Mechanisms Of Induced Systemic Resistance(ISR) In Cucumber Against Botrytis Cinerea By Bacillus Subtilis BSD-2

Bacillus subtilis can produce endospores tolerance to drought,salt, UV radiation and organic solvents.It has inhibitory effect on many disease pathogens. So It is an ideal bio-control bacterium. Bacillus subtilis BSD-2 was isolated and screened in precedent experiments.This strain showed strong inhibition effect on the pathogens of gray mold of vegetable,leaf mold and blight.The paper studied the separation of antibacterial substances,disease prevention effects,disease resistance mechanism and so on.The antibacterial material of Bacillus subtilis BSD-2 strains was separated using hydrochloric acid and sephadex.The best conditions were determined for using hydrochloric acid adjust p H to 2.5, 60% ethanol extracting with material. sephadex G-50 worked better in the separation.The material collected in the second peak has antibacterial activity of Botrytis cinerea.The prevention of Bacillus subtilis BSD-2 and its antibacterial were studied. The best concentration and diluted multiples which have effect were determined.The result shows that fermented liquid has a better prevention effect.Fermented liquid diluted 50 times, antibiotics diluted 10 times and spores solution which index is 108 work best, which the number of prevention reached 62.3%, 52.9%, 56.1%,respectively.Defense enzymes related to induced disease resistance and chitinase incucumber leaves treated by Bacillus subtilis BSD-2 and its antibacteria were determinated. The results showed that the enzyme activity of POD、PPO、PAL and SOD improved and reached the peak at 5 days after induced with fermented liquid, antimicrobial, spore dilute solution. POD enzyme activity were 1.23, 1.06, 1.51 times than that of control. PPO enzyme activity has obvious change trend at the beginning of the fermented liquid and antimicrobial treatment, The peak activity reached more than three times as much as that of control. PAL enzyme activity reached 1.68、1.67、1.68 times than that of control. SOD enzyme activity in the treatments were 1.31, 1.28, 1.23 times than that of control. CAT enzyme activity of the fermented liquid and antimicrobial treatment reached the peak after 3 days, which were 103.5U/g·min and 107.5U/g·min. The chitinase activity reached the peak after 11 days,which were 1.99, 1.49, 1.95 times that of control. Bacillus subtilis BSD-2 and its antibacterial material can improve related defense enzymes(POD, PPO, PAL, SOD, CAT), induced the expression of disease related protein(chitinase),then accelerated the synthesis disease resistance related material and induced resistance of cucumber against grey mould.The determination results of isozyme and intercellular space protein showed that the expression of POD isozyme changed after the treatments of fermentation liquid, antimicrobial, spore solution. The expression of POD isozyme changed, isozyme types increased at the same time. After each treatment chitinase in intercellular space liquid enzyme activity increased, which were 1.32, 1.39, 1.46 times than that of control.The electrophoresis showed that new protein which number was about 27 k D appeared after treated by fermented liquid, antimicrobial and spore solution. Thus it can be seen that BSD-2 and its antibacterial substances can induce the expression of defense genes and the related protein, then induced disease resistance of cucumber.The study established two-dimensional electrophoresis system which is suitable for the protein of cucumber leaf, then analyzed the changes leaf protein treated by Bacillus subtilis BSD-2 and its antibacterial material.The condition optimization has been reached,which to extact total protein using TCA/acetone precipitation method,separating the proteins with p H3-7 IPG strips,with 300 ug sample to electrophoresis, isoelectric focusing program is focusing protein on 25000 Vhr. Difference map shows part of the protein appeared the rise and fall phenomenon, that Bacillus subtilis BSD-2 and its antibacterial material after processing block and weaken the normal expression of gene expression and the expression of related genes start and had induced effect. It provides a good foundation for explaining the internal relationship between changes in protein including structure and functions and induced resistance disease mechanism in plants.