Both Leaf Properties and Microbe-Microbe Interactions Influence Within-Species Variation in Bacterial Population Diversity and Structure in the Lettuce ( Lactuca Species) Phyllosphere

Abstract

Morphological and chemical differences between plant genera influence phyllosphere microbial populations, but the factors driving within-species variation in phyllosphere populations are poorly understood. Twenty-six lettuce accessions were used to investigate factors controlling within-species variation in phyllosphere bacterial populations. Morphological and physiochemical characteristics of the plants were compared, and bacterial community structure and diversity were investigated using terminal restriction fragment length polymorphism (T-RFLP) profiling and 16S rRNA gene clone libraries. Plant morphology and levels of soluble carbohydrates, calcium, and phenolic compounds (which have long been associated with plant responses to biotic stress) were found to significantly influence bacterial community structure. Clone libraries from three representative accessions were found to be significantly different in terms of both sequence differences and the bacterial genera represented. All three libraries were dominated by Pseudomonas species and the Enterobacteriaceae family. Significant differences in the relative proportions of genera in the Enterobacteriaceae were detected between lettuce accessions. Two such genera (Erwinia and Enterobacter) showed significant variation between the accessions and revealed microbe-microbe interactions. We conclude that both leaf surface properties and microbial interactions are important in determining the structure and diversity of the phyllosphere bacterial community.The phyllosphere (the aerial parts of plants) is known to support large and diverse naturally occurring microbial communities (19, 51), of which bacteria, living both epiphytically and endophytically, are the most numerous and diverse (2,6,44). A range of environmental factors such as temperature, rainfall, wind, and solar radiation have been shown to play an important role in determining patterns of bacterial phyllosphere colonization (19). Much less is known of the role of plant factors in determining the diversity and dynamics of phyllosphere microbial communities. Plant species (20), gross plant morphology, the position and height of leaves (45), and leaf age (16) have all been associated with variation in the size of phyllosphere bacterial populations. Furthermore, various leaf surface features such epidermal cell wall junctions (12) and grooves along the veins, stomata, and the base of trichomes (29) and near hydathodes (32) have all been identified as preferential bacterial attachment sites, resulting in uneven bacterial distribution on leaf surfaces. The microbial population already resident in the phyllosphere is also likely to impact significantly on the ability of incoming organisms to successfully colonize the leaf surface. Variation in such resident populations between different plant species has been attributed to differences in a range of plant physiochemical characteristics, including the water and phosphorus contents of the leaves, levels of phenolic compounds (which may be inhibitory toward many b...

Bacterial Communities Associated with Surfaces of Leafy Greens: Shift in Composition and Decrease in Richness over Time

“…In contrast, Xanthomonas was present in only 33 of 88 samples in a survey in California (14). Pantoea, which is a member of the Enterobacteriaceae family, is also found to be one of the major genera in other studies of the leafy-green phyllosphere (5,11,14). The genus Pantoea includes several species that are generally associated with plants as either epiphytes or pathogens, and some species can cause disease in humans (38).…”

“…Bacterial communities associated with leafy greens have already been described by several culture-independent studies. "First-generation" molecular techniques have been used to describe variation in community structure in the context of leaf surface properties and microbial interactions (5), seasonal variation in the community structure (6), and monitoring of bacterial communities in the food chain (7). The introduction of culture-independent methods, in particular, microbial profiling using high-throughput sequencing to study microorganisms, has revealed more complexity and diversity of the phyllosphere microbiota and has dramatically changed the landscape of microbial ecology (5,(7)(8)(9)(10).…”

“…"First-generation" molecular techniques have been used to describe variation in community structure in the context of leaf surface properties and microbial interactions (5), seasonal variation in the community structure (6), and monitoring of bacterial communities in the food chain (7). The introduction of culture-independent methods, in particular, microbial profiling using high-throughput sequencing to study microorganisms, has revealed more complexity and diversity of the phyllosphere microbiota and has dramatically changed the landscape of microbial ecology (5,(7)(8)(9)(10). The 454 pyrosequencing platform has been used in different studies of bacterial communities associated with leafy greens (11)(12)(13)(14)(15), but Illumina MiSeq has lately been the dominant platform for microbial profiling.…”

High-Level Culturability of Epiphytic Bacteria and Frequency of Biosurfactant Producers on Leaves

“…Recent culture-independent community analyses have confirmed the apparent selectivity of leaf surfaces, finding that leaf surfaces harbor less diverse communities than other environments, such as the immediate subtending soil (3,4). Furthermore, plant species identity also appears to be a factor that can influence the composition of associated phyllosphere bacterial communities (5)(6)(7)(8)(9). While epiphytes are apparently distinct in their ability to grow and survive on leaves, their adaptations needed to thrive in this habitat remain largely unknown.…”

The Microbiological Status of (Ready to Eat) Lettuce Before and After Standard Washing

“…Experiments have shown that the presence of Erwinia can affect negatively E.coli O157:H7 inoculum on lettuce leaves (32), and this is explained as competition for food, where better adapted species suppress competition. It was determined in previous experiments that Enterobacter adversely affect the adherence and colonization of Erwinia species in lettuce (31). However, some recent research have confirmed the opposite, as in the case of pectinovorum species, which makes lesions on the surfaces as additional sites for pathogenic microorganisms to adhere and penetrate their hosts more easily (40).…”