Current Microbiology (2019) 76:855–862

https://doi.org/10.1007/s00284-019-01699-4

Role of Maize Root Exudates in Promotion of Colonization of Bacillus

velezensis Strain S3‑1 in Rhizosphere Soil and Root Tissue
Yeqing Jin1 · Hangfei Zhu1 · Si Luo1 · Wenwu Yang1 · Li Zhang1 · Shanshan Li1 · Qing Jin1 · Qin Cao1 · Shurong Sun1 ·
Ming Xiao1

Received: 2 February 2019 / Accepted: 29 April 2019 / Published online: 9 May 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract
Bacillus velezensis strain S3-1 has a broad range of hosts and is used as a biocontrol agent and biofertilizer. However, the
interaction of maize root exudates and colonization of the strain S3-1 has not yet been investigated. In our study, strain
S3-1 effectively colonized both rhizosphere soil and root tissue. Collected maize root exudates significantly induced the
chemotaxis, cluster movement, and biofilm formation of strain S3-1, showing increases of 1.43, 1.6, and 2.08 times, respec-
tively, compared with the control. In addition, the components of root exudates (organic acids: citric acid, malic acid, and
oxalic acid; amino acids: glycine, proline and phenylalanine; sugars: glucose, fructose, and sucrose) were tested. Each of
these compounds could induce chemotactic response, swarming motility, and biofilm formation significantly. The strongest
chemotactic response and swarming motility were found when malic acid was applied, but maximal ability of biofilm for-
mation was stimulated by proline. Furthermore, we found that these compounds of root exudates stimulated the population
of S3-1 adhering to the maize root surface, especially in the presence of malic acid. These results indicate that maize root
exudates play an important role in the colonization of S3-1, and provide a deeper understanding of the interaction between
plants and microorganisms.

Introduction source of nutrition and a signal that attracts microorganisms

Plant growth-promoting rhizobacteria (PGPR) is widely
applied in agriculture, which can promote plant growth and
control the growth of some plant pathogens [8, 27]. Most
of Bacillus spp. are members of PGPR, having been com-
mercially used as biofertilizers and biocontrol agents [9, 41],
such as Bacillus subtilis GB03, Bacillus subtilis MB1600,
and Bacillus subtilis QST713 [25]. Extensive past studies
have led to an understanding of effective root colonization,
which is crucial to suppression effects [13, 20]. Therefore,
it is imperative to investigate the deeper processes related to
colonization of microorganisms in root.
The abundance and activity of microorganisms will be
increased by releasing large amounts of low molecular
weight carbon compounds from plant root exudates to rhizo-
sphere soil [29, 42]. Root exudates are composed of amino
acids, carbohydrates, and carboxylic acids, which represent a
* Ming Xiao
xiaom88@shnu.edu.cn
1
College of Life Sciences, Shanghai Normal University,
Shanghai 200234, People’s Republic of China
to the rhizosphere [6, 19], known as a bacterial chemotac-
tic response. Chemotaxis is required to establish the initial
host-micro interaction as a basic step towards the root sys-
tems [34]. Swarming is dependent on single polar flagel-
lum which enables the cell to swim in low-agar media [21].
The flagellum and the chemotaxis system can allow bacte-
ria to respond to attractants and repellents [32]. Twitching
motility is believed to propagate at surface interfaces [10].
Furthermore, biocontrol microorganisms can form biofilms
to prevent infection from pathogens. Biofilms are assem-
blages of bacterial cells involved in a matrix composed of
exopolysaccharides and some proteins that contribute to the
adherence to the surface of root systems [7]. In addition, the
biocontrol agent can take advantage of nutrients from root
exudates to reproduce and proliferate, facilitating the biofilm
formation [38].
Bacillus velezensis strain S3-1 (registration number
CP016371) was isolated from the rhizosphere soil of toma-
toes. It has obvious antifungal effects on gray mold of tomato
(surfactant, itrin and fengycin), and induces systemic resist-
ance in the tomatoes [30]. Our previous report showed that
strain S3-1 could increase the root length, shoot length, and

