Nitrate, Nutrient Content and Growth Parameters of Komatsuna (Brassica Rapa L.) in Response to Manure Application Depending on EMN (Estimated Mineralizable Nitrogen)

Journal of Plant Nutrition

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Nitrate, nutrient content and growth parameters of komatsuna (Brassica rapa L.) in response to manure application depending on EMN (estimated mineralizable nitrogen)

Kyi Moe, Seinn Moh Moh, Aung Zaw Htwe & Takeo Yamakawa

To cite this article: Kyi Moe, Seinn Moh Moh, Aung Zaw Htwe & Takeo Yamakawa (2019) Nitrate, nutrient content and growth parameters of komatsuna (Brassicarapa L.) in response to manure application depending on EMN (estimated mineralizable nitrogen), Journal of Plant Nutrition, 42:15, 1726-1739, DOI: 10.1080/01904167.2019.1643366 To link to this article: https://doi.org/10.1080/01904167.2019.1643366

Published online: 23 Jul 2019.

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Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=lpla20 JOURNAL OF PLANT NUTRITION 2019, VOL. 42, NO. 15, 1726–1739 https://doi.org/10.1080/01904167.2019.1643366

Nitrate, nutrient content and growth parameters of komatsuna (Brassica rapa L.) in response to manure application depending on EMN (estimated mineralizable nitrogen)

Kyi Moea,b, Seinn Moh Moha, Aung Zaw Htwea,b, and Takeo Yamakawac aPlant Nutrition Laboratory, Graduate School of Bioresource and Bioenvironmental Sciences, Faculty of Agriculture, Kyushu University, Fukuoka, Japan; bDepartment of Agronomy, Yezin Agricultural University, Nay Pyi Taw, Myanmar; cPlant Nutrition Laboratory, Division of Molecular Biosciences, Department of Biosciences & Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, Japan

ABSTRACT ARTICLE HISTORY We investigated the effects of poultry manures (PM) and cow manures Received 22 August 2018 (CM) using estimated mineralizable nitrogen (EMN) method on nitrate, Accepted 25 October 2018 nutrient content and growth parameters of komatsuna and compared with KEYWORDS chemical fertilizer (CF) in an abandoned soil. We applied manures as EMN Estimated-mineralizable-N based on their total N content. The 100% of EMN by PM or CM enhanced (EMN); growth-parameters; dry matter (DM) but depressed nitrate content. The PM-Keifun (PMK) with komatsuna; manures; total N (4.87%) produced greater growth parameters, DM, nutrient content nitrates; nutrient-content and lower nitrate content but manures with total N (<2%) could not. The solo CF150 did not promote leaf number but increased nitrate and Na content. In conclusion, a higher total N (>4%) concentration of manure led to increase availability and nutrient contents, DM, and depressed nitrate content of komatsuna, comparable with solo CF in an abandoned soil.

1. Introduction In modern vegetable production, inorganic fertilizer is exclusively used to maximize the production of foliage. The application of an excessive amount of chemical fertilizer (CF) decreases the performance of plants because of soil acidification, reduction of soil biological activity, decreased soil physical properties, and lack of micronutrients in fertilizer (Ededirant et al. 2004). In most of cases, CF application causes environmental pollution and ecological problems, which increased production costs (Barth, De Tullio, and Conklin 1998). The source of nutrients and rate of fertilizer used can affect nutrient composition and quality of crops, especially leafy vegetables (Riahi et al. 2009). In green vegetables, fertilizer application not only influences the vegetative growth of the crop but also changes quality aspects such as nitrates, vitamin and antioxidant activities (Seung and Adel 2000). Nitrogen in plant cells can be in the form of nitrate or ammonium. Using CF, nitrate could be postulated to produce detrimental effect to human health (Winchester, Huskins, and Ying 2009). However, without CF, it is not possible to fulfill consumer demand for increased growth or yield of vegetables. Wise use of CF is necessary, especially for vegetable production with minimal negative effects. Organic manures are alternative sources for chemical fertilizer substitution. Organic manures contain higher nitrogen (N), phosphorus (P), potassium (K), and trace element contents. In addition, crops grown with organic manures resulted in a higher total

CONTACT Kyi Moe [email protected] Plant Nutrition Laboratory, Graduate School of Bioresource and Bioenvironmental Sciences, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. ß 2019 Taylor & Francis Group, LLC JOURNAL OF PLANT NUTRITION 1727

Table 1. Mineralization efficiency of manures. Mineralization (%) of nutrients from applied manures

