A major bottleneck in plant biotechnology is the inefficient and genotype-dependent regeneration of callus, which severely limits genetic transformation and functional studies across many species. This barrier is acutely exemplified in the study of beneficial plant-microbe interactions, such as the Epichloë –grass symbiosis—a system conferring remarkable stress tolerance to its host but hindered by a lack of efficient genetic tools. To address this, we established a chromosome-scale genome for an Epichloë native host grass Achnatherum inebrians . We discovered that the expression dynamics of evolutionarily conserved cell pluripotency regulators (CPRs) including ARF5/7/19 , BBM , WUS/WOX5 and CUC1/2 serve as a precise molecular predictor for callus regenerative capacity, revealing that pluripotency is dynamic and peaks within a narrow, definable time window. Harnessing this predictable window enabled the development of a highly efficient transformation system for A. inebrians (49.4% efficiency). Crucially, this CPR-based strategy proved generalizable: applied to wheat and the legume sainfoin, it pinpointed species-specific optimal regeneration windows, boosting shoot regeneration rates to 65.7% and 87.5%, respectively. Collectively, our work provides an integrated research system and a rational design principle that removes a key barrier to uncovering molecular mechanisms in plant systems, particularly the Epichloë –enhanced stress tolerance symbiosis. ### Competing Interest Statement The authors have declared no competing interest. National Natural Science Foundation of China, 32300241, 32441036

Drunken horse grass (Achnatherum inebrians) plays a vital role in ecological restoration and grassland sustainability in Northwest China, but its ecological functions are increasingly threatened by emerging fungal diseases. In 2024, a leaf spot disease characterized by brown lesions with yellow halos was observed on drunken horse grass in Gansu Province, China. The causal pathogens were identified as Alternaria alternata and Alternaria infectoria based on morphological characterization, pathogenicity tests, and multi-locus phylogenetic analysis (ITS, TEF, GPD, RPB2, Alt a 1, endoPG, and OPA10-2). Preliminary fungicide sensitivity assays revealed that tetramycin and difenoconazole had the strongest inhibitory effects against mycelial growth in vitro. The EC50 values for tetramycin were 0.0755 mg/L (A. alternata) and 0.2175 mg/L (A. infectoria), while for difenoconazole, they were 0.1023 mg/L (A. alternata) and 0.0599 mg/L (A. infectoria). To our knowledge, this is the first report of Alternaria species infecting the host plant, drunken horse grass, providing an essential basis for the effective management of this disease and the protection of grassland ecosystems.

Fungal endophytes are universally present in plant tissues to enhance stress resilience and growth of plants. They can change the microbial communities and functional characteristics of the rhizosphere without harming the host. However, the effect of their colonization on the distribution of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in rhizosphere soil remains largely unexplored. In the present study, the impact of the Epichloë bromicola fungal endophyte on the rhizosphere resistome structure and environmental factors in wild barley (Hordeum brevisubulatum) and bluegrass (Poa pratensis) plants was investigated. The rhizosphere ARGs and MGEs communities were characterized through metagenome analysis. Simultaneously, their relationship with key rhizosphere environmental variables was evaluated using redundancy analysis (RDA) and Mantel tests. The results revealed that infection altered the taxonomic distribution of ARGs and MGEs-carrying bacteria. Besides, it reduced the prevalence of the predominant genera Sphingomonas and Nocardioides, while increasing the contribution of the less prevalent genera Bradyrhizobium and Rubrivivax to multidrug efflux and macrolide resistance mechanisms. Co-occurrence network analysis showed decreased modularity, indicating a less compartmentalized resistome-mobilome under infection. The findings revealed that endophyte infection fundamentally restructured the rhizosphere resistome by changing environmental pressures and favoring stress-response mechanisms. The study provides novel insights into how fungal endophytes influence microbial resistome assembly and HGT processes, their role in environmental antibiotic resistance dissemination, and One Health resistome dynamics in the rhizosphere.

