苹果早期落叶病生物防治关键技术研究及其田间应用
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摘要
目前,苹果早期落叶病已成为我国苹果生产中最严重的病害之一,在我国各苹果主产区均有发生,且发病率逐年上升,直接影响苹果的产量和质量,危害巨大。
本研究对广谱拮抗菌株分离筛选、菌株分类鉴定、拮抗菌株对病原菌的抑制作用、抑菌物质的分离纯化和研究、发酵工艺优化和田间防治等苹果早期落叶病生物防治关键技术进行了系统地研究,以期为苹果早期落叶病的生物防治提供理论依据和奠定应用基础,旨在探索一条绿色高效、可持续的苹果早期落叶病病害防治途径。
采用稀释分离法从不同来源的11份样品中分离纯化得到细菌菌株173株,以8种植物病原菌为靶标、采用平板对峙法、以抑菌效果明显和菌株生长迅速为筛选标准,进行植物病害广谱拮抗菌株的筛选,共筛选得到广谱拮抗菌株20株,其中有7株菌株对苹果斑点落叶病病原菌(Alternaria alternata f.sp.Mali)和苹果褐斑病病原菌(Marssoninacoronaria(Ell Et Davis)Davis)均有明显抑制作用。然后以Alternaria alternata f.sp.Mali和Marssonina coronaria(Ell Et Davis)Davis为靶标、采用平皿叶片法,对7株广谱拮抗菌株进行苹果早期落叶病广谱拮抗菌株的复筛,复筛结果显示菌株BS24和菌株D4的抑制效果最好,病叶抑制率分别为100%和90.48%。
结合传统细菌分类鉴定方法和16S rDNA序列分析对拮抗菌株BS24和D4进行分类鉴定,结果表明菌株BS24应为枯草芽孢杆菌和菌株D4应为巨大芽孢杆菌,Genbank登录号分别为GQ213991和FJ755786。
系统研究了拮抗菌株BS24对病原菌的作用方式。菌株BS24可显著抑制Alternariaalternata f.sp.Mali、Marssonina coronaria(Ell Et Davis)Davis的菌丝生长,抑菌带宽度分别为9.5 mm和9.2 mm;可在苹果叶片上迅速定殖、占据生存空间,使病原菌无法在叶片上定殖,从而有效地阻断了病原菌对苹果叶片的侵染。菌株无细胞培养滤液同样可明显抑制病原菌的生长,对Alternaria alternata f.sp.Mali和Marssonina coronaria(Ell EtDavis)Davis的抑制率分别为85.48%和82.86%;可使病原菌菌丝生长畸形,出现扭曲变形、明显交联、部分部位断裂、细胞质聚集以及内含物外溢等现象;还可导致芽管生长畸形,明显抑制病原菌分生孢子的萌发。通过酸沉淀、甲醇抽提和RP-HPLC等步骤,分离得到抑菌物质BSP,经研究初步确定其为一个小分子抑菌多肽。BSP抑菌谱较广,对Alternaria alternata f.sp.Mali、Marssonina coronaria(Ell Et Davis)Davis、Phyllostictapirina、Bolyosphoma berengeriana、Fusarim oxysporium f.sp.Melonis、Glanerella cingulata、Alternaria alternata fries Keissler和Alternaria solani等病原真菌和Escherichia coli均具有抑菌活性,其中对Alternaria alternata f.sp.Mali、Marssonina coronaria(Ell Et Davis)Davis的EC_(50)分别为5.03μg/mL和5.60μg/mL。BSP具有良好的热稳定性、pH稳定性和蛋白酶稳定性。菌株BS24可分泌胞外蛋白酶。
综合考虑工业化生产要求,以发酵液活菌数为检测标准,从6种供试发酵培养基中选出初始发酵培养基。采用Plackett-Burman设计法、最陡爬坡试验、中心组合设计法进行进一步的优化,得到拟合良好的回归方程,R~2=0.96。模型验证显示预测值与实测值之间具有较好的拟合性,预测模型可靠。未优化前发酵液活菌数为8.6×10~9 cfu/mL,优化后为15.08×10~9 cfu/mL,增加了75.35%。优化后发酵工艺为淀粉1.5g,黄豆粉2.16 g,酵母膏0.3 g,蔗糖0.5 g,尿素0.2 g,MnSO_4 0.01 g,MgSO_4 0.01 g,K_2HPO_4 0.2 g,蒸馏水100mL,pH 7.0,转速300 r/min,接种量7.8%。
田间试验结果表明,拮抗菌剂三个不同浓度的处理均对苹果早期落叶病有较好的防治效果,拮抗菌剂原液、拮抗菌剂50倍液和拮抗菌剂100倍液防治效果分别为83.14%、79.21%和76.99%,均显著高于10%多抗霉素WP 1000倍的70.04%(p＜0.05)。供试拮抗菌剂在试验浓度范围内对供试果树无任何不良影响,使用安全。拮抗菌剂处理组果实感官评价、农药残留、重金属残留均符合国家无公害食品的要求。
Nowadays Apple early defoliation diseases have becoming the most serious diseases in most apple-growing areas in China,decreasing the yield and quality of apple.And the incidence was rising year by year.Until now,there is no a very effective control methods for the disease,the chemical pesticides were employed to suppress the diseases.However,the overuse of chemical pesticides inevitably brought about pesticide residues in apple fruit and environmental pollution,directly or indirectly affecting the health of human.Aimed at obtaining the broad-spectrum antagonistic strains and exploring a green and efficient,and sustainable control methods towards Apple early defoliation diseases,the present study was undertaken in antagonistic Bacillus stains screening,identification,inhibitory action, purification and characteristic of antifungal substance,optimization of fermentation process and field experiment.
