Variation in local carrying capacity and the individual fate of bacterial colonizers in the phyllosphere

To better characterize the bacterial community members capable of biosurfactant production on leaves, we distinguished culturable biosurfactant-producing bacteria from nonproducers and used community sequencing to compare the composition of these distinct cultured populations with that from DNA directly recovered from leaves. Communities on spinach, romaine, and head lettuce leaves were compared with communities from adjacent samples of soil and irrigation source water. Soil communities were poorly described by culturing, with recovery of cultured representatives from only 21% of the prevalent operational taxonomic units (OTUs) (>0.2% reads) identified. The dominant biosurfactant producers cultured from soil included bacilli and pseudomonads. In contrast, the cultured communities from leaves are highly representative of the culture-independent communities, with over 85% of the prevalent OTUs recovered. The dominant taxa of surfactant producers from leaves were pseudomonads as well as members of the infrequently studied genus Chryseobacterium. The proportions of bacteria cultured from head lettuce and romaine leaves that produce biosurfactants were directly correlated with the culture-independent proportion of pseudomonads in a given sample, whereas spinach harbored a wider diversity of biosurfactant producers. A subset of the culturable bacteria in irrigation water also became enriched on romaine leaves that were irrigated overhead. Although our study was designed to identify surfactant producers on plants, we also provide evidence that most bacteria in some habitats, such as agronomic plant surfaces, are culturable, and these communities can be readily investigated and described by more classical culturing methods. IMPORTANCEThe importance of biosurfactant production to the bacteria that live on waxy leaf surfaces as well as their ability to be accurately assessed using culture-based methodologies was determined by interrogating epiphytic populations by both culture-dependent and culture-independent methods. Biosurfactant production was much more frequently observed in cultured communities on leaves than in other nearby habitats, such as soil and water, suggesting that this trait is important to life on a leaf by altering either the leaf itself or the interaction of bacteria with water. While pseudomonads were the most common biosurfactant producers isolated, this habitat also selects for taxa, such as Chryseobacterium, for which this trait was previously unrecognized. The finding that most epiphytic bacterial taxa were culturable validates strategies using more classical culturing methodologies for their study in this habitat. The phyllosphere is recognized to be a selective habitat in which epiphytic bacteria must be able to access limited and spatially heterogeneous nutrient supplies and endure frequent fluctuations in moisture availability on a water-repellent surface (1, 2). Recent culture-independent community analyses have confirmed the apparent selectivity of leaf surfaces, finding that leaf sur...

Section: Discussionmentioning

confidence: 99%

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

Burch

Paulina

Sbodio

et al. 2016

“…Such variability may be due to small differences in the environment, such as relative humidity and temperature, that may affect the plant-pathogen interaction (7). It may also be due to stochastic events introduced by the localization of the tested antagonist and/or the pathogen in heterogeneous microniches (40) or to effects occurring as a consequence of phenotypic variation of one of the three partners.…”

Section: Discussionmentioning

confidence: 99%

Forward Genetic In Planta Screen for Identification of Plant-Protective Traits of Sphingomonas sp. Strain Fr1 against Pseudomonas syringae DC3000

Vogel

Innerebner

Zingg

et al. 2012

Sphingomonas sp. strain Fr1 has recently been shown to protect Arabidopsis thaliana against the bacterial leaf pathogen Pseudomonas syringae DC3000. Here, we describe a forward genetic in planta screen to identify genes in Sphingomonas sp. Fr1 necessary for this effect. About 5,000 Sphingomonas sp. Fr1 mini-Tn5 mutants were assayed for a defect in plant protection against a luxCDABE-tagged P. syringae DC3000 derivative in a space-saving 24-well plate system. The bioluminescence of the pathogen was used as the indicator of pathogen proliferation and allowed for the identification of Sphingomonas sp. Fr1 mutants that had lost the ability to restrict pathogen growth before disease symptoms were visible. Potential candidates were validated using the same miniaturized experimental system. Of these mutants, 10 were confirmed as plant protection defective yet colonization competent. The mutants were subsequently evaluated in a previously described standard microbox system, and plants showed enhanced disease phenotypes after pathogen infection relative to those inoculated with the parental strain as a control. However, the disease severities were lower than those observed for control plants that were grown axenically prior to pathogen challenge, which suggests that several traits may contribute to plant protection. Transposon insertion sites of validated mutants with defects in plant protection were determined and mapped to 7 distinct genomic regions. In conclusion, the established screening protocol allowed us to identify mutations that affect plant protection, and it opens the possibility to uncover traits important for in planta microbe-microbe interactions.

