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Quantitative proteomics profiling of endophyte-specific response to colonization of L. perenne with epichloid strains. (A-C) Principal components analysis of L. perenne colonized with epichloid strains (AR1, AR37, E+, NEA2); dark bars denote 400 ppm CO 2 , light bars denote 800 ppm CO 2 . Shown are the means and standard errors. (D) Heat map of hierarchical clustering by Euclidean distance for plant proteins that differed among the epichloid strains and at normal (400 ppm) and elevated (800 ppm) CO 2 levels. (E) Heat map of hierarchical clustering by Euclidean distance for fungal proteins that differed among the epichloid strains and at normal (400 ppm) and elevated (800 ppm) CO 2 levels. (F,G) Venn diagrams of common and unique differences in plant and fungal proteins at 400 ppm CO 2 . (H,I) Venn diagrams of the number of common and unique differences in plant and fungal proteins at 800 ppm CO 2 . The numbers in F-I indicate the number of proteins with large differences in abundance, as evaluated by Student's t-tests, p < 0.05, FDR = 0.05, S0 = 1. Below each graph are the results from the corresponding ANOVA. We also report the Shannon information transformation, s.
Source publication
Perennial ryegrass (Lolium perenne) is the most cultivated cool-season grass worldwide with crucial roles in carbon fixation, turfgrass applications, and fodder for livestock. Lolium perenne forms a mutualism with the strictly vertically transmitted fungal endophyte, Epichloë festucae var lolii. The fungus produces alkaloids that protect the grass...
Contexts in source publication
Context 1
... 63 proteins (16 fungal and 47 plant) account for 36.5% (13.2%, 12.8%, and 10.5%) of all the protein abundance variation. Figure 6A-C shows separation of the fungal strains by CO 2 concentrations. For example, AR1 shows large separation by CO 2 concentrations on PC2 but not PC3, while NEA2 segregates by CO 2 concentrations on PC1 but not PC2 or PC3. ...
Context 2
... we examined the univariate responses of individual proteins that differed between CO 2 concentrations for at least one endophyte strain. Hierarchical clustering by Euclidean distance produced a heatmap demonstrating variability in protein abundance associated with CO 2 conditions and endophyte for the plant-derived proteins Figure 6D as well as the endophyte derived proteins Figure 6E. We performed FDR-corrected Student's t-test comparisons of the differences in protein abundances for each fungal strain at 400 and 800 ppm CO 2 . ...
Context 3
... we examined the univariate responses of individual proteins that differed between CO 2 concentrations for at least one endophyte strain. Hierarchical clustering by Euclidean distance produced a heatmap demonstrating variability in protein abundance associated with CO 2 conditions and endophyte for the plant-derived proteins Figure 6D as well as the endophyte derived proteins Figure 6E. We performed FDR-corrected Student's t-test comparisons of the differences in protein abundances for each fungal strain at 400 and 800 ppm CO 2 . ...
Context 4
... performed FDR-corrected Student's t-test comparisons of the differences in protein abundances for each fungal strain at 400 and 800 ppm CO 2 . We found a total of 133 different unique proteins, including 98 plant proteins and 35 fungal proteins ( Figure 6 and Appendix B: Tables A3 and A4). Of the 35 fungal proteins that changed abundances between ambient and elevated CO 2 , 27 changed only in a single endophyte strain, 14 of these in AR1. ...
Context 5
... generally, we examined overlap by constructing Venn diagrams using only the differently abundant proteins among all comparisons from both the plant and endophyte perspectives ( Figure 6F-I). Here, considering plant protein abundance at both CO 2 levels, we consistently observed the most common responses in the AR37 and NEA2 comparison, as well as several combinatory categories showing unique responses with identification of only one protein (e.g., AR1 and NEA2 and AR37 and E+). ...
Context 6
... the proteomes of the host plant-fungal strain combinations different from each other and how are they altered by elevated CO 2 ? Yes, proteomes differed between endophyte strains and there was evidence of substantial interaction between endophyte strains and CO 2 levels (Tables A2-A4, Figures 6 and 7). ...
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Although Epichloë endophytes are present only in aboveground tissues of grasses they indirectly influence soil biological characteristics through increased litter incorporation and root exudation. Epichloë endophytes have been reported to affect the decomposition rates of litter by altering litter quality and microbial decomposers to affect soil ch...
