agronomy

Article
Application of Homemade Organic Fertilizer for Improving
Quality of Apple Fruit, Soil Physicochemical Characteristics,
and Microbial Diversity
Xiuzhang Wang 1,2 , Qi Bao 1,2 , Guotao Sun 1,2, * and Jianming Li 3, *

1 College of Mechanical and Electronic Engineering, Northwest A&F University, Xianyang 712100, China
2 Western Scientific Observing and Research Station for Development and Utilization of Rural Renewable Energy,
Ministry of Agriculture and Rural Affairs, Xianyang 712100, China
3 College of Horticulture, Northwest A&F University, Xianyang 712100, China
* Correspondence: guotao_sun@nwsuaf.edu.cn (G.S.); lijianming66@nwsuaf.edu.cn (J.L.)

Abstract: Application of the organic fertilizers can improve soil properties and agricultural product
quality, while the in-depth effect of organic fertilizer needs further exploration. In this study, the
apple fruit (Malus pumila Mill.) quality, soil physicochemical characteristics, and microbial commu-
nity under different fertilizers including control without fertilizer (CK), composted manure (CM),
biogas slurry (BS), and chemical fertilizer (CF) were systematically investigated, and each treatment
was repeated three times in completely random block plots with equal NPK content of fertilizer
applied in each treatment. The CM treated apple fruit were observed with the best vertical diameter
(78.72 ± 4.32 mm), transverse diameter (87.51 ± 1.3 mm), and color index (L* = 55.48 ± 5.48,
b* = 18.96 ± 1.86). Meanwhile, the CM and BS treated apple fruit had higher flesh firmness than
that of CK and CF treatment, implying that the organic fertilization can improve the storability and
crispy taste apple fruit. Furthermore, the total sugars, essential amino acid, crude protein, total
Citation: Wang, X.; Bao, Q.; Sun, G.;
acids, and soluble solid contents of apple fruit were also significantly improved by using CM and BS
Li, J. Application of Homemade fertilizer due to the resulting higher N, P, and organics content in soil. The application of organic
Organic Fertilizer for Improving fertilizer also dramatically enhanced the bacterial and fungi diversity, which may contribute to better
Quality of Apple Fruit, Soil soil respiration. The results obtained in this study reveals the insight effect of organic fertilizer on
Physicochemical Characteristics, and improvement of fruit quality and soil health and can supply technical assistance to organic cultivation
Microbial Diversity. Agronomy 2022, of apple.
12, 2055. https://doi.org/10.3390/
agronomy12092055 Keywords: organic fertilizer; composted manure; apple fruit quality; soil fertility; soil microbial
Academic Editor: Masoud Hashemi diversity

Received: 13 August 2022

Accepted: 25 August 2022
Published: 29 August 2022
1. Introduction
Publisher’s Note: MDPI stays neutral China possesses the largest apple orchard planting area of 2.04 million hectares with
with regard to jurisdictional claims in apple fruit production of 42.42 million tons in the world [1]. As the largest apple fruit
published maps and institutional affil-
producing region of China, Shaanxi Province produced 26.7% of total apple fruit yield in
iations.
China [1]. Due to the high altitude origin of Loess Plateau and abundant solar resources, the
apple fruit that originated from Shaanxi Province has been accepted for its various health
benefits and fantastic texture [2,3]. However, the apple fruit production in Shaanxi Province
Copyright: © 2022 by the authors.
was also confronted with a situation in which apple fruit quality regarding nutritional
Licensee MDPI, Basel, Switzerland.
value and flesh firmness evidently decreased due to the constant application of chemical
This article is an open access article
fertilizer [4]. In order to improve the current difficulties in apple fruit production, exploring
distributed under the terms and better fertilization strategy to improve apple fruit quality and soil properties has become
conditions of the Creative Commons the focus of research.
Attribution (CC BY) license (https:// Various fertilization methods have been studied to improve crop yield, fruit quality,
creativecommons.org/licenses/by/ and nutritional value [5]. Chemical fertilizer has been extensively used to maintain soil
4.0/). fertility and improve crop yields, especially in China. However, their long-term use has

Agronomy 2022, 12, 2055. https://doi.org/10.3390/agronomy12092055 https://www.mdpi.com/journal/agronomy

Agronomy 2022, 12, 2055 2 of 16

also led to soil deterioration because the chemical fertilizer builds up inside the soil and
eventually exceeds what the soil can actually handle. The deterioration of soil environment
may adversely affect soil fertility, soil microbial diversity, and then the nutrient quality of
the agricultural product [6,7]. In order to avoid further deterioration of the soil environment,
the animal manure derived fertilizers such as composted manure and biogas slurry have
been widely used in farmland instead of chemical fertilizer. The composted manure is
produced by aerobic microorganisms under artificial controlled conditions [8], and the
application of it as part of the green agriculture concept has been widely recognized because
of its potential benefits [9,10]. As a byproduct of anaerobic fermentation, biogas slurry
contains large quantities of trace elements and amino acids which can increase crop yield
and improve soil physicochemical characteristics, which have been broadly utilized as
pesticides and organic fertilizer additives [11].
The intensive studies by researchers have reported that using organic fertilizer in-
stead of chemical fertilizer can improve soil microbial community and physicochemical
characteristics, and enrich the plant production quality. For example, Safaei Khorram et al.
concluded that organic fertilizer could improve soil nutrient content and reduce soil bulk
density [12]. Li et al. discovered that organic fertilizer could improve apple fruit output
in the Loess Plateau area and increase soil organic carbon [13]. Chen et al. demonstrated
that addition of pig manure compost could improve bacterial richness on rice yield [14].
Milošević et al. indicated that fertilization could significantly increase apple fruit weight
and firmness, but none of the fertilizer treatments showed significant difference on fruit
size and size ratio [2]. Leff et al. discovered that excessive application of nitrogen fertilizer
would acidify soil, thus reducing soil pH value [15]. Different fertilization conditions and
crop types had different effects on the structure, quantity, and activity of soil microbial
communities. In turn, different soil microbial communities have different effects on soil
physical and chemical properties as well as crop growth, yield, and quality [16]. However,
it was found that there are many studies on the effects of fertilizer on crop quality, soil
physicochemical properties, and soil microbial diversity, but effects of different fertilizers on
the combination of apple fruit quality, soil physicochemical properties, and soil microbial
community in apple orchards were rarely reported. For example, Wang et al. illustrated
that application of bioorganic fertilizer could increase apple fruit yield and soil organic
matter content, and changed the relative abundance of dominant bacteria, but there was
no further study on apple fruit quality [17]. Similarly, Milošević & Milošević only studied
the effect of fertilizer on apple fruit quality, but did not elaborate on soil fertility and soil
microbial community [3]. Furthermore, to the best of our knowledge, there have been many
studies investigating the influence of organic fertilizer on apple fruit quality, but very few
studies were done to elucidate the effect of organic fertilizer on soil microbial community
and the co-relationship between soil condition and fruit quality.
In order to alter the situation of apple orchard regarding the soil degradation and
decreased apple fruit quality, the insight study of the organic fertilizer on soil microbial
community might be the breakthrough point. Therefore, this study aimed to systemati-
cally investigate the influence of different fertilizer treatments on apple fruit quality, soil
fertility, and soil microbial community, in order to provide theoretical reference for nutrient
management and sustainable development of apple orchards.

2. Materials and Methods

2.1. Experimental Conditions
The experimental site is located in Yanchuan County, Shanxi Province, China (36◦ 520 N
latitude and 110◦ 420 E longitude). This region has a northern warm temperate zone conti-
nental semi-arid monsoon climate, with an average annual temperature of 10.4 ◦ C, average
annual frost-free period of 170 days, and a mean annual precipitation of 564 mm. The
soil type was identified to be a typical calcareous soil in beige color formed from Malan
loess. The soil texture in selected experimental plot is featured with high porosity and
uniform particles. The pH of the soil is in the range of 8.2–8.3. The experiment was
Agronomy 2022, 12, 2055 3 of 16

conducted in a plot of appropriate-density (3 m distance between trees and 4 m between

