烟草疫霉菌拮抗细菌的筛选鉴定及发酵条件优化
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摘要
为了筛选对烟草疫霉菌具有拮抗作用的生防细菌菌株,本研究从烟草黑胫病病圃地采集健康烟株的根际土样,采用对峙培养法筛选得到8株对烟草黑胫病具有拮抗作用的细菌菌株,其中编号为LB-9的菌株对烟草疫霉菌的抑制作用最强且效果稳定,抑菌率为60.6%,且抑菌谱广,对其进行16S rRNA基因序列分析后将该菌株鉴定为多粘芽孢杆菌。采用单因素优化试验初步探究了多粘芽孢杆菌LB-9的最适发酵条件,明确了LB-9的最适发酵培养基、最适发酵温度、最适发酵时间和最适发酵初始pH值,对其生防制剂的开发应用具有重要意义。
In order to screen the bacterial strains which have antagonistic effects on Phytophthora nicotianae and provide favorable resources for the biological control of tobacco black shank disease in Henan tobacco areas, the rhizosphere soil samples of healthy tobacco plants were collected from tobacco black shank disease nursery and eight bacterial strains were screened by confrontation culture method. Among them, the strain LB-9 had the strongest and stable inhibitory effect on P. nicotianae with a bacteriostatic rate of 60.6% and a broad spectrum. The strain was identified as Paenibacillus polymyxa by 16 S rRNA gene sequencing analysis. The optimum fermentation medium, temperature, time and initial pH value of LB-9 were determined by single factor optimization test which has great significance to the development and application of biocontrol agents.
In order to screen the bacterial strains which have antagonistic effects on Phytophthora nicotianae and provide favorable resources for the biological control of tobacco black shank disease in Henan tobacco areas, the rhizosphere soil samples of healthy tobacco plants were collected from tobacco black shank disease nursery and eight bacterial strains were screened by confrontation culture method. Among them, the strain LB-9 had the strongest and stable inhibitory effect on P. nicotianae with a bacteriostatic rate of 60.6% and a broad spectrum. The strain was identified as Paenibacillus polymyxa by 16 S rRNA gene sequencing analysis. The optimum fermentation medium, temperature, time and initial pH value of LB-9 were determined by single factor optimization test which has great significance to the development and application of biocontrol agents.
引文
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[25]曹琦琦,周登博,郑丽,等.水稻纹枯病菌拮抗菌的筛选、鉴定及培养条件探索[J].中国生物防治学报,2013,29(2):270-276.CAO Q Q,ZHOU D B,ZHENG L,et al.Screening,identification and cultivation conditions of microbes antagonistic to rice sheath blight fungus Rhizoctonia solani[J].Chinese Journal of Biological Control,2013,29(2):270-276.
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[27]赵德立,曾林子,李晖,等.多粘芽孢杆菌JW-725抗菌活性物质及其发酵条件的初步研究[J].植物保护,2006,32(1):48-51.ZHAO D L,ZENG L Z,LI H,et al.Fermentation of Bacillus polymyxa JW-725 and preliminary extraction of the antifungal substances[J].Plant Protection,2006,32(1):48-51.
[2]赖传雅.农业植物病理学:南方本[M].北京:科学出版社,2003:157-161.LAI C Y.Agricultural Plant Pathology:Southern version[M].Beijing:Science Press,2003:157-161.
[3]王志愿,姜清治,霍沁建.烟草黑胫病的研究进展[J].中国农学通报,2010,26(21):250-255.WANG Z Y,JIANG Q Z,HUO Q J.Research progress on tobacco black shank disease[J].Chinese Agricultural Science Bulletin,2010,26(21):250-255.
[4]罗华元,王绍坤,常寿荣,等.12种农药在烟叶中残留及烟气中转移试验初报[J].云南农业大学学报(自然科学版),2010,25(5):636-641.LUO H Y,WANG S K,CHANG S R,et al.Determination of 12 pesticides residues in tobacco leaf and shifting to smoke[J].Journal of Yunnan Agricultural University,2010,25(5):636-641.
[5]李义强,孙惠青,王秀国,等.防治烟草黑胫病常用农药残留量与降解的研究[J].中国烟草学报,2011,17(4):67-73.LI Y Q,SUN H Q,WANG X G,et al.The degradation dynamics and residue level of different germicides against tobacco black shank[J].Acta Tabacaria Sinica,2011,17(4):67-73.
