菌核病生防菌小盾壳霉的固体发酵及几个菌株的生物学特性研究
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摘要
在国内首次以多种农产品材料(小麦、谷子、麸皮等)作为生防菌小盾壳霉(Coniothyrium minitans)的固体发酵基质，以从德国引进小盾壳霉Cm-02菌株为发酵对象，对影响发酵的多种条件进行研究，并对确定出的各单因素较佳条件进行正交实验，最终确定出各单因素条件的最佳组合，为小盾壳霉生防制剂的工业化生产提供理论依据。此外，从四川地区分离到两株小盾壳霉Cm-c1，Cm-b2，与采自德国的两个菌株Cm-02和Cm-315一起作不同小盾壳霉菌株的生物学特性对比研究。结果如下：
采用小麦作为固体发酵基质，20℃培养，加水比例为基质：水分=1∶1(g／ml)，基质灭菌前添加发酵营养液，酸碱度调PH6是适合小盾壳霉Cm-02菌株固体发酵的各最佳单因素条件。各处理下基质产孢量最大值均在第20d出现。选取基质加水比例、营养液添加时间和酸碱度中的较佳水平各3个进行正交试验，得到的最佳发酵条件为：发酵营养液调PH6，按基质与其比例1∶1(g∶ml)于灭菌前加入，小盾壳霉Cm-02的产孢量在第20d达到最大，为18.43×10~9个／g。
四个菌株在生物学特性方面存在明显差异。Cm-c1在形态特征上和其它菌株差异最大。各菌株在5℃-30℃的范围内均能生长，生长和产孢的最适温度皆为20℃；在PH2-PH12内均能生长，生长和产孢的最适PH值Cm-c1为PH4，其余菌株为PH6；四菌株均在全光照下生长最快，全黑暗下产孢量最大；PDA培养基对小盾壳霉生长和产孢较为适宜；各种处理下Cm-c1均生长很快，产孢量却最低；此外，随着发酵时间的延长，小盾壳霉产生的孢子的萌发能力在降低，而一定的外源营养能促进其孢子萌发；对峙培养时四菌株对核盘菌(Sclerotinia sclerotiorum)Ya菌株的生长抑制率均在50％以上；各菌株对核盘菌所产菌核的数量和质量均有明显抑制作用，菌核寄生致腐率均在80％以上，其中，Cm-02、Cm-315、Cm-b2均在90％左右。总的来说，国外与国内的四个盾壳霉菌株对核盘菌作用的能力没有较大差异。在油菜离体叶片接种试验中，四菌株对核盘菌M08叶面病斑的扩展没有抑制作用。
The wheat, rice and wheat bran were firstly treated as the solid state fermentation media of biological control fungus Coniothyrium minitans. On the present study, strain Cm-02 of Coniothyrium minitans introduced from Germen was fermented under various conditions to determine the best single fermentation factor, and then they were subjected to orthogonal experiment to determine the best cross fermentation conditions, aiming to provide theoretical base for the industrial production of biological agents originated from Coniothyrium minitans. In addition, the biological characters of two Coniothyrium minitans strains (Cm-c1 and Cm-b2) from Sichuan eco-region were compared with two Germanic strains, Cm-02 and Cm-315. The results were provided in the following sections.
The strain Cm-02 was fermented under 20℃on solid wheat fermentation medium in which concentration was 50% (g/ml), and nutritional materials were added before fermentation. The results showed that pH 6.0 was the best for all of the single factors when strain Cm-02 was fermented. The sporulation of each treatment reached the peak in 20 d after fermentation. The orthogonal experiment was conducted with three treatments of each of the three conditions (desirable fraction of water added in the solid medium, time for adding nutrition and pH value). PH 6.0 was the best fermentation condition for srtain Cm-02, and its sporulation reached to the peak in 20 d after fermentation (18.43×10~9 cfu/ml).
The biological characteristics of the four strains were significantly different, and the morphological character of strain Cm-c1 was apparently differ from the others. All of the four strains could grown between 5 to 30℃under the pH value range of 2-12, but the best one was pH 6.0 except for strain Cm-c1 for which desirable pH value was 4.0. They were grown best under full lighting while they sporulated best in complete darkness. PDA medium was desirable for the growth and sporulation of Coniothyrium minitans. Strain Cm-c1 of all treatments grown best but its sporulation was the smallest. In addition, the germination capability of Coniothyrium minitans spores reduced with longer fermentation time while promoted with additional nutrition. The inhibition rate of the four fungal strains against Sclerotinia sclerotiorum strain Ya was over 50% on dual culture. The sclerotic parasitism rotting capability of three fungi (Cm-02, Cm-315, Cm-b2) was around 90% and the last one was over 80%, revealing apparent inhibition on the formation of sclerotia. In short, the inhibition capability of the four fungi exhibited nearly the same level. The four fungi could not exhibit the enlargement of oil-rape leaf spots of sclerotinia sclerotiorum M08 strain in vitro.
