Culturable Leaf-Associated Bacteria on Tomato Plants and Their Potential as Biological Control Agents

Culturable leaf-associated bacteria inhabiting a plant have been considered as promising biological control agent (BCA) candidates because they can survive on the plant. We investigated the relationship between bacterial groups of culturable leaf-associated bacteria on greenhouse- and field-grown tomato leaves and their antifungal activities against tomato diseases in vitro and in vivo. In addition, the isolated bacteria were analyzed for N-acyl-homoserine lactone (AHL) and indole-3-acetic acid (IAA) production, which have been reported to associate with bacterial colonization, and resistance to a tomato alkaloid (α-tomatine). Leaf washings and subsequent leaf macerates were used to estimate the population size of epiphytic and more internal bacteria. Bacterial population sizes on leaves at the same position increased as the leaves aged under both greenhouse and field conditions. Field-grown tomatoes had significantly larger population sizes than greenhouse-grown tomatoes. Analysis of 16S rRNA gene (rDNA) sequencing using 887 culturable leaf-associated bacteria revealed a predominance of the Bacillus and Pseudomonas culturable leaf-associated bacterial groups on greenhouse- and field-grown tomatoes, respectively. Curtobacterium and Sphingomonas were frequently recovered from both locations. From the 2138 bacterial strains tested, we selected several strains having in vitro antifungal activity against three fungal pathogens of tomato: Botrytis cinerea, Fulvia fulva, and Alternaria solani. Among bacterial strains with strong in vitro antifungal activities, Bacillus and Pantoea tended to show strong antifungal activities, whereas Curtobacterium and Sphingomonas were not effective. The results indicated the differences in antifungal activity among predominant bacterial groups. Analysis of α-tomatine resistance revealed that most bacterial strains in the dominant groups exhibited moderate or high resistance to α-tomatine in growth medium. Furthermore, some Sphingomonas and Pantoea strains showed AHL and IAA production activities. Strain 125NP12 (Pantoea ananatis) showed particular α-tomatine resistance, and AHL and IAA production had the highest protective value (91.7) against gray mold. Thus, the differences of these physiological properties among dominant bacteria may be associated with the disease suppression ability of BCAs on tomato plants.     

Leaf Elongation in Relation to Leaf Water Potential in Soybean

Leaf water potential, turgor pressure, and leaf elongation ratewere measured in soybeans growing in controlled environmentchambers, greenhouses, and outdoors. Plants in chambers hadthe highest water potentials and turgor pressures, and plantsoutdoors the lowest. In all three environments there was a linearrelationship between elongation rate and turgor pressure. Leavesof plants in drier environments required less turgor for elongation,and showed a greater increase in elongation rate per unit increasein turgor. Elongation rates over a 72 h period were equal inthe three environments. Leaves reached the largest final sizein the greenhouse (intermediate in water potential). Epidermalcells were larger in chamber- and greenhouse-grown leaves thanin leaves of plants grown outdoors. The number of epidermalcells per leaf was greater in the greenhouse and outdoors thanin the chamber. Leaf elongation characteristics of greenhouseplants were duplicated by mildly stressing chamber plants, andleaf elongation characteristics of field plants were duplicatedby more severely stressing chamber plants. Leaves of mildlystressed chamber plants also reached a larger final size thanleaves of more severely stressed chamber plants, or leaves ofcontrol plants in the chamber. Water stress in the chamber increasedthe number of epidermal cells per leaf. More severe water stressin the chamber reduced epidermal cell size. Based on the waterstress experiments it is concluded that the differences in plantwater status in the chamber, greenhouse, and field caused differencesin elongation characteristics, and were responsible for thedifferences in leaf size.     

Aggregates of Resident Bacteria Facilitate Survival of Immigrant Bacteria on Leaf Surfaces

The fate of immigrant bacterial cells on leaves under stressful conditions was determined as a function of the anatomical features and the local spatial density of resident cells at their landing site. Pantoea agglomerans 299R was established on bean leaves and the survival of immigrant cells of Pseudomonas fluorescens A506 and Pseudomonas syringae B728a, as well as P. agglomerans itself, was determined by epifluorescence microscopy following subsequent exposure of plants to desiccation stress. Resident and immigrant bacterial strains constitutively expressed the cyan and the green fluorescent protein, respectively, and the viability of individual cells was assessed directly on leaf surfaces following propidium iodide staining. Although only a small fraction of the immigrant cells landed on established bacterial aggregates, their fate was usually strongly influenced by the presence of indigenous bacteria at the site at which they landed. Immigrants of P. agglomerans 299R or P. fluorescens A506 that arrived as solitary cells had about double the probability of survival when landing on aggregates formed by P. agglomerans 299R than when landing on uncolonized areas of the leaf surface. In contrast, the survival of P. syringae B728a was similar irrespective of whether it landed on colonized or uncolonized parts of a leaf. The nature of plant anatomical features at which immigrant bacteria landed also strongly influenced the fate of immigrant bacteria. The fraction of immigrant cells of each species tested that landed on veins, glandular trichomes, or epidermal cells altered by P. agglomerans that died was always less than when they landed on normal epidermal cells or at the base of hooked trichomes. Depending on the process by which immigrants arrive at a leaf, only a small fraction of cells may be deposited on existing bacterial aggregates. Although uncolonized sites differed greatly in their ability to influence the survival of immigrant cells, the fate of an immigrant bacterium will depend on the nature of the leaf structure on which it is deposited, and apparently indirectly on the amount of nutrients and water available at that site to support the development of bacterial aggregates.     

