Microbiological Research 231 (2020) 126373

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Microbiological Research
journal homepage: www.elsevier.com/locate/micres

Biochar amendment controlled bacterial wilt through changing soil T

chemical properties and microbial community
Shu Chen, Gaofu Qi, Gaoqiang Ma, Xiuyun Zhao*
College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China

A R T I C LE I N FO A B S T R A C T

Keywords: Long-term continuous cropping has led to epidemic of bacterial wilt disease in Southern China. Bacterial wilt
Bacterial wilt disease is caused by Ralstonia solanacearum and difficult to control. In order to control bacterial wilt, rice hull
Ralstonia solanacearum biochar was applied to soil with different doses (0, 7.5, 15, 30 and 45 t ha-1) in a field trial. After three years, the
Biochar influence of biochar on soil properties, incidence of bacterial wilt and microbial community were characterized.
Soil properties
Biochar amendment significantly suppressed bacterial wilt through changing soil chemical properties and mi-
Microbial community
crobial composition. Compared with control, disease incidence and index of biochar amendments (7.5, 15, 30,
and 45 t ha-1) significantly decreased. Disease incidence and index of biochar amendment (15 t ha-1) were the
lowest. Compared to the unamended control, contents of soil organic matter in biochar amendments (15, 30 t ha-
1
), available nitrogen in biochar amendment (15 t ha-1), and urease activity in biochar amendments (7.5, 15 t ha-
1
) significantly increased. Biochar amendments (15, 30, and 45 t ha-1) increased the relative abundances of
potential beneficial bacteria (Aeromicrobium, Bacillus, Bradyrhizobium, Burkholderia, Chlorochromatium,
Chthoniobacter, Corynebacterium, Geobacillus, Leptospirillum, Marisediminicola, Microvirga, Pseudoxanthomonas,
Telmatobacter). Biochar amendments (7.5, 30, and 45 t ha-1) reduced the relative abundances of denitrifying
bacteria (Noviherbaspirillum, Reyranella, Thermus). Biochar amendments (7.5, 15, and 45 t ha-1) significantly
decreased pathogen Ralstonia abundance. Overall, application of biochar effectively controlled bacterial wilt
through sequestering more carbon and nitrogen, enriching specific beneficial bacteria and decreasing pathogen
abundance. This study revealed the potential of biochar in control of bacterial wilt.

1. Introduction River, disease incidence of tomato bacterial wilt ranged from 10 % to

Bacterial wilt disease is caused by Ralstonia solanacearum and results
in severe economic loss (Mansfield et al., 2012). R. solanacearum is
pathogenic on more than 200 plant species belonging to 54 different
families (e.g. banana, eggplant, peanut, potato, tobacco, tomato). R.
solanacearum enters the plant through the roots and colonizes the vas-
cular system and ultimately leads to whole plant wilting and death
(Genin and Denny, 2012). R. solanacearum can survive in soil for many
years in the absence of susceptible crop and can spread through water,
rhizosphere contact and farming. So it is difficult to control bacterial
wilt disease (Van Elsas et al., 2000).
Long-term continuous cropping has led to epidemic of bacterial wilt
in China. Bacterial wilt disease has been reported in 30 provinces of
China, especially in southern provinces, and has caused great economic
losses in the recent years. In China, R. solanacearum infected more than
90 plant species belonging to 39 botanical families (Jiang et al., 2017;
Li et al., 2017). For instance, in the southern provinces of the Yangtze
80 % (Wei et al., 2015). Potatoes planted in more than 10 provinces of
China are infested by bacterial wilt with estimated yield losses ranging
from 10 to 100% (Chen et al., 2005). Tobacco bacterial wilt occurs in
14 out of the 22 tobacco growing regions, with 15–75% disease in-
cidence, and causes 50–100% yield reduction (Liu et al., 2017). Bac-
terial wilt affects about 800,000 ha of peanut land in China and causes
10–100% yield losses (Yu et al., 2011). Therefore, bacterial wilt disease
is one of the most important diseases in China due to its wide dis-
tribution and cumulative losses on many crops, ornamental and med-
icinal plants. There are still no effective strategies to control bacterial
wilt. Currently, antimicrobial substances produced by bacteria and
chemical pesticides (e.g. zinc thiazole, bismerthiazol, and saisentong)
are the main methods used to control bacterial wilt, which have limited
efficacy and can’t effectively prevent the occurrence of bacterial wilt
(Bai et al., 2016). In addition, long-term use of chemical pesticides
results in heavily environmental pollution and induces the emergence
of pesticide-resistant pathogens (Fujiwara et al., 2011). Thus, there is

⁎
Corresponding author.
E-mail address: xiuyunzh@mail.hzau.edu.cn (X. Zhao).

