CN114933977A - 一株用于烟草土传病害的生防篮状菌及其应用 - Google Patents
一株用于烟草土传病害的生防篮状菌及其应用 Download PDFInfo
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- CN114933977A CN114933977A CN202210762564.3A CN202210762564A CN114933977A CN 114933977 A CN114933977 A CN 114933977A CN 202210762564 A CN202210762564 A CN 202210762564A CN 114933977 A CN114933977 A CN 114933977A
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Abstract
本发明涉及生物防治技术领域，具体涉及一株用于烟草土传病害的生防篮状菌及其应用，本发明公开一种对烟草土传病原真菌具有明显拮抗效果的篮状菌属（Talaromyces C.R.Benj.）YC0121，保藏号CCTCC NO：M 20211577，并对其防病促生特性进行了初步分析。该菌株生长缓慢，但产孢量大，繁殖快，适应能力强，易受风、雨、气流、人为因素等传播与定殖，具有较好的促生能力和水解酶活性，对烟草种子根长和烟苗根系活力具有显著的促进作用，以烟草尖孢镰刀菌为例，该菌具有较好的无菌发酵滤液抑菌活性和盆栽防治效果，生防潜力较大，具有较好的研究与应用价值，为烟草根茎类病害的生物防治提供优质资源和理论基础。
Description
技术领域
本发明涉及生物防治技术领域，具体涉及一株用于烟草土传病害的生防篮状菌及其应用。
背景技术
烟草是我国重要的经济作物之一。近年来，随着烟田作物布局和栽培耕作制度的改变，烟草黑胫病、烟草根黑腐病、烟草镰刀菌根腐病等土传病害的发生日益严重，不但影响了烟叶的外观质量和内在品质，而且影响烟叶的商品性能和经济效益，已成为制约烟草生产的重要因素。烟草土传病害绿色防控要遵从生态学、经济学和综合治理技术原理，全面贯彻落实我国“预防为主，综合防治”的植保方针，将抗病品种利用、保健栽培、生物防治与化学防治有机结合，提高烟叶生产的经济效益与生态效益。
但是，化学防治仍是目前烟草根茎类病害最主要、最有效的防治措施，常用药剂有75％甲基硫菌灵可湿性粉剂、50％福美双可湿性粉剂、25％甲霜灵可湿性粉剂、25％甲霜灵可湿性粉剂与65％代森锌可湿性粉剂的复配剂等，通过喷施、灌根或蘸根处理可达到较好的防治效果。但长期、大量地使用化学农药所带来的药物残留增加、病虫害抗药性增强，农田微生态平衡破坏、环境污染以及对生物健康的威胁等问题也是不容小觑的。
因此，生产一种具有较好的无菌发酵滤液抑菌活性和盆栽防治效果，生防潜力较大，具有较好的研究与应用价值，为烟草根茎类病害的生物防治提供优质资源和理论基础的用于烟草土传病害的生防篮状菌及其应用，具有广泛的市场前景。
发明内容
针对现有技术的不足，本发明提供一种具有较好的无菌发酵滤液抑菌活性和盆栽防治效果，生防潜力较大，具有较好的研究与应用价值，为烟草根茎类病害的生物防治提供优质资源和理论基础的用于烟草土传病害的生防篮状菌及其应用，用于克服现有技术中缺陷。
本发明采用的技术方案为：一株用于烟草土传病害的生防篮状菌，所述的篮状菌属真菌菌株的保藏名称为篮状菌属YC0121，保藏编号：CCTCC NO：M 20211577。
包括以下步骤：
步骤一：提供制备生防篮状菌的原材料材料；
步骤二：篮状菌筛选；
步骤三：篮状菌的鉴定；
步骤四：篮状菌对烟草土传病害的抑菌测定。
步骤三中篮状菌的鉴定的方法为：(1)、篮状菌属YC0121的形态学鉴定，(2)、篮状菌属YC0121的分子鉴定。
