (endophytes-pathogens interactions) and indirect mechanism (enhanced plant
defense). In the direct mechanism, endophytes directly produce antibiotics which help in suppressing
pathogens. However, direct endophyte-pathogen interactions are compounded and responsive to
species-specific antagonism (Arnold et al., 2003). Some examples of direct mechanisms used by
endophytes are discussed below.
Antibiotics Produced by Endophytes
Most endophytes have been reported to produce some secondary metabolites and some of them
exhibit antibacterial and antifungal properties which help in inhibiting the growth of phytopathogenic
microorganisms (Gunatilaka, 2006). Many types of research are still ongoing in a bid to identify
endophyte metabolites for possible commercial use. Different bioactive compounds have been

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studied for their ability to inhibit many phytopathogens (Suryanarayanan, 2013; Daguerre et
al., 2016). Also, many metabolites with antimicrobial properties have been discovered from
endophytes, some recently reviewed one are flavonoids, peptides, quinones, alkaloids, phenols,
steroids, terpenoids, and polyketides (Mousa and Raizada, 2013; Lugtenberg et al., 2016). When
many microbial species are present in the same plant, the association propels the secretion of
metabolites by the endophytes or the host to inhibit the growth of microbes that are harmful (Kusari
et al., 2012). In some instances, the endophytes and the host plant do use some distinct pathways in
enhancing the production of metabolites, some use induced metabolism which helps in metabolizing
the product of the other (Kusari et al., 2012; Ludwig-Müller, 2015). It was later concluded that many
endophytic strains cannot produce the compounds independently (Heinig et al., 2013).
An endophyte isolated from Cassia spectabilis, named Phomopis cassia was able to synthesize five
substances similar to 3,11,12-trihydroxycadalene and cadinane sesquiterpenes in which one of the
five derivatives produced the most active antifungal metabolite against Cladosporium
cladsporioides and Cladosporium sphaerospermum (Silva et al., 2006). Alkaloids were reported to
have strong potential in inhibiting the proliferation of microbes, for instance, altersetin, a novel
alkaloid which was isolated from the endophyte Alternaria spp., exhibited a strong antibacterial
effect against many gram-positive bacteria that are pathogenic (Hellwig et al., 2002). Another
metabolite which exhibited antibiosis is a volatile oil. An endophytic fungus from the tropical trees
known as Muscodor albus produced many volatile organic compounds, including aciphyllene, 2-
butanone and 2-methyl furan which were reported to produce antibiotic properties (Atmosukarto et
al., 2005). Also, fungal endophytes isolated in vitro from Artemisia annua can suppress the growth of
most phytopathogenic organisms by the production of antifungal compounds such as n-butanol and
ethylacetate (Liu et al., 2001). Tian et al. (2017) assessed the role of anti-fungal protein produced
by Epichloë festucae in controlling Sclerotinia homoeocarpa in Festuca rubra. The result presented
this attribute by fescues as one of the unique ones. The mechanism of plant protection used by
Paraconiothyrium strain SSM001 linked with the production of taxol from yew tree (Taxus spp.)
against dangerous wood-decaying fungi was investigated by Rafiqi et al. (2013) and Soliman et al.
(2015). A summary of related studies on the antimicrobial properties of endophytes is presented in
Table-1.
Table-1: Summary of studies on the antimicrobial activities of endophytes.
Endophytes Plant host Activity Compounds Class of References
compound
Endophytic fungi
Phoma sp. Cinnamomum Antifungal 5-hydroxyramulsin Polyketides Santiago et
mollissimum al., 2012
Geotrichum candidum, Phyllanthus Antibacterial – Pai and
Cylindrocladium sp. reticulatusPoir and Chandra, 2018
Fusarium sp. antifungal
Cladosporium
cladosporioides sp., Mucor
pusillus, Rhizopus sp.,
and Alternaria alternata
Phompsis sp. Aconitum carmichaeli Antifungal Gavodermside and Steroids Wu et
Clavasterols al., 2013
Xylaria sp. F0010 Abies holophylla Antifungal Griseofulvin – Park et
al., 2005
Chaetomium globosum Ginkgo biloba Antifungal Chaetomugilin A and D Azaphilone Qin et
derivative al., 2009

140
Pestalotiopsis mangiferae Mangifera Antibacterial 4-(2,4,7-trioxa-bicyclo[4,10]- Phenols Subban et

indica Linn heptan-3-yl) al., 2013
Aspergillus sp. Bauhinia guianensis Antibacterial Fumigaclavine C and Alkaloids Pinheiro et
Pseurtotin C al., 2013
Phomopsis sp., Garcinia sp. Antibacterial – – Phongpaichit
Botryosphaeria sp. and et al., 2006
antifungal
Nigrospora sphaerica Indigofera Antibacterial – – Santos et
(URM- suffruticosa Miller al., 2015
6060) and Pestalotiopsis
maculans (URM-6061)
MR1B and MRB.2 Catharanthus Antibacterial Citreoisocoumarin, paxilline, Isocoumarin Akpotu et
roseus and Euphorbia and nigricinol, fatty acid, derivative al., 2017
hirta antifungal sceptrin, cladosporin
Endophytic actinomycetes
Streptomyces noursei – Antifungal Nystatin Steroids Fjærvik and
Zotchev, 2005
Streptomyces sp. – Antibacterial Harmaomycin Peptide Bae et
derivatives al., 2015
Streptomyces remosus – Antifungal Tetracyclin Steroids Nelson, 2001
Streptomyces sp. Grevillea pteridifolia Antibacterial Kakadumycin A Peptides Castillo et
Echinodermycin al., 2003
Streptomyces sp. TP- Allium tuberosum Antifungal 6-Prenylindole Alkaloids Singh and
A0595 Dubey, 2018
Aeromicrobium ponti Vochysia divergens Antibacterial 1-Acetyl-β-carboline, Indole- Alkaloids Gos et
3-carbaldehyde, 3- al., 2017
(Hydroxyacetyl)-Indole,
Brevianamide F, and Cyclo-
(L-Pro-L-Phe)
Streptomyces sp. neau-D50 Glycine max Antifungal 3-Acetonylidene-7- Alkaloids Zhang et
Prenylindolin-2-one and 7- al., 2014
Isoprenylindole-3-carboxylic
acid
Actinosynnema pretiosum Maytenus serrata Antibacterial Ansamitocin Polyketides Siyu-
Mao, 2013
Streptomyces sp. TP- Aucuba japonica Antibacterial Cedarmycin A and B Terpenes Sasaki et
A0456 and al., 2001
Terpenoids
Streptomyces Zingiber officinale Antifungal 5,7-Dimethoxy-4- Coumarins Taechowisan
aureofaciensCMUAc130 pmethoxylphenylcoumarin; et al., 2007
5,7-Dimethoxy-4-
phenylcoumarin
Streptomyces sp. BT01 Boesenbergia Antibacterial 7-Methoxy-3, 3′,4′,6- Flavonoids Taechowisan
rotunda (L.) tetrahydroxyflavone and 2′,7- et al., 2014
Dihydroxy-4′,5′-
Dimethoxyisoflavone,
Fisetin, Naringenin, 3′-
Hydroxydaidzein,
Xenognosin
Streptomyces sp. DSM Alnus glutinosa Antibacterial Alnumycin Quinones Singh and
1175 Dubey, 2018
Dactylosporangium sp. Cucubalus sp. Antifungal Streptol Tannins Singh and
strain SANK 61299 Dubey, 2018
Verrucosispora maris AB- Sonchus oleraceus Antibacterial Proximicin Peptides Fiedler et
18-032 al., 2008

141
Lytic Enzymes Secretion

Most microorganisms secrete lytic enzymes for the hydrolysis of polymers (Gao et al., 2010). About
1,350 compounds can be secreted; among them are cellulose, hemicellulose, proteins, DNA, and
chitin (Tripathi et al., 2008). For endophytes to colonize the surface of plants, they produce
numerous enzymes which successively aid the hydrolysis of the plant cell wall. These enzymes help
in reducing phytopathogens indirectly and also aid the fungi cell wall degradation. There are
numerous types of enzymes some of which are chitinases, cellulases, hemicellulases, and 1, 3-
glucanases. Application of mutagenesis to the genes of 1, 3-glucanase present in a strain
of Lysobacter enzymogenes reduced the biocontrol activity toward the damping-off disease of sugar
beet caused by Pythium and tall fescue leafspot disease (Gao et al., 2010). The lytic enzymes
produced by Streptomyces have a strong effect on antagonizing cacao witches broom disease
(Macagnan et al., 2008). Even though enzymes may not be solely effective as an antagonizing agent,
they may enhance antagonistic activities when combined with other mechanisms. Pectinase was also
reported to aid the reduction of pathogenesis in a plant (Babalola, 2007).
Production of Phytohormone
Endophytes produce phytohormone which enhances plant growth promotion and changes the
morphology and structure of the plant. As a result of this attribute, endophytes have gained ground in
the area of agricultural sustainability (Sturz et al., 2000). The mechanism adopted by endophytes in
the production of phytohormones in the host plant is related to the mechanism used by rhizobacteria
in plant growth promotion. They help in growth promotion and protection of non-leguminous plants
by the secretion of gibberellic acid (Khan et al., 2014), auxins (Dutta et al., 2014), indole acetic acid
(Khan et al., 2014; Patel and Patel, 2014), and ethylene (Babalola, 2010; Kang et al., 2012).
Indole acetic acid (IAA) triggers plant cell division, differentiation and extension; stimulates of seed
and tuber germination; increases the rate at which root and xylem develop, enhances lateral
initiation, controls the rate of vegetative growth, and the formation of adventitious root formation;
aw well as the formation of pigments and biosynthesis of metabolites, controls responses to gravity,
light, and fluorescence, affects photosynthesis and resistance to extreme conditions (Gao et
al., 2010). IAA secreted by plant growth-promoting bacteria sometimes slows down the
physiological processes listed above by affecting the level of auxin secretion by the plant. Also, the
IAA produced by endophytic bacteria has the capacity to increase the root length and surface area,
thereby giving room for the plant to have better access to nutrients from the soil. Additionally, IAA
production expands bacteria cell walls and increases the secretion of exudates alongside providing
more nutrients for growth enhancement of other beneficial bacteria present in the rhizosphere.
Therefore, the IAA produced by endophytic bacteria is recognized as the major effector molecule in
phyto stimulation, pathogenesis, and plant-microbe interaction (Gao and Tao, 2012). Several have
studies demonstrated that endophytic actinomycetes also produce plant growth-promoting
compounds such as IAA which have been reported to enhance the formation and elongation of plant
adventitious roots in a plant (de Oliveira et al., 2010; Shimizu, 2011).
Phosphate Solubilization
The third most important nutrient for plant growth is potassium (K) and endophytes are capable of
solubilizing forms of potassium that are insoluble. Most soil-related microorganisms are capable of
solubilizing insoluble phosphate to enhance the production of P, thus making it available for plant
use (Alori et al., 2017). The most common mechanism used for inorganic phosphate solubilization is

142
the dissolution of mineral compounds such as organic acids, protons, siderophores, carbon dioxide
(CO2), and hydroxyl ions (Olanrewaju et al., 2017). The existence of microorganisms that solubilizes
potassium might have opened our eyes to an alternative means of making potassium available for
plant uptake (Rogers et al., 1998). Endophytes also introduce organic acids into the soil which help
to solubilize the phosphate complexes and change them into ortho-phosphates for plant absorption
and usage. Numerous bacteria species namely Bacillus mucilaginosus, B. circulans,
Pseudomonas sp., Burkholderia, Paenibacillus sp., Acidothiobacillus ferrooxidans, and Bacillus
edaphicus were identified in the release of the accessible form of potassium from potassium-bearing
minerals in soils (Yadav, 2018). As abundant as phosphorus is in the soil, unfortunately, many of its
remains do not exist in an insoluble form (Miller et al., 2010). Many studies have shown the role of
endophytic microorganisms as a biofertilizer and biocontrol agent. For example, endophytes isolated
from the root nodule for peanut, identified as Pantoea spp. was reported to have strong solubilizing
activity (Yadav et al., 2018). Similarly, endophytic actinomycetes have been reported to perform an
important role in phosphate solubilization and also enhances its availability to plants through
chelation, acidification, and mineralization and redox changes of organic phosphorus (Singh and
Dubey, 2018). Solubilization of phosphate alongside secretion of phytase was demonstrated by an
endophytic actinomycete, Streptomyces sp., which significantly improve plant growth (Jog et
al., 2014).
Siderophore Production-
Siderophores are small molecular compounds which are capable of chelating iron which can be
produced by endophytes and can make iron available for plant use while starving pathogens of iron
(Yadav, 2018). Some of the siderophores known to be produced by endophytes can confer biocontrol
activities such as hydroxymate, phenolate and/or catecholate types. Also, the iron-deficient plant is
enhanced by siderophores which help in the fixing of nitrogen since diazotrophic organisms require
Fe2+and Mo factors for the functioning and synthesis of nitrogenase (Kraepiel et al., 2009). There are
many literature evidences to support the insecticidal properties of endophytes (Azevedo et al., 2000).
Some endophytes reduce pest penetration of the stele by thickening the endodermal cell wall (Gao et
al., 2010). Others destroy insects by producing secondary metabolites. Though some toxic
metabolites are traceable to endophytes some of these metabolites are pyrrolizidine, alkaloids,
pyrrolopyrazine alkaloid, peramine ergot alkaloid, and ergovaline (Wilkinson et al., 2000).
In the case of plant growth-promoting bacteria, Fe2+ is oxidized to Fe3+-siderophore complex in the
bacterial membrane, which is later introduced into the cell by endophytes through a gating
mechanism (Gao et al., 2010). The concentration of soluble metals increases when siderophores bind
to the metal surface. Once the level of heavy metal contaminants is removed, different mechanisms
are employed by plants to ingest iron from bacterial siderophores, for example, iron chelates aid the
direct absorption of siderophore-Fe complexes, or ligand exchange (Schmidt, 1999). A sider. Some
endophytic actinomyces such as Streptomyces sp. GMKU 3100, Streptomyces sp.
mhcr0816, Streptomyces sp. UKCW/B, and Nocardia sp. have been reported to produce siderophores
(Singh and Dubey, 2018). Similarly, S. acidiscabies E13 was also reported as a superb producer of
siderophore which enhances the growth of Vigna unguiculata under nickel stress conditions
(Sessitsch et al., 2013).

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1-Aminocyclopropane-1-Carboxylate (ACC) Utilization

Generally, ethylene is an essential metabolite for the normal growth and development of plants
(Khalid et al., 2006). This important hormone known for enhancing plant growth is secreted by
almost all plants and is affected by different abiotic and biotic activities in the soil which improve
physiological changes in most plants. The occurrence of extreme conditions such as pathogenicity,
drought, salinity, and heavy metals increases the level of ethylene which has side effects on the
growth of the plant; this may result in alteration of the cellular processes and defoliation which
affects the yield of the crop (Bhattacharyya and Jha, 2012). Many endophytic bacterial species that
can produce ACC deaminase have been discovered in genera like Achromobacter, Agrobacterium,
Acinetobacter, Bacillus, Enterobacter, Pseudomonas, Serratia, Ralstonia, Rhizobium, Alcaligenes,
Burkholderia etc. (Kang et al., 2012). Most of the bacterial endophytes trap the ethylene precursor of
ACC and change it into ammonia and 2-oxobutanoate (Arshad et al., 2007). Lugtenberg and
Kamilova (2009) reported that some stresses like radiation, heavy metals, flooding resistance due to
stress coming from polyaromatic hydrocarbons, high light intensity, wounds, high salt concentration,
insect predation, draft, and extreme temperature can be overcome by plants that can produce ACC
deaminase.
Competition With Pathogens
Competition is a strong mechanism used by endophytes in preventing pathogens from colonizing the
host tissue (Martinuz et al., 2012). Endophytes possess the ability to colonize many plant tissues
systemically or locally (Latz et al., 2018). For example, they act through colonization and the lurking
of nutrients that are available and by occupying the position that is available for pathogens to carry
out their activities (Rodriguez et al., 2009). This can be further buttressed using a study by
Mohandoss and Suryanarayanan (2009), who discovered that destruction of endophytes in mango
leaves by the application of fungicides in its treatment allows other fungi to inhabit the niche,
especially pathogenic fungi.
The mechanism used for competition by most endophytes usually takes place in combination with
other mechanisms, instead of acting independently. Since the control method employed by
endophytes is often local, they will, however, need to systematically colonize the part of the host
where most pathogens may attack. The colonization of the root of oilseed rape with
endophyte Heteroconium chaetospira could not successfully prevent clubroot symptoms (Lahlali et
al., 2014). The result, therefore, indicates the limitations that may be encountered with competition
as a biocontrol method, as it may be inactive when there is a high presence of microorganisms
causing disease. The symptoms of Phytophthora sp. were successfully reduced when treated through
a foliar application with mixtures of endophytes from leaves of cacao tree leaves, thus showing
competition as one mechanism of disease suppression in a plant. However, some of the strains were
also observed to produce other active metabolites which is an indication that, competition might not
be the only mechanism used in controlling the disease (Arnold et al., 2003).
Indirect Mechanisms of Plant Protection from Pathogens
Plants employ several mechanisms to survive in extreme conditions such as drought, salt stress, and
cold. Some of the rapid noticeable biochemical and morphological changes observed include the
hypersensitive response, cellular necrosis and phytoalexin production. In long term evolution, non-
specific (general) resistance and specific resistance are examples of innate resistance developed for
pathogen resistance (Kiraly et al., 2007). Those that possess specific resistance can resist infection

144
from one or a few pathogens while the non-specific resistance is active against many pathogens.
Endophytes increase the plant defense mechanism through the production of secondary metabolites
and enhanced resistance.
Induction of Plant Resistance
For over 20 years now, many studies have concentrated on the way plants respond to attack from
parasites and pathogens using various categories. Induced systemic resistance (ISR) and Systemic
acquired resistance (SAR) are the two resistance patterns which have attracted the most attention of
researchers. ISR, which is induced by some non-pathogenic rhizobacteria, is moderated by ethylene
or jasmonic acid which cannot be linked with the building up of pathogenesis-related (PR) proteins.
SAR, which is caused by infections from pathogens is mediated by salicylic acid and linked with the
building up of PR proteins (Tripathi et al., 2008). These PR proteins have many enzymes, such as 1,
3-glucanases and chitinases which help in the direct lysing of invading cells, and strengthening of
cell wall boundaries to build resistance against infection and cell death (Gao et al., 2010). ISR
produced by endophytes can also be linked with the enhancement of genes that are expressed in
pathogenesis. The root of tomato harbors important endophytes called Fusarium solani which
prompt ISR against Septoria lycopersici, the causative agent of tomato foliar pathogens and activate
PR genes, PR7, and PR5 activities in the roots (Kavroulakis et al., 2007). Redman et al. (1999)
reported that the inoculation of a non-pathogenic mutant strain of Colletotrichum magna on Cucumis
sativus and Citrullus lanatus produced a high amount of peroxidase, lignin deposition, and
phenylalanine ammonialyase which help in protecting the plant against diseases which are caused
by Fusarium oxysporum and Colletotrichum orbiculare. Reduction in the lesions on leaves was
observed when Neotyphodium lolii engaged against four different pathogens, which could be
attributed to enhanced peroxidase and superoxide dismutase activities of the host plant (Tian et
al., 2008).
Plant Secondary Metabolites Stimulation
Secondary metabolites from plants are compounds which have limited functions in the life cycle of
the plant but are of great importance in its adaption to different environments (Bourgaud et
al., 2001). Notable among all the secondary compounds produced by a plant is an antimicrobial
molecule with a low molecular weight called phytoalexins (Gao et al., 2010). It has many substances
in it, some of which are terpenoids and flavonoids among many others. Orchis
morio and Loroglossum hircinum were the first to produce phytoalexins in response to a fungal
attack initiated by a French botanist called Noel Bernard, outcomes of other studies showed that
phytoalexins can now be produced through some abiotic stress factors such as heavy metals ion, salt
stress and UV light (Gao et al., 2010). Some studies have concentrated on the production of
phytoalexins when triggered by pathogens (Pedras et al., 2008). The production of plant secondary
metabolism moderated by endophytes is still a new research area. Findings revealed that the elicitors
of Fusarium E5 could propel triterpene and dipertene production in cell suspensions of E. pekinensis.
Li and Tao (2009) reported a similar result in Taxus cuspidate culture suspensions, in which culture
supernatants of endophytes resulted in increased production of paclitaxel when compared with the
control. It is suspected that the co-culturing with elicitor endophytes is a likely way of increasing
plant secondary metabolites and boosting plant resistance. Endophytic colonization induced the
production of hydrolase for plant cells to reduce the growth of fungi, therefore making endophytes
act as elicitors through hydroxylation production (Gao et al., 2010). Some elicitors like glycoprotein,

145
polysaccharides and lipopolysaccharides trigger plant defense mechanisms and increase secretion of
plant secondary metabolites which effectively reduce attack by pathogens. However, there is limited
information as regards the way in which endophytes survive in the host plant when producing large
quantities of secondary metabolites are produced (Gao et al., 2010).
Promotion of Plant Growth and Physiology
Endophytes sometimes support the host plant defense mechanism against plant pathogenic
microorganisms by taking over the plant physiology (Gimenez et al., 2007). As the growth of the
plant increases, it develops vigor and resistance to different stresses both abiotic and biotic, this is
considered as one of the strategies used by the plant for defense against pathogens (Kuldau and
Bacon, 2008). Several studies have shown that plants inoculated with endophytes recorded an
increase in growth, drought resistance (Gao et al., 2010), and tolerance to any type of soil
(Malinowski et al., 2004). Plant growth can be enhanced by several compounds, an
endophyte, Colletotrichum sp., isolated from A. annua produces a substance called indole acetic acid
(IAA) which helps in regulating plant physiology (Lu et al., 2000). Dai et al. (2008) reported that
extracts from Fusarium sp. E5 produced auxin. Another mechanism adopted by endophytes can be
said to be the release of phytohormones (Dai et al., 2008). We can, therefore, believe that plant
growth promotion when triggered by endophytes will indirectly protect the plant against pathogens.
Hyperparasites and Predation
Hyperparasites is another mechanism endophyte use to protect their host ecologically. In this
mechanism, endophytes directly attack identified pathogens or their propagules (Tripathi et
al., 2008). Endophytic fungi capture the pathogens by twisting and penetrating their hyphae and by
the production of lyase which destroys the cell wall of the pathogen. For instance, Trichoderma sp.
was able to capture and penetrate the hyphae of Rhizoctonia solani, a known plant pathogen; the
observation was linked to biocontrol activities (Grosch et al., 2006). Another mechanism is microbial
predation; this entails a general way of reducing pathogens of plants. Most endophytes exhibit their
predatory characteristics in nutrient-deficient conditions. As an example, a variety of enzymes
attacking the cell wall of fungal pathogens directly are produced by Trichoderma sp. (Gao et
al., 2010).
Future Prospects
Concise studies on a specific population of endophytes active in a host are required before bulk
production can be carried out which often requires research with advanced technology. Studies are
also needed as regards getting plant-specific inoculum doses of endophytes, this will help in reducing
bulk production and also enhance productivity by reducing our dependence on synthetic fungicides,
pesticides, and fertilizers. Development of endophytes that can be sprayed just like most chemical
pesticides will help in the acceptance of endophytes in integrated pest management.
Future studies will need to take into account the development of genomic tools and metabolomics
tools for further studies on how endophytes colonize the plant and plant-microbe interaction. There is
still a need to study the compounds produced by endophytes and their activities in reducing diseases.
This will help in developing efficient markers for some important and distinct biocontrol agents and
assessing the effects of plant genotypes, innate microbe community, and most importantly the
environment. This structured approach will also help in discovering new endophytes with important
traits.

146
Molecular study of these endophytes is important in order to improve drug research. Also,
metagenomics study will be very important in order to showcase the diversity of endophytes and the
functions they are capable of performing through a detailed analysis of their genes. Molecular
biology techniques can be used for the isolation and identification of the different types of genes
present in the biosynthetic pathways and this will further open our eyes to new bioactive compounds
at a commercial level as well as in the laboratory. Future studies should focus on the biosynthetic
pathways which might be responsible for the secretion of numerous important bioactive compounds
by endophytes.
Also, future studies can look into the development of endophytic nanoparticles which will help in
improving the plant growth. Transfer of genes can also be employed in order to detect more efficient
species. The idea of manipulating genes can help the host plants in developing new traits like
phytoremediation and herbicide resistance, among others, which could more suitably regulate
metabolism. There is no microbial technology that can be considered successful until it has been
commercialized. The specificity of endophytes within a plant is one of the limitations in its large-
scale production.
Conclusion
The study attempts to appreciate the diverse mechanisms used by endophytes in protecting plants
from diseases for sustainable agriculture. Endophytic microbes support the plant and accelerate plant
growth by employing different mechanisms of action, both direct and indirect. The major benefit of
embracing such beneficial microorganisms in the field of agriculture is to bring about reduction in
the use of different agrochemicals such as pesticides, chemical fertilizers, other artificial chemicals
etc. and this would make agriculture more productive and sustainable. Endophytes can still be very
useful in the biomedical field because endophytes can synthesize and secrete chemicals which may
be used for the development of antibiotics of importance for human use. Many studies are still
ongoing toward assessing the ability of endophytes to secrete novel bioactive compounds which will
be of great importance in the treatment of human diseases.
Besides the numerous applications of endophytes in medicine, therapeutics, and mining, some novel
metabolites may be useful in sustainable agriculture and in enhancing plant growth. These
metabolites can also confer insecticidal, and pest control activities, alongside enhance plant nutrient
uptake under extreme conditions such as drought, salinity, and waterlogging. Taken as a whole,
novel bioactive compounds secreted by endophytes especially endophytic actinomycetes could offer
immense contributions in address the present and future challenges in agriculture, environment and
medicine. Finally, the application of metagenomics combined with next-generation sequencing
technologies is expected to open up the numerous unexplored pools of antimicrobials secreted by yet
uncultivated endophytic microbes.
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***
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Chapter 15
A COMPREHENSIVE REVIEW ON
BIOLUMINESCENT FUNGI
Dr. Mehtab Yasmeen
A bstract
Some of the fungi in the diverse group of Fungi Kingdom glow at night by a mechanism known as
‘Bioluminescence’. All known luminescent species of fungi found to be mushrooms that form thin-
walled white spores for dispersal, and all are white rot fungi capable of digesting both the cellulose
and lignin in plant debris. The greatest diversity occurs in the tropics, although a few species grow in
temperate habitats. The luminescent fungi glow continually, emitting a yellowish-green light at a
wavelength of 520-530 nanometers. Not all parts of the mushroom glow in some species, it is only
the cap or the gills that glow; in others, only the stem or the mycelium. Bioluminescence is a
luciferin-luciferase mediated reaction that emits light when water and oxygen are present. It is
similar to but different from that functioning in luminescent bacteria, dinoflagellates and animals.
The study of evolution of luminescence in fungi shows that there are four distinct lineages of fungi
that glow. Commonly known glowing fungi from North America like the Jack-o-Lantern Mushroom
(Omphalotus spp.) and the Honey Mushroom (Armillaria spp.) belong to two different lineages. The
most diverse of the four lineages are the mycenoid fungi (Mycena and allies). The reasons that these
fungi glow is to attract nocturnal animals to aid in spore dispersal etc. This is especially adaptive in
closed-canopy forests where wind dispersal is hindered. Others glow to attract the predators of
insects that eat the mushrooms.
Keyword: Fungi, Bioluminescence, Mushrooms, Luciferin, Luciferase, Glow, Omphalotus,

Armillaria, Mycena.
Faculty, Department of Botany, Telangana Mahila Viswavidyalayam, Koti, Hyderabad,

Telangana, India. myashu77@gmail.com
Dr. Mehtab Yasmeen

161
I ntroduction
Bioluminescence is derived from the Greek word combination ‘bios’ means living form and Latin
word ‘lumen’ means light. So, bioluminescence means living organisms emitting light or glowing.
Bioluminescence is considered to be a process through which light is produced and emitted by a
living organism resulting from a chemical change occurring within the body of an organism. These
organisms use a variety of body parts to emit light in several colors and for various purposes. A
unique feature of biological light that differentiates it from other types of light is that it is not hot but
a ‘cold light’. Contradicting to the light of a candle, bioluminescent light is produced with little or no
heat radiation (Weitz, 2004).
The natural phenomenon of bioluminescence is the emission of visible light by living organisms
mediated by an enzyme-catalyzed (‘luciferase’) reaction of molecular oxygen with a substrate
(‘luciferin’). There is biodiversity of bioluminescent organisms with wide distribution in nature.
They occur in many organisms like bacteria, dinoflagellates, insects, fungi etc. The enzyme
‘luciferases’ occur in different forms in different organisms and even the substrate ‘luciferins’ show
no chemical similarity. Molecular oxygen is the only common feature of bioluminescence reactions,
concluding that the luminescent mechanism in different organisms may have evolved independently
(Wilson & Hastings, 1998).
History of Bioluminescence
The Greeks and Romans were the first to report the characteristics of luminous organisms. Aristotle
(384-322 BC) described 180 marine species and was the first to recognize "cold light." The Greeks
also made reference to sea phosphorescence (about 500 BC) (Harvey, 1957). In the fifteenth century
voyagers commented on the "burning sea" phenomenon. Christopher Columbus (1492) referred to
mysterious lights in the water before reaching San Salvador Tropical fireflies in the east were seen
by Sir Frances Drake (1540-1596).
In the sixteenth century references to bioluminescence were found in literature such as Shakespeare
(1564 -1616) in Hamlet who talked of the "uneffectual fire" of the glow-worm. English explorers
apparently mistook the light from fire beetles for the lights of Spanish campfires and decided to
avoid landing on Cuba in 1634, perhaps altering the history of the new world (Jacobs, 1974). The
first book devoted to bioluminescence and chemiluminescence was published in 1555 by Conrad
Gesner (1555; Carter and Kricka, 1982: Harvey, 1957).
In 1667 Robert Boyle (1667) documented the air requirement for luminescence. Oxygen had not yet
been discovered, but we now recognize that this air requirement was, in reality, an oxygen
requirement of the process. This represented a new era in the characterization of bioluminescence
rather than just its documentation. The nineteenth century brought scientific voyages such as that of
H.M.S. Challenger (1873). Later in that century Raphael Dubois performed a significant experiment
where he extracted the two key components of a bioluminescent reaction and was able to generate
light. He coined the terms "luciferine" and the heat labile "luciferase".
One of the most eminent scientists of the twentieth century was a "Princeton Professor" E.Newton
Harvey (1887-1959). He spent much of his life looking for the existence of a luciferin-luciferase
system in virtually every luminous organism that he could find (Harvey, 1952). The first luciferin
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was isolated in 1956 (Green and McElroy, 1956). The first photoprotein to be isolated was the
calcium-activated photoprotein aequorin in the 1960's (Shimomura et al., 1962). This report also
noted that a "green protein" was present in the extracts from which the aequorin was purified,
although the green-fluorescent protein (GFP) itself was not recognized as the source of the
bioluminescence in the jellyfish at that time.
Roman scholar Pliny the Elder observed the effect of fungal bioluminescence when they described
the glowing light of the cold “fire” of damp wood, this probably later became known as “foxfire”
cause in old French “fois” means “false”. The first mention that the light of luminous wood was due
to fungi occurred from a study of luminous timbers used as supports in mines by Bishoff in 1823. A
Dutch consul in 1700 reported that Indonesian people used fungal fruits to illuminate forest
pathways. From the 1850’s to the early part of the 20th century the identification of the majority of
fungal species exhibiting bioluminescent traits was completed.
The research of bioluminescent fungi stagnated from the 1920’s till 1950’s (Newton, 1952 and
Herring, 1978). After which extensive research began involving the mechanisms of bioluminescence
and is being still carried out.
Types of Bioluminescent Fungi
Amongst fungi, the majority of bioluminescence occurs in the Basidiomycetes and only one
observation has been made involving the Ascomycetes; specifically in the Ascomycete genus Xylaria
(Harvey 1952). Presently, 109 luminescence fungi are recognized which would be classified into
four molecular lineages 12 within the Omphalotus lineage, 10 within the Armillaria lineage, 85
within the Mycenoid lineage (mostly Mycenaceae) and two within the Lucentipes lineage (Ke and
Tsai, 2022).
The essential parameters of bioluminescence fungi are:
1. Bioluminescent fungi emit a greenish light with a maximum intensity at 520-530 nm (O’ Kane
et al., 1990).
2. Light without heat.
3. Light ceased during a vacuum, in hydrogen, and carbon dioxide.
4. The light was independent of humidity, temperature, and didn’t burn any brighter in pure
oxygen (Vladimir et al., 2012).
5. Luminescence may not confer a significant selective advantage as there are both luminescent
and nonluminescent strains of the same species and species that only have luminescent
mycelium (Herring, 1994).
6. Luminescence may occur in both mycelia and fruiting bodies, as for example in P. stipticus and
O. olearius, or only in mycelia and young rhizomorphs as in A. mellea (Wassink, 1978).
7. Some luminescent fungi like A. mellea, rceportedly exhibit diurnal periodicity (Berliner, 1961;
Calleja & Reynolds, 1970) and seasonal variation of bioluminescence (Kamzolkina, 1982).
The intriguing diversity and evolution of bioluminescent fungi have always been a topic of interest to
mycologists. This diversity and evolution cover diverse fungi that exhibit different emission patterns,
including emitted tissues.
There are more than 40 species in the Armillaria lineage, most of which are saprotrophs or tree root
pathogens. All 10 of the species investigated were confirmed to have luminescent properties. An
interesting finding from this lineage is that luminescence only occurs in mycelium, not fruiting
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bodies; this is believed to be because the substrate of the luminescent reaction is not synthesized in
the fruiting bodies of hot and cold extracts.
Conversely, the Omphalotus lineage contains a historically unresolved taxonomic group belonging to
Neonothopanus, Nothopanus, Omphalotus, and Panus. The lineage is known for large mushrooms,
and the bioluminescent mechanisms of N. gardneri and N. nambi are well characterized.
The Mycenoid lineage includes over 600 species diverse in not only color and shape but also habitat,
leading to a wide spectrum of nutritional strategies, including saprotrophic, parasitic, and
mycorrhizal. Their luminescence also shows diverse patterns among tissues. For instance, in Mycena
jingyinga, luminescence is observed in the mycelium only, while in M. luxarboricola, light can be
seen in both the mycelium and the entire fruiting body, though in M. kentingensis, luminescence is
present in mycelium and pileus, but not the stipe.
The two important genera that display bioluminescence are Pleurotus which has at present 12 species
which occur in continents of Europe and Asia and genus Mycena which has 19 species identified to
date with a worldwide distribution range. Panellus stipticus is unusual in that luminescence is
exhibited only by the North American strains of P. stipticus and not by the Eurasian strains (Peterson
& Bermudes, 1992).
Distribution of Bioluminescent Fungi
Bioluminescent fungi are mostly found in a tropical and temperate climate, Xylaria hypoxylon being
the exception are prevalent in the dense forests of South America, Europe and a few Asian countries.
Seven novel species of Mycena have been outlined as luminescent by Desjardin et al. (2012).
Mycena is a ruling genus exhibiting luminous properties. They have depicted particularly the
taxonomy and morphology of the new species collected. Earlier scientists have described a wood
sample from an Indian state that showed luminescence during the monsoons and have studied its
requirements for growth by culture samples.
Desjardin et al. (2005) have characterized a new species of Gerronema viridilucens collected from
the bark of living Eugenia trees from Brazil. The fungus emitted yellowish-green light that appeared
bluish to the unaided eye. The mycelium and fruit bodies of the fungus are bioluminescent.
Seas and Avalos (2013) collected samples from litter, decaying trunks, broken branches of Costa
Rica forests and found a noticeable relationship between the types of fungi and soil and deciphered
that they are affiliated. Chew et al. (2014) determined the phylogeny of fifteen surveyed specimens
of luminescent fungi from Malaysia using molecular data.
Aravind akshan and Manimohan (2015) have given details on Mycena genus from India. Amongst
wide varieties of fungi, there are bioluminescent fungi one can found in the Western Ghats of Goa and
Kerala.
A new variety of mushroom has been discovered in Northeast India’s Meghalaya state. Nearly 600
varieties of a bioluminescent mushroom have been found in the jungles of Meghalaya and because of
its light-emitting nature, the newly found species has been named Roridomyces phyllostachydis.
In North America only five species of bioluminescent basiodiomycetes have been reported. These
include the Honey mushroom Armillaria mellea, the common Mycena- Mycena galericulata, the
Jack O’Latern - Omphalotus olearius, Panellus stipticus and Clitocybe illuden. Terashima et al.
(2016) reported eight new species of bioluminescent mushrooms from Japan. Cortés-Pérez et al.
(2019) reported seven species bioluminescent mushrooms from Mexico.
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Three species of bioluminencent fungi have been identified in Singapore: Filoboletus

manipularis, Mycena chlorophos and Neonothopanus nambi.
Three species of bioluminencent fungi have been identified in Singapore: Filoboletus
manipularis, Mycena chlorophos and Neonothopanus nambi.
Three species of bioluminencent fungi have been identified in Singapore (Lee, 2022): Filoboletus
manipularis, Mycena chlorophos and Neonothopanus nambi. Until now, 12 species of
bioluminescent fungi have been reported from Taiwan, including Armillaria mellea, Mycena
chlorophos, M. haematopus, M. inclinata, M. kentingensis, etc (Chang et al, 2020).
Distribution of bioluminescent fungi depends on the nature of the substrates they colonize (Mata,
1997). Fungi from the order Agaricales, which comprise the majority of bioluminescent fungi
(Desjardin et al. 2008), are associated with different habitat type (Alexopoulus et al. 1996). The
abundance of bioluminescent fungi in a region is influenced by humidity, temperature, exposure to
light (Bermudes et al. 1990) and presence of streams. Interaction of forest type and soil type may
influence the distribution of bioluminescence fungi; with soil type is considered as the most
important variable.
PARTS EXHIBITING BIOLUMINESCENCE IN FUNGI
In fungi, the bioluminescent part is sometimes present in the fruiting body though it is restricted
mostly either in the mycelium or in the fruiting body (Shih et al., 2014). Lack of substrate for
luminescence reaction is the principal cause for the absence of luminosity in fruit bodies. Puzyr et al.
(2017) have studied biochemical changes that followed a non-luminous fruit body in Armillaria
borealis. A segment of the bioluminescent system in Mycena chlorophos is limited to the cell
membrane. It gave-off brilliant green light from its pileus for around two days at raised humidity
levels (Teranishi, 2016a). Light potency in the cap and upper portion of the gill was substantial than
the bottom piece. At microscopic levels, the light is produced from the membrane of hymenium and
basidia cells on the gill.
Mogilnaya et al. (2018) treated mycelium of Neonothopanus nambi with beta-glucosidase and found
that such treated specimens attained maximum light emission strength shortly. Light emitters from
the pileus of Mycena chlorophos were investigated. Luminescent gills in pileus had riboflavin, FAD,
riboflavin 5’-monophosphate as green fluorescent components (Teranishi, 2016b). Flavins are
known to be light emitters.
A special procedure is to be followed to separate the mycelium culture of glowing mushrooms and
found that moisture is the critical element. Mycelium found to have perpetual bright luminescence
when raised on solid media. High humidity levels are essential for elevated wood luminescence
(Puzyr et al., 2017). This was confirmed by their work on collected wood samples that inhabited
glowing mycelium.
Armillaria mellea shows limited luminescence as only its mycelium is luminous under natural habitat
as well as on artificial media but the fruiting bodies is non-luminous (Purtov et al., 2016). A group of
enzymes and substrates are essential to render luminosity in the mycelium and under the influence of
free oxygen accessibility. The formation of luciferin precursors in fruit bodies was found to be
thwarted therefore; the formation of luciferin was not initiated. Fungal luminescence is also found to
be a temperature-dependent process.
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Physiology of Bioluminescence in Fungi

