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ANTIFUNGAL POTENTIAL OF MARINE SOIL ACTINOBACTERIA

ANTIFUNGAL POTENTIAL OF MARINE SOIL ACTINOBACTERIA
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ANTIFUNGAL POTENTIAL OF
MARINE SOIL ACTINOBACTERIA
LIPUN KUMAR PRADHAN

K. KOLANJINATHAN
SUBHASHREE JENA
Monograph Book
ANTIFUNGAL POTENTIAL OF
MARINE SOIL
ACTINOBACTERIA
Editors
Mr. Lipun Kumar Pradhan
Department of Microbiology, Faculty of Science, Annamalai University, Annamalai Nagar,
Chidambaram, Tamil Nadu, India.
Dr. K. Kolanjinathan
Ms. Subhashree Jena

Published by
JPS Scientific Publications

India
i
Copyright©2019 by JPS Scientific Publications. All Rights Reserved
Published by
JPS Scientific Publications, Tamil Nadu, India.

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Published in India.
International Standard Book Number (ISBN): 978-81-940316-5-9
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ii
Contents
Chapter Chapter Title Page

Number Number
1 Introduction 1
2 Review of Literature 5
3 Objectives 16
4 Materials and Methods 17
5 Results 22
6 Discussion 28
7 Conclusion 30
8 References 31
iii
Antifungal Potential of Marine Soil Actinobacteria 1
1
INTRODUCTION
Of the total sea surface, only 7 – 8 % is coastal area and the rest is deep sea, of
which 60 % is covered by water more than 2000 m deep. The deep sea is a unique
and extreme environment characterized by high pressure, low temperature, lack of
light and variable salinity and oxygen concentration. Though, the deep-sea area is
geographically vast, scientific knowledge and research on deep sea microbial
diversity is meagre (Das et al. 2006). However, it has been shown to be a good source
of novel microorganisms for the discovery of new antibiotics (Bull et al. 2000).
Marine environments were recently found to be one of the important sources for the
isolation of new Actinomycetes with potentiality to produce chemically diverse
compounds with a wide range of biological activities (Bredholt et al., 2002). It is a
boon in marine bio prospecting for the exploration and exploitation of the rich
biological and chemical diversity found in marine organisms that inhabits the
oceans.
Marine microorganisms are increasingly becoming an important source in the

search for industrially important molecules. Today both academic and industrial
interests in marine microorganisms are on the rise, because unique and biologically
active metabolites have been reported from marine organisms (Jensen and William,
1994; Zhang et al., 2005; Imada et al., 2007). Marine environment contains a wide
range of distinct microorganisms that are not present in the terrestrial environment.
Though some reports are available on antibiotic and enzyme production by marine
Actinomycetes, the marine environment is still a potential source for new
Actinomycetes, which can yield novel bioactive compounds and industrially important
enzymes (Sharma and Pant, 2001).
Microorganisms are the main colonizers of the earth, bestowed with inherent
physiological and functional diversity and have found applications in agriculture,
medicine, industry and environment. Actinomycetes are frequently filamentous and
sporulating with DNA rich in G + C from 55 – 75 % (Ho et al., 2002). Actinomycetes
are Gram-stain positive saprophytic bacteria that are widely distributed in soil and
other terrestrial environments. They contribute significantly to the turnover of
©JPS Scientific Publications, India

complex biopolymers of organic matter, such as chitin and lignocellulose in eco

systems (Cao et al., 2005). There are two characteristics that distinguish Actinomycetes
from fungi (Bull A.T et al., 2005).
a) Absence of cell nucleus, thus resembling prokaryotes.

b) They form hyphae 0.5-1.0 μm in diameter, which is smaller than the fungal
hyphae.
For discovering novel bioactive compounds of biotechnological interest is

important.
Several factors such as:
• Choice of screening source
• Pre-treatment of soil
• Selective medium
• Culture condition and
• Recognition of Actinomycetes colonies on a primary isolation plates, are used
for identification of unique Actinomycetes. (Baskaran et al., 2011)
The important genera of Actinomycetes are Streptomyces, Nocardia,

Micromonospora, Thermomonospora, Actinoplanes, Microbispora, Streptosporangium,
Actinomadura, Actinosynnema, Dactylosporangium, Rhodococcus, Gordona,
Intrasporangium and Streptoalloteichus. Actinomycetes from the genera Actinoplane,
Streptomyces and Actinopolysporahave been reported to produce a number of broad-
spectrum antibiotics. Actinomycetes are also exist in symbiotic association with plants
and animals and other living organisms.
Around 23 000 bioactive secondary metabolites produced by microorganisms

have been reported and over 10 000 of these compounds are produced by
Actinomycetes, representing 45 % of all bioactive microbial metabolites discovered
(Vimal et al., 2009). Among the different types of drugs prevailing in the market,
antifungal antibiotics are a very small but significant group of drugs and have an
important role in the control of mycotic diseases. Fungal infections have been
gaining prime importance because of the morbidity of hospitalized patients (Beck-
Sague and Jarvis, 1993). Microbial diseases are increasing year by year and they are
becoming a big threat to public health (Binder et al., 1999; Smolinski et al., 2003;
Morens et al., 2004; Jones et al., 2008). In particular, Candidiasis and Aspergillosis
have remained the opportunist fungal infections that occur most frequently.

Presently, they represent a major area of concern in the medical field;

however, the occurrences of invasive fungal diseases, particularly in AIDS and other
immune compromised patients, are life-threatening and they increase economic
burden (Beck-Sague and Jarvis, 1993; Talbot et al., 2006). Fungi are found to be
important pathogens and among which Candida albicans are the most common
pathogen in several nosocomial infections and control efforts should target such
fungal infections (Beck-Sague and Jarvis, 1993). Banana Fusarium wilt, also called
banana Panama wilt, is a destructive disease that affects bananas in tropical and
subtropical areas worldwide. Fusarium oxysporum f. sp. cubense race 4 (FOC4) is one
of the major pathogens causing banana Fusarium wilt (Sun, 2014). The report reveals
that fungal infections have been gaining prime importance because of the morbidity
of hospitalized patients (Hwang et al., 1994). Extensive application of chemical
pesticides in agriculture has led to numerous side effects for the environment and
human health. In search for environmentally friendly and safe substitute, biological
pesticides are more appropriate compared to chemical pesticides.
Although, more than 30,000 diseases have been clinically described, less than
one third of these can be treated symptomatically and fewer can be cured (Schultz
and Tsaklakidis et al., 1997). New therapeutic agents are urgently needed to fulfil the
medical needs that are currently unmet (Wright and Sutherland, 2007). Natural
products once played a major role in drug discovery (Demain and Zhang, 2005;
Zhang, 2005). Although, the exploitation of marine Actinomycetes as a source for
discovery of novel secondary metabolites is at an early stage, numerous novel
metabolites have been isolated in the past few years (Lam, 2006). The need for new,
safe and more effective antifungal is a major challenge to the pharmaceutical
industry today, especially with the increase in opportunistic infections in the immune
compromised host. The history of new drug discovery shows that novel skeletons
from natural sources (Bevan et al., 1995). Most of these agents were reported to
exhibit excellent therapeutic efficacy in experimental dermatophytic animals as well
as in patients with dermatophytosis, so that mycological cure can be achieved.
(Gupte et al., 2002). Among Actinomycetes, around 7 600 compounds are produced by
Streptomyces species. Many of these secondary metabolites are potent antibiotics,
which has made streptomycetes the primary antibiotic-producing organisms
exploited by the pharmaceutical industry (Bonjar et al.., 2004; Berdy, 2005)

