Mixed Intercropping of Wheat and White Clover to Enhance the Sustainability of the Conventional Cropping System: Effects on Biom

sustainability

Article Mixed Intercropping of Wheat and White Clover to Enhance the Sustainability of the Conventional Cropping System: Effects on Biomass Production and Leaching of Mineral Nitrogen

Antonín Kintl 1, Jakub Elbl 1,2 , Tomáš Lošák 3, Magdalena Daria Vaverková 4,* and Jan Nedˇelník 1

1 Agriculture Research, Ltd., Zahradní 1, 664 41 Troubsko, Czech Republic; [email protected] (A.K.); [email protected] (J.E.); [email protected] (J.N.) 2 Department of Agrosystems and Bioclimatology, Faculty of AgriSciences, Mendel University in Brno, Zemˇedˇelská 1, 613 00 Brno, Czech Republic 3 Department of Environmentalistics and Natural Resources, Faculty of Regional Development and International Studies, Mendel University in Brno, Zemˇedˇelská 1, 613 00 Brno, Czech Republic; [email protected] 4 Department of Applied and Landscape Ecology, Faculty of AgriSciences; Mendel University in Brno, Zemˇedˇelská 1, 613 00 Brno, Czech Republic * Correspondence: [email protected]; Tel.: +420-545-132-484

Received: 3 August 2018; Accepted: 16 September 2018; Published: 20 September 2018

Abstract: The main goal of our research was to compare the mixed intercropping (MC) of winter wheat (Triticum aestivum) and white clover (Trifolium repens) with the sole cropping of Triticum aestivum, during two growing seasons (2012–2014), in a lysimetric experiment. We aimed to investigate the effect of the above growing system on total biomass production, grain yield and leaching of mineral nitrogen (Nmin). Four variants of intercropping (0 kg N/ha; 112 kg N/ha; 112 kg N/ha + 1.25 L of humic acids/ha; 70 kg N + 0.65 L of humic acids/ha) and two variants of sole cropping (0 kg N/ha; 140 kg N/ha) were used. Research results showed a good potential for growing wheat in the mixed cropping system without any negative effect on grain and total biomass production. No signiﬁcant differences were found between the variants where winter wheat was grown with white clover (70 and 112 kg N/ha), and variants with sole wheat (140 kg N/ha), in both years of the experiment. The loss of Nmin from the soil was affected by the application of N fertilizer and mixed intercropping system. During the experiment, the loss of Nmin was higher by 20% in the variant using the sole winter wheat (140 kg N/ha), than in the MC variants.

Keywords: nitrogen; mixed cropping; grain yield; wheat; clover

1. Introduction The global challenge to meet the increased food demand and to protect the quality of the environment will be won or lost by the growing systems producing corn, rice and wheat [1]. Intensive agriculture provides high yields but can also cause serious environmental problems [2]. Over the past century, the development of new agricultural practices to meet the growing global demand for food disrupted the N cycle within agro-ecosystems [3]. Reactive nitrogen (all oxidizable N forms) is not only an important nutrient to ensure human nutrition but also an agent signiﬁcantly disturbing the agro-ecosystem [4–6]. The excessive application of N mineral fertilizers causes serious problems [5]. During the 20th century, farmers around the world replaced leguminous crops as a

Sustainability 2018, 10, 3367; doi:10.3390/su10103367 www.mdpi.com/journal/sustainability Sustainability 2018, 10, 3367 2 of 13 natural source of N with synthetic fertilizers [7,8]. Today’s food and energy production are not feasible without the use of N synthetic fertilizers [8]. To sustain global food supply, nearly 100 million tons of N are produced annually using the Haber-Bosch process [9]. Current methods for determining the need for N fertilizer in winter wheat are based on agronomic assumptions of yield targets and on the fixed dose of N taken from the grain unit produced [10,11]. The problem with the “optimization of the fertilization plan” is that plants absorb only a small percentage of applied mineral nutrients. A great amount of nutrients (30–50%) can subsequently be irretrievably lost, especially in the case of mineral fertilizers [12]. The applied N, which is not taken up by the plants or immobilized in the soil, is susceptible to volatilization, denitrification, and loss by leaching. Total N-use efficiency in the growing system can be increased by the higher efficiency of N from N inputs, or by reducing the amount of N lost from soil resources [1]. For agricultural systems to remain productive, the supply of nutrients removed or lost from the soil has to be replenished [13]. As to nitrogen, inputs to the agricultural system may be partially replaced by the biological fixation of atmospheric N2 (BFN) provided by leguminous species [13–15]. The use of leguminous plants in the rotation or mixed crop is, nowadays, regarded as an alternative to the sustainable way of introducing N into the growing systems [16–18]. One way to reintroduce leguminous plants into crop rotation is to use mixed cultures. A mixed culture means growing two different crops on the same plot at the same time, during the growing season. These are mainly mixtures of legumes and non-legumes. The need for incorporating these crops into grain crops, to increase the soil fertility and sustainability of agricultural systems, is often neglected, but the positive influence of leguminous crops in the crop rotation is generally recognized [19,20]. The main objective of the presented research study was to evaluate the possibility of growing wheat in the mixed culture with legumes (white clover), and the influence of this method on grain yield, and leaching of mineral nitrogen Nmin.

2. Materials and Methods

2.1. Design of the Experiment Winter wheat (Triticum aestivum, cv. Golem) and white clover (Trifolium repens, cv. Dolina 4n) were used as model plants in the presented experiment. Seed crops were purchased each year prior to the planned-sowing of cv. Golem ELITA (a company) semenarian, a.s., and cv. Dolina 4n from the Agriculture Research, Ltd., (Troubsko, Czech Republic). Germination of Triticum aestivum, cv. Golem was 93% and Trifolium repens, cv. Dolina 4n was 82%. The experiment consisted of six variants, each one with three replications. These were divided into two groups: sole crops (SC) and mixed crops (MC), with different doses of fertilizers (Table1). Fertilizers used during the experiment were as follows: DAM 390 (liquid nitrogen fertilizer) and Lignohumate B (LG B). DAM 390 (nitrogen fertilizer—NF) is a solution of ammonium N and urea, with a weight average N content of 30%. It contains 39 kg of N in 100 L. LG B represents water solution obtained by the hydrolytic-oxidative decomposition of technical lignosulfonates, and can be defined as a mixture of humic and fulvic acids (ratio 1:1), and their salts. NF and LG B were applied as foliar fertilization during the two years of the experiment, DAM as a source of N for plants and LG B to support the soil microbial activity. The fertilizers were applied in three doses each year. The BBCH (Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie) scale was used to identify vegetation stage of winter wheat. The first dose was applied at the end of tillering (BBCH 27–29), the second one during the stage of stem elongation growth (BBCH 30) and the third one after the flag leaf stage (BBCH 39). Winter wheat and white clover were sown directly (seeds set without pre-germination) into individual pots, seeding rates being 220 kg/ha and 15 kg/ha, respectively. Winter wheat was sown as a sole crop or together with clover as a mixed crop. In both cases, the seed rates were identical. Sustainability 2018, 10, x FOR PEER REVIEW 3 of 13

