Microbial phyllosphere populations are more complex than previously realized

Abstract

Phyllosphere microbial communities were evaluated on leaves of field-grown plant species by culture-dependent and -independent methods. Denaturing gradient gel electrophoresis (DGGE) with 16S rDNA primers generally indicated that microbial community structures were similar on different individuals of the same plant species, but unique on different plant species. Phyllosphere bacteria were identified from Citrus sinesis (cv. Valencia) by using DGGE analysis followed by cloning and sequencing of the dominant rDNA bands. Of the 17 unique sequences obtained, database queries showed only four strains that had been described previously as phyllosphere bacteria. Five of the 17 sequences had 16S similarities lower than 90% to database entries, suggesting that they represent previously undescribed species. In addition, three fungal species were also identified. Very different 16S rDNA DGGE banding profiles were obtained when replicate cv. Valencia leaf samples were cultured in BIOLOG EcoPlates for 4.5 days. All of these rDNA sequences had 97-100% similarity to those of known phyllosphere bacteria, but only two of them matched those identified by the culture independent DGGE analysis. Like other studied ecosystems, microbial phyllosphere communities therefore are more complex than previously thought, based on conventional culture-based methods.A ll plant species in natural habitats have associated epiphytic microflora comprising the so-called phyllosphere (1, 2). The composition and quantity of nutrients, including carbohydrates, organic acids, and amino acids that support the growth of epiphytic bacteria, are affected by the plant species, leaf age, leaf physiological status, and the presence of tissue damage (3). Similarly, host plants, leaf age, leaf position, physical environmental condition, and availability of immigrant inoculum have also been suggested to be involved in determining species of microbes in the phyllosphere (4-7).There has been much interest in life forms that inhabit extreme environments such as the phyllosphere. With the repeated, rapid alteration of environmental conditions occurring on leaf surfaces, the phyllosphere has been recognized as a hostile environment to bacteria (8). Leaf surfaces are often dry and temperatures can reach 40-55°C under intense sunlight. During the night, however, leaves are frequently wet with dew and at relatively cool temperatures (5-10°C). Strong UV radiation during the day and sparse nutritional (oligotrophic) conditions also contribute to stressful conditions in the phyllosphere (8). More than 85 different species of microorganisms in 37 genera have been reported in the phyllospheres of rye, olive, sugar beet, and wheat, all by culture-based methods (8-10). Most of these bacteria establish large populations with no apparent effect on the plant, but a few of them can infect the leaves and cause disease (1).Microbial ecologists have devoted much effort to investigating microbial diversity and studying biological interactions between species in the environment. Microor...

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