Pantoea stewartii subsp. stewartii: lessons learned from a xylem-dwelling pathogen of sweet corn

Pantoea stewartii subsp. stewartii is a Gram-negative enteric bacterium that primarily infects sweet corn. Studies of this bacterium have provided useful insight into how xylem-dwelling bacteria establish themselves and incite disease in their hosts. Pantoea stewartii subsp. stewartii is a remarkable bacterial system for laboratory studies because of its relative ease of propagation and genetic manipulation, and the fact that it appears to employ a minimal number of pathogenicity mechanisms. In addition, P. stewartii subsp. stewartii produces copious amounts of its quorum sensing (QS) signal, acyl-homoserine lactone (AHL), making it an excellent organism for studying QS-controlled gene regulation in a plant-pathogenic bacterium. In fact, P. stewartii subsp. stewartii has become the microbial paradigm for QS control of gene expression by both repression and activation via a QS regulator that binds DNA in the absence and dissociates in the presence of the signal ligand. Moreover, P. stewartii subsp. stewartii is a member of the Enterobacteriaceae, and lessons learned from its interaction with plants may be extrapolated to other plant-associated enterics, such as Erwinia, Dickeya and Pectobacterium spp., or enteric human pathogens associated with plants, such as Escherichia coli and Salmonella spp. TAXONOMY: Bacteria; Gammaproteobacteria; family Enterobacteriaceae; genus Pantoea; species stewartii (Mergaert et al., 1993). MICROBIOLOGICAL PROPERTIES: Gram-negative, motile, yellow pigmented, mucoid, facultative anaerobe. HOST RANGE: Pantoea stewartii subsp. stewartii (Smith, 1898) Dye causes Stewart's wilt of corn (Zea mays). Early-maturing sweet corn varieties and some elite inbred maize lines are particularly susceptible. DISEASE SYMPTOMS: There are two major phases of Stewart's wilt disease: (i) wilt and (ii) leaf blight. The wilt phase occurs when young seedlings are infected with P. stewartii subsp. stewartii (Fig. 1A). Water-soaked lesions first appear on the young expanding leaves and, later, seedlings may become severely wilted (Fig. 1B). The plants usually die when infected at the seedling stage. The leaf blight phase occurs when mature plants are infected (Fig. 1C). The bacteria enter the xylem and cause long linear yellow-grey lesions with a wavy margin that run parallel to the leaf veins. These lesions later turn necrotic and dark in colour. The leaf blight phase is most apparent after tasselling and does not generally cause death of the plant. In addition, the bacteria can sometimes break out of the xylem and cause pith rot in mature sweet corn plants. In resistant varieties, lesions are usually limited to only a few centimetres depending on the level of resistance of the particular hybrid (Claflin, 2000; Pataky, 2003). USEFUL WEBSITES: http://www.apsnet.org/publications/apsnetfeatures/Pages/StewartsWilt.aspx.     

Bacteria in the plant tissue culture environment

Bacteria and plants are joined in various symbiotic relationships that have developed over millennia and have influenced the evolution of both groups. Bacteria inhabit the surfaces of most plants and are also present inside many plant organs. These bacteria may have positive, neutral or negative impacts on their plant hosts. Probiotic effects may improve plant nutrition or increase resistance to biotic and abiotic stresses. Conversely pathogenic bacteria may kill or reduce the vigor of plant hosts. In addition some bacteria inhabit plants and profit from excess metabolites or shelter while not injuring the plant. Micropropagation of plants is based on the stimulation of organogenesis or embryogenesis from explants that are superficially decontaminated and placed into a sterile environment. If successful, this process removes bacteria from surfaces, but those inhabiting inner tissues and organs are usually not affected by these steriliants. In vitro conditions are designed for optimal plant growth and development, however these conditions are also often ideal for bacterial multiplication. The presence of bacteria in the in vitro environment was almost universally considered negative for plant culture, but more recently this view has been questioned. Certain bacteria appear to have a beneficial effect on the explants in culture; increasing multiplication and rooting, increasing explant quality, and organo- and embryogenesis of recalcitrant genotypes. The most important role of beneficial bacteria for micropropagated plants is likely to be during acclimatization, when growth is resumed under natural conditions. This review includes the role of bacterial interactions in plants, especially those grown in vitro.     

