| Wheat sharp eyespot and rice sheath blight are worldwide distribution of soil-borne fungi diseases, which are mainly infected by Rhizoctonia cerealis and R. solani. From1970s, the replacement of wheat varieties and high-yield cultivation measures (such as early sowing, high density, high-fat, etc.) promoted the diseases in China. Nowadays, the diseases have become among the most important diseases in the middle and lower reaches of Yangtze River and the Huang-Huai Plain. Owing to few varieties with good resistance to the diseases available, application of fungicides has been the most effective measurement to prevent and control the two diseases.Thifluzamide, belonging to thiazole carboxamides, is succinate dehydrogenase inhibitors (SDHI), whose target was the succinate dehydrogenase (SDH) complex in the respiratory chain, referred to as complex II or succinate:ubiquinone oxidoreductase (SQR).A total of120R. cerealis isolates were collected from Jiangsu, Anhui, Shandong, Henan to determine baseline sensitivity and its risk for developing resistance to thifluzamide. The results revealed that all the120isolates, which were never exposed to thifluzamide, were sensitive to this fungicide, with effective concentrations for50%inhibition of mycelial growth (EC50) of0.025to0.36μg/ml and a mean of0.064±0.013μg/ml, indicating a skewed unimodal distribution. The two thifluzamide-resistant mutants of R. cerealis were obtained by domestication of thifluzamide. The resistance factor (RF) of the R. cerealis mutants for thifluzamide was120and40, respectively. The phenotype of resistance of the mutants could be stabe after10th culture transfer, and exhibited almost equal fitness compared to their wild-type parents in mycelial growth, sclerotia production, and virulence. Intersting, the mutants had different cross resistance between the SDHIs. One mutant, WX14-1, had low resistance to carboxin, while the other mutant, WX14-2, had low level of resistance to flutolanil, not carboxin. The frequent rate of resistance selection averaged2×10-4.Therefore, the data revealed that the potential risk of R. cerealis was moderate in nature. Bacillus subtilis has been extensively used as a biocontrol agent to control various crop diseases. B. subtilis strain NJ-18was isolated from the soil of oilseed rape field in our lab, with a broad spectrum inhibitive activity and great potential prospect of a bio-agent. In order to determine the plantation of the strain, NJ-18-GFP was constructed. Owing to self-modification system in the plasmid, NJ-18was applied to remove the plasmid by sodium dodecyl sulfate (SDS), and then GFP gene was transformed into the resulting mutants (plasmid-free) by Escherichia coli-B. subtilis shuttle vector. Via Escherichia coli-B. subtilis shuttle vector, GFP gene was sucessfully transformed into B. subtilis strain NJ-18and C136, whose plasmid was removed from NJ-18. The transformation rate of C136could be up to3.42×105cfu/ug of plasmid DNA, nine times higher than that of NJ-18. Compared with NJ-18, C136had the same inhibitive activity against R. cerealis and R. solani in vitro, but the summit of the growth curve for C136delayed about12h and the summit value decreased29.4%when compared with that of NJ-18. Colonization of the NJ-18-GFP mutants in the wheat roots was observed with laser scanning confocal microscope. The NJ-18-GFP mutants could attach on mycelia of the pathogen, resulting in their mycelial enlargement, malformations, and colonize in the phloem and xylem of wheat roots after the7th days of inoculation.To mix thifluzamide and B. subtilis NJ-18, the compatibility between the fungicide and NJ-18was determined in vitro. The results showed that compatibility between thifluzamide and NJ-18was good. The EC50values of R. solani to thifluzamide was only0.023μg/ml, and B. subtilis NJ-18at the concentration of1.0×108cfu ml-1exhibited high activity against R. solani. The mixture of thifluzamide (15ml24%suspension/667m2) with B. subtilis (1.0×108cfu ml-1in the spraying liquid) offered67.4%control efficacy against rice sheath blight in the field, which was significantly higher than that of the single conponent. |
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