Glomalin‐related soil protein in French temperate forest soils: interference in the Bradford assay caused by co‐extracted humic substances

Thermostable soil protein, known as glomalin, is an important component of soil carbon stocks. Thought to originate from endomycorrhizal fungi, Glomales, this operationally‐defined fraction of soil organic matter contains proteins of diverse origin as well as non‐protein material, including humic substances. Accumulation results from the balance between production/release and subsequent degradation. Quantification of the protein is subject to uncertainty because of the co‐extraction of other components that interfere with the Bradford assay. We studied 10 topsoils from French temperate forests, taken from the national forest monitoring network (Renecofor). Two fractions were extracted, easily extractable (EE) at neutral pH and total extractable (T) at pH 8. Protein was quantified with the colorimetric Bradford method, either by direct calibration using bovine serum albumin (BSA) or by extrapolation of the standard addition plot of BSA. Solubilized organic matter was characterized by using absorbance at 465 and 665 nm and by three‐dimensional fluorescence excitation‐emission spectroscopy. Neither soil properties nor forest cover influenced glomalin‐related soil protein (GRSP) content. Direct assay gave the GRSP_EE to be about 1 g kg^?1 soil, and GRSP_T in the range 3–10 g kg^?1, accounting for about 2% of soil organic carbon and about 15% of soil nitrogen. Standard addition plots indicated a two to sixfold under‐estimation of protein in total extracts, caused by negative interference with the Bradford assay. The GRSP_EE was correlated significantly with both estimates of GRSP_T. Under‐estimation of GRSP_T by direct assay was not related to the E4:E6 ratio but was correlated significantly with the intensity of absorbance at either 460 or 660 nm and with one of the fluorescence peaks. We conclude that GRSP_EE is not necessarily more recent than GRSP_T and that both fractions may be probes of protein content, but that absolute contents may be under‐estimated because of co‐extracted humic substances.     

Losses of glomalin-related soil protein under prolonged arable cropping: A chronosequence study in sandy soils of the South African Highveld

Residues of arbuscular mycorrhizal fungi (AMF) may be important for agroecosystem functioning due to their ability to promote soil aggregation, especially in coarse textured soils with little biomass input and low capacity to conserve soil organic matter (SOM). Our aim was to assess the fate of AMF residues with prolonged arable cropping in coarse textured soils in a subtropical savannah assuming that glomalin-related soil protein (GRSP), especially the MAb32B11-immunoreactive fraction, mainly constitutes material of AMF origin. In three agroecosystems on the South African Highveld, surface soils were sampled. The former grassland soils had a history of up to 98 yr of cropping. We measured four GRSP fractions: Bradford-reactive soil protein (BRSP) and immunoreactive soil protein (IRSP), and easily extractable fractions of both. The primary grassland sites exhibited generally low contents of SOM and low GRSP contents. Prolonged arable land use of former grassland soils reduced the content of GRSP further. The decline could be described with a mono-exponential function with rate constants ranging from 0.04 to 0.41 yr⁻¹. Depending on the GRSP fraction, steady-state conditions were reached after 11-92 yr on a level of 39-69% of the initial contents. We conclude that even though GRSP fractions had the same hypothesized origin, they comprised pools with different stability or replacement rate. Easily extractable IRSP was lost most rapidly. In contrast to carbon, nitrogen and microbial residue dynamics, GRSP contents were not reduced below a certain steady-state level, despite potentially negative management effects on AMF, such as tillage, inclusion of fallows into crop rotation and fertilization with inorganic phosphorus. The steady-state GRSP contents coincided with low, but steady agroecosystem yields under the given cropping management.     

Is glomalin an appropriate indicator of forest soil reactive nitrogen status?

