Biological Control 178 (2023) 105145

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Biological Control
journal homepage: www.elsevier.com/locate/ybcon

Volatile compounds produced by endophytic bacteria adversely affect the

virulence traits of Ralstonia solanacearum
Maryam Yousefvand , Behrouz Harighi *, Abdolbaset Azizi
Department of Plant Protection, Faculty of Agriculture, University of Kurdistan, Sanandaj, Iran

H I G H L I G H T S

• Several endophytic bacteria were identified as Bacillus species using molecular analysis.
• Endophytic bacteria identified have antagonistic activity against Ralstonia solanacearum.
• These strains produce VOCs with antibacterial activity.
• These VOCs inhibited virulence traits of R. solanacearum in vitro.
• VOCs reduced potato wilt incidance caused by R. solanacearum in greenhouse.

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

Keywords: In this study, we investigated the effects of volatile organic compounds (VOCs) produced by endophytic bacteria
Bacterial wilt belonging to the Bacillus genus on the virulence traits of Ralstonia solanacearum R32, the causal agent of potato
Biological control bacterial wilt disease. The results of gas chromatography-mass spectrometry analysis showed that strains
Endophytic bacteria
B. safensis Har267, B. pumilus Fer469, B. zhangzhouensis Kh690, B. aerius Kh867, and B. wiedmannii Ah945 able to
Potato
Ralstonia solanacearum
produce 10, 4, 18, 6, and 12 VOCs with high quality, respectively. All endophytic bacteria produced VOCs that
Virulence traits significantly reduced wilting symptoms in planta, inhibited the cell population of R. solanacearum R32 in vitro,
Volatile compounds and reduced the cell population both in the shoots and roots of inoculated plants at different levels. The VOCs
significantly reduced the attachment of R. solanacearum R32cells to root tissues of potato plants, as well as
chemotaxis motility towards root extract. Our results revealed that VOCs could reduce swarming-, swimming-,
twitching motility, and biofilm formation by R. solanacearum R32 which are important for pathogenicity.
Scanning electron microscopy analysis revealed various morphological abnormalities in the R. solanacearum R32
cells exposed to the VOCs produced by B. safensis Har267 and B. aerius Kh867 strains. Results presented in this
study showed that the VOCs produced by endophytic bacteria investigated can reduce bacterial wilt disease.
Investigation of the mechanisms of biocontrol activities increases our knowledge to design better methods to
control bacterial pathogens.

1. Introduction 2014). Upon the infection of plants, R. solanacearum multiplies and

Ralstonia solanacearum as a soil-borne bacterium causes vascular wilt
disease in over 200 plant species worldwide and has been ranked as the
second most important bacterial pathogen (Peeters et al., 2013). To
locate plant roots the bacterial cells required sensing, chemotaxing, and
flagellar motility toward root exudates (Tans-Kersten et al., 2001; Yao
and Allen, 2006). R. solanacearum cells attach to the root surface via the
production of exopolysaccharides, enter the roots through wounds or
natural openings, and finally reach the xylem vessels (Dalsing and Allen,
produce exopolysaccharides, and other virulence factors resulting in
development of the wilting symptoms (Genin and Denny, 2012). Inside
the xylem vessels, some R. solanacearum cells use twitching motility to
move along the vessel wall (Liu et al., 2001).
Vascular wilt disease is known as one of the most limiting factors in
the cultivation of potato plants worldwide (Elphinstone, 2005). The
wide host range of the causal agent and the adaptability to survive in
various environmental conditions for long periods make control very
difficult. The management of the disease with different strategies

* Corresponding author.
E-mail address: bharighi@uok.ac.ir (B. Harighi).

