Plant availability of trace elements in sewage sludge-treated soils: methodology¹

Synthetic root exudates were formulated based on the organic acid composition of root exudates derived from the rhizosphere of aseptically grown corn plants, pH of the rhizosphere, and the background chemical matrices of the soil solutions. The synthetic root exudates, which mimic the chemical conditions of the rhizosphere environment where soil-borne metals are dissolved and absorbed by plants, were used to extract metals from sewage-sludge treated soils 16 successive times. The concentrations of Zn, Cd, Ni, Cr, and Cu of the sludge-treated soil were 71.74, 0.21, 15.90, 58.12, and 37.44 mg kg-1, respectively. The composition of synthetic root exudates consisted of acetic, butyric, glutaric, lactic, maleic, propionic, pyruvic, succinic, tartaric, and valeric acids. The organic acid mixtures had concentrations of 0.05 and 0.1 mol L-1 -COOH. The trace elements removed by successive extractions may be considered representative for the availability of these metals to plants in these soils. The chemical speciation of the metals in the liquid phase was calculated; results showed that metals in sludge-treated soils were dissolved and formed soluble complexes with the different organic acid-based root exudates. The most reactive organic acid ligands were lactate, maleate, tartarate, and acetate. The inorganic ligands of chloride and sulfate played insignificant roles in metal dissolution. Except for Cd, free ions did not represent an important chemical species of the metals in the soil rhizosphere. As different metals formed soluble complexes with different ligands in the rhizosphere, no extractor, based on a single reagent would be able to recover all of the potentially plant-available metals from soils; the root exudate-derived organic acid mixtures tested in this study may be better suited to recover potentially plant-available metals from soils than the conventional extractors.

Brachiaria species affecting soil nitrification

Nitrification can lead to substantial losses of the applied N through nitrate leaching and N2O emission. The regulation of nitrification may be a strategy to improve fertilizer N recovery and increase its agronomic efficiency. The objective of this study was to evaluate the inhibiting capacity of nitrification in soil by Brachiaria species. The greenhouse experiment was conducted using pots with 10 dm³ of a Red Latosol sample. The treatments consisted of the cultivation of three forage species (Brachiaria brizantha, B. ruziziensis and B. decumbens) and four n rates (0, 100, 200, and 300 mg/pot), and the control (without plants). In the absence of the forage plants, all N fertilization levels raised the N-NO3- soil levels, as a result of nitrification. The mineralization of organic matter supplied much of the N requirement of the forage plants and nitrification was influenced in the rhizosphere of B. brizantha; however, this effect was not high enough to alter the N-NH4+ level in the total soil volume of the pot.

Influence of plant species on disease suppression by Pseudomonas fluorescens strain CHA0 with enhanced antibiotic production

Introduction of the recombinant cosmid pME3090 into Pseudomonas fluorescens strain CHAO, a good biocontrol agent of various diseases caused by soilborne pathogens, increased three- to five-fold the production of the antibiotic metabolites pyoluteorin (Pit) and 2,4-diacetylphlorogIucinol (Phi) in vitro. Strain CHAO/pME3090 also overproduced Pit and Phi in the rhizosphere of wheat infected or not infected with Pythium ultimum. The biocontrol activity of the wild-type and recombinant Straitis was compared using various plant pathogen-host combinations in a gnotobiotic system. Antibiotic overproduction affected neither the protection of wheat against P. ultimum and Gaeumannomyces graminis var. tritici nor the growth of wheat plants. In contrast, strain CHA0/pME3090 showed an increased capacity to protect cucumber against Fusarium oxysporum f. sp. cucumerinum and Phomopsis sclerotioides, compared with the wild-type strain CHAO, The antibiotic overproducing strain protected tobacco roots significantly better against Thielaviopsis basicola than the wild-type strain but drastically reduced the growth of tobacco plants and was also toxic to the growth of sweet com. On King's B agar and on malt agar, the recombinant strain CHA0/pME3090 inhibited all pathogens more than did the parental strain CHAO. Synthetic Pit and Phi were toxic to all fungi tested. Tobacco and sweet com were more sensitive to synthetic Pit and Phi than were cucumber and wheat. There was no correlation between the sensitivity of the pathogens to the synthetic antibiotics and the degree of disease suppression by strain CHAO pME3090. However, there was a correlation between the sensitivity of the plants and the toxicity of the recombinant strain. We conclude that the plant species rather than the pathogen determines whether cosmid pME3090 in P. fluorescens strain CHAO leads to improved disease suppression.