https://doi.org/10.1016/j.micres.2019.126373
Received 5 July 2019; Received in revised form 18 October 2019; Accepted 10 November 2019
Available online 11 November 2019
0944-5013/ © 2019 Elsevier GmbH. All rights reserved.
S. Chen, et al.
an urgent need to find alternative environmentally friendly measures to
effectively control bacterial wilt disease in China.
Biochar is produced via pyrolysis of biomass wastes (e.g. crop re-
sidues, manure, wood wastes etc.) (Sohi et al., 2010). Rice hull is an
agricultural waste annually accumulating for a large amount in
Southern China, and the disposal of rice hulls poses significant public
health and environmental risks. In this study, biochar was prepared by
rice hull for its recycling use and suppressing bacterial wilt. Biochar
contains multiple nutrient elements such as carbon, nitrogen, phos-
phorus, potassium, calcium and magnesium (Cao and Harris, 2010;
Huang et al., 2013). Biochar addition has been shown to improve soil
physical and biochemical properties, suppress plant disease, improve
plant growth and stimulate soil microbial activity (Elad et al., 2010;
Graber et al., 2010; Lehmann et al., 2011). Therefore, biochar is widely
used as a soil amendment. Studies have evidenced the potential for
biochar in improving soil fertility and crop productivity through en-
hancing soil organic carbon storage, nutrients and moisture availability,
ameliorating acidic soils, and stimulating microbial activity and di-
versity (Lehmann and Joseph, 2009; Gwenzi et al., 2015; Ippolito et al.,
2015). Biochar amendments alter soil microbial biomass, activity and
community composition (Graber et al., 2010; Xu et al., 2014; Jaiswal
et al., 2017).
Most importantly, biochar reduces plant disease severity and in-
duces plant resistance to disease possibly by increasing nutrient reten-
tion, alleviating soil acidity, altering microbial community as well as a
better soil structure (Elad et al., 2010; Graber et al., 2014; Jaiswal et al.,
2017). For example, biochar helps to control cucumber damping-off
caused by Rhizoctonia solani (Jaiswal et al., 2014). Biochar amendments
are effective in inhibiting bacterial wilt and significantly reduce the
disease index (Lu et al., 2016; Zhang et al., 2017). Therefore, biochar
amendment can serve as a management tool for suppressing plant
diseases (Lehmann and Joseph, 2009; Jaiswal et al., 2017). However,
most trials about biochar application have been carried out in the la-
boratory over short time periods. The long-term effects (i.e. > 12
months) of biochar application on soil microbial community, soil bio-
chemical properties and bacterial wilt had been poorly understood in
field experiment (Jones et al., 2012). Effect of biochar on inhibiting
plant disease varied with different doses of biochar applied (Jaiswal
et al., 2014, 2015; Copley et al., 2015; Huang et al., 2015). In this
study, action of different doses of biochar amendment treatments was
compared in order to find the optimal dose of biochar for control of
bacterial wilt disease. The experiment was set up in a bacterial wilt-
infected tobacco field in Enshi State of Southern China. After amending
biochar for three years, effect of different doses of biochar on incidence
of bacterial wilt and soil properties and microbial communities were
investigated. We hypothesized that application of biochar would alter
soil properties and microbial abundance, with an increase in some
beneficial bacteria, which would lead to a decrease in bacterial wilt
incidence. Different doses of biochar would have different control ef-
ficiency on bacterial wilt disease. These study would find the optimal
dose of biochar for effectively inhibit bacterial wilt.
2. Materials and methods
2.1. Biochar preparation
Biochar was produced by the Nanjing Institute of Soil Science,
Chinese Academy of Sciences, China. Briefly, dry rice hull was pyr-
olysed at 400 °C for 3 h under oxygen-limited conditions. The physio-
chemical characteristics of the biochar were detected as follows: pH
9.2, total nitrogen 13.5 g/kg, total carbon 630 g/kg, total phosphate
4.5 g/kg, total potassium 21.5 g/kg, and ash content 140 g/kg. Fourier
transform infrared spectroscopy (FTIR) and scanning electron micro-
scope (SEM) images were shown in Figure S1. FTIR analysis showed
biochar with the following characteristic absorption peaks: 3338 cm-1
representing the stretching vibration of −OH, 1592 cm-1 representing
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Microbiological Research 231 (2020) 126373
the stretching vibration of -C = O carboxyl, and 1061 cm-1 representing
phenolic hydroxyl group Ar−OH. SEM image showed that there were
many microspores (< 1 μm in diameter) and small pores (1−5 μm in
diameter) on the surface of biochar.
2.2. Biochar amendment field trials
The field trial was established at Xuanen County, Hubei Province,
China (29°97′N, 109°39′E). The replicated (n = 3) trial plots
