Advances in Research on Epichloë endophytes in Chinese Native Grasses

Epichloë fungal endophytes are broadly found in cool-season grasses. The symbiosis between these grasses and Epichloë may improve the abiotic and biotic resistance of the grass plant, but some Epichloë species produce alkaloids that are toxic for livestock. Therefore, it is important to understand the characteristics of the grass-Epichloë s symbiosis so that the beneficial aspects can be preserved and the toxic effects to livestock can be avoided. Since the 1990s, Chinese researchers have conducted a series of studies on grass-Epichloë symbiosis. In this review, we describe the current state of Epichloë endophyte research in Chinese native grasses. We found that more than 77 species of native grasses in China are associated with Epichloë endophytes. In addition, we review the effects of various Epichloë species on native grass responses to abiotic and biotic stress, phylogeny, and alkaloid production. We provide an overview of the study of Epichloë species on native grasses in China and directions for future research.

Various Epichloë species have been discovered in China, but have not been formally taxonomically described. There are two reasons for this lack of taxonomic data: (i) the limited number of researchers in this field and (ii) insufficient knowledge on the identification and classification of Epichloë species. To address the latter issue, Chinese researchers are establishing collaborations with international institutes. The topic of hybrid occurrence in Chinese Epichloë species is not discussed in-depth in this manuscript because few Epichloë species are confirmed to be of hybrid origin. However, known hybrid species from native grasses appear to have the same two ancestors, for two main reasons. First, researchers have only confirmed some Epichloë species crosses for the Epichloë bromicola × Epichloë typhina complex. Second, hybrid species are distributed in the same and similar natural and geographic environments. Accordingly, these hybrid species underwent the same hybridization process, but are hosted by different grasses. This topic will be discussed in future reviews when more data are available on hybrid endophytes.
We have built a long-term collaboration with Prof. Christopher L. Schardl from the University of Kentucky and Prof. German Spangenberg from the Australian Academy of Technological Sciences and Engineering. With their help, two kinds of Epichloë endophytes in drunken horse grass were confirmed. The whole genome sequencing of an Epichloë endophyte in Festuca sinensis is near completion. These studies will push Epichloë research to a new level in China. We firmly believe that the research prospects with respect to Epichloë species are bright in our country.
Interestingly, Epichloë endophytes in natural grasses are morphologically diverse, e.g., the species that infect Achnatherum sibiricum (Wei et al., 2007) and Elymus species (Song et al., 2015b). Ren et al. (2009) isolated 484 Epichloë endophytes from seven populations of A. sibiricum in Inner Mongolia, China and detected five morphotypes that also exhibited different magnitudes of inhibition of Rhizoctonia solani, Fusarium oxysporum, Curvularia lunata, Cladosporium cucumerium, and Phomopsis vexans. Researchers have also detected morphological differences along an altitudinal gradient. Epichloë isolates from populations of Elymus above 3000 m present similar morphological traits, while Epichloë populations below 3000 m are morphologically variable (Song et al., 2015b). Asexual Epichloë endophytes below 3000 m tend to grow faster on potato dextrose agar than asexual Epichloë endophytes above 3000 m (Song et al., 2015b). In addition, a phylogenetic analysis showed that Epichloë endophytes above 3000 m form a clade, but isolates from regions below 3000 m belong to several clades (Song et al., 2015b).

Salt Stress
Plant cells are harmed by salt stress and do not intake sodium as an essential element for their physiology. Although plants have evolved several strategies to adapt to salt stress (Zhu, 2003;Dinneny, 2015), only a few studies have confirmed that Epichloë endophytes can increase salt tolerance in a grass host (Reza Sabzalian and Mirlohi, 2010). When Hordeum brevisubulatum was infected with Epichloë (EI), the grass exhibited significantly increased N, P, and K + concentrations, which led to an increase in total biomass. The Epichloë infection also reduced Na + accumulation in the EI plants compared to Epichloë-free plants (EF) (Song et al., 2015d). Based on this work, we inferred that salt tolerance could be further increased in grass-Epichloë symbiosis, which potentially provide a valuable resource for improved salt tolerance in crops.

