Effects of Organic Fertilizer on Bok Choy Growth and Quality in Hydroponic Cultures

: Effects of corn steep liquor (organic fertilizer, OF) and conventional chemical fertilizer (CF) on the growth and yield of bok choy ( Brassica rapa var. chinensis ) in summer and autumn hydroponic growing systems were compared. When OF and CF were applied with the same amount of total nitrogen in summer cultivation, there was no signiﬁcant difference between yields; however, the growth rate in OF was slower than in CF. When OF was applied with twice the amount of nitrogen in CF (OF2), bok choy growth and yield were signiﬁcantly inhibited in summer cultivation, likely owing to dissolved oxygen deﬁciency and different rates of nitriﬁcation and nitrogen absorbance by the plant root. Although the contents of potassium, calcium, and magnesium in bok choy showed no difference among the three treatments in both cultivation seasons, the carbon/nitrogen ratio tended to be higher in OF and OF2 than in CF. Lower nitric acid and higher ascorbic acid content was found in OF and OF2 than in CF. Overall, our results suggest that a comparable yield is expected by using the same nitrogen amount with a conventional recipe of chemical fertilization in autumn cultivation. However, further improvement of hydroponic management is needed in summer cultivation. Contributions:


Introduction
Chemical fertilizers are commonly applied in hydroponics cultivation systems for Chinese vegetables including bok choy (Brassica rapa var. chinensis) [1] during summer and autumn. However, the overuse of chemical fertilizers causes fertilizer-derived waste, growth inhibition, and soft rot damage to the cultivated vegetables. Organic fertilizers have been expected to control such problems through providing an environmentally friendly growing environment. Nevertheless, the decomposition of organic fertilizers in hydroponic solutions is often incomplete, leading to an accumulation of phytotoxic compounds such as ammonium ions and other substances with high molecular weight. In addition, the application of organic fertilizers in hydroponic solutions leads to the decline culture, Ibaraki University, Japan. Bok choy "Seitei Chingensai" (Sakata seeds, Yokohama, Japan) seedlings were grown for two weeks in plastic trays before planting. The treatments were chemical fertilizer (CF), organic fertilizer (OF), and organic fertilizer with twice the application rate of nitrogen (OF2). The CF treatment was conventional hydroponics with a half unit of Otsuka A formula (N-P 2 O 5 -K 2 O: 260-120-405 mg kg −1 ; Otsuka Agri Techno, Japan, current name: OAT A formula (CF), OAT Agrio, Tokyo, Japan). A half unit is commonly applied in hydroponics for leaf vegetables in Japan. The OF treatment was organic hydroponics with corn steep liquor (CSL) (Nature Aid, N-P 2 O 5 -K 2 O: 3-3-2%; Sakata seeds, Japan) in which the applied nitrogen amount (260 mg kg −1 initial rate) was identical to that in the CF treatment. The OF2 treatment was organic hydroponics with CSL in which the nitrogen amount was twice (520 mg kg −1 initial rate) that in CF treatment. Each treatment was conducted in triplicate using independent hydroponic lines.

Hydroponic System
The hydroponic system was based on the deep flow technique. Lines were 720 × 65 × 6.5 cm (length × width × depth) with no slope. Nutrient solution was continuously circulated between a 300-L cultivation vessel and a 200-L underground tank by a sludge pump at 7 L min −1 . Bok choy seedlings were transplanted in the top panel of the cultivation tank at a density of 15 cm × 18 cm in 4 lines. Water lines were washed every week in OF and OF2 treatments to eliminate the clogging caused by CSL. Electric conductivity (EC) and pH of the nutrient solution in each tank were measured using an EC meter (CM-14P; TOA DKK, Tokyo, Japan) and a pH meter (HI 98129; Hanna Instruments, Woonsocket, RI, USA), respectively. Values of EC and pH were in the range suggested by Singh and Dunn [23] for hydroponic production of leafy greens (Table 1).

Cultivation
Seeding and planting were conducted on 6 and 20 July 2011, for summer cultivation, and on 12 and 26 October 2011, for autumn cultivation, respectively. The side windows and doors of the greenhouse were fully open except in storm conditions. Leaf length was monitored on randomly selected plants. When mean leaf length reached 20-25 cm in each treatment, all plants in all treatments were harvested. When leaves did not reach the required length, plants were harvested at the end of the growth period.

