Soil microbiological properties and available nitrogen for corn in monoculture and intercropped with forage

The objective of this work was to evaluate the effects of corn (Zea mays) in monoculture and intercropped with forage on soil microbiological properties and nitrogen availability under no-tillage in the Cerrado (Brazilian savanna). The experiment was carried out from the 2007/2008 to the 2010/2011 crop season, in a Latossolo Vermelho-Amarelo distrófico (Typic Haplustox). A randomized complete block design with three replicates was used, with the following treatments: corn in monoculture; corn intercropped with Panicum maximum; corn intercropped with Urochloa humidicola; P. maximum in monoculture; and U. humidicola in monoculture. Soil samples were taken at 0.00–0.05, 0.05–0.10, 0.10–0.20, and 0.20–0.30-m soil depths, in the begining and in the end of the last crop season. The intercropping systems of corn increased soil nitrogen availability, but did not alter total nitrogen and organic carbon contents in the soil, when compared to corn in monoculture. Corn intercropped with P. maximum increased soil microbial biomass nitrogen and microbial nitrogen quotient, in comparison to corn in monoculture, as well soil as microbial biomass carbon in the surface soil layer, when compared to corn intercropped with U. humidicola.


Introduction
The adoption of no-tillage (NT) practices when intercropping forages with cash crops has great potential to improve soil quality through carbon sequestration, water and nutrient cycling, and improvement of soil biological diversity (Lemaire et al., 2014).The Cerrado (Brazilian savanna) biome is occupied with approximately 26% of pasture lands, with several degrees of degradation (Sano et al., 2010), and accounts for 45% of corn production in the Midwest region of Brazil (Acompanhamento..., 2015).Therefore, the use of diversified systems in the Cerrado, such as cash crop-forage intercropping, should be encouraged and better understood.
Soil organic matter (SOM) is considered a key indicator of soil quality because it is the most important component of soil fertility in highly weathered soils (Lopes et al., 2013).Studies have reported increases in SOM under NT practices with intercropping of forages and cash crops, mainly due to higher inputs of plant residues, which can increase soil nutrient availability and soil C sequestration (Boeni et al., 2014).Increments in soil organic C are commonly related to improvements in chemical and biological properties (Araújo et al., 2007;Lopes et al., 2013).
Increases in soil microbial biomass (SMB), under NT, are well documented (Figueiredo et al., 2007;Ferreira et al., 2011).However, differences in plant residue quality and chemical composition may affect SMB growth (Ferreira et al., 2011;Cong et al., 2015), which, in turn, may be positively related to N (NO 3 - and NH 4 + ) mineralization potential in the soil and, consequently, to its uptake by SMB (Burger & Jackson, 2003).Dourado-Neto et al. (2010) highlighted that, in tropical agroecosystems, soil N pool can provide up to 79% of the total N uptake by plants, showing the importance of SOM preservation and of plant residue inputs for N availability to crops.
To reduce environmental impacts on the Cerrado, one of the most threatened biomes in the world (Hunke et al., 2015), intercropping systems have been increasingly adopted.However, there is still a lack of information on the effects of different combinations of plant species on soil microbiota and on N availability in these systems for the Cerrado.Plant species differ in the way they allocate biomass and, therefore, in the quantity and quality of their residues (Carvalho et al., 2012), which may affect C input and the levels of N and microbial biomass in the soil.
The objective of this work was to evaluate the effects of corn (Zea mays L.) in monoculture and intercropped with forage on soil microbiological properties and N availability under no-tillage in the Cerrado (Brazilian savanna).
