Changes in chemical fractionation of copper and zinc in soil as a function of incubation moisture content and organic matter amendments.

Copper and zinc are essential micronutrients that are potentially toxic when present in excess in soils. Their bioavailability depends on their speciation in soil, but this may vary with environmental conditions. Aeration and hence redox conditions, and organic matter amendments are among the factors likely to cause variation on metal fractionation. We have monitored the chemical fractionation of both native and added copper and zinc in a clay loam top soil during a 5-month laboratory incubation. The effects of aeration (moist soil or flooded) and addition of two organic matter amendments, alfalfa straw or leaf compost, were studied. Metal spike was more labile than legacy metal, and was slowly redistributed over the incubation period. Organic matter caused short-lived flushes of metals, attributed to metal chelation with soluble organic matter. This effect was greater for straw than for more stable compost. There was no evidence that added organic matter increased the capacity of soil organic matter to immobilise metal. Flooding solubilized soil metal (hydr)oxides, releasing legacy Cu and Zn, but with less effect on the capacity to immobilise metal spike. Effects of flooding and organic matter addition were not additive. Both metals appear to be precipitated as sulphides under reducing conditions, and accounted for in the acid soluble phase. Monitoring the dynamics of metal distribution gives a more comprehensive understanding of underlying processes than would a single measurement, and is closer to in campo conditions than slurry microcosms.

pH-Dependent Changes in Structural Stabilities of Bt Cry1Ac Toxin and Contrasting Model Proteins following Adsorption on Montmorillonite.

The environmental fate of insecticidal Cry proteins, including time-dependent conservation of biological properties, results from their structural stability in soils. The complex cascade of reactions involved in biological action requires Cry proteins to be in solution. However, the pH-dependent changes in conformational stability and the adsorption-desorption mechanisms of Cry protein on soil minerals remain unclear. We used Derjaguin-Landau-Verwey-Overbeek (DLVO) calculation and differential scanning calorimetry to interpret the driving forces and structural stabilities of Cry1Ac and two contrasting model proteins adsorbed by montmorillonite. The structural stability of Cry1Ac is closer to that of the "hard" protein, α-chymotrypsin, than that of the "soft" bovine serum albumin (BSA). The pH-dependent adsorption of Cry1Ac and α-chymotrypsin could be explained by DLVO theory, whereas the BSA adsorption deviated from it. Patch-controlled electrostatic attraction, hydrophobic effects, and entropy changes following protein unfolding on a mineral surface could contribute to Cry1Ac adsorption. Cry1Ac, like chymotrypsin, was partly denatured on montmorillonite, and its structural stability decreased with an increase in pH. Moreover, small changes in the conformational heterogeneity of both Cry1Ac and chymotrypsin were observed following adsorption. Conversely, adsorbed BSA was completely denatured regardless of the solution pH. The moderate conformational rearrangement of adsorbed Cry1Ac may partially explain why the insecticidal activity of Bt toxin appears to be conserved in soils, albeit for a relatively short time period.

Arbuscular Mycorrhizal Fungi and Soil Quality Indicators in Eucalyptus genotypes With Different Drought Tolerance Levels.

Silviculture has great importance worldwide, and the use of Eucalyptus species, which account for 75% of the local planted forest in Brazil, is one of the factors that contributes to the success of this activity in the country. Despite its adaptability, the yield of Eucalyptus is often affected by climate change, particularly water deficiency. Plants have developed strategies to mitigate water stress, for example, through their association with mycorrhizal fungi. The genus Eucalyptus, particularly in the plant domain, establishes symbioses with arbuscular mycorrhizal fungi (AMF) and ectomycorrhizal fungi (ECMF). The influence of Eucalyptus species on AMF and soil quality indicators is not well understood. Our aim was to conduct a preliminary evaluation of the various responses of soil AMF communities and soil nutrient dynamics in the presence of Eucalyptus species with different degrees of drought tolerance. A field experiment was established containing six Eucalyptus species, E. brassiana, E. camaldulensis, E. citriodora, E. cloeziana, E. grandis, and E. urophylla, all of which were planted in large plots. Soil and root samples were taken when the plants were 1.7 and 2.2 years old. We found that Eucalyptus species with low (E. grandis and E. urophylla) and intermediate drought tolerance (E. citriodora and E. cloeziana) showed stronger correlations with the AMF community than Eucalyptus species with high drought tolerance (E. brassiana and E. camaldulensis). Differences were also found between Eucalyptus species for AMF spore numbers and root colonization percentages, which was most evident for E. urophylla. The microbiological attributes found to be most responsive to Eucalyptus species were soil enzyme activities, AMF spore numbers, root colonization percentages, and fungal abundance. Soil organic carbon, phosphorus, potassium, zinc, copper, and iron were the main chemical drivers related to the soil AMF community structure in the presence of E. brassiana.

