Behavior of Bradford-reactive substances is consistent with predictions for glomalin

There is considerable controversy concerning detection in soils of the protein, glomalin, which is produced by arbuscular mycorrhizal fungi. Glomalin was originally defined as a substance that cross reacts with a monoclonal antibody formed against a substance in the cell walls of an arbuscular mycorrhizal fungus. Thus, one can use an immunological approach to detect glomalin. However, it was recently shown that other proteins cross react with the antibody. The other, more common, approach involves assay of soil protein using the Bradford reaction. This approach assumes that the Bradford assay is specific to protein, and that the assayed protein is largely glomalin, either because other proteins are in low concentration, or because the extraction process eliminates the possibility of their detection. These assumptions, however, have been called into question recently. One way to test whether the Bradford assay can be useful in quantifying glomalin is to determine whether the concentrations of Bradford-reactive substances are consistent with predictions for glomalin. For example, if recently produced glomalin is more labile than older glomalin, the concentrations of the two fractions should not be highly correlated. Moreover, when a contrast is established between mycorrhizal and nonmycorrhizal vegetation, recently produced glomalin should soon occur in higher concentration in soils supporting mycorrhizal vegetation. Older glomalin should not be found in higher concentrations in the soils of mycorrhizal vegetation until some time later. We tested these predictions by employing the Bradford assay during the course of a three-year field experiment in which canola (nonmycorrhizal) and soy (mycorrhizal) were grown in separate plots in year 1, both of which were followed by maize (mycorrhizal) in years 2 and 3. The correlation between the concentrations of fraction 1 Bradford-reactive substances (also known as easily extractable glomalin and frequently assumed to be recently produced) and fraction 2 (the more difficult-to-extract fraction and frequently assumed to be older glomalin), was very poor. In year 1, the concentration of fraction 1 was significantly greater in soy plots than in canola plots. Finally, fraction 2 was only significantly higher in the former soy plots than in former canola plots in years 2 and 3. These data support the hypothesis that the Bradford assay was useful in detecting glomalin in this case.     

Glomalin extraction and measurement

We investigated extraction from soil of glomalin, a glycoprotein produced by arbuscular mycorrhizal fungi, and we examined its measurement. The most commonly used protocols for extracting glomalin require autoclaving of soil in citrate solution, followed by centrifugation to separate the supernatant, and then measurement by either Bradford protein assay or enzyme-linked immunosorbent assay (ELISA). We found that lengthening the time of autoclaving increased easily extractable glomalin extraction. Delay of centrifugation after autoclaving, however, diminished Bradford-reactive substances in the supernatant, suggesting that extracted substances might be reversibly immobilized on soil particles. Surprisingly, increasing the volume of extraction solution did not accelerate extraction of “total glomalin”, but instead, substantially increased the amount extracted. Multiple autoclave cycles nevertheless denature glomalin, which may not be as heat-resistant as thought. Repeated 1-h autoclaving of supernatant diminished both its Bradford-reactive substances (7.3% h^?1) and immunoreactive protein (22% during the first hour and 9.5% h^?1 of the remainder thereafter), although a large initial volume of extractant could reduce the loss of immunoreactive protein. Proteins and polyphenols that survive the extraction process are measured non-specifically by the Bradford assay. When we added other glycoproteins to dry soils, we recovered a maximum 34% bovine serum albumin and 22% bovine mucin, primarily in the first two, 1-h extraction cycles. These added proteins may adhere to soil organic matter and thereby be protected from denaturation. In addressing the endpoint of glomalin extraction, we found that the Michaelis–Menten equation closely fits cumulative glomalin per extraction cycle such that its asymptote provides an objective estimate of total extractable glomalin for a given set of extraction conditions. Additionally, the equation provides a curvature parameter that reflects the soil-specific efficiency of an extraction protocol. Although the soils that we investigated with 7.6% or more soil organic matter had the most asymptotic total glomalin, they were extracted the least efficiently.     

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.     

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.     

Differential decomposition of arbuscular mycorrhizal fungal hyphae and glomalin

Arbuscular mycorrhizal fungi (AMF) are obligate symbionts of most higher plants. In addition to being a major component of soil microbial biomass, AMF hyphae produce glomalin, a recalcitrant glycoproteinaceous substance highly correlated with soil aggregate water stability. This study addresses the lack of knowledge concerning the decomposition of hyphae and glomalin. We used an experimental design that exploited the lack of saprobic capabilities of AMF hyphae by incubating field soil samples in the dark, and hence in the absence of plant or AMF hyphal growth. In 150 days, hyphal length decreased 60%, while glomalin, quantified by the Bradford protein assay, declined only 25%. Immuno-reactive glomalin decreased 46%. This study serves as a proof-of-concept for further examination of factors that influence decomposition of AMF hyphae using similar experimental designs.     

