Pantoea ananatis, a plant growth stimulating bacterium, and its metabolites isolated from Hydrocotyle umbellata (dollarweed)

Abstract

A bacterium growing on infected leaves of Hydrocotyle umbellata, commonly known as dollarweed, was isolated and identified as Pantoea ananatis. An ethyl acetate extract of tryptic soy broth (TSB) liquid culture filtrate of the bacterium was subjected to silica gel chromatography to isolate bioactive molecules. Indole was isolated as the major compound that gave a distinct, foul odor to the extract, together with phenethyl alcohol, phenol, tryptophol, N-acyl-homoserine lactone, 3-(methylthio)-1-propanol, cyclo(L-pro-L-tyr), and cyclo(dehydroAla-L-Leu). This is the first report of the isolation of cyclo(dehydroAla-L-Leu) from a Pantoea species. Even though tryptophol is an intermediate in the indoleacetic acid (IAA) pathway, we were unable to detect or isolate IAA. We investigated the effect of P. ananatis inoculum on the growth of plants. Treatment of Lemna paucicostata Hegelm plants with 4 × 10⁹ colony forming units of P. ananatis stimulated their growth by ca. five-fold after 13 days. After 13 days of treatment, some control plants were browning, but treated plants were greener and no plants were browning. The growth of both Cucumis sativus (cucumber) and Sorghum bicolor (sorghum) plants was increased by ca. 20 to 40%, depending on the growth parameter and species, when the rhizosphere was treated with the bacterium after germination at the same concentration. Plant growth promotion by Pantoea ananatis could be due to the provision of the IAA precursor indole.

Full text

RESEARCH PAPER
Pantoea ananatis, a plant growth stimulating bacterium, and its metabolites isolated
from Hydrocotyle umbellata (dollarweed)
Kumudini M. Meepagala
a
, Caleb M. Anderson
a,b
, Natascha Techen
c
, and Stephen O. Duke
c
a
United States Department of Agriculture, Agricultural Research Service, Natural Products Utilization Research Unit, University, USA;
b
Department of
Microbiology and Molecular Medicine, University of Geneva, Geneva, Switzerland;
c
National Center for Natural Products Research, School of
Pharmacy, University of Mississippi, University, USA
ABSTRACT
A bacterium growing on infected leaves of Hydrocotyle umbellata, commonly known as dollarweed, was
isolated and identied as Pantoea ananatis. An ethyl acetate extract of tryptic soy broth (TSB) liquid
culture ltrate of the bacterium was subjected to silica gel chromatography to isolate bioactive molecules.
Indole was isolated as the major compound that gave a distinct, foul odor to the extract, together with
phenethyl alcohol, phenol, tryptophol, N-acyl-homoserine lactone, 3-(methylthio)-1-propanol, cyclo
(L-pro-L-tyr), and cyclo(dehydroAla-L-Leu). This is the rst report of the isolation of cyclo(dehydroAla-
L-Leu) from a Pantoea species. Even though tryptophol is an intermediate in the indoleacetic acid (IAA)
pathway, we were unable to detect or isolate IAA. We investigated the eect of P. ananatis inoculum on
the growth of plants. Treatment of Lemna paucicostata Hegelm plants with 4 × 10
9
colony forming units of
P. ananatis stimulated their growth by ca. ve-fold after 13 days. After 13 days of treatment, some control
plants were browning, but treated plants were greener and no plants were browning. The growth of both
Cucumis sativus (cucumber) and Sorghum bicolor (sorghum) plants was increased by ca. 20 to 40%,
depending on the growth parameter and species, when the rhizosphere was treated with the bacterium
after germination at the same concentration. Plant growth promotion by Pantoea ananatis could be due
to the provision of the IAA precursor indole.
ARTICLE HISTORY
Received 30 October 2023
Revised 12 January 2024
Accepted 22 January 2024
KEYWORDS
Pantoea ananatis; indole;
hydrocotyle umbellata; plant
growth stimulant; quorum
sensing
Introduction
Plant-microbe interactions in the rhizosphere are an important
mechanism for overall health and growth of plants, particularly in
crop production. Utilization of plant growth promoting bacteria
(PGPB) and nitrogen-fixing microorganisms are useful and eco-
nomical alternatives to synthetic chemical fertilizers in crop pro-
duction and crop yields. Efforts are underway to improve the
efficacy of PGPB that are available to farmers as alternatives to
expensive and environment-damaging fertilizers.