Fifteen experimental plots were established in October 2007, in an area where Andropogon gayanus Kunth 'Planaltina' had been grown for a period of six years.Each plot had 80 m 2 (10x8 m), of which 48 m 2 were evaluated.The experiment consisted of the following treatments under NT: corn in monoculture; corn intercropped with Panicum maximum Jacq.'Aruana'; corn intercropped with Urochloa humidicola (Rendle) Morrone & Zuloaga; P. maximum in monoculture; and U. humidicola in monoculture.A randomized complete block design, with three replicates, was used.The experimental plots were kept as previously described, and agricultural practices were repeated in the following cash crop seasons: 2007/2008, 2008/2009, 2009/2010, and 2010/2011.In November 2010, prior to corn sowing, a herbicide mixture of glyphosate [N-(phosphonomethyl) glycine], with 1.08 kg ha -1 a.i.(3 L ha -1 ), and of 2.4-dichlorophenoxyacetic acid (2.4-D), with 1.1 kg ha -1 a.i.(1.5 L ha -1 ), was applied for forage and weed desiccation.
The early-maturing triple-cross corn hybrid 'BG7055' was sown in December 2010, in eight rows spaced 0.9 m apart, with six plants per meter, totaling 65,000 plants per hectare.For fertilization, 30 kg ha -1 N, 100 kg ha -1 P 2 O 5 , 70 kg ha -1 K 2 O, and 66 kg ha -1 fritted trace elements (FTE) BR-12 were applied.Forty-four days after corn emergence, when plants had around eight leaves, 70 kg ha -1 N were also applied in the form of ammonium sulfate.
The species P. maximum and U. humidicola were sown in the 2007/2008 and 2009/2010 seasons with 30 kg ha -1 seed, considering the pure live seed percentage.The same procedure was adopted in the intercropping and monoculture plots.The seeds were sown in between corn rows, after corn seeding.The average amount of plant biomass (dry matter) produced by the P. maximum and U. humidicola forages in consortium, at the end of the 2009/2010 season, was 2.17 and 2.12 Mg ha -1 , respectively.In the Pesq.agropec.bras., Brasília, v.51, n.9, p.1660-1667, set.2016 DOI: 10.1590/S0100-204X2016000900066 plots with monoculture forages, the obtained values were 1.74 Mg ha -1 for P. maximum and 2.10 Mg ha -1 for U. humidicola.The plots with monoculture forage received 60 kg ha -1 P 2 O 5 and 60 kg ha -1 K 2 O, and N was divided into two side-dressing doses of 30 kg ha -1 , totaling 60 kg ha -1 N.
Soil samples were collected at the beginning (before corn planting) and at the end (after harvest) of the 2010/2011 crop season, in December 2010 and April 2011, respectively, at the depths of: 0.00-0.05,0.05-0.10,0.10-0.20,and 0.20-0.30m.For each plot, 15 subsamples were collected at three locations within the plot.At each location, one sample was collected in the planting row and another four samples were collected in between corn rows (equidistant lines).
Total organic carbon (TOC) was determined by oxidation with potassium dichromate in the presence of acid without external heat source (Walkley & Black, 1934).Total soil nitrogen (TN) was estimated by the Kjeldahl method, according to Bremner & Mulvaney (1982).
To measure available N, the extraction method Na 3 PO 4 /borax buffer pH 11.2 + NO 3 -was used (Serra, 2006).Calculations were performed from the calibration curve obtained with the distillation of standard solutions containing 0, 15, 30, 45, and 60 µg mL -1 N. The extracted N was determined by colorimetric spectrophotometry at 440 nm.
Microbial biomass nitrogen (MBN) was obtained with the method of chloroform fumigation-extraction, described by Brookes et al. (1985) and Vance et al. (1987), using a correction factor of 0.54 (Wardle, 1994), and the microbial nitrogen quotient (qMIN) was determined by the ratio between MBN and TN.In addition, microbial biomass carbon (MBC) was calculated according to Vance et al. (1987), using a correction factor of 0.38 (Wardle, 1994), whereas the microbial carbon quotient (qMIC) was obtained by the ratio between MBC and TOC.
Data were subjected to the analysis of variance, and the means were compared by Tukey's studentized range test, at 5% probability.Data from all treatments, depths, and sampling periods were further analyzed with Pearson's linear correlation analysis.Analyses were performed using the Sisvar software, version 5.3 (Universidade Federal de Lavras, Lavras, MG, Brazil).