Measurements
Data on air temperature and sunshine hours were obtained from the Meteorological Agency for the Automated Meteorological Data Acquisition System located at the Tsuchiura observation point located 7.9 km north of the experimental greenhouse. Nitric ion and ammonium ion concentrations in the nutrient solutions were measured every week using a reflection photometer (RQ flex 2; Merck, Darmstadt, Germany). At harvest, the dry weights of the leaf and root of bok choy were determined from three plants per plot, and the values were calculated as the leaf/root ratio. Chlorophyll content was measured as a SPAD reading using a chlorophyll meter (SPAD-502 plus; Konica Minolta, Tokyo, Japan). Nitrate ion and ascorbic acid content in the leaves was measured using ion-exchanged water and 5% metaphosphoric acid as extracting solutions, respectively. Briefly, plant leaf samples were homogenized to pulp with the same amount of extracting solution immediately after harvest and centrifuged at 2240× g for 10 min at room temperature. Nitrate ion and ascorbic acid were then measured from the supernatant. Chlorophyll, nitrate ion, and ascorbic acid content were determined in triplicate.
Carbon (C) and N content in leaves and their ratio (C/N) were measured as the total organic C and total N of 60 • C dried and pulverized samples using an automatic C/N analyzer (JM3000N C/N; J-SCIENCE, Kyoto, Japan). The content of K, calcium (Ca), and magnesium (Mg) in the bok choy leaves was measured using the wet ash method in an atomic absorption spectrophotometer (SPCA-6210; Shimadzu, Kyoto, Japan). Briefly, leaf samples (200 mg) were dissolved in a solution containing 5 mL nitric acid, 2 mL perchloric acid, and 1 mL sulfuric acid. This solution was diluted 50-fold with the ion exchange water, and 1 mL of this diluted solution was then added to 1 mL acetone and 8 mL pure water; the final mixture was used to measure K, Ca, and Mg content. The levels of amino acids were evaluated in the third mature leaf from the outside of each plant. The leaves were frozen in liquid N and immediately hand-ground by mortar and pestle to obtain a powder, which was then mixed with 1% sulfosalicylic acid at a 50-fold dilution ratio. The mixture was centrifuged at 16,610× g and 5 • C for 10 min, and the resulting supernatant was filtered through a syringe filter (pore size: 0.45 µm); the filtrate was analyzed in a fully automatic amino acid analyzer (JLC-500/V2; JEOL, Tokyo, Japan). C, N, K, Ca, Mg, and amino acids were measured in triplicate per sample.
The concentration of dissolved oxygen was measured in a dissolved oxygen meter (DO-24P; TOA DKK, Japan) at four randomly selected points of the cultivation vessel from 6 to 27 August 2011 (summer) and from 10 November to 8 December 2011 (autumn), thus including the post-cultivation periods.

Inoculation Test of Gray Mold
The inoculation method of Chinta et al. [16] was applied to some plants after the autumn harvest. A mycelium disc of gray mold (Botrytis cinerea) was inoculated on the largest leaf of each of the 10 plants selected per treatment on 15 January 2012. The longitudinal (L) and transverse (T) diameters of each lesion were measured and disease incidence was calculated as (L + T)/2.

Statistical Analyses
All statistical analyses were conducted using the Ekuseru-Toukei 2010 software package (Social Survey Research Information Co., Ltd., Tokyo, Japan). Data were first screened for homogeneity of variances using Bartlett's test and then tested for statistical differences using Tukey's multiple range. The Student's t-test was also employed for pairwise comparison. The significance level for all tests was set at 0.05.

