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.

Forms of inorganic phosphorus in soil under different long term soil tillage systems and winter crops

The cultivation of crops with different capacity of P uptake and use under long-term soil tillage systems can affect the distribution of P cycling and inorganic forms in the soil, as a result of higher or lower use efficiency of P applied in fertilizers. The purpose of this study was to evaluate the effect of long-term cultivation of different winter species under tillage systems on the distribution of inorganic P forms in the soil. In 1986, the experiment was initiated with six winter crops (blue lupin, hairy vetch, oat, oilseed radish, wheat and fallow) on a Rhodic Hapludox in southwestern Paraná, under no-tillage (NT) and conventional tillage (CT). The application of phosphate fertilizer in NT rows increased inorganic P in the labile and moderately labile forms, and soil disturbance in CT redistributed the applied P in the deeper layers, increasing the moderately labile P concentration in the subsurface layers. Black oat and blue lupin were the most efficient P-recyclers and under NT, they increased the labile P content in the soil surface layers.

Carbon balance and crop residue management in dynamic equilibrium under a no-till system in Campos Gerais

The adoption of no-tillage systems (NT) and the maintenance of crop residues on the soil surface result in the long-term increase of carbon (C) in the system, promoting C sequestration and reducing C-CO2 emissions to the atmosphere. The purpose of this study was to evaluate the C sequestration rate and the minimum amount of crop residues required to maintain the dynamic C equilibrium (dC/dt = 0) of two soils (Typic Hapludox) with different textural classes. The experiment was arranged in a 2 x 2 x 2 randomized block factorial design. The following factors were analyzed: (a) two soil types: Typic Hapludox (Oxisol) with medium texture (LVTM) and Oxisol with clay texture (LVTA), (b) two sampling layers (0-5 and 5-20 cm), and (c) two sampling periods (P1 - October 2007; P2 - September 2008). Samples were collected from fields under a long-term (20 years) NT system with the following crop rotations: wheat/soybean/black oat + vetch/maize (LVTM) and wheat/maize/black oat + vetch/soybean (LVTA). The annual C sequestration rates were 0.83 and 0.76 Mg ha-1 for LVTM and LVTA, respectively. The estimates of the minimum amount of crop residues required to maintain a dynamic equilibrium (dC/dt = 0) were 7.13 and 6.53 Mg ha-1 year-1 for LVTM and LVTA, respectively. The C conversion rate in both studied soils was lower than that reported in other studies in the region, resulting in a greater amount of crop residues left on the soil surface.

Stabilization of soil hydraulic properties under a long term no-till system

The area under the no-tillage system (NT) has been increasing over the last few years. Some authors indicate that stabilization of soil physical properties is reached after some years under NT while other authors debate this. The objective of this study was to determine the effect of the last crop in the rotation sequence (1st year: maize, 2nd year: soybean, 3rd year: wheat/soybean) on soil pore configuration and hydraulic properties in two different soils (site 1: loam, site 2: sandy loam) from the Argentinean Pampas region under long-term NT treatments in order to determine if stabilization of soil physical properties is reached apart from a specific time in the crop sequence. In addition, we compared two procedures for evaluating water-conducting macroporosities, and evaluated the efficiency of the pedotransfer function ROSETTA in estimating the parameters of the van Genuchten-Mualem (VGM) model in these soils. Soil pore configuration and hydraulic properties were not stable and changed according to the crop sequence and the last crop grown in both sites. For both sites, saturated hydraulic conductivity, K0, water-conducting macroporosity, εma, and flow-weighted mean pore radius, R0ma, increased from the 1st to the 2nd year of the crop sequence, and this was attributed to the creation of water-conducting macropores by the maize roots. The VGM model adequately described the water retention curve (WRC) for these soils, but not the hydraulic conductivity (K) vs tension (h) curve. The ROSETTA function failed in the estimation of these parameters. In summary, mean values of K0 ranged from 0.74 to 3.88 cm h-1. In studies on NT effects on soil physical properties, the crop effect must be considered.

Rapid disease emergence through horizontal gene transfer between eukaryotes.

