CN113558069A - 一株捕食植物病原菌的黏细菌h56d21及其应用 - Google Patents
一株捕食植物病原菌的黏细菌h56d21及其应用 Download PDFInfo
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Abstract
本发明公开了一株捕食植物病原菌的黏细菌H56D21及其应用，属于微生物技术与生物防治领域。分析菌株H56D21与相近模式菌株的ANI值和dDDH值，结果表明本发明H56D21菌株是玻囊菌属(Hyalangium）的新物种。本发明的Hyalangium sp.H56D21，于2020年11月17日保藏于广东省微生物菌种保藏中心，地址为广东省广州市越秀区先烈中路100号大院59号楼5楼，邮编为：510070，保藏编号为：GDMCC No：61294，其能捕食植物病原菌，并具有广谱的捕食特性，可以用于植物病害的生物防治。
Description
技术领域
本发明涉及微生物技术与生物防治领域，具体涉及一种捕食植物病原菌的黏细菌H56D21及其应用。
背景技术
植物病害是制约农作物优质高产的重要因素之一，给农作物的产量造成严重损失的同时，也增加了农业生产的投入成本，制约着农产品品质的提升。目前防控植物病害的主要途径有化学农药防治、抗性品种选育、生物防治等手段。目前广泛使用的化学农药见效快，但易诱发病原菌耐药性，且容易造成对环境的二次污染；抗性品种选育往往周期较长、技术难度大；而生物防治具有防治效果好、不易诱导产生耐药性、绿色环保、无二次污染等特点，随着国家农业发展政策的调整，近年来受到了越来越多的重视，逐渐成为植物病害防治领域的重要发展方向。
目前应用较多的植物病害生防菌主要包括了木霉、青霉、芽孢杆菌、假单胞菌、链霉菌等。黏细菌能产生抗逆性极强的黏孢子，广泛分布于土壤中，是一类特殊的具有群体捕食行为的“高等细菌”。它们能通过“狼群”捕食方式，分泌多种裂解酶对土壤中的细菌和真菌进行捕食，从而控制土壤中其他菌群的数量，维持土壤微生态平衡，是土壤有机物代谢转化的重要参与者。黏细菌也是重要的药源微生物，能产生丰富多样、结构新颖的抗菌活性次级代谢产物。因此，利用黏细菌的捕食和抑菌功能来防治植物病原菌繁殖和传播有望获得良好的防治效果。而目前关于黏细菌在植物病原菌的生物防治上应用报道不多，且主要集中在黏球菌（Myxococcus）、珊瑚球菌（Corallococcus）、多囊菌（Polyangium）、孢囊杆菌（Cystobacter）等属，目前专利数据库中仅能检索到3个相关专利：一株黏细菌及其应用（202010469601.2）、一株捕食植物病原细菌的叶柄黏球菌及其在细菌性病害生物防治中的应用（201711363218.3）、黏细菌在制备捕食和抑制植物病原细菌的药物中的应用（201611095485.2），关于玻囊菌属（Hyalangium）的黏细菌捕食植物病原菌还未见报道。黏细菌往往具有菌株特异性，即同种不同株型的黏细菌产生的裂解酶、活性次级代谢产物差异较大。因此，积极探索和发掘自然界中蕴藏的黏细菌新资源，利用其裂解和抑菌特性用于植物病原的生物防治，对于提升我国的生物防治能力，开发和创制高效的生防制品，促进我国农业的绿色高质量发展具有重要战略意义。
发明内容
本发明的目的是提供一株捕食植物病原菌的黏细菌Hyalangium sp. H56D21，该菌株于2020年11月17日保藏于广东省微生物菌种保藏中心，地址为广东省广州市越秀区先烈中路100号大院59号楼5楼，邮编为：510070，保藏编号为：GDMCC No：61294。
本发明的黏细菌主要生理生化特征为：革兰氏阴性，好氧，子实体呈粉红色椭球状或者豌豆状，营养细胞为杆状，菌落在VY/2培养基上为粉红色或棕黄色，辐射状向外扩展并形成薄膜，生长温度为20-37 ℃，pH为4.0-10.0，NaCl耐受范围为0-0.5%（w/v）。
本发明黏细菌H56D21的16S rRNA基因序列长度为1536 bp，如SEQ ID NO：1所示。将该序列在NCBI及EzBioCloud网站上进行同源性比对分析，其与Cystobacter gracilisDSM 14753T最高相似性为98.83%，系统发育分析表明其属于玻囊菌属（Hyalangium）。基于基因组分析结果表明，菌株H56D21与进化树上同一簇的亲缘关系较近的已发表模式菌株的平均核苷酸一致性为79.5%-85.6%，对应的模拟DNA杂交值为22.7%-29.8%，根据目前公认的对新种的定义（ANI值小于95%-96%、dDDH值小于70%），可以判定菌株H56D21为玻囊菌属的潜在新物种。
