CN109112071B - 一种木霉菌及其应用 - Google Patents
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Abstract
本发明公开了一种木霉菌及其应用。本发明的木霉菌为棘孢木霉(Trichoderma asperellum)菌,菌株号:HTTA‑Z0002,保藏编号CGMCC No.15677。本发明的木霉菌能够应用在防治植物病害、抑菌以及农药中。本发明的菌株生物学特性和农药活性稳定,容易培养,培养周期短,操作方法简便可行,在抑菌活性、液体培养产分生孢子等特性上较野生菌株有较大的提高,克服了木霉菌株液体培养产孢量少的劣势,对农产品和环境没有污染,对植物多种病原菌有广而强的抑制作用。
Description
技术领域
本发明属于微生物技术领域,具体涉及一种木霉菌及其在农药中的应用。
背景技术
栽培植物化学农药污染问题,是一个世界性关注的热点。随着设施栽培蔬菜瓜果技术的日益普及,这一问题显得更加突出。温室的高温、高湿的环境满足了人们对蔬菜瓜果四季的需求,也造成了病害不分季节的严重发生。超量的农药和激素对蔬菜和瓜果造成严重的污染,其毒性和潜在的致癌作用对人体健康构成很大的危胁。生物农药以其对非靶标生物比较安全和环境相容性好的突出优点而符合现代社会对农业生产及农药的要求。
在所有的生物资源中,微生物以其繁殖快,成本低,野生菌株较易改造从而快速提高产量的特点,成为农药研制的首选资源。目前微生物农药销售额达数亿美元,且年产值每年上升10%~20%。为解决陆源微生物获得新农药性状日益减少的现状,极端海洋微生物新资源开始受到关注。极地独特的地理位置,形成了高盐、酷寒、强紫外辐射等自然环境,造就了生物的多种适应性生存,具备独特的分子生物学机制和生理生化特性,产生新型的代谢物质可以被人类所利用。
我国土壤盐渍化较严重,盐渍化土壤约占耕地的1/3,鉴于人多地少的特点,这些盐渍化土壤仍被人们所利用栽种较耐盐的作物,目前国内外开发微生物农药由于生防菌都来源于陆地,在含盐量较高的土壤中难以存活、定殖,在盐渍地土传病害的防治效果较差,陆地生防菌由于生境原因,限制了其发挥作用。海洋微生物高盐的原始生境,更易在盐渍土壤发挥作用,在新农药开发领域显现其生境优势。
生物农药的活菌制剂是一类相对开发周期较短,资金需求相对较少,农药功能较全的产品。在已获登记的陆地微生物活菌制剂在生物农药中占有较大的比例,并在植物病害的防治中起了重要作用。农药菌株为了更好的为人类所利用和产业化生产,菌株要满足农药活性及生产发酵性状均达标的条件,微生物菌株通过各种筛选模型获得后,通常都进行诱变育种,如操作的成功,其经济价值可在野生菌株的基础上提高几百甚至上千倍。微生物液体发酵是体现低成本和容易工业化生产的基础,但木霉在液态培养产孢量少,固态培养又制约了大规模生产,而木霉菌株的活菌制剂施用的是分生孢子,即分生孢子数量与药效成正比。
本发明主要针对木霉农药菌株最重要的抑菌活性和液体培养产孢量特性,诱变育种获得的菌株HTTA-Z0002,不但对植物危害较大的病原菌具有优良和广谱的抑菌特性,还具有良好的人工发酵培养生产的特性。
发明内容
本发明的目的在于提供一种木霉菌及其应用。
为了实现上述目的,本发明提供如下技术方案。
一种木霉菌,所述木霉菌为棘孢木霉(Trichoderma asperellum)菌,菌株号:HTTA-Z0002,保藏编号CGMCC No.15677。
本发明的棘孢木霉(Trichoderma asperellum)菌保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏地址:北京市朝阳区北辰西路1号院3号;保藏日期2018年5月17日。
上述木霉菌在防治植物病害中的应用。
