CN116656521A - 一种具有抗病、杀虫作用的云南微球菌SfA3菌株及其应用 - Google Patents
一种具有抗病、杀虫作用的云南微球菌SfA3菌株及其应用 Download PDFInfo
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Abstract
本发明公开了一种具有抗病、杀虫作用的云南微球菌SfA3菌株及其应用。SfA3菌株于2022年4月29日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:62446。本发明研究显示SfA3菌株对稻瘟病菌、香蕉枯萎病菌、番茄酸腐病菌、番石榴果腐病菌、菜心炭疽病菌和水鬼蕉叶斑病菌等植物病原真菌具有良好的拮抗效果;同时SfA3菌株具有增效AcMNPV病毒杀虫活性作用,能显著提高对鳞翅目害虫的死亡率,降低害虫半数生存期,提高病毒AcMNPV在防治草地贪夜蛾等害虫的杀虫效率。SfA3菌株能用于蔬菜害虫的防控以及病原菌的抑制,可作为微生物杀虫剂或增效剂来加强对害虫的防控。
Description
技术领域
本发明属于农业微生物技术领域。更具体地,涉及一种具有抗病、杀虫作用的云南微球菌SfA3菌株及其应用。
背景技术
草地贪夜蛾(Spodoptera frugiperda)属于鳞翅目(Lepidoptera)夜蛾科(Noctuidae),是一种世界性的重要农业害虫。主要危害玉米、马铃薯、甘蔗等重要经济作物,具有很强的迁飞能力。草地贪夜蛾对多种农药和不同类型的转基因玉米产生不同程度的抗性。目前,传统农药如有机磷酸酯类、拟除虫菊酯类等农药对入侵并定殖我国的草地贪夜蛾并没有较好的防效,广东地区的草地贪夜蛾也对新型农药如甲维盐和氯虫苯甲酰胺产生中等水平的抗性。因此,许多学者把研究的目光转向了生物防治,从长远来看,可持续防治草地贪夜蛾应更侧重于使用迁飞监测、生态调控的手段,运用性信息素引诱剂、天敌昆虫(如卵黑蜂Telenomus remus、异色瓢虫(Harnonia axyridis)、病原微生物(如莱氏绿僵菌Metarhizium riley、苏云金芽胞杆菌Bacillus thuringiensis,Bt)进行生物防治也是防控草地贪夜蛾的关键措施。
核型多角体病毒Nucleopolyhedrovirus(NPV)是昆虫杆状病毒当中发现较早,研究较深的一类病毒。通过昆虫取食植物叶片时吞入病毒包涵体OBs,随后在昆虫肠道进行感染。其中,苜蓿银纹夜蛾核型多角体病毒Autographa californica multiplenucleopolyhedrovirus(AcMNPV)是一种宿主范围较广的α-杆状病毒,主要用于鳞翅目害虫的防治,主要症状表现为初期体色变浅,多为白色或浅黄色,食欲减退,行动迟缓,后期体内组织液化死亡,体壁极易戳破,并伴随着乳白色脓液流出。野外中常见感染病毒的虫子悬挂而死的现象。草地贪夜蛾幼虫对AcMNPV口服感染有较强的抗性,但由于病毒单剂存在见效慢、活性低的问题,因此在田间防治上常常以复配药剂的方式施药。同时杆状病毒杀虫剂杀虫谱窄、作用时间长等局限性限制了其推广应用。因此缩短作用期、扩大杀虫谱成了研究杆状病毒增效的研究重点。
而利用宿主自身特性如体内微生物的协同作用来增强病毒杀虫效果是目前杆状病毒増效途径的研究方向之一。昆虫肠道微生物主要包括细菌、真菌、原生生物和古生菌。不同种类的昆虫由于取食习惯和栖息环境不同,体内的肠道菌群也相同。这些肠道微生物与昆虫共同进化,相互作用,昆虫为肠道微生物提供生存场所及养料,肠道微生物对昆虫的生长发育、消化分解、新陈代谢以及免疫防御等方面产生极其重要的影响。且农药的长期使用也会改变草地贪夜蛾肠道微生物群落构成,并存在能够降解农药的肠道共生菌。
