芽孢杆菌拮抗苹果树腐烂病菌的筛选、鉴定及抑菌活性初探
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摘要
【背景】苹果树腐烂病发生面积广、危害严重,目前对于该病的防治以化学防治为主,但存在一定的弊端,因此迫切需要寻找一种对该病高效、安全的防治措施。【目的】从敦煌地区盐碱土壤中筛选出对苹果树腐烂病有良好抑制作用的芽孢杆菌菌株。【方法】采用平板对峙培养法和菌丝生长速率法对盐碱土中分离筛选的芽孢杆菌进行拮抗苹果树腐烂病菌的筛选,并对优良拮抗效果菌株进行离体组织防效测定、发酵培养基选择以及结合培养形态、生理生化及16SrRNA基因序列分析进行菌种鉴定。【结果】初筛阶段菌株7-2-1-2对病原菌的抑制率为86.18%,复筛试验中发酵液对病原菌的抑制率为73.2%;在离体枝条创面涂抹菌株7-2-1-2发酵原液后,苹果树腐烂病菌的生长可被完全抑制;菌株7-2-1-2的最优生长培养基为NBY培养基;经初步鉴定,菌株7-2-1-2为枯草芽孢杆菌亚种(Bacillus subtilis subsp. spizizenii)。【结论】菌株7-2-1-2对苹果树腐烂病有较好的生防效果,为该病生防菌剂的选择提供了必要的菌种资源。
[Background] Apple tree canker is widespread and dangerous. Currently, chemical control is the main way to prevent and cure it. However, there are some drawbacks. Therefore, it is urgent to find an effective and safe prevention and control measure. [Objective] In order to select Bacillus isolated in Saline-alkali soil from Dunhuang that showed significantly antagonistic effect on the Valsa mali growth. [Methods] The antifungal activities of selected Bacillus were investigated by dual culture assar and colony diameter assay, respectively. In addition, the control effect of antagonistic bacteria strains against Valsa mali in detached apple twigs in vitro and selection for medium were determined and the antagonistic Bacillus was identified based on morphological, physiological and biochemical characteristics and 16 S rRNA gene phylogenetic analysis. [Results] The results revealed that strain 7-2-1-2 exhibited a strong antagonistic activity against V. mali, inhibiting significantly the germination of conidia and the growth of hyphae and especially the inhibitory rate of strain 7-2-1-2 was 86.18% and 73.2% in second screening. Strain 7-2-1-2 fermentation broth applied prior to wound inoculation of apple twigs with V. mali resulted in total inhibition of infection. The optimum growth medium of strain 7-2-1-2 was NBY. Strain 7-2-1-2 was identified as Bacillus subtilis subsp. spizizenii. [Conclusion] These results indicated that the strain 7-2-1-2 promised to provide a new biological control method in containing apple canker.
[Background] Apple tree canker is widespread and dangerous. Currently, chemical control is the main way to prevent and cure it. However, there are some drawbacks. Therefore, it is urgent to find an effective and safe prevention and control measure. [Objective] In order to select Bacillus isolated in Saline-alkali soil from Dunhuang that showed significantly antagonistic effect on the Valsa mali growth. [Methods] The antifungal activities of selected Bacillus were investigated by dual culture assar and colony diameter assay, respectively. In addition, the control effect of antagonistic bacteria strains against Valsa mali in detached apple twigs in vitro and selection for medium were determined and the antagonistic Bacillus was identified based on morphological, physiological and biochemical characteristics and 16 S rRNA gene phylogenetic analysis. [Results] The results revealed that strain 7-2-1-2 exhibited a strong antagonistic activity against V. mali, inhibiting significantly the germination of conidia and the growth of hyphae and especially the inhibitory rate of strain 7-2-1-2 was 86.18% and 73.2% in second screening. Strain 7-2-1-2 fermentation broth applied prior to wound inoculation of apple twigs with V. mali resulted in total inhibition of infection. The optimum growth medium of strain 7-2-1-2 was NBY. Strain 7-2-1-2 was identified as Bacillus subtilis subsp. spizizenii. [Conclusion] These results indicated that the strain 7-2-1-2 promised to provide a new biological control method in containing apple canker.
