2.1. Location and Greenhouses
The work was conducted in two identical plastic greenhouses in the Research Station of Cajamar in El Ejido, Almería, in SE Spain (36°48′ N, 2°43′ W). The greenhouses were representative of those used in this region [18
]. The greenhouses had an asymmetrical, shallow, inverted V-shaped roof, and the structure was of stainless-steel tubes and wires; the cladding was 200 µm-thick colorless low-density polyethylene film. Each greenhouse measured 24 × 18 m. The two greenhouses had an east–west orientation, and were adjacent to one another along the east–west axis. They had passive ventilation and no heating system. Each greenhouse was divided approximately in half along the east-west axis by a 2 m wide concrete path; this work was conducted in the northern half of each greenhouse.
The greenhouses had an artificial soil system known locally as “enarenado” soil, which is commonly used in this vegetable production system [18
]. The soil consisted of a 30 cm layer of imported clay soil, obtained from a local quarry, which was placed over the naturally occurring loam soil; a 10 cm layer of coarse river sand was placed over the imported clay soil as a mulch. The 10 cm sand mulch layer over the soil surface substantially reduces evaporation and weed growth. Generally, in this artificial soil system, roots are mostly concentrated in the imported soil layer. Relevant properties of the soil at the beginning of the study are given in Table 1
. All soil depths in Table 1
, and referred to subsequently, are relative to the surface of the imported layer of clay soil. This artificial soil system was formed when the greenhouse was constructed in 1995.
In June 2003, sheep manure was applied to the surface layer of imported clay soil at a rate of 73 t ha−1
, supplying 1270 kg N ha−1
. The application of manure at these rates, at the formation of the artificial soil system and thereafter every 2–5 years, is common practice in this vegetable production system in response to the low organic matter contents of the sub-soils that are imported, from quarries, into the greenhouses for cropping [18
All plants were grown in north–south aligned rows, with 1 m spacing between rows, and 0.5 m between adjacent plants within rows. All plants were vertically supported using nylon guides.
2.2. Crops and Crop Management
Muskmelon (Cucumis melo, L., cv. ‘Deneb’) type Galia and sweet pepper (Capsicum annum, L., cv. ‘Vergasa’) crops were grown sequentially in both 2005 and 2006. The crops grown in 2005 are hereafter referred to as the 2005 muskmelon and 2005 sweet pepper crops, and those grown in 2006 as the 2006 muskmelon and 2006 sweet pepper crops. All crops were grown following transplanting as 6-week old seedlings.
The 2005 muskmelon crop was grown from 17 March to 1 June 2005 (76 days) and the 2006 muskmelon crop from 14 February to 18 May 2006 (93 days). The 2005 sweet pepper crop was grown from 21 July to 20 December 2005 (152 days), and the 2006 sweet pepper crop from 20 July 2006 to 2 February 2007 (201 days).
Above-ground drip irrigation was used with one 2.8 L h−1
emitter immediately adjacent to each plant and separated by approximately 5 cm. Complete nutrient solutions were applied by fertigation in all irrigations after the first 2 weeks following transplanting; previously, only water was applied. Examples of the nutrient solutions used for the conventionally managed muskmelon and sweet pepper crops are presented in Supplementary Table S1
. All cultural practices (crop pruning and guiding, pollination, fruit harvesting) and pest management operations followed established local practices. Sweet pepper fruit, in both crops, was collected in five harvests conducted at 1–2 week intervals over approximately 2-month period. Muskmelon fruit was harvested at the end of the crop. Irrigation and N management are described subsequently in sub-Section 2.4
, and in Table 2
and Table 3
. Monthly average climatic data inside the greenhouses from January 2005 to January 2007 are presented in Figure 1
2.4. Treatments–N and Irrigation Management
For the conventional management treatment, irrigation followed local practices with respect to volume and timing of irrigation. Published [29
] and unpublished data (M.D. Fernández, Research Station of Cajamar, El Ejido, Almeria, Spain) relating irrigation volumes to calculated crop evapotranspiration (ETc) were used to characterize conventional irrigation management. Crop evapotranspiration was calculated using the PrHo model developed for vegetable crops in this system [30
]. For conventional management, volumes of applied nutrient solution were in excess of crop evapotranspiration (ETc) until maximum crop coefficient (kc
) values [29
] were reached, and then, were similar to ETc [29
]. In the improved management systems, irrigation was based on estimated daily ETc [30
] and adjusted to maintain soil matric potential (SMP) at 15 cm depth (measured with tensiometers) between −10 and −40 kPa; the timing of irrigation was in response to SMP.
