Populations of the spinach wilt pathogen, Fusarium oxysporum f. sp. spinaciae, in the root tissues, rhizosphere, and soil in the field.

Populations of Fusarium oxysporum f. sp. spinaciae in root tissues and rhizosphere soil of diseased spinach plants were higher than in the root tissues and rhizosphere soil of healthy plants. Populations in soil rhizosphere were higher than in nonrhizosphere soil. The fungus populations were very low in the root tissues of the nonsusceptible strawberry, broccoli, chinese cabbage, and mustard grown in the infested field. The populations were low at the beginning of the season, increased, and remained high during the summer, then dropped in the fall. The fungus populations ranged from 1600 to 2600 propagules/g in the top 10 cm of soil, declined sharply between 11 and 20 cm, and were nondetectable between 41 and 60 cm.     

Identification of QuiP, the product of gene PA1032, as the second acyl-homoserine lactone acylase of Pseudomonas aeruginosa PAO1

The relevance of the acyl homoserine lactone (acyl-HSL) quorum signals N-3-oxododecanoyl-homoserine lactone (3OC12HSL) and N-butanoyl-homoserine lactone to the biology and virulence of Pseudomonas aeruginosa is well investigated. Previously, P. aeruginosa was shown to degrade long-chain, but not short-chain, acyl-HSLs as sole carbon and energy sources (J. J. Huang, J.-I. Han, L.-H. Zhang, and J. R. Leadbetter, Appl. Environ. Microbiol. 69:5941-5949, 2003). A gene encoding an enzyme with acyl-HSL acylase activity, pvdQ (PA2385), was identified, but it was not required for acyl-HSL utilization. This indicated that P. aeruginosa encodes another acyl-HSL acylase, which we identify here. A comparison of total cell proteins of cultures grown with long-acyl acyl-HSLs versus other substrates implicated the involvement of a homolog of PvdQ, the product of gene PA1032, for which we propose the name QuiP. Transposon mutants of quiP were defective for growth when P. aeruginosa was cultured in medium containing decanoyl-HSL as a sole carbon and energy source. Complementation with a functional copy of quiP rescued this growth defect. When P. aeruginosa was grown in buffered lysogeny broth, constitutive expression of QuiP in P. aeruginosa led to decreased accumulations of the quorum signal 3OC12HSL, relative to the wild type. Heterologous expression of QuiP was sufficient to confer long-chain acyl-HSL acylase activity upon Escherichia coli. Examination of gene expression patterns during acyl-HSL-dependent growth of P. aeruginosa further supported the involvement of quiP in signal decay and revealed other genes also possibly involved. It is not yet known under which "natural" conditions quiP is expressed or how P. aeruginosa balances the expression of its quorum-sensing systems with the expression of its acyl-HSL acylase activities.     

Mannityl opine accumulation and exudation by transgenic tobacco

Three genes from the T_R region of pTi15955 were introduced into tobacco (Nicotiana tabacum L.) to direct the synthesis of the mannityl opines from hexose sugars and glutamine or glutamate. Opines were present in all tissue types tested and accumulated to levels of 100 to 150 micrograms per milligram dry weight in root, stem, and leaf tissues. Opine-producing plants appeared normal with respect to morphology and development. Transgenic plants grown for 60 days under sterile autotrophic conditions produced up to 540 micrograms of the mannityl opines per milligrams dry weight of tissue as root exudates. Opines were also detected in leaf and seed washes from soil-grown plants.     

Glutamate synthase isoforms in rice: immunological studies of enzymes in green leaf, etiolated leaf, and root tissues

Rabbit antiserum was raised against ferredoxin-dependent glutamate synthase (EC 1.4.7.1) purified from green leaves of Oryza sativa L. cv Delta. Ferredoxin-dependent glutamate synthase, detected in green leaf, etiolated leaf, and root tissues cross-reacted completely with the antiferredoxin glutamate synthase immunoglobulin G. In contrast, the immunoglobulin G did not cross-react with NADH-dependent (EC 1.4.1.14) and NADPH-dependent (EC 1.4.1.13) glutamate synthases found in nonphotosynthetic etiolated leaf and root tissues. In addition, ferredoxin-dependent glutamate synthase was separated and distinguished by its affinity to ferredoxin from NAD(P)H-dependent glutamate synthase on ferredoxin-Sepharose affinity chromatography. Based on the immunological studies, it is suggested that ferredoxin-dependent glutamate synthases in green leaf and etiolated leaf tissues are closely related proteins; in contrast, ferredoxin-dependent glutamate synthase in root tissue is a distinct protein from the leaf enzymes.     

