Journal of Plant Biology (2020) 63:63–71

https://doi.org/10.1007/s12374-020-09236-8

RESEARCH ARTICLE

Enhanced Drought and Salt Stress Tolerance in Arabidopsis

by Flavobacterium crocinum ­HYN0056T
Jeong‑eun Kim1 · Og‑Geum Woo1,2 · Yoowon Bae1 · Hye Lim Keum3 · Sunglan Chung4 · Woo Jun Sul3 ·
Jae‑Hoon Lee1 

Received: 21 November 2019 / Revised: 26 December 2019 / Accepted: 3 January 2020 / Published online: 21 February 2020
© Korean Society of Plant Biologists and Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
Plant growth-promoting bacteria (PGPB) are indigenous to the plant rhizosphere and largely affect many events occur-
ring during the plant life cycle, through either a direct or an indirect mechanism such as regulation of hormonal balance,
facilitation of nutrients uptake and improvement of stress tolerance. Since drought stress, a representative abiotic stress,
is one of the main reasons limiting plant growth, the identification of useful PGPB involved in drought stress resistance in
plants and its application into the agricultural field could be utilized as a strategy to facilitate crop productivity. To obtain a
useful PGPB that is involved in drought stress resistance, we checked the expression patterns of drought-inducible marker
genes such as RD29A and RAB18 in Arabidopsis after application of 16 Flavobateria obtained from various environmental
sources. After screening, a PGPB known as Flavobacterium crocinum ­HYN0056T, which contributes to more than twofold
upregulation of drought-inducible marker genes, was finally selected for this study. Application of H ­ YN0056T enhanced the
tolerance against both drought stress, possibly via induction of stomatal closure, the highly related salt stress, in the pres-
ence of ­HYN0056T. Moreover, treatment of H ­ YN0056T under drought and salt stresses resulted in enhanced upregulation
of various drought- and salt-inducible genes in Arabidopsis. ­HYN0056T was responsible for the development of lateral roots
under nonstress condition, implying that it may be involved in effective uptake of water/inorganic nutrients. Based on these
results, we suggest that this bacterium could be used as a useful biocontrol agent to improve plant productivity, especially
under drought/salt stress conditions.

Keywords  Flavobacterium crocinum ­HYN0056T · PGPB · Drought stress · Salt stress · Arabidopsis

Introduction

PGPB largely affect plant growth via both direct and indirect
Electronic supplementary material  The online version of this mechanisms (Backer et al. 2018). PGPB directly improve
article (https​://doi.org/10.1007/s1237​4-020-09236​-8) contains plant growth by regulating the level of various phytohor-
supplementary material, which is available to authorized users.
mones, increasing siderophore production and enhancing
* Woo Jun Sul nutrient availability via nitrogen fixation and phosphorus/
sulwj@cau.ac.kr iron solubilization (Glick 2012; Vejan et al. 2016). Moreo-
* Jae‑Hoon Lee ver, enhanced tolerances to biotic and abiotic stresses of
jhlee72@pusan.ac.kr plants by PGPB indirectly promote plant growth (Vejan et al.
2016). The beneficial role of PGPB for plants is potentially
1
Department of Biology Education, Pusan National achieved by production of phytohormones such as IAA,
University, Busan 46241, Korea
cytokinin and abscisic acid (ABA), production of bacterial
2
Department of Integrated Biological Science, Pusan National exopolysaccharides and ACC deaminase, and regulation of
University, Busan 46241, Korea
the level of various organic compounds such as proline, free
3
Department of Systems Biotechnology, Chung-Ang amino acid and sugar (Kumar and Verma 2018).
University, Anseong 17546, Korea
Plants consistently encounter various abiotic stresses
4
Underwood International College, Yonsei University, such as drought, salt, heat, cold, UV-B and heavy metals
Seoul 03722, Korea

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Vol.:(0123456789)

64
throughout their life cycles, and these stresses adversely
influence plant development, growth and productivity (Zhu
2016). Among these stresses, especially water-deficit stress
resulting from drought and high salinity is a major factor
that decreases crop productivity on irrigated lands (Cushman
and Bohnert 2000). By 2050, both stresses are projected to
adversely affect crop productivity on more than 50% of all
arable lands (Vinocur and Altman 2005). Therefore, under-
standing the detailed process of plant response to such abi-
otic stresses is essential to ameliorate their adverse effects
on the growth and productivity of useful crops. Properly
addressing this issue has rapidly raised the potential value to
be gained by applying PGPB, which contribute to resistance
against the stresses, into agricultural fields.