Total N content (DW) NP2O5 K2O <2% 20 100 65 2–4 % 30 100 65 4% 50 100 65 Data source: Nishio (2007); DW ¼ Dry Weight basis. antioxidant capacity in cabbage (Bimova and Pokluda 2009) and vegetables (Ramesh, Shivana, Santa, and Ram 2011); more vitamin C, iron, magnesium, and phosphorus in fruits, vegetables, and grains; and a significantly lower nitrate content than crops grown with CF (Worthington 2001). On the other hand, an excessive amount of organic manure should not be applied to crops (Liang et al. 2003) because it risks the toxic effects of reduced metabolic intermediates. Miah (1994) reported that only using organic sources could not provide the nutritional requirements of crops due to the slow release of plant nutrients from organic matter. When used appropriately, manure has nutritive and economic values (Khaliq, Abbasi, and Hussain 2006). Manures contain a large amount of nitrogenous compounds, which are easily mineralized to ammonia or nitrate (Jose and Michael 2012). Then, applications of manures stimulate macro- and micro-nutrient uptake, such as N, P, K, Ca, Mg, Fe, Mn, Zn, and Cu in crops (Adesemoye, Torbert, and Kloepper 2010). Thus, one potential way to decrease the negative environmental impacts and nutrient losses resulting from extreme use of CF is application of manures and a supplemental CF, to enhance macro-and micro-nutrient contents and the productivity of komatsuna. Commonly, many researchers apply manures on a weight basis and never consider the total nutrient (NPK) content or the mineralizable nutrients in them. Consequently, the nutrient demand of crops is not fully met by the applied manures. Because the manure has to undergo mineralization – process following utilized by the plant. A plant can get only mineralizable N (i.e., NH4 Nand – NO3 N converted from organic N by different soil microbes). Nutrient mineralization from applied manure depends on soil temperature, soil moisture, soil properties, manure characteristics, and micro- bial activity (Eghball et al. 2002). Since these environmental factors are difficult to predict for crop cultivation, an estimate of the rate of mineralization is useful for crop cultivation (Pettygrove, Heinrich, and Crohn 2009). Nishio (2007) reported that the amount of mineralizable nutrients in manure depends on its total N content (Table 1). In this experiment, we calculated the amount of three poultry manures (PM) and three cow manures (CM) (Table 2) based on the estimated mineralizable N (EMN) dependent on their total N according to Nishio (2007). Importantly, few researchers have considered the application of manures by EMN method with a supplemental CF. Thus, we hypothesized that the EMN from manures could supply enough N to komatsuna without adverse effects. Application of PM or CM in different quantities by EMN and two levels of the supplemental CF was intended to provide a greater advantage than that of CF alone in terms of growth parameters and DM of komatsuna (Brassica rapa L. var. wakana komatsuna) in an abandoned soil. This study also aims to evaluate the contents of nitrates, N, P, K, Ca, Mg, and Na in komatsuna for the safety of consumer in response to different PM or CM application by EMN and compare with solo CF.

2. Materials and methods 2.1. Experimental site A pot experiment was conducted in a glasshouse at Kyushu University, Japan (33370N, 130250E, 3 m above the sea level) from April to May (spring season) in 2017 (day length 13:28–14:10 h; temperature range 15–20 C min, 22–35 C max). 1728 K. MOE ET AL.

Table 2. Weight of manure, total NPK and EMN applied from manures and a supplemental chemical fertilizer. Total NPK applied from manure and CF (g pot–1) Mineralizable N (g pot–1) Manure applied –1 No. Treatments (g pot )DW NP2O5 K2O Manures (EMN) CF

1 NPK0 0.00 0.00 0.00 0.00 0.0 0.00 2CF50 0.00 0.25 0.25 0.25 0.0 0.25 3CF100 0.00 0.50 0.50 0.50 0.0 0.50 4CF150 0.00 0.75 0.75 0.75 0.0 0.75 5CF100PMH50 22.28 1.33 0.99 0.87 0.25 0.50 6CF100PMK50 10.27 1.00 0.73 0.61 0.25 0.50 7CF100PME50 38.58 1.33 2.39 1.28 0.25 0.50 8CF100CMG50 34.87 1.33 0.83 0.76 0.25 0.50 9CF100CMN50 40.65 1.33 0.85 0.80 0.25 0.50 10 CF100CMH50 39.87 1.33 0.91 0.76 0.25 0.50 11 CF50 PMH100 44.56 1.92 1.22 0.99 0.50 0.25 12 CF50 PMK100 20.53 1.25 0.72 0.47 0.50 0.25 13 CF50 PME100 77.16 1.92 4.04 1.82 0.50 0.25 14 CF50 CMG100 69.74 1.92 0.92 0.78 0.50 0.25 15 CF50 CMN100 81.30 1.92 0.96 0.86 0.50 0.25 16 CF50 CMH100 79.74 1.92 1.07 0.76 0.50 0.25 Subscript numbers of treatments show the amount of N or EMN applied as a percentage based on 0.5 g N pot1.DW¼ Dry weight basis, CF ¼ Chemical fertilizer, EMN ¼ Estimated mineralizable N.

