拮抗菌Paenibacillus polymyxa Cp-S316菌株的分离鉴定与活性物质的提取纯化及性质研究
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摘要
从泰山林地土壤中分离获得一株多粘类芽孢杆菌（Paenibacillus polymyxa），该菌株对板栗干腐病菌（Diplodia maydis）、茶云纹叶枯病菌（Colletorot richam camelliae）、茶轮斑病菌（Physatospora piricola Nose.）、棉花立枯病菌（Rhizoctomia sola）玉米圆斑病菌（Bipolaris zeicola shoemaker）、辣椒疫病菌（Phyto phthora Casici Leon）、黄瓜枯萎病菌（Fusarium vasin fectum）、家蚕白僵菌（Beauveria bassiana）、苏云金芽孢杆菌（Bacillus thuringiensis var.kurstaki）、白假丝酵母（Candida albicans）和大肠杆菌（E.coli k12）等都有强烈拮抗作用。通过对该菌株进行形态观察、生理生化实验、G+Cmol%测定和16SrRNA碱基序列测定，鉴定该菌株为类芽孢杆菌属中的一株新的多粘类芽孢杆菌，定名为Paenibacillus polymyxa Cp_S316，编码其16SrRNA的基因为一新基因，已经提交Gene Bank注册，注册号为AY292989（2003年6月4日）。
对该菌株发酵条件的研究表明：该菌株用马铃薯葡萄糖液体培养基发酵培养， 28℃，200rpm摇瓶中振荡培养72h可获得高活性的发酵产物，用苏云金芽孢杆菌HD-1做指示菌，将发酵液稀释40倍生测仍可形成明显的抑菌圈。以8000rpm对发酵液离心收集菌体。用蒸馏水对菌体稀释；加入适量吸附树脂在150rpm、28℃下振荡吸附4h， 80%的丙酮解吸，过滤解吸液得到活性物质的澄清溶液，旋转蒸发仪旋转蒸发去处丙酮，经SephadexG-75分子筛层析得单一活性峰 ，收集峰值部分样品液经冷冻干燥得到淡褐色粉末，该活性物质用丙酮充分洗涤、甲醇-乙醚重结晶获得略带微黄的白色粉末，该活性物质50μg/ml仍可对苏云金芽孢杆菌HD-1产生明显的抑制作用。
该菌株产生的活性物质耐高温，121℃加热25min活性基本不丧失；对丙酮、乙醚、氯仿等有机溶剂不敏感。在酸性条件下，活性基本不变，但在强碱条件下没有活性（调回PH值可恢复活性）。
A bacterium named Paneibacillus polymyxa is screened from soil sample of Mountain Tai. It secrets a large amounts of active substance which can inhibit against several plant and animal diseases，such as Candiada albicans 、Aspergillus flavus Link and Nomuraea rileyi Farlow .The determination show that it is a strain of paenibacillus polymyxa through measuring its G+Cmol% and the sequences of bases of 16SrRNA. The cultural conditions such as temperature, fermentation period and the compound of medium are studied. The result of test show that suitable factors for both bacterium to grow and active substance to produce are 28℃,200rpm and 72 hours. The bacterium is gotten through centrifuge with 8000rpm for 20min.Then the bacteria is diluted and colophony named S-8 is put into and used to absorbed active substance for 4 hours. The active substance is unshiped from colophony through acet. After the acet is vaporized, The active substance in water is gotten. And which is vaporized at low temperature .Then the crude active substance is purified by column chromatography on sephadex G-75.After a series of purifications again ,we could get some white powder at last .Though the active substance is diluted to50μg/ml, The activity is still checkeded-up through Phyto phtnora casicileon.The purified active substance is insensitive to heat, resistant to chloroform、ethanol and the orhers.In addition, the active substance is sensitive to high pH(10~14),but it is not sensitive to low pH(1~5).Furthermre, when the pH is made to low again , The activity of it's comes back.
对该菌株发酵条件的研究表明：该菌株用马铃薯葡萄糖液体培养基发酵培养， 28℃，200rpm摇瓶中振荡培养72h可获得高活性的发酵产物，用苏云金芽孢杆菌HD-1做指示菌，将发酵液稀释40倍生测仍可形成明显的抑菌圈。以8000rpm对发酵液离心收集菌体。用蒸馏水对菌体稀释；加入适量吸附树脂在150rpm、28℃下振荡吸附4h， 80%的丙酮解吸，过滤解吸液得到活性物质的澄清溶液，旋转蒸发仪旋转蒸发去处丙酮，经SephadexG-75分子筛层析得单一活性峰 ，收集峰值部分样品液经冷冻干燥得到淡褐色粉末，该活性物质用丙酮充分洗涤、甲醇-乙醚重结晶获得略带微黄的白色粉末，该活性物质50μg/ml仍可对苏云金芽孢杆菌HD-1产生明显的抑制作用。
该菌株产生的活性物质耐高温，121℃加热25min活性基本不丧失；对丙酮、乙醚、氯仿等有机溶剂不敏感。在酸性条件下，活性基本不变，但在强碱条件下没有活性（调回PH值可恢复活性）。
A bacterium named Paneibacillus polymyxa is screened from soil sample of Mountain Tai. It secrets a large amounts of active substance which can inhibit against several plant and animal diseases，such as Candiada albicans 、Aspergillus flavus Link and Nomuraea rileyi Farlow .The determination show that it is a strain of paenibacillus polymyxa through measuring its G+Cmol% and the sequences of bases of 16SrRNA. The cultural conditions such as temperature, fermentation period and the compound of medium are studied. The result of test show that suitable factors for both bacterium to grow and active substance to produce are 28℃,200rpm and 72 hours. The bacterium is gotten through centrifuge with 8000rpm for 20min.Then the bacteria is diluted and colophony named S-8 is put into and used to absorbed active substance for 4 hours. The active substance is unshiped from colophony through acet. After the acet is vaporized, The active substance in water is gotten. And which is vaporized at low temperature .Then the crude active substance is purified by column chromatography on sephadex G-75.After a series of purifications again ,we could get some white powder at last .Though the active substance is diluted to50μg/ml, The activity is still checkeded-up through Phyto phtnora casicileon.The purified active substance is insensitive to heat, resistant to chloroform、ethanol and the orhers.In addition, the active substance is sensitive to high pH(10~14),but it is not sensitive to low pH(1~5).Furthermre, when the pH is made to low again , The activity of it's comes back.
