A Novice Achromobacter Sp. EMCC1936 Strain Acts As a Plant- Growth-Promoting Agent

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

1 1 2 H. M. Abdel-Rahman • A. A. Salem • Mahmoud M. A. Moustafa • Hoda A. S. El-Garhy2

Received: 20 May 2016 / Revised: 10 January 2017 / Accepted: 14 January 2017 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2017

Abstract Fifteen bacterial isolates were isolated from a inoculation with the obtained isolate and of its role in watering canal at Al Hadady-Damrou, Kafr El-Sheikh increasing soil enzymatic activity. These features fulfill Governorate, Egypt (31.3°N 30.93°E). The screening the isolate to be used as a PGPR for various crops. process was achieved based on nitrogenase activity. The most potent bacterial isolate (B9) was tested as plant- Keywords Sustainable agriculture Á Achromobacter sp Á growth-promoting rhizobacteria (PGPR). Ultrastructural, 16S rRNA gene Á Scanning electron microscopy (SEM) Á cultural, biochemical characteristics and 16S rDNA par- Transmission (TEM) Á Plant-growth-promoting tial sequence were used for the isolate identification and rhizobacteria (PGPR) characterization. From the 16S rRNA gene sequencing results, the nearest bacterial species to our isolate was Achromobacter marplatensis B2 (T), EU150134.1, with Introduction 97% matching. The sequence was submitted to the NCBI website with the accession number GenBank: Sustainable agricultural production can be achieved using KM491552.1. In vitro analysis revealed that the isolate bio-resources as a supplement to chemical fertilizers for under study is non-pathogenic (virulence factors-free) minimizing environmental damage and health hazards. and capable of producing indole acetic acid (IAA), Many rhizobacterial strains that have been proven useful as a gibberellin (GA3) and solubilizing rock phosphate. major source of various bio-agents are being applied to Under greenhouse conditions, tomato inoculation with improve both soil biochemical processes and plant perfor- the obtained Achromobacter sp. EMCC1936 significantly mance. Plant-growth-promoting rhizobacteria (PGPR) are increased vegetative growth, yield parameters and highly diverse and act as biofertilizers, where biofertilizers endogenous phytohormones content as compared with are less expensive and more safe relative to chemical fertil- common free diazotrophic PGPR, Azotobacter izers (Abd El-Aal and Abd El-Rahman 2014; Krishnaraj and chroococcum EMCCN1458. It was deposited in Micro- Dahale 2014). Nitrogen fixing PGPR application promises to biological Resource Center for public use with number be an effective tool in biofertilization, which in turn will (EMCC1936). Data revealed the importance of soil enhance productivity and sustainability of agriculture (Si- vasakthi et al. 2014). In addition to nitrogen fixation, PGPR Communicated by MJ Reigosa. may also have other plant-growth-promoting activities such as facilitating mineral uptake, helping in phosphorus solu- & Hoda A. S. El-Garhy bilization process, producing phytohormones, and stimu- [email protected] lating disease resistance mechanisms (Dobbelaere et al. 2003; Vacheron et al. 2013). The application of PGPR results 1 Agricultural Microbiology, Agricultural Botany Department, Faculty of Agriculture, Benha University, Moshtohor, in a reduction of soil pH which positively influences the Qalyubia 13736, Egypt solubility of some nutrients such as P, Fe, Zn, Mn and Cu, 2 Genetics Department, Faculty of Agriculture, Benha which increase nutrient uptake by plants (Abd El-Aal and University, Moshtohor, Qalyubia 13736, Egypt Abd El-Rahman 2014). 123 61 Page 2 of 15 Acta Physiol Plant (2017) 39:61