步骤四中篮状菌对烟草土传病害的抑菌测定方法为：(1)、篮状菌属YC0121的促生能力及水解酶活性测定，(2)篮状菌属YC0121对烟草种子促生及根系活力的测定，(3)篮状菌属YC0121对盆栽烟草促生作用的测定，(4)篮状菌属YC0121无菌发酵滤液的抑菌活性测定，(5)篮状菌属YC0121对盆栽烟草的防效测定。
一种如上所述的用于烟草土传病害的生防篮状菌的应用，采用平板对峙培养法筛选对不同病原菌的抑菌活性。在距离培养基平板中心2.5cm的相对2点上分别接种已纯化的拮抗菌和病原菌5mm菌饼，以不接拮抗菌只接病原菌为对照，重复3次，倒置放入25℃恒温光照箱培养至对照组病菌菌落生长至平板面积2/3时，采用十字交叉法测量菌落半径，计算菌丝生长抑制率，并调查统计真菌拮抗系数。
所述的真菌拮抗系数分级标准：Ⅰ级：待测真菌菌丝平板覆盖率100％；Ⅱ级：待测真菌菌丝平板覆盖率2/3以上；Ⅲ级：待测真菌菌丝平板覆盖率1/3～2/3；Ⅳ级：待测真菌菌丝平板覆盖率1/3以下；Ⅴ级：病原菌丝平板覆盖率100％。
本发明有益效果是：本发明公开一种对烟草土传病原真菌具有明显拮抗效果的篮状菌属(Talaromyces C.R.Benj.)YC0121，保藏号CCTCC NO：M 20211577，并对其防病促生特性进行了初步分析。该菌株生长缓慢，但产孢量大，繁殖快，适应能力强，易受风、雨、气流、人为因素等传播与定殖，具有较好的促生能力和水解酶活性，对烟草种子根长和烟苗根系活力具有显著的促进作用，以烟草尖孢镰刀菌为例，该菌具有较好的无菌发酵滤液抑菌活性和盆栽防治效果，生防潜力较大，具有较好的研究与应用价值，为烟草根茎类病害的生物防治提供优质资源和理论基础。
附图说明
图1为菌株YC0121对不同病原真菌的平板抑制效果图。
图2为菌株YC0121的形态学性状效果图。
图3为基于18S rDNA基因序列构建的系统发育树效果图。
具体实施方式
以下是本发明的具体实施例：一株用于烟草土传病害的生防篮状菌，所述的篮状菌属真菌菌株的保藏名称为篮状菌属YC0121，保藏编号：CCTCC NO：M 20211577。
包括以下步骤：
步骤一：提供制备生防篮状菌的原材料材料，其中包括(1)、供试病原菌：烟草尖孢镰刀菌(Fusarim oxysporum)、烟草茄病镰刀菌(F.solani)、烟草疫霉菌(Phytophthoranicotianae)、根串珠霉菌(Thielaviopsis basicola)、链格孢菌(Alternariaalternata)、拟茎点霉(Phomopsis sp.)、烟草炭疽菌(Colletotrichum nicotianae)、立枯丝核菌(Rhizoctonia solani)、葡萄座腔菌(Botryosphaeria dothide)和灰葡萄孢菌(Botrytis cinerea)均由河南省农业科学院烟草研究所菌源库提供；(2)、供试土样：根际土壤样品采集于许昌市油菜花田，经纬度(34°04′26″N，113°50′41″E)，海拔61.31m；(3)、供试培养基：马铃薯葡萄糖琼脂培养基(PDA)：马铃薯200.0g/L，葡萄糖20.0g/L，琼脂15g/L，自然pH，121℃，20min，用于真菌菌株的培养和平板抑菌试验，PD培养基同PDA培养基(不含琼脂)，用于真菌的液体培养及发酵液的制备，燕麦培养基(OA)：燕麦粉60.0g/L，琼脂12.5g/L，PH 7.0～7.4，121℃，15min，用于烟草疫霉菌的培养和平板抑菌试验，有机磷培养基：葡萄糖10.0g/L，(NH4)2SO4 0.5g/L，NaCl 0.3g/L，MgSO4 0.3g/L，MnSO4 0.03g/L，K2SO4 0.3g/L，FeSO4 0.03g/L，Ca3(PO4)2 5.0g/L，卵磷脂0.2g/L，琼脂15.0g/L，pH 7.0～7.5，用于解有机磷活性测定，无机磷培养基同有机磷培养基(不含卵磷脂)，用于解无机磷活性测定，固氮培养基：KH2PO4 