蔬菜根结线虫生防菌的筛选与鉴定
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摘要
采用河北农业大学植物病害生物防治研究室已经筛选到的、对多种病原菌具有颉颃作用的120株链霉菌和50株芽孢杆菌，筛选对根结线虫有防治作用的菌株，并利用形态学特征、生理生化性质和16S rDNA的保守性对筛选得到的菌株进行鉴定。研究初步筛选到Z-L-2、Z-L-17、Z-L-19、Men-myco-93-19、Men-myco-93-38、Men-myco-93-63等6株链霉菌，其发酵液稀释4倍后对根结线虫二龄幼虫的致死率均在90％以上。
上述6株链霉菌发酵液在不同浓度和不同处理时间下对根结线虫二龄幼虫分别测定，表现出不同的致死率。Z-L-17发酵液的稀释2倍的致死率最高，为100％，其次是Z-L-2的为96％；Z-L-2发酵液的稀释4倍液致死率最高，为95.2％；在稀释8×、16×和32×下菌株Z-L-2发酵液的致死率均为最高。处理4h时Z-19发酵液的致死率最高，为85.6％；处理8h、12h、24h后Z-L-2均为最高，依次为87.4％、94.6％、95.2％。
6株链霉菌发酵液用于盆栽防治黄瓜根结线虫病，结果菌株Z-L-2发酵液的防效为最高，达到75％，地上部和地下部鲜重分别增加89.4％和66.67％。0.5％阿维菌素1000×的防效为8.3％，地上部和地下部鲜重分别增加了46.3％和63.9％。
菌株Z-L-2用于防治根结线虫病的温室田间试验，与空白对照相比，1.8％虫螨克星3000倍可增产49.9％，菌株Z-L-2发酵液稀释4倍增产了51.3％；菌株Z-L-2发酵液稀释10倍增产42.9％，三个处理之间差异不显著，但均与对照存在显著差异。
通过对链霉菌菌株Z-L-2形态特征、生理生化特征及16S rDNA进行鉴定，结果可以将其初步分类到粉红孢C类群，弗氏Fradiae亚群，玫瑰黄链霉菌Streptomyces roseflavus。
通过16S rDNA序列，菌株Z-L-2与模式菌株S．roseoflavus同源性达到了99.80％，仅相差3个碱基。与本实验室的刘占良定名的玫瑰黄链霉菌Men-myco-93-63相差3个碱基。Z-L-2与已有数据的120 bpγ可变区比较，与S．roseoflavus和玫瑰黄链霉菌Men-mycg-93-63均相差一个碱基。系统发育树表明，菌株Z-L-2与和玫瑰黄链霉菌Men-myco-93-63遗传距离最小，菌株Z-L-2可能为它的一个变种。
One hundred and twenty Streptomyces spp. strains and fifty Bacillus spp. strains were used to screen biocontrol agents to J2 of root-knot nematodes. Six Streptomyces spp. strains were gotten in the first step, such as Z-L-2, Z-L-17, Z-L-19, Men-myco-93-19, Men-myco-93-38 and Men-myco-93-63. All of the 4-fold diluted ferment filtrates of the above six strains had more than 90% kill-down efficiency to J2.
The six strains ferment filtrates were used to measure the death-rates of J2 of root-knot nematodes respectively in different concentration and treated time. The ferment filtrate showed strong nematicidal activity to J2. The 2-fold diluted ferment filtrates of Z-L-17 and Z-L-2 had 100% and 96% kill-down efficiency respectively, but 4-fold diluted ferment filtrate of Z-L-2 had trong nematicidal activity. At 8-fold, 16-fold and 32-fold, the ferment filtrate of isolate Z-L-2 expressed more effective to the J2 than others. When treated time reached to 4h, isolate Z-19 show strong nematicidal activity to J2, the kill-down rate reached to 85.6%. The 4-fold diluted ferment filtrate of isolate Z-L-2 had 87.4%, 94.6% and 95.2% respectively kill-down efficiency to the J2 when treated for 8 hours, 12 hours and 24 hours.
The six strains ferment filtrate were all used to control cucumber root-knot nematodes disease, the control efficiency of Z-L-2 ferment filtrate reached to 75%. The fresh weight of plants above grand and under grand are increased in 89.4% and 66.7% compared with CK. The control result of avilamycin was 8.3%. Their fresh weight of above grand and under grand were 46.3% and 63.9% respectively.
The ferment filtrate of Z-L-2 strain was used in greenhouse to control the pumpkin root-knot nematodes. The results showed that 1.8% Chongmankexing diluted 3000-fold increased production 49.9%, the Z-L-2 4-fold diluted ferment filtrate increased 51.3% production, the Z-L-2 10-fold diluted ferment filtrate increased 42.9% production compared with CK. The control effects of the three treatments on root-knot nematodes are not significantly difference, but are all significantly difference to control.
The strain Z-L-2 DNA was extracted, and was amplificated with general primers of 16S rDNA. The amplification was detected, cloned and sequenced by Dalian Takara company. The sequence was compared with the known sequences in the database of
GenBank. The phylogenetic tree drew and analyzed according the sequence homology. BLAST analysis result suggests that Z-L-2 is 99.80% homology with S. roseoflavus , with only 3bp difference. There is only Ibp different compared with S'. roseoflavus and Men-myco-93-63 at the 120bp- Y regions of 16S rDNA. And the phylogenetic tree shows the genetic distance between Z-L-2 and Men-myco-93-63 is the shortest than any others. The strain Z-L-2 may be a mutation of Men-myco-93-63.
