Journal of Applied Microbiology, 2023, 134, 1–14

https://doi.org/10.1093/jambio/lxad036
Advance access publication date: 25 February 2023
Research Article

Biostimulation of Salicornia europaea L. crops with plant

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growth-promoting bacteria in laboratory and field
conditions: effects on growth and metabolite profile
Maria J. Ferreira1 , I. Natalia Sierra-Garcia1 , Javier Cremades2 , Carla António3 , Ana M. Rodrigues3 ,
Diana C.G.A. Pinto4 , Helena Silva1 , Ângela Cunha1,*
1
CESAM & Department of Biology, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal
2
Centre for Advanced Scientific Research (CICA), University of A Coruna, 15071 A Coruna, Spain
3
Plant Metabolomics Lab Portugal, Forest Research Centre (CEF), School of Agriculture University Lisbon (ISA/ULisbon), Tapada da Ajuda,
1349-017 Lisboa, Portugal
4
LAQV-REQUIMTE & Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal
∗
Corresponding author. Department of Biology, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal. E-mail: acunha@ua.pt

Abstract
Aim: The objective of the work was to assess the effect of biostimulation with selected plant growth-promoting bacteria on growth and metabo-
lite profile of Salicornia europaea.
Methods and results: Salicornia europaea seeds were inoculated with different combinations of plant growth-promoting bacteria Brevibacterium
casei EB3, Pseudomonas oryzihabitans RL18, and Bacillus aryabhattai SP20. Plants germinated from inoculated seeds were grown either in
laboratory conditions or in a saline crop field. Fresh and dry weight were determined at the end of the experiment, for biomass quantification.
The microbiological quality of fresh shoots for human consumption as salad greens was assessed, and the persistence of the inoculated strains
in the plant rhizosphere was confirmed by next-generation sequencing (Illumina) of the 16S rDNA gene. The primary metabolite profile of
biostimulated plants was characterized by GC–TOF-MS.
In laboratory conditions, inoculation with the two strains Br. casei EB3 and Ps. oryzihabitans RL18 caused the most significant increase in biomass
production (fresh and dry weight), and caused a shift in the central metabolic pathways of inoculated plants toward amino acid biosynthesis.
In the field experiment, no significant biostimulation effect was detected with any of the tested inoculants. Seed inoculation had no significant
effect on the microbiological quality of the edible parts. The persistence of inoculants was confirmed in both experiments.
Conclusions: Manipulation of the plant microbiome can trigger primary metabolic reconfiguration and modulate the plant metabolism while
promoting plant growth.

Significance and impact of the study

Rhizosphere engineering is a powerful tool to manipulate plant growth and the metabolite composition of plants. However, the outcome of
biostimulation of crops in field conditions can be very different from that observed in laboratory trials. Understanding the factors that hinder the
successful scaling up of biostimulation assays to field conditions, is a critical step toward the widespread application of biostimulants in saline
agriculture.
Keywords: biostimulants, halophytes, PGPB, primary metabolites, rhizosphere engineering

Introduction has recently raised interest in the pharmaceutical industry for

Agricultural soils are under increasing pressure from climate
change and anthropogenic factors. Maintaining or even in-
creasing crop productivity and simultaneously, minimizing
harmful impacts of intensive agriculture on the environment,
requires a shift toward more sustainable agricultural practices
(Chen et al. 2017).
On coastal agricultural lands, the use of halophyte plants as
crops is becoming a promising alternative to traditional salt-
sensitive crops (Lombardi et al. 2022). Salicornia europaea
L. (syn. S. ramosissima) is a succulent halophyte present in
saltmarshes throughout Europe (Silva et al. 2007). This plant
has been subject of growing interest, not only for cultiva-
tion as crop species due to their nutritional composition, but
also for their potential to recover contaminated and salinized
soils (Pessoa et al. 2022, Sanjosé et al. 2022). Moreover, it
its content in secondary metabolites with antioxidant, anti-
inflammatory, and antimicrobial properties (Silva et al. 2021).
Although halophytes can tolerate a wide range of salt
concentrations, adaptations imply a physiological effort that
translates into reduced germination, and low growth rate
and yield (Pan et al. 2020). Mitigation of salt-stress in tradi-
tional crops has been successfully achieved by plant growth-
promoting bacteria (PGPB) (Yoo et al. 2019) but the applica-
tion of this approach to stimulate growth of crop halophytes
is still virtually unexplored.
To reduce possible negative impacts on the native soil mi-
crobial communities, the use of PGPB native to the plant mi-
crobiome is thought to be preferable (Armada et al. 2018). The
formulation of cocktails of autochthonous PGPB, with dif-
ferent growth promoting traits, ideally impacts plant growth,

Received: December 22, 2022. Revised: January 27, 2023. Accepted: February 24, 2023

C The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International. All rights reserved. For permissions, please

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2 Ferreira et al.

E—endosphere; R—rhizosphere; Q—chitinase; A—amylase; L—lipase; P—protease; C—cellulase; Av3–Boco (Aveiro); Tg—Tagus estuary (Lisbon); + positive; − negative; +∗ visible growth on solid DF+ACC medium.
access to nutrients as well as stress responses, and metabo-

EPS (OD540 )

1.24 ± 0.03
0.40 ± 0.01
0.80 ± 0.01
lite production, without disrupting the structure of soil bacte-
rial communities (Nephali et al. 2020). These cocktails, used
as biofertilizers, represent a rhizosphere engineering approach
with the purpose of remodeling the microbiome of inoculated

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plants, favoring desired microbial functions (Armada et al.

N-fixation
2018). Ultimately, the goal is to enhance crop productivity and

+
−
−
soil fertility while reducing the need for chemical agrochemi-
cals.
Information on the composition of bacterial communities

10.87 ± 0.48
39.55 ± 1.01
(μgmL−1 )
naturally associated with S. europaea is still limited (Shi et al.

IAA

−
2015, Figueira et al. 2019, Camacho-Sanchez et al. 2022), and

Plant growth-promoting traits

plant–bacteria interactions have been primarily explored in
the perspective of using these plants for microbe-assisted phy-
toremediation approaches (Oliveira et al. 2015, Pérez-Romero

ACC deaminase (nm

+∗ (44.09 ± 4.33)
+ (17.63 ± 1.35)
et al. 2016). Moreover, plant–bacteria interactions have been

mg−1 h−1 )
studied mostly in greenhouse or controlled laboratory condi-
tions. Studies conducted under realistic production conditions

−
are less available, although considered as essential to the de-
velopment and effective application of PGPB-based biostimu-
lants for saline agriculture.
Inoculation with PGPB has been successfully tested to
improve halophyte seed germination (Pérez-Romero et al.

Siderophore
2016, Figueira et al. 2019, Mesa-Marín et al. 2019, 2020)

+
+
+
and plant growth (Kearl et al. 2019). In our previous work

Table 1. Summary of the plant growth-promotion activities and salt tolerance of the tested bacterial strains (Ferreira et al. 2021).
(Ferreira et al. 2021), bacteria associated with the roots of
S. europaea from different sites along the Portuguese coast
were isolated and screened for plant growth-promotion traits.

P-solubilization
Here, we aimed at evaluating the impact of the inoculation
of selected PGPB, originally isolated from roots S. europaea

−
+
+
in wild populations, on plant growth and metabolic profile
of S. europaea, cultivated either in laboratory or in field
conditions.

+
+
+
C
Extracellular enzymes

+
−
+
P

Material and methods

−
−
+
L

Preparation of bacterial inoculants

Bacteria isolated from the rhizosphere and endosphere of S.
−
+
+
A

europaea plants, collected in three different estuaries along

the Portuguese coast (Ria de Aveiro, Tagus River, and Algarve
Q

−
−
+

Coast), were previously screened for plant growth-promoting

tolerance (g L−1 )

traits (Ferreira et al. 2021). Considering all PGPB traits, three

Limit salt

highly salt-tolerant isolates were selected: Bacillus aryabhattai

100
100
50

SP20, Pseudomonas oryzihabitans RL18, and Brevibacterium

casei EB3 (Table 1). Brevibacterium casei EB3 and Ps. oryzi-
habitans RL18 tolerate salinity up to 100 g L−1 of salt (NaCl),
whereas Ba. aryabhattai SP20 can tolerate a maximum of 50 g
L−1 . The isolates were grown separately in Tryptic Soy Broth
Ps. oryzihabitans
Ba. aryabhattai
Identification

(TSB; Liofilchem, Roseto degli Abruzzi) modified by addition

Br. casei

of NaCl 25 g L−1 . Cultures were incubated for 24–48 h in

a rotary shaker (150 rpm, 30◦ C ± 2◦ C). Cells were collected
by centrifugation at room temperature (5000 g; 10 min). Pel-
lets were washed twice with sterile saline solution (NaCl 9 g
Isolates

L−1 ), and finally re-suspended in sterile physiological saline

solution. For the inoculation of seeds, the concentration of
Source

the suspension was adjusted to 108 CFU mL−1 (OD600 ∼1)

R
R
E

(Mesa-Marín et al. 2019).

