龙牙百合腐败菌的分离鉴定、生物学特性及抑制剂筛选
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摘要
利用常规组织分离法对湖南隆回地区龙牙百合鳞茎优势腐败菌进行分离纯化,并应用科赫法则验证了分离菌株的致病性,运用形态学和分子生物学技术对腐败菌进行鉴定,同时,探究了9种食品级防腐剂对优势腐败菌的抑制效果。结果表明:引起隆回百合鳞茎腐烂的优势菌是尖孢镰刀菌,该菌菌丝生长和产孢的最适温度分别为30,25℃,菌丝致死温度为66℃处理10min;菌丝在pH 7,8时生长最快,在pH为7时产孢量最大;可利用多种碳氮源,其中,在以葡萄糖和D-果糖为碳源,牛肉膏为氮源的培养基上生长最块;在以D-木糖为碳源、牛肉膏和L-苯丙氨酸为氮源的条件下更利于产孢;光照有利于菌丝生长和产孢。采用9种食品级防腐剂对该优势菌的抑菌试验结果表明,纳他霉素、脱氢乙酸钠和焦亚硫酸钠对该菌具有显著抑菌效果(P<0.05),最低抑菌浓度(MIC)分别为150,125,100mg/L。
The pathogenicity of isolated strains of Lilium brownii var.Viridulum Baker was studied by using conventional tissue separation method.The pathogenicity of the isolated strains was verified by Koch's law.The morphological and molecular biology techniques were used to identify the spoilage bacteria.Moreover,the inhibitory effects of nine food-grade preservatives on dominant spoilage bacteria were explored.The results showed that the dominant bacteria causing the decay of lily bulbs was Fusarium oxysporum.The optimal temperature for growth and sporulation of the mycelium was 30 and 25℃,respectively.The lethal temperature of hyphae was 66 ℃for 10 min.The growth rate was the fastest at pH 7 and 8,and the sporulation was the highest at pH 7.A variety of carbon and nitrogen sources could be used,which were grown on a medium with glucose and Dfructose as the carbon source and beef extract as the nitrogen source.In the case of D-xylose as a carbon source,with beef extract and L-phenylalanine as a nitrogen source,it was more conducive to sporulation;light was conducive to mycelial growth and sporulation.The antibacterial test results of 9 kinds of food-grade preservatives against the dominant bacteria showed that natamycin,sodium dehydroacetate and sodium metabisulfite had a significant antibacterial effect against the bacteria(P<0.05),and the minimum inhibitory concentration(MIC)were 150,125,100 mg/L,respectively.
The pathogenicity of isolated strains of Lilium brownii var.Viridulum Baker was studied by using conventional tissue separation method.The pathogenicity of the isolated strains was verified by Koch's law.The morphological and molecular biology techniques were used to identify the spoilage bacteria.Moreover,the inhibitory effects of nine food-grade preservatives on dominant spoilage bacteria were explored.The results showed that the dominant bacteria causing the decay of lily bulbs was Fusarium oxysporum.The optimal temperature for growth and sporulation of the mycelium was 30 and 25℃,respectively.The lethal temperature of hyphae was 66 ℃for 10 min.The growth rate was the fastest at pH 7 and 8,and the sporulation was the highest at pH 7.A variety of carbon and nitrogen sources could be used,which were grown on a medium with glucose and Dfructose as the carbon source and beef extract as the nitrogen source.In the case of D-xylose as a carbon source,with beef extract and L-phenylalanine as a nitrogen source,it was more conducive to sporulation;light was conducive to mycelial growth and sporulation.The antibacterial test results of 9 kinds of food-grade preservatives against the dominant bacteria showed that natamycin,sodium dehydroacetate and sodium metabisulfite had a significant antibacterial effect against the bacteria(P<0.05),and the minimum inhibitory concentration(MIC)were 150,125,100 mg/L,respectively.
引文
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[9]巩慧玲,孙爱洁,李茜,等.兰州百合鳞茎贮藏腐烂病原菌的鉴定及生物学特性[J].食品工业科技,2015,36(20):97-101.
[10]梁巧兰,徐秉良,刘艳梅.观赏百合根腐病病原鉴定及药剂筛选[J].甘肃农业大学学报,2004,39(1):25-28.
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[12]尹乐斌,雷志明,孔彦卓,等.一株高产果胶酶的真菌分离鉴定及酶学特性[J].中国酿造,2017,36(2):93-97.
[13]WHITE T J.Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[J].PCR Protocols,1994(38):315-322.
[14]丁文姣,于安芬,李瑞琴,等.定西市黄芪根腐病优势病原菌生物学特性研究[J].甘肃农业科技,2018(3):33-36.
[15]方中达.植病研究法[M].3版.北京:中国农业出版社,1998:124.
[16]王凡,胡奎,陈然,等.8种杀菌剂对核桃根腐病菌室内毒力测定[J].长江大学学报:自然科学版,2013,10(23):4-6.
