Dehydrogenase and mycorrhizal colonization: Tools for monitoring agrosystem soil quality

Applied Soil Ecology 100 (2016) 144–153

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Applied Soil Ecology journal homepage: www.elsevier.com/locate/apsoil

Dehydrogenase and mycorrhizal colonization: Tools for monitoring agrosystem soil quality Fuentes-Ponce Marielaa,* , Moreno-Espíndola Iván Pávela , Sánchez-Rodríguez Luis Manuela , Ferrara-Guerrero María Jesúsb , López-Ordaz Reyesa a b

Universidad Autónoma Metropolitana-Xochimilco, Departamento de Producción Agrícola y Animal, Mexico Universidad Autónoma Metropolitana-Xochimilco, Departamento el Hombre y su Ambiente, Mexico

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 June 2015 Received in revised form 17 December 2015 Accepted 18 December 2015 Available online xxx

In order to improve food production while reducing environmental impact, the redesign of agrosystems to incorporate monitoring tools for decision-making is a fundamental requirement. Chemical and physical indicators of medium and long-term soil quality have been developed in order to monitor changes in agroecosystems; however, it is crucial to develop parameters that can predict the short-term trajectory of the system. For this reason, the objective of the present study was to determine whether certain soil biological parameters, such as the activities of microbial enzymes (dehydrogenase (DH), acid phosphatase (ACP), urease (URE) and protease (PRO)) and mycorrhizal colonization, could be useful as indicators of soil biological quality, given their sensitivity to different agricultural management practices. An evaluation was conducted over two years in five different agrosystems of Valle de México in Mexico. The activity of DH presented greater sensitivity to changes in agricultural management produced by types of tillage and input (organic or synthetic) and topological arrangement, compared to that of URE, ACP and PRO, which did not present a clear pattern with respect to the different agrosystems or to sampling date (based on the agricultural practices). Mycorrhizal colonization was sensitive to the type of inputs used, but not to tillage type or crop rotation. It is therefore considered that DH and mycorrhizal colonization could represent useful parameters for measuring soil quality and the environmental impact of the use of agrochemicals in agriculturally managed soils. Based on DH and mycorrhization, the agrosystem with the highest quality soil was the Mesoamerican system known as “Milpa” (typified by minimum tillage, intercropping and organic inputs). ã 2015 Elsevier B.V. All rights reserved.

Keywords: Soil biological indicators Agriculture management Organic inputs Enzymatic activities

1. Introduction The biological components of the soil can be used as short-term agrosystem quality indicators, given their high sensitivity to any alteration of the system or change in the environment (Bending et al., 2004), as well as their close relationship with plant root systems, stress tolerance, productivity and adaptability, among other agrosystem characteristics (Rodriguez and Redman, 2008; Schnitzer et al., 2011; Lau and Lennon, 2011). Different agricultural management practices that imply various types of tillage, fertilization and weed control, among others, can generate physical and chemical conditions in the soil that affect the activity and composition of the microbiota and thus its enzymatic activity

* Correspondence author at: Laboratorio de Fisiología Vegetal, Calzada del Hueso 1100, Col. Villa de la Quietud, Coyoacán, CP 04960, México D.F., Mexico. E-mail address: [email protected] (F.-P. Mariela). http://dx.doi.org/10.1016/j.apsoil.2015.12.011 0929-1393/ ã 2015 Elsevier B.V. All rights reserved.

(Acosta-Martínez et al., 2008; Schipanski and Drinkwater, 2012; Vasseur et al., 2013). The dynamics of release, flow and absorption of nutrients through the activity of extra- and intracellular enzymes are of great interest from an agronomic perspective (Ceja-Navarro et al., 2010; Kumar and Varma, 2011) and control the recycling of soil organic materials, thus dictating the availability of nutrients (Kohler et al., 2009). Microbial enzymatic activities that are affected by the type of agricultural management include those of dehydrogenase, which is important for the decomposition of soil organic matter (SOM) and in N dynamics (García et al., 1993; Nannipieri et al., 2003; Vepsäläenen et al., 2004; De Varennes et al., 2007); protease and urease, which participate in the hydrolysis of peptide bonds and release of NH4+ (Banik and Prakash 2004; Wang et al., 2008); and acid phosphatase, which catalyzes the hydrolysis of esters and anhydrides of phosphoric acid under different conditions of pH (Gianinazzi et al., 1992). Similarly, microbial enzymatic activity varies according to the associated plant species, since each has a