Table 1. Variants and nutrient amounts applied to the lysimeters. Sustainability 2018, 10, 3367 3 of 13 Total N Applied Humic and Fulvic Acids Treatment Description Fertilizers (kg N/ha) (L/ha) C1 SoleTable Crop 1. (Control)Variants and nutrient amounts0 applied to the lysimeters. 0 - C2 Sole Crop 140 0 DAM 390 Total N Applied Humic and Fulvic Acids Treatment Description Fertilizers A1 Mixed Cropping (kg N/ha) 112 (L/ha) 0 DAM 390 A2 Mixed Cropping 112 1.25 DAM 390 + LG B A3C1 Sole Mixed Crop Cropping (Control) 0 70 0 0.65 DAM - 390 + LG B C2 Sole Crop 140 0 DAM 390 A4 Mixed Cropping (Control) 0 0 - A1 Mixed Cropping 112 0 DAM 390 A2 Mixed Cropping 112 1.25 DAM 390 + LG B WinterA3 wheat Mixed and Cropping white clover were 70 sown directly (seeds 0.65 set without pre-germination) DAM 390 + LG B into Mixed Cropping individualA4 pots, seeding rates being 220 kg/ha0 and 15 kg/ha, respectively. 0 Winter wheat was - sown as a sole crop or together(Control) with clover as a mixed crop. In both cases, the seed rates were identical.

2.2.2.2. Soil Soil Lysimeter Lysimeter TheThe above-mentioned above-mentioned objectiveobjective ofof thethe presentedpresented workwork waswas studiedstudied inin aa lysimetriclysimetric experiment.experiment. AA lysimetric lysimetric stationstation waswas installedinstalled in the buffer buffer zone zone of of the the underground underground source source of of drinking drinking water water in inBř Bˇrezovezová ánadnad Svitavou Svitavou (Bohemian–Moravian (Bohemian–Moravian Highlands; Highlands; lat. lat. 49°39 49◦39′41.82041.82”″ N; N;long. long. 16°30 16◦′1.913001.91”″ E; the E; thearea area of interest of interest is marked is marked with witha red asquare) red square) where where forty-year forty-year average average (1962–2012) (1962–2012) of rainfall of is rainfall 588.47 ismm 588.47 and mm annual and air annual temperature air temperature 7.9 °C. Monthly 7.9 ◦C. Monthly average averagetemperatures temperatures and precipitation and precipitation amounts amountsduring the during experiment the experiment are shown are in shownFigure in1. Above Figure 1all,. Above the overview all, the overviewand location and of location the experiment of the experimentare shown arein Figure shown 2. in Figure2.

Figure 1. Weather conditions at the lysimetric station: (A) Average monthly temperature during Figure 1. Weather conditions at the lysimetric station: (A) Average monthly temperature during 2012– 2012–2014; (B) average monthly precipitation during 2012–2014. 2014; (B) average monthly precipitation during 2012–2014. Lysimeters (AppendixA) were ﬁlled with 25 kg of topsoil and 25 kg of subsoil; the soil was Lysimeters (Appendix A) were filled with 25 kg of topsoil and 25 kg of subsoil; the soil was sampled in the area of our interest. The topsoil and subsoil samples were processed separately sampled in the area of our interest. The topsoil and subsoil samples were processed separately by by homogenization and sieving (sieve size 10 mm). Selected properties of soils (luvisol modal; homogenization and sieving (sieve size 10 mm). Selected properties of soils (luvisol modal; sandy sandy loam), used in the experiment, are listed in Table2. loam), used in the experiment, are listed in Table 2. Table 2. Selected physical and chemical properties of experimental arable soil (topsoil = 0–0.25 m; subsoil = 0.25–0.40 m).

Ctot Ntot Plant Available Nutrient (mg/kg) Sample C/N pH (CaCl2) (g/kg) (g/kg) P K Ca Mg Topsoil 13.32 1.41 9.44 233 349 1801 69 5.7 Subsoil 0.597 0.052 11.48 143 56 1451 46 6.1

Sustainability 2018, 10, 3367 4 of 13 Sustainability 2018, 10, x FOR PEER REVIEW 4 of 13

Figure 2.2. Location of thethe lysimetriclysimetric experimentexperiment (source(source ofof mapmap data:data: mapsopensource.com):mapsopensource.com): (A) thethe position of the individual lysimeters, in the ﬁeld,field, together with the control shaft that has been used to collect the soil eluate; ((B)) LysimeterLysimeter preparedprepared forfor soilsoil placement.placement.

2.3. LeachingTable 2. ofSelected Mineral physical Nitrogen and chemical properties of experimental arable soil (topsoil = 0–0.25 m; subsoil = 0.25–0.40 m). The leaching of Nmin was measured as a concentration of ammonium and nitrate N in the soil leachate, fromCtot lysimeters (AppendixA). ThePlant soil leachateAvailable was Nutrient monitored (mg/kg) three times per week. Sample Ntot (g/kg) C/N pH (CaCl◦ 2) When a leachate(g/kg) was detected, it was immediatelyP collected,K transportedCa andMg stored at 3.5 C, before analysis.Topsoil The analysis13.32 of Nmin 1.41 concentration 9.44 was 233 made 349after each 1801 sampling 69 of the soil 5.7 leachates. The distillation–titrationSubsoil 0.597 method 0.052 by Muñoz-Huerta 11.48 143 et al. [21 56] was used 1451 to determine 46 the 6.1 N min in the soil leachate with the use of the Distillation Unit Behr S3 (Behr Labor Technik, Düsseldorf, Germany). The2.3. Leaching concentration of Mineral of NNitrogenmin was always determined in the particular volume of the soil leachate; therefore, the loss of Nmin from the individual lysimeters was expressed in mg/L. The leaching of Nmin was measured as a concentration of ammonium and nitrate N in the soil 2.4.leachate, Analysis from of lysimeters Plant Biomass (Appendix A). The soil leachate was monitored three times per week. When a leachate was detected, it was immediately collected, transported and stored at 3.5 °C, before analysis.At the The end analysis of each of N growingmin concentration season (2013; was made 2014), after grain each production sampling andof the total soil productionleachates. The of above-grounddistillation–titration biomass method were by measured Muñoz-Huer by takingta et al. all [21] the was plants, used andto determine their parts, the (stems,Nmin in the leaves, soil husks,leachate etc.) with from the theuseindividual of the Distillation lysimeters, Unit for Behr dry S3 weight (Behr determination. Labor Technik, After Düsseldorf, the determination Germany). ofThe production concentration parameters, of Nmin was the grainalways and determined straw samples in the were partic subjectedular volume to the of determinationthe soil leachate; of ◦ totaltherefore, nitrogen the loss content of N (Nmin totfrom) by the high-temperature individual lysimeters (1000 wasC) dryexpressed combustion in mg/L. method, according to Reference [22], using the automatic analyzer LECO CNS 2000 (St. Joseph, MI, USA). The values of Ntot were2.4. Analysis used to of calculate Plant Biomass the N content in the grain and the straw, which was necessary for determining N use efﬁciency. At the end of each growing season (2013; 2014), grain production and total production of above- 2.5.ground Soil Propertiesbiomass were measured by taking all the plants, and their parts, (stems, leaves, husks, etc.) from the individual lysimeters, for dry weight determination. After the determination of production The selected physical and chemical properties of topsoil and subsoil were determined in parameters, the grain and straw samples were subjected to the determination of total nitrogen content homogenized soil samples, after a twenty-day incubation period, at 19 ◦C. Nutrients available to (Ntot) by high-temperature (1000 °C) dry combustion method, according to Reference [21], using the plants (P, K, Ca, Mg) were extracted using the Mehlich III reagent, and the concentrations of individual automatic analyzer LECO CNS 2000 (St. Joseph, MI, USA). The values of Ntot were used to calculate nutrientsthe N content were indetermined the grain and according the straw, to which Reference was necessary [23]. Ctot forand determining Ntot of the N soiluse efficiency. samples were

