agronomy

Article
Short-Term Effects of Bio-Organic Fertilizer on Soil Fertility and Bacterial
Community Composition in Tea Plantation Soils

Zhenmin Hu 1,†, Lingfei Ji 2,† , Qing Wan 1, Huan Li 1 , Ronglin Li 1 and

Yiyang Yang 1,*

1 Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement,

Institute of Leisure Agriculture, Jiangsu Academy of Agricultural Sciences,
Nanjing 210014, China
2 Key Laboratory of Biology, Genetics and Breeding of Special Economic
Animals and Plants, Ministry of
Agriculture and Rural Affairs, Hangzhou 310008, China
* Correspondence: yangyiyang_yyy@126.com; Tel.: +86-025-8439-1693
† These authors contributed equally to this work.

Citation: Hu, Z.; Ji, L.; Wan, Q.; Li, H.; Li, R.; Yang, Y. Short-Term Effects of
Bio-Organic Fertilizer on Soil Fertility and Bacterial Community Composition in
Tea Plantation Soils. Agronomy 2022, 12, 2168. https://
doi.org/10.3390/agronomy12092168

Academic Editor: David Houben

Received: 8 August 2022

Accepted: 8 September 2022
Published: 13 September 2022

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in

published maps and institutional affil- iations.
Abstract: Overuse of chemical fertilizers to maintain tea production has caused
many adverse effects in tea plantations and largely hampers the sustainable
development of the tea industry. Applying bio-organic fertilizer (BOF) to achieve
the goal of sustainable agriculture has become popular be- cause of its
advantages, such as its pollution-free nature, considerable amount of beneficial
microbes and soil-friendly organic materials. However, the effects of BOF
application on tea plantation soil remain an open question. Herein, we carried
out a 3-year pot experiment with four treatments, including control without
fertilization (CK), 100% chemical fertilizer (CF), 50% chemical fertilizer
+50% BOF (CFOF) and 100% BOF (OF), to explore the effects of BOF application
on soil fertility and bacterial community in tea plantations. The results showed
that BOF application could increase soil fertility in both bulk and rhizosphere
soils and improve the biomass of tea leaves. In addition, the nutrient level
change caused by BOF application significantly changed bacterial community
diversity and composition and accounted for 74.91% of the community variation.
CFOF and OF treatments significantly increased the bacterial Chao1 and Shannon
indices compared to CF treatment (p < 0.05). Moreover, bacterial community
composition was dominated by Betaproteobacteria (46.88%), Acidobacteria (11.29%),
Alphaproteobacteria (9.69%) and Gammaproteobacteria (9.59%). BOF application
increased the relative abundance of Alphaproteobacteria, Acidobacteria,
Deltaproteobacteria and plancto- mycetes and decreased the relative abundance of
Betaproteobacteria (p < 0.05). Furthermore, bacterial function prediction revealed
that BOF application improved the N and C cycling processes and enhanced the co-
occurrence network complexity in the bulk soils. Bacterial community functions and
co-occurrence networks in the rhizosphere did not show similar results,
indicating that rhizosphere bacterial communities were more affected by the
rhizosphere effect than BOF application. All these findings verified our hypothesis
that applying BOF in tea plantations could increase the biomass of tea plants by
improving soil fertility and influencing the soil bacterial function groups. In
summary, we suggested that BOF application could be a promising way to achieve
the sustainable development of the tea industry.

Keywords: bio-organic fertilizer; bacterial community; bulk soils; rhizosphere;

tea plantation soils

Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This

article is an open access article distributed under the terms and conditions of
the Creative Commons Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
Tea plants are one of the most popular beverage crops worldwide. Large quantities
of synthetic fertilizers (e.g., urea, potassium sulphate and superphosphate) are
applied in tea plantations to achieve economic value [1,2]. Inevitably, excessive
synthetic fertilizers application has resulted in many environmental problems,
such as aggravating and acceler- ating soil acidification [1,3], leaching risk
[4], loss of soil microbial diversity [5] and so on. However, in recent years,
applying organic fertilizer in tea plantations has been promoted because organic
substitution in tea plantations can significantly mitigate soil acidification,

Agronomy 2022, 12, 2168. https://doi.org/10.3390/agronomy12092168

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Agronomy 2022, 12, 2168
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improve soil fertility, increase soil microbial diversity and maintain tea yield
[5–7]. Nev- ertheless, the application of animal-derived organic fertilizers also
has the risk of heavy metal, antibiotic and pathogen microorganism contamination
[8–10]. For example, chicken manure treatment significantly increased available
As, Pb, Cu and Zn concentrations com- pared to chemical fertilizer treatment in
tea plantation [11]. Thus, bio-organic fertilizer (BOF) has raised much attention
because it is pollution-free and contains considerable beneficial microbes and
soil-friendly organic materials [12,13]. After BOF application in a pigeon pea
field, the electrical conductivity and bulk density were drastically reduced while
porosity, organic carbon and water holding capacity were significantly elevated
[14]. In a field trial and continuous pot experiments in tomatoes, BOF produced
tomato yields equivalent to those obtained using the 100% chemical fertilizer and
improved tomato quality. This may be due to improved soil fertility and soil
microbial activity [15]. The application of BOF in degraded red soil improved soil
nutrients, soil microbial diversity and plant growth [16].
Soil bacteria have been proven to play vital roles in soil nutrient cycling
and are directly or indirectly involved in nitrogen (N) cycling processes and
phosphorus (P) miner- alization [17,18]. Previous studies have demonstrated that
BOF application can significantly influence soil bacterial communities through the
specific action of microbial inoculants or synergistic interaction with the
resident soil bacterial communities, thus impacting rhizosphere microbial
activity and plant growth [19,20]. For instance, BOF application can dramatically
increase plant biomass and fruit yield in pomegranate plants by improving
rhizosphere activity and soil fertility [19]. In addition, a recent study has
revealed that BOF application could stimulate indigenous soil Pseudomonas
populations to enhance plant disease suppression [20]. Moreover, BOF application
could also accelerate the metabolism of specific microorganisms and restore soil’s
natural nutrient cycling ability [21–23]. There- fore, we hypothesized that
applying BOF in tea plantations may increase the biomass of tea plants by
improving soil fertility and influencing the soil bacterial function groups.
In addition, the rhizosphere microbiome is considered the second genome of plants
and exerts a critical role in plant growth [24–26]. For example, after
inoculation with Bacillus licheniformis MH48, which was isolated from rhizosphere
soil, Camellia japonica seedling development was improved in coastal lands
[27]. The application of BOF in potato field experiments reduced the use of
chemical fertilizers and promoted potato growth [28]. Studies have also shown
that the nutrient preference of plants can significantly alter their rhizosphere
microbiome [29]. Tea plants prefer absorbing ammonia to maintain their growth,
which will result in rhizosphere acidification [30]. However, whether the
ammonia preference of tea plants will influence the rhizosphere microbiome is
still an open question. In the meantime, some studies have proved that long-term
fertilization could change the rhizosphere microbiome. For example, recent
studies have reported that long- term fertilization rather than plant species
shapes the rhizosphere microbiome and will reduce the dependence of the
rhizosphere microbiome on plant-derived carbon [31,32]. In previous studies, soil
pH has also been proven to be one of key determinant of soil microbial diversity
[17,33]. High N fertilization reduced the diversity of soil fungi and shifted
community composition in tea gardens. These changes were partially due to
alterations in soil pH [34]. Nevertheless, whether BOF application can manipulate
the rhizosphere bacterial community in tea plantation soils remains unclear.
Thus, understanding the effects of BOF application on rhizosphere bacterial
communities remains a pressing need. We hypothesized that BOF application could
shape the rhizosphere bacterial communities through the ammonia preference of tea
plants will result in a strong environmental filter because of the low pH.
In this study, a three-year pot experiment was conducted to investigate (1) how
soil fertility and tea biomass respond to BOF application; (2) how the bulk soil
bacterial commu- nity responds to BOF application, and (3) whether BOF
application can alter rhizosphere bacterial community composition.
Agronomy 2022, 12, 2168
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2. Materials and Methods

