CN115918680A - 湿地链霉菌菌株及其发酵液在防治双孢蘑菇疣孢霉病中的应用 - Google Patents
湿地链霉菌菌株及其发酵液在防治双孢蘑菇疣孢霉病中的应用 Download PDFInfo
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- CN115918680A CN115918680A CN202211504308.0A CN202211504308A CN115918680A CN 115918680 A CN115918680 A CN 115918680A CN 202211504308 A CN202211504308 A CN 202211504308A CN 115918680 A CN115918680 A CN 115918680A
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Abstract
本发明涉及一种湿地链霉菌菌株13‑3在防治双孢蘑菇疣孢霉病中的应用，所述湿地链霉菌菌株13‑3保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.22830，保藏日期：2021年7月6日。本发明的优点在于：湿地链霉菌菌株13‑3对引起双孢蘑菇疣孢霉病的3种病原菌有害疣孢霉、红丝疣孢霉和M.xinjiangensis均具有拮抗作用，其发酵液可以显著抑制引起双孢蘑菇疣孢霉病的3种病原菌有害疣孢霉、红丝疣孢霉和M.xinjiangensis的菌丝生长，另外发酵液对双孢蘑菇疣孢霉病具有优良的防治效果，且对蘑菇安全。
Description
技术领域
本发明涉及农作物病害防治技术领域，具体为湿地链霉菌菌株及其发酵液在防治双孢蘑菇疣孢霉病中的应用。
背景技术
疣孢霉病是为害双孢蘑菇最重要的病害之一，其致病菌为疣孢霉属 (MycogoneLK.ex Chev.)真菌。据报道引起我国疣孢霉病的病原菌主要有3种，分别为有害疣孢霉M.perniciosa、红丝疣孢霉M.rosea和M.xinjiangensis。咪鲜胺锰盐和噻菌灵是目前仅有的2种在我国登记用于防治双孢蘑菇疣孢霉病的杀菌剂，其中咪鲜胺锰盐是蘑菇生产上最常用的杀菌剂，在我国已有近20年的使用历史，这无疑大大增加了病原菌产生抗性的风险。因此为了更好地防治疣孢霉病，需要开发生物防治。
放线菌是一类基因组中G+C比例高于55％的丝状革兰阳性菌，其次级代谢产物种类繁多，是重要的微生物资源。放线菌已有较长的研究历史，从中发现了许多重要的抗菌药物(两性霉素B、红霉素、万古霉素)、抗癌药物(柔红霉素、博莱霉素、丝裂霉素)和免疫抑制剂(雷帕霉素、他克莫司)。Yuan等从海南文昌红树林沉积物中的链霉菌Streptomycessp.211726中发现一系列具有广泛抗菌活性的9个新的大环内酯类化合物(3～11)和3个已知azalomycin类似物 (12～14)，该系列化合物均对白色念珠菌(Candida albicans)表现出较为明显的抗菌活性，其MIC为1.56～12.5μg/mL；5～11对金黄色葡萄球菌、枯草芽孢杆菌和大肠埃希菌表现出明显的抗菌活性，其MIC范围分别为0.39～1.56μg/mL、 0.20～0.78μg/mL和3.13～25.00μg/mL。Zeng等从海南东寨港红树林沉积物中分离得到的白色链霉菌Streptomyces albogriseolus HA10002中分离得到一种新的抗根结线虫活性成分fungichromin B(A22-1)，该化合物具有广泛的抗真菌谱，10、 1、0.1μg/mL下对真菌酵母菌(Saccharomyces cerevisiae)、尖孢镰孢菌(Fusarium oxysporum)和黑曲霉菌(Aspergillus niger)抑菌圈MIC大小在12～22mm范围。
放线菌作为生物防治的主要来源之一，在生物防治中发挥着巨大作用。寻求新的菌种资源成为一个新的重要难题。
