Molecular identification of tobacco leaf curl disease in Sichuan province of China

Background Tobacco leaf curl disease (TLCD) is caused by begomoviruses in Geminiviridae, and infected plants exhibit leaf thickening, downward leaf curling, vein swelling as well as stunting symptoms. It is one of the economically important diseases in tropical and subtropical tobacco-growing areas. Seven monopartite begomoviruses have been identified causing TLCD in China. Findings In this study, two begomoviruses were identified, characterized and polygenetically analyzed to be responsible for TLCD in Sichuan province, China. The complete genomes of two isolates SC230 and SC379 from diseased tobacco samples were cloned and sequenced to be 2738 nucleotides (nts) and 2748 nts in size, respectively. Sequence alignment indicated that SC230 and SC379 were most closely related to Tomato yellow leaf curl China virus (TYLCCNV-CN[CN:Sc226:Mal:12]) and Papaya leaf curl China virus (PaLCuCNV-CN[CN:Gx30:Lyc:03]), with a sequence identity of 99.2 and 99.2 %, respectively. The infection rate of TYLCCNV and PaLCuCNV was 100 and 34.78 %, respectively and the co-infection rate was 34.78 % in fields. Betasatellites of SC230 and SC379 share the highest sequence identity with Tomato yellow leaf curl China betasatellite (TYLCCNB-CN[CN:Sc176:Malva:12]) and TYLCCNB-CN[CN:Yn149:Tom:09], with a sequence identity of 95.2 and 97.2 % respectively. Sequence identity between betasatellites of SC230 and SC379 was 89.6 %. And TYLCCNB was detected in all the samples. Conclusion Co-infection of TYLCCNV and PaLCuCNV was identified in tobacco plants with typical symptoms of TLCD from Sichuan province in China, and this is the first report of PaLCuCNV infecting tobacco in China. TYLCCNV/TYLCCNB disease complex is widespread in tobacco-growing areas in Panzhihua city of Sichuan. Electronic supplementary material The online version of this article (doi:10.1186/s12985-015-0461-7) contains supplementary material, which is available to authorized users.

Recently, the occurrence of begomoviruses in Sichuan is more and more frequent. TbCSV was firstly reported infecting pepper. The co-infection of TYLCCNV/TYLCCNB and PaLCuCNV was identified in tomato, which caused serious tomato yellow leaf curl disease (TYLCD), and TYLCCNV/TYLCCNB was found infecting Malva rotundifolia Linn. Malvastrum coromandelianum, a widespread weed in tropical regions, was infected by TYLCCNV, Malvastrum yellow vein Yunnan virus (MYVYNV) and Malvastrum yellow vein virus (MYVV) associated betasatellite [7,[16][17][18]. However none of begomoviruses was found on tobacco. In this study we report the occurrence of TLCD associated begomoviruses in Panzhihua city of Sichuan province, and the infection of PaLCuCNV in tobacco for the first time in China.

Methods
Twenty three samples of tobacco plants with typical leaf thickening, downward leaf curling, vein swelling, yellow vein and stunting symptoms were collected in two fields at a distance of about 80 kilometers in Panzhihua city of Sichuan province (southwestern China) in August, 2012, including eight samples showing very severe leaf thickening, leaf crinkling, enation and stunting symptoms ( Fig. 1) [see Additional file 1]. In addition, four symptomless tobacco plants were randomly collected from the same fields.
Total DNA was extracted using CTAB method. Degenerate primer pair PA/PB specific for members of the genus Begomovirus was used for detection [19]. The PCR products were cloned into pGEM-T Easy Vector (Promega, Madison, WI, USA) and four clones were randomly selected and sequenced. The sequencing of clones was performed by DNA sequencer in Beijing Genomics Institute. Sequences were assembled and analyzed with the aid of the DNAStar software version 6.0 (DNAStar Inc., Madison, WI, USA) and MEGA 6.0.6.