https://doi.org/10.1016/j.biocontrol.2022.105145
Received 22 November 2022; Received in revised form 20 December 2022; Accepted 29 December 2022
Available online 7 January 2023
1049-9644/© 2022 Elsevier Inc. All rights reserved.
M. Yousefvand et al.
including physical, chemical, cultural, and biological methods has been
extensively investigated (Yuliar et al., 2015). No control method seems
to be completely suitable against bacterial wilt. In this regard, biological
control using bacteria with antagonistic activity seems to be a safe and
cost-effective method for the management of bacterial wilt disease
(Álvarez et al., 2019). Several studies indicated the potential of bacteria
as biological control agents, including nonpathogenic strains of
R. solanacearum, plant growth-promoting rhizobacteria, and endophytic
bacteria in controlling bacterial wilt (Yuliar et al., 2015).
Endophytic bacteria colonize plants internally as plant pathogens
and might have the potential to compete and suppress the virulence of
pathogenic organisms (Rosenblueth and Martinez-Romero, 2006). The
increasing interest in endophytic bacteria for plant disease control is
based on their ability to be biologically active against the pathogen
through competition for nutrient uptake, antibiosis, production of
siderophore, production of enzymes, and/or induction of systemic
resistance (Compant et al., 2005).
Bacteria have been reported to produce and release various volatile
organic compounds (VOCs) with significant biological activities
including growth inhibition of phytopathogens (Garbeva and Weisskopf,
2020). VOCs are defined as small molecules less than 300 Dalton with
low boiling point and high vapor pressure. These molecules can easily
spread in the environment even at distances (Chowdhury et al., 2019).
The direct antibacterial activity of bacterial VOCs has been reported.
Many biocontrol agents like Pseudomonas and Bacillus species have been
reported to emit VOCs with antibacterial actions against bacterial plant
pathogens (Rajer et al., 2017; Xie et al., 2018). It was shown that VOCs
emitted by Bacillus amyloliquefaciens SQR-9 and Pseudomonas fluorescens
WR-1 can reduce the virulence traits of Ralstonia solanacearum (Raza
et al., 2016a; Raza et al., 2016b). In addition, VOCs released by Bacillus
spp. were found to have inhibitory effects and cause morphological
abnormalities in R. solanacearum cells (Tahir et al., 2017). In addition,
the effects of bacterial VOCs in the induction of systemic resistance in
plants have been reported. VOCs produced by Bacillus and Pseudomonas
strains were demonstrated to improve resistance in plants against
Erwinia carotovora subsp. carotovora and R. solanacearm (Ryu et al.,
2004; Tahir et al., 2017; Kashyap et al., 2022).
The major goals of the current study were to identify endophytic
bacteria as potential antagonistic agents against R. solanacearum R32,
evaluate the effects of VOCs produced by these endophytic bacteria on
the growth rate, structural change, and virulence traits such as motility,
chemotaxis, attachment, and biofilm formation. Moreover, the major
VOCs produced by endophytic bacteria against R. solanacearum R32
were identified using gas chromatography–mass spectrophotometry
(GC–MS).
2. Materials and methods
2.1. Bacterial strains and growth conditions
The endophytic bacteria previously isolated from the different
plants, as well as Ralstonia solanacearum R32 (GenBank Acc. No
OP713766), which exhibited wilting disease in potato plants obtained
from department of plant protection, University of Kurdistan were used
in this study. Accordingly, Endophytic bacteria and bacterial pathogen
were grown on nutrient agar (NA) or Triphenyltetrazolium chloride
(TTC) media, respectively, and then stored at 4–6 ◦ C as a working stock
or grown in nutrient broth (NB) medium for 24 h at 26–28 ◦ C with
shaking. Finally, sterile glycerol was added to the final concentration of
20 % and then stored at − 20 ◦ C for long-term storage.
2.2. Molecular identification of the endophytic bacteria
Endophytic bacterial strains were identified by partial nucleotide
sequencing of the 16S rRNA gene using PCR with two universal primers,
fD2 (5′ -AGA GTT TGA TCA TGG CTC AG-3′ , position 8–27) and rP1 [5′ -
2
Biological Control 178 (2023) 105145
ACG GTT ACC TTG TTA CGA CTT-3′ , position 1512–1492 (Escherichia
coli)] (Weisburg et al., 1991), and gyrB gene using primers gyrBF (5′ -
TTA TCT ACG ACC TTA GAC G − 3′ ) and gyrBR (5′ - TAA ATT GAA GTC
TTC TCC G − 3′ ) based on the method previously described (Liu et al.,
2013). The PCR products were sequenced using an ABI3730XL DNA
sequencer (Applied Biosystems). The 16S rRNA and gyrB gene sequences
obtained were aligned and manually adjusted where necessary by using
BioEdit Sequence Alignment Editor 7.0.9.0 software (Hall, 2011).
