microorganisms

Article
Bacillus velezensis BV01 Has Broad-Spectrum Biocontrol
Potential and the Ability to Promote Plant Growth
Ting Huang 1,2 , Yi Zhang 1 , Zhihe Yu 2 , Wenying Zhuang 1 and Zhaoqing Zeng 1, *

1 State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences,

Beijing 100101, China; h_ting17@163.com (T.H.); zhangyicrazy@163.com (Y.Z.); zhuangwy@im.ac.cn (W.Z.)
2 College of Life Sciences, Yangtze University, Jingzhou 434025, China; zhiheyu@hotmail.com
* Correspondence: zengzq@im.ac.cn

Abstract: To evaluate the potential of a bacterial strain as a fungal disease control agent and plant
growth promoter, its inhibitory effects on phytopathogens such as Bipolaris sorokiniana, Botrytis cinerea,
Colletotrichum capsici, Fusarium graminearum, F. oxysporum, Neocosmospora rubicola, Rhizoctonia solani,
and Verticillium dahliae were investigated. The results showed that the inhibitory rates in dual-culture
and sterile filtrate assays against these eight phytopathogens ranged from 57% to 83% and from
36% to 92%. The strain was identified as Bacillus velezensis based on morphological and physiological
characterization as well as phylogenetic analyses of 16S rRNA and the gyrase subunit A protein (gyrA)
regions. The results demonstrated that B. velezensis was able to produce fungal cell-wall-degrading
enzymes, namely, protease, cellulase, and β-1,3-glucanase, and the growth-promotion substances
indole-3-acetic acid (IAA) and siderophore. Furthermore, B. velezensis BV01 had significant control
effects on wheat root rot and pepper Fusarium wilt in a greenhouse. Potted growth-promotion
experiments displayed that BV01 significantly increased the height, stem diameter, and aboveground
fresh and dry weights of wheat and pepper. The results imply that B. velezensis BV01, a broad-
spectrum biocontrol bacterium, is worth further investigation regarding its practical applications
in agriculture.

Keywords: Bacillus; antifungal activity; fungal phytopathogens; wheat root rot; Fusarium wilt;
Citation: Huang, T.; Zhang, Y.; Yu, Z.; greenhouse pot experiment
Zhuang, W.; Zeng, Z. Bacillus velezensis
BV01 Has Broad-Spectrum Biocontrol
Potential and the Ability to Promote
Plant Growth. Microorganisms 2023, 1. Introduction
11, 2627. https://doi.org/10.3390/
Crop diseases caused by phytopathogens have resulted in a decrease in agricultural
microorganisms11112627
yields and quality, leading to significant economic losses [1]. In particular, soil-borne
Academic Editors: Chetan Keswani fungal infections of important crops such as wheat, corn, rice, and pepper cause large
and Rainer Borriss economic losses [2]. The United Nations 2030 Sustainable Development Goals suggested
Received: 12 September 2023
that the world should ensure sustainable consumption and production patterns, promote
Revised: 23 October 2023
sustainable agriculture, and reduce environmental pollution [3]. For a long time, synthetic
Accepted: 23 October 2023 chemical pesticides were commonly used in traditional agriculture to combat plant dis-
Published: 25 October 2023 eases, but they often caused environmental pollution and residual toxic effects in animals
and humans [4]. Thus, discovery of eco-friendly, long-lasting, and effective methods are
required for disease prevention and management in agriculture. The use of microbial and
biochemical agents has been explored as a practical alternative approach [5].
Copyright: © 2023 by the authors. The plant-growth-promoting rhizobacteria (PGPRs) are often used for the production
Licensee MDPI, Basel, Switzerland. of bioactive substances that can protect plants by suppressing pathogens, inducing sys-
This article is an open access article
temic resistance, or improving resistance to environmental stresses, by facilitating nutrient
distributed under the terms and
acquisition and modulating phytohormone levels in plants [6,7]. In recent years, Bacillus
conditions of the Creative Commons
subtilis and its closest relatives B. amyloliquefaciens, B. velezensis, B. cereus, and B. licheni-
Attribution (CC BY) license (https://
formis have been widely used as biofertilizers and biofungicides [8,9]. Bacillus velezensis
creativecommons.org/licenses/by/
FZB42, the classical PGPR strain, was successfully used as a biocontrol agent in potato,
4.0/).

Microorganisms 2023, 11, 2627. https://doi.org/10.3390/microorganisms11112627 https://www.mdpi.com/journal/microorganisms

Microorganisms 2023, 11, 2627 2 of 13

strawberry, wheat, and cabbage [10–14]. The most prevalent plant fungal diseases, such
as grey mold, Fusarium head blight, anthracnose, and root rot, etc., are mainly caused by
species of Botrytis, Fusarium, Colletotrichum, and Rhizoctonia [15]. This can be attributed to
their broad host range, genetic diversity, rapid adaptation to plant disease resistance, and
production of toxins [16]. Previous studies have shown that B. velezensis is a promising
agent for control of Rhizoctonia solani [17], Gaeumannomyces graminis var. tritici [18], Fusarium
oxysporum f. sp. niveum [19], Botrytis cinerea, Colletotrichum gloeosporioides, and Phytophthora
infestans [20], and it has attracted widespread attention in agricultural disease research.
Nevertheless, studies on its biocontrol mechanism, screening of excellent strains, analyses
of transcriptomics, proteomics, metabolomics, and research on industrial and commercial
applications of B. velezensis are needed [21].
In this study, we aimed to assess the potential of a newly isolated bacterial strain, B.
velezensis BV01, as a broad-spectrum biocontrol agent and investigate its capacity to control
plant diseases and promote wheat and pepper development. The findings are of great
significance for reducing the use of chemical fungicides to control soil-borne fungal diseases,
thereby improving the ecological environment, and for providing technical support for
food safety and sustainable development.