In this paper we address total glomalin‐related soil protein (T‐GRSP) as a possible indicator of differences in forest soils related to reactive nitrogen and forest composition. We focused especially on the relationship between T‐GRSP (g kg⁻¹), soil organic carbon (SOC), and reactive nitrogen (N_r) availability among different categories of temperate forests and different horizons. Our study included 105 sampling sites divided into 5 categories, which vary in elevation and tree species composition (coniferous, deciduous, mixed). We detected significantly higher T‐GRSP and SOC in the F+H horizon under conifers. We assume that this observation might be attributed to suppression of decomposition of T‐GRSP and SOC by nature of coniferous litter. The lack of significant differences in T‐GRSP/SOC among the categories and the positive correlations between T‐GRSP and SOC in most of the categories confirmed the strong relationship of T‐GRSP with SOC. We found a significantly higher content of T‐GRSP in the F+H horizon for all studied forest categories. However, the contribution of T‐GRSP to SOC is significantly higher in the A horizon, which might be caused by stabilization of glomalin by mineral fraction, including clay minerals or by the belowground origin of glomalin. We found the increase of SOC with increasing N_r in the A horizon for most categories of forest. T‐GRSP follows this trend in the case of deciduous forests (decid), mixed forest (mixed), and mountain forests (mount). On the other hand, we detected a decrease of T‐GRSP with increasing N_r in the F+H horizon of coniferous forests (conif). Moreover the T‐GRSP/SOC decreases with the increase of N_r in the A horizon of conif, mixed and mount, which points to the higher sensitivity of forest with prevalence of coniferous trees. Our observations have confirmed an ecosystem‐specific relationship between T‐GRSP, SOC and N_r. We concluded that T‐GRSP in combination with T‐GRSP/SOC has the potential to reveal qualitative changes in soil organic matter (SOM) connected with increasing N_r.     

Chemical characteristics of glomalin-related soil protein (GRSP) extracted from soils of varying organic matter content

Glomalin is described in the literature as a N-linked glycoprotein and the putative gene product of arbuscular mycorrhizal fungi (AMF). Since the link between glomalin and various protein fractions in soil is not yet clearly defined, glomalin-related soil protein (GRSP) more appropriately describes glomalin's existence in natural organic matter (NOM). The objective of this study was to examine the chemical characteristics of GRSP present in several mineral and organic soils of varying organic carbon content. GRSP was isolated using high temperature sodium citrate extraction followed by either trichloroacetic acid (TCA) or hydrochloric acid (HCl) precipitation. GRSP was characterized by quantitative solid-state ¹³C DPMAS NMR, infrared (IR) spectroscopy, elemental analysis, and the Bradford assay for protein content. GRSP accounted for 25% and 52% of total C in the mineral soils and organic soil, respectively. Molar C/N and H/C ratios reveal that GRSP has less nitrogen than bovine serum albumin (BSA), and that GRSP extracted from the Pahokee peat soil possessed a more unsaturated, and thus aromatic character relative to the mineral soil GRSP, respectively. GRSP's high aromatic (42-49%) and carboxyl (24-30%) carbon contents and low aliphatic (4-11%) and carbohydrate-type carbon contents (4-16%) suggests that GRSP does not resemble a typical glycoprotein. In fact, the NMR spectra of GRSP closely resemble that of humic acid. GRSP extracted from mineral and organic soils possessed the same NMR fingerprint regardless of the precipitation method used (i.e., either TCA or HCl). It is likely that the current GRSP extraction methods, because of their similarity to the method used to extract humic acid, are coextracting both materials.     

Bradford-reactive soil proteins and aggregate stability under abandoned versus tilled olive groves in a semi-arid calcisol

Glomalin, a substance produced by arbuscular mycorrhizal fungi, is reported to play a role in soil aggregation, but this role has been questioned in soils rich in calcium carbonate. We studied the relationship between aggregation stability and glomalin in a Haplic Calcisol comparing abandoned and active cultivation of olive groves. Abandonment was associated with increases in soil organic carbon, the percentage of water stable aggregates (WSA_1-2mm), and easily extractable and total Bradford-reactive soil protein. WSA_1-2mm was strongly positively correlated with both easily extractable and total Bradford-reactive soil protein. While easily extractable Bradford-reactive soil protein measured in both stable and unstable aggregates did not show any significant differences, Bradford-reactive soil protein was twice as high in stable than in unstable aggregates under both tillage and abandonment. Our results suggest that Bradford-reactive soil protein influences aggregate stability, even in soils with low organic matter and high calcium carbonate contents. However, more research is needed to elucidate the role of easily extractable Bradford-reactive soil protein in soil aggregation.     

