CN1291008C - 一种真菌菌株及其用途 - Google Patents
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Abstract
本发明公开了一种真菌菌株，该菌株为三线镰刀菌(Fusarium tricinctum)真菌菌株(ZJUF0998)，保藏单位：中国微生物菌种保藏管理委员会普通微生物中心，保藏日：2005年3月16日，保藏号：CGMCC1331，属于真菌界(Fungi)，子囊菌门(Ascomycota)，子囊菌纲(Ascomycetes)，粪壳亚纲(Sordariomycetidae)，肉座菌目(Hypocreales)，丛赤壳科(Nectriaceae)，镰刀菌属(Fusarium)。本发明还公开了该菌株的内转录间隔区核糖体DNA全序列。本发明的真菌菌株，可产生挥发性芳香气体、并能抑制植物病原菌的生长。
Description
技术领域
本发明涉及一种真菌及其抗植物病原真菌的用途，属于微生物及微生物应用领域。
背景技术
很多真菌都能散发出一些低浓度的气体物质，这些气体物质很多都有令人不愉快的气味。有些真菌产生的挥发性气体物质具有抗菌作用，如木霉产生的挥发性物质能抑制立枯丝核菌(Rhizoctonia solani)、尖孢镰刀菌(Fusariumoxysporum)、终极腐霉(Pythium ultimum)等的生长。目前对真菌产生的挥发性气体并没有进行很深入的研究，一般认为挥发性气体的成分之一是乙醛。Muscodor albus是从一种樟属植物锡兰肉桂(Cinnamomum zeylanicum)中分离到的一种新内生真菌，研究发现这种内生真菌可产生具有抗菌作用的挥发油成分，包含27种组分，其中，主要成分是3-甲基丁酸乙酯(22.24％)、2，3-二甲基-丙酸丁酯(1.53％)、3-甲基-1-丁醇(22.99％)。尽管Muscodor albus产生的挥发性气体对部分病原菌具有强列的致死作用，但其挥发性气体没有芳香性。
发明内容
针对现有技术中存在的不足之处，本发明提供一种可产生挥发性芳香气体、并能抑制植物病原菌生长的真菌。
本发明为达到以上目的，是通过这样的技术方案来实现的：提供一种真菌菌株，该菌株为三线镰刀菌Fusarium tricinctum真菌菌株ZJUF0998，保藏单位：中国微生物菌种保藏管理委员会普通微生物中心，保藏日：2005年3月16日，保藏号：CGMCC1331，属于真菌界Fungi，子囊菌门Ascomycota，子囊菌纲Ascomycetes，粪壳亚纲Sordariomycetidae，肉座菌目Hypocreales，丛赤壳科Nectriaceae，镰刀菌属Fusarium；在PDA上F0998的气生菌丝呈棉絮状，白至草珠红、淡绛红，培养基不变色；有桔橙色分生孢子座；大型分生孢子镰状，弯曲，顶胞渐细，基胞有足跟；产孢细胞单瓶梗。
上述三线镰刀菌系从我国江西官山自然保护区采集的南方红豆杉树皮中分离得到。
本发明的三线镰刀菌CGMCC1331，该菌株的内转录间隔区核糖体DNA(ITS rDNA)全序列如序列表所示。
本发明的三线镰刀菌CGMCC1331，可用于抗植物病原真菌。测定方法如下：
选择齐整小核菌(Sclerotium rolfsii Saccardo)、立枯丝核菌(Rhizoctoniasolani Kuhn)、尖孢镰刀菌(Fusarium oxysporum Schlecht.)及灰葡萄孢(Botrytiscinerea)等4个植物病原真菌菌株作为指示菌株。在直径9cm的培养皿中加入预先溶化的PDA培养基制成平板，用无菌刀片挖去平板中央1.0cm的琼脂条。将三线镰刀菌菌株和上述4种指示菌株均用打孔器打成直径0.4cm的菌丝饼。将三线镰刀菌菌丝饼接种在平板一侧，24℃培养1周后将指示菌菌丝饼接种在平板另一侧，以不接种三线镰刀菌为对照。用Parafilm将培养皿包裹后24℃培养，肉眼观察内生真菌产生的挥发性气体对指示菌的抑制作用。三线镰刀菌菌株CGMCC1331(在图中简称为F0998)挥发性芳香气体抗菌作用的结果见附图3。
本发明的三线镰刀菌CGMCC1331，其挥发性芳香气体成分分析：
本挥发性芳香气体经固相微萃取、GC/MS分析，得到各成分的总离子流图，见附图4。根据标准图谱和有关文献确定各组分的化学成分，并按峰面积归一法计算各组分的相对百分含量，结果表明菌株F0998的挥发性芳香气味中包含了14种单体化合物，包括9种酯，4种酸和角鲨烯。其中，十六酸甲酯(22.06％)、十六烷酸(19.94％)、9-十六烯酸(6.87％)、十四酸(6.66％)、十六酸乙酯(6.13％)为主要成分，共占总成分的61.66％。以上物质均具有香味，是本挥发性气体具有芳香味的来源。
