七彩神仙鱼水霉病的鉴定及其防治方法的研究

发布时间：2018-09-05 21:18

【摘要】：水霉病(Saprolegniasis)是淡水养殖的常见病害,其发病地域广,对宿主无选择性,感染品种多,在5℃-30℃均可发生。国内外众多真菌学家和水产学家对其进行了研究,取得了一定的进展。但是这类疾病至今仍没有很好的解决,目前还没有确定是哪些种类的水霉菌导致七彩神仙鱼发病。七彩神仙鱼水霉菌病原种类的鉴定及其生活史的观察,以及其防治方法的研究亟待解决。鉴此,本论文通过提取患有水霉病的七彩神仙鱼鱼卵,并通过人工感染实验验证,获得一株致病菌株YL2,并对其使用传统形态学和ITS相结合的现代分子学方法对其种类进行鉴定,结果显示七彩神仙鱼水霉病的致病菌为寄生水霉(S aprolegnia parasitica);进一步对其防治方法进行了探究,使用不同药物对YL2菌丝的抑制效果进行了比较,发现卡松对其的抑制效果较好,然后探讨分析其对七彩神仙鱼的安全性;最后通过人工感染试验对其抑菌效果进行验证。本文的研究结果为:1.菌株YL2的分离鉴定取实验室的患有水霉病七彩神仙鱼卵,通过分离纯化的方法,得到一株致病水霉菌,命名为YL2,采用现在最常用的水霉菌种类鉴定的方法,即传统形态学和分子生物ITS序列分析技术作为菌株种类的鉴定方法。试验除常用方法外,使用现在最新的微生物裂解液,可以直接释放核酸用于PCR检测反应,作为YL2菌株DNA的提取方法以上两种方法,均鉴定菌株YL2为寄生水霉(Saprolegnia parasitica),相比而言后一种方法更加高效的提取DNA,能够达到微生物快速进行PCR检测的目的,可作为水霉菌DNA提取研究的新方法。2.菌株YL2的生活史YL2菌株菌丝外观为白色棉絮状,经乳酸棉兰染液固定,于显微镜下观察到是无隔透明的管状结构;恒温水培于倒置显微镜下观察其生活史,在不同温度下培养时25℃时生长最好,25℃时最适合菌株YL2的生长,这与七彩神仙鱼生长孵化的温度比较接近,猜测此是七彩神仙鱼在孵化时易被感染的主要原因;在20℃-25℃恒温条件下,第3d有棒状孢子囊形成,第4d开始释放孢子,释放的方式为典型的水霉属菌模式,在15℃恒温条件下能够产生大量有性器官,第5d时有大量的球形藏卵器形成,与雄器主要为同体或者同枝,是水霉属的典型形态特征。3.菌株YL2的药敏特性通过采用四种不同类型的药物对水霉菌杀菌效果进行研究,药物包括益生菌,消毒剂类、防腐剂类和酶类。通过比较分析四类药物对于水霉菌YL2菌丝的生长抑制力和对水霉菌孢子萌发的的抑制浓度间的差异,发现:益生菌和酶类对于抑制菌丝生长的效果不明显,防腐剂和消毒剂类均可抑制菌丝生长,通过高、中、低3个浓度组之间组内比较,发现四种药物抑制效果为:卡松聚维酮碘溴硝丙二醇=过氧乙酸,且浓度增大抑菌能力增强,得到卡松的抑菌效果最好;益生菌和酶类在对于水霉菌孢子萌发无抑制效果,防腐剂和消毒剂类则可以抑制其孢子萌发,其中浓度为16mg/L即可抑制80%的水霉菌孢子萌发,而聚维酮碘、过氧乙酸和溴硝丙二醇,能够抑制80%的水霉孢子萌发的浓度均比常用高浓度大。因此,得出卡松对于水霉菌具有较好的抑制效果。4.卡松对七彩神仙鱼的防治效果卡松是国际上公认的日用品广谱防腐剂,且在安全范围内长期,反复使用是安全的。试验测定了卡松对七彩神仙鱼半数致死浓度(LC50)和安全浓度(SC),并在之前卡松对水霉菌YL2菌丝抑菌力和孢子萌发的抑菌浓度基础上,通过人工反感染试验,探索了卡松对七彩神仙鱼水霉病预防效果的研究。最后,发现浓度为16mg/L的卡松能够有效地抑制水霉菌的菌丝生长和孢子的萌发,从而达到抑制水霉菌的效果,可以作为水霉病的预防药物。
[Abstract]:Saprolegniasis is a common disease in freshwater aquaculture. It has a wide incidence area, no selectivity to the host and many kinds of infection. It can occur at 5-30 C. Many mycologists and aquatic scientists at home and abroad have studied it and made certain progress. The identification of the pathogenic species of Narcissus and the observation of its life history as well as the study of its prevention and treatment methods need to be solved urgently. Its species were identified by modern molecular methods combined with traditional morphology and ITS. The results showed that the pathogenic bacteria of Narcissus diversicolor was S. parasitica, and its control methods were further explored. The results of this study are as follows: 1. Isolation and identification of strain YL2 from the laboratory eggs of the seven-colored immortal fish infected with water mold, through the method of isolation and purification, a pathogenic water mold was obtained. Named YL2, traditional morphological and molecular biological ITS sequence analysis techniques are used to identify strains. In addition to the common methods, the latest microbial lysates can be used to directly release nucleic acids for PCR detection reaction, which can be used as the DNA extraction method of YL2 strain. Two methods were used to identify strain YL2 as Saprolegnia parasitica. Compared with the latter method, it is more efficient to extract DNA, which can achieve the purpose of rapid PCR detection of microorganisms. It can be used