The invasion of pea roots, pisum sativum l., by soil microorganisms, Acanthamoeba palestinensis (reich) and pseudomonas SP

Pea roots (Pisum sativum L. cultivar Tall Sugar White) were inoculated with Pseudomonas sp., isolated from the roots of Timothy grass (Phleum pratensis L.). Microscopic examination showed that the bacterium had invaded many epidermal and outer cortical cells of the root. The presence of the soil amoeba, Acanthamoeba palestinensis (Reich), which readily ingests Pseudomonas sp., did not alter the root damage. The amoebae were also found in the epidermis and outer cortex of pea roots. No amoebae were found inside pea roots when Pseudomonas sp. was absent. Bacterial invasion also occurred in pea roots grown in garden soil previously sterilized by γ-irradiation and inoculated with Pseudomonas sp., but not in the same soil inoculated and unsterilized. Timothy grass roots were similarly uninfected in inoculated unsterilized garden soil.     

The model predator Acanthamoeba castellanii induces the production of 2,4, DAPG by the biocontrol strain Pseudomonas fluorescens Q2-87

Fluorescent pseudomonads show great potential as biological control agents due to their capability to produce antifungal toxins such as 2,4-diacetylphloroglucinol (DAPG). DAPG is synthesized from the precursor monoacetyl-phloroglucinol (MAPG) and its production depends on the metabolic state of the bacteria as well as on their interaction with other organisms. In this study we show that Pseudomonas fluorescens responds to the bacterivorous amoeba Acanthamoeba castellanii by upregulating the production of phloroglucinol derivates in a density-dependent manner. Living amoebae caused moreover a distortion of the MAPG to DAPG balance in favor of the latter, suggesting that amoebae may interfere with the first steps of DAPG synthesis. Predator-prey interactions appear thus to be an important factor for the regulation of antibiotics production in biocontrol microorganisms.     

Soil fungi as food for giant amoebae

Feeding trials were carried out to assess the ability of a giant vampyrellid soil amoeba to attack and lyse spores of fungi. Of 24 species of fungi studies, 15 were perforated in the same manner as was reported for Cochliobolus sativus. This giant amoeba is nutritionally versatile and can feed on bacteria, flagellates, blue green algae, diatoms and nematodes. Seven other soil amoebae failed to lyse conidia of C. sativus.     

A new method for determining the metabolic activity of specific bacterial populations in soil using tritiated leucine and immunomagnetic separation

 A new assay, using immunomagnetic separation and uptake of tritiated leucine ([³H]-Leu), was developed for measuring the in situ metabolic activity of specific bacterial populations in soil. Such assays are needed to assess the role individual species play in diverse microbial soil communities. The method was optimized using Pseudomonas putida KT2440 : :Tc⁺/TOL::gfp inoculated into soil microcosms. Inoculated soil samples were incubated with [³H]-Leu followed by an immunomagnetic separation to recover the target bacteria. Radiolabel incorporated by the target bacteria was then measured. Incubation time with immunomagnetic beads was not critical for optimal target cell recovery, but samples needed to be washed at least 5 times during the immunomagnetic separation to reduce unspecific binding of the indigenous soil bacteria to the magnetic beads. Soil absorption of the polyclonal antibody further reduced this unspecific binding, resulting in <1% contamination by indigenous soil bacteria relative to numbers of recovered target cells. The assay was tested by investigating the effect of different incubation temperatures on the metabolic activity of the target cells. As expected, a linear relationship between activity and temperature was observed, demonstrating the sensitivity of the assay. The method was applied to compare activities of the target strain in bulk soil and in the rhizosphere of barley. Contrary to what was anticipated, no significant difference in metabolic activity was observed. Received: 4 November 1999     

Seasonal distribution of air-borne pathogenic and free-living amoebae in Mexico City and its suburbs

