Microsphere stem blockage as a screen for nitrogen-fixation drought tolerance in soybean.

Symbiotic nitrogen-fixation of soybean (Glycine max [Merr.] L) commonly decreases in response to soil drying in advance of other plant processes. While a few soybean lines express nitrogen-fixation drought tolerance, breeding for genetic variation is hampered by laborious phenotyping procedures. The objective of this research was to explore the potential of an initial screen for nitrogen-fixation drought-tolerant genotypes based on a possible relationship with xylem-vessel diameter. The hypothesis was that nitrogen-fixation drought tolerance might result from fewer, large-diameter xylem vessels in the stem that are vulnerable to disrupted flow as water deficit develops. The disrupted flow could cause nitrogen products to accumulate in nodules resulting in negative feedback on nitrogen-fixation rate. The proposed screen involved exposing de-rooted shoots to a suspension containing microspheres (45-53 µm diameter) and recording the decrease in transpiration rate as a result of microsphere xylem-blockage. Two soybean populations were tested. One population was progeny derived from mating of two parents with high and low nitrogen-fixation drought sensitivity. A high correlation (R2  = 0.68; P < 0.001) was found in this population between decreasing transpiration rate resulting from the microsphere treatment and increasing sensitivity of nitrogen-fixation to soil drying. The second tested population consisted of 16 genotypes, most of which had been previously identified in germplasm screens as expressing nitrogen-fixation drought tolerance. Nearly half of the lines in this second population were identified in the screen as showing minimum blockage of transpiration when exposed to the microspheres. Overall, these results showed the potential of using the microsphere screen to identify candidate genotypes expressing nitrogen-fixation drought tolerance.

Quorum sensing communication: Bradyrhizobium-Azospirillum interaction via N-acyl-homoserine lactones in the promotion of soybean symbiosis.

Quorum-sensing (QS) mechanisms are important in intra- and inter-specific communication among bacteria. We investigated QS mechanisms in Bradyrhizobium japonicum strain CPAC 15 and Azospirillum brasilense strains Ab-V5 and Ab-V6, used in commercial co-inoculants for the soybean crop in Brazil. A transconjugant of CPAC 15-QS with partial inactivation of N-acyl-homoserine lactones (AHLs) was obtained and several parameters were evaluated; in vitro, CPAC 15 and the transconjugant differed in growth, but not in biofilm formation, and no differences were observed in the symbiotic performance in vivo. The genome of CPAC 15 carries functional luxI and luxR genes and low amounts of three AHL molecules were detected: 3-OH-C12-AHL, 3-OH-C14-AHL, and 3-oxo-C14-AHL. Multiple copies of luxR-like genes, but not of luxI are present in the genomes of Ab-V5 and Ab-V6, and differences in gene expression were observed when the strains were co-cultured with B. japonicum; we may infer that the luxR-genes of A. brasilense may perceive the AHL molecules of B. japonicum. Soybean symbiotic performance was improved especially by co-inoculation with Ab-V6, which, contrarily to Ab-V5, did not respond to the AHLs of CPAC 15. We concluded that A. brasilense Ab-V5, but not Ab-V6, responded to the QS signals of CPAC 15, and that the synergistic interaction may be credited, at least partially, to the QS interaction. In addition, we confirmed inter- and intra-species QS communication between B. japonicum and A. brasilense and, for Azospirillum, at the strain level, impacting several steps of the symbiosis, from cell growth to plant nodulation and growth.

Selection of host-plant genotype: the next step to increase grain legume N2 fixation activity.

Symbiotic N2 fixation research thus far has been primarily focused on selection of bacteria. However, little progress in impacting crop yields has resulted from this approach. Bacteria introduced in field soils rarely compete well with indigenous bacteria, including mutated lines selected for high nitrogen fixation capacity. Consequently, introduction of 'elite' bacteria in fields commonly does not result in crop yield increase. This review highlights that the primary regulation of N2 fixation is a result of response of integrated physiological activity at the plant level. Nitrogen feedback from the host plant plays an important role in regulating the N2 fixation rate. Rapid sequestration of fixed nitrogen by the plant is especially important for high N2 fixation activity. In addition, water cycling in the plant between the shoot and nodules plays a key role in sustaining high N2 fixation activity. Therefore, attention in selecting the host-plant genotype is suggested to be the next step to increasing N2 fixation activity of grain legumes.

