| Mango(Mangifera indica L.)is a major tropical and subtropical cash crop.,It is widely planted in Hainan Province,China,has an annual yield up to 700,000 tons.Mango stem-end rot is one of the main postharvest diseases of mango,which seriously damages the yield and quality of mango.Lasiodiplodia theobromae is an important pathogen causing mango stemend rot and postharvest fruit rot.This fungus may become established asymptomatically in the field and remain in a quiescent state.The frequent use of chemical fungicides in mango orchards increases selection pressure and resistance of L.theobromae.In order to determine the pyraclostrobin-resistance of L.theobromae in Hainan,and to explore the biochemical and molecular mechanisms of resistance to pyraclostrobin,this study has carried out related research as follows: The resistance level of 91 isolates of L.theobromae to pyraclostrobina was determined,and the cross resistance of the isolates to other four fungicides was detected;The resistance stability,ATP content and AOX enzyme content of the different pyraclostrobin-resistance isolates were determined to explore the biochemical mechanism of resistance;The gene sequences of Cytb gene and AOX gene of isolates were cloned and analyzed,and the expression levels of these two genes were assayed to explore the molecular mechanism of pathogen resistance.The results showed that:1.Among the 91 L.theobromae isolates,87 isolates were resistant to pyraclostrobin,with a resistance frequency of 95.6%,among them,28.6% were highly resistant.The results indicated that L.theobromae field isolate from mango in Hainan has a high resistance risk to pyraclostrobin.2.The cross resistance test showed that pyraclostrobin-resistant isolates were generally resistant to azoxystrobin,a kind of Qo Is fungicide,and there was cross resistance between pyraclostrobin and azoxystrobin.Some isolates showed high resistance to carbendazim,a kind of benzimidazole fungicide,but many were sensitive to phenylpyrrole fungicide fludioxonil and triazole fungicide difenoconazole.These results indicated that there was no cross resistance between pyraclostrobin and the three fungicides above.3.After subjected to 10 successive transfers on fungicide-free PDA medium,the mycelial growth rate changed little and the resistance could be inherited stably when cultured on PDA containing pyraclostrobin.With the increase of pyraclostrobin concentration,Compared with pyraclostrobin-sensitive isolates,the ATP content in resistant isolates was significantly different(p < 0.05).The higher the resistance level of the isolates,the higher the ATP content in isolates.This indicated that the resistance of pyraclostrobin may be related to the ATP synthesis.4.The content of AOX in highly resistant isolates was significantly higher than that in sensitive and low resistant isolates.AOX enzyme was inhibited to some extent by salicylhydroxamic acid(SHAM),and the AOX enzyme content of resistant isolate was significantly higher than that of sensitive isolate(p < 0.05).5.Sequence alignment of Cytb gene and AOX gene showed that there were no mutations at 129,137 and 143 codons of Cytb gene in resistant isolates.However,there were25 differences in amino acid codons of AOX gene in resistant isolates when compared with that of sensitive isolates.There was no significant difference in the relative expression of Cytb gene among the different resistance isolates,but the relative expression of AOX gene in the highly resistant isolate was significantly higher than that in the low resistant isolate and the sensitive isolate.Conclusion: L.theobromae was seriously resistant to pyraclostrobin;There was cross resistance between pyraclostrobin and azoxystrobin.The pyraclostrobin-resistance of the L.theobromae was not caused by the mutation at 129,137 and 143 of potential resistance mutation sites of Cytb gene,but it might be related to the expression of AOX gene and the mutation of AOX gene. |
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