Acta Physiol Plant (2017) 39:61
DOI 10.1007/s11738-017-2360-6
ORIGINAL ARTICLE
A novice Achromobacter sp. EMCC1936 strain acts as a plant-
growth-promoting agent
H. M. Abdel-Rahman1 • A. A. Salem1 • Mahmoud M. A. Moustafa2
Hoda A. S. El-Garhy2
Received: 20 May 2016 / Revised: 10 January 2017 / Accepted: 14 January 2017 / Published online: 30 January 2017
Ó Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2017
Abstract Fifteen bacterial isolates were isolated from a
watering canal at Al Hadady-Damrou, Kafr El-Sheikh
Governorate, Egypt (31.3°N 30.93°E). The screening
process was achieved based on nitrogenase activity. The
most potent bacterial isolate (B9) was tested as plant-
growth-promoting rhizobacteria (PGPR). Ultrastructural,
cultural, biochemical characteristics and 16S rDNA par-
tial sequence were used for the isolate identification and
characterization. From the 16S rRNA gene sequencing
results, the nearest bacterial species to our isolate was
Achromobacter marplatensis B2 (T), EU150134.1, with
97% matching. The sequence was submitted to the NCBI
website with the accession number GenBank:
KM491552.1. In vitro analysis revealed that the isolate
under study is non-pathogenic (virulence factors-free)
and capable of producing indole acetic acid (IAA),
gibberellin (GA3) and solubilizing rock phosphate.
Under greenhouse conditions, tomato inoculation with
the obtained Achromobacter sp. EMCC1936 significantly
increased vegetative growth, yield parameters and
endogenous phytohormones content as compared with
common free diazotrophic PGPR, Azotobacter
chroococcum EMCCN1458. It was deposited in Micro-
biological Resource Center for public use with number
(EMCC1936). Data revealed the importance of soil
Communicated by MJ Reigosa.
& Hoda A. S. El-Garhy
hoda.algarhy@fagr.bu.edu.eg
1 2003; Vacheron et al. 2013). The application of PGPR results
Agricultural Microbiology, Agricultural Botany Department,
Faculty of Agriculture, Benha University, Moshtohor,
Qalyubia 13736, Egypt
2
Genetics Department, Faculty of Agriculture, Benha
University, Moshtohor, Qalyubia 13736, Egypt
•
inoculation with the obtained isolate and of its role in
increasing soil enzymatic activity. These features fulfill
the isolate to be used as a PGPR for various crops.
Keywords Sustainable agriculture  Achromobacter sp 
16S rRNA gene  Scanning electron microscopy (SEM) 
Transmission (TEM)  Plant-growth-promoting
rhizobacteria (PGPR)
Introduction
Sustainable agricultural production can be achieved using
bio-resources as a supplement to chemical fertilizers for
minimizing environmental damage and health hazards.
Many rhizobacterial strains that have been proven useful as a
major source of various bio-agents are being applied to
improve both soil biochemical processes and plant perfor-
mance. Plant-growth-promoting rhizobacteria (PGPR) are
highly diverse and act as biofertilizers, where biofertilizers
are less expensive and more safe relative to chemical fertil-
izers (Abd El-Aal and Abd El-Rahman 2014; Krishnaraj and
Dahale 2014). Nitrogen fixing PGPR application promises to
be an effective tool in biofertilization, which in turn will
enhance productivity and sustainability of agriculture (Si-
vasakthi et al. 2014). In addition to nitrogen fixation, PGPR
may also have other plant-growth-promoting activities such
as facilitating mineral uptake, helping in phosphorus solu-
bilization process, producing phytohormones, and stimu-
lating disease resistance mechanisms (Dobbelaere et al.
in a reduction of soil pH which positively influences the
solubility of some nutrients such as P, Fe, Zn, Mn and Cu,
which increase nutrient uptake by plants (Abd El-Aal and
Abd El-Rahman 2014).
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Traditional phenotypic profile-based analyses for identifying
Achromobacter species have been demonstrated to be inade-
quate in differentiating them from other similar Gram-negative,
aerobic and non-fermenting species (Wayne et al. 1987; Gomila
et al. 2011; Vandamme et al. 2013; Wittmann et al. 2014). The
use of 16Sr RNA as a biomarker gene in genetic fingerprinting
proved to be decisive in identifying different isolates in many
laboratories (Wayne et al. 1987; Spilker et al. 2013; Gomila
et al. 2011; Vandamme et al. 2013; Gomila et al. 2014).
Production of different phytohormones has taken place in
many PGPR belonging to Achromobacter sp. (Abd El-Azeem
2007; Vacheron et al. 2013; Mareque et al. 2015). Achro-
mobacter sp. diazotrophic endophytic bacteria, fixes Nitrogen
more efficiently than rhizospheric organisms, presumably due
to the low concentration of Oxygen molecule in the interior of
plants, as well as direct accessibility of the fixed nitrogen to the
plants (Prabhat and Kumar 2009; Mareque et al. 2015).
Therefore, this research was designed to isolate new friendly
diazotrophic endophytic bacterial isolates from an agricultural
canal. Our study seems to be the first report on the isolation of
A. marplatensis from an aquatic environment. In addition to
diazotrophic nature, other beneficial features including P
solubilization, GA3 and IAA production by this bacterium
have been evaluated. Also, tomato (Lycopersicon esculentum
var. commune) was used to study the effect of the obtained
isolate on different plant growth parameters as a promising
PGPR under greenhouse conditions.
Materials and methods
Samples’ collection
Water samples were collected from the agricultural canal at
Kafr Elsheikh governorate (31.3°N 30.93°E) (Fig. 1). Sam-
ples of water were transferred to sterile plastic bags by using