Fungal endophytes, by living entire or part of their life inside a plant, are potentially useful in natural ecosystems and agroecosystems to confer multiple benefits to their hosts. These include improvement of water and nutrientional status, higher tolerance against biotic/abiotic stresses. In this review, the consequences of infection by Epichloë...
Epichloë endophytes are filamentous fungi (family Clavicipitaceae) that live in symbiotic associations with grasses in the sub family Poöideae. In New Zealand, E. festucae var. lolii confers significant resistance to perennial ryegrass (Lolium perenne) against insect and animal herbivory and is an essential component of pastoral agriculture, where...
Citations
... (Syn. Schedonorus arundinaceus) and Lolium perenne plants associated with endophytes, but the greenhouse gas did not affect the production of reproductive tillers or seed in symbiotic plants [24][25][26]. Similar beneficial effects of CO 2 on plant growth were documented in endophyte-symbiotic Brachypodium sylvaticum and L. perenne plants that grew in soils with high nutrient contents [27,28]. ...
There is an increasing interest in determining the influence of global change on plant–microorganism interactions. We review the results of experiments that evaluated the effects of the global change factors carbon dioxide, ozone, temperature, drought, flooding, and salinity on plant symbioses with beneficial Epichloë endophytes. The factors affected the performance of both plants and endophytes as well as the frequency of plants symbiotic with the fungus. Elevated carbon dioxide levels and low temperatures differentially influenced the growth of plants and endophytes, which could compromise the symbioses. Furthermore, we summarise the plant stage in which the effects of the factors were quantified (vegetative, reproductive, or progeny). The factors ozone and drought were studied at all plant stages, but flooding and carbon dioxide were studied in just a few of them. While only studied in response to ozone and drought, evidence showed that the effects of these factors on symbiotic plants persisted trans-generationally. We also identified the putative mechanisms that would explain the effects of the factors on plant–endophyte associations. These mechanisms included the increased contents of reactive oxygen species and defence-related phytohormones, reduced photosynthesis, and altered levels of plant primary metabolites. Finally, we describe the counteracting mechanisms by which endophytes would mitigate the detrimental effects of the factors on plants. In presence of the factors, endophytes increased the contents of antioxidants, reduced the levels of defence-related phytohormones, and enhanced the plant uptake of nutrients and photosynthesis levels. Knowledge gaps regarding the effects of global change on plant–endophyte associations were identified and discussed.
... For example, the vertically transmitted fungal endophyte, Epichloë spp. produces alkaloids that protect grasses from herbivory and confer protection from drought and nutrient stress [3]. For perennial ryegrass (Lolium perenne), a cool-season grass with global distribution for its roles in carbon fixation, turfgrass applications, and fodder for livestock, the interaction with endophytes may protect the grass from rising atmospheric CO 2 associated with climate change. ...
The beauty within biological systems can be uncovered using a variety of advanced technological platforms for in-depth profiling. Improvements in genome, transcriptome, proteome, and metabolome investigations, as well as data integration, are moving our understanding of diverse biological systems forward at a rapid rate. Combined with publicly available and customizable bioinformatics tools, we comprehensively profile biological changes under a plethora of circumstances. For fungal pathogens innovation is driven by our ability to explore mechanisms of antifungal resistance, reveal new relationships and interactions between a host and pathogen, improve our characterization of virulence determinants, and discover new antifungal targets. In this Special Issue dedicated to “Systems biology in fungal research”, we explore each of these factors and more, highlighting the multitude of avenues and strategies available to study fungal pathogens and how they impact our environment.
... lolii and E. coenophiala strains are now available commercially in their host grass cultivars (Fletcher et al. 1991;Tapper and Latch 1999;Bouton and Easton 2005). Endophyte strain affects multiple characteristics in the host grass, including alkaloid profiles (Popay and Gerard 2007;Soto-Barajas et al. 2019), volatile organic compound quantities (Qawasmeh et al. 2015), root exudate chemical composition (Guo et al. 2015;Patchett and Newman 2021), metabolite profiles (Rasmussen et al. 2009;Geddes-McAlister et al. 2020), and protein production (Geddes-McAlister et al. 2020). Thus, host grasses from the same genetic background but with different Epichloë strains may differ in their allelopathic effects, if such effects occur. ...
