CN116426444B - 一种斜纹夜蛾准霍氏肠杆菌XWc1菌株及其应用 - Google Patents
一种斜纹夜蛾准霍氏肠杆菌XWc1菌株及其应用 Download PDFInfo
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Abstract
本发明公开了一种斜纹夜蛾准霍氏肠杆菌XWc1菌株及其应用，所述菌株保藏于广东省微生物菌种保藏中心，生物保藏号：GDMCC NO:63411。本发明的斜纹夜蛾准霍氏肠杆菌XWc1菌株可以提高斜纹夜蛾抗性的产生，通过能够在体外降解双酰胺类农药溴虫氟苯双酰胺；本发明的斜纹夜蛾准霍氏肠杆菌XWc1菌株为斜纹夜蛾抗性机理的研究提供依据，同时由于其具有农药降解方面的应用，为生物防治的应用提供了基础。
Description
技术领域
本发明属于农业微生物技术领域，具体涉及一株斜纹夜蛾肠杆菌Enterobacterquasihormaechei XWc1菌株及其应用。
背景技术
斜纹夜蛾（Spodoptera litura）属鳞翅目（Lepidoptera）夜蛾科（Noctuidae），是世界性分布的广食性害虫。具有很强的迁徙能力，对棉花、玉米、大豆和花生等栽培作物造成严重危害。由于斜纹夜蛾的高繁殖率，田间不得不依赖大量的化学农药进行防治，长期不合理大量使用化学农药，已经使得斜纹夜蛾进化出对许多杀虫剂的高抗药性，包括有机氯、有机磷、拟除虫菊酯、氨基甲酸酯、阿维菌素等。抗性谱不断扩大，斜纹夜蛾的抗药性治理成为世界性的难题；因此对于斜纹夜蛾的抗性研究具有必要性。
发明内容
本发明的目的在于针对现有技术的不足，提供一种斜纹夜蛾准霍氏肠杆菌菌株XWc1及其应用，所述菌株能够在只含溴虫氟苯双酰胺为唯一营养源的培养基中生存，且能在体外对该农药进行降解。
为了达到上述目的，本发明采用如下技术方案：
一种斜纹夜蛾准霍氏肠杆菌，命名为准霍氏肠杆菌Enterobacterquasihormaechei XWc1，保藏单位：广东省微生物菌种保藏中心；保藏地址：广州市先烈中路100号大院59号楼5楼；保藏日期：2023年4月27日；生物保藏号：GDMCC No：63411。
本发明的准霍氏肠杆菌Enterobacter quasihormaechei XWc1菌株为圆形白色菌落，表面光滑有光泽，半透明状边缘整齐。呈单个或链状排列。最适生长温度为35℃，最适PH为6.0-7.0，兼性厌氧，无芽孢。
本发明的准霍氏肠杆菌Enterobacter quasihormaechei XWc1菌株对d-半乳糖、d-葡萄糖、麦芽糖、d-甘露醇、阿拉伯糖、蜜二糖、d-果糖、d-木糖、海藻糖、棉子糖、蔗糖、d -龙胆二糖、L-鼠李糖、甘油、L-山梨醇都能利用。
优选的，作为一个较佳的实施方式，本发明所述的准霍氏肠杆菌Enterobacterquasihormaechei XWc1菌株可以提高斜纹夜蛾的抗药性。
优选的，作为一个较佳的实施方式，本发明所述的准霍氏肠杆菌Enterobacterquasihormaechei XWc1菌株可以在体外降解双酰胺类农药溴虫氟苯双酰胺。
本发明的另一目的在于提供一种准霍氏肠杆菌Enterobacter quasihormaecheiXWc1菌株用于降解农药的应用。
与现有技术相比，本发明的有益效果为：
本发明的研究结果表明，准霍氏肠杆菌Enterobacter quasihormaechei XWc1菌株具备提高斜纹夜蛾对于溴虫氟苯双酰胺的抗药性，同时能在体外降解溴虫氟苯双酰胺农药，可以减少农药残留；准霍氏肠杆菌Enterobacter quasihormaechei XWc1菌株可以作为抗性机理研究的基础生物材料，用于害虫抗性机理研究，也可以作为生物降解材料，应用于减少农药残留，具有很好的应用前景。
附图说明
图1示出了准霍氏肠杆菌Enterobacter quasihormaechei XWc1菌株分离平板图；
图2示出了准霍氏肠杆菌Enterobacter quasihormaechei XWc1菌株在MS培养基中的生长折线；
