CN116376720B - 一种金色毛壳菌及其在防治烟草叶斑病中的应用 - Google Patents
一种金色毛壳菌及其在防治烟草叶斑病中的应用 Download PDFInfo
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Abstract
本发明涉及微生物生物防治技术领域,特别的涉及金色毛壳菌或其孢子在抗烟草叶斑病中的应用,以及抗烟草叶斑病的生物制剂。本发明公开了一种金色毛壳菌菌株,分类学命名Arcopilusaureus YZXR,所述金色毛壳菌的保藏于2022年5月7日,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心,保藏地址北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,保藏编号为CGMCC NO:40190。能够有效的抑制角斑病病菌、野火病菌、破烂叶斑病引起的烟草叶斑病菌,具有制备微生物防治菌剂的潜力,对于提高烟草品质,促进烟农增收具有十分重要的作用。
Description
技术领域
本发明涉及微生物生物防治技术领域,特别的涉及金色毛壳菌或其孢子在抗烟草叶斑病中的应用,以及抗烟草叶斑病的生物制剂。
背景技术
烟草属双子叶植物纲,管状花目,茄科,烟草属,是我国重要的经济作物之一。然而,长期的单一品种的种植导致导致资源退化,而且长期在同一片地里种植也导致病虫害的累积,我国烟草的发病率有增高的趋势。叶斑类病害是危害大田期烟草的主要病害中的一种,其大多由真菌侵染所致,在大面积大片种植的情况下,病害易暴发流行,严重影响烟草品质。烟草叶斑病有多种类型,包括烟草角斑病、野火病、破烂叶斑病、靶斑病、赤星病等。
金色毛壳菌(Chaetomium aureum)具有很强的拮抗效果和生防潜力。早在1975年,即有科学家就对金色毛壳菌的固氮作用开始了研究工作。也有文献报道,从核桃叶片上分离得到金色毛壳菌对辣椒疫霉的菌丝生长具有很强的抑制作用,能防治辣椒疫病的发生;金色毛壳菌对水稻稻瘟病也有较好的防治作用。金色毛壳菌、渐狭蜡蚧菌和少根根霉菌可用于烟蚜的生物防治。
目前,烟草叶斑病主要使用一些化学农药和配合偏保护性质的药剂,例如苯醚甲环唑、苯甲溴菌腈(炭洁),43%戊唑醇,配合80%代森锰锌,25%嘧菌酯、50%吡唑嘧菌酯、苯甲嘧菌酯等。近年来,多种微生物农药兴起,生物农药具有安全、有效、无污染、不易产生抗药性等特点,现已成为烟草绿色防控重要措施之一。生物农药主要有芽孢杆菌制剂和木霉制剂,芽孢杆菌制剂涉及的菌种种类较多,具有广谱性的抗菌性能,尤其对于土传性的真菌具有较好的防治效果,已经应用在烟草的青枯病、黑胫病等根茎病害的防治中。木霉菌等主要是哈茨木霉生防菌剂产品,已经用于烟草的根腐病、炭疽病、赤星病等病菌的防治中。CN202011506161.X公开了通过筛选到一种烟草赤星病拮抗放线菌为黄三素链霉菌,经过测试其对赤星病的防治效率可达60%。
发明内容
进一步筛选和研发的能够防治烟草叶斑病的微生物,对于提高烟草品质,促进烟农增收具有十分重要的作用。通过长期的筛选和测试,本发明发现了一种金色毛壳菌能够有效的抑制烟草叶斑病菌,具有制备微生物防治菌剂的潜力。为此,本发明提供如下技术方案:
本发明的一个方案,本发明公开了金色毛壳菌或及其代谢产物在制备抗烟草叶斑病生物制剂中的应用,所述生物制剂中含有金色毛壳菌。优选的,所述烟草叶斑病由角斑病病菌、野火病、破烂叶斑病病菌所引起的。