173 isolates of bacterial strains were isolated from 11 samples of different sources by Plate Dilution method.20 isolates with broad-spectum antagonistic activity against eight phytopathogens such as Alternaria alternata f.sp.Mali,Marssonina coronaria(Ell Et Davis) Davis,Phyllosticta pirina,Bolyosphoma berengeriana and so on were screening from these 173 isolates by Plate inhibition method.10%of the twelve bacterial strains exhibited obvious antagonistic activity against all of the eight phyopathogens and 70%exhibited obvious antagonistic activity against at least 3 phytopathogens.Seven strains could effectively inhibit Alternaria alternata f.sp.Mali and Marssonina coronaria(Ell Et Davis) Davis,the width of inhibition zone both exceeded 5 mm.The potential antagonistic strains against Alternaria alternata f.sp.Mali and Marssonina coronaria(Ell Et Davis) Davis were screening from the seven broad-spectum antagonistic strains by In vitro leavesf plate method.Compared with the control,the rate of diseased leaves in treatment group were significantly decreased(p＜0.05), and strain BS24 and D4 were proved to be the most effective ones with inhibition rate of 100%and 90.48%.This is the first report on the antagonistic strain with broad-spectum antagonistic activity against Alternaria alternata f.sp.Mali and Marssonina coronaria(Ell Et Davis) Davis.
Strain BS24 and D4 were respectively identified as Bacillus subtilis and Bacillus megaterium though the traditional bacterial systemic identification method and the analysis of 16S rDNA sequence.Genbank accession number was GQ213991 and FJ755786,repectively. This is the first report on Bacillus megaterium with broad-spectum antagonistic activity against kinds of phyopathogens.
Antagonism of antagonistic strains on phyopathogens was always achieved by the synergistic effect of various action modes.Strain BS24 could strongly inhibit the mycelial growth of Alternaria alternata f.sp.Mali and Marssonina coronaria(Ell Et Davis) Davis, and the width of inhibition zone were 9.5 mm and 9.2 mm,respectively.It could colonize rapidly on apple leaves in vitro and occupy the living space,which inhibit the colonization of phyopathogens and effectively block their invasion of apple leaves.Cell-free culture filtrate could also strongly inhibit the growth of Alternaria alternata f.sp.Mali and Marssonina coronaria(Ell Et Davis) Davis,the inhibition rate was 85.48%and 82.86%,respectively.It could also result in the abnormity and tortuosity of mycelial,cytoplasmic aggregation,the leak of cytoplasmic substances,the abnormal growth of germ tube and the inhibition of Conidia germination.
