Wheat fungal endophyte communities are inseparable from the host and influence plant development.

Plants harbor complex and highly diverse fungal endophyte communities (FECs), making it difficult to evaluate the functional role of individual taxa, subsets of the community, or the FEC as a whole. To reduce the complexity of this system, we aimed to produce fungi-null wheat (Triticum aestivum) plants. To this end, we treated seeds with heat and fungicides and generated plants from rescued embryos and callus tissue. A culture-based approach and reverse transcription PCR analysis were negative, indicating that all treatments produced plants apparently free of fungi. However, the analysis of DNA using digital droplet PCR and next-generation sequencing revealed that tissues from all treatments retained low levels but diversity-rich FECs. While the FECs varied in composition across treatments and tissues, they all included core taxa of the mycobiome. The reduced fungal biomass, along with the changes in FEC composition, negatively affected plant development, supporting a FEC contribution to proper plant development and fitness. Our discovery that a large part of the FEC cannot be separated from plants and can be transmitted through seeds and tissue culture calls for reevaluation of particular microbiome paradigms, such as core taxa concepts, transmission modes, and functional species.IMPORTANCEThe native microbiome in a given plant must be considered when evaluating the effect of a single taxon or synthetic community. The pre-existing microbiome can interact with artificially added microbial cargo, which affects the final outcome. Such issues can be at least partially solved by the use of endophyte-free plants, which provide a clean background that should be useful in determining the effect of a single taxon, taxa combinations, or the entire microbiome on plant performance. Previous reports regarded plants as endophyte-free or axenic by the lack of fungal growth on culture media or the generation of plants from tissue cultures. We showed here that while fungi could not be isolated from fungicide-treated or tissue culture-regenerated plants, nevertheless, all plants contained rich fungal endophyte communities; namely, it was impossible to create fungi-free wheat plants. Our results call for rethinking fundamental microbiome-related concepts, such as core taxa, transmission mode, and functional species.

Distinct Features Based on Partitioning of the Endophytic Fungi of Cereals and Other Grasses.

Endophytic fungi form a significant part of the plant mycobiome. Defining core members is crucial to understanding the assembly mechanism of fungal endophytic communities (FECs) and identifying functionally important community members. We conducted a meta-analysis of FECs in stems of wheat and five wild cereal species and generated a landscape of the fungal endophytic assemblages in this group of plants. The analysis revealed that several Ascomycota members and basidiomycetous yeasts formed an important compartment of the FECs in these plants. We observed a weak spatial autocorrelation at the regional scale and high intrahost variations in the FECs, suggesting a space-related heterogeneity. Accordingly, we propose that the heterogeneity among subcommunities should be a criterion to define the core endophytic members. Analysis of the subcommunities and meta-communities showed that the core and noncore members had distinct roles in various assembly processes, such as stochasticity, universal dynamics, and network characteristics, within each host. The distinct features identified between the community partitions of endophytes aid in understanding the principles that govern the assembly and function of natural communities. These findings can assist in designing synthetic microbiomes. IMPORTANCE This study proposes a novel method for diagnosing core microbiotas based on prevalence of community members in a meta-community, which could be determined and supported statistically. Using this approach, the study found stratification in community assembly processes within fungal endophyte communities (FECs) in the stems of wheat and cereal-related wild species. The core and noncore partitions of the FECs exhibited certain degrees of determinism from different aspects. Further analysis revealed abundant and consistent interactions between rare taxa, which might contribute to the determinism process in noncore partitions. Despite minor differences in FEC compositions, wheat FECs showed distinct patterns in community assembly processes compared to wild species, suggesting the effects of domestication on FECs. Overall, our study provided a new approach for identifying core microbiota and provides insights into the community assembly processes within FECs in wheat and related wild species.

Jasmonic acid pathway is required in the resistance induced by Acremonium sclerotigenum in tomato against Pseudomonas syringae.

