Open Access

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

  • Chenchen Jing1,
  • Chunyan Wang1,
  • Ke Li1,
  • Gentu Wu1,
  • Xianchao Sun1 and
  • Ling Qing1Email author
Virology Journal201613:4

https://doi.org/10.1186/s12985-015-0461-7

Received: 8 September 2015

Accepted: 30 December 2015

Published: 6 January 2016

Abstract

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.

Keywords

Tobacco Tomato yellow leaf curl China virus Papaya leaf curl China virus Co-infection

Background

Geminiviruses are a large family of plant viruses with circular, single-stranded DNA genome encapsidated in unique twinned particles. They consist of seven genera (Mastrevirus, Curtovirus, Topocuvirus, Begomovirus, Becurtovirus, Eragrovirus and Turncurtovirus) on the basis of genome structure, host range and insect vector [1, 2]. Most of the economically important geminiviruses belong to the genus Begomovirus, which are transmitted by Bemisia tabaci to a wide range of dicotyledonous plants [3]. Begomoviruses cause significant economic losses to many crops, including common bean, cotton, cucurbits, okra, tomato, pepper and tobacco [47]. In China, TLCD caused by begomoviruses was first observed in Yunnan and Fujian province in 1982 [8]. Recently, TLCD has occurred in Guangxi and Guangdong [9, 10]. So far seven distinct monopartite begomoviruses have been identified causing TLCD in China, including TYLCCNV, Tobacco leaf curl virus (TbLCV), Tomato leaf curl China virus (ToLCCNV), Tobacco leaf curl Yunnan virus (TLCYNV), Tobacco curly shoot virus (TbCSV), Ageratum yellow vein virus (AYVV) and Papaya leaf curl Guangdong virus (PaLCuGuV) [915].

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, 1618]. 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.
Fig. 1

The typical symptoms of tobacco plant infected by tobacco leaf curl disease in the field. a The symptoms of tobacco plant infected by TYLCCNV + TYLCCNB; b The symptoms of tobacco plant infected by PaLCuCNV + TYLCCNV + TYLCCNB

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.

To identify the possible viruses in these tobacco samples, the specific primer pairs of begomoviruses which reported occurring in Sichuan province were either designed based on their conserved region obtained from multiple sequence alignment of the corresponding isolates in GenBank or cited from previous studies. These primer pairs are specific for PaLCuCNV (YM1P-F: 5′-TCACAAACAAAAGGAGGTCA-3′/YM1P-R: 5′-GAATATGTAACACATTCAA-3′), MYVV (MY-F: 5′-GCCCACTAACTACTGTCTG-3′/MY-R: 5′-TTAGAGGCATGGGTACATGC-3′), MYVYNV (MYV-F: 5′-GTTCTTTGGTTGAGGCAG-3′/MYV-R: 5′-TTAGAGGCATGGGTACATGC-3′), TbCSV (TbCSV-F/YnR) and TYLCCNV (TYLCCNV-F/YnR) [20], respectively.

Overlapping primers SC379-Pa-F (5′-GTTGTATATGGCATGTACTCATGC-3′) SC379-Pa-R (5′-CCGATACATCCTGGGCTTTCGGTA-3′) and TYLCCNV-F (5′-ACCGGATGTACAGAAGCCCTGA-3′)/TYLCCNV-R (5′-ATGTACCGGAAGCCCATGATGTAC-3′) were designed for amplification of full length DNA of PaLCuCNV and TYLCCNV, respectively. The universal abutting primers beta01 and beta02 were used to amplify the DNA molecules of betasatellites [21]. The primer pairs Y10beta (5′-CGGCATTATTTTGAGGCAGT-3′)/beta02 were used for specific amplification of Tomato yellow leaf curl China betasatellite (TYLCCNB). Products of PCR amplification of viral DNA and betasatellite were cloned and sequenced. All of the sequences used for comparison were obtained from GenBank. The sequences of begomoviruses and betasatellites, occurred in Sichuan and causing TLCD in China, were selected for construction of phylogenetic tree [see Additional file 2 & Additional file 3]. Phylogenetic trees were constructed by the neighbour-joining method with 1000 bootstrap replications using MEGA6.0.6.