0.2g/L，MgSO4 0.2g/L，NaCl 0.2g/L，CaCO3 5.0g/L，CaSO40.1g/L，甘露醇10.0g/L，琼脂15.0g/L，pH 6.9～7.1，用于固氮活性测定，嗜铁素培养基：铬天青S(CAS)60.5mg/L，十六烷基三甲基溴化铵(HDTMA)72.9mg/L，FeCl3·6H2O 2.645mg/L，NaH2PO4·2H2O 295.25mg/L，Na2HPO4·12H2O 1213.5mg/L，NH4Cl 125.0mg/L，KH2PO437.5 mg/L，NaCl 62.5mg/L，琼脂9.0g/L，pH 6.7～6.9，用于产嗜铁素活性测定，解钾硅酸盐培养基：蔗糖5.0g/L，MgSO4 0.5g/L，CaCO3 0.1g/L，Na2HPO4 2.0g/L，FeCl3 0.005g/L，玻璃粉1.0g/L，琼脂15.0g/L，pH 6.9～7.1，用于解钾硅酸盐活性测定，蛋白酶培养基：干酪素10.0g/L，琼脂20.0g/L，pH 7.2，用于蛋白酶活性测定，纤维素水解培养基：蛋白胨10.0g/L，KH2PO4 1.0g/L，酵母浸粉10.0g/L，NaCl 5.0g/L，羧甲基纤维素钠10.0g/L，琼脂15.0g/L，pH 7.2，用于纤维素酶活性测定，几丁质酶培养基：胶体几丁质10mL/L，KH2PO4 0.5g/L，MgSO4 0.5g/L，NaCl 0.5g/L，FeSO4·7H2O 0.01g/L，ZnSO4·7H2O 0.01g/L，琼脂15.0g/L，pH 6.5～7.0，用于几丁质酶活性测定，淀粉水解培养基：蛋白胨1.0g/L，可溶性淀粉20.0g/L，牛肉膏1.0g/L，NaCl 5.0g/L，K2HPO4 1.0g/L，MgSO4 0.1g/L，CaCl2 0.05g/L，MnSO40.001g/L，琼脂20.0g/L，pH 7.0～7.2，用于解淀粉酶活性测定；
步骤二：篮状菌筛选，包括拮抗真菌的分离与筛选，将取回的根际土壤样品自然风干并挑除杂物，10目筛子过筛后用四分法收集备用。采用常规的梯度稀释涂布平板法，在PDA平板上培养真菌菌落，25℃倒置培养3～7d，纯化培养后于4℃保存备用；采用平板对峙培养法筛选对不同病原菌的抑菌活性。在距离培养基平板中心2.5cm的相对2点上分别接种已纯化的拮抗菌和病原菌5mm菌饼，以不接拮抗菌只接病原菌为对照，重复3次，倒置放入25℃恒温光照箱培养至对照组病菌菌落生长至平板面积2/3时，采用十字交叉法测量菌落半径，计算菌丝生长抑制率，并调查统计拮抗系数，其公式为：
真菌拮抗系数分级标准：Ⅰ级：待测真菌菌丝平板覆盖率100％；Ⅱ级：待测真菌菌丝平板覆盖率2/3以上；Ⅲ级：待测真菌菌丝平板覆盖率1/3～2/3；Ⅳ级：待测真菌菌丝平板覆盖率1/3以下；Ⅴ级：病原菌丝平板覆盖率100％，(2)、
步骤三：篮状菌的鉴定；
步骤四：篮状菌对烟草土传病害的抑菌测定。
步骤三中篮状菌的鉴定的方法为：(1)、篮状菌属YC0121的形态学鉴定，篮状菌属菌株的形态学观察需将菌株接种于PDA、MEA、CYA、CYAS、YES、DG-18、OA、CREA培养基上，25℃黑暗培养7d后观察并记录菌落的颜色、质地、产孢程度和色素分泌等特性。使用在MEA培养基上生长7d后的菌落进行观察玻片的制备，观察并记录分生孢子梗、产孢结构、分生孢子等的特征，(2)、篮状菌属YC0121的分子鉴定，使用Ezup柱式真菌基因组DNA抽提试剂盒，提取待测真菌基因组DNA为模板，应用真菌鉴定通用引物ITS1和ITS4进行PCR扩增真菌18S rDNA片段，PCR扩增体系和反应条件见表1。
表1真菌通用引物及PCR反应信息
Tab.1 General primers and PCR reaction information for fungi
步骤四中篮状菌对烟草土传病害的抑菌测定方法为：
