Quantification of a novel biotrophic mycoparasitic fungus using genus specific real-time PCR for biocontrol of phytopathogenic Fusarium graminearum in wheat root under controlled conditions
Fusarium species are well-known causal agents of Fusarium root-rot, Fusarium head blight (FHB), and Fusarium damaged kernels (FDK) diseases in Saskatchewan and other provinces of Canada. Our goal is to develop quantitative real-time PCR techniques to determine and evaluate interactions between Fusarium-associated biotrophic mycoparasitic fungus SMCD 2220 and 3-acetyldeoxynivalenol (3-ADON) producing Fusarium graminearum Schwabe – in and surrounding wheat roots. ITS1F/ITS4 (internal transcribed spacer) sequences from SMCD 2220 biotrophic mycoparasitic fungal isolate and 20 different Fusarium strains were aligned, and consensus sequences were verified. Four candidate primer sets from ITS regions were designed based on the non-conserved regions of the consensus sequences. Using the primer set SmyITSF/R, the biotrophic mycoparasite genomic DNAs were amplified from SMCD 2220. This primer set was developed for assessing and quantifying the interactions between SMCD 2220 biotrophic mycoparasite and F. graminearum. Well-known necrotrophic T. harzianum T-22, was used as the positive control. During in vitro studies, only SMCD 2220 was observed to improve wheat seed germination, whereas T-22 induced post-emergence damping-off symptoms. Under controlled phytotron conditions, both SMCD 2220 and T. harzianum strains were able to reduce the quantity of F. graminearum in spring wheat root, as well as improving the survival and growth of the spring wheat seedlings. However, amount of SMCD 2220 DNA detected was no significantly difference between wheat inoculated with F. graminearum and without Fusarium. In contrary, the amount of T. harzianum DNA monitored in the treatment inoculated with F. graminearum was observed to reduce significantly, as compared to non-Fusarium treatment.
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