A survey was carried out over a one-year period to isolate amoebae suspended in the air of Mexico City and its suburbs. Sampling stations were placed at the four cardinal points of the metropolitan area. Selective media were used to culture the amoebae isolated. Specialized taxonomic keys and physical and physiological tests were used for identification, and a statistical analysis was performed to determine the correlations between physico-chemical and biological parameters. 108 strains were isolated, of which 19 were pathogenic via intracerebral inoculation and 9 via intranasal inoculation. Species of the generaAcanthamoeba, Vahlkampfia andHartmannella were most abundant.Acanthamoeba polyphaga showed the highest abundance. Several times during the period of the study SO₂ O₃, CO, NO and NO₂ exceeded the permissible levels established by the Mexican government. The ability of amoebae to form cysts and cyst size were important factors for their presence, survival, abundance and diversity in the atmosphere. The main source of air-borne amoebae was the soil. Factors that favored the incidence and diversity of the isolates were wind speed and direction, low relative humidity, generation of frequent dust-storms, resuspension of amoebae by vehicular traffic, proximity to garbage dumps and large extensions of bare soil. Soil cover was a factor associated with a reduction in the incidence and diversity of the aerial amoebae. This study demonstrates that there are viable cysts of amoebae in the atmosphere of Mexico City, that may have potential importance in the case of certain kinds of human allergies and diseases. Further research is needed to find out the aerial presence of viable cysts of obligatory, amphizoic or opportunistic amoebic parasites, and to clarify the qualitative and quantitative effects of the local meteorological and physico-chemical environment on the free-living amoebae present in the atmosphere.     

Infection of Mayorella penardi (Gymnamoebia) in oak-hornbeam forest soil by the ectoparasitic fungus Amoebophilus simplex (Zygomycota)

During research on naked amoebae in terrestrial habitats in the oak-hornbeam forests of Malé Karpaty Mts. (Western Slovakia), a mass fungal infection in Mayorella penardi Page, 1972 (Euamoebida: Paramoebidae) was observed. In one of the 243 soil and moss samples examined a remarkably high percentage (80%) of amoebae were infected by the ectoparasitic soil fungus Amoebophilus simplex Barron, 1983 (Zygomycota: Zoopagales), as revealed by detailed light microscopical observations of infected specimens. The parasitic fungus displayed characteristic cylindrical conidia (length 10 μm, breadth 3 μm) projecting out from the posterior part of the amoeba cell. Infecting conidium frequently produced a chain of two to three conidia and, rarely, secondary chains from the distal end. Two to four infections in a single amoeba were frequently observed. From the present study, and from earlier records, it can be deduced that Amoebophilus simplex is specialized in parasitizing mayorellid amoebae, maybe even Mayorella penardi exclusively. Two other species (Korotnevella diskophora and Korotnevella stella) from the family Paramoebidae were recorded in the same sample, but neither of them showed signs of infection.     

Effects of silver nanoparticle size, concentration and coating on soil quality as indicated by arylsulfatase and sulfite oxidase activities

Recently, the nanotechnology industry has seen a growing interest in integrating silver nanoparticles(AgNPs) into agricultural products, which increases soil exposure to these particles. This demands an investigation into the effect of AgNPs on soil health. Changes in soil enzyme activities upon exposure to AgNPs can serve as early indicators of any adverse effects that these particles may have on soil quality. This study aimed to determine the effects of AgNP size, concentration, coating, and e...     

Distribution of and insights from soil protozoa of the Olympic coniferous rain forest

Decomposition occurs in the surface litter and soil to support temperate rainforests, but little is known about the protozoa that stimulate bacterial activity and turnover. I examined surface litter and top soils, fallen logs, and epiphytes within 2 m from the soil surface in Olympic National Park, USA, of the Pacific Northwest Temperate Coniferous Rain Forest. Ciliates in surface litter numbered 180-580 g⁻¹ dry weight, but were reduced by 20-60% in the underlying soils. Testate amoebae numbered 18,000-77,000 g⁻¹ dry weight in both litter and soil although they were often more abundant in underlying soils. Rotting logs, essential for tree regeneration, supported similar numbers of ciliates, but twice the numbers of testate amoebae. In three epiphytic soils, ciliates numbered 350-550, and testate amoebae 35,000-195,000 g⁻¹ dry weight of soil. In these soils, 26 species of gymnamoebae, 64 species of ciliates, and 113 species of testate amoebae were found. About 65% of the individuals in ciliate and 45% in testate amoebae populations were small, r-selected taxa. Rain forest soil protozoa have distinct testate amoebae populations, and are characterized by enormous biodiversity, the dominance of acrostome species, the proliferation of Euglypha and Nebela species, and the appearance of aquatic taxa. Ecological succession of ciliates and testate amoebae follows an additive (non-replacement) pattern according to a neutral model. The large numbers of persistent r-selected species respond to ecosystem disturbances by mobilizing quickly to resume the bacterivory necessary to help restore the recovering above-ground plant community.     