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fresh weight, while also increasing the quality of tomatoes. It
can also be used as a biofertilizer and biocontrol agent [30].
However, the potential interaction of maize root exudates
and colonization of the strain S3-1 have not yet been inves-
tigated. In this work, we try to reveal the excellent ability of
colonization of Bacillus velezensis strain S3-1 and the pos-
sible role of maize root exudates in promoting colonization
of strain S3-1 [24‚ 40].
Materials and Methods
Microorganism and Culture Conditions
Bacillus velezensis strain S3-1 was isolated from rhizos-
phere soil (Hebei Province, China) and showed outstanding
suppression effects against Fusarium graminearum, Capsi-
cum Wilt, Rhizoctonia solani, and Botrytis cinerea [3‚ 30].
When required, S3-1 was incubated in Luria–Bertani (LB)
liquid medium at 28 °C, 200 rpm. Screening of antibiotic-
resistant strain S3-1 was performed by streaking the colo-
nies of S3-1 onto the LB agar plates containing rifampicin
[2]. The rifampin concentration gradient was designed to be
0–100 μg/mL. The colonies able to grow at 100 μg/mL were
selected and designated S3-1-Rif.
Plant Growth Conditions and Preparation of Maize
Root Exudates
The collection of maize root exudates was performed as
described [33]. Briefly, maize seeds were surface sterilized
by 70% ethanol for 1 min and rinsed three times with sterile
water, then immersed in 3% sodium hypochlorite (NaClO)
for 5 min, followed by washing six times and germinated on
wet sterile filter paper at 25 °C for 3 or 4 days until the main
roots of maize at least were 2 cm long. The 50 germinated
seedlings were transferred into a sterile conical tube contain-
ing sterile double-distilled water. The tubes were incubated
at 25 °C for 24 h to collect root exudates. The root exudates
of maize were sterilized by passing them through a 0.45 μm
filter, then 100 μL samples were checked for contamination
by spreading on LB medium, indicating that the collected
root exudates were not contaminated. The filter-sterilized
root exudates were lyophilized and divided into two parts.
One part was stored at − 80 °C until further study.
Colonization of Strain S3‑1 in Rhizosphere Soil
and Root Tissue
Antagonistic experiment was performed in order to assess
the effects of S3-1-Rif on Botrytis cinerea [1]. Briefly,
the Botrytis cinerea was incubated on the center of PDA
medium, the S3-1-Rif strain ­(OD600 of 1.2, 1 μL) and
13
Y. Jin et al.
sterile water (as control) were cultured around the fungi for
5–6 days at 25 °C.
As a colonization experiment, the germinated maize
seedlings were transferred to pots containing sterile soil and
incubated at 25 °C. The 10 mL of S3-1-Rif (­ 107 CFU/mL)
was poured into the soil. Rhizosphere soil was collected and
the population density of S3-1-Rif was determined every
7 days for 28 days. Rhizosphere soil (0.5 g) was added to
sterile water and shaken for 15 min. The serial dilutions of
the samples were incubated on the LB plate supplemented
with rifampicin and the colonies were counted. In order to
determine the colonization ability of the strain S3-1 in root
tissue, the roots were washed and surface sterilized with
0.3% NaClO solution for 20 min, rinsed and weighed. The
roots were ground fully, serially diluted, and the colonies
were counted.
Chemotaxis Assay
A drop assay was performed as described in a previous
report [14] for quantification of chemotaxis of the strain
S3-1 response to maize root exudates or artificial root exu-
dates with slight modifications. Briefly, the strain S3-1 was
incubated on LB medium at 28 °C, 200 rpm for 8 h until
reaching logarithmic phase (­ OD600 of 1.0). Then the 40 mL
of cell suspensions collected by centrifugation (600×g,
10 min) were resuspended in the 12 mL of chemotaxis
buffer [100 mM ­K3PO4 (pH 7.0) with 20 μM EDTA] and
were added to 4 mL of 1% hydroxypropylmethycellulose
(a viscosity of about 4000 cP). Root exudates or artificial
root exudates (10 μL for a drop, 0.1 M) containing citric
acid, malic acid, oxalic acid, glycine, proline, phenylalanine,
glucose, fructose, and sucrose were added to the center of a
60-mm-diameter dish containing cell suspensions. The plate
was inspected to see whether a ring appeared after 0–30 min
at room temperature.
The modified capillary assay was performed based on a
procedure [38] for quantitative measurement. As a chemot-
axis capillary, a needle attached to a syringe containing root
exudates or artificial root exudates (100 μL, 10 μM) was
inserted into a pipette tip with a 100 μL cell suspension
­(OD600 of 1.0). After incubation for 2 h at room temperature,
the needle was washed with sterile water and transferred into
an Eppendorf tube. By serial dilution, the suspensions were
plated on LB plates and the colonies were counted. Each