2. Materials and Methods

2.1. Tested Strains
Information on the 12 bacterial and fungal strains used is listed in Table 1. Bacillus
velezensis BV01 was isolated from a contaminated potato dextrose agar (PDA) plate in the
laboratory. Bacillus velezensis JDF and B. subtilis L01 and BS208 were isolated from three
commercially available bacterial agents NongBaoShengWu® , LvLong® , and GuanLan® ,
respectively, and the eight fungal plant pathogen strains were provided by colleagues
from Beijing Academy Agriculture and Forestry Sciences, China Academy Agricultural
Sciences, Guangxi Academy Agricultural Sciences, Nanjing Agricultural University, and
our institute (Table 1). All strains were deposited in the China General Microbiological
Culture Collection Center (CGMCC) and the State Key Laboratory of Mycology, Institute
of Microbiology, Chinese Academy of Sciences.

Table 1. The bacterial and fungal strains tested in this study.

Strain Characteristics Relevant to This Work Source

Isolated from the State Key Laboratory of
Bacillus velezensis CGMCC Mycology, Institute of Microbiology, Chinese
This work
1.60184 Academy of Sciences (116.38982◦ E,
40.01076◦ N) on 6 November 2019
Registration of broad-spectrum antagonism,
B. velezensis JDF stress resistance and growth-promoting Isolated from NongBaoShengWu® bacterial agent
ability, used as a positive control
Registration of antimicrobial activity and
B. subtilis L01 strong stress resistance, used as a positive Isolated from LvLong® bacterial agent
control
Registration of prevention and control of
B. subtilis BS208 gray mold and powdery mildew, used as a Isolated from GuanLan® bacterial agent
positive control
Provided by Prof. Niu Yongchun of Chinese
Bipolaris sorokiniana PP12 Causes wheat root rot
Academy of Agricultural Sciences
Botrytis cinerea PP1 Causes tomato gray mold Provided by Prof. Qiu Jiyan of Beijing Academy of
Colletotrichum capsici PP6 Causes ring rot disease Agricultural Sciences
Provided by Prof. Ma Zhengqiang of Nanjing
Fusarium graminearum PP15 Causes Fusarium head blight
Agricultural University
Microorganisms 2023, 11, 2627 3 of 13

Table 1. Cont.

Strain Characteristics Relevant to This Work Source

Provided by Prof. Li Qili of Guangxi Academy of
F. oxysporum F6 Causes blight disease
Agricultural Sciences
Provided by Dr. Fu Shenzhan of Institute of
Neocosmospora rubicola PP23 Causes root and stem rot
Microbiology, Chinese Academy of Sciences
Rhizoctonia solani PP11 Causes wilt disease Provided by Prof. Niu Yongchun of Chinese Academy of
Verticillium dahliae PP8 Causes greensickness Agricultural Sciences

2.2. Evaluation of Antagonistic Abilities In Vitro

All bacterial strains were tested for their antagonistic abilities against eight phy-
topathogens in dual-culture assays [18]. The bacterial strains were grown in nutrient broth
(NB: peptone 10.0 g, beef paste 3.0 g, NaCl 5.0 g, dH2 O 1000 mL) at 28 ◦ C for 2 d, and
the cell suspension was adjusted to 5 × 108 CFU/mL. The fungal strains were grown on
PDA at 25 ◦ C for 5 d. Mycelial plugs (5 mm in diam.) were placed in the center of each
new PDA plate (90 mm in diam.); then, four sterile filter papers (5 mm in diam.), which
contained 6 µL of a bacterial suspension, were placed evenly around individual mycelial
plugs. Sterile water was used as the control. All plates were incubated at 25 ◦ C for 5 d. Each
treatment had three replicates. Measurements were calculated with the following formula:
y = (A − B)/A × 100% (y: percentage of inhibition, A: colony diameter of pathogen on
control plate, and B: colony diameter of pathogen in experimental group) [22].

2.3. Assessment of Antifungal Activity of Bacterial Sterile Filtrate

The antifungal activity of bacterial sterile filtrate was evaluated by measuring the
diameter of the fungal colony [23]. Four bacterial strains were incubated in NB with
shaking at 180 rpm at 28 ◦ C for 2 d, and then the cultures were centrifuged at 8000 rpm at
4 ◦ C for 20 min to collect the supernatant. The supernatant was filtered through a 0.22 µm
filter and then mixed into molten PDA at 45–50 ◦ C with a concentration of 20% (v/v).
The mycelium plug (5 mm in diam.) of each pathogen was placed at the center of each
PDA plate with bacterial filtrate and incubated for 5 d at 25 ◦ C. The PDA plate without
bacterial filtrate was used as the control. Three replicates were set up for each treatment.
The inhibition percentage (y%) was calculated with the following formula: y = (A − C)/A
× 100% (A: growth radius of pathogen in control and C: growth radius of pathogen in
different treatments).

