CN111621435B - 一株粘细菌及其应用 - Google Patents
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Abstract
本发明公开了一株粘细菌及其应用。本发明的粘细菌M34,其保藏编号为GDMCC No:61026。本发明的粘细菌M34能够明显抑制植物病原真菌的生长,包括引起水稻纹枯病的立枯丝核菌和引起香蕉枯萎病的尖孢镰刀菌、香蕉盘长孢菌,并且能够捕食丁香疫病病菌丁香假单胞菌、番茄青枯病病菌茄劳尔氏菌、甘蓝黑腐病病菌野油菜黄单胞菌、菜豆萎蔫病病菌萎蔫短小杆菌。该菌具有对植物病原菌的广谱抑制效果,且抑制策略多样,包括对真菌生长的直接抑制和对细菌的捕食,在制备微生物肥料添加剂和生防菌剂方面具有较好的应用价值。
Description
技术领域
本发明属于微生物领域,具体涉及一株粘细菌及其应用。
背景技术
植物病害是作物生产的重要限制因素之一,据估计每年的直接经济损失高达数千亿美元。植物病原菌种类繁多,引起的病害种类也比较多,导致控制难度大。化学农药的应用对控制农作物病虫害、提高作物产量和品质方面做出了巨大贡献,然而,当前农药滥用导致生态环境破坏并威胁人类健康。此外,几乎每种作物都有几种甚至几十种真菌和细菌病害,这就导致单一化学防治效果差;而作物抗病品种选育困难,抗病性往往具有菌株专化性,病原菌变异产生抗性的速度快于品种选育速度。因此,广谱抗菌对植物病害防控具有重要价值。生物防治法是利用生物物种之间的生态关系,主要包括竞争、捕食、寄生等,以一种或一类生物抑制另一种或另一类生物,具有安全、对环境污染小、病原物不易产生抗性等优点,成为替代化学法防治植物病害的理想手段。
目前已有许多生防细菌得到筛选和应用,主要包括芽孢杆菌、假单胞菌、放射土壤杆菌、巴氏杆菌等,但是也存在定殖能力弱、作用机制有限、防效差等问题。粘细菌是一类具有复杂群体行为和广泛存在的捕食菌,能够在饥饿条件下聚集形成抗逆性的子实体,在捕食过程中通过产生多种抗菌物质和胞外酶,以多种策略杀死猎物细胞,并获取营养物质用于生长。粘细菌能够产生丰富多样、结构新颖、作用机制独特、功能多效的次级代谢产物,但是目前大部分的研究都集中在代谢产物作为药物先导化合物的研究上,关注粘细菌代谢产物抗植物病原菌的研究和应用不多。粘细菌作为一类捕食性细菌,以胞外酶裂解猎物细胞获取营养也是其掠食策略,粘细菌分泌的胞外囊泡中含有大量水解酶,包括蛋白酶、磷酸酶、核酸酶、纤维素酶、几丁质酶、淀粉酶、酯酶等。另外,已有多项研究报道,粘细菌是一种广谱性的捕食菌,捕食谱比较宽泛,能够裂解多种微生物细胞。总之,粘细菌丰富多样的捕食策略、良好的定殖能力、广泛的猎物谱使得其能够成为一种良好的生防菌。
发明内容
本发明的目的是发掘具有广谱抗菌活性的生防粘细菌新资源,提供了一株具有抑制植物病原真菌生长和捕食植物病原细菌的粘细菌潜在新物种Corallococcus sp.M34。
本发明的粘细菌Corallococcus sp.M34菌学特性如下:
形态特征:
观察在MD1液体培养基和VY/2固体培养基培养5天后的聚集生长特征和菌落形态,在VY/2平板上表现为滑动扩展的浅柠檬黄色透明菌落,整体菌落湿润;在培养5天后营养细胞开始聚集形成子实体,成熟的子实体呈球形、浅柠檬黄色、无柄。结晶紫染色在100倍油镜下观察该菌株营养细胞呈长杆状,粘孢子呈球形。总之,该菌菌落形态、营养细胞、能聚集形成子实体等特征表明该菌符合粘细菌的特征。
生理生化特征:
粘细菌M34能够强烈水解淀粉和脱脂牛奶,能够在以干酪素、脂类为唯一营养来源的培养基上扩展和生长,而不能利用纤维素、几丁质和木聚糖。
分子生物学特征:
测得的Corallococcus sp.M34的16S rRNA基因序列,与在EzBioCloud(https://www.ezbiocloud.net/)上BLAST得到的高度相似的16S rRNA基因序列进行同源性比对,并构建系统发育树。比对结果显示该菌株与模式菌株Corallococcus coralloides DSM 2259具有最高相似性,序列相似性为97.79%。
