Cotton leaf curl Multan virus newly reported to be associated with cotton leaf curl disease in China

Cotton (family Malvaceae, genus Gossypium) is one of the most important crops in China.In October 2008 and July 2009, some cotton plants were found to develop upward leaf curl symptoms accompanied by thickening and often thick dark greening of leaf veins, leafy enations and stunting in a field of the suburb of Nanning city, Guangxi, China (Figs. 1, 2). Leaf samples of three individual cotton plants exhibiting leaf curl symptoms were collected in 2009 and tested for the presence of geminiviruses. Using the universal begomovirus-specific primers A and B (Liu et al., 1998) which amplify the partial intergenic region and V2 gene, all three samples gave approximately 500bp PCR products which were cloned and sequenced. Their sequences were almost identical. Full-length DNA-A of isolate GX1 was amplified by PCR using additional primers designed on the basis of the determined sequences and cloned. The complete DNA-A sequence of isolate GX1 (CLCuMV-[CN:GX1:09]) was determined to be 2738 nucleotides (GenBank Accession No. GQ924756). A comparison with other begomoviruses showed that DNA-A of isolate GX1 has more than 99.7% nucleotide sequence identity to Cotton leaf curl Multan virus (CLCuMV) isolates G6 (EF465535) and Okra06 (FJ770370) and shares 93.7% to 95.6% nucleotide sequence identity to other CLCuMV isolates CLCuMV (GenomeNet NC_004607), CLCuMV-26 (AJ002458), CLCuMV-62 (AJ002447) and CLCuMV-Ok ( AJ002459).

The previously characterized CLCuMV isolates have been shown to be associated with DNA β molecules (Briddon et al., 2001; Mao et al., 2008). To test whether a satellite molecule was associated with this isolate, PCR was performed with a pair of DNA β conserved primers (Briddon et al., 2002). About 1.3 kb PCR products were obtained from all three samples, cloned and sequenced, revealing that the sequences from all samples were almost identical. Full-length DNA β of isolate GX1 was amplified using additional primers designed on the basis of the determined sequences and cloned. The 1346 bp full-length sequence of DNA β of isolate GX1 (CLCuMB-[CN:GX1:09]) was submitted to GenBank (GQ906588). A comparison with other satellite molecules showed that the DNA β of isolate GX1 has more than 99.4% nucleotide sequence identity to CLCuMV satellite DNA β of isolates G6 (EF465536) and Okra06 (FJ770371), and 91.4% and 92.4% nucleotide sequence identity to CLCuMB-[PK:Fai1:96] (AJ298903) and CLCuMB-[IN:Bha:05] (DQ191161), respectively. Based on the symptoms and sequence information, the cotton leaf curl disease in China is associated with CLuCMV. To our knowledge, this is the first report of a virus associated with cotton disease in China .