CN110317739B - 一株仙人掌有孢汉逊酵母菌株及其分离方法和应用 - Google Patents
一株仙人掌有孢汉逊酵母菌株及其分离方法和应用 Download PDFInfo
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Abstract
本发明公开了一株仙人掌有孢汉逊酵母菌株,该菌株于2019年6月24日保藏在中国典型培养物保藏中心,保藏号为CCTCC NO:M 2019482。采用本发明所提供的一株仙人掌有孢汉逊酵母菌,对番木瓜炭疽病病原菌具有良好的拮抗作用,可用于番木瓜炭疽病病害的防治,且该菌株易于培养,容易保存,便于工业化生产。
Description
技术领域
本发明涉及微生物病害防治领域,尤其涉及一株仙人掌有孢汉逊酵母菌株JM-1及其分离方法和应用。
背景技术
在果实成熟采摘及贮藏期间,由真菌引起的果实腐烂尤为严重。由于国内采后保鲜贮藏技术尚未成熟,全国番木瓜采后损失高达40%-93%,番木瓜炭疽病已成为番木瓜采前落果和采后烂果现象严重的主要病害之一。目前在番木瓜炭疽病的防治方面多采用化学杀菌剂,如特克多、多菌灵、苯来特和甲基托布津等。然而长期采用化学杀菌剂使某些病原菌产生抗药性,并且由此引起的食品安全性及环境污染等问题日益严峻。
多年来,利用拮抗微生物来防治果蔬病害一直是研究热点,随着生防菌抑病机制的揭示,酵母菌越来越受到研究者的青睐,成为防治果蔬采后病害的主要拮抗菌。拮抗酵母具有对病原菌拮抗效果好、不产生毒素、繁殖能力强、可与化学物质配合使用等多种优势,在控制果蔬采后腐烂的应用上有较大的应用潜力。以拮抗酵母菌制成的生物制剂取代化学菌剂也将成为果蔬保鲜绿色环保防治措施的必然趋势,目前报道常用的拮抗酵母有膜醭毕赤酵母(Pichia membranefaciens)、罗伦隐球酵母(Cryptococcus laurentii)、海洋红酵母(Rhodosporidium paludigenum)、美极梅奇酵母(Metschnikowia pulcherrima)、柠檬形克勒克酵母(Kloeckera apiculata)等,大多数主要用于防治果蔬采后贮藏时期的青霉病、绿霉病、灰霉病及其它腐烂病害。但对于采后番木瓜炭疽菌(Colletotrichum)病害的防治研究鲜有报道,尤其是还未有从大自然中成功分离出对番木瓜炭疽菌具有良好拮抗作用的仙人掌有孢汉逊酵母(Hanseniaspora opuntiae)的研究报道。
发明内容
有鉴于此,本发明提供了一株仙人掌有孢汉逊酵母菌株,该菌株于2019年6月24日保藏在中国典型培养物保藏中心,保藏号为CCTCC NO:M 2019482。
本发明还提供了一株仙人掌有孢汉逊酵母菌株在防治番木瓜炭疽菌病害的应用。
本发明还提供了一株仙人掌有孢汉逊酵母菌株的筛选方法,包括以下步骤:分别切取5g番木瓜果皮,加入90mL YPD培养液中,于28℃120r/min下摇床振荡2~3h,采用平板稀释法将培养液分别稀释至10-3、10-4、10-5和10-6,取100μL稀释培养液涂布于YPDA固体培养基上并在28℃恒温培养箱培养48h,挑取形态各异的单菌落进行平板划线分离,直至获得单一菌落的拮抗菌菌饼;以培养5d的番木瓜炭疽病病原菌为靶标,通过平板对峙法对拮抗菌进行初筛,用直径为5mm的无菌打孔器分别取番木瓜炭疽病病原菌菌饼和拮抗菌菌饼,将其放置于PDA培养基平板上,两菌饼间距约30mm,平板于28℃恒温培养箱培养5d,初步筛选有抑菌带的拮抗菌为初筛菌株;采用滤纸片法对初筛菌株进行复筛,首先将初筛拮抗菌用无菌水制备成浓度为1×108CFU/mL的菌悬液,将番木瓜炭疽病病原菌菌饼放置于PDA培养基平板中央,然后取4片无菌滤纸片浸泡初筛拮抗菌悬液后,分别放置于距病原菌菌饼约30mm处,平板于28℃恒温培养箱培养5d,通过测量抑菌带半径为11.33mm的拮抗菌为仙人掌有孢汉逊酵母菌株。
优选地,YPD培养液由酵母浸出粉10g,蛋白胨10g,葡萄糖20g和1000mL无菌蒸馏水制得;YPDA固体培养基包括酵母浸出粉10g,蛋白胨10g,葡萄糖20g,琼脂20g和1000mL无菌蒸馏水制得;PDA培养基由新鲜去皮马铃薯200g,葡萄糖20g,琼脂20g和1000mL无菌蒸馏水于121℃灭菌20min后制得。