引文
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[2]Adams GC,Roux J,Wingfield MJ.Cytospora species(Ascomycota,Diaporthales,Valsaceae):introduced and native pathogens of trees in South Africa[J].Australasian Plant Pathology,2006,35(5):521-548
[3]Ke XW.Histocytology and transcriptomics studies on infection of Malus domestica Cv.Fuji by Valsa mali[D].Xi’an:Doctoral Dissertation of Northwest A&F University,2013(in Chinese)柯希望.黑腐皮壳侵染苹果的组织细胞学及转录组学研究[D].西安:西北农林科技大学博士学位论文,2013
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[5]Abe K,Kotoda N,Kato H,et al.Genetic studies on resistance to Valsa canker in apple:genetic variance and breeding values estimated from intra-and inter-specific hybrid progeny populations[J].Tree Genetics&Genomes,2011,7(2):363-372
[6]Lee DH,Lee SW,Choi KH,et al.Survey on the occurrence of apple diseases in Korea from 1992 to 2000[J].Plant Pathology Journal,2006,22(4):375-380
[7]Wang L,Gao ZP,Huang LL,et al.Screening fungicide for pathogen inhibition and disease control of apple tree Valsa canker[J].Acta Phytopathologica Sinica,2009,39(5):549-554(in Chinese)王磊,郜佐鹏,黄丽丽,等.防治苹果树腐烂病杀菌剂的室内筛选[J].植物病理学报,2009,39(5):549-554
[8]Yin ZY,Ke XW,Huang DX,et al.Validation of reference genes for gene expression analysis in Valsa mali var.mali using real-time quantitative PCR[J].World Journal of Microbiology and Biotechnology,2013,29(9):1563-1571
[9]Bessho H,Soejima J,Tsuchiya S.Screening methods of apple trees for resistance to Valsa canker[A]//Progress in Temperate Fruit Breeding[M].Netherlands:Springer,1994:49-52
[10]Liu XY,Lyu S,Wang Y,et al.Evaluation of resistance of malus germplasms to apple canker(Valsa ceratosperma)[J].Journal of Fruit Science,2011,28(5):843-848(in Chinese)刘欣颖,吕松,王忆,等.苹果种质资源对苹果树腐烂病抗性评价[J].果树学报,2011,28(5):843-848
[11]Guo SQ,Cui ZT,Fu QM.Idea and suggestions on saline-alkali soil status quo and managements in Gansu province[J].Chinese Journal of Agricultural Resources and Regional Planning,2013,34(4):75-79(in Chinese)郭世乾,崔增团,傅亲民.甘肃省盐碱地现状及治理思路与建议[J].中国农业资源与区划,2013,34(4):75-79
[12]Jacobsen BJ,Zidack NZ,Larson BJ.The role of Bacillus-based biological control agents in integrated pest management systems:plant diseases[J].Phytopathology,2004,94(11):1272-1275
[13]Pérez-García A,Romero D,de Vicente A.Plant protection and growth stimulation by microorganisms:biotechnological applications of Bacilli in agriculture[J].Current Opinion in Biotechnology,2011,22(2):187-193
[14]Wang WX,Xu BL,Xue YY,et al.Identification and antifugla activity of the antagonistic bacteria of Cytospora spp.[J].Chinese Journal of Eco-Agriculture,2014,22(10):1214-1221(in Chinese)王卫雄,徐秉良,薛应钰,等.苹果树腐烂病拮抗细菌鉴定及其抑菌作用效果测定[J].中国生态农业学报,2014,22(10):1214-1221
[15]Ma R,Liu XL,Wang XW,et al.Screening and identification of the antagonistic bacteria apple canker in Xinjiang[J].Journal of Xinjiang Agricultural University,2015,38(2):136-139(in Chinese)马荣,刘晓琳,王晓炜,等.新疆苹果树腐烂病拮抗细菌的筛选与初步鉴定[J].新疆农业大学学报,2015,38(2):136-139
[16]Zhang JX,Gu YB,Chi FM,et al.Bacillus amyloliquefaciens GB1can effectively control apple Valsa canker[J].Biological Control,2015,88:1-7
[17]Schaad NW.Laboratory Guide for Identification of Plant Pathogenic Bacteria[M].Guiyang:Guizhou People’s Publishing House,1986(in Chinese)Schaad NW.植物病原细菌鉴定实验指南[M].贵阳:贵州人民出版社,1986