图3示出了准霍氏肠杆菌Enterobacter quasihormaechei XWc1菌株对溴虫氟苯双酰胺的72 h降解率；
图4示出了准霍氏肠杆菌Enterobacter quasihormaechei XWc1菌株的进化树。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范围。下面具体的实施方式对本发明作进一步的描述。
如无特殊说明，本发明实施例所使用的试剂和材料均可以通过市售手段购买的得到。
一、菌株的分离培养
1、培养基的配制
LB培养基：10 g蛋白胨，5 g酵母提取物，10 gNaCl,15 g琼脂，溶于1 L无菌水中，调pH至7.0。
2、菌株的分离纯化
2.1解剖并涂板
无菌条件下，在超净工作台内解剖斜纹夜蛾三龄幼虫并取出中肠组织，放入研磨机中，用无菌水稀释5个浓度梯度，按梯度分别涂布LB培养基平板，放置于30℃恒温培养箱培养，每隔24 h观察一次。
2.2 纯化培养并拍照
培养基中有单菌落生长出后，根据菌落颜色，形态和菌落大小不同挑取单菌落，每个单菌落至少在LB平板上连续划线五次，然后对分离菌株的划线平板进行拍照（图1），随后转接至LB液体培养基中，把菌液保存于25%的甘油水溶液中，冻存于-80℃冰箱备用。
3、菌株的鉴定
3.1形态鉴定
从形态学来看，为圆形白色菌落，表面光滑有光泽，半透明状边缘整齐。呈单个或链状排列。最适生长温度为35℃，最适PH为6.0-7.0，兼性厌氧，无芽孢。
3. 2生理生化特征
生理生化测定参考《常见细菌鉴定手册》的方法进行，采用细菌微量生化反应管。对可以利用的碳源测试结果，对d-半乳糖、d-葡萄糖、麦芽糖、d-甘露醇、阿拉伯糖、蜜二糖、d-果糖、d-木糖、海藻糖、棉子糖、蔗糖、d -龙胆二糖、L-鼠李糖、甘油、L-山梨醇都能利用。
3.3.3 分子鉴定
使用天根生物公司的细菌基因组DNA提取试剂盒提取单菌落菌株基因组DNA,并以提取的DNA为模板，使用通用引物27F（5'-AGAGTTTGATCCTGGCTCAG-3'）和1492R（5'-GGTTACCTTGTTACGACTT-3'）为上下引物扩增细菌16S rDNA，PCR反应体系如表1。用离心管混匀，短暂离心。PCR设置程序，95℃预热5 min，94℃30 s，54℃ 30 s，72℃延伸30 s，30个循环，72℃ 5 min，4℃保存。PCR产物经1 %琼脂糖凝胶检测并切胶回收纯化送擎科生物技术有限公司测序。
根据测序引物27FA及1492R的结果，分析测序质量，将该序列与NCBI中的rRNA/ITS数据库进行blast比对。选取下载整理XWc1菌株的边缘序列后用MEGA7.0软件进行多序列比对分析和构建系统进化树，调整bootstrap值并检验进化树可靠性。结果如图4所示，可知XWc1菌株与Enterobacter quasihormaechei strain WCHEs120003相似性最高，将其命名为斜纹夜蛾准霍氏肠杆菌（Enterobacter quasihormaechei）XWc1菌株。分类地位为细菌界(Bacteria),变形菌门(Pseudomonadota), γ-变形菌纲(Gammaproteobacteria),肠杆菌目(Enterobacterales),肠杆菌科(Enterobacteriaceae),肠杆菌属(Enterobacter), 准霍氏肠杆菌(Enterobacter quasihormaechei)。所述菌株已于2023年4月27日保藏于广东省微生物菌种保藏中心(GDMCC)，菌种保藏号为GDMCC No:63411，分类命名为Enterobacterquasihormaechei WXc1，保藏地址为广东省广州市先烈中路100号。
二、菌株体外降解试验
1、测定斜纹夜蛾肠道细菌对溴虫氟苯双酰胺的耐药性
