Non-systemic fungal endophytes of grasses
Salud SÁNCHEZ MÁRQUEZa, Gerald F. BILLSb, Noemí HERREROa, Íñigo
ZABALGOGEAZCOAa,*
a
Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA-CSIC).
Apartado 37071, Salamanca, Spain
b
Fundación MEDINA, Parque Tecnológico de Ciencias de la Salud, 18100 Armilla,
Granada, Spain
Corresponding author: email: i.zabalgo@irnasa.csic.es , phone: +34 923219606
FUNGAL ECOLOGY 2011. in Press
Abstract
Many fungi behave as endophytes in grasses. Unlike the well known
Epichloë/Neotyphodium species, most other endophytes are not capable of systemic
colonization of plant organs, or seed transmission. The species diversity of the nonsystemic endophytic mycobiota of grasses is large, dominated by ascomycetes. The
relative abundance of species is very unequal, a few dominant taxa like Acremonium,
Alternaria, Cladosporium, Epicoccum, and Penicillium spp., occur in many grasses and
locations. In contrast, many rare species are isolated only once in endophyte surveys.
The possible ecological functions of endophytes are diverse, and often unknown. Latent
pathogens represent a small fraction of endophytic mycobiotas, indicating that many
non pathogenic fungal taxa have the capability to penetrate plants overriding defence
reactions. Some dominant species behave as latent saprophytes, sporulating when the
host tissue dies. Endofungal viruses and bacteria occur among endophytic species, but
their effect in their hosts is largely unknown.
Index descriptors: Poaceae; endophytic; symbiosis; biodiversity; grasses; fungus-plant
interactions; latent saprophytes; latent pathogens; mycovirus.
Introduction
After Neotyphodium species were found to be responsible for the toxicity of
pasture grasses used for animal production (Bacon et al. 1977; Fletcher & Harvey
1981), surveys of endophytes in other plant families showed that this group of fungi and
their Epichloë teleomorphs are exclusive to a small number of grass species. However,
these studies further revealed the existence of a large number of fungal species capable
of infecting plants without causing symptoms, and at the present time, no endophytefree plant species has been reported (Stone et al. 2004; Arnold 2007; Sieber 2007; Hyde
& Soytong 2008).
Depending on the extent of plant colonization and transmission mechanisms,
several schemes for classifying endophytic species have been proposed (e.g. White
1988; Saikkonen et al. 1998; Schulz & Boyle 2005; Rodriguez et al. 2009). Interactions
between Epichloë and Neotyphodium species and grasses, sometimes referred as
systemic, clavicipitaceous, balansiaceous, type 1, or epichloë endophytes, constitute
well studied and documented examples of plant-fungal symbioses (Clay & Schardl
2002; Schardl et al. 2004; Kuldau & Bacon 2008; Rodriguez et al. 2009). These
endophytes are mutualistic, colonize the intercellular space of leaves and stems in a
systemic manner, and are vertically transmitted by seed. In contrast, little is known
about symbiotic interactions in a large number of other endophytic species which appear
to be non-systemic, not vertically transmitted, and have been reported in wild and
cultivated cereals and grasses. Although there are some examples of non
clavicipitaceous endophytes that systemically infect their hosts (i.e. Fusarium
verticillioides; Bacon & Hinton 1996), in this article we will use the name “nonsystemic endophytes” to refer to all those endophytic species which do not belong to the
Epichloë or Neotyphodium genera. Our objective is to review and synthesize some of
the current knowledge about the diversity and characteristics of non-systemic
endophytic species associated with grasses.
Incidence, species richness and abundance in assemblages of endophytes
Many surveys have shown that most or all individuals of a grass species are
infected by fungi causing visible external symptoms and by non systemic endophytes
(Sprague 1950; Peláez et al. 1998; Wirsel et al. 2001; Sánchez Márquez et al. 2007;
2008; 2010; Porras Alfaro et al. 2008; Higgins et al. 2010). However, in habitats
inhospitable for plant or fungal life, like polar ecosystems, the incidence of endophyte
infections seems to be relatively lower in grasses and in other plants (Arnold & Lutzoni
2007; Rosa et al. 2009). Plants in environments where exposure to aerial fungal
inoculum is reduced (i.e. indoors) may also show a lowered incidence of endophyte
infections.
The results of endophyte surveys depend on the methods of fungal detection or
isolation used. Until the recent application of direct sampling of host plant DNA and
amplification of fungal marker sequences (i.e. Vandenkoornhuyse et al. 2002; Neubert
et al. 2006; Porras Alfaro et al. 2008; Higgins et al. 2010), most surveys had been
designed to detect species diversity via culturing, and this generally precludes the
detection of unculturable species, such as obligate parasites, e.g, rusts, powdery
mildews. Also, the culture media and dissection protocols used in a particular study may
exclude some culturable fungi, and fast growing fungi may be over represented (Bills &
Polishook 1991; Hyde & Soytong 2008).
