39-Kisiel Et Al., 2016

Planta (2016) 243:1169–1189
DOI 10.1007/s00425-016-2469-7
ORIGINAL ARTICLE
Medicago truncatula Gaertn. as a model for understanding

the mechanism of growth promotion by bacteria from rhizosphere
and nodules of alfalfa
Anna Kisiel1 • Ewa Ke˛pczyńska1
Received: 25 August 2015 / Accepted: 14 January 2016 / Published online: 10 February 2016
Ó The Author(s) 2016. This article is published with open access at Springerlink.com
Abstract truncatula root development involves also root interac-

Main conclusion The present study showed all the 16 tion with pseudomonads, known to produce 2,4-di-
strains isolated and identified from the alfalfa rhizo- acetylphloroglucinol (DAPG), a secondary metabolite
sphere and nodules, and registered in GenBank, to be reported to alter the root architecture by interacting with
good candidates for targeted use in studies addressing an auxin-dependent signaling pathway. Inoculation of
the rather weak known mechanism of plant growth seedlings with Pseudomonas brassicacearum KK 5, a
promotion, including that of Medicago truncatula, a bacterium known for its lowest ability to produce indolic
molecular crop model. compounds, the highest ACCD activity and the presence
of the phlD gene responsible for DAPG precursor syn-
Based on physiological, biochemical and molecular
thesis, resulted in a substantial promotion of root
analysis, the 16 isolates obtained were ascribed to the
development. Inoculation with the strain increased the
following five families: Bacillaceae, Rhizobiaceae,
endogenous IAA level in M. truncatula leaves after
Xantomonadaceae, Enterobacteriaceae and Pseudomon-
inoculation of 5-week-old seedlings. Three other strains
adaceae, within which 9 genera and 16 species were
examined in this study also increased the IAA level in
identified. All these bacteria were found to significantly
the leaves upon inoculation. Moreover, several other
enhance fresh and dry weight of root, shoots and whole
factors such as mobilization of phosphorus and zinc to
5-week-old seedlings. The bacteria were capable of the
make them available to plants, iron sequestration by
in vitro use of tryptophan to produce indolic compounds
siderophore production and the ability to ammonia pro-
at various concentrations. The ability of almost all the
duction also contributed substantially to the phytostim-
strains to enhance growth of seedlings and individual
ulatory biofertilizing potential of isolated strains. There
roots was positively correlated with the production of the
is, thus, evidence that Medicago truncatula growth
indolic compounds (r = 0.69; P = 0.0001), but not
promotion by rhizobacteria involves more than one
with the 1-aminocyclopropane-1-carboxylate deaminase
mechanism.
(ACCD) activity (no correlation). For some strains, it
was difficult to conclude whether the growth promotion
Keywords ACC deaminase Bacteria identification
was related to the production of indolic compounds or to
Growth promotion Indole acetic acid (IAA) content
the ACCD activity. It is likely that promotion of M.
PGPR traits PhlD gene detection
Electronic supplementary material The online version of this Abbreviations

article (doi:10.1007/s00425-016-2469-7) contains supplementary
material, which is available to authorized users. ACC 1-Aminocyclopropane-1-carboxylate acid
ACCD 1-Aminocyclopropane-1-carboxylate deaminase
& Ewa Ke˛pczyńska DAPG 2,4-Diacetylphloroglucinol
ekepcz@univ.szczecin.pl
IAA Indole acetic acid
1
Department of Plant Biotechnology, Faculty of Biology, PGPR Plant growth promoting rhizobacteria
University of Szczecin, Wa˛ska 13, 71-415 Szczecin, Poland
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1170 Planta (2016) 243:1169–1189
Introduction (Khan et al. 1990; Glick 1995; Gholami et al. 2009;