Sequence comparisons were conducted with other sequences deposited
in the NCBI database using the BlastN program. The Maximum Likeli­
hood phylogenetic analysis was carried out using MEGA version 6.0
software (Tamura et al., 2013) and a phylogenetic tree was constructed
(bootstrap analysis with 1000 replicates was conducted).
2.3. Effect of VOCs on wilt development
Potato tubers (Sprite cultivar) were sown in plastic pots containing
steam-sterilized soil (consisting of 50 % sand, 20 % clay, 30 % peat, pH
7.2). The suspension of R. solanacearum R32 cells with or without
exposure to the VOCs of endophytic bacterial strains for three days at
28–30 ◦ C was prepared in sterile water (density of about 1 × 108 CFU.
mL− 1). The stems of potato plantlets were punctuated with a sterile
toothpick and 50 µL was inoculated (between the third and fourth in­
ternodes) using a sterile syringe. Plantlets were incubated in a green­
house (85 % humidity, 28–30 ◦ C, 12 h/12 h day/night photoperiod).
The relative of disease incidence and biocontrol efficacy was calculated
using the following formula (Kheirandish and Harighi, 2015).
Disease incidence = (number of wilted leaves per pot/ total number
of leaves per pot) × 100.
Biocontrol efficacy = [(disease incidence in control pots - disease
incidence in antagonist treated pots) /disease incidence in control] ×
100. Control and antagonist treated pots includes plantlets inoculated
with non-exposed or pre-exposed bacterial pathogen with VOCs,
respectively.
Inoculated Plants were then cut above ground, the fresh weight of
shoots was measured, then oven-dried at 80 ◦ C for 72 h and dry weight
was measured. Greenhouse experiments were carried out with a facto­
rial arrangement in a completely randomized design with three repli­
cations and three individual experiments. Inoculated plants with non-
exposed R. solanacearum R32 and sterile water were used as positive
and negative control respectively. Data analysis was performed using
the SAS software program and comparison of means was done with the
least significant difference (Duncan) test at a 5 % probability level.
2.4. Evaluation of the antibacterial activity of VOCs produced by
endophytic bacteria
The antibacterial activity of VOCs against R. solanacearum R32 was
assessed on Casamino acid-Peptone-Glucose (CPG) medium using
divided Plates. The overnight growth of the endophytic bacteria
(adjusted to the concentration of about 1 × 108 CFU.mL− 1) was streaked
on one compartment of the plate, while the other compartment was spot
inoculated with 50 µL of the pathogen (1 × 107 CFU.mL− 1). In the
control, the pathogen was cultured alone. The plates were sealed with
parafilm and then incubated at 28–30 ◦ C for 3 days. Thereafter, the cell
population of the R. solanacearum R32 was measured per plate (Tahir
et al., 2017). Three replications were performed for each treatment.
2.5. Effect of VOCs on R. solanacearum R32 colonization
The endophytic bacteria (1 × 108 CFU.mL− 1) was streaked on one
compartment of the divided plate containing Nutrient Agar medium
(NA), while the other compartment (CPG medium) was spot inoculated
with 50 µL of the R. solanacearum R32 (1 × 107 CFU.mL− 1). The plates
were then incubated at 28–30 ◦ C for 48 h, the R. solanacearum R32 cells
were suspended in sterile distilled water, the concentration adjusted to
M. Yousefvand et al.
108 CFU.mL− 1 with a spectrophotometer, and 1 mL was added to each
pot containing potato tubers previously prepared as described in section
4.2. Pots without bacterial suspension serve as controls. The pots were
kept in a greenhouse with a light/dark cycle of 12:12 h at 28–30 ◦ C and
irrigated with sterile water at two-day intervals. After 28 days, roots and
shoots were selected, surface sterilized with 1 % sodium hypochlorite for
3–5 min, and washed several times with sterile water. Plant tissues were
then homogenized aseptically with a pestle and mortar in 1 mL of
sterilized-distilled water. Aliquots of 50 µL were spread onto CPG media,
incubated at 26–28 ◦ C for 48 h, and colonies were counted as CFU.g− 1 of
fresh plant tissues. All experiments were repeated three times with three
independent replicates.
2.6. Potato root attachment assay
R. solanacearum R32 cell suspensions with or without exposure to
VOCs of endophytic bacteria for 72 h at 26–28 ◦ C were prepared
(adjusted to about 1 × 107 CFU.mL− 1). Thereafter, unwounded potato
roots were submerged in pathogen suspensions for 1 h. Then, inoculated
root segments were placed individually in 15 mL of sterile distilled
water. After stirring for 10 s, the excess water was removed gently. One
gram was separated from the root tips and ground, serially diluted in
sterile distilled water, and plated to quantify CFU.g− 1 of the root
(Dalsing and Allen, 2014). Three replications were considered for each
