Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Ochrobactrum sp. Pv2Z2 exhibits multiple traits of plant growth promotion, biodegradation and N-acyl-homoserine-lactone quorum sensing

Ochrobactrum sp. Pv2Z2 exhibits multiple traits of plant growth promotion, biodegradation and... Ann Microbiol (2014) 64:1797–1806 DOI 10.1007/s13213-014-0824-0 ORIGINAL ARTICLE Ochrobactrum sp. Pv2Z2 exhibits multiple traits of plant growth promotion, biodegradation and N-acyl-homoserine-lactone quorum sensing Asma Imran & Marryam Jumma Abdulla Saadalla & Sami-Ullah Khan & Muhammad Sajjad Mirza & Kauser Abdulla Malik & Fauzia Yusuf Hafeez Received: 21 November 2013 /Accepted: 22 January 2014 /Published online: 19 February 2014 Springer-Verlag Berlin Heidelberg and the University of Milan 2014 Abstract Rhizosphere bacteria play a vital role in plant solubilized zinc sulphate and zinc oxide in the LG1 medium. growth, pathogen control, biodegradation and rhizosphere The production of AHLs (e.g. 3O-C7-HSL, 3OH-C7-HSL) signaling. A motile, rod-shaped bacterium, Pv2Z2, isolated was detected with biosensor strains CV026 using reverse from the nodules of the common bean grown in Tanzanian soil phase thin layer chromatography. The bacterium was able to was characterized using a polyphasic approach. The traits grow in minimal salt medium supplemented with 100 mg/L assessed included the production of indole-3-acetic acid and each of phenol, 2-bromophenol, 2,4-diamino phenol hydro- N-acyl homoserine lactone (AHL) molecules, solubilization chloride, 3,4-dimethoxy benzyl alcohol and 4-methoxy ben- of insoluble phosphate and zinc compounds and biodegrada- zyl alcohol. Phenol degradation was recorded up to a level of tion of a number of toxic compounds. The 16S rRNA se- 94 % within 12 days. Inoculation of common bean plants quence of Pv2Z2 (EU399793) showed 99 % homology to resulted in a significant increase in plant height, fresh/dry Ochrobactrum anthropi isolates (Accession no. AJ867292, weight and nitrogen uptake as compared to non-inoculated AJ867291, AJ867290) from soil samples of wheat root. Phy- plants. The data suggest that the plant growth-promoting and logenetic analysis showed relatedness to nodulating strain biodegradation potential of this bacterium may be exploited Ochrobactrum cytisi rather than to the clinical/pathogenic type on a large scale. The capacity to produce AHL molecules by strain of O. anthropi. Moreover, it showed unique fingerprints members of the Ochrobactrum genus has not been previously in the randomly amplified polymorphic DNA (RAPD) and reported and needs to be explored in detail. two primers-RAPD assays which were different from those of the pathogenic type strain of O. anthropi. The bacterium . . . Keywords Legumes Ochrobactrum Biodegradation -1 produced 6.68 μg/mL indoleacetic acid in the presence of Phosphate solubilization AHLs -1 tryptophan, released 25.7 μg/mL phosphorus from inorganic tri-calcium phosphate in the Pikoviskaya’smediumand Introduction : : : : A. Imran (*) M. J. A. Saadalla S.<U. Khan M. S. Mirza K. A. Malik F. Y. Hafeez (*) Although nitrogen (N) and phosphorus (P) are vital nutrients National Institute for Biotechnology and Genetic Engineering, for plant growth and development, only 30–50 % of exoge- P.O. Box 577, Jhang Road, Faisalabad, Pakistan nously applied N and 10–45 % of P are taken up by plants e-mail: asmaaslam2001@yahoo.com e-mail: fauzia_y@yahoo.com (Adesemoye et al. 2009; Garnett et al. 2009). The rest of the fertilizer fraction becomes unavailable either due to leaching M. J. A. Saadalla or nutrient run-off. In addition, long-term use of synthetic Ministry of Agriculture, Natural Resources, fertilizers negatively affects the overall biogeochemical cycles Environment and Co-operatives, Zanzibar, Tanzania and ecosystem (Steinshamn et al. 2004) and has become a K. A. Malik global environmental concern (Tilman et al. 2002). An alter- FC College