Study On Population Dynamics Of Endophytic Bacteria In Processing Tomato And Inhibitory And Growth-promoting Effect Of Endophytic Bacillus

In order to clarify population dynamic distribution of endophytic bacteria in different parts of processing tomato varieties in north of Xinjiang cultivated regions, and provide the source for screening and developping bacteria biological control agent on effectively prevention of processing tomato diseases. Using tissue dilution culture method, endophytic bacteria in different parts of processing tomato samples from the seventh division and the eighth division in north of Xinjiang were isolated and counted, and endophytic bacteria isolated were identified by the morphological characteristics, physiological and biochemical and molecular biological methods in order to clarify population dynamic distribution of endophytic bacteria in different parts of processing tomato varieties and plantting areas. Antagonistic endophytic bacteria were evaluated by PDA plates for selection training with antimicrobial activity, and two strains of endophytic Bacillus TEB-01 and TEB-31 with high antagonistic activity were identified by conventional identification methods and molecular biological methods( 16 S rDNA and gry A gene sequence analysis). By PCR based on the known lipopeptide genes, RP-HPLC and MALDI-TOF-MS methods, two strains of endophytic bacteria antibacterial lipopeptide were identified, and the stability of antibacterial lipopeptide were studied. Finally, growth-promoting effects on two endophytic strains were evaluated, and the mechanism of the growth-promoting and mode of colonization pattern were studied and analyzed. The main results obtained are listed below:1. Endophytic bacteria in processing tomato were initially identified by six groups, and ten species in seventh and eighth divisions that were Pseudomonas sp., Xanthomonas sp., Aagrobacterium sp., Erwina sp., Entenobacter sp., Bacillus sp. five species were identified as Bacillus cereus, Bacillus megaterium, Bacillus pumilus, Bacillus mojavensis and Bacillus amyloliquefacien in Bacillus sp. Bacillus sp. and Pseudomonas sp.were main dominant population in endophytic bacteria of processing tomato, because that were the most among the 285 endophytic bacteria, and distributed in different varieties of processing tomato and different organs and their quantities occupied the dominant position.2. The groups and quantities of endophytic bacteria in processing tomato were different in different cultivated regions, varieties, tissues, growth period. There were five group, eights pecies among endophytic bacteria in different processing tomato varieties in seventh division cultivated regions. In the four processing tomato varieties, Hengshi3402 within five bacterial species was at least, The remaining three varieties that Shifan35, Shihong 303 and Ligeer 87-5 were seven endophytic bacterial species. There were six group, nine species among endophytic bacteria in different processing tomato varieties in eighth division cultivated regions. In the three processing tomato varieties, Ligeer 87-5 within nine bacterial species was at most, The remaining two varieties that Shifan35 and Jiayi6 were seven endophytic bacterial species. Population dynamics of endophytic bacteria in different growing period of processing tomato Ligeer 87-5 was that from seedling stage to flowering stage, the species of endophytic bacteria were increasing, and the species of endophytic bacteria remained stable during flowering stage, the fruiting stage and the mature stage.3. As the quantities of endophytic bacteria, there were different in different processing tomato varieties, but the overall quantities of endophytic bacteria was in 104cfu/g.fw; Quantities of endophytic bacteria were different in different organs of processing tomato, but generally the root was the most, followed by stems, leaves, fruit. In different growth periods, the quantities of endophytic bacteria in processing tomato Ligeer 87-5 increased from seedling stage to flowering stage, decreased from fruiting stage to mature stage.4. Two endophytic bacteria strains TEB-01 and TEB-31 that obtained by antibacterial test had good antagonistic effect on five kinds of pathogenic fungi, that were Alternaria solani, Fusarium oxysporum f.sp. Lycopersici Snyder et Hansen, Rhizoctonia solani, Fusarium oxysporum f. sp. Vasinfectum, Verticillium dahliae Kleb. The strains TEB-01 and TEB-31 were identified respectively as Bacillus mojavensis and Bacillus amyloliquefaciens by morphological characteristics, physiological and biochemical determination, 16 S rDNA and gry A gene sequences analysis.5. By PCR with four primers designed based on the known lipopeptide genes, RP-HPLC and MALDI-TOF-MS methods, surfactin and fengycin were detected in Bacillus mojavensis TEB-01, and there were four kinds of homologues that C15-surfactin(1058.67and1074.61), C16-fengycin A, C-17 fengycin A. In Bacillus amyloliquefaciens TEB-31, IturinA, mycosub tilin, surfactin and fengycin were detected, and there were eight kinds of homologues that C15-iturinA, C15-surfaction, C18-mycosubtilin, C14-fengycinA, C15-fengycinA, C16-feng ycinA, C17-fengycinA(or C15-fengycinB), C16-fengycin.6. The lipopeptide extracts from strains TEB-01 and TEB-31 were harmful to hypha morphogenesis of five kinds of pathogenic fungi, which could cause the fungal hyphae to distortion, deformation, distort and form the structure of the vesicles or even rupture. its charateristics of antagonistic activity on lipopeptides of strains TEB-01 and TEB-31 had high stability to pH, heat and ultraviolet irradiation, and were not sensitive to proteinase K, relatively sensitive to trypsin.7. The results on the growth promoting effect of strains TEB-01 and TEB-31 showed that the free cell filtrate of 100 times dilution had best promoting effect on seed root growth of processing tomato and cotton. Different concentrations of bacteria by soaking with 106cfu/ml had best promoting effect on height, fresh weight, dry weight of processing tomato and cotton seedling. Three inoculation methods, that seed soaking, root irrigation, spray, seed soaking method had ignificant growth promoting effect, spray inoculation method with CK had no promoting effect on processing tomato and cotton seedling.8. Preliminary study on mechanism of promoting growth about strains TEB-01 and TEB-31 showed that two strains could produce siderophore, and also produc plant growth hormone which GA and IAA were in strain TEB-01, GA within strain TEB-31. In addition, TEB-01 and TEB-31 strains by the appropriate concentration(106cfu/ml) increased the chlorophyll content of processing tomato and enhanced its root strength reduction to promote the growth of processing tomato.9. By GFP marker gene, successfully Bacillus subtilis shuttle vector pGFP4412 plasmid was imported into strains TEB-01 and Bacillus TEB-31, through acupuncture injury into the processing tomato stem base, two taggeg strains were in colonization of stem tissue with slitting stem tissue slice by fluorescence microscope observation. The labeled strain recovery in different tissues of processing tomato showed that two strains could colonize in root and stem tissues.