颉颃细菌对根结线虫病的生防作用研究
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摘要
南方根结线虫(Meloidogyne incognita Kofoid & White)是重要的土传病害病原物,具有寄主范围广、抗逆性强、难以根除等特点,防治上既缺乏优良的抗病品种,又缺乏低毒、高效的化学防治药剂。生物防治克服了上述弊病,因而成为研究的热点,越来越受到人们的重视。
我们选取了26株颉颃细菌,测定它们对南方根结线虫二龄幼虫致死率和卵孵化的抑制率,26株颉颃细菌对南方根结线虫二龄幼虫致死率最高的为77.8%,最低的为1.3%,对卵孵化的抑制率最高为87.8%,最低为-16.5%。根据以上室内测定结果,我们挑选13个菌株(12株对二龄幼虫致死率达到40%以上的菌株和1个随机挑选作为对照的菌株)用于温室防效测定。其中,防效最低为30.0%,最高达到65.6%。此外,我们又测定了26株颉颃细菌产蛋白酶和几丁质酶的活性,结果显示20株菌株具有蛋白酶活性,而14株具有几丁质酶的活性。经过分析,13个菌株的温室防效和蛋白酶活性相关系数高达0.92,但温室防效与几丁质酶活性相关系数仅为0.06。因此细菌产蛋白酶活性作为一个指标用于筛选根结线虫颉颃菌具有一定的可行性。本实验选取了在温室试验中防效最好和产蛋白酶活性高的菌株AR156分别在江苏和山东两地做田间试验。在江苏淮安,AR156防效为73.4%；在山东枣庄,AR156防效为71.0%,菌株AR156的防效都显著高于农药阿维菌素的防效。
我们对芽孢杆菌制剂AR156采用了四种不同的大田使用方法防治黄瓜根结线虫,分别是AR156的堆肥处理(A)、AR156的灌根处理(B)、AR156的喷雾处理(C)、AR156与化学农药辛硫磷的协同使用处理(D)、AR156采用堆肥处理、灌根处理和与化学农药辛硫磷的协同使用处理,90天后和对照相比,对根结线虫防治效果分别为74.4%、74.9%和73.3%,增产率分别达到194.8%,193.1%和189.7%；AR156喷雾处理和对照相比的防治效果为21.7%,增产率为37.9%,而化学农药辛硫磷防治效果却达到61.1%,增产率达155.2%。
生防菌AR156是本实验室从镇江的森林根围土分离得到,经初步鉴定为芽孢杆菌(Bacillus spp.)。该菌株具有产蛋白酶、几丁质酶、纤维素酶和嗜铁素的活性,并具有解磷固氮能力；AR156在根围具有很强的定殖能力,处理前种子的带菌量为每粒种子9.6×106CFU,14天后AR156rif在番茄根围的定殖量为8.9×105CFU/g土样。AR156处理5天后,青枯病原菌HB10挑战接种；病原菌接种14天和21天后,AR156对青枯病的防效分别为75.3%和50.7%。
枯草芽孢杆菌(Bacillus subtilis (Ehrenberg) Cohn)的SpoOA是非常重要的转录因子,可以调控枯草芽孢杆菌生物膜(biofilm)的形成。本实验通过单交换的方式,构建了spoOA基因的突变株mBs,又通过双交换产生了突变体mBs的互补菌株CmBs。PopW是来源于青枯病菌的一种harpin蛋白,具有诱导植物抗病性和促进植物生长的作用。我们构建了含有p43强启动子和nprB信号肽的PopW基因的表达载体pHTPOW,将表达载体pHTPOW分别转入B. subtilis 168和突变体mBs,得到两株工程菌株BsW、mBsW。SDS-PAGE和Western blot结果表明BsW和mBsW都能分泌PopW蛋白。工程菌株BsW、mBsW,在离体试验中,处理24 h和48 h后,J2死亡率分别为53.4%、79.6%、59.0%和89.7%；工程菌Bsw和mBsw的菌悬液处理番茄植株,6周后根系上的卵块和根结数目明显减少,防效分别为60.0%和54.3%。
复合菌剂GJ23在已经完成的室内离体试验和温室实验中,表现出对根结线虫很好的防治作用。大田试验表明使用复合菌剂GJ23能有效防治黄瓜根结线虫,同时对黄瓜有一定的促进生长和增产效应。90天后,复合菌剂GJ23对根结线虫防治效果为63.3%,增产率达到39.4%,均显著高于1.8%阿维菌素乳油。
Meloidogyne incognita (Kofoid & White) is an important plant soil-borne pathogen which can infect hundreds of hosts, and it is difficult to control because of its high tolerance and ineradicable traits. No appropriate disease-resistant cultivar and chemical pesticides with low toxicity and high efficacy were used commonly. Meanwhile, using of biological control agents (BCAs) may provide a friendly alternative to chemical pesticides for plant soil-borne disease control. Therefore, it would be necessary to explore new biological control agents to control these plant pathgens.
