Between September 2019 and May 2020, we investigated the cultivation of Norabona (Brassica napus L.) using organic and conventional farming methods in an upland field at Ikuta Campus, Meiji University. Generally, norabona is cultivated with chemical fertilizers and synthetic pesticides, but in our study, no pesticides were used in either the organic or conventional farming treatments. The field has been managed with chemical fertilizers and synthetic pesticide management for about eight years. The norabona seeds were sown on a seedling bed in a greenhouse in September. Then, in October when they had reached the development stage of two to three main leaves, they were potted up in 9 cm pots. Then, in November they were planted in the field in rows, with 60 cm between plants and 70 cm between rows. The soil surface was then covered then with black mulch. In the conventional farming treatment, the chemical fertilizer was applied according to typical rates for high yielding growers in the local area. Specifically, basal fertilizer (N:P₂O₅:K₂O = 150:200:150 kg∙ha⁻¹) was incorporated into the field soil in September, before planting. Then subsequent fertilizer was applied on two occasions in December and January: (N:P₂O₅:K₂O = 120:0:120 kg∙ha⁻¹) on each occasion. The organic fertilizer was commercial cattle manure (N:P₂O₅:K₂O = 270:200:270 kg∙ha⁻¹, Oobari Corporation, Tochigi, Japan), and applied in September.

The soil was a light andosol, and particle size analysis was conducted by sieve analysis to determine the soil texture; sand (2 - 0.02 mm), silt (0.02 - 0.002 mm), and clay (less than 0.002 mm), with particles of less than 75 μm measured by sedimentation [18].

Soil samples (top 15 cm layer, excluding the top 2 - 3 cm surface crust) were taken near the base of five selected norabona plants in each treatment, and mixed to provide one composite sample for each treatment. The following chemical properties were analyzed: TC, TN, ammonium-nitrogen ( NH 4 + -N ), nitrate-nitrogen ( NO 3 − -N ), TP, available phosphoric acid (SP), TK, and exchangeable potassium (SK). The TC content was analyzed with a TOC analyzer (Model: SSM-5000A, Shimadzu, Kyoto, Japan). NH 4 + -N and NO 3 − -N were analyzed by extracting the soil sample with 1 M KCl, followed by the indophenol blue and brucine methods, respectively [19]. SP and SK were analyzed by shaking a soil-water suspension (1:20, w/v) at 100 rpm for 1 h and analyzing the extract with the molybdenum blue method [20] and atomic absorption spectrophotometry, respectively. The TN, TP, and TK contents were analyzed by digesting soils in a Kjeldahl Therm digestion unit (Gerhardt, Königswinter, Germany) with H₂SO₄ and H₂O₂; NH 4 + -N , SP, and SK contents in the digest were determined. The pH of a soil-water suspension (1:2.5, w/v) was analyzed using a pH meter (Model: LAQUA F-72, Horiba Scientific, Kyoto Japan).

Organic nitrogen substances such as proteins in soil are decomposed by soil microorganisms into ammonia nitrogen ( NH 4 + ) → nitrite nitrogen ( NO 2 − ) → nitrate nitrogen ( NO 3 − ). The NH 4 + oxidation activity ( NH 4 + → NO 2 − ), NO 2 − oxidation activity ( NO 2 − → NO 3 − ), and bacterial biomass were determined. Bacterial biomass was determined by eDNA analysis, which establishes an accurate and simple measurement by extracting microbial DNA from soil. NH 4 + oxidation activity, NO 2 − oxidation activity, and the number of microorganisms were quantified with a triangular radar chart, and the ability of the soil to convert organic nitrogen to NO 3 − was evaluated as “N circulation activity.” The larger the area of the triangle, the more active the nitrogen circulation in the soil, and vice versa. In addition, phytic acid (organic phosphate) must be broken down into inorganic phosphate (phytic acid-degrading activity) before the plant can absorb phosphate. Therefore, the ability to convert phytic acid into organic phosphate was evaluated as “P circulation activity”.

Soils in which all phytic acid was converted to phosphoric acid, and there was no mineral chemisorption were assigned a P circulation activity of 0 (zero). A P circulation activity of 100 points would indicate that there is little mineral content. Therefore, soil with a moderate mineral content and abundant microorganisms (due to phosphoric acid being supplied) was assigned a P circulation activity value of 40 - 60. The following biological properties were analyzed: total bacterial biomass, NH 4 + oxidation activity, NO 2 − oxidation activity, N circulation activity, and P circulation activity. Total bacterial biomass was estimated by quantifying environmental DNA (eDNA) using the slow-stirring method following the procedures of Aoshima et al. (2006) [21]. The N circulation activity was analyzed by pooling the values of NH 4 + , NO 2 − oxidation activities, and total bacterial number. The NH 4 + and NO 2 − oxidation activities were estimated by analyzing the percent reduction in N in soil sample with added ammonium sulfate and sodium nitrate, respectively, that were incubated for 3 days at 25˚C, as described by Matsuno et al. (2013) [22] and Adhikari et al. (2014) [12]. Similarly, P circulation activity was determined by analyzing the rate at which soluble P was released from phytic acid (a dominant form of organic P in soil) over a three-day incubation period [23].