[18]Sun Y.Laboratory Manual for Microbiology[M].Xi’an:Shaanxi Normal University General Publishing House Co.LTD.,2015:154-157(in Chinese)孙燕.微生物学实验指导[M].西安:陕西师范大学出版总社,2015:154-157
[19]Wang JF,Qi KB,He YM.Experimental Course of Genetics[M].Beijing:Higher Education Press,2008:131(in Chinese)王金发,戚康标,何炎明.遗传学实验教程[M].北京:高等教育出版社,2008:131
[20]Zhang XZ,Xu JH,Li SP.Isolation,screening and preliminary identification of plant pathogenic fungi biocontrol strains of Bacillus spp.[J].Soils,2005,37(1):85-88(in Chinese)张晓舟,徐剑宏,李顺鹏.植病生防芽孢杆菌的分离筛选与初步鉴定[J].土壤,2005,37(1):85-88
[21]Wang AJ,Chai ZX,Li JH,et al.Screening and identification of antagonistic Bacillus strains against pathogens of fusarium dry rot and black scurf in potato[J].Chinese Journal of Biological Control,2013,29(4):586-594(in Chinese)王爱军,柴兆祥,李金花,等.马铃薯干腐病菌和黑痣病菌拮抗芽胞杆菌的筛选及鉴定[J].中国生物防治学报,2013,29(4):586-594
[22]Wang B,Niu SQ,Da WY,et al.Screening of Actinomyces on Antagonism to Rhizoctonia solani Isolated from Saline-alkali Soils in Hexi Corridor[J].Biotechnology Bulletin,2014(1):156-160(in Chinese)王蓓,牛世全,达文燕,等.河西走廊盐碱土壤中抗立枯丝核菌的放线菌筛选[J].生物技术通报,2014(1):156-160
[23]Wei JL,Huang LL,Gao ZP,et al.Laboratory evaluation methods of apple Valsa canker disease caused by Valsa ceratosperma sensu Kobayashi[J].Acta Phytopathologica Sinica,2010,40(1):14-20(in Chinese)韦洁玲,黄丽丽,郜佐鹏,等.苹果树腐烂病室内快速评价方法的研究[J].植物病理学报,2010,40(1):14-20
[24]Noble R,Coventry E.Suppression of soil-borne plant diseases with composts:A review[J].Biocontrol Science&Technology,2005,15(1):3-20
[25]Sharma RR,Singh D,Singh R.Biological control of postharvest diseases of fruits and vegetables by microbial antagonists:Areview[J].Biological Control,2009,50(3):205-221
[26]Piggot PJ,Hilbert DW.Sporulation of Bacillus subtilis[J].Current Opinion in Microbiology,2004,7(6):579-586
[27]Tiago I,Teixeira I,Silva S,et al.Metabolic and genetic diversity of mesophilic and thermophilic bacteria isolated from composted municipal sludge on poly-ε-caprolactones[J].Current Microbiology,2004,49(6):407-414
[28]Niu SQ,Long Y,Li HY,et al.Microbial diversity in saline alkali soil from Hexi Corridor analyzed by Illumina MiSeq high-throughput sequencing system[J].Microbiology China,2017,44(9):2067-2078(in Chinese)牛世全,龙洋,李海云,等.应用Illumina MiSeq高通量测序技术分析河西走廊地区盐碱土壤微生物多样性[J].微生物学通报,2017,44(9):2067-2078
[29]Wang CX,Zhang QM,Li GF,et al.Identification of the antagonistic bacteria BJ1 and its antifungal activity against Valsa ceratosperma[J].Acta Phytophylacica Sinica,2012,39(5):431-437(in Chinese)王彩霞,张清明,李桂舫,等.苹果树腐烂病拮抗细菌菌株BJ1的鉴定及其抑菌作用[J].植物保护学报,2012,39(5):431-437
[30]Ongena M,Jourdan E,Adam A,et al.Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants[J].Environmental Microbiology,2007,9(4):1084-1090
[31]Niu HJ,Li H,Wang NN,et al.Isolation and purification of antifungal protein from Bacillus subtilis E1R-j against Valsa mali[J].Journal of Northwest A&F University(Natural Science Edition),2016,44(9):135-142(in Chinese)牛焕杰,李辉,王娜娜,等.苹果树腐烂病菌拮抗枯草芽孢杆菌E1R-j抗菌蛋白的分离纯化[J].西北农林科技大学学报:自然科学版,2016,44(9):135-142
[2]Adams GC,Roux J,Wingfield MJ.Cytospora species(Ascomycota,Diaporthales,Valsaceae):introduced and native pathogens of trees in South Africa[J].Australasian Plant Pathology,2006,35(5):521-548