2.2. Experimental design and treatments In a randomized complete block design (RCB) with three replications, three types of PM; Hakkou keifun (PMH), Keifun (PMK), and Ekono hakkou keifun (PME) and three types of CM; Gyufun (CMG), Neobi-ru (CMN), and Hakkou gyufun taihi (CMH) were integrated with either 100% or 50% CF. The experiment had 15 treatments (Table 2): 50%, 100%, and 150% CF; 100% CF þ 50% (PMH or PMK or PME), 100% CF þ 50% (CMG or CMN or CMH), 50% CF þ 100% (PMH or PMK or PME); and 50% CF þ 100% (CMG or CMN or CMH). The 100% CF treatment is þ equivalent to a rate of 0.5 g N (as (NH4)2SO4) and 0.5 g P2O5 and K2O (as KH2PO4 K2HPO4) pot 1. To obtain 0.5 g NPK per 20 mL, all CFs were dissolved thoroughly into a solution and were applied uniformly in all pots. The quantity of applied manure (Table 2) was calculated based on the estimated mineralization (%) of individual manures in the following equation according to Nishio (2007)(Table 1). In the NPK0 treatment, no fertilizers were applied. Application rate NðÞ g 104 Wt:ðÞg of manure ¼ Total NðÞ % MineralizationðÞ % Initially, the soil was sieved (2 cm) and mixed homogenously, and 4.0 kg of soil (dry basis) was weighed into a/5000 Wagner pot (AS ONE Corporation, Japan) for komatsuna cultivation. The soil was a Futsukaichi soil (Japanese Society of Pedologist 1984). Since komatsuna grows well on approximately neutral soil, the soil pH was adjusted to 6.5 by mixing 1.34 g of CaMg(CO3)2 < powder (size 0.25 mm) into each pot. Eventually, the CF solution, manures and CaMg(CO3)2 were thoroughly mixed with the soil and added to the pots. The soil in each pot was allowed to moisten for 2 days before planting. The amount of CaMg(CO3)2 was calculated based on the pH buffering capacity analysis of the soil using the buffering curve method (Date 1986).

2.3. Soil and organic manures analysis Before the experiment, the Futsukaichi soil was air-dried at room temperature, crushed by hand, and sifted through a 2 mm mesh sieve for analysis. Soil pH (using the 1:2.5 water extraction H2O method) (Thomas 1996) was measured using a pH meter (pH Meter HM-10P, DKK-TOA Corporation, Tokyo, Japan). For a total N and P analysis of the soil and manures, the samples – – were first digested using the salicylic acid H2SO4 hydrogen peroxide (H2O2) digestion method JOURNAL OF PLANT NUTRITION 1729

Table 3. Chemical compositions of poultry and cow manures. Total % (dry basis)

No. Sample Ash content (%) Moisture (%) NNH4–NNO3–NP2O5 K2ONH4–N/total N (%) 1. PMH 22.06 19.22 3.74 0.33 0.00 4.36 3.34 8.82 2. PMK 37.37 21.86 4.87 0.75 0.00 4.56 2.14 15.40 3. PME 61.95 17.60 2.16 0.06 0.00 9.82 4.06 2.78 4. CMG 31.48 46.40 2.39 0.19 0.00 1.91 1.52 7.95 5. CMN 31.21 51.12 2.05 0.08 0.00 1.74 1.50 3.90 6. CMH 29.60 54.98 2.09 0.07 0.00 2.06 1.28 3.35 Note: PMH ¼ Poultry manure (Hakkou keifun), PMK ¼ Poultry manure (Keifun), PME ¼ Poultry manure (Ekono hakkou keifun), CMG ¼ Cow manure (Gyufun), CMN ¼ Cow manure (Neobi-ru), CMH ¼ Cow manure ¼ (Hakkou gyufun taihi).

(Ohyama et al. 1991). Then, total N was analyzed using the indophenol method (Cataldo, Schrader, and Youngs 1974) and total P by the ascorbic acid method (Murphy and Riley 1962). For the manures, total K, calcium (Ca), magnesium (Mg), and sodium (Na) were analyzed using a digestion solution by an atomic absorption spectrophotometer (Z-5300, Hitachi, Tokyo, Japan). In addition, – manures were extracted using the hot water extraction method (Curtin et al. 2006), and NH4 Nwas – measured by the indophenol method (Cataldo, Schrader, and Youngs 1974)andNO3 N by the nitration of salicylic acid method (Cataldo et al. 1975)(Table 3). For cation exchange capacity and exchangeable cations of soil, the ammonium acetate shaking extraction method (Muramoto, Goto, and Ninaki 1992) was used, and the CEC and exchangeable cations were measured by an atomic absorption spectrophotometer (Z-5300, Hitachi). An analysis of mineralizable N was performed using the soil incubation method (Sahrawat 1983) followed by the indophenol method (Cataldo, Schrader, and Youngs 1974). The available P of soil samples was analyzed using Truog’smethod(Truog1930), followed by the ascorbic acid method (Murphy and Riley 1962).

2.4. Crop management Komatsuna (Brassica rapa L. var. wakana komatsuna), a typical Japanese leafy vegetable, was cultivated in this study. For each pot, 5 holes were dug and sown with three seeds in each hole. Then, the seeds were covered by a thin layer of fine soil and sprayed with water. At 10 days after sowing (DAS), the seedlings were thinned out to get one plant per hole. We maintained the moisture content at approximately 40% by adjusting the weight of soil and the volume of water until 21 DAS. At the later stage, as the water consumption by plants increased under hot weather, all pots were equally irrigated twice a day. However, there was no flooding or water draining out of pots. The moisture content in all pots did not exceed the maximum water holding capacity of soil (68.40 mL 100 g 1 dry soil).

