| Rice bacterial leaf blight is one of the four major diseases in rice production in China, mainly caused by Xanthomonas oryzae pv. oryzae largely breeding and extension in vascular bundle through the hole and wound of rice leaf. Applying chemical pesticides such as phenazine oxide drug or breeding resistant varieties become the main prevention and treatment methods at present. But applying the chemical pesticide too much will easily caused pesticide residues, ecological environment destruction and beneficial organisms killed.Endophytic fungi refer to the kind of fungi living in healthy plant leaf, but not producing obvious infection for the host formation,which commonly exist in the aquatic plants and terrestrial plants studied in the present. In terms of biomass in plants, proportion of endophytic fungi is slight, but endophytic fungi plays a vital role in the whole development process of agriculture. In addition to enhance plant resistance and promote plant growth having been widely recognized, endophytic fungi can produce antimicrobial active ingredients such as fragrance beans, alkaloids, insecticidal, quinones, peptides and phenolic substances. These antimicrobial active substances can specifically inhibit the growth and breeding speed of some pathogenic bacteria so as to achieve the aim of reducing crop diseases. Therefore, screening a strain of endophytic fungi showing efficient antagonism effect to rice bacterial leaf blight from natural environment is an important direction at the present stage.A strain Bo-1with antimicrobial activity to Xanthomonas oryzae pv. oryzae was screened from rice in the suburb of Jinhua. Strain Bo-1was initially identified as Bionectria ochroleuca by the morphological features, ITS rDNA sequence analyses and phylogenetic tree construction. The results of physiological and biochemical test showed that Bo-1strain did not show special selectivity of carbon source, nitrogen source and can hydrolyze macromolecular materials such as gelatin, starch, urea, casein. In addition, Bo-1strain showed strong tolerance ability to the external environment.The results of antimicrobial spectrum experiments demonstrated that it had a strong antagonistic ability against bacterial pathogens especial for Monilia albican with the inhibition circle of38.9mm. The antimicrobial thermal stability, acid and alkali stability and light stability of ethyl acetate extracts from culture liquid of strain Bo-1was tested by using Xanthomonas oryzae pv. oryzae as the indicator bacteria. Experiments showed that as the temperature rose, the antibacterial activity of ethyl acetate extracts produced by strain Bo-1decreased. The inhibition zone diameter reduced to17.6mm at121℃. The antimicrobial activity decreased with the acid and alkaline enhancement, but their inhibition zone diameters at pH2.5and pH10.5were still32.2mm and35.1mm separately. In addition, the inhibition zone diameter was35.6mm after it was irradiated with ultraviolet light of40W for7h. The antibacterial activity of ethyl acetate extracts irradiated with ordinary light of4000Ix for18h had no significant difference from the control.Based on the index of antimicrobial activity of ethyl acetate extracts from culture liquid of Bionectria ochroleuca strain Bo-1to Xanthomonas oryzae pv. oryzae, the optimal carbon source, nitrogen source and salt for the production of antimicrobial substance by strain Bo-1were tested by single factor experiments. The medium composition and fermentation conditions were optimized by orthogonal experiments. The results showed that the optimal carbon source, nitrogen source and salt were starch, peptone, MgSO4·7H2O, respectively. The optimal composition of the medium was30g/L starch,2g/L peptone,0.5g/L MgSO4·7H2O. The optimal fermentation conditions were the combination of temperature30℃, agitation speed150r/min, medium volume80mL/250mL, initial pH6.5.Applying pot experiments determined the biocontrol effect of ethyl acetate extracts from culture liquid of Bionectria ochroleuca strain Bo-1to Xanthomonas oryzae pv. oryzae. Using artificial cut leaf methods infected four varieties of rice. In addition, using method1(prevention), method2(treatment), method3(P6control) and method4(sterile water as blank control) dealed with rice leaves, respectively.The results showed that the maximal of lesion length reduction of rice leaves was up to86.38%, the disease indexes reduced41.62%and the best control efficiency was59.67%. Besides, by comparison of the three methods, it could be known that the control effect of method1that mainly focused on the prevention was superior to other two methods. In conclusion, taking preventive measures was more important in the process of prevention and treatment of rice bacterial leaf blight.Using Xoo race P6as the indicator bacteria, active monomer compound was isolated and purified by means of fractional extraction, silica gel column chromatography separation, high performance liquid chromatography purification. Through structural analysis and comparison, target compound was identified as usnic acid with molecular weight of344.0835and formula of C18H16O7on the basis of mass specturm and nuclear magnetic resonance analyses. Using20%Bismerthiazol and3%Zhongshengmycin as control, antimicrobial activity to Xoo race P6of usnic acid was tested by96-well plate method. Assay illustrated that usnic acid showed better antibacterial activity to Xoo race P6with MIC value of200μg/mL, while lower than3%Zhongshengmycin and equal to20%Bismerthiazol. |
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