... lolii and E. coenophiala strains are now available commercially in their host grass cultivars (Fletcher et al. 1991;Tapper and Latch 1999;Bouton and Easton 2005). Endophyte strain affects multiple characteristics in the host grass, including alkaloid profiles (Popay and Gerard 2007;Soto-Barajas et al. 2019), volatile organic compound quantities (Qawasmeh et al. 2015), root exudate chemical composition (Guo et al. 2015;Patchett and Newman 2021), metabolite profiles (Rasmussen et al. 2009;Geddes-McAlister et al. 2020), and protein production (Geddes-McAlister et al. 2020). Thus, host grasses from the same genetic background but with different Epichloë strains may differ in their allelopathic effects, if such effects occur. ...
Background and aims
Host-specific Epichloë spp. are endophytic fungal symbionts of pooid grasses that produce herbivore-deterring alkaloids and alter the host’s metabolite and protein profiles. Early observations suggested that Epichloë may have negative allelopathic effects on neighbouring plant species, particularly Trifolium spp. clovers, but subsequent allelopathy experiments produced variable results. We examined two hypotheses: (1) Epichloë strains differ in allelopathic effect, and (2) Epichloë allelopathy negatively affects other plant species.
Methods
We performed a greenhouse experiment using root exudates from Lolium perenne L. hosting different E. festucae var. lolii (Latch, M.J. Chr. & Samuels) C.W. Bacon & Schardl strains to compare their allelopathic effects on native legumes and forbs. We then used meta-analysis to examine the evidence to date for allelopathic effects of Epichloë endophytes.
Results
We found little difference in effect among E. festucae var. lolii strains and very little evidence for negative allelopathic effects of Epichloë in cool-season grasses across a range of methodologies, target plant species, and response measures. Negative allelopathic effects were detected only for root hair measures, which were from a single study. Positive effects on biomass were found for some experimental subgroups, including legumes. However, the majority of response variables showed no evidence for Epichloë allelopathy.
Conclusions
Although there is currently little evidence for negative Epichloë allelopathic effects, our meta-analysis identified several research gaps. Experiments testing the functional belowground effects of Epichloë presence may help to determine its effects on non-host plant performance via plant-soil feedbacks.
... Nevertheless, we know that the endophyte has many impacts on the host, prompting Rasmussen, Parsons, Popay, et al. (2008) to ask whether there was more to this mutualism than just alkaloids? We know from metabolomic and proteomic studies that these endophytes have profound effects on the plant's metabolism, biochemistry (see, e.g., Rasmussen, Parsons, Fraser, et al. 2008;Rasmussen et al. 2007;Patchett and Newman 2021;Geddes-McAlister et al. 2020) and nutrient uptake (see, e.g., Soto-Barajas et al. 2016, and references therein). Another empirically well-supported advantage conferred by the endophyte is drought resistance (see, e.g., Hosseini et al. 2016, and references therein). ...
The species comprising the fungal endophyte genus Epichloë are symbionts of cool season grasses. About half the species in this genus are strictly vertically transmitted, and evolutionary theory suggests that these species must be mutualists. Nevertheless, Faeth and Sullivan (e.g., 2003) have argued that such vertically transmitted endophytes are "usually parasitic," and Müller and Krauss (2005) have argued that such vertically transmitted en-dophytes fall along a mutualism-parasitism continuum. These papers (and others) have caused confusion in the field. We used a systematic review to show that close to half of the published papers in this field incorrectly categorize the interaction. Here, we develop the argument that advantages and disadvantages are not the same things as mutualism and parasitism and that experimental evidence must be interpreted in the context of theory. We argue that, contra Faeth and Sullivan, it is highly unlikely that such strictly vertically transmitted endophytes can be parasites, and that, contra Müller and Krauss, it is inappropriate to apply the continuum concept to strictly vertically transmitted endophytes. We develop a mathematical model to support and illustrate our arguments. We use the model to clarify that parasitism requires some degree of horizontal transmission, and that while it is appropriate to use the continuum concept when referring to the genus as a whole, or to species that possess horizontal transmission, we argue that it is inappropriate to use the concept when referring to species that are strictly vertically transmitted.
