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Much nitrogen (N) is lost in high-input protected cropping systems mainly via leaching of not only nitrate-N but also extractable organic N (EON), but the role of EON in this process is poorly appreciated. A consecutive 3-year plot experiment was conducted to investigate the impact of co-application of manures with chemical N fertilizer on N accumulation and loss in a greenhouse soil rotationally planted with cucumber or tomato and lettuce. Application of manures significantly enhanced the average contents and stocks of NO3 -N, EON, and total N (TN) in 0to 60-cm soil layer, although EON accumulated within growing season, while NO3 -N accumulated with fluctuation, and TN accumulated gradually throughout the 3-year experiment. With application rate at 120 or 180 t dry manures per hectare per 3 years, the corresponding apparent N surplus was 2710 or 3924 kg·ha per 3 years. Due to little increase of biomass N uptakes during vegetable seasons, the accumulated N in soil profile would be a potential loss source, largely via leaching of both nitrate and EON. Application of manures slowed soil acidification but intensified secondary salinization of the greenhouse soil. Considering the manures-induced high soil N accumulation and loss, well-balanced evaluation of the role of manures in high-input agricultural ecosystems is needed. Nitrogen is an essential nutrient for plant growth. Nitrogen fertilization is the major feature of intensive agricultural management to improve crop yields (Zhao et al., 2014; Zhu and Chen, 2002). Generally, manures are applied together with chemical N fertilizer to buffer soil acidification, improvemicrobial activity and functional diversity, provide nutrients, and maintain the sustainable productivity (Hati et al., 2008; Shen et al., 2010; Zhao et al., 2012). The relatively low cost of manures and the increasing demand for cheap food have encouraged the overuse and misuse of manures in the last several decades (Tian et al., 2011), resulting in the accumulation of superfluous N in soil (Fan et al., 2010; Zhao et al., 2014), and its migration to a deeper soil layer or groundwater when the conditions are satisfied (Bergstr€om and Kirchmann, 1999). High-input cropping systems, especially greenhouse cultivation, develop quickly in China to meet consumer demand for vegetables throughout the whole year (Qiu et al., 2010; Yan et al., 2012). However, closed growth environment and high-rate fertilization easily degrade a greenhouse soil within a period of several years (Lei et al., 2010). Co-application of organic fertilizers such as manures with chemical fertilizers may alleviate the deterioration of soil quality but maintain high yield. However, application of manure N, typically in the range of 1000– 3046 kg N/ha/year (Ju et al., 2007; Shi et al., 2008; Thompson et al., 2007) and accounting for 51% to 65% of TN inputs in greenhouse vegetable cultivation systems (Gao et al., 2012; Ju et al., 2006), increases the overall rate of N fertilization, making greenhouse cropping systems accumulate superfluous N subject to loss via denitrification and leaching. The averaged N surplus in 0to 90-cm soil layer was up to 3327 kg N/ha/year in vegetable cultivation greenhouses (n = 56) in Huiming, North China (Ju et al., 2006). Nitrate as the main form of mobile N in greenhouse soil highly accumulate and easily migrate to shallow aquifers causing very serious nitrate pollution in nearby shallow wells (Ju et al., 2006). The extent of nitrate pollution in soil and shallow aquifers will continue to be worsened when greenhouse cultivation continue (Zhang et al., 2010). Available evaluations on N leaching risk in greenhouse soils are mainly based on accumulation and migration of nitrate in soil profile (Ju et al., 2006; Zhang et al., 2010), while the leaching loss via other forms of leachable N is not well appreciated. Other forms of leachable N, especially EON, have been suggested to be taken into account when evaluating N leaching risk due to its large contribution to N migration in the soil profile (Bhogal et al., 2000; Khalili and Nourbakhsh, 2012; Siemens and Kaupenjohann, 2002). Especially in greenhouse soils, often-applied manures may be a direct source of EON subject to leaching loss (Goyne et al., 2008). Hence, simultaneous investigation of accumulation and migration of nitrate and EON is needed to better understand the scale and the dynamics of N migration and leaching loss in greenhouse soils. In this study, a 3-year experiment was conducted in greenhouse soil rotationally planted with cucumber or tomato and lettuce with and without manures to investigate the spatial (vertical) and temporal dynamics of nitrate, EON, and TN in soil, and estimate the leaching-dominated N loss based on N balance in soil. Materials and Methods Site description. A long-term microplot experiment was conducted in a traditional solar greenhouse (41 31# N, 123 22# E) near Shenyang city from Dec. 2010 (Quan et al., 2014). From 2006 to 2010, this field was used to