In 2019, we selected 12 differentially productive apple orchards under differing fertilizer, pesticide, and tillage management systems in Aomori and Nagano Prefectures. Experimental treatments are shown in Table 1. The soils in these orchards are volcanic ash soils. Natural farming involves management systems that do not use fertilizers, pesticides, or tillage; apple organic farming uses only

compost and organic materials as fertilizers, and pesticides as prescribed by the Japanese Agricultural Standard system are sprayed 1 - 2 times each year. Hybrid apple farming uses a combination of chemical and organic fertilizers, plus synthetic chemical pesticides sprayed 5 - 7 times each year, and conventional chemical methods of apple cultivation use chemical fertilizers and synthetic pesticides that are sprayed 11 - 14 times each year. As many pests infest apple orchards, a total of 40 or more synthetic chemical pesticides are sprayed every 10 days over the entire growing season. Synthetic chemical pesticides, irrigation, and cultivation operations are applied as per the recommendations for Aomori and Nagano Prefectures by the Japanese Government [16]. For example, in Aomori Prefecture, fertilization rates of 0.15 t∙ha⁻¹ of N, 0.05 t∙ha⁻¹ of P₂O₅, and 0.05 t∙ha⁻¹ of K₂O are recommended. In turn, recommended fertilization rates in Nagano Prefecture are 0.12 - 0.20, 0.04 - 0.06, and 0.10 - 0.14 t∙ha⁻¹ of N, P₂O₅, and K₂O, respectively. The experiment treatments have been under the same farming methods for five years. All 12 selected orchards contain 10 - 20-year-old Fuji/Marubakaidou (Malus prunifolia Borkh. Var. ringo Asami) trees. The climate in Aomori and Nagano Prefecture is humid temperate; July is the warmest month, while January is the coolest.

Fruit sugar content and acidity were analyzed using a pocket sugar-acidity meter (PAL-BX/ACID5; Atago, Tokyo, Japan). Sugar content was determined by dripping undiluted apple juice onto the sensor of the pocket sugar-acidity meter; each apple juice sample was measured thrice, and the average was calculated. Acidity was measured by diluting 1 mL of apple juice to 50 mL by gently stirring in purified water (100 rpm, 5 min) and placing a drop of the diluted juice on the pocket sugar-acidity meter sensor. Again, each apple juice sample was measured thrice, and the average was calculated. The sugar/acid ratio was calculated as sugar content divided by acidity.

Composite soil samples (top 15 cm layer, excluding the top 2 - 3 cm surface crust) were collected near the base of five randomly selected trees in each orchard. Soil sampling was performed in July when soil microorganisms were active [14]. The following chemical properties of the composite soil samples were analyzed: TC, TN, NH+ 4, NO− 3, TP, SP, TK, and exchangeable potassium (SK). TC content was analyzed with a TOC analyzer (SSM-5000A; Shimadzu, Kyoto, Japan). NH+ 4 and NO− 3 were analyzed by extracting the soil sample with 1 M KCl, followed by the indophenol blue method and brucine methods [17]. A soil-water suspension (1:20, w/v) was reciprocally shaken at 100 rpm for 1 h, and the extracts were analyzed using the molybdenum blue method [18] and atomic absorption spectrophotometry for quantitative determination of SP and SK, respectively. TN, TP, and TK contents were analyzed by digesting the soil samples 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 the soil-water suspension (1:2.5, w/v) was analyzed using a pH meter (Model: LAQUA F-72; Horiba Scientific, Kyoto, Japan).

Nitrogenous organic substances, such as proteins, are decomposed in soil as follows: NH+ 4 → NO− 2 → NO− 3, after protein → peptide → amino acid and low-molecular-weight molecules derived from amino acid decomposition are rendered by soil microorganisms. During these processes, 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 environmental DNA (eDNA) analysis, which allows an accurate and simple measurement by extracting microbial DNA from the 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 N in organic matter to NO− 3 was evaluated as “N circulation activity” [19]. 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 phosphate before the plant can absorb phosphate (phytic acid-degrading activity). Therefore, the ability to convert phytic acid into organic phosphate was evaluated as “P circulation activity” [19].

Different soils were assigned a certain score: soils in which all phytic acid changed to phosphoric acid without chemisorption with minerals were given a score of 100 points, while soils in which phosphoric acid was not produced at all were assigned a score of 0 points. However, a P circulation activity score of 100 points indicated a low mineral content. Therefore, soils with a moderate mineral content and abundant microorganisms (due to phosphoric acid being supplied) were assigned a score of 40 - 60 points.

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 eDNA using the slow-stirring method [20]. The N circulation activity was analyzed using NH+ 4 and NO− 2 oxidation activity values and total bacterial number, as described by Matsuno et al. (2013) [21] and Adhikari et al. (2014) [13]. 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 [14] [15] [22].

