CN112980733A - 一株只对病原细菌有抑制作用、对病原真菌无抑制作用的蜡样芽孢杆菌n15-2及其应用 - Google Patents
一株只对病原细菌有抑制作用、对病原真菌无抑制作用的蜡样芽孢杆菌n15-2及其应用 Download PDFInfo
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- bacillus cereus
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/02—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing liquids as carriers, diluents or solvents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/30—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests characterised by the surfactants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
本发明涉及一种生防菌剂,其中有效活性成分由蜡样芽孢杆菌(Bacillus cereus)N15‑2组成。其特征在于:该生防菌只对病原细菌有抑制作用、对病原真菌无抑制作用;该生防菌剂是水剂,其组成与重量百分含量为:蜡样芽孢杆菌N15‑2:89.5‑99.8%;十二烷基磺酸钠:0.1‑10%;山梨酸钾:0.1‑0.5%。应用时,将上述水剂灌根用于防治大白菜软腐病和辣椒青枯病,水稻叶面均匀喷施用于防治水稻白叶枯病和水稻细菌性条斑病。其优点是:通过引进对人畜安全的蜡样芽孢杆菌N15‑2,达到利用生防菌剂防治植物细菌病害;减少化学药剂用药量;同时改善生态环境、减少化学药剂在农产品中残留量的目的。
Description
技术领域
本发明涉及一种生防菌剂及其应用,尤其是一株蜡样芽孢杆菌N15-2及其在防治植物细菌性病害中的应用。
背景技术
植物细菌性病害是农业生产中的重要病害,其危害程度已超越病毒,成为仅次于真菌的第二大病害。由于细菌性病害预防措施复杂、传播方式多样、防治药剂缺乏,加上长期连作的种植模式,导致防治难度日益加大。据不完全数据统计,我国植物细菌性病害目前每年的发生面积在1.2亿亩次。细菌性病害是由病原细菌侵染所致的病害,如软腐病、青枯病、白叶枯病等。侵害植物的细菌可通过自然孔口(气孔、皮孔、水孔等)和伤口侵入,借流水、雨水、昆虫等传播,在病残体、种子、土壤中过冬。细菌性病害的主要症状是腐烂、坏死、萎蔫和穿孔等。细菌性病害发病后期遇潮湿天气,在病害部位溢出细菌粘液,有明显恶臭味,是其主要特征。
生产上防治植物细菌性病害主要依靠各种铜制剂(噻菌铜、王铜、氢氧化铜、络氨铜、柠檬酸铜、噻森铜、噻霉酮等)和少数几种抗生素(如农用硫酸链霉素、春雷霉素、中生菌素等)。农用链霉素对多数细菌性病害都有很好的防治效果,过去曾被广泛应用。2016年6月14日,我国最后一个农用硫酸链霉素登记证件到期,不再续展,这意味着农用硫酸链霉素正式退出防治细菌性病害的舞台,细菌性病害的防治也由其它药剂所取代。农业作物上禁用硫酸链霉素,主要原因是硫酸链霉素是人畜共用的抗生素,应用到作物上后,会通过食物链进入人畜体内,从而产生抗性风险。而铜制剂等化学农药的长期、大量使用不仅造成生态环境严重污染,而且直接增加农产品中有毒化学物质的残留,对人类健康带来严重危害。为了达到有效防治细菌性病害、减少化学农药使用量的目的,使用生物防治技术控制植物病害的方法越来越受到各国政府和人民的关注。生物防治技术主要是利用生物多样性,通过在自然界释放或引进有益微生物,可持续控制植物病害。1950年以来国内外均开展了利用生防菌剂防治植物细菌性病害的研究,主要包括芽孢杆菌(Bacillus)和假单胞菌(Pseudomonads)两类生防菌。