Several studies have investigated the PGPB involved in
tolerance to drought and salt stresses in diverse plant spe-
cies (Kumar and Verma 2018). Burkholderia phytofirmans
PsJN and PGPB strain IG 3 (Klebsiella sp.) improved wheat
growth by alleviating harmful effects under drought condi-
tions (Naveed et al. 2014; Gontia-Mishra et al. 2016). Four
PGPB strains (including Pseudomonas fluorescens, Entero-
bacter hormaechei, and Pseudomonas migulae) isolated
from foxtail millet (Setaria italica L.), a drought-tolerant
crop, positively regulated seed germination and seedling
growth under drought stress conditions, possibly by pro-
ducing a high level of exopolysaccharide (Niu et al. 2018).
Furthermore, inoculation of several drought-tolerant Bacil-
lus spp. mitigated negative effects from drought stress, by
reducing the activity of various antioxidant enzymes in
maize (Vardharajula et al. 2011). Inoculation of Azospiril-
lum brasilense Sp 245 also ameliorated the drought toler-
ance of Arabidopsis and this response was correlated with
an increased level of ABA in Arabidopsis by the microbial
treatment (Cohen et al. 2015). On the other hand, Pseu-
domonas putida N21 and three Pseudomonas (MK1, Mk20
and Mk25) improved plant growth under salt stress condi-
tion in wheat and mungbean, respectively, via their ACC
deaminase activities to lower ethylene level (Zahir et al.
2009; Ahmad et al. 2011). In addition, Bacillus amylolique-
faciens NBRISN13 conferred salt stress tolerance through
modulation of the gene expression profiles in rice, and
Hartmannibacter diazotrophicus ­E19T positively regulated
the growth of barley under salt stress condition via its ACC
deaminase production (Nautiyal et al. 2013; Suarez et al.
2015). Although several studies have shown that PGPB from
diverse genera are widely involved in the responses of host
plants exposed to drought and salt stresses (Vurukonda et al.
2016; Ilangumaran and Smith 2017), the reports on the role
of the genus Flavobacterium (a genus of gram-negative and
rod-shaped bacteria), which has been noted as one of PGPB,
for plant responses to drought and salt stresses have been
limited (Gontia-Mishra et al. 2016).
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Journal of Plant Biology (2020) 63:63–71
In this study, we have found a PGPB, Flavobacterium
crocinum ­HYN0056T, whose application enhanced the tol-
erance against drought and salt stresses in Arabidopsis. Its
treatment also resulted in upregulation of various drought-
and salt-inducible genes in Arabidopsis. As well as improv-
ing the tolerance to drought and salt stresses, F. crocinum
­HYN0056T also positively regulated the developmental pro-
cess of lateral roots. Collectively, these findings show that
F. crocinum ­HYN0056T, investigated herein, is an effective
PGPB for improving plant growth under unfavorable envi-
ronmental conditions, and is therefore a promising candidate
as a useful biological resource for agricultural applications
into crop plants.
Results and Discussion
Identification of a Flavobacterium that Upregulates
Drought‑Inducible Genes in Arabidopsis
To identify a Flavobacterium strain that contributes to the
tolerance of plants to drought stress, we used 16 Flavoba-
teria obtained from various biological resources (Table S1)
(Shin et al. 2014, 2017; Choi et al. 2017; Baek et al. 2018).
For screening the related PGPB, microbes were appli-
cated into the soil around the growth area of Arabidopsis
as a model plant and the transcript levels of drought stress
marker genes such as RD29A and RAB18 in the resulting
Arabidopsis were checked. During the screening process,
among candidate microbes whose application resulted in
upregulation of the drought-responsive genes by more than
two times under nonstress conditions, one stain Flavobac-
terium crocinum ­HYN0056T (KY077156), which has been
reported by Baek et al. (2018), was selected for this study.
