| Poplar anthracnose is one of the most destructive diseases on Poplus sp.and it developed seriously in local region of our country in recent years,causing serious economic losses.So,this research is based on the target pathogen Colletotrichum gloeosporioides and C.aenigma and study its sensitivity to the four kinds of commonly used bactericides,assessmenting the risk of Colletotrichum gloeosporioides to carbendazim and studying preliminarily the resistant molecular mechanism of Colletotrichum gloeosporioides to carbendazim.The results provide scientific guidance for medical treatment scientifically and making reasonable resistance strategy in the field,and laid the foundation for establishing a molecular detection technology of resistant strain.The main results and concllusions were outlined as follows:1.Sensitivities of 59 Colletotrichum gloeosporioides isolates and four C.aenigma isolates causing poplar anthracnose to four fungicides(carbendazim,difenoconazole,tebuconazole and triadimefon)were determined by measuring the mycelium growth.The results showed the frequency distribution of EC50 values of C.gloeosporioides for carbendazim was described by continuous,unimodal curve,indicating the absence of carbendazim-resistant subpopulation among these isolates.The frequency distribution of EC50 values of C.gloeosporioides for difenoconazole and tebuconazole were bimodal curves,implying the appearance of resistant subpopulation.There was no significantly difference among the sensitivities of C.gloeosporioides isolates from different poplar hosts(P>0.05).2.Five resistant mutants of C.gloeosporioides to carbendazim and four resistant mutants of C.aenigma to carbendazim were obtained by selection for resistance to fungicide with the range of EC50 values of were 6.5852?39.0251pg/mL and the range of mutation frequency were 115?573.High mutation frequency and resistant level showed the fact that it is easy for C.gloeosporioides and C.aenigma developing resistance to carbendazim.After 10 successive transfers on a fungicide-free medium,the sensitivity to five resistant mutants of C.gloeosporioides to carbendazim was relatively stable and the sensitivity to three resistant mutants of C.aenigma to carbendazim was falling.3.According to the result of fitness parameters of the mutants of C.gloeosporioides,showed lower than the sensitive strains.The hyphal growth showed significantly lower than their parental isolates and sporulation,spore germination and pathogenicity of resistant mutants were similar to their parental isolate.The main biological characters of the resistant mutants of C.aenigma have different degree changes compared with the parent strain.4.In order to test cross resistance,sensitivities of five parental isolates and resistant mutants of C.gloeosporioides to carbendazim to seven frequently used fungicides belonging to other chemical groups(difenoconazole,tebuconazole,triadimefon,azoxystrobin,chlorothalonil,diethofencarb and zoxamide)were determined by measuring the mycelium growth.With the result of cross resistance test,carbendazim has no cross resistance with the botryticides in common use like difenoconazole,tebuconazole,triadimefon,azoxystrobin,chlorothalonil and diethofencarb;carbendazim has significant positive cross resistance with zoxamide.5.?-tubulin of five carbendazim resistant strains were sequenced,and alignment were carried out among these sequences with parental isolates.Result showed no mutation was detected in ?-tubulin of four resistant strains based on DNA sequence alignment with corresponding sequences of parental isolates.The multiple sequence alignment for ?-tubulin2 revealed a mutation(E198 K)at the codon 198 in 2C5-3-D1.Relative expression levels of(3-tubulin of resistant strain were detected using real-time quantitative PCR(qPCR).Result showed no significant change was detected in p-tubulin of 2C5-3-D1 and resistant strain(Y3-6-D1,Y3-6-D2,BH9-1-D4,YP1-7-D1)have a significant increase. |
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