Grain size represents a key agronomic trait and a major determinant of barley grain yield potential. We observed that the grain size of Epichloë bromicola-inoculated barley plants were significantly larger than those of the uninoculated barley plants. However, the mechanisms and functions by which the endophyte fungus E. bromicola modulates barley grain size have not been investigated. In this study, we compared grain size between uninoculated (KE-) and E. bromicola-inoculated (KE+) barley plants to elucidate the association between E. bromicola colonization and barley grain size. Quantitative grain morphometric analysis further showed that grain width and grain area increased in KE+ by 6.8% and 5.2% compared with KE-, respectively. Structural analyze revealed that E. bromicola increased starch granule density in seeds. Phytohormone analyze showed that IAA enhanced nutrient transport by activating endosperm transfer cells, while ABA increased starch and storage protein synthesis for better grain filling. Integrated transcriptomic, and metabolomic analyses revealed that E. bromicola activated multiple hormone pathways, and enhanced the antioxidant system to regulate grain size. These findings suggest that inoculation with E. bromicola may improve barley grain yield performance. This study provides mechanistic insights into E. bromicola-mediated regulation of major seed agronomic traits and proposes a promising strategy for optimizing plant architecture by E. bromicola inoculation in sustainable agriculture.

Grassland degradation has impacted the Qinghai-Tibet Plateau over recent decades, with bare patches and Ligularia virgaurea-degraded grasslands serving as typical examples. The effects of grassland degradation on soil nutrients, microbial communities, seed banks, and vegetation restoration potential were investigated to identify restoration techniques for degraded grasslands. The contents of total nitrogen, phosphorus, potassium, water, and organic carbon in degraded grasslands decreased significantly, whereas pH and nitrate nitrogen content increased. CaCO3 content was highest in bare patches, followed by L. virgaurea-degraded grasslands, which were higher than that in non-degraded grasslands by 76.46% and 54.09%, respectively (P < 0.05). Water content in the two types of degraded grasslands was significantly lower than that in non-degraded grasslands, by 18.4-22.85%. Brassinosteroid content in bare patches and L. virgaurea-degraded grasslands decreased by 44.18% and 80.66%, respectively. The soil seed bank composition in degraded areas changed significantly, shifting toward higher forb abundance, while Poaceae decreased by 55.21-78.13%. The germination rate of Elymus nutans seeds treated with soil extracts from degraded grasslands did not change significantly. Bacteria in non-degraded grasslands were significantly enriched for brassinosteroid biosynthesis pathway function. Bacteria associated with brassinosteroid biosynthesis were classified as Beggiatoa Trevisan 1842 (Approved Lists 1980) and members of the class Deltaproteobacteria Kuever et al. 2006 and order Chromatiales Imhoff 2005. Compared with non-degraded grasslands, soil microorganisms in degraded grasslands were unfavorable to seed germination. Therefore, artificial restoration of the grassland soil seed bank, optimization of the soil microbial community, and maintenance of soil moisture are crucial to the restoration of the aboveground plant community.

Fire is widely used to reduce plant disease in grasslands, yet whether its effects vary across local spatial gradients within burned patches, and the underlying mechanisms, remain poorly understood. We combined a three-year prescribed burning experiment with an edge-to-interior spatial framework in an alpine grassland to test whether fire-driven changes in plant growth type (grass vs. forb) regulate foliar fungal disease suppression. Prescribed fire significantly reduced foliar fungal pathogen loads across divergent pathogen life histories (necrotrophic vs. biotrophic). However, this suppressive effect was spatially structured, becoming progressively stronger from the burned boundary toward the patch interior. Structural equation modeling showed that the edge-to-interior gradient did not directly explain variation in pathogen load. Instead, it was primarily associated with shifts in plant growth type, characterized by declining grass cover (β = -0.393, P < 0.001) and increasing forb cover (β = 0.412, P < 0.001) toward the patch interior. These shifts mediated pathogen suppression: grass cover was positively associated with pathogen load (necrotrophic: β = 0.367, P = 0.001; biotrophic: β = 0.378, P < 0.001), whereas forb cover showed negative associations (necrotrophic: β = -0.299, P = 0.006; biotrophic: β = -0.411, P < 0.001). In contrast, variation in aboveground biomass, species richness, and community-weighted mean height did not directly explain pathogen load. By linking edge-to-interior spatial variation with post-fire shifts in plant growth type, our findings show that spatially structured vegetation responses—not merely fire occurrence—are central to disease regulation in fire-managed grasslands.

