Yan-Zhong Li

Alfalfa Verticillium wilt is a soil‐borne disease that severely limits alfalfa yield and quality, yet effective control measures remain scarce. In this study, four Bacillus strains (i.e., Bacillus amyloliquefaciens LYZ1125, B. velezensis LYZ1126, B. atrophaeus LYZ1127 and B. subtilis LYZ1128) were used singly or in combination to evaluate their biocontrol efficacy. In greenhouse, the strains reduced disease incidence by approximately 60%, decreased the disease index by about 80%, and achieved 80% control efficacy, significantly outperforming the chemical fungicides 0.0052 mg L ⁻¹ trifloxystrobin‐tebuconazole (60%) and 0.0446 mg L ⁻¹ azoxystrobin (30%). The biocontrol treatments promoted alfalfa growth, increasing plant height, stem diameter, root length, and dry weight per plant by 25%–37%, 20%–27%, 17%–48% and 39%–88%, respectively. Used singly or in combination, they also induced systemic resistance, with salicylic acid and jasmonic acid increasing by 15%–86% and over 33%, respectively. Net photosynthetic rate and chlorophyll content increased by approximately twofold and 4%–83%, respectively, along with enhanced peroxidase activity, while hydrogen peroxide and malondialdehyde accumulation decreased compared with the control. Alfalfa quality improved, with crude protein, soluble sugar, magnesium, and potassium increasing by 8%–30%, 3%–16%, 9%–22%, and 3%–14%, respectively, and reductions in acid and neutral detergent fibre. In the field, alfalfa Verticillium wilt occurred only in the control and thiram‐treated plots, with an incidence rate of approximately 1%. Bacillus mixture (1:1:1:1) outperformed single strains and controls, effectively managing wilt and promoting alfalfa growth via resistance and photosynthesis.

Although Alternaria gansuense causes yellow stunt and root rot (YSRR)—a destructive disease of the leguminous forage Astragalus adsurgens in northern China—systematic investigations of this pathogen at the protein level remain scarce. In this study, we establish an optimized proteomic workflow for A. gansuense by comparing two protein extraction methods. Using data-independent acquisition (DIA) mass spectrometry with a DIA-NN search against the Alternaria protein database, we construct the first comprehensive proteome reference map of A. gansuense, then perform functional annotation via Gene Ontology, KOG, KEGG, InterPro domain, and subcellular localization analyses. A total of 5052 proteins were identified from vegetative mycelia, and the proteome was found to be predominantly composed of proteins involved in primary metabolism, signal transduction, secondary metabolism, and stress responses. Notably, a set of putative pathogenicity-associated proteins, including two-component regulators (SSK1p), protein kinases, cytochrome P450 enzymes, and ABC transporters, was identified. These proteins are homologs of well-characterized virulence factors in other pathogenic fungi, suggesting a potential coordinated signaling—metabolism—defense network that may contribute to fungal virulence. Subcellular localization further shows that cytoplasmic and nuclear proteins together account for over 50% of the annotated proteome. This study presents the first comprehensive proteomic reference map for A. gansuense, providing a valuable resource for functional genomics. Subsequent experimental validation of the bioinformatically predicted candidate molecular targets presented here may help to dissect the pathogenic mechanisms of YSRR and enable the development of novel disease management strategies.

Grassland ecosystems harbor diverse plant-associated fungal communities that contribute to ecosystem functioning and plant health. However, large-scale culture-based inventories of root-invading fungi across multiple grassland regions in China remain scarce. In this study, we conducted a multi-region survey of culturable root-invading fungi isolated from surface-sterilized, discolored root and basal stem tissues in contact with the soil. We sampled 33 herbaceous plant species, representing 19 genera and 9 families, across 17 counties in Qinghai, Xinjiang, Inner Mongolia, and Xizang. A total of 273 fungal isolates were preserved and identified using ITS sequence analysis complemented by morphological observations. These isolates were classified into 40 genera and 53 species-level taxa spanning several major fungal lineages. A limited number of genera, including Fusarium , Darksidea , and Preussia , accounted for a substantial proportion of the isolates, whereas numerous low-frequency taxa were also recovered. Fungi were consistently isolated from both root and basal stem tissues, indicating extensive internal colonization of symptomatic tissues across these plant compartments. Significant differences were observed between plant-level and tissue-level isolation rates within both Poaceae and Cyperaceae. Functional guild assignment revealed the presence of endophytic, saprotrophic, and pathogenic taxa, with relative proportions varying among plant groups. Analysis revealed strong fungus-host associations, with Cramer’s V of 0.850 indicating a high overall association strength and indicator species analysis identifying eight host-associated taxa. This study provides a culture-based baseline inventory of root-invading fungi associated with natural grassland plants in China and establishes a curated isolate collection for future ecological and functional investigations.

