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 descri- bed 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.