黄颡鱼卵水霉病病原的分离鉴定及其无性繁殖特性

Identification and asexual reproduction characterization of a Saprolegnia ferax pathogen from the egg of Yellow catfish (Pelteobagrus fulvidraco)

摘要:

【目的】对黄颡鱼卵水霉病病原进行分离鉴定, 并对其无性繁殖特性进行研究。【方法】采用传统方法从患水霉病的黄颡鱼卵上进行丝状真菌的分离, 然后通过人工感染实验证实分离菌株的致病性, 通过形态学观察和ITS rDNA序列分析对致病菌株进行鉴定, 并进一步通过单因子法研究其无性繁殖特性。【结果】从患水霉病的黄颡鱼卵上分离了4株丝状真菌, 经人工感染试验证实其中一株丝状真菌HP对黄颡鱼卵具有致病性, 并进一步研究了其形态与无性繁殖特性, 开展了ITS rDNA序列分析。实验结果表明, 菌株HP菌丝为透明管状结构, 中间无横隔, 分枝较少; 游动孢子囊多数呈棒状, 游动孢子发育成熟后从孢子囊中释放出来, 并迅速游离; 能够产生第二孢孢子; 新孢子囊以内层出的方式产生; 藏卵器呈球形, 与雄器同枝或异枝。菌株HP的ITS rDNA序列与GenBank基因库中水霉属菌株自然聚类, 同源性高达99%, 与多子水霉菌株Arg4S (GenBank登录号GQ119935)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HP为多子水霉(Saprolegnia ferax)。此外, 菌株HP在5 °C?35 °C、pH 4?10范围内均能产生游动孢子, 产生游动孢子的最适温度和pH分别为20 °C和7, 而且5?25 mg/L福尔马林和0.25?1.25 mg/L二硫氰基甲烷对菌株HP产生游动孢子具有明显的抑制作用。【结论】分离鉴定了黄颡鱼卵水霉病病原, 并确定了其无性繁殖特性, 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 eggs suffering from saprolegniasis, and study its asexual reproduction characteristics. [Methods] Filamentous fungal strains were first isolated from the Pelteobagrus fulvidraco eggs suffering from saprolegniasis using the traditional method. The pathogenic strain was further confirmed through artificial infection experiment, and identified by using morphological observation and phylogenetic analysis based on its ITS rDNA sequence. Additionally, its asexual reproduction characteristics was studied using single factor method. [Results] Four filamentous fungal strains were isolated form Pelteobagrus fulvidraco eggs with saprolegniasis, and strain HP was proved to be pathogenic to Pelteobagrus fulvidraco eggs by artificial infection. Therefore, morphology and asexual reproduction characteristics of strain HP were studied, and the phylogenetic analysis based on its ITS rDNA sequence was further conducted. The experimental results showed that the hyphae of strain HP were aseptate, transparent and seldom branched. Its zoosporangia were often clavate and renewed internally. Primary zoospore was discharged in Saprolegnia fashion. New sporangium generated from the base of old sporangium by the way of internal proliferation. Spherical oogonia were attached by monoclinous or diclinous antheridium hyphae. The ITS rDNA sequences of strain HP were naturally clustered with ITS rDNA sequences of Saprolegnia sp. in GenBank with 99% of homology, and had closest relationship with Saprolegnia ferax strain Arg4S (GenBank accession number: GQ119935). Combined morphological characterization with phylogenetic analysis based on ITS rDNA sequence, strain HP was identified as Saprolegnia ferax. In addition, strain HP could produce zoospores at 5 °C?35 °C and pH 4?10, its optimum temperature and pH for the zoospore production were 20 °C and 7, respectively. It was greatly inhibited by 5?25 mg/L formalin and 0.25?1.25 mg/L dithiocyano-methane. [Conclusion] The pathogen of Pelteobagrus fulvidraco egg saprolegniasis was isolated and identified in the present study, and its asexual reproduction characteristics was also determined, which could serve as a foundation for the control of Pelteobagrus fulvidraco egg saprolegniasis.