CN115820454B - 一株湿地链霉菌（Streptomyces paludis）菌株13-3及其应用 - Google Patents
一株湿地链霉菌（Streptomyces paludis）菌株13-3及其应用 Download PDFInfo
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- CN115820454B CN115820454B CN202210799659.2A CN202210799659A CN115820454B CN 115820454 B CN115820454 B CN 115820454B CN 202210799659 A CN202210799659 A CN 202210799659A CN 115820454 B CN115820454 B CN 115820454B
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Abstract
本发明涉及一株湿地链霉菌菌株13‑3，所述湿地链霉菌(Streptomycespaludis)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。本发明的首要目的在于针对大豆炭疽病发生严重，阻碍大豆产业的发展，杀菌剂的大量使用造成环境污染等问题，提供一株湿地链霉菌菌株13‑3。本发明的优点在于：湿地链霉菌菌株13‑3对引起大豆炭疽病的3种病原菌均具有拮抗作用，其发酵液可以显著抑制大豆炭疽病3种病原菌的菌丝生长和孢子萌发，且所制得的发酵液对大豆炭疽病具有优良的的防治效果。
Description
技术领域
本发明涉及农作物病害生物防治技术领域，具体为一株湿地链霉菌(Streptomyces paludis)菌株13-3及其应用。
背景技术
大豆是一种重要粮食、油料作物，是人类植物蛋白和水产畜牧饲料的重要来源，是全球食物链的重要组成部分。我国年产大豆1千多万吨，而需求量达到1亿多吨，缺口较大，长期依赖大量进口。由炭疽菌属(Colletotrichum)真菌侵染引起的大豆炭疽病(soybeananthracnose)在世界范围内发生，轻者减产，重者绝收，是大豆生产中重要的真菌病害。大豆炭疽病从苗期至收获期均可发病，苗期为害种子和子叶引起大豆死亡，成株期为害茎秆、豆荚，严重影响大豆的产量和品质。
目前防治大豆炭疽病主要依靠抗病品种和使用杀菌剂，而鲜食大豆中缺乏对炭疽病高抗的品种，杀菌剂仍然是防控大豆炭疽病的主要手段。炭疽菌属不同种对杀菌剂的敏感性上存在差异，这给杀菌剂的使用带来了一定的困难。随着杀菌剂的大量连续使用，许多问题也随之出现，其中最为主要的是抗药性问题。抗药性问题出现后，农民为提高防效通常加大杀菌剂使用剂量，这对环境生态构成了威胁。
放线菌是一类代谢产物含有大量生物活性物质的微生物资源，其产生的生物活性物质具有活性好且不易产生抗性等优点，已广泛应用于农业生产或作为新型杀菌剂的前体化合物。此外，放线菌的次生代谢产物在甾体转化、石油脱蜡、污水处理及生物固氮等方面也应用广泛。由吸水链霉菌井冈变种产生的井冈霉素是我国开发的最成功的生物农药，它对水稻纹枯病的防效好、持效期长，能够有效控制病害的发生，现已广泛用于水稻纹枯病的防治。Zambrano等从水稻周围土壤中分离到放线菌Streptomyces spp.M2A2，能抑制水稻纹枯病菌菌丝的生长外，进而可以延迟病症的出现，并且可影响易感品种Fedearroz 68中病原菌的扩展，与苯醚甲环唑的防治效果相当。
生物防治具有对环境无污染，对作物无毒副作用及病原菌不易产生抗性等多种优点，利用生物措施防治大豆炭疽病符合农业可持续发展的策略。
发明内容
本发明的首要目的在于针对大豆炭疽病发生严重，阻碍大豆产业的发展，杀菌剂的大量使用造成环境污染等问题，提供一株湿地链霉菌(Streptomyces paludis)菌株13-3，对引起大豆炭疽病的3种病原菌均有拮抗作用。
本发明的再一目的在于提供湿地链霉菌(Streptomyces paludis)菌株13-3的发酵液在防治大豆炭疽病中的应用。
本发明的目的通过如下技术方案实现：
一株湿地链霉菌(Streptomyces paludis)菌株13-3，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，保藏地址：北京市朝阳区北辰西路1号院，保藏编号为CGMCC No.22830，保藏日期：2021年7月6日。
湿地链霉菌(Streptomyces paludis)菌株13-3对引起大豆炭疽病的胶孢炭疽菌(Colletotrichum gloeosporioides)、平头炭疽菌(C.truncatum)和C.plurivorum均具有拮抗作用。
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。
湿地链霉菌(Streptomyces paludis)菌株13-3在高氏一号培养基上基内菌丝北瓜黄色，气生菌丝白色，无可溶性色素产生。孢子丝直、柔曲、钩状、松敞与紧密螺旋形，孢子椭圆形、柱形。