treatment.
2.7. Effect of VOCs on EPS production by R. solanacearm
Extraction and quantification of EPS were performed using the
method previously described (Dubois et al., 1956). Briefly, the
R. solanacearum R32 cells with and without exposure to the VOCs of
endophytic bacteria for 72 h were suspended in sterilized distilled water
and shaked at 40 ◦ C for half an hour to solubilize EPS present in the
solution and then centrifuged at 12,000g for 10 min. Thereafter, two
volumes of ice-cold ethanol were added to the collected supernatant and
incubated at 4 ◦ C for 24 h. The precipitated EPS was obtained by
centrifugation at 12,000g for 10 min at 4 ◦ C, dissolved in sterilized
distilled water and after two days, the EPS content was quantified by the
phenol–sulfuric acid method. These experiments had three replicates.
2.8. Biofilm formation assay
The biofilm formation ability of R. solanacearum R32 cells with or
without exposure to the VOCs of endophytic bacteria was tested in
polypropylene tubes. Overnight growth of endophytic bacterial strains
(1 × 108 CFU.mL− 1) were prepared and 10 μL were cultured onto one
compartment of the divided plates containing NA medium. In the other
compartment, a microtube containing 190 μL of CPG broth medium that
was inoculated with 10 μL of R. solanacearum R32 (1 × 107 CFU.mL− 1)
was placed vertically. The plates were then sealed with parafilm and
kept at 28–30 ◦ C for 48 h. Thereafter, 25 μL of 1 % crystal violet solution
was added to each tube and kept at room temperature for 15 min. After
that, the tubes were rinsed twice with phosphate buffer. Subsequently, 2
× 200 μL of 95 % ethanol was added to each tube, the resulting volume
was brought to 1 mL with sterile-distilled water and the absorbance was
measured at 540 nm using a spectrophotometer (SPECORD 210, Ana­
lytik Jena, Germany). The experiment was performed in a completely
randomized design with three replications (O’Toole and Kolter, 1998).
2.9. Swarming, swimming, and twitching motility behaviors
For the swarming motility assay, Overnight growth of
R. solanacearum R32 (approximate concentration of 107 CFU.mL− 1) was
prepared and 2 µL was spotted onto one compartment of the divided
plates containing CPG medium plus 0.7 % agar. In the other compart­
ment, 30 µL of the endophytic bacteria (approximate concentration of
3
Biological Control 178 (2023) 105145
108 CFU.mL− 1) was streaked on NA medium. For the swimming and
twitching motility assays, 2 µL of freshly prepared R. solanacearum R32
was spotted on plates containing CPG medium plus 0.2, and 1.6 % agar,
respectively. In the other plates, 30 µL of the endophytic bacteria were
streaked on NA medium and both plates were placed on opposite sides.
Then, all plates were sealed with parafilm and incubated at 26–28 ◦ C.
The halo diameters of swarming, swimming, and twitching motility
were measured at each 12 h interval. For the twitching motility, the
morphology of the colony edges was observed using a light microscope.
The experiment was done in three replications (Tahir et al., 2017).
2.10. Chemotaxis assay
Endophytic bacteria were streaked onto one compartment of the
divided plates containing NA medium as described earlier. In the other
compartment, chemotaxis buffer medium (0.1 mM EDTA, 10 mM
K2HPO4, 0.35 % agar, pH 7.2) was prepared, the hole of 5 mm of the
medium was removed and then refilled with 50 µL of root extract of
potato (cultivar Sprite). Then, freshly prepared R. solanacearum R32
cells were spot inoculated at a distance of 15 mm from the hole. The
plates were sealed with parafilm and then incubated at 28–30 ◦ C. The
movement of the R. solanacearum R32 cells toward the root extract was
measured by counting the CFU.mL− 1 of the cell on CPG medium. This
experiment was performed in three replicates (Tahir et al., 2017).
2.11. Scanning electron microscopy (SEM)
Scanning electron microscopy (SEM) was conducted to observe
external morphological changes in the R. solanacearum R32 cells. Bac­
terial cells with or without exposure to the VOCs of endophytic bacteria
for two days at 26–28 ◦ C were collected into Eppendorf tubes, washed
with sterilized-distilled water, and centrifuged (10 min, 3600g, 4 ◦ C).
Afterward, the pelleted cells were spread onto the clean slide. After
fixation in 2.0 % glutaraldehyde solution for 2 h at room temperature,
the sample was washed two times with sterilized-distilled water. Serial
dehydration was done in ethanol solutions of 20 %, 40 %, 60 %, 80 %,
and 100 % for 10 min each time, followed by 100 % ethanol for 1 h. The
samples were then placed at − 20 ◦ C overnight followed by the freeze-
drying process at − 40 ◦ C for 3 h. Finally, the samples were coated
with gold, and electron micrographs were taken using a TSCAN SEM
system (TSCAN SEM, TSCAN, Czechoslovakia).