Antagonism between inoculant strains was ruled out by
Av3

Av1
Site

Tg

testing all possible strain combinations by the cross-streaking

method (Lertcanawanichakul and Sawangnop 2011), per-
RL18
Code

formed in Tryptic Soy Agar plates (TSA; Liofilchem, Roseto

SP20
EB3

degli Abruzzi).
Biostimulation of Salicornia crops
Inoculation of S. europaea seeds
Salicornia europaea seeds, collected in early autumn in a crop
cultivation facility (Horta dos Peixinhos, Aveiro, Portugal),
were stored in the dark for 4 months at 4◦ C until the be-
ginning of the experiment to simulate dormancy stage (Un-
gar 1979). Before inoculation, S. europaea seeds were surface-
disinfected by immersion (2 min) in 1 mL of a 1:1 proportion
of two commercial solutions of hydrogen peroxide (30%) and
ethanol (96%), and rinsed three times with sterile distilled wa-
ter (Piernik et al. 2017). A factorial inoculation experiment
was designed to test the three selected bacterial inoculants, ap-
plied individually, or combined as inoculant cocktails. In total,
eight formulations were tested: (a) EB3 Br. casei; (b) RL18 Ps.
oryzihabitans; (c) SP20 Ba. aryabhattai; (d) EB3 Br. casei +
RL18 Ps. oryzihabitans; (e) EB3 Br. casei + SP20 Ba. aryab-
hattai; (f) RL18 Ps. oryzihabitans + SP20 Ba. aryabhattai; (g)
EB3 Br. casei + RL18 Ps. oryzihabitans + SP20 Ba. aryabhat-
tai; and (h) noninoculated control.
For each inoculation condition, sets of ∼150 seeds were
transferred to sterile microtubes, and the appropriate bac-
terial suspension was added. For inoculation with cocktails
of strains, equal volumes of bacterial suspensions were com-
bined. Seeds were kept immersed in the bacterial suspension
for 2 h in a rotary shaker (150 rpm, 30 ± 2◦ C) and, finally,
pelleted by low-speed centrifugation (5000 g; 2 min). The liq-
uid was discarded, and seeds were transferred onto sterile fil-
ter paper, and partially air-dried for 1 h in the laminar flow
hood. Seeds exposed with sterile saline solution served as the
noninoculated control.
Pot experiments
Inoculated seeds were germinated in 1% water agar plates
(Komaresofla et al. 2019, Mesa-Marín et al. 2019) (25 seeds
per plate) in a Sanyo MLR 350 H Versatile Environmental
Test Chamber (Moriguchi, Osaka, Japan) programmed for a
16/8 h light/dark regime for 15 days at 24◦ C.
The obtained seedlings were carefully transferred from the
agar to individual plastic pots (5.5 cm high × 5.5 cm diame-
ter). The pots were previously disinfected with 75% sodium
hypochlorite solution, rinsed several times in sterile distilled
water, and filled with perlite. Pots were placed in shallow trays
and watered with 20% Hoagland’s solution. Salinity of the ir-
rigation solution was progressively increased by adding ma-
rine salt, at a step of 2.5 g L−1 every second day, up to a final
concentration of 10 g L−1 of salt (171 mM), that is considered
the optimal for S. europaea (Ameixa et al. 2016). The cultures
were maintained for 2 months (Pérez-Romero et al. 2019a)
in a removable greenhouse, under natural sunlight irradia-
tion. Temperature and humidity were not controlled. Grown
plants were later transferred to pots (9 cm high × 9 cm diam-
eter) containing a substrate composed by a mixture of perlite
and autoclaved saltmarsh sediment (1:1). Pots were placed in
shallow trays and watered as previously described, with 20%
Hoagland’s solution amended with 10 g L−1 of marine salt.
Irrigation solution was changed every week and maintained
a nearly constant level of water in the trays. Re-inoculations
were performed every 15 days by adding 20 mL of suspension
of bacteria in sterile water (OD600 ∼1) directly to the substrate
in each pot. Noninoculated control plants received 20 mL of
sterile water. The pots were acclimatized in the same remov-
able greenhouse for 30 days and finally transferred to a cli-
mate chamber (Fitoclima D1200, Aralab, Sintra, Portugal) un-
3
der controlled conditions (16/8 h photoperiod; 25/20◦ C tem-
perature; 40% relative humidity; 500 μmol m−2 s−1 photon
flux density). These conditions were maintained for 35 days
to avoid a decline in the physiological conditions due to ag-
ing (Pérez-Romero et al. 2019b). In total, the duration of the
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experiment was ∼5 months, from the laying of seeds to the
harvesting of plants.
Field experiment
The field experiment was conducted in a crop area, within
the estuarine system Ria de Aveiro, held by a local private
company. Sets of 100 seeds, corresponding to each inoculation
condition, were laid in the center of 10-cm diameter sediment
beds (13 replicates by treatment), delimited by a PVC rim.
Seeds and subsequent germinated plants were subjected to the
natural regime of light, temperature, and weekly tidal irriga-
tion with brackish water 24–28 ‰ (Dias et al. 2021), as were
the surrounding crops. Re-inoculation was performed every 2
weeks directly to the sediment inside the rims by adding 20 mL
of the suspension of bacteria in sterile water (OD600 ∼1), as
previously described. Noninoculated control plants received
20 mL of sterile water. After 2 months, the number of plants
in each rim was reduced to a maximum of three. Plants were
grown for 5 months before harvest.
Harvesting and determination of growth
parameters
At the end of the experiments, plants corresponding to each in-
oculation condition were collected. Sets of five replicate spec-
imens (pot experiments) and 10 specimens (field trial) were
immediately used for the determination of fresh weight (FW)
as a descriptor of plant growth. Dry weight (DW) was deter-
mined after drying at 60◦ C, until constant weight. Plants were
individually weighted, and the values were averaged.
Microbial load of edible parts
Three replicate plants from each pot and field experimental
condition were used to assess the microbiological quality of
fresh shoots, according to the Portuguese guidelines for ready-
to-eat green salads (Saraiva et al. 2019). Fresh shoots were cut,
washed with tap water, and clipped in small pieces (∼3 cm).
Subsamples of 10 g were mixed with 90 mL of Ringer solution
(Merck, Rahway, NJ, USA), and homogenized in a stomacher.
The homogenate was further diluted in Ringer solution and
analyzed for total coliforms, Escherichia coli, Ba. cereus, to-
tal aerobic mesophilic bacteria, yeasts, and molds, according
to standard food microbiology protocols (Pérez-Romero et al.
2019b). Aerobic mesophilic bacteria were quantified in Plate
Count Agar (Merck, Rahway, NJ, USA) by the pour-plate tech-
nique (in triplicate). Total coliforms and E. coli were enumer-
ated by pour-plating triplicates of each dilution in Chromocult
Coliform Agar (Merck, Rahway, NJ, USA). Bacillus cereus
were enumerated by the spread-plating technique (five repli-
cates) on Mannitol Yolk Polymyxin Agar (OXOID). Yeasts
and molds were analyzed by the spread-plating technique (five
replicates) on Dicloran Rose Bengal Chloramphenicol Agar
(Merck, Rahway, NJ, USA) (Spencer and de Spencer 2001).
Colony counts were averaged, corrected for the dilution fac-
tor and aliquot volume, and expressed as colony-forming units
per gram FW (CFU gfw−1 ).
4 Ferreira et al.
Presence of inoculants in grown plants
At the end of experiments, the presence of the inoculant PGPB
strains in the plant endosphere and rhizosphere was assessed
by next-generation sequencing. In the pot experiment, the rhi-
zosphere was analyzed for all inoculation conditions. The en-
dosphere was analyzed only for the condition corresponding
to the highest effect on plant growth (EB3+RL18). In the field
experiment, only the rhizospheres of the EB3+RL18 condi-
tion, the noninoculated controls, and the surrounding crop
plants (outside the rims) were analyzed. Rhizosphere and en-
dosphere samples were obtained as described in Szymańska et
al. (2018) and Barillot et al. (2013). DNA extraction followed
the instructions of FastDNA™ SPIN Kit for Soil (MP Biomed-
icals, France). The quality of the DNA extracts was checked
by amplification of the 16S ribosomal RNA (rRNA) gene by
PCR. DNA extracts (three replicates per treatment) were an-
alyzed by next-generation sequencing (Illumina) of the hyper-
variable V3–V4 region of 16S rDNA gene (Herlemann et al.
2011, Klindworth et al. 2013) at Genoinseq (Cantanhede, Por-
tugal).
Bioinformatic analyses were conducted with QIIME2 pack-
age version 2020.8 and its plug-ins (Bolyen et al. 2019). The
DADA2 plug-in (Callahan et al. 2016) was used for the re-
moval of chimeras (consensus method) and grouping into
representative sequences called amplicon sequence variants
(ASVs). Downstream analyses were performed on R v4.0
(R Core Team 2014) and the Phyloseq package (McMur-
die and Holmes 2013). Taxonomy was assigned to ASVs us-
ing a naïve Bayes taxonom classifier against the Silva 138
99% OTUs reference sequence (Quast et al. 2012). The 16S
rRNA sequence of the isolates EB3 Br. casei (GeneBank acces-
sion number MT981731), RL18 Ps. oryzihabitans (GeneBank
accession number: MT981740), and SP20 Ba. aryabhattai
(GeneBank accession number: MG583717) were used in a
blast alignment against all the ASVs in the dataset. ASVs
matching 100% identity were considered as detection of the
inoculant.
GC–TOF-MS primary metabolite profiling
The analysis of primary metabolites was conducted only in the
EB3+RL18 inoculation condition, and respective noninocu-
lated controls, from the pot experiment. Material from the
aerial parts was collected, rinsed in running water, dried at
