Enhancement of Trichoderma Harzianum Activity Against Sclerotinia sclerotiorum by Overexpression of Chit42

  Backgoround: Plant diseases, caused by a wide range of phytopathogenic fungi, could be managed using of Trichoderma sp, as a biocontrol agent. Cell wall degrading enzymes like chitinase from T. harzianum are important means for fungal pathogen inhibition. Overexpression of these chitinase enzymes can improve the antagonistic potential of Trichoderma sp. strains. Objectives: This study aimed to produce a new enhanced biocontrol system of Trichoderma harzianum, overexpressing chit42 gene. The improved T. harzianum could be an appropriate biocontrol agent for controlling the stem rot disease of canola caused by Sclerotinia sclerotiorum. Materials and Methods: T. harzianum protoplast cotransformation was carried out by pLMRS3-Chit42 and p3SR2 plasmids. The transformants were selected based on their growth on colloidal chitin containing medium. The improvement of transformants was investigated by quantification of mRNA using real-time quantitative polymerase chain reaction (RT-PCR) and measurement of chitinase activity in the medium containing colloidal chitin as the carbon source. Furthermore, the antagonistic activity of transformants against S. sclerotiorum was assessed by dual culture experiments. Results: The overexpressing transformants of Chit42 displayed higher levels of chitinase activity to inhibit S. sclerotiorum growth compared with the wild type. The results indicated that the value of the chitinase activity (126.42 + 0.07 U/mL) of Chit42-9 increased 64.17 fold. Transcriptomic analysis demonstrated that Chit42-9 transformant expressed 5.2 fold of Chit42 transcript as compared with the parent strain. Biocontrol inhibition of this transformant was 4.98-fold more compared with the non-transformant type. Conclusion: The improved Chit42-9 transformant can be used for biocontrolling S. sclerotiorum, cause of stem rot disease in canola.