Root colonization by phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 is essential for biocontrol of tomato foot and root rot

The phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicislycopersici. To test whether root colonization is required for biocontrol, mutants impaired in the known colonization traits motility, prototrophy for amino acids, or production of the site-specific recombinase, Sss/XerC were tested for their root tip colonization and biocontrol abilities. Upon tomato seedling inoculation, colonization mutants of strain PCL1391 were impaired in root tip colonization in a gnotobiotic sand system and in potting soil. In addition, all mutants were impaired in their ability to control tomato foot and root rot, despite the fact that they produce wild-type levels of phenazine-1-carboxamide, the antifungal metabolite previously shown to be required for biocontrol. These results show, for what we believe to be the first time, that root colonization plays a crucial role in biocontrol, presumably by providing a delivery system for antifungal metabolites. The ability to colonize and produce phenazine-1-carboxamide is essential for control of F. oxysporum f. sp. radicis-lycopersici. Furthermore, there is a notable overlap of traits identified as being important for colonization of the rhizosphere and animal tissues.     

Interactions in the tomato rhizosphere of two Pseudomonas biocontrol strains with the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici

The fungus Fusarium oxysporum f. sp. radicis-lycopersici causes foot and root rot of tomato plants, which can be controlled by the bacteria Pseudomonas fluorescens WCS365 and P. chlororaphis PCL1391. Induced systemic resistance is thought to be involved in biocontrol by P. fluorescens WCS365. The antifungal metabolite phenazine-1-carboxamide (PCN), as well as efficient root colonization, are essential in the mechanism of biocontrol by P. chlororaphis PCL1391. To understand the effects of bacterial strains WCS365 and PCL1391 on the fungus in the tomato rhizosphere, microscopic analyses were performed using different autofluorescent proteins as markers. Tomato seedlings were inoculated with biocontrol bacteria and planted in an F. oxysporum f. sp. radicis-lycopersici-infested gnotobiotic sand system. Confocal laser scanning microscope analyses of the interactions in the tomato rhizosphere revealed that i) the microbes effectively compete for the same niche, and presumably also for root exudate nutrients; ii) the presence of either of the two bacteria negatively affects infection of the tomato root by the fungus; iii) both biocontrol bacteria colonize the hyphae extensively, which may represent a new mechanism in biocontrol by these pseudomonads; and iv) the production of PCN by P. chlororaphis PCL1391 negatively affects hyphal growth and branching, which presumably affects the colonization and infecting ability of the fungus.     

Tomato seed and root exudate sugars: composition, utilization by Pseudomonas biocontrol strains and role in rhizosphere colonization

The role of tomato seed and root exudate sugars as nutrients for Pseudomonas biocontrol bacteria was studied. To this end, the major exudate sugars of tomato seeds, seedlings and roots were identified and quantified using high-performance liquid chromatographic (HPLC) analysis. Glucose, fructose and maltose were present in all studied growth stages of the plant, but the ratios of these sugars were strongly dependent on the developmental stage. In order to study the putative role of exudate sugar utilization in rhizosphere colonization, two approaches were adopted. First, after co-inoculation on germinated tomato seeds, the root-colonizing ability of the efficient root-colonizing P. fluorescens strain WCS365 in a gnotobiotic quartz sand-plant nutrient solution system was compared with that of other Pseudomonas biocontrol strains. No correlation was observed between the colonizing ability of a strain and its ability to use the major exudate sugars as the only carbon and energy source. Secondly, a Tn5lacZ mutant of P. fluorescens strain WCS365, strain PCL1083, was isolated, which is impaired in its ability to grow on simple sugars, including those found in exudate. The mutation appeared to reside in zwf, which encodes glucose-6-phosphate dehydrogenase. The mutant grows as well as the parental strain on other media, including tomato root exudate. After inoculation of germinated sterile tomato seeds, the mutant cells reached the same population levels at the root tip as the wild-type strain, both alone and in competition, indicating that the ability to use exudate sugars does not play a major role in tomato root colonization, despite the fact that sugars have often been reported to represent the major exudate carbon source. This conclusion is supported by the observation that the growth of mutant PCL1083 in vitro is inhibited by glucose, a major exudate sugar, at a concentration of 0.001%, which indicates that the glucose concentration in the tomato rhizosphere is very low.     