Paraphoma radicina is a newly reported pathogen causing alfalfa Paraphoma root rot (APRR). Although alfalfa cultivars with different resistance levels to P. radicina have been identified, the field occurrence of APRR and the host responses associated with resistance remain unclear. This study investigated APRR occurrence and its effect on alfalfa biomass, the histological, physiological, and biochemical responses in roots of resistant and susceptible cultivars after P. radicina infection, and the effect of root exudates from different cultivars on P. radicina growth. Field investigations showed that APRR occurrence tended to be higher in older stands and was associated with reduced alfalfa biomass during spring. Inoculation with P. radicina caused a greater reduction in plant growth and dry weight in the susceptible cultivar than in the resistant cultivar. Histological observations revealed severe phloem cell damage, as well as protoplast shrinkage and detachment from the cell wall in cambial cells of the susceptible cultivar at 49 dpi. Infection also caused significant changes in photosynthetic and chlorophyll fluorescence parameters in the susceptible cultivar. The activities of defense enzymes were increased after infection, especially in the resistant cultivar. Root exudates from the resistant cultivar more effectively inhibited P. radicina growth. These findings suggest that the resistant alfalfa cultivar enhances APRR resistance through physiological and biochemical defense responses and root exudate suppression of P. radicina growth.

Pigeonpea (Cajanus cajan) shoot tip dieback was recently observed in Danzhou, Hainan Province, China, with disease incidence of 30–100% in affected fields, but its causal agent and management options remained unclear. This study aimed to isolate and identify the fungal agent associated with the disease, verify its pathogenicity, characterize its biological traits and effects on host growth, and screen candidate fungicides. Fungal isolates were obtained from symptomatic shoot margins and purified by hyphal-tip and single-spore isolation. Representative isolates LYZ0717, LYZ0718, and LYZ0719 were characterized by colony morphology, pycnidial and conidial traits, and combined phylogenetic analysis of ITS and TEF1-α sequences. These analyses identified the isolates as Lasiodiplodia theobromae. Spray inoculation of 20-day-old pigeonpea seedlings reproduced typical shoot tip dieback symptoms within 6 days, and the inoculated fungus was reisolated from symptomatic tissues, fulfilling Koch’s postulates. The pathogen grew over a pH range of 3–11 and at temperatures of 5–35 °C, with optimal mycelial growth at 25–30 °C and maximum sporulation under acidic conditions. At 20 days after inoculation, infection reduced plant height, dry weight, and total flavonoid content by 45.21%, 75.88%, and 42.19%, respectively. Among the seven fungicides tested, trifloxystrobin–tebuconazole and thiophanate-methyl showed the strongest in vitro inhibition, with EC₅₀ values of 0.0270 and 0.0614 ppm, respectively. In greenhouse pot experiments, thiophanate-methyl achieved the highest disease control efficacy, reaching 64.35% after the second application. These findings demonstrate that L. theobromae is the causal agent of pigeonpea shoot tip dieback in China and provide a basis for disease diagnosis, biological understanding, and fungicide-based management.

Lespedeza davurica is an important leguminous forage species widely distributed in China’s natural grasslands, where it contributes significantly to ecological stability and sustainable animal husbandry. However, a severe root rot disease, with disease incidence up to 100% has been observed in surveyed areas of Huan County, Gansu Province, China, causing substantial damage to the host plant, while the causal agent remains uncharacterized. Four fungal isolates recovered from symptomatic roots were identified as a novel species of Monosporascus based on morphological characteristics and phylogenetic analyses of ITS, LSU, and Tub2 sequences. The isolates were formally described as Monosporascus lespedezae sp. nov. Pathogenicity tests confirmed high virulence, with inoculated plants showing 100% disease incidence and significant reductions in root length (81.3%) and plant height (63.0%) compared to non-inoculated controls ( P < 0.001). In vitro fungicide sensitivity assays showed differential responses among tested compounds: diniconazole exhibited the lowest EC ₅₀ value (0.5198 μg/mL), whereas carbendazim completely inhibited mycelial growth at 10 μg/mL (EC ₅₀ = 2.791 μg/mL). Both fungicides show promise for further valuation. This study represents the first report of M. lespedezae sp. nov. as a pathogen of L. davurica , establishes its taxonomic position, and provides baseline in vitro fungicide sensitivity data, thereby laying a scientific foundation for developing effective disease management strategies.

