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.     

Monitoring the colonization of sugarcane and rice plants by the endophytic diazotrophic bacterium Gluconacetobacter diazotrophicus marked with gfp and gusA reporter genes

Aims: To evaluate the colonization process of sugarcane plantlets and hydroponically grown rice seedlings by Gluconacetobacter diazotrophicus strain PAL5 marked with the gusA and gfp reporter genes. Methods and Results: Sugarcane plantlets inoculated in vitro with PAL5 carrying the gfp::gusA plasmid pHRGFPGUS did not present green fluorescence, but β‐glucuronidase (GUS)‐stained bacteria could be observed inside sugarcane roots. To complement this existing inoculation methodology for micropropagated sugarcane with a more rapid colonization assay, we employed hydroponically grown gnotobiotic rice seedlings to study PAL5–plant interaction. PAL5 could be isolated from the root surface (10⁸ CFU g^?1) and from surface‐disinfected root and stem tissues (10⁴ CFU g^?1) of inoculated plants, suggesting that PAL5 colonized the internal plant tissues. Light microscopy confirmed the presence of bacteria inside the root tissue. After inoculation of rice plantlets with PAL5 marked with the gfp plasmid pHRGFPTC, bright green fluorescent bacteria could be seen colonizing the rice root surface, mainly at the sites of lateral root emergence, at root caps and on root hairs. Conclusion: The plasmids pHRGFPGUS and pHRGFPTC are valid tools to mark PAL5 and monitor the colonization of micropropagated sugarcane and hydroponic rice seedlings. Significance and Impact of the Study: These tools are of use to: (i) study PAL5 mutants affected in bacteria–plant interactions, (ii) monitor plant colonization in real time and (iii) distinguish PAL5 from other bacteria during the study of mixed inoculants.     

Studies on Endophytic Colonization Ability of Two Upland Rice Endophytes, <Emphasis Type="Italic">Rhizobium</Emphasis> sp. and <Emphasis Type="Italic">Burkholderia</Emphasis> sp., Using Green Fluorescent Protein Reporter

Colonization ability of the two endophytic bacteria, isolated from surface sterilized roots of upland cultivated rice viz., Rhizobium sp. and Burkholderia sp., was compared after genetically tagging them with a constitutively expressing green fluorescent protein gene (gfp/gusA). Confocal laser scanning microscopy (CLSM) of gnotobiotically grown seedlings of Narendradhan 97, inoculated with gfp/gusA-tagged endophytes, revealed that both Rhizobium sp. and Burkholderia sp. colonized the intercellular spaces in the root cortex when inoculated separately. Colonization by gfp/gusA-tagged Rhizobium sp. was severely inhibited when co-inoculated with an equal number (10⁶ cfu ml⁻¹) of wild type Burkholderia sp. Burkholderia sp. was a more aggressive endophytic colonizer of rice than Rhizobium sp. The potential of using gfp/gusA reporter and CLSM as tools in evaluating competitive ability of colonization among endophytes is demonstrated in this study.     

Enhancement of Chilling Resistance of Inoculated Grapevine Plantlets with a Plant Growth-Promoting Rhizobacterium, Burkholderia phytofirmans Strain PsJN

In vitro inoculation of Vitis vinifera L. cv. Chardonnay explants with a plant growth-promoting rhizobacterium, Burkholderia phytofirmans strain PsJN, increased grapevine growth and physiological activity at a low temperature. There was a relationship between endophytic bacterial colonization of the grapevine plantlets and their growth at both ambient (26°C) and low (4°C) temperatures and their sensitivities to chilling. The major benefits of bacterization were observed on root growth (11.8- and 10.7-fold increases at 26°C and 4°C, respectively) and plantlet biomass (6- and 2.2-fold increases at 26°C and 4°C, respectively). The inoculation with PsJN also significantly improved plantlet cold tolerance compared to that of the nonbacterized control. In nonchilled plantlets, bacterization enhanced CO₂ fixation and O₂ evolution 1.3 and 2.2 times, respectively. The nonbacterized controls were more sensitive to exposure to low temperatures than were the bacterized plantlets, as indicated by several measured parameters. Moreover, relative to the noninoculated controls, bacterized plantlets had significantly increased levels of starch, proline, and phenolics. These increases correlated with the enhancement of cold tolerance of the grapevine plantlets. In summary, B. phytofirmans strain PsJN inoculation stimulates grapevine growth and improves its ability to withstand cold stress.     

