广东省番茄白尖曲叶病病原鉴定及其致病性分析OA北大核心CSTPCD

[Objective]Lisianthus enation leaf curl virus(LELCV)is a new species of the genus Begomovirus discovered in 2015.The objective of this study is to explore the molecular characteristics,genetic relationship,pathogenicity and effective transmission vector of the first isolate Tomato-2022 of LELCV invading mainland China,and to provide a theoretical basis for the prevention and control of the virus disease.[Method]In 2022,anew disease of tomato occurred in Zengcheng District,Guangzhou City,Guangdong Province.The diseased plants exhibited small leaf,curling and leaf tips whitening symptoms.Total DNA was extracted from two diseased samples,respectively,and used as template for PCR detection with degenerate Begomovirus primers AV494/CoPR.The full genome sequence of isolate Tomato-2022 was obtained by RCA amplification,gene cloning and sequencing from positive samples by PCR detection.The virus sequence was analyzed with BLASTn program and the representative isolate sequences with high similarity were downloaded.Further the sequence similarity was compared using MUSCLE alignment of SDTv1.2 software.Phylogenetic analysis between isolate Tomato-2022 and the reported representative isolates with high similarity was performed using MEGA7.0.Infectious clone pGreenⅡ0229-1.6A of isolate Tomato-2022 was constructed using restriction enzyme digestion and ligation.The infectious clone was inoculated to Nicotiana benthamiana leaves and tomato stems by Agrobacterium-mediated injection to determine its pathogenicity.The efficiency of Bemisia tabaci MEAM1 cryptic species transmitting isolate Tomato-2022 was determined by indoor artificial virus transmission method.PCR specific primers of LELCV were used to detect 135 diseased tomato samples collected from the main tomato producing areas of Guangdong Province from 2021 to 2023 to clarify the distribution of LELCV in Guangdong Province.[Result]The PCR detection result showed that two diseased tomato samples with small leaf,curling and leaf tips whitening symptoms were infected by begomoviruses.Gene cloning and sequence analysis indicated that the genome of the virus isolate Tomato-2022 contained only DNA-A,with a size of 2 757 nt,and encoded six ORFs.The comparison of sequence similarity showed that the genomic sequence of isolate Tomato-2022 had high similarity with LELCV isolates registered in GenBank,and had the highest similarity with LELCV Taiwan,China Eustoma grandiflorum isolates(LC091539,LC091538)at 98.33％.Phylogenetic analysis indicated that Tomato-2022 and 19 isolates of LELCV were clustered in a separate branch and had a close relationship.The constructed Tomato-2022 infectious clone pGreenⅡ0229-1.6A was inoculated into N.benthamiana and tomato by Agrobacterium-mediated injection.At 10 days post-inoculation(dpi),the upper leaf margins of N.benthamiana showed slight upward curling symptoms,and new leaves of one tomato plant showed slight curling symptoms.At 18 dpi,N.benthamiana and tomato plants showed more obvious curling symptoms.As time went by,the symptoms of the inoculated plants became more and more obvious.At 30 dpi,N.benthamiana showed veins swelling and leaves severe curling symptoms,and the leaves of tomato plants showed curling and leaf tips whitening symptoms.The results of PCR showed that LELCV could be detected in both the inoculated plants.The indoor virus transmission experiments proved that B.tabaci MEAM1 cryptic species could efficiently transmit Tomato-2022.When each tomato plant was inoculated with 1,5 and 10 MEAM1 B.tabaci fed LELCV for 48 h,the average virus transmission efficiency was 26.67％,93.33％and 96.67％,respectively.LELCV was detected from 8 of 135 diseased tomato samples by PCR using specific primer,indicating that LELCV had been colonized in Guangdong Province.[Conclusion]Tomato-2022 is a new isolate of LELCV,and also the first isolate found in Chinese mainland.Its genome is a single-stranded circular DNA with a total length of 2 757 nt,encoding six ORFs.The virus isolate is the pathogen of tomato curl leaf and white tip disease in Guangdong Province,and can be efficiently transmitted by B.tabaci MEAM1 cryptic species.