Endophytes are prevalent in plants and significantly contribute to plant growth and development. In the present study, Epichloë bromicola endophyte strain WBE1 was artificially inoculated into wild barley (Hordeum brevisubulatum, natural host) and cultivated barley (Hordeum vulgare, novel host) to obtain endophyte-barley symbionts. Physiological traits, such as callus formation, lignin content, cell mortality, early signaling molecules, second messenger endogenous signaling molecules, and the expression patterns of differential genes at different time periods were studied. The colonization rate of E. bromicola was 54.21% in wild barley and 9.91% in cultivated barley. Artificial endophytic infection enhanced callus growth, lignin content, and cell mortality in both hosts, with cultivated barley showing stronger resistance than wild barley. The infection induced the expression of early signaling molecules, and the O2- production rate as well as H2O2 and NO contents were increased in both hosts. During the early infection stage, mitogen-activated protein kinase (MAPK) activity in cultivated barley increased by 12.24% and 54.60% compared with wild barley at 2 and 4 days post-infection, respectively. Transcriptomic analysis revealed that cultivated barley triggered an earlier and targeted defense response than wild barley, characterized by the stage-specific upregulation of genes involved in resistance-related secondary metabolite biosynthesis and key signaling molecules. Expression patterns showed upregulation of signaling molecules alongside downregulation of genes associated to oxylipin biosynthesis, lipid oxidation, cellular responses to environmental stimuli, oxidoreductase activity, and heme binding. These findings indicated that E. bromicola infection effectively triggered an enhanced and timely defense response in cultivated barley.

Background: Artificial inoculation of Epichloë endophytes into elite forage germplasm aims to establish beneficial symbioses for developing high-yield, high-quality, and stress-tolerant cultivars, but host specificity of the fungi often causes compatibility issues in non-natural hosts. Methods: The E. bromicola isolated from native wild barley was inoculated into cultivated wild barley (Hordeum brevisubulatum) and cultivated barley (Hordeum valgare), forming Hb+Eb and Hv+Eb. The NHb+Eb (native wild barley naturally infected with E. bromicola) served as a control. We analyzed fungal colonization patterns and symbiotic gene regulation to clarify the compatibility between E. bromicola and non-natural hosts. Results: Compared with NHb+Eb and Hb+Eb, E. bromicola in Hv+Eb showed obvious hyphal vacuolization. E. bromicola colonization altered host trichome morphology and induced stomatal closure. Correspondingly, expression of the siderophore biosynthesis gene sidN and the NADPH oxidase complex genes (NoxA, NoxB, NoxR, RacA) was significantly lower (p < 0.05) in Hv+Eb than in Hb+Eb and NHb+Eb. Conclusions: This study reveals that the incompatibility between cultivated barley and E. bromicola is characterized by altered hyphal morphology, which is linked to the downregulation of sidN and Nox. These findings provide a critical theoretical foundation for developing highly compatible cereal-Epichloë germplasms.

Italian ryegrass (Lolium multiflorum Lam.) is widely cultivated as a vital forage crop in southern China due to its exceptional quality and yield, good palatability, and rapid early growth. A survey found that black leaf spot (BLS), caused by Pestalotiopsis species, is a prevalent and emerging disease of Italian ryegrass, typically manifesting as black spots or net blotches. This study isolated 70 Pestalotiopsis strains from diseased leaves of Italian ryegrass planted in the Chongqing, Yunnan, and Guizhou provinces of southwestern China between 2021 and 2022. Based on morphological features and multilocus phylogenetic analyses (ITS, TUB2, and TEF1), five new records and one unknown species were identified in association with Italian ryegrass. These are Pestalotiopsis australasiae, P. biciliata, P. telopeae, P. trachycarpicola, P. tumida, and Pestalotiopsis sp.1. Together with the isolation rates and geographical distributions of these species, pathogenicity tests showed that P. trachycarpicola is the most important causal agent of BLS on Italian ryegrass in southwestern China. These findings form the basis for the diagnosis, detection, pathogen identification, and scientific control of BLS on Italian ryegrass.