Drought Stress
Compared to salt stress, crop plants are inclined to suffer from drought stress (Boyer, 1982). Studies have confirmed Epichloë endophytes play a vital role in increasing drought tolerance in EI grasses (Richardson et al., 1992;Clay and Schardl, 2002;Schardl et al., 2004). A relationship between increased drought tolerance and EI has been well documented in five EI grasses that are native to China. Under drought stress, EI Leymus chinensis had significantly more total biomass than EF L. chinensis, regardless of fertilizer levels . Peng et al. (2013) found that seed hydropriming treatment is an effective strategy to improve seed germination and plant growth in EI F. sinensis. Epichloë infection also increased the germination of Elymus dahuricus under different osmotic potential pressures, but germination success was variable among populations (Zhang and Nan, 2010). Several studies have shown that Epichloë infection can improve the relative fitness of grasses under drought stress (Faeth, 2002;Faeth et al., 2004;Iannone et al., 2012). Zhang and Nan (2007b) showed that EI E. dahuricus produced more biomass, more tillers, and taller plants under low water treatment, but EI had no influence on plant biomass in the high water treatment. However, in a study of EI A. sibiricum, the addition of fertilizer resulted in greater plant growth, but this advantage decreased under reduced water and/or nutrient availability (Ren et al., 2011). Moreover, Song et al. (2015e) demonstrated that asexual Epichloë endophyte infection can increase resistance to waterlogging stress in H. brevisubulatum. The effect of EI on drought tolerance seems to differ among grass species. It remains to be determined whether these effects are caused by the species of infectious Epichloë, the grass species, or other factors.

Other Abiotic Stress
Epichloë endophytes confer stress tolerance to native grasses in China and play a significant role in the survival of some plants in high-stress environments, such as cadmium (Cd)-contaminated soils and nutrient-depleted soils. Epichloë-infected A. inebrians (Zhang et al., 2010a,b) and E. dahuricus (Zhang et al., 2012a) had higher germination rates, more tillers, longer shoots and roots, and more biomass compared to EF plants in high Cd 2+ concentrations. There was no significant difference between EI and EF plants under low Cd 2+ concentrations, indicating that Epichloë infection was only beneficial to the growth and development of A. inebrians and E. dahuricus exposed to high Cd 2+ concentrations. Studies of nutrient acquisition in EI grasses have focused on the influence of nitrogen (N), since this element is a constituent of alkaloids in infected plants and is also one of the most important limiting resources for plant growth in general . It has been documented that increased N availability may change the relative availability of other nutrients, such as phosphorus (P) (Van Der Wouder et al., 1994). Li et al. (2012a) found that A. sibiricum-Epichloë associations are conditional on both N and P availability, but are more conditional on N than P. Changes in N allocation increase the photosynthetic ability of EI plants and also significantly increase their biomass. In addition, the benefits of Epichloë infection decline when nutrient availability decreases (Ren et al., 2011). Epichloë infection tends to reduce overall nitrogen concentration in A. sibiricum leaves, but causes the host to allocate significantly higher fractions of N to the photosynthetic machinery (Ren et al., 2011). Thus, EI plants have higher photosynthetic N use efficiency and shoot biomass than that of EF plants when fertilizer is limited . Song et al. (2015d) confirmed that EI H. brevisubulatum has lower ratios of C:N, C:P, Na + :K + and a higher ratio of N:P than EF plants under salt stress. According to Jia et al. (2014), the effects of EI on A. sibiricum suggest that the A. sibiricum host genotype has a stronger influence on the response to stress than the influence of Epichloë. They found that Epichloë infection did not positively affect general growth, physiology, or nutrient content of A. sibiricum, before or after clipping.

