CN111996125B - 一种球毛壳菌在防治小麦茎基腐病中的应用 - Google Patents
一种球毛壳菌在防治小麦茎基腐病中的应用 Download PDFInfo
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Abstract
本发明提供一种球毛壳菌在防治小麦茎基腐病中的应用,所述球毛壳菌的保藏名称为12XP1‑2‑3,保藏编号为CGMCC No.17183,保藏单位为中国微生物菌种保藏管理委员会普通微生物中心,保藏日期为2019年1月23日。田间试验结果表明,用球毛壳菌12XP1‑2‑3进行小麦种子包衣处理,可以使病茎率、病级和病情指数分别降低19.0%‑41.3%、43.7%和4.7%‑45.4%。
Description
技术领域
本发明涉及一种球毛壳菌在防治小麦茎基腐病中的应用。
背景技术
小麦茎基腐病是小麦的重要根部病害,能够降低穗粒重及千粒重,在小麦苗期、分蘖期、拔节期以及成株期都可以侵染,典型症状为小麦茎基部褐变呈酱油颜色,灌浆期田间出现散点状白穗。
对于小麦茎基腐病,目前没有显著效果的防治药剂,现有化学药剂的使用不仅会破坏土壤微生态环境,加重环境污染,有些拌种剂还会使小麦出苗期延迟、影响出苗。因此,病害防治重点逐步转向生物防治和农业防治措施上。在农作物植物保护领域,利用作物的健株根际土壤筛选对病原菌具有显著生防效果的土壤微生物,并制成微生物菌剂是生物防控研究的重要手段之一,也是有益微生物资源开发利用的重要途径。毛壳菌属(Chaetomiumsp.)真菌广泛分布于土壤及植物体内。该类微生物能产生毛壳素、球毛壳素等多种抗生素,作为植物病原菌的生物防治菌被广泛研究。目前利用分离到的球毛壳菌在防治小麦茎基腐病未见报道。
发明内容
本发明提供一种球毛壳菌在防治小麦茎基腐病中的应用,田间试验结果表明使用球毛壳菌12XP1-2-3对小麦种子包衣处理后,既可以将小麦病茎率降低19-41.3%,也能使病级降低43.7%、病情指数降低4.7-45.4%。
本发明提供一种球毛壳菌在防治小麦茎基腐病中的应用;
其中,所述球毛壳菌的保藏名称为12XP1-2-3,保藏编号为CGMCC No.17183,保藏单位为中国微生物菌种保藏管理委员会普通微生物中心,保藏日期为2019年1月23日。
本发明还提供一种含上述球毛壳菌的生防菌剂在防治小麦茎基腐病中的应用。
该生防菌剂是按照以下方法制备得到的:对所述球毛壳菌菌株进行扩繁培养,得到孢子悬浮液,再将所述孢子悬浮液分散至羧甲基纤维素钠溶液中,即得所述生防菌剂。
优选地,所述羧甲基纤维素钠溶液的质量分数为4%,所述孢子悬浮液与所述羧甲基纤维素钠溶液的体积比为3:1。
上述生防菌剂在具体应用时,是以包衣浓度为5.0-5.3×104个子囊孢子/每颗种子对小麦种子进行包衣处理。
生物保藏说明
生物材料:球毛壳菌12XP1-2-3;分类命名:球毛壳菌(拉丁文名称:Chaetomiumglobosum),于2019年01月23日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏中心地址为:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所;保藏编号为CGMCC No.17183。
对比现有技术,本发明的有益效果为:
本发明提供的球毛壳菌12XP1-2-3能够用于防治小麦茎基腐病,田间试验结果表明使用球毛壳12XP1-2-3拌种,使小麦病茎率减少19-41.3%,也能使病级降低43.7%、病情指数降低4.7-45.4%。
附图说明
图1是球毛壳菌12XP1-2-3对小麦茎基腐病菌G14LY24-2的平板拮抗效果。
具体实施方式
以下结合具体实施例对本发明做进一步详细描述,这些实施例用于理解而不是限制本发明的保护范围。
实施例1
球毛壳菌株12XP1-2-3的分离