目前还鲜少关于增效AcMNPV杀虫活力的研究报道,以此来防控草地贪夜蛾,更加缺乏能用于增效AcMNPV杀虫活力以及能防控草地贪夜蛾的微生物菌株。本研究为了对草地贪夜蛾的有效防控提供更多的方法和手段,还需研究更多具有抗性或具有更多更好作用的菌株作为微生物杀虫剂或增效剂用来加强对害虫的防控。
发明内容
本发明要解决的技术问题是克服现有上述问题的缺陷和不足,提供一种具有抗病、杀虫作用的云南微球菌SfA3菌株及其应用。
本发明的目的是提供具有抗病性和增效AcMNPV杀虫活力的云南微球菌SfA3菌株。
本发明另一目的是提供所述菌株的应用。
本发明再一目的是提供一种抑制植物病原真菌和/或防控鳞翅目害虫的微生物菌剂。
本发明又一目的是提供一种AcMNPV病毒增效杀虫剂。
本发明上述目的通过以下技术方案实现:
本发明提供一种具有抗病、杀虫作用的云南微球菌(Micrococcus sp.)SfA3菌株,该菌株于2022年4月29日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:62446。SfA3菌株分离自草地贪夜蛾中肠中,其16S rDNA的核苷酸序列如SEQ ID NO.1所示,经形态学特征和16S rRNA系统发育分析鉴定为云南微球菌(Micrococcus yunnanensis)同源性达到99.08%。SfA3菌株形态特征为:球状,圆形,凸起,光滑,不透明,黄色,外周无淡色黄晕,属于革兰氏阳性菌G+。
本发明研究显示SfA3菌株对甜菜夜蛾、小菜蛾、菜青虫、棉铃虫、斜纹夜蛾等都有很好地杀虫效果,具有广谱的杀虫作用,能用于防治其害虫;同时采用SfA3菌与AcMNPV一起处理后,其杀虫活性得到显著提升,显示SfA3菌株具有增效AcMNPV病毒杀虫活性作用,可以进一步增强AcMNPV的杀虫活性。把SfA3菌株和AcMNPV病毒混合添食可快速致死草地贪夜蛾,AcMNPV病毒对草地贪夜蛾的死亡率能达到100%,死亡时间有10天降低到7天;本发明将草地贪夜蛾肠道菌群清除后,肠道菌SfA3的回补可显著提高草地贪夜蛾幼虫对AcMNPV病毒的敏感性(P<0.0001),而且SfA3细菌的回补均降低了半数生存期(Median SurvivalTime),使草地贪夜蛾半数生存期由5.0天降低至3.5天。
同时SfA3无菌滤液产生的抑菌物质可以有效抑制大部分病原真菌的生长,如对香蕉枯萎病菌(Fusarium oxysporum f.sp.cubense)、菜心炭疽病菌(Colletotrichumhigginsianum Sacc.)、水鬼蕉叶斑病菌(Botrytis cinerea)、番茄酸腐病菌(Geotrichumcandidum)和番石榴果腐病菌(Pstalotiopsis cocuuli Guba)具有很好的抑菌性。
本发明提供云南微球菌SfA3菌株或其发酵液在抑制植物病原真菌和/或防治鳞翅目害虫、在制备抑制植物病原真菌和/或防控鳞翅目害虫的微生物菌剂、在制备AcMNPV病毒增效杀虫剂中的应用。
本发明提供一种抑制植物病原真菌和/或防控鳞翅目害虫的微生物菌剂,含云南微球菌SfA3菌株或其发酵液。
优选地,所述发酵液为发酵上清液。
优选地所述发酵上清液浓度为1.0×105~1.0×1010CFU/mL。
更优选地,发酵上清液浓度为1.0×107CFU/mL。
优选地,所述发酵液为发酵培养基10h后的上清液。
本发明提供一种AcMNPV病毒增效杀虫剂,含所述云南微球菌SfA3菌株或其发酵液。