After acid precipitation,methanol extraction and RP-HPLC,BSP,an antifungal substance, was obtained and initially determined as a small antifungal peptide.BSP had broad-spectrum angonistic activity,such as Alternaria alternata f.sp.Mali,Marssonina coronaria(Ell Et Davis) Davis,Phyllosticta pirina,Bolyosphoma berengeriana,Fusarim oxysporium f.sp. Melonis,Glanerella cingulata,Alternaria alternata fries Keissler and Alternaria solani and Escherichia coli.And the EC_(50) towards Alternaria alternata f.sp.Mali and Marssonina coronaria(Ell Et Davis) Davis was 5.03μg/mL and 5.60μg/mL,respectively.BSP had good thermal stability,pH stability and protease stability.Strain BS24 can produce protease.
Considering viable count in fermentation broth and the requirements for industrial production,the initial fermentation medium was screening from six fermentation medium. The further optimization was undertaken though Plackett-Burman Design,path steepest ascent and Central Composite Design.And a good regression equation was obtained;the determination coefficient(R~2) was 0.96,which ensure an adequate credibility of the model. Verification of the predicted values was conducted by using optimal conditions in inoculation experiments.Result showed that the predicted value was much closer to practical value, which corroborated the validity and the effectiveness of this model.The viable count in fermentation broth after optimization was 15.08×10~9 cfu/mL,which increased by 75.35% compared with initial count 8.6×10~9 cfu/mL.The fermentation medium after optimization was as followed,corn flour 1.5 g,bean flour 2.16 g,yeast extract 0.3 g,sucrose 0.5 g,urea 0.3 g, MnSO_4 0.01 g,MgSO_4 0.01 g,K_2HPO_4 0.2 g,distilled water 100 mL,pH 7.0,300 r/min and 7.8%of inoculation.
Field experiment showed that the three concentration of antagonistic inoculum have both achieved the desirable control efficacy towards Apple early defoliation diseases.The control efficacy were 83.14%、79.21%and 76.99%respectively,which both exceeded that of 10% multiantimycin.The antagonistic inoculum had no harmful effects on plants under the three testing concentration.Residues of chemical pesticides and heavy metals were in all accord with national request for food.Due to its high control efficacy and safety,the antagonistic agent had great potential of application in the field control of Apple early defoliation diseases.
本研究对广谱拮抗菌株分离筛选、菌株分类鉴定、拮抗菌株对病原菌的抑制作用、抑菌物质的分离纯化和研究、发酵工艺优化和田间防治等苹果早期落叶病生物防治关键技术进行了系统地研究,以期为苹果早期落叶病的生物防治提供理论依据和奠定应用基础,旨在探索一条绿色高效、可持续的苹果早期落叶病病害防治途径。
采用稀释分离法从不同来源的11份样品中分离纯化得到细菌菌株173株,以8种植物病原菌为靶标、采用平板对峙法、以抑菌效果明显和菌株生长迅速为筛选标准,进行植物病害广谱拮抗菌株的筛选,共筛选得到广谱拮抗菌株20株,其中有7株菌株对苹果斑点落叶病病原菌(Alternaria alternata f.sp.Mali)和苹果褐斑病病原菌(Marssoninacoronaria(Ell Et Davis)Davis)均有明显抑制作用。然后以Alternaria alternata f.sp.Mali和Marssonina coronaria(Ell Et Davis)Davis为靶标、采用平皿叶片法,对7株广谱拮抗菌株进行苹果早期落叶病广谱拮抗菌株的复筛,复筛结果显示菌株BS24和菌株D4的抑制效果最好,病叶抑制率分别为100%和90.48%。
结合传统细菌分类鉴定方法和16S rDNA序列分析对拮抗菌株BS24和D4进行分类鉴定,结果表明菌株BS24应为枯草芽孢杆菌和菌株D4应为巨大芽孢杆菌,Genbank登录号分别为GQ213991和FJ755786。
