Studies On Resistance And Mechanism Of Magnapothe Grisea To Enestrobulin In Liaoning Province

Because of fungicide resistance of Magnapothe grisea,the effect of fungicides was decreasing on rice blast.In this paper,the sensitivies of Magnapothe grisea to enestrobilurin were determined;the enestrobilurin-resistance mutants of Magnapothe grisea were induced; Biological feature of resistant isolates were conducted indoor,cross resistance between enestrobilurin,tricyclazole,azoxystrobin,isoprothiolane,carbendazim,kresoxirn-methyl, prochloraz,EBP were conducted indoor and molecular resistance mechanism of Magnapothe grisea to enestrobilurin-resistance was detected.The results derived from this study would preliminarily evaluate the risk of Magnapothe grisea to enestrobilurin-resistance.A theoretical basis was provided for the scientific application of enestrobilurin and the development of managing strategies when isolates of Magnapothe grisea to enestrobilurin-resistance were occurred.Examination of sensitivity:The sensitivities of 151 Magnapothe grisea isolates collected from 8 different geographical regions of Liaoning Province in different years were examined in vitro.The results showed that all the isolates were sensitive to enestrobilurin.The EC₅₀ value ranged from 0.6567 to 1.9375μ.g·mL^-1.The baseline-sensitivity of Magnapothe grisea to enestrobilurin was found,with an everage EC₅₀value 1.3772μg·mL^-1.Enestrobilurin susceptibility differences between strains collected from different areas in the same year were not large.However,enestrobilurin susceptibility differences between strains collected from different areas in 2005-2007 exist.The most sensitive isolates are from Fushun with EC₅₀ 1.2613μg·mL^-1,the least sensitive ones are from Yingkou with EC₅₀1.5264μg·mL^-1 respectively.Biological activities of Magnapothe grisea to enestrobilurin:The influence of mycelia growth,spore production,melanin biosynthsis of sensitive isolate B2.In the spot,protective and curative activity was examined.Effect of SHAM,Ascorbic Acid and mannitol on inhibition of enestrobilurin to B2 mycellia growth.The result indicated enestrobilurin can inhibited mycelia growth,spore production,but it can not inhibited melanin biosythesis.In the spot,enestrobulin had better protective and curative activity.Resistance induce:Magnapothe grisea isolates were gradually adapted to the fungicide or were exposed to either fungicide or UV light for mutation induction and then screened,resulting in the resistant mutants MRA5-f,MRB2-f,MRB6-f,MRC4-f,MRF3-f,MRF8-f,LRB6-f,LRF8-f,LRF3-UV+f,LRA5-UV+f.Tow lower lever mutant of Magnapothe grisea with enestrobilurin-resistance was isolated by treating mycelium with enestrobulin and ultraviolet radiation.The resistance level of mutant was 12.10 and 14.14 times as original isolates.By validating resistance,the result suggested that the resistance to enestrobulin of other 6 mid-resistance mutants of Magnapothe grisea was obtained.The resistance level of mutant MRB6-f is the highest(27.24).Results of the chemical inducement treatment were basically the same.Two low resistance mutants LRB6-f and LRF8-f were obtained by chenmical inducement.Mutation induction with UV light and the chemical inducement treatment produced a higher frequency of resistant mutants.The chemical inducement was better than both fungicide and UV light.The biological feature of resistant isolates were examined,including mycelial growth rate of fungi,spore production,Competitiveness,Producing toxin ability,Osmotic sensitivity (including different centigrade degree,different concentration of Nacl,different dextrose):the competitiveness of the resistant and susceptible strains was determined in potted plant tests in order to more reliably evaluate fitness of the resistant strain.The resistant and susceptible spores were inoculated with a 1:1 mixture.After 8-10d,the disease index of the resistant and susceptible strains were got.The The result indicated that,the pertinence coefficient between fungi growth rate and the EC₅₀of isolates in 0.7847,it is obvious in 0.01 value,that is to say,more the isolate sensitive to enestrobilurin,faster the fungi growth rate;the pertinence coefficient between spore amount and the EC₅₀of isolate is 0.5818,it is not obvious in 0.05 value;mycelium the pertinence coefficient between fresh weight the EC₅₀of isolate is 0.9661,it is obvious in 0.01.pathogenicity:in order to reflect field conditions.Thus, competitiveness of the resistant and susceptible strains was determined in potted plant test in order to more reliably evaluate fitness of the resistant strains.After inoculation with a 1:1 mixture of resistant and susceptible spores,which indicates that the pathogenicity of resistant spores is more weaker than sensitive ones,the susceptible ones became the dominant strains.Dis.index is obvious in 0.05 value.The resistant mutants can't pass down stably,they can become sensitive isolates after they were continuously transferred in YPSA medium without fungicide.The result of Osmotic sensitivity was that the regulative ability of sensitive strains was better than resistance ones.More the isolate sensitive,faster the fungi growth rate. Producing toxin ability:as for F3,producing toxin ability of sensitive strain was better than mutants.By contrast,the EC₅₀value of F3 was 30.3592μg·mL^-1,MRF3 was 50.3925μg·mL^-1, LRF3 was 42.5826μg·mL^-1.Cross resistance.In order to investigate the existence of cross resistance between enestrobilurin and other classes of fungicides,cross resistance was determined between enestrobilurin and tricyclazole,carbendazim,kresoxim-methyl,azoxystrobin,prochloraz, isoprothiolane,EBP fungicides.Results indicated that enestrobilurin is cross with these two fungicides(kresoxim-methyl,azoxystrobin)belonged to strobilurins.enestrobilurin is not cross with other two classes of fungicides,such as tricyclazole,carbendazim,prochloraz, isoprothiolane,EBP.Strobilurins belong to the high resistance risk fungicides,they are easy to produce resistance.The major mechanism of resistance is the amino acid substitution of glycine with alanine at position 143(G143A).A second target site mutation,the substitution of phenylalanine with leucine at position 129 of the cytochrome b(F129).To characterize this resistance,the cyt b gene was amplified from enestrobilurin-sensitive isolates and induced enestrobilurin-resistant isolates.We obtained the amino acid and nucletide sequence of the partial cyt b gene from enestrobilurin-sensitive and enestrobilurin-resistant strains of M.grisea..Three differences were founded in the necletide of the three sensitive isolates.Bue the amino acid sequence was the same.None of the mutation site was found.The induced enestrobilurin-resistant isolates,target site mutation,the substitution of phenylalanine with leucine at position 129 of the cytochrome b(F129).(TTT toTTA).The result of this chapter was similar to the result reported by other people.