Bhattacharyya and Jha 2012). PGPR used include genera
The family Fabaceae, called also Leguminosae, is the third such as Bacillus, Pseudomonas, Erwinia, Serratia,
largest family among angiosperms and second only to the Arthrobacter, Micrococcus, Flavobacterium, Azotobacter,
Gramineae in their importance for humans (Graham and Azospirillum, Rhizobium and Agrobacterium (Verma et al.
Vance 2003). The family’s most commercially important 2010; Glick 2012). Although the mechanisms used by
species worldwide include the soybean (Glycine max), the PGPR to directly or indirectly promote plant growth are not
garden pea (Pisum sativum), the peanut (Arachis hypo- fully understood yet, several have been suggested. Based
gaea) and the alfalfa (Medicago sativa). Those plants are on their mode of action, PGPR are classified as biofertil-
the most important source of protein and oil for humans izers, phytostimulants and biopesticides, although many
and animals, and enrich the soil with nitrogen. They are PGPR often use more than one mode of plant growth
also important as a fuelwood and with respect to carbon enhancement (Bhattacharyya and Jha 2012; Glick 2012). A
(C) sequestration (Abberton 2010). Among forage crops, direct phytostimulation may include the production of
cultivation of M. sativa covers the largest acreage world- phytohormones, e.g., gibberellins, cytokinins and auxins as
wide and produces the largest profit. Furthermore, the well as modulation of ethylene level, a gaseous phytohor-
alfalfa, one of the most important small legumes, is highly mone, by bacterial ACC deaminase (ACCD) or enhanced
adaptable to different climatic and soil conditions, which provision of nutrients. Indole acetic acid (IAA) produced
facilitate its cultivation worldwide. Cultivation of alfalfa by bacteria colonizing the plant rhizosphere is considered
has, for many years, been known to bring benefits for the to act in conjunction with the endogenous plant IAA to
soil, e.g., structure improvement, protection against wind stimulate root proliferation and elongation resulting in a
and water erosion, nitrogen and organic enrichment, and more branched root system architecture, which enhances
weed population reduction. The increasing concerns about the host’s uptake of minerals and nutrients from the soil
a decline in soil organic matter content and fertility as well (Mantelin and Touraine 2004; Mantelin et al. 2006;
as the rising costs of energy and nitrogen fertilizers have Spaepen et al. 2007; Shokri and Emtiazi 2010). However,
renewed the interest in legumes. PGPR which do not produce auxin are known to be able to
Recently, the role of legumes and their contribution to modify the endogenous transport of plant IAA or to regu-
both the sustainable intensification of agricultural produc- late auxin homeostasis by, e.g., production of volatile
tion and the livelihoods of small-holder organic farmers in organic compounds (VOC), resulting in the same root
many parts of the world have featured on the research and architecture effects (Zhang et al. 2007; Contesto et al.
economic agendas. Alfalfa is grown for hay, pasture, seed 2010; Zamioudis et al. 2013). Some PGPR belonging to
and—in some areas—as a major crop for the dehydration fluorescent pseudomonads are well-known producers of the
industry. It also finds its use in many medical formulae, and antimicrobial compound 2,4-diacethylphloroglucinol
the available preparations such as alfalfa tonic and malt are (DAPG) which, at a low concentration, can enhance root
known for their health benefits on account of a variety of branching (Brazelton et al. 2008; Couillerot et al. 2011).
bioactive natural products the alfalfa-based preparations One of the most important factors in seeking an increase
contain (Gholami et al. 2014). The seed production in the of the PGPR efficacy is to find the best bacteria available.
temperate climate is limited mainly by the too short Bhattacharyya and Jha (2012) listed the commercially
growing season due to the low temperature and insufficient available products used worldwide in plant (mainly cereal)
sunlight resulting in delay of the flowering time. Healthy growth promotion, but the list seems to be too short. This is
and well-rooted plants obtained in a relatively short time due, i.a., to the fact that neither all the mechanisms the
ensures their withstanding adverse conditions and the bacteria use for promoting plant growth have been fully
possibility of the perennial alfalfa producing seeds in the elucidated, nor the plant response mechanisms during
year of sowing. The measures to improve plant quality and PGPR treatment are completely known (Vacheron et al.
health include the use of synthetic fertilizers and pesticides. 2013).
In the context of organic farming, however, it is mandatory Thus, it is essential to explain these mechanisms at
to look for other, more efficient and also environmentally various levels, including molecular. Such studies should
safe methods to improve germination, seedling emergence avoid using Arabidopsis thaliana, a weed serving to date as
and plant health. One such method can be the use of rhi- a molecular model, but should instead use a crop plant,
zosphere microorganisms, including the plant growth— Medicago truncatula Gaertn. known as the barrel medic. In
promoting rhizobacteria (PGPR) (Chandler et al. 2008; recent years has this plant gained great interest; its attrac-
Glick 2012). Numerous authors have reported application tiveness compared with alfalfa (M. sativa) is related to its
of PGPR, via seed coating or as inoculants after seedlings more rapid vegetative growth (12–16 weeks from seed to
have emerged, to improve seedling growth of many plants seed) and a high seed production, associated with low
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Planta (2016) 243:1169–1189 1171
requirements for light, temperature, and substrate compo- 20 ml of TSB or 2xYT medium and incubated for another
sition. In contrast to the alfalfa, it does not require polli- 24 h. Following the two incubations, 1 ml of both sus-
nators such as bees (Cook 1999; Rose 2008). Moreover, M. pensions each was transferred to 20 ml of minimal medium
truncatula belongs to the phylogenetic group which also and DF salts (Dworkin and Foster 1958), respectively,
includes alfalfa, peas, beans, chickpeas and clover. It can, supplemented with ammonium sulfate or M9 salts sup-
thus serve as a model plant because it has a small diploid plemented with ammonium chloride as a nitrogen source.
genome which is already known (alfalfa is tetraploid). After incubation, a 1 ml aliquot was removed from the
Furthermore, genetic mapping of M. truncatula and M. culture and transferred to 20 ml DF salts or M9 salts with
sativa showed a high level of synteny. This fact will allow 5 mM ACC as a source of nitrogen, and was grown for
future use of genetic and genomic tools, created on the 24 h under conditions identical as those used in the first
basis of M. truncatula, to improve productivity and culti- incubation. A 100 ll portion of the final culture was plated
vation of alfalfa by, i.a., understanding of plant growth onto solid DF or M9 salts minimal medium containing
promotion mechanisms by PGPR. It is a model plant for 5 mM ACC and 2 % Bacto-Agar, and was incubated for
molecular and genetic studies of legumes mainly because 72 h at 28 °C. Morphologically different colonies appear-
of its symbiotic association with rhizobia, but also on ing on the medium were isolated and purified by streaking
account of its interaction with other non-symbiotic growth- on plates with the same medium, placed in cryovials
promoting bacteria (PGPR), arbuscular mycorrhiza (AM) (Scharlau Cryoinstant) and kept at -80 °C for further
fungi or pests and pathogens (Barker et al. 1990; Hohnjec characterization and identification.
et al. 2006; Rose 2008).
To date, no reports on the application of rhizobacteria to Phenotypic characterization of bacterial isolates
promote the development of M. truncatula have been
published, and no data on bacteria species identified from Physiological and biochemical characteristics of the bac-
the M. sativa rhizosphere. Therefore, the work reported terial isolates were examined according to the methods
here was aimed at: (1) isolating bacteria from the rhizo- described in Bergey’s Manual of Determinative Bacteri-
sphere of the legume M. sativa L.; (2) evaluating their ology (Holt et al. 1994). The bacterial isolates were grown
physiological and genetic characteristics; (3) screening on Tryptic Soy Agar (TSA) at 28 °C for 24 h. Phenotypic
them for their promoting capacities with respect to M. characterization of the isolates was conducted based on
truncatula Gaertn., and finally evaluating their application their colony morphology. Subsequently, the cellular mor-
as environmentally friendly adjuncts to agriculture prac- phology of pure cultures of the isolate (after Gram staining)
tice—as biostimulants and biofertilizers. was analyzed by light microscopy. PGPR motility was
determined and some standard biochemical tests (starch
hydrolysis, lactose fermentation, resistance to streptomycin
Materials and methods and ability to fluorescence) were applied following meth-
ods described in Bergey’s Manual of Determinative Bac-
Isolation of rhizobacteria teriology (Holt et al. 1994).
Strains of bacteria were isolated from the rhizosphere and PCR amplification and sequencing
nodules of M. sativa L. from the region of Western
Pomerania in Poland. The Medicago roots were washed All the strains isolated were characterized based on 16S
with 5 ml of sterile distilled water and the soil solution, ribosomal RNA (rRNA) gene sequences and additionally
thus, obtained was used for isolation of non-symbiotic on the rpoD (coding RNA polymerase sigma factor) and
bacteria using the method of Penrose and Glick (2003) with gyrB (coding gyrase B) genes of the strains assigned to the
some modifications. The root nodules were separated from genus Pseudomonas (Cladera et al. 2004). The bacteria
the roots and sterilized in 95 % ethanol for 5 min; they were cultured in TSB or 2xYT medium at 28 °C (the
were then rinsed with sterile water and homogenized using shaker set at 200 rpm). A 1-ml portion of the cell sus-
a micro-pestle. The root nodule homogenate was used for pension was centrifuged at 5000g for 10 min. The cell
isolation of symbiotic bacteria. The soil solution and the pellets were resuspended in 300 ll lyse buffer and used to
nodule homogenate were placed in 20 ml of Tryptic Soy purify the genomic DNA using the DNA Extraction Kit
Broth (TSB) and 2xYT (Liquid Yeast Tryptic) medium, (Eurx, Gdansk, Poland). The following primers were used
respectively, in 50-ml Falcon tubes. The suspensions were for the polymerase chain reaction (PCR) amplification of
incubated in a shaker incubator (200 rpm) at 28 °C. After the 16S ribosomal RNA: 16S rRNA-F (50 -AGCGGCG
24 h, a 1 ml of the growing culture was transferred to GACGGGTGAGTAATG-30 ) and 16S rRNA-R (50 -AAG
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1172 Planta (2016) 243:1169–1189
GAGGGGATCCAGCCGCA-30 ) (Young et al. 2004) and were inoculated with 10 ml bacteria inoculum (density of
UP-1E (50 -CAGGAAACAGCTATGACCAYGSNGGNG 108 CFU ml-1) or 10 mM MgSO4. The inoculum of non-
GNAARTTYRA-30 ) and APrU (50 -TGTAAAACGACGG symbiotic and symbiotic bacteria was prepared by growing
CCAGTGCNGGRTCYTTYTCYTGRCA-30 ) for gyrB bacterial cells in 20 ml of TSB and 2xYT medium,
gene amplification and 70Fs (50 -ACGACTGACCCGGT respectively, and incubated in a shaker incubator (200 rpm)
ACGCATGTA-30 ) and 70Rs (50 -ATAGAAATAACCAGA at 28 °C. The density of each culture was measured in a
CGTAAGTT-30 ) for rpoD gene amplification (Yamamoto Shimadzu UV–Vis 1800 spectrophotometer at 600 nm. The
et al. 2000; Cladera et al. 2004). The all gene amplification medium was subsequently separated from the culture by
was performed in 25 ll of the reaction mixture containing centrifugation (8000g/10 min/4 °C). The cells were sus-
1 lM of primers, 0.2 lM of each dNTP, 4 mM MgCl2, 1U pended in 20 ml of sterile 10 mM MgSO4. Following
Taq DNA Hot Start polymerase (Promega) and 1 ll of centrifugation (8000g/10 min/4 °C), the supernatant was
DNA template. PCR was performed in a thermal cycler discarded and the washing procedure was repeated twice.
(Biometra) with an initial denaturation (96 °C for 2 min) The cell suspension was diluted fivefold by the addition of
followed by 35 cycles of amplification (denaturation at sterile 10 mM MgSO4, and 10 ml portions of the dilution
96 °C for 15 s, annealing at 58 °C for 16S rRNA and were used for seedling inoculation with a pipette, at a
50 °C for rpoD and gyrB for 15 s, extension at 72 °C for distance of 1 cm from the stem. The plants were irrigated
1 min 30 s for 16S rRNA and 45 s for rpoD and gyrB) and with distilled water. The soil was fertilized once a week
the single final extension (72 °C for 5 min). A 2-ll aliquot with N-low fertilizer (pH 5.8) containing (mg l-1): KNO3
of amplified PCR product was separated by gel elec- (13), KH2PO4 (17), CaCl2 9 H2O (46), MgSO4 9 7H2O
trophoresis in 1 % agarose with ethidium bromide in TBE (15), K2SO4 (27), EDTA2Na2Fe (5) and microtraces
buffer for 30 min and viewed under UV light. The 1500, (lg l-1) of: H3BO3 (371), MnSO4 9 H2O (209), KCl
786 and 849 bp-long products of 16S rRNA, rpoD and (337), ZnSO4 9 7H2O (33), (NH4)6Mo7O24 9 4H2O (33),
gyrB, respectively, were purified using the QIAquick PCR CuSO4 9 5H2O (17), H2SO4 (17). The shoot, root fresh
Purification Kit (Qiagen) and sequenced by Genomed and dry (dried at 105 °C for 24 h) weights were determined
