葡萄果穗附生微生物区系分析及葡萄灰霉病拮抗菌筛选
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摘要
葡萄灰霉病是国内外重要的葡萄病害之一,每年由此病造成的产量损失很大。现在的防治方法主要是农业防治和化学防治,但是这两种措施均存在一定的局限性。本文对葡萄果穗附生微生物区系分析、附生拮抗菌的筛选、鉴定以及附生拮抗细菌的定殖和防治试验的进行研究,得出以下主要结论:
1葡萄果穗附生微生物区系分析
本试验采用稀释平板分离法对处于幼果膨大期、着色期、成熟期的抗病品种巨峰、中抗品种红地球、感病品种京亚果穗的附生微生物区系进行分析。结果表明:葡萄果穗附生微生物的数量随生育期及品种的不同而有差异,抗感病品种间附生微生物数量差异显著。自膨大期到成熟期真菌和酵母菌数量逐渐增加;细菌数量先增加后下降,总菌数也在着色期达到最大值,而后呈下降趋势,但数量变化不大;自幼果膨大期到成熟期,细菌数量一直占优势。病果穗附生的真菌、细菌和酵母菌的数量均高于健果穗,果穗发病后真菌的种类减少,细菌和酵母菌的种类变化不大。
2优势拮抗细菌的筛选
在葡萄果穗上分离出的13种附生细菌经平板对峙试验初筛,筛选出11种对葡萄灰霉病有抑制作用的拮抗菌。
将11种具有拮抗作用的附生细菌对灰霉孢子抑制试验、对灰霉病菌菌丝生长的抑制试验及离体果的防治试验结果均表明,B5和B12菌株的抑制能力最强,是优势拮抗菌株,而B4和B6菌株的抑制能力最差,其它7种拮抗细菌的抑菌能力均在它们之间,波动不大。通过离体果的防治试验表明了先喷施拮抗菌发酵液后接种病原菌的防治效果与先接种病原菌而后喷施拮抗菌发酵液的防治效果有显著差异,这说明了拮抗菌的保护作用好于治疗作用。
3优势拮抗细菌B5、B12的16srDNA的鉴定
本试验采用传统分类方法即菌落大小和形态、革兰氏染色和分子生物学方法16SrDNA相结合,将拮抗菌B5、B12菌株进行了PCR扩增、测序,将测出的菌16SrDNA序列用Blast软件、Clustal(1.8)软件及Mega软件对其序列进行了同源性分析和系统发育分析,初步确立了菌株在细菌系统发育学上的地位。B5属于芽孢杆菌属(Bacillus sp.);B12属于枯草芽孢杆菌(Bacillus subtilis.)。
4拮抗菌B5、B12在葡萄果穗表面的定殖及日光温室防治试验
定殖试验结果表明经抗药性标记后B5、B12菌株均能在葡萄果穗表面定殖,B5菌株定殖周期为19d,B12菌株在葡萄表面定殖周期为25d。
日光温室自然发病防效试验的结果表明B5、B12菌株的防治效果均达到了50%以上,达到了比较理想的效果。
Grape grey mold, which can make a great deal of loss of grape production, is one of the important grape disease in the world. Now, agronomic control and chemical control are important control measures, but both of them showed some limitations。
The research to analysis on the adnascent microorganism flora of grape fruit spike、screening and identification of antagonistic strain、antagonistic strains colonization and prevention experiment in the sunlight greenhouse,the conclusion shows that:
1 Analysis on the adnascent microorganism flora of grape fruit spike
In this test the epiphytic microorganism flora on the fruit spike of three grape breeds (jingya、hongdiqiu、jufeng) are studied in the time of young fruit enlargement, pigmentation and maturity. The results showed that amount of filamentous fungus and yeasts increased gradually from the inflation stage to the maturity stage, the amount of germs increased at the beginning and then decreased. Total count rise to the highest during the pigmentation, then shows declining tendency, but changed unobviously. The germs had predominance in the inflation stage and the pigmentation stage.
The account of filamentous fungus, germs and yeasts on the diseasing fruit is more than that on the normal fruit. The varieties of filamentous fungus become less but germs and yeasts had no obviously change.
2 Screening of antagonistic strain
In experiment of flat confront, filtrate 11 kinds of epiphytic bacterium with anti-function to the grape grey mold. Then through the inhibition germination experiment of disease germ spore, inhibition test in growth experiment of mycelium and the test of control effect on vitro fruit, finally filtrated B5 and B12 strains to be the best controlling efficiency on the grape grey mold, but B4 and the B6 strains inhibiting ability is worst, other 7 kinds of oppress between them, it changed a little.
3 16srDNA identification of B5 and B12 strains
This experiment uses the tradition taxonomic approach is the colony size and the shape, Gelanshi dyes and molecular biology method 16SrDNA unifies, will oppress antibacterial B5, the B12 strain to carry on PCR to increase, the sequence, which determine the fungus 16SrDNA sequence with the Blast software, Clustal (1.8) the software and the Mega software has carried on the homology analysis and the phyletic evolution analysis to its sequence, has established the strain initially in the bacterium phyletic evolution study on status. 16srDNA identification shows that the B5 strain is:Bacillus sp. and the B12 strain is:Bacillus subtilis.
4 B5 and B12 strains colonization on the surface of fruit and the sunlight greenhouse prevention experiment
Both B5 and B12 strain can breed on grape surface, the B5 strain′setting period is 19d, B12 strain′setting period is 25d, and they are quite stable.