An emerging energetic soil contaminant,CL-20, can affect the soil invertebrate community in a sandy loam soil

We investigated the effects of nitramine explosive CL-20 (China Lake compound 20) on the indigenous soil invertebrate community in Sassafras sandy loam (SSL) soil using a 12-week soil microcosm assay. Freshly collected SSL soil was amended with CL-20 to prepare multiple treatment concentrations ranging from 0 (acetone control) to 10,300 mg kg⁻¹. The selected concentration range of CL-20 adequately assessed the concentration–response relationships for total microarthropods, and for individual microarthropod groups. The overall composition of microarthropod community in SSL soil was not affected by exposure to CL-20, based on the number of taxonomic groups present in the individual treatments after 12 weeks. However, community structure analysis revealed greater sensitivity to CL-20 by predatory mesostigmatid mites. Microarthropod and nematode communities showed contrasting sensitivities to CL-20 in SSL soil. Total numbers of nematodes were either unaffected or significantly (p < 0.05) increased in CL-20 treatments compared with control. Only predator group among nematodes was consistently adversely affected by exposure to CL-20. The abundance of predatory nematodes decreased in a concentration-dependent manner throughout the 12-week exposure. Microcosm assay with corresponding community structure analysis can provide the means for validating the ecotoxicity data from standardized laboratory tests, both complimenting and expanding upon the ecotoxicological significance of data from standardized single-species toxicity tests.     

Exposure and effects assessments of Bt-maize on non-target organisms (gastropods, microarthropods, mycorrhizal fungi) in microcosms

Potential differences between Bt-maize (MEB307 expressing the insecticidal Cry1Ab protein) and a near-isogenic non-Bt variety (Monumental) in their influence on the garden snail (Helix aspersa), soil microarthropods (Collembola, Actinedida, Acaridida, Gamasida and Oribatida) and mycorrhizal fungi were studied. Growing snails were caged in microcosms allowing the development of Bt or non-Bt-maize (Zea mays L.) on a sandy loam soil. After 3 months exposure, survival and growth of snails were similar in both treatments. Cry1Ab protein was detected in the Bt-maize leaves (22–42.2 μg Bt protein g⁻¹ dry wt), in the snail tissues (0.04–0.11 μg Bt-protein g⁻¹ dry wt) and in their faeces (0.034–5 μg Bt-protein g⁻¹ dry wt). Total soil microarthropod abundance and diversity were similar between control (non-Bt-maize) and the genetically modified (GM) Bt-maize microcosms. The mycorrhizal colonization of roots did not differ between Bt and non-Bt-maize (frequency of mycorrhizal roots was 88.7% and 83.3% respectively). The mycorrhizal infectivity of soils, expressed as MI₅₀ (minimum soil dry weight required to colonize 50% of plants) was measured using red clover. MI₅₀ was similar for soils where Bt or non-Bt-maize was cultivated for 4 months. The detection of Cry1Ab protein in the viscera and faeces of H. aspersa exposed to Bt-maize indicates that snails contribute to the transfer of the Bt-protein from plant to soil or snail predators. This may constitute an alternative route of exposure for Bt-protein in soil, but this was without a negative influence on mycorrhizal fungi or microarthropods. Results showed that Bt-maize was not toxic for the selected non-target species exposed for 3 or 4 months. The microcosms and analyses used in this study represent new methods for assessing effects of chronic exposure to GM plants of several diverse, yet ecologically and temporally associated species. As the soil organisms we studied can also be used in standardized ecotoxicological tests (XP X31-205-2 for mycorrhizal fungi, ISO 11267 for Collembola and ISO 15952 for snails), microcosm exposures represent a way to link laboratory and field methods for the ecotoxicological evaluation of GM plants.     

Effects of Silver Nanoparticles on the Activities of Soil Enzymes Involved in Carbon and Nutrient Cycling

Soils are continuously exposed to large amounts of engineered nanoparticles, especially silver nanoparticles (AgNPs), which can affect the activity, stability, and specificity of microbial enzymes. Therefore, the measurement of specific enzyme activity can be used to identify major changes in soil environments. Accordingly, the aim of the present study was to investigate the effects of AgNPs on soil enzymes that play critical roles in mineralizing carbon and nutrients in soil. Soil samples (silt loam and sandy loam) were collected from the surface layer (0–15 cm) of a field at the George Washington Carver Farm, Lincoln University of Missouri, USA. The soils were then treated with AgNP solutions at 0, 1 600, or 3 200 μg Ag kg~(-1) dry soil, using either 10- or 50-nm AgNPs and a randomized complete block design, with three replicates per treatment. The AgNP-treated soil samples were homogenized and incubated for one month, and soil acid phosphatase, β-glucosaminidase, β-glucosidase, and arylsulfatase activities were measured after one hour, one week, and one month of incubation. The activities of all four enzymes were reduced by AgNP treatment after one hour and one week.However, AgNP size had no effect. After one month of incubation, the AgNP treatments had mixed effects, which suggests that soil enzymes are only affected on a short-term basis. Further studies are required to determine the mechanisms by which AgNPs reduce soil enzyme activity.     

