Other strains belonging to this species have been isolated from soils and rhizospheres. Cells are gram-negative, non-sporulating rods that grows in the presence of various sugars. The strain does not produce nitrate or nitrite. PsJN produces ACC deaminase activity probably contributing to the plant growth promoting abilities of the strain. It produces the quorum signaling compound 3-hydroxy-C8-homoserine lactone. Strain PsJN colonizes varios plants endophytically and in the rhizospheres and plant growth promotion have been reported with various plants such as potato, tomato, grapevine Frommel et al.
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Plant innate immunity serves as a surveillance system by providing the first line of powerful weapons to fight against pathogen attacks. Beneficial microorganisms and Microbial-Associated Molecular Patterns might act as signals to trigger this immunity. Burkholderia phytofirmans PsJN, a highly efficient plant beneficial endophytic bacterium, promotes growth in a wide variety of plants including grapevine.
Further, the bacterium induces plant resistance against abiotic and biotic stresses. However, no study has deciphered triggered-mechanisms during the tripartite interaction between grapevine, B.
Herein, we showed that in contrast with classical rhizobacteria, which are restricted in the root system and act through ISR, B. Nevertheless, considering the endophytic level of PsJN in leaves, the plant protection efficacy of B. The presence of PsJN modulated changes in leaf carbohydrate metabolism including gene expression, sugar levels, and chlorophyll fluorescence imaging after Botrytis challenge. Our findings indicated that protection induced by B.
Plants have to face a broad range of invading pathogens. In response, they can deploy a large set of defense responses including constitutive pre-existing physical and chemical barriers as well as an innate immunity activated after pathogen perception Zipfel, ; Boller and Felix, One of the earliest responses at the time of pathogen assault is the production of ROS, which plays a crucial role to restrain pathogen development through programmed cell death at the site of infection Torres et al.
The second stage of perception corresponds to the direct or indirect recognition of pathogen effectors by intracellular immune receptors leading to effector-triggered immunity ETI; Jones and Dangl, Pathogen attack not only affects plant defenses reactions but can also lead to changes in photosynthesis rates and consequently carbohydrates metabolism.
Indeed, during the resistance response, the production of defense-related compounds becomes the high priority of the plant leading to reduced photosynthetic rates until the end of the pathogen growth Rojas et al. The photosynthesis decreases through the infection process as a result of leaf metabolism perturbation attributed to sugar-mediated repression of photosynthetic gene expression Bonfig et al. Cell wall invertase Cw-Inv catalyzes the cleavage of the sucrose into glucose and fructose, and supply sink organs with carbohydrates, playing thus a crucial role in the regulation of carbohydrate partitioning Roitsch et al.
Additionally, starch reserves may also be converted to soluble sugars Chou et al. The use of plant growth-promoting rhizobacteria PGPR to induce plant resistance is one of the alternatives developed to protect crops against damages caused by various forms of stress Yang et al. Among the plant-growth promoting bacteria, Burkholderia phytofirmans strain PsJN is able to colonize a variety of genetically unrelated plants such as potato and tomato Conn et al. In addition to colonize grapevine tissues Compant et al.
In addition, during the interaction between B. Moreover, Trda et al. The endophytic presence of B. It has also been shown that this strain reduces damages caused by chilling in grapevine through a priming of plant defense responses and changes in primary metabolism, particularly an increase of soluble sugars concentration and an accumulation of proline Ait Barka et al. In addition, the bacterium improves tolerance against biotic stress as Verticillium sp. However, the mechanisms involved beyond the observed induced resistance are not elucidated.
To decipher the mechanisms induced by B. Plantlets of Vitis vinifera cv. Chardonnay clone were micro-propagated by nodal explants grown on 15 ml of agar medium in 25 mm-culture tubes as described by Ait Barka et al. For the inoculum preparation, conidia of B. Control and bacterized plantlets were then grown for an additional week before their transfer aseptically into sterile Magenta boxes containing 60 g of soil.
This protocol was used for measures of necrosis diameter. Leaves were then sampled at different time points after B. Leaves of control and root-bacterized plantlets collected 24, 48, 72 hpi with B. The mycelium development was then observed using 3D Keyence, France or epifluorescence microscope. To determine rhizoplane colonization of B. Leaves were surface sterilized with 0. The samples were then ground with 1 ml of PBS. Germ tube growth was observed 24 h after challenge using inverted light microscopy Leica, Wetzlar, Germany.
To test the effect of different soluble sugars sucrose, glucose, fructose on B. Germ tubes were observed by inverted light microscopy Leica, Wetzlar, Germany 24h later. Both experiments were repeated twice each in triplicates. The 4-week-old grapevine leaves of plantlets were sprayed with different concentration of B. The H 2 O 2 content was also evaluated according to Theocharis et al.
The absorbance of the solution was read at nm, and H 2 O 2 content was calculated according to a standard curve. Callose deposition was observed as described in Schenk et al. Detached leaves were collected at 24 h post infection with B. This experiment was repeated twice and each replicate consisted of six leaves.
For each sample, 50 mg of leaves were ground in liquid nitrogen. Identical thermal cycling conditions were used for all targets. For each experiment, PCR reactions were performed in duplicate and 3 independent experiments were analyzed.
GI is the gene of interest. EF1a and 60RSP are used as internal controls. Fresh leaves were collected at 24 h post infection with B. After three rinses 5 min each with the phosphate buffer containing 0. The samples were then dehydrated in an alcohol series, transferred to acetone, and finally, they were embedded in Araldite.