The endophytic mycobiota associated with a given grass species usually consists
of a relatively large number of fungal species (Table 1; Supplementary Table I). A very
unequal distribution of isolate richness among fungal species is typical of endophyte
surveys (Figure 1). A few dominant endophytic taxa are commonly found in different
plants and locations. In temperate grasses, among the group of dominant endophytic
taxa there are Alternaria, Acremonium, Cladosporium, Penicillium, Epicoccum, and
Aureobasidium spp. (Table 1; Supplementary Table I). Dominant species of temperate
and tropical grasses seem to have low host specificity, because they occur on multiple
grass species, as well as in non-grass hosts (Stone et al. 2004; White & Backhouse
2007; Porras Alfaro et al. 2008; Sánchez Márquez et al. 2008; Higgins et al. 2010;
Khidir et al. 2010).
At the other extreme of the species abundance distribution in a survey, there are
rare species that are represented by single isolates (Figure 1). These fungi are found
only occasionally in a given plant species. Such interactions between plants and rare
species possibly only occur when a given plant and fungal phenotype are confronted
and may represent unstable associations. Such rare species often constitute more than
half of the endophytic species identified in a survey (Neubert et al. 2006; Sánchez
Márquez et al. 2007; 2008; 2010; Herrera et al. 2010; Higgins et al. 2010)
(Supplementary Table I). The existence of a large number of rare species in endophyte
assemblages complicates the detection of host specific endophytes. Only species
isolated from several individuals and absent from other sympatric plant species can
give inferences of host-specificity (White & Backhouse 2007; Sánchez Márquez et al.
2008).
The number of species identified in an endophytic assemblage depends on the
number of plants and locations analysed. This relationship can be visualized in species
accumulation curves, where the rate of identification of endophytic species in
proportion to the number of plants analysed can be estimated. Curves plotted with data
from grass endophyte surveys are usually non-asymptotic (Figure 2) (Sánchez Márquez
et al. 2007; 2008; 2010; White & Backhouse 2007; Higgins et al. 2010), a characteristic
of very species-rich systems, like insects in tropical forests (Novotny et al. 2004). These
curves indicate that an increment in the number of plants analysed would yield
additional fungal species. Species accumulation curves with steep slopes (see Figure 2)
suggest that a new fungal species would be discovered even when one or a few more
plants are analysed. Furthermore, extrapolations of the total number of species of
endophytes from survey data may be unreliable. Estimators of the total number of
species (i.e. Chao1, Jacknife, etc.) often fail to attain an asymptotic shape that would
give an estimate of the maximum number of species of a community (Colwell &
Coddington 1994; Sánchez Márquez et al. 2008).
The non asymptotic shape of species accumulation curves is caused by rare
species: if rare species are excluded from a survey data set, and used to plot a species
accumulation curve, the resulting curve approaches an asymptote (Figure 2). This
change in curve shape shows that rare species drive upwards species accumulation
curves in endophyte surveys. Therefore, after an intensive initial sampling, new species
found in a survey by increasing sampling effort are likely to be rare or coincidental
species. In addition, the asymptotic shape of plural species accumulation curves
indicates that most species commonly associated with a grass species can be identified
in a survey.
Regarding taxonomy, except for a few basidiomycetes and zygomycetes, most
known endophytic taxa of grasses belong to the Ascomycota (i.e. all references from
Table 1). This phylogenetic bias is also common to endophytic assemblages of other
plant families (Stone et al. 2004; Arnold 2007). Within the Ascomycota, the orders
Pleosporales and Hypocreales dominate in terms of species abundance in temperate
grasses (Figure 3; Porras Alfaro et al. 2008). In contrast, in tropical grasses Xylariales
and Phyllachorales were the most common taxa (Higgins et al. 2010), these orders are
also common in the mycobiota of trees and woody plants (Arnold & Lutzoni 2007;
Sieber 2007). Although mycorrhizal species belonging to the Glomeromycota have been
extensively studied as fungal symbionts of plants, several studies indicate that
endophytes belonging to the Ascomycota and Basidiomycota are much more diverse
and frequent in grass roots than mycorrhizae (Vandenkoornhuyse et al. 2002; Porras
Alfaro et al. 2008; Sánchez Márquez et al. 2010).
A significant proportion of the isolates obtained in endophyte surveys are
difficult to classify into a known taxonomic group; these isolates do not sporulate in
culture, and nucleotide sequences used for their classification do not match those of
known taxa present in nucleotide databases. This suggests that many of these isolates
may constitute unknown species, and that endophytic ecosystems may harbor an
important fraction of the numerous undocumented fungal species (Hawksworth &
Rossman 1997). Alternatively, many of these grass-associated fungi may have been
described during the 19th and 20th centuries (Sprague 1950; Farr et al. 1989; Hyde et al.
2002), but because the majority of the fungi described from grass species still have not
been characterized by contemporary methods, phylogenetic marker sequences are
lacking from databases, and this can lead to a false perception of taxonomic novelty.
