| Saprolegnia was the main fungal pathogen of Saprolegniasis. Almost all freshwater aquatic animals were susceptible to this organism and it had a huge impact to aquaculture industry. Malachite green once be considered a specific drugs for Saprolegniasis, but some researcher later found its causing liver cell cancer when it went into the human food chain. With the forbidden using of malachite green, Saprolegniasis became a big problem to the healthy development of aquaculture again. So far, antibiotics, salt and formalin were used to prevent and treat Saprolegniasis. The existence of a problem was the poor control effects and not suitable for large area pond culture and other issues.Chinese herbal medicine were favored by the researchers for its wide collecting sources, low costs, and not easy to produce drug resistance. So far, Chinese herbal medicine were rarely used in aquaculture to treat saprolegniasis. Research work for screening the antifungal herbs and finding its main ingredients would not only help to solve the lack of effective clinical drugs to prevent saprolegniasis but also increase the types of drugs of our country and reduce the risk of aquatic food safety.Thirty Chinese herbs which had antifungal activity were found from dozens of references in present study. Antifungal activities were evaluated by using extraction, concentration methods and those that had sure antifungal function to Saprolegnia were found. Systematic solvent method was used to find different antifungal active parts of Magnolia Officinalis and a preliminary study was developed to analyze the active ingredient of the most active part using different checking methods.1. A pathogenic fungi strain CCF1301 was isolated from infected grass carp after several times purification by using PDA medium culture method. Hyphae smear were made to confirm the morphological features under the microscope. Purified hyphae were selected to extract DNA and 26S rDNA D1/D2 gene and ITS gene were amplified using some commercial kits and its’sequences were acquired by sequencing company. The obtained sequences were submitted to NCBI, and homology were analyzed using the Blast alignment tool. Phylogenetic tree also made by using Mega5.1 software. The results showed that the collected filamentous fungi were transparent tubular, non-septa, sparingly branched. Clavate sporangiums and zoospores existed in asexual reproduction and spherical archegonium appeared in sexual reproduction. The strain was initially classified as Saprolegnia according to the genus Saprolegnia description in handbook of fungi and those feature above. Two fragment around 800bp were amplified using commercial kit and the size of 26S rDNA D1/D2 and the ITS gene were 839bp and 798bp respectively. Submit those sequences to NCBI and the NCBI accession numbers were KJ577573 and KF871196 respectively. Using the blast tool of NCBI to compare ITS sequence with other sequences in GenBank database, it found homology of this strain and other Saprolegnia sp. was highest, the similarity rate was 99%. Phylogenetic trees were extablished using Mega 5.1 software and found 26S rDNA D1/D2 sequence and the sequence of JN400036 went to the same branch. It supported the conclusion that CCF1301 were Saprolegnia sp. On the same time. The sequence between ITS and JN400035 went to the same branch. It supported the conclusion that CCF1301 were Saprolegnia ferax.2. Thirty candidate herbs were crushed, sieved, extracted with 95% ethanol by refluxing liquid ratio 1:15 for 1.5h. Repeat it for three times and combined all the filtrate. The extracts then were filtered and evaporated in a vacuum rotary evaporator with 120 rpm/min,0.09Mpa at 75℃ until it can not flow and dissolved with solvent as a stocking solution. Plate method which included different drugs was developed to check the antifugal activity of different extracts at 2.0g/L(according to raw-herb count). Herbs which had good antifungal effect were selected to check the MIC to Saprolegnia spores using microdilution method. The results showed that Syzygium aromaticum, Magnolia officinalis, Rhus chinensis, Euphorbia fischeriana Steud, Sophora flavescentis Ait. exhibited the better inhabitation. The MIC values of those herbs were 0.5 g/L(according to raw-herb count), 62.5×10-3 g/L(according to raw-herb count),2.5×10-1 g/L(according to raw-herb count), 62.5×10-3 g/L(according to raw-herb count),2.5×10-1g/L(according to raw-herb count) respectively. The MIC of the other twenty-five medicine were ≥2.0 g/L(according to raw-herb count). It also showed can be seen, Magnolia officinalis and Euphorbia fischeriana Steud had a good resistance to Saprolegnia. Due to the natural toxic of Euphorbia fischeriana Steud, Magnolia officinalis was considered as the object of further study.3. Systematic solvent method was used to extract different active part of Magnolia officinalis using petroleum ether (60～90), ethyl acetate, ethanol, acetic acid, and water. The solvent in the extracts were evaporated using a rotary evaporator and the dryness were dissolved with a small amount of ethanol. Meanwhile, MIC of five extract parts were checked using micro-dilution method and confirmed the best effective parts. Use different methods to identify the possible constituents of the most active site. The results showed that compared with other parts of the extraction, petroleum ether parts of Magnolia officinalis had the most active antifungal activity; MIC of inhibiting Saprolegnia spore was 5.0×10-2 g/L(according to raw-herb count) and the main ingredients this site were the lipids, flavonoids, alkaloids, phenols and organic acids. |
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