Twenty-six bacterial strains that had demonstrated antagonism to some fungal and bacterial pathogens were evaluated for their ability to inhibit the root-knot nematode. In the in vitro test, the 26 strains caused the J2 mortality rates of M. incognita varying from 1.3% to 77.8% and the inhibition rates of egg-hatching ranging from -16.5% to 87.4%. The 12 strains inducing J2 mortality over 40% were chosen for further greenhouse experiments and one of the remaining strains was randomly selected as a control, in which their biocontrol efficacy reached from 30.0% to 65.6%. On the other side, the 26 strains were tested for their protease and chitinase activities, which revealed that 20 of them had protease activities and 14 of them had chitinase activities. We investigated the relationships between the biocontrol efficacy and protease or chitinase activities. The biocontrol efficacy against M. incognita of the antagonistic strains were highly correlated to their in vitro protease activities (r=0.92), but not to their chitinase activities (r=0.06). We proposed that the in vitro protease activity of an antagonistic strain could be used as a parameter for selecting the BCAs against the root-knot disease. We further assessed the biocontrol efficacy of strains Bacillus sp. AR156 in field. Consequently, the biocontrol efficacy of AR156 reached 74.3%, in the field in Huai-an, Jiangsu, and 71.0% in Zao-zhuang, Shandong, respectively, which were significantly higher than that of abamectin:45.4% in Huai-an and 42.2% in Zao-zhuang.
Biocontrol efficacy of Bacillus sp. AR156 with diferent application methods towards Meloidogyne root-knot were tested under field conditions. The application methods included (A) composting AR156 with rapeseed residue and made compost before application, (B) watering diluted AR156 into rhizosphere of plants, (C) spraying diluted AR156 into field, and (D) watering plants with dilution of AR156 preparation after plants treated by phoxime for 3 days. In field trials at Xuzhou in 2007, the total average control efficacy for treatments A, B, and D reached 74.4%,74.9% and 73.3%. And the yield increased 194.8%,193.1%, and 189.7%, respectively. However, method C, spraying, showed worse disease control efficacy than the other methods and the control efficacy was only 21.7% and the yield increased 39.7%. The average control efficacy of phoxime was 61.1% and the yield increased 155.2%.
Strain AR156(Bacillus sp.) was isolated from Zhen Jiang forest soil in Nanjing, Jiangsu province. In order to investigate the mechanism of this biocontrol agent against plant disase, we studied its colonization and other activities. This strain could produce protease, cellulase, chitinase and siderophore. It also has the functions of phosphate-dissolving and nitrogen-fixing. Fourteen days after seed inoculation with 10 ml AR156 suspension with the concentration of 9.6×109 CFU/ml, the density of this strain in rhizosphere of tomato was 8.9×105 CFU/g soil. In addition, strain AR156 could induce the resistance of tomato against the diseases caused by Ralstonian solanacearum HB10. In green house experiments, on the 14th and 21th day after tomato plants were treated with strain AR156, the average biocontrol efficacy were 75.3% and 50.7%, respectively.