While the main stem was still at the immature stage, before stem elongation and having about 15 to 17 main leaves and before stem elongation, the top of the main stem was cut off to leave the main stems on all plants with 10 to 12 leaves. After cutting off the top of the main stem, the flower stalks of all side branches were harvested to leave the primary side branches with 0 to 4 leaves and the other side branches with 0 to 3 leaves, based on management methods reported by Nomura [24] and Odawara et al. [25].

The number of leaves was counted on each plant. Also, shoot height, shoot with and the width and length of the four largest leaves on each plant were measured with a ruler. SPAD values of the four leaves were measured by chlorophyll meter (SPAD-502Plus, Konica Minolta Co., Osaka, Japan).

Eight replicate samples of 5 plants each were harvested for each fertilizer treatment. Average values were calculated on a per plant basis. The harvest period started on 4 March 2020 and ended on 26 May 2020. Main stems and primary lateral branches were harvested in March, secondary lateral branches in April, and tertiary lateral branches in May. Flower stems that reached 30 cm or more in length to the leaf tip were harvested on the primary lateral branches, and flower stems that had emerged on the secondary lateral branches and beyond were harvested based on the report by Tsuge et al. [26].

The roots and shoots of the 5 plants × 8 replicates (= 40 plants) from each fertilizer treatment were collected. The roots and shoots from the 8 replicates of 5 plants each, were harvested every month, and the roots were washed carefully. The total dry weights of shoots and roots were measured after oven drying at 80˚C for 3 days. The number of lateral branches was defined as the number of flower stems harvested from one plant. The lateral branch saleable yield was defined as the amount of flower stems generated from one plant, and the weight of all harvested flower stems. Based on the reports of Yoshida [27] and Tsuge et al. [28], flower stalks exceeding 30 cm length were cut to 30 cm in length and weighed. The saleable weight per lateral branch is a value related to the quality of the flower stalk, with higher values indicating better quality, and was determined by dividing the lateral branch saleable yield by the number of lateral branches.

The sugar content was analyzed using a pocket sugar meter (Model: PAL-S, Atago, Tokyo, Japan). Sugar content was determined by dropping undiluted norabona juice onto the sensor of the pocket sugar meter. Each norabona juice sample was measured three times, and the average was calculated.

Data are presented as the mean ± standard deviation (SD) values and analyzed using BellCurve for Excel 2016 for Windows (Social Survey Research Information Co., Ltd., Tokyo, Japan). All data were analysed using ANOVA followed by Fisher’s least significant difference test where appropriate. All statistical analysis was conducted at a significance level of α = 0.05 (p < 0.05).

The soil grain size of the field site was 53.2% sand, 29.0% silt, and 17.8% clay, meaning the soil had a clay loam texture. Soil chemical properties before norabona cultivation in September 2019, and in the organic and chemical treated soils in May 2020 (after harvest) are shown in Table 1. The soil TC, TN, TP, and TK content values before cultivation were 25862, 2806, 2137, and 2746 mg kg⁻¹, respectively. The C/N ratio value was 9.2. In addition, NO 3 − -N , NH 4 + -N , SP, SK content values were 40, 2, 2, and 245 mg∙kg⁻¹, respectively. Furthermore, pH, EC, and volumetric water content values were 6.3, 0.66 mS∙cm⁻¹, and 24.7%, respectively. After harvesting the norabona in May 2020, the soil TC, TN, TP, TK contents in the organic fertilizer were 49,033, 3584, 2721, and 3394 mg∙kg⁻¹, respectively, giving a C/N ratio of 13.7. The NO 3 − -N , NH 4 + -N , SP, SK contents were 2, 11, 15, and 48 mg∙kg⁻¹, respectively, and the pH, EC, and volumetric water contents were 6.9, 0.71 mS∙cm⁻¹, and 42.5%. In comparison, the chemically fertilized soil had TC, TN, TP, TK contents of 28,733, 1,793, 1,504, and 3,107 mg∙kg⁻¹, respectively. giving a C/N ratio of 14.4. The NO 3 − -N , NH 4 + -N , SP, SK contents were 5, 18, 37, and 172 mg∙kg⁻¹, respectively, and the pH, EC, and volumetric water contents were 5.6, 0.65 mS∙cm⁻¹, and 33.5%.

The TN and TP contents were significantly higher after the organic fertilizer soil than in the chemical fertilizer soil or before cultivation, as was soil pH. The TC content and volumetric water content were significantly higher after the organic fertilizer treated soil than before cultivation, but SK content was significantly lower. On the other hand, the C/N ratio, NH 4 + -N , and SP contents were significantly higher after the chemical fertilizer treated soil than before cultivation. However, NO 3 − -N content was significantly in the organic and chemical fertilizers treated soils than before cultivation, but there was no significant difference for TK or EC.

Soil biological properties before norabona cultivation in September 2019, and in the organic and chemical fertilizer treated soils in May 2020 (after harvest) are shown in Table 2. Bacterial biomass number, NH 4 + oxidation activity, NO 2 − oxidation activity, N circulation activity, and P circulation activity values before cultivation were 0.3 × 10⁸ cells∙g⁻¹, 51.3, 66.0, 13.3, and 0.7 points, respectively. After harvest in the organic fertilizer treated soil, the bacterial biomass number,