Application of Landfill Leachate Improves Wheat Nutrition and Yield but Has Minor Effects on Soil Properties.

Landfill leachates, which are potential pollutants, may also carry significant amounts of nutrients that can be recycled by plants. We assessed the nutritional status and yield of wheat ( L.) and properties of a Rhodic Kandiudult soil (depths of 0-10, 10-20, 20-40, and 40-60 cm) after 11 applications of landfill leachate over 4 yr. In the last application, wheat received 0, 32.7, 65.4, 98.1, or 130.8 m ha (875 mg L of nitrogen, N) of leachate and a positive control (90 kg ha of N as urea) 15 d after sowing. Urea increased nitrate (>160 mg kg) in the topsoil (down to 40 cm), whereas landfill leachate increased nitrate (>60 mg kg) only at 40 to 60 cm with the highest dose, suggesting leaching. Urea-treated soil had less negative ΔpH, which might have led to greater retention of nitrate in the topsoil. Sodium (0.02-0.26 cmol Na kg), potassium (0.18-0.82 cmol K kg), and electrical conductivity (0.05-0.14 dS m) all increased with leachate dosage. Treatments did not affect resistance to penetration and clay dispersion. Basal respiration increased with leachate dosage, whereas dehydrogenase activity decreased, suggesting effects on soil microbial metabolism. Microbial biomass and soil enzyme activities were not affected by addition of leachate. Nitrogen nutrition (15.1-22.7 g N kg in flag leaves) and grain yield (1381-2378 kg grain ha) increased with leachate dosage so that the highest dose gave results similar to those for urea-treated plants (2563 kg grain ha). Landfill leachate showed strong potential as source of N for wheat but caused none, or transient, effects on soil properties. However, nitrate from leachate was more leachable than nitrate from urea.

Effect of Landfill Leachate on Cereal Nutrition and Productivity and on Soil Properties.

Landfill leachates carry nutrients, especially N and K, which can be recycled in cropping systems. We applied doses of landfill leachate (0 [Control], 32.7, 65.4, 98.1, and 130.8 m ha) three times in 2008 and three times in 2009 on a clay Rhodic Kandiudult soil. In 2009, black oat ( L.) and corn ( L.) were cropped in succession and assessed for concentration of nutrients in leaves and for shoot biomass and grain yield, respectively. As a positive control, an additional treatment with urea (120 kg ha of N) was studied in corn. Soil was sampled at four depths (down to 60 cm) in three sampling dates to assess chemical and biochemical properties. Concentration of nutrients in leaves, oat biomass (8530-23,240 kg ha), and corn grain yield (4703-8807 kg ha) increased with increasing doses of leachate. There was a transient increase in the concentration of nitrate in soil (3-30 mg kg), increasing the risk of N losses by leaching at doses above 120 kg ha N, as revealed by an estimated N balance in the cropping system. Sodium and K in soil also increased with increasing doses of leachate but decreased as rainfall occurred. The activity of dehydrogenase decreased about 30% from the control to the highest dose of leachate and urea, suggesting an inhibitory effect of mineral N on microbial metabolism. Landfill leachate was promising as a source of N and K for crop productivity and caused minor or transient effects on soil properties.

Dosage-dependent shift in the spore community of arbuscular mycorrhizal fungi following application of tannery sludge.