Fig. 1 Sites of collected water samples, watering canal at Al Hadady-Damrou, Kafr El-Sheikh Governorate, Egypt (31.3°N 30.93°E)
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Acta Physiol Plant (2017) 39:61
150 ml sterile syringes and were delivered to the laboratory
within 4 h after their collection under aseptic conditions.
Isolation and selection
Serial dilution and spread plate method was used to isolate
the desired bacteria as described by (Aneja 2003). To get
10-1 to 10-6, 0.1 ml concentration range of each dilution,
10-ml sample of water was diluted using sterilized distilled
water, each sample was uniformly spread on plates con-
taining Modified Ashby’s medium agar which was incu-
bated at 35 °C for 24–72 h (Abd El–Malek and Ishac
1986). The single colonies were sub-cultured to get a pure
culture. Bacterial isolates were screened according to
nitrogenase activity. The obtained bacterial isolates were
kept on tryptic soya agar slants at 4 °C.
Light microscopy and characterization
The more potent bacterial isolate was examined using a
light microscope to check for the Gram stain reaction,
shape, sporulation, motility and flagella-staining proce-
dures. Hanging drop method was used to determine the
motility of bacteria (Bertrand et al. 2001). Spore-forming
bacteria were determined according to Harrigan and Mac-
Cance (1976). The cultural characteristics were recorded as
colony morphology, i.e., color, shape, size, nature of the
colony and pigmentation according to the method descri-
bed by Hucker and Conn (1923).
Scanning electron microscopy (SEM)
Samples of the selected bacterial isolate were fixed in 2.5%
glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, for 2 h at
room temperature and washed in the same buffer. The
samples were post-fixed in 1% osmium tetroxide in the same
Acta Physiol Plant (2017) 39:61
buffer for 2 h at room temperature. In 2 h, the cells were
rinsed with PBS for three times. The specimens were dehy-
drated in a series of graded ethanol (50, 60, 70, 80, 90 and
100%) for a period of 15 min in each series. Fixed samples
were dried under carbon dioxide using a critical point dryer
(EMS 850, USA) and sputter covered with gold in a sputter
coater system (SPI-Module, USA). Morphological changes
observed and photomicrographs were then carried out with a
JEOL JSM-5500LV scanning electron microscope (JEOL
Instruments Inc., Japan) at an accelerating voltage of 20 kV
at the Regional Center for Mycology and Biotechnology
(RCMB), Al-Azhar University, Egypt.
Transmission electron microscopy (TEM)
Biofilm was prepared by using freeze-substitution and
conventional embedding methods, and then it was thin-
sectioned on a Reichert-Jung Ultracut E ultra-microtome.
Before thin-sectioning, the sapphire disks were removed
from the Epon, where the bacterial sample was kept
embedded in the plastic resin, and later it was sectioned.
Sections were ridden on Formvar and carbon-coated
200-mesh copper grids. To improve contrast, sections were
post-stained in 2% (wt/vol) uranyl acetate. A transmission
electron microscope (Philips, CM10) operating at 80 kV
under standard operating conditions was used to perform
electron microscopy. All observations reported in this
paper are based on interpretations from 5 to 10 thin sec-
tions from numerous biofilm samples (Hunter and Bev-
eridge 2005). Photomicrographs were then carried out with
a JOEL1010 model at the Regional Center for Mycology
and Biotechnology (RCMB), Al-Azhar University.
APIÒ 20 methods
The selected bacterial isolate was characterized biochem-
ically using the APIÒ 20 E system strips that consist of 20
micro-tubes which contain dehydrated substrates. The
conventional tests were inoculated with a saline bacterial
suspension which reconstitutes the media. The metabolism
of the organism under investigation caused color changes
during incubation period by the addition of substances in
response to a specific enzymatic reaction. Samples were
read following the reading table included with the API kit;
in addition, the identification was achieved according to the
analytical profile index or by using the identification
software.
Antibiotics susceptibility test
Susceptibility test of the most potent isolate was carried out
by using the method of disk diffusion and read according to
Matuschek et al. (2014). Antibiotics that were tested in this
Page 3 of 15 61
part of study were: b-lactams (cefotaxime, ceftazidime,
oxacillin, meropenem, aztreonam, amoxicillin ? clavu-
lanic acid; augmentin; imipenem, trazobactam and ampi-
cillin), aminoglycosides (amikacin and gentamicin),
glycopeptide (vancomycin), cephalosporin (cephradine,
cefodizime, cefoxitin, cefixime, cefadroxil and cefepime),
oxazolidinone (linezolid), fluoroquinolone (ofloxacin and
levofloxacin), and ansamycins (rifamycin).
DNA isolation
For PCR amplifications, bacterial genomic DNA was
extracted by inoculating LB broth media with the selected
bacterial isolate and incubated overnight at 37 °C with
shaking. A 5 ml sample of the bacterial suspension was
centrifuged. Following discarding of the supernatant, 1 ml
TE buffer was used for washing the pellet three times, and
then it was re-suspended with 500 ll TE buffer. A 10 ll
Proteinase K (20 mg ml-1), 20 ll lysozyme (50 mg ml-1)
and 100 ll 10% SDS were added to the cell suspension.
The bacterial cell suspension was incubated overnight;
100 ll 5 M NaCl and 100 ll 10% CTAB were added and
well mixed, and this mixture was incubated for 30 min at
70 °C, and then it was incubated for 10 min on ice. The
tubes were centrifuged at high speed for 15 min. The upper