本发明的金色毛壳菌,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心,保藏地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,保藏编号为:CGMCC NO:40190,分类学命名Arcopilus aureus YZXR。
本发明的一个方案,本发明公开了金色毛壳菌或代谢产物在制备抗烟草叶斑病生物制剂中的应用,所述生物制剂含有金色毛壳菌的代谢产物,所述代谢产物来自金色毛壳菌发酵培养上清,包括孢子和次级代谢产物。
生物制剂为的类型可以包括发酵液、冻干粉剂、可湿粉剂、颗粒剂、悬浮剂、固定化菌剂。发酵液是将金色毛壳菌在培养基中发酵后获得的含菌或孢子以及其他代谢物的产物,发酵可以采用发酵瓶、发酵罐,发酵的培养基为PDB培养基,其组成为马铃薯200g,蔗糖20g,蒸馏水1000mL;冻干粉剂,是将金色毛壳菌在培养基中培养或发酵后获得含菌或孢子的产物过滤后添加冻干保护剂例如海藻糖、甘油等通过冷冻干燥来获得粉末状产品,冷冻干燥有助于保持生物制剂的稳定性,在另一个方面也有助于保存和运输。可湿性粉剂是指有效成分在水中分散成悬浮液的粉状制剂。
本发明的一个方案,本发明的生物制剂包括药学上可以接受的助剂。优选的,所述助剂包括载体、表面活性剂、保护剂、营养助剂中的一种或多种。至于具体的载体、表面活性剂、保护剂、营养助剂的种类,其是本领域技术人员在微生物农药的制备中所常规使用的,例如载体中使用硅藻土、海泡石、吸附树脂、活性炭,表面活性剂选择机硅类表面活性剂,保护剂选择紫外保护剂以避免微生物在施加到植物后被阳光中的紫外线所杀伤,营养助剂提供微生物生长所需的碳源、氮源。
本发明的一个方案,本发明的生物制剂还包括其他的抗烟草叶斑病的微生物或抗烟草叶斑病化学药剂。
本发明的一个方案,本发明公开了金色毛壳菌或代谢产物在抑制烟草叶斑病菌中的应用。
有益效果
本发明通过筛选和试验发现金色毛壳菌能够对烟草叶斑病中野火病菌、角斑病菌、破烂叶斑病菌产生抑制作用,在1×107CFU/mL的浓度下能够起到比较明显的抑制效果,为烟草叶斑病菌的生物防治提供了新的思路。
附图说明
图1:平板对峙法验证金色毛壳菌对烟草角斑病的拮抗作用。
图2:平板对峙法验证金色毛壳菌对烟草野火病的拮抗作用。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。本发明所述技术方案,如未特别说明均为本领域的常规方式,所述试剂或材料,如未特别说明,均为常规试剂,来源于商业渠道。
实施例1金色毛壳菌对烟草叶斑病病菌的拮抗作用
按照常规方法玉竹叶片上分离得到金色毛壳菌,将金色毛壳菌培养基在PD B培养基(马铃薯蔗糖培养基)中培养,PDB培养基配方组成马铃薯200g,蔗糖20g,蒸馏水1000mL,pH7.0。所述菌株在显微镜下观察,从形态学上确认为金色毛壳菌。进一步的,提取真菌基因组,用18s通用引物(ITS1和ITS4)扩增18S rDNA序列并测序,将序列在Genbank中进行Blast比对,进一步确认为金色毛壳菌中的一种,并进行菌种保藏,保藏日期2022年5月7日,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心,保藏地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,保藏编号为:CGMCC NO:40190,分类学命名Arcopilusaureus YZXR。