Rapid acyl-homoserine lactone quorum signal biodegradation in diverse soils

Signal degradation impacts all communications. Although acyl-homoserine lactone (acyl-HSL) quorum-sensing signals are known to be degraded by defined laboratory cultures, little is known about their stability in nature. Here, we show that acyl-HSLs are biodegraded in soils sampled from diverse U.S. sites and by termite hindgut contents. When amended to samples at physiologically relevant concentrations, 14C-labeled acyl-HSLs were mineralized to 14CO2 rapidly and, at most sites examined, without lag. A lag-free turf soil activity was characterized in further detail. Heating or irradiation of the soil prior to the addition of radiolabel abolished mineralization, whereas protein synthesis inhibitors did not. Mineralization exhibited an apparent Km of 1.5 microM acyl-HSL, ca. 1,000-fold lower than that reported for a purified acyl-HSL lactonase. Under optimal conditions, acyl-HSL degradation proceeded at a rate of 13.4 nmol x h(-1) x g of fresh weight soil(-1). Bioassays established that the final extent of signal inactivation was greater than for its full conversion to CO2 but that the two processes were well coupled kinetically. A most probable number of 4.6 x 10(5) cells . g of turf soil(-1) degraded physiologically relevant amounts of hexanoyl-[1-14C]HSL to 14CO2. It would take chemical lactonolysis months to match the level of signal decay achieved in days by the observed biological activity. Rapid decay might serve either to quiet signal cross talk that might otherwise occur between spatially separated microbial aggregates or as a full system reset. Depending on the context, biological signal decay might either promote or complicate cellular communications and the accuracy of population density-based controls on gene expression in species-rich ecosystems.     

Metabolism of acyl-homoserine lactone quorum-sensing signals by Variovorax paradoxus

Acyl-homoserine lactones (acyl-HSLs) serve as dedicated cell-to-cell signaling molecules in many species of the class Proteobacteria. We have addressed the question of whether these compounds can be degraded biologically. A motile, rod-shaped bacterium was isolated from soil based upon its ability to utilize N-(3-oxohexanoyl)-L-homoserine lactone as the sole source of energy and nitrogen. The bacterium was classified as a strain of Variovorax paradoxus. The V. paradoxus isolate was capable of growth on all of the acyl-HSLs tested. The molar growth yields correlated with the length of the acyl group. HSL, a product of acyl-HSL metabolism, was used as a nitrogen source, but not as an energy source. Cleavage and partial mineralization of the HSL ring were demonstrated by using radiolabeled substrate. This study indicates that some strains of V. paradoxus degrade and grow on acyl-HSL signals as the sole energy and nitrogen sources. This study provides clues about the metabolic pathway of acyl-HSL degradation by V. paradoxus.     

Fate of polycyclic aromatic hydrocarbons (PAH) in the rhizosphere and mycorrhizosphere of ryegrass

Polycyclic aromatic hydrocarbons (PAH) can be degraded in the rhizosphere but may also interact with vegetation by accumulation in plant tissues or adsorption on root surface. Previous studies have shown that arbuscular mycorrhizal (AM) fungi contribute to the establishment and maintenance of plants in a PAH contaminated soil. We investigated the fate of PAH in the rhizosphere and mycorrhizosphere including biodegradation, uptake and adsorption. Experiments were conducted with ryegrass inoculated or not with Glomus mosseae P2 (BEG 69) and cultivated in pots filled with soil spiked with 5 g kg⁻¹ of anthracene or with 1 g kg⁻¹ of a mixture of 8 PAH in a growth chamber. PAH were extracted from root surfaces, root and shoot tissue and rhizosphere soil and were analysed by GC-MS. In both experiments, 0.006 – 0.11‰ of the initial extractable PAH concentration were adsorbed to roots, 0.003 – 0.16‰ were found in root tissue, 0.001‰ in shoot tissue and 36 – 66% were dissipated, suggesting that the major part of PAH dissipation in rhizosphere soil was due to biodegradation or biotransformation. With mycorrhizal plants, anthracene and PAH were less adsorbed to roots and shoot tissue concentrations were lower than with non mycorrhizal plants, which could contribute to explain the beneficial effect of AM fungi on plant survival in PAH contaminated soils. This revised version was published online in June 2006 with corrections to the Cover Date.     

