CN116286393A - 一株高效抑制灰霉菌和炭疽菌的生防菌jsnl-c1及其应用 - Google Patents
一株高效抑制灰霉菌和炭疽菌的生防菌jsnl-c1及其应用 Download PDFInfo
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Abstract
本发明公开了一种草莓灰霉病和炭疽病生防菌及其应用,所述生防菌命名为TalaromycespurpureogenusJSNL‑C1,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCCNO.40439,所述生防菌TalaromycespurpureogenusJSNL‑C1能够有效防治由B.cinerea引起的草莓灰霉病和由C.siamense引起的草莓炭疽病。本发明提供的生防菌TalaromycespurpureogenusJSNL‑C1具有广谱抗菌性,培养简单,适应性强,抗菌能力稳定,可以广泛应用于多种植物真菌病害的防治。
Description
技术领域
本发明属于草莓草莓灰霉病和炭疽病生防技术领域,涉及一株高效抑制灰霉菌和炭疽菌的生防菌JSNL-C1及其应用。
背景技术
草莓因其色、香、味俱佳,享有“水果皇后”之美誉,是我国重要的经济作物,但每年因草莓病害都会造成不同程度的损失。其中草莓灰霉病是影响草莓产量和品质的重要病害之一。该病害是由灰葡萄孢菌(Botrytis cinerea)引起的,主要侵染草莓、番茄、葡萄、黄瓜、茄子、辣椒等多种植物,寄主范围广泛。灰霉病分布广、扩展速度快、危害严重,给全球果蔬的产量和品质带来巨大损失,在全球十大植物病害中排名第二位。灰霉病是草莓开花后危害最严重的一种真菌性病害,该病害主要侵染果实,也侵染叶片、果梗、花萼、花瓣及叶柄,但以果实受害时最为严重,造成的损失最大,感病品种的病果率一般在30%-60%,严重的情况下甚至绝收,给草莓生产造成巨大的损失。
由炭疽菌(刺盘孢)属(Colletotrichum spp.)引起的草莓炭疽病是近几年草莓的主要病害,在草莓整个生育期都可以发生危害,主要发生在草莓育苗期和定植初期。炭疽菌寄主范围广,除了可侵染草莓,还可侵染葡萄、苹果、辣椒、芒果、柑橘、桃、梨和铁皮石斛等植物。草莓炭疽病发生时,草莓叶片、叶柄、托叶、匍匐茎、花瓣和果实均可受害。该病一般造成草莓减产25%-30%,严重时达80%,甚至绝收,给草莓育苗和生产带来毁灭性的危害。草莓炭疽病是制约中国草莓产业的第三大病害。
现有技术中草莓灰霉病和炭疽病的防治主要包括以下方式:①选育抗病品种。选育抗病品种是防控草莓灰霉病和炭疽病最经济有效的途径。但一方面由于我国草莓主栽品种大多是来自国外抗病性较差的品种,草莓品种的抗病资源筛选工作还不完善;另一方面由于引起该病害的病原菌致病群体复杂,致使草莓品种的抗性容易丧失。②加强化学防治。化学药剂虽然能够在一定程度上防治草莓灰霉病和炭疽病,但是大量使用化学药剂一方面给生态环境和食品安全带来极大威胁;另一方面会导致病原菌抗药性不断增强,出现农药防治无效的现象。
因而,找到一种环境友好且行之有效的灰霉病和炭疽病防治措施是现今亟待解决的问题。生防菌所产生的抑菌物质通常直接针对相应的病原菌起作用,特异性强,因此不会对农业生态系统其它有益微生物产生不利影响,有利于保持生态平衡。
综上所述,现有技术还缺少一种预防草莓灰霉病和炭疽病的绿色、高效的益生菌。
发明内容
针对上述存在的问题,本发明提供了一种能够有效防治草莓灰霉病和炭疽病的生防菌Talaromycespurpureogenus JSNL-C1及其应用。
为实现上述目的,本发明采用下述技术方案:
第一方面,本发明提供了一种防治草莓灰霉病菌(Botrytis cinerea)引起的草莓灰霉病和炭疽病菌(Colletotrichum siamense)引起的草莓炭疽病的生防菌,所述生防菌命名为Talaromycespurpureogenus JSNL-C1,分离自南京市灰飞虱虫体上。
Talaromyces purpureogenus JSNL-C1,分类命名为Talaromycespurpureogenus,已于2022年12月8日在中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC)登记保藏,保藏编号为CGMCCNO.40439,保藏地址为北京市朝阳区北辰西路1号院3号。
第二方面,本发明提供了一种工程菌,所述工程菌包含本发明第一方面所述的Talaromycespurpureogenus JSNL-C1的核酸片段。
进一步地,所述核酸片段的碱基序列包括SEQ ID NO.1、SEQ ID NO.2和SEQ IDNO.3。
