土传植物病害拮抗细菌根围生态学研究
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摘要
从浙江大学华家池蔬菜所和抗州市蔬菜所的老熟菜地土壤中分离到大量土著细菌，然后从中筛选出对Pythium aphanidermatum和Fusarium oxysporum f.sp.cucumerinum分别具有离体拮抗作用细菌分离物6株ZJ7、ZJ14、ZJ31、ZJ32S1、ZJ45和A2，进一步通过促芽实验和无菌土盆栽测定，发现菌株ZJ14和ZJ32S1能促进黄瓜种子的萌发；6要菌株都能促进黄瓜苗的生长、增加苗干物质重量。在接种引起黄瓜苗期猝倒病(Pythium spp.)和中期枯萎病(Fusarium oxysporum f.sp.cucumerinum)的盆栽测定中，发现它们能明显提高防病效果。
根据格兰氏染色反应、生理生化测定和16S rDNA序列同源性比对，鉴定了生防效果相对稳定的4个菌株，分别为ZJ14(Bacillus subtilis)，ZJ32S1(Bacillus subtilis)，ZJ45(Pseudomonas aeruginosa)和A2(Paenibacillus polymyxa)。
相对于革兰氏阴性细菌定殖研究的蓬勃发展，革兰氏阳性细菌缺乏稳定、高效的标记系统。而枯草芽孢杆菌(Bacillus subtilis)由于其具有较强的抗逆性，在生物防治研究中越来越受到重视。为了进一步研究生防枯草芽孢杆菌在植物根际的定殖情况，对其进行标记是有必要的。因此，根据绿色荧光蛋白基因和枯草芽孢杆菌木糖诱导型启动子PxylR序列，分别设计两对特异引物xylR-F／R和GFPuv-F／R，扩增得到完整的xylR启动子序列和GFPuv基因序列后分别对上述产物做克隆，获得pGEM-xylR和pGEM-gfp质粒。对上述质粒分别经HindⅢ／KpnI双酶切后连接，获得质粒pGEM-xylRGFP。经SphI／KpnI双切质粒pGEM-xylRGFP后，回收片段xylR-gfp，插入穿梭载体pRP22，得到重组子pRP22-GFP。经原生质体转化法转化B.subtilis 168和野生菌株CC41和ZJ32S1均得到有良好发光表型的转化子。并且，室内平板显示，工程菌株抑菌效果与原始菌株无明显差异，并且在紫外灯下易于区别。
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 under
365nm UV light. There were no obvious difference between the transformants of CC41 and ZJ32S1 and then- wild type strains in plate.
根据格兰氏染色反应、生理生化测定和16S rDNA序列同源性比对，鉴定了生防效果相对稳定的4个菌株，分别为ZJ14(Bacillus subtilis)，ZJ32S1(Bacillus subtilis)，ZJ45(Pseudomonas aeruginosa)和A2(Paenibacillus polymyxa)。
相对于革兰氏阴性细菌定殖研究的蓬勃发展，革兰氏阳性细菌缺乏稳定、高效的标记系统。而枯草芽孢杆菌(Bacillus subtilis)由于其具有较强的抗逆性，在生物防治研究中越来越受到重视。为了进一步研究生防枯草芽孢杆菌在植物根际的定殖情况，对其进行标记是有必要的。因此，根据绿色荧光蛋白基因和枯草芽孢杆菌木糖诱导型启动子PxylR序列，分别设计两对特异引物xylR-F／R和GFPuv-F／R，扩增得到完整的xylR启动子序列和GFPuv基因序列后分别对上述产物做克隆，获得pGEM-xylR和pGEM-gfp质粒。对上述质粒分别经HindⅢ／KpnI双酶切后连接，获得质粒pGEM-xylRGFP。经SphI／KpnI双切质粒pGEM-xylRGFP后，回收片段xylR-gfp，插入穿梭载体pRP22，得到重组子pRP22-GFP。经原生质体转化法转化B.subtilis 168和野生菌株CC41和ZJ32S1均得到有良好发光表型的转化子。并且，室内平板显示，工程菌株抑菌效果与原始菌株无明显差异，并且在紫外灯下易于区别。
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 under
365nm UV light. There were no obvious difference between the transformants of CC41 and ZJ32S1 and then- wild type strains in plate.
引文
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