Scientia Horticulturae 312 (2023) 111841

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Antifungal activity of Bacillus mojavensis D50 against Botrytis cinerea

causing postharvest gray mold of tomato
Lining Zheng a, Xuehu Gu a, Yufeng Xiao a, Shengyi Wang a, Ling Liu a, Hongyu Pan b,
Hao Zhang a, *
a
College of Plant Protection, Jilin Agricultural University, Changchun 130118, PR China
b
College of Plant Sciences, Jilin University, Changchun 130062, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: Botrytis cinerea causes considerable economic losses to the tomato processing industry. Hence, the study eval­
Antifungal mechanism uated the antifungal activity of eight biocontrol strains isolated from tomato rhizosphere soil against B. cinerea.
Cherry tomato These strains demonstrated potent antifungal activity with an inhibition rate ranging from 19.30% to 69.88%.
Gray mold
Among them, a strain named D50 showed the highest effect with an inhibition rate of 69.88%. D50 strain was
Bacillus mojavensis
identified as Bacillus mojavensis by morphological, physiological, and biochemical analyses. The Bacillus moja­
Botrytis cinerea
vensis D50 fermentation supernatant (BMFS) was used for subsequent experiments to unveil antimicrobial ac­
tivity of its secondary metabolites. BMFS (5%, v/v) reduced the growth of B. cinerea by inhibiting mycelial
growth, conidial production, and mycelial dry weight by 51.7%, 45.9%, and 53.7%, respectively. Detailed
analysis revealed that the antifungal effect of BMFS decreased when it was heated above 80 ◦ C and adjusted to
pH 2, 8, and 10. The antifungal effect also decreased when exposed to UV for more than 40 min. Further, the in
vivo effect of BMFS in preventing gray mold was evaluated using cherry tomatoes. After B. cinerea inoculation for
5 days, the disease incidence and disease severity of cherry tomato treated with BMFS decreased by 33.5% and
31.5%, respectively. BMFS treatment enhanced the activities of peroxidase (POD), catalase (CAT), polyphenol
oxidase (PPO), superoxide dismutase (SOD), and phenyl alanine lyase (PAL) and increased the content of
malondialdehyde (MDA) in cherry tomatoes during infection. Moreover, BMFS prevented tomato gray mold by
enhancing the transcript levels of systemic acquired resistance-related marker genes (SlLoxD, SlMyc2, SlPR1-a,
SlPAL5, SlEIN2, and SlEIN3). SlLoxD and SlMyc2 had high expression levels among the defense-related genes,
indicating activation of the jasmonic acid signal pathway. Thus, the study demonstrates the great potential of
BMFS in managing the postharvest gray mold of tomato caused by B. cinerea.

1. Introduction
The cherry tomato is accepted for its nutritional value due to its high
vitamin C and lycopene content (Bu et al., 2021). However, the occur­
rence of gray mold during growing seasons, storage, and transport cause
huge economic losses (Zheng et al., 2021b). Gray mold, caused by
Botrytis cinerea, affects tomato plants worldwide (Abro et al., 2013;
Yang et al., 2021). Therefore, immense effort and money are invested to
control this devastating disease (Dean et al., 2012; Zheng et al., 2021b).
In recent years, synthetic fungicides have been used to control gray
mold due to their low cost and high effectiveness (Gao et al., 2018; Li
et al., 2020; Raynaldo et al., 2021). However, the widespread use of
these fungicides has disadvantages, such as environmental pollution,
pathogen resistance, and potential health risks (Angelini et al., 2014;
Cortes et al., 2019). Several studies have reported the adverse effects of
pesticides (Nong et al., 2021). Therefore, it’s urgent to find an envi­
ronmentally friendly way to control tomato gray mold and reduce the
usage of chemical fungicides (Palou et al., 2016).
Biological control is an environmentally friendly and efficient
approach to control diseases, wherein another organism is introduced to
control the pathogen rather than chemical pesticides. Biological control
mechanisms (assisted by antagonistic microorganisms) include space or
nutrient competition, inducible resistance, and metabolite production.
Bacillus species are gram positive and exist in soil, water and air. The
Bacillus species typically act by producing various antifungal and anti­
microbial compounds (Kang et al., 2020; Santos da Silva et al., 2015; Wu

* Corresponding author.
E-mail address: zhanghao100@jlau.edu.cn (H. Zhang).

