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

Abstract

<span style="color: windowtext;">Backgoround: </span><span style="color: windowtext;">Plant diseases, caused by a wide range of phytopathogenic fungi, could be managed using of <em>Trichoderma</em> sp, as a biocontrol agent. Cell wall degrading enzymes like chitinase from <em>T. harzianum</em> are important means for fungal pathogen inhibition. Overexpression of these chitinase enzymes can improve the antagonistic potential of <em>Trichoderma</em> sp. strains.</span> <span style="color: windowtext;">Objectives: </span><span style="color: windowtext;">This study aimed to produce a new enhanced biocontrol system of Trichoderma harzianum, overexpressing <em>chit42</em> gene. The improved <em>T. harzianum</em> could be an appropriate biocontrol agent for controlling the stem rot disease of canola caused by <em>Sclerotinia sclerotiorum</em>.</span> <span style="color: windowtext;">Materials and Methods: </span><em><span style="color: windowtext;">T. harzianum</span></em><span style="color: windowtext;"> 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 <em>S. sclerotiorum</em> was assessed by dual culture experiments. </span> <span style="color: windowtext;">Results: </span><span style="color: windowtext;">The overexpressing transformants of <em>Chit42</em> displayed higher levels of chitinase activity to inhibit <em>S. sclerotiorum</em> growth compared with the wild type. The results indicated that the value of the chitinase activity (126.42 + 0.07 U/mL) of <em>Chit42-9</em> increased 64.17 fold. Transcriptomic analysis demonstrated that <em>Chit42-9</em> transformant expressed 5.2 fold of <em>Chit42</em> transcript as compared with the parent strain. Biocontrol inhibition of this transformant was 4.98-fold more compared with the non-transformant type.</span> <span style="color: windowtext;">Conclusion: </span><span style="color: windowtext;">The improved <em>Chit42-9</em> transformant can be used for biocontrolling S. sclerotiorum, cause of stem rot disease in canola.</span>