2.1. Experimental Design and Sample Collection
The pot experiment consisted of four different fertilization regimes: control
without fertilization (CK), 100% chemical fertilizer (CF), 50% chemical
fertilizer + 50% BOF (CFOF) and 100% BOF (OF). The fertilization scheme of each
treatment is shown in Table 1.

Table 1. Different experimental groups and fertilizer dosages.

Treatment Fertilization Regime

Amount of Fertilizer Application (g/Basin)

CK No fertilizer
0
CF 100% Chemical fertilizer
Urea 4.67
CFOF 50% Chemical fertilizer + 50% Bio-organic fertilizer
Urea 2.34 + Bio-organic fertilizer 53.75
OF 100% Bio-organic fertilizer
Bio-organic fertilizer 107.5
The total nitrogen application rate of each treatment was the same (calculated as
pure N), which was 350 kg/hm2.

One-year-old cutting seedlings of Camellia sinensis cultivar ‘Longjing 430 were

selected for this study. The seedlings were planted in 3-gallon plastic
flowerpots (the upper diameter, lower diameter and height of pots were 28 cm,
23 cm and 25 cm, respectively). The fertilization amount was calculated
according to the area of the upper diameter. In addition to CK, 2.15 g of pure N
per flowerpot was applied to each treatment (the total N amount was equivalent to
350 kg/hm2). The N content of BOF was determined to be 2%, and the N content of
urea was 46%. The amount of urea and BOF was calculated according to the N
content. Four seedlings were planted in each flowerpot, and each treatment
included four flowerpots.
The soil was taken from a newly reclaimed tea garden in the Jiangsu Academy of
Agricultural Sciences. The properties of the soil are listed in Table 2.

Table 2. The physicochemical properties of soil.

Index
Value

Soil type
yellow-brown soil pH (H2 O)
5.99
Total N(g/kg) 1.0
Organic matter (g/kg) 13.3
Available P (mg/kg) 141.6
Available K (mg/kg) 183.5

BOF was firstly mixed with soil before planting tea seedlings in flowerpots. A
week later, urea, P and K fertilizer (K2HPO4 1.0 g/pot), Mg fertilizer (MgSO4·7H2O
1.0 g/pot) and Zn fertilizer (ZnSO4·7H2O 0.25 g/pot) were dissolved in water and
applied in each flowerpot. After application, tea seedlings were placed in the
greenhouse. The experiment was started in the middle of April 2016. Fertilization
was carried out in April 2017 and 2018 according to the above scheme. The
experiment ended in May 2019, and then tea roots were carefully uprooted from the
pot and slightly shaken to remove the soil loosely combined with the root. The
soil closely adhered to the tea root system was taken as the rhizosphere soil.
Other soil was taken as the non-rhizosphere soil. One part of the rhizosphere soil
was air-dried and grounded for analysis of the soil’s physical and chemical
properties. The other part of the fresh rhizosphere soil was frozen at −80 ◦C for
determination of the soil’s inorganic nitrogen and microbial high-throughput
sequencing.
The tea seedlings in each pot were separated by root, stem and leaf, and then the
fresh weight of each part was measured by a scale.

2.2. Soil Property Analysis and Soil Fertility Calculation

Soil pH was determined in a 1:2.5 (w/v) soil:deionized water suspension with a pH
meter (ORION 3 STAR, Thermo Fisher, Waltham, MA, USA). Soil organic carbon (SOC)
Agronomy 2022, 12, 2168
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and total nitrogen (TN) were measured by an element analyzer (Vario Max,
Elementar, Hanau, Germany). Soil organic matter (SOM) was calculated from SOC by
a conversion factor of 1.724. The soil ammonium and nitrate were extracted by 2 M
KCl solution, and concentrations in the extracts were determined by the flow
injection analyzer (SAN++, Skalar, Breda, The Netherlands). The available P, K, Mg
and Ca in the soil were extracted using the Mehlich 3 method [35] and then
measured using an inductive coupled plasma emission spectrometer (ICAP6300, Thermo
Fisher, Waltham, MA, USA). The soil chemical properties mentioned in this
paragraph were conducted following the methods in the soil analysis handbook [36].
The soil fertility index in this study was calculated according to our previous
study. The present study used the total data set (TDS) to calculate the soil
fertility index. Briefly, the weight of each soil factor was calculated, and
then the score of each soil factor was calculated using the standard score
function [37]. Finally, the fertility index was calculated by using the
following equation:

n
Fertility index = ∑ Wi ∗ Si
i=1

where Wi is the weight value of each soil parameter, Si is the score of each
parameter, and
n is the number of parameters in the TDS [38].