进一步地,所述植物病害包括茄早疫病(Alternaia solani)、番茄灰霉病(Botrytis cinerea)、马铃薯晚疫病(Phytophthora infestans)、小麦赤霉病(Fusariumgraminearum)、大豆锈病(hakopsora pachyrhizi)、小麦叶斑病(Zymoseptoria tritci)、甜瓜枯萎病(Muskmelon blight)、甘蓝白粉病(Cabbage powdery mildew) 中的一种。本发明的木霉菌株对上述病害具有良好的广谱防治效果,尤其对小麦赤霉病防治效果非常好达100%,对番茄灰霉病防效达到80.1%-87.9%,发病比较严重后的治疗效果也在52.5%,对甜瓜枯萎病、甘蓝白粉病预防能达到 68.87%-72.26%和59.49%-60.76%,结果较传统的化学药剂效果好。
上述木霉菌在抑菌中的应用。
进一步的,所述木霉菌对稻瘟霉菌(Pyricularia oryzae)、茄链孢菌(Alternariasalani)、镰孢霉菌(Fusarium oxysporrum)、灰葡萄孢菌(Botrytis cinerea)、大丽轮枝菌(Verticillium dahliae)、芽枝霉菌(Cladosporium fulvum)、辣椒疫霉菌 (Phytophthoracapsici)、丝核菌(Rhizoctonia solani)、赤霉菌(Fusarium graminearum)、菌核菌(Sclerotina sclerotiorum)中的一种或多种具有抑制作用。本发明棘孢木霉HTTA-Z0002菌株基因检测证明,菌株具有几丁质酶和抗菌肽合成的关键基因,即可通过产生抗菌肽和几丁质酶抑制病原真菌,对上述农业常见和危害严重的10种植物病原真菌具有强和广谱的抑菌作用,抑菌圈都30mm 以上,抑菌率达79.0-95.4%。
一种农药,包含上述木霉菌。
上述木霉菌的培养方法,该菌株在固体MPDA培养基上,于温度20℃-23℃,培养3-5天,菌丝体转入液体MPDA培养基震荡培养5-7天。震荡培养3-7天。
进一步地,所述固体MPDA培养基由以下原料组成:马铃薯浸汁200-250ml, 蛋白胨1-2g,葡萄糖25-30g,NaCl 5-8g,MgCl2·6H2O 0.1-0.3g,KCl 0.1g,琼脂 18g,水1000ml。本发明的菌株很容易人工培养,在MPDA培养基上生长良好,培养基材料价格低廉,易于获得;菌株在MPDA人工培养基生长旺盛,产孢层与分生孢子梗密集如毡状,青绿色,间或白色,分生孢子梗有隔膜,产生侧向分枝,分枝垂直对生,基部较宽,末端较细,呈瓶状,分生孢子单孢,球形至卵形,无色,在尖端形成较明显的分生孢子团。
本发明具有如下特点:
1)本发明获得的棘孢木霉菌(Trichoderma asperellum)人工培养方法简单,培养材料价格低廉,易于获得。
2)本发明获得的棘孢木霉菌(Trichoderma asperellum),在抑菌活性、液体培养产分生孢子等特性上较野生菌株有较大的提高,克服了木霉菌株液体培养产孢量少的劣势,最大产孢量达到4.25×108cfu/cm2。
3)棘孢木霉菌(Trichoderma asperellum)对农业常见和危害严重的植物病原真菌具有强和广谱的抑菌作用,同时对相关植物病害具有良好防治作用,防治效果明显优于传统化学药剂。
附图说明
图1亲缘关系分枝树。
具体实施方式
以下具体实施例是对本发明提供的方法与技术方案的进一步说明,但不应理解成对本发明的限制。
1.木霉菌株的分类鉴定
棘孢木霉HTTA-Z0002菌株,在固体MPDA培养基(马铃薯浸汁200-250ml, 蛋白胨1-2g,葡萄糖25-30g,NaCl 5-8g,MgCl 26H2O 0.1-0.3g,KCl 0.1g,琼脂18g水 1000ml),20℃-23℃,培养3-5天,连续观察记录菌落形态、颜色等变化,挑取菌丝于光学显微镜下观察产孢结构。菌株在MPDA人工培养基生长旺盛,产孢层与分生孢子梗密集如毡状,青绿色,间或白色,分生孢子梗有隔膜,产生侧向分枝,分枝垂直对生,基部较宽,末端较细,呈瓶状,分生孢子单孢,球形至卵形,无色,在尖端形成较明显的分生孢子团。
菌株经上述方法培养,利用真菌ITS基因的通用引物,常规方法PCR扩增产物测序,并通过NCBI的BLAST程序进行对比,分析序列同源性及做亲缘关系分枝树比较近缘种类(见图1)。结合生物学菌落形态、颜色及产孢结构的显微构造,菌株的分子生物学信息,参考国内外相关木霉分类鉴定专著,鉴定菌株为棘孢木霉Trichoderma asperellum。
2.木霉菌株的诱变育种
