Method for simple and rapid enumeration of total epiphytic bacteria in the washing solution of rice plants

The phyllosphere is one of the most common habitats for terrestrial bacteria. However, little is known about the populations of bacteria, including unculturable bacteria, that thrive on plant surfaces. Here, we developed a fluorescent nuclear staining technique to easily and rapidly observe and enumerate populations of total and living epiphytic bacteria, with particular emphasis on the concentration by centrifugation and fixation of the epiphytic bacteria. An investigation on the optimal conditions for centrifugation and fixation revealed that centrifugation at 20 400g for 2 min and fixation with 0.5% glutaraldehyde solution were the optimum conditions for observation of the bacteria. Using this technique, we assessed the populations of the total and living bacteria on the surface of rice plants. When epiphytic bacteria were recovered from rice seeds (Oryza sativa 'Koshihikari'), the number of total and living bacterial cells was 7.36 and 6.85 log??·g?1 (fresh mass) in the seed washing, respectively. In contrast, the numbers of total and living bacterial cells in the leaf sheath washings were 5.5-5.8 and 5.3-5.7 log??·g?1, respectively. Approximately 5%-30% of the total bacteria in the washing solution of rice plant were culturable. The usefulness of the enumeration method and the amount of bacteria on the plant surfaces are discussed.