青枯无致病力菌株对烟草青枯病的室内控病研究
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摘要
烟草青枯病是由劳尔氏菌属的茄假单胞杆菌Ralstonia solanacearum引起的一种系统性侵染病害,对烟草的产量和质量影响极大,是烟草上一大毁灭性病害,其防治十分困难。生物防治对烟叶生产的可持续发展具有较好的应用前景。利用青枯无致病力菌株作为生防菌,它们能够在与病原菌相似的生态条件下发挥作用,且对生态环境的影响很小。本研究从重庆不同区县采集茄、番茄、烟草和辣椒青枯病株分离青枯无致病力菌株,并通过紫外光诱变和连续转代青枯致病菌获取无致病力菌株。对这些菌株进行平板拮抗筛选及温室控病试验,并对它们诱导烟草植株体内防御酶系活性及病程相关蛋白变化进行了测定。
1.从2006年6月初至9月底,在重庆市璧山县、北碚区、九龙坡区白市驿镇、江津区、酉阳县和涪陵区采集青枯病株样本共184份,其中茄青枯病株67份,番茄青枯病株42份,烟草青枯病株38份,辣椒青枯病株37份。分离纯化保存并初步鉴定菌株共138株,其中青枯致病菌株22株,占获得的青枯菌株总数的16%。22株青枯致病菌株中,茄青枯菌13株,烟草青枯菌6株,番茄青枯菌2株,辣椒青枯菌1株。无致病力菌株116株,占获得的青枯菌株总数的84%,其中番茄青枯菌41株,茄青枯菌41株,辣椒青枯菌21株,烟草青枯菌13株。
2.将直接分离得到的青枯无致病力菌株116株,通过紫外光诱变得到的青枯无致病力菌株42株,及连续转代获得的青枯无致病力菌株19株,对它们进行烟草青枯菌的平板抑菌试验。初筛得到对烟草青枯菌有抑菌活性的菌株共62株,其中61株为直接分离得到的菌株,1株为紫外光诱变得到。进行复筛后得到抑菌带宽≥1cm的菌株21株;抑菌带宽为0.5～1cm的菌株13株;抑菌带宽度≤0.5cm的菌株28株。将抑菌带宽≥1cm的菌株采用喷雾法和含菌培养基法两种筛选方法比较。结果表明:采用喷雾法得到的抑菌带宽普遍大于含菌培养基法得到的,只有Tmt3-2-1和TbYYk1-1两个菌株是例外。通过两种方法比较,认为喷雾法比含菌培养基法操作简便、快捷,比较适用于平板筛选试验。
3.平板筛选出的抑菌带宽≥1cm的21株菌株,对它们进行烟草青枯病的温室控病试验。初测结果表明:这21株菌株大部分能够使烟草推迟7d发病,CKB只灌接待测菌株的烟草苗未表现明显异常,初步说明它们对烟草是安全的。在接种20d后,相对防效＜70%有6株,其余的相对防效＞70%。结合菌株在NA培养基上的生长势,将其中的6株进行复测。结果表明:L6-3、Tmjd1-3、Au004-1、TbYYk1-1和Aujd8-2-1处理分别较CKRs推迟发病14d、6d、5d、2d和1d。L6-3、Au004-1和Tmjd1-3处理对烟草青枯病的温室控病效果较好。在接种30d后,它们的相对防效分别达到80.83%、77.5%和75%,其中L6-3处理的相对防效最好。
4.在NA培养基上的生长势较好且温室控病效果较好的青枯无致病力菌株Au004-1和Tmjd1-3,用它们分别处理烟草后测定植株体内的苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)及多酚氧化酶(PPO)活性,并通过电泳分析烟草植株体内病程相关蛋白(PRP)的变化。结果表明:它们处理烟草后,植株体内的PAL、POD及PPO活性增强,PRP含量也有提高。表明青枯无致病力菌株对烟草青枯病的控病机制除了对致病菌的直接抑制作用外,很可能诱导烟草植株产生了抗病性。
Tobacco bacterial wilt,which is a kind of bacteria disease caused by Ralstonia solanacearum and greatly affects the yield and quality of tobacco production,is one of the most destructive diseases on the tobacco crop and is hard to control.Biocontrol is an impromising way to control this tobacco disease. Avirulent strains of Ralstonia solanacearum has been found to be effective to the pathogen under similar ecological conditions,while it exerts a little negative influence on entironment.In the study several avirulent isolates were got from disease samples in eggplant,tomato,tobacco and pimiento planting area in Chongqing,and they were acquired by ultraviolet mutation and culturing the pathogen in succession. These avirulent isolates were screened for antagonistic bacteria aginst the pathogen,and were tested to control the disease in greenhouse.Meanwhile the activities of defensive enzymes and the content of pathogensis-related prcteins(PRP)in the tobacco plants inoculated by these avirulent strains were measured.The main results are listed as follows.