Bioluminescence in fungi is an oxygen-dependent process involving substrates generally called
as ‘luciferans’, which are catalyzed by one or more of an assortment of unrelated enzymes named as
‘luciferases’. But, in fungi both the luciferans and luciferases are not largely identified. During
luciferic-luciferase reaction, unstable chemical intermediates are produced. With the decomposition
of these intermediates, excess energy is released in the form of light emission, making the tissues in
which this reaction occurs to glow or luminesce (Perry, 2007). Recent studies indicate that
bioluminescent fungi emit a greenish light with a maximum wavelength of 520-530nm.
The reaction involves the following elements-
1. Enzymes (luciferase) – a biological catalyst that accelerates and control the speed of chemical
process in cells.
2. Photons – packs of light energy.
3. ATP – Adenosine triphosphate, the energy storing molecule of all living organisms.
4. Substrates (luciferin) – a selected molecule that undergoes a chemical change when affixed by an
enzyme.
5. Oxygen – as a catalyst.
A simplified formula for the bioluminescent reaction is: -
ATP (energy) + luciferin (substrate) + luciferase (enzyme) + O2 (oxidizer) = light (photon)
The Bioluminescent Reaction Occurs in Two Stages
1. The reaction involves a substrate (D – luciferin), combining with ATP and oxygen, which is
controlled by the enzyme (luciferase).
2. The energy in stage 1 excites a particular molecule (the luminescent molecules; the combining of
luciferase and luciferin). The thrill is caused by the increased energy state of that of the
luminescent molecule. The result of this excitement is decay which is manifested within the form of
photon emissions that produces the light. The light given off does not rely on external light or other
energy taken in by the organism and is simply the by-product of the chemical changes and is
therefore cold light.
The energy in photons can vary with the frequency (color) of the light. Different types of substrates
(luciferins) in organisms produce different colors. Hence, the assorted luminous organisms have their
own different colors.
Ecological Significance of Bioluminescence in Fungi
To explain the ecological importance of bioluminescence in fungi two hypotheses are laid.
According to first hypothesis, role of luminescence in fungi is to attract invertebrates to assist fungal
spore dispersal (Sivinski, 1981, 1998). The presence of luminescence is observed more prominently
in the gills (e.g., P. stipticus) or spores (e.g. Mycena rorida var. lamprospora) may support this
hypothesis (Bermudes et al., 1992).
Sivinski (1981) also discusses other possible reasons for luminescence that include the attraction of
predators of fungivores, the repulsion of negatively phototropic fungivores, and as a warning signal
to nocturnal fungivores.
Luminescence in Neonothopanus gardneri is controlled by circadian rhythm. The substrate
(luciferin), reductase, and luciferase engaged in luminescence in fungi are at great heights during the
night. Shaw (2004) also hypothesized that insects lend a helping hand in the process of spore
dispersal at places in forests covered by dense canopies where wind cannot blow with a speed such
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that it carries along with the spores to distant places. He further hypothesized that fungi need wind
for spore dispersal, as a result, it could be speculated that bioluminescence is responsible for the
spore dispersal mechanism.
It was observed that N. gardneri fruit bodies crowded by beetles (family-Staphylinidae). The mycelia
of fungi are also a potential source of food to certain invertebrates and the fruit bodies are sometimes
grazed by animals so fungi have evolved these processes to repel these predators (Mamiya, 2006).
Night-time clock-controlled luminosity makes it a subject of insects and provides an advantage to it
in spore dispersal form. Karunarathna et al. (2020) reported that Roridomyces genus fungi are moist
loving and seems as if they have co-evolved with some insects which assist in their spore-dispersal.
The second hypothesis suggests that bioluminescence is a by-product of a biochemical reaction and
has no ecological value. For example, a relationship of biolumenescenc to lignin degradation has
been suggested where it may act to detoxify peroxides that are formed during lignolysis. (Bermudes
et al., 1992; Lingle, 1993).
Current Research
Luminescence is the only biochemical process which possess a ‘visible indicator’ than can be
measured. Luminometer is a device used to measure luminescence which can detect traces of light
emitting out in the bioluminescent reaction. The process can be utilized in scientific research for
biological applications. Bioluminescence can be used as ‘biosensors’ in in detecting heavy metal ions
like mercury and aluminium. It was found that by adding luciferin and testing for light production
with a luminometer, the presence of the metal ion in the solution can be detected. This technique is
suitable for testing water pollution, when the concentrations of ions is too low to detect by
conventional means (Herring, 1978).
Biosensors would also be used in testing tuberculosis in patients in early stages. Testing for
tuberculosis was a lengthy procedure as pathogenic bacteria (Mycobacterium) were taking long time
to grow to a size that is detectable. With the introduction of bioluminescence in the TB test it was
found that the diagnosis time was shortened to only two days. The technique involves inserting the
gene through a viral vector that codes for luciferase into the genome of the TB bacterial culture taken
from the patient. The bacteria now start producing luciferase. When luciferins are added the amount
of light produced needed to code for enough luciferase to produce a detectable amount of light, is
reduced to only 2-3 days. By reducing the time needed to prescribe the correct drugs for treatment,
this application of bioluminescence will someday be ready to save some of the 3 million killed each
year by tuberculosis (Tarr et.al, 1997).
Fungal luciferin chemically differs from other known luciferins because it exhibits a different
mechanism of light emission. This attribute of it is used in photochemistry, biochemistry and
evolution. Bioluminescence is also used in scientific research including evolution, ecology,
histology, physiology, biomedical applications, cytology and taxonomy.
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***
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Chapter 16
MYCOLOGY A DETAILED EXAMINATION: IN
CONTRAST OF PRESENT AND FUTURE SCOPE OF
MYCOLOGY
Deepesh Lall1 & Nisha Verma2
A bstract
In the new bioeconomy, fungi play a veritably important part in addressing major global challenges,
being necessary for bettered resource effectiveness, making renewable backups for products from
reactionary coffers, upgrading waste aqueducts to precious food and feed constituents, neutralizing
lifestyle conditions and antibiotic resistance through strengthening the gut biota, making crop
shops more robust to survive climate change conditions, and performing as host organisms for
product of new natural medicines. This range of new uses of fungi all stand on the shoulders of the
sweats of mycologists over generations the scientific discipline mycology has erected comprehensive
understanding within fungal biodiversity, bracket, elaboration, genetics, physiology, ecology,
pathogenesis, and nutrition. Applied mycology couldn't make progress without this platform. To
unfold the full capabilities of what fungi can do for both terrain and man we need to strengthen the
field of mycology on a global scale. The current charge statement gives an overview of where we
are, what needs to be done, what obstacles to overcome, and which capabilities are within reach. It
further provides a vision for how mycology can be strengthened, the time is right to make the world
apprehensive of the immense significance of fungi and mycology for sustainable global
development, where land, water and natural accoutrements are used in a more effective and more
sustainable manner. This is an occasion for profiling mycology by chronicling the part played by
fungi in the bioeconomy. Greater mindfulness and appreciation of the part of fungi can be used to
make support for mycology around the world. Support will attract further gift to our field of study,
empower mycologists around the world to induce further finances for necessary introductory
exploration, and strengthen the global mycology network. The use of fungi for unleashing the full
capabilities of the bioeconomy relies on similar progress. The fungal area can be an alleviation for
indeed more.
Keyword: Bioeconomy, Fungal Research, Fungal Products, Resource Efficiency, Global Solutions,
Funding Opportunities.
Deepesh Lall & Nisha Verma

171
1Department of Pharmaceutics, Bhagwant University, Ajmer, Rajasthan, India.

deepesh.lall95@gmail.com
2Department of Pharmacology, LCIT School of Pharmacy, Bilaspur, Chhattisgarh, India
I ntroduction
Fungi are eukaryotic microorganisms. Fungi can do as provocations, molds, or as a combination of

both forms. Some fungi are able of causing superficial, cutaneous, subcutaneous, systemic or
antipathetic conditions. provocations are bitsy fungi conforming of solitary cells that reproduce by
expiring. Molds, in discrepancy, do in long fibers known as hyphae, which grow by apical extension.
Hyphae can be sparsely septate to regularly septate and retain a variable number of capitals. Anyhow
of their shape or size, fungi are all heterotrophic and digest their food externally by releasing
hydrolytic enzymes into their immediate surroundings (absorptive nutrition). Other characteristics of
fungi are the capability to synthesize lysine by the L- α- adipic acid biosynthetic pathway and
possession of a chitinous cell wall, tube membranes containing the sterol ergosterol, 80S rRNA, and
microtubules composed of tubulin.
Fig.1: Representing the Trametes versicolor- Coriolus versicolor, Polyporus versicolor.

Applied mycology has traditionally concentrated on areas where fungi beget damage. largely
advanced mycological exploration has generated knowledge, abstract understanding and sapience
within fungal factory conditions, mortal mycoses, inner climates, and decays in wood, feed and food.

172
There's also expansive knowledge about the added value fungi contribute to beer, chuck, wine, spirit,
food preservation, and food taste. Much lower attention has been given to fungi and fungal products
for adding value for assiduity, husbandry, health, and medicinal. The last many decades, still, have
witnessed veritably intriguing developments regarding use of fungi for new processes, products and
results of significance for the world, and most importantly for increased abstract understanding of the
area Fungi at large (Hibbett et al. 2007, James et al. 2006, Schoch et al. 2006). It is now well proved,
indeed in gauged up practice, that fungi can induce palpable and substantial value through bettered
resource effectiveness, performing in dropped pollution and hothouse gas emigrations. similar
progress stands on the shoulders of the benefactions made by mycologists over decades and indeed
centuries to make up mycology as an independent field of wisdom. Importantly, value added use of
fungi was innovated not only on applied studies but also on introductory exploration aimed at
understanding fungal biodiversity, growth, nutrition, physiology, genetics, metabolism, and ecology.
The shift from chemical processes to natural processing, achieved by using fungal (and bacterial)
enzymes rather of chemical processes in diligence, similar as fabrics, leather, paper and pulp, has
significantly reduced negative impacts on the terrain. Use of enzymes in the food and feed assiduity,
similar as beast feed, baking, brewing, and wine and juice, has significantly bettered what we get out
of natural raw accoutrements. Microbial enzymes added to cleansers, washing laundry clean indeed
at low temperatures, has significantly reduced CO2 emigrations. The newest chapter in the artificial
biotechnology period is to substitute reactionary coffers with renewable coffers.
Structural and Physiological Studies of Mycology
Fungi can use a number of different carbon sources to meet their carbon needs for the conflation of
carbohydrates, lipids, nucleic acids, and proteins. Oxidation of sugars, alcohols, proteins, lipids, and
polysaccharides provides them with a source of energy. Differences in their capability to use
different carbon sources, similar as simple sugars, sugar acids, and sugar alcohols, are used, along
with morphology, to separate the colorful provocations. Fungi bear a source of nitrogen for
conflation of amino acids for proteins, purines and pyrimidines for nucleic acids, glucosamine for
chitin, and colorful vitamins. Depending on the fungus, nitrogen may be attained in the form of
nitrate, nitrite, ammonium, or organic nitrogen; no fungus can fix nitrogen. utmost fungi use nitrate,
which is reduced first to nitrite (with the aid of nitrate reductase) and also to ammonia. Nonfungal
organisms, including bacteria, synthesize the amino acid lysine by the meso-- diaminopimelic acid
pathway (DAP pathway), whereas fungi synthesize lysine by only the L- α- adipic acid pathway
(AAA pathway). Use of the DAP pathway is one of the reasons microorganisms preliminarily
considered to be fungi, similar as the myxomycetes, oomycetes, and hypothyroid, are no longer
classified as fungi. The DAP and AAA biosynthetic pathways for lysine conflation represent
dichotomous elaboration.
Fig.2: Colonies of Penicillium and Aspergillus

173
Cell Wall
The rigid cell wall of fungi is a stratified structure conforming of chitinous microfibrils bedded in a
matrix of small polysaccharides, proteins, lipids, inorganic mariners, and colors that provides
cadaverous support and shape to the enclosed protoplast. Chitin is a (β1 – 4)- linked polymer of N-
acetyl- D- glucosamine (GlcNAc). It is produced in the cytosol by the transfer of GlcNAc from
uridine diphosphate GlcNAc into chains of chitin-by-chitin synthetase, which is located in the
cytosol in organelles called chitosomes. The chitin microfibrils are transported to the plasmalemma
and latterly integrated into the new cell wall. The major polysaccharides of the cell wall matrix
correspond of noncellulosic glucans similar as glycogen- suchlike composites, mannans (polymers of
mannose), chitosan (polymers of glucosamine), and galactans (polymers of galactose). Small
quantities of fucose, rhamnose, xylose, and uronic acids may be present. Glucan refers to a large
group of D-glucose polymers having glycosidic bonds. Of these, the most common glucans
composing the cell wall have the β- configuration. Polymers with (β1 – 3)- and (β1 – 6)- linked
glucosyl units with colorful proportions of 1 – 3 and 1 – 6 liaisons are common. undoable β- glucans
are supposedly unformed in the cell wall. In Paracoccidioides brasiliensis, the hyphal cell wall
consists of a single, 80- to 150- nm subcaste composed of chitin and β- glucan. In discrepancy, the
200- to 600- nm-thick incentive cell wall has three layers. The inner face is chitinous, containing
some α- glucan, and the external subcaste contains α- glucan. It has been suggested that the (α1 – 3)-
glucan occurs in a microfibrillar form in P brasiliensis and Histoplasma capsulatum. numerous
fungi, especially the provocations, have answerable peptidomannans as an element of their external
cell wall in a matrix of α- and β- glucans. Mannans, galactomannans, and, less constantly,
rhamnomannans are responsible for the immunologic response to the medically important
provocations and molds. Mannans are polymers of mannose or heteroglucans with α- D- mannan
chines. Structurally, mannan consists of an inner core, external chain, and base- labile
oligomannosides. The external- chain region determines its antigenic particularity. Determination of
mannan attention in serum from cases with circulated candidiasis has proven a useful individual
fashion. Cryptococcus neoformans produces a capsular polysaccharide composed of at least three
distinct polymers glucuronoxylomannan, galactoxylomannan, and mannoprotein. On the base of the
proportion of xylose and glucuronic acid remainders, the degree to which mannose has side- chain
substituents, and the chance of O- acetyl attachments of the capsular polysaccharides, isolates of C
neoformans can be separated into four antigenic groups designated A, B, C, and D. The capsule is
antiphagocytic, serves as a acridity factor, persists in body fluids, and allows the incentive to avoid
discovery by the host vulnerable system.
Fig.3: Representing Penicillium, ascomycetous fungi are of major importance in tissue culture.

174
In addition to chitin, glucan, and mannan, cell walls may contain lipid, protein, chitosan, acid
phosphatase, α- amylase, protease, melanin, and inorganic ions similar as phosphorus, calcium, and
magnesium. The external cell wall of dermatophytes contains glycopeptides that may elicit both
immediate and belated cutaneous acuity. In the incentive Candida albicans, for illustration, the cell
wall contains roughly 30 to 60 percent glucan, 25 to 50 percent mannan(mannoprotein), 1 to 2
percent chitin (located primarily at the cub scars in the parent incentive cell wall), 2 to 14 percent
lipid, and 5 to 15 percent protein. The proportions of these factors vary greatly from fungus to
fungus.
Plasma Membrane
Fungal tube membranes are analogous to mammalian tube membranes, differing in having the
nonpolar sterol ergosterol, rather than cholesterol, as the top sterol. The tube membrane regulates the
passage of accoutrements into and out of the cell by being widely passable. Membrane sterols give
structure, modulation of membrane fluidity, and conceivably control of some physiologic events.
The tube membrane contains primarily lipids and protein, along with small amounts of
carbohydrates. The major lipids are the amphipathic phospholipids and sphingolipids that form the
lipid bilayer. The hydrophilic heads are toward the face, and the hydrophobic tails are buried in the
innards of the membrane. Proteins are interspersed in the bilayer, with supplemental proteins being
weakly bound to the membrane. In discrepancy, integral proteins are tightly bound. The lipoprotein
structure of the membrane provides an effective hedge to numerous types of motes. motes cross the
membrane by either prolixity or active transport. The point of commerce for utmost antifungal agents
is the ergosterol in the membrane or its biosynthetic pathway. Polyene antifungal agents similar as
amphotericin B bind to ergosterol to form complexes that permit the rapid-fire leakage of the cellular
potassium, other ions, and small motes. The loss of potassium results in the inhibition of glycolysis
and respiration. Several antifungal agents intrude with ergosterol conflation. The first step in the
conflation of both ergosterol and cholesterol is demethylation of lanosterol. The necessary enzymes
are associated with fungal microsomes, which contain an electron transport system similar to the one
in liver microsomes. Cytochrome P450 catalyzes the 1 4- α- demethylation of lanosterol, an essential
step in the conflation of ergosterol. The imidazole and triazole antifungal agents intrude with
cytochrome P450-dependent 1 4- α- demethylase, which inhibits the conformation of ergosterol. This
results in tube membrane permeability changes and inhibition of growth. Ergosterol may also be
involved in regulating chitin conflation. Inhibition of ergosterol conflation by antifungal agents can
affect in a general activation of chitin synthetase zymogen, leading to inordinate chitin product and
abnormal growth.
Microtubules
Fungi retain microtubules composed of the protein tubulin. This protein consists of a dimer
composed of two protein subunits. Microtubules are long, concave cylinders roughly 25 nm in
periphery that do in the cytoplasm as a element of larger structures. These structures are involved in
the movement of organelles, chromosomes, capitals, and Golgi vesicles containing cell wall
precursors. Microtubules are the top factors of the spindle filaments, which help in the movement of
chromosomes during mitosis and meiosis. When cells are exposed to antimicrotubular agents, the
movement of capitals, mitochondria, vacuoles, and apical vesicles is disintegrated. Griseofulvin,
which is used to treat dermatophyte infections, binds with microtubule- associated proteins involved
in the assembly of the tubulin dimers. By snooping with tubulin polymerization, griseofulvin stops

175
mitosis at metaphase. The destruction of cytoplasmic microtubules interferes with the transport of
secretory accoutrements to the cell fringe, which may inhibit cell wall conflation. The fungal nexus is
bounded by a double nuclear envelope and contains chromatin and a nucleolus. Fungal capitals are
variable in size, shape, and number. The DNA and associated proteins do as long fibers of chromatin,
which condenses during nuclear division. The number of chromosomes varies with the particular
fungus. Within the cell, 80 to 99 percent of the inheritable material occurs in chromosomes as
chromatin, and roughly 1 to 20 percent in the mitochondria. In some isolates of Saccharomyces
cerevisiae, over to 5 percent of their DNA can be set up in nuclear plasmids. When the DNA helix
unwinds, one beachfront serves as the template for the conflation of rRNA, tRNA, and mRNA.
mRNA passes into the cytoplasm and attaches to one of the ribosomes, which are complexes of RNA
and protein that serve as spots for the conflation of protein.
Classification
In mycology, fungi are classified on the base of their capability to reproduce sexually, asexually, or
by a combination of both. Asexual reproductive structures, which are appertained to as anamorphs,
are the base for one of the sets of criteria. Because the criteria are grounded upon asexual
morphologic forms, this system does not reflect phylogenetic connections. It exists so that we can
communicate in a simple and harmonious manner by using names grounded upon analogous
morphologic structures. The alternate set of criteria is grounded upon sexual reproductive structures,
which are appertained to as teleomorphs. Ascospores, basidiospores, oospores, and zygospores, as
well as any technical structures associated with their development, are the base of the alternate set of
criteria. These criteria reflect phylogenetic connections because they are grounded upon structures
that form following meiosis. The term holomorph is used to describe the whole fungus, which
consists of its teleomorph and anamorphs.
Table-1: Showing classification of Phyla, sexual and asexual region
Classification
Phyla Sexual Asexual
Chytodiomycota Oospores Spores and conidia
Zygomycota Zygospores Spores and conidia
Ascomycota Ascospores Conidia
Basidiomycota Basidiospores Conidia
Fungi Imperfection Absent Conidia
Importance of Current Mycology

Applied mycology has traditionally concentrated on areas where fungi beget damage. largely
advanced mycological exploration has generated knowledge, abstract understanding and sapience
within fungal factory conditions, mortal mycoses, inner climates, and decays in wood, feed and food.
There's also expansive knowledge about the added value fungi contribute to beer, chuck, wine, spirit,
food preservation, and food taste. Much lower attention has been given to fungi and fungal products
for adding value for assiduity, husbandry, health, and medicinal. The last many decades, still, have
witnessed veritably intriguing developments regarding use of fungi for new processes, products and
results of significance for the world, and most importantly for increased abstract understanding of the
area Fungi at large (Hibbett et al. 2007, James et al. 2006, Schoch et al. 2006). It is now well proved,
indeed in gauged up practice, that fungi can induce palpable and substantial value through bettered

176
resource effectiveness, performing in dropped pollution and hothouse gas emigrations. similar
progress stands on the shoulders of the benefactions made by mycologists over decades and indeed
centuries to make up mycology as an independent field of wisdom. Importantly, value added use of
fungi was innovated not only on applied studies but also on introductory exploration aimed at
understanding fungal biodiversity, growth, nutrition, physiology, genetics, metabolism, and ecology.
The shift from chemical processes to natural processing, achieved by using fungal (and bacterial)
enzymes rather of chemical processes in diligence, similar as fabrics, leather, paper and pulp, has
significantly reduced negative impacts on the terrain. Use of enzymes in the food and feed assiduity,
similar as beast feed, baking, brewing, and wine and juice, has significantly bettered what we get out
of natural raw accoutrements. Microbial enzymes added to cleansers, washing laundry clean indeed
at low temperatures, has significantly reduced CO2 emigrations. The newest chapter in the artificial
biotechnology period is to substitute reactionary coffers with renewable coffers (Fig.4).
Fig.4: The value aggregate of biomass conversion at the bottom, with smallest value, is the bulk use
of biomass for combustion, making heat and electricity. Coming subcaste is using biomass for
biofuel, an important demanded renewable volition to reactionary transport energy. Further up the
biomass value aggregate, is product of technical and advanced value products (accoutrements,
chemicals, feed, food and pharma). similar products are not only substituting for fuds but also
making use of the complex structures of new biomass, making products not possible to make from
fossilized biomass. All uses except for burning, gasification., of the biomass involve use of microbial
(primarily fungal) conversion and upgrade. Courtesy of Peter Westermann.
The role of fungi and mycology in addressing major global challenges
Discovery of novel fungal merchandise for specific industrial needs, builds on simple research of
fungal biodiversity, and on the usage of the experimental molecular
mycology device container for figuring out probably fascinating genes and proteins, synthesis
pathways, and metabolites. For industrial use it is the secretome of the fungi which
is focused for each metabolite and enzyme discovery1.Most molecular research inside fungi
have targeted on what the fungi are and solely much less on what fungi do and how they have
interaction with different organisms and substrates. The molecular technology rest targeted on

177
elucidating phylogenetic relationships and species identification. Now the time is ripe for
intensifying the find out about of the secretome with regard to composition, development, and
evolution. Molecular research of the secretome (genomics and transcriptomics of secreted proteins)
will add new conceptual appreciation of the function of the secretome in substrate specialization and
organismal speciation. We will research extra about the mechanisms of secretome evolution,
going past mutation and decision
Inspiration and Learning from Nature
The want for extra sorts of enzyme blends for efficient decomposition of similarly types of
lignocellulosic biomass (not solely wheat straw and corn stover) has revitalized the complete eld
of research on fungal degradation of lignocellulose, along with ascomycetous saprophytes and
basidiomycetous white rot and brown rot fungi. This quest has additionally led to new research of
ecological habitats, the place biomass conversion takes region in nature, developed over evolutionary
time. The enzymes from the termite larvae intestine channel and from the cow rumen,
investigated by using recreation screening of cDNA meta-libraries, have been already studied a long
time in the past by means of some biotechnology companies. Description of the biomass conversion
of the ectomycorrhizal Paxillus involutus (Rineau et al. 2012), studied by using mass spectrometry
(MS) for the documentation of bioconversion and Transposon Assisted Signal Trapping (TAST) of
the cDNA library for discovery (Hamann & Lange 2006, Rineau et al. 2012), gave new insights into
the viable twin characteristic of this ectomycorrhizal fungus. A most fascinating meta-study
examined the enzymes produced and secreted in the fungus backyard of the leaf cutter ant (Grell et
al. 2013). This confirmed that Leucoagaricus gongylophorus, farmed by means of leaf cutter ants,
expressed the complete spectrum of enzymes wanted for breaking down the cellulose bres of
the inexperienced leafy biomass which the ants deliver to the fungal backyard in their nest (Fig. 2).
The ants pretreat by using chewing the biomass. The fungus expresses the wished regime of enzyme
proteins. The enzymes even continue to exist (in an intact and energetic form)
passage via the intestine channel of the ants. Redistribution of the enzymes to the newly harvested
leafy biomass is completed via the ants putting their enzyme-holding fecalia on newly harvested
leaves on the pinnacle of the fungus garden. This whole state-of-the-art mutualistic machine has been
comprehensively described (Kooij 2013). In a current evaluation of the distinguished position of
fungal enzymes for the success of the leaf cutter ants (Lange & Grell 2014), the gadget is described
from a new point of view that sees the fungal accomplice in the symbiosis no longer only as being
passively farmed however additionally as an energetic partner. In this interpretation the
Leucoagaricus fungus is an instance of a fungal adaptation course toward growing attractants to
recruit bugs to disperse the fungal spores (as, for example, this are attracted to the spore gleba of
Phallus impudence’s and for this reason lured into disseminating the fungal spores).
Conclusion
It is evident from the above descriptions that funding in strengthening mycology globally is
worthwhile. The following efforts are urgently wanted to allow this to occur:
✓ Understanding the fungal kingdom, thru phylogeny and phylogenomic:
Help for international participation and international insurance of traces used for resolving
the Fungal Tree of Life.
✓ Strengthening the mycological platform for classification, identification
and pressure collection: Guide implementation of one identifies one fungus nomenclature;

178
recollection and sequencing of fungi no longer protected in molecular database, inclusive

of neo- or epitypication the place appropriate; increase concepts and structures geared
up for shooting and categorizing sequence discoveries of novel fungal genes no
longer originating from any acknowledged species.
✓ Building an international mycology network: boost globally allotted databases,
with world enter and access: Set up a traveling professor programmed and
a younger investigator worldwide publicity programmed; set up an IMA delegate
participation programmed, permitting all areas to take part equally in IMA
ExCo conferences and different organizational IMA activities.
✓ Open get admission to mycology: Set up international access/regional collections of
bioeconomically-relevant traces and organic materials, special in
that traces and substances are accompanied via searchable statistics about uses,
recipes/protocols on secure and quickly use for development of the bioeconomy, e.g., for
upgrading biowaste to new biobased products.
✓ The function of fungi in nature, interactions, and the secret me:
Furnish international help to mining sequence
databases, quicker and greater characteristic targeted, for recognizing useful proteins and
metabolites; reinforce world discovery and stimulate collaboration between enterprise and
academia in all components of the world.
Reference
1. Kwon-chung KJ, Bennett JE: Medical Mycology. Lea and Febiger, Philadelphia, 1992.
2. McGinnis MR: Laboratory Handbook of Medical Mycology. Academic Press, San Diego,
1980.
3. McGinnis MR, Borgers M (eds): Current Topics in Medical Mycology. Springer-Verlag,
New York, 1989.
4. Murphy JW, Friedman H, Bendinelli M (eds): Fungal Infections and Immune Responses.
Plenum Press, New York, 1993.
5. San-Blas G: Paracoccidioides brasiliensis: cell wall glucans, pathogenicity, and dimorphism.
p. 235. In McGinnis MR (ed): Current Topics in Medical Mycology. Vol.1. Springer-Verlag,
New York, 1985.
6. Shepherd MG: Morphogenetic transformation of fungi. p.278. In McGinnis MR (ed): Current
Topics in Medical Mycology. Vol. 2. Springer-Verlag, New York, 1988.
7. Vanden Bossche H, Odds FC, Kerridge D (eds): Dimorphic Fungi in Biology and Medicine.
Plenum Press, New York, 1993.
8. Yoshida Y: Cytochrome P-450 of fungi: primary target for azole antifungal agents. p. 388. In
McGinnis MR (ed): Current Topics in Medical Mycology. Vol. 2. Springer-Verlag, New
York, 1988.
9. Kubicek CP, Starr TL, Glass NL (2014) Plant cell wall-degrading enzymes and their
secretion in plant-pathogenic fungi. Annual Review Phytopathology 52: 427–451.
10. Lange L, Grell MN (2014) The prominent role of fungi and fungal enzymes in the ant-fungus
biomass conversion symbiosis. Applied Microbiology and Biotechnology 98: 4839-4851.