(1)、篮状菌属YC0121的促生能力及水解酶活性测定，通过对不同培养基的利用情况定性检测菌株的促生能力和部分水解酶活性，包括产IAA能力、解磷能力、固氮能力、解钾能力、产NH3能力、产嗜铁素活性、产蛋白酶活性、产纤维素酶活性、产几丁质酶活性和产淀粉酶活性；
(2)篮状菌属YC0121对烟草种子促生及根系活力的测定，种子经孢子悬浮液(1×107孢子/mL)浸种16h后，整齐摆放在铺有灭菌沙子和滤纸保湿的培养皿中，以无菌水浸种为空白对照，每皿25粒(5×5)，重复3次，第14d统计种子萌发率和烟苗根长，并计算增长率，其公式为：
灭菌基质与拮抗菌发酵液(5×107孢子/mL)按质量体积比10∶1均匀混合，采用漂浮育苗法播种育苗(7×10孔)，以等体积PD培养基混拌基质为空白对照，重复3次，期间温度与水肥管理一致，第43d采用氯化三苯基四氮唑(TTC)法测定根系活力，根系活力测定具体步骤：
1)、TTC标准曲线的制作。取0.4％TTC溶液0.2mL放人10mL容量瓶中，加少许Na2S2O4摇匀后立即产生红色的甲攒，再用乙酸乙酯定容至刻度并摇匀。然后分别取此液0.25mL、0.50mL、1.00mL、1.50mL、2.00mL和2.50mL置10mL容量瓶中，用乙酸乙酯定容至刻度，即得到含甲攒25μg、50ug、100ug、150ug、200ug和250μg的标准比色系列，以空白作参比，在485nm波长下测定吸光度，绘制标准曲线。
2)、取43d的烟苗样品，小心清洗掉根系的基质。称取根尖样品0.5g，放人10mL烧杯中，加人0.4％TTC溶液和磷酸缓冲液的等量混合液10mL，把根尖样品充分浸没在溶液内，在37℃暗保温3h，此后加人1mol/L硫酸2mL，以停止反应。以先加硫酸再加根样品，之后37℃暗保温再加硫酸为空白对照。
3)、把根取出，吸干水分后与乙酸乙酯3～4mL和少量石英砂一起在研钵内磨碎，以提出甲攒，红色提取液移人试管。并用少量乙酸乙酯把残渣洗涤3次，皆移人试管，用乙酸乙酯定容至10mL，用分光光度计在波长485nm下比色，以空白试验作参比测出吸光度，通过标准曲线求出四氮唑还原量，进而计算四氮唑还原强度，其公式为：
(3)篮状菌属YC0121对盆栽烟草促生作用的测定，采用温室盆栽法，温度(26±0.5)℃、相对湿度50％～60％、光暗周期12L/12D，选择40d苗龄长势一致的烟苗进行移栽，缓苗后每株灌根20mL拮抗菌发酵液(5×107孢子/mL)，每处理10株，重复3次，共灌根2次，间隔10d，以等体积PD培养基灌根为空白对照，期间温度与水肥管理一致，30d后根据烟草行业标准：烟草农艺性状调查测量方法(YC/T142—2010)测量烟株农艺性状，并计算最大叶的叶面积，其公式为：
叶面积(cm2)＝0.6345×叶长(cm)×叶宽(cm)；
(4)篮状菌属YC0121无菌发酵滤液的抑菌活性测定，以烟草尖孢镰刀菌为例，测定菌株无菌发酵滤液的抑菌活性。于PD培养基中28℃、180r/min振荡培养7d，收集培养液离心保留上清液，用直径为0.22μm的过滤器过滤除菌得到无菌发酵滤液。采用菌丝生长速率法，将无菌发酵滤液按1％、5％、10％的比例制备成含药平板，中心位置上接种病原菌菌饼，以不加发酵滤液的平板接种病原菌为空白对照，重复3次，25℃恒温倒置培养6～8d，测量病原菌落直径并计算抑菌率；
(5)篮状菌属YC0121对盆栽烟草的防效测定，以烟草尖孢镰刀菌为例，测定菌株的盆栽防效。选择60d苗龄长势一致的烟苗进行移栽，缓苗后每株灌根20mL拮抗菌发酵液(5×107孢子/mL)，3d后每盆接种20g烟草尖孢镰刀菌麦粒培养物(打取新鲜的菌丝块，接种于高温灭菌过的麦粒培养基，28℃培养7d，制成带菌麦粒培养物)，施足水后，(28±0.5)℃保湿培养、光暗周期12L/12D，10d后再次灌根，每处理10株，重复3次，以不接病菌为阴性对照(CK1)，以只接种病菌为阳性对照(CK2)，以阳性对照组盆栽均出现典型的矮小萎蔫、根部坏死症状为标准，在接种病原20d时以株为单位，按照烟草国家标准：烟草病虫害分级及调查方法(GB/T23222—2008)调查病害发生情况，并计算发病率、病情指数和相对防效，其公式为：