The controlled disposal of tannery sludge in agricultural soils is a viable alternative for recycling such waste; however, the impact of this practice on the arbuscular mycorrhizal fungi (AMF) communities is not well understood. We studied the effects of low-chromium tannery sludge amendment in soils on AMF spore density, species richness and diversity, and root colonization levels. Sludge was applied at four doses to an agricultural field in Rolândia, Paraná state, Brazil. The sludge was left undisturbed on the soil surface and then the area was harrowed and planted with corn. The soil was sampled at four intervals and corn roots once within a year (2007/2008). AMF spore density was low (1 to 49 spores per 50 cm(3) of soil) and decreased as doses of tannery sludge increased. AMF root colonization was high (64%) and unaffected by tannery sludge. Eighteen AMF species belonging to six genera (Acaulospora, Glomus, Gigaspora, Scutellospora, Paraglomus, and Ambispora) were recorded. At the sludge doses of 9.0 and 22.6 Mg ha(-1), we observed a decrease in AMF species richness and diversity, and changes in their relative frequencies. Hierarchical grouping analysis showed that adding tannery waste to the soil altered AMF spore community in relation to the control, modifying the mycorrhizal status of soil and selectively favoring the sporulation of certain species.

Compatibility of Azospirillum brasilense with Pesticides Used for Treatment of Maize Seeds.

Seed treatment with chemical pesticides is commonly used as an initial plant protection procedure against pests and diseases. However, the use of such chemicals may impair the survival and performance of beneficial microorganisms introduced via inoculants, such as the plant growth-promoting bacterium Azospirillum brasilense. We assessed the compatibility between the most common pesticide used in Brazil for the treatment of maize seeds, composed of two fungicides, and one insecticide, with the commercial strains Ab-V5 and Ab-V6 of A. brasilense, and evaluated the impacts on initial plant development. The toxicity of the pesticide to A. brasilense was confirmed, with an increase in cell mortality after only 24 hours of exposure in vitro. Seed germination and seedling growth were not affected neither by the A. brasilense nor by the pesticide. However, under greenhouse conditions, the pesticide affected root volume and dry weight and root-hair incidence, but the toxicity was alleviated by the inoculation with A. brasilense for the root volume and root-hair incidence parameters. In maize seeds inoculated with A. brasilense, the pesticide negatively affected the number of branches, root-hair incidence, and root-hair length. Therefore, new inoculant formulations with cell protectors and the development of compatible pesticides should be searched to guarantee the benefits of inoculation with plant growth-promoting bacteria.

Sporulation and diversity of arbuscular mycorrhizal fungi in Brazil Pine in the field and in the greenhouse.

The aim of this work was to assess the sporulation and diversity of arbuscular mycorrhizal fungi (AMF) at different forest sites with Araucaria angustifolia (Bert.) O. Ktze. (Brazil Pine). In addition, a greenhouse experiment was carried out to test the use of traditional trap plants (maize + peanut) or A. angustifolia to estimate the diversity of AMF at each site. Soil samples were taken in two State Parks at southwestern Brazil: Campos do Jordão (Parque Estadual de Campos do Jordão [PECJ]) and Apiaí (Parque Estadual Turístico do Alto Ribeira [PETAR]), São Paulo State, in sites of either native or replanted forest. In PECJ, an extra site of replanted forest that was impacted by accidental fire and is now in a state of recuperation was also sampled. The spore densities and their morphological identification were compiled at each site. In the greenhouse, soil samples from each site were used as inoculum to promote spore multiplication on maize + peanut or A. angustifolia grown on a sandy, low-fertility substrate. Plants were harvested, respectively, after 4 months or 1 year of growth and assessed for mycorrhizal root colonization. Spore counts and identification were also performed in the substrate, after the harvest of plants. Twenty-five taxa were identified considering all sites. Species richness and diversity were greater in native forest areas, being Acaulospora, the genus with the most species. Differences in number of spores, diversity, and richness were found at the different sites of each State Park. Differences were also found when maize + peanut or A. angustifolia were used as trap plants. The traditional methodology using trap plants seems to underestimate the diversity of the AMF. The use of A. angustifolia as trap plant showed similar species richness to the field in PECJ, but the identified species were not necessarily the same. Nevertheless, for PETAR, both A. angustifolia and maize + peanut underestimated the species richness. Because the AMF sporulation can be affected by many conditions, it is impossible to draw detailed conclusions from this kind of survey. More precise experiments have to be set up to isolate the different factors that modulate the ecophysiological interactions between host plant and endophyte.