经多次农药活性测试筛选出的木霉菌株,采用常规紫外-微波复合诱变,选择生长旺盛的单克隆菌落,以茄链孢菌Alternaria salani作为病原靶标菌,采用对峙培养法测试抑菌活性,计算抑菌率:抑制率(%)=(对照组靶标菌落直径—处理组靶标菌落直径)/对照组靶标菌落直径×100%,并与野生菌株的抑制率加以比较,选择抑菌率提高25%以上的菌株,先用固态MPDA培养基培养5天,淘汰生长异常的菌株,然后接入液态MPDA,分别在20℃、23、25℃培养7天,显微计数并比较其产孢数量,选择产孢数量提高50%的菌株,选出的菌株连续液体培养5代,采用上述同样活性和产孢数量测试方法,观察其抑菌和产孢的稳定性,最终选出的HTTA-Z0002木霉菌株,抑菌率较野生菌提高了67.2%,最大产孢量为4.25×108cfu/cm2,较野生菌提高了150.7%。
3.实验室植物离体抑菌活性测试及基因验证测试
菌株HTTA-Z0002,经MPDA培养基7天,采用上述同样的对峙培养法对农业常见和危害严重的10种植物病原真菌:稻瘟霉菌Pyricularia oryzae、茄链孢菌Alternariasalani、镰孢霉菌Fusarium oxysporrum、灰葡萄孢菌Botrytis cinerea、大丽轮枝菌Verticillium dahliae、芽枝霉菌Cladosporium fulvum、辣椒疫霉菌Phytophthoracapsici、丝核菌Rhizoctonia solani赤霉菌Fusarium graminearum,菌核菌(Sclerotinasclerotiorum)测试抑菌活性及计算抑菌率。在 NCBI(http://www.ncbi.nlm.nih.gov/)上检索到木霉β-1,4-乙酰葡糖胺糖苷酶基因 Nag1与Peptaibols合成酶基因Tex1序列,利用Premier 5.0设计对应的引物 JDZ-F/JDZ-R、KJT-F/KJT-R,JDZ-F(5′-CAGAACCGAATCCAGAGC-3′)/JDZ-R (5′-GAAGGAGTGTAGACGAGC-3′),PCR常规方法扩增,对菌株产生抑菌活性的特性进行验证,结果如下:
表1 HTTA-Z0002对植物病原菌抑菌活性
注释:T1:对峙培养测试,以抑菌圈大小表示菌株活性,抑菌圈30mm以上表示抑制活性强;T2:对病原菌的抑制率=(对照菌落半径-处理菌落半径)/对照菌落半径×100%植物病原菌:Po:Pyricularia oryzae;As:Alternaria salani;Fo:Fusarium oxysporum;Bc:Botrytis cinerea;Vd:Verticillium dahliae;Cf:Cladosporium fulvum;Pc:Phytophthora capsici; Rs:Rhizoctonia solani;Fg:Fusarium graminearum;Ss:Sclerotina sclerotiorum。
由上表数据可知,木霉HTTA-Z0002菌株对10种农业常见和危害严重的病原真菌,具有很好的抑菌效果,在对峙培养法中,对病原菌的活性都达到强抑制作用,抑菌圈都30mm以上,抑菌率达79.0-95.4%,显示强抑菌活性,具有研制新杀菌剂的潜力。从HTTA-Z0002菌株PCR扩增到Nag1基因、Tex1基因,特征条带分别约为800bp、250bp,说明菌株具有几丁质酶和抗菌肽合成的关键基因,即可通过产生抗菌肽和几丁质酶抑制病原真菌。
4.植物活体防治病害测试(植物盆栽试验及田间小区试验)
植物活体防治病害测试采用常规方法进行,盆栽植物苗培养至3-4片真叶,每次盆栽试验用苗不少于60棵,田间小区试验小区面积25m2以上,田间盐渍土壤小区防治试验,盐渍土壤选择即墨靠海近的自然盐渍耕地,氯化物盐约0.2- 0.3%,盆栽盐渍土壤采用人工培制的模拟盐渍土,每个处理不少于3个重复,接种发病或自然发病,用木霉活菌孢子稀释液进行防治,以农业生产防治该病常用的传统农药做为阳性对照,同浓度的无菌发酵液处理做为空白对照,6-7 天后调查病情,计算防治效果。
表1 HTTA-Z0002菌株对盆栽番茄灰霉病的防治效果
表2 HTTA-Z0002菌株对田间小区番茄灰霉病的防治效果