2.5. Plant growth parameters Plant growth parameters (number of leaves, leaf length and SPAD value) were recorded at 3 day intervals after thinning until harvest. The SPAD value was measured using a chlorophyll meter (SPAD-502, Konica Minolta Sensing Inc., Osaka, Japan). One day before harvesting, the plant growth parameters were measured as a final record. At 32 DAS, the plants in each pot were harvested manually at the cotyledonary node above ground and oven-dried at 70 C for 72 h to determine shoot dry weight (g).

2.6. Nutrient content analysis The oven-dried plant samples were ground into fine powder using a Cyclotec 1093 sample mill (100–120 mesh, Tecator AB, Hoedanaes, Sweden). Approximately 100 mg of each sample was 1730 K. MOE ET AL.

– – digested using the salicylic acid H2SO4 hydrogen peroxide (H2O2) digestion method (Ohyama et al. 1991), and the total N and P were analyzed using the same methods described in the soil analysis. Total K, Ca, Mg, and Na were analyzed by the nitric acid digestion method (Niazi, Littlejohn, and Halls 1993) and measured by atomic absorption spectrophotometer (Z-5300, Hitachi). The nitrate contents of komatsuna were analyzed using a rapid colorimetric method with nitration of salicylic acid (Cataldo et al. 1975).

2.7. Statistical analysis The data were subjected to an analysis of variance (ANOVA). The mean values of treatments were compared using Tukey’s honestly significant difference (HSD) test at a 5% probability level using Statistix software (ver. 8.0; Analytical Software, Tallahassee, FL, USA).

3. Results 3.1. Soil analysis According to the results of the soil analysis, the Futsukaichi abandoned soil was slightly acidic, with a pH of 6.11, and there were low levels of total N (0.68 g kg 1), mineralizable N (0.06 mg H2O 1 1 1 100 g ), and total P2O5 (0.37 g kg ). Additionally, the available P (5.42 mg 100 g ) was low, 1 1 CEC was moderate (12.55 c molc kg ), and exchangeable K was low (0.37 c molc kg ). Thus, the Futsukaichi soil (sandy loam) displayed poor fertility due to it having been abandoned for approximately 10 years.

3.2. Plant growth parameters In all treatments, the number of komatsuna leaves gradually increased with time (Figure 1). The plants from all manure application showed a higher number of leaves than those from the CF treatment during the crop period. Throughout the crop period, with a supplemental CF100 or CF50, PMK maintained the highest leaf numbers. Similarly, the CF50CMG100 treatment also had higher leaf numbers. At harvest, the number of leaves was highest in the CF100PMK50 and CF50PMK100 treatments at 9.33 and 9.30 leaves per plant, respectively, followed by CF100CMG50 at 8.70, and the values were significantly different at p < 0.01 (Table 4). Regardless of containing þ the maximum NPK level, the CF150 produced a lower leaf number, 8.13 than in the CF100 PM50 or CM50. Because of the low fertility of the soil, the plants from the NPK0 treatment showed the lowest number of leaves. In the measurements of leaf length, no significant differences were observed before 23 DAS (Figure 1). After that stage, CF100PMK50,CF50PMK100,andCF100CMG50 dominated all the treatments in terms of leaf length. However, a similar value was recorded in CF100PMH50. Compared with all integrated treatments, in the CF100 and CF50 treatments, the shorter leaf lengths were seen in the plants of them. At harvest, the plants grown with CF150 showed the longest leaf length at 29.03 cm, which was similar to that in the CF50PMK100,CF100PMK50,andCF100CMG50 treatments, which were 28.19, < 26.88, and 26.87 cm, respectively (p 0.01) (Table 4). With the general application rate of CF100,the leaf length became shorter at 22.31 cm, and the shortest leaf length was 6.84 cm in the NPK0 treatment. The SPAD values, based on the chlorophyll content of the leaf, were obviously influenced by PM or CM throughout the crop period (Figure 1). Prior to 20 DAS, no differences in SPAD values were actually observed, but after that stage, the CF150 treatment showed a higher SPAD value, followed by similar values in the CF100PMK50 and CF50PMK100 treatments. At the rate of CF100, the komatsuna plants showed lower SPAD values on abandoned soil. One day before harvest, the JOURNAL OF PLANT NUTRITION 1731

Figure 1. Changes of leaf number per plant, leaf length (cm) and SPAD values of komatsuna as affected by manure application depending on EMN and a supplemental CF.