(10 m × 4 m) were laid out in a randomized block design in an existing
flat agricultural field where tobaccos have been continuously planted
during the last 20 years, and the outbreak of tobacco bacterial wilt
disease was recorded in recent years. In March 2015, the site was
ploughed, and biochar was spread on the surface soil at different rates
of 0 (control, BC1), 7.5 (BC2), 15 (BC3), 30 (BC4) and 45 (BC5) t ha-1,
respectively. The biochar was then harrowed into the topsoil
(0−30 cm). In May 2015, 2016, and 2017, total 60 tobacco seedlings at
six-leave stage were transplanted into each plot following standard
farming practices, respectively.
2.3. Soils sampling and bacterial wilt recording
Soil samples were collected in August 2017 (90 d after trans-
planting). At this time, bacterial wilt disease was at its peak. In each
plot, soil sample was taken from 10 different sites, 100 g of soil was
collected from each site, then mixed together to form one composite
sample. The soil samples were used for analyzing soil chemical prop-
erties and enzymatic activities and microbial community.
At the same time, tobacco height, stem circumference, the incidence
and severity of bacterial wilt (n = 60 plants per plot) was recorded.
Disease incidence was calculated by the percentage of diseased to-
baccos. Disease index was evaluated using a disease score method: 0=
no symptom; 1= less than a half of tobacco leaves are wilted; 3= one
half to two-thirds of leaves are wilted; 5 = more than two thirds of
leaves are wilted; 7 = all leaves are wilted; and 9 = stems collapsed or
tobaccos are dead. Disease index was calculated using the formula (Yi
et al., 2007):
Disease index = [∑(r × N) / (n × R)] × 100
Where r is the disease severity, N is the number of infected tobaccos
with a rating of r, n is the total number of tobaccos tested, and R is the
value of the highest disease severity in each plot.
2.4. Analysis of soil chemical properties and enzyme activities
Soil pH was determined using a 1:2.5 (w/v) soil : distilled water
ratio. Contents of available potassium (AK), available phosphorus (AP),
alkaline nitrogen (AN), and soil organic matter (SOM) were determined
as the methods described previously (Wang et al., 2017). Activities of
soil phosphatase, urease, invertase, and catalase activity were de-
termined as the methods described previously (Tabatabai and Bremner,
1969; Sinha, 1972; Kandeler and Gerber, 1988; Schinner and Wvon,
1990).
2.5. Assay of microbial community
Soil DNA was extracted from 0.4 g of each soil sample following the
instructions of FastDNA Spin Kit (MP Biomedicals, USA). The extracted
DNA was used as template to amplify the bacterial 16S ribosomal RNA
(rRNA) gene. Hypervariable V3 and V4 regions of 16S rRNA gene were
amplified by forward primer 338 F (5′-ACTCCTACGGGAGGCAGCA-3′)
and reverse primer 806R (5′-GGACTACHVGGGTWTCTAAT-3′) (Wang
et al., 2017). Amplicons were sequenced on Illumina Miseq platform
(Illumina, San Diego, USA) at SHBIO Technology (Shanghai, China).
Raw sequences were processed with QIIME quality filter (Sun et al.,
S. Chen, et al.
2014), then analyzed via UPARSE pipeline (Caporaso et al., 2010).
Paired-end clean reads were merged using FLASH (V1.2.11) according
to the relationship of the overlap between the paired-end reads. Se-
quences were assigned to each sample based on their unique barcode
and primer using Mothur software (V1.35.1), after which the barcodes
and primers were removed and got the effective clean tags.
Sequences analysis was performed by usearch software (V10).
Sequences with ≥97 % similarity were assigned to the same opera-
tional taxonomic unit (OTU). An OTU is thought to possibly represent a
species. Alpha diversity is applied for analyzing complexity of bacterial
specie diversity of a sample including observed species, Chao1,
Shannon and Simpson index, which were calculated with QIIME
(V1.9.1) and displayed with R software (V2.15.3) (Caporaso et al.,
2011). Beta diversity analysis was used to evaluate differences between
samples of bacterial specie complexity. Sample cluster analysis was
performed as an UPGMA (Unweighted Pair-group Method with Ar-
ithmetic Means) method to interpret the distance matrix using average
linkage and was conducted by upgma_cluster.py script in QIIME soft-
ware based on the unweighted unifrac distance matrix (Lozupone et al.,
2006).
2.6. Statistical analysis
Treatment differences in soil and tobacco properties, alpha diversity
and microbial abundance were compared by one-way analysis of var-
iance (ANOVA) with Tukey pair-wise comparisons (P < 0.05, P <
0.01). Correlation between disease index and microbial abundance was
analyzed by Pearson correlation analysis with SPSS 20 software (Wang
et al., 2017).
3. Results
3.1. Disease severity index of bacterial wilt
The disease incidence and indices of biochar treatments (BC2, BC3,
