摘要
油茶内生拮抗菌Y13是一株对油茶炭疽病原菌具有较强的抑制效果的细菌,该菌经鉴定为枯草芽孢杆菌。为开发该菌株作为生防菌剂,本文主要开展Y13菌株发酵条件、菌剂可湿性粉剂加工工艺,定殖能力及林间应用研究。主要研究结果如下：
(1)生防细菌Y13菌株发酵条件的优化
采用单因素实验和正交优化实验,确定了Y13发酵培养的最佳培养基配方为：葡萄糖2.0%,酵母膏1.0%,牛肉膏0.3%,NaCl浓度为1.5%；最佳发酵条件为：温度30℃,初始pH 6～7,摇瓶装液量为50 mL/250mL,接种量5%,摇床转速160 r/min,发酵周期36 h。
(2)生防细菌Y13菌株可湿性粉剂的研制
通过测定润湿时间、悬浮率及热稳定性得到了Y13可湿性粉剂的较佳组合为硅藻土、STPP、SDBS。采用L9(34)正交表安排实验,结果表明,Y13菌株可湿性粉剂的最佳配方为：硅藻土37.17%,STPP 4.13%,SDBS8.26%,腐植酸0.41%,Y13原粉50%。对Y13菌株可湿性粉剂的质量指标进行了测定,其含水量、润湿时间、pH值、细度悬浮率等指标达到了有关国家标准。菌株Y13经急性经口毒性实验和急性经皮毒性试验,小白鼠均无明显中毒表现。连续观察14d,小白鼠无死亡发生,说明生防细菌菌株Y13为微毒性。
(3)生防菌剂Y13定殖及林间应用研究
通过耐利福平标记菌株回收,研究其在油茶叶片和果实上的定殖能力及其影响因素。研究表明,该拮抗菌能在油茶叶部和果部良好定殖。较高的菌悬液浓度(>108cfu/mL),无炭疽病原菌干扰,温度较高(25℃～35℃)、湿度较大(RH95%-100%)对该拮抗菌的定殖有利。对油茶炭疽病进行了林间防治试验,结果表明：生防菌剂对油茶炭疽病有较好的防治效果(70%左右),与对照药剂百菌清的防治效果相当。
Endophytic bacillus subtilis strain Y13, which was obtained from oil-tea leaves through isolation and screening, possess strong antagonistic action to oil-tea anthracnose. In order to manufacture the biological control agent of Y13, fermented condition, wettable powder, colonization ability, garden tested were studied.
(1) Optimizing fermentation condition of strain Y13
Through culture condition and orthogonal tests, the optimal medium composition was obtained:glucose 2.0%, yeast extract 1.0%, beef extract 0.3%, concentration of NaCl 1.5%,30℃, initial pH6-7, the optimal fermentation condition was obtained:shake bottle fluid volume 50 mL/250 mL, inoculation volume 5%, speed 160 r/min, fermentation period 36h.
(2) Development on wettable Powders of strain Y13
The WP of Y13 was studied by orthogonal test, the additives'dosages of the wettable powder of Y13 were optimized:diatomaceous earth 37.17%,STPP 4.13%, SDBS 8.26%,humic acid 0.41%, original powder of Y13 50%.The quality of the formulation included water content, moisiturizing time, pH can meet national standards. Through the experiment of cute oral toxieity of biocontrol baeteria strain Y13, no one death case was observed, which showed that the strain Y13 had slight toxieity.
(3) Application of bioeontrol microbial inoculum Y13
The rifampin-marked strain Y13 was used to study its colonization ability in leaves and fruits of oil-tea plant. The results showed the introduced antagonistic bacteria were able to in leaves and fruits of oil-tea plant. Higher concentration＞108cfu/mL), noninterference of pathogen of oil-tea anthracnose, higher temperature (25℃～35℃), higher relative humidity (RH 95%～100%) were favorable to the colonization of the antagonistic bacteria. The microbial inoculum was tested to have good effect of preventing Camellia oleifera anthracnose (70%) in oil-tea garden, which was appropriate to the effect of Chlorothalonil as a control.
引文
[1]程亮,游春平,肖爱萍.拮抗细菌的研究进展[J].江西农业大学学报,2003,25(5)：732-737.
[2]Kenneth F, Baker. Evolving concepts of biological control of plant pathogens[J]. Ann Rev Phytopathol.1987,25:67-85.