1,2
Pantoea ananatis is a Gram-negative bacterium of the
Enterobacteriacea family that occurs in plant tissues mostly
as a phytopathogen.
3
Previous studies have established the
taxonomy of this species, placing it in the class
Gammaproteobacteria and family Enterobacteriaceae, under
the diverse genus Pantoea, which contains approximately 20
different species with varying applications and properties.
3–5
It
can co-exist with other microbial communities that cause dis-
ease symptoms, or it can occur as an endophyte without caus-
ing any disease symptoms.
3
P. ananatis spp. has been widely
characterized and explored for its varying metabolites and
pathogenic properties. Pantoea spp. isolates are unique in
that they have been found to infect both plants and animals,
are present in soil and water, and have applications ranging
from therapeutics to biocontrol and bioremediation.
4
Some species of P. ananatis has been demonstrated as both
a plant growth-promoting and -inhibiting species.
6
P. ananatis
has been reported to epiphytically colonize rice and pineapple,
as well as to parasitically colonize multiple crops, including rice
and corn.
5,7–9
Consistent with other species of the Pantoea
genus, P. ananatis was also found to colonize insects such as
tobacco thrips (Frankliniella fusca) and even to cause diseases
in humans, such as corneal infiltration and bacteremia.
10–12
P. ananatis was first isolated in the Philippines in 1928 as
a phytopathogen that caused fruitlet rot in Ananas comosus
(pineapple) and, thus, given its species epithet as ananas and
was originally named as Erwinia ananas.
13
Re-examination of
the taxonomy of Erwinia ananas placed this bacterium in the
genus Pantoea, and it was renamed Pantoea ananas.
7
The
name Pantoea ananas was corrected in 1997 by Truper and
Declari as Pantoea ananatis.
14
P. ananatis causes disease symp-
toms in major crops such as rice, maize, melon, Eucalyptus spp.
and Sudangrass (Sorghum sudanese). It can also exist as an
epiphyte, endophyte, or symbiont. Some strains of Pantoea
have been found to contaminate aviation jet fuel, and this
categorizes it as an “unconventional pathogen” and an inter-
esting microbe to study.
3
A bacterium growing on fungal pathogen-infected leaves of
Hydrocotyle umbellata, commonly known as dollarweed, was
CONTACT Kumudini M. Meepagala kumudini.meepagala@usda.gov United States Department of Agriculture, Agricultural Research Service, Natural Products
Utilization Research Unit, University, MS 38677, USA
PLANT SIGNALING & BEHAVIOR
2024, VOL. 19, NO. 1, e2331894 (9 pages)
https://doi.org/10.1080/15592324.2024.2331894
This work was authored as part of the Contributor’s official duties as an Employee of the United States Government and is therefore a work of the United States Government. In accordance with
17 U.S.C. 105, no copyright protection is available for such works under U.S. Law.
This is an Open Access article that has been identified as being free of known restrictions under copyright law, including all related and neighboring rights (https://creativecommons.org/
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isolated and identified as P.ananatis (Figures 1). This bacter-
ium coexisted in the leaves of H. umbellata with the plant
pathogenic fungus Diaporthe ceratozamiae. In this paper, we
show that this bacterium stimulates the growth of three plant
species and that compounds from this microbe affect quorum
sensing of Chromobacterium violaceum.
Materials and methods
Instrumentation and chemicals
Solvents used were of reagent grade without any purification.
TLC (silica gel thin layer chromatography) was performed on
250-µm, glass-backed plates with GF (Gypsom binder with
Fluoroscent indicator) (Analtech, Newark, DE). I
2
vapor, UV
light (254 and 365 nm), and anisaldehyde spray reagent were
used to detect and visualize spots on TLC plates. Column
chromatography fractionation was carried out using a Biotage
(Biotage Inc., Charlottesville, Virginia) flash chromatography
system with an Isolera pump and a dual wavelength detector
(254 and 280 nm) and using silica gel SNAP flash columns
(particle size 40–65 µm). Melting points were determined
with an Optimelt melting point apparatus (Stanford Research
System, Sunnyvale, California). NMR spectra were recorded on
Bruker NMR spectrometers (Billerica, Massachusetts) operat-
ing at 400 MHz for
1
H NMR and at 100 MHz for
13
C NMR.