Numerous antibiotics have been isolated from a variety of microorganisms;

however, studies are still being conducted to identify novel antibiotics effective
against pathogenic fungi (Atlas and Bartha, 1986). Marine actinobacteria are useful
biological tools for the production of antifungal substances against fungi (Okami and
Hotta, 1988). Marine Actinobacteria are widely distributed in biological sources such
as fishes, mollusks, sponges, seaweeds, mangroves, besides seawater and sediments.
These organisms are importance not only for their taxonomic and ecological
perspectives, but also for their production of novel bioactive compounds like
antibiotics, antitumor agents, immunosuppressive agents, enzymes, enzyme
inhibitors, pigments (Dharmaraj, 2010), growth promoting substances for plant and
animals, immunomodifiers, enzyme inhibitor and many other compounds of use of
man. They have provided about two-thirds (more than 4000) of naturally occurring
antibiotics including many of those important in medicine, such as aminoglycosides,
anthracyclines, chloramphenicol, beta lactams, macrolide, tetracycline etc.
(Shadomy, 1987).

2
REVIEW OF LITERATURE .
2.1 ANTIMICROBIAL EFFECTS OF Actinomycetes

2.1.1 ANTIBACTERIAL ACTIVITY
Mustafa Oskayet al. (2004) identified A total of 50 different Actinomycetes
strains were recovered from farming soil samples collected from Manisa Province
and its surrounding. These were then assessed for their antibacterial activity against
four phytopathogenic and six pathogenic bacteria. Results indicated that 34% of all
isolates are active against, at least, one of the test organisms; Agrobacterium
tumefaciens, Erwinia amylovora, Pseudomonas viridiflova, Clavibactermichiganensis subsp.
michiganensis, Bacillus subtilis ATTC 6633, Klebsiella pneumoniae ATTC 10031,
Enterococcus feacalisATCC 10541, Staphylococcus aureus ATCC 6538, Esherichia coli
ATCC 29998 and Sarcina lutea ATCC 9341. According to antibacterialactivity and
spectrum broadness, seven of the isolates were selected and characterized by
conventional methods. The unusual antibiotic profile of these isolates underlined
their potential as a source of novel antibiotics.
Sathiyaseelan and Stella (2011) isolated five Actinomycetes strains from soil
collected in two different regions of parangipattai. The physico-chemical
characteristics of soil samples are analysed. Morphological studies indicated that the
strains belonged to the genera Streptomyces spectabilis, Actinomadura roseale,
Streptomyces platensis, Streptomyces kavamyceticus and Streptomyces citricolor. Out of five
isolated Actinomycetes species, one was selected for antimicrobial activity against five
human pathogens. Streptomyces citricolor showed the best level of antibacterial effect
against Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Salmonella typhi
and Vibrio cholerae whereas did not showed any antifungal effect against Aspergillus
niger, Fusarium oxysporum, Rhizoctonia solani, Candida albicans and Penicillium citrinum.
Valli et al. (2012) screened twenty-one strains of Actinomycetes were isolated

from sample Royapuram, Muttukadu, Mahabalipuram sea shore and Adyar estuary,
Preliminary screening was done using cross-streak method against two Gram
positive and eight Gram negative bacteria. The most potent strains C11 and C12

were selected from which antibacterial substances were extracted. The antibacterial
activities of the extract were performed using Kirby-Bauer method. Molecular
identification of those isolates was done. All the 21 isolates were active against at
least one of the test organisms. Morphological characters were recorded. C11
showed activity against Staphylococcus species (13.0±0.5 mm), Vibrio harveyi
(11.0±0.2 mm), Pseudomonas species (12.0±0.3 mm). C12 showed activity against
Staphylococcus species (16.0±0.4 mm), Bacillus subtilis (11.0±0.2 mm), Vibrio harveyi
(9.0±0.1 mm), Pseudomonas species (10.0±0.2 mm). 16S rRNA pattern strongly
suggested that C11 and C12 strains were Streptomyces species. They revealed that
the marine Actinomycetes from coastal environment are the potent source of novel
antibiotics.
Muharram et al. (2013) screened thirty-three soil-isolates of Actinomycetes for

their antimicrobial activities. Three potent isolates, (SA110, SC110 and SF202),
showed antibacterial and antifungal activity against one or more strain of Escherichia
coli, ATCC 8739; Candida albicans ATCC 66027; Candida albicans ATCC 2091,
Aspergillus niger, ATTC 16404 Staphylococcus aureus, ATCC 29213; Bacillus subtilis,
ATCC 11774 and Streptococcus epidermidis ATCC 12228. Screening of the
antimicrobial activity was conducted by Cross-streak, Cork-borer and agar well
methods. The isolates of SA110, SC110 and SF202 were identified as Streptomyces
samposonii, Streptomyces albidoflavus and Streptomyces roche, respectively based on a
variety of morphological, physiological and molecular analysis of 16S rDNA gene
sequence isolates. Factors affecting the biosynthesis of antimicrobial agent like
different inoculum size, pH values, temperatures, incubation period, and different
carbon and nitrogen sources were analysed.
Sandeep Rana and Menaka Devi Salam (2014) determined the antibacterial
and antifungal potential of Actinomycetes isolated from soil samples collected from
two different locations of Punjab, India. The Actinomycetes isolates showed various
types of color pigments like pink, yellow, orange, red and brown which may have
potential use in industries. Out of 23 Actinomycetes isolates, 7 of them showed
significant inhibition of different Gram positive and Gram-negative bacteria and also
some fungi. Actinomycetes isolate no. A4 inhibited Klebsiella pneumoniae and
Salmonella enterica, Salmonella typhimurium efficiently. Actinomycetes isolate no. A5
inhibited Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Salmonella
enterica typhimurium and the fungi Alternaria alternata and Trichoderma viride. The
crude antimicrobial compound from the best isolates A4 and A5 were obtained by
ethyl-acetate extraction and the MIC against two different bacteria Klebsiella

