Colonization of Vitis vinifera by a green fluorescence protein-labeled,gfp-marked strain of Xylophilus ampelinus,the causal agent of bacterial necrosis of grapevine

The dynamics of Xylophilus ampelinus were studied in Vitis vinifera cv. Ugni blanc using gfp-marked bacterial strains to evaluate the relative importance of epiphytic and endophytic phases of plant colonization in disease development. Currently, bacterial necrosis of grapevine is of economic importance in vineyards in three regions in France: the Cognac, Armagnac, and Die areas. This disease is responsible for progressive destruction of vine shoots, leading to their death. We constructed gfp-marked strains of the CFBP2098 strain of X. ampelinus for histological studies. We studied the colonization of young plants of V. vinifera cv. Ugni blanc by X. ampelinus after three types of artificial contamination in a growth chamber and in a greenhouse. (i) After wounding of the stem and inoculation, the bacteria progressed down to the crown through the xylem vessels, where they organized into biofilms. (ii) When the bacteria were forced into woody cuttings, they rarely colonized the emerging plantlets. Xylem vessels could play a key role in the multiplication and conservation of the bacteria, rather than being a route for plant colonization. (iii) When bacterial suspensions were sprayed onto the plants, bacteria progressed in two directions: both in emerging organs and down to the crown, thus displaying the importance of epiphytic colonization in disease development.     

Bleeding Sap and Old Wood Are the Two Main Sources of Contamination of Merging Organs of Vine Plants by Xylophilus ampelinus, the Causal Agent of Bacterial Necrosis

The spatial distribution of vine plants contaminated by Xylophilus ampelinus, the agent responsible for bacterial necrosis, was studied over a 5-year period within two vineyards in the Cognac area. Both vineyards were planted with Vitis vinifera cv. Ugni blanc but were different in age and agronomic location. The emission of X. ampelinus in contaminated bleeding sap was observed during vine sprouting. Contaminated bleeding sap is an important source of inoculum for external contamination due to the high susceptibility of young merging shoots to the pathogen. X. ampelinus emission by bleeding sap was not affected by the age of the plants or the location of the vineyards. However, its emission was irregular with time, and it varied between two fruit canes from individual plants and between plants as well as between years. Moreover, the two vineyards appeared to be entirely contaminated. Consequently, the behavior of the pathogen is not predictable. The distribution of the pathogen inside vine plant organs was analyzed through the four growing seasons. The old wood was contaminated throughout the year and constituted a stock inoculum for endophytic contamination of crude sap during the winter and the spring. Despite the fact that most of the young green shoots were contaminated in May, X.ampelinus was not found in green shoots in June and September, refuting the hypothesis of an epiphytic life of the pathogen under natural conditions. Although all plants were entirely contaminated in both vineyards, symptoms were rare and were observed on different plants each year.     

Endophytic Colonization of Vitis vinifera L. by Plant Growth-Promoting Bacterium Burkholderia sp. Strain PsJN

Patterns of colonization of Vitis vinifera L. cv. Chardonnay plantlets by a plant growth-promoting bacterium, Burkholderia sp. strain PsJN, were studied under gnotobiotic conditions. Wild-type strain PsJN and genetically engineered derivatives of this strain tagged with gfp (PsJN::gfp2x) or gusA (PsJN::gusA11) genes were used to enumerate and visualize tissue colonization. The rhizospheres of 4- to 5-week-old plantlets with five developed leaves were inoculated with bacterial suspensions. Epiphytic and endophytic colonization patterns were then monitored by dilution plating assays and microscopic observation of organ sections. Bacteria were chronologically detected first on root surfaces, then in root internal tissues, and finally in the fifth internode and the tissues of the fifth leaf. Analysis of the PsJN colonization patterns showed that this strain colonizes grapevine root surfaces, as well as cell walls and the whole surface of some rhizodermal cells. Cells were also abundant at lateral root emergence sites and root tips. Furthermore, cell wall-degrading endoglucanase and endopolygalacturonase secreted by PsJN explained how the bacterium gains entry into root internal tissues. Host defense reactions were observed in the exodermis and in several cortical cell layers. Bacteria were not observed on stem and leaf surfaces but were found in xylem vessels of the fifth internode and the fifth leaf of plantlets. Moreover, bacteria were more abundant in the fifth leaf than in the fifth internode and were found in substomatal chambers. Thus, it seems that Burkholderia sp. strain PsJN induces a local host defense reaction and systemically spreads to aerial parts through the transpiration stream.     

