枯草芽孢杆菌(Bacillus subtilis)活性物质的分离和功能测定
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摘要
林果病害是一类重要的生物灾害,生物防治作为是林果病害管理中的一种有效持续控制方法,是现代林业重大生物灾害可持续控制系统中的重要组成部分。枯草芽孢杆菌(Bacillus subtilis)是一种重要的生防内生菌。本研究从石榴果实中分离获得一株内生菌-枯草芽孢杆菌sdau08-96菌株,研究结果表明该菌对多种林果病原菌有明显的抑制作用。本研究通过对最佳发酵条件,抗菌物质的理化性质、分离纯化及功能测定,初步探讨了枯草芽孢杆菌sdau08-96菌株生防机理,为该菌的开发利用提供依据,主要结果如下:
1.产生抗菌物质的最佳培养基为LB培养基,装液量为100/250mL,初始pH值为7.0、温度28℃、接种量10%、培养时间72h和转速200r/m时菌体生长良好,产生的抗菌物质较多,抑菌活性高。
2.抗菌谱测定表明,sdau08-96菌株产生的抗菌蛋白和挥发性物质对多种病原菌都有较强的抑制作用,菌丝形态的变化相差不多,主要表现为,菌丝分支增多,扭曲,部分菌丝细胞出现断裂和原生质浓缩现象,菌丝顶端和中部细胞膨大成球形或椭圆形,部分菌丝变粗或纤细。抗菌蛋白较耐高温、耐盐、耐有机溶剂、耐酸碱性较好,挥发性物质还可抑制病原菌菌丝色素产生的速度。
3.抗菌蛋白的分离采用的方法为SDS-PAGE电泳找出胞外目的蛋白条带,切下条带测序,比对结果显示,胞外蛋白A条带与枯草芽孢杆菌膜关联的蛋白激酶的覆盖率为19%,胞外蛋白B条带与枯草芽孢杆菌脂蛋白的覆盖率为25%,可以断定A、B条带为目的蛋白条带。
4.挥发性物质的分离纯化采用顶空固相微萃取-气相色谱-质谱联用技术(HS-SPME-GC-MS)进行,经过GC-MS分析,挥发性活性物质共有21种不同组分,这些物质分属于烷烃、醇、酸、酯、酚和醛等几类物质。
5.挥发性活性物质组分的功能测定结果表明,十五烷、二苯并呋喃、邻苯二甲酸二乙酯、十四酸、2-丙烯酸十四烷酯和三聚乙醛6种物质的抑菌作用比较高,其中,二苯并呋喃和三聚乙醛的抑菌率可以高达80%;十四烷、4-羟基丁基丙烯酸酯、月桂醇、月桂酸、十六烷酸和硬脂酸6种物质的抑菌作用在20%以下或基本没有抑菌作用。
Tree and Fruit disease is a kind of important biological disasters, biological control is an important disease management method and component of sustainable control major biological disasters in modern forestry system. Bacillus subtilis is an important biocontrol bacterium. Bacillus subtilis strain of sdau08-96 is isolated from pomegranate fruit, the results indicated that it has obvious bacteriostatic action to a variety of tree and fruit disease pathogens. In this study, the best fermentation condition was determined through optimizing test,the purification and characteristics of the antifungal substances produced by Bacillus subtilis strain of sdau08-96 were studied in this article. The separation, purification and function identification of antimicrobial components were studied. The biological action mechanisms of sdau08-96 were also investigated. The results were as follows:
1. The optimum fermentation condition: LB medium, pack amount 100/250mL triangle bottle, initial pH value was 7.0 of medium, temperature was 28℃, inoculation amount was 10%, time was 72h, speed was 200r/m.
2. The antifungal substances of sdau08-96 had strong antagonistic activity on many kinds of plant pathogenic fungi. The antagonistic characteristic showed that it can obviously inhibit different pathogens growth. It can caused mycelia branch to increase and distort, partly cell of mycelia can produce fracture and protoplasm to condense, top and middle cells of mycelia can intumescentia into sphere and oval or bead-like, some top cells of mycelia obvious intumescentia to clusters, partly mycelia became intumescentia or slimmer. The substance was found to be thermostable, more resistant to salt, organic solvents, acid and alkali. The pigment production speed of mycelia was also inhibited by the volatile substances.
3. The separation method of antimicrobial protein was that identify the extracellular protein band by SDS-PAGE electrophoresis, cut it and sequence. The results show that the homology of the extracellular proteins B strip and bacillus subtilis antimicrobial protein is very high. It can be concluded that the band B protein band was the purpose protein.
4. The volatile substances was separated by head space solid-phase microextraction (HS-SPME) coupled to capillary gas chromatography-mass spectrometry(GC-MS). After GC-MS analysis, there were 21 different components in volatile substances. These substances points belong to paraffin, alcohol, acids, esters, phenolic and aldehydes and several other kind materials.
5. Function identification of volatile substances showed that the role of different components were different. For Pentadecane, Dibenzofuran, Diethyl Phthalate, Tetradecanoic acid , 2-Propenoic acid, tetradecyl ester and 2,4,6-tripropyl-,1,3,5-Trioxane, the bacteriostasis is higher. And the bacteriostatic rate of Dibenzofuran and 2,4,6-tripropyl-, 1,3,5-Trioxane can reach 80%. For Tetradecane, 4-Hydroxybutyl acrylate, Dodecyl alcohol, Dodecanoic acid, Hexadecanoic acid and Octadecanoic acid, the bacteriostasis is below 20%, or basically no bacteriostasis.