60◦ C until constant weight, and ground to a fine homogenized
powder with liquid nitrogen.
The primary metabolome was obtained with gas chro-
matography time-of-flight mass spectrometry (GC–TOF-MS),
using five biological replicates for each experimental condi-
tion. Primary metabolites were extracted from 20 mg DW
of finely homogenized vegetative material in 700 μL ice-cold
methanol with 30 μL of internal standard ribitol (0.2 mg
mL−1 ribitol in water) as previously described (Lisec et al.
2006). Samples were vortex-mixed and incubated in a shaker
(ThermoMixer, Eppendorf, Hamburg, Germany) for 15 min at
70◦ C and 950 rpm, and subsequently centrifuged at 12 000 g
for 10 min. The supernatant was collected, mixed with 370 μL
chloroform followed by 750 μL water, and vortex-mixed.
After centrifugation at 2200 g for 15 min, an aliquot of
150 μL of the polar aqueous/methanol phase was evaporated
to dryness for 3 h at 30◦ C using a centrifugal concentrator
(Vacufuge Plus, Eppendorf, Hamburg, Germany), and stored
at −80◦ C prior to GC–TOF-MS analysis. Dried polar ex-
tracts were subsequently derivatized with 40 μL of 20 mg
mL−1 methoxyamine hydrochloride in pyridine, followed
by 70 μL of N-methyl–N-(trimethylsilyl)trifluoroacetamide
(TMS derivatization) and 20 μL mL−1 of a mixture of fatty
acid methyl esters (FAMES). Primary metabolite profiling
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analysis of the derivatized samples (1 μL injection) was per-
formed on an Agilent 6890 N gas chromatograph (Agilent
Technologies, Böblingen, Germany) coupled to a LECO Pe-
gasus III TOF-MS running in electron ionization (EI) mode
(LECO Instrumente, Mönchengladbach, Germany). The chro-
matographic separation was performed on a VF-5MS col-
umn (Varian Inc., 30 m length, 0.25 mm inner diameter, and
0.25 mm film thickness). The temperature program was set
as follows: isothermal for 2 min at 85◦ C, followed by a 15◦ C
min−1 ramp to 360◦ C, and hold at this temperature for 6 min.
The injector and transfer line temperatures were set to 230◦ C
and 250◦ C, respectively, and the helium carrier gas flow set
to 2 mL min−1 . After a solvent delay of 180 s, mass spec-
tra were scanned from m/z 70–600 with acquisition rate of
20 spectra s−1 and a detector voltage between 1700 and
1850 V. GC–TOF-MS data were evaluated using AMDIS (Au-
tomated Mass Spectral Deconvolution and Identification Sys-
tem) software (v. 2.71). Primary metabolites were annotated
using the TagFinder 4.0 software (Luedemann et al. 2008)
and a reference library of ambient mass spectra and reten-
tion indices from the Golm Metabolome Database (Kopka
et al. 2005, Schauer et al. 2005). Metadata information
of this experiment following minimum reporting standard
guidelines of the Metabolomics Standard Initiative (Fiehn et
al. 2007, Sumner et al. 2007, Fernie et al. 2011, Alseekh
et al. 2021) can be found as Supplementary Information
(Table S1).
Statistical analyses
Statistical differences in plant biomass were estimated using
ordinary one-way ANOVA and corrected with Dunnett’s mul-
tiple comparison test in GraphPad Prism version 8 for Mac
(GraphPad Software, Inc., San Diego, CA, USA. Normality
was confirmed by Shapiro–Wilk test.
Metabolomics statistical analyses were performed in R Stu-
dio software (RStudio Team 2020) using the “Agricolae”
(Mendiburu and Yaseen 2020), “gplots” (Warnes et al. 2021),
and “mixOmics” (Rohart et al. 2017) packages. The relative
abundance of primary metabolite levels was normalized to the
internal standard (ribitol) and the DW of the samples. One-
way ANOVA at a 95% confidence level was used to assess dif-
ferences between treatments. Subsequently, fold-changes were
determined and log10-transformed for heatmap plotting. Su-
pervised partial least squares discriminant analysis (PLS-DA)
was performed using the leave-one-out cross-validation em-
bedded in the “mixOmics” package.
Results
Plant growth
The average FW of plants corresponding to the different inoc-
ulation conditions tested in pot experiments varied between
5.9 ± 3.04 g for noninoculated plants, and 25.9 ± 3.95 g
for plants inoculated with Br. casei EB3 and Ps. oryzihabi-
tans RL18 (Fig. 1). Except for the inoculation conditions
EB3+SP20 and RL18+SP20, the differences between the FW
of bio-stimulated plants and noninoculated controls were all
Biostimulation of Salicornia crops
Figure 1. FW and DW of Salicornia europaea plants under different biostimulation conditions, in the pot experiment. Values represent the average of five
biological replicates and the error bars represent the standard deviation. Significant differences in relation to the noninoculated controls are indicated as
∗
P < .05; ∗∗∗ P < .001. C—noninoculated control; EB3—Br. casei EB3; RL18—Ps. oryzihabitans RL18; and SP20—Ba. aryabhattai SP20.
statistically significant (Fig. 1). For visual comparison, repre-
sentative specimens are depicted in Fig. 2.
DW varied between 0.6 ± 0.36 g for noninoculated plants,
and 2.90 ± 0.75 g for plants co-inoculated with Br. casei
EB3 and Ps. oryzihabitans RL18 (Fig. 1). However, differ-
ences in relation to the noninoculated control were only sig-
nificant for the inoculation conditions EB3, EB3+RL18, and
EB3+RL18+SP20.
In general, plants grown in the field were visibly smaller
than those obtained in the pot experiment, and no signifi-
cant differences between inoculation conditions were detected
(Fig. 3). The average FW of plants grown in field conditions
was 2.3 ± 0.52 g and the DW was 0.3 ± 0.09 g.
Microbial load in plants from pot and field
experiments
The microbiological characterization of fresh shoots of plants
corresponding to all tested inoculation conditions, summa-
rized in Table 2, was assessed using 2019 Portuguese guide-
lines for ready-to-eat green salads issued by the national refer-
ence laboratory Instituto Nacional de Saúde Dr Ricardo Jorge
(Saraiva et al. 2019). Acceptance limits (worst tolerable limit
values, beyond which food cannot be considered safe) and sat-
isfaction limits (limit values corresponding to nearly absolute
safety) are established in this document for molds, yeasts, aer-
obic mesophilic bacteria, total coliforms, Ba. cereus, and E.
coli. The concentration of molds varied between 6.0 ± 8.94 ×
101 and 1.7 ± 0.61 × 104 CFU gFW−1 , and the satisfaction
(5 × 102 CFU gFW−1 ) and acceptance (103 CFU gFW−1 ) lim-
its were frequently exceeded. In the pot experiment, noninoc-
ulated controls, and inoculation treatments SP20, RL18, and
EB3+SP20, developed molds above the acceptance limit. The
satisfaction limit was exceeded in all inoculation conditions,
except for EB3+RL18+SP20. In the field experiment, the ac-
ceptance limit was exceeded for plants corresponding to inoc-
ulation conditions EB3, EB3+RL18, and EB3+RL18+SP20,
and the satisfaction limit was exceeded in both SP20 and
RL18+SP20 inoculation conditions.
The concentration of yeasts varied between 1.6 ± 0.49 ×
102 and 2.7 ± 0.50 × 103 CFU gFW−1 , but all values were
below the satisfaction limit (105 CFU gFW−1 ). Similarly, the
acceptance limit for the concentration of aerobic mesophilic
5
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bacteria (106 CFU gFW−1 ) was not exceeded. The concentra-
tions ranged from 3.6 ± 0.53 × 102 to 3.0 × 105 CFU gFW−1 .
Total coliforms were only detected in the pot experiment, in
the inoculation conditions C (9.1 ± 0.24 × 102 CFU gFW−1 )
and EB3+RL18 (1.3 ± 0.05 × 10 CFU gFW−1 ). Bacillus
cereus (1.4 ± 0.80 × 102 CFU gFW−1 ) was also detected only
in the pot experiment, in the inoculation condition RL18. Es-
cherichia coli was not detected in any of the inoculation con-
ditions.
Detection of the bacterial inoculants
The results of the detection of Ba. aryabhattai SP20, Ps. oryz-
ihabitans RL18, and Br. casei EB3 in the rhizosphere and en-
dosphere of plants from the different inoculation conditions
are summarized in Table 3.
In the pot experiment, Br. casei EB3 was detected in the en-
dosphere and rhizosphere of plants corresponding to all the
treatments in which this strain was inoculated. Pseudomonas
oryzihabitans RL18 was not detected in any of the inoculated
treatments. Bacillus aryabhattai SP20 was detected in the rhi-
zosphere of the condition SP20, where it was the sole inocu-
lant, but not in the co-inoculations. It was detected in very low
relative abundance in the EB3 treatment. In the field experi-
ment, Br. casei EB3 was detected in the rhizosphere of plants
that had been inoculated with this strain, and also in neigh-
boring crop plants, growing outside the rims.
GC–TOF-MS primary metabolite profiling
GC–TOF-MS analysis allowed to identify 51 primary metabo-
lites, including 24 amino acids and derivatives, 10 organic
acids, 12 sugars and sugar alcohols, the polyamine putrescine
(among other metabolites) (Fig. 4, Supplementary Table S2).
Primary metabolite profiling revealed that S. europaea plants
inoculated with EB3+RL18 where characterized mainly by
a significant increase (up to 2-fold) in the levels of several
amino acids (e.g. asparagine, γ -aminobutyric-acid (GABA),
glycine, homoserine, and serine), in the polyamine putrescine,
and in glycerol-3-phosphate. On the other hand, these plants
showed a significant decrease in the levels of sugars, particu-
larly glucose, fructose, trehalose, fucose, sugar alcohol galacti-
nol, amino acid arginine, and organic acids citrate and quinate
(Fig. 4, Supplementary Table S2).
6 Ferreira et al.

