Tolerance And Resistance Mechanism Of Several Main Pathogenic Fungi Of Vegetable Base On Tubulin

Carbendazim and diethofencarb that target on fungus β-tubulin, are two important fungicides for controlling various crop diseases. Carbendazim shows a broaden-spectrum active efficiency on inhibiting the mycelia expension of ascomycetes and most of deuteromycetes, but exerts no effect on mastigomycotina and few deuteromycetes that might possess tolerant properties to carbendazim.Diethofencarb has a negative cross-resistant spectrum of carbendazim and usually been used to control carbendazim-resistant isolates. In this dissertation, the carbendazim-tolerant mechanism was explored on several carbendazim-resistant fungi by cloning β-tubulin gene, analyzing and comparing the mutant amino acid sequences. Meanwhile, the resistance of Corynespora cassiicola to carbendazim and diethofencarb was also studied systematically. The research progresses were shown as follows:1.The changes of amino acid at the position167of β-tubulin may be responsible for the sensitivity of plant pathogenic fungi from different genus to carbendazim.(1)Three Alternaria, two Stemphylium and two-Oomycetes species have been shown carbendazim-tolerant properties.The sensitivities of23pathogenic fungi from20genus were determined to carbendazim. The results showed that1μg/ml carbendazim inhibited thoroughly the mycelium growth of Botrytis cinerea, but100μg/ml carbendazim revealed no significant effect on Alternaria cucumerina, Alternaria solani, Alternaria Alternate, Stemphylium solani, Stemphylium spinaciae, Phytophthora capsici and Pythium debaryanum, indicating that these fungi were tolerant to carbendazim.(2)Tyrosine at the position167of P-tubulins in three Alternaria, two Stemphylium, two-Oomycetes species play an important role on these fungi’s tolerance to carbendazim.P-tubulin genes of A. cucumerina, A.solani, A Alternate, S.solani, S.spinaciae, P.capsici, and P.debaryanum were cloned and their amino acid sequences were analysed. The results showed that the amino acid at position167were changed from phenylalanine in carbendazim-sensitive isolate totyrosine in carbendazim-tolerant isolate, indicating that the mutation of the amino acid at positon167of β-tubulins caused the tolerance of plant pathogenic fungi to carbendazim.2.The resistance mechanisms of Chinese C.cassiicola to carbendazim and diethofencarb have been clarified explicitly. Meanwhile, the characters of the tolerant strains have been elucidated clearly on bases of the resistance frequency, the fitness and the cross-resistant property.(1)Most of163C.cassiicola isolates revealed a strong resistance to carbendazim and a low resistant frequency to diethofencarb, including145isolates that expressed high resistance to carbendazim with MIC value greater than10μg/ml and18isolates that were sensitive to carbendazim. The resistant frequency among the total isolates was89.0%to carbendazim and14.7%to both carbendazim and diethofencarb.(2)The fitness of MBCHR and MBCHRNPCR isolates had been reduced insignificantly. Under normal cultured condition, MBCS isolates developed55.17mm-50.83mm of round colony, produced1.78×105/ml-2.50×105/ml of spores, and displayed79.50-82.80of disease index. However, MBCHR and MBCHRNPCR isolates showed50.17mm-59.83mm of round colony,1.89×105/ml and2.94×105/ml of spores,81.40and85.40of disease index, respectively.(3)MBCHR and MBCHRNPCR isolates exibited no cross resistance to boscalid, fludioxonil, prochloraz, cyprodinil and procymidone. For two MBCS isolates, the EC50values of the above five fungicides were0.49μg/ml and0.65μg/ml,0.26and0.20μg/ml,0.14and0.19μg/ml,0.71and0.75μg/ml, and0.45μg/ml and0.37μg/ml, respectively. For MBCHR and MBCHRNPCR isolates,the EC50values of the five fungicides were about0.36μg/ml-0.71μg/ml,0.15μg/ml-0.27μg/ml,0.14μg/ml-0.26μg/ml,0.67μg/ml-0.83μg/ml and0.37μg/ml-0.50μg/ml, respectively.(4)The resistance mechanism of C.cassiicola to carbendazim and diethofencarb were taken into in-depth investigated. Sequence comparison revealed that a point mutation at the position198had replaced MBCS alanine with MBCHR glutamic, and the amino acids at the position198and200have been changed from MBCS glutamic and phenylalanine to MBCHRNPCR lysine and tyrosine.In this dissertation, the amino acid at the position167of β-tubulinwas found to be relative to the sensitivity of plant pathogenic fungi to carbendazim at the first time.The MBCHR and MBCHRNPCR isolates were also first reported in China. The resistance mechanism of C.cassiicola to carbendazim and diethofencarb had been studied systematically. The results laid a foundation todevelop new fungicide and fungicide screening modeland provide better strategy for the chemical control and molecular detection of resistant strains.