本发明的三线镰刀菌CGMCC1331，可产生具有挥发性的芳香气体，并具有抑制病原菌生长的作用，因此可作为真菌薰剂在农业、卫生等行业使用，具有极高应用价值。十六酸甲酯、十六酸乙酯、十六烷酸、十五酸、十八酸甲酯皆为烟用香料；十四酸、十六烷酸是鸢尾根茎净油成分之一，鸢尾净油是一种高品质的香料产品，广泛用于食品和化妆品，十六酸乙酯还是无花果香料香味成分之一。角鲨烯是具有香味的油液体，是胆甾醇生物合成的中间体，角鲨烯能向细胞供应大量氧气，使细胞恢复活力，提高身体的自然治愈能力。角鲨烯的功能主要分为四方面：1)、促进血液循环，帮助预防及治疗因血液循环不良而引致的病变；2)、活化身体机能细胞，帮助预防及治疗因机能细胞缺氧而引致的病变；3)、消炎杀菌，帮助预防及治疗细菌引致的疾病；4)、修复细胞，加快伤口愈合，在工业上可用于成药物、表面活性剂、有机色素等。本发明的三线镰刀菌CGMCC1331，产生的挥发性芳香气体具有抑制齐整小核菌(Sclerotium rolfsii)、立枯丝核菌(Rhizoctonia solani)、尖孢镰刀菌(Fusarium oxysporum)及灰葡萄孢(Botrytis cinerea)等植物病原真菌生长的作用，具有从中开发出新型、高效、无毒的天然杀菌剂的巨大潜力。
附图说明
图1为本发明的三线镰刀菌CGMCC1331在PDA培养基上的照片；
图2为本发明的三线镰刀菌CGMCC1331的大型分生孢子；
图3为本发明的三线镰刀菌CGMCC1331(在图中简称为F0998)挥发性芳香气体抗菌作用结果；
图4为本发明的三线镰刀菌CGMCC1331的挥发性成分的总离子流图。
具体实施方式
参照上述附图，对本发明的具体实施方式进行详细说明。
实施例1、菌株的分离培养：
在实验室中按下列条件分离培养，即可获得本发明所述的三线镰刀菌。从我国江西官山自然保护区采集南方红豆杉(Taxus mairei)树皮及树枝，将采集的南方红豆杉的树皮及树枝在5％次氯酸钠表面消毒2min，无菌水漂洗后置于无菌滤纸上吸干水，用无菌刀片去除外皮层，并将其切成1cm左右大小组织片，将组织片移接在2％含100μg·ml-1氨苄青霉素和链霉素的水琼脂平板上，24℃培养，待菌丝长出后，将菌丝顶端移接到PDA培养基上，连续重复纯化3次，获得纯培养。
实施例2、挥发性芳香气体的抗菌生物测定：
马铃薯葡萄糖琼脂培养基(potato dextrose agar，PDA)：马铃薯200g，葡萄糖20g，琼脂20g，水1000ml。
选择齐整小核菌(Sclerotium rolfsii Saccardo)、立枯丝核菌(Rhizoctoniasolani Kuhn)、尖孢镰刀菌(Fusarium oxysporum Schlecht.)及灰葡萄孢(Botrytiscinerea)等4个植物病原真菌菌株作为指示菌株。在直径9cm的培养皿中加入预先溶化的PDA培养基制成平板，用无菌刀片挖去平板中央1.0cm的琼脂条。将三线镰刀菌菌株和指示菌株用打孔器打成直径0.4cm的菌丝饼，将三线镰刀菌菌丝饼接种在平板一侧，24℃培养1周后将指示菌菌丝饼接种在平板另一侧，以不接种三线镰刀菌为对照。用Parafilm将培养皿包裹后24℃培养，肉眼观察内生真菌产生的挥发性气体对指示菌的抑制作用。三线镰刀菌菌株F0998挥发性芳香气体抗菌作用的结果见附图3。本菌株产生的挥发性芳香气体具有明显的抑制齐整小核菌(Sclerotium rolfsii)、立枯丝核菌(Rhizoctonia solani)、尖孢镰刀菌(Fusarium oxysporum)及灰葡萄孢(Botrytiscinerea)等植物病原真菌生长的作用。
实施例3、挥发性气体采集及成分分析：
将三线镰刀菌菌株F0998在PDA培养基中活化培养，备用。取活化培养的三线镰刀菌菌株F0998菌丝块(直径4em)接种在含100ml PDA培养基的250ml三角瓶中，24℃培养1周后用parafilm将三角瓶瓶口包裹，24℃继续培养1周。
将固相微萃取装置插入待测三角瓶中，萃取头探出不绣钢保护针管后浸入三角瓶，但不要碰到菌丝，持续萃取20分钟。待达到萃取平衡后，将萃取头缩回保护针管，立即插入已准备好的GC-MS的进样室，推出萃取头，利用气化室的高温解析目标分析物后，进行色谱测定。
通过GC/MS分析，得到各成分的总离子流图(附图4)，根据标准图谱和有关文献确定各组分的化学成分，并按峰面积归一法计算各组分的相对百分含量，结果(表1)表明菌株F0998的挥发性芳香气味中包含了14种单体化合物，包括9种酯，4种酸和角鲨烯。其中，十六酸甲酯(22.06％)、十六烷酸(19.94％)、9-十六烯酸(6.87％)、十四酸(6.66％)、十六酸乙酯(6.13％)为主要成分，共占总成分的61.66％。
仪器、试剂及GC/MS条件：固相微萃取装置(美国Supelco公司)，萃取探头涂层为85μm聚丙烯酸酯(PA)；气相色谱仪-质谱仪(Finnigantrans2000GC-Voyager MS)，石英毛细管柱HP-5MS(0.25mm×30m，0.25μm)；数据检索应用2004的NIST数据库。