采用小麦作为固体发酵基质，20℃培养，加水比例为基质：水分=1∶1(g／ml)，基质灭菌前添加发酵营养液，酸碱度调PH6是适合小盾壳霉Cm-02菌株固体发酵的各最佳单因素条件。各处理下基质产孢量最大值均在第20d出现。选取基质加水比例、营养液添加时间和酸碱度中的较佳水平各3个进行正交试验，得到的最佳发酵条件为：发酵营养液调PH6，按基质与其比例1∶1(g∶ml)于灭菌前加入，小盾壳霉Cm-02的产孢量在第20d达到最大，为18.43×10~9个／g。
四个菌株在生物学特性方面存在明显差异。Cm-c1在形态特征上和其它菌株差异最大。各菌株在5℃-30℃的范围内均能生长，生长和产孢的最适温度皆为20℃；在PH2-PH12内均能生长，生长和产孢的最适PH值Cm-c1为PH4，其余菌株为PH6；四菌株均在全光照下生长最快，全黑暗下产孢量最大；PDA培养基对小盾壳霉生长和产孢较为适宜；各种处理下Cm-c1均生长很快，产孢量却最低；此外，随着发酵时间的延长，小盾壳霉产生的孢子的萌发能力在降低，而一定的外源营养能促进其孢子萌发；对峙培养时四菌株对核盘菌(Sclerotinia sclerotiorum)Ya菌株的生长抑制率均在50％以上；各菌株对核盘菌所产菌核的数量和质量均有明显抑制作用，菌核寄生致腐率均在80％以上，其中，Cm-02、Cm-315、Cm-b2均在90％左右。总的来说，国外与国内的四个盾壳霉菌株对核盘菌作用的能力没有较大差异。在油菜离体叶片接种试验中，四菌株对核盘菌M08叶面病斑的扩展没有抑制作用。
The wheat, rice and wheat bran were firstly treated as the solid state fermentation media of biological control fungus Coniothyrium minitans. On the present study, strain Cm-02 of Coniothyrium minitans introduced from Germen was fermented under various conditions to determine the best single fermentation factor, and then they were subjected to orthogonal experiment to determine the best cross fermentation conditions, aiming to provide theoretical base for the industrial production of biological agents originated from Coniothyrium minitans. In addition, the biological characters of two Coniothyrium minitans strains (Cm-c1 and Cm-b2) from Sichuan eco-region were compared with two Germanic strains, Cm-02 and Cm-315. The results were provided in the following sections.
The strain Cm-02 was fermented under 20℃on solid wheat fermentation medium in which concentration was 50% (g/ml), and nutritional materials were added before fermentation. The results showed that pH 6.0 was the best for all of the single factors when strain Cm-02 was fermented. The sporulation of each treatment reached the peak in 20 d after fermentation. The orthogonal experiment was conducted with three treatments of each of the three conditions (desirable fraction of water added in the solid medium, time for adding nutrition and pH value). PH 6.0 was the best fermentation condition for srtain Cm-02, and its sporulation reached to the peak in 20 d after fermentation (18.43×10~9 cfu/ml).
The biological characteristics of the four strains were significantly different, and the morphological character of strain Cm-c1 was apparently differ from the others. All of the four strains could grown between 5 to 30℃under the pH value range of 2-12, but the best one was pH 6.0 except for strain Cm-c1 for which desirable pH value was 4.0. They were grown best under full lighting while they sporulated best in complete darkness. PDA medium was desirable for the growth and sporulation of Coniothyrium minitans. Strain Cm-c1 of all treatments grown best but its sporulation was the smallest. In addition, the germination capability of Coniothyrium minitans spores reduced with longer fermentation time while promoted with additional nutrition. The inhibition rate of the four fungal strains against Sclerotinia sclerotiorum strain Ya was over 50% on dual culture. The sclerotic parasitism rotting capability of three fungi (Cm-02, Cm-315, Cm-b2) was around 90% and the last one was over 80%, revealing apparent inhibition on the formation of sclerotia. In short, the inhibition capability of the four fungi exhibited nearly the same level. The four fungi could not exhibit the enlargement of oil-rape leaf spots of sclerotinia sclerotiorum M08 strain in vitro.
引文
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