Growth of Bok Choy
Mean air temperature and hours of sunshine were 26.1 • C and 5.5 h, respectively, in summer cultivation and 13.3 • C and 5.3 h, respectively, in autumn cultivation ( Figure 1). The initial height and number of leaves of bok choy plants were 3.6 cm and 3.0, respectively, in summer cultivation and 4.1 cm and 3.0, respectively, in autumn cultivation ( Figure 2). Growth was slower in OF than CF in summer cultivation, and harvest in OF (30 August) was one week later than in CF (23 August) (Figure 2A). The plants in OF2 never reached harvest size by the end of summer cultivation; therefore, no plants were left in this treatment at the end of the experiment. In the autumn cultivation, plant growth was not different among treatments ( Figure 2B); harvest size was reached at the same time (23 November) in all three treatments. longitudinal (L) and transverse (T) diameters of each lesion were measured and disease incidence was calculated as (L+T)/2.

Statistical Analyses
All statistical analyses were conducted using the Ekuseru-Toukei 2010 software package (Social Survey Research Information Co., Ltd. Tokyo, Japan). Data were first screened for homogeneity of variances using Bartlett's test and then tested for statistical differences using Tukey's multiple range. The Student's t-test was also employed for pairwise comparison. The significance level for all tests was set at 0.05.

Growth of Bok Choy
Mean air temperature and hours of sunshine were 26.1 °C and 5.5 h, respectively, in summer cultivation and 13.3 °C and 5.3 h, respectively, in autumn cultivation ( Figure 1). The initial height and number of leaves of bok choy plants were 3.6 cm and 3.0, respectively, in summer cultivation and 4.1 cm and 3.0, respectively, in autumn cultivation ( Figure 2). Growth was slower in OF than CF in summer cultivation, and harvest in OF (30 August) was one week later than in CF (23 August) ( Figure 2A). The plants in OF2 never reached harvest size by the end of summer cultivation; therefore, no plants were left in this treatment at the end of the experiment. In the autumn cultivation, plant growth was not different among treatments ( Figure 2B); harvest size was reached at the same time (23 November) in all three treatments.  In the summer cultivation, the dry weights of bok choy leaves and roots were significantly lower in OF2 than in OF and CF (Table 2). Nonetheless, there was no difference in the leaf/root ratio and chlorophyll content because of the considerable variations between individuals, especially in OF2. Leaf length (cm) In the summer cultivation, the dry weights of bok choy leaves and roots were significantly lower in OF2 than in OF and CF (Table 2). Nonetheless, there was no difference in the leaf/root ratio and chlorophyll content because of the considerable variations between individuals, especially in OF2. In the autumn cultivation, treatments did not affect the dry weight of leaves. On the other hand, OF significantly enhanced the root dry weight compared with CF; OF2 showed intermediate values of root dry weight. According to the effects on leaves and roots, the leaf/root ratio was highest in CF, followed by that in OF2 and OF. Chlorophyll content was highest in OF2 followed by OF and CF.

Chemical Composition of Harvested Bok Choy
In the summer cultivation, the bok choy plants in CF showed higher nitrate content than those in the other treatments (Table 3). Ascorbic acid was highest in OF2, followed by OF and CF, and these differences were significant. There was no difference in K, Ca, and Mg among treatments. The OF and OF2 treatments resulted in high and low content of C and N, respectively, leading to high C/N ratios, compared with CF. During the autumn cultivation, nitrate and ascorbic acid content were increased and decreased, respectively, in CF compared with the other treatments. Similar to the summer cultivation, treatment had no significant effect on K, Ca, and Mg content. Although there was no difference in C content among treatments, N content varied. Thus, the C/N ratio was highest in OF followed by OF2 and CF.
The six amino acids selected (aspartic acid, threonine, serine, glutamic acid, glycine, and valine) were significantly different among the three treatments ( Figure 3). In the summer cultivation, OF2 increased the content of these six amino acids compared with the other two treatments, except for glutamic acid ( Figure 3A). Glutamic acid was significantly higher in OF2 than in the other two treatments in the autumn cultivation ( Figure 3B).

Nitrogen Concentration in the Hydroponic Solution
Changes in N concentration (i.e., considering nitrate and ammonium ions) during each cultivation period revealed that in both cultivation periods, nitrate concentration was higher in CF than in OF and OF2 ( Figure 4A,C). Ammonium ion was detected only in OF2, wherein its concentration fluctuated ( Figure 4B,D).
Agronomy 2021, 11, x FOR PEER REVIEW 10 of 17 Changes in N concentration (i.e., considering nitrate and ammonium ions) during each cultivation period revealed that in both cultivation periods, nitrate concentration was higher in CF than in OF and OF2 ( Figure 4A,C). Ammonium ion was detected only in OF2, wherein its concentration fluctuated ( Figure 4B,D).  The level of dissolved oxygen in OF2 was significantly decreased compared with the other two treatments in both summer and autumn cultivation periods ( Figure 5). The level of dissolved oxygen in OF2 was significantly decreased compared with the other two treatments in both summer and autumn cultivation periods ( Figure 5).