色谱条件：载气为氦气，柱流量1.0mL·min-1；汽化室温度：280℃，色谱柱初始温度50℃，保持2min，以5℃·min-1升温速率升至240℃，最后保持10min。
质谱条件：EI电离源，电离电压70eV，离子源温度230℃，扫描范围：30-500amu，进样量1.0μL，分流比10∶1。
表1.F998挥发性成分SPME-GC-MS鉴定结果及各化合物的质量分数
| 编号No. | tR/min | 化合物Compound | 分子式Formula | Mw | w(％) |
| 12 | 29.7931.78 | 十六酸甲酯Hexadecanoic acid，methylester十四酸Tetradecanoic acid | C17H34O2C14H28O2 | 270228 | 22.066.66 |
| 34567891011121314 | 31.9332.8733.8634.9335.3935.7937.1338.2838.3838.7643.3851.37 | 十六酸乙酯Hexadecanoic acid，ethylester异丙基棕榈酸酯Isopropylpalmitate十五酸Pentadecanoic acid9，12十八二烯酸甲酯9，12-Octadecadienoic acid，methyl ester9-十六烯酸9-Hexadecenoic acid十六烷酸n-Hexadecanoic acid9-十八烯酸乙酯9-Octadecenoic acid，ethyl ester10-十八烯酸甲酯10-Octadecenoicacid，methyl ester16-十八烯酸甲酯16-Octadecenoicacid，methyl ester十八酸甲酯Octadecanoic acid，methylester二异辛基乙二酸酯Diisooctyladipte角鲨烯Spinacen | C18H36O2C19H38O2C15H30O2C19H34O2C16H30O2C16H32O2C20H38O2C19H36O2C19H36O2C19H38O2C22H42O2C30H50 | 284298242294254256310296296298338410 | 6.132.872.993.326.8719.942.181.370.480.392.171.14 |
最后，还需要注意的是，以上列举的仅是本发明的一个具体实施例。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。
三线镰刀菌(Fusarium tricinctum)真菌菌株ZJUF0998 ITS序列表
ccgagtttac aactcccaaa cccctgtgaa cataccttaa tgttgcctcg gcggatcagc 60
ccgcgccccg taaaacggga cggcccgcca gaggacccaa actctaatgt ttcttattgt 120
aacttctgag taaaacaaac aaataaatca aaactttcaa caacggatct cttggttctg 180
gcatcgatga agaacgcagc aaaatgcgat aagtaatgtg aattgcagaa ttcagtgaat 240
catcgaatct ttgaacgcac attgcgcccg ctggtattcc ggcgggcatg cctgttcgag 300
cgtcatttca accctcaagc ccccgggttt ggtgttgggg atcggctctg cccttctggg 360
cggtgccgcc cccgaaatac attggcggtc tcgctgcagc ctccattgcg tagtagctaa 420
cacctcgcaa ctggaacgcg gcgcggccat gccgtaaaac cccaacttct gaatg 475
Claims (2)
1、一种真菌菌株，其特征在于该菌株为三线镰刀菌Fusarium tricinctum真菌菌株ZJUF0998，保藏单位：中国微生物菌种保藏管理委员会普通微生物中心，保藏日：2005年3月16日，保藏号：CGMCC1331，属于真菌界Fungi，子囊菌门Ascomycota，子囊菌纲Ascomycetes，粪壳亚纲Sordariomycetidae，肉座菌目Hypocreales，丛赤壳科Nectriaceae，镰刀菌属Fusarium；在PDA上ZJUF0998的气生菌丝呈棉絮状，白至草珠红、淡绛红，培养基不变色；有桔橙色分生孢子座；大型分生孢子镰状，弯曲，顶胞渐细，基胞有足跟；产孢细胞单瓶梗。
2、一种如权利要求1所述的真菌菌株用于抗植物病原真菌的用途。
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