进一步地,所述植物病原真菌为香蕉枯萎病菌(Fusarium oxysporumf.sp.cubense)、菜心炭疽病菌(Colletotrichum higginsianum Sacc.)、水鬼蕉叶斑病菌(Botrytis cinerea)、番茄酸腐病菌(Geotrichum candidum)和番石榴果腐病菌(Pstalotiopsis cocuuli Guba)中的一种或多种。
进一步地,所述鳞翅目害虫为草地贪夜蛾、斜纹夜蛾、甜菜夜蛾、菜青虫、小菜蛾中的一种或多种。
本发明具有以下有益效果:
本发明提供一种具有增效AcMNPV杀虫活力的SfA3菌株,该菌株能够在体外发酵培养,具有增效AcMNPV病毒作用,能提高AcMNPV在蔬菜害虫草地贪夜蛾的杀虫效率,与AcMNPV病毒混合添食可快速致死草地贪夜蛾,降低了草地贪夜蛾的半数生存期;同时,SfA3菌株对多种害虫以及病原真菌都有很好的抑制作用效果,对甜菜夜蛾幼虫、小菜蛾幼虫、菜青虫幼虫、棉铃虫幼虫、斜纹夜蛾幼虫等都有很好地杀虫效果,具有广谱的杀虫作用,能用于防治其害虫;而SfA3无菌滤液产生的抑菌物质可以有效抑制香蕉枯萎病菌、菜心炭疽病菌、水鬼蕉叶斑病菌、番茄酸腐病菌和番石榴果腐病菌的生长,具有很好地抑菌性。因此,SfA3菌株作为新的生防微生物菌株,用于蔬菜害虫的防控以及病原菌的抑制,具有很好的生物防治潜力和应用前景;可作为微生物杀虫剂或增效剂来加强对害虫的防控,为蔬菜害虫的防治提供新的微生物菌株和防治方法策略。
附图说明
图1为草地贪夜蛾幼虫侵染症状效果图;
图2为AcMNPV侵染后的草地贪夜蛾肠道分离细菌的分离结果图(注:图片分别为AcMNPV侵染后24h、72h、96h、168h的草地贪夜蛾肠道分离菌在LB平板、NA平板、EB平板及SS平板的生长状态图);
图3为病毒侵染后的草地贪夜蛾肠道SfA3分离菌48h平板培养图;
图4为草地贪夜蛾SfA3肠道可培养细菌的16S rDNA系统发育分析图(注:通过Mega7.0软件的邻接法(Neighbor-Joining)构建系统发育树,标尺长度0.0050代表遗传距离,各个菌株的序列GenBank登录号在靠近进化枝的位置);
图5为SfA3菌株发酵液的平板对扣菌落直径差异分析图(注:*和**分别表示在p<0.05和p<0.01水平上差异显著,***代表p<0.001,****代表p<0.0001,ns代表无显著差异,CK为对照);
图6为SfA3+AcMNPV对鳞翅目害虫的广谱性测定结果图;
图7为草地贪夜蛾肠道细菌的清除效果图(48h);
图8为菌株SfA3回补对AcMNPV病毒毒力的影响结果图(注:无菌草地贪夜蛾幼虫(n=60)经AcMNPV病毒处理的存活率分析;实验进行三次生物学重复,误差棒表示标准差;采用log-rank(Mantel-Cox)检验,p<0.01视为具有显著性差异)。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
实施例1草地贪夜蛾肠道微生物的分离纯化
1、草地贪夜蛾AcMNPV病毒感染
挑选长势一致的2龄草地贪夜蛾幼虫放于24孔板中,饥饿处理12h。每1g饲料加入约500μL含10%蔗糖的AcMNPV溶液溶液,再滴入1-2滴可食用蓝色素,搅拌均匀后取适量饲喂草地贪夜蛾幼虫,处理两天后用正常饲料进行喂养。饲养过程放置在人工气候箱中,光照、温湿度等条件均与正常人工饲料饲养一致。饲养期间观察虫子形态变化并在体视镜下拍照保存。
结果如图1所示,发现幼虫感染病毒后,初期症状不明显,第4天出现食欲下降、虫体肿大、腹部瘫痪等特征,相比于正常饲料饲养的幼虫体色变浅发白,虫体软化,大约一周后组织液化死亡,体壁极易戳破,流出乳白色脓液,肠道溃烂,无臭味。
2、供试昆虫解剖
整个解剖过程需在超净工作台中进行,所有的解剖用具需在紫外灯照射处理下灭菌30min。苜蓿银纹夜蛾核型多角体病毒侵染后24h、72h、96h、168h进行肠道菌群的分离。先用70%酒精迅速将虫体表面进行简单消毒,再用PBS溶液洗去虫体表面残余的酒精,然后用解剖剪小心剪去幼虫的臀足,用镊子缓慢拉出肠道,加入200μL PBS,磨成匀浆。
