| Fusarium graminearum, which is the overriding pathogen of Fusarium head blight (FHB) on wheat, is a leading cause of economic loss in these crops. In addition to reducing seed mass and quality, the fungus contaminates grain with toxic metabolites such as DON and NIV that are a threat to human and other mammals'health. Grey mold (Botrytis cinered) is an important plant disease on several economic crops in China, especially results in severe loss to vegetables and strawberrys in protected fields, inducing fruit rot.Carbendazim and other benzimidazole fungicides have been used to control FHB and grey mold for more than 40 years since Shenyang Research Institute of Chemical Industry industrialized manufacture in 1970 led by Zhang Shaoming. Benzimidazole fungicides combine withβ-tubulin in plant pathogenic fungi's cell, preventing the formation of spindle fiber, thus inhibiting mitosis. Plant pathogenic fungi retain the tubulin gene's random mutation in evolutionary process to adapt to infaust medium. Therefore, most resistance of plant pathogenic fungi to benzimidazole fungicides owes to some definite amino acid mutation ofβ-tubulin, which causes decreased affinity between benzimidazole fungicides and the target. Amino acid mutation is located inβ-tubulin site 198 or 200 generally in most field resistant strains. The site-mutation ofβ-tubulin at codon198 (Glu to Ala or Lys or Val) may lead to high resistance while mutation at codon167(Phe to Tyr) and codon200(Phe to Tyr) cause medium resistance to MBC in Botrytis cinerea. Fusarium graminearum containsβ1-tubulin andβ2-tubulin. Besides,β1-tubulin in strains of dirrenernt sensitivity patterns is identical, without any mutation. However, site mutation ofβ2-tubulin at codon 73,167,198 or 200 can result in distinct resistance, Q73R or E198L to high level while F167Y or F200Y to medium level. Data above shows that the mechanism of resistance to MBC in Fusarium graminearum to Botrytis cinerea.In this article we adopted Double-joint PCR to construct the vector containing MBC resistantβ-tubulin gene in vitro, using PEG-mediated protoplast transformation to homologous replaceβ1-tubulin gene andβ2-tubulin gene in Fusarium graminearum. Furthermore, we deleted the originalβ1-tubulin gene andβ2-tubulin gene of mutants we got above. Accordingly, we obtained mutant of Fusarium graminearum thatβ1/β2-tubulin gene knock-out mutants, MBC-resistantβ-tubulin gene replacingβ1/β2-tubulin gene transformants respectively and corresponding knock-outs.We assayed the sensitivity to MBC, mycelial growth rate, conidia productivity, perithecium productivity, pathogenicity and other biological characteristics of different Fusarium graminearum transformants by testing the expression of MBC-resistantβ-tubulin gene via homologous replacement withβ1-tubulin gene and P2-tubulin gene in Fusarium graminearum using semiquantitative RT-PCR to study whetherβ-tubulin gene of different plant pathogenic fungi can be substituted and if the MBC-resistantβ-tubulin gene of Botrytis cinerea can express in Fusarium graminearum which provided reference to further research the resistance mechanism of Fusarium graminearum to MBC and the mechanism of action between MBC andβ-tubulin. Below are our main findings.We adopted Double-joint PCR to construct the vector containing MBC resistantβ-tubulin gene in vitro, using PEG-mediated protoplast transformation to homologous replaceβ1-tubulin gene andβ2-tubulin gene in Fusarium graminearum, and successfully got the transformants which proved thatβ-tubulin gene of different plant pathogenic fungi can be substituted.The test of sensitivity to MBC of mutant strains containingβ-tubulin gene substituted forβ1-tubulin gene andβ2-tubulin gene respectively showed that the mutant strains had lower sensitivity to MBC, however, without resistance.The test of the expression of MBC-resistantβ-tubulin gene via homologous replacement withβ1-tubulin gene andβ2-tubulin gene in Fusarium graminearum using semiquantitative RT-PCR indicated thatβ-tubulin gene can express on mRNA level in Fusarium graminearum, and there were no significant deviation between the expression ofβ-tubulin gene after homologous replacement withβ1-tubulin gene andβ1-tubulin gene while notable reduction afterβ-tubulin gene replacedβ2-tubulin gene than P2-tubulin gene.The test of the sensitivity to MBC, mycelial growth rate, conidia productivity, perithecium productivity, pathogenicity and other biological characteristics of Fusarium graminearum transformants of MBC-resistantβ-tubulin gene replacingβ1-tubulin gene andβ2-tubulin gene separately manifested thatβ1-tubulin gene andβ2-tubulin gene of Fusarium graminearum had no inhibition to the expression of MBC-resistantβ-tubulin gene of Botrytis cinerea in Fusarium graminearum and the mutant remained sensitive to MBC.The test of the sensitivity to MBC, mycelial growth rate, conidia productivity, perithecium productivity, pathogenicity and other biological characteristics of Fusarium graminearumβ1/β2-tubulin gene knockout mutant strains, MBC-resistantβ-tubulin gene replacingβ1-tubulin gene andβ2-tubulin gene separately and their corresponding knockouts proclaimed thatβ2-tubulin gene was essential for Fusarium graminearum whileβ1-tubulin gene was not, nevertheless, two were complementary, and MBC-resistantβ-tubulin gene replacingβ1-tubulin gene andβ2-tubulin gene separately can both repair the sexual and asexual reproduction capability and pathogenicity of Fusarium graminearum.
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