Calcareous soils are alkaline with high CaCO3 and low zinc available for the plant. Glomalin-related soil protein (GRSP), a mycorrhizal glycoprotein, produced in soil increases nutrients availability. A column laboratory experiment was performed for 66 days using four GRSP treatments (0%, 2.5%, 5% and 10% of GRSP), subtreatments by using different Zn concentrations (5, 10, 20 and 30 mg L-1) were added at 0.5 ml h-1. Each column filled with 70 g calcareous soil. Available zinc significantly increased with increasing GRSP at the top 4 cm depth. The highest adsorption was observed in the 5% GRSP treatments. As a result, Zn in leachates decreased with increasing GRSP contents. The Zn bound to Fe-Mn oxide fraction was highly found the 5% GRSP, treated with 30 mg L-1, followed by Zn bound to carbonate, residuals, and Zn bound to organics. In conclusion, the GRSP increased the availability of Zn in calcareous soils.
Calcareous soils are generally alkaline with high CaCO3 contents and characterize by the low organic compounds and nutrients available for plant growth (Quirantes et al., 2016; Mihoub et al., 2017). The low availability of nutrients occurs due to their high adsorption capacity under alkaline conditions. Moreover, the hardness of soil surface crust formed on due to the abundance of CaCO3 may restrict the root proliferation thus minimizing nutrients availability in the rhizosphere (Alloway, 2008). Zinc is one of the most micronutrients essential for soil-plant interaction. Zinc is highly adsorbed to carbonate and oxides in calcareous soils and became unavailable for the plant (Antoniadis et al., 2017). To overcome the insufficiently available zinc in these soils under traditional agricultural practices some chemical fertilizers containing zinc such as zinc sulfate (ZnSO4) or zinc oxide (ZnO) can be applied for their high solubility and in some circumstances may be associated with acidification to facilitate the nutrient uptake by plants (Jensen et al., 2011). However, the intensive and continuous addition of these chemicals is generally correlated with soil chemical degradation, groundwater contamination, loss of biodiversity and negative impacts on the environment and human health. Moreover, these solutions did not positively contribute to obtaining sustainable agricultural management system.
One of the most effective methods for sustainable agricultural management leading to increase soil nutrients availability (Pardini et al., 2017) and facilitate their uptake for plant growth is the application of biofertilizers such as mycorrhizal additions based on the symbiotic associations occur with more than 90% of plant species (Wang and Qiu, 2006). In this laboratory experimental work, a mycorrhizal glycoprotein, operationally identified as glomalin-related soil protein, was applied for its high adsorption capacity to soil micronutrients such as iron, zinc, manganese, and copper (Emran et al., 2017). Glomalin is a mycorrhizal glycoprotein produced in soil able to increase soil macronutrients such as carbon, nitrogen and phosphorus and micronutrients such as iron, zinc, manganese, and copper (Chen et al., 2017; Wang et al., 2017; Gispert et al., 2018). Mycorrhizal glomalin increases soil carbon via photosynthesis and assimilation processes. Moreover, it chelates many ions such as Fe or releases some others from minerals by ion exchange. Similarly, mycorrhizal symbiosis enhances Zn availability for plant growth (Göhre and Paszkowski, 2006). Emran et al. (2012), Luna et al. (2016), and Gispert et al. (2018) showed a significant increase in glomalin concentrations with the increasing stability of soil aggregates for its role as a binding agent able to bind soil nutrients with clay and soil particles. Arbuscular mycorrhizal fungi are widespread and have agronomical important as plant symbiont and often encourage plant uptake of nutrients in poor soils (Liu et al., 2000) and improved tolerance of plants to heavy metals and salts (Colla et al., 2008). several studies reported high Zn retention in the roots of mycorrhizal plants such as maize and clover (Chen et al., 2003; Zhu et al., 2001). Emran et al. (2017) found that zinc was highly sequestered by glomalin in soils with moderately alkaline conditions intercropped with legumes and received annual manure additions. It was noticed that zinc was added in its soluble form as a foliar spray for promoting plant growth that was then precipitated in the soil. In addition, Chen et al. (2017) showed that the presence of Glomus intraradices, one of mycorrhizal fungal species, improved the morphological and physiological plant characteristics and soil nutrients uptake especially zinc in zinc-deficient soil. The key question has arisen when found that zinc was highly abundant in the glomalin extracts of those soils with high zinc contents (Emran et al., 2017).