[3]Ke XW.Histocytology and transcriptomics studies on infection of Malus domestica Cv.Fuji by Valsa mali[D].Xi’an:Doctoral Dissertation of Northwest A&F University,2013(in Chinese)柯希望.黑腐皮壳侵染苹果的组织细胞学及转录组学研究[D].西安:西北农林科技大学博士学位论文,2013
[4]Wang L,Zang R,Huang LL,et al.The investigation of apple tree Valsa canker in Guanzhong region of Shaanxi Province[J].Journal of Northwest Sci-Tech University of Agriculture and Forestry(Natural Science Edition),2005,33(S1):98-100(in Chinese)王磊,臧睿,黄丽丽,等.陕西省关中地区苹果树腐烂病调查初报[J].西北农林科技大学学报:自然科学版,2005,33(S1):98-100
[5]Abe K,Kotoda N,Kato H,et al.Genetic studies on resistance to Valsa canker in apple:genetic variance and breeding values estimated from intra-and inter-specific hybrid progeny populations[J].Tree Genetics&Genomes,2011,7(2):363-372
[6]Lee DH,Lee SW,Choi KH,et al.Survey on the occurrence of apple diseases in Korea from 1992 to 2000[J].Plant Pathology Journal,2006,22(4):375-380
[7]Wang L,Gao ZP,Huang LL,et al.Screening fungicide for pathogen inhibition and disease control of apple tree Valsa canker[J].Acta Phytopathologica Sinica,2009,39(5):549-554(in Chinese)王磊,郜佐鹏,黄丽丽,等.防治苹果树腐烂病杀菌剂的室内筛选[J].植物病理学报,2009,39(5):549-554
[8]Yin ZY,Ke XW,Huang DX,et al.Validation of reference genes for gene expression analysis in Valsa mali var.mali using real-time quantitative PCR[J].World Journal of Microbiology and Biotechnology,2013,29(9):1563-1571
[9]Bessho H,Soejima J,Tsuchiya S.Screening methods of apple trees for resistance to Valsa canker[A]//Progress in Temperate Fruit Breeding[M].Netherlands:Springer,1994:49-52
[10]Liu XY,Lyu S,Wang Y,et al.Evaluation of resistance of malus germplasms to apple canker(Valsa ceratosperma)[J].Journal of Fruit Science,2011,28(5):843-848(in Chinese)刘欣颖,吕松,王忆,等.苹果种质资源对苹果树腐烂病抗性评价[J].果树学报,2011,28(5):843-848
[11]Guo SQ,Cui ZT,Fu QM.Idea and suggestions on saline-alkali soil status quo and managements in Gansu province[J].Chinese Journal of Agricultural Resources and Regional Planning,2013,34(4):75-79(in Chinese)郭世乾,崔增团,傅亲民.甘肃省盐碱地现状及治理思路与建议[J].中国农业资源与区划,2013,34(4):75-79
[12]Jacobsen BJ,Zidack NZ,Larson BJ.The role of Bacillus-based biological control agents in integrated pest management systems:plant diseases[J].Phytopathology,2004,94(11):1272-1275
[13]Pérez-García A,Romero D,de Vicente A.Plant protection and growth stimulation by microorganisms:biotechnological applications of Bacilli in agriculture[J].Current Opinion in Biotechnology,2011,22(2):187-193
[14]Wang WX,Xu BL,Xue YY,et al.Identification and antifugla activity of the antagonistic bacteria of Cytospora spp.[J].Chinese Journal of Eco-Agriculture,2014,22(10):1214-1221(in Chinese)王卫雄,徐秉良,薛应钰,等.苹果树腐烂病拮抗细菌鉴定及其抑菌作用效果测定[J].中国生态农业学报,2014,22(10):1214-1221
[15]Ma R,Liu XL,Wang XW,et al.Screening and identification of the antagonistic bacteria apple canker in Xinjiang[J].Journal of Xinjiang Agricultural University,2015,38(2):136-139(in Chinese)马荣,刘晓琳,王晓炜,等.新疆苹果树腐烂病拮抗细菌的筛选与初步鉴定[J].新疆农业大学学报,2015,38(2):136-139
[16]Zhang JX,Gu YB,Chi FM,et al.Bacillus amyloliquefaciens GB1can effectively control apple Valsa canker[J].Biological Control,2015,88:1-7
