一株杜英疫病菌拮抗真菌的鉴定及生物学特性研究

    Identification and Biological Characteristics of One Antagonistic Fungal Strain Against Pseudocryphonectria elaeocarpicola

    • 摘要:
      目的 由杜英生假隐丛赤壳菌引起的杜英疫病是近年来在杜英属植物上发现的新病害。目前,有关该病害的研究较少,也缺乏有效的防控措施。通过稀释分离法对酒厂排污口污水样品进行菌株分离,以期筛选出有效拮抗杜英疫病菌的生防菌,并明确拮抗菌的分类地位及生物学特性,为该菌株的开发利用提供科学依据。
      方法 以杜英疫病菌为靶标菌,采用平板对峙法筛选具有较强抑菌活性的菌株。基于形态学、ITS-rpb2-tef1序列分析方法明确菌株的分类地位,采用生长速率法测定该菌株最适生长培养基、碳源、氮源、温度、pH值等生物学特性。
      结果 从污水样品中分离获得8株菌株,通过离体对峙拮抗筛选试验筛选出2株对杜英疫病菌具有拮抗效果的菌株。复筛结果表明,菌株P45-1对杜英疫病菌的抑菌率最佳、为67.25%。菌株P45-1挥发性物质对杜英疫病菌无抑制作用,其发酵液对杜英疫病菌的抑制率为52.58%。通过形态学特征和rpb2-tef1双基因联合构建系统发育树,鉴定拮抗菌株P45-1为绿木霉(Trichoderma virens)。培养基对菌株P45-1生长具有显著影响,该菌株最适培养基为MEA培养基。其最适碳源为甘露醇,最适氮源为L-天门冬酰胺,最适无机盐为硝酸钾或不添加无机盐,最适温度为25℃,且温度低于20℃或高于35℃时菌丝生长均受到明显抑制。此外,该菌株能耐受较广的酸碱度范围,在pH 4.0~7.0范围内均生长良好,最适pH为5.0。
      结论 从污水样品中分离的绿木霉P45-1对杜英疫病菌有较好的抑制效果,可用于杜英疫病菌的生物防治。生物学特性的试验结果明确了其菌丝生长的最适条件。

       

      Abstract:
      Objective Elaeocarpus spp. stem blight is a newly discovered branch disease on Elaeocarpus spp. caused by Pseudocryphonectria elaeocarpicola in recent years. At present, there is limited research on this disease and there is still a lack of effective prevention and control measures. This study aims to isolate and screen for effective biocontrol strains from wastewater samples at the distillery's discharge outlet that antagonize P. elaeocarpicola by using dilution separation method, and to clarify taxonomic status, and biological characteristics of the antagonistic strain, and to provide reference for further utilization of this strain as a biocontrol agent.
      Method The inhibitory effect of antagonistic strains on P. elaeocarpicola was studied using plate two-point confrontation method. Based on morphology and ITS-rpb2-tef1 sequence analysis, the taxonomic status of the strains was determined. The growth rate method was used to determine the optimal growth medium, carbon source, nitrogen source, inorganic salt, temperature, pH value of biological characteristics of the strain.
      Result Two antagonistic fungal strains P45-1 and P45-2 were selected by confrontation in vitro from 8 strains isolated from wastewater samples. After the secondary screening, the strain P45-1 showed the better antifungal efficacy, with the inhibition rates of 67.25% against P. elaeocarpicola. The volatile metabolites of the strain P45-1 have no inhibitory effect on P. elaeocarpicola, the inhibition rate of the fermentation broth of the strain P45-1 against P. elaeocarpicola was 52.58%. The strain P45-1 was identified as Trichoderma virens based on morphological characteristics and rpb2-tef1 sequence analysis. There existed significant differences among media for this fungal growth, the optimal growth medium was MEA. The optimal carbon was mannitol. The optimal nitrogen source was L-asparagine. The optimal inorganic salt were KNO3 and no inorganic salt. The optimal temperature was 25℃, and the hypha was inhibited significantly when the temperature fell below 20℃ or rose above 35℃. This fungus also tolerated very wide range of pH, and grew well under pH 4.0 to 7.0. The optimal pH was 5.0.
      Conclusion T. virens P45-1 isolated from wastewater samples at the distillery's discharge outlet can be used for the biocontrol of P. elaeocarpicola. The experimental results of biological characteristics of the strain P45-1 clarified the optimal conditions for mycelial growth.