CN108179125B - 赤杆菌h7及其应用 - Google Patents
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Abstract
赤杆菌H7(Erythrobacter citreus H7)及其应用,该菌株已在国家知识产权局指定的保藏单位保藏,保藏日期为2017年11月06日,保藏单位名称:中国典型培养物保藏中心,保藏编号:CCTCC NO:M2017660。该菌株能有效抑制尖孢镰刀菌的生长,且对盐有较好的耐受性。
Description
技术领域
本发明属于微生物技术领域,尤其涉及一种赤杆菌H7(Erythrobacter citreusH7)及其应用。
背景技术
尖孢镰刀菌(Fusarium oxysporum)是镰刀菌属(Fusarium)真菌的一个重要种,可侵染多种植物,并引起植物维管束萎蔫病害,严重可造成植株枯死,且侵染在植株的全生育期均可发生。目前世界范围内已报道的被严重危害的经济作物种类繁多,常见的黄瓜枯萎病在我国各地均有发生,对农业生产造成了严重威胁。
针对黄瓜枯萎病的防治,人们通过对瓜苗实施高温闷棚、种子消毒、轮作,加强田间管理等农技方面的措施,能降低枯萎病的发病率,但这些方法始终存在耗费人力物力的弊端;另外,化学药剂能有效杀死致病菌的同时在一定程度上可破坏生态环境,污染土壤和水体等自然环境。因此,很多学者都把注意力转向了利用有益微生物防治各种作物的病害。微生物繁殖速度快,生命力强,应用性广,大量研究结果表明,很多细菌都能产生拮抗物质,并且一种细菌菌株可能对多个病原菌产生拮抗作用;从经济和社会的实际情况着眼:细菌菌株发酵以及生产工艺简单且成本较低,是理想的生防微生物。因此,宜于作为拮抗菌进而大规模的发酵制成生物肥料。
近年来,生物防治被各国广泛采用,我国目前也已筛选出了较多对土传病害有良好防效的生防菌株,国外学者利用枯草芽孢杆菌防治丝核菌、腐霉菌、镰刀霉菌引起的病害方面,也取得了很好的效果。目前,虽有大量尖孢镰刀菌拮抗菌筛选方面的报道,但是分离获得的抗病菌株大多是从患病菌株根际土壤获得,随着研究深度的日益加深,新型抗病成分的开发面临严峻的挑战。同时,耐药现象的持续出现使得寻找新型抗菌活性物质变得更加迫切。
海洋微生物因高压、高盐、低营养及低温等生长环境,造就了其独特的代谢途径,能够产生完全不同于陆地微生物的新颖的生物活性物质,Tareq等报道了从海洋芽孢杆菌(Bacillus subtilis)中分离出的3种新型线性脂肽gageostatins A~C,研究表明,该化合物对尖孢炭疽病菌、水稻纹枯病菌及番茄灰霉病菌等病原性真菌显示出良好的抑菌活性。自弗莱明发现青霉素以来,人们已从微生物中发现了一万多种生物活性物质,其中八千多种具有抗菌活性,而且越来越多的研究表明许多具有开发前景的生物活性物质是由海洋微生物产生的,因此海洋微生物资源已成为国内外研究的热点。
发明内容
解决的技术问题:针对目前土传病害防治过程中农药残留超标,对农产品品质、食品安全、人类健康乃至生态平衡都造成了严重影响的现状,本发明提供一种赤杆菌H7及其应用,它能有效的抑制尖孢镰刀菌的生长。
技术方案:一株赤杆菌(Erythrobacter citreus)H7,该菌株已在国家知识产权局指定的保藏单位保藏,保藏日期为2017年11月06日,保藏单位名称:中国典型培养物保藏中心,保藏编号:CCTCC NO:M2017660。
上述赤杆菌H7的菌落形态为黄色,不透明、圆形、表面光滑,中心凸出、边缘整齐;严格需氧,革兰氏阴性杆菌,过氧化氢酶阳性,氧化酶阴性,MR、VP阴性,最适生长pH为6.8~7.2,最适生长温度为28~30℃。
上述赤杆菌(Erythrobacter citreus)H7在抑制尖孢镰刀菌中的应用。该菌株能够有效抑制病原菌菌丝的正常生长,造成其菌丝畸变,末端分枝增多,膨大、肿胀现象严重。