2.3. DNA Extraction, High-Throughput Sequencing

Total soil DNA was extracted from 0.25 g of fresh soil by the DNA Isolation Kit
(Pow- erSoil, MOBIO, Carlsbad, CA, USA) according to the product’s protocol. The
quality of extraction was tested by the DNA concentration measurement using a nano
spectropho- tometer (ND2000, Thermo Scientific, Waltham, MA, USA).
The bacterial 16s rRNA (V4-V5 region) was amplified using the primers 515F/907R
[39]. The PCR reaction mixture (25 µL) contained 1 µL of the purified template DNA,
2.5 µL of
10 × PCR Mg2+ free buffer, 2.0 µL of 25 mM dNTPs, 2.5 µL of 2.5 mM Mg2+, 0.5 µL
(10 µM)
of each primer, and 0.5 µL (1.25 U) of Taq polymerase, and sterilized ultrapure
water up to
25 µL. The bacterial V4-V5 region amplification started with an initial
denaturation at 94 ◦C for 5 min, 15 cycles of 94 ◦C for 60 s, 54 ◦C for 30 s, 72
◦C for 90 s, and a final extension step at 72 ◦C for 10 min. The PCR was
performed by a Thermal Cycler (ABI 2720, Thermo Fisher Scientific, Waltham, MA,
USA). The PCR products were then purified with a Gel Extraction Kit (QIAquick,
Duesseldorf, Germany).
The purified PCR products were sequenced using the high throughput sequencing
platform (MiSeq PE250, Illumina, San Diego, CA, USA). Raw sequence data were
deposited in the National Center for Biotechnology Information (NCBI) database
with the accession number PRJNA831366.

2.4. Bioinformatics and Statistical Analysis

USEARCH (v 11.0.667) was employed to process the raw bacterial data [40]. In
brief, paired raw sequences were merged and re-oriented by comparing them to
the RDP database [41]. Then sequences with expected error > 1 and lengths <
250 bp were discarded. Next, fastx_uniques and Unosie3 commands were
implemented to remove redundant sequences and chimeras, and representative
sequences were obtained in this step. The otutab command was employed to generate
the ZOTU table. The representative sequences were aligned against the RDP
database with a cutoff value of 0.97 by using the sintax command. The ZOTU
table with taxonomic information was used to align against the FAPROTAX
database using a python script to predict metabolic or other ecologically
relevant functions (e.g., nitrification, denitrification or fermentation) [42].
In order to compare the relative difference between samples, a randomly selected
subset of
24,900 sequences per sample was performed for downstream analyses.
Bacterial Chao1 and Shannon indices were calculated by using the alpha_div
command in USEARCH. The differences in bacterial Chao1 and Shannon indices among
treatments
Agronomy 2022, 12, 2168
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were detected by Kruskal–Wallis’s rank-sum test at p < 0.05. Principal coordinates

analysis (PCoA) was performed using Bray–Curtis distance to evaluate the overall
differences in bacterial community structure under different treatments and
different compartments, and one-way permutational analysis of variance (PERMANOVA)
was used to analyze the effects of treatments and compartments on the community
structure of bacterial by using the function “adonis” in the R package “vegan”.
The relative abundance of the bacterial community was displayed at the phylum
level, and the Proteobacteria was divided into Alphaproteobacteria,
Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria, and other sub-
divisions were classified as Proteobacteria. Random forest (RF) analysis was
employed to detect the impacts of soil properties on bacterial community
composition in the R packages “randomForest”, “rfPermute”, and “rfUtilities”.
STAMP was implemented to test the differences in functional profiles between bulk
and rhizosphere in BOF treatments and between no BOF input treatments and BOF
input treatments. Co-occurrence network analysis was performed in the R package
“igraph” to detect the effects of BOF input on bulk and rhizosphere bacterial co-
occurrence networks. The visualization of co-occurrence was performed by Gephi (v
0.9.2).
A partial least squares path model (PLS-PM) analysis was applied by using the R
package “plspm” to investigate the possible causal relationships between organic
input, soil pH, fertility, plant biomass, bacterial community and diversity.
Differences in soil properties, plant biomass and soil fertility index among
treatments were tested by LSD t-test at p < 0.05.
All the statistical analysis in the present study was performed in R software
(version
4.1.1, R Development Core Team, https://www.r-project.org/, accessed on 7 August
2022).

3. Results
3.1. Bulk and Rhizosphere Soil Properties, Fertility and Tea Biomass Response to
Different
Fertilization Treatments
Our results found that in both bulk and rhizosphere soils, soil pH decreased
sharply in CF treatment compared to CK treatment (p < 0.05), while CFOF and OF
treatments showed mitigation of soil acidification compared to CF treatment
(Table 3). In addition, the measured soil properties, including Ca, AK, AP, NO3-
N, TN and SOM, revealed relatively higher values in CFOF and OF treatments than
in CF treatment (Table 3). However, several soil properties, such as soil pH,
TN, NH4+-N and available Ca, displayed relatively higher values in CK treatment
compared to those fertilization treatments in bulk soils (Table 3). The soil
fertility index of bulk and rhizosphere soils showed a similar variation, i.e.,
CF displayed the lowest soil fertility index and was significantly lower than CK,
CFOF and OF treatments (p < 0.05), while BOF application (CFOF and OF) increased
soil fertility both in both bulk and rhizosphere soils (Figure 1).

Table 3. Bulk and rhizosphere soil properties of different fertilization modes.

Treatment Ca (mg/kg) Mg (mg/kg)

AK (mg/kg) AP (mg/kg) NH4 +-N (mg/kg)
NO3• -N (mg/kg)

pH SOM (g/kg) TN (g/kg)

Bulk soil

Rhizosphere soil

CK 4183.8 ± 319.45 a 382.22 ± 7.91 de

203.40 ± 5.76 c 167.48 ± 6.95 a 14.76 ± 2.28 ab
38.24 ± 1.48 cd 5.57 ± 0.06 a 17.06 ± 0.15 ab
1.63 ± 0.35 a CF 3105.0 ± 497.57 bc 445.10 ± 30.08 abcd
203.32 ± 6.04 c 43.52 ± 6.73 d 9.02 ± 0.41 c
33.94 ± 1.98 d 4.87 ± 0.34 bc 14.82 ± 2.05 b
0.83 ± 0.11 b CFOF 3812.6 ± 179.04 abc 372.36 ± 6.00 e
230.32 ± 10.04 abc 184.14 ± 6.26 a 10.15 ± 0.18 c
39.79 ± 2.31 cd 5.35 ± 0.09 ab 17.25 ± 0.38 ab
1.15 ± 0.07 b OF 3284.0 ± 294.03 abc 474.06 ± 41.7 ab
249.22 ± 7.86 a 114.12 ± 31.81 bc 11.37 ± 0.40 bc
60.01 ± 7.25 a 5.30 ± 0.14 ab 18.86 ± 1.56 a
1.06 ± 0.08 b
CK 4020.4 ± 224.1 ab 414.38 ± 6.40 bcde
208.40 ± 17.14 bc 162.18 ± 6.06 ab 9.52 ± 0.45 c
38.99 ± 3.58 cd 5.54 ± 0.04 a 17.42 ± 0.32 ab
1.03 ± 0.09 b CF 2986.4 ± 352.22 c 472.08 ± 20.88 abc
158.58 ± 12.35 d 38.34 ± 6.31 d 8.42 ± 0.33 c
32.94 ± 2.65 d 4.60 ± 0.39 c 14.76 ± 1.93 b
0.80 ± 0.06 b CFOF 3694.6 ± 178.9 abc 409.48 ± 9.72 cde
230.48 ± 11.86 abc 168.48 ± 2.16 a 10.98 ± 0.53 c
51.11 ± 4.54 ab 5.19 ± 0.11 abc 19.80 ± 0.09 a
1.22 ± 0.09 ab OF 3194.6 ± 436.82 bc 490.38 ± 19.40
a 237.60 ± 12.86 ab 105.7 ± 30.85 c
16.95 ± 2.36 a 44.92 ± 1.52 bc 5.10 ± 0.16 abc
19.64 ± 1.42 a 1.15 ± 0.07 b
CK, control treatment; CF, 100% chemical fertilizer; CFOF, 50% chemical
fertilizer + 50% bio-organic fertilizer; OF,
100% bio-organic fertilizer. Different letters in the same column represent
significant differences among treatments
at a significance level of p < 0.05.