Role of gluconic acid production in the regulation of biocontrol traits of Pseudomonas fluorescens CHA0.

The rhizobacterium Pseudomonas fluorescens CHA0 promotes the growth of various crop plants and protects them against root diseases caused by pathogenic fungi. The main mechanism of disease suppression by this strain is the production of the antifungal compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT). Direct plant growth promotion can be achieved through solubilization of inorganic phosphates by the production of organic acids, mainly gluconic acid, which is one of the principal acids produced by Pseudomonas spp. The aim of this study was to elucidate the role of gluconic acid production in CHA0. Therefore, mutants were created with deletions in the genes encoding glucose dehydrogenase (gcd) and gluconate dehydrogenase (gad), required for the conversion of glucose to gluconic acid and gluconic acid to 2-ketogluconate, respectively. These enzymes should be of predominant importance for rhizosphere-colonizing biocontrol bacteria, as major carbon sources provided by plant root exudates are made up of glucose. Our results show that the ability of strain CHA0 to acidify its environment and to solubilize mineral phosphate is strongly dependent on its ability to produce gluconic acid. Moreover, we provide evidence that the formation of gluconic acid by CHA0 completely inhibits the production of PLT and partially inhibits that of DAPG. In the Deltagcd mutant, which does not produce gluconic acid, the enhanced production of antifungal compounds was associated with improved biocontrol activity against take-all disease of wheat, caused by Gaeumannomyces graminis var. tritici. This study provides new evidence for a close association of gluconic acid metabolism with antifungal compound production and biocontrol activity in P. fluorescens CHA0.

Effects of tillage systems on physical properties of a cohesive yellow argisol in the northern state of Espírito Santo, Brazil

Tillage systems are a key element of the technology of crop production, both with a view to crop yield and from the perspective of soil conservation and sustainability of the production system. The aim of this paper was to evaluate the effects of five tillage systems on the physical properties of a cohesive Yellow Argisol. The experiment was installed in the field on January 21, 2011 and lasted 260 days, in an area previously used as pasture with Brachiaria grass without liming or fertilization, but irrigated by a low pressure spray system. The treatments, in five replications and in a randomized block design, consisted of: 1) disk plow (twice) + disk harrow + ridge-furrow tillage (raising a ridge along the planting row), 135 days after transplanting (DP + RID); 2) disk plow (twice) + disk harrow (DP no RID); 3) subsoiler (SB); 4) disk plow (twice) + disk harrow + scarification with three shanks along the plant row (DP + SPR); and 5) disk plow (twice) + disk harrow + scarification with three shanks in the total area (DP + STA). In all tillage systems, furrows were mechanically opened for the papaya plants. After the treatments, the mechanical resistance to penetration was determined, followed by soil moisture, mean weight diameter (MWD), geometric mean diameter (GMD), bulk density (BD), macroporosity (Ma), microporosity (Mi), and number of fruits per plant. There were differences in penetration resistance (PR) between treatments. The subsoiler was more effective to decrease RP to a distance of 0.35 m from the plants, perpendicular to the plant row. The scarifier resulted in a lower PR than DP or SB, even at the depth of 0.40 m, and it was more effective at greater distances perpendicular to the plant. All tillage systems induced a PR between 2.0 and 3.0 MPa at the depth with the highest concentration of papaya tree roots (0-0.25 m), improving the physical conditions to this depth. There was no statistical difference among the treatments for BD, Ma, Mi, MWD, and GMD at a depth of 0.20 m. The disk plow changed the physical properties of the soil most intensely to a depth of 0.20 m. The use of scarification, reduced tillage with a forest subsoiler, or ridge-furrow tillage did not improve the physical properties in the rhizosphere. Reduced tillage with a forest subsoiler resulted in a lower number of fruits per plant than all other treatments, which did not differ from each other.