pneumoniae and Salmonella entericatyphimurium were 15 μg/ml and 12 μg/ml

respectively. Determination of the nature of the antimicrobial compound by TLC
analysis indicated that the compound may be alcohol, phenol or steroid in nature.
The antimicrobial compound obtained from isolate A5 was stable even above 60 °C
and in the presence of proteinase K enzyme (5 mg/ml). The results showed that
isolate A5 which produced dark orange colour pigment has excellent antimicrobial
characteristics with very low MIC value and stability at high temperatures and high
concentration of proteinase K.
Saraswathi et al. (2015) isolated a total of sixteen isolates of Actinomycetes from

marine sediments of Chennai, Tamil Nadu, India. Each isolate was tested against
four pathogenic bacteria and also against pathogenic fungi. Among the sixteen
isolates, one of the isolates showed potent activity against specific bacteria. The
selected isolate appears to produce high anti-bacterial compounds on starch casein
agar medium and nutrient agar medium respectively, by using the cross-streak
method. The potent Actinomycetes were characterized by morphological methods
consist of macroscopic and microscopic methods. The mycelium colour and the
appearance were observed as well as various biochemical tests such as starch
hydrolysis, carbohydrate utilization, catalase test, etc. was performed. Further
purification of the crude compound resulted in three distinct bands in different
solvent system. Also, the anti-bacterial activity was effective for the crude
compound.
Pathalam Ganesan et al. (2017) revealed that Microbial diseases are increasing
year by year and they are becoming a big threat to public health. There are more than
200 known diseases transmitted by bacteria, fungi, viruses, prions, rickettsia and
other microbes to humans. The emergence of drug resistance to chemical drugs is the
biggest threat in controlling human pathogens. Hence novel antimicrobial agents
from Actinomycetes are timely needed for the control of several human pathogens.
The aim was to find some Actinomycetes with antimicrobial metabolites. Methods:
Soil samples were collected from Nilgiris district in Western Ghats of Tamil Nadu,
India. Actinomycetes were isolated using serial dilution and plating techniques on
Actinomycetes Isolation Agar. Streptomycin and Ketoconazole (25 µg/disc) were used
as reference controls. The active strains were identified by 16S rRNA and
phylogenetic tree was constructed; the sequences were submitted in the GenBank.
Totally, 106 Actinomycete strains were isolated and cross streaked against various
human microbial pathogens. Only 44 (41.50 %) exhibited good antimicrobial activity
against different pathogenic microbes. Five isolates (FMS-20, TGH-30, TGH-31,
TGH-31-1 and IS-4) were chosen for secondary screening using filtrate. Among them

FMS-20 filtrate showed good inhibition on the 16th day against all tested microbial
pathogens. Further the intracellular methanol extract of FMS- 20 showed maximum
zone of inhibition against A. brasiliensis (22 mm) at 5 mg/disc.Similarly, the
extracellular ethyl acetate extract of FMS-20 showed maximum zone of inhibition
against Bacillus subtilis (25 mm).
Paul NjengaWaithaka et al. (2017) isolated and screened Actinomycetes for

antimicrobials from Menengai Crater in Kenya. The Actinomycetes were isolated
using Starch Casein Agar, Luria Bertani Agar and Starch Nitrate Agar. Primary
screening for antagonism was carried out using perpendicular method while
secondary screening was done using agar disk technique. Extraction of the
antimicrobials was carried out using ethyl acetate. Sensitivity testing of the crude
extracts against Staphylococcus aureus, Bacillus subtili, Escherichia faecalis, Escherichia coli,
Klebsiella pneumoniae, Salmonella typhi, Xanthomonas campestris, Erwinia carotovora,
Candida albicans, Alternaria alternate and Fusarium oxysporum were carried out using
Agar well technique. Biochemical tests and carbon source requirements were used in
characterization of the selected antimicrobial producers. M1 was the best agar
medium for isolation of Actinomycetes. The number of Actinomycetes from regions A,
B, C, and D in the crater varied significantly (F = 27.50 P = 0.000). Out of the 156
Actinomycetes isolates, 20 isolates were positive for both primary and secondary
screening for antimicrobials. There was no significant difference in the zones of
inhibition in primary screening of the Actinomycetes for antagonistic properties against
the test pathogens (F = 1.6957 P = 0.0838). The zones of inhibition after secondary
screening varied significantly (F = 2.4473 P = 0.0089). Likewise, there was a
significant difference (F = 6.6046 P = 0.001338) in the zones of inhibition after
exposing the pathogens to ethyl extracts of the selected antagonistic Actinomycetes.
There is need to purify and characterize the antimicrobials obtained from their study.
Monisha Khanna Kapur et al. (2018) discussed pathogenic microbes are

becoming resistant to antibiotics used for their treatment. There is therefore an
urgency to discover and develop new antimicrobial compounds with altered modes
of action. Natural products from microorganisms are good source of new drugs.
Among microbes, actinomycete bacteria are major producers of existing antibiotics.
In their study, Actinomycetes were isolated from diverse ecological habitats and
subjected to antimicrobial analyses. During primary screening, it was found that
maximum number of isolates showed activity against Bacillus cereus (17.28 %)
followed by Fusarium oxysporum (14.11 %), Candida albicans (11.26 %), Staphylococcus
aureus (2.65 %) and Escherichia coli (2.24 %). Isolates 196, 51 and 173 were selected

for further analyses on the basis of their maximum and broad-spectrum activity
during primary screening. Activities of these cultures were quantified during
secondary screening. It was revealed by bioautography that single bioactive fractions
were present in ethyl acetate extracts of isolates 196 (Rf: 0.48) and 173 (Rf: 0.90)
whereas the ethyl acetate extract of isolate 51 (Rf: 0.90, 0.51) had two bioactive
fractions. Bioactive fractions present in ethyl acetate extracts of isolates 196 and 173
showed activity against Bacillus cereus and Fusarium oxysporumrespectively, whereas
methanolic extract of isolate 51 showed broad spectrum activity.
Md. Ajijur Rahman et al. (2011) studied actinomycete colonies having

antibacterial activity from soil samples collected from differentplaces around
Rajshahi, Bangladesh. Thirty Actinomycete colonies were isolated in pure culture
from five soil samples using Starch Casein – Nitrate Agar Medium. The isolates were
grouped in five colour series based on their aerialmycelia colour and screened for
their antibacterial activity against a range of test bacteria. Sixteen isolates (53.3%)
were found to have moderate to high activity against four gram-positive and four
gram-negative bacteria. Since many isolates showed inhibitory activity against
indicator bacteria, it wassuggested that Bangladeshi soil could be an interesting
source to explore for antibacterial secondary metabolites.
2.1.2 ANTIFUNGAL ACTIVITIES OF Actinomycetes