The use of biologically based phosphorus fractions to evaluate soil P availability in reduced P‐input paddy soils

Chemical soil phosphorus (P) extraction has been widely used to characterize and understand changes in soil P fractions; however, it does not adequately capture rhizosphere processes. In this study, we used the biologically based phosphorus (BBP ) grading method to evaluate the availability and influencing factors of soil P under four P fertilizer regimes in a typical rice–wheat cropping rotation paddy field. Soil P was assessed after seven rice‐growth seasons at multiple growth stages: the seedling, the booting and the harvest stage. Soil CaCl₂‐P, citrate‐P and HC l‐P (inorganic P, Pi) as well as enzyme‐P (organic P, Po) were not significantly different between soil treated with P fertilizer during the wheat season only (PW ) and during the rice season only (PR ) compared with soil treated during both the rice and the wheat seasons (PR +W) at all three rice‐growth stages. No P fertilizer application during either season (Pzero) significantly reduced the concentration of soil citrate‐P and HC l‐P at the rice‐seedling and harvest stages. Significant correlations were observed between the HC l extraction and Olsen‐P (R ² = 0.823, P  <  0.001), followed by enzyme‐P (R ² = 0.712, P  <  0.001), citrate‐P (R ² = 0.591, P  <  0.001) and CaCl₂‐P (R ² = 0.133, P  <  0.05). Further redundancy analysis (RDA ) suggested that soil alkaline phosphatase (S‐ALP ) activity played a role in soil P speciation changes and was significantly correlated with enzyme‐P, citrate‐P and HC l‐P. These results may improve our ability to characterize and understand changes in soil P status while minimizing the overapplication of P fertilizer.     

An investigation of the pathogenic and non-pathogenic free-living amoebae from the root zone method of wastewater treatment

Pathogenic and non-pathogenic free-living amoebae from the Root Zone Method (RZM) of wastewater treatment were investigated. Ten reed beds planted on four different substrate types were compared for their efficiency of removal of free-living amoebae. Reed beds planted on coarse substrates generally gave the highest removal rates (up to 100%) while those planted in soil gave the lowest (60.4%). Possible reasons for differences in removal efficiencies are discussed. Thirteen species representing nine genera of free-living amoebae were isolated; these included three species of Acanthamoeba (A. astronyxis, A. polyphaga and A.rhysodes) all of which were pathogenic for mice. The implications for RZM design of the presence of potentially pathogenic free-living amoebae in the effluent are discussed.     

Density,metabolic activity,and identity of cultivable rhizosphere bacteria on Salix viminalis in disturbed arable and landfill soils

The objective of this study was to provide fundamental data for a subsequent selection of willow growth and soil remediation promoting bacterial strains. The rhizosphere of willows (Salix viminalis) was screened for cultivable bacteria with high enzymatic activity (proteolytic, pectolytic, cellulolytic, amylolytic) and production of siderophores at four test sites with broad spectrum of anthropogenic soil disturbance: sewage‐sludge application, impoverishment by unfavorable arable use, ash dumping, and household‐waste depositing. The density of bacteria in the rhizosphere ranged from 7.92 to 8.56 log₁₀ of colony‐forming units per gram dry weight of soil and varied in a site‐ and willow‐clone‐specific manner. Within the 240 bacterial strains, a high diversity of metabolic activities was observed but was rarely combined in one strain (1.2% having six and 5.8% having five out of seven metabolic activities, respectively). The majority of strains (79.2%) revealed just one or two metabolic activities. Most common was a combination of lipolytic, proteolytic activities, and siderophore production as found in 13.8% of the bacterial strains. The 50 strains with the highest metabolic activity belonged predominantly to the Gammaproteobacteria (66%), the others to Flavobacteria (18%), Betaproteobacteria (8%), Actinobacteria (4%), and Bacilli (4%). The highest portion of cultivable strains of rhizosphere bacteria with high metabolic activities belonged to the genera Pseudomonas, Serratia, and Flavobacterium. We hypothesize that these genera include strains that support willow growth and soil remediation. Therefore, the described strain collection from the rhizosphere of S. viminalis provides a valuable basis for a subsequent selection of these candidates for applications in improvement of site adaptation of plants or remediation of soils.     

