一株香樟炭疽病拮抗菌的鉴定及其发酵条件优化
|
摘要
为寻获香樟炭疽病生物防治的优良菌种资源,通过形态学观察、生理生化检测和基于16S rDNA系统发育分析,对一株香樟炭疽病拮抗菌SWUJ1进行了菌种鉴定;通过单因素试验优化该菌株产生抑菌活性物质的发酵条件,并釆用抑菌圈法检测其发酵液抑菌活性;进而利用菌丝生长速率法测定该菌株的抑菌谱.菌种鉴定结果表明SWUJ1为革兰氏阳性杆状菌株、产芽孢,在LB固体培养基上菌落呈圆形、边缘整齐光滑、湿润、呈乳白色黏稠状,过氧化氢酶呈阳性且具运动性;基于16S rDNA序列的系统发育分析结果显示该菌株与登录号为NR116240的甲基营养型芽孢杆菌(Bacillus methylotrophicus)的亲缘关系最近,且处于系统发育树的同一分枝,故将SWUJ1菌株鉴定为甲基营养型芽孢杆菌,命名为B.methylotrophicus SWUJ1;发酵条件优化结果表明该菌株产抑菌活性物质的最佳氮源为酵母粉,碳源为乳糖,无机盐离子为MgSO_4,初始pH值为5.0,培养温度为25℃,接种量为1.0%,发酵时间为96 h,优化后拮抗细菌B.methylotrophicus SWUJ1等量发酵上清液对香樟炭疽病菌的拮抗作用显著提高,且其发酵上清液对核盘菌(Sclerotinia sclerotiorum)及旋孢腔菌(Cochiobolus sativus)等10余种常见植物病原菌具不同程度的抑制作用.研究结果表明,B.methylotrophicus SWUJ1菌株可作为开发香樟炭疽病生防制剂的候选菌株.
In order to prepare strain resources for the biological control of camphor anthracnose, an antagonistic bacterium SWUJ1 against camphor anthracnose was identified by morphological features, physiological and biochemical characteristics and 16 S rDNA phylogenetic analysis. Further, the fermentation conditions and medium composition were optimized through a single-factor experiment, and the antimicrobial activity of the cell-free fermentation supernatant was determined with the inhibition zone method. Moreover, the antimicrobial spectrum of the cell-free fermentation supernatant was assayed with the method of mycelia growth rate. The results of identification showed that SWUJ1 was Gram-positive, rod-shaped and able to form spores. The colony of SWUJ1 was round, neat, smooth, moist, milky white and viscous on the LB solid medium. Its catalase and motility were positive. Based on 16 S rDNA phylogenetic analysis, SWUJ1 was close to Bacillus methylotrophicus and in the same minimal clade with B.methylotrophicus(accession number: NR116240). Therefore, strain SWUJ1 was identified as a strain of B. methylotrophicus, and named B. methylotrophicus SWUJ1. The results of fermentation optimization showed that optimal nitrogen source for the antibacterial substances of strain SWUJ1 was yeast powder, the carbon source was lactose, and the inorganic ion was MgSO4 and the optimal culture conditions were inoculation size of 1.0% for 94 h at 25 ℃ with an initial pH of 5.0. Equal fermentation supernatant of the antagonistic bacterium B. methylotrophicus SWUJ1 significantly improved its antagonistic activity against camphor anthracnose via fermentation optimization and showed antagonistic activity in different degrees against more than 10 plant pathogens such as Sclerotinia sclerotiorum and Cochiobolus sativus. The above results indicated that B. methylotrophicus SWUJ1 could be a candidate strain for future biological control of camphor anthracnose.
In order to prepare strain resources for the biological control of camphor anthracnose, an antagonistic bacterium SWUJ1 against camphor anthracnose was identified by morphological features, physiological and biochemical characteristics and 16 S rDNA phylogenetic analysis. Further, the fermentation conditions and medium composition were optimized through a single-factor experiment, and the antimicrobial activity of the cell-free fermentation supernatant was determined with the inhibition zone method. Moreover, the antimicrobial spectrum of the cell-free fermentation supernatant was assayed with the method of mycelia growth rate. The results of identification showed that SWUJ1 was Gram-positive, rod-shaped and able to form spores. The colony of SWUJ1 was round, neat, smooth, moist, milky white and viscous on the LB solid medium. Its catalase and motility were positive. Based on 16 S rDNA phylogenetic analysis, SWUJ1 was close to Bacillus methylotrophicus and in the same minimal clade with B.methylotrophicus(accession number: NR116240). Therefore, strain SWUJ1 was identified as a strain of B. methylotrophicus, and named B. methylotrophicus SWUJ1. The results of fermentation optimization showed that optimal nitrogen source for the antibacterial substances of strain SWUJ1 was yeast powder, the carbon source was lactose, and the inorganic ion was MgSO4 and the optimal culture conditions were inoculation size of 1.0% for 94 h at 25 ℃ with an initial pH of 5.0. Equal fermentation supernatant of the antagonistic bacterium B. methylotrophicus SWUJ1 significantly improved its antagonistic activity against camphor anthracnose via fermentation optimization and showed antagonistic activity in different degrees against more than 10 plant pathogens such as Sclerotinia sclerotiorum and Cochiobolus sativus. The above results indicated that B. methylotrophicus SWUJ1 could be a candidate strain for future biological control of camphor anthracnose.