2.1.2.1 ISOLATED FROM MARINE SOIL
Subramani Ramesh and Narayanasamy Mathivanan (2009) screened a total
of 288 marine samples from different locations of the Bay of Bengal starting from
Pulicat lake to Kanyakumari, and 208 isolates of marine Actinomycetes were isolated
using starch casein agar medium. The growth pattern, mycelial coloration,
production of exopolysaccharides and diffusible pigment and abundance of
Streptomyces spp. were documented. Among marine Actinomycetes, Streptomyces spp.
were present in large proportion (88 %). Among 208 marine Actinomycetes,111
isolates exhibited antimicrobial activity against humanpathogens, and 151 showed
antifungal activity against two plant pathogens. Among 208 isolates, 183, 157, 116,
72 and 68 isolates produced lipase, caseinase, gelatinase, cellulase and amylase,
respectively. The results of diversity, antimicrobial activity and enzymes production
have increased the scope of finding industrially important marine Actinomycetes from
the Bay of Bengal and these organisms could be vital sources for the discovery of
industrially useful molecules/enzymes.

Lalitha (2015) investigated that infection caused by Dermatophytic fungi

in man and animals is common throughout the world. Dermatophytoses poses a
serious concern to the economically poor population of India. Fungi cause both
superficial and internal mycoses. The mycoses, though normally not lethal, is
unpleasant and difficult to cure and cause considerable economic loss. Majority
of superficial infections are caused by a closely related group of keratinophilic
fungi called dermatophytes, which cause ringworm infection or tinea infection.
Their aim was to develop an antibiotic (Streptomyces species) against
dermatophytic fungi. A total of 25 soil samples were used for the isolation of
soil microorganisms. They were subjected to primary screening by Cross Streak
Plate Assay Method against Dermatophytic fungi. Then they were subjected to
secondary screening by Cross Streak Plate Assay Method to further test the
capabilities of primarily screened organisms. Finally, 5 isolates were selected for
further study on the basis of (a) Broad spectrum activity and (b) Larger zone of
inhibition in comparison to others. The antimicrobial substances were extracted
with ethyl acetate from isolate- inoculated Kuster’s broth fermented for 7 days at
28ºC by solvent extraction method. Minimum Inhibitory Concentration (MIC)
was determined.
2.1.2.2 ISOLATED FROM MNGROVE SOIL
Janaki et al. (2016) suggested thattotally 25 Actinomycetes were isolated by dry

heat (70 ℃) pre-treatment method on Starch casein agarmedia, from the soil sample
nearer to the root region of the mangrove Avicennia marina from the back-water area,
Ariyankuppam, Puducherry (UT). All the 25 Actinomycetes were subjected for
primary screening against the test fungi by agar plug method. Broad spectrum
antifungal activity was confirmed by cross streak method for selected antagonistic
Actinomycetes. Only 2 Actinomycetes were selected from A. marina and that were
subjected for secondary screening. Finally, very active isolate M-20 was selected for
further study.
Mary SulakshanaPallaet al. (2018) isolated and characterized the Actinomycetes

from Koringa mangrove soilsamples near Kakinada, Andhra Pradesh, India. Isolated
Actinomycetes were tested for antagonistic activity against various bacteria and fungi.
The potent strain KMFA-1 having activity against dermatophytes Candida albicans
and Pectinotrichum llanense was characterized. Based on physiological, morphological
characteristics and 16s rRNA gene sequencing the isolated strain was identified as
Streptomyces hydrogenans. Extraction was done with different solvents and the
activity was evaluated for each solvent extract. The antibiotic that is responsible for

activity is found to be highly water soluble. The bioactive metabolite present in crude
supernatant was found to be stable at 80 °C for 1 hrs, 90 % activity was retained after
boiling at 100 °C for 1 hrs, but 53.4 % activity was reduced after autoclaving (at 121
°C for 15 min). The activity was not lost when the crude supernatant was subjected
to wide range of pH (2–10). Furthermore, analytical techniques must be employed to
isolate themolecule of interest. The result of their study revealed that the
Streptomyces hydrogenansproduced an interesting antifungal metabolite.
2.1.2.3 ISOLATED FROM SOIL
Akansha Saxena et al. (2013) isolated from eight samples of two different
habitats, i.e., garden soil and cultivated field soil. Isolationof Actinomycetes was
carried out on soil extract agar medium using serial dilution method and forty-seven
isolates were obtained. All these Actinomycetes were then assessed for their antifungal
activity against two fungal pathogens i.e., Candida albicans and Aspergillus nigerby
cross streak method. Eleven Actinomycetes were found to inhibit A. niger. Out of 11
Actinomycetes, only 6 showed antifungal activity against both test organisms. On the
basis of screening, one potential actinomycete GS 22 was selected for further studies.
The antibitiotic was extracted with methanol from cellular extract of actinomycete
GS 22. The extract of GS 22 culture filtrate produced a clear zone of 27 mm against
C. albicans by agar well diffusion method. Actinomycete strain GS 22 was further
characterized on the basis of morphological, biochemical and physiological
characteristics and identified using probabilistic identification of bacteria (PIBWin)
software as Streptomyces phaeochromogenes.
Alpana Bharti et al. (2010) identified total of 316 Actinomycetes from 69 soil
samples from different localities of Garhwal region, Uttarakhand, India. The growth
pattern, mycelial colouration were documented. Among 316 isolates, 98 (31.01%)
isolates exhibited antifungal activity against one or more pathogens. Out of 98 active
isolates, 19, 67, 42, 37, 18 and 25 isolates showed activity against C. albicans, T.
rubrum, M. canis, M. gyseum, A. flavus, A. fumigatus respectively, while 7 isolates
showed activity against all the fungal pathogens. The antagonistic potential of
Streptomyces was found dominant as compare to other genera. These results have
increased the scope of finding industrially important antifungal antibiotics from
Actinomycetes isolated from unexplored Garhwal region.

