氮肥分次施用对小白菜镉积累及品质的作用

Effects of split applications of nitrogen fertilizers on the Cd level and nutritional quality of Chinese cabbage # # 氮肥分次施用对小白菜镉积累及品质的作用 first-author corresp ‡Corresponding Author ‡Corresponding Author †E-mail:jincw@zju.edu.cn †E-mail:jincw@zju.edu.cn jincw@zju.edu.cn College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China Cadmium (Cd) contamination in soil is an increasingly serious problem. Management of plant nutrients has been proposed as a potentially promising strategy for minimizing Cd accumulation in crops grown in contaminated soil. This study investigated the effects of split applications of nitrogen (N) fertilizers on the Cd concentration in Chinese cabbage ( Brassica chinensis L.) plants grown in Cd-contaminated soil. Compared with single applications, split applications of ammonium or urea resulted in significantly lower Cd concentrations, and higher biomass production and antioxidant-associated nutritional quality in the edible plant parts. However, when nitrate was used as the N fertilizer, there were no significant differences between the split and single applications for the same parameters. We conclude that a split application could be more beneficial than a single application method when ammonium or urea is used as the N fertilizer for vegetable cultivation in Cd-contaminated soil. 目的 2 研究不同形态氮肥的分次施用对小白菜镉积累、产量及品质的影响，并初步分析了其作用机制。 创新点 2 首次提出尿素、铵态氮和硝态氮肥料的分次施用方式可提升镉污染土壤上的作物安全生产。 方法 2 选用尿素、硫酸铵和硝酸钙等3种常用氮肥，通过盆栽试验，比较了这3种肥料的分次施用方法和一次性施用方法对小白菜的镉含量、生物量以及营养品质的影响。 结论 2 在镉污染的条件下，与一次性施肥相比，尿素和硫酸铵的分次施肥方法显著降低了小白菜可食部位的镉含量，增加了可食部位的生物量并改善了抗氧化相关的营养品质指标；然而，当硝酸钙作为氮肥时，小白菜可食部位的镉含量、生物量以及抗氧化相关的营养品质指标在两种不同的施肥方法之间并无统计学上的显著差异。因此，在镉污染的土壤上，当硫酸铵或尿素作为氮肥时，分次施肥方法比一次性施肥方法更加有利于保障蔬菜的安全生产与品质改善。 Fertilization method Cadmium (Cd) Nitrogen (N) Nutritional quality 施肥方法 镉污染 氮肥 营养品质 Introduction 1 Contamination of soil by heavy metals is an increasingly serious problem due to various anthropogenic activities, such as the application of sewage sludge, mining and smelting of metalliferous ores, and overuse of agrochemicals (Kirkham, 2006 2006 ; Wei and Yang, 2010 2010 ). Cadmium (Cd) is one of the most toxic heavy metals. In the human body, it adversely affects kidneys and bones (Clemens et al., 2013 2013 ). The primary route of Cd entry into the body is through the consumption of crops grown in Cd-contaminated soil (Clemens et al., 2013 2013 ). A recent survey indicated that vegetables account for about 40% of the total Cd exposure of residents of Shanghai, China’s largest city (He et al., 2013 2013 ). Therefore, measures should be developed to minimize the amount of Cd entering vegetables through contaminated soil. It has been proposed that the management of plant nutrients is potentially a promising strategy for minimizing Cd accumulation in crops grown in contaminated soil (Sarwar et al., 2010 2010 ). Growers have already applied nutrients, particularly macronutrients, to obtain a higher crop yield. Many nutrients have direct and indirect effects on Cd bioavailability in soil and Cd uptake through the roots of plants. For instance, phosphate favors precipitation to decrease Cd availability in soil (Dheri et al., 2007 2007 ; Dong et al., 2007 2007 ) and divalent cations, such as Fe 2+ and Zn 2+ , compete with Cd 2+ for the same membrane transporters thereby reducing Cd entry into plants (Lux et al., 2011 2011 ). Accordingly, if growers keep in mind the interactions between nutrients and Cd, with the proper management of nutrients, they could have a cost-effective and time-saving strategy for reducing Cd accumulation in crops. Management of nitrogen (N) is one of the most frequent agronomic practices. Nitrogen is taken up by plants mainly in the form of ammonium (NH 4 + ) and nitrate (NO 3 − ). Physiologically, NO 3 − uptake by roots is accompanied by a simultaneous uptake of protons (H + ), resulting in an increase in rhizosphere pH (Crawford and Glass, 1998 1998 ). Conversely, when NH 4 + is taken up, the H + is released into the rhizosphere, resulting in acidification of the rhizosphere soil (Hinsinger et al., 2003 2003 ). The nitrification of NH 4 + by microorganisms also produces H + , further increasing the soil acidification caused by the release of H + by roots during ammonium uptake (Herman et al., 2006 2006 ). The soil pH