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PubMed2006

The dctA gene of Pseudomonas chlororaphis O6 is under RpoN control and is required for effective root colonization and induction of systemic resistance.

Transcription from the dctA gene, which encodes an organic acid transporter in the root-colonizing bacterium Pseudomonas chlororaphis O6, is under complex regulatory control. Promoter sequence analysis revealed an RpoN binding site. The regulation of transcript accumulation by the level of ammonium ions in the growth medium confirmed RpoN regulation, even in the presence of glucose. A dctA mutant colonized tobacco roots to a lesser extent than the wild-type mutant during early seedling development. Colonization by the dctA mutant, as compared to the wild type, also reduced the level of systemically induced resistance against the soft rot pathogen Erwinia carotovora SCC1. We ascribe this reduced colonization to the inability of the mutant to utilize certain organic acid components in the root exudates. The dctA mutant failed to grow on succinate and fumarate, and showed reduced growth on malate. All altered properties of the mutant were complemented by the full-length dctA gene. We propose that organic acids in root exudates may provide important nutrient sources for the beneficial root-colonizing pseudomonad.

Ammonium Chloride↗
PubMed2000

Antibiotic susceptibility of bacteria detected from the root canal exudate of persistent apical periodontitis.

Intracanal exudates in persistent endodontic cases were sampled for detecting bacteria using a preculture method, and the antibiotic susceptibility of the bacteria identified was examined. Twelve species were recovered from 15 cases. The species most commonly detected were alpha-Streptococcus and Enterococcus. The results of the antibiotic susceptibility tests revealed that Enterococcus was highly resistant to antibiotics tested, especially to cephalosporin products. Enterococcus is known as one of the bacteria that show multidrug resistance. In cases of prolonged endodontic treatment, a specific antibiotic prescription would play an important role for efficient treatment. Collecting data about antibiotic susceptibility could be helpful. Identification, however, take approximately 2 wk and a further study to develop the method that rapidly informs us about the antibiotic susceptibility that might be needed.

Anti-Bacterial Agents↗
PubMed2001

Selection of a plant-bacterium pair as a novel tool for rhizostimulation of polycyclic aromatic hydrocarbon-degrading bacteria.

We developed a novel procedure for the selection of a microbe-plant pair for the stable and efficient degradation of naphthalene. Based on the rationale that root exudate is the best nutrient source available in soil, the grass (Lolium multiflorum) cultivar Barmultra was selected because of its abilities to produce a highly branched root system, root deeply, and carry a high population of Pseudomonas spp. bacteria on its roots. Starting with a mixture of total rhizobacteria from grass-like vegetation collected from a heavily polluted site and selecting for stable naphthalene degradation as well as for efficient root colonization, Pseudomonas putida strain PCL1444 was isolated. The strain's ability to degrade naphthalene was shown to be stable in the rhizosphere. Moreover, it had superior root-colonizing properties because, after the inoculation of grass seedlings, it appeared to colonize the root tip up to 100-fold better than the efficient root colonizer Pseudomonas fluorescens WCS365. Strain PCL1444 uses root exudate as the dominant nutrient source because the presence of grass seedlings in soil results in up to a 10-fold increase of PCL1444 cells. Moreover, the root colonized by strain PCL1444 was able to penetrate through an agar layer, resulting in the degradation of naphthalene underneath this layer. In addition, the inoculation of grass seeds or seedlings with PCL1444 protected them against naphthalene phytotoxicity. Finally, this plant-microbe combination appeared able to degrade naphthalene from soil that was heavily polluted with a complex mixture of polycyclic aromatic hydrocarbons. To our knowledge, this is the first time that a naturally occurring bacterium has been selected for the combination of the abilities to degrade a pollutant and colonize plant roots. We suggest that the principle described here, to select a bacterium which combines efficient root colonization with a beneficial activity, also can be used to improve the selection of other more efficient plant-bacterium pairs for beneficial purposes such as biocontrol, biofertilization, and phytostimulation.

Biodegradation, Environmental↗
PubMed2001

Transcriptional responses in the hemiparasitic plant Triphysaria versicolor to host plant signals.

