CN117363489B - 一株具备黄瓜促生和抗病功能的长尾柄孢壳菌及其应用 - Google Patents
一株具备黄瓜促生和抗病功能的长尾柄孢壳菌及其应用 Download PDFInfo
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Abstract
一株具备黄瓜促生和抗病功能的长尾柄孢壳菌及其应用,保藏于中国普通微生物菌种保藏管理中心,其保藏编号为CGMCC NO.40741。本发明提供的长尾柄孢壳菌NNUF2能有效抑制尖孢镰刀菌黄瓜专化型(Fusarium oxysporum f.sp.Cucumerinum)、腐皮镰孢菌(Fusarium solani)、茄科劳尔氏菌(Ralstonia solanacearum)的生长。应用长尾柄孢壳菌NNUF2制备的微生物菌剂,能够有效防治黄瓜枯萎病、立枯病的发生,同时促进黄瓜生长,是一株多功能菌株。
Description
技术领域
本发明涉及农业微生物领域,具体涉及一株具备黄瓜促生与抗病功能的长尾柄孢壳菌NNUF2及其应用。
背景技术
黄瓜枯萎病和立枯病是黄瓜生产中最常见的土传病害,在世界范围内广泛分布,在我国各黄瓜产区均有发生。黄瓜枯萎病是由尖孢镰刀菌黄瓜专化型(Fusariumoxysporum f.sp.Cucumerinum)引起的一种真菌性土传病害,病原菌直接侵入黄瓜植株的根颈部并寄生于维管束内,阻碍植株对水分和养分的吸收,最终引起植株萎蔫枯黄。黄瓜立枯病又称死苗、猝苗,是由立枯丝核菌(Rhizoctonia solani)侵染黄瓜幼苗引起的另一种真菌性土传病害,多发生在育苗的中、后期,造成大量死苗甚至毁床。黄瓜枯萎病和立枯病,病程短、传播快,防治极为困难。
目前生产上应对土传病害主要采用农业防治与化学防治相结合的办法,具体包括培育抗性品种、抗性钻木嫁接及施用化学药剂等。此类防治方法虽然具有一定的防治效果,但也存在着优质抗性品种资源缺乏、培育时间长,嫁接育苗成本高且影响果实口感、化学药剂污染环境等诸多弊端。
近年来,生物防治已成为国内外防治土传病害的研究热点,相比而言,生物防治是利用一种或多种有益微生物来抑制病原菌生命活力和繁殖能力的方法,具有低成本、无药害残留、环境友好等特点。有益微生物可以通过抑制病原菌的菌丝生长及孢子萌发,同时与病原菌竞争生存空间和营养物质,诱导植物产生抗病原菌的化合物或其本身产生抗生素等物质,从而抵御病原菌入侵植物,增强植物的抗病能。此外,许多生防菌还能够通过固氮、融磷、解钾、产生吲哚-3-乙酸(IAA)等植物生长调节物质以促进植物生长和提高作物产量。然而,受菌株自身特性和环境条件等因素的影响,可供选择用于土传病害防治生产实际的微生物资源仍然匮乏,挖掘有效的生防微生物资源对生物防治领域的发展至关重要。
发明内容
解决的技术问题:本发明针对传统防治黄瓜枯萎病、立枯病等土传病害的手段存在着效率不高或危害环境等问题,提供一株具备黄瓜促生和抗病功能的长尾柄孢壳菌及其应用。
技术方案:一株长尾柄孢壳菌(Zopfiella longicaudata)NNUF2,2023年7月24日保藏于中国普通微生物菌种保藏管理中心,其保藏编号为CGMCC NO.40741,保藏地址为中国北京市朝阳区北辰西路1号院3号。
上述长尾柄孢壳菌NNUF2在制备抑制黄瓜专化型尖孢镰刀菌(Fusariumoxysporum f.sp.Cucumerinum)、腐皮镰孢菌(Fusarium solani)、茄科劳尔氏菌(Ralstonia solanacearum)菌剂中的应用。
