Resistance Monitoring And The Resistant Mechanism Of Rhixocotonia Solani To Tebuconazole In Sichuan Province,China

Rice sheath blight,caused by R.solani,is an important fungal disease in the rice production and cultivation.At present,rice sheath blight is mainly managed by chemical control with fungicides,such as triazole fungicides,including hexaconazole,tebuconazole,epoxiconazole and so on,along with the new fungicide such as thifluzamide.However,with the extension of its use and the increase of dosage,the susceptibility of pathogens to the fungicides have been decreased and fungicide-resistant strains have been developed,that finally resulted in the great difficulties for disease control.In this paper,the minimum inhibitory concentration（MIC）method and mycelium growth rate method were used to determine the resistance level and frequency of R.solani against thifluzamide,hexaconazole,tebuconazole,epoxiconazole.Part of the physiological mechanism of resistance between resistant and sensitive strains was determined by osmotic pressure and conductivity method.The CYP51 gene was amplified and sequenced to identify the molecular mechanism of resistance to R.solani against tebuconazole.The preliminarily transcriptomic analysis of susceptible and resistant strains of R.solani towards thifluzamide induction elucidated the molecular mechanism of thifluzamide induction over the gene expression and difference between R.solani strains,so as to explore the resistance mechanism of R.solani against fungicides in Sichuan Province.The main research results of this paper are listed as follows:1.Resistance frequency and field resistance distribution of R.solani to four fungicides in Sichuan ProvinceThe minimum inhibitory concentration（MIC）method was used to determine the resistance frequency of 122 strains of R.solani that were isolated from 7 counties and 5different places（cities）of Sichuan Province against thifluzamide,hexaconazole,tebuconazole and epoxiconazole.The results showed that the resistance frequencies of the four fungicides were 65.57%,45.08%,47.54%and 36.07%,respectively.The EC₅₀values of the representative sensitive strains from region of the Sichuan Province against thifluzamide,hexaconazole,tebuconazole and epoxiconazole were determined by the mycelium growth rate method.The results showed that the EC₅₀mean values of R.solani to the four fungicides were 0.061μg/m L,0.06μg/m L,0.109μg/m L and 0.058μg/m L,respectively,which can be used as a sensitivity baseline of rice sheath blight in the Sichuan Province.The mycelia growth rate method was used to determine the toxicity of representative strains to the four fungicides respectively.The results showed that the range of EC₅₀was 0.15～0.69μg/m L,0.12～0.18μg/m L,0.54～3.1μg/m L and 0.14～0.39μg/m L,respectively.Most strains of R.solani in the Sichuan Province were sensitive to thifluzamide and epoxiconazole,and few ones showed low level of resistance;all strains of R.solani in Sichuan Province were sensitive to hexaconazole;six strains of R.solani from Sichuan Province showed moderate resistance to tebuconazole and one strain showed low resistance.2.Physiological mechanism of resistance of R.solani to tebuconazole in Sichuan ProvinceThe osmotic pressure sensitivity and membrane permeability of R.solani with different resistance levels to tebuconazole were determined by colony diameter method and conductivity method.The results showed that the colony diameters of the sensitive and resistant strains are decreased with the increase of glucose concentration.In addition,the change in diameter of the mycelium growth of the resistant strain was significantly smaller than that of the sensitive strain;at concentrations ranging from 1.25 g/L to 5 g/L,the colony growth diameter of the sensitive strains were decreased with the increase of Na Cl concentration,while the colony growth diameter of the resistant strains were increased with the increase of Na Cl concentration.However,at medium concentrations of Na Cl culture,ranging from 0 g/L to 80 g/L,the inhibition rate of hyphae growth of sensitive and resistant strains was irregular.At different concentrations of tebuconazole,the leakage rate of sensitive and resistant strains of R.solani was increased with the increase of tebuconazole concentration.At the same concentration of tebuconazole,the leakage rate of the resistant strains was generally higher than that of the sensitive strains.3.Molecular mechanism of resistance of R.solani to tebuconazole in Sichuan ProvinceThe amplification and sequencing of CYP51 gene of four resistant strains and four sensitive strains were conducted.The results of amino acid sequence comparison showed that the resistant strains had two mutation types,CYP51^{S94A,N406S,H793R}and CYP51^{S94A,N406S,L750P,H793R},respectively.The comprehensive score of the resistant mutation type and the optimal model of tebuconazole was lower than that of the sensitive model,while the score of the optimal combination model of CYP51^{S94A,N406S,L750P,H793R}mutants was lower than that of the mutant of CYP51^{S94A,N406S,H793R},which was negatively correlated with the toxicity results.In the optimal docking model between 14α-sterol demethylase（CYP51）of the sensitive strain and tebuconazole,three hydrogen bonds formed by the three amino acid residues of GLY299,TRP300 and GLU691 with tebuconazole were found;in the optimal docking model of CYP51^{S94A,N406S,L750P,H793R}mutants and tebuconazole,ARG603 amino acid residues formed hydrogen bonds with tebuconazole;in the optimal docking model of CYP51^{S94A,N406S,H793R}mutants and tebuconazole,ILE605 amino acid residues formed hydrogen bonds with tebuconazole.4.Transcriptome sequencingA total of 54.59GB of Clean Data was obtained by the transcriptome analysis of R.solani that was sensitive to thifluzamide and resistant to thifluzamide-induced mutants by high-throughput sequencing techniques.A total of 344 differentially expressed genes were obtained,of which 160 were significantly up-regulated and 184 were significantly down-regulated.Functional annotation and enrichment analysis were carried out on the differentially expressed genes,and their results showed that the differentially expressed genes were mainly enriched in the pathways of fat metabolism,glycometabolism and biosynthesis of secondary products.Two differentially expressed genes related to ABC transporters were selected by analyzing the differentially expressed genes related to resistance.