Assessment of soil erosion at large watershed scale using RUSLE and GIS: a case study in the Loess Plateau of China

Soil erosion is a serious problem in the Loess Plateau of China, and assessment of soil erosion at large watershed scale is urgently need. This study used RUSLE and GIS to assess soil loss in the Yanhe watershed. All factors used in the RUSLE were calculated for the watershed using local data. RUSLE‐factor maps were made. The mean values of the R‐factor, K‐factor, LS‐factor, C‐factor and P‐factor were 970 209 MJ km⁻² h⁻¹ a⁻¹, 0·0195 Mg h MJ⁻¹ mm⁻¹, 10·27, 0·33359 and 0·2135 respectively. The mean value of the annual average soil loss was found to be 14 458 Mg km⁻² per year, and the soil loss rate in most areas was between 5000 and 20 000 Mg km⁻² per year. There is more erosion in the centre and southeast than in the northwest of Yanhe watershed. Because of the limitations of the RUSLE and spatial heterogeneity, more work should be done on the RUSLE‐factor accuracy, scale effects, etc. Furthermore, it is necessary to apply some physical models in the future, to identify the transport and deposition processes of sediment at a large scale. Copyright © 2005 John Wiley & Sons, Ltd.     

Tracking the Transport of Silver Nanoparticles in Soil: a Saturated Column Experiment

Silver nanoparticles (AgNPs) can enter the environment when released from products containing them. As AgNPs enter soil, they are often retained in the soil profile and/or leached to the groundwater. This research assessed the transport of AgNPs in their “particle form” through the soil profile using a series of columns. Three soil types were put into soil columns: LSH (loam with high organic matter (OM)), LSL (loam with low OM), and Sand (no OM). The results showed that AgNP transport and retention in soil as well as particle size changes are affected by soil organic matter (OM) and the cation exchange capacity (CEC) of soil. OM affected the transport and retention of AgNPs. This was evident in the LSH columns where the OM concentration was the highest and the AgNP content the lowest in the soil layers and in the effluent water. The highest transported AgNP content was detected in the Sand columns where OM was the lowest. CEC had an impact on the particle size of the AgNPs that were retained in the soil layers. This was clear in columns packed with high CEC-containing soils (LSL and LSH) where AgNP particle size decreased more substantially than in the columns packed with sand. However, the decrease in AgNP sizes in the effluent water was less than the decrease in particle size of AgNPs transported through but retained in the soil. This means that the AgNPs that reached the effluent were transported directly from the first layer through the soil macropores. This work highlights the ability to track AgNPs at low concentrations (50 μg kg^?1) and monitor the changes in particle size potential as the particles leach through soil all of which increases our knowledge about AgNP transport mechanisms in porous media.     

Use of the fluorescent antibody technique to characterize equilibrium survival concentrations of Bradyrhizobium japonicum strains in soil

Summary The ability of Bradyrhizobium strains to survive saprophytically in soil was studied by means of fluorescent antibodies (FA). It was found that the recovery rate may be considered a constant value although the limit of detection by the FA technique is approximately 10³ bacteria g^–1 soil. By studying the survival kinetics of B. japonicum strains introduced into soils, we observed that whichever soil-strain combination was tested in a given soil during the incubation all the different populations of a strain reached the same survival balance level, generally about 10³–10⁴ Bradyrhizobium g^–1 soil. When we reintroduced strains into a soil containing rhizobia of the same specificity, the new inhabitants reached the same equilibrium level as that of the initial population. The balance threshold level does not appear to be a very sensitive way to classify, strains on their saprophytic ability. We suggest that survival kinetics should be characterized by the rate at which the population reaches equilibrium.     