综合菌株形态特征、生理生化特征和16S rRNA基因序列分析结果,本发明的粘细菌Corallococcus sp.M34属于粘球菌科的一个潜在新种。其16S rRNA基因序列如SEQ IDNO.1所示。
因此,本发明的第一个目的是提供一株粘细菌M34,其保藏编号为GDMCC No:61026。
本发明还提供所述的粘细菌M34的培养物。
本发明还提供一种菌剂,其以所述的粘细菌M34作为活性成分。
本发明还提供一种生物肥料,其含有所述的粘细菌M34。
本发明还提供所述的粘细菌M34在抑制植物病原菌中的应用。
优选,所述的植物病原菌为植物病原真菌。
优选,所述的植物病原真菌为立枯丝核菌(Rhizoctonia solani Kühn)、尖孢镰刀菌(Fusarium oxysporum f.sp.cubense race 4)或香蕉盘长孢菌(Colletotrchummusarum Cooke et Mass)。
优选,所述的植物病原菌为植物病原细菌。
优选,所述的植物病原细菌为丁香假单胞菌(Pseudomonas syringae GIM1.330)、茄劳尔氏菌(Ralstonia solanacearum GIM 1.70)、野油菜黄单胞菌(Xanthomonascampestris JCM 20466)或萎蔫短小杆菌(Curtobacterium flaccumfaciens GIM 1.343)。
本发明的有益效果:
(1)从原始森林土壤中分离并纯化到一株粘细菌Corallococcus sp.M34,其对多种植物病原真菌和细菌具有捕食和拮抗效应,因此,该菌具有对植物病原菌的广谱抑制效果,且抑制策略多样,在制备微生物肥料添加剂和生防菌剂方面具有较好的应用价值。
(2)粘细菌分布在各类农业土壤中,并且能够产生具有抗逆性的子实体,子实体内包含大量粘孢子,能够在适宜条件下萌发形成营养细胞,并且子实体长期保存仍可萌发,因此,有利于保存和运输,以及在实际应用中环境定殖,有利于快速实现产业化。
保藏说明:
本发明的粘细菌Corallococcus sp.M34于2020年5月6日保藏于广东省微生物菌种保藏中心(GDMCC),地址:广州市先烈中路100号大院59号楼5楼,广东省微生物研究所,保藏编号:GDMCC No:61026。
附图说明
图1是粘细菌Corallococcus sp.M34形态特征;A:Corallococcus sp.M34在MD1液体培养基生长5天的菌液;B:Corallococcus sp.M34在VY/2固体培养基生长5天的菌落;C:体视显微镜下观察Corallococcus sp.M34菌落;D:Corallococcus sp.M34子实体;E:Corallococcus sp.M34杆状营养细胞和球形粘孢子结晶紫染色。
图2是粘细菌Corallococcus sp.M34水解淀粉、水解脱脂牛奶生长。
图3是基于16S rRNA基因的菌株Corallococcus sp.M34系统发育树。
图4是粘细菌Corallococcus sp.M34对致病病原真菌的抑制。
图5是粘细菌Corallococcus sp.M34对植物病原细菌的捕食。
图6是培养2-8天粘细菌Corallococcus sp.M34捕食植物病原细菌形成透明圈半径大小。
具体实施方式
以下实施例是对本发明的进一步说明,而不是对本发明的限制。
实施例1:菌株的分离与纯化
1.1样品采集和前处理
从南岭国家森林公园炉田采集10-20cm土层土壤约200g,去除可见的根系、石子和其他杂物后过2mm筛混匀土壤,置于室温下摊平自然风干,以减少后续分离纯化过程中霉菌、原生生物和其他杂菌的污染。