https://doi.org/10.1016/j.scienta.2023.111841
Received 3 July 2022; Received in revised form 3 January 2023; Accepted 6 January 2023
Available online 15 January 2023
0304-4238/© 2023 Elsevier B.V. All rights reserved.
L. Zheng et al.
et al., 2018). Many studies have reported that the Bacillus sp. has been
used in the biocontrol of several pathogens, such as Fusarium oxysporum
f. sp. lycopersici (Guimarães Pacifico et al., 2021; Jangir et al., 2018),
Alternaria alternata (Xie et al., 2021), and B. cinerea (Paz et al., 2018).
Although Bacillus species-mediated biological control has been proposed
for a long time, biocontrol strains are mostly used for experiments, and
the main biocontrol strains are Bacillus velezensis, Bacillus amyloliquefa­
ciens and Bacillus subtilis (Erdogan and Benlioglu, 2009; Mizumoto et al.,
2007). Furthermore, the biocontrol strains which are used to control
postharvest pathogens of fruit, have low stability, and their inhibitory
effects on pathogens are closely related to the environment, and nutri­
ents (Gu et al., 2022; Ma et al., 2015). Metabolites, produced by
biocontrol strains, can inhibit the growth of pathogens and it also has
great stability (Le Han et al., 2022; Li et al., 2020). Therefore, there is a
need to explore and isolate more other Bacillus sp. to combat the post­
harvest gray mold of tomato.
Our present study was devoted to explore the potential of biocontrol
agent and its fermentation supernatant (BMFS) to delay tomato gray
mold in postharvest stage. Moreover, the potential mechanism related to
BMFS-induced disease resistance was investigated. Therefore, the spe­
cific aims were: 1) Isolate and characterize bacteria with possible bio­
logical control potential; 2) to assess potential of the biocontrol agent
fermentation supernatant in reducing postharvest gray mold of tomato;
and 3) to explore the main mechanisms of biological control of post­
harvest gray mold of tomato.
2. Materials and methods
2.1. Microbial strains, culture conditions, and fruit materials
The plant pathogenic fungus Botrytis cinerea was kindly provided by
the Laboratory of Pesticide Bioassay, Plant Protection College at Jilin
Agricultural University. The Nutrient Agar (NA; 0.003 kg beef extract,
0.01 kg peptone, 0.005 kg NaCl, and 0.02 kg agar in 1 L of distilled
water) was used to isolate biocontrol strains, while the bacteria were
cultured in Luria-Bertani (LB; 0.005 kg yeast powder, 0.01 kg tryptone,
and 0.002 kg NaCl in 1 L distilled water). Meanwhile, B. cinerea was
cultured in Potato Dextrose Agar (PDA; 0.02 kg glucose, 0.02 kg agar,
and 0.2 kg potato in 1 L distilled water).
Cherry tomatoes (Solanum lycopersicum L. cv. Ailsa Craig) were
collected at mature-green phase (40 d posterior to flowering) from a
commercially vegetable planting base (Gongzhuling, Jilin, China) and
immediately sent to the laboratory. The tomato fruit which had uniform
shape, size, color and without pathogen infection or physical injury were
harvested at commercial maturity and used in this study. The selected
cherry tomatoes were sterilized with 0.1% sodium hypochlorite for 3
min, rinsed with distilled water, and then air-dried at room temperature.
2.2. Isolation of the bacterial antagonistic strain
The antagonistic strains were isolated as described by Zheng et al.
with slight modification (Zheng et al., 2021b). Approximately 10 g of
the soil sample, collected from the tomato rhizosphere in the Agricul­
tural Science Experimental Station of Jilin Agricultural University, was
added into a 250 mL flask with 100 mL NB broth, and then incubated at
30 ◦ C and 150 rpm for 12 h. The supernatant was used to isolate the
strains. After serial dilution (10− 6–10− 8), the supernatant was incubated
in LB medium for 2 days at 30 ◦ C. The strains were then purified
following the streak plate method. The flat-stand method was used to
examine the antagonistic effects of strains. Briefly, a plug (5 mm-dia­
meter) of B. cinerea, which was taken from the edge of a 5-day-old fungal
culture, was inoculated on the center of PDA medium, and then the
bacterial suspension (0.5 μL) was dropped with 3.5 cm away from the
plug of B. cinerea in four different directions as treatment, while a plug
(5 mm-diameter) of B. cinerea was inoculated on the center of PDA
medium, and then the sterile water (0.5 μL) was dropped with 3.5 cm
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Scientia Horticulturae 312 (2023) 111841
away from the plug of B. cinerea in four different directions as control.
After that, the plates were cultured at 25 ◦ C for 7 days (Cui et al., 2020).
The inhibitory rate was calculated as follows:
Inhibitory rate (%) = [colony radius of control group – colony radius
of treatment group/colony radius of control group] × 100.
2.3. Characterization and identification of the bacterial antagonistic
strain
2.3.1. Morphological, physiological, and biochemical studies
The morphological characteristics of the strain were examined using
a scanning electron microscope (100 kV, microscopic magnification of
×14,000). The physiological and biochemical characteristics, including
gram reaction, catalase reaction, contact enzyme reaction, liquefaction