2.2 ANTIFUNGAL ACTIVITY OF Actinomycetes AGAINST

PHYTOPAHOGENS
Soybeans are a particularly important legume crop because of their high

protein contents. Soybean has many benefits for human and animal nutrition.
Fungal phytopathogens cause serious problem worldwide in agriculture. The
objective of the present study was to isolate antifungal metabolites from Actinomycetes
isolated from different regions of Madhya Pradesh. Total 80 strains of Actinomycetes
isolated from the soils of different habitats of Chambal region, Madhya Pradesh,
were evaluated for their ability to inhibit plant pathogens Macrophomina phaseolina,
Fusarium oxysporum, Collectotrichum truncatum, Rhizoctonia solani in vitro. Entire isolates
were screened for their antifungal activity by agar overlay method against
phytopathogenic fungi. After screening, out of these only one actinomycetae
ACITM-1 showed antifunagal activity against Macrophomina phaseolina and
Collectotrichum truncatum by Charu Singh et al. (2016).
A study was done by Lanying Wang et al. (2015) on Banana Fusarium wilt
caused by Fusarium oxysporum f. sp. cubenserace 4 (FOC4) is destroying numerous
banana plantations in southern China. In order to select an effective biocontrol agent
for this devastating disease, eighty-nine actinomycete isolates were collected from
soil samples in the Botanical Garden of Chinese Academy of Tropical Agricultural
Sciences in the tropical Hainan Province, China. These isolates were evaluated for
their antagonistic activity against FOC4. Our results showed that eight isolates
exhibited strong anti-FOC4 activity. One of the isolates, HN6, resulted in an
inhibition zone of 35 mm in diameter in the antagonistic test. The mycelia of HN6
were extracted with methanol, and the extract was tested against eight indicator
pathogens by the myceliumgrowth rate method. The HN6 extract demonstrated
broad-spectrum antifungal activity, with an EC50 less than 0.08 mg/ml. Based on
the morphological, biochemical, physiological and cultural characteristics and the
16S rRNA gene sequence, the HN6 isolate was identified as Streptomyces
aureoverticillatus. HN6 isolate can be potentially developed into a biocontrol agent for
banana Fusarium wilt and other plant diseases.
In Thailand, Eucalyptus plantations rapidly expand across the country. Leaf

and shoot blight caused by Cryptosporiopsis eucalypti, Cylindrocladium sp.
andTeratosphaeria destructans is a serious disease in Euca-lyptus plantations. A total of
477 actinomycete strains were successfully isolated by Winanda Himaman et al.
(2016) from roots and rhizosphere soil of Eucalyptus. Four hundred and thirty-nine
isolates were classified as Streptomycetes and 38 isolates were Non-Streptomycetes.
Among these isolates, 272 (57.0 %), 118 (24.7 %) and 241 (50.5 %) isolates were

antagonistic to Cryptosporiopsis eucalypti, Cylindrocladium sp. and Teratosphaeria

destructans, respectively. All isolates were tested for their abilities to produce
Siderophores, Indole acetic acid (IAA) and solubilize phosphate. Most isolates (464,
97.3 %) produced Siderophores. The majority of isolates (345, 72.3 %) solubilized
phosphate. In addition, almost half of these isolates (237, 49.7 %) produced indole
acetic acid. Strain EUSKR2S82 which showed the strongest inhibitory effect against
all tested fungi with plant growth promoting ability was selected to test with
Eucalyptus. Thisstrain could colonize plant roots and increase Eucalyptus roots
length. In a detached leaves bioassay, the disease severity of EUSKR2S82-inoculated
Eucalyptus leaves was only 30 % compared to 95 % in the controltreatment. The 16S
rRNA gene sequence analysis revealed that the strain EUSKR2S82 was related to
Strep-tomycesramulosus NRRL-B 2714T (99.44 % similarity). Identification of non-
streptomycete isolates using16S rRNA gene sequences classified them into 9 genera:
Actinoallomurus, Actinomadura, Amycolatopsis, Cryptosporangium, Microbispora,
Micromonospora, Nocardia, Nonomuraea and Pseudonocardia. It is evidentthat
Eucalyptus tree harbored several genera of Actinomycetes. The selected isolate,
EUSKR2S82 showedpotential as a candidate for biocontrol agent of leaf and shoot
blight of Eucalyptus and to promote growth.
2.3 BIOACTIVE COMPOUNDS PRODUCED BY Actinomycetes
Antibiotic producing Actinomycetes can be identified from soil from different

locations including various geographical locations. But antibiotic producing
Actinomycetes are yet to be identified from hospital wasteland soil or drainage soil.
ShouvikSahaet al (2012) identified 11 Actinomycetesisolate from five different hospital
wasteland and drainage soil of West Bengal, India. Only 1 isolate out of 11 isolates is
found to be antibiotic producer and this antibiotic is showing high level of
antibacterial activity against both grams positive and gram-negative micro-
organisms. The crude extract of the antibiotic is found to be more active against
gram positive microorganisms whereas ethyl acetate fractionated antibiotics is
showing almost two-fold more activity than its crude counterpart against gram
negative microorganisms. Ethyl acetate fractionated antibiotic is demonstrating more
actives than standard antibiotic, chloramphenicol against all the test microorganisms.
MIC of the antibiotic is estimated to be 18μg against Bacillus cereus. Ethyl acetate
fractionated antibiotic shows a single band on TLC plate.
A study was done to isolate some antibiotic producing Actinomycetes strains

from soil by Stephen Kugbere Agadagba (2014) The soil sample used was dark
brown and sandy, with no vegetation covering (bare). Isolation of soil Actinomycetes
was done by culture-dependent methods and isolates were tested for antibiotic

production on selected indicator bacteria plates. The results indicated that a total of
3.56 × 105 Actinomycetes colonies were isolated per gram of dry soil. Furthermore,
microscopic examination of the isolates indicated 6 major colony types (CTs) in the
soil. Only 3 CTs were found to be active against one or more indicator bacteria, with
inhibition zones that ranged from 7 mm to 12.5 mm in diameter. From the results, it
was suggested that the low yield of antibiotic producing Actinomycetes isolates
obtained in their study, could be improved by employing a combination of several
molecular analysis methods.
Actinomycetes have provided many industrially important bioactive

