CN103642725A - 拮抗植物病原菌的生防菌株及其用途 - Google Patents

拮抗植物病原菌的生防菌株及其用途 Download PDF

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CN103642725A
CN103642725A CN201310627298.4A CN201310627298A CN103642725A CN 103642725 A CN103642725 A CN 103642725A CN 201310627298 A CN201310627298 A CN 201310627298A CN 103642725 A CN103642725 A CN 103642725A
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王威
惠俊渊
张雪洪
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Shanghai Jiaotong University
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Abstract

本发明公开了一株拈抗植物病原菌的生防菌株及其用途;所述菌株为灰红链霉菌Y1B(Streptomyces griseoruber Y1B)CCTCC N0:M2013358。采用GYM培养基对该灰红链霉菌Y1B进行发酵,收集发酵液,萃取并减压蒸馏,制得发酵粗产品。该发酵粗产品,对水稻纹枯病菌和西瓜枯萎病菌有较好的抑菌效果。

Description

拮抗植物病原菌的生防菌株及其用途
技术领域
本发明属于生物防治和生物工程技术领域,具体涉及一株拈抗植物病原菌的生防菌株及其用途。
背景技术
伴随着农业经济的迅猛发展,农业生产中的植物病虫害防治越来越依赖于化学农药的广泛应用。但是在化学农药大量使用的同时,环境污染问题也随之而来,由此人们对现代农业的防治技术提出了更高的要求。生防农药作为一种新型的绿色农药,以其安全可靠、不污染环境、抗菌效果好的特点,近年来受到世界各国的广泛关注,因此寻找有效的生防菌株也成为人们日益关注的热点。
链霉菌可以产生多种抗生素,目前世界上已经发现的抗生素中,约有80%是由放线菌产生,其中又以链霉菌属列居放线菌中最大的菌属,该属可产生1000多种抗生素。这些抗生素除了可以广泛应用于医药领域,在农作物生产方面,还具有植物保护的用途。
链霉菌是一类活跃在植物根际的微生物,土壤中的许多链霉菌具有抑制植物病害、促进植物生长的作用,属于植物根际促生菌。生防农药中大部分是农用抗生素,具有毒性低、残留少的特点,可以有效抑制植物病原菌的生长和繁殖。因此,链霉菌产生的农用抗生素在生防农药中占有重要地位,多种链霉菌的次级代谢产物被研制成农用抗生素在生产上大力推广,如井冈霉素、春雷霉素、中生菌素等在植物病虫害防治方面取得的效果得到了人们的公认。
发明内容
本发明的目的在于提供一株拈抗植物病原菌的生防菌株及其用途;通过发酵该菌株制备粗产品,对植物病原菌有较好的防治效果。
本发明的目的是通过以下技术方案来实现的:
第一方面,本发明涉及一株拈抗植物病原菌的生防菌株,所述菌株为灰红链霉菌Y1B(Streptomyces griseoruber Y1B)CCTCC NO:M2013358。
优选地,所述灰红链霉菌Y1B具有SEQ ID NO:1所示的16SrDNA序列。
第二方面,本发明涉及一种上述的拈抗植物病原菌的生防菌株在制备防治植物枯萎病生物农药中的用途。
第三方面,本发明涉及一种生防菌株发酵粗产品的制备方法,采用GYM培养基对如权利要求1所述的灰红链霉菌Y1B进行发酵,收集发酵液,萃取并减压蒸馏,即得所述发酵粗产品。
优选地,每1000mL GYM培养基中含有葡萄糖3~5g、酵母提取物2~6g、麦芽提取物8~12g、余量为水,pH7.0~8.0。
优选地,所述灰红链霉菌Y1B是通过如下步骤获得的:采集水稻土壤样品,土样碾碎后放置在烘箱烘焙,称取烘焙后的土样溶于无菌水中,充分振荡,制成不同浓度的土壤悬浮液,涂布于添加有0.05%~0.25%K2Cr2O7的高氏1号培养基平板上,待长出菌落后,挑单菌落于SFM固体培养基上纯化,即得灰红链霉菌Y1B(Streptomyces griseoruberY1B)CCTCC NO:M2013358。
优选地,每1000mL高氏1号培养基中含有可溶性淀粉10~30g、KNO30.8~1.2g、K2HPO40.3~0.6g、MgSO4·7H2O0.4~0.8g、FeSO4·7H2O0.008~0.012g、NaCl0.4~0.6g、琼脂18~24g、余量为水,pH7.0~8.0。
优选地,每1000mL SFM培养基的制备包括如下步骤:取15~25g黄豆饼粉,加蒸馏水沸水煮20~40min,过滤黄豆饼粉液体,滤液中加甘露醇15~25g,固体培养基另加琼脂18~25g,蒸馏水定容至1000mL,pH7.0~8.0。
第四方面,本发明涉及一种上述的制备方法制得的生防菌株发酵粗产品在制备防治植物枯萎病生物农药中的用途。
优选地,所述植物枯萎病为水稻枯萎病菌或西瓜枯萎病菌。
本发明具有的有益效果为:本发明制得的拈抗植物病原菌的生防菌株发酵粗产品,对水稻纹枯病菌和西瓜枯萎病菌有较好的抑菌效果,相对抑菌率分别达到47.1%和30.9%。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述,本发明的其它特征、目的和优点将会变得更明显:
图1为本发明菌株灰红链霉菌Y1B(Streptomyces griseoruber Y1B)的菌落形态图;
图2为本发明菌株的系统发育树。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明,但不以任何形式限制本发明。应当指出的是,对本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干调整和改进。这些都属于本发明的保护范围。
本发明的灰红链霉菌Y1B(Streptomyces griseoruber Y1B),该菌株已在中国典型培养物保藏中心(简称CCTCC)保藏,保藏单位地址为:中国.武汉.武汉大学,邮编为:430072,保藏日期为:2013年8月1日,保藏编号为:CCTCC NO:M2013358。
本发明菌株的形态特征、培养特征、16SrDNA序列和菌种分类结果如下:
1、形态特征和培养特征:灰红链霉菌Y1B革兰氏染色呈阳性,孢子椭圆形,孢子丝紧密螺旋形。在SFM固体培养基上形成圆形灰色菌落,菌落表面凸起,光滑干燥,菌落可产生红色色素(如图1所示)。
2、本发明所述的灰红链霉菌Y1B的16SrDNA序列如SEQ ID NO:1所示。
图2为本发明菌株的系统发育树,根据菌落形态和16SrDNA序列分析,该菌株属于灰红链霉菌(Streptomyces griseoruber),故命名为灰红链霉菌Y1B。
本发明的菌株发酵粗产品的制备方法具体如下:
1、菌株的获得:从江苏省无锡市采集水稻土壤样品若干,将土样碾碎后,放置在50℃~70℃烘箱烘焙30~60min。称取烘焙后的土样溶于无菌水中,充分振荡,制成不同浓度梯度的土壤悬浮液,涂布于添加有0.05%~0.25%K2Cr2O7的高氏1号培养基平板上,在28℃~32℃恒温培养箱中培养5~10天。挑单菌落于SFM固体培养基上纯化,得到灰红链霉菌Y1B(Streptomyces griseoruber Y1B),CCTCC M2013358。
2、生防菌株发酵粗产品的制备:用SFM固体培养基活化冷冻保藏的CCTCC M2013358菌株,然后将上述培养物接种到SFM液体培养基摇瓶中,28℃~32℃条件下培养2~3天,作为种子液;再将种子液按1%~5%的接种量接种到GYM培养基摇瓶中,28℃~32℃条件下发酵6~8天。发酵结束后,将发酵液离心收集发酵上清液,用乙酸乙酯等体积萃取发酵上清液,合并收集有机相,减压蒸馏有机相,浓缩蒸干后,得到发酵粗产品。具体见以下各实施例:
实施例1
菌株的获得:从江苏省无锡市采集水稻土壤样品若干,将土样碾碎后,放置在60℃烘箱烘焙30min。称取烘焙后的土样5g加入盛有45mL无菌水的摇瓶中,振荡30min,制成10-1浓度的土壤悬浮液,将土壤悬浮液充分混匀,依次稀释制成10-2、10-310-4、10-5浓度的土壤悬浮液。然后分别取10-2、10-3、10-4、10-5浓度的土壤悬浮液各200μL,涂布于添加有0.1%K2Cr2O7的高氏1号培养基平板上,在28℃恒温培养箱中培养7天。挑单菌落于SFM固体培养基上纯化,得到灰红链霉菌Y1B(Streptomyces griseoruberY1B)。
上述各种培养基的组分分别为:
高氏1号培养基:可溶性淀粉20g,KNO3lg,K2HP040.5g,MgSO4·7H2O0.5g,FeSO4·7H2O0.0lg,NaCl0.5g,琼脂20g,蒸馏水定容至1000mL,pH7.2~7.4。
SFM培养基:取20g黄豆饼粉,加蒸馏水沸水煮30min,用纱布过滤,滤液中加甘露醇20g,固体培养基另加琼脂20g,蒸馏水定容至1000mL,pH7.2~7.3。
实施例2
生防菌株发酵粗产品的制备:用SFM固体培养基活化冷冻保藏的CCTCC M2013358菌株,然后将上述培养物接种到SFM液体培养基摇瓶中,28℃、180rpm条件下培养2天,作为种子液;再将种子液按2%的接种量接种到GYM培养基摇瓶中,28℃、180rpm条件下发酵7天。发酵结束后,将发酵液离心收集发酵上清液,用乙酸乙酯等体积萃取发酵上清液3次,合并收集有机相,40℃减压蒸馏有机相,浓缩蒸干后,得到发酵粗产品。
上述GYM培养基的组分为:葡萄糖4g,酵母提取物4g,麦芽提取物10g,蒸馏水定容至1000mL,pH7.2。
实施例3
生防菌株发酵粗产品对植物病原菌的抑制作用:称取适量粗产品溶于二甲基亚砜(DMSO),配制成1000mg/L的溶液,按一定比例加入融化好的PDA培养基中,混合后倒入灭菌的培养皿中,分别制成粗产品终浓度为50mg/L的PDA培养基平板;同时,分别将等体积的无菌水和DMSO溶剂添加到融化好的PDA培养基中,混合后倒入灭菌的培养皿中,制成空白对照平板和溶剂对照平板。用直径为8mm的打孔器打取水稻纹枯病菌和西瓜枯萎病菌的菌饼,接种到上述各平板中央,在28℃恒温培养箱中培养5天,用十字交叉法测定各平板菌落的生长直径,按以下公式计算相对抑菌率(初始接种的菌落直径为8mm):
Figure BDA0000425076240000041
结果表明,本发明所述的生防菌株发酵粗产品,对水稻纹枯病菌和西瓜枯萎病菌有较好的抑制作用,相对抑菌率分别达到47.7%和31.0%。
PDA培养基:取200g去皮马铃薯,切成小块后沸水煮30min,用纱布过滤,滤液中加葡萄糖20g,琼脂15g,蒸馏水定容至1000mL,自然pH值。
以上对本发明的具体实施例进行了描述。需要理解的是,本发明并不局限于上述特定实施方式,本领域技术人员可以在权利要求的范围内做出各种变形或修改,这并不影响本发明的实质内容。
Figure IDA0000425076340000021