Among different species of eukaryotes, the extent and evolutionary significance of horizontal gene transfer remains poorly understood. A newly published study by Friesen and colleagues indicates that a recent gene transfer between two species of fungi has enabled the recipient to rapidly acquire high virulence on wheat. The study highlights a mechanism by which diseases can suddenly emerge, but also brings up the controversial issues of how horizontal gene transfer occurs and whether fungal incompatibility barriers to gene flow are more 'leaky' than was previously thought.

PHYTOAVAILABILITY OF COPPER IN INDUSTRIAL BY-PRODUCTS AND MINERAL FERTILIZERS

Alternative copper (Cu) sources could be used in fertilizer production, although the bioavailability of copper in these materials is unknown. The objective of this study was to evaluate the extractants neutral ammonium citrate (NAC), 2 % citric acid, 1 % acetic acid, 10 % HCl, 10 % H2SO4, buffer solution pH 6.0, DTPA, EDTA, water, and hot water in the quantification of available Cu content in several sources, relating them to the relative agronomic efficiency (RAE) of wheat grown in a clayey Latossolo Vermelho eutrófico (Oxisol) and Neossolo Quartzarênico (Typic Quartzipsamment). Copper was applied at the rate of 1.5 mg kg-1 as scrap slag, brass slag, Cu ore, granulated copper, and copper sulfate. The extractants 10 % HCl, 10 % H2SO4, and NAC extracted higher Cu concentrations. The RAE values of brass slag and Cu ore were similar to or higher than those of Cu sulfate and granulated Cu. Solubility in the 2nd NAC extractant, officially required for mineral fertilizers with Cu, was lower than 60 % for the scrap slag, Cu ore, and granulated copper sources. This fact indicates that adoption of the NAC extractant may be ineffective for industrial by-products, although no extractant was more efficient in predicting Cu availability for wheat fertilized with the Cu sources tested.

WATER INFILTRATION IN TWO CULTIVATED SOILS IN SOUTHERN BRAZIL

Infiltration is the passage of water through the soil surface, influenced by the soil type and cultivation and by the soil roughness, surface cover and water content. Infiltration absorbs most of the rainwater and is therefore crucial for planning mechanical conservation practices to manage runoff. This study determined water infiltration in two soil types under different types of management and cultivation, with simulated rainfall of varying intensity and duration applied at different times, and to adjust the empirical model of Horton to the infiltration data. The study was conducted in southern Brazil, on Dystric Nitisol (Nitossolo Bruno aluminoférrico húmico) and Humic Cambisol (Cambissolo Húmico alumínico léptico) soils to assess the following situations: simulated rains on the Nitisol from 2001 to 2012 in 31 treatments, differing in crop type, sowing direction, type of soil opener on the seeder, amount and type of crop residue and amount of liquid swine manure applied; on the Cambisol, rains were simlated from 2006 to 2012 and 18 treatments were evaluated, differing in crop, seeding direction and crop residue type. The constant of the water infiltration rate into the soil varies significantly with the soil type (30.2 mm h-1 in the Nitisol and 6.6 mm h-1 in the Cambisol), regardless of the management system, application time and rain intensity and duration. At the end of rainfalls, soil-water infiltration varies significantly with the management system, with the timing of application and rain intensity and duration, with values ranging from 13 to 59 mm h-1, in the two studied soils. The characteristics of the sowing operation in terms of relief, crop type and amount and type of crop residue influenced soil water infiltration: in the Nitisol, the values of contour and downhill seeding vary between 27 and 43 mm h-1, respectively, with crop residues of corn, wheat and soybean while in the Cambisol, the variation is between 2 and 36 mm h-1, respectively, in soybean and corn crops. The Horton model fits the values of water infiltration rate into the soil, resulting in the equation i = 30.2 + (68.2 - 30.2) e-0.0371t (R2 = 0.94**) for the Nitisol and i = 6.6 + (64.5 - 6.6) e-0.0537t (R2 = 0.99**) for the Cambisol.