treatment contained three replicates.
Swarming Assay
The swarming assay was performed as described previously
with a minor modification [26]. Briefly, a solid medium (1%
agar) containing 1 mL of root exudates or 10 μM of artificial
root exudates was prepared. An 8-mm-diameter filter paper
Role of Maize Root Exudates in Promotion of Colonization of Bacillus velezensis Strain S3-1…
was put on the center of the plate. The cells of strain S3-1
were grown at 28 °C until reaching O­ D600 of 0.4 and resus-
pended in 2 mL sterile water. The suspensions (5 μL) were
added into the paper and incubated for 2 days. The colony
diameters were measured in three different directions. Each
treatment was replicated 3 times.
Biofilm Formation Assay
To quantify the effects of root exudates or artificial root exu-
dates on biofilm formation by the strain S3-1, the biofilm
formation assay was performed as described previously in
the 96-well microtiter plates with a slight modification [33].
Briefly, the strain S3-1 was incubated until O ­ D600 of 1.0
and was resuspended by 1/10 LB medium containing root
exudates or artificial root exudates (25 μM) reaching the
density of 1­ 05 CFU/mL. The suspensions were added to the
96-well plates and incubated for 2 days. The strain S3-1
was removed and the plate wells were washed 3 times with
sterile water and air-dried. The cells were then stained by
1% crystal violet (CV) for 30 min at room temperature. After
excess CV solution was taken, the CV bound to cells was
washed three times, and solubilized with 200 μL acetic acid
(33%). The data were measured by the multi-function plate
reader at A570 nm [39].
In vitro Assay
Using a modified method as described [23], the germinated
maize seeds were transferred in the center of tissue bottles
containing 100 mL of nutrient medium and incubated at
25 °C with a 16-h light regime for 2 days. The bacterial
suspensions were injected into the nutriment medium 2 mm
apart from the main root. Then 20 μL of artificial root exu-
dates (10 μM) was dropped onto the surface of the roots.
As the control, sterile water was dropped onto the surface
of the roots. After being incubated 14 days, the maize roots
were collected from the nutriment medium, from which
500 mg of root tissues was taken. The tissues were washed
six times with sterile distilled water, cut into small pieces,
then immersed in 1 mL of sterile water for 5 min to extract
bacteria from the rhizosphere. To count effectively, the sus-
pensions were serially diluted and plated on LB medium.
Statistical Analysis
A completely randomized design with three replications was
used in this study. The data of Log CFUs were represented
as log10 values before statistical analysis. For statistical
analysis, data were subjected to the analysis of variance
(ANOVA) using SPSS Stat Program. Least significant dif-
ference (LSD) at 5% level of probability was computed, and
857
Ducan’s Multiple test was performed to make comparisons
among different means.
Results
Assessment of Colonization of Strain S3‑1
in Rhizosphere Soil and Root Tissue
S3-1-Rif demonstrated outstanding suppression effects on
Botrytis cinerea. In order to investigate the degree of colo-
nization of strain S3-1-Rif, the population density was taken
in the rhizosphere soil and root tissues. The results indi-
cated that the population trend of S3-1 decreased, reaching
7.2 × 106 CFU/g on the 7th day, then the trend increased until
28th day, increasing up to 9.3 × 106 CFU/g (Fig. 1). Fur-
ther analyses indicated that the population densities on the
7th, 14th, 21th, and 28th day were not significantly different
(Fig. 1). In the root tissue, the trend was similar to the sam-
ple of rhizosphere soil. A bacterial population of 1­ 06 CFU
was observed at 7 days post-inoculation, which decreased
during the following period (7–14 days post-inoculation) per
gram of root tissue. Next, the population of S3-1 increased
up to 1.6 × 106 CFU in each gram of root tissue. Until the
28th day, the bacterial population remained stable at about
2.2 × 106 CFU. All the bacterial populations were not sig-
nificantly different from each other (Fig. 1).
Fig. 1  Colonization of Bacillus velezensis strain S3-1 in the rhizos-
phere soil and root tissue of maize. Maize germinated seedlings and
S3-1-Rif ­(107 CFU/mL) were co-introduced into the rhizosphere soil.
The population density of S3-1-Rif was determined in the rhizosphere
soil and root tissue of maize at indicated time points. As shown in
Fig. 1, after 28 days, the number of S3-1 in the rhizosphere soil was
observed and compared with in the root tissue of maize. Each value
is means of three replicates, and the error bars indicate the standard
deviations from the mean. Duncan’s test was used to examine differ-
ences
13
858 Y. Jin et al.

