Screening Of Antagonistic Bacteria Against Sphaeropsis Sapinea And Mechanism Of Antagomism

Pine shoot blight is a kind of worldwide pine diseases caused by Sphaeropsis sapinea,which badly influences the pinewoods in many countries around the world.The disease does harm to varieties of trees in a large scale where the speed of diffusion is quite fast,and the loss caused by Sphaeropsis sapinea is no less than that by Bursaphelenchus xylophilu.At present,it is main way to curb this trend by improving the forest management measures and using chemical pesticides.However,biological control has become a significant strategy and direction to prevent plant disease.Some microorganisms which were isolated from plants and plant rhizospheric soil have the capacity of controling pests and diseases,while they could promot the growth of plants.An endophytic bacteria strain(called JK-AH7)was isolated from Pinus elliottii which was resistant to Sphaeropsis sapinea.Then,the strain was identified as Bacillus amyloliquefaciens.The mechanism of resistance to Sphaeropsis sapinea,the condition of colonization inside pines and the optimizing fermentation of metabolic product are studied in succession.Besides,the project developed the field application with some other bacterial strains.There are some main research results as follows:(1)The strain JK-AH7 which is one of 11 strains of endophytic bacteria isolated from the stem of healthy Pinus elliottii has the strong and sustainable ability of resistance to twelve pathogenic fungi such as Phomopsis sp.and Phytophthora palmivora.The strain JK-AH7 was identified as Bacillus amyloliquefaciens by morphological observation,physiological and biochemical,16 s rDNA sequence alignment and specific primer amplification.(2)The mechanism of strain JK-AH7 to resistant to the pathogen was explored by observing and counting the mycelium growth,spore germination and the inhibitory rate of fermentation products of different concentrations under the antagonism,as well as their ability of producing antibacterial substances and induced resistance of Pinus elliottii and enzyme activity determination.The results showed that pathogen hyphae produced top enlargement,branch,twist etc under the effect of strain JK-AH7.After adding the same amount of fermentation product in the pathogen spore suspension,no spore did germination after24 h cultivation,only a small number of spores germination in 48 h.The inhibition rate of 1% fermented production concentration to Sphaeropsis sapinea was 18.6%,and the inhibition rate could reach 89.7% in 10% concentration.JK-AH7 strain could secrete cellulase,phosphatase and protease,which had certain phosphate-solubilizing ability.After the Sphaeropsis sapinea invading,the activity of the defense enzymes SOD,POD and PPO in the slash pine(Pinus elliottii)which processed ahead by strain JK-AH7 increased evidently,but the disease-resistant material MDA and Pro remained low level.It was revealed strain JK-AH7 could enhance resistance of the slash pine to shoot blight disease.The strain JK-AH7 could be used as a candidate biocontrol strain for preventing the disease.(3)The ability of colonization of JK-AH7 strain in the pine was verified by using GFP marker method and scanning electron microscope.Laser confocal observation results showedthat the GFP-markered strain could be detected in Pinus elliottii roots after 30 days of inoculation.Scanning electron microscopy(SEM)observation results on pine inoculated by JK-AH7 showed that the JK-AH7 could colonize in Pinus elliottii stem section.Two validation results showed uniformly that JK-AH7 strain could be good colonizated in the pine trees and a longer duration.(4)The cell amount of strain JK-AH7 and antibacterial ability of fermentation metabolites were improved by method optimization of the single factor method,the Plackett-Burman experiment,the Box-Behnken central composite experiment and response method to optimize the fermentation culture medium and fermentation conditions.The best culture medium and fermentation condition: maltose 3 g/L,pancreatic peptone 20 g/L,magnesium sulfate12 g/L,the initial pH6,60 h of incubation time,incubation temperature 28?,the quantity of 6%,fluid volume 80 ml and speed is 200 rpm.(5)In Baima base and Laojiashan forest farm,the strain of JK-AH7 and several other benificial strains were used to determine field application effect through composite application,concentration ratio,strains distribution and so on.In Baima experimental base and Laojiashan forest farm,the annual pine seedling,P.thunbergii,P.massoniana and P.elliottii were managed root dipping treatment,the effect of JZ-GX1 to P.massoniana and P.elliottii were remarkable.In the next year after retreatment of NSM-05,JW-JS1,JZ-GX1,JK-JS7 most of them had different promoting effect,besides the effect of ZB42 to P.massoniana was not obvious.In Wang jiaba testing area,three-year old slash pine seedlings were treated by different concentration of strain spray processing.According to the results of two years of monitoring,except of 100 ml/tree of NSM-05 strain,the concentration of NSM-05,JK-JS7 and NSM-05+JK-JS7 had different degree of promoting action,and the single fertilization effect was better.When the three-years old slash pine were treated by different adding bacteria ways,after two years the monitoring results showed that the strain NSM-05 grew better by spray processing,the effect of JK-JS7+NSM-05 was better by filling root or dry injection,but there was no obvious difference in the single fertilization of strains.In Laojiashan forest farm,the three-year old seedlings of Pinus elliottii had been added bacteria treatment through spraying and beating root methods,the promotion growth effection of FZB42+JK-AH7,FZB42+NSM-05 and JZ-GX1+NSM-05 were obvious,and the better control effect to Diplodia pinea would be JZ-GX1+JK-AH7,FZB42+JK-JS7,FZB42+JK-SH007 and FZB42+NSM-05.Through two years of monitoring to Pinus elliottii in Yu wan of Lao jiashan forest farm showed that JZ-GX1,JW-JS1,FZB42,NSM-05+JZ-GX1,NSM-05,NSM-05+JW-JS1 had better continuously growth effect,but each processing of plant height and ground diameter had emphasized particularly on the growth.