| Pepper Phytophthora blight, caused by the oomycete pathogen Phytophthora capsici, is a devastating soil spread disease. It seriously threats the planting and yeild of the world’s pepper because of the strong resistance and survival. So far, chemical is still a main means of control of the disease in the production, but the long-term use of systemic fungicides may result in occurrence of prominent resistance of P. capsici to fungicides. Fluopicolide is a novel benzamide class fungicide that has good effect on oomycete pathogens. It has not been used at large scale. The risk of P. capsici to fluopicolide needs to be assessed. In this paper baseline-sensitivity of P. capsici to fluopicolide was set up and the resistance-risk of P. capsici to fluopicolide was assayed. At the same time, physiological and biochemical mechanism of resistance risk and phenotype of resistance mutant were studied systematically. The results were showed as follows:1. Establishment of a sensitive baseline for the sensitivity of P. capsici to fluopicolide. 110 isolates of P. capsici collected from six districts of Chongqing were tested for the sensitivity of P. capsici to fluopicolide by the method of mycelia growth rate. The results showed that the 110 isolates were all sensitive to fluopicolide and a mean EC50 value was(0.3222±0.1080) μg/mL. Low differential of EC50 value was tested among different regions and the EC50 values were 0.1028 to 0.5719μg/mL, representing a range-of-variation factor of 5.6. The sensitivity frequency of P. capsici to fluopicolide distributed as a unimodal curve, so this sensitive baseline was suitable for resistance monitoring of P. capsici to fluopicolide.2. Three mutants resistant to fluopicolide were acquired by UV irradiating mycelia with a mutation frequency is 0.86%. The resistant strains’ resistance ratios respectively reached up to 69.5, 98.5, and 94.2. Based on the standard of resistance created by FAO and the sensitive baseline and the resistance level of resistant mutants, 3 MR mutants were obtained. According to the result of biological characteristic of some mutants, the mycelial growth, sporulation and pathogenicity, showed was similar with parental isolate, some mutants were significantly lower than the parental strain though they had the capability of sporulation and pathogenicity. The resistant levels of three resistant mutants were stable. Besides, with the result of cross resistance test, fluopicolide has no cross resistance with metalaxyl, cymoxanil, dimethomorph, azoxystrobin and pyrimorph. To sum up, there could be a certain risk of field population of P. capsici developing resistance to fluopicolide. Therefore, it was suggested that the fluopicolide should be used alternately with other fungicides without cross-resistance.3. Comparison physiological and biochemical indexes of P. capsici isolates’ mutants resistant to the fluopicolide with parental isolates. The results showed that all isolates were sensitive to high, medium and low osmotic pressure. And the mutants’ ability of the osmotic pressure regulation was higher than that of the parental strain. Mutants in the glucose and NaCl concentration showed different adjustment mode of osmotic pressure that glucose could be as a nutrient for pathogens, but NaCl couldn’t be as a nutrient. Fluopicolide in low concentrations all isolates in vivo electrolyte to leak out more. Mutants’ conductivity was higher than parental isolates in a certain range, and lower than that of parent strains at higher concentrations. With the treated time goes on, the β-1,3-glucanase activity of resistant and parental strains both tended to decrease, while resistant strains kept much lower than that of parental ones. But the changes of the soluble protein content of the different mutants and parental strains were not regular.4. This study developed a Phenotype microarray protocol PM1-3 and PM21-22 to assess the utilization of carbon and nitrogen sources and the sensitivity of chemical substances in the fluopicolide-resistant P. capsici isolates and parental isolates. The results showed that almost all positive assays for C-source utilization using the PM1 screen and few signals from the PM2 screen. Utilization of carbon sources by resistant strains was similar with the parental strains, with no significant difference. The maximum utilization of 95 nitrogen sources of resistant mutant BS11-5-1 and its parental isolate could be achieved. But mutants JLP11-4-2 and JLP11-4-3, and their parental strains had a low utilization of nitrogen sources. In the test for sensitivity measurement to chemical substances, it was found that the sensitivity of mutants and parental strains to the substances in PM21-PM22 existed differences, i.e. mutants showed more sensitive comparison with parental strains. This study suggested that the information of sensitive substance can provide a theoretical basis for the research and development of new fungicides even control strategy making for P. capsici disease. |
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