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 SC228, SC230, SC232, SC236, SC238, SC240, SC242 and SC379) were detectable, and the co-infection rate of PaLCuCNV and TYLCCNV was 34.78 % [see Additional file 1].

The complete DNA sequences of isolate SC230 and SC379 were determined to be 2738 nts [GenBank: KF640689] and 2748 nts [GenBank: KF373768], respectively. A comparison with other begomoviruses showed that DNA of SC230 was closely related to TYLCCNV-CN [CN:Sc226:Mal:12] (99.2 % identity), and SC379 was closely related to PaLCuCNV-CN[CN:Gx30:Lyc:03] (99.2 % identity). The genomic organization of two DNA sequences were typical of the Old World begomoviruses, with two ORFs (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[CN:Sc230:Tob:14] and PaLCuCNV-CN[CN:C379:Tob:12]. The relationship dendrogram of the complete nucleotide sequences of PaLCuCNV-CN[CN:C379:Tob:12], TYLCCNV-CN[CN:Sc230:Tob:14] and other begomoviruses indicates that PaLCuCNV-CN[CN:C379:Tob:12] was grouped into a single cluster together with PaLCuCNV-CN[CN:Gx2:Pap:04] and PaLCuCNV-CN[CN:Gx4:Pap:04], and TYLCCNV-CN[CN:Sc230:Tob:14] was grouped into a single cluster together with TYLCCNV-CN[CN:Sc226:Mal:12] (Fig. 2).
Fig. 2

Phylogenetic tree of complete DNA of TYLCCNV-CN[CN:Sc230:Tob:12] and PaLCuCNV-CN[CN:Sc379:Tob:12]. The phylogenetic tree was constructed with neighbour-joining method with 1000 bootstrap replications and viewed with the help of MEGA 6.0.6. Triangle symbol indicates the position of TYLCCNV-CN[CN:Sc230:Tob:12] and PaLCuCNV-CN[CN:Sc379:Tob:12]

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 [GenBank: KF640695] and had the highest sequence identity with Tomato yellow leaf curl China betasatellite (TYLCCNB-CN[CN:Yn149:09]) 97.2 % identity), and the betasatellite from SC230 is 1337 bp long [GenBank: KF640694] and had the highest sequence identity with TYLCCNB-CN[CN:Sc176:Malva:12] (95.2 % identity). These results suggested that both of betasatellites are isolates of TYLCCNB. The sequence identity between TYLCCNB-CN[CN:Sc379:12] and TYLCCNB-CN[CN: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.
Fig. 3

Phylogenetic tree of betasatellites associated with TYLCCNV-CN[CN:Sc230:Tob:12] and PaLCuCNV-CN[CN:Sc379:Tob:12]. The phylogenetic tree was constructed with neighbour-joining method with 1000 bootstrap replications and viewed with the help of MEGA 6.0.6. Triangle symbol indicates the positions of TYLCCNB-CN[CN:Sc379:Tob:12] and TYLCCNB-CN[CN:Sc230:Tob:12]

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, 2633]. 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.

Declarations

Acknowledgements

This research work was supported by National Natural Science Founding of China (Grant No. 31272013) and Program for New Century Excellent Talents in University by the Ministry of Education in China (NCET-12-0931).

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Chongqing Key Laboratory of Plant Disease Biology, College of Plant Protection, Southwest University