2.4. Morphological Observation and Molecular Identification of Strain BV01

The morphological characteristics of strain BV01 were recorded after incubation on a
nutrient agar (NA: peptone 10.0 g, beef paste 3.0 g, NaCl 5.0 g, agar 18.0 g, dH2 O 1000 mL)
plate at 28 ◦ C for 2 d. A single colony was then taken and evenly spread onto a glass
slide, dry fixed, and Gram stained for 1 min [24]. The microscopic photographs were taken
with a Zeiss AxioCam MRc 5 digital camera (Carl Zeiss, Jena, Germany) attached to Zeiss
Axioskop 2 plus microscope (Carl Zeiss, Göttingen, Germany).
Genomic DNA was extracted from fresh cultures using a bacterial genomic DNA kit
(ZOMAN, Beijing, China) according to the manufacturer’s instructions. The 16S rRNA
and the gyrase subunit A protein (gyrA) regions were amplified using the primer pairs
of 27F (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 1492R (50 -GGTTACCTTGTTACGACTT-
30 ) and gyrA-F (50 -CAGTCAGGAAATGCGTACGTCTT-30 ) and gyrA-R (50 -CAAGGTAAT
GCTCCAGGCATTGCT-30 ), respectively. PCR reactions were conducted using an ABI
2720 Thermal Cycler (Applied Biosciences, Foster City, CA, USA), and the products were
then sequenced using an ABI3730XL DNA Sequencer (Applied Biosciences, Foster City,
CA, USA). The newly obtained sequences and those of the ex-type strain as well as the
related ones retrieved from GenBank were aligned using BioEdit 7.2 [25] and analyzed with
the neighbor-joining (NJ) method [26] via MEGA X [27]. The topological confidence of the
resulting tree and the statistical supports of the branches were tested using the neighbor-
Microorganisms 2023, 11, 2627 4 of 13

joining bootstrap proportion (NJBP) with 1000 replications, each with 10 replicates of
random addition of taxa [28].

2.5. Determination of Enzyme Activity and Secondary Metabolites

Protease, cellulase, and β-1,3-glucanase were detected on skim milk, carboxymethyl
cellulose (CMC), and glucose agar, respectively [29]. Siderophore and indole-3-acetic acid
(IAA) were tested using modified chrome azurol S and Salkowski reagent agar, respec-
tively [30]. Enzyme activity and production of metabolites were observed based on the
presence of clear zones around bacterial colonies after incubation at 28 ◦ C for 3 d. All
treatments were repeated three times.

2.6. Biocontrol Activity of Strain BV01 In Vivo

After evaluation of the antagonistic abilities of the four bacterial strains, two strains,
namely, JDF and BV01, were further tested for their biocontrol and plant-growth-promotion
abilities in a pot experiment. Four germinated wheat seeds were sowed per pot containing
a mixture of podsolic soil and vermiculite (v:v = 1:1). On the 14th day, the roots of the plants
were punctured and inoculated nearby the wounds with pathogen mycelium blocks (5 mm
in diam.). After 24 h, the roots were irrigated with 30 mL (2.5 × 108 CFU/mL) of bacterial
suspensions per wheat plant, and the same amount of NB was used as the control. The
plants were kept in a greenhouse with a 14 h/10 h photoperiod/dark period at 26 ± 1 ◦ C.
The incidence of disease of wheat treated with BV01, JDF, and the non-treated control was
recorded and calculated at 20 d after B. sorokiniana inoculation. Infection types (ITs) of B.
sorokiniana on wheat were evaluated based on the area of a brown or black lesion at the
stem base, with scores varying from 0 to 4 (IT0: no lesion, IT1: coverage of necrotic lesion
less than 1/4, IT2: lesion coverage between 1/4 and 1/2, IT3: coverage between 1/2 and
2/3, and IT4: coverage between 2/3 and total). The disease index = ∑ (di × li ) / (L × dimax )
× 100% (di : infection type, li: number of plants with each infection type, and L: number of
wheat plants investigated) [31].
Three true leaves at the age of 40 d were detached from pepper seedlings and punc-
tured in two places with a sterile needle on both sides of the leaf. Mycelium blocks (5 mm
in diam.) of F. graminearum were placed on the two wounds and then sprayed with 2 mL of
either BV01 or JDF fermentation broth at a concentration of 2.5 × 108 CFU/mL, and the
same volume of NB was used as the non-inoculated control at 24 h after F. graminearum
inoculation. The treatments were kept in a greenhouse at 26 ± 1 ◦ C with a 14 h/10 h
photoperiod/dark period for 6 d; sterile water was added once to keep the treatment
moist. There were 3 leaves in each pot and 3 replicates in each treatment. The diameters of
diseased spots were recorded and calculated at the 6th day after F. graminearum inoculation.
The inhibition rate was calculated as follows: (DCK − Di )/DCK × 100% (DCK : the control
group’s average colony diameter and Di: the treatment group’s average colony diameter).

2.7. Plant-Growth-Promoting Assays in a Greenhouse

Sterilized wheat and pepper seeds were soaked in suspensions of BV01 or JDF at a
concentration of 2.5 × 108 CFU/mL and then in sterile water for 10 min. NB was used
as the control. The plants were kept in a greenhouse with a 14 h/10 h photoperiod/dark
period at 26 ± 1 ◦ C. The wheat and peppers were harvested at the 21st and 35th days,
respectively. Plant height, fresh weight, dry weight, and leaf width were recorded, and the
strong seedling index (SSI) was calculated [32].

