CN117126770A - 一种阿氏肠杆菌av1在防治烟草辣椒脉斑驳病毒中的应用 - Google Patents
一种阿氏肠杆菌av1在防治烟草辣椒脉斑驳病毒中的应用 Download PDFInfo
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Abstract
本发明公开了一种阿氏肠杆菌AV1在防治烟草辣椒脉斑驳病毒中的应用,所述阿氏肠杆菌(Enterobacter asburiae)AV1于2022年7月27日保藏在中国典型培养物保藏中心,保藏号为:CCTCC NO:M 20221183。该阿氏肠杆菌AV1对烟草辣椒脉斑驳病毒的钝化作用突出,荧光定量PCR对菌剂处理后的烟草进行辣椒脉斑驳病毒定量检测,处理后的烟株病毒含量明显低于对照,并且阿氏肠杆菌AV1是筛选出的天然病毒接抗菌对环境无污染,不会破坏生物多样性。
Description
技术领域
本发明涉及烟草辣椒脉斑驳病毒防治技术领域,具体涉及一种阿氏肠杆菌AV1在防治烟草辣椒脉斑驳病毒中的应用。
背景技术
辣椒脉斑驳病毒(Chilli veinal mottle virus,ChiVMV)属马铃薯Y病毒科(Potyviridae)马铃薯Y病毒属(Potyvirus),是茄科作物的主要病毒种类之一,严重危害茄科作物的生产。ChiVMV主要为害辣椒,而2011年首次在中国报道了它对烟草的生产也造成危害。感染ChiVMV的烟草植株出现叶片点状坏死、采收前烟叶表现穿孔、局部坏死症状,严重影响烤后烟叶质量和品质,更严重者叶片变形、叶茎系统性坏死,最终导致植株死亡。
病毒属于细胞内专性寄生物,烟株一旦感染,生产上尚无有效的药剂进行彻底控制。因此,筛选天然微生物拮抗菌防治烟草病毒病符合国家绿色防控发展需求,减少化学农药对土壤及环境的污染。
发明内容
本发明为了筛选天然微生物拮抗菌防治烟草病毒病,减少化学农药对土壤及环境的污染,提供了一种阿氏肠杆菌AV1在防治烟草辣椒脉斑驳病毒中的应用。
本发明通过以下技术方案来实现上述目的:
一种阿氏肠杆菌AV1在防治烟草辣椒脉斑驳病毒(Chilli veinal mottle virus,ChiVMV)中的应用,所述阿氏肠杆菌(Enterobacter asburiae)AV1于2022年7月27日保藏在中国典型培养物保藏中心,保藏号为:CCTCC NO:M 20221183。
所述阿氏肠杆菌AV1菌落呈椭圆形、平滑均匀,乳白色,革兰氏染色阴性。纯化培养时,在NA培养基(牛肉膏3.0g/L、蔗糖10.0g/L、蛋白胨5.0g/L、酵母提取物1.0g/L,琼脂15.0g/L,pH=7.1)上采用涂布平板法,28℃培养24h。
所述阿氏肠杆菌AV1的16S rDNA基因序列为SEQ ID No.1所示的核苷酸序列。
所述阿氏肠杆菌AV1的16S rDNA基因引物为:
P0:GAGAGTTTGATCCTGGCTCAG;和
P6:CTACGGCTACCTTGTTACGA。
所述阿氏肠杆菌AV1作为拮抗菌株,用于对烟草辣椒脉斑驳病毒((Chilli veinalmottle virus,ChiVMV))侵染的抑制。
本发明的有益效果在于:该阿氏肠杆菌AV1对辣椒脉斑驳病毒的钝化作用突出,荧光定量PCR对菌剂处理后的烟草进行辣椒脉斑驳病毒定量检测,处理后的烟株病毒含量明显低于对照,并且阿氏肠杆菌AV1是筛选出的天然病毒接抗菌对环境无污染,不会破坏生物多样性。
附图说明
图1为本发明阿氏肠杆菌AV1的菌落形态图;
图2为阿氏肠杆菌AV1与病毒共同接种烟草的症状示意图;其中,左侧ChiVMV接种烟草后喷施AV1(AV1+ChiVMV)的症状图;右侧是ChiVMV和培养基共同接种烟草(培养基+ChiVMV)的阴性对照症状图。
图3为荧光定量PCR检测处理和阴性对照的病毒含量结果。
具体实施方式
下面结合附图对本申请作进一步详细描述,有必要在此指出的是,以下具体实施方式只用于对本申请进行进一步的说明,不能理解为对本申请保护范围的限制,该领域的技术人员可以根据上述申请内容对本申请做出一些非本质的改进和调整。
一种用于防治烟草花叶病毒病的阿氏肠杆菌AV1,所述阿氏肠杆菌(Enterobacterasburiae)AV1于2022年7月27日保藏在中国典型培养物保藏中心,保藏号为:CCTCC NO:M20221183。
