CN116676221B - 一株高效毒杀植物寄生线虫的巨大芽胞杆菌及其应用 - Google Patents
一株高效毒杀植物寄生线虫的巨大芽胞杆菌及其应用 Download PDFInfo
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Abstract
本发明提供了一株高效毒杀植物寄生线虫的巨大芽胞杆菌及其应用,属于农业微生物技术领域。该菌株为巨大芽胞杆菌(Priestiamegaterium)BMB25109,其在中国典型培养物保藏中心的保藏编号为CCTCCNO:M2022898。本发明菌株可高效毒杀植物寄生线虫的活性在实验室发酵条件下及工业培养基发酵条件下均以盆栽实验的方式进行了验证,同时更进一步采用田间实验的方式证明了该菌株工业发酵液可以很好地控制南方根结线虫的定殖,其防效与市售药物路富达相当,存在显著的开发价值。
Description
技术领域
本发明属于农业微生物技术领域,具体涉及一株新菌株巨大芽胞杆菌及其在植物寄生线虫尤其是根结线虫、孢囊线虫防治方面的应用。
背景技术
植物寄生线虫是引起我国乃至世界农作物病害的重要病原微生物之一,具有隐蔽不可见、生活周期短、繁殖系数大、种类繁多、分布传播广和危害难以辨识等特点。植物寄生线虫宿主范围广泛,对小麦、玉米、水稻、烟草、马铃薯、大豆、蔬菜等粮食和经济作物造成严重减产,严重阻碍我国粮食、经济作物安全生产。据估计,全球农业生产中因其危害造成的经济损失每年高达1570亿美元,我国每年由于植物寄生线虫造成的损失也超过35亿美元,其中又以根结线虫和孢囊线虫为最。
根结线虫为专性活体营养寄生线虫,主要分布于3-10厘米的表层土壤中,通过在宿主根系建立由巨大细胞形成的取食位点来获取营养。而孢囊线虫是仅次于根结线虫的一类重要植物病原线虫,其孢囊为贮卵机构,由成熟雌虫经过角质膜逐渐增厚、***和颜色加深后直接转变形成,从而抵抗不良环境,保护内部贮卵。这两类植物寄生线虫分布广泛,寄主种类多,对我国的农业生产造成巨大损失。针对植物寄生线虫带来的病害,传统防治的措施有轮作、土壤改良、高温闷棚、暴晒耕作土、土壤药剂消毒等,但效果有限,无法根治。化学防治仍是目前防治线虫病的主要手段,然而大量施用化学药剂不仅使线虫抗药性问题愈发突出,更对人体健康及环境污染带来不容忽视的潜在威胁。
目前,生物农药作为新兴的农药类型,具有长效、稳定、安全低毒和环境友好等特点,为国家所重视,是现阶段农药开发的重点研究方向。
发明内容
鉴于现有资源及技术存在的不足,本发明的首要目的在于提供一株对植物寄生线虫具有高毒杀活性的生防菌,且此菌株对南方根结线虫等根结线虫属及大豆孢囊线虫等具有高毒杀作用,可用于植物寄生线虫病的生物防治。
为了实现上述技术目的,本发明人长期致力于芽胞杆菌杀线虫的资源挖掘及研究,从多个生态中分离、鉴定并保存了上千株有杀虫潜力的芽胞杆菌,发现多株具有高毒杀线虫活力的芽胞杆菌菌株,并对这些有杀虫活性的芽胞杆菌进行了深入研究,由此开展了对植物寄生线虫具有防治价值的芽胞杆菌资源的挖掘。通过大量的实验室生测、温室大棚盆栽及田间实验,筛选并鉴定出一株对防治植物寄生线虫具有显著效果的生防芽胞杆菌。根据其菌落形态特征、分子生物学实验及基因组测序后,发明人最终确定该菌为芽胞杆菌属,具体为巨大芽胞杆菌(Priestia megaterium),并命名为BMB25109。该菌株于2022年6月15日保藏于中国湖北省武汉市洪山区武汉大学中国典型培养物保藏中心,其保藏编号为CCTCC M 2022898。
需要说明的是,本发明分离得到的巨大芽胞杆菌(Priestia megaterium)具有如下形态学特征:
形态学特征:巨大芽胞杆菌BMB25109为革兰氏阳性菌,一种大形的产芽孢、以周生鞭毛运动的革兰氏阳性杆菌,杆状,末端圆,单个或呈短链状排列。其芽胞为圆形,中生或次端生。菌落呈液滴状,乳白色或淡黄色,不透明,边缘较为圆整,表面光滑、***,挑起时有粘连情况(参见图1(a)和图1(b))。
