Studies On Relationships Between The Diversity Of Bacterial Community, Ketone Synthase Genes (ks) In Different Cucumber Rhizosphere Soil And Fusarium Wilt

The relationships between the diversity of bacterial community in cucumber rhizosphere soil, ketone synthase genes (KS) and the Fusarium wilt have been studied in this paper. This experiments have been finished by traditional isolation methods, PCR-DGGE molecular technology and molecular clone technology. According to the diversity of microbial populations and encoding polyketide ketone synthase (KS) genes from different cucumber rhizosphere soil samples, studied the relevance with cucumber wilt.This experiments analyzed the differences of diversity about microbial and KS in cucumber rhizosphere soil. PCR-DGGE molecular technology was used to analyze the microbial populations diversity from different soil, the results showed that the species number of bacterial community in cucumber rhizosphere soil from infected cucumber by F. oxysporum f.sp. cucumerinum significantly higher than from healthy cucumber, it is inconsistency with the result that the health cucumber soil had richer microbials by previously literature. Meanwhile, the diversity of KS gene in different disease severity of rhizosphere soil of cucumber was construed using the target gene cloning technology and sequence analysis technology, the results were consistent with the diversity analysis of the species number of bacterial community, namely the KS gene in rhizosphere soil of diseased cucumber showed more abundant.This paper designed genomic DNA extraction methods of different soil samples-alkaline lysis method, and compared with the other three methods. The results showed that potassium acetate and magnesium chloride were used in alkaline lysis method to remove the precipitated impurities, and high DNA yields were obtained. The DNA showed good quality and big molecular size and suitable for PCR amplification as well. And it provided a good template DNA for later experiments. It is proved that alkaline lysis method was innovation DNA extraction method, the method has been applied for a patent (application No. 2010101032923).Actinomycetes from different soil samples were isolated by traditional isolation methods, and the stocks differentia of culturable actinomycetes from various samples were compared. The results showed that the types of actinomycetes were higher from rhizosphere soil of diseased cucumber than from rhizosphere soil of no diseased cucumber.The PCR-DGGE method was optimized, and the primer with "GC clamp" was conducted to amplify the expected KS gene gragements, and changes of bacterial populations in different levels of diseased cucumber rhizosphere soil were analyzed. The results showed that the detection rate of DGGE was greatly improved by this method. Bacterial populations from four rhizosphere soil samples were analyzed. The four rhizosphere soil samples included pre-planting soil, pre-inoculation soil, rhizosphere soil from diseased cucumber and healthy cucumber. The result showed that the soil samples from diseased cucumber had richer bacterial populations.The extracted DNA from the rhizosphere soil from diseased and healthy cucumber were as template for PCR amplification, expected KS gene fragments were amplified by degenerate primers which were designed basing on type I polyketide synthase gene. Expected KS gene fragments were cloned and sequenced,205 gene sequences were submitted to GenBank and obtained GenBank Accession number, used for similar studies. The number of the KS gene from four rhizosphere soil samples were statistical analysis. The results showed that it can obtained more positive clones from rhizosphere soil of diseased cucumber and more abundant KS gene diversity, the results were consistent with the diversity analysis of the species number of bacterial community by PCR-DGGE.