Strain ­HYN0056T Improves the Drought Stress
Tolerance of Arabidopsis
­ YN0056T triggered upregu-
Since the inoculation of strain H
lation of the drought marker genes, we next checked whether
this bacterium could alter the sensitivity in response to
drought stress. As shown in Fig. 1a, the survival rate of
mock-treated plants after drought stress was 10%, while that
of ­HYN0056T-treated plants was 52%, indicating that the
application of ­HYN0056T increases the drought stress toler-
ance of Arabidopsis. Since the sensitivity to drought stress
is highly related to the stomatal aperture of plant leaves in
many cases, the stomatal aperture of Arabidopsis followed
by mock or H ­ YN0056T treatments was investigated. The
stomatal aperture of ­HYN0056T-treated plants was only
about 42% of that of mock-treated samples (Fig. 1b). Taken
together, these data show that strain H ­ YN0056T could
Journal of Plant Biology (2020) 63:63–71 65
Fig. 1  Enhanced drought and salt tolerance of Arabidopsis
by strain ­ HYN0056T application. a Increased survival rate of
­HYN0056T-treated Arabidopsis under drought stress condition.
Values indicate the means ± SD (n = 3). A Student’s t test was con-
ducted to identify significant differences between mock-treated and
­HYN0056T-treated samples; **P < 0.01. b Enhanced stomatal clo-
sure of H ­ YN0056T-treated Arabidopsis plants. The apertures of at
improve the drought stress tolerance of Arabidopsis, pos-
sibly through induction of stomatal closure.
Strain ­HYN0056T Improves the Salt Stress Tolerance
of Arabidopsis
The drought- and salt stress-signaling pathways largely
share the protein components that are used to overcome
such stresses (Lee and Kim 2011). Since strain H ­ YN0056T
altered the sensitivity to drought stress in Arabidopsis, we
investigated if the tolerance against salt stress could be
­ YN0056T in the plant. Figure 1c shows that the
altered by H
survival rate of Arabidopsis co-incubated with ­HYN0056T
was 1.8 times higher than that of the mock-treated one in the
presence of salt stress, indicating that ­HYN0056T is able to
enhance tolerance against salt stress in Arabidopsis. Appli-
cation of drought and salt stresses into plants commonly
triggers accumulation of endogenous ABA, and the result-
ing elevated ABA level is largely responsible for transduc-
ing both stress signalings into downstream processes that
display the stress tolerance response (Agarwal et al. 2006).
Therefore, the biological role of strain ­HYN0056T to endow
drought and salt stress tolerance may be involved in the
increase of ABA accumulation in plants and/or alteration of
plant sensitivity in response to ABA.
least 117 stomata for each sample were measured in three independ-
ent experiments. The average value of stomatal apertures from mock-
treated samples was considered to be 1.0. c Enhanced salt tolerance
of Arabidopsis by ­HYN0056T application. The images were retrieved
12 days after the final salt treatment. Values indicate the means ± SD
(n = 3). *P < 0.05
Strain ­HYN0056T Did Not Affect the Sensitivity
of Arabidopsis to UV‑B and Heavy Metal Stresses
Several reports show that a diverse range of abiotic stress
signalings could crosstalk through mediation of phyto-
hormones and converge at a certain point in the stress
signaling networks (Larkindale et al. 2005; Fujita et al.
2006; Bandurska et al. 2013). Moreover, the application of
Paenibacillus yonginensis ­DCY84T as PGPB into a plant
commonly enhances its tolerance against a diverse range
of abiotic stresses such as salt, drought and heavy metal
stresses (Sukweenadhi et al. 2015). These findings raise a
possibility that strain ­HYN0056T might be involved in the
tolerant process against a wide range of abiotic stresses.
For UV-B or heavy metal stress treatments, mock- or
­H YN0056 T-treated Arabidopsis plants were exposed to
UV-B light of 20  μmol  m −2  s −1 irradiance or 250  mM
­Al2(SO4)3, respectively, and their phenotypes were moni-
tored. As shown in Fig. 2a, b, UV-B and aluminum (heavy
metal) stress challenges did not lead to phenotypic change
between ­HYN0056T- and mock-treated samples, indicat-
ing that it did not alter the sensitivity of Arabidopsis to
those stresses.
