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First Report of Tobacco curly shoot virus Infecting Pepper in China

In recent years, whitefly-transmitted begomovirues (family Geminiviridae) have caused severe leaf curl disease on tobacco and tomato in southern China, but have not been found on pepper. In August 2009, pepper plants (Capsicum frutescens) grown in the field in Panzhihua City of Sichuan Province (southwestern China), from where the occurrence of begomoviruses has not been reported previously, showed stunting, leaf yellowing, and mild curling symptoms. To identify possible begomoviruses, total DNA was extracted from three infected pepper plants (SC117, SC118, and SC119) with typical symptoms. Using degenerate primer pair PA/PB specific for members of the genus Begomovirus (2), a 500-bp DNA fragment covering parts of the intergenic region and V2 gene of the genome of begomoviruses was amplified from all samples. No amplification was observed from healthy plant extracts. The PCR product from SC118 was cloned and two clones were chosen to be sequenced. Alignment of the partial DNA sequences revealed that the cloned products from isolate SC118 were nearly identical (98.5%) and most closely related to Tobacco curly shoot virus isolate Y35 (TbCSV-[China:Yunnan 35:2001]; Accession No. AJ420318) (96.9 and 97.3% identity, respectively). Therefore, the entire genome of isolate SC118 was sequenced. Overlap primers TbCSV-F(5′-CCGCCGTCTCAACTTCGACAG-3′) and TbCSV-R(5′-ATCTGCTGGTCGCTTCGACAT-3′) were designed to amplify the full-length genome of SC118. The complete genome sequence of SC118 was determined to be 2,746 nucleotides (Accession No. GU001879) long, with two open reading frames (ORFs) in the virion-sense strand and four ORFs in the complementary-sense strand, typical of the Old World begomoviruses. A comparison with other reported sequences of begomoviruses shows that the genome of SC118 shares the highest nucleotide sequence identity (99.7%) with TbCSV-[China:Yunnan 35:2001]. When PCR was used to detect TbCSV from the other two isolates (SC117 and SC119) with TbCSV specific primer pair Y35F1 and Y35+10R (4), which amplified the fragment covering the whole C2 and C3 genes and the partial C1 and V1 genes of the genome of TbCSV, an amplicon of approximately 1.0 kb was obtained from all samples. To determine whether a satellite molecule was associated with the three virus isolates, a universal betasatellite abutting primer pair (beta01 and beta02) was used (1). No amplification product was detected. In previous studies, it was demonstrated that only 11 isolates were associated with betasatellites among 39 TbCSV-infected, field-collected samples (3), and betasatellites could be associated with noncognate begomoviruses (4). Therefore, the three isolates examined in this study are too few to come to a conclusion that betasatellites are not associated with TbCSV infection of pepper plants. A detailed search for the presence of betasatellites needs to be conducted to draw a definitive conclusion. The above results confirmed that samples SC117, SC118, and SC119 were infected by TbCSV. To our knowledge, this is the first report of TbCSV on pepper in China.

References: (1) R. W. Briddon et al. Mol. Biotechnol. 20:317, 2002. (2) D. Deng et al. Ann. Appl. Biol. 125:327, 1994. (3) Z. Li et al. Phytopathology 95:902, 2005. (4) L. Qing et al. Phytopathology 99:716, 2009.