七彩神仙魚水霉病的鑒定及其防治方法的研究
發(fā)布時間：2018-09-05 21:18
【摘要】：水霉病(Saprolegniasis)是淡水養(yǎng)殖的常見病害,其發(fā)病地域廣,對宿主無選擇性,感染品種多,在5℃-30℃均可發(fā)生。國內外眾多真菌學家和水產學家對其進行了研究,取得了一定的進展。但是這類疾病至今仍沒有很好的解決,目前還沒有確定是哪些種類的水霉菌導致七彩神仙魚發(fā)病。七彩神仙魚水霉菌病原種類的鑒定及其生活史的觀察,以及其防治方法的研究亟待解決。鑒此,本論文通過提取患有水霉病的七彩神仙魚魚卵,并通過人工感染實驗驗證,獲得一株致病菌株YL2,并對其使用傳統(tǒng)形態(tài)學和ITS相結合的現(xiàn)代分子學方法對其種類進行鑒定,結果顯示七彩神仙魚水霉病的致病菌為寄生水霉(S aprolegnia parasitica);進一步對其防治方法進行了探究,使用不同藥物對YL2菌絲的抑制效果進行了比較,發(fā)現(xiàn)卡松對其的抑制效果較好,然后探討分析其對七彩神仙魚的安全性;最后通過人工感染試驗對其抑菌效果進行驗證。本文的研究結果為:1.菌株YL2的分離鑒定取實驗室的患有水霉病七彩神仙魚卵,通過分離純化的方法,得到一株致病水霉菌,命名為YL2,采用現(xiàn)在最常用的水霉菌種類鑒定的方法,即傳統(tǒng)形態(tài)學和分子生物ITS序列分析技術作為菌株種類的鑒定方法。試驗除常用方法外,使用現(xiàn)在最新的微生物裂解液,可以直接釋放核酸用于PCR檢測反應,作為YL2菌株DNA的提取方法以上兩種方法,均鑒定菌株YL2為寄生水霉(Saprolegnia parasitica),相比而言后一種方法更加高效的提取DNA,能夠達到微生物快速進行PCR檢測的目的,可作為水霉菌DNA提取研究的新方法。2.菌株YL2的生活史YL2菌株菌絲外觀為白色棉絮狀,經乳酸棉蘭染液固定,于顯微鏡下觀察到是無隔透明的管狀結構;恒溫水培于倒置顯微鏡下觀察其生活史,在不同溫度下培養(yǎng)時25℃時生長最好,25℃時最適合菌株YL2的生長,這與七彩神仙魚生長孵化的溫度比較接近,猜測此是七彩神仙魚在孵化時易被感染的主要原因;在20℃-25℃恒溫條件下,第3d有棒狀孢子囊形成,第4d開始釋放孢子,釋放的方式為典型的水霉屬菌模式,在15℃恒溫條件下能夠產生大量有性器官,第5d時有大量的球形藏卵器形成,與雄器主要為同體或者同枝,是水霉屬的典型形態(tài)特征。3.菌株YL2的藥敏特性通過采用四種不同類型的藥物對水霉菌殺菌效果進行研究,藥物包括益生菌,消毒劑類、防腐劑類和酶類。通過比較分析四類藥物對于水霉菌YL2菌絲的生長抑制力和對水霉菌孢子萌發(fā)的的抑制濃度間的差異,發(fā)現(xiàn):益生菌和酶類對于抑制菌絲生長的效果不明顯,防腐劑和消毒劑類均可抑制菌絲生長,通過高、中、低3個濃度組之間組內比較,發(fā)現(xiàn)四種藥物抑制效果為:卡松聚維酮碘溴硝丙二醇=過氧乙酸,且濃度增大抑菌能力增強,得到卡松的抑菌效果最好;益生菌和酶類在對于水霉菌孢子萌發(fā)無抑制效果,防腐劑和消毒劑類則可以抑制其孢子萌發(fā),其中濃度為16mg/L即可抑制80%的水霉菌孢子萌發(fā),而聚維酮碘、過氧乙酸和溴硝丙二醇,能夠抑制80%的水霉孢子萌發(fā)的濃度均比常用高濃度大。因此,得出卡松對于水霉菌具有較好的抑制效果。4.卡松對七彩神仙魚的防治效果卡松是國際上公認的日用品廣譜防腐劑,且在安全范圍內長期,反復使用是安全的。試驗測定了卡松對七彩神仙魚半數(shù)致死濃度(LC50)和安全濃度(SC),并在之前卡松對水霉菌YL2菌絲抑菌力和孢子萌發(fā)的抑菌濃度基礎上,通過人工反感染試驗,探索了卡松對七彩神仙魚水霉病預防效果的研究。最后,發(fā)現(xiàn)濃度為16mg/L的卡松能夠有效地抑制水霉菌的菌絲生長和孢子的萌發(fā),從而達到抑制水霉菌的效果,可以作為水霉病的預防藥物。
[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.
【學位授予單位】：上海海洋大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S943
本文編號：2225490
[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.
【學位授予單位】：上海海洋大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S943
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