The aim of this work was to increase the mobility and availability of zinc in calcareous soils under different glomalin-related soil protein additions. Different Zn concentrations (5, 10, 20 and 30 mg L-1) were applied to calcareous soils with various GRSP contents (0%, 2.5%, 5% and 10%).
Materials and methods
The soil used for the laboratory experiment was sampled from the experimental farm of SRTA-City, New Borg El-Arab City (30°53’33.24″N, 29°32’49.61″E), Alexandria Governorate, Egypt. Soils were collected from the plough layer and transported into the laboratory in sterile plastic bags.
Soil samples were air-dried, grounded and sieved at 2 mm. Particle size analysis was determined by hydrometer method. The total CaCO3 contents were quantified by CO2 release from soil upon acidification with 10% HCl. Soil pH was measured in 1:2.5 (w/v) soil suspensions. Electrical conductivity (EC) was measured in a 1:1 (w/v) aqueous extracts. Total soil organic carbon (SOC) was determined by dichromate oxidation (Walkley-Black) method. Cation exchange capacity (CEC) was determined by ammonium acetate extraction method. Available zinc, iron, and manganese were extracted using diethylenetriaminepenta acetic acid (DTPA) solution and then measured by the Agilent 4100 Microwave Plasma-Atomic Emission Spectrometer (MP-AES) (USA) (Emran et al., 2017). All analyses were carried out following the manual of soil analysis (Ryan et al., 2007). Total glomalin (GRSP) was extracted from soil by 50 mM trisodium citrate solution at pH 8.0 through sequential extractions in the autoclave at 121 °C for 1 h. Concentrations of glomalin were quantified by Bradford protein assay (Bradford, 1976).
The column laboratory experiment was carried out as described by Rashad et al. (2010) using polypropylene columns with a diameter of 2.5 cm and a height of 12.5 cm, each column was filled with 70 g of air-dried soil. A filter paper was placed at the bottom of each column.
For obtaining sufficient quantity of GRSP to conduct the column experiment, 20 mM trisodium citrate solution pH 7 (1:8 w/v) were added to 200 g of the soil sample and autoclaved at 121 °C for 30 min (Wright and Upadhyaya, 1998). The supernatant was precipitated by 20% trichloroacetic acid overnight at 4 °C and the pellet was re-suspended in 20 mM trisodium citrate solution. Glomalin concentrations were quantified by the Bradford protein assay (Bradford, 1976) to obtain the GRSP amount for each treatment. Four treatments (control, G2.5%, G5% and G10%) were performed in this experiment based on different glomalin concentrations (0%, 2.5%, 5% and 10% of GRSP (v/w) for each treatment, respectively). The GRSP with different concentrations was added to the top layer of the packed soil aggregates in each column (Rashad et al., 2010).
Different zinc concentrations (5, 10, 20 and 30 mg L-1) were continuously added on the top of the packed aggregates using a peristaltic plumb with a flow rate of 0.5 ml h-1 (Rashad et al., 2010).
Scheduled soil analysis
During 66 days the leachates were collected every 48 hours to measure their contents of the extractable Zn and total organic carbon (TOC). After 66 days, end of the experiment, soils at each column were divided to three depths (0-4 cm for D1, 4-8 cm for D2 and 8-12 cm for D3). Soils of D1-D3 were air-dried and analyzed for their contents of available Zn, TOC and GRSP contents.
Sequential fractionation of zinc forms was performed. Sequential extraction was performed in the following order using one gram of soil: i) the exchangeable fraction, ions bound to soil particles by electrical charges, was extracted with 25 ml of 1 M MgCl at pH 7.01 under shaking for 1 h at 25°C. ii) Zn bound to carbonate fraction was extracted with 25 ml of 1 M Na-acetate at pH 5.04 under shaking for 6 h at 25°C. iii) the fraction of Zn bound to Fe and Mn oxide was extracted with 25 ml of 0.04 M hydroxylamine hydrochloride in acetic acid (25% v/v) under shaking for 6 h at 96°C. iv) organic bound fraction (ions adsorbed, chelated or complexed with organic ligands) was extracted with 6 ml of 0.02 M HNO3 and 9 ml of 30% H2O2 under shaking for 2 h at 85°C followed by adding of 9 ml of 30% H2O2 under shaking for 3 h at 85°C. v) the residual fraction was extracted by acid digestion (concentrated HNO3/HCl 1:3 v/v) (Chen and Ma, 2001). For all fractions, supernatants were collected after centrifugation at 3000 rpm for 15 min. Zn concentrations were measured in each supernatant using the Agilent 4100 Microwave Plasma-Atomic Emission Spectrometer (MP-AES) (USA) (Emran et al., 2017).