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Figure 2. Specimens of S. europaea, representative of different inoculation conditions, tested in the pot experiment. C—noninoculated control; EB3—Br.
casei EB3; RL18—Ps. oryzihabitans RL18; and SP20—Ba. aryabhattai SP20.

Figure 3. FW and DW of S. europaea plants under different biostimulation conditions, in the field experiment. Values represent the average of 10
biological replicates and the error bars represent the standard deviation. C—noninoculated control; EB3—Br. casei EB3; RL18—Ps. oryzihabitans RL18;
and SP20—Ba. aryabhattai SP20.

Central metabolic pathways representing the changes in the
relative levels of primary metabolites in vegetative tissues of S.
europaea plants inoculated with EB3+RL18 are represented
in Fig. 5.
Central metabolic pathways of inoculated plants re-
vealed marked differences in energy metabolism, TCA
cycle, and biosynthesis of amino acids. PLS-DA anal-
ysis allowed to clearly discriminate between noninocu-
lated controls and the inoculation condition EB3+RL18
(Fig. 6a), even with few samples. The correlation cir-
cle plot (Fig. 6b) identifies the metabolites most re-
sponsible for the separation between the metabolite pro-
file of the control and of the inoculation condition
EB3+RL18.
The contribution plots (Fig. 7) show that among those
metabolites, homoserine and glycerol-3-phosphate (compo-
nent 1) and tryptophan (component 2) contributed the most
to the discrimination between plants grown from inoculated
seeds and the noninoculated controls.
Discussion
The primary objective of this research was to design an ef-
fective biostimulant for the crop cultivation of S. europaea,
 Biostimulation of Salicornia crops 7

C—noninoculated control; EB3—Br. casei EB3; RL18—Ps. oryzihabitans RL18; SP20—Ba. aryabhattai SP20; EB33+RL18—Br. casei EB3 and Ps. oryzihabitans RL18; EB3+SP20—Br. casei EB3 and Ba. aryabhattai SP20;
RL18+SP20—Ps. oryzihabitans RL18 and Ba. aryabhattai SP20; EB3+RL18+SP20—Br. casei EB3, Ps. oryzihabitans RL18, and Ba. aryabhattai SP20; and nd—not detected. Values represent the average ± SD of five (molds
meeting the requirements of effective enhancement of growth
with reduced risk of introduction of nonautochthonous mi-

Absent
Absent
E. coli
crobes to the native microbial communities of soils. For that,

nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
bacterial strains from an existing collection of PGPB, iso-
lated from the endosphere and rhizosphere of several S. eu-

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ropaea populations, along the Portuguese coast, were used.
The selection of three PGPB strains was intended to cover
the widest possible range of PGPB traits, such as extracel-
0.1 ± 0.08 × 103

lular enzymes (extracellular enzymes cellulase, lipase, pro-

Ba. cereus

tease, amylase, and chitinase) and exopolysaccharide produc-

Absent
Absent
nd
nd

nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
tion, stress-attenuation enzymes (ACC deaminase), phytohor-
mones (indole-3-acetic acid), siderophore production, phos-
phate solubilization, and nitrogen fixation activity (Ferreira
et al. 2021). The strains were applied individually, or in cock-
tails of all possible combinations, to also represent different
biotic interactions between bacteria.
0.01 ± 0.001 × 103
1.0 ± 0.02 × 103
Total coliforms

106
108

nd
nd
nd

nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
Growth promotion in laboratory and field
Table 2. Microbiological load of fresh shoots of S. europaea corresponding to the different inoculation conditions tested in pot and field experiments.

conditions
In the pot experiment, under more controlled abiotic condi-
tions, several inoculants significantly promoted biomass pro-
duction. Plants responded to the cocktails EB3+RL18 and
EB3+RL18+SP20 by increasing FW and DW up to five times
Total aerobic mesophilic

compared to noninoculated plants. Isolates EB3 and RL18 are

0,2 ± 0,09 × 103

20,0 ± 0,4 × 103

10.0 ± 0.4 × 103

22.0 ± 0.6 × 103

5.4 ± 0.03 × 103

0.4 ± 0.06 × 103

11.0 ± 7.0 × 103

0.4 ± 0.06 × 103

15.0 ± 2.7 × 103

1,0 ± 0,2 × 103

1.5 ± 0.2 × 103

4.6 ± 0.2 × 103

0.5 ± 0.1 × 103

2.7 ± 0.7 × 103

the common components of the two formulations. Brevibac-

>30 × 103

>30 × 103
bacteria

terium casei EB3 was the only bacterium to show nitrogen-

106
108

fixing capacity among the selected inoculants. Since nitrogen

is a limiting element in estuarine sediments (Alldred et al.
2017), the contribution of Br. casei EB3 to the nutrient sup-
ply to the plant might have been the most relevant benefit of
and yeasts) or three (total aerobic mesophilic bacteria) analytical replicates of three biological replicates per treatment.

inoculation. It has been demonstrated that nitrogen fertiliza-

tion enhances the development of roots and shoots of halo-
phytes, and increases salinity tolerance (Jun-Feng et al. 2010,
0.2 ± 0.05 × 103
0.2 ± 0.14 × 103

0.4 ± 0.17 × 103

0.3 ± 0.08 × 103

0,2 ± 0,2 × 103

0,8 ± 0,4 × 103
0,5 ± 0,3 × 103
1.3 ± 0.5 × 103
1.8 ± 0.6 × 103
1.0 ± 0.3 × 103
2.7 ± 0.5 × 103
1.1 ± 0.7 × 103
1.0 ± 0.5 × 103
1.1 ± 0.5 × 103

2.0 ± 0.6 × 103

0.8 ± 0.2 × 103

Jeong et al. 2013). Brevibacterium casei lacked phosphate sol-

Yeasts

ubilization capacity that, in the case of the most successful

105
106

formulation (EB3+RL18), could have been delivered by Ps.

oryzihabitans RL18. Pseudomonas oryzihabitans RL18 is also
a prolific producer of IAA, a phytohormone that activates
growth, and ACC-deaminase that contributes to the reduc-
tion of ethylene levels and attenuation of stress (Fahad et al.
2015). However, Ps. oryzihabitans RL18 could not be detected
2.1 ± 0.40 × 103

17.0 ± 0.6 × 103

0.6 ± 0.07 × 103

0.1 ± 0.08 × 103

1.0 ± 0.2 × 103

2.2 ± 0.4 × 103

0.5 ± 0.2 × 103
2.1 ± 0.3 × 103
1.1 ± 0.2 × 103
0.1 ± 0.1 × 103
0.2 ± 0.2 × 103
1.1 ± 0.3 × 103
0.1 ± 0.1 × 103

1.3 ± 0.5 × 103

0.7 ± 0.3 × 103

1.1 ± 0.4 × 103

in the plants, at the end of the experiments. This may indicate

5 × 102
Molds

that Ps. oryzihabitans RL18 was outcompeted by other PGPB

103

and that the plant growth promotion effect may have been
triggered by a beneficial shift in the composition of the plant
microbiome rather than by the direct activity of the inoculant
strain.
The inoculant Ba. aryabhattai SP20 had a minor effect
on plant growth, although this strain has previously demon-
EB3+RL18+SP20

EB3+RL18+SP20
Satisfaction limit
Acceptance limit

strated a positive effect on the germination of S. europaea

RL18+SP20

RL18+SP20
EB3+RL18

EB3+RL18
EB3+SP20

EB3+SP20

seeds in high-salinity medium (Figueira et al. 2019). In addi-

RL18

RL18
SP20

SP20
EB3

EB3
C

tion, Ba. aryabhattai SP20 produces several lytic extracellular

Values are expressed as CFU gFW−1 .

enzymes that may contribute to biocontrol activity and facili-

tate the access to the colonization of internal plant tissues (Fer-
reira et al. 2021). This step is regarded as vital to the establish-
ment of a long-term plant–bacteria relation, because the endo-
sphere is a more protected environment than the rhizosphere
(Walitang et al. 2017). All isolates produced exopolysaccha-
Field experiment
Pot experiment

rides that protect the plant against osmotic stress and pro-
Guidelines

mote the colonization of the root surface (Kaur et al. 2017,

Morcillo and Manzanera 2021) as well as cellulases that fa-
cilitate the translocation to the endosphere (Walitang et al.
8 Ferreira et al.