本发明黏细菌H56D21在捕食植物病原细菌实验中，对青枯劳尔氏菌（Ralstonia solanacearum）GDMCC 1.1561和香豌豆带化病菌（Rhodococcusfascians）GDMCC 1.839有较强的捕食能力，对甘蓝黑腐病菌（Xanthomonas campestris）GDMCC 1.857捕食能力稍弱；在捕食植物病原真菌实验中，菌株H56D21对四种植物病原真菌腐皮镰刀菌（Fusarium solani）GDMCC 3.501、黄瓜枯萎病菌（Fusarium oxysporum Schlechtendal）GDMCC 3.392、禾谷镰刀菌（Fusarium graminearum）GDMCC 3.507、香蕉枯萎病菌4号生理小种（Fusarium oxysporum）GDMCC 3.482均能捕食，不仅能抑制新的病原真菌菌丝的生长，也能裂解原已长出的病原真菌菌丝。基于上述实验结果表明，黏细菌H56D21在植物病原菌的生物防治方面具有重要的应用潜力。
本发明的第二个目的是提供玻囊菌属(Hyalangium）菌株在以下任一方面中的应用：
（1）植物病原菌的生物防治；
（2）制备抑制植物病原菌的药物；
（3）制备食品添加剂或动物饲料添加剂；
优选地，所述玻囊菌属菌株为黏细菌（Hyalangium sp.）H56D21，其保藏编号为：GDMCC No：61294；
优选地，所述的植物病原菌为腐皮镰刀菌（Fusarium solani）、黄瓜枯萎病菌（Fusarium oxysporum Schlechtendal）、禾谷镰刀菌（Fusarium graminearum）、香蕉枯萎病菌4号生理小种（Fusarium oxysporum）、青枯劳尔氏菌（Ralstonia solanacearum）、甘蓝黑腐病菌（Xanthomonas campestris）和香豌豆带化病菌（Rhodococcus fascians）的一种或多种。
本发明的第三个目的是提供一种微生物制剂，以本发明黏细菌H56D21培养物或其发酵液作为活性成分。
本发明的第四个目的是提供一种培养黏细菌H56D21的方法，是将黏细菌H56D21接于VY/2固体培养基上进行扩大培养，获得黏细菌H56D21的菌体。
本发明的第五个目的是提供一种本发明黏细菌H56D21的代谢产物，所述产物可应用于植物病原菌的生物防治。
与现有技术相比，本发明具有如下有益效果：
（1）黏细菌H56D21是玻囊菌属(Hyalangium）的新物种，该属目前仅有1株模式菌株并且还未曾报道过能捕食植物病原菌；
（2）黏细菌H56D21能捕食7种植物病原菌，具有广谱的捕食特性，可以用于植物病害的生物防治。
本发明的Hyalangium sp. H56D21，于2020年11月17日保藏于广东省微生物菌种保藏中心，地址为广东省广州市越秀区先烈中路100号大院59号楼5楼，邮编为：510070，保藏编号为：GDMCC No：61294。
附图说明
图1为黏细菌H56D21形态特征图，其中，a: 菌株H56D21在ST21平板上子实体图（500 μm）；b: 菌株H56D21在VY/2平板上菌落图（2 mm）；c: 菌株H56D21在光学显微镜下营养细胞图（100×, 5 μm ）。
图2 为黏细菌H56D21基于16S rRNA基因序列构建的系统进化树（NJ法）。
图3 为黏细菌H56D21捕食植物病原细菌图，其中，1、2、3为菌株H56D21捕食植物病原细菌的三个重复，CK为不接黏细菌的对照。
图4 为黏细菌H56D21捕食植物病原真菌图，其中，a为只接种禾谷镰刀菌对照，b为菌株H56D21捕食禾谷镰刀菌，c为只接种腐皮镰刀菌对照，d为菌株H56D21捕食腐皮镰刀菌，e为只接种黄瓜枯萎病菌对照，f菌株H56D21捕食黄瓜枯萎病菌，g为只接种香蕉枯萎病菌对照，h菌株H56D21捕食香蕉枯萎病菌。
具体实施方式
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
下面结合实施例对本发明作进一步说明，下述实施例中试验方法如无特殊说明，均为常规试验方法，下述实施例中所述的实验试剂及耗材如无特殊说明，均来自常规生化试剂公司。
