Preface

Preface
Ancient plant species Ficus gomelleira Kunth & C.D. Bouché (Moraceae) inside the endangered
Tropical Rain Forest fragment at Parque Estadual do Rio Doce (PERD), Minas Gerais state, Brazil
(Photo credits: L.H. Rosa)
v
vi Preface
The Neotropical biogeographic region (Fig. 1), also known as Neotropics, includes

Central America, the Caribbean, and South America and represents one of the eight
biogeographic realms of the Earth’s land surface. The major Neotropical regions
include Amazonia, Caribbean, Central America, Central Andes, Eastern South
America, Northern Andes, Orinoco, and Southern South America. Neotropics is
recognized to shelter a rich biological diversity of animal, plants, and microorgan-
isms. However, these knowledge is insufficient and many species still hidden and
unknown by the science. In addition, the Neotropical biodiversity represents an
important genetic heritage for humanity able to origin different bioproducts for
industrial and agricultural sectors.
Neotropics is vast and comprises several distinct biomes and habitats such as
seasonally dry forests, arid zones, high-elevation grasslands, young and old moun-
tain systems, and the Atlantic Forests and Amazonia rainforests (Hoorn et al. 2010).
In all these biomes shelter rich and diverse biological communities, many of them
endemics and endangered.
Fig. 1 Perspective view of the Neotropical realm ranging from Southern Mexico to the Southern
portion of South America. (Source: Google Maps)
Preface vii
Kingdom Fungi includes eukaryotic organisms that are present in nearly all envi-
ronments on Earth. However, different fungal groups occur mainly in the tropical
regions of the World. However, recent polyphasic taxonomic studies proposed new
phyla. Tedersoo et al. (2018) proposed the following phyla for Fungi:
Aphelidiomycota, Basidiobolomycota, Calcarisporiellomycota, Glomeromycota,
Entomophthoromycota, Entorrhizomycota, Kickxellomycota, Monoblepharomycota,
Mortierellomycota, and Olpidiomycota. According to Hawksworth (1993), the
complexity of tropical forests of the World associated with the high diverse habitats,
available organic matter, and warm climate make it a region with large numbers, if
not the majority, of undescribed fungal species.
Among the different fungal groups, those characterized as endophytes have been
call attention in the last decades. Fungal endophytes virtually can be found all
plants, including trees, grasses, and herbaceous species. Mostly of the endophytic
fungi belong to the Ascomycota and their anamorphic stages, which live asymptom-
atically in plant tissues (Petrini et at. 1991). Fungal endophytes are recognised as a
repository of different bioactive compounds, including novel metabolites of phar-
maceutical and agricultural importance. The broad diversity and taxonomic spec-
trum exhibited by these fungal group make them especially interesting for ecological,
evolutionary, and bioprospecting studies programs.
Despite the ecological and biotechnological importance of the Neotropical
realm, few mycological studies have been performed to date, in special those with
focus on the endophytes, perhaps due its extensive size and the diverse biomes. The
aim of this book is to present the vanguard aspects of diversity, ecology, and bio-
technology of Neotropical fungi. Experts in tropical mycology and chemistry of
natural products from several countries (i.e. Argentine, Brazil, Chile, Colombia,
Costa Rica, Ecuador, Spain, and United States) describe and discuss fungal occur-
rence in the different biomes of Neotropics.
We are grateful to Joao Pildervasser (Publishing Editor) and Arun Siva
Shanmugam and G. Chitra (Production Editor) for their support and patience during
the development of this book. We acknowledge the financial support from Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Fundação de Amparo à
Pesquisa do Estado de Minas Gerais (FAPEMIG); Ibero-American Programme on
Science and Technology for Development (Cyted); Bio-organizaciones Research
Group (University Simón Bolívar, Barranquilla-Colombia); Diseño Biosintético de
Fungicidas Research Group (University of Cádiz, Spain); the Center for Marine and
Limnological Research of the Caribbean CICMAR (Barranquilla, Colombia); Dr.
Giorgio Anfuso (University of Cádiz, Spain) for the design of the map; Luis Diego
Vargas, Luis Guillermo Acosta, Jorge Blanco, Catalina Murillo, Myriam Hernandez,
Silvia Soto, Loengrin Umaña, and Nefertiti Campos, Allan Jimenez and Ana Lorena
Guevara. Grant NIH U01 TW007404 supported the data generated at the former
National Institute of Biodiversity; authorities from Absolute Natural Reserve Cabo
Blanco; Carara, Braulio Carrillo, Guanacaste, Santa Rosa, Palo Verde, Rincón de la
Vieja, and Tenorio National Parks, and to CONAGEBIO for granting the sample
collecting permits (307-2003-OFAU, R-012-2005-OT-CONAGEBIO, R-CM-
viii Preface
INBio-03-2006-OT, R-CM-INBio-06-2006-OT, R-CM-INBio-30-2007-OT, R-CM-

INBio-059-2008-OT); ANPCyT of Argentina for the financial support to our
projects (PICT-2018-01593); Mariana Valente for her expert assistance in making
the figures; the National Fund for Scientific and Technological Development,
FONDECYT Grant Nº 11190754 (Iniciación); Project High Altitude Laboratory
ATA #1799 Ministry of Education of Chile; FONDECYT Postdoctoral Grant Nº
3140279 and the INACH G22-11 Grant of Chilean Antarctic Institute (INACH); the
Ministry of Environment of Ecuador; Dr Paul Brickle (director), staff and scientists
of the South Atlantic Environmental Research Institute (SAERI) and the Falkland
Islands Government.
Departamento de Microbiologia Luiz Henrique Rosa
Instituto de Ciências Biológicas,
Universidade Federal de Minas Gerais
Belo Horizonte, Minas Gerais, Brazil

References
Hawksworth DL (1993) The tropical fungal biota: census, pertinence, prophylaxis, and progno-
sis. In: Isaac S, Frankland JC, Watling R, Whalley AJS (Eds) Aspects of Tropical Mycology.
Cambridge University Press, UK, pp.265-293
Hoorn C, Wesselingh FP, ter Steege H et al. (2010) Amazonia through time: Andean uplift, climate
change, landscape evolution, and biodiversity. Science 330:927-931
Kirk PM, Cannon PF, Minter DW, Stalpers JA (2008) Dictionary of the Fungi, 10th ed. CAB
International: Wallingford, UK.
Petrini O (1991) Fungal endophyte of tree leaves. In: Andrews J, Hirano SS (Eds) Microbial
Ecology of Leaves. Springer, New York, pp.179–197
Tedersoo L, Sánchez-Ramírez S, Köljalg U, Bahram M, Döring M, Schigel D, May T, Ryberg M,
Abarenkov K (2018) High-level classification of the Fungi and a tool for evolutionary ecologi-
cal analyses. Fungal Div 90:135-159
Contents
1 Ecology of Neotropical Endophytic Fungi �� 1

Camila Rodrigues de Carvalho, Mariana Costa Ferreira,
and Luiz Henrique Rosa
2 Diversity, Ecology, and Applications of Epichloë
Fungal Endophytes of Grasses in South America�� 11
Leopoldo J. Iannone, M. Victoria Novas, Patricia D. Mc Cargo,
Andrea C. Ueno, and Pedro E. Gundel
3 Trypanocidal and Herbicidal Activities of Endophytic
Fungi Associated with Medicinal Plant Lafoensia pacari
Living in Neotropical Wetland Pantanal of Brazil�� 37
Soraya Sander Amorim, Camila Rodrigues de Carvalho,
Jéssica Catarine Silva de Assis, Carlos Leomar Zani,
Tânia Maria de Almeida Alves, Policarpo Ademar Sales Junior,
Marcos Antônio Soares, and Luiz Henrique Rosa
4 Advances in Research on Biodiversity and Bioprospecting
of Endophytic Fungi in Chile�� 53
Rómulo Oses-Pedraza, Víctor Hernández, Leonardo Campos,
José Becerra, Dánae Irribarren-Riquelme, Paris Lavín,
and Jaime Rodríguez
5 Endophytic Fungal Community Associated
with Colombian Plants�� 93
Hernando José Bolívar-Anillo, Ezzanad Abdellah,
Gesiane da Silva Lima, Inmaculada Izquierdo-Bueno,
Javier Moraga, and Gabriel Franco dos Santos
6 Fungal Endophytes and Their Bioactive Compounds
in Tropical Forests of Costa Rica�� 109
Keilor Rojas-Jimenez and Giselle Tamayo-Castillo
ix
x Contents
7 What Do We Know About Fungal Endophyte Diversity

in a Mega Diverse Country? An Appeal for Increased
Conservation and Research�� 131
Alexandra Narvaez-Trujillo, María R. Marchán-Rivadeneira,
Eliana Veloz-Villavicencio, and Carolina E. Portero
8 Diversity, Ecology, and Bioprospecting of Endophytic
Fungi in the Brazilian Biomes of Rupestrian Grasslands,
Caatinga, Pampa, and Pantanal �� 151
Camila Rodrigues de Carvalho, Alice Ferreira-D’Silva,
Soraya Sander Amorim, and Luiz Henrique Rosa
9 Endophytic Fungi Associated with Medicinal Plants
of Amazonian Forest�� 177
Eskálath Morganna Silva Ferreira, Tatiana Maracaípe Corrêia,
Juliana Fonseca Moreira da Silva, and Raphael Sanzio Pimenta
10 Association of Endophytic Fungi with Ancient
Neotropical Plants�� 199
Marina Bahia and Luiz Henrique Rosa
11 Bioprospecting of Neotropical Endophytic Fungi
in South America Applied to Medicine�� 213
Mariana Costa Ferreira, Denise de Oliveira Scoaris,
Soraya Sander Amorim, Betania Barros Cota,
Emerson de Castro Barbosa, Jaquelline Germano de Oliveira,
Carlos Leomar Zani, and Luiz Henrique Rosa
12 Bioactive Compounds Produced by Neotropical
Endophytic Fungi Applied to Agriculture�� 257
Débora Luiza Costa Barreto, Rafaela Nogueira de Azevedo,
Charles Lowell Cantrell, Stephen Oscar Duke,
13 Neotropical Plant-Associated Endophytic Fungi: A Source
of Promising Macromolecules for Use in Biotechnology�� 297
Mariana de Lourdes Almeida Vieira and Luiz Henrique Rosa
14 Bioprospecting of Bioactive Secondary Metabolites
of Endophytic Fungi of Carapichea ipecacuanha
(Rubiaceae), a Neotropical Medicinal Species�� 321
Rafaela Nogueira de Azevedo, Mariana Costa Ferreira,
Jéssica Catarina Silva de Assis, Policarpo Ademar Sales Junior,
Daniela Nabak Bueno Maia, Tânia Maria de Almeida Alves,
Markus Kohlhoff, Carlos Leomar Zani, and Luiz Henrique Rosa
Contents xi
15 Effectiveness of Endophytic Fungi from Baccharis dracunculifolia

Against Sucking Insect and Fungal Pathogens �� 337
Yumi Oki, Isabela M. Nascimento, Naíla B. da Costa,
Renata Aparecida Maia, Jacqueline A. Takahashi, Vany Ferraz,
Ary Corrêa Júnior, and G. Wilson Fernandes
16 Bioprospecting of Secondary Bioactive Metabolites Produced
by Endophytic Fungi of the Medicinal Piper
sp. in the Brazilian Tropical Rain Forest �� 351
Raissa Hellen da Silva Florindo, Mariana Costa Ferreira,
Carlos Leomar Zani, Tânia Maria de Almeida Alves,
Policarpo Ademar Sales Junior, Emerson de Castro Barbosa,
Jaquelline Germano de Oliveira, Fernanda Ruth França Cavalcanti,
Antoniana Ursine Krettli, Isabela Penna Ceravolo,
17 Diversity of Endophytic Fungi of Empetrum rubrum
Vahl ex Willd (Ericaceae): A Medicinal Plant
from Austral South America �� 375
Mariana Costa Ferreira, Pedro Henrique Rodrigues Loureiro,
Jéssica Catarina Silva de Assis, Micheline Carvalho-Silva,
Paulo Eduardo Aguiar Saraiva Câmara, Diego Knop Henriques,
Index�� 387
Contributors
Ezzanad Abdellah Organic Chemistry Department, Faculty of Science,University

of Cádiz, Puerto Real, Cádiz, Spain
Soraya Sander Amorim Departamento de Microbiologia, Instituto de Ciências
Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
Marina Bahia Departamento de Microbiologia, Instituto de Ciências Biológicas,
Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
Débora Luiza Costa Barreto Departamento de Microbiologia, Instituto de
Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas
Gerais, Brazil
José Becerra Laboratory of Natural Products Chemistry, Department of Botany,
Faculty of Natural and Oceanographic Sciences, Universidad de Concepcion,
Concepcion, Chile
Hernando José Bolívar-Anillo Microbiology Department, Faculty of Basic and
Biomedical Sciences, Universidad Simón Bolívar, Barranquilla, Colombia
Paulo Eduardo Aguiar Saraiva Câmara Departamento de Botânica, Universidade
de Brasília, Distrito Federal, Brasília, Brazil
Leonardo Campos Environmental Microbiology and Biotechnology Unit –
Zenobia Group SpA, Chillan, Chile
Charles Lowell Cantrell Natural Products Utilization Research Unit, Agricultural
Research Service, United States Department of Agriculture, Oxford, MS, USA
Micheline Carvalho-Silva Departamento de Botânica, Universidade de Brasília,
Distrito Federal, Brasília, Brazil
Fernanda Ruth França Cavalcanti Instituto René Rachou, FIOCRUZ-MG, Belo
Horizonte, Minas Gerais, Brazil
Isabela Penna Ceravolo Instituto de Pesquisa René Rachou, FIOCRUZ-MG,
Belo Horizonte, MG, Brazil
xiii
xiv Contributors
Tatiana Maracaípe Corrêia Laboratorio de Microbiologia Geral e Aplicada,

Universidade Federal do Tocantins, Palmas, Tocantins, Brazil
Betania Barros Cota Instituto René Rachou, FIOCRUZ-MG, Belo Horizonte,
Minas Gerais, Brazil
Naíla B. da Costa Departamento de Genética Ecologia & Evolução, Instituto de
Gerais, Brazil
Raissa Hellen da Silva Florindo Departamento de Microbiologia, Universidade
Federal de Minas Gerais, Belo Horizonte, MG, Brazil
Gesiane da Silva Lima Departamento de Química, Universidade Federal de Minas
Gerais, Belo Horizonte, Minas Gerais, Brazil
Juliana Fonseca Moreira da Silva Laboratório de Microbiologia Geral e Aplicada,
Tânia Maria de Almeida Alves Instituto René Rachou, FIOCRUZ-MG, Belo
Horizonte, Brazil
Jéssica Catarine Silva de Assis Departamento de Microbiologia, Instituto de
Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
Rafaela Nogueira de Azevedo Departamento de Microbiologia, Instituto de
Gerais, Brazil
Camila Rodrigues de Carvalho Instituto René Rachou, FIOCRUZ-MG, Belo
Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade
Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
Emerson de Castro Barbosa Instituto René Rachou, FIOCRUZ-MG, Belo
Mariana de Lourdes Almeida Vieira Departamento de Química, Centro Federal
de Educação Tecnológica de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
Denise de Oliveira Scoaris Fundação Ezequiel Dias, Belo Horizonte, Brazil
Jaquelline Germano de Oliveira Instituto René Rachou, FIOCRUZ-MG, Belo
Gabriel Franco dos Santos Departamento de Química, Universidade Federal de
Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
Stephen Oscar Duke National Center for Natural Products Research, School of
Pharmacy, University of Mississippi, Oxford, MS, USA
Contributors xv
G. Wilson Fernandes Departamento de Genética Ecologia & Evolução, Instituto

de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte,
Vany Ferraz Departamento de Química, Instituto de Ciências Exatas, Universidade
Alice Ferreira-D’Silva Unidade Superior de Ensino de Feira de Santana, Feira de
Santana, Bahia, Brazil
Eskálath Morganna Silva Ferreira Laboratório de Microbiologia Geral e
Aplicada, Universidade Federal do Tocantins, Palmas, Tocantins, Brazil
Mariana Costa Ferreira Instituto René Rachou, FIOCRUZ-MG, Belo Horizonte,
Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade
Pedro E. Gundel IFEVA-Facultad de Agronomía (UBA)/CONICET, Cátedra de
Ecología, Buenos Aires, Argentina
Diego Knop Henriques Departamento de Botânica, Universidade de Brasília,
Distrito Federal, Brasília, Brazil
Víctor Hernández Laboratory of Natural Products Chemistry, Department of
Botany, Faculty of Natural and Oceanographic Sciences, Universidad de Concepcion,
Concepcion, Chile
Leopoldo J. Iannone DBBE-FCEN-UBA & INMIBO-CONICET, Buenos Aires,
Argentina
Dánae Irribarren-Riquelme Environmental Microbiology and Biotechnology
Unit – Zenobia Group SpA, Chillan, Chile
Inmaculada Izquierdo-Bueno Department of Biomedicine, Biotechnology and
Public Health, Faculty of Marine and Environmental Sciences, University of Cadiz,
Puerto Real, Cádiz, Spain
Ary Corrêa Júnior Departamento de Microbiologia, Instituto de Ciências
Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais,
Brazil
Policarpo Ademar Sales Junior Instituto René Rachou, FIOCRUZ-MG, Belo
Horizonte, Brazil
Markus Kohlhoff Instituto René Rachou, FIOCRUZ-MG, Belo Horizonte, Minas
Gearis, Brazil
Antoniana Ursine Krettli Instituto de Pesquisa René Rachou, FIOCRUZ-MG,
Belo Horizonte, MG, Brazil
xvi Contributors
Paris Lavín Environmental Microbiology and Biotechnology Unit – Zenobia

Group SpA, Chillan, Chile
Pedro Henrique Rodrigues Loureiro Departamento de Microbiologia, Instituto
MG, Brazil
Daniela Nabak Bueno Maia Instituto René Rachou, FIOCRUZ-MG, Belo
Horizonte, Minas Gearis, Brazil
Renata Aparecida Maia Departamento de Genética Ecologia & Evolução,
Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo
María R. Marchán-Rivadeneira Biotechnology Institute, Ohio University,
Athens, OH, USA
Patricia D. Mc Cargo DBBE-FCEN-UBA & INMIBO-CONICET, Buenos Aires,
Argentina
Javier Moraga Department of Biomedicine, Biotechnology and Public Health,
Faculty of Marine and Environmental Sciences, University of Cadiz, Puerto Real,
Cádiz, Spain
Alexandra Narvaez-Trujillo Center for Research on Health in Latinamerica
(CISeAL) – Plant Biotechnology Research Group, Pontificia Universidad Catolica
del Ecuador (PUCE), Quito, Ecuador
Isabela M. Nascimento Departamento de Genética Ecologia & Evolução, Instituto
M. Victoria Novas DBBE-FCEN-UBA & INMIBO-CONICET, Buenos Aires,
Argentina
Yumi Oki Departamento de Genética Ecologia & Evolução, Instituto de Ciências
Brazil
Rómulo Oses-Pedraza Vicerrectoria de Investigacion y Postgrado (VRIP), Centro
Regional de Investigacion y Desarrollo Sustentable de Atacama (CRIDESAT) –
Universidad de Atacama, Copiapo, Chile
Environmental Microbiology and Biotechnology Unit – Zenobia Group SpA,
Chillan, Chile
Raphael Sanzio Pimenta Laboratório de Microbiologia Geral e Aplicada,
Carolina E. Portero Center for Research on Health in Latinamerica (CISeAL) –
Plant Biotechnology Research Group, Pontificia Universidad Catolica del Ecuador
(PUCE), Quito, Ecuador
Contributors xvii
Jaime Rodríguez Center of Biotechnology, Universidad de Concepcion,