Traditional phenotypic profile-based analyses for identifying 150 ml sterile syringes and were delivered to the laboratory Achromobacter species have been demonstrated to be inade- within 4 h after their collection under aseptic conditions. quate in differentiating them from other similar Gram-negative, aerobic and non-fermenting species (Wayne et al. 1987;Gomila Isolation and selection et al. 2011;Vandammeetal.2013;Wittmannetal.2014). The use of 16Sr RNA as a biomarker gene in genetic fingerprinting Serial dilution and spread plate method was used to isolate proved to be decisive in identifying different isolates in many the desired bacteria as described by (Aneja 2003). To get laboratories (Wayne et al. 1987; Spilker et al. 2013;Gomila 10-1 to 10-6, 0.1 ml concentration range of each dilution, et al. 2011;Vandammeetal.2013;Gomilaetal.2014). 10-ml sample of water was diluted using sterilized distilled Production of different phytohormones has taken place in water, each sample was uniformly spread on plates con- many PGPR belonging to Achromobacter sp. (Abd El-Azeem taining Modified Ashby’s medium agar which was incu- 2007; Vacheron et al. 2013; Mareque et al. 2015). Achro- bated at 35 °C for 24–72 h (Abd El–Malek and Ishac mobacter sp. diazotrophic endophytic bacteria, fixes Nitrogen 1986). The single colonies were sub-cultured to get a pure more efficiently than rhizospheric organisms, presumably due culture. Bacterial isolates were screened according to to the low concentration of Oxygen molecule in the interior of nitrogenase activity. The obtained bacterial isolates were plants, as well as direct accessibility of the fixed nitrogen to the kept on tryptic soya agar slants at 4 °C. plants (Prabhat and Kumar 2009; Mareque et al. 2015). Therefore, this research was designed to isolate new friendly Light microscopy and characterization diazotrophic endophytic bacterial isolates from an agricultural canal. Our study seems to be the first report on the isolation of The more potent bacterial isolate was examined using a A. marplatensis from an aquatic environment. In addition to light microscope to check for the Gram stain reaction, diazotrophic nature, other beneficial features including P shape, sporulation, motility and flagella-staining proce- solubilization, GA3 and IAA production by this bacterium dures. Hanging drop method was used to determine the have been evaluated. Also, tomato (Lycopersicon esculentum motility of bacteria (Bertrand et al. 2001). Spore-forming var. commune) was used to study the effect of the obtained bacteria were determined according to Harrigan and Mac- isolate on different plant growth parameters as a promising Cance (1976). The cultural characteristics were recorded as PGPR under greenhouse conditions. colony morphology, i.e., color, shape, size, nature of the colony and pigmentation according to the method described by Hucker and Conn (1923). Materials and methods Scanning electron microscopy (SEM) Samples’ collection Samples of the selected bacterial isolate were fixed in 2.5% Water samples were collected from the agricultural canal at glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, for 2 h at Kafr Elsheikh governorate (31.3°N30.93°E) (Fig. 1). Sam- room temperature and washed in the same buffer. The ples of water were transferred to sterile plastic bags by using samples were post-fixed in 1% osmium tetroxide in the same

Fig. 1 Sites of collected water samples, watering canal at Al Hadady-Damrou, Kafr El-Sheikh Governorate, Egypt (31.3°N 30.93°E)

123 Acta Physiol Plant (2017) 39:61 Page 3 of 15 61 buffer for 2 h at room temperature. In 2 h, the cells were part of study were: b-lactams (cefotaxime, ceftazidime, rinsed with PBS for three times. The specimens were dehy- oxacillin, meropenem, aztreonam, amoxicillin ? clavu- drated in a series of graded ethanol (50, 60, 70, 80, 90 and lanic acid; augmentin; imipenem, trazobactam and ampi- 100%) for a period of 15 min in each series. Fixed samples cillin), aminoglycosides (amikacin and gentamicin), were dried under carbon dioxide using a critical point dryer glycopeptide (vancomycin), cephalosporin (cephradine, (EMS 850, USA) and sputter covered with gold in a sputter cefodizime, cefoxitin, cefixime, cefadroxil and cefepime), coater system (SPI-Module, USA). Morphological changes oxazolidinone (linezolid), fluoroquinolone (ofloxacin and observed and photomicrographs were then carried out with a levofloxacin), and ansamycins (rifamycin). JEOL JSM-5500LV scanning electron microscope (JEOL Instruments Inc., Japan) at an accelerating voltage of 20 kV DNA isolation at the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Egypt. For PCR amplifications, bacterial genomic DNA was extracted by inoculating LB broth media with the selected Transmission electron microscopy (TEM) bacterial isolate and incubated overnight at 37 °C with shaking. A 5 ml sample of the bacterial suspension was Biofilm was prepared by using freeze-substitution and centrifuged. Following discarding of the supernatant, 1 ml conventional embedding methods, and then it was thin- TE buffer was used for washing the pellet three times, and sectioned on a Reichert-Jung Ultracut E ultra-microtome. then it was re-suspended with 500 ll TE buffer. A 10 ll Before thin-sectioning, the sapphire disks were removed Proteinase K (20 mg ml-1), 20 ll lysozyme (50 mg ml-1) from the Epon, where the bacterial sample was kept and 100 ll 10% SDS were added to the cell suspension. embedded in the plastic resin, and later it was sectioned. The bacterial cell suspension was incubated overnight; Sections were ridden on Formvar and carbon-coated 100 ll 5 M NaCl and 100 ll 10% CTAB were added and 200-mesh copper grids. To improve contrast, sections were well mixed, and this mixture was incubated for 30 min at post-stained in 2% (wt/vol) uranyl acetate. A transmission 70 °C, and then it was incubated for 10 min on ice. The electron microscope (Philips, CM10) operating at 80 kV tubes were centrifuged at high speed for 15 min. The upper under standard operating conditions was used to perform phase—should not be viscous—was transferred to a 1.5 ml electron microscopy. All observations reported in this clean micro-centrifuge tube; equal volume from phe- paper are based on interpretations from 5 to 10 thin sec- nol:chloroform:isoamyl (25:24:1) was added and mixed tions from numerous biofilm samples (Hunter and Bev- well by inverting the tubes. The mixtures were centrifuged eridge 2005). Photomicrographs were then carried out with at 15,000 rpm for 15 min. Clean micro-centrifuge tubes a JOEL1010 model at the Regional Center for Mycology were used to transfer the upper phase in it, and then an and Biotechnology (RCMB), Al-Azhar University. equal volume of 24:1 (chloroform-isoamyl) was added followed by centrifugation at 15,000 rpm for 15 min. The APIÒ 20 methods upper phase was moved to the clean micro-centrifuge tube followed by adding twofold volume of absolute ethanol The selected bacterial isolate was characterized biochem- and incubated overnight at -20 °C. For increasing the ically using the APIÒ 20 E system strips that consist of 20 DNA yield, 50 ll 5 M sodium acetate was added. The micro-tubes which contain dehydrated substrates. The tubes were centrifuged at high speed for 20 min. Finally, conventional tests were inoculated with a saline bacterial 1 ml of 70% ethanol was used for washing the DNA pellet suspension which reconstitutes the media. The metabolism three times, and then it was re-suspended in 50 ll sterilized of the organism under investigation caused color changes TE Buffer which was kept at -20 °C for further use. during incubation period by the addition of substances in response to a specific enzymatic reaction. Samples were PCR reaction read following the reading table included with the API kit; in addition, the identification was achieved according to the Polymerase chain reaction (PCR) analysis was performed analytical profile index or by using the identification using 16S rRNA gene according to (Jiang et al. 2006). The software. primers used were universal 27F (50-AGAGTTTG- GATCMTGGCTCAG-30) and 1492R (50-CGGTTACC Antibiotics susceptibility test TTGTTACGACTT-30). The PCR reaction was amplified in 50 ll mixtures that contained 0.4 lM of each primer,

