Antagonistic Activity And Mechanism Of Anti-saprolegnia Bacteria

Saprolegniasis is a common disease of aquatic animals caused by some fungi thatbelong to Oomycetes Saprolegnia. Saprolegnia infections cause fish death,hatchability of eggs reduction, among them Saprolegnia parasitica spores germinatefaster, invasionstrong, particularly serious harm to freshwater fish, causing hugelosses to annual freshwater aquaculture.Whether in the field of medicine, agriculture or aquaculture, fungal infection hasbeen considered difficult to control microbial diseases. Malachite green, which rely ona long time for the prevention and treatment of Saprolegniasis-an effectivebroad-spectrum bactericidal chemical dyes, but due to its high carcinogenic,teratogenic, high residual hazards to the human body, has been the world-wide wasbanned aquaculture for food grade. A lot of literatures report that chemical drugs,antibiotics, vaccines and herbal can combat Saprolegnia infections, find there are alsoa variety of shortcomings and deficiencies.Thus for prevention and control ofsaprolegniasis,it’s still lack a security and effective method.As the core of biological antagonistic biological control method has theadvantages of green, environmentally friendly, safe and efficient. In recent yearsbiological control of rapid progress represents Saprolegniasis prevention and controlof mainstream direction in the aquatic field. With the increasing levels of nationalfood quality and safety,the fewer alternative drug source of new drugs, and screeningnew drugs has not to know whether there is potential safety hazard. With the growingmaturity and popularity of the technology of genetic engineering, to carry outbiological control or ecological control based on genetic engineering, and to becomethe preferred direction of the disease prevention and treatment Research.Its non-pathogenic and is considered to be Generally Recognized as Safe microorganisms, has developed into a highly anticipated host bacteria of theBacillus.This research compared several Bacillus strains that antagonize againstSaprolegnia to determine a Bacillus strain that inhibited Saprolegnia growthsignificantly-TCCC11322. In the experiment we determine the supernatant offermentation broth have Saprolegnia suppressing effect, through further experiment ofprotein, polysaccharide, polypeptide composition of the fermentation supernatant wasseparated, and research the inhibition activity of each component. The results showthat for a significant inhibition activity against Saprolegnia is the protein component.And it’s suppressing effect can achieve the same effect as100μl10ppm malachitegreen, with a further development and application value.Many literature reported that a variety of Bacillus spp strains can secrete proteaseto the surrounding environment, wherein the neutral protease and alkaline protease,which often have the effect of anti-fungal.Many neutral protease and alkaline proteasegene sequence of a variety of Bacillus strains were found and compared, and find thatthe sequence homology is very high. We designed degenerate primers on the basis ofhomologous sequences, the alkaline protease gene sequence of TCCC11322wereobtained, including signal peptide and the mature peptide sequence.According to the sequencing of TCCC11322strains alkaline protease genesequence design primers,and using pBSA43plasmid construct the recombinantexpression vector. Transforming Bacillus subtilis expression strain WB600,constructed Bacillus subtilis alkaline protease expression strain, for secretoryexpression and study the activity of expressed product. The results show that,Recombinant expression strain secreted alkaline protease with Saprolegnia growthinhibitory activity. The study provides a theory of reference for the biocontrol ofsaprolegniasis.