[17]程桂林,徐淑芳,刘凤华.大蒜挥发油对猪鸡四种常见致病菌的抗菌效果[J].动物营养学报,2009,21(4):554-560.
[18]曾富春,黄云,赵艳琴,等.草莓枯萎病菌的生物学特性[J].四川农业大学学报,2006,24(2):156-160.
[19]潘虹,关凤芝,吴广文,等.尖孢镰刀菌亚麻专化型生物学特性研究[J].东北农业大学学报,2011,42(7):50-56.
[20]耿丽华,李常保,迟胜起,等.番茄颈腐根腐病病原鉴定及不同条件对其生长的影响[J].植物病理学报,2012,42(5):449-455.
[21]赵杰.山东省烟草镰刀菌根腐病病原及生物学特性的研究[D].青岛:中国农业科学院,2013:19-23.
[22]李忠平,周岩民,陈敏,等.脱氢醋酸钠防霉效果的研究[J].粮食与饲料工业,2008(7):32-34.
[23]隋莎莎,李志文,张平,等.纳他霉素结合柠檬酸处理对葡萄采后灰霉菌的抑制活性[J].食品科学,2013,34(24):299-303.
[24]李永才,毕阳.钼酸铵和焦亚硫酸钠对采后苹果青霉病和黑斑病的控制[J].食品科技,2008,33(12):238-241.
[2]胡才.百合:抗癌润肺[J].养生月刊,2015(1):42-43.
[3]赵秀玲.百合的营养成分与保健作用[J].中国野生植物资源,2010,29(1):44-46.
[4]SARA L M,ERLICH O,MAYMON M,et al.Bulb and root rot in lily(Lilium longiflorum)and onion(Allium cepa)in Israel[J].Journal of Phytopathology,2014,162(7/8):466-471.
[5]王艳,杜彼,连中学,等.百合鳞茎带真菌检测[J].甘肃中医学院学报,2011,28(2):48-51.
[6]LAWSON R H,HSU H T.Lily diseases and their control[J].Acta Horticulturae,1996(414):175-188.
[7]PRADOS-LIGERO A M,BASALLOTE-UREBA M J,ME-LERO-VARA J M.First report of Fusarium oxysporum f.sp.lilii and F.proliferatumaffecting Liliumcrops in Spain[J].Trop Plant Pathology,2008,33(3):235-236.
[8]朱海燕,夏花,高必达.湖南省食用百合生长期鳞茎腐烂病的病原鉴定[J].植物病理学报,2012,42(5):525-527.
[9]巩慧玲,孙爱洁,李茜,等.兰州百合鳞茎贮藏腐烂病原菌的鉴定及生物学特性[J].食品工业科技,2015,36(20):97-101.
[10]梁巧兰,徐秉良,刘艳梅.观赏百合根腐病病原鉴定及药剂筛选[J].甘肃农业大学学报,2004,39(1):25-28.
[11]魏景超.真菌鉴定手册[M].上海:上海科学技术出版社,1979:1-780.
[12]尹乐斌,雷志明,孔彦卓,等.一株高产果胶酶的真菌分离鉴定及酶学特性[J].中国酿造,2017,36(2):93-97.
[13]WHITE T J.Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[J].PCR Protocols,1994(38):315-322.
[14]丁文姣,于安芬,李瑞琴,等.定西市黄芪根腐病优势病原菌生物学特性研究[J].甘肃农业科技,2018(3):33-36.
[15]方中达.植病研究法[M].3版.北京:中国农业出版社,1998:124.
[16]王凡,胡奎,陈然,等.8种杀菌剂对核桃根腐病菌室内毒力测定[J].长江大学学报:自然科学版,2013,10(23):4-6.
[17]程桂林,徐淑芳,刘凤华.大蒜挥发油对猪鸡四种常见致病菌的抗菌效果[J].动物营养学报,2009,21(4):554-560.
[18]曾富春,黄云,赵艳琴,等.草莓枯萎病菌的生物学特性[J].四川农业大学学报,2006,24(2):156-160.
[19]潘虹,关凤芝,吴广文,等.尖孢镰刀菌亚麻专化型生物学特性研究[J].东北农业大学学报,2011,42(7):50-56.
[20]耿丽华,李常保,迟胜起,等.番茄颈腐根腐病病原鉴定及不同条件对其生长的影响[J].植物病理学报,2012,42(5):449-455.
[21]赵杰.山东省烟草镰刀菌根腐病病原及生物学特性的研究[D].青岛:中国农业科学院,2013:19-23.
[22]李忠平,周岩民,陈敏,等.脱氢醋酸钠防霉效果的研究[J].粮食与饲料工业,2008(7):32-34.
[23]隋莎莎,李志文,张平,等.纳他霉素结合柠檬酸处理对葡萄采后灰霉菌的抑制活性[J].食品科学,2013,34(24):299-303.
[24]李永才,毕阳.钼酸铵和焦亚硫酸钠对采后苹果青霉病和黑斑病的控制[J].食品科技,2008,33(12):238-241.