Results and Discussions
The corn/U.humidicola intercropping provided the highest contents of TOC and TN in the 0.00-0.05-msoil layer, whereas U. humidicola in monoculture, the lowest ones (Figure 1).These results indicate the benefits of integrating this forage with corn, with increases of 13% in TOC and 9% in TN in the uppermost soil layer, when compared to sole U. humidicola.Corn fertilization may have increased forage root development -which, in some species, can reach up to 4 Mg ha -1 in the 0.00-0.40-mlayer (Saraiva et al., 2014) -and affected soil TOC and TN.Moreover, under NT systems, corn roots, which are more concentrated in surface soil layers (Silva et al., 2000), together with the deposition of plant residues, may have also contributed to increases in TOC and TN.It should be noted, that although the enhanced levels of TN may also be attributed to N fertilization in corn, the simultaneous increase in TOC suggests the possibility of synergy between C and N, whereby C sequestration enhances N sequestration, and vice versa (Cong et al., 2015).Diógenes et al. (2013) found higher TOC values for corn intercropped with U. ruziziensis, when compared to this forage in monoculture.
Four years after the establishment of the experiment, no differences were found in TOC content between U. humidicola and P. maximum, nor between the intercropping systems and corn in monoculture.The largest compartment of organic C, approximately 67%, in Oxisols in the Cerrado region is allocated in organomineral complexes, an interaction that minimizes microbial attack and decomposition, reducing the turnover rate of organic matter (Boemi et al., 2014).This may explain the lack of significant differences between some of the management systems.Changes in C concentration from particulate organic matter, which accounts for 33% of TOC and plays a very important role in biological activity, are more evident following shifts in soil management and cropping systems (Coser et al., 2012;Boeni et al., 2014).
The corn/P.maximum intercropping system showed the highest soil N availability (AN) in the 0.00-0.05-mlayer and prior to corn planting, followed by corn/U.humidicola, P. maximum, corn, and U. humidicola (Table 1).Corn/P.maximum also increases C content of the particulate organic matter fraction, indicating that this may be the reason for higher AN under this system (Burger & Jackson, 2003;Coser et al., 2012).
Figure 1.Total organic carbon and nitrogen in soil under monoculture of corn (Zea mays), Panicum maximum, and Urochloa humidicola, as well as under integrated systems with these species, at different soil depths.Means followed by equal letters do not differ by Tukey's test, at 5% probability.
Table 1.Available nitrogen and its relation to total nitrogen in soil under monoculture of corn (Zea mays), Panicum maximum, and Urochloa humidicola, as well as under integrated systems with these species, at different soil depths, in two sampling periods: before planting of corn and after corn harvest (1) .
The content of available N under the intercropping systems decreased from the beginning to the end of the season, possibly indicating net N mineralization of N-organic forms during the rainy season or uptake of N (NO 3 --N and NH 4 + -N) by the plants during this period.
Considering all the cropping systems, depths, and sampling periods, the percentage of AN compared to TN (AN/TN) ranged from 2.75 to 3.94%, with no significant differences (Table 1).These ranges indicate that only a small fraction of TN is easily decomposable by soil microorganisms.
MBN ranged from 10.47 to 48.31 mg kg -1 , which represented between 0.67 and 2.79% of TN (Table 2).Corn/P.maximum showed the highest soil MBN in the 0.00-0.05-mlayer, prior to corn sowing, which was significantly higher than that of the sole cropping systems (Table 2).The species U. humidicola and P. maximum in monocultures did not differ from each other regarding MBN and qMIN, in the 0.00-0.05m layer.The introduction of P. maximum as an intercrop in corn production systems favors SMB growth in this soil layer.Higher qMIN and MBN in the corn/P.maximum intercropping system may represent higher N cycling efficiency and availability in short-term periods (Xavier et al., 2006).
After corn harvest, corn/U.humidicola showed higher MBN and qMIN in the 0.00-0.05-mlayer than corn in monoculture.This may be explained by root exudates released by U. humidicola, which inhibit soil nitrification (Subbarao et al., 2006) and may reduce N losses in the form of NO 3 --N, increasing soil microbial biomass N. The soil nitrification process under P. maximum was between 47-73% lower than under U. humidicola (Ipinmoroti et al., 2008).