Optical rotations were measured using an Autopol IV
Automatic Polarimeter model 589–546 (Rudolph Research
Analytical, Hackettstown, NJ). GC-MS analyses were carried
out using GC/MSD on a 7890GC system coupled to
a DB5975C InertXL MSD (Agilent) equipped with a DB-5
fused silica capillary column) 30 m x 0.25 mm, film thickness
0.25 µm) using the following temperature program: injector
temp at 240°C; column temperature increased from 60–240°C
at 3°C/min and held at 240°C for 5 min. He was used as the
carrier gas, and the injection volume was 1 uL (splitless). High
resolution mass analysis was carried out on JMS-T1000LC
AccuTOF system (JOEL).
Isolation and identication of the bacterium
An infected leaf of Hydrocotyle umbellata (dollarweed) was
placed directly onto a potato dextrose agar (39 g/L; potato
starch (from infusion) 4 g/L, dextrose 20 g/L, agar 15 g/L; BD
Difco
TM
) plate for microbial isolation. A single bacterial colony
was isolated and streaked onto tryptic soy agar (TSA) (40 g/L;
pancreatic digest of casein 15 g/L, papaic digest of soybean 5 g/
L, NaCl 5 g/L, agar 15 g/L; BD Difco
TM
), and 16s rRNA gene
sequencing was used for molecular identification of the bacter-
ium. The Qiagen DNeasy extraction kit (Thermo Fisher
Scientific) was used to prepare a DNA sample for 16s rRNA
amplification and consequent Sanger sequencing that was per-
formed by GeneWiz, LLC (South Plainfield, NJ). The sequence
data obtained from GeneWiz was aligned and referenced
against the NCBI DNA database, and the bacterium was iden-
tified as Pantoea ananatis based on 100% sequence identity to
CP020943.1, KY604955.1, MF407400.1 and other. The same
procedure for isolation was done to a surface-sterilized piece of
leaf (approx.2 cm × 2 cm) (using 5% Clorox™ that contains 6%
sodium hypochlorite), followed by thoroughly rinsing with
sterile deionized water (SDW), and the same bacterium was
isolated.
Cultivation and extraction
An inoculum (250 mL) was prepared in tryptic soy broth
(TSB) (30 g/L; pancreatic digest of casein 17 g/L, papaic
digest of soybean 3 g/L, dextrose 2.5 g/L, sodium chloride 5
g/L, dipotassium phosphate 2.5 g/L; BD Difco
TM
) by addi-
tion of the bacterium grown on tryptic soy agar (TSA) plate
using a sterile loop and this culture was grown for 48 h at
37°C in an orbital shaker (200 rpm). This inoculum was used
to inoculate 30 flasks (2-L Erlenmeyer flasks containing 1 L
of sterile TSB medium in each). Inoculated cultures were
allowed to grow at 37°C in an orbital shaker (200 rpm) for
72 h before extraction of the metabolites. The liquid culture
was centrifuged for 20 min at 3836 g, and the cell free culture
broth was extracted twice with ethyl acetate (2:1; medium:
solvent). The ethyl acetate extract was concentrated in
a rotary evaporator under reduced pressure at 40°C to
about 1 L, and the extract was dried over anhydrous Na
2
SO
4
. The solvent was evaporated at 40°C under reduced
pressure to produce 5.2 g of crude extract as a brownish
(A)
(B)
Figure 1. (A): infected hydrocotyle umbellata (dollarweed) plant with necrotic
lesions on the leaves. (B): colonies of pantoea ananatis grown on tryptic soy agar
plate (BD difco).
e2331894-2 K. M. MEEPAGALA ET AL.