拮抗菌株YC0121对不同病原菌的抑制作用，经分离筛选，获得1株拮抗真菌YC0121对烟草常见土传病原菌均有较好的抑菌效果。对10种病原菌的抑菌范围在16.56％～69.96％，对尖孢镰刀菌的抑制率最高，可达69.96％，拮抗系数Ⅱ级(表2)，平板抑菌效果见图1。
表2菌株YC0121对烟草土传病原真菌的抑制作用
Tab.2 Inhibitory effect of strain YC0121 on tobacco soil-bornepathogenic fungi
拮抗菌株YC0121的鉴定，拮抗菌株YC0121的形态特征，如图2所示，菌株YC0121菌落在培养基上菌丝生长缓慢，产孢量较大，分生孢子易分散产大量单菌落，后期聚连成一片。菌落在PDA上25℃7d直径约4.43cm，中等厚度，前期发散出卷曲缠绕的黄色菌丝，后期菌落颜色自中心向外由灰褐色向白色过度，边缘规则，菌落背面红褐色向外扩散变浅。菌落在MEA上25℃7d直径约3.45cm，质地绒状，菌落中心刈安色向外扩散至孔雀绿色，边缘规则、菌丝白色，菌落背面淡橙红色向外扩散变浅，中心赭色，渗出液少量或无；分生孢子梗帚状单轮生或不规则生，瓶梗安瓿形排列紧密，分生孢子暗绿色或黄绿色。菌落在CYA上25℃7d直径约2.76cm，生长缓慢，菌落中心孔雀绿色粉状孢子层，外圈菌丝绳状、灰白色，菌落背面淡黄绿色；37℃7d直径约1.40cm，与25℃菌落形态相似，质地絮状，菌落背面烧土褐色；菌落在CYAS上25℃7d直径约1.07cm，几乎不生长。菌落在YES上25℃7d直径约4.07cm，表面平坦，粉状孢子层，中心橘黄色呈同心轮纹状向外扩散至浅黄色和白色，边缘整齐，产孢量大，可溶性色素及渗出液无，菌落背面血红色向外扩散至白色。菌落在OA上25℃7d直径约4.00cm，较薄，平坦，粉状，墨绿色，质地絮状或绳状，产孢量大，可溶性色素及渗出液无；菌落在DG18上25℃7d直径约1.97cm，菌落分层，中心桔灰色，边缘菌丝白色，不整齐，菌落背面浅黄绿色。菌落在CREA上25℃7d直径约4.40cm，质地绒状，菌丝稀疏，白色或浅黄色，产孢量少。
拮抗菌株YC0121的分子鉴定，将菌株YC0121的18S rDNA基因测序结果(566bp)进行序列分析及同源性比对，并构建系统发育树，如图3所示，结果表明菌株YC0121的基因序列与Talaromyces sp.XJ3(MG745313.1)等同源相似性为100.00％，亲缘关系最近，且位于同一进化分支，结合形态学性状可将其鉴定为篮状菌属(Talaromyces C.R.Benj.)。但篮状菌的鉴定还需要基于β-微管蛋白基因(BenA)、钙调蛋白基因(CaM)和RNA多聚酶II第二大亚基基因(Rpb2)进行多基因分子种系学分析，扩增测序后经比对分析发现，该菌株与多个篮状菌属的种相似度较高，且由于篮状菌属真菌鉴定的复杂性，目前只能将其初步鉴定到属。
篮状菌属YC0121的促生能力及产酶活性，促生能力及产酶活性测定结果表明，菌株YC0121不产生IAA，不产NH3或能力极弱，能在解钾硅酸盐培养基上生长，菌丝稀疏，产孢量较少，具有解磷、固氮、产嗜铁素、产蛋白酶和解淀粉酶活性，无解纤维素酶和几丁质酶活性，如表3所示。
表3菌株YC0121对烟草土传病原真菌的抑制作用
Tab.3 Inhibitory effect of strain YC0121 on tobacco soil-bornepathogenic fungi
注：IAA：产IAA；NPA：解无机磷；OPA：解有机磷；NFb：固氮；Sil：解钾硅酸盐；NH3：产氨；Sid：产噬铁素；Pro：产蛋白酶；Amy：产淀粉酶；Cel：产纤维素酶；Chi：产几丁质酶；+：阳性；－：阴性；A：可以生长。