BC4, and BC5) were all significantly (P < 0.05) lower than those of
control (Table 1). Among all treatments, disease index and incidence of
BC3 was the lowest, the disease incidence and the severity of disease
was decreased by 58.72 % and 69.81 %, respectively when compared to
the control. These results indicated that biochar amendment could
potentially suppress bacterial wilt disease, and 15 t ha-1 of biochar
showed the best control effect on bacterial wilt. No significant differ-
ence in tobacco height and stem girth was found among biochar
treatments and control.
3.2. Influences of biochar amendment on soil properties
Biochar amendment significantly (P < 0.05) affected soil nutrient
status. Compared to the control, the concentration of alkaline nitrogen
in biochar amendment (BC3) increased by 16 %, soil organic matter in
biochar amendments BC3 and BC4 increased by 46.7 % and 46.8 %,
respectively (Table 2). These results suggested that amendment with
15−30 t ha-1 biochar could increase the soil nitrogen and organic
matter contents. No significant difference was observed for available
phosphorus and potassium contents and soil pH among biochar
Table 1
Influence of biochar on tobacco properties and bacterial wilt.
Tobacco properties BC1 BC2
Plant height (cm) 95.56 ± 4.44 a 93.44 ± 6.55 a
Stem girth (mm) 9.33 ± 0.29 a 9.33 ± 0.29 a
Disease incidence (%) 20.18 ± 0.40 a 10.09 ± 0.29 c
Disease index 3.08 ± 0.12 a 1.12 ± 0.23 c
All data are represented as the means ± SE. Values with different letters in the same row are significantly (P < 0.05) different.
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Microbiological Research 231 (2020) 126373
treatments and the control.
Compared to the control, the urease activity in biochar amendments
(BC2 and BC3) significantly (P < 0.05) increased (Table 2), suggesting
amendment with 7.5−15 t ha-1 biochar could increase soil urease ac-
tivity. No significant difference was observed for invertase and catalase
activities among biochar treatments and the control. Phosphatase ac-
tivity in BC5 was significantly (P < 0.05) lower than that in BC1, BC2,
BC3 and BC4, suggesting that amendment with excessive biochar (45 t
ha-1) decreased phosphatase activity.
3.3. Influence of biochar amendment on bacterial communities
At three years after biochar amendment, no significant difference in
the observed species, Chao1, Shannon, and Simpson index was found
between biochar treatments and control (Table S1), suggesting that
biochar had no effect on bacterial α-diversity. 5186 OTUs (53.1 %)
were shared by all soil samples (Fig. 1A). Each biochar treatment
possessed its unique bacterial species. BC3 had the most unique OTUs
(1578 OTUs), followed by BC4 (1241 OTUs), suggesting that 15−30 t
ha-1 biochar could increase the number of unique soil bacterial species.
UPGMA analysis showed that BC1 and BC5 were clustered together;
while BC2 and BC4 were clustered together (Fig. 1B). BC3 sample was
different from other samples, suggesting that the bacterial community
structure of BC3 was changed most.
3.4. Abundance of different phyla
Total 43 bacterial phyla were found in the soil samples.
Actinobacteria, Chloroflexi and Proteobacteria were the most abundant
phyla and together occupied 70 % of bacterial abundance (Fig. 1B).
Biochar amendments significantly (P < 0.05) changed the relative
proportions of seven bacterial phyla. Compared to control, relative
abundances of Armatimonadetes, Firmicutes, Saccharibacteria and
Verrucomicrobia in biochar amendment (BC3), Cyanobacteria and
Verrucomicrobia in biochar amendment (BC4), Planctomycetes in bio-
char amendment (BC5) significantly (P < 0.05) increased (Table 3),
Gemmatimonadetes in biochar amendments (BC3 and BC4) sig-
nificantly (P < 0.05) decreased. These results suggested that biochar
amendments changed bacterial community composition, which was
related to the biochar doses.
3.5. Abundance of bacterial genera
The biochar treatments significantly enriched potential beneficial
bacteria such as Aeromicrobium, Bacillus, Bradyrhizobium, Burkholderia,
Chlorochromatium, Chthoniobacter, Corynebacterium, Geobacillus,
Leptospirillum, Marisediminicola, Microvirga, Pseudoxanthomonas and
Telmatobacter in soil (Fig. 2). Compared to the control, relative abun-
dances of Aeromicrobium, Bacillus, Bradyrhizobium, Chthoniobacter,
Corynebacterium, Geobacillus, Microvirga and Pseudoxanthomonas in BC3,
Chthoniobacter and Marisediminicola in BC4, Burkholderia, Chlor-
ochromatium, Leptospirillum and Telmatobacter in BC5 significantly
(P < 0.05 or P < 0.01) increased.
In contrast, biochar treatment caused the significant (P < 0.05)
decrease of the relative abundances of denitrifying bacteria (e.g.
BC3 BC4 BC5
93.00 ± 4.18 a 96.78 ± 6.99 a 96.11 ± 3.56 a
9.17 ± 1.04 a 8.83 ± 0.29 a 9.00 ± 0.50 a
8.33 ± 0.66 d 10.09 ± 1.06 c 14.47 ± 0.23 b
0.93 ± 0.13 c 1.12 ± 0.20 c 2.00 ± 0.30 b
S. Chen, et al. Microbiological Research 231 (2020) 126373

