Identification of Soybean Bush Dwarf Disease Associated with Co-infection of a Phytoplasma and Cowpea Mild Mottle Virus in Guangxi, China

Phytoplasmas and viruses are important pathogens of soybean (Glycine max) and other legumes, posing a severe threat to global legume yields. In this study, three soybean plants exhibiting severe bush dwarfing, stem necrosis, leaf deformation and wrinkling were collected from Nanning, Guangxi Zhuang Autonomous Region, China. High-throughput sequencing (HTS) coupled with reverse transcription-PCR (RT-PCR) were employed for virome analysis, while nested-PCR and conventional PCR with 16S ribosomal RNA (16S rRNA) universal and tuf gene-specific primers were used for the specific detection of phytoplasmas. All three samples were confirmed to be co-infected with Cowpea mild mottle virus (CPMMV) and Candidatus Phytoplasma. The complete genome sequence of CPMMV was determined, which showed the highest nucleotide identity with the CPMMV isolate GX NiuDaLi, and the putative new isolate was named CPMMV GX-NN. Biological inoculation assays showed that CPMMV GX-NN could infect Glycine max, Vigna sinensis and Nicotiana benthamiana, inducing typical viral symptoms. Phylogenetic analysis based on 16S rRNA sequences and virtual restriction fragment length polymorphism (RFLP) via iPhyClassifier were further conducted and the phytoplasma was classified into the 16SrII Group. This is the first report of co-infection by CPMMV and a phytoplasma in soybean plants in Guangxi, China, providing critical insights for diagnosing and managing soybean diseases caused by these pathogens.