烟草青枯病生防菌的筛选、定殖与应用研究
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摘要
烟草青枯病是由茄科罗尔氏菌(Ralstonia solanacearum E. F Smith)引起的细菌性土传病害,其分布广、危害严重。它不仅危害烟草,而且还危害许多茄科作物,迄今还没有一种有效的药剂防治它。因此发掘有效的生防菌来防治该病,具有重要的现实意义。
本研究采用抑菌圈-定殖力双重测定法,筛选出FT12、F08、Y05三个对青枯菌抑菌作用较强的菌株。抑菌谱测定表明,三个菌株有较宽的抑菌谱。依据三个菌株的形态学、生物学和生理生化特性对它们进行分类鉴定,初步确定FT12为枯草芽孢杆菌(Bacillus subtilis),F08、Y05为侧孢芽孢杆菌(Bacillus laterosporus)。此外,三个菌株有很强的定殖能力。在温室防病试验中,其平均防病效果分别达70.21%(FT12)、70.70%(F08)和60.69%(Y05)。
用双抗标记法测定了3株生防菌,内生枯草芽孢杆菌(Bacillus subtilis)B001、内生短短芽孢杆菌(Bacillus brevis) B011和根围侧孢芽孢杆菌(Bacillus laterosporus) 2-Q-9,在烟株根茎叶及根表土壤中的定殖和消长动态。结果表明,3株生防菌具有很强的定殖能力,其中以B011的定殖能力最强。平板拮抗试验表明,3株生防菌相互间无拮抗作用,且混合菌群的抑菌活性有所增强。生防菌B001、B011和2-Q-9以不同组合混合接种烟株后,生防菌两两混接总体上可提升其定殖力,但3株混接对B011和2-Q-9有抑制作用；在烟株接种移栽后的第60d,各混接组合的定殖总量均达到最大,其中B001+B011和B011+2-Q-9组合的定殖总量分别达7.28、7.231g-cfu/g,远高于其它处理。
将生防菌B001、B011和2-Q-9浸根,灌根,注射,拌饼肥的生物防治法与喷雾农用链霉素的化学防治法相结合,筛选出了对青枯病防效较好的防治组合。其中,联合处理的防治效果均优于72%农用链霉素单独处理。拌饼肥联合处理要好于对应的不拌饼肥处理,注射法联合处理好于对应的非注射法处理；根围生防菌2-Q-9与农用链霉素联合处理优于对应的单用2-Q-9处理,但链霉素与两株内生菌B001、B011的联合处理却对2株生防菌产生抑制作用。同时,筛选出的内生生防菌拌饼肥-灌根-注射联合处理的施用组合,其防效分别达51.37%(B001),71.20%(B011)；2-Q-9的菌饼肥-菌灌根-喷雾Agr-SM组合,其防效可达62.32%。
将3株生防菌B001、B011和2-Q-9混合接种,表明多功能菌群协同防治烟草青枯病具有有效性和可行性。生防菌B001、B011和2-Q-9的不同组合混接烟株后,其防治效果均优于72%的农用链霉素单独处理；两菌混接要优于对应的单菌处理,其中以B011+2-Q-9组合防效最好,达83.39%,其次是B001+B011组合,防效为78.34%；但三株混接的防效仅为45.49%,低于所有生防处理。
Tobacco bacteria wilt caused by Ralstonia solanacearum is the soil-born disease which is destructive to tobacco. There are no effective and environmentally harmonious methods to control it as yet. In the study, some bacterial strains were studied to control the disease.
Screening of biocontrol bacteria showed the stains FT12, F08, Y05 had strongly colonizing capacity and antagonistic action to R. solanacearum. And bio-control agent against bacterial wilt in greenhouse indicated that the average control effects of three strains were 70.21%(FT12),70.70%(F08) and 60.69%(Y05), respectively. Meanwhile strains FT12, F08, Y05 had a wide spectrum of inhibiting plant pathogen. According to morphological, physiological and biochemical characteries, strains FT12, F08 and Y05 were preliminarily identified and FT12 belongs to Bacillus subtilis,F08 and Y05belong to Bacillus laterosporus.
