四川番茄黄化曲叶病病原分子鉴定及变异分析

Molecular Identification and Variation Analysis of the Pathogen Causing Tomato Yellow Leaf Curl Disease in Sichuan Province

【目的】明确引起四川番茄黄化曲叶病(Tomato yellow leaf curl disease,TYLCD)的病原。【方法】对采自四川攀枝花市田间表现矮化、黄化和曲叶症状的番茄植株SC64-67,通过PCR、克隆及测序等技术获得病毒及卫星DNA的全基因组序列,并对序列进行变异分析。【结果】利用双生病毒简并引物PA/PB从4个样品中均扩增得到约500 bp的片段,随机选择SC65进行DNA-A全基因组扩增和序列测定,该DNA分子全长为2 732 nts,系统进化分析表明,其与已报道的中国番茄黄化曲叶病毒(Tomato yellow leaf curl China virus,TYLCCNV)来自云南元谋的菜豆分离物(TYLCCNV-Bean-YM)的核苷酸序列相似性最高,为96.0%。检测发现,所有分离物均伴随有卫星DNAβ分子。全序列测定表明SC65 DNAβ全长1 338 nts,与分离自云南楚雄番茄上的TYLCCNV-Y25伴随的卫星DNAβ亲缘关系最近,核苷酸序列相似性为77.5%。【结论】研究表明四川番茄黄化曲叶病样品均受到TYLCCNV/DNAβ病害复合体的侵染,但其病毒DNA-A和卫星DNAβ分子来源于TYLCCNV的不同分离物。

【Objective】In this study,pathogenic agents associated with tomato yellow leaf curl disease in Panzhihua,Sichuan Province in China were identified.【Method】Diseased tomato plants exhabiting typical stunting,yellowing and leaf-curling symptoms were collected in Panzhihua,Sichuan Province.Using PCR,cloning,and sequencing techniques a begomovirus disease complex associated with tomato was identified for phylogenetic analysis.【Result】500 bp fragments were amplified from four samples（SC64-67） using the begomovirus degenerate primer pair PA/PB.The complete nucleotide sequence of isolate SC65 was determined to be 2 732 nts and shared the highest sequence identity（96.0%） with an isolate of Tomato yellow leaf curl China virus（TYLCCNV-Bean-YM） isolated from kidney bean in Yuanmou,Yunnan Province.All tomato samples were found to be associated with the satellite DNAβ molecules.The satellite DNAβ molecule associated with SC65（SC65 DNAβ） consisted of 1 338 nts and shared 77.5% nucleotide sequence identity with DNAβ associated with TYLCCNV-Y25 isolated from tomato plants in Chuxiong,Yunnan Province.【Conclusion】These results suggested that tomato yellow leaf curl disease in Sichuan Province is associated with the TYLCCNV/DNAβ disease complex which may originate from DNA-A and DNAβ molecules of different TYLCCNV isolates.