梨灰霉病菌拮抗细菌Pseudomonas extremorientalis的鉴定、发酵条件优化及防效评价
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摘要
灰霉病是梨贮藏期的主要病害,发病率高,为害严重。为了有效控制该病害发生,利用平皿稀释法从梨根际土壤中分离内生细菌,并采用琼脂平板扩散法进行抑菌活性初筛,筛选出1株拮抗细菌L21,基于形态特征、生理生化特性及16S rRNA和gyrB序列分析,鉴定为Pseudomonas extremorientalis。采用生长速率法测定菌株L21的无菌发酵液对12种病原真菌均有抑制作用,采用Salkowski比色法测定菌株L21能够分泌吲哚-3-乙酸(IAA),采用酪蛋白培养基平板检测菌株L21可以产生胞外蛋白酶。在单因素试验的基础上,通过Plackett-Burman试验设计筛选出pH、硫酸锰和转速3个影响发酵的重要因素,利用最陡爬坡试验和Box-Behnken试验设计,获得菌株L21的最佳发酵条件为蔗糖20 g/L、牛肉膏20 g/L、硫酸锰3.00 g/L、甘油1 g/L、K2HPO4 1.50 g/L、接种量8%、温度34℃、pH 7.31、转速200.66 r/min和培养时间15h。处理96h后,菌株L21对梨灰霉病的防效为74.23%,与多菌灵的防效差异不显著。本研究表明菌株L21具有广谱抑菌活性,为植物真菌病害的生物防治提供新的生防资源。
Pear gray mold, an important postharvest disease with high incidence, causes serious damage during the storage period. To control this disease efficiently, endophytic bacteria strains were isolated from pear rhizosphere soil by plating dilution method, and screened based on antifungal activity by using agar plate diffusions method.Antagonistic bacterium strain L21 was obtained, and identified as Pseudomonas extremorientalis based on the morphological, physiological and biochemical characteristics, and 16 S rRNA and gyrB sequence analysis. The antifungal effects of cell-free fermentation broth of strain L21 on twelve pathogenic fungi were obvious by using mycelial growth rate method. Strain L21 could produce indoleacetic acid(IAA) by Salkowski colorimetric method.Strain L21 could produce extracellular by using casein medium plate. Based on single factor tests, three important factors of pH, temperature and yeast powder were screened by Plackett-Burman experimental design. The optimal fermentation conditions for sucrose 20 g/L, beef extract 20 g/L, manganese sulfate 3.00 g/L, glycerol 1 g/L, K2 HPO41.50 g/L, inoculation amount 8%, cultural temperature 34 ℃, pH 7.31, rotational speed 200.66 r/min and cultural time 15 hours were determined by steepest ascent experiment and Box-Behnken experimental design. At 96 hours after application, the control efficacy of strain L21 was 74.23%, and no significant difference compared with that of carbendazim. Strain L21 showed strong inhibition effect and wide antagonistic spectrum, which has the potential to be the novel biocontrol agent for plant fungal diseases control.
Pear gray mold, an important postharvest disease with high incidence, causes serious damage during the storage period. To control this disease efficiently, endophytic bacteria strains were isolated from pear rhizosphere soil by plating dilution method, and screened based on antifungal activity by using agar plate diffusions method.Antagonistic bacterium strain L21 was obtained, and identified as Pseudomonas extremorientalis based on the morphological, physiological and biochemical characteristics, and 16 S rRNA and gyrB sequence analysis. The antifungal effects of cell-free fermentation broth of strain L21 on twelve pathogenic fungi were obvious by using mycelial growth rate method. Strain L21 could produce indoleacetic acid(IAA) by Salkowski colorimetric method.Strain L21 could produce extracellular by using casein medium plate. Based on single factor tests, three important factors of pH, temperature and yeast powder were screened by Plackett-Burman experimental design. The optimal fermentation conditions for sucrose 20 g/L, beef extract 20 g/L, manganese sulfate 3.00 g/L, glycerol 1 g/L, K2 HPO41.50 g/L, inoculation amount 8%, cultural temperature 34 ℃, pH 7.31, rotational speed 200.66 r/min and cultural time 15 hours were determined by steepest ascent experiment and Box-Behnken experimental design. At 96 hours after application, the control efficacy of strain L21 was 74.23%, and no significant difference compared with that of carbendazim. Strain L21 showed strong inhibition effect and wide antagonistic spectrum, which has the potential to be the novel biocontrol agent for plant fungal diseases control.