Colonization Pattern of the Biocontrol Strain Pseudomonas chlororaphis MA 342 on Barley Seeds Visualized by Using Green Fluorescent Protein

Pseudomonas chlororaphis MA 342 is a potent biocontrol agent that can be used against several seed-borne diseases of cereal crops, including net blotch of barley caused by the fungus Drechslera teres. In this study, strain MA 342 was tagged with the gfp gene (encoding the green fluorescent protein) in order to study the fate of cells after seed inoculation. The gfp-tagged strain, MA 342G2, had the same biocontrol efficacy as the wild type when it was applied at high cell concentrations to seeds but was less effective at lower cell concentrations. By comparing cell counts determined by microscopy to the number of CFU, we found that the number of culturable cells was significantly lower than the total number of bacteria on seeds which were inoculated and dried for 20 h. Confocal microscopy and epifluorescence stereomicroscopy were used to determine the pattern of MA 342G2 colonization and cell aggregation on barley seeds. Immediately after inoculation of seeds, bacteria were found mainly under the seed glume, and there was no particular aggregation pattern. However, after the seeds were sown, irregularly distributed areas of bacterial aggregation were found, which reflected epiphytic colonization of glume cells. There was a trend towards bacterial aggregation near the embryo but never within the embryo. Bacterial aggregates were regularly found in the groove of each seed formed by the base of the coleoptile and the scutellum. Based on these results, we suggest that MA 342 colocalizes with the pathogen D. teres, which facilitates the action of the fungistatic compound(s) produced by this strain.     

Characterization of Epiphytic Bacterial Communities from Grapes,Leaves, Bark and Soil of Grapevine Plants Grown,and Their Relations

Despite its importance in plant health and crop quality, the diversity of epiphytic bacteria on grape berries and other plant parts, like leaves and bark, remains poorly described, as does the role of telluric bacteria in plant colonization. In this study, we compare the bacterial community size and structure in vineyard soils, as well as on grapevine bark, leaves and berries. Analyses of culturable bacteria revealed differences in the size and structure of the populations in each ecosystem. The highest bacteria population counts and the greatest diversity of genera were found in soil samples, followed by bark, grapes and leaves. The identification of isolates revealed that some genera – Pseudomonas, Curtobacterium, and Bacillus – were present in all ecosystems, but in different amounts, while others were ecosystem-specific. About 50% of the genera were common to soil and bark, but absent from leaves and grapes. The opposite was also observed: grape and leaf samples presented 50% of genera in common that were absent from trunk and soil. The bacterial community structure analyzed by T-RFLP indicated similarities between the profiles of leaves and grapes, on the one hand, and bark and soil, on the other, reflecting the number of shared T-RFs. The results suggest an interaction between telluric bacterial communities and the epiphytic bacteria present on the different grapevine parts.     

Contrasting colonization and plant growth promoting capacity between wild type and a gfp-derative of the endophyte Pseudomonas putida W619 in hybrid poplar

This study aims to investigate the colonization of poplar by the endophyte Pseudomonas putida W619 and its capacity to promote plant growth. Poplar cuttings were inoculated with P. putida W619 (wild-type or gfp-labelled). The colonization of both strains was investigated and morphological, physiological and biochemical parameters were analyzed to evaluate plant growth promotion. Inoculation with P. putida W619 (wild-type) resulted in remarkable growth promotion, decreased activities of antioxidative defence related enzymes, and reduced stomatal resistance, all indicative of improved plant health and growth in comparison with the non-inoculated cuttings. In contrast, inoculation with gfp-labelled P. putida W619 did not promote growth; it even had a negative effect on plant health and growth. Furthermore, compared to the wildtype strain, colonization by the gfp-labelled P. putida W619::gfp1 was much lower; it only colonized the rhizosphere and root cortex while the wild-type strain also colonized the root xylem vessels. Despite the strong plant growth promoting capacity of P. putida W619 (wild-type), after gfp labelling its growth promoting characteristics disappeared and its colonization capacity was strongly influenced; for these reasons gfp labelling should be applied with sufficient caution.     