of gelatin, inositol, hippurate reaction, were assayed using the methods
by Zheng et al. (Zheng et al., 2021b).
2.3.2. 16 s rDNA sequencing
The genomic DNA was extracted from the strain using TIANamp
Bacteria DNA Kit (Tiangen Biotech, Beijing, China). The 16S rDNA gene
was amplified using 27F (5′ -AGAGTTTGATCMTGGCTCAG-3′ ) and
1492R (5′ -TACGGYTACCTTGTTACGACTT-3′ ) primers in a thermal
cycler as follows: 94 ◦ C for 4 min; 35 cycles of 94 ◦ C for 30 s, 56 ◦ C for 30
s, and 72 ◦ C for 90 s; and a final extension step at 72 ◦ C for 10 min
(Frank et al., 2008). The PCR products were purified and sequenced by
Shanghai Biological Engineering Co., Ltd. (Shanghai, China). The BLAST
program from the GenBank database was used for the homology anal­
ysis. Phylogenetic and molecular evolutionary analyses were carried out
using MEGA version 6.0.
2.4. Preparation of Bacillus mojavensis D50 fermentation supernatant
(BMFS) and PDA medium containing BMFS
2.4.1. Preparation of the seed liquid
To prepare the Bacillus mojavensis D50 seed liquid, the bacterium was
inoculated with an inoculating loop in a 250 mL flask containing 100 mL
LB broth and incubated at 150 rpm for 16 h at 30 ◦ C. Then, the culture
broth was centrifuged at 7104 g for 5 min to obtain bacteria. The bac­
teria were washed with PBS three times, and the concentration of the
bacterial suspension was adjusted to 1.0 × 108 CFU/mL following
hemacytometer-based counting.
2.4.2. Preparation of BMFS
Approximately, 0.5 mL of the seed liquid (1.0 × 108 CFU/mL) was
added into a 500 mL flask containing 250 mL LB broth and then incu­
bated at 150 rpm for 7 days at 30 ◦ C. The culture broth was centrifuged
at 11,100 g for 10 min. The supernatant was collected and passed
through 0.22-μm filter membranes thrice to obtain the BMFS (Bunge
et al., 2008; Zhang et al., 2020).
2.4.3. Preparation of PDA containing BMFS
Different concentrations of BMFS (1%, 5%, 10%, v/v) were added
into PDA medium (100 mL) when the temperature of PDA medium was
around 40 ◦ C. The medium containing BMFS at different concentrations
were poured into plates (90 mm-diameter) for solidification and used in
the subsequent experiments (Zheng et al., 2021b).
2.5. Antifungal activity of BMFS in vitro
2.5.1. Antifungal activity of BMFS at different concentrations on B. cinerea
mycelial growth
The effect of BMFS on mycelial growth of B. cinerea was measured
following the methods by Zheng et al. (2021a). Meanwhile, the
dual-culture method was used to observe the effect of BMFS at different
concentrations on B. cinerea morphology (Cozzolino et al., 2020). The
mycelium in contact with BMFS was observed under a microscope
L. Zheng et al.
(Microscopic magnification of × 400) (Heilman et al., 2013). After that,
the B. cinerea mycelium was scraped off with a knife and weighed to
determine the effect of BMFS on the mycelial dry weight. The percentage
inhibition of mycelial dry weight was calculated as follows:
Percentage inhibition (%) = [mycelial dry weight of control group –
mycelial dry weight of treatment group/ mycelial dry weight of control
group] × 100.
2.5.2. Spore production by B. cinerea treated with BMFS on PDA plates
Here, B. cinerea was grown on PDA at 25 ◦ C until the colony was
about 4 cm in diameter. Then, 10 mL of BMFS (1%, 5%, 10%, v/v) was
added into the plate. Plates treated with 10 mL sterile water containing
different concentrations of LB broth (1%, 5%, 10%, v/v) were used as a
control. Hyphae was scraped off with a cotton-tipped. Liquid in the
plates was collected in a tube and incubated at 25 ◦ C for 96 h. Conidia
from each treatment were counted using the cell count technique (Li
et al., 2020).
2.6. Antifungal activity of BMFS in vivo
Uniform wounds (3-mm diameter× 3-mm deep) were made at the
equator of each cherry tomato using a sterile needle to determine the in
vivo antifungal activity of BMFS. Approximately 50 μL of BMFS (1%, 5%,
10%) or sterile water (containing different concentrations of LB broth)
was added to the wound. After 2 h air drying, 10 μL of the B. cinerea
sporangial suspension (5 × 104 spores/mL) was inoculated into the
wound. Thirty tomatoes were maintained per concentration, and three
replicates with 90 tomatoes were maintained per treatment. Finally, the
fruit were kept at 25 ◦ C for 5 days, and the disease incidence and disease
severity were calculated (Raynaldo et al., 2021; Wang et al., 2010).
2.7. Stability of BMFS
The BMFS was treated at different temperatures, pH, and UV expo­
sure durations to determine stability. The temperature stability of BMFS
was measured following the methods of Li et al. with modifications (Li
et al., 2020). Acid-base stability of BMFS was performed as described by
Zheng et al. (Zheng et al., 2021a). Briefly, the BMFS was treated in a
metal bath at 4, 20, 40, 60, 80, and 100 ◦ C or autoclaved (121 ◦ C) for 20
min to determine the temperature stability, and the pH of BMFS was
adjusted to 3, 4, 5, 6, 7, 8, 9, and 10 with 0.1 M HCl or NaOH to
determine the acid-base stability. The BMFS was irradiated for 10, 20,
30, and 60 min by placing it under an ultraviolet lamp (200 μW/ cm2) at
5 cm distance to determine the radiation stability. After that, the PDA