Susceptibility test of the most potent isolate was carried out 400 lM of dNTP mix, 2 mM MgCl2,5llof109 PCR by using the method of disk diffusion and read according to buffer, 2.5 units from TAKARA Taq DNA polymerase (Cat. Matuschek et al. (2014). Antibiotics that were tested in this #:R001AM) and 1 lg DNA. Master cycler (Eppendorf) was 123 61 Page 4 of 15 Acta Physiol Plant (2017) 39:61 used for amplification under the following conditions: initial Detection of virulence factors denaturation step at 95 °C for 3 min, 35 cycles of amplification (95 °C for 50 s, 52 °C for 1 min and 72 °C for 1 min), The selected bacterial strain was grown in tryptic soy broth and followed by a final extension at 72 °C for 10 min. The up to five days. The isolate cells were harvested by cen- obtained PCR product was examined using electrophoresis trifugation at 10,000 rpm for 5 min, the supernatant was on 1.2% agarose gel stained with ethidium bromide using filtered aseptically using filters with pore size 0.22 lm, GeneRulerTM 1 kb DNA ladder (Cat. #: SM0313), and then thereafter this harvest was used for detection of virulence visualized under UV transilluminator. factors. Elastase, chitinase, rhamno lipid and protease activities were assayed in supernatants as described by Cloning and sequencing Jakobsen et al. (2013). Chitinase and elastase activities were measured by a spectrophotometer (SCO-Tech, SPUV- The expected DNA band, almost 1500 bp, was eluted 19, Germany) at 550 and 495 nm, respectively. from agarose gel and purified according to the manufacturer’s QIAquick Gel Extraction Kit (Cat.#: 28704). The Plant-growth-promoting features of Achromobacter purified PCR fragment was ligated in pGEMÒ-T sp. EMCC1936 Easy Vector Systems (Cat. #: A1360) according to the manufacturer. The competent cells from the top 10 strains Nitrogenase activity was measured as a guide for nitrogen were prepared and transformed as described by Inoue fixation using the acetylene reduction technique given by et al. (1990). The white colonies were picked up from Diloworth (1970). The acetylene reduction activity of LB/Amp/Xgal plates and inoculated on LB/Amp broth isolate was measured using Gas–liquid chromatography media. Then it was incubated overnight at 33 °C with GLC. shaking for stabilizing the plasmid inside the transformed The ability of the isolate to solubilize phosphate was cells. The alkaline method of Birnboim and Doly (1979) screened on Pikovskaya’s (PVK) media, according to was used to isolate the plasmid. The purified plasmids Nguyen et al. (1992). Phosphate solubilization was evalu- were examined using electrophoresis on 1.2% agarose gel ated quantitatively on the National Botanical Research using GeneRulerTM 1 kb DNA Ladder (Cat. #: SM0313) Institute’s Phosphate (NBRIP) growth and broth media to confirm the recombinant plasmids. The recombinant (Nautiyal 1999). Results were expressed according to plasmids were sequenced by Macrogen Company (South (Naik et al. (2008). Korea). Moreover, Yeast Extract Mannitol broth medium was used to determine qualitatively the isolate ability to pro- Phylogenetic analysis duce gibberellins (GA3) and indole acetic acid (IAA) according to Pandya and Desai (2014) and Sarwart et al. The obtained sequence for 16S rRNA gene was examined (1992) methods, respectively. for vector contamination using VecScreen tool (http:// www.ncbi.nlm.nih.gov/tools/vecscreen/). Also, NEbcuter Evaluation of Achromobacter sp. EMCC1936 V2.0wasusedtocreatearestrictionmapandtoidentify inoculum on tomato production the GC content of the obtained sequence (Vincze et al. 2003, http://nc2.neb.com/NEBcutter2/). Possible ORFs Plant-growth-promoting potential of Achromobacter sp. of the resulted sequence were obtained by using ORF EMCC1936 was evaluated and compared with common finder software. Also, Jalview software was used to show free diazotrophic PGPR reference strain named Azotobac- single nucleotide polymorphisms (SNPs) and consensus ter chroococcum EMCCN1458 that obtained from Micro- resulted from the alignment of our bacterial isolate biological Resource Center (MIRCEN) Cairo, Egypt. obtained sequence and the nearest bacterial strain in A 3-month pot experiment (Marsh to May, 2015) was EzTaxon-e database (http://www.ezbiocloud.net/eztaxon) carried out in a greenhouse, Faculty of Agriculture, Benha (Kim et al. 2012). Construction of the phylogenetic tree University –Qalyubia Governorate, Egypt. Plastic pots was done using Clustal Omega and MEGA6 software. (20 9 27 cm) were filled with a mix of 20 kg soil (clay The sequence was registered in NCBI database under soil, pH 8.03, organic matter 3.1%, bulk density accession number KM491552.1. Also, the obtained iso- 1.36 g cm-3, field capacity 51.1%, wilting point 17.05%) late was deposited in Microbiological Resource Center and 10 g Compost [herbal plant residues as well as cattle (MERCIN), Egypt. Microbial Culture Collection manure (50:50) are: pH 7.6, electrical conductivity (EC) (EMCC) belonging to the WDCM with number (EMCC 3.1 dS m-1, total organic matter values 32.7%, total-N 1936). 1.21%, total-P 0.91% and the porosity 62.67%].