phase—should not be viscous—was transferred to a 1.5 ml
clean micro-centrifuge tube; equal volume from phe-
nol:chloroform:isoamyl (25:24:1) was added and mixed
well by inverting the tubes. The mixtures were centrifuged
at 15,000 rpm for 15 min. Clean micro-centrifuge tubes
were used to transfer the upper phase in it, and then an
equal volume of 24:1 (chloroform-isoamyl) was added
followed by centrifugation at 15,000 rpm for 15 min. The
upper phase was moved to the clean micro-centrifuge tube
followed by adding twofold volume of absolute ethanol
and incubated overnight at -20 °C. For increasing the
DNA yield, 50 ll 5 M sodium acetate was added. The
tubes were centrifuged at high speed for 20 min. Finally,
1 ml of 70% ethanol was used for washing the DNA pellet
three times, and then it was re-suspended in 50 ll sterilized
TE Buffer which was kept at -20 °C for further use.
PCR reaction
Polymerase chain reaction (PCR) analysis was performed
using 16S rRNA gene according to (Jiang et al. 2006). The
primers used were universal 27F (50 -AGAGTTTG-
GATCMTGGCTCAG-30 ) and 1492R (50 -CGGTTACC
TTGTTACGACTT-30 ). The PCR reaction was amplified in
50 ll mixtures that contained 0.4 lM of each primer,
400 lM of dNTP mix, 2 mM MgCl2, 5 ll of 109 PCR
buffer, 2.5 units from TAKARA Taq DNA polymerase (Cat.
#:R001AM) and 1 lg DNA. Master cycler (Eppendorf) was
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61 Page 4 of 15
used for amplification under the following conditions: initial
denaturation step at 95 °C for 3 min, 35 cycles of amplifi-
cation (95 °C for 50 s, 52 °C for 1 min and 72 °C for 1 min),
and followed by a final extension at 72 °C for 10 min. The
obtained PCR product was examined using electrophoresis
on 1.2% agarose gel stained with ethidium bromide using
GeneRulerTM 1 kb DNA ladder (Cat. #: SM0313), and then
visualized under UV transilluminator.
Cloning and sequencing
The expected DNA band, almost 1500 bp, was eluted
from agarose gel and purified according to the manufac-
turer’s QIAquick Gel Extraction Kit (Cat.#: 28704). The
purified PCR fragment was ligated in pGEMÒ-T
Easy Vector Systems (Cat. #: A1360) according to the
manufacturer. The competent cells from the top 10 strains
were prepared and transformed as described by Inoue
et al. (1990). The white colonies were picked up from
LB/Amp/Xgal plates and inoculated on LB/Amp broth
media. Then it was incubated overnight at 33 °C with
shaking for stabilizing the plasmid inside the transformed
cells. The alkaline method of Birnboim and Doly (1979)
was used to isolate the plasmid. The purified plasmids
were examined using electrophoresis on 1.2% agarose gel
using GeneRulerTM 1 kb DNA Ladder (Cat. #: SM0313)
to confirm the recombinant plasmids. The recombinant
plasmids were sequenced by Macrogen Company (South
Korea).
Phylogenetic analysis
The obtained sequence for 16S rRNA gene was examined
for vector contamination using VecScreen tool (http://
www.ncbi.nlm.nih.gov/tools/vecscreen/). Also, NEbcuter
V2.0 was used to create a restriction map and to identify
the GC content of the obtained sequence (Vincze et al.
2003, http://nc2.neb.com/NEBcutter2/). Possible ORFs
of the resulted sequence were obtained by using ORF
finder software. Also, Jalview software was used to show
single nucleotide polymorphisms (SNPs) and consensus
resulted from the alignment of our bacterial isolate
obtained sequence and the nearest bacterial strain in
EzTaxon-e database (http://www.ezbiocloud.net/eztaxon)
(Kim et al. 2012). Construction of the phylogenetic tree
was done using Clustal Omega and MEGA6 software.
The sequence was registered in NCBI database under
accession number KM491552.1. Also, the obtained iso-
late was deposited in Microbiological Resource Center
(MERCIN), Egypt. Microbial Culture Collection
(EMCC) belonging to the WDCM with number (EMCC
1936).
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Acta Physiol Plant (2017) 39:61
Detection of virulence factors
The selected bacterial strain was grown in tryptic soy broth
up to five days. The isolate cells were harvested by cen-
trifugation at 10,000 rpm for 5 min, the supernatant was
filtered aseptically using filters with pore size 0.22 lm,
thereafter this harvest was used for detection of virulence
factors. Elastase, chitinase, rhamno lipid and protease
activities were assayed in supernatants as described by
Jakobsen et al. (2013). Chitinase and elastase activities
were measured by a spectrophotometer (SCO-Tech, SPUV-
19, Germany) at 550 and 495 nm, respectively.
Plant-growth-promoting features of Achromobacter
sp. EMCC1936
Nitrogenase activity was measured as a guide for nitrogen
fixation using the acetylene reduction technique given by
Diloworth (1970). The acetylene reduction activity of
isolate was measured using Gas–liquid chromatography
GLC.
The ability of the isolate to solubilize phosphate was
screened on Pikovskaya’s (PVK) media, according to
Nguyen et al. (1992). Phosphate solubilization was evalu-
ated quantitatively on the National Botanical Research
Institute’s Phosphate (NBRIP) growth and broth media
(Nautiyal 1999). Results were expressed according to
(Naik et al. (2008).
Moreover, Yeast Extract Mannitol broth medium was
used to determine qualitatively the isolate ability to pro-
duce gibberellins (GA3) and indole acetic acid (IAA)
according to Pandya and Desai (2014) and Sarwart et al.
(1992) methods, respectively.
Evaluation of Achromobacter sp. EMCC1936
inoculum on tomato production
Plant-growth-promoting potential of Achromobacter sp.
EMCC1936 was evaluated and compared with common
free diazotrophic PGPR reference strain named Azotobac-