Factors affecting the distribution of endophytic species
The composition of the mycobiota of grasses varies substantially among
individual plants and locations. For example, in a survey of the grass Ammophila
arenaria across 12 locations, the mean number of endophytic species per plant was 1.8,
the species number per location was 12.5, and in all locations 75 species were identified
(Sánchez Márquez et al. 2008). Similar variation among sites and habitats has been
detected in the endophytic mycobiota of other grasses (Wirsel et al. 2001; Wilberforce
et al. 2003; Neubert et al. 2006; White & Backhouse 2007). The positive correlation
between spatial complexity and species richness indicates that -diversity, a diversity
component related to the amount of change in species composition from one individual
or location to another, is an important factor determining the structure of endophytic
mycobiota (Whittaker et al. 2001). Clay (1995) measured a positive correlation between
the geographical ranges of cereal and forage species and the numbers of their associated
fungal pathogens. Similar geographic correlations appear to operate with the endophyte
richness of grass species.
Differences in endophytic species richness can be observed between different
plant organs. Leaves and roots, or leaves and rhizomes did not differ significantly in
species richness in some grasses (Sánchez Márquez et al. 2008; 2010; Herrera et al.,
2010), but roots were richer than leaves in others (Neubert et al. 2006; Su et al. 2010).
However, the similarity of the mycobiota from different locations was greater for aerial
than for underground plant organs (Neubert et al. 2006; Sánchez Márquez et al. 2010).
This suggests that -diversity may be greater for the mycobiota of underground organs,
and different soils, with their biotic and abiotic components, may have more favourable
or restrictive influences than the above ground environment for some endophytic
species. In other words, different soils could act as different habitat filters for
endophytes (Saunders et al. 2010).
There is some evidence indicating that differences in leaf dimensions,
anatomical structure (i.e. degree of lignification), or chemical compounds between and
within grass species might affect the diversity of fungi found in them (Wong & Hyde
2001; Sánchez Márquez et al. 2008; Saunders et al. 2010).
During a survey of four temperate grasses, the number of species isolated was
greater in winter than in other seasons (Figure 4; Sánchez Márquez 2009). This seasonal
pattern might be caused by the accumulation in time of horizontally transmitted
endophytes in aerial tissues. In temperate grasses, leaves tend to die with summer
drought or the onset of winter. The first endophytic infections would start in new leaves
produced in the spring, then, as these leaves grow older, their exposure to aerial and
rain-dispersed endophytic inoculum increases, resulting in an accumulation of
endophytes in older leaves. In cocoa it has been shown that as leaves age their
endophyte load increases (Arnold et al. 2003). The longer life of roots compared to that
of leaves in arid grasslands might also explain the greater colonization of underground
tissues observed in Stipa grandis (Su et al. 2010).
Ecological functions of endophytes
Little is known about the ecological role or the outcome of the interactions
between most non systemic endophytic species and their grass hosts. Given the large
number of species of both plants and fungi involved, a wide spectrum of interaction
types, ranging from antagonism to mutualism can be expected. The possibility of
endophytes being latent pathogens, latent saprophytes, or mutualists is discussed next.
Latent pathogens
During their period of latency plant pathogens behave as endophytes. How many
latent pathogens are present in the endophytic mycobiota of a given plant species can be
estimated by comparing lists of its pathogens (Sprague 1950; Farr et al. 1989) and
endophytes. Out of 109 taxa identified as endophytes of Dactylis glomerata, only 12
coincided with a list of 68 pathogens of this grass (Sánchez Márquez et al. 2007). In
Phragmites australis only Fusarium and Phoma were common to endophyte and
pathogen lists (Peláez et al. 1998; Wirsel et al. 2001), and about 7% of the endophytic
taxa identified in roots of Bouteloua gracilis corresponded to known pathogens (Porras
Alfaro et al. 2008). Similarly, only a few of the hundreds of fungal species described on
bamboo are pathogens (Hyde et al. 2002). Although the pathogen lists compiled for the
above grasses might be incomplete, the relatively small overlap between pathogens and
endophytes suggests that pathogens constitute only a small fraction of endophytic
mycobiotas. A similar conclusion can be derived from studies of the mycobiota
associated to non grass hosts (Bills & Polishook 1986). Therefore, the capability to
penetrate and infect plant hosts is not a feature unique to pathogens, but seems to be
widespread among fungal species.
The capability of many fungal species to penetrate and establish within plant
tissues, and the ubiquity of endophytes somehow contradict current models of plant
defence against fungi. In these models, plant receptors help to detect fungi through
several different mechanisms, including some which are non-specific, like chitin
receptors (Jones & Dangl 2006). After a fungus is detected, plant defence mechanisms
are activated, although some fungi might have effectors capable of inhibiting plant
defence responses (de Jonge et al. 2010). The ubiquity of endophytic infections
suggests that the capability to override plant detection or defence mechanisms, at least
temporarily, might be widespread among fungi. Schulz & Boyle (2005) proposed that a
balance between fungal virulence and host defence permits endophytic infections;
however, mechanisms responsible for modulating these processes remain unknown.