Table 4. No. of leaf per plant, leaf length (cm) and SPAD values of komatsuna at harvest. Treatments No. of leaf per plant Leaf length (cm) SPAD values

NPK0 3.63 e 6.84 f 29.26 d CF50 6.60 d 17.97 e 42.06 c CF100 8.07 abc 22.31 de 47.50 abc CF150 8.13 abc 29.03 a 54.36 a CF100 PMH50 8.37 abc 25.07 abcd 47.36 abc CF100 PMK50 9.33 a 26.88 abc 51.16 ab CF100 PME50 8.37 abc 23.97 bcd 48.90 abc CF100 CMG50 8.70 abc 26.87 abc 49.10 abc CF100 CMN50 8.43 abc 24.47 bcd 45.50 bc CF100 CMH50 8.63 abc 24.87 abcd 45.00 bc CF50 PMH100 8.07 abc 26.12 abcd 47.60 abc CF50 PMK100 9.30 ab 28.19 ab 51.86 ab CF50 PME100 8.20 abc 24.97 abcd 45.20 bc CF50 CMG100 8.63 abc 24.74 abcd 47.60 abc CF50 CMN100 7.47 cd 23.94 bcd 46.66 abc CF50 CMH100 7.93 bcd 22.39 cde 48.56 abc Tukey value 0.05 1.38 4.55 8.14 Source of variance (Pr > F) Integrated treatment <0.0001 <0.0001 <0.0001 CV (%) 5.69 6.32 5.73 Means followed by the same letter in each column are not significantly different in Tukey’s HSD tests (p < 0.05). Subscript numbers of treatments show the amount of N or EMN applied as a percentage based on 0.5 g N pot1.

CF150 treatment had the highest SPAD value at 54.36 (Table 4), which was statistically similar to þ the CF100PMK50 and CF50PMK100 treatments at 51.16 and 51.86, respectively. Generally, CF100 PM50 showed higher values. CF100CMG50 also showed a similar SPAD value, 49.10. However, severe N deficiency symptoms occurred on the plants in the CF50 and NPK0 treatments, which had lower SPAD values, 42.06 and 29.26, respectively. 1732 K. MOE ET AL.

Figure 2. Dry matter (g pot1) of komatsuna as affected by manure application depending on EMN and a supplemental CF. The histograms with the same letter are not significantly different by the Tukey HSD test (p < 0.05). EMN ¼ Estimated mineralizable N.

< With increased NPK levels from CF, DM increased until CF100 (p 0.01) (Figure 2). Over this level, no further DM increase was seen between CF100 and CF150. The maximum DM values, 1 29.76 and 30.06 g pot were obtained from CF100PMK50 and CF50PMK100, respectively. þ Generally, a higher DM was obtained in CF100 PM50 or CM50 compared with that of the CF150 1 þ treatment at 21.06 g pot . However, in the integration of CF50 PM100 or CM100, the DM was obviously low, except for the PMK and CMG treatments. On an abandoned soil, a general appli- 1 cation rate of CF100 showed lower DM, 20.36 g pot .

3.3. N, P, K, Ca, Mg, Na and nitrate contents of komatsuna The N content of komatsuna leaves linearly increased with the increase in CF levels (Table 5). The 1 highest N content, 23.81 mg g accumulated in the leaves in the CF150 treatment. However, similar contents were achieved by the plants in the CF100PMK50 and CF100PMH50 treatments, which had 21.66 and 19.23 mg g 1, respectively. The CF þ PM treatment generally could supply a higher N con- þ tent to komatsuna than CF CM, except for PME. Integrated with CF100 or CF50, the plants grown in 1 þ PME had lower leaf N contents of 14.62 or 16.85 mg g , respectively. Nonetheless, CF50 PM100 1 þ obtained higher values, similar to the 23.81 mg g observed in the CF150 treatment, but the CF50 1 CM100 treatment did not. Clearly, the plants from CF100 had lower N contents with 15.72 mg g . The P content results showed similar trends as the N contents (Table 5). The CF150 supplied 1 more P to komatsuna, showing a greater content of 13.50 mg P g .CF100PMH50 and 1 CF100PMK50 also showed higher P contents at 12.91 and 12.61 mg g , respectively. However, PME, which contained the highest total P (9.82%), did not. Integrated with CF50, PMH100 and 1 PMK100 also showed higher P at 12.92 and 12.58 mg g , respectively. Generally, the plants grown JOURNAL OF PLANT NUTRITION 1733

Table 5. Contents of N, P, K, Ca, Mg, and Na (mg g1) of komatsuna affected by manure application depending on EMN and a supplemental CF. Treatments N P K Ca Mg Na