湿地链霉菌(Streptomyces paludis)菌株13-3的生理生化特征为：不能产生黑色素；不能还原硝酸盐；可水解淀粉；能利用葡糖糖、甘露糖、甘露醇、乳糖、半乳糖、山梨醇、麦芽糖、蔗糖、蜜二糖、鼠李糖、棉籽糖、海藻糖、果糖、木糖、核糖、淀粉、阿拉伯糖、纤维二糖、苦杏仁苷、赤藓醇、肌醇、甘油、柠檬酸钠、苹果酸钠、琥珀酸钠；不能利用山梨糖、松三糖、菊糖、水杨苷、卫矛醇、酒石酸钠和葡萄糖酸钠；能利用赖氨酸；不能利用苏氨酸、丙氨酸和脯氨酸。
湿地链霉菌(Streptomyces paludis)菌株13-3在防治大豆炭疽病中的应用。
湿地链霉菌(Streptomyces paludis)菌株13-3的发酵液的制备方法，湿地链霉菌(Streptomyces paludis)菌株13-3接种于ISP2固体培养基，发酵温度为26-28℃，发酵培养时间7-8d，并采用乙酸乙酯萃取，提取液减压浓缩蒸干后再用DMSO溶解，得发酵液。
湿地链霉菌(Streptomyces paludis)菌株13-3的发酵液的应用，湿地链霉菌(Streptomyces paludis)菌株13-3的发酵液在防治大豆炭疽病中的应用。
湿地链霉菌(Streptomyces paludis)菌株13-3的发酵液用于抑制大豆炭疽病病原菌的菌丝生长和孢子萌发；
所述大豆炭疽病病原菌为胶孢炭疽菌(Colletotrichum gloeosporioides)、平头炭疽菌(C.truncatum)和C.plurivorum。
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对引起大豆炭疽病的3种病原菌均具有拮抗作用，其发酵液可以显著抑制大豆炭疽病3种病原菌的菌丝生长和孢子萌发，且所制得的发酵液对大豆炭疽病具有优良的的防治效果。
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对峙培养条件下对胶孢炭疽菌(Colletotrichum gloeosporioides)、平头炭疽菌(C.truncatum)和C.plurivorum的抑制效果。注：A为胶孢炭疽菌，B为平头炭疽菌，C为C.plurivorum，CK为对照组。
图3为湿地链霉菌(Streptomyces paludis)菌株13-3发酵液对胶孢炭疽菌(C.gloeosporioides)、平头炭疽菌(C.truncatum)和C.plurivorum的菌丝生长的抑制效果。注：A为胶孢炭疽菌，B为平头炭疽菌，C为C.plurivorum，CK为对照组。
图4为湿地链霉菌(Streptomyces paludis)菌株13-3发酵液对胶孢炭疽菌(C.gloeosporioides)、平头炭疽菌(C.truncatum)和C.plurivorum的分生孢子萌发的抑制效果。注：A为湿地链霉菌(Streptomyces paludis)菌株13-3发酵液处理后的胶孢炭疽菌分生孢子的萌发，B为湿地链霉菌(Streptomyces paludis)菌株13-3发酵液处理后的平头炭疽菌分生孢子的萌发，C为湿地链霉菌(Streptomyces paludis)菌株13-3发酵液处理后的C.plurivorum的分生孢子的萌发，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9mL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，参见表1。光学显微镜下菌株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)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)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)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湿地链霉菌(Streptomyces paludis)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，将所得序列提交至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)。
实施例二：湿地链霉菌(Streptomyces paludis)菌株13-3对3种大豆炭疽菌的拮抗作用测定。
采用平板对峙培养法，将湿地链霉菌(Streptomyces paludis)菌株13-3对胶孢炭疽菌、平头炭疽菌、C.plurivorum进行拮抗作用测定。首先在PDA培养基靠边缘两侧划线接种菌株13-3，3d后在平板中央接入直径5mm供试病原菌菌饼，28℃下培养5d测量菌株13-3对供试病原菌的抑菌带宽度(表3)，以不接拮抗菌的供试病原菌为对照，结果表明菌株13-3对胶孢炭疽菌、平头炭疽菌和C.plurivorum均具有非常强的拮抗作用(图2)。
表3菌株13-3对3种大豆炭疽菌的拮抗作用
实施例三：湿地链霉菌(Streptomyces paludis)菌株13-3发酵液对3种大豆炭疽菌的抑制作用。
菌株13-3在高氏一号培养基上活化，28℃恒温培养5d，将菌株13-3的孢子接入ISP2固体培养基中，26-28℃发酵7-8d，乙酸乙酯萃取3次，提取液减压浓缩蒸干后用DMSO溶解，制成20mg/mL的发酵液，备用。