发明内容
本发明的首要目的在于针对疣孢霉病的发生阻碍了双孢蘑菇产业的健康发展，杀菌剂大量使用造成环境污染和病原菌抗药性等问题，提供湿地链霉菌 (Streptomycespaludis)菌株13-3在防治双孢蘑菇疣孢霉病中的应用。
本发明的另一目的在于提供湿地链霉菌(Streptomyces paludis)菌株13-3发酵液在防治双孢蘑菇疣孢霉病中的应用。
本发明的目的通过如下技术方案实现：
湿地链霉菌(Streptomyces paludis)菌株13-3在防治双孢蘑菇疣孢霉病中的应用，所述湿地链霉菌(Streptomyces paludis)菌株13-3保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏中心地址：北京市朝阳区北辰西路1号院3号。保藏编号为CGMCCNo.22830，保藏日期： 2021年7月6日。
湿地链霉菌(Streptomyces paludis)菌株13-3对引起双孢蘑菇疣孢霉病的有害疣孢霉(Mycogone perniciosa)、红丝疣孢霉(M.rosea)和M.xinjiangensis 均有非常强的拮抗作用。
湿地链霉菌(Streptomyces paludis)菌株13-3发酵液在防治双孢蘑菇疣孢霉病中的应用，将湿地链霉菌(Streptomyces paludis)菌株13-3接种于ISP2液体培养基，发酵温度为26-28℃，发酵培养时间为7-8d，过滤后得发酵液。
所述湿地链霉菌(Streptomyces paludis)菌株13-3发酵液在抑制有害疣孢霉(Mycogone perniciosa)、红丝疣孢霉(M.rosea)和M.xinjiangensis的菌丝生长中的应用。
较之现有技术而言，本发明的优点在于：湿地链霉菌(Streptomyces paludis) 菌株13-3对引起双孢蘑菇疣孢霉病的3种病原菌有害疣孢霉(Mycogone perniciosa)、红丝疣孢霉(M.rosea)和M.xinjiangensis均具有拮抗作用，其发酵液可以显著抑制引起双孢蘑菇疣孢霉病的3种病原菌有害疣孢霉(Mycogone perniciosa)、红丝疣孢霉(M.rosea)和M.xinjiangensis的菌丝生长，另外发酵液对双孢蘑菇疣孢霉病具有优良的的防治效果，且对蘑菇安全。
此外，湿地链霉菌(Streptomyces paludis)菌株13-3是从土壤中获得的，与自然环境和谐相容，具有良好的应用前景。
附图说明
图1为湿地链霉菌(Streptomyces paludis)菌株13-3在高氏一号培养基上的培养特征。
图2为湿地链霉菌(Streptomyces paludis)菌株13-3与双孢蘑菇疣孢霉病的 3种病原菌的对峙培养。注：A为有害疣孢霉(Mycogone perniciosa)，B为红丝疣孢霉(M.rosea)，C为M.xinjiangensis，CK为对照组。
图3为湿地链霉菌(Streptomyces paludis)菌株13-3发酵液在离体条件下对双孢蘑菇疣孢霉病的3种病原菌的抑制效果。注：A为有害疣孢霉(Mycogone perniciosa)，B为红丝疣孢霉(M.rosea)，C为M.xinjiangensis，CK为对照组。
具体实施方式
下面结合说明书附图和实施例对本发明内容进行详细说明：
实施例一：湿地链霉菌(Streptomyces paludis)菌株13-3的分离及鉴定。
1、土壤样品的采集
从江西井冈山采集土样3份，去除表面的土壤，采集5-20cm深处的土样，标记后带回实验室自然风干。
2、放线菌的分离
采用平板稀释法进行分离。将风干土样用研钵磨碎，称取样品1g悬浮于9 mL无菌水中，于40℃、180rpm震荡30min后静置5min，依次稀释10倍，分别配制成10-2、10-3、10-4的悬浮液，分别吸取不同浓度的悬浮液各0.1mL加入到改良的HVA培养基(加入终浓度为100-200ppm的重铬酸钾)平板上，均匀涂布后倒置于28℃培养观察，5-7天后挑取不同的单菌落划线纯化，纯化后的菌株采用甘油法保藏于-80℃冰箱。