Characterization of glomalin as a hyphal wall component of arbuscular mycorrhizal fungi

Arbuscular mycorrhizal fungi (AMF) produce a protein, glomalin, quantified operationally in soils as glomalin-related soil protein (GRSP). GRSP concentrations in soil can range as high as several mg g⁻¹ soil, and GRSP is highly positively correlated with aggregate water stability. Given that AMF are obligate biotrophs (i.e. depending on host cells for their C supply), it is difficult to explain why apparently large amounts of glomalin would be produced and secreted actively into the soil, since the carbon could not be directly recaptured by the mycelium (and benefits to the AMF via increased soil structure would be diffuse and indirect). This apparent contradiction could be resolved by learning more about the pathway of delivery of glomalin into soil; namely, does this occur via secretion, or is glomalin tightly bound in the fungal walls and only released after hyphae are being degraded by the soil microbial community? In order to address this question, we grew the AMF Glomus intraradices in in vitro cultures and studied the release of glomalin from the mycelium and the accumulation of glomalin in the culture medium. Numerous protein-solubilizing treatments to release glomalin from the fungal mycelium were unsuccessful (including detergents, acid, base, solvents, and chaotropic agents), and the degree of harshness required to release the compound (autoclaving, enzymatic digestion) is consistent with the hypothesis that glomalin is tightly bound in hyphal and spore walls. Further, about 80% of glomalin (by weight) produced by the fungus was contained in hyphae and spores compared to that released into the culture medium, strongly suggesting that glomalin arrives mainly in soil via release from hyphae, and not primarily through secretion. These results point research on functions of glomalin and GRSP in a new direction, focusing on the contributions this protein makes to the living mycelium, rather than its role once it is released into the soil.     

Decomposition and the contribution of glomalin-related soil protein (GRSP) in heavy metal sequestration: Field experiment

Glomalin is a metal-sorbing glycoprotein excreted by arbuscular mycorrhizal fungi (AMF). One method of estimating glomalin in soils is as glomalin-related soil protein (GRSP). In this study the role of GRSP in sequestering Pb and Cd was investigated in an in situ field experiment. The effect of metal sequestration on the subsequent decomposition of GRSP was also investigated. GRSP was determined using the Bradford method as total glomalin-related soil protein (T-GRSP) and as easily extractable glomalin-related soil protein (EE-GRSP). After 140 days, GRSP bound Pb accounted for 0.21–1.78% of the total Pb, and GRSP bound Cd accounted for 0.38–0.98% of the total Cd content in the soil. However when compared on a soil organic matter (SOM) basis, only 4% of the Pb or Cd was bound to the GRSP fraction of the SOM compared with 40–54% of the Pb or Cd bound to the humin and fulvic acids in the SOM fraction. In soils contaminated with the highest levels of Pb and Cd, the T-GRSP (EE-GRSP) decomposition after 140 days was reduced by 8.0 (6.6)% and 7.0 (7.5)%, respectively, when compared with the controls. In the high Pb or Cd treatment groups we found that the fraction of metal bound to GRSP increased even though the total GRSP content declined over time. The mass ratio between Pb and GRSP-carbon changed from 2.3 to 271.4 mg (100 g)⁻¹ in all Pb levels soil, while with the high-Cd treatment group the mass ratio between Cd and GRSP-carbon (0.36 mg (100 g)⁻¹) was higher than the mass ratio seen with Cd-bound humic acid fractions. Our in situ field study shows that while GRSP does bind Pb and Cd, in the soils we investigated, the levels are insignificant compared to soil organic matter such as humic and fulvic acids.     

