Fungicide Resistance In Peach Anthracnose Fungi Colletotrichum Species In China

Anthracnose,mainly caused by species complexes Colletotrichum gloeosporioides and Colletotrichum acutatum,is a devastating disease of peach in China.The chemical control has been widely used for years and management failures have been increased towards commonly used fungicides.Emerging resistance is a serious problem,and new alternatives should be found timely in order to keep efficient disease control in practice.Therefore,sensitivity screening of Colletotrichum species to different fungicides is required to make proper management strategies against peach anthracnose.In this study,a total of 220 Colletotrichum spp.isolates including 140 isolates of C.nymphaeae,41 of C.siamense and 39 of C.fructicola were collected to test sensitivity to fungicides from six major fungicide groups.At the same time,fitness parameters of resistant isolates were investigated along with molecular studies including expression analysis of transporters,various point mutations in fungicide target genes,molecular docking analysis.The main results are as follow:1)Sensitivity of 80 C.gloeosporioides isolates including C.fructicola and C.siamense was conducted for pyraclostrobin,procymidone,prochloraz and fludioxonil based on mycelial growth inhibition at discriminatory doses.Results showed that C.fructicola and C.siamense isolates were highly resistant(EC50>100)to procymidone and fludioxonil,but sensitive to prochloraz.For pyraclostrobin,74%of C.fructicola isolates showed high resistance and 26%were low resistant,all of the C.siamense isolates were low resistant.No positive cross-resistance was observed between members of the same quinone outside inhibitor(Qo I)fungicide group(pyraclostrobin and azoxystrobin)and between Qo I and non-Qo Is.Resistant isolates to Qo I fungicides were evaluated for the fitness penalty.Results showed that no significant differences except for mycelial growth rates were detected between highly resistant and lowly resistant isolates of C.fructicola.Molecular characterization of Cyt b genes revealed that the G143A point mutation was the determinant of the high resistance in C.fructicola.2)It was found that the C.nymphaeae sp.was highly resistant to methyl benzimidazole carbamate(MBC)fungicide carbendazim,succinate dehydrogenase inhibitor(SDHI)fungicide boscalid and dicarboximide fungicide(DCF)procymidone in current study.In addition,C.nymphaeae isolates exhibited differential sensitivities to phenylpyrrole(PP)fungicide fludioxonil,approximately 14%fludioxonil resistance(Flu~R)frequency was recorded in this study.Luckily,C.nymphaeae isolates did not show resistance to Qo I fungicide pyraclostrobin and demethylation inhibitor(DMI)fungicide prochloraz.The mean values of effective concentration to cause 50%growth inhibition(EC50)of C.nymphaeae for pyraclostrobin and prochloraz were 0.0083?g/ml and 0.002?g/ml respectively,and 0.0194?g/ml for fludioxonil in sensitive populations.No cross-resistance was observed between fungicides from different groups(carbendazim,procymidone,boscalid,pyraclostrobin and prochloraz).Mycelial growth rate,control efficacy and osmotic stress responses were significantly different(P<0.05)between fludioxonil sensitive(Flu~S)and resistant isolates(Flu~R)of C.nymphaeae,but no significant difference was observed(P>0.05)in virulence and sporulation between Flu~Sand Flu~R isolates.No mutation was detected in Cn Os-1,Cal,Hk1,Hog1,TPI and Mrr1genes.Interestingly,with fludioxonil treatment,the expression of ABC transporter gene atr B was significantly over-expressed in some resistant isolates.However,over-expression of the atr B gene was not detected in one moderately and one highly resistant isolate,indicating that other unknown mechanisms may be involved.3)From C.fructicola,which has been described as natural resistant species to fludioxonil,a sensitive isolate was detected surprisingly.No cross-resistance was found for fludioxonil to pyraclostrobin,procymidone and prochloraz.The significant difference of fitness was observed between sensitive and resistant isolates,i.e.significantly different mycelial growth rate and osmotic sensitivity were observed but no significant difference was found for sporulation.A novel mutation I880V was detected in Cf OS1 gene from the sensitive isolate GDHY6-1.Molecular docking based on the mutation I880V was used to explain reversal behaviour from inherent tolerance(natural resistance)to sensitivity in C.fructicola to fludioxonil.Results suggested that the I880V mutation in Cf OS-1 gene changed the conformation of Cf OS1 protein in the fludioxonil-binding pocket,resulting in the deceased affinity between Cf OS1 protein and fludioxonil.These findings are remarkable to demonstrate the fludioxonil resistance mechanism,and further studies such as genetic transformation are needed to make a firm conclusion in future.4)In this study,sensitivity screening revealed that pyraclostrobin(Qo I)effectively controlled C.nymphaeae population but could not control C.fructicola and C.siamense spp.At the same time,it was suggested that C.fructicola and C.siamense have acquired carbendazim(MBC)resistance and C.nymphaeae showed natural resistance to carbendazim.Likely,fludioxonil(PP)was less effective chemical to control C.nymphaeae populations.On the other hand,C.fructicola and C.siamense showed inherent tolerance to fludioxonil.Procymidone(DCF)and boscalid(SDHI)were also not effective candidates to control Colletotrichum species.Fortunately,C.nymphaeae,C.fructicola and C.siamense isolates did not show resistance to demethylation inhibitor(DMI)fungicide prochloraz.Current findings uncovered the effectiveness of several chemicals and provided the foundation to design management strategies to control peach anthracnose in practice.DMI fungicide prochloraz is the best option among different chemicals to control peach anthracnose in China.