Parasitic plants in the Scrophulariaceae use chemicals released by host plant roots to signal developmental processes critical for heterotrophy. Haustoria, parasitic plant structures that attach to and invade host roots, develop on roots of the hemiparasitic plant Triphysaria versicolor within a few hours of exposure to either maize (Zea mays) root exudate or purified haustoria-inducing factors. We prepared a normalized, subtractive cDNA library enriched for transcripts differentially abundant in T. versicolor root tips treated with the allelopathic quinone 2,6-dimethoxybenzoquinone (DMBQ). Northern analyses estimated that about 10% of the cDNAs represent transcripts strongly up-regulated in roots exposed to DMBQ. Northern and reverse northern analyses demonstrated that most DMBQ-responsive messages were similarly up-regulated in T. versicolor roots exposed to maize root exudates. From the cDNA sequences we assembled a unigene set of 137 distinct transcripts and assigned functions by homology comparisons. Many of the proteins encoded by the transcripts are predicted to function in quinone detoxification, whereas others are more likely associated with haustorium development. The identification of genes transcriptionally regulated by haustorium-inducing factors provides a framework for dissecting genetic pathways recruited by parasitic plants during the transition to heterotrophic growth.

Benzoquinones↗
PubMed1999

Inducers of nod genes of Rhizobium ciceri.

Induction of nodABC genes of R. ciceri was studied by constructing nodABC-lacZ fusion. The root exudates of the homologous hosts induced the expression of nodABC genes but those of heterologous hosts failed to do so. The HPLC analysis of the root exudates of C. arietinum showed the presence of 6-7 compounds with retention times matching to flavonoids like naringenin, hesperetin, daidzein, naringin, 7 OH coumarin and luteolin. Induction studies using the standard flavonoids showed naringenin, followed by daidzein, as most potent inducer of the nodABC genes of R. ciceri. Naringenin in combination with daidzein showed a synergistic effect on the expression of nodABC genes.

Artificial Gene Fusion↗
PubMed2005

Root responses and metal accumulation in two contrasting ecotypes of Sedum alfredii Hance under lead and zinc toxic stress.

Sedum alfredii Hance has been reported to be a Zn-hyperaccumulator plant species. In this study, root morphological and physiological response of the hyperaccumulating ecotype of S. alfredii H. (HE) from the mined area and the non-hyperaccumulating ecotype of S. alfredii H. (NHE) from the agricultural area to supplied levels of Zn and Pb were investigated. The results showed that Zn concentrations in the leaves and the stems of the HE were 34 and 41 times higher, whereas lead concentrations were 1.9 and 2.4 times greater, respectively, than those of the NHE when grown at 1224 microM Zn and/or 200 microM Pb. At combined supply of 1224 microM Zn with 200 microM Pb, however, zinc concentrations in the stems and leaves of the, HE decreased, while lead concentrations in the stems increased significantly, as compared with those of single metal treatment. Lead uptake of the HE was enhanced by Zn addition. Root activity of the HE decreased by Pb treatment in the first two days, but recovered afterward and close to the control at day 10 of the treatment. However, root activity of the NHE decreased by each metal treatment, and was not recovered with the advance of treatment time. Root length, root surface area, and root volumes increased obviously due to Zn and/or Pb/Zn combined treatments for the HE, but significantly decreased due to Pb, Zn, or Pb/Zn combined treatment for the NHE. Zinc and Pb concentrations in both ecotypes of S. alfredii H. were positively correlated with root length, root surface area, and root volumes. Root exudates of the HE, especially treated with Zn, increased the extractability of Pb and Zn from the mined soil. At the Zn supply level of 1224microM, the extractability of root exudates on soil Pb was 3-12 times greater for the HE than for the NHE. These results imply that the tolerance and hyperaccumulation of the hyperaccumulating ecotype of S. alfredii H. to Zn and Pb appear to be closely related to its high adaptation of root growth, morphology, and physiology to Pb and Zn toxicity, and through its root excretion of some special substances that can activate Pb and Zn in the mined soil, thus increasing their mobilization and bioavailability.