SEQ ID NO.1:
TACGGAGTGAGGGGCCCTCGCGGCCCACCTCCCACCCTTGTCTCCAA
CACCTGTTGCTTCGGCGGGCCCACCGGGGCCACCCGGTCGCCGGGGG
ACATCCGTCCCCGGGCCCGCGCCCGCCGAGGCGCTCTGTGAACCCTG
ATGAAGATGGGCTGTCTGAGTGATATGAAAATTGTCAAAACTTTCAAC
AATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGA
TAAGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATCTTTGAACG
CACATTGCGCCCCCTGGCATTCCGGGGGGCATGCCTGTCCGAGCGTCA
TTTCTGCCCTCAAGCACGGCTTGTGTGTTGGGTGTGGTCCCCCTGGGG
ACCTGCCCGAAAGGCAGCGGCGACGTCCGTCTGGTCCTCGAGCGTAT
GGGGCTCTGTCACTCGCTCGGGAAGGACCTGCGGGGGTTGGTCACCA
CCACATCTTTTTACAAGGTTGACCTCGGATCAGGTAGGAGTTACCCGC
TGAACTTAAGCTATCATAAGGCGGAGGAAGCCTTATTTATTTACSEQ ID NO.2:
TTCAGCTTCATCTGCCTGCATCATTGTTTGGGTATGTTGGTTGGTCGGTTATCTAACTAGCCCGTTTGGACGAGTAGGACAAGGATGGTGATGGTGAGTTCACCCGAACACGCAGCAATAAACGATAGGACTCTGAACAGGATATTTACTATATCGATTAGGTCAAATCACAACCAAGGAACTGGGCACCGTCATGCGCTCCCTCGGCCAGAACCCCTCCGAATCCGAATTGCAGGACATGATCAACGAAGTTGACGCTGACAACAACGGCACAATCGATTTCCCTGGTATGATGACTCTCGCTACAATCTACTGTGGATAGGTAACTGATTGATAATGGATAGAATTCTTGACAATGATGGCCCGCAAAATGAAGGATACCGACTCCGAGGAAGAGATCCGTGAGGCTTTCAAGGTGTTTGACCGTGACAACAATGGATTCATCTCTGCAGCTGAATTGCGTCACGTCATGACTTCGATTGGCGAGAAGTTGACCGATGACGAGGTTGATGAGATGATTCGTGAGGCTGATCAGGATGGTGATGGAAGGATTGACTGTGAGTTCCTCCTATAATGATTCAGAATGTGGGACGAAGCTGTTCTAATTAGTGATTGTGTTTCTAGACAACGAGTCTCATGTGTTTGGAGGAAAAAAAAAAAAAAAAGCCGCCGGCC
SEQ ID NO.3:
CAGTTCAGCTTCATCTGCCTGCATCATTGTTTGGGTATGTTGGTTGGTCGGTTATCTAACTAGCCCGTTTGGACGAGTAGGACAAGGATGGTGATGGTGAGTTCACCCGAACACGCAGCAATAAACGATAGGACTCTGAACAGGATATTTACTATATCGATTAGGTCAAATCACAACCAAGGAACTGGGCACCGTCATGCGCTCCCTCGGCCAGAACCCCTCCGAATCCGAATTGCAGGACATGATCAACGAAGTTGACGCTGACAACAACGGCACAATCGATTTCCCTGGTATGATGACTCTCGCTACAATCTACTGTGGATAGGTAACTGATTGATAATGGATAGAATTCTTGACAATGATGGCCCGCAAAATGAAGGATACCGACTCCGAGGAAGAGATCCGTGAGGCTTTCAAGGTGTTTGACCGTGACAACAATGGATTCATCTCTGCAGCTGAATTGCGTCACGTCATGACTTCGATTGGCGAGAAGTTGACCGATGACGAGGTTGATGAGATGATTCGTGAGGCTGATCAGGATGGTGATGGAAGGATTGACTGTGAGTTCCTCCTATAATGATTCAGAATGTGGGACGAAGCTGTTCTAATTAGTGATTGTGTTTCTAGACAACGAGTCTCATGTGTTTGGAGGAAAAAAAAAAAAAAAAGCCGCCGGCC