Endophytic colonization of Vitis vinifera L. by Burkholderia phytofirmans strain PsJN: from the rhizosphere to inflorescence tissues

The colonization pattern of Vitis vinifera L. by Burkholderia phytofirmans strain PsJN was determined using grapevine fruiting cuttings with emphasis on putative inflorescence colonization under nonsterile conditions. Two-week-old rooted plants harbouring flower bud initials, grown in nonsterile soil, were inoculated with PsJN:gfp2x. Plant colonization was subsequently monitored at various times after inoculation with plate counts and epifluorescence and/or confocal microscopy. Strain PsJN was chronologically detected on the root surfaces, in the endorhiza, inside grape inflorescence stalks, not inside preflower buds and flowers but rather as an endophyte inside young berries. Data demonstrated low endophytic populations of strain PsJN in inflorescence organs, i.e. grape stalks and immature berries with inconsistency among plants for bacterial colonization of inflorescences. Nevertheless, endophytic colonization of inflorescences by strain PsJN was substantial for some plants. Microscopic analysis revealed PsJN as a thriving endophyte in inflorescence organs after the colonization process. Strain PsJN was visualized colonizing the root surface, entering the endorhiza and spreading to grape inflorescence stalks, pedicels and then to immature berries through xylem vessels. In parallel to these observations, a natural microbial communities was also detected on and inside plants, demonstrating the colonization of grapevine by strain PsJN in the presence of other microorganisms.     

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.     

L-Tryptophan-dependent biosynthesis of indole-3-acetic acid (IAA) improves plant growth and colonization of maize by <Emphasis Type="BoldItalic">Burkholderia phytofirmans</Emphasis> PsJN

Burkholderia phytofirmans PsJN is a well-known plant growth-promoting bacterium that establishes rhizospheric and endophytic colonization in different plants. PsJN inoculation promotes growth of different horticultural crops. L-Tryptophan (L-TRP) application may further improve its effectiveness, due to substrate (L-TRP)-dependent inoculum (PsJN)-derived auxins in the rhizosphere. In the present study, the substrate (L-TRP)-dependent response of PsJN inoculation to maize growth and auxin biosynthesis was evaluated under pot conditions. In vitro auxin biosynthesis by PsJN was determined in the absence and presence of L-TRP, a physiological precursor of auxins. Surface-disinfected seeds were treated with peat-based inoculum and L-TRP solutions (10^?4 and 10^?5 M). Results revealed that L-TRP application and PsJN inoculation, when applied separately, significantly increased the growth parameters of maize compared to untreated control. However, PsJN inoculation supplemented with L-TRP (10^?5 M) gave the most promising results and significantly increased plant height, photosynthesis, chlorophyll content, root biomass and shoot biomass up to 18, 16, 45, 62 and 55 %, respectively, compared to the uninoculated control. Similarly, higher values of N, P and IAA content were observed with precursor (L-TRP)–inoculum (PsJN) interaction. The inoculant strain efficiently colonized maize seedlings and was recovered from the rhizosphere, root and shoot of plants. The results imply that substrate (L-TRP)-derived IAA biosynthesis in the rhizosphere by PsJN inoculation could be a useful approach for improving the growth, photosynthesis and nutrient content of maize plants.     