Table 2
Influence of biochar on soil chemical properties and enzymatic activities.
Soil properties BC1 BC2 BC3 BC4 BC5

AN (mg/kg) 120.25 ± 6.28 b 121.11 ± 12.79 b 139.56 ± 4.75 a 127.50 ± 9.93 ab 112.38 ± 4.67 b
AK (mg/kg) 464.73 ± 111.25 a 520.60 ± 24.92 a 616.39 ± 169.93 a 553.86 ± 198.76 a 588.45 ± 69.59 a
AP (mg/kg) 44.45 ± 7.38 a 63.41 ± 12.70 a 71.01 ± 42.21 a 53.17 ± 31.62 a 53.85 ± 13.68 a
SOM (g/kg) 25.41 ± 5.29 b 32.00 ± 4.33 ab 37.28 ± 6.19 a 37.31 ± 1.71 a 30.21 ± 9.14 ab
pH 5.20 ± 0.45 a 5.24 ± 0.37 a 5.43 ± 0.46 a 5.48 ± 0.35 a 5.08 ± 0.40 a
Urease (mg/g) 0.39 ± 0.05 b 0.57 ± 0.09 a 0.68 ± 0.11 a 0.35 ± 0.07 b 0.35 ± 0.03 b
Invertase (mg/g) 14.71 ± 5.59 a 18.59 ± 2.81 a 16.94 ± 5.98 a 19.17 ± 4.55 a 15.60 ± 0.79 a
Phosphatase (mg/g) 1.05 ± 0.02 a 1.03 ± 0.06 a 1.05 ± 0.02 a 1.08 ± 0.18 a 0.84 ± 0.03 b
Catalase (mg/g) 5.11 ± 0.58 a 4.91 ± 0.46 a 4.81 ± 0.60 a 4.89 ± 0.39 a 4.28 ± 0.75 a