Tannic acid reduces recovery of water-soluble carbon and nitrogen from soil and affects the composition of Bradford-reactive soil protein

Tannins are plant-derived polyphenolic compounds that precipitate proteins, bind to metals and complex with other compounds. Solutions of tannic acid, or other phenolic compounds, were added to soil samples to determine if they would affect recovery of soluble soil carbon (WSC) or –nitrogen (WSN) or influence the extraction and composition of Bradford-reactive soil protein (BRSP), associated with glomalin. Tannic acid-C added with water was not completely recovered from samples and the amount of total net WSC and WSN recovered was reduced, suggesting formation of insoluble complexes. By comparison, non-tannin phenolics like gallic acid, or methyl gallate, had little effect on extraction of WSC or WSN while a simple gallotannin derived from tannic acid, 1,2,3,4,6-penta-O-galloyl-d-glucose (PGG), inhibited extraction most. The C and N concentrations in BRSP increased when soil samples were treated with tannic acid or PGG before extraction, a procedure that includes autoclaving. Increases were greatest in the 10–20 cm compared to 0–5 cm depth. Accompanying these were declines in the ratio of absorbance at 465 and 665 nm (E4/E6 ratio) of BRSP extracts suggesting formation of larger or heavier molecules. In contrast, C and N composition in lyophilized BRSP was unaffected or even slightly reduced when tannic acid or PGG were added to the BRSP extract solution after the extraction process. We conclude that some tannins can reduce the solubility of labile soil C and N, at least temporarily and given unpredictability of response associated with phenolic substances, the Bradford assay should not be relied on to quantify pools or composition of soil proteins like glomalin.     

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.     

A comparison of two colorimetric assays,based upon Lowry and Bradford techniques,to estimate total protein in soil extracts

Soil extracts usually contain large quantities of dissolved humified organic material, typically reflected by high polyphenolic content. Since polyphenols seriously confound quantification of extracted protein, minimising this interference is important to ensure measurements are representative. Although the Bradford colorimetric assay is used routinely in soil science for rapid quantification protein in soil-extracts, it has several limitations. We therefore investigated an alternative colorimetric technique based on the Lowry assay (frequently used to measure protein and humic substances as distinct pools in microbial biofilms). The accuracies of both the Bradford assay and a modified Lowry microplate method were compared in factorial combination. Protein was quantified in soil-extracts (extracted with citrate), including standard additions of model protein (BSA) and polyphenol (Sigma H1675-2). Using the Lowry microplate assay described, no interfering effects of citrate were detected even with concentrations up to 5 times greater than are typically used to extract soil protein. Moreover, the Bradford assay was found to be highly susceptible to two simultaneous and confounding artefacts: 1) the colour development due to added protein was greatly inhibited by polyphenol concentration, and 2) substantial colour development was caused directly by the polyphenol addition. In contrast, the Lowry method enabled distinction between colour development from protein and non-protein origin, providing a more accurate quantitative analysis. These results suggest that the modified-Lowry method is a more suitable measure of extract protein (defined by standard equivalents) because it is less confounded by the high polyphenolic content which is so typical of soil extracts.     

Spatial distribution of arbuscular mycorrhizal fungi, glomalin and soil enzymes under the canopy of Astragalus adsurgens Pall. in the Mu Us sandland, China

To measure and manage plant growth in arid and semi-arid sandlands, improved understanding of the spatial patterns of desert soil resources and the role of arbuscular mycorrhizal (AM) fungi is needed. Spatial patterns of AM fungi, glomalin and soil enzyme activities were investigated in five plots located in the Mu Us sandland, northwestern China. Soils to 50 cm depth in the rhizosphere of Astragalus adsurgens Pall. were sampled. The study demonstrated that A. adsurgens Pall. could form strong symbiotic relationships with AM fungi. Arbuscular mycorrhizal fungal status and distributions were significantly different among the five studied plots. Correlation coefficient analysis demonstrated that spore density was significantly and positively correlated with soil organic carbon (SOC), soil acid phosphatase and to two Bradford-reactive soil protein (BRSP) fractions (P < 0.01). Colonization of arbuscules and vesicles were positively correlated with protease activity. The BRSP fractions were also significantly and positively correlated to edaphic factors (e.g. SOC, available nitrogen, and Olsen phosphorus) and soil enzymes (e.g. soil urease and acid phosphatase). The means of total BRSP and easily extractable BRSP were 0.95 mg g⁻¹ and 0.5 mg g⁻¹ in all data, respectively. The levels of BRSP in the desert soil were little lower than those in native and arable soils, but the ratios of BRSP to SOC were much higher than farmland soils. The results of this study support the conclusion that glomalin could be an appropriate index related to the level of soil fertility, especially in desert soil. Moreover, AM fungal colonizations and glomalin might be useful to monitor desertification and soil degradation.     

