Increased killing of Bacillus subtilis on the hair roots of transgenic T4 lysozyme-producing potatoes

Transgenic potato plants expressing the phage T4 lysozyme gene which are resistant to the plant-pathogenic enterobacterium Erwinia carotovora subsp. carotovora have been constructed. The agricultural growth of these potatoes might have harmful effects on soil microbiota as a result of T4 lysozyme release into the rhizosphere. To assess the bactericidal effect of roots, we have developed a novel method to associate the cells of Bacillus subtilis with hair roots of plants and to quantify the survival of cells directly on the root surface by appropriate staining and fluorescence microscopy. With this technique, we found that the roots of potato plants (Desiree and transgenic control lines) without T4 lysozyme gene display measurable killing activity on root-adsorbed B. subtilis cells. Killing was largely independent of the plant age and growth of plants in greenhouse or field plots. Roots from potato lines expressing the T4 lysozyme gene always showed significantly (1.5 to 3.5-fold) higher killing. It is concluded that T4 lysozyme is released from the root epidermis cells and is active in the fluid film on the root surface. We discuss why strong negative effects of T4 lysozyme-producing potatoes on soil bacteria in field trials may not be observed. We propose that the novel method presented here to study interactions of bacteria with roots can be applied not only to bacterial killing but also to interactions leading to growth-sustaining effects of plants on bacteria.