The Monitor On Sensitivity Of Botrytis Cinerea To Fungicides In Shandong Province And The Research On Detection Technology Of Microtiter Method

Gray mould, one of major fungal diseases on vegetable and fruit crops, can cause severe losses of vegetables. The control of gray mould relies on application of chemical synthesized fungicides. But the contradict between chemical control and occurrence of the fungal resistance to the fungicides is standing out increasingly. So it is necessary to test the resistance of Botrytis cinerea fast and efficiently. In order to provide the basis for field spraying, the mycelium growth rate method was used to determine the sensitivity of B. cinerea strains from different areas of Shandong Province to commonly used fungicides. As a new method for determinating the virulence of fungicides, microtiter method has demonstrated a good potential in the relevant reports. Based on the microtiter method of Stammler G., and compared with traditional method, a rapid and efficient method for testing the sensitivity of B. cinerea to fungicides fast was established.1.138strains of B. cinerea were collected from23tomato protected areas of Shandong Province, the sensitivities of them to six fungicides and two quaternary ammonium cationic surfactants were evaluated by detecting the inhibitory on the mycelium growth rate. Results showed that high-resistance to carbendazim emerged with a frequency of78.26%, and total resistance frequency of91.30%. Meanwhile, the resistance of B. cinerea to diethofencarb was not significant, and the medium effective concentrations (EC₅₀) values were less than10μg/mL. The sensitivity of the strains to iprodione was significantly higher than procymidone, including resistance to procymidone was a frequency of73.88%, and to iprodione was a frequency of34.78%. The tested strains produced resistance to pyrimethanil in general with a resistance frequency of95.65%. By measuring the sensitivities of the53strains of B.cinerea to C8-10and1227, the mean EC₅₀ of12.98±10.70μg/mL and22.91±15.32μg/mL could be regarded as the baseline sensitivity of B.cinerea to two quaternary ammonium cationic surfactants. The mean EC₅₀ of1.70±1.36μg/mL of108strains of B. cinerea could be regarded as the baseline sensitivity to boscalid.2. A rapid and efficient method for testing the sensitivity of B. cinerea to fungicides was established by using the enzyme-labelling measuring instrument. With△OD value as an evaluation index, the conditions of microtiter method to test the sensitivity of B. cinerea to fungicides were optimized. The effect of eight widely-used fungicides （including boscalid, azoxystrobin, diethofencarb, thiophanate-methyl, iprodione and difenoconazole） against B. cinerea spore germination and mycelial growth was tested, and the results were compared with the conclusions of spore germination method and mycelium growth rate method. The conditions of microtiter method were optimized:the determination wavelength was630nm, the medium was PDE, the concentration of conidial suspension was8×10⁵-1.6×10⁶spores/mL, the optimum culture conditions were under shaking culture（150r/min） at21℃in the dark, the text time for the inhibition of spore germination was24h after adding fungicide and conidial suspension in the wells, the text time for the inhibition of mycelial growth was12-24h after adding fungicide（conidial suspension in wells was incubated for12h before adding fungicide）. The conclusion was almost consistent with that from conventional spore germination method or mycelium growth rate method.57isolates of B. cinerea from three regions of Shandong Province were tested by this methed, the mean EC₅₀ values were1.9311and5.6488μg/mL for inhibition of conidial germination and mycelial growth, respectively. Microtiter method combines the advantages of both conventional methods, ang it has characteristics of fast testing speed, high measuring accuracy and good reproducibility, so it possesses better practicability.