引文
[1] 陈梅婷等.微生态制剂-活菌制剂现状[J].中国微生态学杂志，1992，4（1）：36~40
[2] 陈延诈.口服非致病性活菌制剂概述 [J] .中国微生态学杂志.，1993，5（3）：51~59
[3] 陈天寿.微生物培养基的制造与应用.中国农业出版社，1995
[4] 陈延熙，陈壁、潘贞德.增产菌作用机理研究进展[J] .生物防治通报，1985，1（2：）22~24
[5] 陈志谊等.拮抗细菌分泌拮抗素JK-91-b对水稻纹枯病抗生活性试验[J].中国水稻科学，1994，8（4）：250~252
[6] 东秀珠，蔡妙英.常见细菌系统鉴定手册.科学出版社.，2001
[7] 黄健斌，周蓓云等.紫云英根瘤胞外多糖合成基因exol的序列分析[J].生物化学生物物理学报，1997，9（5）：483~488
[8] 胡剑等.拮抗菌BS-98分泌抗菌蛋白的条件及其发酵液的特性[J].微生物学通报，1996，23（6）：445~449
[9] 胡学智等.高温α-淀粉酶生产菌种选育的研究[J].微生物学报，1991，31（4）：267~273
[10] J.萨姆布鲁克等著，金冬雁译.分子克隆实验指南 ，第二版.北京，科学出版社，1992.
[11] 李红叶，黎军英等，脱乙酰壳多糖对桃软腐病、褐腐病菌的抑制和采后软腐病的防治研究[J].浙江农业学报.1997，9（2）：87~92
[12] 李洪连.根际微生物多样性与棉花品种对黄萎病关系的研究[J].植物病理学报，1999，29（3）：242~246
[13] 刘开启.苹果树腐烂病浸染来源的研究[J].山东农业大学学报，1996，27（3）：281~283
[14] 黎起秦、林纬.芽孢杆菌对水稻纹枯病的防治效果[J].中国生物防治，2000，16（4）：160~162
[15] 刘建国.新型抗菌多肽APS的抗真菌研究[J]. 中国生物防治 ，1999，15（3）：108~110
[16] 李少霞等.细菌半乳葡聚糖的研究[J]. 食品发酵工业，2000，（3）：56~60
[17] 刘进元，刘玮等.拮抗菌A014的筛选及其分泌抗菌蛋白的条件[J].植物病理学报，1991，33（2）：157~161
[18] 刘训理等.农用抗生素S-921有效组分的分离纯化及部分理化性质研究[J]. 山东农业大学学报，1994，4 26（2）：160~162
[19] 刘颖等.天麻中一种凝集素类似蛋白对植物病原真菌的抑制作用[J]. 微生物学报，
1999，39（5）：441~445
[20] 彭于发等 ，重组农业微生物的安全性评价.生物安全与防止污染（朱守一主编），北京：化学工业出版社，1990，54~64
[21] 彭于发.荧光假单胞菌Tn5诱变防病菌增产研究初报[J] .中国农业科学， 1990，23（1）：88~90
[22] 裴炎等.抗真菌多肽APS-1的分离纯化与特性 微生物学报[J].1999，15（3），114~117
[23] 齐涵等.抑菌生的研究总结报告[R].中国微生态学杂志，1991，3（4）：1~11
[24] 丘建华、白峰等.多粘菌素研究现状及应用[J]. 福建畜牧兽医学报，2002，（2）：p2
[25] 邱秀宝等.嗜碱性芽孢杆菌碱性蛋白酶的研究Щ.酶的性质及应用[J].微生物学报，1990，30（6）445~449
[26] 焦瑞身 .今日的微生物学， 复旦大学出版社 ，1987
[27] 荣建燕，于智勇.真菌多糖的研究利用[J].扬州教育学院报，2000，（3）301~304
[28] 宋大新、范长胜、徐德强.微生物学实验技术教程[Z].上海复旦大学出版社，1993：254
[29] 孙明、喻子牛.苏云金芽孢杆菌华中亚种CT-43菌株伴胞晶体蛋白的特征.微生物学报[J].1996，81（4）：303~306
[30] 涂玉琴等.生物农药的研究和应用进展[J]. 江西植保，1998，（4）：32~35
[31] 童蕴慧等.灰葡萄孢拮抗细菌的筛选[J]. 中国生物防治，1999 ，16（3）：123~126
[32] 王雅平等.枯草芽孢杆菌A014菌株防治小麦赤霉病的初步研究[J]..生物防治通报，1992，82（2）：54~57
[33] 王金生，何晨阳 . 转座子Tn诱变水稻白叶枯病菌毒性基因突变体及其生化行为.[J]. 植物病理学报 1992 22（3）：217~222
[34] 王金生、张学君、钱显明.小麦纹枯病生防菌株的筛选及小区实验[J].生物防治通报，1993，9（3）：102~105
[35] 王文仲.应用微生物学-现代生物技术.中国医药科技出版社[R].，1996
[36] 许光辉、郑洪远 .土壤微生物分析方法手册.农业出版社 ，1986
[37] 许其放，黄秀梨.八株芽孢杆菌菌株的分类及固氮活性的测定[J]..微生物学通报，1998，25（5）：253~258
[38] 徐丽华、姜成林. 微生物资源学 .科学出版社 ，1987
[39] 徐同.喀麦隆木霉种对芋艿根腐病菌的拮抗作用[J]. 植物病理学报， 1997，27（3）：
256
[40] 徐同.哈茨木霉c184对喀麦隆香蕉及大蕉根部病原菌的体外拮抗作用[J]. 浙江大学学报， 2002，28（4）：407~410