实施例1 黏细菌H56D21的分离、纯化
1.1 菌株H56D21分离
培养基：（1）ST21培养基（溶液 A: K2HPO4 1.0 g，Yeast extract 0.02 g，Agar14.0 g，Water 600 mL；溶液 B: KNO3 1.0 g，MgSO4·7H2O 1.0 g，CaCl2·2H2O 1.0 g，MnSO4·7H2O 0.1 g，Water 300 mL；溶液 C: FeCl3 0.2 g，Water 100 mL。溶液A、B、C单独灭菌，加热至50℃左右混合后倒培养基）。（2）VY/2培养基：安琪酵母5 g， CaCl2 .2H2O 1.36g, Agar 15 g，水1L，将各成分混合均匀，调pH 7. 2。灭菌后加入VB12 50 μg/mL。（3）LB培养基：胰蛋白胨10 g，NaCl 10 g，酵母粉5 g，pH 7.2。
称取风干的森林土壤样品10 g加入适量的无菌水（添加终浓度为100 µg/mL的放线菌酮），浸湿约10 h，将土样点接于铺有无菌滤纸的ST21培养基上，置于30℃培养5天后，体视镜下连续用无菌的注射器针头挑取子实体或者菌膜，转接于新鲜的VY/2培养基上。
1.2 菌株H56D21纯化与保存
通过反复转接子实体或者菌膜于新鲜的VY/2培养基上，直至无杂菌生长。将疑似已纯化的菌落转接LB液体培养基中，30℃、200 r/min振荡培养12 h，如果培养基变浑浊，说明有杂菌生长；如果LB培养基澄清说明菌株已纯。将已纯化菌株转接于30 %甘油中，置于-80℃长期保存。由此获得黏细菌H56D21。
实施例2 黏细菌H56D21的分类鉴定
2.1 形态学鉴定
黏细菌H56D21子实体、菌落形态颜色、营养细胞形态特征见图1。该菌在ST21培养基上子实体单生，粉红色，椭球状或者豌豆状，有丰富的黏液包裹（a）；在VY/2培养基上菌落为粉色或者黄棕色，圆形，呈辐射的薄膜状，边缘火焰状凸起（b）；营养细胞为革兰氏阴性、两端钝圆的杆状细胞（c）。
2.2 分子鉴定
用改良的CTAB法提取菌株H56D21的基因组DNA，采用细菌通用引物F27/1492R扩增16S rRNA基因，扩增产物送至上海美吉生物医药科技有限公司（广州分公司）进行测序分析。用SeqMan软件拼接获得16S rRNA基因序列长度为1536 bp，如SEQ ID NO：1所示。将序列在NCBI和Ezbiocloud (http://www.ezbiocloud.net)进行对比分析，其与Cystobacter gracilis DSM 14753T序列相似性最高（98.83%），其次为Hyalangium minutum DSM 14724T(98.70%)。选取相近的模式菌株16S rRNA基因序列，采用MEGA 7.0软件以NJ构建系统发育树（图2）。根据系统进化树及形态特征分析表明，菌株H56D21从属于玻囊菌属（Hyalangium），且与Cystobacter gracilis DSM 14753T和Hyalangium minutum DSM14724T亲缘关系最近。
用细菌基因组提取试剂盒（Magen）提取菌株H56D21的基因组，由上海美吉生物有限公司进行基因组测序，所用测序平台为Illumina Hiseq。所得的数据采用SPAdesv3.13.0软件完成基因组组装并用Checkm检测基因组完整度。基于基因组序列，采用在线工具http://www.ezbiocloud.net/tools/ani和http://ggdc.dsmz.de/distcalc2.php分别计算菌株H56D21与相近模式菌株的平均核苷酸一致性（ANI）和模拟DNA-DNA杂交值（dDDH）。计算结果如表1所示，菌株H56D21与亲缘关系最近的模式的ANI值为79.6%-86.6%、dDDH值为22.7%-29.8%，均低于目前所公认的ANI值95%-96%、dDDH值70%的物种界限的阈值，这些结果表明菌株H56D21代表玻囊菌属（Hyalangium）的新物种。
表1 菌株H56D21与5株近缘模式种的比较基因组分析
2.3 生理生化鉴定
在VY/2平板上测定其生长温度（10℃、15℃、20℃、25℃、30℃、37℃、40℃）；以VY/2为基础培养基测定其pH值（4.0-10.0），NaCl耐受性（0-3%，w/v），抗生素耐受性（抗生素终浓度50 µg/mL）。使用API 20 NE、ZYM试剂条测定其酶活力指标。
