Study On Ecology Of Antagonistic Bacteria To Soil-borne Plant Pathogens In The Rhizosphere Of Vegetables.

Abundant bacterial isolates were obtained from vegetable-field in the farms of Zhejiang University and Hangzhou Institution of Vegetable. Six strains of those bacteria ZJ7, ZJ14, ZJ31, ZJ32S1, ZJ45 and A2, were showed an antagonize to Pythium aphanidermatum caused seedling damping-off and Fusarium oxysporum f.sp. cucumerinum caused stem wilt of cucumber in plate. Among them, ZJ7, ZJ14, ZJ31, ZJ32S1 and ZJ45 antagonized to P. aphanidermatum, and ZJ45 and A2 to F.oxysporum f.sp. cucumerinum. Then, those bacteria were effective on germination of cucumber seeds in plate and pot testing in sterile soil. Two strains called , ZJ14 and ZJ32S1, largely increased the length of root and hypocotyls of cucumber after seed bacterization, and all 6 strains above promoted growth of cucumber seedlings, including increasing incidence of emergence and plant dry weight. These 6 antagonists were used to test the effect of bio-control on the cucumber damping-off and Fusarium wilt at pot testing respectively. The results showed that while the growth of seedlings was promoted, the diseases were suppressed too.6 strains, ZJ14, ZJ32S1, ZJ45 and A2 were identified from Gram-stain, Physiological and biochemical characteristics, and combined with 16S rDNA sequence cluster. The results were showed that ZJ14 and ZJ32S1 was as Bacillus subtilis, ZJ45 as Pseudominas aeruginosa, and A2 as Paenibacillus polymyxa respectively.There were lack of stabile and effective marker approaches for Gram-posotive bacteria relatively to Gram-nagative bacteria. Bacillus subtilis plays more and more important roles in biological control, due to its resistance to terrible environment. So it is nessary to construct a vector to mark wild type strains of Bacillus subtilis for their colonization hi rhizosphere.The full length of the promoter and GFPuv gene were obtained by PCR with two pairs unique primers xylR-F/R and primers GFPuv-F/R respectively, which were designed according to the GFPuv gene and the sequence of xylose operon from Bacillus subtilis 168, and the DNA template chromosomal DNA of B.subtilis 168 and plasmid pGFPuv. Furthermore, grp gene were inserted into pGEM-xylR, after plasmids pGEM-xylR and pGEM-gfp were digested by Hind III and Kpn I, which were clones of above the PCR production. Then, the DNA fragment xylR-gfp which were obtained after pGEM-xylRGFP being digested by Kpn I and Sph I , was inserted into Kcoli-B.subtilis shuttle vector pRP22, and the resulted recombinant plasmid was named at pRP22-GFP. The recombinant plasmid was transferred into their protoplasts of B.subtilis lab strain 168 and wild type strain CC41 and ZJ32S1 respectively, and the resulted transformants were showed the bright green under365nm UV light. There were no obvious difference between the transformants of CC41 and ZJ32S1 and then- wild type strains in plate.