Using electrical signals of microbial fuel cells to detect copper stress on soil microorganisms

A method based on microbial fuel cells (MFCs) was used to evaluate the effects of copper (Cu²⁺) on soil microorganisms. Soil spiked with 50–400 mg kg^?1 of Cu²⁺ as CuCl₂ was incubated for 24 hours before being packed into the MFC anode chambers and assayed for dehydrogenase activity (DHA), substrate‐induced respiration (SIR) and microbial biomass carbon (C_mic). Soil was amended with 5% (w/w) glucose to accelerate ‘start‐up’ and improve power generation, followed by 150 hours of operation. Anode biofilm and soil was extracted to recover total nucleic acids and the 16S rRNA gene was subjected to PCR‐DGGE, sequencing and phylogenetic analysis. Results showed that increases in soil Cu²⁺ concentrations reduced voltage and postponed start‐up. The quantity of generated electrons within 48 hours was 32.5 coulomb (C) in the without‐Cu control and decreased with increasing Cu²⁺ concentrations (11.7, 7.7, 2.0 and 1.3 C under 50, 100, 200 and 400 mg kg^?1 Cu²⁺, respectively). Cyclic voltammetry identified decreased soil electrochemical activity with increasing Cu²⁺ concentrations. The results indicate that Cu²⁺ reduced electrical signals by inhibiting the electrochemical activity, metabolic activity and biomass of microorganisms. The 16S sequences of recovered anodic bacteria were assigned to Firmicutes, including Bacillaceae, Acetobacteraceae, Clostridium, Bacillus and Sporolactobacillus. In general, the DGGE band intensity of anodic bacteria decreased with increasing Cu²⁺ concentrations, except for bands assigned to Firmicutes and Bacillus, which increased with increasing Cu²⁺ concentrations. We suggest that the short‐term electrical signals generated from MFCs with contaminated soil can be used to assess the toxic effect of heavy metal pollutants on soil microorganisms.     

Modeling of the fine‐scale temperature response of arylsulfatase activity in soil

Fine‐scale (1.0–2.2 °C) temperature dependence of soil arylsulfatase activity (arylsulfate sulfohydrolase, EC 3.1.6.1) was measured at 0 to 75 °C in a Danish sandy, arable soil. Assays were done with field‐moist soil samples in the absence of toluene as plasmolytic agent – a procedure that primarily measures the extracellular enzymes. The aim was to evaluate the use of temperature models to describe the temperature response of soil arylsulfatase activity. In addition, we searched for increases in activity at high temperatures (e.g., 50–60 °C), which might be associated with unmasking (exposure) of intracellular enzymes. Arylsulfatase activities ranged from 1.1 to 60.3 μg p‐nitrophenol (g dry weight soil)^–1 h^–1, with an optimum temperature at 58.1 °C. The temperature response below 58.1 °C could be described by the Arrhenius equation (r² = 0.978, n = 83) and the simple Ratkowsky equation (r² = 0.977, n = 83). The expanded Ratkowsky equation, which covered the entire temperature range (0–75 °C), was less satisfactory (r² = 0.958, n = 90) because the model underestimated the reaction rates near the optimum temperature. The activation energy (E_a) calculated from the Arrhenius equation was 42.2 kJ mol^–1. This was higher than previously found for other soils (16.5–34.7 kJ mol^–1), possibly due to the use of toluene in these studies. Further analysis of the temperature response showed that no increase in activity occurred due to potential unmasking of intracellular enzymes by disintegration of bacterial cell membranes at high temperatures. Thus, the use of high incubation temperatures did not facilitate the differentiation between intra‐ and extracellular enzyme activity.     

The ability of the DGT soil phosphorus test to predict pasture response in Australian pasture soils – a preliminary assessment

Diffusive gradients in thin‐films (DGT) technology provides an alternative assessment of available phosphorus (P) for a range of crops, suggesting a preliminary examination of the performance of the new DGT‐P test, compared to existing bicarbonate extractable Olsen and Colwell P tests, for pastures is justified. This study utilized historic data from the Australian National Reactive Phosphate Rock (NRPR) study (1992–1994) that included 25 experimental sites representing a wide range of soil types and climates used for pasture production. Stored (~19 yr) soil samples were analysed for DGT‐P, Olsen P and a single point P buffering index (PBI) and re‐analysed for Colwell P. Results showed the traditional bicarbonate extractable Colwell (r² = 0.45, P < 0.001) and Olsen P (r² = 0.27, P < 0.001) methods predicted relative pasture P response more accurately, compared to the novel DGT‐P test (r² = 0.09, P = 0.03) when all 3 yr of data were examined. We hypothesize that the harsher bicarbonate extraction used for the Olsen and Colwell methods more accurately reflects the ability of perennial pasture roots to access less labile forms of P, in contrast to the DGT‐P test, which does not change the soil pH or dilute the soil and appears unable to fully account for a plants ability to solubilize P. Further studies are needed to compare the capacity of DGT‐P to measure P availability in perennial pasture systems and to better understand the soil chemical differences between pasture and cropping systems.