2.8. Statistical Analysis

Statistical analysis was performed using SPSS 21 (Armonk, NY, USA). ANOVA was
performed, and mean values were compared using Duncan’s multiple range test with
p < 0.05 as the level of significance. All analyses were conducted using GraphPad Prism
8 (San Diego, CA, USA).
Microorganisms 2023, 11, x FOR PEER REVIEW 5 of 14

Microorganisms 2023, 11, 2627 5 of 13

3. Results
3.1. Inhibitory Effects of Four Tested Bacterial Strains against Eight Fungal Phytopathogens
Strain BV01 exhibited varying degrees of antagonism against different phytopatho-
3. Results
gens,
3.1. and theEffects
Inhibitory inhibition rates
of Four ranged
Tested fromStrains
Bacterial 61% toagainst
83% (Table
Eight2)Fungal
with the highest potential
Phytopathogens
inhibitory effects against B. cinerea PP1 (Figure 1, Treatment 1). The inhibition rates
Strain BV01 exhibited varying degrees of antagonism against different phytopathogens,
showed that BV01 had significantly higher inhibitory effects than
and the inhibition rates ranged from 57% to 83% (Table 2) with the highest JDF, L01, andpotential
BS208 on
six of the eight
inhibitory effectstested
againstfungal phytopathogens.
B. cinerea PP1 (Figure 1, Treatment 1). The inhibition rates showed
that BV01 had significantly higher inhibitory effects than JDF, L01, and BS208 on six of the
Tabletested
eight 2. Antifungal
fungalactivities of four bacterial strains.
phytopathogens.
Dual-Culture Inhibition Rate (%) Fermentation Broth Inhibition Rate (%)
Strains CGMCCTable 2. Antifungal activities of four bacterial strains. CGMCC
JDF L01 BS208 JDF L01 BS208
1.60184 1.60184
Dual-Culture Inhibition Rate (%) Fermentation Broth Inhibition Rate (%)
B. sorokiniana
Strains
PP12 80.68 ± 1.23 a 79.55 ± 0.93 a 77.27 ± 1.05 b 27.27 ± 2.47 c 92.26 ± 0.68 a 76.77 ± 0.23 b 70.32 ± 1.03 c 8.39 ± 1.89 d
CGMCC CGMCC
B. cinerea PP1 82.95 ± 1.23 b 85.23 JDF± 2.23 a 85.23L01± 2.14 a 0.00 BS208
± 0.83 c 81.82 ± 0.82 a 64.77JDF ± 1.00 c 71.59L01 ± 0.58 b 2.27 BS208
± 1.44 d
1.60184 1.60184
C. capsiciPP12
B. sorokiniana PP6 79.55
80.68 ±± 0.64
1.23 a
a 75.00±±0.93
79.55 1.25
a
b 77.27±±1.05
77.27 1.07
b
b 26.14± ±2.47
27.27 1.68
c
c 72.73
92.26 ±±0.68
0.75a
a 71.59
76.77 ±±0.23
0.86b
a 72.73
70.32 ±±1.03
0.42c a 4.55
8.39 ±±1.89
1.45d b
a a a b c c a a c c
F. graminearum
B. cinerea PP1 PP15 82.95
61.36±±1.23 b b 65.91±±2.23
1.09 85.23 0.49 85.23
62.50±±2.14
0.62 0.00 0.00±±0.83
0.62 59.0981.82 ±±0.82
1.66 43.1864.77 ±±1.00
0.52 47.53
71.59 ±±0.58
0.66 13.64
b b 2.27 ±±1.44
0.97
d d

C. capsici PP6 79.55 ± 0.64 a a 75.00 ± 1.25 b b 77.27 ± 1.07 b c 26.14 ± 1.68 c d 72.73 ± 0.75 a a 71.59 ± 0.86 a b 72.73 ± 0.42 a b 4.55 ± 1.45 b c
F. oxysporum F6 61.36
F. graminearum PP15
65.91 ± 0.66 65.91
± 1.09 b
62.50±±0.49
1.03
a 52.27 ± 0.71 32.95
62.50 ± 0.62 b
± 1.27 56.79
0.00 ± 0.62 c
± 1.23 13.58
59.09 ± 1.66 a
± 1.06 12.04
43.18 ± 0.52 c
± 0.69 13.64
47.53 ± 0.66 b
8.95 ±± 0.97
0.46d
N. rubicola F6
F. oxysporum PP23 65.91
56.52±± 1.02
0.66 a a 50.72 ± 0.93 50.72±±0.71
62.50 ± 1.03 b b 52.27 1.23
c b 42.03 ± 1.24
32.95 ± 1.27 d c 36.13
56.79 ±±1.23
1.19a a 12.18 ± 0.99
13.58 ± 1.06 b b 12.18
12.04 ±±0.69
0.52b b 4.19
8.95 ±±0.46
0.74c c
a b b c a b b c
N. rubicola PP23
R. solani PP11 56.52 ± 1.02 50.72 ±
69.32 ± 0.71 54.55 ± 0.86
a 0.93 b 50.72 ± 1.23
53.41 ± 1.23 b 42.03 ± 1.24
0.00 ± 1.7 c 36.13 ± 1.19
46.59 ± 1.23 a 12.18 ± 0.99
0.00 ± 1.08c c 12.18 ± 0.52
0.00 ± 1.21c c 4.19 ± 0.74
5.68 ± 0.56b b
R. solani PP11 69.32 ± 0.71 a 54.55 ± 0.86 b 53.41 ± 1.23 b 0.00 ± 1.7 c 46.59 ± 1.23 a 0.00 ± 1.08 0.00 ± 1.21 5.68 ± 0.56
V. dahliae
V. dahliae PP8
PP8 70.15
70.15 ±± 1.18
1.18 a a 58.21 ± 0.73
58.21 ± 0.73 b
b 55.22 ± 1.35
55.22 ± 1.35 c
c 32.84 ± 0.88
32.84 ± 0.88 d
d 71.72 ± 1.18
71.72 ± 1.18 a
a 59.07 ± 0.73
59.07 ± 0.73 b
b 61.03 ± 1.35
61.03 ± 1.35 b
b 33.95 ± 0.88
33.95 ± 0.88 c
c

Theinhibition
The inhibition rates
rates (%)(%)
(n (n
= 3,= mean
3, mean ± SE).
± SE). Different
Different letters
letters indicate
indicate significantly
significantly different
different groupsgroups
(p < 0.05,(p
< 0.05, ANOVA,
ANOVA, Tukey HSD).
Tukey HSD).

