Study On Resistance Mechanisms To Dicarboximide Fungicide Dimethachlone In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum is the causal organism of the destructive stem rot disease in oilseed rape. Due to the lack of highly resistant cultivars, the disease is generally controlled by using systemic, site-specific modern fungicides. The dicarboximide fungicide(DCF) dimethachlone which belongs to the FRAC E3 group of fungicides, target osmotic signal transduction, is used in China for more than 12 years to control the disease. First signs of resistance in the field are reported at low frequency. But, multiple, consecutive and extensive use of the fungicide has led to the emergence of fungicide resistance in Sclerotinia sclerotiorum. In this study, one resistant field isolate and 35 laboratory mutants were used to explore still unknown mechanisms leading to dimethachlone resistance.In the first experiment, we tested fungicide cross resistance, different fitness parameters such as EC50 values, mycelial growth rate, sclerotial production rate, pathogenicity test, osmotic pressure, oxidative stress and electrolyte test, and molecular study of four resistant isolate/mutants compare with their sensitive parents. Cross-resistance was confirmed between dimethachlone and procymidone, iprodione and fludioxonil for four resistant isolate/mutants. The resistant isolate/mutants had significantly higher EC50 values compared with the sensitive control isolates. The resistant isolate/ mutants revealed a decreased mycelial growth rate, were less pathogenic on leaves of oilseed rape, were more sensitive to osmotic pressure and oxidative stress and released more electrolytes compared with the sensitive isolates. No significant differences were found in case of sclerotial production between resistant isolate/mutants and their sensitive mutants. Only in one lab mutant did we find a point mutation(V238A) in the SsOs1 gene of the high-osmolarity glycerol(HOG) signalling pathway. The expression of this gene was lost in the field resistant isolate HN456-1-JBJ and decreased in mycelium that was subjected to either high osmotic pressure or dimethachlone; however, another key gene in the HOG pathway, SsHog1, could be induced in the resistant isolate and mutants with NaCl treatment.cDNA library transformation was performed to screen the gene related to the resistance of dimethachlone in the second experiment. We constructed cDNA library using dimethachlone resistant isolate HN456-1-JBJ and then transferred the library into sensitive isolate HN404-2-J through agrobacterium mediated transformation. Inspite of our best efforts, no cDNA fragments were inserted into sensitive isolate through series of transformations, only two transformants were confirmed containing empty vector, other 30 resistant transformants did not contain any heterogenous insertions and considered as spontaneous mutants during the cDNA library transformation and fungicide screening.Compared to their parental isolate HN404-2-J, the EC50 values were significantly higher in 30 dimethachlone resistant mutants. The resistance of the mutants were stable after ten generations of transfers on dimethachlone free PDA. To investigate the molecular mechanisms of the dimethachlone resistance, the expression of SsOs1 gene in mutants was conducted by real-time PCR, results indicated that 19 mutants amplified SsOs1 gene, but interestingly 11 mutants failed to express this gene. A comparison of the nucleotide sequences of the SsOs1 gene between 19 dimethachlone resistant mutants and their parental sensitive isolate revealed that 14 mutants contained single-point mutations at different positions; one mutant(P-3-3) contained two point mutations at independent 322 and 406 amino acid positions; two mutants(P-3-26 and P-3-8) changed amino acid to stop codon at amino acid positions 279 and 553, respectively; one mutant(P-3-20) contained an insertion of 3 amino acids at position 542-544, and finally one mutant(No.As-2nd-17) contained a deletion of 7 amino acids at positions 657-663.This study demonstrates that resistance to DCF fungicide dimethachlone in Sclerotinia sclerotiorum is emerging in China. Several fitness parameters, including mycelial growth rate, sclerotia formed in vitro, aggressiveness on leaves, osmotic and H2O2 sensitivity, indicated that the resistant strains might not effectively compete with sensitive isolates in the field in the absence of selection pressure. Lost expression or non-specific located point mutations in the SsOs1 gene might confer resistance to DCF fungicides in Sclerotinia sclerotiorum.