Oats are among the most significant cereal grains globally, valued for their diverse applications. In China, oat cultivation predominantly occurs in the northern and northwestern regions, particularly in higher-elevation areas where cooler climates and fertile soils create optimal growing conditions. However, the production of oats is severely threatened by various biotic stresses, mainly diseases, which adversely affect both yield and quality. Additionally, abiotic stresses such as drought, frost and unfavourable soil conditions further complicate production efforts. In oat-growing regions of China, rainfall is limited and primarily concentrated between July and September. The timing and intensity of this rainfall significantly influences disease prevalence, as persistent rainfall during the harvest season, coupled with high humidity and elevated temperatures, fosters ideal conditions for the spread of oat diseases. These environmental factors contribute to the increased incidence and severity of diseases and abiotic stresses, necessitating the development of breeding strategies to produce varieties that exhibit disease resistance and stress tolerance. Recent advancements in molecular marker-assisted selection are being used to enhance the efficiency of breeding programmes, facilitating the identification of desirable traits linked to specific genetic markers. This review aims to address the existing gaps in the literature regarding oat production in China by examining the significant diseases and abiotic stresses affecting crops and exploring the development of resistant varieties for effective disease management. Ultimately, this work seeks to enhance understanding of the challenges faced in oat production, providing valuable insights for researchers, agronomists and policymakers.

The wild barley, Hordeum brevisubulatum (Trin.) Link, is a high-quality forage grass and exhibits enhanced stress resistance when infected by endophytes. For this symbiotic system, a systematic assessment was conducted to examine the relationships among growth traits, nutritional quality, and salt tolerance across different samples, with the final aim to identify superior germplasm resources. Ecotypes collected from different regions were studied. Field experiments were conducted to evaluate agronomic traits and nutritional quality. Concurrently, greenhouse experiment subjected the ecotypes under salt stress treatment to monitor their growth. Comprehensive evaluation and correlation analysis were employed to identify superior ecotypes. Significant variations in agronomic traits, forage quality and salt tolerance were detected between different ecotypes. Crown breadth, acid detergent fiber, and relative cell membrane permeability had the significant direct path coefficient on relative feeding value. Significant negative correlations between quality and agronomic traits and between quality and salt tolerance were found, whereas significant positive correlation between agronomic traits and salt tolerance was detected. Our efforts identified three ecotypes with good agronomic traits and salt tolerance that may be used for developing new varieties for ecological restoration and forage.