University, Lahore, Pakistan native to the use of chemical fertilizers is the application of biological fertilizer technology. This technology not only F. Y. Hafeez COMSTAS Institute of Information Technology, Islamabad, Pakistan benefits plants in terms of growth and yield but improves soil 1798 Ann Microbiol (2014) 64:1797–1806 health and the environment, reduces reliance on chemical and soybean to trap the indigenous rhizobia specific to these fertilizers and provides economic benefits. hosts. After screening a number of isolates, we identified one Biological fertilizers composed of plant growth-promoting nodule isolate having the ability to produce AHL compounds rhizobacteria (PGPR) are often described as having multiple and multiple traits of plant growth promotion and biodegra- traits of plant growth promotion. They convert atmospheric N dation. The isolate, when characterized in detail, showed great to ammonia, thereby making N available to plants, transform promise for use in the detoxification of hazardous chemicals unavailable phosphate or zinc compounds to plant-available and as a biofertilizer. The AHL quorum-sensing system might fractions, produce different growth hormones and amino be involved in regulating its function in the rhizosphere and acids, produce signal compounds, such as N-acyl homoserine environment. lactone (AHL) molecules, to interact with the host and coor- dinate their activities in the rhizosphere, exhibit 1- aminocyclopropane-1-carboxylate-deaminase activity to mit- Materials and methods igate the stress-induced negative impacts and produce differ- ent antifungal compounds to combat pathogen attack Strain isolation (Compant et al. 2005). In addition to the biotic stress caused by pathogen attack, The strain was isolated from soil collected from the Maruhubi plant growth and yield are seriously affected by abiotic stress- region of Tanzania, which was a sandy-loam soil having a pH -1 es, such as salinity, drought, frost and the presence of toxic of 5.69, electrical conductivity of 10.2 dS/m ,11%Ccon- compounds in water or soil. Each day huge amounts of tent, 2.2 % organic matter content, 0.08 % total N content, 10 organic compounds are released as industrial by-products, mg/kg P, 0.72 mg/100 g calcium, 0.48/100 g magnesium, 0.50 pesticides or agrochemicals into the environment, of which mg/100 g potassium and a C/N ratio of 13. The last crop phenolic compounds are very hazardous (Afzal et al. 2007). grown in the soil was common bean. Serial eightfold dilutions Microbes found in natural waters and soils have an amazing, were prepared from 1 g of soil (Somasegaran and Hoben wide-ranging ability to utilize many of these toxic compounds 1994), and 1 mL of each dilution was applied to the base of as carbon (C) and energy sources and have a great potential for the root tip of 3-day-old seedlings of common bean (var. use in large-scale bioremediation programs. Biodegradation Lyamungu 90). The plants were irrigated with N-free ability has been reported in many soil bacteria, particularly Hoagland solution and observed for nodule formation. Nod- PGPR [e.g. Rhizobium sp. (Amer 2008; Wei et al. 2008), ules were detached from the root, flame sterilized, crushed on R. leguminosarum bv. Trifolli (Parke et al. 1991), R. meliloti a sterile glass surface and streaked onto YEM-Congo red (Frassinetti et al. 1998), Ralstonia taiwanensis (Chen et al. plates as described by Shah et al. (1995) to isolate the bacteria. 