Separation Of The Active Small Molecules Of Pseudomonas Putida A3 Strain Against TMV And Study On Its Mechanism

Tobacco mosaic virus (TMV) is one of the most important viruses causing severe damage to yield and serious economic losses. The control effects and security of antiviral agent are not as good as people's demands till now. However, the extensive use of chemicals causes a series of alarming environmental and social problems. Developing antivirus products with high efficiency and high activity is the key mission of plant disease biocontrol for sustainable development. There are several control strategies of biological pesticides, one of which is the application of microbial secondary metabolites.Bacterial strain A3 which has strong antagonist activity against TMV was selected from healthy Tobacco (K326) Field from the area infected TMV in Jimo. Combined the results of the physiological, biochemical characteristics and 16S rDNA sequence analysis, strain A3 was identified as Pseudomonas putida. Its culture medium and fermentation conditions were investigated. The results showed that the optimal fermentation conditions were the following: NB culture medium, original pH 7.0, the culture temperature 28℃, ventilation volume 100 mL/250 mL, the culture time 60-72 h. Glucose was the suitable carbon-source nutrition, and peptone or yeast was the suitable nitrogen-source nutrition as well. Active constituents from bacterial strain A3 was isolated and purified through solvent extraction. GC/MS was used to analyze the chemical composition and structure identification from the extract. The results showed that 38 kinds of compounds were separated and identified by GC/MS. Ketones and alcohols were the major portions.We made preliminary research on the mechanism of the antagonistic strain A3 using Nicotiana tabacum var.samsun NN and NC89 as host. The results of the mechanism showed that TMV particles could be inactivated by the activity small molecular substances in A3 broth. The activity small molecular substances could induce the production of PR protein and induce resistance to TMV in the host.Different concentrations of Tween-20 as a surfactant were mixed with the fermentation liquid spraying NC89 and inoculated at different times to determine the most suitable bacteria for the production of formulations of the type and concentration. The study showed that 0.25% Tween -20 compared with A3 could be more suitable for the leaf surface, and TMV inoculated by one day after spraying pharmacy could be inhibited. In this study, soil samples collected from Tengchong of Yunnan province and Longlin of Guangxi province were quantitatively detected by the way of real-time PCR assary, and combined with the incidence and disease index of virus diseases. The results indicated that P. putida A3 strain could reduce the amount of TMV in soil planting tobacco through bio-remediation, however, without bio-remediation by P. putida A3 strain, the TMV amount in soil were maintenanced high level. And the incidence and disease index of virus diseases were also reduced through bio-remediation by A3 strain. Therefore, P. putida A3 strain could bio-remediated the soil polluted by TMV, which showed the new method of comprehensive treatment of tobacco virus diseases.