Sensitivity Determination And Resistance Risk Assessment Of Alternaria Alternata F. Sp. Mali To Tebuconazole

Apple alternaria blotch, which caused by Alternaria alternata f. sp. Mali, is a serious disease on the apple trees. The disease can cause large amount of deciduous leaves in apple trees, and result in induce seriously of the yield and the quality., The chemical control is still the important measure for management of A. alternata f. sp. mali in the production at present, but the long-term use of the same fungicides may lead to resistance. Tebuconazole is a SBIs fungicide with the broad-spectrum, high activity and low toxicity, which have a good effect to control A. alternata f. sp. mali. The demand of the fungicide is growing up gradually year by year, and there is no report on the resistace research of A. alternata f. sp. mali to tebuconazole so far. This research tested the sensitivity of A. alternata f. sp. mali to tebuconazole and the sensitivity baseline has been established. On this basis, the tebuconazole-resistant mutants were induced. The fitness, the biological characteristics and the physiological and biochemical characteristics of mutant-isolates were studied, looking forward to provide some certain theoretical guides for the rational use of tebuconazole in A. alternata f. sp. mali.1. The sensitivity baseline of A. alternata f. sp. mali to tebuconazole was established. The 64 isolates of A. alternata f. sp. mali from the five areas of Shanxi province, where tebuconazole and the similar pesticides haven’t used, were collected, isolated and determined sensitivity. The results showed that the EC50 values ranged from 0.7687μg/mL to 2.6738μg/mL, and the mean value is 1.6224±0.5582μg/mL. The sensitivity frequency of the 64 isolates was distributed as a single peak cure, so the EC50 values could be used as the relative baseline sensitivity of tebuconazole to A. alternata f. sp. mali.2.9 tebuconazole-resistant mutants were induced by ultraviolet irradiation and drug domestication, which 6 mutants were obtained by UV irradiation and the other 3 ones were obtained by drug domestication. It’s showed that under the continuous selection pressure of pesticides, resistant mutants can be obtained.3. The determination to the fitness of resistant mutants indicated that the mycelium growth, spore production, spore germination and conidial competition ability of tebuconazole-resistant mutants were declined in comparison to the sensitive isolates, so it be infered that it is difficult to become the dominant isolates for the resistant mutants. The result of Pathogenicity showed that, sensitive isolates have stronger pathogenic ability than resistant ones without tebuconazole,. But in the press of tebuconazole, there was the tolerance in the resistant strains. The research of the genetic stability indicated that part of the mutants could inherit stablely; the sensitivite of the other mutants was little changed in some selective pressures of tebuconazole, while the one reduced without tebuconazole. There was cross-resistance between tebuconazole and myclobutanil, which both belong to the triazole fungicide.4. The influence of the nutrient conditions, pH value, temperature, and humidity to tebuconazole-resistant mutants and sensitive isolates was determined. The results showed that the utilization of maltose as carbon sources, peptone as nitrogen source were the highest in both resistant and sensitive isolates, and both of them grow better in the meta-acid or leaning alkalinity conditions. Sensitive isolates grow well in temperature range of 25-30℃, and the optimum temperature for resistant isolates is 30℃. Higher humidity is suitable for the sensitive isolates growth, but the optimum humidity for mutants is irregular.5. The chemistry characteristics of tebuconazole-resistant mutants and sensitive strains were determined, including the sensitivity to osmotic stress, the membrane permeability and the ergosterol content. The results showed that the sensitivity of mutants and sensitive isolates to different osmolarity has no regularity, and resistance level of mutants has no direct link to osmotic pressure sensitivity; The relative leakage of middling tebuconazole-resistant isolates were higher the resistant isolates, but the low tebuconazole-resistant isolates were lower; Concentration of ergosterol was measured by HPLC, the results showed that the ergosterol content from high to low in the press of tebuconazole is middling tebuconazole-resistant isolates, low tebuconazole-resistant isolates, sensitive isolates. The inhibitions of CYP51 is larger on the sensitive isolates than on the resistant isolates.