Concepcion, Chile
Keilor Rojas-Jimenez Escuela de Biologia, Universidad de Costa Rica, San Jose,
Costa Rica
Luiz Henrique Rosa Departamento de Microbiologia, Instituto de Ciências
Brazil
Marcos Antônio Soares Departamento de Botânica e Ecologia, Universidade
Federal do Mato Grosso, Cuiabá, Brazil
Jacqueline A. Takahashi Departamento de Química, Instituto de Ciências Exatas,
Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
Giselle Tamayo-Castillo Escuela de Química, Universidad de Costa Rica, San
José, Costa Rica
Centro de Investigaciones en Productos Naturales (CIPRONA), Universidad de
Costa Rica, San José, Costa Rica
Andrea C. Ueno IFEVA-Facultad de Agronomía (UBA)/CONICET, Cátedra de
Ecología, Buenos Aires, Argentina
Eliana Veloz-Villavicencio Center for Research on Health in Latinamerica
(CISeAL) – Plant Biotechnology Research Group, Pontificia Universidad Catolica
del Ecuador (PUCE), Quito, Ecuador
Carlos Leomar Zani Instituto René Rachou, FIOCRUZ-MG, Belo Horizonte,
Minas Gearis, Brazil
Chapter 1
Ecology of Neotropical Endophytic Fungi

Abstract Endophytic fungi are a diverse group of microorganisms that have

different effects on the ecology of the host plant in its healthy state and on its evolu-
tion. Different hypotheses have been proposed to explain how endophytic fungi
manage to infect and often even grow within their hosts without causing visible
disease symptoms. The initial hypothesis was that the asymptomatic colonization is
based on a balanced antagonism between fungal virulence factors and host defense
responses. The more recent proposal is that fungal endophytes, besides maintaining
a balanced antagonism with the plant, also maintain a balance with bacterial and
other fungal inhabitants of the host. In this chapter, the authors discuss the current
knowledge on ecological interaction and signal transduction between Neotropical
plants and their endophytic fungal, in addition to some examples of benefits that
these fungi can offer to their host plants.
Keywords Fungi · Host plants · Balanced antagonism · Tropical
1.1 Introduction
The community of microorganisms associated with a diversity of unique

environments can be defined as “microbiome” (Gilbert et al. 2014). Identifying and
understanding plant microbiomes may contribute to improvement of agricultural
practices and development of novel biotechnological approaches for diagnostic and
therapeutic purposes (Rosier et al. 2016). According to Yan et al. (2019), plants
Camila Rodrigues de Carvalho and Mariana Costa Ferreira contributed equally with all other
contributors.
C. R. de Carvalho (*) · M. C. Ferreira

Instituto René Rachou, FIOCRUZ-MG, Belo Horizonte, Minas Gerais, Brazil
Departamento de Microbiologia, Instituto de Ciências Biológicas,
L. H. Rosa
Departamento de Microbiologia, Instituto de Ciências Biológicas,
© Springer Nature Switzerland AG 2021

L. H. Rosa (ed.), Neotropical Endophytic Fungi, 1
https://doi.org/10.1007/978-3-030-53506-3_1
2 C. R. de Carvalho et al.
h arbor a wide diversity of microorganisms such as fungi, bacteria, algae, archaea,

and protists, both within and outside their tissues. Endophytic fungi are among those
microorganisms that inhabit plant organs that, at some time in their life, can colonize
internal plant tissues without causing apparent harm to the host (Petrini 1991). These
fungi show a complex relationship with their hosts, which are widely studied in
some instances and could be beneficial for the plant. These benefits have been widely
studied and could be used in agriculture, medicine, and other industries.
1.2 Endophytic Fungi: Definition
Over the years, several definitions of endophytes have been proposed (endo = within;
phyte = plant). Originally, the term was introduced by de Bary (1866), who defined
endophyte as any organism that can colonize internal plant tissues. However, the
most commonly used definition over the years is that proposed by Petrini (1991) as
all the organisms inhabiting plant organs that at some time in their life, can colo-
nize internal plant tissues without causing apparent harm to the host. Petrini’s defi-
nition includes those endophytic organisms that have lengthy epiphytic phases as
well as latent pathogens that may live symptomless in their hosts during a certain
time in their life. The term endophyte may include protists (Peters 1991), bacteria
(Kobayashi and Palumbo 2000), and fungi (Stone et al. 2000; Carvalho et al. 2012;
Ferreira et al. 2017, 2020); most often, it refers to fungi that are isolated most fre-
quently (Strobel and Daisy 2003; Strobel et al. 2004; Tan and Zou 2001). All plants
in a natural ecosystem seem to have some interactions with endophytic or some
other associated fungi (Rodriguez et al. 2008).
1.3 Balanced Antagonism Hypothesis
Compant et al. (2016) reported that despite extensive studies on endophytic com-
munities, the interactions established among the endophytes and their host plants
are still poorly understood. Over the last decades, several studies have been con-
ducted with the purpose of drawing hypotheses about the establishment of this inter-
action. They aimed to understand how fungal endophytes manage to infect and
often even grow within their hosts without causing visible disease symptoms
(Schulz and Boyle 2005; Kusari et al. 2012; Schulz et al. 2015). The underlying
mechanisms of these interactions, such as the influence of environmental, genetic,
and phenological factors that may apparently determine the establishment of spe-
cific associations of microbial communities found in different plant tissues, are
some interesting points that are currently under study (Compant et al. 2016).
Figure 1.1 shows the history of our understanding of the mechanisms of endophytic
fungi-host plant interaction proposed by different authors in the last decades.
1 Ecology of Neotropical Endophytic Fungi 3
Fig. 1.1 Representative diagrams of evolution of endophytic-host plant interactions. (a)

Hypothesis of balanced antagonism proposed by Schulz and Boyle (2005); (b) hypothesis of bal-
anced antagonism proposed by Kusari et al. (2012). a. Balanced antagonism hypothesis is shown.
b. Plant disease caused by pathogenic fungi is presented. c. Endophyte-pathogen reciprocity is
demonstrated. The question mark (?) indicates that this phenomenon might not be universal, and
further research is necessary for verification. d. Endophyte survival strategy is illustrated. e.
Balanced synergism is shown. (c) Hypothesis of balanced antagonism proposed by Schulz et al.
(2015)
The relationship between endophytic fungi and their host plants is quite com-
plex; it involves interactions with other endophytic species and the influence of
biotic and abiotic factors. Although the asymptomatic nature of colonization by an
endophyte may lead to its classification as a mutualistic relationship, it is still con-
sidered that while these microorganisms are enormously diverse, some of them may
be saprobes or opportunistic pathogens (Strobel and Daisy 2003). This hypothesis
is reinforced by the fact that some phytopathogens have an endophytic origin, as
well as that some of such microorganisms can cause symptomatic infections in an
old plant or under stress conditions (Tan and Zou 2001; Firáková et al. 2007).
According to Schulz and Boyle (2005), fungi that are accidental opportunists can
usually be found on diverse other substrates and are not specifically adapted to their
hosts. For example, some coprophilous species could be detected in plant tissues.
According to Kogel et al. (2006), mutualistic interaction does not mean the
absence of plant defense. These relationships require a sophisticated balance
between plant defense responses and endophytic nutrient demand. Schulz and Boyle
(2005) reported that asymptomatic colonization of plant tissue or organ by an endo-
phyte would result from a balanced antagonistic relationship, where an equilibrium
is reached between the microorganism virulence and host defense responses. The
hypothesis of the balanced antagonism was initially proposed to demonstrate how
an endophyte could prevent the activation of host defense mechanisms, thus
p rotecting itself before being incapacitated by the host’s toxic metabolites, and
keeps growing in plant tissues without causing visible manifestations of infection or
disease (Fig. 1.1a) (Schulz and Boyle 2005).
Host specificity requires a close adaptation between the host plant and the endo-
phyte, suggesting that a mutual influence develops during cohabitation and coevolu-
tion over thousands of years (Kusari et al. 2012). Over time, this association
becomes permanently engraved in the genetic material of both organisms, which
therefore develop complementary genetic systems. This emphasizes that the rela-
tionship between endophytic fungi and their hosts is much more complex than ini-
tially assumed (Moricca and Ragazzi 2008). For example, several metabolites
produced by endophytic fungi are also found in their host plants, suggesting that an
event of horizontal transfer of the genes that regulate synthesis of these compounds
might occur from the host plants to the fungi (Venugopalan and Srivastava 2015).
The hypothesis established by Kusari et al. (2012) proposes that asymptomatic col-
onization is an equilibrium of antagonisms between the endophyte and the host. The
interaction could occur by the following scenarios: (i) if fungus virulence and plant
defense strategies are balanced, the association remains apparently asymptomatic,
and this phase is only a transitional period where environmental factors play an
important role in destabilizing the established delicate equilibrium; (ii) if the plant’s
defense mechanisms completely neutralize the fungal virulence factors, it may die,
and in contrast, if the plant succumbs to the fungus virulence, the fungal invasion
can lead to a disease. The variability in this interaction would depend not only on
the adaptation of the endophytic microorganism to a host tissue or organ but also on
such factors as innate endophyte virulence, plant defense responses, and environ-
mental conditions (Fig. 1.1b). Further, plant-endophyte interaction may be not just
a balance between fungal virulence and host defense, but a much more complex and
precisely controlled interaction.
According to Zipfel and Oldroyd (2017), plants are able to perceive the signals
emitted by microbes and respond appropriately by activating the plant immune sys-
tem. Plants rely on the innate immunity to recognize microbial signal molecules,
which results in two different defense systems: one is based on the detection of
microbe-associated molecular patterns (MAMPs) via cell surface-localized pattern
recognition receptors and leads to the MAMP-triggered immunity. The other is
mediated by recognition of the molecules produced by microorganisms (termed
effectors) via intracellular receptors; it activates effector-triggered immune response
(Mendoza-Mendoza et al. 2018; Yan et al. 2019). Kusari et al. (2012) also suggest
that the plant is colonized by high diversity of endophyte microorganisms that inter-
act directly or indirectly (fungus/fungus, fungus/bacteria, bacteria/bacteria) and
such microbial interactions play an important role in the production of secondary
metabolites by endophytes. Schulz et al. (2015) showed that for endophytic fungi to
be able to grow and survive, they must maintain a balanced antagonism not only
with the host plant but also with the competing microbiota, whether endophytic or
pathogenic. These authors suggest that secondary metabolites play an important
role in maintaining this balance. The plethora of antibacterial and antifungal
etabolites that endophytic fungi produce is crucial for equilibrating the antago-
m
nisms with microbial competitors, resulting in a compatible multipartite symbiosis
(Fig. 1.1c).
Thus, the balanced antagonism hypothesis postulates that a balance exists
between plant defensive responses into endophytes and the toxic effect of endo-
phytes on their host plants. Endophytes resist the host defense mechanisms, avoid
being incapacitated by the toxic metabolites, and survive within their hosts without
causing visible infection or disease symptoms. In this way, plant-endophyte rela-
tionships are asymptomatic as long as there is a balanced antagonism between the
host defense and fungal virulence (Yan et al. 2019). However, further studies are
still needed to better understand the metabolic pathways and signaling mechanisms
involved in these interactions.
1.4 E
ndophytic Fungi: Classification, Benefits,
and Interaction
Rodriguez et al. (2009) published an important study where they classified endo-
phytic fungi in two major groups according to differences in their evolutionary ori-
gins, plant hosts, taxonomy, and ecological functions, as follows: C-endophytes or
clavicipitaceous endophytes, which infect some grasses, and NC-endophytes or
nonclavicipitaceous endophytes, which can be isolated from asymptomatic tissues
of nonvascular plants, ferns and allies, conifers, and angiosperms.
C-endophytes (endophytes of grasses) may constitute a monophyletic clade with
the fungal family Clavicipitaceae (Ascomycota) (Clay and Schardl 2002) that are
fastidious in culture and are limited to some cool- and warm-season grasses
(Bischoff and White 2005). Traditionally, this group of endophytes could be found
within plant shoots, where they form systemic intercellular infections, and may be
transmitted vertically through seeds and horizontally (Rodriguez et al. 2009).
According to Clay and Schardl (2002), the grass/endophyte associations are based
primarily on the protection of the host from biotic and abiotic stresses. Furthermore,
the endophyte infection can provide some benefits, such as enhanced drought toler-
ance, photosynthetic rate, and growth, as well as defense of the host plant against
pests, herbivores, and plant pathogens.
NC-endophytes are highly diverse fungi that predominantly belong to the
Ascomycota phylum, while some minor species belong to Basidiomycota. They
may include at least three distinct functional classes based on the host colonization
patterns, mechanism of transmission between host generations, in planta biodiver-
sity levels, and ecological role (Rodriguez et al. 2009). Rodriguez et al. (2009)
describe the host range and tissue colonized by different classes. They show that
Classes 2, 3, and 4 have broad host ranges; however, Class 2 endophytes may grow
in above- and underground tissues (roots, stems, and leaves), Class 3 endophytes are
restricted to aboveground tissues, and Class 4 is restricted to roots. Colonization of
host tissues (in planta colonization) also differs: Class 3 endophytes form highly
localized infections, while Class 2 and 4 endophytes are capable of extensive tissue
colonization. When the in planta diversity is analyzed for each class of endophytes
within a host plant’s organs or tissues, the diversity of Class 2 endophytes is usually
low; the one of Class 3 endophytes is extremely high; and in contrast, the diversity
of Class 4 endophytes is unknown (Rodriguez et al. 2009). The authors still report
that differences in in planta biodiversity of Class 2 and 3 endophytes may reflect the
variability in transmission patterns: although both classes are transmitted horizon-
tally, Class 2 endophytes are also transmitted vertically. Transmission of Class 4
endophytes is only horizontal.
An important trait of the Class 2 endophytes is related to the fitness benefits.
Only the Class 2 endophytes have shown the ability to confer habitat-specific stress
tolerance to the host plants (Rodriguez et al. 2008, 2009). According to Rodriguez
et al. (2009), endophyte-conferred fitness benefits are defined as habitat-adapted if
the benefits are the result of habitat-specific selective pressures such as tempera-
ture, pH, and salinity or as nonhabitat-adapted if the benefits are common among
endophytes regardless of habitat. As reported by Busby et al. (2015), the coloniza-
tion of plants by fungal endophytes has beneficial effects, which are mainly repre-
sented by the increase of the plant resistance to biotic and abiotic stresses. Few
studies reported the beneficial relationship between the Neotropical plants and
their endophytic fungi host. Some examples reported in the literature are
discussed below.
According to Pietro-Souza et al. (2020), endophytic microorganisms are instru-
mental for metal remediation, or they could assist their hosts in phytoremediation
processes. Root endophytic fungal assemblages of Polygonum acuminatum Kunth.
and Aeschynomene fluminensis Vell., collected in Poconé, Mato Grosso, Brazil, in
areas characterized as wetland were studied by Pietro-Souza et al. (2017). These
plants were collected in places with and without mercury (Hg+2). The authors
observed higher endophytic fungi in hosts in soil contaminated with mercury.
Besides, the frequency of colonization, the abundant distribution of taxa of endo-
phytic fungi, and the structure and community function were influenced by mercury
contamination. When the strains were identified, the authors observed a predomi-
nance of Sordariomycetes for hosts collected in contaminated areas and an abun-
dance of Dothideomycetes in uncontaminated areas, and the order of Pleosporales
was proportional and larger in all analyzed communities. The selected strains of
endophytic fungi were evaluated in host growth promotion in the presence of mer-
cury. In short, the seeds of A. fluminensis were germinated in vases, the seedlings
were posteriorly transferred to new vases, and then, the selected strains were inocu-
lated. After a period of cultivation in the absence of mercury, doses of Hg+2 were
applied at the final concentration of 120 mg kg−1 of Hg+2. The authors suggested that
the use of mercury-tolerant fungal strains positively influenced the growth of A. flu-
minensis in mercury contamination conditions since nine strains promoted the
growth of the host with growth promotion efficiency (GPE) (dry biomass and
length) greater than that obtained for uninoculated plants and those without addition
of mercury to the substrate. The authors concluded that the inoculation of
A. fluminensis with certain strains of stress-tolerant endophytic fungi contributes to