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2. Materials and methods

the production of native maize (“chalqueño”), and were based mainly on tillage type (minimum and conventional), crop management (rotation, monoculture and intercropping) and use of organic and synthetic inputs. Organic management in 2012 consisted of the following treatments: ZTIO + r and ZTRO + r, which were fertilized with composted cow dung added to each plant (4.4 t ha1); CTMO30r, in which two fertilizations were carried out, the first consisting of composted cow dung (1.166 t ha1) and the second with a different composted manure (2.733 t ha1)—in 2013, however, the first fertilization in the ZTIO + r and ZTRO + r treatments consisted of an application of mountain microorganisms and 500 kg ha1 basalt rock dust (187.5 kg), while the second consisted of composted sheep dung (3.3 t ha1) and chicken manure (1.1 t ha1); CTMO30r, in which the first fertilization consisted of dry sheep manure (3.3 t ha1) and basalt rock dust (0.5 t ha1), while the second used chicken manure (1.1 t ha1). Weeds were removed manually. In 2012 and 2013, synthetic management consisted of fertilization with urea (243.5 kg ha1) and calcium triple superphosphate (50 kg ha1), while herbicide (Hierbamina (2,4-D)) was applied at a rate of 12.5 l y1 ha1. Composite soil samples were obtained from each experimental plot at depths 0–10 and 10–20 cm of the soil profile. Samples were stored at 4
C until subsequent analysis. In order to determine enzymatic activity, sampling was conducted on seven different dates in the years 2012 (10th of July and 8th of September) and 2013 (14th of January, 3rd of May, 4th of June, 11th of September and 2nd of October). There were five sampling dates in the case of the mycorrhizae: June 4th, July 19th, September 11th and October 2nd, all in 2013. The dates were chosen according to both the occurrence of rain and the different treatments in the plots (Table 2) in order to evaluate the potential of enzymatic activity and mycorrhizal colonization in both dry and wet periods, before and after the application of agrochemicals or organic inputs, according to treatment.

2.1. Experimental site and soil sampling

2.2. Enzymatic activities

The experimental site is located in the municipality of Cocotitlán in the east of Estado de México, in Mexico (19
1201800 –19
140 3300 N; 98
490 4600 –98
520 5200 W: 2300 masl). The climate is of type C(w1)(w), temperate sub-humid, with summer rains (García, 2004). The wet season extends from May to October at the experimental site, with a mean annual precipitation of 784 mm. The soil is Vitric eutric epiarenic (WRB classification). In 2011, the area in which the experimental plots were established (1 ha) was subdivided into plots of 6.6  30 m (198 m2), in which five different treatments were established (Table 1). In the case of the treatments with rotation (ZTRO + r and ZTRQ + r), we have presented two versions in order to visualize the performance of both crops (maize = m and oats = o), using a random block experimental design with three replicates, giving a total of 15 experimental plots. All of the management models featured

The enzymatic activities considered for evaluation of potential were those of dehydrogenase (DH), acid phosphatase (ACP), urease (URE) and protease (PRO). All of these enzymes participate in processes of SOM decomposition (Das and Varma, 2011). Measurement of the potential of each enzyme activity was performed following the spectrophotometric methods described by García et al. (2003). The activity of dehydrogenase, the only intracellular enzyme considered in this study, was determined by measuring the iodonitrotetrazolium formazan (INTF) formed after incubating 1 g of soil for 20 h at 20
C in darkness. A Shimadzu dualbeam spectrophotometer was used at wavelength 490 nm (García et al., 2003). Acid phosphatase activity was determined following the method of Tabatabai (1994), which is based on quantification of the p-nitrophenol released after incubating 1 g of soil at 37
C for 1 h in a buffered solution of p-nitrophenyl phosphate. The

different effect on the microbiota according to the nature and quantity of its root exudates (Nannipieri et al., 1990; Czarnes et al., 2000; Bending et al., 2004). There are microorganisms, such as arbuscular mycorrhizal fungi (AMF) (Nielsen and Winding, 2002), that are key to soil biological quality. These fungi associate with the roots of plants, contributing to nutrient (P) acquisition, resistance to pests and diseases and tolerance to drought and heavy metals, as well as improving soil structure (Gosling et al., 2006). Abundance of AMF in the roots can be a biological indicator of the impact of different agricultural management practices, since the degree of AMF colonization is related to practices such as type of rotation, proportion of organic material and intensity of tillage. It can also be an indicator of the physico-chemical characteristics of the soil (Hijri et al., 2006; Miller and Jackson, 1998). Soil biological properties are closely related to agrosystem management and quality and monitoring of these properties can provide basic tools for evaluating these systems. As part of the task of redesigning these agrosystems from a holistic and co-innovatory perspective, an aspiration toward more closed cycles of energy and materials is required, while simultaneously reducing the use of synthetic products and considering renewable energy sources that respect the living organisms of the system (Baars and Baars, 2007; Guzmán and González de Molina, 2009). The objectives of this study were: (i) to evaluate the biological quality of five agrosystems through measuring the potential of key soil enzymatic activities and the percentage of mycorrhizal colonization in production systems of native maize under different management strategies (type of tillage, fertilization, weed management and rotation), in the southeast of Valle de México, in Mexico; and (ii) to determine which of the assessed biological indicators could be monitored to obtain reliable info about the impact of management strategies on soil quality.

