黄颡鱼卵致病性绵霉的分离鉴定与药敏特性

Identification and drug susceptibility of a pathogenic Achlya klebsiana strain from the eggs of yellow catfish (Pelteobagrus fulvidraco)

摘要:

【目的】对黄颡鱼水霉病病原进行分离鉴定, 并对其药敏特性进行研究。【方法】参照传统方法对患水霉病的黄颡鱼卵上的丝状真菌进行分离, 通过人工感染实验证实分离菌株的致病性, 然后根据形态学特征和ITS rDNA序列分析对致病菌株进行鉴定, 并进一步采用倍比稀释法研究其药敏特性。【结果】从患水霉病的黄颡鱼卵上分离到8株丝状真菌, 经人工感染试验证实菌株YC对黄颡鱼卵具有致病性, 并进一步研究了其形态与药敏特性, 开展了ITS rDNA序列分析。结果表明, 菌株YC为透明管状结构, 中间无横隔; 游动孢子囊多呈圆筒形、棍棒形或穗状, 游动孢子发育成熟后不断从孢子囊顶端释放出来, 成团聚集在游动孢子囊口, 并经过一个时期的静休后, 成团脱落或直接分散在水中游动; 次生孢子囊具有典型的侧生现象; 藏卵器呈球形或梨形, 大多与雄器异枝, 少数与雄器同枝, 含1?15个卵孢子; 成熟卵孢子中生或亚中生, 偏生一个大油球。其ITS rDNA序列与GenBank基因库中绵霉属菌株自然聚类, 同源性高达99%, 与异丝绵霉(Achlya klebsiana)菌株CBS101.49 (GenBank登录号AF119579)的亲缘关系最近。结合形态特征与ITS序列鉴定的结果, 判定菌株YC为异丝绵霉(Achlya klebsiana)。此外, 在实验选用的中草药和消毒剂中, 黄连和异噻唑啉酮分别对菌株YC的抑菌效果最好, 其对菌株YC的最小抑菌浓度分别为256 mg/L和2 mg/L。【结论】首次分离了黄颡鱼卵致病性异丝绵霉菌株YC, 并确定了其药敏特性, 可以作为该病防治用药的依据。

Abstract:

[Objective] The aim of the present study was to isolate and identify the pathogen of Pelteobagrus fulvidraco egg saprolegniasis, and study the drug susceptibility characteristics of the pathogen. [Methods] Filamentous fungal strains were first isolated using the traditional method from the Pelteobagrus fulvidraco eggs suffering from saprolegniasis, and the pathogenic strain was further confirmed through artificial infection experiment, identified using morphological observation and phylogenetic analysis based on its ITS rDNA sequence. In addition, its drug susceptibility was studied using the doubling dilution method. [Results] Eight filamentous fungal strains were isolated form Pelteobagrus fulvidraco eggs with saprolegniasis, and strain YC was proved to be pathogenic to Pelteobagrus fulvidraco eggs by artificial infection. Thus, morphological characterizations of strain YC were studied, the phylogenetic analysis based on its ITS rDNA sequence and drug susceptibility assay were also further conducted. The experimental results showed that the hyphae of strain YC were aseptate and transparent, its zoosporangia were cylindrical, clavate or fusiform, its zoospores were discharged from the tip of zoosporangia, clumped together and fell off or directly swam into the water after a period of time. The zoosporangia were renewed in basipetalous succession, infrequently in sympodial arrangement. Its oogonia were spherical or obpyriform with frequently diclinous and occasionally monoclinous antheridial branches, and produced one to fifteen oospores, which were centric or subcentric, and laterally generated a big oil body. The ITS rDNA sequences of strain YC was naturally clustered with ITS rDNA sequences of Achlya sp. submitted to GenBank with the homology of 99%, and had closest relationship with Achlya klebsiana strain CBS101.49 (GenBank accession numberAF119579). Combined the morphological characterization with phylogenetic analysis based on ITS rDNA sequence, strain YC was identified as Achlya klebsiana. In addition, strain YC was well inhibited by Coptis chinensis and isothiazolinone, whose minimum inhibitory concentrations to strain YC were respectively 256 mg/L and 2 mg/L. [Conclusion] A pathogenic Achlya klebsiana strain YC was isolated for the first time from Pelteobagrus fulvidraco eggs suffering from saprolegniasis, and its drug susceptibility was also determined, which could serve as a foundation for its infection in Pelteobagrus fulvidraco eggs.