ter chroococcum EMCCN1458 that obtained from Micro-
biological Resource Center (MIRCEN) Cairo, Egypt.
A 3-month pot experiment (Marsh to May, 2015) was
carried out in a greenhouse, Faculty of Agriculture, Benha
University –Qalyubia Governorate, Egypt. Plastic pots
(20 9 27 cm) were filled with a mix of 20 kg soil (clay
soil, pH 8.03, organic matter 3.1%, bulk density
1.36 g cm-3, field capacity 51.1%, wilting point 17.05%)
and 10 g Compost [herbal plant residues as well as cattle
manure (50:50) are: pH 7.6, electrical conductivity (EC)
3.1 dS m-1, total organic matter values 32.7%, total-N
1.21%, total-P 0.91% and the porosity 62.67%].
Acta Physiol Plant (2017) 39:61
Prior to transplantation, tomato seedlings L. esculentum
(c.v. Super Strain B) were obtained from a special nursery in
Qaha city. Seedlings were divided into three groups, two
were soaked by dipping the root system in mixture of 10%
Arabic gum as an adhesive for inocula of either Achro-
mobacter sp. EMCC1936 or A. chroococcum EMCCN1458
(7 9 109) cell suspension for 30 min before transplanting
and one was a control (not treated with bio-inoculants). Pots
were transplanted (2 seedlings/pot) and organized in a ran-
domized complete plot design with one factor and ten
replicates. Tomato plants were watered weakly and the boost
doses of PGPR strains were added, 30 and 50 DAT. Chemical
fertilizers were supplemented to all treatments with 1/4 of the
recommended dose (50 kg N fed-1 as ammonium sulfate
(20.5% N), 25 kg P2O5 fed-1 as super phosphate (15.5%
P2O5) and 40 kg K2O fed-1 as potassium sulfate (48%
K2O). The activity of each of dehydrogenase, phosphatase
and nitrogenase was determined at 30, 60 and 90 DAT. At the
flowering stage (60 DAT), some plants were collected to
measure different growth parameters and phytohormones’
content. A total number of fruits/plant was recorded 90 DAT.
Measurements of microbial enzymatic activity
The microbial enzymatic activities were determined in
rhizosphere at 15, 30 and 60 DAT. The activities of
dehydrogenase (DH) and alkaline phosphatase (Alp) were
measured using spectrophotometer (SCO-Tech, SPUV-19,
Germany) at 464 and 400 nm, respectively, as described by
Schinner et al. (1996). However, nitrogenase (N2-ase)
activity was measured as previously mentioned with some
modification by Okafor and MacRae (1973).
Morphological, endogenous phytohormones
and yield characteristics
Vegetative growth parameters were evaluated on ten plants
which were randomly taken from each treatment, stem
length (cm), branches/plant number, shoot dry weight (g),
root size (cm3) and number of flowers/plant. For each
treatment, total number of fruits/plant was recorded as well
as fruits setting percentage was calculated. Endogenous
Phytohormones (IAA, GA3 and abscisic acid) were eval-
uated quantitatively in tomato shoots at 60 DAT using
high-performance liquid chromatography (HPLC; YL9100
HPLC System, Korea) according to the procedure of
Koshioka et al. (1983). Cytokinins was determined by
HPLC according to Nicander et al. (1993).
Statistical analysis
The obtained data were statistically analyzed according to
the methods outlined by Gomez and Gomez (1984). For
Page 5 of 15 61
comparison between means, Duncan’s multiple range test
was used (Duncan, 1955). Means followed by the same
alphabetical letters were not significantly different at 5%
level of significance.
Results
Isolation and screening of bacterial isolates
Fifteen different bacterial isolates were detected on Ash-
by’s medium after 48–72 h of incubation at 35 °C. Bac-
terial isolates were screened based on nitrogenase activity
(Table 1) and the more potent bacterial isolate (B9)
(p \ 0.05) was selected for the subsequent experiments.
Diazotrophic nature of this obtained isolate was confirmed
by its ability to produce nitrogenase.
Morphological characters of the more potent
bacterial isolate
After 48 h of incubation on nitrogen free Ashby’s medium,
at 35 °C, the colonies of the selected isolate ranged in size
from 0.5 to 2.25 mm in diameter, become opaque and
mucoid. Continued incubation resulted in a colony with
diameter from 3.0 to 4.8 mm. After incubation at 35 °C for
12 h, the tryptic soya broth culture exhibited a barely
visible turbidity that after an additional 12 h, it increased to
approximately 2.0 9 109 cfu ml-1. The growth was uni-
formly distributed throughout the broth. A small amount of
sediment was produced by the isolate that rose upward by
rotation. Continued incubation led to increasing turbidity
near the surface and the formation of a slimy pellicle.
Regarding, microscopic morphology, the obtained bacterial
isolate was straight rods, 0.6–1.2 lm with rounded ends
(Table 2). It was Gram negative, single and diploid, non-
spore forming and motile with peritrichously-arranged
flagella. Electron micrographs (SEM and TEM) images
revealed the accumulation of extracellular material sur-
rounding one pole of the Achromobacter isolate cells and
forming a polar cap as shown in (Fig. 2). These accumu-
lations add irregular contours so that many of the cells had
a coryneform appearance. On Gram stain, curved (solid
arrowhead) and hooked-end (arrow) forms were also
presented.
Biochemical characterization
The bacterial isolate grew on nitrogen free Ashby’s med-
ium showing obligatory aerobic features. Phenotypic
characterization based on API system (Table 3), showed
that it was oxidase-positive and gelatinase-negative. It
utilized glucose as sole carbon source and produced acid
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Table 1 Screening of bacterial