Heritability of,and relationships between phosphorus and nitrogen concentration in shoot,stolon and root of white clover (Trifolium repens L.)

Ninety eight white clover genotypes were cloned and grown in pots at two levels of phosphorus (P) supply in soil. After harvest the nitrogen (N) and P content of shoot (leaf, petiole and unrooted stolon), stolon and root tissue was determined. Broad sense heritabilities for %N, %P, and proportion of total N or P in each tissue type were calculated. Heritabilities ranged from 0.22 to 0.68. They were generally higher for %P than %N; and higher in shoot and stolon tissue than root tissue for %P, %N, and proportion of N or P. Level of P in which plants were grown had little effect on heritability values. Genotypes from bred cultivars differed from those collected from hill country pastures for plant size, and partitioning of N and P to shoot, stolon and root. Relationships between plant characters were examined to determine the consequences of selection.     

The liberation of amino acids and reducing compounds by plant roots

Summary Drying and remoistening of different plants grown in sand and in a sand-soil mixture resulted in the liberation of appreciable amounts of amino acids and detectable amounts of reducing compounds. Small quantities of amino acids were produced even under normal conditions of growth. The amino acids produced could be utilized for growth by amino acid requiring bacteria isolated from soil.Peas grown under aseptic conditions also yielded small amounts of ninhydrin-positive substances. Wheat under similar conditions produced less of such materials but in addition liberated detectable amounts of a reducing compound.These results are discussed in relation to the incidence of amino acid requiring bacteria in the rhizosphere.Contribution No. 385     

The Ti plasmid of Agrobacterium tumefaciens harbors an attM-paralogous gene,aiiB, also encoding N-Acyl homoserine lactonase activity

The Agrobacterium tumefaciens C58 genome contains three putative N-acyl homoserine lactone (acyl-HSL) hydrolases, which are closely related to the lactonase AiiA of BACILLUS: When expressed in Escherichia coli, two of the putative acyl-HSL hydrolases, AttM and AiiB, conferred the ability to degrade acyl-HSLs on the host. In Erwinia strain 6276, the lactonases reduced the endogenous acyl-HSL level and the bacterial virulence in planta.     

Quorum-Sensing Signals and Quorum-Sensing Genes in Burkholderia vietnamiensis

Acyl-homoserine lactone (acyl-HSL) quorum sensing is common to many Proteobacteria including a clinical isolate of Burkholderia cepacia. The B. cepacia isolate produces low levels of octanoyl-HSL. We have examined an environmental isolate of Burkholderia vietnamiensis. This isolate produced several acyl-HSLs. The most abundant species was decanoyl-HSL. Decanoyl-HSL in B. vietnamiensis cultures reached concentrations in excess of 20 microM. We isolated a B. vietnamiensis DNA fragment containing a gene for the synthesis of decanoyl-HSL (bviI) and an open reading frame that codes for a putative signal receptor (bviR). A B. vietnamiensis bviI mutant did not produce detectable levels of decanoyl-HSL.     

