CN116716211A - 一种生防微白黄链霉菌及其应用 - Google Patents
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Abstract
本发明涉及农业微生物生防技术领域,具体涉及一种生防微白黄链霉菌及其应用,通过菌种筛选、分离以及鉴定确认其具体基因组序列,其制备的防菌剂可应用在防治花生白绢病、土传病害、根腐病病害中,具体步骤如下:将微白黄链霉菌的菌种在32℃的环境中,使用马铃薯葡萄糖固体培养基培养,培养时间3天;得到的菌种利用液体培养,液体培养72小时后转接到固体培养,干燥得生防菌剂。该防菌剂的发酵液和固体发酵体能对花生白绢病、土传病害、根腐病有明显的抑制和防治作用,可应用在花生土传白绢病和根腐病的预防和治疗,也可应用在促进植物茄子的种子萌发上,不会对生态环境造成影响,也不会对人体健康造成伤害。
Description
技术领域
本发明涉及农业微生物生防技术领域,具体涉及一种生防微白黄链霉菌及其应用。
背景技术
土传病害和花生白绢病是花生生产过程中的主要病害,其病原菌为齐整小核菌(Sclerotium rolfsii),菌落在马铃薯葡萄糖培养基上为近圆形,菌丝白色,菌落呈辐射状生长。花生白绢病在世界主要花生产区普遍发生,中国、印度、美国、阿根延、印度尼西亚、菲律宾、泰国、越南和南非等国家均有花生白绢病发生的报道。花生白绢病也是我国花生主产区的主要病害之一,在高温和多雨的年份更易发生,造成的危害也更大。
目前花生白绢病的防治主要以化学药物为主,同时采用一些“闷棚”类的操作,以求杀死线虫。从实际效果来看,化学药物对线虫的杀灭效果大约在20-60%,难以从根本上解决线虫危害。并且随着化学农药使用伴随而来的对土壤生态系统的损伤、线虫耐药性的增强和对人体健康的潜在危害越来越严重,急需环境友好和生物安全的生物制剂来应对花生白绢病的发生。
发明内容
为解决上述技术问题,本发明提供一种生防微白黄链霉菌,通过菌种筛选、分离以及鉴定确认其具体基因组序列,具体步骤如下:
1)菌种筛选、分离:从花生田内多次采集发生白绢病病株的根际土壤样品,分离病原菌作为测试菌株,选取同一地块内的健康植株根际土壤作为候选菌株来源,通过有限稀释获得单菌落,通过平板拮抗试验筛选到一株有效抑制线虫发生、没有耐药性的链霉菌,并将其编号:编号为Y1124;
2)对上述(1)中的链霉菌-Y1124进行菌种的鉴定:
(1)细菌基因组的提取:采用天根细菌基因组提取试剂盒提取链霉菌的细菌DNA;
(2)PCR扩增体系;
(3)PCR产物割胶纯化;PCR产物经2%琼脂糖凝胶电泳,割取目的条带,按照天根回收试剂盒(DP214-03)纯化回收;
(4)采用sanger法进行测序;
(5)数据分析:使用3730XL DNA序列分析仪进行测序,用DNA Sequencinganalysis软件分析测序结果,用Sequencing Analysis 5.2.0软件进行解读,经鉴定上述链霉菌为微白黄链霉菌。
另外,本发明还公开了上述微白黄链霉菌的具体应用,其对花生白绢病、土传病害、根腐病有明显的抑制和防治作用,微白黄链霉菌可应用在防治花生白绢病、土传病害、根腐病病害中,土传病害和花生白绢病的病原菌为齐整小核菌。
优选的:所述微白黄链霉菌的应用,其利用微白黄链霉菌制备的防菌剂的发酵液或固体发酵剂进行应用,其中,固体发酵剂还可制成水剂、颗粒或粉剂。
本发明还提供一种农用微生物菌剂,其包括所述的微白黄链霉菌。
优选的:所述农用微生物菌剂包括所述微白黄链霉菌制备的防菌剂的发酵液或固体发酵剂。
还有,本发明中还公开了一种生防微白黄链霉菌制备防菌剂的方法,具体步骤如下:
(1)将微白黄链霉菌的菌种在32℃的环境中,使用马铃薯葡萄糖固体培养基培养,培养时间3天;
(2)将步骤(2)中得到的菌种利用液体培养,方法为:在32℃、溶氧量不低于50%的液体培养基中培养2天;
液体培养72小时后转接到固体培养,固体培养方法为:在30℃的房间内,以浅盘方式或固体发酵罐通气方式进行培养,保持湿度在70%-85%之间,培养10天后干燥得生防菌剂。
优选的:所用液体培养基配方为:玉米淀粉1%,葡萄糖2%,蛋白胨1%,磷酸二氢钾0.2%,磷酸氢二钾0.018,硫酸镁0.002%。
优选的:所述固体培养基配方为:麸皮50%,玉米面15%,糖蜜2%,蛋白胨5%,硫酸镁0.002%,硫酸钙0.02%。
本发明的技术效果和优点:
本发明的微白黄链霉菌所得到的防菌剂,其发酵液和固体发酵体能对花生白绢病、土传病害、根腐病有明显的抑制和防治作用,可应用在花生土传白绢病和根腐病的预防和治疗,同时又不会对生态环境造成影响,也不会对人体健康造成伤害,是一种良好的生防安全制剂,而且,该菌剂也可以应用在促进植物茄子的种子萌发上,提升出芽率。
附图说明
图1是本申请实施例提供的链霉菌Y1124的16S rDNA的序列;
图2是本申请实施例提供的Y1124的基于16S rDNA序列构建的系统发育进化树。
具体实施方式
下面结合附图和具体实施方式对本发明作进一步详细的说明。本发明的实施例是为了示例和描述起见而给出的,而并不是无遗漏的或者将本发明限于所公开的形式。很多修改和变化对于本领域的普通技术人员而言是显而易见的。选择和描述实施例是为了更好说明本发明的原理和实际应用,并且使本领域的普通技术人员能够理解本发明从而设计适于特定用途的带有各种修改的各种实施例。
实施例1
在本实施例中提供一种生防微白黄链霉菌,首先对该微白黄链霉菌进行菌种的筛选分离,再对其进行菌种鉴定,确认其为微白黄链霉菌,具体操作如下:
1)菌种筛选、分离:从河南驻马店区域、辽宁绥中区域花生田内多次采集发生白绢病病株的根际土壤样品,分离病原菌作为测试菌株,选取同一地块内的健康植株根际土壤作为候选菌株来源,通过有限稀释获得单菌落,通过平板拮抗试验筛选到一株有效抑制线虫发生、没有耐药性的链霉菌,并将其编号:编号为Y1124;
2)对上述(1)中的链霉菌-Y1124进行菌种的鉴定:
(1)细菌基因组的提取:采用天根细菌基因组提取试剂盒(DP302-02)提取链霉菌的细菌DNA;
(2)PCR扩增体系:
成分 | 体系(20ul) |
Premix Taq | 10ul |
上下游引物 | 各1ul |
模板 | 1ul |
ddH20 | 7ul |
反应条件:
(3)PCR产物割胶纯化:
PCR产物经2%琼脂糖凝胶电泳,割取目的条带,按照天根回收试剂盒(DP214-03)纯化回收;
(4)采用sanger法进行测序:测序用的试剂盒为:BigDye v3.1 Chemistrykit(Applied Biosystems,Foster City,CA);
(5)数据分析:使用3730XL DNA序列分析仪进行测序,用DNA Sequencinganalysis软件分析测序结果,用Sequencing Analysis 5.2.0软件进行解读,经鉴定上述链霉菌为微白黄链霉菌(Streptomyces albidoflavus),其DNA序列如图1所示。
实施例2
在本实施例中,提供一种生防微白黄链霉菌制备防菌剂的方法,具体步骤如下:
(1)将微白黄链霉菌的菌种在32℃的环境中,使用马铃薯葡萄糖固体培养基培养,培养时间3天;
(2)将步骤(2)中得到的菌种利用液体培养,方法为:在32℃、溶氧量不低于50%的液体培养基中培养2天;
其中所用液体培养基配方为:玉米淀粉1%,葡萄糖2%,蛋白胨1%,磷酸二氢钾0.2%,磷酸氢二钾0.018,硫酸镁0.002%;
液体培养72小时后转接到固体培养,固体培养方法为:在30℃的房间内,以浅盘方式或固体发酵罐通气方式进行培养,保持湿度在70%-85%之间,培养10天后干燥得生防菌剂;
其中,固体培养基配方为:麸皮50%,玉米面15%,糖蜜2%,蛋白胨5%,硫酸镁0.002%,硫酸钙0.02%。
实施例3
本实施例中,对实施例2中制备的防菌剂进行防病害实验,将防菌剂制成所需的发酵液和固体发酵剂,其中,固体发酵剂可制备成水剂、颗粒和粉剂,可根据实际使用进行选择,方便保存;
实验:选择1亩样品花生田,对花生田内发生白绢病病株、土传病害、根腐病的病株进行标记,每种病株标记60株用于实验,然后将发酵液、固体发酵剂稀释后作用于病株上,同时采用清水作为对照组进行实验,实验结果如下:
由上述表格数据可知:该菌剂的发酵液和固体发酵剂对土传病害和花生白绢病、根腐病有明显的抑制和防治作用,发酵液稀释500倍、固体发酵制剂稀释1000倍后对花生白绢病的抑制率分别为81.3%、89.5%,对土传病害、根腐病的防治率分别为79.1%、85.3%,从而可得到,该菌剂可安全的用于花生土传白绢病和根腐病的预防和治疗。
实施例4
在链霉菌-Y1124制备的防菌剂能对细菌的病害具有较好的防治作用的前提下,容易想到,一般作物的种子在发芽时,其埋入到土壤中,也会受到细菌的影响,该菌剂是否能对作物种子的生长(即是发芽率)起到促进作用,从而设计如下实验:
取100粒茄子种子,分为相同数量的A/B两组,每组50粒,A组使用稀释500后的防菌剂发酵液浸泡,B组使用清水浸泡,浸泡30min后晾干,然后再按照茄子种植培育的常规方法进行播种、培育,然后计算其出芽率:
A组:出芽数48,出芽率=48/50*100%=96%;
B组:出芽数44,出芽率=44/50*100%=88%;
由上述数据可知,通过防菌剂浸泡后的茄子种子在发芽率上有明显的提升,从而能知道,该防菌剂可以应用在促进茄子的种子萌发上,可作为拌种剂使用,以拌种方式使用时,可有效促进茄子的生长,同理,其也可应用在其他植物上,提升出芽率,促进植物的生长。
显然,所描述的实施例仅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,本领域及相关领域的普通技术人员在没有作出创造性劳动的前提下所获得的所有其他实施例,都应属于本发明保护的范围。本发明中未具体描述和解释说明的结构、装置以及操作方法,如无特别说明和限定,均按照本领域的常规手段进行实施。
Claims (10)
1.一种生防微白黄链霉菌,其特征在于,通过菌种筛选、分离以及鉴定确认其具体基因组序列,具体步骤如下:
1)菌种筛选、分离:从花生田内多次采集发生白绢病病株的根际土壤样品,分离病原菌作为测试菌株,选取同一地块内的健康植株根际土壤作为候选菌株来源,通过有限稀释获得单菌落,通过平板拮抗试验筛选到一株有效抑制线虫发生、没有耐药性的链霉菌,并将其编号:编号为Y1124;
2)对上述(1)中的链霉菌-Y1124进行菌种的鉴定:
(1)细菌基因组的提取:采用天根细菌基因组提取试剂盒提取链霉菌的细菌DNA;
(2)PCR扩增体系;
(3)PCR产物割胶纯化;PCR产物经2%琼脂糖凝胶电泳,割取目的条带,按照天根回收试剂盒(DP214-03)纯化回收;
(4)采用sanger法进行测序;
(5)数据分析:使用3730XL DNA序列分析仪进行测序,用DNA Sequencing analysis软件分析测序结果,用Sequencing Analysis 5.2.0软件进行解读,经鉴定上述链霉菌为微白黄链霉菌。
2.一种如权利要求1所述的微白黄链霉菌在防治花生白绢病、土传病害、根腐病病害中的应用。
3.根据权利要求2所述的应用,其特征在于,所述土传病害和花生白绢病的病原菌为齐整小核菌。
4.根据权利要求2所述的应用,其特征在于,所述微白黄链霉菌利用微白黄链霉菌制备的防菌剂的发酵液或固体发酵剂进行应用。
5.根据权利要求4所述的应用,其特征在于,所述固体发酵剂包含水剂、颗粒或粉剂。
6.一种农用微生物菌剂,其特征在于,其包括权1中所述的微白黄链霉菌。
7.根据权利要求6所述的农用微生物菌剂,其特征在于,农用微生物菌剂包括所述微白黄链霉菌制备的防菌剂的发酵液或固体发酵剂。
8.一种生防微白黄链霉菌制备防菌剂的方法,其特征在于,具体步骤如下:
(1)将微白黄链霉菌的菌种在32℃的环境中,使用马铃薯葡萄糖固体培养基培养,培养时间3天;
(2)将步骤(2)中得到的菌种利用液体培养,方法为:在32℃、溶氧量不低于50%的液体培养基中培养2天;
液体培养72小时后转接到固体培养,固体培养方法为:在30℃的房间内,以浅盘方式或固体发酵罐通气方式进行培养,保持湿度在70%-85%之间,培养10天后干燥得生防菌剂。
9.根据权利要求8所述的制备防菌剂的方法,其特征在于,所用液体培养基配方为:玉米淀粉1%,葡萄糖2%,蛋白胨1%,磷酸二氢钾0.2%,磷酸氢二钾0.018,硫酸镁0.002%。
10.根据权利要求8所述的制备防菌剂的方法,其特征在于,所述固体培养基配方为:麸皮50%,玉米面15%,糖蜜2%,蛋白胨5%,硫酸镁0.002%,硫酸钙0.02%。
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CN117821341A (zh) * | 2024-02-29 | 2024-04-05 | 云南省农业科学院农业环境资源研究所 | 一种白黄链霉菌菌剂及其应用 |
CN117821341B (zh) * | 2024-02-29 | 2024-05-28 | 云南省农业科学院农业环境资源研究所 | 一种白黄链霉菌菌剂及其应用 |
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