Colonization of Madagascar periwinkle (Catharanthus roseus), by endophytes encoding gfp marker

This study reports the introduction of gfp marker in two endophytic bacterial strains (Pantoea agglomerans C33.1, isolated from cocoa, and Enterobacter cloacae PR2/7, isolated from citrus) to monitor the colonization in Madagascar perinwinkle (Catharanthus roseus). Stability of the plasmid encoding gfp was confirmed in vitro for at least 72 h of bacterial growth and after the colonization of tissues, under non-selective conditions. The colonization was observed using fluorescence microscopy and enumeration of culturable endophytes in inoculated perinwinkle plants that grew for 10 and 20 days. Gfp-expressing strains were re-isolated from the inner tissues of surface-sterilized roots and stems of inoculated plants, and the survival of the P. agglomerans C33:1gfp in plants 20 days after inoculation, even in the absence of selective pressure, suggests that is good colonizer. These results indicated that both gfp-tagged strains, especially P. agglomerans C33.1, may be useful tools to deliver enzymes or other proteins in plant.     

Role of Leaf Surface Sugars in Colonization of Plants by Bacterial Epiphytes

The relationship between nutrients leached onto the leaf surface and the colonization of plants by bacteria was studied by measuring both the abundance of simple sugars and the growth of Pseudomonas fluorescens on individual bean leaves. Data obtained in this study indicate that the population size of epiphytic bacteria on plants under environmentally favorable conditions is limited by the abundance of carbon sources on the leaf surface. Sugars were depleted during the course of bacterial colonization of the leaf surface. However, about 20% of readily utilizable sugar, such as glucose, present initially remained on fully colonized leaves. The amounts of sugars on a population of apparently identical individual bean leaves before and after microbial colonization exhibited a similar right-hand-skewed distribution and varied by about 25-fold from leaf to leaf. Total bacterial population sizes on inoculated leaves under conditions favorable for bacterial growth also varied by about 29-fold and exhibited a right-hand-skewed distribution. The amounts of sugars on leaves of different plant species were directly correlated with the maximum bacterial population sizes that could be attained on those species. The capacity of bacteria to deplete leaf surface sugars varied greatly among plant species. Plants capable of supporting high bacterial population sizes were proportionally more depleted of leaf surface nutrients than plants with low epiphytic populations. Even in species with a high epiphytic bacterial population, a substantial amount of sugar remained after bacterial colonization. It is hypothesized that residual sugars on colonized leaves may not be physically accessible to the bacteria due to limitations in wettability and/or diffusion of nutrients across the leaf surface.     

Herbaspirillum-plant interactions: microscopical, histological and molecular aspects

Diazotrophic species in the genus Herbaspirillum (e.g. H. frisingense, H. rubrisubalbicans and H. seropedicae) associate with several economically important crops in the family Poaceae, such as maize (Zea mays), Miscanthus, rice (Oryza sativa), sorghum (Sorghum bicolor) and sugarcane (Saccharum sp.), and can increase their growth and productivity by a number of mechanisms, including nitrogen fixation. Hence, the improvement and use of these plant growth-promoting bacteria could provide economic and environmental benefits. We review the colonization processes of host plants by Herbaspirillum spp., including histological aspects and molecular mechanisms involved in these interactions, which may be epiphytic, endophytic, and even occasionally pathogenic. Herbaspirillum can recognize plant signals that modulate the expression of colonization traits and plant growth-promoting factors. Although a large proportion of herbaspirilla remain rhizospheric and epiphytic, plant-associated species in this genus are noted for their ability to colonize the plant internal tissues. Endophytic colonization by herbaspirilla begins with the attachment of the bacteria to root surfaces, followed by colonization at the emergence points of lateral roots and penetration through discontinuities of the epidermis; this appears to involve bacterial envelope structures, such as lipopolysaccharide (LPS), exopolysaccharide (EPS), adhesins and the type three secretion system (T3SS), but not necessarily the involvement of cell wall-degrading enzymes. Intercellular spaces are then rapidly occupied, proceeding to colonization of xylem and the aerial parts of the host plants. The response of the host plant includes both the recognition of the bacteria as non-pathogenic and the induction of systemic resistance to pathogens. Phytohormone signaling cascades are also activated, regulating the plant development. This complex molecular communication between some Herbaspirillum spp. and plant hosts can result in plant growth-promotion.     