[6]彭阁,姜乾坤,谭军,等.烟草黑胫病拮抗真菌的筛选及活性评价[J].中国烟草科学,2018,39(1):77-84.PENG G,JIANG Q K,TAN J,et al.Selection and field evaluation of antagonistic fungi against tobacco black shank[J].Chinese Tobacco Science,2018,39(1):77-84.
[7]徐同伟,周建云,祖庆学,等.两株烟草黑胫病拮抗菌的筛选、鉴定和促生防病潜力评价[J].中国烟草科学,2017,38(3):44-50.XU T W,ZHOU J Y,ZU Q X,et al.Screening,identification,biocontrol and growth-promoting potential evaluation of two bacterial strains against tobacco black shank[J].Chinese Tobacco Science,2017,38(3):44-50.
[8]杨兵,刘慧美,龙章富.微生物防治烟草黑胫病的研究进展[J].农药,2015,54(9):629-634.YANG B,LIU H M,LONG Z F.Recent advances in microbiological control of Phytophthora parasitica var.nicotianae[J].Agrochemicals,2015,54(9):629-634.
[9]杨珍福,吴毅歆,陈映岚,等.烟草拮抗内生细菌的筛选与防病促生长效果[J].中国烟草科学,2014(6):48-53.YANG Z F,WU Y X,CHEN Y L,et al.Screening,growth promotion,and disease control of antagonistic and endophytic bacteria in tobacco[J].Chinese Tobacco Science,2014(6):48-53.
[10]王静,孔凡玉,张成省,等.放线菌F8对烟草黑胫病的拮抗作用及其产酶活性[J].中国烟草科学,2013(2):49-53.WANG J,KONG F Y,ZHANG C X,et al.Antagonism effect and enzyme-producing comparison of Actinomycetes F8 against tobacco black shank[J].Chinese Tobacco Science,2013(2):49-53.
[11]陈志谊.芽孢杆菌类生物杀菌剂的研发与应用[J].中国生物防治学报,2015,31(5):723-732.CHEN Z Y.Research and application of bio-fungicide with Bacillus spp.[J].Chinese Journal of Biological Control,2015,31(5):723-732.
[12]陈志谊,刘荣,刘永锋.水稻纹枯病拮抗细菌B-916的选育[J].中国生物防治学报,2003,19(1):15-18.CHEN Z Y,LIU R,LIU Y F.The screening of antagonistic bacteria strain B-916 against rice sheath blight[J].Chinese Journal of Biological Control,2003,19(1):15-18.
[13]郭荣君,刘杏忠,杨怀文,等.芽孢杆菌BH_1防治大豆根腐病的效果及机制[J].中国生物防治学报,2003,19(4):180-184.GUO R J,LIU X Z,YANG H W,et al.Mechanism of rhizobacteria BH1(Bacillus sp.)to suppress soybean root rot disease caused by Fusarium spp.[J].Chinese Journal of Biological Control,2003,19(4):180-184.
[14]欧雄常,柳凤,詹儒林.拮抗辣椒疫病菌的红树内生细菌筛选及RS261菌株鉴定[J].微生物学通报,2009,36(2):175-180.OU X C,LIU F,ZHAN R L.Screen of mangrove endophytic bacteria antagonists against Phytophthora capsici and identification of strain RS261[J].Microbiology,2009,36(2):175-180.
[15]冯志珍.解淀粉芽孢杆菌FB-16的筛选及其对作物疫霉菌的抑制作用[D].咸阳:西北农林科技大学,2012.FENG Z Z.Screening and inhibitory effect of Bacillus amyloliquefaciens FB-16 against crop Phytophthora sp.[D].Xianyang:Northwest A&F University,2012.
[16]张荣胜,王晓宇,罗楚平,等.解淀粉芽孢杆菌Lx-11产脂肽类物质鉴定及表面活性素对水稻细菌性条斑病的防治作用[J].中国农业科学,2013,46(10):2014-2021.ZHANG R S,WANG X Y,LUO C P,et al.Identification of the lipopeptides from Bacillus amyloliquefaciens Lx-11 and biocontrol efficacy of surfactin against bacterial leaf streak[J].Scientia Agricultura Sinica,2013,46(10):2014-2021.
