The Genetic Diversity And Mechaniam Of Prochloraz Resistance In Magnaporthe Oryzae

Rice blast,caused by filamentous ascomycete Magnaporthe oryzae,is classified as one of the three major diseases on rice.M.oryzae has a typical invasive way to infect rice.The conidia germinate into germ buds on the leaf surface and differentiate into a special infective structure known as appressoria.The normally mature appressoria use glycerol as solutes,and dark melanin pigment around the appressorium cells,which can osmotically generate a 8 MPa turgor pressure to drive an actin-rich cellular protuberance through the surface of a rice leaf.It develops into secondary hyphae and forms disease lesions.Prochloraz is an imidazole fungicide belonging to sterol biosynthesis inhibitors(SBIs)with a broad bactericidal spectrum.It is mainly used for the control of powdery mildew and leaf spot of cereal crops,which has both therapeutic and protective effects.Prochloraz is also used to control rice blast more than 20 years in China.Not only is there no report on the resistance of rice blast fungus to prochloraz in production,but there is still no systematic study on the risk of prochloraz resistance.In this paper,the wild sensitive strain S-1 of M oryzae was used as material to induce prochloraz resistance in PDA medium containing sublethal dose of prochloraz.3000 plates were inoculated and cultured in the dark at 28℃ for 20 days.A total of 30 resistant mutants of prochloraz were obtained with 1%mutation frequency.According to the level of resistance,we classified the resistance of M.oryzae to prochloraz into three types:low resistance,medium resistance and high resistance.30 resistant mutants obtained from domestication culture were low resistance,which are stable to culture whithout selective pressure.The CYP51A and CYP51B genes of 30 resistance mutants and S-1 strain were amplified by PCR.All the prochloraz-resistant mutants have 1 to 2 amino acid base mutations in CYP51A compared with sensitive strain S-1.According to the amino acid changes,30 mutants can be divided into type I:F496L,type II:L218R and F496L and typeⅢ:P271L and F496L.There were 7 strains,1 strain,and 22 strains of type Ⅰ,type Ⅱ,and type Ⅲ,respectively.The biological phenotypes of 30 prochloraz-resistant mutants such as mycelial growth rate,asexual reproduction,and virulence on barley leaves were studied.It was found that the growth rate of most prochloraz-resistant mutants was lower than the original strain S-1,the pathogenicity decreased significantly on barley leaves,even lost pathogenicity and conidia production decreased;fitness of resistant strains to temperature,pH,and osmotic pressure decreased compared with their sensitive strains.The prochloraz resistant mutants had no cross-resistance to epoxiconazole,tebuconazole,hexaconazole and azoxystrobin.By the homologous exchange the CYP51A gene of the parental susceptible strain S-1 and the resistant strain,we found that the replacement mutant with the resistant fragment showed resistance and the replacement mutant with the sensitive fragment showed sensitive to prochloraz.These results indicate that point mutations in the CYP51A gene are the genetic mechanisms of the resistance of M oryzae to prochloraz.The qRT-PCR analysis demonstrated that the resistance of the 30 resistant mutants of M.oryzae obtained from domestication was not related to the transcriptional levels of CYP51A and CYP51B genes.The docking energy of the CYP51A protein of the sensitive strain S-1 and the prochloraz molecule forms a hydrogen bond with LYS137 with a score of 2.9158.In the resistant strains RA,RB,and RC,the docking energy of the CYP51A protein with the prochloraz molecule can form hydrogen bonds with TYR126,LYS137,and TYR112,and the scores below the sensitive strains were 2.3105,1.9991 and 1.9315 respectively,Which indicate that point mutations in the CYP51A protein reduce or prevent the prochloraz molecule from binding to the CYP51A protein.This study showed that the CYP51A gene of M oryzae can undergo 1%low resistance point mutation under the selective pressure of prochloraz,but the fitness of resistant mutants decreased significantly and the risk of prochloraz resistance was low.The results of this study have theoretical and practical value in guiding the rational use of prochloraz and the management of resistance.