Figure1.1.Inhibitory
Figure Inhibitoryeffects
effectsofofBV01
BV01against
againstfungal
fungalphytopathogens.
phytopathogens. CK:
CK: only
only pathogen
pathogen on onPDA
PDAatat
25◦°C
25 for 55 d;
C for d; Treatment
Treatment1:1: dual
dual culture
cultureof
ofBV01
BV01against
againstpathogen
pathogenon onPDA
PDAat 25◦°C
at25 for 55 d;
C for d; Treatment
Treatment
2:2: pathogen
pathogenon onPDA
PDAamended
amendedwith withfermentation
fermentationbroth
brothof
ofBV01
BV01atat25
25◦°C for55d.
C for d.

Antifungal assay
Antifungal assay by
by fermentation
fermentation broth
broth test
test showed
showed that
that BV01
BV01 had
had relatively
relatively high
high
inhibitory effects
inhibitory effects against
against different
different pathogens
pathogens (Table
(Table 2),
2), and
and the
the highest
highest inhibitory
inhibitory rate
rate
reached 92%
reached 92% (against
(against B. sorokiniana
sorokiniana PP12)
PP12) (Figure
(Figure 1,1,Treatment
Treatment2). 2).Overall,
Overall,the effects
the of
effects
BV01
of BV01were
werebetter than
better thanthose of of
those JDF and
JDF L01
and L01and
and were
weresignificantly superior
significantly superiorto to
those
thoseof
BS208.
of BS208.

3.2.
3.2. Identification
Identification of
of Strain
StrainBV01
BV01
The colony of BV01
The colony of BV01 was was ivory
ivorywhite
whiteandandnon-transparent
non-transparentwith with a rough
a rough surface
surface on
on NA medium (Figure 2A–C). The cells were Gram-positive (Figure
NA medium (Figure 2A–C). The cells were Gram-positive (Figure 2D), rod-shaped, 1.43–2D), rod-shaped,
1.43–2.53 µm long,
2.53 µm long, 0.66–0.88
0.66–0.88 µmµm
wide,wide,
andand occurred
occurred singly,ininpairs,
singly, pairs,or
or occasionally
occasionally in
in short
short
chains. The analysis of 16S rRNA sequences showed that strain BV01 shared 99% identity
with the type strain of B. velezensis (CR502) according to a BLAST search. The resulting NJ
2023, 11, x FOR PEER REVIEW 6 of 14

Microorganisms 2023, 11, x FOR PEER REVIEW 6 of 14

chains. The analysis of 16S rDNA sequences showed that strain BV01 shared 99% identity
with
Microorganisms 2023,the type
strain of B. velezensis (CR502) according to a BLAST search. The resulting NJ 6 of 13
11, 2627
chains. Theofanalysis
trees based on sequences of 16Sand
16S rRNA rDNA sequences
gyrA (Figureshowed that strain
3) showed that BV01
BV01shared 99% identity
clustered
with Bacillus species and grouped with the type strain of B. velezensis, which confirmedresulting
with the type strain of B. velezensis (CR502) according to a BLAST search. The its NJ
trees
taxonomic position. based on sequences of 16S rRNA and gyrA (Figure 3) showed that BV01
trees based on sequences of 16S rRNA and gyrA (Figure 3) showed that BV01 clustered clustered
with
with Bacillus
Bacillus species
species and
and grouped
grouped with
with the
the type
type strain
strain of
of B.
B.velezensis,
velezensis,which
whichconfirmed
confirmedits
its
taxonomic
taxonomic posi
position.
tion.

Figure 2. Colony and microscopic

Figure
Figure 2.
2. Colony characteristics
Colonyand microscopicof
andmicroscopic BV01. (A–C)
characteristics
characteristicsof General
ofBV01. colonies
BV01.(A–C)
(A–C) oncolonies
General
General nutrient
colonies onagar;
on nutrient
nutrientagar;
agar;
(D) Gram-stained cells.
(D) Bar: D = 10 cells.
(D) Gram-stained
Gram-stained µm. Bar:
cells. Bar: DD==10
10µm.
µm.

Figure 3. Phylogenetic trees generated based on sequences of 16S rRNA (A) and gyrA (B) regions of
Bacillus species. NJBP values greater than 75% are shown at the nodes.