In vivo and in vitro 31P-NMR Study of the Phosphate Transport and Polyphosphate Metabolism in Hebeloma cylindrosporum in Response to Plant Roots Signals.

We used in vivo and in vitro phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy to follow the change in transport, compartmentation and metabolism of phosphate in the ectomycorrhizal fungus Hebeloma cylindrosporum in response to root signals originating from host (Pinus pinaster) or non-host (Zea mays) plants. A device was developed for the in vivo studies allowing the circulation of a continuously oxygenated mineral solution in an NMR tube containing the mycelia. The in vitro studies were performed on fungal material after several consecutive treatment steps (freezing in liquid nitrogen; crushing with perchloric acid; elimination of perchloric acid; freeze-drying; dissolution in an appropriate liquid medium).

A Method for Radioactive Labelling of Hebeloma cylindrosporum to Study Plant-fungus Interactions.

In order to quantify P accumulation and P efflux in the ectomycorrhizal basidiomycete fungus Hebeloma cylindrosporum, we supplied 32P to mycelia previously grown in vitro in liquid medium. The culture had four main steps that are 1) growing the mycelium on complete medium with P, 2) transfer the mycelia into new culture solution with or without P, 3) adding a solution containing 32P and 4) rinsing the mycelia before incubation with or without plant. The main point is to rinse very carefully the mycelia after 32P supply in order to avoid overestimation of 32P efflux into the medium.

Establishing a Symbiotic Interface between Cultured Ectomycorrhizal Fungi and Plants to Follow Fungal Phosphate Metabolism.

In ectomycorrhizal plants, the fungal cells colonize the roots of their host plant to create new organs called ectomycorrhizae. In these new organs, the fungal cells colonize the walls of the cortical cells, bathing in the same apoplasm as the plant cells in a space named the 'Hartig net', where exchanges between the two partners take place. Finally, the efficiency of ectomycorrhizal fungi to improve the phosphorus nutrition of their host plants will depend on the regulation of phosphate transfer from the fungal cells to plant cells in the Hartig net through as yet unknown mechanisms. In order to investigate these mechanisms, we developed an in vitro experimental device mimicking the common apoplasm of the ectomycorrhizae (the Hartig net) to study the phosphorus metabolism in the ectomycorrhizal fungus Hebeloma cylindrosporum when the fungal cells are associated or not with the plant cells of the host plant Pinus pinaster. This device can be used to monitor 32Phosphate efflux from the fungus previously incubated with 32P-orthophosphate.

The host plant Pinus pinaster exerts specific effects on phosphate efflux and polyphosphate metabolism of the ectomycorrhizal fungus Hebeloma cylindrosporum: a radiotracer, cytological staining and 31 P NMR spectroscopy study.