Soil nitrogen dynamics after Brachiaria desiccation

Brachiaria species, particularly B. humidicola, can synthesize and release compounds from their roots that inhibit nitrification, which can lead to changes in soil nitrogen (N) dynamics, mainly in N-poor soils. This may be important in crop-livestock integration systems, where brachiarias are grown together with or in rotation with grain crops. The objective of the present study was to determine whether this holds true in N-rich environments and if other Brachiaria species have the same effect. The soil N dynamics were evaluated after the desiccation of the species B. brizantha, B. decumbens, B. humidicola, and B. ruziziensis, which are widely cultivated in Brazil. The plants were grown in pots with a dystroferric Red Latosol in a greenhouse. Sixty days after sowing, the plants were desiccated using glyphosate herbicide. The plants and soil were analyzed on the day of desiccation and 7, 14, 21 and 28 days after desiccation. The rhizosphere soil of the grasses contained higher levels of organic matter, total N and ammonium than the non-rhizosphere soil. The pH was lowest in the rhizosphere of B. humidicola, which may indicate that this species inhibits the nitrification process. However, variations in the soil ammonium and nitrate levels were not sufficient to confirm the suppressive effect of B. humidicola. The same was observed for B. brizantha, B. decumbens and B. ruziziensis, thereby demonstrating that, where N is abundant, none of the brachiarias studied has a significant effect on the nitrification process in soil.

Using flow cytometry for in situ monitoring of antimicrobial compound production in the biocontrol bacterium Pseudomonas fluorescens CHA0

Pseudomonas fluorescens strain CHA0 is able to protect plants against a variety of pathogens, notably by producing the two antimicrobial compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT). The regulation of the expression of these compounds is affected by many biotic factors, such as fungal pathogens, rhizosphere bacteria as well as plant species. Therefore, the influence of some plant phenolic compounds on the expression of DAPG and PLT biosynthetic genes has been tested using GFP-based reporter, monitored by standard fluometry and flow cytometry. In situ experiments were also performed with cucumber plants. We found that several plant metabolites such as IAA and umbelliferone are able to modify significantly the expression of DAPG and PLT. The use of flow cytometry with autofluorescents proteins seems to be a promising method to study rhizobacteria-plant interactions.

Plants respond to pathogen infection by enhancing the antifungal gene expression of root-associated bacteria.

Plant health and fitness widely depend on interactions with soil microorganisms. Some bacteria such as pseudomonads can inhibit pathogens by producing antibiotics, and controlling these bacteria could help improve plant fitness. In the present study, we tested whether plants induce changes in the antifungal activity of root-associated bacteria as a response to root pathogens. We grew barley plants in a split-root system with one side of the root system challenged by the pathogen Pythium ultimum and the other side inoculated with the biocontrol strain Pseudomonas fluorescens CHA0. We used reporter genes to follow the expression of ribosomal RNA indicative of the metabolic state and of the gene phlA, required for production of 2,4-diacetylphloroglucinol, a key component of antifungal activity. Infection increased the expression of the antifungal gene phlA. No contact with the pathogen was required, indicating that barley influenced gene expression by the bacteria in a systemic way. This effect relied on increased exudation of diffusible molecules increasing phlA expression, suggesting that communication with rhizosphere bacteria is part of the pathogen response of plants. Tripartite interactions among plants, pathogens, and bacteria appear as a novel determinant of plant response to root pathogens.

Detection of plant-modulated alterations in antifungal gene expression in Pseudomonas fluorescens CHA0 on roots by flow cytometry.

The biocontrol activity of the root-colonizing Pseudomonas fluorescens strain CHA0 is largely determined by the production of antifungal metabolites, especially 2,4-diacetylphloroglucinol. The expression of these metabolites depends on abiotic and biotic environmental factors, in particular, elements present in the rhizosphere. In this study, we have developed a new method for the in situ analysis of antifungal gene expression using flow cytometry combined with green fluorescent protein (GFP)-based reporter fusions to the phlA and prnA genes essential for the production of the antifungal compounds 2,4-diacetylphloroglucinol and pyrrolnitrin, respectively, in strain CHA0. Expression of phlA-gfp and prnA-gfp in CHA0 cells harvested from the rhizosphere of a set of plant species as well as from the roots of healthy, leaf pathogen-attacked, and physically stressed plants were analyzed using a FACSCalibur. After subtraction of background fluorescence emitted by plant-derived particles and CHA0 cells not carrying the gfp reporters, the average gene expression per bacterial cell could be calculated. Levels of phlA and prnA expression varied significantly in the rhizospheres of different plant species. Physical stress and leaf pathogen infection lowered phlA expression levels in the rhizosphere of cucumber. Our results demonstrate that the newly developed approach is suitable to monitor differences in levels of antifungal gene expression in response to various plant-derived factors. An advantage of the method is that it allows quantification of bacterial gene expression in rhizosphere populations at a single-cell level. To our best knowledge, this is the first study using flow cytometry for the in situ analysis of biocontrol gene expression in a plant-beneficial bacterium in the rhizosphere.

