关键词: 中国南瓜曲叶病毒, 广东省, 分子特征, 侵染性克隆, 致病性

Abstract:

【Objective】Squash leaf curl China virus (SLCCNV) is one of the main viruses infecting Cucurbitaceae crops, which can infect pumpkin, cucurbits, wax gourd, cucumber, muskmelon and honeydew melon, and severely affect Cucurbitaceae crop production. The objective of this study is to investigate the molecular characteristics, genetic relationship and pathogenicity of SLCCNV Guangdong isolates, and to provide a theoretical basis for the prevention and control of SLCCNV.【Method】Fifty-three suspected samples infected by Begomovirus were collected from Leizhou City and Xuwen County of Zhanjiang City, Boluo County of Huizhou City, Yuancheng District of Heyuan City and Gaoming District of Foshan City, Guangdong Province. Total DNA was extracted from all suspected samples, respectively, and used as template for PCR amplification with degenerate begomovirus primers AV₄₉₄/CoPR. The full-length DNA-A sequence of each isolate was obtained by RCA amplification, gene cloning and sequencing from positive samples by PCR detection. The full-length DNA-B sequence of each isolate was obtained by PCR amplification using designed abutting primers. All full-length sequences of SLCCNV obtained from Guangdong Province were analyzed with BLAST in NCBI. The similarity was compared using MUSCLE alignment of SDT1.2 software. Phylogenetic analysis between SLCCNV Guangdong isolates and the other SLCCNV isolates was performed using MEGA7. Infectious clone of SLCCNV isolate HYNG was constructed using homologous recombination technology. The infectious clone was inoculated to the cotyledon of pumpkin plants by agro-inoculation.【Result】The PCR detection result showed that 52 out of 53 suspected samples were infected by Begomovirus. The full-length sequences of eight isolates of SLCCNV from Guangdong Province were obtained from pumpkin, cucurbits, wax gourd and white melon, respectively. The full DNA-A sequences of eight isolates ranged from 2 735 to 2 739 nt in size, contained six ORFs, which were identical to the reported SLCCNV. The full DNA-B sequences of eight Guangdong isolates of SLCCNV ranged from 2 701 to 2 721 nt in size, contained two ORFs, encoding proteins associated with viral movement BC1 and BV1, respectively. The similarity analysis of the full sequence showed that the DNA-A of eight isolates from Guangdong shared >94.9% nt identities with each other, and shared >88% nt identities with other isolates of SLCCNV in GenBank database, and the DNA-B of eight isolates from Guangdong shared >86.7% nt identities with each other, and shared >83% nt identities with isolates of SLCCNV in GenBank database. Phylogenetic analysis indicated that the eight Guangdong isolates clustered with SLCCNV isolates from Guangxi, Henan, Hainan of China, Vietnam, Thailand, Philippines, Indonesia to form one branch, and the isolates from India clustered to form the other branch. Pathogenicity results showed that the new leaves of pumpkin inoculated with infectious clones of SLCCNV displayed crimple symptoms at 15 days post-inoculation (dpi), and more severe symptoms at 30 dpi. SLCCNV could be detected by PCR from the symptomatic plants, Western blot detection further verified the above results.【Conclusion】SLCCNV was detected infecting Cucurbitaceae crops in Guangdong Province. The SLCCNV is the pathogen causing pumpkin curl leaf disease in Guangdong Province. The SLCCNV isolates from Guangdong are closely related to isolates from Guangxi, Hainan etc., belonging to the same branch, while it is more distantly related to isolates from India.

Key words: squash leaf curl China virus (SLCCNV), Guangdong Province, molecular characteristic, infectious clone, pathogenicity