Sustainability 2018, 10, 3367 5 of 13 determined according to Reference [24,25] using the LECO CNS 2000 automatic analyzer (St. Joseph, MI, USA).

2.6. Data Treatment Exploratory data analysis was used to assess the measured data of individual parameters to verify the homogeneity and normality of the data collected. Potential differences in Nmin leaching, biomass production and grain yield between the individual experiment variants were analyzed by using a one-way analysis of variance (ANOVA), combined with post-hoc Tukey’s HSD test, at p < 0.05 level of signiﬁcance. Principal Component Analysis (PCA) and regression analysis were used to identify potential relationships between the respective parameters. All analyses were performed using the Statistica 12 software (StatSoft, Dell Software, Aliso Viejo, CA, USA).

3. Results and Discussion

3.1. Plant Biomass—Biomass Production Biomass was harvested from the model plants of Triticum aestivum, in 2013 and 2014, at the stage of their harvest maturity (BBCH 89). Total above-ground plant biomass production of the respective variants, i.e., straw, grain, and husk, is shown in Figure3. Grain production is shown separately in FigureSustainability4. 2018, 10, x FOR PEER REVIEW 6 of 13

Figure 3. Total plant biomass production of winter wheat from the lysimetric experiment (0.07 m2), Figure 3. Total plant biomass production of winter wheat from the lysimetric experiment (0.07 m2), in in 2013 and 2014. Mean values (n = 3) are shown, different letters (lowercase for 2013 and uppercase 2013 and 2014. Mean values (n = 3) are shown, different letters (lowercase for 2013 and uppercase for for 2014) indicate significant differences in total plant biomass production, at the level of significance 2014) indicate significant differences in total plant biomass production, at the level of significance p < p < 0.05. 0.05. In the first year of the experiment (2013), the total production of biomass in variants C2, A1–A3 was atThe the same same course level of (± values50 g) and was nofound significant also in the statistical second differencesyear of the wereexperiment found (2014) between but thesethere −1 variants.was no difference On the other between hand, thevariants demonstrably C2 (SC—112 lowest kg productionN ha ) and of MC plant (A1–A4). biomass The was demonstrably found in the controllowest value variant of (C1total = plant 34 g), biomass compared production to all other was variants.found again The in second variant lowest C1 (33 valueg). The was results found show in variantthat the A4 non-fertilized (38 g), where variant MC was A4 usedproduced, without approximately, the addition ofthe fertilizers. same amount Biomass of biomass production (44 g) in as this the variantfertilized was variants significantly (average lower, C2; asA1; compared A2; A3 = 46 to theg), in fertilized 2014. In control addition, C2 the and values MC variants, were analyzed where NF for fertilizationthe detection (112 of andpotential 70 kg differences N/ha year) between and LG B the (1.25 years and (paired 0.62 L of t-test, LG B/ha) P < 0.05). were The used, results i.e., variants of total A2biomass and A3. production On the otherindicated hand, that no the significant model plants difference in variant was foundA4 obtained between the thesufficient variants amount A1, A2, of andN, through A3. Thus, BFN no from significant legumes. influence This is because of LG B if application they would on not the have production had sufficient of biomass N, they could would be considered.not have been However, able to produce if we compared the amount the values of biomass of variants nearing A1, the A2, level and of A3, the we fertilized could see variant that theC2, and higher than the unfertilized variant C1, which is consistent with [19,26–29]. On the other hand, they point to a potential problem of the Nmin addition, in terms of its usability, as variants A1–A3 were applied from 112 to 72 kg N ha−1, presenting quite large amounts of Nmin, without any positive effect.

3.2. Grain Yield Grain production (Figure 4) was assessed in 2013 and 2014. The highest grain production was found in variants C2, A1, A2, and A3, as compared to the control variant of C1 (SC—0 kg N/ha year) and variant A4 (MC—0 kg N/ha year), in both years of the experiment (2013 and 2014). The yield was about 15 g in 2013, and 16 g in 2014, in the variants C2–A3. The lowest values (±10 g) of grain production were found in variants C1 and A4 in both years, i.e., in the variants where there was no application of fertilizers (NF + LG B).

Sustainability 2018, 10, 3367 6 of 13 higher N dose in variant A1 (+20% as compared to A2 and A3) had a rather negative inﬂuence on the biomassSustainability production 2018, 10, x FOR in this PEER variant. REVIEW 7 of 13