系统研究了拮抗菌株BS24对病原菌的作用方式。菌株BS24可显著抑制Alternariaalternata f.sp.Mali、Marssonina coronaria(Ell Et Davis)Davis的菌丝生长,抑菌带宽度分别为9.5 mm和9.2 mm;可在苹果叶片上迅速定殖、占据生存空间,使病原菌无法在叶片上定殖,从而有效地阻断了病原菌对苹果叶片的侵染。菌株无细胞培养滤液同样可明显抑制病原菌的生长,对Alternaria alternata f.sp.Mali和Marssonina coronaria(Ell EtDavis)Davis的抑制率分别为85.48%和82.86%;可使病原菌菌丝生长畸形,出现扭曲变形、明显交联、部分部位断裂、细胞质聚集以及内含物外溢等现象;还可导致芽管生长畸形,明显抑制病原菌分生孢子的萌发。通过酸沉淀、甲醇抽提和RP-HPLC等步骤,分离得到抑菌物质BSP,经研究初步确定其为一个小分子抑菌多肽。BSP抑菌谱较广,对Alternaria alternata f.sp.Mali、Marssonina coronaria(Ell Et Davis)Davis、Phyllostictapirina、Bolyosphoma berengeriana、Fusarim oxysporium f.sp.Melonis、Glanerella cingulata、Alternaria alternata fries Keissler和Alternaria solani等病原真菌和Escherichia coli均具有抑菌活性,其中对Alternaria alternata f.sp.Mali、Marssonina coronaria(Ell Et Davis)Davis的EC_(50)分别为5.03μg/mL和5.60μg/mL。BSP具有良好的热稳定性、pH稳定性和蛋白酶稳定性。菌株BS24可分泌胞外蛋白酶。
综合考虑工业化生产要求,以发酵液活菌数为检测标准,从6种供试发酵培养基中选出初始发酵培养基。采用Plackett-Burman设计法、最陡爬坡试验、中心组合设计法进行进一步的优化,得到拟合良好的回归方程,R~2=0.96。模型验证显示预测值与实测值之间具有较好的拟合性,预测模型可靠。未优化前发酵液活菌数为8.6×10~9 cfu/mL,优化后为15.08×10~9 cfu/mL,增加了75.35%。优化后发酵工艺为淀粉1.5g,黄豆粉2.16 g,酵母膏0.3 g,蔗糖0.5 g,尿素0.2 g,MnSO_4 0.01 g,MgSO_4 0.01 g,K_2HPO_4 0.2 g,蒸馏水100mL,pH 7.0,转速300 r/min,接种量7.8%。
田间试验结果表明,拮抗菌剂三个不同浓度的处理均对苹果早期落叶病有较好的防治效果,拮抗菌剂原液、拮抗菌剂50倍液和拮抗菌剂100倍液防治效果分别为83.14%、79.21%和76.99%,均显著高于10%多抗霉素WP 1000倍的70.04%(p＜0.05)。供试拮抗菌剂在试验浓度范围内对供试果树无任何不良影响,使用安全。拮抗菌剂处理组果实感官评价、农药残留、重金属残留均符合国家无公害食品的要求。
Nowadays Apple early defoliation diseases have becoming the most serious diseases in most apple-growing areas in China,decreasing the yield and quality of apple.And the incidence was rising year by year.Until now,there is no a very effective control methods for the disease,the chemical pesticides were employed to suppress the diseases.However,the overuse of chemical pesticides inevitably brought about pesticide residues in apple fruit and environmental pollution,directly or indirectly affecting the health of human.Aimed at obtaining the broad-spectrum antagonistic strains and exploring a green and efficient,and sustainable control methods towards Apple early defoliation diseases,the present study was undertaken in antagonistic Bacillus stains screening,identification,inhibitory action, purification and characteristic of antifungal substance,optimization of fermentation process and field experiment.
173 isolates of bacterial strains were isolated from 11 samples of different sources by Plate Dilution method.20 isolates with broad-spectum antagonistic activity against eight phytopathogens such as Alternaria alternata f.sp.Mali,Marssonina coronaria(Ell Et Davis) Davis,Phyllosticta pirina,Bolyosphoma berengeriana and so on were screening from these 173 isolates by Plate inhibition method.10%of the twelve bacterial strains exhibited obvious antagonistic activity against all of the eight phyopathogens and 70%exhibited obvious antagonistic activity against at least 3 phytopathogens.Seven strains could effectively inhibit Alternaria alternata f.sp.Mali and Marssonina coronaria(Ell Et Davis) Davis,the width of inhibition zone both exceeded 5 mm.The potential antagonistic strains against Alternaria alternata f.sp.Mali and Marssonina coronaria(Ell Et Davis) Davis were screening from the seven broad-spectum antagonistic strains by In vitro leavesf plate method.Compared with the control,the rate of diseased leaves in treatment group were significantly decreased(p＜0.05), and strain BS24 and D4 were proved to be the most effective ones with inhibition rate of 100%and 90.48%.This is the first report on the antagonistic strain with broad-spectum antagonistic activity against Alternaria alternata f.sp.Mali and Marssonina coronaria(Ell Et Davis) Davis.