All data are represented as the means ± SE. Values with different letters in the same row are significantly (P < 0.05) different.

Fig. 1. Analysis of bacterial community. (A) Venn showed the shared and unshared OTUs among biochar treatments and control; (B) UPGMA clustering tree of
bacterial community in different treatments.

Noviherbaspirillum, Reyranella, Thermus) and Ralstonia (pathogen of
bacterial wilt). Compared to the control, relative abundances of
Noviherbaspirillum in BC5, Reyranella in BC2, Thermus in biochar treat-
ments (BC2 and BC4), Ralstonia in biochar treatments (BC2, BC3 and
BC5) significantly (P < 0.05) decreased. Compared to the control, the
abundance of Ralstonia in biochar treatments (BC2, BC3, BC4 and BC5)
decreased by 56.7 %, 69.9 %, 46.3 % and 68.8 %, respectively.
abundances of Gemmatimonadetes, Ralstonia, Reyranella and Thermus
were significantly (P < 0.05) positively correlated with the disease
index. Thereby, a higher relative abundance of these microbes in soils is
possible to promote the outbreak of bacterial wilt.
4. Discussion

3.6. Correlation between bacterial abundance and severity of bacterial wilt
The relative abundances of Armatimonadetes, Bacillus,
Chthoniobacter and Saccharibacteria were significantly (P < 0.05) ne-
gatively correlated with the disease index of bacterial wilt (Fig. 3).
Thereby, a higher abundance of these bacteria in biochar treatments
might be helpful for inhibiting bacterial wilt. In contrast, the relative
This study found that rice hull biochar had potential for controlling
bacterial wilt disease. Biochar amendments significantly suppressed
tobacco bacterial wilt in the field trial through changing soil chemical
properties and microbial abundances. The lowest disease incidence and
index was observed in biochar treatment BC3, followed by BC2 and
BC4, suggesting that 30 t ha-1 biochar had the most positive effect on
disease suppression, and appropriate biochar doses (7.5−30 t ha-1)
could more effectively suppress bacterial wilt disease, which was

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S. Chen, et al. Microbiological Research 231 (2020) 126373