2004, Bradyrhizobium japonicum (Radwan et al. 2007), The plates were incubated at 28±2 °C for 3–4 days until the Sinorhizobium (Seo et al. 2007), Mesorhizobium (Feng and appearance of separate colonies. Lee 2009)]. Bacterial activities (either beneficial or pathogenic) are Biochemical characterization mainly controlled by different signal molecules which are produced by bacteria to regulate the expression of specific Colony morphology, size, color, shape, gum production and genes and to interact with the environment. The AHLs are growth pattern were recorded after 24 hofgrowthonYeast signal compounds that are produced specifically by Gram- Extract Mannitol (YEM) and Luria–Bertani(LB)agarplatesat negative bacteria and are involved in signaling processes such 28±2 °C as described by Somasegaran and Hoben (1994). Cell as antibiotic production and conjugal plasmid transfer. This size and motility was observed by light microscopy. Acid/alkali kind of signaling has been identified in Pseudomonas, production was tested on LB-agar plates containing 0.025 % Agrobacterium and Rhizobium (An et al. 2006; Abbas et al. (w/v) bromothymol blue as pH indicator. The Gram reaction 2007; Antunes and Ferreira 2009). The members of family was performed as described by Vincent and Humphrey (1970). Brucellaceae are known to produce signal molecules Amino-peptidase and cytochrome oxidase tests were per- (Taminiau et al. 2002), but to date this activity has not been formed by using commercially available strips (Merck, Darm- reported for the genus Ochrobactrum. Quorum-quenching stadt, Germany). Catalase production was checked by placing a activity, however, has been reported for one member of this drop of H O onto the bacterial colony on a glass slide. Resis- 2 2 genus (Jafra et al. 2006; Czajkowski et al. 2011). tance to the antibiotics chloromphenicol (30 μg), erythromycin The aim of this study was to isolate the nodulating bacteria (15 μg), aztreonam (30 μg), carbenicillin (100 μg), ampicillin that can be used for inoculum production for common bean, (10 μg), gentamycin (10 μg), amikacin (30 μg), streptomycin cowpeas and soybean in Zanzibar, Tanzania. For that purpose, (10 μg), doxicycline (30 μg), neomycin (30 μg) and soil was collected from different regions of Zanzibar, Tanza- trimethoprim/sulfamethoxazole (25 μg) was determined on nia, and soil inoculum was applied to common bean, cowpea antibiotic sulphonamide sensitivity-test agar (Merck) plates Ann Microbiol (2014) 64:1797–1806 1799 using commercially available discs (Bioanalyse®, Ankara, Tur- Solubilization of tri-calcium phosphate, zinc oxide and zinc key). The utilization of different carbon sources and enzymatic sulphate reactions were performed using the QTS-24 kit (DESTO, Ka- rachi, Pakistan) following the manufacturer‘s protocol. The Aliquots (10 μL) of Pv2Z2, after overnight growth in LB capacity to utilize mannitol, maltose, fructose, sucrose, D-glu- medium, were spot inoculated onto Pikovskaia’sagar cose, levulose, sorbitol, myo-inositol, glycerol and yeast extract (Pikovskaia 1948) containing tri-calcium phosphate as the insol- as carbon sources was determined as described by uble P source and onto LGI medium (Cavalcante and Somasegaran and Hoben (1994). Döbereiner 1988) supplemented with 0.1 % zinc oxide and zinc sulphate. The plates were incubated at 28±2 °C for 10–14 days and examined for the formation of a clear zone around the Molecular characterization bacterial growth spot daily. The appearance of a clear zone was considered to be a positive indication for phosphate and zinc Total genomic DNA of the bacterium was isolated by the solubilization activities. P solubilization was quantified using the alkaline lysis method (Maniatis et al. 1982) and used to phosphomolybdate blue colour method on a spectrophotometer amplify the 16S rRNA gene with primers P1/P6 (Tan et al. (λ=882 nm) as described by Murphy and Riley (1962) 1997). The PCR assay was carried out in a thermal cycler (Eppendorf, Hamburg, Germany) as described by Imran Antifungal activity et al. (2010). The amplified PCR product was purified using the QIA Quick PCR Purification kit (Qiagen, Hilden, Antifungal activity was tested using a dual-culture assay as Germany), ligated in TA cloning vector pTZ57R/T described by Sakthivel and Gnanamanickam (1986). A drop (Fermentas, Thermo Fisher Scientific, Waltham, MA) and of the exponentially grown bacterial culture (approx. 