| Tobacco virus disease is one of most destructive diseases to tobacco leaf production, and more than 30 virus species could infect tobacco. Mosaic disease in field is very common, while the incidence of leaf curl disease is increasing. In order to elucidate virus types in tobacco plants in Yunnan, China, tobacco mosaic and leaf curl disease samples are collected in Yunnan and virus types were tested by serological and molecular methods, virus disease control measures are evaluated.Tobacco leaf samples showing mosaic symptoms collected from Yunnan province were tested for virus types by using antigen direct coated, double antibody sandwich or triple antibody sandwich (TAS) enzyme-linked immunosorbent assay (ELISA), and TAS-ELISA was then used for subgrouping of Cucumber mosaic virus (CMV) samples. Among 520 tobacco samples collected in Yunnan province, 71.74%, 55.01% and 6.35% samples were infected by Tobacco mosaic virus (TMV), CMV and Potato virus Y (PVY), respectively. Among 64 CMV samples from Yunnan, Hunan and Fujian province, 89.1% samples were infected by subgroup I isolates, and 15.6% were infected by subgroup II isolates.The leaf curl samples were tested for virus types by PCR using three primer specific to Tobacco curly shoot virus (TbCSV), Tobacco leaf curl Yunnan virus (TbLCYNV) and Tomato yellow leaf curl China virus (TYLCCNV), respectively. The representative samples were amplified using begomoviruses universal primers PA and PB, and PCR products were cloned and sequenced. PCR detections demonstrate that tobacco leaf curl disease in Yunnan is associated with three begomoviruses including TbCSV, TbLCYNV and TYLCCNV. Among 109 leaf curl tobacco samples collected, 41.3%, 16.5% and 53.2% samples were infected by TbCSV, TbLCYNV and TYLCCNV, respectively, 8.2% samples were mix-infected by two or three virus. Alignment of the 500 bp sequences showed that nucleotide sequence identities among these samples ranged from 77%～100%. TYLCCNV samples could be further divided into two groups based on geographic distribution, samples collected from Baoshan form one group with 93% nucleotide sequence identity, while those from Wenshan and Honghe form another group with 95% nucleotide identity. However, nucleotide sequence identity between thetwo groups is only 89%.Based on the 500 bp sequence, isolate Y264 was chosen to be sequenced completely. Sequence of Y264 comprised 2741 nucleotides (nts), having a genomic organization typical of begomoviruses originating from the Old World. Y264 is most closely related to TYLCCNV-[Y10] with 91.3 % nucleotide sequence identity for DNA-A and 87.6%～95.7% amino acid sequence identities for the encoded proteins, therefore Y264 should be considered as a isolate of TYLCCNV.Tobacco float seedlings with or without mosaic symptom collected before transplant were tested for virus types by TAS-ELISA, among 1400 float seedling samples collected in Yunnan province, 95% positive samples were infected by TMV, while less than 5% positive samples were infected by CMV or PVY. Tobacco leaf samples showing mosaic symptoms were collected in field plants at rosette period when mosaic incidence was above 20% and tested for virus types, among 480 samples, 48%～100% samples were infected by TMV, and 0%～12.2% samples were infected by CMV, PVY or Tobacco etched virus, indicating TMV is the dominant virus infecting tobacco float seedling in Yunnan province.Inactivation effects to TMV of 9 chemicals were tested and their injury symptom and dosage to tobacco seedling leaf were evaluated. The local lesion assay indicated when the following chemical solutions were mixed (1/1,V/V) with TMV infected leaf extracts diluted in 1:10000 for 3 min, TMV was inactivated with 100% efficiency, these chemicals were 1:10 solution of 30% chloride of lime, 1:10 solution of 98% Trisodium phosphate, 1:150 solution of 7% sodium hypochloride, 1:125 solution of anonymity disinfectant used in USA and 75% alcohol solution. 1:200 solution of 10% chlorine dioxide and 1:150 solution of 40% Yubao could inactivate TMV with 100% and 96.3% efficiencies, respectively, when the solutions were mixed with diluted TMV leaf extracts for 30 min, and the following chemicals had only less than 80% efficiency in the same time for mixture: 1:125 solution of 0.6% Jundujin, 1:800 solution of 3.95% Bingdubike and 1:800 solution of 24% Duxiao. Except for 0.6% Jundujing, the other tested chemicals in normal dosage could induce seedling leaf injury with chlorotic spot, necrotic spot and disorder symptoms which varied among different chemicals.Tests were also carried out for evaluation the inactivation effect to TMV and influence to tobacco seed germinate and seedling growth of chemicals used in water pool for tobacco float seedling growth. The local lesion assay indicated when the following chemical solutions were mixed (1/1,V/V) with TMV infected leaf extracts diluted in 1:10000 for 12 h, they could inactivate TMV with more than 80% efficiency, these chemicals were 30% chloride of lime at 15 mg/L and 150 mg/L, 7% sodium hypochlorite at 2μl/L and 20μl/L, anonymity disinfectant used in USA at 5μl/L and 50μl/L. The seed germinate rate and seedling rate show no obvious difference between treatment and control, but seedlings grew slowly in later growth for some chemicals. The following chemicals could promote seedling growing with recommended concentrations, these chemicals were 15 mg/L～150 mg/L Trisodium phosphate, 50 mg/L～100 mg/L copper sulfate, 10 mg/L～40 mg/L potassium permanganate and 10μl/L Duxiao. The following chemicals show no influence to seedling growth at low concentration but inhibite seedling growth at high concentration, these chemicals were 40% Yubao, 30% chloride of lime, 7% sodium hypochlorite, anonymity disinfectant used in USA and 0.6% Jundujing. To avoid negative effects to seedling growing, the following concentrations were recommended: 40% Yubao at 100μl/L, 30% chloride of lime at 300mg/L, 7% sodium hypochlorite at 20μl/L, anonymity disinfectant used in USA at 50μl/L and 0.6% Jundujing at 100μl/L.
|
PDF Full Text Request