Bacillus subtilis (Ehrenberg) Cohn have long been exploited for industrial and biotechnological applications. The completion of the sequencing and annotation of the B. subtilis 168 strain genome provieded a complete view of the Bacillus subtilis protein machinery. spoOA-deficient mutant of B. Subtilis 168 (mBs) contructed by using the pBGSCsp vector which could be integrated into spoOA locus within the B. subtilis 168 chromosome by single-crossover could not form biofilm. Meanwhile, another vector, pSGspOA, was integrated into amyE locus of the mutant (mBs) by double crossover, and we got the complementary strain named CmBs. PopW from Ralstonia solanacearum, is a member of the harpin group of proteins, eliciting hypersensitive cell death in non-host plants, inducing disease resistance in plants and enhancing plant growth. To expression and secreting PopW protein in Bacillus subtilis, we constructed recombinant expression vector pHTPOW, which carried p43 promoter, signal peptide element nprB, and the total PopW gen. The shuttle vector pHTPOW was transformed into competent cell of B. subtilis 168 and mBs separately, and two genetically modified strains, BsW and mBsW were constructed. The expression of PopW in two genetically modified strains was confirmed by SDS-PAGE and Western-blot analysis. In the in vitro test, on the 24 and 48 hous after treated with genetically modified strains, the J2 mortality rates of M. incognita were 53.4%, 79.6%,59.0%, and 89.7%, separately. On the 42th day, the biocontrol efficacy of strains (BsW and mBsW) reached 60.0% and 54.3%, respectively.
In this study, bacteria GJ23 was used to control root-knot nematodes Meloidogyne spp., and it achieved a good efficacy. Previously, strain GJ23 showed great mortality of J2, in vitro. In the greenhouse tests, GJ23 could effectively inhibit gall formation in the roots of tomato. Under field conditions, the average biocontrol efficacy investigated on the 90th day after cucumber plants were treated with strain GJ23 was 63.3%, and yield increasement was 39.4%. As for control, the biocontrol efficacy and yield increasement of 1.8% abamectin were 35.4% and 13.2%, seperately.
我们选取了26株颉颃细菌,测定它们对南方根结线虫二龄幼虫致死率和卵孵化的抑制率,26株颉颃细菌对南方根结线虫二龄幼虫致死率最高的为77.8%,最低的为1.3%,对卵孵化的抑制率最高为87.8%,最低为-16.5%。根据以上室内测定结果,我们挑选13个菌株(12株对二龄幼虫致死率达到40%以上的菌株和1个随机挑选作为对照的菌株)用于温室防效测定。其中,防效最低为30.0%,最高达到65.6%。此外,我们又测定了26株颉颃细菌产蛋白酶和几丁质酶的活性,结果显示20株菌株具有蛋白酶活性,而14株具有几丁质酶的活性。经过分析,13个菌株的温室防效和蛋白酶活性相关系数高达0.92,但温室防效与几丁质酶活性相关系数仅为0.06。因此细菌产蛋白酶活性作为一个指标用于筛选根结线虫颉颃菌具有一定的可行性。本实验选取了在温室试验中防效最好和产蛋白酶活性高的菌株AR156分别在江苏和山东两地做田间试验。在江苏淮安,AR156防效为73.4%；在山东枣庄,AR156防效为71.0%,菌株AR156的防效都显著高于农药阿维菌素的防效。
我们对芽孢杆菌制剂AR156采用了四种不同的大田使用方法防治黄瓜根结线虫,分别是AR156的堆肥处理(A)、AR156的灌根处理(B)、AR156的喷雾处理(C)、AR156与化学农药辛硫磷的协同使用处理(D)、AR156采用堆肥处理、灌根处理和与化学农药辛硫磷的协同使用处理,90天后和对照相比,对根结线虫防治效果分别为74.4%、74.9%和73.3%,增产率分别达到194.8%,193.1%和189.7%；AR156喷雾处理和对照相比的防治效果为21.7%,增产率为37.9%,而化学农药辛硫磷防治效果却达到61.1%,增产率达155.2%。