20 μL) cloned in Escherichia coli strain DH5α as described by was spotted onto potato dextrose agar (PDA) plates close to the Maniatis et al. (1982). Cloned PCR products were se- walls of the petri dish on both sides and the plate left in laminar quenced commercially from Macrogen (Seoul, Korea). The air flow cabinet until dry. A 6-mm agar disk of each of three gene sequence was compared with others in the GenBank fungal species, namely, Fusarium solani, F. oxysporum and database using the NCBІ BLASTn. Multiple sequence align- F. moniliforme (obtained from the lab collection), were sepa- ments were performed by ClustalX, and phylogeny was rately placed at the centre of PDA plates. The plates were determined by neighbor-joining method. Random amplified incubated at 28±2 °C for 5 days to measure the inhibition of polymorphic DNA (RAPD) analysis was performed as de- radial fungal growth between fungal and bacterial colonies. scribed by Williams et al. (1990) using primers OPC-13 and Percentage inhibition was determined by the reduction in fungal OPC-15 (Operon Technologies, Alameda, CA). Two primers growth compared to control (un-inoculated bacterial strain). (TP)-RAPD was performed as described by Imran et al. (2010). The DNA of the O. anthropi type strain LMG3331 The AHL assay and detection by reverse-phase thin-layer and other type strains of the genus obtained from DSMZ chromatography (German Collection of Microorganisms and Cell Cultures) were used as reference. The Pv2Z2 isolate was streaked onto the center of a Tryptone Yeast extract (TY) (Beringer 1974) agar plate and grown overnight at 28 °C to achieve appreciable growth. The indica- Bioassays for plant growth-promoting activities tor strain Chromobacterium violaceum CV026 (obtained from John Innes Centre, Norwich, UK) was grown separately in LB Production of indole-3-acetic acid broth at 30 °C with constant shaking overnight up to an OD of 10 CFU/mL. AHLs were detected by the plate overlay assay The production of indole-3-acetic acid (IAA) was determined as described by McLean et al. (2004). The reference strain A34 using the colorimetric method of Gordon and Weber (1951). (obtained from the John Innes Centre) was used as the positive Quantitative estimation was done by growing Pv2Z2 in LB- control. A34 is a Rhizobium leguminosarum 8401 containing broth supplemented with 100 mg/L tryptophan as IAA pre- pRLIJ1. Plates were incubated at 30±2 °C overnight, and cursor for 5 days. IAA was extracted with an equal volume of purple pigmentation produced by the indicator strain on the ethyl acetate as described by Tien et al. (1979) and finally plates was considered to indicate AHL production. collected in ethanol. A 100-μL sample of the extract was AHLs were extracted from the culture supernatant analysed on high-performance liquid chromatograph with an equal volume of ethyl acetate (acidified with equipped with Turbochrom software (Perkin Elmer Life Sci- 0.1 % v/v glacial acetic acid).Water residues were re- ence, Boston, MA) and on a C-18 column at a flow rate of moved by treating the extract with anhydrous Na SO . 2 4 0.5 mL/min. The extract was filtered, concentrated in a rotary evaporator 1800 Ann Microbiol (2014) 64:1797–1806 (Rota vapor R-210; Buchi Corp, New Castle, DE) and re- of indicator strain CV026, and AHLs were identified as purple confirmed by reverse plate assay as described by McLean spots on the off-white background. et al. (2004). The AHL extract (10 μL) was separated on glass-backed C18 reverse-phase thin-layer chromatography Plant inoculation assay (RP-TLC) plates (Merck) in methanol/water (60:40 v/v) along with the AHL extract of A34 as reference. The TLC plate was The Pv2Z2 strain, which had been grown in yeast-extract- developed with the overlay of an exponentially grown culture mannitol (YEM) medium to a concentration of up to 10 cells/ Table 1 