[17]Schaad NW.Laboratory Guide for Identification of Plant Pathogenic Bacteria[M].Guiyang:Guizhou People’s Publishing House,1986(in Chinese)Schaad NW.植物病原细菌鉴定实验指南[M].贵阳:贵州人民出版社,1986
[18]Sun Y.Laboratory Manual for Microbiology[M].Xi’an:Shaanxi Normal University General Publishing House Co.LTD.,2015:154-157(in Chinese)孙燕.微生物学实验指导[M].西安:陕西师范大学出版总社,2015:154-157
[19]Wang JF,Qi KB,He YM.Experimental Course of Genetics[M].Beijing:Higher Education Press,2008:131(in Chinese)王金发,戚康标,何炎明.遗传学实验教程[M].北京:高等教育出版社,2008:131
[20]Zhang XZ,Xu JH,Li SP.Isolation,screening and preliminary identification of plant pathogenic fungi biocontrol strains of Bacillus spp.[J].Soils,2005,37(1):85-88(in Chinese)张晓舟,徐剑宏,李顺鹏.植病生防芽孢杆菌的分离筛选与初步鉴定[J].土壤,2005,37(1):85-88
[21]Wang AJ,Chai ZX,Li JH,et al.Screening and identification of antagonistic Bacillus strains against pathogens of fusarium dry rot and black scurf in potato[J].Chinese Journal of Biological Control,2013,29(4):586-594(in Chinese)王爱军,柴兆祥,李金花,等.马铃薯干腐病菌和黑痣病菌拮抗芽胞杆菌的筛选及鉴定[J].中国生物防治学报,2013,29(4):586-594
[22]Wang B,Niu SQ,Da WY,et al.Screening of Actinomyces on Antagonism to Rhizoctonia solani Isolated from Saline-alkali Soils in Hexi Corridor[J].Biotechnology Bulletin,2014(1):156-160(in Chinese)王蓓,牛世全,达文燕,等.河西走廊盐碱土壤中抗立枯丝核菌的放线菌筛选[J].生物技术通报,2014(1):156-160
[23]Wei JL,Huang LL,Gao ZP,et al.Laboratory evaluation methods of apple Valsa canker disease caused by Valsa ceratosperma sensu Kobayashi[J].Acta Phytopathologica Sinica,2010,40(1):14-20(in Chinese)韦洁玲,黄丽丽,郜佐鹏,等.苹果树腐烂病室内快速评价方法的研究[J].植物病理学报,2010,40(1):14-20
[24]Noble R,Coventry E.Suppression of soil-borne plant diseases with composts:A review[J].Biocontrol Science&Technology,2005,15(1):3-20
[25]Sharma RR,Singh D,Singh R.Biological control of postharvest diseases of fruits and vegetables by microbial antagonists:Areview[J].Biological Control,2009,50(3):205-221
[26]Piggot PJ,Hilbert DW.Sporulation of Bacillus subtilis[J].Current Opinion in Microbiology,2004,7(6):579-586
[27]Tiago I,Teixeira I,Silva S,et al.Metabolic and genetic diversity of mesophilic and thermophilic bacteria isolated from composted municipal sludge on poly-ε-caprolactones[J].Current Microbiology,2004,49(6):407-414
[28]Niu SQ,Long Y,Li HY,et al.Microbial diversity in saline alkali soil from Hexi Corridor analyzed by Illumina MiSeq high-throughput sequencing system[J].Microbiology China,2017,44(9):2067-2078(in Chinese)牛世全,龙洋,李海云,等.应用Illumina MiSeq高通量测序技术分析河西走廊地区盐碱土壤微生物多样性[J].微生物学通报,2017,44(9):2067-2078
[29]Wang CX,Zhang QM,Li GF,et al.Identification of the antagonistic bacteria BJ1 and its antifungal activity against Valsa ceratosperma[J].Acta Phytophylacica Sinica,2012,39(5):431-437(in Chinese)王彩霞,张清明,李桂舫,等.苹果树腐烂病拮抗细菌菌株BJ1的鉴定及其抑菌作用[J].植物保护学报,2012,39(5):431-437
[30]Ongena M,Jourdan E,Adam A,et al.Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants[J].Environmental Microbiology,2007,9(4):1084-1090
[31]Niu HJ,Li H,Wang NN,et al.Isolation and purification of antifungal protein from Bacillus subtilis E1R-j against Valsa mali[J].Journal of Northwest A&F University(Natural Science Edition),2016,44(9):135-142(in Chinese)牛焕杰,李辉,王娜娜,等.苹果树腐烂病菌拮抗枯草芽孢杆菌E1R-j抗菌蛋白的分离纯化[J].西北农林科技大学学报:自然科学版,2016,44(9):135-142