LEAF SURFACE WAXES AND THE PROCESS OF COLONIZATION BY BACTERIA
Lise M. Marcell1 and Gwyn A. Beattie2, Departments of 1Plant Pathology and 2Microbiology, Iowa State University, Ames, IA, 50011-3211

The first point of contact of a bacterial immigrant with a leaf is usually at the plant cuticle. The plant cuticle, which is composed primarily of waxes and cutin, thus may be a defining component of the leaf surface as a habitat for bacteria. The quantity, composition, and structure of leaf surface waxes can vary with plant species, plant growth stage, and environmental factors such as water availability and pollution exposure. To identify how properties of the plant cuticle influence bacterial colonization of leaves, we examined near isogenic maize mutants that were altered in loci involved in the biosynthesis of cuticular waxes on juvenile stage plants. These mutants varied in the morphology and distribution of their epicuticular wax crystals, in their leaf surface hydrophobicity, and in the amount of extractable waxes they produced. We examined the population dynamics of both Pantoea agglomerans and Clavibacter michiganensis subsp. nebraskensis (Cmn) on several of these mutants. Although the mutants exhibited differences in the number of bacteria that they retained following spray inoculation, for studies on the population dynamics, we attained similar initial population sizes by applying inocula with distinct cell densities to each mutant. We found that the leaf surface waxes influenced bacterial colonization in a bacterial species-dependent manner. For example, one maize mutant, gl1, fostered significantly larger population sizes of Cmn than did wild-type maize, but similar population sizes of P. agglomerans as the wild type. We also found that leaf surface waxes influenced bacterial colonization in an environment-dependent manner. For example, mutant gl5,gl20 fostered significantly smaller population sizes of P. agglomerans than did the wild-type maize under wet conditions (95% RH), but not under dry conditions (45% RH). Surprisingly, the mutant that had the most dramatic effect on bacterial colonization was the mutant that was the most similar to the wild-type maize in all of the measured leaf surface properties. This mutant, gl4, fostered significantly larger populations of both P. agglomerans and Cmn under all of the conditions tested. In contrast to both the wild type, which produced a dense stand of epicuticular wax crystals, and several mutants that were practically devoid of epicuticular wax crystals, mutant gl4 produced epicuticular crystals that were similar in morphology to those of the wild type but were simply less densely distributed. Thus, we hypothesized that the distribution of epicuticular wax crystals on mutant gl4 is optimal for bacterial growth. In support of this hypothesis, we found that when we mechanically removed wax crystals from very localized regions of wild-type leaves, and then verified their removal and the absence of cellular damage by scanning electron microscopy, these small changes in wax distribution resulted in the leaves supporting significantly larger populations of P. agglomerans. These results suggest that factors that are known or predicted to change leaf surface waxes, including global warming, air pollution, and plant breeding for drought tolerance, are likely to influence the populations of one or more of the resident bacterial species.