Colonization ability of three biocontrol agents Bacillus subtilis B001, B. brevis B011 and B. laterosporus 2-Q-9 were determined by the method of dual-resistant marker. The result showed that three biocontrol bacteria had a strong colonizing ability and the best was B011 in strain's single inoculations. The antagonistic test showed that mutual antagonism between three bio-control strains had no occurrence but the antibacterial activities against R. soanacearum were improved. And colonizing abilities of two strains'associative inoculations in tobacco were generally increased but B011 and 2-Q-9 in mixture inoculation of three strains were inhibited. Furthermore, the total populations of mixed strains reached the maximum values on 60th d after dipping tobacco roots treatment.
In order to further stabilize and improve the control effects of three biocontrol agents B001, B011 and 2-Q-9 on tobacco bacterial wilt, the control effects of B001, B011 and 2-Q-9 were tested by the combined inoculation ways of root-soaking, root-watering, stem-injecting, fermented rapeseed-cake and chemical way of streptomycin-spraying. The field trial indicated that effects of different combined treatments on tobacco bacterial wilt were all better than that of single 72% agricultural streptomycin treatment. Impacts of the biocontrol bacteria mixed with fermented rapeseed-cake on tobacco bacteria wilt were better than that of the non-mixed, so did combined injection treatments vs. the corresponding non-injection; and combined treatments of 2-Q-9 plus agricultural streptomycin were superior to the single 2-Q-9 treatment, but the joint treatments of streptomycin plus B001 or BO 11 inhibited the two strains'antibacterial activities against R. soanacearum. Furthermore, The control effects of B001 and B011,which were treated by combined ways of fermented rapeseed-cake, root-watering and stem-injecting, reached 51.37%(B001), 71.20%(B011), respectively, and efficiencies of fermented rapeseed-cake, root-watering of 2-Q-9 plus streptomycin-spraying were 62.32%
The combination test of bio-control strains B001, B011 and 2-Q-9 indicated that effects of single bio-strain and mixtures on tobacco bacterial wilt were all better than that of 4000X of 72% agricultural streptomycin, and the mixed treatments of the two strains were also superior to corresponding single bacterium's. Particularly, efficiencies of B011+2-Q-9 and B004+B011 against the disease reached 83.39% and 78.34%, respectively, but effect of B001+B011+2-Q-9 was only 45.49%.
本研究采用抑菌圈-定殖力双重测定法,筛选出FT12、F08、Y05三个对青枯菌抑菌作用较强的菌株。抑菌谱测定表明,三个菌株有较宽的抑菌谱。依据三个菌株的形态学、生物学和生理生化特性对它们进行分类鉴定,初步确定FT12为枯草芽孢杆菌(Bacillus subtilis),F08、Y05为侧孢芽孢杆菌(Bacillus laterosporus)。此外,三个菌株有很强的定殖能力。在温室防病试验中,其平均防病效果分别达70.21%(FT12)、70.70%(F08)和60.69%(Y05)。
用双抗标记法测定了3株生防菌,内生枯草芽孢杆菌(Bacillus subtilis)B001、内生短短芽孢杆菌(Bacillus brevis) B011和根围侧孢芽孢杆菌(Bacillus laterosporus) 2-Q-9,在烟株根茎叶及根表土壤中的定殖和消长动态。结果表明,3株生防菌具有很强的定殖能力,其中以B011的定殖能力最强。平板拮抗试验表明,3株生防菌相互间无拮抗作用,且混合菌群的抑菌活性有所增强。生防菌B001、B011和2-Q-9以不同组合混合接种烟株后,生防菌两两混接总体上可提升其定殖力,但3株混接对B011和2-Q-9有抑制作用；在烟株接种移栽后的第60d,各混接组合的定殖总量均达到最大,其中B001+B011和B011+2-Q-9组合的定殖总量分别达7.28、7.231g-cfu/g,远高于其它处理。
将生防菌B001、B011和2-Q-9浸根,灌根,注射,拌饼肥的生物防治法与喷雾农用链霉素的化学防治法相结合,筛选出了对青枯病防效较好的防治组合。其中,联合处理的防治效果均优于72%农用链霉素单独处理。拌饼肥联合处理要好于对应的不拌饼肥处理,注射法联合处理好于对应的非注射法处理；根围生防菌2-Q-9与农用链霉素联合处理优于对应的单用2-Q-9处理,但链霉素与两株内生菌B001、B011的联合处理却对2株生防菌产生抑制作用。同时,筛选出的内生生防菌拌饼肥-灌根-注射联合处理的施用组合,其防效分别达51.37%(B001),71.20%(B011)；2-Q-9的菌饼肥-菌灌根-喷雾Agr-SM组合,其防效可达62.32%。
将3株生防菌B001、B011和2-Q-9混合接种,表明多功能菌群协同防治烟草青枯病具有有效性和可行性。生防菌B001、B011和2-Q-9的不同组合混接烟株后,其防治效果均优于72%的农用链霉素单独处理；两菌混接要优于对应的单菌处理,其中以B011+2-Q-9组合防效最好,达83.39%,其次是B001+B011组合,防效为78.34%；但三株混接的防效仅为45.49%,低于所有生防处理。
Tobacco bacteria wilt caused by Ralstonia solanacearum is the soil-born disease which is destructive to tobacco. There are no effective and environmentally harmonious methods to control it as yet. In the study, some bacterial strains were studied to control the disease.