第三方面,本发明提供了一种能够防治草莓灰霉病菌(B.cinerea)引起的草莓灰霉病和草莓炭疽病菌(C.siamense)引起的草莓炭疽病的生防菌剂,其活性成分为如下一种或多种:
A1)本发明第一方面所述的Talaromycespurpureogenus JSNL-C1;
A2)本发明第一方面所述的Talaromycespurpureogenus JSNL-C1的菌丝、孢子或次生代谢物中的一种或多种;
A3)本发明第二方面任一所述的工程菌。
进一步地,所述Talaromyces purpureogenus JSNL-C1的菌丝由Talaromycespurpureogenus JSNL-C1接种于PDA和OA培养基培养制得。
进一步地,所述生防菌剂包括生防化肥或生防农药。
第四方面,本发明还提供了第三方面所述的生防菌剂的制备方法,其特征在于,包括以下步骤:
S1、制备无菌基质;
S2、向S1所述基质中加入Talaromycespurpureogenus JSNL-C1的菌丝、孢子或次生代谢物;
S3、将S2得到的基质密封培养数天即得生防菌剂。
进一步地,所述方法包括如下步骤:
A1:将基质装入带有透气孔的无菌三角瓶(250mL)中,每瓶放入200mL基质,在121℃环境下高温灭菌20min。
A2:Talaromycespurpureogenus JSNL-C1接种于直径9cm的含有15mL的PDA培养基中,于25℃、避光培养3-5d。
A3:将长满Talaromycespurpureogenus JSNL-C1菌落边缘切取3mm×3mm的菌丝块,无菌条件下转移到液体基质中,封口后置于25℃,160rpm培养7d,制得Talaromycespurpureogenus JSNL-C1的液体发酵菌剂。
第五方面,本发明还提供了上述的生防菌、上述的工程菌或上述的生防菌剂在防治草莓灰霉病菌(B.cinerea)引起的草莓灰霉病和草莓炭疽病菌(C.siamense)引起的草莓炭疽病害中的应用。
第六方面,本发明还提供了一种植物灰霉病和炭疽病的防治方法,所述方法包括向植物生长环境中施用本发明所提供的生防菌、工程菌或生防菌剂。
与现有技术相比,本发明提供的菌株Talaromyces purpureogenus JSNL-C1分泌的胞外代谢物对B.cinerea和C.siamense均有较高的抑制效果,且随着胞外代谢物浓度的增加,抑制效果有增加的趋势;当Talaromyces purpureogenus JSNL-C1胞外代谢物浓度为20mL/100mL时,对B.cinerea抑制率达到了83.33%,对C.siamense抑制率达到了100%;当JSNL-C1胞外代谢物浓度为40mL/100mL时,对B.cinerea和C.siamense抑制率均达到了100%。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式描述中所需要使用的附图作简单地介绍。
图1为实施例1中Talaromycespurpureogenus JSNL-C1在平板上抑制B.cinerea菌丝生长的效果图;
图2为实施例1中Talaromycespurpureogenus JSNL-C1在平板上抑制C.siamense菌丝生长的效果图;
图3为实施例1中Talaromycespurpureogenus JSNL-C1对B.cinerea的抑制活性;
图4为实施例1中Talaromycespurpureogenus JSNL-C1对C.siamense的抑制活性;
图5为实施例2中Talaromycespurpureogenus JSNL-C1分泌的胞外代谢物对B.cinerea的影响;
图6为实施例2中Talaromycespurpureogenus JSNL-C1分泌的胞外代谢物对C.siamense的影响。
具体实施方式
下面将结合附图对本发明技术方案的实施例进行详细的描述。以下实施例仅用于更加清楚地说明本发明的技术方案,因此只是作为示例,而不能以此来限制本发明的保护范围。需要注意的是,除非另有说明,本申请使用的技术术语或者科学术语应当为本发明所属领域技术人员所理解的通常意义。
如本发明中所述,术语“生防菌”指有益微生物,可以防治植物病害,主要有细菌、真菌和放线菌。
如本发明中所述,术语“工程菌”指采用现代生物工程技术加工出来的新型微生物,具有多功能、高效和适应性强等特点。
如本发明中所述,术语“草莓灰霉病菌(B.cinerea)”分类地位:真菌界(Fungi)、子囊菌门(Ascomycota)、锤舌菌纲(Leotiomycete)、柔膜菌目(Helotiales)、核盘菌科(Sclerotiniaceae)、孢盘菌属(Botryotinia)。主要分布在美国、瑞典、德国、法国、中国、巴西、日本、韩国、阿根廷、印度、智利等国家。
