Studies On Microbial Diversity Of Vegetables Rhizosphere Soil And Regulation Of Antagonistic Actinomycetes

Bacterial community structure and diversity of rhizosphere soil in infected plants pepperphytophthora blight and cucumber Fusarium wilt in Dalian of Liaoning were studied, usingboth the16S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) and the16S rDNAgene clone library. And the relationship between the rhizosphere bacterial communitystructure and soil-borne disease was initially revealed. The co-operative and synergisticcombination ACT-1and ACT-2were screening from a large number of actinomyces strains,and the fermentation condition was optimized. Furthermore, the control efficiency and theeffect on the diversity of bacterial community after applying the biological agents wereanalysed. The results were as follows:1. Bacterial community structure and diversity of rhizosphere soil in infected plantspepper phytophthora blight were studied. There are five rhizosphere soil samples, includingpre-planting soil, pre-inoculation soil, rhizosphere soil from diseased pepper, healthy pepperand not inoculated as a control. They were analysed by DGGE and the16S rDNA gene clonelibrary. A total of38to49bands were separated from five samples, respectively, and there aresome differences among each sample. Among them, the healthy pepper rhizosphere soil hasthe most DNA bands. It indicates that the dominant bacterial community in healthy pepperrhizosphere soil sample was more than that in the infected pepper rhizosphere soil sample.Based on the analysis of16S rDNA gene clone library, a total of89clones and52cloneswere sequenced from healthy pepper rhizosphere soil and diseased pepper rhizosphere soilsample respectively. The bacterial community in the two samples displayed a highlydiversified composition including members from11bacterial phylum and some unclassifiedsequences. Proteobacteria, Acidobacteria and Bacteroidetes were the rather dominant groups.The bacterial community in the infected pepper rhizosphere soil sample was slightly lessdiversity than those in the healthy pepper rhizosphere soil sample, and the clone number thatwere most affiliated with Sphingomonas sp. in the health soil sample was more than diseasedsoil sample.2. Bacterial community structure and diversity of type I ketosynthase of rhizosphere soil in infected cucumber Fusarium wilt and healthy cucumber were studied.There are five rhizosphere soil samples, including pre-planting soil, pre-inoculation soil,rhizosphere soil from diseased cucumber, healthy cucumber and not inoculated as a control.They were analysed by DGGE and the16S rDNA gene clone library. A total of15to24bandswere separated from five samples, respectively, and there are some differences among eachsample. The results of nucleotide sequence analysis for the dominant bands demonstrated thatthe major phylogenetic groups identified by DGGE belong to Proteobacteria, Bacteroidetes,Firmicutes, Acidobacteria, Gemmatimonadetes and Actinobacteria. Firmicutes was dominantphylum. Bacteroidetes and Firmicutes were the unique phylum in the healthy cucumberrhizosphere soil.Based on the analysis of16S rDNA gene clone library, a total of118clones and81clones were sequenced from healthy cucumber rhizosphere soil and diseased cucumberrhizosphere soil sample respectively. The bacterial communities in these two samplesdisplayed a highly similarity with the pepper samples. The bacterial community in theinfected cucumber rhizosphere soil sample was slightly less diversity than those in the healthycucumber rhizosphere soil sample, and the clone number that were most affiliated withSphingomonas sp. in the health soil sample was more than diseased soil sample.On the base of these results, the KS gene library was constructed by the specific primersof KS.80and102clones were obtained from the healthy cucumber rhizosphere soil sampleand diseased cucumber rhizosphere soil sample, respectively. According to the phylogeneticanalysis of KS amino acid (AA) sequences indicate that the KS genes in the rhizosphere soilsamples were clustered into diverse seven clades. Some KS AA sequences come form oneclade, and this clade was peculiar to healthy cucumber rhizosphere soil sample, moreover,these KS genes showed high homology with that from Streptomyces by Blast program.3. Optimization of fermentation conditions of antagonistic actinomycetes combination.Four antagonistic strains were screened from26strains with fungus cake method andthere have no interference among the four strains. According to the different target pathogens,2antagonistic actinomycetes combinations were designed, that is ACT-1and ACT-2. Theeffect of single factor on fermentation conditions of ACT-1and ACT-2and orthogonal experiment was studied. It was demonstrated that the optimum fermentation conditions ofACT-1were as follows: the proportion of GAOQING-17, YH91and YH23was3:3:2, pH wasnatura1, inoculum volume was3%, respectively. The ACT-1was cultured in50ml of culturemedium in250ml shaking flasks with rotation speed180r/min at25℃for5d. The optimumfermentation conditions of ACT-2were as follows: the proportion of GAOQING-17, YH91,YH23and YH6was3:1:3:2, pH was natura1, inoculum volume was3%, respectively. TheACT-2was cultured in50ml of culture medium in250ml shaking flasks with rotation speed180r/min at25℃for5d. The antimicrobial activity of ACT-1and ACT-2increased14.03%and15.10%after the optimization fermented the condition.4. Control efficiencies to cucumber Fusarium wilt of antagonistic actinomycetescombination and the effect on cucumber rhizosphere soil bacterial community weredetermined.The results of control effect to cucumber Fusarium wilt of antagonistic actinomycetescombination of ACT-1and ACT-2showed better control efficiencies. Furthermore, thediversity of these samples after appling ACT-1and ACT-2(including diseased cucumberrhizosphere soil sample, healthy sample), control sample (including diseased cucumberrhizosphere soil sample, healthy sample) were analysed by PCR-DGGE. The results showedthat the bacterial community is similarity among samples. The bacterial species and quantityhave change after applying ACT-1and ACT-2. So, it indicates that the rhizosphere soilmicroorganisms community was changed by antagonistic actinomycetes combination. Inparticular, there is obvious difference between the diseased sample and the healthy sample. Italso declares that antagonistic actinomycetes combination effect cucumber whether or notdisease.