Studies On Fungicide Resistance And Detection Of Dominant Species Of Colletotrichum Acutatum Species Complex In China

Anthracnose not only affects the yield,but also reduces the quality of peach fruit,causing huge economic losses to the peach industry.Application of chemical fungicides is a common method to control peach anthracnose,but the sensitivity of different anthracnose pathogens to fungicides is different,leading to difficulties for anthracnose management in fields.In this study,the sensitivity of Colletotrichum acutatum species complex to commonly used fungicides were detected.The control efficiency of phenylpyrrole fungicide fludioxonil on C.acutatum species complex was investigated,and the resistance mechanism of fludioxonil was preliminarily explored.A simple detection technique for Colletotrichum nymphaeae,the dominant species of Colletotrichum spp.in China,was developed.The research results are as follows:（1）The sensitivity of 113 isolates of C.acutatum species complex collected from five provinces of China to eight fungicides was determined by mycelial growth inhibition method.The average EC₅₀ values of different fungicides to tested isolates from low to high were fludioxonil,prochloraz,difenoconazole,tebuconazole,azoxystrobin,propiconazole,flutriafol,iprodione.Correlation analysis of EC₅₀ values showed that there was a strong positive correlation among DMI fungicides,there was a certain degree of correlation between azoxystrobin and tebuconazole,and the correlation coefficient was0.36.No correlation was observed between fludioxonil and other tested fungicides,but there was a strong negative correlation between iprodione and other fungicides except fludioxonil.In addition,there were significant differences for the sensitivity to prochloraz,tebuconazole,propiconazole and fludioxonil among C.nymphaeae,C.fioriniae and C.godetiae.（2）Fludioxonil had an obvious effect on C.acutatum species complex.Under the treatment of 0.05μg/m L,the mycelium shrank and bent.Fludioxonil had an inhibitory effect on sporulation of pathogens,but the inhibition of spore germination was not obvious.We found that the isolates of C.fioriniae were differentiated.In addition to the two subgroups reported,in the phylogenetic tree established in this study,GZTR 2-1,GZTR 2-2,GZTR 5-1,GZTR 5-2,GZTR 6-1,GZTR 6-2,GZTR 7-1 and GZTR 7-2isolates were grouped into a separate cluster.The colony diameters of this cluster isolates on low concentration fludioxonil plate was larger than that on high concentration fludioxonil plate.In the indoor control experiment,fludioxonil could effectively control the anthracnose caused by C.nymphaeae and C.godetiae,and the control efficiency was100%,but the efficiency on C.fioriniae was not good,there were still small lesions on the fruit under the treatment of 500μg/m L.（3）Eight fludioxonil-resistant mutants with stable resistance were obtained by fludioxonil taming.Compared with the parental isolate,the resistant mutants were significantly more sensitive to Na Cl and slightly less pathogenic.There was no cross-resistance between fludioxonil and azoxystrobin or difenoconazole.The two fungicides could effectively control fludioxonil-resistant mutants.Genome resequencing analysis of the sensitive isolate FJNP 11-1-2,the resistant mutants FJNP 1-1-2R and FJNP20-1-2R showed that there was a frame shift mutation（A-AT）in the Cn Os1 gene in the mutant FJNP 1-1-2R,and an early termination codon（CAG-TAG）was observed in the Cn Os1 gene of the mutant FJNP 20-1-2R.These results indicated that the mutations of the Cn Os1 gene were the main reason for the resistance of C.nymphaeae to fludioxonil.（4）Based on the RPA/Cas12a method,a rapid field detection technique was developed for C.nymphaeae.In this study,three pairs of RPA primers were designed based on the 202 bp gene fragment amplified by C.nymphaeae specific primer pair PCNF/PCNR.It was found that the primer pair Cn RPA F3/Cn RPA R3 could specifically amplify the target sequence,which was an ideal RPA amplification primer pair.A crRNA was designed based on the unique PAM site of C.nymphaeae target sequence.Due to the ss DNA cleavage activity of Cas12a,FQ-reporter probe was added to the CRISPR/Cas12 a reaction system,and the detection results could be visualized by a miniature ultraviolet flashlight.After optimizing the parameters of the reaction system,the specificity test was carried out.The results showed that the technology could specifically recognize C.nymphaeae from related species and other peach disease pathogens.The technology is universal,and C.nymphaeae strains from different provinces could be accurately detected.The detection limit of this technique was 5 pg/μL sample DNA,which was lower than that of conventional PCR.Finally,the peach fruit was artificially inoculated with spore suspension to simulate the disease in the field,and the DNA of the sample at the lesion was extracted by a simple boiling method for RPA/Cas12a detection.The results showed that only the fruit inoculated with C.nymphaeae showed positive results,and it took only1 h 30 min from DNA extraction to complete detection.