strongly affects Cd availability, which is increased when the pH drops (Kirkham, 2006 2006 ). Accordingly, it has often been suggested that NH 4 + fertilizers could result in higher Cd availability in rhizosphere soil than NO 3 − fertilizers (Sarwar et al., 2010 2010 ). This suggestion has been supported by many studies (Florijn et al., 1992 1992 ; Tsadilas et al., 2005 2005 ; Zaccheo et al., 2006 2006 ), in which N fertilizers were applied to the soil as a single fertilizer treatment. Nevertheless, in practical crop cultivation, a split application of N fertilizers at different growth stages is often recommended by agronomists, since this method of fertilization favors plant growth (López-Bellido et al., 2005 2005 ). A change in plant growth may affect Cd uptake by roots, thereby altering the Cd accumulation in plant tissues. In addition, NH 4 + has a high rate of nitrification in soil (Zerulla et al., 2001 2001 ). Accordingly, in comparison with a single application, split fertilization theoretically decreases the rate of NH 4 + nitrification in the soil, thereby increasing N uptake by the roots in the form of NH 4 + . Since more protons are released from NH 4 + nitrification than from NH 4 + uptake by the root cells, a split application of NH 4 + might be expected to alleviate the acidification-increased Cd solubility, compared with a single application. Considering the approach described above, the method of N fertilization may dramatically affect Cd accumulation in vegetables and other crops. However, little information is available to support this speculation. Currently, three forms of N fertilizers, ammonium, nitrate, and amide, are most commonly used in practical crop production. Nitrate is generally the preferred form for most vegetable plants (Chen et al., 2005 2005 ). However, which forms of N fertilizer are used in practical vegetable cultivation in a region depends largely on the traditional practice of the local growers and the fertilizer provided by local fertilizer dealers. Therefore, it is necessary to investigate the effects of the method of fertilization application (single or split applications) on Cd accumulation in the edible parts of vegetables when using different forms of the N nutrient. In this study, the Chinese cabbage ( Brassica chinensis L.), one of the most popular vegetables in China, was used to address this issue. The quality of the nutritional characteristics of vegetables, such as the levels of soluble sugar, soluble protein, and antioxidants, is also very important for human health. Previous studies have found that Cd contamination in the growth medium clearly affects the nutritional quality of vegetables (Prince et al., 2002 2002 ). Therefore, we also investigated the effects of the N fertilization method on the nutritional quality of Chinese cabbage grown in Cd-contaminated soil. Materials and methods 1 Soil and plants 2 The soil used for the pot experiment was collected from Jianggan District, Hangzhou, China (30°16′ N, 120°12′ E). The basic properties of the soil, determined by standard procedures (Bao, 2008 2008 ), are summarized in Table 1 1 . After the air-dried soil had been ground to pass through a 2-mm sieve, chemical fertilizers were mixed into the soil: K 2 SO 4 at 160 mg K/kg soil and KH 2 PO 4 at 150 mg P/kg soil. Then, half of the soil was mixed with CdCl 2 at 10 mg Cd/kg soil. Plant pots, 2.5 L in volume, were then each filled with 3 kg of soil. The Chinese cabbage vegetable plant B . chinensis L. cv. Changfeng was used in this study. Sample pH CEC (cmol/kg) EC (mS/cm) Clay (%) Silt (%) Sand (%) Organic C (g/kg) NH 4 + -N (mg/kg) NO 3 − -N (mg/kg) Total Cd (mg/kg) Pot-culture soil 7.2 7.9 0.7 6.2 28.8 65.0 11.5 2.5 13.4 0.2 Plant cultivation and nitrogen treatments 2 The pot experiment was conducted in a greenhouse. The soil in the pots was watered to 60% field water holding capacity (FWHC; 22% water content, w/w). Chinese cabbage seeds were sown at a depth of 1 cm, with ten seeds per pot. The pots were divided into two groups, with one group given a single N application, and the other a split N application. For the single N application, at the sowing stage, the soil was fertilized with either (NH 4 ) 2 SO 4 , Ca(NO 3 ) 2 , or CO(NH 2 ) 2 (urea) at 400 mg N/kg soil. For the split N