Latent and facultative saprophytes
The apparently quiescent lifestyle of some non systemic endophytes may transition as a
consequence of the senescence and death of the host tissue. For example in Spain, after
the grass growing season ends with summer drought, and with the onset of winter rains.
Under those circumstances mycelia of some endophytes may colonize larger areas of
tissue and sporulate, completing their life cycle. Such endophytes could be considered
“latent saprophytes”, and it has been shown that they constitute a fraction of endophytic
mycobiotas in non grass hosts (Promputtha et al. 2007).
In Dactylis glomerata, Lolium perenne, and other grasses some of the taxa
sporulating during the first phases of senescence are Cladosporium, Epicoccum,
Alternaria, and Phaeosphaeria, which often sporulate over a significant extent of the
aerial plant (Webster, 1956; Thomas & Shattock 1986; Wong & Hyde 2001). The same
fungi are also some of the dominant endophytes of Dactylis and other grasses (Table 1;
Sánchez Márquez et al. 2007). This pattern indicates that these dominant endophytic
species of grasses have an intimate relationship with the host plant and become more
active after host senescence, behaving as latent saprophytes (Wicklow & Poling 2009).
An analogous set of latent saprophytes exist in trees; wood decay fungi have been
detected as endophytes in several tree species (Crozier et al. 2006; Parfitt et al. 2010).
Endophytes respond differently to active or inactive tissues, Abdellatif et al. (2009)
have shown that the morphology of endophytic hyphae and its cells, as well as the
patterns of root colonization change remarkably in living or dead wheat roots.
The spores of latent saprophytes such as Alternaria, Cladosporium, and
Penicillium are ubiquitous components of air particles in numerous environments (Aira
et al. 2007; Bogomolova & Kirtsideli 2009), and have been linked to respiratory
diseases in humans (Portnoy et al. 2008). Therefore, in order to respond to this threat, it
is important to understand that some of the most abundant fungal species in air samples
worldwide begin their life cycle as endophytes and sporulate as their host tissues die.
Several endophytic species associated with roots of Bouteloua gracilis were
common coprophilic fungi (Porras Alfaro et al. 2008), and endophytes of non-grass
hosts were shown to be viable after passing through the gut of insect herbivores
(Devarajan & Suryanarayanan 2006). These observations suggest that the coprophilous
habit is an alternate phase of the life cycle for some endophytic fungi, and that certain
coprophilic fungi might have coevolved with grazing animals and plants to establish an
endophytic life cycle bridge in stems, seeds or roots (Porras Alfaro et al. 2008).
Similarly, some endophytic Fusarium species isolated from grass stems can persist in
soil, making a transition to a saprobic phase in the life cycle that will allow the
inoculation of new hosts (Walsh et al. 2010).
Mutualistic interactions
Some of the best known mutualistic interactions between plants and fungi are
those of grasses and clavicipitaceous endophytes. In these symbioses, fungal alkaloids
and other factors increase plant performance under biotic and abiotic stress (Clay &
Schardl 2002; Schardl et al. 2004; Kuldau & Bacon 2008; Rodriguez et al. 2009). These
cases initiated an interest in the application of systemic endophytes for agricultural plant
improvement. In contrast, few naturally occurring mutualistic interactions between
grasses and non systemic endophytes have been revealed so far, but there is enough
evidence from other plant-endophyte systems to consider that the search for interactions
of this type might be fruitful (Zabalgogeazcoa 2008).
Several studies support the idea of endophytes having a role in the adaptation of
grasses to suboptimal or early succession environments. For example, the survival in
geothermal soils of the grass Dichanthelium lanuginosum is possible because of its
association with a Curvularia root endophyte (Redman et al. 2002). An increment in
drought and salt tolerance in Leymus mollis, a coastal grass, is due to its association
with an endophytic strain of Fusarium culmorum. This endophyte was also mutualistic
when inoculated in tomato and rice (Rodriguez et al. 2008). Increased biomass
production was observed in Phragmites australis infected by a Stagonospora sp.
capable of systemic colonization and seed transmission (Ernst et al. 2003). Acremonium
implicatum is another seed transmitted endophyte of Brachiaria species, which might
be implicated in leaf spot resistance (Abello et al. 2008). Fusarium verticilliodes is a
widespread symptomless endophyte of maize and other plants. The fungus is capable of
transforming and detoxifying benzoxazolinone allelochemicals that provide to maize
natural resistance from pathogenic bacteria, fungi, and insects (Glenn et al. 2001). This
mechanism may allow for an endophytic coexistence with maize, resulting in a
competitive advantage for resistant fungi (Glenn et al. 2003). In some cases, the
artificial infection of cultivated grasses with non specific endophytes obtained from
plants of other families has yielded beneficial effects. For instance, several species of
root endophytes artificially inoculated in barley effectively competed with pathogens for
root colonization, and increased the tolerance of this crop species to salinity, and leaf
and root fungal pathogens (Waller et al. 2004; Maciá Vicente et al. 2008; 2009).