Bacterial leaf symbiosis in angiosperms: host specificity without co-speciation

Bacterial leaf symbiosis is a unique and intimate interaction between bacteria and flowering plants, in which endosymbionts are organized in specialized leaf structures. Previously, bacterial leaf symbiosis has been described as a cyclic and obligate interaction in which the endosymbionts are vertically transmitted between plant generations and lack autonomous growth. Theoretically this allows for co-speciation between leaf nodulated plants and their endosymbionts. We sequenced the nodulated Burkholderia endosymbionts of 54 plant species from known leaf nodulated angiosperm genera, i.e. Ardisia, Pavetta, Psychotria and Sericanthe. Phylogenetic reconstruction of bacterial leaf symbionts and closely related free-living bacteria indicates the occurrence of multiple horizontal transfers of bacteria from the environment to leaf nodulated plant species. This rejects the hypothesis of a long co-speciation process between the bacterial endosymbionts and their host plants. Our results indicate a recent evolutionary process towards a stable and host specific interaction confirming the proposed maternal transmission mode of the endosymbionts through the seeds. Divergence estimates provide evidence for a relatively recent origin of bacterial leaf symbiosis, dating back to the Miocene (5-23 Mya). This geological epoch was characterized by cool and arid conditions, which may have triggered the origin of bacterial leaf symbiosis.     

Evolutionary pathways of an ancient gene recX

RecX is a regulator of RecA activity by interacting with RecA protein or RecA filaments. Genes encoding RecX were found in genomes of a wide diversity of bacteria and some plants (e.g., Arabidopsis thaliana and Oryza sativa). Our comparative genome analysis showed that although members of the RecX family are found in many bacterial species, they are not found in archaea and the only gene found in eukaryotes is likely derived from bacteria genomes. It is therefore proposed that RecX is of bacterial origin, and the gene had presented in the common ancestor of bacteria. Moreover, bacterial RecX and plant RecX domain are homologues, and RecX domain in plants may have derived from bacteria via unknown pathways. Plant RecX-like protein was formed by a gene fusion event between a unique N-terminal domain of unknown origin and RecX domain within plant cells. Finally, three possible evolutionary pathways from bacteria to plant were discussed.     

Are drought-resistance promoting bacteria cross-compatible with different plant models?

The association between plant and plant growth promoting bacteria (PGPB) contributes to the successful thriving of plants in extreme environments featured by water shortage. We have recently shown that, with respect to the non-cultivated desert soil, the rhizosphere of pepper plants cultivated under desert farming hosts PGPB communities that are endowed with a large portfolio of PGP traits. Pepper plants exposed to bacterial isolates from plants cultivated under desert farming exhibited a higher tolerance to water shortage, compared with untreated control. This promotion was mediated by a larger root system (up to 40%), stimulated by the bacteria, that enhanced plant ability to uptake water from dry soil. We provide initial evidence that the nature of the interaction can have a limited level of specificity and that PGPB isolates may determine resistance to water stress in plants others than the one of the original isolation. It is apparent that, in relation to plant resistance to water stress, a feature of primary evolutionary importance for all plants, a cross-compatibility between PGPB and different plant models exists at least on a short-term.     

The Rhizobium--legume symbiosis.

The rhizobia are soil microorganisms that can interact with leguminous plants to form root nodules within which conditions are favourable for bacterial nitrogen fixation. Legumes allow the development of very large rhizobial populations in the vicinity of their roots. Infections and nodule formation require the specific recognition of host and Rhizobium, probably mediated by plant lectins. Penetration of the host by a compatible Rhizobium species usually provokes host root cell division to form the nodule, and a process of differentiation by both partners then ensues. In most cases the rhizobia alter morphologically to form bacteroids, which are usually larger than the free-living bacteria and have altered cell walls. At all stages during infection, the bacteria are bounded by host cell plasmalemma. The enzyme nitrogenase is synthesized by the bacteria and, if leghaemoglobin is present, nitrogen fixation will occur. Leghaemoglobin is a product of the symbiotic interaction, since the globin is produced by the plant while the haem is synthesized by the bacteria. In the intracellular habitat the bacteria are dependent upon the plant for supplies of energy and the bacteroids, in particular, appear to differentiate so that they are no longer able to utilize the nitrogen that they fix. Regulation of the supply of carbohydrate and the use of the fixed nitrogen thus appear to be largely governed by the host.     