(Warszawa, Poland). 4 weeks after inoculation.
Multiple-sequence alignments of 16S rRNA, rpoD and
gyrB sequences were constructed with CLUSTAL W2 Plant growth-promoting traits
software, and maximum likelihood trees were constructed
with the neighbour-joining technique (Saitou and Nei All the strains used in M. truncatula growth experiments
1987) using MEGA v. 6.0 software subjected to BLAST were analyzed for their ability to produce indolic com-
analysis (Tamura et al. 2013) with those deposited in the pounds, their ACCD activity, siderophore, ammonia and
GenBank in NCBI database (http://www.blast.ncbi.nlm. organic acid production as well as for their ability to sol-
nih.gov). Bootstrap tests using 1000 replicates were per- ubilize phosphate and zinc compounds. Additionally, the
formed to test the robustness of each phylogeny (Felsen- phlD gene detection in pseudomonads was analyzed. In
stein 1985). addition, the IAA content in leaves after 3 and 7 days from
inoculation of 5-week-old seedlings with selected strains
Growth room pot experiment was determined.
The soil mixture containing sand and perlite (1:1, w/w) was Bacterial indolic compounds production
sterilized in an autoclave under pressure for 30 min during
two consecutive days, and the mixture was placed in The indole compounds quantification was performed with
9 9 9 cm plastic pots. The seeds of M. truncatula Gaertn. Salkowski’s reagent (Khalid et al. 2004). The non-symbi-
Jemalong J5 ecotype (provided by INRA, Versailles, otic and symbiotic bacteria were cultured overnight in
France) were sterilized and scarified using 95 % H2SO4 for 10 ml of TSB and 2xYT medium, respectively, in a shaker
10 min, rinsed thoroughly with sterile distilled water, incubator (200 rpm) in the dark at 28 °C. Subsequently,
placed on water-saturated Whatman No. 1 disks in Petri 20 ll aliquots were transferred to 8 ml of DF or M9 salts
dishes, and incubated at 4 °C for 4 days. Subsequently, the minimal medium supplemented with L-tryptophan
seeds were incubated at 20 °C for 24 h and the seedlings (500 lg ml-1). The density of each culture was measured
were planted in the pots. The seedlings were grown for 1 spectrophotometrically at 600 nm (ca. 5 9 108
week under controlled growth room conditions with light– CFU ml-1). Subsequently, the bacterial cells were
dark and temperature cycles (16 h light at 24 °C; 8 h dark removed from the culture medium by centrifugation
at 20 °C). The light density was 150 lmol m-2 s-1 (Green (8000g/10 min). A 1-ml aliquot of the supernatant was
Power LED modules, Philips). The one-week-old seedlings mixed vigorously with 4 ml of Salkowski’s reagent
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(150 ml of concentrated H2SO4, 250 ml of distilled water, Raaijmakers et al. (1997). PhlD gene amplification was
7.5 ml of 0.5 M FeCl3 9 6H2O) and incubated at 20 °C for performed in 25 ll reaction mixture containing 1 lM of
20 min. Indole production was measured spectrophoto- each primer, 0.2 lM of DNA template each dNTP, 4 mM
metrically using absorbance at 535 nm. The concentration MgCl2, 1U Tag Hot Start polymerase (Promega) and 1 ll
of indolic compounds in each culture medium was deter- of DNA template. This reaction mixture was incubated in a
mined by comparison with a standard curve plotted for thermal cycler (Biometra) with initial denaturation (96 °C
pure IAA (Sigma-Aldrich) and reported as lg IAA ml-1 of for 2 min) and then cycled 35 times through the following
bacterial culture (density ca. 5 9 108 CFU ml-1). temperature profile: denaturation at 96 °C for 15 s,
annealing at 60 °C for 15 s, extension at 72 °C for 1 min,
ACC deaminase activity and the single final extension at 72 °C for 5 min. A 2-ll
aliquot of the amplified PCR product was separated by gel
The ACCD activity was measured by a modified ACCD electrophoresis in 1 % agarose with ethidium bromide in
activity assay following Penrose and Glick (2003) and TBE buffer for 30 min and was visualized with a UV
Honma and Shimomura (1978). The bacterial cells were transilluminator. The 720-bp PCR product was sequenced
incubated overnight in 20 ml of TSB or 2xYT medium in a by Genomed. The phylogenetic tree was inferred by the
shaker incubator (200 rpm) at 28 °C. After incubation, the neighbour-joining method using MEGA v 6.0.
bacterial culture was centrifuged (8000g/10 min/4 °C).
The supernatant was removed and the cells were washed Phosphate and zinc compounds solubilization
with 10 ml DF or M9 salts minimal medium, and cen-
trifuged again. The cells were suspended in 7.5 ml DF or The solubilization was detected by the formation of
M9 salts minimal medium with 5 mM ACC. The bacterial transparent halos (mm) surrounding bacterial colonies on
cells were incubated at 28 °C for 24 h. The culture was the Pikovskaya medium containing insoluble CaHO13P3 or
centrifuged (8000g/10 min/4 °C), the supernatant was ZnO after 10 days at 28 °C (Pikovskaya 1948).
removed, and the cells were washed with 0.1 M Tris–HCl
(pH 7.6). The washing procedure was repeated twice. A Medium acidification
1-ml bacterial suspension was centrifuged at 16,000g for
5 min and the supernatant was removed. The pellet was To detect the ability of the bacterial strains to acidify
suspended in 600 ll 0.1 M Tris–HCl (pH 8.0) to which medium, the isolates were spotted on the TSA medium (pH
300 ll toluene were added, and the cell suspension was 7.5) with bromothymol blue, which allowed to detect
vortexed for 30 s. A 100-ll aliquot of protein extract was acidification (DuPree and Wilcox 1977). The medium
kept from each sample to determine protein concentration surrounding the organic acid-producing strains changed
of each sample using the Bradford method (Bradford colour from blue to green as pH dropped below 7.
1976). Toluenized cells (200 ll) were placed in micro-
centrifuge tubes with 20 ll of 0.5 M ACC and incubated at Siderophore production
30 °C for 15 min. Following the addition of 1 ml of
0.56 M HCl and mixing, the cell suspension was cen- The siderophore production was detected by the production of
trifuged (16,000g/5 min/RT). Subsequently, 800 ll of orange halos surrounding the bacterial colonies on a standard
0.56 M HCl was added to 1 ml of the supernatant and the Chrome Azurol-S (CAS) agar plates after 72 h at 28 °C
mixture was vortexed. The dinitrophenylhydrazine reagent (Schwyn and Neilands 1987; Alexander and Zuberer 1991).
(0.2 % 2,4-DNP in 2 M HCl) in the amount of 300 ll was
added to the glass tube and incubated at 30 °C for 30 min. Ammonia production
Before the measurement, 2 ml of 2 N NaOH was added
and the absorbance was measured at 540 nm. The ACCD The ammonia production by the bacterial strains was
activity was determined by measuring the production of a- determined after their incubation in peptone water for 72 h
ketobutyrate and comparing the result with a standard at 28 °C (Cappuccino and Sherman 1992). Nessler’s
curve using an a-ketobutyrate (Honma and Shimomura reagent (0.5 ml) was added to each tube. The development
1978). The enzyme activity was expressed as micromoles of brown to yellow colour was a positive test for the
of a-KB mg protein-1 h-1. ammonia production.
PCR analysis with specific phlD primers Protein content determination
Bacterial DNA was used for PCR amplification with The protein content of the enzyme extract was determined
specific phlD gene primers (Phl2a and Phl2b) designed by by the dye binding method using the Bradford reagent
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(Sigma-Aldrich) and bovine serum albumin (Sigma- Colonies formed by most isolates (75 %) were circular, the
Aldrich) as standard (Bradford 1976). remaining 25 % were irregular in shape. All the colonies
had smooth edges, their texture being smooth as well. As
IAA determination in seedling leaves by UPLC-MS/ many as 81 % of the colonies were convex, one (KK 3)
MS was umbonate, one (KK 11) was crater-like, and one (KK
13) was flat. Twelve of the 16 isolates formed creamy
Extraction and purification of indole-3-acetic acid (IAA) colonies, 2 being whitish and 2 yellowish. All the 16 iso-
was conducted as described by Novák et al. (2012) with lates were found to be rod-shaped (data not shown).
minor modifications. Frozen leaves (20 mg FW) of The second step in bacteria identification involved
5-week-old seedlings previously inoculated with bacterial determination of physiological and biochemical charac-
suspension of the strains selected during the study (density teristics of the isolates. Almost all the isolates, except KK
of 108 CFU ml-1) or treated with 10 mM MgSO4 (control) 8a, were able to move (Table 1). Seven isolates (KK 1a,
were homogenized using a MixerMill (Retsch GmbH, KK 1b, KK 2, KK 3, KK 4, KK 9a, KK 11) reacted pos-
Haan, Germany) and extracted in 1 ml 50 mM sodium itively to Gram staining. Most of the isolates tested were
phosphate buffer (pH 7.0) containing 1 % sodium able to hydrolyse starch, while only three isolates (KK 6,
diethyldithiocarbamate and stable isotope-labeled internal KK 9a, KK 10) were able to hydrolyse lactose. As few as 4
standard (5 pmol of 13C-IAA per sample). The pH was isolates tested positively in the resistance to streptomycin
adjusted to 2.7 with 1 M hydrochloric acid, and the sam- test. Three isolates (KK 5, KK 7, KK 9b) were able to
ples were purified by solid phase extraction. The extracts fluoresce.
were purified on Oasis HLB columns (30 mg, Waters Based on the morphological, physiological and bio-
Corp., Milford, MA, USA), conditioned with 1 ml metha- chemical characteristics, only the isolates KK 1a, KK 1b,
nol, 1 ml water and 0.5 ml sodium phosphate buffer (pH KK 2, KK 3, KK 4, KK 9a could be assigned to the families
2.7). After sample application, the column was washed Bacillaceae and Listeriaceae, whereas 9 isolates (KK 5, KK
with 2 ml 5 % methanol and then eluted with 2 ml 80 % 6, KK 7, KK 8a, KK 8b, KK 9b, KK 10, KK 12, KK 13)
methanol. Eluates were evaporated to dryness and dis- were placed in the families Pseudomonadaceae, Enter-
solved in 30 ll of mobile phase prior to mass analysis obacteriaceae, Moraxellaceae, Xantomonadaceae,
using an Acquity UPLCÒ System and triple quadrupole Burkholderiaceae and Rhizobiaceae. 16S rRNA gene
mass spectrometer XevoTM TQ MS (Water) (Floková et al. sequencing was used to fine-tune the identification of these
2014). bacteria. An about 1.5 kb fragment of 16S rRNA of all the
isolates was amplified by PCR products. The PCR products
Statistical analysis were purified, cloned and sequenced. Nucleotide sequences
of the isolates were compared with sequences available in
Each experiments was run in five replicates and repeated NCBI GenBank. The percentage of 16S rRNA gene
twice. Analysis of variance (ANOVA), Duncan’s multiple sequence similarities (97.7–99.8 %) of all the isolates to
range test and Pearson’s correlation coefficients were cal- the closest type strains is shown in Table 2. The data
culated using Statistica for Windows v. 9.0 (StatSoft Inc., allowed also to create a dendrogram with 5 separate tax-
Tulsa, OK, USA). onomic groups (Fig. 1). Seven of the 16 isolates isolated
from the M. sativa rhizosphere and nodules were assigned
to the family Bacillaceae, one to Rhizobiaceae, two to
Results Xantomonadaceae, three to Enterobacteriaceae and three to
Pseudomonadaceae. The family Bacillaceae was repre-
Isolation and identification of bacterial isolates sented by 3 genera: Bacillus (strains KK 1b, KK 11),
Lysinibacillus strains (KK 2, KK 3) and Paenibacillus
Sixteen bacterial isolates were obtained from the rhizo- strains (KK 1a, KK 4, KK 9a) (Table 2; Fig. 1). All strains
spheric soil and root nodule samples from alfalfa. The first identified as representing the family Bacillaceae are Gram-
step in characterizing and identifying a bacterial culture positive, the remaining 9 strains being Gram-negative.
involved examination of colony morphology and cell Only one isolate, KK 13, obtained from the M. sativa
shape. After 24-h incubation of isolates on the TSA med- nodules belongs to the family Rhizobiaceae and the genus
ium, the size, shape, edge, texture, height, colour and Sinorhizobium (Table 2; Fig. 1). The next two Gram-neg-
optical properties of bacterial colonies were determined. ative isolates, KK 8b and KK 9b, obtained from the M.
Most isolates (85 %) formed small (1–2 mm) and medium- sativa rhizosphere belong to the family Xantomonadaceae
sized (3 mm) colonies. Only two isolates: KK 8b and KK and the genus Stenotrophomonas (Table 2; Fig. 1). The
11, formed large colonies, about 4 mm in diameter. further 3 Gram-negative bacterial isolates, KK 8a, KK 10
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Table 1 Physiological and biochemical characteristics of isolates from Medicago sativa rhizosphere and nodules
Strain code Motility ability Gram staining Starch hydrolysis Fermentation of lactose Streptomycin resistance Fluorescence
KK 1a ? ? ? – ? –
KK 1b ? ? ? – ? –
KK 2 ? ? ? – ? –
KK 3 ? ? ? – – –
KK 4 ? ? ? – ? –
KK 5 ? – ??? – – ?
KK 6 ? – ?? ? – –
KK 7 ? – ??? – – ?
KK 8a – – ?? – – –
KK 8b ? – – – – –
KK 9a ? ? ?? ? – –
KK 9b ? – – – – ?
KK 10 ? – – ? – –
KK 11 ? ? ? – – –
KK 12 ? – – – – –
KK 13a ? – – – – –
Reaction index: -, negative; ?, low; ??, medium; ???, high
a
Isolated from nodules
Table 2 16S rRNA gene sequencing-based identification of bacteria isolated from Medicago sativa rhizosphere and nodules
Strain code Families Genus Closest relative (GenBank no.) Similarity % GenBank accession no.
KK 1b Bacillaceae Bacillus B. niacini (AB021194) 99.7 KP858911