The tripartite interplay among plants, bacteria, and fungal endophytes is crucial for maintaining host plant fitness. However, how Epichloë endophytes influence plant-associated bacterial communities in cool-season grasses, particularly in ecologically important yet understudied species, such as Achnatherum inebrians, remains unclear. Using phylogenetic molecular ecological network analysis, we determined that seed-borne (seed epiphytic) and phyllosphere bacterial communities of Epichloë-infected (EI) A. inebrians exhibited reduced network complexity compared to Epichloë-free (EF) plants. Across all samples, Proteobacteria and Firmicutes dominated the keystone taxa, with Pseudomonas (OTU744 and OTU8264) consistently identified as a hub genus in both seed-borne and phyllosphere networks. Culture-based analysis revealed that endophyte-infected plants had a significantly (P < 0.05) higher relative abundance of Pseudomonas and Bacillus than EF A. inebrians, especially Pseudomonas comprised 13, 35, and 33% of isolates from the seed, leaf, and rhizosphere of A. inebrians, respectively. To capture potential functional diversity, we selected two phylogenetically distant Pseudomonas strains from each of the three ecological niches for further analysis. Inoculation of A. inebrians seedlings with these strains consistently promoted plant growth, enhanced forage quality (total nitrogen content), and improved nutritional value (ether extract). Whole-genome sequencing combined core-genome phylogenetic tree of the six Pseudomonas strains and confirmed that five strains belong to P. atacamensis, whereas Pse19 was P. cucumis. Our findings reveal that Epichloë endophytes modulate bacterial network stability and enrich plant-associated Pseudomonas, which synergistically enhance host performance. Collectively, this study provides a mechanistic framework for manipulating keystone taxa and beneficial isolates to improve grass productivity in grassland agricultural ecosystems.IMPORTANCEAlthough the tripartite interplay between plants, bacteria, and fungal endophytes, such as Epichloë, is recognized as vital for host fitness, the specific mechanisms through which these endophytes shape associated bacterial communities, particularly in ecologically significant grasses, such as Achnatherum inebrians, remain poorly understood. This study provides crucial mechanistic insights by revealing that Epichloë endophytes reconfigure the structure and stability of both seed-borne and phyllosphere bacterial networks in A. inebrians, leading to reduced complexity but enrichment of specific keystone taxa. We identify Pseudomonas as a consistently dominant hub genus across these niches. Notably, functional validation shows that diverse Pseudomonas isolates, representative of those enriched by the endophyte, significantly enhance host plant growth, forage quality, and nutritional value. Our findings reveal a synergistic mechanism where Epichloë endophytes modulate bacterial network stability to favor beneficial Pseudomonas populations, collectively boosting host performance.

Epichloë endophytes enhance plant defense and biotic stress resistance through mutualistic interactions in natural hosts. However, whether these protective effects are retained in annual non-native hosts such as barley (Hordeum vulgare L.) remains unclear. An integrated multi-omics approach was used to characterize defense responses in Epichloë bromicola-infected barley (LD1) and endophyte-free barley (CK) following pathogen challenge. The results show that LD1 had 20.0% lower disease incidence and 24.1% lower disease index than CK. LD1 also exhibited 21.1% lower malondialdehyde (MDA) content and higher antioxidant enzyme activities, indicating more effective control of oxidative damage and improved redox homeostasis. Integrated transcriptomic and metabolomic analyses revealed distinct defense responses between LD1 and CK. LD1 showed stronger activation of glutathione metabolism and antioxidant pathways than CK after 6 h of endophyte inoculation. This was consistent with more efficient control of early reactive oxygen species (ROS) dynamics. By 48 h, LD1 preferentially enriched phenylpropanoid biosynthesis and mitogen-activated protein kinase (MAPK) signaling, accompanied by increased accumulation of defense-related metabolites and enhanced structural and chemical defenses. Collectively, the study demonstrates that Epichloë bromicola enhances disease resistance in barley by reprogramming host defense dynamics from early redox regulation to late structural and chemical reinforcement.

Abstract Background Epichloë endophytes form beneficial symbioses with cool-season grasses, enhancing host tolerance to abiotic stresses such as drought while maintaining normal plant growth. However, the molecular mechanisms underpinning this symbiosis, particularly the role of fungal-secreted protein, remain largely unexplored. Results In this study, we identify EgSPE, a secreted protein from Epichloë gansuensis , as a key regulator of symbiotic establishment and host drought adaptation in drunken horse grass ( Achnatherum inebrians ). Transcriptome profiling during host colonization revealed EgSPE as a strongly induced gene encoding a secreted protein. Functional characterization facilitated by a substantially improved transformation system demonstrates that EgSPE is critical for fungal growth and efficient host colonization, as its deletion severely disrupted symbiotic establishment. Notably, EgSPE activates the host drought-responsive signaling by inducing the marker gene RD29A in a heterologous system ( Nicotiana benthamiana ) and upregulating stress-related genes ( AiRD22 , AiNAC5 , and AiABA1 ) in its native host ( A. inebrians ). Consistently, only the E. gansuensis wild-type and OE- EgSPE strains enhanced host drought resistance, whereas the Δ egspe mutants failed to confer this benefit. Conclusions In summary, our research findings identify EgSPE as a fungal protein that plays an important role in the establishment of symbiosis and in the host’s drought response, providing strong evidence for how E. gansuensis promotes abiotic stress tolerance in grasses.