NPK0 11.76 d 8.78 bc 21.13 f 23.58 abc 2.76 abc 0.02 d CF50 12.10 cd 8.22 c 25.44 ef 20.96 bc 2.01 cd 1.22 bcd CF100 15.72 bcd 9.49 abc 24.72 ef 23.61 abc 1.83 d 1.35 bcd CF150 23.81 a 13.50 a 38.35 bcdef 28.44 a 2.14 bcd 4.54 a CF100 PMH50 19.23 abc 12.91 ab 50.27 abcd 23.76 abc 2.36 abcd 1.55 bcd CF100 PMK50 21.66 ab 12.61 ab 31.53 cdef 26.85 ab 2.39 abcd 2.98 ab CF100 PME50 14.62 bcd 10.31 abc 44.30 abcde 19.86 c 2.62 abc 1.02 bcd CF100 CMG50 16.58 abcd 11.24 abc 49.81 abcd 23.69 abc 2.28 abcd 2.49 abc CF100 CMN50 16.73 abcd 11.08 abc 51.27 abc 27.08 ab 2.57 abcd 1.49 bcd CF100 CMH50 13.74 cd 10.50 abc 42.39 abcde 18.67 c 2.37 abcd 0.80 cd CF50 PMH100 18.72 abcd 12.92 a 55.76 ab 21.17 bc 2.15 bcd 1.37 bcd CF50 PMK100 18.41 abcd 12.58 ab 30.33 def 28.37 a 2.40 abcd 2.75 abc CF50 PME100 16.85 abcd 10.12 bc 55.24 ab 19.13 c 2.98 a 1.96 bcd CF50 CMG100 16.17 bcd 11.71 abc 45.83 abcd 20.16 c 2.31 abcd 2.26 bc CF50 CMN100 18.88 abcd 11.18 abc 61.06 a 20.14 c 2.81 ab 2.11 bcd CF50 CMH100 14.07 cd 10.86 abc 40.98 abcdef 17.74 c 2.53 abcd 0.75 cd Tukey value 0.05 7.29 4.13 20.19 6.65 0.79 2.11 Source of variance (Pr > F) Integrated treatments <0.0001 0.0002 <0.0001 <0.0001 0.0006 <0.0001 CV (%) 14.27 11.83 15.89 9.64 10.88 8.79 Means followed by the same letter in each column are not significantly different in Tukey’s HSD tests (p < 0.05). Subscript numbers of treatments show the amount of N or EMN applied as a percentage based on 0.5 g N pot1.CF¼ Chemical fertilizer, EMN ¼ Estimated mineralizable N. with CF þ PM accumulated higher P contents than those from the CF þ CM treatment. Inferior P contents were observed in the leaves from the CF100,CF50 and NPK0 treatments at 9.49, 8.22, and 8.78 mg g 1, respectively. Regarding the K content of komatsuna, the above tendency was changed (Table 5). The solo 1 application of CF, especially CF150, resulted in lower K contents, 38.35 mg g , and the integrated treatments accumulated higher K values in komatsuna. However, the K contents were signifi- 1 1 cantly lower at 31.53 mg g in CF100PMK50 and 30.33 mg g in CF50PMK100, but the other integrated treatments significantly increased the K content of komatsuna. For the Ca content of komatsuna, a linear increase was observed with increased CF levels (Table 5). Statistically, the highest Ca content, 28.44 mg g 1 was seen in the plants from the CF150 treatment, and the CF100PMK50,CF100CMN50, and CF50PMK100 treatments had similar val- 1 þ ues: 26.85, 27.08, and 28.37 mg g , respectively. However, CF50 PM100 or CM100 tended to accumulate less Ca, except for the PMK. Since the soil had a moderate content of exchangeable Ca, the plants in the NPK0 treatment showed a relatively higher Ca content, 23.58, which was 1 similar to the 23.61 mg g in the CF100 treatment. The Mg content of komatsuna resulted in a different tendency (Table 5). The application of CF alone resulted in a lower Mg content compared with integrated treatments. Since PME contained a higher Mg concentration (1.25%), the 1 plants could have had higher Mg contents at 2.62 and 2.98 mg g when integrated with CF100 or 1 CF50, respectively. Similarly, the NPK0 plants also had a similar value, 2.76 mg g due to the higher Mg content in the soil. Evidently, all integrated treatments showed higher Mg contents in komatsuna, but individual applications of CF resulted in a lower value. Surprisingly, the Na contents of komatsuna were superficially increased with the increased CF level (Table 5). The CF150 treatment had the highest Na content, 4.54 mg g 1 in the plants, but not all integrated treatments did. Among the integrated treatments, CF þ PMK had a relatively higher Na content, 2.98 and 2.75 mg g 1 in the plants, followed by CF þ CMG. Since the Futsukaichi soil has low exchange- 1 able Na, the plants in the NPK0 treatment showed a lower Na content at 0.02 mg g . As a quality parameter, the nitrate content of komatsuna was significantly decreased by manure application, compared with CF only (p < 0.01) (Figure 3). The nitrate content linearly increased with the increase in CF level, resulting in the highest nitrate content (0.75 mg g 1)in 1734 K. MOE ET AL.

1 Figure 3. NO3–N content (mg g ) of komatsuna as affected by manure application depending on EMN and a supplemental CF. The histograms with the same letter are not significantly different by the Tukey HSD test (p < 0.05). EMN ¼ Estimated mineralizable N. the plants from the CF150 treatment. Obviously, all integrated treatments showed lower nitrate contents than treatments with CF. The CF100PMK50 and CF100CMN50 treatments accumulated the lowest nitrate contents, 0.55 and 0.50 mg g 1 in the plants, respectively. Obviously, nitrate contents were lower when PM100 or CM100 were applied with CF50, especially CMG. Application of PMs or CMs and a supplemental CF could significantly suppress nitrate accumulation in komatsuna.