1.Among June to September 2006,a number of 184 samples were collected in vegetable or tobacco planting area in Bishan county,Beibei borough,Baishiyi town Jiulongpo brough,Jiangjin brough,Youyang county and Fuling brough,including 67 samples of eggplant,42 ones of tomato,38 ones of tobacco,37 ones of pimiento which were infected by the pathogen.A total number of isolates that were conserved and identified primarily was 138,including 22 virulent isolates accounting for 16%of the total isolates and was composed by 13 ones from diseased eggplant,6 from diseased tobacco,2 from diseased tomato,1 from diseased pimiento,and 116 avirulent isolates accounting for 84%of the total isolates and composed by 41 ones from diseased tomato,41 from diseased eggplant,21 from diseased pimiento,13 from diseased tobacco.
2.One hundred and sixteen avirulent isolates which were isolated directly,42 ones which were obtained throng ultraviolet radiation treatment,and 19 ones which were acquired by culturing the pathogen in succession,had been assayed for antagonistic activity in the study.Sixty two of the avirulent isolates constituted by 61 from direct isolation and 1 from ultraviolet mutation,were found antagonistic against Rsolanacearum solanacearum.Afer the second screening,21 had an inhibition zone =1cm in width,13 had an inhibition zone between 0.5cm and 1cm in width,28 had an inhibition zone =0.5cm in width.Employed the 21 isolates,spaying and fixing bacterium for screening were compared.The results showed that most of the isolates inhibition zones gained by spaying were wider than those from fixing bacterium,except numbered Tmt3-2-1 and TbYYk1-1.The conclusion was that the method of spaying was handier and quicher than the other,and was more applicable for antagonizing test.
3.The greenhouse experiment on tobacco plants was carried out with the 21 isolates,the results indicated that most of them could postpone the disease occurrence 7 days,obvious abnormity did not appear on young tobacco plants that showed the 21 isolates may be harmless to tobacco plants.Of 5 isolates,the relative control efficacy was =60%,the others' was＞60%.Six isolates were chose for the second greenhouse experiment,according the results of the first test and development on media,the results showed that comparing to trentment with CKRs,each treatment with L6-3、Tmjd1-3、Au004-1、TbYYk1-1 and Aujd8-2-1 could postpone the disease occurrence 14d、6d、5d、2d and 1d,respectively.L6-3,Au004-1 and Tmjd1-3 presented better controlling effect on tobacco bacterial wilt,the relative control efficacy was 80.83%、77.5%and 75%respectively at 30 days after treatment,and the relative control efficacy of L6-3 was the highest.
4.The activities of phenylalanineammonium-lyase(PAL),peroxidase(POD),polyphe-noloxidase (PPO)and the content of pathogensis-related proteins(PRP)in tobacco were determined,after treated by Au004-1 and Tmjd1-3 growing better on NA media and owning the better control effect.It was found the the activities of PAL,POD,PPO and the content of PRP in tobacco increased.Then the results suggested that the impacting mechanism of avirulent strains of Ralstonia solanacearum on tobacco bacterial wilt was directly antagonistic action besides,might be induced resistance.
1.从2006年6月初至9月底,在重庆市璧山县、北碚区、九龙坡区白市驿镇、江津区、酉阳县和涪陵区采集青枯病株样本共184份,其中茄青枯病株67份,番茄青枯病株42份,烟草青枯病株38份,辣椒青枯病株37份。分离纯化保存并初步鉴定菌株共138株,其中青枯致病菌株22株,占获得的青枯菌株总数的16%。22株青枯致病菌株中,茄青枯菌13株,烟草青枯菌6株,番茄青枯菌2株,辣椒青枯菌1株。无致病力菌株116株,占获得的青枯菌株总数的84%,其中番茄青枯菌41株,茄青枯菌41株,辣椒青枯菌21株,烟草青枯菌13株。
2.将直接分离得到的青枯无致病力菌株116株,通过紫外光诱变得到的青枯无致病力菌株42株,及连续转代获得的青枯无致病力菌株19株,对它们进行烟草青枯菌的平板抑菌试验。初筛得到对烟草青枯菌有抑菌活性的菌株共62株,其中61株为直接分离得到的菌株,1株为紫外光诱变得到。进行复筛后得到抑菌带宽≥1cm的菌株21株;抑菌带宽为0.5～1cm的菌株13株;抑菌带宽度≤0.5cm的菌株28株。将抑菌带宽≥1cm的菌株采用喷雾法和含菌培养基法两种筛选方法比较。结果表明:采用喷雾法得到的抑菌带宽普遍大于含菌培养基法得到的,只有Tmt3-2-1和TbYYk1-1两个菌株是例外。通过两种方法比较,认为喷雾法比含菌培养基法操作简便、快捷,比较适用于平板筛选试验。
3.平板筛选出的抑菌带宽≥1cm的21株菌株,对它们进行烟草青枯病的温室控病试验。初测结果表明:这21株菌株大部分能够使烟草推迟7d发病,CKB只灌接待测菌株的烟草苗未表现明显异常,初步说明它们对烟草是安全的。在接种20d后,相对防效＜70%有6株,其余的相对防效＞70%。结合菌株在NA培养基上的生长势,将其中的6株进行复测。结果表明:L6-3、Tmjd1-3、Au004-1、TbYYk1-1和Aujd8-2-1处理分别较CKRs推迟发病14d、6d、5d、2d和1d。L6-3、Au004-1和Tmjd1-3处理对烟草青枯病的温室控病效果较好。在接种30d后,它们的相对防效分别达到80.83%、77.5%和75%,其中L6-3处理的相对防效最好。
4.在NA培养基上的生长势较好且温室控病效果较好的青枯无致病力菌株Au004-1和Tmjd1-3,用它们分别处理烟草后测定植株体内的苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)及多酚氧化酶(PPO)活性,并通过电泳分析烟草植株体内病程相关蛋白(PRP)的变化。结果表明:它们处理烟草后,植株体内的PAL、POD及PPO活性增强,PRP含量也有提高。表明青枯无致病力菌株对烟草青枯病的控病机制除了对致病菌的直接抑制作用外,很可能诱导烟草植株产生了抗病性。