colonization and establishment of the host and may be promising microorganisms
in bioremediation programs.
Pietro-Souza et al. (2020) examined the capacity to promote mercury bioreme-
diation of 30 mercury-resistant endophytic fungi isolated from the A. fluminensis
and P. acuminatum root systems in their previous study (Pietro-Souza et al. 2017).
Among the tested strains, Aspergillus sp. A31, Curvularia geniculata P1,
Lindgomycetaceae P87, and Westerdykella sp. P71 remediated more than 97% of
Hg+2 added to culture media and were selected to evaluate for mercury bioremedia-
tion and bioaccumulation in vitro, as well as for growth promotion of A. fluminensis
and Zea mays in the presence or absence of the metal. Those endophytic fungi
strains removed up to 100% of mercury from the culture medium in a species-
dependent manner, and they promoted A. fluminensis and Z. mays growth in sub-
strates with or without mercury. According to the authors, the increase in the host
biomass correlated with the reduction in soil mercury concentration due to the metal
bioaccumulation in host tissues and its possible volatilization. The soil mercury
concentration was decreased by 7.69% in A. fluminensis plants inoculated with
Lindgomycetaceae P87 + Aspergillus sp. A31 and by 57.14% in plants inoculated
with Lindgomycetaceae P87. The authors report that the resistance mechanisms of
mercury volatilization and bioaccumulation in plant tissues mediated by these endo-
phytic fungi can contribute to bioremediation programs.
According to Vergara et al. (2017, 2018), the use of dark septate endophytic
(DSE) fungi to promote plant growth can be beneficial to agriculture, and these
organisms are important allies in the search for sustainable agriculture practices.
Vergara et al. (2017) investigated the effects of inoculation with the DSE fungi iso-
lates A101, A104, and A105 on nutrient recovery efficiency, nutrient accumulation,
and growth of tomato plants (cv. Santa Clara I-5300) fertilized with organic and
inorganic N sources, Canavalia ensiformis (L.), and ammonium sulfate, respec-
tively. The effects of inoculation were evaluated under greenhouse conditions where
all the three isolates colonized the root tissue of tomato plants and promoted tomato
growth without causing symptoms of apparent disease, using finely ground
C. ensiformis-15N as the organic N source. Tomato plants exhibited significant
increases in aboveground dry biomass, plant height, and leaf number, relative to the
uninoculated treatment. Besides, the 15N, N, P, K, Ca, Mg, Fe, Mn, and Zn contents
increased. In contrast, the only positive effects observed in the presence of an inor-
ganic N source were fertilizer-K recovery efficiency, content of K, and leaf area
when inoculated with the fungus A104. The authors concluded that, especially when
an organic N source is used, tomato plants inoculated with DSE fungi acquired
macro- and micronutrients more efficiently, resulting in increased plant growth.
Vergara et al. (2018) studied the contribution of four dark septate fungi (A101,
A103, A104, and A105) to the absorption of nutrients by rice plants and their ensu-
ing growth. The four isolates were inoculated to the rice plants. The colonization
was significant by all of the fungi, mainly the isolate A103 (Pleosporales) that
increased the fresh and dry biomass of the shoots and the number of tillers per plant,
amino-N, and soluble sugars as well as the N, P, K, Mg, and S contents in c omparison
with the control treatment.
1.5 Conclusion
In Neotropical biomes, endophytic fungi can have different effects on the ecology
of their host plant in its healthy state and during its evolution. They help the host
plant to adapt to the environment and to establish tolerance to biotic and abiotic
stresses. Such microorganisms should be widely explored for better characteriza-
tion of their species diversity, interactions with host plants, and biotechnological
applications, among others. However, further ecological studies have to be con-
ducted to understand the impact of endophytic fungal assemblages associated with
rich plant communities inhabiting the Neotropical biomes.
References
Bischoff JF, White JF (2005) Evolutionary development of the Clavicipitaceae. In: Dighton J,
White JF, Oudemans P (eds) The fungal community: its organization and role in the ecosystem.
Taylor & Francis, Boca Raton, pp 505–518
Busby PE, Ridout M, Newcombe G (2015) Fungal endophytes: modifiers of plant disease. Plant
Mol Biol 90:645–655
Carvalho CR, Gonçalves VN, Pereira CB, Johann S, Galliza IV, Alves TMA, Rabello A, Sobral
MEG, Zani CL, Rosa CA, Rosa LH (2012) The diversity, antimicrobial and anticancer activity
of endophytic fungi associated with the medicinal plant Stryphnodendron adstringens (Mart.)
Coville (Fabaceae) from the Brazilian savannah. Symbiosis 57:95–107
Clay K, Schardl C (2002) Evolutionary origins and ecological consequences of endophyte symbio-
sis with grasses. Am Nat 160:99–127
Compant S, Saikkonen K, Mitter B, Campisano A, Mercado-Blanco J (2016) Editorial special
issue: soil, plants and endophytes. Plant Soil 405:1–11
De Bary A (1866) Morphologie und Physiologie der Pilze, Flechten und Myxomyceten.
Hofmeister’s handbook of physiological botany, vol II. Engelmann, Leipzig
Ferreira MC, Cantrell CL, Wedge DE, Gonçalves VN, Jacob MR, Khan S, Rosa CA, Rosa LH
(2017) Diversity of the endophytic fungi associated with the ancient and narrowly endemic
neotropical plant Vellozia gigantea from the endangered Brazilian rupestrian grasslands.
Biochem Syst Ecol 71:163–169
Ferreira MC, de Assis JCS, Rosa LH (2020) Diversity of endophytic fungi associated with
Carapichea ipecacuanha from a native fragment of the Atlantic Rain Forest. S Afr J Bot 57:1–5
Firáková S, Sturdíková M, Múcková M (2007) Bioactive secondary metabolites produced by
microorganisms associated with plants. Biologia 62:251–257
Gilbert JA, Jansson JK, Knight R (2014) The earth microbiome project: successes and aspirations.
BMC Biol 12:69
Kobayashi DY, Palumbo JD (2000) Bacterial endophytes and their effects on plants and uses in
agriculture. In: Bacon CW, White JF (eds) Microbial endophytes. Marcel Dekker, New York,
pp 199–236
Kogel K-H, Franjken P, Huckelhoven R (2006) Endophyte or parasite what decides. Curr Opin
Plant Biol 9:358–363
Kusari S, Hertweck C, Spiteller M (2012) Chemical ecology of endophytic fungi: origins of sec-
ondary metabolites. Chem Biol 19:792–798
Mendoza-Mendoza A, Zaid R, Lawry R, Hermosa R, Monte E, Horwitz BA, Mukherjee PK (2018)
Molecular dialogues between Trichoderma and roots: role of the fungal secretome. Fungal Biol
Rev 32:62–85
Moricca S, Ragazzi A (2008) Fungal endophytes in Mediterranean oak forests: a lesson from
Discula quercina. Phytopathology 98:380–386
Peters AF (1991) Field and culture studies of Streblonema macrocystis new species (Ectocarpales,
Phaeophyceae) from Chile, a sexual endophyte of giant kelp. Phycologia 30:365–377
Petrini O (1991) Fungal endophytes of tree leaves. In: Andrews JH, Hirano SS (eds) Microbial
ecology of leaves. Springer, New York, pp 179–197
Pietro-Souza W, Mello IS, Vendruscullo SJ, Silva GF, Cunha CN, White JF, Soares MA (2017)
Endophytic fungal communities of Polygonum acuminatum and Aeschynomene fluminensis are
influenced by soil mercury contamination. PLoS One 12:1–24
Pietro-Souza W, Pereira FC, Mello IS, Stachack FFF, Terezo AJ, Cunha CN, White JF, Li H,
Soares MA (2020) Mercury resistance and bioremediation mediated by endophytic fungi.
Chemosphere 240:1–12
Rodriguez RJ, Henson J, Volkenburgh EV, Hoy M, Wright L, Beckwith F, Kim Y-O, Redman RS
(2008) Stress tolerance in plants via habitat-adapted symbiosis. ISME J 2:404–416
Rodriguez RJ, White Jr JF, Arnold AE, Redman RS (2009) Fungal endophytes: diversity and func-
tional roles. New Phytol 182:314–330
Rosier A, Bishnoi U, Lakshmanan V, Sherrier DJ, Bais HP (2016) A perspective on inter-kingdom
signaling in plant–beneficial microbe interactions. Plant Mol Biol 90:537–548
Schulz B, Boyle C (2005) The endophytic continuum. Mycol Res 109:661–686
Schulz B, Haas S, Junker C, Andrée N, Schobert M (2015) Fungal endophytes are involved in
multiple balanced antagonisms. Curr Sci 109:39–45
Stone JK, Bacon CW, White JF (2000) An overview of endophytic microbes: endophytism defined.
In: Bacon CW, White JF (eds) Microbial endophytes, vol 30. Marcel Dekker, New York,
pp 3–30
Strobel G, Daisy B (2003) Bioprospecting for microbial endophytes and their natural products.
Microbiol Mol Biol Rev 67:491–502
Strobel G, Daisy B, Castillo U, Harper J (2004) Natural products from endophytic microorgan-
isms. J Nat Prod 67:257–268
Tan RX, Zou WX (2001) Endophytes: a rich source of functional metabolites. Nat Prod Rep
8:448–459
Venugopalan A, Srivastava S (2015) Endophytes as in vitro production platforms of high value
plant secondary metabolites. Biotechnol Adv 33:873–887
Vergara C, Araujo KEC, Urquiaga S, Schultz N, Balieiro FC, Medeiros OS, Santos LA, Xavier
GR, Zilli JE (2017) Dark septate endophytic fungi help tomato to acquire nutrients from ground
plant material. Front Microbiol 8:1–12
Vergara C, Araujo KEC, Alves LS, Souza SR, Santos LA, Santa-Catarina C, Silva K, Pereira
GMD, Xavier GR, Zilli JE (2018) Contribution of dark septate fungi to the nutrient uptake and
growth of rice plants. Braz J Microbiol 49:67–78
Yan L, Zhu J, Zhao X, Shi J, Jiang C, Shao D (2019) Beneficial effects of endophytic fungi colo-
nization on plants. Appl Microbiol Biotechnol 103:3327–3340
Zipfel C, Oldroyd GE (2017) Plant signalling in symbiosis and immunity. Nature 543:328–336
Chapter 2
Diversity, Ecology, and Applications
of Epichloë Fungal Endophytes of Grasses
in South America
Leopoldo J. Iannone , M. Victoria Novas , Patricia D. Mc Cargo ,

Andrea C. Ueno , and Pedro E. Gundel
Abstract Epichloë fungal endophytes are a conspicuous group of fungi

(Ascomycota, Hypocreales, Clavicipitaceae) that form persistent symbiosis with
certain cool-season grasses (Pooideae) worldwide. The symbiosis is not vital for the
plants, but it seems to be associated with fitness benefits, a basic condition for being
favorably selected. Epichloë endophytes infect systemically green tissues, and
while sexual stages reduce fertility of the host plant, their asexual forms persist
asymptomatically through generations by means of vertical transmission (from
plant to seeds). Host plants are endowed by a suite of fungal alkaloids that can be
toxic to livestock (such as ergot alkaloids and lolitrem B) or protect plants against
herbivorous insects (lolines and peramine). Mainly studied in the Northern
Hemisphere, where species with sexual or asexual stages are found, Epichloë in
South America appears to present its own characteristics. Only asexual vertically
transmitted Epichloë has been detected in South America from Venezuela to
Argentina. Although research in genetic biodiversity of Epichloë fungi in South
America is in the dawn and mostly restricted to Argentina, we know that most of the
endophytes from South America evolved from hybridization events among species
from the Northern Hemisphere not found in this region. Interestingly, a few strains
or fungal species are associated to more than one host grass species, which contrasts
with what is known from the Northern Hemisphere. Regional surveys of grass-
endophyte associations indicate that some environmental conditions promote the
symbiosis while others don’t (e.g., aridity). However, variation among closely
related plant species also evidences phylogenetic constrains. Fungal endophytes are
being used in breeding programs of forage crops with two main goals: (i) replace
those wild toxic endophytes and (ii) inoculate endophytes that protect host plants
against agricultural plagues (as agents of biological control). The knowledge of the
L. J. Iannone (*) · M. V. Novas · P. D. Mc Cargo

DBBE-FCEN-UBA & INMIBO-CONICET, Buenos Aires, Argentina
A. C. Ueno · P. E. Gundel
IFEVA-Facultad de Agronomía (UBA)/CONICET, Cátedra de Ecología,
Buenos Aires, Argentina
© Springer Nature Switzerland AG 2021 11

L. H. Rosa (ed.), Neotropical Endophytic Fungi,
https://doi.org/10.1007/978-3-030-53506-3_2
12 L. J. Iannone et al.
diversity of Epichloë fungi, the host grasses they infect, and the ecophysiological
impact on plant fitness opens a big potential to advance eco-friendly tools for the
development of a more sustainable agriculture.
Keywords Endophytic fungi · Distribution · Neotropical · Taxonomy
2.1 Introduction
rigin and Life History of Asexual Epichloë Fungal

2.1.1 O
Endophytes
Grasses, as most plants on earth, host microorganisms that during part or the entire
life cycle grow asymptomatically within their tissues and are commonly termed
“endophytes” (Hyde and Soytong 2008; Wilson 1995). Different fungal species of
the genera Acremonium, Alternaria, Epicoccum, Phoma, Phomopsis, and
Stemphylium (König et al. 2018; Sánchez Márquez et al. 2008, 2010; Zabalgogeazcoa
et al. 2013) have been found growing endophytically in grasses as well as in other
plant groups (D’Jonsiles et al. 2019; Suryanarayanan 2013; Vega et al. 2010). Most
of them colonize via spores and leaf tissues of seedlings and adult plants establish-
ing local or systemic infections. A particular group of fungal endophytes are those
in the genus Epichloë (Clavicipitaceae, Hypocreales, Ascomycota) which show a
close coevolutionary relationship with certain species of cool-season grasses in the
subfamily Pooideae (family: Poaceae) (Schardl 2010; Schardl et al. 2008). That is
the reason why fungi of the genus Epichloë are commonly known as “the fungal
endophytes of grasses.” Apart from being a model system to study species interac-
tions in ecology and evolution, grass endophytes are important due to their presence
in two of the most economically important forage and turf species (i.e., tall fescue
(Festuca arundinacea) and perennial ryegrass (Lolium perenne L.)) as well as in
wild grasses of grasslands around the world.
During vegetative stages of the host, Epichloë fungal endophytes grow systemi-
cally in the intercellular spaces (the apoplast) of aerial plant tissues. In the apoplast,
the fungus obtains simple sugars and amino acids for nutrition (Kuldau and Bacon
2008). The fungal hyphae colonize the apical meristems and grow firmly attached
to the cell walls of the host. The growth in the meristems provides accession to
elongation zones of new leaves and lateral buds that will originate new vegetative or
reproductive tillers. As plant cells enlarge, the hyphae are stretched out and grow
intercalary (Christensen et al. 2008). As a result, the endophyte grows coordinately
with the host without causing a defensive response of the plant. In parenchymal tis-
sues, hyphae are non-branched and parallel oriented to the longitudinal axes of host
cells. The mechanisms that regulate the hyphal growth and systemic colonization of
the plant without causing an antagonist reaction have been thoroughly studied and
include intercalary growth, balanced ROS production, and secretion of small
2 Diversity, Ecology, and Applications of Epichloë Fungal Endophytes… 13
p eptides (see, e.g., Scott et al. 2018). Even though these endophytes grow mostly
endophytically, epiphytic mycelia with potential to produce conidia have been also
detected (Craven et al. 2001; Moy et al. 2000; Tadych et al. 2014; White et al. 1996).
The mechanisms by which endophytes can grow between epidermal cells and cross
throughout the cuticle have been carefully studied by Becker et al. (2016).
The species of Epichloë differ in their life cycles and reproductive strategies,
which determine the mechanisms by which these endophytes colonize new plants.
These differences become evident when the host plants enter in the reproductive
phase. In those species that can accomplish sexual reproduction, the hyphae of the
endophyte grow epiphytically and profusely forming a stroma with conidiogenous
cells and conidia on the phylloplane of reproductive tillers (culms) of the host plant.
The development of the stroma arrests the expansion of the flag leaf preventing the
emergence of the inflorescence; thus, flowers will not be formed in this culm.
Limited to some Epichloë species, this is considered the pathogenic manifestation
of the fungus, a syndrome known as “choke disease” (Bucheli and Leuchtmann
1996; Kirby 1961). The conidia formed in the stroma may act like spermatia, and
female flies of the genus Botanophila (Anthomyiidae, Diptera) that feed, oviposit,
and defecate on the stroma transport conidia from one stroma to another. Slugs that
feed on the stroma and the ascospores are also mechanisms for transferring sperma-
tia among stromata (Alderman and Rao 2008; Hoffman and Rao 2014). When
conidia are deposited in a stroma of the opposite mating type, fertilization takes
place and perithecia with ascospores are formed. When the perithecia mature, the
stromata become bright yellow (Fig. 2.1a, b). The ascospores are forcibly discharged,
and when they reach a host plant, the spore germinates producing conidia that will
germinate and colonize a new healthy plant.
Fig. 2.1 (a, b) Picture of Dactylis glomerata grass exhibiting the stromata or choke disease caused
by Epichloë typhina. (c, d) Endophyte mycelium growing in culture media. (e, f) Detail of fungal
endophyte mycelium and conidia in culture
Some Epichloë species do not reproduce sexually, and stromata are never formed
(or if formed, perithecia are not produced). These fungal endophytes, the vertically
transmitted endophytes of grasses, present a life cycle tightly associated to that of
the host plant (Fig. 2.2). These endophytes pass from mother plant to seeds by
growing hyphae into flowers and ovaries of host plants (Philipson and Christey
1986). Once in the seed, the endophyte mycelium is found in the embryo, aleurone
layer, or coats (Liu et al. 2017; Zhang et al. 2017). After germination, endophytic
mycelium is then found in leaf tissues of young seedlings. During plant vegetative
stages, each tiller can be colonized by the endophyte. During culm elongation and
reproductive structure appearance (either spikes or panicles), the endophyte can
colonize each flower and developing ovary (Liu et al. 2017; Sugawara et al. 2004;
Fig. 2.2 Schematic drawing showing a simplified life cycle of a grass plant with the stages (seed,
seedling, vegetative and reproductive tillers) and vital rates (germination, tillering, flowering, and
fecundity). Below each plant stage, it is explained where the fungal endophyte is in the different
plant parts, and with the curly brackets, the eventual failures in the endophyte vertical transmission
are highlighted (mechanism by which an endophyte-symbiotic plant can produce nonsymbiotic
progeny)
Zhang et al. 2017). In this way, the asexual endophytes are vertically transmitted to
the next generation via the seeds of the host plant (Fig. 2.2).
Despite the high synchronization between the fungus and grass, the endophyte
can be lost at several steps in the plant growing cycle (Gundel et al. 2011). Failures
in colonization of tillers and/or flowers can lead to imperfect vertical transmission,
and thus, endophyte-symbiotic plants can produce endophyte-free seeds (Afkhami
and Rudgers 2008; Gagic et al. 2018). On the other hand, an endophyte-symbiotic
seed can give rise to an endophyte-free seedling due to the anticipated death of the
fungus (Fig. 2.2). The longevity of the endophyte in the seeds depends among other
factors on the temperature and humidity during seed dormancy or storage. High
temperatures and relative humidity significantly reduce the survival of the endo-
phyte in the seeds (Gundel et al. 2009, 2010, 2012; Hill and Roach 2009; Rolston
et al. 1986; Welty et al. 1987; Wheatley et al. 2007).
Accordingly to the life cycle pattern, (Rodriguez et al. 2009; White 1988) estab-
lished three types of associations. Type I associations are those established by some
Epichloë spp. that develop stromata in all the culms of the host plants; thus, these
plants will be unable to produce seeds, as occurs in the association between E. typh-
ina and Dactylis glomerata (Fig. 2.1a, b). In the type II associations, the stromata
are formed in some of the culms; thus, the host plant yet produces seeds. Type III
associations involve those Epichloë species that do not produce stromata on the host
plant and are vertically transmitted via seeds. Even though the ascospores are
responsible of horizontal transmission, it has been proved that conidia produced on
the phylloplane of asymptomatic plants could be transported by water drops and are
able to infect new plants.
Most of Epichloë species can grow in standard culture media as potato dextrose
agar or malt extract agar; thus, they can be isolated from superficially disinfected
tissues of the host plant. In culture, they present slow-growing colonies with whit-
ish mycelium and reverse tan (Fig. 2.1c, d). Conidiogenous cells are phialidic and
solitary and produce one to four allantoid, reniform, or uncinated conidia
(Fig. 2.1e, f).
A remarkable feature of most of the asexual Epichloë is their evolutionary
origin. First sequence analyses of nuclear genes revealed the presence of two or
three alleles in most of the studied genes (Craven et al. 2001; Gentile et al. 2005;
Moon et al. 2004; Tsai et al. 1994). The phylogeny of each allele of different
genes of an asexual species relates it with a different sexual species suggesting
that asexual species evolved from hybridization events among endophytes from
different parental species. Nowadays, genome analyses indicate that most of the
hybrids are allopolyploids since they retain most of the genome of each ancestor
(Campbell et al. 2017).
At the present time, the known Epichloë endophytes associated with native
grasses in South America do not form stromata and reproduce asexually by growing
agamic hyphae into the seeds (Iannone et al. 2012a). Because of this, we will focus
hereafter on vertically transmitted fungal endophytes of grasses.
2.1.2 Fitness Effects in an Intimate Relationship
The frequency of endophyte-symbiotic individuals greatly varies across species

populations and environment (Semmartin et al. 2015). The symbiosis persistence
relays mostly on the mutual benefits between partners and the efficiency with which
the fungus is transmitted to progeny (Gundel et al. 2008, 2011). Benefits for verti-
cally transmitted endophyte fungi are clear, as they obtain nutrition, protection, and
a way of dispersion via the host seeds. Despite that endophyte-conferred benefits on
the host fitness have been widely documented (for a review, see Clay and Schardl
2002), the effects can be variable as they are controlled by genetic and environmen-
tal factors, hence leading to the idea of the mutualism-parasitism continuum
(Rodriguez et al. 2009; Saikkonen et al. 1998).
Historically, the symbiosis between grasses and Epichloë was considered a
defensive mutualism, since the endophyte produces four main classes of alkaloids
that are deterrents or cause toxicity to herbivores (Clay 1988; Clay and Schardl
2002; Saikkonen et al. 2013). The alkaloids lolines and peramine are mainly active
against invertebrate animals. Alternatively, the indole-diterpenes (i.e., lolitrem B)
and ergot alkaloids (i.e., ergovaline) are tremorgenic or produce metabolic disorders
in vertebrate animals such as sheep, livestock, and horses (Philippe 2016; Saikkonen
et al. 2013; Schardl et al. 2013a). With exception of peramine, the genes involved in
the biosynthesis of the alkaloids are arranged in clusters. Different total or partial
deletions in these clusters are observed among Epichloë species or even among
strains of the same species (Schardl et al. 2012, 2013b; Shymanovich et al. 2014),
which determine differences in alkaloid profile and in the level of toxicity of the
plant. In many cases (most prominent in domestic agronomic grasses), the associa-
tion with Epichloë also provides the host plants with increased growth, higher seed
production, and tolerance to abiotic stress factors such as drought (Clay and Schardl
2002; Gundel et al. 2013; Iannone et al. 2012b, 2017; Iannone and Cabral 2006;
Novas et al. 2003; Vignale et al. 2013). Interestingly, it has been also well demon-
strated that under certain environmental conditions, the endophyte can become a
cost for the grass (Faeth et al. 2004; Faeth and Sullivan 2003). Therefore, the out-
come of grass-endophyte symbiosis is most likely to be context dependent.
2.1.3 S
pecificity and Maintenance of the Grass-Endophyte
Association
In general, each species of Epichloë is associated with one or a group of closely

related species of grasses. On the other hand, each host species is associated with
one or a few species of Epichloë. These patterns suggest the existence of certain
host specificity and a coevolution between the host and its endophytes. Studies on
the coevolution of plants and endophytes suggest that the symbiosis between
Epichloë and grasses arose early in the evolutionary radiation of the Pooideae
(Schardl et al. 2008). Additionally, a similar co-evolutive pattern can be appreciated