另外,本发明的第二个目的在于提供一株生防芽胞杆菌的应用,即上述的生防芽胞杆菌巨大芽胞杆菌BMB25109在防治农业线虫病害中的应用。以及,上述生防芽胞杆菌在制备防治农业线虫病害的生物农药和/或生物有机肥中的应用。
最后,本发明的第三个目的在于提供一种对植物寄生线虫具有高毒杀活性的制剂,该制剂的活性成分由上述巨大芽胞杆菌(Priestia megaterium)的发酵液制备而成。
相较于现有技术,本发明提供的生防芽胞杆菌BMB25109具有以下显著优点:
(1)该菌株对南方根结线虫及大豆孢囊线虫有高毒杀作用,与此同时,其对秀丽隐杆线虫毫无活性,杀虫专一性优异,对环境友好。
(2)相较于其它生物农药,该菌株的发酵上清液毒杀活性生效快,其5倍稀释液对南方根结线虫及大豆孢囊线虫的生物活性测定时1h内具有100%致死活性。
(3)该菌株发酵产生杀线虫活性物质所需时间短,在实验室内发酵10h后即产生了大量的活性物质,具有很好的毒杀效果。
(4)将本发明菌株BMB25109的发酵液施用于根结线虫侵染严重的烟草幼苗,结果显示在盆栽试验中对根结线虫有很好的防效,与化学农药效果相当。
(5)该菌株在发酵期间产生了丙酰胺、乙酸、异丙酸、异丁酸等多种对根结线虫具有毒杀活性的酰胺类及小分子酸类化合物。
附图说明
图1:(a)为巨大芽胞杆菌BMB25109菌株的菌落形态;(b)为巨大芽胞杆菌BMB25109菌株于光学显微镜下的细胞形态(目镜10×,物镜100×);
图2:(a)为本发明的巨大芽胞杆菌(Priestia megaterium)BMB25109发酵上清处理组培养1h的根结线虫;(b)为空白对照清水处理组培养48h后的根结线虫;(c)为为本发明的巨大芽胞杆菌(Priestia megaterium)BMB25109发酵上清处理组培养48h的秀丽隐杆线虫。
图3:不同浓度发酵上清液处理4h的杀线虫活性曲线图,其中Mi:南方根结线虫;Hg:大豆孢囊线虫;Ce:秀丽隐杆线虫。
图4:(a)为等量ICPM培养基空白对照组盆栽实验根样本;(b)为路富达对照组盆栽实验根样本;(c)为巨大芽胞杆菌BMB25109菌株摇瓶发酵液处理组盆栽实验根样本。
图5:巨大芽胞杆菌BMB25109菌株工业发酵液对盆栽烟草根系根结数的影响,其中**为在t检验中P值<0.005,***为P值<0.001;
图6:(a)为市售农药路富达处理组与空白对照田间实验根样本对比图,其中左根样本为空白对照,右根样本为路富达处理组;(b)为巨大芽胞杆菌BMB25109菌株工业发酵液处理组与空白对照田间实验根样本对比图,其中左根样本为空白对照,右根样本为发酵液300ml/株处理组。
具体实施方式
以下为本发明的具体实施例,对本发明的技术方案做详细描述,实施例中未注明具体技术操作或条件者,均以本领域文献所述的技术或条件进行操作,所用试剂或仪器未注明生产厂商者,均为可正常购买的常规产品。
实施例1:菌株的分离和活性检测
1.实验方法
1.1根结线虫的培养及虫卵孵化
1)温室条件下根结线虫的养殖及虫卵孵化
(1)将易感品种烟草种子播种于营养土与沙土2:1比例混合的土壤中,待其适宜条件下发芽生长;
(2)待烟草幼苗株高长至10cm时从土壤中拔出,流水洗净根上的土壤;
(3)移液器吸取南方根结线虫接种于烟草根表,再移栽到大盆的土壤中,每盆接种量约为2000-3000头;
(4)约30-45天后烟草根系受根结线虫侵染的程度适宜,可拔取病株挑卵块;
(5)先将卵块挑取到装有灭菌ddH2O中的瓶中,置于20℃培养箱孵化3天,时间适当延长可收获更多根结线虫J2s;
(6)如果想要获取更干净的根结线虫J2s,可以选取被根结线虫侵染严重、根结大且多的烟草根系,将其尽可能的剪碎;