一种菌剂,含有上述长尾柄孢壳菌NNUF2,含菌量≥1×108CFU/mL。
上述菌剂的制备方法,步骤为:将活化后的长尾柄孢壳菌NNUF2接种至PDA液体培养基中培养,培养条件为:25℃~28℃,搅拌速度170r/min,培养时间96h;培养完成后,破碎菌体,使用离心机10000r/min离心10分钟,弃去上清液,使用无菌水清洗两遍,并稀释至含菌量约1×108CFU/mL。
上述长尾柄孢壳菌NNUF2在促进黄瓜幼苗生长中的应用。
上述应用具体步骤为:将上述菌剂按1×108CFU/kg浓度接种至育苗基质。
上述长尾柄孢壳菌NNUF2在防治黄瓜枯萎病中的应用。
上述应用的具体步骤为:将上述菌剂按1×108CFU/kg、辅料以10g/kg的浓度一起加入枯萎病病土;所述辅料由苜蓿、水稻或小麦秸秆粉碎制成,并添加尿素调节其碳氮比至25~30:1。
有益效果:本发明提供的长尾柄孢壳菌NNUF2能有效抑制尖孢镰刀菌黄瓜专化型(Fusarium oxysporum f.sp.Cucumerinum)、腐皮镰孢菌(Fusarium solani)、茄科劳尔氏菌(Ralstonia solanacearum)的生长。应用长尾柄孢壳菌NNUF2制备的微生物菌剂,能够有效防治黄瓜枯萎病、立枯病的发生,同时促进黄瓜生长,是一株多功能菌株。
目前生产上对土传病害的防治措施主要包括嫁接技术、培育抗性品种,高温闷棚以及化学熏蒸等。虽然这些方式具有一定的防治效果,但是这些措施也都存在一定的缺陷。例如,高温闷棚受环境条件限制大、效果不佳;抗病品种的选育时间长,影响作物品质;土壤化学熏蒸会将有益微生物群落连同病原微生物一并杀死,且大部分熏蒸剂对生态环境破坏性较强。相对而言,应用本发明提供的长尾柄孢壳菌NNUF2进行生物防治,具有低成本、高效、无药害残留、绿色环保等优点,有利于农业可持续发展。
现有的柄孢壳菌多被报道具有抗炎和抗肿瘤活性等功能,应用于农业微生物领域的柄孢壳菌报道较少,即便有报道也存在抑制病原菌能力较弱或不具备广谱抑病能力等缺点。CN108179115A(一株亳菊内生柄孢壳菌及其应用)、CN111592988A(一株桑树内生拮抗菌新种柄孢壳菌及其应用)专利文献报道了应用于农业领域的两株柄孢壳属菌株,但其中未见具备在土壤中抑制植物病害的结果。此外,目前尚无本发明中所述的长尾柄孢壳菌(种水平)应用于生物防治的相关报道。本发明提供的长尾柄孢壳菌不仅具有促生效果,同时能够抑制真菌与细菌性土传病害。尚未见现有技术中公开同属菌株具有抑制细菌性病原菌(茄科劳尔氏菌)的效果。
本发明还提供了一种长尾柄孢壳菌NNUF2的菌剂及其防控作物病害的应用方法。一方面应用此方法能够有效提高防病效果,在本发明的一个实施例中,与单独施用NNUF2菌剂相比,NNUF2菌剂与作物秸秆制备成的辅料混合施用对黄瓜立枯病的防治效果第一茬和第二茬分别提高了62.96%和55.56%。另一方面,使用该方法,还能够提高作物秸秆利用率,减少环境污染,缓解农业有机废弃物处理压力。
附图说明
图1为本发明提供的长尾柄孢壳菌NNUF2在PDA培养基的正面及反面形态;
图2为本发明提供的长尾柄孢壳菌NNUF2的系统发育树;
图3为本发明提供的长尾柄孢壳菌NNUF2对两种病原真菌的抑制效果。A,尖孢镰刀菌黄瓜专化型(左侧)和NNUF2(右侧);B,腐皮镰孢菌(左侧)和NNUF2(右侧);