Mutualistic interactions might also occur with several entomopathogenic taxa
like Beauveria, Torrubiella, Metarrhizium, and Tolypocladium, which have been
frequently isolated as endophytes from grasses and other plant species (Vega et al.
2008; Sánchez Márquez et al. 2010). Whether these endophytic entomopathogenic
fungi play a role protecting plants against insect herbivores is being questioned under
the bodyguard hypothesis, which postulates that plants may favour or retain natural
enemies of their herbivore pests (Quesada Moraga et al. 2009; Vega et al. 2009).
Endofungal microorganisms in endophytes
Associations of plants with endophytes are ubiquitous, and associations of
endophytes with microorganisms like bacteria or viruses also seem to be quite frequent.
The presence of RNA viruses is relatively common among endophytic species of
grasses, and in some fungal species the incidence and cellular concentrations of virus
are very high (Romo et al. 2007; Herrero et al. 2009). Unlike animal or plant viruses,
which are often pathogenic, mycoviral infections rarely have been associated to obvious
phenotypic changes in fungal hosts. The only exceptions are viruses that cause
hypovirulence in some pathogens (i.e. hypoviruses in Cryphonectria parasitica) or
disease in a mushroom species (Romaine & Goodin 2002; Ghabrial & Suzuki 2009). In
endophytes an interesting exception to the cryptic nature of mycoviral infection was
found in the case of a mutualistic mycovirus infecting the root endophyte Curvularia
protuberata. Virus infection of the endophyte was shown to be beneficial for its host
grass, increasing its thermal tolerance (Marquez et al. 2007).
Endofungal bacteria have been detected in several endophytic species isolated
from different hosts. How these bacteria affect their hosts fungi and the association with
a plant host is not known yet, but some endophytes were unable to grow in artificial
media containing antibiotics (Hoffman & Arnold, 2010). In addition, some fungal
toxins originally thought to be produced by fungi have been found to be produced by
endofungal bacteria (Lackner et al. 2009). Therefore, the presence of bacteria in
endophytes might have a prominent role in plant-endophyte symbioses.
Conclusions, questions and future directions
The study of non systemic grass-endophyte associations presents a unifying
challenge to biologists striving to understand the interdependency of fungal and plant
biology. Until relatively recently, pathogens and mycorrhizae have been the most
studied plant-associated fungi. However, endophytes seem to represent a larger, more
diverse and ubiquitous group which challenges our perception of how common and
intimate are the associations between the earth’s plants and fungi.
Field surveys suggest that possibly thousands of fungal species, mostly
ascomycetes, behave as non systemic endophytes in grasses. In contrast to the well
known Epichloë and Neotyphodium associations with grasses, there is lagoon in the
knowledge about the life cycles of most non-systemic endophytes and the effects of
their presence in their grass hosts. The elucidation of the life cycles of endophytic
species will help to understand their ecological functions. Life cycle elucidation starts
with field observations of fungi, their phenology and symptoms on their host plant, and
culturing them when possible. Hosts can be reinfected to satisfy Koch’s postulates.
Many species seem to have alternative lifestyles as saprophytes, coprophiles,
invertebrate pathogens, latent pathogens, or pathogens of other host genotypes or
species, and their ecological role might be significant. Recognizing how and when these
fungi sporulate and are transmitted to other hosts is essential to understanding their life
cycles and ecosystem function. For example, the spores of some dominant and
generalist grass endophytes like Alternaria, Cladosporium, Penicillium and other taxa
are ubiquitous in the earth’s atmosphere; these fungi characteristically sporulate when
host tissues senesce and die. It has been hypothesized that fungi might have been a
selective force in the evolution of avian and mammalian endothermy (Robert &
Casadevall 2009; García-Solache & Casadevall 2010), and the massive atmospheric
spore load from the above mentioned endophytes could be involved in such processes.
Presently, very few examples of the beneficial effects of non-systemic
endophytic species are known. Further research on endophyte effects on host
performance might produce applications useful for plant improvement and potential
alternatives to chemical disease and pest control. Plant pathogens only represent a small
fraction of the endophytic mycobiota of grasses. The fact that a myriad of endophytic
species are able to override plant defence mechanisms challenge the generalities and
accuracy of current plant defence models. In this context several interesting questions
remain to be answered. Why and how generalist taxa like Alternaria or Cladosporium
can infect so many hosts and species? Does host specialization drive speciation within
these taxa? Are rare endophytes particular genotypes capable of infecting a particular
host, or are they simply accidents of spore dispersion and functionally dead ends to the
life cycle? Research into this area will surely be of interest for the fields of plant
pathology and fungal natural history.