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66
Fig. 2  Comparison of UV-B (a) and heavy metal (b) stress responses between mock-treated and ­HYN0056T-treated Arabidopsis plants. The
images were taken 3 days after UV-B treatments and 7 days after the final treatment of heavy metal
Application of Strain ­HYN0056T Under Drought
and Salt Stress Leads to Enhanced Upregulation
of Various Drought‑ and Salt‑Inducible Genes
in Arabidopsis
To check if drought- and salt-stress tolerant phenotypes of
­ YN0056T are
Arabidopsis induced by application of strain H
functionally related to the expression of the stress-related
Fig. 3  Enhanced upregulation of several drought- and salt-respon-
sive genes in strain H­ YN0056T-treated Arabidopsis plants, under
drought and salt stress conditions. a Expression patterns of various
drought-responsive genes in the absence or presence of H ­ YN0056T,
under (−) or ( +) drought treatment. Eleven-day-old Arabidopsis
plants were treated with mock or ­HYN0056T. After 14  days, Arabi-
dopsis plants were kept in the air until loss of approximately 20%
of the total fresh weight or without drought stress. The expression
levels of various genes were normalized using the expression levels
of ACTIN2. The average value of each gene expression from mock-
treated samples under (−) drought condition was considered to be
1.0. Values represent means ± SD (n ≥ 4). A Student’s t test was con-
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Journal of Plant Biology (2020) 63:63–71
genes, the expression patterns of various drought- and salt-
responsive genes were monitored in the absence or presence
of drought/salt stresses. As shown in Fig. 3a, ­HYN0056T
enhanced the upregulation of several drought-inducible
genes such as RD29A, RAB18, ADH1 and NCED3 under
drought stress condition, correlating with enhanced drought
tolerance by ­HYN0056T. Since the drought-inducible genes
used in Fig. 3a have also been known as ABA-inducible
ducted to identify significant differences between mock-treated and
­HYN0056T-treated samples; *P < 0.05; **P < 0.01; ***P < 0.001. b
Expression patterns of various salt-responsive genes in the absence
or presence of H­ YN0056T, under (−) or ( +) salt treatment. Eleven-
day-old Arabidopsis plants were treated with mock or ­HYN0056T.
From 1 week after inoculation with bacteria, the plants in a pot were
exposed to 50  ml of either a 250  mM NaCl solution or water, two
times at a 3-day interval over the following 3 days. The samples were
harvested 3 days after the final treatment. The average value of each
gene expression from mock-treated samples under (−) salt condi-
tion was considered to be 1.0. Values represent means ± SD (n ≥ 6).
**P < 0.01; ***P < 0.001
Journal of Plant Biology (2020) 63:63–71 67
genes, the enhanced tolerance could be mediated through
ABA-dependent process. This explanation is supported by
the data that ­HYN0056T enhances the ABA-induced sto-
matal closure (Fig. 1b). Moreover, it triggered upregulation
of NCED3, a representative ABA biosynthesis gene (Tan
et al. 2003), in the presence of drought stress, showing that
its role for drought tolerance is connected to the increase of
endogenous ABA level in Arabidopsis (Fig. 3a). Consistent
with the data that strain ­HYN0056T contributes to the salt-
stress tolerance of Arabidopsis, its application also led to
the hyper-induction of several salt-inducible genes such as
RD29A, RD17, RAB18 and WRKY8 under salt stress condi-
tion (Fig. 3b).