阿氏肠杆菌AV1的制备:
一、取样:在已有烟草花叶病发生的烟田中,选取仍然生长健康的烟株,在烟株根部20厘米深度选取烟株根际土壤,备用;
二、研磨:将备用的土壤10g置于灭过菌的三角瓶中,加入20ml无菌水,震荡待液体浑浊,室温静止10分钟,备用;
三、纯化:用无菌枪头吸取静止后的液体上清200微升,在NA培养基(牛肉膏3.0g/L、蔗糖10.0g/L、蛋白胨5.0g/L、酵母提取物1.0g/L,琼脂15.0g/L,pH=7.1)上采用涂布平板法,28℃培养24h,待分离平皿上菌落长出后,根据微生物菌落的形态、大小、表面结构、边缘结构、质地、光泽、透明度、颜色和产生的可溶性色素等方面特征,用接种环将孤立的单个菌落一一挑出,挑取形态不同的单菌落在相应的空白NA培养基平板上进行划线纯化,并在28℃恒温培养箱中倒置培养24h,得纯化菌株,备用;
如图1所示,AV1菌落呈椭圆形、平滑均匀,乳白色,革兰氏染色阴性。利用Wi zard基因组DNA纯化试剂盒(Promega,Madison,WI)提取病原菌DNA,其16S rD NA的核苷酸序列如SEQ ID No.1所示,16S rDNA基因引物为:P0(GAGAGTTTG ATCCTGGCTCAG)和P6(CTACGGCTACCTTGTTACGA)
利用引物进行PCR扩增,反应体系为50μL(10×PCR buffer 5μL、dNTP 1μL、FD12μL、FD2 2μL、Tag酶1μL、模板DNA 2μL、dd H2O 37μL),扩增条件为:94℃5min;94℃30s,55℃1min,72℃90s,30个循环;72℃1min;10℃20min。将菌株的16SrDNA基因测序结果于NCBI网站上用BLAST软件进行同源性比对,初步判断病原菌为Enterobacter asburiae。
四、筛选:挑选单菌落细菌于已经灭菌的盛有20ml培养基的100ml锥形瓶中,振荡培养(28℃,200r/min)24h,使菌液含量足够大,作发酵备用菌种。
辣椒脉斑驳病毒ChiVMV的侵染抑制试验:
本试验利用本生烟(Nicotiana benthamianais)为接种对象。将充分发病的ChiVMV毒源幼嫩叶片以1:10(质量比体积)加蒸馏水研磨过滤后,作为病毒供试样品。病毒供试样品摩擦本氏烟。接种7天和14天后,将待测AV1菌液喷施于接种的本氏烟,三次重复。喷施NA培养基的为对照。
接种21天后,提取接种本氏烟系统叶RNA,利用荧光定量PCR的方法检测样本中ChiVMV病毒含量,通过比对明确AV1钝化病毒的效果。引物如下:
qRT-ChiVMV-274F:CATTGATTGACCATGCCAAG
qRT-ChiVMV-274R:CTACCGTCCAGTCCGAACAT
从图2所示的阿氏肠杆菌AV1与病毒共同接种烟草的症状示意图(其中,左侧ChiVMV接种烟草后喷施AV1(AV1+ChiVMV)的症状图;右侧是ChiVMV和培养基共同接种烟草(培养基+ChiVMV)的阴性对照症状图)可以看出菌株AV1对ChiVMV拮抗作用明显更好,三次重复试验得出,AV1发酵液对ChiVMV的抑制率可达70%,具体的防治效果见图3,CK为健康烟株。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。
Claims (4)
1.一种阿氏肠杆菌AV1在防治烟草辣椒脉斑驳病毒(Chilli veinal mottle virus)中的应用,其特征在于,所述阿氏肠杆菌(Enterobacter asburiae)AV1于2022年7月27日保藏在中国典型培养物保藏中心,保藏号为:CCTCC NO:M 20221183。
2.根据权利要求1所述的应用,其特征在于,所述阿氏肠杆菌AV1作为拮抗菌株,用于对烟草辣椒脉斑驳病毒侵染的抑制。
3.根据权利要求1所述的应用,其特征在于,所述阿氏肠杆菌AV1的16S rDNA基因序列为SEQ ID No.1所示的核苷酸序列。
4.根据权利要求1所述的应用,其特征在于,所述阿氏肠杆菌AV1的16S rDNA基因引物为:
P0:GAGAGTTTGATCCTGGCTCAG;和
P6:CTACGGCTACCTTGTTACGA。
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