N management in the conventionally managed treatments was based on applying fixed NO3−
concentrations in the applied nutrient solutions following the recommendations of local technical advisors. N management in the treatments with improved management was based on a prescriptive-corrective management system [12
] that was developed with these crops. The prescriptive component developed for N management was based on applying N to match simulated daily crop N uptake that was estimated using the Nup simulation model [22
]. Nup calculates daily crop N uptake in vegetable crops grown in plastic greenhouses in SE Spain. Inputs are daily values of maximum and minimum air temperatures and the integral of solar radiation, within the greenhouse. It was calibrated and validated for muskmelon and sweet pepper [22
]. Brief [22
] and detailed [34
] descriptions are available. The corrective component for N management was based on maintaining the soil solution NO3−
concentration, in the immediate root zone, within a specified range [12
]. The specific N management practices used in each crop are described subsequently.
In the 2005 IM muskmelon crop, the concentration of applied N in the IM treatment was similar to the CM treatment; the 16% reduction in irrigation volume was associated with an 18% reduction in the amount of N applied (Table 2
). In the 2005 IM sweet pepper crop, N management was based on maintaining the soil solution NO3−
concentration, at 15 cm depth, within the range of 8–12 mM
. With the reductions of 17 and 30% in irrigation volume and N concentrations, the amount of applied N was reduced by 42% (Table 3
In the 2006, IM muskmelon crop, N management was based on estimated crop N uptake, which was simulated using the Nup model [22
]. With the reductions of 11% and 15% in irrigation volume and applied N concentration, the amount of applied N was reduced by 28% (Table 2
). In the 2006 IM sweet pepper crop, N management was based on simulated crop N uptake using the Nup model [22
] and maintaining soil solution NO3−
concentration, at 15 cm depth at 8–12 mM
. With the reductions of 16 and 24% in irrigation volume and N concentrations, the amount of applied N was reduced by 36% (Table 3
The concentrations of the nutrients other than N applied in the complete nutrient solutions followed established local practice. From two weeks after transplanting, the crops received complete nutrient solutions in all irrigations, with 92–96% of N as NO3−
, and the rest as NH4+
and Table 3
) following local practice. Irrigation volumes were measured daily with a volume meter. Samples of applied nutrient solutions were analyzed weekly for NO3−
concentrations with an automatic continuous segmented flow analyzer (model SAN++, Skalar Analytical B.V., Breda, The Netherlands).
2.5. 15N Applications
In each of the two treatments of each of the 2005 and 2006 muskmelon and sweet pepper crops, four replicate individual plants, one in each replicate plot, directly received nutrient solution containing 15
(16–17 atom% excess 15
N in muskmelon crops, 18 atom% excess 15
N in sweet pepper crops) on each of three consecutive days. The 15
N-labeled nutrient solution was applied to the crops at two different developmental stages: (i) during rapid vegetative growth (VG) and (ii) during the fruit production phase (FP). Detailed descriptions of the 15
N applications in the muskmelon and sweet pepper crops are given in Table 4
and Table 5
The vegetative growth phase was defined as being when there was predominantly vegetative growth, and the fruit production phase when there was predominantly fruit growth. The fruit production phase was considered to start when there was the full canopy and the exponential growth of fruit had commenced. In muskmelon, the transition between the vegetative growth and fruit production phases occurred when fruit biomass was approximately 1% of final fruit production. In the sweet pepper crops, it occurred at 12–15% of final fruit production.
For both muskmelon and sweet pepper, the 15N applications during vegetative growth were made in 2005, and during fruit production in 2006. The fruit production applications were made in 2006 and not in 2005, as was originally planned, because localized virus infections in the latter part of the 2005 muskmelon and sweet pepper crops created uncertainty about the value of applying 15N in the latter part of the 2005 crops.
In muskmelon, the 15N application during vegetative growth was applied to the 2005 crop, 41–43 days after transplanting (DAT), on 26, 27 and 28 April 2005. The 15N application in the fruit production phase was made 71–73 DAT in the 2006 muskmelon crop, on 25, 26 and 27 April 2006. In sweet pepper, 15N was applied during vegetative growth 70–72 DAT in the 2005 sweet pepper crop, on 27, 28 and 29 September 2005, and in the fruit production phase at 105–107 DAT in the 2006 sweet pepper crop, on 1, 2 and 3 November 2006.