菌株H56D21能在20-37℃，pH值为4.0-10.0，NaCl浓度为0-0.5%（w/v）的条件下生长；最适生长条件是28-30℃，pH值为7.0-8.0，无NaCl添加。该菌对氨苄西林、萘啶酮酸、多粘菌素B、安普霉素、杆菌肽B有抗性，而对庆大霉素、红霉素、卡那霉素、利福平、链霉素、四环素、甲氧苄啶、氯霉素、新霉素、土霉素敏感。该菌氧化酶和过氧化氢酶阳性，能水解酪蛋白、吐温20、80，淀粉、七叶苷和明胶；不能水解几丁质和纤维素。其碱性磷酸酶，酯酶(C4)，类酯酶(C8)，类肪酶(C14)，亮氨酸芳香基酰胺酶，缬氨酸芳香基酰胺酶，胱氨酸芳香基酰胺酶，胰蛋白酶，α-胰凝乳蛋白酶，酸性磷酸酶，萘酚-AS-Bl-磷酰胺酶，β-葡萄糖苷酶，N-乙酰-β-葡萄糖胺酶为阳性；其α-半乳糖苷酶，β-半乳糖苷酶，β-葡萄糖醛酸酶，α-葡萄糖苷酶，α-甘露糖苷酶，β-岩藻糖苷酶，精氨酸水解酶，脲酶为阴性。
因此结合形态学、分子生物学和生理生化特征，将菌株H56D21命名为Hyalangiumsp. H56D21，于2020年11月17日保藏于广东省微生物菌种保藏中心，地址为广东省广州市越秀区先烈中路100号大院59号楼5楼，邮编为：510070，保藏编号为：GDMCC No：61294。
实施例3 黏细菌H56D21捕食植物病原细菌
将菌株H56D21接于VY/2固体培养基上，30 ℃培养7 d。将植物病原细菌青枯劳尔氏菌（Ralstonia solanacearum）GDMCC 1.1561、甘蓝黑腐病菌（Xanthomonas campestris）GDMCC 1.857、香豌豆带化病菌（Rhodococcusfascians）GDMCC 1.839分别转接于液体营养肉汤培养基中，30 ℃、180 r/min振荡培养48 h后，8000 r/min离心5 min，收集菌体，用MMC缓冲液洗涤两次，将菌体重悬于MMC缓冲液，菌体浓度稀释至1×1011 cfu/mL，各取150 µL菌液点接于TPM培养基（Tris-HCl (pH 7.6) 10 mM，K2HPO4 1 mM，MgSO4 8 mM，Agar 15g，Water 1 L）中心，使得菌斑为圆形。待菌体干后，在黏细菌H56D21菌落上用蓝枪头打孔，将菌块接于菌斑中心，设置三个重复。以不接黏细菌、只接植物病原细菌的TPM平板作为对照（CK），将平板置于30 ℃培养，3天后观察捕食情况。培养3天后，可观察到病原菌菌斑变薄，而黏细菌H56D21在病原菌菌斑上生长；随着时间延长，黏细菌H56D21可以扩展到病原菌菌斑边缘，说明黏细菌H56D21能捕食病原细菌以获得营养物质（图3）。这些结果表明，黏细菌H56D21对青枯劳尔氏菌和香豌豆带化病菌有较强的捕食能力，对甘蓝黑腐病菌捕食能力稍弱。
实施例4 黏细菌H56D21捕食植物病原真菌
将菌株H56D21接于VY/2固体培养基上，30 ℃培养7 d。将植物病原真菌腐皮镰刀菌（Fusarium solani）GDMCC 3.501、黄瓜枯萎病菌（Fusarium oxysporum Schlechtendal）GDMCC 3.392、禾谷镰刀菌(Fusarium graminearum) GDMCC 3.507、香蕉枯萎病菌4号生理小种（Fusarium oxysporum）GDMCC 3.482分别转接于PDA培养基上，置于28 ℃培养直至病原真菌长满平板，用蓝色枪头沿同心圆在PDA培养基上打孔菌块，将植物病原真菌菌块接于VY/2培养基中心位置，置于28 ℃培养，待植物病原真菌长至直径约3 cm大小时，在黏细菌H56D21菌落上用蓝枪头打孔，将菌块接种至植物病原真菌菌落边缘处。每个处理三个重复，以只接植物病原真菌、不接黏细菌为对照。将平板置于28 ℃培养，每隔24 h观察植物病原真菌与黏细菌的生长情况。培养7天时进行拍照观察，结果如图4所示，菌株H56D21能够捕食腐皮镰刀菌、黄瓜枯萎病菌、禾谷镰刀菌、香蕉枯萎病菌4号生理小种，菌株H56D21不仅能抑制新的病原真菌菌丝的生长，也能裂解原已长出的病原真菌菌丝。
序列表
<110> 广东省科学院微生物研究所（广东省微生物分析检测中心）
<120> 一株捕食植物病原菌的黏细菌H56D21及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1536
<212> DNA
<213> 黏细菌H56D21(Hyalangium sp.)