2.12. Identification of VOCs produced by endophytic bacteria using
GC–MS analysis
In order to collect the VOCs produced by each endophytic bacteria, a
three-compartment plate was used. One compartment, containing NA
medium was streaked with 100 µL (1 × 108 CFU.mL− 1) freshly growth of
each endophytic bacteria, and the second compartment containing CPG
medium was spot inoculated with 5 µL (1 × 107 CFU.mL− 1) of
R. solanacearum R32, and in the third compartment, 0.3 g of sterile
activated charcoal was placed to adsorb the VOCs. As well, the same
experimental design without endophytic bacteria was used as a control.
Subsequently, the plates were sealed with parafilm and then incubated
at 28–30 ◦ C for 72 h. Thereafter, the activated charcoal traps were
transferred into glass vials and ethyl acetate (1: 1.25 W/V) was added.
The adsorbed VOCs were extracted by shaking for 20 min, followed by
centrifugation (2500g, 5 min) and the supernatants were analyzed by
using a gas chromatography device connected to a mass spectrometer
(Agilent 7890B GC System / 5977A MSD).
One microliter of the collected sample was injected into HP-5 ms
column (30 m × 0.25 mm, 0.25 µm), the initial column temperature was
set at 60 ◦ C, which was then increased to 260 ◦ C at a rate of 7 ◦ C.min− 1,
and held for 5 min. The mass spectrometer was operated in the electron
ionization mode at 70 eV, with continuous scanning from 50 to 550 m/z.
Helium carrier gas with a purity of 99.999 %, a 34 psi pressure, and a
M. Yousefvand et al.
flow rate of 1 mL.min− 1 was used at this stage. The compounds were
identified by comparing their mass spectra with the databases of the
device, including the National Institutes of Standards and Technology
(NIST) and Wiley databases, along with comparing the inhibition indices
and the failure pattern reported for them (Dewanjee et al., 2015).
2.13. Statistical analysis
The data from each experiment were statistically evaluated by using
analysis of variance (ANOVA), followed by Least-Significant Difference
(LSD) and Duncan test (P = 0.05), employing SAS ver. 9.1 software. All
the experiments were conducted in a completely randomized design.
Graphs and figures were plotted using the Excel program.
3. Results
3.1. Identification of the endophytic bacteria
Nucleotide sequences obtained were submitted to the NCBI nucleo­
tide sequence database under accession numbers OP217053-OP217057,
and OP267569-OP267573 for the 16S rRNA and gyrB genes, respec­
tively. Results of the partial nucleotide sequencing of the 16S rRNA and
gyrB genes revealed that endophytic bacterial strains clustered into the
Bacillus genus. Blast search results revealed that strains Har267, Fer469,
Kh690, Kh867, and Ah945 had 99–100 % similarity to B. safensis, B.
pumilus, B. zhangzhouensis, B. aerius, and B. wiedmannii, respectively. The
Fig. 1. Phylogenetic tree of partial 16S rRNA and gyrB gene sequences showing the position of endophytic bacterial strains (shown in bold) in addition to taxo­
nomically similar selected reference strains. The analysis was conducted by the Maximum Likelihood method with Tamura-Nei calculation model in MEGA version
6.0. The scale bar represents the number of substitutions per site. Numbers at branching points indicate bootstrap value derived from 1000 replicates.
4
Biological Control 178 (2023) 105145
phylogenetic tree was constructed which shows the position of strain
amongst the type strains of the genera Bacillus species (Fig. 1).
3.2. Effect of VOCs on bacterial wilt disease development
Statistical analysis revealed significant differences between the
treatments in terms of the inhibition of wilting development (F =
848.34; P < 0.0001) by R. solanacearum R32 exposed to VOCs produced
by endophytic bacteria compared to the control (Table 1). The obtained
results reveal that VOCs produced by the strains B. wiedmannii Ah945
and B. zhangzhouensis Kh690 with about 100 % had the highest
decreasing effects on wilting development by R. solanacearum R32
(Fig. 2A,D). Statistical analysis revealed significant differences between
the treatments in terms of the increasing of fresh weight (F = 21.55; P <
0.0001) and dry weight (F = 6.18; P = 0.0025) of shoots in plant
inoculated by R. solanacearum R32 exposed to VOCs produced by
endophytic bacteria compared to the non-treated control (Table 1).
Strains B. zhangzhouensis Kh690 and B. wiedmannii Ah945 with about 81
% and 79 %, had the highest increasing effects on the fresh weight of the
shoot (Fig. 2B). Similarly, Strains B. zhangzhouensis Kh690 and Bacillus
wiedmannii Ah945 with about 49 % and 43 %, had the highest increasing
effects on the dry weight of the shoot (Fig. 2C).
3.3. Antibacterial activity of VOCs against R. solanacearum R32
According to the statistical analysis, significant differences were
M. Yousefvand et al. Biological Control 178 (2023) 105145