[41] 袁虹霞等.利用土壤拮抗微生物防治棉花枯萎病[J].中国生物防治，1998，14（4）：156~158
[42] 周德庆.微生物学试验手册.上海.上海科学技术出版社，1996
[43] 周和毅.微生物杀虫剂怎样面对机遇和挑战[J] .化工管理 ，2001，（2）：14~16
[44] 张龙翔 张庭芳 李令媛. 生化实验方法和技术. 第二版，北京，高等教育出版社，1997.
[45] 占新华等.微生物制剂促进植物生长机理的研究进展[J].植物营养与肥料学报，1999，5（2）：97~105
[46] 曾林、任改新.苏云金芽孢杆菌杀虫晶体蛋白Cry基因研究的现状[J].微生物学通报，1998，25（1）：49~51
[47] 周瑞霞等.乳酸菌胞外多糖的研究进展[J].中国乳品工业，1999，（2）：84~89
[48] Arase,-S;Miyake,-K;Honda,-Y;Nozu.M.Production of anti-fungal substances in the "Sekiguchi lesion" induced by Pyricularria oryzae infection on rice Oryza sativa]cv. Sekiguchi-asahi. [J]. Bullentin-of -the-Faculty-of Agriculture-Shimane1，993,27p7~14
[49] AhimouF,.Jacques and Deleu..surfactin and iturin A effects on Bacillus subtilis surface hydrophobicity[J]. Microbiol Technol, 2000，27:749~754
[50] Benkerroum N, Sandine W.E.Inhibitory action of nisin against Listeria monocy to genes[J].Dairy Sci, 1988;77:3237~3251
[51] Baker CJ,Starvely JR and Thomas CA et al..Inhibitory effect of Bacillus on Uromyces phaseoli and on development of rust pustules on bean lesves[J].Phytopathology
1983，73:1148~1152
[52] Bell DK,Wells HD and Markham CR..IN vitro antagonism of Trichoderma species against six fungal plant pathogens. Phytopathology[J],.1982，60:1058~1061
[53] Blakeman JP, and Fokkema NJ. potential for biological control of plant disease on the phyllosphere.Ann.Rev[J]. Phytopathol. 1982，20:167~192
[54] Broadbent P,Baker KF and Franks and N et al.. Effect of Bacillus spp.on increased growth of seeding in steamed and otreated soil.Phytopathol. 1997，67: 1027~1034
[55] Buyer JS. and Leong J..Iron transport-mediated antagonism between plant growth-promoting and plant-deleterious seudomonas P strains [J].Biol.ogy，1986，261:791~794
[56] Buysens S, Heungens. K, and Poppe . Involvement of pyochelin and pyoverdin in suppression of Pythium-induced damping-doff tomato by Pseudomonans aeruginosa TNSK2. Environ[J].Microbiol. 1996，62:865~871
[57] Chatterjee A,Valasubramanian R,Ma WL et al.. Isolation of Pseudomonas strain pf7-14 altered in antibiotic production,cloning of ant DNA and evaluation of the role of antibiotic production in cintrol of blast and sheath blight of rice[J].Biological Control, 1996，7:185~195.
