Simple and Portable Magnetic Immunoassay for Rapid Detection and Sensitive Quantification of Plant Viruses

Plant pathogens cause major economic losses in the agricultural industry because late detection delays the implementation of measures that can prevent their dissemination. Sensitive and robust procedures for the rapid detection of plant pathogens are therefore required to reduce yield losses and the use of expensive, environmentally damaging chemicals. Here we describe a simple and portable system for the rapid detection of viral pathogens in infected plants based on immunofiltration, subsequent magnetic detection, and the quantification of magnetically labeled virus particles. Grapevine fanleaf virus (GFLV) was chosen as a model pathogen. Monoclonal antibodies recognizing the GFLV capsid protein were immobilized onto immunofiltration columns, and the same antibodies were linked to magnetic nanoparticles. GFLV was quantified by immunofiltration with magnetic labeling in a double-antibody sandwich configuration. A magnetic frequency mixing technique, in which a two-frequency magnetic excitation field was used to induce a sum frequency signal in the resonant detection coil, corresponding to the virus concentration within the immunofiltration column, was used for high-sensitivity quantification. We were able to measure GFLV concentrations in the range of 6 ng/ml to 20 μg/ml in less than 30 min. The magnetic immunoassay could also be adapted to detect other plant viruses, including Potato virus X and Tobacco mosaic virus, with detection limits of 2 to 60 ng/ml.     

Phosphorus efficiency of plants

Föhse et al. (1988) have shown that P influx per unit root length in seven plant species growing in a low-P soil varied from 0.6×10^-14 to 4.8×10^-14 mol cm^-1s^-1. The objective of this work was to investigate the reasons for these differences. No correlation was found between P influx and root radius, root hairs, cation-anion balance and Ca uptake. However, when root hairs were included in mathematical model calculations, the differences of P influx could be accounted for. These calculations have shown that in soils low in available P, contribution to P uptake by root hairs was up to 90% of total uptake. The large contribution of root hairs to P uptake was partly due to their surface area, which was similar to that of the root cylinder. However, the main reason for the high P uptake efficiency of root hairs was their small radius (approx. 5×10^-4 cm) and their perpendicular growth into the soil from the root axis. Because of the small radius compared to root axes, P concentration at root hair surfaces decreased at a slower pace and therefore P influx remained higher. Under these conditions higher I_max (maximum influx) or smaller K_m values (Michaelis constant) increased P influx. The main reasons for differences found in P influx among species were the size of I_max and the number and length of root hairs. In a soil low in available P, plant species having more root hairs were able to satisfy a higher proportion of their P demand required for maximum growth.     

Mobilization of different phosphate fractions in the rhizosphere

Availability of soil P fractions and mechanisms of acquisition by plants were studied. Plants mobilize soil P by desorption via depletion of P solution concentration around roots. In an oxisol, the process was enhanced by nitrate N nutrition of ryegrass, which increased soil pH, and by carboxylate release by white lupin. Ligand exchange and Fe/Al solubilization are assumed to be the mechanisms. Ammonium N nutrition of ryegrass decreased pH and allowed P mobilization in a luvisol but had no such effect in an oxisol, due to acid solubility of P in these soils. Organic P dissolved in soil solution contributed one third to the P uptake of field-grown barley on a luvisol. Laboratory experiments suggest that organic P is hydrolyzed by phosphatases at the root surface and replenished by micro-organisms.     

Mobilization of phosphate in different soils by ryegrass supplied with ammonium or nitrate

Mobilization of soil P as the result of plant-induced changes of soil pH in the vicinity of plant roots was studied. Seedlings of ryegrass were grown in small containers separating roots from soil by a 30-μm meshed nylon screen which root hairs could penetrate but not roots. Two soils were used, a luvisol containing P mainly bound to calcium and an oxisol containing P mainly bound (adsorbed) to iron and aluminum. Plant-induced changes of soil pH were brought about by application of ammonium-or nitrate-nitrogen. After plants had grown for 10 d the soil was sliced in thin layers parallel to the root mat which had developed on the screen, and both soil pH and residual P determined. Mobilization of P was assessed by P-depletion profiles of the rhizosphere soil. Soil pH at the root surface decreased by up to 1.6 units as the result of ammonium N nutrition and it increased by up to 0.6 units as the result of nitrate N nutrition. These changes extended to a distance between 1 and 4 mm from the root surface depending on the type of soil and the source and level of nitrogen applied. In the luvisol, compared to zero-N treatment, P mobilization increased with the NH₄-induced decrease in pH, whereas the NO₃-induced pH increase had no effect. In contrast, in the oxisol a similar pH decrease caused by NH₄ nutrition had no effect, whereas the pH increase caused by NO₃ increased markedly the mobilization of soil P. It is concluded that in the luvisol calcium phosphates were dissolved by acidification, whereas in the oxisol adsorbed phosphate was mobilized by ligand exchange.     