1.2大肠杆菌诱导法诱导粘细菌子实体的形成
取风干的土壤样品大约10g置于50mL离心管,并加入15mL 100μg/mL的已过滤除菌的放线菌酮,浸泡处理过夜,离心后倒掉液体。用大肠杆菌为底物的水琼脂培养基(即WCX培养基,含有CaCl2·2H2O 1g/L和琼脂15g/L,余量为水;pH 7.2)分离噬细菌粘细菌,将收集的大肠杆菌菌体在WCX培养基表面画“田”字,在“田”字的四个空格中分别接种黄豆粒大小的土壤。将上述分离平板置于30℃培养,培养过程中及时用体视显微镜观察并分离挑取粘细菌子实体至纯化培养基上。
1.3粘细菌的纯化与保藏
纯化培养基为VY/2(含有酵母5g/L,CaCl2·2H2O 1g/L,VB12 0.5mg/L和琼脂15g/L,余量为水;pH 7.2),挑取将子实体或者菌落边缘用VY/2反复转接,直至肉眼观察到纯菌株。得到的纯菌株接种于营养肉汤培养基(含有蛋白胨10g/L,牛肉浸粉3g/L和NaCl 5g/L,余量为水;pH 7.2),30℃过夜培养验纯,若培养液澄清表明该粘细菌为纯菌株。已纯化的粘细菌菌株转接至VY/2固体培养基上,待子实体成熟后刮下用于甘油种和冻干种的制作。
实施例2:菌株的鉴定
1、菌株形态鉴定
图1展示了分离到的粘细菌M34的形态特征,其中图1A和图1B分别是该菌株在MD1液体培养基(含有酪蛋白胨6g/L,可溶性淀粉2g/L,MgSO4.7H2O 2g/L和CaCl2·2H2O 0.4g/L,余量为水;pH 7.2)和VY/2固体培养基培养5天后的聚集生长特征和菌落形态,在VY/2平板上表现为滑动扩展的浅柠檬黄色透明菌落,整体菌落湿润;在培养5天后营养细胞开始聚集形成子实体(图1C和图1D),成熟的子实体呈球形、浅柠檬黄色、无柄。结晶紫染色在100倍油镜下观察该菌株营养细胞呈长杆状,粘孢子呈球形(图1E)。总之,该菌菌落形态、营养细胞、能聚集形成子实体等特征表明该菌符合粘细菌的特征。
2、生理生化
配制双层培养基,其中底层为WCX培养基,待底层琼脂凝固后加5mL含有各类底物的上层琼脂培养基(上层琼脂培养基含有MgSO4.7H2O 1g/L,K2HPO4 0.2g/L和琼脂15g/L;另加底物5g/L),底物包括淀粉、纤维素、几丁质、木聚糖、干酪素、脱脂牛奶或棕榈酸对硝基苯酯。接种粘细菌M34于双层培养基,30℃培养。该菌水解利用各种生物大分子的情况表明,分离到的粘细菌M34能够强烈水解淀粉和脱脂牛奶(图2),能够在以干酪素、脂类为唯一营养来源的培养基上扩展和生长,而不能利用纤维素、几丁质和木聚糖。
3、菌株的分子鉴定
从VY/2培养基上刮取菌体,并加入少量无菌水,置于90℃处理10min,以此获得PCR反应的模板。PCR反应体系(25μL):buffer 12.5μL,16S rRNA基因上下游引物各1μL,模板1μL,无菌去离子水9.5μL。反应程序:95℃5min,95℃45s,60℃45s,72℃90s,30个循环,72℃10min。PCR产物进行琼脂糖电泳确定无误后交由广州天一辉远生物技术有限公司测序。
将测得的Corallococcus sp.M34的16S rRNA基因序列,与在EzBioCloud(https://www.ezbiocloud.net/)上BLAST得到的高度相似的16S rRNA基因序列进行同源性比对,并构建系统发育树(图3)。比对结果显示该菌株与模式菌株Corallococcuscoralloides DSM 2259具有最高相似性,序列相似性为97.79%。
综合菌株形态特征、生理生化特征和16S rRNA基因序列分析结果,本发明的粘细菌Corallococcus sp.M34属于粘球菌科的一个潜在新种,其16S rRNA基因序列如SEQ IDNO.1所示(Corallococcus sp.M34 16S rRNA基因序列)。
实施例3:粘细菌Corallococcus sp.M34菌株抗植物病原菌活性研究