rows), fruit trees grow strong, and there is medium tree potential and planted varieties
of red Fuji (Malus pumila Mill). The soil of 0–20 cm depth layer had the characteristics
including total organic matter of 7.5 g kg−1 , total nitrogen of 0.64 g kg−1 , available nitro-
gen of 38.71 mg kg−1 , available phosphorus of 2.33 mg kg−1 , and available potassium of
1.82 g kg−1 . The soil of the 20–40 cm depth layer had the characteristics including to-
tal organic matter of 4.44 g kg−1 , total nitrogen of 0.31 g kg−1 , available nitrogen of
23.15 mg kg−1 , and available phosphorus of 0.43 mg kg−1, and available potassium
of 1.21 mg kg−1.
Tested fertilizers include composted manure (CM), biogas slurry (BS) and chemical
fertilizer (CF). The CM was prepared by homemade composting of pig manure and apple
sawdust with wood vinegar (1%) and biochar (5%) as coupling additives. The physicochem-
ical characteristics of CM were analyzed and contained organics of 61.02%, total nitrogen of
1.78%, total phosphorus of 2.23%, and total potassium of 1.89%. The BS were collected from
the biogas plant (Liangjiahe) managed by CGN New Energy Co. Ltd (Beijing, China). with
organics of 0.53%, total N of 0.11%, total P of 0.02%, and total K of 0.13%. CF is purchased
from the Yanchuan Run Sheng Agricultural Science and Technology Co., Ltd (Yanchuan,
China). with a mixture of urea (N 46%), superphosphate (P2 O5 16%) and potassium sulfate
(K2 SO4 50%).
On beginning of October 2017, the experiment was established as a completely ran-
domized block design with four treatments and three replicates for each treatment. The
experiment was conducted under four treatments including control group with no fertilizer
application (CK), chemical fertilizer (CF), composted manure (CM), and biogas slurry (BS).
Taking 100 square meters as a treatment plot, each plot selected 9 apple fruit trees with
basically the same growth trend and more than 7 years of age as experimental fruit trees.
Therefore, the total 1200 square meters of apple fruit orchard were selected for the fertiliza-
tion experiment. Apple orchard management measures (irrigation, weeding, pruning, pest
control, spraying, bagging, harvesting, etc.) are consistent with previous years. CF, CM,
and BS were applied based on the consistency of total nitrogen content and supplemented
with superphosphate and potassium sulfate as needed to achieve the same total nitrogen,
phosphorus, and potassium content applied at all stages. Fertilizer application rates of
different treatments are presented in Table 1. The base fertilizers were buried in a ditch
(1.2 m length, 40 cm width, 35 cm depth) 1 m from the trunk of each tree in October 2017.
The topdressing fertilizer was applied in a circle ditches (20 cm width and 20 cm depth)
with 3 m distance to the tree trunk in March and July 2018. The soil sample and fruit sample
were collected at the same time when the apple fruit ripen in October 2018.

Table 1. Fertilizer application procedure of different treatments.

N P2 O5 K2 O
Treatment Fertilization Time
(kg ha−1 ) (kg ha−1 ) (kg ha−1 )
October 2017 (Base fertilize)
CK March 2018 (Topdressing)
July 2018 (Topdressing)
October 2017 (Base fertilize) 150 188 188
CF March 2018 (Topdressing) 150 188 188
July 2018 (Topdressing) 75 135
October 2017 (Base fertilize) C150 C188 C159 + 29
CM March 2018 (Topdressing) C150 C188 C159 + 29
July 2018 (Topdressing) 75 135
October 2017 (Base fertilize) C150 C29 + 159 C188
BS March 2018 (Topdressing) C150 C29 + 159 C188
July 2018 (Topdressing) 75 135
C stands for the corresponding content of N, P2 O5 and K2 O present in biogas slurry and composted manure
CK: Control Group, CF: Chemical Fertilizer, CM: Composted Manure, BS: Biogas Slurry.
Agronomy 2022, 12, 2055 4 of 16

2.2. Sampling and Determination of Apples

After apple fruit ripening (October 2018), 30 fruits were randomly selected from each
treatment to determine the fruit quality. Apple fruit size (vertical and transverse diameter,
both in mm) was determined with caliper gauge Sttarret 727 (Athol, MA, USA). The weight
of apple fruit samples was measured using electronic scales. Fruit firmness was determined
with Gy-1 fruit durometer (Jiangsu, China), and the color index (L*, a*, b*) of apple
fruit skin coordinates was determined using Minolta chromomete (Shenzhen, China) [18].
Soluble solid content was measured with a Carl Zeiss 32-G hand refractometer (Carl Zeiss,
Germany) at 25 ◦ C. The total acidity (TA, %) was measured by using an acid–based titration
method. Fruit juice (1 mL) and distilled water (50 mL) were added to a conical flask and
titrated with aqueous sodium hydroxide to pH 8.1 by using a Metrohm 719S titrimeter
(Titrino, Switzerland). The total acid content was calculated as the equivalent of citric
acid, and the mean values were recorded in triplicate [19]. After harvesting, apples were
homogenized in a blender, and total sugar (TS, %) was determined by the Lane–Eynon’s
method. The apple homogenate sample was placed in a 250 mL volumetric flask and
extracted with ethanol. The extracted solution was titrated with Fehling reagent using
methylene blue as an indicator until the blue solution disappeared. Total sugar/total acid
ratio (TS/TA) and vertical diameter/ transverse diameter ratio (fruit shape index) were
calculated. The crude protein content of apple fruit was determined by using the Method
of Kelvin [20]. The amino acid content in apple fruit was determined by automatic amino
acid analyzer (SYKAM S-433D, Germany) after acidifying the apple fruit with hydrochloric
acid (6 mol/L).

2.3. Soil Physicochemical Analysis

Soil samples were collected at 0–20 cm and 20–40 cm depth using a five-point method [21].
The sampling sites were selected to avoid the fertilization ditches. The soil samples at the
same depth were mixed together and sieved (2 mm) to remove debris such as gravel and
plant roots, sample quartering. The soil samples were divided into two groups, in which
one group was air-dried for soil physical and chemical analysis, and the other one was
stored at −80 ◦ C for DNA extraction and microbial community analysis.
To determine the levels of available nitrogen, the dried soil samples were extracted
with 50 mL 1 mol/L potassium chloride solution (1:10 (w/v)) and analyzed using a seg-
mented flow analyzer (Technicon Auto-analyzer II System, Norderstedt, Germany). The
available phosphorus was analyzed using a segmented flow analyzer (Technicon Auto-
analyzer II System, Germany) after extracting the soil samples with 50 mL 0.5 mol/L
Sodium Bicarbonate solution (1:20 (w/v)). The available potassium was determined by a
flame photometer (M410 blue notes, City, UK) after the extraction with 50 mL ammonium
acetate solution (1 mol/L) [22]. The Kjeldahl method was utilized for total nitrogen (TN)
estimation [23]. Mo-Sb Colorimetry was used for total phosphorus (TP) detection, and
total potassium (TK) was determined with flame photometry [24]. Soil organic matter was
measured using an SSM-5000A total organic carbon analyzer (Shimadzu, Kyoto, Japan).
The soil samples collected from different soil depth and fertilization treatment were
used for total genomic DNA extraction, which was conducted using an E.Z.N.A.® Soil
DNA Kit (D5625-01, Omega BIO-TEK, USA). The obtained DNA samples were amplified
using 16S rRNA universal primers 515F (50 -GTGCCAGCMGCCGCGG-30 ) and 806R (50 -
GGACTACHVGGGTWTCTAAT-30 ), and fungi ITS primers ITS1F (50 - CTTGGTCATTTAGA
GGAAGTAA-30 ) and ITS2 (50 -GCTGCGTTCTTCATCGATGC-30 ). The amplified DNA
samples were then analyzed using an Illumina MiSeq platform (Biozeron, Shanghai, China).
The obtained pyrosequencing data were analyzed by USEARCH (version 7.0, Robert C.
Edgar, CA, USA http://drive5.com/uparse, accessed on 19 November 2018).
Agronomy 2022, 12, 2055 5 of 16

2.4. Statistical Analysis

Significant differences between fertilization treatments were determined with a one-
way (ANOVA) to compare the least significance difference (LSD) at p = 0.05 using SPSS
software version 20.0 (Armonk, USA).

3. Results
3.1. Physical Properties of the Fruit
As shown in Table 2, compared to the CK treatment, the vertical diameter of BS, CM,
and CF treatment increased by 3.94%, 8.12%, and 2.84%, respectively, and the transverse
diameters also increased by 2.39%, 7.35%, and 0.01%, separately. Meanwhile, CM treat-
ment obtained maximum transverse (87.51 mm) and vertical diameters (78.72 mm) with
significant difference from CK treatment. Moreover, it should be noted that fertilization
treatments can change the vertical and transverse dimensions of apples, but there were
no significant differences in the fruit shape index among different fertilizer treatments.
The weight of single apple fruit in the treatments of the CF, CM, and BS treatments were
raised in contrast to CK, increased significantly (p < 0.05) by 8.42%, 14.47%, and 8.66%,
respectively. In addition, the maximum value was observed in CM treatment. Meanwhile,
the application of BS could improve the size and weight of apples, but the effect was less
than that of CM treatment.

Table 2. Physical properties of apple fruit response to different fertilizers application.

Shape Color
Flesh
Fertilizer Vertical Transverse Weight of Single
Fruit Shape Firmness
Treatment Diameter Diameter Fruit (g) L* a* b*
Index (kg cm−2 )
(mm) (mm)
CK 72.81 ± 1.79 b 81.33 ± 2.07 b 0.89 ± 0.04 a 246.16 ± 9.18 c 54.20 ± 1.55 ab 32.52 ± 1.06 ab 17.72 ± 0.53 b 6.86 ± 0.24 b
CF 74.88 ± 4.09 ab 81.38 ± 1.52 b 0.92 ± 0.05 a 267.48 ± 13.98 b 50.93 ± 4.56 b 33.97 ± 5.56 a 17.24 ± 2.07 b 6.71 ± 0.23 b
CM 78.72 ± 4.32 a 87.51 ± 1.3 a 0.90 ± 0.04 a 281.79 ± 19.3 a 55.48 ± 5.48 a 29.70 ± 6.42 c 18.96 ± 1.86 a 7.43 ± 0.32 a
BS 75.68 ± 3.62 ab 83.37 ± 1.35 ab 0.89 ± 0.03 a 266.88 ± 17.63 b 52.20 ± 2.2 b 31.98 ± 3.03 b 17.50 ± 0.65 b 7.51 ± 0.34 a
Different letters in the same column indicate significant difference at (p < 0.05), L*: brightness; a*: red–green axis;
b*: yellow–blue axis. CK: Control Group, CF: Chemical Fertilizer, CM: Composted Manure, BS: Biogas Slurry.