medium containing 5% (v/v) BMFS was made as described in Section
2.4.3, and the effect of BMFS on B. cinerea mycelial growth was deter­
mined as described in Section 2.4.
2.8. Measurement of defense-related enzyme activity and the content of
malondialdehyde (MDA) in tomato fruit
The activity of defense-related enzymes and the content of malon­
dialdehyde (MDA) were measured in cherry tomatoes under four
treatments: 1: sterile water (contained 5% v/v LB broth), 2: B. cinerea, 3:
BMFS (5%, v/v), 4: B. cinerea + BMFS. BMFS (5%, v/v, 50 μL) or sterile
water (containing 5% v/v LB broth, 50 μL) was added to the wound (3
mm diameter × 3 mm deep) of cherry tomatoes. After 2 h air drying, 10
μL of the sporangial suspension of B. cinerea (5 × 104 conidia/mL) or
sterile water was also inoculated into the wound. The tomato fruit were
collected at 0, 12, 24, 48, 72, and 96 h post-pathogen infection (hppi) to
determine the enzyme activity and the content of malondialdehyde
(MDA). The tissues around the wound were collected from cherry to­
matoes to extract MDA, peroxidase (POD), catalase (CAT), superoxide
dismutase (SOD), phenylalanine ammonia-lyase (PAL). The enzyme
activity and content of MDA were assessed by spectrophotometry
(Mojtaba and Homayoon, 2002; Xu et al., 2013, 2021a).
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Scientia Horticulturae 312 (2023) 111841
One unit (U) of PPO, POD, CAT, and PAL was defined as the amount
of enzyme extract that caused an increase in absorbance of 0.01 per
minute at A525, A470, A240, and A290, respectively; these enzyme activ­
ities were expressed as U kg− 1 min− 1. One unit of SOD activity was
defined as the change of 0.01 absorbance per gram of fresh weight at
A560, and the enzyme activity was expressed as U kg− 1. One unit of MDA
was defined as a change in MDA concentration (mol) per kilogram of
fresh weight, and the content of MDA was expressed as mol/kg.
2.9. Defense-related gene expression analysis
The expression of defense-related genes was analyzed by quantita­
tive real-time PCR (qRT-PCR). The treatments and methods used were
the same as those mentioned in Section 2.8. In the induced systemic
resistance experiments, the collected tissue for RNA extraction was
around the wound using Trazol up Plus RNA Kit (Transgen Biotech.).
The ultraviolet spectrophotometer was used to determine the purity of
the total RNA, and RNA integrity was evaluated from the 28S and 18S
ribosomal RNA (rRNA) bands on an agarose gel. The cDNA was gener­
ated following the manufacture’s protocol (Transgen all-in-one first-
strand cDNA synthesis SuperMix for qPCR, Transgen Biotech.). The
defense-related genes were selected according to the previous studies
(Tao et al., 2022; Xu et al., 2021b). The genes SlPR1-a and SlPAL5
related to the salicylic acid signal pathway, SlEIN2 and SlEIN3 related to
the ethylene signal pathway, and the key genes SlLoxD and SlMyc2
related to the jasmonic acid signal pathway were analyzed. The primers
were designed using Primer 6 software (Table S1), and the quantitative
real-time PCR was carried out using PerfectStart Green qPCR SuperMix
on a Roche Light Cycler® 96 (Roche Diagnostics Corporation, Indian­
apolis, IN, USA). The qRT-PCR program used was as follows: 95 ◦ C for 2
min, followed by 39 cycles of 95 ◦ C for 30 s, 60 ◦ C for 30 s, 72 ◦ C for 30 s
and 78 ◦ C for 11 s. The melting curve cycle program was as follows:
95 ◦ C for 10 s, 60 ◦ C for 1 min, and 97 ◦ C for 1 s. The relative expression
levels of the genes were determined using the 2− ΔΔCT method, and the
gene of Actin was used as a reference gene. Data are reported on a log­
arithmic scale as a relative gene expression ratio (RQ) after calibration
on values obtained at 0 h.
2.10. Statistical analysis
In this study, all the treatment had three replicates, and all the
experiment was conducted three times. The data (including the per­
centage of data of disease incidence, and inhibition rate) were statisti­
cally analyzed using analysis of variance (ANOVA) in SPSS 26.0 (SPSS
Inc., USA). Significant differences between the means were analyzed
using the LSD test. Differences were considered statistically significant
at P < 0.05. Respective significant differences were denoted using
different letters (a, b, c, etc.).
3. Results
3.1. Isolation and screening of bacterial antagonistic strains
Various strains were isolated from the rhizosphere soil of tomato
plants in the Agricultural Science Experimental Station of Jilin Agri­
cultural University. Among them, eight strains showed antagonistic
activity against B. cinerea with an inhibitory rate ranging from 19.30%
to 69.88% (Table 1). Thus, our observations revealed that strain D50
had the strongest antagonistic activity with an inhibitory rate of 69.88%
(Table 1). Therefore, D50 was used for further experiments.
3.2. Characterization and identification of bacterial antagonistic strains
Further, the morphological characteristics were analyzed to identify
D50. The single colony of D50 was milky white on LB medium. With
aging, the margin of the colony became folded with a flat and weak
L. Zheng et al. Scientia Horticulturae 312 (2023) 111841