Isolates number Isolates code Nitrogenase activity (lg C2H4 h l-1)
isolates according to
nitrogenase activity 1 B1 24.6k
2 B2 37.2f
3 B3 28.6hi
4 B4 42.8d
5 B5 21.4l
6 B6 33.7g
7 B7 46.1c
8 B8 26.8j
9 B9 53.4a
10 B10 47.5b
11 B11 29.7h
12 B12 33.1g
13 B13 41.6e
14 B14 38.2f
15 B15 27.8ij
Means followed by the same alphabetical letters were not significantly different at 5% level of probability
according to Duncan test

Table 2 Morphological and

Characteristics Results
cultural characteristics of the
obtained Achromobacter Shape Straight rods, 0.6–1.2 lm with rounded ends
bacterial isolate
Gram stain Gram negative
Spore formation Non-spore forming
Motility Motile by peritrichous flagella
Respiration Aerobic
Colonies
Color Non-pigment
Size 0.5–2.25 mm in diameter
Opacity Opaque and mucoid
Growth on Ashby’s medium Positive

derived from glucose, mannitol, inositol, sorbitol, rham-
nose, saccharose, melibiose, amygdalin, arabinose and
xylose which caused indicator color change. Data showed
that the obtained isolate was capable of using denitrifica-
tion as a respiratory process with nitrate electron acceptor.
Antibiotic susceptibility test
Antibiotic susceptibility patterns of the obtained Achro-
mobacter sp. EMCC1936 isolate demonstrated a susceptibil-
ity profile characteristic (Table 4). The isolate was resistant to
ceftazidime, amoxicillin ? clavulanic acid (augmentin),
linezolid and rifamycin. It was categorized as intermediate
resistant to cefotaxime, oxacillin, meropenem, trazobactam,
ampicillin, cefodizime, cefoxitin, cefixime, cefadroxil, cefe-
pime and ofloxacin. It remained susceptible to aztreonam,
imipenem, amikacin, gentamicin, vancomycin, levofloxacin
and trimethoprim/sulfamethoxazole (Bactrim).
DNA and phylogenetic analysis
The obtained PCR amplified fragment for 16S rRNA gene
was &1500 bp (Fig. 3). The PCR product sequencing
result was registered in NCBI database under accession
number, KM491552.1. Analysis of the obtained sequence
via VecScreen database showed no contamination with
vector sequence. The FASTA homology showed that
the16S rRNA gene sequence of the current isolate (ACC.
No. KM491552.1) had 97% nucleotide similarity with that
of Achromobacter marplatensis B2 (T) strain recorded in
EzTaxon-e database (ACC. No. EU150134). This result
was confirmed by the phylogenetic position of the obtained
isolate, forming polyphyletic clade with A. marplatensis
B2 (T), but with an obvious phylogenetic distance (Fig. 4).
Also, the restriction Map of the obtained 16S rRNA partial
sequence was done as shown in Fig. 5a. By calculating
pairwise alignment analysis, data exhibited 16 SNPs

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Acta Physiol Plant (2017) 39:61
Fig. 2 Ultrastructure images of the obtained Achromobacter sp. EMCC1936 from current study: a scanning electron microscopy image (SEM).
b–f Transmission electron microscopy images (TEM)
between the sequence of the obtained isolate and the
nearest registered bacterial strain, A. marplatensis B2 (T),
(EU150134), for 16S rRNA gene (Fig. 5b).
Detection of virulence factors
The ability of the obtained isolate, Achromobacter sp.
EMCC1936, to produce protease on agar plates containing
5% skim milk was studied. A clearing zone is considered as
a proteolytic activity being present. Chitinase was mea-
sured by degradation of chitin azure, while elastase activity
was measured by degradation of elastin Congo red. Also,
the production of rhamnolipid was determined by lysing of
the red blood cells. From the obtained results it was clear
that none of investigated virulence factors were
detectable under the conditions used in this study.
In vitro plant-growth-promoting features
of Achromobacter sp. EMCC1936
Under laboratory conditions, Achromobacter sp.
EMCC1936 was bio-assayed for its growth-promoting
potentiality through nitrogenase activity and phytohor-
mones production such as auxins and gibberellins
(Table 5). It had 55.03 lg C2H4 h l-1 nitrogenase activity
indicating its ability to fix atmospheric N2. It also pro-
duced 50.8 lg ml-1 of IAA and 10.35 lg ml-1 of GA3.
Moreover, it had the ability to solubilize rock phosphate
Page 7 of 15 61
on PVK agar medium, and it showed 104% phosphate
solubilization efficiency. The quantitative assessment of
phosphate solubilization on NBRIP broth medium was
23.7 lg ml-1.
Effects of inoculation with Achromobacter sp.
EMCC1936 on tomato production
Effect of bio-inoculants on enzymes activity of tomato
rhizosphere
Soil enzymes activity has been suggested to be an index for
soil fertility, whereas soil microbial activity has been used
as an index of fertility. Data in Table 6 showed that during
the experimental periods, the effects of bio-inoculants (A.
chroococcum and Achromobacter sp. EMCC1936) tended
to be stronger on dehydrogenase, alkaline phosphatase and
nitrogenase activities when compared with un-inoculated
treatment (control). The overall data revealed that the
inoculation with Achromobacter sp. EMCC1936 had sig-
nificant effect on the previous soil enzymes activity com-
pared with A. chroococcum EMCCN1458 inoculation. At
flowering stage (60 DAT), inoculation with Achromobacter
sp. EMCC1936 showed about fourfold, sixfold and fivefold
increase in dehydrogenase, alkaline phosphatase and
nitrogenase activities, respectively, when compared to
initial period. However, control showed about twofold
increase in all enzymes’ activity.
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Vegetative growth and yield characteristics

mannitol, INO fermentation of inositol, SOR fermentation of sorbitol, RHA fermentation of rhamnose, SAC fermentation of saccharose, MEL fermentation of melibiose, AMY fermentation of
OX

ONPG b-galactosidase (ortho nitrophenyl-bD-galactopyranosidase), ADH arginine dihydrolase, LDC lysine decarboxylase, ODC ornithine decarboxylase, CIT citrate utilization, H2S H2S
production, URE urease, TDA tryptophane deaminase, IND indole production, VP acetoin production (Voges Proskauer), GEL gelatinase, GLU fermentation of glucose, MAN fermentation of
1
NO3
Inoculation of tomato with Achromobacter sp. EMCC1936

1
affected plant length, number of branches/plant, stems and
leaves dry weight, root size and number of flower/plant as