Strain ­HYN0056T Treatment Leads to an Increase
in the Number of Lateral Roots in Arabidopsis
Since several studies have suggested that PGPB strains that
endow plants with tolerance to abiotic stresses could also
be involved in the regulation of plant growth, we firstly
Fig. 4  Growth pattern of Arabidopsis by strain ­ HYN0056T appli-
cation. a Germination pattern of control and H ­ YN0056T-treated
Arabidopsis plants. Seeds were germinated on MS plates in the
absence or presence of ­HYN0056T, and then further grown for the
indicated times. Germination rates were determined with an aver-
age of ≥ 100 seeds from each of three replicates. Values are rep-
resented as means ± SD (n = 3). b Comparison of fresh weights of
shoots and roots between control and H ­ YN0056T-treated Arabidop-
sis plants. Six-day-old seedlings grown on MS plates were trans-
ferred on MS plates in the absence or presence of H ­ YN0056T, and
then further grown for 12 days. The average value of shoot/root fresh
investigated whether strain ­HYN0056T affects the growth
pattern of Arabidopsis under nonstress conditions. Although
­HYN0056T triggered the increased tolerance to drought
stress mediated by ABA, its application did not alter the
germination pattern, which is also regulated by ABA, under
normal growth condition (Fig. 4a). As shown in Figs. 3a and
S1, ­HYN0056T induced the upregulation of ABA-responsive
genes such as RD29A and RAB18, but not NCED3, AAO3,
ABA1, ABA2 and ABA3 as ABA biosynthesis genes in the
absence of drought stress, implying that the strain may be
involved in the enhancement of ABA sensitivity, and not
in the accumulation of ABA under nonstress conditions.
Moreover, a part of downstream signaling components par-
ticipating in the ABA-mediated germination process in ABA
signaling are different from those in the ABA-mediated
drought response, indicating that the two processes are both
partially overlapping and partially distinct from each other
(Finkelstein et al. 2005; Kim 2006; Yoshida et al. 2010;
Kim et al. 2016). Based on these results, we cannot exclude
the possibility that the enhanced ABA-sensitivity triggered
weights from control plants was considered to be 1.0. Values indicate
the means ± SD (n = 7). **P < 0.01. c Comparison of primary root
lengths between control and H ­ YN0056T-treated Arabidopsis plants.
Five-day-old seedlings grown on MS plates were transferred on MS
plates in the absence or presence of ­HYN0056T, and then further
grown for the indicated times. Values indicate the means ± SD (n ≥ 3).
d Increased number of lateral roots in ­HYN0056T-treated Arabidopsis
plants. Five-day-old seedlings grown on MS plates were transferred
on MS plates in the absence or presence of H­ YN0056T, and then fur-
ther grown for the indicated times. Values indicate the means ± SD
(n ≥ 4). **P < 0.01
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68
by ­HYN0056T in the absence of drought stress may not be
effective or sufficient for altering the germination process.
Although ­HYN0056T did not affect the growth of aerial
parts, its application slightly increased the root fresh weight
(Fig. 4b). Furthermore, the microbial application increased
the number of lateral roots, but did not increase the primary
root length (Fig. 4c, d). Since the primary root lengths were
comparable between control and ­HYN0056T-treated plants,
the increased root fresh weight mediated by H ­ YN0056T
may have been caused by the increased number of lateral
roots, at least in part (Fig. 4c, d). A diverse range of PGPB
strains have been implicated in root development and growth
(Zamioudis et al. 2013; Zhao et al. 2016; Zhou et al. 2016;
Lu et al. 2018). Furthermore, it has been suggested that root
weight is increased under drought stress condition, and that
drought resistance varieties display improved root develop-
ment relatively to susceptible varieties, which supports the
connection between drought stress response and root devel-
opment (Naseem et al. 2018). Similar with the results from
F. crocinum ­HYN0056T, Bacillus megaterium BOFC15,
Bacillus amyloliquefaciens FZB42 and Azospirillum bra-
silense Sp 245 increase the tolerance to drought stress and
root growth, including lateral root development (Cohen et al.
2015; Zhou et al. 2016; Lu et al. 2018). These results imply
that increased lateral root formation by ­HYN0056T may
be correlated to improved tolerance against drought stress.
Enhanced lateral root development induced by H ­ YN0056T is
expected to improve the assimilation of water and inorganic
nutrients, which might help to properly cope with an unfa-
vorable environment from drought and salt stress conditions.
The initiation and development of lateral roots are triggered
by auxin (Boerjan et al. 1995), and the growth of lateral root
by PGPB could be regulated via auxin produced by PGPB
and/or plant auxin level altered by PGPB (Dodd et al. 2010).