Surface colonization of lodgepole pine (Pinus contorta var. latifolia [Dougl. Engelm.]) roots by Pseudomonas fluorescens and Paenibacillus polymyxa under gnotobiotic conditions

Indirect immunofluorescence techniques and confocal scanning laser microscopy were used to identify rhizobacterial strains on the root surfaces of pine seedlings, which were grown from seeds under gnotobiotic conditions. Conifer plant growth promoting rhizobacterial strains Paenibacillus polymyxa L6 and Pw-2, and the forest soil isolate Pseudomonas fluorescens M20, were inoculated onto surface-disinfested pine seeds, singly, or in dual combinations: strains L6 + M20, or strains Pw-2 + M20. Segments containing particular root microsites (root tip, root hair zone, or areas of lateral root emergence) were sampled randomly from roots 7 or 13 weeks after inoculation, and the colonization of roots by each bacterium was observed. Root segments were also sampled from individual roots at six different points along the length of the root, and the qualitative colonization of younger areas, closer to the root tip, contrasted with that of older areas, closer to the root base. The ability of strain M20 to colonize root areas adjacent to sites of lateral root emergence improves in the presence of either P. polymyxa strain, while the ability of the P. polymyxa strains to colonize these areas was not affected. More rhizobacteria were also generally observed on younger root tissues than on areas closer to the root base.     

Flagella-driven chemotaxis towards exudate components is an important trait for tomato root colonization by Pseudomonas fluorescens

Motility is a major trait for competitive tomato root-tip colonization by Pseudomonas fluorescens. To test the hypothesis that this role of motility is based on chemotaxis toward exudate components, cheA mutants that were defective in flagella-driven chemotaxis but retained motility were constructed in four P. fluorescens strains. After inoculation of seedlings with a 1:1 mixture of wild-type and nonmotile mutants all mutants had a strongly reduced competitive root colonizing ability after 7 days of plant growth, both in a gnotobiotic sand system as well as in nonsterile potting soil. The differences were significant on all root parts and increased from root base to root tip. Significant differences at the root tip could already be detected after 2 to 3 days. These experiments show that chemotaxis is an important competitive colonization trait. The best competitive root-tip colonizer, strain WCS365, was tested for chemotaxis toward tomato root exudate and its major identified components. A chemotactic response was detected toward root exudate, some organic acids, and some amino acids from this exudate but not toward its sugars. Comparison of the minimal concentrations required for a chemotactic response with concentrations estimated for exudates suggested that malic acid and citric acid are among major chemo-attractants for P. fluorescens WCS365 cells in the tomato rhizosphere.     

The two-component colR/S system of Pseudomonas fluorescens WCS365 plays a role in rhizosphere competence through maintaining the structure and function of the outer membrane

Pseudomonas fluorescens strain PCL1210, a competitive tomato root tip colonization mutant of the efficient root colonizing wild type strain WCS365, is impaired in the two-component sensor-response regulator system ColR/ColS. Here we show that a putative methyltransferase/wapQ operon is located downstream of colR/colS and that this operon is regulated by ColR/ColS. Since wapQ encodes a putative lipopolysaccharide (LPS) phosphatase, the possibility was studied that the integrity of the outer membrane of PCL1210 was altered. Indeed, it was shown that mutant PCL1210 is more resistant to various chemically unrelated antibiotics which have to pass the outer membrane for their action. In contrast, the mutant is more sensitive to the LPS-binding antibiotic polymyxin B. Mutant PCL1210 loses growth in competition with its wild type when grown in tomato root exudate. Mutants in the methyltransferase/wapQ operon are also altered in their outer membrane permeability and are defective in competitive tomato root tip colonization. A model for the altered outer membrane of PCL1210 is discussed.     