Strain BS24 and D4 were respectively identified as Bacillus subtilis and Bacillus megaterium though the traditional bacterial systemic identification method and the analysis of 16S rDNA sequence.Genbank accession number was GQ213991 and FJ755786,repectively. This is the first report on Bacillus megaterium with broad-spectum antagonistic activity against kinds of phyopathogens.
Antagonism of antagonistic strains on phyopathogens was always achieved by the synergistic effect of various action modes.Strain BS24 could strongly inhibit the mycelial growth of Alternaria alternata f.sp.Mali and Marssonina coronaria(Ell Et Davis) Davis, and the width of inhibition zone were 9.5 mm and 9.2 mm,respectively.It could colonize rapidly on apple leaves in vitro and occupy the living space,which inhibit the colonization of phyopathogens and effectively block their invasion of apple leaves.Cell-free culture filtrate could also strongly inhibit the growth of Alternaria alternata f.sp.Mali and Marssonina coronaria(Ell Et Davis) Davis,the inhibition rate was 85.48%and 82.86%,respectively.It could also result in the abnormity and tortuosity of mycelial,cytoplasmic aggregation,the leak of cytoplasmic substances,the abnormal growth of germ tube and the inhibition of Conidia germination.
After acid precipitation,methanol extraction and RP-HPLC,BSP,an antifungal substance, was obtained and initially determined as a small antifungal peptide.BSP had broad-spectrum angonistic activity,such as Alternaria alternata f.sp.Mali,Marssonina coronaria(Ell Et Davis) Davis,Phyllosticta pirina,Bolyosphoma berengeriana,Fusarim oxysporium f.sp. Melonis,Glanerella cingulata,Alternaria alternata fries Keissler and Alternaria solani and Escherichia coli.And the EC_(50) towards Alternaria alternata f.sp.Mali and Marssonina coronaria(Ell Et Davis) Davis was 5.03μg/mL and 5.60μg/mL,respectively.BSP had good thermal stability,pH stability and protease stability.Strain BS24 can produce protease.
Considering viable count in fermentation broth and the requirements for industrial production,the initial fermentation medium was screening from six fermentation medium. The further optimization was undertaken though Plackett-Burman Design,path steepest ascent and Central Composite Design.And a good regression equation was obtained;the determination coefficient(R~2) was 0.96,which ensure an adequate credibility of the model. Verification of the predicted values was conducted by using optimal conditions in inoculation experiments.Result showed that the predicted value was much closer to practical value, which corroborated the validity and the effectiveness of this model.The viable count in fermentation broth after optimization was 15.08×10~9 cfu/mL,which increased by 75.35% compared with initial count 8.6×10~9 cfu/mL.The fermentation medium after optimization was as followed,corn flour 1.5 g,bean flour 2.16 g,yeast extract 0.3 g,sucrose 0.5 g,urea 0.3 g, MnSO_4 0.01 g,MgSO_4 0.01 g,K_2HPO_4 0.2 g,distilled water 100 mL,pH 7.0,300 r/min and 7.8%of inoculation.
Field experiment showed that the three concentration of antagonistic inoculum have both achieved the desirable control efficacy towards Apple early defoliation diseases.The control efficacy were 83.14%、79.21%and 76.99%respectively,which both exceeded that of 10% multiantimycin.The antagonistic inoculum had no harmful effects on plants under the three testing concentration.Residues of chemical pesticides and heavy metals were in all accord with national request for food.Due to its high control efficacy and safety,the antagonistic agent had great potential of application in the field control of Apple early defoliation diseases.
引文
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