篮状菌属YC0121对烟草种子促生及根系活力的影响，子促生试验结果表明，浸种处理的种子萌发率高于空白对照组，种子根长显著长于对照，种子根长增长率为10.88％，如表4所示，根系活力测定结果表明，基质拌菌后可显著提高烟苗的根系活力，较对照组增加了91.98％，如表4所示，综合说明篮状菌属YC0121具有较好的根系促生作用。
表4菌株YC0121对烟草种子生长及根系活力的影响
Tab.4 Effects of strain YC0121 on tobacco seed growth and rootvitality
注：表中数据为Mean±SE，每列不同小写字母表示处理间差异有统计学意义(P≤0.05)，下同。
篮状菌属YC0121对烟株生长发育的影响，菌株YC0121灌根处理的盆栽烟草与空白对照组相比，在各农艺性状指标上均有一定的促进作用，其中在茎围和有效叶片数上差异显著，如表5所示，但盆栽试验的影响因素较多，需要对试验环境严格把控，对菌株的发酵条件进行优化，对施用量和施用次数进行严格测定，以期达到最好的促生效果。试验结果说明该菌株对烟草植株无致病性，且具有一定程度的促生作用。
表5菌株YC0121对盆栽烟草的促生效果
Tab.5 Growth-promoting effect of strain YC0121 on tobacco
篮状菌属YC0121无菌发酵滤液对烟草尖孢镰刀菌的抑菌活性，拮抗菌资源在烟草病害防治领域的研究和应用备受关注，目前，已有关于烟草黑胫病、根黑腐病、赤星病、青枯病等生物防治的相关报道，但对于烟草镰刀菌根腐病菌生物防治的研究鲜见报道。本实施例选择烟草尖孢镰刀菌为研究对象，采用菌丝生长速率法测定篮状菌属YC0121无菌发酵滤液不同浓度下对病原菌的影响，结果表明1％、5％和10％浓度对烟草尖孢镰刀菌的平板抑菌率分别为18.00％、25.00％和35.00％，说明该菌株发酵液活性较好。
篮状菌属YC0121对烟草尖孢镰刀菌的防治效果，以烟草尖孢镰刀菌为实施例进行盆栽防效试验，菌株YC0121发酵液处理的盆栽较阳性对照组(CK2)发病轻，病情指数显著低于阳性对照组；处理组的根系受侵染程度明显较阳性对照组轻，相比阴性对照组(CK1)根系有一定程度受损，植株整体较矮小黄化，如表6所示，结果表明篮状菌属YC0121对烟草尖孢镰刀菌具有较好的防治效果。
表6菌株YC0121对烟草尖孢镰刀菌的盆栽防效
Tab.6 Potted control effect of strain YC0121 against F.oxysporum
篮状菌是一类适应性非常强的腐生霉菌，广泛分布于自然界的土壤、空气、水体和人类活动场所及植物叶际和根际。篮状菌是自然界的重要分解者，如嗜松篮状菌、产紫篮状菌、小疣篮状菌等能产生高活性的木质纤维素酶类，从而增加土壤腐殖质；黄色篮状菌、沃特曼篮状菌等分泌磷酸酯酶等有机物，对植物矿物质吸收、抗病、抗逆和生长起促进作用；暗玫瑰篮状菌、白双轮篮状菌等可产生大量颜色鲜艳的黄色或红色色素而不产生任何真菌毒素，可用于食用色素的工业化生产。但篮状菌的某些种是人类和动物的条件致病菌，如马尔尼菲篮状菌是免疫缺陷病的继发感染病原；大孢篮状菌、杆孢篮状菌等是食品工业生产的常见污染霉菌，严重威胁世界粮食和饲料的安全。
篮状菌属分布广泛，与人体健康密切相关，在多相分类框架下，我国篮状菌属的物种资源有望得到更加系统的研究，近年来，也有越来越多的新种被报道。但因其生长缓慢，菌落较小，在分离、筛选和鉴定过程中往往被忽视或混淆，且关于篮状菌用于生物防治的研究鲜有报道，篮状菌属用于烟草土传病害的生物防治系属国内首次报道。由于篮状菌属复杂的分布特点、分类体系、作用机制和研究应用价值，后续需要对篮状菌属YC0121的分类更加细化，分类到种，同时加强对其促生防病作用机制和次生代谢物质的开发与应用研究。