Utilization of organic phosphorus in calcium chloride extracts of soil by barley plants and hydrolysis by acid and alkaline phosphatases

Organic phosphorus is often a major part of total phosphorus in soil solution. The role of this fraction as a P source for plants and the mechanism involved in its transfer from soil to plant is still unclear. We studied the utilization of organic phospharus in 0.01 M calcium chloride extracts by barley and its hydrolysis by isolated acid and alkaline phosphatases. Calcium chloride extracts were used as a nutrient solution in 24 hrs assays. Concentration of organic and inorganic P in equilibrium calcium chloride extracts was 7.8 and 1.8 µmol P L^-1, respectively, which was similar to the soil solution P concentration. When soil microbial biomass was destroyed by autoclaving, organic P concentration increased to 64.8 µmol P L^-1 whereas the inorganic P was hardly changed. Inoculation of the autoclaved soil with non-sterile soil and incubation for 5 days decreased the organic P concentration to 27.9 µmol P L^-1 but did not change inorganic P. In this study barley plants utilized organic P from all extracts. The greatest reduction of organic P concentration occurred in fresh extracts of the autoclaved soil. Inorganic P was depleted to traces in all extracts. Organic P was hydrolyzed by isolated acid and alkaline phosphatases. We conclude that organic P in soil solution is a heterogeneous pool of organic P compounds originating from microbial biomass. Its initial availability to plants was nigh but its susceptibility to phosphatase hydrolysis was quickly reduced but not completely lost.     

Influence of phosphate and nitrate supply on root hair formation of rape,spinach and tomato plants

Summary Experiments with tomato, rape and spinach in nutrient solutions have shown that the formation of root hairs is strongly influenced by phosphate and nitrate supply. Decreasing the phosphate concentration of the nutrient solution from 100 to 2 M P resulted in an increase of root hair length from 0.1–0.2 to 0.7 mm of the three plant species. Root hair density also increased by a factor of 2–4 when the P concentration was lowered from 1000 to 2 M. The variation of these two root properties raised the root surface area by a factor of 2 or 3 compared to plants well supplied with P. Root hair length was closely related to the phosphate content of the root and shoot material. On the other hand, spinach plants grown in a split-root experiment produced root hairs in solutions of high P concentration (1000M P) if the major part of the total root system was exposed to low P concentration (2 M P). It is therefore concluded that the formation of root hairs does not depend on directly the P concentration at the root surface but on the P content of the plant.Similar experiments with nitrate also resulted in an increase in length and density of root hairs with the decrease of concentration below 1000 M. In this case marked differences between plant species occurred. At 2 M compared to 1000 M NO₃ root hair length of tomato increased by a factor of 2, of rape by a factor of 5 and of spinach by a factor of 9. Root hair length was correlated, but not very closely, to the total nitrogen content of the plants. It is concluded, that the influence of nutrient supply on the formation of root hairs is a mechanism for regulating the nutrient uptake of plants.Dedicated to Prof. Dr. E. Welte on the occasion of his 70th anniversary.     

Impact of post-surgical freezing delay on brain tumor metabolomics

Metabolomics - The identification of frequent acquired mutations shows that patients with oligodendrogliomas have divergent biology with differing prognoses regardless of histological...     

A new approach to quantify the utilization of non-exchangeable soil potassium by plants

To predict the contribution of soil K fractions of different mobility to K supply of plants, the kinetics of K release from soil was related to the kinetics of K uptake of young sugar beet and wheat plants. For this purpose K release rates from soil were measured by continuously percolating samples of a luvisol with 0.01 M CaCl₂ solution and effective diffusion coefficients, D_e, were determined. Two soil K fractions of different mobility were obtained. D_e values of the more mobile exchangeable K and the less mobile non-exchangeable K fraction were found to be 58.9 × 10^–9 and 8.2 × 10^–9 cm² s^–1, respectively. In a pot experiment, sugar beet and wheat plants were grown, for 15 days and both root growth and K uptake were measured. K uptake kinetics of both crops was determined in a separate experiment using flowing solution culture. To integrate these data quantitatively, the simulation model of Claassen et al. (1986) was applied. Results show that calculated total K uptake agreed closely with real K uptake of the plants. On this basis, 64 and 79% of the K taken up by wheat and sugar beet plants was derived from the rapidly released exchangeable and 21–36% from the less mobile non-exchangeable soil K fraction.     

A method for determining concentration profiles at the soil-root interface by thin slicing rhizospheric soil

Summary A method is described for the determination of concentration gradients in the vicinity of plant roots. Plants are grown in small containers in which the roots are separated from the soil by a screen of nylon cloth. Root hairs but not roots penetrate the screen into the soil. In order to investigate the rhizospheric soil, the soil is frozen by liquid nitrogen and sliced into layers about 0.06 mm thick by means of a refrigerated microtome.