The use of fungal endophytes is considered as a new tool to confer resistance in plants against stresses. However, the mechanisms involved in colonization as well as in the induction of resistance by the endophytes are usually unclear. In this work, we tested whether a fungal endophyte isolated from an ancestor of wheat could induce resistance in plants of a different class from the ones that were isolated from the beginning. Seeds of Solanum lycopersicum were inoculated with Acremonium sclerotigenum and after four weeks, seedlings were inoculated with the bacterium Pseudomonas syringae pv tomato. Plants inoculated with endophytes showed significantly lower symptoms of infection as well as lower levels of colony forming units compared with control plants. Moreover, the presence of the endophytes induced an enhancement of Jasmonic acid (JA) upon inoculation with P. syringae compared with endophyte free plants. To ascertain the implication of JA in the resistance induced by A. sclerotigenum, two mutants defective in JA were tested. Results showed that the endophyte is not able to induce resistance in the mutant spr2, which is truncated in the first step of JA biosynthesis. On the contrary, acx1 mutant plants, which are unable to synthesize JA from OPC8, show a phenotype similar to wild type plants. Moreover, experiments with GFP-tagged endophytes showed no differences in the colonization in both mutants. In conclusion, the jasmonic acid pathway is required for the resistance mediated by the endophyte A. sclerotigenum in tomato against the biotrophic bacterium P. syringae but is not necessary for the colonization.

Significant host- and environment-dependent differentiation among highly sporadic fungal endophyte communities in cereal crops-related wild grasses.

Endophytic fungi compose a significant part of plant microbiomes. However, while a small number of fungal taxa have proven beneficial impact, the vast majority of fungal endophytes remain uncharacterized, and the drivers of fungal endophyte community (FEC) assembly are not well understood. Here, we analysed FECs in three cereal crops-related wild grasses - Avena sterilis, Hordeum spontaneum and Aegilops peregrina - that grow in mixed populations in natural habitats. Taxa in Ascomycota class Dothideomycetes, particularly the genera Alternaria and Cladosporium, were the most abundant and prevalent across all populations, but there was also high incidence of basidiomyceteous yeasts of the class Tremellomycetes. The fungal community was shaped to large extent by stochastic processes, as indicated by high level of variation even between individuals from local populations of the same plant species, and confirmed by the neutral community model and Raup-Crick index. Nevertheless, we still found strong determinism in FEC assembly with both incidence and abundance data sets. Substantial differences in community composition across host species and locations were revealed. Our research demonstrated that assembly of FECs is affected by stochastic as well as deterministic processes and suggests strong effects of environment heterogeneity and plant species on community composition. In addition, a small number of taxa had high incidence and abundance in all of the 15 populations. These taxa represent an important part of the core FEC and might be of general functional importance.

Endophytes from wild cereals protect wheat plants from drought by alteration of physiological responses of the plants to water stress.

Endophytes contribute to plant performance, especially under harsh conditions. We therefore hypothesized that wild plants have retained beneficial endophytes that are less abundant or not present in related crop plants. To test this hypothesis, we selected two endophytes that were found in Sharon goatgrass, an ancestor of wheat, and tested their effect on bread wheat. Both endophytes infected wheat and improved sustainability and performance under water-limited conditions. To determine how the endophytes modify plant development, we measured parameters of plant growth and physiological status and performed a comparative metabolomics analysis. Endophyte-treated wheat plants had reduced levels of stress damage markers and reduced accumulation of stress-adaptation metabolites. Metabolomics profiling revealed significant differences in the response to water stress of endophyte-treated plants compared with untreated plants. Our results demonstrate the potential of endophytes from wild plants for improvement of related crops and show that the beneficial effects of two endophytes are associated with alteration of physiological responses to water-limited conditions.

Diversity of fungal endophytes in recent and ancient wheat ancestors Triticum dicoccoides and Aegilops sharonensis.

Endophytes have profound impacts on plants, including beneficial effects on agriculturally important traits. We hypothesized that endophytes in wild plants include beneficial endophytes that are absent or underrepresented in domesticated crops. In this work, we studied the structure of endophyte communities in wheat-related grasses, Triticum dicoccoides and Aegilops sharonensis, and compared it to an endophyte community from wheat (T. aeastivum). Endophytes were isolated by cultivation and by cultivation-independent methods. In total, 514 intergenic spacer region sequences from single cultures were analyzed. Categorization at 97% sequence similarity resulted in 67 operational taxonomic units (OTUs) that were evenly distributed between the different plant species. A narrow core community of Alternaria spp. was found in all samples, but each plant species also contained a significant portion of unique endophytes. The cultivation-independent analysis identified a larger number of OTUs than the cultivation method, half of which were singletons or doubletons. For OTUs with a relative abundance >0.5%, similar numbers were obtained by both methods. Collectively, our data show that wild grass relatives of wheat contain a wealth of taxonomically diverse fungal endophytes that are not found in modern wheat, some of which belong to taxa with known beneficial effects.