... Untargeted metabolomic study approaches have been used to understand the metabolic potential of Epichloë strains relating to antifeeding (insect/invertebrate) activity [127][128][129]. Other studies have investigated the effect of endophytes on the root exudate metabolome [130] and in response to different field nutrient or environmental conditions [128,131]. Untargeted metabolite fingerprinting coupled to spectral data analysis software could be the solution to understanding the complex process of endophyte-host plant-phytopathogen relationships and the metabolic response or production of bioactive metabolites in the presence of a pathogen (Figure 2d). Green et al. (2020) studied the Lolium perenne apoplast to identify Epichloë festucae-derived novel metabolites [73] and recently Fernando et al. (2021) conducted bioassay guided extraction of antifungal metabolites and metabolite annotation of bioactive extracts from Epichloë strains [36]. ...
Asexual species of the genus Epichloë (Clavicipitaceae, Ascomycota) form endosymbiotic associations with Pooidae grasses. This association is important both ecologically and to the pasture and turf industries, as the endophytic fungi confer a multitude of benefits to their host plant that improve competitive ability and performance such as growth promotion, abiotic stress tolerance, pest deterrence and increased host disease resistance. Biotic stress tolerance conferred by the production of bioprotective metabolites has a critical role in an industry context. While the known antimammalian and insecticidal toxins are well characterized due to their impact on livestock welfare, antimicrobial metabolites are less studied. Both pasture and turf grasses are challenged by many phytopathogenic diseases that result in significant economic losses and impact livestock health. Further investigations of Epichloë endophytes as natural biocontrol agents can be conducted on strains that are safe for animals. With the additional benefits of possessing host disease resistance, these strains would increase their commercial importance. Field reports have indicated that pasture grasses associated with Epichloë endophytes are superior in resisting fungal pathogens. However, only a few antifungal compounds have been identified and chemically characterized, and these from sexual (pathogenic) Epichloë species, rather than those utilized to enhance performance in turf and pasture industries. This review provides insight into the various strategies reported in identifying antifungal activity from Epichloë endophytes and, where described, the associated antifungal metabolites responsible for the activity.
... root exudate chemical composition, 49,50 metabolite profiles,51,52 and protein production.52 Thus, host grasses from the same genetic background but with different Epichloë strains may differ in their allelopathic effects, if such effects occur.We performed an experiment and a meta-analysis to examine the evidence for allelopathic effects of Epichloë endophytes. ...
... root exudate chemical composition, 49,50 metabolite profiles,51,52 and protein production.52 Thus, host grasses from the same genetic background but with different Epichloë strains may differ in their allelopathic effects, if such effects occur.We performed an experiment and a meta-analysis to examine the evidence for allelopathic effects of Epichloë endophytes. ...
Host-specific Epichloë spp. endophytic fungal symbionts of pooid grasses that produce herbivore-deterring alkaloids and alter the grass host's metabolite and protein profiles. Early observations suggested that Epichloë may have negative allelopathic effects on neighbouring plant species, particularly Trifolium spp. clovers, but subsequent allelopathy tests produced variable results. We examined two hypotheses: (1) Epichloë strains differ in allelopathic effect, and (2) Epichloë allelopathy negatively affects other plant species. We performed a greenhouse experiment using root exudates from Lolium perenne L. hosting different E. festucae var. lolii (Latch, M.J. Chr. & Samuels) C.W. Bacon & Schardl strains to compare their allelopathic effects on native legumes and forbs. We then used meta-analysis to examine the evidence to date for allelopathic effects of Epichloë endophytes. We found little difference in effect among E. festucae var. lolii strains and very little evidence for negative allelopathic effects of Epichloë in cool-season grasses across a range of methodologies, target plant species, and response measures. Negative allelopathic effects were detected only for root hair measures, which were from a single study. Positive effects on biomass were found for some experimental subgroups, including legumes. However, the majority of response variables showed no evidence for Epichloë allelopathy. Although there is currently little evidence for negative Epichloë allelopathic effects, our meta-analysis identified several research gaps. Experiments testing the functional belowground effects of Epichloë presence may help to determine its effects on non-host plant performance via plant-soil feedbacks.
... We found that NEA2 and AR37 infected plants performed better than E+ or AR1 infected plants. Nevertheless, Geddes-McAlister et al. [55] used the same L. perenne cultivar (Alto) and the same endophyte strains as we did and they did not find strain specific effects on biomass production. Other experimental details may explain this discrepancy, but minimally these differences suggest there is much still to understand about this plant-fungal interaction. ...
... We found that NEA2 infected plants had lower endophyte concentrations than E+ or AR37 plants (AR1 had intermediate concentrations; see Figure 3). These results are very similar to those found by Geddes-McAlister et al. [55]. ...