Compared with the CK treatment, the ‘L*’ value of representing color brightness of
apple fruit outer epidermis was significantly decreased by applying chemical fertilizer, but
the ‘L*’ and ‘b*’ value of organic fertilizer was increased. Meanwhile, the lowest ‘a*’ value
was obtained by CM treatment. Compared with the CK and CF treatments, CM and BS
treatments could significantly increase the flesh firmness of apple fruit by 8.31% and 9.48%,
respectively (Table 2).

3.2. Chemical Properties of the Fruit

As shown in Table 3, the BS and CM treatment significantly (p < 0.05) increased the
total acid of apple fruit compared to the CK treatment, and CM treat apple fruit had highest
total acid (0.35%), followed by BS (0.30%), CF (0.245%), and CK (0.25%). In this study,
the application of organic fertilizer evidently reduced the TS/TA ratio with the minimum
TS/TA ratio (37.9) by CM treatment. Among all treatments, the lowest soluble solid content
was observed in the CK treatment (11.63 ◦ Brix), whereas BS treated apple fruit showed
maximum soluble solid (13.25 ◦ Brix) followed by CM (13.15 ◦ Brix) and CF (12.44 ◦ Brix).
Compared to the CK treatment, the vitamin C content of CF, CM, and BS treatment clearly
(p < 0.05) increased by 78.0%, 59.1%, and, 99%, respectively. However, the vitamin C content
of CM treatment was lower than that of CF and BS treatment. Meanwhile, the content of
crude protein in apple fruit treated with BS and CM was significantly increased compared
with the CK and CF treatment.
Agronomy 2022, 12, 2055 6 of 16

Table 3. Effects of different fertilizers on chemical properties of apple fruit.

Total Sugars Vitamin C Soluble Solid Crude Protein

Treatment Total Acid (%) TS/TA Ratio
(%) mg/100 g (◦ Brix) (%)
CK 12.70 b 0.25 c 53.0 a 1.81 d 11.63 c 0.24 b
CF 12.30 b 0.24 c 51.3 a 3.22 b 12.44 b 0.19 c
CM 13.25 ab 0.35 a 37.9 c 2.88 c 13.15 a 0.29 a
BS 13.85 a 0.30 b 46.2 b 3.61 a 13.25 a 0.27 a
Different letters in the same column indicate significant difference at (p < 0.05). TS: total sugars, TA: total acid. CK:
Control Group, CF: Chemical Fertilizer, CM: Composted Manure, BS: Biogas Slurry.

As shown in Table 4, compared with the CK, BS, and CM treatments significantly
increased the total amino acid concentration by 22.93% and 10.24%, respectively. As part
of the total amino acids, the essential amino acids in BS and CM treated apple fruit were
increased by 8.89% and 17.18%, and medicinal amino acids were increased by 7.64% and
24.84%, respectively.

Table 4. The amino acids content (%) of in apple fruit under different fertilizer treatments.

Amino Acid CK CF CM BS
Thr * 0.008 0.008 0.009 0.01
Vla * 0.007 0.006 0.008 0.007
Met *# 0.001 0 0 0
Ile *# 0.006 0.006 0.007 0.007
Leu *# 0.009 0.009 0.011 0.010
Phe *# 0.003 0.005 0.006 0.004
Lys *# 0.011 0.01 0.012 0.011
His 0.003 0.003 0.005 0.004
Arg # 0.009 0.004 0.005 0.004
Pro 0.01 0.011 0.013 0.013
Gly # 0.007 0.006 0.007 0.007
Ala 0.008 0.008 0.009 0.009
Cys 0 0 0 0
Asp # 0.093 0.081 0.099 0.134
Ser 0.009 0.008 0.01 0.01
Glu # 0.018 0.017 0.022 0.019
Tyr 0.003 0.003 0.003 0.003
E 0.045 c 0.044 c 0.053 a 0.049 b
N 0.16 c 0.141 d 0.173 b 0.203 a
T 0.205 c 0.185 d 0.226 b 0.252 a
M 0.157 c 0.138 d 0.169 b 0.196 a
T: Total amino acid, E: Total essential amino acid, N: Total nonessential amino acid, M: Medicinal amino acid,
* represents essential amino acid, # represent medicinal amino acid. Different letters in the same column indicate
significant difference at (p < 0.05). CK: Control Group, CF: Chemical Fertilizer, CM: Composted Manure, BS:
Biogas Slurry.

3.3. Soil Physicochemical Characteristics

In this work, the utilization of different fertilizers resulted in the variation of soil
physicochemical properties. As shown in Table 5, the addition of fertilizer significantly
(p < 0.05) improved the total nitrogen content in the 0–20 cm soil layer, and CM treatment
achieved the maximum total nitrogen (1.12 g kg−1 ). Furthermore, compared to the CM
treatment, the total nitrogen content of BS treatment decreased significantly. The fertilizer
application also increased soil total P content, and the CM treatment acquires the highest
total P content of 0.84 g kg−1 and 0.61 g kg−1 in 0–20 cm soil depth and 20–40 cm soil
depth, respectively. Notably, the total K content (0–20 cm soil layer) in BS treated soil had
no significant difference to the CK treatment. In addition, total N and total P decreased
with increasing soil depth. Meanwhile, the CM treatment presented the highest available P,
N, and K concentrations at different depths, while the minimum available P content came
from CK treatment. Furthermore, available P and available K decreased with the increase
Agronomy 2022, 12, 2055 7 of 16

of soil depth. Meanwhile, the fertilization can also increase soil organic matter content at
different depths, and CM treated soil achieved the best results. In addition, the soil organic
matter content decreased with increasing soil depth.

Table 5. Soil chemical properties under different fertilization treatments.

Soil Depth Total N Total P Total K Available N Available P Available K Organics

Fertilizers
(cm) (g kg−1 ) (g kg−1 ) (g kg−1 ) (mg kg−1 ) (mg kg−1 ) (mg kg−1 ) (%)
CK 0.60 d 0.52 d 23.25 a 50.19 b 2.31 d 167 c 0.74 d
CF 0.67 c 0.57 c 21.39 b 51.92 b 2.65 c 196 b 1.06 c
0–20
CM 1.12 a 0.84 a 23.20 a 58.84 a 26.93 a 487 a 1.64 a
BS 0.84 b 0.68 b 23.17 a 51.92 b 8.60 b 188 b 1.24 b
CK 0.32 c 0.57 b 19.27 c 46.72 b 1.39 c 116 b 0.45 c
CF 0.34 c 0.54 c 23.07 ab 41.53 c 1.27 d 114 b 0.45 c
20–40
CM 0.70 a 0.61 a 24.08 a 82.12 a 3.82 a 358 a 0.92 a
BS 0.47 b 0.56 b 22.40 b 38.07 d 1.55 b 101 c 0.63 b
Different letters in the same column indicate significant difference at (p < 0.05). CK: Control Group, CF: Chemical
Fertilizer, CM: Composted Manure, BS: Biogas Slurry.

3.4. Diversity of the Microbial Community

Table 6 shows the microbial diversity and abundance indices of all treatments. The
coverage estimator of all samples was greater than 0.98, indicating that these libraries
well represented the true situation of microbes in the samples. For bacterial community,
compared with CK treatment, Chao 1 index of CF treatment in a 0–20 cm depth layer
decreased significantly (p < 0.05). Inversely, an increase of Chao 1 and Shannon index was
observed in the BS and CM treatments. For the fungal community, compared with CK
treatment, the Shannon index of BS and CM treatment increased significantly (p < 0.05) and
Simpson index decreased significantly (p < 0.05). Compared with CK treatment, the CF
treatment could significantly (p < 0.05) decrease the Chao 1 index of bacteria in 0–20 cm
depth layer and could increase the Shannon index of fungi.

Table 6. Species diversity and abundance index at the different treatment and soil depth.

Soil Death (cm) Treatment Simpson Chao1 Shannon Coverage

Bacteria
CK 0.009 b 2079 b 6.25 a 0.99 a
CF 0.030 a 1849 c 5.50 b 0.99 a
0–20
CM 0.006 d 2239 a 6.48 a 0.99 a
BS 0.008 c 2091 b 6.32 a 0.98 a
CK 0.046 a 1921 b 4.98 b 0.99 a
CF 0.006 c 2121 a 6.34 a 0.99 a
20–40
CM 0.007 b 2204 a 6.36 a 0.99 a
BS 0.004 d 2110 a 6.42 a 0.99 a
Fungal
CK 0.107 a 297 c 3.31 d 1a
CF 0.061 b 256 d 3.68 c 1a
0–20
CM 0.036 c 412 b 3.98 b 1a
BS 0.025 d 464 a 4.38 a 1a
CK 0.173 a 266 c 2.54 c 1a
CF 0.045 b 407 a 3.97 b 1a
20–40
CM 0.025 c 340 b 4.23 a 1a
BS 0.044 b 354 b 4.1 ab 1a
Different letters in the same column indicate the significant difference at (p < 0.05). CK: Control Group,
CF: Chemical Fertilizer, CM: Composted Manure, BS: Biogas Slurry.