图4为本发明提供的长尾柄孢壳菌NNUF2对茄科劳尔氏菌的抑制效果。CK为对照;NNUF2为添加NNUF2过滤上清液处理;
图5为本发明应用长尾柄孢壳菌NNUF2菌剂促进黄瓜幼苗生长试验效果;
图6为本发明应用长尾柄孢壳菌NNUF2菌剂对黄瓜枯萎病的防治效果;
图7为本发明实施例应用长尾柄孢壳菌NNUF2防治黄瓜立枯病的试验效果。
具体实施方式
下面通过具体实施例进一步对本发明进行详细描述,但不以任何方式限制本发明的范围。本发明所述技术方案,如未特别说明,均为常规技术。
实施例1、长尾柄孢壳菌NNUF2菌株的分离与鉴定
1.1菌株分离
采集广东省设施蔬菜栽培土壤,将5g土样置于盛有45mL无菌水的三角瓶中,200r/min、28℃振荡30min制成土壤悬液。吸取100μL土壤悬液置于PDA培养基平板并涂布,放置于28℃培养箱中培养48h,观察平板上菌落生长情况,挑选差异菌落纯化,将纯化后的菌株接种到新的PDA培养基上28℃培养3天后置于4℃冰箱保存待用。
1.2长尾柄孢壳菌NNUF2的鉴定
将菌株长尾柄孢壳菌NNUF2接种至PDA培养基上,28℃培养5天,结果如图1所示,菌落圆整,中心呈褐色,从中心向菌落边缘颜色逐渐变为黑色,新生菌丝呈乳白色绒毛随后逐渐向黑灰色转变,菌落扁平呈毛绒状,气生菌丝致密。对上述菌株进行测序:采用ITS通用引物ITS1/ITS4:5'-TCCGTAGGTGAACCTGCGG-3'/5'-TCCTCCGCTTATTGATATGC-3'对菌株的ITS基因序列进行扩增和测序,然后在NCBI中进行BLAST比对,并构建系统发育树图,长尾柄孢壳菌NNUF2的ITS序列如SEQ NO.1所示。系统发育树如图2所示,菌株NNUF2与长尾柄孢壳菌(Zopfiella longicaudata)聚类到一支。结合菌株形态综合判定菌株NNUF2属于长尾柄孢壳菌种。
实施例2、长尾柄孢壳菌NNUF2对病原菌的抑制效果
在PDA培养基平板上,在平板左侧接种活化后的尖孢镰刀菌黄瓜专化型菌株菌饼(直径6mm),在平板右侧接种活化后的长尾柄孢壳属NNUF2菌株菌饼(直径6mm),在28℃条件下培养96h,观察菌株生长情况,具体如图3A所示。在PDA培养基平板上,在平板左侧接种活化后的腐皮镰孢菌菌株菌饼(直径6mm),在平板右侧接种活化后的长尾柄孢壳属NNUF2菌株菌饼(直径6mm),在28℃条件下培养96h,观察菌株生长情况,具体如图3B所示。
将活化后的NNUF2接种至PDA液体培养基中,28℃,170r/min振荡培养96h。培养完成后,10000r/min离心10分钟,取其上清液,过0.22μm除菌滤膜。量取50mLNNUF2过滤上清液至150mL NA培养基中,接种活化后的茄科劳尔氏菌,30℃、170r/min振荡培养24h,使用分光光度测定其在600nm波长处的吸光度。另设置150mL NA培养基添加50mL PDA液体培养基接种茄科劳尔氏菌作为对照(CK),具体如图4所示。
其中所用PDA液体培养基配制方法:200g土豆削皮后切成小块放入水中煮沸20min,过滤,滤液中加入20g普通葡萄糖,定容至1L,pH值自然,120℃灭菌20min。其中所用NA培养基配制方法:胰蛋白胨(Tryptone)10g,氯化钠(NaCl)5g,牛肉膏3g,定容至1L,使用NaOH调节pH至7.3,120℃灭菌20min。