BACKGROUND:Perennial ryegrass (Lolium perenne L.) is a widely cultivated turfgrass and forage species. AP2/ERF transcription factors play indispensable roles in plant growth and development, particularly in responses to biotic and abiotic stresses. However, comprehensive knowledge of this family in perennial ryegrass remains underexplored. RESULTS:In this study, a total of 172 LpAP2/ERF genes were identified in the perennial ryegrass genome and renamed according to their chromosomal distribution. Phylogenetic and synteny analyses were performed using rice to investigate the evolution of the AP2/ERF genes in perennial ryegrass. These 172 LpAP2/ERF genes were categorized into four distinct subfamilies: the AP2 subfamily (25), the ERF subfamily (84), the DREB subfamily (59), and the RAV subfamily (4). Within each subfamily, gene structure and motif compositions were conserved. The analysis of cis-acting elements in LpAP2/ERF gene promoters suggests that these genes may play crucial roles in plant growth, development, and stress responses. Analysis of the expression patterns of AP2/ERF family genes in endophyte-infected perennial ryegrass under low nitrogen induction showed that a total of 44 LpAP2/ERF genes had significantly different expression levels. 39 genes responded strongly to nitrogen starvation in both endophyte-infected and endophyte-free perennial ryegrass, while 21 genes were expressed in response to infection by endophytic fungi under normal and low nitrogen conditions. Importantly, through GO, KEGG enrichment pathway analysis and protein interaction network prediction, two key core genes (LpAP2/ERF64 and LpAP2/ERF164) were finally identified, which play an important role in enhancing perennial ryegrass's response to nitrogen starvation by endophytic fungi. CONCLUSIONS:This study is the first to identify the characteristics of the AP2/ERF family in perennial ryegrass, providing invaluable information for further evolutionary and functional studies of AP2/ERF. It also contributes to a better understanding of the molecular basis of development and stress tolerance in this species and others.

Taraxacum mongolicum, which is valued for its both feeding and medicinal values (Lin et al. 2022; Wang et al. 2022), is one of the main associated species in the alpine meadow of China. In August 2023, a new leaf spot disease of T. mongolicum with an incidence rate of about 90% was observed on natural grassland in Hongyuan County, Aba (Ngawa) Tibetan and Qiang Autonomous Prefecture, China (32°48' N, 102°33' E, alt. 3500 m). The symptoms appeared as grayish white spots in the center with dark brown or purple black margins. For isolation, 18 tissue pieces (5mm × 5mm) from 2 symptomatic plants were surface sterilized with 70% ethanol for 30 s and rinsed three times with sterilized distilled water. Then, these tissues were placed on potato dextrose agar (PDA) at 25°C and incubated in the dark for 2 to 7 days. Finally, six pure strains with consistent colony characteristics were obtained from hyphal tips. Colonies on PDA were dark brown or green black with abundant aerial mycelia on the upper side, and black on the reverse side. Hyphae were hyaline to brown, septate, 3 to 6 μm wide. Conidia were hyaline, cylindrical, 10.7 to 15.4 × 3.0 to 4.9 μm (average 12.8 × 3.8 μm; n = 50), guttulate. For further identification, the internal transcribed spacer region (ITS), 28S nrRNA gene (LSU), partial beta-tubulin (tub2) and RNA polymerase II subunit 2 (rpb2) genes of the six strains were amplified with primers described by Valenzuela-Lopez et al. (2018). Sequences were deposited in GenBank (PP345804, PV840064-PV840068 for ITS; PQ002493, PV840076-PV840080 for LSU; PQ015093, PV854533-PV854537 for rpb2; and PQ015094, PV854538-PV854542 for tub2). A maximum likelihood (ML) phylogenetic tree based on the combined ITS, LSU, rpb2, and tub2 alignments showed the six strains, and UTHSC: DI16-352 and ex-type CBS 142456 of N. cylindrispora (Valenzuela-Lopez et al. 2018) formed a subclade with 97% bootstrap support. Based on the above morphological and molecular features, the six strains were finally confirmed as Neoascochyta cylindrispora. For pathogenicity tests, 7-week-old healthy plants were obtained by growing T. mongolicum seeds in pots (two plants per pot). Above six pure cultures were subsequently used to inoculate healthy plants as follows: for each strain, four pots were spray inoculated with a conidial suspension (about 1 × 10 6 conidia/ml); in addition, four pots considered as non-inoculated controls were sprayed with sterilized distilled water. All pots were individually covered with transparent polyethylene bags for 5 days to maintain high relative humidity and placed in a greenhouse with a temperature of 23 to 29°C, humidity of 60 to 70%, 12 hours of light and 12 hours of darkness. After incubation for 10 days, plants developed symptoms similar to those observed in the field. No symptoms were observed on the non-inoculated control plants. Pathogenicity tests were conducted thrice using the same method. The same fungus was re-isolated from the lesions and confirmed by the morphological and molecular methods described in this note, thus fulfilling Koch’s postulates. N. desmazieri, closely phylogenetically related to N. cylindrispora, has been recorded on Lolium perenne (Poaceae) in Germany (Chen et al. 2015; Valenzuela-Lopez et al. 2018). To our knowledge, this is the first report of N. cylindrispora as pathogen on T. mongolicum worldwide. This information would be benefit for the diagnosis, detection and pathogen identification of leaf spot disease on T. mongolicum in natural grassland.