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11. Martinez D, Challacombe J, Morgenstern I, Hibbett D, Schmoll M, et al. (2009) Genome,

transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique
mechanisms of lignocellulose conversion. Proceedings of the National Academy of Sciences,
USA 106: 1954–1959.
12. Murphy C, Powlowski J, Wu M, Butler G, Tsang A (2011) Curation of characterized
glycoside hydrolases of fungal origin. Database: bar020 doi:10.1093/database/bar020.
13. Nielsen PH, Kragelund C, Seviour R J, Nielsen JL (2009) Identity and ecophysiology of
lamentous bacteria in activated sludge. FEMS Microbiology Reviews 33: 969–998.
14. Nobre T, Eggleton P, Aanen DK (2010) Vertical transmission as the key to the
colonization of Madagascar by fungus-growing termites? Proceedings of the Royal Society of
London, Biological Sciences 277: 359–365.
15. Pitt JI, Hocking AD (2009) Fungi and Food Spoilage. 3rd edn. New York: Springer.Rineau
F, Roth D, Shah F, Smits M, Johansson T, et al. (2012) The ectomycorrhizal fungus Paxillus
involutus converts organic matter in plant litter using a trimmed brown-rot mechanism
involving Fenton chemistry. Environmental Microbiology 14: 1477–1487.
16. Schoch CL, Shoemaker RA, Seifert KA, Hambleton S, Spatafora JW, Crous PW (2006) A
multigene phylogeny of the Dothideomycetes using four nuclear loci. Mycologia 98: 1041–
1052.
17. Seifert KA, Samson RA, Chapela IH (1995) Escovopsis aspergilloides, a rediscovered
hyphomycete from leaf-cutting ant nests. Mycologia 87: 407–413.
18. Taylor JW (2006) Evolution of human-pathogenic fungi: phylogenies and species. In:
Molecular Principles of Fungal Pathogenesis (Heitman J, Filler SG, Edwards JE jr, Mitchell
AP, eds): 113-132.
19. Washington DC: American Society of Microbiology Press. Zajc J, Liu Y, Dai W, Yang Z, Hu
J, Gostinčar C, Gunde-Cimerman N (2013) Genome and transcriptome sequencing of the
halophilic fungus Wallemia ichthyophaga: haloadaptations present and absent. BMC
Genomics 14: 1–21.
***
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Chapter 17
ARBUSCULAR MYCORRHIZAL FUNGI ACT AS
NATURAL BIOFERTILIZERS IN SUSTAINABLE
AGRICULTURE
Ranjana Singh & G. S. Shailaja Sharma
A bstract
Mycorrhiza is the keystone among microorganisms that form a critical linkage between the plant root
and soil. This symbiotic association is characterized by bi-directional movement of nutrients where
carbon flows to the fungus and inorganic nutrients move to the plants. Arbuscular mycorrhizal fungi
(AMF) are soil fungi that form a symbiosis with the roots of plant. A variety of host benefits have
been attributed to mycorrhiza, most commonly increasing uptake of immobile soil nutrients,
particularly phosphorus and nitrogen. Understanding the impact of agronomic practices on these
fungal communities would help ensure that symbiosis can be harnessed and can contribute to the
success of sustainable agriculture. The co-evolution of mycorrhizae with plants represents an
important evolutionary adaptation to the terrestrial environment. As a bio-inoculum, arbuscular
mycorrhizal fungi (AMF) play a useful role in sustainable agriculture by symbiotically attaching to
many crops and fungi, which provide the plant with nutrients such as P, Zn, Cu, etc., protection from
water stress, toxic effects of heavy metals, and the infection of some soil diseases with increased
plant growth. In addition, this association favors the formation of stable soil aggregates that improve
soil structure stability, especially in granular soils, and reduces soil erosion. The production of the
mycorrhizal fungal inoculum is complex and expensive due to its absolutely biotrophic nature. AMF
spores in the soil can be collected by wet sieving and decanting, and a pure culture of mycorrhizal
fungi can be prepared by the soil method or the single-spore method. The spores are then
characterized according to their shape, size, color, and hyphal attachment and classified according to
their morphology. Each type of mycorrhizal fungus has specific cell wall characteristics that can be
used for identification. In this chapter, recent research advancement on the influence of AMF in
promoting the morphological and physiological aspects of agricultural crops is reviewed. Successful
AMF colonization could be the most organic way to significantly aid agricultural crop productivity
and discuss briefly the exploitation of arbuscular mycorrhiza in sustainable agriculture.
Keyword: Mycorrhiza, Symbiosis, Keystone, Sustainable Agriculture.
Ranjana, Singh & Shailaja Sharma

181
Department of Botany, Bangalore University, Bangalore, Karnataka, India

dr.singhranjana1978@gmail.com
I ntroduction
The 20th century witnessed an augmentation in agricultural as well as floricultural production,

mainly through the progress and use of pesticides, fertilizers containing nitrogen and phosphorus,
and developments in plant breeding and genetic skills. Agriculture is the largest industry in the
world. It employs over a billion people and generates over $1.3 trillion in food annually. Grasslands
and croplands cover about 50% of the planet's habitable surface and provide shelter and food for
many species. When managed sustainably, agricultural activities can protect and restore important
habitats, help protect watersheds, and improve soil and water quality. However, unsustainable
practices have serious impacts on people and the environment. The need for sustainable resource
management is becoming ever more pressing. Demand for agricultural products is growing rapidly as
the world population grows. Agriculture's close ties to the global economy, human societies and
biodiversity make it one of the world's most important wildlife sanctuaries. Agriculture has played a
major role in the development of different human civilizations worldwide. In fact, agricultural
practices have evolved on parallel lines with those of human civilizations and both can be regarded
as interdependent.
The economic growth of a nation is also dependent on agriculture, and it is estimated that agriculture
contributes around 40 to 60 percent of a country’s economy [54]. Agriculture can be defined as an
art of cultivating the soil to grow and harvest crops. It involves amending the soil to make it fertile
and suitable for the growth of a specific type of crop.
Prehistorically, the newly established human societies cleared forest fields to grow specific crops of
choice. After several years of repeated farming on that land, as they observed a reduction in the
yield due to the decreased soil fertility, they abandoned that field and the entire society shifted to a
different place and cleared a new patch of forest land for agriculture. During the course of
development, as human societies became civilized, they reduced nomadic and wandering lifestyles,
they learnt the art of mixed farming where they simultaneously raised stock animals along with
growing crops on fields. They realized that the animal dung could be mixed with soil to regain soil
fertility, and mastered this art of organic farming for several centuries [57]. Further, as human
societies became well established and more competitive, to fulfil the food needs of an exuberantly
growing society, they industrialized agricultural practices. Production and utilization of chemical
fertilizers to increase the vegetative growth and yield of the crops is one such outcome. Since the
trends in agricultural practices have witnessed drastic changes.
Agricultural practices in the coming decades of the 21st century will face tremendous challenges due
to a fragile economy, climate change and biodiversity degradation to produce enough healthy food to

182
meet the needs of the world population. The world's rising inorganic fertilizer prices will continue to
increase, and the demand for chemical fertilizers in conventional agriculture will continue to grow,
which directly affects the increase in production costs and its high energy intensity. Furthermore, it is
quite disappointing that the maximum yields of the main crops are decreasing relative to the ever-
increasing human population [36, 42, 112]. For about 30 years, our country has been accustomed to
traditional methods of organic farming or integrated with organic fertilizers, biological or biological
control with predators, integrated fisheries, etc. and sustainable agriculture and an ecosystem with
healthy biodiversity. Over the years, the scenario has shifted towards an agriculture dependent on
chemical products that disturb the balance of the soil, which also affects water and the environment,
with consequent health risks. Fertilizers supplement the soil's nutrient needs for optimal plant
growth. The dose of fertilizer must be adjusted to the nutrient deficiency in the soil and the needs of
the crop [86].
Most developed and developing countries today choose to produce, consume and import organic
food. Even developing countries are using organic farming for export and profit purposes. Public
awareness of the negative impacts of modern agriculture on biodiversity, food security, the
environment and the economy has increased in recent years [103]. This gives rise to the concept of
sustainable agriculture, where sufficient agricultural production is possible without harming the
environment and human health [4, 78]. Organic farming is an organic agro-systematic alternative that
maintains safe, profitable, and environmentally friendly food production. The transition from
conventional, high-input farming to organic farming should never level out crop yields overnight.
Organic fertilizer is processed and used in the presence and function of various soil microbes.
The use of arbuscular mycorrhizal fungi (AMF) as bio-inoculation could represent an effective
alternative, offer significant benefits for long-term soil fertility, plant nutrition and protection, and
have promising potency in sustainable agriculture [23, 93]. Mycorrhiza is an important link between
two kingdoms, Plantae and Fungi. Arbuscular mycorrhizae (AM), a common endotrophic symbiont,
are taxonomically and functionally diverse [58] and members of the monophyletic tribe,
Glomeromycota [88], are found in more than 90% of land plants [30]. They form two unique
structures: finely branched arbuscular hyphal tips for nutrient exchange; and balloon-shaped vesicles
for storing nutrients in host plant root cortex cells [77]. AM Fungi can also be used as a biological
control agent to protect host plants from soil-borne pathogens [98].
Mycorrhiza
The term mycorrhiza was coined by A. B. Frank, a scientist in Germany, more than 100 years ago
[44]. It literally means fungus root and describes the mutualistic association existing between a group
of soil fungi and higher plants. He recorded roots with such fungi that lacked root hair and he
proposed that these fungi took up the job of root hair; such associations between the roots or other
underground plant parts with certain soil fungi are common and they show low pathogenicity
towards the host [70].
Brundrett, in 2004, has defined the mycorrhizal association as an essential symbiotic association
(essential for either one or both the involved organisms), between a plant root of a living plant (or
any other plant organ that is in contact with soil or substrate) and a soil fungus, and shows nutrient
transfer from one to the other organism. Mycorrhizal associations with plant roots are in general non-
pathogenic and beneficial to either one or both the associated partners. Mycorrhizae differ from other
epiphytic/endophytic soil fungi by the following features [16, 14] –
1. Mycorrhizal fungal growth and host plant productivity are correlated: the host plant with
mycorrhizal association may show dominance amongst other non- associated plants in its
habitat, or may show high yield and reproductive rates. Similarly, the fungal partner may also
show high growth and sporulation rates after being associated with higher plant roots in

183
comparison with that of unassociated fungi or the same species in the same habitat. The plant
and the fungal partners of the mycorrhizal association may also show negative growth rates
(like low plant or fungal reproductive or vegetative growth rates) when not associated with each
other.
2. Some mycorrhizal fungi are incapable of surviving on their own as saprophytes when not
associated with plant roots. These fungi may even show specificity towards the host with which
they associate, while certain mycorrhizal fungi that express host specificity may cause
antagonism or allelopathy when they try to get associated with non-host plants.
3. The host plant and its mycorrhizal fungal partners show simultaneous and reciprocal exchange
of organic/inorganic nutrients, metabolites, and may even display a correlation between the area
of mycorrhizal association with plant roots and the nutrient exchange or accumulation rates.
The associated plant and fungal partners may even exhibit co-dispersal of their reproductive
propagules. Physiological interdependence and synchronous development of both partners are
often recorded.
Classification of Mycorrhiza
Harley in 1961 classified the mycorrhizal fungi into two categories based on their location on plant
roots - Ectotheraphic (or Ectomycorrhiza) and Endotheraphic (or Endomycorrhiza). Further
researchers have added the third category - Ectendomycorrhiza [16]. Several other workers have
separated AM fungi, mycorrhizae of ericales and mycorrhizae of orchids as three distinct categories
and have recorded their structural and functional differences [14, 70].
Ectomycorrhiza: Few members of Basidiomycetes, ascomycetes and zygomycetes fall under this
category. They colonize the intercellular spaces between the epidermal cells and form a mesh like
structure called-Hartig network through which they exchange the metabolites with the host plant.
They remain within the intercellular spaces and do not enter the host cells.
Endomycorrhiza: These fungi mostly belong to Zygomycetes. They penetrate the root cells and
hence are called endomycorrhiza.
Ectendomycorrhiza: The mycorrhizae which show both a-Hartig net-like structure (in the
intercellular spaces between epidermal cells) and penetrative intracellular hyphae are categorized
into this group.
Arbuscular Mycorrhizal (AM) Fungi: These fungal genera were initially kept under the category
endomycorrhiza. Unlike other forms of endomycorrhiza, these fungi can form special structures
called arbuscules and vesicles and hence they are grouped into a separate class. The arbuscules are
finger-like projections formed within cells as a result of successive divisions of fungal cells and they
are involved in the function of metabolite exchange between plant and fungal cells. Vesicles are
formed by the swelling of the tip of the fungal cell and have the appearance of a sack or bag like
structure. They function as storage and resting organs; accumulation of nutrients and lipid droplets
can be seen in such vesicles. Few endomycorrhiza are known to produce only arbuscules and few
others are known to have both vesicles and arbuscules.
Mycorrhizae of Ericales: They colonize the last part of the root branches called “hair roots”, which
are very thin and lack root hairs; they are further divided into two sections - Arbutoid and Ericoid
mycorrhizae. Both these categories penetrate the host cells and form hyphal loops. However, the
Arbutoid mycorrhizae form a Hartig network on the surface of the roots and the Ericoid type lacks
this structure, while these two sections of mycorrhizae also differ in their host specificity.
Mycorrhizae of Orchids: These mycorrhizae belong to Basidiomycetes and associate specifically
with the members of Orchidaceae; they usually do not penetrate into deeper regions of host tissue,
their hyphae penetrate the cells, loops and the coils. These special structures are called Peletons.

184
These are usually degraded by the metabolic reactions occurring in the host cell. In some cases, at the
end of the life cycle, peletons collapse and aggregate to form fungal tissue without any specific
shape.
Establishment of the Mycorrhizal Symbiosis
AM fungi are considered to be obligate biotrophs as they are unable to grow and reproduce in
regions of the soil where the host plant roots are absent [68]. Mycorrhizae refers to an association or
symbiosis between plants and fungi that colonize the cortical tissue of roots during periods of active
growth [91]. AMF are obligate biotrophic symbionts with a lifecycle divided into two distinct stages.
On the one hand, the resting and reproductive stages (spores, sporocarps and possibly also vesicles)
are independent of the plant. On the other hand, vegetative stages are involved in complex
interaction, colonization and nutrient exchange. The stages are represented by the development of
external hyphae in soil and hyphae, arbuscles and vesicles within the root (Fig.1). Mycorrhizae are
involved in associations between fungi of different taxonomic groups with most plant species and
form mutual symbiosis, although plants of some families like Brassicaceae, Cyperaceae, Juncaceae,
Urticaceae and Chenopodiaceae inhibit mycorrhizal colonization [61, 80]. Variations in plant root
colonization have divided mycorrhizae into two major categories: endomycorrhizae (phylum
Glomeromycota) and ectomycorrhizae (phylum: Basidiomycota, Ascomycota, and Mucoromycota).
The symbiotic relationship results in enhanced uptake of nutrients (e.g., phosphates and nitrates)
from the soil by partner plants, and in turn allows AMF to absorb photosynthetically fixed carbon
sources (e.g., sugars) necessary for their survival (Fig.2) and reproduction [93, 97]. This lineage of
fungi is known to affect the functioning and biodiversity of entire ecosystems, creating vast
underground networks of hyphae and spores connecting a number of individuals and unrelated plant
species. These networks also serve as important reservoirs of atmospheric carbon dioxide and are
important underground "food highways" that benefit entire plant and microbial communities. Indeed,
AMF spores and hyphae are also a valuable food source for many soil microorganisms (e.g., bacteria,
other fungi and nematodes) and because of their beneficial effects on terrestrial ecosystems, AMFs
are used extensively in organic farming and nurseries to enhance the growth of economically
important species. This symbiosis can be used as a tool to predict the sustainability of production
systems. Mycorrhizal fungi have been shown to mitigate the salinity, drought and toxicity of HM,
thereby enhancing host plant growth. To assess the potential role of AM fungi in improving soil
fertility, research projects on exotic and native species of AM fungi (AMF) should be conducted
using the best approaches in sustainable agricultural systems.
Fig.1. Characteristic structures of AMF inside Fig.2. Physiological relationship between plants
and outside of plant roots. Modified figure by and mycorrhiza.
Mark Kelly, Heartspring.net © 2012.

185
In terms of abundance, arbuscular mycorrhizal fungi colonize a large number of plant species (80-
90%) and only 10% of plants form ectomycorrhizae. Both species play an important role in
sustainable agriculture and forestry [72]. The symbiotic interaction occurs through a series of
chemical signals exchanged between the plant and the fungus, and the plant's basic genetic
determinants identify mycorrhiza as a beneficial organism [15, 100]. In the pre-symbiotic phase,
plant roots produce strigolactones, which lead to the germination of fungal spores [12], while
mycorrhizae produce "Myc factors" (lipo-chitooligosaccharides and chitooligosaccharides) [55], thus
initiating a symbiosis. Subsequently, internalization of arbuscular mycorrhizae occurs through the
formation of hypophodia, followed by arbuscules and vesicles. In parallel, ectomycorrhizae form a
coat and the Hartig net intercellularly connects to the host roots [46, 80]. The best-known and most
visible ectomycorrhizae form symbiotic relationships with temperate forest plant species (mainly
trees) and their endomycorrhizae with crops. The fungus plays its important role either by modifying
plant metabolism or by increasing the uptake area of plant roots through the development of fine
hyphae of the fungus when the plant is under normal or stressful environmental conditions [72].
Fig.3. Positive effects of arbuscular mycorrhizal (AM) colonization.
Maintenance of soil structure by AMF

AMFs are widespread among plants and produce different colonization (Fig.3) patterns and show
remarkable diversity in terms of host plants, climate, latitude, soil C:N:P ratio, soil types, soil acidity,

186
soil, rhizosphere and aerial organisms [37, 90]. Host specificity and colonization rate variability can
be identified within genotypes of the same species [107]. For example, variation in AMF
colonization has been identified in the roots of different sexes of dioecious plants [96]. Also, in the
photosynthetic behavior of plants (e.g.C3 and C4 plants), the contribution of AMF to drought
resistance is different. Due to the specific physiological mechanisms of C4 plants, they are better
adapted to drought conditions than C3 plants and are less susceptible to severe damage. Therefore, C3
plants utilize the AMF symbiosis more when the plant is in drought [96]. Therefore, in a no-till crop
rotation system, the various underground AMF compounds add value to the excavation by
minimizing accumulation of pest populations, improving soil health, creating a common mycorrhizal
network, etc. [107].
Variables such as pH, soil N:P ratio, annual temperature and growing season length vary with
latitude. Carrenho et al. [22] found that clay soil causes less AMF colonization than sandy soil in
corn, peanut, and sorghum due to higher nutrient concentrations, higher suberin deposition on plant
epidermal cells, and less space between soil particles in clay soil that reduces AMF colonization in
plants. Increasing temperatures have been shown to reduce AMF colonization and have a positive
impact on biomass content in montane grasslands with Mediterranean temperature gradients in the
Pacific Northwest [105]. Soil structure plays an important role in plant root penetration, soil-plant
water relations, flow of nutrients, soil microflora etc. Well - aggregated soil is known to support
plant growth. Fine soil and clay particles are aggregated together by the bacterial cells. These
microstructures are bound together to form micro aggregates by AMF and saprophytic fungi.
Furthermore, they are combined together with plant debris and fungal fragments to form larger
microaggregates. These are further glued together to form macro aggregates by the clay and
polysaccharides derived from plants, bacteria and fungi. It is recorded that up to 50% of the total
soil biomass is made up of AMF [75]. Thus, AMF has a major role in contributing to the temporary
binding agents like polysaccharides and thereby stabilizes soil structure. They also increase the fiber
content of the soil, making it more porous [69].
Soil quality depends on the physical and chemical properties as well as the diversity, distribution and
activity of soil flora and fauna [18, 52, 81]. Soil health refers to the soil's ability to function as a
living ecosystem. Soil health depends on soil quality and therefore affects human health by
improving crop quality. As a useful biological tool, AMF maintains soil quality and health by
influencing three main factors: soil structure, plant physiology, and ecological interactions [34, 43].
Maintaining soil structure is one of the most important processes in agriculture. AMFs form water-
stable soil microaggregates by secreting a hydrophobic glycoprotein, glomalin, which serves as a
long-term binder [60]. AMF deposits glomalin between the outer walls of hyphae and adherent soil
particles to form microaggregates and other macroaggregates, thus forming the backbone of soil
aggregation [52, 65]. This helps increase water holding capacity, airflow, soil volume and organic
matter content. On agricultural land, less disturbance and unploughed fields are required to get more
benefit from tending to these aggregates [13].
Glomalin is a hydrophobic glycoprotein and a mucilaginous substance produced by AMF, which
helps in binding of the soil particles. This further helps in water retention in the soil pores and
increases hydraulic conductivity in the soil. Up to 5% of the soil carbon content is made of Glomalin
and has a retention time of 6 to 42 years. They further attract other soil microbial communities [38,
104]. AMF maintain both positive and negative ecological interactions in the rhizosphere. AMF
positively interacts with beneficial microorganisms including phosphate-lysing bacteria (PSB),

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nitrogen-fixing bacteria and plant growth-promoting rhizobacteria (PGPR). Mycorrhiza acts

negatively to suppress both soil pathogens and pests [47, 73]. AMF identifies pathogens using chitin-
like compounds, e.g., chito-oligosaccharides (CO) and lipo-chito-oligosaccharides (LCO), as well as
plants producing specific microbial molecular patterns in their receptor complexes. First, all chitin-
related molecules are identified by the LysM-RLK complex. By combining this complex with other
proteins, identification of microbes is achieved.
Improvement of mineral nutrition in Agriculture land by AMF: as natural
biofertilizers
Nitrogen (N), Phosphorous (P) and Potassium (K) are the important major nutrients that affect plant
growth. AMF is known to enhance the phosphorous and nitrogen uptake by host plants. One of the
main reasons is that the extraradicular hyphae of AMF can grow 100 times longer than the host roots
and reach distant soil, from where the hyphal cells absorb the nutrients and transport them to the
plant's host cells. The second reason is that AMF enhances colonization of saprophytic microbes that
can decompose large complex organic molecules (in which the N and P may be present in complex
states which are non-absorbable by plants) and convert them to simpler nitrates and phosphates that
can be easily absorbed and assimilated by host plants [38]. In general, AMF affects nutrient uptake in
the soil and improves plant growth, biomass and ultimately productivity [85]. In mycorrhizal plants,
the root architecture changes with the formation of recognizable lateral root branches, root hairs and
a characteristic tip [35]. Changes in radical cell division and differentiation are altered by molecular
mechanisms.
Plants without mycorrhiza take up nutrients directly from the soil via nutrient transporters located in
the root hairs and epidermis [17]. In AMF symbiosis, nutrient uptake occurs first by transporters
from extra-sporular to intra-root mycelium and is then transported to the plant cell by mycorrhizal-
induced transporters located in the peri-arbuscular membrane [76]. The efficiency of nutrient uptake
is related to the degree of distribution and colonization of the hyphae [64]. Plants produce different
types of plant nutrient transporters for direct pathways and mycorrhizal-mediated nutrient uptake
pathways [50]. For example, rice plants use OsPT2 and OsPT6 for direct pathways and OsPT11 for
mycorrhizal-mediated pathways. Once AMF colonizes a plant, it downregulates a gene encoding
plant nutrient transporters involved in the direct nutrient uptake pathway. However, nutrient
transporters located in mycorrhizal membranes are also species-specific; for example, Glomus
versiforme produces GvPT and G. intraradices produces GiPT as Pi transporters [87, 94]. Further
when the plants are inoculated by AMF in combination with phosphate solubilizing bacteria like
Bascillus circulans or saprophytic fungi like Penicillium thomii a synergistic effect is recorded by
several researchers [19, 79]. It is also recorded that legumes inoculated with AMF show increased
nitrogen fixation and phosphorous uptake. It is speculated that the AMF release high amounts of
carbon that help in higher interaction with lage populations of nitrogen fixing bacteria and hence
indirectly increases nitrogen uptake by plants [38].
During phosphorus transport, Pi is adsorbed as negatively charged polyphosphate granules by the
phosphate importers found in the extraradical mycelium ERM [56]. During nitrogen uptake, nitrogen
importers take up nitrogen as NH4+ and NO3− and then convert it to glutamine, producing positively
charged arginine [41]. The negatively charged polyphosphate granules and positively charged
arginine then form a complex and are transported to the intraradicular mycelium (IRM). In IRM,
arginine is broken down into urea and ornithine. The hydrolysis products of ammonia and urea are

188
then transported to the plant cells via a symbiotic interface. Polyphosphate granules are also
transported to the IRM with the help of arginine, releasing more Pi at the symbiotic interface. The
molecular mechanisms involved in some of these events are still poorly understood [99]. Researchers
showed that AMF symbiosis in a field bean/wheat cover crop system increased root length, density
and biomass in an all-wheat system and increased uptake of P and micronutrients such as Fe and Zn.
Interestingly, mixing wheat with broad beans increased P uptake by the wheat plant. MACs have no
effect on P uptake by broad beans (faba beans), neither in a pure system nor in a catch crop system
[24].
Role of AMF in stress tolerance
AMFs respond to both biotic and abiotic stresses that plants experience. Plant pathogens act as biotic
stress. Abiotic stresses such as drought, salinity, flooding, heavy metals and extreme temperatures
can affect plant physiology and ultimately productivity [74]. Due to these conditions, plant
communities and AMF undergo changes in their composition [108]. As both organisms are exposed
to these stresses independently, the frequency and diversity of AMF is decreasing. Changing their
diversity can result in more resilient species communities, and AMF provides a feedback effect to
restore species diversity and plant productivity [53].
Exposure of plants to a combination of drought and salinity leads to increased production of reactive
oxygen species that can be very harmful to plants [10]. Reactive oxygen species (ROS)
detoxification is performed by enzymes that typically include superoxide dismutase (SOD), catalase
(CAT), peroxidase (POD), and glutathione reductase (GR) [3]. Furthermore, the combined
application of drought and salinity on tomato plants inoculated with Scolecobasidium constrictum
showed improved biomass production, leaf water ratio, stomatal conductance and Fv/Fm compared
to uninoculated plants [31]. Therefore, AMFs are crucial to improving plant growth and yield under
stressed conditions [1]. Very few research reports are available in the literature showing the role of
AMF in mitigating the combined effects of two or more exposures. AMF symbiosis protects plants
from various abiotic stresses through various processes such as photosynthetic rate enhancement,
mineral uptake and accumulation, osmoprotectant accumulation, enzyme activity upregulation,
antioxidant activity, and rhizosphere ecosystem modification [7, 20, 109].
Several studies have shown an improvement in the nutritional status of AMF plants under conditions
of osmotic stress [5, 59] after irrigation or salt starvation. Similarities between tolerance mechanisms
may emerge in response to AMF-mediated combined adaptations to stress. It is hypothesized that
AMF-induced changes in phytohormone profile, mineral uptake and assimilation, accumulation of
compatible osmolytes and secondary metabolites, and upregulation of the antioxidant system are
common mechanisms induced during various stresses. However, specific mechanisms such as
compartmentalization and sequestration of toxic ions, phytochelatin production, and protein
expression can be specific and show significant changes depending on the type of stress and the
AMF species involved. Changes in root properties, such as hydraulic conductivity, can significantly
improve tolerance to osmotic stress. Zhang et al. (2018b) [111] showed that AMF protects castor
beans from salt stress by altering the gas exchange properties and the concentration of some key
metabolites. These properties of AMF can improve the nutraceutical quality of crops and can be of
great agronomic importance for the production and management of several potential crops. However,
more research is needed to discover the role of AMF in combating the effects of combined stressors.

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Increased salinity decreases water uptake by plants due to osmotic changes. In salt-stressed plants,
AMF employs multiple morphological, biochemical, and physiological strategies to overcome this
condition through increased water uptake. Morphologically, AMF increases water and nutrient
uptake by increasing root volume, by increasing root length, projected area and surface area [33,
106]. Modification of the biochemical pathways of plants therefore leads to modifications of their
physiology. To balance the concentrations of Na+ and Cl- ions, AMF facilitate the uptake of more
nutrient ions (NO3-, PO43-, Ca2+ and Mg2+) from the soil solution into plants, thereby increasing plant
growth and plant stress productivity and maintaining salinity. Meanwhile AMFs induce the
production of osmoregulators (proline, amides, glycine betaines, polyamines, sugars, and mannitol)
[108] and balance ion homeostasis [33, 106] to maintain water levels in the plant. Upretti et al. [27,
95] showed that AMF maintains salt stress in grapevine plants (Vitis vinifera L.) by inducing
morphophysiological responses including K+: Na+ ratio, phosphorus, spermidine, polyamine-
spermine and abscisic acid accumulation.
Pathogen biocontrol by AMF
A large number of pathogens, including aerial and soil organisms, cause biotic stress that impairs
plant vigor and functionality, resulting in significant crop losses in cultivated fields. Like other
beneficial microorganisms, mycorrhizae have the ability to biologically control pathogens and serve
as a pioneering system in pathogen resistance. Studies have shown that the AMF can control several
diseases caused by pathogenic fungi, bacteria and nematodes like Fusarium root rot of French beans
and Asparagus, Verticillium wilt of solanaceae members like brinjal and tomato, root and stem rot of
Vigna radiata caused by Rhizoctonia, blackleg disease of potato caused by Pectobacterium, bacterial
wilt caused by Ralstonia spp. in tomatoes and potatoes, various nematodal diseases in citrus, peach,
kidney beans, soayabeans, tomatoes, cucumber etc., [2, 6, 26, 40, 92, 102].
AMF identifies pathogens using chitin-like compounds such as chito-oligosaccharides (CO) and lipo-
chito-oligosaccharides (LCO). Plants also produce specific microbial molecular patterns on their
receptor complexes. First, all chitin-related molecules are identified by the LysM-RLK complex. By
combining this complex with other proteins, the identification of microbes is achieved. In addition,
mycorrhiza confers resistance to plant pathogens through direct and indirect mechanisms. AMF can
indirectly induce induced systemic resistance (ISR) in plants. In this way, plants alter gene
expression levels, lignification, and hormone levels [84], thus increasing resistance to pathogens.
Rice plants inoculated with Rhizophagus intraradices increased pathogen resistance by regulating
host plant gene expression and inducing ISR. In addition, signal transduction genes and leaf calcium-
mediated signaling genes are also upregulated in the absence of a pathogen [21].
As direct mechanisms, AMFs are involved in plant nutrient improvement, integration with other
beneficial organisms, direct competition, morphological, biochemical and physiological changes in
plants to control biological pathogens. By creating competition for space, sites of infection, and
nutrients for the pathogen, as well as through photosynthesis, AMF controls the invasion of plant
pathogens [43]. For example, AMF competed with the major plant pathogen nematode Meloidogyne
incognita to colonize the root nodules of the plant Prunus perscisa and inhibit gall formation [25].
Several different mechanisms have been proposed by different researchers by which the AMF
inhibits plant disease. (1) Colonization and competition. (2) Induction of systemic resistance by
induction of specific secondary metabolite production. (3) Alteration or enhancement of root
exudation and indirectly change the composition of microflora in the mycorrhizosphere. (4) AMF

190
interacts with other beneficial plant microbes and enhances the resistance against plant diseases. (5)
Anatomical changes in the host root.
Role of AMF in drought resistance
Drought is a condition in which plants do not have water to carry out their physiological functions.
Environmental conditions are also known as water stress (Subramanian and Charest 1998).
Fluctuating transpiration rates produce reactive oxygen species (ROS) and thus accelerate oxidative
stress in plants. Mycorrhiza can promote plant growth and development by improving the root
system and thickness, plant biomass, as well as nutrient uptake and transport under drought
conditions [29]. Mycelial root culture facilitates the synthesis of denser mycelium networks and
secretion of glumaline which increases water and nutrient uptake, thereby improving soil quality
[39].
AMF symbiosis affects many biochemical and physiological processes, such as (1) enhanced osmotic
regulation, (2) enhanced gas exchange, (3) uptake and transport of water and nutrients, and (4) better
protection against oxidative stress [67]. A study in Zea mays infested with mycorrhiza Glomus
intraradices reported the expression of two aquaporin genes (Gint AQPF1 and Gint AQPF2) in
water-pressurized root cortex cells containing arbuscules [71, 62, 63]. AMF-mediated enhancement
of drought resistance demonstrated by (Li et al. 2019) in C3 plants (Leymus chinensis) and C4 plants
(Hemarthria altissima) was observed due to the altered expression of the antioxidant enzyme.
Role of AMF in Sustainable Agriculture
The term "sustainability" is derived from the Latin word "sustinere" which means "performance or
long-term support" or "to sustain". Therefore, sustainable agriculture can be considered as
agricultural practices that can sustain soil productivity for a long time without affecting the quality
and structural integrity of the soil. Sustainable farming practices include the following: (1) Inculcate
methods that can increase field efficiency resulting in less requirement for pesticides, fertilizers, etc.,
(2) Use of methods that are less damaging to the soil and environment, such as the use of bio-
fertilizers, biological pest control methods, etc. (3) Rehabilitation of agricultural land by natural
methods such as polyculture, crop rotation, etc.(4) Promote organic farming at the market level by
bringing bio-fertilizers to market at a low cost. The population, composition, and diversity of soil
microbial communities are influenced by plant communities, soil texture and type, temperature,
seasonal variation, soil pH, and soil moisture. Among the diversity of soil microbiota, beneficial
plant bacteria such as root nodule bacteria, phosphate and potassium solubilizing bacteria,
saprophytic fungi and mycorrhizal fungi play a major role in maintaining soil fertility. They play an
important role in nutrient absorption, making efficient use of the water available through plant roots.
They naturally alter the microbial community in the rhizosphere and suppress the growth of plant
pathogens, helping to manage abiotic and biotic stress by activating the production of
phytohormones by plants, and also act as biological control agents against plant pests.
AMF absorbs nutrients and water as well as plays an important role in improving soil structure and
quality, as the external mycelium network promotes soil agglomeration by how to create skeleton
structure in the fungal rhizosphere. CMA improves soil structure by releasing various protein and
non-protein organic compounds; glomalin protein was most effective for binding soil particles and
these aggregates remained stable six months after the disappearance of the network. Arbuscular
mycorrhization improved the organic matter content and water holding capacity of the soil [11, 110],
which helps to maintain soil ecosystem conservation. Expanded mycelium plays an important role in
overcoming water shortage in dry soil and reducing evaporation [51]. AM symbiosis is a potential
component of sustainable agricultural systems as it has been found to have beneficial effects on host
plant nutrition, mineral cycling and growth [93].

191
Symbiosis also increases chlorophyll, carotenoids, phenols, etc., early chlorophyll and growth
improvements maintain plant vigor and reproductive health and increase yields. Plant growth and
productivity have been found to be improved with the use of AM inoculum [32]. Several recent
studies with various crops such as tomatoes, rice, wheat, corn, yams, potatoes, etc. have shown
positive effects on plant growth and productivity [48, 49, 66, 82]. In addition, it has been reported
that by AMF colonization, the food quality of the crop in terms of antioxidants, flavonoids, vitamin
C, etc. enhances [45, 66]. AM can be used as a potential change to improve soil fertility, crop
productivity and quality, and revitalize agroecosystems [25]; by harnessing the beneficial effects of
AM-inoculated plants, enhancing plant growth and the quality of their host products, it can be
integrated into sustainable agricultural systems [9]; they also increase nodulation in legumes and also
free nitrogen- fixation [101]. AMF can be used as bio-fertilizer and replaces the need for chemical
fertilizer in agricultural production. AM plants produce phytochemicals such as carotenoids,
flavonoids, etc. that reduce oxidative damage, which benefits human health [83].
Conclusion
Agriculture is a major economic sector that helps in the development of a nation. However, chemical
farming methods involving the usage of chemical fertilizers, insecticides, pesticides are not
environmentally friendly and can render the agricultural land barren within a few years use, not
sustainable. Organic or bio-farming is the solution to attain agricultural sustainability. These
methods encompass the usage of naturally formed organic manure or plant beneficial organisms that
can act as bio-used fertilizers and bio-pesticides. AMF plays an important role in plant vigor,
growth, productivity and ultimately yield by mitigating all stresses and other ecosystem components
that plants are exposed to. As an effective bio-inoculant in sustainable agriculture, AMF ensures
greater food production while preserving the ecosystem without affecting the environmental balance.
AMF also interacts with other plant beneficial microorganisms and synergistically increases the
beneficial effects.
Furthermore, AMF can act as soil conditioners by helping in structuring the soil maintenance, which
is necessary for root penetration and development, flow of nutrients from soil to plant roots and
maintaining soil, water and plant root consortium. Since AMF has multiple beneficial effects, it
forms the best candidate to be used as an all-rounder or all-in-one biofertilizer. Studies on AMF,
AMF-PBM associations can enhance specific secondary metabolite production that can increase the
nutritional and organoleptic values, flavor, odor or color of the crop. The behavior of mycorrhizae is
highly dependent on ecosystem fluctuations. The use of AMF on agricultural land leads to a
diversification of its occurrence, colonization and functionality. In order to optimize the productivity
of AMF application in sustainable agriculture, it is much more important to produce appropriate
fungal inoculants by examining all field parameters and selecting the plants with the highest AMF
colonization potential, thus increasing the sustainability performance of current cropping systems.
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Chapter 18
PREDACEOUS FUNGI: AN OVERVIEW
Dr. Kirankumar Khandare
A bstract
Predaceous fungi develop an extensive hyphal system in the substratum; the hyphae produce trapping
devices, either nets or constricting rings or adhesive pegs which are used to attract and capture
nematodes and rotifers. Some preliminary evidence indicates that several species of predatory fungi
capture both bdelloid and loricate rotifers. The first predaceous fungus to be recognised was
Arthrobotrys oligospora Fresenius (1852) but honour goes to Lohde (1974) of being the first to
record a fungus Harposporium anguillulae attacking eelworms as internal parasite. Predacious fungi
form specialized hyphae structures to trap nematodes and other microscopic animals. Among the six
kinds of trapping devices, the constricting ring is the only one that actively captures nematodes.
Keywords: Predaceous Fungi, Hyphae, Nematodes, Rotifers.
Associate Professor, Department of Botany, Maratha Vidya Prasarak Samaj’s S.S.S.M Arts,
Science and Commerce College Saikheda, Nashik-42200, Maharashtra, India
drkhandarekr@gmail.com

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I ntroduction
Predaceous fungi are fungi that derive some or most of their nutrients from trapping and eating
microscopic or other minute animals. More than 200 species have been described, belonging to the
phyla Ascomycota, Mucoromycotina, and Basidiomycota. Predacious fungi form specialized hyphae
structures to trap nematodes and other microscopic animals. Among the six kinds of trapping
devices, the constricting ring is the only one that actively captures nematodes. When a nematode
enters the aperture of the ring, which is formed by three cells, the cells rapidly triple their volume,
close the aperture and hold the nematode in place. Hyphae then penetrate and consume the
nematode.
Predaceous fungi develop an extensive hyphal system in the substratum; the hyphae produce trapping
devices, either nets or constricting rings or adhesive pegs which are used to attract and capture
nematodes and rotifers. Some preliminary evidence indicates that several species of predatory fungi
capture both bdelloid and loricate rotifers. The procedures used to loor adhesive pegs cate and
recover fungal predators are similar to those used for endoparasitic species. If a predator is present, it
will capture rotifers and anchor itself to the plate by hyphal growth into the agar. Plates that have
been used to collect endoparasites can be washed off under running tap water to remove loose debris
and then can be inspected under a dissecting microscope for clusters of rotifer bodies. The clusters
indicate zones where predation has occurred. Alternatively, debris from ponds, ditches, puddles, and
so forth can be transferred to stackable, nested glass dishes and examined periodically under a
dissecting scope for evidence of rotifer predators.
Fig. Nematode tapering fungi.
The predaceous fungi can be classified for convenience as either predatory or endo-parasitic. The
predator group produces an extensive hyphal system in the environment and at intervals along its
length. trapping devices are produced to catch and hold nematodes or prey alive. The captured
victim is then penetrated and its entire body Contents are rapidly consumed. In case of endoparasites
no extensive hyphal development outside the body of the host and the fungus exists in the
environment as spores. They lie around the substrate until they come in contact of a prey and adhere