Ectomycorrhizal (ECM) association can improve plant phosphorus (P) nutrition. Polyphosphates (polyP) synthesized in distant fungal cells after P uptake may contribute to P supply from the fungus to the host plant if they are hydrolyzed to phosphate in ECM roots then transferred to the host plant when required. In this study, we addressed this hypothesis for the ECM fungus Hebeloma cylindrosporum grown in vitro and incubated without plant or with host (Pinus pinaster) and non-host (Zea mays) plants, using an experimental system simulating the symbiotic interface. We used 32 P labelling to quantify P accumulation and P efflux and in vivo and in vitro nuclear magnetic resonance (NMR) spectroscopy and cytological staining to follow the fate of fungal polyP. Phosphate supply triggered a massive P accumulation as newly synthesized long-chain polyP in H. cylindrosporum if previously grown under P-deficient conditions. P efflux from H. cylindrosporum towards the roots was stimulated by both host and non-host plants. However, the host plant enhanced 32 P release compared with the non-host plant and specifically increased the proportion of short-chain polyP in the interacting mycelia. These results support the existence of specific host plant effects on fungal P metabolism able to provide P in the apoplast of ectomycorrhizal roots.

Fate of insecticidal Bacillus thuringiensis Cry protein in soil: differences between purified toxin and biopesticide formulation.

BACKGROUND: Bacillus thuringiensis produces insecticidal proteins known as Cry, and its efficiency and absence of side effects make it the most widely used biopesticide. There is little information on the role of soils in the fate of Cry proteins from commercial biopesticide formulations, unlike toxins from genetically modified crops, which have been intensively studied in recent years. The persistence of Cry in soil was followed under field and laboratory conditions. RESULTS: Sunlight accelerated loss of detectable Cry under laboratory conditions, but little effect of shade was observed under field conditions. The half-life of biopesticide proteins in soil under natural conditions was about 1 week. Strong temperature effects were observed, but they differed for biopesticide and purified protein, indicating different limiting steps. CONCLUSION: For the biopesticide, the observed decline in detectable protein was due to biological factors, possibly including the germination of B. thuringiensis spores, and was favoured by higher temperature. In contrast, for purified proteins, the decline in detectable protein was slower at low temperature, probably because the conformational changes of the soil-adsorbed protein, which cause fixation and hence reduced extraction efficiency, are temperature dependent. © 2016 Society of Chemical Industry.

Adsorption of alexa-labeled Bt toxin on mica, glass, and hydrophobized glass: study by normal scanning confocal fluorescence.

We studied the kinetics of adsorption of alexa-labeled Bt toxin Cry1Aa, in monomer and oligomer states, on muscovite mica, acid-treated hydrophilic glass, and hydrophobized glass, in the configuration of laminar flow of solution in a slit. Normal confocal fluorescence through the liquid volume allows the visualization of the concentration in solution over the time of adsorption, in addition to the signal due to the adsorbed molecules at the interface. The solution signal is used as calibration for estimation of interfacial concentration. We found low adsorption of the monomer compared to oligomers on the three types of surface. The kinetic adsorption barrier for oligomers increases in the order hydrophobized glass, muscovite mica, acid-treated hydrophilic glass. This suggests enhanced immobilization in soil if toxin is under oligomer state.

Adsorption and desorption of 85Sr and 137Cs on reference minerals, with and without inorganic and organic surface coatings.

The adsorption properties of reference minerals may be considerably modified by the presence of the inorganic and organic coatings that are ubiquitous in soils. It is therefore important to assess the effect of such coatings to evaluate the relevance of adsorption studies on pure minerals. The adsorption of trace amounts of (85)Sr and (137)Cs has been studied in dilute suspensions for various minerals that are common components of soils: quartz, calcium carbonate, kaolinite, montmorillonite and illite. We studied the effect of coatings with either Fe or Al oxide with varying additions of soil-extracted humic or fulvic acid. Both adsorption and desorption were measured and data presented as distribution coefficients, Kd. No adsorption was detected on quartz and it was not possible to coat this surface. Adsorption on calcium carbonate was small and not influenced by coatings. Adsorption of Sr on the three clay minerals was very similar, enhanced by the Al-coating, but not affected by Fe and organic coatings. The presence of organic coatings decreased Cs adsorption on illite. Similar but smaller effects were seen on montmorillonite and kaolinite. Aluminum coating enhanced Cs adsorption on illite, whereas both inorganic coatings caused decreases in adsorption on montmorillonite, and there was no effect on kaolinite. Effects were not additive with mixed, organic-inorganic coatings. Adsorption of both Cs and Sr on all minerals was strongly irreversible, with Kd (desorption) being up to four-times greater than adsorption Kd. The ratio of desorption and adsorption Cs Kd values (an assessment of irreversibility) was inversely related to adsorption Kd. This is consistent with a decreasing contribution of high-affinity adsorption as adsorption increases, but may also reflect the partial loss of organic coatings during desorption.