采用活体抑菌验证试验说明一株仙人掌有孢汉逊酵母菌JM-1可有效抑制炭疽病致病菌在番木瓜果实伤口处的生长。离体抑菌实验表明,当菌悬液浓度达到1×108CFU/mL及以上时,即可显著降低番木瓜炭疽病病原菌孢子的萌发(P<0.05)。酵母菌JM-1对番木瓜果实的防效应用试验分为果实刺伤和浸泡两种方式,结果均表明酵母菌JM-1处理后有效的降低了番木瓜果实的发病率,延长了番木瓜的贮藏时间。综上,本发明的仙人掌有孢汉逊酵母菌株在低浓度下(1×109CFU/mL)对番木瓜炭疽病病原菌具有良好的抑制作用,灵敏性高,可用于番木瓜炭疽病病害的防治,且该菌株易于培养,容易保存,便于工业化生产。
附图说明
图1为本发明的仙人掌有孢汉逊酵母菌JM-1的菌落形态及细胞形态图;
图2为本发明的仙人掌有孢汉逊酵母菌JM-1的26S rDNA电泳图。
图3为实施例一的仙人掌有孢汉逊酵母菌JM-1在分离筛选培养中的抑菌效果图,其中右图为第5天时的抑菌效果图,左图为未浸泡初筛拮抗菌悬液的空白对比实验图;
图4为实施例三的仙人掌有孢汉逊酵母菌JM-1对番木瓜炭疽病病原菌抑菌效果,其中A组:无菌水;B组:无菌水+病原菌孢子悬液;C组:无菌水+酵母菌悬液;D组:酵母菌悬液+病原菌孢子悬液;
图5为实施例五的不同浓度仙人掌有孢汉逊酵母菌悬浮液对番木瓜果实伤口处防效应用试验;
图6为实施例六的不同浓度仙人掌有孢汉逊酵母菌悬浮液对番木瓜果实自然发病情况的影响。
具体实施方式
以下对本发明的原理和特征进行描述,所举实施例只用于解释本发明,并非用于限定本发明的范围。
先特制培养基如下:
酵母浸出粉胨葡萄糖琼脂(Yeast Extract Peptone Dextrose Agar,YPDA)培养基具体为:酵母浸出粉10g,蛋白胨10g,葡萄糖20g,琼脂20g,1000mL无菌蒸馏水。
酵母浸出粉胨葡萄糖(Yeast Extract Peptone Dextrose,YPD)培养液:酵母浸出粉10g,蛋白胨10g,葡萄糖20g,1000mL无菌蒸馏水。
马铃薯葡萄糖琼脂(potato dextrose ager,PDA)培养基:新鲜去皮马铃薯200g,葡萄糖20g,琼脂20g,1000mL无菌蒸馏水。于121℃灭菌20min后待用。
材料:从海南省海口市南北水果市场选购7~8成熟“日升”番木瓜,挑选健康、大小一致果实作为抑菌试验材料。
实施例一:一株仙人掌有孢汉逊酵母菌株JM-1的分离筛选
从海南番木瓜种植地采集番木瓜果实作为试验样品,用无菌小刀分别切取5g番木瓜果皮,加入90mLYPD培养液中,于28℃120r/min下摇床振荡2~3h,采用平板稀释法,将培养液稀释至10-3、10-4、10-5和10-6,取100μL稀释培养液涂布于YPDA固体培养基上并在28℃恒温培养箱培养48h,挑取形态各异的单菌落进行平板划线分离,直至获得单一菌落的拮抗菌菌饼,于4℃冰箱保藏备用。
以培养5d的番木瓜炭疽病病原菌为靶标,通过平板对峙法对拮抗菌进行初筛,用直径为5mm的无菌打孔器分别取番木瓜炭疽病病原菌菌饼和纯化后的拮抗菌菌饼,将其放置于PDA培养基平板上,两菌饼间距约30mm,平板于28℃恒温培养箱培养5d,初步筛选有明显抑菌带的拮抗菌为初筛菌株;
采用滤纸片法对初筛菌株进行复筛,首先将初筛拮抗菌用无菌水制备成浓度为1×108CFU/mL的菌悬液,将番木瓜炭疽病病原菌菌饼放置于PDA培养基平板中央,然后取4片无菌滤纸片浸泡初筛拮抗菌悬液后(不经过浸泡初筛拮抗菌悬液作为空白对比实验),分别放置于距病原菌菌饼约30mm处,平板于28℃恒温培养箱培养5d,抑菌效果和空白对比结果如图3,将抑菌带半径为11.33mm的菌株并进行保存。
实施例二:一株仙人掌有孢汉逊酵母菌株JM-1的鉴定
1、首先通过光学显微镜观察拮抗菌形态特征,如图1,实施例一的拮抗菌株JM-1在YPDA培养基上菌落呈乳白色、圆形,表面光滑、湿润、有光泽,边缘整齐,质地粘稠,有芳香味气息。通过显微镜观察菌体细胞呈柠檬形或椭圆形,不形成假菌丝和掷孢子,菌体两端芽殖。
2、其次参照《酵母菌的特征与鉴定手册》进行生理生化试验,实施例一的拮抗菌株JM-1JM-1可发酵葡萄糖、棉子糖和果糖;对于碳源同化测定,JM-1可同化葡萄糖、蔗糖、棉子糖、麦芽糖、可溶性淀粉和果糖,不能同化L-鼠李糖、D半乳糖、乳糖和柠檬酸。氮源同化试验中不能同化硫酸铵和硝酸钾。其他生理生化实验测试结果表明酵母菌JM-1可产生酯类物质,但不能产酸以及类淀粉化合物,结果如表1。