3、肠道微生物的分离
上述内容物用PBS按10-5进行稀释中肠原液,稀释后取10μL分别涂布于LB平板、NA平板、EB平板及SS平板,用涂布棒涂抹均匀,置于30℃培养。期间每24h观察一次。结果如图2所示,可知选择性培养基因营养成分不同,菌落的颜色、大小也有所区别。相比于LB平板,NA平板上的菌落更小,EB平板上菌落的颜色偏黄。病毒侵染后在SS平板上无菌落生长。在不同培养基上观察在不同时间点取样处理后的肠道细菌分离培养也无显著差异。由于在肉眼观察,挑取形态不一的菌落纯化培养过程中受人为因素影响较大,需通过测序确定肠道可培养细菌的分离结果。
4、细菌分离物的纯化培养
用接种环挑取具有代表性的单菌落,在新的LB平板上划线后倒置于30℃培养,待新的菌落长出后进行4-5次的连续划线得到纯化后的菌种。将单菌落接入LB液体培养基摇菌培养至对数生长期,在超净工作台中将菌液与50%甘油按3:7混合于1.5mL离心管中,使甘油菌终浓度为15%,置于-40℃保存。
实施例2菌株的鉴定
1、菌株的形态鉴定
在LB固体培养基上,菌株呈球状,圆形,凸起,光滑,不透明,黄色,外周无淡色黄晕,属于革兰氏阳性菌G+,命名为SfA3菌株,菌株SfA3的平板照片如图3所示,可知菌株SfA3在平板上划线后菌株呈圆形,凸起、光滑、整齐,不透明,黄色菌落。
2、16S rDNA鉴定
挑取纯化后的菌种于LB液体培养基过夜培养,并对纯化后的肠道分离菌进行编号,提取细菌DNA(TIANamp Bacteria DNA Kit,天根生物公司)。取5μLDNA溶液进行1.5%的琼脂糖凝胶电泳检测,并用NanoDrop测定DNA浓度,使其浓度保持在20~1000ng,OD260/280在1.8~2.0范围内。以提取的DNA为模板,16S rDNA通用引物27F:5’-AGTTTGATCMTGGCTCAG-3’和1492R:5’-GGTTACCTTGTTACGACTT-3’,为上下游引物进行PCR扩增,PCR扩增的反应体系如表1所示,配制好反应体系后轻轻混匀后,短暂离心,置于PCR仪上;PCR扩增的程序为:98℃预变性30s;98℃变性10s,55℃退火30s,72℃延伸20s,35个循环;72℃,5min;4℃保存。
表1细菌16S rDNA PCR扩增体系
PCR产物经1%琼脂糖凝胶检测并切胶回收纯化后送广州擎科生物技术有限公司测序。
3、菌株的系统发育分析
根据测序结果进行分析DNA双向测序质量并用SeqMan(DNAStar)拼接序列,然后将该序列与美国国家生物技术信息中心(NCBI)中rRNA/ITS数据库(https://www.ncbi.nlm.nih.gov/)使用BLAST进行序列比对。用Chromas软件拼接序列,然后将该序列与NCBI中rRNA/ITS数据库进行blast比对。GenBank序列号以及与数据库中已知细菌比对信息见下表2所示,SfA3菌株的序列信息如SEQ ID NO.1所示。
表2草地贪夜蛾肠道分离菌SfA3 16S rDNA序列比对
选取SfA3菌株的近缘序列后用ClustalW软件进行多序列比对分析,选取各个菌株的近缘序列用Mega7.0软件的邻接法(Neighbor-Joining)构建系统发育进化树,系统树的自展值为1000次重复,调整bootstrap值并检验进化树的可靠性。结果如图4所示,可知SfA3与云南微球菌(Micrococcus yunnanensis)的相似度最高,推测可能为同一菌种。
4、菌株SfA3的生理生化特征测定