among the Epichloë that is horizontally transmitted and their host species (Schardl
et al. 1997). The same co-evolutive pattern is not easy to detect among asexual
hybrid endophytes, which are largely more diverse than those sexual or asexual
haploid Epichloë; it could be explained by events of endophyte species jumping
among host grass species (Craven 2012). Host plants are usually infected with one
strain of Epichloë, and if they are coinfected, different endophytes segregate in dif-
ferent tillers, or in the end, just one endophyte will prevail in the entire plant
(Christensen et al. 2000; Soto-Barajas et al. 2019; Wille et al. 1999). It has been
proposed that in these plants, simultaneous infection by two Epichloë fungi opens
the opportunity for new hybrid to appear by means of hyphal anastomosis. If this
hybrid outperforms the asexual haploids (Selosse and Schardl 2007) or if it is more
efficient in being transmitted through the seeds, it would be fixed and spread.
Symbiosis specificity – which seems to work at population and species level –
can be evidenced by low capability of establishing a stable interaction in terms of
endophyte persistence (which include transmission) and effects on plant fitness
(Gundel et al. 2010; Saikkonen et al. 2004). Host specificity has been mainly stud-
ied through artificial inoculations, crossing endophytes from one host into another
(either population or species) but also through controlled cross-pollination experi-
ments (Gundel et al. 2012; Leuchtmann 1992; Piano et al. 2005; Saikkonen et al.
2010). Fungal endophytes can be artificially cross inoculated by injection of myce-
lium in meristems of seedlings or callus in tissue culture (Christensen 1995; Latch
and Christensen 1985). The success and the effects of the cross inoculations on
plant fitness depend highly on the phylogenetic proximity of the plants from where
the endophyte was isolated and the plant on which the endophyte will be inoculated
(Gundel et al. 2012; Oberhofer and Leuchtmann 2014; Saikkonen et al. 2010;
Shymanovich and Faeth 2019).
2.2 D
iversity and Evolutionary Origin of Epichloë Fungal
Endophytes in South America
The discovery of endophyte-infected grasses in South America dates back to the

beginning of the twentieth century, when Rivas and Zanolli studied the “tembladera”
syndrome, an intoxication in domestic cattle grazing on the grass Festuca fiebrigii
in northern Argentina. They discovered a fungus that was growing and producing
conidia inside the plants (then, an endophyte). Although without providing a formal
description of the species, they associated the presence of this fungus with the toxic-
ity of the plant and named it Endoconidium tembladerae (Rivas and Zanolli 1908).
Unfortunately, Rivas and Zanolli’s discovery remained almost ignored until the
beginning of the nineties when Dr. D. Cabral and collaborators started a systematic
study of the fungal endophytes of grasses in Argentina.
Grasses of the subfamily Pooideae are widely spread in South America, with
more than 900 species from Venezuela to Tierra del Fuego. In tropical regions from
Venezuela to Bolivia, they are commonly found at high altitudes (over 2500m a.s.l.)
along the Cordillera de los Andes. In the Southern Cone of South America, includ-
ing “Argentina, Uruguay, Southern Brazil, Paraguay, and Chile,” Pooideae is the
dominant subfamily of the Poaceae and has been recognized as 691 species of cool-
season grasses (Biganzoli and Zuloaga 2015). In this part of the subcontinent, the
Pooideae spreads from the Atlantic to the Pacific oceans (Fig. 2.3).
The study of endophytes of grasses in South America is mostly restricted to
Argentina and Uruguay. Up to now, 45 taxa of grasses native to South America
(Fig. 2.3; Table 2.1) have been detected to be associated with Epichloë endophytes
(Bertoni et al. 1993; Iannone et al. 2012a; Lugo et al. 1998), a number that repre-
sents less than 10% of the species of Pooideae described for the region. Although
Epichloë-associated grasses are more frequently found along or close to the
Cordillera de los Andes, many host species inhabit in subtropical forests of the
Selva Paranaense, grasslands in the Pampas and Patagonia, and seashores of
Argentina and Uruguay (Fig. 2.3). Sexual stromata of Epichloë have not been
detected in South America; thus, the detection of endophytes must be achieved by
microscopic analysis of the endophyte in seeds and parenchymatic tissues of the
potential host species. This result strongly suggests that the Epichloë species associ-
ated with grasses from South America reproduce only asexually and that endophyte
fungi are transmitted from the mother plant to the seeds.
The asexual condition of the Epichloë from South America is also evidenced by
phylogenetic analyses of sequences of nuclear genes. In general, most of the isolates
bear two alleles from each gene, and each allele is phylogenetically associated with
one species that presents sexual reproduction (Fig. 2.4). These sexual species are
considered to be the evolutionary ancestors that, by means of interspecific hybrid-
ization, gave place to the endophytes from South America. Hybridization events
between E. festucae and E. typhina subsp. poae gave origin to E. tembladerae and
E. pampeana. Hybridization events between E. typhina subsp. poae and a common
ancestor of E. amarillans and E. baconii established the hybrid E. cabralii. In other
cases, only one allele associated with E. typhina subsp. poae was detected, suggest-
ing that some endophytes evolved by the loss of sexual reproduction as is the case
of E. typhina subsp. poae var. aonikenkana (Mc Cargo et al. 2014). In spite of these
clear different evolutionary histories, the different endophytes cannot be easily dif-
ferentiated by its morphological characteristics in culture. Slight differences could
be found in the growth rate and in the size of the conidia and conidiogenous cells
(Fig. 2.1e, f), but these characters are very variable, even among strains of the same
species. Thus, differences in the morphological characteristics are not consistent
enough to clearly differentiate species, and the identification of Epichloë species in
South America mostly relies on phylogenetic analyses.
As mentioned above, the association between the endophyte and the host is
highly specific, and in general, one host species is associated with one or two spe-
cies of Epichloë. However, one species of Epichloë could be associated with more
than one host species. Particularly, E. tembladerae is the most ubiquitous asexual
Fig. 2.3 Map of the Neotropical ecoregion, which includes the south of North America, Caribbean
islands, and Central and South America, showing grass species identified for harboring Epichloë
fungal endophytes. The numbers connect the grass species with the site of collection. The species
are just listed in alphabetical order
Table 2.1 List of grass species, their site of collection (country), and the associated Epichloë
fungal endophyte. Argentina (Arg.), Brazil (Bra.), Chile (Chi.), Colombia (Col.), Ecuador (Ecu.),
México (Mex.), Paraguay (Par.), Peru (Per.), Venezuela (Ven.), and Uruguay (Uru.). Countries in
“bold” indicate the fungus was isolated, “underlined” indicates that the observation was made on
herbarium specimens, and “plain font” indicates countries where they are present but have not
been studied
Case Grass species Country Fungal endophyte
1 Briza paleapilifera Arg. Epichloë tembladerae
2 Bromus auleticus Arg., Bra., Uru. E. tembladerae
Epichloë pampeana
3 Bromus brachyanthera Arg., Bra., Uru. Epichloë sp.
4 Bromus pictus Arg., Chi. E. tembladerae
Epichloë typhina subsp. poae
5 Calamagrostis alba Arg., Bra., Uru. E. tembladerae
6 Calamagrostis bogotensis Col., Ven.
7 Calamagrostis ecuadoriensis Ecu.
8 Calamagrostis sclerantha Per.
9 Calamagrostis tarmensis Arg. Epichloë sp.
10 Festuca argentina Arg. E. tembladerae
11 Festuca breviglumis Mex.
12 Festuca dissitiflora Arg.
13 Festuca fiebrigii Arg. E. tembladerae
14 Festuca fimbriata Arg., Bra., Par., Uru. Epichloë sp.
15 Festuca hieronymi Arg. E. tembladerae
16 Festuca horridula
17 Festuca linigluma Arg. Epichloë sp.
18 Festuca magellanica Arg. E. tembladerae
19 Festuca parodiana Arg. E. tembladerae
20 Festuca sp. Arg. Epichloë sp.
21 Festuca simplisiuscula Arg.
22 Festuca superba Arg. Epichloë sp.
23 Festuca tucumanica Arg.
24 Festuca ulochaeta Arg., Bra., Par.
25 Festuca weberbaueri Arg.
26 Hordeum comosum Arg., Chi. E. tembladerae
27 Melica macra Arg.
28 Melica stuckertii Arg. E. tembladerae
29 Phleum alpinum Arg., Chi. E. tembladerae-E. cabralii
30 Poa alopecurus subsp. alopecurus Arg., Chi.
31 Poa alopecurus subsp. fuegiana Arg., Chi.
32 Poa alopecurus subsp. shuka Arg., Chi.
33 Poa bergii Arg. E. tembladerae
34 Poa bonariensis Arg., Uru. E. tembladerae
35 Poa calchaquiensis Arg. E. tembladerae
36 Poa durifolia Arg. E. tembladerae
(continued)
Table 2.1 (continued)
Case Grass species Country Fungal endophyte
37 Poa holciformis Arg.
38 Poa huecu Arg. E. tembladerae
39 Poa gymnantha Arg., Chi.
40 Poa lanigera Arg., Bra., Uru. E. tembladerae
41 Poa lanuginosa Arg. E. tembladerae
42 Poa ligularis Arg. E. tembladerae
43 Poa lillioi Arg.
44 Poa plicata Arg.
45 Poa spiciformis Arg. E. tembladerae
Epichloë sp.
46 Polypogon elongatus Arg., Uru., Bra. E. tembladerae
species of Epichloë in the world, being present in at least 20 grass species in

Argentina (Table 2.1) and in two species from the United States (Charlton et al.
2014; Iannone et al. 2012a; Mc Cargo et al. 2014). Some host species could be
associated with different endophytes, and many of the grasses associated with
E. tembladerae (e.g., Bromus auleticus, Bromus pictus, and Phleum alpinum) are
also associated with another endophytic species (Table 2.1), although different
endophytes coinhabiting in the same individual plant have not been detected.
Even though asexual endophytes are considered to be exclusively vertically
transmitted, some characteristics of the endophytes from South America are not
consistent with this assumption. The presence of hybrid endophytes whose parental
species are not present in the region, the wide host range of E. tembladerae, and
other seed-transmitted endophytes lead us to consider the existence of events of
horizontal transmission of endophytes between sympatric host species. Conidia
formed on the epidermis of several host species (e.g., Poa spiciformis, Bromus aule-
ticus) would suggest the occurrence of this strategy (Iannone et al. 2009; White
et al. 1996).
Some of the host species associated with E. tembladerae are toxic to cattle.
Festuca argentina and Poa huecu in Patagonia and Festuca fiebrigii in the north of
Argentina cause some disorders known as “huecu” and “tembladera” in sheep,
horses, and donkeys (Cabral et al. 1999; Rivas and Zanolli 1908). Interestingly,
other forage species associated with E. tembladerae such as Bromus auleticus,
Bromus pictus, Hordeum comosum, or Poa spp. have not been reported to produce
toxicosis in domestic animal. These observations would suggest the existence of
toxic and nontoxic strains of E. tembladerae; however, genetic characterization
indicates that E. tembladerae isolates from toxic and nontoxic grasses do not have
genes for ergot alkaloids or lolitrem B biosynthesis (Iannone et al. 2012a) and these
alkaloids have not been detected in any of the toxic grasses from Argentina (Pomilio
et al. 1989; Rivas and Zanolli 1908; Scuteri et al. 1992).
Fig. 2.4 Phylogenetic tree of calM gene of Epichloë fungal endophytes isolated from grasses from
Argentina. Boxes in different colors highlight the different endophytes. The names in the branches
indicate the host plant from where the endophyte was isolated. The numbers in the nodes represent
bootstrap support and probabilities of each node
2.3 Ecology of Grass-Endophytes Symbiosis
Determining selection forces – either biotic or abiotic – that drive endophyte inci-
dence in populations has been central to understand the ecology and evolution of the
grass-Epichloë symbiosis. The most common experimental approaches are of at
least one of the following types: (i) field surveys searching for associations between
the endophyte incidence (proportion of endophyte-symbiotic plants in a population)
and a given environmental variation factor either discrete (e.g., with or without her-
bivory) or continuous (e.g., aridity or precipitation) and (ii) controlled condition
experiments where the performance of endophyte-symbiotic and endophyte-free
plants is compared under different conditions of a given environmental factor (e.g.,
herbivory, drought). When performed together, the combination of both approaches
provides, occasionally, robust results.
2.3.1 A
ssociations Between the Endophyte Incidence
and Environmental Conditions
As we stated in the previous section, many grass species from a wide range of envi-
ronments are associated with Epichloë fungal endophyte species (Iannone et al.
2011, 2015). It offers a variety of model systems to study the role of this symbiosis
in the adaptation of plants to different ecological settings and/or environmental con-
ditions. In this section, we summarize the current knowledge regarding the ecology
of the grass-Epichloë symbiosis in South America.
Patagonia encompasses 60 million ha in southern Argentina covering an exten-
sive area of South America. Within this region, fungal endophytes of grasses are
preceded by a negative reputation since Epichloë tembladerae has been associated
to the “huecu” disease in sheep and cattle grazing on Festuca argentina (Speg.)
Parodi and Poa huecu Parodi (Cabral et al. 1999; Parodi 1950). However, the fungal
endophyte symbiosis in many other Patagonian grasses seems not to be related to
animal toxicity (Iannone et al. 2011; Novas et al. 2007; Wilson 2007). Nonetheless,
the confirmation of toxicity requires a combination of genetic and biochemical anal-
yses to determine the alkaloid genes in the fungus and alkaloid concentration in the
plant that have, in addition, to be complemented with animal bioassays. Despite the
alkaloids, the real threat for grazing animals will depend on the abundance a given
grass represents in the whole vegetation community or as part of its diet.
The association between the incidences of Epichloë fungal endophytes in natural
populations of Bromus pictus Hook. f. (Bromus setifolius) and abiotic or biotic fac-
tors was explored in two contrasting ecological frames. Firstly, a survey was con-
ducted in the Andes Mountains near Las Leñas town, Mendoza province (35°12´
S/69°45´ W), between 2000 and 4000 meters above sea level. While the endophyte
incidence was not associated with the altitude, the incidence was higher in those
populations located in areas with high abundance of leaf-cutting ants (Acromyrmex
sp.) (Fig. 2.5) (White et al. 2001). Considering that E. tembladerae was found to
produce two different alkaloids (ergovaline and peramine), which are known to be
toxic for herbivores, it was suggested that the endophyte presence would dissuade
the ants from attacking the plants. Thus, it provided an evidence of the defensive
mutualism hypothesis (White et al. 2001). In another work on B. pictus populations
Fig. 2.5 Summary picture highlighting the main environmental factors or bioclimatic variables
driving the symbiosis incidence for the studied native grass species and Epichloë fungal
endophytes
in Southern Patagonia, the association between endophyte incidence and environ-

mental variables and soil conditions was explored (Novas et al. 2007). The study
included 36 sites along a northwest-southeast 400 km transect, covering the con-
spicuous ecological areas of the region (Novas et al. 2007). The incidence of
Epichloë endophytes in the surveyed populations ranged from 0% to 100% (none
and all individuals infected, respectively), showing a positive and significant corre-
lation with mean annual precipitation (Fig. 2.5). The incidence did not show an
association with the analyzed soil parameters. The factor herbivory was not included
in the analysis as neither ants nor other herbivorous insects were observed during
the work period.
In Tierra del Fuego island, the southernmost region of Patagonia, the association
between Epichloë incidence in natural populations of Phleum alpinum L. and Poa
spiciformis Hauman and Parodi and environmental variables was explored (Novas
et al. 2007). Phleum alpinum and P. spiciformis are perennial grasses with no
records of toxicity to livestock. Phleum alpinum plants were sampled in 30 sites

along a southwest-northeast transect, including the Nothofagus forests, grasslands,
Chiliotrichum dense scrub, and “vegas.” The incidence of Epichloë endophytes in
populations of Ph. alpinum varied from 0% to 100%. The ecological analysis
revealed that populations with lower endophyte incidence were found in community
stands dominated by evergreen forests of Nothofagus pumilio, humid areas charac-
terized by the highest mean annual precipitation in the transect (Novas et al. 2007).
In the case of P. spiciformis, all the populations presented plants associated with
Epichloë, and the incidence ranged from 40% to 100%. The association between
endophyte incidence and plant communities was negatively correlated with the
abundance of Empetrum rubrum (Fig. 2.5; Novas et al. 2007), a species indicative
of acidic and infertile soils (Collantes et al. 1989). Although endophyte incidence
diminished in these stressful soils, P. spiciformis is one of the scarce grasses that
grow in those poor habitats and is one of the principal taxa consumed by sheep
throughout the year (Posse et al. 1996). At least for Ph. alpinum, the environmental
conditions that promotes the symbiosis seem to support the idea that fungal endo-
phytes improve plant tolerance to drought.
Based on a previous survey which covered several species (Vila-Aiub et al.
2001), 14 wild populations of Hordeum comosum J. Presl were sampled in north-
western Patagonia to explore the incidence of Epichloë in association with environ-
mental variables (Iannone et al. 2015). The Epichloë incidence ranged from 0% to
100%, and while endophyte-free populations were restricted to a humid meadow
and grassy steppes from the Sub-Andean district, the populations with the higher
proportion of endophyte-infected individuals were collected in shrub steppes
(Fig. 2.5). The results evidenced a negative correlation between endophyte inci-
dence and the mean annual precipitation (Iannone et al. 2015). The endophyte spe-
cies E. tembladerae associated to H. comosum (Iannone et al. 2015) is predicted to
synthetize the alkaloids peramine and terpendole C (Yi et al. 2018). Although this
can imply that the endophyte confers protection to this highly preferred grass by
herbivores, there is no study so far that confirmed the previous expectation based on
genetic analysis. In fact, the endophyte incidence in H. comosum populations sub-
jected to intense grazing regimes was low compared to those in moderate or
ungrazed conditions (Hernández-Agramonte and Semmartin 2016).
An endophyte-mediated increased tolerance to drought in some plant species is
suggested to be behind the positive association between symbiosis incidence and
aridity (Lewis et al. 1997; Malinowski and Belesky 2019). This would be explained
by the patterns of endophyte incidence in relation to precipitation for H. comosum
and Ph. alpinum (Iannone et al. 2015; Novas et al. 2007). However, opposite pat-
terns have been described for the grass B. pictus (Novas et al. 2007) and for H. como-
sum in a new exhaustive study (Casas et al. 2015). The tendency to present a very
low level of – or not at all – fungal endophytes in populations located in sites with
very low mean annual precipitation (<400 mm y−1) may result from the very high
energy expenditure that implies maintenance of symbiont fungi for host plants in
such extremely dry or stressful environments (Gundel et al. 2016; Semmartin et al.
2015). Despite this, more studies are required to understand the controls of
e ndophyte symbiosis in grass populations as the puzzling results may arise from
differences in the endophyte-specific effects in relation to the environment where
host plant species have evolved.
Other studies have tried to relate the endophyte incidence in host populations not
just to a specific bioclimatic variable but to the vegetation communities or type of
vegetation. Populations of the native perennial grasses Poa bonariensis Kunth and
P. lanuginosa Poir., both dioecious and rhizomatous, were sampled during spring
and summer of 2005, 2006, 2007, and 2008 (Iannone et al. 2013). The sampled area
extended between 35° and 42° south latitude and from the Atlantic coast in the east
to the Andes in the west, covering almost the whole distribution area of both species
in Argentina. Populations were characterized according to plant community (domi-
nant plant species), ecological characteristics of the environment, and recalculated
variables as the ratio between average rainfall in winter and summer. The communi-
ties surveyed were identified as grasslands, xerophytic forests, hills in the Humid
Pampas, coastal dunes, shrub steppes, and arid grassy steppes. Poa lanuginosa, a
species that inhabits sandy soils from Southern Brazil to Patagonia, endophyte-
symbiotic plants were found in 8 of 46 populations. Ecological analyses showed
that endophyte-infected populations tended to be located on those coastal dune
communities characterized by the highest mean annual precipitation (Fig. 2.5).
Thus, endophyte incidence correlated positively with a winter precipitation regime
and with a higher water availability in soil during winter (Iannone et al. 2013). Poa
bonariensis is a species that inhabits grasslands and xerophytic forests in central
Argentina and Uruguay. Thirteen out of 28 sampled populations presented
endophyte-symbiotic plants. While no clear association was found between the inci-
dence of endophytes and any climatic or soil variables, infected populations seemed
to be associated with those xerophytic forests characterized by the presence of
Celtis ehrenbergiana trees of the northern limit of the distribution area for this spe-
cies (Fig. 2.5) (Iannone et al. 2012a).
In summary, Epichloë incidence was correlated with at least an environmental
factor (Fig. 2.5). Whereas, in most cases, the driving variable was precipitation or a
bioclimatic variable associated with water availability, contrasting trends were also
observed among different grass-endophyte associations. There is also some support
for the evidence of the defensive role of fungal endophytes in protecting host grasses
against herbivores (Fig. 2.3). However, more studies are needed in order to cover all
the ecological variants (plant and endophyte species and ecological settings), espe-
cially considering other plant traits (not necessarily associated to fungal endophytes)
that may determine the plant conditions to growth:
(i) Environment of evolution: certain species that are adapted to arid conditions
may not display any endophyte-mediated response to, for example, precipita-
tion gradient.
(ii) Animal preference: some plant species are preferred while others are not by
grazing animals.
(iii) Representation in the vegetation community: the herbivory pressure on a given
species will depend on its relative abundance.
The incorporation of such relevant information into explicatory models may help
to understand the full range of effect of the Epichloë fungal endophytes in host plant
adaptation to different environmental conditions and ecological settings. Last but
not least, we urge the scientific community to conduct manipulative experiments
where the performance of endophyte-symbiotic and endophyte-free plants is evalu-
ated under different environmental conditions (e.g., drought vs control). By comple-
menting correlational surveys with results from manipulative experiments, we will
have more solid conclusion on the fungal endophyte effects on plant adaptation to
the environment.
2.3.2 E
ffect of Epichloë on the Performance of Native Grasses
from South America
In spite of the broad range of grasses associated with Epichloë in South America,
the number of studies conducted in order to establish the role of the endophyte in
the association is still scarce and restricted to only two native species, Bromus
pictus and Bromus auleticus. So far, there are no records of toxicosis caused by
grazing these species. In addition, the association with Epichloë seems not to pro-
vide tolerance to hydric stress since the incidence of Epichloë in populations of
Bromus pictus increases with precipitation (Fig. 2.5). Therefore, the question here
is what benefits the symbiosis with fungal endophyte offer to this specific host spe-
cies. An experimental study was performed to compare natural populations of
B. pictus differing in the incidence of Epichloë endophytes. Plants from E+ popula-
tions produced heavier seeds, and under greenhouse conditions, E+ seedlings pre-
sented higher emergence and growth parameters, dry weight, and leaf length and
lower mortality than the E− ones (Novas et al. 2003), suggesting an adaptive
advantage of infected plants, at least related with plant growth, as a consequence
of endophyte association. However, no further studies were performed on this plant
species, so there is no clear evidence of other benefits associated with its fungal
symbiont.
On the other hand, the outfits of the association between grasses native from
South America and Epichloë are better known in Bromus auleticus, a species that
inhabits grasslands from central Argentina, Uruguay, and Southern Brazil. Some
breeding programs are being conducted in order to improve forage properties of
this perennial grass; however, the presence of the endophytes and their role in the
response of this host to biotic and environmental stresses have not been considered.
Most of the populations of this species present high levels of infection with
Epichloë. As we have mentioned before, this species is associated with at least two
Epichloë species. Greenhouse and field experiments indicated that B. auleticus
plants associated with Epichloë produce more biomass and seeds and are more
resistant to continuous defoliation than endophyte-free plants (Iannone et al.
2012b; Iannone and Cabral 2006) (Fig. 2.6). In vitro micropropagation is also
Fig. 2.6 Severity (culms affected by the pathogen) of the smut fungal disease caused by Ustilago
bullata on Bromus auleticus plants with (E+) and without (E−) Epichloë fungal endophyte
enhanced; e ndophyte-infected seeds presented a higher callus induction, higher