1.产生抗菌物质的最佳培养基为LB培养基,装液量为100/250mL,初始pH值为7.0、温度28℃、接种量10%、培养时间72h和转速200r/m时菌体生长良好,产生的抗菌物质较多,抑菌活性高。
2.抗菌谱测定表明,sdau08-96菌株产生的抗菌蛋白和挥发性物质对多种病原菌都有较强的抑制作用,菌丝形态的变化相差不多,主要表现为,菌丝分支增多,扭曲,部分菌丝细胞出现断裂和原生质浓缩现象,菌丝顶端和中部细胞膨大成球形或椭圆形,部分菌丝变粗或纤细。抗菌蛋白较耐高温、耐盐、耐有机溶剂、耐酸碱性较好,挥发性物质还可抑制病原菌菌丝色素产生的速度。
3.抗菌蛋白的分离采用的方法为SDS-PAGE电泳找出胞外目的蛋白条带,切下条带测序,比对结果显示,胞外蛋白A条带与枯草芽孢杆菌膜关联的蛋白激酶的覆盖率为19%,胞外蛋白B条带与枯草芽孢杆菌脂蛋白的覆盖率为25%,可以断定A、B条带为目的蛋白条带。
4.挥发性物质的分离纯化采用顶空固相微萃取-气相色谱-质谱联用技术(HS-SPME-GC-MS)进行,经过GC-MS分析,挥发性活性物质共有21种不同组分,这些物质分属于烷烃、醇、酸、酯、酚和醛等几类物质。
5.挥发性活性物质组分的功能测定结果表明,十五烷、二苯并呋喃、邻苯二甲酸二乙酯、十四酸、2-丙烯酸十四烷酯和三聚乙醛6种物质的抑菌作用比较高,其中,二苯并呋喃和三聚乙醛的抑菌率可以高达80%;十四烷、4-羟基丁基丙烯酸酯、月桂醇、月桂酸、十六烷酸和硬脂酸6种物质的抑菌作用在20%以下或基本没有抑菌作用。
Tree and Fruit disease is a kind of important biological disasters, biological control is an important disease management method and component of sustainable control major biological disasters in modern forestry system. Bacillus subtilis is an important biocontrol bacterium. Bacillus subtilis strain of sdau08-96 is isolated from pomegranate fruit, the results indicated that it has obvious bacteriostatic action to a variety of tree and fruit disease pathogens. In this study, the best fermentation condition was determined through optimizing test,the purification and characteristics of the antifungal substances produced by Bacillus subtilis strain of sdau08-96 were studied in this article. The separation, purification and function identification of antimicrobial components were studied. The biological action mechanisms of sdau08-96 were also investigated. The results were as follows:
1. The optimum fermentation condition: LB medium, pack amount 100/250mL triangle bottle, initial pH value was 7.0 of medium, temperature was 28℃, inoculation amount was 10%, time was 72h, speed was 200r/m.
2. The antifungal substances of sdau08-96 had strong antagonistic activity on many kinds of plant pathogenic fungi. The antagonistic characteristic showed that it can obviously inhibit different pathogens growth. It can caused mycelia branch to increase and distort, partly cell of mycelia can produce fracture and protoplasm to condense, top and middle cells of mycelia can intumescentia into sphere and oval or bead-like, some top cells of mycelia obvious intumescentia to clusters, partly mycelia became intumescentia or slimmer. The substance was found to be thermostable, more resistant to salt, organic solvents, acid and alkali. The pigment production speed of mycelia was also inhibited by the volatile substances.
3. The separation method of antimicrobial protein was that identify the extracellular protein band by SDS-PAGE electrophoresis, cut it and sequence. The results show that the homology of the extracellular proteins B strip and bacillus subtilis antimicrobial protein is very high. It can be concluded that the band B protein band was the purpose protein.
4. The volatile substances was separated by head space solid-phase microextraction (HS-SPME) coupled to capillary gas chromatography-mass spectrometry(GC-MS). After GC-MS analysis, there were 21 different components in volatile substances. These substances points belong to paraffin, alcohol, acids, esters, phenolic and aldehydes and several other kind materials.
5. Function identification of volatile substances showed that the role of different components were different. For Pentadecane, Dibenzofuran, Diethyl Phthalate, Tetradecanoic acid , 2-Propenoic acid, tetradecyl ester and 2,4,6-tripropyl-,1,3,5-Trioxane, the bacteriostasis is higher. And the bacteriostatic rate of Dibenzofuran and 2,4,6-tripropyl-, 1,3,5-Trioxane can reach 80%. For Tetradecane, 4-Hydroxybutyl acrylate, Dodecyl alcohol, Dodecanoic acid, Hexadecanoic acid and Octadecanoic acid, the bacteriostasis is below 20%, or basically no bacteriostasis.
引文
别小妹,吕凤霞,陆兆新,等. Bacillus subtilis fmbJ脂肽类抗菌物质的分离和鉴定[J],生物工程学报,2006,22(4):644-649.
曹君,高智谋,潘月敏,等.枯草芽孢杆菌BS菌株和哈茨木霉TH-1对棉花黄萎病菌的拮抗作用[J],植物病理学报,2005,35(6):170-172.
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.
陈华,郑之明,余增亮.枯草芽孢杆菌JA脂肤类及挥发性物质抑菌效应的研究[J],微生物学通报,2008,35(l):l-4.
陈华,袁成凌,蔡克周,等.枯草芽孢杆菌JA产生的脂肽类抗生素-iturin A的纯化及电喷雾质谱鉴定[J],微生物学报,2008,48(1):116-120.
陈文君,吴剑波,全启光.农用抗生素11371各组分的分离纯化和鉴别[J],中国抗生素杂志,1990,15(1): 42-48.
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.
杜立新,冯书亮,曹克强,等.枯草芽孢杆菌BS-08和BS-209菌株防治番茄灰霉病研究[J],农药学学报,2004,6(3):37-42.
惠明,窦丽娜,田青,等.枯草芽孢杆菌的应用研究进展[J],安徽农业科学, 2008, 36(27): 11623-11624,11627.
范青,田世平,李永兴,等.枯草芽孢杆菌(B-912)对桃和油桃褐腐病的抑制效果[J],植物学报,2000,42(11):1137-1143.
高学文,姚仕义,Huong P,等.枯草芽孢杆菌B2菌株产生的抑菌活性物质分析[J],中国生物防治,2003,19(4):175-179.