compounds having great economic importance and always being a curious organism
for secondary metabolite production. Actinomycetes have long been recognized as
prolific producers of enzymes, antibiotics, anti -cancerous agents and play important
role in recycling of organic matter. Numerous methods have been advocated for
isolation of Actinomycetes to facilitate the discovery of natural compounds specially
antibiotics. Isolation of Actinomycetes can fulfil all the novel essentialities associated
with Actinomycetes. Actinomycetes are widely distributed in the natural habitats, hence
various methods like pre-treatments, enrichment, combinations of antibiotics,
specific isolation media and some novel methods has been adapted for isolation.
Marine Actinomycetes were also isolated by various methods and found to be
important source for novel secondary metabolites. Rare Actinomycetes isolated by
providing combinations of methods or high throughput screening methods.
Endophytic Actinomycetes can be isolated by mostly chemical treatment for surface
sterilization followed by serial dilution. Strategy for a range of isolation methods
have been mentioned in this review for discovery of various genera of Actinomycetes
(Ravi Ranjan Kumar and Vasantba Jadeja., 2016).
Manikandan Madheslu et al. (2019) revealed that the Streptomyces species are
predominant in marine sediments and gain more attention for their antagonistic
potential. Therefore, assessment and evaluation of marine microbial diversity were
done, which could serve as a potential source for novel antibiotics.
Manikkam Radhakrishnan et al. (2016) reported that bioactive potential of

actinobacteria isolated from certain under-studied regions in India. Soil and sediment
samples and mangrove leaves were collected from 16 different under-studied regions
in India. Actinobacteria was isolated by adopting selective isolation methods. Totally
158 actinobaterial cultures were selected from the collected terrestrial, marine and
plant samples. More number of colonies was isolated from magnesite area, Kolli
Hills and forest area in Himachal Pradesh. Majority of the isolates produced
powdery (40 %) and leathery (25 %) colonies with white (38 %) or Grey (37 %)

colour aerial mycelium. Bioactive compound from all the isolates were produced by
agar surface fermentation and its activity was tested by agar plug method against
Staphylococcus aureus, Escherichia coli and Candida albicans. About 64 out of 158
cultures showed antibacterial activity in which 62 cultures was active against S.
aureus whereas 26 were active against Escherichia coli. Twenty-three actinobacterial
cultures were exhibited antifungal activity. About 11 actinobacterial cultures were
active against both Staphylococcus aureus and Escherichia coli. In antifungal testing
whereas fourteen actinobacterial cultures were found to be active against
Staphylococcus aureus, Escherichia coli and Candida albicans. Maximum of 21
antibacterial cultures and antifungal cultures were obtained from Magnesite soil
followed by 12 antibacterial and 7 antifungal cultures were obtained from the soil
sample collected from Himachal Pradesh. This evidenced that the under-studied
ecosystems in India are the promising source for bioactive actinobacteria with broad
spectrum antibacterial and antifungal activity. Further studies on the potential
actinobacterial strains result in the isolation of broad-spectrum antimicrobial
metabolites.

3
OBJECTIVES
• Collection of sediment sample from Puducherry Rock Beach.
• Isolation of pure culture of Actinomycetes from marine soil sample.
• Phenotypic characterization of cultures sample of Actinomycetes.
• Antifungal testing of all strains of Actinomycetes.

4
MATERIALS AND METHODS
4.1. SAMPLING
A study was done by the collection of two soil samples from two different sitesof
Rock Beach of Puducherry.
4.2. LOCATION OF SAMPLE COLLECTION
Latitude: 11.9313̊ N
Longitude: 79.8358̊ E

Table – 1: Geographical area of Sample collection
District Site Ecological Geographica Area

importance l Location
Puducherry Rock Estuaries, brackish 11.9313̊ N 1.2 Km stretch in
Beach water lagoons 79.8358̊ E Puducherry
4.3. COLLECTION OF SAMPLES:
The samples were collected on 6th February 2019 between 4:05 to 04:11 PM
from top 4 cm soil profile, where most of the microbial activity takes place, and thus
where most of the bacterial population is concentrated. The sediments and water
samples were collected by using clean, sterile zip polythene bags (approx. 500 g) and
screw cap bottles along with sterile spatula, marking pen, and other accessories. The
site selection was done by taking care of the point where widely varying
characteristics as possible with regard to, moisture content, and particle size and
colour of soil and to avoid contamination as far as possible. Samples were stored in
iceboxes and transported to the laboratory where they were kept in refrigerator at 4
℃ until analysis.
4.4. SAMPLE PROCESSING
Non-actinomycetes bacteria prevent the growth of Actinomycetes as a pure

culture and hence, selective isolation of Actinomycetes was developed using six
approaches:
a) Nutritional selection, where media are formulated with nutritional
components, which are preferentially utilized by Actinomycetes.
b) Pretreatment of sample, in which soil, marine sample or plant parts were
treated with physical or chemical method in order to decrease the number of
non-Actinomycetes bacteria or fungi.
c) Enrichment method, in which nutrient media can be enriched with certain
additional supplements, which favors the growth of Actinomycetes or inhibit
the growth of other microbes.
d) Integrated method, in which any combination of different approaches can be
applied.
Soil samples were air-dried under room temperature for about 15 days before
isolation and then kept in the hot air oven under 55 °C for 30 min to eliminate other
vegetative bacterial flora. Pre-treatment of soil can stimulate the isolation of
Actinomycetes by either promoting growth of Actinomycetes or eliminating most

unwanted gram-negative bacteria (Matsukawa et al., 2007 and Hong et al., 2009).
Various pretreatment techniques have been developed for different genera of
actinomycetes.
4.5. ISOLATION OF Actinomycetes FROM MARINE SOIL SAMPLE
One gram of dried marine sediment sample was serially diluted up to 10 4 in 50

% marine water before plating on media. The starch casein agar (Hi Media) and
Actinomycete isolation agar (Hi Media) were prepared in filtered marine water and
autoclaved at 121°C in 15 lbs. for 15 min. The 0.1ml of each dilution were inoculated
on the media by spread plate technique and incubated all the inoculated plates along
with control plate in room temperature for 7 to 14 days. At the end of the incubation
period, all plates were observed for the complete growth of actinobacterial colonies
and were selected for further studies.
4.6. PHENOTYPIC CHARACTERIZATION
The appearance and growth of marine Actinomycetes were observed every day
on SCA plates and the colonies were recognized by their characteristic chalky to
leathery appearance. Further, they were observed using a light microscope for their
filamentous nature, spores, width of hyphae and spiral sporophores. Individual
colonies were picked up, and sub cultured on SCA and International Streptomyces
Project medium 2 (ISP2) to ascertain their purity.
4.7. GRAM STAINING
The culture was smeared in a microscopic slide and then subjected to primary
staining which involves staining with crystal violet for 1 min, followed by washing
with tap water. Then gram’s iodine mordant was added on the slide and left for 30
seconds, which is followed by addition of 95% ethanol or acetone. The safranin was
added on the slide and kept for 1 min. The slide was washed with tap water and the
slide was blotted on tissue paper in order to make it dry. The slide was observed
under microscope.
Table – 2: Media composition of Starch Casein Agar
Ingredients Grams/Litre
Casein powder 1.00
Starch 10.00
Sea Water 37.00
Agar 15.00
pH (at 25 ℃) – 7.2±0.2

