| Xanthomonas oryzae pv. oryzae is the phytopathogen of bacterial blight of rice, which has always been thought as one of the most serious agriculture diseases pathogens. This disease can make a reduction in production of rice. Using chemical fungicide is principal pathway in agriculture. However, the chemicals could cause toxic residues, and using excess chemical fungicide cause results that phytopathogens mutate and get drugs resistance. The research of the antibiotic phenomenon showed that natural biofungicide may exit in entironment. A natural fungicide could play an important role in production of agriculture, which could be more specific and less harmful than chemical fungicide.The actinomycetes especially the strains of Streptomyces are the most common antibiotic producing microorganisms, which widely exist in soil, air and water. Evidences of study supported that Streptomyces can produce many kinds of antibiotics, which are secondary metabolites, and could be biodegraded in entironment. Streptomyces are prokaryotic gram-positive, which produce an extensive branch mycelia that rarely fragment. Streptomyces could produce different kinds of antibiotics, which could inhibit the growth and proliferation of the phytopathogen, specifically. These antibiotics could be used to produce biological fungicide. It might play an important role in prevention and cure plant diseases, which were caused by opposite phytopathogen.1. The actinomycetes strain ACT-2was isolated from the soil of rice experimental field in the suburb of Jinhua. The morphologic,16S rDNA gene sequence and phylogenetic tree observation results demonstrated that this strain showed close homology to Streptomyces termitum (98.6%). The tests of physiological and biochemical experiments showed that Streptomyces termitum strain ACT-2appeared a good adaptive capacity to surroundings, which could use the most carbon source and nitrogen source. The strain ACT-2could live in the high temperature, pH and some other extreme environments. Streptomyces termitum strain ACT-2and its fermentation fluid were showed obvious antimicrobial activity to Xanthomonas oryzae pv. oryzae physiological strain P6, simultaneously. Strain ACT-2could efficaciously hold inhibition zone, which diameter was38.00mm. Fermentation fluid could hold bacteriostatic action and the diameter of inhibition zone was21.73mm. After optimization of fermentation conditions, the optimal fermentation conditions for the strain:50ml culture fluid in250ml Erlenmeyer flasks; fermented for72h at28℃on a rotary shaker (180r/min); the original pH was7.5prior to sterilization. The diameter of inhibition zone was26.20mm.2. The results of bacteriostasis experiments demonstrated that strain ACT-2suppress a wide range of bacterial pathogens in vitro, while no obvious antimicrobial activities towards to fungal pathogens. Antibacterial stability of Streptomyces termitum strain ACT-2fermentation fluid was studied while Xanthomonas oryzae pv. oiyzae physiological strain P6was chosen as indicator bacteria. The fermentation fluid could hold obvious bacteriostatic activity when the tempreture was below80℃or exposured in the4000lx illuminant or UV(40w); the diameter of inhibition zone reduced when the tempreture was up to90℃. The fermentation fluid was sensitive to acidity and alkalinity, the activity ruduced after treatment.3. Biological control experiment:determine the control effect of the fermentation fluid of the Streptomyces termitum strain ACT-2, which was used in control experiment of bacterial blight of rice. The leaves of the four kinds of sensitivity rice varieties were artificially infected by Xanthomonas oryzae pv. oryzae. Using method①(To be dealt with fermentation fluid firstly, then artificial infection) and method②(Artificial infection at first, then the leaves were dealt with fermentation fluid) to deal with samples. The results of experiments showed that the maximal of the lesion length reduction was70.97%, the disease indexes reduced43.04%which was maximal, and the most obvious control efficiency of bacterial blight of rice was80.80%. The results showed that method①was more effectual than method②in control efficiency, and method①was good at preventing disease. So, it meant that preventive measures were more effectual than treatments.4. Petroleum ether, dichloromethane, ethyl acetate and butanol were used for extracting antimicrobial ingredient from fermentation fluid, which was choosen after antimicrobial efficacy tests. Ethyl acetate was the best extracting agent. Silica gel column chromatography was used to separate mixture, which pore space was200-300mesh. Methylene dichloride was used as eluent in gradient elution research, which was choosen after antimicrobial tests. LH-20gel column chromatography was used to subtly separate antimicrobial ingredient. The methanol was used as eluent. The pure antimicrobial component was obtained and crystallized after static placing at25℃for48h. The crystallized antimicrobial component was yellow or greenish orange, irregular shape. It was hardly dissolve in petroleum ether, slightly soluble in methylene dichloride, dichloromethan and chloroform, easily soluble in methanol, acetone and dimethyl sulfoxide. The smell was slight, similar to antibiotics and none pungent odor. Their structures have been characterized by ESI-MS and1H NMR spectra. The result were that1H NMR (400.13MHz, d6-DMSO):7.72(t, J=8.0Hz,1H),7.63(d, J=1.8,1H),7.45(d, J=8.0Hz,1H),7.32(d, J=8.0,1H),7.04(d, J=1.8Hz,1H),2.69(s,3H). ESI-MS:253.1([M-1]-). After compared with known substances, this antibacterial substance was confirmed Aloesaponarin Ⅱ(1-Methyl-3,8-dihydroxyanthraquinone), which molecular formula was C15H10O4.The results of this study showed that Streptomyces termitum ACT-2cuold produce Aloesaponarin II, which showed highly antibacterial activity to Xanthomonas oryzae pv. oryzae. The results of biological control experiments showed that Aloesaponarin II could alse have good preventive and therapeutic effect on bacterial blight of rice. |
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