综上所述，本发明公开一种对烟草土传病原真菌具有明显拮抗效果的篮状菌属(Talaromyces C.R.Benj.)YC0121，保藏号CCTCC NO：M 20211577，并对其防病促生特性进行了初步分析。该菌株生长缓慢，但产孢量大，繁殖快，适应能力强，易受风、雨、气流、人为因素等传播与定殖，具有较好的促生能力和水解酶活性，对烟草种子根长和烟苗根系活力具有显著的促进作用，以烟草尖孢镰刀菌为例，该菌具有较好的无菌发酵滤液抑菌活性和盆栽防治效果，生防潜力较大，具有较好的研究与应用价值，为烟草根茎类病害的生物防治提供优质资源和理论基础。
上述实施例仅仅是为清楚地说明本发明所做的举例，而并非对实施方式的限定，因试剂耗材规格、品牌、厂家等的不同，试验结果也会存在一定的误差，所以实施例展示的也并非最优结果。同时，对于所属领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换，这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (6)
1.一株用于烟草土传病害的生防篮状菌，其特征在于：所述的篮状菌属真菌菌株的保藏名称为篮状菌属YC0121，保藏编号：CCTCC NO：M 20211577。
2.一种如权利要求1所述的用于烟草土传病害的生防篮状菌的制备方法，其特征在于：包括以下步骤：
步骤一：提供制备生防篮状菌的原材料材料；
步骤二：篮状菌筛选；
步骤三：篮状菌的鉴定；
步骤四：篮状菌对烟草土传病害的抑菌测定。
3.根据权利要求2所述的用于烟草土传病害的生防篮状菌的制备方法，其特征在于：步骤三中篮状菌的鉴定的方法为：（1）、篮状菌属YC0121的形态学鉴定，（2）、篮状菌属YC0121的分子鉴定。
4.根据权利要求2所述的用于烟草土传病害的生防篮状菌的制备方法，其特征在于：步骤四中篮状菌对烟草土传病害的抑菌测定方法为：（1）、篮状菌属YC0121的促生能力及水解酶活性测定，（2）篮状菌属YC0121对烟草种子促生及根系活力的测定，（3）篮状菌属YC0121对盆栽烟草促生作用的测定，（4）篮状菌属YC0121无菌发酵滤液的抑菌活性测定，（5）篮状菌属YC0121对盆栽烟草的防效测定。
5.一种如权利要求1所述的用于烟草土传病害的生防篮状菌的应用，其特征在于：采用平板对峙培养法筛选对不同病原菌的抑菌活性，在距离培养基平板中心2.5 cm的相对2点上分别接种已纯化的拮抗菌和病原菌5 mm菌饼，以不接拮抗菌只接病原菌为对照，重复3次，倒置放入25℃恒温光照箱培养至对照组病菌菌落生长至平板面积2/3时，采用十字交叉法测量菌落半径，计算菌丝生长抑制率，并调查统计真菌拮抗系数。
6.根据权利要求5所述的用于烟草土传病害的生防篮状菌的应用，其特征在于：所述的真菌拮抗系数分级标准：Ⅰ级：待测真菌菌丝平板覆盖率100%；Ⅱ级：待测真菌菌丝平板覆盖率2/3以上；Ⅲ级：待测真菌菌丝平板覆盖率1/3～2/3；Ⅳ级：待测真菌菌丝平板覆盖率1/3以下；Ⅴ级：病原菌丝平板覆盖率100%。
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| CN115029249A (zh) * | 2022-05-10 | 2022-09-09 | 黑龙江省农业科学院经济作物研究所 | 一种拮抗马铃薯疮痂病病菌的真菌及其应用 |
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| CN115029255A (zh) * | 2022-07-28 | 2022-09-09 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | 一株生防内生真菌褐红篮状菌及其应用 |
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