引文
[1] 王丽贞.芳香樟炭疽病的研究 [D].福州:福建农林大学,2007.
[2] 许东新,杨学军,唐东芹,等.上海外环林带森林结构的优化模式 [J].东北林业大学学报,2002,30(3):118-122.
[3] 秦霞.香樟的功用及主要栽培技术 [J].中国林副特产,2001(3):18.
[4] 葛建明,张伟,管丽琴,等.香樟炭疽病菌生物学特性及其植物源农药的筛选 [J].上海交通大学学报(农业科学版),2005,23(4):401-405,442.
[5] 何思瑶,吴道军,任慧爽,等.5种杀菌剂对香樟炭疽病菌的室内药效试验 [J].绿色科技,2017(13):21-23.
[6] 彭琼,卢宗荣,何传统,等.恩施市城区樟树主要病虫害的研究 [J].湖北林业科技,2014,43(2):35-37.
[7] 杨彦辉,龙珠平.樟树炭疽病防治措施初探 [J].湖南林业科技,2007,34(5):81-82.
[8] 单体江,冯皓,艾彩霞,等.樟树病害及其防治研究综述 [J].湖南林业科技,2014,41(4):75-77,85.
[9] 任建国,黄思良,晏卫红,等.拮抗微生物防治芒果炭疽病研究 [J].西南农业学报,2002,15(4):82-85.
[10] 唐玉清,殷允广.微生物功能菌生物防治葡萄炭疽病试验报告 [J].新疆农业科技,2015(3):41-42.
[11] SANTHANAM R,BALDWIN I T,GROTEN K.In Wild Tobacco,Nicotiana Attenuata,Variation Among Bacterial Communities of Isogenic Plants is Mainly Shaped by the Local Soil Microbiota Independently of the Plants' Capacity to Produce Jasmonic Acid [J].Communicative & Integrative Biology,2015,8(2):e1017160-1-e1017160-5.
[12] PANKE-BUISSE K,POOLE A C,GOODRICH J K,et al.Selection on Soil Microbiomes Reveals Reproducible Impacts on Plant Function [J].The ISME Journal,2015,9(4):980-989.
[13] SANGUIN H,SARNIGUET A,GAZENGEL K,et al.Rhizosphere Bacterial Communities Associated with Disease Suppressiveness Stages of Take-All Decline in Wheat Monoculture [J].New Phytologist,2009,184(3):694-707.
[14] EDWARDS J,JOHNSON C,SANTOS-MEDELLíN C,et al.Structure,Variation,and Assembly of the Root-Associated Microbiomes of Rice [J].Proceedings of the National Academy of Sciences,2015,112(8):E911-E920.
[15] MENDES R,KRUIJT M,DE BRUIJN I,et al.Deciphering the Rhizosphere Microbiome for Disease-Suppressive Bacteria [J].Science,2011,332(6033):1097-1100.
[16] SANTOYO G,DEL CARMEN OROZCO-MOSQUEDA M,GOVINDAPPA M.Mechanisms of Biocontrol and Plant Growth-Promoting Activity in Soil Bacterial Species of Bacillus and Pseudomonas:A Review [J].Biocontrol Science and Technology,2012,22(8):855-872.
[17] YANG H W,LI J,XIAO Y H,et al.An Integrated Insight into the Relationship Between Soil Microbial Community and Tobacco Bacterial Wilt Disease [J].Frontiers in Microbiology,2017,8:2179-2190.
[18] 张晖,宋圆圆,吕顺,等.香蕉根际促生菌的抑菌活性及对作物生长的促进作用 [J].华南农业大学学报,2015,36(3):65-70.
[19] 喇文军,贾书娟,吴小丽,等.抑制辣椒炭疽菌的生防芽孢杆菌菌株筛选和鉴定 [J].深圳职业技术学院学报,2016,15(3):48-52.
[20] 祝福元,吴小丽,吕风青,等.菜心炭疽病菌拮抗细菌的筛选及鉴定 [J].微生物学通报,2009,36(9):1350-1355.