Table 1 Different agro-management experimental treatments conducted in Cocotitlán, México, Mexico. Treatment

Tillage

Residues management

Fertilization

Weed management

Rotation

ZTIO + r

Minimum

Retention (100%)

Organic

Intercropping (maize, squash, beans, vetch)

ZTRO + r

Minimum

Retention (100%)

Organic

Manual Forage crop Manual

ZTRQ + r

Minimum

Retention (100%)

Chemical

Chemical

CTMO30r CTMQ  r

Conventional Conventional

Incorporate (30%) Without residues

Organic Chemical

Manual Plow and chemical

Rotation (maize -m-, oats -o-) Rotation (maize -m-, oats -o-) Monoculture (maize) Monoculture (maize)

ZTI = minimum tillage with intercrop, ZTR = minimum tillage with rotation, CTM = conventional tillage with monoculture, O = organic inputs, Q = chemical inputs, +r = with residues, r = without residues, 30r = with 30% residues.

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Table 2 Dates of the different agricultural practices conducted in 2012 and 2013 in the experimental plots. Year

Practices

CTMQ  r

CTMO30r

ZTRQ + r

ZTRO + r

ZTIO + r

2012

Sowing maize oat Fertilization

May 15

May 15

Herbicide

August 12 September 3

May 15 June 19 Manual

May 15 July 5 May 15

May 15

June 19

May 15 July 5 May 15 August 12 September 3

Manual

Manual

Sowing maize oat Fertilization

April 8

April 8

Herbicide

June 18 August 8 September 4

May 2 July 18 Manual

May 3 August 7 May 2 July 17 Manual

May 3

July 18

May 3 August 7 May 3 July18 June 18 August 8 August 28 September 14

2013

May 15

May 2 July 17 Manual

CTM = conventional tillage with monoculture, ZTR = minimum tillage with rotation, ZTI = minimum tillage with intercrop, Q = chemical inputs, O = organic inputs, r = without residues, +r = with residues, 30r = with 30% residues.

wavelength used was 400 nm. Urease activity was determined following the method of Kandeler et al. (1999), which is based on the colorimetric determination of ammonium released after incubation of 1 g of soil in the presence of urea. The wavelength used was 610 nm. Protease activity was estimated by determining the degradation of casein, based on the colorimetric quantification (through the Folin reaction) of soluble peptides released in trichloroacetic acid after incubation of 1 g of soil in a solution with 1% casein at 52
C. The wavelength used was 578 nm (García et al., 2003). Corresponding calibration curves were generated for each enzymatic activity and each test featured a control and six replicates.

2.3. Colonization of maize roots by arbuscular mycorrhizal fungi (AMF) In order to estimate the level of colonization of the maize roots by AMF, fine roots of 6 plants per treatment at each sampling date were digested with 10% KOH and stained with Trypan blue (Phillips and Hayman, 1970). Thirty root segments of 1 cm in length from each plant were placed on a slide with lactoglycerol and observed under the microscope. Levels of mycorrhization (M%) and abundance of arbuscules (A%) were calculated for each treatment using the package Mycocalc (Trouvelot et al., 1986).

Fig. 1. The potential enzymatic activities of dehydrogenase in the soils, in the different systems: ZTR = minimum tillage with rotation, ZTI = minimum tillage with intercrop CTM = conventional tillage with monoculture, O = organic inputs, Q = chemical inputs, +r = with residues, r = without residues, 30r = with 30% residues, m = maize and o = oats. Bars are  one standard error for triplicates.

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2.4. Statistical analysis Data pertaining to the acid phosphatase activities were transformed to square root +0.5 in order to satisfy the assumptions of normality and homogeneity of variance. The effects of the treatments were analyzed using a general linear model (GLM) procedure. The variables urease and protease did not fit the assumptions and analysis was therefore conducted using a nonparametric procedure (Kruskal–Wallis), while a Tukey test (SAS version 9.0) was used for multiple comparison of the means. In order to analyze levels of mycorrhization and abundance of arbuscules, a one-way ANOVA was performed along with a Tukey test to compare the means (SAS version 9.0). 3. Results and discussion 3.1. Enzymatic activities Throughout the 15 months of the experiment, the potential enzymatic activities of dehydrogenase (DH), urease (URE) and protease (PRO) in the soils of depth 0–10 cm of the different systems were significantly affected (p < 0.05) by the agricultural management practices adopted (DH and URE < 0.0001 and PRO < 0.0003), except for the activity of acid phosphatase (ACP). The only potential activity significantly affected at soil depth 10–20 cm was DH (p 0.0006). This supports the notion that variation in soil biological quality is more evident in the topsoil (depth 0–10 cm), given the fact that the chemical and physical characteristics of this layer seem to be more affected. For example, in conventional agriculture, disturbance by tillage is greater in this layer than further down the profile, while in systems where the soil is not disturbed and harvest residues are left on the surface, such as in conservation agriculture, there is a greater presence of organic material in this layer than at greater depth (Fuentes et al., 2009;