如本发明中所述,术语“草莓炭疽病菌(C.siamense)”分类地位:真菌门(Eumycota)、半知菌亚门(Deuteromyeotina)、腔孢纲(Coelomycetes)、黑盘孢目(Melanconiales)、黑盘孢科(Melanconiaceae)、炭疽属(Colletotrichum)。主要分布在美国、加拿大、澳大利亚、日本、英国、中国、比利时、德国、印度、越南、韩国、巴西、巴拿马、墨西哥、俄国、以色列等国家。
实施例1菌Talaromycespurpureogenus JSNL-C1的筛选
1、分离潜在的生防菌
1.1、获取试验材料:从染病的灰飞虱虫体上分离获得菌株。
1.2、灰飞虱染病虫体上真菌的分离及培养:取染病的灰飞虱虫体,用70%(v/v)乙醇处理30s,2.5%(v/v)次氯酸钠溶液处理2min,最后用无菌水冲洗3-4遍,用无菌吸水纸吸干表面水分。在无菌条件下将灰飞虱放置在含有氨苄(50mg/L)和利福平(50mg/L)的PDA平板培养基上,每皿放置5头。将培养皿密封,在25℃、黑暗条件下培养1-2d。当菌丝体从虫体上中出现时,将生长在培养基边缘的小块培养基连同菌丝体一起小心地转移到一个新的PDA平板上。
2、平板对峙法筛选拮抗菌
采用PDA平板对峙法,在直径9cm的含有15mL的PDA培养基平板距离边缘2cm处分别对称接种培养3-5d的B.cinerea和C.siamense菌株菌饼(直径5mm)和供试真菌菌饼,以只接种B.cinerea和C.siamense菌株菌饼的平板作为对照。平板置于25℃、黑暗条件下培养。5d后测量B.cinerea和C.siamense菌落半径并计算抑制率。抑制率按照公式计算:抑制率=(对照组菌落半径-处理组菌落半径)/对照组菌落半径×100%。
最终筛选得到1株在平板上对B.cinerea和C.siamense均有很好拮抗活性的菌株,具体表现为强烈的抑制病原菌菌丝生长,编号为JSNL-C1。
如图1所示,为JSNL-C1与B.cinerea菌株对峙,与对照相比,实验组B.cinerea菌株的菌落明显更小,抑制率达到了72.11%,如图2所示,为JSNL-C1与C.siamense菌株对峙,与对照相比,实验组C.siamense菌株的菌落明显更小,抑制率达到了64.04%,说明B.cinerea和C.siamense菌株的菌丝生长均受到了抑制。
如图3所示,光学显微观察结果显示:正常B.cinerea菌丝外形规则,内含物在菌丝细胞壁内分布均一、较透明;与JSNL-C1对峙7天后,B.cinerea菌丝肿胀变形、内部物质瓦解、凝聚,不能正常生长。
如图4所示,正常C.siamense菌丝外形规则,生长顺直,菌丝细胞壁内容物分布均一;与JSNL-C1对峙7天后,C.siamense菌丝内部物质凝聚、混乱、粗细不均,不能正常生长。
3、筛选所得菌株的分子生物学鉴定
3.1、采用TIANGEN公司的试剂盒(DP320-03)提取菌株JSNL-C1的基因组DNA。
3.2、分别扩增基因组DNA的ITS基因、BenA基因和CAL基因。DNA扩增采用30μL的反应体积,包含15μL 2×EasyTaq PCR SuperMix(+dye),引物F/R各1μL(10μM),2μL模板DNA和11μL ddH2O,PCR扩增程序条件如下:94℃预变性5min;94℃变性30s,54℃退火45s,72℃延伸60s,共35个循环;最后72℃延伸10min。
3.3、扩增所得PCR产物经过1%琼脂糖凝胶电泳后,紫外灯下观察,将有目标条带的PCR产物送至南京擎科生物科技有限公司测序,测序结果为:步骤2筛选得到的菌株JSNL-C1的ITS基因序列为SEQ ID NO.1,BenA基因序列为SEQ ID NO.2,CAL基因序列为SEQ IDNO.3。
3.4、在NCBI数据库网站上比对SEQ ID NO.1、SEQ ID NO.2和SEQ ID NO.3三条序列。比对结果如表1所示。根据表1结果可以推定本发明的生防菌JSNL-C1为Talaromycespurpureogenus,命名为Talaromyces purpureogenus JSNL-C1。
表1SEQ ID NO.1、SEQ ID NO.2和SEQ ID NO.3序列比对结果
实施例2Talaromycespurpureogenus JSNL-C1胞外代谢物对B.cinerea和C.siamense的影响
1、将B.cinerea和C.siamense与Talaromycespurpureogenus JSNL-C1分别接种于直径9cm的含有15mL PDA培养基的培养皿中,置于25℃培养箱,避光培养5d。待病原菌长满培养皿,用打孔器(直径5mm)在菌落边缘打孔,制备菌碟。