高学文,姚仕义,Huong P,等.枯草芽抱杆菌B2菌株产生的表面活性素变异体的纯化和鉴定[J].微生物学报,2003,5(43):647-752.
高芬,马利平,乔雄梧,等.芽孢杆菌BC98_I抗真菌物质的初步分离及特性[J],植物保护学报,2004,31(4):365-370.
郝华昆,韩俊华,李为民,等.棉花黄、枯萎病拮抗菌株B110的鉴定及其抑菌作用方式[J],植物保护,2007,33(2):77-80.
黄丽丹,陈玉惠.生防菌及相关生物技术在植物病害防治中的应用[J],西南林学院学报, 2006,26(1):85-89.
华菊玲,李湘民,罗任华.拮抗细菌B-77的种类鉴定及对水稻恶苗病的防治效果[J],江西农业学报,2004,16(3):62-64.
韩艳霞,王刚,王俊芳,等.枯草芽孢杆菌B1-41对小麦全蚀病及其病原的防治和抑制作用[J],开封大学学报,2005,19(1):83-84.
黄河清,孔波,林庆梅,等. MALDI-TOF质谱技术研究铁蛋白蛋白壳表层的电荷分布[J],生物物理学报,2002,18(1):99-104.
纪兆林,唐丽娟,张清霞,等.地衣芽孢杆菌W10抗菌蛋白的分离纯化及其理化性质研究[J],植物病理学报,2007,37(3):260-264.
金华勇,曾灿伟,康旭,等.顶空固相微萃取-气-质联用技术分析传统甜面酱中挥发性风味成分[J],中国酿造,2009,(5):152-154.
姜莉莉,陈彦闯,辛明秀.枯草芽孢杆菌在防治植物病害上的应用及研究进展[J].安徽农学通报,2009.15(7):37-39.
刘伊强,王雅平,潘乃穟,等.拮抗菌TG26的鉴定及其抗菌蛋白Bl的纯化和部分特性[J].植物学报,1994,36(3):197-203.
李晶,杨谦.生防枯草芽孢杆菌的研究进展[J].安徽农业科学,2008,36(1):106-111, 132.
刘静,王军,姚建铭,等.枯草芽孢杆菌JA抗菌物特性的研究及抗菌肽的分离纯化[J].微生物学报,2004,44(4):511-514.
李德全,陈志谊,聂亚锋.生防菌Bs_916及高效突变菌株抗菌物质及其对水稻抗性诱导作用的研究[J].植物病理学报,2008,38(2):192-198.
黎海彬,李小梅.大孔吸附树脂及其在天然产物研究中的应用[J].广东化工,2005(3): 22- 25.
李州,寥史书,雷文.制药工业中溶剂萃取的机制和应用发展方向[J].中国医药工业杂志, 1996,(2):89-94.
刘颖,徐庆,陈章良.抗真菌肽LP-1的分离纯化及特性分析[J].微生物学报,1999, 39(5): 441-447.
李宝庆,鹿秀云,郭庆港,等.枯草芽孢杆菌BAB-1产脂肽类及挥发性物质的分离和鉴定[J].中国农业科学,2010,43(17):3547-3554.
刘军锋,昝俊峰,陈家春,等.固相微萃取技术在中药挥发性成分分析中的应用[J].湖北中医学院学报,2006,8(3):29-31.
刘振宇,柳韶华,赵青春,等.枯草芽孢杆菌对桑炭疽病菌的抑制作用[J].蚕业科学, 2005, 31(4):409-413.
李晶,孙宇峰,张淑梅,等.枯草芽孢杆菌水剂防治黄瓜枯萎病田间防效研究[J].生物技术, 2004,14(15):77-78.
林福呈,李德葆.枯草芽孢杆菌( Bacillus subtilis)S9对植物病原真菌的溶菌作用[J].植物病理学报,2003,33(2):174-177.
林多多,居正英,王明江,等.茄子黄萎病生防内生细菌Jaas edl产生的抑菌物质特性初步分析[J].江苏农业科学,2009,(6):153-156.
刘会香,周翠,金静,等.一株拮抗细菌的鉴定及其抗菌特性[J].林业科学,2010,46(9): 110 - 114.
刘永锋,陈志谊,周明国,等.枯草芽孢杆菌Bs-916的抑菌活性及其抑菌物质初探[J].农药学学报,2007,9(1):92-95.
穆常青,潘玮,陆庆光,等.枯草芽孢杆菌对稻瘟病的防治效果评价及机制初探[J].中国生物防治,2006,22(2):158-160.
牛术敏,郭晓军,李术娜,等.枯草芽孢杆菌BS_26菌株纤溶酶的性质分析及活性组分的分离纯化[J].微生物学报,2008,48(10):1387-1392.
童有仁,马志超,陈卫良,等.枯草芽孢杆菌B034拮抗蛋白的分离纯化及特性分析[J].微生物学报,1999,399(4):339-394.
汪家政,范明.蛋白质技术手册[M].北京:科学出版社,2002.
吴清平,蔡正荷,等.表面活性素的发酵生产及应用[J].微生物学通报,1999,26(1):14-16.
吴艳,闫豫君,等.组合芽孢杆菌抑菌物质特性及其抑菌效果研究[J].西北农业学报,2007, 16(5):266-270.
王星云,宋卡魏,张荣意,等.枯草芽孢杆菌菌剂的开发应用[J].广西热带农业,2007,(2):32- 34.
王玉霞,李晶,张淑敏.枯草芽孢杆菌对黄瓜根腐病的防治效果[J].生物技术,2004, 14(3): 57.
王明林,乔鲁芹,张莉,等.固相微萃取-气相色谱/质谱测定植物叶片中的挥发性物质[J].色谱,2006,24(4):343-346.
王芳,纪明山,谷祖敏,等.枯草芽孢杆菌B36抑菌活性成分的初步提取[J].湖北农业科学, 2009,48(4):874-876.