细菌生理生化测定参考《常见细菌系统鉴定手册》的方法进行,采用细菌微量生化反应管。对可利用的碳源测试结果如表3所示。细菌SfA3的生理生化测定结果显示SfA3是革兰氏阳性、好氧菌,能利用甘油,硝酸盐还原,淀粉水解,不分解纤维素;不能利用赤癣醇、D-阿拉伯糖、L-阿拉伯糖、核糖、D-木糖、L-木糖、阿东醇、半乳糖、果糖、甘露糖、山梨糖、鼠李糖、密二糖、D-松二糖、蔗糖、海藻糖、菊糖、松叁糖、棉子糖、淀粉和肝糖、木糖醇均不能利用。在温度4~45℃、pH值5.0~11.0均能生长。在没有NaCl,的培养基上生长缓慢,在NaCl含量为1%~15%的培养基上均生长良好,在NaCl含量为20%的培养基上仍可生长,可见菌株SfA3具有一定的需盐性和耐碱性。比较SfA3和云南微球菌标准菌株,发现二者主要生理生化特征完全一致。
表3 SfA3生理生化鉴定
注:“+”为阳性反应,“﹣”为阴性反应。
通过以上鉴定结果可以确定菌株SfA3分类单元属于细菌界,放线菌门,微球菌属(Micrococcus sp.),云南微球菌(Micrococcus yunnanensis)。因此本发明筛选获得的菌株鉴定为云南微球菌(Micrococcus sp.)SfA3,并于2022年4月29日保存于广东省微生物菌种保藏中心,保藏号为GDMCC No:62446,保藏地址:广州市先烈中路100号大院59号楼5楼。
实施例3 SfA3菌株的抑菌活性
本发明采用平板法测定SfA3菌株对植物病原真菌的抑菌能力。所用固体培养基为PDA培养基;其配制方法为:46g马铃薯葡萄糖琼脂培养基(不含抗生素)制成1L,将培养基加热至完全溶解,121℃高压灭菌20分钟。
1、SfA3菌液的制备方法
将保存于-80℃的甘油菌以1:100的体积比接入新鲜的液体LB培养基中,在220rpm,37℃条件下活化12h获得种子菌液,再以1:100的体积比取种子菌液接入新鲜的液体LB培养基,220rpm,37℃摇10h,发酵液于8000r/min离心10min后收集上清。
2、SfA3无菌滤液处理
将收集的上清,在无菌条件使用0.22μm细菌微孔滤膜过滤上清液除菌,采用生长速率法,按滤液:培养基1:5、1:10、1:20的比例配置培养基,即按比例取一定量的滤液与15mL灭菌冷却至60℃的PDA培养基混合均匀制成平板,以添加等量无菌水代替滤液的平板作为对照,将直径为0.5cm生长旺盛的植物病原菌菌饼接种在平板上。每个处理重复3次。置于28℃恒温培养,待对照长满后,测量对照和处理的菌落直径,检测抑菌效果。
3、SfA3无菌滤液的抑菌活性
分别将病原菌稻瘟病菌(Magnaporthe oryzae)、香蕉枯萎病菌(Fusariumoxysporum f.sp.cubense)、菜心炭疽病菌(Colletotrichum higginsianum Sacc.)、水鬼蕉叶斑病菌(Botrytis cinerea)、番茄酸腐病菌(Geotrichum candidum)和番石榴果腐病菌(Pstalotiopsis cocuuli Guba)接种到以不同比例(1:5、1:10、1:20)的SfA3菌株的无菌滤液制成的培养基中作为处理组,以不添加菌的培养基作为CK对照组,培养7d后统计对照组和三种不同比例的处理组的病原菌直径,进行差异分析。
结果如图5所示,处理组的香蕉枯萎病菌、菜心炭疽病菌、水鬼蕉叶斑病菌、番茄酸腐病菌和番石榴果腐病菌的菌落直径均小于对照组,达到了极显著水平,稻瘟病菌的处理组和对照组之前差异不显著,表明SfA3无菌滤液产生的抑菌物质可以有效抑制大部分病原真菌的生长。
实施例4 SfA3和AcMNPV对不同鳞翅目害虫的毒性测定