Xyl

1
compared to the control (p \ 0.05) (Table 7). Relative to
ARA A. chroococcum EMCCN1458 and the control, Achro-
1
mobacter sp. EMCC1936 strain caused 24 and 162% more
fruits number/plant; it had relatively more 12 and 35%, for
AMY

percentage of fruit setting respectively.

amygdalin, ARA fermentation of arabinose, Xyl fermentation of xylose, NO3 nitrous reduction, OX cytochrome-oxidase, (?) developed color, (-) colorless
1

Endogenous phytohormones content of tomato shoot

MEL

Levels of major tomato shoot endogenous phytohor-

SAC

mones—both growth promoting and inhibiting ones—were

1

all maintained at satisfactory concentrations (p \ 0.05) due

to Achromobacter sp. EMCC1936 inoculation (Table 8).
RHA

Relative to the control shoots, concentrations of each of

gibberellins (GA), auxins (IAA), and cytokinins were 79,
SOR

226, and 177%, respectively. These concentrations dropped

1

to differentials of 5, 15, and 16%, respectively, if compared

INO

to those resulted from A. chroococcum EMCCN1458

1

inoculant. For growth-inhibiting abscisic acid (ABA), the

Achromobacter sp. EMCC1936 inoculant caused compar-
MAN

ative reductions of 51 and 18%, respectively. Finally, all

1

the obtained results qualified the isolated bacterial strain,

GLU

Achromobacter sp. EMCC1936, to be added to Egypt.

1

Microbial Culture Collection (EMCC), at Cairo MERCIN

under number (EMCC 1936) to be available for
GEL
Table 3 The APIÒ 20 E strip results for the isolated Achromobacter bacterial isolate

application.
VP

Discussion
IND

Plant-growth-promoting rhizobacteria (PGPR), particularly

TDA

Achromobacter marplatensis involved positively in dif-

2

ferent biotic activities in the soil which enhance plant

URE

growth as a result of nutrient obtainability in the soil as

1

well as producing various plant growth regulators (Ahemad

and Kibret 2014; Wu et al. 2014). It is oxidase, catalase-
H2S

positive, Gram-negative and an aerobic bacilli that lives in

aqueous environments and contaminated soils (Gomila
CIT

et al. 2011; Vandamme et al. 2013). Morphological and

biochemical examination of the obtained isolate revealed
ODC

several distinctive features related to genus Achromobac-

2

ter. Our isolate, Achromobacter sp. EMCC1936 was tested

LDC

both morphologically and biochemically to provide definite

1

information for using in bacterium identification. By using

a light microscope, most cells were seen as typical rod-
ADH

shaped and Gram-negative bacilli, but some of them

2

apparently had curved forms. Moreover, bacterial cells

ONPG

were seen with a swollen at one end as well as electron

microscopy was used in addition to the previous methods
?

123
Acta Physiol Plant (2017) 39:61 Page 9 of 15 61

Table 4 Antibiograms of the obtained Achromobacter isolate by disk diffusion method

Antibiotics Antibiotic abbreviation Antibiotic (lg disk-1) Inhibition zone per mm

b-Lactams
Cefotaxime CTX 30 8.0
Ceftazidime CAZ 30 0
Oxacillin OX 11
Meropenem MEM 10 14
Aztreonam ATM 15 27
Amoxicillin ? clavulanic acid; augmentin AMC 30 0
Imipenem IPM 10 26
Trazobactam TPZ 10 20
Ampicillin AMP 20 16
Aminoglycosides
Amikacin AK 30 31
Gentamicin CN 10 27
Glycopeptide
Vancomycin VA 30 24
Cephalosporin
Cephradine CE 30 21
Cefodizime CDZ 0
Cefoxitin FOX 5 15
Cefixime CFM 30 19
Cefadroxil CFR 19
Cefepime FEP 30 13
Oxazolidinone
Linezolid LZD 30 0
Fluoroquinolone
Ofloxacin OFX 5 17
Levofloxacin LEV 5 23
Ansamycins
Rifamycin RF 20
Trimethoprim/sulfamethoxazole; Bactrim SXT 25 30

to show bacteria in great detail in their natural environ-
ment. Electron micrographs revealed the accumulation of
extracellular material on many of the cells often at one pole
(Fig. 2). Apparently these accumulations added irregular
contours to the cell, and therefore in Gram stain, appear as
curved and hooked forms which supported by Chester and
Cooper (1979). Achromobacter isolate was the exceedingly
small colonies produced at 24 h and the subsequent rapid
increase in colony size after continued incubation due to
the elaboration of large amounts of mucoid material.
Phenotypic characterization of Achromobacter sp.
EMCC1936 using API 20NE test showed change in indi-
cator color, where the isolate utilized its carbon sources, D-
xylose, D-gluconate and D-glucose. Data of morphological
and biochemical examination of the obtained isolate agree
with previous findings (Hugh 1970; Chester and Cooper
1979; Kersters and De Ley 1984; Yabuuchi et al. 1998;
Vandamme et al. 2013). They reported that the genus
Achromobacter is a Gram-negative, oxidase-positive,
obligate aerobic, non-fermenting bacilli with peritrichous
flagella, and is able to oxidize maltose, mannitol, and
sucrose. Although, the biochemical studies using API
20NE revealed core of reactions useful in the identification
of Achromobacter; however, they were not enough to fully
identify the isolated strain.
Regarding antibiograms of Achromobacter strain,
results from previous studies reported that isolates
belonging to A. xylosoxidans exhibited susceptibility to
piperacillin (95.6%), ceftazidime (91.1%), imipenem
(97.2%) and trimethoprim-sulfamethoxazole (78.6%),
while they were resistant to most cephalosporins, amino-
glycosides, ampicillin and aztreonam (Legrand and