[17]黄玲玲,裘纪莹,唐琳,等.解淀粉芽胞杆菌NCPSJ7对采后苹果轮纹病的生物防治作用[J].中国食物与营养,2015,21(2):20-24.HUANG L L,QIU J Y,TANG L,et al.Biological control effect of Bacillus amylus NCPSJ7 on postharvest apple ring rot[J].Food and Nutrition in China,2015,21(2):20-24.
[18]周晓梅,刘强,王瑛璐.拮抗菌生物防治农作物病害的研究进展[J].吉林师范大学学报(自然科学版),2010(4):36-39.ZHOU X M,LIU Q,WANG Y L.Research progress on biocontrol of crop diseases by antagonistic bacteria[J].Journal of Jilin Normal University(Natural Science Edition),2010(4):36-39.
[19]常冬梅.芽孢杆菌(Bacillus spp.)根际接种促进辣椒(Capsicum annuum L.)生长的作用机理[D].北京:中国农业科学院,2010.CHANG D M.Mechanisms of Bacillus spp.rhizosphere inoculation promoting growth of pepper(Capsicum annuum L.)[D].Beijing:Chinese Academy of Agricultural Sciences,2010.
[20]Ryu C M,Kim J,Choi O,et al.Improvement of biological control capacity of Paenibacillus polymyxa E681 by seed pelleting on sesame[J].Biological Control,2006,39(3):282-289.
[21]吴秉奇,梁永江,丁延芹,等.两株烟草根际拮抗菌的生防和促生效果研究[J].中国烟草科学,2013(1):66-71.WU B Q,LIANG Y J,DING Y Q,et al.Study on disease-preventing and growth-promoting effects of two antifungal bacteria from tobacco rhizosphere[J].Chinese Tobacco Science,2013(1):66-71.
[22]Beatty P H,Jensen S E.Paenibacillus polymyxa produces fusaricidin-type antifungal antibiotics active against Leptosphaeria maculans,the causative agent of blackleg disease of canola[J].Canadian Journal of Microbiology,2002,48(2):159-169.
[23]王波,周涧楠,黄忠勤,等.一株多粘类芽孢杆菌Paenibacillus polymyxa对甘薯黑斑病的生物防治效果及作用机理初探[J].江西农业学报,2017,29(10):40-43.WANG B,ZHOU J N,HUANG Z Q,et al.Biocontrol efficacy of a Paenibacillus polymyxa strain against black rot of sweet potato and its action mechanism[J].Acta Agriculturae Jiangxi,2017,29(10):40-43.
[24]马夙静.多粘类芽孢杆菌ZYPP18的分离鉴定与促生防病效果测定[D].泰安:山东农业大学,2018.MA S J.Identification and determination the plant growth promoting and biocontrol properties of Paenibacillus polymyxa ZYPP18[D].Tai’an:Shandong Agricultural University,2018.
[25]曹琦琦,周登博,郑丽,等.水稻纹枯病菌拮抗菌的筛选、鉴定及培养条件探索[J].中国生物防治学报,2013,29(2):270-276.CAO Q Q,ZHOU D B,ZHENG L,et al.Screening,identification and cultivation conditions of microbes antagonistic to rice sheath blight fungus Rhizoctonia solani[J].Chinese Journal of Biological Control,2013,29(2):270-276.
[26]张镜,牟利辉,刘志伟,等.Act0988拮抗菌株鉴定及培养条件与菌株生长关系研究[J].生物技术通报,2016,32(7):112-118.ZHANG J,MOU L H,LIU Z W,et al.Identification of antagonistic strain act0988,and relationships between cultivation conditions and its growth[J].Biotechnology Bulletin,2016,32(7):112-118.
[27]赵德立,曾林子,李晖,等.多粘芽孢杆菌JW-725抗菌活性物质及其发酵条件的初步研究[J].植物保护,2006,32(1):48-51.ZHAO D L,ZENG L Z,LI H,et al.Fermentation of Bacillus polymyxa JW-725 and preliminary extraction of the antifungal substances[J].Plant Protection,2006,32(1):48-51.
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