Chemical and Biological Properties of Apple Orchard Soils under Natural, Organic, Hybrid, and Conventional Farming Methods

Apples in Japan are generally cultivated under management systems that use chemical fertilizers and synthetic chemical pesticides. However, the continuous use of these fertilizers and pesticides damages the soil environment and reduces the number of soil microorganisms. In this study, we compared the chemical and biological properties of 12 soils from apple orchards in Aomori and Nagano Prefectures under four types of management systems, namely, natural conditions, with no cultivation, fertilizers, or pesticides; organic farming methods, using organic materials and pesticides approved by the Japanese Agricultural Standard organic certification system; hybrid farming methods, using a mix of organic and chemical fertilizers; and conventional farming, using chemical fertilizers and pesticides. Soil total carbon (TC), total nitrogen (TN), total phosphorus (TP), nitrate-nitrogen (NO − 3 ), and available phosphoric acid (SP) contents were generally found to be the highest where organic farming methods were used. Similarly, bacterial biomass, nitrification (N) circulation activity, ammonia (NH + 4 ) oxidation activity, nitrite (NO − 2 ) oxidation activity, and phosphoric (P) circulation activity were the highest under organic farming, especially in comparison with conventional farming. This study indicated that the differences in apple sugar content, acidity, and sugar/acidity ratio between different orchard management systems were due to different soil conditions, and soil conditions under organic farming management system in apple cultivation increased bacterial biomass while enhancing N and indicated that soil conditions under organic farming management are the most suitable for increasing microbial numbers and enhancing N and P circulation activity.


Introduction
Apples (Malus domestica Borkh.) are believed to have originated in the region between the northern Caucasus and the Tianshan mountains in west and central Asia [1]. Currently, there are about 15,000 apple varieties worldwide, mainly in the subtropical, temperate, and subarctic regions, including those in China, Korea, North America, and Australia, and about 2000 in Japan [2]. The development of new varieties and discoveries of changes in branching architecture have allowed apples to become one of the most widely cultivated and productive fruit crops in the world [3]. The Fuji apple variety was developed in Fujisaki City, Aomori Prefecture, in 1962, and it is currently the most commonly produced Japanese apple variety, with large volumes of export to other countries. Cool climate regions with average annual temperatures of 6˚C -14˚C, low annual rainfall, and wide temperature differences between day and night are best suited for apple cultivation [4]. Therefore, Japanese apples are mainly cultivated in Aomori, Nagano, Iwate, Yamagata, and Fukushima Prefectures, among which Aomori and Nagano Prefectures account for approximately 77% of the total national production [5]. The overall mean annual apple yield in Japan is 21.2 t•ha −1 per year, which is lower than the yields obtained in other developed countries, e.g., 77.9 t•ha −1 per year in Australia, 47.4 t•ha −1 per year in New Zealand, and 31.9 t•ha −1 per year in the United States [6]. Poor soil fertility seems to be one of the most important reasons for the prevalent low productivity of this highly important fruit crop in Japan [2].
Most of the apple production in Japan relies on the use of chemical fertilizers and synthetic pesticides [7]. Chemical fertilizers have made it possible to effectively supply nutrients to the soil because they are soluble in water and have an immediate effect [7], thereby reducing the labor needed for fertilizer application and greatly increasing the yield per unit area. Therefore, the use of chemical fertilizers and synthetic pesticides has increased apple productivity. However, the continuous use of these chemical fertilizers and synthetic chemical pesticides demonstrably deteriorates the soil environment and reduces soil microorganism function and diversity. Although organic production systems might reduce soil damage, previous studies suggest that organic farming systems typically show 20% to 30% lower productivity than conventional systems that make heavy use of chemical fertilizers and pesticides [8] [9]. Journal of Agricultural Chemistry and Environment Although, as stated before, poor soil fertility is thought to be one of the major reasons for the lower apple productivity of apple orchards in several prefectures, information on the relationship between soil properties and apple productivity is scarce [10] [11] [12].
Previously, we reported that organic fertilizer-based systems can effectively support stable and high crop productivity by maintaining suitable bacterial biomass levels, N circulation activity, and P circulation activity by controlling TC and TN contents and the C/N ratio in the soil [13]. Excessive levels of TC, TN, TP, and total potassium (TK) in Japanese orchards that use conventional chemical fertilizer management systems can reduce yields [14]. In addition, apple orchards are relatively rich in TC, TN, TP, and TK, compared to annual croplands such as paddy fields and uplands [15]. Therefore, to assess the characteristics of organic soil condition under four agricultural methods, we compared the chemical and biological properties of soils under natural farming, organic farming, hybrid agricultural methods using both organic and chemical fertilizers, and conventional chemical fertilizer methods.

Study Sites
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  in Aomori and Nagano Prefecture is humid temperate; July is the warmest month, while January is the coolest.

Fruit Sugar, Acidity, and Sugar/Acidity Ratio
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.

Soil Chemical Properties
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 ana-  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

Soil Biological Properties
Nitrogenous organic substances, such as proteins, are decomposed in soil as follows: NH 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

Statistical Analysis
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 dif-T. Kai, M. Kubo Journal of Agricultural Chemistry and Environment ference test where appropriate. All statistical analyses were conducted at a significance level of α = 0.05 (P < 0.05).

Comparison of Fruit Content and Acidity
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.

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

Comparison of Soil Biological Properties
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.

Discussion
To assess the characteristics of soil conditions for apple cultivation under four  [7] reported that the recommended carbon and nitrogen contents and C/N ratio for orchard soils are 25,000 mg•kg −1 or higher, 1500 mg•kg −1 or higher, and 10 -25, respectively. In this study, these recommended values were not reached in two orchards under natural farming systems nor in two orchards using hybrid farming methods (Figure 3). In the natural farming orchards, soils might have been deficient in TN because no fertilizer had been added for nearly 5 years. As for the hybrid farming orchards, the soils might have been deficient in TC or TN. Meanwhile, the conventional apple orchard soils closely matched the recommended values, indicating that the fertilizer applied in accordance with the guidelines of the prefectures and municipalities is appropriate. In turn, the TC and TN values in the organic orchards under study were also all within the recommended range, although very large variation among these orchards was observed.
This finding suggests that apple cultivation in these organic orchards depends on the experience and intuition of each farmer and that at present, its reproducibility is low. Therefore, it is necessary to assess the soil conditions in each orchard, as well as the components of compost and organic materials used. With this information, it should be possible to decide more accurately which nutrients are needed and how much organic fertilizer to apply. This will promote soil conditions in which microorganisms thrive, improving soil biodiversity and nutrient cycling. Overall, the effect should be a highly reproducible organic agriculture in harmony with the local environment.
The relationship between soil TC and soil bacterial biomass in orchard soils we have surveyed under the four farming management strategies is shown in   This study indicated that the differences in apple sugar content, acidity, and sugar/acidity ratio between different orchard management systems were due to different soil conditions, and soil conditions under organic farming management system in apple cultivation increased microbial biomass while enhancing N and P cycle activity and high TC.