| Rice blast caused by Magnaporthe grisea (Hebert) Barr is one of the most important rice diseases, which caused serious loss of rice production in Southwest China. Currently, the major strategy to control this disease is to use chemical fungicides, but risk of fungiciede-resistance of the pathogen may occur if a fungicide is continuously used for a long period of many years. In the present study,18 counties of Guizhou, Sichuan and Chongqing provinces were surveyed in 2013-2014 to collect rice samples with blast symptoms and 902 isloates of Magnaporthe grisea were obtained to measure their resistance to isoprothiolane and pyraclostrobin. One isoprothiolane-resistant isolate was found from the 902 isolates. Ahighly resistant mutant to pyraclostrobin was obtained by artificial induction using this chemical. Biological characteristics, resistance mechanism and cross-resistance of this mutantwas also analysed.1. Sensitivity baseline and resistance of M. grisea to isoprothiolane. A number of 100 M. grisea isolates were collected from Sichuan and Chongqing provinces in 2013 and their sensitivity to isoprothiolane was measured using the mycelial growth diameter method. The EC50 values of these isolates were between 0.8746 μg/mL to 8.1430 μg/mLand the highest EC50 for the least sensitive isolates was 9.3 times higher than that for the most sensitive isolates. The everage value of EC50 was 3.0764±1.5909 μg/mL, which was used as the sensitivity baseline to evaluate the resistance of M. grisea to isoprothiolane. The minimum inhibitory concentration (MIC) values of isoprothiolane to 50 wild isolates of M. grisea were measured and the values were less than or equal to 45 μg/mL. Based on the MIC values,50 μg/mL was assigned as the differential dose of isoprothiolane-resistant and isoprothiolane-sensitive isolates of M. grisea in monitoring the resistance. Tests of the 902 isolates using the differential value showed that only one isolate was resistant to isoprothiolane and the other 901 isolates were all sensitive. This isoprothiolane-resitant isolate was isolated from Longtan Town, Fuling District, Chongqing and its resistance index was measured as 3.13.2. Isoprothiolane-resistant mutant of M. grisea and their stability of resistance. Five intermediate and one high isoprothiolane-resistant mutants of M. grisea were obtained through induction using isoprothiolane containing culture medium. The mutants were cultured on PDA medium and their resistance to isoprothiolane decreased to sensitivive level after 8 generations of cultivation. The results implied that the isoprothiolane-resistance of the M. grisea mutants was not stable genetically. Because no isoprothiolane-resistant mutant was obtained, mechanism of the resistance was not further studied in the present work.3. Sensitivity baseline and resistance of M. grisea to pyraclostrobin. The sensitivity of 100 M. grisea isolates were collected from Sichuan and Chongqing provinces in 2013 and their sensitivity to pyraclostrobin were measured by mycelial growth diameter method. The EC50 values were between 0.00313 μg/mLto 0.02448μg/mL. Highest EC50 for the least sensitive isolates was 7.8 times than that of the most sensitive isolat. The everage value of EC50 was 0.00917±0.00385 μg/mL, which was used as the sensitivity baseline to evaluate the resistance of M. grisea to pyraclostrobin. The MIC values of pyraclostrobin to 50 wild isolates of M. grisea were measured and the values were less than or equal to 0.8 μg/mL. Based on the MIC values,1.0μg/mL was assigned as the differential dose of pyraclostrobin-resistant and pyraclostrobin-sensitive isolates of M. grisea in monitoring resistance of M. grisea. Tests of the 902 isolates using the differential value showed that there was no isolate resistant to pyraclostrobin. 4. Pyraclostrobin-resistant mutants of M. grisea. Three intermediate and one high pyraclostrobin-resistant mutants of M. grisea were obtained through mutation using pyraclostrobin containing culture medium. After cultured on PDA plates without the fungicide continuously for 8 generations, only one mutant PYC40-Mut remained resitant whilst the other 3 mutants became sensitive to pyraclostrobin. PYC40-Mut was a highly resistant mutant to pyroclostrobin and the resistance was 370 times of that of its parent isolate PYC40. The pyraclostrobin-resistance to PYC40-Mut was very stable and its single-cell offspring could grow normally on PDA and YPSA plates. Inoculation with PYC40-Mut in vitro, vivo and compound fitness, respectively, resulted in significantly lower disease severity than that of inoculation with its parent isolate. This indicated that the pathogenicity or infectivity of the mutant was decreased. 5. The mechanism of pyraclostrobin-resistance of PYC40-mut. The genomes DNA of PYC40-Mut and its parent PYC40 were gained using the CTAB method and the partial nucleotide sequences of Cyt b gene were obtained by PCR amplification using primers Pgcytb-F1 (5’-AGTCCTAGTGTAATGGAAGC-3’) and Pgcytb-R1 (5’-ATCTTCAACGTGTTTAGCACC-3’) of the gene. Comparingthe Cyt b gene sequences of PYC40-Mut and PYC40 found that the base G at site 428 in PYC40 sequence was mutated to base C in PYC40-Mut sequence. This mutation led to a related change of amino acid from glycine to alanine at site 143 in the translated protein. Because of the change of this protein the normal combination of pyraclostrobin and mitochondrial complex III could be impeded, resulting in the occurrence of the resistance of the pathogen to pyraclostrobin.6. Cross-resistance of M. grisea to different fungicides. The results of tests showed thatcross-resistance of M. grisea to diferent fungicedes existed. There was a positive cross-resistance between pyraclostrobing and kresoxim-methyl and between pyraclostrobing and azoxystrobin. However, there were no cross-resistance found among difenoconazole, tebuconazloe, tricyclazole and isoprothiolane.Fungicide-resistance of fungal pathogens may impose a sew ere impact on crop production. From results of the present study, no obvious resistance to isoprothiolane and pyraclostrobin was detected in the field isolates of M. grisea. A mutant with high and stable resistance to pyraclostrobin was obtained successfully, indicating that the risk of forming pyraclostrobin-resistance existed. Additionally, the pyraclostrobin-resistant mutant was also found resistant to other strobilurins, there exised a positive cross-resistance between the strobilurin fungicides. Therefore, isoprothiolane and pyraclostrobin may still be applied safely to control rice blast in most rice cropping areas of southwest China, but fungicide-resistance of the pathogen needs to be observed and monitored frequently in order to prevent the formation of fungicide-resistant M. grisea populations. |
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