Single and combined colonization of Sclerotinia sclerotiorum sclerotia by the fungal mycoparasites Coniothyrium minitans and Microsphaeropsis ochracea

Abstract

Potential enhancement of mycoparasitic efficacy of Coniothyrium minitans and Microsphaeropsis ochracea through concomitant colonization of Sclerotinia sclerotiorum sclerotia was investigated, following observation that the two mycoparasites did not exhibit any mutual antagonism in dual culture assays. Simultaneous application of both mycoparasites increased sclerotia mortality in a temperature range from 16 to 26 degrees C compared to single application, indicating a predominantly additive interaction. With increasing temperature the efficacy of M.ochracea decreased, but C.minitans was unaffected. Degradation of sclerotia by C.minitans proceeded slightly faster than with M.ochracea. Simultaneous colonization of sclerotia was studied at the histopathological level with mycoparasite strains transformed via Agrobacterium tumefaciens-mediated transformation (ATMT) with reporter genes encoding for DsRed and GFP. Sclerotia colonization followed by fluorescence microscopy revealed effective penetration of the sclerotial rind, growth and formation of pycnidia in the cortex and medulla by both antagonists, resulting in complete degradation of sclerotia within 25days after single inoculation. Upon simultaneous inoculation, both antagonists concomitantly colonized the sclerotial tissue and independently formed pycnidia in the sclerotial medulla and on the sclerotial rind, demonstrating their ability to co-colonize the same host fungus. Although the individual growth of the two mycoparasites in dual inoculations was slightly delayed, the sclerotia degrading effects were additive, suggesting a complementary antagonistic interaction. The combined application of two different species of mycoparasites cooperating on the same host fungus and differing in temperature requirements may be advantageous for making biocontrol applications in the field less sensitive to varying environmental and host conditions.