Mycorrhizal association of maritime pine, Pinus pinaster, with Rhizopogon roseolus has contrasting effects on the uptake from soil and root-to-shoot transfer of 137Cs, 85Sr and 95mTc.

The beneficial role of mycorrhizal association on plant nutrition and water supply is well-known, however, very little information exists with respect to the availability of radionuclides. We have measured the effect of controlled mycorrhizal association on the root uptake from soil and accumulation in leaves of three radionuclides. The radionuclides have contrasting chemical and biological properties: Cs is strongly adsorbed on soil, has no biological role and is a close analogue of potassium; Sr is less strongly adsorbed on soil and behaves very similarly to calcium; and Tc is very mobile in soil as pertechnetate, but immobilised when reduced to Tc(IV), it is also considered to be easily assimilated by biological systems. We found that mycorrhizal association had no effect on root-to-needle transfer of Cs, but increased root uptake and that this increase could not be explained by improved potassium nutrition. In contrast, the symbiotic relation decreased Tc soil-to-needle transfer, but this resulted from complex dynamics of root uptake and rapid immobilisation of Tc in soil. No effect of mycorrhizal association on Sr, like its stable analogue Ca, was observed. The addition of a phytotoxic metal, Cu, inhibited mycorrhizal association, thus eliminating the effects observed for non-contaminated plant-fungus couples, but had no additional effect on radionuclide dynamics.

Fate of prions in soil: adsorption and extraction by electroelution of recombinant ovine prion protein from montmorillonite and natural soils.

Prions, the infectious agents thought to be responsible for transmissible spongiform encephalopathies, may contaminate soils and have been reported to persist there for years. We have studied the adsorption and desorption of a model recombinant prion protein on montmorillonite and natural soil samples in order to elucidate mechanisms of prion retention in soils. Clay minerals, such as montmorillonite, are known to be strong adsorbents for organic molecules, including proteins. Montmorillonite was found to have a large and selective adsorption capacity for both the normal and the aggregated prion protein. Adsorption occurred mainly via the N-terminal domain of the protein. Incubation with standard buffers and detergents did not desorb the full length protein from montmorillonite, emphasizing the largely irreversible trapping of prion protein by this soil constituent. An original electroelution method was developed to extract prion protein from both montmorillonite and natural soil samples, allowing quantification when coupled with rapid prion detection tests. This easy-to-perform method produced concentrated prion protein extracts and allowed detection of protein at levels as low as 0.2 ppb in natural soils.

Evolution of Sr distribution coefficient as a function of time, incubation conditions and measurement technique.

A thorough understanding of the dynamics of radiostrontium in soil is required to allow accurate long-term predictions of its mobility. We have followed the soil solution distribution of 85Sr as a function of time under controlled conditions over 4 months and studied the effect of soil moisture content and organic matter amendments. Data have been compared to redox conditions and soil pH. To fuel the ongoing debate on the validity of distribution coefficient (K(d)) values measured in dilute suspension, we have compared values obtained from the activity concentration in soil solution obtained by centrifugation to data obtained in suspension with or without air-drying of the soil samples after incubation. The 85Sr adsorption properties of soil, incubated without prior contamination were also measured. There is some time-dependent adsorption of Sr. This is partly due to changing soil composition due to the decomposition of added organic matter and anaerobic conditions induced by flooding. There is also a kinetic effect, but adsorption remains largely reversible. Most of the observed effects are lost when soil is suspended in electrolyte solution.