Table 3. Quantification of ASVs corresponding to Br. casei EB3, Ps. oryzihabitans RL18, and Ba. aryabhattai SP20 in the rhizosphere or endosphere bacterial
communities of S. europaea, corresponding to the different inoculation conditions tested in the pot and field experiments.

ASVs
Experimental condition Br. casei EB3 Ps. oryzihabitans RL18 Ba. aryabhattai SP20

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Pot experiment Rhizosphere EB3 0.31% ± 0.05 nd 0.005% ± 0.009
RL18 nd nd nd
SP20 nd nd 0.01% ± 0.02
EB3+RL18 0.14% ± 0.08 nd nd
EB3+SP20 0.23% ± 0.007 nd nd
RL18+SP20 nd nd nd
EB3+RL18+SP20 0.70% ± 0.06 nd nd
Control (noninoculated) nd nd nd
Endosphere EB3+RL18 0.07% ± 0.001 nd nd
Field experiment Rhizosphere EB3+RL18 0.06% ± 0.05 nd nd
Crop (natural) 0.03% ± 0.02 nd nd
C (noninoculated) nd nd nd

The values correspond to the means of the relative abundance of the replicates ± SD; nd—not detected. C—noninoculated control; Crop—plants growing at
the production site, outside the rims (field experiment); EB3—Br. casei EB3; RL18—Ps. oryzihabitans RL18; and SP20—Ba. aryabhattai SP20.

2017) and improve soil fertility by degrading plant debris
(Han and He 2010) and siderophores involved in the supply
of iron.
The enhanced development of the root system and of the
aerial parts, in treatments EB3+RL18 and ALL, depicted in
Fig. 1, is possibly the result of interactive effects of these traits.
It was established that IAA can enhance ethylene production
by stimulating the activity of the ACC synthase in plant tissues
(Glick et al. 1998). In this case, ACC deaminase action is of the
outmost importance in balancing the stimulation of ethylene
production by high levels of IAA, and therefore, in controlling
stress.
The association EB3+SP20 resulted in one of the poor-
est performances in the pot experiment. The formulation
RL18+SP20 lacks nitrogen-fixing ability, a key feature in
plant development. Furthermore, different combinations of
inoculants resulted in different relative abundances in the rhi-
zosphere of the grown plants. Antagonism between the inoc-
ulant strains was ruled out but antagonism and competition
between inoculants and native bacteria of the seed, negative
selection from the plant host or insufficient rhizo-competence
of some PGPB may still have a determining effect on the suc-
cess of the use of PGPB as biofertilizers (Morales-García et al.
2019).
The biostimulation effect of the EB3+RL18 cocktail, ob-
served in plants in the pot experiments, was not observed in
field conditions, and this cannot be immediately attributed
to the lack of persistence of the inoculated strains. Brevibac-
terium casei EB3, that seems to have a major effect on growth,
was detected in the rhizosphere of plants that were inoculated
with this strain in the field and pot experiment, and in the root
endosphere of plants of the EB3+RL18 condition.
The loss of biostimulation effect upon transposition from
the laboratory to the field, is not a rare observation. The lack
of success in the scaling up of biostimulant applications can
be attributed to different factors. In addition to the different
behavior of plants in laboratory and field conditions (De Vries
1980, Poorter et al. 2016), the variability seems to be associ-
ated with the environmental conditions (Bacilio et al. 2017).
Although the germination efficiency was not calculated, the
number of germinated seeds was visibly lower in the field than
in the pot experiment. In some rims, only four to five seeds ger-
minated, which is less than half the number of seeds, pointing
to an unfavorable interaction of environmental factors.
The effect of the inoculation on overall structure and com-
position of the plant microbiome is being further examined in
order to better why formulations that performed well in the
pot experiment, failed in field conditions. The reduced sur-
vival of bacterial inoculants has been attributed to the buffer-
ing effect of the natural host plant microbiome, that tends to
regulate and ultimately balance, the relative abundance of in-
oculated bacteria (Ishizawa et al. 2020). Pseudomonas and
Bacillus are ubiquitous in soils and sediments and the species
Ps. oryzihabitans and Ba. aryabhattai have, in fact, been de-
tected in the microbiome of Salicornia in different popula-
tions, along the Portuguese coast (Ferreira et al. 2021). There-
fore, it is possible that the inoculated stains (Ba. aryabhattai
SP20 and Ps. oryzihabitans RL18) were quickly outcompeted
by native strains. In natural soils, competition with the res-
ident microbiota and predation by protozoa and nematodes
can lead to a rapid decrease of the relative abundance of in-
oculated strains, by orders of magnitude per week (Martínez-
Viveros et al. 2010). In field conditions, re-inoculations should
probably have been done more frequently than in the pot ex-
periments (every 2 weeks).
In the present study, field cultures followed a saline agri-
culture practice, with weekly irrigation with estuarine water.
Tidal irrigation entails periods of flooding and drainage, that
impose physical forcing on the seeds and plantlets and may
have an erosion/dilution effect on the inoculants. For an effi-
cient use in the field, it may become necessary to combine bac-
terial cells with carriers or coadjutants, that enhance binding
of bacteria to the seeds. Additionally, in the field, both bacte-
ria and the host seeds or plantlets experience strong short-term
variations of salinity, temperature, and solar radiation. The in-
corporation of osmoprotectants in the biostimulants formula-
tions, could also contribute to a higher survival of the inocu-
lants.
Effect of inoculation on microbiological quality and
metabolite profile of Salicornia
Because S. europaea is an edible plant, that is actually gaining
acceptance as a salad green or as a substitute of salt (Cardoso
et al. 2022), there is the concern on whether inoculation could
jeopardize the microbiological quality. Microbiological qual-
ity of fresh shoots was assessed using 2019 Portuguese guide-
lines for ready to eat green salads by Instituto Nacional de
Biostimulation of Salicornia crops
Figure 4. Heatmap representing the changes in the relative levels of
primary metabolites in aerial vegetative tissues of S. europaea plants
corresponding to the inoculation condition EB3+RL18 of the pot
experiment, in relation to the noninoculated control. Relative values (as
the average of n = 5 independent biological replicates) were normalized
to the internal standard (ribitol) and DW of the samples. False color
imaging was performed on log10-transformed GC–TOF-MS primary
metabolite data. Fold changes were calculated in relation to the control
(Supplementary Table S2). Significant changes using one-way ANOVA are
indicated as ◦ (P < .05). Heatmap was performed in RStudio software
(RStudio Team 2020) using the “gplots” package (Gregory et al. 2021).
Metabolites grouped in several classes: amino acids and derivatives (AA),
organic acids (OA), sugars and sugar alcohols (SS), and others (O).
9
Saúde Dr Ricardo Jorge (Saraiva et al. 2019). The results in-
dicate an overall satisfactory quality of freshly harvested ma-
terial. However, in some treatments both in pot and field ex-
periments, the concentration of molds exceeds the acceptance
limit. In this case, the development of molds could be related
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to the contamination of the irrigation water and to the long
flooding imposed on the plants, in the pot experiment, and in-
side the PVC rims, in the field experiment. The occurrence of
Br. cereus in plants from the RL18 pot treatment might have
been caused by the contamination of the water in the irriga-
tion trays, although the values were very low. Interestingly,
plants inoculated with the cocktail EB3+RL18+SP20 culti-
vated in the field, presented one of the highest mold loads,
whereas plants from the pot experiment inoculated with the
same mixture, revealed the lowest. These differences seem to
support the idea of an environment-modulated effect of the
plant-associated microbiome.
Several studies of microbiome–metabolome relations in
wild and crop plants have been producing evidence that in-
oculation with bacteria influences plant metabolite composi-
tion (Pang et al. 2021). Also, in halophytes, inoculation of S.
bigelovii seeds with Vibrio species increased the plant biomass
and its carboxylic acids content (Bashan et al. 2000). In this
work, the analysis of the effects of inoculation with the most
effective biostimulants formulation, in terms of plant growth,
on the primary metabolite profile of the plant, was conducted
with the dual purpose of detecting changes in the expression
of nutritionally interesting molecules, and inferring on the
biochemical pathways involved in the enhancement of plant
growth.
Primary metabolite profiling data showed that inocula-
tion with Br. casei EB3 and Ps. oryzihabitans (EB3+RL18)
had a profound effect on the carbohydrate and amino
acid metabolism. Inoculated plants have significantly lower
amounts of sugars and higher amounts of amino acids, in com-
parison to control plants.
Plants exposed to saline stress activate protective mecha-
nisms by down regulating the energy metabolism and protein
synthesis (Bandehagh and Taylor 2020). This could be related
to an efficient strategy to catabolize sugars to increase ATP
production and increased amino acid de-novo synthesis. The
inoculation with a nitrogen-fixing bacteria could explain the
accumulation of amino acids as well as a decrease in glucose
and other monosaccharides, as nitrogen is assimilated into car-
bon skeletons obtained from glycolysis and tricarboxylic acid
(TCA)-cycle intermediates (Gilbert et al. 1998).
Biotic and abiotic stress are also responsible for the accu-
mulation of GABA (Shelp et al. 2012). GABA is involved in
the C:N equilibrium maintenance of under a range of abiotic
stresses, oxidative stress and biotic defense, osmoregulation,
and cytosolic pH regulation (Bandehagh and Taylor 2020).
The levels of GABA and proline are well-known to often in-
crease for cellular stress protection, primarily as osmolytes,
ROS scavengers, and signaling molecules (Carillo et al. 2008).
It has been demonstrated that low tissue nonstructural car-
bohydrate levels in plants is related to putrescine and amino
acid accumulation. The polyamine putrescine is synthesized
via arginine and proline metabolism (Ravi et al. 2020), which
could explain the significantly lower amounts of arginine and
significantly higher levels of putrescine in treated plants.
Glycerol-3-phosphate (G3P) is significantly higher in inocu-
lated plants when compared to control plants. This molecule,
important in carbohydrate and lipid metabolism, participates
10 Ferreira et al.