Factors promoting the exposure to bioaerosols among crop workers

Agricultural workers are among the professional groups most at risk of developing acute or chronic respiratory problems. Despite this fact, the etiology of these occupational diseases is poorly known, even in important sectors of agriculture such as the crops sector. A chronic exposure to multiple microorganisms, such as different bacterial and fungal species, has been proposed to be the cause of these multiple respiratory pathologies. Nevertheless, these microbial communities are still partially known. The aim of this study is to characterize all fungal species inhaled by the crops workers during different grain related activities and identify the abiotic and biotic factors that reduce the growth of the toxigenic, irritative or allergenic microbial species. Here, we are presenting the factors promoting the exposure to bioaerosols during different wheat related activities: harvesting, grain unload, baling straw, the cleaning of harvesters and silos. Total dust has been quantified following NIOSH 0500 method. Reactive endotoxin activity has been determined with Limulus Amebocyte Lysate Assay. All molds have been identified by the pyrosequencing of ITS2 amplicons generated from bioaerosol.

Do Rates and Splitting of Phosphogypsum Applications Influence the Soil and Annual Crops in a No-Tillage System?

ABSTRACT Applications of phosphogypsum (PG) provide nutrients to the soil and reduce Al3+ activity, favoring soil fertility and root growth, but allow Mg2+ mobilization through the soil profile, resulting in variations in the PG rate required to achieve the optimum crop yield. This study evaluated the effect of application rates and splitting of PG on soil fertility of a Typic Hapludox, as well as the influence on annual crops under no-tillage. Using a (4 × 3) + 1 factorial structure, the treatments consisted of four PG rates (3, 6, 9, and 12 Mg ha-1) and three split applications (P1 = 100 % in 2009; P2 = 50+50 % in 2009 and 2010; P3 = 33+33+33 % in 2009, 2010 and 2011), plus a control without PG. The soil was sampled six months after the last PG application, in stratified layers to a depth of 0.8 m. Corn, wheat and soybean were sown between November 2011 and December 2012, and leaf samples were collected for analysis when at least 50 % of the plants showed reproductive structures. The application of PG increased Ca2+ concentrations in all sampled soil layers and the soil pH between 0.2 and 0.8 m, and reduced the concentrations of Al3+ in all layers and of Mg2+ to a depth of 0.6 m, without any effect of splitting the applications. The soil Ca/Mg ratio increased linearly to a depth of 0.6 m with the rates and were found to be higher in the 0.0-0.1 m layer of the P2 and P3 treatments than without splitting (P1). Sulfur concentrations increased linearly by application rates to a depth of 0.8 m, decreasing in the order P3>P2>P1 to a depth of 0.4 m and were higher in the treatments P3 and P2 than P1 between 0.4-0.6 m, whereas no differences were observed in the 0.6-0.8 m layer. No effect was recorded for K, P and potential acidity (H+Al). The leaf Ca and S concentration increased, while Mg decreased for all crops treated with PG, and there was no effect of splitting the application. The yield response of corn to PG rates was quadratic, with the maximum technical efficiency achieved at 6.38 Mg ha-1 of PG, while wheat yield increased linearly in a growing season with a drought period. Soybean yield was not affected by the PG rate, and splitting had no effect on the yield of any of the crops. Phosphogypsum improved soil fertility in the profile, however, Mg2+ migrated downwards, regardless of application splitting. Splitting the PG application induced a higher Ca/Mg ratio in the 0.0-0.1 m layer and less S leaching, but did not affect the crop yield. The application rates had no effect on soybean yield, but were beneficial for corn and, especially, for wheat, which was affected by a drought period during growth.

Tn5-directed cloning of pqq genes from Pseudomonas fluorescens CHA0: mutational inactivation of the genes results in overproduction of the antibiotic pyoluteorin.

Pseudomonas fluorescens CHA0 produces several secondary metabolites, e.g., the antibiotics pyoluteorin (Plt) and 2,4-diacetylphloroglucinol (Phl), which are important for the suppression of root diseases caused by soil-borne fungal pathogens. A Tn5 insertion mutant of strain CHA0, CHA625, does not produce Phl, shows enhanced Plt production on malt agar, and has lost part of the ability to suppress black root rot in tobacco plants and take-all in wheat. We used a rapid, two-step cloning-out procedure for isolating the wild-type genes corresponding to those inactivated by the Tn5 insertion in strain CHA625. This cloning method should be widely applicable to bacterial genes tagged with Tn5. The region cloned from P. fluorescens contained three complete open reading frames. The deduced gene products, designated PqqFAB, showed extensive similarities to proteins involved in the biosynthesis of pyrroloquinoline quinone (PQQ) in Klebsiella pneumoniae, Acinetobacter calcoaceticus, and Methylobacterium extorquens. PQQ-negative mutants of strain CHA0 were constructed by gene replacement. They lacked glucose dehydrogenase activity, could not utilize ethanol as a carbon source, and showed a strongly enhanced production of Plt on malt agar. These effects were all reversed by complementation with pqq+ recombinant plasmids. The growth of a pqqF mutant on ethanol and normal Plt production were restored by the addition of 16 nM PQQ. However, the Phl- phenotype of strain CHA625 was due not to the pqq defect but presumably to a secondary mutation. In conclusion, a lack of PQQ markedly stimulates the production of Plt in P. fluorescens.