江苏省农业科学院从江苏省南京市六合豌豆地采集的土样中,分离纯化后获得一株新颖的生防菌株N15-2。前期研究结果表明:生防菌株N15-2对生产上多种细菌病害(软腐病、青枯病、白叶枯病、细菌性条斑病、黑腐病等)有很好的防治效果。经中国科学院微生物研究所检测鉴定,根据细胞形态、生理生化特征、16S rRNA基因序列等实验数据综合分析,菌株N15-2鉴定为蜡样芽孢杆菌(Bacillus cereus)。蜡样芽孢杆菌N15-2已于2021年1月6日在中国微生物菌种保藏管理委员会普通微生物中心登记入册,地址是北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,保藏编号为CGMCC No.21599。
近年来已有报道的蜡样(有时也称为蜡质)芽孢杆菌除了作为根围有益促生菌(Plant growth-promoting rhizobacteria,PGPR)外(Gao Tantan等,2020年,《AppliedMicrobiology and Biotechnology》;E1-Sayed M.Desoky等,2020年,《Biocatalysis andAgricultural Biotechnology》;王春娟等,2012年,《农业生物技术学报》),主要防治真菌病害,包括小麦锈病(Asghari Bano等,2020年,《Jouranl of Plant Pathology》)、茎点霉(Pankaj Kumar等,2020年,《Scientific Reports》)、番茄枯萎病和早疫病(Karthika S等,2020年,《Microbial Pathogenesis》)、黄瓜枯萎病菌(孙向平等,2019年,《江苏农业科学》)、番茄灰霉病(潘晓梅等,2019年,《西北农业学报》)、根结线虫(廖梦婕等,2019年,《微生物学通报》;蒋春号,2016年博士学位论文;刘元兵等,2015年,《农业科学与管理》)、水稻稻曲病(王晓荣,2018年,《农业开发与装备》)、杨树腐烂病(田爱霞,2017年,《浙江农林大学学报》)、小麦白粉病(龚双军等,2016年,《中国生物防治学报》)、禾谷孢囊线虫(Zhang Jie等,2016年,《Biological Control》)、花生曲霉(Navya HM等,2015年,《Annals of AppliedBiology》)、水稻纹枯病(陈刘军等,2014年,《中国生物防治学报》)、玉米小斑病(Chien-JuiHuang等,2010年,《Annals of Applied Biology》)等。蜡样(蜡质)芽孢杆菌AR156除了对上述的水稻纹枯病(陈刘军等,2014年,《中国生物防治学报》)和根结线虫(廖梦婕等,2019年,《微生物学通报》;蒋春号,2016年博士学位论文)有防效外,对辣椒青枯病也有一定的防治作用(王勇等,2014年,《植物病理学报》)。已有蜡样(蜡质)芽孢杆菌申请和授权专利主要防治对象也是真菌病害,包括水稻稻瘟病、水稻纹枯病、水稻恶苗病、水稻胡麻叶斑病、水稻稻曲病、草莓白粉病、根结线虫等(专利申请号:CN201610799877.0;CN201611027048.7;CN201410510935.4;CN201810472191.X;CN201710837023.1)。还有一些含有蜡样(蜡质)芽孢杆菌的组合型肥料,具有良好的促生、防病作用,对土壤中的根结线虫、纹枯病和枯萎病具有良好的防效(专利申请号:CN201610694583.1;CN201410012023.4)。此外,蜡样(蜡质)芽孢杆菌通过体内的SOD酶,提高作物对病菌和逆境危害引发体内产生氧的清除能力,调节作物细胞微生境,维护细胞正常的生理代谢和生化反应,提高抗逆性,加速生长,提高产量和品质(专利申请号:CN201910264931.5)。