[3]Walters S A, Wehner T C, Barker K R. Effects of Root Decay on the Relationship between Meloidogyne spp. Gall Index and Egg Mass Number in Cueumber and Homed Cucumber[J]. Supplement to Journal of Nematol,1992,24(4):707-711.
[4]张炳欣,张平,陈晓斌.影响引入微生物根部定殖的因素[J].应用生态学报,2000,11(6)：951-953.
[5]蔡学清,何红.双抗标记法测定枯草芽孢杆菌BS-2和BS-1在辣椒体内的定殖动态[J].福建农林大学学报,2003,32(1)：41-45.
[6]吴蔼民,顾本康,傅正擎,等.内生菌73a在不同抗性品种棉花体内的定殖和消长动态研究[J].植物病理学报,2001,31(4)：289-294.
[7]刘忠梅,王霞,赵金焕,等.有益内生细菌B946在小麦体内的定殖规律[J].中国生物防治,2005,21(2)：113-116.
[8]任小平,谢关林,王笑.铜绿假单胞菌zJ1999对水稻纹枯病的防治及其在水稻上的定殖.中国生物防治.2006,22(1)：54-57.
[9]冯永君,宋未.水稻内生优势成团泛菌GFP标记菌株的性质与标记丢失动力学[J].中国生物化学与分子生物学报,2002,18(1)：85-91.
[10]高增贵,庄敬华,陈捷等.应用免疫胶体金银染色技术定位玉米内生细菌[J].植物病理学报,2005,35(3)：262-266.
[11]张炳欣,张平.植物根围外来微生物定殖的检测方法[J].浙江大学学报(农业与生命科学版),2000,26(3)：624-628.
[12]李杨苹.土壤中枯草芽孢杆菌B908的分子检测[D].西北农村科技大学.2004.
[13]田涛,元雪晨,王琦,等.芽孢杆菌绿色荧光蛋白标记及其在小麦体表定殖的初探[J].植物病理学报,2004,34(4)：346-351.
[14]Kloepper J W. Plant growth promoting rhizobacteria as biological control agelts. In:Soil Mcorbai 1 Ecology:Application in Agricultral and environmental management,metting,F.B.,Jr.,Ed.,Marcel Deckker, New York,1992,pp.255-274.
[15]王平,胡正嘉,李阜棣.荧光假单胞菌群根部定殖的研究进展[J].应用与环境生物学报,1996,2(4)：408-414.
[16]Pillay V K and Nowak J. Inoculum density, temperature and genotype effects on in vitro growth promotion and epiphytic and endophytic colonization of tomato (lycopersicon esculentum L.) seedlings inoculated with a pseudomonad bacterium [J].Can.J.Microbiol.,1997, 43:354-361.
[17]Samish,Z, Etinger-Tulczynska, R., Bick M. Microflora within healthy tomatoes [J].Appl.Microbiol,1961,9:20-25.
[18]Mahaffee W. F., Kloepper J. W. et al. Improving Plant Productivity with Rhizosphere Bacteria. Ryder M.H., Stephense P.M., Bowen G.D. ed. [M]. CSIRO, Australia,1994:180.
[19]Sonenshein A.L., Losiek R., Hoch J. A.. Bacillus Subtilis and the Gram-Positive Bacteria-Biochemistry, Physiology and Molecular Geneties[M].Washington D.C:American Society for Microbiology, 1993.11-12.
[20]陈延熙,陈璧,潘贞德,等.增产菌的应用与研究[J].生物防治通报,1985,1(2)：22-23.
[21]李华荣,肖建国,颜思齐.蜡质芽孢杆菌R2防治水稻纹枯病研究[J].植物病理学报,1993,23(2)：101-105.
[22]湛晓曦,陈卫良,李德葆.357细菌拮抗物质的提取和生物活性检测[J].浙江大学学报(农业与生命科学版),2000,26(5)：489-492.
[23]张中鸽,彭于发,张玉勋.多粘芽孢菌B48菌株抑菌作用[J].中国生物防治,1994,(2)：85-86.
[24]崔云龙,姬金红,衣海青.短小芽孢杆菌D82对小麦根腐病原菌拮抗的研究[J].中国生物防治,1995,11(3)：114-118.
