Screening Of The Antagonistic Bacteria Against PVY And Study On Its Mechanism

Potato virus Y (PVY) is one of the most important viruses causing huge damage to yield and quality of tobacco in recent years. This study screened of antagonistic bacteria against PVY for the first time, which provides a new theoretical basis and technical support for biological control of tobacco virus disease through in-depth research.A quantitative method using real-time PCR technique based on fluorescence dye SYBR Green was adopted to detect PVY in tobacco in this study using actin gene as the reference gene. PVY genome sequence alignments logged in GenBank were analyzed using DNAMAN software. Primer 5.0 software was used to design specific primers of PVY gene and actin gene. We found that the amplification curve had flat baseline, distinct exponential area, large and stable slope and the coefficient of variation was very little. There was a linear relationship between threshold cycle values at which samples crossed threshold and the logarithmic values of template concentration. The results showed that this system can be used for quantitative detection of potato Y virus rapidly, sensitively and efficiently.We isolated 103 bacterial strains from Jimo farm and obtained some strains which have antagonistic effect of PVY using the system. The NO.1 and NO.22 strain have stronge antagonistic activity. The inhibition rates of these strains to the PVY are 67.68% and 55.28%. The NO.1 strain was isolated from the surface of tobacco leaves, while the NO.22 strain was separated from the inside of diseased tissue. There are also some strains separated from soil and inside diseased tissue of tobacco whose inhibition rates to PVY are between 10% and 20%. C10 is the strain that separated from the tobacco rhizosphere soil, which has the strongest antagonistic activity. The inhibition rate of C10 broth to the PVY is 88.19%. C10 strain identified as Chryseobacterium sp. by 16S rDNA sequence analysis.Ammonium sulfate fractionation was used for the deposition of the protein in C10 broth. Three kinds of antagonistic activity of PVY proteins were isolated and purified using DEAE-SepharoseTM Fast Flow anion-exchange chromatography and Sephadex-G75 gel filtration chromatography combined with PVY Real-time PCR system for quantitative detection of the antagonistic activity of the eluate. Their molecular sizes are 40.6KDa, 42.4KDa and 37.8KDa respectively detected by SDS-PAGE. Their suppression efficiency on PVY are 67.94%,55.27%,47.06%. This study showed that the antagonistic activity of C10 was multivariate by three low molecular sizes protein in C10 broth.We made preliminary research on the mechanism of the antagonistic strain C10 using Nicotiana tabacum var.samsun NN as host. Electron microscopy indicated that PVY particles can be inactivated by the activity protein in C10 broth. We found that inducing tobacco with C10 broth can greatly improve the resistance of tobacco against PVY infection. In addition, sprayeding C10 broth on tobacco that have been infected by PVY can delay the development of the disease.The results showed that the biocontrol mechanism of strain C10 is complicated which mainly manifested as followes. First of all, the active proteins from the C10 broth have interaction with the PVY particles directly and destruct the force between the coat protein subunits, which lead to fracture of PVY virus particles and other damaging effects reducing the power of PVY infection. Secondly, the activity of two important defense enzymes POD and PPO in tobacco were increased induced by C10 broth. At the same time, the activity of these enzymes can be controlled in a longer period. Therefore it improved systemic resistance of the host plant, inhibit the replication of PVY and its spread in the host, and finally reduced the harm of PVY. Thirdly, sprayeding C10 broth on tobacco that has been infected by PVY can inhibit the replication of PVY in 72 hours, which indicated that there may be some complex mechanism playing a therapeutic effect.