Small RNA-dependent expression of secondary metabolism is controlled by Krebs cycle function in Pseudomonas fluorescens.

Pseudomonas fluorescens CHA0, an antagonist of phytopathogenic fungi in the rhizosphere of crop plants, elaborates and excretes several secondary metabolites with antibiotic properties. Their synthesis depends on three small RNAs (RsmX, RsmY, and RsmZ), whose expression is positively controlled by the GacS-GacA two-component system at high cell population densities. To find regulatory links between primary and secondary metabolism in P. fluorescens and in the related species Pseudomonas aeruginosa, we searched for null mutations that affected central carbon metabolism as well as the expression of rsmY-gfp and rsmZ-gfp reporter constructs but without slowing down the growth rate in rich media. Mutation in the pycAB genes (for pyruvate carboxylase) led to down-regulation of rsmXYZ and secondary metabolism, whereas mutation in fumA (for a fumarase isoenzyme) resulted in up-regulation of the three small RNAs and secondary metabolism in the absence of detectable nutrient limitation. These effects required the GacS sensor kinase but not the accessory sensors RetS and LadS. An analysis of intracellular metabolites in P. fluorescens revealed a strong positive correlation between small RNA expression and the pools of 2-oxoglutarate, succinate, and fumarate. We conclude that Krebs cycle intermediates (already known to control GacA-dependent virulence factors in P. aeruginosa) exert a critical trigger function in secondary metabolism via the expression of GacA-dependent small RNAs.

Salicylic Acid Biosynthetic Genes Expressed in Pseudomonas fluorescens Strain P3 Improve the Induction of Systemic Resistance in Tobacco Against Tobacco Necrosis Virus.

ABSTRACT Application of salicylic acid induces systemic acquired resistance in tobacco. pchA and pchB, which encode for the biosynthesis of salicylic acid in Pseudomonas aeruginosa, were cloned into two expression vectors, and these constructs were introduced into two root-colonizing strains of P. fluorescens. Introduction of pchBA into strain P3, which does not produce salicylic acid, rendered this strain capable of salicylic acid production in vitro and significantly improved its ability to induce systemic resistance in tobacco against tobacco necrosis virus. Strain CHA0 is a well-described biocontrol agent that naturally produces salicylic acid under conditions of iron limitation. Introduction of pchBA into CHA0 increased the production of salicylic acid in vitro and in the rhizosphere of tobacco, but did not improve the ability of CHA0 to induce systemic resistance in tobacco. In addition, these genes did not improve significantly the capacity of strains P3 and CHA0 to suppress black root rot of tobacco in a gnotobiotic system.

Contribution of the Global Regulator Gene gacA to Persistence and Dissemination of Pseudomonas fluorescens Biocontrol Strain CHA0 Introduced into Soil Microcosms.

Structural and regulatory genes involved in the synthesis of antimicrobial metabolites are essential for the biocontrol activity of fluorescent pseudomonads and, in principle, amenable to genetic engineering for strain improvement. An eventual large-scale release of such bacteria raises the question of whether such genes also contribute to the persistence and dissemination of the bacteria in soil ecosystems. Pseudomonas fluorescens wild-type strain CHA0 protects plants against a variety of fungal diseases and produces several antimicrobial metabolites. The regulatory gene gacA globally controls antibiotic production and is crucial for disease suppression in CHA0. This gene also regulates the production of extracellular protease and phospholipase. The contribution of gacA to survival and vertical translocation of CHA0 in soil microcosms of increasing complexity was studied in coinoculation experiments with the wild type and a gacA mutant which lacks antibiotics and some exoenzymes. Both strains were marked with spontaneous resistance to rifampin. In a closed system with sterile soil, strain CHA0 and the gacA mutant multiplied for several weeks, whereas these strains declined exponentially in nonsterile soil of different Swiss origins. The gacA mutant was less persistent in nonrhizosphere raw soil than was the wild type, but no competitive disadvantage when colonizing the rhizosphere and roots of wheat was found in the particular soil type and during the period studied. Vertical translocation was assessed after strains had been applied to undisturbed, long (60-cm) or short (20-cm) soil columns, both planted with wheat. A smaller number of cells of the gacA mutant than of the wild type were detected in the percolated water and in different depths of the soil column. Single-strain inoculation gave similar results in all microcosms tested. We conclude that mutation in a single regulatory gene involved in antibiotic and exoenzyme synthesis can affect the survival of P. fluorescens more profoundly in unplanted soil than in the rhizosphere.