The biomasses of roots and total plant were significantly increased after
fertilization compared to CK treatment (p < 0.05), and no significant difference
was found between CF, CFOF and OF treatments (Figure 2b,c). However, leaf
biomass displayed the highest
Agronomy 2022, 12, 2168
6 of 17

Agronomy 2022, 12, x FOR PEER REvVaIEluWe in CFOF treatment and was significantly
higher than that in CK and CF treatm6enofts19
(p < 0.05) (Figure 2a).

Agronomy 2022, 12, x FOR PEER REVIEW

8 of 19

The biomasses of roots and total plant were significantly increased after
fertilization
cFoiFmgiugpruearre1e.1d.SStooililCffeKerrtttiilrliiettyaytiimndeenxtii(nnpbb<uu0lkl
.k0a5an)nd,darnhrhidzioznsoopsphsihegreenresifosiicolaiulnnutdndedirfedfreifrdfeeif
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idiccaiasllpfeflearrtyitlieilzidzeertrh+e+5h50i0%g%hbeibosi-too-vrogaralgunaeincic
ifefretirltiizliezre; rO; FO, F1,001%00b%iob-oior-
goarngiacnfeicrtfielirzteilri.zDeri.ffDerieffnetrleenttelresttinerrsoiwnsrionwdsici
anteddictahtedsigthneifisciagntifdicifafnetrednicfefesr-
ences at p < 0.05.
05) (Figure 2a).

Figure 2. Biomass of leaf (a), root (b) and total tea plants (c) as affected by
different fertilization tFreigautmreen2t.s.BCioKm,
caossntorfolletraefa(tam),ernoto; tC(Fb,)1a00n%d tcohteaml tieca
pfelartnitlisz(ecr); CasFOafFfe, c5t0e%d
cbhyedmiifcfearlefnerttifleirzteirliz+a5t0io%n btiroe-aotmrgeanntisc.
fCerKti,lcizoenrt;rOolFt,r1ea0t0m%ebnito; -CoFr,g1a0n0ic
%fecrhteilmiziecra.lDfeirffteilrieznert;lCetFteOrFs,o5n0%eachebmaricraelpfreertsiel
inztesrig+n5i0fi%-
cbainot-odrigffaenriecnfceerstilaimzeor;nOgFt,r1e0a0tm%ebnitos-
oartgaasniigcnfiefritcialinzceer.lDevifefleorefnpt <le0tt.e0r5s. on each bar
represent significant
differences among treatments at a significance level of p < 0.05.
3.2. Bulk and Rhizosphere Soil Bacterial Diversity and Community Composition Change
under
Different Fertilization Treatments
In the present study, the bacterial Chao1 index in bulk soils did not show
significant differences among different fertilization treatments, whereas
significant differences were found in rhizosphere soils (Figure 3a). In addition,
the Chao1 index increased after ferti-
Agronomy 2022, 12, 2168
7 of 17

3.2. Bulk and Rhizosphere Soil Bacterial Diversity and Community Composition Change
under
Different Fertilization Treatments
Agronomy 2022, 12, x FOR PEER REVIInEWthe present study, the bacterial Chao1
index in bulk soils did not show significan9t of 19 differences among different
fertilization treatments, whereas significant differences were found in
rhizosphere soils (Figure 3a). In addition, the Chao1 index increased after
fertilizer inputcodmecmreuasneitdywchitahntghee (iFnicgruearesin4g). aTmheournemt
oafinBiOngF isnopilupt r(oFpigeurtriees3am).aTdheeliSthtlaencnoonntriinbduetxion to
in rhitzhoesbpahcetreerisaolilcsosmhmowuenditythcehsaanmge.variation trend as the
Chao1 index in bulk soils and
displayed significant differences among treatments (Figure 3b).

FigurFei3g.uArelp3h. aA-dlpivhear-sdiitvyeirnsditiyceins
diniccelusdininclguCdihnago1C(haa)oa1n(da)SahnadnnSohnan(bn)oinn(bu) likn
abnudlkrhanizdosrphhizeorsepshoeilre soil
bacterbia. tBearicat.eBrialtceormialmcuomnimtyusntriutyctsutre c(tu)
raen(dc)caonmdpcoosmitipoons(iti)ocnh(adn)gceh
ndeerudnidffeerrdeniftfeferertniltifzearttiiolinzation
c d
treatments. CK, control treatment; CF, 100% chemical fertilizer; CFOF, 50% chemical
fertilizer +50%
eatments. CK, control treatment; CF, 100% chemical fertilizer; CFOF, 50% chemical
fertilizer +50%
tr bio-organic fertilizer; OF, 100% bio-organic fertilizer. Different letters
in rows indicated significant
bio-organic fertilizer; OF, 100% bio-organic fertilizer. Different letters in rows
indicated significant
differences at p < 0.05.
differences at p < 0.05.

The bacterial community composition showed that Betaproteobacteria, Acidobacteria,

Alphaproteobacteria and Gammaproteobacteria were the dominant phyla in this study,
with an average relative abundance of 46.88%, 11.29%, 9.69% and 9.59%,
respectively (Figure 3d). Moreover, the relative abundance of Betaproteobacteria,
Acidobacteria, Alphaproteobacteria, Planctomycetes, Deltaproteobacteria and
Gemmatimonadetes displayed a significant change among treatments in bulk soils.
In rhizosphere soils, only the relative abundance of Acti- nobacteria and
Gemmatimonadetes was significantly changed among treatments (Figure S1).
Specifically, CFOF and OF treatments notably increased the relative abundance of
Alphapro-
Agronomy 2022, 12, 2168
8 of 17

teobacteria, Acidobacteria, Deltaproteobacteria and Planctomycetes while

significantly decreasing the relative abundance of Betaproteobacteria (Figure S1).
Bacterial community structure also displayed a notable difference among
treatments (PERMANOVA test: R2 = 0.43, p < 0.001) (Figure 3c). The RF analysis
showed that soil properties explained 74.91% of the community change (R2 =
0.74, p < 0.001), and soil available K (24.1%, p < 0.01), Ca (15.3%, p < 0.01), P
(12.1%, p < 0.05), Mg (10.5%, p < 0.05),
Agronomy 2022, 12, x FOR PEER RE pH (14.4%, p < 0.05) and total N (8.53%, p < 0.05)
contributed significantly to the bacterial
community change (Figure
4). The remaining soil properties made little contribution to the
bacterial community change.