KK 11 B. megaterium (GU252112) 98.8 KP858923
KK 2 Lysinibacillus L. fusiformis (AJ310083) 99.8 KP858912
KK 3 L. fusiformis (AJ310083) 99.7 KP858913
KK 1a Paenibacillus P. odorifer (AJ223990) 97.8 KP858910
KK 4 P. borealis (AJ011322) 98.3 KP858914
KK 9a P. amylolyticus (AB073190) 97.7 KP858920
KK 13 Rhizobiaceae Sinorhizobium S. meliloti (AL591688) 98.8 KP858909
KK 8b Xantomonadaceae Stenotrophomonas S. maltophilia (AB294553) 99.2 KP858919
KK 9b S. maltophilia (CP001111) 98.6 KP858921
KK 10 Enterobacteriaceae Citrobacter C. murliniae (AF025369) 99.4 KP858922
KK 6 Leclercia L. adecarboxylata (JN175338) 98.8 KP858916
KK 8a Raoultella R. planticola (AB680712) 98.9 KP858918
KK 5 Pseudomonadaceae Pseudomonas P. brassicacearum (CP002585) 99.8 KP858915
KK 7 P. corrugata (HM190230) 99.5 KP858917
KK 12 P. corrugata (HM190230) 99.8 KP858924
and KK 6 represent 3 genus of the family Enterobacteri- Effects of the bacteria identified on M. truncatula
aceae: Citrobacter, Leclercia and Raoultella (Table 2; seedling growth
Fig. 1). Finally, the family Pseudomonadaceae is repre-
sented by 3 isolates (KK 5, KK 7 and KK 12) identified as Inoculation of 1-week-old M. truncatula seedlings
representing the genus Pseudomonas (Table 2; Fig. 1). The (Fig. 3a), grown in soil mixture under controlled condi-
use of house-keeping genes rpoD (Fig. 2a) and gyrB tions, with a suspension of all the strains identified had a
(Fig. 2b) in the taxonomic study of the 3 Pseu- positive effect on the seedling development 4 weeks after
domonas strains confirmed that the strains KK 5, KK 7 inoculation (Fig. 3c–r) compared with that of the control
and KK 12 belong to the species listed in Table 2. (Fig. 3b). Six of the 16 strains showed a particularly high
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1176 Planta (2016) 243:1169–1189
KK7
Pseudomonas sp. UW4
86 Pseudomonas kilonensis strain 520-20
Pseudomonas corrugata CFBP 5454
78 KK12
Pseudomonas corrugata NCPPB 2445 1
63 Pseudomonas fluorescens NCIMB 11764
Pseudomonas fluorescens A506
Pseudomonas fluorescens F113
90
76 Pseudomonas fluorescens SBW25
68 Pseudomonas fluorescens strain LBUM223
Pseudomonas fluorescens strain PCL1751