Root Colonization by Pseudomonas sp. DSMZ 13134 and Impact on the Indigenous Rhizosphere Bacterial Community of Barley

Over the last few decades, the ability of rhizosphere bacteria to promote plant growth has been considered to be of scientific, ecological, and economic interest. The properties and mechanisms of interaction of these root-colonizing bacteria have been extensively investigated, and plant protection agents that are based on these bacterial strains have been developed for agricultural applications. In the present study, the root colonization of barley by Pseudomonas sp. DSMZ 13134, that is contained in the commercially available plant protection agent Proradix®, was examined using the fluorescence in situ hybridization method with oligonucleotide probes and specific gfp-tagging of the inoculant strain in combination with confocal laser scanning microscopy. In the first phase of root colonization, the inoculant strain competed successfully with seed and soil-borne bacteria (including Pseudomonads) for the colonization of the rhizoplane. Pseudomonas sp. DSMZ 13134 could be detected in all parts of the roots, although it did not belong to the dominant members of the root-associated bacterial community. Gfp-tagged cells were localized particularly in the root hair zone, and high cell densities were apparent on the root hair surface. To investigate the impact of the application of Proradix® on the structure of the dominant root-associated bacterial community of barley, T-RFLP analyses were performed. Only a transient community effect was found until 3 weeks post-application.     

Development of a high-efficient transformation system of Bacillus pumilus strain DX01 to facilitate gene isolation via gfp-tagged insertional mutagenesis and visualize bacterial colonization of rice roots

A Tn5 transposition vector, pMOD-tet-egfp, was constructed and used for the random insertional mutagenesis of Bacillus pumilus. Various parameters were investigated to increase the transformation efficiency B. pumilus DX01 via Tn5 transposition complexes (transposome): bacterial growth phase, type of electroporation buffer, electric field strength, and recovery medium. Transformation efficiency was up to 3?×?10⁴?transformants/μg of DNA under the optimized electroporation conditions, and a total of 1,467 gfp-tagged transformants were obtained. Fluorescence-activated cell sorting analysis showed that all gfp-tagged bacterial cells expressed GFP, indicating that foreign DNA has been successfully integrated into the genome of B. pumilus and expressed. Finally, flanking DNA sequences were isolated from several transformants and colonization of rice roots by B. pumilus DX01 was also studied. The method developed here will be useful for creating an insertion mutant library of gram-positive bacteria, thus facilitating their molecular genetic and cytological studies.     

Colonization of Maize and Rice Plants by Strain Bacillus megaterium C4

Bacillus megaterium C4, a nitrogen-fixing bacterium, was marked with the gfp gene. Maize and rice seedlings were inoculated with the, GFP-labeled B. megaterium C4 and then grown in gnotobiotic condition. Observation by confocal laser scanning microscope showed that the GFP-labeled bacterial cells infected the maize roots through the cracks formed at the lateral root junctions and then penetrated into cortex, xylem, and pith, and that the bacteria migrated slowly from roots to stems and leaves. The bacteria were mainly located in the intercellular spaces, although a few bacterial cells were also present within the xylem vessels, root hair cells, epidermis, cortical parenchyma, and pith cells. In addition, microscopic observation also revealed clearly that the root tip in the zone of elongation and differentiation and the junction between the primary and the lateral roots were the two sites for the bacteria entry into rice root. Therefore, we conclude that this Gram-positive nitrogen-fixer has a colonization pattern similar to those of many Gram-negative diazotrophs, such as Azospirillun brasilense Yu62 and Azoarcus sp. As far as we know, this is the first detailed report of the colonization pattern for Gram-positive diazotrophic Bacillus.     