生防菌AR156是本实验室从镇江的森林根围土分离得到,经初步鉴定为芽孢杆菌(Bacillus spp.)。该菌株具有产蛋白酶、几丁质酶、纤维素酶和嗜铁素的活性,并具有解磷固氮能力；AR156在根围具有很强的定殖能力,处理前种子的带菌量为每粒种子9.6×106CFU,14天后AR156rif在番茄根围的定殖量为8.9×105CFU/g土样。AR156处理5天后,青枯病原菌HB10挑战接种；病原菌接种14天和21天后,AR156对青枯病的防效分别为75.3%和50.7%。
枯草芽孢杆菌(Bacillus subtilis (Ehrenberg) Cohn)的SpoOA是非常重要的转录因子,可以调控枯草芽孢杆菌生物膜(biofilm)的形成。本实验通过单交换的方式,构建了spoOA基因的突变株mBs,又通过双交换产生了突变体mBs的互补菌株CmBs。PopW是来源于青枯病菌的一种harpin蛋白,具有诱导植物抗病性和促进植物生长的作用。我们构建了含有p43强启动子和nprB信号肽的PopW基因的表达载体pHTPOW,将表达载体pHTPOW分别转入B. subtilis 168和突变体mBs,得到两株工程菌株BsW、mBsW。SDS-PAGE和Western blot结果表明BsW和mBsW都能分泌PopW蛋白。工程菌株BsW、mBsW,在离体试验中,处理24 h和48 h后,J2死亡率分别为53.4%、79.6%、59.0%和89.7%；工程菌Bsw和mBsw的菌悬液处理番茄植株,6周后根系上的卵块和根结数目明显减少,防效分别为60.0%和54.3%。
复合菌剂GJ23在已经完成的室内离体试验和温室实验中,表现出对根结线虫很好的防治作用。大田试验表明使用复合菌剂GJ23能有效防治黄瓜根结线虫,同时对黄瓜有一定的促进生长和增产效应。90天后,复合菌剂GJ23对根结线虫防治效果为63.3%,增产率达到39.4%,均显著高于1.8%阿维菌素乳油。
Meloidogyne incognita (Kofoid & White) is an important plant soil-borne pathogen which can infect hundreds of hosts, and it is difficult to control because of its high tolerance and ineradicable traits. No appropriate disease-resistant cultivar and chemical pesticides with low toxicity and high efficacy were used commonly. Meanwhile, using of biological control agents (BCAs) may provide a friendly alternative to chemical pesticides for plant soil-borne disease control. Therefore, it would be necessary to explore new biological control agents to control these plant pathgens.
Twenty-six bacterial strains that had demonstrated antagonism to some fungal and bacterial pathogens were evaluated for their ability to inhibit the root-knot nematode. In the in vitro test, the 26 strains caused the J2 mortality rates of M. incognita varying from 1.3% to 77.8% and the inhibition rates of egg-hatching ranging from -16.5% to 87.4%. The 12 strains inducing J2 mortality over 40% were chosen for further greenhouse experiments and one of the remaining strains was randomly selected as a control, in which their biocontrol efficacy reached from 30.0% to 65.6%. On the other side, the 26 strains were tested for their protease and chitinase activities, which revealed that 20 of them had protease activities and 14 of them had chitinase activities. We investigated the relationships between the biocontrol efficacy and protease or chitinase activities. The biocontrol efficacy against M. incognita of the antagonistic strains were highly correlated to their in vitro protease activities (r=0.92), but not to their chitinase activities (r=0.06). We proposed that the in vitro protease activity of an antagonistic strain could be used as a parameter for selecting the BCAs against the root-knot disease. We further assessed the biocontrol efficacy of strains Bacillus sp. AR156 in field. Consequently, the biocontrol efficacy of AR156 reached 74.3%, in the field in Huai-an, Jiangsu, and 71.0% in Zao-zhuang, Shandong, respectively, which were significantly higher than that of abamectin:45.4% in Huai-an and 42.2% in Zao-zhuang.