References

  1. Brown JK, Fauquet CM, Briddon RW, Zerbini M, Moriones E, Navas-Castillo J. Geminiviridae. In: AMQ AM CE K, Lefkowitz EJ, editors. Virus taxonomy Ninth Report of the International Committee on Taxonomy of Viruses. London: Elsevier Publication; 2012.Google Scholar
  2. Varsani A, Navas-Castillo J, Moriones E, Hernandez-Zepeda C, Idris A, Brown JK, et al. Establishment of three new genera in the family Geminiviridae: Becurtovirus, Eragrovirus and Turncurtovirus. Arch Virol. 2014;159:2193–203.View ArticlePubMedGoogle Scholar
  3. Harrison BD, Robinson DJ. Natural genomic and antigenic variation in whitefly-transmitted geminiviruses (begomoviruses). Annu Rev Phytopathol. 1999;37:369–98.View ArticlePubMedGoogle Scholar
  4. Briddon RW, Markham PG. Cotton leaf curl virus disease. Virus Res. 2000;71:151–9.View ArticlePubMedGoogle Scholar
  5. Moffat AS. Geminiviruses emerge as serious crop threat. Science. 1999;286:1835.View ArticleGoogle Scholar
  6. Moriones E, Navas-Castillo J. Tomato yellow leaf curl virus, an emerging virus complex causing epidemics worldwide. Virus Res. 2000;71:123–34.View ArticlePubMedGoogle Scholar
  7. Qing L, Xiong Y, Sun X, Yang S, Zhou C. First report of Tobacco curly shoot virus infecting pepper in China. Plant Dis. 2010;94:637.View ArticleGoogle Scholar
  8. Gong Z, Shen J, Zheng Q, Chen Z, Cao T, Chen R, et al. The first geminiviruses in China-tobacco leaf curl virus isolation and identification by electrical microscopy. Chin Sci Bull. 1982;27:1393–5.Google Scholar
  9. Meng J, Li Z, Wei M. Molecular identification of the causal agents causing tobacco leaf curl disease in some regions of Guangxi. Plant Prot. 2012;2:37–41.Google Scholar
  10. Yang C, Zheng L, Wu Z, Xie L. Papaya leaf curl Guangdong virus and ageratum yellow vein virus associated with leaf curl disease of tobacco in China. J Phytopathol. 2013;161:201–4.View ArticleGoogle Scholar
  11. Gong Z, Shen J, Zheng Q, Chen Z, Cao T, Chen R, et al. The first case of geminivirus in China-tobacco leaf curl virus. Chin Sci Bull. 1983;28:1249.Google Scholar
  12. Li B, Li Z, Wei M, Lu Y, Liao Y, Chen B, et al. Genomic characterization of DNA-A and associated satellite DNA molecule of an isolate of tomato leaf curl China virus infecting tobacco. Gen App Biol. 2011;30:281–7.Google Scholar
  13. Liu Z, Yang C, Jia S, Zhang P, Xie L, Xie L, et al. First report of ageratum yellow vein virus causing tobacco leaf curl disease in Fujian Province. China Plant Dis. 2008;92:177.View ArticleGoogle Scholar
  14. Zhou X, Xie Y, Zhang Z. Molecular characterization of a distinct begomovirus infecting tobacco in Yunnan, China. Arch Virol. 2001;146:1599–606.View ArticlePubMedGoogle Scholar
  15. Xie Y, Zhou XP, Zhang ZK, Qi YJ. Tobacco curly shoot virus isolated in Yunnan is a distinct species of Begomovirus. Chin Sci Bull. 2002;47:197–200.View ArticleGoogle Scholar
  16. Xiong Y, Zhou C, Li Y, Wang C, Sun X, Qing L. The More Severe Tomato Yellow Leaf Curl Disease is Caused by Co-infection of PaLCuCNV and TYLCCNV. Acta Hortic Sin. 2014;41:268–76.Google Scholar
  17. Li K, Zhang J, Jing C, Wu G, Sun X, Qing L. First report of tomato yellow leaf curl China virus infecting malva rotundifolia in China. J Plant Pathol. 2015;97:547.Google Scholar
  18. Ruan T, Yu Y, Bao L, Zhang J, Qing L. Identification and detection of mix-infection of whitefly-transmitted geminiviruses on Malvastrum coromandelianum in Miyi County of Sichuan Province. Acta Phytopathol Sin. 2011;5:419–24.Google Scholar
  19. Deng D, McGrath PF, Robinson DJ, Harrison BD. Detection and differentiation of whitefly-transmitted geminiviruses in plants and vector insects by the polymerase chain reaction with degenerate primers. Ann Appl Biol. 1994;125:327–36.View ArticleGoogle Scholar