application, one of the above three N fertilizers was applied to the soil at rates of 120, 120, and 160 mg N/kg soil at the sowing, seedling, and vegetative growth stages, respectively. When the second true leaves were observed, the seedlings in each pot were thinned to three plants of similar size. During the experimental period, water was added to compensate for evaporation and transpiration, and soil moisture content was maintained at about 60% of FWHC. The edible parts of the 8-week-old plants were harvested for further analysis. Measurements of the concentrations of soluble sugar, soluble protein, ascorbate, total phenolics, and total flavonoids, and DPPH free radical-scavenging activity 2 The concentrations of soluble sugar, soluble protein, ascorbate, total phenolics, and total flavonoids, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging activity were analyzed as described in our previous study (Jin et al., 2013 2013 ). Analysis of cadmium content 2 Plant tissues were dried at 80 °C for 48 h, and then the dried samples were wet-digested, as previously described (Luo et al., 2012 2012 ). The digestates were diluted with ultrapure water, and the concentrations of Cd in the digestates were analyzed using an absorption spectrometer (Thermo Scientific AAS, iCE 3300), following a standard procedure. The same procedure without samples was used as a control and three replications were conducted for each sample. Quality assurance and quality control of the analysis included the recovery of spiked Cd (better than 95%) from an independent standard source, the analysis of blank and standard references, and the use of duplicates for every set of ten samples. Statistics 2 All statistical analyses were conducted with IBM SPSS Version 20 (IBM Corp., Armonk, NY, USA). Means were compared using Duncan’s multiple range test with a significance level of P < 0.05 in all cases. Results 1 Effects of the N fertilization methods on the yield of the edible parts 2 The effect of the fertilization method on the biomass production of Chinese cabbage was highly dependent on the form of N fertilizer in both the control (no Cd added) and the Cd-added soils (Fig. 1 1 ). When ammonium ((NH 4 ) 2 SO 4 ) or amide (urea) was used as the N source, the biomass of the edible parts of the plants from the split dressing treatment was increased by 46% and 30% in the control soil, respectively, and by 47% and 24% in the Cd-added soil, respectively, compared with that of the plants from the single dressing treatment. However, when nitrate (Ca(NO 3 ) 2 ) was used as the N source, there was no statistically significant difference in the biomass of the edible parts between the single and split dressings in either the control or Cd-added soils. The above results indicate that the impact of the N fertilization method on the biomass production of Chinese cabbage was independent of Cd contamination in the soil. Note that, within the same fertilization method, the edible-part biomass of the plants fed with nitrate was greater than that of the plants fed with ammonium or amide in either the control or the Cd-added soil. The plants were cultured in pots. For single N applications, the soil was fertilized with (NH 4 ) 2 SO 4 (ammonium), Ca(NO 3 ) 2 (nitrate), or CO(NH 2 ) 2 (urea) at 400 mg N/kg soil at the sowing stage. For split N applications, each of the above three N fertilizers was applied to the soil at rates of 120, 120, and 160 mg N/kg soil at the sowing, seedling, and vegetative growth stages, respectively. The edible parts of 8-week-old plants were harvested for analysis. Data are expressed as mean±standard deviation (SD) ( n =6). Different letters represent significantly different values (Duncan’s test, P < 0.05) Effects of the N fertilization methods on the Cd levels in edible parts 2 No Cd was detected in the edible parts of the Chinese cabbage grown in the control soil following any treatment (data not shown). In the Cd-added soil, the N fertilization method clearly affected the Cd concentration in the edible parts, but the effect was dependent on the form of the N fertilizer. The Cd concentrations in the edible parts following split dressing treatments of ammonium and amide were about 40% and 60% lower, respectively, than those following single dressing treatments of