The similarity and overlap of OTUs number composition in different treatments

samples can be intuitively expressed via Venn diagram. As shown in Figure 1a, more than
 Agronomy 2022, 12, x FOR PEER REVIEW 8 of 16

Agronomy 2022, 12, 2055 8 of 16

The similarity and overlap of OTUs number composition in different treatments sam-
ples can be intuitively expressed via Venn diagram. As shown in Figure 1a, more than
two-thirds of the common bacterial OTUs were present in all treatment. The data dis-
two-thirds of the common bacterial OTUs were present in all treatment. The data distinctly
tinctly indicated that 174 fungal OUTs were common for different treatment, and more
indicated that 174 fungal OUTs were common for different treatment, and more than one
than one third of fungal OTUs exists only in specific treatments (Figure 1b).
third of fungal OTUs exists only in specific treatments (Figure 1b).

Figure 1. Venn diagram for bacterial (a) and fungal (b) operational taxonomic units (OTUs) found
Figure 1. Venn diagram
under different for treatments.
fertilization bacterial (a)CK:
andControl
fungal Group,
(b) operational taxonomic
CF: Chemical unitsCM:
Fertilizer, (OTUs) found
Composted
under
Manure,different fertilization
BS: Biogas Slurry. treatments. CK: Control Group, CF: Chemical Fertilizer, CM: Com-
posted Manure, BS: Biogas Slurry.
3.5. Microbial Community Structures
3.5. Microbial Community
In this study, a total Structures
of five fungi phyla were detected among all samples (Figure 2a).
The Inpreponderant
this study, a total ofwere
phyla Ascomycota
five fungi (91.8–56.8%),
phyla were followed
detected among by Mucoromycota
all samples (Figure 2a).
The preponderant phyla were Ascomycota (91.8–56.8%), followed byChytridiomycota
(41.09–1.21%), Basidiomycota (5.6–0.8%), Unclassified (7.3–1.3%), and Mucoromycota
(2.4–0%). The fungi
(41.09–1.21%), species Chytridiomycota
Basidiomycota with the (7.3–1.3%),
(5.6–0.8%), Unclassified function forand lignocellulose degra-
Chytridiomycota
dation was
(2.4–0%). Theonly
fungidetected
speciesinChytridiomycota
CM and BS treated soil.
with theNotably,
functiontheforrelative abundance
lignocellulose of
degra-
Basidiomycota
dation was onlyofdetected
fertilizer
in treated
CM andsoil BS increased compared
treated soil. Notably,with the CK.abundance
the relative Furthermore,of
Agronomy 2022, 12, x FOR PEER REVIEWthe diversity of fungi varied greatly at different soil depths. The relative abundance of 9 of 16
Basidiomycota of fertilizer treated soil increased compared with the CK. Furthermore, the
Ascomycota
diversity and Basidiomycota
of fungi varied greatly at decreases
differentwith
soilincreasing
depths. Thesoil depth.abundance of Asco-
relative
mycota and Basidiomycota decreases with increasing soil depth.
Figure 2b is showing the relative abundance of bacteria at phylum level. The common
dominant bacterial phyla, including Cyanobacteria (35.3–0.4%), Actinobacteria (31.4–
14.1%), Proteobacteria (29.8–15.8%), Firmicutes (31.6–5.3%), and Acidobacteria (20.9–
5.6%), were diffusely reported to present in orchard soil, and plays an important role in
promoting plant growth, inhibiting disease, and improving carbon availability[16]. Nota-
bly, compared with CK treatment, the addition of fertilizer not only changed the relative
abundances of the main bacterial phylum, but also changed the composition of the main
bacterial phylum. The relative abundance of Proteobacteria significantly increased from
23.7% in CK treatment to 28.0% in CF treatment and 28.3% in CM treatment in the 0–20
cm depth layer. The similar results were also obtained in 20–40 cm soil layer. In the 0–20
cm depth layer, the most dominant bacterial phylum in CK treatment were Actinobacteria
(28.2%), followed by Proteobacteria (23.7%) and Firmicutes (15.3%). In contrast, Acidobac-
teria became predominant species for CM and BS treated soil with relative abundance of
Figure
9.2% 2.and
Figure The soilsoil
2. 10.1%,
The microbial community
respectively.
microbial community composition
compositionunder
underdifferent
differentfertilization
fertilization treatments in phy-
treatments in
lumphylum
levels of fungi (a) and bacteria (b). 1: 0–20 cm depth layer; 2: 20–40 cm depth layer.
levels of fungi (a) and bacteria (b). 1: 0–20 cm depth layer; 2: 20–40 cm depth layer. CK: Control
Group, CF: Chemical
CK: Control Fertilizer,
Group, CF: ChemicalCM: Composted
Fertilizer, Manure, BS:
CM: Composted Biogas
Manure, BS:Slurry.
Biogas Slurry.

Figure 2b is showing the relative abundance of bacteria at phylum level. The common
4. Discussion
dominant bacterial phyla, including Cyanobacteria (35.3–0.4%), Actinobacteria (31.4–14.1%),
The attractiveness of apples to consumers is impacted by visual factors that contain
Proteobacteria (29.8–15.8%), Firmicutes (31.6–5.3%), and Acidobacteria (20.9–5.6%), were
shape, color, size, weight, firmness, etc. These appearance attributes of apple fruit are not
diffusely reported to present in orchard soil, and plays an important role in promoting plant
only influenced by inheritance,
growth, inhibiting disease, andbut also related
improving carbontoavailability
environmental growth conditions
[16]. Notably, compared [4].
Fruit diameter and fruit weight are important parameters affecting the commercial rate of
fruit and determining the market price of apple [25]. In our study, fertilizer application
can increase apple size and individual fruit weight compared with CK treatment. The re-
sults about variations of the fruit size to fertilizer treatment were consistent with findings
Agronomy 2022, 12, 2055 9 of 16

with CK treatment, the addition of fertilizer not only changed the relative abundances
of the main bacterial phylum, but also changed the composition of the main bacterial
phylum. The relative abundance of Proteobacteria significantly increased from 23.7% in
CK treatment to 28.0% in CF treatment and 28.3% in CM treatment in the 0–20 cm depth
layer. The similar results were also obtained in 20–40 cm soil layer. In the 0–20 cm depth
layer, the most dominant bacterial phylum in CK treatment were Actinobacteria (28.2%),
followed by Proteobacteria (23.7%) and Firmicutes (15.3%). In contrast, Acidobacteria
became predominant species for CM and BS treated soil with relative abundance of 9.2%
and 10.1%, respectively.