2 FigureFigure 4. GrainGrain yield yield of of winter winter wheat wheat from from the the lysimetric lysimetric experiment experiment (0.07 (0.07 m2), min 2013), in and 2013 2014. and Mean 2014. Meanvalues values (n = 3) (n are= 3)shown, are shown, different different letters letters (lowercase (lowercase for 2013 for2013 and anduppercase uppercase for 2014) for 2014) indicate indicate the p thesignificant significant differences differences in grain in grain yiel yield,d, at the at thelevel level of significance of significance p < 0.05.< 0.05. The same course of values was found also in the second year of the experiment (2014) but there In 2013, significant differences were found between variants C1 and C2, and between variants was no difference between variants C2 (SC—112 kg N ha−1) and MC (A1–A4). The demonstrably A4 and C2. No significant differences were found between variants C2, A1, A2, and A3. In the lowest value of total plant biomass production was found again in variant C1 (33 g). The results show following year of 2014, the results were more proving, as significant differences were found between that the non-fertilized variant A4 produced, approximately, the same amount of biomass (44 g) as the variants where there was no application of NF (C1 and A4), and variants where NF (C2) or the fertilized variants (average C2; A1; A2; A3 = 46 g), in 2014. In addition, the values were analyzed for combination of fertilization and MC (A1, A2, A3) was applied. the detection of potential differences between the years (paired t-test, P < 0.05). The results of total The results confirm the generally known effect of mineral nitrogen fertilization on the promotion biomass production indicated that the model plants in variant A4 obtained the sufficient amount of of grain production in Triticum aestivum [28], and subsequently also the fact that using MC with the N, through BFN from legumes. This is because if they would not have had sufficient N, they would lower dose of N fertilizer (A1—112 kg N/ha year) did not affect negatively the grain production of not have been able to produce the amount of biomass nearing the level of the fertilized variant C2, Triticum aestivum, as compared to the conventional variant (C2—140 kg N/ha year). Furthermore, the and higher than the unfertilized variant C1, which is consistent with [19,26–29]. On the other hand, results confirm a possibility for growing wheat in the MC system, with a decreased N input, in the they point to a potential problem of the N addition, in terms of its usability, as variants A1–A3 were form of mineral fertilizers, which is in accordancemin with findings of other authors [26,27], and also in applied from 112 to 72 kg N ha−1, presenting quite large amounts of N , without any positive effect. accordance with the working hypotheses stated in the goals of our paper.min It can be suggested that 3.2.there Grain is a Yieldpotential for growing wheat in the MC system without any negative effect on the grain production. Grain production (Figure4) was assessed in 2013 and 2014. The highest grain production was found3.3. Leaching in variants of Mineral C2, A1, Nitrog A2,en and from A3, Experimental as compared Containers to the control variant of C1 (SC—0 kg N/ha year) and variant A4 (MC—0 kg N/ha year), in both years of the experiment (2013 and 2014). The yield The loss of Nmin was measured as a concentration of Nmin in the soil leachate, from September was about 15 g in 2013, and 16 g in 2014, in the variants C2–A3. The lowest values (±10 g) of grain 2012 to October 2014, when the lysimetric experiment was terminated. A complete overview of the production were found in variants C1 and A4 in both years, i.e., in the variants where there was no measured values is presented in Figure 5. application of fertilizers (NF + LG B). In the first year of the experiment (2013), the highest Nmin leaching was noticed in the MC variant In 2013, significant differences were found between variants C1 and C2, and between variants A4 (A1 = 67 mg/L) with the fertilization of 112 kg N/ha. In this variant, a statistically significant difference and C2. No significant differences were found between variants C2, A1, A2, and A3. In the following was found, as compared to all other variants. The second highest leaching (compared to all other year of 2014, the results were more proving, as significant differences were found between variants variants) was found in variant C2 (55.31 mg/L), fertilized only with NF (140 kg N/ha). Statistically where there was no application of NF (C1 and A4), and variants where NF (C2) or the combination of significant differences were found also between the respective MC variants (A1–A4), where the loss fertilization and MC (A1, A2, A3) was applied. of Nmin decreased gradually from A1 to A4. The lowest loss of Nmin, by leaching from the soil was The results confirm the generally known effect of mineral nitrogen fertilization on the promotion identified in variant A4 (15.38 mg/L), which was significant when compared to all the other variants. of grain production in Triticum aestivum [29], and subsequently also the fact that using MC with the The second lowest concentration of Nmin was found in the control variant C1 (28.97 mg/L) and in lower dose of N fertilizer (A1—112 kg N/ha year) did not affect negatively the grain production of variant A3 (35.90 mg/L). These values were significant as compared with the other variants.

Sustainability 2018, 10, 3367 7 of 13

Triticum aestivum, as compared to the conventional variant (C2—140 kg N/ha year). Furthermore, the results conﬁrm a possibility for growing wheat in the MC system, with a decreased N input, in the form of mineral fertilizers, which is in accordance with ﬁndings of other authors [27,28], and also in accordance with the working hypotheses stated in the goals of our paper. It can be suggested that there is a potential for growing wheat in the MC system without any negative effect on the grain production.

3.3. Leaching of Mineral Nitrogen from Experimental Containers

The loss of Nmin was measured as a concentration of Nmin in the soil leachate, from September 2012 to October 2014, when the lysimetric experiment was terminated. A complete overview of the measuredSustainability values 2018, 10 is, x presentedFOR PEER REVIEW in Figure 5. 8 of 13

Figure 5. Concentrations of N (mean values ± SE) in the soil leachate, in 2013 and 2014. Mean values Figure 5. Concentrations of Nminmin (mean values ± SE) in the soil leachate, in 2013 and 2014. Mean values (n(n= = 3)3) areareshown, shown, differentdifferentletters letters(lowercase (lowercase forfor 20132013and anduppercase uppercasefor for2014) 2014)indicate indicatesignificant significant differences in N leaching. Significant differences between the respective years are asterisked (*) at differences in Nminmin leaching. Significant differences between the respective years are asterisked (*) at thethe level level of of significance significancep p< <0.05. 0.05. In the first year of the experiment (2013), the highest N leaching was noticed in the MC variant In the second year of the experiment (2014) statisticallymin significant differences were found again. (A1 = 67 mg/L) with the fertilization of 112 kg N/ha. In this variant, a statistically significant difference The lowest leaching of Nmin was recorded in the control variant C1 (15.05 mg/L), and in the non- was found, as compared to all other variants. The second highest leaching (compared to all other fertilized variant A1 with MC (15.51 mg/L). Conversely, the absolutely highest loss in 2014 was found variants) was found in variant C2 (55.31 mg/L), fertilized only with NF (140 kg N/ha). Statistically in variant C2 (59.67 mg/L). This variant was fertilized only with NF (140 kg N/ha) and wheat was significant differences were found also between the respective MC variants (A1–A4), where the loss cultivated in the SC system. The value of Nmin loss, measured in this variant, was demonstrably the of N decreased gradually from A1 to A4. The lowest loss of N , by leaching from the soil was highest,min as compared with the other variants. On the other hand,min variants A1–A3 showed the same identified in variant A4 (15.38 mg/L), which was significant when compared to all the other variants. level of Nmin loss (±40 mg/L), which was approximately 40% lower, as compared to the variant C2 The second lowest concentration of N was found in the control variant C1 (28.97 mg/L) and in (application of NF, wheat grown as SC).min variant A3 (35.90 mg/L). These values were significant as compared with the other variants. The values found in the two years (2013 and 2014) of the experiment were influenced by two In the second year of the experiment (2014) statistically significant differences were found factors: (1) application of nitrogen fertilizers; (2) use of MC. Relations between the most important again. The lowest leaching of N was recorded in the control variant C1 (15.05 mg/L), and in parameters are presented in Figuremin 6; two main factors were determined. The first group of variables the non-fertilized variant A1 with MC (15.51 mg/L). Conversely, the absolutely highest loss in 2014 (PC 1) described 83.29% and 83.69% of the variance, in 2013 and 2014, respectively. This factor or PC was found in variant C2 (59.67 mg/L). This variant was fertilized only with NF (140 kg N/ha) 1 correlated inversely with Nmin leaching (R = −0.89 in 2013, and R = −0.87 in 2014), and biomass and wheat was cultivated in the SC system. The value of N loss, measured in this variant, was parameters correlated inversely to (R = −0.89 in 2013, and minR = −0.90 in 2014). Overall, PC1 could demonstrably the highest, as compared with the other variants. On the other hand, variants A1–A3 characterize the influence of growing technology, on the loss of Nmin from the soil, and the production showed the same level of N loss (±40 mg/L), which was approximately 40% lower, as compared to of grain or total biomass. minMeasured values of the total biomass showed small differences between the variant C2 (application of NF, wheat grown as SC). variants C2 to A3 but were relatively significant in the loss of Nmin. This assumption could confirm a The values found in the two years (2013 and 2014) of the experiment were influenced by two regression analysis that was performed independent of the PCA, for each year of the experiment. factors: (1) application of nitrogen fertilizers; (2) use of MC. Relations between the most important Total biomass production correlated inversely with Nmin leaching (R = −0.71 in 2013 and R = −0.61 in 2014). The second group (PC 2) described additional 11.90% in 2013, and 12.11% in 2014. The second factor (PC2) had a correlation with the grain yield near zero (R = 0.05 in 2013, and R = 0.07 in 2014). Correlation of PC 2 with other parameters, i.e., biomass production and Nmin leaching, was variable and weak (inconclusive): (a) 2013—PC 2 correlated with Nmin leaching (R = 0.41) and, inversely, with the total plant biomass production (R = −0.42); (b) 2014—there was Nmin leaching that correlated inversely (R = −0.46) with PC 2, and positively with plant biomass production (R = 0.37). PC 2 is unlikely to be related to the growing technology used because no proven correlation with the selected parameters has been identified. Therefore, it can only be assumed that this factor could be affected by seasonal fluctuations, for example, rainfall or temperatures. Their variability in the individual years of the experiment was evident from the measured data from the local meteorological station (Figure 1).