表1酵母菌JM-1生理生化测试结果
注:“+”表示阳性,“-”表示阴性
3、酵母菌菌株JM-1经26S rDNA扩增结果如图2所示,获得的序列长度约587bp,并由生工生物工程(上海)股份有限公司对拮抗菌进行测序,其DNA序列表如SEQ ID No.1所示。
结合形态学和生理生化结果对拮抗菌进行菌株分类学鉴定,将实施例一的拮抗酵母菌菌株JM-1命名为仙人掌有孢汉逊酵母菌株(Hanseniaspora opuntiae)JM-1,该菌株于2019年6月24日保藏在中国典型培养物保藏中心,保藏号为CCTCC NO:M 2019482,保藏地址为湖北省武汉市武昌珞珈山武汉大学。
实施例三:仙人掌有孢汉逊酵母菌JM-1对番木瓜炭疽病病原菌抑菌试验
选取健康、大小一致的番木瓜用1%(v/v)的次氯酸钠消毒1min后用自来水冲净、晾干。用打孔器(直径为10mm)在番木瓜表面刺2个1mm深的伤口,将果实分为4组:A组:只接种50μL无菌水,作为对照;B组:接种25μL无菌水和25μL浓度为1×107CFU/mL病原菌孢子悬液;C组:接种25μL无菌水和接种25μL浓度为1×108CFU/mL酵母菌悬液;D组:接种25μL浓度为1×108CFU/mL酵母菌悬液和25μL浓度为1×107CFU/mL病原菌孢子悬液。将处理后的3组果实放置于25℃、相对湿度为90%的恒温恒湿培养箱中,观察并记录果实的病斑直径和发病率,结果如表2和图3所示,未接种炭疽病病原菌孢子悬液的A组和C组与接种病原菌孢子悬液的B组和D组间存在显著差异,且C组发病率明显低于A组,说明酵母菌JM-1对果实无致病性,与B组相比较,D组的病斑直径明显降低了15.71mm,发病率也显著低于B组,说明本发明仙人掌有孢汉逊酵母菌株JM-1可明显抑制炭疽病致病菌在番木瓜果实伤口处的生长。
表2果实伤口处的病斑直径和发病率
实施例四:不同浓度的仙人掌有孢汉逊酵母菌悬浮液对番木瓜炭疽病病原菌抑菌试验
在10mL离心管中加入100μL用无菌蒸馏水配置的107CFU/mL病原菌孢子悬浮液,再分别加入等量浓度为1×106、1×107、1×108和1×109CFU/mL酵母菌悬液,以加入等量无菌水作为对照(CK)组,于28℃、100r/min振荡培养,分别在4h和6h后通过显微镜下观察孢子萌发情况,一般孢子芽管长度为孢子长度的1.5倍时视为萌发。结果如表3所示,与对照组相比,酵母菌JM-1对病原菌孢子的萌发具有一定的抑制作用。处理4h后,4个浓度酵母菌悬液对病原菌孢子的相对抑制率为100%,6h后,4个浓度酵母菌悬液处理的孢子萌发率分别为CK组的0.76、0.79、0.06和0.05倍,当酵母菌悬液浓度为1×108和1×109CFU/mL时,对孢子萌发抑制效果最强,抑制率显著大于其他浓度(P<0.05)。
表3不同浓度酵母菌JM-1对病原菌孢子萌发的影响
实施例五:不同浓度的仙人掌有孢汉逊酵母菌悬浮液对番木瓜果实伤口处防效应用试验
选取健康、大小一致的番木瓜用1%(v/v)的次氯酸钠消毒1min后用自来水冲净、晾干。用打孔器(直径为10mm)在番木瓜表面刺2个1mm深的伤口,试验分为5组,首先分别接种25μL 1×106、1×107、1×108和1×109CFU/mL酵母菌悬液,以加入等量无菌水作为对照(CK)组,1h后所有果实再接种25μL由无菌水配置的1×107CFU/mL番木瓜炭疽病病原菌孢子悬液。所有处理果实室温晾干后,放置于25℃、相对湿度90%的培养箱中贮藏,第7d时记录发病情况。
发病率和相对防治效果计算公式如下:
发病率(%)=发病刺伤伤口数/总刺伤伤口数;
相对防治效果(%)=(对照组发病率-处理组发病率)×100/对照组发病率
结果如表4和图5所示。与对照组相比,其他处理组番木瓜果实伤口处发病率明显降低,各组发病率分别为对照组的0.78、0.92、0.50和0.43倍。除酵母菌悬液浓度为1×107CFU/mL处理组的相对防效较低以外,其它浓度处理组防治效果较好,其中浓度为1×106CFU/mL处理组的相对防效为21.60%,浓度为1×108和1×109CFU/mL酵母菌悬液防治效果达50%以上,相对防效分别为50.00%和56.82%。
