养殖日本鳗鲡腐皮病真菌性病原的分离与鉴定

ISOLATION AND IDENTIFICATION OF FUNGAL PATHOGEN FROM DERMAL ULCERATION DISEASE ON CULTURED ANGUILLA JAPONICA

针对近年来人工养殖日本鳗鲡(Anguilla japonica)在越冬过程中广泛发生、传染速度快、死亡率高的腐皮病进行了真菌性病原的分离与鉴定。以麦粒培养法从患病鳗鲡的病灶处分离获得1株真菌菌株Js80122;通过孢子悬液背部肌肉注射、培养物创伤浸泡、孢子创伤涂抹、孢子与细菌混合注射等方法人工感染健康鳗鲡,证实了Js80122为日本鳗鲡腐皮病的病原菌。于不同培养温度条件下,以载玻片培养观察法和盖玻片插片培养观察法对Js80122进行生活史观察与形态学鉴定,结果显示Js80122为丝状真菌,菌丝无横隔,分枝发达,具无性与有性两种生殖方式,动孢子囊呈棍棒形或纺锤形,新生孢子囊以层出或聚伞状方式生长,孢子具两游现象,藏卵器中有1个以上的卵孢子,卵孢子光滑无皱缩。依据其形态学特征,鉴定Js80122为水霉科(Saprolegniaceae)、原绵霉属(Protoachlya Coker)、原绵霉(Protoachlya paradoxa)。

In recent years,dermal ulceration disease is one of the most serious diseases on cultured Japanese eels（An-guilla japonica）,which occurs widely,spreads quickly and has caused great expense through the winter.In the begin-ning,the grume of the diseased eels are hyperplastic and then broken off.Following the evolvement of the illness,the derma is bareness and the muscle is necrotic.Eventually,the derma and the muscle are rotted.The isolation and identification of the fungal pathogen of this disease was studied.The fungal strain Js80122 was isolated from diseased eels by cultured on kernels of wheat.Strain Js80122 was confirmed as the causative pathogen of the dermal ulceration disease by artificial infection tests,which lasted for 2 weeks.In these tests,healthy eels were in-fected with four kinds of different methods,namely,injected with spores（0.3 mL/ind.） on dorsal muscle,marinated with 3 dish of culture after ravaged,daubed with spores（1.0×108 cell/mL）on wound and injected with spores and bacteria（0.3 mL/ind.） on dorsal muscle.The results showed that dermal ulceration disease could be recurred well on the experimental eels by any infection method;the symptom and process were similar to the naturally diseased eels.During the course of the artificial infection tests,the mortality were up to 100% in the group injected with spores（5.0×107 cell/mL） and bac-teria（5.0×107 CFU/mL） on dorsal muscle,80% in the group injected with spores（1.0×108 cell/mL） on dorsal muscle,60% in the group daubed with spores on wound,20% in the group marinated with culture after ravaged,respectively.Observation on the morphology and life history of strain Js80122 cultured on slide and cover glass showed that strain Js80122 was the filamentous fungi and could grow well at 15℃,20℃ and 25℃.Its hyphae were non-aseptate with plentiful branches,the reproductive modes were asexual and sexual.The asexual structure of Js80122 could be well produced at 20℃ or 25℃,the sexual structure could not be produced at those temperatures hereinbefore,even cultured for 4 weeks,but it could be produced at 15℃ when the strain was cultured for more than 3 weeks.Zoosporangium was clavate or spindly and renewed by internal or cymose proliferation,zoospores were diplanetic.Every oogonium could form more than one oospore,which were smooth.Flagella stain with AgNO3 revealed that there were two flagella with almost equal length on the zoospore.According to the characteristics of morphology and life history,such as the modal-ity of the hyphae,the reproductive modes,the modality and the mode of movement of the zoospores,the amount and modality of the oospores,etc,strain Js80122 was identified as Saprolegniaceae,Protoachlya Coker,Protoachlya para-doxa.Epidemiological investigation showed that the epidemic temperature of the dermal ulceration disease was under 20℃,but it was nonoccurrence when the aquatic temperature exceeded to 23℃,and it could not be prevented and con-trolled by using multifarious disinfectors or antibacterial medicaments in breed aquatics of Japanese eels,contrarily,it would be more serious if the antibacterial medicaments were used in the beginning.Additionally,the fungal hyphae could be easily observed under the microscope even at the initial stage of the disease,when the derma of the diseased eels was unworn and the muscle had not been rotted.Strain Js80122 could cause dermal ulceration disease even the experimental eels were infected with spores alone as already noted.According to the results of epidemiological investi-gation and this study,it could be confirmed that the primary pathogen of dermal ulceration disease on cultured Japanese eels was not any pathogenic bacteria but fungi,in the concrete,it was Protoachlya paradoxa,the bacteria caused sec-ondary infection,and they brought on nothing more than the worse illness.