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1、微生物学通报 APR 20, 2012, 39(4): 579 587 Microbiology China 2012 by Institute of Microbiology, CAS 基金项目：国家 863 计划项目(No. 2011AA10A216); 现代农业产业技术体系建设专项资金项目(No. CARS-46-12); 公益性行业(农业)科研专项(No. 201203085) 共同第一作者 *通讯作者：Tel: 86-21-61900453; Fax: 86-21-61900452; : 收稿日期：2011-09-23; 接受日期：2011-11-11 主编点评文章 黄颡鱼卵水霉
2、病病原的分离鉴定及 其无性繁殖特性 许佳露 曹海鹏 欧仁建 杨先乐* (上海海洋大学 国家水生动物病原库 上海 201306) 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
3、 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 C35
4、C、pH 410 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 525 mg/L 福尔马林和0.251.25 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。 关键词: 黄颡鱼卵, 多子水霉, 鉴定, 无性繁殖 580 微生物学通报 2012, Vol.39, No.4 http:/ Identification and asexual reproduction characterization of a Saprolegnia fe
5、rax pathogen from the egg of Yellow catfish (Pelteobagrus fulvidraco) XU Jia-Lu CAO Hai-Peng OU Ren-Jian YANG Xian-Le* (National Pathogen Collection Center for Aquatic Animals, Shanghai Ocean University, Shanghai 201306, China) Abstract: Objective The aim of the present study was to isolate and iden
6、tify the pathogen of Pelteobagrus fulvidraco eggs suffering from saprolegniasis, and study its asexual reproduction characteristics. Methods Filamentous fungal strains were first isolated from the Pelteoba- grus fulvidraco eggs suffering from saprolegniasis using the traditional method. The patho- g
7、enic 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 filamento
8、us 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 ana
9、lysis 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
10、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
11、 had closest relationship with Saprolegnia ferax strain Arg4S (GenBank acces- sion number: GQ119935). Combined morphological characterization with phylogenetic analy- sis based on ITS rDNA sequence, strain HP was identified as Saprolegnia ferax. In addition, strain HP could produce zoospores at 5 C3
12、5 C and pH 410, its optimum temperature and pH for the zoospore production were 20 C and 7, respectively. It was greatly inhibited by 525 mg/L formalin and 0.251.25 mg/L dithiocyano-methane. Conclusion The pathogen of Pelteobagrus fulvidraco egg saprolegniasis was isolated and identified in the pres
13、ent study, and its asexual reproduction characteristics was also determined, which could serve as a foun- dation for the control of Pelteobagrus fulvidraco egg saprolegniasis. Keywords: Pelteobagrus fulvidraco eggs, Saprolegnia ferax, Identification, Asexual reproduction 黄颡鱼(Pelteobagrus fulvidraco)
14、是一种小型名优淡水鱼类, 味道鲜美, 营养丰富, 深受广大消费者的喜爱, 在日本、韩国、东南亚等国家具有巨大的市场, 是我国出口创汇的优质特种鱼类1。然而, 黄颡鱼在鱼卵孵化过程中因水温等环境因素的变化易患水霉病而大批死亡, 严重影许佳露等: 黄颡鱼卵水霉病病原的分离鉴定及其无性繁殖特性 581 http:/ 响了苗种生产的规模化进程2。因此, 开展黄颡鱼卵水霉病的病原学研究对其苗种繁殖生产具有重要的意义。2011 年 6 月份, 仙桃市沙湖水产技术推广站人工繁殖的黄颡鱼卵发生了严重的水霉病, 因生长迅速的水霉菌丝在一定程度上导致鱼卵周围的水流减缓, 阻碍氧气的运输, 引起鱼卵死亡, 使孵化率
15、降低。鉴于此, 本实验从患水霉病的黄颡鱼卵上分离了一株致病性水霉菌HP, 并观察了温度、pH、福尔马林、二硫氰基甲烷对其游动孢子产生的影响, 以期为黄颡鱼卵水霉病的病原学研究与有效预防提供理论参考。 1 材料与方法材料与方法 1.1 菌株的分离与纯化 将发病鱼卵用 75%酒精浸洗 23 s, 然后用无菌蒸馏水冲洗数次, 置于加有 100 mg/L 链霉素青霉素的马铃薯葡萄糖琼脂培养基(PDA)平板上, 在 25 C 恒温培养 24 h后立即在长出的菌落边缘放置无菌油菜籽, 在25 C恒温继续培养, 待油菜籽上覆盖菌丝后将其取出并置于灭菌过滤河水中, 于 25 C 培养直至游动孢子释放。无菌吸取 100 L 孢子悬液至 PDA 平板上均匀涂布, 25 C 恒温培养并切取单菌落琼脂块到 PDA平板上进行纯化培养, 于 4 C 保存待用。 1.2 分离菌株的人工感染试验 将长有菌丝的油菜籽放到灭菌过滤河水中, 于 25 C 恒温培养直至产生大量游动孢子, 然后用三层无菌纱布过滤制成终浓度为 5103个/mL的孢子悬液。分别取 40 粒黄颡鱼受精卵放入装有孢子悬液的培养皿中, 用吸管多次吹打以充氧, 以放入灭菌过滤河水(不加孢子)的受精卵为对照。黄颡鱼受精卵在无菌水中清洗后, 镜检没有菌丝或孢子附着才用于试验。在显微镜下观察和记录卵的感染情况, 并