[21] 孙卓,杨利民.人参灰霉病拮抗细菌的筛选及鉴定 [J].植物保护学报,2016,43(6):935-942.
[22] 周德庆.微生物学教程 [M].2版.北京:高等教育出版社,2002:356-357.
[23] 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.
[24] 沈萍.微生物学 [M].北京:高等教育出版社,2000.
[25] WEISBURG G,BARNS M,PELLETIER A,et al.16S Ribosomal DNA Amplification for Phylogenetic Study [J].Journal of Bacteriology,1991,173(2):697-703.
[26] 谢洁,夏天,林立鹏,等.一株桑树内生拮抗菌的分离鉴定 [J].蚕业科学,2009,35(1):121-125.
[27] 方中达.植病研究方法 [M].北京:中国农业出版社,1998.
[28] 王军,何大敏,陈廷智,等.大蒜与3种药用作物对烟草炭疽病菌的抑菌效果 [J].西南大学学报(自然科学版),2018,40(2):1-7.
[29] 吕倩,胡江春,王楠,等.南海深海甲基营养型芽孢杆菌SHB114抗真菌脂肽活性产物的研究 [J].中国生物防治学报,2014,30(1):113-120.
[30] 魏新燕,黄媛媛,黄亚丽,等.甲基营养型芽孢杆菌BH21对葡萄灰霉病菌的拮抗作用 [J].中国农业科学,2018,51(5):883-892.
[31] 康林玉,刘周斌,欧立军,等.土壤微生物促进作物生长发育研究进展 [J].湖南农业科学,2017(3):113-116.
[32] 任慧爽,徐伟芳,王爱印,等.桑树内生细菌多样性及内生拮抗活性菌群的研究 [J].西南大学学报(自然科学版),2017,39(1):36-45.
[33] STEIN T.Bacillus subtilis Antibiotics:Structures,Syntheses and Specific Functions [J].Molecular Microbiology,2005,56(4):845-857.
[34] 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.
[35] GOND S K,BERGEN M S,TORRES M S,et al.Endophytic Bacillus spp.Produce Antifungal Lipopeptides and Induce Host Defence Gene Expression in Maize [J].Microbiological Research,2015,172:79-87.
[36] 常征,王蓉,李洪潮,等.菌株YS(r)-19分离鉴定及对三七根腐病菌的抑菌活性 [J].江苏农业科学,2017,45(6):92-95.
[37] 孙卓,杨利民.人参锈腐病拮抗细菌的筛选鉴定 [J].中国农业大学学报,2016,21(2):73-81.
[38] 尹向田,苏玲,吴新颖,等.芽孢杆菌GSBM05对葡萄白腐病菌的抑菌活性及其鉴定 [J].中国农学通报,2018,34(1):134-141.
[2] 许东新,杨学军,唐东芹,等.上海外环林带森林结构的优化模式 [J].东北林业大学学报,2002,30(3):118-122.
[3] 秦霞.香樟的功用及主要栽培技术 [J].中国林副特产,2001(3):18.
[4] 葛建明,张伟,管丽琴,等.香樟炭疽病菌生物学特性及其植物源农药的筛选 [J].上海交通大学学报(农业科学版),2005,23(4):401-405,442.
[5] 何思瑶,吴道军,任慧爽,等.5种杀菌剂对香樟炭疽病菌的室内药效试验 [J].绿色科技,2017(13):21-23.
[6] 彭琼,卢宗荣,何传统,等.恩施市城区樟树主要病虫害的研究 [J].湖北林业科技,2014,43(2):35-37.
[7] 杨彦辉,龙珠平.樟树炭疽病防治措施初探 [J].湖南林业科技,2007,34(5):81-82.
[8] 单体江,冯皓,艾彩霞,等.樟树病害及其防治研究综述 [J].湖南林业科技,2014,41(4):75-77,85.
[9] 任建国,黄思良,晏卫红,等.拮抗微生物防治芒果炭疽病研究 [J].西南农业学报,2002,15(4):82-85.
[10] 唐玉清,殷允广.微生物功能菌生物防治葡萄炭疽病试验报告 [J].新疆农业科技,2015(3):41-42.
[11] SANTHANAM R,BALDWIN I T,GROTEN K.In Wild Tobacco,Nicotiana Attenuata,Variation Among Bacterial Communities of Isogenic Plants is Mainly Shaped by the Local Soil Microbiota Independently of the Plants' Capacity to Produce Jasmonic Acid [J].Communicative & Integrative Biology,2015,8(2):e1017160-1-e1017160-5.
[12] PANKE-BUISSE K,POOLE A C,GOODRICH J K,et al.Selection on Soil Microbiomes Reveals Reproducible Impacts on Plant Function [J].The ISME Journal,2015,9(4):980-989.
