CN116555091B - 一株耐盐性黏细菌及其应用 - Google Patents
一株耐盐性黏细菌及其应用 Download PDFInfo
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Abstract
本发明公开了一株耐盐性黏细菌及其应用。一株耐盐性黏球菌MxC21，其保藏号为GDMCCNo：63059。本发明所述的黏球菌MxC21能够在2％的盐浓度下正常生长，具有良好的耐盐性。共培养实验显示黏细菌MxC21能够拮抗包括稻瘟病菌，尖孢镰刀菌，禾谷镰刀菌，葡萄孢菌和大丽轮枝菌在内的多种植物病原真菌的生长，显示出黏细菌MxC21广谱的抗病原真菌能力。盆栽实验显示，黏细菌处理能够显著提高水稻和玉米在盐胁迫条件下的存活率，提高植物耐盐性和生长状态，可用于盐渍地土壤改良和盐胁迫下作物种植，提高粮食产量，实现盐碱土地资源开发与利用。
Description
技术领域
本发明属于应用微生物学领域，涉及一株具有耐盐特性的黏球菌及其在拮抗植物病原菌和提高植物耐盐性中的应用。
背景技术
自20世纪初期，国内外专家围绕盐碱地的治理与改良开展了大量理论和实践研究。目前，全世界盐碱地改良利用已形成两类主导技术：灌排技术和发展耐盐植物。随着研究的发展，利用微生物的作用实现盐碱地改良与资源利用已成为一种可选的手段，其中利用微生物与植物的相互作用提高植物耐盐性以及利用耐盐微生物实现盐碱地的改良已取得较好的研究与应用进展。目前已报道的用于盐碱地改良和提高植物耐盐性的微生物主要包括假单胞菌属(202111349055.X)、芽孢杆菌(201910838043.X)、黄杆菌(202011100077.8)、栖盐田菌(202111339361.5)以及相关的微生物混合菌剂(201910838043.X)等。
黏细菌是一类具有多细胞行为的土著微生物，具有良好的环境适应性和土壤微生物菌群调控能力。目前，国内外所获取的黏细菌资源主要来源于常规环境样品，且分离纯培养野生型黏细菌的纯度验证方法主要为LB培养基培养法。由于LB培养基中含有1％NaCl，因此常规方法分离获得的黏细菌一般不具有耐盐特性。因此，耐盐性黏细菌资源及其应用并未引起太多的关注。目前，国内仅少数研究单位已开展了耐盐和嗜盐性黏细菌的研究工作，仅限于资源分离和耐盐特性研究等方面，其在农业生产过程中的应用尚未涉及，且耐盐性黏细菌资源及其应用无相关专利报道。
发明内容
本发明提供一株耐盐性黏细菌Myxococcus sp.MxC21在拮抗植物病原菌和提高植物耐盐性中的应用。
一株拮抗植物病原菌的粘细菌MxC21，分类命名为黏球菌(Myxococcus sp.)，保藏于广东省微生物菌种保藏中心，保藏地址为广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所，保藏日期为2022年12月20日，其微生物保藏号为GDMCC No：63059。
本发明所述的黏细菌菌株MxC21的培养物、菌株菌液。
本发明所述的黏细菌MxC21为活性成分的生物菌剂和生物肥料。
本发明所述的黏细菌MxC21、其培养物或菌株发酵培养液或以其为活性成分的生物菌剂或生物肥料在盐渍地土壤修复和/或提高植物耐盐性中的应用，其中所述的植物为水稻或玉米。
所述的应用，优选将所述的黏细菌MxC21进行培养，得到菌株培养物、菌株菌液或菌株发酵培养液，所制备的黏细菌MxC21菌悬液细胞浓度为5х108CFU/mL；用所得到的菌株培养物、菌株菌液或菌株发酵培养液应用于所述的改良盐渍地土壤和/或提高植物耐盐性。