①将其全部浸入10%次氯酸钠溶液中,搅拌处理8min左右;
②将悬液分别过60目、100目、200目、500目的套筛,根结线虫散卵粒于500目筛网层收集;
③收集到的根结线虫卵悬液在500目膜上进行孵化,置于20℃培养箱孵化3天,时间适当延长可收获更多根结线虫J2s。
1.2菌种培养方法及培养基
菌种活化:将斜面保藏的菌种(巨大芽胞杆菌BMB25109)转接到LB平板上,37℃培养24h备用;种子液的制备:挑取一环活化好的菌种,接入50mL LB液体培养基中,37℃条件下220rpm振荡培养12h备用;发酵液摇瓶培养:将菌种子液按1%的接种量接种到发酵培养基中,37℃,220rpm振荡培养指定时间。
种子培养基(LB液体培养基):酵母膏0.5%,蛋白胨1%,NaCl 1%,pH7.0,121℃,灭菌30min。
菌种保藏培养基(LB固体培养基):酵母膏0.5%,蛋白胨1%,NaCl 1%,琼脂2%,pH7.0,121℃,灭菌30min。
发酵培养基(ICPM培养基):葡萄糖0.5%,蛋白胨0.5%,KH2PO40.05%,MgSO40.05%,115℃,灭菌20min。
1.3发酵液处理
将发酵液于4℃,10000rpm;下离心10min后吸取上清,0.22μm孔径过滤器过滤得到上清液,获取发酵上清液,将上清原液2倍、5倍、10倍、50倍稀释,分别测定对线虫的致死率。
1.4植物寄生线虫生物活性测定
利用96孔板进行植物寄生线虫体外生物测定的具体步骤如下:
(1)吸取孵化3天的线虫J2s于1.5ml离心管中,5000rpm离心1min,弃上清;
(2)适量ddH2O重悬线虫,显微镜镜检确定虫体密度,线虫终密度约为30头/μL;
(3)配制不同浓度的待测发酵液上清,在超净工作台中用移液器将发酵液和线虫加入96孔生物测定板中对应的孔中(每孔20-40头J2s),将灭菌ddH2O及ICPM培养基作为空白对照,每孔总体积为100μL;每个处理重复三次;
(4)将96孔生测板用封口膜封好后放置在20℃培养箱中,4h后统计每孔线虫总数和死亡数。
1.5BMB25109发酵上清液对根结线虫有高毒杀活性
据附图2及附图3得,芽胞杆菌菌株BMB25109的发酵上清液对南方根结线虫(Mi)的二龄幼虫及大豆孢囊线虫有强致死活性。原液在30min时即可达到对南方根结线虫100%的致死活性,随着稀释倍数的增加,致死活性降低,杀死全部根结线虫所需时间增长,但直至10倍稀释液仍含有较强活性,可在4h内毒杀样品孔内约90%的根结线虫。
实施例2:菌株BMB25109防治烟草根结线虫的盆栽实验
1、实验室培养基摇瓶发酵
1)挑选长势、大小一致的健康烟草幼苗作为供试幼苗。在烟草苗根部接种1000头二龄幼虫,加摇瓶发酵液原液20mL,对照组接种等体积的ICPM培养基和路富达稀释液。处理完成后置于合适环境下培养。30-40天后统计根结数量。盆栽实验共设3个处理,分别为:CK(等量ICPM培养基)、BMB25109的ICPM菌悬液、阳性对照路富达稀释液(30ul路富达以无菌水定容至20ml),每个处理设置3个重复。
2)统计根结数量:轻轻拔起烟草植株,取出完整根系。后将根于流水下冲洗干净并目测计数根结数量。根结数量表示方法采用每株烟草根结数。
以BMB25109菌株发酵液处理为处理组,路富达为对照组,ICPM培养基为CK进行盆栽实验。结果如图4所示:相较于CK组,含菌液的处理组烟草植株的根结系数显著减少,且与市面上现存防治线虫的化学农药路富达的防治效果对比也基本相当。证明了菌株发酵液在盆栽过程中发挥了其杀虫活性,抑制了根结线虫对植株的侵染和繁殖,存在着应用于农业植物寄生线虫防治的潜力。
2、工业50L发酵罐发酵
1)工业发酵所用培养基配方以1L培养基为标准,具体为:玉米浆36g;玉米粉5g;黄豆饼粉18g;葡萄糖5g;鱼粉3g;K2HPO42.16 g;MgSO4·7H2O 0.75g;CaCO31.5 g;(NH4)2SO42g;蒸馏水定容至1L;调pH至7.0)。