cultivate vegetables by conventional practices, and theretofore it was a corn field. The soil in the field is meadow burozem (Inceptisols in U.S. soil taxonomy). Soil basic properties are listed in Table 1. Rotation of two vegetables in 1 year was arranged in the microplots (3.4 · 3.8 m): September to December for lettuce (Lactuca sativa L.) season, and January to June for cucumber (Cucumis sativus L.) or tomato (Lycopersicon esculentum Mill.) season. There were summer fallows ( 3 months) and winter fallows ( 20 d) between the two consecutive vegetable production seasons. Experimental design. Three treatments were set up according to the application rate of manure (dry basis): no manure (Control), 20 t·ha per season (M 20), and 30 t·ha per season (M 30, local normal rate). Each treatment had three replications. The normal application rate that farmers chose was based on the need of organic matter in soil to Received for publication 23 July 2015. Accepted for publication 2 Sept. 2015. This research was supported by grant from the National Natural Science Foundation of China (grant number 41001176) and Liaoning Science and Technology Project Program (grant number 2011215003; 2014215016). Corresponding author. E-mail: microyan76@ 126.com. 1688 HORTSCIENCE VOL. 50(11) NOVEMBER 2015 improve soil properties and provide nutrients for plants. The experimental microplots (3 · 3) were arranged as randomized block design in the central area of the greenhouse. Manures were broadcasted to the fields as basal dressing evenly after composting and drying (chicken manure for cucumber or tomato season, and cattle manure for lettuce season). Carbon (C) and N contents of the manures are listed in Table 2. Compound chemical fertilizer (N:P2O5:K2O =15:15:15) was broadcasted at 1 t·ha. Tillage was thereafter done using a small plough (0–20 cm) and vegetable seedlings were transplanted at densities of 110, 72, and 72 plants/plot for lettuce, cucumber, and tomato, respectively. Amino acid liquid fertilizer was applied to the root area to facilitate the survival of seedlings after transplanting. During the six consecutive vegetable seasons, irrigation was carried out regularly in the furrows (Fig. 1). Irrigation water was pumped from a well near the greenhouse, and the irrigation rate was 730 mm annually (TN in irrigated water <3 mg·L). No rain water enters the greenhouse owing to the plastic film covering all year-round. All aboveground vegetable residues were moved out of the greenhouse after harvest. Nitrogen inputs in the system from the studied period of 2011 to 2013 are listed in Table 3. Soil sampling. Background soil was sampled on 20 Dec. 2010. Soil samples of the six vegetable growth periods (four in cucumber/ tomato season, and two in lettuce season) were taken at the depths of 0–10, 10–20, 20– 40, and 40–60 cm with a 2.5-cm-diameter soil drill. Soil samples were taken almost 1 week after irrigation to weaken the impact from irrigation except for the background soil samples. Six soil cores were taken from each plot to make a composite sample. After removal of stones, visual fauna, and plant residues, fresh soil samples were mixed homogeneously and sieved (<2 mm). Subsamples were stored at –18 C in the refrigerator for NH4 -N, NO3 -N, and total extractable N (TEN) analysis. The rest samples were airdried, ground, and sieved (<0.15) mm for TN analysis. Plant sampling. Fruit samples (cucumbers and tomatoes) were collected from the beginning of fruiting to the end of vegetable growing season. Six aboveground plant samples from each plot, partitioned into stems and leaves, were sampled at the end of vegetable growing seasons. Aboveground biomass (stems and leaves) and fresh vegetable yield were recorded when samples were taken. Subsamples were oven-dried at 70 C for 48 h to calculate dry weight and plant N absorption. Sample analysis. Field-moist soil samples of 20 g were weighed and extracted by shaking with 100 mL, 2 mol·L KCl for 1 h. Extractable NO3 -N and NH4 -N in extracts were determined by continuous flow analyzer (AA3; Seal, UK). Total extractable N in extracts was determined by TOC-TN analyzer (Multi N/C 3100; Analytik Jena, Germany) (extracts pass through 0.45-mm filter membrane before injection). Extractable organic N = TEN – NO3 -N – NH4 -N. The extracts were kept in polypropylene bottles in a –18 C refrigerator before analysis. Soil and manure pH (1:2.5, w/v) and electrical conductivity (EC) (1:5, w/v) were determined separately by a pH electrode (Model 868; Thermo Electron, China) and an electrical conductivity meter (DDSJ-308A; Leici, China) to evaluate acid and salt accumulation. Soil, plant, andmanure total C, TN, total phosphorus, and total potassium were determined by combustion, Kjeldahl, molybdenum, and flame photometer methods. Soil N loss estimation. The estimated soil N loss was calculated by the following formula: Nitrogen loss = input N components compound fertilizer

Nitrogen Accumulation and Loss in a High-input Greenhouse Vegetable Cropping System Elevated by Application of Manures