Molecular Resistance Mechanism Of Fusarium Fujikuroi Against Carbendazim And Diagnosis Techniques For Detecting Genotypes With Carbendazim-Resistance Alleles

Bakanae disease of rice, an important rice disease in rice-growing regions worldwide, is a threat to food safety due to its ability to produce kinds of mycotoxins. Among Gibberella fujikuroi species complex, three mating populations are associated with bakanae disease, including （1） mating type population A （anamorph, Fusarium verticillioides= F. moniliforme）, （2） mating type population C （anamorph, F. fujikuroi） and （3） mating type population D （anamorph, F.proliferatum）. The filamentous fungus F. fujikuroi was firstly isolated from infected rice plants and identified as the causative agent of "bakanae" or foolish seedling disease in 1890.Benzimidazole fungicides are a group of broad spectrum systematic fungicides interacting with tubulin genes, especially β-tubulin, inhibiting the formation of spindle body, thus inhibiting mitosis. The fungicides, including benomyl, carbendazim （methyl benzimidazole-2-yl-carbamate, MBC）, thiabendazole and thiophanate-methyl, have been extensively used to control this disease with excellent efficacy since 1960s. However, after being used for years, these fungicides have generally lost their efficacy because of benzimidazole-resistance. However, the resistance mechanism of F. fujikuroi against benzimidazoles is not well understood. In this thesis, the molecular resistance mechanism of F. fujikuroi against carbendazim has been revealed by constructing of β2tub-deletion and-complementation mutants. Besides, F. fujikuroi strains with different resistant levels against MBC were detected rapidly by molecule-based PCR diagnosis techniques. Previous research of our laboratory indicated that the MIC value of MBC sensitive strains was less than 5μg/ml, MBC moderately resistant strain between 50-100μg/ml, MBC highly resistant strain>100μg/ml. No mutations at β1-tubulin （β1tub） gene of F. fujikuroi were detected concerning with resistance to carbendazim. The main results of this thesis present as follows:1. The mutations at β2-tubulin （β2tub） conferred to the resistance of F. fujikuroi to MBC. According to the sequence of β2tub from F. verticillioides （Broad Institute acccession number:FVEG₀5512.3）, β2tub of F. fujikuroi was cloned and analyzed. The ORF of β2tub encodes a putative protein of 448 amino acid residues, with a calculated molecular weight of 50.16 kDa, a G+C content of 52.46%, and an estimated iso-electric point of 4.38. Beisides, the β1tub and β2tub of F. fujikuroi was highly homologous （77%）. Comparison between β2tubs of 12 MBC-sensitive strains and 56 MBC-resistent strains, substitutions [TTC （phe）�'TAC（tyr） at codon 200 and GGC（gly）�'GGT（gly） at codon 235] were found in moderately MBC-resistant strain, and substitutions [GAG（glu）�'GTG（val） at codon 198 and GGC（gly）�'GGT（gly） at codon 235] were detected in MBC highly resistant strain.To known whether the MBC-resistance of F. fujikuroi is conferred by the mutations of β2tub, the β2tub-deletion and-complementation mutants were constructed using the hph and hsv-tk, and the MBC-sensitive and biological phenotypes of parental strains and mutants were detected in viro. The results showed that:the β22tab-deletion （Δβ2tub） mutants were supersensitive to MBC （EC50 values of MBCS, MBCMR and MBCHR were 1.08�'0.302 μg/ml、2.41�'0.300μg/ml and 3.32�'0.306μg/ml, respectively; MIC values of all Δβ2tub mutants were 1μg/ml）, sporulation reduced 50-80%, virulence decreased 20-30% when inoculated on tomato fruits and the mycelial growth rate decreased at 6.5-17.1%. Complementation of the Δβ2tub mutants with a copy of the whole β2tub locus from their own parent strains restored their corresponding MBC-resistance or MBC-sensitivity.2. PIRA-PCR can rapid distinguish genotypes of strains Tested. Based on the mutation sites of β2tub among F. fujikuroi strains differ in carbendazim-resistent level. PCR template of each isolate was amplified by one outer primer pair. The AccⅡ and PmaC I restriction enzyme recognition sites were introduced artificially by inner primers to MBC-sensitive and MBC-resistant strains, respectively. The sensitivity of each strain to MBC was indirectly determined by analyzing electrophoresis patterns of the resulting amplified fragments after simultaneous digested by both Acc Ⅱ and PmaC I. PIRA-PCR not only received the same result as that analyzed by the conventional method, but also costed only 10 hours for assay, while the conventional method required 7-8 days.3. ASO-PCR can rapid distinguish MBC-sensitive strains from MBC-resistant strains. The sequences of 68 strains differed in MBC resistance have shown a big difference at codon 235 （nucleotide 1006）. Monitoring of MBC resistant frequency in fields has shown that the MBC highly resistant strains were predominant （55%）. Besides, MBC resistant frequency increased rapidly in the past few years. Thus, rapid detection of resistance frequency is important to the agricultural production safety. Based on the nucleotide mutation at 1006 （codon 235） of β2tub in MBC-resistent strains. ASO-PCR could distinguish MBC-sensitive strains from MBC-resistent strains. Compare with onventional method, ASO-PCR costs only 6-7 hours and the accuracy was 100%.