Effect of alkaline and saline substrates on ABA contents,distribution and transport in plant roots

The distribution of the phytohormone abscisic acid (ABA) between plant and soil and within plants growing on an alkaline substrate has been studied in order to separate the true effect of high soil pH from any effects that might be a result of the high salinity normally observed in alkaline soils. Leaves of a range of plants grown in an alkaline and saline solid substrate (municipal solid waste incinerator bottom slag) exhibited higher ABA levels than leaves of control plants. In contrast, roots of most plants grown on alkaline and saline substrates, particularly those without an exodermis (various species of Fabaceae), had slightly lower than or comparable ABA contents to control roots. However, in corn roots (Zea maysL. cv. Garant FAO 240) which possess a well-developed exodermis, alkaline and saline conditions in the rhizosphere did not reduce the endogenous ABA concentration, because the leaching of ABA from corn roots into the rhizosphere was lower than that from Vicia faba (variety Dreifache Weisse) roots. ABA efflux from corn and Vicia roots into the soil solution was observed only during the first days of the experiments and thereafter became substantially decreased. Because the leaching of ABA from Vicia faba roots into the rhizosphere was higher than that from corn roots, the leaves of Vicia plants grown in alkaline soil at low salinity no longer exhibited an elevated ABA concentration. However, whilst the roots of corn plants grown on desalted slag retained ABA levels that were higher than those of the control, the ABA content of leaves was not significant higher than the controls. For this reason, root ABA retention must be enough to induce tolerance to alkalinity in corn plants and there is no need to implicate changes in ABA concentrations in the aerial parts of the plant as having a role in this tolerance. In alkaline soil substrates, considerable portions of the ABA synthesised in the roots leached out into the soil solution of the rhizosphere according to the anion trap concept. An exodermis substantially reduces this leakage. The transient nature of ABA efflux into the rhizosphere was a result of the fact that the salt stress itself was only a transient phenomenon due to a washout of salt by irrigation. The results match predictions of mathematical models describing the effect of alkaline pH on the distribution of abscisic acid within plants and between roots and the rhizosphere. Species that can retain root ABA in the face of its tendency to leach into the more alkaline compartment are able to tolerate these normally harmful sites. This revised version was published online in June 2006 with corrections to the Cover Date.     

Long-term hydraulic acclimation to soil texture and radiation load in cotton

The concept of root contact hypothesizes that the absorbing roots grown in sandy soil are only partially effective in water uptake. Co-ordination of water supply and demand in the plant requires that the capacity for water uptake from the soil should correspond to an operational rate of water loss from the leaves. To examine how the plant hydraulic system responds to variations in soil texture or evaporative demand through long-term acclimation, an experiment was carried on cotton plants (Gossypium herbaceum L.), where three grades of soil texture and three grades of evaporative demand were applied for the whole life cycle of the plants. Plants were harvested 50 and 90 d (fully grown) after sowing and root length and leaf area measured. At 90 d hydraulic conductance was measured as the ratio of sap flow (measured with sap flow sensors or gravimetrically) and water potential. Results showed that for plants grown at the same evaporative demand, those in sandy soil, where root-specific hydraulic conductance was low, developed more absorbing roots than those grown in heavy-textured soil, where root specific conductance was high. This resulted in the same leaf specific hydraulic conductance (1.8 × 10⁻⁴ kg s⁻¹ Mpa⁻¹ m⁻²) for all three soils. For plants grown in the same sandy soil, those subjected to strong evaporative demand developed more absorbing roots and higher leaf-specific hydraulic conductance than those grown under mild evaporative demand. It is concluded that when soil texture or atmospheric evaporative demand varies, plants co-ordinate their capacities for liquid phase and vapour phase water transport through long-term acclimation of the hydraulic system, or plastic morphological adaptation of the root/leaf ratio.     

Bacterial populations in the rhizosphere of tobacco plants producing the quorum-sensing signals hexanoyl-homoserine lactone and 3-oxo-hexanoyl-homoserine lactone

A tobacco line genetically modified to produce two N-acyl homoserine lactones and its non-transformed parental line were grown in non-sterile soil. Microbial populations inhabiting the bulk soil, and those colonizing the root system of the two tobacco lines, were analyzed using cultivation-independent (phospholipid fatty acid and denaturing gradient gel electrophoresis) and cultivation-based assays. The cell density of total cultivable bacteria, fluorescent pseudomonads, sporulated, and thermotolerant bacteria was also determined in a time-course experiment (15 weeks). A possible "rhizosphere effect" related to the development of the plant was seen. However, no dissimilarities in cell population densities or population ratios of the microbial groups were detected in the rhizosphere of the two plant lines. Similarly, bacterial communities that either produced N-acyl homoserine lactone or degraded the signal hexanoyl homoserine lactone were enumerated from the two plant lines. No noticeable differences were evidenced from one plant genotype to the other. Whilst the transgenic plants released detectable amounts of the quorum-sensing signal molecules and efficiently cross-talked with the surrounding microbial populations, the bias generated by these signals in the reported experimental conditions therefore appears to remain weak, if not non-existent.     