吴冠芸,潘华珍,吴晕.生物化学与分子生物学实验常用数据手册[M].北京:科学出版社, 2000,161-163.
徐素平,曹广霞,彭远义.抗生素分离纯化技术[J].安徽农学通报,2009,15(7):79-81.
辛玉成.枯草芽孢杆菌BL03对苹果霉心病和棉苗病害田间防治效果[J].中国生物防治, 2000,16(1):47.
辛玉成,秦淑莲,刘希光.几种拮抗菌株对苹果霉心病菌抑制作用的研究[J].中国果树, 1999,(3):14-15.
许瑛华,朱炳辉,钟秀华,等.顶空气相色谱法测定化妆品中15种挥发性有机溶剂残留[J].色谱,2010,28(1):73-77.
易有金,刘如石,孙吉康,等.内生枯草芽孢杆菌B-001菌株抗菌肽纯化与性质[J].植物病理学报,2007,37(5):556-560.
杨佐忠.枯草芽孢杆菌PRS5抗生作用的初步研究[J].西南林学院学报,1992,12(2): 167- 173.
余桂荣,张敏,叶华智.小麦赤霉病的生物防治研究1.拮抗芽孢杆菌的分离、筛选、鉴定和防病效果[J].四川农业大学学报,1998,16(3):314-419.
尹向田,范素素,徐亮,等.桑树内生枯草芽孢杆菌TasA基因的克隆及序列分析与表达产物的抑菌作用[J].蚕业科学,2010,36(2):0319-0320.
张成省,孔凡玉,等.枯草芽孢杆菌Tpb55挥发物对烟草的防病促生效应[J].中国生物防治, 2009,25(3):245-249.
朱茂山,赵奎华,刘长远,等.枯草芽孢杆菌B18菌株酶类抑菌物质分析研究初报[J].辽宁农业科学,2003,(2):41-42.
张秀玉,孔凡玉,王静,等.枯草芽孢杆菌SH7抑菌蛋白的分离纯化及对烟草青枯病菌的抑制作用[J].中国烟草科学,2010,31(1):13-15.
赵秀香,赵柏霞,马镝,等.枯草芽孢杆菌SN-02抑菌物质的分离、纯化及结构测定[J].植物保护学报,2008,35(4):322-326.
张彦杰,罗俊彩,武燕萍,等.生防枯草芽孢杆菌研究进展[J].生命科学仪器,2009,7(4): 19- 23.
朱茂山,赵奎华,刘长远,等.枯草芽孢杆菌B18菌株对黄瓜枯萎病抑菌作用研究[J].辽宁农业科学,2003,(3):39.
赵达,傅俊范,裘季燕,等.枯草芽孢杆菌在植病生防中的作用机制与应用[J].辽宁农业科学,2007(1):46-48.
朱金勇,黄河清. MALDI TOF质谱技术分析细胞与组织多肽组成的研究进展[J].分析仪器,2004,(2)2-7.
Amparoc M., Fred G., Juan F., et al. Histopathological effects and growth reduction in a suscep tible and a resistance of heliothis virescens caused by sublethal doses of pure CrylA crystal proteins from Bacillus thuringiesis[J]. Biocontrol Science and Technology,1999,9(2): 239-246.
Asaka O., Shoda M.. Biocontrol of the Rhizoctonia solani damping of tomato with Bacillus subtilis RB-14[J]. Apply Environment Microbioloy,1996,62(11):4081-4085.
Broich J. R., Hoffman D. B., Goldner S. J., et al. Liquid-solid extraction of lyophilized biological material for forensic analysis: I. Application to urine samples for detection of drugs of abuse[J]. Journal of Chromatography A. 1971, 63(2):309-312.
Chitarra G. S., Breeuwer P., Nout M. J.,et al.An antifungal compound produced by Bacillus subtilis YM10-20 inhibits germination of Penicillium roqueforti conidiospores[J]. Apply Microbiology, 2003, 94 (2): 159-166.
Chaurasia B., Pandey A., Palni L. M. S., et al. Diffusible and volatile compounds produced by an antagonistic Bacillus subtilis strain cause structural deformations in pathogenic fungi in vitro[J]. Microbiological Research,2005,160(1):75-81.
Charles W. B, Ida E. Y., Dorothy M. H., et al. Biological Control of Fusarium moniliforme in Maize[J]. Environmental Health Perspective, 2001,109(2):325-332.
Disney R. D., Danielle M. V., Marialice P. C. S., et al. Alkaline protease from Bacillus sp. isolated from coffee bean grown on cheese whey[J]. World Journal of Microbiology and Biotechnology. 2008, 24(10): 2027-2034.
Emmert E. A. B., Handelsman J.. Biocontrol of plant diease:a(Gram-)positive perspective[J]. FEMS Microbiology Letters,1999,171(1):1-9.
Fernando W. G. D., Ramarathnam R., Krishnamoorthy A. S., et al. Identification and use of potential bacterial organic antifungal volatiles in biocontrol[J]. Soil Biology and Biochemistry, 2005,37(5): 955-964.
Zheng G.. Isolation, partial purification and characterization of a bacteriocin produce by a newly isolated Bacillus subtilis strain[J]. Letter in Applied Microbiology, 1999, 28 (5): 363-367.
Handelsman J., Stabb E. V.. Biocontrol of soilborne plant pathogens [J]. Plant Cell, l996, 8 (10):1855-1869.
Hyronimus B., Le M. C.and Urdaci M. C.. Coagulina bactereiocin-like inhibitory substance produced by Bacillus coagulans I4[J].Apply Microbiology,1998,85(1):42-50.
J.Eleazar B. C., Toma′s O. R., Normadela F. S., et al. Hyperproduction of chitinase influences crystal toxin synthesis and sporulation of Bacillus thuringiensis[J]. Antonie van Leeuwenhoek, 2009,96(1):31-42.
Junge H., Kerbs B.and Kilian. Strain selection production and formulation of the biological plant vitality enhancing agent FZB24 Bacillus subtilis[J]. Pflanzenschutz-Nachtichten Bayer, 2000,53(1):94-104.