Results and discussion
General soil characteristics
The soil used for the experiment showed sandy clay loam texture with 20.72% of Clay, 15.21% of silt and 64.11% of sand and reported by Rashad et al. (2018) as Typic Calciorthids. It is an alkaline calcareous soil with pH 8.41 and 31% of CaCO3. Soil showed slight salinity with an electrical conductivity (EC) of 2.14 dS m–1 and CEC of 11.85 cmol+ kg-1. The soil is very poor in organic compounds as indicated by its low contents of SOC (0.43%) and GRSP (111 mg kg–1). The concentrations of the available micronutrients such as Fe, Zn, Mn, and Cu were small 4.61 mg kg?1, 1.06 mg kg?1, 6.21 mg kg?1 and 0.63 mg kg?1 respectively. All these parameters may depict a poor soil structure with high sensitivity to soil chemical degradation. Application of glomalin treatments in this experimental work was based on the increasing of soil nutrients availability, specifically, zinc depending on the sequestration capacity of glomalin. Glomalin increased significantly among all treatments (Control, G2.5%, G5% and G10%) and revealed an average of 109, 124, 137, 149 mg kg-1 for D1 (F=5.31, p<0.05), 102, 124, 153, 122 mg kg-1 for D2 (F=8.17, p<0.01) and 120, 143, 152, 142 mg kg-1 for D3 (F=6.08, p<0.01), respectively (Fig. 1). In D1, the highest values were in G10% while in D2 and D3 the highest values were in G5%. The one-way ANOVA showed significant variability within and between treatments as can be seen in Table 1. Fig. 1. Table 1. Glomalin as an organic carbon source Glomalin is a glycoprotein containing 15% of organic carbon (Rillig et al., 2001) thus it can be considered as a reserve of SOC pool. Glomalin carbon (G-C) represented 28-42% of glomalin content in rainforest (Lovelock et al., 2004), 28-43% in soil under forests and grasslands (Nichols and Wright, 2005), 52% in organic soils (Schindler et al., 2007), 12-59% in weakly acidic soils under vines, forests, abandoned shrubs and pasture (Emran, 2012; Gispert et al., 2017), 26-30% in agricultural alkaline calcareous soils (Emran et al., 2017). Glomalin was positively correlated with soil organic carbon (Gispert et al., 2017; Emran et al., 2017) and thus it can be considered as a stabilizing agent biochemically bounded to soil particles and increasing the stability of soil aggregates. The GRSP additions in the column experiment, in this work, increased the SOC contents by 3% in the G10% treatment when compared to the control treatment. Despite that, this slight increase of SOC contents did not show any significant data variability among glomalin treatments (Control-G10%). Consequently, no significant variability was observed in pH and EC data among all treatments. It should be taken into consideration that incubation of glomalin additions under laboratory temperature around 25 °C did not show any significant effects on the metabolization processes of glomalin units. As a result, application of glomalin additions in the field may allow the stabilization of organic compounds in soil. Stabilized compounds in soil lead to increase the organic matter contents in the soil thus increasing the binding sites with metals. In this work, zinc was added to soil columns with glomalin additions and resulted that glomalin acted as a binding agent for sequestering Zn and may increase its availability in soil. Table 2 Available Zn in soil The recommended concentration of zinc in soil should be within 10-300 mg kg-1 (Kiekens, 1995), equivalent to 5-100 kg per hectare (Mengel and Kirkby, 2001). Due to the low zinc concentrations in the initial soils, glomalin was added to increase Zn availability (Fig. 1). The control treatment did not show a clear trend along Zn additions. However, all glomalin treatments revealed the pronounced increase of Zn concentrations along with Zn solution added to the soil. This may explain the role of glomalin in increasing the available Zn in the soil. The highest values of available Zn were found at the first layer of the column (D1). In D1, Zn availability increased proportionally with zinc additions from 5 mg L-1 to 30 mg L-1. However, no significant data variability among with all glomalin treatments was found. The highest concentration of all treatments was found in Zn 30 mg L-1. Zinc in leachate For each treatment, a total of 720 ml leached from each soil column during the 66 days. From Fig. 2, it can be noticed that the cumulative concentrations of Zn in these leachates represented with very low values. The total Zn leached from the soil column in the control treatment were 0.081, 0.040, 0.049 mg L-1 under Zn 5 mg L-1, Zn 10 mg L-1, Zn 20 mg L-1, respectively and reached 0.334 mg L-1 under Zn 30 mg L-1. The cumulative concentrations of