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Figure 5. Central metabolic pathways representing the changes in the relative levels of primary metabolites in aerial vegetative tissues of S. europaea
plants corresponding to the inoculation condition EB3+RL18 of the pot experiment. Relative values (average of five independent biological replicates)
were normalized to the internal standard (ribitol) and DW of the samples. False color imaging was performed on log10-transformed GC–TOF-MS primary
metabolite data. Fold changes were calculated in relation to the control (Supplementary Table S2). Significant changes using one-way ANOVA are
indicated as ◦ (P < .05).

Figure 6. PLS-DA score plot (a) and correlation plot (b) of the primary metabolite profile in vegetative tissues of S. europaea plants corresponding to the
inoculation condition EB3+RL18 of the pot experiment. PLS-DA was performed using the leave-one-out cross-validation embedded in the “mixOmics” R
package (Rohart et al. 2017).

in mitochondrial respiration through G3P shuttle and plays
an important role in modulating the NADH/NAD+ ratio and
cellular redox state in plant cells (Shen et al. 2006). G3P also
enhances pathogen defense mechanisms (Chanda et al. 2008).
As a plant defense mechanism, G3P serves as an inducer of
systemic acquired resistance at a very early time point, con-
tributes to systemic acquired resistance against stripe rust in
wheat and is required for stability of defense proteins (Chanda
et al. 2008, Yang et al. 2013, Yu et al. 2013). Defense mech-
anisms in treated plants may be enhanced due to inoculation
Biostimulation of Salicornia crops
Figure 7. PLS-DA contribution plots relative to component 1 and component 2 of the primary metabolite profile in vegetative tissues of S. europaea
plants corresponding to the inoculation condition EB3+RL18 of the pot experiment.
with Ps. oryzihabitans RL18, since this strain displays bio-
control activity against Fusarium oxysporum (Vagelas et al.
2003, Ferreira et al. 2021). Biocontrol activity has also been
described for Br. casei (Hashim et al. 2020).
Overall, this analysis clearly indicates inoculation with iso-
lates Br. casei EB3 and Ps. oryzihabitans RL18 caused a shift in
plant metabolism, directed mainly to enhance growth, result-
ing in biomass production increased up to five times compared
to noninoculated plants.
Genetics and chemistry in holobionts are intimately tangled
(Etalo et al. 2018) in such a way that the impact of a microbe-
mediated alteration of the plant metabolome, must necessar-
ily include the study of the relation between the microbiome,
its functions and the way it affects the plant metabolome dy-
namics. Inoculations with PGPB must also take this intricate
network of relations into account, as it will not only affect the
plant, but also its microbial community.
In summary, PGPB inoculation of S. europaea significantly
enhanced biomass production in controlled laboratory condi-
tions. Primary metabolite profiling unveiled a metabolic shift
that may explain this effect, confirming that the microbiome
can indeed trigger metabolic reconfiguration and modulate
the metabolite profile of the plant. Rhizosphere engineering
with selected microbes, emerges as a powerful tool to manip-
ulate not only plant growth, but also the metabolite compo-
sition of inoculated plants. However, inoculation of plants in
natural environments can lead to very different results from
those obtained in controlled conditions. Consequently, the
lack of understanding of the factors that hinder the successful
scaling up of biostimulation assays to field conditions still re-
11
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stricts the possibility of widespread applications of biostimu-
lants in saline agriculture studies not only regarding the effect
of the inoculation on the native soil microbial community, but
also the structure of the microbial-root-associated community.
Acknowledgements
We are grateful to Horta dos Peixinhos for their help and
support during sampling and seed collection. We also thank
Glória Pinto for the use of the climate chambers during the
final months of the experiment, and Enrique Mateos-Naranjo
and Jennifer Mesa-Marín of the Departamento de Biología
Vegetal y Ecología of the University of Sevilla, for their ad-
vice on the cultivation of Salicornia plants in laboratory and
greenhouse conditions.
Supplementary data
Supplementary data are available at JAMBIO Journal online.
Conflict of interest
No conflict of interest declared.
Funding
This work was supported by project Rhizomis PTDC/BIA-
MIC/29736/2017, Halius PTDC/BIA-MIC/3157/2020 fi-
nanced by the Fundação para a Ciência e a Tecnologia (FCT)
through the Regional Operational Program of the Center
12
(02/SAICT/2017) with FEDER funds (European Regional
Development Fund, FNR, and OE) and by the FCT through
CESAM (UIDP/50017/2020+UIDB/50017/2020), LAQV-
REQUIMTE (UIDB/50006/2020). We also acknowledge
FCT/FSE for the financial support to Maria João Ferreira
through a PhD grant (PD/BD/150363/2019).
Author contributions
Maria J. Ferreira (Investigation, Methodology, Writing – orig-
inal draft), I. Natalia Sierra-Garcia (Data curation, Investi-
gation, Methodology, Writing – original draft), Javier Cre-
mades (Conceptualization, Supervision, Writing – original
draft), Carla António (Conceptualization, Formal analysis, In-
vestigation, Methodology, Writing – original draft), Ana M.
Rodrigues (Investigation, Methodology), Diana C. G. A. Pinto
(Conceptualization, Formal analysis, Writing – original draft),
Helena Silva (Conceptualization, Formal analysis, Supervi-
sion, Writing – original draft), and Ângela Cunha (Concep-
tualization, Data curation, Formal analysis, Funding acquisi-
tion, Project administration, Supervision, Writing – original
draft)
Data availability
The data that support the findings of this study are available
from the corresponding author upon reasonable request.
References
Alldred M, Liberti A, Baines SB. Impact of salinity and nutrients on salt
marsh stability. Ecosphere 2017;8:e02010. https://doi.org/10.1002/
ecs2.2010
Alseekh S, Aharoni A, Brotman Y et al. Mass spectrometry-based
metabolomics: a guide for annotation, quantification and best re-
porting practices. Nat Methods 2021;18:747–56. https://doi.org/10
.1038/s41592-021-01197-1
Ameixa OM, Marques B, Fernandes VS et al. Dimorphic seeds of Sal-
icornia ramosissima display contrasting germination responses un-
der different salinities. Ecol Eng 2016;87:120–3. https://doi.org/10
.1016/j.ecoleng.2015.11.019
Armada E, Leite MF, Medina A et al. Native bacteria promote plant
growth under drought stress condition without impacting the rhi-
zomicrobiome. FEMS Microbiol Ecol 2018;94:fiy092. https://doi.or
g/10.1093/femsec/fiy092
Bacilio M, Moreno M, Lopez-Aguilar DR et al. Scaling from the
growth chamber to the greenhouse to the field: demonstration
of diminishing effects of mitigation of salinity in peppers inoc-
ulated with plant growth-promoting bacterium and humic acids.
Appl Soil Ecol 2017;119:327–38. https://doi.org/10.1016/j.apsoil.2
017.07.002
Bandehagh A, Taylor NL. Can alternative metabolic pathways and
shunts overcome salinity induced inhibition of central carbon
metabolism in crops? Front Plant Sci 2020;11:1072. https://doi.org/
10.3389/fpls.2020.01072
Barillot CD, Sarde C-O, Bert V et al. A standardized method for the
sampling of rhizosphere and rhizoplan soil bacteria associated to a
herbaceous root system. Ann Microbiol 2013;63:471–6. https://doi.
org/10.1007/s13213-012-0491-y
Bashan Y, Moreno M, Troyo E. Growth promotion of the seawater-
irrigated oilseed halophyte Salicornia bigelovii inoculated with man-
grove rhizosphere bacteria and halotolerant Azospirillum spp. Biol
Fertil Soils 2000;32:265–72. https://doi.org/10.1007/s003740000
246
Bolyen E, Rideout JR, Dillon MR et al. Reproducible, interactive, scal-
able and extensible microbiome data science using QIIME 2. Nat
Ferreira et al.