Metabolic footprint of epiphytic bacteria on Arabidopsis thaliana leaves

The phyllosphere, which is defined as the parts of terrestrial plants above the ground, is a large habitat for different microorganisms that show a high extent of adaption to their environment. A number of hypotheses were generated by culture-independent functional genomics studies to explain the competitiveness of specialized bacteria in the phyllosphere. In contrast, in situ data at the metabolome level as a function of bacterial colonization are lacking. Here, we aimed to obtain new insights into the metabolic interplay between host and epiphytes upon colonization of Arabidopsis thaliana leaves in a controlled laboratory setting using environmental metabolomics approaches. Quantitative nuclear magnetic resonance (NMR) and imaging high-resolution mass spectrometry (IMS) methods were used to identify Arabidopsis leaf surface compounds and their possible involvement in the epiphytic lifestyle by relative changes in compound pools. The dominant carbohydrates on the leaf surfaces were sucrose, fructose and glucose. These sugars were significantly and specifically altered after epiphytic leaf colonization by the organoheterotroph Sphingomonas melonis or the phytopathogen Pseudomonas syringae pv. tomato, but only to a minor extent by the methylotroph Methylobacterium extorquens. In addition to carbohydrates, IMS revealed surprising alterations in arginine metabolism and phytoalexin biosynthesis that were dependent on the presence of bacteria, which might reflect the consequences of bacterial activity and the recognition of not only pathogens but also commensals by the plant. These results highlight the power of environmental metabolomics to aid in elucidating the molecular basis underlying plant–epiphyte interactions in situ.     

Note: Colonization and invasion of leaves of the aquatic macrophyteCeratophyllum demersum L. by epiphytic bacteria

The colonization of leaves of the aquatic macrophyteCeratophyllum demersum L. by epiphytic bacteria, and the hypothesis that bacterial invasion causes leaf senescence, was studied using transmission and scanning electron microscopy and light microscopy. Population densities of epiphytic bacterial communities onCeratophyllum leaves were positively correlated with leaf age. Initial settlement of bacteria on young leaves appeared to favour the boundaries between epidermal cells. On older leaves, large populations of bacteria were present over the whole surface. One third of senescentCeratophyllum leaves examined by transmission electron microscopy showed signs of bacterial invasion. Of these, up to 54% of the leaf's epidermal cells contained bacteria. Areas of cell wall degradation were associated with invasive bacteria in senescent leaves. In healthy, nonsenescent leaves, no bacterial invasion was observed. These results suggest that epiphytic bacteria did not cause leaf senescence but probably colonized the internal tissues of leaves once senescence had occurred.     

In Situ Detection of High Levels of Horizontal Plasmid Transfer in Marine Bacterial Communities

Gene transfer of the conjugative plasmid pBF1 from Pseudomonas putida to indigenous bacteria in seawater was investigated with a detection system for gene transfer based on the green fluorescent protein (GFP) (C. Dahlberg et al., Mol. Biol. Evol. 15:385–390, 1998). pBF1 was tagged with the gfp gene controlled by a lac promoter which is down regulated in the donor cell by a chromosomal repressor (lacI^q). The plasmid donor cells (Pseudomonas putida KT2442) subsequently do not express gfp. Transfer to recipient strains lacking the repressor results in expression of gfp. The transconjugant can subsequently be detected by epifluorescence microscopy on a single-cell level. By using this method, transfer of pBF1::gfp and expression of the gfp gene were first shown to occur during nutrient-limiting conditions to several defined recipient bacteria in artificial seawater. Second, we measured transfer of pBF1 from P. putida to the marine bacterial community directly in seawater samples, on a single-cell level, without limiting the detection of gene transfer to the culturable fraction of bacteria. Plasmid transfer was detected on surfaces and in bulk seawater. Seawater bacteria with different morphologies were shown to receive the plasmid. Gene transfer frequencies of 2.3 × 10⁻⁶ to 2.2 × 10⁻⁴ transconjugants per recipient were recorded after 3 days of incubation.     

