Studies On Resistance Mechanisms Of Sclerotinia Sclerotiorum To Dimethachlone

Sclerotinia stem rot disease is caused by Sclerotinia sclerotiorum(Lib.)de Bary,a fungal plant pathogen with a broad host range.The disease may occur from the seedling to the mature stage of oilseed rape,causing enormous economic losses to oilseed production worldwide.The dicarboximide fungicide dimethachlone has been used to control S.sclerotiorum for more than a decade and resistance to dimethachlone has recently been reported in China.In the present study,the hereditary stability of dimethachlone resistance was determined in field and laboratory-induced resistant isolates,the biological characteristics were measured in the laboratory-induced resistant isolates after four years of cold storage(4℃),and the physiological and biochemical mechanisms for dimethachlone resistance were investigated.The histidine kinase(HK)gene Sshk,response regulator gene Sssk1,his-phosphotransfer gene Ss Ypd,and a mitogen-activated protein kinase(MAPK)cascade genes Sssk2,Ss Pbs,and Ss Hog were cloned and sequenced in three arbitrarily selected sensitive isolates and three dimethachlone resistant ones.The molecular mechanisms for dimethachlone resistance were studied by the RNA interference method.The results were as follows:1.Dimethachlone resistance of field resistant isolates(HLJ3,HLJ4,and HLJ6)remained stable after four years of cold storage at 4℃ and after consecutive transfers for 20 generations on PDA without fungicide.In contrast,dimethachlone resistance of two laboratory-induced resistant isolates SCG7 and LA50 declined dramatically by 99.5% and 98.9%,respectively.Along with the decline of dimethachlone resistance,mycelial growth rate,sclerotia production,and pathogenicity increased significantly,whereas osmotic sensitivity decreased tremendously.Significant negative correlations were detected between dimethachlone resistance levels and mycelial growth rate on PDA(r =-0.980,P = 0.021),and between resistance levels and pathogenicity(r =-0.997,P = 0.002).2.The biochemical characteristics of dimethachlone resistant isolates(HLJ3,HLJ4,and HLJ6)and sensitive isolates(HLJMG2,HLJMG3,and HLJMG5)were measured.The field resistant isolates exhibited significantly higher cell membrane permeability,POD,and PPO activities than dimethachlone sensitive isolates.Dimethachlone at 0.25 μg/m L significantly increased cell membrane permeability and enhanced activity of the two enzymes in both resistant and sensitive isolates.There were no significant differences in glycerol or oxalate content between the resistant and sensitive isolates.Dimethachlone at 0.25 μg/m L increased glycerol content in the resistant isolates,whereas reduced in the sensitive isolates.These findings indicate that the cell membrane permeability,POD and PPO activities might be related to dimethachlone resistance in S.sclerotiorum.3.Histidine kinase(HK)gene Sshk,response regulator gene Sssk1,his-phosphotransfer gene Ss Ypd,and MAPK cascade genes(Sssk2,Ss Pbs,and Ss Hog)were cloned and sequenced in three dimethachlone sensitive and three resistant isolates.Dimethachlone resistant isolate HLJ4 harbored point mutation in the gene Sshk located in the first 90-amino-acid repeat(I232T)and isolate HLJ6 had the mutation(G1087D)between the kinase core domain and the RR domain.Isolate HLJ4 had a point mutation of P96 L in the deduced amino acid sequence of the MAPK gene Ss Pbs.Several synonymous mutations were found in the Sssk1(A759G)and Ss Ypd genes(T324C)in isolates HLJ6 and HLJ3,and in Sssk2 gene(C48T)in isolate HLJ4 and T1736 G in isolate HLJ3.The expression levels of the Sshk gene were higher in the isolate HLJ4 and HLJ6 than in the sensitive isolate HLJMG2 and the resistant isolate HLJ3.The transcription of the Sshk gene was up-regulated by dimethachlone for the three resistant isolates.These findings indicate that upregulations of transcription of the Sshk gene might be involved in dimethachlone resistance.4.A Sshk-silenced vector and an overexpression vector were constructed and the transformants were screened by ATMT technology.Compared with parent isolate HLJ4,the transcription of the Sshk in three silenced transformants was significantly down-regulated by 99.9%,94.1%,and 95.1%,respectively.Dimethachlone resistance ratios dramatically declined by 62.9%,42.5%,and 49.8%,respectively.Compared with parent isolate HLJMG1,the Sshk transcription levels of three over expression transformants were up-regulated 7.7,10.2,and 14.1-fold,respectively,and dimethachlone resistance ratios increased 168.1,189.5,and 221.2-fold,respectively.The three silenced transformants exhibited significantly higher mycelial growth and pathogenicity,lower sclerotia production capability and sensitivity to temperature and osmosis.Transmission electron microscope observations indicated that the cell wall and plasma membrane of most cells shrank in the three transformants,the organelles of partial cells were destroyed and the contents leaked,leading to cell lysis.The over expression transformants had dramatically lower mycelial growth and pathogenicity,higher temperature adaptability and osmotic sensitivity.These results demonstrate that transcription levels of Sshk were correlated with dimethachlone resistance levels in S.sclerotiorum.