根据生测实验计算出苜蓿银纹夜蛾核型多角体病毒AcMNPV对不同鳞翅目害虫的LC50,其中,草地贪夜蛾3龄幼虫的LC50为1.0×105PIB/mL,3龄甜菜夜蛾幼虫LC50为8.0×105PIB/mL,3龄小菜蛾幼虫LC50为6.0×105PIB/mL,3龄菜青虫幼虫LC50为7.0×105PIB/mL,3龄斜纹夜蛾幼虫LC50为7.0×105PIB/mL。
菌液SfA3配制:将保存于-80℃的甘油菌以1:100的体积比接入新鲜的液体LB培养基中,在220rpm,37℃条件下活化12h获得种子菌液,再以1:100的体积比取种子菌液接入新鲜的液体LB培养基,220rpm,37℃摇10h,发酵液于8000r/min离心10min后收集菌体,用PBS清洗3次后,配置成1×108cfu/mL
将配好的SfA3菌液与饲料以1:4的比例混匀(1ml菌液配4g饲料,饲料配置时使用80%水分),最终配置成1×107cfu/g的饲料,对照组以PBS替代菌液。挑选长势相对一致的3龄甜菜夜蛾幼虫、草地贪夜蛾幼虫、小菜蛾幼虫、菜青虫幼虫、斜纹夜蛾幼虫。每种虫子分成3个组,每组三个重复,进行饥饿处理8h。饲喂饲料(1×107cfu/g SfA3)中分别添加10μLAcMNPV(1×107PIB/g)病毒,并将幼虫放入12孔板里进行单头饲养。每24h更换一次饲料,每24h统计死亡率一次,直至所有幼虫死亡或化蛹。并记录数据,使用SPSS统计软件进行分析数据。
对鳞翅目害虫进行毒力生测和Log-rank Mantel-Cox检测结果如图6所示,其中菌株SfA3和AcMNPV对草地贪夜蛾夜蛾致死率7天内达100%以上,对甜菜夜蛾的致死率8天为91.7%;对菜青虫的致死率8天为83.6%,对小菜蛾的致死率8天为77.0%,对斜纹夜蛾的致死率8天为68.7%。且菌株SfA3和AcMNPV混合后对不同的鳞翅目害虫的杀虫效果不同,效率不一样,之间存在显著的统计学差异(p<0.5);采用SfA3能显著增效AcMNPV的杀虫活性,缩短时间,对甜菜夜蛾幼虫、小菜蛾幼虫、菜青虫幼虫、棉铃虫幼虫、斜纹夜蛾幼虫具有广谱的杀虫效果,具有很强的防控作用。
实施例5菌株SfA3对草地贪夜蛾AcMNPV病毒的杀虫活性影响
1、草地贪夜蛾的饲养
草地贪夜蛾饲料的配置方法如下:
1)将7颗九合维生素丸(2.4g)打碎后,倒入搅拌机中;
2)分别称量以下各组分,加入搅拌机中;
3)分别称量以下各组分,放入锅中;加入500μL ddH2O,煮沸后搅拌降温至65℃。
4)将步骤3)中的体系倒入搅拌机中,持续搅拌30s至混匀。将搅拌后的饲料倒入饭盒中,放入超净台灭菌30min,冷却至室温后放入4℃冰箱保存。
2、抗生素溶液的配制
配制母液50g/L(利福平、硫酸庆大霉素、青霉素G钠、硫酸新霉素、链霉素、氯霉素和氨苄青霉素钠)。
3、收集草地贪夜蛾雌虫产下的卵块,加入0.5%的次氯酸钠溶液消毒3min,用清水洗去残余的消毒液,然后用吸水纸吸干卵块表面水分后放入养虫盒中。
4、抗生素饲料的配置与可培养肠道菌清除效果的检测
每100g饲料分别加入50g/L的7种抗生素母液96μL(利福平、硫酸庆大霉素、青霉素G钠、硫酸新霉素、链霉素、氯霉素和氨苄青霉素钠),揽拌均匀后饲喂草地贪夜蛾初孵到三龄幼虫(L3D1),每组30头虫,3次重复。对照组从孵化开始一直饲喂正常饲料。
5、肠道菌清除效果的检测
从处理组与对照组分别随机取10头虫,解剖取中肠内容物,无菌ddH2O梯度稀释103~104倍,取100μL涂板LB固体培养基(无抗性),置于30℃培养箱内培养,48h后检测有无细菌生长。
6、SfA3肠道分离菌的培养
将测序正确和保藏于微生物所的菌种,按照1:1000比例转接于LB液体培养基中过夜活化培养得到种子菌,种子菌按照1:100的比例转接至新鲜LB培养基中,37℃,180r/min培养约6h。培养后将菌液12000g离心2min富集菌体,用0.9%的生理盐水洗涤3次后稀释到OD600=1.0。使用时滴加250μL菌液到1g草地贪夜蛾无防腐剂的人工饲料中。