Figure 3. Phylogenetic trees

Figure generated based
3. Phylogenetic on sequences
trees generated basedof
on16S rRNA of
sequences (A) and
16S gyrA
rRNA (A)(B)
andregions ofregions of
gyrA (B)
Bacillus species. NJBP values
Bacillus greater
species. than
NJBP 75%greater
values are shown at the
than 75% are nodes.
shown at the nodes.
Microorganisms 2023, 11, x FOR PEER REVIEW 7 o
Microorganisms 2023, 11, 2627 7 of 13

3.3. Detection of Antagonism-Related Lytic Enzymes

3.3. Detection of Antagonism-Related Lytic Enzymes
Clear zones detected around the colony of BV01 indicated that the strain produ
Clear zones detected around the colony of BV01 indicated that the strain produced pro-
protease,
tease, cellulase,
cellulase, and β-1,3-glucanase
and β-1,3-glucanase (Figure
(Figure 4A–C) 4A–C)
as well as well as(Figure
as siderophore siderophore
4D) and(Figure
and IAA (Figure 4E), which suggested its high potential in biological control.
IAA (Figure 4E), which suggested its high potential in biological control. The production of The prod
tionreached
IAA of IAA12.17
reached 12.17
mg/mL mg/mL
after afterfor
incubation incubation
6 d. for 6 d.

Figure4.4. Detection
Figure Detectionofofextracellular
extracellular enzyme
enzyme production
production and growth-promotion
and growth-promotion traits oftraits
BV01.of BV01
protease;
(A) protease;(B)
(B)cellulase;
cellulase; (C)(C)β-1,3-glucanase;
β-1,3-glucanase; (D) siderophore;
(D) siderophore; (E) indole-3-acetic
(E) indole-3-acetic acid (IAA); acid
Gp1 : (IAA);
BV01
BV01suspension,
suspension, CKCK1 : 10
1: mg/mL
10 mg/mL IAA, CK
IAA,2 : sterilize
CK 2 : distilled
sterilize water,
distilledCK :
water,
3 NB.
CK 3 : NB.
3.4. Biocontrol Effects of Bacterial Strains BV01 and JDF on Wheat Root Rot
3.4. Biocontrol Effects of Bacterial Strains BV01 and JDF on Wheat Root Rot
Lesions at the stem bases of wheat were obviously brown in the non-treated control,
while Lesions at the
those treated stem
with BV01bases of wheat
and JDF were verywere obviously
slightly infectedbrown
(Figurein theThe
5A). non-treated
disease con
while those treated with BV01 and JDF were very slightly infected (Figure 5A). The dise
indices of CK, BV01, and JDF were 76.4, 40.8, and 53.6, respectively (Figure 5B). In the BV01
treatment,
indices ofinfection with and
CK, BV01, wheat rootwere
JDF rot was significantly
76.4, 40.8, and(p53.6,
< 0.05) reduced, the(Figure
respectively relative 5B). In
control efficacy wasinfection
BV01 treatment, 47% (Figure 5C),wheat
with and the fresh
root and
rot wasdrysignificantly
weights (Figure(p <5D) andreduced,
0.05) plant the
height (Figure 5E) were increased by 24%, 91%, and 34%, respectively.
ative control efficacy was 47% (Figure 5C), and the fresh and dry weights (Figure 5D)
plant
3.5. heightEffect
Biocontrol (Figure 5E) were
of Strain increased
BV01 on Fusarium by
Wilt24%, 91%, and 34%, respectively.
The symptoms on pepper leaves of the control were severe, on those treated with
JDF were moderate, and on those treated with BV01 were weak (Figure 6A). The average
diameter of a spot was 2.31, 0.99, and 1.76 cm in the CK, BV01, and JDF treatments
(Figure 6B). The control effect reached 57% and 24% for the treatments with BV01 and JDF,
respectively (Figure 6C).
ms 2023, 11, x FOR PEER REVIEW 8 of 14
Microorganisms 2023, 11, 2627 8 of 13

Figure 5. Inhibitory effect

Figure of Bacilluseffect
5. Inhibitory strains on wheat
of Bacillus rooton
strains rotwheat
diseaserootcaused by B.
rot disease sorokiniana
caused PP12.
by B. sorokiniana PP12.
(A) Symptoms of(A) B. Symptoms
sorokinianaofon B. wheat rootsonwith
sorokiniana wheat different
roots withtreatments; (B) disease(B)
different treatments; index of B.
disease index of B.
sorokiniana in the sorokiniana
treatmentsinwiththe BV01 and JDF;
treatments with (C)
BV01inhibition
and JDF;rates of BV01 and
(C) inhibition ratesJDF; (D) fresh
of BV01 and(D) fresh
and JDF;
dry weights of wheat; (E) shoot biomass (cm) measured by plant height of wheat. The results
and dry weights of wheat; (E) shoot biomass (cm) measured by plant height of wheat. The results were
Microorganisms 2023, 11, x FOR PEER REVIEW 9 of 14
observed after 40were d ofobserved
incubation.afterValues
40 d ofare the means
incubation. ± SEs,
Values are nthe
= 27 plants,
means ** np =< 27
± SEs, 0.001, and
plants, ** *p p< <0.001, and
0.05. * p < 0.05.

3.5. Biocontrol Effect of Strain BV01 on Fusarium Wilt

The symptoms on pepper leaves of the control were severe, on those treated with JDF
were moderate, and on those treated with BV01 were weak (Figure 6A). The average di-
ameter of a spot was 2.31, 0.99, and 1.76 cm in the CK, BV01, and JDF treatments (Figure
6B). The control effect reached 57% and 24% for the treatments with BV01 and JDF, re-
spectively (Figure 6C).

