Screening, Fermentation Conditions And Inhibiting Mechanisms Of Antagonistic Strains Against Camellia Oleifera Anthracnose Caused By Colletotrichum Gloeosporioides

Camellia oleifera anthracnose caused by Colletotrichum gloeosporioides is one of the major diseases in China's Camellia producing areas.It occurs broadly and causes serious drop in buds and leaves, sometimes resulting in the death of the tree.It occurs up to 20%～40% of the growing regions are infected with this disease, reducing up to 80% of the total yield. Now, the most way of control the disease is combine the breeding variety to chemical control, but the effect is uncomfortable. Biological control has become an important way to plant disease for its safe, efficient and pollution-free features, which was attentioned by people.The most aim of the experiment was isolated and screened the strong antagonistic bacteria against C. gloeosporioides from the healthy leaves of Camellia oleifera, identified the strain, optimised the aimed strain fermented condition and its inhibiting Mechanisms against C. gloeosporioides. The results showed that:(1)Isolating and screening the strong antagonistic bacteria against C. gloeosporioides from the healthy leaves of Camellia oleifera and identifing the strain.156 bacterial strains isolated from the healthy leaves of Camellia oleifera collected from heyang city, Hunan province.9 strains inhibiting Colletotrichum gloeosporioides were screened out by inhibitory zone method and the aseptic filtrate treatment. Through pot experiment test in vivo,it was found that isolated Y13 was the most effective antagonist against Colletotrichum gloeosporioides,which preservation number is CSUFT B0013.The identification of Y13 was performed with traditional bacterial systemic identification method. The result showed that the physiological and biochemical character of Y13 is similar to that of Bacillus subtilis. Then, the molecular identification of Y13 was performed. Through the comparative 16s rDNA sequences analysis, the 16s rDNA sequence of Y13 had high homology with typical Bacillus subtilis (99%).(2)Optimising the aimed strain fermented condition. Through single factor and orthogonal test, the optimal fermentation conditions of producing antibiotic products of Bacillus subtilis Y13 were given out. The optimal culture conditions of the bacteria for the antibacterial protein producing are pH7.0,30℃,7%,140r/min, NYBD medium with shaking for 72h. Orthogonal test show that when Y13 got highest inhibiting activity, composition of the best media were glucose 25g, beef extract 10g, yeast extract 5g, NaCl 1.2%;when Y13 grow best,glucose 15g, beef extract 10g, yeast extract 3g, NaCl 1.5%.(3)Study on its inhibiting Mechanisms against C. gloeosporioides. The strain Y13 always has the function of antifungal after undergo 10 transfer of culture. The effect of antifungal is stabile. Y13 could inhibit the growth of mycelia and conidia germination. Meanwhile,Y13 strain showed better repressing activity on Colletotrichum gloeosporioides. Wide antifungal spectrum was found through the leavening trials, which showed Y13 also had better repressing impact onFusarium oxysporium,Thanatephorus cucumeris,Alternaria solani,PestalotiopsisStey.Their inhibition rates were 88.5%,84.3 %,81.8%,80.0%和80.1%, respectively. Bacillus subtilis Y13 antagonistic against C.gloeosporioides was induced to be rifampicin-resistant and used for monitoring its colonization on leaves of Camellia oleifera. Experiments showed that the introduced antagonistic bacteria could colonize on Camellia oleifera concentration 7.65×103CFU/g. In comparison with inoculation of antagonistic bacteria only, pre-inoculation of C.gloeosporioides had a negative effect on colonization of the antagonists,which indicated that the antagonistic bacteria was better in prevention than curation for grey mould.