as a new method for DNA extraction of water mold. 2. The life cycle of strain YL2 is white cotton flocculent mycelium appearance, after milk. Fixed with acid cotton blue dye solution, the tubular structure without septum was observed under microscope; the life history of the fish was observed under inverted microscope under constant temperature water culture. The strain YL2 grew best at 25 C under different temperatures, and the strain YL2 grew best at 25 C, which was close to the incubation temperature of the fish. The main reason for susceptibility to infection during incubation was that rod-shaped sporangia formed on the 3rd day and released spores on the 4th day under the constant temperature of 20-25 C. The release mode was typical of Hygromycetes. Under the constant temperature of 15 C, a large number of sexual organs could be produced. On the 5th day, a large number of spherical oviducts were formed, and the male organs were mainly homologous or homologous. 3. The antimicrobial susceptibility of strain YL2 was studied by using four different types of drugs, including probiotics, disinfectants, preservatives and enzymes. The inhibitory effect of the four drugs on hyphal growth of Hyphae YL2 and spore germination of Hyphae were compared and analyzed. The results showed that probiotics and enzymes had no obvious effect on inhibiting the growth of mycelium, preservatives and disinfectants could inhibit the growth of mycelium. By comparing the three groups with high, medium and low concentrations, the inhibiting effects of four drugs were found as follows: Casson povidone iodobromopropylene glycol = peroxyacetic acid, and the concentration increased the inhibiting ability. Probiotics and enzymes had no inhibitory effect on the spore germination of Hygromycetes, preservatives and disinfectants could inhibit the spore germination of Hygromycetes. The concentration of 16 mg/L could inhibit 80% of the spore germination of Hygromycetes, while povidone iodine, peracetic acid and bromopropylene glycol could inhibit 80% of the spore germination of Hygromycetes. The germination concentration of Casson is higher than that of common high concentration. Therefore, it is concluded that Casson has better inhibition effect on water mold. 4. Casson is an internationally recognized broad-spectrum antiseptic in daily use, and it is safe to be used repeatedly for a long time in a safe range. On the basis of LC50 and SC, and on the basis of the previous inhibitory concentration of Casson on Hyphae and Spore Germination of Hyphae of Hyphae Hyphae YL2, the preventive effect of Casson on Hyphae and Spore Germination of Hyphae hyphae was studied by artificial anti-infection test. Finally, it was found that the concentration of 16 mg/L Casson could effectively inhibit Hyphae growth and Spore germination of Hyphae hyphae. The germination of spores can inhibit the effect of water mold and can be used as a preventive medicine for water mildew.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S943

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