Based on these previous findings, the possible involvement
of auxin in the enhanced lateral root development displayed
in ­HYN0056T-treated Arabidopsis will be investigated in
our upcoming study.
Identification of Secondary Metabolite BGCs
in Strain ­HYN0056T
Genomes of strain ­HYN0056T have a potential to produce
secondary metabolites, including arylpolyene/resorcinol,
betalactone, lanthipeptides, NRPS/T1PKS, and terpenes.
Among those, carotenoid, flexirubin, and colanic acid was
assigned to the terpene cluster type, arylpolyene/resorcinol
cluster type, NRPS & T1PKS cluster type, respectively
against MIBiG (Minimum Information about a Biosyn-
­ YN0056T genome
thetic Gene cluster) database. The strain H
possessed 55 genes that had 94% genes similarity to the
flexirubin cluster. The following four genes were predicted
to be core biosynthetic genes: one acyl-carrier-protein,
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Journal of Plant Biology (2020) 63:63–71
two beta-ketoacyl synthase, and one StlD/DarB family
beta-ketosynthase.
Flexirubin is the yellow-orange pigment produced by bac-
teria from Bacteroidetes phylum, especially, genera Flexi-
bacter, Flavobacterium, Cytophaga. Because of a limited
distribution among bacteria, flexirubin has acted as a chemo-
taxonomic marker for the bacteria of the Bacteroidetes phy-
lum (Schöner et al. 2014; Fautz et al. 1979). Several studies
have reported on the biological activity of this pigment, such
as antimicrobial activities and antioxidant activities (Rad-
hakrishnan et al. 2016; Umadevi et al. 2013). By controlling
other bacteria in the soil or reducing stress, flexirubin, as the
bacterial secondary metabolite, may be involved in plant
growth and thus is worthy of further experimental studies
(Kumar et al. 2015).
Concluding Remarks
In this study, we found that strain H ­ YN0056T is responsi-
ble for enhancing tolerance of plant to abiotic stresses such
as drought and salt. In spite of its marked contribution to
resistance against drought and salt stresses in Arabidop-
sis, the detailed action mechanism of H ­ YN0056T for the
related response of the plant is still limited. The interaction
of PGPB with host plants affects a variety of plant processes
covering nutrient uptake, phytohormone levels and meta-
bolic processes in plants. Therefore, monitoring alteration
at the levels of gene expression and protein accumulation in
Arabidopsis by the application of H ­ YN0056T could provide
a proper answer to further understand its detailed role as a
PGPR. To utilize a selected PGPR as a useful biological
resource in agricultural fields, our further study will focus on
elucidation of the possibility that ­HYN0056T affects growth,
productivity and abiotic stress tolerance of useful crops.
Several studies that PGPB that positively affect growth and
stress tolerance in Arabidopsis also endow the similar effects
to other crops raise a possibility that the biological role of
­HYN0056T could be applied to crop plants (Zhu et al. 2015;
Sukweenadhi et al. 2015, 2018; Trinh et al. 2018). Bras-
sica crop, a plant genus to be most closely related to Arabi-
dopsis, could be a suitable target to check the possibility
of ­HYN0056T as a suitable biological tool for agricultural
applications.
Materials and Methods
Plant Materials and Growth Conditions
Arabidopsis thaliana ecotype Col-0 was used for this study.
The seeds were surface-sterilized with 75% ethanol and
0.1% Triton X-100 for 5 min, stored at 4 °C for 3 days for
Journal of Plant Biology (2020) 63:63–71 69
stratification, and then grown on 1 × Murashige and Skoog
(MS) agar medium including 1% sucrose and 0.8% bactoagar
(pH 5.8) under long-day condition (16 h light/8 h dark) in a
controlled-environment chamber at 23 °C. For the stress tol-
erance assays, analysis of stomatal apertures and qRT-PCR
analysis, the seedlings grown on MS plates for 10 days were
moved into a pot filled with horticultural soil mix (cocopeat
51.5%, peat moss 10%, vermiculite 13%, perlite 15%, zeolite
10%, humic acid 0.1%, fertilizer 0.4%) and further grown
under long-day condition at 23 °C.