plant regeneration from callus, and increased biomass of the regenerated plantlets
(Regalado et al. 2018).
The association with Epichloë may also confer resistance against fungal patho-
gens. Plants of Bromus auleticus associated with E. pampeana and Epichloë sp.
presented immunity against the smut disease caused by Ustilago bullata Berk.
(Iannone et al. 2017; Vignale et al. 2013). Smut fungi infect the ovary of the host
plants producing a gall with teliospores, causing sterility in the host plant (Fig. 2.6).
In this pathosystem, the smut and the endophyte compete for plant nutrients and the
ovary of the flower. If the endophyte excludes the smut, both the plant and the endo-
phyte are benefited. In addition, Epichloë-infected plants of Bromus auleticus inoc-
ulated with U. bullata present less mortality and produce more seeds than E− plants
(Iannone et al. 2017).
In summary, the results of the experiments conducted to elucidate the effect of
the fungal endophytic symbionts on the fitness of the host plants reveal several ben-
efits. These results are certainly promising to consider the use of fungal endophytes
as biological control agents and as tools for improving forage and turf grasses.
Nonetheless, the successful implementation of endophyte technology does still
have certain challenges to overpass.
2.4 Potential Application of Epichloë Fungal Endophytes

to Plant Breeding
The interest for harnessing microbial symbionts in agriculture is rising as it is

increasing the necessity for using environmentally friendly alternatives, in clear
opposition to the use of potentially toxic agrochemicals (Dent and Cocking 2017;
Gundel et al. 2013; Lawson et al. 2019; Lugtenberg et al. 2016; Wei and Jousset
2017). Historically, the use of microorganisms in agriculture has been mostly cen-
tered on N-fixing bacteria and arbuscular mycorrhizal fungi (AMF), two groups of
root microorganisms that facilitate plant acquisition to soil nutrients (Dent and
Cocking 2017; Gianinazzi et al. 2010; Zhang et al. 2019). Recent efforts, however,
aim to boost other positive aspects of the associated microorganisms such as
enhancement of plant physiology, priming immune system, or taking advantage of
novel secondary metabolites (e.g., alkaloids) or metabolic functions, effects that are
not exclusive of the abovementioned microorganisms but of other members of the
microbiome (Lawson et al. 2019; Pagano et al. 2017; Pankievicz et al. 2019).
Vertically transmitted symbionts are, from the technological perspective, easy to
manage; once inside the plant, they can persist across generations by passing from
mother plants to seeds. Despite this evident advantage, the use of vertically trans-
mitted microbial symbionts is not without limitations.
A symbiotically modified organism, as a breeding strategy, consists of replacing
the common symbiotic microorganism of a plant by a new – selected – one (Gundel
et al. 2013; Kauppinen et al. 2016). In particular, the endophytic technology con-
sists of inoculating a given Epichloë fungal endophyte into plants of forage cultivars
for improving aspects of production (persistence and growth) and for commercial
purposes (Johnson et al. 2013). As an added value to a commercial product, it is
important to assure final consumers that (i) there is no risk of animal toxicity, (ii)
the fungal endophyte persists enough time during seed storage, and (iii) it is able to
persist and transmit successfully to the seed under a wide range of environmental
conditions. In this section, we discuss briefly how the diversity of fungal endo-
phytes found in South America can contribute to solve those challenges this
technology faces.
As we showed in the second section (Diversity and Evolutionary Origin of
Epichloë Fungal Endophytes in South America), the up-to-date information sug-
gests there is a great source of fungal diversity to be explore in this region of the
world. This mostly unexplored diversity could be rich not only in terms of fungal
endophytes differing in alkaloid profile but also in terms of genetic diversity with
potential to establish compatible symbiosis with the cultivated grass species. For
example, the fungal endophyte recently found in the Patagonian grass Hordeum
comosum which is supposedly nontoxic to cattle (Iannone et al. 2015) could be
potentially inoculated in cultivated Hordeum species such as H. vulgare. The native
grass Bromus auleticus which is currently in a breeding program for high forage
production (Condón et al. 2017) could be improved by using any of its two common
fungal endophytes (i.e., E. tembladerae-E. pampeana) (Table 2.1). As previously
shown, these fungal endophytes have shown to improve different aspects of plant
fitness (Iannone et al. 2012b, 2017; Vignale et al. 2013).
Future integral breeding programs should consider all the aspects mentioned
above that may hinder the successful application of Epichloë fungal endophytes to
the forage production. The presented picture indicates that there is a long way to go
before native endophytes can be used in breeding programs, but at the same time,
there are great opportunities to find suitable endophytes to overcome each of the
technological limitations. Thus, it is necessary not only to find the right alkaloid
profile for avoiding toxicity but also to select for high-compatible and low-cost
endophytes to warrant symbiosis persistence and the fitness benefits for host plants
in a wide range of environmental conditions.
2.5 Conclusions
Although very limited in comparison with the ecoregions from the Northern
Hemisphere, our work compiled a significant volume of information regarding the
symbiosis between Epichloë fungal endophytes and cool-season grasses for the
Neotropical ecoregion. We presented data on the distribution of symbiotic grasses
and the diversity of the associated fungal endophytes. Despite this, however, the
different aspects of the grass-Epichloë symbiosis for the whole Neotropical ecore-
gion are still in its dawn. The distribution and genotypic and chemotypic diversity
of fungal endophytes in the whole Neotropical ecoregion have clearly not been
homogeneously inspected. There is a clear bias to studies carried out in Argentina
and less in neighboring countries such as Uruguay, Chile, and Southern Brazil.
There seems to be a great diversity that will allow us to find endophytic fungal vari-
ants with potential to be inoculated into forage cultivars. All this require long-term
projects ideally coordinating multidisciplinary research teams from different coun-
tries as to expand the limits of the field surveys to discover the host grasses and the
associated fungal endophytes.
References
Afkhami ME, Rudgers JA (2008) Symbiosis lost: imperfect vertical transmission of fungal endo-
phytes in grasses. Am Nat 172:405–416
Alderman SC, Rao S (2008) Ascosporic fertilization of Epichloë typhina in Dactylis glomerata
seed production fields in Oregon and implications for choke management. Plant Health Prog
9:47
Becker M, Becker Y, Green K, Barry S (2016) The endophytic symbiont Epichloë festucae estab-
lishes an epiphyllous net on the surface of Lolium perenne leaves by development of an expres-
sorium, an appressorium-like leaf exit structure. New Phytol 211:240–254
Bertoni MD, Cabral D, Romero N, Dubcovsky J (1993) Endofitos fúngicos en especies sudameri-
canas de Festuca (Poaceae). Bol Soc Argent Bot 29:25–34
Biganzoli F, Zuloaga F (2015) Análisis de diversidad de la familia Poaceae en la región austral de

