| Brown spot(BR),caused by Alternaria alternata(Fr.)Keissl.,is an important foliar disease of tobacco.Dicarboximide fungicides and other taditional protective fungicides are commonly used for control BR.In this study,the resistance to the taditional protective fungicides and sensitivity to new fungicides of Alternaria alternata,which collected from xinquan(FQ)and xingyi(XY)in Guizhou province,were studied to providing scientific data for management of BR.Resistance to thiophanate-methyl and procymidone was determined by distinguishing dosage method.Results showed that all the tested strains(n=128)were highly resistant(HR)to thiophanate-methyl(a benzimidazole fungicide).And the ?-Tubulin gene of resistant strains were amplified and sequenced.Sequence alignment indicated that the amino acid tyrosine at codon position 167,which explained that the reason A.alternate is resistant to thiophanate-methyl.The resistance frequency to procymidone(a dicarboximide fungicide)was 8.3% and all resistant isolates(n=9)were low-level(LR).Biological activity of azoxystrobin to A.alternata was evaluated.Without the synergism of salicylhydroxamic acid(an alternative oxidase inhibitor,SHAM),the effect of azoxystrobin on A.alternata spore germination was highly,while mycelia growth was weakly,with the average EC50(effective concentration to inhibit 50% growth)values 10.556 and130.545 mg/m L,respectively.The mean EC50 value of azoxystrobin to spore germination and mycelia growth was 0.140 and 17.760 mg/m L,respectively in the presence of 100 mg/m L SHAM.The protective and therapeutical effects of azoxystrobin were measured on detached leaves.The results showed that the EC50 value of azoxystrobin to inhibited A.alternata spot extent,pre-applied of azoxystrobin at 0h?12h and 24 h,were 0.118,0.084 and 0.001mg/m L respectively.When the concertration of azoxystrobin was greater than 1 mg/m L,A.alternata failed to form a visible lesions in leaves.And azoxystrobin also had the ability to inhibit the extent of lesion with the average value of 0.180 mg/m L 48 h after innoculations.Determination the sensitivity of A.alternata population(n=95)to 25% azoxystrobin SC by using mycelium growth rate method in the absent of SHAM,all the EC50 values were more than 5 mg/m L,74.7% EC50 values of the strains were more than 100 mg/m L.The sensitivity of A.alternata population(n=33)to azoxystrobin were determined by using spore germination method in the presence of 100 mg/m L SHAM.The EC50 values varied from 0.004 to 2.416 mg/m L with the average value of 0.230±0.470 mg/m L.The sensitivity of A.alternata to boscalid(a succinate dehydrogenase inhibitor fungicide)was also determined by using mycelium growth rate method and the iron-sulfur subunit(Sdh B)gene of 12 sensitive and low sensitive strains were amplified and sequenced.The EC50 values for the tested population(n=102)to boscalid ranged from 0.186 to 5.818 mg/m L(average value of 2.157±1.112 ?g /m L)following a unimodal distribution.Further analysis declared that the EC50 values of the two areas were significantly different(p<0.01).Sequence analysis showed that some of the substitutions occurred in sensitive isolates,the deduced amino acid showing at codon 224 was replaced by arginine acid or glycine acid(C224R/G),a substitution of serine acid by proline acid occurred at amino acid position 226(S226P).To manage resistance,the screening of the compound fungicides was carried out.Synergistic effect was found for all the ratio except 7:1 between pyraclostrobin and boscalid against A.atrans.And the best ratio was 7:1 for inhibiting A.alternata spore germination.All the ratio of pyraclostrobin and imazalil had synergistic effect for inhibiting Alternaria sp.mycelium growth and the best ratio for inhibiting A.alternata mycelium growth was 1:7. |
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