Endofungal microorganisms like viruses and bacteria occur in many endophytic
species, and these third partners in endophytic symbioses do not seem to be deleterious
to their hosts. Little is known about fungus-virus or fungus-bacteria interactions,
furthermore, these interactions are likely to be expressed in plant hosts. The study of
such interactions at an organismal and molecular level is largely undeveloped, but it
will very likely shed light in the complex area of tritrophic interactions.
Endophytes have been used as sources of antimicrobials and other biologically
active secondary metabolites with pharmacological and agrochemical applications (Bills
et al. 2009; Suryanarayanan et al. 2009). In spite of the sustained awareness of their
biosynthetic capacity, attempts to develop a hypothetical framework, parallel to that of
the Clavicipitaceae endophytes, explaining their chemical protective benefits to their
plant hosts have been less than satisfying because of difficulties in assessing the in
planta growth and metabolism of cryptic endophyte infections. Colonization of plant
organs and competition with other fungi may be a situation where the production of
antagonistic metabolites is positively selected. For example in grasses, the antimicrobial
activities of pyrrocidines from the maize endophyte Acremonium zeae appear to
augment host defences to fungal and bacterial pathogens (Wicklow et al. 2005;
Wicklow & Poling 2009). These and the previous observations indicate that in general,
fungi have a role in the way grasses and other plants respond and function in different
environments.
Acknowlegements
We are grateful for comments and suggestions to Pedro Gundel and Soledad Sacristán.
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Table 1. Species richness and dominant taxa observed in surveys of the non systemic
endophytic mycobiota in several grass species.
Grass
Ammophila arenaria
Bamboo spp.
Botriochloa macra
Bouteloua gracilis
Dactylis glomerata
Deschampsia anctartica
Elymus farctus
Festuca arizonica
Holcus lanatus
Hypharrhenia hirta
Oryza
Phragmites australis
Phragmites australis
Stipa grandis
Stipa tenacissima
Triticum aestivum
Zea
11 tropical grasses
a
Number
of species
75
60
65
51
109
5
54
14
134
57
>11
32
>20
34
38
213
23
94-124
Three most abundant taxa
Dominant taxaa
Reference
Alternaria , unknown sp., Podospora
Xylariales
Alternaria, Periconia, Phoma
Pleosporales, Sordariales, Agaricales
Cladosporium, Helgardia, Acremonium
Alternaria, Phaeosphaeria, unidentified sp.
Alternaria, Podospora, Acremonium
Neotyphodium, unidentified spp.
Alternaria, Cladosporium, Penicillium
Nigrospora, Periconia, Alternaria
Alternaria, Cladosporium Epicoccum
Alternaria, Sporormiella, Rhizoctonia
Microdochium, Cladosporium, Trichoderma
Pyrenopora,Alternaria, Phialophora
Alternaria, Sporormiella, Rhizoctonia
Alternaria, Epicoccum, Idriella
Alternaria, Aureobasidium, Acremonium
Xylariales, Halosphaeriales, Phyllachlorales
Sánchez Márquez et al. 2008
Morakotkarn et al. 2007
White & Backhouse 2007
Porras Alfaro et al. 2008
Sánchez Márquez et al. 2007
Rosa et al. 2009
Sánchez Márquez et al. 2008
Schultess & Faeth 1998
Sánchez Márquez et al. 2010
White & Backhouse 2007
Fisher & Petrini 1992
Peláez et al., 1998
Wirsel et al. 2001
Su et al. 2010
Peláez et al. 1998
Sieber et al. 1988
Fisher et al. 1992
Higgins et al. 2010
Figure 1. Rank-order plot showing the distribution of 77 species of foliar leaf
endophytes of Holcus lanatus in terms of the number of isolates of each species.
Species represented by more than 10 isolates were considered dominant, species with 2
or more isolates plural, and species represented by a single isolate were considered rare.
This very unequal distribution of isolate richness is typical of grass mycobiotas.
Adapted from Sánchez Márquez et al. (2010)
Number of isolates
30
20
dominant species (>10 isolates)
10
plural species (2 isolates)
rare species (1 isolate)
0
0
10
20
30
40
50
60
Species ranked by their abundance
70
80
Figure 2. Species accumulation curves of a survey of foliar endophytes in Holcus
lanatus. Curve (a) was obtained with data from all 77 taxa; its non asymptotic shape is
typical of species-rich systems, similar curves are routinely obtained in endophyte
surveys of grasses and other plants. When rare species represented by a single isolate
are withdrawn from the data set, the resulting curve (b) is asymptotic, indicating that
most fungal species commonly associated to this grass were identified in the survey,
and that rare species are the factor driving upwards the all species curve. The vertical
arrow indicates the contribution of rare species to the number of species detected.
Adapted from Sánchez Márquez et al. (2010).
90
80
a
Number of fungal species
70
60
50
40
30
b
20
10
0
0
10
20
30
40
50
60
Number of plants analyzed
70
80
Order
Figure 3. Distribution in taxonomic orders of the endophytic species found in a survey
of four cold season grasses (Sánchez Márquez, 2009).