Inoculation Test of Gray Mold
The leaves inoculated with gray mold showed water-soaked lesions that gradually spread from mycelial discs. Four days after inoculation, a clear difference in lesion diameter was noticed between the treatments, with leaves in CF and OF showing the largest and smallest diameters, respectively ( Figure 6A). Whereas leaves cultivated in CF and OF turned yellow, leaves cultivated in OF2 only showed a yellowish ring around the mycelial disk of gray mold ( Figure 6B). on vertical columns indicate standard error (n = 3). Identical letters between bars indicate no significant difference (p < 0.05) among treatments according to Tukey's multiple range test. OF (organic fertilizer): corn steep liquor (CSL) applied as organic fertilizer at the same amount of total nitrogen as that in the half unit of Otsuka A formula; OF2: CSL applied at twice the amount of total nitrogen in OF; CF (chemical fertilizer): half unit of Otsuka A formula.

Inoculation Test of Gray Mold
The leaves inoculated with gray mold showed water-soaked lesions that gradually spread from mycelial discs. Four days after inoculation, a clear difference in lesion diameter was noticed between the treatments, with leaves in CF and OF showing the largest and smallest diameters, respectively ( Figure 6A). Whereas leaves cultivated in CF and OF turned yellow, leaves cultivated in OF2 only showed a yellowish ring around the mycelial disk of gray mold ( Figure 6B).

Nitrification by the Multiple Parallel Mineralization Method
This study used the multiple parallel mineralization method, which has been established by Shinohara [6], by growing bok choy in OF and CF. Organic hydroponics were set up 14 days before bok choy transplantation to allow the nitrification process. The remarkably lower concentrations of nitrate ions in hydroponic solutions with OF and OF2 than in the hydroponic solution with CF in both summer and autumn cultivations indicated that nitrification had occurred. However, most of the N contained in CSL was in forms that had high molecular weights, such as proteins and peptides [9,10]. These N forms are either not absorbed or inefficiently absorbed by plants [18], and thus, the nitric acid and N content of bok choy cultivated in OF and OF2 was considerably lower than that of the bok choy cultivated in CF. However, the addition of a small amount of CSL every three days was expected to enable the development of biofilm on the root surface of plants, allowing the microorganisms that formed the biofilm to mineralize organic compounds and provide absorbable N to plant roots [8]. Moreover, microorganisms forming the biofilm may absorb organic N and mineralize organic N, thereby directly providing absorbable N to plant roots [13], despite N absorption not being upregulated in OF and OF2 compared with CF. Contrary to the nitrate ion, the ammonium ion was only detected in OF2 during summer cultivation. An accumulation of ammonium ions as that shown in OF2 can negatively affect the growth of bok choy. The dissolved oxygen level in