Glomalin-related soil protein contains non-mycorrhizal-related heat-stable proteins, lipids and humic materials

Glomalin is reportedly a stable and persistent protein produced in copious quantities by mycorrhizal fungi and may be an important pool of organic N in soil. Glomalin-related soil protein (GRSP), however, is only operationally defined by its extraction method, and has been only poorly characterized at best. The goal of this study was to characterize the molecular structures within GRSP. Synchrotron-based X-ray absorption near-edge structure (XANES) spectroscopy and pyrolysis field-ionization mass spectrometry (Py-FIMS) revealed that GRSP contains a consortium of proteins along with many impurities. Employing proteomic techniques, we found that glomalin itself may be a thioredoxin-containing chaperone; however, no homologies with proteins or DNA of mycorrhizal origin were detected. Proteomics techniques further revealed that this fraction contains large amounts of soil-related heat-stable proteins and proteins of non-mycorrhizal origin. Results of this research show that the current extraction procedure that defines GRSP yields a mixture of compounds and thereby overestimates glomalin stocks when quantified using the Bradford assay. The chemical nature of glomalin has yet to be conclusively determined; it is unlikely that the chemical structure of glomalin can be elucidated from the mixture extracted as GRSP. Instead, an investigation into the specific biochemistry of immunoreactive assays currently used to define GRSP, followed by proteomic characterization of monoxenic mycorrhizal cultures may be required to advance our understanding of the chemical nature and agronomic significance of GRSP in soils.     

Carbon and nitrogen in operationally defined soil organic matter pools

Humic substances [humic acid (HA), fulvic acid (FA), and insoluble humin], particulate organic matter (POM), and glomalin comprise the majority (ca 75%) of operationally defined extractable soil organic matter (SOM). The purpose of this work was to compare amounts of carbon (C) and nitrogen (N) in HA, FA, POM, and glomalin pools in six undisturbed soils. POM, glomalin, HA, and FA in POM, and glomalin, HA, and FA in POM-free soil were extracted in the following sequence: (1) POM fraction separation from the soil, (2) glomalin extraction from the POM fraction and POM-free soil, and (3) co-extraction of HA and FA from the POM fraction and POM-free soil. Only trace amounts of HA and FA were present in the POM fraction, while POM-associated glomalin (POM-glomalin) and POM alone contributed 2 and 12%, respectively, of the total C in the soil. Mean combined weights for chemically extracted pools from POM and from POM-free soil were 9.92 g glomalin, 1.12 g HA, and 0.88 g FA kg⁻¹ soil. Total protein and C, N, and H concentrations showed that glomalin and HA were, for the most part, separate pools, although protein was detected in HA extracts. Even though percentage carbon was higher in HA than in glomalin, glomalin was a larger (almost nine times) operationally defined pool of soil organic C. Glomalin was also the largest pool of soil N of all the pools isolated, but all pools combined only contained 31% of the total N in the soil.     

Lability of soil organic carbon in tropical soils with different clay minerals

Soil organic carbon (SOC) storage and turnover is influenced by interactions between organic matter and the mineral soil fraction. However, the influence of clay content and type on SOC turnover rates remains unclear, particularly in tropical soils under natural vegetation. We examined the lability of SOC in tropical soils with contrasting clay mineralogy (kaolinite, smectite, allophane and Al-rich chlorite). Soil was sampled from A horizons at six sites in humid tropical areas of Ghana, Malaysian Borneo and the Solomon Islands and separated into fractions above and below 250 μm by wet sieving. Basal soil respiration rates were determined from bulk soils and soil fractions. Substrate induced respiration rates were determined from soil fractions. SOC lability was significantly influenced by clay mineralogy, but not by clay content when compared across contrasting clay minerals. The lability of SOC was lowest in the allophanic and chloritic soil, higher in the kaolinitic soils and highest in the smectitic soil. Our results contrast with conventional concepts of the greater capacity of smectite than of kaolinite to stabilize SOC. Contents of dithionite-citrate-bicarbonate extractable Fe and Al were inversely related to SOC lability when compared across soil types. A stronger inverse correlation between content of ammonium-oxalate extractable Fe and SOC lability was found when considering the kaolinitic soils only and we conclude that the content of active Fe (hydr-) oxides controls SOC stabilization in the kaolinitic soils. Our results suggest that the validity of predictive models of SOC turnover in tropical soils would be improved by the inclusion of soil types and contents of Fe and Al (hydr-) oxides.     