Table – 3: Composition of ISP2 Medium
Ingedients Grams/Litre
Yeast Extract 4.00
Malt Extract 10.00
Dextrose 4.00
Agar 20.00
pH – 7.3
4.8. ISOLATION OF Aspergillusspp and Fusariumspp:

Inoculated of spoiled fruit samples and marine soil on two different Potato
dextrose agar medium plates. Incubated at 25 ℃ for 4 days and identified by
Lactophenol cotton blue staining method.
4.9. SCREENING OF Actinomycetes

The screening method consists of two steps; primary screening and
secondaryscreening.
4.9.1 PRIMARY SCREENING

In primary screening the antimycotic activity of pure isolates were determined
by cross streak method on Potato dextrose agar medium. The test organism used
were, Aspergillusspecie and Fusarium species. All the marine actinomycete isolates
were screened for antifungal activity by cross streak. In cross streak method, the
marine isolates were streaked on the side of Starch casein agar of the Petri plates and
the test organism were kept in the middle of the plates or vice versa. Incubated at
room temperature for 2 to 7 days.
4.9.2 SECONDARY SCREENING

The secondary screening of the selected marine isolates was done by agar well
diffusion or by Disc diffusion method.
4.9.2.1 AGAR WELL DIFFUSION METHOD:

The selected marine isolates were grown in ISP2 broth medium. After 14 days
of fermentation the cultures were used for assay. The fungal cultures were seeded on
Potato dextrose agar media. The wells were prepared by sterile cork borer and
different concentration (75 µl, 100 µl and 150 µl) of culture were taken from the
fermented ISP2 broth. The plates were incubated at 28 ℃ for 24 to 48 hours.

4.9.2.2 DISC DIFFUSION METHOD:

The fermented culture was filtered by Whatman No.1 filter paper. The filtrate
was used for assay. The equal volume of filtrate and ethyl acetate was kept for
overnight. Then the solution was condensed by evaporation.
The crude extracts of potential were screened for antifungal assay by disc
diffusion method. All the crude extracts were impregnated with sterile filter paper
disc and tested against fungal strains (Aspergillus species, Fusarium species). At the
end of the incubation period, the antifungal activities were evaluated by measuring
zone of inhibition.

5
RESULTS
5.1 PRE-TREATMENT OF SOIL SAMPLES
Soil samples were air-dried under room temperature for about 15 days before
isolation and then kept in the Hot air oven under 55 °C for 30 mins.pre-treatment
method was removed the unwanted microbes.
5.2 ISOLATION AND INOCULATION OF SOIL SAMPLE

Sediment sample was inoculated by serial dilution and 6 pure strains were
isolated by streak plate method. Out of 6 Actinomycetes the dominant one was
analysed for their antifungal activities. The pure cultures were maintained on the
same medium that was used for isolation and preserved at 4 ºC.
5.3 PHENOTYPIC CHARACTERIZATION

The light microscopical studies and the growth on starch casein medium
revealed that all the isolates possess characters similar to the Actinomycetes. Six
Actinomycetes were identified by morphological characteristics and it shows gram
positive rod, filamentous shaped under microscope.
TABLE - 4 Colony morphology of Actinomycetes
Colony Colour Appearance
A1 White Chalky Powder
A4 Yellow Mucoid
A5 Grey Leather

5.4. ISOLATION OF Aspergillus spp. and Fusarium spp.

Aspergillus species colonies appeared dark brown colour on PDA medium
plate. After LPCB staining method, when viewed under the microscope, Aspergillus
species appeared as filamentous and colourless conidiophores and spores. On PDA,
Fusarium species appeared white cottony mycelium and a dark purple colour. Septate
hyphae, conidiophores, macroconidia and microconidia are observed
microscopically.
5.5. PRIMARY SCREENING

This study was done by the Cross-streak method against two fungus strains
(Aspergillus species and Fusarium species). Only one strain inhibits these two fungi
moderately
5.6. SECONDARY SCREENING BY WELL DIFFUSION:

In the present study, well-diffusion method was used for the detection for
antifungal activity. In this method, the Actinomycetes grown in ISP2 broth showed
maximum antifungal activity as compared to other media. The zone measurements
against Aspergillus species are in Table - 5.
TABLE – 5: Agar Well Diffusion Test against Aspergillus species
Pathogens Zone of Inhibition (mm in dm)
75 µl 100 µl 150 µl
Aspergillus species 9±0.5 10±0.7 12±0.4

±SD
5.7. DISC DIFFUSION METHOD

The potential Actinomycetes was selected based on the result in primary screening
of Actinomycetes for antifungal activity. In that only one strain which inhibit the fungal
pathogens selected for the fermentation process.
The crude extract prepared from culture filtrate by the solvent ethyl acetate were
analysed by disc diffusion method. The result clearly indicated that the antifungal activity
of potent strains is due to the production of extracellular bioactive compounds.

Table – 6: Disc Diffusion Test against Aspergillus species
Pathogens Zone of inhibition (mm in dm)
30µl
Aspergillus species 12±0.7
Fig. 3 Figure - 1: Isolation of Actinomycetes on Starch Casein Agar
Figure - 2: Grams staining of Actinomycetes

Figure - 3: Microscopic view of Substrate Mycelium
Figure - 4: Primary screening against Aspergillus species

Figure - 5: Primary screening against Aspergillus and Fusarium species
Figure - 6: Fermented filtrate broth with Ethyl acetate (1:1)