7、病毒饲喂处理及回补肠道分离菌
挑取长势一致的二龄草地贪夜蛾幼虫,每组30头,饥饿处理12h,连续两天饲喂含病毒的30%蔗糖水溶液。第三天开始饲喂带菌饲料,带菌饲料处理两天后恢复正常饲料。光照、温湿度等条件与正常人工饲养一致。每12h统计一次死亡率。利用graphPad prism软件和SPSS20.0分析数据。
8、草地贪夜蛾肠道细菌的清除效果
为了获得回补实验所需的无肠道菌种群,草地贪夜蛾初孵幼虫用抗生素混合饲料持续饲喂直至3龄初幼虫,然后解剖取100μL稀释到10-4倍中肠内容物涂布于LB平板,30℃培养48h,平板生长结果如图7所示,正常喂养下幼虫肠道稀释液的菌落遍布平板,而使用抗生素处理的肠道稀释液几乎无菌落长出,说明抗生素混合饲料饲喂处理能够有效清除肠道菌群。
9、肠道分离菌SfA3对AcMNPV杀虫活性的影响
生物测定实验结果如图8所示,单独添加SfA3和AcMNPV到饲料后饲喂草地贪夜蛾,可以杀死草地贪夜蛾,最终SfA3存活率为58%;AcMNPV到饲料后饲喂草地贪夜蛾,存活率10d为5%;但是将SfA3菌株回补到AcMNPV饲料中,草地贪夜蛾存活率6d就存活率变成1.6%,7d 100%死亡。说明SfA3的回补可显著提高草地贪夜蛾幼虫对AcMNPV的敏感性(P<0.0001,log-rank(Mantel-Cox)检验),而且SfA3细菌的回补均降低了半数生存期(MedianSurvival Time),使草地贪夜蛾半数生存期由5天降低至3.5天。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
1.一株云南微球菌(Micrococcus sp.)SfA3菌株,其特征在于,该菌株于2022年4月29日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC NO.62446。
2.权利要求1所述云南微球菌SfA3菌株或其发酵液在抑制植物病原真菌和/或防治鳞翅目害虫中的应用。
3.权利要求1所述云南微球菌SfA3菌株或其发酵液在制备抑制植物病原真菌和/或防治鳞翅目害虫的微生物菌剂中的应用。
4.权利要求1所述云南微球菌SfA3菌株或其发酵液在制备AcMNPV病毒增效杀虫剂中的应用。
5.一种抑制植物病原真菌和/或防治鳞翅目害虫的微生物菌剂,其特征在于,含权利要求1所述云南微球菌SfA3菌株或其发酵液。
6.根据权利要求5所述微生物菌剂,其特征在于,所述发酵液为发酵上清液。
7.根据权利要求6所述微生物菌剂,其特征在于,所述发酵上清液浓度为1.0×105~1.0×1010CFU/mL。
8.一种AcMNPV病毒增效杀虫剂,其特征在于,含权利要求1所述云南微球菌SfA3菌株或其发酵液。
9.根据权利要求2~3任一所述应用或权利要求5~7任一所述微生物菌剂,其特征在于,所述植物病原真菌为香蕉枯萎病菌(Fusarium oxysporum f.sp.cubense)、菜心炭疽病菌(Colletotrichum higginsianum Sacc.)、水鬼蕉叶斑病菌(Botrytis cinerea)、番茄酸腐病菌(Geotrichum candidum)和番石榴果腐病菌(Pstalotiopsis cocuuli Guba)中的一种或多种。
10.根据权利要求2~3任一所述应用或权利要求5~7任一所述微生物菌剂,其特征在于,所述鳞翅目害虫为草地贪夜蛾、斜纹夜蛾、甜菜夜蛾、菜青虫、小菜蛾中的一种或多种。
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