Exploring The Diversity Of Begomoviruses In Whiteflies By Metagenomic Sequencing

Whitefly-transmitted geminiviruses (WTGs), belong to the genus begomovirus of the Geminiviridae, spread by whiteflies, can damage a variety of main economic plants. In this study, we utilize a novel approach of sampling of the polyphagy and highly mobile insect vectors whiteflies, followed by purification of viral particles from whiteflies, metagenomic sequencing and molecular biology methods to explore the viral community and diversity of WTGs in China.From 2011 to 2015, we collected 23 batches of whiteflies from Yunnan, Guangxi, Guangdong, Hainan, Zhejiang and Shandong Provinces. Viral DNA extracted from whiteflies was divided into 9 samples and sequenced by Illumina Miseq platform. A lot of viruses were identified through contigs assembly and sequence alignment. We verified the sequencing results by PCR and the results showed 18 begomoviruses,8 betasatellites,2 alphasatellites,10 defective interfering DNA derived from DNA A and 1 defective interfering DNA derived from betasatellite were found in all whiteflies samples. Among 18 begomoviruses, a new species, Whitefly Transmitted Geminiviruse A (WTGA) and a virus which firstly reported in China, Ludwigia yellow vein Vietnam virus (LuYVVNV), were identified. In Yunnan Province, there identified 11 begomoviruses, containing Malvastrum yellow vein Yunnan virus (MYVYnV), Papaya leaf curl China virus (PaLCuCNV), Sweet potato leaf curl virus (SPLCV), LuYVVNV, Ludwigia yellow vein virus (LuYVV), Tobacco leaf curl Yunnan virus (TbLCYnV), Tomato yellow leaf curl China virus (TYLCCNV), Ageratum yellow vein China virus (AYVCNV), Tomato yellow leaf curl virus (TYLCV), Pepper yellow leaf curl China virus (PepYLCCNV) and WTGA. In Guangxi Province,9 begomoviruses PaLCuCNV, AYVCNV, WTGA, MYVYnV, SPLCV, Squash leaf curl China virus (SLCCNV), Tomato leaf curl China virus (ToLCCNV), Tomato leaf curl Guangxi virus (ToLCGxV) and Pouzolzia golden mosaic virus (PGMV) were found. In Hainan Province,4 begomoviruses SPLCV, SLCCNV, LuYVVNV and Sida leaf curl virus (SiLCV) were identified. In Zhejiang Province,4 begomoviruses TYLCV, SPLCV, Sweet potato leaf curl Georgia virus (SPLCGV) and Sweet potato leaf curl Henan4 virus (SPLCHn4V) were isolated. TYLCV was the only begomoviruse identified in Shandong Provinces. No begomoviruse was found in Guangdong Province. We chose 13 begomoviruses to analyze their identity and variation. Compared with the begomoviruse isolated from plants, the sequence identity among the begomovirus isolated from whiteflies showed more higher, while the variation degree of the begomovirus isolated from whiteflies was lower, and the variation sites of the viral genome were different. Phylogenetic relationships showed that all the begomoviruses from whiteflies and plants cluster into different branchs according to the different sample region.A new begomovirus species was identified from samples collected in Yunnan and Guangxi Provinces. The full length of DNA A was 2744 bp, and shared the highest nucleotide sequence identity (87%) with Croton yellow vein mosaic virus (CYVMV) reported in India, and the name of Whitefly Transmitted Geminiviruse A (WTGA) was proposed. WTGA was a monopartite begomovirus without betasatellite. Sequence analyses suggested that WTGA may have arisen by recombination among begomovirus related to CYVMV, Squash leaf curl Yunnan virus (SLCYnV), Synedrella leaf curl virus (SyLCV), Tobacco curly shoot virus (TbCSV) and Tobacco leaf curl Thailand virus (TbLCTHV). The infectious clones of WTGA was constructed and infected into Nicotiana benthamiana seedlings. Both plants inoculated by WTGA alone or co-inoculated with TYLCCNB/AYVCNB could induce typical symptoms as upward leaf curling, vein swelling, chlorisis and dwarfing. The plants co-inoculated by WTGA and betasatellites developed symptoms three to four days earlier, and showed more serious symptoms than that of plants inoculated by WTGA alone. Southern blot result showed the WTGA DNA accumulation were detected in all plants inoculated by WTGA alone and co-inoculated with betasatellites.LuYVVNV was identified in Yunnan and Hainan Province, and this is the first report of LuYVVNV in China. The full length of LuYVVNV DNA A was 2762 bp and shared 94.2%nucleotide sequence identity with Ludwigia yellow vein Vietnam virus reported in Vietnam. LuYVVNV was a monopartite begomovirus without betasatellite, and has a typical genome organization of geminiviruses. The infectious clones of LuYVVNV was constructed and infected into Nicotiana benthamiana seedlings. Both plants inoculated by LuYVVNV alone or co-inoculated with TYLCCNB/AYVCNB could induce typical symptoms as leaf downward curling, crimping, yellowing and dwarfing. The plants co-inoculated by LuYVVNV and betasatellites showed more serious symptoms than that of plants inoculated by LuYVVNV alone. Southern blot result showed the DNA accumulation of LuYVVNV were detected in all plants inoculated by LuYVVNV alone and co-inoculated with betasatellites.