[25]黄绍宁,沈华山,吴华明.应用地衣芽孢杆菌和木霉防治基质栽培黄瓜苗期碎倒病[J].中国生物防治,1999,15(1)：45.
[26]童蕴慧,徐敬友,陈夕军.番茄灰霉病菌拮抗菌的筛选和应用[J].江苏 农业研究,2001,22(4)：25-28.
[27]纪兆林,童蕴慧,凌筝.拮抗细菌对部分水果产后病原真菌的抑制及防腐作用[J].南京农专学报,2003,19(4)：23-27.
[28]童蕴慧,郭桂萍,徐敬友.拮抗细菌对番茄植株抗灰霉病的诱导[J].中国生物防治,2004,20(3)：187-189.
[29]游春平,肖爱萍,魏金莲,等.稻瘟病拮抗细菌的活性研究[J].江西农业大学学报,2002,24(1)：24-26.
[30]Swadling I.R., Jeffries P..Bioeontrol Science & Technology[J].1998, 8(3):439-448.
[31]Walker R., Powell A. A., Seddon B.. Bacillus isolates from the spermosphere of peas and dwarf french beans with antifungal activity against Botrytis cinerea and Pythium species[J].Journal of Applied Microbiology,1998,84(5):791-801.
[32]Jock S., Volksch B., Mansvelt L., et al. Characterization of Bacillus strains from apple and pear trees in South Africa antagonistic to Erwinia amylovora[J].FEMS Microbiology Letters,2002,211:247-252.
[33]Hassni M. EI., Hadrami A., Daayf F.,et al. Biological control of bayoud disease in date palm:Selection of microorganisms inhibiting the causal agent and inducing defense reactions[J]. Environmental and Experimental Botany,2007,59:224-234.
[34]于淑池,张利平,王立安.拮抗细菌作为生物防治手段研究进展[J].河北农业科学,2004,8(1)：62-65.
[35]彭于发,陈善铭.荧光假单胞菌Tn5诱变菌株防治小麦全蚀病的初步研究[J].植物病理学报,1990,20(3)：229-233.
[36]彭于发.荧光假单胞菌Tns诱变防病增产研究初报[J].中国农业科学,1990.23(1)：88-90.
[37]祝新德,冯镇泰,许煌泉,等.荧光假单胞菌株M18防治甜瓜蔓枯病害[J].上海交通大学学报,2001,35(7)：1062-1065.
[38]Van overbeek L. S., Cassidy M, Kozdroj J., et al. A polyphasic approach for studying the interaction between Ralstonia solanacearum and Potential control agents in the tomato phytosphere[J]. Journal of Microbiological Methods,2002,48:69-86.
[39]Mercado-Blanco J., Rodriguez-Jurado D., Hervas A., et al. Suppression of Verticillium wilt in olive planting stocks by root-associated fluorescent Pseudomonas spp.[J].Biological Control,2004,30:474-486.
[40]Barka E. A., Gognies S., Nowak J., et al. Inhibitory effect of endophyte bacteria on Botrytis cinerea and its influence to promote the grapevine growth[J]. Biological Control,2002,24:135-142.
[41]Safiyazov J.S., Mannanov R.N., Sattarova R. K.. The use of bacterial antagonists for the control of cotton diseases[J]. Field Crops Research,1995,43:51-54.
[42]刘邮洲,陈志谊,刘荣,等.几种拮抗细菌对番茄早疫病的防治效果[J].江苏农业科学,2002,(6)：50-51.
[43]王远山,王平,胡正嘉.绿针假单胞菌PL9菌株对烟草疫霉的拮抗作用研究[J].华中农业大学学报,2002,21(3)：248-251.
[44]Cartwright D. K., Benson D. M.. Biological Control of Rhizoctonia Stem Rot of Poinsettia in Polyfoam Rooting Cubes with Pseudomonas cepacia and Paecilomyces lilacinus [J]. BiologiealControl, 1995,5:237-244.
[45]任小平,谢关林,王笑.铜绿假单胞菌zJ1999对水稻纹枯病的防治及其在水稻上的定殖[J].中国生物防治,2006,22(1)：54-57.
[46]丁中,刘跃.菜丰宁B1浓缩菌粉的防病机理,生产工艺及应用技术研究[J].农药.2000,39(8)：25-27.