Screening of biocontrol bacteria showed the stains FT12, F08, Y05 had strongly colonizing capacity and antagonistic action to R. solanacearum. And bio-control agent against bacterial wilt in greenhouse indicated that the average control effects of three strains were 70.21%(FT12),70.70%(F08) and 60.69%(Y05), respectively. Meanwhile strains FT12, F08, Y05 had a wide spectrum of inhibiting plant pathogen. According to morphological, physiological and biochemical characteries, strains FT12, F08 and Y05 were preliminarily identified and FT12 belongs to Bacillus subtilis,F08 and Y05belong to Bacillus laterosporus.
Colonization ability of three biocontrol agents Bacillus subtilis B001, B. brevis B011 and B. laterosporus 2-Q-9 were determined by the method of dual-resistant marker. The result showed that three biocontrol bacteria had a strong colonizing ability and the best was B011 in strain's single inoculations. The antagonistic test showed that mutual antagonism between three bio-control strains had no occurrence but the antibacterial activities against R. soanacearum were improved. And colonizing abilities of two strains'associative inoculations in tobacco were generally increased but B011 and 2-Q-9 in mixture inoculation of three strains were inhibited. Furthermore, the total populations of mixed strains reached the maximum values on 60th d after dipping tobacco roots treatment.
In order to further stabilize and improve the control effects of three biocontrol agents B001, B011 and 2-Q-9 on tobacco bacterial wilt, the control effects of B001, B011 and 2-Q-9 were tested by the combined inoculation ways of root-soaking, root-watering, stem-injecting, fermented rapeseed-cake and chemical way of streptomycin-spraying. The field trial indicated that effects of different combined treatments on tobacco bacterial wilt were all better than that of single 72% agricultural streptomycin treatment. Impacts of the biocontrol bacteria mixed with fermented rapeseed-cake on tobacco bacteria wilt were better than that of the non-mixed, so did combined injection treatments vs. the corresponding non-injection; and combined treatments of 2-Q-9 plus agricultural streptomycin were superior to the single 2-Q-9 treatment, but the joint treatments of streptomycin plus B001 or BO 11 inhibited the two strains'antibacterial activities against R. soanacearum. Furthermore, The control effects of B001 and B011,which were treated by combined ways of fermented rapeseed-cake, root-watering and stem-injecting, reached 51.37%(B001), 71.20%(B011), respectively, and efficiencies of fermented rapeseed-cake, root-watering of 2-Q-9 plus streptomycin-spraying were 62.32%
The combination test of bio-control strains B001, B011 and 2-Q-9 indicated that effects of single bio-strain and mixtures on tobacco bacterial wilt were all better than that of 4000X of 72% agricultural streptomycin, and the mixed treatments of the two strains were also superior to corresponding single bacterium's. Particularly, efficiencies of B011+2-Q-9 and B004+B011 against the disease reached 83.39% and 78.34%, respectively, but effect of B001+B011+2-Q-9 was only 45.49%.
引文
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