201
to the body wall or are ingested. In both cases spore germinates, penetrates the body cavity and
consumes the victim. The hyphal development of the fungus is within the body of the prey and only
reproductive hyphae break out through predatory and endoparasitic fungi is not clear cut. A few of
endoparasites produce flagellate zoospores which can track down the victims by a chemical gradient
of the body excretions. On reachino prey, the zoospore encysts, penetrates the cuticle and kills the
The difficulty victim. in distinguishing the two groups arises in some cases such as in the genus
Nematoctonus, in which some species are classed as predators and others as endoparasites. It is often
difficult to distinguish between the two groups and some workers prefer to regard all nematode.
destroying fungi as predatory. However, Barron (1977) has divided the nematode-destroying fungi
into two groups, "predatory' and 'endoparasitic' on the basis if they produce external mycelium or
not.
The first predaceous fungus to be recognised was Arthrobotrys oligospora Fresenius (1852) but
honour goes to Lohde (1974) of being the first to record a fungus Harposporium anguillulae
attacking eelworms as internal parasite. They remain in ignorance of the remarkable fact that besides
being able to live as saprophytes they were prone to supplement their diet by attacking eelworms.
The occurrence of predaceous fungi has been investigated by many workers all over the world. They
occur in small proportions of partially decomposed organic matter from moist partially decaved
wood, that is in contact with the soil, freshly collected roots, decomposed leaves as grasses, mosses,
in terrestrial habitats, but their occurance mainly distributed in the surface soil layer. Apart from free
living Protozoa, the dominant soil animals are either nematodes or arthropods. In certain soils,
however, earthworms and enchytraeids constitute a high proportion of the biomass.
In considering the microflora of the soil, one can make a preliminary grouping into, those which
carry out most of their life in soil, those which primarily attack living plant or animal material but
continue growing there at least for a time and lastly those which are there by accident, usually in the
form of a resting propagule which may remain dormant for varying lengths of time. Since they are
different ecological groups of organisms, it is difficult to assign the accurate meaning of a 'soil
organism'. Nematophagous fungi (nematode-destroying), which capture and consume nematodes
leading to their complete destruction, are one of the most interesting groups to be found in soil. The
main attraction of science lies in the promise of reward with the most interesting and unexpected
discoveries.
Nematode constitute one of the largest and most ubiquitous group of the animal kingdom. In terms of
zoologists, they are members of a phylum Nematode often referred to as eelworms or roundworms
and in terms of plant pathologists, as pest causing diseases of several economically important crop
plants. Comparatively small number of plant parasitic species and many of the parasites of animals
spend much of their time in soil. As seen under the microscope they look like eels, moving slowly or
rapidly with vigorous thrashing movements of their body, rather like a heraldic flash of lightening.
Their bodies are elongate, non-segmented, cylindrical, usually tapering at both ends. They live either
as free (saprophytes) in soil or as parasites in plants.
Parasitism on animals, a second direction of adaptation, emerged parallel with the main route of
fungal evolution. Heterotrophic saprophytes, adapted to parasitism on plant debris, be probably
considered as primary forms, from which the semi-parasites and obligatory parasites on animals
developed. However, it seems most probable that the ability to parasitize animals as well as plants
developed independently in various groups of fungi at different times. Naumov (1939) considered
following basic features of phylogenetic recentness in fungi parasitizing animals: (1) the ability to

202
lead a Predatism-a new, unusual or a very rare mode of nutrition has emerged in certain groups of
Zygomycetes and Deuteromycetes is an exceptionally interesting example of physiological and
morphological variability in fungi. It may be considered as lateral branch in the evolution of fungi
adaptation to parasitism in animals. However, adaptation in predatism should have proceeded in a
different way from the evolution towards parasitism on animals. The transition to predatism required
the inherernt appearance in fungi of relatively complex organ for capturing and killing invertebrate
animals. For the over-whelming majority of predaceous fungi, predatism is only a supplementary
mode of nutrition and is compatible with saprophytic existence. Moreover, as saprophytes, these
fungi grow equally well on organic matter of animal and plant origin. AIl the available facts lead us
to believe that predatism in fungi is an independent branch of evolution not linked to phenomenon of
parasitism on animals. There is no clear indication as to the reason for the evolution of the nematode
trapping habit although Cooke (1962, 1964) has suggested that this is a means of escaping
competition for available substances during the decomposition of organic material in the soil.
Predaceous fungi once established have an extensive hyphal development in the substratum. The
amount of growth will depend on the number and frequency of successful captures. Hyphae are
usually sparse, radiate out form the original locus of primary capture with each hypha producing
trapping devices at intervals along its length. Organs of capture of predatory fungi have been
described in detail by Drechsler (1934, 1941) and have been reviewed by Duddington (1962).
Nematodes are captured by adhesive hyphae, adhesive branches, adhesive nets, adhesive knobs, non-
constricting rings and constricting rings.
Cooke (1977) in his book on the biology of symbiotic fungi states "the nematode-trapping fungi have
been known for a considerable period of time but the basis for this habit is not understood. He drew
attention to our lack of understanding of the nematophagous habit and suggested that the great
majority are not obligately predaceous and nematode-destroying fungi are in a simple labile
equilibrium then predaceous activity would be expected to continue as long as the soil contained a
high proportion of nematodes. He observed that these fungi were able to trap nematodes only for a
relatively short period after the onset of decomposition of a soil amendment. He noted that increasing
the amendments above a certain level resulted in a decrease in predaceous activity despite an
increase in nematode population. Based on experimental results, he concluded that nematodes were
an alternative rather a sole nutrient source for these fungi. He also suggested that nematode-trapping
fungi were predaceously active only during competition and further, that the predaceous habit was
adopted to ease or escape competetion for available substrates during decomposition of organic
matter in soil. He considered that in soil there was an intense competition for the carbon energy
sources but that the predaceous fungi had the edge because they could utilize nematodes as an
additional food source and thus evade some of the stress in the competition for food. He also
suggested that the energy stress in the competition for food. He also suggested that the energy from
nematodes could be channeled into obtaining other nutrients from soil which, when depleted,
resulted in a decline in predatory activity. An alternative explanation, however, might consider
nitrogen, not fixed carbon, as the limiting nutrient. Thus, the ability to trap nematodes might allow
the trapping fungi to utilize the carbon sources available more efficiently. Carbohydrate nutrition of
some nematode trapping fungi was reported by Satchuthananthavale and Cooke (1967). had been
suggested by Cooke previously that during the decomposition of organic matter in the soil these
fungi are capable of trapping nematodes only while suitable carbon sources are available to provide

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energy for growth. They were of the view that the loss of competitive ability by these fungi might be
correlated with a loss of nutritional versatility with respect to carbohydrates.
Organ of Capture
Predacious fungi form specialized hyphae structures to trap nematodes and other microscopic
animals. Among the six kinds of trapping devices, the constricting ring is the only one that actively
captures nematodes. When a nematode enters the aperture of the ring, which is formed by three cells,
the cells rapidly triple their volume, close the aperture and hold the nematode in place. Hyphae then
penetrate and consume the nematode. This paper reviews the data and hypotheses on conserving the
evolution of constricting rings and their cytological and molecular mechanisms.
The nematophagous fungi have been placed into two categories, Predatory and endoparasitic
depending upon their mode of attack. The predatory ones have an extensive hyphal system on the
substratum and capture their prey by producing trapping organs. Hyphae are usually sparse and
radiate out from the original point of primary capture with each hypha producing trapping devices at
intervals along its length. Victims are captured either by adhesive or non-adhesive devices. Adhesive
organs of capture have been categorised as hyphae, branches, knobs and nets.
Adhesive Hyphae
They are characteristic of lower fungi such as Zygomycetes and have no cross walls in the active
vegetative hyphae. Nematodes are captured by means of an adhesive produced on the hyphae. There
is no region of a hypha that can be assigned secretary in function or in appearance. Hyphae are either
coated with adhesive along entire length or produce adhesive at any point in response to nematodes.
A common example of such type of trapping device is Stylopage hadra in which the hyphae are
rather sparse and are characterised by an irregular branching. The adhesive hyphae are relatively
stout and a thick yellowish adhesive material is formed at the point of capture. Following this, an
appresorium like structure develops at the point of contact with the nematode. The captured victim
after a prolonged struggle becomes exhausted and is penetrated by the fungus. Following penetration,
elongate unbranched absorptive hyphae grow in both direction along the length of the body. After
complete exploitation of the contents, protoplasm is migrated back from the absorptive hyphae and a
cross wall is formed as it retracts. Related species that capture amoebae, are known to produce
zygospores are placed in Zygomycetes though technically it is an imperfect fungus. The adhesive
produced in Zygomycetes, while similar in function, is different from that produced by members of
Deuteromycetes.
Adhesive Branches
Adhesive branches are found mostly in Deuteromycetes. They are most primitive and
morphologically the simplest forms of capture organs produced in higher fungi. They are mostly few
cells in height and arise as short laterals that grow erect from the prostrate hyphae. A thin film of
adhesive is secreted over the entire surface of the branch and the branches are arranged in a fairly
close array such that a struggling nematode becomes attached at several points along its length.
There are few nematophagous fungi such as Dactylella cionopaga and Dactylella gephyropaga in
which the organs of capture are adhesive branches and even in these there is a tendency to form
simple two dimentional nets. The trap is elevated above the substrate such that even if the hyphae
become partly buried by debris, it does not interfere with its efficiency. At times the nematode breaks
away from the adhesive contact by the violent thrashing action of their struggles and thus escape.
This is partly influenced by environmental conditions. In dry conditions, where only a thin film of

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water covers the substrate, due to the phenomenon of surface tension a much greater leverage takes
place and nematodes break free, while in water they cannot exert sufficient leverage to break the
hold. In D. cionopaga, when two traps are in close proximity, they may form a hoop like trap which
is essentially a primitive net. In D. gephyropaga the sticky branches are more uniform in size and
shape and form a ladder like (scalariform) adhesive networks.
Adhesive Nets
These are the most commonly occurring trapping device as compared to above two. Net-forming
fungi are very aggressive in nature but ubiquitous in occurrence. Nets vary from a single loop-like to
complex multibranched, three dimentional networks as in Arthrobotrys oligospora. The nets become
adhesive very quickly, the adhesive is highly effective and the prey is held fast. Its struggle results in
contact with another net system and consequently the nematode gets attached at several points. The
violence of its efforts to escape quickly exhausts the victim and it becomes moribund the adhesive
produced by the nets has attracted considerable attention of the workers in the area and are of the
view that it is quite remarkable in its properties. Most probably the bait nematode has been
completely eliminated by the fungi or the ecological Conditions are unsatisfactory for its continued
survival.
Adhesive Knobs
Adhesive knobs are morphologically distinct cells, in some species it is sessile on hypha, often borne
on a short erect branch. They are found in Basidiomycetes and Deuteromycetes. Branches produced
at the apex of a slender non-adhesive stalks composed of one to three cells. The knob is separated
from the support stalk by a septum at its point of origin. Nematodes adhere to a knob and eventually
are caught by several knobs with subsequent penetration and destruction. Violent struggle for the
escape usually leads to breaking-off of the knobs from the point of attachment to the stalk. The
occurrence of detachable knobs is a distinct advantage to the parasite. The parasite is not only
transported to a new site for further predation, but arrives with an immediately available food source
and can quickly produce additional trapping devices. Occasionally a predator has both adhesive
knobs and also non constricting rings for the purpose of catching nematodes as in Dactylaria
candida.
The fungi following penetration, form a globes infection bulb, from which the hyphae emerge and
fill the entire body of the host, and finally digest the host contents. The genus Nematoctonous
belonging to Basidiomycetes is unique in having adhesive knobs growing directly from hyphae or
terminally on the ends or erect aerial branches. In endo-parasitic species of this genus the adhesive
knobs are never found. The adhesive device is very distinctive as it consists of a secretary cell shaped
like an hour glass and at maturity is engulfed. Nematoctonus species in their method of capture
behave as Dactylaria, except in the nature of the adhesive. It is produced much more profusely than
in Deuteromycetes, binds the cuticle to the knob so rigidly that even the hypodermis is pulled away
from the cuticle. Conidia are produced singly on short denticles from the vegetative hyphae and are
sausage shaped. Following dispersal, each conidium produces an adhesive knob from its tip, attaches
to a nematode and infects it.
Non-constricting Rings
Members of Deuteromycetes produce these types of rings on lateral branches arising from prostrate
hyphae. They are three celled rings supported on a slender stalk. There is a marked swelling just
above the point of contact with the support stalk. The juncture of the ring and its stalk is particularly

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weak and signs of collapse are often seen in mature rings. Non-constricting rings are passive in
action. The nematode enters the rings at speed and its forward motion causes the ring to wedge
around the body sufficiently tight to cause a marked constriction of the cuticle. During the struggle,
the ring often breaks off and the nematode swims off the ring around its body. This process may be
repeated until several rings are wedged around the host body. Detached rings are viable, the prey is
penetrated and contents consumed in usual manner. These rings are also advantageous as knobs are
for dissemination of the fungus. Production of non-constricting rings is often associated with
adhesive knobs, this fungus produces spindle shaped spores and in the presence of nematodes
germinate directly to produce an adhesive knob. These adhesive knobs can be produced while the
spores are still attached to conidiophores. The spores can be attached to any passing nematode and
transported to a new site where they arrive with a readily available food material in a new
environment.
Constricting Rings
These are most efficient mechanism for trapping nematodes, reported from members of
Deuteromycetes They are produced in the same manner as the non-constricting rings but here the
support stalk is usually shorter and much stouter. Rings are about 30 um in the outer diameter while
the aperture being 20 um. When a nematode happens to pass through the rings, it triggers a response
to the fungus in such a way that the three cells composing the ring swell rapidly inwards with such
force, which grasp the nematodes in a strangle hold from which there is no chance to escape. It takes
about 1/10 of a second for the ring cells to inflate to their maximum size. The ring cells are sensitive
all along their inner edges and when a nematode enters a ring, the friction of its body stimulate the
ring cells to swell rapidly inwards. This almost obliterates the opening of the ring constricting the
body of the animal, events then follow their usual course of action. The rings are extremely
sophisticated mechanism for catching nematodes, however, much depends on chance. Under all
optimum conditions like abundant rings and nematodes, only a small proportion may be caught. The
reasons may be that small nematodes may pass through the rings without touching the inner surfaces,
while large ones may brush them from their way. Normally the medium sized nematodes are caught
as they enter the rings, it snaps, giving little time for them to withdraw.
References
1. Keke Liu, Jianqing Tian, Meichun Xiang, and Xingzhong Liu, (2012), How carnivorous fungi
use three-celled constricting rings to trap nematodes, Protein cell, 3(5): 325–328.
2. Barron G.L. Predators and parasites of microscopic animals. In: Cole G.T., Kendrick B.,
editors. Biology of Conidial Fungi. Vol. 2. New York: Academic Press; 1981. pp. 167–200.
3. Barron G.L. Lignolytic and cellulolytic fungi as predators and parasites. In: Carroll G.C.,
Wicklow D.T., editors. The fungal community: its organization and role in ecosystem. New
York: Marcel Dekker; 1992. pp. 311–326.
4. Liu X.Z., Xiang M.C. The living strategy of nematophagous fungi. Micoscience. 2009; 50:20–
25. doi: 10.1007/S10267-008-0451-3.
5. Rubner A. Revision of predacious hyphomycetes in the Dactylella-Monacrosporium complex.
Stud Mycol. 1996; 39:1–134.
6. Stirling G.R. Biological control of plant parasitic nematodes: progress, problems and prospects.
Wallingford, UK: CAB International; 1991. pp. 56–59.

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7. Yang Y., Yang E.C., An Z.Q., Liu X. Evolution of nematode-trapping cells of predatory fungi
of the Orbiliaceae based on evidence from rRNA-encoding DNA and multiprotein sequences.
Proc Natl Acad Sci U S A. 2007;104: 8379–8384.
8. Ramalingam Karthik Raja, Alagarsamy Arun, Mustapha Touray, Sebnem Hazal Gulsen, Harun
Cimen, Baris Gulcu, Canan Hazir, Dilipkumar Aiswarya, Derya Ulug, Ibrahim Cakmak, Harry
K. Kaya, Selcuk Hazir, (2021). Antagonists and defense mechanisms of entomopathogenic
nematodes and their mutualistic bacteria, Biological Control, Vol. 152: 104452.
***
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Chapter 19
POST-HARVEST FUNGAL DISEASE AND ITS
MANAGEMENT ON ALLIUM CEPA
S. Kaviyalakshmi, S. Srinithiksha, Dr. K. V. Shalini
A bstract
Onion is one of the most widely cultivated and consumed vegetable crops in the world, with a global
production of over 100 million tonnes per year. It is a member of the Allium family, which also
includes garlic, shallots, and chives. Onion bulbs are used in a variety of culinary preparations due to
their distinctive flavour and aroma, as well as their nutritional content. One of the biggest challenges
in onion cultivation and marketing is post-harvest storage, as onion bulbs are susceptible to a variety
of fungal diseases during storage. These diseases can cause significant losses in bulb quality and
quantity, as well as reduce their shelf life. The most common fungal pathogens affecting onion
during storage include Aspergillus, Fusarium, Botrytis, and Penicillium. To mitigate the impact of
fungal diseases on onion storage, various control strategies have been employed including the use of
synthetic fungicides and biological control agents. This review article focused on post-harvest fungal
disease and its preventive measures in onions.
Keywords: Allium cepa, post-harvest diseases, Fungicides, Preventive measures.
Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam Institute for

Home Science and Higher Education for Women, Coimbatore- 43, India
shalinisahaana@gmail.com
S. Kaviyalakshmi, S. Srinithiksha, K. V. Shalini

208
I ntroduction
Onions are an important agricultural crop worldwide providing essential flavour and nutrition to
many cuisines. However, post-harvest losses caused by fungal diseases during storage can
significantly impact the quality and shelf-life of onions, resulting in economic losses for growers and
suppliers. Fungal pathogens such as Aspergillus niger, Botrytis squamosa, and Penicillium spp., are
the main culprits behind onion storage diseases, causing black mold, neck rot, and soft rot
respectively. Chemical fungicides have been widely used to manage these diseases, but their overuse
has led to the emergence of fungicide-resistant strains and environmental concerns (Mahajan et
al.,2018). Therefore, there is a growing interest in developing alternative methods for post-harvest
onion disease management, such as the use of biocontrol agents. Biocontrol agents are naturally
available organisms that can suppress the growth of pathogenic fungi and enhance the quality and
shelf-life of onion bulbs. (Sharma et al., 2009). In this review article the current knowledge on
managing post-harvest fungal diseases in onions and their preventive measures are focused.
Post- Harvest Fungal Diseases in Onion
Botrytis Leaf Blight

Botrytis neck rot is caused by Botrytis allii, a fungus that overwinters on plant debris in soil, on
infected bulbs, and as sclerotia in soil. Botrytis neck rot is seen primarily in storage. In onions, the
disease is more apparent after harvest, while bulbs are in storage. At first, soft neck tissue looks
water soaked, and a yellow discoloration moves down the neck into the scales. Bulbs break down to
a soft mass. A grey mold develops between the onion scales, later producing small to large black
bodies (sclerotia) which develop as a solid layer around the neck of the onions.
Aspergillus niger
Onion black mold rot disease is the most destructive disease of storages and in the field (Wani et al.,
2011). Aspergillus niger is a soil borne fungus that can survive on plant debris in the soil. Infections
spread from bulb to bulb by direct contact, through bruises or wounds, by mechanical means or by
air-borne spores. Spores can germinate within three to six hours under high relative humidity, but
germination is inhibited below 75 per cent relative humidity. The optimum temperature for growth of
A. niger ranges from 28 to 34 °C, and it is inhibited below 17°C and above 47 °C (Sumner, 1995).
Sporulation can take place in 24 h after infection (Salvestrin and Letham, 1994).
Blue Mold
Blue Mold rot of onion may be caused by several Penicillium species. Decay of garlic caused by P.
hirsutum is responsible for decay during storage as well as poor plant growth in the field. Symptoms
in the field include bulb decay soon after cultivating due to wilting, leaves becoming yellow or
stunted seedlings. Infected plants are weak and stands are poor. Other species of Penicillium cause
Blue Mold on onions and may be prevalent on fresh plants. This fungus attacks a wide range of
fruits, vegetables, bulbs, and seeds. Disease symptoms start with pale blemishes, yellow lesions, and
soft spots. A blue-green mold develops on lesions and these pathogens survive in infected bulbs of
the onions. (Jonny et al., 2020).

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White Rot
White rot is one of the most destructive fungal diseases causing heavy losses reducing potential yield
of onion to a considerable degree (Amin et al.,2014). White rot disease caused by Sclerotium
cepivorum Berk. is very destructive to Allium crops including onion, leek, Welsh onion, shallot and
garlic and the fungus attack the plant at any crop stages and often manifests after harvest as bulb rot
and often disseminates on diseased bulbs or seedlings. White rot on shallot, garlic and onion results
in death of the roots and semi watery decay of the scale which is associated with superficial growth
of white fluffy mycelium of the fungus.
Preventive Approaches of Fungal Diseases in Onion

Biocontrol Agents
Trichoderma spp has a wide range of applications in in vitro inhibition of storage pathogens of
horticultural crops. Cherkupally et al., (2017) evaluated the antagonistic activities of seven
Trichoderma species, against fungal pathogen, Fusarium oxysporum in vitro conditions. All the
biocontrol agents showed considerable decrease in the growth of the fungal pathogens. Out of the
seven fungal antagonists studied for their efficacy, T. harzianum showed maximum degree of
inhibition 81.11%, followed by T. koningii 80.00%, T. pseudokoningii and T. viride 78.88% each, T.
virens, T. atroviride, and T. reesei 77.77% each by non -volatile compounds. The results suggested
that T. harzianum had a highly antagonistic potential 81.11% and T. koningii showed least
antagonistic efficacy by 28.88%. (Mahmoud et al.,2011).
Synthetic Fungicide
Futane et al., (2018) evaluated in vitro antifungal activity against fungal pathogens in A. niger and
Fusarium oxysporum. In A. niger pathogen the antifungal activity was most operational with
significantly highest mycelial growth inhibitions were SAFF (A Broad spectrum systemic and
contact fungicide) (94.55 per cent), mancozeb (90.98 per cent) and carbendazim (92.22 per cent). In
Fusarium oxysporum fungicides viz SAFF, mancozeb, hexaconazole and carbendazim were found
most effective with significantly highest mycelial growth inhibition of 91.74 per cent, 91.38 per cent,
90.81 per cent and 88.42 per cent respectively. Studies were undertaken to control black mold rot of
onions through synthetic fungicides in vitro and in vivo. Turkkan and Erper (2014) investigated
efficiency of different sodium salts as substitutes to synthetic fungicides for the control of onion
basal rot caused by Fusarium oxysporum. Srinivasan and Shanmugam (2006) found that Aspergillus
niger was predominantly related with black mold rot of onion during storage showed that among
fungicides used, 0.1% carbendazim concentration was found to the most effective when applied
either as a foliar spray in standing crop or as a post-harvest dip followed by sulphur oxides and acetic
acid.
Fumigation
One effective way to control onion disease is through fumigation treatment. Fumigation treatment
involves the use of chemicals that release gas when applied to soil, which kills or suppresses soil-
borne pathogens. In the case of onion disease, the most commonly used fumigants are methyl
bromide, chloropicrin, and 1,3-dichloropropene. Methyl bromide was widely used for fumigation,
but it is now banned in many countries due to its negative impact. Chloropicrin is a popular
alternative to methyl bromide, but it can cause skin and respiratory irritation. 1,3-dichloropropene is
a relatively new fumigant that is gaining popularity due to its lower toxicity.

210
Onions treated with the thymol solution fumigation method had only a loss of less than 17% from
fungal pathogens while maintaining the storage of the onions for 10 months in a low-temperature
storage house. Compared to the untreated controls, the storage period of the onions treated with
thymol was extended for more than three months. These results confirmed that thymol solution
fumigation is a highly successful candidate for the control of fungal diseases during low-temperature
storage. Additionally, since thymol residues from onions stored for 10 months were not detected,
thymol solution fumigation could be a safe treatment method. Thymol has been shown to be
thermally safe up to 100 °C through the heat stability test. (Sang Hye Ji et al.,2018). This study
proposed that the fumigation treatment method using the thymol solution was ideal for fungal disease
control of long-term storage crops such as onions.
Neem Leaf Extract
Neem Extract has been used to prevent numerous fungal plant Pathogens, such as Aspergillus flavus,
Alternaria alternata, Fusarium oxysporum, Fusarium solani, Sclerotinia Sclerotiorum and
Rhizoctonia solani (Jatav and Mathur, 2005; Wang et al., 2010; Obongoya et al., 2010; Mina, 2012).
Medicinal properties of neem leaf and its constituents have antifungal, anti-inflammatory, and
Antibacterial properties (Subapriya and Nagini, 2005). Krishanti and Prianto (2017) studied five
types of neem Seed oil formulation which was sowing antifungal activity against fungi and they
could prevent the growth of mycelial pathogenic fungi, Fusarium oxysporum, effectively. The results
indicated that neem seed formulations were potential to be developed as bio fungicide and it needs a
further analysis about active compound that shows an important role in fungal overthrow. Different
formulations of neem cake gave the maximum efficacy in inhibition of mycelial growth of fungal
pathogens (77.55%) afterward mustard cake (73.53%) and groundnut cake (67.25%) extracts.
Yadav et al., (2014) revealed that neem cake extract (30%) significantly prevented the fungal
mycelial growth. Shrivastava and Swarnkar (2014) studied antifungal activity of leaf extract of
Azadirachta indica (Neem) on different fungal pathogens of Aspergillus flavus, Alternaria solani and
Cladosporium. Ethanolic and Methanolic extract of this plant in different ranges (25%, 50%, 75%
And 100%) were equipped and proven its efficacy against test organisms by disc diffusion indicating
the development of an inhibition zone.
Among all the extracts the most effective Methanolic extract of Azadirachta indica against
Aspergillus Flavus has been observed. In the same year Olufemi et al. (2014) revealed that the neem
seed extract has maximum antifungal properties in Curvularia sp. (37.76% inhibition) followed by
Aspergillus sp. (20.22% inhibition) and Fusarium Sp. (7.56% inhibition).
Hussein et al. (2014) tested the antagonistic ability of Trichoderma harzianum and Trichoderma
viride against B. Allii, causal agent of umbel “head” blight disease of onions. The treatment of T.
Viride demonstrated 30 to 45% reduction of disease severity in a 2-year test.
Mahmoud et al. (2011) studied the outcome of aqueous extracts of neem formulations on different
fungal growth (Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Aspergillus terreus and
Microsporum gypseum, Candida Albicans) in vitro at different range of concentration 5, 10, 15 And
20%. The neem extracts withdrawn the growth of fungal Pathogens in dose dependent manner.
Ghorbanian et al. (2008) investigated the efficacy of leaf extract (aqueous neem) in varied
concentrations of on development of fungus and due to production of aflatoxin by the fungi
Aspergillus Parasiticus at change in different time of incubation period. The maximum inhibitions
found 80-90% in the existence of 50% formulations. The Inhibitory effect of aflatoxin synthesis by

211
plant-based extracts initiates to be time and dose dependent. In organic alterations of neem cake was
given a favourable form for inhibition of the mycelial growth of storage fungi.
Ali et al. (1992) studied the effect of leaf extract and neem oil from Azadirachta indica along with
Tecto-60 and Boric acid, against Pencillium italium, Alternaria altunala and Aspergillus niger on
decayed vegetables and fruits. The results revealed that neem oil was most effective comparable to
thiabendazole in examination growth of fungi on decayed vegetables and fruits.
Conclusion
There is an emergency need to develop environmentally friendly fungicide against global fungal
pathogens to combat the toxic residual effects of traditional synthetic fungicides on human health.
Fungicide used is important for the control of decay of vegetables and fruits such as green mould,
blue mould and sour rot, which can occur during storage. However, fungicides are one of the tools
required to effectively manage postharvest decay. Fumigants such as sulphur dioxide (SO2) are used
for controlling post-harvest disease in many vegetable and fruit crops during storage. This treatment
results in a residue of 5-18 ppm SO2 in the vegetables is sufficient to control the decay. Several other
management factors such as biocontrol agents, plant extracts can also be used. The use of bacteria as
biocontrol agents (BCA) has been very important in the integrated management of cultivations and
organic production, where their value as a postharvest control of fungal diseases stands out.
Biocontrol methods provide plant protection against fungal diseases, and currently represent a viable
alternative for fruit and vegetable protection against phytopathogens at the postharvest stage. Further
research is needed to develop more effective and sustainable strategies for the management of post-
harvest fungal diseases of onions.
References
1. Ali, T.E.S, Nasir, M.A. and Shakir, A.S. (1992). In vitro evaluation of certain neem products
as mould inhibitors against post-harvest fruit rotting fungi of tomato Pakistan, Journal of
Phytopathology,4(1-2): 58-61.
2. Amin M, Shiberu T & Selvaraj T. (2014). White rot (Sclerotium cepivorum Berk) – an
aggressive pest of onion and garlic in ethiopia. Journal of Agricultural Biotechnology and
Sustainable Development, 6(1): 6-15. Doi: 10.5897/JABSD2013.0210.
3. Cherkupally, R., Hindumathi, A. And Reddy, B.N. (2017). In vitro antagonistic activity of
Trichoderma species Against fusarium oxysporum f. Sp. Melongenae. International Journal
of Applied Agricultural Research, 12(1): 87-95.
4. Futane, A.S. Dandnaik, B.P. Salunkhe, P.P. and Magar, S.J. (2018). Management of storage
diseases of onion by using different fungicides and antibiotics international, Journal of
current microbiology and applied sciences, 7(02): 1149-1158.
5. Ghorbanian, M., Razzaghi-Abyaneh, M. Al, A. Shams-Ghahfarokhi, M. and Qorbani, M.
(2008). Study on the effect of neem (Azadirachta indica A. Juss) leaf extract on the growth of
aspergillus parasiticus and production of aflatoxin by it at different incubation Times.
Mycoses, 51(1): 35-39.
6. Jatav, R.S. and Mathur, K. (2005). Bio-agents and neem-based seed treatment for
management of root rot complex in cluster beans. Indian Phytopathology, 58:235–236.
7. John, Vinny & Kesherwani, Drbhagyashree & Maurya, Amit. (2020). AGRICULTURE &
FOOD: e-Newsletter Blue mold rot disease of onion.

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8. Krishanti, N.P.R.A. and Prianto, A.H. (2017). In-vitro assay of neem seed formulation against
fusarium oxysporum, causal agent of basal plate rot on onion. In the proceeding of the
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9. Mahmoud, D.A., Hassanein, N.M., Youssef, K.A. and Zeid, (2011). Antifungal activity of
different neem leaf extracts and the nimonol against some important human pathogens.
Brazilian Journal of Microbiology, 42(3): 1007-1016.
10. Mahajan B.V, Rawal R.D, Bhattacharyya S, (2018). Onion (Allium cepa L.) postharvest
management practices: A review. J Food Sci Technol. 55:4703-4715.
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plant extracts on soil-borne fungi of common beans. African Crop Science Journal, 18(1).
12. Olufemi OS, Agbaje GO, Olusoji JO, Owolabi OA, Olawuyi OJ, Ogunjobi A.A (2014).
Phytochemical screening, antifungal and antioxidant activities of neem (Azadirachta indica)
seed extracts." Journal of Applied Sciences and Environmental Management. 18(3): 419-423.
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Horticulture, 358: 289-293.
14. Sang Hye Ji, Joo Yeon Park, Jin Gyeong Lee, Seok Ju Park, and Jae-Hyuk Yu.(2018).
Efficacy of thymol and acetic acid vapors against Aspergillus niger and their effects on
postharvest quality of cherry tomatoes." Journal of Food Quality; 2018: Article ID 8073423.
doi: 10.1155/2018/8073423.
15. Sharma RR, Singh D, Singh R. (2009). Biological control of postharvest diseases of fruits
and vegetables by microbial antagonists: A review of Bio control. 50:205-221.
16. Shrivastava, D.K. and Swarnkar, K. (2014). Antifungal activity of leaf extract of neem
(Azadirachta Indica Linn). International Journal of Current Microbiology and Applied
Sciences, 3(5): 305-308.
17. Srinivasan, R. And Shanmugam, V. (2006). Post-harvest management of black mould of
onion. Indian Phytopathology, 59(3): 333-339.
18. Subapriya, R. And Nagini, S. (2005). Medicinal properties of neem leaves: a review. Current
Medicinal Chemistry Anticancer Agents, 5(2): 149–160.
19. Sumner DR. 1995. Diseases of bulbs caused by fungi-Black mold. In: Compendium of onion
and garlic diseases, H. F. Schwartz and S. K. Mohan (eds.) APS Press, St. Paul. Minnesota:
26- 27.
20. Türkkan, M.U.H.A.R.R.E.M. and Erper, I. (2014). Evaluation of antifungal activity of
sodium salts against onion basal rot caused by fusarium oxysporum F.sp. Cepae. Plant
Protection Science, 50(1): 19-25.
21. Yadav AK, Singh AK, Raj A, Singh PK. (2014). Evaluation of neem cake extracts for their
antifungal, nematicidal and insecticidal activities. Journal of Pharmacognosy and
Phytochemistry. 3(1): 53-56.
22. Wang, J., Jian, L., Jiankang. C and Jiang, W. (2010). Antifungal activities of neem
(Azadirachta indica) seed kernel extracts on postharvest diseases.
23. Wani AH and Taskeen-Un-Nisa. (2011). Management of black mold rot of onion. Journal of
Mycology and Plant Pathology, 9 (1): 43-49.
***
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Chapter 20
NEMATOPHAGOUS FUNGI – A REVIEW
S. Gomathi, S. P. Hemavarthini, S. Kaviya, Dr. P. Chitra
A bstract
Nematophagous fungi are usually investigated and used in biocontrol of phytonematodes; recently,
these fungi were found in association to the rhizosphere of crop plants colonizing their roots.
Associated with plants, nematophagous fungi may also synthesize phytohormones or benefit the
absorption of phosphorus by the plants. These fungi positively affect plant growth, plant-disease
antagonism, and rhizosphere colonization. In an increasing demand for more sustainable solutions to
raise yields, plant productivity may be enhanced by exploring the potential of beneficial plant-
entomopathogen interaction, as growth promoters. Microfungi that can capture, kill, and digest
nematodes are known as nematophagous fungi. They use special mycelial structures known as traps
or spores to trap vermiform nematodes or hyphal tips to attack nematode eggs and cysts prior to
cuticle penetration, invasion, and digestion. The saprophytic/parasitic ability of the more than 200
known species varies. Although many trap-forming and egg-parasitic fungi can survive in soil
saprophytically, endoparasites rely heavily on nematodes for nutrition. Molecular data have greatly
improved our understanding of fungi function in terms of taxonomy, physiological/biochemical
activity, and ecology, including their role as biocontrol agents.
Keywords: Nematophagus, Phytohormones, Rhizosphere, Microfungi, Biocontrol.
Department of Zoology, School of Biosciences, Avinashilingam Institute for Home Science and
Higher Education for Women, Coimbatore, Tamil Nadu, India
S. Gomathi, S. P. Hemavarthini, S. Kaviya, P. Chitra

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I ntroduction
Nematophagous fungi are nematode's natural enemies. They are divided into three groups:
nematode-trapping and endoparasitic fungi, which attack vermiform living nematodes with
specialised structures, and egg- and cyst-parasitic fungi, which attack these stages with their hyphal
tips. The persistence of interest in these fungi stems, in part, from their potential as biocontrol agents
against plant- and animal-parasitic nematodes. Because of their potential as biocontrol agents, the
egg- and cyst-parasitic fungi have received special attention in this regard. Another reason for
nematophagous fungi's continued fascination is their remarkable morphological adaptations and
dramatic capture of nematodes by both nematode-trapping and endoparasitic fungi.
Nematophagous fungi are cosmopolitan microorganisms, natural antagonists of nematodes that feed
on several living stages of these animals (Nordbring-Hertz et al., 2006). Many of the nematophagous
fungi are facultative parasites, and in the presence of their hosts they can change from a saprophytic
to a parasitic stage and form infection structures, which are used to classify them according to their
predation characteristics (Lopez-Llorca et al., 2006). These fungi can be classified into four groups:
nematode-trapping/predators, opportunistic or ovicidal, endoparasites, and toxin-producing fungi.
Currently, a new group of nematophagous fungi has been proposed: producers of special attack
devices (Liu et al., 2009; Soares et al., 2018). Nematode-trapping fungi use various types of
modified hyphae, e.g., adhesive networks, adhesive knobs or constricting rings, to capture nematodes
by adhesion and mechanical function, and to digest and to eat them using enzymes (mainly proteases,
chitinases and lipases). The ovicidal group also uses traps/enzymes to prey and to feed on eggs, cysts
and nematode females. Endoparasitic fungi are a group of fungi that use their conidia to infect
nematodes. The fungi from this class have no saprophytic phase or only a limited saprophytic phase,
and produce almost no mycelium in soil, hindering its cultivation and industrial use (Moosavi and
Askary, 2015; Li et al., 2015). Toxin-producing fungi attack their prey by secreting diffusible toxins,
immobilizing them before hyphae penetration through the cuticles (Lopez-Llorca et al., 2008).
Special attacking devices are similar to a weapon, causing damage to the nematode cuticle and
allowing fungal colonization and consumption (Soares et al., 2018).
Nematophagous fungi are used in biocontrol of phyton-ematodes, thus protecting plants from these
parasites. Moreover, it is known that these fungi are found in the rhizosphere of crop plants and can
colonize their roots (Bordallo et al., 2002; Lopez-Llorca et al., 2002). Plant-parasitic nematodes
generally attack plant roots; therefore, the ability of nemato-phagous fungi to colonize roots is very
attractive to explore (Bordallo et al., 2002).
Furthermore, both fungi and nematodes can be easily grown in the laboratory, making them an
excellent model system for interaction studies. Nematode (round worms) (round worms) Nematode-
trapping and endo-parasitic fungi are found in all major taxonomic groups of fungi, and they live in a
variety of soil environments as saprophytes. They benefit nutritionally from their ability to use
nematodes as an additional nutrient source. Fungi enter their parasitic stage when their morphology
changes and traps or mature spores form. The formation of infection structures is required for
nematode trapping. The mechanisms underlying this development, as well as the mechanisms
underlying the capture process, such as nematode attraction, adhesion, penetration, and digestion, are
the primary topics of this Chapter.