Microfluidic devices for studying bacterial taxis,drug testing and biofilm formation

Some bacteria have coevolved to establish symbiotic or pathogenic relationships with plants, animals or humans. With human association, the bacteria can cause a variety of diseases. Thus, understanding bacterial phenotypes at the single-cell level is essential to develop beneficial applications. Traditional microbiological techniques have provided great knowledge about these organisms; however, they have also shown limitations, such as difficulties in culturing some bacteria, the heterogeneity of bacterial populations or difficulties in recreating some physical or biological conditions. Microfluidics is an emerging technique that complements current biological assays. Since microfluidics works with micrometric volumes, it allows fine-tuning control of the test conditions. Moreover, it allows the recruitment of three-dimensional (3D) conditions, in which several processes can be integrated and gradients can be generated, thus imitating physiological 3D environments. Here, we review some key microfluidic-based studies describing the effects of different microenvironmental conditions on bacterial response, biofilm formation and antimicrobial resistance. For this aim, we present different studies classified into six groups according to the design of the microfluidic device: (i) linear channels, (ii) mixing channels, (iii) multiple floors, (iv) porous devices, (v) topographic devices and (vi) droplet microfluidics. Hence, we highlight the potential and possibilities of using microfluidic-based technology to study bacterial phenotypes in comparison with traditional methodologies.     

An ecological perspective on bacterial biodiversity

Bacteria may be one of the most abundant and species-rich groups of organisms, and they mediate many critical ecosystem processes. Despite the ecological importance of bacteria, past practical and theoretical constraints have limited our ability to document patterns of bacterial diversity and to understand the processes that determine these patterns. However, recent advances in molecular techniques that allow more thorough detection of bacteria in nature have made it possible to examine such patterns and processes. Here, we review recent studies of the distribution of free-living bacterial diversity and compare our current understanding with what is known about patterns in plant and animal diversity. From these recent studies a preliminary picture is emerging: bacterial diversity may exhibit regular patterns, and in some cases these patterns may be qualitatively similar to those observed for plants and animals.     

Allelopathic Bacteria and Their Impact on Higher Plants

The impact of allelopathic, nonpathogenic bacteria on plant growth in natural and agricultural ecosystems is discussed. In some natural ecosystems, evidence supports the view that in the vicinity of some allelopathically active perennials (e.g., Adenostoma fasciculatum, California), in addition to allelochemicals leached from the shrub's canopy, accumulation of phytotoxic bacteria or other allelopathic microorganisms amplify retardation of annuals. In agricultural ecosystems allelopathic bacteria may evolve in areas where a single crop is grown successively, and the resulting yield decline cannot be restored by application of minerals. Transfer of soils from areas where crop suppression had been recorded into an unaffected area induced crop retardation without readily apparent symptoms of plant disease. Susceptibility of higher plants to deleterious rhizobacteria is often manifested in sandy or so-called skeletal soils. Evaluation of phytotoxic activity under controlled conditions, as well as ways to apply allelopathic bacteria in the field, is approached. The allelopathic effect may occur directly through the release of allelochemicals by a bacterium that affects susceptible plant(s) or indirectly through the suppression of an essential symbiont. The process is affected by nutritional and other environmental conditions, some may control bacterial density and the rate of production of allelochemicals. Allelopathic nonpathogenic bacteria include a wide range of genera and secrete a diverse group of plant growth-mediating allelochemicals. Although a limited number of plant growth-promoting bacterial allelochemicals have been identified, a considerable number of highly diversified growth-inhibiting allelochemicals have been isolated and characterized. Some species may produce more than one allelochemical; for example, three different phyotoxins, geldanamycin, nigericin, and hydanthocidin, were isolated from Streptomyces hygroscopicus. Efforts to introduce naturally produced allelochemicals as plant growth-regulating agents in agriculture have yielded two commercial herbicides, phosphinothricin, a product of Streptomyces viridochromogenes, and bialaphos from S. hygroscopicus. Many species of allelopathic bacteria that affect growth of higher plants are not plant specific, but some do exhibit specificity; for example, dicotyledonous plants were more susceptible to Pseudomonas putida than were monocotyledons. Differential susceptibility of higher plants to allelopathic bacteria was noted also in much lower taxonomical categories, at the subspecies level, in different cultivars of wheat, or of lettuce. Therefore, when test plants are employed to evaluate bacterial allelopathy, final evaluation must include those species that are assumed to be suppressed in nature. The release of allelochemicals from plant residues in plots of ‘continuous crop cultivation’ or from allelopathic living plants may induce the development of specific allelopathic bacteria. Both the rate by which a bacterium gains from its allelopathic activity through utilizing plant excretions, and the reasons for the developing of allelopathic bacteria in such habitats, are important goals for further research.     