Table 1 and root colonization was assessed quantitatively (CFU.g− 1 of plant

Effect of VOCs produce by endophytic bacteria on wilting development by tissues). Statistical analysis revealed significant differences between the
Ralstonia solanacearum R32 under greenhouse conditions and analysis of vari­ treatments in terms of the root (F = 65.55; P < 0.0001) and shoot
ance (ANOVA) of data. colonization (F = 20.26; P < 0.0001) by R. solanacearum R32 exposed to
Mean of Square VOCs produced by endophytic bacteria compared to the control
Source of df Biocontrol Fresh weight of Dry weight of (Table 2). In roots, strains Bacillus wiedmannii Ah945 and
variation activity (%) shoot (g) shoot (g) B. zhangzhouensis Kh690 with 95 % showed the highest reduction effects
Har267 11.67b 17.23b 3.48abc
followed by B. aerius Kh867, B. safensis Har267, and B. pumilus Fer469
Fer469 6.67b 11.61b 2.11c with 56 % and 50 % reduction effects, respectively (Fig. 3B). Moreover,
Kh690 100a 47.97a 4.93a our finding revealed that VOCs produced by strains B. wiedmannii Ah945
Kh867 3.71b 11.03b 2.38bc and B. zhangzhouensis Kh690 decreased the populations of
Ah945 100a 44.04a 4.46ab
R. solanacearum R32 to about 80.32 %, followed B. safensis Har267,
Ctrl+ 0b 9.51b 2.54bc
Ctrl- 100a 36.79a 4.30ab B. pumilus Fer469, and B. aerius Kh867 by 49 % reduction effect in shoots
Treatment 6 7688.66* 851.30* 3.86* compared with non-exposed control (Fig. 3C).
Error 14 22.07 89.50 0.63
F-value 848.34 21.55 6.18
CV (%) 8.70 14.69 12.91 3.5. Effect of VOCs of endophytic bacteria on R. solanacearum R32 root
attachment
*Significant at 5 % probability level; df = Degrees of Freedom; CV = coefficient
of variation; Significance among treatment means is presented as the different
lowercase letters. Data are presented as the mean of three replicates. The ability of Ralstonia solanacearum R32 cells pre-exposed to VOCs
of endophytic bacteria to attach to the roots of potato plants (cultivar
sprite) was investigated. Based on the results of ANOVA, significant
found between the treatments in terms of reduction of the cell popula­
differences existed among all the treatments (F = 29.37; P < 0.0001)
tion (F = 77.73; P < 0.0001) of the R. solanacearum R32 exposed to VOCs
(Table 2). The highest inhibition belonged to B. wiedmannii Ah945 and
produced by endophytic bacteria compared to the control (Table 2).
B. zhangzhouensis Kh690 with 95.99 % and 91.03 %, respectively, fol­
All endophytic bacteria significantly reduced the cell population of
lowed by B. pumilus Fer469 (56.36 %), B. aerius Kh867 (53.96 %), and
the R. solanacearum R32 at various levels compared to the control.
B. safensis Har267(45.31 %) compared to the control (Fig. 4A).
Strains B. zhangzhouensis Kh690, B. wiedmannii Ah945, and B. safensis
Har267 with about 45.34 %, 44.76 %, and 43.02 % reduction of the cell
population, respectively were found to have the highest effect, followed 3.6. Effect of VOCs on EPS and biofilm production by R. solanacearum
by B. pumilus Fer469, and B. aerius Kh867 with 28.48 %% and 34.30 % R32
reductions, respectively (Fig. 3A).
Based on the results of ANOVA, significant differences existed among
the treatments in terms of EPS production by R. solanacearum R32 (F =
3.4. Effect of VOCs on R. solanacearum R32 colonization 27536.4; P < 0.0001) (Table 2). The VOCs produced by strains Ah945
and Kh690 showed a significant negative effect while the decrease in the
Twenty-eight days after treatment of potato plantlets with EPS production was 55.51 % and 44.38 % respectively, after exposure to
R. solanacearum R32 cells exposed to VOCs of endophytic bacteria, shoot the VOCs compared to the control (Fig. 4B).

