Detection And Biological Characteristics Of Alternaria Alternata From Garlic Field Resistant To Four Fungicides With Different Modes Of Action

Garlic is the main economic crop in Jiangsu and Shandong provinces,making huge contributions to the production of crop and economic development.But,the serious garlic leaf spot caused by Alternaria spp in recent years,causing great loss of garlic production.In addition,the pathogen could infect numerous plants,for example,broccoli,tomato,potato,pear,apple,strawberry,pepper,bean and so on.To date,the most effective and economical method to prevent this disease is to depend on application of fungicides.But,the effectiveness of fungicides decreased because fungicides were used too frequently.In order to understand the resistance of Alternaria spp to four fungicides of azoxystrobin,tebuconazole,boscalid and procymidone,the diseased samples collected from Jiangsu and Shandong Provinces were isolated,purified and identified.At the same time,the biological characteristics of resistant isolates were studied.The main progress was present as follows:(1)Morphological and molecular identificationThe pathogen was preliminarily determined as Alternaria spp according to the colony morphology,the size and shape of the spore.In addition,the ITS and GAPDH genes were amplified from the genomic DNA of the representative strain,then aligned on the NCBI website and the pathogen was identified as Alternaria alternata finally.(2)The resistant detection of Alternaria alternata to strobilurins(QoIs)fungicidesOf the 151 A.alternata isolates,no azoxystrobin-resistant strain was obtained,showing that the pathogen of A.alternata has not generated resistance to azoxystrobin.(3)The resistant detection of Alternaria alternata to demethylation inhibitors(DMIs)fungicidesThe 151 A.alternata isolates were resistant to tebuconazole.(4)Resistance frequency and biological characteristics of A.alternata isolates to Succinate dehydrogenase inhibitor(SDHI)fungicidesOf the 151 A.alternata isolates,113 isolates were resistant to boscalid,and the resistance frequency was 98.26%.There is no significant difference between resistant and sensitive isolates in mycelial growth,but the sporulations of boscalid-resistant isolates were more than that of the sensitive isolates,Positive cross-resistance between boscalid and Y13149 was observed,and no cross-resistance between boscalid and pdiflumentofen.According to sequencing and alignment of boscalid-sensitive and-resistant isolates,mutations were distributed in the SDHC(I21V+A28V+I52V+C169F)and SDHD(A19Q+I30T+A36V+L44F+P46R+T48A+P50Q+R51K+A53T+V74I+A149S+L154K+V155T+A157K+S159A)genes of the resistant isolates.(5)Resistance frequency and biological characteristics of A.alternata isolates to dicarboxyimide fungicidesOf the 151 A.alternata isolates,14 isolates were high resistant(HR)to procymidone and fiudioxonil,and the resistance frequency was 12.17%.There is no significant difference between HR and sensitive isolates in mycelial growth,but the sporulations of HR isolates were more than that of the sensitive isolates,Positive cross-resistance between procymidone and iprodione or fiudioxonil was observed,and no cross-resistance between fiudioxonil and fluazinam or azoxystrobin.In the absence of procymidone,glycerol concentrations in the sensitive and resistant isolates were relatively low.When treated with procymidone at 10 μg/ml,glycerol concentrations in the sensitive isolates were increased significantly,but those in HR isolates were increased slightly.The sensitivities are different to salt stress(1 M NaCl and 1 M KCl)between HR and sensitive isolates,but no difference to cell wall and cytomembrane damage agents.The OS1 genes of HR isolates were modified except HR isolates PZ10,PZ74 and PZ78.The HR isolates PZ40 and PZ70 had single-point mutation S1277L(an amino acid changed from serine to leucine at position 1277)and G512R(an amino acid changed from glycine to arginine at position 512),respectively;the HR isolates PZ71 had five point mutations,G524D(an amino acid changed from glycine to aspartic acid at position 524),R1144T(an amino acid changed from arginine to threonine at position 1144),K1161N(an amino acid changed from lysine to asparaginate at position 1161),A1180P(an amino acid changed from alanine to proline at position 1180)and V1198L(an amino acid changed from valine to leucine at position 1198);the OS1 amino acid of HR isolates of PZ72,PZ75,PZ76 and PZ95 shifted because of the deletion nucleotides of A,G&G,C or T at positions 1175,1566&1578,4120 or 2273,respectively,resulting in early or delayed termination of the OS1 sequence;the nucleotide C at position 886 of HR isolate PZ73 changed into T,leading to a frameshift and a premature stop codon at amino acid position 263;there is a 18bp nucleotide duplication inserted into the OS1 sequence of HR isolate PZ77,resulting in a frameshift and a delayed termination;a nucleotide G inserted into the OS1 sequences of HR isolates PZ80 and PZ92,resulting in early termination.Furthermore,no mutation occurred in AaHOG1 sequence of HR isolates.In addition,the proportion of the 115 strains that are all sensitive to the four fungicides(S)was 0,and the proportion of strains(R1)that were only resistant to boscalid was 1.739%.The proportion of strains(R2)that were resistant to tebuconazole and boscalid was 86.087%,and the proportion of strains(R3)that were resistant to tebuconazole,boscalid and procymidone was 12.174%.