[58] Chet I, and Baker,R..Isolation and biocontrol potential of Trichoderma hamatum from soil naturally suppressive to Rhizotonia solani[J],.Phytonathology. 1981，71:286~290
[59] Chet I, Trichoderma-Application. Mode of action,and potential as a biocontrol agent of soilberne plant pathogenic fungi.In:Inovative approach to plant disease control (ed:Chet I).[J].New York, John wiley and sens，1987：137~156
[60] Chen W, Echandi E and Spur HW..Protection of tobacoo plants from bacterial wilt with avirulent bacteriocin-producing strains of Pseudomons solanacearum[J].Pro.Fifth intCont Plant Bact.Cali，1981：484~492
[61] Cook RJ,Weller DM. Mangement of take-in consecutive crops of wheat or barely.innovative Approaches to Plant Disease Control[J].New York:Wiley. 1987： 41~76
[62] Cook RJ. Making greater use of introduced microorganisms for biological control of plant Pathogens.Sn.Rev[J]. Phytopathol. 1993，31:53~80
[63] Cook RJ,Thomashoe LS, and weller DM et al..Molc\ecular Mechanisma for biological control of plant pathogens[J]. Phytopatholgy1995，31:53~80
[64] Dennis C and Webster.. Aatagonistic properties of species groups of Trichoderma.Trans.Br[J].Mycol.Soc.，1971..57:363~69
[65] Dewit PJGM..Molecular characterization of gene -for-gene systems in plant pathogen.Ann.Rev[J].Phytopathol. 1992，30:308~313
[66] Emmert EAB and Handelsman J..Biocontrol of plant disease :a(gram) positive perpective[J.].FEMS Microbiology letters，1999，171:1~9
[67] Elad Y, and Baker R..The role of competition for iron and carbon in suppression of chlamydospoore germination of Fusarium spp.by Pseudomonas [J]..Phytopathology. 1985，75:1053~59
[68] Elad Y..Biological control of folier pathogens by meanings of Trichoderma hazianum and potential modes of action[J]. Crop Protection 2000，（19）:709~714
[69] Feng T-Yetal. Bccillus thuringiensis biotechnology and Environmental benefits VolI.Taipei[J] :Hua Shiang Yuan Publishing Co, 1995，1:11~17
[70] Ferreria, J.H.,S,F.N.Matthee and A.C. Thomas. Biological control of Eutypa lata on grapevine by an antagoinsistic strain on Bscillus subtillis[J]. Phythopathology. 1991,81
(3):283~87
[71] Ford W.W.Lawrence,J.S. and Laubach, C.A. Studies on aerobic spore-bearing nonpathogenic Bacteria[J]. Journal of Bacteriology, 1996,1:273~316
[72] Gilbson.I. and Topping, L.E.Further studies of the aerobic spore-forming Bactilli.proceedings[J].Society of Agricultural Bacterioldists..1983,43~44
[73] Gillaman H. Brave a new world of pest control: first commercial use of a man made organism. Geres [J].The FAO Review,1992,24(1):3~5
[74] Handelsman J,Raffel S, and Master EH et al.. Biocontrol of damping -off of alfalfa seedings with Bacillus cereus UW85[J]. Appl. Environ. Microbiol. 1990，56:713~18
[75] Handelsman J, and Stabb EV. Biocontrol of soilborne plang pathogens[J]. The plant cell, 1996，18:1855~69
[76] Hardar Y, Harman GE and Taylor AG . Evaluation of Trichoderma Koningii and T.hariaznum from New York soil for biological control of seed rot caused by Pythium spp[J]. Phytopathology. 1984，74:106~110
[77] Frrand SK, Wang CL and Hong SB et al.. Deletion derivatives of pAk84 and their use in the analysis of Agrobacterium plasmid functions[J]. Plasmid, 1992，28(3):201~212
[78] Howell CR, and Stipanovic Rd.1Gliovirin, a new antibiotic from Ggliccladium virens, and its role in the biological control of Pythium ultimum can [J]. Microbiolo, 1983.，29:321~24
[79] Howell. CR, Stipanovic RD and Lumsden RD.. Antibiotic produced by strains of Gliocladium virens and its relation to the biocontrol of cotton seeding disease[J].Biocongtrol Sci.Technol, 1993，3:435~42
[80] Howie wj and Suslow TV. Role of antibiotic biosynthesis in the Pseudomonas fluorescens[J]. Mol.Plant-microbe Interact. 1991，4:393~99
[81] J.Lacadena,.A.Martimez delpozo[R],.Arch-biochem-biophys. 1995, 1324(2): 268~73
[82] Kerr A . Biological control of crown gall through production of agrocin84[J]. Plant Disease1980，64:25~30
[83] Kerr A and Tate ME. Agrocins and the biological control of crown gall [J.]Microbiol Sci. 1984，1:1~4
[84] Koby S., Schickler H, Chet I et al. The chitinase encoding Tn 7 based chiA gene endows Pseudomonas fluorescens with the capacity to control plant pathogens in soil.[J]. Gene ，1994,147:81~83