Green fluorescent protein as a visual marker in a p-nitrophenol degrading Moraxella sp.

The green fluorescent protein gene (gfp) was introduced into a p-nitrophenol-metabolizing strain of Moraxella sp. by chromosomal integration. The gfp-marked transformants, designated Moraxella sp. strains G21 and G25, exhibited green fluorescence under UV light. Molecular characterization by PCR and Southern hybridization showed the presence of gfp in both transformants. Both transformants and the parent strain degraded 720 μM of p-nitrophenol with nitrite release within 4 h after inoculation in minimal medium supplemented with yeast extract. Transformants degraded up to 1440 μM p-nitrophenol and mineralized about 60% of 720 μM p-nitrophenol, both in broth and in soil, to the same extent as the parent strain. Insertion of gfp did not adversely affect the expression of p-nitrophenol-degrading genes in the transformants. Survival studies indicated that individual green fluorescent colonies of transformants can be detected up to 2 weeks after inoculation in soil. These marked strains could be of value in studies on microbial survival in the environment.     

Bleeding sap and old wood are the two main sources of contamination of merging organs of vine plants by Xylophilus ampelinus, the causal agent of bacterial necrosis

The spatial distribution of vine plants contaminated by Xylophilus ampelinus, the agent responsible for bacterial necrosis, was studied over a 5-year period within two vineyards in the Cognac area. Both vineyards were planted with Vitis vinifera cv. Ugni blanc but were different in age and agronomic location. The emission of X. ampelinus in contaminated bleeding sap was observed during vine sprouting. Contaminated bleeding sap is an important source of inoculum for external contamination due to the high susceptibility of young merging shoots to the pathogen. X. ampelinus emission by bleeding sap was not affected by the age of the plants or the location of the vineyards. However, its emission was irregular with time, and it varied between two fruit canes from individual plants and between plants as well as between years. Moreover, the two vineyards appeared to be entirely contaminated. Consequently, the behavior of the pathogen is not predictable. The distribution of the pathogen inside vine plant organs was analyzed through the four growing seasons. The old wood was contaminated throughout the year and constituted a stock inoculum for endophytic contamination of crude sap during the winter and the spring. Despite the fact that most of the young green shoots were contaminated in May, X.ampelinus was not found in green shoots in June and September, refuting the hypothesis of an epiphytic life of the pathogen under natural conditions. Although all plants were entirely contaminated in both vineyards, symptoms were rare and were observed on different plants each year.     

The Role of Pigmentation, Ultraviolet Radiation Tolerance, and Leaf Colonization Strategies in the Epiphytic Survival of Phyllosphere Bacteria

Phenotypic mechanisms that enhance bacterial UVR survival typically include pigmentation and DNA repair mechanisms which provide protection from UVA and UVB wavelengths, respectively. In this study, we examined the contribution of pigmentation to field survival in Clavibacter michiganensis and evaluated differences in population dynamics and leaf colonization strategies. Two C. michiganensis pigment-deficient mutants were significantly reduced in UVA radiation survival in vitro; one of these mutants also exhibited reduced field populations on peanut when compared to the wild-type strain over the course of replicate 25-day experiments. The UVR-tolerant C. michiganensis strains G7.1 and G11.1 maintained larger epiphytic field populations on peanut compared to the UVR-sensitive C. michiganensis T5.1. Epiphytic field populations of C. michiganensis utilized the strategy of solar UVR avoidance during leaf colonization resulting in increased strain survival on leaves after UVC irradiation. These results further demonstrate the importance of UVR tolerance in the ability of bacterial strains to maintain population size in the phyllosphere. However, an examination of several bacterial species from the peanut phyllosphere and a collection of environmental Pseudomonas spp. revealed that sensitivity to UVA and UVC radiation was correlated in some but not all of these bacteria. These results underscore a need to further understand the biological effects of different solar wavelength groups on microbial ecology.