The four individual plants that were directly labeled in each “labeling” of each treatment were in each of the four representative plots of the corresponding treatment. Within each plot, the locations of the 15N applications were chosen to maximize the distance from any previous 15N applications.
The 15N labeled nutrient solutions were prepared firstly as nutrient solutions with a composition of nutrients, other than N, as close as possible to that of the nutrient solutions being applied to the crops. These initial nutrient solutions had 2.0–2.5 mM NO3− and no NH4+. 15N labeling was conducted by adding sufficient 15N labeled Ca(NO3)2 at 60 atom% 15N and unlabeled Ca(NO3)2 to achieve the target NO3− concentrations and 15N enrichments.
The 15N analysis was conducted with elemental analyzer isotope ratio mass spectrometry (EA-IRMS) using an isotope ratio mass spectrometer (IRMS) (Model Europa Scientific 20–20, Sercon Ltd., Crewe, UK) coupled to a Europa Scientific elemental analyzer system (Sercon Ltd., Crewe, UK). Total N content was calculated as the total ion beam area generated by the IRMS. The 15N analyses were conducted by Iso-Analytical Limited Sandbach, UK. 15N enrichment was calculated, as 15N atom% excess by subtracting the 15N natural abundance values in equivalent materials (e.g., nutrient solution, fruit, leaf, stem, pruned material) that were unlabeled.
The nutrient solutions of 15N-labeled Ca (NO3)2 were applied using inverted 1.5 L polyethylene bottles connected by silicon tubing to intra-venous drippers. The inverted bottles were supported on metal stakes and the drippers were positioned 3 cm above the sand layer on the soil surface, directly above where irrigation emitter was normally located. Each day, prior to applying the 15N-labeled solution, unlabeled nutrient solution was applied through the drip irrigation system, to all plants in each greenhouse except those that were to directly receive the labeled nutrient solutions. For plants that were to be directly labeled, 7 L plastic trays were used to collect the unlabeled nutrient solution. This ensured that the only nutrient solution received during the application period of three consecutive days of labeling was the 15N-labeled nutrient solution. The applied volumes and the nutrient concentrations of 15N-labeled nutrient solution were identical to those being applied to the rest of the plants in each greenhouse during the three days labeling periods.
Sheet metal plates 1 m long × 22 cm high were inserted into the soil, to a depth of 22 cm, parallel to and 50 cm from the row of plants being labeled, to limit lateral movement, perpendicular to the rows, and prevent uptake of 15N by plants in adjacent crop rows. One plate was inserted on each side of each labeled plant, parallel to the crop rows. The center part of each plate was adjacent to the directly labeled plant.
2.6. Determinations of Plant/Soil Parameters
2.6.1. Field Sampling and Handling of 15N-Labeled Plant Material
The plants that directly received 15N-labeled nutrient solution (“directly labeled plants”; DLP) together with the two immediately adjacent plants on either side, within the same plant row (“adjacent plants”; AP), were identified as “15N labeled plants”. Following labeling, all pruned shoot material from each subsequent pruning of each 15N labeled plant was collected and oven-dried at 65 °C until constant weight. Similarly, all mature fruit collected from each 15N labeled plant was collected and oven-dried at 65 °C. At the end of each crop (crop maturity), each labeled plant was cut at the ground level, and separated into leaf, stem and immature fruit. Each of these three components was oven-dried at 65 °C until constant weight.
All dried pruned material collected after labeling was bulked for each plant. Similarly, all dried mature fruit was bulked for each plant. Fallen leaf collected from the soil surface below each plant was collected and bulked. For data analysis, the small amounts of 15N in fallen leaf were included in that of pruned material. From each labeled plant, both from directly labeled (DLP) and adjacent (AP) plants, there were samples of bulked pruned material, bulked mature fruit, bulked fallen leaf, and the leaf, stem and immature fruit removed at the end of the crop.
