| Sheath blight (Sheath blight), a worldwide rice disease caused by Rhizoctoniasolani iKühn, is becoming more and severe in recent years. Up to date in China, itscontrol mainly relies on validamycin (jinggangmycin), but long-term and large-scaleapplication of sole fungicide produces ecological and resistance problems. Therefore,in order to improve the effectiveness of integrated control, it is necessary to explorenew control strategies and methods for the control of rice sheath blight. Screening ofefficient antagonistic microorganism strains from soil and identification ofantagonistic activity components or compounds is an important way to control thisdisease, and exhibits broad application prospects.An antagonistic bacterium against rice sheath blight fungus Rhizoctonia solani,named kwkjT4, was screened from vegetable root rhizosphere soil by serial dilutionmethod and dual culture technique on agar plate, and identified based on itsmorphological, physiological characteristics and16S rDNA sequence. For in vitrodetermination of the inhibition against Rhizoctonia solani, growth rate assay was used.Control efficacy against rice sheath blight was evaluated by seed bacterization tests ina green house. The optimal growth conditions of the strain and the optimal conditionsfor accumulating inhibitory substances were also investigated. Several organicsolvents were used to extract active metabolites of the isolatd strain. The antibacterialsubstances were isolated and identified by thin-layer chromatography, silica gelchromatography and gas chromatography-mass spetrum analysis. The results showedthat:1. The ioslated bacterium was preliminarily identified as a strain of Chromobacteriumpseudoviolaceum, the first report on C. pseudoviolaceum strain as an antagonistic bacteriumagainst rice sheath blight fungus Rhizoctonia solani.2. The strain kwkjT4exhibited excellent in vitro inhibition of55.1%against the fungalpathogen. Its control efficacy against rice sheath blight was61.9%, comparable to that ofagro-funcides jinggangmycin. These results suggest that the strain shows a promisingapplication potential for the control rice sheath blight.3. Secretion of antifungal substances, i.e., antibiosis, is one of the antagonisticmechanisms. The optimal growth conditions of the strain were as follows: pH7.0,temperature32°C, incubation time36h. Inconsistence with those for the bacterial growth, theoptimal conditions for accumulating inhibitory substances were pH6.0, temperature28°C,and incubation time48h.4. The effects of several organic solvent with different polarity extraction on the extractefficiency of active substabces were explored. Among them, ethyl acetate was the mostefficient. Most of mebolites with inhibitory activity substance of the strains kwkjT4could be extracted by ethyl acetate. The inhibition rate against R. solani reached53.4%.5. Five components (Fraction1-5) were separeted from the ethyl acetate crude extract bysilica gel chromatography. Active determination indicated that the Fr.2showed the strongestinhibitive activity with67.3%of relative inhibition rate, next was the Fr.4and Fr.3with46.2%and28.5%of relative inhibition rate, respectively, whereas, the Fr.1and Fr.5showedno activity. The Fr.2was further seperated by silica gel chromatography to give threesubfractions (Fr.2-1-3). Among them, the Fr.2-2had the best inhibitive effect against thefungus with the relative inhibition of72.4%.6. Thirteen compounds were detected by GC/MS as follows:Com.1:3-Pyridinecarboxaldehyde, Com.2:1H-Pyrrole-2-carboxylic acid, Com.3:3,4-Methyl-enedioxymandelic acid, Com.4:3-Pyridinecarboxylic acid, Com.5:4,6-Diamino-5-pyrimidinylformamide, Com.6:1H-Imidazole-4-carboxylic acid,Com.7:8-Hydroxy-7-methyl-quinoline, Com.8:3-Pyrrolidin-2-yl-propionic acid,Com.9:4-Amino-6-hydroxy-2-methyl-5-nitrosopyrimidine, Com.10:3-Indolylglyoxylic acid, Com.11:3-Benzyl-6-(hydroxymethyl)-2,5-piperazinedione,Com.12:1,2-Benzenedicarboxylic acid and Com.13: Phenylalanine,2,5-diketo-3-hydroxy-6-piperazinyl ester. |
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