Figure 4. The impacts of soil properties on bacterial community composition by RF

analysis. Soil Figure 4. The impacts of soil properties on bacterial community
composition by RF analysis. Soil properties are ranked in ascending order of
importance to the accuracy of the model. The signifi- properties are ranked in
ascending order of importance to the accuracy of the model. The significance
cance level is indicated by ** (p < 0.01) and * (p< 0.05).
level is indicated by ** (p < 0.01) and * (p< 0.05).
33..33..BBaacctteerriiaallFFuunnccttiioonnGGrroouuppaannddCCoo--
OOccccuurrrreenncceeNNeettwwoorrkkRReessppoonnsseettooBBOOFFAAdddditiitoionn
TTooddeetetectctthteheefefeffcetcotfoBfOBFOiFnpinupt uont
obnacbtearcitaelrfiualncfutinocntaiol ngarol ugproaunpd acnod-occcou-
orrcecnucrerennecte- wnoetrwk,owrke,cwateegcoarteizgeodritzwedo ntwewo
tnreatmtreenattsm, ie.en.t,sn, oi.eB.O, nFo(NBOBOFF()NinBpOuFt)tirneaptumt
etnreta(tinmcelundt i(ning- CclKudainndg
CFKtarenadtmCFenttrse)aatmndenBtsO)FanindpBuOt
Ftreinaptmutentrte(aitnmcleundti(ningcCluFdOinFgaCnFdOOFFatnrdeaOtmFetnretas)t,-
tmo eenxtpsl)o, rtoe etxhpelorersepthonesresopfotnhse
obfatchterbiaalctfeurniacltifounncgtironupgraonudp acnod-occoc-
uorcrceunrcreennceetwneotrwkotrok BtoOBFOadFdaidtidointi.on.
FFoorr tthhee BOF ttrreeaattmenntt,,tthheeFFAAPPRROOTTAAXXfufunncctitoionnaal
lpprerdedicitcitoinosnsidiednetniftiiefided181d8ifdfeifrfeenr--
etniatlilayllpyrpesresetnetleemleemntesnctyscclyincglinbgetwbeetewnebeunlkbuanlkdar
nhdizorhspizhoesrpehseorielssionilBsOinF tBrOeaFtmtreenatmMeonst.t
Mfuonscttifounnscthioadnshhigahdehr
ipgrhoeproprtrioopnosritniobnuslkinsobiulslkexsoceilpstefxocreuprtefoolrysuisreaonl
ydsNis fainxdatiNonfi(xFaitgiuorne
(5Faig).uTrhee5laa)r.gTehstesliagrngiefisctasnitgdniiffifecraenntcdeisffwereernecfe
usnwctieornesfufonrcntiiotrnifsicfaotrionnit,rmifiectahtaionno,l moxeitdhaatnioonl,
omxiedthatyiloontr, ompehtyh,yilnottrraocpehllyu,lianrtrpaacrealsluitleasr,
puarreaosliytseiss,
aunredoNlysfiisxantidonN(Ffiixgautrioen5a(F).igInuraed5dai)t.ioIn
a2d2dditiifofenr,e2n2tidailflfyerfeunntciatilolynsfuwnecrtieoindsewnteirfieeiddebne
tiwfiednbBetOwFeeandBONFBaOnFdiNn BbOulFk isnobilus,lkansodil1s8,
afnudnc1t8iofnusnchtaiodnhsihgahderhpigrhoeproprtrioopnosrtiinonBsOiFn
BtrOeaFtmtreanttms e(Fnitgsu(Freig5ubr)e. 5Tbh)e.
Tlahregleasrtgseisgtnsigincaifin-t cdainfftedriefnfecreesncweesrwe
efruenfcutniocntisonfosrfocrhcehmeomhoehtetoertorotrpohpyh,y,inintrtaracceelllulula
larrppaarraassiitteess,, nitrifificattiion,,
mmeetthhaannoollooxxiiddaattiioonn,,mmeetthhyylolottrroopphhyy, ,uurereoolylysissis
aannddNNfifxixataitoionn(F(Figiguurere5b5b).).
AgroAngormoyno2m0y222,01222,, 21126, 8x FOR PEER REVIEW
11 o9f o1f917

(a)
95% confidence intervals
B-M R_M • B_M • R_M
ritnficallon
,......,
..
1
0.0425
methanol_oxidation
:r*-l 0.0418
melhylouophy ..
•r*-l 0.0418

0.0402
imrac:eHutar_parasites
,....., 0.0458
predatory_or_exopa,-asrtic

dark_oxldatioo_of_sulfur_oompouncts
0.0172
darlUhlosuftate_Olddation

n1t,ate_denitnfication
0.0061 'o
(1)
0.0061 t..,.

nitrite_dcnittlficatiOn
• 0.0061
.(1..)
nltrous oxide denitrificalion
0.0061 ..s.
(1)
:::,
0.0061 -¡¡;
human pattw:,gens sepbeemia

pattw:,gens pneumonll

0.0061
>
a1.
human

plastic_degradation
• 0.0061
aromatic hydrocarbon degradation

hydrocarbon_degfadatoo
•
0.0418
0.0418
0.0248
nitrogen fixation
• 0.0172
o.o 0.1 0.2 0.3
-0.15 -0.10 --0.05
0.00
Mean proportion Difference in mean proportions

(b)

95% confidence intervals

• B-M • B-NM • B-M • B-NM
chemoheterooophy
: 1
0.044
inlrac:eHutar_parasites
11-e-i 0.0374

meltlanot_oxielallon
'1-e-i 0.0374
melhylouophy

predatory_or_exoparasrtic: dark_oxidation_ol_suHur_oompouncts lenneolalion

dark thiosuUate OIUdation

aromatiC_hydrocarbon_degradaliOn
1
0.0259
0.0115
0.044
0.0063 'o
0.0043 t,
• 0.0043 o�

hydrocarbon_degradation

nrtrate_denitnfication

tme_denitnfic:allon

0.004 ..s.
• 0.004 (1)
ni -

• 0.004
:::,

a1.
nttrous_oxide_denitrlficabon
0.004 -¡¡;
>

umanJ)lllhogens_sepücemia
h
0.004
human_pathogens_pneurnonia
• 0.004
plaSlic_degradatiOn
ureolysis
0.004
0.044
tt 0.0382
0.0219

nitrogen lixation
0.0156
o.o o., 0.2 0.3
-0.15 --0.10 --0.05 0.00
0.05
Mean proportion Difference in mean proportions

Figure 5. Functional predictions of bacterial communities between bulk and

rhizosphere soils under different fertilization treatments. (a) Comparison
between bulk and rhizosphere soils in BOF treatment; (b) comparison between BOF
and NBOF in bulk soil bacteria.
Agronomy 2022, 12, x FOR PEER REVIEW
12 of 19

Agronomy 2022, 12, 2168

Figure 5. Functional predictions of bacterial communities between bulk and
rhizosphere soils under different fertilization treatments. (a) Comparison between
bulk and rhizosphere soils in BOF10troefa1t7- ment; (b) comparison between BOF and
NBOF in bulk soil bacteria.