目前利用蜡样(蜡质)芽孢杆菌防治茄子青枯病和番茄青枯病,国内有2个申请专利(专利申请号:CN201810928087.7;CN201811123658.6)和1篇文献报道(乔康等,2012年,《农药科学与管理》),它们都是直接将蜡样(蜡质)芽孢杆菌单剂或复配产品直接用于田间防治试验,无室内生测试验。而申请人分离获得的蜡样芽孢杆菌N15-2作用机理与前人报道完全不同,具体如下:1、不同于以往报道的其他蜡样(蜡质)芽孢杆菌(同时对很多病原真菌和病原细菌均具有较好的抑制作用),蜡样芽孢杆菌N15-2只对病原细菌有抑制作用、对病原真菌无抑制作用。2、有些生防菌在室内抑菌作用明显,但是田间防治效果却不稳定。蜡样芽孢杆菌N15-2对测试的几种细菌性病害具有较好的室内外抑制作用,室内抑菌试验结果和田间防治试验结果趋势一致。3、众所周知,辣椒青枯病菌和番茄青枯病菌、茄子青枯病菌虽然都是劳尔氏菌属,但是侵染不同寄主会产生不同专化型、致病性等特质。申请人分离获得的蜡样芽孢杆菌N15-2对辣椒青枯病等细菌性病害有防治作用,其他蜡样(蜡质)芽孢杆菌未必对辣椒青枯病有防治作用。换句话说,已报道的防治番茄青枯病、茄子青枯病的蜡样(蜡质)芽孢杆菌亦不一定就能防治辣椒青枯病。
蜡样芽孢杆菌N15-2(Bacillus cereus)是由申请人采样分离获得一株新菌株,只对病原细菌有抑制作用、对病原真菌无抑制作用。蜡样芽孢杆菌N15-2能有效防治大白菜软腐病、辣椒青枯病、水稻白叶枯病、水稻细菌性条斑病等多种细菌性病害,国内外未见相关报道,具有创造性和新颖性。
发明内容
本发明的目的是提供一株具有较好应用前景的生防菌株-蜡样芽孢杆菌(Bacillus cereus)N15-2,该菌株已于2021年1月6日在中国微生物菌种保藏管理委员会普通微生物中心登记入册,地址是北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,保藏编号为CGMCC No.21599。
本发明提供了一种生防菌剂,其中有效活性成分由蜡样芽孢杆菌N15-2组成。
本发明所述的生防菌剂,其中蜡样芽孢杆菌N15-2只对病原细菌有抑制作用、对病原真菌无抑制作用。
本发明所述的生防菌剂,其中蜡样芽孢杆菌N15-2分泌的代谢产物中只包括蛋白酶一种抗菌相关物质。
本发明所述的生防菌剂,它是水剂。
本发明所述的水剂,其特征在于:各组成及重量百分含量如下:蜡样芽孢杆菌N15-2:89.5-99.8%;十二烷基磺酸钠:0.1-10%;山梨酸钾:0.1-0.5%;pH 6.5-7.0。
本发明所述的水剂灌根用于防治大白菜软腐病和辣椒青枯病,水稻叶面均匀喷施用于防治水稻白叶枯病和水稻细菌性条斑病。
本发明的优点在于:开发出一株对人畜安全、具有较好应用前景的生防菌株-蜡样芽孢杆菌N15-2,丰富了生防资源;该生防菌剂可以用来防治多种植物细菌病害,减少生产中化学药剂的使用量,同时改善生态环境、减少化学药剂在农产品中的残留量。
附图说明
下面结合附图和实施例对本发明进一步说明。
图1是蜡样芽孢杆菌N15-2对病原细菌的抑制作用
图2是蜡样芽孢杆菌N15-2产生蛋白酶。
图中A:蜡样芽孢杆菌N15-2;B:大白菜软腐病菌;C:辣椒青枯病菌;D:水稻白叶枯病菌;E:水稻细菌性条斑病菌。
具体实施方式:
实施例1
蜡样芽孢杆菌N15-2对病原细菌的抑制作用
A、供试病原细菌