Zn in the leachates collected from the G2.5% treatment were 0.121, 0.064, 0.026 and 0.075 mg L-1, for the G5% were 0.034, 0.040, 0.055 and 0.080 mg L-1 and for the G10% were 0.124, 0.062, 0.039 and 0.172 mg L-1 for the same order of Zn additions, respectively. The Zn leached from soil columns increased significantly (p<0.001) respecting the power equations (Fig. 2). The total Zn leached from the soil column of the G5% treatment showed significant increase indicated by the linear equation (y = 0.0153x + 0.0139, r=0.963) with a significant p-level < 0.01. Fig. 2. Distribution of Zn fractions with soil depth All glomalin treatments showed an increase in Zn fractions at 0-4 cm depth (D1). The last two layers (D2-D3) revealed very low concentrations in carbonate bound fraction, Fe and Mn bound fraction and the residual fraction and consequently the total Zn contents. Exchangeable Zn fraction and organic bound fraction were undetectable. The highest concentrations in all fractions were generally found in G5% treatment. A pronounced increase of the exchangeable fraction was found in D1, where it increased 116%, 140% and 83% for G2.5%, G5% and G10% compared to the control treatment, respectively. Carbonate fraction increased by 68%, 79%, and 69%, Fe and Mn bound fraction increased by 88%, 120% and 69%, organic bound fraction increased by 167%, 224% and 183% and residual fraction increased by 49%, 71% and 37% in the same order of glomalin treatments with respect to the control in the first layer (D1). The total zinc, as a result, showed an increase of 76%, 97% and 66% in glomalin treatments with respect to the control. By comparing each fraction with respect to the total Zn contents, carbonate and organically bound fractions represented their higher ratio in the G10% treatment than in other treatments. Table 3 The exchangeable Zn fraction represented low concentrations in D1 with respect to other fractions such as carbonate, Fe and Mn and residual Zn fractions and was not detectable in the other two layers (D2 and D3) which were in agreement with Jaradat et al. (2006). The high concentrations generally found in carbonate and Fe and Mn bound fractions (G5% at D1) with respect to other fractions. In calcareous soils, zinc was greater sorbed in the presence of the precipitated iron oxides on calcite, under alkaline conditions (Uygur and Rimmer, 2000), and therefore immobilized and became less available to plants. The highest concentration of zinc fractions was the fraction which bound to Fe-Mn oxide at G5% in the top layer. Many observations reported the dominance of Fe and Mn oxide-bound Zn (Kabala and Singh, 2001). The contribution of this fraction decreased in subsurface layers (D2 and D3). The lowest concentration of zinc fractions was the fraction which bound to organic compounds in the first layer and not detected in the two other layers. According to Kumar et al. (2011), the organic fraction of Zn is not considered very mobile or available because of its binding to the stable humic substances with high molecular weight. The residual fraction in D1 at G5% was lower than Zn-bound to Fe-Mn oxide and Zn-bound to carbonate while was higher than these fractions in D2 and D3 indicating the immobilization of the residual fractions in the second two layers (Kabala and Singh, 2001). Highly significant positive correlations were observed among all Zn fractions identified by the sequential fractionation procedure from the first top layer. In the second layer, only Zn bound to carbonate was positively correlated with the fraction bound to Fe and Mn oxides. Table 4. In Table 5, it can be found that glomalin additions increased the soluble and available forms of zinc that were organically-complexed and become mobile and available to plants in soils. Glomalin was highly adsorbed in the first top layers and consequently, the availability of zinc forms in that layers was increased. Table 5. Conclusion Glomalin-related soil protein was highly adsorbed to soil particles at the top 4 cm depth. Consequently, Zn availability increased along the GRSP treatments causing an increase of Zn fraction bound to organics. The highest adsorption was found in the 5% GRSP treatment indicating that the GRSP addition should be added to produce an equilibration in zinc availability. In general, Zn fractions distributed in the following order: Zn bound for Fe-Mn oxide, carbonates, residuals, exchangeable and organic bound fraction. In conclusion, glomalin acted as an organic ligand to increase Zn availability in alkaline calcareous soils. Further researches are needed to develop the efficiency of glomalin to increase soil nutrients availability and their organic bound fractions.