Pseudomonadaceae
100 Pseudomonas brassicacearum PP1 210F
77 KK5
62 Pseudomonas brassicacearum subsp. brassicacearum NFM421
96
Pseudomonas fluorescens Pf0-1
KK6
Enterobacteriaceae Leclercia adecarboxylata CIP 82.92
93
Leclercia adecarboxylata strain LMG 2803
100
Escherichia ludwigii genetic cluster V
100 92 Citrobacter murliniae CDC 2970-59
68
KK10
89 KK8a
Raoultella planticola ATCC 33531 DSM 3069
96
Raoultella planticola strain NBRC 14939
88 KK8b
Xantomonadaceae
KK9b
100 Stenotrophomonas maltophilia R551-3
Stenotrophomonas maltophilia ATCC 13637
95
Stenotrophomonas maltophilia strain NBRC 14161
88
Stenotrophomonas pavanii LMG 25348
Sinorhizobium meliloti LMG 6133
Rhizobiaceae
100 Sinorhizobium meliloti NBRC 14782
Sinorhizobium meliloti 1021
KK13
84 Paenibacillus borealis KK19
77 Paenibacillus typhae strain xj7
61
Paenibacillus odorifer TOD45
100 Paenibacillus borealis KK19 DSM 13188
KK1a
100
84 KK4
KK9a
Paenibacillus amylolyticus NBRC 15957
100
Paenibacillus tundrae A10b
Bacillaceae
Bacillus megaterium QM B1551
100
100 KK11
96 Bacillus megaterium ATCC 14581
Bacillus niacini IFO15566
Bacillus niacini NBRC 15566
100
KK1b
98
72 Lysinibacillus sphaericus DSM 28
Lysinibacillus sphaericus NBRC 15095
100 KK3
KK2
74
Lysinibacillus fusiformis DSM 2898
69
Lysinibacillus fusiformis NBRC 15717
0.02
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Planta (2016) 243:1169–1189 1177
seedling growth promotion ability: Paenibacillus borealis brassicacearum KK 5 (Fig. 3p), Citrobacter murliniae KK
KK 4 (Fig. 3h), Raoultella planticola KK 8a (Fig. 3o), 10 (Fig. 3m) and Bacillus niacini KK 1b (Fig. 3c). These
Sinorhizobium meliloti KK 13 (Fig. 3j), Pseudomonas bacteria induced a high increase in fresh weight of the
roots, 2.2–2.5 times higher than that of the control. Their
beneficial effect was also observed in growth of shoots;
b Fig. 1 16S rRNA gene sequence-based phylogenetic tree (not rooted) their fresh weight was almost 3–4.7 times higher that of the
showing position of 16 isolates from Medicago sativa rhizosphere and
nodules in relation to taxonomically similar bacteria. The analysis
control. The bacterial promotional effect on the seedling
was conducted using the neighbor-joining test. The scale bar growth involves not only their impact on the root weight,
indicates 0.02 changes/site. Bootstraps of 1000 replicates were used but also the root architecture. After the seedlings had been
and are shown at the branch nodes of the phylogenetic tree inoculated with a suspension of P. borealis KK 4 (Fig. 3h)
(a) KK 5
99
Pseudomonas brassicacearum PP1 210F
91
Pseudomonas fluorescens NCIMB 11764
100
74 Pseudomonas sp. UW4
Pseudomonas fluorescens Pf0-1
76 KK 12
100 Pseudomonas corrugata NCPPB 2445
100 Pseudomonas corrugata CFBP 5454
69 KK 7
69 Pseudomonas fluorescens A506
Pseudomonas fluorescens strain LBUM223
Pseudomonas fluorescens SBW25
67 Pseudomonas fluorescens strain PCL1751
0.01
(b) 83 KK 5
99
Pseudomonas brassicacearum PP1 210F
83
KK 7
100 KK 12
77 Pseudomonas corrugata CFBP 5454
69 Pseudomonas corrugata NCPPB 2445
78 Pseudomonas fluorescens NCIMB 11764