Jack R. W, Tagg J. R., Ray B.. Bateriocin of Gram-positive bacteria[J]. Microbiol Rev, 1995,59(2):171-200.
Kilian M., Steiner U., Krebs B., et al. FZB24(r)Bacillus subtilis mode of action of a microbial agent enhancing plant vitality[J]. Journal Pflanzenschutz-Nachrichten Bayer. 2000, 53(1):72-93.
Kai M., Haustein M., Molina F., et al. Bacterial volatiles and their action potential[J]. Applied Microbiology and Biotechnology,2009,81(6):1001-1012.
Kai M., Effmert U., Berg G., et al.Volatiles of bacterial antagonists inhibit mycelial growth of the plant pathogen Rhizoctonia solani[J]. Archives of Microbiology, 2007, 187(5): 351-360.
Leelasupakul W., Aemmanee P.and Chuenchitt S.. Growth inhibitory properties of Bacillus subtilis strains and their metabolites against the green mold pathogen(Penicillium digitatum Sacc.)of citrus fruit[J]. Postharvest Biology and Technology, 2008,48(1): 113-121.
Li L., Mo M. H., Qu Q., et al. Compounds inhibitory to nematophagous fungi produced by Bacillus sp. strain H6 isolated from fungistatic soil [J]. European Journal of Plant Pathology, 2007, 117(4): 329-340.
Liu W. W., Zhao L. J., Wang C., et al. Bioactive evaluation and application of antifungal volatiles generated by five soil bacteria[J].Acta Phytophylacica Sinica. 2009,36(2): 97- 105.
Manjula K., Kishore G. K.and Podile A. R.. Whole cells of Bacillus subtilis AF1 proved more effectivethan cel1-free and chitinase-based formulations in biological control of citrus fruit rot and groundnutrust[J]. Canadian Journal of Microbiology, 2004,50(9): 737-744.
Motoo K., Yuuji K., Keita H.. Antifungal substances reduced by fungal strain Kyu-W63 from wheat leaf and its taxonomic position[J]. Journal of General Plant Pathology, 2004, 70(2): 124-130.
Masatake A., Kinya K., Yuji H., et al. A novelβ-glucanase gene from Bacillus halodurans C- 125[J]. FEMS microbiology letters. 2005,248(1):9-15.
Oscariz J.. Detection and characterization of cerein 7, a new bacteriocin produced by Bacillus cereus with a broad spectrum of activity[J]. Fems Microbial.Lettt. 1999,178 (2): 337-341.
Pierre U.. Sustainable agriculture and chemical control:opponents or components of the same strategy[J].Crop Protetion, 2000,19(8-10):831-836.
Paulitz T. C., Belanger R. R.. Biological control in greenhouse systems[J].Annual Review of phytophthology,2001,39(1):103-133.
Berlardi R., Pawliszyn J., The application of chemically modified fused silica fibers in the extracyion of organics from water matrix samples and their rapid transfer to capillary columns[J]. Water Pollution Research Journal of Canada, 1989, 24(1):179-191.
Ralph W. J , John R. T., Bibek R..Bacteriocins of gram-positive bacteria[J]. Microbiological Review, 1995,59(2):171-200.
Wu S., Jia S., Sun D., et al. Purification and characterization of two novel antimicrobial peptides subpeptin JM4-A and subpeptin JM4-B produced by Bacillus subtilis JM4[J]. Current Microbiology. 2005,51(5):292-296.
Stein T.. Bacillus subtilis antibiotics: structures, syntheses and specific functions[J]. Mol Microbiol, 2005, 56(4): 845-857.
Wang S. L., Lin T. Y., Yen Y. H., et al. Bioconversion of shellfish chitin wastes for the production of Bacilllus subtills W-118 chitinase[J].Carbohydrate Research, 2006,341 (15): 2507-2515.
Shoda M.. Bacterial control of plant diseases[J] .Journal of Bioscience and Bioengineering, 2000,89(6):515-521.
Serpil T., Baoak M.. Effects of lipidic carbon sources on the extracellular lipolytic activity of a newly isolated strain of Bacillus subtilis[J]. Journal of Industrial Microbiology Biotechnology, 2008,35 (9):1019-1025.
Torsten S.. Bacillus subtilis antibiotics:structures, syntheses and specific functions[J]. Molecular Microbiology, 2005,56(4):845-857.
Vespermann A., Kai M.and Piechulla B.. Rhizobacterial volatiles affect the growth of fungi and Arabidopsis thaliana[J]. Applied and Environmenal Microbiology, 2007,73 (17):5639-5641.
Wang H. X., Ng T. B.. Dendrocin, a distinctive antifungal protein from bamboo shoot[J]. Biochemical and Biophysical Research Communications, 2005,307(3):750-755.
Wagner W. C.. Sustainable agriculture:how to sustain a production system in a changing environment[J]. International Journal for Parasitology, 1999.29(1):1-5.
Yazgana A., Ozcengiza G., Ozengizb E., et al. Bacilysin biosynthesis by a partially-purified enzyme fraction from Bacillus subtilis[J].Enzyme and Microbial Technology, 2001,29 (6-7): 400- 406.
曹君,高智谋,潘月敏,等.枯草芽孢杆菌BS菌株和哈茨木霉TH-1对棉花黄萎病菌的拮抗作用[J],植物病理学报,2005,35(6):170-172.
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.
陈华,郑之明,余增亮.枯草芽孢杆菌JA脂肤类及挥发性物质抑菌效应的研究[J],微生物学通报,2008,35(l):l-4.
陈华,袁成凌,蔡克周,等.枯草芽孢杆菌JA产生的脂肽类抗生素-iturin A的纯化及电喷雾质谱鉴定[J],微生物学报,2008,48(1):116-120.
陈文君,吴剑波,全启光.农用抗生素11371各组分的分离纯化和鉴别[J],中国抗生素杂志,1990,15(1): 42-48.