America del Sur. Rodriguésia 66:337–351
Bright M, Bulgheresi S (2010) A complex journey: transmission of microbial symbionts. Nat Rev
Microbiol 8:218–230
Bucheli E, Leuchtmann A (1996) Evidence for genetic differentiation between choke-inducing
and asymptomatic strains of the grass endophyte from Brachypodium sylvaticum. Evolution
50:1879–1887
Cabral D, Cafaro MJ, Saidman B, Lugo M, Reddy PV, White JF (1999) Evidence supporting
the occurrence of a new species of endophyte in some South American grasses. Mycologia
91:315–325
Campbell MA, Tapper BA, Simpson WR, Johnson RD, Mace W, Ram A, Lukito Y, Dupont PY,
Johnson LJ, Scott DB, Ganley ARD, Cox MP (2017) Epichloë hybrida, sp. nov., an emerging
model system for investigating fungal allopolyploidy. Mycologia 109:715–729
Casas C, del Toro C, Deliens E, Casabella MP, García-Martínez GC, Nagahama N, Vignale VM,
Iannone LJ, Schnyder H, Gundel PE (2015) Incidence of Fungal endophyte in the preferred
grass Hordeum comosum associated to environmental stress gradients in Patagonia. 5th
International Symposium of Forage Breeding – Buenos Aires, Argentina
Charlton ND, Craven KD, Afkhami ME, Hall BA, Ghimire SR, Young CA (2014) Interspecific
hybridization and bioactive alkaloid variation increases diversity in endophytic Epichloë spe-
cies of Bromus laevipes. FEMS Microbiol Ecol 90:276–289
Christensen MJ (1995) Variation in the ability of Acremonium endophytes of Lolium perenne,
Festuca arundinacea and F. pratensis to form compatible associations in the three grasses.
Mycol Res 99:466–470
Christensen MJ, Simpson WR, Al Samarrai T (2000) Infection of tall fescue and perennial ryegrass
plants by combinations of different Neotyphodium endophytes. Mycol Res 104:974–978
Christensen MJ, Bennett RJ, Ansari HA, Koga H, Johnson RD, Gregory TB, Simpson WR,
Koolaard JP, Nickless ME, Voisey CR (2008) Epichloë endophytes grow by intercalary hyphal
extension in elongating grass leaves. Fungal Genet Biol 45:84–93
Clay K (1988) Fungal endophytes of grasses: a defensive mutualism between plants and fungi.
Ecology 69:10–16
Clay K, Schardl CL (2002) Evolutionary origins and ecological consequences of endophyte sym-
biosis with grasses. Am Nat 160:99–S127
Collantes M, Anchorena JÁ, Koremblit G (1989) A soil nutrient gradient in Magellanic
Empetrum Heathlands. Vegetation 80:183–193
Condón F, Jaurena M, Reyno R, Otaño C, Lattanzi FA (2017) Spatial analysis of genetic diver-
sity in a comprehensive collection of the native grass Bromus auleticus Trinius (ex Nees) in
Uruguay. Grass Forage Sci 72:723–733
Craven KD (2012) Population studies of native grass-endophyte symbioses provide clues for the
roles of host jumps and hybridization in driving their evolution. Mol Ecol 21:2562–2564
Craven KD, Blankenship JD, Leuchtmann A, Hignight K, Schardl CL (2001) Hybrid fungal endo-
phytes symbiotic with the grass Lolium pratense. Sydowia 53:44–73
D’Jonsiles MF, Carmarán CC, Robles CA, Ceriani-Nakamurakare ED, Novas MV (2019)
Mycorrhizal colonization and soil parameters affected by foliar endophytes in Jatropha curcas
L. J Soil Sci Plant Nutr 19:332–341
Dent D, Cocking E (2017) Establishing symbiotic nitrogen fixation in cereals and other non-
legume crops: the Greener Nitrogen Revolution. Agric Food Secur 6:1–9
Faeth SH, Sullivan T (2003) Mutualistic asexual endophytes in a native grass are usually parasitic.
Am Nat 161:310–325
Faeth SH, Helander ML, Saikkonen KT (2004) Asexual Neotyphodium endophytes in a native
grass reduce competitive abilities. Ecol Lett 7:304–313
Gagic M, Faville MJ, Zhang W, Forester NT, Rolston MP, Johnson RD, Ganesh S, Koolaard JP,
Easton HS, Hudson D, Johnson LJ, Moon CD, Voisey CR (2018) Seed transmission of Epichloë
endophytes in Lolium perenne is heavily influenced by host genetics. Front Plant Sci 9:1580
Gentile A, Rossi MS, Cabral D, Craven KD, Schardl CL (2005) Origin, divergence, and phylogeny
of Epichloë endophytes of native Argentine grasses. Mol Phylogenet Evol 35:196–208
Gianinazzi S, Gollotte A, Binet MN, van Tuinen D, Redecker D, Wipf D (2010) Agroecology: the
key role of arbuscular mycorrhizas in ecosystem services. Mycorrhiza 20:519–530
Gundel PE, Batista WB, Texeira M, Martínez-Ghersa MA, Omacini M, Ghersa CM (2008)
Neotyphodium endophyte infection frequency in annual grass populations: relative importance
of mutualism and transmission efficiency. Proc R Soc London Ser B 275:897–905
Gundel PE, Martínez-Ghersa MA, Garibaldi LA, Ghersa CM (2009) Viability of Neotyphodium
endophytic fungus and endophyte-infected and noninfected Lolium multiflorum seeds. Botany
87:88–96
Gundel PE, Omacini M, Sadras VO, Ghersa CM (2010) The interplay between the effectiveness
of the grass-endophyte mutualism and the genetic variability of the host plant in an agronomic
context. Evol Appl 3:538–546
Gundel PE, Rudgers JA, Ghersa CM (2011) Incorporating the process of vertical transmission into
understanding of host-symbiont dynamics. Oikos 120:1121–1128
Gundel PE, Martínez-Ghersa MA, Omacini M, Cuyeu R, Pagano E, Ríos R, Ghersa CM (2012)
Mutualism effectiveness and vertical transmission of symbiotic fungal endophytes in response
to host genetic background. Evol Appl 5:838–849
Gundel PE, Pérez LI, Helander M, Saikkonen K (2013) Symbiotically modified organisms: non-
toxic fungal endophytes in grasses. Trends Plant Sci 18:420–427
Gundel PE, Irisarri JGN, Fazio L, Casas C, Pérez LI (2016) Inferring field performance from
drought experiments can be misleading: the case of symbiosis between grasses and Epichloë
fungal endophytes. J Arid Environ 132:60e62
Hernández-Agramonte IM, Semmartin M (2016) The role of grazing intensity and preference on
grass-fungal endophyte symbiosis in a Patagonian steppe. J Arid Environ 134:122–124
Hill N, Roach P (2009) Endophyte survival during seed storage: Endophyte-host interactions and
heritability. Crop Sci 49:1425–1430
Hoffman GD, Rao S (2014) Fertilization of Epichloë typhina stromata by mycophagous slugs.
Mycologia 106:1–7
Hyde K, Soytong K (2008) The fungal endophyte dilemma. Fungal Diversity 33:163–173
Iannone L, Cabral D (2006) Effects of the Neotyphodium endophyte status on plant performance of
Bromus auleticus, a wild native grass from South America. Symbiosis 41:61–69
Iannone LJ, Cabral D, Schardl CL, Rossi MS (2009) Phylogenetic divergence, morphological
and physiological differences distinguish a new Neotyphodium endophyte species in the grass
Bromus auleticus from South America. Mycologia 101:340–351
Iannone LJ, White JF, Giussani LM, Cabral D, Novas MV (2011) Diversity and distribution of
Neotyphodium-infected grasses in Argentina. Mycol Prog 10:9–19
Iannone LJ, Novas MV, Young CA, De Battista JP, Schardl CL (2012a) Endophytes of native
grasses from South America: biodiversity and ecology. Fungal Ecol 5:357–363
Iannone LJ, Pinget AD, Nagabhyru P, Schardl CL, De Battista JP (2012b) Beneficial effects of
Neotyphodium tembladerae and Neotyphodium pampeanum on a wild forage grass. Grass
Forage Sci 67:382–390
Iannone LJ, Mc Cargo PD, Giussani LM, Schardl CL (2013) Geographic distribution patterns of
vertically transmitted endophytes in two native grasses in Argentina. Symbiosis 59:99–110
Iannone L, Irisarri J, Mc Cargo PD, Pérez LI, Gundel P (2015) Occurrence of Epichloë fungal
endophytes in the sheep-preferred grass Hordeum comosum from Patagonia. J Arid Environ
115:19–26
Iannone LJ, Vignale MV, Pinget AD, Re A, Mc Cargo PD, Novas MV (2017) Seed-transmitted
Epichloë sp. endophyte alleviates the negative effects of head smut of grasses (Ustilago bul-
lata) on Bromus auleticus. Fungal Ecol 49:45–51
Johnson LJ, de Bonth ACM, Briggs LR, Caradus JR, Finch SC, Fleetwood DJ, Fletcher LR, Hume
DE, Johnson RD, Popay AJ, Tapper BA, Simpson WR, Voisey CR, Card SD (2013) The exploi-
tation of epichloae endophytes for agricultural benefit. Fungal Diversity 60:171–188
Kauppinen M, Saikkonen K, Helander M, Pirttilä AM, Wäli PR (2016) Epichloë grass endophytes
in sustainable agriculture. Nat Plants 2:1–7
Kirby EJM (1961) Host-parasite relations in the choke disease of grasses. Trans Br Mycol Soc
44:493–503
König J, Guerreiro MA, Peršoh D, Begerow D, Krauss J (2018) Knowing your neighbourhood—
the effects of Epichloë endophytes on foliar fungal assemblages in perennial ryegrass in depen-
dence of season and land-use intensity. PeerJ 6:e4660
Kuldau G, Bacon C (2008) Clavicipitaceous endophytes: their ability to enhance resistance of
grasses to multiple stresses. Biol Control 46:47–51
Latch GCM, Christensen MJ (1985) Artificial infections of grasses with endophytes. Ann Appl
Biol 107:17–24
Lawson CE, Harcombe WR, Hatzenpichler R, Lindemann SR, Löffler FE, O’Malley MA, García
Martín H, Pfleger BF, Raskin L, Venturelli OS, Weissbrodt DG, Noguera DR, McMahon KD
(2019) Common principles and best practices for engineering microbiomes. Nat Rev Microbiol
17:725–741
Leuchtmann A (1992) Systematics, distribution, and host specificity of grass endophytes. Nat
Toxins 1:150–162
Lewis GC, Ravel C, Naffaa W, Astier C, Charmet G (1997) Occurrence of Acremonium endo-
phytes in wild populations of Lolium spp. in European countries and a relationship between
level of infection and climate in France. Ann Appl Biol 130:227–238
Liu J, Nagabhyru P, Schardl CL (2017) Epichloë festucae endophytic growth in florets, seeds, and
seedlings of perennial ryegrass (Lolium perenne). Mycologia 109:691–700
Lugo MA, Anton AM, Cabral D (1998) Micofilas en gramíneas Sudamericanas. An Jard Bot
Madrid 56:15–22
Lugtenberg BJJ, Caradus JR, Johnson LJ (2016) Fungal endophytes for sustainable crop produc-
tion. FEMS Microbiol Ecol 92:1–17
Malinowski DP, Belesky DP (2019) Epichloë (formerly Neotyphodium) fungal endophytes increase
adaptation of cool-season perennial grasses to environmental stresses. Acta Agrobot 72:1767
Mc Cargo PD, Iannone LJ, Vignale MV, Schardl CL, Rossi MS (2014) Species diversity of Epichloë
symbiotic with two grasses from southern Argentinean Patagonia. Mycologia 106:339–352
Moon CD, Craven K, Leuchtmann A, Clement S, Schardl C (2004) Prevalence of interspecific
hybrids amongst asexual fungal endophytes of grasses. Mol Ecol 13:1455–1467
Moy M, Belanger F, Duncan R, Freehoff A, Leary C, Meyer WR, Sullivan R, White JF (2000)
Identification of epiphyllous mycelial nets on leaves of grasses infected by clavicipitaceous
endophytes. Symbiosis 28:291–302
Novas MV, Gentile A, Cabral D (2003) Comparative study of growth parameters on diaspores and
seedlings between populations of Bromus setifolius from Patagonia, differing in Neotyphodium
endophyte infection. Flora 198:421–426
Novas MV, Collantes M, Cabral D (2007) Environmental effects on grass-endophyte associations
in the harsh conditions of south Patagonia. FEMS Microbiol Ecol 61:164–173
Oberhofer M, Leuchtmann A (2014) Horizontal transmission, persistence and competition capa-
bilities of Epichloë endophytes in Hordelymus europaeus grass hosts using dual endophyte
inocula. Fungal Ecol 11:37–49
Pagano MC, Azevedo Correa EJ, Duarte NF, Yelikbayev B, O’Donovan A, Gupta VK (2017)
Advances in eco-efficient agriculture: the plant-soil mycobiome. Agriculture 7:1–12
Pankievicz VCS, Irving TB, Maia LGS, Ané JM (2019) Are we there yet? The long walk towards
the development of efficient symbiotic associations between nitrogen-fixing bacteria and non-
leguminous crops. BMC Biol 17:99
Parodi A (1950) Las gramíneas tóxicas para el ganado en la República Argentina. Rev Arg
Agronomía 17:163–227
Philippe G (2016) Lolitrem B and indole diterpene alkaloids produced by endophytic fungi of the
genus Epichloë and their toxic effects in livestock. Toxins 8:1–16
Philipson MN, Christey MC (1986) The relationship of host and endophyte during flowering, seed
formation, and germination of Lolium perenne. N Z J Bot 24:125–134
Piano E, Bertoli FB, Romani M, Tava A, Riccioni L, Valvassori M, Carroni AM (2005) Specificity
of host-endophyte association in tall fescue populations from Sardinia, Italy. Crop Sci
45:1456–1463
Pomilio AB, Rofi RD, Gambino MP, Mazzini CA, Debenedetti de Langenheimc RT (1989)
The lethal principle of Poahuecu (Coiron Blanco): a plant indigenous to Argentina. Toxicon
27:1251–1262
Posse G, Anchorena J, Collantes MB (1996) Seasonal diets of sheep in the steppe region of Tierra
del Fuego, Argentina. J Range Manag Arch 49(1):24–30
Regalado JJ, Berdion V, Vignale MV, Novas MV, Pitta-Alvarez SI, Iannone LJ (2018) The pres-
ence of Epichloë sp. in Bromus auleticus (Trin.) seeds enhances micropropagation and growth
of micropropagated plantlets from these seeds. Plant Cell Tissue Organ Culture 135:279–286
Rivas H, Zanolli M (1908) La tembladera. Enfermedad propia de los animales herbívoros de las
regiones andinas. Rev Fac Agron La Plata 5:5–35
Rodriguez RJ, White JF, Arnold AE, Redman RS (2009) Fungal endophytes: diversity and func-
Rolston M, Hare M, Moore K, Christensen M (1986) Viability of Lolium endophyte fungus in seed
stored at different moisture contents and temperatures. N Z J Exp Agric 14:297–300
Saikkonen K, Faeth SH, Helander M, Sullivan TJ (1998) Fungal endophytes: a continuum of inter-
actions with host plants. Annu Rev Ecol Evol Syst 29:319–343
Saikkonen K, Wäli P, Helander M, Faeth SH (2004) Evolution of endophyte-plant symbioses.
Trends Plant Sci 9:275–280
Saikkonen K, Wäli PR, Helander M (2010) Genetic compatibility determines endophyte-grass
combinations. PLoS One 5:e11395
Saikkonen K, Gundel PE, Helander M (2013) Chemical ecology mediated by fungal endophytes
in grasses. J Chem Ecol 39:962–968
Sánchez Márquez S, Bills GF, Zabalgogeazcoa I (2008) Diversity and structure of the fungal endo-
phytic assemblages from two sympatric coastal grasses. Fungal Divers 33:87–100
Sánchez Márquez S, Bills GF, Domínguez Acuña L, Zabalgogeazcoa I (2010) Endophytic myco-
biota of leaves and roots of the grass Holcus lanatus. Fungal Divers 41:115–123
Schardl CL (2010) The epichloae, symbionts of the grass subfamily poöideae. Ann Mo Bot Gard
97:646–665
Schardl CL, Craven KD, Speakman S, Stromberg A, Lindstrom A, Yoshida R (2008) A novel test
for host symbiont codivergence indicates ancient origin of fungal endophytes in grasses. Syst
Biol 57:483–498
Schardl CL, Florea S, Pan J, Nagabhyru P, Bec S, Calie PJ (2013a) The Epichloae: alkaloid diver-
sity and roles in symbiosis with grasses. Curr Opin Plant Biol 16:480–488
Schardl CL, Leuchtmann A, Chung KR, Penny D, Siegel MR (1997) Coevolution by common
descent of fungal symbionts (Epichloë spp.) and grass hosts. Mol Biol Evol 14:133–143
Schardl CL, Young CA, Faulkner JR, Florea S, Pan J (2012) Chemotypic diversity of epichloae,
fungal symbionts of grasses. Fungal Ecol 5:331–344
Schardl CL, Young CA, Hesse U, a. (2013b) Plant-symbiotic fungi as chemical engineers:
multi-genome analysis of the Clavicipitaceae reveals dynamics of alkaloid loci. PLoS Genet
9:e1003323
Scott B, Green K, Berry D (2018) The fine balance between mutualism and antagonism in the
Epichloë festucae-grass symbiotic interaction. Curr Opin Plant Biol 44:32–38
Scuteri M, Sala de Miguel MA, Blanco Viera J, Planes de Banchero E (1992) Tremorgenic
mycotoxins produced by strains of Penicillium spp. isolated from toxic Poa huecu Parodi.
Mycopathologia 120:177–182
Selosse MA, Schardl CL (2007) Fungal endophytes of grasses: hybrids rescued by vertical trans-
mission? An evolutionary perspective. New Phytol 173:452–458
Semmartin M, Omacini M, Gundel PE, Hernandez Agramonte I (2015) Broad-scale variation in
the incidence of fungal endophytes in temperate grasses. J Appl Ecol 103:184–190
Shymanovich T, Faeth SH (2019) Environmental factors affect the distribution of two Epichloë
fungal endophyte species inhabiting a common host grove bluegrass (Poa alsodes). Ecol Evol
9:6624–6642
Shymanovich T, Saari S, Lovin ME, Jarmusch AK, Jarmusch SA, Musso AM, Charlton ND, Young
CA, Cech NB, Faeth SH (2014) Alkaloid variation among epichloid endophytes of sleepygrass
(Achnatherum robustum) and consequences for resistance to insect herbivores. J Chem Ecol
41(1):1–12
Soto-Barajas MC, Vázquez-de-Aldana BR, Álvarez A, Zabalgogeazcoa I (2019) Sympatric
Epichloë species and chemotypic profiles in natural populations of Lolium perenne. Fungal
Ecol 39:231–241
Sugawara K, Ohkubo H, Yamashita M, Mikoshiba Y (2004) Flowers for Neotyphodium endophytes
detection: a new observation method using flowers of host grasses. Mycoscience 45:222–226
Suryanarayanan TS (2013) Endophyte research: going beyond isolation and metabolite documen-
tation. Fungal Ecol 6:561–568
Tadych M, Bergen MS, White JF (2014) Epichloë spp. associated with grasses: new insights on life
cycles, dissemination and evolution. Mycologia 106:181–201
Tsai HF, Liu JS, Staben C, Christensen MJ, GCM L, Siegel MR, Schardl CL (1994) Evolutionary
diversification of fungal endophytes of tall fescue grass by hybridization with Epichloë species.
PNAS 91:2542–2546
Vega FE, Simpkins A, Aime MC, Posada F, Peterson SW, Rehner SA, Infante F, Castillo A, Arnold
AE (2010) Fungal endophyte diversity in coffee plants from Colombia, Hawai’i, Mexico and
Puerto Rico. Fungal Ecol 3:122–138
Vignale MV, Astiz-Gassó MM, Novas MV, Iannone LJ (2013) Epichloid endophytes confer resis-
tance to the smut Ustilago bullata in the wild grass Bromus auleticus (Trin.). Biol Control
67:1–7
Vila-Aiub MN, Demartin EB, Maseda P, Gundel PE, Ghersa CM (2001) Exploración de la presen-
cia de hongos endofíticos en pastos de la Estepa Patagónica. Actas de la I Reunión Binacional
de Ecología
Wei Z, Jousset A (2017) Plant breeding goes microbial. Trends Plant Sci 22:555–558
Welty RE, Azevedo MD, Cooper TM (1987) Influence of moisture content, temperature, and
length of storage on seed germination and survival of endophytic fungi in seeds of tall fescue
and perennial ryegrass. Phytopathology 77:893–900
Wheatley WM, Kemp HW, Simpson WR, Hume DE, Nicol HI, Kemp DR, Launders TE (2007)
Viability of endemic endophyte (Neotyphodium lolii) and perennial ryegrass (Lolium perenne)
seed at retail and wholesale outlets in south-eastern Australia. Seed Sci Technol 35:360–370
White JF (1988) Endophyte-host associations in forage grasses. A proposal concerning origin and
evolution. Mycologia 80(4):442–446
White JF, Martin TI, Cabral D (1996) Endophyte-host associations in grasses XXII. Conidia for-
mation by Acremonium endophytes on the phylloplanes of Agrostis hiemalis and Poa rigidifo-
lia. Mycologia 88:174–178
White JF, Sullivan RF, Balady GA, Gianfagna TJ, Yue Q, Meyer WA, Cabral D (2001) A fungal
endosymbiont of the grass Bromus setifolius: distribution in some Andean populations, identi-
fication and examination of beneficial properties. Symbiosis 31:241–257
Wilson D (1995) Endophyte: the evolution of a term and clarification of its use and definition.
Oikos 73:274–276
Wilson AD (2007) Clavicipitaceous anamorphic endophytes in Hordeum germplasm. Plant Pathol
J 6:1–13
Wille PA, Aeschbacher RA, Boller T (1999) Distribution of fungal endophyte genotypes in doubly
infected host grasses. Plant J 18:349–358
Yi M, Hendricks WQ, Kaste J, Charlton ND, Nagabhyru P, Panaccione DG, Young CA (2018)
Molecular identification and characterization of endophytes from uncultivated barley.
Mycologia 110:453–472
Zabalgogeazcoa I, Gundel PE, Helander M, Saikkonen K (2013) Non-systemic fungal endophytes

in Festuca rubra plants infected by Epichloë festucae in subarctic habitats. Fungal Diversity
60:25–32
Zhang W, Card SD, Mace WJ, Christensen MJ, McGill CR, Matthew C (2017) Defining the
pathways of symbiotic Epichloë colonization in grass embryos with confocal microscopy.
Mycologia 109:153–161
Zhang S, Lehmann A, Zheng W, You Z, Rillig MC (2019) Arbuscular mycorrhizal fungi increase
grain yields: a meta-analysis. New Phytol 222:543–555
Chapter 3
Trypanocidal and Herbicidal Activities
of Endophytic Fungi Associated
with Medicinal Plant Lafoensia pacari
Living in Neotropical Wetland Pantanal
of Brazil
Soraya Sander Amorim, Camila Rodrigues de Carvalho,

Jéssica Catarine Silva de Assis, Carlos Leomar Zani,
Tânia Maria de Almeida Alves, Policarpo Ademar Sales Junior,
Marcos Antônio Soares, and Luiz Henrique Rosa
Abstract Endophytes are microorganisms that inhabit plant tissues during at least
one stage of their life cycle, and they do not cause any apparent symptom of disease
on their hosts. Medicinal plants represent a particularly promising target for the
studies of the endophytic fungal community as a source of bioactive molecules. In
the present study, we evaluate the medicinal plant Lafoensia pacari present in the
Neotropical wetland of Pantanal Matogrossense, Brazil, as a reservoir of endophytic
fungi capable of producing bioactive metabolites. A total of 222 fungal endophyte
crude extracts were evaluated for trypanocidal activity against a tropical parasite
Trypanosoma cruzi and for herbicidal activity against seeds of Lactuca sativa and
Allium schoenoprasum. Four extracts of Aspergillus aculeatus, Coniothyrium sp.,
and Valsariaceae sp. were active against T. cruzi and presented high efficiency while
maintaining low cytotoxicity against host cells. The herbicidal activity was detected
S. S. Amorim (*) · J. C. S. de Assis · L. H. Rosa

Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de
Minas Gerais, Belo Horizonte, Brazil
C. R. de Carvalho
Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de
Minas Gerais, Belo Horizonte, Brazil
Instituto René Rachou, FIOCRUZ-MG, Belo Horizonte, Brazil
C. L. Zani
Instituto René Rachou, FIOCRUZ-MG, Belo Horizonte, Minas Gearis, Brazil
T. M. de Almeida Alves · P. A. Sales Junior
Instituto René Rachou, FIOCRUZ-MG, Belo Horizonte, Brazil
M. A. Soares
Departamento de Botânica e Ecologia, Universidade Federal do Mato Grosso, Cuiabá, Brazil
© Springer Nature Switzerland AG 2021 37

L. H. Rosa (ed.), Neotropical Endophytic Fungi,
https://doi.org/10.1007/978-3-030-53506-3_3
38 S. S. Amorim et al.
in 46 extracts, and 14 of them completely inhibited the germination of Lactuca

sativa (lettuce) and Allium schoenoprasum (chive) seeds. Our results show that the
medicinal Neotropical plant L. pacari is a rich reservoir of endophytic fungi pro-
ducing bioactive metabolites that can be used for antiparasitic and agricultural
purposes.
Keywords Endophytes fungi · Lafoensia pacari · Trypanosoma cruzi · Pesticidals
3.1 Introduction
Endophytes are microbes capable of colonizing internal tissues of plant organs, at

least for some period of their life cycle, without causing visible symptoms of dis-
ease or morphological alterations of the host plant (Suryanarayanan et al. 2009).
Endophytic fungi are found in almost all plants (Rodriguez et al. 2009). Endophytes
and the host plant establish a harmonious symbiotic interaction in which the micro-
organisms obtain energy, nutrients, and shelter in exchange for protection against
pathogens, herbivores, and insects a well as induction of plant growth and plant
defense mechanisms (Firáková et al. 2007). Various research studies all around the
world have shown that endophytes have the potential to biosynthesize diverse natu-
ral compounds with intriguing biological activities.
Medicinal plants are reported to harbor endophytes (Strobel 2002). Some endo-
phytic fungi could promote the accumulation of secondary metabolites (including
compounds with medicinal properties) originally produced by plants. According to
some resources, these metabolites may be produced by the host plant and their
endophytic fungi (Shwab and Keller 2008). Interestingly, several rare medicinal
plants produce important bioactive compounds to survive in unique environments
and may host novel and diverse fungal endophytes, which have rarely been isolated
and characterized (Strobel 2003).
Endophytic fungi could be used to produce important antiparasitic compounds to
treat neglected tropical diseases such as Chagas disease. Trypanosoma cruzi was
identified as the causative agent of Chagas disease more than 100 years ago, yet the
condition still remains a major social and public health problem in Latin America
and is regarded as a neglected tropical disease by the World Health Organization
(WHO) (Pérez-Molina and Molina 2017). Despite advances in the discovery and
development of plant-derived drugs, neglected tropical diseases continue to cause
the death of hundreds of millions of people, especially in poor areas (Goupil and
McKerrow 2014).
Natural resources, including fungal endophytes, are currently being explored for
bioactive substances including agrochemicals such as plant growth-regulating sub-
stances and bio-herbicides (Strobel et al. 2004). Unlike synthetic herbicides, natural
herbicides are reported to be environmentally friendly and biodegradable. They are
3 Trypanocidal and Herbicidal Activities of Endophytic... 39
also less toxic than man-made counterparts (Phattanawasin et al. 2007). Herbicide-
resistant weed biotypes are the main problem in weed control due to the steady
increase in their number because of the use of the same plant protection products for
years (Green 2014). Due to the emergence of herbicide-resistant varieties and
restrictions regarding the use of synthetic herbicides, researchers are focusing on
natural products that may be used as herbicides (Karthick and Aruna 2012). Such
compounds could be used as models for developing herbicides with new modes of
action (Cimmino et al. 2013; Varejão et al. 2013).
Lafoensia pacari A. St.-Hil. (Lythraceae) is a medicinal plant known in Brazil as
dedaleiro, pacari, or mangava-brava. Its leaves and stem bark are used in folk medi-
cine as antipyretic and antidiarrheal. They are also applied for wound healing and in
the treatment of gastritis, ulcers, and cancer (Solon et al. 2000; Mundo and Duarte
2007). Different pharmacological studies involving extracts from L. pacari have
shown antioxidant, antigenotoxic, anti-inflammatory, analgesic, antiulcer, antimi-
crobial, and antidepressant activities (Solon et al. 2000; Rogerio et al. 2003, 2006,
2008a, b, 2010; Lima et al. 2006, 2013; Matos et al. 2008; Galdino et al. 2009;
Silva-Júnior et al. 2010; Nascimento et al. 2011; Pereira et al. 2011; Tamashiro-
Filho et al. 2012). Lafoensia pacari can be found in Pantanal, a unique Neotropical
wetland area of Brazil, where its macerated stem bark has been used as an excellent
cicatrizing and antiulcer agent (Neto 2006). So far, only a small proportion of the
existing endophytic fungi has been studied, especially of those in a symbiotic rela-
tionship with tropical Brazilian plants. Therefore, this study aims to broaden this
investigation by focusing on the endophytic fungi living in the tissues of L. pacari
present in the Neotropical wetland of Pantanal as potential producers of bioactive
compounds.
3.2 Study Area and Fungal Material
The endophytic fungi used in this study were isolated from the internal leaf and bark
tissues of Lafoensia pacari A. St.-Hil., an angiosperm belonging to the family
Lythraceae (Amorim 2018). Specimens of L. pacari were collected in October 2014
at Pantanal Matogrossense, in the region of Poconé (16°22′10.9″ S; 56°18′31.6″
W). Lafoensia pacari was identified by comparisons with the voucher specimen
deposited at the herbarium of the Institute of Biological Sciences (BHCB) of the
Federal University of Minas Gerais, Brazil, under the code BHCB 173389. The
fungi isolates belong to the Culture of Microorganisms and Cells of the Federal
University of Minas Gerais (UFMGCB), Brazil, and were identified by sequencing
the internal transcribed spacer (ITS) region with universal primers ITS1 and ITS4
(White et al. 1990) as well as β-tubulin gene (Glass and Donaldson 1995). The
sequences were analyzed by Basic Local Alignment Search Tool (BLAST) at the
National Center for Biotechnology Information (NCBI) website.
3.3 Preparation of Fungal and Plant Extracts
All fungal isolates were cultivated according to protocols established by Rosa et al.
(2013). Briefly, a five mm diameter plug of each fungus was placed on 20 mL of
PDA medium at the center of Petri dishes (90 mm diameter) and cultured for 15 days
at 25 ± 2 °C (time enough for fungi to produce secondary metabolites). These fungal
cultures were lyophilized for 72 h, cut in small pieces, and transferred to 150 mL
glass tubes followed by the addition of 50 mL of dichloromethane (Synth, Brazil).
To compare the herbicidal activity among endophytic fungal extracts and L. pacari
extracts, leaves and stems of each plant specimens were collected, macerated with
50 mL of dichloromethane, and incubated for 15 days at 25 ± 2 °C. After 72 h at
room temperature, the organic phase was filtered and the solvent dried at room tem-
perature. An aliquot of each dried extract was dissolved in DMSO (Merck/USA) to
prepare a 20 mg mL−1 stock solution, which was stored at −20 °C.
3.4 I n Vitro Assays with Intracellular Amastigote