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Prior to transplantation, tomato seedlings L. esculentum comparison between means, Duncan’s multiple range test (c.v. Super Strain B) were obtained from a special nursery in was used (Duncan, 1955). Means followed by the same Qaha city. Seedlings were divided into three groups, two alphabetical letters were not significantly different at 5% were soaked by dipping the root system in mixture of 10% level of significance. 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 Results and one was a control (not treated with bio-inoculants). Pots were transplanted (2 seedlings/pot) and organized in a ran- Isolation and screening of bacterial isolates domized complete plot design with one factor and ten replicates. Tomato plants were watered weakly and the boost Fifteen different bacterial isolates were detected on Ash- doses of PGPR strains were added, 30 and 50 DAT. Chemical by’s medium after 48–72 h of incubation at 35 °C. Bac- fertilizers were supplemented to all treatments with 1/4 of the terial isolates were screened based on nitrogenase activity recommended dose (50 kg N fed-1 as ammonium sulfate (Table 1) and the more potent bacterial isolate (B9) -1 (20.5% N), 25 kg P2O5 fed as super phosphate (15.5% (p \ 0.05) was selected for the subsequent experiments. -1 P2O5) and 40 kg K2O fed as potassium sulfate (48% Diazotrophic nature of this obtained isolate was confirmed K2O). The activity of each of dehydrogenase, phosphatase by its ability to produce nitrogenase. and nitrogenase was determined at 30, 60 and 90 DAT. At the flowering stage (60 DAT), some plants were collected to Morphological characters of the more potent measure different growth parameters and phytohormones’ bacterial isolate content. A total number of fruits/plant was recorded 90 DAT. After 48 h of incubation on nitrogen free Ashby’s medium, Measurements of microbial enzymatic activity at 35 °C, the colonies of the selected isolate ranged in size from 0.5 to 2.25 mm in diameter, become opaque and The microbial enzymatic activities were determined in mucoid. Continued incubation resulted in a colony with rhizosphere at 15, 30 and 60 DAT. The activities of diameter from 3.0 to 4.8 mm. After incubation at 35 °C for dehydrogenase (DH) and alkaline phosphatase (Alp) were 12 h, the tryptic soya broth culture exhibited a barely measured using spectrophotometer (SCO-Tech, SPUV-19, visible turbidity that after an additional 12 h, it increased to Germany) at 464 and 400 nm, respectively, as described by approximately 2.0 9 109 cfu ml-1. The growth was uni-