Quantitative and Qualitative Chemotactic Response Table 1  Chemotactic response Chemo-attractant Response
of S3‑1 to Root Exudates and Artificial Root of Bacillus velezensis strain
S3-1 towards artificial root Citric acid ++
Exudates exudates
Malic acid +++
To investigate the effect of root exudates and artificial root Oxalic acid ++
exudates (organic acids, amino acids, and sugars) on chemo- Glycine ++
taxis of the strain S3-1, the drop assay was performed. The Proline ++
results indicated that S3-1 was attracted to root exudates and Phenylalanine +++
artificial root exudates. As shown in Fig. 2a, b, the attraction Glucose ++
of root exudates was more than the control treatment. The Fructose +
artificial root exudates also attracted the strain S3-1 and were Sucrose +
more than chemotaxis buffer (Table 1). The response indicated with +,
++, and +++ refers to artifi-
cial root exudates. +++—fast
response (0–10 min); ++—
moderate response (10–20 min);
+—slow response (20–30 min)

The capillary assay was conducted to detect chemotaxis

quantitatively for root exudates and artificial root exudates.
Root exudates showed 1.43-fold increase compared to the
control (Fig. 2c), and numbers of S3-1 migrated into all indi-
vidual solution were all higher than the control treatment
(Fig. 2d). Malic acid showed the most significant attraction,
as high as 4.34 log CFU/mL. Much lower numbers of S3-1
were found in the sucrose solution and fructose solution and
were statistically non-significant compared with each other.

Assessment of Swarming Motility of Bacillus

velezensis Strain S3‑1 Towards Maize Root Exudates
and Artificial Root Exudates

In order to investigate the effect of maize root exudates and

Fig. 2  Qualitative (a, b) and quantitative (c, d) chemotactic response
of the strain S3-1 towards maize root exudates. Root exudates (10 μL
for a drop, 1 mM) (c) were added to the center of a 60-mm-diameter
dish contained cell suspensions and chemotaxis buffer served as con-
trols (d). Root exudates (a) and artificial root exudates (b) were added
to a pipette and the pipette tip was inserted into cell suspensions.
After 2 h, the population of S3-1 in the pipette was counted by serial
dilution. Data are means of three replicates and error bars represent
standard derivation (SDs). Different letters are significant differences
as analyzed by Duncan’s test (P < 0.05)
artificial root exudates on swarming motility of the strain
S3-1, the colony diameters were measured in three differ-
ent directions. The diameters of the S3-1 swarming areas
were significantly larger in the presence of maize root exu-
dates than the control, increasing 1.6-fold (Fig. 3a). Differ-
ent components of root exudates containing sugars, organic
acids, and amino acids induce different swarming abilities of
the strain S3-1 (Fig. 3b). Furthermore, the largest diameter
associated with swarming, with malic acid, was 1.78-fold
higher than the control. The smallest effect on swarming
motility was in the presence of the fructose solution show-
ing 19.00 mm.
Influence of Maize Root Exudates and Artificial Root
Exudates on Biofilm Formation of Bacillus velezensis
Strain S3‑1
To discover maize root exudates and artificial root exudates
that might influence biofilm formation of S3-1, the absorb-
ance at 570 nm was measured quantitatively. Root exudates