注:稀释200倍液。
表3盐渍化土壤HTTA-Z0002对甜瓜枯萎病的田间小区药效
注:稀释300倍液。
表4盐渍化土壤HTTA-Z0002对甘蓝白粉病的田间小区药效
注:稀释300倍液。
表1-4数据证明,木霉HTTA-Z0002菌株普通土壤盆栽或田间小区防治番茄灰霉病,防治效果分别达到80.1%-87.9%,发病比较严重后的治疗效果也在 52.5%,盐渍土壤防治化学药剂较难控制的甜瓜枯萎病、甘蓝白粉病预防能达到 68.87%-72.26%和59.49%-60.76%的治疗效果,结果都较传统的化学药剂效果好
5.第三方验证结果
为了经第三方确认菌株的农药潜力,与德国BASF合作期间,在双方签署对菌株的保密协议前提下,BASF在德国对上述2株菌株进行了多次植物防治病害测试验证并提供了防治结果。BASF试验的病害包括:茄早疫病Alternaia solani;番茄灰霉病Botrytiscinerea;马铃薯晚疫病Phytophthora infestans,小麦赤霉病Fusarium graminearum;大豆锈病Phakopsora pachyrhizi;小麦叶斑病 Zymoseptoria tritci。
表5 BASF公司在德国的生测结果
上述结果证明HTTA-Z0002棘孢木霉Trichoderma asperellum对上述病害具有良好的广谱防治效果,尤其对小麦赤霉病Fusarium graminearum防治效果非常好达100%。鉴于德国BASF公司在世界农药研制与开发领域的知名度,他们的结果是对HTTA-Z0002木霉菌株农药潜力的最好的验证。
由此,在植物离体测试、活体测试、盆栽试验、田间小区试验、第三方验证等多方面证实了菌株良好的农药潜力。
以上实施例的说明只是用于帮助理解本发明方法及其核心思想。应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以对本发明进行若干改进和修饰,这些改进和修饰也落入本发明权利要求保护范围内。
Claims (6)
1.一种木霉菌,其特征在于,所述木霉菌为棘孢木霉(Trichoderma asperellum)菌,菌株号:HTTA-Z0002,保藏编号CGMCC No.15677。
2.根据权利要求1所述的木霉菌的培养方法,其特征在于,将木霉菌菌株在固体MPDA培养基上,于温度20℃-23℃,培养3-5天,菌丝体转入液体MPDA培养基震荡培养5-7天。
3.根据权利要求2所述的木霉菌的培养方法,其特征在于,所述固体MPDA培养基由以下原料组成:马铃薯浸汁200-250ml, 蛋白胨1-2g,葡萄糖25-30g,NaCl 5-8g,MgCl2·6H2O0.1-0.3g,KCl 0.1g,琼脂18g,水1000ml。
4. 根据权利要求1所述的木霉菌在防治植物病害中的应用,其特征在于,所述植物病害包括马铃薯致病疫霉菌(Phytophthora infestans)造成的马铃薯晚疫病、赤霉菌(Fusarium graminearum)造成的小麦赤霉病、小麦叶枯菌(Zymoseptoria tritici) 造成的小麦叶斑病、甜瓜枯萎病菌(Muskmelon blight)造成的甜瓜枯萎病、甘蓝白粉菌(Cabbage powdery mildew)造成的甘蓝白粉病中的一种。
5. 根据权利要求1所述的木霉菌在抑菌中的应用,其特征在于所述木霉菌对稻瘟霉菌(Pyricularia oryzae)、茄链孢菌(Alternaria salani)、镰孢霉菌(Fusarium oxysporrum)、灰葡萄孢菌(Botrytis cinerea)、大丽轮枝菌(Verticillium dahliae)、芽枝霉菌(Cladosporium fulvum)、辣椒疫霉菌(Phytophthora capsici)、丝核菌(Rhizoctonia solani)、赤霉菌(Fusarium graminearum)、菌核菌(Sclerotina sclerotiorum)中的一种或多种具有抑制作用。
6.一种农药,包含权利要求1所述的木霉菌。
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