供试细菌包括白菜软腐病菌、辣椒青枯病菌、水稻白叶枯病菌和水稻细菌性条斑病菌,由江苏省农业科学院植物保护研究所提供。用LB培养液(蛋白胨10.0g,酵母粉5.0g,NaCl 10g,蒸馏水1000mL)28℃、150rpm振荡培养2d,用无菌水稀释至108cfu/mL,备用。
B、试验方法:
将江苏省农业科学院筛选获得的蜡样芽孢杆菌N15-2在LB培养基上进行活化,然后移入50mL LB培养液中,于28℃、150rpm振荡培养2d,用无菌水稀释至108cfu/mL,备用。
吸取蜡样芽孢杆菌N15-2菌液5μL,点接在LB平板中央,28℃培养48h后用病原细菌稀释液喷雾,28℃培养过夜,调查抑菌圈直径。每处理重复3次,取平均值。设清水对照。
C、试验结果:
试验结果见图1。蜡样芽孢杆菌N15-2菌液对对供试的4种细菌均有一定的抑制作用,对白菜软腐病菌的抑菌圈直径为40.3mm;对辣椒青枯病菌的抑菌圈直径为25.9mm;对水稻白叶枯病菌的抑菌圈直径为36.0mm;对水稻细菌性条斑病菌的抑菌圈直径为39.8mm。
如图1所示。
实施例2
蜡样芽孢杆菌N15-2对多种植物病原真菌的抑制作用
A、供试病原真菌:
17种病原真菌包括水稻稻瘟病菌、水稻纹枯病菌、水稻稻曲病菌、水稻恶苗病菌、水稻胡麻叶斑病、小麦赤霉病菌、小麦根腐病菌、小麦纹枯病菌、小麦全蚀病菌、油菜菌核病菌、番茄灰霉病菌、番茄晚疫病菌、番茄早疫病菌、西瓜枯萎病菌、茄子黄萎病菌、辣椒疫病病菌、苹果腐烂病菌,均由江苏省农业科学院植物保护研究所提供。用PDA培养基(200g马铃薯、20g葡萄糖、20g琼脂和1000mL蒸馏水,pH 6.5-7.0)28℃培养5d,备用。
B、试验方法:
蜡样芽孢杆菌N15-2菌液制备同实施例1。
将病原真菌菌饼(直径为6mm)置于空白PDA平板一侧,距平板边缘30mm。平板另一侧,距平板边缘30mm,点接蜡样芽孢杆菌N15-2菌液5μL。28℃恒温培养,设清水对照,待对照平板病原菌菌落长满时调查各处理病原菌生长直径,计算抑制率。每处理重复3次,取平均值。
病原真菌抑制率=[(对照病原菌生长直径-处理病原菌生长直径)/对照病原菌生长直径]×100%。
C、试验结果:
试验结果表明:蜡样芽孢杆菌N15-2与目前报道的其他蜡样芽孢杆菌不同,不能对供试的17种病原真菌有抑制作用。
实施例3
蜡样芽孢杆菌N15-2代谢分泌物-抗菌相关物质的产生
供试材料:蛋白酶检测培养基:脱脂奶粉100g,琼脂20g,定容至1000mL。嗜铁素检测培养基:CAS 60.5mg,10mL三价铁溶液(1mmol·L-1FeCl3·6H2O),HDTMA 72.9mg,琼脂20g,定容至1000mL,pH 7.0。几丁质酶检测培养基:几丁质15g,MgSO4·7H2O 0.5g,FeSO4·7H2O 0.01g,K2HPO40.7g,KH2PO40.3g,琼脂20g,定容至1000mL,pH 7.0-7.2。纤维素酶检测培养基:蛋白胨10g,酵母粉10g,羧甲基纤维素钠10g,NaCl 5g,KH2PO41g,琼脂20g,定容至1000mL,pH 7.0。
蜡样芽孢杆菌N15-2菌液制备同实施例1。在各检测培养基平板中央点接蜡样芽孢杆菌N15-2菌液5μL,每处理重复3次。28℃恒温培养3-7d,观察。
蛋白酶检测:观察有无透明圈产生。若产生透明圈,说明该拮抗菌可分泌蛋白酶。
嗜铁素检测:28℃恒温培养7-10d后观察有无黄色晕圈产生。若产生黄色晕圈,说明该拮抗菌可分泌嗜铁素。
几丁质酶检测:观察有无透明圈产生。若产生透明圈,说明该拮抗菌可分泌几丁质酶,降解几丁质。
纤维素酶检测:观察菌落周围有无透明圈产生。若产生透明圈,说明该拮抗菌可分泌纤维素酶。
试验结果表明:蜡样芽孢杆菌N15-2只能分泌蛋白酶,利用检测平板中的蛋白质,在菌落周围形成透明圈(图2);不能分泌纤维素酶、嗜铁素和几丁质酶。