... We found large differences among exudate metabolomes from the different endophyte strains for all principal components (Figure 9). This is consistent with such differences found in the plant tissue metabolome by Geddes-McAlister et al. [55] although the exudate contained far fewer metabolites than the plant tissue. ...
Lolium perenne infected with the fungal endophyte Epichloë festucae var. lolii have specific, endophyte strain-dependent, chemical phenotypes in their above-ground tissues. Differences in these chemical phenotypes have been largely associated with classes of fungal-derived alkaloids which protect the plant against many insect pests. However, the use of new methodologies, such as various omic techniques, has demonstrated that many other chemical changes occur in both primary and secondary metabolites. Few studies have investigated changes in plant metabolites exiting the plant in the form of root exudates. As root exudates play an essential role in the acquisition of nutrients, microbial associations, and defense in the below-ground environment, it is of interest to understand how plant root exudate chemistry is influenced by the presence of strains of a fungal endophyte. In this study, we tested the influence of four strains of E. festucae var. lolii (E+ (also known as Lp19), AR1, AR37, NEA2), and uninfected controls (E−), on L. perenne growth and the composition of root exudate metabolites. Root exudates present in the hydroponic water were assessed by untargeted metabolomics using Accurate-Mass Quadrupole Time-of-Flight (Q–TOF) liquid chromatography–mass spectrometry (LC–MS). The NEA2 endophyte strain resulted in the greatest plant biomass and the lowest endophyte concentration. We found 84 metabolites that were differentially expressed in at least one of the endophyte treatments compared to E− plants. Two compounds were strongly associated with one endophyte treatment, one in AR37 (m/z 135.0546 RT 1.17), and one in E+ (m/z 517.1987 RT 9.26). These results provide evidence for important changes in L. perenne physiology in the presence of different fungal endophyte strains. Further research should aim to connect changes in root exudate chemical composition with soil ecosystem processes.
Complex environmental conditions like heavy metal contamination and elevated CO2 concentration may cause numerous plant stresses and lead to considerable crop losses worldwide. Cadmium is a non-essential element and potentially highly toxic soil metal pollution, causing oxidative stress in plants and human toxicity. In order to assess a combination of complex factors on the responses of two genotypes of Festuca arundinacea (75B and 75C), a greenhouse experiment was conducted on plants grown in two Cd-contaminated soil conditions and two soil textures under combined effects of elevated ambient CO2 (700 ppm) and Epichloë endophyte infection. Plant biomass, Cd, Fe, Cu, Zn, and Mn concentrations in the plant shoots and roots, Fv/Fm, chlorophyll (a & b), and carotenoid contents were measured after 7 months of growth in pots. Our results showed that endophyte-infected plants (E+) grown in elevated CO2 atmosphere (CO2+), clay-loam soil texture (H) with no Cd amendment (Cd−) in the genotype 75B had significantly greater shoot and root biomass than non-infected plants (E−) grown in ambient CO2 concentration (CO2−), sandy-loam soil texture (L) with amended Cd (Cd+) in the genotype 75C. Increased CO2 concentration and endophyte infection, especially in the genotype 75B, enabled Festuca for greater phytoremediation of Cd because of higher tolerance to Cd stress and higher biomass accumulation in the plant genotype. However, CO2 enrichment negatively influenced the plant mineral absorption due to the inhibitory effects of high Cd concentration in shoots and roots. It is concluded that Cd phytoremediation can be positively affected by the increased atmospheric CO2 concentration, tolerant plant genotype, heavy soil texture, and Epichloë endophyte. Using Taguchi and AIC design methodologies, it was also predicted that the most critical factors affecting Cd phytoremediation potential were CO2 concentration and plant genotype.
Research on fungal endophytes has demonstrated the ability to improve crop performance and protect host plants against diverse biotic and abiotic stresses. Yet, despite the exponential growth of this topic, a whole outline to reflect the relevance and extent of each study type is missing. Hence, we performed an analysis of all available literature to expose the characteristics and limitations of this research field. Our results suggested that, overall, there is still a tendency to study the most known models in plant-fungal-stress combinations (ascomycetous fungi, grasses, abiotic stress). Fungal endophytes in dicot plants or against biotic stress, though promising, are still quite unexplored. All these data could lead future studies to assess less considered study factors that might help discern the beneficial effects of fungal endophytes with more extent and accuracy.