Ecosystem↗
PubMed1975

Promotion of Sorghum Callus Growth by the s-Triazine Herbicides.

Growth-promoting action of simazine and other s-triazine herbicides was detected by the use of sorghum (Sorghum bicolor [L]. Moench) callus tissue and the chlorophyll retention test. Soil application of simazine [2-chloro-4, 6-bis(ethylamino)-s-triazine] at sublethal levels nearly doubled the growth-promoting action of sorghum root exudates. Treated plants yielded up to 26% more total protein than untreated plants. This indicated that the level of callus growth-promoting action in the root exudate of the plant has a positive effect on its final total protein yield and confirms a positive effect of simazine on total protein content in certain instances. The results may provide a new understanding of the mode of action of s-triazines applied at sublethal levels in increasing protein content and certain enzymic activities of treated plants. It is speculated that the growth-promoting action of these herbicides is hormonal in nature and most likely kinetin-like.

Journal Article↗
PubMed2005

[Surface chemical behavior of 2,4-dichlorophenol in presence of metal oxides].

To understand the surface chemistry of phenolic pollutants in the presence of metal oxides, this paper studied the reactions of 2,4-dichlorophenol with metal oxides in a kinetic and batch experiment. The results showed that amorphous ferric oxyhydroxide, goethite, delta-MnO2 and alpha-MnO2 were the potential oxidizing agents of natural and xenobiotic organic compounds. The oxidation rate of 2,4-DCP on the surface of these metal oxides was in order of delta-MnO2 >> alpha-MnO2 > iron oxides. It was observed from the pseudo-first-order kinetics that the reaction rates of manganese oxides were 3-50 times higher than those of iron oxides, and delta-MnO2 had a 1.5-3.2 times higher reaction rate than alpha-MnO2. Root exudates had a significant effect on the oxidation ability of metal oxides through changing the surface characteristics or involving in the reaction process. Upon the addition of ryegrass root exudates, the oxidation of 2,4-DCP was decreased in the suspension of alpha-MnO2 while increased in that of delta-MnO2. The coexistence of Cu probably influenced the oxidation process of 2,4-DCP on the surface of metal oxides. A greater negative effect of Cu on the oxidation of 2,4-DCP was also observed in the suspension of delta-MnO2 due to its strong adsorption toward Cu, as compared with that of alpha-MnO2.

Environmental Pollutants↗
PubMed2002

Isolation and characterization of a novel ribosome-inactivating protein from root cultures of pokeweed and its mechanism of secretion from roots.

Ribosome-inactivating proteins are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a novel type I ribosome-inactivating protein, termed PAP-H, was purified from Agrobacterium rhizogenes-transformed hairy roots of pokeweed (Phytolacca americana). The protein was purified by anion- and cation-exchange chromatography. PAP-H has a molecular mass of 29.5 kD as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its isoelectric point was determined to be 7.8. Yeast (Saccharomyces cerevisiae) ribosomes incubated with PAP-H released the 360-nucleotide diagnostic fragment from the 26S rRNA upon aniline treatment, an indication of its ribosome-inactivating activity. Using immunofluorescence microscopy, PAP-H was found to be located in the cell walls of hairy roots and root border cells. PAP-H was determined to be constitutively secreted as part of the root exudates, with its secretion enhanced by a mechanism mediated by ethylene induction. Purified PAP-H did not show in vitro antifungal activity against soil-borne fungi. In contrast, root exudates containing PAP-H as well as additional chitinase, beta-1,3-glucanase, and protease activities did inhibit the growth of soil-borne fungi. We found that PAP-H depurinates fungal ribosomes in vitro and in vivo, suggesting an additive mechanism that enables PAP-H to penetrate fungal cells.

Amino Acid Sequence↗
PubMed1990

Rhizobium meliloti nodD genes mediate host-specific activation of nodABC.