3.1粘细菌Corallococcus sp.M34对植物病原真菌的抑制
挑取水稻纹枯病病菌立枯丝核菌(Rhizoctonia solani Kühn)、香蕉枯萎病病菌尖孢镰刀菌(Fusarium oxysporum f.sp.cubense race 4)和香蕉盘长孢菌(Colletotrchum musarum Cooke et Mass)3种植物病原真菌丝块接种于VY/2固体培养基靠近边缘约2cm处,并在平板另一侧相对位置处接种粘细菌M34,每种病原真菌设置5次重复,同时设置不接种粘细菌M34平板5个作为对照。30℃培养,在培养第3-5天时发现其对立枯丝核菌和香蕉盘长孢菌生长的明显抑制,而尖孢镰刀菌由于气生菌丝旺盛而受到粘细菌M34的抑制作用较小(图4)。粘细菌M34对立枯丝核菌、尖孢镰刀菌和香蕉盘长孢菌的抑制率分别为61.52%±0.73%,56.26±0.73%%和32.53%±0.78%。
3.2粘细菌Corallococcus sp.M34对植物病原细菌的捕食
取植物病原细菌丁香疫病病菌丁香假单胞菌Pseudomonas syringae GIM 1.330、番茄青枯病病菌茄劳尔氏菌Ralstonia solanacearum GIM 1.70、甘蓝黑腐病病菌野油菜黄单胞菌Xanthomonas campestris JCM 20466三种革兰氏阴性病原菌、菜豆萎蔫病病菌萎蔫短小杆菌Curtobacterium flaccumfaciens GIM 1.343革兰氏阳性病原菌分别接种于营养肉汤液体培养基中,28℃培养16-18h,7500rpm离心5min收集菌体,用MMC缓冲液(3-吗啉丙磺酸MOPS 20mM,MgSO4 4mM,CaCl2 2mM)重悬,并用血球计数板计数使细胞密度为1010mL/L。取150μL菌悬液滴在TPM平板(含有Tris-HCl 10mM,KH2PO4 1mM,MgSO4 8mM,余量为水;pH7.6)中央。待菌悬液吹干后形成菌斑,接种粘细菌M34菌块于病原细菌菌斑中央,置于30℃培养,并设置不接种粘细菌的对照;另外,还设置无被捕食菌的对照确定粘细菌M34在TPM培养基上无法正常生长,说明在有病原细菌的条件下,粘细菌M34的生长是由于对病原细菌的捕食。培养1-2天后,观察到粘细菌M34的扩展和生长,以及病原细菌菌体的消失,并用游标卡尺测定捕食形成的透明圈半径,可观察到病原菌菌体被捕食后形成的透明圈;培养8天后,病原菌菌体消失,粘细菌M34可将这4株粘细菌完全捕食,说明M34对植物病原细菌的广谱性捕食(图5,图6)。
序列表
<110> 广东省微生物研究所(广东省微生物分析检测中心)
<120> 一株粘细菌及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1402
<212> DNA
<213> 粘细菌M34(Corallococcus sp. M34)
<400> 1
tgcagtcgag cgcgaatggg gaaaccctag tagagcggcg cacgggtgcg taacacgtgg 60
gtaatctgcc tggatgcctg ggataaccag tcgaaagact ggctaatacc ggataagccc 120
acggactctt cggagtctga gggaaaaggt ggcctctgta tacaagctat cacattcaga 180
tgagcccgcg gcccatcagc tagttggcgg ggtaatggcc caccaaggcg acgacgggta 240
gctggtctga gaggacgatc agccacactg gaactgagac acggtccaga ctcctacggg 300
aggcagcagt ggggaatttt gcgcaatggg cgaaagcctg acgcagcaac gccgcgtgtg 360