Table 3
Abundance of bacterial phyla in biochar amended and unamended soils.
Taxonomy BC1 BC2 BC3 BC4 BC5

Acidobacteria 5.65 ± 2.99 a 5.85 ± 1.4 a 8.88 ± 5.33 a 7.28 ± 3.11 a 7.21 ± 2.34 a
Actinobacteria 35.19 ± 9.30 a 34.17 ± 5.08 a 27.55 ± 11.34 a 30.81 ± 8.29 a 33.72 ± 2.97 a
Armatimonadetes 0.18 ± 0.13 b 0.32 ± 0.10 ab 0.41 ± 0.09 a 0.34 ± 0.12 ab 0.18 ± 0.05 b
Bacteroidetes 2.62 ± 0.60 a 2.31 ± 0.27 a 2.73 ± 1.02 a 2.16 ± 0.31 a 2.33 ± 0.10 a
Chloroflexi 18.21 ± 12.05 a 20.78 ± 6.98 a 21.16 ± 9.19 a 23.01 ± 8.23 a 17.27 ± 1.28 a
Cyanobacteria 0.34 ± 0.30 b 0.67 ± 0.30 ab 0.52 ± 0.07 ab 1.04 ± 0.71 a 0.54 ± 0.05 ab
Chlorobi 0.05 ± 0.03 a 0.05 ± 0.01 a 0.05 ± 0.01 a 0.06 ± 0.03 a 0.04 ± 0.02 a
Chlamydiae 0.01 ± 0.00 a 0.01 ± 0.01 a 0.01 ± 0.01 a 0.02 ± 0.02 a 0.01 ± 0.00 a
Deinococcus-Thermus 0.06 ± 0.05 a 0.02 ± 0.01 a 0.02 ± 0.02 a 0.01 ± 0.01 a 0.02 ± 0.03 a
Elusimicrobia 0.03 ± 0.01 a 0.03 ± 0.02 a 0.05 ± 0.05 a 0.03 ± 0.01 a 0.04 ± 0.01 a
Firmicutes 1.57 ± 0.24 b 1.31 ± 0.38 b 2.32 ± 0.32 a 1.61 ± 0.58 b 1.39 ± 0.16 b
Gemmatimonadetes 5.76 ± 2.00 a 4.42 ± 0.35 ab 3.79 ± 0.52 b 3.53 ± 0.43 b 4.87 ± 0.77 ab
Latescibacteria 0.27 ± 0.24 a 0.23 ± 0.03 a 0.56 ± 0.65 a 0.47 ± 0.34 a 0.25 ± 0.25 a
Microgenomates 0.06 ± 0.05 a 0.09 ± 0.03 a 0.07 ± 0.04 a 0.08 ± 0.04 a 0.12 ± 0.02 a
Nitrospirae 0.55 ± 0.24 a 0.42 ± 0.16 a 0.82 ± 0.88 a 0.57 ± 0.26 a 0.43 ± 0.23 a
Parcubacteria 0.02 ± 0.02 a 0.05 ± 0.05 a 0.04 ± 0.02 a 0.03 ± 0 a 0.06 ± 0.04 a
Planctomycetes 0.19 ± 0.15 b 0.45 ± 0.16 ab 0.49 ± 0.24 ab 0.48 ± 0.16 ab 0.53 ± 0.17 a
Proteobacteria 25.37 ± 6.36 a 23.93 ± 3.39 a 22.87 ± 5.61 a 22.35 ± 4.68 a 26.30 ± 0.35 a
Saccharibacteria 3.12 ± 2.27 b 4.13 ± 1.03 ab 6.33 ± 1.93 a 4.80 ± 2.03 ab 3.63 ± 1.22 ab
Thermotogae 0.07 ± 0.07 a 0.04 ± 0.02 a 0.06 ± 0.07 a 0.04 ± 0.03 a 0.04 ± 0.03 a
Verrucomicrobia 0.05 ± 0.05 b 0.11 ± 0.03 ab 0.23 ± 0.08 a 0.25 ± 0.19 a 0.10 ± 0.06 ab

All data are represented as the means ± SE. Values with different letters in the same row are significantly (P < 0.05) different.

consistent with previous studies indicating that relatively low con-
centrations of biochar suppressed plant diseases, higher concentrations
of biochar were mostly ineffective or even accelerated plant diseases
(Jaiswal et al., 2014, 2015; Copley et al., 2015; Huang et al., 2015). As
previously reported, plant resistance to disease was frequently depen-
dent on biochar dose applied, plant disease responses eventually show
an inverted U-shaped dose/response curve if dose of biochar is further
increased (Jaiswal et al., 2014, 2015).
Several studies have demonstrated that biochar amendment makes
soil nutrients more available (Rodrigues et al., 2007; Rondon et al.,
2007; Hernandez-Soriano et al., 2016). Biochar retains more nitrogen in
soils through enhancing nitrogen fixation, ammonia/ammonium re-
tention, reducing N2O emissions and nitrate leaching (Rodrigues et al.,
2007; Rondon et al., 2007). In this study, we found that biochar ap-
plication significantly increased content of alkaline nitrogen and urease
activity. Increase of urease activity enhances the availability of nitrogen

Fig. 2. Heat map showed the relative abundances of bacterial genera in different treatments. Red color represented higher abundance, blue color represented
lower abundance.