Table 2. Microbial biomass nitrogen and microbial nitrogen quotient (qMIN) in soil under monoculture of corn (Zea mays), Panicum maximum, and Urochloa humidicola, as well as under integrated systems with these species, at different soil depths, in two sampling periods: before planting of corn and after corn harvest (1) .

Cropping system
Microbial biomass nitrogen (mg kg (1) Means followed by equal letters, uppercase in the rows and lowercase in the columns, do not differ by Tukey's test, at 5% probability.
MBC ranged from 74.73 to 248.9 mg kg -1 (Table 3).Considering the interpretative classes for microbial indicators in a clayey Oxisol in the Cerrado region (Lopes et al., 2013), the MBC values in the 0.00-0.05and 0.05-0.10-mlayers varied from low to moderate.
The amount of MBC obtained under corn/P.maximum (248.9 mg kg -1 soil) was almost twice as high as with corn/U.humidicola (127.2 mg kg -1 soil).Since both forages produced similar amounts of shoot dry matter in the 2009/2010 season (2.12 and 2.17 Mg ha -1 , respectively), the higher MBC under corn/P.maximum may be related to the quality of its residues, including lower C:N ratios and lignin contents.Plant litter quality can explain decomposition rates and the release of nutrients to the soil, affecting the levels of MBC (Carvalho et al., 2012).
When both forages were compared as a monoculture, P. maximum had higher MBC in the 0.10-0.20-mlayer.Lopes et al. (2010) reported that MBC is greater under P. maximum pastures than in those with Urochloa spp., in the 0.00-0.10-mlayer.These authors associated the higher content of MBC in P. maximum pastures with greater amounts of shoot dry matter and released exudates, when compared to Urochloa spp.However, in the present study, the amounts of shoot dry matter produced by both forages were similar and, therefore, the greatest MBC values should be related to root exudates and to the chemical composition of P. maximum litter.
Comparing sampling periods, MBC ranged from 119.6 to 172 mg kg -1 , prior to corn planting, and from 137.8 to 199.7 mg kg -1 after corn harvest.However, differences between sampling periods were only significant between the layers at 0.05-0.20-msoil depths, in which MBC was greater after corn harvest (Table 3).As also observed for TOC, higher MBC after corn harvest may be associated with the decomposition of plant residues and to nutrient mineralization, which would provide nutrients to SMB during the corn growing season.
The ratio of biomass C to soil organic C (qMIC) reflects the contribution of microbial biomass to soil organic C and, therefore, indicates the quality of SOM (Wardle, 1994).Differences in qMIC among cropping systems were found up to 0.20-m depth (Table 3), with values ranging from 1.03 to 0.33%.As also verified for MBC, corn/P.maximum showed greater qMIC than corn/U.humidicola in the 0.00-0.05and 0.10-0.20-mlayers.Therefore, corn/P.maximum seems to be more efficient in converting TOC in MBC than corn/U.humidicola (Wardle, 1994).
Despite the similarity in the values of qMIN and AN/TN, the lack of correlation between AN and MBN may be an indication that the AN pool is a distinct fraction of MBN (Table 4).However, it is important 78% of the mineralizable N in the soil depends on the size and fraction proportions of SOM (1) Means followed by equal letters, uppercase in the rows and lowercase in the columns, do not differ by Tukey's test, at 5% probability.Pesq.agropec.bras., Brasília, v.51, n.9, p.1660-1667, set.2016 DOI: 10.1590/S0100-204X2016000900066 Furthermore,

Table 3 .
Microbial biomass carbon (MBC) and microbial carbon quotient (qMIC) in soil under monoculture of corn (Zea mays), Panicum maximum, and Urochloa humidicola, as well as under integrated systems with these species, at different soil depths, in two sampling periods: before planting of corn (BPC) and after corn harvest (ACH)(1).
(1) Means followed by equal letters, uppercase in the rows and lowercase in the columns, do not differ by Tukey's test, at 5% probability.