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Taxonomy
The trapping mechanism is also an important diagnostic factor in nematophagous fungi. The majority
of the group's isolation methods rely on the fungus making itself known by capturing or infecting
nematodes. Many of these fungi do not produce trapping organs in pure culture, and the dimensions
of the conidia and conidiophore are frequently different from those examined on baited plates. As a
result, the characters used to identify these fungi are those found on nematode-infected cultures.
Cooke and Godfrey (1964) extensively revised an early key by Dollfus (1946), and while this
contains details on all commonly found species, only 97 species are included, leaving another 50-60
species unaccounted for. Many new species have been described since 1964, and while many are
either scarce or have a limited distribution, some, such as Arthrobotrys pauca, may be far more
abundant than previously thought. The problem with any key is that unless the original description of
fungi is constantly referred to, the user will generally choose the 'best fit' approach to identification.
While Cooke and Godfrey's key is undeniably the best available at the time and is still widely used, a
new key is required that will not only update the taxonomy and include the numerous species not
included in the current key, but will also review certain groups.
Ecology of Nematophagus Fungi
The majority of nematode-trapping fungi are found in the upper part of the soil, pastures, leaf litter,
mangroves, and certain shallow aquatic habitats. They use adhesive hyphal strands, adhesive knobs,
adhesive nets formed from hyphal threads, hyphae loop that tighten around any ensnared nematodes,
and non-constricting loops. When the nematode is restrained, the hyphae penetrate the cuticle and
consume the nematode's internal tissues. It has been discovered that Arthrobotrys oligospora, a
fungus that builds nets, can detect the presence of nematodes in the soil and only builds snares when
they are present. This is presumably due to the high energy cost of constructing the net; the fungus
detects the presence of the nematode by detecting the pheromones with which the worms
communicate. The fungus actively seeks prey by producing olfactory cues similar to those used by
the worm to find food and attract mates. Arthrobotrys dactyloides is a species that uses a hypha loop
to catch nematodes; when the prey tries to pass through the ring, the loop constricts rapidly, trapping
the prey.
Extra cellular enzymes involved in infection against nematodes
Serine proteases
Serine proteases are a class of enzymes that catalytically hydrolyze peptide bonds by using a
specially activated serine residue in the substrate-binding pocket (Schultz and Liebman 1997; Siezen
and Leunissen 1997). Serine proteases perform a wide range of physiological functions and have
been identified as pathogenic factors in bacterial or fungal pathogens against insects, nematodes, and
even humans (Yousef et al. 2003). (e.g., Tunlid et al. 1994; Joshi et al. 1995; Tian et al. 2006).
Serine proteases are major extracellular enzymes produced in large quantities by virulent isolates of
V. lecanii and Metarhizium anisopliae (Jackson et al. 1985). (St Leger et al. 1992). P. rubescens
produced the first pathogenicity-related serine protease, P32. Similar proteases were later discovered
in other nematophagous organisms. Other pathogenic proteases (PII and VCP1) from A. oligospora
(Tunlid et al. 1994) and Pochonia chlamydosporia (syn. V. chlamydosporium; Segers et al. 1994)
were identified in 1994. Recently, more pathogenic serine proteases including pSP-3, Aoz1, Ver112,
Mlx, PrC, and Ds1 were identified from nematophagous fungi Paecilomyces lilacinus (Bonants et al.
1995), A. oligospora (Zhao et al. 2004), L. psalliotae (Yang et al. 2005a), Monacrosporium

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microscaphoides (Wang et al. 2006a), Clonostachys rosea (syn. Gliocladium rosea; Li et al. 2006),
and Dactylella shizishanna (Wang et al. 2006b), respectively.
Chitinases
Chitin is an important structural polymer found in fungi cell walls and invertebrate exoskeletons. It is
an important component of the nematode eggshell's middle layer (Wharton 1980; Bird and Self
1995). To cause infection, egg-parasitic fungi such as P. rubescens and P. chlamydosporia must first
penetrate the nematode eggshell (Lysek and Krajci 1987). Based on ultrastructural studies, there is
substantial evidence that fungal parasites produce chitinases during infectious disease (e.g., Lopez-
Llorca and Robertson 1992; Tikhonov et al. 2002; Khan et al. 2004). Recently, the extracellular
chitinase CHI43 from P. chlamydosporia and P. rubescens was discovered to act as a nematicidal
factor in infecting nematode eggs (Tikhonov et al. 2002). Chitinase activity was also detected in the
culture supernatant of the nematophagous fungus P. lilacinus strain 251 in chitin-containing minimal
medium, and after separation by isoelectric focusing, six proteins with chitinolytic activity were
identified (Khan et al. 2003). Chitinase activity was confirmed using a sandwich assay with glycol
chitin as a substrate on nondenaturing 1D and 2D gels. P. lilacinus extracellular enzyme studies
revealed that the application of protease and chitinase, either individually or in combination,
drastically altered the eggshell structures (Khan et al.200)
Collagenase and other hydrolytic enzymes
Collagenases are enzymes that can catalyse the hydrolysis of collagen and gelatin rather than other
proteinaceous substrates (Maclennan et al. 1953). The nematode cuticle is primarily composed of
collagen (Blaxter and Robertson 1998). Collagenase from these fungi may thus play an important
role in nematode infection. However, reports on collagenase production by nematophagous fungi are
scarce (Schenck et al. 1980; Tosi et al. 2001). Collagenases were recently discovered in Arthrobotrys
spp. (Tosi et al. 2001), and when grown in liquid medium free of proteosepeptone, all Arthrobotrys
species tested positive for collagenase (proteosepeptone induces collagenase production). This
finding indicates that collagenase is a constitutive enzyme in these fungi. Other hydrolytic enzymes
have been implicated in fungi infection of nematodes. For example, Heterodera schachtii eggs
infected with fungi appeared to have their inner lipid layers degraded when compared to uninfected
eggs, which was attributed to the fungus's lipolytic activity (Perry and Trett 1986).
Toxic compounds
Some NF species produce chemical compounds that are toxic to nematodes. These compounds cause
nematode paralysis, and the fungus then consumes them. In some cases, nematode head shrinkage is
observed as a side effect of toxin action (Satou et al., 2008). These NF are known as toxin-producing
fungi. However, most NF research has concentrated on predatory and endoparasitic fungi (Soares et
al., 2018). The majority of toxin-producing fungi are basidiomycetes. In this context, several
Pleurotus genus edible mushrooms produce toxins with nematotoxic activity (Kwok et al., 1992;
Nordbring-Hertz et al., 1995; Satou et al., 2008).
P. ostreatus, for example, produces trans-2-decenoic acid, a linoleic acid-derived compound that is
toxic to nematodes, insects, and other fungi (Kwok et al., 1992). However, it should be noted that
basidiomycetes do not only produce these types of toxins, but there are also some fungi that produce
compounds that are toxic to nematodes but are not nematophagous, meaning they do not consume
the nematode (Soares et al., 2018).The chemical properties of these compounds are also quite
diverse, including simple fatty acids and other organic acids, such as pyrones, lactones,

217
benzoquinones, anthraquinones, furans, alkaloids, cyclodepsipeptides, and peptaibiotics, and hybrid

structures, such as lactam-bearing macrolactones (Degenkolb and Vilcinskas, 2016a).
Interaction with other fungi and plants
Interactions with other plants and fungi Apart from attacking nematodes, nematophagous fungi can
also infect other fungi (act as myco-parasites). Nematode-trapping fungi, such as A. oligospora,
attack their host fungi by coiling their hyphae around the host hyphae, resulting in disintegration of
the host cell cytoplasm without penetration. Using radioactive phosphorus tracing, it was
demonstrated that nutrient transfer occurred between the nematode-trapping fungus A. oligospora
and its host R. solani (Osslon and Persson,1994) Other plants and fungi interactions Nematophagous
fungi can infect other fungi in addition to attacking nematodes (act as myco-parasites). Nematode-
trapping fungi, such as A. oligospora, attack their hosts by coiling their hyphae around the host
hyphae, resulting in cytoplasm disintegration without penetration. Nutrient transfer between the
nematode-trapping fungus A. oligospora and its host R. solani was demonstrated using radioactive
phosphorus tracing (Osslon and Persson,1994) P. chlamydosporia endophytic growth in barley and
wheat roots appeared to increase plant growth while decreasing growth of the plant parasitic take-all
fungus Gaeumannomyces graminis var. tritici (Monfort et al., 2005). Mycoparasitism and plant
endophytism may be important issues for extending the nematophagous fungi's biological control
potential.
Nematode – Fungus interaction mechanism
Recognition and host specificity
The mechanism by which nematophagous fungi recognise their prey is complicated. There is no
simple host specificity in any of the nematode-trapping species, but experiments with the
endoparasite D. coniospora have revealed slightly higher host specificity. Nonetheless, it appears that
there are recognition events in cell-cell communication at several stages of the fungus-nematode
interaction, which may elicit a defined biochemical, physical, or morphological response. Nematodes
are attracted to the mycelia of fungi, where they can induce trap formation, and they are even more
attracted to fully developed traps and spores. Following this is a'short-range' or contact
communication: adhesion. This step could involve a carbohydrate-binding protein (lectin) in the
fungus interacting with a carbohydrate receptor on the nematode. The recognition of the host is
probably also important for the subsequent steps of the infection, such as nematode cuticle
penetration.
Attraction
Compounds released from themycelium and traps of nematode-trapping fungi, as well as the spores
of endoparasites, attract nematodes. Both morphology and, as a result, saprophytic/parasitic ability
have a strong influence on fungi attractiveness. Fungi that are more parasitic appear to be more
attractive than those that are more saprophytic; that is, endoparasitic species infecting nematodes
with conidia appear to be more effective in attracting nematodes than saprophytic species using
various trapping devices.
Adhesion
The contact and adhesion of nematodes to nematophagous fungi traps and spores can be seen under
the electron microscope. Even before the interaction with the nematodes, the three-dimensional nets
in A. oligospora are surrounded by a layer of extracellular fibrils. Following contact, these fibrils
become perpendicular to the host surface, most likely to facilitate nematode anchoring and further

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fungal invasion. Whether or not contact with the nematode has been established, the endo-parasite D.
coniospora exhibits a completely different type of adhesive that appears to be composed of radiating
fibrils. Furthermore, D. con-iospora spores adhere specifically to the sensory organs at the tip of the
nematode's head, preventing nematode attraction. The chemical composition of nematophagous fungi
surface fibrils is unknown, but they do contain proteins and carbohydrate-containing polymers.
Fig. Mode of action: a) Formation of 3 –

dimensional networks 12 hrs. after
inoculation with nematode larvae. b)
Infective larvae after 12hr of interaction
between nematode and fungus. c) captured
and immobilized in 3 dimensional
networks. d) Magnification of area
highlighted. e) Hyphal pressure point on
nematodes cuticle. f) Hyphal penetration on
nematodes cuticle (Source: Falbo et al.,
2012).
Penetration
The fungi are differentiated due to the traps' adhesion to the nematode. A penetration tube forms and
pierces the nematode cuticle in A. oligospora. This step is likely to involve both the activity of
hydrolytic enzymes that dissolve the cuticle's macromolecules and the activation of a mechanical
pressure generated by the penetrating growing fungus. The nematode cuticle is mostly made up of
proteins, including collagen, and several proteases that can hydrolyze cuticle proteins have been
isolated from nematophagous fungi. In all cases, these proteases are members of the family of serine
proteases, and data from sequencing show that they have a high homology to the subtilisin of serine
proteases (Ahman et al., 1996; Bonants et al., 1995). A chymoptrypsin-like protease appears to be
involved in the penetration process of the endoparasite D.coniospora. Proteases are another term for
enzymes. More in-depth studies of the subtilisin PII produced by A. oligospora have revealed that
this type of protease can serve a variety of functions (Ahman et al., 2002). Thus, PII appears to have
nematotoxic activity in addition to being involved in the penetration and digestion of the cuticle and
tissues of infected nematodes.
Digestion and Nutrients
The nematode is digested by the infecting fungus after penetration. Once inside the nematode, A.
oligospora's penetration tube swells to form a large infection bulb (Figure 2d). The bulb and
trophichyphae develop in tandem with dramatic changes in the fungus's ultrastructure and
physiology. The dense bodies are broken down in the trap cells and the bulb. The bulb and the
trophic hyphae typically contain normal cell organelles, endoplasmatic reticulum being particularly
well developed. Later stages see the accumulation of lipid droplets in the trophic hyphae, which are

219
likely involved in the digestion and storage of nutrients obtained from the infected nematode. In
contrast to trap-forming fungi, the endoparasite D. coniospora does not form an infec-tion bulb upon
penetration and lacks dense bodies. Along with the formation of lipid droplets, A. oligospora
produces large amounts of a lectin in the cytoplasm to store nutrients derived from the host (Rosen et
al., 1997). This protein (designated Arthrobotrys oligospora lectin, AOL) belongs to a new family of
low molecular weight lectins with similar primary sequences and binding properties that have only
been found in a few filamentous fungi (Rosen et al., 1996).
AOL is rapidly synthesized in A. oligospora during nematode infection once the nematodes have
been penetrated and digestion has begun. The trophichyphae growing inside the nematode
accumulates a large amount of AOL. Later, the lectin is transported from the infected nematode to
other parts of the mycelium, where it is degraded and helps the fungus grow. Although the
mechanisms are unknown, it has been proposed that AOL, like other lectins, is involved in a
recognition event during nematode interaction. This hypothesis is supported by the fact that the AOL
family of lectins binds to sugar structures found in animal glycoproteins such as nematodes but not in
fungi.
Constricting rings
Although the patterns of nematode infection of other predatory fungi, which use adhesive layers to
capture nematodes (nets, hyphae, or knobs), appear to be largely similar to those described for A.
oligospora. The trapping mechanism of constricting rings, on the other hand, is completely different.
When a nematode enters the ring, it causes the three cells that make up the ring to swell inward and
close around the nematode. Other stimuli, such as a needle touching the inside (luminal) surface of a
ring or heat, can also cause the trap to close. The reaction is rapid (0.1 s), irreversible, and is
accompanied by a large increase in cell volume, resulting in nearly complete closure of the trap's
aperture. Following capture, the fungus produces a penetration tube that pierces the cuticle of the
nematode. A small infection bulb forms inside the nematode, from which trophic hyphae develop.
The mechanism that closes the constricting rings is not fully understood. The outer cell wall of the
ring cells is ruptured along a defined line on the inner surface of the ring during ring-cell expansion,
according to electron microscopy.
This release of wall pressure is thought to cause a rapid uptake of water, followed by an expansion of
the elastic inner wall of the ring cells. In A. dactyloides, the signal transduction pathway involved in
ring cell inflation has been investigated (Chen et al., 2001). The pressure exerted by a nematode on
the ring appears to activate G-proteins in the ring cells in this fungus. The activation causes an
increase in cytoplasmic Ca2+, calmodulin activation, and the opening of water channels. The ring
cells expand to constrict the ring and thus immobilize the nematode.
Biological Control of Nematophagous Fungi
In agriculture, biological control of pests refers to a strategy of pest management that makes use of
herbivory, parasitism, predation, or other natural mechanisms. It may play a significant role in
integrated pest control (IPM) strategies. Species known as parasitoids mature on or within a single
insect host, ultimately killing the host or lethally infecting it. Infection-causing pathogens including
bacteria, fungi, and viruses kill or weaken their own host and are relatively specific.
Natural enemies with varied modes of action that are similar to those being researched by plant
pathologists for the control of soil-borne diseases have been discovered by nematologists. Several
species that are efficient in the field as nematode natural enemies may have limited promise when

220
used by cultivators as biological control agents. Nematophthora gynophila, for instance, is a common
cause of cereal-cyst nematode reduction in many soils, but its potential utility is too limited to
guarantee continuing advancement as a biological control agent.
Fig. Trapping mechanism of constricting rings,

Trapping mechanism of constricting rings of A.
brochopaga. (a–c) Closure of a ring triggered
by applying heat to the trap. The closure is
rapid (0.1 s), irreversible and is accompanied
by a large increase in cell volume leading to an
almost complete closure of the aperture of the
trap. Bars, 5 μm. (d) Nematode firmly captured
in a ring. Bar, 10 μm (Source: Hertz et al.,
2006).
Over 160 species of nematophagous fungi are nematode's primary predators. Global pests in
horticulture and agriculture, plant-parasitic nematodes including root-knot and cyst nematodes
significantly reduce crop yields. They consist of three main groups of fungi: the nematode trapping
and endoparasitic fungi, which use specialised structures to attack vermiform living nematodes in a
variety of soil environments where they primarily survive as saprophytes (Hertz et al., 2006), and the
egg- and cyst-parasitic fungi, which attack these stages with their hyphal tips (Greg et al., 2000).
Possibility of utilizing nematophagous fungi for biological control of plant-parasitic nematodes is a
significant feature of these organisms. New nematode management alternatives are consequently
required because numerous nematicides have been banned due to health and environmental concerns.
Efficacy of Nematophagous fungi in agriculture
Nematophagous fungi are more beneficial than other species, such as bacteria and viruses, for the
biological control of PPNs. These NF are present in the nematode prey habitat. Direct and indirect
impacts of NF can be used to categorize its significance. By parasitizing adults, juveniles, and eggs
as well as by generating poisons or secondary metabolites that make worms immobile, NF is able to
directly influence nematodes. The previously described mechanisms are used by many NF. In order
to have indirect impacts, this mechanism can affect PPN activities, such as egg laying, egg hatching,
and the full life cycle from juveniles to adults, by triggering both plant defences (Lopez-Llorca et al.,
2010) and plant resistance in monocotyledon and dicotyledon (Bordallo et al., 2002).
Verticillium chlamydosporium and Arthrobotrys oligospora, two nematophagous fungus, invaded the
tomato and barley plants (Bordallo et al., 2002). After being used to treat bananas, endophytic
Fusarium oxysporum demonstrated its capacity to lower the population of PPNs Meloidogyne sp
(Waweru et al., 2014). By harming juveniles (an important stage for producing plant disease) and
eggs, some biocontrol fungus, such as Trichoderma, can be helpful in lowering the pathogenicity
activity of nematodes. While T. brevicompactum T-3 may significantly reduce egg production by

221
86%, Trichoderma asperellum T-16 lowered densities of juveniles (second-stage J2s) in roots by
roughly 80%. (Affokpon et al., 2011).
Methods utilizing Nematophagous fungi in sustainable agriculture
Nematodes that parasitize plants are a major source of production and financial losses in global
agriculture. Nematodes' small size, subsurface habitat, and vague indications of plant infection make
them difficult for farmers to detect (Kim, 2015). Many instances of crop loss go unreported or are
frequently attributed to other sources, such as fungus infestations, hydric stress, nutrient deficiency,
or other soil-related issues (Abd-Elgawad and Askary, 2018). The most common method for
managing PPNs is chemical nematocides, but their usage is increasingly being reconsidered due to
their numerous environmental and health risks, as well as their scarcity and high cost (Degenkolb
and Vilcinskas, 2016a, b). The development of resistant nematode races, which might make
management even more challenging, is a major drawback of the repeated use of these drugs (Abd-
Elgawad and Askary, 2018). Since NF are PPNs' natural adversaries and can aggressively consume
them. The research of these fungi's use, shelf life, culture, mass production, compatibility with other
biotic and abiotic variables, and crop management strategies are all part of this trend (Kim, 2015).
Bioproducts having these NF in their formulation have various advantages over chemical
nematicides for more sustainable agriculture, including ease of application, environmental safety,
minimal impact on the soil biota, and no residues in harvested goods.
However, because it is a living system, there are several factors to consider when developing a
commercial bionematicidal product. This study is important for determining both the biotic and
abiotic factors that affect the biocontrol agent and the target parasite in order to achieve effective and
reproducible biological control (Figure 4). Fungi must be able to grow well in the treated field,
taking into account the influence of chemical, physical, and other biological factors in the soil that
act as fungistatic compounds, as well as the fact that these factors can be difficult to predict due to
crop rotations (Yang et al., 2007). As a result, new technology has been incorporated into
biopesticide production. Genetic modification techniques are used to improve virulence, nematode
aggression, UV protectants, heat shock factor expression, immune modulators, and cuticle degrading
enzymes. There are two broad approaches to using NF as a biological control agent. One method is
to add large amounts of fungi to the soil or as an endophytic organism of the plant by coating seeds
to promote rhizosphere colonisation. Before the nematodes are drawn to the plant roots, the fungus
can establish itself (NordbringHertz et al., 2006). The second technique is to improve the
bionematicide formulation with the addition of nematophagous fungus biocontrol agents to improve
the growth, adaptability, and efficiency of NF as an integrated pest management technique (Yang et
al., 2007). In this case, combining a fungus as a biocontrol agent with a lower dose of chemical
pesticide or plant product can yield effective results if the components are compatible (Kim, 2015).
To maximise the action of the biopesticide, various formulations have been developed. A study of
the fungus T. viride in combination with the chemical nematicide carbofuran revealed an increase in
plant height, root length, and a decrease in the population of the nematode M. graminicola in rice
crops (Pankaj et al., 2015). Similarly, an ovicidal fungus P. lilacinus formulation added to the plant
product neem cake exhibits egg parasitism of the nematode N. incognita and plant growth in
tomatoes (Zaki and Maqbool, 1992). Other formulations employ the phytohormone abscisic acid
(ABA), which boosts plant defence and increases the nematode-trapping ability of D. stenobrocha, a
constricting ring-forming fungus (Xu et al., 2011). Several studies have also shown that using

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bacteria P. fluorescens or a combination of several fungi species can be an effective integrated pest
management technique for PPNs (Elgawad and Askary, 2018).
Conclusion
The interaction of fungi and nematodes is intriguing, particularly the use of NF as an alternative to
synthetic chemicals used in the production of nematocides. Techniques for genome sequencing and
transcriptome analysis, as well as techniques for detecting chemicals at the nanoscale, have been
developed in recent years. We know a lot about the relationship between NF and nematodes, which
includes enzymes and secondary metabolites as toxins.
This antagonistic relationship is based on the development of a unique structure that captures the host
and produces a toxin. "-omics" data can provide information on the interactions. The enhancement in
the manufacture of bionematicides is depending on selecting aggressive isolates of NF by
determining the virulence factors to a molecular level for isolates of NF. Efficiently obtaining
bionematicides is a target and requirement for all researchers in the field of agriculture sustainability.
Some information is still lacking, depending on the factor of pathogenicity. Some enzymes, such as
serine proteases, chitinases, and toxins, of NF are more interesting in the process of infection against
parasitic nematodes through their role as virulence factors. The production of various enzymes
during parasitic nematode infection necessitates their penetration of various layers of cuticle and
eggshell. Some strains of NF's success indicate differences in host preference. Some NF, such as the
genus Trichoderma, play an important role in agricultural sustainability. Finally, we consider the
widespread use of NF in the control of PPNs as a viable alternative to synthetic chemicals, and they
may be more useful in sustainable agriculture by reducing harmful chemical residues in the
environment.
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laterosporus G4. Arch Microbiol 186:297–305.
15. Tikhonov VE, Lopez-Llorca LV, Salinas J, Jansson HB (2002) Purification and characterization
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suchlasporium. Fungal Genet Biol 35:67–78.
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52:130–139.
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characterization of a serine protease from the nematophagous fungus, Lecanicillium psalliotae,
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***
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Chapter 21
ROLE OF FUNGI IN BIOREMEDIATION OF
POLLUTANTS
Dr. Jahanarah Khatoon
A bstract
Human conscious attempts to achieve a sustainable approach towards purification and restoration of
polluted habitats by cleaning up contaminated soil and water by use of organisms—including fungi,
bacteria, and their enzymes is a cost-efficient, sustainable, and natural approach (in comparison with
other typical techniques). Incineration is one of the most effective and common remediation
practices, however it is associated with a number of shortcomings. Bioremediation is biologically
mediated process used to remove/ neutralize an environmental pollutant from contaminated sites.
Bioremediation seems to be one of the promising alternative and safer treatment strategy as
compared to incineration. Fungi being natural decomposers of waste matter and secrete several
extracellular enzymes, capable of decomposing lignin and cellulose, (essential components of plant
fiber), are considered as potential candidates of bioremediation. Fungi possess robust and diverse
metabolic capacities to break down complex and recalcitrant organic material present in nature.
Mycoremediation, is term used for remediation by fungi. Mycoremediation, is term used for
bioremediation by fungi. This book chapter provides an overview of bioremediation and focusses on
microbial process because the cycling of organic compounds in the environment is an important part
of bioremediation.
Keywords: Bioremediation, Fungi, Mycoremediation, Enzymes, Environmental Pollutant.
Assistant Professor, Department of Biosciences, Integral University, Lucknow 226026, U.P.

India. jahanarah@iul.ac.in

225
I ntroduction
Fungi is nature’s most dynamic agents for the decomposition of waste matter; perform an essential
role in the decomposition of organic matter and have fundamental role in nutrient cycling and
exchange in environment. Fungi is also involved in providing nourishment for the other biota that
live in the soil and act as an essential component of the soil food web (Rhodes, 2012). Fungi is the
only living organisms present on Earth that can decomposes wood. Mycelium is the vegetative part
of the fungus, which exudes powerful extracellular enzymes and acids that are able to decompose
essential components of plant fibre including lignin and cellulose. Structurally mycelium looks like
as fine, white threads that grow out from dead wood, leaves, etc. Humus is a rich organic material
formed by breakdown of woods and leaves, by fungus. The rate of degradation is directly
proportional to decent supply of nutrients in the soil, e.g., N, P, K and other essential inorganic
elements. (Rhodes, 2013). Aspergillus is one of the oldest genera of kingdom fungi, and it producing
numerous useful extracellular enzymes and organic acids, these moulds also produce secondary
metabolites of importance in biotechnology. Aspergillus and other moulds are highly efficient in
decomposing starches, hemicelluloses, celluloses, pectins and other sugar polymers. Some
Aspergilli are capable of degrading of more refractory compounds such as fats, oils, chitin, and
keratin. Any undesirable change in the properties of a man-made items, such as paper and textiles
(cotton, jute and linen) are readily degraded by these moulds, and this process is often referred to as
bio-deterioration (Rhodes, 2013).
Fungi are among the potential candidates of bioremediation as they are natural decomposers of waste
matter and secrete several extracellular enzymes capable of decomposing lignin and cellulose, the
two essential components of plant fiber. Fungi are relatively more tolerant to higher level of
contamination than algae, archaea, and bacteria (Yadav et al., 2017). The efficiency of fungal
assisted phytoremediation mainly depends on host plant genotype and the type of fungal strain being
used. (Kumar et al., 2020). Mycoremediation has been found very efficient in the decontamination of
multiple contaminates and therefore can serve the purpose effectively. Various fungal species are
generally distinguished by their biochemical, physiological and metabolic capacities to metabolize or
degrade a number of hazardous or persistent chemicals (Barrech et al., 2018). Myco-remediation
could be one of the ideal strategies to clean the contaminated soil and water. It is necessary to
correctly identify and select the fungal species to target a particular pollutant to achieve a successful
mycoremediation. Selection and identification of the correct fungal species to target a particular
pollutant is the key component in process of mycoremediation. A simple screening procedure has
been described for selection of fungi to a particular target. (Kumar et al., 2019).
Myco-remediation is economically efficient and ecological sound strategy to counter the escalating
crisis of aquatic and terrestrial pollution. The benefits employing fungi as a potential candidate for
mycoremediation includes vigorous growth, massive hyphal network, secretion of multipurpose
extracellular enzymes, and increased surface area to volume ratio, skirmish capabilities towards
complex pollutants, compliance to fluctuating pH, temperature, and having metal-binding proteins
(Kumar et al., 2019; Kumar et al., 2020).

226
Bioremediation of Contaminated Land

Polluted groundwater and soil have damaging effect on the flora and fauna of affected habitats
because of accumulation of toxic chemicals in food chains. Even after numerous efforts taken to
counter this problem, still a significant number of known polluted sites exist and new ones are
continually being discovered. Employment of microorganism to decontaminate the soil and water
from pollutants is known as bioremediation (Rhodes, 2013). Bioaccumulation of methylmercury in
shellfish causing poisoning in humans upon consumption is well known example of impact of these
pollutant on each trophic level of food chain. The effective solution to mitigate this problem of
emerging pollution is bioremediation. In situ and ex situ are two methods employed in
bioremediation. In situ methods, the contaminated material is treated onsite, whereas in ex situ
method, the material is physically removed to be treated elsewhere. Decontamination of soil is a
relatively costly procedure, as compared to cleaning done via agents such as chemical methods, or by
incineration. However, bioremediation is used to degrade or convert highly toxic organic materials to
harmless compounds. The end result of bioremediation is the full mineralization of contaminants,
i.e., their transformation to CO2, H2 O, N2, HCl, etc. Heavy metal and radioactive cations, of course,
cannot be decomposed but can be rendered into forms of low solubility, e.g., by a change in
oxidation state, such as U (IV) (in UO2) (Singh et al., 2014), so that they remain less harmfully in the
ground, or might be physically removed by phytoremediation or mycoremediation, which involves
harvesting the plant or fungus.
Bioremediation Potential of Fungi
Emerging contaminants are class of chemicals and compounds that have been identified as
hazardous to environment and ecosystem including mankind. The chief source of emerging
contaminants are plastic polymers, pesticides, pharmaceuticals and personal care products (PPCPs),
heavy metals and industrially-related synthetic dyes and dye-containing pollutants. However, these
emerging pollutants have hostile effects, on every living organism, ranging from its impact on
individual organism to ecosystem level (Rhode.et.al.2012). Fungi plays a vital role in bioremediation
of contaminants such as persistent organic pollutants (POPs), textile dyes, coal, paper leather tanning
effects, pharmaceuticals and personal care products (PPCPs), polycyclic aromatic hydrocarbons
(PAHs), and pesticides .(Prasad 2017, 2018). Fungi from different class including Aspergillus,
Penicillium, and White-red fungi are key players for bioremediation and decolorization of textile
dyes, sugar industry effluent, chemicals used in kraft pulp mills, and leather tanning effluent,
indicating the diverse substrate choices of these fungi (Redman et al. 2001; Redman 2002; Rockne
and Reddy 2003). Removal of petrol and diesel contaminants from soil by incubating Aspergillus
niger and Phanerochaete chrysosporium for short period of time with petroleum hydrocarbons was
shown in conjunction with total organic carbon (TOC) elimination, which helps in bioremediation
(Timmis 2010; Redman and Kim 2011; Echeveria et al. 2020).
Use of Fungal Enzymes in Bioremediation
Fungal enzymes such as cellulases, laccases, peroxidases, xylanases, amylases, proteases, lipases,
catalases, chitinases, etc. have industrial significance and have application in organic waste
management. (Betancor et al. 2013). White rot fungi possess a range of enzymes including releases
of extra-cellular lignin modifying enzymes with a low substrate-specificity, which can act upon
various molecules that are broadly similar to lignin. In addition to this, they can also be used in
bioremediation requiring degradation of numerous xenobiotic compounds, including dyes (Nigam

227
2013). White rot basidiomycetes produce Laccases and certain fungal class II to degrade organic
pollutants (Quintella et al. 2019).
Mycoremediation Using Fungi
Scientific literature shows that about 30% of the bioremediation, is fix by means of fungi (Cruz-
Hernández et al. 2013). Various strains of white rot fungi have differing degree of suitability to
several organic molecules, including untraceable and persistent components such as PAHs and they
also involved in effectively disintegrating the hazardous compounds such as dioxins, pesticides,
phenols, polychlorinated biphenyls and chlorophenols, effluent, dyestuffs, and heavy metals.
(Egamberdieva and Lugtenberg 2014). Decontamination of Soil polluted with crude oil can be done
by mixing it with a sawdust or maize cob (lignocellulose substrate) which allows the fungal species
to proliferate in the soil and decompose the crude oil.
Technological Advancement in Fungal Bioremediation
Recently bioremediation using immobilized fungi in various bioreactors such as rotating biological
contactors and fluidized bed reactors are in trend (Tordoff et al. 2000). Bioremediation of
benzo[a]pyrene under nutrient-enhanced conditions (involving ligninolysis) results in PAH oxidant
monooxygenesis, which was also removed during a subsequent non ligninolytic process (Joutey et al.
2013). Sewage treatment plants, mixed with a filament inoculum in a broad-scale bioreactor, has
been shown to be sustainable and environmentally friendly when performed using a continuous
process (Connell and Staudigel 2013; Yadav et al. 2019). New reactive biobarriers of Trichoderma
longibrachiatum on nylon sponges, shown to remove of 90% of PAHs in a span of 14 days (Tyagi
et al. 2011; Li et al. 2013).
Bioremediation Using Fungal Cytochromes
Complex enzyme detoxification mechanisms are found in bodies of fungi for oxidative and hydrolyte
detoxification apart from this, some possess intracellular xenome networks consisting of cytochrome
P450 monooxygenases and glutathione transferases. P450 monooxygenases and glutathione
transferases enable them to cope with various different pollutants. The fungal cytochrome P450
system is a flexible catalyst for region-specific and stereospecific oxidation of non-activated
hydrocarbons. Generations of molecular instruments such as wide range of yeast expression systems
with a viral vector (Arxula adeninivorans), which are able to generate cytochrome P450
monooxygenases quickly and abundantly, helps in eradication of pollutants (Arxula adeninivorans)
(Tangahu 2011; Singh et al. 2013).
Phytoremediation Using Arbuscular Mycorrhizal Fungi
Bioremediation is a technology that basically employs microbes to treat contaminated soil. The
combination of Arbuscular Mycorrhizal Fungi (AMFs) and plants also plays a role in soil
remediation. AMFs have been found to reduce metal toxicity to plants through a decrease in the rate
of root-to-shoot translocation (Fan and Liu 2011). Phytoremediation is a technique refers to remove
dangerous elements or compounds from soil or water, most usually heavy metals and may further
cultivate and discard or remove heavy metals. Organic pollutants (such as PAHs) are transformed by
the microbial activity that is commonly seen around plant roots (Gianinazzi et al. 2010). PAHs are
degraded by exudates from plant roots, microorganisms, and AMFs. Different methods of soil
quality improvement were assessed, in non-inoculated soil and soil inoculated with a single AMF
mix (indigenous AMFs) were studied (Kumar et al. 2008, 2017; Sim et al. 2019). The AMFs in the
soil inoculated with the indigenous mix were found to be effective in soil quality improvement. The