Theebbaaccteterriaial lcoc-oo-
coccucrurrernecnecneentwetowrkosrkwsewreedreistdiniscttilnycdtliyffedriefnfetriennb
tuilnk banudlkrhainzdosrphhiezroe- ssopihlsearendsoiinlsBaOnFdain
BNOBFOaFndtreNaBtmOeFnttrse(aFtimguenrets6(aFnigduTraeb6lea4n)d.
OTuabrlrees4u).ltOs uforurnedsubltosthfoBuOnFd
abnodthNBBOOFFatnredatNmBeOnFts,trtheaetmbaecntetsr,iatlhceob-oacteurriraelnccoe-
oncectuwrorerknsceinnbetuwlkorskosilsinrebvuelaklesdoimlsorree- nvoedaleesdamndore
ngoedsetshaanndiendrgheisztohsapnhienrerhsioziolss,pahnedrewsoitihls,raenladtiwveit
lhy rheilgahtievrelayvherigahgerdaevgerreaegse,
adveegrraegees,paavtehralegnegptahtshalenndgdthiasmanedtedrsia(mTaebtelers4()T,
awbhleic4h),iwndhiiccahteinddtihcatebdutlhkastobiulslkhsaodilms hoarde
cmoomrpelceoxmnpetlwexonrkestwthoarnksrhthizaonsprhhiezroesspohielsr.eIsnobilus.lkI
nsobiulsl,kthsoeiclso,-tohcecucror-eonccurnreetnwcoernkentwodoerks
annoddesdagnesdiendBgOesFintrBeaOtmF terneta(tnmoednets(=no1d5e4s, e=d1g5e4s,
e=d2g8e1s)=in2c8r1e)aisnecdrecaosmedpacoremdptoarNedBtOoFNtBreOatF-
mtreanttm(neondt e(nso=d9e7s,=ed97g,eesd=g2e2s6=),2w26h)i,lwe
thhieleptrhoepporotipoonrotifon ogfantievgeaetidvgeeesdingeBsOinFBtrOeaFtmtreeantt-
rmeveenatlreedveaadlerdamaadtircamdeactriceadseecrceoamsepacroemdptaoreNdBtOoFN(Bfr
OomF (3fr4o.5m1%34t.5o11%3.1to7%13).1(F7i%g)ur(Fei6gau,rbe; T6aab,ble;
T4a).bHleo4w).eHvoerw, ienvethr,einrhtihzeosrphhizeorsep,
hBeOreF,tBreOaFtmtreenatmdiesnptladyisepdlaayseidmaplseimr cpol-eorcccou-
rorcecnucre- nrentwceornketcwomorpkacreodmtpoaNreBdOtFo
tNreBaOtmFetnrte,aatnmdenaltl, tahnedneatlwl tohrek
npertowpoerkiepsrwopereertdieescrweaesre dien-
tchreaBsOedFitnretahtemBeOntFetxreceaptmt
feonrttehxecedpetnfsoitrythaneddemnosidtyulaanridtym(Foidguulraeri6tyc,d(F;
iTgaubrlee64c),.d; Table 4).

Figure 6. The bacterial co-occurrence networks in bulk soil of NBOF (a: CK and CF)
and BOF (b:
Figure 6. The bacterial co-occurrence networks in bulk soil of NBOF ((a): CK
and CF) and BOF
CFOF and OF) treatments, in rhizosphere soils of NBOF (c: CK and CF) and BOF (d:
CFOF and OF)
(t(rbe)a:tCmFeOntFs.aNndodOeFs)iztreeaistmpreonptso,ritniornhailztoosptherbeestwoie
lsenofnNesBs OceFn(t(rca)l:itCyKofaneadchCFg)eannuds,BaOndF e((ddg):eCthFiOckF-
annedssOisFp) rtroepaotmrtieonntas.l Ntootdhe swizeeigihs tporof
peaocrhtiocnoarrletloattihoenb.
Tethweeceonlnoresosfceeancthraelditgyeorfeeparcehsegnetnsupso, saintidveedangde
thickness is proportional to the weight of each correlation. The color of each
edge represents positive
and negative correlation coefficients: red represents positive correlation, and
blue represents negative correlation. The thickness of each edge is proportional
to the correlation coefficient (p < 0.01).
negative correlation coefficients: red represents positive correlation, and blue
represents negative correlation. The thickness of each edge is proportional to the
correlation coefficient (p  <  0.01).

Table 4. The bacterial co-occurrence network properties.

Agronomy 2022, 12, 2168
11 of 17
Network Properties
Bulk
Bulk
Rhizosphere
Rhizosphere
BOF NBOF
BOF NBOF
Average degree 3.649 4.66 1.2
1.825
The bacterial co-occurr network pr
Average weighted degree 3.616 4.605 1.2
1.807

Network Properties
DiameteBrulk
B1u3lk
R6hizosphere
1 Rhizosphere 4
Average pathBleOnFgth 4N.0B3O7F
2.406BOF
1 NBOF
1.526
Average degree
Density3.649
0.40.6264 0.0491.2
0.041
1.825
0.033
Average weighted degree
Modulari3t.y616
04..764015
0.8321.2
0.907
1.807
0.898
Diameter
Modularity c1la3ss 461 13 1
14 4 19
Average path length 4.037
2.406 1 1.526
weakly
Density
Number of
0.024 03.0749
12 0.041
14 0.033 19
Modularity
Modularity class
connected com0p.7o4n1ents
Average cluste4r1ing
0.832 0.907 0.898
13 14 19
Number of weakly connected components coefficien3t7
0.71522 0.844 14
1 19
0.783
Average clustering coefficient
0.752 0.844
nodes 154
97 1
30 0.783 57
Number of nodes 154
97 30 57
Number of edges
Number of e2d8g1es 228216
226 18
18 52 52
NBOF represents CK and CF treatments and BOF represents CFOF and OF treatments.
NBOF represents CK and CF treatments and BOF represents CFOF and OF treatments.