viscous oil with a strong foul smell. This extract (5 g) was
fractionated using flash column chromatography (50 g KP-
SIL, Biotage, Charlotte, NC) with a gradient from 100%
n-hexane to 30% ethyl acetate in n-hexane over 28 column
volumes, then to 100% ethyl acetate over ten column
volumes, and ending with an isocratic hold at 100% ethyl
acetate for five column volumes. The column was subse-
quently washed with five column volumes of 10% methanol
in ethyl acetate. After TLC analysis, similar fractions accord-
ing to chromatographic profile were combined to produce
nine fractions for further analysis and fractionation.
Isolation of metabolites in the ethyl acetate extract of cell
free culture broth
Indole (1)
Fractions 1 and 2 produced a major spot on TLC plates when
eluted with 10% ethyl acetate in hexane that indicated a dark
yellow spot when sprayed with anisaldehyde spray reagent.
These two fractions were combined (2.6 g) and chromatographed
in 25 g Biotage silica column using 0–10% ethyl acetate in hexane
to afford indole as a white crystalline solid (2.2 g, m.p 52–53°C).
The identity was confirmed by NMR and GC-MS.
Phenol (2)
Fraction 3 (1.1 g) was chromatographed in 25 g Biotage silica
column using 0–15% ethyl acetate in hexane to afford phenol as
a crystalline sold (0.820 g). The identity was confirmed by NMR
and GC-MS.
Phenethyl alcohol (3), Tryptophol (4)
Fraction 4 (0.889 g) was chromatographed in 25 g Biotage
silica column using 0–20% ethyl acetate in hexane to
afford phenethyl alcohol as a colorless liquid (0.65 g).
The identity was confirmed by NMR and GC-MS.
Further elution of the column with 30% ethyl acetate in
hexane to afford tryptophol as an off-white solid (2.1 mg).
The identity was confirmed by HRMS and NMR data.
3-(methylthio)-1-propanol (5)
Fraction 5 (0.065 g) was chromatographed in 10 g Biotage silica
column using 0–40% ethyl acetate in hexane to afford
a colorless liquid (0.055 g) with a distinct, foul smell. The
identity was confirmed by NMR and GC-MS.
(R)-3-isobutyl-6-methylenepiperazine-2,5-dione (6)
[cyclo-(dehydroAla-L-Leu)]
(0.133 g) Fraction 6 was chromatographed in 10 g Biotage silica
column using 10–70% ethyl acetate in hexane to afford a white
powder (0.0.048 g) eluted with 50% ethyl acetate in hexane. [α]
D25
=
−163 (c 1.0 × 10
−4
g/mL, MeOH); DART-HRMS positive mode
showed a molecular ion for [M+H]
+
at 183.113352 which corre-
sponds to a molecular formula C
9
H
15
O
2
N
2
. [α]
D
(-) 37.8° (c =
0.01, CHCl
3
);
1
H NMR (400 MHz, DMSO-d
6
) δ 0.86 (6 H, d, J =
6.4, CH
3
-9 and 10), 1.59 (2 H, m, H-7), 1.79 (1 H septet, J = 6.4,
H-7), 3.97 (1 H, m, H-6), 4.77 (1 H, s, H
b
-11), 5.18 (1 H, s, H
a
-11),
8.45 (1 H, br s NH-5), 10.5 (1 H, br s NH-1); 13C NMR (100 MHz,
DMSO-d
6
) δ 22.0 (C-10), 22.6 (C-9), 23.3 (C-8), 43.5(C-7), 53.6
(C-6), 98.9 (C-11), 134.6 (C-3), 158.2, (C-2), 166.4 (C-5). The
identity of the compound was established as (6) by comparison
of NMR data and specific rotation with those reported in the
literature.