For each labeled plant, the bulked pruned material, bulked mature fruit, bulked fallen leaf, and the leaf, stem and immature fruit, removed at the end of each crop, were individually ground with a knife mill (Model SM100 Comfort, Retsch, Germany). A representative sub-sample was then ground with a ball mill (Model MM200 Comfort, Retsch, Germany) until sufficiently fine to pass through a 0.2 mm mesh.
2.6.2. Calculation of 15N Recovery
For each 15
N-labeled plant, the amount of recovered excess 15
N was calculated as the sum of excess 15
N in each of the components of leaf, stem and immature fruit at the end of the crop, and the bulked mature fruit and bulked pruned material. The percentage crop recovery of 15
N from 15
N-labeled nutrient solutions was calculated using a mass balance method using the equation:
In this equation, R is the percentage 15N recovery of applied 15N by the plant, 15NU is the uptake of enriched 15N by the plant and 15NA is the enriched 15N in the nutrient solution applied to the directly labeled plant. The total 15N recovery by the crop was calculated as the sum of 15N recovery by the DLP plant and of each of the two corresponding AP plants. For each plant, 15N recovery was calculated for leaf, stem, fruit and pruned material to provide information on the relative distribution of recovered 15N within plants. All 15N data were the means of four replicates.
2.6.3. Fruit Production, Dry Matter Production, and Crop N Uptake of Unlabeled Plants
For each treatment of each crop, four areas of 4 m2, each with eight plants were marked to determine fruit production, the mass of pruned material removed during the crop, and final standing biomass (after prior removal of prunings and harvested fruit) at the end of each crop. There was one group of eight plants in each replicate plot. There were several prunings in each crop, one harvest of mature muskmelon fruit and 5–8 harvests of mature sweet pepper fruit. At each fruit harvest, the fruit was separated into commercial and non-commercial fruit using local commercial criteria; fresh and dry weights were determined of both fruit categories. At each pruning, dry matter removed in leaves and stems was determined. Dry matter (DM) content of all crop components was determined by oven drying at 65 °C until constant weight.
At the end of each of crop, the four groups of eight plants in each treatment, were completely removed and separated into leaves, stem and fruit, which were weighed, and the dry matter content determined. Final total shoot dry matter production (final DMP) for each treatment was determined by summing the amount of DM of these sampled plants, the total amount of DM removed in the various prunings, and the total amount of DM harvested as fruit.
Representative samples of leaves, stems, and fruit from the final standing biomass sampling, and of harvested fruit and prunings were ground separately and sequentially with a knife mill and ball mill, as previously described for the equivalent 15N labeled material. The total N content of each sample was determined using a Dumas-type elemental analyzer system (model EA 3000, EuroVector SpA., Milan, Italy). Total crop N uptake was calculated, as the sum of N in the final standing biomass, harvested fruit and prunings for each replicate group of plants for each treatment.
2.6.4. Soil Mineral N
For each treatment of each crop, soil samples were taken at the beginning and end of the crop, at three depths from the surface of the imported clay soil (0–20, 20–40 and 40–60 cm depth), from four replicate locations, one from each replicate plot. In each location of each treatment, the soil was sampled in three positions with respect to the plant and the irrigation emitter, being (1) very close to the plant and emitter, (2) between two adjacent emitters of the same drip-line, and (3) mid-way between adjacent emitters of adjacent lines of emitters. The inorganic N content of the soil was determined following extraction by potassium chloride (KCl) (40 g moist soil: 200 mL 2 M KCl). Concentrations of NO3− and NH4+ in the soil extracts were determined using the automatic segmented flow analyzer previously described for analyzing nutrient solutions. With respect to the sampling positions, soil mineral N (NO3−–N plus NH4+–N) was calculated as: (0.25 × position 1) + (0.25 × position 2) + (0.50 × position 3).
2.6.5. Soil Solution NO3−
Soil solution was obtained weekly using soil solution suction samplers (model 1900L12, Soil Moisture Equipment Co., Santa Barbara, CA, USA) installed at 15 cm depth from the surface of the imported clay soil, 5 cm from the emitter in the perpendicular direction of the drip-line and 8 cm from the plant in the same direction of the drip-line. There were four replicates per treatment. The soil solution samples were taken 24 h after applying vacuum (−60 kPa) to the suction cups. There was a period of at least 24 h after the previous irrigation (and application of nutrient solution) before vacuum was applied. The NO3− concentration in soil solution samples was analyzed using the procedures and equipment previously described for nutrient solutions.