3.4. B3a.4c.teBraiaclteDriiavleDrsivtye,rsCitoym,
mCoumnimtyuSntitryucStturruec,tSuoriel,pSHoi,lFpeHrt,ilFiteyrtailnitdyTaenadBTioema
aBsisomReassoRnesseptoonse to
BOFBInOpFutInput
A PLASPPMLSaPnMalyansiaslywsaiss wemaspelomypedloytoedetotedctetecetctohme
cpolemxprleelxatrieolnastihoinpshaimpsoanmg osonigl psoHil, pH,
fertilfietryt,ibliatyct,ebraiactlecroiaml mcoumnmityundiitvyedrsiivteyr,
ssittryu,csttururec,tuanred,
atenadbtieoambaiossmuanssdeurnBdOerFBtOreFattmreeantmtse. nts. The Tmhoedmelosdheol
wsheodwtehdatthBaOt BFOinFpiunpt
uetxpexlapilnaeinde4d74%7%ofotfhtehevvaariraiannceceininbbiioommaassss with a 0.59
0.59 ggooooddnneessssoofffifit t(F(iFgiugruere7)7. )T.
heThresureltssuslthsoswheodwtehdatthBaOtFBiOnpFuitnhpaudt
phoadsitpivoesiatnivdedainredct ef- direcftecetfsfeocntstoean
bteioambaiossm(apsasth(pcaotheffciocieefnfitci(epnct) (=pc0).5=6)0, .s5o6i)l,
fseoritlilfietyrti(lpitcy=(p0c.5=8)0a.5n8d) baancdterial bactecroimal
mcoumnmityunstirtyucstururcet(uprce =(p0c.3=10) .b3u1)t
baulstoaslshooswheodwdeidredcitrencetgnaetgivaetivefefecfftescotsnobnabctaecrtiearl
iadliver-
divesristiyty(p(pcc==−−0.01.21)2.)I.nInadaditiitoinon, ,bbaactcetreiraial
lccoommmmuunnitiytysstrtruucctuturreenneeggaattiivveellyyaaffffeecctteedd tteeaa
bio-
biommaasss(p(pcc==−−019), while bacterial diversity displlayed an oppossiittee
rressulltt ((pcc = 00..4400)).. Soil Soil fertility (pc = 0..09) and pH ((pc = −
−0.00.90)9)rerveveaealeldedslsilgighhtltylyddirierecct
teefffefecctstsoonnteteaabbioiommaass (Fig-
(Figuurree77aa));;hhoowweevveerr,,ssooiillppHHaallssooeexxhhiibbiitteeddaanniinnddi
irreecctteeffffeecctt((ppcc == −−00.1.199))oonnteteaabbioiommaass (Fig-
(Figuurree77bb)).. Excluding the indiirectt negaattiivvee
eeffffeecctt,,tthheettoottaalleeffffeeccttooffBBOOFFininppuuttwwaass00.4.488(Fig-
(Figuurree77bb))..

Figure 7. The relationship between bacterial diversity, community structure, soil

pH, fertility, BOF input and tea biomass. (a) Path model outputs: the numbers on
the arrows represent standardized path coefficients. The value of each path
coefficient is indicated by the arrow width; blue and red arrows indicate
positive and negative effects, respectively. Values of R2 indicate the variance
explained by the model. (b) The standardized path coefficients for direct and
indirect effects on the bacterial community.
Agronomy 2022, 12, 2168
12 of 17

4. Discussion
4.1. Effect of BOF Application on Soil Properties and Fertility
In the present study, BOF application (CFOF and OF treatments) revealed an
excellent offset effect on soil acidification and a notable improvement in soil
fertility compared to CF treatment (Table 3 and Figure 1). These results are
consistent with previous studies and have been verified in many field
experiments with different plants, such as pigeon pea [14], potatoes [15] and
grains [16]. However, CF treatment displayed the lowest soil fertility, even
lower than CK treatment. We suggested that the strong leaching effect under pure
chemical fertilizer treatments might be the reason for the decrease in soil
fertility [43]. Many studies have proved that pure chemical fertilizer input
can increase the leaching risk, while chemical fertilizer combined with organic
amendments (e.g., organic fertilizer, straw and biochar) has good performance in
reducing soil nutrient leaching [44,45]. In addition, the rhizosphere displayed a
relatively lower soil pH than the bulk soils. This result could be attributed to
the preference for ammonia absorption and the organic acids in root exudates
because both of these could acidify the root-zone pH [46,47].

4.2. Effect of BOF Application on Soil Bacterial Diversity, Structure, Function

Group and
Co-Occurrence Network
Previous research has proved that applying BOF to fields has tremendous influences
on soil bulk and rhizosphere bacterial communities. For example, applying BOF
can increase soil bacterial diversity in tobacco, potato and cucumber soils
compared to pure chemical fertilization treatment [28,48,49]. However, the soil
bacterial Chao1 index in bulk did not show significant change after applying
BOF, and only the Shannon index showed a slight increase in BOF treatment
(Figure 3a,b). We suggested that the short term of this experiment might be the
reason for this result because active microbes in BOF may compete with the local
bacterial community and result in a decline in richness [50,51]. In addition,
the bacterial Chao1 and Shannon index showed a significant decrease under BOF
treatments (CFOF and OF) in the rhizosphere. Previous studies have reported that
applying BOF can promote the formation of beneficial microbial communities in the
rhizosphere, which may also lead to a decrease in microbial diversity because
of the harbouring of beneficial microbes [52]. The co-occurrence network also
confirmed that, i.e., the network of BOF treatment in the rhizosphere showed fewer
nodes and edges (including negative edges) (Figure 6c,d).
Nevertheless, bulk soil bacterial composition was significantly changed under
different treatments, although rhizosphere soil bacterial composition showed no
significant change for most phyla, indicating that the rhizosphere effects
intensely impact bacterial composi- tion [53,54]. Moreover, Proteobacteria and
Acidobacteria dominated at the phylum level. This result was similar to previous
studies in acidic soils [7,55]. Acidobacteria was reported that have the
function of C cycling through degradation [56,57]. BOF treatments significantly
increased Acidobacteria relative abundance compared to CF treatment, which
indicated that BOF could improve the function of C degradation. The functional
prediction results further proved that BOF input did enhance the aromatic
hydrocarbon and hydrocarbon degradation processes compared to NBOF treatment (CK
and CF treatments) (Figure 5b). Proteobacteria constitute the most dominant
fraction of the mangrove bacterial community. All classes of Proteobacteria
(Alpha, Beta, Gamma, Delta, and Epsilonproteobacteria) have been reported from
mangroves across the globe, although the abundance of different classes varied
significantly [58–60]. Since Alphaproteobacteria can convert atmospheric N to
nitrites, making nitrogen usable by other forms of life, the increase in the
relative abundance of Al- phaproteobacteria in BOF treatments indicated that
BOF had a positive effect on N transform processes. Moreover, the functional
prediction results again verified that nitrification and denitrification
processes were enhanced in BOF treatments. Deltaproteobacteria are consid- ered
to have hydrocarbon-degrading abilities [61]. The increase in the relative
abundance of Deltaproteobacteria in BOF treatments indicated that BOF could
increase C degradation. In addition, the contributions of soil properties to
bacterial community structure changes
Agronomy 2022, 12, 2168
13 of 17