4. Discussion 4.1. Effect of manure application (EMN method) and a supplemental CF on growth parameters of komatsuna In this study, we applied PM and CM as EMN calculated from total N content of manure. In fact, the total N applied from manures were same except PMK (Table 2), because mineralizable percentage is the same for total N (2–4%) that contained by other manures. We did not adjust P and K level of each treatment. Obviously, the PMK and a supplemental CF achieved higher growth parameters than CF150, followed by CMG. Before 20 DAS, no differences were seen in growth parameters among integrated treatments because manures released nutrients slowly under mineralization. After that stage, the plants from the CF þ PMK treatment sustained a higher SPAD value, reflective of the mineralizable N supplied. As a result, the plants from the CF þ PMK treatment produced a higher leaf number with a longer average leaf length, followed by those from the CF þ CMG treatment. These two manure has higher total N and higher ratios of NH4-N/Total N. Certainly, the CF þ PMK treatment, which contained high total N (4%) and NH4-N/Total N (15.40%) contents, could provide a steady supply of N, resulting in higher growth parameters and DM than the CF150 treatment on an abandoned soil. Abbasi et al. (2007) also highlighted that manure with a high total N content resulted in a high net N mineralization. JOURNAL OF PLANT NUTRITION 1735

In fact, with the same N rate, all PM or CM and a supplemental CF resulted in higher DM than that the only CF150 treatment. However, the DM of komatsuna decreased when the CF50 was integrated with PM100 or CM100, except if PMH, PMK or CMG were used. These manures have relatively high total N contents. Although we applied manure by equal EMN, nutrient mineralization ratios differed for different types of manure (Eghball et al. 2002). The quantity or ratio of manures is considerable when supplemented with CF in abandoned soil, especially when using manures with low total N. It is well documented that the addition of large amounts of organic manures to soils increases the immobilization process (Barakat, Ismail, and Ehsan 2016), especially in manures with high C:N ratios due to slower decomposition and nitrification (Khalil, Hossain, and Schmidhalter 2005). In this study, the use of CF has both positive and negative effects on plant growth parameters – – in komatsuna. After CF application, the NH4 N and NO3 N concentrations in the soil may increase. At an early growth stage, the young plants could not absorb all dissolved nutrients. As the plants grew, the nutrient requirements increased, but the N concentration in the soil gradually decreased. The nutrients supplied by CF did not coincide with crop requirements. This phenom- enon occurred in the plants from the CF only treatment. Consequently, the komatsuna plants had resulted lower leaf numbers and DM, even in the CF150 treatment. Application of CF alone might result in losses and a low N recovery (Liu et al. 2016), increases in the risk of environmental pollution by eutrophication and economic losses (Akoumianakis et al. 2011).

4.2. Correlation between nutrients contents and growth parameters of komatsuna affected by manure application (EMN method) and a supplemental CF In all treatments, the N content of komatsuna was positively and significantly correlated with the DM of komatsuna at p < 0.05 (Table 6). Because N is the building block for amino acids and pro- tein synthesis as well as chlorophyll composition (Hassan et al. 2012), N is the main element for enhancing P and K absorption by plants (Newbery et al. 1995). As CF is easily dissolved in the soil, the N and P contents in the leaf tissue were increased by CFs. There is a good correlation coefficient (r)¼0.63 between DM and N, and r ¼ 0.54 between DM and P content, when affected by CF only (Table 6). Nonetheless, the increased use of CF created higher nitrate contents in the plants. Goh and Haynes (1977) reported that CF was probably transformed to nitrate in the soils before absorption by plants. When the applied N exceeds the demand of plant, plants are no longer able to absorb it, and N then builds up in the soil, mostly as nitrates (Nosengo 2003). This causes imbalance of nutrients in the soil and increases the nitrate level in ground-water supplies (NAAS 2005) which influences the nitrate content of plants (Dapoigny et al. 2000), especially the leafy vegetables. The CF is readily soluble and absorbed into the plant, which tends to result in higher nitrate/nitrite levels in the leaves of vegetables (Sonmez,€ Kaplan, and Sonmez€ 2007). The ¼ – DM formation was significantly correlated (r 0.80) with NO3 N content in komatsuna when only CF was applied. The finding is in line with Karaman et al. (2000) who found that the nitrate content in vegetables increased with increased N application by CF. However, the nitrate contents resulting from CF in this study are lower than the detrimental level for human beings. Forlani

Table 6. Pearson correlation between dry matter (DM) and nutrients content of komatsuna as affected by CF only, CF þ PM and CF þ CM. Pearson correlation coefficient (r) values between DM and nutrients content

Treatments NP KCaMgNaNO3-N ns ns CF only 0.63 0.54 0.55 0.27 –0.83 0.68 0.80 ns ns ns ns CF þ PM 0.38 0.41 0.29 0.16 –0.17 0.38 0.13 ns ns ns ns CF þ CM 0.31 0.32 0.18 0.18 –0.23 0.35 0.09 Note: **, *significant at 1% and 5% probability, ns ¼ non-significance. CF ¼ Chemical fertilizer, PM ¼ Poultry manure, CM ¼ Cow manure. 1736 K. MOE ET AL.