Increased uptake of putrescine in the rhizosphere inhibits competitive root colonization by Pseudomonas fluorescens strain WCS365

Sequence analysis of the chromosomal Tn5lacZ flanking regions of the Pseudomonas fluorescens WCS365 competitive root colonization mutant PCL1206 showed that the Tn5lacZ is inserted between genes homologous to bioA and potF. The latter gene is the first gene of the potF1F2GHI operon, which codes for a putrescine transport system in Escherichia coli. The position of the Tn5lacZ suggests an effect on the expression of the pot operon. A mutation in the potF1 gene as constructed in PCL1270, however, had no effect on competitive root colonization. The rate of uptake of [1,4-14C]putrescine by cells of mutant PCL1206 appeared to be increased, whereas cells of strain PCL1270 were strongly impaired in the uptake of putrescine. Dansylation of tomato root exudate and subsequent thin-layer chromatography showed the presence of a component with the same Rf value as dansyl-putrescine, which was identified as dansyl-putrescine by mass spectrometric analyses. Other polyamines such as spermine and spermidine were not detected in the root exudate. Growth of mutant strains, either alone or in competition with the wild type, was tested in media containing putrescine, spermine, or spermidine as the sole nitrogen source. The results show that mutant PCL1206 is strongly impaired in growth on putrescine and slightly impaired on spermine and spermidine. The presence of the polyamines had a similar effect on the growth rate of strain PCL1270 in the presence of putrescine but a less severe effect in the presence of spermine and spermidine. We conclude that an increased rate of putrescine uptake has a bacteriostatic effect on Pseudomonas spp. cells. We have shown that putrescine is an important tomato root exudate component and that root-colonizing pseudomonads must carefully regulate their rate of uptake because increased uptake causes a decreased growth rate and, therefore, a decreased competitive colonization ability.     

Bacterial endophyte-mediated naphthalene phytoprotection and phytoremediation

Polyaromatic hydrocarbons (PAHs) are major and recalcitrant pollutants of the environment and their removal presents a significant problem. Phytoremediation has shown much promise in PAH removal from contaminated soil, but may be inhibited because the plant experiences phytotoxic effects from low-molecular-weight PAHs such as naphthalene. This paper describes the construction of a naphthalene-degrading endophytic strain designated Pseudomonas putida VM1441(pNAH7). This strain was found to be an efficient colonizer of plants, colonizing both the rhizosphere and interior root tissues. The inoculation of plants with P. putida VM1441(pNAH7) resulted in the protection of the host plant from the phytotoxic effects of naphthalene. When inoculated plants were exposed to naphthalene, both seed germination and plant transpiration rates were higher than those of the uninoculated controls. The inoculation of plants with this strain also facilitated higher (40%) naphthalene degradation rates compared with uninoculated plants in artificially contaminated soil.     

Do arbuscular mycorrhizal fungi play a role in the ability of rare plant species to colonize abandoned fields?

While some plant species colonize abandoned agricultural fields and dry grasslands with similar frequency (generalists), others are absent or underrepresented in abandoned fields (specialists). We tested if inoculation with dry grassland or abandoned field soil could improve specialist performance in an abandoned field and compared the effects of inoculation in the stage of sown seeds and transplanted seedlings. Arbuscular mycorrhizal fungi from abandoned field had higher root colonization potential. This could explain the higher performance of the sown specialists inoculated with the abandoned field inoculum compared to those inoculated with dry grassland inoculum. This difference disappeared when specialists were transplanted instead of sown. The results do not provide any support for higher performance of specialists inoculated with dry grassland inoculum. Transplantation, however, seems to be an efficient way to introduce specialists into the abandoned fields.     