如图2所示。
实施例4
蜡样芽孢杆菌N15-2水剂的获得
A、菌种准备及活化。
LB培养基(同实施例1)按常规方法加琼脂后灭菌,做成斜面。接种蜡样芽孢杆菌N15-2,28℃培养2d。
B、液体发酵。
一级发酵:将上述培养好的试管斜面,用接种环移一环菌苔入300mL LB培养液。在28℃、150rpm振荡培养2d,备用。
二级发酵:培养基配方:豆饼粉1%,大豆粉0.5%,酵母膏0.15%,玉米粉1%,玉米浆0.5%,蔗糖0.5%,NaCl 0.05%,CaCO30.18%。灭菌前pH 7.0。2吨发酵罐中投料1吨。121℃灭菌30分钟。冷却至30℃左右时接种上述一级发酵菌种液300mL。液体发酵温度32℃,搅拌转速300rpm。通气量1∶1.5。发酵周期为18小时。
三级发酵:培养基配方同二级发酵。15吨发酵罐中投料10吨。121℃灭菌30分钟。冷却至30℃左右时接种上述二级发酵菌种液1吨。液体发酵温度32℃,搅拌转速300rpm。通气量1∶1.5。发酵周期为24小时左右,放罐。
C、水剂配置。
发酵液放入储罐中,加入十二烷基磺酸钠0.1-10%、山梨酸钾0.1-0.5%,搅拌均匀。测得发酵液pH值为6.5-7.0,活菌数为109cfu/mL。灌装成蜡样芽孢杆菌N15-2水剂。
实施例5
蜡样芽孢杆菌N15-2水剂对大白菜软腐病的防治试验
试验地点:江苏省农业科学院植物保护研究所旱地作物试验基地。
试验品种:中狮头大白菜,由江苏省徐淮地区连云港农科所提供。
试验药剂和对照药剂:蜡样芽孢杆菌N15-2水剂由实施例4提供。对照药剂为2%春雷霉素可湿性粉剂,购自吉林省延边春雷生物药业有限公司,按其田间推荐剂量使用。
试验方法:大白菜移栽后10d、17d、25d各灌根1次,每株灌药液250ml。试验设5个处理::蜡样芽孢杆菌N15-2水剂100倍稀释液、蜡样芽孢杆菌N15-2水剂200倍稀释液、2%春雷霉素可湿性粉剂750倍稀释液、2%春雷霉素可湿性粉剂1500倍稀释液和清水对照。4次重复,共20个小区,每个小区面积为20m2,随机排列。药后35d和55d每小区随机取样调查50棵大白菜,调查发病情况,计算发病指数和防效。病害分级标准如下:
0级:无症状;
1级:叶片始见水渍状褐色病斑;
3级:底部1-2叶片基部有明显病斑;
5级:外部叶片有1/3-1/2腐烂;
7级:外部叶片全部腐烂;
9级:整株腐烂。
病情指数=[(各级发病数×各级代表值)/(调查总株数×最高级代表值)]×100
防治效果(%)=[(对照的病情指数-防治的病情指数)/对照的病情指数]×100%
试验结果见表3。用药35d后蜡样芽孢杆菌N15-2水剂100倍稀释液的防效最高,为72.06%,对照药剂2%春雷霉素可湿性粉剂750倍稀释液的防效次之,为65.20%,两者防效无显著性差异。蜡样芽孢杆菌N15-2水剂200倍稀释液与2%春雷霉素可湿性粉剂1500倍稀释液的防效相当。药后55d,清水对照的病情指数增加显著,蜡样芽孢杆菌N15-2水剂100倍稀释液的防效为75.38%,2%春雷霉素可湿性粉剂750倍稀释液的防效为68.68%,两者防效显著性差异。结果表明:蜡样芽孢杆菌N15-2防治大白菜软腐病效果显著。
表3蜡样芽孢杆菌N15-2水剂对大白菜软腐病的田间防治效果
注:同一列不同小写字母表明呈显著性差异。
实施例6
蜡样芽孢杆菌N15-2水剂防治辣椒青枯病的田间小区试验
试验地点:江苏省农业科学院六合试验基地设施辣椒大棚。
试验品种:苏椒17号,江苏省农业科学院蔬菜研究所提供。
试验药剂:蜡样芽孢杆菌N15-2水剂由实施例4提供,对照药剂为3%中生菌素可湿性粉剂,购自深圳诺普信农化股份有限公司,按其田间推荐剂量使用。