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Govaerts et al., 2006). This has a direct repercussion on the biological conditions of the soil (Askari and Holden, 2015) in the different layers. 3.1.1. Potential enzymatic activities of dehydrogenase (DH) 3.1.1.1. Organic and synthetic inputs. The enzymatic activity of DH throughout the experiment (two crop cycles) increased significantly (p < 0.05) at soil depth 0–10 cm in the treatments with organic inputs, compared to those with synthetic inputs (fertilizers and herbicides) (Fig. 1), regardless of the type of tillage or crop established. In the month of September in both 2012 and 2013, there was a marked increase in DH activity in the soils with organic management practices (CTMO30r, ZTRO + ro, ZTRO + rm and ZTIO + r) compared to those with synthetic inputs, including under the conventional agriculture system (CTMQ  r) practiced in the zone (Fig. 1). In the ZTRQ + r and CTMQ  r systems, the herbicide 2,4-dichlorophenoxyacetic acid (2-4-D) was applied in the months of August and September of 2012 and 2013 at 12.5 l ha1 y1 (Table 2), a practice that led to a decline in the presence and function of the microbial community that was reflected in the enzymatic activity. The activity of DH has been proposed as an indicator since it is the product of living cells and is considered to reflect the detrimental or beneficial effects of substances such as herbicides on the structure of the microbial community (Nannipieri et al., 2003). Ratcliff et al. (2006) found that bacterial and fungal populations were modified as a result of the application of various herbicides. Specifically, the 2,4-D herbicide used in the present study can influence the soil microbial communities by altering the equilibrium among populations (Zabaloy et al., 2010). Zhang et al. (2010) showed that the addition of 2,4-D in different doses significantly changed microbial community structure in two agricultural soils; as the dose of herbicide increased the microbial population decreased,

Fig. 2. Soil organic matter and total nitrogen in the soils, in the different systems: ZTR = minimum tillage with rotation, ZTI = minimum tillage with intercrop CTM = conventional tillage with monoculture, O = organic inputs, Q = chemical inputs, +r = with residues, r = without residues, 30r = with 30% residues, m = maize and o = oats. Bars are  one standard error for triplicates.

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with a more marked effect in soils with lower SOM levels and less fertility. Considering the entire soil profile (depth 0–20 cm), the traditional Mesoamerican agricultural system known as the “Milpa” (ZTIO + r), intercropped with a forage crop (vetch), presented the highest DH enzymatic activity as well as the highest percentages of SOM and total nitrogen at soil depth 0–10 cm (Fig. 2). These two parameters are indicators of soil quality (Shukla et al., 2006) and are intimately linked to DH enzymatic activity (De Varennes et al., 2007). The establishment of vetch and the resulting vegetal cover throughout the experimental cycle may have led to improved chemical and physical conditions in the soil that favored the microbial community, as reflected by the DH enzymatic activity. This activity is an indicator of the levels of oxidoreduction in the live and active microbial community (Das and Varma, 2011) and supports that reported by Bastida et al. (2008) in soils under semiarid conditions similar to those of the present study. These authors concluded that, in sites covered with grass, the abundance of roots and proportion of vegetal cover was related to five major indices of enzymatic activity. 3.1.2. Tillage practices In our experiment, conservation of SOM was favored in systems in which minimum tillage was practiced (ZTIO + r, ZTRO + r and ZTRQ + r), compared to the treatments with conventional tillage (CTMO30r and CTMQ  r) involving disturbance of the soil (Fig. 2). It has been reported that reduced tillage protects the soil aggregates and thus the organic material held within these soil structures (Huggins et al., 2007). Under the conventional system, in which tillage was practiced, harvest residues were removed and synthetic inputs including herbicide were used (CTMQ  r). At soil depth 0–10 cm, the enzymatic activity of DH throughout the course