To differentiate among the roles of the three nodD genes of Rhizobium meliloti 1021, we studied the activation of a nodC-lacZ fusion by each of the three nodD genes in response to root exudates from several R. meliloti host plants and in response to the flavone luteolin. We found (i) that the nodD1 and nodD2 products (NodD1 and NodD2) responded differently to root exudates from a variety of hosts, (ii) that NodD1 but not NodD2 responded to luteolin, (iii) that NodD2 functioned synergistically with NodD1 or NodD3, (iv) that NodD2 interfered with NodD1-mediated activation of nodC-lacZ in response to luteolin, and (v) that a region adjacent to and upstream of nodD2 was required for NodD2-mediated activation of nodC-lacZ. We also studied the ability of each of the three R. meliloti nodD genes to complement nodD mutations in R. trifolii and Rhizobium sp. strain NGR234. We found (i) that nodD1 complemented an R. trifolii nodD mutation but not a Rhizobium sp. strain NGR234 nodD1 mutation and (ii) that R. meliloti nodD2 or nodD3 plus R. meliloti syrM complemented the nodD mutations in both R. trifolii and Rhizobium sp. strain NGR234. Finally, we determined the nucleotide sequence of the R. meliloti nodD2 gene and found that R. meliloti NodD1 and NodD2 are highly homologous except in the C-terminal region. Our results support the hypothesis that R. meliloti utilizes the three copies of nodD to optimize the interaction with each of its legume hosts.

Amino Acid Sequence↗
PubMed2001

Overexpression of malate dehydrogenase in transgenic alfalfa enhances organic acid synthesis and confers tolerance to aluminum.

Al toxicity is a severe impediment to production of many crops in acid soil. Toxicity can be reduced through lime application to raise soil pH, however this amendment does not remedy subsoil acidity, and liming may not always be practical or cost-effective. Addition of organic acids to plant nutrient solutions alleviates phytotoxic Al effects, presumably by chelating Al and rendering it less toxic. In an effort to increase organic acid secretion and thereby enhance Al tolerance in alfalfa (Medicago sativa), we produced transgenic plants using nodule-enhanced forms of malate dehydrogenase and phosphoenolpyruvate carboxylase cDNAs under the control of the constitutive cauliflower mosaic virus 35S promoter. We report that a 1.6-fold increase in malate dehydrogenase enzyme specific activity in root tips of selected transgenic alfalfa led to a 4.2-fold increase in root concentration as well as a 7.1-fold increase in root exudation of citrate, oxalate, malate, succinate, and acetate compared with untransformed control alfalfa plants. Overexpression of phosphoenolpyruvate carboxylase enzyme specific activity in transgenic alfalfa did not result in increased root exudation of organic acids. The degree of Al tolerance by transformed plants in hydroponic solutions and in naturally acid soil corresponded with their patterns of organic acid exudation and supports the concept that enhancing organic acid synthesis in plants may be an effective strategy to cope with soil acidity and Al toxicity.

Adaptation, Physiological↗
PubMed2006

An axenic plant culture system for optimal growth in long-term studies.

The symbiotic co-evolution of plants and microbes leads to difficulties in understanding which of the two components is responsible for a given environmental response. Plant-microbe studies greatly benefit from the ability to grow plants in axenic (sterile) culture. Several studies have used axenic plant culture systems, but experimental procedures are often poorly documented, the plant growth environment is not optimal, and axenic conditions are not rigorously verified. We developed a unique axenic system using inert components that promotes plant health and can be kept sterile for at least 70 d. Crested wheatgrass (Agropyron cristatum cv. CDII) plants were grown in sand within flow-through glass columns that were positively pressured with filtered air. Plant health was optimized by regulating temperature, light level, CO2 concentration, humidity, and nutrients. The design incorporates several novel aspects, such as pretreatment of the sand with Fe, graduated sand layers to optimize the air-water balance of the root zone, and modification of a laminar flow hood to serve as a plant growth chamber. Adaptations of several sterile techniques were necessary for maintenance of axenic conditions. Axenic conditions were verified by plating and staining leachates as well as a rhizoplane stain. This system was designed to study nutrient and water stress effects on root exudates, but is useful for assessing a broad range of plant-microbe-environment interactions. Based on total organic C analysis, 74% of exudates was recovered in the leachate, 6% was recovered in the bulk sand, and 17% was recovered in the rhizosphere sand. Carbon in the leachate after 70 d reached 255 microg d(-1). Fumaric, malic, malonic, oxalic, and succinic acids were measured as components of the root exudates.