Involvement of Quinolinate Phosphoribosyl Transferase in Promotion of Potato Growth by a Burkholderia Strain

Burkholderia sp. strain PsJN stimulates root growth of potato explants compared to uninoculated controls under gnotobiotic conditions. In order to determine the mechanism by which this growth stimulation occurs, we used Tn5 mutagenesis to produce a mutant, H41, which exhibited no growth-promoting activity but was able to colonize potato plants as well as the wild-type strain. The gene associated with the loss of growth promotion in H41 was shown to exhibit 65% identity at the amino acid level to the nadC gene encoding quinolinate phosphoribosyltransferase (QAPRTase) in Ralstonia solanacearum. Complementation of H41 with QAPRTase restored growth promotion of potato explants by this mutant. Expression of the gene identified in Escherichia coli yielded a protein with QAPRTase activities that catalyzed the de novo formation of nicotinic acid mononucleotide (NaMN). Two other genes involved in the same enzymatic pathway, nadA and nadB, were physically linked to nadC. The nadA gene was cotranscribed with nadC as an operon in wild-type strain PsJN, while the nadB gene was located downstream of the nadA-nadC operon. Growth promotion by H41 was fully restored by addition of NaMN to the tissue culture medium. These data suggested that QAPRTase may play a role in the signal pathway for promotion of plant growth by PsJN.     

In situ localization of Paenibacillus polymyxa HKA-15 in roots and root nodules of soybean (Glycine max.L.)

Background and aims

Bacterial endophytes can colonize various plants and organs. However, endophytic bacteria (other than rhizobia) colonizing root nodules in legumes have been rarely analyzed. The present study aimed to examine the colonization and spread of gfp-tagged Paenibacillus polymyxa in soybean plants under gnobiotic conditions.

Methods

Inoculation with gfp-tagged Paenibacillus. polymyxa HKA ?15 alone and in combination with Bradyrhizobium japonicum were done on soybean seedlings. In situ localization was detected through confocal microscopy and PCR.

Results

Inoculation with P. polymyxa-gfp strain alone and in combination with B. japonicum DS-1 had a stimulatory effect on the plant growth. There was an increase in shoot (7.2 %) and root dry weights (14.5 %) when the two strains were co - inoculated over that of B. japonicum inoculation alone. In vivo simultaneous visualization using Confocal Laser Scanning Microscopy (CLSM) showed the localization of the gfp-tagged P. polymyxa cells in the root nodules and its spread in the root tissue, both tap as well as lateral roots. Systemic spread into aerial tissue did not occur as indicated by the absence of bacteria. CLSM observations of the presence of gfp-tagged P. polymyxa in the nodule and roots tissues was corroborated with PCR amplification of the gfp-gene from these tissues.

Conclusions

CLSM and PCR methods confirmed that P. polymyxa invades roots and root nodules of soybean, but the spread is restricted to root tissue only. The strain improves plant growth when inoculated singly or in combination with B. japonicum.     

Sites of gibberellin biosynthesis in pea seedlings

Potential sites of gibberellin biosynthesis in 10-day-old `Alaska' pea (Pisum sativum L.) seedlings were investigated using a cell-free ezyme system capable of incorporating [¹⁴C]-mevalonic acid into ent-kaurene. In peas, ent-kaurene is assumed to be a committed intermediate in the gibberellin biosynthetic pathway. Comparative results from enzyme assays using extracts from shoot tips, leaf blades, internodes, and root tips indicate that the highest capacity for ent-kaurene (and presumably gibberellin) synthesis is in those tissues with the greatest potential for growth. The highest rates were obtained with extracts prepared from the fifth (youngest) internode, the fourth (youngest) expanded leaf, and the shoot tip itself. This report represents the first direct evidence that the enzymes responsible for early stages in gibberellin biosynthesis occur in internode tissues with potential for rapid elongation.     