Arbuscular mycorrhizal-like fungi and glomalin-related soil protein drive the distributions of carbon and nitrogen in a large scale

Purpose

Arbuscular mycorrhizal-like fungi (AM-like fungi) are crucial for ecosystem functioning and soil organic matter (SOM) is an indicator of soil quality. However, the spatial distribution of arbuscular mycorrhizal-like fungi, glomalin-related soil protein (GRSP) and SOM in a large scale is still unclear. The objectives of this study were to investigate the spatial distribution of SOM, arbuscular mycorrhizal-like fungi and GRSP, and reveal the potential relationship among them in a large scale across China.

Materials and methods

Soil samples (different in vegetation type, climate, and soil variables) were collected from 26 sites in a large scale across China. The soil properties including pH, total carbon (TC), total nitrogen (TN), and SOM were determined. Quantitative PCR amplification of the 18S rRNA gene was conducted to evaluate the abundance of arbuscular mycorrhizal-like fungi. The contents of easily extractable GRSP (EE-GRSP), difficultly extractable GRSP (DE-GRSP), and total GRSP (T-GRSP) were measured.

Results and discussion

Arbuscular mycorrhizal-like fungi abundance was significantly affected by the vegetation type and dramatically correlated with the soil TN and mean annual precipitation (MAP). EE-GRSP and DE-GRSP were more associated with the TC and TN content, respectively. The abundance of arbuscular mycorrhizal-like fungi significantly but weakly correlated with the T-GRSP and EE-GRSP. The SOM content positively correlated with the DE-GRSP and T-GRSP. Those results suggested that the arbuscular mycorrhizal-like fungi are a larger contributor to regulating the content of GRSP, which is an important indicator of the soil organic carbon pool.

Conclusions

Our results indicated that arbuscular mycorrhizal-like fungi abundance has a greater contribution to driving the distribution of soil C and N in a large scale by affecting the content of glomalin-related soil protein.

     

Arbuscular Mycorrhizal Fungi and Glomalin‐Related Soil Protein as Potential Indicators of Soil Quality in a Recuperation Gradient of the Atlantic Forest in Brazil

This study aimed at surveying arbuscular mycorrhizal fungi (AMF) species and glomalin‐related soil protein (GRSP) to understand their role as presumable biological indicators of soil quality in an undisturbed forest site (NT) and three sites with different management histories, soil textures, and different ages of recovery after reforestation for 20 (R20), 10 (R10) and 5 years (R05). Our objective was to determine how physical, chemical and microbiological soil attributes influence AMF species distribution, total‐GRSP (T‐GRSP) and easily extractable‐GRSP (EE‐GRSP). Glomus and Acaulospora were related to impacted sites, Gigaspora rosea to sites R10 and R20 that have different management histories and soil textures and Glomus geosporum to sites NT and R10, suggesting some influence of texture on its distribution. Scutellospora pellucida and other species were found only in one season. Correlations between EE‐GRSP and T‐GRSP on the one hand and total carbon and nitrogen, dehydrogenase and urease activity, microbial biomass carbon and microbial biomass nitrogen, on the other, reached values of 40–70% and were especially strong in summer. Soil bulk density had a negative and macroporosity a positive effect only on EE‐GRSP, suggesting the necessity to choose either EE‐GRSP or T‐GRSP as biological indicator depending on the soil characteristics and management. This study demonstrates the effect of recovery age, seasonality and other soil attributes on AMF and GRSP distribution and shows that these biological attributes may be used as indicators of soil quality in the Atlantic forest in Brazil. Copyright © 2013 John Wiley & Sons, Ltd.     

Plant invasion of native grassland on serpentine soils has no major effects upon selected physical and biological properties

Plant invasions alter soil microbial community composition; this study examined whether invasion-induced changes in the soil microbial community were reflected in soil aggregation, an ecosystem property strongly influenced by microorganisms. Soil aggregation is regulated by many biological factors including roots, arbuscular mycorrhizal fungal hyphae, and microbially-derived carbon compounds. We measured root biomass, fungal-derived glomalin-related soil protein (GRSP), and aggregate mean weight diameter in serpentine soils dominated by an invasive plant (Aegilops triuncialis (goatgrass) or Centaurea solstitialis (yellow starthistle)), or by native plants (Lasthenia californica and Plantago erecta, or Hemizonia congesta). Root biomass tended to increase in invaded soils. GRSP concentrations were lower in goatgrass-dominated soils than native soils. In contrast, starthistle dominated soil contained a higher amount of one fraction of GRSP, easily extractable immunoreactive soil protein (EE-IRSP) and a lower amount of another GRSP fraction, easily extractible Bradford reactive soil protein (EE-BRSP). Soil aggregation increased with goatgrass invasion, but did not increase with starthistle invasion. In highly aggregated serpentine soils, small increases in soil aggregation accompanying plant invasion were not related to changes in GRSP and likely have limited ecological significance.     