本发明还提供2种提高植物耐盐性的方法，优选的，将制备的MxC21菌悬液按照每植株3-10mL进行灌根处理，或者室温条件下将植物幼苗根部浸泡到所制备的菌悬液中3-5h，后进行移栽。
作为本发明的一种优选，每植株5mL进行灌根处理。
本发明中黏细菌Myxococcus sp.MxC21具有良好的耐盐特性，盆栽实验中，黏细菌MxC21能够较好的定殖到植物根部，在盐胁迫下促进水稻或玉米的生长。
本发明所述的黏细菌MxC21及其培养物、菌株菌液或菌株发酵培养液在防治植物病原菌中的应用。其中所述的病原真菌包括稻瘟病菌(Magnaporthe oryzae)，尖孢镰刀菌(Fusarium oxysporum)，禾谷镰刀菌(Fusarium graminearum)，葡萄孢菌(Botrytiscinerea)和大丽轮枝菌(Verticillium dahlia)等。
本发明中黏细菌Myxococcus sp.MxC21在平板对峙培养中能够显著抑制供试病原菌的生长。
有益效果
本发明采用兔粪诱导法成功的从所采集的土样中筛选到一株黏细菌菌株MxC21，经16S rDNA鉴定为黏球菌(Myxococcus sp.)，该菌能够在含有2％NaCl条件下生长，具有良好的耐盐特性。同时该菌能够抑制多种植物病原真菌的生长，表现出黏细菌MxC21广谱的抗病原菌活性。盆栽实验表明本发明涉及的黏细菌Myxococcus sp.MxC21在盐渍地改良和提高植物耐盐能力和生长状态方面具有巨大的应用潜力。
附图说明
图1菌株纯化流程图
图2菌株的扫描电镜图
图3基于16s rDNA序列比的菌株鉴定
图4黏细菌MxC21具有良好的耐盐特性。
a图为菌株MxC21在含有不同盐浓度的固体平板生长；b图为菌株MxC21在含有不同盐浓度的液体环境下生长；
图5黏细菌Myxococcus sp.MxC21对多种病原真菌具有良好的抗菌能力
图6水稻盆栽实验
盆栽实验中菌株MxC21浸泡处理(a)和灌根处理(b)提高盐胁迫下水稻苗株的存活率。左侧为植株表型观察，右侧为存活率
图7玉米盆栽实验
盆栽实验中菌株MxC21浸泡处理(a)和灌根处理(b)提高盐胁迫下玉米植株的存活率。左侧为植株表型观察，右侧为存活率
生物材料保藏信息
Myxococcus sp.MxC21，分类命名为Myxococcus sp.，保藏于广东省微生物菌种保藏中心，保藏地址为广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所，保藏日期为2022年12月20日，其微生物保藏号为GDMCC No：63059。
具体实施方式
实施例1.菌株的分离纯化及驯化培养
将从新疆和江苏等地采集的土壤样品用100μg/mL的放线酮溶液浸泡过夜，后去除残留液。将灭菌的食草兔粪接种到浸泡土壤中，使得兔粪球的一半被包埋在土壤中。30℃恒温培养，3天后开始观察子实体的形成。用毛细管挑取所观察到的子实体，接种至VY/4平板上，待菌落扩散及时挑取菌落边缘的菌体，反复转接于VY/4培养基(干酵母0.25％，CaCl20.1％，pH至7.2)的固体平板。将纯化后的细菌接种于LB培养基(tryptone 1％，NaCl1％，yeast extract 0.5％)中，30℃振荡培养过夜培养液澄清即表明该细菌为纯菌株，命名为MxC21。
将纯化获得菌株按照3％的接种量重新接种到LB培养基中，30℃连续培养8天，待菌悬液变浑浊以后，按照3％的接种量再次接种到LB培养基中，30℃接续培养3天。待接种的菌株能够在LB培养基较好的生长时，通过终浓度为16％的甘油管进行低温冷冻保藏(图1)。
实施例2.菌株的鉴定及其耐盐特性