  20. Liu Y, Xie Y, Liao B, Mugiira RB, Zhou X. Occurrence and distribution of geminiviruses in tobacco in Yunnan province of China. Acta Phytopathol Sin. 2007;37:566–71.Google Scholar
  21. Briddon RW, Bull SE, Mansoor S, Amin I, Markham PG. Universal primers for the PCR-mediated amplification of DNA beta: a molecule associated with some monopartite begomoviruses. Mol Biotechnol. 2002;20:315–8.View ArticlePubMedGoogle Scholar
  22. Heyraud F, Matzeit V, Schaefer S, Schell J, Gronenborn B. The conserved nonanucleotide motif of the geminivirus stem-loop sequence promotes replicational release of virus molecules from redundant copies. Biochimie. 1993;75:605–15.View ArticlePubMedGoogle Scholar
  23. Brown J, Zerbini FM, Navas-Castillo J, Moriones E, Ramos-Sobrinho R, Silva JF, et al. Revision of Begomovirus taxonomy based on pairwise sequence comparisons. Arch Virol. 2015;160:1593–619.View ArticlePubMedGoogle Scholar
  24. Wang X, Xie Y, Zhou X. Molecular characterization of two distinct begomoviruses from Papaya in China. Virus Genes. 2004;29:303–9.View ArticlePubMedGoogle Scholar
  25. Zhou X, Xie Y, Tao X, Zhang Z, Li Z, Fauquet CM. Characterization of DNA? 2 associated with begomoviruses in China and evidence for co-evolution with their cognate viral DNA-A. J Gen Virol. 2003;84:237–47.View ArticlePubMedGoogle Scholar
  26. Zhang H, Ma X, Qian Y, Zhou X. Molecular characterization and infectivity of papaya leaf curl china virus infecting tomato in china. J Zhejiang Univ Sci B. 2010;11:109–14.View ArticlePubMedPubMed CentralGoogle Scholar
  27. Jiao X, Gong H, Liu X, Xie Y, Zhou X. Etiology of Ageratum Yellow Vein Diseases in South China. Plant Dis. 2013;97:1497–503.View ArticleGoogle Scholar
  28. Cai J, Qin B, Xie Y, Chen Y. Occurrence,transmission via whitefly and intermediate hosts of Papaya leaf curl China virus in Nanning. Plant Prot. 2007;33:57-9.Google Scholar
  29. Xiong Y, Yang S, Qing L, Zhou C, Sun X, Yang S. Molecular identification and variation analysis of the pathogen causing tomato yellow leaf curl disease in Sichuan Province. Sci Agric Sin. 2011;44:477–84.Google Scholar
  30. Xu Y, Zhou X. Genomic characterization of Tomato yellow leaf curl China virus and its associated satellite DNA infecting tobacco in Guangxi. Acta Microbiol Sin. 2006;46:358–62.Google Scholar
  31. Ding M, Yang C, Luo Y, Yue N, Fang Q, Su X, et al. Molecular characterization of Tomato yellow leaf curl China virus and its associated satellite DNA?2 infecting Datura stramonium L. Acta Phytopathol Sin. 2008;4:364–9.Google Scholar
  32. Peng Y, Xie Y, Zhou X. Molecular characterization of Tomato yellow leaf curl China virus and its associated satellite DNA infecting Siegesbeckia orientalis. Acta Microbiol Sin. 2004;44:29–33.Google Scholar
  33. Liao B, Liu Y, Xie Y, Zhou X. Genomic characterization of DNA-A and associated satellite DNA molecule of an isolate of Tomato yellow leaf curl China virus in Solanum aculeatissimum. Acta Phytopathol Sin. 2007;37:138–43.Google Scholar
  34. Saunders K, Salim N, Mali VR, Malathi VG, Briddon R, Markham PG, et al. Characterisation of Sri Lankan Cassava Mosaic Virus and Indian Cassava Mosaic Virus: Evidence for Acquisition of a DNA B Component by a Monopartite Begomovirus. Virology. 2002;293:63–74.View ArticlePubMedGoogle Scholar
  35. Qing L, Zhou X. Trans-replication of, and competition between, DNA beta satellites in plants inoculated with Tomato yellow leaf curl China virus and Tobacco curly shoot virus. Phytopathology. 2009;99:716–20.View ArticlePubMedGoogle Scholar

Copyright

© Jing et al. 2016

Advertisement