Data in tables and figures are means ± SD analyzed using Bell Curve for Excel 2016 for Windows (Social Survey Research Information Co., Ltd., Tokyo, Japan). All data were analyzed using ANOVA followed by Fisher’s least significant difference test where appropriate. All statistical analyses were conducted at a significance level of α = 0.05 (P < 0.05).

Sugar content, acidity, and sugar/acidity ratio of apple fruits grown under the four farming management strategies are summarized in Table 2. Sugar content was the highest in apples from organic orchards, although it did not significantly differ from the other treatments. In contrast, acidity was highest in apples from hybrid orchards.

Furthermore, sugar/acidity ratios were significantly higher in the natural and organic orchards than under hybrid or conventional management. Fuji apples are judged most delicious when their sugar content is ≥14%, acidity is 0.4%, and sugar/acid ratio is 30 - 40 [23]. Apples from organic orchards showed a sugar content of 13.7%, acidity of 0.4%, and sugar/acidity ratio of 34.3, implying that these apples were the closest to the established “delicious taste” requirements. Overall, our data suggested that the differences in apple sugar content, acidity, and sugar/acidity ratio between the different orchard management systems were attributed to differences in soil conditions.

Chemical properties of the orchard soils sampled in July under natural, organic, hybrid, and conventional management systems are shown in Table 3.

TC contents of the natural, organic, hybrid, and conventional system soils were 22,270 - 78,210, 31,290 - 143,700, 21,600 - 58,300, and 28,740 - 49,490 mg∙kg⁻¹, respectively. In turn, TN content ranges were 1340 - 3240, 2190 - 12,310, 690 - 2600, and 2030 - 3070 mg∙kg⁻¹, respectively. TP contents were 1060 - 2500, 6820 - 24,280, 3400 - 11,300, and 1070 - 2360 mg∙kg⁻¹, and TK contents ranges were 1820 - 8110, 2590 - 5130, 4700 - 14,500, and 7050 - 8390 mg∙kg⁻¹, respectively. Furthermore, NO− 3-N content values were 6 - 18, 17 - 64, 0 - 9, and 6 - 9 mg∙kg⁻¹, NH+ 4-N content values were 0 - 5, 1 - 3, 0 - 2, and 3 - 25 mg∙kg⁻¹. while SP contents were 46- 495, 2196 - 13,017, 10 - 81, and 66 - 1234 mg∙kg⁻¹,

^zMean ± standard deviation of a sample (sugar content, acidity, sugar/acidity ratio; n = 5 - 16). ^yDifferent letters within columns are significantly different at the P < 0.05 level, according to the Tukey-Kramer method.

respectively. SK contents were 87 - 2120, 1116 - 1457, 29 - 156, and 1075 - 2100 mg∙kg⁻¹, respectively. Overall, TC, TN, TP, NO− 3-N, and SP contents were the highest in organic farming orchards. The soil in one of the organic orchards (No. 4) showed excess TC, TN, and TP, while the highest TK content was recorded in soils under hybrid management. The C/N ratios in natural, organic, hybrid, and conventional orchard soils were 16.6 - 24.1, 11.7 - 16.8, 22.4 - 31.3, and 14.2 - 16.4, respectively; thus, the highest C/N ratio was registered in orchards under hybrid farming. However, as soil inorganic nitrogen is readily used by microorganisms while decomposing organic matter, in such high C/N ratio soils, N might soon become deficient and microorganisms might become N starved, thereby inhibiting apple growth.

Biological properties of the soils sampled in July from the four farming management strategies are summarized in Table 4, and radar charts of average N circulation activity from the natural, organic, hybrid, and conventional farming systems are shown in Figure 1. Average P circulation activity from the natural, organic, hybrid, and conventional farming systems are shown in Figure 2.

Ranges of bacterial biomass under these four farming methods were 2.6 - 10.0, 4.5 - 12.4, 4.8 - 7.2, and (1.8 - 5.1) × 10⁸ cells g⁻¹, respectively, while N circulation activities were 22- 75, 39 - 100, 30 - 48, and 1 - 10 points. In turn, NH+ 4 oxidation activities were 15 - 82, 75 - 100, 23 - 40, and 4 - 10, respectively, while NO− 2 oxidation activities were 52 - 99, 40 - 100, 36 - 90, and 0 - 51. Moreover, P circulation activities were 10 - 23, 16 - 40, 1 - 32, and 0 - 1, respectively. These results indicated that bacterial biomass, N circulation activity, NH+ 4 oxidation activity, NO− 2 oxidation activity, and P circulation were the highest in organic farming

orchards and the lowest in conventional orchards. Thus, apple orchard soils under organic farming methods showed increased bacterial biomass and enhanced N and P circulation activities respectively. These results indicated that bacterial biomass, N circulation activity, NH+ 4 oxidation activity, NO− 2 oxidation activity, and P circulation were the highest in organic farming orchards and the lowest in conventional orchards. Thus, apple orchard soils under organic farming methods showed increased bacterial biomass and enhanced N and P circulation activities.