由图3可见NNUF2具有繁殖迅速的特点,在相同时间能够抢占更多的生长空间与营养,出现包围病原菌的抑菌现象,同时在菌落边缘形成明显抑菌圈,抑制尖孢镰刀菌黄瓜专化型和腐皮镰孢菌菌丝生长。根据图4,添加NNUF2过滤上清液处理的茄科劳尔氏菌浓度在培养24h后显著低于CK,长尾柄孢壳菌NNUF对茄科劳尔氏菌的抑制率达到70.0%。
实施例3、长尾柄孢壳菌NNUF2对黄瓜幼苗的促生效果
3.1 NNUF2微生物菌剂的制备
长尾柄孢壳菌NNUF2菌株接种到PDA培养基平板上,25℃~28℃,培养3~4d,获得长尾柄孢壳菌NNUF2菌饼。在长尾柄孢壳菌NNUF2菌饼表面刮取菌丝与孢子,接种至PDA液体培养基中液体培养,培养条件为:25℃~28℃,搅拌速度170r/min,培养时间96h。培养完成后,使用破碎仪将菌丝打碎,离心机10000r/min离心10分钟,弃去上清液,使用无菌水清洗两遍后将菌体稀释至1×108CFU/mL。
3.2NNUF2微生物菌剂对黄瓜幼苗的促生效果试验
促生试验供试黄瓜品种为天津科润津冬58号。将黄瓜种子在50℃-60℃温水中浸泡6h后,放置在湿润的纱布上于28℃培养箱中催芽12h-24h,待75%以上种子萌发后,选取露白的种子移至72孔育苗穴盘中,育苗穴盘提前均匀铺装按照1×108CFU/kg浓度接入NNUF2菌剂的育苗基质。设置不含NNUF2的普通育苗基质为对照组(CK)。播种30d后测量植株生物量,促生效果如表1和图5所示。
表1 NNUF2菌剂对黄瓜幼苗促生效果
结合表1与图5,种植30d后,添加NNUF2菌剂与使用普通育苗基质(CK)进行育苗,在黄瓜株高、地上部鲜重、地上部干重、叶面积(选取倒数第二片真叶)、叶绿素含量(选取倒数第二片真叶进行测定)方面均有显著性差异。与CK相比,添加NNUF2菌剂培育的黄瓜幼苗在株高、地上部鲜重、地上部干重、叶绿素方面,分别增加了25.4%、80.18%、29.28%、89.84%、97.74%,表明使用NNUF2菌剂对黄瓜幼苗期生长具有明显的促进作用。
实施例4、长尾柄孢壳菌NNUF2对黄瓜枯萎病和立枯病的防治效果
4.1 NNUF2对黄瓜枯萎病的防治效果
盆栽土壤采集自一块黄瓜多年连作地块,该地块黄瓜连作障碍严重,枯萎病高发。供试黄瓜品种为天津科润津冬58号。将黄瓜种子浸泡在50℃-60℃温水中浸泡6h后,放置在湿润的纱布上于28℃培养箱中催芽12h-24h,待75%以上种子萌发后,选取露白的种子移至育苗穴盘育苗,待长出2片真叶后挑选长势一致幼苗移栽至盆中。设置两个处理:①按照1×108CFU/kg干土添加NNUF2菌剂至盆栽土壤;②使用未添加菌剂土壤作为对照(CK)。每个处理3盆,每盆种植6株黄瓜幼苗,每盆装土2kg。所述①处理中,NNUF2菌剂加入盆栽土壤后充分混匀并等待菌株定殖4d后开始种植黄瓜幼苗。盆栽种植期间正常水肥管理,种植8周后统计发病率与植株生物量指标。
表2NNUF2对黄瓜枯萎病的防治效果
发病率=(病株数/株数总和)×100%;
防治效果=[(CK发病率-NNUF2发病率)/CK发病率]
结合表2与图6,添加长尾柄孢壳菌NNUF2菌剂显著降低了黄瓜枯萎病发病率58.35%,对黄瓜枯萎病防效达到87.5%。添加NNUF2菌剂处理黄瓜在株高、地上部鲜重、地上部干重方面较对照也有显著增加,分别增加了26.16%、18.81%和56.94%。综上,说明应用NNUF2菌剂能够有效防治黄瓜枯萎病,同时具有促进黄瓜生长的效果。
4.2NNUF2对黄瓜立枯病的防治效果
