Risk Assessment Of Resistance To Carbendazim,tebuconazole And The Function Of Resistance Gene CfCRZ1 In Colletotrichum Fructicola

Camellia oleifera,a unique woody oil species in China,occupies an important position in China’s regional economy.Anthracnose-based diseases have become increasingly serious,resulting in serious economic losses.Colletotrichum fructicola is the main pathogen of Camellia oleifera anthracnose.We have previously examined that anthrax strains have developed different degrees of resistance to carbendazim and tebuconazole in oil tree nurseries.However.the resistance risk is unclear.Therefore,we assessed the risk of resistance to these two fungicides,further investigated the biology of the zinc finger transcription factor CfCrz1 in C.fructicola,and located the approach to control the resistance of bacteria to fungicides.It thus provides potential target sites for the development of new fungicides against this pathogen.The main results are stated as follows;(1)Risk assessment of resistance to carbendazim in C.fructicola.The sensitivities to carbendazim of 114 C.fructicola across the country were determined.Three resistant strains GD-6(Guangdong),HNWZS-26-1(Hainan Wuzhishan)and HNFM-8(Hainan Fengmu)were isolated from the field where such elements as resistance lever,resistance stability,growth rate,sporulation,spore germination rate and pathogenicity of the resistant strains were determined.The results demonstrated that all of the mutants presented a high resistance level to carbendazim,while the resistance phenotype could be inherited stably.Bio-fitness test displayed that the growth rate,sporulation and appressorium formation rate of the resistant strains remained stable,with the spore germination rate and pathogenicity significantly higher than those of sensitive strains.These results indicate that the risk of resistance to C.fructicola to carbendazim was at a high level.As a result,it is not recommended to employ this kind of chemical to control Camellia anthracnose.(2)Risk assessment of resistance to tebuconazole in C.fructicola.The sensitivities to tebuconazole of 116 C.fructicola across the country were determined.Five resistant strains HNLY-2-2(Hunan Liuyang),HNYX-4(Hunan Youxian),JX-19(Jiangxi),GZGY-22(Guizhou Guiyang)and GX-18-1(Guangxi)were isolated from the field where such factors as resistance lever,resistance stability,growth rate,sporulation,spore germination rate,pathogenicity and external stress response were determined.The results demonstrated that these strains had lower levels of resistance to tebuconazole,while the resistance phenotype could be inherited stably.The growth rate,spore germination,appressorium formation rate,and pathogenicity of the resistant strains witnessed no downward trend,while the sporulation rate was significantly higher than those of sensitive strains.The correlation between the susceptibility of resistant strains to tebuconazole and metconazole,epoxiconazole,and difenoconazole were very low,indicating that there was no cross-resistance between tebuconazole and these three others.These results reveal that the resistance of C.fructicola to tebuconazole was at a low level,which suggested that this type of fungicide can be used to control Camellia oleifera anthracnose.(3)The study of the biological function of transcription factor CfCrz1 of C.fructicola.By the homologous recombination method,a gene knockout mutant ΔCfcrz1 and complementary strain ΔCfcrz1/CRZ1 of C.fructicola were generated.The biological phenotypes of the mutant strain ΔCfcrz1,compared with a control group made up ofΔCfcrzl/CRZ1 and the wild-type strain,were measured in growth,pathogenicity,and resistance to azole fungicides.The result illustrated that the mutant ΔCfcrz1 had a significant decrease in the growth rate of hyphae,while the production of conidia and appressorium was significantly reduced.The inhibition rates of the mutant strain ΔCfcrz1 of C.fructicola were 100.0%and 46.3%respectively in a PDA medium containing 0.3 mmol·L-1 CaCl2 and 5 mmol·L-1 dithiothreitol.The mutant ΔCfcrz1 was remarkably weakened in virulence to Ca.olivera.The sensitivity test of sterol demethylation inhibitor(DMI)fungicides such as tebuconazole disclosed that the mycelial growth of the mutant strain ΔCfcrz1 was significantly inhibited.The study revealed that the transcription factor CfCrz1 of C.fructicola is involved in regulating growth,conidiation,the response to external stress,pathogenicity,and resistance to DMI fungicides.It provided a potential target site for the development of new fungicides against the pathogen.