| Saprolegnia parasitica often leads to fungal infection in freshwater fish and roe, thus causing saprolegniasis. Frequent occurrence of saprolegniasis will result in the widespread mortality, which will severely affect the healthy development of aquaculture. Current drugs for the treatment of saprolegniasis in aquaculture animals that have been infected are not ideal. Euphorbia ebracteolata and Stellera chamaejasme stellera had a good inhibitory effect on fungus, but there is no effect on Saprolegnia parasitica. To explore the influence on Saprolegnia parasitica, this study uses the Systematic solvent extraction and ultrasonic extraction were performed to obtain Euphorbia ebracteolata and Stellera chamaejasme. The minimal inhibitory concentrations of both Langdu active ingredients on Saprolegnia parasitica were compared, in order to explore their influence on the growth of Saprolegnia parasitica. Ethanol crude extract of stellera chamaejasme was studied in Saprolegnia parasitica growth, spore germination rate, the influence of the morphology and ultrastructure of hyphae. Treatment process and by simulating explores the ethanol extract of crucian carp parasitic of stellera chamaejasme treatment effect of saprolegniasis.1. A strain of Saprolegnia was isolated from infected crucian carp, which was purified and cultured. The disease-inducing strain was identified through morphology and molecular biology as Saprolegnia parasitica.2. According to the size of the solvent polarity.petroleum ether, ethyl acetate, acetone, ethanol and water were selected as the solvent system, followed by ultrasonic extraction of the active ingredients of Euphorbia ebracteolata and Stellera chamaejasme root powders under optimal conditions. The results indicate that:water solvent for Euphorbia ebracteolata has the highest extraction rate was 21.38%, the acetone solvent extraction yield of Euphorbia ebracteolata lowest is 0.98%, the rest of the solvent extraction yield of Euphorbia ebracteolata respectively:2.19%,1.91%,1.51%;Ethyl acetate solvent extraction yield of 12.03% is the highest of Stellera chamaejasme and petroleum ether extraction of Stellera chamaejasme lowest is 0.50%, the rest of the solvent extraction of stellera respectively:4.22%,1.45%,3.26%.3. Microdilution was performed to determine the minimum inhibitory concentration of extracts obtained by petroleum ether, ethyl acetate, acetone, ethanol and water solvents. Among them, the minimum inhibitory concentration of extracts for Euphorbia ebracteolata root powder was 2.5mg/mL, and the optimum solvent was ethanol; the minimum inhibitory concentration of extracts for Stellera chamaejasme root powder was 0.15625mg/mL, and the optimum solvent was ethanol.4. By measuring the effects of Stellera chamaejasme alcohol extract on the growth of Saprolegnia parasitica colonies, we discovered that experimental group colonies growth blank diameter was significantly lower than control group (P<0.05). The inhibition rate of Stellera chamaejasme alcohol extract at a concentration of 0.3125mg/mL on Saprolegnia parasitica growth was 100%, The toxicity regression equation was:Y=3.1503X+9.9012, where EC50 was 0.0278 1mg/mL,the ethanol crude extract of Stellera chamaejasme mold inhibitory effect of the Saprolegnia parasitica close to the methylene blue mold inhibitory effect. After culturing at 20℃ for 24h, the mean germination rate of Saprolegnia parasitica spores in the blank control group was 86.67%. When the concentration of Saprolegnia parasitica alcohol extract was 0.625mg/mL, the mean germination rate of Saprolegnia parasitica spores was 0.00%. The regression equation for the extract concentration and germination rate of Saprolegnia parasitica spores was Y=290.45X2 —— 304.45X+78.106. Optical microscopy revealed transparent Saprolegnia parasitica mycelium with inhomogeneous staining after treatment with Saprolegnia parasitica alcohol extract. SEM showed that the surface of Saprolegnia parasitica mycelium was rough and wrinkled after treatment with Stellera chamaejasme alcohol extract. Observations under TEM indicated that Saprolegnia parasitica spores treated with Stellera chamaejasme alcohol extract showed cell wall,cytoplasm electron density is low, the cell contents appear a large number of vacuoles, cell structures are destroyed. In addition, Saprolegnia parasitica mycelium treated with Stellera chamaejasme alcohol extract showed the cell walls of fuzzy, shrivel, or even disappear, cytoplasm electron density is low, the cell contents, adjacent cell membrane appear some adhesion phenomenon.The ethanol crude extract of stellera chamaejasme can by destroying Saprolegnia parasitica spore silk, cellular structure effectively restrain Saprolegnia parasitica growth.5. Simulation studies of the effects of treatment process of crucian carp, only 0.625 mg/mL group treatment effect is significantly higher than blank control group (P< 0.05). Simulation treatment trials observed after sterile knife treatment experiment fish have no obvious symptoms; saprolegniasis crucian carp swimming slow response, imbalances and even death phenomenon, observed in the experiment of death fish eye dot haemorrhage, Similar to crucian carp’s poisoning symptoms observation test results. In the statistical analysis of the test fish mortality, Uninfected saprolegniasis test fish no deaths. Infection saprolegniasis crucian carp,test 24h, only 0.625mg/mL solution soak group had a significantly higher mortality in blank control group (P<0.05); Test 48h, drug concentration is 0.15625mg/mL crucian carp mortality rate immersion group were significantly higher than that of blank control group (P< 0.05); Test 72h,0.15625mg/mL, 0.625mg/mL solution soak group had a significantly higher mortality in blank control group (P<0.05).The ethanol extract of stellera chamaejasme could have therapeutic effect on Saprolegnia parasitica, but leads to crucian carp with saprolegniasis poisoning or even death, and thus affect the treatment effect. |
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