123
61 Page 10 of 15
Fig. 3 PCR products for 16S RNA partial-length gene (1500 bp) of
the obtained diazotrophic bacterial isolate. -C, negative control, no
DNA sample. M refers to GeneRulerTM 1 kb DNA ladder (Cat. #:
SM0313)
Fig. 4 Phylogenetic trees recovered from maximum likelihood
analyses of the 16S rRNA gene partial sequence for the obtained
isolate, Achromobacter sp. EMCC1936 (KM491552.1). The trees
show the phylogenetic position of recovered Achromobacter species
123
Acta Physiol Plant (2017) 39:61
Anaissie 1992; Duggan et al. 1996; Knippschild et al.
1996; Gomez-Cerezo et al. 2003). The above-mentioned
results are not in harmony with our results except the
susceptibility to imipenem. This is evident from the fact
that the obtained isolate is a novice strain.
For confirming the isolate identification, ribosomal genes
sequencing was carried out, which is a more effective iden-
tification approach concerning non-fermenting Gram-nega-
tive bacilli than traditional phenotypic procedures
(Wellinghausen et al. 2006; Van Hal et al. 2008; Gomila et al.
2011; Mareque et al. 2015). In the current study, sequencing
analysis results confirmed that the obtained bacterial strain
has similar characteristics to A. marplatensis B2 (T), which
was obtained from contaminated soil and characterized by
Gomila et al. 2011. Based on the sequence of 16S rRNA
gene, it was monitored at an accurate position of the phylo-
genetic tree. This gene has been proven to have quite slow
rates of evolution, indicating its genetic stability. An indi-
cation of this gene stability was confirmed by the GC content
and of its slow evolutionary rate by the presence of 16 SNPs.
within the phylogenetic branches of Alcaligenaceae family. Average
bootstrap values, of compared algorithms, are indicated at the branch
roots. The bar represents 0.02 changes per nucleotide. Accession
numbers of database extracted sequences are in brackets
Acta Physiol Plant (2017) 39:61
Fig. 5 a Restriction map of the partial sequence of 16S rRNA gene of
interest with available commercially restriction enzymes. b Single
nucleotide polymorphism (SNPs) showed 16 SNPs between the
obtained isolate, Achromobacter sp. EMCC1936 (KM491552.1), and
Table 5 Evaluation of the obtained Achromobacter sp. EMCC1936 bacterial strain as plant-growth-promoting rhizobacteria
IAA production GA3 production Phosphate solubilization
(lg ml-1) (lg ml-1) efficiency (%)
50.8 10.35 103.7
In this respect, many studies were conducted using 16S
rDNA method for proving identity and characterization of
unknown isolates, especially Achromobacter sp. (Drancourt
et al. 2000; Janda and Abbott 2007; Petti 2007; Gomila et al.
2011; Abyar et al. 2012; Mazumdar and Deka 2013; Bhosale
et al. 2014; Ramasamy et al. 2014; Das et al. 2014; Srini-
vasan et al. 2015; Alhamlan et al. 2015; Mareque et al. 2015).
One prominent feature of Achromobacter sp.
EMCC1936 which was noticed in this study is the capa-
bility to reduce nitrogen molecules from the atmosphere
Page 11 of 15 61
the nearest one on EzTaxon-e database, Achromobacter marplatensis
B2 (T), EU150134.1 (Kim et al. 2012) based on pairwise alignment
analysis method
Phosphate concentration Nitrogenase activity
(lg ml-1) (lg C2H4 h l-1)
23.7 55.03
into ammonia (diazotrophy), which was confirmed by
estimating nitrogenase activity. Besides diazotrophy, other
beneficial characteristics are IAA production and solubi-
lized high amount of phosphate. Growth-promoting char-
acters were exhibited by many endophytic diazotrophic
bacteria including A. xylosoxidans and A. marplatensis
(Prabhat and Kumar 2009; Wedhastri et al. 2013; Wu et al.
2014). The obtained results from the current study are in
harmony with the previous studies but are novel especially
for the bacterium Achromobacter sp. EMCC1936 where it
123
61 Page 12 of 15 Acta Physiol Plant (2017) 39:61

Table 6 Effect of tomato (Lycopersicon esculentum var. commune) inoculation with Achromobacter sp. EMCC1936 on microbial enzymes
activity
Treatments Dehydrogenase activity Alkaline phosphatase activity Nitrogenase activity
(lg TPF g-1 dw h-1) (lg qNP g-1 h-1) (ll C2H4 g-1 dw h-1)
Initial 30 60 90 days Initial 30 60 90 days Initial 30 60 90 days

Control 10.3h 14.9g 17.8f 14.4g 3.2h 4.4g 6.1f 5.6f 9.2h 15.2g 19.7e 17.0f
A. chroococcum 10.8h 23.5e 28.01c 25.4d 3.4h 7.8e 9.3c 8.7d 8.9h 39.2d 42.9a 40.2c
Achromobacter sp. EMCC1936 10.4h 38.7b 40.6a 39.5ab 3.2h 19.2b 20.7a 19.4b 9.3h 40.3c 43.6a 41.3b
Means followed by the same alphabetical letters were not significantly different at 5% level of probability according to Duncan test

Table 7 Effect of tomato (L. esculentum var. commune) inoculation with Achromobacter sp. EMCC1936 on vegetative growth and yield
characteristics
Treatments Vegetative growth characteristics Yield characteristics
Stem length Number of Shoot dry weight Root size Flower Fruits Setting
(cm) branches/plant (g) (cm3) no./plant no./plant (%)

Control 37.3c 5.3c 14.3c 12.3c 9.3c 6.0c 64.3c

b b b b b b
A. chroococcum 54.7 8.0 33.7 34.7 16.3 12.7 77.6b
a a a a a a
Achromobacter sp. 65.7 10.7 41.3 41.3 18.0 15.7 87.0a
EMCC1936
Means followed by the same alphabetical letters were not significantly different at 5% level of probability according to Duncan test

Table 8 Effect of tomato (L. esculentum var. commune) inoculation with Achromobacter sp. EMCC1936 on endogenous phytohormones
content
Treatments Promoters (lg g-1 F.W) Inhibitors abscisic acid Promoters/
(lg g-1 F.wt.) inhibitors
Gibberellins Auxins Cytokinins Total
promoters

