CN106676034B - 具有抗三七根腐病作用的细菌及其用途 - Google Patents
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Abstract
本发明公开了一种细菌Pseudomonas aeruginosa PAO1 strain PAO1,为铜绿假单胞菌PA01 Pseudomonas aeruginosa PAO1,其保藏号为CCTCC NO:M 2016575。本发明还同时公开了上述细菌Pseudomonas aeruginosa PAO1 strain PAO1的用途:预防三七根腐病的发生。
Description
技术领域
本发明涉及一种具有抗三七根腐病作用的细菌及其作用--有效的预防三七根腐病的发生。
背景技术
三七(学名:Panax notoginseng)为五加科人参属植物,在我国已有400多年的栽培历史,其作为一种名贵中药,具有化瘀止血、消肿止痛等功效,三七的药理作用主要体现在对血液系统、心血管系统、脑血管系统、神经系统、代谢、免疫调节系统等的影响。三七又名田七、滇三七、参三七、血参、田三七等。三七已有很多年的药用历史,其根、茎、叶、花均可入药,是复方丹参滴丸、云南白药、片仔癀、复方三七口服液等常见中药制剂的主要成分之一,目前以三七为配方进入《国家基本药物目录》和《国家中药保护品种目录》的制剂达20余种。作为我国传统的名贵药材,三七生物学特性方面的研究也已十分详实。
三七的市场需求正逐步增加,但就目前而言,三七的野生资源非常有限,而且采挖野生三七会破坏自然环境的生态平衡,三七资源远远不能满足人们需求;最主要的是在不同的地区三七药材的质量差别非常大。因此通过大棚内大批量种植三七就会解决这一系列的问题。但是,在大棚内大批量种植三七会受到根腐病的影响,根腐病在各个季节都有发生,其发病比较集中在8月份,它的危害常常导致三七产量、质量降低,严重者甚至全园绝收。
由于连年大面积单一种植,加之三七为多年生草本植物,性喜温暖阴湿,因此病害问题十分突出,特别是根腐病问题,目前已成为限制三七种植业发展的严重障碍,根腐病常年发病率一般在5%~20%,严重的可损失70%以上。对该病的防治目前主要依靠轮作倒茬和化学药剂灌根。但轮作年限一般要求8年以上,生产上难以大面积推行。药剂灌根虽有效,但农药残留问题严重。对于用细菌来作用于三七根腐病的防治研究并没有相关报道。
发明内容
本发明要解决的技术问题是提供一种细菌--Pseudomonas aeruginosaPAO1strain PAO1,该菌株可预防三七根腐病的发生。
为了解决上述技术问题,本发明提供一种细菌Pseudomonas aeruginosaPAO1strain PAO1,为铜绿假单胞菌PA01Pseudomonas aeruginosa PAO1,其保藏号为CCTCCNO:M 2016575。
本发明还同时提供了上述细菌Pseudomonas aeruginosa PAO1strain PAO1的用途:预防三七根腐病的发生。
该菌株Pseudomonas aeruginosa PAO1strain PAO1是从半夏植株中采用平板稀释法分离得到一株细菌,经鉴定其对由病原菌G3B引起的三七根腐病有拮抗作用。
具体分离方法为:
1)、将半夏经表面消毒处理后,加入无菌水进行研磨稀释,涂布于营养琼脂上,于28~32℃培养1~3天;
营养琼脂配方:蛋白胨10g,牛肉膏3g,NaCl 5g,琼脂18g和蒸馏水1000mL,pH=7.2~7.4。
2)、挑取单菌落于相同的营养琼脂平板划线纯化,于28~32℃培养1~3天;
3)、重复步骤2),直至获得纯培养物。
具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此。
实施例1、拮抗菌(Pseudomonas aeruginosa PAO1strain PAO1)的分离培养方法,依次进行以下步骤:
1、采集浙江省的半夏植株,洗净后25KHz超声波处理5分钟;
2、在无菌条件下,对步骤1所得的供试材料用灭菌去离子水冲洗3次,并依次用75%(v/v)的酒精和3%(m/V)次氯酸钠溶液表面消毒;
3、在无菌环境中,继续将步骤2所得供试材料用无菌水冲洗3次,取100μl最后1次冲洗的无菌水冲洗液涂布接种于营养琼脂上,30℃培养24h后,进行无菌验证。
如无菌落产生,则继续后续的步骤4;如有菌落产生,则返回步骤2继续表面消毒处理。
4、在超净台中,取处理好的半夏样品1~2g,在无菌研钵中充分研磨,并加入5mL无菌水,混匀,静置15min,取100μl上清稀释涂布于营养琼脂培养基中,置于28~32℃(较佳30℃)培养1~3天;
5、挑取步骤4中所产的单菌种于相同培养基划线纯化分离,置于28~32℃(较佳30℃)培养1~3天,继续挑取单菌落;
重复上述操作直至得到纯培养为止。
实施例2、拮抗菌(Pseudomonas aeruginosa PAO1strain PAO1)的初筛,依次进行以下步骤:
1、拮抗菌的初筛在平板上进行,采用平板对峙培养方法进行筛选:用打孔器沿着病原真菌菌丝生长旺盛的地方打孔获得菌碟。
2、将植物病原菌G3B的菌碟接种到PDA培养皿的中央,在其四周与植物病原菌G3B等距约3cm的四个点上,粘上直径为7mm的滤纸四片,每片滤纸上点接上10μl的供试菌(浓度约为108cfu/ml)。所述供试菌为实施例1中分离纯化出来的所有细菌。
上述植物病原菌G3B来源于CCTCC,其保藏号为:CCTCC AF 2016007。
