Endophytic fungi in green manure crops; friends or foe?

Astragalus sinicus and Vicia villosa; are frequently applied green manure plants used in China. However, there is poor knowledge of the fungal endophytic community and the mycobiome of green manure crops. Field surveys were conducted during 2017-2019 in four provinces in China. Asymptomatic plant parts were collected. Using a culture-dependent method, 517 fungal isolates were obtained from Astragalus sinicus and Vicia villosa. These isolates were further identified using a combination of morphological and multi -loci phylogenetic analyses and were differentiated into 30 species in 15 genera in ten families belonging to only Ascomycota. Most isolated strains belonged to Sordariomycetes. The most dominant genus was Fusarium, with 381 isolates from both crops, while all other taxa were isolated less than 40 times. The similarity search on the Fusarium MLST database showed the 370 strains belonged to seven Fusarium complexes and one subclade. Eleven strains could not be assigned to any complex. The remaining 136 isolates were identified and assigned to 23 known and seven novel species. A total of 178 Operational Taxonomic Units (OTUs) were obtained from Illumina analysis and mainly classified into five phyla (Ascomycota, Basidiomycota, Chytridiomycota, Cryptomycota, and Mucoromycota). Overall OTUs were further assigned to 21 classes, 48 orders, 66 families, and 74 genera. Based on overall OTUs, the most abundant species was Alternaria alternata, which was also isolated from the culture -dependent method. Most species and genera recorded from the High Throughput Sequencing (HTS) approach were not obtained in the culture -dependent method (Boeremia, Cladosporium, Filobasidium, Magnoporthe, Mucor, Rhizoctonia, Sporidiobolus). Functional annotation reveals that all Ascomycetes genera obtained in both approaches comprised several plant pathogenic species. Potential beneficial and/or biocontrol strains were also identified. The common green manure crops used in China harbors a hidden, underexplored mycobiome which may comprise potential for application. These results will increase awareness of green manure practices. Precautions need to be in place when incorporating green manure crops in the soil, as these could facilitate inoculum sources for the next disease cycle of the main crop.