| Broccoli,cabbage and peanut black spot caused by Alternaria alternata and peanut web blotch caused by Phoma arachidicola have become more and more serious,which has be-come a restricting factor for the industrial development.At present,due to lack of resistant varieties,pesticide is the main emergency prevention and control measure in disease control.However,due to the frequent and long-term use of fungicides,the fungicide-resistance in the field is unclear.For this purpose,the two pathogens were isolated and identified from samples collected in Jiangsu,Shandong and Hebei Provinces to detect the resistance of azoxystrobin,boscalid,tebuconazole,procymidone and pyrimethanil with different modes of action,fre-quency of resistance to the five fungicides and the biological characteristics of resistant iso-lates were determined.The main results were present asfollows:（1）Biological characteristics and frequency of resistance to five fungicides in A.alter-nata from broccoli.199 isolates were sensitive to azoxystrobin and pyrimethanil,however,different degrees of resistance to boscalid,tebuconazole and procymidone were observed.The frequency of single resistance was 12.55%,including frequency of procymidone-single resistance was5.02%,frequency of boscalid-single resistance was 3.01%,and frequency of tebuconazole-single resistance was 4.52%;The frequency of dual resistance was 68.34%,including fre-quency of procymidone and boscalid-dual resistance was 16.58%,frequency of procymidone and tebuconazole-dual resistance was 27.64%,and frequency of tebuconazole and boscalid-dual resistance was 24.12%;The frequency of procymidone,tebuconazole and boscalid-tri-ple resistance was 6.54%.Biological characteristics of procymidone-highly resistant(Pro HR)A.alternata isolates from broccoli:Most Pro HR isolates in the fitness were reduced compared with procymidone-sensitive（ProS）isolates;Correlation analysis between fungicide sensitivities revealed a pos-itive cross-resistance between procymidone and iprodione or fludioxonil;The most ProHRisolates displayed high sensitivity to osmotic stress;When treated with 10μg/m L pro-cymidone for 5 h,the net increases of MIGAs in ProHR isolates were significantly lower than that in ProS isolates;Sequence alignment results of Aa HK1 gene indicated that five types of mutations were found in 6 ProHR isolates,including 3 types of single point-mutations and 2types of deletions.Biological characteristics of boscalid and tebuconazole-dual resistant isolates of A.al-ternata from broccoli:The fitness of resistant isolates was similar to sensitive isolates.When treated with boscalid for 24 h,increased gene expressions of Aa Sdh B,Aa Sdh C or Aa Sdh D from sensitive isolates were observed compared with those on boscalid-free condition,fur-thermore,the net reduction of intracellular Succinate Dehydrogenase（SDH）activity of the sensitive isolate was higher than those of low-resistant isolates.Sequence alignment results of Aa Sdh B,Aa Sdh C,Aa Sdh D or Aa Cyp51 genes showed that point-mutations were found in Aa Sdh B,Aa Sdh C and Aa Cyp51.When treated with tebuconazole for 24 h,the gene expres-sion of Aa Cyp51 from low-resistant isolates was significantly increased compared with that on tebuconazole-free condition.（2）Biological characteristics and frequency of resistance to five fungicides in A.alter-nata from cabbage.85 isolates were sensitive to azoxystrobin and pyrimethanil,however,different degrees of resistance to boscalid,tebuconazole and procymidone were observed.The frequency of single resistance was 21.18%,including frequency of procymidone-single resistance was7.06%,frequency of boscalid-single resistance was 5.88%,and frequency of tebuconazole-single resistance was 8.24%.The frequency of dual resistance was 61.18%,including fre-quency of procymidone and boscalid-dual resistance was 20.00%,frequency of procymidone and tebuconazole-dual resistance was 20.00%,and frequency of tebuconazole and boscalid-dual resistance was 21.18%.The frequency of procymidone,tebuconazole and boscalid-triple resistance was 11.76%.Biological characteristics of ProHR A.alternata isolates from cabbage:The Pro HR iso-lates in the fitness were reduced compared with ProS isolates;The ProHR isolates displayed high sensitivity to osmotic stress;Correlation analysis between fungicide sensitivities re-vealed a positive cross-resistance between procymidone and iprodione or fludioxonil;When treated with 10μg/m L procymidone for 5 h,the net increases of MIGAs in ProHR isolates were significantly lower than that in ProS isolates;Sequence alignment results of Aa HK1gene indicated that 3 types of mutations were found in the 3 ProHR isolates,including point mutation,deletion and insertion.（3）Biological characteristics and frequency of resistance to five fungicides in A.alter-nata from peanut.155 isolates were sensitive to azoxystrobin and pyrimethanil,however,different de-grees of resistance to boscalid,tebuconazole and procymidone were observed.The fre-quency of single resistance was 50.97%,including frequency of procymidone-single re-sistance was 40.00%,frequency of boscalid-single resistance was 3.22%,and frequency of tebuconazole-single resistance was 7.74%.The frequency of dual resistance was 36.78%,including frequency of procymidone and boscalid-dual resistance was 4.52%,frequency of procymidone and tebuconazole-dual resistance was 3.22%,and frequency of tebuconazole and boscalid-dual resistance was 29.03%.The frequency of procymidone,tebuconazole and boscalid-triple resistance was 3.22%.Biological characteristics of boscalid and tebuconazole-dual resistant isolates of A.al-ternata from peanut:The fitness of resistant isolates was similar to sensitive isolates;When treated with boscalid for 24 h,increased gene expressions of Aa Sdh B,Aa Sdh C or Aa Sdh D from sensitive isolates were observed compared with those on boscalid-free condition,fur-thermore,the net reduction of intracellular SDH activity of the sensitive isolate was higher than those of low-resistant isolates.Sequence alignment results of Aa Sdh B,Aa Sdh C,Aa Sdh D and Aa Cyp51 genes showed that point mutations were found in Aa Sdh D and Aa Cyp51 gene.When treated with tebuconazole for 24 h,the gene expression of Aa Cyp51 from low-resistant isolates was significantly increased compared with those on tebuconazole-free condition.（4）Biological characteristics and detection of resistance to five fungicides in P.arachi-dicola from peanut.30 isolates were sensitive to azoxystrobin,boscalid and pyrimethanil,and were low-resistant to tebuconazole and procymidone.No significant difference of tebuconazole and procymidone-dual resistant isolates of P.arachidicola was observed in mycelial growth,sporulation capacity or virulence. |
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