Figure - 7: Secondary screening by Well diffusion method

6
DISCUSSION
In the present study, sediment samples were collected from the Puducherry,
Rock Beach. By serial dilution technique six Actinomycetes colonies were isolated on
the starch casein agar medium. A total of 288 marine samples were collected from
different locations of the Bay of Bengal starting from Pulicat lake to Kanyakumari,
and 208 isolates of marine Actinomycetes were isolated using starch casein agar
medium by Subramani Ramesh and Narayanasamy Mathivanan (2009). Marine
sediment samples are good for the isolation of Actinomycetes; Goodfellow and Hynes
(1984) reviewed the literature on isolation of Actinomycetes from marine sediments
and suggested that the marine sediment may be valuable for the isolation of novel
Actinomycetes.
In the present study, the aerial mass colour of almost all strains were White
and grey colour as well as chalky powder appearance colonies and microscopically
gram-positive rod and filamentous shape. Saraswathi (2015) have also reported that
all the plates were observed with dried powdery shape colonies after an incubation of
five days. The microscopic image of the strain revealed that the isolate was
filamentous in nature with spiral shaped spores. Valli et al. (2012) identified the
Actinomycetes by the presence of powdered colonies on the surface of agar plate. All
the isolated colonies were characterized and identified by microscopical and
macroscopical observations. Identification of the isolates revealed that all isolates
belong to the genus Streptomyces by Gunasekaran Mohanraj and Thangavel Sekar
(2013).
In present study, one colony, out of six colonies was screened for antifungal
activity against Aspergillus species and Fusarium species by cross streak method on
media. Palla et al. (2018) revealed that the preliminary screening by cross streak and
agar overlay method showed that the selected isolate KMFA-1 was a good antifungal
producer against Candida albicans and P. llanense. Pathalam Ganesan et al. (2017)
found that 106 Actinomycetes strains were isolated from five different soil samples.
These strains were cross streaked against microbial pathogens. The preliminary and
secondary screening results clearly showed that FMS-20 alone possessed very high
antimicrobial activity against all the tested human pathogens.

In the present study, after the fermentation process of Actinomycetes in ISP2

broth, the broth is subjected to well diffusion method and showed antifungal activity
against the Aspergillus species. Silambarasan et al. (2012) have also reported thatin
well diffusion method, the crude culture filtrate of actinobacterial strain (RA5)
showed good activity against all the tested bacterial strains and fungal strains.
The crude extracts prepared from culture filtrates were analysed by

Silambarasan et al. (2012) for their antimicrobial activity by Disc diffusion method
and reported that the chloroform extract showed good activity against all the test
pathogens. But in present study, the Disc diffusion method by using ethyl acetate
extract with filtrate showed good activity against Aspergillus species. Lalitha (2015)
has also stated that the antimycotic compound from PR01, PR05, KA07, SU09, and
AN10 were extracted from the supernatant using ethyl acetate solvent. Most of the
antifungal antibiotics are extracted using ethyl acetate. Ganesan et al. (2017) found
thatextra cellular ethyl acetate crude extract of active strain FMS- 20 showed good
activity against all the tested human pathogenic bacteria and candida. They did not
show any activity against fungal pathogens. Intracellular methanol extract of FMS-
20 also showed good activity against human fungal pathogens and it did not show
any activity against bacterial and candidal pathogens.

7
CONCLUSION
In conclusion, the extra cellular ethyl acetate crude extract of
Actinomycetes showed good activity against Aspergillus species. The present study
suggests that the isolated Actinomycetes could be used as antifungal agent and
also a biocontrol agent against the tested fungal pathogens. This also provided a
new insight towards the development of good candidates for pharmaceuticals
and bioactive natural products.

8
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ANTIFUNGAL POTENTIAL OF MARINE SOIL ACTINOBACTERIA

Book · May 2021

Lipun Kumar Pradhan Subhashree Jena

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LIPUN KUMAR PRADHAN

Ms. Subhashree Jena

JPS Scientific Publications

JPS Scientific Publications, Tamil Nadu, India.

International Standard Book Number (ISBN): 978-81-940316-5-9

No part of this Monograph book (E – book) may be reproduced in any form, by

Chapter Chapter Title Page

4 Materials and Methods 17

Marine microorganisms are increasingly becoming an important source in the

©JPS Scientific Publications, India

complex biopolymers of organic matter, such as chitin and lignocellulose in eco

a) Absence of cell nucleus, thus resembling prokaryotes.

For discovering novel bioactive compounds of biotechnological interest is

The important genera of Actinomycetes are Streptomyces, Nocardia,

Around 23 000 bioactive secondary metabolites produced by microorganisms

©JPS Scientific Publications, India

Presently, they represent a major area of concern in the medical field;

©JPS Scientific Publications, India

Numerous antibiotics have been isolated from a variety of microorganisms;

©JPS Scientific Publications, India

2.1 ANTIMICROBIAL EFFECTS OF Actinomycetes

Valli et al. (2012) screened twenty-one strains of Actinomycetes were isolated

©JPS Scientific Publications, India

Muharram et al. (2013) screened thirty-three soil-isolates of Actinomycetes for

©JPS Scientific Publications, India

pneumoniae and Salmonella entericatyphimurium were 15 μg/ml and 12 μg/ml

Saraswathi et al. (2015) isolated a total of sixteen isolates of Actinomycetes from

©JPS Scientific Publications, India

Paul NjengaWaithaka et al. (2017) isolated and screened Actinomycetes for

Monisha Khanna Kapur et al. (2018) discussed pathogenic microbes are

©JPS Scientific Publications, India

Md. Ajijur Rahman et al. (2011) studied actinomycete colonies having

2.1.2 ANTIFUNGAL ACTIVITIES OF Actinomycetes

©JPS Scientific Publications, India

Lalitha (2015) investigated that infection caused by Dermatophytic fungi

2.1.2.2 ISOLATED FROM MNGROVE SOIL

Janaki et al. (2016) suggested thattotally 25 Actinomycetes were isolated by dry

Mary SulakshanaPallaet al. (2018) isolated and characterized the Actinomycetes

©JPS Scientific Publications, India

2.1.2.3 ISOLATED FROM SOIL

©JPS Scientific Publications, India

2.2 ANTIFUNGAL ACTIVITY OF Actinomycetes AGAINST

Soybeans are a particularly important legume crop because of their high

In Thailand, Eucalyptus plantations rapidly expand across the country. Leaf

©JPS Scientific Publications, India

antagonistic to Cryptosporiopsis eucalypti, Cylindrocladium sp. and Teratosphaeria

2.3 BIOACTIVE COMPOUNDS PRODUCED BY Actinomycetes

Antibiotic producing Actinomycetes can be identified from soil from different

A study was done to isolate some antibiotic producing Actinomycetes strains

©JPS Scientific Publications, India

Actinomycetes have provided many industrially important bioactive

Manikkam Radhakrishnan et al. (2016) reported that bioactive potential of

©JPS Scientific Publications, India

©JPS Scientific Publications, India

©JPS Scientific Publications, India

4.2. LOCATION OF SAMPLE COLLECTION

©JPS Scientific Publications, India

Table – 1: Geographical area of Sample collection