Ustilaginales
Saccharomycetales
Rhytismatales
Phyllachorales
Onygenales
Mortierellales
Microascales
Cystofilobasidiales
Corticiales
Agaricostilbales
Trichosphaeriales
Tremellales
Polyporales
Phyllachorales
Chaetosphaeriales
Sporidiobolales
Dothideales
Capnodiales
Agaricales
Incertae sedis
Helotiales
Eurotiales
Xylariales
Diaporthales
Sordariales
Hypocreales
Pleosporales
0
5
10
15
20
25
Number of species
30
35
Figure 4. Seasonal distribution of the number of endophytic species per plant sample
identified in four grasses. The trend to higher number of species in winter might be due
to the older age of the leaves in this season (Sánchez Márquez, 2009).
Endophytic species / plant
4
1: spring
2: summer
3: fall
4: winter
4
3
4
2
3
3
4
1
2 3
2
4
1 2
3
1
1
1
0
Dactylis
glomerata
Ammophila
arenaria
Elymus
farctus
Holcus
lanatus
Supplementary material
Supplementary table I. Fungal taxa and number of isolates obtained as endophytes in
surveys of four temperate grass species in Spain (Sánchez Márquez, 2009; Sánchez
Márquez et al. 2007, 2008, 2010).
Fungal species
Ascomycetes
Alternaria spp.
Cladosporium spp.
Penicillium spp.
Acremonium spp.
Epicoccum spp.
Podospora spp.
Phaeosphaeria spp.
Aureobasidium pullulans
Arthrinium spp.
Acremonium strictum
Drechslera spp.
Chaetomium spp.
Curvularia inaequalis
Leptosphaeria spp.
Helgardia sp.
Cordyceps bassiana
Preussia australis
Microdochium bolleyi
Phomopsis spp.
Pestalotiopsis spp.
Aspergillus tubingensis
Fusarium spp.
Fusarium oxysporum
Gaeumannomyces cylindrosporus
Phialemonium dimorphosporum
Helotiales spp.
Chaetomium globosum
Coniothyrium cereale
Epichloë typhina
Stemphylium solani
Acremonium alternatum
Gliomastix murorum
Microdochium phragmitis
Nigrospora oryzae
Torrubiella confragosa
Fusarium culmorum
Glomerella sp.
Hypoxylon sp.
Leptodontidium orchidicola
Microdochium sp.
Periconia macrospinosa
Trichoderma viride
Ulocladium sp.
Pleosporales spp
Chaetomium funicola
Colletotrichum spp.
Diaporthe viticola
Emericellopsis sp.
Epichloë clarkii
Host grass
Dactylis Ammophila
Elymus
glomerata
arenaria
farctus
15
21
34
3
15
13
17
0
5
17
5
8
0
3
18
3
0
0
2
0
0
8
3
0
0
0
0
5
8
2
0
0
6
0
1
4
3
0
5
0
1
3
1
0
1
0
0
0
0
62
5
6
27
6
17
1
2
9
4
0
2
4
4
0
4
2
8
2
4
0
0
0
5
1
5
1
0
0
2
1
5
0
2
2
0
0
0
0
0
0
2
0
2
0
0
0
0
0
66
1
4
16
1
7
2
2
3
6
2
4
4
8
0
8
14
5
1
7
0
0
0
1
0
4
0
1
0
2
2
0
0
0
3
0
0
5
0
0
0
0
0
1
0
0
0
1
0
Holcus
lanatus
Total isolates
92
68
47
22
33
16
12
27
13
2
18
9
14
4
0
2
0
0
7
0
10
2
6
3
8
0
7
2
0
2
3
1
0
4
0
1
2
0
0
5
4
0
4
2
3
4
4
3
4
235
95
91
68
55
53
32
31
30
29
25
23
22
19
18
17
16
13
12
11
10
10
9
9
9
9
8
8
8
8
6
6
6
6
6
5
5
5
5
5
5
5
5
5
4
4
4
4
4
Fusarium equiseti
Fusarium poae
Fusarium sporotrichioides
Fusarium tricinctum
Leptodontidium sp.
Lophodermium sp.
Paecilomyces spp.
Stagonospora spp.
Thielavia sp.
Sordariales sin identificar
Acremonium cyanophagus
Discula quercina
Drechslera biseptata
Drechslera dactylidis
Eupenicillium spp.
Nigrospora sp.
Plectosphaerella cucumerina
Podospora decipiens
Xylaria sp.
Coelomycete spp.
Aspergillus terreus
Botryosphaeria dothidea
Cochliobolus sativus
Coniochaeta ligniaria
Cyathicula sp.
Dactylaria sp.
Davidiella tassiana
Debaryomyces hansenii
Engyodontium album
Gaeumannomyces graminis
Helgardia anguioides
Lachnum sp.