228
presence of AMF nodules in the soil increased plant growth, water infiltration, and soil aeration
through soil agitation (Francis and Read 1995).
Conclusion
Bioremediation is a flexible and environmental friendly remedy to counter emerging and persistent
environmental pollutants and is rapidly growing practice. Some microbes possess inherent property
to deal with environmental pollutants, they detoxify or convert them into less hazardous forms (US
Environmental Protection Agency 1999). The mycoremediation capabilities are species specific for
individual or group of contaminants. The identification of pollutant specific species and its growth
influencing factor such as temperature, nutrient condition etc. is critical for enhancing the
remediation process. Recent research focusing on understanding of bioremediation mechanisms and
genomic developments has shown that whole-genome studies can help to explain and explore
bioremediation pathways. Polluted/contaminated land (unfit for agriculture) can be remediated via
use of arbuscular mycorrhizal fungi (AMFs) to make it suitable for agriculture. The yield and
nutritional value of crops are also increased by use of AMFs. AMFs habours strong degraders of
polycyclic aromatic hydrocarbons. AMFs can also be used in bioassays to test soil and its toxicity
levels, because of their sensitivity to a great variety of pollutants.
References
1. Rhodes CJ (2012) Feeding and healing the world: through regenerative agriculture and
permaculture. Sci Prog 95(4):345–446.
2. Rhodes CJ (2013) Applications of bioremediation and phytoremediation. Sci Prog 96(4):417–
427.
3. Kumar M, Prasad R, Goyal P, Teotia P, Tuteja N, Varma A, Kumar V (2017) Environmental
biodegradation of xenobiotics: role of potential microflora. In: Hashmi MZ, Kumar V, Varma A
(eds) Xenobiotics in the soil environment: monitoring, toxicity and management. Springer,
Cham, pp 319–334.
4. Yadav AN, Mishra S, Singh S, Gupta A (eds) (2019) Recent advancement in white
biotechnologythrough fungi. Springer, ChamZengguang X, Yanlong L, Junrui C, Ronggao Q,
Rong Y (2015) Bioremediation modelling of anaquifer contaminated by benzene using the
slow-release oxygen source technique. Arab J SciEng 40(9):2457–2463.
5. Kumar V, Dwivedi SK (2019) Hexavalent chromium reduction ability and bioremediation
potential of Aspergillus flavus CR500 isolated from electroplating wastewater. Chemosphere
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6. Singh P, Raghukumar C, Parvatkar RR, Mascarenhas-Pereira MB (2013) Heavy metal tolerance
inthe psychro tolerant Cryptococcus sp. isolated from deep-sea sediments of the Central
IndianBasin. Yeast 30(3):93–101.
7. Timmis KN (ed) (2010) Handbook of hydrocarbon and lipid microbiology. Springer, Berlin
8. Echeveria L, Gilmore S, Harrison S, Heinz K, Chang A, Nunz-Conti G, Cosi F, Singh P, BondT
(2020) Versatile bio-organism detection using microspheres for future biodegradation
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9. Redman RS, Kim YO (2011) Increased fitness of rice plants to abiotic stress via habitat
adaptedsymbiosis: a strategy for mitigating impacts of climate change. PLoS One 6(7): e14823.
10. Betancor L, Johnson GR, Luckarift HR (2013) Stabilized laccases as heterogeneous bioelectro -
catalysts. Chem Cat Chem 5(1):46–60.
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12. Quintella CM, Mata AM, Lima LC (2019) Overview of bioremediation with technology
assessment and emphasis on fungal bioremediation of oil contaminated soils. J Environ
Manag241:156–166.
13. Cruz-Hernández A, Tomasini-Campocosio A, Pérez-Flores LJ, Fernández-Perrino FJ,
Gutiérrez-Rojas M (2013) Inoculation of seed-borne fungus in the rhizosphere of Festuca
arundinaceapromotes hydrocarbon removal and pyrene accumulation in roots. Plant Soil 362(1–
2):261–270.
14. Tordoff GM, Baker AJ, Willis AJ (2000) Current approaches to the revegetation and
reclamationof metalliferous mine wastes. Chemosphere41(1–2):219–228.
15. Joutey NT, Bahafid W, Sayel H, El Ghachtouli N (2013) Biodegradation: involved
microorganismsand genetically engineered microorganisms. In: Chamy R (ed) Biodegradation:
life of science.InTechOpen, London, pp 289–320.
16. Connell L, Staudigel H (2013) Fungal diversity in a dark oligotrophic volcanic ecosystem
(DOVE)on Mount Erebus, Antarctica. Biology 2(2):798–809.
17. Tyagi M, da Fonseca MM, de Carvalho CC (2011) Bioaugmentation and biostimulation
strategiesto improve the effectiveness of bioremediation processes. Biodegradation 22(2):231–
241.
18. Li Y, Fu K, Gao S, Wu Q, Fan L, Li Y, Chen J (2013) Increased virulence of
transgenicTrichodermakoningi strains to the Asian corn borer larvae by over expressing
heterologous chit42 gene withchitin-binding domains. J Environ Sci Health 48(5):376–383.
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phytore-mediation. Int J Chem Eng 2011:939161.
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Chapter 22
FUNGAL METABOLITES WITH ANTICANCER
ACTIVITY
V. Naveen Sahith1 & V. Sai Sruthi2
A bstract
Cancer is one of the most prevalent and deadly diseases in the world. According to the World Health
Organization, cancer is the second leading cause of death globally, responsible for approximately 9.6
million deaths in 2018. Traditional cancer therapies, such as chemotherapy and radiotherapy, often
have debilitating side effects, leading researchers to investigate alternative treatments.
Fungi have long been known to produce secondary metabolites with pharmacological properties,
including anticancer activity. These metabolites have the potential to be developed into new cancer
therapies with fewer side effects than traditional treatments. In this review, we will explore recent
research on fungal metabolites with anticancer activity and their potential as cancer therapies.
Fungi are a well-known and valuable source of compounds of therapeutic relevance, in particular of
novel anticancer compounds. Although seldom obtainable through isolation from the natural source,
the total organic synthesis still remains one of the most efficient alternatives to resupply them.
Furthermore, natural product total synthesis is a valuable tool not only for discovering new complex
biologically active compounds but also for developing innovative methodologies in enantioselective
organic synthesis.
Keywords: Fungal Metabolites, Anticancer Activity.
1Lecturer in Microbiology, Smt. N. P. Savithramma women’s degree college, Chittoor, Andhra

Pradesh, India. naveensahith30@gmail.com
2Lecturer in Microbiology, Sudha vocational college, Kakinada, Andhra Pradesh, India
V. Naveen Sahith & V. Sai Sruthi

231
I ntroduction
Cancer is a generic term for a large group of diseases that can affect any part of the body. Other
terms used are malignant tumors and neoplasms. One defining feature of cancer is the rapid creation
of abnormal cells that grow beyond their usual boundaries, and which can then invade adjoining
parts of the body and spread to other organs; the latter process is referred to as metastasis.
Widespread metastases are the primary cause of death from cancer.
Cancer Epidemiology
According to the Current Cancer report garnered by World Health Organization (WHO), cancer is a
leading cause of death worldwide, accounting for nearly 10 million deaths in 2020 (1). The most
common in 2020 (in terms of new cases of cancer) were:
• breast (2.26 million cases)
• lung (2.21 million cases)
• colon and rectum (1.93 million cases)
• prostate (1.41 million cases)
• skin (non-melanoma) (1.20 million cases)
• stomach (1.09 million cases)
The most common causes of cancer death in 2020 were-
• lung (1.80 million deaths)
• colon and rectum (916 000 deaths)
• liver (830 000 deaths)
• stomach (769 000 deaths)
• breast (685 000 deaths)
Each year, approximately 400 000 children develop cancer. The most common cancers vary between
countries. Cervical cancer is the most common in 23 countries.
Cancer is a leading cause of death worldwide, accounting for nearly 10 million deaths in 2020, or
nearly one in six deaths. The most common cancers are breast, lung, colon and rectum and prostate
cancers.
Tobacco use, alcohol consumption, unhealthy diet, physical inactivity and air pollution are risk
factors for cancer and other non-communicable diseases. Some chronic infections are risk factors for
cancer; this is a particular issue in low- and middle-income countries. Approximately 13% of cancers
diagnosed in 2018 globally were attributed to carcinogenic infections, including Helicobacter pylori,
human papillomavirus (HPV), hepatitis B virus, hepatitis C virus, and Epstein-Barr virus.
Although cancer remains a devastating diagnosis, advances in cancer biology and biotechnology
have recently led to the successful development of a number of drugs that improve survival. Among
all the strategies used for combatting various types of cancers, cytotoxic agents remain powerful
weapons. In addition to these, recent additions include, among others, targeted therapies, therapies
based on cancer epigenetics, photodynamic therapy, immunotherapy, and antiangiogenic agents.

232
The Role of Natural Products in Cancer Drug Discovery

Natural products and their derivatives have traditionally been a major source of new anticancer
agents. According to the analysis of Newman and Cragg, in the timeframe from the 1940s to date, of
the 175 small molecule cancer drugs, 131 (or 75%) belong to the category of “other than synthetic”.
1 Of these, 85 (or 49%) are actually natural products or their direct derivatives.1 Natural products
derived from bacteria and plants play a leading role in cancer drug discovery, which is demonstrated
by the large number of approved anticancer agents derived from these natural sources. These drugs
include such effective chemotherapeutic agents as doxorubicin, daunomycin, mitomycin C, and
bleomycin, all obtained from Streptomyces, or etoposide, teniposide, topotecan, paclitaxel, and the
vinca alkaloids (vincristine, vinorelbine, .) derived from plant-based natural products.
Fungal derived metabolites as potential anti-cancer drugs

Fungi-derived natural products have been an excellent source of pharmaceuticals as well. The
anticancer metabolites are grouped into three categories based on the fungal source from which they
are isolated. These include metabolites produced by (i) phytopathogenic, (ii) toxigenic, and (iii) non-
toxigenic fungi
A number of fungal metabolites and/or their analogues have progressed to various stages of cancer
clinical trials.
Anguidine
It is also known as 4,15-diacetoxyscirpenol, is a well-known mycotoxin belonging to the group of
trichothecenes produced by several Fusarium species, including F. roseum, F. sambucinum, F.
tricinctum, F. equiseti, F. lateritium, F. graminarium, F. semitectum, F. sulfureum, F. diversisporum,
F. scirpi, and Giberella intrigans. 18, 20 Anguidine irreversibly blocks protein synthesis by
inhibiting the protein chain initiation through degradation of polyribosomes. Early preclinical studies
at the National Cancer Institute (NCI) revealed >100% lifespan extension of P-388 leukemia-bearing
mice with chronic administration of anguidine. However, based on a broad Phase II study conducted
at the M. D. Anderson Hospital and Tumor Institute and the University of Kansas (the Southeastern
Cancer Study Group) from November 1977 to June 1981 , no meaningful activity was found in six
tumor categories and the conclusion was made that anguidine did not warrant additional evaluation
for the treatment of solid tumors
Phenylahistin
Phenylahistin is a fungal diketopiperazine metabolite consisting of phenylalanine and isoprenylated
dehydrohystidin, which was isolated from Aspergillus ustus as a racemic mixture.22 Later, the
enantiomers were separated by chiral HPLC and their structure was assigned by physical and
spectroscopic data.88 Of interest to this review was that (−)-phenylahistin was found to exhibit
antitumor activity in vitro against eight tumor cell lines (A431 dermal, A549 lung, Hela ovary, K562
leukemia, MCF-7 breast, TE-671 CNS, WiDr colon) with IC50 values ranging from 0.18 to 3.7 μM.
However, the (+)-enantiomer exhibited 33–100× less-potent activity. (−)-Phenylahistin also showed
antitumor activity against P-388 leukemia and Lewis lung carcinoma cells in vivo.88 This natural
product has been shown to have potent vascular disrupting properties through binding to the
colchicine site on beta-tubulin.89 Currently its semisynthetic analogue, NPI-2358 (plinabulin,), is in
clinical trials involving patients with NSCLC . A Phase I study of NPI-2358, in combination with
docetaxel, revealed this combination to be quite tolerable with promising indications of antitumor

233
activity. Currently, this combination is being assessed in a randomized Phase II clinical trial in
previously treated patients with advanced and metastatic NSCLC.
Cytochalasins
Cytochalasins from Pyrenophora and Phoma species Cytochalasins are part of a larger group of
metabolites named cytochalasans and they incorporate diverse polyketide–amino acid hybrid
structures with a wide range of distinctive biological functions.Up to now, more than 60 different
cytochalasans from several species of fungi have been puried, identied and sub-grouped as described
in a comprehensive review by the Hertweck group. The most thoroughly investigated biological
effects of cytochalasans in cell culture involve the capping of actin filaments, which results in
cytokinesis impairment during cell division and also affects cancer cell migration properties.While
most cytochalasins are cytotoxic at their IC50 concentrations, some members of this group of fungal
metabolites, including for example cytochalasin A are cytostatic, a feature that could be explained,
at least partly, by the targeting of the Kv1.5 ion channel by cytochalasin A.Because of their actin-
binding properties cytochalasins have been broadly used to study actin microfilaments and their
involvement in cell motility, ruffling, cell division, contraction and cell stiffness.5 Furthermore,
because tumor invasiveness and metastatic potential are tightly associated with deregulation of the
actin cytoskeleton, there is an appreciation of actin-targeting agents
Tryprostatins
Tryprostatins from Aspergillus fumigatus Tryprostatins A and B are indole alkaloidal fungal
products isolated from Aspergillus fumigatus, among other sources. They have also been obtained by
total synthesis. Tryprostatin A was first demonstrated to be an inhibitor of the mitogen activated
protein (MAP)-kinase-dependent microtubule assembly and, through the disruption of the
microtubule. Tryprostatins from Aspergillus fumigatus Tryprostatins A and B are indole alkaloidal
fungal products isolated from Aspergillus fumigatus, among other sources. They have also been
obtained by total synthesis. Tryprostatin A was first demonstrated to be an inhibitor of the mitogen
activated protein (MAP)-kinase-dependent microtubule assembly and, through the disruption of the
microtubule.
Halenaquinones
Halenaquinones from Xestospongia cf. carbonaria Halenaquinone was isolated from two Indo-
Pacific collections of the sponge Xestospongia cf. carbonaria. Additional metabolites isolated from
this sponge include tetrahydrohalenaquinone B, 14-methoxyhalenaquinone , xestoquinolide A,
xestoquinolide B, halenaquinol, halenaquinol sulfate, xestoquinone, and tetrahydrohalenaquinone A.
These natural products, along with halenaquinone derivatives, were tested for their ability to inhibit
the activity of various protein tyrosine kinases (PTKs); while halenaquinone inhibited EGF
(Epidermal Growth Factor) receptor activity (IC50 ¼ 19 mM), it was not active against protein
kinase C, a result suggesting that halenaquinone is not a general kinase inhibitor. Its congener 14-
methoxyhalenaquinone showed a similar activity (IC50 ¼ 5 mM), while synthetic derivatives were
less active. SAR analyses revealed the minimum structural requirements for strong PTK inhibitory
activity of halenaquinone derivatives involving the presence of a pentacyclic skeleton with
electrophilic sites at each end (e.g., the A/E- and D-rings of halenaquinone). The planar
polyunsaturated framework with a quinone end ring was found to be a necessary but not a sufficient
condition for PTK activity.

234
Fumagillin
Fumagillin is a complex metabolite first isolated from the liquid culture of Aspergillus fumigatus
strain H-3. Its structure was assigned based on chemical studies that included the preparation of a
number of key derivatives. Of interest was that biosynthetic studies showed that fumagillin in part
arises by a terpene route and in part by the acetate pathway. Fumagillin was also produced by other
species of Aspergillus, including A. flavus and A. parasiticus. Recently, a chlorinated derivative of
fumagillin (ligerine) was isolated from a marine-derived Penicillium strain, showing strong
inhibitory activity against an osteosarcoma cell line.Synthetically derived fumagillin analogues—
TNP-470, PPI-2458, and CKD-732 have also been evaluated in human cancer clinical trials.
Together with fumagillin, these synthetic analogues disrupt tumor vasculature by targeting the
enzyme methionine aminopeptidase type 2, which cleaves the N-terminal methioninyl residue of
newly synthesized proteins. Although Medicinal Research promising results for TNP-410 were
reported in patients with prostate cancer, NSCLC, and melanoma, neurotoxicity was observed to be
an important side effect, which seemingly limits its clinical usefulness. Understanding the
mechanism of neurotoxicity and developing strategies to overcome this dose-limiting side effect
would be required before this agent could be widely used in a clinical setting. The newer analogues,
PPI-2458 and CKD-732, appear to alleviate the neurotoxicity issues associated with TNP-470, but
their therapeutic effects remain to be demonstrated in the clinic
Bisorbicillinoids
Bisorbicillinoids from Trichoderma citronoviride The in vitro growth-inhibitory activity of 14
metabolites isolated from terrestrial (including phytopathogenic and toxigenic) fungi were
characterized in six cancer cell lines. The natural products were classified as those metabolites
associated with IC50 concentrations < 100 mM in all six cancer cell lines analyzed as opposed to
those metabolites associated with IC50 concentrations > 100 mM in at least one out of the six cancer
cell lines analyzed. Two metabolites, namely bislongiquinolide and 20, 30 -dihydrotrichodimerol
(bisorbibutenolide) were found with IC50 concentrations < 100 mM in the six cancer cell lines
analyzed. These two natural products were isolated from the biomass of fungus Trichoderma
citrinoviride on settling and feeding preference of the aphid Schizaphis graminum.
Bislongiquinolide is also named trichotetronine.The cytotoxic activity of 20, 30 -
dihydrotrichodimerol had been reported previously;20 ,30 -dihydrotrichodimerol was also shown to
activate the peroxisome proliferator-activated receptor-Y (PPAR-Y), which exerts major roles in
cancer cell biology. This metabolite has also been reported to suppress the production of tumor
necrosis factor-alpha (TNF-alpha) and nitric oxide in LPS (lipopolysaccharide)-stimulated
RAW264.7 cells. The remaining 12 compounds displayed weak cytotoxic activity, if any, in the six
cancer cell lines analysed. These 12 metabolites include phyllostin, seiricardines B and C, cavoxin,
cyclopaldic acid, fusapyrone, scytolide, seiricuprolide, seiridin, verrucarin E and 16,17-
dihydrobislongiquinolide (for structures,). The origin of each of these compounds is detailed in Balde
et al.
Fusarisetin-A
Fusarisetin A produced by Fusarium sp. FN080326 Fusarisetin A is produced by a strain of
Fusarium sp. FN080326 isolated from a soil sample collected in Daejeon, Korea. Fusarisetin A is an
acinar morphogenesis inhibitor that possesses both an unprecedented carbon skeleton and a new

235
pentacyclic ring system. Fusarisetin A is a potent cancer migration inhibitor and ex vivo studies
show that this compound is able to inhibit different types of cell migration
Pintulin
Pintulin from Penicillium vulpinum Pintulin is produced in racemic form together with
mhydroxybenzylic alcohol and isopatulin in the liquid culture filtrates of Penicillium vulpinum, a
strain F-4148 isolated from a soil sample collected at Towada city, Aomori prefecture, Japan.
Pintulin displays 40 times weaker cytotoxic activity against various cultured cancer cell lines than
adriamycin. Also, the administrations of 1.25 mg kg1 of pintulin on days 1 and 3 provided weak
therapeutic benefits in mice transplanted with p388 leukemia cells, while the continuous
administration for 5 days at 3.13 mg kg1 did not prolong the survival time of mice and resulted in
toxicity
Conclusion
cancer is a devastating disease that affects millions of people worldwide and remains a leading cause
of death. Despite the development of several effective chemotherapeutic agents, cancer remains a
challenging health problem, and natural products derived from bacteria, fungi, and plants have long
been a source of potential anti-cancer agents. Fungal metabolites have been found to possess anti-
cancer properties and have progressed to various stages of clinical trials. However, not all
compounds have been successful in clinical trials. Therefore, further research is needed to discover
and develop effective anti-cancer agents to combat this devastating disease.
References
1. https://gco.iarc.fr/today/data/factsheets/cancers/39-All-cancers-fact-sheet.pdf
2. https://www.who.int/news-room/fact-
sheets/detail/cancer#:~:text=Cancer%20is%20a%20leading%20cause,and%20rectum%20and
%20prostate%20cancers.
3. Fungal metabolites with anticancer activity Antonio Evidente,Alexander Kornienko, Alessio
Cimmino, Anna Andolfi, Florence Lefranc,Veronique Mathieu and Robert Kiss
4. Investigations of Fungal Secondary Metabolites with Potential Anticancer Activity ElHadj
Saidou Balde, Anna Andolfi, Ce´line Bruye`re, Alessio Cimmino,Delphine Lamoral-Theys,§
Maurizio Vurro,⊥ Marc Van Damme,Claudio Altomare,⊥ Ve´ronique Mathieu,Robert
Kiss,and Antonio Evidente
5. Fungal Anticancer Metabolites: Synthesis Towards Drug Discovery Margherita Barbero,
Emma Artuso and Cristina Prandi: Department of Chemistry, University Degli Studi di
Torino, via P. Giuria 7-10125 Torino, Italy
6. Fungal metabolites with anticancer activity Antonio Evidente, Alexander Kornienko, Alessio
Cimmino,Anna Andolfi, Florence Lefranc,c Veronique Mathieu and Robert Kiss
7. Anticancer compounds derived from fungal endophytes: their importance and future
challenges Ravindra N. Kharwar,Ashish Mishra,a Surendra K. Gond,a Andrea Stierle and
Donald Stierleb

236
8. Toward a Cancer Drug of Fungal Origin Alexander Kornienko, Antonio Evidente, Maurizio
Vurro, Veronique Mathieu, ´ Alessio Cimmino,2 Marco Evidente,Willem A. L. van Otterlo,
Ramesh Dasari,1 Florence Lefranc and Robert Kiss
9. Can Some Marine-Derived Fungal Metabolites Become Actual Anticancer Agents? Nelson
G. M. Gomes 1,2, Florence Lefranc, Anake Kijjoa 1,2 and Robert Kiss.
***
237
Chapter 23
FUNGAL TANNASE: THE MOST EMINENT
BIOCATALYST FOR INDUSTRIAL APPLICATIONS
S. Jayashri Pawar1, A. Ashwini Sanap2, A. Rupali Nikam3
A bstract
Tannase is one of the versatile biocatalysts which plays significant role in different bioconversion
under protein precipitating reaction. Fungal tannase are industrially important enzymes belongs to
the family hydrolases which cleave esters and lateral bonds of tannins such as tannic acid, gallo-
tannin, epigallocathechin-3-gallate, gallic acid esters with releasing glucose and gallic acid as end
product. The advantages of bioconversion process are high yields, environmental safety and process
reproducibility using biocatalyst (enzyme or whole cells of microorganisms) have been well
established over chemical technologies. Tannins are polyphenolic secondary metabolites of higher
plants (such as chestnut, Oak wood, Trillo, Valonea, Myrobalan, Divi-Divi), classified as
hydrolysable and non-hydrolysable tannins (condensed tannins). They are considered as the classic
defense compounds of plants against herbivores and have a broad-spectrum distribution in the plant
kingdom which includes pteridophytes, gymnosperms and angiosperms and generally accumulate in
large amounts in roots, bark, leaves and fruits. Microorganisms, especially filamentous fungi are the
more potent producers of tannase. Inspite of wastewater treatment and gallic acid production, tannase
have wide application in processing food, beverage and animal feed and also possess many
pharmaceutical applications.
Keywords: Tannase, Tannic acid, Gallic acid, Industrial Applications.
1Assistant Professor, Department of Microbiology, Sanjivani Arts, Commerce and Science
College Kopargaon, India. jayashripawar0912@gmail.com
2Quality Control Executive Microbiologist, Varun Beverages Pvt Ltd (PepsiCo), Aurangabad
3Quality Assurance Officer, Nilons Enterprises Pvt Ltd, Jalgaon, India
Jayashri Pawar, Ashwini Sanap, Rupali Nikam

238
I ntroduction
Enzymes are the biocatalyst that produced by living organisms which responsible to carry out a
specific biochemical reaction. They are employed for large number of reactions in different industrial
processes because they offer many advantages over chemical catalyst particularly the microbial
enzymes have many uses in various industries. Microbial enzymes are more stable and active than
plants and animal enzymes and also acts as an alternative source of chemical catalyst because they
can be cultured in short time in a large quantity by fermentation. By the use of specifically selected
strain now it is possible to produce purified, well characterized enzymes on large scale. Microbial
enzymes are growing more desirably for the industry because of their diversity and susceptibility to
gene manipulation (1).
The production of enzymes can be done by recombinant DNA technology that were not produced
commercially by meliorating the production processes. Here enzymes play an important role in
various biotechnology products and the processes that are commonly encountered in the production
of food and beverage, cleaning supplies, paper products, pharmaceuticals monitoring devices and
transportation fuels. Enzymes also play a key role in evolution and microbial adaptation (2).
Tannase
Tannase or tannin acyl hydrolase (E.C.3.1.1.20) is a microbial enzyme which is inducible and
catalyzes the hydrolysis of ester and depside bonds between substrates like gallo-tannin,
epigallocathechin-3-gallate, gallic acid esters and hydrosable tannins to liberate gallic acid and
glucose. The molecular weight of tannase is 0.5-3.0kDa (7).
Mechanism of Action
Tannase (tannin acyl hydrolase; EC 3.1.1.20) is one of the hydrolases that have been reported to
contain dual activities catalysing the hydrolysis of ester bonds (galloyl ester of an alcohol moiety)
and depside bonds (galloyl ester of gallic acid) present in gallotannins, complex tannins, and gallic
acid esters but they are not able to affect the carbon-carbon bonds i.e., this enzyme is known to have
two different activities. The first activity is an esterase activity; with the help of which it can
hydrolyze ester bonds of gallic acid esters with the glucose (galloyl-glucose) or alcohols (e.g. methyl
gallate) The second activity is called as depsidase activity; by which it cause the hydrolysis of
depside bonds of m-digallic acid. Tannase have different sources hence it has different molecular
masses. Such as in the case of fungi and yeast are forms hetero- or homo-oligomers having a two to
eight subunits. The dual activities of tannase are responsible to have a wide range of substrate
specificity. This specificity mainly depends on the source and the methods utilized for it am
production and isolation. Tannase acts as an isoenzyme. The gallic acid monomers are utilised to
simple aliphatic acids by oxidative breakdown, which enters into the citric acid cycle. Gallate
decarboxylase is responsible for conversion of gallic acid to pyrogallol before ring cleavage.

239
Fig.1: Hydrolytic Pathway of tannic acid by tannase
Fig.2: Diagram Representing Tannic acid degradation

240
Attributes of Tannase
Fungal tannase shows good activity at an optimum pH range of about 5.0-7.5 and the stability of the
enzyme could be observed at a wide range of pH of about 3.5-8.0 with an optimal temperature range
of 30-60ºC for different genus of fungus, whereas the bacterial tannase has an optimum activity at a
pH range of 4.5-5.5 with an optimal temperature range of 30-40ºC. The plant tannase isolated from
penduculate oak was shown to be active over a wide pH range with an optimum of approximately 5.0
at a temperature of 35ºC and 40ºC (4).
Limited molecular analysis has been made in fungal tannase when compared to bacterial tannase.
The molecular weight of tannase from Aspergillus strains, ranges between 150,000 Da to 350,000
Da. whereas in the plant pendunculate oak it was about 300,000 Da. The gene sequence
of Aspergillus oryzae codes for 588 amino acids with no introns and the tannase produced has a
molecular weight of 64,000 Da. Tannase from A. oryzae has four pairs of two subunits with one
subunit having molecular weight of 30,000 Da and the other with 33,000 Da that are linked by
disulphide bond, which form hetero-octamer with a molecular weight of about 300,000 Da. Tannase
from Candida sp. K1 also contains two subunits of 120,000 Da each that could be separated after
treatment with SDS and 2.mercaptoethanol (8).
Fungal Tannase
Fungi is the most dominant tannase producer among all microbes. Tannase is produced as a
membrane bound enzyme in most of the organisms and not all tannase is equally active against the
different tannin substrates. Fungal tannases have a better activity in degrading hydrolysable tannins,
whereas yeast tannases degrade tannin better and has a lower affinity for naturally occurring
tannins.5 Among the fungal species the best producer of tannase were found to be Aspergillus oryzae,
A. niger, A. japonicus, A. awamori, whereas the moderate producers were A. flavus,
Penicillium species, Trichoderma sp., Helicostylum sp., and Cunnighamella sp. Tannin degradation
by yeasts has not been studied to its full potential. Aoki et al. 1976 isolated and reported the
enzymatic degradation of gallotannins by yeast species belonging to Candida that was able to
produce tannase. The tannase from this yeast was able to hydrolyze the ester and depside linkages
from tannic acid. Production of tannase has been noted in Candida sp., Pichia sp., Debaryomyces
hansenii, Mycotorula japonica (3), (12) and Saccharomyces cerevisiae (11).
Tannase Substrate
Tannins are plant phenolic compounds which acts as a substrate for tannase. It is distributed in
various parts of vascular plants including stems, leaves, bark etc. Tannins are water soluble,
polyphenolic secondary metabolites of plants which are fourth most abundant plant constituents.
They come after cellulose, hemicellulose and lignin. Tannin provides an immunity to plants from
microbial attack, due to their accumulation in vulnerable parts of plants. Tannins are considered to be
antimicrobial in nature which inhibits the microbial growth. They are responsible to precipitate the
macromolecules such a proteins, cellulose, gelatin and starch by forming non reversible complexes
with them. Tannins currently have been classified into four groups as follows (7)(1).
1. Hydrolysable tannins or gallotanin-
These are the simplest form of polyphenols characterized by presence of many molecules of organic
acid such as digallic, Gallic and chebulic acid esterified to a molecule of glucose.

241
2. Ellagitannins-
They have building block made up of ellagic acid units linked to glucosides. These are more stable
than gallotannins.
3. Complex tannins-
Catechin-gallate with hydrolysable and condensed bonds is an example of this type of tannins.
Reaction between several units of Gallic or ellagic acids with catechin and glucosides responsible for
generation of complex tannins.
4. Condensed tannins-
Condensed tannins are also knowns as proanthocyanidins, which are oligomeric and polymeric
complex compounds. The catechin derivative such as cyanidin and delphinidin are responsible for
the astringent taste of wine and fruit.
Sources of Tannins
The word tannins are so old and it reflects traditional technology “Tanning” (waterproofing and
preserving) word was used to describe the process of the transforming animal hides into leather with
the help of plant extracts from different plant parts of different plant species. Tannins are abundantly
found in natural plants such as monocots, tea, coffee, sorghum, berries, nuts, pomegranates, legumes,
some herbes and spices like cloves and cinnamon, palm kernel and other different species of plants
or plant products which are used for human consumption.
Isolation, Screening and selection of potential strains
For isolation of Tannase producer Sample should be collected from tannin rich environment such as
agro industrial wastes, tannery effluent, slaughter house, pickle and beverages etc. Tannase
producing fungi are screened by using Tannic acid agar (TAA) medium which contained (g/L):
NaNO3, 3.0; KH2PO4, 1.0; MgSO4.7H2O, 0.5; KCl, 0.5; FeSO4.7H2O, 0.01; Tannic acid, 10;
Agar, 30; pH 4.5 as described by (Bradoo et al) (10). Autoclave medium at 121 °C for 15 min and
incubate for 96 hours at 30°C. After incubation observe for zone of tannic acid hydrolysis. For
further study select colonies showing large zone of tannic acid degradation. Incubate selected strain
for 5 days at room temperature with constant agitation at 120 rpm in an orbital shaker.
Harvesting of Tannase
After incubation for harvesting of Tannase, filter the mycelial suspension through Whatman No.1
filter paper and the centrifuge culture filtrate at 12,000 g for 30 min at 4 °C (10).
Tannase Assay
Tannase assay can be done by using following method described by Sharma et al. (9). based on the
formation of chromogen between gallic acid (released by the action of tannase on methyl gallate) and
rhodanine (2-thio-4-ketothiazolidine). Prepare standard curve using Gallic acid. One unit of tannase
activity is defined as the amount of enzyme required to liberate 1 μM of Gallic acid/min under
defined conditions (13).
Synopsis of Purification of tannase from various fungi
Fungal strain Tannase extraction and purification method Specific activity References
Aspergillus niger LCF 8 Mechanic Hydrolysis of mycelia followed by 1010 nkat/mg protein [19]
Ultrafiltration

242
Aspergillus japonicus Extracellular enzyme extracted using PEG and 91.89 U/mg [20]
Tannic acid for precipitation
643.12 U/mg (finally)
1. At pH 3.0
2. At pH 4.5
Aspergillus niger MTCC Crude extract isolated, dialysed and freeze 355.6 U/mg protein [17]
2425 dried. Reconstituted with Chromatography
Sephadex G-150 followed by DEAE Sephadex
A-50
Aspergillus awamori Acetone precipitated fraction of extracellular 3.23 U/ml (total [21]
nakazawa tannase, run through Gel Filtration 250 column activity)
(with pore size 4µm) with the solvent 0.2 M
acetate buffer
Penicillium variable Ultrafiltration, followed by Gel Filtration G-200 2055 U/mg [18]
Industrial application of tannase

❖ Preparation of instantaneous Tea-
After the water tea is second most highly consumed beverage. During the production of tea
beverages, hot and clear tea infusions turbid precipitate are formed. These precipitates are known as
a tea cream which is formed by a complex mixture of polyphenols. The cream is form due to tea
polyphenols which forms the hydrogen bonds with caffeine. When there is a cream formation, it is a
quality problem, and may have the anti-nutritional effects. Tannase have potential to hydrolyze the
ester bonds of catechins to release free Gallic acid and water-soluble compounds having lower
molecular weight, which reduces turbidity and responsible to increase the solubility of tea beverage
in cold water. This treatment of tea beverage gives a better colour appearance, low cream formation,
good taste, feeling of mouth, and overall acceptance. The green tea treated with tannase shows a
higher antioxidant property than normal green tea or black tea (14).
❖ Beverage clarification-
Recently new fruit juices (pomegranate, cranberry, raspberry, etc.) acclaimed for their health
benefits, in particular because of their antioxidant properties. The high tannin contents in those fruits
causes haze and sediment formation not only for colour, bitterness, but also astringency of the juice
upon storage. The quality of juices improves when it is enzymatically treated with tannase. Tannase
is acts as a clarifying agent in refreshing drinks with the coffee flavour and nowadays, this process
for the enrichment of antioxidant properties of coffee by using tannase and other enzymes has also
been patented (22).
❖ Gallic acid production for pharmaceutical industry-
Tannase have major application in the production of Gallic acid. Gallic acid is nothing but the 3, 4,
5-trihydroxybenzoic acid. It is the phenolic compound and it contains the monomeric units of the
Gallo tannins and complex tannins. Gallic acid and its compounds have therapeutic properties
including anticancer and antimicrobial properties. It has a major application in the manufacture of the
trimethoprim. It is also used in the leather industry, in the manufacturing gallic acid esters, such as a
propyl gallate. The propyl gallate is a potent antioxidant utilized as an antioxidant in fats and oils.
Gallic acid is helpful in the manufacture of pyrogallol. It is also used in assemble of the ink and
photographic developers. It acts as a photosensitive resin in semiconductor production (16)(13).