Morphological and biochemical characters of Ochrobactrum sp. Pv2Z2 Cultural/Morphological characters Growth on Colony morphology on LB agar Circular, Nutrient agar + Low convex, entire LB agar + Smooth, shining YEM agar + Optimum growth temperature on LB, NA, YEM agar 28°C Colony color on LB agar Light-Yellow Colony color on Nutrient agar Off-white to creamy Pigment production on LB, NA, YEM & TY agar No Cell shape in Nutrient agar Small rods Gum production on LB, NA, TY & YEM agar + Cell motility in LB and Nutrient broth + Biochemical characters Reaction/test PV Z Reaction/test PV Z 2 2 2 2 Cytochrome oxidase + Catalase + Aminopeptidase + Nitrate reduction + β-galactosidase production + Production of acid from Glucose + Sodium citrate utilization UI Production of acid from Maltose + Sodium malonate fermentation - Production of acid from Sucrose - Lysine decarboxylase - Production of acid from Mannitol + Arginindihydrolase - Production of acid from Arabinose + Ornithine decarboxylase - Production of acid from Rhamnose + H S production - Production of acid from Sorbitol + Urea hydrolysis - Production of acid from Inositol + Tryptophanedeaminase + Production of acid from Adonitol - Indole production - Production of acid from Melibiose + Acetion formation + Production of acid from Raffinose - (Voges-Proskauer test) Gelatin hydrolysis - Utilization of carbon source Mannitol +++ Yeast extract ++ Maltose ++ Myo-inositol +++ Sucrose +++ Sorbitol +++ Fructose + Levulose +++ D-Glucose - Glycerol +++ Resistant to antibiotic Erythromycin (15 μg) + Kanamycin (10 μg) + Ampicillin (100 μg) + Spectinomycin(50 μg) + Aztreonam (30 μg) + Chloramphenicol (30 μg) + Carbenicillin (100 μg) + Gentamycin (10 μg) - Streptomycin (10 μg) - Neomycin (10 μg) - LB=Luria-Bertani; YEM=Yeast Extract Mannitol, TY=Trypton Yeast medium. - shows the reaction/test is negative + shows that reaction is positive +++/++ shows that growth is sufficiently good on agar plates containing respective carbon source UI reaction/test cannot be categorized as positive or negative Ann Microbiol (2014) 64:1797–1806 1801 mL, was inoculated (1 mL inoculum) onto the base of the yellow colonies in TY and LB medium. Colonies are root tips of common bean (var. Lyamungu 90) in growth generally convex and shiny, with smooth margins, and pouches (Asad et al. 1991). The plants were harvested after produce high amounts of gum. It is an acid-producing 30 days, and the effectiveness of nodules was determined by strain and showed complete resistance to chloromphenicol the acetylene reduction assay as described by Hafeez et al. (30 μg), erythromycin (15 μg), aztreonam (30 μg) and (2005). Total N content of the plants was determined by the carbenicillin (100 μg), but was highly sensitive to ampi- micro-Kjeldahal method (Yoshida et al. 1976) on a Kjeldahal cillin (10 μg), gentamycin (10 μg), amikacin (30 μg), system (Rapid Kjeldahl System; Labconco Corp., Kansas streptomycin (10 μg), doxicycline (30 μg), neomycin City, MO), and the results were statistically analysed using (30 μg) and trimethoprim/ sulfamethoxazole (25 μg). the MSTAT Version C statistical package. Significance at the The almost full-length 16S rRNA (1,461 bp) fragment of 5 % level was tested by New Duncan’s Multiple Range. PV2Z2 showed its maximum homology (99 %) to Ochrobactrum anthropi strain CLM6 (AJ867292). The nucle- otide accession number for strain PV2Z2 is EU399793. Full- Biodegradation potential length 16S rRNA gene sequences were obtained from the NCBI (www.ncbi.nlm.nih.gov) site and were aligned with A 1 mL sample of bacterial culture, after overnight growth in LB medium, was inoculated into 100 mL of minimal salts PV2Z2 to generate a phylogenetic tree. This phylogenetic -1 tree (Fig. 1) showed that the strain falls in the same cluster medium (MSM) supplemented with 100 mg L each of the phenol, 2-bromo-phenol, 4-nitrophenol, 2, 4-diamino phenol with the type strains of