Molecular identification of pathogens infecting Millettia speciosa Champ. mosaic disease in Guangxi and complete genome sequence analysis based on small RNA deep sequencing

【Objective】 To clarify the viral pathogens which caused Millettia speciosa Champ. exhibiting mottled mosaic symptom in Nanning and Qinzhou of Guangxi，which could provide scientific basis for prevention and control of Millettia speciosa Champ. mosaic disease. 【Method】 During 2022-2024，leaf samples of Millettia speciosa Champ. suspected infected by viruses were collected from Nanning and Qinzhou. The methods of small RNA deep sequencing（RNA-Seq）， reverse transcription polymerase chain reaction（RT-PCR），segmental cloning，sequence analysis and alignment，phylogenetic tree construction were applied for virus identification and phylogenetic analysis on suspected infectious samples. 【Result】 The results showed that 49 contigs obtained by RNA-seq were annotated as viral genome，of which 25 contigs were annotated as cowpea mild mottle virus（CpMMV），2 contigs were annotated as cucurbit chlorotic yellows virus （CCYV）and 22 contigs were annotated as lettuce chlorosis virus（LCV）. These results above indicated that the Millettia speciosa Champ. samples might infected by the 3 viruses：CpMMV，CCYV and LCV. The RT-PCR test results showed that only the target band of CpMMV could be amplified in the samples，while LCV and CCYV failed to amplify the target band，confirming that the collected Millettia speciosa Champ. samples were infected by CpMMV. One sample of Millettia speciosa Champ. from Nanning with positive CpMMV detection was randomly selected. A CpMMV complete genome sequence with a total length of 8194 bp was successfully obtained by the method of segmental cloning and named CpMMV_GX_NiuDaLi. BLASTn analysis of the sequence in GenBank showed that the sequence had a high nucleotide similarity with each isolate of CpMMV registered in GenBank，nucleotide similarity of CpMMV_NJ_JS（GenBank accession number： OK625819.1） and CpMMV_JS_NJ （GenBank accession number：MW354944.1）was the highest （97.46%）. Analysis of the phylogenetic evolutionary tree constructed based on the complete genome sequence of CpMMV and the sequence of the CP gene showed that the isolates used for the analysis were divided into 2 large branches，the isolates from China in a big branch and all foreign isolates，except for the New Delhi isolate from New Delhi，India（GenBank accession number：OQ791180.1），were in another major branch. It indicated that these isolates have regional distribution characteristics. CpMMV_GX_NiuDaLi was in a large branch with various isolates from China， and was also in a small branch with CpMMV_NJ_JS and CpMMV_JS_NJ，indicating that there was close genetic relationship among the 3 isolates. 【Conclusion】 The samples of Millettia speciosa Champ. with mottled leaves in Nanning and Qinzhou of Guangxi are infected by CpMMV.