[47]陈志谊,陆凡,刘春祥,等.水稻纹枯病拮抗细菌B-916培养条件与发酵配方的研究[J].西南农业学报,1999,01,12-14
[48]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)：47-50.
[49]宋卡魏.枯草芽孢杆菌B68发酵培养的优化及两种剂型的初步研究[D].华南热带农业大学,2007.
[50]申继忠.微生物农药剂型加工研究进展[J].中国生物防治,1998,14(3)：229-1133.
[51]赵岩.浅谈微生物农药的发展前景[J].科技咨询导报,2006,9：246.
[52]刘萍萍.微生物农药研究进展[J].山东农业科学,2005,2：78-80.
[53]陈晓娟.木霉菌可湿性粉剂的研制[J].重庆工贸职业技术学院学 报,2007,4：28-32.
[54]刘步林.农药剂型加工技术[M].化学工业出版社,1998,10.
[55]韩熹莱.农药概论[M].北京农业大学出版社.1995.
[56]曹红娣.农药可湿性粉剂生产工艺的改进[J].上海农业科技2005，6：139.
[57]湘元.农药粉剂与可湿性粉剂有何区别[J].农村实用技术2007，10：54.
[58]钱长英.浅谈农药可湿性粉剂的配方研制及加工工艺[J].安徽化工,2005,1：37-38.
[59]洪永聪.生防菌株TL2的菌剂研制及大田防病试验[J].中国农学通报,2009,25(17)：181-185.
[60]梁志宏,王慈敏,李金云,等.菌药合剂中杀菌剂对生防菌株E26的影响[J],中国生物防治,2008,24(增刊)：62-65.
[61]吴齐.行业标准简介[J].化工标准化与质量监督,1990,7.
[62]中华人民共和围国家标准GB/T 17980.26-2000.农药田间药效试验准则.
[63]朱昌雄,孙东园,蒋细良.我困微生物农药产业化标准及产业化对策建议[J].现代化工,2004,24(3)：6-10.
[64]沈萍.微生物学[M].北京：高等教育出版社,2000.
[65]孔建,王文夕.枯草芽孢杆菌B2903菌株的研究[J].中国生物防治,1999,15(4)：157-161.
[66]戈住,海恩斯,帕格(著),蔡妙英,戈立克(译).芽孢杆菌属[M].北京：农业出版社,1983,41-45.
[67]Monica LE, Elizabeth A D J, William E B J, et al. Viabilityand stability of biological control agents on cotton and snap bean seeds [J].Pest Management Science,2001,57(8):695-706.
[68]Chitarra G S, Breeuwer P, Nout M J R, et al. An antifungal compound produced by Bacillus subtils YM10-20 ihinbits germination of pencillium roqueforti conidiospoers [J].Applied Microbiology, 2003,94:159-166.
[69]Grosch R, Junge H. Use of Bacillus subtilis as a biocontrol agellt III Influence of Bacillus subtilis on fungal root diseases and on yield in soilless culture [J]. Zeilschrift fuer Pflanzenkrankrankheiten and Pflanzenschutz,1999,106(6):568-580.
[70]The Bacillus Subtilis Story[EB/OL].2005/06/12.http://www. upwardquest.com/web-site-positioning.html
[71]Gutter Y, Littauer F. Antagonistic action of Bacillus subtilis against citrus fruit pathogens [J].Bull Res.Counc.Israel.1953,3:192-197.
[72]Bhatt D D, Vaughan E K. Preliminary investigations on biological control of grey mold(Botrytis cinerea)of strawberries [J].Plant Dis.Rep, 1962,46:342-345.
[73]Mari M, Guizzardi, M and Pratella P C. Biological control of grey mold in pears by antiagonistic bacteria [J].Biological Control,1996,7:30-37.
[74]Office of Pesticide Program:biopesticide[DB].2001,http://www.epa. gov/pesticides/biopesticides.
[75]Paulitz T C,Belanger R R. Biological control in greenhouse systems [J].Annu Rev.Phytopathol,2001,39:103-133.
[76]Hirotsugu Yoshiya Asano Onishi Kentaro T. Flagellin as a biomarker for Bacillus subtilis strains:application to the DB9011 strain and the study of interspecific diversity in amino-acid sequences [J].Bioscience, Biotechnology, and Biochemistry,2001,65:p.1218-1222.