于小麦灌浆期,在河南省西平县采集小麦全蚀病田中未发病的植株装纸袋中。样品置于4℃的冰箱保存。将样品根部冲洗干净,然后取小麦根部组织2-3cm,用质量分数75%无水乙醇表面消毒10-30s,质量分数1%NaCIO消毒1.5min,无菌水冲洗3次,灭菌滤纸吸干水分后,剪成0.5cm左右的小段,用无菌的镊子放置在马铃薯葡萄糖琼脂培养基(PDA,原料组成为:去皮马铃薯300g、葡萄糖20g、琼脂20g及蒸馏水1000ml)平板上,在20℃下培养5-7天后,挑去边缘菌丝于新的PDA平板上生长,同样条件下5-7天后,挑去菌落均匀的菌丝块置于PDA斜面上培养,置于4℃保存。
实施例2
球毛壳菌株12XP1-2-3的DNA提取
将球毛壳菌株12XP1-2-3挑取至PDA培养基上,20℃培养5-7天后,挑取5块边缘菌丝块均匀地放置到铺有灭菌玻璃纸的PDA平板上。3-5天后用灭菌的小铁铲刮取菌丝,并用液氮速冻,存放在-20℃的冰箱里。提取DNA时,取出20-25mg菌丝入预冷的1.5ml的EP管中;加入少许液氮用预冷的铁钉在EP管中将菌丝研磨至粉末;加入500μl Extraction Buffer(50mM Tris-Cl(pH 8.0),150mM NaCl,100mM EDTA(8.0)),在漩涡振荡器上振荡悬浮沉淀并混匀;然后加入预热的25μl(质量分数为20%)的十二烷基磺酸钠,颠倒混匀,在37℃水浴锅中放置1-3小时;然后加入75μl的5M NaCl,颠倒混匀;加入65μl的CTAB/NaCl(10%CTAB,0.7M NaCl)溶液,65℃水浴30分钟;加入等体积的(700μl)Tris-酚:氯仿:异戊醇(25:24:1)的混合液,混匀,10000rpm,4℃离心10分钟,将上清(550μl)转移至另一个EP管中;然后加入0.6倍体积的预冷的异丙醇(330μl),-20℃沉淀10分钟,10000rpm,4℃离心10分钟,弃上清;沉淀用质量分数70%的乙醇洗涤两次,放入工作台风干5-10分钟;干燥后的沉淀溶于适量的无菌ddH2O中,-20℃保存。
实施例3
球毛壳菌株12XP1-2-3的分子鉴定
本试验采用引物ITS1(5’-TCC GTA GGT GAA CCT GCG G-3’)和ITS4(5’-TCC TCCGCT TAT TGA TAT GC-3’)扩增待测菌株的包含ITS1、5.8S rDNA和ITS2的DNA片段。体系参考Daval et al.(2010)。扩增反应条件:PCR反应程序为:95℃3min;95℃45s,50℃30s,72℃1min,35个循环;72℃10min。PCR产物测序后,在NCBI中进行BLAST序列比对分析。序列比对结果为生防菌12XP1-2-3为球毛壳。
表1.PCR反应体系
成分 | 浓度 | 用量(μl) |
10×PCR缓冲液 | 10mM | 5.0 |
dNTP混合物 | 10mM | 1.0 |
TaqDNA酶 | 5u | 0.5 |
引物1 | 20μM | 1.0 |
引物2 | 20μM | 1.0 |
DNA模板 | 10-50ng/μl | 1.0 |
双蒸馏水 | 纯水 | 40.5 |
总体积(μl) | 50.0 |
实施例4
球毛壳菌12XP1-2-3对小麦茎基腐病菌的平板拮抗效果
将球毛壳菌12XP1-2-3用无菌牙签挑取在PDA上活化,培养条件为25℃,12h光照12h黑暗;将小麦茎基腐病菌G14LY24-2在PDA上活化;培养条件为25℃,无光照;待菌落接近培养皿边缘时(7天后)用内径0.5cm打孔器分别把毛壳菌、小麦茎基腐病菌菌落边缘打成若干菌饼。在PDA培养皿距离圆心2.5cm处两段做标记;分别在所做标记处放入球毛壳菌和小麦茎基腐病菌菌饼,两者中心相距5cm;空白对照只放置小麦茎基腐病菌菌病,培养条件为25℃,无光照。3天后划线测定小麦茎基腐病菌生长情况,结果如图1所示。结果表明,球毛壳菌12XP1-2-3在平板上对小麦茎基腐病菌G14LY24-2的抑制率为30.3%。
实施例5
球毛壳菌12XP1-2-3种子包衣防治小麦茎基腐病的田间效果
1、球毛壳菌对小麦茎基腐病的田间防治效果
1.1、小麦茎基腐病菌接种物的培养
小麦粒浸泡12h,然后灭菌60min,放置2天后,再灭菌30-60分钟,冷却后使用。选取小麦茎基腐病菌G14LY24-2在PDA平板上,生长3天后,挑取边缘菌丝在新鲜的PDA平板上生长,3天后将菌丝块接入装好灭好菌的麦粒聚乙烯袋中,室温生长2个星期直至病菌长满整个麦粒,风干后存放于4℃冰箱。
