Molecular Resistant Mechanism Of Fusarium Fujikuroi Against Carbendazim

Rice bakanae is one of the important diseases in the rice-growing regions worldwide. The disease not only causes serious yield losses, but also reduces the quality of rice. The pathogen of rice bakanae was used to be considered as Fusarium moniliforme Sheld （teleomorph Gibberella fujikuroi）. However, F. moniliforme is a broad "species" concept, whose teleomorph usually belongs to the G. fujikuroi species complex, and that F. fujikuroi （teleomorph G. fujikuroi MP-C） is the major pathogen of rice bakanae.Benzimidazole fungicides are characterized by broad spectrum, high efficiency, single action position and systemic transportation. Carbendazim was widely used to control rice bakanae. The fungicides bind to β-tubulin, inhibiting the formation of spindle body, thus inhibiting mitosis. Due to its single action position and high selectivity, the resistance of pathogens against carbendazim occurred easily. In recent years resistance to benzimidazoles in fungal pathogens has been attributed to single amino acid changes in the β-tubulin. The majority of these changes were located in β-tubulin site198or200in field benzimidazoles-resistant isolates, however, site mutations of β2-tubulin lead to distinct resistance in F. graminearum.14suspected F. fujikuroi strains were collected from Yancheng, Jiangsu Province in2010-2011. The strains were identified based on results of morphological characteristics, rDNA-ITS and TEF-la gene sequence. In the phylogenetic tree based on rDNA-ITS sequence, five tested strains gathered in a group with F. fujikuroi, F. veticillioides and F. proliferaium, the result showed that rDNA-ITS could not be used for the identification of these three Fusarium species. However, in the phylogenetic tree based on TEF-1α gene sequence, five tested strains gathered in a group with F. fujikuroi （98%confidence）. This suggested that TEF-la gene can be reliably used for identification of Fusarium species. Based on TEF-1α gene sequence and morphological characteristics, the strains were identified as F. fujikuroi.The whole nucleotide sequence of β1-tubulin gene was sequenced and analyzed from different sensitive strains by the primers designed according to the nucleotide sequence of β1-tubulin gene （Broad Institute Accession Number:FVEG₀4081.3） from the sequenced strain7600of F. verticillioides. Real-time quantitative RT-PCR （qRT-PCR） was applied to analyze the expression pattern of β1-tubulin gene from different sensitive strains treated with or without carbendazim. The full-length nucleotide sequence of β1-tubulin gene （Accession Number:JQ026022） was cloned from different sensitive strains of F. fujikoroi, which spanned1671bp with4introns, encoding447amino acids and showed100%nucleotide sequence homology between sensitive and resistant strains. The expression of the β1-tubulin gene in2sensitive strains was significantly higher than3resistant strains when these strains were cultured on carbendazim-free media （p=0.05）. Carbendazim at the concentration of their EC50caused the expression, of the gene from the five strains increasing significantly （p=0.05） in comparison with that of strains treated without carbendazim. However, there was no significant difference between strains when treated with carbendazim. The results showed that the resistance of F. fujikuroi against carbendazim was irrelative toβ1-tubulin gene.The whole nucleotide sequence of β2-tubulin gene was also analyzed from different sensitive strains by the primers designed according to the nucleotide sequence of β2-tubulin gene （Broad Institute Accession Number:FVEG₀5512.3） from the sequenced strain7600of Fusarium verticillioides. The full-length nucleotide sequence of β2-tubulin gene is1704bp, with6introns, encoding448amino acids. The homology of amino acids of β2-tubulin in F. fujikuroi with that of other benzimidalzole resistanceβ2-tubulin was between75%-76%, while it was93%with the β2-tubulin of F. graminearum. Comparing to the sequence of β2-tubulin from MBC strains, there was only one mutation at the1006nucleotide of the β2-tubulin from MBCMR and MBCHR strains, but not leading to mutation at the codon235of amoni acid; The site mutation of β2-tubulin at codon200（Phe→Tyr） in F. fujikuroi may confer carbendazim （MBC） medium resistance; Site-mutation of P2-tubulin at its codon198（Glu→Val） may confer MBC high resistance.