Biocontrol efficacy of Bacillus sp. AR156 with diferent application methods towards Meloidogyne root-knot were tested under field conditions. The application methods included (A) composting AR156 with rapeseed residue and made compost before application, (B) watering diluted AR156 into rhizosphere of plants, (C) spraying diluted AR156 into field, and (D) watering plants with dilution of AR156 preparation after plants treated by phoxime for 3 days. In field trials at Xuzhou in 2007, the total average control efficacy for treatments A, B, and D reached 74.4%,74.9% and 73.3%. And the yield increased 194.8%,193.1%, and 189.7%, respectively. However, method C, spraying, showed worse disease control efficacy than the other methods and the control efficacy was only 21.7% and the yield increased 39.7%. The average control efficacy of phoxime was 61.1% and the yield increased 155.2%.
Strain AR156(Bacillus sp.) was isolated from Zhen Jiang forest soil in Nanjing, Jiangsu province. In order to investigate the mechanism of this biocontrol agent against plant disase, we studied its colonization and other activities. This strain could produce protease, cellulase, chitinase and siderophore. It also has the functions of phosphate-dissolving and nitrogen-fixing. Fourteen days after seed inoculation with 10 ml AR156 suspension with the concentration of 9.6×109 CFU/ml, the density of this strain in rhizosphere of tomato was 8.9×105 CFU/g soil. In addition, strain AR156 could induce the resistance of tomato against the diseases caused by Ralstonian solanacearum HB10. In green house experiments, on the 14th and 21th day after tomato plants were treated with strain AR156, the average biocontrol efficacy were 75.3% and 50.7%, respectively.
Bacillus subtilis (Ehrenberg) Cohn have long been exploited for industrial and biotechnological applications. The completion of the sequencing and annotation of the B. subtilis 168 strain genome provieded a complete view of the Bacillus subtilis protein machinery. spoOA-deficient mutant of B. Subtilis 168 (mBs) contructed by using the pBGSCsp vector which could be integrated into spoOA locus within the B. subtilis 168 chromosome by single-crossover could not form biofilm. Meanwhile, another vector, pSGspOA, was integrated into amyE locus of the mutant (mBs) by double crossover, and we got the complementary strain named CmBs. PopW from Ralstonia solanacearum, is a member of the harpin group of proteins, eliciting hypersensitive cell death in non-host plants, inducing disease resistance in plants and enhancing plant growth. To expression and secreting PopW protein in Bacillus subtilis, we constructed recombinant expression vector pHTPOW, which carried p43 promoter, signal peptide element nprB, and the total PopW gen. The shuttle vector pHTPOW was transformed into competent cell of B. subtilis 168 and mBs separately, and two genetically modified strains, BsW and mBsW were constructed. The expression of PopW in two genetically modified strains was confirmed by SDS-PAGE and Western-blot analysis. In the in vitro test, on the 24 and 48 hous after treated with genetically modified strains, the J2 mortality rates of M. incognita were 53.4%, 79.6%,59.0%, and 89.7%, separately. On the 42th day, the biocontrol efficacy of strains (BsW and mBsW) reached 60.0% and 54.3%, respectively.
In this study, bacteria GJ23 was used to control root-knot nematodes Meloidogyne spp., and it achieved a good efficacy. Previously, strain GJ23 showed great mortality of J2, in vitro. In the greenhouse tests, GJ23 could effectively inhibit gall formation in the roots of tomato. Under field conditions, the average biocontrol efficacy investigated on the 90th day after cucumber plants were treated with strain GJ23 was 63.3%, and yield increasement was 39.4%. As for control, the biocontrol efficacy and yield increasement of 1.8% abamectin were 35.4% and 13.2%, seperately.
引文
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