引文
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[3]孙平平,崔建潮,贾晓辉,等.贝莱斯芽孢杆菌L-1对梨灰霉和青霉病菌的抑制作用评价及全基因组分析[J].微生物学报,2018,58(9):1637-1646.
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[6]纪军建,张小风,韩秀英,等.8种杀菌剂对番茄灰霉病菌的毒力及田间番茄灰霉病菌对咯菌腈的敏感性[J].植物保护,2012,38(6):144-146.
[7]赵建江,陈治芳,韩秀英,等.啶酰菌胺与唑胺菌酯混配对灰葡萄孢毒力的增效作用[J].农药学学报,2015,17(4):417-424.
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[9]赵建江,王文桥,马志强,等.啶酰菌胺与吡唑醚菌酯混配对灰葡萄孢的增效作用[J].农药,2016,55(3):211-213.
[10]刘圣明,海飞,车志平,等.4种杀菌剂及其复配剂对番茄灰霉病菌的毒力[J].植物保护,2017,43(2):230-234.
[11]Saito S,Michailides T,Xiao C L.Fungicide resistance profiling in Botrytis cinerea populations from blueberries in California and Washington and their impact on control of gray mold[J].Plant Disease,2016,100(10):2087-2093.
[12]Nunes C,Usall J,TeixidóN,et al.Biological control of postharvest pear diseases using a bacterium,Pantoea agglomerans CPA-2[J].International Journal of Food Microbiology,2001,70(1-2):53-61.
[13]Sun P P,Cui J C,Jia X H,et al.Isolation and characterization of Bacillus amyloliquefaciens L-1 for biocontrol of pear ring rot[J].Horticulture Plant Journal,2017,3(5):183-189.
[14]孙平平,崔建潮,贾晓辉,等.藤黄灰链霉菌Pear-2的拮抗性及对梨果实防御相关酶的诱导效应[J].植物保护学报,2017,44(5):869-870.
[15]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001.
[16]Guo Y,Guo J,Bin L I.Advance of taxonomy of Pseudomonas based on 16S rRNA sequence[J].Journal of Microbiology,2004,24(2):38-41.
[17]Yamamoto S,Harayama S.PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains[J].Applied and Environmental Microbiology,1995,61(3):1104-1109.
[18]Glickmann E,Dessaux Y.A critical examination of the specificity of the salkowski reagent for indolic compounds produced by phytopathogenic bacteria[J].Applied and Environmental Microbiology,1995,619(2):793-796.
[19]习柳,田世平.酵母拮抗菌与碳酸氢钠配合对番茄果实采后病害的防治效果研究[J].中国农业科学,2005,38(5):950-955.
[20]刘邮洲,梁雪杰,乔俊卿,等.梨果贮藏期病害拮抗细菌的筛选、鉴定和评价[J].中国生物防治学报,2017,33(1):121-127.
[21]郝晓娟,刘波,谢关林,等.铜绿假单胞菌FJAT-346对番茄枯萎病的生防作用[J].山西农业大学学报(自然科学版),2011,31(1):39-43.
[22]张亚,苏品,刘双清,等.拮抗假单胞菌SU8对几种植物病原真菌的抑制作用[J].农药,2013,52(12):917-920.
[23]Couillerot O,Prigent-Combaret C,Caballero-Mellado J,et al.Pseudomonas fluorescens,and closely-related fluorescent pseudomonads as biocontrol agents of soil-borne phytopathogens[J].Letters in Applied Microbiology,2009,48(5):505-512.
[24]刘力伟,刘丽媛,胡同乐,等.荧光假单胞菌SS101对苹果再植病害的生防效果[J].植物保护学报,2016,43(5):812-818.
[25]熊欣,侯博文,金雪洁,等.生防绿针假单胞菌HT66中脂肽的鉴定与功能[J].微生物学通报,2017,44(3):611-619.
