Abstract
Resveratrol belongs to the stilbenes family which is commonly found in grapes, blueberries, peanut, willow, and other plants. It acts as a natural polyphenol phytoalexin that responds and helps mitigate microbial attacks within grape plants. A methylated form of resveratrol, pterostilbene, shows 6-fold higher inhibition on fungal growth. Our hypothesis is that increased production of stilbenes in transgenic American chestnut will enhance resistance to the chestnut blight. This research first evaluated the effects of resveratrol and pterostilbene in growth medium plate experiments. At concentrations ranging from 2.5mM to 20mM resveratrol significantly reduced mycelium growth of Cryphonectria parasitica by 22% to 90%, respectively, and resulted in significant morphological changes to the hyphae. The concentration of 2.5mM pterostilbene decreased the mycelial growth by 92%, supporting its more potent inhibitory effect. In minimum inhibition concentration assays, concentrations of 10mM of resveratrol and 2.5mM of pterostilbene, both significantly inhibited the germination of conidia (asexual spores of C. parasitica). RTqPCR showed American chestnut plants transformed with the stilbene synthase (EC 2.3.1.95) gene, Vst1, overexpressed their transcripts in both tissue culture plants and greenhouse plants. Direct fungal inoculation tests, such as leaf assays, micro stem assays, and small stem assays showed the plants overexpressing the Vst1 gene had intermediate levels of pathogen resistance, which is significantly higher than the resistance of the wild type American chestnut (blight-susceptible control) but significantly lower than the resistance of the Chinese chestnut (blight-resistance control). We also detected the accumulation of stilbenes in the transgenic American chestnut through HPLC-MS, the concentration of resveratrol ranging from 0.11μg/g to 26.93μg/g of plant tissue, similar to amounts used in medium plate assays. Therefore, over expression of the Vst1 gene can enhance pathogen resistance, but will likely need to be used in combination with other resistance-enhancing genes to develop fully blight resistant American chestnut trees.