Alfalfa Verticillium wilt, caused by Verticillium alfalfae , is a destructive disease spreading through the Northwest and central Gansu province, severely impacting alfalfa production. This study compared the community composition of rhizosphere soils from alfalfa varieties with different resistance using high-throughput sequencing. The relative abundances of Devosia and Novosphingobium were higher in the rhizosphere of the highly resistant Gannong No.4 than in Saranac. Redundancy analysis revealed that the abundances of these two bacterial genera were positively correlated with soil total nitrogen, available potassium, organic matter, and pH. Alpha diversity analysis further revealed that the rhizosphere soil of Gannong No.4 had a higher Simpson diversity index. LefSe analysis indicated that Bacillota, Bacillales, Bacillaceae, and Actinobacteria were key bacterial taxa in the rhizospheres of varieties with differing resistance. A total of 50 bacterial and 6 fungal strains were isolated from the stems and rhizosphere soils of different alfalfa varieties. Molecular characterization revealed that the bacteria comprised 48 species from 18 genera, of which 60% belonged to Bacillota and 26% belonged to Actinobacteria. Fifty bacterial strains were tested for antagonistic activity against V. alfalfae LYZ0257. Among these strains, seven exhibited an inhibition rate greater than 60%, and five ( Streptomyces galilaeus LYZ1124, Bacillus amyloliquefaciens LYZ1125, Bacillus velezensis LYZ1126, Bacillus atrophaeus LYZ1127, and Bacillus subtilis LYZ1128) were as effective as the control strain ( B. amyloliquefaciens LYZ69). Additionally, B. amyloliquefaciens LYZ1125, B . velezensis LYZ1126, B. atrophaeus LYZ1127, and B. subtilis LYZ1128 demonstrated notable capabilities in potassium solubilization, protein dissolution, and nitrogen fixation. Consequently, the rhizosphere soil of highly resistant alfalfa varieties may recruit abundant beneficial microbial communities against V. alfalfae .

Climate change‐induced grassland degradation has exacerbated the spread of toxic plants, yet many potentially toxic species remain overlooked, undermining rangeland management and causing significant economic losses. Quantifying the toxicity and distribution of potential toxic plants under climate change is critical for mitigating biogeographic risks. As a case study, taking Sphaerophysa salsula , a leguminous plant distributed in Asia and the Americas, historically utilized for erosion control but recently associated with livestock poisoning, this research integrated toxicity identification, species distribution modeling (SDM), and risk assessment to evaluate its biogeographic threats in China. Results suggested for the first time that S. salsula can function as a high‐toxicity (chemotype 1) locoweed due to swainsonine (mean content 0.373%), produced by its endophyte Alternaria oxytropis (23.46 pg/ng), which is implicated in locoism‐like syndromes in livestock. The Maximum Entropy model identified temperature annual range (43.22°C), mean temperature of the driest quarter (−6.29°C), and soil pH (8.61) as key distribution drivers. Currently, suitable habitats are concentrated in Northern China (Xinjiang, Inner Mongolia, Ningxia). By the 2070s, these habitats are projected to decline by 6.3%–9%, shifting westward toward pastoral regions. Risk assessments integrating grazing intensity revealed high‐risk zones in Gansu, Ningxia, and Inner Mongolia, with future scenarios predicting declining risks in eastern Inner Mongolia but increasing threats in western Tibet. These findings clarify S. salsula 's toxic mechanism and biogeographic risks, providing a framework for targeted management of overlooked toxic plants under climate change.

Plant endophytes, often termed the “second genome”, critically shape host adaptability. However, the complexity of their interactions, regulated by microbial traits, host species, and environment, has limited both our understanding of symbiosis and the application of beneficial endophytes. The symbiosis between locoweeds (Oxytropis and Astragalus species) and the endophyte Alternaria sect. Undifilum, which produces the neurotoxin swainsonine, serves as an ideal model for investigating these relationships. Through extensive national surveys (2021–2023) across China’s major locoweed habitats, combining field sampling with cultivation, molecular, quantitative, and modeling approaches, a central question emerged: To what extent are the distribution and function of this symbiosis shaped by the contemporary environment versus host evolutionary history? The results showed that: (1) Among 32 surveyed species of Oxytropis, Astragalus, and Sphaerophysa, the endophyte Alternaria sect. Undifilum colonized 11 species. In colonized plants, endophyte loads ranged from 0.02 to 58.87 pg/ng total DNA, and swainsonine concentrations varied from 0.00003% to 1.00%. (2) Environmental factors, rather than host phylogeny, were the key driver governing the geographical distribution and expression of the symbiosis. (3) Low temperature and drought stress regulated the symbiotic relationship and chemical defense through both direct effects on the symbionts and indirect pathways involving grazing pressure. This study demonstrates that the environment is the core force dominating the geographical pattern and functional expression of the locoweed–endophyte symbiosis at ecological scales. These findings provide new perspectives for understanding the general principles of plant–endophyte symbiosis and establish a scientific foundation for predicting and utilizing endophyte resources in changing environments.