将培养好的菌株MxC21通过扫描电镜观察其菌体形态，发现MxC21细胞呈长杆状，无鞭毛(图2)。通过对该菌株16S rDNA(SEQ ID NO.1)扩增与序列测序获取基因序列，基于NCBI(www.ncbi.nlm.nih.govPblastP)数据库序列比及常规的生理生化分析，将该菌株初步鉴定为Myxococcus sp.(图3)。将黏细菌MxC21接种到含有0.5％，1％，2％和2.5％NaCl的CTT(1％酪胨，10mM Tris-HCl[pH7.6],1mM KH2PO4[pH 7.6],8mM MgSO4)固体培养基和液体培养基上，并以不含NaCl的CTT培养基作为对照，30℃接续培养3天，通过OD600测定黏细菌MxC21在不同条件下的生物量变化。结果发现。在固体(图4a)和液体(图4b)环境条件下，菌株MxC21均能够在含有2％的CTT培养基中生长良好，其中在固体平板上能够耐受2.5％的盐浓度(图4a)。结果表明通过对分离获得野生型黏细菌进行驯化培养获得的黏细菌MxC21具有良好的耐盐特性。
实施例3.黏细菌(Myxococcus sp.MxC21)拮抗植物病原真菌的研究
利用PDA培养基(200g土豆煮沸30min，过滤收集上清，加20g葡糖糖，1g酵母提取物，定容至1L，琼脂为15g)对选取的植物病原真菌进行培养，包括稻瘟病菌(Magnaportheoryzae)，尖孢镰刀菌(Fusarium oxysporum),禾谷镰刀菌(Fusarium graminearum)，葡萄孢菌(Botrytis cinerea)和大丽轮枝菌(Verticillium dahlia)等；随后将活化的真菌转接至VY/4固体培养基(干酵母0.25％，CaCl20.1％，pH至7.2，1.5％琼脂)上，待真菌菌落直径长出1-2cm左右时，将培养好的黏细菌MxC21接种到真菌菌落的两边，28℃条件下培养3天，观察菌落的生长状态。与对照真菌相比，共培养处理组黏细菌MxC21能够显著抑制植物病原真菌的生长(图5)。结果表明耐盐性黏细菌MxC21对多种植物病原真菌均显示出良好的拮抗作用，显示其良好的广谱抗病原真菌能力。
实施例4.耐盐性黏细菌MxC21提高植物耐盐能力的应用评估
4.1水稻盆栽实验
挑选大小一致，饱满的水稻种子置于去离子水中浸泡12h，将水倒掉，再用去离子水将水稻种子冲洗两遍，然后将处理后的水稻种子平铺在放有湿润滤纸的培养皿中，接着在黑暗的25℃的培养箱内萌发3天。选择出芽一致的水稻种子分别进行水培和土培(营养土与蛭石，2：1)培养，待水稻幼苗生长至三叶一心期时，进行后续处理。
将菌株MxC21接种到LB培养基摇瓶发酵2天(30℃，180rpm)，制备OD600为1的菌悬液。水稻盆栽实验通过2种黏细菌施用方式(根部浸泡和灌根)，并分别设置正常组，正常组+盐，正常组+盐+MxC21，正常组+MxC21组。根部浸泡处理：将三叶期水稻幼苗根部浸泡到含有OD600为1的MxC21菌悬液中4h，取出水稻苗移栽到含有营养土与蛭石的盆栽中；灌根处理：将出芽一致的水稻种子土培培养至三叶期，后将MxC21菌悬液进行灌根处理。所有处理组中正常组为浇灌自来水作为对照，加盐组为浇灌100mM NaCl溶液作为处理。实验设置每组15棵水稻幼苗，每组3个重复，处理20天后计算水稻苗株的存活率。整个培养过程在光照培养箱中进行，白天26±5℃及夜间21±5℃，相对湿度：白天50％±10％及夜间60％±6％。结果发现水稻在100mM NaCl胁迫下植株明显死亡，存活率低于60％，而黏细菌MxC21处理组在浸泡(图6a)和灌根(图6b)两种方式下，水稻苗株的存活率分别为88％和92％，水稻植株的生长明显得到改善。表明MxC21能够提高水稻的耐盐能力，提高盐胁迫条件下水稻的存活率。