Schinner et al. (1996). However, nitrogenase (N2-ase) formly distributed throughout the broth. A small amount of activity was measured as previously mentioned with some sediment was produced by the isolate that rose upward by modification by Okafor and MacRae (1973). rotation. Continued incubation led to increasing turbidity near the surface and the formation of a slimy pellicle. Morphological, endogenous phytohormones Regarding, microscopic morphology, the obtained bacterial and yield characteristics isolate was straight rods, 0.6–1.2 lm with rounded ends (Table 2). It was Gram negative, single and diploid, non- Vegetative growth parameters were evaluated on ten plants spore forming and motile with peritrichously-arranged which were randomly taken from each treatment, stem flagella. Electron micrographs (SEM and TEM) images length (cm), branches/plant number, shoot dry weight (g), revealed the accumulation of extracellular material sur- root size (cm3) and number of flowers/plant. For each rounding one pole of the Achromobacter isolate cells and treatment, total number of fruits/plant was recorded as well forming a polar cap as shown in (Fig. 2). These accumu- as fruits setting percentage was calculated. Endogenous lations add irregular contours so that many of the cells had Phytohormones (IAA, GA3 and abscisic acid) were eval- a coryneform appearance. On Gram stain, curved (solid uated quantitatively in tomato shoots at 60 DAT using arrowhead) and hooked-end (arrow) forms were also high-performance liquid chromatography (HPLC; YL9100 presented. HPLC System, Korea) according to the procedure of Koshioka et al. (1983). Cytokinins was determined by Biochemical characterization HPLC according to Nicander et al. (1993). The bacterial isolate grew on nitrogen free Ashby’s med- Statistical analysis ium showing obligatory aerobic features. Phenotypic characterization based on API system (Table 3), showed The obtained data were statistically analyzed according to that it was oxidase-positive and gelatinase-negative. It the methods outlined by Gomez and Gomez (1984). For utilized glucose as sole carbon source and produced acid 123 61 Page 6 of 15 Acta Physiol Plant (2017) 39:61

Table 1 Screening of bacterial Isolates number Isolates code Nitrogenase activity (lgCH hl-1) isolates according to 2 4 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- DNA and phylogenetic analysis nose, saccharose, melibiose, amygdalin, arabinose and xylose which caused indicator color change. Data showed The obtained PCR amplified fragment for 16S rRNA gene that the obtained isolate was capable of using denitrifica- was &1500 bp (Fig. 3). The PCR product sequencing tion as a respiratory process with nitrate electron acceptor. result was registered in NCBI database under accession number, KM491552.1. Analysis of the obtained sequence Antibiotic susceptibility test via VecScreen database showed no contamination with vector sequence. The FASTA homology showed that Antibiotic susceptibility patterns of the obtained Achro- the16S rRNA gene sequence of the current isolate (ACC. mobacter sp. EMCC1936 isolate demonstrated a susceptibil- No. KM491552.1) had 97% nucleotide similarity with that ityprofilecharacteristic(Table 4). The isolate was resistant to of Achromobacter marplatensis B2 (T) strain recorded in ceftazidime, amoxicillin ? clavulanic acid (augmentin), EzTaxon-e database (ACC. No. EU150134). This result linezolid and rifamycin. It was categorized as intermediate was confirmed by the phylogenetic position of the obtained resistant to cefotaxime, oxacillin, meropenem, trazobactam, isolate, forming polyphyletic clade with A. marplatensis ampicillin, cefodizime, cefoxitin, cefixime, cefadroxil, cefe- B2 (T), but with an obvious phylogenetic distance (Fig. 4). pime and ofloxacin. It remained susceptible to aztreonam, Also, the restriction Map of the obtained 16S rRNA partial imipenem, amikacin, gentamicin, vancomycin, levofloxacin sequence was done as shown in Fig. 5a. By calculating and trimethoprim/sulfamethoxazole (Bactrim). pairwise alignment analysis, data exhibited 16 SNPs

123 Acta Physiol Plant (2017) 39:61 Page 7 of 15 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 on PVK agar medium, and it showed 104% phosphate nearest registered bacterial strain, A. marplatensis B2 (T), solubilization efficiency. The quantitative assessment of (EU150134), for 16S rRNA gene (Fig. 5b). phosphate solubilization on NBRIP broth medium was 23.7 lgml-1. Detection of virulence factors Effects of inoculation with Achromobacter sp. The ability of the obtained isolate, Achromobacter sp. EMCC1936 on tomato production EMCC1936, to produce protease on agar plates containing 5% skim milk was studied. A clearing zone is considered as Effect of bio-inoculants on enzymes activity of tomato a proteolytic activity being present. Chitinase was mea- rhizosphere sured by degradation of chitin azure, while elastase activity was measured by degradation of elastin Congo red. Also, Soil enzymes activity has been suggested to be an index for the production of rhamnolipid was determined by lysing of soil fertility, whereas soil microbial activity has been used the red blood cells. From the obtained results it was clear as an index of fertility. Data in Table 6 showed that during that none of investigated virulence factors were the experimental periods, the effects of bio-inoculants (A. detectable under the conditions used in this study. chroococcum and Achromobacter sp. EMCC1936) tended to be stronger on dehydrogenase, alkaline phosphatase and In vitro plant-growth-promoting features nitrogenase activities when compared with un-inoculated of Achromobacter sp. EMCC1936 treatment (control). The overall data revealed that the inoculation with Achromobacter sp. EMCC1936 had sig- Under laboratory conditions, Achromobacter sp. nificant effect on the previous soil enzymes activity com- EMCC1936 was bio-assayed for its growth-promoting pared with A. chroococcum EMCCN1458 inoculation. At potentiality through nitrogenase activity and phytohor- flowering stage (60 DAT), inoculation with Achromobacter mones production such as auxins and gibberellins sp. EMCC1936 showed about fourfold, sixfold and fivefold -1 (Table 5). It had 55.03 lgC2H4 hl nitrogenase activity increase in dehydrogenase, alkaline phosphatase and indicating its ability to fix atmospheric N2. It also pro- nitrogenase activities, respectively, when compared to duced 50.8 lgml-1 of IAA and 10.35 lgml-1 of GA3. initial period. However, control showed about twofold Moreover, it had the ability to solubilize rock phosphate increase in all enzymes’ activity.