上述赤杆菌(Erythrobacter citreus)H7的发酵培养基,组成为:蛋白胨10g/L,酵母粉2.5g/L,NaCl 30g/L,琼脂15g/L,蒸馏水1L,pH 7.0。
有益效果:本发明的赤杆菌H7,是一种分离的全新的菌株,该菌株能有效抑制尖孢镰刀菌的生长,且对盐有较好的耐受性。查阅有关资料,尚无有关赤杆菌属有抑制其他微生物生长的报道。本发明为尖孢镰刀菌植物病害的防治提供了有用菌源,拓宽了赤杆菌功能方面的应用,具有较强的应用价值。
附图说明
图1为菌落形态图;
图2为菌株显微形态图;
图3为菌株电泳图谱;
图4为菌株系统发育树;
图5为菌株对植物致病菌的抑菌效果;A-A’组马铃薯炭疽病菌;B-B’组草莓尖孢镰刀菌;C-C’组黄瓜枯萎病原菌;D-D’组小麦禾谷镰刀菌;E-E’组水稻立枯丝核菌;F-F’组板栗围小丛壳菌。
具体实施方式
以下实施例进一步说明本发明的内容,但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改和替换,均属于本发明的范围。
若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1:赤杆菌H7的筛选分离、纯化鉴定
(一)材料准备
1、采样:
海洋沉积样品取自江苏省连云港市东西连岛。
2、培养基:
筛选培养基:Zobell 2216E(蛋白胨5g/L,酵母粉1g/L,FeSO4·7H2O 0.1g/L,琼脂15g/L,陈海水1L,pH 7.0)
发酵培养基:优化2216E(蛋白胨10g/L,酵母粉2.5g/L,NaCl 30g/L,琼脂15g/L,蒸馏水1L,pH 7.0)
3、实验仪器和设备
赛福智能生化培养箱、恒温振荡器(IS-RDV1)、立式压力蒸汽灭菌器、光学显微镜、pH计(sarporiusPD-10)、超净工作台、PTC200型PCR仪、电泳仪及电泳槽、UVP凝胶紫外观测仪。
(二)菌株的筛选分离与纯化
1、筛选分离:
称取1g海泥样品,用灭菌水10梯度系列稀释之后用无菌移液管吸取100μL涂布在Zobell2216E混菌平板上,28℃恒温培养3天,挑取有明显抑菌圈的菌落准备纯化;
2、纯化:
将有明显抑菌圈的菌落在Zobell 2216E固体培养基上反复划线培养三次直至获得单一菌株,并于Zobell 2216E斜面培养基上保存。
(三)菌落形态特征及生理生化特征
菌株赤杆菌H7(E.citreus H7)培养48h后的菌落形态:菌落呈黄色、圆形、不透明、表面光滑、中心凸出、边缘整齐(图1),在显微镜下呈杆状(图2);严格需氧,革兰氏阴性,甲基红(M.R.)、乙酰甲基甲醇(V.P.)阴性,菌株能产过氧化氢酶,产硫化氢和硝酸盐还原反应呈阳性,最适生长pH为6.8~7.2,最适生长温度为28~30℃。菌株E.citreus H7的理化指标反应特性和《常见细菌系统鉴定手册》中描述的赤杆菌理化特性基本一致,因此,菌株E.citreus H7可以被初步鉴定为赤杆菌。
表1菌株E.citreus H7的生理生化特性分析
(四)菌株分子鉴定
细菌基因组DNA的提取和PCR产物的回收采用OMEGA的Bacterial DNA kit(50)试剂盒,按说明书操作。16S rRNA序列的PCR扩增采用通用引物对(正向引物:5’-AGAGTTTGATCATGGCTCAG-3’,反向引物:5’-GGTACCTTGTTACGACTT-3’)。PCR反应体系(50μL):10×PCR缓冲液0.5μL,dNTPs(20mmol/L)2.5μL,MgCL2(25mmol/L)4μL,引物(20μmol/L)各1μL,DNA模板0.5μL,Taq酶(175U/μL)0.5μL,双蒸水40μL。扩增程序为:94℃预变性5min,94℃变性1min,55℃退火1min,72℃延伸1.5min,循环30次,72℃后延伸5min。扩增后吸取2μLPCR反应液,于1%琼脂糖凝胶电泳中进行电泳分析检测,引物合成和测序由华大基因完成。