2)以培养基体积的0.5%接种,发酵时长为32~36h。
3)盆栽所用植株约为3~4周龄,长势一致的烟草幼苗,于幼苗根部接种800头J2期南方根结线虫,发酵液处理组分别以50ml、150ml、300ml为基准,同时以等体积无菌培养基作为对照,以路富达稀释液为阳性对照(每株施加药物量为30ul,以无菌水适当稀释),实验组及对照组各设5个重复,5周后统计各组根结数量。
以工业培养基发酵该菌株,得到菌株发酵液,以无菌水作为CK,分50ml/株、150ml/株、300ml/株3组处理组进行工业发酵液的盆栽实验,5周后统计根结数情况。如图5所示,发酵液的处理,尤其是150ml/株和300ml/株的处理显著降低了植株的根结数量,与上述实验室发酵液的结果相吻合,为开展田间试验提供了的数据支撑。
实施例3:菌株BMB25109防治番茄根结线虫的田间实验
田间试验采用的发酵液与上述工业发酵液一致,于武汉市蔬菜所根结线虫侵染严重的土病地中进行,以清水处理作为CK,市售药物路富达为对照处理(用量同上),发酵液分为150ml/株及300ml/株两组处理,以上各组处理均有10个重复。待灌根处理40d后收集样本,对实验数据进行收集处理。
以同样的工业发酵液施用到田间,并采用盆栽实验中效果显著的150ml/株、300ml/株两个处理组,每组采用十个重复,采用长势一致且株高合适(约为10cm)的番茄幼苗为处理对象,具体防治效果的评判指标及结果见下表1及表2。
表1:根结指数分级标准
根结指数=[10A+9B+8C+7D+6E+5F+4G+3H+2I+1J+0K/10N]×100上式中A~K为对应级别的植株数量,N为调查总植株数,N=A+B+C+D+E+F+G+H+I+J+K。
防治效果(%)=[(对照组根结指数-处理组根结指数)/对照组根结指数]×100%。
表2:BMB25109发酵液处理后的田间防治效果
根结指数 | 相对防效 | |
实施处理组1(150mL/株) | 14 | 81.08% |
实施处理组2(300mL/株) | 9 | 87.84% |
路富达 | 10 | 86.49% |
对照组 | 74 | - |
实施例4:菌株BMB25109发酵液的活性物质探究
将处理并浓缩后的发酵上清(发酵液上清处理方法同实施例3中1.3节)通过安捷伦的GC-MS气相质谱进行物质鉴定,使用DB-WAX极性柱。
通过气相色谱质谱联用分析后发现,BMB25109菌株在发酵过程中产生了多种小分子物质,诸如丙酰胺、乙酸、异丙酸、2-甲基丁酸等。
本发明从天然土样中分离鉴定出一株对植物寄生线虫尤其是南方根结线虫与大豆孢囊线虫具有高毒杀活性的巨大芽胞杆菌BMB25109,该芽胞杆菌在培养基条件下发酵,其上清可对上述两种线虫产生极强的毒杀活性。该活性在实验室发酵条件下及工业培养基发酵条件下均以盆栽实验的方式进行了验证,同时更进一步采用田间实验的方式证明了该菌株工业发酵液很好的控制了南方根结线虫的定殖,其防效与市售药物路富达相当,存在显著的开发价值。
Claims (4)
1.一种巨大芽胞杆菌(Priestia megaterium)BMB25109,其在中国典型培养物保藏中心的保藏编号为CCTCC NO:M 2022898。
2. 权利要求1所述的巨大芽胞杆菌(Priestia megaterium)BMB25109在防治农业线虫病害中的应用,所述的农业线虫选自如下的一种或两种以上:根结线虫属、孢囊线虫属;所述的根结线虫属线虫为南方根结线虫,所述的孢囊线虫属线虫为大豆孢囊线虫。
3. 权利要求1所述的巨大芽胞杆菌(Priestia megaterium)BMB25109在制备防治农业线虫病害的生物农药和/或生物有机肥中的应用,所述的农业线虫选自如下的一种或两种以上:根结线虫属、孢囊线虫属;所述的根结线虫属线虫为南方根结线虫,所述的孢囊线虫属线虫为大豆孢囊线虫。