Pest Resistance
The grass-Epichloë symbiosis provides the grass host protection from herbivorous insects by producing alkaloids in the form of secondary metabolites (García Parisi et al., 2014;Thom et al., 2014). Aphid populations exhibit slow growth when feeding on grass infected with Epichloë species (Hartley and Gange, 2009;Saikkonen et al., 2010). However, Börschig et al. (2014) concluded that the effect of Epichloë endophytes on herbivores is generally weak and depends on the regional environmental context. They posit that more field research is necessary to detect the relative importance of Epichloë endophytes and environmental context on biotic interactions in grasslands (Börschig et al., 2014).
To date, insect resistance has only been reported for L. chinensis-E. bromicola, A. sibiricum-E. sibirica and A. inebrians-E. gansuensis associations in China. Jia et al. (2013) concluded that L. chinensis-E. bromicola and A. sibiricum-E. sibirica symbioses could diminish the negative effects of infection by Meloidogyne incognita. The researchers used a 72-h exposure to undiluted culture filtrates of the two endophytes and found L. chinensis infected with E. bromicola had an especially strong antagonistic effect on Meloidogyne infection. Similarly, Zhang et al. (2012b) found that A. inebrians infected with E. gansuensis reduced the survival of the aphids Rhopalosiphum padi, Tetranychus cinnabarinus, Oedaleus decorus, and Messor aciculatus under laboratory and field conditions. Additionally, they demonstrate that EI had an anti-herbivore effect on a wide range of arthropod groups (Zhang et al., 2012b).

Pathogen Resistance
Reports that EI grasses are resistant to diseases and pathogens are limited compared to evidence that EI increases pest resistance. Epichloë endophytes negatively impact the in vitro growth of plant fungal pathogens (White and Cole, 1985;Siegel and Latch, 1991). However, Sabzalian et al. (2012) found that EI tall fescue was not more resistant to powdery mildew (Blumeria graminis) than EF tall fescue. Yue et al. (2000) demonstrated that extracts from a wide range of Epichloë endophytes exhibited various degrees of antifungal activity and the greatest antifungal activity was detected from extracts of E. festucae and E. tembladerae. Li et al. (2007) confirmed that the fungi Bipolaris sorokiniana, Curvularia lunata, Fusarium acuminatum, and Alternaria alternate cause lesions on detached A. inebrians leaves, regardless of their status as EI or EF. When leaves were EF, the number and size of lesions caused by all pathogens were reduced compared to those on EI leaves. In addition, Xia et al. (2015) demonstrated that, in greenhouse conditions, EI reduced the ability of Blumeria graminis to colonize A. inebrians and enhanced the photosynthetic performance of host plants under pathogen stress or ameliorated host plant damage, to some degree (Xia et al., 2016). Zhou et al. (2015b) found that EI F. sinensis produced secondary metabolites that inhibited fungal pathogens, including Alternaria alternata, Bipolaris sorokiniana, Curvularia lunata, and Fusarium acuminatum. They found significant reductions in disease incidence and lesion size on EI detached leaves compared to EF leaves (Zhou et al., 2015b). Song et al. (2015f) found that E. bromicola from Elymus tangutorum exhibits antifungal activities against Alternaria alternata, Fusarium avenaceum, Bipolaris sorokiniana, and Curvularia lunata.