菌株16S rRNA扩增图见图3,经测序,H7菌株的16S rRNA序列全长为1338bp,与目标条带大小相符,采用BLAST分析法对菌株H7的16SrRNA基因序列与GenBank数据库比较分析发现,菌株H7与赤杆菌属的同源性最高。构建系统发育树(图4),结果表明,菌株H7与模式菌株Erythrobacter citreus RE35F/1(T)聚类在同一分支,遗传距离最近,结合形态学及生理生化特征对H7菌株的鉴定结果为,该分离菌株应属赤杆菌。
同时,该菌株与三株模式菌株(CGMCC1.8703CGMCC1.8708CGMCC1.7715)的DNA-DNA杂交同源性均低于70%,因此该菌株为新菌株。综合以上鉴定结果,菌株H7命名为Erythrobacter citreus H7。该菌株已于2017年11月06日送交位于湖北省武汉市武昌区八一路299号武汉大学校内的中国典型培养物保藏中心(简称CCTCC)保藏,保藏号为CCTCCNO.M2017660。
实施例2:赤杆菌H7的应用。
(一)材料准备
1、供试病原菌:黄瓜尖孢镰刀菌cfcc 82185(Fusarium oxysporumf.sp.Cucumerinum)由南京农业大学海洋生物学重点实验室提供。尖孢镰刀菌以菌核的形式保藏在-70℃冰箱中。
2、供试拮抗菌:本发明提供的赤杆菌H7(Erythrobacter citreus H7)。
3、培养基:真菌传代培养采用马铃薯葡萄糖琼脂(PDA)培养基;细菌发酵培养采用优化后的2216E培养基。
4、实验仪器和设备
赛福智能生化培养箱、恒温振荡器(IS-RDV1)、立式压力蒸汽灭菌器、超净工作台。
(二)赤杆菌H7发酵液的制备
1、菌悬液的制备
将保存在2216E斜面培养基上的赤杆菌H7于培养箱中培养活化24h,备用。将活化后的供试斜面培养基用10mL无菌生理盐水稀释制成含菌量为106~108cfu/mL的菌悬液。
2、发酵液的制备
取上述菌悬液200μL加到事先准备好的2216E液体培养基中,180r/min,30℃培养2d。
(三)平板抑菌试验
取菌株H7发酵液200μL涂布在PDA培养基表面,对照组分别在培养基中加入等量的无菌水。真菌在PDA平板上培养5d,自菌落边缘用直径5mm的灭菌打孔器,取圆形的病原菌菌苔,移接到涂有拮抗菌的PDA平板中央,30℃培养3d,直到对照组的病原菌长满平板时,用十字交叉法分别测量对照组平板和供试组平板的真菌菌落直径,计算抑菌率。
供试菌抑菌率=(对照平板真菌直径-供试平板真菌直径)/(对照平板真菌直径)×100%
从结果中(表2、图5)可以看出,菌株对不同植物病原菌表现出不同的抑菌活性,菌株对草莓尖孢镰刀、黄瓜枯萎病菌、水稻立枯丝核菌有较为显著的抑活性,抑菌率超过了70%;对马铃薯炭疽病菌和小麦禾谷镰刀菌的抑菌效果不明显,抑菌率只有30%左右。
表2菌株E.citreus H7对不同植物致病菌的抑菌活性
注:字母a,b,c,d表示经Duncan氏新复极差法检验差异显著(p<0.05)。
序列表
<110> 中国科学院南京土壤研究所
<120> 赤杆菌H7及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
agagtttgat catggctcag 20
<210> 2
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ggtaccttgt tacgactt 18
Claims (2)
1.一株菌,该菌株已在国家知识产权局指定的保藏单位保藏,保藏日期为2017年11月06日,保藏单位名称:中国典型培养物保藏中心,保藏编号:CCTCC NO:M2017660。
2.权利要求1所述菌在制备抑制尖孢镰刀菌试剂中的应用。
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