89 Pseudomonas fluorescens Pf0-1
Pseudomonas sp. UW4
84 Pseudomonas fluorescens A506
Pseudomonas fluorescens strain LBUM223
100 Pseudomonas fluorescens SBW25
99 Pseudomonas fluorescens strain PCL1751
0.02
Fig. 2 Phylogenetic tree (not rooted) based on rpoD (a) and gyrB The scale bar indicates 0.01 (a) and 0.02 (b) changes/site. Bootstraps
(b) gene sequence showing position of 3 Pseudomonas isolates from of 1000 replicates were used and are shown at the branch nodes of the
Medicago sativa rhizosphere in relation to taxonomically similar phylogenetic tree
bacteria. The analysis was conducted using the neighbor-joining test.
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1178 Planta (2016) 243:1169–1189
Fig. 3 Effects of bacteria

isolated from Medicago sativa
rhizosphere and nodules on
Medicago truncatula shoot and
root growth 4 weeks after
inoculation (c–r). Two-way
ANOVA with Duncan’s
multiple range test was used to
detect significant differences.
Means denoted with different
letters (a–g) are significantly
different (P B 0.05)
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Planta (2016) 243:1169–1189 1179
and P. odorifer KK 1a (Fig. 3g), they produced the same produce indolic compounds and examined them with
root fresh weight (ca. 4.0 g), but the architecture of the respect to the ACCD responsible for modulation of the
roots was different. The roots of the seedlings inoculated rhizosphere ethylene content. All the strains were capable
with P. borealis KK 4 were more branched than the roots of indolic compounds biosynthesis in the presence of L-
of seedlings inoculated with P. odorifer KK 1a and the tryptophan, the amount produced being found to range
fresh shoot weight of the seedlings was twice of that of the from about 4–47 lg per 1 ml of suspension with a density
latter. Similarly to P. borealis KK 4, other bacteria (R. of 5 9 108 cells ml-1 (Table 3). The highest amount of
planticola KK 8a, S. meliloti KK 13, P. brassicacearum these compounds produced resulted from effects of S.
KK 5, C. murliniae KK 10 and B. niacini KK 1b) which meliloti KK 13 (47.2 lg ml-1), P. corrugata KK 12
had a positive impact on the shoot weight increase, dis- (46.8 lg ml-1), P. borealis KK 4 (41.1 lg ml-1) and R.
tinctly stimulated root branching (Fig. 3o, j, p, m, c). The planticola KK 8a (41.1 lg ml-1); the lowest amounts
weakest promotion of root and shoot growth was observed being induced by P. brassicacearum KK 5 (4.0 lg ml-1).
after seedling inoculation with suspensions of KK 8b strain The ability to produce indolic compounds depends not only
closest relative to Stenotrophomonas maltophilia, Pseu- on a species, but also on a strain; P. brassicacearum (KK
domonas corrugata KK 7 and Lysinibacillus fusiformis KK 5) produced the lowest amount of these compounds among
2 (Fig. 3k, q, e). Four weeks after inoculation, the seedlings all the strains tested. However, even different strains within
treated with these bacteria showed the least branched roots a species differed in their ability to produce the indolic
and the lowest root weight (between 1.89 and 2.05 g), compounds: while P. corrugata KK 7 produced
although the weight was still higher than that in the non- 24.8 lg ml-1, P. corrugata KK 12 produced almost twice
inoculated controls. The beneficial effect of inoculation that, 46.8 lg ml-1 (Table 3). The same observation was
with all the strains was confirmed by data on dry weights of made when the production of these compounds by three
shoots (Fig. 4a), roots (Fig. 4b) and whole seedlings species of Paenibacillus was analyzed; P. amylolyticus KK
(Fig. 4c) which were all higher than those of the untreated 9a, P. odorifer KK 1a and P. borealis KK 4 produced 29.7,
seedlings. The shoot dry weight was the highest in plants 34.5 and 41.1 lg ml-1, respectively. On the other hand,
inoculated with R. planticola KK 8a, P. borealis KK 4, P. very similar amounts of indolic compounds were synthe-
brassicacearum KK 5, S. meliloti KK 13 and B. niacini KK sized by both Bacillus species: B. niacini KK 1b and B.
1b, which were 6.5, 6.3, 5.0, 4.8 and 4.6 times higher, megaterium KK 11 produced 37.1 and 36.8 lg ml-1,
respectively, than in control plants (Fig. 4a). Inoculation respectively.
with the remaining 11 strains also substantially (from about Of the 16 strains, 14 were able to grow on the ACC-
2.0–3.0 times) increased the shoot dry weight, compared to containing medium as the sole nitrogen source (Table 3).
the control. The strain C. murliniae KK 10 was the most These strains, representing 13 species, expressed the
effective promoter of root biomass (Fig. 4b). The isolate ACCD activity at levels ranging from 0.4 to about
increased the root dry weight 7 times (299 mg), compared 1469.2 lmol a-KB mg protein-1 h-1. The highest ACCD
with the control (43 mg). The strains S. meliloti KK 13, activity (1469.2 lmol a-KB mg protein-1 h-1) was
Paenibacillus odorifer KK 1a, P. borealis KK 4, L. fusi- exhibited by P. brassicacearum KK 5, followed by B.
formis KK 3 and R. planticola KK 8a produced also a megaterium KK 11 (345.3 lmol a-KB mg protein-1 h-1)
marked positive effect on the root dry weight, compared and P. corrugata KK 7 (108.2 lmol a-KB mg protein-1
with the non-inoculated control seedlings; the root weight h-1). The ACCD activity in 8 strains was very low. Those
of the treated seedlings was more than 5 times higher strains include all the three strains representing the Enter-
(223–250 mg) than that of controls. Four weeks after obacteriaceae, 4 from the Bacillaceae and one from the
inoculation with all the strains, whole seedling biomass Xantomonadaceae. Two strains: P. amylolyticus KK 9a and
was several times higher than that of the non-inoculated S. meliloti KK 13 showed no ACCD activity.
plants (Fig. 4c). The most effective strains were again P. The analyses described above were conducted to assess
borealis KK 4, R. planticola KK 8a, S. meliloti KK 13, P. the potential of the strains to be used as phytostimulants.
brassicacearum KK 5, C. murliniae KK 10 and B. niacini The seedlings inoculated with strains which did not pro-
KK 1b; the seedling dry weights 6.0, 5.8, 5.2, 4.8, 4.6 and duce ACCD, but differed in their ability to produce phe-
4.5 times higher than those of the control, respectively. nolic compounds showed a different reaction in terms of
the root and shoot growth. S. meliloti KK 13 producing 2
Bacterial indolic compounds production, ACC times higher amounts of indolic compounds
deaminase activity and the presence of phlD gene (47.2 lg ml-1), compared to S. maltophilia KK 9b
(23.2 lg ml-1; Table 3) induced also a nearly twofold
We also evaluated the ability of the strains showing a increase in the fresh weight of both roots and shoots
phytostimulation effect on M. truncatula seedlings to (Fig. 3j, l) as well as in their dry weights (Fig. 4a, b). The
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1180 Planta (2016) 243:1169–1189
Fig. 4 Effects of bacteria

isolated from Medicago sativa
rhizosphere and nodules on dry
mass of Medicago truncatula
shoots (a), roots (b) and
seedlings (c) 4 weeks after
inoculation. Vertical bars
indicate ±SD. Two-way
ANOVA with Duncan’s
multiple range test was used to
detect significant differences.
Means denoted with different
letters (a–g) are significantly
different (P B 0.05)
whole seedling weight doubled (the seedling dry weight The ACCD activity in R. planticola KK 8a and KK 6
was 2.4 times that of the seedlings treated with KK 9b) closest relative to L. adecarboxylata was at a similar, very
(Fig. 4c). Similarly, a beneficial effect on the seedlings low, level (0.42 and 0.45 lmol a-KB mg protein-1 h-1),
growth involving the amount of indolic compounds pro- but KK 8a showed an about 1.6 times higher ability to
duced by the bacteria was observed in two Enterobacteri- produce indolic compounds (41.1 lg ml-1) than strain KK
aceae bacteria strains: R. planticola KK 8a and KK 6 6 (25.7 lg ml-1; Table 3), and induced an about 1.4 and
closest relative to Leclercia adecarboxylata. To summa- 1.9 times higher fresh weight of roots and shoots, respec-
rize, the ability of almost the all strains (except for P. tively (Fig. 3o, n) the dry weight of roots, shoots, and
brassicacearum KK 5) to enhance root growth (dry weight) whole seedlings being higher by the factor of 1.9, 2.4 and
was positively correlated with the indolic compounds 2.2, respectively (Fig. 4a–c). A very interesting effect on
production (r = 0.69; P = 0.0001) (data not shown). seedling growth was observed after inoculation with
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Planta (2016) 243:1169–1189 1181
Table 3 Indolic compounds production and ACC deaminase activity in bacteria isolated from Medicago sativa rhizosphere and nodules
Families Strain name Indolic compounds (lg IAA ml-1) ACCD (lmol a-KB mg protein -1 -1
h )
Bacillaceae Bacillus niacini KK 1b 37.1 ± 2.6bc 0.6 ± 0.01h