123 61 Page 8 of 15 Acta Physiol Plant (2017) 39:61 S

2 Vegetative growth and yield characteristics OX H S 2 3 H Inoculation of tomato with Achromobacter sp. EMCC1936 affected plant length, number of branches/plant, stems and fermentation of fermentation of leaves dry weight, root size and number of ﬂower/plant as compared to the control (p \ 0.05) (Table 7). Relative to AMY MAN A. chroococcum EMCCN1458 and the control, Achro-

citrate utilization, mobacter sp. EMCC1936 strain caused 24 and 162% more fruits number/plant; it had relatively more 12 and 35%, for CIT percentage of fruit setting respectively.

Endogenous phytohormones content of tomato shoot ) colorless - fermentation of glucose,

fermentation of melibiose, Levels of major tomato shoot endogenous phytohor-

GLU mones—both growth promoting and inhibiting ones—were MEL all maintained at satisfactory concentrations (p \ 0.05) due ornithine decarboxylase, to Achromobacter sp. EMCC1936 inoculation (Table 8).

gelatinase, Relative to the control shoots, concentrations of each of ODC ) developed color, (

? gibberellins (GA), auxins (IAA), and cytokinins were 79, GEL 226, and 177%, respectively. These concentrations dropped to differentials of 5, 15, and 16%, respectively, if compared to those resulted from A. chroococcum EMCCN1458

fermentation of saccharose, inoculant. For growth-inhibiting abscisic acid (ABA), the Achromobacter sp. EMCC1936 inoculant caused compar- lysine decarboxylase, SAC ative reductions of 51 and 18%, respectively. Finally, all cytochrome-oxidase, (

LDC the obtained results qualiﬁed the isolated bacterial strain, OX Achromobacter sp. EMCC1936, to be added to Egypt. Microbial Culture Collection (EMCC), at Cairo MERCIN under number (EMCC 1936) to be available for application. acetoin production (Voges Proskauer), VP nitrous reduction, 3 fermentation of rhamnose, arginine dihydrolase,

NO Discussion bacterial isolate RHA ADH Plant-growth-promoting rhizobacteria (PGPR), particularly Achromobacter marplatensis involved positively in dif- indole production, ferent biotic activities in the soil which enhance plant IND Achromobacter growth as a result of nutrient obtainability in the soil as well as producing various plant growth regulators (Ahemad fermentation of xylose,

S URE TDA IND VP GEL GLU MAN INO SOR RHA SAC MEL AMYand ARA Kibret Xyl NO 2014; Wu et al. 2014). It is oxidase, catalase- 2 -galactopyranosidase), fermentation of sorbitol, D Xyl

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

SOR aqueous environments and contaminated soils (Gomila et al. 2011; Vandamme et al. 2013). Morphological and biochemical examination of the obtained isolate revealed tryptophane deaminase, several distinctive features related to genus Achromobac-

TDA ter. Our isolate, Achromobacter sp. EMCC1936 was tested both morphologically and biochemically to provide deﬁnite 20 E strip results for the isolated Ò

urease, information for using in bacterium identiﬁcation. By using fermentation of arabinose,

fermentation of inositol, a light microscope, most cells were seen as typical rod- URE ARA

111121111 1111 111111 212121211211 shaped and Gram-negative bacilli, but some of them INO The API -galactosidase (ortho nitrophenyl-

b apparently had curved forms. Moreover, bacterial cells were seen with a swollen at one end as well as electron ? production, ONPG ONPG ADH LDC ODC CIT H Table 3 amygdalin, mannitol, 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- 1979; Kersters and De Ley 1984; Yabuuchi et al. 1998; ment. Electron micrographs revealed the accumulation of Vandamme et al. 2013). They reported that the genus extracellular material on many of the cells often at one pole Achromobacter is a Gram-negative, oxidase-positive, (Fig. 2). Apparently these accumulations added irregular obligate aerobic, non-fermenting bacilli with peritrichous contours to the cell, and therefore in Gram stain, appear as flagella, and is able to oxidize maltose, mannitol, and curved and hooked forms which supported by Chester and sucrose. Although, the biochemical studies using API Cooper (1979). Achromobacter isolate was the exceedingly 20NE revealed core of reactions useful in the identification small colonies produced at 24 h and the subsequent rapid of Achromobacter; however, they were not enough to fully increase in colony size after continued incubation due to identify the isolated strain. the elaboration of large amounts of mucoid material. Regarding antibiograms of Achromobacter strain, Phenotypic characterization of Achromobacter sp. results from previous studies reported that isolates EMCC1936 using API 20NE test showed change in indi- belonging to A. xylosoxidans exhibited susceptibility to cator color, where the isolate utilized its carbon sources, D- piperacillin (95.6%), ceftazidime (91.1%), imipenem xylose, D-gluconate and D-glucose. Data of morphological (97.2%) and trimethoprim-sulfamethoxazole (78.6%), and biochemical examination of the obtained isolate agree while they were resistant to most cephalosporins, amino- with previous findings (Hugh 1970; Chester and Cooper glycosides, ampicillin and aztreonam (Legrand and