13
Role of Maize Root Exudates in Promotion of Colonization of Bacillus velezensis Strain S3-1…
Fig. 3  Swarming motility of Bacillus velezensis strain S3-1 towards
maize root exudates and artificial root exudates. The suspensions
were added into the paper which was put on the semisolid medium
containing root exudates or artificial root exudates. The colony diam-
eters were measured in three different directions. a Maize root exu-
dates; b artificial root exudates. Error bars indicate SDs based on the
replicates. These different letters are statistical differences on the top
of columns according to Duncan’s test (P < 0.05)
significantly stimulated biofilm formation by the strain S3-1
which showed an increase of 2.08-fold compared to the con-
trol (Fig. 4a). Organic acids, amino acids, and sugars showed
that the biofilm formation of the bacteria was higher than the
control at 25 μM concentration (Fig. 4b). Biofilm intensity
in the proline was the highest, which showed an increase of
2.78-fold. Although much lower biofilm intensity was found
in the sucrose, fructose, and phenylalanine, they significantly
induced the biofilm formation of the strain S3-1 compared
to the control.
Investigation of Recruitment Ability of Strain S3‑1
to the Rhizoplane of Maize Induced by Artificial
Root Exudates
To further confirm the effects of artificial root exudates on
strain S3-1 (chemotaxis, swarming motility, and biofilm
formation), a vitro assay was performed to investigate the
colonization ability to the rhizoplane of maize induced by
different components of root exudates. As shown in Fig. 5,
859
Fig. 4  Effects of root exudates or artificial root exudates on biofilm
formation by Bacillus velezensis strain S3-1. The bacterial suspen-
sions were resuspended by 1/10 LB medium containing root exudates
or artificial root exudates and incubated at 96-well plate. Biofilm
intensity was measured at A570 nm. a maize root exudates; b artifi-
cial root exudates. Error bars devote SDs based on three experimental
values. Column with different letters represent significantly difference
following Duncan’s test (P < 0.05)
the population of S3-1 recruited to the rhizoplane of maize
was much higher in the presence of malic acid and glucose
than others, being as high as 8.61 log CFU/mL and 8.52 log
CFU/mL, respectively. Of nine components of root exudates,
only fructose exhibited the lowest performance about colo-
nization ability, but it was still an increase of 1.24-fold com-
pared to the control, which suggested that the root exudates
induced significantly the bacteria to colonize the rhizoplane
of maize relative to the control.
Discussion
The interaction of root exudates with PGPR has attracted
wide attention, because these bacteria have significant
inhibitory effects on agricultural diseases and demonstrate
13
860
Fig. 5  The colonization ability of Bacillus velezensis strain S3-1 to
the rhizoplane of maize induced artificial root exudates. The bac-
terial suspensions were injected into medium 2 mm apart from the
main roots and artificial root exudates were dropped onto the roots of
maize. The population of S3-1 recruited to the rhizoplane of maize
was assessed by serial dilution. Error bars indicate SDs from three
different experimental values. Numbers on top of bars represent sig-
nificant differences between different treatments following Duncan’s
test (P < 0.05)
great potential for field application, such as Pseudomonas
aurantiaca JD37 against Bipolaris maydis [15] and Bacillus
amyloliquefaciens PG12 against B. dothidea [12]. In order to
apply to the agricultural industry, Bacillus velezensis strain
S3-1, revealing outstanding suppression effects against
Fusarium graminearum, Capsicum Wilt, Rhizoctonia solani,
and Botrytis cinerea, showed an enhanced colonization of
the maize root and rhizosphere soil (Fig. 1) [4]. Extensive
past studies have highlighted the role of root exudates for
PGPR in root colonization [16].
Positive chemotactic response in a lot of PGPR was
induced in the presence of root exudates, which was an
essential feature of root colonization [22]. In our experi-
ments, root exudates induced strong chemotaxis on the strain
S3-1 with an increase of 1.43-fold compared to the control
(Fig. 2c). The result was consistent with other reports such
as Pseudomonas fluorescens [14] and Rhizobium legumi-
nosarum (Gaworzewska and Carlile [17]). Pseudomonas
fluorescens was attracted by tomato root exudates [14].
Similarly, Rhizobium leguminosarum showed strong chemo-
