Biochemically active humic substances in contrasting agricultural managements

Because their crucial role in several soil biochemical cycles and their fast response to changes in soil management, extracellular enzymes activities are widely used as sensitive indicators of ecological change and soil quality. The aim of this work was to determine the effects of soil management on the stable pool of soil carbon cycling enzymes as indicators of essential functions. For this, extracellular β-glucosidase enzymes bounded by humic acids (C higher than 104 Da) were used to compare four long-term contrasting agricultural managements in a rainfed olive orchard representative of semi-arid Mediterranean habitats. The study was conducted for 30 years by designing a random-block of four treatments (nude vs. covered soils) and four replicates. Maintaining cover crops through fall, winter and early spring provoked a more stable and active pool of extracellular β-glucosidase in soils only if spontaneous vegetation was managed with mechanical methods. When herbicides were used during 30 years, the pattern of the molecular composition and activity of humus β-glucosidase complexes were similar in covered and nude soils, although higher activity was retrieved in the former. Tillage management increased carbon mineralization and the level of humic substances and the activity of β-glucosidase humic-bound were quite lower than in the rest of treatments. Given the ecological role of extracellular soil carbon cycling enzymes, the characterization of humus β-glucosidase complexes could be an adequate indicator of sustainability of agricultural management systems. additional key words: Olea europaea; β-glucosidase enzyme; humic substances; sustainability; cover spontaneous vegetation. abbreviations used: CH (cover crop + herbicides); CM (cover crops + mower); IEF (isoelectric focusing); NC (non-tillage and no-cover); PNG (4-nitro-phenyl-β-D-glucanopyranoside); PNP (p-nitrophenol); T (tillage). authors’ contributions: Conceived and designed the experiments: EB, RN and BM. Analyzed and interpreted the data: EB, SD and BM. Critical revised the manuscript for important intellectual content: EB, RN, GM and BM. Contributed reagents/materials/ analysis tools: RN, SD, GM and BM. Wrote the paper: EB and BM. Coordinated the research project: EB. citation: Benitez, E.; Nogales, R.; Doni, S.; Masciandaro, G.; Moreno, B. (2016). Short communication: Biochemically active humic substances in contrasting agricultural managements. Spanish Journal of Agricultural Research, Volume 14, Issue 3, e03SC01. http://dx.doi.org/10.5424/sjar/2016143-8431. received: 07 Aug 2015. accepted: 22 Jun 2016 copyright © 2016 inia. This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial (by-nc) Spain 3.0 Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Funding: Cofinanced ERDF / Spanish Ministry of Economy and Competitiveness (Grant CGL2009-07907). competing interests: The authors have declared that no competing interests exist. correspondence should be addressed to Emilio Benitez: emilio.benitez@eez.csic.es


introduction
Microbial extracellular enzymes can remain active in soil for a long time bound and protected by humic substances. The ecological importance of humic-enzyme complexes is that their activity is not affected by microbial stresses, therefore playing an important role in a large number of biochemically mediated processes under adverse conditions for soil microorganisms (Nannipieri et al., 1996;Doni et al., 2012). Humic-bound enzymes extracted from soils or other organic materials can be separated by isoelectric focus-2 for 2 h and then quantified according to Yeomans & Bremner (1988). This C fraction was used to determine extracellular β-glucosidase activity as described by Garcia et al. (1993), and it is based on the use of 0.05 M 4-nitro-phenyl-β-D-glucanopyranoside (PNG) as a substrate (Hayano & Tubaki, 1985). The p-nitrophenol (PNP) released by β-glucosidase activity was extracted and determined spectrophotometrically at 398 nm (Tabatabai & Bremner, 1969).
Isoelectric focusing was carried out in cylindrical gel rods (0.5×8 cm) containing polyacrylamide gel (5% w/v) and carrier ampholines in the pH 4-6 range (Bio-Rad Laboratories, Richmond, CA, USA) at a final concentration of 2% (Ceccanti et al., 1986;1989). N,N,N′,N′-tetramethyl-1,2-diaminomethane and ammoniumperoxy-disulfate were also added in gel solution at a concentration of 0.03%. Organic material (100 μL of pyrophosphate extract derived fraction >10 4 Da) at 4.4% of glycerine was applied at the top of the gel rod (cathode). A little amount (5 μL) of glycerine at 2.2% was added on the top of the sample to avoid the mixing with the cathodic solution (0.02 N NaOH); 0.01 M H 3 PO 4 was used for the anodic cell. A pre-run of 1 h at the same current intensity and voltage used for the samples run were carried out for each gel tube (1.5 mA for each tube, 100-800 V); subsequently, the samples run was carried out for 2 h or longer until a stable IEF banding was reached. The electrophoretic bands were scanned by a Bio-Rad GS 80 densitometer, obtaining a typical IEF profile for each soil investigated. The IEF peak area was determined for each soil IEF profile, assuming as 100% the area under the entire IEF profiles (representative of the total loaded C). Gel pH was measured at 0.5 cm intervals with an Orion microprocessor (model 901, Orion research) connected to a microelectrode gel-pHiler (Bio-Rad Laboratories, Richmond, CA, USA). To determine β-glucosidase activity of the humic bands obtained by IEF, the gel was gently removed from the glass tubes. The bands were cut and pre-washed for 1 h with 2 mL of 0.1 M phosphate buffer, pH 6.4, at 37 °C (pre-washing removes the carrier ampholytes, salts, and other impurities from the gel, without releasing the gel-trapped humic matter). After removal of the buffer, 2 mL of fresh 0.1 M phosphate buffer, pH 6.4, and 0.5 mL of 0.05 M PNG were added and the mixture was incubated at 37 °C under shaking for 17 h.
Results are the means of 12 replicates (three per plot). The results were subjected to a factorial analysis of variance (ANOVA) using the STATISTICA software program (StatSoft Inc., Tulsa, OK, USA). The post hoc Tukey HSD test in a one-way ANOVA was used. to be evidence of statistical significance p-values of under 0.05 were considered. functions in a highly vulnerable −and economically vital− agroecosystem as rainfed olive farming. Within this framework, the aim of this work was to determine the effects of soil management on the stable pool of soil carbon cycling enzymes as indicators of essential functions.