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.
杜立新,冯书亮,曹克强,等.枯草芽孢杆菌BS-08和BS-209菌株防治番茄灰霉病研究[J],农药学学报,2004,6(3):37-42.
惠明,窦丽娜,田青,等.枯草芽孢杆菌的应用研究进展[J],安徽农业科学, 2008, 36(27): 11623-11624,11627.
范青,田世平,李永兴,等.枯草芽孢杆菌(B-912)对桃和油桃褐腐病的抑制效果[J],植物学报,2000,42(11):1137-1143.
高学文,姚仕义,Huong P,等.枯草芽孢杆菌B2菌株产生的抑菌活性物质分析[J],中国生物防治,2003,19(4):175-179.
高学文,姚仕义,Huong P,等.枯草芽抱杆菌B2菌株产生的表面活性素变异体的纯化和鉴定[J].微生物学报,2003,5(43):647-752.
高芬,马利平,乔雄梧,等.芽孢杆菌BC98_I抗真菌物质的初步分离及特性[J],植物保护学报,2004,31(4):365-370.
郝华昆,韩俊华,李为民,等.棉花黄、枯萎病拮抗菌株B110的鉴定及其抑菌作用方式[J],植物保护,2007,33(2):77-80.
黄丽丹,陈玉惠.生防菌及相关生物技术在植物病害防治中的应用[J],西南林学院学报, 2006,26(1):85-89.
华菊玲,李湘民,罗任华.拮抗细菌B-77的种类鉴定及对水稻恶苗病的防治效果[J],江西农业学报,2004,16(3):62-64.
韩艳霞,王刚,王俊芳,等.枯草芽孢杆菌B1-41对小麦全蚀病及其病原的防治和抑制作用[J],开封大学学报,2005,19(1):83-84.
黄河清,孔波,林庆梅,等. MALDI-TOF质谱技术研究铁蛋白蛋白壳表层的电荷分布[J],生物物理学报,2002,18(1):99-104.
纪兆林,唐丽娟,张清霞,等.地衣芽孢杆菌W10抗菌蛋白的分离纯化及其理化性质研究[J],植物病理学报,2007,37(3):260-264.
金华勇,曾灿伟,康旭,等.顶空固相微萃取-气-质联用技术分析传统甜面酱中挥发性风味成分[J],中国酿造,2009,(5):152-154.
姜莉莉,陈彦闯,辛明秀.枯草芽孢杆菌在防治植物病害上的应用及研究进展[J].安徽农学通报,2009.15(7):37-39.
刘伊强,王雅平,潘乃穟,等.拮抗菌TG26的鉴定及其抗菌蛋白Bl的纯化和部分特性[J].植物学报,1994,36(3):197-203.
李晶,杨谦.生防枯草芽孢杆菌的研究进展[J].安徽农业科学,2008,36(1):106-111, 132.
刘静,王军,姚建铭,等.枯草芽孢杆菌JA抗菌物特性的研究及抗菌肽的分离纯化[J].微生物学报,2004,44(4):511-514.
李德全,陈志谊,聂亚锋.生防菌Bs_916及高效突变菌株抗菌物质及其对水稻抗性诱导作用的研究[J].植物病理学报,2008,38(2):192-198.
黎海彬,李小梅.大孔吸附树脂及其在天然产物研究中的应用[J].广东化工,2005(3): 22- 25.
李州,寥史书,雷文.制药工业中溶剂萃取的机制和应用发展方向[J].中国医药工业杂志, 1996,(2):89-94.
刘颖,徐庆,陈章良.抗真菌肽LP-1的分离纯化及特性分析[J].微生物学报,1999, 39(5): 441-447.
李宝庆,鹿秀云,郭庆港,等.枯草芽孢杆菌BAB-1产脂肽类及挥发性物质的分离和鉴定[J].中国农业科学,2010,43(17):3547-3554.
刘军锋,昝俊峰,陈家春,等.固相微萃取技术在中药挥发性成分分析中的应用[J].湖北中医学院学报,2006,8(3):29-31.
刘振宇,柳韶华,赵青春,等.枯草芽孢杆菌对桑炭疽病菌的抑制作用[J].蚕业科学, 2005, 31(4):409-413.
李晶,孙宇峰,张淑梅,等.枯草芽孢杆菌水剂防治黄瓜枯萎病田间防效研究[J].生物技术, 2004,14(15):77-78.
林福呈,李德葆.枯草芽孢杆菌( Bacillus subtilis)S9对植物病原真菌的溶菌作用[J].植物病理学报,2003,33(2):174-177.
林多多,居正英,王明江,等.茄子黄萎病生防内生细菌Jaas edl产生的抑菌物质特性初步分析[J].江苏农业科学,2009,(6):153-156.
刘会香,周翠,金静,等.一株拮抗细菌的鉴定及其抗菌特性[J].林业科学,2010,46(9): 110 - 114.
刘永锋,陈志谊,周明国,等.枯草芽孢杆菌Bs-916的抑菌活性及其抑菌物质初探[J].农药学学报,2007,9(1):92-95.
穆常青,潘玮,陆庆光,等.枯草芽孢杆菌对稻瘟病的防治效果评价及机制初探[J].中国生物防治,2006,22(2):158-160.
牛术敏,郭晓军,李术娜,等.枯草芽孢杆菌BS_26菌株纤溶酶的性质分析及活性组分的分离纯化[J].微生物学报,2008,48(10):1387-1392.
童有仁,马志超,陈卫良,等.枯草芽孢杆菌B034拮抗蛋白的分离纯化及特性分析[J].微生物学报,1999,399(4):339-394.
汪家政,范明.蛋白质技术手册[M].北京:科学出版社,2002.
吴清平,蔡正荷,等.表面活性素的发酵生产及应用[J].微生物学通报,1999,26(1):14-16.
吴艳,闫豫君,等.组合芽孢杆菌抑菌物质特性及其抑菌效果研究[J].西北农业学报,2007, 16(5):266-270.