4. Discussion
The attractiveness of apples to consumers is impacted by visual factors that contain
shape, color, size, weight, firmness, etc. These appearance attributes of apple fruit are not
only influenced by inheritance, but also related to environmental growth conditions [4].
Fruit diameter and fruit weight are important parameters affecting the commercial rate of
fruit and determining the market price of apple [25]. In our study, fertilizer application can
increase apple size and individual fruit weight compared with CK treatment. The results
about variations of the fruit size to fertilizer treatment were consistent with findings by
Milošević and Mladenović, in which the effect of different fertilizers treatments on apple
fruit quality of ‘Idared’ was studied [2]. Other studies revealed that nitrogen and potassium
fertilizers were important factors affecting the weight and size of apple fruits, and fruit
weight can be increased with the increase of N application [26,27]. As confirmed by our
data, the available nitrogen and potassium components of the soil treated with CM were
significantly higher than those of the other treatments (Table 5), and the CM treatment
achieved the largest apple size and individual fruit weight, showing good commercial
characteristics. This may be due to the CM contains rich organic materials to promote
nitrogen transformation, and microporous structure of biochar in CM to reduce soil nitrogen
loss and improve plant nitrogen uptake [28]. Möller and Müller found that the nutrients in
BS could be easily absorbed by plants, but most nitrogen in BS would be lost in the form of
ammonia, indicating that the application of BS only had a significant effect on crop yield
and quality in a short period after application [29]. On the whole, the soil treated with
organic fertilizer can accelerate the transformation between soil mineralization and plant
absorption, improve the absorption efficiency of plant nutrients, and make organic fertilizer
treatment have better fruit weight and size. Meanwhile, there was no significant difference
in the fruit shape index between different fertilization treatments, possibly due to the fact
that fruit shape index was determined by the genetic level [30].
Color is one of the most important quality parameters for evaluating apple fruit, and
the color of apples can be attributed to many factors, including ripeness, ecology, genotype,
soil type, climate, etc. [31]. The results showed that, compared with CK treatment, the
application of chemical fertilizer and biogas slurry reduced the brightness (L*) color of fruit
epidermis, but there was no significant difference in CM treatment. This may be related
to the higher concentration of organic matter and potassium in the soil treated with CM.
Nava et al. and Kilic et al. found that the soil with high organic matter and potassium
level was more conducive to the development of fruit color [18,32]. Meanwhile, the ‘a*’
value of color index indicates the intensity of fruit color. The lower ‘a*’ value indicates
the lower red intensity [18]. CM treatment showed the lowest ‘a*’ value, representing the
lowest red intensity. This can be attributed to fact that CM treated apple tree absorbed high
nitrogen content and then resulted in a decrease in anthocyanin involved in red pigment
formation [33]. The variations in color ‘a*’ value and weight of single fruit were similar
to the findings by Amiri and Fallahi, who used animal manure to treat ‘Golden Delicious’
apple [34]. In addition, the synthesis of nitrogen and anthocyanins in red Fuji apple fruits
can be regulated by adding exogenous abscisic acid in order to improve the problem of
poor pigmentation caused by excessive nitrogen content in the fruit at the later stage of
apple development [35].
 high nitrogen content and then resulted in a decrease in anthocyanin involved in red pig-
ment formation [33]. The variations in color ‘a*’ value and weight of single fruit were sim-
ilar to the findings by Amiri and Fallahi, who used animal manure to treat ‘Golden Deli-
cious’ apple [34]. In addition, the synthesis of nitrogen and anthocyanins in red Fuji apple
Agronomy 2022, 12, 2055 fruits can be regulated by adding exogenous abscisic acid in order to improve the problem 10 of 16
of poor pigmentation caused by excessive nitrogen content in the fruit at the later stage of
apple development [35].
The storage ability of the same apple fruit variety depends not only on maturity and
The storage ability of the same apple fruit variety depends not only on maturity and
storage conditions, but also on firmness of the fruit [36]. As shown in Table 2, CM and BS
storage conditions, but also on firmness of the fruit [36]. As shown in Table 2, CM and
treatments could significantly increase the flesh firmness of apple fruit. This finding is
BS treatments could significantly increase the flesh firmness of apple fruit. This finding is
also in good agreement with the previous studies by Milošević et al. and Lin et al. [2,37].
also in good agreement with the previous studies by Milošević et al. and Lin et al. [2,37].
This phenomenon can be explained by reason that the organic fertilizer such as BS and
This phenomenon can be explained by reason that the organic fertilizer such as BS and
CM contain Ca, Mg, and other trace elements, which provide the foundation for cell wall
CM contain Ca, Mg, and other trace elements, which provide the foundation for cell wall
and membrane nutrient formation to improve the flesh firmness [38]. Küçükyumuk and
and membrane nutrient formation to improve the flesh firmness [38]. Küçükyumuk and
Erdal reported
Erdal reported thatthat spraying
spraying CaClCaCl2 solution on apple fruits could improve fruit weight,
2 solution on apple fruits could improve fruit weight,
size, firmness,
size, firmness, andand delay
delay fruit
fruit ripening
ripening under
under thethe condition
condition of of soil
soil calcium
calcium deficiency
deficiency [39].
[39].
In addition, the decrease in fruit firmness may also be related to excess
In addition, the decrease in fruit firmness may also be related to excess potassium fertilizer. potassium ferti-
lizer. Potassium
Potassium increases
increases fruitwhile
fruit size, size, while fruit growth
fruit growth is usually
is usually causedcaused
by theby the expansion
expansion of the
cell wall, which leads to a decrease in fruit density and a decrease in fruit fruit
of the cell wall, which leads to a decrease in fruit density and a decrease in firmness
firmness [32].
[32]. may
This This bemay thebereason
the reason
why thewhyapple
the apple
hardnesshardness
of CM oftreatment
CM treatment was slightly
was slightly lowerlower
than
thanof
that that
BS of BS treatment
treatment because because
the soilthe soil potassium
potassium concentration
concentration of BS treatment
of BS treatment was lowerwas
lower than that of CM
than that of CM treatment. treatment.
A PLS-DA
A PLS-DA multivariate
multivariatestatistical
statisticalmethod
methodwas wasconducted
conductedtoto display
display thethe differences
differences in
various physical properties of apple fruit under different fertilization treatments. FigureFig-
in various physical properties of apple fruit under different fertilization treatments. 3a
ure 3a the
shows shows the PLS-DA
PLS-DA score plotscore
withplotthewith
first the first principal
principal component component
(Component (Component
1) and the 1)
and the principal
second second principal
component component
(Component(Component 2) accounting
2) accounting for 70.9%for 70.9%
and 5.2%and of5.2%
theoftotal
the
total variability,
variability, respectively.
respectively. As shown
As shown in Figure
in Figure 3a, there
3a, there is slight
is slight separation
separation among among all
all the
the groups.
groups. TheThe variable
variable importance
importance in projection
in projection VIP VIP
scoresscores of different
of different traitstraits
was was calcu-
calculated
lated based
based on the onPLS-DA
the PLS-DA mode.mode. TheThe traits
traits withwith VIPscores
VIP scoreswere
weredisplayed
displayed in in Figure 3b. 3b.
Furthermore, using
Furthermore, usingaacutoff
cutoffofofp-value
p-value < 0.05
< 0.05 andand
VIPVIPscorescore
> 1,>physical
1, physical
traitstraits of apple
of apple fruit
fruit including
including vertical
vertical diameter,
diameter, ‘a*’ value,
‘a*’ value, and firmness
and firmness werewere determined
determined as theasvariables
the variables
that
thatmost
are are most influenced
influenced by different
by different fertilizer
fertilizer treatments.
treatments.

3. PLS-DA score plot (a) and VIP plot

Figure 3. plot of
of the
the potential
potential physicochemical
physicochemical markers (b) for the
apples under different fertilizer treatments. VD: VD: Vertical
Vertical diameter,
diameter, TD:
TD: Transverse
Transverse diameter, W:
Weight of
Weight of single
single fruit,
fruit, a*:
a*: red–green
red–green axis,
axis, b*:
b*: yellow–blue
yellow–blue axis.
axis. CK: Control Group, CF: Chemical
Chemical
Fertilizer, CM:
Fertilizer, CM: Composted
Composted Manure,Manure, BS:
BS: Biogas
BiogasSlurry.
Slurry.

acid is
Total acid is aa major
major index
index for
for predicting
predicting apple
apple fruit
fruit taste
taste because
because the
the consumers
consumers
generally have different preferences for sour or sweet apples [4]. We found that organic
fertilizer treatment can increase the total acid content of apple fruit, which was similar to
the results presented by Kai and Adhikari that the organic fertilizer can improve the total
acid content in contrast to CF [40]. As for the effect of fertilizers on acid content in apple
fruits, however, a previous literature work by Ernani et al. reported the organic fertilizer
reduced the content of total acid in apple (Royal Gala) fruit [41]. The effect of organic
fertilizer on total acid may be related to the difference in acid metabolism caused by the
specific knowledge of fertilizers, as well as other reasons such as different agricultural
climate conditions, quality factors, planting methods, tree age, etc. [3]. Meanwhile, some
Agronomy 2022, 12, 2055 11 of 16

studies reported that fruit acidity was not significantly affected by nitrogen fertilizer, and
was positively and linearly correlated with soil K content. The titratable acidity of fruit
juice increased linearly with the increase of potassium application rate [31]. In addition,
TS/TA is another key factor to evaluate fruit taste, which can be further applied as the
basis for apple fruit classification [2]. Specifically, apple fruit with a TS/TA ratio lower than
20 are appropriate for by-product processing and cider generation, while fruit varieties with
a TA/TS ratio higher than 20 are suitable for direct consumption. In this study, the results
proved that the CM and BS treatment could improve the acidity of apples, suggesting that
the CM and BS treated apples can be used for direct consumption and be more suitable for
consumers who prefer sour taste.
Soluble solids refer to all compounds dissolved in water in liquid or liquid food, in-
cluding sugars, acids, vitamins, proteins, minerals, etc., which are also important indicators
to evaluate the quality of apple fruits [40]. Compared to the CK treatment, the addition
of fertilizer could increase the soluble solid of apple fruit, possibly due to the fertilizers
containing a lot of nutrients that promote the formation of soluble solid [33]. Furthermore,
the soluble solid content of CM and BS treatment was higher than that of CF treatment,
likely due to the high amount of organic matter that could supply balanced nutrient to
soil respiration and further improve the nutrient transformation [42]. This finding is in
good agreement with the published literature by Milošević et al. in exploring the effects of
organic and mineral fertilizer on soil biochemical characteristics and fruit nutrient content
in apple orchards attributing this phenomenon to the fact that organic fertilizer can pro-
vide various nutrients to apple trees, improve soil pH value, and stimulate soil microbial
activity [2]. The effect of different fertilization on fruit vitamin C content was similar to that
found by Zhao et al., who explored the effects of different proportions of organic fertilizer
on apple fruit quality [43]. The results indicated that the increase of the proportion of
organic fertilizer resulted in the vitamin C content showing a trend of first increasing and
then decreasing, to the point lower than that of CF. Meanwhile, compared with CK and
CF treatment, organic fertilizer treatment has higher crude protein. This indicated that
organic fertilizer treatment could further promote nitrogen uptake by fruits and then result
in higher nutritional value, which may be because application of organic fertilizer could
promote the transformation of nitrogen by increasing the content of nitrate nitrogen, thus
improving the activity of nitrate reductase and improving the protein content of fruits.
Fertilization is one of the main factors affecting soil nutrient content in apple
orchards [17]. Long-term fertilization can improve soil fertility of apple orchard, but
excessive application of N, P, and K fertilizer will cause problems such as soil settling and
acidification [44]. In our study, both organic matter and total nitrogen in the soil treated with
fertilizer were increased, and the increase of organic matter and total N concentration in
the soil treated with CM was greater than that in the soil treated with BS and CK treatment;
Zhu et al. and Zhong et al. reported the similar conclusions [16,45]. Over the past decades,
the consistent application of organic fertilizers to apple orchards has improved soil fertility
and soil microbial community diversity. Meanwhile, long-term application of N fertilizer
could increase total N and available N in 0–20 cm soil depth of apple orchards [44]. Mean-
while, our study found that the soil nutrient reserve of BS treatment was lower than that
of CM treatment, likely due to the fact that nitrogen in BS is easily lost and volatilized
in the form of ammonia [46]. The total amount of nutrient content level can reflect the
reserve of soil nutrients and the available nutrient content level can reflect the dynamic
balance between soil mineralization and plant absorption [17]. Our results indicated that
CM treatment can effectively promote the increase of soil available nutrients, and the CM
treatment was better than BS and CF treatment. The results reported above were quite
similar to that of Wen et al., who attributes this to the fact that the application of organic
fertilizers increases the content of organic matter in the soil, thus speeding up the nutrient
cycling between the soil and the plants [6]. However, some scholars have found that
the content of available nitrogen, phosphorus, and potassium in fertilization experiments
Agronomy 2022, 12, 2055 12 of 16

