CN114507614A - 一种降解秸秆并抑制植物病原真菌的芽孢杆菌及其应用 - Google Patents
一种降解秸秆并抑制植物病原真菌的芽孢杆菌及其应用 Download PDFInfo
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Abstract
本发明涉及一种降解秸秆并防治植物真菌病害的高效芽孢杆菌(Bacillus)。本发明所述芽孢杆菌保藏在中国普通微生物菌种保藏管理中心,保藏编号为CGMCC NO.22829。本发明涉及的芽孢杆菌具有高效降解秸秆的功能,并且可以显著抑制植物病原真菌生长,具有不污染环境和生态安全的优势,能够广泛应用到植物病原真菌,特别是针对梨黑斑病、马铃薯早疫病等多种链格孢属真菌和大斑凸脐孺孢属真菌的防治。此外,将该发明菌株添加到饲料中,进行发酵处理,所形成的生物饲料可有效防止饲料病原真菌污染,达到替抗功效。
Description
技术领域
本发明涉及微生物和发酵饲料添加剂领域,特别是涉及芽孢杆菌,具体而言,本发明涉及一种高效降解秸秆并防治植物真菌病害的芽孢杆菌及其在发酵饲料添加剂中的应用。
背景技术
秸秆是农作物生产中的一项重要生物资源,也是工农业生产的重要材料,更是当今世界上仅次于煤炭、石油和天然气的第四大能源物质。秸秆组成成分复杂,其主要由纤维素、半纤维素和木质素作为基本组分,通过与少量粗蛋白和粗脂肪交联缔结共同组成。在自然条件下,秸秆具有很强的抗分解能力,从而影响资源再生与利用。
自然界中,部分微生物通过自身代谢产生纤维素、木质素等多种酶类,使其具有高效降解秸秆的能力。它们凭借反应条件温和、降解效率高等优点,成为秸秆降解的首选对象。然而,微生物生长繁殖需要适宜外界环境条件,但是自然环境条件无法满足其数量增殖需求,故无法快速降解秸秆,这成为在秸秆还田过程中存在的主要问题。因此,筛选快速分解秸秆的微生物并研制高效的生物菌剂,实现缩短秸秆分解时间,成为秸秆还田有效途径之一。
此外,秸秆中病原菌的残留,会造成新一轮作物病害发生,对作物生长具有严重威胁,影响秸秆还田效果。因此,筛选既能够高效降解作物秸秆,又能够拮抗抑制病原真菌生长的菌株,对秸秆高效、安全还田及其资源开发再利用具有重要生产实践意义。
近年来,芽孢杆菌降解秸秆起着极为重要作用。芽孢杆菌通过自身代谢活动产生多种纤维素降解酶类,对秸秆中木质纤维素具有很强的降解能力,从而提高秸秆利用率,增加蛋白质含量(李立波等,2017 )。此外,秸秆含有众多微量元素,作为发酵饲料原料可以补充动物体内缺乏的微量元素,从而提高动物的产量。本申请发明人经过研究发现一株既能高效降解秸秆,又能高效抑制多种植物病原真菌的芽孢杆菌。
发明内容
本发明的目的在于提供一种新的芽孢杆菌菌株,所述菌株对多种植物病害的防治有明显效果,且具有较高的稳定遗传性。
本发明所述的芽孢杆菌已经于2021年7月6日保藏在中国普通微生物菌种保藏管理中心,保藏编号为CGMCC NO.22829。
进一步地,本发明的芽孢杆菌具有高效降解纤维素和木质素的能力。
进一步地,本发明提供上述芽孢杆菌防治植物真菌病害的用途。
优选地,导致所述植物真菌病害的植物真菌病原体为梨黑斑病、马铃薯早疫病等多种链格孢属真菌和大斑凸脐孺孢属真菌。
本发明所提供的芽孢杆菌是从农业废弃物中分离得到的,其实验室命名为JY214,已经于2021年7月6日保藏在中国普通微生物菌种保藏管理中心,保藏编号为CGMCCNO.22829。
菌株JY214在LB固体培养基(培养基组成为:胰蛋白胨10 g/L,酵母浸粉5 g/L,氯化钠10 g/L,琼脂20 g/L)上菌落呈圆形,边缘光滑,乳白色,透明,凸起,湿润粘稠。利用16SrDNA基因特异引物对16S rDNA进行扩增,将扩增产物回收并送测序,将测序结果进行Blast分析,结果发现与菌株JY214高度同源的均为芽孢杆菌属。
本发明的效果
1.本发明的菌株分别点到水琼脂-苯胺蓝和羧甲基纤维素钠(CMC-Na)培养基上,均可以形成较明显褪色水解圈,说明本发明的菌株兼具有降解纤维素和木质素的能力;
2.在平板对峙法中,本发明的芽孢杆菌对梨黑斑、马铃薯早疫等多种链格孢属真菌和大斑凸脐孺孢属真菌具有良好的抑制效果,说明本发明具有较广的抑菌谱;
3.本发明的芽孢杆菌接种多代后,对梨黑斑、马铃薯早疫等多种链格孢属真菌和大斑凸脐孺孢属真菌的抑制效果未发生显著变化,说明本发明菌株具有很好的遗传稳定性。
附图说明
图1本发明的芽孢杆菌菌株的稀释分离过程示意图
图2本发明的芽孢杆菌菌株降解纤维素和木质素能力的测定
图3本发明的芽孢杆菌菌株对植物病原真菌的抑菌效果图
图4本发明的芽孢杆菌菌株的16S rDNA的聚类分析
具体实施公式
本发明公开一种高抗真菌活性的芽孢杆菌,本领域技术人员可以借鉴本文内容,适当改进工艺参数来实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说显而易见,这些都被视为包括在被发明内。本发明所述的芽孢杆菌已经通过较规范的实施例进行描述,相关人员明显能在不摆脱发明内容、精神和范围内对本文所述的方法和应用进行改动和适当的变更与组合,来实现和应用本发明技术。