and Trypomastigote Forms of Trypanosoma cruzi
and Analysis of L929 Cell Viability
In vitro assays with amastigote and trypomastigote forms of T. cruzi were performed
according to protocols established by Romanha et al. (2010). Each extract was
tested in duplicate at 20 μg mL−1. Benznidazole at its IC50 (1 μg mL−1 = 3.8 μM) was
used as positive control. The active extracts were tested at decreasing concentrations
to determine the IC50. The results were expressed as the percentage of growth inhibi-
tion. The active extracts were tested in vitro for determination of cytotoxic concen-
tration that reduces viability of the L929 cells by 50% (CC50) using the alamarBlue®
dye. The cells were exposed to extracts at increasing concentrations starting at IC50
value for T. cruzi. The cell viability was expressed as the percentage of difference in
the reduction between treated and untreated cells (Romanha et al. 2010). IC50 and
CC50 values were calculated by linear interpolation, and the selectivity index (SI)
was determined based on the ratio of the CC50 value in the host cell divided by the
IC50 value of the parasite. Quadruplicates were run in the same plate, and the experi-
ments were repeated at least once.
3.5 Herbicidal Assay
The herbicidal activities of fungal extracts at 1 mg mL−1 against Lactuca sativa (let-
tuce) and Allium schoenoprasum (chive) were measured using methods according
to Gomes et al. (2018). Briefly, seeds of both plants were disinfected with ethanol at
70% for 1 min and sodium hypochlorite at 2–2.5% for 7.5 min, washed with
sterilized distilled water four times, and dried on sterilized filter paper at room tem-
perature. Herbicidal activity was quantitatively evaluated in 24-well microtiter plate
by visually comparing the amount of germination in each well between untreated
and treat samples. Samples were incubated at 26 °C in a germination incubator 710
(Thoth, Brazil) under continuous light conditions for 10 days for L. sativa and
14 days for A. schoenoprasum. Test samples were dissolved in acetone to a final
concentration of 10% acetone. The control wells contained 400 μL of deionized
water. The solvent control well contained 360 μL of water and 40 μL of the solvent.
Glyphosate at 3 mg mL−1 (Sigma, USA) was used as a positive herbicide control.
All sample wells contained 360 μL of water and 40 μL of the appropriate dilution of
the sample. A quantitative estimate of herbicidal activity was evaluated using a rat-
ing scale of 0–5, where 0 = no effect and 5 = no growth or no germination of the
seeds. All assays were performed in duplicate. All the extracts were evaluated in
duplicate, and those considered active were submitted to a new test to confirm the
activity.
3.6 Results and Discussion
A total of 30 plant specimens of L. pacari were collected from the tropical Pantanal
wetland. Antiparasitic activity tests against T. cruzi as well as herbicidal activity
tests were performed on 282 extracts. In the initial screening, 33 extracts (11.7%)
displayed trypanocidal activity (Table 3.1). Among them, there were 21 (7.4%) fun-
gal extracts and 12 (4.25%) plant extracts. All the active isolates from the initial
screening were retested in order to confirm their activity. The retest validated the
activity of 24 (8.5%) extracts, including 14 (4.96%) fungal extracts and 10 (3.5%)
plant extracts. The isolates with confirmed activity were tested in vitro for their half
maximal inhibitory concentration (IC50) and the 50% cytotoxic concentration (CC50).
Bioactive fungi were cultured under the same initial conditions described in
methods section to produce new extracts and evaluate the reproducibility of the IC50
and CC50 tests. Valsariaceae sp. UFMGCB 11294, Coniothyrium sp. UFMGCB
11339, Aspergillus aculeatus UFMGCB 114111, and UFMGCB 11413 extracts
could inhibit the growth of the trypomastigote and amastigote forms of T. cruzi in
both rounds of cultivation to a similar degree as the control drug. The two isolates
of A. aculeatus that displayed trypanocidal activity were obtained from L. pacari
bark. All plant extracts that showed trypanocidal activity also came from bark,
emphasizing the medicinal importance of this plant tissue. The stem bark of L. pac-
ari is the most commonly used tissue of this plant not only by the Pantanal popula-
tion but also by researchers in the chemical studies of the plant (Pereira et al. 2018).
Members of the Aspergillus genus are considered to be efficient producers of
secondary bioactive metabolites, such as alkaloids, polyketides, terpenoids, xantho-
nes, and steroids, which have already been shown to have antibacterial, antifungal,
and cytotoxic properties (Sun et al. 2012; He et al. 2012). Carvalho et al. (2015)
isolated an endophytic fungus Aspergillus calidoustus recovered from leaves of
Table 3.1 Trypanocidal activities of extracts obtained from cultures of fungal endophytes species isolated from Lafoensia pacari
42
Percentage Percentage reduction IC50 over T. cruzi growth CC50 on

Fungal species UFMGCBa reduction of T. cruzi in new cultivation at (20 μg mL−1)b mouseL929 cellc Selectivity indexd
Aspergillus 11205 100 ± 0 90 ± 2 – – –
aculeatus
Lasiodiplodia 11212 81 ± 5 93 ± 11 – – –
sp. 2
Lasiodiplodia 11214 61 ± 6 – – – –
sp. 2
Aspergillus 11215 100 ± 2 95 ± 3
aculeatus
Colletotrichum 11221 58 ± 1 – – – –
sp. 2
Phyllosticta 11236 58 ± 1 – – – –
sp. 1
Diaporthe 11237 57 ± 8 – – – –
inconspicua
Diaporthe 11245 63 ± 16 91 ± 7 _ _ –
inconspicua
Phyllosticta 11263 62 ± 4 – – – –
sp. 1
Diaporthe 11282 68 ± 12 82 ± 5 49.2 ˃80 ˃1.6
inconspicua
D. inconspicua 11282e – – – – –
Diaporthe sp. 1 11291 56 ± 0 – – – –
Valsariaceae sp. 11294 76 ± 11 84 ± 2 25.5 80 3.1
Valsariaceae sp. 11294e – – 37.1 ˂80 ˂2.1
Coniothyrium 11339 60 ± 2 71 ± 11 ˃5 ˂10 ˂2
sp.
S. S. Amorim et al.
Coniothyrium 11339e – – 27.8 ˃80 ˃2.9
sp.
Aspergillus 11369 72 ± 8 99 ± 2 – – –
aculeatus
Lasiodiplodia 11394 96 ± 4 – – – –
sp. 1
Aspergillus 11409 73 ± 8 97 ± 3 – – –
aculeatus
Aspergillus 11411 61 ± 5 90 ± 6 9.1 >80 >9.1
aculeatus
A. aculeatus 11411e – – ˂5 >80 >16
Aspergillus 11412 76 ± 5 99 ± 3 ˂5 5 ˃1
aculeatus
A. aculeatus 11412e – – – – –
Aspergillus 11413 73 ± 4 93 ± 7 ˂5 5 ˃1
3 Trypanocidal and Herbicidal Activities of Endophytic...
aculeatus
A. aculeatus 11413e – – 16.4 20 1.2
Lasiodiplodia 11421 100 ± 5 101 ± 1 5 ˂10 ˂2
sp. 1
Lasiodiplodia 11421e – – – – –
sp. 1
NI 11431 52 ± 3 92 ± 8 – – –
Lafoensia Lp 8 bark 58 ± 4 96 ± 1 6.9 80 11.6
pacari
43
Table 3.1 (continued)
44

Lp 9 bark 80 ± 5 97 ± 5 7.5 40 5.3
Lp 12 bark 63 ± 7 96 ± 2 7 40 5.7
Lp 13 bark 64 ± 9 97 ± 1 7.7 40 5.2
Lp 14 bark 64 ± 1 96 ± 2 ˂5 40 ˂8
Lp 22 bark 55 ± 3 100 ± 1 11.3 40 3.5
Lp 23 bark 65 ± 1 94 ± 1 8.6 40 4.6
Lp 28 leaf 68 ± 14 – – – –
Lp 28 bark 97 ± 2 82 ± 3 14.1 >80 >5.7
Lp 29 bark 85 ± 3 82 ± 11 14.5 >80 >5.5
Lp 30 leaf 87 ± 3 – – – –
Lp 30 bark 96 ± 4 79 ± 5 9.2 >80 >8.7
Benzonidazolef – – 1 625 625
Lp Lafoensia pacari specimen, − = not assayed
a
UFMGCB = Federal University of Minas Gerais collection of microorganisms and cell
b
Extract concentration that inhibits 50% of Trypanosoma cruzi growth (IC50)
c
Cytotoxic concentration that reduces viability of the L929 cells by 50% (CC50)
d
CC50 L929 cell/IC50 parasite, assay performed on MTT, in Vero cells
e
New fungus cultivation
f
Positive control
S. S. Amorim et al.
Acanthospermum australe (Asteraceae), a medicinal plant native to the Brazilian

savanna. Two substances, ophiobolin K and 6-epi-ophiobolin K, isolated from this
endophyte displayed trypanocidal activity against T. cruzi with IC50 values of 13 and
9.62 μM, respectively. However, these compounds showed a relatively low selectiv-
ity index against the parasite and were cytotoxic to the host cells. In the case of
A. aculeatus, the fungal genome has been recently sequenced by the Joint Genome
Institute; however, none of its genes have yet been correlated with the production of
active metabolites (Petersen et al. 2015). Despite this fact, it has already been shown
that A. aculeatus is able to produce important secondary metabolites including acu-
leacine AG (antibiotics and antifungals); CJ-15,183 (squalene synthase inhibitor
and antifungal agent); aspergillusol A (α-glucosidase inhibitor); secalonic acids A,
D, and F (toxins); asperparaline A; cytotoxic aculeatusquinones A–D; and okara-
mines H and I (okaramine alkaloids) (Ingavant et al. 2009). According to our knowl-
edge, the current work is the first report of the species A. aculeatus producing
trypanocidal metabolites.
In relation to the other two bioactive taxa, it has been reported that the Valsariaceae
family contains species of Myrmaecium that are able to produce secondary metabo-
lites (Jaklitsch et al. 2015) and a species of the genus Coniothyrium, isolated from
Pinus wallichiana (Himalayan Blue Pine) that showed activity against Candida
albicans with IC50 of 17 μg mL−1 (Qadri et al. 2014). However, no trypanocidal
activity was reported for these taxa.
Chagas disease affects approximately seven to eight million individuals, with
50,000 new cases diagnosed each year in Latin America, North America, and
Europe. It is estimated that more than 90 million individuals are at risk of infection
with the disease causative agent T. cruzi (Coura and Dias 2009; WHO 2014; Vazquez
et al. 2015). Conventional treatment is based on benzimidazole and nifurtimox,
drugs developed over a century ago, which have strong adverse effects, such as loss
of appetite, vomiting, polyneuropathy, and dermopathy. Many patients give up on
the treatment because, in addition to the strong side effects of these drugs, there is a
need for long-term therapy (Guedes et al. 2011). In addition, benzimidazole and
nifurtimox are more effective in the acute phase of the disease, when the parasite
remains in an extracellular blood form, and have low efficacy against intracellular
forms of T. cruzi during the chronic phase of the disease (Muellas-Serrano
et al. 2002).
The results obtained in the present study are promising for the investigation of
new bioactive molecules isolated from the three taxa as their trypanocidal activity
was reproduced after recultivation. Since there are no reports of A. aculeatus being
a producer of trypanocidal metabolites and the fungal species of Valsariaceae sp.
UFMGCB 11294 and Coniothyrium sp. UFMCCB 11339 have probably not been
described yet, these fungi are an especially attractive target for the discovery of
trypanocidal substances.
Fourteen isolates displayed strong and selective herbicidal activity against both
monocotyledonous (A. schoenoprasum) and dicotyledonous (L. sativa) targets with
100% inhibition of the seed germination (Table 3.2). The isolates included A. acu-
leatus, Chaetomiaceae sp., Diaporthe sp. 4, D. insconspicua, Lasiodiplodia sp. 1,
Table 3.2 Herbicidal activities of extracts obtained from cultures of fungal endophytes species isolated from Lafoensia pacari
Herbicidal activity Herbicidal activity
46
UFMGCBa Fungi Lactuca sativa Allium schoenoprasum UFMGCBa Fungi Lactuca sativa Allium schoenoprasum
11205 Aspergillus aculeatus – 5 ± 0 11433 Penicillium sp. 1 – 4 ± 1
11215 A. aculeatus – 5 ± 0 11435 Penicillium sp. 3 – 5 ± 0
11369 A. aculeatus 5 ± 0 5 ± 0 11436 Penicillium sp. 4 3.5 ± 0.5 4.5 ± 0.5
11409 A. aculeatus 5 ± 0 5 ± 0 11432 Penicillium wotroi 5 ± 0 4.5 ± 0.5
11410 A. aculeatus 5 ± 0 5 ± 0 11250 Phyllosticta sp. 1 5 ± 0 5 ± 0
11412 A. aculeatus 5 ± 0 5 ± 0 11256 Phyllosticta sp. 1 – 5 ± 0
11413 A. aculeatus 5 ± 0 5 ± 0 11264 Phyllosticta sp. 1 – 5 ± 0
11357 Beltroniella sp. 3.5 ± 0.5 3.5 ± 0.5 11311 Phyllosticta sp. 1 3 ± 0 3 ± 0
11430 Chaetomiaceae sp. 5 ± 0 5 ± 0 11313 Phyllosticta sp. 1 3 ± 0 –
11207 Colletotrichum sp. 2 – 3 ± 0 11331 Phyllosticta sp. 1 – 3.5 ± 0.5
11221 Colletotrichum sp. 2 – 5 ± 0 11332 Phyllosticta sp. 1 – 4 ± 1
11316 Colletotrichum sp. 2 – 4 ± 1 11333 Phyllosticta sp. 1 – 4 ± 0
11237 Diaporthe insconspicua – 4 ± 1 11294 Valsariaceae sp. 5 ± 0 5 ± 0
11245 D. insconspicua 3.5 ± 0.5 4.5 ± 0.5 11208 NI 5 ± 0 5 ± 0
11282 D. insconspicua – 5 ± 0 11277 Neofusicoccum sp. – 5 ± 0
11321 S. insconspicua 5 ± 0 5 ± 0 11270 NI – 5 ± 0
11322 Diaporthe sp. 4 5 ± 0 5 ± 0 11279 NI – 3 ± 0
11213 Lasiodiplodia sp. 1 5 ± 0 5 ± 0 11439 NI 5 ± 0 5 ± 0
11394 Lasiodiplodia sp. 1 5 ± 0 5 ± 0 Lp 1 leaf – – 3 ± 0
11395 Lasiodiplodia sp. 1 – 3 ± 0 Lp 22 leaf – – 3.5 ± 0.5
11399 Lasiodiplodia sp. 1 – 5 ± 0 Lp 29 leaf – – 3 ± 0
11418 Lasiodiplodia sp. 1 – 4 ± 1 NC – 0 ± 0 0 ± 0
11419 Lasiodiplodia sp. 1 5 ± 0 5 ± 0 PC – 5 ± 0 5 ± 0
11209 Lasiodiplodia sp. 2 – 3 ± 0 SC – 0 ± 0 0 ± 0
11397 Lasiodiplodia sp. 2 5 ± 0 5 ± 0
Lp Lafoensia pacari specimen, NC negative control, PC positive control, SC solvent control
a
UFMGCB = Culture of Microorganisms and Cells from the Federal University of Minas Gerais. The qualitative estimate of phytotoxicity was evaluated by
S. S. Amorim et al.
using a rating scale of 0–5, where 0 = no effect and 5 = no growth or no germination of the seeds. Plant targets: L. sativa = Lactuca sativa (lettuce) and
A. schoenoprasum = Allium schoenoprasum (chive)
Lasiodiplodia sp. 2, Phyllosticta sp. 1, and Valsariaceae sp. Several phytotoxins,

such as phyllosinol, have been previously identified in Phyllosticta species (Wikee
et al. 2011). In the present study, eight extracts from Phyllosticta showed herbicidal
activity for both or only one of the studied plants. Species of this genus have fre-
quently been isolated as endophytes and have been considered to be excellent
sources of novel lead bioactive compound structures (Rodrigues-Heerklotz et al.
2001). Diaporthe spp. are known as producers of secondary metabolites with the
potential for herbicidal and anti-herbivory activity (Ash et al. 2010; Andolfi
et al. 2015).
Five isolates of A. aculeatus showed total inhibition of both A. schoenoprasum
and L. sativa seed germination, and two isolates selectively inhibited only the chive
seed germination. Four Penicillium specimen showed herbicidal activity, and
Penicillium sp. 4 UFMGCB 11436 and P. wotroi UFMGCB 11432 inhibited the
germination of the seeds of both plant models used in the assay. Khattak et al.
(2014) have shown that crude ethyl acetate extracts from Aspergillus and Penicillium
have phytotoxic activity against Lemna minor (water lentil) and may also delay seed
germination of the weed Silybum marianum. Zhang et al. (2013) isolated a bioactive
compound from the endophytic fungus A. fumigatus obtained from the stem bark of
a medicinal plant Melia azedarach. The compound was named brevianamide F and
represented a potentially new class of broad-spectrum herbicides as it showed
greater activity than the positive control of glyphosate herbicide. Brevianamide F
also inhibited the turnip (Raphanus sativus) root and amaranth (Amaranthus man-
gostanus) seedlings growth.
Brazil is one of the largest consumers of agrochemicals in the world, and glypho-
sate is the most commercialized herbicide in the country (Brazil, IBAMA 2018).
Glyphosate-based herbicides are currently among the most widely used agricultural
chemicals in the world (Gianessi and Reigner 2006). The impact of this class of
herbicides on modern farming practices is undeniable and includes the development
of resistant crop varieties as well as environmental and ecological implications
(Gilbert 2013). The use of natural herbicides in place of traditional chemical herbi-
cides can provide a large number of environmental and socioeconomic benefits. It
could favor better management and lead to the reduced impact of agriculture on the
environment as well as on human health. Moreover, it could contribute to the con-
trol of the weeds that are resistant to current herbicides. In our study, we obtained
extracts from the A. aculeatus UFMGCB 11413 and Valsariaceae sp. UFMGCB
11294 fungi, which displayed antiparasitic activity and inhibited the germination of
all lettuce and chive seeds. These results point to the promising potential of these
fungi for the future isolation of substances with herbicidal properties.
3.7 Conclusion
The present study has contributed to the knowledge about the community of endo-
phytic fungi associated with Brazilian medicinal plants and allowed them to be
considered as a potential source of bioactive metabolites. The obtained results con-
firm the importance of medicinal plants present in the rarely studied Brazilian
Neotropical environments, such as the Pantanal wetlands, which are potential reser-
voirs of known and still unknown endophytic fungi. These as yet undiscovered sym-
bionts are likely to produce bioactive secondary metabolites, which could be used
in the future for agricultural and therapeutic purposes.
References
Amorim SS (2018) Taxonomia, diversidade e bioprospecção de fungos endofíticos associados à

