Engineering melanin on mycoparasitic fungus Trichoderma spp. to enhance antistress tolerance and virulence for biocontrol

Abstractrichoderma, a destructive mycoparasite, has been studied and being used to biocontrol plant fungal disease for more than 70 years. Nevertheless, the control efficacy in fields sometime was lower than anticipation, mainly due to the biotic or abiotic stress encountered. Previously melanin was proved unequivocally with the capacity to enhance immense organisms to counteract the stressed conditions or increase virulence toward animal or plant hosts. Attempt to circumvent the obsticals encountered in biocontrol, the melanin biosynthesis genes encoding polyketide synthase（PKS）, scytalone dehydratase（SCD）, and 1, 3, 8-trihydroxynaphthalene reductase (THN) cloned from Alternaria alternata were engineered into the Trichoderma harzianum and T. reesei by Agrobacterium tumefaciens mediated transformation（ATMT）. To carry out transformantion, two shuttle vectors were constructed, using pCAMBIA 1300 binary vector as backbone, one vector（pCAM-GPD-GFP-PKS）inserted with PKS and green fluorescence protein（GFP）, while the other（pCAM-GPD-HYG-Tri-Scy）harbors SCD, THN and hygromycin-B phosphotransferase（hygromycinr）；both vectors using common Aspergillus GPD promoter and Trpc terminator to drive the transcription. Totally, seven T. harzianum transformants derived from protoplast were obtain, and five T. reesei transformants from protoplasts or conidia were secured. Transformants T. harzianum 3-3 and T. reesei 3-1 possess one copy of PKS, SCD and THN gene, respectively, whereas transformants T. harzianum 11-1 harbors 2 coppies of PKS and one copy of SCD and THN. The T. harzianum transformant exhibited two-fold tolerance toward UV-B irradiation than wild type, and also showed higher growth rate at 35℃, and at lower water activity（aw）at 0.945. Additionally, the transformant also possessed higher virulence toward fungal pathogens Colletotrichum gloeosporioides, Phellinus noxius, and Phytophthora parasitica compared with wild type. While the T. reesei transformant after irradiation with UV-B at the dosage of 3744 mJ/cm2 for 10 min, exhibited 10 times more germination rate than wild type, so as to the germination rate at lower water activity（aw 0.929）, with 35.68% versus 13% between the two strains. However, there are no significant difference in regarding the virulence between the transformant and wild type strains.