Detection Of Avirulence Gene Mutations In The Field Population Of Rice Blast Fungus In Fujian Province

Rice blast is the major disease that threatens rice production in Fujian.At present,the two commonly used measures for the control of rice blast are the breeding of disease-resistant varieties and the use of fungicides.Particularly,breeding of new disease-resistant varieties and the appropriate rotation of the resistance varieties is the most environment-friendly and cost-effective method to control rice blast disease,which had long been used and promoted broadly.However,due to the fast-evolving genome and the rapid variation of avirulence genes(AVR genes)of the Magnaporthe oryzae population,newly applicated disease-resistant varieties often lose the resistance 3-5 years after released,resulting in the outbreak of blast disease.Therefore,monitoring the mutation frequency of AVR genes can guide the appropriate application and rotation of resistance varieties for efficient controlling of this devastating blast disease.In this study,we investigated the pathogenicity and genetic variation of 8 AVR genes in 113 M.oryzae single spores isolated from field populations collected from three different rice planting regions in Fujian Province,which may facilitate the appropriate planting of the resistance cultivars next year.For pathogenicity and virulence assays of these strains,spray inoculation was performed using 24 monogenic lines carrying 24different major blast resistance genes.The results showed that the pathogenic frequency of 23 strains isolated from Jianyang were ranged from 24.50%to 95.83%,and the strong pathogenic strains were dominant in the population.Among them,?70%strains,which belong to strong virulence,are virulent on 16 resistance genes namely Pia,Pii,Pik~s,Piz,Pita,Pib,Pit,Pish,Pi3,Pi5,Pi7,Pi12,Pi19,Pi20,Pita~2and Pi11.The results indicated that rice varieties containing these resistance genes could not be planted in this region.The pathogenic frequency of 68 strains isolated from Ninghua area ranged from 24.97%to 100%,and strong pathogenic strains are dominant in this area.Among them,?70%of the strains are virulent on 16 resistance genes namely Pia,Pii,Pik~s,Piz,Piz~t,Pita,Pib,Pit,Pi3,Pi5,Pi7,Pi12,Pi19,Pi20,Pita~2,Pi11,which indicated that rice varieties carrying these resistance genes could not be used next year in the region.The pathogenic frequency of 22 strains isolated from Shanghang area ranged from 24.5%to 86.36%,and moderately pathogenic strains were dominant in this area.Among them,?70%of the strains were virulent on monogenic lines carrying 4resistance genes,namely Pik~s,Pib,Pi3 and Pi12.This indicated that the 4rice varieties containing these resistance genes could not be planted in the region.To investigate the genetic variation of AVR genes in the above strains,we designed specific primers for 8 AVR genes:Avr-Pia,Av r-Pita,Avr-Pib,Avr-Pik,Avr-Pizt,Avr-Pii,Avr-Pi9,Avr-Pi54,and Av r1-Co39.Whole genome DNA prepare from the 113 strains were us ed as template for the PCR-based genotyping.As expected,we coul d not amplify any target band for Avr1-Co39 in all these strains,in dicating that all the strains collected in the three regions of Fujian Province did not carry this avirulence genes.For Avr-Pib,the ampli cons showed different sizes were obtained,indicating that mutation events occurred on Avr-Pib of these strains.Accordingly,the sequen cing results confirmed that the deletion,substitution and insertion o ccurred in promoter next to the start codon Avr-Pik?Avr-Pii and Av r-Pi54 are the 3 AVR genes showing high presence frequency in al l the 113 strains,although the PCR amplification results of some st rains are inconsistent with the pathogenicity results.The sequencing results showed that single nucleotide polymorphism also presented in Avr-Pik and Avr-Pita,which may lead to the loss of the function of the two genes according to the pathogenicity assay results.