utilizing CM and BS is lower than that of CF treatment [14], possibly due to the different
raw materials of aerobic compost and soil acidity and alkalinity.
Soil organic matter plays an important role in plant growth and is considered an im-
portant indicator of soil health and productivity, and high levels of soil organic matter not
only increase plant productivity but also improve soil microbial community diversity [40,47].
Some studies have shown that the application of biochar and organic fertilizer is conducive
to the winding and condensation of soil particles in the topsoil, and promotes the formation
of organic carbon [48]. The same results were obtained in our data, and the soil organic
matter content of CM treatment was higher than that of BS treatment. At the same time,
the application of chemical fertilizers also increased soil organic matter level, which may
be related to the promotion of plant root growth and the increase of organic matter input
in the rhizosphere [49]. In addition, the level of most soil properties decreased with the
increase of soil depth, and some studies have shown that 0–20 cm is an effective depth
system for simultaneous detection of soil fertility changes during apple production [44].
Soil microorganisms are important drivers of agro-ecosystem function, and their
diversity and biomass are critical to soil quality and health [50]. In our experiment, the
application of different types of fertilizers in apple orchards not only greatly changed the
physical and chemical properties of soil, but also affected the composition and structure
of bacterial communities. Generally, both Chao 1 index and Shannon index can reflect the
diversity and richness of microbial community, respectively. The higher Shannon index
represents a more complex microbial community, and the higher Chan 1 index represents a
more species richness [51]. Compared with the CK, the addition of CF clearly decreased
species richness in the 0–20 cm soil layer, likely due to the fact that the soil were acidified
because of long-term application of fertilizer [16,52]. Inversely, the BS or CM could improve
the richness and diversity of soil bacteria [17], suggesting that a possible benefit of organic
fertilizer application can increase abundance and activity of soil biota [53]. Compared with
CK, the diversity and abundance of fungi in BS and CK soil were increased, possibly due
to the fact that CM and BS contain more balanced nutrients [16]. Some studies have shown
that soil with balanced nutrients contains more soil microorganisms, and available nitrogen
and potassium are the key factors for the growth of soil microorganisms [54]. Meanwhile,
compared with CK treatment, the CF treatment could significantly (p < 0.05) decrease the
Chao 1 index of bacteria in the 0–20 cm depth layer and could increase the Shannon index
of fungi, which explained that the utilization of CF showed a positive effect on fungal
growth, but a negative effect on soil bacteria abundance [50]. The results showed that the
bacterial community variations were mainly reflected by ratio, while the fungal community
differences were mainly reflected by species variety. Meanwhile, according to the similarity
and overlap of OTU number composition of different processed samples in Figure 1, our
study found that the bacterial community variations were mainly reflected by ratio, while
the fungal community differences were mainly reflected by species variety.
Fertilization alters the distribution of major phyla of bacteria and fungi. In our study,
Chytridiomycota was only detected in soil treated with CM and BS. It is reposted that
Chytridiomycota can be used for plant absorption and nutrient cycling by converting
organic sources of nitrogen and phosphorus into inorganic forms such as nitrate and
ammonia phosphate, and can also help degrade cellulose and chitin in materials [55,56].
These results indicated that the soil treated by CM and BS may be more beneficial to the
growth of apple trees and the improvement of apple fruit quality. Compared with the CK
treatment, the relative abundance of Basidiomycetes increased under fertilization, probably
because soil organic matter provided energy for Basidiomycetes reproduction. Kjøller
and Rosendahl discovered that Basidiomycota plays a significant role in decomposition
of soil organic matter, and they can decompose complex organic matter under the action
of enzymes [57]. Furthermore, the relative abundance of Ascomycota and Basidiomycota
decreases with increasing soil depth, indicating that the change of microbial community
also has a relationship with soil depth. This phenomenon can be attributed to the fact that
Agronomy 2022, 12, 2055 13 of 16

the different fertilization resulted in the variation of root exudates, which are the main
nutrients of the rhizosphere microbes and may be affected by plant nutrient status [58].
The results of our study indicated that fertilization resulted in significant changes
in bacterial community structure. Previous literature has showed that Proteobacteria
are Gram-negative bacteria with an effective function of nitrogen fixation in symbiotic
legumes [17]. Our study found that fertilizer application could increase the relative abun-
dance of Proteobacteria, possibly due to the fact that nutrient-rich soil is beneficial to the
growth of Proteobacteria [59]. The Bacteroideds play an important role in the transforma-
tion of lipids, proteins, and other organic materials. The Actinobacteria was participated
in cellulose and chitin degrading for organic material active turnover [60]. The phylum
Acidobacteria is an oligotrophic bacteria and favors the conditions with lower soil organic
C, their members are adapted to malnourished soils and are associated with turnover of soil
organic carbon and nutrient availability [17]. Firmicutes are the most dynamic group that
is associated with disease inhibition [17]. Compared with the CK, the relative abundance of
Firmicutes in BS and CM treatment was relatively low, which may be because the addition
of BS and CM could supply a healthy soil environment for the growth of the root system.
Furthermore, the bacterial community composition varies with the changes of soil depth
due to the difference of the physicochemical characteristics (nutrients and soil organics)
in various soil layers as shown in Table 5. On the whole, CM treatment can significantly
improve the diversity and community structure of the soil microbial community, which
is beneficial for improving the ability of plant organic matter degradation, plant growth,
disease resistance, and nitrogen fixation.

5. Conclusions
In this study, the influence of chemical fertilizer (CF), biogas slurry (BS) and composted
manure (CM) on the quality of apple fruits, soil nutrients, and soil microbial diversity were
evaluated. The results showed that fertilizers played an important role in the physico-
chemical properties of apple fruit and soil. Compared with the control group, the levels of
vertical diameter, transverse diameter, weight of single fruit, and soluble solid increased by
8.42–14.47%, 0.01–7.35%, 8.84–14.47%, and 6.96–13.93%, respectively, for CF, CM, and BS
treatments. At these levels, CM treatment obtains the best results. Meanwhile, the CM and
BS treatments could significantly increase fruit hardness, essential amino acid, and total
acid compared to CK and CF treatments. Among all treatments, the application of CM sig-
nificantly increased the accumulation of soil organic matter and total nitrogen, accelerated
the nutrient transformation between plants and soil, and improved the functional diversity
of soil communities, thus ensuring more stable and higher apple quality. The results ob-
tained in this study reveals the insight effect of organic fertilizer on improvement of fruit
quality and soil health and could potentially promote organic cultivation of apple fruit and
green agricultural development. However, the further investigation of organic fertilization
affection on root nutrient metabolism and the activities of rhizospheric microorganism is
needed in future study.

Author Contributions: Conceptualization, X.W. and G.S.; software, X.W.; investigation, G.S.;
resources, G.S. and J.L.; data curation, X.W. and Q.B.; writing—original draft preparation, X.W.;
writing—review and editing, X.W., J.L. and G.S.; project administration, J.L. and G.S. All authors
have read and agreed to the published version of the manuscript.
Funding: The current work is supported by the Key R&D Program of Shaanxi Province (2022ZDLNY02-09,
2019TSLNY01-03), China Agriculture Research System (CARS-23-C-05), and the Post-doctoral Foun-
dation of the Shaanxi Province (2018BSHEDZZ20).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The dataset analyzed in this article is available upon request to the
corresponding authors.
Agronomy 2022, 12, 2055 14 of 16

Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.