Myrothecium sp.
Neofabraea alba
Oidiodendron sp.
Periconiella sp.
Phialocephala spp.
Phialophora sp.
Phoma pinodella
Phoma sp.
Pyrenochaeta sp.
Schizothecium sp.
Sordaria macrospora
Trichocladium sp.
Valsa fabianae
Valsa sordida
Valsa sp.
Verticillium nigrescens
Xylariales sp.
Anthostomella eucalyptorum
Ascochyta sp.
Aspergillus fumigatus
Aspergillus niger
Aspergillus versicolor
Auxarthron conjugatum
Biscogniauxia mediterranea
Botryosphaeria australis
Calycina herbarum
Chaetosphaeria sp.
Chloridium sp.
1
1
0
0
0
0
2
2
0
0
0
1
3
3
3
1
0
3
0
0
1
0
0
0
2
0
2
0
1
0
0
0
0
0
1
0
0
0
0
1
0
0
2
0
0
0
2
0
0
0
1
1
0
0
1
0
0
1
0
1
0
0
0
0
0
3
0
2
1
4
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
2
0
1
1
0
1
0
0
0
0
2
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
0
0
0
0
0
0
0
1
0
2
3
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
1
0
0
2
0
0
1
1
1
0
0
0
0
0
0
0
0
1
0
3
3
4
4
4
1
2
0
0
0
3
2
0
0
0
2
1
0
1
0
1
2
2
2
0
0
0
1
0
2
1
2
2
1
1
0
1
2
2
1
1
1
0
2
0
2
0
1
1
0
0
0
0
0
0
1
1
0
0
0
4
4
4
4
4
4
4
4
4
4
3
3
3
3
3
3
3
3
3
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
Coniochaeta sp.
Cordyceps sinensis
Creosphaeria sassafras
Cryptodiaporthe salicella
Cryptosporiopsis sp.
Cylindrotrichum sp.
Diaporthe melonis
Didymella bryoniae
Discostroma sp.
Drechslera andersenii
Drechslera erythrospila
Eurotium amstelodami
Eutypella cerviculata
Fimetariella rabenhorstii
Fusarium solani
Fusarium subglutinans
Gabarnaudia sp.
Gibberella avenacea
Glarea sp. / Cordyceps sp.
Gnomonia petiolorum
Guignardia philoprina
Helicosporium pallidum
Hormonema sp.
Hypoxylon fuscum
Kabatiella sp.
Lachnum pygmaeum
Leptosphaerulina chartarum
Lophiostoma sp.
Macrophomina phaseolina
Microdochium nivale
Minimidochium sp.
Mycoarthris corallinus
Petriella guttulata
Phaeoacremonium rubrigenum
Phialophora alba
Phoma exigua
Phoma herbarum
Phoma terrestris
Phyllosticta pyrolae
Pleurophoma cava
Podospora tetraspora
Podospora coprophila
Podospora tetraspora
Preussia isomera
Preussia minima
Preussia sp.
Pseudeurotium bakeri
Sagenomella sp.
Sarea sp.
Sordaria fimicola
Sporormia subticinensis
Stagonospora arenaria
Sydowia polyspora
Tolypocladium cylindrosporum
Trichocladium opacum
Verticillium sp.
Volutella ciliata
Xylariaceae sin identificar
Basidiomycetes
Cryptococcus victoriae
1
0
1
0
0
1
0
0
0
1
0
1
0
0
0
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
1
0
1
0
0
0
0
1
1
0
0
0
0
1
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
0
1
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
1
1
0
1
1
1
0
1
0
1
0
1
1
1
0
1
1
1
1
0
1
0
0
1
0
0
1
1
1
1
0
1
0
1
1
0
1
0
0
1
1
1
1
0
0
0
1
1
0
0
1
1
0
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
2
0
4
6
Cryptococcus spp.
Coprinellus disseminatus
Laetisaria arvalis
Pseudozyma aphidis
Trametes versicolor
Agrocybe pediades
Ceratobasidium sp.
Coprinellus radians
Coprinus micaceus
Cryptococcus paraflavus
Cryptococcus podzolicus
Cystofilobasidium macerans
Dioszegia hungarica
Kondoa aeria
Mastigobasidium intermedium
Meira sp.
Mycena sp.
Phlebia radiata
Rhodotorula bacarum
Rhodotorula glutinis
Rhodotorula minuta
Rhodotorula slooffiae
Tilletiopsis pallescens
Ustilago sp.
Zygomycetes
Cunninghamella elegans
Mortierella alpina
Mortierella sp. / Umbelopsis sp.
Mucor hiemalis
1
0
2
0
1
0
0
0
0
1
0
1
0
0
0
0
1
0
1
0
1
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
3
2
0
2
1
1
1
0
1
0
1
0
1
0
1
0
0
0
0
1
0
1
1
0
4
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
Unidentified species
18
39
11
71
139
TOTAL
311
270
213
661
1455