Bacterial Culture and Application into the Plants
T
Strain ­HYN0056 was grown on R2A agar media over-
night at 28 °C. After collection, the cultured ­HYN0056T
were diluted to an ­OD600 of 0.04 (stress tolerance assays and
qRT-PCR analysis) or 1.0 (shoot/root growth analysis and
germination assay) with MS liquid. The resulting bacterial
diluent was supplied into MS plate or soil around Arabidop-
sis growth area.
Abiotic Stress Treatments
Eleven-day-old Arabidopsis plants were initially treated
with mock or strain H ­ YN0056T, and then used for further
treatment of various abiotic stresses. For drought stress, two
weeks after H­ YN0056T treatment, the plants were kept with-
out irrigation for a further 10 days, followed by re-watering
for 2 days. For salt stress, from 1 week after inoculation
with the bacteria, each plant in a pot was exposed to 50 ml
of either a 250 mM NaCl solution or water, three times at
3-day intervals over the following 6 days. For UV-B stress,
after 13 days of the bacterial inoculation, the plants were
treated with UV-B light of 20 μmol m−2 s−1 irradiance for
the indicated times. For heavy metal stress, 11-day-old
plants treated with H ­ YN0056T were exposed to 250 mM
­Al2(SO4)3 solutions, twice (at 14th and 21st days after bac-
teria inoculation).
Stomatal Aperture Analysis
Detached leaves from 25-day-old Arabidopsis plants were
­ YN0056T ­(OD600 = 0.3) for 2 h,
treated with mock or strain H
and the stomatal apertures from epidermal peels of the sam-
ples were determined using a microscope (DM750, Leica).
RNA Isolation and RT‑qPCR Analysis
Total RNA was isolated from Arabidopsis plants using
Trizol reagent (Ambion) according to the manufacturer’s
instructions. After DNase I (Promega) treatment, cDNA
was synthesized from 1 µg of RNA and using RevertAid™
Reverse Transcriptase (Fermentase). RT-qPCR analyses
were performed as previously described (Kim et al. 2016).
They were conducted with the Solg™ 2 × real-time PCR
Smart Mix (SolGent) according to the manufacturer’s pro-
tocol and the Rotor-Gene Q system (Qiagen). Relative gene
expression fold was obtained by the delta–delta Ct method.
Data were normalized to an internal control gene, ACTIN2.
The information of primer sequences for the analysis is
shown in Table S2.
Secondary Metabolite Biosynthetic Gene Clusters
(BGCs) Analysis of Strain ­HYN0056T
The whole-genome sequence of strain ­HYN0056 T was
obtained from the RefSeq database (O’Leary et al. 2015)
under accession number CP029255.1. To predict the sec-
ondary metabolite BGCs of H­ YN0056T, the antibiotics, and
secondary metabolite analysis shell, antiSMASH v5.0 (Blin
et al. 2019) was conducted using standard parameters.
Statistical Analysis
Statistical calculations were performed using GraphPad
QuickCalcs calculator (https​://www.graph​pad.com/quick​
calcs​/).
Acknowledgements  We thank Dr. Hana Yi for kindly providing ten
Flavobaterium strains ­(EM1308T, ­EM1321T, ­HYN0048T, ­HYN0049T,
­HYN0056T, ­HYN0059T, HYN0086, LPB0074, ­LPB0076T, HYN0034).
This work was supported by the National Research Foundation of
Korea (NRF) grant funded by the Korea government (MSIT) (no.
2019R1F1A1041226), and by the Strategic Initiative for Microbiomes
in Agriculture and Food, Ministry of Agriculture, Food and Rural
Affairs, Republic of Korea (916007021HD040).
Author Contributions  WJS and JHL conceived the study. WJS and
JHL designed the experiments and analyzed the data. JK, OGW, YB
and HLK carried out the experimental work and analyzed the data.
OGW and JHL revised the manuscript. JHL wrote the manuscript with
contributions of WJS and SC.
Compliance with Ethical Standards 
Conflict of interest  All authors have approved the manuscript and de-
clare no conflict of interests.
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