Regulation of the type III secretion system in phytopathogenic bacteria

The type III secretion system (TTSS) is a specialized protein secretion machinery used by numerous gram-negative bacterial pathogens of animals and plants to deliver effector proteins directly into the host cells. In plant-pathogenic bacteria, genes encoding the TTSS were discovered as hypersensitive response and pathogenicity (hrp) genes, because mutation of these genes typically disrupts the bacterial ability to cause diseases on host plants and to elicit hypersensitive response on nonhost plants. The hrp genes and the type III effector genes (collectively called TTSS genes hereafter) are repressed in nutrient-rich media but induced when bacteria are infiltrated into plants or incubated in nutrient-deficient inducing media. Multiple regulatory components have been identified in the plant-pathogenic bacteria regulating TTSS genes under various conditions. In Ralstonia solanacearum, several signal transduction components essential for the induction of TTSS genes in plants are dispensable for the induction in inducing medium. In addition to the inducing signals, recent studies indicated the presence of negative signals in the plant regulating the Pseudomonas syringae TTSS genes. Thus, the levels of TTSS gene expression in plants likely are determined by the interactions of multiple signal transduction pathways. Studies of the hrp regulons indicated that TTSS genes are coordinately regulated with a number of non-TTSS genes.     

Microbial mediation of fruit fly–host plant interactions: is the host plant the “centre of activity”?

Insects utilize resources in their environment with the aid of mutualistic or symbiotic mediation by microorganisms. Some insect species such as ants and termites often have complex ecological and evolutionary associations with their symbionts, while the nature and functional significance of such associations in non-social insects is often unclear. In the Dacinae (Diptera: Tephritidae), specific Enterobacteriaceae ( Erwinia herbicola , Enterobacter cloacae , Klebsiella oxytoca ) are believed to mediate interactions between the adult fruit flies and the larval host plant. This bacterial mediation is hypothesized as being integral to the larval host plant being the "centre of activity" of the fly. Using a non-pest, monophagous fruit fly ( Bactrocera cacuminata [Hering]), we tested this hypothesis by manipulating the fruiting state of its larval host plant ( Solanum mauritianum Scopoli) and subsequently assessing insect behaviour and phylloplane microflora on those hosts. On host plants that had never fruited, few flies or bacterial colonies were recorded, consistent with hypothesis expectations. On fruiting host plants or plants that had had their fruit removed, bacterial colonies were present; again consistent with expectation. However, few flies were recorded on fruit-removed plants and all fly behaviours, other than resting or oviposition, were rare or absent on any hosts; inconsistent with expectation. The general pattern of results suggested that female flies coming to oviposit on fruiting hosts were spreading Enterobacteriaceae, but such spread was incidental and not part of some mutualistic interaction between fruit flies and bacteria.     

SHR5: a novel plant receptor kinase involved in plant-N2-fixing endophytic bacteria association

Endophytic nitrogen-fixing bacteria have been isolated from graminaceous plants such as maize, rice, and sugarcane. They are thought to promote plant growth, not only by fixing nitrogen, but also by the production of plant hormones. The molecular mechanisms involved in this interaction are not yet clear. In this work, the identification of a receptor-like kinase (RLK), named SHR5, which may participate in signal transduction involved in the establishment of plant-endophytic bacteria interaction is described for the first time. SHR5 seems to be part of a novel subclass of RLKs present in a wide range of plant species. The expression of this gene is down-regulated in sugarcane plants associated exclusively with beneficial endophytic bacteria and is not a general response caused by micro-organisms or abiotic stress. In addition, more successful sugarcane-endophytic bacteria associations have a more pronounced decrease in SHR5 expression, suggesting that SHR5 mRNA levels in plant cells are inversely related to the efficiency of the association.     