A detailed urinary excretion time course study of captan and folpet biomarkers in workers for the estimation of dose, main route-of-entry and most appropriate sampling and analysis strategies

Captan and folpet are two fungicides largely used in agriculture, but biomonitoring data are mostly limited to measurements of captan metabolite concentrations in spot urine samples of workers, which complicate interpretation of results in terms of internal dose estimation, daily variations according to tasks performed, and most plausible routes of exposure. This study aimed at performing repeated biological measurements of exposure to captan and folpet in field workers (i) to better assess internal dose along with main routes-of-entry according to tasks and (ii) to establish most appropriate sampling and analysis strategies. The detailed urinary excretion time courses of specific and non-specific biomarkers of exposure to captan and folpet were established in tree farmers (n = 2) and grape growers (n = 3) over a typical workweek (seven consecutive days), including spraying and harvest activities. The impact of the expression of urinary measurements [excretion rate values adjusted or not for creatinine or cumulative amounts over given time periods (8, 12, and 24 h)] was evaluated. Absorbed doses and main routes-of-entry were then estimated from the 24-h cumulative urinary amounts through the use of a kinetic model. The time courses showed that exposure levels were higher during spraying than harvest activities. Model simulations also suggest a limited absorption in the studied workers and an exposure mostly through the dermal route. It further pointed out the advantage of expressing biomarker values in terms of body weight-adjusted amounts in repeated 24-h urine collections as compared to concentrations or excretion rates in spot samples, without the necessity for creatinine corrections.

Effect of management practices on mycorrhizal infection, growth and dry matter partitioning in field-grown bean

The experiment was carried out on unsterilized field soil with low phosphorus availability with the objective of examining the effect of cultural practices on mycorrhizal colonization and growth of common bean. The treatments were: three pre-crops (maize, wheat and fallow) followed by three soil management practices ("ploughing", mulching and bare fallow without "ploughing" during the winter months). After the cultural practices, Phaseolus vulgaris cv. Canadian Wonder was grown in this soil. Fallowing and soil disturbance reduced natural soil infectivity. Mycorrhizal infection of the bean roots occurred more rapidly in the recently cropped soil than in the fallow soil. Prior cropping with a strongly mycorrhizal plant (maize) increased infectivity even further.

Rearing the southern green stink bug using an artificial dry diet and an artificial plant

Laboratory and greenhouse studies were conducted with an artificial dry diet to rear nymphs, and with an artificial plant as substrate for egg laying by the southern green stink bug, Nezara viridula (L.). The artificial diet was composed of: soybean protein (15 g); potato starch (7.5 g); dextrose (7.5 g); sucrose (2.5 g); cellulose (12.5 g); vitamin mixture (niacinamide 1 g, calcium pantothenate 1 g, thiamine 0.25 g, riboflavin 0.5 g, pyridoxine 0.25 g, folic acid 0.25 g, biotin 0.02 mL, vitamin B12 1 g - added to 1,000 mL of distilled water) (5.0 mL); soybean oil (20 mL); wheat germ (17.9 g); and water (30 mL). Nymphs showed normal feeding behavior when fed on the artificial diet. Nymphal development time was longer than or similar to that of nymphs fed on soybean pods. Total nymphal mortality was low (ca. 30%), both for nymphs reared on the artificial diet, and for nymphs fed on soybean pods. At adult emergence, fresh body weights were significantly (P<0.01) less on the artificial diet than on soybean pods. Despite the lower adult survivorship and fecundity on artificial plants than on soybean plants, it was demonstrated for the first time that a model simulating a natural plant, can be used as a substrate for egg mass laying, in conjunction with the artificial diet.

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.