Role of allophanes in the accumulation of glomalin‐related soil protein in tropical soils (Martinique,French West Indies)

Thermo‐stable, operationally defined soil protein, known as glomalin, may make an important contribution to carbon storage in soils. The term glomalin is used because this putative protein, or group of proteins, was originally thought to be produced only by Glomus fungi. There is currently little information on the glomalin‐related soil protein (GRSP) content of tropical soils, particularly allophanic soils that are known to have different carbon dynamics to temperate climate soils. We have measured the Bradford‐reactive GRSP content of soils sampled from forests and grasslands on the tropical island of Martinique and compared the observations with soil composition. Two operationally defined fractions of GRSP were measured, namely easily‐extractable and total GRSP. The contents of GRSP in moist soils were in the range of 2–36 g kg^?1, accounting for about 8% of soil organic carbon, and were greater in topsoils than in corresponding subsoils. Both the GRSP contents and the fraction of soil organic carbon attributed to GRSP were greater than those reported for temperate climate soils. Both total and easily extractable GRSP contents were positively correlated to soil organic carbon content. The fraction of soil organic carbon that could be attributed to soil protein decreased with increasing allophane content for allophanic soils. No other trends of GRSP content with soil properties or land use were found. GRSP extraction was decreased about seven‐fold by air‐drying of soils, confirming the irreversible change in the soil microstructure of allophanic soils. Total and easily extractable GRSP were correlated and we conclude that both are good probes of thermo‐stable soil protein content for these soils. No attempt was made to verify the fungal origin of the protein detected.     

接种AM真菌对采煤沉陷区文冠果生长及土壤特性的影响

煤炭井工开采往往造成地表塌陷,导致了土壤养分贫瘠和水分缺乏,土壤沙化和水土流失,从而限制了当地矿区植被生长,而丛枝菌根真菌(arbuscular mycorrhiza fungi,AM真菌)对植被生长有促进作用。以文冠果为宿主植物,采用野外原位监测和室内分析方法,研究了未接种和接种丛枝菌根真菌对采煤沉陷区复垦植物文冠果生长和土壤特性的影响。结果表明:与未接种AM真菌处理相比,接种AM真菌显著提高了文冠果根系菌根侵染率和土壤根外菌丝密度,7月接种AM真菌文冠果的株高、冠幅和地径提高了31.89%,23.07%,9.89%。同时,9月接种AM真菌处理的根际土壤全氮、碱解氮和有机碳含量分别比对照组增加0.29g/kg、13.0mg/kg和1.4g/kg,接种AM真菌显著提高了根际土壤的含水率、总球囊霉素和易提取球囊霉素,而速效磷和速效钾的含量显著降低。相关分析结果表明,菌根侵染率、土壤根外菌丝密度与根际土壤理化性质之间存在协同反馈效应。因此,接种AM真菌促进了采煤沉陷区复垦植被文冠果的生长和土壤的改良,这对矿区水土保持、维持生态系统稳定性和持续性具有重要意义。     

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.     

Dynamics of arbuscular mycorrhizal fungi and glomalin in the rhizosphere of Artemisia ordosica Krasch. in Mu Us sandland, China

