Monitoring And Molecule Mechanisms Of Resistance To Tebuconazole By Magnaporthe Grisea In Southwest China

Rice blast is one of the most important rice disease, which caused serious loss of rice production and quality. Currently, the major strategy to control this disease was chemical fungicides, and Southwest China was the region which occurred rice blast frequently. Tricyclazole and isoprothiolane were the popular fundicides, and it brought about potential safety hazard for a fungicide was continuously used for a long period of many years. Tebuconazole still not widely used in Southwest China is a new systemic triazole fungicide which has excellent control efficacy in many fungus has already used in other region. In this study, we established the baseline of Magnaporthe grisea to tebuconazole and monitored the resistance of isolates collected from Southwest China, compared the biological characteristics between tebuconazole-resistance isolates which induced by fungicide in labor and their parent isolates and studied the preliminary molecular mechanisms which tebuconazole-resistance isolates occurring resistance. And we wanted provide theoretical guidance for controlling rice blast of Southwest China by tebuconazole. 1. Sensitivity baseline of Magnaporthe grisea to tebuconazoleIn this study, the sensitivity to 14α-demethylation inhibitors fungicide tebuconazole of 150 isolates of Magnaporthe grisea was tested by mycelium growth rate method. Statistical analysis showed that the EC50 values of these isolates were between 0.0475 μg/mL to 0.5996 μg/mL, the average value of EC50 was 0.2154±0.1497μg/mL and the highest EC50 for the least sensitive isolates was 12.62 times higher than the most sensitive isolates. Sensitivity distribution to tebuconazole was unimodal and continuous with only one susceptibility. So, we used 0.2154±0.1497μg/mL as the sensitivity baseline of Magnaporthe grisea to tebuconazole in Southwest China.The sensitivity of Magnaporthe grisea isolates to tebuconazole in 10 counties of Southwest China was analyzed by SPSS. Statistical analysis showed that there was significant difference in EC50 values to tebuconazole between populations from different extents. And the average value of EC50 of isolates from Mianyang in Sichuan province was 0.0872±0.0461μg/mL was the most sensitive. The EC50 of isolates from Xuanhan in Sichuan province with a mean value of 0.4570±0.0855μg/mL and isolates from Liuzhitequ in Guizhou province with a mean value of 0.4436±0.1289μg/mL were the most non-sensitive. 2. Monitoring of resistant isolates in Southwest China.30 wild strains of Magnaporthe grisea were selected for testing the minimum inhibitory concentration(MIC) value by mycelium growth rate method. The MIC value was used to monitor all isolates collected in Sichuan, Guizhou and Chongqing province of China. The values of MIC range from 1.5 to 4.0 μg/mL, so 4.0μg/mL was assigned as the differential dose of tebuconazole-resistant and tebuconazole-sensitive isolates of Magnaporthe grisea in monitoring the resistance. The result showed that there were 2 strains come from Xuanhan and Fuling respectively can grow up in 4.0μg/ml tebuconazole. Testing EC50 of hypothetic resistant isolates and calculating resistance factor. The result showed that their EC50 were 0.6512μg/ml and 0.6557μg/ml, the resistance frequency were 3.02 and 3.04, respectively. So the most of isolates of Magnaporthe grisea in Southwest China were sensitivity and a few of them were low-resistance. 3. Biological characteristics of tebuconazole-resistance isolates.In this study, we obtained two mid-resistance isolates PWS2-1 and PMY30-1 by fungicide taming. After cultured on PDA plates without the fungicide continuously for 8 generations, they remained resistance, so the resistance could be inherited stable.We compared the biological characteristics between resistance isolates and their parent isolates, the result showed that the growth rate and conidial production of resistant isolates were below their parent isolates. Inoculating the spore suspension on the rice leaves, the result showed that the pathogenicity of resistant isolates were less than their parent isolates. By testing mycelia growth rate of different temperature and pH values on the resistant strains and sensitive strain was determined. The result showed that there was no significantly difference in the effect on the sensitive and resistant strain of pH value and temperature.Testing the sensitivity of isolates to six fungicides which have different mechanism of action, the result showed that the tebuconazole-resistance isolates have positive cross-resistance to fungicides of DMIs. But they don’t have cross-resistance to isoprothiolane and tricyclazole which have been widely applied in controlling of rice blast, so tebuconazole could substitute or alternate these fungicides. 4. Molecular mechanisms of tebuconazole-resistance isolates.The genomes DNA of PWS2-1, PMY30-1and their parent isolates PWS2, PMY30 were gained using the CTAB method and the partial nucleotide sequences of cyp51-Ⅰand cyp51-Ⅱgene were obtained by PCR amplification using specific primers Cyp51-ⅠF:5’-ATGGGCCTTCTACAGG ACACC-3 ’ /Cyp51- Ⅰ R:5 ’-TTAGCGCCTCTCCCAGTAAATT-3 ’ and Cyp51- Ⅱ F:5 ’-CTACGCAGTCTTCGGGCT-3’/Cyp51-ⅡR:5’-ATGGCTTTCTTCTTCCC-3’. The result showed that resistant isolates both have mutation sites but not unanimous. Compared cyp51 gene sequences between PWS2-1 and PWS2, the result showed that, cyp51-Ⅰ have not locus mutation and three mutation sites in cyp51-Ⅱ gene, R163 C,F253I and V257 A. Compared cyp51 gene sequences between PMY30-1 and PMY30, the result showed that, there have one mutation site in cyp51-Ⅰgene, R23 G and have not locus mutation in cyp51-Ⅱ gene.Testing expression level of cyp51-Ⅰand cyp51-Ⅱ gene by real-time quantitative PCR between resistant isolates and sensitive isolates, the result showed that the trend of cyp51-Ⅰ was not consistency between PWS2-1 and PMY30-1, the expression level of PWS2-1 was higher than PWS2 and the expression level of PMY30-1 was less than PMY30. And the trend of cyp51-Ⅱ expression level of resistant isolates were less than their parent isolates. So the molecule mechanisms of tebuconazole-resistance isolates which obtained by fungicide taming maybe connect with mutation sites, and did not related to expression of cyp51 gene, but further study is necessary to confirm it.