these two fertilizers (Fig. 2 2 ). However, when nitrate was used as the N source, the Cd concentration in the edible parts following the split dressing treatment was similar to that following the single dressing treatment. The N form also significantly affected the Cd concentration in the edible parts when the fertilizers were applied using the same method (Fig. 2 2 ). With single applications, the Cd concentration was much lower in the nitrate treatment than in the ammonium or amide treatment. With split applications, the Cd concentrations in the nitrate and amide treatments were similar, but were clearly lower than those in the ammonium treatment. Treatments are the same as in Fig. 1 1 . The data are expressed as mean±SD ( n =5). Different letters represent significantly different values (Duncan’s test, P < 0.05) Effects of the N fertilization methods on the nutritional quality of the edible parts 2 The fertilization method had little effect on the level of soluble sugar in the edible parts of Chinese cabbage grown in the control soil for all three N forms of fertilizer. In the Cd-added soil, the soluble sugar levels with the split dressing method were higher in the ammonium treatments but lower in the nitrate and amide treatments, compared with the levels in the single dressing method (Fig. 3a 3a ). We also determined the level of soluble protein in the edible plant parts. In the control soil, it was higher following the split dressing than following the single dressing regardless of the N form, but the difference was not statistically significant in the plants fed with nitrate. However, in the Cd-added soil, the split dressing method resulted in a slightly lower level of soluble protein in the plants fed with ammonium or urea, but a significantly higher level (by about 35%) in the plants fed with nitrate, compared with the single dressing (Fig. 3b 3b ). (a) The levels of soluble sugar in the edible plant parts. (b) The levels of soluble protein in the edible plant parts. Data are expressed as mean±standard deviation (SD) ( n =6). Different letters represent significantly different values (Duncan’s test, P < 0.05) To evaluate the antioxidant capacity in the edible parts of the Chinese cabbage, we first measured the levels of antioxidation-associated compounds. In both the control and Cd-added soils, the application method barely affected the ascorbate level in the edible plant parts for all three forms of N treatment (Table 2 2 ). The application method also had little effect on the levels of total phenolics and flavonoids in the edible parts of the plants grown in the control soil. However, in the Cd-added soil, the total phenolic level in the edible plant parts following the split dressing was significantly higher for all three forms of N treatment, compared with the single dressing. In addition, the split dressing also had a significantly higher level of flavonoids than the single dressing with the nitrate treatment. We then analyzed the total antioxidant capacity. The results show that, in the control soil, the application method had little effect on the DPPH radical-scavenging activity of the edible plant parts for all three forms of N treatment. In the Cd-added soil, the DPPH radical-scavenging activity of the edible plant parts with the ammonium and urea treatments was significantly higher following a split dressing compared with a single dressing, whereas with the nitrate treatment, this activity was not affected (Fig. 4 4 ). The plants were cultured in pots. For single N applications, the soil was fertilized with (NH 4 ) 2 SO 4 (ammonium), Ca(NO 3 ) 2 (nitrate), or CO(NH 2 ) 2 (urea) at 400 mg N/kg soil at the sowing stage. For split N applications, each of the above three N fertilizers was applied to the soil at rates of 120, 120, and 160 mg N/kg soil at sowing, seedling, and vegetative growth stages, respectively. The edible parts of 8-week-old plants were harvested for analysis. The data are expressed as mean±SD ( n =4). Different letters represent significantly different values in the same column (Duncan’s test, P < 0.05). GAE: gallic acid equivalent; DAE: 3,4-dihydroxybenzoic acid equivalent; DW: dry weight 2 Treatment 2 Total phenolics (mg GAE/g DW) 2 Ascorbate (μmol/g DW) 2 Flavonoids (μmol DAE/g DW) N form Application