3、湿地链霉菌(Streptomyces paludis)菌株13-3的鉴定
(1)形态特征观察
湿地链霉菌(Streptomyces paludis)菌株13-3在多数培养基上生长良好，都不产可溶性色素(表1)。光学显微镜下湿地链霉菌(Streptomyces paludis)菌株 13-3孢子丝直、柔曲、钩状、松敞及紧密螺旋形，孢子椭圆形、柱形。
表1湿地链霉菌(Streptomyces paludis)菌株13-3的培养特征
(2)生理生化特征
参照《链霉菌鉴定手册》中所述的方法测定湿地链霉菌(Streptomyces paludis)菌株13-3的淀粉水解、硝酸盐还原及碳、氮源的利用等特性，结果见表2。
表2湿地链霉菌(Streptomyces paludis)菌株13-3的生理生化特征
(3)序列分析
采用细菌基因组提取试剂盒提取菌株13-3基因组DNA后，分别进行16S rRNA和rpoB基因扩增，得到的序列全长分别为1410bp和767bp，将所得序列提交到GenBank数据库进行BLAST比对，经形态特征、生理生化特征及16S rRNA和rpoB基因分析得出菌株13-3为湿地链霉菌(Streptomyces paludis)。
湿地链霉菌(Streptomyces paludis)菌株13-3的16s rRNA序列及rpoB基因序列分别如SEQ ID NO:1和SEQ ID NO:2所示。
实施例二：湿地链霉菌(Streptomyces paludis)菌株13-3对3种疣孢霉菌的拮抗作用测定。
采用平板对峙培养法，将湿地链霉菌(Streptomyces paludis)菌株13-3对有害疣孢霉、红丝疣孢霉及M.xinjiangensis进行拮抗测定。首先在PDA培养基靠边缘两侧划线接种湿地链霉菌(Streptomyces paludis)菌株13-3，3d后在平板中央接入直径5mm供试病原菌菌饼，25℃下培养5d测量湿地链霉菌 (Streptomyces paludis)菌株13-3对供试病原菌的抑菌带宽度和病原菌直径(表 3)，以不接拮抗菌的供试病原菌为对照。结果表明，湿地链霉菌(Streptomyces paludis)菌株13-3对有害疣孢霉、红丝疣孢霉和M.xinjiangensis均具有非常强的拮抗作用(图2)。
表3湿地链霉菌菌株13-3对3种疣孢霉菌的拮抗作用
实施例三：湿地链霉菌(Streptomyces paludis)菌株13-3发酵液对3种疣孢霉菌的抑制作用。
将湿地链霉菌(Streptomyces paludis)菌株13-3在高氏一号培养基活化，28℃恒温培养5d，将湿地链霉菌(Streptomyces paludis)菌株13-3的孢子接入ISP2 液体培养基中，26-28℃发酵7-8d，过滤得发酵液，然后0.22μm微孔滤膜过滤除菌，备用。
取湿地链霉菌(Streptomyces paludis)菌株13-3的发酵液5mL加入到50mL 冷却至约50℃(不烫手)PDA培养基中，混匀后倒入培养皿中，将直径5mm 供试病原菌菌饼接入PDA培养基平板中央，置于25℃下培养5d后测量菌落直径。以空白的PDA为对照，计算发酵液对3种疣孢霉菌的菌丝生长的抑制率(表 4)。结果表明，湿地链霉菌(Streptomycespaludis)菌株13-3的发酵液对3种疣孢霉菌的菌丝生长具有较强的抑制效果(图3)。
表4湿地链霉菌菌株13-3发酵液对3种疣孢霉菌的菌丝生长的抑制作用
实施例四：湿地链霉菌(Streptomyces paludis)菌株13-3发酵液在防治双孢蘑菇疣孢霉病中的应用。
供试的有害疣孢霉、红丝疣孢霉和M.xinjiangensis 25℃培养5d，用直径5 mm的打孔器在菌落边缘打取菌饼，将菌饼转入PDB液体培养基中，于150 r/min、25℃黑暗振荡培养7d，用血球计数板检测厚垣孢子浓度，制成浓度为 1×105个孢子/mL的孢子混合悬浮液，备用。