15,16
Cyclo(L-Pro-L-Tyr) (7) (maculosin)
Fraction 8 (122 mg) eluted with 80% ethyl acetate in hexane
yielded a white crystalline solid (0.061 g): m.p = 142–146°C; [α]
D25
= −54 (c 0.7, EtOH)
1
H NMR (400 MHz, CDCl
3
) δ 1.86 (1
H, m), 1.98 (1 H, m), 2.31 (1 H, m), 2.81 (1 H, dd, J = 14.4, 9.3),
3.41 (1 H, dd, J = 10.5, 3.96), 3.55 (1 H, dd, J = 8, 3 Hz), 3.63 (2
H, m), 4.07 (1 H, t, J = 7.4), 4.23 (1 H, dd J = 10.5, 2.8), 6.2 (1 H br
Figure 2. Structures of the compounds indole (1), phenol (2), Phenethyl alcohol (3), Tryptophol (4), 3-(methylthio)-1-propanol (5), cyclo-(dehydroAla-L-Leu) (6), Cyclo
(L-Pro-L-Tyr) (7) isolated from cell-free culture broth of pantoea ananatis.
PLANT SIGNALING & BEHAVIOR e2331894-3

s NH), 7.67 (1 H, br s OH), 6.78 (2 H, d, J = 8 Hz), 7.04 (2 H, d, J =
8 Hz);
13
C NMR (100 MHz, CDCl
3
) δ 22.6, 28.5, 36.2, 45.6, 56.5,
59.3, 116.2, 126.8, 130.6, 156.0, 165. 5, 169.9. DART-HRMS posi-
tive mode showed a molecular ion for [M + 1]
+
at 261.123917
which corresponds to a molecular formula C
14
H
17
N
2
O
3
. Based
on NMR data, and also comparison of specific rotation data and
other physical data with those reported in the literature, the
identity of the compound was confirmed as cyclo(L-Pro-
L-Tyr).
15–17
Inoculum preparation for bioassays
Bacterium inoculum for bioassays was prepared by an overnight
culture of P. ananatis. A glycerol freezer stock of P. ananatis (30%
glycerol in TSB) was inoculated onto a TSA plate and allowed to
grow at 37°C for 24 h. This 24-h culture was used to inoculate 1 L
of TSB and then allowed to grow at 37°C in an orbital shaker
(200 rpm) for 24 h. The culture was centrifuged for 15 min at 3836
g, and the pellet was washed three times with SDW before resus-
pending in either filter sterilized Hoagland medium (1.63 g/L;
NH
4
H
2
PO
4
115.03 mg/L, boric acid 2.86 mg/L, calcium nitrate
656.4 mg/L, CuSO
4
.5 H
2
O 0.08 mg/L, Na
2
EDTA .2 H
2
O 3.35
mg/L, FeSO
4
.7 H
2
O 2.5 mg/L, anhydrous MgSO
4
240.76 mg/L,
MgCl
2
.4 H
2
O 1.81 mg/L, MoO
3
0.016 mg/L, KNO
3
606.6 mg/L,
ZnSO
4.
7 H
2
O 0.22 mg/L; Phyto Technology Laboratories, LLC)
for the lemna assay or in SDW for the plant growth assay. The
final inoculum was adjusted to an optical density of 1.0 at 600 nm,
corresponding to 2.0 × 10
9
CFU/mL as determined by dilution
plating.
Bioassays with Lemna paucicostata
Inoculum was prepared as described above in normal strength,
filter sterilized Hoagland media (Phyto Technology Laboratories,
LLC), then serially diluted 1:5 for total of five test concentrations.
The method of Michel et al. was used for this bioassay.
18
Six
replicate wells of each concentration were tested with two duck-
weed plants (with 2 fronds each) per well in 5 mL of inoculum.
The control wells contained only the Hoagland solution. The
growth of L. paucicostata plants were monitored by measuring
change in the area of fronds using a Lemnatec Scanalyzer PL
with LemnaLauncher and Lemna Miner software (LemnaTec
GmbH, Schumanstr 19, 52146 Würselen Germany) at day 0
and at 1, 2, 3 6, 7, 8, 10,12 and 13 days after treatment, and
the percent increase in frond area was plotted against time.
Plant growth assay with cucumber (cucumis sativus) and
sorghum (sorghum bicolor)
Cucumber (60335A, Burpee, Warminster, Pennsylvania) and
sorghum (Pioneer 83P17) seeds were surface sterilized in a 5%
A
B
Figure 3. Duckweed plants 12 days after treatment. A: plants growing in bacter-
ium inoculum in Hoagland solution; B: control plants growing in Hoagland
solution.