of the experiment was lower in this system than in the other treatments. We can therefore state that the lower organic matter content of the soil amplifies, or at least cannot buffer, the negative effect of the herbicide on the microbial populations. The reduced buffering capacity of the soil acts to impede the maintenance of stable microbial activity. The low values of DH indicate a lack of suitable conditions for microbial activity, since their concentration level reflects the initial stages of organic matter (OM) oxidation (Vepsäläenen et al., 2004; De Varennes et al., 2007). This phenomenon decreases in systems with herbicide; however, in soils with minimum tillage (ZTRQ + r), where the SOM at depth 0– 10 cm was greater (Fig. 2) than in the tilled soils, the negative effect of the herbicide is buffered to a greater extent (Zhang et al., 2010. The other system that featured tillage (CTMO30r), a practice that reduces the conservation of SOM relative to no-tillage systems (Huggins et al., 2007) with organic management, presented higher DH enzymatic activity at soil depth 0–20 cm (Fig. 1) than the conventional system with synthetic inputs (CTMQ  r). This was judged to be the result of not applying the 2,4-D herbicide, since the SOM content was similar under both treatments (Fig. 2). Likewise, enzymatic activity was found to increase in systems with minimum soil disturbance, more organic inputs and retention of residues (ZTIO + r y ZTRO + r). Since several authors state that the use of organic fertilizers increases the soil biochemical activity associated with the SOM (Kandeler et al., 1999; Trasar-Cepeda et al., 2008), the increased DH activity was probably due to both the absence of 2,4-D and the increased inputs of organic material to the soil. After two crop cycles, the treatment that presented significantly higher DH enzymatic activity at soil depth 10–20 cm than the others was CTMO30r, a trend that peaked in September of 2013 (Fig. 1). The treatments ZTIO + r y CTMQ  r presented an

Fig. 3. The potential enzymatic activities of urease in the soils, in the different systems: ZTR = minimum tillage with rotation, ZTI = minimum tillage with intercrop CTM = conventional tillage with monoculture, O = organic inputs, Q = chemical inputs, +r = with residues, r = without residues, 30r = with 30% residues, m = maize and o = oats. Bars are  one standard error for triplicates.

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intermediate DH enzymatic activity, while the rest of the minimum tillage treatments presented less activity (Fig. 1). With the lack of tillage at 10–20 cm, possible compaction may have reduced the quantity of oxygen in the soil, restricting microbial and thus enzymatic activity (Kandeler et al., 1999). 3.1.3. The potential activity of DH as an agrosystem quality indicator The objective of our study was to evaluate the use of enzymatic activity to monitor evolution of the soil and changes brought about by the particular type of agricultural management (types of inputs, tillage, crops). We found that the most sensitive enzymatic activity in this respect was that of DH, an enzyme that is considered part of intact cells and does not present extracellular accumulation in the soil (Kandeler et al., 1999). This enzyme participates in the decomposition of SOM and the transfer of protons and electrons related to processes of microorganism respiration. These activities are governed by soil type and condition, including air and water availability, which is related to the physical properties of the soil. Activity of this enzyme thus constitutes an indicator of the health and fertility of the soil (De Varennes et al., 2007; Kumar and Varma, 2011). Likewise, the activity of DH is affected by the inputs used in agriculture and by soil contamination (Nannipieri et al., 2003), for which reason it can also be a tool with which to measure environmental impacts. Different studies show that SOM or organic C is one of the most commonly used indicators to determine soil quality (Shukla et al., 2006); however, we consider that DH activity can also be used as an indicator to monitor the results of change in the agricultural management of agrosystems and can provide a more dynamic and integral perspective. Bastida et al. (2008) state that biological indicators offer more information regarding the dynamics of the soil than physical and chemical indicators, since biological indicators are closely linked to the nutrient cycle, particularly in terms of the impact of different uses

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and management practices of the soil. Furthermore, biological indicators make it possible to obtain information specifically pertaining to short-term processes. 3.1.4. Potential enzymatic activities of urease (URE) and protease (PRO) 3.1.4.1. Organic and synthetic inputs. On analysis of the entire crop cycle, the enzymatic activities of URE and PRO presented significant differences among treatments, but only at soil depth 0–10 cm (p < 0.0001 and p 0.003, respectively). However clear differences were not found among the treatments, according to the management and sampling data (Figs. 3 and 4). Throughout the experiment (two crop cycles), the enzymatic activities of PRO and URE were significantly higher (p < 0.05) in the soil of ZTIO + r (the traditional Mesoamerican agricultural system “Milpa”) at 0–10 cm in depth (Figs. 3 and 4). The enzymatic activity of PRO was similar among the remaining treatments. However, the enzymatic activity of URE differed among treatments, with the lowest activity found in the conventional management (CTMQ  r), despite the fact that fertilization had been carried out with urea. In the system ZTIO + r, the highest activity of both enzymes, which participate in the hydrolysis of peptide bonds and release of NH4+ (Banik and Prakash, 2004; Wang et al., 2008), may occur because between the years 2012 and 2013 this soil presented a higher quantity of total N and a greater proportion of organic matter at 0–10 cm soil depth (Fig. 2). With a higher proportion of soil organic matter, the urease hydrolyzes urea to form ammonium, which could increase the availability of nutrients and, in turn, stimulate the secretion of radical enzymes (Sujoy and Aparna, 2013). It has been shown that the activity of hydrolytic enzymes (PRO and URE) increases with organic inputs (Kandeler et al., 1999; Kremer and Li, 2003). This was demonstrated in the present study,

Fig. 4. The potential enzymatic activities of protease in the soils, in the different systems: ZTR = minimum tillage with rotation, ZTI = minimum tillage with intercrop CTM = conventional tillage with monoculture, O = organic inputs, Q = chemical inputs, +r = with residues, r = without residues, 30r = with 30% residues, m = maize and o = oats. Bars are  one standard error for triplicates.