243
❖ Animal feed preparation-

Animal nutrition is affected by the high levels of dietary tannase because tannin binds to
macromolecule. Tannins are responsible for making the strong complex with enzymes, minerals, and
other nutrients. They give bitter taste, which is responsible for less feed intake. Tannins are widely
distributed in nature i.e. Have ubiquitous nature. They mostly found in feedstuffs, forages, fodder
and agro-industrial waste, affecting livestock production. Tannase or tannase producing
microorganisms can reduced the anti-nutritional effect of tannins. There is release of similar amount
of the reducing sugars from the all flours used as animal feed (barley, bran, maize, oat, rye, soya, and
wheat flour) when it treated with the enzymatic extract of tannase compared with commercial
enzymatic additives used in animal feeding (23).
❖ Bioremediation of Tannin- contaminated Wastewaters-
Tannins mostly found in effluent derived from several agro-industries one such include tanneries.
This kind of wastewater is difficult to be treated, because tannins are highly soluble and also, they
are responsible for inhibition of many microorganisms. The tannins in these effluents can be
degraded by the tannase. The tannery effluent treatment can be done by enzyme tannase which help
in removal of 42% of tannin content and 20% of the colour (23).
❖ Other potential Application of tannase-
In recent years ethanol as a fuel production from agro-industries wastes gained attention. Tannase
can cause degradation of oligomeric phenolic. Tannase gene and tannase activity can be used as an
indicator of colon cancer. For the therapeutic application the tannase can be utilized, for ex. Some
esters derived from pyrunioside with the anti- inflammatory activity. Tannase have other potential
application such as in the manufacture of laundry detergent, in cosmetology, and in leather industry
for the preparation of high-grade leather tannins (22)(23).
References
1. Landete, J. M. (2011). Ellagitannins, ellagic acid and their derived metabolites: A review about
source, metabolism, functions and health. Food Research International, 44(5), 1150–1160.
2. Archana D. Tripathi, Kajal Sharma and Laxmi B. (2016): Study on Tannase producing Bacillus
megaterium isolated from tannery effluent. International Journal of Advanced Research in
Biological Sciences 3, 28-35.
3. Deschamps A.M. and Lebeault J.M., Let., 6, 237–242 (1984).
4. Sharma, S., Bhat, T. and Dawra, R., 2000, “A Spectrophotometric Method for Assay of
Tannase Using Rhodamine” Anal. Biochem., 279(1) pp. 85-89.
5. Albertse E.K., M.Sc. thesis, Faculty of Natural and Agricultural Sciences, Department of
Microbiology and Biotechnology, University of the Free State Bloemfontein, South Africa
(2002).
6. Natarajan K, Rajendran A, Thangavelu V. Tannase enzyme: The most promising biocatalyst for
food processing industries. Biosci Biotechnol Res Asia 2008;5.
7. Dhruvil Brahmbhatt, H.A.Modi(2015): Comparative studies on methods of tannase assay.
International Journal for Research in Applied Science and Engineering Technology (IJRASET)
3, 13-98.
8. Aoki K. Shinke R. and Nishira H., Biol. Chem., 2, 297–302 (1976).

244
9. Niehaus J.U. and Gross G.G., Phytochem, 45, 1555–1560 (1997).

10. Bradoo, S., Gupta, R. and Saxena, R.K., 1996, “Screening of extracellular tannase-producing
fungi: Development of a rapid and simple plate assay”, J. Gen. Appl. Microbiol., 42(4), pp.
325- 329
11. Zhong X. Peng L. Zheng S. Sun Z. Ren Y. Dong M. and Xu A., Protein. Expr. Purif., 36, 165–
169 (2004).
12. Balmers R. Contreras-Esquivel J.C. Rodriguez-Herraera R. Ramirez Coronel A. and Aguilar
C.N., Wissenschaftund. Technologies, 37, 857–864 (2004).
13. Neethu R S, Pradeep N S Isolation and Characterization of potential Tannase producing fungi
from Mangroves and Tanneries Volume 21 Number 3 October - December 2018, pp. 1-13.
14. Shajitha, G and Nisha M.K. (2018): Tannase production from agro -wastes as a substrate by
Trichoderma viride. International Journal of Current Research in Life Sciences 7, 1994-1997.
15. Beniwal, V., Kumar, A., Sharma, J., & Chhokar, V. (2013). Recent Advances in Industrial
Application of Tannases: A Review. Recent Patents on Biotechnology, 7(3), 228–233.
16. Sharma, S., and Gupta, M.N. (2003): Synthesis of antioxidant propyl gallate using tannase from
Aspergillus niger van Teighem in nonaqueous media. Bioorganic and Medicinal Chemistry
Letters.
17. Bharadwaj R. Singh B. and Bhat T.K., J. Basic. Microbiol., 43(6), 449–461 (2003).
18. Sharma S. Agarwal L. and Saxena R.K., Bioresource. Technol., 99(7), 2544-2551 (2008).
19. Barthomeuf C. Regerat F. and Pourrat H., J. Ferment. Technol., 77, 137–142 (1994).
20. Gupta R. Bradoo S. and Saxena R.K., Appl. Microbiol., 24, 253 – 255 (1997).
21. Mahapatra K. Nanda R.K. Bag S.S. Banerjee R. Pandey A. and Szakacs G., Process. Biochem.,
40, 3251–3254 (2005)
22. Kumar, S. S., Sreekumar, R., & Sabu, A. (2019). Tannase and Its Applications in Food
Processing. Springer EBooks, 357–381.
23. Aharwar, A., & Parihar, D. K. (2018). Tannases: Production, properties,
applications. Biocatalysis and Agricultural Biotechnology, 15, 322–334.
***
245
Chapter 24
SPECIAL EMPHASIS ON THE STUDY OF
MEDICINALLY IMPORTANT GENERA -PERMELIA,
CETRARIA, CLADONIA AND USNEA
Dr. Alka Rajput, Dr. Varsha Sharma, Dr. Renu Yadav
A bstract
Fourteen genera belong to a monophyletic core of cetrarioid lichens, Ahtiana, Allocetraria,

Arctocetraria, Cetraria, Cetrariella, Cetreliopsis, Flavocetraria, Kaernefeltia, Masonhalea,
Nephromopsis, Tuckermanella, Tuckermannopsis, Usnocetraria and Vulpicida. A total of 71 samples
representing 65 species (of 90 worldwide) and all type species of the genera are included in
phylogentic analyses based on a complete ITS matrix and incomplete set of group-I intron, -tubulin,
GAPDH and mtSSU sequences. Eleven of the species included in the study are analysed
phylogenetically for the first time, and of the 178 sequences, 67 are newly constructed. Two
phylogenetic trees, one based solely on the complete ITS-matrix and a second based on total
information, are similar, but not entirely identical. About half of the species are gathered in a
strongly supported clade composed of the genera Allocetraria, Cetraria s. str., Cetrariella and
Vulpicida. Arctocetraria, Cetreliopsis, Kaernefeltia and Tuckermanella are monophyletic genera,
whereas Cetraria, Flavocetraria and Tuckermannopsis are polyphyletic. The taxonomy in current use
is compared with the phylogenetic results, and future, probable or potential adjustments to the
phylogeny are discussed. The single non-DNA character with a strong correlation to phylogeny
based on DNA-sequences is conidial shape. The secondary chemistry of the poorly known species
Cetrariaannae is analyzed for the first time; the cortex contains usnic acid and atranorin, whereas
isonephrosterinic, nephrosterinic, lichesterinic, protolichesterinic and squamatic acids occur in the
medulla. Notes on the anatomy of Cetrariaannae and Flavocetrariaminuscula are also provided.
Cladonia relates to the five predominant lichen-associated bacteria belonged to the phyla
Proteobacteria (31.84%), Planctomycetota (17.08%), Actinobacteriota (15.37%), Verrucomicrobiota
(12.17%), and Acidobacteriota (7.87%). The diversity analysis metric showed that Heterodermia
highest bacterial contained the species richness. Within the lichens, Ramalinaconduplicans and
Alka Rajput, Varsha Sharma, Renu Yadav

246
Cladoniarappii showed a distinct bacterial community from the other lichen species. The community
of lichen-associated actinobacteria was investigated as a potential source of synthesized biologically
active compounds. m. Evaluation of the pretreatment method (heat, air-drying, phenol, and fooding)
and isolation media used for the culture-dependent actinobacterial isolation revealed that
thecombined with different isolation mediadiferentpretreatments were effective in obtaining several
species of actinobacteria. However, metagenomics analyses revealed that there were still several
strains, including rare actinobacterial species, that were not isolated.
Keywords: Medicinal Genera, Permelia, Cetraria, Cladonia, Usnea.
Department of Botany, School of Bioscience, RIMT University, Mandi Gobindgarh, Punjab, India
dr.rajputminni@gmail.com
I ntroduction
The genus Usnea Adans. (Parmeliaceae; lichenized Ascomycetes) is a typical group of mostly pale
grayish-green fruticose lichens that grow like leafless mini-shrubs. More than 360 species of Usnea
are known in the world. Usnea has long been thought to have treating power for various illness
together with its use as dye, cosmetics, preservative, and deodorants in centuries particularly in
eastern countries such as China, Japan, Taiwan, India and Europe, (Pyrczak-
Felczykowskaet.al.,2023). The current review focuses on the traditional uses and phytochemistry
aspects of different Usnea species, and it discusses the pharmacological findings and toxicology of
their extracts and isolated compounds. The available compilation of data will provide a new base for
future perspective and needs of further studies of this potent herbal source to harvest more beneficial
therapeutic drugs. Nineteen species of the genus Usnea are found to be important folk medicines in
all over the world. It is well evident from the comparative analysis of available searched literature
shows that the genus Usnea has been used for its various purposes for centuries and its long
traditional medicinal history was well documented in the past. As per ancient records and recent
scientific literatures, the species of genus Usnea have been used as a promising traditional medicine
exerting an array of therapeutic properties for relieve sore throat, bronchitis, cold, flu, infection, and
indigestion. Phytochemical analysis confirms the general presence of a wide range of metabolites,
polysaccharides, fatty acids, phenolic acids, flavonoids, terpenes, sterols, depsides, depsidones, and
benzofurans, (Rosentretor. Roger and Ann. De. Bolt., 2023). As specific constituents, usnic acid,
polyphenols, and depsides have been considered as main efficacy component for antibacterial and

247
antifungal activities. (Osipov and Kuznetsov.2022), While pharmacological analysis also revealed
that other pure compounds and crude extracts of Usnea species prove to be significant anti-cancer,
anti-proliferative, anti-oxidant, anti-viral, anti-inflammatory, anti-ulcer, hepatoprotective, and anti-
genotoxic agents. However, there is a need for more precise investigations to examine the clinical
value of both isolated pure compounds and crude extracts and to elucidate their mechanisms of
action. Apart from clinical validation and elucidation of mechanism of action, biosafety studies of
the compounds are also important to use legitimately the potential bioactive compounds for further
development of future lead drugs, (Vonsceloz.et.al.,2023).
Fig. (a) Parmelia biota (b) Parmelia dioscora

Lichens are an obligate mutualism between a fungus (mycobiont) and one or more photosynthetic
organisms, an alga or cyanobacterium (photobiont).1,2 Typically the fungal partner delivers most of
the composite organism’s structure and mass, hence trading physical protection for carbohydrates
manufactured by the photosynthetic partner. These organisms represent a unique division in the plant
kingdom and are the most successful symbiotic organisms in nature, dominating 8% or more
(Bisht.K.et.al.,2023) of the earth’s terrestrial area.2–4 Lichens have been shown to produce a number
of primary and secondary metabolites that may protect them against physical stresses or biological
attack.5,6 Some lichen species and their metabolites have been used for medicinal and industrial
purposes.7–10 Among the medicinal lichens, the genus Usnea Adans. (Parmeliaceae; lichenized
Ascomycetes) is edible and is utilized in the preparation of traditional foods and medicines in both
Eastern and Western countries.11,12 This genus is regarded as one of the taxonomically most
difficult genera of macrolichens13, (Soares.R.N.et.al.,2023). Most of the species are globally
distributed with more than 350 species and highly variable in morphology. Many species are also
very variable in chemistry, and may include several chemotypes.14 In India, 57 species of Usnea are
known which grow luxuriantly in higher regions of Western Ghats and Himalaya.15,16 Many
species are also very variable in chemistry, and may include several chemotypes. The species of
Usnea are known to be used in traditional medicines, in dyeing and in spices in various parts of the
country.15,17 The rst recorded use of the Usnea species in traditional Chinese medicine dates to
101 B.C., when it was used as an antimicrobial agent under the Chinese name of Song Lo. Song Lo
tea or its decoction (Aziz. F.H. and Toma.F.M.2020) has also been recorded for internal and external
detoxication of the liver, treatment of malaria, wounds, snake bite, and cough.18 In Unani literature,
the medicinal uses of Usnea species are mentioned as astringent, antidote, analgesic, cardiotonic,
resolvent and stomachic.
Along with its emerging position in the herbal market, primary as well as secondary metabolites
(extrolites), the chemical constituents of Usnea species have been broadly investigated. Usnic acid,

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protocetraric acid, barbatic acid, norstictic acid, salazinic acid, and stictic acid were characterized as
the main bioactive chemical constituents in Indian species.17,22,23 All the major secondary
metabolites in Usnea belong to aromatic products formed from b-orcinol units, while ceparatic acid
and protolichesterinic acid belong to higher aliphatic acids. Most of these metabolites are unique to
Usnea lichens (fujjikama and Hayakawa.C.et.al.,2023) being of great significance for systematics
and phylogeny, and are employed at different taxonomic levels from species and subspecific to
generic and higher ranks.24,25 Furthermore, these bioactive metabolites play important ecological
roles in nature such as UV protection and defense against predators and pathogenic microorganisms.
Significant research has been done on Usnea and its metabolites which confirm various biological
activities including anti-microbial, anti-oxidant, anti-tumor, anti-viral, anti-inflammatory,
cardiovascular protective, and hepatoprotective properties.17,20,22,27–33 These are closely
correlated with the ethno-medicinal uses. Recent pharmacological studies have revealed significant
anti-cancer, anti-genotoxic, antiproliferative, and anti-neoplastic activities and these potentials have
further put Usnea under the spotlight (29, 34–36). The aim of this review is to summarize the recent
advances in photochemistry and pharmacology of the genus Usnea. Phylogenetic and toxicological
aspects are also given in brief.
Recognize Usnea in fields by its shrubby to pendent greenish yellow thallus, radial symmetry and
presence of central cartilaginous axis.15 There are three forms of fruticose thallus in Usnea,
erect/bushy when the thallus is small (U. orientalis), a long pendulous thallus hanging from tree
branches (U. angulate and U. longissima) or a sub-pendent thallus of intermediate length (U.
aciculifera and U. rubicunda). Dominant branching patterns including dichotomous, sub-
dichotomous and sympodial are observed,(Mallen.Cooper.M.et.al.,2023).The basic characteristics
used for identification of Usnea up to species level include morphological features like the habit of
the thallus, branching pattern, pigmentation of basal part, presence or absence of sorelia together
with its morphology, isidia, pseudocyphellae, papillae, tubercles, brils, faveolae and shape of
branches; anatomical features like the ratio of thickness of the cortex (C), medulla (M) and central
axis (A), the compactness of fungal hyphae in medulla and the presence or absence of specific
secondary metabolites. A combination of morphological, anatomical and chemical characters can be
used to delimit species.
Usnea sensulato comprises of an assemblage of approximate 350 species worldwide.15 The species
belonging to the genus Usnea contain usnic acid, a bioactive compound, which imparts a yellow
colour to the thalli, (Ozimac.S.and Prlic.D.2022). Using modern concepts, the taxonomist divided
Usnea into three genera, i.e., genus Dolichousnea, genus Eumitria and genus Usnea sensustricto. The
genus Dolichousnea is characterized by an annular pseudocyphellae between the segments, solid
central axis and positive iodine reaction of central axis. The genus Eumitria is characterized by a
stulose central axis whereas genus Usnea sensustricto is characterized by the absence of annular
pseudocyphellae and a solid, I – central axis.
Cladonia
Cladonia is a genus of moss-like lichens in the family Cladoniaceae. They are the primary food
source for reindeer/caribou. Cladonia species are of economic importance to reindeer-herders, such
as the Sami in Scandinavia or the Nenets in Russia. Antibiotic compounds are extracted from some
species to create antibiotic cream. The light green species Cladonia stellaris is used

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in flower decorations. Although the phylogeny of the genus Cladonia is still under investigation, two
main morphological groups are commonly differentiated by taxonomists: the Cladonia morpho-type
andthe Cladina morpho-type. The Cladonia morpho-type has many more species, and is generally
described as a group of squamulose (grow from squamules), cup-bearing lichens.
The Cladina morpho-types are often referred to as forage lichens, mat-forming lichens, or reindeer
lichens due to their importance as caribou winter forage (Kumari.M. et.al.,2022).
Fig. (a) Cladonia perforate (b) Cladonia gracilis

Cladoniaperforata ("perforate cladonia") is one of two on the U.S. Endangered Species List, and it
should never be collected, (Dziurowickz.P.et.al.,2023). It exists only in a few small populations
in Florida.
Fig. (a) Cladonia uncilais (b) Cladonia asahinae
Several Cladonia species grow on sand dunes. The presence, and luxuriant carpet-like growth,
of Cladonia species is one of the defining characters of grey dune, a priority habitat for conservation
under the E.U. Habitats Directive.Species of lichen C. mitis and bryophyte P. schreberihas been
randomly collected in BoryTucholskie National Park within the lichen Scots pine forest community
(Cladonio-Pinetum Ass.) in February 2022 (Węgrzyn et al, 2020; 2021a; 2021b).
As treatment within containers, there was two types of artificial light conditions we used 1) full light
(FL) – 100% of artificial light; 2) reduction light (RL) – 30% of artificial light. The reduction was
achieved by covering the glass of the aquarium with a dark film. By THE application of artificial
lighting both light conditions were created which was generated by lamps with the following

250
specifications: type GROWY LED 354, light PAR − 224.3 µmol/m2s and wavelength range 50%
max. 450 nm and 50% max. 650 nm. F
Cladonia species are used as food plants by the larvae of some Lepidoptera species
including Chionodescontinuella.
Selected species of Cladonia include-
• Cladoniaamaurocraea – quill lichen
• Cladoniaarbuscula – reindeer lichens
• Cladoniaasahinae – pixie cup lichen
• Cladonia borealis – boreal cup lichen
• Cladoniacardiosa – split-leg lichen
• Cladoniacenotea – powdered funnel lichen
• Cladoniachlorophaea – mealy pixie cup
• Cladoniacoccifera – madam's cup
• Cladoniaconiocraea – common powderhorn
• Cladoniacristatella – British soldier’s lichen
• Cladoniadeformis – lesser sulphur-cup lichen
• Cladonia fimbriata – trumpet lichen
• Cladoniagracilis – smooth horn lichen
• Cladoniamacilenta – lipstick powderhorn
• Cladoniamacrophyllodes – large-leaved cladonia
• Cladonia mitis – green reindeer lichen, yellow reindeer lichen
• Cladoniamongkolsukii
• Cladoniamultiformis – sieve lichen
• Cladoniaparasitica
• Cladoniapeziziformis – cup lichen
• Cladoniaphyllophora – felt horn lichen
• Cladoniapleurota – red-fruited pixie cup
• Cladoniaportentosa – reindeer lichen
• Cladoniapyxidata – pebbled pixie lichen
• Cladoniarangiferina – grey reindeer lichen, true reindeer lichen
• Cladonia rei – wand lichen
• Cladonia squamosa – dragon horn
• Cladonia stellaris – northern reindeer lichen, star-tipped reindeer lichen
• Cladoniaturgida – crazy scale lichen
• Cladoniauncialis – thorn lichen
• Cladoniaverticillata – ladder lichen
Parmelia
Humans' need on plants for things like food, housing, clothing, and even maintenance and restoration
of health dates back to the dawn of humanity. Unique among plants, lichens are made up of a fungus
and an alga (which are not even closely related to each other) that thrive in symbiosis. It is a very
successful population that has mastered a variety of ecosystems and taken over around 8% of the
planet's terrestrial ones.

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Ancient civilizations like China and Egypt relied on them for their healing properties. In Rigveda,
the earliest genuine record of 'Aushadhi' (medicine) has been detailed, a lichen was used as an
example of the knowledge of ancient Indian medicinal plants preserved in literature. In addition,
lichen's medicinal uses in folklore have been documented in several international pharmacopoeias.
The approximately 2450 different types of lichens in India demonstrate the country's remarkable
biodiversity. They may be found in great numbers in Peninsular India's hilly areas and the temperate
Himalayas.
They have been used medicinally for thousands of years, and scientists have recently discovered over
700 physiologically active components inside them that are structurally distinct from those found in
higher plants. 3-5. Lichens are famous for their high concentrations of secondary metabolites, which
have been linked to a wide variety of beneficial biological effects, including antiviral, antibacterial,
antifungal, anticancer, antioxidant, and immunomodulatory effects. This article aims to explain the
lichen Permaliaperlata (Huds.) Ach., a member of the Parmeliaceae family also known as Chharila
and, perhaps due to its traditional medicinal activity on ashmari (urinary stone) in Ayurvedic Indian
medicine, by a variety of other common names. For the same reason that Didymocarpus pedicellata9
is called a stone crusher, Saxifraga ligulata8 is likewise known as Pashanbheda (stone breaker).
Usnic acid, a primary component of parmelia species, has been shown to have potent antimitotic,
antitumor, and antimycobacetrial effects; other unique constituents, including diffractic acid,
gyrophoric acid, caperatic acid, etc., have been isolated and are well-known for their analgesic,
antipyretic, antispasmodic potential and beneficial effects against hyperproliferative skin disease like
psoriasis, 10-12.
There are three types of parmelia, and they are P. perlata (L.) Ach. [Synonym: Parmotremachinense
(Osbeck) Hale & Ahti], P. perofrata (L.) (Wulf.) The acronym Ach. [Synonym:
Parmotremaperforatum (Ach.) Mass.]
In India, chharila refers to both P. sanacti-angeliiLynge and P. sanactifoliaBenth. Tridecyl myristate,
3-ketooleanane, icosan-1-ol, usnic acid12, parmelanostene permelabdone14, atranorin, lecanoric
acid, orcin, erythrolein, azolitmin, and spaniolitmint15 are only a few of the chemical components
found in it. In most cases, P. perlata is added to food as a spice to improve its flavour. In addition to
its purported aphrodisiac qualities, it is astringent, resolvent, laxative, and carminative. In addition to
relieving pain, it may help with conditions such as bronchitis, hypersalivation, toothache, boils,
inflammation, seminal deficiency, spermatorrhea, amenorrhea, dyspepsia, calculi, anaemia,
cardiovascular disease, stomach problems, scabies, leprosy, piles, and enlarged spleen. Powdered
medicine is used to treat wounds, and a good cephalic snuff is said to alleviate headaches. The
primary purpose of this manuscript is to summarise the findings of numerous scientific studies
conducted on this medication, as well as related information such as its common names, taxonomic
classification, botanical descriptions, traditional uses, ayurvedic properties, pharmacological
activities, chemical constituents, marketed formulations, etc. In addition, this publication is the first
scientific account of such a wide range of characteristics of the lichen P. perlata, making it of great
relevance to researchers interested in investigating any such characteristics of this fungus.
isidia and soralia, both of which are bud-like vegetative structures found on the thallus's top surface,
may reach a maximum height of 0.5 mm and have a cylinder-like shape with a straight, simple or
branching stem. The thallus's bottom surface is somewhat rhizinate. The rootlets, or rhizines, that
anchor the lichen to its host surface are thin, uncomplicated, and very slightly furcated. Adnate and
ranging in size from 1 to 3 millimetres, apothecia are part of an isidiate amphithecia.

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Traditional Uses
In most cases, P. perlata is added to food as a spice to improve its flavour. Sores, bronchitis,
excessive salivation, vomiting, tooth-ache, boils, inflammations, etc. may benefit from its astringent,
carminative, demulcent, bitter, resolvent, emollient, laxative, sporofic, sedative, diuretic properties.
Indicated in cases of seminal weakness, spermatorrhoea, nocturnal emission, amenorrhea,
leucorrhoea, etc., it is also a very effective aphrodisiac. Dyspepsia, calculi, blood problems, heart
illnesses, stomach disorders, enlarged spleen, bleeding piles, scabies, and leprosy are only some of
the other conditions for which it might be helpful. The renal and lumbar regions might benefit from
this when applied topically. It's also helpful for alleviating general aches and pains, such those
associated with the liver and the womb. In addition to its other uses, the powdered medication is
widely regarded as an effective cephalic snuff and an effective agent for enhancing digestion. It
strengthens the urinary system and prevents stones from forming. It prevents respiratory diseases and
helps keep your body at a healthy temperature. Inflammations may be reduced with the use of the
medication paste. Smoking the substance is said to alleviate headache pain. It is also an integral part
of many other kinds of beauty products (Rola.K.et.al.,2023)
The Ayurvedic Pharmacopoeia of India notes that P. perlata has therapeutic applications for a wide
variety of conditions, including kandu (itching), kustha (skin diseases), asmari (calculi), daha
(burning sensation), visa (poison), hrllasa (angina pectoris), trsna (thirst), varna (ulcer), hrdaya-roga.
Ayurvedic Formulations
Several herbal preparations used in various medical systems use P. perlata as a key ingredient. Neeri
(Aimil Pharmaceuticals India Ltd.), Calcury (Charak Pharma Pvt. Ltd.), Pathrina (Shri
BaidyanathAyurved Bhawan Pvt. Ltd.), etc. are only a few examples of the many Ayurvedic and
Allopathic medicines that rely on it.
These preparations find particular use in the treatment of renal stones and the recovery of normal
kidney function. Several popular medications for sexual dysfunction and enhancement, including as
Confido, Speman, V-Gel, and Speman Vet (all manufactured by The Himalayan Drug Company),
contain this active component. Dawa-ul-miskmutadil, Dawa-ul-miskmutadil, Erqember, Laboob-e-
kabirmuqquawie bah, Ma-ul-leham, Majun shababawar, Majun muqwwimumsik,
Mufarrheyaqutimotadil, Mumsik be nazir, Roghan-e-surkh, Sharbat mufarrhe mu Same use may be
seen in Mahanarayan Oil.
Cetrelia
Cetrelia is a genus of leafy lichens in the large family Parmeliaceae. They are commonly known as
sea-storm lichens, alluding to the wavy appearance of their lobes. The name of the genus,
circumscribed in 1968 by the husband and wife lichenologists William and ChicitaCulberson, alludes
to the former placement of these species in the genera Cetraria and Parmelia.
The main characteristics of the genus are the broad, rounded lobes of the greyish-green lichen body,
and the presence of tiny pores in the outer surface (the cortex) that enable gas exchange. The lower
surface of the lichen is brown to black with few root-like rhizines to act as holdfasts, resulting in a
fairly loose attachment to the surface the lichen is on. Sexual reproductive structures are usually not
present, so the physical characteristics used to distinguish between Cetrelia species include the
presence or absence of asexual reproductive structures such as isidia, soredia, and lobules. Cetrelia

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lichens are chemically diverse and produce several secondary chemicals: atranorin is the main
compound produced in the cortex, while substances known as depsides or depsidones are compounds
in the medulla (the tissue layer under the cortex) that can be used to help distinguish between
species.
The number of species in Cetrelia depends on how the chemical variation in the genus is categorized:
there are five distinct forms (morphotypes), and additional morphologically identical but chemically
unique species (chemotypes). Although 19 Cetrelia species have been formally described, some
authorities prefer to consider the morphologically similar taxa as chemotypes of the same species.
Molecular phylogenetic analyses suggest that the chemical races are best treated as distinct species.
Known predominantly from the Northern Hemisphere, most Cetrelia species are found in eastern or
southern Asia. They prefer somewhat moist, cool habitats, and are most commonly found on tree
trunks, but sometimes on rocks or on bryophytes over shaded boulders. The type species,
Cetreliacetrarioides, is widely distributed, having been recorded in Asia, Europe, and North America.
Most Cetrelia species that occur in Europe are considered rare or threatened, and appear on the
Regional Red Lists of several countries. In Asia, some Cetrelia lichens are used in the preparation of
natural dyes, for the production of material with antibiotic-like properties used in traditional
medicine, or in the preparation of litmus reagent.
Taxonomic History
The generic name Cetrelia combines Parmelia and Cetraria, the two genera in which most of the
species were originally classified.(Furmanek.L.et.al.,2022)The genus was circumscribed by
lichenologists William and ChicitaCulberson in 1968. In 1960 they started a study of the genus
Cetraria in the broad sense, as it had been defined by Alexander Zahlbruckner in his 1930 work
CatalogusLichenum Universalis. The genus had become a wastebasket taxon, a repository for all
parmeliaceous lichens with apothecia on the margins. The Culbersons started by studying the broad-
lobed species that had traditionally been placed in Cetraria, a group they called the "parmelioid"
Cetrariae. This group included Cetrariasanguinea, C. japonica, and C. collata, as well as a group
referred to as the Cetraria glauca group, including C. norvegica and C. chrysantha. This latter group
of species had been previously identified by Norwegian botanist Eilif Dahl, who had suggested that
Cetraria was polyphyletic in a 1952 study. The Culbersons noticed that Cetrariasanguinea and
C. japonica were very similar in morphology to two Parmelia species, Parmeliacetrarioides and
P. olivetorum. As a result of their investigations, they transferred three species to the new genus
Asahinea, created genus Platismatia for 10 members of the Cetraria glauca group, and Cetrelia for 14
members of the Parmeliacetrarioides group.According to a 2022 review of the family Parmeliaceae,
the creation of these three new more natural genera by the Culbersons initiated "a trend of splitting
that continued for more than three decades"(Regner.A.et.al2023).
Fig. Cetrelia olivetorum

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In the early 1970s Josef Poelt developed the concept of "species-pairs"—taxa that are
morphologically and chemically similar (if not identical), but differ primarily in their reproductive
modes: one taxon reproduces sexually, the other vegetatively. Using Cetrelia as an example to
explain his ideas, he suggested that both members of these pairs should be assigned the rank of
species (da.Rosa.et.al.,2022). In 1976 the Culbersons introduced the idea of morphological groups,
or morphotypes with the genus Cetrelia. They named these groups with the Latin epithets of the most
prominent species of that group, thus: cetrariodes (thallus with soredia), isidiatadavidiana(thallus
with isidia), sinensis (thallus with marginal dorsiventral lobules), collata (thallus without soredia,
isidia, or lobules, often without apothecia and large pseudocyphellae), and (thallus without
vegetative propagules and frequently with apothecia, but pseudocyphellae small). Similarly, the
genus contains a number of chemotypes related to the content of the major substances in the medulla.
Species with the same chemotype always have the same one or two major constituents, while the
complex of minor substances may vary somewhat. Six chemotypes have been recognized in Cetrelia.
In a 2019 phylogenetic analysis, these chemotypes were named according to their major medullary
substance(s): imbricaric, olivetoric, anziaic, perlatolic, microphyllinic, and alectoronic + α-collatolic.
An example of changing taxonomic affinities is given by the four European species of Cetrelia
(C. cetrarioides, C. chicitae, C. monachorum, and C. olivetorum). Several studies have suggested
accepting only one species in the group with four chemotypes, (Popovici.V.et.al.,2022),separated
according to the major medullary substances, which can be detected by standard thin-layer
chromatography methods,while others have considered the different chemotypes as separate species.
Cetrelia species are commonly known as sea-storm lichens; according to author Colin Rees, "the
greenish-gray uplifted edges of its lobes are reminiscent of foam on ocean waves".
Phylogeny
Prior to the advent of molecular phylogenetics, Cetrelia was grouped together as part of the
morphological group "cetrarioid lichens" – species with erect, foliose thalli with marginal apothecia
and pycnidia. However, DNA evidence from several studies showed that the cetrarioid lichens were
not a monophyletic group of species, and that Cetrelia clustered together outside the cetrarioid
lichens, in a group called the "parmelioidCetrariae", along with the genera Asahinea and
Platismatia.(Tripathi.A.H.et.al.,2022) Morphological characteristics that unite these three genera
include the presence of large, broad, rounded lobes, and thalli that are usually oriented horizontally.
Similar to several other groups of parmelioid taxa, Cetrelia lichens have the polysaccharide
isolichenan in their cell wall, which is absent in the cetrarioid group. In a later analysis (2010),
Cetrelia is resolved as the only member of the Cetrelia clade, one of nine major groups within the
parmelioid clade of Parmeliaceae that share similar morphological and chemical characteristics.
A 2019 study investigating the usefulness of secondary metabolites in molecular taxonomy of
Cetrelia demonstrated a clear correlation between lichen chemistry and phylogeny, suggesting that it
is useful to include information from secondary metabolites when identifying taxa. Cetrelia produces
a constant set of polyphenolic compounds of still unknown function, specifically orcinol-type
depsides and depsidones. Character state analyses showed that metabolites in Cetrelia seem to be
evolving towards more (Rutherford.R.D.and Robertus.A.2022) complex substances, indicating
possibly their evolutionary importance in the survival or functioning of the species. This research
corroborated prior DNA-based evidence that also supported the use of chemotaxonomy to delineate
species in Cetrelia.

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Description
Closeup of the lobes of Cetrelia cetrarioides. White spots and lines are pseudocyphellae.
Closeup shows continual marginal soralia on the lobe margin of Cetrelia cetrarioides
Cetrelia is characterized by its foliose (leafy), greyish-green thallus that is loosely attached to the
substrate. The thallus has laminal pseudocyphellae – these are small pores in the cortex through
which the medulla is directly visible. It has a prosoplectenchymatous upper cortex, meaning that it is
made of densely agglutinated hyphae that are not swollen and have tiny lumina.The lower cortex is
black, at least in parts, with sparse rhizines, and pycnidia (asexual fruiting bodies) on the margin.
The ascospores made by Cetrelia lichens are colourless with an ellipsoid shape, numbering eight per
ascus (Otero.et.al.,2023),Their length ranges from 11 to 22 μm, with a width of 6–12 μm. Conidia
(asexual spores that are produced in the pycnidia) are rod-shaped and measure 3–6 μm long.
Atranorin is the main secondary chemical in the cortex, and is present in all species. Cetrelia makes
several orcinol-type depsidones in the medulla that can be used as diagnostic substances to help
identify specimens to species level.Other secondary chemicals that have been identified in this genus
are: alectoronic, anziaic, collatolic, imbricaric, microphyllinic, olivetoric, perlatolic, and physodic
acids.
In a study of the carotenoids present in six Cetrelia species found in Eurasia, only violaxanthin was
found to be present in all samples of all species collected in a range of environments. Of the twenty

256
different carotenoids identified in this study, most are common, although a few are rare in lichens.
For example, flavoxanthin, detected in the thalli of C. japonica, had previously only been found in
three other instances(Seaward.M.R and Amrani.S.2022),although it is not uncommon in the blossom
and fruit of higher plants. The presence of a single carotenoid common to all species suggests that in
these species, carotenoid biosynthesis is to a large extent dependent upon the conditions of the
environment.(Irinyi.et.al.,2023)The presence or absence of reproductive propagules such as isidia,
soredia, and lobules are important physical characters in the taxonomy of Cetrelia. Isidia are rare in
the genus, being associated only with C. braunsiana and C. isidiata. Soredia are common; with the
exception of C. alaskana, all species are sorediate. The soredia are fine and powdery, and produced
on the edge of lobes as continual marginal soralia, which often need magnification to be seen.
Lobulae are tiny lobes on the upper surface and margins of the thallus. They are found in
C. japonica, C. sinensis, and C. pseudolivetorum.
Morphological characters or chemical characters alone are not enough to distinguish between species
in Cetrelia and they should be considered together. For example, C. pseudolivetorum and C. japonica
are difficult to distinguish with only a brief physical examination, and are often incorrectly labeled in
collections in locations where both species occur,(Cernajov.I and Skaloud.P.2019).However, they
can be identified by checking the colour reaction of C (sodium hypochlorite): a positive reaction
(C+), indicates the presence of olivetoric acid, which only occurs in C. pseudolivetorum. In contrast,
C. chicitae and C. braunsiana produce the same secondary compounds, and so cannot be
distinguished by spot tests or thin-layer chromatography. However, they are readily distinguishable
through examination: C. chicitae has soredia on its margin, while C. braunsiana has isidia (Fatima.M.
and Bhatt.M.2022).
Cetrelia lichens resemble somewhat those in genus Parmotrema, but species of the latter never have
pseudocyphellae and usually have cilia on the margin.[14]Platismatia is another morphologically
similar genus, but it differs from Cetrelia in always lacking caperatic acid.[25]Cetreliopsis is a genus
segregated from Cetreliopsis in 1980. Although superficially similar, Cetreliopsis lichens have a
distinct secondary chemistry: they contain usnic acid in the upper cortex, and orcinol depsidones in
the medulla (Fraser.M.W.et.al.,2023).
Altogether, 19 species have been included in Cetrelia, either described as new species, or transferred
from other genera. Morphotypes are distinguished primarily by the vegetative propagules: presence
or absence of soredia, isidia and lobulae.Although there are only five distinct morphologies
(morphotypes) in Cetrelia, 18 species are generally recognized based on differences in secondary
compounds (chemotypes), in combination with morphology. Based on the distribution of the
majority of the chemotype and morphotype combinations, the centre of speciation in Cetrelia is
thought to be southern and eastern Asia.we determined the quantitative content of antioxidants of
low molecular weight and antimicrobial activity of 40% of the water ethyl extracts
of Cetrariaislandica and Cladoniaarbuscula lichens depending on the layers’ treatment methods and
the ratio of dilution of the dry extract with water. Live parts of dried lichens collected in an
ecologically clean area of Yakutia were used for extraction. The antioxidant activity of the water-
ethanol extract of lichens was performed using a spectrophotometric method.