MOLECULAR IDENTIFICATION OF CHINESE EPICHLOË SPECIES
In the past, taxonomic identification of Epichloë endophytes relied on morphological features, e.g., colony morphology, colony growth rate, and spore type and size. Currently, allozyme profiles and molecular methods have been applied to Epichloë research and greatly aid in identification. Recent research combines morphological features and molecular data to identify Epichloë endophytes.
Although new Epichloë endophytes have been identified based on traditional morphology, this method has limitations when determining whether the Epichloë endophytes experienced hybridization events. Fortunately, DNA sequencing can help resolve this problem. To date, all putative Epichloë hybrids contain more than one copy of tubB and can be detected by allozyme analysis (Moon et al., 2004;Oberhofer and Leuchtmann, 2012;Leuchtmann et al., 2014;Iannone et al., 2015). For example, E. chisosa and E. coenophiala each have three copies , indicating they experienced multiple ancient hybridization events or subsequent gene duplication. Oberhofer and Leuchtmann (2012) found four new Epichloë species in Hordelymus europaeus using five enzymes; two were interspecific hybrids and the others were of nonhybrid origin.
Molecular markers can be used to identify new species and to estimate evolutionary relationships with phylogenetic trees. Molecular studies on Chinese Epichloë species have mainly been applied to identify new species. Various Epichloë species, e.g., E. stromatolonga (Li et al., 2006b;Ji et al., 2009), E. sinica ), E. sinofestucae ), E. liyangensis , and E. sp. (Han et al., 2012), have been described and exhibit natural symbioses with R. kamoji, Calamagrostis epigeios, Roegneria spp., F. parvigluma, P. pratensis ssp. pratensis, and F. myuros. These Epichloë species are native to China and were described based on host specificity, morphology, and molecular phylogenetic evidence. Zhang et al. (2009) identified a new Epichloë endophyte, E. sibirica (A. sibiricum), and three morphotypes based on morphological and phylogenetic analyses. They found that its ancestor was probably derived from E. sibirica (Zhang et al., 2009). Zhu et al. (2013) analyzed L. chinensis and found that its Epichloë associate is E. bromicola, which was classified into three morphotypes based on morphological features and phylogenetic analyses of tubB, tefA, and actG sequences. Additionally, a molecular phylogenetic study showed that E. gansuensis var. inebrians from Chinese A. inebrians is a unique and novel non-hybrid species (Moon et al., 2007).
Although some studies have examined the evolutionary relationships among Epichloë species, few have examined the phylogeny or co-evolution of Chinese Epichloë species and hosts. In the southern hemisphere, most asexual Epichloë species are the result of hybridization events between two sexual species (e.g., E. festucae and E. typhina) from the northern hemisphere (Gentile et al., 2005). These studies have looked at the extent of Epichloë gene flow between the Northern and Southern Hemispheres based on molecular data (Moon et al., 2002). Iannone et al. (2009) studied South American Epichloë endophytes from Bromus auleticus and found that E. tembladerae was a hybrid of the Northern E. festucae and E. typhina, but the ancestral E. typhina genotype was distinguished based on tubB and tefA. Schirrmann et al. (2015) used 15 microsatellites to assess the population structure of sympatric species in the E. typhina complex and found that host specificity and maladaptation of Epichloë hybrids to host grasses may act as reproductive isolation barriers in asexual Epichloë and therefore promote their speciation.
Notably, Kang et al. (2011b) analyzed the asymptomatic symbiosis between Roegneria and E. sinica and found no relationship between phylogeny and morphology in the E. sinica isolates. They concluded that E. sinica is a species complex that resulted from multiple, independent hybridization events (Kang et al., 2011b). In a comparison of genetic diversity in Epichloë species and their host plants, Zhang et al. (2010c) found approximately 4-7-fold greater diversity among Epichloë endophytes than among host plants based on SSR markers. This indicates more gene flow of Epichloë endophytes than hosts. The authors also state that Epichloë infection might confer selective advantages to A. sibiricum under certain conditions, which could help to maintain high-EI frequencies, even when their population structure would not suggest selection for EI (Zhang et al., 2010c). Song et al. found that Epichloë species likely originated in Eurasia, and Epichloë gene flow between the Western and Eastern hemispheres is common based on phylogenetic and network analyses (Song and Nan, 2015;Song et al., 2015a). They suspect that migratory birds or humans might have aided the dispersal of Epichloë endophytes from Eurasia to other continents (Song and Nan, 2015). Furthermore, Song et al. (2015c) analyzed Hordeumendophytes and Elymus-endophytes and found that Chinese Hordeum species likely contain two Epichloë endophyte species. One is also found in North American Elymus species and the other endophyte is found in Chinese Elymus species, indicating that Epichloë endophytes isolated from Chinese Hordeum are not host-specific. They proposed that Epichloë endophytes spread among different grass hosts by plant hybridization, and this could likely transform the hybrid offspring from EF status to EI status (Song et al., 2015c). This needs to be tested in future studies, but it would add further evidence to the hypothesis that asexual Epichloë endophytes are horizontally transmitted (Tadych et al., 2012;Wiewióra et al., 2015). Moreover, molecular phylogenetic studies based on tubB and tefA intron sequences have confirmed that E. gansuensis infected A. sibiricum and A. inebrians in China, indicating the potential of conidia cultures to mediate horizontal transmission .