Single-plant,Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

Rhizobial bacteria form symbiotic, nitrogen-fixing nodules on the roots of compatible host legume plants. One of the most well-developed model systems for studying these interactions is the plant Medicago truncatula cv. Jemalong A17 and the rhizobial bacterium Sinorhizobium meliloti 1021. Repeated imaging of plant roots and scoring of symbiotic phenotypes requires methods that are non-destructive to either plants or bacteria. The symbiotic phenotypes of some plant and bacterial mutants become apparent after relatively short periods of growth, and do not require long-term observation of the host/symbiont interaction. However, subtle differences in symbiotic efficiency and nodule senescence phenotypes that are not apparent in the early stages of the nodulation process require relatively long growth periods before they can be scored. Several methods have been developed for long-term growth and observation of this host/symbiont pair. However, many of these methods require repeated watering, which increases the possibility of contamination by other microbes. Other methods require a relatively large space for growth of large numbers of plants. The method described here, symbiotic growth of M. truncatula/S. meliloti in sterile, single-plant microcosms, has several advantages. Plants in these microcosms have sufficient moisture and nutrients to ensure that watering is not required for up to 9 weeks, preventing cross-contamination during watering. This allows phenotypes to be quantified that might be missed in short-term growth systems, such as subtle delays in nodule development and early nodule senescence. Also, the roots and nodules in the microcosm are easily viewed through the plate lid, so up-rooting of the plants for observation is not required.     

Endophytic bacteria in toxic South African plants: identification, phylogeny and possible involvement in gousiekte

Background

South African plant species of the genera Fadogia, Pavetta and Vangueria (all belonging to Rubiaceae) are known to cause gousiekte (literally ‘quick disease’), a fatal cardiotoxicosis of ruminants characterised by acute heart failure four to eight weeks after ingestion. Noteworthy is that all these plants harbour endophytes in their leaves: nodulating bacteria in specialized nodules in Pavetta and non-nodulating bacteria in the intercellular spaces between mesophyll cells in Fadogia and Vangueria.

Principal Findings

Isolation and analyses of these endophytes reveal the presence of Burkholderia bacteria in all the plant species implicated in gousiekte. Although the nodulating and non-nodulating bacteria belong to the same genus, they are phylogenetically not closely related and even fall in different bacterial clades. Pavetta harborii and Pavetta schumanniana have their own specific endophyte – Candidatus Burkholderia harborii and Candidatus Burkholderia schumanniana – while the non-nodulating bacteria found in the other gousiekte-inducing plants show high similarity to Burkholderia caledonica. In this group, the bacteria are host specific at population level. Investigation of gousiekte-inducing plants from other African countries resulted in the discovery of the same endophytes. Several other plants of the genera Afrocanthium, Canthium, Keetia, Psydrax, Pygmaeothamnus and Pyrostria were tested and were found to lack bacterial endophytes.

Conclusions

The discovery and identification of Burkholderia bacteria in gousiekte-inducing plants open new perspectives and opportunities for research not only into the cause of this economically important disease, but also into the evolution and functional significance of bacterial endosymbiosis in Rubiaceae. Other South African Rubiaceae that grow in the same area as the gousiekte-inducing plants were found to lack bacterial endophytes which suggests a link between bacteria and gousiekte. The same bacteria are consistently found in gousiekte-inducing plants from different regions indicating that these plants will also be toxic to ruminants in other African countries.     

Modifications of Xanthomonas axonopodis pv. citri lipopolysaccharide affect the basal response and the virulence process during citrus canker

