Detection And Biological Characteristics Of Alternaria Alternata Resistant To Four Fungicides From Commercial Field Of Paris Polyphylla Var.Chinensis,an Indigenous Medical Herb

Disease life cycle and chemical control for Alternaria alternata on Paris polyphylla Smith var.chinensis(Franch.)Hara,an indigenous medical herb,have seriously lagged and restrained the development of the industry.The yield of wild P.polyphylla Smith var.chinensis(Franch.)Hara is seriously limited.However,the disease booms when the herb grows on a large-scale and commercial plantation.The black spot caused by A.alternata on P.polyphylla Smith var.chinensis(Franch.)Hara has caused serious damage to yield.The pathogen can infect other crops,including pear,apple,strawberry,potato and wheat.Owing to disease-resistant varieties unavailable;systematic researches on disease occurrence and prevention and control are rare;and no government-registered fungicides on this herb are registered.Furthermore,the fungicides registered in other crops are widely used to control black spot on P.polyphylla Smith var.chinensis(Franch.)Hara.However,these fungicides have been used for decades,but the resistance status of A.alternata to them is unclear.Therefore,the diseased samples were collected from Mianyang City,Sichuan Province,China.These isolates were separated and single-conidium strains were obtained.Resistance frequencies to difenoconazole,procymidone,pyrimethanil or azoxystrobin with different modes of action were determined and biological properties of A.atlernata resistant to difenoconazole,procymidone and pyrimethanil were revealed,respectively.The main progress was present as follows:(1)Morphological and molecular identificationCombined by colony morphology on the PDA plate,the size and shape of the spores,the pathogen was microscopically identified as Alternaria sp..The ITS,GAPDH and RPB2 genes were amplified from the genomic DNA of the representative strain R20,then aligned on the NCBI website,and constructed a phylogenetic tree.Finally,the pathogen was identified as Alternaria alternata.(2)Resistance frequency and biological properties of strains from A.alternata to demethylation inhibitors(DMIs)Of the 22 strains of A.alternata,20 strains were resistant to difenoconazole,and the resistance frequency was 90.91%.The mycelial growth rates of difenoconazole-resistant strains were lower than that of the sensitive strain,but the sporulation of the difenoconazole-resistant strains were more than that of the sensitive strain.There was difference significantly in pathogenicity between low-resistant and high-resistant strains.The virulence of resistant strains were higher than that of the sensitive strain on tobacco and pear,but they were opposite on tomato.No positive cross-resistance between difenoconazole and propiconazole or tebuconazole was observed.Mutations were distributed in the P450 domain of AaCYP51 gene of the resistant strains based on sequencing and alignment of difenoconazole-sensitive and-resistant strains.The mutations can be divided into two types[(K715R+Y781C);(K715R+D1140G+T1628A)].The docking score of the sensitive strain was 5.6020,higher than 3.8651 of the resistant strains R19(three-point mutation),or 4.4599 of the resistant strain R37(double point mutation).Therefore,the affinity of the sensitive strain to difenoconazole was less than the resistant strains.(3)Resistance frequency and biological properties of strains from A.alternata to dicarboximides(DCFs)Of the 22 strains of A.alternata,8 high-resistant strains and 14 low-resistant strains to procymidone,and the resistance frequencies were 36.36%and 63.64%,respectively.The mycelial growth rates of procymidone-high resistant strains were lower than that of the low resistant strains,but the sporulation of the procymidone-high resistant strains were more than that of the the low resistant strains.There was difference significantly in pathogenicity between sensitive and resistant strains.The intracellular glycerol contents of the procymidone-high resistant strains were higher than that of the low resistant strains,and the intracellular glycerol content of the high resistant strains increased significantly after treatment with 1.0 M NaCl for 2 h.Sensitivity of the procymidone-high resistant strains to different stress factors differented.No positive cross-resistance between procymidone and iprodione was observed.Point mutation at the AaOS1 gene from strains HR12-1 and HR40-2 were found,including serine to leucine(S1277L)and valine to leucine(P894L),respectively,and no point mutation of other strains was discovered.No mutations were detected in the AaHOGl and AaTPI1 gene from the procymidone-resistant strains.(4)Resistance frequency and biological properties of strains from A.alternata to anilino-pyrimidines(AP fungicides)Of the 22 strains of A.alternata,14 strains were resistant to pyrimethanil,and the resistance frequency was 63.64%.The mycelial growth rates of pyrimethanil-sensitive strain was lower than that of the resistant strains,but the sporulation of pyrimethanil-sensitive strain was more than that all of the resistant strains.The virulence of resistant strains was more than sensitive strain.No positive cross-resistance between pyrimethanil and cyprodinil was observed.Three point mutations were harbored only at the resistant strain R39 MDL2 gene:threonine to methionine(T130M),cysteine to glutamine(C357Q)and lysine to arginine(K494R),respectively.Futhermore,point mutations were determined at the POS5 target gene from the pyrimethanil-resistant strains,including leucine to valine(I46V)from the resistant strain R4,valine to leucine point mutation(P214L)from resistant strain R15,and two point mutations[valine to leucine(V7M)and alanine to valine(A393V)]from resistant strain R39.(5)Resistance frequency of strains from A.alternata to azoxystrobinOf the 22 strains of A.alternata,no azoxystrobin-resistant strain was detected.