Ascending Migration of Endophytic Rhizobia, from Roots to Leaves, inside Rice Plants and Assessment of Benefits to Rice Growth Physiology

Rhizobia, the root-nodule endosymbionts of leguminous plants, also form natural endophytic associations with roots of important cereal plants. Despite its widespread occurrence, much remains unknown about colonization of cereals by rhizobia. We examined the infection, dissemination, and colonization of healthy rice plant tissues by four species of gfp-tagged rhizobia and their influence on the growth physiology of rice. The results indicated a dynamic infection process beginning with surface colonization of the rhizoplane (especially at lateral root emergence), followed by endophytic colonization within roots, and then ascending endophytic migration into the stem base, leaf sheath, and leaves where they developed high populations. In situ CMEIAS image analysis indicated local endophytic population densities reaching as high as 9 × 10¹⁰ rhizobia per cm³ of infected host tissues, whereas plating experiments indicated rapid, transient or persistent growth depending on the rhizobial strain and rice tissue examined. Rice plants inoculated with certain test strains of gfp-tagged rhizobia produced significantly higher root and shoot biomass; increased their photosynthetic rate, stomatal conductance, transpiration velocity, water utilization efficiency, and flag leaf area (considered to possess the highest photosynthetic activity); and accumulated higher levels of indoleacetic acid and gibberellin growth-regulating phytohormones. Considered collectively, the results indicate that this endophytic plant-bacterium association is far more inclusive, invasive, and dynamic than previously thought, including dissemination in both below-ground and above-ground tissues and enhancement of growth physiology by several rhizobial species, therefore heightening its interest and potential value as a biofertilizer strategy for sustainable agriculture to produce the world's most important cereal crops.     

Enhancement of chilling resistance of inoculated grapevine plantlets with a plant growth-promoting rhizobacterium, Burkholderia phytofirmans strain PsJN

In vitro inoculation of Vitis vinifera L. cv. Chardonnay explants with a plant growth-promoting rhizobacterium, Burkholderia phytofirmans strain PsJN, increased grapevine growth and physiological activity at a low temperature. There was a relationship between endophytic bacterial colonization of the grapevine plantlets and their growth at both ambient (26 degrees C) and low (4 degrees C) temperatures and their sensitivities to chilling. The major benefits of bacterization were observed on root growth (11.8- and 10.7-fold increases at 26 degrees C and 4 degrees C, respectively) and plantlet biomass (6- and 2.2-fold increases at 26 degrees C and 4 degrees C, respectively). The inoculation with PsJN also significantly improved plantlet cold tolerance compared to that of the nonbacterized control. In nonchilled plantlets, bacterization enhanced CO(2) fixation and O(2) evolution 1.3 and 2.2 times, respectively. The nonbacterized controls were more sensitive to exposure to low temperatures than were the bacterized plantlets, as indicated by several measured parameters. Moreover, relative to the noninoculated controls, bacterized plantlets had significantly increased levels of starch, proline, and phenolics. These increases correlated with the enhancement of cold tolerance of the grapevine plantlets. In summary, B. phytofirmans strain PsJN inoculation stimulates grapevine growth and improves its ability to withstand cold stress.     

Colonization pattern of plant root and leaf surfaces visualized by use of green-fluorescent-marked strain of <Emphasis Type="Italic">Methylobacterium suomiense</Emphasis> and its persistence in rhizosphere

The localization of bacterial cell, pattern of colonization, and survival of Methylobacterium suomiense CBMB120 in the rhizosphere of rice and tomato plants were followed by confocal laser scanning, scanning electron microscopy, and selective plating. M. suomiense CBMB120 was tagged with green fluorescent protein (gfp), and inoculation was carried out through seed source. The results clearly showed that the gfp marker is stably inherited and is expressed in planta allowing for easy visualization of M. suomiense CBMB120. The colonization differed in rice and tomato—intercellular colonization of surface-sterilized root sections was visible in tomato but not in rice. In both rice and tomato, the cells were visible in the substomatal chambers of leaves. Furthermore, the strain was able to compete with the indigenous microorganisms and persist in the rhizosphere of tomato and rice, assessed through dilution plating on selective media. The detailed ultra-structural study on the rhizosphere colonization by Methylobacterium put forth conclusively that M. suomiense CBMB120 colonize the roots and leaf surfaces of the plants studied and is transmitted to the aerial plant parts from the seed source.     