王星云,宋卡魏,张荣意,等.枯草芽孢杆菌菌剂的开发应用[J].广西热带农业,2007,(2):32- 34.
王玉霞,李晶,张淑敏.枯草芽孢杆菌对黄瓜根腐病的防治效果[J].生物技术,2004, 14(3): 57.
王明林,乔鲁芹,张莉,等.固相微萃取-气相色谱/质谱测定植物叶片中的挥发性物质[J].色谱,2006,24(4):343-346.
王芳,纪明山,谷祖敏,等.枯草芽孢杆菌B36抑菌活性成分的初步提取[J].湖北农业科学, 2009,48(4):874-876.
吴冠芸,潘华珍,吴晕.生物化学与分子生物学实验常用数据手册[M].北京:科学出版社, 2000,161-163.
徐素平,曹广霞,彭远义.抗生素分离纯化技术[J].安徽农学通报,2009,15(7):79-81.
辛玉成.枯草芽孢杆菌BL03对苹果霉心病和棉苗病害田间防治效果[J].中国生物防治, 2000,16(1):47.
辛玉成,秦淑莲,刘希光.几种拮抗菌株对苹果霉心病菌抑制作用的研究[J].中国果树, 1999,(3):14-15.
许瑛华,朱炳辉,钟秀华,等.顶空气相色谱法测定化妆品中15种挥发性有机溶剂残留[J].色谱,2010,28(1):73-77.
易有金,刘如石,孙吉康,等.内生枯草芽孢杆菌B-001菌株抗菌肽纯化与性质[J].植物病理学报,2007,37(5):556-560.
杨佐忠.枯草芽孢杆菌PRS5抗生作用的初步研究[J].西南林学院学报,1992,12(2): 167- 173.
余桂荣,张敏,叶华智.小麦赤霉病的生物防治研究1.拮抗芽孢杆菌的分离、筛选、鉴定和防病效果[J].四川农业大学学报,1998,16(3):314-419.
尹向田,范素素,徐亮,等.桑树内生枯草芽孢杆菌TasA基因的克隆及序列分析与表达产物的抑菌作用[J].蚕业科学,2010,36(2):0319-0320.
张成省,孔凡玉,等.枯草芽孢杆菌Tpb55挥发物对烟草的防病促生效应[J].中国生物防治, 2009,25(3):245-249.
朱茂山,赵奎华,刘长远,等.枯草芽孢杆菌B18菌株酶类抑菌物质分析研究初报[J].辽宁农业科学,2003,(2):41-42.
张秀玉,孔凡玉,王静,等.枯草芽孢杆菌SH7抑菌蛋白的分离纯化及对烟草青枯病菌的抑制作用[J].中国烟草科学,2010,31(1):13-15.
赵秀香,赵柏霞,马镝,等.枯草芽孢杆菌SN-02抑菌物质的分离、纯化及结构测定[J].植物保护学报,2008,35(4):322-326.
张彦杰,罗俊彩,武燕萍,等.生防枯草芽孢杆菌研究进展[J].生命科学仪器,2009,7(4): 19- 23.
朱茂山,赵奎华,刘长远,等.枯草芽孢杆菌B18菌株对黄瓜枯萎病抑菌作用研究[J].辽宁农业科学,2003,(3):39.
赵达,傅俊范,裘季燕,等.枯草芽孢杆菌在植病生防中的作用机制与应用[J].辽宁农业科学,2007(1):46-48.
朱金勇,黄河清. MALDI TOF质谱技术分析细胞与组织多肽组成的研究进展[J].分析仪器,2004,(2)2-7.
Amparoc M., Fred G., Juan F., et al. Histopathological effects and growth reduction in a suscep tible and a resistance of heliothis virescens caused by sublethal doses of pure CrylA crystal proteins from Bacillus thuringiesis[J]. Biocontrol Science and Technology,1999,9(2): 239-246.
Asaka O., Shoda M.. Biocontrol of the Rhizoctonia solani damping of tomato with Bacillus subtilis RB-14[J]. Apply Environment Microbioloy,1996,62(11):4081-4085.
Broich J. R., Hoffman D. B., Goldner S. J., et al. Liquid-solid extraction of lyophilized biological material for forensic analysis: I. Application to urine samples for detection of drugs of abuse[J]. Journal of Chromatography A. 1971, 63(2):309-312.
Chitarra G. S., Breeuwer P., Nout M. J.,et al.An antifungal compound produced by Bacillus subtilis YM10-20 inhibits germination of Penicillium roqueforti conidiospores[J]. Apply Microbiology, 2003, 94 (2): 159-166.
Chaurasia B., Pandey A., Palni L. M. S., et al. Diffusible and volatile compounds produced by an antagonistic Bacillus subtilis strain cause structural deformations in pathogenic fungi in vitro[J]. Microbiological Research,2005,160(1):75-81.
Charles W. B, Ida E. Y., Dorothy M. H., et al. Biological Control of Fusarium moniliforme in Maize[J]. Environmental Health Perspective, 2001,109(2):325-332.
Disney R. D., Danielle M. V., Marialice P. C. S., et al. Alkaline protease from Bacillus sp. isolated from coffee bean grown on cheese whey[J]. World Journal of Microbiology and Biotechnology. 2008, 24(10): 2027-2034.
Emmert E. A. B., Handelsman J.. Biocontrol of plant diease:a(Gram-)positive perspective[J]. FEMS Microbiology Letters,1999,171(1):1-9.
Fernando W. G. D., Ramarathnam R., Krishnamoorthy A. S., et al. Identification and use of potential bacterial organic antifungal volatiles in biocontrol[J]. Soil Biology and Biochemistry, 2005,37(5): 955-964.
Zheng G.. Isolation, partial purification and characterization of a bacteriocin produce by a newly isolated Bacillus subtilis strain[J]. Letter in Applied Microbiology, 1999, 28 (5): 363-367.