4.2玉米盆栽实验
挑选大小一致，饱满的玉米种子，用70％的乙醇对玉米种子进行表面消毒1min，用无菌水冲洗3次，用3％的次氯酸钠溶液浸泡5min，用无菌水清洗6次，将玉米种子平铺在25℃的湿无菌滤纸上发芽3到4天，直至玉米主根长2cm。选择出芽一致的玉米种子分别进行水培和土培(白马实验基地土壤)培养，待玉米生长至三叶期时，进行后续处理。
将菌株MxC21接种到LB培养基摇瓶发酵2天(30℃，180rpm)，制备OD600为1的菌悬液。玉米盆栽实验通过2种黏细菌施用方式(根部浸泡和灌根)，并分别设置正常组，正常组+盐，正常组+盐+MxC21，正常组+MxC21组。根部浸泡处理：将三叶期玉米幼苗根部浸泡到含有OD600为1的MxC21菌悬液中4h，取出玉米幼苗移栽到含有营养土与蛭石的盆栽中；灌根处理：将出芽一致的玉米种子土培培养至三叶期，后将MxC21菌悬液进行灌根处理。所有处理组中正常组为浇灌自来水作为对照，加盐组为浇灌100mM NaCl溶液作为处理。实验设置每组15棵玉米幼苗，每组3个重复，处理20天后计算玉米苗株的存活率。整个培养过程在光照培养箱中进行，白天26±5℃及夜间21±5℃，相对湿度：白天50％±10％及夜间60％±6％。结果发现玉米在100mM NaCl胁迫下植株明显死亡，存活率低于88.9％，而黏细菌MxC21处理组在浸泡(图6a)和灌根(图6b)两种方式下，玉米苗株的存活率分别为100％和100％，玉米植株的生长明显得到改善。表明MxC21能够提高玉米的耐盐能力，提高盐胁迫条件下玉米的存活率。
Claims (8)
1.一株耐盐性黏细菌(Myxococcus sp.)MxC21，保藏于广东省微生物菌种保藏中心，保藏地址为广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所，保藏日期为2022年12月20日，其微生物保藏号为GDMCC No：63059。
2.权利要求1所述的黏细菌MxC21为活性成分的生物菌剂。
3.权利要求1所述的黏细菌MxC21为活性成分的生物肥料。
4.权利要求1所述的黏细菌MxC21、权利要求2所述的生物菌剂、权利要求3所述的生物肥料在提高水稻和/或玉米耐盐性中的应用，其特征在于，权利要求1所述的黏细菌MxC21、权利要求2所述的生物菌剂、权利要求3所述的生物肥料能够提高盐胁迫条件下水稻和/或玉米的存活率，或减缓盐害现象。
5.权利要求1所述的黏细菌MxC21、权利要求2所述的生物菌剂、权利要求3所述的生物肥料在防治植物病原真菌中的应用，其中所述的植物病原真菌选自稻瘟病菌(Magnaporthe oryzae)，尖孢镰刀菌(Fusarium oxysporum)，禾谷镰刀菌(Fusarium graminearum)，葡萄孢菌(Botrytis cinerea)和大丽轮枝菌(Verticillium dahlia)中的任意一种。
6.权利要求1所述的黏细菌MxC21、权利要求2所述的生物菌剂、权利要求3所述的生物肥料在水稻和/或玉米盐渍土壤修复中的应用。
7.权利要求1所述的黏细菌MxC21、权利要求2所述的生物菌剂、权利要求3所述的生物肥料在盐胁迫下促进水稻或玉米生长中的应用。
8.一种提高水稻或玉米耐盐能力的方法，其特征在于，将权利要求1所述MxC21菌株的菌悬液通过根部浸泡或灌根至水稻或玉米苗根际土壤中，所述菌悬液终浓度为5х108CFU/mL。
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