References
1. Huo, X.; Liu, T.; Liu, X.; Wei, Y.; Yao, X.; Ma, X.; Lu, F. 2020 China Apple Industry Development Report (Simplified Version). China
Fruit Veg. 2022, 42, 6. [CrossRef]
2. Milošević, T.; Milošević, N.; Mladenović, J. Tree Vigor, Yield, Fruit Quality, and Antioxidant Capacity of Apple (Malus ×
Domestica Borkh.) Influenced by Different Fertilization Regimes: Preliminary Results. Turk. J. Agric. For. 2019, 43, 48–57.
[CrossRef]
3. Milošević, T.; Milošević, N. Influence of Mineral Fertilizer, Farmyard Manure, Natural Zeolite, and Their Mixture on Fruit Quality
and Leaf Micronutrient Levels of Apple Trees. Commun. Soil Sci. Plant Anal. 2017, 48, 539–548. [CrossRef]
4. Nour, V.; Trandafir, I.; Ionica, M.E. Compositional Characteristics of Fruits of Several Apple (Malus Domestica Borkh.) Cultivars.
Not. Bot. Horti Agrobot. Cluj-Napoca 2010, 38, 228–233. [CrossRef]
5. Mi, S.; Zhang, X.; Wang, Y.; Ma, Y.; Sang, Y.; Wang, X. Effect of Different Fertilizers on the Physicochemical Properties, Chemical
Element and Volatile Composition of Cucumbers. Food Chem. 2022, 367, 130667. [CrossRef]
6. Wen, Y.C.; Li, H.Y.; Lin, Z.A.; Zhao, B.Q.; Sun, Z.-B.; Yuan, L.; Xu, J.K.; Li, Y.Q. Long-Term Fertilization Alters Soil Properties and
Fungal Community Composition in Fluvo-Aquic Soil of the North China Plain. Sci. Rep. 2020, 10, 7198. [CrossRef]
7. Chen, J.; Lü, S.; Zhang, Z.; Zhao, X.; Li, X.; Ning, P.; Liu, M. Environmentally Friendly Fertilizers: A Review of Materials Used
and Their Effects on the Environment. Sci. Total Environ. 2018, 613–614, 829–839.
8. Wang, Q.; Li, R.; Cai, H.; Awasthi, M.K.; Zhang, Z.; Wang, J.J.; Ali, A.; Amanullah, M. Improving Pig Manure Composting
Efficiency Employing Ca-Bentonite. Ecol. Eng. 2016, 87, 157–161. [CrossRef]
9. Khorram, M.S.; Wang, Y.; Jin, X.; Fang, H.; Yu, Y. Reduced Mobility of Fomesafen through Enhanced Adsorption in Biochar-
Amended Soil. Environ. Toxicol. Chem. 2015, 34, 1258–1266. [CrossRef]
10. Schulz, H.; Dunst, G.; Glaser, B. Positive Effects of Composted Biochar on Plant Growth and Soil Fertility. Agron. Sustain. Dev.
2013, 33, 817–827. [CrossRef]
11. Liang, F.; Xu, L.; Ji, L.; He, Q.; Wu, L.; Yan, S. A New Approach for Biogas Slurry Disposal by Adopting CO2-Rich Biogas Slurry as
the Flower Fertilizer of Spathiphyllum: Feasibility, Cost and Environmental Pollution Potential. Sci. Total Environ. 2021, 770, 145333.
[CrossRef] [PubMed]
12. Safaei Khorram, M.; Zhang, G.; Fatemi, A.; Kiefer, R.; Maddah, K.; Baqar, M.; Zakaria, M.P.; Li, G. Impact of Biochar and Compost
Amendment on Soil Quality, Growth and Yield of a Replanted Apple Orchard in a 4-Year Field Study. J. Sci. Food Agric. 2019, 99,
1862–1869. [CrossRef] [PubMed]
13. Li, Z.; Wang, M.; Yang, Y.; Zhao, S.; Zhang, Y.; Wang, X. Effect of Composted Manure plus Chemical Fertilizer Application
on Aridity Response and Productivity of Apple Trees on the Loess Plateau, China. Arid Land Res. Manag. 2017, 31, 388–403.
[CrossRef]
14. Chen, Z.; Wang, Q.; Ma, J.; Chapman, S.; Zou, P.; Ye, J.; Yu, Q.; Sun, W.; Lin, H.; Jiang, L. Soil Microbial Activity and Community
Composition as Influenced by Application of Pig Biogas Slurry in Paddy Field in Southeast China. Paddy Water Environ. 2020, 18,
15–25. [CrossRef]
15. Leff, J.W.; Jones, S.E.; Prober, S.M.; Barberán, A.; Borer, E.T.; Firn, J.L.; Harpole, W.S.; Hobbie, S.E.; Hofmockel, K.S.; Knops, J.M.H.;
et al. Consistent Responses of Soil Microbial Communities to Elevated Nutrient Inputs in Grasslands across the Globe. Proc. Natl.
Acad. Sci. USA 2015, 112, 10967–10972. [CrossRef]
16. Zhu, Z.; Bai, Y.; Lv, M.; Tian, G.; Zhang, X.; Li, L.; Jiang, Y.; Ge, S. Soil Fertility, Microbial Biomass, and Microbial Functional
Diversity Responses to Four Years Fertilization in an Apple Orchard in North China. Hortic. Plant J. 2020, 6, 223–230. [CrossRef]
17. Wang, L.; Li, J.; Yang, F.; Yaoyao, E.; Raza, W.; Huang, Q.; Shen, Q. Application of Bioorganic Fertilizer Significantly Increased
Apple Yields and Shaped Bacterial Community Structure in Orchard Soil. Microb. Ecol. 2017, 73, 404–416. [CrossRef]
18. Kilic, N.; Burgut, A.; Gündesli, M.A.; Nogay, G.; Ercisli, S.; Kafkas, N.E.; Ekiert, H.; Elansary, H.O.; Szopa, A. The Effect of Organic,
Inorganic Fertilizers and Their Combinations on Fruit Quality Parameters in Strawberry. Horticulturae 2021, 7, 354. [CrossRef]
19. Kafkas, E.; Koşar, M.; Paydaş, S.; Kafkas, S.; Başer, K.H.C. Quality Characteristics of Strawberry Genotypes at Different Maturation
Stages. Food Chem. 2007, 100, 1229–1236. [CrossRef]
20. Wang, H.; Zhang, Q.; Weng, B.; Li, F.; An, M.; Luo, T. Effect of Different Fertilizer on Accumulation, Distribution and Assimilation
of Nitrate in Radish. Nongye Gongcheng Xuebao/Trans. Chin. Soc. Agric. Eng. 2016, 32, 148–154. [CrossRef]
21. He, Y.; Zhang, C.; Zhang, X.; Xv, G.; Hei, G. Key Points of Sample Collection and Preparation for Detailed Investigation of Soil
Pollution in Agricultural Land. Soush China Agric. 2021, 15, 230–231. [CrossRef]
22. Zhang, S.; Yang, W.; Muneer, M.A.; Ji, Z.; Tong, L.; Zhang, X.; Li, X.; Wang, W.; Zhang, F.; Wu, L. Integrated Use of Lime with Mg
Fertilizer Significantly Improves the Pomelo Yield, Quality, Economic Returns and Soil Physicochemical Properties under Acidic
Soil of Southern China. Sci. Hortic. 2021, 290, 110502. [CrossRef]
23. Chen, Y.; Wen, X.; Sun, Y.; Zhang, J.; Wu, W.; Liao, Y. Mulching Practices Altered Soil Bacterial Community Structure and
Improved Orchard Productivity and Apple Quality after Five Growing Seasons. Sci. Hortic. 2014, 172, 248–257. [CrossRef]
Agronomy 2022, 12, 2055 15 of 16

