Molecular Variability And Synergism Of Potato Virus X And Tobacco Vein Banding Mosaic Virus

Potato virus X (PVX) is one of the most common plant viruses infecting solanaceous plants, including potato, tobacco, eggplant and tomato. PVX alone causes mild symptoms in solanaceous plants. However, it causes much more severe symptoms when it mixedly infects with potyviruses. Tobacco vein banding mosaic virus (TVBMV) is a species of the genus Potyvirus and has been detected frequently in tobacco plants in Shandong, Henan, Anhui and Yunnan provinces. The losses caused by TVBMV had been increasing in the past few years. In this paper, we reported the complete genomic sequence of two Chinese PVX isolates and the first complete genomic sequence of TVBMV in the world, confirmed synergism between PVX and TVBMV. And analyzed the effects of site-directed mutagenesis in HC-Pro protein on PVX/HC-Pro synergism. Finally, we investigated the use of plant growth-promoting rhizobacteria (PGPR) to induce resistance to infection by plant viruses.The complete genomic sequences of two Chinese PVX isolates (PVX-FX21 and PVX-1985) were determined from four overlapping cDNA clones. The genome of both isolates are 6435 nucleotides (nt) in length excluding the poly(A) tail and contains five open reading frames (ORFs). Both the entire sequences shared higher identities with nine European isolates (group I), and lower identities with four South American isolates (group II). Phylogenetic analysis of complete genomic sequence of 15 isolates and the coat protein genes of 38 isolates revealed two major groups. All PVX isolates from Asia are clustered to group I, while isolates from Europe and America are clustered to both groups. PVX-FX21 had the highest identities of 96.9% and 100.0 % with PVX-X3 (group I) at nt and amino acid (aa) levels, respectively. While PVX-1985 was clustered with two isolates (Roth1 and XS ) from United Kingdom, sharing identities of 97.6% and 97.3% at nt level, respectively, and identity of 97.5% at aa level. Nucleotide sequence diversity analyses showed that constraint on the ORF encoding RdRp is much higher than those on the other four ORFs. The central coding region of RdRp was under positive seletion, although the encoding RdRp was under negative selection.The complete genomic sequence of one Tobacco vein banding mosaic virus isolate from Yunnan China, TVBMV-YND, was determined by sequencing overlapping cDNA fragments obtained by RT-PCR with degenerate and/or specific primers. The genome is composed of 9,570 nt excluding the 3′-terminal poly(A) tail and contains one single open reading frame of 9,240 nt encoding a large polyprotein of 3,079 amino acids with predicted Mr of 348.6 kDa. Phylogenetic analysis of complete genomic sequences confirmed that TVBMV is a distinct species of the genus Potyvirus. Different parts of TVBMV-YND genome shared different levels of identity with other potyviruses, while most parts showed greatest identity with Chilli veinal mottle virus among the potyviruses with available complete genomic sequences. TVBMV-YND had a rare Q/N cleavage site for NIb/CP and uncommon RITC motif in HC-Pro that is crucial for aphid transmission of potyviruses.Mixed infections of Nicotiana tabacum cvs NC89 and Samsun plants by PVX and TVBMV resulted in synergistic interaction. NC89 and Samsun plants coinfected by both viruses displayed remarkably inhibited plants growth and significantly decreased photosynthesis. N. benthamiana plants coinfected by both viruses also displayed stronger synergistic interaction, progressing from severe mosaic and eventually to plant death. Differenting from previous study, the concentration of both viruses did not increase significantly in any of the infected tobacco plants. When the HC-Pro of potato virus A (PVA) was transiently expressed in N. benthamiana via potato virus X (PVX) vector, a highly synergistic response of leaf necrosis and plant death was observed in PVX-HC infected plants. Twelve sites in the HC-Pro of PVX-HC were mutated successfully through site-directed mutagenesis. Eleven of these mutants loss the synergism function. However, mutation lysine (K) to alanine acid (A) in the"KITC"motif, which is involved in aphid transmission of potyviruses, had no obvious influence on synergism.Two hundred and ninety-five bacterial isolates were obtained from rhizosphere of plants.Among which five potential biocontrol isolates showed broad and strong antagonistic effects on plant pathogens. One bacterial strain, Lyc2, was selected for further study. It showed remarkable inhibition to the mycelial growth of multiple pathogenic fungi in dual culture. Lyc2 showed 48.8% efficiency to control cotton seedling damping-off in greenhouse, and significantly increased the fresh weight and dry weight of cotton seedlings in solution culture experiment. It could also induce resistance to TMV in Samsun plants. In field, Lyc2 could promote the growth of tobacco plants, and showed 33.7% efficiency to control plant viral disease. The morphological, physio-chemical characteristics, homology of 16S rDNA and ITS sequences showed that Lyc2 was closely related to Burkholderia cepacia. Further comparison of recA gene sequence showed that Lyc2 belonged to B. pyrrocinia, genomovar IX of B. cepacia complex.