material and methods
Humic-β-glucosidase complexes were extracted and then characterized from soils under four agricultural managements in a rainfed olive orchard representative of semi-arid Mediterranean habitats. The main characteristics of the experiment and soils are described in Moreno et al. (2009). The soil has been classified as an Anthropic Regosol (FAO, 1998). The study was conducted for 30 years in Jaen (southeastern Spain) by designing a random-block of four treatments and four replicates (plots). The treatments tested were: − Tillage (T): Bare soils where weeds were eliminated by 3-4 annual passes with a disk harrow (at 30 cm deep) and/or a cultivator in spring, followed by a tine harrow in summer. − Non-tillage and no-cover (NC): Bare soils where weeds were eliminated by applying the preemergence herbicide oxyfluorfen in autumn. Initially, simazine and diuron were used until 12 years ago. In the spring, glyphosate was applied locally. − Cover crop + herbicides (CH): Covered soils where weeds were left to grow each year to be eliminated in spring with herbicides. Initially, diquat/paraquat was used until prohibition, being replaced by glyphosate at a later date. − Cover crops + mower (CM): weeds were eliminated with various passes of a chain mower in spring. Two samples were collected in the centre of each plot by a modified soil-sample ring kit (Eijkelkamp) which includes a cylinder of 20 cm (depth of sampling) specifically manufactured for this purpose and then bulked. The samples were stored at 4ºC until analyses were made.
Humic substances were extracted from soil at 37 °C for 24 h under shaking, using Na 2 P 4 O 7 (0.1 M, pH 7.1) in a 1:10 w/v dry soil/extract ratio (Ceccanti et al., 1978). Then, the suspension was centrifuged (12,400×g) and filtered through a 0.22-μm Millipore membrane. The C-derived fraction >10 4 Da was obtained through ultrafiltration on AMICON PM10 membrane of the C extract. The C content was determined by acid digestion with K 2 Cr 2 O 7 and H 2 SO 4 at 140 °C 3 Short communication: Biochemically active humic substances in contrasting agricultural managements substances, both total and those corresponding to each IEF band. Humic C focused at pH 4.2 was higher in all the treatments, confirming the stability of the humic substances isolated (Govi et al., 1994;Doni et al., 2012).
The β-glucosidase activity was also quite higher in all the four humic bands of CM soils (Fig. 2a). For T, NC and CH soils, the higher β-glucosidase activity was measured in the lower pH where band focalized. The trend was just the opposite for CM soils, and the higher activity was found at pH 4.5, pH range where enzymatic proteins generally are stabilized by humus molecules Ceccanti & Masciandaro (2003). However, the specific β-glucosidase activity, i.e. the ratio between β-glucosidase activity and C>10 4 Da, was higher at this pH except for soil where tillage was the main management (Fig. 2b).
In this work, the stable pool of soil β-glucosidase enzymes were used to compared four long-term contrasting agricultural managements. Maintaining cover crops through fall, winter and early spring provoked a more stable and active pool of extracellular β-glucosidase in soils only if spontaneous vegetation was managed with mechanical methods.

results and discussion
Extracellular β-glucosidase was higher in soils under cover vegetation (Fig. 1), especially when these were managed with mechanical methods, evidencing the effect of sustainable managements on the soil biochemical activity. No significantly differences were found between soils where spontaneous vegetation was regularly eliminated by tillage (T) or herbicide applications (NC). The above agrees with previous experimental works where extracellular enzyme production has been linked to those agricultural managements increasing the quantity, quality and chemical diversity of cover crops residues (Tiemann et al., 2015).
The humic carbon in the bands after IEF humic carbon calculated from the IEF peak areas focused in the different selected bands (1, 2, 3 and 4) is shown in Table 1. The sum of the humic carbon in the IEF bands represent the C>10 4 Da fraction of high molecular weight heteropolymers traditionally described as humic acids. CM soils showed the greater levels of humic table 1. Humic carbon (mg C/kg) in the pH range 4.5-4.2 after isoelectric focusing in tillage (T), non-tillage and no-cover (NC), cover vegetation + herbicides (CH), and cover vegetation + mower (CM) soils. Significant differences are indicated by different letters (p<0.05, ANOVA, Tukey post-hoc).  When herbicides were used during 30 years, the pattern of the molecular composition and activity of humus-β-glucosidase complexes were similar in covered and nude soils, although higher activity was retrieved in the former. This fact is probably related with the higher vegetal biomass incorporated and also with the different mode of action of the herbicides applied during the whole experiment, with different effects on both microbial activity and CO 2 fixation (Fedtke, 1982;Riah et al., 2014;Kane, 2015). Tillage management increased carbon mineralization. As a consequence, the level of humic substances and the activity of β-glucosidase humicbound were quite lower than in the rest of treatments. Given the ecological role of extracellular soil carbon cycling enzymes, the characterization of humus β-glucosidase complexes could be an adequate indicator of sustainability of agricultural management systems.