planta medicinal Lafoensia pacari A. St. Hil. (mangava-brava) presente no Pantanal Mato-
grossense. PhD Thesis. Universidade Federal de Minas Gerais, Belo Horizonte
Andolfi A, Boari A, Evidente M, Cimmino A, Vurro M, Ash G, Evidente A (2015) Gulypyrones A
and B and Phomentrioloxins B and C produced by Diaporthe gulyae, a potential mycoherbi-
cide for saffron thistle (Carthamus lanatus). J Nat Prod 78:623–629
Ash GJ, Stodart B, Sakuanrungsirikul S, Anschaw E, Crump N, Hailstones D, Harper JDI (2010)
Genetic characterization of a novel Phomopsis sp., a putative biocontrol agent for Carthamus
lanatus. Mycologia 102:54–61
Brasil, Instituto Brasileiro do Meio Ambiente-IBAMA (2018) Acesso em 20/06/2018. MMA/
IBAMA, Brasilia. http://www.ibama.gov.br. Accessed 20 Apr 2020
Carvalho CR, Vieira MLA, Cantrell CL, Wedge DE, Alves TMA, Zani CL, Pimenta RS, Junior
PAS, Murta SMF, Romanha AJ, Rosa CA, Rosa LH (2015) Biological activities of ophiobolin
K and 6-epi-ophiobolin K produced by the endophytic fungus Aspergillus calidoustus. Nat
Prod Res 30(4):478–481
Cimmino A, Andolfi A, Zonno MC, Boari A, Troise C, Motta A, Vurro M, Ash G, Evidente A
(2013) Phomentrioloxin, afungal phytotoxin with potential herbicidal activity, and its deriva-
tives: a structure–activity relationship study. J AgricFood Chem 61(40):9645–9649
Coura JR, Dias JCP (2009) Epidemiology, control and surveillance of Chagas disease – 100 years
after its discovery. Men Inst Oswaldo Cruz 104(Suppl 1):31–40
Firáková S, Sturdíková M, Múcková M (2007) Bioactive secondary metabolites produced by
microorganisms associated with plants. Biologia 62:251–257
Galdino PM, Nascimento MVM, Sampaio BL, Ferreira RN, Paula JR, Costa EA (2009)
Antidepressant-like effect of Lafoensia pacari A. St.-Hil. ethanolic extract and fractions in
mice. J Ethnopharmacol 124:581–585
Gianessi LP, Reigner N (2006) Pesticide use in U.S. Crop protection: 2002.CropLife Foundation.
Washington, D.C.
Gilbert N (2013) A hard look at GM crops. Nature 497:24–26
Glass NL, Donaldson GC (1995) Development of primer sets designed for use with the PCR to
amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol 61:1323–1330
Gomes EC, Godinho VM, Silva DAS, Paula MTR, Vitoreli GA, Zani CL, Alves TMA, Junior
PAS, Murta SMF, Barbosa EB, Oliveira JG, Oliveira FS, Carvalho CR, Ferreira MC, Rosa CA,
Rosa LH (2018) Cultivable fungi present in Antarctic soils: taxonomy, phylogeny, diversity,
and bioprospecting of antiparasitic and herbicidal metabolites. Extremophiles 22(3):381–393
Goupil LS, McKerrow JH (2014) Introduction: drug discovery and development for neglected
diseases. Chem Rev 114:11131–11137
Green JM (2014) Current state of herbicides in herbicide-resistant crops. Pest Manag Sci
70:1351–1357
Guedes PMM, Silva GK, Gutierrez FRS, Silva JS (2011) Current status of Chagas disease chemo-
therapy. Expert Rev Anti-Infect Ther 9(5):609–620
He F, Sun YL, Liu KS, Zang XY, Qian PY, Wang YF, Qi SH (2012) Indole alkaloids from marine-
derived fungus Aspergillus sydowii SCSIO 00305. J Antibiot 65:109–111
Ingavant N, Dobereiner J, Wiyakrutta S, Ruchirawast S, Kittakoop P (2009) Aspergillusol A, an
α-glucosidase inhibitor from the marine-derived fungus Aspergillus aculeatus. J Nat Prod
72:2049–2052
Jaklitsch WM, Fournier J, Dai DQ, Hyde KD, Voglmayr H (2015) Valsaria and the Valsariales.
Fungal 73(1):59–202
Karthick RN, Aruna A (2012) Necrosis inducing herbicidal protein from cowpea pathogenic iso-
late Fusarium oxysporum and its herbicidal activity against common weed Peporomia wighti-
ana. Global J Biosci Biotechnol 1:248–252
Khattak SU, Iqbal Z, Lutfullah G, Bacha N, Khan AA, Saeed M, Ali M (2014) Phytotoxic and
herbicidal activities of Aspergillus and Penicillium species isolated from rhizosphere and soil.
Pak J Weed Sci Res 20:293–303
Lima MRF, Ximenez ECPA, Luna JS, Santana AEG (2006) The antibiotic activity of some
Brazilian medicinal plants. Rev Bras Farmacogn 16:300–306
Lima DCS, Silva CR, Sampaio BL, de Paula JR, Chen-Chen L (2013) Antigenotoxic, and anticy-
totoxic activities of an ethanolic extract of Lafoensia pacari (Lythraceae) stem bark in bacteria
and mice. Genet Mol Res 12:3887–3896
Matos LG, Santos SR, Ferreira RN, Pontes IS, Paula JR, Costa EA (2008) Anti-inflammatory, anti-
nociceptive and sedating effects of Lafoensia pacari aqueous extract. Pharm Biol 46:341–346
Muellas-Serrano S, Le-Senne A, Fernandez-Portillo C, Nogal JJ, Ochoa C, Gomez-Barrio A
(2002) In vitro and in vivo anti-Trypanosoma cruzi activity of a novel nitro-derivative. Mem
Inst Oswaldo Cruz 97:553–557
Mundo SR, Duarte MR (2007) Morfoanatomia Foliar e Caulinar de Dedaleiro: Lafoensia pacari
A. St.-Hil. (Lythraceae). Lat Am J Pharm 26:522–529
Nascimento MVM, Galdino PM, Florentino IF, Sampaio BL, Vanderlinde FA, Paula JR, Costa EA
(2011) Antinociceptive effect of Lafoensia pacari A. St.-Hil. Independent of anti-inflammatory
activity of ellagic acid. J Nat Med 65:448–454
Neto GG (2006) O saber tradicional pantaneiro: as plantas medicinais e a educação ambien-
tal. Revista Eletrônica do Mestrado em Educação Ambiental – Universidade Federal do Rio
Grande (17)
Pereira EMR, Gomes RT, Freire NR, Aguiar EG, Brandão MGL, Santos VR (2011) In vitro anti-
microbial activity of brazilian medicinal plant extracts against pathogenic microorganisms of
interest to dentistry. Planta Med 77:401–404
Pereira LOM, Vilegas W, Tangerina MMP, Arunachalam K, Balogun SO, Orlandi-Mattos PE,
Martins DTO (2018) Lafoensia pacari A. St.-Hil.: wound healing activity and mechanism of
action of standardized hydroethanolic leaves extract. J Ethnopharmacol 219:337–350
Pérez-Molina JA, Molina IM (2017) Chagas disease. Lancet 391(10115):82–94
Petersen L, Frisvald JC, Kundsen PB, Rholfs MR, Gotsfredsen CH, Larsen TO (2015) Induced
sclerotium formation exposes new bioactive metabolites from Aspergillus sclerotiicarbonar-
ius. J Antibiot 68:603–608
Phattanawasin P, Pojchanakom K, Sotanaphun U, Piyapolrungroj N, Zungsontiporn S (2007)
Weed growth inhibitors from Aspergillus fischeri TISTR 3272. Nat Prod Res 21:1286–1291
Qadri M, I Rajput R, Abdin MZ, Vishwakarma RA, Riyaz-Ul-Hassan S (2014) Diversity, molecu-
lar phylogeny, and bioactive potential of fungal endophytes associated with the Himalayan
Blue Pine (Pinus wallichiana). Microb Ecol 67(4):877–887
Rodrigues-Heerklotz KF, Drandarov K, Heerklotz J, Hesse M, Werner C (2001) Guignardic acid,

a novel type of secondary metabolite produced by the endophytic fungus Guignardia sp.: isola-
tion, structure elucidation, and asymmetric synthesis. Helv Chim Acta 84:3766–3772
Rodriguez RJ, White JF, Arnold AE, Redman RS (2009) Fungal endophytes: diversity and func-
Rogerio AP, Sá-Nunes A, Albuquerque DA, Anibal FF, Medeiros AI, Machado ER, Souza AO,
Prado-Junior JC, Faccioli LH (2003) Lafoensia pacari extract inhibits IL- 5 production in
toxocariasis. Parasite Immunol 25:393–400
Rogerio AP, Fontanari C, Melo MC, Ambrosio SR, Souza GE, Pereira PS, Franca SC, Costa FB,
Albuquerque DA, Faccioli LH (2006) Anti-inflammatory, analgesic and anti-oedematous
effects of Lafoensia pacari extract and ellagic acid. J Pharm Pharmacol 58:1265–1273
Rogerio AP, Fontanari C, Borducchi E, Keller AC, Russo M, Soares EG, Albuquerque DA, Faccioli
LH (2008a) Anti-inflammatory effects of Lafoensia pacari and ellagic acid in a murine model
of asthma. Eur J Pharmacol 580:262–270
Rogerio AP, Sá-Nunes A, Albuquerque DA, Soares EG, Faccioli LH (2008b) Anti-eosinophilic
effect of Lafoensia pacari in toxocariasis. Phytomedicine 15:348–357
Rogerio AP, Sá-Nunes A, Faccioli LH (2010) The activity of medicinal plants and secondary
metabolites in eosinophilic inflammation. Pharmacol Res 62:298–307
Romanha AJ, de Castro SL, Soeiro MNC, Lannes-Vieira J, Ribeiro I, Talvani A, Bourdin B,
Blum B, Olivieri B, Zani C, Spadafora C, Chiari E, Chatelain E, Chaves G, Calzada JE,
Bustamante JM, Freitas-Junior LH, Romero LI, Bahia MT, Lotrowska M, Soares M, Andrade
SG, Armstrong T, Degrave W, Andrade Zde A (2010) In vitro and in vivo experimental models
for drug screening and development for Chagas disease. Mem Inst Oswaldo Cruz 105:233–238
Rosa LH, Queiroz SCN, Moraes RM, Wang X, Techen N, Pan Z, Cantrell CL, Wedge DE (2013)
Coniochaeta ligniaria: antifungal activity of the cryptic endophytic fungus associated with
autotrophic tissue cultures of the medicinal plant Smallanthus sonchifolius (Asteraceae).
Symbiosis 60:133–142
Shwab EK, Keller NP (2008) Regulation of secondary metabolites production in filamentous asco-
mycetes. Mycol Res 112:225–230
Silva-Júnior IF, Raimondi M, Zacchino S, Cechinel Filho V, Noldin VF, Rao VS, Lima JCS,
Martins DTO (2010) Evaluation of the antifungal activity and mode of action of Lafoensia pac-
ari A. St.-Hil., Lythraceae, stem-bark extracts, fractions and ellagic acid. Braz J Pharmacogn
20:422–428
Solon S, Lopes L, Sousa-Junior PT, Schmeda-Hirschmann G (2000) Free radical scavenging activ-
ity of Lafoensia pacari. J Ethnopharmcol 72:173–178
Strobel GA (2002) Rainforest endophytes and bioactive products. Crit Rev Biotechnol
22(4):315–333
Strobel GA (2003) Endophytes as sources of bioactive products. Microbes Infect 5:535–544
Strobel G, Daisy B, Castillo U, Harper J (2004) Natural products from endophytic microorgan-
isms. J Nat Prod 67:257–268
Sun LL, Shao CL, Chen JF, Guo ZY, Fu XM, Chen M, Chen YY, Li R, Voogd NJ, She ZG,
Lin YC, Wang C, Bioorgs Y (2012) New bisabolane sesquiterpenoids from a marine-derived
fungus Aspergillus sp. isolated from the sponge Xestospongia testudinaria. Med Chem Lett
22:1326–1329
Suryanarayanan TS, Thirunavukkarasu N, Govindarajulu MB, Sasse F, Jansen R, Murali TS
(2009) Fungal endophytes and bioprospecting. Fungal Biol Rev 23:9–19
Tamashiro-Filho P, Olaitan BS, Almeida DAT, Lima JCS, Marson-Ascêncio PG, Ascêncio SD,
Rios-Santos F, Martins DTO (2012) Evaluation of antiulcer activity and mechanism of action
of methanol stem bark extract of Lafoensia pacari A. St.-Hil. (Lythraceae) in experimental
animals. J Ethnopharmacol 144:497–505
Varejão EVV, Demuner AJ, Barbosa LCA et al (2013) The search for new natural herbicides – stra-
tegic approaches for discovering fungal phytotoxins. Crop Prot 48:41–50
Vazquez BP, Vazquez TP, Miguel CB et al (2015) Inflammatory responses and intestinal injury
development during acute Trypanosoma cruzi infection are associated with the parasite load.
Parasit Vectors 8:206
White TJ, Bruns T, Lee S, Taylor JW (1990) Amplification and direct sequencing of fungal
ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ
(eds) PCR protocols: a guide to methods and applications. Academic Press, Inc, New York,
pp 315–322
Wikee S, Udayanga D, Crous PW, Chukeatirote E, Mckenzie EHC, Bahkali AH, Dai D, Hyde
KD (2011) Phyllosticta – an overview of current status of species recognition. Fungal Divers
51:43–61
World Health Organization (WHO) (2014) Chagas disease (American trypanosomiasis)
Zhang Q, Wang SQ, Tang HY, Li XJ, Zhang L, Xiao J, Gao YQ, Zhang AL, Gao JM (2013)
Potential allelopathic indole diketopiperazines produced by the plant endophytic Aspergillus
fumigatus using the one strain-many compounds method. J Agric Food Chem 67:11447–11452

Preface

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Preface

Uploaded by

Copyright:

Available Formats

Preface

The Neotropical biogeographic region (Fig. 1), also known as Neotropics, includes

INBio-03-2006-OT, R-CM-INBio-06-2006-OT, R-CM-INBio-30-2007-OT, R-CM-

1 Ecology of Neotropical Endophytic Fungi �������������������������������������������� 1

7 What Do We Know About Fungal Endophyte Diversity

15 Effectiveness of Endophytic Fungi from Baccharis dracunculifolia

Ezzanad Abdellah Organic Chemistry Department, Faculty of Science,University

Tatiana Maracaípe Corrêia Laboratorio de Microbiologia Geral e Aplicada,

G. Wilson Fernandes Departamento de Genética Ecologia & Evolução, Instituto

Paris Lavín Environmental Microbiology and Biotechnology Unit – Zenobia

Jaime Rodríguez Center of Biotechnology, Universidad de Concepcion,

Camila Rodrigues de Carvalho, Mariana Costa Ferreira,

Abstract Endophytic fungi are a diverse group of microorganisms that have

Keywords Fungi · Host plants · Balanced antagonism · Tropical

The community of microorganisms associated with a diversity of unique

C. R. de Carvalho (*) · M. C. Ferreira

© Springer Nature Switzerland AG 2021

h­ arbor a wide diversity of microorganisms such as fungi, bacteria, algae, archaea,

1.2 Endophytic Fungi: Definition

1.3 Balanced Antagonism Hypothesis

Fig. 1.1 Representative diagrams of evolution of endophytic-host plant interactions. (a)

A. ­fluminensis with certain strains of stress-tolerant endophytic fungi contributes to

Leopoldo J. Iannone , M. Victoria Novas , Patricia D. Mc Cargo ,

Abstract Epichloë fungal endophytes are a conspicuous group of fungi

L. J. Iannone (*) · M. V. Novas · P. D. Mc Cargo

© Springer Nature Switzerland AG 2021 11

Keywords Endophytic fungi · Distribution · Neotropical · Taxonomy

 rigin and Life History of Asexual Epichloë Fungal

2.1.2 Fitness Effects in an Intimate Relationship

The frequency of endophyte-symbiotic individuals greatly varies across species

In general, each species of Epichloë is associated with one or a group of closely

(Schardl et al. 2008). Additionally, a similar co-evolutive pattern can be appreciated

The discovery of endophyte-infected grasses in South America dates back to the

species of Epichloë in the world, being present in at least 20 grass species in

2.3 Ecology of Grass-Endophytes Symbiosis

in Southern Patagonia, the association between endophyte incidence and environ-

records of toxicity to livestock. Phleum alpinum plants were sampled in 30 sites

enhanced; e­ ndophyte-­infected seeds presented a higher callus induction, higher

2.4 Potential Application of Epichloë Fungal Endophytes

The interest for harnessing microbial symbionts in agriculture is rising as it is

Biganzoli F, Zuloaga F (2015) Análisis de diversidad de la familia Poaceae en la región austral de

Zabalgogeazcoa I, Gundel PE, Helander M, Saikkonen K (2013) Non-systemic fungal endophytes

Soraya Sander Amorim, Camila Rodrigues de Carvalho,

S. S. Amorim (*) · J. C. S. de Assis · L. H. Rosa

© Springer Nature Switzerland AG 2021 37

in 46 extracts, and 14 of them completely inhibited the germination of Lactuca

Keywords Endophytes fungi · Lafoensia pacari · Trypanosoma cruzi · Pesticidals

Endophytes are microbes capable of colonizing internal tissues of plant organs, at

3.2 Study Area and Fungal Material

3.3 Preparation of Fungal and Plant Extracts

3.4 I n Vitro Assays with Intracellular Amastigote

3.5 Herbicidal Assay

3.6 Results and Discussion

Percentage Percentage reduction IC50 over T. cruzi growth CC50 on

Percentage Percentage reduction IC50 over T. cruzi growth CC50 on

Acanthospermum australe (Asteraceae), a medicinal plant native to the Brazilian

Lasiodiplodia sp. 2, Phyllosticta sp. 1, and Valsariaceae sp. Several phytotoxins,

Amorim SS (2018) Taxonomia, diversidade e bioprospecção de fungos endofíticos associados à

Rodrigues-Heerklotz KF, Drandarov K, Heerklotz J, Hesse M, Werner C (2001) Guignardic acid,

You might also like

1 Ecology of Neotropical Endophytic Fungi �� 1

7 What Do We Know About Fungal Endophyte Diversity

15 Effectiveness of Endophytic Fungi from Baccharis dracunculifolia

h arbor a wide diversity of microorganisms such as fungi, bacteria, algae, archaea,

1.2 Endophytic Fungi: Definition

1.3 Balanced Antagonism Hypothesis

A. fluminensis with certain strains of stress-tolerant endophytic fungi contributes to

rigin and Life History of Asexual Epichloë Fungal

2.1.2 Fitness Effects in an Intimate Relationship

2.3 Ecology of Grass-Endophytes Symbiosis

enhanced; e ndophyte-infected seeds presented a higher callus induction, higher

2.4 Potential Application of Epichloë Fungal Endophytes

3.2 Study Area and Fungal Material

3.3 Preparation of Fungal and Plant Extracts

3.4 I n Vitro Assays with Intracellular Amastigote

3.5 Herbicidal Assay

3.6 Results and Discussion