Nitrification by the Multiple Parallel Mineralization Method
This study used the multiple parallel mineralization method, which has been established by Shinohara [6], by growing bok choy in OF and CF. Organic hydroponics were set up 14 days before bok choy transplantation to allow the nitrification process. The remarkably lower concentrations of nitrate ions in hydroponic solutions with OF and OF2 than in the hydroponic solution with CF in both summer and autumn cultivations indicated that nitrification had occurred. However, most of the N contained in CSL was in forms that had high molecular weights, such as proteins and peptides [9,10]. These N forms are either not absorbed or inefficiently absorbed by plants [18], and thus, the nitric acid and N content of bok choy cultivated in OF and OF2 was considerably lower than that of the bok choy cultivated in CF. However, the addition of a small amount of CSL every three days was expected to enable the development of biofilm on the root surface of plants, allowing the microorganisms that formed the biofilm to mineralize organic compounds and provide absorbable N to plant roots [8]. Moreover, microorganisms forming the biofilm may absorb organic N and mineralize organic N, thereby directly providing absorbable N to plant roots [13], despite N absorption not being upregulated in OF and OF2 compared with CF. Contrary to the nitrate ion, the ammonium ion was only detected in OF2 during summer cultivation. An accumulation of ammonium ions as that shown in OF2 can negatively affect the growth of bok choy. The dissolved oxygen level in OF2 was significantly lower than that in OF and CF in both summer and autumn cultivation periods, leading to the disruption of the nitrification process by aerobic nitrifiers. The high air temperature in summer was assumed to increase nutrient solution temperature, which then leads to increased plant root growth and higher respiration rate. This higher respiration rate may induce microorganisms in the root biofilm to consume more oxygen, thereby reducing the levels of dissolved oxygen in OF2 required for nitrification to occur [24]. Although the dissolved oxygen level in OF2 was similar between summer and autumn, the possibility of oxygen deficiency should be considered for improving bok choy cultivation in summer.

Effect of Fertilizer Types and CSL Application Rates on Bok Choy Growth
Bok choy was successfully grown hydroponically using CSL at the same N level as in CF (one application rate, OF) in summer and autumn, but not at twice the application rate in CF (OF2) in summer. Autumn cultivation did not show differences in bok choy growth regarding fertilizer type or application rate. However, the nitrification deficiency in OF and OF2 under the high air and nutrient solution temperature in summer caused a lower plant yield and growth rate compared with CF. To fulfill N demand, bok choy may absorb organic N [18] as shown for tomato, which can increase its N uptake by 15-40% [25]. In the present study, the glutamic acid concentration was significantly higher in OF2 than in OF and CF in autumn, suggesting that bok choy cultivated in OF2 absorbed a high amount of glutamic acid. This agreed with the results of Nihei [19], who found that the addition of glutamic acid promotes bok choy growth. In summer, the concentrations of aspartic acid, threonine, serine, glycine, and valine in bok choy were higher in OF2 than CF, and OF showed intermediate values. In bok choy cultivation, aspartic acid effectively replaced the nitrate ion, while glycine and serine had no effect on growth; threonine and valine inhibited bok choy growth when compared to no N application [19]. Thus, the double application rate of CSL in summer might have stimulated the absorption of organic N. However, this stimulation was not sufficient for increasing plant N content. High C concentration and low N concentration resulted in the high C/N ratios observed in OF and OF2, suggesting that C-containing N sources such as amino acids were directly absorbed. Further studies are needed to clarify this issue. There was no difference in growth rates among OF, OF2, and CF, possibly due to the rhizobacteria in the root biofilm that act as growth promoters. One possible pathway leading to the greater root development in OF and OF2 compared with CF in autumn is auxin production [26]. However, the identical leaf yield observed in all treatments led to lower leaf/root ratios in OF and OF2 than in CF. It remains unclear how root development may be positively affected by the beneficial rhizobacteria and contribute to the growth of the aerial part of bok choy. Thus, the function of rhizosphere microbiota in the nitrification process and biofilm formation warrants further study to elucidate the role of microorganisms in the absorption mechanisms of different N forms and in growth inhibition.

Effects of Fertilizer Types and CSL Application Rates on Bok Choy Quality
Content of nitrate ion and ascorbic acid in bok choy leaves in OF and OF2 differed from those in CF. Specifically, higher nitrate ion concentrations were accompanied by lower ascorbic acid concentrations. This trend is commonly observed not only in soilless cultures using CSL [8] but also in soil cultures using organic fertilizers [27]. It has been suggested that ascorbic acid concentration and total N content are negatively correlated in spinach mustard (B. rapa var. perviridis) [27] and broccoli [28]. Kodashima [29] suggested that the application of OF lowered the nitrogen content of spinach compared to the application of CF without inhibiting its growth. Rozek et al. [30] showed that ammonium sulfate fertilizer resulted in a higher content of free amino acids in spring cabbage when compared with potassium nitrate fertilizer. Amino acid supplementation in a nitrogen-deficient nutrient solution was also reported to decrease nitrate concentration and enhance the antioxidant capacity of green lettuce [31]. The present results suggest that the absorption of amino acids and ammonium ion derived from CSL may lead to lower nitrate ion concentration and higher ascorbic acid concentration in OF and OF2. Thus, bok choy quality, which is determined by its high content of ascorbic acid, was improved by OF and OF2 in autumn cultivation. Although amino acid concentration may affect the taste of bok choy and values in summer were remarkably lower than in autumn, the difference in such concentrations among fertilizer types does not seem to be a determining factor of bok choy quality.