[26]胡菁.恶臭假单胞菌A1在番茄青枯病生物防治中的应用[D].福州:福建农林大学,2017.
[27]Egamberdieva D.Survival of Pseudomonas extremorientalis TSAU20 and P.chlororaphis TSAU13 in the rhizosphere of common bean(Phaseolus vulgaris)under saline conditions[J].Plant Soil and Environment,2011,57(3):122-127.
[28]Egamberdieva D,Jabborova D,Mamadalieva N.Salt-tolerant Pseudomonas extremorientalis able to stimulate growth of Silybum marianum under salt stress[J].Medicinal and Aromatic Plant Science and Biotechnology,2013,7(1):7-10.
[29]布婷婷,周国英,刘君昂,等.油茶内生细菌Y13对油茶抗性相关酶的诱导作用[J].植物保护学报,2012,39(3):285-286.
[30]李然,邢介帅,韩立英,等.枯草芽孢杆菌胞外蛋白酶突变株的筛选及其拮抗作用[J].应用与环境生物学报,2008,14(2):231-234.
[31]邢介帅,李然,赵蕾,等.生防芽孢杆菌T2胞外蛋白酶的纯化及其抗真菌作用[J].植物病理学报,2008,38(4):377-381.
[32]范丽霞,胡晓苹,刘文波,等.响应面试验优化链霉菌Z331-A的发酵条件[J].中国酿造,2018,37(4):77-82.
[33]葛启隆,岳秀萍,王国英.一株苯酚降解菌的分离鉴定及响应面法优化其固定化[J].中国环境科学,2014,34(2):518-525.
[34]尹萌萌,贺婷停,王超,等.响应面法优化短小芽孢杆菌SCU11发酵产碱性蛋白酶及关键基因转录调控分析[J].应用与环境生物学报,2016,22(3):371-376.
[2]Lutz M C,Lopes C A,Rodriguez M E,et al.Efficacy and putative mode of action of native and commercial antagonistic yeasts against postharvest pathogens of pear[J].International Journal of Food Microbiology,2013,164(2-3):166-172.
[3]孙平平,崔建潮,贾晓辉,等.贝莱斯芽孢杆菌L-1对梨灰霉和青霉病菌的抑制作用评价及全基因组分析[J].微生物学报,2018,58(9):1637-1646.
[4]Romanazzi G,Smilanick J L,Feliziani E,et al.Integrated management of postharvest gray mold on fruit crops[J].Postharvest Biology and Technology,2016,113(3):69-76.
[5]孙平平,贾晓辉,崔建潮,等.梨灰霉病拮抗放线菌L-30的筛选、鉴定及作用机制研究[J].园艺学报,2016,43(12):2335-2346.
[6]纪军建,张小风,韩秀英,等.8种杀菌剂对番茄灰霉病菌的毒力及田间番茄灰霉病菌对咯菌腈的敏感性[J].植物保护,2012,38(6):144-146.
[7]赵建江,陈治芳,韩秀英,等.啶酰菌胺与唑胺菌酯混配对灰葡萄孢毒力的增效作用[J].农药学学报,2015,17(4):417-424.
[8]黄海,张鑫,邹杭,等.番茄灰霉病菌室内毒力测定及药效试验[J].西北农林科技大学学报(自然科学版),2015,43(2):184-190.
[9]赵建江,王文桥,马志强,等.啶酰菌胺与吡唑醚菌酯混配对灰葡萄孢的增效作用[J].农药,2016,55(3):211-213.
[10]刘圣明,海飞,车志平,等.4种杀菌剂及其复配剂对番茄灰霉病菌的毒力[J].植物保护,2017,43(2):230-234.
[11]Saito S,Michailides T,Xiao C L.Fungicide resistance profiling in Botrytis cinerea populations from blueberries in California and Washington and their impact on control of gray mold[J].Plant Disease,2016,100(10):2087-2093.
[12]Nunes C,Usall J,TeixidóN,et al.Biological control of postharvest pear diseases using a bacterium,Pantoea agglomerans CPA-2[J].International Journal of Food Microbiology,2001,70(1-2):53-61.