24. GAO, X.-s.; XIAO, Y.; DENG, L.-j.; LI, Q.-q.; WANG, C.-q.; LI, B.; DENG, O.-p.; ZENG, M. Spatial Variability of Soil Total Nitrogen,
Phosphorus and Potassium in Renshou County of Sichuan Basin, China. J. Integr. Agric. 2019, 18, 279–289. [CrossRef]
25. Milošević, T.; Milošević, N.; Mladenović, J. The Influence of Organic, Organo-Mineral and Mineral Fertilizers on Tree Growth,
Yielding, Fruit Quality and Leaf Nutrient Composition of Apple Cv. ‘Golden Delicious Reinders’. Sci. Hortic. 2022, 297, 110978.
[CrossRef]
26. Wang, C.; Zhu, F.; Liu, L.; Xv, D.; Xv, B. Effects of Nitrogen, Phosphorus and Potassium Fertilizer on Yield, Quality and Content of
Mineral Elements in Leaves of Red Fuji Apple. China Fruit 2009, 2, 14–17. [CrossRef]
27. Raese, J.T.; Drake, S.R. Nitrogen Fertilization and Elemental Composition Affects Fruit Quality of “Fuji” Apples. J. Plant Nutr.
1997, 20, 1797–1809. [CrossRef]
28. Purnomo, E.; Sinaga, F.; Amanda, I.P.; Putra, R.D.P. Balance of Nitrogen in Plant-Soil System with the Presence of Com-
post+Charcoal. Pertanika J. Trop. Agric. Sci. 2018, 41, 833–844.
29. Möller, K.; Müller, T. Effects of Anaerobic Digestion on Digestate Nutrient Availability and Crop Growth: A Review. Eng. Life Sci.
2012, 12, 242–257.
30. Cao, K.; Chang, Y.; Sun, R.; Shen, F.; Wu, T.; Wang, Y.; Zhang, X.; Han, Z. Candidate Gene Prediction via Quantitative Trait Locus
Analysis of Fruit Shape Index Traits in Apple. Euphytica 2015, 206, 381–391. [CrossRef]
31. Hernández-Rodríguez, A.; Montaño-Carrasco, M.; Ojeda-Barrios, D.; Martínez-Rosales, A.; Cruz-Álvarez, O.; Martínez-Damián,
M.T.; Rodríguez-Roque, M.J. Foliar Nutritional Content and Apple Fruit Quality as Affected by Organic, Conventional, or
Integrated Management. J. Plant Nutr. 2021, 44, 1886–1902. [CrossRef]
32. Nava, G.; Dechen, A.R.; Nachtigall, G.R. Nitrogen and Potassium Fertilization Affect Apple Fruit Quality in Southern Brazil.
Commun. Soil Sci. Plant Anal. 2008, 39, 96–107. [CrossRef]
33. Jakopic, J.; Schmitzer, V.; Veberic, R.; Smrke, T.; Stampar, F. Metabolic Response of ‘Topaz’ Apple Fruit to Minimal Application of
Nitrogen during Cell Enlargement Stage. Horticulturae 2021, 7, 266. [CrossRef]
34. Amiri, M.E.; Fallahi, E. Impact of Animal Manure on Soil Chemistry, Mineral Nutrients, Yield, and Fruit Quality in “golden
Delicious” Apple. J. Plant Nutr. 2009, 32, 610–617. [CrossRef]
35. Wang, F.; Sha, J.; Chen, Q.; Xu, X.; Zhu, Z.; Ge, S.; Jiang, Y. Exogenous Abscisic Acid Regulates Distribution of 13C and 15N and
Anthocyanin Synthesis in ‘Red Fuji’ Apple Fruit Under High Nitrogen Supply. Front. Plant Sci. 2020, 10, 1738. [CrossRef]
36. Skendrović Babojelić, M.; Ivančić, K.; Družić, J.; Kovač, A.; Voća, S. Chemical and Sensory Characteristics of Three Apple Cultivarz
(Malus x Domestica Borkh.). Agric. Conspec. Sci. 2007, 72, 317–322.
37. Lin, R.U.I.; Yu-ying, Y.; Ti-jiang, R.U.I.; Li-chun, L.I.U.; Peng-fei, Z.; Chun-xiang, Y.O.U.; Yu-jin, H.A.O. Influence of Biogas Slurry
on the Growth and Fruit Quality of Red Fuji Apple Trees in Low Latitude Plateau. J. Shandong Agric. Univ. 2020, 51, 985–991.
[CrossRef]
38. Murtić, S.; Oljača, R.; Koleška, I.; Čivić, H. Apple Quality and Calcium Content as Affected by Fertilizer Treatment. Polish J.
Environ. Stud. 2017, 26, 2107–2111. [CrossRef]
39. Küçükyumuk, Z.; Erdal, İ. Effect of Calcium on Mineral Nutrient Concentrations and Fruit Quality in Different Apple Tree
Varieties. J. Elem. 2022, 27, 75–85. [CrossRef]
40. Kai, T.; Adhikari, D. Effect of Organic and Chemical Fertilizer Application on Apple Nutrient Content and Orchard Soil Condition.
Agriculture 2021, 11, 340. [CrossRef]
41. Ernani, P.R.; Rogeri, D.; Proença, M.M.; Dias, J. Addition of Nitrogen Had No Effect on Yield and Quality of Apples in an High
Density Orchard Carrying a Dwarf Rootstock. Rev. Bras. Frutic. 2008, 30, 1113–1118. [CrossRef]
42. Crop, A.; Society, S. Use of Mineral, Organic, Slow Release and Biofertilizers for Anna Apple Trees in a Sandy Soil. In Proceedings
of the 8th African Crop Science Society Conference, El-Minia, Egypt, 27–31 October 2007; Volume 8, pp. 265–271.
43. Zhao, Z.P.; Yan, S.; Liu, F.; Ji, P.H.; Wang, X.Y.; Tong, Y.A. Effects of Chemical Fertilizer Combined with Organic Manure on Fuji
Apple Quality, Yield and Soil Fertility in Apple Orchard on the Loess Plateau of China. Int. J. Agric. Biol. Eng. 2014, 7, 45–55.
[CrossRef]
44. Hou, L.; Liu, Z.; Zhao, J.; Ma, P.; Xu, X. Comprehensive Assessment of Fertilization, Spatial Variability of Soil Chemical Properties,
and Relationships among Nutrients, Apple Yield and Orchard Age: A Case Study in Luochuan County, China. Ecol. Indic. 2021,
122, 107285. [CrossRef]
45. Zhong, W.; Gu, T.; Wang, W.; Zhang, B.; Lin, X.; Huang, Q.; Shen, W. The Effects of Mineral Fertilizer and Organic Manure on Soil
Microbial Community and Diversity. Plant Soil 2010, 326, 511–522. [CrossRef]
46. Möller, K.; Stinner, W.; Deuker, A.; Leithold, G. Effects of Different Manuring Systems with and without Biogas Digestion on
Nitrogen Cycle and Crop Yield in Mixed Organic Dairy Farming Systems. Nutr. Cycl. Agroecosystems 2008, 82, 209–232. [CrossRef]
47. Wang, L.; Yang, F.; Yao, E.; Yuan, J.; Raza, W.; Huang, Q.; Shen, Q. Long-Term Application of Bioorganic Fertilizers Improved
Soil Biochemical Properties and Microbial Communities of an Apple Orchard Soil. Front. Microbiol. 2016, 7, 1893. [CrossRef]
[PubMed]
48. Duan, Y.; Yang, J.; Song, Y.; Chen, F.; Li, X.; Awasthi, M.K.; Li, H.; Zhang, L. Clean Technology for Biochar and Organic Waste
Recycling, and Utilization in Apple Orchard. Chemosphere 2021, 274, 129914. [CrossRef]
49. Tan, B.; Fan, J.; He, Y. Effect of Long-Term Application of Chemical Fertilizer on Soil Organic Carbon Content in Top Layer of
Paddy Fields in South China. Acta Pedol. Sin. 2014, 51, 96–103.
Agronomy 2022, 12, 2055 16 of 16

50. Sabir, M.S.; Shahzadi, F.; Ali, F.; Shakeela, Q.; Niaz, Z.; Ahmed, S. Comparative Effect of Fertilization Practices on Soil Microbial
Diversity and Activity: An Overview. Curr. Microbiol. 2021, 78, 3644–3655. [CrossRef]
51. Shao, Z.; Guo, X.; Qu, Q.; Kang, K.; Su, Q.; Wang, C.; Qiu, L. Effects of Chlorine Disinfectants on the Microbial Community
Structure and the Performance of Anaerobic Digestion of Swine Manure. Bioresour. Technol. 2021, 339, 125576. [CrossRef]
52. Tamilselvi, S.M.; Chinnadurai, C.; Ilamurugu, K.; Arulmozhiselvan, K.; Balachandar, D. Effect of Long-Term Nutrient Manage-
ments on Biological and Biochemical Properties of Semi-Arid Tropical Alfisol during Maize Crop Development Stages. Ecol. Indic.
2015, 48, 76–87. [CrossRef]
53. Liu, W.; Wang, Q.; Wang, B.; Wang, X.; Franks, A.E.; Teng, Y.; Li, Z.; Luo, Y. Changes in the Abundance and Structure of
Bacterial Communities under Long-Term Fertilization Treatments in a Peanut Monocropping System. Plant Soil 2015, 395, 415–427.
[CrossRef]
54. Kumar, U.; Shahid, M.; Tripathi, R.; Mohanty, S.; Kumar, A.; Bhattacharyya, P.; Lal, B.; Gautam, P.; Raja, R.; Panda, B.B.;
et al. Variation of Functional Diversity of Soil Microbial Community in Sub-Humid Tropical Rice-Rice Cropping System under
Long-Term Organic and Inorganic Fertilization. Ecol. Indic. 2017, 73, 536–543. [CrossRef]
55. Hanrahan-tan, D.G.; Henderson, L.; Kertesz, M.A.; Lilje, O. The Effects of Nitrogen and Phosphorus on Colony Growth and
Zoospore Characteristics of Soil Chytridiomycota. J. Fungi 2022, 8, 341. [CrossRef]
56. Zhao, B.; Xing, P.; Wu, Q.L. Microbes Participated in Macrophyte Leaf Litters Decomposition in Freshwater Habitat. FEMS
Microbiol. Ecol. 2017, 93, fix108. [CrossRef]
57. Kjøller, R.; Rosendahl, S. Cultivated and Fallow Fields Harbor Distinct Communities of Basidiomycota. Fungal Ecol. 2014, 9,
43–51. [CrossRef]
58. Singh, B.; Pandey, R. Differences in Root Exudation among Phosphorus-Starved Genotypes of Maize and Green Gram and Its
Relationship with Phosphorus Uptake. J. Plant Nutr. 2003, 26, 2391–2401. [CrossRef]
59. Shange, R.S.; Ankumah, R.O.; Ibekwe, A.M.; Zabawa, R.; Dowd, S.E. Distinct Soil Bacterial Communities Revealed under a
Diversely Managed Agroecosystem. PLoS ONE 2012, 7, e40338. [CrossRef]
60. Duan, Y.; Zhang, L.; Yang, J.; Zhang, Z.; Awasthi, M.K.; Li, H. Insight to Bacteria Community Response of Organic Management
in Apple Orchard-Bagasse Fertilizer Combined with Biochar. Chemosphere 2022, 286, 131693. [CrossRef]