上述6株链霉菌发酵液在不同浓度和不同处理时间下对根结线虫二龄幼虫分别测定，表现出不同的致死率。Z-L-17发酵液的稀释2倍的致死率最高，为100％，其次是Z-L-2的为96％；Z-L-2发酵液的稀释4倍液致死率最高，为95.2％；在稀释8×、16×和32×下菌株Z-L-2发酵液的致死率均为最高。处理4h时Z-19发酵液的致死率最高，为85.6％；处理8h、12h、24h后Z-L-2均为最高，依次为87.4％、94.6％、95.2％。
6株链霉菌发酵液用于盆栽防治黄瓜根结线虫病，结果菌株Z-L-2发酵液的防效为最高，达到75％，地上部和地下部鲜重分别增加89.4％和66.67％。0.5％阿维菌素1000×的防效为8.3％，地上部和地下部鲜重分别增加了46.3％和63.9％。
菌株Z-L-2用于防治根结线虫病的温室田间试验，与空白对照相比，1.8％虫螨克星3000倍可增产49.9％，菌株Z-L-2发酵液稀释4倍增产了51.3％；菌株Z-L-2发酵液稀释10倍增产42.9％，三个处理之间差异不显著，但均与对照存在显著差异。
通过对链霉菌菌株Z-L-2形态特征、生理生化特征及16S rDNA进行鉴定，结果可以将其初步分类到粉红孢C类群，弗氏Fradiae亚群，玫瑰黄链霉菌Streptomyces roseflavus。
通过16S rDNA序列，菌株Z-L-2与模式菌株S．roseoflavus同源性达到了99.80％，仅相差3个碱基。与本实验室的刘占良定名的玫瑰黄链霉菌Men-myco-93-63相差3个碱基。Z-L-2与已有数据的120 bpγ可变区比较，与S．roseoflavus和玫瑰黄链霉菌Men-mycg-93-63均相差一个碱基。系统发育树表明，菌株Z-L-2与和玫瑰黄链霉菌Men-myco-93-63遗传距离最小，菌株Z-L-2可能为它的一个变种。
One hundred and twenty Streptomyces spp. strains and fifty Bacillus spp. strains were used to screen biocontrol agents to J2 of root-knot nematodes. Six Streptomyces spp. strains were gotten in the first step, such as Z-L-2, Z-L-17, Z-L-19, Men-myco-93-19, Men-myco-93-38 and Men-myco-93-63. All of the 4-fold diluted ferment filtrates of the above six strains had more than 90% kill-down efficiency to J2.
The six strains ferment filtrates were used to measure the death-rates of J2 of root-knot nematodes respectively in different concentration and treated time. The ferment filtrate showed strong nematicidal activity to J2. The 2-fold diluted ferment filtrates of Z-L-17 and Z-L-2 had 100% and 96% kill-down efficiency respectively, but 4-fold diluted ferment filtrate of Z-L-2 had trong nematicidal activity. At 8-fold, 16-fold and 32-fold, the ferment filtrate of isolate Z-L-2 expressed more effective to the J2 than others. When treated time reached to 4h, isolate Z-19 show strong nematicidal activity to J2, the kill-down rate reached to 85.6%. The 4-fold diluted ferment filtrate of isolate Z-L-2 had 87.4%, 94.6% and 95.2% respectively kill-down efficiency to the J2 when treated for 8 hours, 12 hours and 24 hours.
The six strains ferment filtrate were all used to control cucumber root-knot nematodes disease, the control efficiency of Z-L-2 ferment filtrate reached to 75%. The fresh weight of plants above grand and under grand are increased in 89.4% and 66.7% compared with CK. The control result of avilamycin was 8.3%. Their fresh weight of above grand and under grand were 46.3% and 63.9% respectively.
The ferment filtrate of Z-L-2 strain was used in greenhouse to control the pumpkin root-knot nematodes. The results showed that 1.8% Chongmankexing diluted 3000-fold increased production 49.9%, the Z-L-2 4-fold diluted ferment filtrate increased 51.3% production, the Z-L-2 10-fold diluted ferment filtrate increased 42.9% production compared with CK. The control effects of the three treatments on root-knot nematodes are not significantly difference, but are all significantly difference to control.
The strain Z-L-2 DNA was extracted, and was amplificated with general primers of 16S rDNA. The amplification was detected, cloned and sequenced by Dalian Takara company. The sequence was compared with the known sequences in the database of
GenBank. The phylogenetic tree drew and analyzed according the sequence homology. BLAST analysis result suggests that Z-L-2 is 99.80% homology with S. roseoflavus , with only 3bp difference. There is only Ibp different compared with S'. roseoflavus and Men-myco-93-63 at the 120bp- Y regions of 16S rDNA. And the phylogenetic tree shows the genetic distance between Z-L-2 and Men-myco-93-63 is the shortest than any others. The strain Z-L-2 may be a mutation of Men-myco-93-63.
引文
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