Figure 4. Duckweed frond growth assay indicating bacterium inoculum-treated plants exhibit significant growth promotion. Error bars represent ± one standard error of
the mean.
e2331894-4 K. M. MEEPAGALA ET AL.

Clorox™ bleach solution (approx. 0.6% sodium hypochlorite)
for 15 min, then washed thoroughly with SDW. The seeds were
then planted in sterilized soil (Miracle-Gro potting mix) and
after germination (7 days), 50 mL of freshly prepared inoculum
in SDW was added to the rhizosphere of each plant. The
control group received 50 mL of SDW. The plants were
grown for two weeks in a growth chamber at 25°C, with
artificial light (about 120 µmol s
−1
m
−2
average PAR) (photo-
synthetically active radiation) for 16 h per day. All plants
received 20 mL of SDW every two days after the start of the
treatment. Two weeks after the treatment, the average fresh
mass of the shoots and dry mass of the roots were taken.
Quorum sensing inhibition assays with chromobacterium
violaceum ATCC 12,472
A quorum sensing inhibition assay was carried out according
to the protocol described by Chenia.
19
Violacein-producing
Chromobacterium violaceum Bergonzini (ATCC 12,472) was
streaked from a freezer stock onto nutrient agar (23 g/L; beef
extract 3 g/L; peptone 5 g/L; agar 15 g/L; BD Difco
TM
) and
allowed to incubate overnight at 30°C. Overnight culture was
used to inoculate nutrient broth (8 g/L: beef extract 3 g/L;
peptone 5 g/L; BD Difco
TM
) 250 mL and was incubated over-
night at 30°C. Soft agar, 5%, (5 g agar powder in 100 mL SDW
and autoclave at 121°C for 15 min) was heated until fully
dissolved, cooled to approx. 40°C, and 20 mL of soft agar was
mixed with 2 mL of overnight broth culture. This was overlaid
onto a pre-warmed (about 35°C) nutrient agar plate and
allowed to set for 30 min at room temperature. Whatman
paper disks (diameter 6 mm, made from hole puncture) were
loaded with 1 mg, 0.5 mg, and 0.25 mg of crude P. ananatis
ethyl acetate extract in methanol (20 µL) and allowed to dry.
An untreated blank disk and a disk with 0.5 mg cinnamalde-
hyde (Sigma Aldrich) were used as negative and positive con-
trols. The disks were placed onto the soft agar overlay and
incubated for 12 h at 30°C.
Results and discussion
Indole (1) was identified as the major constituent in the crude
ethyl acetate extract, making up over 44% of the crude extract
by mass. From 5 g of crude extract 2.2 g (44% by mass) of
indole was isolated. This compound gives a distinct, foul
odor to the bacterial culture and the ethyl acetate extract of
the culture broth. At low concentrations, indole, as well as the
extract, smelled like jasmine flowers. Phenethyl alcohol (2),
phenol (3), tryptophol (4), 3-(methylthio)-1-propanol (5),
(R)-3-isobutyl-6-methylenepiperazine-2,5-dione (6) and cyclo
(L-Pro-L-Tyr) (7) were also isolated and identified as metabo-
lites present in the ethyl acetate extract (Figure 2). N-hexanoyl-
homoserine lactone (8) was detected as a minor metabolite in
the extract. Compound (8) is a known quorum sensing (QS)
metabolite of a P. ananatis strain (P. ananatis Serrano CCT
6481T [= ATCC 33,244 type strain]) that has been isolated
from pineapple.
20
Other acyl-homoserine lactones (AHLs),
namely N-heptanoyl-homoserine lactone and N-octanoyl-
homoserine lactone, have also been reported from Pantoea
species, but we were not able to detect them in this extract.
21
AHLs are produced by gram-negative bacteria as cell-signaling
molecules. They also act as anti-microbials against other
bacteria.
19
Quorum sensing, particularly in gram-negative bac-
teria, plays an important role in pathogenicity, formation of
biofilm, biosynthesis of exopolysaccharides, cell aggregation,
and extracellular hydrolytic enzyme production.