tactic response towards root exudates of Pisum sativum [17].
Furthermore, the result from this study showed that strain
S3-1 was attracted by artificial root exudates (organic acid,
amino acid, and sugars) (Fig. 2d). Among all the detected
components of root exudates, the maximal chemotactic
response was observed in the presence of malic acid and
phenylalanine. The result was similar to previous studies of
malic acid [23], which suggested that malic acid activated
P. polymyxa SQR-21 chemotaxis. In addition, oxalic acid
13
Y. Jin et al.
also significantly induced the S3-1 chemotactic response,
which differed from other reports [5‚ 23]. But Kai Wu et al.
suggested that the population of R. solanacearum attracted
by oxalic acid was significantly higher than in the control,
attributing it to the nutritional and signaling characteristics
[37]. Similar results of fructose and sucrose were reported
in Phytophthora sojae zoospores [31, 36].
Swarming motility was also an essential mechanism of
Bacillus strain in the colonization of roots and rhizosphere
soil. It was found that root exudates of maize significantly
improved the swarming motility relative to the control
(Fig. 3c). Watermelon root exudates also induced Paeniba-
cillus polymyxa SQR-21 swarming motility [23]. Further-
more, we found that sugars, organic acids, and amino acids
also significantly induced the motility of the strain S3-1 and
the maximal inducing ability was obtained with malic acid
(Fig. 3d). The strain S3-1 might utilize these components of
root exudates as energy and nutrients to help colonization.
Citric acid, oxalic acid, and fructose had large effects on
swarming activity of P. corrugata CCR04 [28].
Results from the current study showed that maize root
exudates significantly promoted S3-1 biofilm formation
(Fig. 4a). Exudates of tomato roots strongly stimulated
biofilm formation of B. subtilis, which was dependent on
the sensor histidine kinase KinD [11]. In addition, these
components of root exudates all induced biofilm formation,
especially oxalic acid and proline (Fig. 4b). Among artificial
root exudates, glucose enhanced the biofilm formation by
promoting the growth of Bacillus amyloliquefaciens SQR9
and citric acid stimulated biofilm formation by promoting
growth and inducing the expression of epsD and tapA genes
[35‚ 41]. Therefore, we further investigated the potential
mechanism of biofilm formation.
To further confirm the effects of these root exudate com-
pounds on S3-1, a colonization assay was performed to
investigate the recruitment ability induced by these compo-
nents. Among these compounds, sugars, organic acids, and
amino acids also stimulated an increase in the population
of S3-1 attached on the surface of the maize, especially in
the presence of malic acid and glucose (Fig. 5). Malic acid
and citric acid, acting as chemo-attractants, could stimulate
the recruitment of SQR9 and N11 to the hosts [18‚ 39]. The
result was roughly consistent with the data of chemotac-
tic response and swarming motility but not biofilm forma-
tion. This could indicate that the ability of chemotaxis and
swarming motility were higher than biofilm formation.
This study focused on the effects of root exudates and
their compounds on the rhizosphere colonization of strain
S3-1. And the exudates collected from the roots of maize
seedlings showed obvious chemotaxis, population mobility,
and the ability to form biofilm. In addition, we also found
that malic acid has the strongest chemotaxis and cluster
motility, but proline has the greatest ability to stimulate
Role of Maize Root Exudates in Promotion of Colonization of Bacillus velezensis Strain S3-1…
biofilm formation. Furthermore, these root exudate com-
pounds stimulated the population of S3-1 adhering to the
maize root surface, especially in the presence of malic acid
was confirmed in this article. Therefore, the research can
provide the theoretical evidence that S3-1 exhibits outstand-
ing suppression effects to soil-borne diseases.
Acknowledgements This work was supported by Shanghai Munici-
pal Science and Technology Commission (Grant No. 16391902100),
the Foundation of Key Laboratory of Urban Agriculture (Grant No.
UA201705), Ministry of Agriculture of the People’s Republic of China
and Shanghai Engineering and Technical Research Center of Plant
Germplasm Resources (Grant No. 17DZ2252700), and the National
Key R&D Program of China (Grant No. 2016YFC0502702).
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