Plant protection and rhizosphere colonization of barley by seed inoculated herbicide degrading Burkholderia (Pseudomonas) cepacia DBO1(pRO101) in 2,4-D contaminated soil

The 2,4-dichlorophenoxyacetic acid (2,4-D) degrading bacterium, Burkholderia cepacia (formerly Pseudomonas cepacia) DBO1(pRO101) was coated on non-sterile barley (Hordeum vulgare) seeds, which were planted in two non-sterile soils amended with varying amounts of 2,4-D herbicide. In the presence of 10 or 100 mg 2,4-D per kg soil B. cepacia DBO1(pRO101) readily colonized the root at densities up to 10⁷ CFU per cm root. In soil without 2,4-D the bacterium showed weak root colonization. The seeds coated with B. cepacia DBO1(pRO101) were able to germinate and grow in soils containing 10 or 100 mg kg^–1 2,4-D, while non-coated seeds either did not germinate or quickly withered after germination. The results suggest that colonization of the plant roots by the herbicide-degrading B. cepacia DBO1(pRO101) can protect the plant by degradation of the herbicide in the rhizosphere soil. The study shows that the ability to degrade certain pesticides should be considered, when searching for potential plant growth-promoting rhizobacteria. The role of root colonization by xenobiotic degrading bacteria is further discussed in relation to bioremediation of contaminated soils.     

The sss colonization gene of the tomato-Fusarium oxysporum f. sp. radicis-lycopersici biocontrol strain Pseudomonas fluorescens WCS365 can improve root colonization of other wild-type pseudomonas spp.bacteria

We show that the disease tomato foot and root rot caused by the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici can be controlled by inoculation of seeds with cells of the efficient root colonizer Pseudomonas fluorescens WCS365, indicating that strain WCS365 is a biocontrol strain. The mechanism for disease suppression most likely is induced systemic resistance. P. fluorescens strain WCS365 and P. chlororaphis strain PCL1391, which acts through the production of the antibiotic phenazine-1-carboxamide, were differentially labeled using genes encoding autofluorescent proteins. Inoculation of seeds with a 1:1 mixture of these strains showed that, at the upper part of the root, the two cell types were present as microcolonies of either one or both cell types. Microcolonies at the lower root part were predominantly of one cell type. Mixed inoculation tended to improve biocontrol in comparison with single inoculations. In contrast to what was observed previously for strain PCL1391, mutations in various colonization genes, including sss, did not consistently decrease the biocontrol ability of strain WCS365. Multiple copies of the sss colonization gene in WCS365 improved neither colonization nor biocontrol by this strain. However, introduction of the sss-containing DNA fragment into the poor colonizer P. fluorescens WCS307 and into the good colonizer P. fluorescens F113 increased the competitive tomato root tip colonization ability of the latter strains 16- to 40-fold and 8- to 16-fold, respectively. These results show that improvement of the colonization ability of wild-type Pseudomonas strains by genetic engineering is a realistic goal.     

The inoculation method affects colonization and performance of bacterial inoculant strains in the phytoremediation of soil contaminated with diesel oil

Plants in combination with microorganisms can remediate soils, which are contaminated with organic pollutants such as petroleum hydrocarbons. Inoculation of plants with degrading bacteria is one approach to improve remediation processes, but is often not successful due to the competition with resident microorganisms. It is therefore of high importance to address the persistence and colonization behavior of inoculant strains. The objective of this study was to determine whether the inoculation method (seed imbibement and soil inoculation) influences bacterial colonization, plant growth promotion and hydrocarbon degradation. Italian ryegrass was grown in non-sterilized soil polluted with diesel and inoculated with different alkane-degrading strains Pantoea sp. ITSI10, Pantoea sp. BTRH79 and Pseudomonas sp. MixRI75 individually as well as in combination. Inoculation generally had a beneficial effect on plant biomass production and hydrocarbon degradation, however, strains inoculated in soil performed better than applied by seed imbibement. Performance correlated with the colonization efficiency of the inoculated strains. The highest hydrocarbon degradation was observed in the treatment, in which all three strains were inoculated in combination into soil. Our study revealed that besides the degradation potential and competitive ability of inoculant strains the inoculation method plays an important role in determining the success of microbial inoculation.     