将从抗性品系斜纹夜蛾分离出的XWc1菌株，分别接种于30ml以溴虫氟苯双酰胺为唯一碳源的MS培养基(1.5 g k2HPO4、0.5 g KH2PO4、0.5 g（NH4）2SO4、0.5 g NaCl、0.2 gMgSO4、0.05 g CaCl2、0.02 g FeSO4、1 L H2O，加入几滴二甲基亚砜助溶，溴虫氟苯双酰胺浓度配置为100 mg/L放置于摇床培养，每隔24 h测定并记录OD600，并以未接菌的MS培养基作空白对照。
2、肠道细菌对溴虫氟苯双酰胺的降解
将XWc1菌株接种于LB培养基中，摇床培养48 h，接着离心去除培养基，将得到富集的菌加入以溴虫氟苯双酰胺为唯一碳源的MS培养基中，摇床培养。每培养完成后，再通过1500rpm离心菌体，去除LB培养基液体，然后在最小盐溶液中洗涤两次。将富集的细菌加入150 mL以溴虫氟苯双酰胺为唯一碳源的MS培养基中，溴虫氟苯双酰胺为100 mg/L，调节OD600=1.0后进行培养。培养条件：37℃，140 rpm摇床培养24 h，72 h，108 h。每次分别取出取出20 ml以溴虫氟苯双酰胺为唯一碳源的MS培养液，1500 rpm离心15 min后，去除上清溶液，加入20 ml乙腈和5m水，然后在离心管中加入足量的NaCl至饱和，并且立马剧烈震荡混匀，混匀后紧接着放入50°℃恒温水浴水浴8min，等待形成清晰的界面后，用移液枪取出液面上层乙腈。取出的乙腈通过装有无水硫酸镁的净化管脱水，然后将处理好的液体经滤膜过滤注入样品瓶，进行HPLC分析。以未接种细菌的等量培养基作对照测得异戊醇对溴虫氟苯双酰胺的降解率。
3、菌株对昆虫抗药性的影响
Enterobacter quasihormaechei XWc1菌株放入LB液体培养基中培养，调节OD600=1.0，将新鲜人工饲料切成小块状浸泡在菌液中10 min，取出饲料自然晾干，置于培养皿中，放入龄期一致、健康良好的敏感品系斜纹夜蛾。无菌水浸泡的作为空白对照。
待饲喂细菌的斜纹夜蛾存活率稳定后，将处理得到的加菌饲喂的斜纹夜蛾、敏感品系进行生物毒力测定。采用浸叶法，按梯度配置溴虫氟苯双酰胺溶液，将甘蓝叶片浸泡在药液中10 s，取出叶片晾干，置于培养皿中，放入龄期一致、健康良好的加菌饲喂斜纹夜蛾。无菌水浸泡的作为空白对照。24 h和48 h分别观察别记录结果。存活率统计结果如表2所示，从最高浓度0.2 mg/L，加菌饲喂48 h存活率为40 %，空白处理48 h存活率0%。最低浓度0.0125 mg/L，加菌饲喂48 h存活率93%，空白处理48 h存活率90 %。在各浓度下加菌饲喂的存活率均高于空白处理。结果用spss分析，计算毒力回归方程、LC50值及置信区间如表3所示，加菌处理的LC50为 0.139 mg/L，空白处理的LC50为0.025 mg/L，加菌饲喂后抗性提高5.56倍（抗性LC50/敏感LC50），饲喂菌后斜纹夜蛾对溴虫氟苯双酰胺抗性有显著提升。
本发明分离得到的Enterobacter quasihormaechei XWc1菌株具有提高斜纹夜蛾对溴虫氟苯双酰胺的抗性，本发明分离得到的Enterobacter quasihormaechei XWc1菌株可以作为研究抗性机理的基础材料。也可用于农药降解方面，具有很好的应用应用前景。
上述实施例为本发明较佳的实现方案，除此之外，本发明还可以其他方式实现，在不脱离本发明构思的前提下任何显而易见的替换均在本发明的保护范围之内。
Claims (2)
1.一种菌株，其特征在于，所述菌株为准霍氏肠杆菌XWc1菌株，保藏单位：广东省微生物菌种保藏中心；保藏日期：2023年4月27日；生物保藏号：GDMCC NO:63411。
2.根据权利要求1所述的菌株用于降解农药的应用，其特征在于，所述农药为溴虫氟苯双酰胺农药。
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| CN110199995A (zh) * | 2019-06-19 | 2019-09-06 | 江西正邦作物保护有限公司 | 一种含有溴虫氟苯双酰胺的农药组合物 |
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