盆栽土壤采集自江苏省常州市某温室大棚,该大棚土壤富含立枯丝核菌,黄瓜立枯病高发。供试黄瓜品种为天津科润津冬58号。将黄瓜种子浸泡在50℃-60℃温水中浸泡6h后,放置在湿润的纱布上于28℃培养箱中催芽12h-24h,待75%以上种子萌发后,选取露白的种子移至育苗穴盘育苗,待长出2片真叶后挑选长势一致幼苗移栽至盆中。试验设置三个处理:①按照1×108CFU/kg干土添加NNUF2菌剂至盆栽土壤;②按照NNUF2菌剂1×108CFU/kg干土与1%辅料混合后添加至盆栽土壤;③使用未处理土壤作为对照(CK)。每个处理3盆,每盆种植9株黄瓜幼苗,每盆装土2.5kg。所述①②处理中,NNUF2菌剂及辅料添加至盆栽土壤后充分混匀并等待菌株定殖4d后开始黄瓜幼苗种植。所述②处理中,辅料由苜蓿、水稻、小麦秸秆等比例混合而成,并添加尿素调节其碳氮比至27:1。盆栽种植期间正常水肥管理,连续种植两茬,每一茬种植30天后统计发病率。
黄瓜发病率如图7所示。由图可知,添加长尾柄孢壳菌NNUF2菌剂处理与长尾柄孢壳菌NNUF2结合辅料均能降低黄瓜立枯病发病率,且应用菌剂结合该辅料的方法具有更好的防治效果。其中,与对照相比,NNUF2菌剂结合辅料处理,第一茬、第二茬分别降低了发病率77.78%、55.56%;仅添加NNUF2菌剂处理,第一茬、第二茬分别降低了发病率14.82%、22.23%。综上,说明应用NNUF2结合本发明所述辅料的方法能够提高菌株生防效果,更加高效防治黄瓜立枯病。
上述技术方案仅体现了本发明技术方案的优选技术方案,本技术领域的专业人员对其中某些部分所可能做出的一些变动均体现了本发明的原理,属于本发明的保护范围之内。
Claims (8)
1.一株长尾柄孢壳菌(Zopfiella longicaudata)NNUF2,2023年7月24日保藏于中国普通微生物菌种保藏管理中心,其保藏编号为CGMCC NO.40741。
2.权利要求1所述长尾柄孢壳菌NNUF2在制备抑制黄瓜专化型尖孢镰刀菌(Fusarium oxysporum f. sp. Cucumerinum)、腐皮镰孢菌(Fusarium solani)或茄科劳尔氏菌(Ralstonia solanacearum)的菌剂中的应用。
3.权利要求1所述长尾柄孢壳菌NNUF2在促进黄瓜幼苗生长中的应用。
4.权利要求1所述长尾柄孢壳菌NNUF2在防治黄瓜枯萎病中的应用。
5.一种菌剂,其特征在于,含有权利要求1所述长尾柄孢壳菌NNUF2,含菌量≥1×108 CFU/mL。
6.权利要求5所述菌剂的制备方法,其特征在于,步骤为:将活化后的长尾柄孢壳菌NNUF2接种至PDA液体培养基中培养,培养条件为:25℃~28℃,搅拌速度170 r/min,培养时间96h;培养完成后,破碎菌体,使用离心机10000 r/min离心10分钟,弃去上清液,使用无菌水清洗两遍,并稀释至含菌量≥1×108 CFU/mL。
7.权利要求5所述菌剂在促进黄瓜幼苗生长中的应用,其特征在于,步骤为:将所述菌剂按1×108 CFU/kg浓度接种至育苗基质。
8.权利要求5所述菌剂在防治黄瓜枯萎病中的应用,其特征在于,步骤为:将所述菌剂按1×108 CFU/kg、辅料以10 g/kg的浓度一起加入枯萎病病土;所述辅料由苜蓿、水稻或小麦秸秆粉碎制成,并添加尿素调节其碳氮比至25~30:1。
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