Control 39.4c 19.44c 32.35c 91.19c 2.08b 43.84c

A. chroococcum 67.2b 55.25b 77.92b 200.37b 1.22a 164.23b
Achromobacter sp. 70.6a 63.48a 89.57a 223.65a 1.19a 187.94a
EMCC1936
Means followed by the same alphabetical letters were not significantly different at 5% level of probability according to Duncan test

has been isolated from aquatic environment and able to
produce GA3.
Application of Achromobacter sp. EMCC1936 as a
PGPR strain with tomato had a great effect on microbial
community and their activities especially their extracellular
enzymes. These extracellular enzymes show a crucial
effect in soil biogeochemical cycles of C, N, and P that are
play role in nutrient conversion (Burns and Dick 2002; Wu
et al. 2014). Therefore, significant reductions in both soil
enzymatic activity and soil microbial biomass should be
counted as signs of soil quality and land use effectiveness
(Mganga et al. 2015). Results showed the importance of
soil inoculation with PGPR strains and their role in
increasing the activities of soil enzymes especially the
distinctive role of the isolated Achromobacter sp.
EMCC1936 in this respect. These results may be attributed
to the role of intercellular substances of Achromobacter sp.
(i.e., plant-growth-promoting substances and microbial
enzymes) in promoting soil microbial proliferation and
their activity. Moreover, increasing the population dosage
of Achromobacter sp. was beneficial to both the plant, as it
fixes more Nitrogen, and to the population of rhizospheric
organisms on plant roots.
The encouraged growth of inoculated plants with
Achromobacter sp. EMCC1936 more than A. chroococcum
EMCCN1458 may be attributed to the vital role of this
strain in enhancing the obtainability as well as acquisition
of nutrients such as phosphorus. Microorganism solubilize
phosphate (P) to provide it as a major nutrient to plants in
soils lacking P and improve the overall growth as well as

123
Acta Physiol Plant (2017) 39:61
plants development is considered another important free-
living soil microbiota (Khan et al. 2014; Wu et al. 2014).
Moreover, more fixed nitrogen has been provided through
the application of endophytic diazotrophic bacteria com-
pared to rhizospheric one where the interior of plants is a
more appropriate for nitrogen fixation as an explanation of
low partial oxygen pressure (pO2) and direct accessibility
of the fixed nitrogen to the plants (Prabhat and Kumar
2009; Mazumdar and Deka 2013; Wu et al. 2014). Fur-
thermore, Achromobacter sp. EMCC1936 has the ability to
produce IAA and GA3 that play role in increasing the root
length surface area and responsible for branching of root
hair hence nutrients up-taking from the soil increase. Zaidi
et al. (2009) reported that different phytohormones, i.e.,
IAA, gibberellins and cytokinins, are essential for shoot
growth and root morphogenesis of numerous plants. The
IAA has an impact on tissue development, the root growth,
as well as light and gravity responses. Intense growth and
high yielding biomass as well as grain production resulted
from the absorption of more nutrients and water from the
soils by roots with larger surface area hence translocate
them to different parts of the plants. On the other hand,
gibberellins affected seed germination, stem development,
flowering, as well as plants fruit setting.
Generally, inoculation with Achromobacter sp.
EMCC1936 resulted from current study, promote signifi-
cantly the phytohormones level. Also, there was an
increase of endogenous hormones in tomato may refer to
the improvement of growth features. For example, cyto-
kinins accumulation could support the increasing of the
branches number which could increase transverse growth
more than longitudinal one. Inoculation with Achro-
mobacter sp. EMCC1936 increased the content of
endogenous promoting phytohormones of tomato shoot
which may attributed to the positive effect of this isolate in
PGPs production.
Moreover, the quantities of total promoter elements
relative to the abscisic acid (ABA) as an inhibitor were
relatively higher in case of Achromobacter sp.
EMCC1936 inoculation. These treatments enhanced the
internal metabolically features of tomato causing an
increase of promoting hormones content which maxi-
mized its growth. These results are accordance with that
obtained by (Hosseny and Ahmed 2009; Abd El-Aal and
Abd El-Rahman 2014; Wu et al. 2014) who reported that
maximizing growth and productivity of lettuce, sweet
ananas melon and sugar beet were related to high pro-
moting hormones content.
In summary, both traditional and molecular characteri-
zation approaches were integrated in this study for
obtaining new friendly diazotrophic bacterial strains from
an agricultural canal. The obtained isolate, Achromobacter
sp. EMCC1936 (KM491552.1) could be exploited as plant-
Page 13 of 15 61
growth-promoting rhizobacteria (PGPR) for various crops
since it exhibits reasonable potential characteristics.
Moreover, it could be recommended as a new effective
rhizobacteria to promote plant growth, increase crop pro-
duction, decrease production costs and reduce environ-
mental pollution. Considering these facts as well as other
features of PGPR new strains, further investigations are
ongoing in our laboratories.
Author contribution statement AHM and SAA: Iso-
lated the bacterial isolate form aquatic environment,
conceived the greenhouse experiment work, performed
the experiments to measure the physiological effects of
the bacterial isolate as a PGPR, drafted the corre-
sponding sections of the manuscript and revised the
complete manuscript. MMAM and HASE: Isolated
DNA from the isolated bacterial strains, performed PCR
using 16SrRNA gene, cloned and sequenced the PCR
product for the used gene, analyzed the sequencing
results, as well as they submitted the results in NCBI
data base, also they drafted the corresponding sections
of the manuscript and revised the complete manuscript.
Acknowledgements We would like to thank Prof. Dr. M. Hany
Tageldin, Horticulture Dept., Faculty of Agriculture, Benha Univer-
sity, for his kind help and assistance.
Compliance with ethical standards
Conflict of interest There is no conflict of interest.
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