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causing reduced activity in the only treatment in which no organic material (compost or stubble) was added (CTMQ  r). The peak of enzymatic activity of URE was detected in the treatments with organic additives (ZTIO + r, ZTRO + rm and CTMO30r) in the same month as these were applied (Table 2 and Fig. 3). Despite the existence of some differences in the PRO and URE enzyme activities among treatments, these proved to be less sensitive than the DH activity as indicators of change in the soil related to agricultural management. With respect to the PRO activity, only one treatment could be differentiated from the others, while with the URE activity there was a marked division between the activities of soils that were fertilized with organic inputs and those managed with synthetic inputs. 3.1.5. Tillage practices No clear evidence was found to discern any effect of tillage type on the enzymatic activities of PRO and URE. 3.1.6. Potential enzymatic activities of acid phosphatase (ACP) No significant differences in ACP activity were presented throughout the agricultural cycle (at soil depth 0–10 cm, p = 0.70 and depth 10–20 cm, p = 0.82) or in a detailed analysis per sampling date among treatments (Fig. 5). It is possible that there was no P deficiency in the soil in any of the treatments and that the type of fertilization, whether synthetic or organic, was suitable, for which reason the availability of P did not constitute a limiting factor for the development of the crops in any of the treatments. The ACP activity increased when there was a deficiency of P, the plant roots secrete this enzyme in order to improve the solubilization and remobilization of phosphate (Mudge et al., 2002; Versaw and Harrison, 2002).

Trasar-Cepeda et al. (2008) state that not all of the enzymes have a direct relationship with increased use of the soil or with the application of certain synthetic inputs used in the agricultural process, but that they did have a relationship with the SOM and its dynamics. These authors therefore suggest the need to identify an enzymatic activity that is directly related to the change or use of soil, and highlight the need for studies surrounding the different activities in agricultural uses. 3.2. Mycorrhizal colonization Throughout the 2013 agricultural cycle beginning of 2013, with the rains in June, the synthetic input treatments (those with application of fertilizers and herbicides) in both the conventional (CTMQ  r) and conservation (ZTRQ + r) agriculture presented the lowest indices of mycorrhizal colonization in the maize roots (Table 3). The trend was similar in terms of abundance of arbuscules, with these structures completely absent in the conservation agriculture treatment with synthetic inputs (Table 4). Different agricultural management practices determined the soil dynamics with respect to biogeochemical cycles and the dynamics of the microbiota, including colonization of the maize roots by native arbuscular mycorrhizal fungi (AMF). Hijri et al. (2006) state that rotation has a significant effect on AMF; the presence of different host plants helps to increase the presence and diversity of the fungi, compared to monocultures (Karasawa et al., 2002). Throughout the agricultural cycle in the present study, the maize plants that presented the lowest percentage of colonization and arbuscules were those established in a monoculture of maize managed in a conventional manner (CTMQ  r). However, the plants of the plot managed with CTMO30r, a maize monoculture with organic inputs, presented a similar percentage of

Fig. 5. The potential enzymatic activities of acid phosphatase in the soils, in the different systems: ZTR = minimum tillage with rotation, ZTI = minimum tillage with intercrop CTM = conventional tillage with monoculture, O = organic inputs, Q = chemical inputs, +r = with residues, r = without residues, 30r = with 30% residues, m = maize and o = oats. Bars are  one standard error for triplicates.

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Table 3 Mycorrhizal colonization (standard error) in the maize roots cultivated in five different agrosystems. Sampling 2013 treatments

4th of June

ZTIO + r* ZTRO + r* ZTRQ + r* CTMO30r** CTMQ  r**

a

4.26 3.95 bc 0.45 ab 2.94 c 0.05 a

    

0.95 0.62 0.20 0.69 0.05

19th of July b

0.32  0.28 0.50  0.46 ab 0.83  0.30 a 1.25  0.59 b 0.00  0.00 ab

26th of July b

0.25  0.22 2.73  0.45 b 0.65  0.49 ab 0.79  0.65 b 0.16  0.13 a

11th of September a

4.19  0.76 0.25  0.05 b 0.05  0.03 b 0.65  0.40 b 0.09  0.01 b

2nd of October a

2.12  0.63 1.97  1.20 1.20  0.35 a 2.93  0.76 ab 1.78  0.26 a

b

*Treatments sowing May 3 and **treatments sowing April 8. ZTI = minimum tillage with intercrop, ZTR = minimum tillage with rotation, CTM = conventional tillage with monoculture, O = organic inputs, Q = chemical inputs, +r = with residues, r = without residues, 30r = with 30% residues. The different letters (a–c) differ significantly at 5% based on least square difference grouping.