To understand the ecological significance of arbuscular mycorrhizal (AM) associations in semi-arid and arid lands, the temporal and spatial dynamics of AM fungi and glomalin were surveyed in Mu Us sandland, northwest China. Soil samples in the rhizosphere of Artemisia ordosica Krasch. were collected in May, July and October 2007, respectively. Arbuscular, hyphal and total root infection and spore density of AM fungi peaked in summer. The mean contents of total Bradford-reactive soil proteins (T-BRSPs, TG) and easily extractable Bradford-reactive soil proteins (EE-BRSPs, EEG) reached maximal values in spring. Spore density and two BRSPs fractions were the highest in the 0-10 cm soil layer, but the ratios of two BRSPs fractions to soil organic carbon (SOC) were the highest in the 30-50 cm soil layer. Hyphal infection was negatively correlated with soil enzymatic activity (soil urease and acid phosphatase) (P < 0.05). Arbuscular infection was negatively correlated with soil acid phosphatase (P < 0.01). Spore density was positively correlated with edaphic factors (soil available N, Olsen P, and SOC) and soil enzymatic activity (soil acid and alkaline phosphatase) (P < 0.01). Two BRSPs fractions were positively correlated with edaphic factors (soil available N and SOC) and soil enzymatic activity (soil urease, acid and alkaline phosphatase) (P < 0.01). TG was positively correlated with soil Olsen P (P < 0.05). We concluded that the dynamics of AM fungi and glomalin have highly temporal and depth patterns, and influenced by nutrient availability and enzymatic activity in Mu Us sandland, and suggest that glomalin are useful indicators for evaluating soil quality and function of desert ecosystem on the basis of its relationship to AM fungal community, soil nutrient dynamics and carbon cycle.     

Aggregate stability and glomalin in alternative crop rotations for the central Great Plains

 Land productivity, along with improvement or maintenance of soil health, must be evaluated together to achieve sustainable agricultural practices. Winter wheat-fallow (W-F) has been the prevalent cropping system in the central Great Plains for 60 years where moisture is a limitation to crop production. Alternative cropping systems show that producers can crop more frequently if residue management and minimum tillage are used. The impact of different crops, crop rotations and tillage management practices on soil quality was assessed by measuring aggregate stability and glomalin production by arbuscular mycorrhizal (AM) fungi in soil from cropping trials established in 1990. Crops were wheat (W), corn (C), proso millet (M), and sunflower (S). Rotations sampled were W-F, W-C-M, W-C-M-F, W-C-F, and W-S-F. In the same area as the cropping trials, soils were taken from a perennial grass (crested wheatgrass) and from a buffer area that had been planted to Triticale for the past 2 years but prior to that had been extensively plowed for weed control. We found that aggregate stability and glomalin were linearly correlated (r=0.73, n=54, P<0.001) across all treatments sampled. Highest and lowest aggregate stability and glomalin values were seen in perennial grass and Triticale soils, respectively. Aggregate stability in W-S-F was significantly lower than in the other crop rotations (P≤0.03), while W-C-M had significantly higher glomalin than the other rotations (P<0.05). Differences between crop rotations and the perennial grass indicate that selected comparisons should be studied in greater detail to determine ways to manage AM fungi to increase glomalin and aggregate stability in these soils. Received: 16 March 1999     

Response of Arbuscular Mycorrhizal Fungi in Different Soil Tillage Systems to Long‐Term Swine Slurry Application

Swine slurry is a common agricultural fertilizer in many countries. However, its long‐term use in large amounts can cause excess nutrient accumulation, alter soil compounds, and potentially influence critical microbial populations such as arbuscular mycorrhizal fungi, which have important roles in plant nutrition and soil sustainability. This work determined if arbuscular mycorrhizal status, external mycelium, and glomalin‐related soil protein content were affected by long‐term swine slurry application to different soil tillage systems. The experiment was conducted on a clayey oxisol, in southern Brazil. Swine slurry (0, 30, 60, 90, and 120 m³ ha⁻¹ y⁻¹) was applied for 15 years to conventional tillage and no tillage soil prior to the summer (soybean or maize) and winter (wheat or oats) crop seasons. Swine slurry decreased mycorrhizal root colonization, spore number, and total external mycelium. Swine slurry increased active external mycelium and both easily extractable and total glomalin‐related soil protein. No‐tillage soil had more glomalin‐related soil protein than conventional tillage soil. The most significant response variables were root colonization, easily extractable glomalin‐related soil protein, and total external arbuscular mycorrhizal mycelia. Long‐term application of swine slurry in this environment, even at high rates, did not adversely affect crop yield but did influence arbuscular mycorrhizae fungi and their products in the soil environment. Benefits of swine slurry application for crop nutrition must be weighed against potential adverse consequences for the size, activity, and benefits of the mycorrhizal community to subsequent annual crops. Copyright © 2014 John Wiley & Sons, Ltd.     