Glomalin amount and compositional variation,and their associations with soil properties in farmland,northeastern China

Glomalin‐related soil protein (GRSP) is well‐known for its soil conditioning functions, but compositional traits are rarely considered. Farmland in northeastern China is the most important commercial grain basis, and soil degradation becomes the bottleneck for keeping crop productivity. The objective of this study was to uncover the possible associations between GRSP (amount and composition) and soil properties, and make suggestions for soil improvement from soil glomalin rehabilitation in northeastern China. Here, spatial variation in GRSP amount (Easily‐extractable‐GRSP, EE‐GRSP; Total‐GRSP, T‐GRSP) and its compositional traits from infrared spectroscopy, UV‐absorbance, X‐ray diffraction (XRD) and 3‐D fluorescence spectroscopy were surveyed in 360 soil samples across northeastern China, and their association with 11 soil properties were also analyzed for finding the possible influence of soil properties on GRSP composition in farmland. There about 3‐fold spatial variation in GRSP amount was observed, while functional group variations were ranged from 1.2‐fold (O–H & N–H stretching) to 2.4‐fold (C–O stretching & O–H bending of –COOH) in different locations. The XRD showed that grain size was 113–180Å and crystallinity was 0.71–1.42%, and GRSP contained seven fluorescent compounds of tyrosine‐like, tryptophan‐like, fulvic acid‐like, soluble microbial byproduct, humic acid‐like, nitrobenzoxadidole‐like, and calcofluor white‐like. Both, EE‐GRSP and T‐GRSP positively associated with soil organic carbon (SOC), soil N (SON), soil P (SOP), alkali‐hydrolyzed N (AN), available P (AP), available K (AK), and soil water, while negatively associated with soil pH and soil bulk density. Structural equation model (SEM) analysis indicates that direct effects on GRSP amounts were mainly from soil bulk density (coefficient: –0.27), soil pH (coefficients: –0.51 to –0.57), SOC (coefficients: 0.51 to 0.69) and AP (coefficients: 0.18 to 0.26), while all other soil properties had indirect effects on GRSP amounts via their close associations with these four parameters. Compared with the GRSP amounts, soil properties laid fewer effects on GRSP compositional traits. Of 16 compositional traits, five of them showed possible regulations from soil properties, which were three infrared functional groups (IR‐II: aliphatic C–H stretching; IR‐V: C–O stretching & O–H bending of –COOH; IR‐VII: O–H binding) and two fluorescent compounds (tyrosine‐like and humic acid‐like). SEM analysis indicates that soil water, pH and EC could directly affect IR‐II, IRV, tyrosine‐like and humic acid‐like, while available nutrients showed more evident influences on infra‐red functional groups than total amounts of N, P and K. Moreover, SOC, as a media of various soil nutrients, gave the strongest influence on GRSP compositional traits. As a supplement to previous studies, we found that GRSP is a mixture of different fluorescent compounds with different functional groups. Our findings highlight that soil properties could strongly change both GRSP accumulation in soil and their compositional traits, and the definition of the most probable soil properties in regulating glomalin amount and composition in this paper could favor good soil management in farmland at northeastern China.     