3、设置只接种病原菌的培养皿为空白对照组,每个处理三个重复,然后置于22℃的培养箱中培养7-10d,观察和测量植物病原真菌的菌落直径。
实施例3、拮抗菌(Pseudomonas aeruginosa PAO1strain PAO1)的复筛,根据初筛结果,将筛选出来的对病原菌G3B具有较强拮抗效果的细菌作为供试菌株,进一步进行复筛,依次进行以下步骤:
1、拮抗菌的初筛在平板上进行,采用平板对峙培养方法进行筛选:用打孔器沿着病原真菌菌丝生长旺盛的地方打孔获得菌碟。
2、将植物病原菌的菌碟接种到PDA培养皿的中央,在其四周与植物病原菌等距约3cm的四个点上,粘上直径为7mm的滤纸四片,每片滤纸上点接上10μl的供试菌(浓度约为108cfu/ml)。
3、设置只接种生防菌或者只接种病原菌的培养皿为空白对照组,每个处理三个重复,然后置于22℃的培养箱中培养7-10d,观察和测量植物病原真菌的菌落直径。
4、测量抑菌条带并按下列公式计算抑制率:菌丝生长抑制率(%)=(单独培养菌落半径-对峙培养菌落半径)/单独培养菌落半径×100。所得的结果如下表1所示。
表1、Pseudomonas aeruginosa PAO1strain PAO1菌丝生长抑制率
将上述复筛所得的菌株Pseudomonas aeruginosa PAO1strain PAO1进行保藏,保藏信息具体如下:
保藏单位:中国典型培养物保藏中心(CCTCC),保藏地址:中国武汉武汉大学;保藏名称:铜绿假单胞菌PA01Pseudomonas aeruginosa PAO1,保藏号为CCTCC NO:M 2016575,保藏日期2016.10.18。
实施例4、拮抗菌(Pseudomonas aeruginosa PAO1strain PAO1,即保藏号为CCTCCNO:M 2016575的铜绿假单胞菌PA01Pseudomonas aeruginosa PAO1)的三七切片复筛,依次进行以下步骤:
1、菌液制备:①病原真菌G3B菌液制备:将从三七病根分离的病原真菌G3B,在PDA固体斜面培养基上活化,22℃恒温培养箱培养7~14d,用无菌ddH2O洗下孢子并制成107cfu/mL病原菌悬浮液。
②接抗菌液制备:用无菌水将培养24h的斜面生长(培养条件为30℃恒温培养箱培养2d)的拮抗细菌洗下,制成拮抗菌悬液;拮抗菌悬液中拮抗菌浓度为108cfu/mL。
2、切片制备:将健康的三七根用流水冲洗30min;70%酒精浸泡20s;3%次氯酸钠溶液浸泡3min;用无菌ddH2O冲洗5次;用无菌手术刀片将表面消毒的根切成每片5mm厚的薄片,置于含有灭菌滤纸的培养皿中,培养皿中添加1mL无菌水保湿。
3、接种:拮抗菌采用菌液浸泡的方式接种(即,放入5ml拮抗菌悬液中浸泡),浸泡时间分别为20min,病原菌采用点接的方式接种,每个薄片的接种量为20ul。先接拮抗菌再接病原菌,间隔时间为24h。
4、处理方式:
设置①CK0(接无菌水)、②CK1(接病原菌G3B)、③接拮抗菌(Pseudomonasaeruginosa PAO1strain PAO1)、④接拮抗菌(Pseudomonas aeruginosa PAO1strainPAO1)+病原菌G3B四个处理。每个处理三个重复。
5、培养:22℃的培养箱内培养,接种7d后调查发病情况,统计病情指数,计算防治效果。所得的结果如下表2所示。
表2、拮抗菌(Pseudomonas aeruginosa PAO1strain PAO1)对不同离体材料根腐病的抑制效果
实施例5、拮抗菌(Pseudomonas aeruginosa PAO1strain PAO1,即保藏号为CCTCCNO:M 2016575的铜绿假单胞菌PA01Pseudomonas aeruginosa PAO1)的盆栽试验,依次进行以下步骤:
1、在云南文山制备三七盆栽植株,每盆3株;将拮抗菌液(108cfu/ml)接种于三七植株根基部,5mL/株;
2、移栽24h后,采用灌根法,将病原菌的孢子液分别加入已移栽好的三七盆栽植株的根部,根部事先适当给予针刺。接种病原菌液(107cfu/ml),5mL/株。
3、设置①CK0(接无菌水)、②CK1(接病原菌G3B)、③接拮抗菌(Pseudomonasaeruginosa PAO1strain PAO1)、④接拮抗菌(Pseudomonas aeruginosa PAO1strainPAO1)+病原菌G3B四个处理,各处理重复3次,置于室外大棚基地培养,7d后观察、记录发病情况。所得的结果如下表3所示。
表3、内生拮抗细菌的温室盆栽防治效果
对比实验、将以下的菌株A~菌株D替代拮抗菌(Pseudomonas aeruginosaPAO1strain PAO1)按照实施例5所述方法进行+病原菌G3B的实验(菌液浓度和用量均等同于实施例5),所得结果如下表4:
表4、不同菌株的温室盆栽防治效果
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
Claims (2)
1.一种铜绿假单胞菌(Pseudomonas aeruginosa )PAO1,其特征是:保藏号为CCTCCNO:M 2016575。
2.如权利要求1所述的菌株的用途,其特征是:预防保藏号为CCTCC AF 2016007的病原菌G3B引起的三七根腐病的发生。
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