Tobacco bacterial wilt,which is a kind of bacteria disease caused by Ralstonia solanacearum and greatly affects the yield and quality of tobacco production,is one of the most destructive diseases on the tobacco crop and is hard to control.Biocontrol is an impromising way to control this tobacco disease. Avirulent strains of Ralstonia solanacearum has been found to be effective to the pathogen under similar ecological conditions,while it exerts a little negative influence on entironment.In the study several avirulent isolates were got from disease samples in eggplant,tomato,tobacco and pimiento planting area in Chongqing,and they were acquired by ultraviolet mutation and culturing the pathogen in succession. These avirulent isolates were screened for antagonistic bacteria aginst the pathogen,and were tested to control the disease in greenhouse.Meanwhile the activities of defensive enzymes and the content of pathogensis-related prcteins(PRP)in the tobacco plants inoculated by these avirulent strains were measured.The main results are listed as follows.
1.Among June to September 2006,a number of 184 samples were collected in vegetable or tobacco planting area in Bishan county,Beibei borough,Baishiyi town Jiulongpo brough,Jiangjin brough,Youyang county and Fuling brough,including 67 samples of eggplant,42 ones of tomato,38 ones of tobacco,37 ones of pimiento which were infected by the pathogen.A total number of isolates that were conserved and identified primarily was 138,including 22 virulent isolates accounting for 16%of the total isolates and was composed by 13 ones from diseased eggplant,6 from diseased tobacco,2 from diseased tomato,1 from diseased pimiento,and 116 avirulent isolates accounting for 84%of the total isolates and composed by 41 ones from diseased tomato,41 from diseased eggplant,21 from diseased pimiento,13 from diseased tobacco.
2.One hundred and sixteen avirulent isolates which were isolated directly,42 ones which were obtained throng ultraviolet radiation treatment,and 19 ones which were acquired by culturing the pathogen in succession,had been assayed for antagonistic activity in the study.Sixty two of the avirulent isolates constituted by 61 from direct isolation and 1 from ultraviolet mutation,were found antagonistic against Rsolanacearum solanacearum.Afer the second screening,21 had an inhibition zone =1cm in width,13 had an inhibition zone between 0.5cm and 1cm in width,28 had an inhibition zone =0.5cm in width.Employed the 21 isolates,spaying and fixing bacterium for screening were compared.The results showed that most of the isolates inhibition zones gained by spaying were wider than those from fixing bacterium,except numbered Tmt3-2-1 and TbYYk1-1.The conclusion was that the method of spaying was handier and quicher than the other,and was more applicable for antagonizing test.
3.The greenhouse experiment on tobacco plants was carried out with the 21 isolates,the results indicated that most of them could postpone the disease occurrence 7 days,obvious abnormity did not appear on young tobacco plants that showed the 21 isolates may be harmless to tobacco plants.Of 5 isolates,the relative control efficacy was =60%,the others' was＞60%.Six isolates were chose for the second greenhouse experiment,according the results of the first test and development on media,the results showed that comparing to trentment with CKRs,each treatment with L6-3、Tmjd1-3、Au004-1、TbYYk1-1 and Aujd8-2-1 could postpone the disease occurrence 14d、6d、5d、2d and 1d,respectively.L6-3,Au004-1 and Tmjd1-3 presented better controlling effect on tobacco bacterial wilt,the relative control efficacy was 80.83%、77.5%and 75%respectively at 30 days after treatment,and the relative control efficacy of L6-3 was the highest.
4.The activities of phenylalanineammonium-lyase(PAL),peroxidase(POD),polyphe-noloxidase (PPO)and the content of pathogensis-related proteins(PRP)in tobacco were determined,after treated by Au004-1 and Tmjd1-3 growing better on NA media and owning the better control effect.It was found the the activities of PAL,POD,PPO and the content of PRP in tobacco increased.Then the results suggested that the impacting mechanism of avirulent strains of Ralstonia solanacearum on tobacco bacterial wilt was directly antagonistic action besides,might be induced resistance.
引文
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