257
Cetrelia chicitae; scale bar is 1 cm
As of September 2021, Species Fungorum accepts five species of Cetrelia: C. cetrarioides,

C. chicitae, C. monachorum, C. olivetorum, and C. sayanensis.
Habitat and distribution
Cetrelia monachorum; scale bar is 0.5 cm

Cetrelia lichens usually grow on bark, but sometimes they are found on rocks and bryophytes. They
have been found at a wide altitudinal range, from 1,000 to 4,500 m (3,300 to 14,800 ft).[46] In
general, the genus prefers somewhat moist, cool habitats, and is often found on tree trunks or
bryophytes over shaded boulders.[47] Cetrelia species found in Europe are largely epiphytic (growing
on plants), usually encountered in old natural or seminatural forests on tree bark, and sometimes on

258
mossy rocks. They are usually in areas of high humidity, such as swamp forests, or forests that are
surrounded by a lake or river or on hillsides near lakes or streams.[48] The observation that Cetrelia
lichens are commonly found among bryophytes or on bark-growing bryophytes suggests that this
association provides better moisture conditions for the lichens.Cetrelia usually prefers deciduous
trees, although on rare occasions it is recorded growing on conifers.[48] A study of several hundred
collections of the genus from locations largely in the eastern European Alps revealed the trees most
preferred as substrates: sycamore (34%), European beech (14%), alder (mostly grey alder, 13%), oak
sp. (8%), willow sp. (6%), and European ash (6%).The substrate selectivity of Cetrelia is not always
consistent through broad geographical regions such as Europe, because the range of the substrate
determines whether it will be present in a certain area.[49]
Most Cetrelia species are found in eastern or southeastern Asia. None are found in Africa, Australia,
New Zealand, or South America, (Williams.Monsey.et.al.,2023). Four species have been recorded
from Europe: Cetreliacetrarioides, C. chicitae, C. monachorum, and C. olivetorum.In a 2021 study of
Cetrelia species in Hungary, the use of chemical analysis to determine the lichen secondary
metabolites revealed that all four of those species are found in that country, double the number
previously thought to be part of the Hungarian lichen flora.Ten species are found in India, [46] nine
species from northeast China, and four from South Korea. Five species are known from North
America,while eight species have been recorded in the expansive geographic area formerly known as
the Soviet Union. Of the five morphotypes, only cetrarioides is widely distributed in the
world.[12]Cetreliacetrarioides is found throughout the Northern Hemisphere, with a few records from
the Southern Hemisphere.[53]
Uses
In India, Cetreliabraunsiana is used as a source of a natural dye to colour different fibres such as silk,
tussar silk, and cotton. Depending on the type of method used to extract the lichen dye, it produces a
final colour of ivory yellow or light yellowish oliveCrittenden, (P.D. et.al.,2023) Atranorin is one
dye-producing substance found in this species; alectoronic and α-collatolic acid are additional
secondary chemicals that occur in C. braunsiana,(Loanurit.N.et.al.,2023).
In China, Cetreliacetrarioides, C. olivetorum, and C. pseudolivetorum are collected in bulk for the
preparation of material with antibiotic-like properties used in traditional medicine. These three
species are also used as a raw material in the production of litmus reagent.
Usnea
Botanical Characterization and Distribution
The genus Usnea is highly diverse, with more than 350 estimated species, distributed in polar,
temperate and tropical regions. This genus is characterized by fruticose habit and especially by the
presence of a cartilaginous central axis. Dillenius (1742) rust proposed the name Usnea in Historia
muscorum. 37 The genus was placed in family Usneaceae until studies on apothecial ontogeny and
ascus apical structures proved that Usnea belongs to the family Parmeliaceae.38 One can easily
Usnea is a cosmopolitan genus occurring on all continents. Species diversity, however, is low in arid
and arctic areas and is highest in humid regions of temperate latitudes. U. aciculifera is found in
Eastern Asia and U. angulate is distributed in Australia, America and West Africa. U. baileyi is
found in pantropical countries in world. U. compressa is widely distributed in India and Nepal. U.
fragilis is known in the South-East Asian region. U. himalayana is from the Himalayas, Western
Ghats and Africa. U. indica is endemic to North-West Himalayas and found in Uttarakhand in India.

259
U. ghattensis is endemic to Western Ghats. U. luridorufa is found in North and South Asia. U.
nepalensis is found in Himalayas and Western Ghats in India. Several species including U. orientalis,
U. pangiana, U. sinensis, and U. perplexans are widely distributed in North-East Asia.
(Cateura.P.J.A.2022), U. pseudosinensis and U. robusta are restricted only to the Himalayas. U.
subforida is distributed in East Africa and North Asia, while U. suboridana is found in Europe and
North East Asia. U. undulata is found in South and East Africa.
U. longissima is distributed throughout the Northern temperate zones, such as the sub-arctic and the
coastal rainforests of Europe, Asia and North America.41,42 In India, the species is distributed in
North-Eastern Himalayan regions between 1500–4000 m altitudes in moist old mixed forests of
Quercus and Pinus. Seven chemosyndromes of U. longissima are reported from India which include
barbatic acid, squamatic acid, diffractaic acid, evernic acid, fumaroprotocetraric acid and usnic acid
strains. U. longissima is characterized by fruticose, a pendulous thallus of 60 cm long or more, of
pale yellow-greyish green to light brown, a 0.5–1.0 mm diameter main branch, a 2–5 cm long, dense
perpendicular usually decorticated or pulverulent to powdery lateral branches, sorediate or isidiate,
with a colorless central lattice. Apothecia range from being rare to up to 5 mm in diameter, with
ciliate margin. The species has more than seven chemotypes containing barbatic, squamatic,
diffractiatic, evernic, fumarprotocetraric, and usnic acids.
Phylogeny and Classification
The phylogeny and classification of Usnea have been a matter of debate, given the lack of
phenotypic characters to describe phylogenetic clades and the low degree of resolution of
phylogenetic trees.37 Motyka (1936–38) proposed a classification of Usnea, in which all fruticose
lichens with an inner, cartilaginous tissue are included. He identified six subgenera: Euusnea,
Protousnea, Lethariella, Chlorea, Neuropogon, and Eumitria. 43 Later Protousnea and Lethariella
(including Chlorea) were elevated to generic rank by Krog (1976).44 The position of Neuropogon as
a subgenus to Usnea was accepted by several authors.43Krog (1982) suggested a classification of
genera (usneoide.g.Neuropogon, Protousnea, Evernia, Letharia, Lethariella).45 In this hypothesis
Neuropogon and Usnea are sister groups. Protousnea and Evernia form together the sister to the
clade, comprising of Neuropogon and Usnea. Finally, Letharia and Lethariella form the sister group
to the other usneoid genera. But gradually many diverse classications have been proposed due to a
lack of phenotypic characters.
A study based on the ITS sequence data supported the subgenera Eumitria and Usnea, and revealed a
new subgenus, Dolichousnea. 46 The authors also concluded that Usnea contains at least three taxa
at subgeneric level, Usnea, Eumitria, and Dolichousnea. Neuropogon was not included in this study
and the position remained unclear. Molecular phylogenies based on the ITS-LSU nrDNA and part of
the b-tubulin region have been used to examine the position of Neuropogon in Usnea s. lat.43
Bayesian inference and maximum parsimony strongly supported the monophyly of Neuropogon.
Subgenus Usnea and Neuropogon form a strongly supported group with subg. Eumitria and subg.
Dolichousnea is a consecutive monophyletic sister group. The following generic classification was
proposed: Usnea (subgenus Usnea only), Neuropogon, Eumitria, and Dolichousnea. Dolichousnea is
elevated to generic rank. The following new combinations are made: DolichousneaArticus, D.
longissima (Ach.) Articus, D. trichodeoides (Vain.) Articus, D. diffracta (Vain.) Articus, and
Eumitriapectinata (Taylor) Articus.43 Recently, the phylogenetic relationships of 52 Usnea species
from across the genus, based on ITS rDNA, nuLSU, and two protein-coding genes RPB1 and MCM7
have been investigated. The phylogeny based on the concatenated dataset revealed that the genus

260
Usnea is subdivided into four highly-supported clades, corresponding to the traditionally

circumscribed subgenera Eumitria, Dolichousnea, Neuropogon and Usnea. 47 However,
characteristics that have been used to describe these clades are often homoplasious within the
phylogeny and their parallel.
Evolution is suggested. The study has suggested that combinations of phenotypic characters are
suitable discriminators for delimitating species, but are inadequate to describe generic subdivisions.
Traditional Uses and Ethnopharmacology
The species of lichen genus Usnea is used for the treatment of various diseases such as diarrhea,
ulcer, urinary infection, tuberculosis, pneumonia, stomachache, anti-fungal, human pathogens, and
cattle fungal diseases.48–51 Some other uses of the species are for strengthening, hair growth,
sterility cure, avoring agent, pulmonary disease, antiseptic, antituberculosis and anti-viral diseases
are summarized in Table 1.52–60 The Usnea species are the most common source of antibiotic and
antifungal lichen acids, particularly usnic acid. The species have widespread potential for medicinal
applications. Usnea is used for weight loss, pain relief, fever control, and wound healing; and to
make phlegm easier to cough up.61,62 It was recorded that Usnea had been used directly on the skin
for sore mouth and throat.
U. longissima grows commonly on bark, mostly on the twigs of trees, bushes and over soil and rock
in temperate and alpine regions of India, it is known locally as “Syara” by the Bhotia and Garhwalis
of remote areas of Uttarkashi district of Uttarakhand, India and is used for making pillows. The
Baiga tribes of Madhya Pradesh, India used the species along with other ingredients for treating bone
fractures.62,63 Likewise the ancient Greeks used lichen as medicines. Hippocrates recommended a
lichen, perhaps U. barbata, for uterine complaints. The Chinese used U. longissima as an expectorant
and as a powder application to heal external ulcers in the name “Sun-Lo”. 1 It is also a major
ingredient of Chinese medicine.57 In China, this species of Usnea is also called as “Lao-tzu’s” beard,
“Pine gauze” and used for stopping sweating dizziness cold, pain and phlegm. U. longissima is still
utilizing today as a tincture to treat tuberculosis lymphadenitis. In the Bolivian Andes, U. longissima
is commercially sold as a folk medicine for cough and hoarseness.7,12 The Nitinaht Indians of
Vancouver also used the species for wound dressing in Turkey.63 U. longissima is used in the
treatment of gastric ulcers by the Anatolians as a folk medicine. This species is also used as to strain
impurities from hot pitch before the pitch was used as medicine. In Unani medicine, it has been
described to stimulate menstruation or induce abortion, taken orally and inserted into the vagina.63
However, it was used for treating cancer, tuberculosis, and ulcers in Turkey.64 It has also been used
as a decongestant and for the local treatment of ulcers and tuberculosis by Chinese people.
Usnea glabrata

261
U. barbata is used to treat mammary infections in cattle.(Dymov.et.al.,2023 The udder is washed

several times with a decoction of lichen and used for indigestion in humans, where the tincture or
decoction taken orally several times daily.65 In the Philippines, it has been used for wounds,
chopped, and mixed with coconut oil, spread over the wound and for abdominal pain where the
decoction is used as a drink.66 However, in Europe it was used for internal and external bleeding,
whooping cough, jaundice, and growing hair.67 Spanish people used it as drying agent and an
antiseptic for cracks and irritations of the feet.68 In China U. aciculifera was used to treat bladder
infection, painful urination, urinary retention, swelling, and edema in the heart and kidneys.57 In
Tanzania, U. articulate was used for the treatment of stomachache.69 A handful of U. articulate and
U. gigas are chewed fresh and the bitter juice swallowed, relieving pain per a time. In China, U.
ceratina was used for coughs, inamed lungs, pulmonary tuberculosis, hepatitis, heat related
headaches, infection due to injury, inamed lymph channels, mastitis, and snakebites.57
Usnea barbata
In traditional Argentinian medicine, teas of U. densirostra and U. durietzii were used externally as
astringents, antiseptics, and anti-inammatory agents.70,71. In China, U. diffracta has been applied to
treat a range of problems such as cough, tuberculosis of neck or lungs, headache, dizziness, sweating,
dim vision, swelling, pus oozing from breasts or sores, burns and scalds, snakebite, traumatic
injuries, bone fracture. Higher indication and higher toxicity of HG aloneas, e.g., 100 µM Cd had no
effect on phenols in the foliose lichen Hypogymnia. This may indicate that specific interaction
between Hg and SNP modulates final response, but no relevant data was found to discuss in detail.
We can only think that, owing to the complex chemistry of SNP, more SNP was consumed for NO
donating in the presence of Hg and this might produce more residue of SNP, leading to this side
effect. bleeding from external injuries, vomiting blood, blood in faeces, bleeding from uterus,
menstrual disorders, vaginal discharge, swelling of female genitalia, urinary tract afflictions, and
ascarid or schistosoma parasitic infections. (Marty.C.et.al.,2023)72,73 .The same species of Usnea
were used to cure fevers of the lungs, liver, and heart and fever caused by poisoning in Tibet74 while
in Korea, the species was used to induce menstruation (Pusan) and treat tuberculosis of the neck.75.
The traditional Chinese herbal medicine, U. orida has been used for aching in sinews and bones,
stopping bleeding or infection from external injuries, skin diseases, painful urination, coughs,
tuberculosis of lungs or neck, heart palpitations, and edema.76 The decoction of U. orida was also
used for colds.

262
and coughs in Europe,77 while in Chile, its infusion is used for diarrhea.49 U. himalayana is burned
as a “lichen cigarette” in Japan.78 U. hirta has been used by European people to heal wounds and to
prevent hair loss.77 U. laevis has been widely used to treat different kind of microbial infections
including dermatosis, fungal infections, tuberculosis, and pneumonia.50 In Canada, most of the
Usnea species were used for wound dressing, but U. longissima is preferred by wrapping around the
wound.79 It was recorded that U. pectinata had been used in China for stopping bleeding from
external injuries, relieving pain, bloody feces, and swelling (76).
Usnea plicata Usnea trichodoides

U. plicata used as an astringent for internal and external use,80 also for whooping cough,81 jaundice,
strengthening stomach and abdominal cavity, and restraining abortion in Europe.82 As the traditional
Indian herbal medicine, U. sikkimensis has been used for treating lung troubles, hemorrhages and
asthma.83 It has also been used to bandage surface wounds, skin eruptions, and boils.84 The
concoction of U. strigosa was taken orally for the treatment of headaches.58 Moreover, in Ireland U.
suboridana was used to treat sore eyes. In China, it was used to treat painful and reddened eyes,
bleeding from external injuries, and swelling.76 The traditional Chinese herbal medicine, U.
trichodeoides has been used to treat coughs, pulmonary tuberculosis, headaches, blurred vision,
inamed cornea, swellings, sores, and pus discharge, bleeding from external injuries, bloody feces,
uterine bleeding, menstrual disorders, and vaginal discharge.
Conclusions and Future Prospects
Within the fungus kingdom, lichens produce a wide array of both primary (intracellular) and
secondary (extracellular) compounds with different biological properties. However, the knowledge
of the biological potential of many lichens and their metabolites is very narrow compared to other
fungi. Moreover, this knowledge is very recent and limited investigations have been conducted for a
deeper understanding of the mechanism and cellular sites of action of lichen substances responsible
for the different pharmacological properties described so far. Thus, the aim of this review is to
provide up-to-date information about traditional uses, phylogeny, phytochemistry, pharmacology,
and toxicology of the most numerous and widespread genus of lichens, Usnea which comprises of
approximately 350 species based on scientist literatures.
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APPENDIX
A
1. A. Ashwini Sanap Chapter-23 238
2. A. Pramila Chapter-2 08
3. A. Rupali Nikam Chapter-23 238
4. A. Shajahan Chapter-4 32
5. Adrija Mukherjee Chapter-8 74
6. Alka Rajput Chapter-24 246
7. Anju Rao Chapter-1 01
8. Ankita Pokhriyal Chapter-11,14 102,136
9. Arijit Das Chapter-8 74
B
10. B. Sowbakkiyalakshmi Chapter-7 63
11. Barot Jagruti K Chapter-3 23
12. Bikram Saha Chapter-5 40
C
13. C. Kanimozhi Chapter-12 111
D
14. D. Swarna Bharathi Chapter-12 111
15. Debraj Chakraborty Chapter-8 74
16. Deepesh Lall Chapter-16 171
G
17. G. S. Shailaja Sharma Chapter-17 181
18. Gargee Dhar Purkayastha Chapter-5 40
I
19. Intwala Siddhi M. Chapter-3 23
iv
J
20. Jahanarah Khatoon Chapter-21 225
K
21. K. Kolanjinathan Chapter-7 63
22. K. V. Shalini Chapter-19 208
23. Kirankumar Khandare Chapter-18 200
24. Kumud Das Chapter-9 80
M
25. M. I. Zahir Hussain Chapter-4 32
26. Malay Duyari Chapter-6 54
27. Mayuri Saini Chapter-11,14 102,136
28. Mehtab Yasmeen Chapter-15 161
N
29. Nisha Verma Chapter-16 171
30. Nripen Kumar Gogoi Chapter-9 80
P
31. P. Chitra Chapter-20 214
32. Poonam Sabrwal Chapter-10 89
33. Prabir Sahoo Chapter-6 54
R
34. R. Marivignesh Chapter-4 32
35. Ranjana Singh Chapter-17 181
36. Renu Yadav Chapter-24 246
S
37. S. Gomathi Chapter-20 214
38. S. Jayashri Pawar Chapter-23 238
39. S. Kaviya Chapter-20 214
40. S. Kaviyalakshmi Chapter-19 208
41. S. P. Hemavarthini Chapter-20 214
42. S. Srinithiksha Chapter-19 208
v
43. S. Sujatha Chapter-12 111

44. Salman Hussain Chapter-9 80
45. Shiwani Latwal Chapter-1 01
46. Sourish Dasgupta Chapter-5 40
V
47. V. Naveen Sahith Chapter-22 231
48. V. Sai Sruthi Chapter-22 231
49. Varsha Sharma Chapter-24 246
Y
50. Yusra Mohaddis Khatib Chapter-13 127
Z
51. Zarin Zarrar Kuwari Chapter-13 127
52. Zohra Zarrar Kuwari Chapter-13 127
vi
Editor-1 Level-1
Mr. Balwant Singh, M.Sc.-AAS, M.Sc.-Botany

Ph.D. Research Scholar
Department of Botany
K. S. Saket P.G. College Ayodhya
Dr. Ram Manohar Lohia Avadh University, Ayodhya, India
balwantsingh1642@gmail.com
+91 7408600478
He is a Young and Active Researcher and Academician as well as

Pursuing Ph.D. (Botany) in the Department of Botany from K. S. Saket P.G.
College Ayodhya, Uttar Pradesh affiliated to Dr. Ram Manohar Lohiya Avadh
University Ayodhya, Uttar Pradesh, India. He is also working as Guest Faculty,
Department of Botany, B. P. P.G. College Narayanpur, Maskanwa, Gonda (UP)
India. He has 7 Years of Teaching and 5 Years Research Experience. He has
completed dual master’s degrees as M.Sc. in Applied Animal Science from
Babasaheb Bhimrao Ambedkar Central University Lucknow, Uttar Pradesh in
2015 and M.Sc. in Botany from Acharya Narendra Deo Kisan P.G. College
Babhnan, Gonda, Uttar Pradesh in 2018. He has published more than 10
Research Articles in National and International Journals and more than 10 Book
Chapters and also 6 Books in his credits. He has also participated in more than 75
National and International Seminar, Conferences and Workshops. Editor is the
Life Members of National Bodies like Indian Science Congress Association, Asian
Biological Research Foundation, Indian Academy of Science and Technology
Society and Editorial Board Member of Journal of Emerging Trends in Medicinal
Plants Research. He also received some prestigious awards like- YOUNG
SCIENTIST AWARD, INCREDIBLE PUBLICATION AWARD- 2022, YOUNG
BOTANIST AWARD.
vii
Editor-2 Level-1
Mr. Mukul Machhindra Barwant M.Sc. Botany

Yashavantrao Chavan Institute of Science, Satara – 415 001,
Maharashtra, India
mukulbarwant97@gmail.com
+91 7057529221
The Editor is well known for his great work in the field of Botany
especially in Phycology and Biodiversity and Phytochemistry in Researcher. He
has more than 04 years research experiences in their field. He has completed their
M.Sc. in 2017 and done the registration for Ph.D. through Qualified by PET of
Shivaji University Kolhapur the Year 2018. He is currently Ph.D. Pursuing
(Research Scholar) Department of Botany, Yashavantrao Chavan Institute of
Science, Satara. He has published more than 30 National and International
Research Articles in reputed Journals. He has also published 12 Books and more
than 10 Book Chapters. Editor also has participated more than 40 National and
International Seminar, Conference and Workshops. Beside this, the editor also
works as several National and International Journals Member of Reviewer Board
and Editorial Board also work Deputy Chief Editor. He also received award like
BEST PRESENTER AWARD-2021, YOUNG RESEARCHER AWARD 2021, AIB-VSC-
BEST YOUNG SPEAKER AWARD-2021, BEST YOUNG SCHOLAR, AWARD OF
BEST BOOK CHAPTER, BEST RESEARCHER AWARD. An Indian Patent also in
his credit for HEAVY METAL IDENTIFICATION GLOVES USING NANO-
TECHNOLOGY.
viii
Editor-3 Level-1
Ms. Shivangi Tripathi, M.Sc.-AAS

Department of Bioscience, Integral University Lucknow
Uttar Pradesh, India
Former Research Assistant (DST SERB)
Scientist, King George’s Medical University, Lucknow (UP),
India. shivangii4.1993@gmail.com
She is an Active Researcher and Pursuing Ph.D. (Biosciences) in the

Department of Biosciences from Integral University, Lucknow, Uttar Pradesh,
India. She is also working as Young Scientist post in Department of Microbiology,
King George’s Medical University, Lucknow, Uttar Pradesh, India. She has 5 Years
of Research Experience in Mycology Laboratory. He has completed master’s
degrees as M.Sc. in Applied Animal Science from Babasaheb Bhimrao Ambedkar
Central University Lucknow, Uttar Pradesh in 2015. She has published more than
7 Research and Review Papers in National and International Journals and more
than 10 Book Chapters. He has also participated in more than 25 National and
International Seminar, Conferences and Workshops. Editor is the Life Members of
National Bodies like Indian Science Congress Association. She has working as
Research assistant in project (2018-2022) funded by Department of Science and
Technology, Science and Engineering Research Board (SERB) New Delhi, India.
She also received young Scientist Fellowship from Department of Health Research
(DHR), New Delhi, India in 2023. She has done research basically pathogenic
fungus.
ix
Editor-1 Level-2
Dr. Vinay Kumar Singh, M.Sc., Ph.D.

Associate Professor
K. S. Saket P.G. College Ayodhya, Uttar Pradesh, India
Dr. Ram Manohar Lohia Avadh University, Ayodhya,
vksingh77saket@gmail.com
The Editor is well known for his great work in the field of Botany
especially in Mycology and Plant Pathology as Academician and Researcher. He
has more than 14 years teaching and 18 years of research experiences in their
field. He has completed their M.Sc. in 1998 and awarded their Ph.D. in the Year
2001. He is currently working as Associate Professor in Department of Botany at
K. S. Saket P.G. College Ayodhya affiliate to Dr. Ram Manohar Lohiya Avadh
University Ayodhya, Uttar Pradesh, India. He has published more than 20
National and International Research Articles in reputed Journals. He has also
published 7 Books and more than 10 Book Chapters. Editor also has participated
more than 60 National and International Seminar, Conference and Workshops.
Beside this, the editor has Guide of several Ph.D. Students as Supervisor and also
has member of Board of Studies. Editor is the Life Members of National Bodies like
Indian Science Congress Association, Asian Biological Research Foundation and
Editorial Board Member of Journal of Emerging Trends in Medicinal Plants
Research.
x
Editor-2 Level-2
Dr. Vanita Chandrashekhar Karande

M.Sc., Ph.D.
Former Head
P. G. Department of Botany
Yashavantrao Chavan Institute of Science, Satara – 415 001,
Maharashtra, India
vaneetachandra@gmail.com
The Editor is well known for her great work in the field of Botany,
especially in Phycology, Biodiversity, Cell Biology, and Ecology Taxonomy as
Academician and Researcher. She was Head, the P. G. Department of Botany,
Yashavantrao Chavan Institute of Science, Satara. She is a recognized Guide for
M.Phil. and Ph.D. of Shivaji University Kolhapur. 5 Students were awarded Ph.D.
under her guidance She has published more than 25 Research Articles in reputed
Journals. She has the Co-authored of 5 Books and presented more than 40 Papers
presented in Conferences, Seminars, Workshops and Symposia. She also
completed 4 research projects funded by UGC during the period of 2000-2011 She
was a member of BOS in Botany Shivaji University Kolhapur.
xi
Editor-3 Level-2
Dr. Aisha Kamal, M.Sc., Ph.D.

Professor
Department of Bioengineering, Integral University Lucknow
Area of Specialization: -
Plant Biotechnology
Agricultural Biotechnology
Environmental Biotechnology
She is a Professor in the Department of Bioengineering, Integral

University Lucknow. She has 20 years of research and teaching experience. She
has 39 research and review papers, 12 book chapters and 2 books to her credit.
She has guided 6 Ph.D. and 15 Postgraduate thesis. Currently, she is actively
involved in investigating the role of different phytohormones and other natural
products in amelioration of various stress generated growth disorders in plant.
She successfully established that seed priming with SA and GA3 prevents damage
to the photosynthetic machinery by enhancing antioxidant activity, as well as
reducing oxidative stress in pea plant. She is also working on elucidation of
protective role of different plant extracts in oxidative stress induced hepatotoxicity
and successfully formulated liposomal vesicles from cyanobacterial lipid. The
study revealed the better efficacy of encapsulated thymoquinone as compared to
thymoquinone alone indicating cyanosome as a promising candidate for drug
carrier. Importantly, the use of natural lipids from cyanobacteria will circumvent
the need for the costly synthetic lipid for the preparation of liposome.
xii
Editor-1 Level-3
Dr. Shailendra Kumar, M.Sc., M.Phil., Ph.D.

Professor and Head
Department of Microbiology
Dr. Ram Manohar Lohia Avadh University Ayodhya
shailendra.microbio@gmail.com
shailendrakumar@rmlau.ac.in
Sir is well known Professor and Researcher in the field of Microbiology

with the 22 years of teaching and 26 years of research experiences. He has
published more than 30 research articles in National and International reputed
journals. He has authored 12 book chapters and 3 Books. He has delivered several
guest lectures, invited talks, and participated in more than 25 National and
International seminars, conferences and workshops. He has organized several
conferences, workshops and training programmes and received research grants
from UGC, SERB and Government of Uttar Pradesh. He is member of professional
bodies, viz., Microbiologists Society of India, Society of Biochemists of India,
Society for Agriculture Innovation & Development and Society for Environmental
Sustainability. He is also member of editorial board and reviewer of many
Journals. Beside this, the Editor of Honour has guided of several M.Sc. and Ph.D.
Students. Sir is Honoured by several awards like Distinguished Scientific Award,
Young Scientist Award, Excellence Teaching Award, INSA-Visiting Scientist
Programme Fellowship, and many more.
xiii
Editor-2 Level-3
Dr. Belle Damodara Shenoy, M.Sc., Ph.D.

Scientist
CSIR-NIO Regional Centre, Visakhapatnam,
Andhra Pradesh, India
Marine Biodiversity
Marine Bioinformatics
Biotechnology & Molecular Biology
Sir is a fungal taxonomist by training. After completing his Bachelor’s

and Master’s degrees in India, he pursued his PhD in fungal taxonomy at the
University of Hong Kong under the guidance of Prof. Kevin Hyde and Dr. Rajesh
Jeewon. His PhD thesis titled “Multigene phylogeny of selected anamorphic
ascomycetes” dealt with the evolutionary relationships of asexual genera such as
Sporidesmium complex and provided insights into the implications of their
polyphyly in the fungal taxonomy. Dr. Shenoy, for a brief period of time, worked as
a post-doctoral fellow with Dr. Keith Seifert at the Agriculture and Agri-Food
Canada, Ottawa on DNA barcoding of Fusarium. In 2008, Dr. Shenoy returned to
India and joined the Microbial Type Culture Collection (MTCC), CSIR-Institute of
Microbial Technology, Chandigarh (CSIR-IMTECH). He supervised two PhD
students at CSIR-IMTECH and published several high-impact papers on fungal
taxonomy, phylogeny, DNA barcoding and comparative genomics. In 2013, Dr.
Shenoy moved to CSIR-National Institute of Oceanography, Goa and initiated
research on the diversity and taxonomy of microbes associated with tarballs
(crude-oil derivatives). He is presently working at CSIR-NIO Regional Centre,
Visakhapatnam, Andhra Pradesh. Dr. Shenoy has so far published 45 papers in
reputed international journals. He is the founding member and presently the
managing editor of a new mycology journal, MycoAsia – Journal of modern
mycology (www.mycoasia.org). Dr. Shenoy has been a member of the International
Sub-commission on Colletotrichum taxonomy. Dr. Shenoy is on the Advisory Board
of Amity Institute of Oceanography and Atmospheric Sciences, India. His non-
scientific hobbies include travelling and exploring India and writing poems. Based
on their contributions to mycology, sir is also known “The Fungi Man of India”.
xiv
Editor-3 Level-3
Dr. Gopa Banerjee, MBBS, MD

Professor
Department of Microbiology, King George Medical University
Lucknow, Uttar Pradesh, India
Medical Microbiology
Medical Mycology
Medical Parasitology
She is a Professor in the Department of Microbiology, King George’s

Medical University Lucknow. She has completed their MBBS in 1985 and MD in
1992. She has awarded with Platinum Jubilee Gold Medal for presenting the best
thesis in MD and completed in first attempt in Medical Microbiology. She has 3
years working experience as Senior Resident from Sanjay Gandhi Post Graduate
Institute of Medical Sciences, Lucknow. She also works as a consultant in
Shushila Tiwari Memorial Hospital Haldwani from 2000-2002. She has 31 years of
research and teaching experience. She has more 53 Research and Review papers
in National and International reputed Journals, 10 book chapters. She has guided
more than 26 MD thesis and 11 PhD thesis as Supervisor and Co-Supervisor.
Currently, she is actively involved in various ongoing project with various
organization viz. Indian Council of Medical Research and Department of Health
Research in Mycology section. She Completed 2 Projects with UPCST and 2
Projects with DST SERB, New Delhi. She also working as a Co-Nodal Officer in
National One Health Programme for Prevention and Control of Zoonotic Diseases
(NOHPCZ) funded by NCDC, New Delhi. Editor also has participated more than 30
National and International Seminar, Conference and Workshops. Editor is the Life
Members of International Society for Human and Animal Mycology (ISHAM) and
Reviewer of various reputed journals like Indian Journal of Medical Microbiology
and many more.
xv
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Research in Mycology Series-II

Book · June 2023

Mukul Barwant Shivangi Tripathi

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Mr. Balwant Singh

Year of Publication: 2023

Printed at: Sheen Graphics

CHAPTER TITLE OF PAGE

Chapter-01 UNDERSTANDING THE ROLE OF ANTIFUNGAL METABOLITES FROM

Chapter-02 THE STUDY OF FUNGAL SPECIES ISOLATED FROM AMARANTHUS

Chapter-03 MYCOREMEDIATION: ANNIHILATION OF ENVIRONMENTAL

Chapter-04 INVITRO ANTAGONISTIC ACTIVITY OF ENDOPHYTIC BACTERIA OF

Chapter-05 REDEFINING THE RELEVANCE AND EFFICACY OF MICROBIAL

Chapter-06 DIVERSITY AND DISTRIBUTION OF ENDOPHYTIC FUNGI IN VELLAR

Chapter-07 AN OVERVIEW OF ENDOPHYTIC FUNGI SECONDARY METABOLITES

Chapter-08 MYCELIAL BRICKS- BUILDING BLOCKS FOR THE FUTURE 74-79

Chapter-09 ISOLATION OF ENDOPHYTIC MICROBES FROM ACACIA PENNATA (L.)

Chapter-10 DIVERSITY OF CORTICIOID FUNGI BELOLNGING TO ORDER

Chapter-11 BENEFICIAL EFFECT OF MUTUALISTIC INTERACTION BETWEEN

Chapter-12 FUNGAL CULTURE, HABITAT WITH SPECIAL REFERENCE TO DISEASE

Chapter-13 NUTRITIONAL VALUE OF AGARICUS BISPORUS, ITS POTENTIAL USE

Chapter-14 MECHANISMS OF ENDOPHYTIC FUNGI USED IN PLANT PROTECTION 136-160

Chapter-15 A COMPREHENSIVE REVIEW ON BIOLUMINESCENT FUNGI 161-170

Chapter-16 MYCOLOGY A DETAILED EXAMINATION: IN CONTRAST OF PRESENT

Chapter-17 ARBUSCULAR MYCORRHIZAL FUNGI ACT AS NATURAL

Chapter-18 PREDACEOUS FUNGI: AN OVERVIEW 200-207

Chapter-19 POST-HARVEST FUNGAL DISEASE AND ITS MANAGEMENT ON

Chapter-20 NEMATOPHAGOUS FUNGI – A REVIEW 214-224

Chapter-21 ROLE OF FUNGI IN BIOREMEDIATION OF POLLUTANTS 225-230

Chapter-22 FUNGAL METABOLITES WITH ANTICANCER ACTIVITY 231-237

Chapter-23 FUNGAL TANNASE: THE MOST EMINENT BIOCATALYST FOR

Chapter-24 SPECIAL EMPHASIS ON THE STUDY OF MEDICINALLY IMPORTANT

ABOUT THE EDITORS vii-xv

Keywords: Antifungal Metabolites, Bryophytes, Plant Bioactive Compounds, Liverworts.

Department of Botany, Panjab University, Chandigarh, India shiwanilatwal@gmail.com

Shiwani Latwal & Anju Rao

Shiwani Latwal & Anju Rao

7- Bazzania trilobata Lepidoziaceae Botrytis cinerea Scher et al., 2004

Shiwani Latwal & Anju Rao

Isoriccardin C Plagiochasma Aytoniaceae Candida albicans Asakawa, 2017

Secondary metabolites from Bryophytes showing Anti-fungal properties

Shiwani Latwal & Anju Rao

Shiwani Latwal & Anju Rao

3. Asakawa, Y. (2017). Polyphenols in Bryophytes: Structures, Biological Activities, and Bio‐and

Shiwani Latwal & Anju Rao

Keywords: Rhizoplane, Rhizosphere, Phylloplane, Amaranthus viridis, Hibiscus cannabinus.

a) Potato Sucrose-Agar (PSA)

Table-1: List of Fungal Species isolated from Amaranthus viridis (Field)

Table-2: List of Fungal Species isolated from Amaranthus viridis (Market)

Table-4: List of Fungal Species isolated from Hibiscus cannabinus (Market)

Abundance Of Funal Colonies In Comparision To Field And Market

Population Dynamics of Mycoflora in different Soil Ecological Niches

Intwala Siddhi M.1 & Barot Jagruti K.2

Keywords: Mycoremediation, Heavy Metals, Xenobiotic Toxicity.

1Naran Lala College of Professional and Applied Sciences, Navsari.

Intwala Siddhi M. & Barot Jagruti K. 23

Mycoremediation is the form of bioremediation in which fungi-based technology is used to

Intwala Siddhi M. & Barot Jagruti K. 24

Intwala Siddhi M. & Barot Jagruti K. 25

Intwala Siddhi M. & Barot Jagruti K. 26

Intwala Siddhi M. & Barot Jagruti K. 27