[77]Baker K F. Evolving concepts of biological control of plant pathogens [J].Ann Rev Phytopathol.1987,25:67-85.
[78]Asaka O, Shoda M. Biocontrol of Rhizoctonia solani damping-off of tomato with Bacillus subtilis RBI4 [J].Appl. Environ. Microbiol.1996, 62(11):4081-4085.
[79]Cavaglieri L, Orlando J, Rodriguez M I et al. Biocontrol of Bacillus subtilis against Fusarium verticillioides in vitro and at the maize root level [J].Research in Microbiology.2005,156:748-754.
[80]陈志谊,陆凡,刘永锋.防治水稻病害微生物农药纹曲宁的研制及产业化[J].江苏农业学报,2003,19(2)：108.
[81]张学君,凌宏通,李洪连,生物农药麦丰宁B-3对小麦纹枯病菌的抑制作用[J].植物病理学报,1994,04：23.
[82]方丽萍.农用生物杀虫剂.[J/OL].2001/10/07(20072)http：// enews.url.com.tw/enews/8384
[83]李军.新型微生物农药-百抗[J].农业知识,2009,16：52.
[84]洪伟,吴承祯,闰淑君.对种群增长模型的改进[J].应用与环境生物学报.2004,10(1)：23-26.
[85]朱昌雄,蒋细良,姬军红,等.我国生物农药的研究进展及对未来发展建议[J].现代化工,2003,23(7)：1-4.
[86]刘波微,彭化贤,曹玉桃,等.稻瘟病拮抗菌xi-55菌株鉴定及其发酵条件[J]中国生物防治,2006,22(增刊)：124-128.
[87]梅汝鸿.增产菌[J].技术与市场,1997,7(1)：15-16.
[88]崔云龙.B-921菌株及其发酵液对苹果腐烂病菌作用的试验研究[J].生物防治通报,1992,8(1)：26-28.
[89]朱天辉,罗孟军,杨佐忠.枯草芽孢杆菌对金花梨果腐病控制的研究[J].四川林业科技,2000,21(3)：14-17.
[90]杨佐忠.菌株筛选及其抗菌活性测定[J].森林病害通讯,1990,2：13-14.
[91]齐冬梅,惠明,梁启梅.等.芽孢杆菌H110对苹果采后梨青霉病和黑斑病的抑制效果[J].应用与环境生物学报,2005,11(2)：171-174.
[92]靳爱仙,周国英,李河.油茶炭疽病的研究现状、问题与方向[J].中国森林病虫,2009,28(3)：27-31.
[93]陈守常,曾大鹏.油茶病害及其防治[M].北京：中国林业出版社，1989.13-28.
[94]何小慧,常见油茶有害生物防治技术[J].湖南林业,2009,(2)：28-29.
[95]杨光道,束庆龙,段琳,等.主要油茶品种对炭疽病的抗性研究[J].安徽农业大学学报,2004,31(4)：480-483.
[96]段琳,杨光道,束庆龙,等.油茶果皮颜色对炭疽病的抗性的影响[J].经济林研究,2005,23(2)：9-12.
[97]傅开睿.修枝防治油茶病虫试验[J].森林病虫通讯,1990,3：23-25.
[98]陈绍红,孙思,王军.14种杀菌剂对油茶炭疽病的防治研究[J].广东林业技,2007,23(2)：42-45.
[99]王军,陈绍红,黄永芳,等.水杨酸诱导油茶抗炭疽病的研究[J].林业科学研究,2006,19(5)：629-632.
[100]刘君昂,周国英,陈或,壳聚糖对油茶炭疽病菌的抑制作用[J].中南林业科技大学学报,2008,28(6)：46-50.
[101]叶锋.在抗油茶炭疽病中的作用研究[J].江西农业大学学报,2001,2(5)：160-163.
[102]曾大鹏,贺正兴,雷章甫,等.油茶炭疽病生物防治的研究[J].林业科学,1987,23(2)：144-150.
[103]周国英,卢丽俐,刘君昂,等.油茶炭疽病拈抗内生细菌的筛选[J].湖南农业大学学报(自然科学版),2008,34(6)：698-700.
[104]程洪斌,刘晓桥,陈红漫.枯草芽孢杆菌防治植物真菌病害研究进展[J].上海农业学报,2006,22(1)：109-112.