下面结合实施例,进一步阐明本发明。
实施例1 微生物从土样中的分离
菌种分离样品采于河北唐山、沧州、邯郸大名、石家庄正定、行唐等市县农村,农作物生长区土壤及田间废弃秸秆。取2 g农田土壤,加入到含有20 mL无菌蒸馏水的三角瓶中,25℃ 180 rpm摇培20 min。取1 mL土壤悬浊液,将其加入到1.5 mL EP管中,25℃离心10,000×g,1 min。取100 μL离心后的土壤上清液,加入到含有900 μL无菌蒸馏水的1.5 mL EP管中,进行梯度稀释,此时稀释度为10-1;取稀释后溶液100 μL,加入到含有900 μL无菌蒸馏水的1.5 mL EP管中,继续进行梯度稀释,此时稀释度为10-2,以此类推;取稀释度为10-3、10-4、10-5的溶液进行平板涂布,涂布至羧甲基纤维素钠平板上,24℃倒置培养,至有大量菌落产生(如图1所示);从平板菌落上挑取少许菌种,根据其菌落形态初步划分菌种类型,并将其接种至相应培养基中,24℃倒置培养;如此根据菌种特征进行多次转接,直至得到纯培养菌落,并对其进行编号。
实施例2 纤维素降解菌和木质素降解菌的筛选
木质素降解菌的筛选:取纯培养的单克隆菌株,接种至5 mL LB液体培养基中,24℃,150 rpm摇培48 h。取5 μL菌液,点至水琼脂-苯胺蓝培养基中,晾干,24℃倒置培养24 h(如图2 A所示)。观察菌液处苯胺蓝是否发生褪色并形成透明区域。
纤维素降解菌的筛选:取纯培养的单克隆菌株,接种至5 mL LB液体培养基中,24℃,150 rpm摇培48 h。取5 μL菌液,点至CMC-Na培养基中,晾干,24℃倒置培养24~48 h,至菌落生长至适当大小。用1% 刚果红染液对上述平板染色15 min,弃去刚果红染液。用0.9%NaCl溶液对平板脱色15 min,弃去NaCl溶液(如图2 B所示)。记录菌落生长区域及其周围是否出现明显褪色水解圈。
实施例3拮抗菌株的筛选
防治植物真菌病害的拮抗细菌筛选:将直径5 mm的病原真菌菌盘接种至双层PDA固体培养基中央(培养基组成为:土豆200 g/L,葡萄糖20 g/L,琼脂13 g/L),24℃倒置培养60 h。在距菌落边缘5 mm处打孔(直径2.5 mm),弃上层培养基。取8 μL菌液加入孔中,24℃正置培养72 h。观察在真菌周围是否形成抑菌圈(如图3所示)。
实施例4 菌株的16S rDNA的序列测定和同源比对分析
从菌株基因组上PCR扩增16S rDNA片段,使用通用引物:27F(AGAGTTTGATCCTGGCTCAG)和1492R(TACGGCTACCTTGTTACGACTT)。
PCR反应体系(50 μL)为:模板(菌液) 1.0 μL、引物R(10 μM) 2.0 μL、引物R(10 μM) 2.0 μL、dNTP Mix(10 μM)0.5 μL、10×long PCR buffer 5.0 μL、Pfu 0.4 μL、0.5%BSA 1.5 μL、ddH2O 37.6 μL。PCR扩增程序为95℃ 5 min,94℃ 30 s,55℃ 30 s,72℃ 1.5min,33个循环,72℃ 10 min。扩增产物送往上海生工生物科技有限公司进行测序。
将测序得到的16S rDNA序列输入GenBank,应用BLAST软件进行同源性搜索。选取不同菌株的16S rDNA序列并与GenBank中已知的16S rDNA进行同源性比较。
对筛选得到的具有拮抗功能以及可以同时降解纤维素和木质素的菌株进行16SrDNA测序,利用NCBI(http://www.ncbi.nlm.nih.gov)数据库进行序列比对分析,下载不同菌株相似度较高的1~3条序列,使用MEGA 6.0软件构建系统进化树。
同源比较结果如下:
将该PCR扩增产物在GenBank中进行BLAST比对。比对结果表明,本发明的菌株JY214与其高度同源的均为芽孢杆菌属(如图4所示)。
Claims (6)
1.一种芽孢杆菌,保藏编号为CGMCC NO.22829。
2.根据权利1的要求,其特征在于,具有高效降解纤维素和木质素的能力。
3. 一种保藏编号为CGMCC NO.22829的芽孢杆菌在防治植物真菌病害的用途。
4.根据权利要求3的用途,其特征在于,所述植物真菌病害的真菌病原体为梨黑斑病、马铃薯早疫病等多种链格孢属真菌和大斑凸脐孺孢属真菌。
5. 一种微生物菌剂,其特征在于,将权利要求1所述的芽孢杆菌的在LB液体中培养,培养温度为37℃,转速为220 rpm,培养12 h,然后过滤并调节菌液细胞数为109个/mL,即制得微生物菌剂。
6.一种发酵饲料添加剂,其特征在于,根据权利要求1所述的芽孢杆菌,是一种在发酵饲料添加剂中常用菌株。
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