硅酸盐细菌NBT菌株在小麦根际定殖的初步研究

对硅酸盐细菌NBT菌株进行了耐药性标记,得到稳定的链霉素抗性标记菌NBT菌株,采用选择性培养基分离计数,通过琼脂平板和盆栽试验,研究了标记菌NBT在小麦根际的定殖动态及影响因素。结果表明,在灭菌土盆栽中,播种后9d左右NBT菌株在小麦根际的定殖水平达最高(1．4×10^8cfu·g^-1根土),播种后54d左右趋向稳定,NBT菌株细胞数量为2．4×10^3cfu·g^-1根土;未灭菌土盆栽中,播种后9d左右NBT菌株的定殖水平达最高(3．8×10^8cfu·g^-1根土),60d左右趋向稳定,菌数为3．1×10^3cfu·g^-1根土,牛物和非牛物因素对NBT菌株定殖小麦根系有影响。     

Ecology of a plant growth-promoting strain of Pseudomonas fluorescens colonizing the maize endorhizosphere in tropical soil

Pseudomonas fluorescens strain BR-5 stimulated the growth of maize in a natural soil and inhibited fungal root pathogens in vitro. Strain BR-5 was detected inside plant cells, indicating that it is able to colonize the endorhizosphere. No significant effect was detected on soil or ectorhizosphere microbial population after inoculation of strain BR-5 onto seeds.     

Biosurfactant-Mediated Biodegradation of Polycyclic Aromatic Hydrocarbons—Naphthalene

The present study is aimed at the naphthalene degradation with and without biosurfactant produced from Pseudomonas aeruginosa isolated from oil-contaminated soil. The present study was carried out to isolate the bacterial strains for the naphthalene degradation and also for biosurfactant production. The isolated strains were screened for their ability to degrade the naphthalene by the methods of optimum growth rate test and for the production of biosurfactants by cetyltrimethylammonium bromide, blood agar medium, and thin-layer chromatography. The present study also focused on the effect of biosurfactant for the degradation of naphthalene by isolate-1. Two bacterial strains were isolated and screened, one for biodegradation and another for biosurfactant production. The second organism was identified as Pseudomonas aeruginosa by 16S rRNA analysis. The purified biosurfactant reduces the surface tension of water and also forms stable emulsification with hexadecane and kerosene. The end product of naphthalene degradation was estimated as salicylic acid equivalent by spectrophotometric method. The results demonstrated that Pseudomonas aeruginosa has the potential to produce biosurfactant, which enhances the biodegradation of naphthalene. The study reflects the potential use of biosurfactants for an effective bioremediation in the management of contaminated soils.     

Colonization of Tomato Plants by Two Agrocin-Producing Strains of Agrobacterium tumefaciens

For a bacterium to be a successful biocontrol agent against crown gall disease, it must produce an effective agrocin specific for Agrobacterium tumefaciens and be able to colonize host plants efficiently. The colonization abilities of K84 and J73, successful and potential biocontrolling strains, respectively, were compared both in vivo and in vitro. Both strains produced fibrils attaching them to tomato root surfaces and had similar colonization efficiencies up to 14 days after inoculation. However, the ability of J73 to colonize plants for longer periods was significantly less than that of K84. Thus, the presence of fibrils is not sufficient to ensure colonization. No correlation was found between hydrophobicity and colonization.     