Sustainability 2018, 10, 3367 8 of 13 Sustainability 2018, 10, x FOR PEER REVIEW 9 of 13 parametersRelations are presentedbetween the in Figurerespective6; two variables main factors (in were2013 determined.and 2014) are The presented first group as of blue variables lines, (PCprecisely 1) described as cosines 83.29% of their and angles 83.69% on of Figure the variance, 6. The smallest in 2013 andangle 2014, was respectively.between variables This factor with strong or PC correlations, and vice-versa. When we added a graphical representation using the 3D surface plot 1 correlated inversely with Nmin leaching (R = −0.89 in 2013, and R = −0.87 in 2014), and biomass parameters(Appendix B, correlated Figures A2 inversely and A3) to to (R the = − PCA0.89 analysis in 2013,, andit was R =evident−0.90 that in 2014). variants Overall, with PC1the highest could grain production showed the highest loss of Nmin, from the soil. The results indicated that the high characterize the influence of growing technology, on the loss of Nmin from the soil, and the production ofproduction grain or total variants biomass. featured Measured the excessive values amount of the total of N biomassmin. While showed Nmin was small used differences primarily betweenfor yield production, rather than for the production of plant biomass (straw, husk, etc.), it could not be stored variants C2 to A3 but were relatively significant in the loss of Nmin. This assumption could confirm ain regression the plants analysisand was thatleached was instead. performed In the independent area, it would of be the more PCA, appropriate for each year to use of the wheat experiment. variants, which were able to produce, not only enough grains but also enough straw, as it could represent an Total biomass production correlated inversely with Nmin leaching (R = −0.71 in 2013 and R = −0.61 inimportant 2014). “pool” to store the surplus N-substances, in order to prevent their loss from the soil.

(A) (B)

FigureFigure 6.6.Projection Projection ofof thethe variablesvariables onon thethe planeplane ofof factors.factors. TheThe graphgraph showsshows thethe resultsresults ofof thethe PCA:PCA: ((AA)) forfor 2013; 2013; ( B(B)) for for 2014. 2014.

TheThe secondlowest values group (PCof N 2)min describedleaching, found additional in variants 11.90% A4 in and 2013, C1, and were 12.11% not surprising in 2014. The since second these factorvariants (PC2) were had not a correlationsubsidized withwith the NF, grain at all. yield If we near compared zero (R = these 0.05 in variants 2013, and (C1 R vs. = 0.07 A4) in among 2014). Correlationthemselves, of we PC would 2 with find other that parameters, in 2013 the i.e., variant biomass with production MC showed and a N lowermin leaching, or steep was loss variable of Nmin andfrom weak the soil, (inconclusive): with a higher (a) total 2013—PC of plant-biomas 2 correlateds production. with Nmin Lowerleaching values (R = in 0.41) the and,variant inversely, A4, as withcompared the total to plantthe variant biomass C1, production indicated that (R = the−0.42); use of (b) MC 2014—there had a positive was Neffectmin leaching on reducing that correlatedthe loss of inverselyNmin from (R the = soil.−0.46) This with fact PCwas 2, confirmed and positively and expl withained plant by biomass Reference production [27] who (Rreport = 0.37). the MC PC root 2 is unlikelysystem to bebe relatedseveral to times the growing larger than technology that in used SC. becauseIt was additionally no proven correlation supplemented with thewith selected AMF, parametersbacteria, and has actinomycetes, been identiﬁed. creating Therefore, a space it can with only a strong be assumed microbial that thisactivity factor and could a large be affectedcapacity by to seasonalcapture, ﬂuctuations,process, and for use example, the N rainfallsubstances, or temperatures. leading to the Their elimination variability of in N themin individualloss from yearsthe soil of theenvironment. experiment The was positive evident influence from the of measured MC and datapresen fromce of the on local reducing meteorological the loss of N station substances (Figure from1). the soilRelations has also between been confirmed the respective by other variables authors—e. (in 2013g., and Reference 2014) are [30,30]. presented On the as contrary, blue lines, the precisely values asmeasured cosines of in their variants angles A1–A3 on Figure were6. Theinconsistent smallest anglewith the was above between information. variables with Thus, strong it is correlations,necessary to andput vice-versa.them in a broader When context. we added The a higher graphical loss representation of Nmin in variants using A1–A3 the 3D (compared surface plot to control (Appendix C1 andB, Figuresvariant A2A4) and could A3) have to the been PCA caused analysis, by it the was combin evidentation that of variants NF addition with the (A1 highest and A2—112 grain production kg N/ha, showedA3—70 thekg highestN ha−1), lossand of the N minprocess, from of the biological soil. The fixation results indicated of N2 (BFN). that the According high production to some variantsauthors featured[30,31], legumes the excessive can deliver amount approximately of Nmin. While 40 N kgmin Nwas ha−1 used, through primarily BFN, forto the yield rhizosphere production, of ratherarable thanland, for during the production one growing of season. plant biomass The surplus (straw, of husk,reactive etc.), N (due it could to the not NF be application stored in the and plants the BFN) and wasin the leached soil and instead. the insufficiently In the area, it closed would stand be more of MC appropriate in the first to useyear wheat of the variants, experiment, which could were have able resulted in the increased loss of Nmin from the soil, in variants A1–A3. It is necessary to note that the decreased loss of Nmin in variants A2–A3 was probably due to the application of LG B, with dissolved Corg (A2—1.25 L/ha year; A3—0.65 L/ha year), to support the microbial activity. Differences between