Control +Cd Control +Cd Control +Cd Ammonium Single 18.42±0.98 a 17.85±0.78 bc 14.92±5.30 a 14.42±2.54 a 3.14±0.27 a 2.75±0.05 b Split 17.10±0.63 ab 20.07±0.29 a 12.95±1.82 a 14.80±1.86 a 2.89±0.12 a 3.12±0.53 ab Nitrate Single 16.17±0.85 b 17.50±0.09 c 14.28±1.18 a 11.06±2.43 a 2.77±0.18 a 2.65±0.17 b Split 16.86±0.80 ab 18.68±0.15 b 14.55±4.17 a 11.91±1.48 a 2.84±0.22 a 3.20±0.38 a Urea Single 17.47±0.88 ab 18.76±0.68 b 12.89±2.10 a 12.22±2.66 a 3.11±0.45 a 3.13±0.10 a Split 16.15±1.48 b 20.63±1.26 a 15.65±2.89 a 11.42±2.61 a 2.91±0.51 a 2.98±0.47 ab Discussion 1 Usually, a split application of N fertilizer gives a greater yield in crop cultivation than a single application, since the split fertilization favors a greater increase in N use efficiency (de Ruijter et al., 2010 2010 ). However, in China, growers often use single fertilizer applications in the cultivation of short life-cycle crops like Chinese cabbage. In this study, we found that split applications of ammonium and urea clearly resulted in a higher yield and a lower Cd concentration in the edible parts of Chinese cabbage, compared with single applications of these fertilizers (Figs. 1 1 and 2 2 ). Note that, although the application method for the nitrate treatment did not affect the Cd level or biomass of edible parts (Figs. 1 1 and 2 2 ), the split fertilization did have the potential for higher nitrogen use efficiency and could reduce the emission of greenhouse gases such as nitrous oxide (López-Bellido et al., 2005 2005 ; Gillam et al., 2008 2008 ). Therefore, in Cd-contaminated soil, we recommend split fertilization for vegetable cultivation, regardless of the type of N fertilizer. The Cd uptake by the roots of plants can be affected by a number of factors, including soil pH, chelation, plant nutrients, root exudates, plant species, and microorganisms (Sarwar et al., 2010 2010 ; Clemens et al., 2013 2013 ). There are two main ways by which these factors affect the Cd uptake in plants: (1) by altering the bioavailability of Cd in the growth media; (2) by modulating the root uptake capacity for Cd. Soil acidification resulting from ammonium nitrification is more pronounced than that caused by ammonium uptake by root cells (Tudoreanu and Phillips, 2004 2004 ), and thus ammonium nitrification should increase the Cd solubility in the soil more than ammonium uptake. The behavior of urea in soil is similar to that of ammonium as urea can be decomposed into ammonium by urease. However, in our study, we found that the fertilization method had little effect on the pH of the soil (Fig. S1 S1 ). This may be because the soil used in the present study had a higher buffering capacity against acidification (Fig. S2 S2 ). Therefore, soil acidification resulting from ammonium nitrification cannot explain why the split applications of ammonium or urea had lower Cd levels in the edible parts of the Chinese cabbage plants compared with the single applications of these fertilizers. Our previous study showed that the presence of nitrate in the growth medium facilitates the uptake of Cd by root cells (Luo et al., 2012 2012 ; Mao et al., 2014 2014 ). In this context, the decreased nitrate generation from ammonium and urea due to the decreased nitrification from split fertilization may prevent the effect of nitrate in promoting the uptake of Cd by plant roots. This may explain why split applications of ammonium and urea resulted in lower Cd levels in the edible parts of the Chinese cabbage, compared with single applications of these fertilizers. In addition, because the split applications of ammonium and urea resulted in a higher biomass than the single applications (Fig. 1 1 ), the dilution effect may be another key reason for the lower Cd level in the edible parts with the split fertilization method. In recent decades, controlled-release fertilizers (CRFs) have been rapidly developed (Shaviv, 2001 2001 ). CRFs enable nutrients to be released over an extended period of time, regulating the nutrient release time through the excipients, and therefore are more efficient than conventional fertilizers (Sempeho et al., 2014 2014 ). Theoretically, application of ammonium-or urea-based CRFs could further decrease the rate of ammonium nitrification in soil. Therefore, CRFs may be a promising fertilizer