Agropyron↗
PubMed2001

Periplasmic PQQ-dependent glucose oxidation in free-living and symbiotic rhizobia.

The expression of the pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) of Rhizobium tropici CIAT899 and Sinorhizobium meliloti RCR2011 was investigated under different nutrient-limiting conditions in continuous cultures, under different conditions of phosphate availability, and in S. meliloti bacteroids. The presence of free PQQ in alfalfa root exudates has also been assayed. It was shown that apo-GDH or holoenzyme was actively synthesized by these rhizobia, with the concomitant production of gluconate from glucose, under certain environmental conditions. GDH activity was also detected in bacteroids from alfalfa root nodules inoculated with either S. meliloti RCR2011 or 102F34. It was also shown that free PQQ was present in root exudates of alfalfa, but its production is ascribed to the activity of Erwinia sp., a normal contaminant of these seeds.

Culture Media↗
PubMed2005

Flux and turnover of fixed carbon in soil microbial biomass of limed and unlimed plots of an upland grassland ecosystem.

The influence of liming on rhizosphere microbial biomass C and incorporation of root exudates was studied in the field by in situ pulse labelling of temperate grassland vegetation with (13)CO(2) for a 3-day period. In plots that had been limed (CaCO(3) amended) annually for 3 years, incorporation into shoots and roots was, respectively, greater and lower than in unlimed plots. Analysis of chloroform-labile C demonstrated lower levels of (13)C incorporation into microbial biomass in limed soils compared to unlimed soils. The turnover of the recently assimilated (13)C compounds was faster in microbial biomass from limed than that from unlimed soils, suggesting that liming increases incorporation by microbial communities of root exudates. An exponential decay model of (13)C in total microbial biomass in limed soils indicated that the half-life of the tracer within this carbon pool was 4.7 days. Results are presented and discussed in relation to the absolute values of (13)C fixed and allocated within the plant-soil system.

Biomass↗
PubMed1980

Evidence Suggesting Protozoan Predation on Rhizobium Associated with Germinating Seeds and in the Rhizosphere of Beans (Phaseolus vulgaris L.).

Changes in populations of microorganisms around germinating bean (Phaseolus vulgaris L.) seeds, in the rhizosphere of bean, and in a model rhizosphere were studied. Strains of Rhizobium phaseoli that were resistant to streptomycin and thiram were used, and as few as 300 R. phaseoli cells per g of soil could be enumerated with a selective medium that was devised. A direct role was not evident for bacterial competitors, lytic bacteria, antibiotic-producing microorganisms, bacteriophages, and Bdellovibrio in the suppression of R. phaseoli around germinating seeds and in the rhizosphere. Protozoa increased in numbers in the soil upon planting of the seeds. The extent of colonization of soil by R. phaseoli was inversely related to the presence of large numbers of bacteria and protozoa. Colonization of R. phaseoli was improved upon suppression of protozoa with thiram and also when the soil was amended with other protozoan inhibitors and mannitol to simulate seed and root exudation. The data support the view that the decrease in numbers of R. phaseoli is caused by an increase in protozoan predation, the protozoa increasing in number because they prey on bacteria that proliferate by using seed and root exudates as nutrients.

Journal Article↗
PubMed2003

[Phosphorus transfer between mixed poplar and black locust seedlings].

