水霉菌的形态及ITS区分子鉴定
SAPROLEGNIA IDENTIFICATION BASED ON THEIR MORPHOLOGICAL CHARACTERISTICS AND ITS rDNA REGION
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摘要: 研究利用形态学和分子生物学相结合的方法, 对从患病黄颡鱼病灶处分离的水霉菌株(HSY)的分类地位进行了研究。结果显示, 该菌在18—23℃均能形成游动孢子囊, 但只在20℃才大量释放游动孢子; 在15℃培养约3 周出现大量的藏卵器, 多数顶生, 形态与Saprolegnia litoralis 和Saprolegnia ferax 的均非常相似。序列比对分析显示, HSY 菌株与S. litoralis、S. bulbosa、S. oliviae、S. longicaulis、S. ferax、S. mixta 和S.anomalies 的ITS 区(包括5.8S rDNA)序列相似性均高于99%。系统发育分析显示, 所选的真菌分为3 个群,HSY 落于第一个群, MP 树显示HSY 菌株与S. litoralis、S. bulbosa、S. oliviae、S. longicaulis、S. ferax 以及S. mixta 枝系均互相平行, 而NJ 树显示其与S. ferax 和S. mixta 更近。综合形态学、ITS 区序列及系统发育分析, 将HSY 菌株暂定为多子水霉(Saprolegnia ferax)。Abstract: Saprolegnia is one of the main genus of water molds responsible for fungal infections of freshwater fish and their eggs. These infections are usually termed “saprolegniasis”, which can cause severe losses of freshwater fish in both nature and commercial fish farms. However, identification of the causative agent is often unmanageable, especially to those isolated from fish lesions. Traditionally, the generic definition was mainly based on the asexual characters, especially the mode of zoospores discharge, and species differentiation was mainly on the features of sexual reproductive organs including oogonia, antheridia, antheridial origin and oospore. However, many of these characters may be variable in one species or similar in different species and some strains either lose the ability to produce sexual reproduction or cannot be induced to form them under laboratory conditions. So, it is difficult to make definitive identification only using traditional morphological criteria in species level.In this study, we identified one saprolegnia isolate HSY obtained from yellow catfish (Pelteobagrus fulvidraco) based on the morphological and molecular characteristics. Results showed Isolate HSY grew luxuriantly on hemp seeds in water at 18—23℃ and were measured 1.5—4.0cm in diameter. The hyphae were stout, sparingly branched and measured between 20—58μm in diameter. Zoosporangia had formed abundantly and were often fusiform, clavate, straight or bent, lateral or terminal and renewed internally, measured 110—490 × 25—62μm. Primary zoospore discharge was typically saprolegnoid, pyriform; secondary zoospores emerged from the encysted, reniform; encysted zoospores were globose and measured 9—12μm in diameter. Oogonia were formed abundantly within three weeks at 15℃, not 18℃ and 23℃, mainly terminal, rarely lateral, and measured 75—108μm in diameter. Oogonial walls were sooth, and stalks were stout and bent. The attachment of antheridial cells was moderate (mainly androgynous and monoclinous, rarely diclinous). The oosproes were centric or subcentric, 3—25 per oogonium, and meansured 18—30μm. These morphological were very similar to S. litoralis and S. ferax.Comparing 744 base pairs of internal transcribed spacer (ITS) region and the 5.8S rDNA, we found a group of currently identified saprolegnia species including S. litoralis and S. ferax shared an almost identical ITS sequence (above 99% similarity) with that of isolate HSY. Then, thirty-nine available sequences for representative Saprolegnia spp. formed three phylogenetically separate clades. Isolate HSY fell into clade I which also comprised a group of isolates showing high similarities among ITS sequences but different in their morphological features. Moreover, both NJ and MP tree showed that almost all these species in clade I were parallel to each other. These suggested the isolates in clade I might be the same or closely related phylogenntic species and showed how unsatisfactory oogonium morphology appeared to be as a predictor of genetic relatedness.Consequently, in our opinions, HSY should be identified as S. ferax because S. litoralis and S. ferax could not be differentiated only by morphology; secondly, S. ferax was the first named one which has the similar sexual organs with isolate HSY; finally, the similarity of the ITS regions of S. ferax and HSY was above 99%. Meanwhile, the present results evidently suggested that Saprlegnia classification only based on sexual organs were likely to lead to artificial species. Many species once identified as different might be just one thing in fact.
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Keywords:
- Oomycetes /
- Saprolegnia /
- Taxonomy /
- ITS rDNA /
- Phylogenetics
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