for use in Cd-contaminated soil. Note that, in single applications, the Cd concentration in the edible plant parts was much lower with the nitrate treatment than with the ammonium or amide treatment. One explanation may be the dilution effect, because the nitrate fertilizer produced a higher biomass than the other two fertilizers (Fig. 1 1 ). In addition, the nitrate fertilizer used in the present study was Ca(NO 3 ) 2 . The Ca 2+ and Cd 2+ would compete for the same Ca 2+ channels during their uptake by roots cells (Perfus-Barbeoch et al., 2002 2002 ). Therefore, the antagonism of Ca to Cd may be another reason for the lower Cd level in the edible plant parts following nitrate treatment. Our results showed that the nutritional quality of Chinese cabbage grown in Cd-added soil was affected by different fertilization methods. Soluble sugar levels are used to evaluate the taste characteristics of vegetables and soluble protein accumulation as an indicator of value for human nutrition (Hurrell, 2003 2003 ; Jin et al., 2009 2009 ). The split applications produced a higher level of soluble sugar in the ammonium treatment, and a higher level of soluble protein in the nitrate treatment, compared with the single applications. The level of antioxidants is another important factor for evaluating vegetable nutritional quality (Shyamala et al., 2005 2005 ). A higher intake of vegetables containing antioxidants including phenolics, flavonoids, and ascorbic acid may contribute to protecting against oxidative damage, thus lowering cancer, cardiovascular disease, and other chronic disease risks. For instance, it is reported that human serum and low-density lipoprotein oxidation can be inhibited by phenolic compounds while ascorbic acid can protect against DNA damage in human sperm and improve the efficacy of antineoplastic drugs such as paclitaxel (Fraga et al., 1991 1991 ; Frankel et al., 1995 1995 ; Abu-Amsha et al., 1996 1996 ; Kurbacher et al., 1996 1996 ). In addition, flavonoids can increase plasma antioxidant capacity in humans and protect human retinal pigment epithelial cells from oxidative-stress-induced death (Hanneken et al., 2006 2006 ; Lotito and Frei, 2006 2006 ). Here, we found that, following any of the three forms of N treatments, the split application resulted in higher levels of phenolics in the edible parts of the plants grown in Cd-added soil, but had little effect on the level of ascorbic acid, compared with the single application. Only the split application of nitrate to Cd-added soil resulted in higher levels of flavonoids in edible plant parts. The above results indicate that the fertilization method differentially affected the production of antioxidants in edible plant parts. Therefore, we further investigated the effect of the fertilization method on the total antioxidant capacity in the edible parts of Chinese cabbage. The DPPH antioxidant assay is used widely to estimate total antioxidant capacity in vegetable extracts (Sanchez-Moreno, 2002 2002 ). We showed that, in the Cd-added soil, the DPPH radical-scavenging activity in the edible plant parts following the split application was higher than that following the single application for the ammonium or urea treatments. However, there was no statistical difference between the two application methods when nitrate was used as the sole N source. Interestingly, the effects of the application methods on elevating DPPH radical-scavenging activity correlated well with their effects in either decreasing Cd levels or increasing the biomass of edible parts, indicating that an appropriate fertilization method not only reduces the human health risk of Cd exposure from crops grown in Cd-contaminated soil, but also improves the nutritional quality and yield of the crops. 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This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1600272 Project supported by the National Key Research and Development Project of China (No. 2016YFD0200103), the National Natural Science Foundation of China (Nos. 31622051 and 31670258), and the Zhejiang Provincial Natural Science Foundation of China (No. LR13C130001) 2016-06-13 2017-01-16 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany 2017 2017 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 10 18