Evaluating the Growth Potential of Pathogenic Bacteria in Water

The degree to which a water sample can potentially support the growth of human pathogens was evaluated. For this purpose, a pathogen growth potential (PGP) bioassay was developed based on the principles of conventional assimilable organic carbon (AOC) determination, but using pure cultures of selected pathogenic bacteria (Escherichia coli O157, Vibrio cholerae, or Pseudomonas aeruginosa) as the inoculum. We evaluated 19 water samples collected after different treatment steps from two drinking water production plants and a wastewater treatment plant and from ozone-treated river water. Each pathogen was batch grown to stationary phase in sterile water samples, and the concentration of cells produced was measured using flow cytometry. In addition, the fraction of AOC consumed by each pathogen was estimated. Pathogen growth did not correlate with dissolved organic carbon (DOC) concentration and correlated only weakly with the concentration of AOC. Furthermore, the three pathogens never grew to the same final concentration in any water sample, and the relative ratio of the cultures to each other was unique in each sample. These results suggest that the extent of pathogen growth is affected not only by the concentration but also by the composition of AOC. Through this bioassay, PGP can be included as a parameter in water treatment system design, control, and operation. Additionally, a multilevel concept that integrates the results from the bioassay into the bigger framework of pathogen growth in water is discussed. The proposed approach provides a first step for including pathogen growth into microbial risk assessment.Pathogenic bacteria can survive and also grow in low-nutrient aquatic environments, such as surface waters or man-made water treatment systems (2, 17, 30). Studies on pathogen survival and/or die-off (including disinfection) in water are common, but little is known about the fundamental factors governing their growth in the environment (34, 35). Understanding the growth of pathogenic bacteria in aquatic ecosystems is essential for a holistic approach to microbial risk assessment as well as for improving drinking water treatment design and operation.A key factor governing growth of all organisms is nutrient availability. All human pathogens are heterotrophs, utilizing organic compounds as their carbon and energy source. Natural organic matter in water comprises a broad spectrum of many different compounds; it is usually determined as a bulk parameter, such as dissolved organic carbon (DOC). Only a fraction (0.1 to 44%) of this DOC pool is readily available for bacterial growth (18, 33). This bioavailable fraction is quantified using bioassays, such as the biodegradable dissolved organic carbon (BDOC) assay (27) or the assimilable organic carbon (AOC) assay (31). Typically, AOC represents small molecules readily available for growth, whereas BDOC can also include larger molecular compounds, which require predegradation before they can be taken up by microbial cells. Results from both of these assays are commonly used as indicators for bacterial growth potential and have previously been associated with regrowth and biofilm formation in drinking water distribution systems (7, 20, 32).Previous studies have pointed toward an apparent correlation between the concentration of AOC and the presence of enteric bacteria. For example, during two large surveys of drinking water treatment systems across North America, the occurrence (presence/absence) of coliform bacteria was found to be elevated above an AOC concentration of 100 μg liter⁻¹ (4, 21). Other studies also found that AOC concentrations were directly correlated to growth of pathogenic bacteria (30, 34, 35). However, AOC is a bulk parameter, which includes many different substrates (e.g., amino acids, sugars, and fatty acids) readily available for heterotrophic growth. Hence, its composition can differ distinctly, and it is assumed that every aquatic environment carries a complex and unique “fingerprint” of utilizable organic carbon compounds (22). Moreover, the spectrum of growth-supporting substrates (carbon compounds) of individual bacterial strains is specific—a fact also used for the classification of bacteria for taxonomic purposes. This principle has been integrated into conventional AOC assays, where the specific substrate spectrum of different pure cultures can be used to quantify different types of compounds present in water (26, 33). The term “pathogenic bacteria” is a collective term for many different bacterial species that can all cause disease in humans but their individual substrate spectra are unique for each species. Thus, we have hypothesized that the total concentration of AOC alone is not a sufficient parameter for describing the growth potential of pathogenic bacteria; the quality of the available carbon compounds has to be considered as well.There is no existing method that is capable of fractionating organic carbon in a way that allows for the quantification of individual compounds that support growth of specific pathogens. In this study, we have developed a pathogen growth potential (PGP) assay by combining the conventional AOC assay (31) with flow cytometric quantification of bacterial growth (11) and using pathogens as inocula. The PGP assay yields two main results, namely, (i) the extent of pathogen growth, and (ii) the relative fraction of AOC consumed by a pathogen. With this approach, we investigated the growth potential of three model pathogens from three different genera, namely, Escherichia coli O157, Vibrio cholerae O1, and Pseudomonas aeruginosa, in a broad range of water samples, differing considerably in their origin and quality.     