ALKALOIDS
From an agronomic point of view, a negative aspect of the grass-Epichloë symbiosis is that some Epichloë produce ergot and indole-diterpene fungal alkaloids that are highly toxic for livestock (Clay and Schardl, 2002). Variability in the profile and level of alkaloids has allowed researchers to inoculate grass cultivars with selected Epichloë endophytes that are not toxic to livestock and still confer benefits to host plants. This has become a key strategy for breeding drought-, salt-, and pestresistant forage grasses (Gundel et al., 2013b;Johnson et al., 2013). A. inebrians is widely distributed in northern China and is commonly known as drunken horse grass because of its longrecognized toxic and narcotic effects on livestock, especially horses. Additionally, owing to the toxicity to livestock, recent research has shown that A. inebrians can protect biodiversity (Yao et al., 2015). These toxins are apparently caused by E. gansuensis (Li et al., 2004;Zhang et al., 2014). Epichloë-infected drunken horse grass contains high levels of the ergot alkaloids, ergine, and ergonovine (Miles et al., 1996;Li et al., 2006a). High alkaloid levels have also been confirmed in EI A. inebrians under salt or drought stress, with higher levels of ergonovine than ergine . Cytotoxic effects to animal muscle tissue have been described after the consumption of ergonovine and ergine (Zhang et al., 2014). The EI E. dahuricus only produces the alkaloid peramine. Production is seasonal; the concentration of peramine are highest in October and below detectable levels in June (Zhang and Nan, 2007a). Recently, Zhou et al. (2015a) evaluated the effects of temperature on ergot alkaloid production in three F. sinensis ecotypes and found that concentrations of ergine and ergonovine differed considerably in the three endophyte-infected ecotypes. They also found the ecotypes varied in their production of secondary metabolites, the bioprotective alkaloids ergine and ergonovine, in response to short-term cold stress. However, compared to recent research abroad , little is known about alkaloid production in Chinese native grasses using molecular methods. We hope to increase research in this area in the future.

CONCLUSIONS AND PERSPECTIVES
In this review, we briefly summarized progress in Epichloë endophyte research in China in the past 25 years. We found that more than 77 species of native grasses in China were infected with Epichloë species. To date, nine Epichloë species have been identified from Chinese native grasses. Additionally, seven have been confirmed as new Epichloë endophytes. Epichloë species originated in Eurasia based on the high species diversity in the area (Song and Nan, 2015). Unfortunately, many isolates from Chinese native grasses have not been identified to the species level. Therefore, to apply this precious resource, Chinese research should focus on taxonomical evaluations of Epichloë species from Chinese native grasses. In addition, Chinese studies have extensively examined abiotic and biotic resistance using Epichloë endophytes. However, little is known about Epichloë evolution, functional genomics, and comparative genomics. Nevertheless, we believe that Chinese researchers will intensify their efforts in these areas in the future.

AUTHOR CONTRIBUTIONS
HS wrote the article. ZN served as the principal investigator, facilitated the project, and assisted in manuscript preparation. QS and CX wrote and revised the paper. XL, XY, WX, YK, PT, and QZ explored literature and modified the article.