Sustainability 2018, 10, 3367 9 of 13 to produce, not only enough grains but also enough straw, as it could represent an important “pool” to store the surplus N-substances, in order to prevent their loss from the soil. The lowest values of Nmin leaching, found in variants A4 and C1, were not surprising since these variants were not subsidized with NF, at all. If we compared these variants (C1 vs. A4) among themselves, we would find that in 2013 the variant with MC showed a lower or steep loss of Nmin from the soil, with a higher total of plant-biomass production. Lower values in the variant A4, as compared to the variant C1, indicated that the use of MC had a positive effect on reducing the loss of Nmin from the soil. This fact was confirmed and explained by Reference [28] who report the MC root system to be several times larger than that in SC. It was additionally supplemented with AMF, bacteria, and actinomycetes, creating a space with a strong microbial activity and a large capacity to capture, process, and use the N substances, leading to the elimination of Nmin loss from the soil environment. The positive influence of MC and presence of on reducing the loss of N substances from the soil has also been confirmed by other authors—e.g., Reference [30,31]. On the contrary, the values measured in variants A1–A3 were inconsistent with the above information. Thus, it is necessary to put them in a broader context. The higher loss of Nmin in variants A1–A3 (compared to control C1 and variant A4) could have been caused by the combination of NF addition (A1 and A2—112 kg N/ha, A3—70 kg N ha−1), and the process of biological fixation of N2 (BFN). According to some authors [31,32], legumes can deliver approximately 40 kg N ha−1, through BFN, to the rhizosphere of arable land, during one growing season. The surplus of reactive N (due to the NF application and the BFN) in the soil and the insufficiently closed stand of MC in the first year of the experiment, could have resulted in the increased loss of Nmin from the soil, in variants A1–A3. It is necessary to note that the decreased loss of Nmin in variants A2–A3 was probably due to the application of LG B, with dissolved Corg (A2—1.25 L/ha year; A3—0.65 L/ha year), to support the microbial activity. Differences between the respective variants A1 > A2 > A3 were significant. The decrease was caused probably by the increased microbial activity promoted by the addition of LG B (with the content of Corg), and by the root exudates produced by the legumes, in MC. The growing of MC according [33] supports microbial activity, which increases the processing of N substances in the soil by up to 40%, as compared with the conventional systems. Furthermore reference [34] arrived at the same conclusions. The measured Nmin losses from the soil pointed again to the combined effect of fertilization and MC cultivation, in 2014. The lowest loss of Nmin was found in variants C1 and A4, again. The values in variant A4 indicated a potential stability in the use of N substances and the ability of MC to utilize the available N substances. The values are consistent with [27,31] where the authors report similar findings from their MC experiments. Furthermore, the Nmin losses, in 2013 and 2014, can be compared: significant differences were found only between the variants C1 and A1. A decrease, by 35%, was found in variant A1, as compared to 2013. This state was confirmed by the assumption that the MC was not yet sufficiently closed, in 2013, and its potential could not have been used. The reason is that Trifolium repens, as a part of the MC, was sown when the experiment was established in 2012 and was only mulched in the other years (no additional sowing). Thus, it could be assumed that the plants were not closed enough in 2013, but in 2014 they could use their potential for increasing the soil capacity to capture and utilize the Nmin, as reported by Reference [28].

4. Conclusions The aim of the paper was to gain new knowledge about the elimination of negative phenomena during the cultivation of winter wheat. In particular, there is a need to reduce the excessive use of N fertilizers and over-limit loss of mineral nitrogen leached from the soil, into other environments, such as watercourses or water reservoirs. The innovated agricultural technology was used to test winter wheat (Triticum aestivum), growing in the system of mixed culture (MC) with white clover (Trifolium repens). Based on the results, we can conclude that growing winter wheat in the MC system significantly reduced the Nmin concentration in the soil leachate. Moreover, it was found that the Sustainability 2018, 10, 3367 10 of 13 cultivation of wheat in MC makes it possible to reduce the applied N dose, by more than 20%, without any impact on grain and biomass production. The use of such N doses makes the usability of N substances in the MC system higher than that in the SC (monoculture) system, which was confirmed by the minimal differences in biomass production between MC and SC. In general, growing winter wheat in the MC system led to the higher sustainability of its cultivation, as compared with the SC system. Less mineral fertilizer was applied, and the natural benefits of the system were used instead, namely the biological fixation of N2. We can conclude with a statement that results of our experiment confirm the potential of sustainable winter wheat production with minimum negative effects on the environment, even with the reduced input of mineral N.

Author Contributions: A.K. and J.E. organized the lysimetric experiment; T.L. and J.N. reviewed the manuscript; J.E. and M.D.V. performed the statistical analysis and approved it for submission, reviewed the manuscript. Funding: This research was supported by the Ministry of Agriculture of the Czech Republic, institutional support MZE-RO1718. Conﬂicts of Interest: The authors declare no conﬂicts of interest.

Appendix A

SustainabilityDetailed 2018 display, 10, x FOR of PEER experimental REVIEW lysimeter used in the experiment. 11 of 13

Figure A1. Soil lysimeter. Description: 1—model plant; 2—soil surface (0.07 m 2);); 3—lysimeter 3—lysimeter body; body; 4—topsoil layer (25 kg); 5—subsoil layer (25 kg); 6—drain hole; 7—plastic hose (to intercept and transport soil leachate).

Appendix B Appendix B

Graphical representation of relationships between the selected parameters (Nmin leaching; Graphical representation of relationships between the selected parameters (Nmin leaching; grain andgrain total and biomass total biomass yield) yield)in all variants, in all variants, using using3D surface 3D surface plots, plots,in 2013 in and 2013 2014. and 2014.

Figure A2. A 3D surface plot of relationships between the individual parameters for 2013. The graph

is a 3D projection of all measured values, i.e., Nmin leaching, grain yield, and total plant biomass production.

Sustainability 2018, 10, x FOR PEER REVIEW 11 of 13

Figure A1. Soil lysimeter. Description: 1—model plant; 2—soil surface (0.07 m2); 3—lysimeter body; 4—topsoil layer (25 kg); 5—subsoil layer (25 kg); 6—drain hole; 7—plastic hose (to intercept and transport soil leachate).