红壤区林下侵蚀劣地土壤碳氮及球囊霉素对接种AM真菌的响应

南方红壤区林下侵蚀劣地近地表植被覆盖度低,导致林下水土流失严重。接种AM真菌能够促进植被生长,改善土壤肥力,进而可以减少土壤侵蚀。以马尾松退化林地为对象,设置引种灌木(S)和引种灌木并接菌(S+AMF)2个处理,研究AM真菌接种对林下侵蚀劣地土壤碳氮及球囊霉素的影响。结果表明:接菌近1年后,菌根侵染率(MCR)在S+AMF处理的坡上部和全坡位上显著S处理(P0.05);SOC、TN、SMBC、EE-GRSP和T-GRSP含量在S+AMF处理的坡下部均显著坡中部(P0.05),而在S处理下各坡位差异均不显著(P0.05);AN、SMBN和pH在各处理不同坡位差异均不显著(P0.05);与未接菌S处理相比,接菌(S+AMF)处理对MCR、SOC、TN、SMBC、SMBN、EE-GRSP、T-GRSP、AN和pH的平均贡献率分别为43.83%±15.10%,5.33%±1.57%,14.69%±7.92%,27.88%±4.89%,39.25%±4.82%,6.90%±2.56%,12.47%±7.95%,-13.18%±6.63%和-0.71%±2.74%。简单相关和逐步回归分析表明,MCR、SOC、TN、SMBC、SMBN和球囊霉素之间呈显著正相关(P0.05),TN、SMBC和MCR解释了SOC 80.5%的变异,SOC、SMBC、SMBN和MCR共同解释了TN 90.4%的变异,而TN、SMBN、pH和MCR解释了AN 48.9%的变异,说明接菌提高了紫穗槐根系的菌根侵染率,从而间接促进了林下土壤碳氮及球囊霉素的增加,为有效改善林下侵蚀劣地土壤质量和促进植被恢复有重要的意义。     

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.     

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.     

Revision of two colorimetric methods to quantify glomalin-related compounds in soils subjected to different managements

The aim of this study was to analyze two colorimetric methods used to determine easily extracted glomalin-related soil proteins (EE-GRSP). The historically and most commonly used method for measurement of EE-GRSP as total protein has been the Bradford assay. After some troubles/inconsistencies with this method, we carefully analyzed the Bradford assay, measuring a dilution series of the EE-GRSP fraction and analyzing the time stability of the product. In addition, we did similar analysis of another colorimetric method that quantifies total protein, the bicinchoninic acid (BCA) assay. Unexpectedly, we found that the EE-GRSP concentration values determined by Bradford assay were dependent and variable with the dilution level of the soil extract; moreover, the Bradford assay shows a great instability with the time when soil samples were analyzed but not when protein solution as bovine serum albumin (BSA) was used as control. On the contrary, the BCA assay was independent of the dilution levels of the soil extract and showed stability in the time either for soil samples or BSA protein quantification. These results were consistent and independent on the different type of soils corresponding to different locations and with different textures.     

Changes in soil aggregation and glomalin-related soil protein content as affected by the arbuscular mycorrhizal fungal species Glomus mosseae and Glomus intraradices

Arbuscular mycorrhizal (AM) fungi are key organisms of the soil/plant system, influencing soil fertility and plant nutrition, and contributing to soil aggregation and soil structure stability by the combined action of extraradical hyphae and of an insoluble, hydrophobic proteinaceous substance named glomalin-related soil protein (GRSP). Since the GRSP extraction procedures have recently revealed problems related to co-extracting substances, the relationship between GRSP and AM fungi still remains to be verified. In this work the hypothesis that GRSP concentration is positively correlated with the occurrence of AM fungi was tested by using Medicago sativa plants inoculated with different isolates of Glomus mosseae and Glomus intraradices in a microcosm experiment. Our results show that (i) mycorrhizal establishment produced an increase in GRSP concentration - compared to initial values - in contrast with non-mycorrhizal plants, which did not produce any change; (ii) aggregate stability, evaluated as mean weight diameter (MWD) of macroaggregates of 1-2 mm diameter, was significantly higher in mycorrhizal soils compared to non-mycorrhizal soil; (iii) GRSP concentration and soil aggregate stability were positively correlated with mycorrhizal root volume and weakly correlated with total root volume; (iv) MWD values of soil aggregates were positively correlated with values of total hyphal length and hyphal density of the AM fungi utilized.The different ability of AM fungal isolates to affect GRSP concentration and to form extensive and dense mycelial networks, which may directly affect soil aggregates stability by hyphal enmeshment of soil particles, suggests the possibility of selecting the most efficient isolates to be utilized for soil quality improvement and land restoration programs.