“…It is known that the leaf surface is a heterogeneous environment where there are microsites where nutrients such as fructose, sucrose, and iron are spatially variable (20,28,34). The population of bacteria on leaves has been described as the sum of those subpopulations in relatively isolated microsites on leaves that differ in availability of carbon (35). A contribution of nitrate to overall population size would thus not likely be seen until the bacteria consumed the preferred nitrogen sources before consuming the available nitrate, and only then at sites where carbon was relatively high in proportion to the available nitrogen compounds.…”

Section: Discussionmentioning

confidence: 99%

Contribution of Nitrate Assimilation to the Fitness of Pseudomonas syringae pv. syringae B728a on Plants

Parangan-Smith

Lindow

2013

The ability of Pseudomonas syringae pv. syringae to use nitrate as a nitrogen source in culture and on leaves was assessed. Substantial amounts of leaf surface nitrate were detected directly and by use of a bioreporter of nitrate on bean plants grown with a variety of nitrogen sources. While a nitrate reductase mutant, P. syringae ⌬nasB, exhibited greatly reduced growth in culture with nitrate as the sole nitrogen source, it exhibited population sizes similar to those of the wild-type strain on leaves. However, the growth of the ⌬nasB mutant was much less than that of the wild-type strain when cultured in bean leaf washings supplemented with glucose, suggesting that P. syringae experiences primarily carbon-limited and only secondarily nitrogenlimited growth on bean leaves. Only a small proportion of the cells of a green fluorescent protein (GFP)-based P. syringae nitrate reductase bioreporter, LK2(pOTNas4), exhibited fluorescence on leaves. This suggests that only a subset of cells experience high nitrate levels or that nitrate assimilation is repressed by the presence of ammonium or other nitrogenous compounds in many leaf locations. While only a subpopulation of P. syringae consumes nitrate at a given time on the leaves, the ability of those cells to consume this resource would be strongly beneficial to those cells, especially in environments in which nitrate is the most abundant form of nitrogen. N itrate assimilation is a central metabolic process that contributes to bacterial growth in a variety of habitats such as the ocean (1), rhizosphere (2), and soils (3). While there are numerous studies of bacterial nitrate assimilation in such locations, very little is known about the importance of bacterial nitrate assimilation in other habitats such as the aerial portions of plants known as the phyllosphere. The phyllosphere is considered to be a relatively stressful habitat due to nutrient limitation, desiccation stress, low and variable water availability, and high fluxes of UV radiation; nonetheless, it is colonized by specialized microbes (4). The fitness of epiphytic bacteria is associated with their ability to tolerate or avoid these various stresses and to utilize the limited nutrients available to them on leaves.Various studies have suggested that the phyllosphere is generally a nutrient-limited habitat. A wide variety of both inorganic and organic compounds are found on a given plant species. Amino acids, organic acids, and carbohydrates were detected in all of the species tested, although the quantity and abundance of a given compound varied from species to species (5, 6). Inorganic nitrogen is also found on the surface of plants, but its form has generally not been determined. Many factors, such as leaf age and the hydrophobic properties of the leaf, affect the amount and composition of leaf leachates. Younger leaves are typically more hydrophobic than older leaves, and their thicker cuticle restricts nutrient flux. Leaves that are more wettable, such as those of beans (Phaseolus vulgaris), experience m...