Role of the beneficial phyllosphere microbiome in the defense against red clover anthracnose caused by Colletotrichum americae-borealis.

Red clover (Trifolium pratense), a high-quality forage plant, faces significant threats from anthracnose in northeastern China, but the pathogen responsible remains unidentified. The phyllosphere microbiota is crucial in plantpathogen interactions, yet its role in the resistance of red clover to anthracnose is poorly understood. Using morphological, molecular, and multigene phylogenetic analyses, we identified Colletotrichum americae-borealis (Cab) as the pathogen that causes anthracnose in red clover in China. We also investigated changes in the phyllosphere microbiomes of highly resistant (XJ) and susceptible (SC) red clover materials after Cab infection, via 16S rRNA gene sequencing. The results revealed significant differences in bacterial α- and β-diversity, with novel microbial taxa and a complex microbial network emerging postinfection. Notably, after Cab inoculation, the Shannon diversity index in XJ exhibited more pronounced changes, manifested as an increase in the abundance of beneficial microorganisms such as Bacillus, Pantoea, and Pseudomonas. Network analysis revealed that the XJ microbiome was more complex and stable than the SC microbiome was, regardless of infection status. Bacillus J2, the dominant bacterium, significantly inhibited Cab growth in vitro and reduced the disease index by 33.4-47.7 % when it was reapplied to the leaf surface, suggesting its role in enhancing disease resistance. This study is the first to report that C. americae-borealis causes anthracnose in red clover in China, and demonstrates the potential of the beneficial bacterium J2 in enhancing disease resistance, providing insights into disease resistance mechanisms and the role of the phyllosphere microbiome in pathogen challenge.