Glomalin-related soil protein: Assessment of current detection and quantification tools

Despite the widely acknowledged importance of arbuscular mycorrhizal fungi (AMF) in soil ecology, quantifying their biomass and presence in field soils is hindered by tedious techniques. Hence biochemical markers may be useful, among which glomalin-related soil protein (GRSP) could show a particular promise. Presently GRSP is operationally defined, its identification resting solely on the methods used to extract it from soil (citric acid buffer and autoclaving) and the assays (Bradford/enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody) utilized to detect it. The current assumption is that most non-heat stable soil proteins except glomalin are destroyed during the harsh extraction procedure. However, this critical assumption has not been tested. The purpose of this research was to challenge the GRSP extraction process to determine the accuracy of the Bradford method as a measure of glomalin; and to provide some assessment of the specificity of the ELISA monoclonal antibody. In two studies we spiked soil samples either with known quantities of a glycoprotein (BSA: bovine serum albumin) or with leaf litter from specific sources. After extraction 41-84% of the added BSA was detected with the Bradford method. This suggests that the currently used extraction procedure does not eliminate all non-glomalin proteins. Also, ELISA cross-reactivity against BSA was limited, ranging from 3% to 14%. Additions of leaf litter also significantly influenced GRSP extraction and quantification suggesting that plant-derived proteins, as would occur in the field, had a similar effect as BSA. Litter additions decreased the immunoreactive protein values, suggesting interference with antibody recognition. We conclude that the use of GRSP, especially Bradford-based detection, in the assessment of AMF-derived substances within field soils is problematic, it may be inappropriate in situations of significant organic matter additions.     

接种丛枝菌根真菌的种植紫花苜蓿土壤中球囊霉素含量与去除的关系

以菲和芘为多环芳烃（PAHs）代表物,以紫花苜蓿（Medicago sativa L.）为宿主植物,选用幼套球囊霉（Glomus etunicatum, Ge）、摩西球囊霉（Glomus mosseae,Gm）和层状球囊霉（Glomus lamellosum,Gla）3种丛枝菌根真菌（AMF）,研究了接种AMF下土壤中AMF菌丝密度、球囊霉素含量与PAHs去除率的关系。35~75 d,接种Ge、Gm、Gla处理的土壤中菌丝密度、总球囊霉素含量、易提取球囊霉素含量均随时间延长而显著增大,与不接种对照相比,75 d时接种Ge、Gm、Gla处理的土壤中易提取球囊霉素含量提高了48.58%、55.99%和50.23%,总球囊霉素含量则提高了38.75%、50.95%和46.12%。接种AMF促进了土壤中菲和芘的去除,随着时间（35~75 d）延长,接种Ge、Gm、Gla处理的土壤中菲去除率分别高达83.4%~92.7%、82.1%~93.8%、86.9%~93.4%,芘去除率达42.2%~63.5%、43.7%~69.2%、44.6%~66.4%。接种Ge、Gm和Gla处理土壤中AMF菌丝密度、总球囊霉素含量均与土壤中菲和芘的去除率之间存在极显著正相关关系,表明接种AMF提高了土壤中AMF菌丝密度和总球囊霉素含量,并促进了土壤中PAHs的去除。研究结果为阐明丛枝菌根修复PAHs污染土壤的规律及机理提供了依据。     

Soil organic matter chemistry in allophanic soils: a pyrolysis-GC/MS study of a Costa Rican Andosol catena

Soil organic matter (SOM) in allophanic soils is supposed to accumulate due to protection caused by binding to allophane, aluminium and iron. We investigated a catena of allophanic and non‐allophanic soils in Costa Rica to determine the effect of such binding mechanisms on SOM chemistry. These soils contain no contribution of black carbon. Molecular characterization of litter, extractable and dispersed organic matter was done by Curie‐point pyrolysis‐GC/MS. The molecular chemistry of the organic fractions indicates a strong decomposition of plant‐derived organic matter and a strong contribution of microbial sugars and N‐compounds to SOM. Both the decomposition of plant‐derived SOM – including that of relatively recalcitrant compounds – and the relative contribution of microbial SOM were greater in allophanic samples than in non‐allophanic ones. This suggests that chemical protection does not act on primary OM, although it may influence the accumulation of secondary OM in these soils. The effect of allophane on SOM contents in such perhumid soils is probably through incorporation of decomposition products and microbial SOM in very fine aggregates that – in a perhumid environment – remain saturated with water during much of the year. Greater concentrations of aliphatics are found in allophanic residues, but there is no evidence of any specific mineral‐organic binding. The results do not support the existing theory of chemical protection of plant‐derived components through binding to allophane, iron and aluminium.     

