Agrobacterium Tumefaciens-mediated Transformation (ATMT) Of Trichoderma Atroviride T23 And Research On Screening And Application Of Mutants With High Dichlorvos-degrading Capacity

A protocol for efficient Agrobacterium tumefaciens-mediated transformation (ATMT) of biocontrol fungus Trichoderma atroviride strain T23 was developed to construct mutants with improved dichlorvos-degradation ability. A transformation frequency of 5×10-6 was achieved. Among 110 genetically stable T-DNA transformants of T. atroviride T23, two transformants, AMT-12 and AMT-28, confirmed by Southern blot analysis to have single-copy inserts of T-DNA, showed an increase in dichlorvos-degradation ability of more than 10% compared to that of the wild type, exhibited similar tolerance to the pesticide, but lower spore formation ability. Five transformants exhibited a reduction in degradation of more than 70%, exhibited wild-type spore formation, and tolerated up to 800μg/mL of dichlorvos. The left-flanking sequence of the insertion site in AMT-12 and AMT-28 were cloned as a 1845-bp and 1730-bp fragments and shown to have 89% and 88% identity to the DNA from T. atroviride IMI 206040, respectively; however, the involvement of these DNA fragments in dichlorvos degradation remains still to be determined. This study can promote both a more efficient isolation of DNA sequence flanking T-DNA integration site in T. atroviride mutants and a more rational utilization of these transformants in dichlorvos degradation.An immobilizing conidia approach was successfully established to study the degradation ability of dichlorvos in AMT-28, and the biodegradation mechanism of DDVP in this fungus was also investigated. The beads immobilized 10'conidia per 100 mL of Na-alginate solution exhibited the highest degradation rate compared to that of 105 and 109 conidia under the experimental conditions. The immobilized AMT-28 conidia showed improved degradation abilities than immobilized or free mycelia. The beads immobilized cells (conidia and mycelia) of AMT-28 kept good storage stability and reutilization capacity, the degradation abilities of them did not decrease, but there were somewhat degree of increase in five bathes of samples through one-month determination test. The dichlorvos in Burk medium with mycelia of AMT-28 was confirmed to be completely removed using HPLC analysis. The dichlorvos degradation in auxotrophic Burk media (designated as N-, P- and C-, respectively) varied with different nitrogen, phosphorus and carbon sources, and it was found to be dramatically affected by nitrogen sources. Dichlorvos could be adopted as a sole carbon or phosphorus source of AMT-28. Meanwhile, The results revealed that the major reason for dichlorvos removal in AMT-28 should be attributed to the fungal biodegradation and there was no detectable biosorption in this study. Inducible intracellular degrading enzyme of dichlorvos could be promoted by a small amount of dichlorvos at the initial stage of this organophosphorus compound stress. Overall, the dichlorvos degradation in AMT-28 was likely to be a kind of Biomineralization process.