<400> 1
caattggaga gtttgatcct ggctcagaac gaacgctggc ggcgtgccta acacatgcaa 60
gtcgagcgcg aatggagcaa tcctagtaga gcggcgcacg ggtgcgtaac acgtgggtaa 120
tctgcctaga agatgaggat aaccagtcga aagattggct aatactcgat aagcccacgg 180
ggacttcggt ccctgaggga aaaggtggcc tctgtataca agctatcact tctagatgag 240
cccgcggccc atcagctagt tggcggggta atggcccacc aaggcgacga cgggtagctg 300
gtctgagagg acgatcagcc acactggaac tgagacacgg tccagactcc tacgggaggc 360
agcagtgggg aattttgcgc aatgggcgaa agcctgacgc agcaacgccg cgtgtgtgat 420
gaaggtcttc ggattgtaaa gcactttcga ccgggacgaa aaccctgaag ctaacatctt 480
caggtttgac ggtaccggga gaagaagcac cggctaactc tgtgccagca gccgcggtaa 540
tacagagggt gcaagcgttg ttcggaatta ttgggcgtaa agcgcgtgta ggcggctttg 600
caagtcgggt gtgaaagccc tcagctcaac tgaggaagtg cgcccgaaac tgcagagctt 660
gagtgccgga gagggtggcg gaattcccca agtagaggtg aaattcgtag atatggggag 720
gaacaccggt ggcgaaggcg gccacctgga cggtaactga cgctgagacg cgaaagcgtg 780
gggagcaaac aggattagat accctggtag tccacgccgt aaacgatgag aactaggtgt 840
cgtgggagtt gacccccgcg gtgccgtagc taacgcatta agttctccgc ctgggaagta 900
cggtcgcaag actaaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt 960
ggtttaattc gacgcaacgc gcagaacctt acctggtctt gacatcctcg gaacctttca 1020
gagatgagag ggtgcccgca agggaaccga gagacaggtg ctgcatggct gtcgtcagct 1080
cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca accctcgcct ttagttgctc 1140
cgcaaggaga tctctagagg gactgccggt gttaaaccgg aggaaggtgg ggatgacgtc 1200
aagtcctcat ggcctttatg accagggcta cacacgtgct acaatggccg gtacagaacg 1260
ctgcaaaccc gcgaggggga gccaatcgta gaaaaccggt ctcagttcag attggagtct 1320
gcaactcgac tccatgaagg cggaatcgct agtaatcgca gatcagcacg ctgcggtgaa 1380
tacgttcccg ggccttgtac acaccgcccg tcacaccatg ggagtcgatt gctccagaag 1440
tcacctcacc aagaggtgcc caaggagtga tcggtaactg gggtgaagtc gtaacaaggt 1500
agccgtaggg gaacctgcgg ctggatcacc tccttt 1536
Claims (4)
1.黏细菌（Hyalangium sp.）H56D21在以下任一方面中的应用：
（1）植物病原菌的生物防治；
（2）制备抑制植物病原菌的药物；
（3）制备食品添加剂或动物饲料添加剂；
所述的黏细菌（Hyalangium sp.）H56D21，其保藏编号为：GDMCC No：61294。
2.根据权利要求1所述的应用，其特征在于，所述的植物病原菌为腐皮镰刀菌（Fusarium solani）、黄瓜枯萎病菌（Fusarium oxysporum Schlechtendal）、禾谷镰刀菌（Fusarium graminearum）、香蕉枯萎病菌4号生理小种（Fusarium oxysporum）、青枯劳尔氏菌（Ralstonia solanacearum）、甘蓝黑腐病菌（Xanthomonas campestris）和香豌豆带化病菌（Rhodococcus fascians）的一种或多种。
3.一种微生物制剂，其特征在于，以黏细菌H56D21培养物或其发酵液作为活性成分，所述的黏细菌H56D21，其保藏编号为：GDMCC No：61294。
4.一种培养黏细菌H56D21的方法，其特征在于，是将黏细菌H56D21接于VY/2固体培养基上进行扩大培养，获得黏细菌H56D21的菌体，所述的黏细菌H56D21，其保藏编号为：GDMCCNo：61294。
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