Handelsman J., Stabb E. V.. Biocontrol of soilborne plant pathogens [J]. Plant Cell, l996, 8 (10):1855-1869.
Hyronimus B., Le M. C.and Urdaci M. C.. Coagulina bactereiocin-like inhibitory substance produced by Bacillus coagulans I4[J].Apply Microbiology,1998,85(1):42-50.
J.Eleazar B. C., Toma′s O. R., Normadela F. S., et al. Hyperproduction of chitinase influences crystal toxin synthesis and sporulation of Bacillus thuringiensis[J]. Antonie van Leeuwenhoek, 2009,96(1):31-42.
Junge H., Kerbs B.and Kilian. Strain selection production and formulation of the biological plant vitality enhancing agent FZB24 Bacillus subtilis[J]. Pflanzenschutz-Nachtichten Bayer, 2000,53(1):94-104.
Jack R. W, Tagg J. R., Ray B.. Bateriocin of Gram-positive bacteria[J]. Microbiol Rev, 1995,59(2):171-200.
Kilian M., Steiner U., Krebs B., et al. FZB24(r)Bacillus subtilis mode of action of a microbial agent enhancing plant vitality[J]. Journal Pflanzenschutz-Nachrichten Bayer. 2000, 53(1):72-93.
Kai M., Haustein M., Molina F., et al. Bacterial volatiles and their action potential[J]. Applied Microbiology and Biotechnology,2009,81(6):1001-1012.
Kai M., Effmert U., Berg G., et al.Volatiles of bacterial antagonists inhibit mycelial growth of the plant pathogen Rhizoctonia solani[J]. Archives of Microbiology, 2007, 187(5): 351-360.
Leelasupakul W., Aemmanee P.and Chuenchitt S.. Growth inhibitory properties of Bacillus subtilis strains and their metabolites against the green mold pathogen(Penicillium digitatum Sacc.)of citrus fruit[J]. Postharvest Biology and Technology, 2008,48(1): 113-121.
Li L., Mo M. H., Qu Q., et al. Compounds inhibitory to nematophagous fungi produced by Bacillus sp. strain H6 isolated from fungistatic soil [J]. European Journal of Plant Pathology, 2007, 117(4): 329-340.
Liu W. W., Zhao L. J., Wang C., et al. Bioactive evaluation and application of antifungal volatiles generated by five soil bacteria[J].Acta Phytophylacica Sinica. 2009,36(2): 97- 105.
Manjula K., Kishore G. K.and Podile A. R.. Whole cells of Bacillus subtilis AF1 proved more effectivethan cel1-free and chitinase-based formulations in biological control of citrus fruit rot and groundnutrust[J]. Canadian Journal of Microbiology, 2004,50(9): 737-744.
Motoo K., Yuuji K., Keita H.. Antifungal substances reduced by fungal strain Kyu-W63 from wheat leaf and its taxonomic position[J]. Journal of General Plant Pathology, 2004, 70(2): 124-130.
Masatake A., Kinya K., Yuji H., et al. A novelβ-glucanase gene from Bacillus halodurans C- 125[J]. FEMS microbiology letters. 2005,248(1):9-15.
Oscariz J.. Detection and characterization of cerein 7, a new bacteriocin produced by Bacillus cereus with a broad spectrum of activity[J]. Fems Microbial.Lettt. 1999,178 (2): 337-341.
Pierre U.. Sustainable agriculture and chemical control:opponents or components of the same strategy[J].Crop Protetion, 2000,19(8-10):831-836.
Paulitz T. C., Belanger R. R.. Biological control in greenhouse systems[J].Annual Review of phytophthology,2001,39(1):103-133.
Berlardi R., Pawliszyn J., The application of chemically modified fused silica fibers in the extracyion of organics from water matrix samples and their rapid transfer to capillary columns[J]. Water Pollution Research Journal of Canada, 1989, 24(1):179-191.
Ralph W. J , John R. T., Bibek R..Bacteriocins of gram-positive bacteria[J]. Microbiological Review, 1995,59(2):171-200.
Wu S., Jia S., Sun D., et al. Purification and characterization of two novel antimicrobial peptides subpeptin JM4-A and subpeptin JM4-B produced by Bacillus subtilis JM4[J]. Current Microbiology. 2005,51(5):292-296.
Stein T.. Bacillus subtilis antibiotics: structures, syntheses and specific functions[J]. Mol Microbiol, 2005, 56(4): 845-857.
Wang S. L., Lin T. Y., Yen Y. H., et al. Bioconversion of shellfish chitin wastes for the production of Bacilllus subtills W-118 chitinase[J].Carbohydrate Research, 2006,341 (15): 2507-2515.
Shoda M.. Bacterial control of plant diseases[J] .Journal of Bioscience and Bioengineering, 2000,89(6):515-521.
Serpil T., Baoak M.. Effects of lipidic carbon sources on the extracellular lipolytic activity of a newly isolated strain of Bacillus subtilis[J]. Journal of Industrial Microbiology Biotechnology, 2008,35 (9):1019-1025.
Torsten S.. Bacillus subtilis antibiotics:structures, syntheses and specific functions[J]. Molecular Microbiology, 2005,56(4):845-857.
Vespermann A., Kai M.and Piechulla B.. Rhizobacterial volatiles affect the growth of fungi and Arabidopsis thaliana[J]. Applied and Environmenal Microbiology, 2007,73 (17):5639-5641.
Wang H. X., Ng T. B.. Dendrocin, a distinctive antifungal protein from bamboo shoot[J]. Biochemical and Biophysical Research Communications, 2005,307(3):750-755.
Wagner W. C.. Sustainable agriculture:how to sustain a production system in a changing environment[J]. International Journal for Parasitology, 1999.29(1):1-5.
Yazgana A., Ozcengiza G., Ozengizb E., et al. Bacilysin biosynthesis by a partially-purified enzyme fraction from Bacillus subtilis[J].Enzyme and Microbial Technology, 2001,29 (6-7): 400- 406.