[13] SANGUIN H,SARNIGUET A,GAZENGEL K,et al.Rhizosphere Bacterial Communities Associated with Disease Suppressiveness Stages of Take-All Decline in Wheat Monoculture [J].New Phytologist,2009,184(3):694-707.
[14] EDWARDS J,JOHNSON C,SANTOS-MEDELLíN C,et al.Structure,Variation,and Assembly of the Root-Associated Microbiomes of Rice [J].Proceedings of the National Academy of Sciences,2015,112(8):E911-E920.
[15] MENDES R,KRUIJT M,DE BRUIJN I,et al.Deciphering the Rhizosphere Microbiome for Disease-Suppressive Bacteria [J].Science,2011,332(6033):1097-1100.
[16] SANTOYO G,DEL CARMEN OROZCO-MOSQUEDA M,GOVINDAPPA M.Mechanisms of Biocontrol and Plant Growth-Promoting Activity in Soil Bacterial Species of Bacillus and Pseudomonas:A Review [J].Biocontrol Science and Technology,2012,22(8):855-872.
[17] YANG H W,LI J,XIAO Y H,et al.An Integrated Insight into the Relationship Between Soil Microbial Community and Tobacco Bacterial Wilt Disease [J].Frontiers in Microbiology,2017,8:2179-2190.
[18] 张晖,宋圆圆,吕顺,等.香蕉根际促生菌的抑菌活性及对作物生长的促进作用 [J].华南农业大学学报,2015,36(3):65-70.
[19] 喇文军,贾书娟,吴小丽,等.抑制辣椒炭疽菌的生防芽孢杆菌菌株筛选和鉴定 [J].深圳职业技术学院学报,2016,15(3):48-52.
[20] 祝福元,吴小丽,吕风青,等.菜心炭疽病菌拮抗细菌的筛选及鉴定 [J].微生物学通报,2009,36(9):1350-1355.
[21] 孙卓,杨利民.人参灰霉病拮抗细菌的筛选及鉴定 [J].植物保护学报,2016,43(6):935-942.
[22] 周德庆.微生物学教程 [M].2版.北京:高等教育出版社,2002:356-357.
[23] 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.
[24] 沈萍.微生物学 [M].北京:高等教育出版社,2000.
[25] WEISBURG G,BARNS M,PELLETIER A,et al.16S Ribosomal DNA Amplification for Phylogenetic Study [J].Journal of Bacteriology,1991,173(2):697-703.
[26] 谢洁,夏天,林立鹏,等.一株桑树内生拮抗菌的分离鉴定 [J].蚕业科学,2009,35(1):121-125.
[27] 方中达.植病研究方法 [M].北京:中国农业出版社,1998.
[28] 王军,何大敏,陈廷智,等.大蒜与3种药用作物对烟草炭疽病菌的抑菌效果 [J].西南大学学报(自然科学版),2018,40(2):1-7.
[29] 吕倩,胡江春,王楠,等.南海深海甲基营养型芽孢杆菌SHB114抗真菌脂肽活性产物的研究 [J].中国生物防治学报,2014,30(1):113-120.
[30] 魏新燕,黄媛媛,黄亚丽,等.甲基营养型芽孢杆菌BH21对葡萄灰霉病菌的拮抗作用 [J].中国农业科学,2018,51(5):883-892.
[31] 康林玉,刘周斌,欧立军,等.土壤微生物促进作物生长发育研究进展 [J].湖南农业科学,2017(3):113-116.
[32] 任慧爽,徐伟芳,王爱印,等.桑树内生细菌多样性及内生拮抗活性菌群的研究 [J].西南大学学报(自然科学版),2017,39(1):36-45.
[33] STEIN T.Bacillus subtilis Antibiotics:Structures,Syntheses and Specific Functions [J].Molecular Microbiology,2005,56(4):845-857.
[34] 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.
[35] GOND S K,BERGEN M S,TORRES M S,et al.Endophytic Bacillus spp.Produce Antifungal Lipopeptides and Induce Host Defence Gene Expression in Maize [J].Microbiological Research,2015,172:79-87.
[36] 常征,王蓉,李洪潮,等.菌株YS(r)-19分离鉴定及对三七根腐病菌的抑菌活性 [J].江苏农业科学,2017,45(6):92-95.
[37] 孙卓,杨利民.人参锈腐病拮抗细菌的筛选鉴定 [J].中国农业大学学报,2016,21(2):73-81.
[38] 尹向田,苏玲,吴新颖,等.芽孢杆菌GSBM05对葡萄白腐病菌的抑菌活性及其鉴定 [J].中国农学通报,2018,34(1):134-141.