Table 1 Table 2
The inhibition rate given by isolated bacterial strains to B. cinerea in vitro. Nu­ The physiological and biochemical characteristics of
merical values were mean ± SD of three replicates. B. mojavensis D50. Symbol “+” indicates positive and “–”
Strain Colony diameter ± SE*/mm Inhibition rate(%)
indicates negative.
Characteristics D50
D3 68.67 ± 0.31 22.10
D6 71.14 ± 0.32 19.30 Gram reaction +
D7 53.07 ± 0.14 39.80 Catalase reaction +
D16 64.61 ± 0.22 26.70 Contact enzyme reaction +
D28 46.01 ± 0.09 47.79 Liquefaction of gelatin +
D32 37.73 ± 0.03 57.20 Inositol –
D48 54.12 ± 0.26 38.60 Hippurate reaction –
D50 26.55 ± 0.01 69.88
Control 88.15 ± 0.12 –
by altering the mycelial morphology (Fig. 3B). With the increase in the
concentration of BMFS from 1% (v/v) to 10% (v/v), the mycelium
surface (Fig. 1A). Scanning electron microscopy showed that D50 was
became branched and shorter, with smaller hyphal compartments
rod-shaped (Fig. 1B). In addition, strain D50 was positive in gram
(Fig. 3B, b-d). Mycelium, in the control group, was full, with a smooth
staining, catalase reaction, contact enzyme reaction, and gelatin lique­
cell wall; the mycelium grew and expanded normally (Fig. 3B, a).
faction, while it was negative in inositol and hippurate reaction
Compared with the control group, the B. cinerea mycelium, treated with
(Table 2). Subsequent 16S rDNA amplification (Fig. 2A) and sequencing
BMFS (1%), changed obviously. As shown in Fig. 3B (b), the mycelium
revealed 99% similarity of D50 with Bacillus mojavensis 15718 (Acces­
was swollen, deformed and grew slowly (Fig. 3B, b). Meanwhile, at 5%
sion No. NR118290.1; GenBank). Phylogenetic analysis confirmed the
(v/v) BMFS, most hyphae showed cell wall thickening (Fig. 3B, c), and at
strain as Bacillus mojavensis D50 (Accession No. MZ165352) (Fig. 2B).
10% (v/v) BMFS, the top of the mycelium expanded and finally broke
(Fig. 3B, d).
3.3. Antifungal activity of BMFS in vitro
3.4. Antifungal activity of BMFS in vivo
The colony diameter, mycelial dry weight, and spore production by
the pathogen treated with different concentrations of BMFS were Furthermore, BMFS at different concentrations was applied on
measured to determine the effect of BMFS on B. cinerea. We found that cherry tomatoes for 2 h before inoculating B. cinerea to assess the anti­
the BMFS could inhibit the growth of B. cinerea in vitro. Moreover, the fungal activity. Five days after the pathogen inoculation, the disease
inhibitory rate increased with the increasing concentrations of BMFS incidence and severity attained lower values than that in control group
(Fig. 3). In all three experiments, the analysis revealed that the con­ at 1% (v/v) BMFS, and the disease incidence and severity were 47.9 and
centration of 5% (v/v) had the higher inhibition rate than the concen­ 34.2, respectively, (Fig. 4). The result indicated that the low concen­
tration of 1% (v/v). There was significant difference in inhibition of tration of BMFS could inhibit postharvest gray mold of tomato. When
colony diameter, mycelial dry weight, and spore production when the the concentration of BMFS was 10% (v/v), the disease incidence and
concentrations of BMFS were 1% (v/v) and 5% (v/v) (P<0.05). How­ severity attained the lowest values (35.4%, Fig. 4A and 27.6%, Fig. 4B,
ever, when the concentrations of BMFS were 5% (v/v) and 10% (v/v), respectively). And the disease incidence and severity were not signifi­
there was no significant difference in inhibition of colony diameter, cantly different between 5% (v/v) and 10% (v/v) of BMFS (P>0.05).
mycelial dry weight, and spore production (P > 0.05). When the con­ According to the results of antifungal activity in vitro, the optimum
centration of BMFS was 5% (v/v), it could effectively inhibit the colony application concentration of BMFS was 5% (v/v).
diameter, mycelial dry weight, and spore production with the inhibitory
rates of 51.7%, 45.9%, and 53.7%, respectively (Fig. 3A).
The B. cinerea mycelial morphology and structural details were also
observed to understand BMFS antagonism. BMFS could inhibit B. cinerea