[85] koch . E..Evalution of commercial products for microbial control of soil-brone plant disease[J] . Crop Protection, 1999，18:119~25
[86] Lechevalier,N.A. and Solotorovsky, M.Three centuries of microbiology[R].NEW York:Mc Graw-Hill Book Co.1965:1~24
[87] Leong,J.. Siderophores; their biochemistry and possible role in the biocontrol of plant pathogens. Ann. Rev[J]. Phytopathol. 1986，24:187~209
[88] Levy et al. Suppression of Septoria tritici and puccinia recondite of wheat by an antibiotic-procucing fluorescent pseudomonad[J]. Plant Pathol, 1989，38:564~70
[89] Mekeen CD. Reiliy CC and Pusey PL.. Production and partial characterization of antifungal substances antagonistic to Monlinia fructicola from Bacillus subtilics,[J].Phytopathology 1986，76:136~39
[90] Mew T W. Current status and future prospects of resesch on bacteria blight of rice.Annu Rev[J]. phytopathol,1987,25:359~382
[91] Mhammedi A,Peypoux F,Besson Fet al.[J]. Antibiotics,1982,35(3):306~11
[92] Nelson EB. Exudate Molecules initiating fungal response to seed and roots.[J]. Plant and soil. 1990，129:61~73
[93] Oakley,B.R.Kirsch, D.P. an Morris,N.RAsimplifiedal transensitive silver stain for
detectinf proteins in polyacrylam ide gel[J] Anal. Biochem, 1980，105:361
[94] Ortlepp S A,Ollington JF,Mc Connell D J.Molecular cloning in Bacillus subtilis of a Bacillus lichenifoemis gene encoding a thomastable alpha amylase[J]. Gene,1983,23:267~276
[95] Pieterse CM, Van Wees SG; Hoffland E, Van Pelt. JA, an Loon LC..systemic resistance in Arabidopsis induced by biocontrol bacteria is independent of salicylic acid accumnation and pathogenesis-related gene expression[J]. Plant Cell. 1996，8(8)1225~1237
[96] R.E..Gordon.In.The Aerobie Endospore-forming Bacteria: Classification and Indenfitication (Eds.R.cC.W.Berkeley and M.G.ood fellow). London: Academic Press Inc. 1981,1~16
[97] Rolert E,Hancock W, and Chapple DS. Peptide antibiotics. Antimicrob. Agents chemother. 1999，43:1317~1323
[98] Ryosuk Iilima. Shoichirokurata and Sunji Natori[J]., The Journal of Biological Chemistry, 1993,268(16):12055~12061
[99] Sivan A, and Chet I..The possible role of competition between Trichoderma hazianum and Fusarium oxysporium on rhizospere colonization[J]. Phytology. 1989，79:1998~2003
[100] Slepecky, R.A, In:spores IV(Eds.P. Gerhardt, H..L. Sand off and R.N.Costilow).Washington:Am erican Society for Microbioligy, 1975,297~313
[101] Smith, N.R,Gordon, R.e and Claik F.E.Aerobic Mesophilic Sporeforming BECTERIA. Washington.D.C: United States Despartment of Agr.1996.1~359
[102] Stackebandt, E. and W.Liesak. In:Hand book of New Bcterial systematics (Eds.Good fellow,M and A.G.O. Donnell)London:Press Ltd. 1993,63~78
[103] Tang K, Sun, X and Hu et al.. Transgenic rice plants expressing the ferredox-like protein(AP1) from the sweat pepper show enhanced resistance to xanthomonas orgzae[J], Plant science 2001，160:1035~43
[104] Turner and Backman PA ..Factors relating to peanut yield increase after seed treat with Bacillus subtilis [J].Plant Disease. 1991，75:347~53
[105] World Health Organization.TDR/BCV/sphaericus.[R]. World Health organization
Geneva.1985,.3
[106] Vanittanakon N,Loeffler W.J Antobiotics[R]. 1986,39(7):888~901
[107] Yapici,-B.M;Karaboz,-I.The effect of two anti-fungal compounds on the growth of molds that frequently appear on tanned leather[J]. The -Jounal of the America-Leather-C hemists-Association (USA) (Feb-Mar 1997).v92(2) p:38~45
[108] Wilson C.L Wisniiewski M.E.. Biological control of postharrest disease of fruits and regetables:an emerging technology.annu Rev[J] .Phytopathol. 1989，27425~41
[2] 陈延诈.口服非致病性活菌制剂概述 [J] .中国微生态学杂志.，1993，5（3）：51~59
[3] 陈天寿.微生物培养基的制造与应用.中国农业出版社，1995
[4] 陈延熙，陈壁、潘贞德.增产菌作用机理研究进展[J] .生物防治通报，1985，1（2：）22~24
[5] 陈志谊等.拮抗细菌分泌拮抗素JK-91-b对水稻纹枯病抗生活性试验[J].中国水稻科学，1994，8（4）：250~252
[6] 东秀珠，蔡妙英.常见细菌系统鉴定手册.科学出版社.，2001
[7] 黄健斌，周蓓云等.紫云英根瘤胞外多糖合成基因exol的序列分析[J].生物化学生物物理学报，1997，9（5）：483~488
[8] 胡剑等.拮抗菌BS-98分泌抗菌蛋白的条件及其发酵液的特性[J].微生物学通报，1996，23（6）：445~449
[9] 胡学智等.高温α-淀粉酶生产菌种选育的研究[J].微生物学报，1991，31（4）：267~273
[10] J.萨姆布鲁克等著，金冬雁译.分子克隆实验指南 ，第二版.北京，科学出版社，1992.