Figure 6. Effect of Bacillus strains on disease symptoms caused by F. graminearum PP15 on leaves.
Figure 6. Effect of Bacillus strains on disease symptoms caused by F. graminearum PP15 on leaves.
(A)
(A) Symptoms
Symptoms of F. F. graminearum
ofgraminearum on leaves
on leaves withwith different
different treatments;
treatments; (B)diameter
(B) spot spot diameter after treatment
after treat-
with BV01 or JDF; (C) inhibition rates of BV01 and JDF. Values are the means ±
ment with BV01 or JDF; (C) inhibition rates of BV01 and JDF. Values are the means ± SEs, n = 9 leaves,n = 9 leaves,
SEs,
****
p <p 0.001, andand
< 0.001, * p <* 0.05.
p < 0.05.

3.6. Growth-Promotion Effects of Strain BV01 on Wheat and Pepper

Wheat treated with BV01 exhibited an increase in height of 13% (Figure 7A), while the
fresh weight and dry weight were improved by 10% and 5% (Figure 7B). Pepper treated
with BV01 exhibited increases in the fresh weight, leaf width, and stem thickness of 20%,
 Microorganisms 2023, 11, 2627 9 of 13

3.6. Growth-Promotion Effects of Strain BV01 on Wheat and Pepper

Wheat treated with BV01 exhibited an increase in height of 13% (Figure 7A), while the
fresh weight and dry weight were improved by 10% and 5% (Figure 7B). Pepper treated
with BV01 exhibited increases in the fresh weight, leaf width, and stem thickness of 20%,
9%, and 9%, respectively. The dry weight and plant height were improved by 12% and 2%
23, 11, x FOR PEER REVIEW 10 of 14
(Figure 7C,D). The SSI for treatment with BV01 and JDF increased by 19% and 10%. These
findings suggest that strain BV01 was more effective in promoting plant growth than JDF.

Figure 7. Growth-promotion effects of BV01 and

Figure 7. Growth-promotion JDF
effects of on
BV01wheat andonpepper.
and JDF Wheat
wheat and growth
pepper. Wheat(A) and (A) and
growth
experimental conditions (B) in CK, BV01, and JDF pot experiments, n = 12 plants. Pepper growth
experimental conditions (B) in CK, BV01, and JDF pot experiments, n = 12 plants. Pepper growth
(C) and experimental
(C)conditions (D) inconditions
and experimental CK, BV01, and
(D) JDFBV01,
in CK, pot experiments, n = 9 plants.
and JDF pot experiments, n =Values
9 plants.are
Values are
the means ± SEs, different letters
the means show
± SEs, significant
different differences
letters show (Fisher’s
significant LSD,
differences p < 0.05).
(Fisher’s LSD, p < 0.05).

4. Discussion
4. Discussion
For a long time, Bacillus amyloliquefaciens and B. subtilis were known to have biocontrol
For a long time, Bacillus
functions amyloliquefaciens
against various plant and B. subtilis
pathogens [33]. were known
Recently, to havewas
B. velezensis biocon-
reported as
trol functions against variousagent
a biocontrol plantagainst
pathogensmany[33]. Recently, B. For
phytopathogens. velezensis
example, wasB. reported as F21
velezensis strain
can control Fusarium wilt on watermelon [19], and strain
a biocontrol agent against many phytopathogens. For example, B. velezensis strain F21 can BR-01 has strong antagonistic
effects
control Fusarium wilt onon rice pathogens
watermelon [34],
[19], andwhile strain
strain CE100
BR-01 hasincreases fruit yield of strawberries
strong antagonistic effects by
controlling fungal diseases [35]. The star strain FZB42 was initially established in 1998,
on rice pathogens and[34],successive
while strain CE100 increases fruit yield of strawberries by control-
studies on its antimicrobial substances, interactions between plants and
ling fungal diseases [35]. The
bacteria, star strain
regulatory smallFZB42
RNAs, andwasbiocontrol
initially established
enzymes have inbeen
1998, and suc-
carried out [33]. In
cessive studies onprevious
its antimicrobial substances,
studies, antagonistic interactions
strains between
of B. velezensis were often plants and
isolated bacteria,
from water, soil, air,
regulatory small RNAs, andplant
plant roots, biocontrol
surfaces,enzymes
and animal have been [7].
intestines carried
In theout [33].study,
present In previous
strain BV01 was
derived from a PDA plate in the laboratory and speculated to
studies, antagonistic strains of B. velezensis were often isolated from water, soil, air, plantbe an air source strain. Based
on morphological characteristics and phylogenetic evidence, strain BV01 was identified
roots, plant surfaces, and animal intestines [7]. In the present study, strain BV01 was de-
as B. velezensis; further exploration of its biological control potential was then performed.
rived from a PDAIts plate in the laboratory
dual-culture and speculated
inhibition rates to bepathogens
against different an air source strain. than
were greater Based 56%, and
on morphologicalthe characteristics and phylogenetic evidence, strain BV01 was
fermentation broth inhibition rates were reduced by more than 36% when compared identified
as B. velezensis; further
to the exploration of its biological
control. The results indicate thatcontrol potentiala was
BV01 produces specialthen performed.
antibacterial substance.
extract components of B. amyloliquefaciens
Its dual-culture inhibition rates against different pathogens were greater than 56%, and as key
Some lipopeptide have been demonstrated
substances
the fermentation broth in controlling
inhibition the growth
rates were reducedof Xanthomonas
by more than citri subsp.
36% when citri [36]. Zhou et al. [34]
compared
to the control. The results indicate that BV01 produces a special antibacterial substance.
Some lipopeptide extract components of B. amyloliquefaciens have been demonstrated as
key substances in controlling the growth of Xanthomonas citri subsp. citri [36]. Zhou et al.
[34] proved that the relative inhibition rate of B. velezensis BR-01 against F. fujikuroi was
Microorganisms 2023, 11, 2627 10 of 13