Transverse ultrathin sections 80 nm nominal thickness were cut Reichert Jung Ultracut E from the Araldite-embedded block and mounted on mesh copper grids. For this experiment, 5 leaves from five plants were used. The spectrophotometer was zeroed with the control blank , and absorbance was read at nm. The data were collected out of necrosis area. In order to test the capacity of B. Assays performed on detached leaves from bacterized plantlets inoculated with drops of B.
In addition, whole potted-plant infection was carried out to quantify the gray mold disease symptoms in control versus bacterized plants. Therefore, whole plants were sprayed with B. As for detached leaves, disease symptoms were significantly reduced in bacterized plants, confirming the protective impact of B. Further, fungal growth was monitored in planta at 2, 8, 24, 48, and 72 hpi by analyzing the transcript levels of the B. While no significant differences were observed between control and PsJN root-inoculated plants at 2, 8, and 24 hpi Supplementary Figure S1A , the Bc-Actin transcript level in bacterized plantlets was approximately fold and fold lower compared to non-bacterized plantlets at 48 and 72 hpi, respectively.
In order to monitor that the induced resistance of plantlets toward B. Results showed that no protection was conferred by the presence of E. These data clearly indicated that the significantly enhanced resistance toward Botrytis infection is related to the presence of B.
Burkolderia phytofirmans PsJN protects grapevine against Botrytis cinerea. A Lesion diameter in detached leaves of plantlets inoculated or not with B. B Grapevine vitroplants inoculated or not with B. Chardonnay 24, 48, 72, and 96 hpi with B. Arrows indicate drops of B. In addition, B. As shown in Supplementary Figure S1, the development of the fungus was moderately reduced in bacterized plantlets at 24 hpi compared to non-bacterized ones. However, fungal hyphae growth was clearly inhibited in bacterized plantlets 72 hpi with B.
Interestingly, while PsJN was not observed at the leaves surface in the absence of the pathogen, the bacteria were detected at the surface, surrounding the fungal mycelium in botrytized leaves Figure 2. Interaction between B. Microscopic observations of infected grapevine leaves root-inoculated or not with B. Observations were realized with a microscope 3D and an epifluorescence microscope, respectively. Representative pictures of three independent experiments are shown. Arrows indicate B.
To test if the bacterium could act via an antimicrobial effect, we estimated its effect on fungal spore germination and also the ability of B. For spore germination assay, the conidial concentration was adjusted to 5. Direct effect of B. A Effect of B. Germ tubes were observed by inverted light microscopy 24 h later. B Protection due to antifungal effect. Expression analysis of B. To establish the role of the direct antifungal effect of B.
The quantification of BcActin in leaves, as an indicator of the rate of fungal growth in planta , was then realized 2, 24, 48, and 72 hpi with B. Our results showed a net dose dependent impact of B.
H 2 O 2 production is an important part of grapevine defense system Boubakri et al. No significant H 2 O 2 production was observed in response to bacterium or fungus inoculation Figure 4A. However, when bacterized plantlets were inoculated with Botrytis , the H 2 O 2 production was primed.
In control and bacterized plants without the pathogen, no H 2 O 2 generation was visualized except in veins Figures 4B,C , which may probably correspond to the lignification process.
Burkholderia Phytofirmans PsJN Acclimates Grapevine to Cold by Modulating Carbohydrate Metabolism
A Gram-negative, non-sporulating, rod-shaped, motile bacterium, with a single polar flagellum, designated strain PsJN T , was isolated from surface-sterilized onion roots. This isolate proved to be a highly effective plant-beneficial bacterium, and was able to establish rhizosphere and endophytic populations associated with various plants. Seven related strains were recovered from Dutch soils. Analysis of whole-cell protein profiles and DNA-DNA hybridization experiments confirmed that all eight strains belonged to a single species. Qualitative and quantitative differences in fatty acid composition between strain PsJN T and closely related species were identified. Isolate PsJN T showed high 1-aminocyclopropanecarboxylate deaminase activity and is therefore able to lower the ethylene level in a developing or stressed plant. Production of the quorum-sensing signal compound 3-hydroxy-C8-homoserine lactone was detected.
The use of plant-associated bacteria has received many scientific and economic attention as an effective and alternative method to reduce the chemical pesticides use in agriculture. The genus Burkholderia includes at least 90 species including pathogenic strains, plant pathogens, as well as plant beneficial species as those related to Paraburkholderia , which has been reported to be associated with plants and exerts a positive effect on plant growth and fitness. Paraburkholderia phytofirmans PsJN, a beneficial endophyte able to colonize a wide range of plants, is an established model for plant-associated endophytic bacteria. Indeed, in addition to its plant growth promoting ability, it can also induce plant resistance against biotic as well as abiotic stresses. Here, we summarized an inventory of knowledge on PsJN-plant interaction, from the perception to the resistance mechanisms induced in the plant by a way of the atypical colonization mode of this endophyte. We also have carried out an extensive genome analysis to identify all gene clusters which contribute to the adaptive mechanisms under different environments and partly explaining the high ecological competence of P. Historically, plant diseases have been controlled by the application of chemical pesticides, commonly leading to residual contamination and pathogen resistance Couderchet,
Low temperatures damage many temperate crops, including grapevine, which, when exposed to chilling, can be affected by symptoms ranging from reduced yield up to complete infertility. We have previously demonstrated that Burkholderia phytofirmans PsJN, a plant growth-promoting rhizobacteria PGPR that colonizes grapevine, is able to reduce chilling-induced damage. We hypothesized that the induced tolerance may be explained at least partly by the impact of bacteria on grapevine photosynthesis or carbohydrate metabolism during cold acclimation. Bacterization affects photosynthesis in a non-stomatal dependent pattern and reduced long-term impact of chilling on P n.