[11] 李红叶，黎军英等，脱乙酰壳多糖对桃软腐病、褐腐病菌的抑制和采后软腐病的防治研究[J].浙江农业学报.1997，9（2）：87~92
[12] 李洪连.根际微生物多样性与棉花品种对黄萎病关系的研究[J].植物病理学报，1999，29（3）：242~246
[13] 刘开启.苹果树腐烂病浸染来源的研究[J].山东农业大学学报，1996，27（3）：281~283
[14] 黎起秦、林纬.芽孢杆菌对水稻纹枯病的防治效果[J].中国生物防治，2000，16（4）：160~162
[15] 刘建国.新型抗菌多肽APS的抗真菌研究[J]. 中国生物防治 ，1999，15（3）：108~110
[16] 李少霞等.细菌半乳葡聚糖的研究[J]. 食品发酵工业，2000，（3）：56~60
[17] 刘进元，刘玮等.拮抗菌A014的筛选及其分泌抗菌蛋白的条件[J].植物病理学报，1991，33（2）：157~161
[18] 刘训理等.农用抗生素S-921有效组分的分离纯化及部分理化性质研究[J]. 山东农业大学学报，1994，4 26（2）：160~162
[19] 刘颖等.天麻中一种凝集素类似蛋白对植物病原真菌的抑制作用[J]. 微生物学报，
1999，39（5）：441~445
[20] 彭于发等 ，重组农业微生物的安全性评价.生物安全与防止污染（朱守一主编），北京：化学工业出版社，1990，54~64
[21] 彭于发.荧光假单胞菌Tn5诱变防病菌增产研究初报[J] .中国农业科学， 1990，23（1）：88~90
[22] 裴炎等.抗真菌多肽APS-1的分离纯化与特性 微生物学报[J].1999，15（3），114~117
[23] 齐涵等.抑菌生的研究总结报告[R].中国微生态学杂志，1991，3（4）：1~11
[24] 丘建华、白峰等.多粘菌素研究现状及应用[J]. 福建畜牧兽医学报，2002，（2）：p2
[25] 邱秀宝等.嗜碱性芽孢杆菌碱性蛋白酶的研究Щ.酶的性质及应用[J].微生物学报，1990，30（6）445~449
[26] 焦瑞身 .今日的微生物学， 复旦大学出版社 ，1987
[27] 荣建燕，于智勇.真菌多糖的研究利用[J].扬州教育学院报，2000，（3）301~304
[28] 宋大新、范长胜、徐德强.微生物学实验技术教程[Z].上海复旦大学出版社，1993：254
[29] 孙明、喻子牛.苏云金芽孢杆菌华中亚种CT-43菌株伴胞晶体蛋白的特征.微生物学报[J].1996，81（4）：303~306
[30] 涂玉琴等.生物农药的研究和应用进展[J]. 江西植保，1998，（4）：32~35
[31] 童蕴慧等.灰葡萄孢拮抗细菌的筛选[J]. 中国生物防治，1999 ，16（3）：123~126
[32] 王雅平等.枯草芽孢杆菌A014菌株防治小麦赤霉病的初步研究[J]..生物防治通报，1992，82（2）：54~57
[33] 王金生，何晨阳 . 转座子Tn诱变水稻白叶枯病菌毒性基因突变体及其生化行为.[J]. 植物病理学报 1992 22（3）：217~222
[34] 王金生、张学君、钱显明.小麦纹枯病生防菌株的筛选及小区实验[J].生物防治通报，1993，9（3）：102~105
[35] 王文仲.应用微生物学-现代生物技术.中国医药科技出版社[R].，1996
[36] 许光辉、郑洪远 .土壤微生物分析方法手册.农业出版社 ，1986
[37] 许其放，黄秀梨.八株芽孢杆菌菌株的分类及固氮活性的测定[J]..微生物学通报，1998，25（5）：253~258
[38] 徐丽华、姜成林. 微生物资源学 .科学出版社 ，1987
[39] 徐同.喀麦隆木霉种对芋艿根腐病菌的拮抗作用[J]. 植物病理学报， 1997，27（3）：
256
[40] 徐同.哈茨木霉c184对喀麦隆香蕉及大蕉根部病原菌的体外拮抗作用[J]. 浙江大学学报， 2002，28（4）：407~410
[41] 袁虹霞等.利用土壤拮抗微生物防治棉花枯萎病[J].中国生物防治，1998，14（4）：156~158
[42] 周德庆.微生物学试验手册.上海.上海科学技术出版社，1996
[43] 周和毅.微生物杀虫剂怎样面对机遇和挑战[J] .化工管理 ，2001，（2）：14~16
[44] 张龙翔 张庭芳 李令媛. 生化实验方法和技术. 第二版，北京，高等教育出版社，1997.
[45] 占新华等.微生物制剂促进植物生长机理的研究进展[J].植物营养与肥料学报，1999，5（2）：97~105
[46] 曾林、任改新.苏云金芽孢杆菌杀虫晶体蛋白Cry基因研究的现状[J].微生物学通报，1998，25（1）：49~51
[47] 周瑞霞等.乳酸菌胞外多糖的研究进展[J].中国乳品工业，1999，（2）：84~89
[48] Arase,-S;Miyake,-K;Honda,-Y;Nozu.M.Production of anti-fungal substances in the "Sekiguchi lesion" induced by Pyricularria oryzae infection on rice Oryza sativa]cv. Sekiguchi-asahi. [J]. Bullentin-of -the-Faculty-of Agriculture-Shimane1，993,27p7~14
[49] AhimouF,.Jacques and Deleu..surfactin and iturin A effects on Bacillus subtilis surface hydrophobicity[J]. Microbiol Technol, 2000，27:749~754
[50] Benkerroum N, Sandine W.E.Inhibitory action of nisin against Listeria monocy to genes[J].Dairy Sci, 1988;77:3237~3251
[51] Baker CJ,Starvely JR and Thomas CA et al..Inhibitory effect of Bacillus on Uromyces phaseoli and on development of rust pustules on bean lesves[J].Phytopathology
1983，73:1148~1152
[52] Bell DK,Wells HD and Markham CR..IN vitro antagonism of Trichoderma species against six fungal plant pathogens. Phytopathology[J],.1982，60:1058~1061
[53] Blakeman JP, and Fokkema NJ. potential for biological control of plant disease on the phyllosphere.Ann.Rev[J]. Phytopathol. 1982，20:167~192
[54] Broadbent P,Baker KF and Franks and N et al.. Effect of Bacillus spp.on increased growth of seeding in steamed and otreated soil.Phytopathol. 1997，67: 1027~1034
[55] Buyer JS. and Leong J..Iron transport-mediated antagonism between plant growth-promoting and plant-deleterious seudomonas P strains [J].Biol.ogy，1986，261:791~794
[56] Buysens S, Heungens. K, and Poppe . Involvement of pyochelin and pyoverdin in suppression of Pythium-induced damping-doff tomato by Pseudomonans aeruginosa TNSK2. Environ[J].Microbiol. 1996，62:865~871
[57] Chatterjee A,Valasubramanian R,Ma WL et al.. Isolation of Pseudomonas strain pf7-14 altered in antibiotic production,cloning of ant DNA and evaluation of the role of antibiotic production in cintrol of blast and sheath blight of rice[J].Biological Control, 1996，7:185~195.