tgatgaaggt ctttggattg taaagcactt tcgaccggga agaaaacccg tagcctaaca 420
tgctacggct tgactgtacc gggagaagaa gcaccggcta actctgtgcc agcagccgcg 480
gtaatacaga gggtgcaagc gttgttcgga attattgggc gtaaagcgcg tgtaggcggc 540
tttgcaagtc gggtgtgaaa gccctcagct caactgagga agtgcgcccg aaactgcaga 600
gcttgagtgc cggagagggt ggcggaattc cccaagtaga ggtgaaattc gtagatatgg 660
ggaggaacac cggtggcgaa ggcggccacc tggacggcaa ctgacgctga gacgcgaaag 720
cgtggggagc aaacaggatt agataccctg gtagtccacg ccgtaaacga tgagaactag 780
gtgtcgtggg agttgacccc cgcggtgccg aagctaacgc attaagttct ccgcctggga 840
agtacggtcg caagactaaa actcaaagga attgacgggg gcccgcacaa gcggtggagc 900
atgtggttta attcgacgca acgcgcagaa ccttacctgg tcttgacatc ctcggaatct 960
ctcagagatg agggagtgcc cgcaagggaa ccgagagaca ggtgctgcat ggctgtcgtc 1020
agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctc gcctttagtt 1080
gccacgcaag tggatctcta gagggactgc cggtgttaaa ccggaggaag gtggggatga 1140
cgtcaagtcc tcatggcctt tatgaccagg gctacacacg tgctacaatg gccggtacag 1200
agcgctgcaa acccgcgagg gggagctaat cgcagaaaac cggtctcagt tcagattgga 1260
gtctgcaact cgactccatg aaggcggaat cgctagtaat cgcagatcag cacgctgcgg 1320
tgaatacgtt cccgggcctt gtacacaccg cccgtcacac catgggagtc gattgctcca 1380
gaagtcatct caccaagaga tg 1402
Claims (8)
1.一株粘细菌(Corallococcus sp.)M34,其保藏编号为GDMCC No:61026。
2.一种菌剂,其特征在于,以权利要求1所述的粘细菌M34作为活性成分。
3.一种生物肥料,其特征在于,含有权利要求1所述的粘细菌M34。
4.权利要求1所述的粘细菌M34在抑制植物病原菌中的应用。
5.根据权利要求4所述的应用,其特征在于,所述的植物病原菌为植物病原真菌。
6.根据权利要求5所述的应用,其特征在于,所述的植物病原真菌为立枯丝核菌、尖孢镰刀菌或香蕉盘长孢菌。
7.根据权利要求4所述的应用,其特征在于,所述的植物病原菌为植物病原细菌。
8.根据权利要求7所述的应用,其特征在于,所述的植物病原细菌为丁香假单胞菌、茄劳尔氏菌、野油菜黄单胞菌或萎蔫短小杆菌。
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