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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 identification approach concerning non-fermenting Gram-negative 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 phylogenetic tree. This gene has been proven to have quite slow Fig. 3 PCR products for 16S RNA partial-length gene (1500 bp) of rates of evolution, indicating its genetic stability. An indi- the obtained diazotrophic bacterial isolate. -C, negative control, no cation of this gene stability was confirmed by the GC content DNA sample. M refers to GeneRulerTM 1 kb DNA ladder (Cat. #: and of its slow evolutionary rate by the presence of 16 SNPs. SM0313)

Fig. 4 Phylogenetic trees recovered from maximum likelihood within the phylogenetic branches of Alcaligenaceae family. Average analyses of the 16S rRNA gene partial sequence for the obtained bootstrap values, of compared algorithms, are indicated at the branch isolate, Achromobacter sp. EMCC1936 (KM491552.1). The trees roots. The bar represents 0.02 changes per nucleotide. Accession show the phylogenetic position of recovered Achromobacter species numbers of database extracted sequences are in brackets

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Fig. 5 a Restriction map of the partial sequence of 16S rRNA gene of the nearest one on EzTaxon-e database, Achromobacter marplatensis interest with available commercially restriction enzymes. b Single B2 (T), EU150134.1 (Kim et al. 2012) based on pairwise alignment nucleotide polymorphism (SNPs) showed 16 SNPs between the analysis method 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 Phosphate concentration Nitrogenase activity -1 -1 -1 -1 (lgml ) (lgml ) efﬁciency (%) (lgml ) (lgC2H4 hl )

50.8 10.35 103.7 23.7 55.03

In this respect, many studies were conducted using 16S into ammonia (diazotrophy), which was conﬁrmed by rDNA method for proving identity and characterization of estimating nitrogenase activity. Besides diazotrophy, other unknown isolates, especially Achromobacter sp. (Drancourt beneﬁcial characteristics are IAA production and solubi- et al. 2000; Janda and Abbott 2007; Petti 2007; Gomila et al. lized high amount of phosphate. Growth-promoting char- 2011; Abyar et al. 2012; Mazumdar and Deka 2013; Bhosale acters were exhibited by many endophytic diazotrophic et al. 2014; Ramasamy et al. 2014; Das et al. 2014; Srini- bacteria including A. xylosoxidans and A. marplatensis vasan et al. 2015; Alhamlan et al. 2015; Mareque et al. 2015). (Prabhat and Kumar 2009; Wedhastri et al. 2013;Wuetal. One prominent feature of Achromobacter sp. 2014). The obtained results from the current study are in EMCC1936 which was noticed in this study is the capa- harmony with the previous studies but are novel especially bility to reduce nitrogen molecules from the atmosphere for the bacterium Achromobacter sp. EMCC1936 where it

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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 -1 -1 -1 -1 -1 -1 (lg TPF g dw h ) (lg qNP g h ) (llC2H4 g dw h ) 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 A. chroococcum 54.7b 8.0b 33.7b 34.7b 16.3b 12.7b 77.6b Achromobacter sp. 65.7a 10.7a 41.3a 41.3a 18.0a 15.7a 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 (lgg-1 F.W) Inhibitors abscisic acid Promoters/ (lgg-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 EMCC1936 in this respect. These results may be attributed produce GA3. to the role of intercellular substances of Achromobacter sp. Application of Achromobacter sp. EMCC1936 as a (i.e., plant-growth-promoting substances and microbial PGPR strain with tomato had a great effect on microbial enzymes) in promoting soil microbial proliferation and community and their activities especially their extracellular their activity. Moreover, increasing the population dosage enzymes. These extracellular enzymes show a crucial of Achromobacter sp. was beneficial to both the plant, as it effect in soil biogeochemical cycles of C, N, and P that are fixes more Nitrogen, and to the population of rhizospheric play role in nutrient conversion (Burns and Dick 2002;Wu organisms on plant roots. et al. 2014). Therefore, significant reductions in both soil The encouraged growth of inoculated plants with enzymatic activity and soil microbial biomass should be Achromobacter sp. EMCC1936 more than A. chroococcum counted as signs of soil quality and land use effectiveness EMCCN1458 may be attributed to the vital role of this (Mganga et al. 2015). Results showed the importance of strain in enhancing the obtainability as well as acquisition soil inoculation with PGPR strains and their role in of nutrients such as phosphorus. Microorganism solubilize increasing the activities of soil enzymes especially the phosphate (P) to provide it as a major nutrient to plants in distinctive role of the isolated Achromobacter sp. soils lacking P and improve the overall growth as well as