[58] Chet I, and Baker,R..Isolation and biocontrol potential of Trichoderma hamatum from soil naturally suppressive to Rhizotonia solani[J],.Phytonathology. 1981，71:286~290
[59] Chet I, Trichoderma-Application. Mode of action,and potential as a biocontrol agent of soilberne plant pathogenic fungi.In:Inovative approach to plant disease control (ed:Chet I).[J].New York, John wiley and sens，1987：137~156
[60] Chen W, Echandi E and Spur HW..Protection of tobacoo plants from bacterial wilt with avirulent bacteriocin-producing strains of Pseudomons solanacearum[J].Pro.Fifth intCont Plant Bact.Cali，1981：484~492
[61] Cook RJ,Weller DM. Mangement of take-in consecutive crops of wheat or barely.innovative Approaches to Plant Disease Control[J].New York:Wiley. 1987： 41~76
[62] Cook RJ. Making greater use of introduced microorganisms for biological control of plant Pathogens.Sn.Rev[J]. Phytopathol. 1993，31:53~80
[63] Cook RJ,Thomashoe LS, and weller DM et al..Molc\ecular Mechanisma for biological control of plant pathogens[J]. Phytopatholgy1995，31:53~80
[64] Dennis C and Webster.. Aatagonistic properties of species groups of Trichoderma.Trans.Br[J].Mycol.Soc.，1971..57:363~69
[65] Dewit PJGM..Molecular characterization of gene -for-gene systems in plant pathogen.Ann.Rev[J].Phytopathol. 1992，30:308~313
[66] Emmert EAB and Handelsman J..Biocontrol of plant disease :a(gram) positive perpective[J.].FEMS Microbiology letters，1999，171:1~9
[67] Elad Y, and Baker R..The role of competition for iron and carbon in suppression of chlamydospoore germination of Fusarium spp.by Pseudomonas [J]..Phytopathology. 1985，75:1053~59
[68] Elad Y..Biological control of folier pathogens by meanings of Trichoderma hazianum and potential modes of action[J]. Crop Protection 2000，（19）:709~714
[69] Feng T-Yetal. Bccillus thuringiensis biotechnology and Environmental benefits VolI.Taipei[J] :Hua Shiang Yuan Publishing Co, 1995，1:11~17
[70] Ferreria, J.H.,S,F.N.Matthee and A.C. Thomas. Biological control of Eutypa lata on grapevine by an antagoinsistic strain on Bscillus subtillis[J]. Phythopathology. 1991,81
(3):283~87
[71] Ford W.W.Lawrence,J.S. and Laubach, C.A. Studies on aerobic spore-bearing nonpathogenic Bacteria[J]. Journal of Bacteriology, 1996,1:273~316
[72] Gilbson.I. and Topping, L.E.Further studies of the aerobic spore-forming Bactilli.proceedings[J].Society of Agricultural Bacterioldists..1983,43~44
[73] Gillaman H. Brave a new world of pest control: first commercial use of a man made organism. Geres [J].The FAO Review,1992,24(1):3~5
[74] Handelsman J,Raffel S, and Master EH et al.. Biocontrol of damping -off of alfalfa seedings with Bacillus cereus UW85[J]. Appl. Environ. Microbiol. 1990，56:713~18
[75] Handelsman J, and Stabb EV. Biocontrol of soilborne plang pathogens[J]. The plant cell, 1996，18:1855~69
[76] Hardar Y, Harman GE and Taylor AG . Evaluation of Trichoderma Koningii and T.hariaznum from New York soil for biological control of seed rot caused by Pythium spp[J]. Phytopathology. 1984，74:106~110
[77] Frrand SK, Wang CL and Hong SB et al.. Deletion derivatives of pAk84 and their use in the analysis of Agrobacterium plasmid functions[J]. Plasmid, 1992，28(3):201~212
[78] Howell CR, and Stipanovic Rd.1Gliovirin, a new antibiotic from Ggliccladium virens, and its role in the biological control of Pythium ultimum can [J]. Microbiolo, 1983.，29:321~24
[79] Howell. CR, Stipanovic RD and Lumsden RD.. Antibiotic produced by strains of Gliocladium virens and its relation to the biocontrol of cotton seeding disease[J].Biocongtrol Sci.Technol, 1993，3:435~42
[80] Howie wj and Suslow TV. Role of antibiotic biosynthesis in the Pseudomonas fluorescens[J]. Mol.Plant-microbe Interact. 1991，4:393~99
[81] J.Lacadena,.A.Martimez delpozo[R],.Arch-biochem-biophys. 1995, 1324(2): 268~73
[82] Kerr A . Biological control of crown gall through production of agrocin84[J]. Plant Disease1980，64:25~30