Leptosphaerulina trifolii (Didymellaceae) is a widespread phytopathogen commonly associated with leaf spot diseases on legumes. However, its occurrence on Poaceae hosts has rarely been documented. In this study, leaf spot symptoms on Elymus plants were observed in Gansu and Qinghai Provinces, China. Morphological characterization, combined with multi-locus phylogenetic analyses (ITS, LSU, and RPB2) and pathogenicity assays, confirmed L. trifolii as the causal agent. Phylogenetic reconstruction demonstrated that newly obtained isolates clustered with ex-type and reference strains of L. trifolii with high support, while inoculation trials reproduced typical field symptoms and fulfilled Koch’s postulates. Growth condition assays further revealed that the fungus exhibited optimal proliferation at 20 °C, with KNO3 and D-maltose as the most favorable nitrogen and carbon sources, respectively, and under either continuous darkness or a 12 h light/12 h dark regime. To our knowledge, this is the first report of L. trifolii causing leaf spot on Elymus spp. in China. This study provides the first evidence of L. trifolii on Elymus species, thereby expanding its known host range. Identification was confirmed through field surveys, morphological and molecular analyses, pathogenicity tests, and fungicide sensitivity, supporting the validity of this host record.

Many Epichloë endophytes are characterized by their mutualistic relationship with Poaceae grasses, which not only enhances the host’s resilience to a range of biotic and abiotic stresses, but also increases its ecological competitiveness. Alkaloids produced by Epichloë spp. play crucial roles in these mutualistic interactions. Numerous studies have systematically characterized four primary classes of Epichloë-derived alkaloids: ergot alkaloids, pyrrolopyrazines (including peramine), indole-diterpenes, and 1-aminopyrrolizidines (including lolines). However, emerging empirical evidence challenges the traditional alkaloid-centric paradigm by demonstrating that the defensive traits mediated by Epichloë in host plants are not exclusively governed by alkaloids. The defensive potential of other metabolites derived from Epichloë remains inadequately understood. Further metabolite profiling could enhance our understanding of the utilization value of Epichloë strains for agricultural benefit. In this study, we employed Liquid Chromatography-Mass Spectrometry (LC-MS) to analyze the metabolites of five Epichloë strains (IB8, QG6, ED11, AD16 and LE7) across three Epichloë species (Epichloë sibirica, E. sinensis, E. bromicola) isolated from five grass species (Achnatherum sibiricum, Psathyrostachys lanuginosa, Festuca sinensis, Elymus cylindricus, and E. dahuricus). Our analysis sought to identify both common and strain-specific metabolites. The findings revealed that 1752 compounds, encompassing 15 distinct classes, were detected across five endophytic fungal strains. The predominant components comprised lipids and lipid-like substances (25.63%), organic acids and their derivatives (24.49%), and organic heterocyclic compounds (12.90%). Although strains with varying alkaloid-producing potential generated similar categories of metabolites, they exhibited quantitative differences. Unique metabolites from strains IB8, AD16, LE7, and ED11 were associated with at least one specific functional pathway each. These pathways corresponded to penicillin and cephalosporin biosynthesis (IB8), folate biosynthesis (AD16), sesquiterpene and triterpenoid biosynthesis (LE7), and the sulfur relay system (ED11). The next phase of this research will focus on elucidating functional linkages between these characterized metabolites and defensive phenotypes in endophyte-symbiotic grasses.