4. 一种对南方根结线虫和大豆孢囊线虫具有高毒杀活性的制剂,其特征在于,该制剂的活性成分由权利要求1所述巨大芽胞杆菌(Priestia megaterium)BMB25109的发酵液制备而成。
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5733544A (en) * | 1996-11-18 | 1998-03-31 | University Of Saskatchewan | Nematicidal bacillus strain and metabolite and methods of use thereof |
WO2014121650A1 (zh) * | 2013-02-05 | 2014-08-14 | 中国农业大学 | 一株短小芽孢杆菌及其在防治小麦禾谷孢囊线虫病中的应用 |
CN109868237A (zh) * | 2019-02-15 | 2019-06-11 | 湖南农业大学 | 一种巨大芽孢杆菌及其应用 |
CN110669691A (zh) * | 2019-10-17 | 2020-01-10 | 青岛农业大学 | 一种防治植物线虫病害的巨大芽孢杆菌及其应用 |
CN115572688A (zh) * | 2022-07-18 | 2023-01-06 | 华中农业大学 | 一种对植物寄生线虫高毒杀活性的蜡状芽胞杆菌及其应用 |
-
2023
- 2023-05-25 CN CN202310596668.6A patent/CN116676221B/zh active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5733544A (en) * | 1996-11-18 | 1998-03-31 | University Of Saskatchewan | Nematicidal bacillus strain and metabolite and methods of use thereof |
WO2014121650A1 (zh) * | 2013-02-05 | 2014-08-14 | 中国农业大学 | 一株短小芽孢杆菌及其在防治小麦禾谷孢囊线虫病中的应用 |
CN109868237A (zh) * | 2019-02-15 | 2019-06-11 | 湖南农业大学 | 一种巨大芽孢杆菌及其应用 |
CN110669691A (zh) * | 2019-10-17 | 2020-01-10 | 青岛农业大学 | 一种防治植物线虫病害的巨大芽孢杆菌及其应用 |
CN115572688A (zh) * | 2022-07-18 | 2023-01-06 | 华中农业大学 | 一种对植物寄生线虫高毒杀活性的蜡状芽胞杆菌及其应用 |
Non-Patent Citations (2)
Title |
---|
Hadeel Badri Dawood等.Effects of bacterial isolate,Priestia megaterium HB8 and its filtrate on Controlling the root-knot nematode,Meloidogyne incognita.《Caspian Journal of Environmental Sciences》.2023,第21卷(第1期),第105-114页. * |
生防菌K-8对南方根结线虫的防治及其鉴定;梁建根等;《中国农学通报》;20111231;第27卷(第21期);第282-286页 * |
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