in the present study were similar to our former study in tea plantations, i.e.,
soil nutrients and pH change can remarkably change the bacterial community
structures [5,62]. After organic fertilizer substitution in a tea plantation for
ten years, the soil pH (p = 0.005) and SOC (p = 0.017) were the predominant soil
characteristics that accounted for the structural changes in the soil bacterial
community [5]. In an eleven-year field experiment with differ- ent N applications,
the variation of bacterial community composition was largely explained (~50%) by
the soil properties of pH, exchangeable magnesium, exchangeable potassium and
exchangeable hydrogen [62].
BOF application usually has significant positive effects on suppressing pathogens
to prevent plants from contracting diseases in previous studies [48–50]. In
addition, BOF application also revealed notable influences on soil bacterial
functional profiles in previous studies [16,23]. For instance,
carbohydrate/lipid metabolism and the biosynthesis of other secondary metabolites
were improved after BOF addition in banana plantations [23]. Moreover, the
application of BOF in degraded red soil also revealed positive impacts on N
cycling, enhanced recalcitrant C degradation, and inhibited labile C degradation
[16]. These results were similar to ours, i.e., BOF significantly enhanced N
cycling and C degrading processes (Figure 5). Interestingly, our results also
showed that rhizosphere soil ureolysis and N fixation processes were enhanced
compared to bulk soil after the addition of BOF. This result is in line with
the recent research showing that genes involved in organic compound conversion
and nitrogen fixation were strongly enriched in the rhizosphere [63].
Previous studies showed that when organic fertilizer was added to soils, a more
com- plex co-occurrence network developed than with the addition of inorganic
fertilizer [64,65]. A similar result was found in our study, where the
bacterial network modularity and negative edges increased with the OSRs (Figure 4
and Table 3). However, the bacterial co-occurrence network in the rhizosphere
soil displayed a reverse result; we suggested that the bacterial community in the
rhizosphere was more affected by the rhizosphere effect than fertilization
[53,54].

4.3. Effect of the Application of BOF on Improving Tea Biomass

It is well demonstrated that the application of BOF could improve soil fertility,
sup- press soil-borne diseases in agricultural production, and increase crop
yields or plant biomass [28,49]. For example, BOF application decreased disease
incidence of tobacco bacterial wilt and cotton Verticillium wilt; contributing
factors to this were the reduction of the abundance of potentially pathogenic
microbes and the recruitment of more beneficial bacteria or fungi in rhizosphere
soil, thus improving pathogenic bacteria resistance and promoting plant growth
[48,52]. In the present study, we found that BOF substituted for
50% of chemical fertilizer displayed the highest leaf and total biomasses. This
result was similar to the recent research, indicating that partial substitution of
chemical fertilizers with BOF is a promising fertilization practice for banana
production in acid soil ecosystems [23].
In addition, PLSPM analysis showed that BOF input and bacterial diversity have
direct positive effects on tea biomass. This result could be attributed to the
improvement of soil fertility and bacterial functions. A previous study also found
a similar result because BOF input could increase soil fertility and enhance
the bacterial function profiles in tea plantation soils [66]. However, soil
fertility showed a negligible positive effect on tea biomass in our study. We
suggested that fertilization treatments could supply adequate nutrients to
maintain the tea plant growth in such a short-term pot experiment, though CF
treatment had a lower soil fertility index because of the leaching.
Therefore, total plant biomass showed no difference between fertilization
treatments after a 3-year experiment, which might be the reason for the negligible
positive effect on tea biomass in our study.

5. Conclusions
Overall, the present study proved that BOF application could increase soil
fertility in both bulk and rhizosphere soils and improve the biomass of tea
leaves. In addition, the nutrient level changes caused by BOF application
significantly changed bacterial com-
Agronomy 2022, 12, 2168
14 of 17

munity diversity and composition and accounted for 74.91% of the community
variation. Furthermore, BOF notably improved the N and C cycling processes and
enhanced the co-occurrence network complexity in the bulk soils, whereas bacterial
community functions in the rhizosphere were more affected by the rhizosphere effect
than BOF application. All these findings verified our hypothesis that applying BOF
in tea plantations could increase the biomass of tea plants by improving soil
fertility and influencing the soil bacterial function groups. In addition, BOF
application could influence the rhizosphere bacterial communities, but the
rhizosphere effects affect more.

Supplementary Materials: The following supporting information can be downloaded at:

https:// www.mdpi.com/article/10.3390/agronomy12092168/s1, Figure S1. Variance
analysis of the top 9 bacterial relative abundance in bulk and rhizosphere soil
in phylum level.

Author Contributions: Conceptualization, Z.H., Q.W. and Y.Y; Methodology, Z.H.

and H.L.; Soft- ware, L.J.; Validation, R.L. and Y.Y.; Formal analysis, Z.H.
and L.J.; Investigation, Q.W. and Z.H.; Visualization, L.J.; Writing—Original
Draft Preparation, Z.H. and L.J.; Writing—Review and Editing, R.L., H.L. and
Y.Y.; Funding Acquisition, Y.Y. All authors have read and agreed to the
published version of the manuscript.

Funding: This research was funded by the National Natural Science Foundation of
China (31800590), the Jiangsu Earmarked Fund for Modern Agro-industry Technology
Research System (tea) (JATS (2022)275), the Pilot Project of Collaborative
Extension Plan of Major Agricultural Technologies in Jiangsu Province (2020-SJ-
047-02-1), the Open Fund of State Key Laboratory of Tea Plant Biology and
Utilization (SKLTOF20200121) and Breeding Industry Revitalization Project in
Jiangsu Province (JBGS (2021)85).

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: Publicly available datasets were analyzed in this

study. These data can be found here: https://www.ncbi.nlm.nih.gov/sra/PRJNA831366
(accessed on 24 April 2022).

Acknowledgments: In this section, you can acknowledge any support given which is
not covered by the author contribution or funding sections. This may include
administrative and technical support, or donations in kind (e.g., materials used
for experiments).

Conflicts of Interest: The authors declare no conflict of interest.

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