bc
Bacillus megaterium KK 11 36.8 ± 3.3 345.3 ± 49.2b
d
Lysinibacillus fusiformis KK 2 32.9 ± 2.4 3.1 ± 0.1g
Lysinibacillus fusiformis KK 3 25.4 ± 0.1e 3.0 ± 0.07g
cd
Paenibacillus odorifer KK 1a 34.5 ± 2.3 0.7 ± 0.04h
b
Paenibacillus borealis KK 4 41.1 ± 3.0 35.3 ± 0.5d
cd
Paenibacillus amylolyticus KK 9a 29.7 ± 4.9 21.6 ± 0.9e
a
Rhizobiaceae Sinorhizobium meliloti KK 13 47.2 ± 2.8 n.d.
Xantomonadaceae Stenotrophomonas maltophilia KK 8b 35.4 ± 0.8c 5.6 ± 0.1f
ef
Stenotrophomonas maltophilia KK 9b 23.2 ± 1.6 n.d.
Enterobacteriaceae Citrobacter murliniae KK 10 30.1 ± 3.8cd 3.3 ± 0.04g
Leclercia adecarboxylata KK 6 25.7 ± 2.1de 0.5 ± 0.01h
Raoultella planticola KK 8a 41.1 ± 2.0b 0.4 ± 0.01h
g
Pseudomonadaceae Pseudomonas brassicacearum KK 5 4.0 ± 0.1 1469.2 ± 136.6a
Pseudomonas corrugata KK 7 24.8 ± 1.0e 108.2 ± 2.5c
a
Pseudomonas corrugata KK 12 46.8 ± 1.9 34.6 ± 1.2d
Two-way ANOVA with Duncan’s multiple range test was used to detect significant differences. Means denoted with different letters (a–h) are
significantly different (P B 0.05)
n.d. not detected
bacteria from two different species of the genus Paeni- amplification of DNAs was performed using primers
bacillus: P. odorifer KK 1a (Fig. 3g) and P. borealis KK 4 specifically designed for the phlD gene. The presence of
(Fig. 3h). The amounts of indolic compounds produced by the gene fragment was confirmed for two (out of 3)
those bacteria did not differ much, but the strains differed Pseudomonas strains: P. brassicacearum KK 5 and P.
strongly in their ACCD activity. In P. borealis KK 4, the corrugata KK 12 (Supplemental Fig. S1). Sequencing of
ACCD activity was 52 times higher that of P. odorifer KK the phlD gene fragments from both strains confirmed the
1a (Table 3). However, the difference had no effect on the expected size of 720 bp. Moreover, as seen in Fig. 5, the
root weight (the fresh and dry weights were almost iden- similarity to other DAPG precursor genes from fluorescent
tical; Figs. 3g, h, 4b), but did affect the root architecture. pseudomonads group is evident.
Roots of the seedlings inoculated with P. borealis KK 4
(Fig. 3h) were more branched. Also the shoot fresh weight Biofertilizing potential of the bacteria
was twice that of the shoot weight of seedlings pre-treated
with P. odorifer KK 1 (Fig. 3g). An identical pattern was Several tests were used to evaluate the potential of all the
observed in the shoot dry weight (Fig. 4a). Generally, the isolates to be used as biofertilizers. The ability of all the
ACCD activity did not correlated with the root dry weight strains examined to convert insoluble inorganic phosphorus
(data not shown). (P) and zinc (Zn) compounds, such as tricalcium phosphate
A very surprising result of seedling growth promotion by P. and zinc oxide, to bioavailable forms was studied
brassicacearum KK 5 (Fig. 3p) was observed with respect of (Table 4). Six out of the 16 strains were not able to solu-
the strain’s ability to produce indolic compounds and its bilize the insoluble inorganic P compounds. Three strains
ACCD activity. Among all the 16 strains examined, the bac- only (P. brassicacearum KK 5, P. corrugata KK 7, R.
teria in question showed the lowest capacity for indolic planticola KK 8a), when tested in vitro, formed clear halo
compounds production (4.0 lg ml-1) and the highest ACCD zones around the colonies kept on Pikovskay’a agar media
activity (1469.2 lmol a-KB mg protein-1 h-1) (Table 3), but (high response), while the remaining 7 strains showed a
the 4-week-old seedlings inoculated with the bacterium very low P-solubilizing activity. On the other hand, half of
showed a well-developed root architecture (Fig. 3p) and high the strains tested displayed the ability to dissolve the
dry weights of both shoot sand whole seedlings (Fig. 4b, c). inorganic zinc compounds. Inorganic zinc oxide was sol-
To find out whether our three Pseudomonas strains ubilized by all the three Pseudomonadaceae and Enter-
contain the phlD gene responsible for the synthesis of obacteriaceae strains as well as by one strain each
monoacetylphloroglucinol, the DAPG precursor, PCR representing the Xantomonadaceae and the Bacillaceae.
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1182 Planta (2016) 243:1169–1189
Fig. 5 Phylogenetic tree (not 73 Pseudomonas sp. S7-42

rooted) of 2,4- 86 Pseudomonas brassicacearum PP1 210F
diacetylophloroglucinol
Pseudomonas fluorescens strain FTAD1R34
biosynthetic protein (PhlD)
fragments of two strains (KK 5 Pseudomonas brassicacearum strain NFM421
and KK 12) inferred by the KK 5
neighbor-joining method. 78
Pseudomonas fluorescens strain MVP1-6
Bootstraps of 1000 replicates
Pseudomonas sp. K94.31
were used and are indicated at
the nodes. Scale bar indicates 100 Pseudomonas sp. P97.30
0.005 changes/site Pseudomonas fluorescens strain A6SM7
Pseudomonas sp. C6-11
97
KK 12
99 Pseudomonas sp. P12
Pseudomonas sp. C10-197
80 99 Pseudomonas sp. K94.38
96 Pseudomonas fluorescens strain 1MA1

89 Pseudomonas fluorescens strain M1-96
Pseudomonas fluorescens strain 5AT2
99 Pseudomonas fluorescens F113
Pseudomonas fluorescens strain B6RN
Pseudomonas fluorescens strain ALEB 7B
0.005
Thirteen out of 16 strains tested positive for the production truncatula seedling development, whereas one (P. corru-
of siderophores, iron chelating compounds, as evidenced gata KK 7) showed the weakest ability to promote the root
by the yellow zone on the CAS agar medium. The effect and shoot growth (Fig. 3h, j, p, q). When selecting the
occurred because of iron removal from the blue CAS- strains as representative pseudomonads, their ability to
FE(III) complex during siderophore production. The most produce indolic compounds, the ACCD activity and the
efficient siderophore producers were the following 5 presence of the phlD gene were taken into account
strains: B. megaterium KK 11, P. brassicacearum KK 5, P. (Table 3; Supplemental Fig. S1). The IAA amount in
corrugata KK 7, KK 8b and KK 6 closest relative to S. leaves of 5-week-old seedlings 3 days after soil inoculation
maltophilia and L. adecarboxylata. with all the bacterial strains (Fig. 6) was observed to have
Ability for produce ammonia (NH3) was a trait common increased. The highest increase was recorded after inocu-
to all the 16 strains (Table 4). Nessler’s reaction used to lation with P. brassicacearum KK 5, characterized by the
detect ammonia emission by bacteria growing on peptone lowest capacity for the production of indolic compounds
media showed the highest ammonia amount to have been and the highest ACCD activity (Table 3), and in which the
produced by L. fusiformis KK 3. phlD gene was detected (Supplemental Fig. S1). On the
Six isolates (L. fusiformis KK 2, S. maltophilia KK 8b other hand, S. meliloti KK 13, the most efficient producer
and KK 9b, R. planticola KK 8a, P. brassicacearum KK 5, of indolic compounds but lacking the ACCD activity also
P. corrugata KK 7) showed the ability to acidify TSA increased the endogenous IAA content in the leaves, but
medium (Table 4). the increase was lower than that induced by P. brassi-
cacearum KK 5. After 7 days from inoculation of seedlings
Inoculation with selected rhizobacteria increased with all the strains discussed above, the endogenous IAA
the IAA content in M. truncatula leaves level in leaves was similar and higher than that in leaves of
non-inoculated plants.
To investigate whether the inoculation with bacteria iso-
lated from the rhizospere and nodules of M. sativa affects
the IAA content in M. truncatula, we assayed IAA in Discussion
leaves of inoculated and non-inoculated plants. For this
experiment, we selected four strains. Three of them (P. Beneficial effects of PGPR on plant growth and yield of
borealis KK 4, S. meliloti KK 13 and P. brassicacearum many agricultural crops have been shown by numerous
KK 5) proved to be the most effective promoters of M. studies (Hayat et al. 2010; Bhattacharyya and Jha 2012;
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Planta (2016) 243:1169–1189 1183
Table 4 Some biochemical traits of bacteria isolated from Medicago sativa rhizosphere and nodules
Families Strain name P-sol Zn- Siderophore NH3 Acidification of
sol production production medium
Bacillaceae Bacillus niacini KK 1b – ? – ? –

Bacillus megaterium KK 11 ? – ??? ?? –
Lysinibacillus fusiformis KK 2 ? – – ?? –
Lysinibacillus fusiformis KK 3 – – ? ??? ?
Paenibacillus odorifer KK 1a ? – – ? –
Paenibacillus borealis KK 4 ? – ? ? –
Paenibacillus amylolyticus KK 9a ? – ?? ? –
Rhizobiaceae Sinorhizobium meliloti KK 13 ? – ? ?? –
Xantomonadaceae Stenotrophomonas maltophilia KK – – ??? ? ?
8b
Stenotrophomonas maltophilia KK – ? ?? ? ?
9b
Enterobacteriaceae Citrobacter murliniae KK 10 – ??? ?? ?? –
Leclercia adecarboxylata KK 6 – ? ??? ?? –
Raoultella planticola KK 8a ?? ? ? ? ?
Pseudomonadaceae Pseudomonas brassicacearum KK 5 ??? ??? ??? ?? ?
Pseudomonas corrugata KK 7 ?? ?? ??? ?? ?
Pseudomonas corrugata KK 12 ? ? ?? ? –
Reaction index: -, negative; ?, low; ??, medium; ???, high
Based on morphological, physiological and biochemical