[13]Sun P P,Cui J C,Jia X H,et al.Isolation and characterization of Bacillus amyloliquefaciens L-1 for biocontrol of pear ring rot[J].Horticulture Plant Journal,2017,3(5):183-189.
[14]孙平平,崔建潮,贾晓辉,等.藤黄灰链霉菌Pear-2的拮抗性及对梨果实防御相关酶的诱导效应[J].植物保护学报,2017,44(5):869-870.
[15]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001.
[16]Guo Y,Guo J,Bin L I.Advance of taxonomy of Pseudomonas based on 16S rRNA sequence[J].Journal of Microbiology,2004,24(2):38-41.
[17]Yamamoto S,Harayama S.PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains[J].Applied and Environmental Microbiology,1995,61(3):1104-1109.
[18]Glickmann E,Dessaux Y.A critical examination of the specificity of the salkowski reagent for indolic compounds produced by phytopathogenic bacteria[J].Applied and Environmental Microbiology,1995,619(2):793-796.
[19]习柳,田世平.酵母拮抗菌与碳酸氢钠配合对番茄果实采后病害的防治效果研究[J].中国农业科学,2005,38(5):950-955.
[20]刘邮洲,梁雪杰,乔俊卿,等.梨果贮藏期病害拮抗细菌的筛选、鉴定和评价[J].中国生物防治学报,2017,33(1):121-127.
[21]郝晓娟,刘波,谢关林,等.铜绿假单胞菌FJAT-346对番茄枯萎病的生防作用[J].山西农业大学学报(自然科学版),2011,31(1):39-43.
[22]张亚,苏品,刘双清,等.拮抗假单胞菌SU8对几种植物病原真菌的抑制作用[J].农药,2013,52(12):917-920.
[23]Couillerot O,Prigent-Combaret C,Caballero-Mellado J,et al.Pseudomonas fluorescens,and closely-related fluorescent pseudomonads as biocontrol agents of soil-borne phytopathogens[J].Letters in Applied Microbiology,2009,48(5):505-512.
[24]刘力伟,刘丽媛,胡同乐,等.荧光假单胞菌SS101对苹果再植病害的生防效果[J].植物保护学报,2016,43(5):812-818.
[25]熊欣,侯博文,金雪洁,等.生防绿针假单胞菌HT66中脂肽的鉴定与功能[J].微生物学通报,2017,44(3):611-619.
[26]胡菁.恶臭假单胞菌A1在番茄青枯病生物防治中的应用[D].福州:福建农林大学,2017.
[27]Egamberdieva D.Survival of Pseudomonas extremorientalis TSAU20 and P.chlororaphis TSAU13 in the rhizosphere of common bean(Phaseolus vulgaris)under saline conditions[J].Plant Soil and Environment,2011,57(3):122-127.
[28]Egamberdieva D,Jabborova D,Mamadalieva N.Salt-tolerant Pseudomonas extremorientalis able to stimulate growth of Silybum marianum under salt stress[J].Medicinal and Aromatic Plant Science and Biotechnology,2013,7(1):7-10.
[29]布婷婷,周国英,刘君昂,等.油茶内生细菌Y13对油茶抗性相关酶的诱导作用[J].植物保护学报,2012,39(3):285-286.
[30]李然,邢介帅,韩立英,等.枯草芽孢杆菌胞外蛋白酶突变株的筛选及其拮抗作用[J].应用与环境生物学报,2008,14(2):231-234.
[31]邢介帅,李然,赵蕾,等.生防芽孢杆菌T2胞外蛋白酶的纯化及其抗真菌作用[J].植物病理学报,2008,38(4):377-381.
[32]范丽霞,胡晓苹,刘文波,等.响应面试验优化链霉菌Z331-A的发酵条件[J].中国酿造,2018,37(4):77-82.
[33]葛启隆,岳秀萍,王国英.一株苯酚降解菌的分离鉴定及响应面法优化其固定化[J].中国环境科学,2014,34(2):518-525.
[34]尹萌萌,贺婷停,王超,等.响应面法优化短小芽孢杆菌SCU11发酵产碱性蛋白酶及关键基因转录调控分析[J].应用与环境生物学报,2016,22(3):371-376.