Phytoextraction of weathered p,p'-DDE by zucchini (Cucurbita pepo) and cucumber (Cucumis sativus) under different cultivation conditions

Previous studies have shown that zucchini (Cucurbita pepo) and cucumber (Cucumis sativus) under field conditions are good and poor accumulators, respectively, of persistent organic pollutants from soil. Here, each species was grown under three cultivation regimes: dense (five plants in 5 kg soil): nondense (one plant in 80 kg soil): and field conditions (two to three plants in approximately 789 kg soil). p,p'-DDE and inorganic element content in roots, stems, leaves, and fruit were determined. In addition. rhizosphere, near-root, and unvegetated soil fractions were analyzed for concentrations of 11 low-molecular-weight organic acids (LMWOA) and 14 water-extractable inorganic elements. Under field conditions, zucchini phytoextracted 1.3% of the weathered p,p'-DDE with 98% of the contaminant in the aerial tissues. Conversely, cucumber removed 0.09% of the p,p'-DDE under field conditions with 83% in the aerial tissues. Under dense cultivation, cucumber produced a fine and fibrous root system not observed in our previous experiments and phytoextracted 0.78% of the contaminant, whereas zucchini removed only 0.59% under similar conditions. However. cucumber roots translocated only 5.7% of the pollutant to the shoot system, while in zucchini 48% of the p,p'-DDE in the plant was present in the aerial tissue. For each species, the concentrations of LMWOA in soil increased with increasing impact by the root system both within a given cultivation regime (i.e., rhizosphere > near-root > unvegetated) and across cultivation regimes (i.e., dense > nondense > field conditions). Under dense cultivation, the rhizosphere concentrations of LMWOAs were significantly greater for cucumber than for zucchini; no species differences were evident in the other two cultivation regimes. To enable direct comparison across cultivation regimes, total in planta p,p'-DDE and inorganic elements were mass normalized or multiplied by the ratio of plant mass to soil mass. For cucumber, differences in total p,p'-DDE and inorganic element content among the cultivation regimes largely disappear upon mass normalization, indicating that greater uptake of both types of constituents in the dense condition is due to greater plant biomass per unit soil. Conversely, for zucchini the mass normalized content of p,p'-DDE and inorganic elements is up to two orders of magnitude greater under field conditions than under dense cultivation, indicating a unique physiological response of C. pepo in the field. The role of cultivation conditions and nutrient availability in controlling root morphology, organic acid exudation, and contaminant uptake is discussed.     

Chilli rhizosphere fungus Aspergillus spp. PPA1 promotes vegetative growth of cucumber (Cucumis sativus) plants upon root colonisation

A rhizosphere fungus was isolated from roots of chilli plants and identified as Aspergillus spp. PPA1. The fungus was tested for its ability to promote the growth of cucumber plants in a pot experiment. Cucumber seeds were sown in sterilised field soil amended with wheat grain inoculum (WGI) of PPA 1 at the rate of 0.5, 1 and 1.5% w/w, and plants were grown for 21 days in a net house. The treatment with PPA1 significantly increased shoot length, shoot fresh weight, shoot dry weight, root length, root fresh weight, root dry weight, plant length, leaf area and leaf chlorophyll content of cucumber plants compared to non-treated control. The growth promotion rate increased with the increasing concentration of inoculum of PPA1 applied to the soil. The fungus was re-isolated from the roots of cucumber plants at higher frequencies. These results suggest that Aspergillus spp. PPA1 is a root colonising plant-growth promoting fungus for cucumber.