Stress resistance and C1 metabolism involved in plant colonization of a methanotroph Methylosinus sp. B4S

Methanotrophs are widespread and have been isolated from various environments including the phyllosphere. In this study, we characterized the plant colonization by Methylosinus sp. B4S, an α-proteobacterial methanotroph isolated from plant leaf. The gfp-tagged Methylosinus sp. B4S cells were observed to colonize Arabidopsis leaf surfaces by forming aggregates. We cloned and sequenced the general stress response genes, phyR, nepR and ecfG, from Methylosinus sp. B4S. In vitro analysis showed that the phyR expression level was increased after heat shock challenge, and phyR was shown to be involved in resistance to heat shock and UV light. In the phyllospheric condition, the gene expression level of phyR as well as mmoX and mxaF was found to be relatively high, compared with methane-grown liquid cultures. The phyR-deletion strain as well as the wild-type strain inoculated on Arabidopsis leaves proliferated at the initial phase and then gradually decreased during plant colonization. These results have shed light firstly on the importance of general stress resistance and C1 metabolism in methanotroph living in the phyllosphere.     

Concomitant colonization of nifH positive endophytic Burkholderia sp. in rice (Oryza sativa L.) promotes plant growth

Six diazotrophic bacteria were isolated from surface-sterilized roots of rice variety HUR-36, which is grown with very low or no inputs of nitrogen fertilizer. Out of six bacteria one isolate, RREM25, showed appreciable level of nitrogenase activity, IAA production, and Phosphate solubilization ability, and was further characterized with a view to exploiting its plant growth promoting activity. Based on 16S rRNA gene sequence analysis, this isolate was identified as Burkholderia cepacia. Diazotrophic nature of this particular isolate was confirmed by Western blot analysis of dinitrogenase reductase and amplification of nifH. Microscopic observation confirmed colonization of gfp/gusA-tagged RREM25 in the intercellular spaces of cortical as well as vascular zones of roots. Inoculation of RREM25 to rice plants resulted in significant increase in plant height, dry shoot and root weight, chlorophyll content, nitrogen content and nitrogenase activity. Plant growth promoting features suggest that this endophytic bacterium may be exploited in rice cultivation after a thorough and critical pathogenicity test.     

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.     

Root colonisation of Pseudomonas aeruginosa strain UPMP3 and induction of defence‐related genes in oil palm (Elaeis guineensis)

Pseudomonas aeruginosa strain UPMP3 labelled with β‐glucuronidase (gusA) and green fluorescent protein (gfp) by electrotransformation yielded ca 1 × 10⁷ transformants µg^?1 DNA. The data obtained from the dilution plate count showed that over 28 days both epiphytic and endophytic populations of P. aeruginosa strain UPMP3 increased from 5.76 log₁₀ [colony forming unit (CFU) + 1] g^?1 fresh weight (FW) to 8.19 log₁₀ (CFU + 1) g^?1 FW and 4.10 log₁₀ (CFU + 1) g^?1 FW to 6.23 (CFU + 1) g^?1 FW, respectively. Confocal laser scanning microscopic analysis of oil palm roots treated with gusA:gfp‐tagged P. aeruginosa strain UPMP3 showed intense root colonisation over the sampling period. The root surface colonisation by P. aeruginosa strain UPMP3 was followed by a second stage, characterised by cortical infection, and a third stage, which involves xylem ingression. The colonisation of oil palm roots by the gusA:gfp‐tagged strain was concentrated on root areas potentially rich in nutrients such as the elongation zones, ridges between epidermal cells and points of secondary adventitious root emergence. Different expression levels of defence‐related genes, namely, chitinase and β‐1,3‐glucanase in the strain UPMP3–host interaction recorded over 28 days, suggested the potential role of P. aeruginosa strain UPMP3 in triggering the defence mechanism in oil palm. This is the first report on root colonisation and upregulation of defence‐related genes on oil palm roots by P. aeruginosa strain UPMP3 and shows the potential of this strain to be used as a biocontrol agent in oil palm.