The prevention test result in greenhouse of B5 and B12 strain shows that:The prevention efficiency of B5 and B12 strains in nature have achieved above 50%, has achieved the quite ideal effect.
1葡萄果穗附生微生物区系分析
本试验采用稀释平板分离法对处于幼果膨大期、着色期、成熟期的抗病品种巨峰、中抗品种红地球、感病品种京亚果穗的附生微生物区系进行分析。结果表明:葡萄果穗附生微生物的数量随生育期及品种的不同而有差异,抗感病品种间附生微生物数量差异显著。自膨大期到成熟期真菌和酵母菌数量逐渐增加;细菌数量先增加后下降,总菌数也在着色期达到最大值,而后呈下降趋势,但数量变化不大;自幼果膨大期到成熟期,细菌数量一直占优势。病果穗附生的真菌、细菌和酵母菌的数量均高于健果穗,果穗发病后真菌的种类减少,细菌和酵母菌的种类变化不大。
2优势拮抗细菌的筛选
在葡萄果穗上分离出的13种附生细菌经平板对峙试验初筛,筛选出11种对葡萄灰霉病有抑制作用的拮抗菌。
将11种具有拮抗作用的附生细菌对灰霉孢子抑制试验、对灰霉病菌菌丝生长的抑制试验及离体果的防治试验结果均表明,B5和B12菌株的抑制能力最强,是优势拮抗菌株,而B4和B6菌株的抑制能力最差,其它7种拮抗细菌的抑菌能力均在它们之间,波动不大。通过离体果的防治试验表明了先喷施拮抗菌发酵液后接种病原菌的防治效果与先接种病原菌而后喷施拮抗菌发酵液的防治效果有显著差异,这说明了拮抗菌的保护作用好于治疗作用。
3优势拮抗细菌B5、B12的16srDNA的鉴定
本试验采用传统分类方法即菌落大小和形态、革兰氏染色和分子生物学方法16SrDNA相结合,将拮抗菌B5、B12菌株进行了PCR扩增、测序,将测出的菌16SrDNA序列用Blast软件、Clustal(1.8)软件及Mega软件对其序列进行了同源性分析和系统发育分析,初步确立了菌株在细菌系统发育学上的地位。B5属于芽孢杆菌属(Bacillus sp.);B12属于枯草芽孢杆菌(Bacillus subtilis.)。
4拮抗菌B5、B12在葡萄果穗表面的定殖及日光温室防治试验
定殖试验结果表明经抗药性标记后B5、B12菌株均能在葡萄果穗表面定殖,B5菌株定殖周期为19d,B12菌株在葡萄表面定殖周期为25d。
日光温室自然发病防效试验的结果表明B5、B12菌株的防治效果均达到了50%以上,达到了比较理想的效果。
Grape grey mold, which can make a great deal of loss of grape production, is one of the important grape disease in the world. Now, agronomic control and chemical control are important control measures, but both of them showed some limitations。
The research to analysis on the adnascent microorganism flora of grape fruit spike、screening and identification of antagonistic strain、antagonistic strains colonization and prevention experiment in the sunlight greenhouse,the conclusion shows that:
1 Analysis on the adnascent microorganism flora of grape fruit spike
In this test the epiphytic microorganism flora on the fruit spike of three grape breeds (jingya、hongdiqiu、jufeng) are studied in the time of young fruit enlargement, pigmentation and maturity. The results showed that amount of filamentous fungus and yeasts increased gradually from the inflation stage to the maturity stage, the amount of germs increased at the beginning and then decreased. Total count rise to the highest during the pigmentation, then shows declining tendency, but changed unobviously. The germs had predominance in the inflation stage and the pigmentation stage.
The account of filamentous fungus, germs and yeasts on the diseasing fruit is more than that on the normal fruit. The varieties of filamentous fungus become less but germs and yeasts had no obviously change.
2 Screening of antagonistic strain
In experiment of flat confront, filtrate 11 kinds of epiphytic bacterium with anti-function to the grape grey mold. Then through the inhibition germination experiment of disease germ spore, inhibition test in growth experiment of mycelium and the test of control effect on vitro fruit, finally filtrated B5 and B12 strains to be the best controlling efficiency on the grape grey mold, but B4 and the B6 strains inhibiting ability is worst, other 7 kinds of oppress between them, it changed a little.
3 16srDNA identification of B5 and B12 strains
This experiment uses the tradition taxonomic approach is the colony size and the shape, Gelanshi dyes and molecular biology method 16SrDNA unifies, will oppress antibacterial B5, the B12 strain to carry on PCR to increase, the sequence, which determine the fungus 16SrDNA sequence with the Blast software, Clustal (1.8) the software and the Mega software has carried on the homology analysis and the phyletic evolution analysis to its sequence, has established the strain initially in the bacterium phyletic evolution study on status. 16srDNA identification shows that the B5 strain is:Bacillus sp. and the B12 strain is:Bacillus subtilis.
4 B5 and B12 strains colonization on the surface of fruit and the sunlight greenhouse prevention experiment
Both B5 and B12 strain can breed on grape surface, the B5 strain′setting period is 19d, B12 strain′setting period is 25d, and they are quite stable.
The prevention test result in greenhouse of B5 and B12 strain shows that:The prevention efficiency of B5 and B12 strains in nature have achieved above 50%, has achieved the quite ideal effect.
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