characteristics, 7 isolates could be pre-assigned to the
families Bacillaceae and Listeriaceae, 9 isolates could be
identified as representing the Pseudomonadaceae, Enter-
obacteriaceae, Moraxellaceae, Xantomonadaceae,
Burkholderiaceae and Rhizobiaceae. However, by phylo-
genetic analysis of the 16S rRNA gene sequences it was
found that the strains represented 5 rather than 8 families
(Bacillaceae, Pseudomonadaceae, Enterobacteriaceae,
Xantomonadaceae and Rhizobiaceae). Most strains (7)
isolated from the alfalfa rhizosphere belonged to the
Bacillaceae. Three strains each were assigned to the
Pseudomonadaceae and Enterobacteriaceae, two to the
Fig. 6 IAA content in leaves of M. truncatula seedlings 3 and 7 days
after soil inoculation of 5-week-old seedlings with Paenibacillus
Xantomonadaceae and one to the Rhizobiaceae. The
borealis KK 4, Pseudomonas brassicacearum KK 5, P. corrugata KK Bacillaceae and Pseudomonadaceae species are common
7, Sinorhizobium melilotii KK 13. IAA content in leaves of 5-week- rhizospheric bacteria, and have been isolated from the
old non-inoculated control seedlings at T=0 was rhizosphere of different agricultural crops, although there
52.68 ± 3.24 pmol/g FW
are few data on bacteria isolated from the Fabaceae rhi-
zosphere. The literature contains data on the presence of
Glick 2012; Vacheron et al. 2013). However, to date no the Bacilleaceae bacteria [B. megaterium strain B153-2-2,
data have been published on the positive effects of PGPR Bacillus fusiformis (PM-5)] in the soybean rhizosphere
isolated from the rhizosphere of M. sativa (alfalfa) and (Liu and Sinclair 1992; Park et al. 2005).
their application to promote the development of seedlings The species dealt with in this work have not been
of M. truncatula (medic barrel). In this study, we isolated reported to be associated with the alfalfa rhizosphere
bacterial strains from the alfalfa rhizosphere, identified before. Although there is admittedly some information
them and evaluated their potential as PGPR for M. about bacteria isolated from the alfalfa rhizosphere, the
truncatula. authors did not identify the bacteria isolates, but provided
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1184 Planta (2016) 243:1169–1189
codes only (Bharucha et al. 2013a). Similarly, Bharucha compounds production by bacterial strains, except that by
et al. (2013b) reported a bacterium identified from the P. brassicacearum KK 5, can be responsible for stimula-
alfalfa rhizosphere, but provided the genus identification tion of M. truncatula root development; the indolic com-
only (Bacillus sp.). Earlier, Guiñazú et al. (2010) also pounds content was positively correlated with the root dry
reported the isolation of bacteria from the alfalfa rhizo- mass (r = 0.69; P = 0.0001). P. borealis KK 4, R. plan-
sphere, but for molecular identification of these isolates ticola KK 8a, S. meliloti KK 13, C. murliniae KK 10, B.
they used a too short DNA region (318 bp) for precise niacini KK 1b and P. odorifer KK 1a, which promoted root
taxonomic identification. The authors found the presence of growth most effectively, are characterized by the highest
strains assigned to the genera Pseudomonas and Bacillus. capacity for indolic compounds production in the presence
The present work demonstrated, for the first time, that of tryptophan, the plant IAA precursor. Indolic compounds
all the bacteria from the M. sativa rhizosphere identified production by these strains ranged from about
could be effective in enhancing the M. truncatula seedling 30–47 lg ml-1. That indolic compounds are mainly
development in soil (sand and perlite mixture) under con- responsible for the stimulation of M. truncatula seedling
trolled growth room conditions. The main effects observed growth can be clearly seen when comparing effects of two
4 weeks after inoculation of 1-week-old seedlings involved bacterial strains: S. meliloti KK 13 (Rhizobiaceae) and S.
an increase in root fresh and dry weights as well as root maltophilia KK 9b (Xantomonadaceae). Showing no
branching. deaminase activity, they differ in the ability to produce
A positive effect on shoot development was observed as indolic compounds; KK 13 which produced 2 times more
well. Our results are in line with findings of many other these compounds (47.2 lg ml-1) than KK 9b
workers who assessed effects of inoculation with non- (23.3 lg ml-1) was more effective in stimulating the roots
pathogenic plant growth promoting bacteria (PGPR) on mass and branching, and the shoot and seedling weight. A
growth of many other plants (Glick 1995, 2012; Wissuwa positive correlation between auxin production and growth
et al. 2009; Martinez-Viveros et al. 2010; Rashid et al. promoting activity of diverse PGPR has been also reported
2011). Seven out of the 16 bacteria species examined in Brassica juncea, wheat and soya bean (Asghar et al.
proved to be most effective promoters of the M. truncatula 2002; Khalid et al. 2004; Wahyudi et al. 2011).
seedling development. In terms of the seedling fresh and To date, however, no information about such correla-
dry weight, those bacteria may be arranged in the following tions in M. truncatula under in vivo conditions is available.
order: P. borealis KK 4 (Bacillaceae), R. planticola KK 8a Earlier Nolan et al. (2003) showed that when M. truncatula
(Enterobacteriaceae), S. meliloti KK 13 (Rhizobiaceae), P. leaf explant tissues were cultured in vitro in an auxin-
brassicacearum KK 5 (Pseudomonadaceae), C. murliniae containing medium, they produced numerous roots. How-
KK 10 (Enterobacteriaceae), B. niacini KK 1b (Bacil- ever, our study did not always demonstrate such a positive
laceae) and P. odorifer KK 1a (Bacillaceae). correlation between high levels of bacterial indolic com-
The approach used in this study included screening of pounds and root development. When the M. truncatula
the isolated bacteria for their in vitro indolic compounds seedlings were inoculated with P. brassicacearum KK 5, it
biosynthesis and ACCD activity. The production of phy- proved markedly effective in promoting root system
tohormones, including auxins, by PGPR is now considered development (particularly the lateral roots), but was one of
to be one of the most important direct mechanisms by the lowest indolic compounds producers (as little as
which many rhizobacteria promote plant growth (Spaepen 4 lg ml-1). The promotional activity of this bacteria on
et al. 2007; Spaepen and Vanderleyden 2011; Glick 2012). the root architecture resulted from a mechanism other than
Among indolic compounds, IAA is mainly known as an the bacterial indolic compounds. Some strains of fluores-
inducer of lateral and adventitious root formation (Casi- cent pseudomonads are known to produce a secondary
miro et al. 2001; Sorin et al. 2005). Indolic compounds metabolite, 2,4-diacetylphloroglucinol (DAPG) (Dwivedi
were produced, in the presence of its precursor L-trypto- and Johri 2003). DAPG can be a signal molecule for plants,
phan (500 lg ml-1), by all the 16 isolates from the M. as enhancement of root branching in tomato and wheat
sativa rhizosphere. Production of these compounds was seedlings has been reported (Brazelton et al. 2008; Couil-
quantified as ranging from 4.0 to 47.2 lg ml-1. In the lerot et al. 2011). Application of exogenous DAPG can
presence of tryptophan (1 %), bacterial strains representing alter the root architecture of tomato seedlings by interact-
three genera: Pseudomonas, Bacillus and Azospirillum ing with an auxin-dependent signaling pathway (Brazelton
produced IAA in amounts ranging from 1.2 to et al. 2008; Couillerot et al. 2011). These authors observed
44.4 lg ml-1, but the production was negatively correlated also that roots of the auxin-resistant diageotropica tomato
with the root length, while correlating positively with the mutant showed a reduced DAPG sensitivity with regard to
number of roots in 15-day-old wheat seedlings (Hussain the inhibition of the primary root growth and induction of
and Hasnain 2011). Our results may suggest that indolic root branching.
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Planta (2016) 243:1169–1189 1185
In the present study, we used electrophoresis of the PCR could be among the potentially M. truncatula growth
products amplified from DNA of three Pseudomonas promoting bacteria. However, our results show that indolic
strains with primers for the phlD gene responsible for the compounds production by most of the strains isolated from
synthesis of monoacethylphloroglucinol (MAPG), the the M. sativa rhizosphere, rather than the ACCD activity, is
DAPG precursor. The detection of the phlD gene in P. responsible mainly for stimulating M. truncatula seedling
brassicacearum KK 5, and also in P. corrugata KK 12, development (roots and shoots). No correlation between
could suggest DAPG production by these bacteria and the ACCD activity and the root dry mass of roots was
probably their potential contribution to the observed strong observed. In general, the highest M. truncatula seedling
stimulation of root branching in M. truncatula seedlings. growth promotion was obtained after inoculation with
However, to verify this hypothesis, DAPG quantification strains which produced most of the IAA (between 30 and
has to be performed, and phlD mutants should be 47 lg ml-1), with simultaneous absence or very low
generated. activity of ACCD. Previously, we also suggested that IAA
The PGPR which do not produce auxins are also known production rather than ACCD activity in two Agrobac-
for their ability to modify the endogenous plant IAA terium rhizogenes strains, 15834 and LBA1334, might be
transport or for regulation of auxin homeostasis by, e.g., responsible for the stimulation of the grass Festuca rubra
production of volatile organic compounds (VOC), resulting L. seed germination, seedling emergence and development
in the same root architecture effects (Zhang et al. 2007; upon seed inoculation; mainly by increasing growth of
Contesto et al. 2010; Zamioudis et al. 2013). Our results lateral roots and more complex architecture of the
suggest that endogenous plant IAA, the content of which in branching root system (Król et al. 2014). We compared the
seedling leaves increased after 3 and 7 days following soil effects of seed inoculation of the two strains tested with the
inoculation with rhizobacteria, may be probably involved ability to produce IAA and of exogenous application of
in M. truncatula seedling growth promotion. Thus, we IAA at concentrations corresponding to IAA production by
provide evidence that some PGPR strains can trigger an these strains. The presence of exogenous IAA during seed
increase of the endogenous auxin content in leaves, the treatment produced a stimulatory effect on seedlings
main site of auxin production. Contesto et al. (2010) growth, but the effect was lower compared to that of a
showed that up-regulation of several genes involved in treatment with bacterial strains. Improvement of F. rubra
tryptophan and IAA biosynthesis took place in shoots of L. seedling development, particularly visible in the root
12-day-old A. thaliana seedlings inoculated with Phyl- architecture, is achieved because the two bacteria strains
lobacterium brassicacearum STM196. Consistent with the are able to produce IAA and have active ACCD which
increased expression levels of genes involved in tryptophan probably prevents the root growth-inhibiting levels of
biosynthesis, the inoculated shoots contained higher ethylene induced by bacterial or exogenous IAA. However,
amounts (80 %) of this IAA precursor compared to the the exact mechanism of growth stimulation by bacteria is
non-inoculated shoots. However, the IAA level in Ara- still unclear. This is distinctly visible on the example of the
bidopsis shoots remained essentially unchanged upon P. brassicacearum KK 5, a strain that is an excellent
inoculation with the strain in question. promoter of M. truncatula seedling growth: the strain
Numerous studies have suggested that bacterial IAA and exerted a particularly positive effect on the root system
ACCD synergistically promote plant growth (Glick et al. development, but was also least able to produce IAA (only
2007; Glick 2012). The main visible effect of root inocu- 4 lg ml-1) among the strains examined, and was charac-
lation with ACCD-producing bacteria is the enhancement terized by the highest ACCD activity (up to 1467 lmol a-
of plant root elongation. Patten and Glick (2002) suggested KB mg protein-1 h-1). However, Contesto et al. (2008)
that IAA and ACCD work in concert to stimulate root described that the effects of the acds-deficient mutants of
elongation. Exogenous IAA is known to increase tran- Phyllobacterium brassicacearum STM196, P. putida
scription and activity of ACC synthase (Peck and Kende UW4, Rhizobium leguminosarum bv. viciae 128C53K and
1995), an enzyme which catalyzes the ACC synthesis in Mesorhizobium loti MAFF303099 on the root system
peas. The plant ACC stimulates ACCD activity in bacteria architecture of Arabidopsis seedlings were not significantly
(Li and Glick 2001). An activity of 20 nmol a-KB mg different from those of their wild-type counterparts. The
protein-1 h-1 has been reported (Penrose and Glick 2003) only exception found concerned the acdS mutant of P.
to be sufficient for plant growth promotion. In our study putida UW4 which triggered an increase in lateral root
the 16 strains for which the ACC-containing selec- number, as opposed to the WT strain which did not affect
tion medium was used as the sole nitrogen source, 14 this number. In turn, P. brassicacearum strain Am3 pro-
showed the ACCD activity to range from 0.2 to about ducing ACCD (ca. 10 lmol a-KB mg protein-1 h-1)
1469.2 lmol a-KB mg protein-1 h-1. It appears that some showed both pathogenic and growth promoting properties
of the M. sativa rhizosphere strains analyzed in this study in its interaction with tomato seedlings (Belimov et al.
123
1186 Planta (2016) 243:1169–1189
2007), depending on the density of the inoculant used. At a enhance the phosphorus status of plants (Van Veen et al.
low bacterial concentration (106 cells ml-1), increased 1997). Thus, it is likely that many of the so-called biofer-
in vitro root elongation and root biomass of soil-grown tilizers exert a dual effect that is mediated by direct solu-
tomato were recorded; however, a higher concentration of bilization of inorganic P by organic acids synthesized and
the strain mentioned produced a negative effect on root excreted by soil bacteria, mineralization of organic P car-
elongation. The ACCD-deficient mutant of strain Am3 ried out by phosphohydrolases such as acid phosphatases,
(T8) decreased root elongation and biomass production, and a stimulatory effect by IAA production (Khan et al.
compared to the wild-type Am3. In our experiments, the 2009). It has been suggested that PGPR which decrease the
density of P. brassicacearum KK5 inoculant was high (108 medium pH during growth through their ability to produce
cells ml-1) and positive effects on root and seedling organic acids are efficient P solubilizers (Nautiyal et al.
development were observed. Similarly, it was found earlier 2000). Seven out of the 16 isolates from the alfalfa rhi-
that P. brassicacearum Am3 increased rape and pea plant zosphere were able to acidify the TSA medium. Three of
growth in pot experiments when the plants were inoculated these were representatives of the Pseudomonadaceae (KK
with a high density suspension (5 9 107 cells ml-1) 5, KK 7, KK 12), two were members of the Xantomon-
(Safronova et al. 2006). Thus, it is becoming increasingly adaceae (KK 8b, KK 9b) and one each belonged to the
apparent that each rhizobacterium can promote plant Enterobacteriaceae (KK 8a) and Bacillaceae (KK 3).
growth by several mechanisms. In the case of M. truncat- In addition, some of the bacteria examined can help to
ula seedlings and P. brassicacearum KK 5, a low level of increase the micronutrient (Zn) supply by solubilizing
bacterial indolic compounds (4 lg ml-1), a high ACCD inorganic zinc compounds (Wissuwa et al. 2009). Among
activity (1467 lmol a-KB mg protein-1 h-1), the presence the strains tested, those representing the Enterobacteriaceae
of phlD gene responsible for biosynthesis of the DAPG and Pseudomonadaceae showed the highest ZnO dissolving
precursor and an increase in endogenous levels of IAA in potential.
leaves may probably be involved in promoting the devel- Iron, essential for cellular growth and metabolism,
opment of seedlings. There is clear evidence that plant has—like phosphorus—a low mobility and is not easily
growth promotion by rhizobacteria involves more mecha- available to plants, but may be supplied by bacteria. Many
nisms than one. plants are known to use various bacterial siderophores as
Moreover, PGPR can additionally help plants by an iron source, although the total concentrations are
increasing their uptake of nutrient elements such as P and probably too low to contribute substantially to plant iron
Zn on account of their ability to solubilize the unavailable uptake (Martinez-Viveros et al. 2010). Siderophores, as
nutrient forms, which is one of the essential criteria in iron Fe-chelating agents, are produced by various types of
facilitating the transport of most nutrients, including P and bacteria including the Gram-negative Pseudomonas and
Zn (Wissuwa et al. 2009). The low availability of Gram-positive Bacillus (Bhattacharyya and Jha 2012). In
macronutrient P to plants results from the fact that the vast our experiments, all the strains isolated from the alfalfa
majority of soil P is in insoluble forms (both organic and rhizosphere did produce siderophores which were detected
inorganic), and plants can only absorb P in two soluble using the chrome azurol S assay, a general test for side-
forms: the monobasic (H2 PO 2
4 ) and the diabasic (HPO4 )
rophores. The most positive reaction in this test was shown
ions. Strains representing the genera Pseudomonas, Bacil- by the bacteria representing the genera Pseudomonas,
lus and Rhizobium are among the most powerful phosphate Bacillus, Xanthomonas and Enterobacter.
solubilizers (Richardson et al. 2009; Hayat et al. 2010). In Ammonia (NH3) produced by rhizobacteria is an additional
our study, all the three Pseudomonas strains were capable N source in soil (McNeill and Unkovich 2007). All the bac-
of solubilizing the insoluble tri-calcium phosphate teria from the M. sativa rhizosphere were able to produce NH3
[Ca3(PO4)2], P. brassicacearum KK 5 showing the highest under in vitro condition in the presence of peptone. Fourteen
solubilizing ability. Five of the seven Bacillaceae strains strains are also potential ammonia producers because of the
were characterized by a low ability to solubilize this presence of ACCD which degrades ACC to ammonia and a-
compound in question. Jorquera et al. (2008) isolated ketobutyrate. When ammonia accumulates in the plant cells at
P-solubilizing bacteria from the rhizospheres of the culti- levels higher than 0.1 mM, it can inhibit some processes such
vated Fabaceae plants such as Trifolium repens and Lupi- as seed germination and seedling establishment, but it can also
nus luteus. Cattelan et al. (1999) found only two of five stimulate root branching. So the beneficial effect of P. bras-
rhizospheric isolates that tested positive for solubilization sicacearum KK 5 observed is likely to be additionally related
had actually a positive effect on soybean seedling growth. to the very high activity of ACCD in ammonia production. All
In earlier studies, phosphate (P)-solubilizing bacteria such these attributes of the bacteria examined may affect the
as Bacillus and Paenibacillus sp. were applied to soils to nutrient uptake abilities of M. truncatula.
123
Planta (2016) 243:1169–1189 1187
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Author contribution statement EK initiated and designed
M (2001) Auxin transport promotes Arabidopsis lateral root
research, interpreted the results and wrote the manuscript. initiation. Plant Cell 13:843–852
AK conducted experiments and statistical analysis. Both Cattelan AJ, Hartel PG, Fuhrmann JJ (1999) Screening of plant
authors approved the manuscript. growth promoting rhizobacteria to promote early soybean
growth. Soil Sci Soc Am J 63:1670–1680
Chandler D, Davidson G, Grant WP, Greaves J, Tatchell GM (2008)
Acknowledgments This work was supported by the National Sci-
Microbial biopesticides for integrated crop management: an
entific Centre (NCN) Grant No. NN310784140. We are indebted to
assessment of environmental and regulatory sustainability.
Dr. Teresa Radziejewska for linguistic assistance.
Trends Food Sci Tech 19:275–283
Cladera AM, Bennasar AB, Barceló M, Lalucat J, Garcı́a-Valdés E
Open Access This article is distributed under the terms of the
(2004) Comparative genetic diversity of Pseudomonas stutzeri
Creative Commons Attribution 4.0 International License (http://crea
genomovars, clonal structure, and phylogeny of the species.
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
J Bacteriol 16:5239–5248
distribution, and reproduction in any medium, provided you give
Contesto C, Desbrosses G, Lefoulon C, Béna G, Borel F, Galland M,
appropriate credit to the original author(s) and the source, provide a
Gamet L, Varoquaux F, Touraine B (2008) Effects of rhizobac-
link to the Creative Commons license, and indicate if changes were
terial ACC deaminase activity on Arabidopsis indicate that
made.
ethylene mediates local root responses to plant growth-promot-
ing rhizobacteria. Plant Sci 175:178–189
Contesto C, Milesi S, Mantelin S, Zancarini A, Desbrosses G,
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Planta (2016) 243:1169–1189

Medicago truncatula Gaertn. as a model for understanding

Abstract truncatula root development involves also root interac-

Electronic supplementary material The online version of this Abbreviations

Introduction (Khan et al. 1990; Glick 1995; Gholami et al. 2009;

PCR analysis with specific phlD primers Protein content determination

KK 1b Bacillaceae Bacillus B. niacini (AB021194) 99.7 KP858911

Pseudomonas fluorescens strain PCL1751

78 Pseudomonas fluorescens NCIMB 11764

Fig. 3 Effects of bacteria

Fig. 4 Effects of bacteria

Bacillaceae Bacillus niacini KK 1b 37.1 ± 2.6bc 0.6 ± 0.01h

Fig. 5 Phylogenetic tree (not 73 Pseudomonas sp. S7-42

96 Pseudomonas fluorescens strain 1MA1

Bacillaceae Bacillus niacini KK 1b – ? – ? –

Based on morphological, physiological and biochemical

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