1.2、建造田间病圃
按照种子量和接种物体积比为1:1的比例来建造病圃。接种位于种子上面3-5cm,先将待播种的种子和接种物进行混合,混合均匀后,使用播种机将混合好的播种物进行播种。
1.3、球毛壳菌种子包衣制作
将挑选的球毛壳菌菌株在新鲜的PDA上活化,然后分别挑取菌丝块到100个9cm的PDA培养皿,15天后加入无菌水用涂布棒将子囊壳研磨,将子囊孢子悬浮液控制在100mL左右,浓度在1.0-2.0×107个/mL;如果浓度太低,用3000rpm5min离心,然后去水重新悬浮。75mL的菌液(1.3×107个/mL)加入25mL的质量分数4%羧甲基纤维素盐的溶液,混合成100mL1%的羧甲基纤维素盐的菌液(1.0×107个/mL)。100mL1×107个/mL 1%的羧甲基纤维素盐的菌液加入5kg小麦种子混合均匀。球毛壳菌12XP1-2-3包衣种子冲洗后涂平板测定种子表面的孢子数量,为5.0-5.3×104个子囊孢子/每颗种子。2017-2018年小麦品种为“百农207”,2018-2019年小麦品种为“矮抗58”,对照组药剂为苯醚甲环唑30g/L,药种比为1:333进行拌种。
1.4、数据调查和记录
田间样品取样方法:苗期调查出苗率,并测定株高、主根长、鲜重、干重、球毛壳菌定殖能力;分蘖期调查发病率,并测定株高、主根长、鲜重、干重、分蘖数,球毛壳菌定殖能力;拔节期调查发病率;灌浆期调查白穗率,调查方式为每个小区调查1m2白穗数和总穗数,计算得到小区白穗率;成熟期调查病茎率,在每个小区中五点取样选取20个茎,每个处理共100个茎带回实验室,将叶鞘剥离干净后测定病级。
成株期分级标准为:0=无病;1=第1节变褐;2=第2节变褐;3=第3节变褐;4=穗下节变褐或灌浆期白穗;5=植株发病未成穗。收获期测定产量。结果见表2。
表2 2017-2018年度和2018-2019年度小麦灌浆期球毛壳种子包衣防治小麦茎基腐病的效果
由表2可知,灌浆期调查,2017-2018年度小麦品种为“百农207”,空白对照组的病茎率为57.8%,平均病级为0.7,球毛壳菌12XP1-2-3种子包衣处理病茎率分别为33.9%,平均病级为0.7;2018-2019年度小麦品种为“矮抗58”,空白对照组的病茎率为50.4%,平均病级为1.6,球毛壳菌12XP1-2-3种子包衣处理病茎率分别为40.8%,平均病级为1.6。
提示本发明提供的球毛壳菌12XP1-2-3将种子包衣处理后,既可以减少白穗率,也能降低病级和病情指数。
需要说明的是,本发明权利要求书中采用的步骤方法与上述实施例相同,为了防止赘述,本发明的描述了优选的实施例,但本领域内的技术人员一旦得知了基本创造性概念,则可对这些实施例作出另外的变更和修改。所以,所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (5)
1.一种球毛壳菌在防治小麦茎基腐病中的应用;
其中,所述球毛壳菌的保藏名称为12XP1-2-3,拉丁文名称为Chaetomiumglobosum12XP1-2-3,保藏编号为CGMCC No.17183,保藏单位为中国微生物菌种保藏管理委员会普通微生物中心,保藏日期为2019年1月23日。
2.含有权利要求1所述球毛壳菌的生防菌剂在防治小麦茎基腐病中的应用。
3.根据权利要求2所述的应用,其特征在于,所述生防菌剂是按照以下方法制备得到的:对所述球毛壳菌菌株进行扩繁培养,得到孢子悬浮液,再将所述孢子悬浮液分散至羧甲基纤维素钠溶液中,即得所述生防菌剂。
4.根据权利要求3所述的应用,其特征在于,所述羧甲基纤维素钠溶液的质量分数为4%,所述孢子悬浮液与所述羧甲基纤维素钠溶液的体积比为3:1。
5.根据权利要求2-4任一项所述的应用,其特征在于,是以包衣浓度为5.0-5.3×104个子囊孢子/每颗种子对小麦种子进行包衣处理。
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