123 Acta Physiol Plant (2017) 39:61 Page 13 of 15 61 plants development is considered another important free- growth-promoting rhizobacteria (PGPR) for various crops living soil microbiota (Khan et al. 2014; Wu et al. 2014). since it exhibits reasonable potential characteristics. Moreover, more fixed nitrogen has been provided through Moreover, it could be recommended as a new effective the application of endophytic diazotrophic bacteria com- rhizobacteria to promote plant growth, increase crop pro- pared to rhizospheric one where the interior of plants is a duction, decrease production costs and reduce environ- more appropriate for nitrogen fixation as an explanation of mental pollution. Considering these facts as well as other low partial oxygen pressure (pO2) and direct accessibility features of PGPR new strains, further investigations are of the fixed nitrogen to the plants (Prabhat and Kumar ongoing in our laboratories. 2009; Mazumdar and Deka 2013; Wu et al. 2014). Fur- Author contribution statement AHM and SAA: Iso- thermore, Achromobacter sp. EMCC1936 has the ability to lated the bacterial isolate form aquatic environment, produce IAA and GA3 that play role in increasing the root conceived the greenhouse experiment work, performed length surface area and responsible for branching of root the experiments to measure the physiological effects of hair hence nutrients up-taking from the soil increase. Zaidi the bacterial isolate as a PGPR, drafted the corre- et al. (2009) reported that different phytohormones, i.e., sponding sections of the manuscript and revised the IAA, gibberellins and cytokinins, are essential for shoot complete manuscript. MMAM and HASE: Isolated growth and root morphogenesis of numerous plants. The DNA from the isolated bacterial strains, performed PCR IAA has an impact on tissue development, the root growth, using 16SrRNA gene, cloned and sequenced the PCR as well as light and gravity responses. Intense growth and product for the used gene, analyzed the sequencing high yielding biomass as well as grain production resulted results, as well as they submitted the results in NCBI from the absorption of more nutrients and water from the data base, also they drafted the corresponding sections soils by roots with larger surface area hence translocate of the manuscript and revised the complete manuscript. them to different parts of the plants. On the other hand, gibberellins affected seed germination, stem development, Acknowledgements We would like to thank Prof. Dr. M. Hany flowering, as well as plants fruit setting. Tageldin, Horticulture Dept., Faculty of Agriculture, Benha Univer- Generally, inoculation with Achromobacter sp. sity, for his kind help and assistance. EMCC1936 resulted from current study, promote signifi- Compliance with ethical standards cantly the phytohormones level. Also, there was an increase of endogenous hormones in tomato may refer to Conflict of interest There is no conflict of interest. the improvement of growth features. For example, cytokinins accumulation could support the increasing of the branches number which could increase transverse growth more than longitudinal one. Inoculation with Achro- References mobacter sp. EMCC1936 increased the content of endogenous promoting phytohormones of tomato shoot Abd El-Aal MM, Abd El-Rahman HM (2014) Impact of PGPR and inorganic fertilization on growth and productivity of sweet which may attributed to the positive effect of this isolate in Ananas Melon. Int J Agric Sci Res (IJASR) 4:11–26 PGPs production. Abd El-Azeem SAM (2007) Some plant growth promoting traits of Moreover, the quantities of total promoter elements rhizobacteria isolated from Suez Canal region, Egypt. Afr Crop relative to the abscisic acid (ABA) as an inhibitor were Sci Conf Proc 8:1517–1525 Abd El-Malek Y, Ishac YZ (1986) Evaluation of methods used in relatively higher in case of Achromobacter sp. counting Azotobacter. J Appl Bacteriol 31:267–269 EMCC1936 inoculation. These treatments enhanced the Abyar H, Safahieh A, Zolgharnein H, Zamani I (2012) Isolation and internal metabolically features of tomato causing an identification of Achromobacter denitrifcans and evaluation of increase of promoting hormones content which maxi- its capacity in cadmium removal. Pol J Environ Stud 12:1523–1527 mized its growth. These results are accordance with that Ahemad M, Kibret M (2014) Mechanisms and applications of plant obtained by (Hosseny and Ahmed 2009; Abd El-Aal and growth promoting rhizobacteria: current prespective. J King Abd El-Rahman 2014; Wu et al. 2014) who reported that Saud Univ Sci 26:1–20 maximizing growth and productivity of lettuce, sweet Alhamlan FS, Al-Qahtani AA, Al-Ahdal MN (2015) Recommended advanced techniques for waterborne pathogen detection in ananas melon and sugar beet were related to high pro- developing countries. J Infect Dev Ctries 9:128–135. doi:10. moting hormones content. 3855/jidc.6101 In summary, both traditional and molecular characteri- Aneja KR (2003) Experiments in microbiology plant pathology and zation approaches were integrated in this study for biotechnology, 4th edn. New Age International (P) Ltd. Pub- lishers, New Delhi, p 320 obtaining new friendly diazotrophic bacterial strains from Bertrand H, Nalin R, Bally R, Marel JCC (2001) Isolation and an agricultural canal. The obtained isolate, Achromobacter identification of the most efficient plant growth promoting sp. EMCC1936 (KM491552.1) could be exploited as plant- bacteria associated with canola. 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