Although drunken horse grass (Achnatherum inebrians) can be simultaneously infected by the foliar endophyte Epichloë gansuensis and colonized by Bacillus subtilis, it remains unclear whether Epichloë endophyte symbiosis influences B. subtilis colonization, as well as how their interaction affects nitrogen fixation and assimilation. The purpose of the present study was to investigate whether E. gansuensis endophyte infection facilitates the colonization of B. subtilis in the roots of host plants, with a focus on understanding the interaction effects of the E. gansuensis endophyte and B. subtilis on plant growth and nutrient absorption. In this study, we measured the colony growth rate of B. subtilis LZU7 when co-cultured with E. gansuensis strains. In addition to an in vitro test, we investigated the root colonization of Epichloë endophyte-infected plants (E+) and Epichloë endophyte-free plants (E−) with the GFP-tagged B. subtilis LZU7 in an inoculation test. Furthermore, we evaluated the interactions between E. gansuensis endophyte symbiosis and B. subtilis LZU7 colonization on the dry weight, nitrogen fixation, nitrogen converting-enzyme activity, and nutrients for E+ and E− plants by labeling with 15N2. The results showed that the growth rates of B. subtilis LZU7 were altered and increased in a co-culture with the E. gansuensis endophyte. A significantly greater colonization of GFP-tagged B. subtilis LZU7 was detected in the roots of E+ plants compared with the roots of E− plants, suggesting that E. gansuensis endophyte symbiosis enhances the colonization of beneficial microorganisms. The combination of E. gansuensis endophyte symbiosis and B. subtilis LZU7 inoculation significantly altered the expression of the nitrogenase (nifH) gene, thereby promoting increased biological nitrogen fixation (BNF). The E. gansuensis endophyte infection and inoculation with B. subtilis LZU7 significantly increased δ15NAir in plants. Co-inoculation with the E. gansuensis endophyte and B. subtilis LZU7 significantly elevated NH4+ accumulation in the roots, depleted the NH4+ availability in the surrounding soil, and showed no measurable impact on the foliar NH4+ content. The observed alterations in the NH4+ content were linked to nitrogen-fixing microorganisms that promoted nitrogen fixation, thereby enhancing nitrogen uptake and contributing to greater biomass production in A. inebrians. Our findings highlighted the fact that a foliar symbiosis with the E. gansuensis endophyte enhances the recruitment of beneficial bacteria, and that the resulting interaction significantly impacts nitrogen fixation, assimilation, and allocation in host plants.

Grassland degradation leads to biodiversity loss and shifts in soil microbial communities, increasing the risk of disease outbreaks. This study aimed to identify how changes in plant and soil microbial communities influence plant diseases in degraded grasslands. A 2-year field study of plant communities across the Tibetan Plateau was conducted to examine how grassland degradation influences disease severity. Soil microbial communities were characterized by amplicon sequencing. The ITS1-5.8S-ITS2 region of fungi and the V1–V9 regions of the bacterial 16S rRNA gene were amplified. This observational study explored potential associations among vegetation characteristics, soil microbial communities, and foliar fungal disease severity. Disease severity increased with the degree of grassland degradation by 13.08