从患叶斑病的烟草上分离病原菌:将已经鉴定为烟草角斑病、野火病、破烂叶斑病的叶斑叶片从烟草植株上剪下,表面清洗,75%乙醇中浸泡30s,1%次氯酸钠消毒3min,无菌水冲洗3次,将处理过的样品病害处剪成小片薄片,接种于PDA培养基(马铃薯葡萄糖琼脂培养基)中,PDA培养基组成:马铃薯200g,葡萄糖20g,琼脂20g,蒸馏水1000mL,pH7.0。使部分组织插入培养基中,充分与培养基接触,放入28℃恒温培养箱中培养5d。待长出菌丝后,挑取前端菌丝置于新鲜的PDA培养基培养。对分离的真菌培养物进行致病性鉴定,确定分离的病原菌培养物能够产生典型的烟草叶斑相关病症的症状。
平板对峙法验证金色毛壳菌对烟草叶斑病菌的抑制作用。即将各菌用直径为5mm的打孔器制成菌饼,烟草角斑病病原菌接种在PDA平板中间,四周放上金色毛壳菌(拮抗菌)。拮抗菌距离培养皿周围约为10mm,两种菌接种距离为30mm,每种处理重复3次。于28℃培养4天。同时,以只接种病原菌的平皿作为对照组,每个处理重复3次。待对照组病原菌菌落长满培养皿时,用毫米测度尺测定菌落直径。根据实验组两菌落的生长速度、菌落之间有无抑制带、菌落边缘的菌丝是否发生稀疏和萎缩的现象来判断拮抗菌(金色毛壳菌)对病原菌是否有拮抗作用,基于实验组和对照组的测量数据计算金色毛壳菌对于致病菌的生长抑制率。参见图1,金色毛壳菌对于各种叶斑致病菌的生长起到了较强的抑制作用。经过测量计算,金色毛壳菌对于烟草角斑病、野火病、破烂叶斑病菌的生长抑制率达到了分别63.8%,69.7%,75.3%有明显的抑制作用。
实施例2金色毛壳菌对抗烟草叶斑致病菌引起的烟草叶斑病的作用
将金色毛壳菌接种到PDB培养基中,28℃振荡培养10天,将发酵液用纱布过滤,保存在三角瓶中,密封,4℃冰箱保存,备用。
调整金色毛壳菌发酵液的菌液浓度为约1×106CFU/mL、1×107CFU/mL、1×108CFU/mL,对长出3-4片真叶的烟苗进行整株喷雾,每个浓度分为6组,每组10株烟苗。重复3次,间隔6d,第2次喷施金色毛壳菌发酵液6h后,每个浓度选3组均匀喷施浓度为约1×106CFU/mL烟草野火病病菌、选3组均匀喷施浓度为约1×106CFU/mL烟草野火病病菌,药剂对照采用多菌灵可湿性粉剂500倍喷雾,另设清水处理为空白对照。将上述处理置于密闭环境中保湿48h后,按照常规温室管理,第9天调查发病率及发病严重度,严重度分级按照烟草病害分级调查方法(GB/T23222-2008)。
金色毛壳菌对于烟草叶斑病具有较强的拮抗抑制作用,在1×107cfu/mL的浓度下,即可以达到70%以上的防治效果,可以用于烟草叶斑病的防治。
以上内容是结合具体实施方式对本发明作进一步详细说明,不能认定本发明具体实施只局限于这些说明,对于本发明所属技术领域的普通技术人员来说,在不脱离本发明的构思的前提下,还可以做出若干简单的推演或替换,都应当视为属于本发明所提交的权利要求书确定的保护范围。
Claims (4)
1.一种金色毛壳菌在制备抗烟草叶斑病的制剂中应用,其特征在于,所述烟草叶斑病为角斑病菌、野火病菌或破烂叶斑病菌所引起的;所述金色毛壳菌于2022年5月7日保藏,保藏单位中国科学院微生物研究所,保藏地址北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC NO:40190,分类学命名为Arcopilus aureus YZXR。
2.根据权利要求1所述的应用,所述制剂为金色毛壳菌的发酵液、冻干粉剂、颗粒剂、乳液或固定化制剂。
3.根据权利要求2所述的应用,其特征在于,所述制剂还包括药学上可以接受的助剂。
4.根据权利要求3所述的应用,所述助剂包括载体、表面活性剂、保护剂中的一种或多种。
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