Table 4 Effect of five different agricultural management types on the abundance of arbuscules (standard error) in maize roots. Sampling 2013 treatments

4th of June

19th of July

26th of July

11th of September

2nd of October

ZTIO + r* ZTRO + r* ZTRQ + r* CTMO30r** CTMQ  r**

0.17  0.16 0.04  0.03 0.00  0.00 0.03  0.03 0.00  0.00

0.00  0.00 0.00  0.00 0.01  0.00 0.28  0.00 0.00  0.00

0.00  0.00 0.24  0.18 0.00  0.00 0.17  0.17 0.07  0.05

0.09  0.09 0.00  0.00 0.00  0.00 0.01  0.00 0.00  0.00

0.04 0.03 0.01 0.01 0.02

    

0.02 0.03 0.01 0.00 0.02

*Treatments sowing May 3 and **treatments sowing April 8. ZTI = minimum tillage with intercrop, ZTR = minimum tillage with rotation, CTM = conventional tillage with monoculture, O = organic inputs, Q = chemical inputs, +r = with residues, r = without residues, 30r = with 30% residues.

mycorrhization as that found in systems with rotation. Likewise, the maize plants managed with conservation agriculture and agrochemicals (ZTRQ + r), despite being cultivated in a rotation system, presented low percentages of colonization compared to those treatments in which organic inputs were used, regardless of whether these were monocultures or rotation systems. For this reason, we consider that the determinant factor for the development and colonization of AMF in the present study was the type of inputs applied to the soil. Incorporation of organic inputs to the soil stimulates mycorrhizal colonization since the mycorrhizae require C as a raw material (Gosling et al., 2006). In addition, the input of soluble P was lower than that implicit in the application of soluble synthetic fertilizers. Excess available P affects the root-fungus relationship, reducing AMF colonization (Kogelmann et al., 2004). Kahiluoto et al. (1999) showed that colonization and density of mycorrhizal fungal spores both decreased in soils with different crops fertilized with soluble P at low and intermediate doses. In the present study, the two plots fertilized with synthetic inputs (conventional agriculture – CTMQ-r – and conservation agriculture with agrochemicals – ZTRQ + r –) presented plants with low mycorrhizal colonization and reduced abundance of arbuscules (Tables 3 and 4). Herbicides can also have an indirect effect on mycorrhizal colonization; however, the greatest effect is seen with the elimination of weeds that could act as host plants (Gosling et al., 2006). Some authors state that the mycorrhizal fungi contribute to the degradation of biocides by creating conditions that stimulate the activity of the enzymes involved in this process (Huang et al., 2009; Wu et al., 2008). There is debate regarding whether or not the use of tillage has an influence over the colonization or persistence of AMF: there are studies that show that intensive agriculture can diminish the presence of mycorrhizae, while non-tillage acts to stimulate it (Oliveira and Sanders, 1999; Galvez et al., 2001; Borie et al., 2006). However, Jansa et al. (2003) argue that the majority of the AMF of agricultural systems are Glomus spp. (Daniell et al., 2001), which form spores that can easily survive the process, implying that intensive tillage merely acts to dissipate them. These authors also argue that addition of agrochemicals to the soil, rather than tillage, may be the factor that reduces the dispersion of the hyphae. This coincides with the results obtained in the present study, since in

the case of the treatment CTMO30r, where the soil was disturbed and organic inputs were used, there was a higher level of colonization compared to the non-tillage treatment with the synthetic inputs (ZTRQ + r) described above. Mirás-Avalos et al. (2011) reiterate this idea, proposing that AMF have a considerable capacity for reestablishing colonization, for which reason tillage has no impact in this sense, and is likely to only affect fungal diversity. In the present study, the time that elapsed between agrochemical (fertilizer and herbicide) applications was shorter between the first and fourth sampling dates than between the fourth and fifth sampling dates (Table 2). During this final stage, AMF colonization increased in those treatments with chemical inputs (Table 3). This may demonstrate the effect of agrochemicals on the development of AMF colonization. In the first sample, 42 days after sowing when the rains began, the maize roots that presented the greatest mycorrhizal colonization were those that developed in the systems “Milpa” (ZTIO + r) and conservation agriculture with organic inputs (ZTRO + r) (Table 2). This could be an indicator of the presence of spores in the soil during the dry season, which did not occur in the other systems. In the second sample, a fall in the percentage of mycorrhizal colonization was observed in all of the treatments relative to the first sample (Table 3). This was attributed to reduced soil O2 as a result of the increased precipitation in the study zone and the formation of a crust in various plots, but was a dynamic that changed when the rains diminished in the fourth and fifth samples (11th of September and 2nd of October). Mycorrhizal colonization can be considered an indicator of soil biological, chemical and physical quality (Khade and Adholeya, 2007; Kohler et al., 2009). Considering this, it is notable that all of the organic systems on the five sample dates presented the highest percentages of mycorrhization, indicating that, three years after initial establishment, these systems present a higher quality soil compared to those systems that use chemical inputs, regardless of soil management practice, rotation or retention of residues. 4. Conclusions In the agroecological region of the Valle de México, the potential microbial dehydrogenase activity in the soil was more