In this paper, the 32P radio-tracer technique was applied to study the ways of phosphorus transfer between poplar (Populus euramericana cv. 'I-214') and black locust (Robinia pseudoacacia). A five compartment root box (18 cm x 18 cm x 26 cm) was used for testing the existence of the hyphal links between the roots of two tree species when inoculated with vesicular-arbuscular (VA) mycorrhizal fungus (Glomus mosseae). Populus I-214 (donor) and Robinia pseudoacacia (receiver) were grown in two terminal compartments, separated by a 2 cm root-free soil layer. The root compartments were lined with bags of nylon mesh (38 microns) that allowed the passage of hyphae but not roots. The top soil of a mixed stand of poplar and black locust, autoclaved at 121 degrees C for one hour, was used for growing seedlings for testing. In 5 compartment root box, mycorrhizal root colonization of poplar was 34%, in which VA mycorrhizal fungus was inoculated, whereas 26% mycorrhizal root colonization was observed in black locust, the other terminal compartment, 20 weeks after planting. No root colonization was observed in non-inoculated plant pairs. This indicated that the mycorrhizal root colonization of black locust was caused by hyphal spreading from the poplar. Test of tracer isotope of 32P showed that the radioactivity of the treatment significantly higher than that of the control (P < 0.05), 14 days from the tracer applied, to 27 days after, when VA mycorrhizal fungus was inoculated in poplar root. Furthermore, mycorrhizal interconnections between the roots of poplar and black locust seedlings was observed in situ by binocular in root box. All these experiments showed that the hyphal links was formed between the roots of two species of trees inoculated by VA mycorrhizal fungus. Four treatments were designed according to if there were two nets (mesh 38 microns), 2 cm apart, between the poplar and black locust, and if the soil in root box was pasteurized. Most significant differences of radioactivity among four treatments appeared 44 days after feeding 32P, the radioactivity of the day was applied to estimating the contribution of the various possible transfer ways to the total amount of nutrient transfer. Level of 32P radioactivity was found to be significantly (P < 0.05) higher in leaves of the treatment of "no separated and soil non-pasteurized" (17.1 pulse.g-1.s-1) than in leaves of "net separated and soil non-pasteurized" (5.3 pulse.g-1.s-1), and also significantly higher in leaves of "no net separated and soil pasteurized" (11.5 pulse.g.s-1) than in leaves of "net separated and soil pasteurized" (2.3 pulse.g-1.s-1), and very significantly (P < 0.01) higher in leaves of "no net separated and soil non-pasteurized" than in leaves of "net separated and soil pasteurized", whereas the levels of 32P radioactivity were not significantly different between the other treatments. The results showed that root contact and root exudations were the main ways of phosphorus transfer between the two species and the amount of phosphorus transfer through these two ways accounted for 62% of the total. The activity of the microorganisms including VA mycorrhizal fungi and the interaction between the microorganisms and root contact and root exudations made up 38% of total amount of phosphorus. The effect of mycorrhizal hyphal links in the direct nutrient transfer between poplar and black locust through separate mesh (38 microns) was little.

Biological Transport↗
PubMed1985

The nodD gene of Rhizobium leguminosarum is autoregulatory and in the presence of plant exudate induces the nodA,B,C genes.

To analyse nod gene expression in Rhizobium leguminosarum, a broad host-range lacZ protein fusion vector was constructed. Two protein fusions, nodC-lacZ and nodD-lacZ, were used to measure the regulation of expression of the promoters of the nodA,B,C and the nodD transcripts by measuring the induced levels of beta-galactosidase activity in R. leguminosarum. In the absence of plant root exudate the nodD-lacZ hybrid was expressed but the nodC-lacZ hybrid was not. The expression of the nodD-lacZ hybrid was repressed in R. leguminosarum strains containing an intact cloned nodD gene indicating that the nodD gene is autoregulatory. The induction of the nodC-lacZ hybrid required both the nodD gene and a component present in plant root exudate. Therefore the nodD gene acts both as a repressor and as an activator of gene expression. The nodD gene is adjacent to nodA and transcribed divergently from nodA,B,C with only approximately 300 nucleotides between the coding regions of nodA and nodD. Within this intergenic region is a unique BclI site and, using nodC-lacZ or nodD-lacZ translational fusions with this BclI site as an end point, no induction of nodC-lacZ or nodD-lacZ was observed. Therefore the promoters of nodD and nodA,B,C overlap at least at this region, and the regulation of these overlapping promoters appears to be controlled by the nodD protein which becomes an activator only in the presence of a component from plant exudate.

Journal Article↗