Effect of Bacteria on Survival and Growth of Acanthamoeba castellanii

The growth and survival of Acanthamoeba castellanii in the presence of Gram-negative bacteria such as Pseudomonas aeruginosa, Escherichia coli, Serratia marcescens, and Stenotrophomonas maltophilia varied with the densities and species of bacteria. All species of bacteria suspended in a buffered saline at densities of 10⁵ to 10⁶/ml supported the growth and survival of 10⁶/ml trophozoites of Acanthamoeba castellanii in a buffered saline solution. At densities of bacteria to amoebae of 100:1 or greater, growth and survival of A. castellanii were suppressed, particularly by P. aeruginosa. In an enrichment medium, the rapid growth of most co-inoculated bacteria inhibited the growth and survival of the amoeba. Received: 12 September 1996 / Accepted: 20 September 1996     

Survival of Bacteria and Fungi in Relation to Water Activity and the Solvent Properties of Water in Biopolymer Gels

Survival of bacteria (Rhizobium, Agrobacterium, and Arthrobacter spp.), fungal spores (Penicillium sp.), and yeasts (Saccharomyces sp.) was studied in relation to water activity (a_w) and the presence of nutritive solutes. The cells were entrapped in polysaccharide gels, as is done to immobilize cells or enzymes, and then dehydrated. The number of living cells (10¹⁰ g of dry polymer⁻¹) remained constant for periods of storage of >3 years at 28°C when the inocula were kept at an a_w of <0.069. At a_w values between 0.069 and 0.83 the number of survivors diminished more and more rapidly as the a_w was raised. For a given a_w and organism, there were large differences in survival rate as a function of the nutritive solutes used to culture the microorganisms. Low-molecular-weight compounds (with three or five carbon atoms) had a deleterious effect on survival, whereas compounds of higher molecular weight (C₆ to C₁₂) had a protecting effect. Thus, the a_w alone was not a sufficient explanation for the deterioration of the inocula. Survival seemed to be more directly related to some properties of the water in the biopolymer. New concepts such as the discontinuity of properties of water and the point of mobilization of solutes, already proposed by Duckworth and Kelly (J. Food Technol. 8:105-113, 1973) and Seow (J. Sci. Food Agric., 26:535-536, 1975), have been taken into consideration to explain the interactions of water with the biopolymer and their specific effects on the microorganisms.     

The Intracellular Water Activity of Bacteria in Relation to the Water Activity of the Growth Medium

Intracellular water activities ( a _w) calculated from the solute composition of various bacterial cells, are in good agreement with values derived from intracellular freezing point data. Further, and confirming literature results based on freezing points, the intracellular a _w was found to be generally equal to or lower than that of the growth medium.     

Medium for Isolation and Growth of Bacteria Associated with Plum Leaf Scald and Phony Peach Diseases

Rickettsia-like bacteria associated with plum leaf scald and phony peach diseases were isolated from diseased but not from healthy tissues and cultured on charcoal-yeast extract medium (BCYE) buffered with ACES (2-[(2-amino-2-oxoethyl) amino]-ethanesulfonic acid). Optimum conditions for isolation and growth on BCYE medium were pH 6.5 to 6.9 at 20 and 25°C under normal atmosphere. Growth of primary colonies and first-passage subcultures was restricted, and colonies reached a maximum diameter of 0.6 mm in 60 days. After 12 passages, subcultures reached maximum growth in 21 days. The rickettsia-like bacteria from BCYE cultures were gram negative, serologically the same as those present in diseased peach and plum, and composed of rod-shaped cells measuring 0.35 by 5 μm (average diameter and maximum length) in a matrix of filamentous strands of similar width but of variable length.     

Variation in Selection for. Leaf Water Conductance in Relation to Growth and Stomatal Dimensions in Lolium perenne L.

A simple, rapid technique for direct selection for leaf waterconductance (LWC) in two populations of Lolium perenne L. isdescribed. Measurements were made with a diffusion porometerin growth rooms on the youngest fully expanded leaf of eachtiller. Considerable variation in LWC was found between 100genotypes of each population. Most of the variation in totalLWC was attributed to variation in adaxial LWC and it was shownthat ignoring abaxial LWC gave very similar ranking of genotypesto those using total LWC. Selection criteria were then establishedwhich maximised the repeatability of LWC measurements and allowedselection of groups of plants significantly different from oneanother in LWC. The variation in LWC was not related eitherto stomatal length or number, or to growth rate. Lolium perenne L., perennial ryegrass, abaxial and adaxial, leaf water conductance, selection, diurnal rhythms, ontogenetic changes, growth, stomatal dimensions