表4不同浓度酵母菌JM-1在果实伤口处对炭疽病病原菌的抑制效果
实施例六:不同浓度的仙人掌有孢汉逊酵母菌悬浮液对番木瓜果实自然发病情况的影响
选取健康、大小一致的番木瓜用1%(v/v)的次氯酸钠消毒1min后用自来水冲净、晾干。将果实分为5组,分别用浓度为1×106、1×107、1×108和1×109CFU/mL酵母菌悬液浸泡果实15min,以清水浸泡作为对照(CK)。所有处理果实室温晾干后,放置于25℃、相对湿度90%的培养箱中贮藏,第12d时记录发病情况。根据果实表面腐烂面积的大小进行分级,0级(无病变),1级(病变面积<1/10),2级(病变区域1/10至1/5),3级(病变区域1/5至1/2),4级(病变区域>1/2)。病情指数和相对防治效果计算公式如下:
病情指数(DI)=∑(各级病果×该病级)×100/(总调查数×最高病情级数);
相对防治效果(%)=(CK病情指数-处理组病情指数)×100/CK病情指数
防治结果如表5和图6所示,对照组的病情指数72.20%,显著大于其他处理组,且用相对较高浓度的酵母菌悬液浸泡果实,所对应的病情指数相对较低。4个浓度的酵母菌悬液浸泡果实,相对防治效果分别为15.37%、19.25%、46.12%和38.50%,在浓度为1×108和1×109CFU/mL酵母菌悬液浸泡下果实相对防治效果相对较好。
表5不同浓度酵母菌JM-1对果实自然发病的影响
综上所述,采用本发明所提供的一株仙人掌有孢汉逊酵母菌,对番木瓜炭疽病病原菌具有良好的抑制作用,可用于番木瓜炭疽病病害的防治,且该菌株易于培养,容易保存,便于工业化生产。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明保护的范围之内。
序列表
<110> 海南大学
<120> 一株仙人掌有孢汉逊酵母菌株及其分离方法和应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 587
<212> DNA
<213> 仙人掌有孢汉逊酵母菌株(Hanseniaspora opuntiae)
<400> 1
ggaggaaaag aaaccaactg ggattacctt agtaacggcg agtgaagcgg taaaagctca 60
aatttgaaat ctggtacttt cagtgcccga gttgtaattt gtagaatttg tctttgatta 120
ggtccttgtc tatgttcctt ggaacaggac gtcatagagg gtgagaatcc cgtttggcga 180
ggataccttt tctctgtaag actttttcga agagtcgagt tgtttgggaa tgcagctcaa 240
agtgggtggt aaattccatc taaagctaaa tattggcgag agaccgatag cgaacaagta 300
cagtgatgga aagatgaaaa gaactttgaa aagagagtga aaaagtacgt gaaattgttg 360
aaagggaagg gcatttgatc agacatggtg ttttttgcat gcactcgcct ctcgtgggct 420
tgggcctctc aaaaatttca ctgggccaac atcaattctg gcagtaggat aaatcattaa 480
gaatgtagct acctcggtag tgttatagct tattggaata ctgctagctg ggattgagga 540
ctgcgcttcg gcaaggatgt tggcataatg gttaaatgcc gcccgtc 587
Claims (2)
1.一株仙人掌有孢汉逊酵母菌株,其特征在于,该菌株于2019年6月24日保藏在中国典型培养物保藏中心,保藏号为CCTCC NO:M 2019482。
2.权利要求1所述的一株仙人掌有孢汉逊酵母菌株在防治番木瓜炭疽菌病害的应用。
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