Biotechnol 2019;37:852–7. https://doi.org/10.1038/s41587-019-0
209-9
Callahan BJ, McMurdie PJ, Rosen MJ et al. DADA2: high-resolution
sample inference from Illumina amplicon data. Nat Methods
2016;13:581–3. https://doi.org/10.1038/nmeth.3869
Camacho-Sanchez M, Camacho M, Redondo-Gómez S et al. Bacterial
Downloaded from https://academic.oup.com/jambio/article/134/3/lxad036/7058244 by Universidade de Aveiro: Serviços de Documentação user on 20 March 2023
assemblage in Mediterranean salt marshes: disentangling the relative
importance of seasonality, zonation and halophytes. Sci Total En-
viron 2022;846:157514. https://doi.org/10.1016/j.scitotenv.2022.1
57514
Cardoso M, Silva H, Patinha C et al. From the saltpan to the plate: an
evaluation of the use of the edible halophyte Salicornia ramosissima
in catering. Ann Appl Biol 2022;180:99–108. https://doi.org/10.111
1/aab.12714
Carillo P, Mastrolonardo G, Nacca F et al. Nitrogen metabolism in du-
rum wheat under salinity: accumulation of proline and glycine be-
taine. Funct Plant Biol 2008;35:412–26. https://doi.org/10.1071/FP
08108
Chanda B, Venugopal SC, Kulshrestha S et al. Glycerol-3-phosphate
levels are associated with basal resistance to the hemibiotrophic
fungus Colletotrichum higginsianum in Arabidopsis.
Plant Physiol 2008;147:2017–29. https://doi.org/10.1104/pp.1
08.121335
Chen J, McCarl BA, Thayer A. Climate change and food se-
curity: threats and adaptation. World Agric Resour Food Se-
cur 2017;17:69–84. https://doi.org/10.1108/S1574-871520170000
017006
De Vries M. How reliable are results of pot experiments? Commun
Soil Sci Plant Anal 1980;11:895–902. https://doi.org/10.1080/0010
3628009367090
Dias JM, Pereira F, Picado A et al. A comprehensive estuarine
hydrodynamics-salinity study: impact of morphologic changes on
Ria de Aveiro (Atlantic Coast of Portugal). J Mar Sci Eng
2021;9:234. https://doi.org/10.3390/jmse9020234
Etalo DW, Jeon J-S, Raaijmakers JM. Modulation of plant chemistry by
beneficial root microbiota. Nat Prod Rep 2018;35:398–409. https:
//doi.org/10.1039/C7NP00057J
Fahad S, Hussain S, Bano A et al. Potential role of phyto-
hormones and plant growth-promoting rhizobacteria in abi-
otic stresses: consequences for changing environment. Environ
Sci Pollut Res 2015;22:4907–21. https://doi.org/10.1007/s11356-0
14-3754-2
Fernie AR, Aharoni A, Willmitzer L et al. Recommendations for report-
ing metabolite data. Plant Cell 2011;23:2477–82. https://doi.org/10
.1105/tpc.111.086272
Ferreira MJ, Cunha A, Figueiredo S et al. The root microbiome of Sal-
icornia ramosissima as a seedbank for plant growth-promoting halo-
tolerant bacteria. Appl Sci 2021;11:2233. https://doi.org/10.3390/
app11052233
Fiehn O, Sumner LW, Rhee SY et al. Minimum reporting standards
for plant biology context information in metabolomic studies.
Metabolomics 2007;3:195–201. https://doi.org/10.1007/s11306-0
07-0068-0
Figueira C, Ferreira MJ, Silva H et al. Improved germination efficiency
of Salicornia ramosissima seeds inoculated with Bacillus aryabhattai
SP1016–20. Ann Appl Biol 2019;174:319–28. https://doi.org/10.1
111/aab.12495
Gilbert GA, Gadush MV, Wilson C et al. Amino acid accumulation in
sink and source tissues of Coleus blumei Benth. during salinity stress.
J Exp Bot 1998;49:107–14. https://doi.org/10.1093/jxb/49.318.1
07
Glick BR, Penrose DM, Li J. A model for the lowering of plant ethylene
concentrations by plant growth-promoting bacteria. J Theor Biol
1998;190:63–8. https://doi.org/10.1006/jtbi.1997.0532
Warnes GR, Bolker B, Bonebakker L et al. gplots: various R program-
ming tools for plotting data. R Package Version 3.0.3 (software).
http://github.com/talgalili/gplots 2021.
Han W, He M. The application of exogenous cellulase to im-
prove soil fertility and plant growth due to acceleration of straw
Biostimulation of Salicornia crops
decomposition. Bioresour Technol 2010;101:3724–31. https://doi.
org/10.1016/j.biortech.2009.12.104
Hashim A, Ismail SI, Alsultan W et al. Control of gray mold dis-
ease of tomato caused by Botrytis cinerea using bacterial secondary
metabolites. Malays Appl Biol 2020;49:89–97. https://doi.org/10.5
5230/mabjournal.v49i5.1641
Herlemann DP, Labrenz M, Jürgens K et al. Transitions in bacterial
communities along the 2000 km salinity gradient of the Baltic Sea.
ISME J 2011;5:1571–9. https://doi.org/10.1038/ismej.2011.41
Ishizawa H, Kuroda M, Inoue D et al. Community dynamics of
duckweed-associated bacteria upon inoculation of plant growth-
promoting bacteria. FEMS Microbiol Ecol 2020;96:fiaa101. https:
//doi.org/10.1093/femsec/fiaa101
Jeong J, Kim T, Choi W et al. Optimum salinity concentration and nitro-
gen fertilization for Salicornia herbaecea growth in reclaimed land.
J Korean Soc Int Agric 2013;2:62–7. https://doi.org/10.12719/KSI
A.2013.25.1.062
Jun-Feng Y, Gu F, Hai-Yan M et al. Effect of nitrate on root develop-
ment and nitrogen uptake of Suaeda physophora under NaCl salin-
ity. Pedosphere 2010;20:536–44.
Kaur C, Selvakumar G, Ganeshamurthy A. Rhizocompetence of applied
bioinoculants. In: Singh D, Singh H, Prabha R (eds.), Plant–Microbe
Interactions in Agro-Ecological Perspectives. Singapore: Springer,
2017, 501–11.
Kearl J, McNary C, Lowman JS et al. Salt-tolerant halophyte rhizo-
sphere bacteria stimulate growth of alfalfa in salty soil. Front Mi-
crobiol 2019;10:1849. https://doi.org/10.3389/fmicb.2019.01849
Klindworth A, Pruesse E, Schweer T et al. Evaluation of general 16S
ribosomal RNA gene PCR primers for classical and next-generation
sequencing-based diversity studies. Nucleic Acids Res 2013;41:e1.
https://doi.org/10.1093/nar/gks808
Komaresofla BR, Alikhani HA, Etesami H et al. Improved growth
and salinity tolerance of the halophyte Salicornia sp. by co-
inoculation with endophytic and rhizosphere bacteria. Appl Soil
Ecol 2019;138:160–70. https://doi.org/10.1016/j.apsoil.2019.02.0
22
Kopka J, Schauer N, Krueger S et al. GMD@CSB.DB: the Golm
Metabolome Database. Bioinformatics 2005;21:1635–8. https://do
i.org/10.1093/bioinformatics/bti236
Lertcanawanichakul M, Sawangnop S. a Comparison of two
methods used for measuring the antagonistic activity of
Bacillus species. Walailak J Sci Technol (WJST) 2011;5:
161–71.
Lisec J, Schauer N, Kopka J et al. Gas chromatography mass
spectrometry–based metabolite profiling in plants. Nat Protoc
2006;1:387–96. https://doi.org/10.1038/nprot.2006.59
Lombardi T, Bertacchi A, Pistelli L et al. Biological and agronomic traits
of the main halophytes widespread in the Mediterranean region as
potential new vegetable crops. Horticulturae 2022;8:195. https://do
i.org/10.3390/horticulturae8030195
Luedemann A, Strassburg K, Erban A et al. TagFinder for the
quantitative analysis of gas chromatography–mass spec-
trometry (GC–MS)-based metabolite profiling experiments.
Bioinformatics 2008;24:732–7. https://doi.org/10.1093/bioinforma
tics/btn023
McMurdie PJ, Holmes S. Phyloseq: an R package for reproducible inter-
active analysis and graphics of microbiome census data. PLoS One
2013;8:e61217. https://doi.org/10.1371/journal.pone.0061217
Martínez-Viveros O, Jorquera MA, Crowley DE et al. Mechanisms and
practical considerations involved in plant growth promotion by rhi-
zobacteria. J Soil Sci Plant Nutr 2010;10:293–319. https://doi.org/
10.4067/S0718-95162010000100006
Mendiburu F, Yaseen M. Agricolae: statistical procedures for agricul-
tural research. http://.tarwi.lamolina.edu.pe/∼fmendiburu/ R Pack-
age Version 14.0. 2020.
Mesa-Marín J, Pérez-Romero JA, Mateos-Naranjo E et al. Effect of
plant growth-promoting rhizobacteria on Salicornia ramosissima
seed germination under salinity, CO2 and temperature stress. Agron-
omy 2019;9:655. https://doi.org/10.3390/agronomy9100655
13
Mesa-Marín J, Pérez-Romero JA, Redondo-Gómez S et al. Impact of
plant growth promoting bacteria on Salicornia ramosissima eco-
physiology and heavy metal phytoremediation capacity in estuarine
soils. Front Microbiol 2020;11:553018. https://doi.org/10.3389/fm
icb.2020.553018