Fig. 2. Effects of VOCs produced by B. safensis Har267, B. pumilus Fer469, B. zhangzhouensis Kh690, B. aerius Kh867, and B. wiedmannii Ah945 on (a) disease
incidence, (b) fresh weight of shoot, (c) dry weight of shoot, and (d) representative inoculated potato plants by treated-R. solanacearum R32 compared to the non-
exposed control (Ctrl+) or inoculated by sterile water (Ctrl-). Error bars indicate the SE of the three replicates. Different letters indicate significant differences (P
= 0.05).

5
M. Yousefvand et al. Biological Control 178 (2023) 105145

Table 2
Effect of VOCs produced by endophytic bacteria on cell population, root and shoot colonization, attachment, and exopolysaccharide (EPS) production of
R. solanacearum R32 and analysis of variance (ANOVA) of data.
Mean of Square

Source of df Antibacterial activity (OD600 Colonization (CFU × 107/g of Colonization (CFU × 104/g of Attachment (CFU × 104/g of EPS
variation nm) root) shoot) root) (µg/ml/
OD)

Har267 0.98c 80b 3b 17.7b 1.98a

Fer469 1.24b 83b 2.9b 14.2b 1.68b
Kh690 0.94c 7.8c 1.2c 2.9c 1.09c
Kh867 1.13b 71b 3b 14.9b 2.01a
Ah945 0.96c 8.8c 1.2c 1.3c 0.88d
Ctrl 1.73a 160a 5.8a 32.4a 1.97a
Treatment 5 0.269** 9.35** 846952338** 3.842** 0.740**
Error 12 0.0034 1.427 41,797,055 1.308 0.000026
F-value 77.73 65.55 20.26 29.37 27536.4
CV (%) 5.07 17.52 22.77 19.98 0.32

** Significant at 1 % probability level; df = Degrees of Freedom; CV = coefficient of variation; Significance among treatment means is presented as the different
lowercase letters. Data are presented as the mean of three replicates.

Fig. 3. Effects of VOCs produced by B. safensis Har267, B. pumilus Fer469, B. zhangzhouensis Kh690, B. aerius Kh867, and B. wiedmannii Ah945 on cell population of
R. solanacearum R32 (a) in dual culture assay, (b) inoculated potato roots, and (c) inoculated potato shoots compared to the non-exposed control (Ctrl). Error bars
indicate the SE of the three replicates. Different letters indicate significant differences (P = 0.05).