– et al. (1997) found that the maximum limit for NO3 N in fresh vegetables that is safe for human consumption is 167 mg kg 1. In addition, a strong relationship (r ¼ 0.69) between DM and Na content shows that the application of CF can increase not only DM but also the Na content. Using CF only, the K supply might be low, and the K content of komatsuna in the CF only treatment was lower. On the other hand, the application of PM or CM treatments showed good correlations with r ¼ 0.38 and r ¼ 0.31, respectively, between DM and N content (Table 6). The N amounts that are mineralized from PMK and CMG matched the crop requirement, resulting in higher DM. In addition to N, the DM was also positively associated with the P content of plants. As PMK and CMG include a moderate amount of P, the plants accumulated higher P contents in the leaves and obtained a higher DM. The finding is consistent with Toor and Bahl (1997) who found that soil incorporation of manure and CF causes a continuous increase in available P in soil for a longer period, thus increasing DM of the crop. However, mineralization of P and K in manures also depends on the total N in them. Despite the highest total P (9.82%) and K (4.06%), PME provided lower P and K contents in plant. Because the mineralization of N, P, and K was lowered due to having a low total N (2.16%) in PME. K contents in komatsuna are not directly related to DM, showing no correlation in any treatment. This result is explained by the CF þ PMK treatment, in which the K content of leaves was low but the DM was maximized. In fact, K has many physiological functions that are indirectly related to DM accumulation in plants, such as the acti- vation of K-dependent enzymes assisting permeation of membranes and transportation of sub- stances, the promotion of photophosphorylation, the neutralization of electrical charge by anions in cells, the regulation of water movement and the maintenance of cell turgor and stomatal open- ing (Horiguchi 1984). At the same time, the CF þ PM or CM suppressed accumulation of nitrate in the leaves of komatsuna, especially the PMK, CMN, and CMG treatments. There is no correlation between þ DM and NO3-N contents after CF PM or CM treatment applications. This result is in agree- ment with Winchester, Huskins, and Ying (2009) who found that the nitrate content was statistically lower in the treatment with organic fertilizer compared to inorganic fertilizer. Organic manures that mineralize slowly lead to less nitrate accumulation in vegetables than materials that mineralize more rapidly and therefore release more nitrate (Barker 1975). Hammad et al. (2007) demonstrated that N sources or fertilizers could affect the accumulation of nitrate in vegetables. Toxicity of nitrate to humans can manifest as headaches, syncope, vertigo and discoloration that is visible in the fingers or lips (Winchester, Huskins, and Ying 2009). Therefore, CF should be wisely applied and should be together with manures in vegetables, considering human health as well as environmental pollution, safety and sustainability.

4.3. Effect of manure application (EMN method) and a supplemental CF on micro-nutrient contents in komatsuna In this study, the Ca and Na contents of komatsuna gradually increased with the increase in CF, but the Mg content did not. At the same time, the plants grown with CF þ PMK had a Ca con- þ tent as high as the CF150 treatment. For Mg, all CF PM or CM treatments show a higher content than the CF treatment but a similar content to the NPK0 treatment. As the findings of Marschner (1995) indicate, since the used soil had a low K concentration, it may encourage an increase in the uptake of Mg and Ca. Hasegawa and Yoneyama (1995) reported that when the K supply decreased, komatsuna showed a drastic decrease in K concentrations and increase in Na concentrations in the plant tissues and a slight increase in Mg and Ca contents in the leaf. Significantly, the integrated CF and PM or CM treatments resulted in higher Ca and Mg contents compared with the solo application of CF and suppressed nitrate and Na contents. Plants grown with organic fertilizers are reported to have higher micronutrient contents than those grown with JOURNAL OF PLANT NUTRITION 1737

CF (Ibrahim and Jaafar 2011). Additionally, plants either directly or indirectly need macro- and micro-nutrients for many chemical reactions in the cells; thus, the integrated organic and inorganic fertilizers provided enough nutrients for crop growth and development, ensuring higher production (Bimova and Pokluda 2009).

5. Conclusion By application of three PM and three CM depending on EMN and a supplemental CF, growth parameter and dry matter was highest in the plants grown with PMK (total N > 4%), whereas nutrients content were also highest in komatsuna cultivated in an abandoned soil. The PMK that – has higher NH4 N/Total N (15.40%) suppressed nitrate content of komatsuna. In the first cultivation of komatsuna on an abandoned soil, the 50% of PM or CM supplemented with 100% of CF generated the higher DM of komatsuna and enhanced macro- and micro-nutrients. However, the plants grown with CF accumulated higher nitrate content and provided lower nutrient contents and DM than those of PMK. For these results, a higher total N (>4%) concentration of manure led to increase nutrient availability and promoted nutrient contents, DM, and depressed nitrate content of komatsuna, comparable with solo CF in an abandoned soil. The C:N ratio was obviously important for mineralization of nutrient in manure. Further studies on different manures or compost or organic fertilizer in solid form or liquid form should perform using EMN method for enhancing nutrient content and dry matter, highlighting quality parameters in different vegetable crops cultivated in various soil types.

Acknowledgments This study was supported by the Japanese Government (MEXT) Scholarship Program 2016–2019, Japan.

Disclosure statement

I have disclosed that there are no conflicts of interest regarding publication of this article.

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