20,22–24
Bioassays revealed potent plant growth promoting proper-
ties when the bacterium inoculum was tested on L. paucicostata
(duckweed), with more than a several-fold increase in growth
of the treated plants as compared to the controls (Figures 3 and
4). In addition, no necrosis of older plants was observed in the
plants treated with the bacterial inoculum (OD
600 nm
of 1.0),
even after 12 days, and the plants appeared darker green and
healthier compared to the control plants (Figure 3). Frond area
of the plants was over five-fold greater than the control after 13
days when treated with 4 × 10
9
CFU/mL P. ananatis (Figure 4).
The experiment was repeated several times, and the effect of
the bacterium did not become evident until about a week after
inoculation in all experiments. We extracted the treated duck-
weed plants and the liquid medium to see if IAA is involved in
this growth promotion effect, but we were unable to detect the
presence of IAA by TLC and LC-MS (liquid chromatography
coupled mass spectroscopy).
B
A
Figure 5. Effect of bacterial inoculum on the growth of cucumber plants and
sorghum seedlings. 5A: cucumber plants (top) and after washing off the soil-
(bottom). 5B: sorghum plants (top) and after washing off the soil(bottom). Three
plants on the left are the treated plants and the three plants on the right are the
control plants in each figure.
PLANT SIGNALING & BEHAVIOR e2331894-5

P. ananatis was also tested for growth-promoting prop-
erties on Cucumis sativus (cucumber) and Sorghum bicolor
(sorghum) plants. The rhizosphere of these plants was
inoculated after germination with 50 mL of freshly pre-
pared bacterial solution (1.0 × 10
9
CFU/mL), and the
plants were grown for three weeks. The leaves of treated
plants were notably larger than those of control plants
(Figures 5(a,b)). The harvested treated and control sor-
ghum plants had average fresh shoot masses of 4.8 and
3.5 g, respectively, and the dry masses of the roots of
treated and control plants were 305 and 199 mg, respec-
tively (Figure 6(a,b)). The harvested treated and control
cucumber plants had average fresh shoot masses of 6.6
and 5.6 g, respectively, and the dry massess of the roots
of treated and control plants were 202 and 143 mg, respec-
tively (Figure 6(a,b)).
Pantoea species have been reported to produce QS mole-
cules and to also possess antibacterial activity. A QSI (quorum
sensing inhibition) assay was carried out using C. violaceum
ATCC 12,472 to screen for potential quorum sensing inhibitors
Dry mass per plant (mg) Fresh mass per plant (g)
Figure 6. Average shoot fresh mass (A) and root dry mass (B) of cucumber and sorghum plants when treated with an inoculum of P. ananati. error bars represent ± one
standard error of the mean.
e2331894-6 K. M. MEEPAGALA ET AL.

and promoters. Paper disks were loaded with 0.25 mg, 0.5 mg,
and 1 mg of crude ethyl acetate extract (disks C, B and A,
respectively), and 0.5 mg cinnamaldehyde (as a positive con-
trol, QS inhibitor) were used (Figure 7). Inhibition zones
indicating QS inhibition was present around an immediate
circular zone of translucent rings of 4, 2 and 0.5 mm width
for C, B and A, respectively, and a clear inner zone and
a translucent ring for the positive control, indicating antibac-
terial activity and/or quorum sensing inhibition, respectively
(Figure 7). A dark violet zone was also observed for C and B in
a concentration-dependent manner, possibly due to quorum
sensing promotion. Cyclic peptides such as dehydro-
ketopiperazines (6) isolated in this study have also been iso-
lated from Pseudomonas aeruginosa in which they act as signal
ligands in bacterial QS promoters.
16
Quorum sensing is a mechanism by which bacteria can
communicate chemically to change gene expression, often
linked to pathogenicity.
25
Biofilm formation can be mediated
by quorum sensing systems, and the inhibition of this process
could potentially be beneficial for disrupting the pathogenicity
of certain bacterial plant pathogens. The protective and defen-
sive nature of biofilms can significantly decrease the effective-
ness of antibacterial agents, depending on their mechanism of
action. Thus, quorum sensing inhibitors in conjunction with
antimicrobial compounds can be a better alternative in con-
trolling bacterial pathogenicity and infections.