Fig. 1. Morphological observation of strain D50. (A) LB solid medium plate exhibiting isolated colonies following 16 h of cultivation at 30 ◦ C. (B) Scanning
electron microscopy of intact cells at 100 kV (Microscopic magnification of ×14,000).

4
L. Zheng et al. Scientia Horticulturae 312 (2023) 111841

Fig. 2. Phylogenetic tree of strain D50. (A) 16S rDNA amplification electrophoresis result. (B) Phylogenetic tree base on maximum-likehood analysis of 16S rDNA
sequence data.

Fig. 3. Antifungal activity of B. mojavensis D50 fermentation supernatant (BMFS) at different concentrations (1%, 5%, 10%, v/v) on mycelial growth,
condical production, dry weight of mycelium and mycelial morphology of B. cinerea on PDA medium at 25 ◦ C for 6 days. (A) antifungal activity of BMFS at
different concentrations (1%, 5%, 10%, v/v) on mycelial growth, condical production and dry weight of mycelium of B. cinerea. (B) antifungal activity of BMFS at
different concentrations on mycelial morphology of B. cinerea. (a) treated with sterile water. The mycelium of B. cinerea was full, the cell wall was smooth, and the
mycelium could grow and expand normally. (b) treated with BMFS (1%, v/v). The mycelium of B. cinerea changed obviously, the mycelial branches increased, the
mycelium was deformed and grew slowly. (c) treated with BMFS (5%, v/v). Most hyphae also had the phenomenon of cell wall thickening. (d) treated with BMFB
(10%, v/v). The top of the mycelium expands and the mycelium breaks. Letters show significance according to LSD test within each group (P < 0.05). Error bars
indicate SD of three experiments. .

Fig. 4. Antifungal activity of B. mojavensis D50 fermentation supernatant (BMFS) in tomato fruit. Tomato fruit were treated with different concentrations of
BMFS (0, 1%, 5%, 10%, v/v) 2 h before inoculation with sporangia suspension of B. cinerea (5 × 104 spores/mL). After 5 d of inoculation, the disease incidence (A)
and disease severity (B) were calculated. Bars indicate standard error ( ± SE). P < 0.05 was considered significantly different from each group.

3.5. Stability of BMFS Fig. 5B, the inhibition rate of BMFB was decreased when treated under
pH 2, 8, and 10 conditions (Fig. 5B). The antifungal effect also decreased
The results of this experiment had shown that the antifungal effect when the ultraviolet irradiation time exceeds 40 min (Fig. 5C).
decreased when BMFS was heated above 80 ◦ C (Fig. 5A). As shown in

5
L. Zheng et al.
Fig. 5. Stability of B. mojavensis D50 fermentation supernatant (BMFS). Stability of BMFS at (A) different temperatures, (B) different pH values, and (C) different
UV irradiation times. Bars indicate standard error ( ± SE). P < 0.05 was considered significantly different from each group.
3.6. Defense-related enzyme activity in tomato fruit
The activity of defense-related enzymes and the content of MDA were
measured to reveal the mechanism of action of BMFS on tomato fruit.
The activities of enzymes were the highest in treatment of BMFS +
B. cinerea. The activities of all enzymes in treatment of B. cinerea were
lower than treatments of BMFS, BMFS + B. cinerea and higher than
control (Fig. 6). The activities of enzymes in the treatment of BMFS +
B. cinerea first rised and then declined with the increase of time (Fig. 6A-
C, E-F). In the treatment of B. cinerea, the content of MDA was the
highest (Fig. 6D). The content of MDA in treatment of BMFS + B. cinerea
was lower than that in the treatment of B. cinerea (Fig. 6D). These results
indicated that the BMFS could control B. cinerea by inhibiting the
pathogen and inducing the activation of defense responses in cherry
tomato.
3.7. Expression of defense-related genes
Furthermore, the expression levels of defense-related genes of to­
mato cherry were analyzed during pathogen-infection. In the treatment
of BMFS + B. cinerea, the expression level of SlPR1-a greatly increased at
96 hppi; the expression level in this treatment was 94.8-fold, which was
more than that in the treatments of B. cinerea (24.4-fold) and BMFS (9.5-
fold) (Fig. 7A). Similarly, SlPAL5 also showed a high expression in the
treatment of BMFS + B. cinerea, which peaked at 96 hppi (9.0-fold)
(Fig. 7B). The expression level of SlEIN-2 transiently declined in treat­
ment of BMFS + B. cinerea at 12–24 hppi, enhanced at 24–96 hppi, and
upregulated (15.8-fold) at 96 hppi (Fig. 7C). Meanwhile, the expression
level of SlEIN-3 significantly increased at 96 hppi in treatment of BMFS
+ B. cinerea (50.6-fold), which was higher than treatments of B. cinerea
and BMFS (13.0-fold and 14.3-fold, respectively) (Fig. 7D). A significant
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Scientia Horticulturae 312 (2023) 111841
increase in SlLoxD expression was observed at 12–96 hppi (203.2-
fold–506.3-fold, respectively) in treatment of BMFS + B. cinerea
compared with treatments of B. cinerea and BMFS (Fig. 7E). In addition,
BMFS induced SlMYC-2 more than the B. cinerea, although there was a
transient and modest enhancement in the treatment of B. cinerea + BMFS
(Fig. 7F). These results showed that the BMFS could induce the
expression of defense-related genes in cherry tomatoes, and then induce
its resistance to B. cinerea.
4. Discussion
This study isolated a strain from tomato rhizosphere soil to test for
antifungal activity. Through morphological, physiological, and
biochemical studies and 16S rDNA sequence analysis, the strain was
identified as Bacillus mojavensis D50 (Fig. 2). Bacillus species have been
used as a biocontrol agent for a long time. Among them, Bacillus marinus
B-9987 (Gu et al., 2017) and Bacillus velezensis SL-6 (Cozzolino et al.,
2020) have been used to protect tomato fruit and various other plants
from pathogens. Cavalcanti et al. isolated a few Bacillus spp. with anti­
fungal properties (Cavalcanti et al., 2020). Bacillus sp. has also been used
to manage the postharvest pathogens of fruit and vegetables (Tian et al.,
2020; Ye et al., 2021). These studies confirmed that the Bacillus sp. could
be used as biocontrol strains to control plant diseases. In this study, we
isolated a Bacillus strain which could manage postharvest gray mold of
tomato.
Biocontrol agents suppress diseases by inhibiting conidial production
and mycelial growth and altering mycelial morphology. Therefore, the
inhibitory effects of B. mojavensis D50 fermentation supernatant (BMFS)
at different concentrations on B. cinerea in vitro were measured. We
found that BMFS inhibited B. cinerea through inhibiting conidial pro­
duction and mycelial dry weight and altering mycelial morphology.
L. Zheng et al. Scientia Horticulturae 312 (2023) 111841