2、菌株JSNL-C1分泌的胞外代谢物的获取
在生长5d的JSNL-C1菌落边缘切取3mm×3mm的菌丝块,无菌条件下将30块菌丝块转移到200mL PDB液体培养基中,25℃、黑暗、160rpm条件下培养7d,先用两层无菌纱布过滤掉培养液中的菌丝块,常温下,10000rpm离心30min后取上清;用0.45μm的细菌过滤器过滤一遍上清液后再用0.22μm细菌过滤器过滤一遍,以完全去除JSNL-C1菌体,得到无菌滤液。
3、无菌操作下,分别量取上述无菌滤液10mL、20mL和40mL,各倒入100mL PDA培养基(冷却至50℃左右)中,迅速混匀,制得浓度为10mL/100mL、20mL/100mL和40mL/100mL的含JSNL-C1无菌滤液的平板,对照以无菌水代替无菌滤液,分别制得相应浓度的无菌水平板。
4、在超净工作台中用接种针挑取各病原菌菌碟接种于上述制得的平板中,注意菌丝面朝下,每种浓度(无菌滤液和无菌水)各设3个重复。
5、将接种后的培养皿放入25℃培养箱,避光培养3-5d,用十字交叉法测量菌落大小。抑制率按照公式计算:抑制率=(对照组菌落直径-处理组菌落直径)/对照组菌落直径×100%。
结果如图5、图6和表2所示
表2菌株JSNL-C1分泌的胞外代谢物对病原菌抑制率
其中,表中菌落直径的数值表达方式为:平均值±标准误差。
从图5、图6和表2数据可知,菌株Talaromycespurpureogenus JSNL-C1分泌的胞外代谢物对B.cinerea和C.siamense均有较高的抑制效果,且随着胞外代谢物浓度的增加,抑制效果有增加的趋势。当Talaromyces purpureogenus JSNL-C1胞外代谢物浓度为20mL/100mL时,对B.cinerea抑制率达到了83.33%,对C.siamense抑制率达到了100%;当JSNL-C1胞外代谢物浓度为40mL/100mL时,对B.cinerea和C.siamense抑制率均达到了100%。
除非另外具体说明,否则在这些实施例中阐述的数值并不限制本发明的范围。在这里示出和描述的所有示例中,除非另有规定,任何具体值应被解释为仅仅是示例性的,而不是作为限制,因此,示例性实施例的其他示例可以具有不同的值。
Claims (9)
1.一种草莓灰霉病和炭疽病生防菌,其特征在于,所述生防菌为TalaromycespurpureogenusJSNL-C1,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCCNO.40439。
2.一种工程菌,其特征在于,所述工程菌包含权利要求1所述的TalaromycespurpureogenusJSNL-C1的核酸片段。
3.根据权利要求2所述的工程菌,其特征在于,所述核酸片段的序列为SEQ ID NO.1、SEQ ID NO.2和SEQ ID NO.3。
4.一种能够防治B.cinerea引起的草莓灰霉病和C.siamense引起的草莓炭疽病的生防菌剂,其特征在于,其活性成分为如下一种或多种:
A1)权利要求1所述的TalaromycespurpureogenusJSNL-C1;
A2)权利要求1所述的TalaromycespurpureogenusJSNL-C1的菌丝、孢子或次生代谢物中的一种或多种;
A3)权利要求2-3任一所述的工程菌。
5.根据权利要求4所述的生防菌剂,其特征在于,所述TalaromycespurpureogenusJSNL-C1的菌丝由TalaromycespurpureogenusJSNL-C1接种于PDA和OA培养基培养制得。
6.权利要求4-5任一所述的生防菌剂的制备方法,其特征在于,包括以下步骤:
S1、制备无菌基质;
S2、向S1所述基质中加入TalaromycespurpureogenusJSNL-C1的菌丝、孢子或次生代谢物;
S3、将S2得到的基质密封培养数天即得生防菌剂。
7.权利要求1所述的生防菌、权利要求2-3任一所述的工程菌或权利要求4-5任一所述的生防菌剂在防治B.cinerea引起的草莓灰霉病和C.siamense引起的草莓炭疽病病害中的应用。
8.根据权利要求7所述的应用,其特征在于,所述草莓病害包括草莓灰霉病和草莓炭疽病。
9.一种草莓灰霉病和草莓炭疽病的防治方法,其特征在于,所述方法包括向植物生长环境中施用权利要求1所述的生防菌、权利要求2-3任一所述的工程菌或权利要求4-5任一所述的生防菌剂。
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