Appendix B

Graphical representation of relationships between the selected parameters (Nmin leaching; grain Sustainability 2018, 10, 3367 11 of 13 and total biomass yield) in all variants, using 3D surface plots, in 2013 and 2014.

Figure A2. A 3D surface plot of relationships between the individual parameters for 2013. The graph is a SustainabilityFigure 2018 A2., A10 ,3D x FOR surface PEER plot REVIEW of relationships between the individual parameters for 2013. The graph 12 of 13 3D projection of all measured values, i.e., N leaching, grain yield, and total plant biomass production. is a 3D projection of all measured values,min i.e., Nmin leaching, grain yield, and total plant biomass production.

FigureFigure A3.A3. A 3D3D surfacesurface plotplot ofof relationshipsrelationships betweenbetween thethe individualindividual parametersparameters forfor 2014.2014. TheThe graphgraph is a 3D projection of all measured values, i.e., N leaching, grain yield, and the total plant is a 3D projection of all measured values, i.e., Nmin leaching,min grain yield, and the total plant biomass biomassproduction. production. References References 1. Cassman, K.G.; Dobermann, A.; Walters, D.T. Agroecosystems, nitrogen-use efﬁciency, and nitrogen 1. Cassman, K.G.; Dobermann, A.; Walters, D.T. Agroecosystems, nitrogen-use efficiency, and nitrogen management. Ambio 2002, 31, 132–140. [CrossRef][PubMed] management. Ambio 2002, 31, 132–140. 2. Joshi, A.K.; Mishra, B.; Chatrath, R.; Ortiz Ferrara, G.; Singh, R.P. Wheat improvement in India: Present 2. Joshi, A.K.; Mishra, B.; Chatrath, R.; Ortiz Ferrara, G.; Singh, R.P. Wheat improvement in India: Present status, emerging challenges and future prospects. Euphytica 2007, 157, 431–446. [CrossRef] status, emerging challenges and future prospects. Euphytica 2007, 157, 431–446. 3. Pelzer, E.; Bazot, M.; Makowski, D.; Corre-Hellou, G.; Naudin, CH.; Rifai, M.A.; Baranger, E.; Bedoussac, L.; 3. Pelzer, E.; Bazot, M.; Makowski, D.; Corre-Hellou, G.; Naudin, CH.; Rifai, M.A.; Baranger, E.; Bedoussac, Biarnés, V.; Boucheny, P.; et al. Pea–wheat intercrops in low-input conditions combine high economic L.; Biarnés, V.; Boucheny, P.; et al. Pea–wheat intercrops in low-input conditions combine high economic performances and low environmental impacts. Eur. J. Agron. 2012, 40, 39–53. [CrossRef] performances and low environmental impacts. Eur. J. Agron. 2012, 40, 39–53. 4. Canfield, D.E.; Glazer, A.N.; Falkowski; P.G. The evolution and future of Earth’s nitrogen cycle. Science 2010, 330, 192–196. 5. Sutton, M.A. The European Nitrogen Assessment: Sources, Effects and Policy Perspectives; Cambridge University Press: Cambridge, UK, 2011; ISBN 9781107006126. 6. Bodirsky, B.L.; Popp, A.; Weindl, I.; Dietrich, J.P.; Rolinski, S.; Scheiffele, L.; Schmitz, C.; Lotze-Campen, H. N2O emissions from the global agricultural nitrogen cycle—Current state and future scenarios. Biogeosciences 2012, 9, 4169–4197.

7. Jensen, E.S.; Hauggaard-Nielsen, H. How can increased use of biological N2 fixation in agriculture benefit the environment? Plant Soil 2003, 252, 177–186. 8. Crews, T.E.; Peoples, M.B. Legume versus fertilizer sources of nitrogen: Ecological tradeoffs and human needs. Agric. Ecosyst. Environ. 2004, 102, 279–297. 9. Biswas, B.; Gresshoff, P.M. The Role of Symbiotic Nitrogen Fixation in Sustainable Production of Biofuels. Int. J. Mol. Sci. 2014, 15, 7380–7397. 10. Fields, S. Global Nitrogen: Cycling out of Control. Environ. Health Perspect. 2004, 112, A556–A563. 11. Lukina, E.V.; Freeman, K.W.; Wynn, K.J.; Thomason, W.E.; Mullen, R.W.; Stone, M.L.; Solie, J.B.; Klatt, A.R.; Johnson, G.V.; Elliott, R.L.; et al. Nitrogen fertilization optimization algorithm based on in-season estimates of yield and plant nitrogen uptake. J. Plant Nutr. 2001, 24, 885–898. 12. Jensen, E.S.; Peoples, M.B.; Robert, M.; Boddey, R.M.; Gresshoff, P.M.; Hauggaard-Nielsen, H.; Alves, B.J.R.; Morrison, M.J. Legumes for mitigation of climate change and the provision of feedstock for biofuels and biorefineries. A review. Agron. Sustain. Dev. 2012, 32, 329–364. 13. Peoples, M.B.; Herridge, D.F.; Ladha, J.K. Biological nitrogen fixation: An efficient source of nitrogen for sustainable agricultural production? Plant Soil 1995, 174, 3–28.

Sustainability 2018, 10, 3367 12 of 13

4. Canﬁeld, D.E.; Glazer, A.N.; Falkowski, P.G. The evolution and future of Earth’s nitrogen cycle. Science 2010, 330, 192–196. [CrossRef][PubMed] 5. Sutton, M.A. The European Nitrogen Assessment: Sources, Effects and Policy Perspectives; Cambridge University Press: Cambridge, UK, 2011; ISBN 9781107006126. 6. Bodirsky, B.L.; Popp, A.; Weindl, I.; Dietrich, J.P.; Rolinski, S.; Scheiffele, L.; Schmitz, C.; Lotze-Campen, H.