试验地点选择在福建省福州市埔党村，双孢蘑菇品种为W192。试验设置3 个处理，分别为：A.50％咪鲜胺锰盐WP1.0克/平方米、B.湿地链霉菌 (Streptomyces paludis)菌株13-3的发酵液、C.清水对照。每处理4次重复，共计12个小区，随机区组排列，每小区4m2。将供试浓度药液定量喷施于待覆土的定量土粒上(每平方米菇床覆盖用土约25kg，喷施药液1.5L)，拌匀，保持土壤含水量约70％，用塑料膜覆盖闷土24h后进行覆土，然后每小区接种上述的孢子混合悬浮液100mL。于覆土7d后分别进行第二次施药，将相同剂量药液均匀喷施于床面土粒上，以土粒湿润且不流进菌料内为度。从采收第一潮菇起开始调查，共调查3潮菇，采取全小区调查方式，于每次采收时调查记录培养料上病菇菇粒数或菌团数(菇粒或菌团≥1cm)，累计各小区的发病菇粒数，计算防治效果。
防治效果(％)＝[(对照区病菇数—处理区病菇数)/对照区病菇数]×100
表5湿地链霉菌菌株13-3的发酵液防治双孢蘑菇疣孢霉病田间药效试验
实验结果表明，A.50％咪鲜胺锰盐WP1.0克/平方米、B.湿地链霉菌(Streptomyces paludis)菌株13-3的发酵液对双孢蘑菇疣孢霉病的防治效果分别为77.06％和75.88％，均对双孢蘑菇安全，未见药害。湿地链霉菌(Streptomyces paludis)菌株13-3的发酵液对双孢蘑菇疣孢霉病的防治效果在5％显著水平上及 1％极显著水平上与50％咪鲜胺锰盐WP1.0克/平方米防效差异均不显著。由此可见，湿地链霉菌(Streptomycespaludis)菌株13-3的发酵液对双孢蘑菇疣孢霉病的防治效果与50％咪鲜胺锰盐WP1.0克/平方米防效相当，具备良好的应用前景。
Claims (4)
1.湿地链霉菌(Streptomyces paludis)菌株13-3在防治双孢蘑菇疣孢霉病中的应用，其特征在于：
所述湿地链霉菌(Streptomyces paludis)菌株13-3保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.22830，保藏日期：2021年7月6日。
2.根据权利要求1所述的湿地链霉菌(Streptomyces paludis)菌株13-3在防治双孢蘑菇疣孢霉病中的应用，其特征在于：
湿地链霉菌(Streptomyces paludis)菌株13-3对引起双孢蘑菇疣孢霉病的有害疣孢霉(Mycogone perniciosa)、红丝疣孢霉(M.rosea)和M.xinjiangensis均有非常强的拮抗作用。
3.湿地链霉菌(Streptomyces paludis)菌株13-3发酵液在防治双孢蘑菇疣孢霉病中的应用，其特征在于：所述发酵液通过将权利要求1中所述的湿地链霉菌(Streptomycespaludis)菌株13-3接种于ISP2液体培养基，发酵温度为26-28℃，发酵培养时间为7-8d，过滤后得发酵液。
4.根据权利要求3所述的湿地链霉菌(Streptomyces paludis)菌株13-3发酵液在防治双孢蘑菇疣孢霉病中的应用，其特征在于：
所述湿地链霉菌(Streptomyces paludis)菌株13-3发酵液在抑制有害疣孢霉(Mycogone perniciosa)、红丝疣孢霉(M.rosea)和M.xinjiangensis的菌丝生长中的应用。
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| CN202210799659.2A Active CN115820454B (zh) | 2022-06-15 | 2022-07-06 | 一株湿地链霉菌（Streptomyces paludis）菌株13-3及其应用 |
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