proved that the relative inhibition rate of B. velezensis BR-01 against F. fujikuroi was 57%,
while the strain showed no antagonistic ability against R. solani. The results of the current
study revealed that strain BV01 possessed very strong antagonistic activity and broad-
spectrum biological ability against B. cinerea, F. oxysporum, C. capsici, V. dahliae, R. solani, B.
sorokiniana, F. graminearum, and N. rubicola.
Many Bacillus species produce a variety of hydrolytic enzymes, such as cellulase, β-1,3-
glucanase, and protease, which are responsible for the degradation of diverse components
of fungal pathogens [35,37]. The detection of cellulase, protease, and β-1,3-glucanase
in BV01 supports its association with the growth suppression of several fungal phy-
topathogens. Our results also revealed that strain BV01 effective in vitro against fungal
pathogens was also able to produce siderophores, which are related to indirect antag-
onistic processes such as plant defenses and growth promotion [30]. Moreover, some
members of Bacillus invade the rhizosphere of plants and promote plant growth by pro-
ducing plant hormones, such as IAA, cytokinins, and gibberellins, and chelating minerals
and siderophores. Many plant-growth-promoting bacteria produce IAA, which promotes
the development of plant roots, and are usually utilized as bioinoculants [38–45]. In a
previous study, B. velezensis BY6 was reported to significantly increase the dry and fresh
mass and plant height of Pdpap poplar seedlings [46]. In the present study, B. velezensis
BV01 produced IAA during its growth. Moreover, our pot experiment results revealed
that pepper and wheat treated with strain BV01 possessed higher fresh weight, dry weight,
plant height, leaf width, stem thickness, and SSI than controls. Both the antifungal activity
assay and greenhouse pot experiment indicated that the strain BV01 has biocontrol and
plant-growth-promotion potential.
Wheat and pepper are two of the most commonly grown crops and vegetables in
the world. Several pathogens cause severe diseases of them and thus reduce significantly
their yields. For example, wheat root rot caused by B. sorokiniana, Fusarium spp., and other
pathogens alone or in combination generally can lead to wheat yield reductions of 20%–
30%, with severe cases of more than 50% [47,48]. Previous studies revealed that B. subtilis
and B. amyloliquefaciens can prevent and control wheat root rot [47]. However, there are
few studies on the effects of B. velezensis on wheat root rot caused by B. sorokiniana. Bacillus
velezensis strains CC09 and NEAU-242-2 could be used as potential biocontrol agents to
control wheat disease [49,50]. In this study, B. velezensis strain BV01 was able to effectively
control wheat root rot caused by B. sorokiniana in a greenhouse, with a control rate of 47%.
The occurrence of pepper wilt is increasing currently and seriously affects the quality of
pepper. For example, the incidence of pepper wilt disease in China is generally 15%–30%,
with severe cases decreasing quality by 70%–80% [51]. The main pathogen, Fusarium
graminearum, is a highly destructive phytopathogen, not only lowering crop yields but also
producing mycotoxins and affecting crop quality. Previous studies have confirmed that B.
velezensis could control pepper root rot [52], wheat spikes [53], corn stalk rot [54], and corn
head blight [55]. To our knowledge, the present study is the first report that B. velezensis can
serve as a potential biocontrol agent for controlling pepper wilt induced by F. graminearum.
Bacillus velezensis BV01 not only promotes the growth of wheat and pepper seedlings but
also significantly controls wheat root rot and pepper wilt. In summary, Bacillus velezensis
BV01 has good control effects in both dual-culture and fermentation broth tests against
B. sorokiniana and F. graminearum, and it obviously reduced the disease symptoms and
promoted the growth of wheat and pepper.

5. Conclusions
Bacillus velezensis BV01 showed protease, cellulase, and β-1,3-glucanase activities,
which are related to phytopathogen cell wall degradation, and produced growth-promotion
substances such as IAA and siderophore. This strain also suppressed the growth of eight
phytopathogens both in dual-culture and sterile filtrate assays and significantly reduced
the disease incidence of wheat root rot and Fusarium wilt in greenhouse settings. More-
over, it significantly promoted wheat and pepper growth. In conclusion, BV01 exhibits
Microorganisms 2023, 11, 2627 11 of 13

broad and effective antagonistic activity against several phytopathogens, promotes plant
growth, and is worthy of further exploration of its biocontrol applications in eco-friendly
agriculture practices.

Author Contributions: Conceptualization, Z.Z. and W.Z.; resources, W.Z. and Z.Z.; data curation,
T.H.; writing—original draft preparation, T.H.; writing—review and editing, Y.Z., W.Z., Z.Y. and Z.Z.;
visualization, T.H. All authors have read and agreed to the published version of the manuscript.
Funding: This research was supported by the National Natural Science Foundation of China (32270009,
31870012, 31750001), the Biological Resources Programme, Chinese Academy of Sciences (KFJ-BRP-017-
082), and the Frontier Key Program of the Chinese Academy of Sciences (QYZDY-SSW-SMC029).
Data Availability Statement: All the data relevant to this manuscript are available on request from
the corresponding author.
Acknowledgments: The authors would like to thank Yongchun Niu, Jiyan Qiu, Zhengqiang Ma, Qili
Li, and Shenzhan Fu for providing the phytopathogens used in this study and Hongjun Chen for
corrections to the language.
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.

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