Cross talk between 2,4-diacetylphloroglucinol-producing biocontrol pseudomonads on wheat roots.

The performance of Pseudomonas biocontrol agents may be improved by applying mixtures of strains which are complementary in their capacity to suppress plant diseases. Here, we have chosen the combination of Pseudomonas fluorescens CHA0 with another well-characterized biocontrol agent, P. fluorescens Q2-87, as a model to study how these strains affect each other's expression of a biocontrol trait. In both strains, production of the antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a crucial factor contributing to the suppression of root diseases. DAPG acts as a signaling compound inducing the expression of its own biosynthetic genes. Experimental setups were developed to investigate whether, when combining strains CHA0 and Q2-87, DAPG excreted by one strain may influence expression of DAPG-biosynthetic genes in the other strain in vitro and on the roots of wheat. DAPG production was monitored by observing the expression of lacZ fused to the biosynthetic gene phlA of the respective strain. Dual-culture assays in which the two strains were grown in liquid medium physically separated by a membrane revealed that Q2-87 but not its DAPG-negative mutant Q2-87::Tn5-1 strongly induced phlA expression in a DeltaphlA mutant of strain CHA0. In the same way, phlA expression in a Q2-87 background was induced by DAPG produced by CHA0. When coinoculated onto the roots of wheat seedlings grown under gnotobiotic conditions, strains Q2-87 and CHA0, but not their respective DAPG-negative mutants, were able to enhance phlA expression in each other. In summary, we have established that two nonrelated pseudomonads may stimulate each other in the expression of an antimicrobial compound important for biocontrol. This interpopulation communication occurs in the rhizosphere, i.e., at the site of pathogen inhibition, and is mediated by the antimicrobial compound itself acting as a signal exchanged between the two pseudomonads.

Regulation of antibiotic production in root-colonizing Peudomonas spp. and relevance for biological control of plant disease.

Certain strains of fluorescent pseudomonads are important biological components of agricultural soils that are suppressive to diseases caused by pathogenic fungi on crop plants. The biocontrol abilities of such strains depend essentially on aggressive root colonization, induction of systemic resistance in the plant, and the production of diffusible or volatile antifungal antibiotics. Evidence that these compounds are produced in situ is based on their chemical extraction from the rhizosphere and on the expression of antibiotic biosynthetic genes in the producer strains colonizing plant roots. Well-characterized antibiotics with biocontrol properties include phenazines, 2,4-diacetylphloroglucinol, pyoluteorin, pyrrolnitrin, lipopeptides, and hydrogen cyanide. In vitro, optimal production of these compounds occurs at high cell densities and during conditions of restricted growth, involving (i) a number of transcriptional regulators, which are mostly pathway-specific, and (ii) the GacS/GacA two-component system, which globally exerts a positive effect on the production of extracellular metabolites at a posttranscriptional level. Small untranslated RNAs have important roles in the GacS/GacA signal transduction pathway. One challenge in future biocontrol research involves development of new strategies to overcome the broad toxicity and lack of antifungal specificity displayed by most biocontrol antibiotics studied so far.

Biocontrol of seed pathogens and growth promotion of common bean seedlings by Trichoderma harzianum

The objective of this work was to evaluate isolates of Trichoderma harzianum regarding biocontrol of common bean seed-borne pathogens, plant growth promotion, and rhizosphere competence. Five isolates of T. harzianum were evaluated and compared with commercial isolate (Ecotrich), Carboxin+Thiram, and an absolute control. Bean seeds of the cultivar Jalo Precoce, contaminated with Aspergillus, Cladosporium, and Sclerotinia sclerotiorum, were microbiolized with antagonists, and seed health tests were carried out. Isolates were evaluated on autoclaved substrate and in field conditions. Ten days after sowing (DAS), plant length was measured. To test rhizosphere competence, isolates were applied in boxes containing autoclaved washed sand, and root colonization was evaluated at 10 DAS, using five plants per box. The most effective isolates in the seed health tests were: CEN287 and CEN289 to control Aspergillus; the commercial isolate to control Cladosporium; and CEN287 and CEN316 to control S. sclerotiorum. Isolates CEN289 and CEN290 promoted bean growth in greenhouse and field. Seed treatment with T. harzianum reduces the incidence of Aspergillus, Cladosporium, and S. sclerotiorum in 'Jalo Precoce' common bean seeds.