Nutrients Can Enhance the Abundance and Expression of Alkane Hydroxylase CYP153 Gene in the Rhizosphere of Ryegrass Planted in Hydrocarbon-Polluted Soil

Plant-bacteria partnership is a promising strategy for the remediation of soil and water polluted with hydrocarbons. However, the limitation of major nutrients (N, P and K) in soil affects the survival and metabolic activity of plant associated bacteria. The objective of this study was to explore the effects of nutrients on survival and metabolic activity of an alkane degrading rhizo-bacterium. Annual ryegrass (Lolium multiflorum) was grown in diesel-contaminated soil and inoculated with an alkane degrading bacterium, Pantoea sp. strain BTRH79, in greenhouse experiments. Two levels of nutrients were applied and plant growth, hydrocarbon removal, and gene abundance and expression were determined after 100 days of sowing of ryegrass. Results obtained from these experiments showed that the bacterial inoculation improved plant growth and hydrocarbon degradation and these were further enhanced by nutrients application. Maximum plant biomass production and hydrocarbon mineralization was observed by the combined use of inoculum and higher level of nutrients. The presence of nutrients in soil enhanced the colonization and metabolic activity of the inoculated bacterium in the rhizosphere. The abundance and expression of CYP153 gene in the rhizosphere of ryegrass was found to be directly associated with the level of applied nutrients. Enhanced hydrocarbon degradation was associated with the population of the inoculum bacterium, the abundance and expression of CYP153 gene in the rhizosphere of ryegrass. It is thus concluded that the combination between vegetation, inoculation with pollutant-degrading bacteria and nutrients amendment was an efficient approach to reduce hydrocarbon contamination.     

Growth response of Afzelia africana Sm. seedlings to ectomycorrhizal inoculation in a nutrient-deficient soil

 The growth and mineral nutrition responses of seedlings of two provenances of Afzelia africana Sm. from Senegal and Burkina Faso, inoculated with four ectomycorrhizal (ECM) fungi (Scleroderma spp. and an unidentified isolate) from the same regions were assessed in a pot experiment in a savanna ECM-propagule-free soil deficient in NPK. There was little variation in the ability of the different fungal species to colonize roots of either provenance of A. africana or to produce external hyphal in soil. Root colonization by ECM fungi and their hyphal development were not related to mineral nutrition or ECM dependency. Differences in P, N, Mg and Ca concentrations in the leaves of inoculated and non-inoculated Afzelia seedlings were not always associated with production of biomass. Only leaf K concentration increased in both provenances after ECM inoculation. However, the Burkina Faso provenance responded better to inoculation with the two fungal isolates than the Senegal provenance in terms of biomass production. This was due to stimulation of root dry weight of the Burkina Faso provenance. Therefore, the hypothesis arises that non-nutritional rather than nutritional effects explain the contribution of ECM inoculation to the growth of A. africana seedlings. Accepted: 27 April 1999     

Growth,root colonization and nutrient status of Helianthemum sessiliflorum Desf. inoculated with a desert truffle Terfezia boudieri Chatin

This study aims to investigate the effects of inoculation using Terfezia boudieri Chatin ascospores (ectomycorrhizal fungus) on growth, root colonization and nutrient status of Helianthemum sessiliflorum Desf. seedlings grown in pots on two-soil types (gypseous and sandy loam). Mycorrhizal seedlings had significantly increased their height and leaf number compared to non-mycorrhizal ones. Regardless of mycorrhizal inoculation treatments, the plants growing on gypseous soil showed higher growth as compared to sandy loam one. It appears that inoculation with T. boudieri changed root morphology, increasing branching of first-order lateral roots of H. sessiliflorum seedlings. The highest root mycorrhizal colonization was recorded in inoculated seedlings on sandy loam soil (89%) when compared to gypseous one (52%). N, P and K concentrations in mycorrhizal seedlings were significantly improved by fungal inoculation. It can be concluded that inoculation of H. sessiliflorum with T. boudieri increased growth attributes and improved plant nutritional status.