Morales-García YE, Baez A, Quintero-Hernández V et al. Bacterial mix-
Downloaded from https://academic.oup.com/jambio/article/134/3/lxad036/7058244 by Universidade de Aveiro: Serviços de Documentação user on 20 March 2023
tures, the future generation of inoculants for sustainable crop pro-
duction. In: Maheshwari D, Dheeman S (eds.), Field Crops: Sus-
tainable Management by PGPR. Sustainable Development and Bio-
diversity. Cham: Springer, 2019, 11–44.
Morcillo RJ, Manzanera M. The effects of plant-associated bacterial
exopolysaccharides on plant abiotic stress tolerance. Metabolites
2021;11:337. https://doi.org/10.3390/metabo11060337
Nephali L, Piater LA, Dubery IA et al. Biostimulants for plant growth
and mitigation of abiotic stresses: a metabolomics perspective.
Metabolites 2020;10:505. https://doi.org/10.3390/metabo101205
05
Oliveira V, Gomes NCM, Almeida A et al. Microbe-assisted phytore-
mediation of hydrocarbons in estuarine environments. Microb Ecol
2015;69:1–12. https://doi.org/10.1007/s00248-014-0455-9
Pan J, Peng F, Tedeschi A et al. Do halophytes and glycophytes differ
in their interactions with arbuscular mycorrhizal fungi under salt
stress? A meta-analysis. Bot Stud 2020;61:1–13. https://doi.org/10
.1186/s40529-020-00290-6
Pang Z, Chen J, Wang T et al. Linking plant secondary metabolites and
plant microbiomes: a review. Front Plant Sci 2021;12:621276. http
s://doi.org/10.3389/fpls.2021.621276
Pérez-Romero JA, Barcia-Piedras J-M, Redondo-Gómez S et al. Impact
of short-term extreme temperature events on physiological perfor-
mance of Salicornia ramosissima J. Woods under optimal and sub-
optimal saline conditions. Sci Rep 2019a;9:1–12. https://doi.org/10
.1038/s41598-018-37346-4
Pérez-Romero JA, Duarte B, Barcia-Piedras J-M et al. Investi-
gating the physiological mechanisms underlying Salicornia
ramosissima response to atmospheric CO2 enrichment un-
der coexistence of prolonged soil flooding and saline excess.
Plant Physiol Biochem 2019b;135:149–59. https://doi.org/10.101
6/j.plaphy.2018.12.003
Pérez-Romero JA, Redondo-Gómez S, Mateos-Naranjo E. Growth and
photosynthetic limitation analysis of the Cd-accumulator Salicornia
ramosissima under excessive cadmium concentrations and optimum
salinity conditions. Plant Physiol Biochem 2016;109:103–13. https:
//doi.org/10.1016/j.plaphy.2016.09.011
Pessoa LGM, Silva LFS, Freire MBGS et al. Effectiveness of soil condi-
tioners to enhance salt extraction ability of Salicornia ramosissima in
saline-sodic soil for different soil moisture contents. Int J Phytorem
2022;24:447–55. https://doi.org/10.1080/15226514.2021.195292
4
Piernik A, Hrynkiewicz K, Wojciechowska A et al. Effect of halotol-
erant endophytic bacteria isolated from Salicornia europaea L. on
the growth of fodder beet (Beta vulgaris L.) under salt stress. Arch
Agron Soil Sci 2017;63:1404–18. https://doi.org/10.1080/036503
40.2017.1286329
Poorter H, Fiorani F, Pieruschka R et al. Pampered inside, pestered out-
side? Differences and similarities between plants growing in con-
trolled conditions and in the field. New Phytol 2016;212:838–55.
https://doi.org/10.1111/nph.14243
Quast C, Pruesse E, Yilmaz P et al. The SILVA ribosomal RNA gene
database project: improved data processing and web-based tools.
Nucleic Acids Res 2012;41:D590–6. https://doi.org/10.1093/nar/gk
s1219
R Core Team. R: A Language and Environment for Statistical Comput-
ing. R Foundation for Statistical Computing. Vienna, Austria. 2014.
http://www.R-projectorg accessed february 2023.
Ravi S, Young T, Macinnis-Ng C et al. Untargeted metabolomics in
halophytes: the role of different metabolites in New Zealand man-
groves under multi-factorial abiotic stress conditions. Environ Exp
Bot 2020;173:103993. https://doi.org/10.1016/j.envexpbot.2020.1
03993
14
Rohart F, Gautier B, Singh A et al. mixOmics: an R package for
‘omics feature selection and multiple data integration. PLoS Com-
put Biol 2017;13:e1005752. https://doi.org/10.1371/journal.pcbi.
1005752
RStudio Team. RStudio: Integrated Development for R. RStudio, PBC:
Boston, MA, 2020.
Sanjosé I, Navarro-Roldán F, Montero Y et al. The bioconcentration
and the translocation of heavy metals in recently consumed Salicor-
nia ramosissima J. Woods in highly contaminated estuary marshes
and its food risk. Diversity 2022;14:452. https://doi.org/10.3390/d1
4060452
Saraiva M, Correia C, Cunha I et al. 2019 Interpretação de resulta-
dos de ensaios microbiológicos em alimentos prontos para consumo
e em superfícies do ambiente de preparação e distribuição alimen-
tar: valores-Guia. INSA, IP—Instituto Nacional de Saúde Doutor
Ricardo Jorge: Lisboa, Portugal.
Schauer N, Steinhauser D, Strelkov S et al. GC–MS libraries for the
rapid identification of metabolites in complex biological samples.
FEBS Lett 2005;579:1332–7. https://doi.org/10.1016/j.febslet.20
05.01.029
Shelp BJ, Mullen RT, Waller JC. Compartmentation of GABA
metabolism raises intriguing questions. Trends Plant Sci
2012;17:57–9. https://doi.org/10.1016/j.tplants.2011.12.006
Shen W, Wei Y, Dauk M et al. Involvement of a glycerol-3-phosphate
dehydrogenase in modulating the NADH/NAD+ ratio provides evi-
dence of a mitochondrial glycerol-3-phosphate shuttle in Arabidop-
sis. Plant Cell 2006;18:422–41. https://doi.org/10.1105/tpc.105.03
9750
Shi Y-w, Lou K, Li C et al. Illumina-based analysis of bacterial diversity
related to halophytes Salicornia europaea and Sueada aralocaspica. J
Microbiol 2015;53:678–85. https://doi.org/10.1007/s12275-015-5
080-x
Silva AM, Lago JP, Pinto D et al. Salicornia ramosissima bioac-
tive composition and safety: eco-friendly extractions approach
(microwave-assisted extraction vs. conventional maceration). Appl
Sci 2021;11:4744.
Ferreira et al.
Silva H, Caldeira G, Freitas H. Salicornia ramosissima population dy-
namics and tolerance of salinity. Ecol Res 2007;22:125–34. https:
//doi.org/10.1007/s11284-006-0008-x
Spencer JF, de Spencer ALR. Food Microbiology Protocols. Humana
Totowa, NJ: Springer Science & Business Media, 2001.
Sumner LW, Amberg A, Barrett D et al. Proposed minimum reporting
Downloaded from https://academic.oup.com/jambio/article/134/3/lxad036/7058244 by Universidade de Aveiro: Serviços de Documentação user on 20 March 2023
standards for chemical analysis. Metabolomics 2007;3:211–21. ht
tps://doi.org/10.1007/s11306-007-0082-2
Szymańska S, Borruso L, Brusetti L et al. Bacterial microbiome of root-
associated endophytes of Salicornia europaea in correspondence to
different levels of salinity. Environ Sci Pollut Res 2018;25:25420–
31. https://doi.org/10.1007/s11356-018-2530-0
Ungar IA. Seed dimorphism in Salicornia europaea L. Bot Gaz
1979;140:102–8. https://doi.org/10.1086/337063
Vagelas I, Gravanis F, Gowen S. Control of Fusarium oxyspo-
rum and Meloidogyne spp. with Pseudomonas oryzihabitans. In:
The BCPC International Congress: Crop Science and Technology,
Volumes 1 and 2. Proceedings of an international congress held at
the SECC, Glasgow, Scotland, UK, 10-12 November 2003, p. 419–
24. Alton: British Crop Protection Council, 2003.
Walitang DI, Kim K, Madhaiyan M et al. Characterizing endophytic
competence and plant growth promotion of bacterial endophytes
inhabiting the seed endosphere of rice. BMC Microbiol 2017;17:1–
13. https://doi.org/10.1186/s12866-017-1117-0
Yang Y, Zhao J, Liu P et al. Glycerol-3-phosphate metabolism in wheat
contributes to systemic acquired resistance against Puccinia stri-
iformis f. sp. tritici. PLoS One 2013;8:e81756. https://doi.org/10.1
371/journal.pone.0081756
Yoo S-J, Weon H-Y, Song J et al. Induced tolerance to salinity stress by
halotolerant bacteria Bacillus aryabhattai H19-1 and B. mesonae
H20-5 in tomato plants. J Microbiol Biotechnol 2019;29:1124–36.
https://doi.org/10.4014/jmb.1904.04026
Yu K, Soares JM, Mandal MK et al. A feedback regulatory loop between
G3P and lipid transfer proteins DIR1 and AZI1 mediates azelaic-
acid-induced systemic immunity. Cell Rep 2013;3:1266–78. https:
//doi.org/10.1016/j.celrep.2013.03.030