Disease Resistance
Hydroponic cultivation using OF and OF2 may induce disease resistance in bok choy. The multiple parallel mineralization technique has been shown to suppress some soilborne pathogens, such as Ralstonia solanacearum in tomato [12] and Fusarium oxysporum in lettuce [13,15] and tomato [14]. In the present study, gray mold disease was suppressed in OF and OF2 compared with that in CF. Fujiwara et al. suggested the existence of antimicrobial factors in the nutrient solution [14] that would work along the physical barrier of root biofilm. Chinta et al. also reported that nutrient solutions upregulated some defense genes and induced cucumber resistance against the gray mold pathogen Botrytis cinerea when sprayed onto the aerial parts [16]. The exogenous application of L-histidine induces plant resistance against Ralstonia solanacearum in tomato and Arabidopsis through the activation of ethylene signaling [32]. The accumulation of amino acids in bok choy may trigger the induction of resistance against gray mold. Furthermore, glutamic acid has been recognized to induce rice plant resistance against Magnaporthe oryzae, the causal agent of rice blast disease [33]. The accumulation of glutamic acid in bok choy in OF2 during autumn cultivation may also induce resistance.

Potential Benefits of Using Organic Fertilizers
As pointed out by Shinohara [6], hydroponics using the multiple parallel mineralization technique has several benefits. CSL, as well as a broad range of organic fertilizers, industrial food wastes, and methane fermentation digest, can be degraded by this method. Even the fermented aerial parts of tomato can be applied to tomato hydroponics. Highly efficient circular agriculture will be implemented if growth suppression under high-temperature conditions is achieved. Multiple additions of small amounts of organic fertilizer after establishing the microecosystem in the hydroponic vessel contributed to the stable nutrient condition in the solution. Establishment of a degrading system of organic compounds enables replanting as phytotoxic compounds or mineral deficiencies are not observed [8]. In addition, inhibition of quorum sensing may prevent soil-borne bacterial diseases [34]. Because there is no need to disinfect vessels before transplanting, year-round continuous cropping may be possible. The consumption of agricultural chemicals can be reduced due to the preventive effect of this method. Low nitrate and high ascorbate content are demanded for vegetables in the human diet [28]. Thus, the use of organic fertilizer in hydroponics can improve the quality of such vegetables in conventional cultivation.

Issues to Be Considered
Although microecosystems in nutrient solutions and biofilms of root and vessel surfaces are key to efficient ammonification, nitrification, and disease prevention, their structure and function are not sufficiently elucidated. In particular, the underlying mechanisms of growth inhibition in summer cultivation should be clarified. If the decreased activities of microecosystems lead to growth inhibition, using a thermotolerant system may be more beneficial than solution chilling in sustainable agriculture. If overdeveloped root biofilm leads to oxygen deficiency, additional aeration or reducing organic input can be tested. Our future study will focus on the relationship between the development of root surface biofilm and nitrogen absorption under high-temperature conditions by implementing long-term trials.

Conclusions
Hydroponic cultivation of bok choy using organic fertilizer is not inferior to chemical fertilizer in the autumn season. Compared to chemical fertilizer, organic fertilizer also resulted in lower nitric acid and higher ascorbic acid content. On the other hand, in summer cultivation, the growth rate was slower with organic fertilizer than with chemical fertilizer likely due to dissolved oxygen deficiency, different rates of nitrification, and nitrogen absorption by the plant root. Accumulated amino acids in the plant body are possible growth inhibitors and also activators of disease resistance against gray mold. Further management techniques should be added to the hydroponic management of summer cultivation. At present, officially certified organic fertilizers tend to be more expensive than chemical fertilizers. The development of a recycling system of crop residue is expected to reduce fertilizer costs and to help establish sustainable production in hydroponics.