Fig. 6. Activity of defensive enzyme and content of Malondialdehyde (MDA) in tomato fruit by B. mojavensis D50 fermentation supernatant (BMFS)
applied as treatment: defense related enzymes (Peroxidase, POD; Catalase, CAT; Polyphenol oxidase, PPO; Superoxide dismutase, SOD; Phenyl­
alanineammonialyase, PAL) and content of MDA at 0, 12, 24, 48 and 96 h after the inoculation with B. cinerea (B. cinerea + BMFS), in comparison with
non-treated (B. cinerea), treated but non-inoculated (BMFS) and neither treated nor inoculated (Control) controls. (A) POD; (B) CAT; (C) PPO; (D) MDA; (E)
SOD; (F) PAL. Bars indicate standard error ( ± SE). P < 0.05 was considered significantly different from each group.

Similarly, Li et al. found that the B. amyloliquefaciens BA17 filtrate tomatoes.

inhibited B. cinerea by reducing conidial germination and mycelial
growth (Li et al., 2020). With the increase in the concentration of BMFS,
the biocontrol effectiveness increased (Fig. 3). Secondary metabolites
secreted by Bacillus sp. could also influent the growth of Puccinia hori­
anna (Dheepa et al., 2016). Similarly, Candida oleophila (strain O), and
Pichia anomala (strain K) inhibited the mycelial growth of B. cinerea and
the effectiveness of biocontrol increased with the increase in the con­
centration of the biocontrol agents (Lahlali et al., 2004). The present
study’s observations are consistent with these earlier reports.
Typically, a biocontrol agent inhibits pathogen growth both in vitro
and in vivo. Researchers have identified many biocontrol agents that
suppress pathogens both in vitro and in vivo (Francés et al., 2006; Zheng
et al., 2019; Zhou et al., 2022). Therefore, we further determined the
effects of BMFS on B. cinerea in cherry tomatoes. We found that the
BMFS reduced the disease incidence and disease severity index in cherry
tomatoes (Fig. 4), confirming the biocontrol capacity of BMFS in cherry
Further, the defense-related enzymes in cherry tomatoes were
measured to determine the mechanism of action of BMFS. Generally,
resistance occurs via the direct inhibition of pathogen and activation of
plant defense responses, including the generation of reactive oxygen
species (ROS) (Yang et al., 2017). Several defense enzymes, such as
peroxidase (POD), superoxide dismutase (SOD), catalase (CAT) and so
on, showed the highest response and activity in cherry tomatoes treated
with BMFS following infection with tomato gray mold (Fig. 6).
Numerous studies have shown that the increased ROS-scavenging en­
zymes, antioxidant enzymes, and defense enzymes indicate disease
resistance (Warabieda et al., 2020). Increased POD and phenylalanine
ammonia-lyase (PAL) protect plant cells against pathogen infection
(Zhang et al., 2016; Zhu et al., 2021). Meanwhile, malondialdehyde
(MDA), a common indicator of oxidative stress, reflects the degree of
plant membrane lipid peroxidation. The activities of enzyme (POD, SOD,
PPO, PAL, CAT) of cherry tomatoes pretreated with BMFS improved to