Xanthomonas axonopodis pv. citri (Xac) is the phytopathogen responsible for citrus canker, one of the most devastating citrus diseases in the world. A broad range of pathogens is recognized by plants through so-called pathogen-associated molecular patterns (PAMPs), which are highly conserved fragments of pathogenic molecules. In plant pathogenic bacteria, lipopolisaccharyde (LPS) is considered a virulence factor and it is being recognized as a PAMP. The study of the participation of Xac LPS in citrus canker establishment could help to understand the molecular bases of this disease. In the present work we investigated the role of Xac LPS in bacterial virulence and in basal defense during the interaction with host and non host plants. We analyzed physiological features of Xac mutants in LPS biosynthesis genes (wzt and rfb303) and the effect of these mutations on the interaction with orange and tobacco plants. Xac mutants showed an increased sensitivity to external stresses and differences in bacterial motilities, in vivo and in vitro adhesion and biofilm formation. Changes in the expression levels of the LPS biosynthesis genes were observed in a medium that mimics the plant environment. Xacwzt exhibited reduced virulence in host plants compared to Xac wild-type and Xacrfb303. However, both mutant strains produced a lower increase in the expression levels of host plant defense-related genes respect to the parental strain. In addition, Xac LPS mutants were not able to generate HR during the incompatible interaction with tobacco plants. Our findings indicate that the structural modifications of Xac LPS impinge on other physiological attributes and lead to a reduction in bacterial virulence. On the other hand, Xac LPS has a role in the activation of basal defense in host and non host plants.     

生防细菌产生的拮抗物质及其在生物防治中的作用

利用生防细菌防治植物病害是生物防治的一个主要内容．生防细菌防治植物病害发生发展的一个重要机制是产生拮抗物质．生防细菌的拮抗物质种类多,作用范围广谱．同一种拮抗物质可以由多种细菌菌株产生,而同一细菌也可以产生多种不同结构的拮抗物质．运用现代分子生物学技术和先进的分析测试手段可以加快对产生拮抗物质生防细菌的研究,了解生防细菌在寄主植物根围和叶围的定植效果。明确拮抗物质在生物防治中的作用．拮抗物质的产生除与细菌基因型有关外,一些外在的生物和非生物因素如病原菌存在与否、温度、pH和C、N营养等也影响拮抗物质产生．文中论述了生防细菌应用中存在的问题。指出混合菌剂的研制对防止病原菌抗性产生具有重要作用,应是今后生防菌剂研制中的重点．     

An Agrobacterium catalase is a virulence factor involved in tumorigenesis

Most plant pathogenic bacteria adopt the type III secretion systems to secrete virulence factors and/or avirulence gene products, which trigger the plant hypersensitive response (HR) and the oxidative burst with hydrogen peroxide (H2O2) as the main component. However, the soil-borne plant pathogen Agrobacterium tumefaciens uses the type IV secretion pathway to deliver its oncogenic T-DNA that causes crown gall tumours on many plant species. A. tumefaciens does not elicit a typical HR on those plants. Here, we report that inactivation of one of A. tumefaciens catalases (which converts H2O2 to H2O and O2) by a transposon insertion highly attenuated the bacterial ability to cause tumours on plants and to tolerate H2O2 toxicity, but not the bacterial viability in the absence of exogenous H2O2. This provides the first genetic evidence that the Agrobacterium-plant interaction involves a plant defence response, such as H2O2 production, and that catalase is a virulence factor for a plant pathogen.     

Decreasing prevalence of rhizosphere IAA producing and seedling root growth promoting bacteria with barley development irrespective of protozoan grazing regime

Barley was grown in soil with either bacteria and a mixed protozoan community (Mixed protozoa) or bacteria and a single vahlkampfiid amoebal species (Single amoeba). We assessed the influence of plant age (day 29, 43 and 57 after sowing) on two aspects of rhizosphere bacterial functioning: (1) the proportion of indole-3-acetic acid (IAA) producing bacteria and (2) the effect of mixed rhizosphere bacterial assemblages on barley seedling root growth in an agar based assay. The proportion of IAA producers was significantly lower at day 57 than at day 29 and 43, and mixed bacterial assemblages extracted from rhizospheres of 29 days old plants were significantly less harmful to seedling growth than bacterial assemblages from older plants. Hence both assays indicated that bacterial communities from rhizospheres of older plants were less beneficial for root growth than bacterial communities from younger plants. Genetic fingerprinting of rhizosphere bacterial communities was compared by use of length heterogeneity polymerase chain reaction (LH-PCR). This analysis showed a clear succession from the inoculum bacterial community with a rather low diversity to a community with much higher diversity at day 29. However, diversity did not change after day 29, and no relationship between protozoan treatment nor plant age and genetic fingerprinting was found. Responsible Editor: Petra Marschner     

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.