N2O emissions from the global agricultural nitrogen cycle—Current state and future scenarios. Biogeosciences 2012, 9, 4169–4197. [CrossRef]

7. Jensen, E.S.; Hauggaard-Nielsen, H. How can increased use of biological N2 fixation in agriculture benefit the environment? Plant Soil 2003, 252, 177–186. [CrossRef] 8. Crews, T.E.; Peoples, M.B. Legume versus fertilizer sources of nitrogen: Ecological tradeoffs and human needs. Agric. Ecosyst. Environ. 2004, 102, 279–297. [CrossRef] 9. Biswas, B.; Gresshoff, P.M. The Role of Symbiotic Nitrogen Fixation in Sustainable Production of Biofuels. Int. J. Mol. Sci. 2014, 15, 7380–7397. [CrossRef][PubMed] 10. Fields, S. Global Nitrogen: Cycling out of Control. Environ. Health Perspect. 2004, 112, A556–A563. [CrossRef] [PubMed] 11. Lukina, E.V.; Freeman, K.W.; Wynn, K.J.; Thomason, W.E.; Mullen, R.W.; Stone, M.L.; Solie, J.B.; Klatt, A.R.; Johnson, G.V.; Elliott, R.L.; et al. Nitrogen fertilization optimization algorithm based on in-season estimates of yield and plant nitrogen uptake. J. Plant Nutr. 2001, 24, 885–898. [CrossRef] 12. Jensen, E.S.; Peoples, M.B.; Robert, M.; Boddey, R.M.; Gresshoff, P.M.; Hauggaard-Nielsen, H.; Alves, B.J.R.; Morrison, M.J. Legumes for mitigation of climate change and the provision of feedstock for biofuels and biorefineries. A review. Agron. Sustain. Dev. 2012, 32, 329–364. [CrossRef] 13. Peoples, M.B.; Herridge, D.F.; Ladha, J.K. Biological nitrogen fixation: An efficient source of nitrogen for sustainable agricultural production? Plant Soil 1995, 174, 3–28. [CrossRef] 14. Olivares, J.; Bedmar, E.J.; Sanjuán, J. Biological Nitrogen Fixation in the Context of Global Change. Mol. Plant Microb. Interact. 2013, 26, 486–494. [CrossRef][PubMed] 15. Sullivan, B.J.; Smith, W.K.; Townsend, A.R.; Nasto, M.K.; Reed, S.C.; Robin, L.; Chazdon, R.L.; Cleveland, C.C. Spatially robust estimates of biological nitrogen (N) fixation imply substantial human alteration of the tropical N cycle. Proc. Natl. Acad. Sci. USA 2014, 111, 8101–8106. [CrossRef][PubMed] 16. Ashworth, A.J.; Taylor, A.M.; Reed, D.L.; Allen, F.L.; Keyser, P.D.; Tyler, D.D. Environmental impact assessment of regional switchgrass feedstock production comparing nitrogen input scenarios and legume-intercropping systems. J. Clean. Prod. 2015, 87, 227–234. [CrossRef] 17. Anglade, J.; Billen, G.; Garnier, J. Relationships for estimating N2 fixation in legumes: Incidence for N balance of legume-based cropping systems in Europe. Ecosphere 2015, 6, 1–24. [CrossRef] 18. Teng, Y.; Wang, X.; Li, L. Rhizobia and their bio-partners as novel drivers for functional remediation in contaminated soils. Front. Plant Sci. 2015, 6, 32. [CrossRef][PubMed] 19. Hauggaard-Nielsen, H.; Jensen, E.S. Facilitative root interactions in intercrops. In Root Physiology: From Gene to Function, 2nd ed.; Lambers, H., Colmer, T.D., Eds.; Plant Ecophysiology; Springer: Dordrech, The Netherlands, 2005; Volume 4, pp. 237–250. ISBN 978-1-4020-4099-3. 20. Wyszkowski, M.; Radziemska, M. The effect of chromium content in soil on the concentration of some mineral elements in plants. Fresenius Environ. Bull. 2009, 18, 1039–1045. 21. Muñoz-Huerta, R.F.; Guevara-Gonzalez, R.G.; Contreras-Medina, L.M.; Torres-Pacheco, I.; Prado-Olivarez, J.; Ocampo-Velazquez, R.V. Review of Methods for Sensing the Nitrogen Status in Plants: Advantages, Disadvantages and Recent Advances. Sensors 2013, 13, 10823–10843. [CrossRef][PubMed] 22. Uzoma, K.C.; Inoue, M.; Andry, H.; Fujimaki, H. Effect of cow manure biochar on maize productivity under sandy soil condition. Soil Use Manag. 2011, 27, 205–212. [CrossRef]

23. Zbíral, J.; Nˇemec,P. Comparison of Mehlich 2, Mehlich 3, CAL, Egner, Olsen, and 0.01 M CaCl2 extractants for determination of phosphorus in soils. Commun. Soil Sci. Plant Anal. 2002, 33, 3405–3417. [CrossRef] 24. Nelson, D.W.; Sommers, L.E. Total carbon, organic carbon, and organic matter. In Methods of Soil Analysis. Part 3, Chemical Methods, 2nd ed.; Sparks, D.L., Ed.; Soil Science Society of America, American Society of Agronomy: Madison, WI, USA, 1996; Volume 5, pp. 961–1010. ISBN 0891188258. 25. Bremner, J.M. Nitrogen Total. In Methods of Soil Analysis. Part 3, Chemical Methods, 2nd ed.; Sparks, D.L., Ed.; Soil Science Society of America, American Society of Agronomy: Madison, WI, USA, 1996; Volume 5, pp. 1085–1122. ISBN 0891188258. Sustainability 2018, 10, 3367 13 of 13

26. Hauggaard-Nielsen, H.; Ambus, P.; Jensen, E.S. The comparison of nitrogen use and leaching in sole cropped versus intercropped pea and barley. Nutr. Cycl. Agroecosyst. 2003, 65, 289–300. [CrossRef] 27. Fustec, J.; Lesuffleur, F.; Mahieu, S. Nitrogen rhizodeposition of legumes. A review. Agron. Sustain. Dev. 2010, 30, 57–66. [CrossRef] 28. Ehrman, J.; Ritz, K. Plant: Soil interactions in temperate multi-cropping production systems. Plant Soil 2014, 376, 1–29. [CrossRef] 29. Haynes, R. Mineral Nitrogen in the Plant-Soil System; Academic Press: Cambridge, MA, USA, 2012; ISBN 978-0-12-334910-1. 30. Tosti, G.; Farneselli, M.; Benincasa, P.; Guiducci, M. Nitrogen Fertilization Strategies for Organic Wheat Production: Crop Yield and Nitrate Leaching. Agron. J. 2016, 108, 770–781. [CrossRef] 31. Fujita, K.; Godfred, O.K.; Ogata, S. Biological nitrogen fixation in mixed legume- cereal cropping systems. Plant Soil 1992, 141, 155–175. [CrossRef] 32. Pirhofer-Walzl, K.; Rasmussen, J.J.; Høgh-Jensen, H. Nitrogen transfer from forage legumes to nine neighbouring plants in a multi species grassland. Plant Soil 2012, 350, 71–84. [CrossRef] 33. Di, H.J.; Cameron, K.C. Nitrate leaching and pasture production from different nitrogen sources on a shallow stoney soil under flood-irrigated dairy pasture. Aust. J. Soil Res. 2002, 40, 317–334. [CrossRef] 34. Zarea, M.J.; Ghalavand, A.; Goltapeh, E.M. Effects of mixed cropping, earthworms (Pheretima sp.), and arbuscular mycorrhizal fungi (Glomus mosseae) on plant yield, mycorrhizal colonization rate, soil microbial biomass, and nitrogenase activity of free-living rhizosphere bacteria. Pedobiologia 2009, 52, 223–235. [CrossRef]

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).