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sensitive to changes in agricultural management produced by the type of tillage, inputs (organic or synthetic) and topological arrangement compared to the activities of urease, protease and acid phosphatase, which did not present any clear pattern with respect to the different agrosystems or to sampling date (based on the agricultural practices). The percentage of mycorrhizal colonization also demonstrated sensitivity to certain changes in agricultural management, especially to the type of inputs utilized, but not to tillage type or crop rotation. Dehydrogenase activity and mycorrhizal colonization were both clearly affected by the use of chemical inputs (fertilizers and herbicide) and the quantity of organic material present in the soil. This indicates that both parameters could represent tools for not only monitoring soil biological quality with respect to nutrient cycling as a function of the crop, but also to determine the level of affectation or buffering with respect to the use of synthetic inputs or contaminants in the soil. According to the dehydrogenase enzymatic activity and mycorrhizal activity of the different agrosystems evaluated, we can conclude that the Mesoamerican agrosystem known as “Milpa”, involving minimum soil movement, intercropping (maize, squash, beans, vetch) and organic inputs, was the system that presented the highest soil biological activity and thus the highest quality. From the agronomic perspective, it may be necessary to determine a manner in which to correlate the parameters studied here with indicators of agrosystem productivity. This could provide tools to the producers and decision-makers with which to manage not only environmental impacts and soil quality, but also the supply of food produced by these soils in both the short and medium term. Acknowledgement This research was funded by the project of Basic Scientific Research 2011, CONACYT, No. 169056 References Acosta-Martínez, V., Acosta-Mercado, D., Sotomayor-Ramírez, D., Cruz-Rodríguez, L., 2008. Microbial communities and enzymatic activities under different management in semiarid soils. Appl. Soil Ecol. 38, 249–260. Askari, M.S., Holden, N.M., 2015. Quantitative soil quality indexing of temperate arable management systems. Soil Till. Res. 150, 57–67. Baars, E., Baars, T., 2007. Towards a philosophical underpinning of the holistic concept of integrity of organisms within organic agriculture. NJAS Wageningen J. Life Sci. 454. Banik, R.M., Prakash, M., 2004. Laundry detergent compatibility of the alkaline protease from Bacillus cereus. Microbiol. Res. 159, 135–140. Bastida, F., Zsolnay, A., Hernández, T., García, C., 2008. Past, present and future of soil quality indices: a biological perspective. Geoderma 147, 159–171. Bending, G.D., Turner, M.K., Rayns, F., Marx, M.C., Wood, M., 2004. Microbial and biochemical soil quality indicators and their potential for differentiating areas under contrasting agricultural management regimes. Soil Biol. Biochem. 36, 1785–1792. Borie, F., Rubio, R., Rouanet, J.L., Morales, A., Borie, G., Rojas, C., 2006. Effects of tillage systems on soil characteristics, glomalin and mycorrhizal propagules in a Chile an Ultisol. Soil Till. Res. 88, 253–261. Ceja-Navarro, J.A., Rivera-Orduña, F.N., Patiño-Zuñiga, L., Vila-Sanjurjo, A., Crossa, J., Govaerts, B., Dendooven, L., 2010. Phylogenetic and multivariate analyses to determine the effects of different tillage and residue management practices on soil bacterial communities. Appl. Environ. Microbiol. 76, 3685–3691. Czarnes, S., Hallett, P.D., Bengough, A.G., Young, I.M., 2000. Root- and microbial16 derived mucilages affect soil structure and water transport. Eur J. Soil Sci. 51, 435–443. Daniell, T.J., Husband, R., Fitter, A.H., Young, J.P.W., 2001. Molecular diversity ofarbuscular mycorrhizal fungi colonising arable crops. FEMS Microbiol. Ecol. 36, 203–209. Das, S.K., Varma, A., 2011. Role of enzymes in maintaining soil health. In: Shukla, G., Varma, A. (Eds.), Soil Enzymology. Springer-Verlag, Berlin, pp. 25–42. De Varennes, A., Torres, M.O., Queda, C., Goss, M.J., Carranca, C., 2007. Nitrogen conservation in soil and crop residues as affected by crop rotation and soil disturbance under Mediterranean conditions. Biol. Fertil. Soils 44, 49–58.

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