Results and discussion
A 500 bp DNA fragment covering parts of the intergenic region (IR) and V2 gene of the genomes of begomoviruses was amplified from all 23 symptomatic samples using degenerate primer pair PA/PB. No positive fragment was detectable in any symptomless samples. Four clones from isolate SC225, SC230, SC378, SC240 were randomly selected to be sequenced [GenBank: KF640690, KF640691, KF640692 and KF640693]. The sequence comparison revealed that the 4 clones were most closely related to TYLCCNV, with a sequence identity ranges from 94.5 to 97.7 %.
In order to identify the occurrence of the other previously reported begomoviruses in Sichuan in symptomatic samples, PCR detection with specific primer pairs was conducted and results showed that only TYLCCNV (in all 23 samples) and PaLCuCNV (in 8 samples of   (AV2 and AV1) in the virion-sense strand and four ORFs (AC1 to AC4) in the complementary-sense strand, which separated by the intergenic region (IR). The IR contains a putative stem-loop structure sequence with the conserved nonanucleotide sequence TAATATTAC in the loop, and this motif contains the nicking site for the initiation of rolling circle replication [22]. Generally, the isolates of begomoviruses showing more than 91 % identity are considered to be strains of the same species [23]. SC230 and SC379 are thus considered to be isolates of TYLCCNV and PaLCuCNV respectively, and we suggested the name that TYLCCNV-CN  (Fig. 2).

A B
With the primers beta01 and beta02 for betasatellite DNA, an amplicon of 1300 bp was obtained from all symptomatic samples and none of fragment was detectable in symptomless samples. Sequence comparison showed that the betasatellite from SC379 is 1339 bp long Sc230:12] was 89.6 %. These two molecules of betasatellite were consistently clustered with the TYLCCNB isolates (Fig. 3). And TYLCCNB was detectable in all symptomatic samples by PCR using specific primer pair Y10beta/beta02.
In general, co-infection of TYLCCNV and PaLCuCNV were detected in tobacco plants with typical symptoms of TLCD, and TYLCCNV/TYLCCNB disease complexes are widespread in infected tobacco plants in Panzhihua city of Sichuan province. These results were consistent with other reports in which PaLCuCNV is a monopartite begomovirus with no satellite, and TYLCCNV is always associated with its cognate satellite TYLCCNB forming begomovirus/betasatellite disease complex [24,25]. TLCD commonly occurs in Yunnan, Fujian, Guangxi and Guangdong provinces in China and has a trend of spreading. In Sichuan province, TYLCD has caused great losses of tomato cultivation, but TLCD has not been found on tobacco so far. To our knowledge, PaLCuCNV commonly infects tomato, Ageratum conyzoides, Corchoropsis tomentosa and papaya, TYLCCNV/TYLCCNB complex infects tomato, tobacco, Datura stramonium L., Siegesbeckia orientalis, Solanum aculeatissimum and Malva rotundifolia Linn. in China [17,[26][27][28][29][30][31][32][33]. In this study, we reported occurrence of TLCD in Sichuan province and PaLCuCNV infecting tobacco for the first time in China, and identified co-infection of PaLCuCNV and TYLCCNV associated with TYLCCNB. Previous studies have revealed that betasatellite could be trans-replicated by noncognate begomoviruses and the host range of begomoviruses could be enlarged while associated with betasatellite [34,35]. In this study, co-infection of TYLCCNV and PaLCuCNV was only detected in the 8 samples with more severe leaf thickening, leaf crinkling, enation and stunting symptoms, which is consistant with the previous report that the tomato yellow leaf curl disease caused by co-infection of TYLCCNV and PaLCuCNV is more severe than that caused by TYLCCNV/TYLCCNB. The results suggested the synergism between TYLCCNV and PaLCuCNV probably occurred. Therefore, it would be interesting to illustrate the role of TYLCCNB on the infection of PaLCuCNV to the new host and the interaction between TYLCCNV and PaLCuCNV in the future.