5
S. Chen, et al.
Fig. 3. Correlation between bacterial genera abundances and disease index of bacterial wilt.
to meet the need of plant growth. In BC3 treatment, biochar amend-
ment increased soil nitrogen level, which was consistent with previous
study (Xu et al., 2014). Biochar has been showed reducing levels of
denitrification via improving soil aeration (Yanai et al., 2007). We
supposed that biochar inhibited denitrifying bacteria and N2O-reducing
bacteria.
Biochar is a product with high carbon content, which may increase
the soil organic matter content after application to soil. Many studies
have showed that biochar improves soil fertility and crop yields
(Shaaban et al., 2018). This study demonstrated that the biochar sig-
nificantly increased the content of organic matter in the soil (Table 2),
which verified the results of previous studies (Liang et al., 2014; Yin
et al., 2014). Biochar can adsorb and promote soil organic molecule
polymerization to form organic matter (Van Zwieten et al., 2010). In
addition, decomposition of biochar contributes to the development of
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Microbiological Research 231 (2020) 126373
humus (Kimetu and Lehmann, 2010). Soil organic matter is an im-
portant indicator of soil fertility and an important source of microbial
nutrients (Cookson et al., 2005). In addition, we supposed that the
higher soil nitrogen and carbon content and urease activity could in-
crease activity of beneficial microorganisms against pathogen such as R.
solanacearum (Bailey and Lazarovits, 2003; Yin et al., 2011; Zhang
et al., 2017). It was speculated that biochar application changed soil
properties that indirectly suppressed pathogen growth by promoting
antagonistic microorganisms.
Our results suggested that biochar might stimulate soil microbial
activity and affect bacterial abundances via increasing soil carbon and
nitrogen, which was consistent with the previous report (Gomez et al.,
2014). The abundances of potential beneficial bacteria, which have the
functions of cellulose-degradation, N-cycling, P-solubilization and pa-
thogen suppression, were improved in biochar amended soils. For
S. Chen, et al.
example, biochar application significantly increased the abundance of
Aeromicrobium, Armatimonadetes, Bacillus, Bradyrhizobium, Burkholderia,
Chthoniobacter, Corynebacterium, Geobacillus, Leptospirillum, Mar-
isediminicola, Microvirga, Pseudoxanthomonas, Saccharibacteria and Tel-
matobacter. These potential beneficial bacteria have positive effects on
soil nutrients cycling. For instance, Chthoniobacter, Geobacillus, Pseu-
doxanthomonas and Telmatobacter can degrade hemicellulose, lig-
nocellulose and cellulose (Sangwan et al., 2004; Pankratov et al., 2012;
Kumar et al., 2015; Daas et al., 2018). Corynebacterium glutamicum
degrades and assimilates a rich spectrum of aromatic compounds in-
cluding lignin-derived aromatic compounds (Shen et al., 2012). Bra-
dyrhizobium, Burkholderia, Leptospirillum and Microvirga are nitrogen-
fixing bacteria, which can increase the nitrogen contents by N2 fixation
(Parro and Moreno-Paz, 2004; Suárez-Moreno et al., 2008; Radl et al.,
2014; Osei et al., 2018). In soils, huge reserves of inorganic or organic
phosphorus are present in immobilized or unavailable form. Bacillus
and Burkholderia are phosphate-mobilizing bacteria, which have the
potential to mineralize and solubilize organic and inorganic phosphorus
in soil (Panhwar et al., 2014). These potential beneficial bacteria can
improve soil nutrients availability for plant uptake through N fixation,
P mobilization and carbon degradation.
Aeromicrobium, Bacillus, Burkholderia and Marisediminicola have the
potential of control plant disease by producing anti-microbial molecules
(Reeves et al., 2004; Wang and Liang, 2014; Rojas-Rojas et al., 2018).
For example, Bacillus amyloliquefaciens effectively controlled bacterial
wilt by producing antimicrobial substances and inducing plant re-
sistance (Tan et al., 2013; Wang and Liang, 2014). We speculated that a
higher abundance of Bacillus in biochar treatment (BC3) could more
effectively inhibit R. solanacearum, which was confirmed by the lowest
disease incidence and index in BC3 treatment, and the negative corre-
lation between abundance of Bacillus and disease index. Arthrobacter
pascens can promote plant growth by producing indole aectic acid (Li
et al., 2018). Arthrobacter is involved in decomposition of aromatic
compounds, which are rich in biochar (O’Loughlin et al., 1999).
Chthonomonas calidirosea belonging to phylum Armatimonadetes, as a
saccharide scavenger, can utilize a wide range of carbohydrates in soil
(Lee et al., 2014). Saccharibacteria species utilize and degrade the
complex carbon sources (cellulose, hemicellulose, pectin, starch and
1,3-β-glucan) from plant root exudates and cell wall (Starr et al., 2018).
We supposed that possibly Armatimonadetes and Saccharibacteria can
compete with R. solanacearum for carbon source to suppress pathogen
growth. These results revealed that biochar enriched more potential
beneficial bacteria, which participated in nutrients cycling and sup-
pressed plant soil-borne pathogens. These findings explained the reason
for higher soil nutrients (C and N) supplying capacity and crop re-
sistance to disease of biochar treatments.
The genera with decreased abundance by biochar treatments were
reported as denitrifying bacteria (Noviherbaspirillum, Reyranella,
Thermus), N2O-reducing bacteria (Gemmatimonadetes) and plant pa-
thogen (Ralstonia). Ralstonia is the pathogen of bacterial wilt, sug-
gesting that biochar could inhibit the growth of pathogen Ralstonia in
soils. This study proved that biochar had the potential to control soil-
borne disease. Denitrification is a stepwise, anaerobic microbial process
reducing nitrate to N2, N2O and NO, which can decrease soil nitrogen
content (Seitzinger et al., 2006). Species of Noviherbaspirillum, Reyra-
nella and Thermus are able to reduce nitrate and nitrite then to NO and
N2O (Kim et al., 2013; Alvarez et al., 2014; Ishii et al., 2017). Gem-
matimonas aurantiaca belonging to Gemmatimonadetes phylum can re-
duce N2O to N2 (Park et al., 2017). We supposed that biochar amend-
ment possibly decreased soil nitrogen losses by reducing the abundance
of denitrifying bacteria. Besides, the abundances of Gemmatimonadetes,
Reyranella and Thermus were positively correlated with the disease
index, suggesting that decreasing denitrifying bacteria by biochar could
help to control bacterial wilt.
In this study, biochar amendment did not elicit augmentation of
bacterial diversity, which was not consistent with the previous studies
7
Microbiological Research 231 (2020) 126373
(Kolton et al., 2017). We supposed that biochar made from different
organic wastes might show different effect on soil microorganisms. On
the other hand, we found that different doses of biochar showed dif-
ferent effects on soil properties and microbial abundances. In this study,
15−30 t ha-1 biochar had a better effect on improving the soil prop-
erties and microbial abundances and controlling bacterial wilt. Ex-
cessive biochar (45 t ha-1 in BC5) could not enhance the enzymatic
activity and soil microbial growth, possibly because large amount of
biochar contains excessive aldehydes and ketones that are not suitable
for microbial growth (Chen et al., 2011). This was consistent with the
reports that lower doses of biochar stimulated positive effects but
higher doses of biochar caused toxicity and inhibition (Jaiswal et al.,
2014; Kammann and Graber, 2015). We suggested that applying
7.5−30 t ha-1 biochar to soil. 30 t ha-1 of biochar was the optimum
application rate for control bacterial wilt.
In conclusions, the results presented here indicated that biochar
additions to soil significantly reduced the disease incidence and severity
of bacterial wilt. The disease incidence and index in biochar amend-
ments (7.5−45 t ha-1) significantly decreased when compared with
control. Amending 15 t ha-1 of biochar showed the best control effect on
bacterial wilt disease. This study provided an alternative potential
method to control bacterial wilt. Biochar amendment increased soil
organic matter, nitrogen and urease activity. Biochar amendment en-
riched potential beneficial bacteria involved in carbon, nitrogen and
phosphorus cycling and bacteria producing anti-microbial compounds.
Biochar also decreased the abundances of denitrifying bacteria and
plant pathogen when compared with control. All these changes in soil
properties and microorganisms might contribute to the control action of
biochar on bacterial wilt disease. However, biochar couldn’t fully in-
hibit bacterial wilt, in BC3 treatment, 8.33 % of tobacco plants was
infected by R. solanacearum. Combination of biochar amendment and
biocontrol agent might further improve the control effect. On the other
hand, the effect of biochar on microbial function genes is still unknown.
Declaration of Competing Interest
The authors declare that they have no competing interests.
Acknowledgments
We wish to thank technicians Tan, Peng, and Xiang for their helps in
collection of soil samples and fieldwork assistance.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.micres.2019.126373.
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