试验方法:选取辣椒青枯病自然发病较重的1个温室大棚为试验大棚。试验用苗按常规操作,经穴盘育苗和定植两个阶段。穴盘播种时,将蜡样芽孢杆菌N15-2水剂按1∶50的比例稀释,每穴浇灌50毫升;定植移栽时,将蜡样芽孢杆菌N15-2水剂按照1∶100的比例进行灌根,每棵苗灌根200毫升;定植后隔15天,将蜡样芽孢杆菌N15-2水剂按照1∶100的比例进行灌根,每棵苗灌根500毫升。试验药剂:蜡样芽孢杆菌N15-2水剂、3%中生菌素可湿性粉剂和清水对照。每个处理小区约500棵苗,重复2次,处理小区随机分布。对照小区番茄、辣椒发病时调查发病情况,计算防效。
发病率=调查小区发病株数/调查总株数*100
防效=(处理小区的发病率-对照小区的发病率)/对照小区的发病率
试验结果见表3。对照病田中辣椒青枯病的发病率为45.72%,蜡样芽孢杆菌N15-2水剂对辣椒青枯病的田间防效为66.91%,3%中生菌素可湿性粉剂的防效为44.31%,两者呈显著性差异。试验结果表明:蜡样芽孢杆菌N15-2水剂能有效防治辣椒青枯病。
表3蜡样芽孢杆菌N15-2水剂对辣椒青枯病的田间防治效果
注:同一列不同小写字母表明呈显著性差异。
实施例7
蜡样芽孢杆菌N15-2水剂防治水稻细菌性条斑病和白叶枯病的田间小区实验
试验地点:江苏省农业科学院植物保护研究所水稻试验田。
试验药剂:蜡样芽孢杆菌N15-2水剂由实施例4提供,对照药剂为20%噻枯唑可湿性粉剂,购自浙江龙湾化工有限公司提供),按药剂说明书使用。
在水稻白叶枯病、细菌性条斑病发病初期(田间零星发病)叶片喷雾施药,间隔10d左右进行第二次防治。调查方法:发病初期每小区随机选取4点,每点5穴,调查每个分蘖最上部3片叶。施药前调查各处理发病基数,初次施药21d后再次调查病情指数。
分级标准:0级,无病斑;1级,病斑占叶片面积<5%;3级,病斑面积占叶片面积6%~15%;5级,病斑面积占叶片面积16%~30%;7级,病斑面积占叶片面积31%~50%;9级,病斑面积占叶片面积>50%。
病情指数=∑(各级发病数×各级代表值)/(调查总叶片数×最高级代表值)×100;
防治效果%=[1-(处理区药后病情指数×对照区药前病情指数)/(处理区药前病情指数×对照区药后病情指数)]×100%。
试验结果见表4。对照药剂-20%噻枯唑可湿性粉剂按照50g/亩的使用剂量对水稻细菌性条斑病和白叶枯病的防效分别为42.89%和45.44%,按照100g/亩的使用剂量对水稻细菌性条斑病和白叶枯病的防效分别为80.15%和76.28%。蜡样芽孢杆菌N15-2水剂按照33.3-50ml/亩的使用剂量,对水稻细菌性条斑病和白叶枯病的防效分别为38.17-44.22%和35.32-42.73%,防效与对照药剂50g/亩的使用剂量相当;按照100ml/亩的使用剂量对水稻细菌性条斑病和白叶枯病的防效分别为78.64%和73.56%,防效与对照药剂100g/亩的使用剂量相当。
表4蜡样芽孢杆菌N15-2水剂对水稻细菌性条斑病和白叶枯病的田间防治效果
Claims (2)
1.一种生防菌剂及其应用,其特征在于:所述生防菌剂中有效活性成分由蜡样芽孢杆菌(Bacillus cereus)N15-2组成;所述蜡样芽孢杆菌N15-2只对病原细菌有抑制作用、对病原真菌无抑制作用;所述蜡样芽孢杆菌N15-2分泌的代谢产物中只包括蛋白酶一种抗菌相关物质;所述生防菌剂为水剂;所述生防菌剂灌根用于防治大白菜软腐病和辣椒青枯病,水稻叶面均匀喷施用于防治水稻白叶枯病和水稻细菌性条斑病。
2.根据权利要求1所述的生防菌剂,其特征在于:各组成及重量百分含量如下:蜡样芽孢杆菌:89.5-99.8%;十二烷基磺酸钠:0.1-10%;山梨酸钾:0.1-0.5%。
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