| Tobacco mosaic virus(TMV), a type member of Tobamovirus, has caused great losses to many plants yields. The previous studies on the population and occurrence frequency of tobacco virus disease in Fujian in our institute(Institute of Plant Virology, Fujian Agriculture and Forestry University) suggested that the ratio of TMV was high in a few main tobacco fields and TMV common strain was dominant strain. Using the cross-protection of attenuated strain to protect plant from infecting by virulent strain of the same virus was proved one of ideal means. On the basis of these results, we studied the methods of obtaining the mild strains of TMV as well as their biology and molecular biology features.TMV commom strain was isolated from Fujian tobacco region, then purified and identified. Two symptomless TMV mutants named TMV-O 17 and TMV- 152 respectively were obtained by treatment of heating, heating and nitrous acid combination. The effects on the effectiveness of TMV mild strain cross-protection, of the inoculation concentration of mild and virulent strains, inoculation interval time between mild and virulent strains, the inoculation position of the virulent strain and the age of tobacco, were observed by orthogonal designing. The inoculation interval time and the age of tobacco were the two most important factors. The cross-protection of mild strains were obvious when inoculation interval time between two strains over 12 days. The root soak was a method to mass inoculation of mild strains.We examined the effects of mild strains of TMV on the tobacco plants. The rate of virus multiplication, the conents of chlorophyll a, chlorophyll b, total chlorophyll and soluble protein were detected, in the inoculated leaves of the tobacco plants infected by virulent and two mild strains of TMV respectively. The rates of replication and the steady concentrations of the two mild strains were less than those of the virulent strain. The contents of chlorophyll a, chlorophyll b, total chlorophyll all decreased in the leaves infected by the virulent and two mild strains respectively, but those of the virulent strain declined the most. Thecontents of soluble protein all changed unconspicuously.With electron microscope techniques, the cytopathological changes of the tobacco leaves infected by TMV-W and TMV-O 17 were observed. The results showed that the chloroplasts of health plant had double-membrance, and the stroma lamellae arranged tight and regular, containing a few of large starch granules. The quantity of chloroplasts was decrease of the leaves infected by TMV-O1 7. The chloroplasts had shaped membrance structure. The stroma lamellae also arranged tight and regular. Some chloroplasts enlarged, containing few little starch granules. The quantity of mitochondrias was increased to 4-6 around the chloroplasts. TMV-O 17 induced ciystal structure in cytoplasts, which appeared around the chloroplasts. Some chloroplasts of the leaves infected by TMV-W were destroyed, the stroma lainellae were caused to be loosed. The quantity of mitochondrias was also increased and enlarged, some even irregular. The rhombic crystal structure were also observed in cytoplasts around the chloroplasts.The sequences of genomic cDNA of TMV-W, TMV-O 17 and TMV- 152 were detected(GenBank accession numbers are AF395127, AF395128, AF395129 respectively). Sequence analysis showed that the whole genomes of the three strains were 6395nt with 4 open reading frames(ORF) encoding for l26kDa protein(69-3419nt, with 11 l6aa), l83kDa protein(69-4919nt, with 1616aa), movement protein(4903-5709nt, with 268aa) and coat protein (5712-6191nt, with lS9aa) respectively. They possessed S抰erminal non-coding regions (NR) 68nt in length( 1 -68nt). There were 1 7nt overlap region between 1 83kDa protein ORE and movement protein ORE. Intercistron regions (IR) between movement protein ORE and coat protein ORE is 2nt. 3扤R has 204nt(6192-6395nt). The nucleotide sequence homology between the genome of TMV-W and TMV-U1 was 98%. TMV-W was proved common str... |
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