| Gray mold has been a great threat in protected vegetable production which is popular in Liaoning Province.Therefore,it is meaningful to implement a thorough study and prevention of this disease with the purpose to make normal vegetable production and to protect the farmers’interest.Taking Botrytis cinerea from tomatoes as the main research object,we evaluated the resistance to procymidone and biological characteristics of the procymidone-resistant isolates in Liaoning Province.Resistance mechanism of Botrytis cinerea to procymidone in Liaoning Province was also studied.Finally,based on the molecular genetic differences,we established a loop-mediated isothermal amplification(LAMP)method for detection of procymidone-resistant mutants in tomato gray mold pathogen Botrytis cinerea,which can provide a technical mean for resistance monitoring and management.Resistance of Botrytis cinerea to procymidone was very general,which has the tendency to aggravate.A total of 134 isolates were collected from 8 cities(Panjin,Huludao,Chaoyang,Dandong,Faku,Fuxin,Pulandian,Shenyang)in Liaoning Province,in which 96(83.48%)isolates were resistant to procymidone.The resistance frequencies of Panjin,Huludao,Chaoyang and Shenyang were more than 90%,and one highly resistant isolate was found separately in Panjin and Fuxin.There was difference in the resistance level of different regions.However,hierarchical clustering analysis of Botrytis cinerea resistance to procymidone showed that the resistance of isolates was not directly related to its region in Liaoning Province..There were differences in the biological characteristics between the procymidone-resistant isolates and procymidone-sensitive isolates of Botrytis cinerea in Liaoning Province.The tests of temperature sensitivity showed that the optimum growth temperature of all the isolates was 20 ~oC or 25 ~oC.In the same temperature,there were no differences in the mycelium growth rate,sporulation and spore germination between the procymidone-resistant isolates and procymidone-sensitive isolates.However,micromorphology of the procymidone-resistant isolates and procymidone-sensitive isolates had certain differences through procymidone treatment.Pathological experiments showed that the procymidone-sensitive isolates exhibited better pathogenicity than the procymidone-resistant ones.There were point mutations in the BcOS1 of the procymidone-resistant isolates of Botrytis cinerea in Liaoning Province.All the gene encoding fragments of BcOS1 were obtained by polymerase chain reaction(PCR)from 115 Botrytis cinerea isolates.Analyses of the sequences of BcOS1 revealed that the procymidone-sensitive isolates showed no point mutation,and the procymidone-resistant isolates had two mutations at position 369 and position373,and the mutation types involved Q369P and Q369P+N373S.Further investigations indicated that there was a conection between mutation type and the location of isolate.The loop-mediated isothermal amplification(LAMP)method was established for detecting the procymidone-resistant mutants of tomato gray mold pathogen Botrytis cinerea in Liaoning Province.On the basis of BcOS1 gene of procymidone-resistant genotype Q369P in Botrytis cinerea,graminearum,the primers were designed and optimized to specially distinguish the procymidone-resistant mutants.Through optimization of the concentration of components,a rapid detection method for procymidone-resistant mutants of Botrytis cinerea based on loop-mediated isothermal amplification was established.In addition,artificial inoculation test was performed.The results further confirmed that the developed LAMP had good reliability,stability and maneuverability and had a broad application prospect in the production. |
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