To the best of our knowledge, this is the first report of
isolation of compound 6 from Pantoea species. It has been
isolated from Penicillium sp. F70614, and the marine bac-
terium Vibrio parahaemolyticus present in the toxic mucus
of the boxfish (Ostracion cubicus), Cellulosimicrobium cel-
lulans, and some limnic bacterium strains.
15,26–28
There is
a report that 6 possesses α-glucosidase inhibitory
activity.
26
It is interesting to note that compound 7, also known as
maculosin, has been isolated from Alternaria alternata and has
been shown to be phytotoxic to spotted knapweed (Centaurea
maculosa), a noxious weed in the northwestern region of the
USA.
29
This compound has not been reported from Pantoea
species. Despite the presence of phytotoxic compound 7 in the
culture, the bacterium inoculum has shown plant growth sti-
mulating activity. In the hands of one of us (SOD) decades ago,
maculosin was a weak phytotoxin (unpublished data). Also,
Stierle et al.
29
reported it to be a host-specific phytotoxin, so it
may not be active on the three species that we tested in this
paper.
Effects of different P. ananatis strains can range from patho-
genic, to no effect, to growth promotion.
30
Several labs have
reported isolates of P. anantis to promote the growth of
plants,
3,31,32
but we have found little effort to connect this
phenomenon to the production of growth-promoting com-
pounds. The growth promotion of plant shoots found on
Arabidopsis thaliana of an isolate of P. anantis from sugarcane
was similar to our results with cucumber and sorghum,
although they found no promotion of root growth.
31
In this
paper, the authors did a cursory evaluation of some of these
common primary metabolites (e.g., amino acids and sugars) of
this strain. Kim et al.,
32
found a P. anantis strain isolated from
the rhizosphere of green onion to promote the growth of
cucumber, pepper, and tomato plants. The promotion of
plant fresh mass was greatest in cucumber (21%), which is
similar to what we found (Figure 7(a)). The isolate of Kim
et al.
33
also increased pepper yields and prevented pathogen
infections by Erwina carotovora in cabbage, carrot, and onion.
Such an affect could have been promoted by the inhibition of
quorum sensing. A nonspecific colorimetric test suggested that
this strain produces IAA. Others have made similar claims
about IAA production by P. ananatis based on colorimetric
assays.
33
This may be due to the colorimetric assays also being
positive for indole (1) and tryptophol (4). Indole produced by
P. ananatis as an endophyte could be used by the plant as an
IAA precursor, possibly contributing to growth stimulation.
Kim et al.
34
reported that there are genes in this same growth-
promoting isolate of P. anantis, but the specific genes were not
clearly identified in the publication.
The modes of action of the metabolites present in the
bacterium that are responsible for plant growth stimula-
tion are not known. Leaves of dollar weed plants were
inoculated with 20 µL of P. ananatis inoculum, but no
disease symptoms could be observed. Since this bacterium
coexisted with a fungus in the Diaporthe species, it is
important to co-culture and co-inoculate the plants to
investigate phytopathogenic properties and to investigate
the secondary metabolite profile of the co-culture. Future
studies will be focused on gene expression of both plants
and the bacterium when they are in a mutualistic envir-
onment to understand the mode of action for plant
growth stimulation.
Acknowledgments
The authors express their gratitude to Robert Johnson, John Andrew
Mulkey and Gloria Hervey for technical assistance. Partial funding was
provided by “Discovery & Development of Natural Products for
Pharmaceutical & Agrichemical Applications” funded by the United
A
C
B
Positive control
negative control
Figure 7. Quorum sensing and antibacterial effect of bacterial extract. Paper disks
were loaded with 0.25 mg, 0.5 mg, and 1 mg of crude ethyl acetate extract (disks
C, B and A respectively), and 0.5 mg cinnamaldehyde as a positive control.
PLANT SIGNALING & BEHAVIOR e2331894-7

States Department of Agriculture, Agricultural Research Service, Specific
Cooperative Agreement No. 58-6060-6-015 to the National Center for
Natural Products Research, University of Mississippi.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
The work was supported by the Agricultural Research Service [58-6060-
6-015].
ORCID
Stephen O. Duke http://orcid.org/0000-0001-7210-5168
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