7
L. Zheng et al. Scientia Horticulturae 312 (2023) 111841

Fig. 7. Induced resistance in tomato fruit by B. mojavensis D50 fermentation supernatant (BMFS) applied as treatment: expression of defense related
genes (SlPR1-a, SlPR5, SlEIN-2, SlEIN-3, SlLoxD, SlMyc-2) at 0, 12, 24, 48 and 96 h after the inoculation with B. cinerea (B. cinerea + BMFS), in comparison
with non-treated (B. cinerea), treated but non-inoculated (BMFS) and neither treated nor inoculated (Control) controls. (A) SlPR1-a, (B) SlPR5, (C) SlEIN-2,
(D) SlEIN-3, (E) SlLoxD, (F) SlMyc-2. Bars represent the average + SEM of three replicates. Data are reported on a logarithmic scale as a relative gene expression ratio
(RQ) after calibration on values obtained at 0 h. Different letters are significantly different from the corresponding control values at each time-point, P < 0.05 was
considered significantly different from each group.

differing degrees than the control, while the content of MDA decreased. enzymes during pathogen-infection (Xu et al., 2021a).
These results indicated that the BMFS controlled B. cinerea by inhibiting Induced resistance (IR) is a kind of stress resistant response produced
the pathogen and inducing the activation of defense responses on tomato by plants under the influence of various environmental factors, such as
fruit (Fig. 6). Similarly, Xu et al. found increased activities of these drought, salt-alkaline, pathogen infection. It has been shown that

8
L. Zheng et al.
endogenous hormone, such as ethylene, jasmonic acid, and salicylic
acid, play important roles in defense response (AbuQamar et al., 2017;
Palmer et al., 2017; Ruan et al., 2019). Ethylene (EFR) and jasmonic acid
(JA) play a synergistic role in resisting dead vegetative pathogens, while
salicylic acid mainly mediates the defense response of plants against
semi living pathogens and living pathogens. EIN2 and EIN3, trans­
membrane protein with a short half-life, are positive regulator of the
EFR signaling pathway (An et al., 2010). Meanwhile, lipoxygenase
(LOX), a nonheme iron-containing enzyme, catalyzes the oxidation of
linolenic acid to 13-hydroperoxylinolenic acid, the first step in jasmonic
acid biosynthesis. Therefore, LOX is important for jasmonic acid
biosynthesis, and its coding gene LoxD is often used as a marker of JA
signaling pathway (Gupta et al., 2020). Myc2 transcription factor is an
important protein involved in the JA signaling pathway (Wasternack,
2019). Meanwhile, salicylic acid induces the related protein
pathogenesis-related protein PR1-a, used as a marker protein of the
salicylic acid (SA) signaling pathway (Nasser et al., 1991). Phenylala­
nine ammonia-lyase (PAL) is the key enzyme of phenylpropane meta­
bolic pathway. Therefore, the expression of defense-related genes
(SlPR1-a, SlPAL5, SlEIN2, SlEIN3, SlLoxD, and SlMyc2) were measured to
determine the mechanism of antifungal of BMFS. In our study, the
defense-related genes rapidly increased in tomatoes treated with BMFS
and B. cinerea, with maximum expression at 96 hppi (Fig. 7). Among
them, the defense-related gene SlLoxD, a marker of the jasmonic acid
pathway, was significantly upregulated (Fig. 7E). These observations
indicate that the inhibition of tomato gray mold by BMFS occurs via the
upregulation of defense-related genes. Notably, BMFS mainly activated
the JA signal pathway to induce tomato disease resistance.
5. Conclusions
In this study, a biocontrol strain of Bacillus mojavensis D50 was iso­
lated and identified. The Bacillus mojavensis D50 fermentation super­
natant (BMFS) could inhibit the growth of mycelium and conidial
production and altered the mycelial morphology, and it also had great
stability. In addition, BMFS treatment enhanced the activities of
defense-related enzymes (POD, CAT, PPO, SOD, PAL) and decreased the
content of MDA in cherry tomatoes. Moreover, the resistance mecha­
nism appeared to be primarily via the increase in the transcript levels of
marker genes of systemic acquired resistance.
CRediT authorship contribution statement
Lining Zheng: Conceptualization, Methodology, Validation, Formal
analysis, Investigation, Resources, Data curation, Writing – original
draft. Xuehu Gu: Methodology, Validation, Formal analysis, Investiga­
tion, Resources, Data curation, Writing – review & editing. Yufeng
Xiao: Methodology, Validation, Formal analysis, Investigation, Re­
sources, Writing – review & editing. Shengyi Wang: Methodology,
Validation, Formal analysis, Investigation, Resources, Writing – review
& editing. Ling Liu: Methodology, Validation, Formal analysis, Inves­
tigation, Resources, Writing – review & editing. Hongyu Pan: Meth­
odology, Validation, Formal analysis, Investigation, Resources. Hao
Zhang: Methodology, Validation, Formal analysis, Investigation, Re­
sources, Writing – review & editing, Supervision, Project administration,
Funding acquisition.
Declaration of Competing Interest
The authors declared that they have no conflicts of interest to this
work. We declare that we do not have any commercial or associative
interest that represents a conflict of interest in connection with the work
submitted.
9
Scientia Horticulturae 312 (2023) 111841
Data availability
Data will be made available on request.
Acknowledgements
This work was supported by the National Key Research and Devel­
opment Program of China [grant numbers 2019YFD1002000].
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.scienta.2023.111841.
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