Claims (10)

1.一株拈抗植物病原菌的生防菌株,其特征在于,所述菌株为灰红链霉菌Y1B(Streptomyces griseoruber Y1B)CCTCC NO:M2013358。
2.如权利要求1所述的拈抗植物病原菌的生防菌株,其特征在于,所述灰红链霉菌Y1B具有SEQ ID NO:1所示的16SrDNA序列。
3.一种如权利要求1所述的拈抗植物病原菌的生防菌株在制备防治植物枯萎病生物农药中的用途。
4.一种生防菌株发酵粗产品的制备方法,其特征在于,采用GYM培养基对如权利要求1所述的灰红链霉菌Y1B进行发酵,收集发酵液,萃取并减压蒸馏,即得所述发酵粗产品。
5.如权利要求4所述的生防菌株发酵粗产品的制备方法,其特征在于,每1000mLGYM培养基中含有葡萄糖3~5g、酵母提取物2~6g、麦芽提取物8~12g、余量为水,pH7.0~8.0。
6.如权利要求4或5所述的生防菌株发酵粗产品的制备方法,其特征在于,所述灰红链霉菌Y1B是通过如下步骤获得的:采集水稻土壤样品,土样碾碎后放置在烘箱烘焙,称取烘焙后的土样溶于无菌水中,充分振荡,制成不同浓度的土壤悬浮液,涂布于添加有0.05%~0.25%K2Cr2O7的高氏1号培养基平板上,待长出菌落后,挑单菌落于SFM固体培养基上纯化,即得灰红链霉菌Y1B(Streptomyces griseoruber Y1B)CCTCC NO:M2013358。
7.如权利要求6所述的生防菌株发酵粗产品的制备方法,其特征在于,每1000mL高氏1号培养基中含有可溶性淀粉10~30g、KNO30.8~1.2g、K2HPO40.3~0.6g、MgSO4·7H2O0.4~0.8g、FeSO4·7H2O0.008~0.012g、NaCl0.4~0.6g、琼脂18~24g、余量为水,pH7.0~8.0。
8.如权利要求6所述的生防菌株发酵粗产品的制备方法,其特征在于,每1000mL SFM培养基的制备包括如下步骤:取15~25g黄豆饼粉,加蒸馏水沸水煮20~40min,过滤黄豆饼粉液体,滤液中加甘露醇15~25g,固体培养基另加琼脂18~25g,蒸馏水定容至1000mL,pH7.0~8.0。
9.一种如权利要求4所述的制备方法制得的生防菌株发酵粗产品在制备防治植物枯萎病生物农药中的用途。
10.如权利要求9所述的用途,其特征在于,所述植物枯萎病为水稻枯萎病菌或西瓜枯萎病菌。
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