Fig. 4. Effects of VOCs produced by B. safensis Har267, B. pumilus Fer469, B. zhangzhouensis Kh690, B. aerius Kh867, and B. wiedmannii Ah945 on (a) root attachment,
(b) exopolysaccharide (EPS) production, and (c) biofilm formation of R. solanacearum R32 compared to the non-exposed control (Ctrl). Error bars indicate the SE of
the three replicates. Different letters indicate significant differences (P = 0.05).

As presented in Table 3, the result of ANOVA showed a significant
difference among all the treatments in biofilm production by
R. solanacearum R32 compared with the non-exposed control (F = 5.23;
P = 0.0089). The VOCs produced by endophytic bacteria revealed a
significant inhibition effect on the biofilm formation by R. solanacearum
R32. The result indicates 58.80 % and 53.84 % decrease in the biofilm
formation by R. solanacearum R32 exposed to VOCs of B. wiedmannii
Ah945 and B. zhangzhouensis Kh690 strains, respectively (Fig. 4C).
3.7. Effect of VOCs on the motility behaviors of R. solanacearum R32
According to statistical analysis, significant differences have existed
between the treatments in the swarming (F = 7.93; P < 0.0001),
swimming (F = 9.20; P < 0.0001), and twitching (F = 7.22; P < 0.0001)
motility assessments (Table 3). Our finding revealed that the swarming
motility of R. solanacearum R32 cells exposed to VOCs of endophytic
bacterial strains was significantly inhibited after 72 h. As illustrated in
Fig. 5A and B, the strains B. safensis Har267, B. zhangzhouensis Kh690,
and B. aerius Kh867 with a mean of 4.4, 4.5, and 4.8 mm respectively,
showed the highest inhibition effects of R. solanacearum R32 cells as
compared to the control with a mean of 6 mm.
Correspondingly, the VOCs produced by B. zhangzhouensis Kh690,
B. aerius Kh867, and B. wiedmannii Ah945 inhibited the swimming
motility of R. solanacearum R32 to 14.5, 15.4, and 16.7 mm, respectively

6
M. Yousefvand et al. Biological Control 178 (2023) 105145

Table 3
Effect of VOCs produced by endophytic bacteria on swimming-, swarming-, twitching- motility, biofilm formation, and chemotaxis of R. solanacearum R32 and analysis
of variance (ANOVA) of data.
Mean of Square

Source of variation df Swarming zone (mm) Swimming zone (mm) Twitching zone (mm) Chemotaxis Biofilm
(107 CFU/ml) (OD540 nm)

Har267 4.4d 17.6b 5.8b 6.4b 0.66ab

Fer469 5.3b 17.3b 5.6b 5.8b 0.67ab
Kh690 4.5d 14.5d 4.4c 4.6b 0.48b
Kh867 4.8cd 15.4cd 5.5b 5.4b 0.73ab
Ah945 5.2bc 16.7bc 5.8b 1.3c 0.48b
Ctrl 6.0a 21.3a 6.6a 9.2a 1.04a
Treatment 5 0.044** 0.73** 0.033** 9.574** 0.12**
Error 12 0.005 0.079 0.004 6.971 0.024
F-value 7.93 9.20 7.22 13.73 5.23
CV (%) 14.66 16.42 12.02 13.99 15

** Significant at 1 % probability level; df = Degrees of Freedom; CV = coefficient of variation; Significance among treatment means is presented as the different
lowercase letters. Data are presented as the mean of three replicates.

Fig. 5. Effects of VOCs produced by B. safensis Har267, B. pumilus Fer469, B. zhangzhouensis Kh690, B. aerius Kh867, and B. wiedmannii Ah945 on swarming motility
of R. solanacearum R32 compared to the non-exposed control (Ctrl). The diameter of the motility zone (a), and representative plate of swarming motility assay (b)
were shown. Three replicates were used for each treatment. Error bars indicate the SE of the three replicates (P = 0.05).

Fig. 6. Effects of VOCs produced by B. safensis Har267, B. pumilus Fer469, B. zhangzhouensis Kh690, B. aerius Kh867, and B. wiedmannii Ah945 on swimming motility
of R. solanacearum R32 compared to the non-exposed control (Ctrl). The diameter of the motility zone (a), and representative plate of swimming motility assay (b)
were shown. Three replicates were used for each treatment. Error bars indicate the SE of the three replicates (P = 0.05).