取菌株13-3的发酵液100μL加入到100mL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28℃下培养7d后测量菌落直径。以加入相同量的DMSO为对照，计算发酵液对3种炭疽菌菌丝生长的抑制率(表4)。结果表明菌株13-3的发酵液对胶孢炭疽菌、平头炭疽菌和C.plurivorum的菌丝生长均具有非常强的抑制作用(图3)。
表4菌株13-3发酵液对3种炭疽菌的抑制作用
实施例四：湿地链霉菌(Streptomyces paludis)菌株13-3发酵液对3种大豆炭疽菌分生孢子萌发的抑制作用。
将直径5mm供试病原菌菌饼接入PDB液体培养基中，于28℃、180rpm震荡培养7d后，过滤收集孢子悬浮液，并将其浓度调至1×105个/mL。取实施例三中的13-3菌株的发酵液1μL加入到1mL的孢子悬浮液中，以加入相同量的DMSO为对照，将胶孢炭疽菌、平头炭疽菌和C.plurivorum的孢子悬浮液分别置于28℃下培养10h、4h和8h，计算13-3菌株的发酵液对3种炭疽菌的孢子萌发的抑制率(表5)。结果表明，菌株13-3的发酵液显著抑制胶孢炭疽菌、平头炭疽菌和C.plurivorum的分生孢子萌发(图4)。
表5菌株13-3发酵液对3种炭疽菌分生孢子萌发的抑制作用
实施例五：湿地链霉菌(Streptomyces paludis)菌株13-3发酵液在防治大豆炭疽病中的应用。
供试的平头炭疽菌、胶孢炭疽菌和C.plurivorum 28℃培养5d，用直径5mm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150r/min、28℃黑暗振荡培养7d，用血球计数板检测分生孢子浓度，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晋安区埔党村，试验设置3个处理，分别为：A.10％苯醚甲环唑WG1500倍液、B.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20m2，按每亩用水量45升喷雾。于大豆(毛豆3号)初荚后分别喷雾施药，喷雾施药24h后接种上述制备的孢子混合悬浮液(加入0.1％的吐温80)于豆荚上，每株植株喷50mL。覆膜保湿2d后打开薄膜两端通风，接种3–5d后移去薄膜。接菌10–15d后调查发病情况。
调查方法：每小区按照对角线五点取样，每点调查相连的3株大豆，每小区调查15株大豆的所有豆荚，记录总荚数、发病荚数，按照以下分级标准统计病情指数：
0级：豆荚无病斑；
1级：豆荚上有褐点型小病斑，病斑面积占整个豆荚面积的5％以下；
3级：豆荚上出现典型病斑，病斑面积占整个豆荚面积的6％～10％；
5级：豆荚上出现典型病斑，病斑面积占整个豆荚面积的11％～25％；
7级：豆荚上出现典型病斑，病斑面积占整个豆荚面积的26％～50％；
9级：豆荚上出现典型病斑，病斑面积占整个豆荚面积的50％以上；
病情指数＝∑(各级病荚数×相对病级数值)÷(调查总荚数×9)×100；
防治效果(％)＝[(CK病情指数-处理病情指数)÷CK病情指数]×100；
表6菌株13-3的发酵液防治大豆炭疽病田间药效试验
实验结果表明，A.10％苯醚甲环唑WG1500倍液、B.菌株13-3的发酵液对大豆炭疽病的防治效果分别为76.48％和75.73％，均对大豆安全，未见药害。菌株13-3的发酵液对大豆炭疽病的防治效果在5％显著水平及1％极显著水平上与10％苯醚甲环唑WG1500倍液防效差异均不显著。由此可见，菌株13-3的发酵液对大豆炭疽病的防效与10％苯醚甲环唑WG1500倍液防效相当，具备良好的应用前景。
Claims (6)
1.一株湿地链霉菌（Streptomyces paludis）菌株13-3，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.如权利要求1所述的湿地链霉菌（Streptomyces paludis）菌株13-3在防治大豆炭疽病中的应用。
3.如权利要求1所述的湿地链霉菌（Streptomyces paludis）菌株13-3的发酵液的制备方法，其特征在于：湿地链霉菌（Streptomyces paludis）菌株13-3接种于ISP2固体培养基，发酵温度为26-28°C，发酵培养时间7-8 d，并采用乙酸乙酯萃取，提取液减压浓缩蒸干后再用DMSO溶解，得发酵液。
4.一种如权利要求3制备方法所得的湿地链霉菌（Streptomyces paludis）菌株13-3的发酵液。
5.如权利要求4所述的湿地链霉菌（Streptomyces paludis）菌株13-3的发酵液的应用，其特征在于：湿地链霉菌（Streptomyces paludis）菌株13-3的发酵液在防治大豆炭疽病中的应用。
6.根据权利要求5所述的湿地链霉菌（Streptomyces paludis）菌株13-3的发酵液的应用，其特征在于：湿地链霉菌（Streptomyces paludis）菌株13-3的发酵液用于抑制大豆炭疽病病原菌的菌丝生长和分生孢子萌发；
所述大豆炭疽病病原菌为胶孢炭疽菌（Colletotrichum gloeosporioides）、平头炭疽菌（C. truncatum）和C. plurivorum。
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