[83] Kerr A and Tate ME. Agrocins and the biological control of crown gall [J.]Microbiol Sci. 1984，1:1~4
[84] Koby S., Schickler H, Chet I et al. The chitinase encoding Tn 7 based chiA gene endows Pseudomonas fluorescens with the capacity to control plant pathogens in soil.[J]. Gene ，1994,147:81~83
[85] koch . E..Evalution of commercial products for microbial control of soil-brone plant disease[J] . Crop Protection, 1999，18:119~25
[86] Lechevalier,N.A. and Solotorovsky, M.Three centuries of microbiology[R].NEW York:Mc Graw-Hill Book Co.1965:1~24
[87] Leong,J.. Siderophores; their biochemistry and possible role in the biocontrol of plant pathogens. Ann. Rev[J]. Phytopathol. 1986，24:187~209
[88] Levy et al. Suppression of Septoria tritici and puccinia recondite of wheat by an antibiotic-procucing fluorescent pseudomonad[J]. Plant Pathol, 1989，38:564~70
[89] Mekeen CD. Reiliy CC and Pusey PL.. Production and partial characterization of antifungal substances antagonistic to Monlinia fructicola from Bacillus subtilics,[J].Phytopathology 1986，76:136~39
[90] Mew T W. Current status and future prospects of resesch on bacteria blight of rice.Annu Rev[J]. phytopathol,1987,25:359~382
[91] Mhammedi A,Peypoux F,Besson Fet al.[J]. Antibiotics,1982,35(3):306~11
[92] Nelson EB. Exudate Molecules initiating fungal response to seed and roots.[J]. Plant and soil. 1990，129:61~73
[93] Oakley,B.R.Kirsch, D.P. an Morris,N.RAsimplifiedal transensitive silver stain for
detectinf proteins in polyacrylam ide gel[J] Anal. Biochem, 1980，105:361
[94] Ortlepp S A,Ollington JF,Mc Connell D J.Molecular cloning in Bacillus subtilis of a Bacillus lichenifoemis gene encoding a thomastable alpha amylase[J]. Gene,1983,23:267~276
[95] Pieterse CM, Van Wees SG; Hoffland E, Van Pelt. JA, an Loon LC..systemic resistance in Arabidopsis induced by biocontrol bacteria is independent of salicylic acid accumnation and pathogenesis-related gene expression[J]. Plant Cell. 1996，8(8)1225~1237
[96] R.E..Gordon.In.The Aerobie Endospore-forming Bacteria: Classification and Indenfitication (Eds.R.cC.W.Berkeley and M.G.ood fellow). London: Academic Press Inc. 1981,1~16
[97] Rolert E,Hancock W, and Chapple DS. Peptide antibiotics. Antimicrob. Agents chemother. 1999，43:1317~1323
[98] Ryosuk Iilima. Shoichirokurata and Sunji Natori[J]., The Journal of Biological Chemistry, 1993,268(16):12055~12061
[99] Sivan A, and Chet I..The possible role of competition between Trichoderma hazianum and Fusarium oxysporium on rhizospere colonization[J]. Phytology. 1989，79:1998~2003
[100] Slepecky, R.A, In:spores IV(Eds.P. Gerhardt, H..L. Sand off and R.N.Costilow).Washington:Am erican Society for Microbioligy, 1975,297~313
[101] Smith, N.R,Gordon, R.e and Claik F.E.Aerobic Mesophilic Sporeforming BECTERIA. Washington.D.C: United States Despartment of Agr.1996.1~359
[102] Stackebandt, E. and W.Liesak. In:Hand book of New Bcterial systematics (Eds.Good fellow,M and A.G.O. Donnell)London:Press Ltd. 1993,63~78
[103] Tang K, Sun, X and Hu et al.. Transgenic rice plants expressing the ferredox-like protein(AP1) from the sweat pepper show enhanced resistance to xanthomonas orgzae[J], Plant science 2001，160:1035~43
[104] Turner and Backman PA ..Factors relating to peanut yield increase after seed treat with Bacillus subtilis [J].Plant Disease. 1991，75:347~53
[105] World Health Organization.TDR/BCV/sphaericus.[R]. World Health organization
Geneva.1985,.3
[106] Vanittanakon N,Loeffler W.J Antobiotics[R]. 1986,39(7):888~901
[107] Yapici,-B.M;Karaboz,-I.The effect of two anti-fungal compounds on the growth of molds that frequently appear on tanned leather[J]. The -Jounal of the America-Leather-C hemists-Association (USA) (Feb-Mar 1997).v92(2) p:38~45
[108] Wilson C.L Wisniiewski M.E.. Biological control of postharrest disease of fruits and regetables:an emerging technology.annu Rev[J] .Phytopathol. 1989，27425~41