Soil structural stability and erosion rates influenced by agricultural management practices in a semi‐arid Mediterranean agro‐ecosystem

Unsuitable agricultural practices can cause loss in soil quality and erodibility to thus increase or trigger desertification under Mediterranean conditions. A field experiment was performed at the El Teularet‐Sierra de Enguera Experimental Station (eastern Spain) to assess the influence during a 5‐yr period of different agricultural practices on physical and chemical indicators of soil quality (total and water‐soluble carbohydrates, glomalin‐related soil proteins (GRSP), total organic carbon, aggregate stability (AS), vegetation cover and soil erosion). The management practices included residual herbicide use, ploughing, ploughing + oats, addition of oat straw mulch and a control (land abandonment). Adjacent soil under natural vegetation was used as a reference for local, high‐quality soil and as a control for comparison with the agricultural soils under different management practices. Oat straw mulching led to higher levels of water‐soluble carbohydrates, GRSP and AS and lower soil erosion rates, resulting in values similar to those in the soil under native vegetation. The lowest levels of carbohydrates and GRSP were for the plots that were treated with herbicide or were ploughed. The maintenance of and increases in stable aggregates promoted by the different agricultural management practices over the years were attributed to increases in labile organic fractions such as carbohydrates and to the GRSP content. The results demonstrate that land abandonment (control plot) or the use of a cover (plants or straw) contributes to increases in soil quality and reduces the risk of erosion. The research also shows that sustainable agricultural management allows soil to recover and that the use of straw mulching is the most effective management strategy.     

Minimal direct contribution of arbuscular mycorrhizal fungi to DOC leaching in grassland through losses of glomalin-related soil protein

Arbuscular mycorrhizal fungi (AMF) have multiple influences on ecosystem C cycling, but most research has focused on ecosystem C gains. We explore here the possibility of direct contributions of AMF to ecosystem C losses, namely via leaching of glomalin-related soil protein (GRSP). We tested the hypothesis that GRSP, an operationally defined SOM pool to which AMF contribute (especially as evidenced with monoclonal antibody MAb32B11-based detection), is mobile in soils and can be lost in leachate. For two New Zealand soils, we showed that only insignificant amounts of GRSP were lost: a maximum of 0.03% of MAb32B11-immunoreactive GRSP present in soils was lost during the week-long experiment, representing a minute fraction of total leachate dissolved organic carbon (0.06%). Our data showed that this pathway of C loss may be relatively unimportant in many soils. However, other indirect contributions of AMF to soil C losses remain yet to be explored.     

Re-examining the glomalin-purity of glomalin-related soil protein fractions through immunochemical,lectin-affinity and soil labelling experiments

Due to analytical similarities with the mycorrhizal glycoprotein glomalin, ubiquitous citrate and heat-extractable soil protein fractions have been assumed to be predominantly glomalin-stabilised within soil. Often termed glomalin-related soil protein (GRSP), little however is actually known of the “glomalin-purity” of these soil fractions. We undertook western and lectin blots and crossed immuno/lectin affinity electrophoresis (CIE/CLAE) analysis of “easily extractible” GRSP fractions, as well as liquid chromatography-tandem mass spectrometry (LC–MS/MS) of “total” GRSP fractions. To further test whether soil saprobes contribute to GRSP production, we amended soil with ¹⁴C-sucrose and examined whether ¹⁴C could be traced in the GRSP pool over a 500-day incubation period.While only four of six bands on SDS–PAGE profiles of easily extracted GRSP reacted with anti-glomalin MAb32B11 and the lectin Con A under our blotting conditions, CIE/CLAE indicated the presence of a single protein moiety in the easily extractible GRSP pool. LC–MS/MS analysis of total GRSP pooled from various soils also showed that although traces of protein tentatively assignable to soil bacteria were present in GRSP, their concentrations were low. Additionally, specific activity of total GRSP in ¹⁴C-labelled soil was relatively depleted compared to the bulk soil and soil microbial biomass. This suggests that little GRSP of heterotrophic origin was laid down over the incubation period, although the potential presence of a pre-existing ¹⁴C-free GRSP background, as well as of low microbial dynamics in the absence of any further substrate inputs to the soil warrant caution with this inference.