O. anthropi LMG3331 , O. lupini T T HCL, 3, 4-dimethoxy benzyl alcohol, 1, 3-dimethoxy benzene LUP21 and O. cytisi ESC1 and that it is more related to and 2(5-bromo-2-pyridylazo)-5-diethylamino phenol. Cultures O. cytisi from an evolutionary point of view than to O. were grown with constant shaking for 12 days at 28 °C, and growth was monitored by measuring the optical density of the culture at λ with a CamSpec M350 double-beam UV- visible spectrophotometer (Spectronic Camspec, Leeds, UK) using non-inoculated MSM medium as blank. Bacte- rial growth was quantified by counting the colony-forming units (CFU) as described (Somasegaran and Hoben 1994). The cells after growth in phenol-supplemented MSM were centrifuged, and the residual phenol concentration in the cell-free supernatant was estimated at λ as described (Greenburg et al. 1985) based upon the reaction between phenol and 4-amino antipyrine in the presence of ferricya- nide under alkaline conditions. Percentage degradation was calculated by the formula %phenoldegradation ¼½ ðÞ C −C =C 100 f 0 0 where C is the final concentration of phenol in medium, and C is the initial concentration of phenol in the medium. Results Physicochemical characters and identification of Pv2Z2 The detailed physicochemical characters of the bacterial strain Pv2Z2are giveninTable 1. Based on the test results, cells of the strain Pv2Z2 are Gram negative (ami- Fig. 1 Neighbour-joining tree, based on 16S rRNA gene sequences nopeptidase positive) and aerobic (catalase and oxidase showing the phylogenetic position of strain Pv2Z2 among recognized members of the genus Ochrobactrum and other related species of the positive), and they appear as highly motile, short rods family Brucellaceae. Bootstrap values of >50 %, based on 1,000 replica- commonly observed as single cells under the light micro- tions, are shown at branch points. The tree was rooted with scope. Growth occurs on LB, YEM, nutrient agar (NA) Pseudochrobactrum asaccharolyticum CCUG 46016 (accession no. and TY media within 10–12 hat28°C, resultinginlight- AM180485). Bar 0.01 substitutions per nucleotide position 1802 Ann Microbiol (2014) 64:1797–1806 that strain Pv2Z2 produces the AHLs 3O-C7-HSL and 3OH- C7-HSL. Plant inoculation assay The inoculation of common bean with Pv2Z2 resulted in increased plant weight, plant height and N uptake as compared to the non-inoculated plants (Fig. 5). Biodegradation assays The strain Pv2Z2 was able to grow on MSM containing Fig. 2 Representative random amplified polymorphic DNA profile of 100 mg/L of each of the phenols 2-bromophenol, 2,4- strain Pv2Z2 and some type strains of genus Ochrobactrum generated with primer OPC-15. Lanes: M DNA marker (1 kb/100 bp), 1 O. ciceri, 2 diamino phenol hydrochloride, 3,4-dimethoxy benzyl alco- T T O. tritici LAIII106, 3O. tritici ScII24 , 4 O. lupini LUP-21 , 5 O. oryzae hol and 4-methoxy benzyl alcohol, demonstrating that it was T T MTCC4195 , 6, 7 O. gallinifaecis ISO196 , 8O. intermedium DSM T T able to utilize these substrates as carbon sources, forming 17986 , 9 O. anthropi DSM 6882 , 10 Ochrobactrum sp. PV Z 2 2 populations of >10 cells per millilitre (Table 2). Maximum and minimum growth was observed in phenol (8.88 CFU) and 3,4-dimethoxy benzyl alcohol (7.53 CFU), respectively. anthropi. The RAPD results (Fig. 2) further validated the Strain Pv2Z2 was unable to grow in MSM containing 4- genetic differences between strains PV2Z2 and O. anthropi nitrophenol and 2 (5-bromo-2-pyridylazo)-5-diethyl amino LMG3331 . phenol. Within 12 days, it degraded 94 % of 100 mg/L phenol (Fig. 6). Bioassay for beneficial plant traits Discussion After 7 days of incubation a clear halo zone around the bacterial growth spot was clearly observed, showing the sol- The beneficial effects of the rhizosphere microbiome have ubilization of zinc oxide (Fig. 3a) and inorganic phosphate been known about for some time, but the wide-spread use of (Fig. 3b). The quantification data of these and other beneficial microbes as a crop supplement and bioremediation has in- planttraitsare giveninTable 2. No antagonistic activity creased in recent years due to the deleterious effects of chem- was observed against Fusarium solani, F. oxysporum and ical fertilizers/pesticides on the environment and non-target F. moniliforme. organisms, the increasing costs of chemical fertilizers and the AHL assay and RP-TLC massive increase in environmental pollution and contaminat- ed water due to rapid industrialization (Agrawal et al. 2010). The production of a violet colour in the TY/LB media detected Beneficial bacteria generally include members of the genera with the biosensor strain after overnight incubation demon- Bacillus, Pseudomonas, Azospirillum, Azotobacter, Serratia, strated the production of AHL molecules in the medium Enterobacter, Arthrobacter, Burkholderia and Ochrobactrum (Fig. 3c). These AHLs after extraction with ethyl acetate were (Lugtenberg and Kamilova 2009; Bhattacharyya and Jha analysed by RP-TLC, and the chromatogram (Fig. 4)showed 2012). These bacteria have been reported to act as plant Fig. 3 Solubilization of zinc oxide (a), tri-calcium phosphate (b) and the production of N-acyl homoserine lactone (AHL) molecules (c)by Ochrobactrum sp. Pv2Z2 in vitro Ann Microbiol (2014) 64:1797–1806 1803 Table 2 Plant growth promoting and biodegradation potential of strain PV2Z2 Beneficial plant traits Growth in the MSM-containing substrate Trait/character Values Substrate/compound Colony-forming units −1 Indole-3-acetic acid production 6.68 μgmL Phenol 8.88 Solubilization of inorganic phosphate 25.7 μg mL 2-Bromophenol 8.17 Production of AHL signal molecule + 2,4-diaminophenol dihydrochloride 8.25 Solubilization of zinc oxide + 4-Nitrophenol 0 Solubilization of zinc sulphate + 2 (5-Bromo-2-pyridylazo)-5-diethyl amino phenol 0 Antagonistic activity - 3,4-Dimethoxybenzyl alcohol 7.53 4-Methoxybenzyl alcohol 7.82 MSM, Minimal salts medium; AHL, N-acyl homoserine lactone -, The reaction/test is negative; +, the reaction/test is positive growth-promoting factors (Hafeez et al. 2006;Kantar et al. biodegradation or bioremediation (Smejkal et al. 2003;Afzal 2007; Mirza et al. 2007; Bhattacharyya and Jha 2012)and in et al. 2007; Anwar et al. 2009). In the study reported here we describe the taxonomic affiliation of a common bean nodule isolate Pv2Z2 and dem- onstrate its beneficial plant traits and biodegradation traits as well as its likely contribution in promoting plant growth. The strain was found to be metabolically diverse, to utilize a number of carbon sources and substrates, to be resistant to different antibiotics and to show the presence of different enzymes. These characteristics show the ecological fitness of the strain in a range of environments. Strain Pv2Z2 was isolated from sterilized nodules, indicating its endophytic nature. Its colony morphology was similar to that of other rhizobia, and nodulation assays were performed to confirm its nodulation ability. The Ochrobactrum strain Pv2Z2 induced nodulation in plant inoculation assay on common bean and -1 - showed ARA activity (3.34 μmol of ethylene g h 1). How- ever we were unable to confirm the identity of the endophyte to our inoculated strain using in situ hybridization or other molecular techniques. The improved N contents in common Fig. 5 Effect of inoculation with Ochrobactrum sp. Pv2Z2 on common -1 bean in growth pouches. Nitrogen uptake is given in units of mg g shoot Fig. 4 Thin-layer chromatogram of AHL extracts of Ochrobactrum sp. dry weight. The results are the average of three replicates. Un-inoculated Pv2Z2 compared with reference strain Rhizobium leguminosarum (A34). control contained sterile water. The concentration of the N+control was HSL Homoserine lactone 1 M potassium nitrate 1804 Ann Microbiol (2014) 64:1797–1806 Fig. 6 Degradation of phenol in comparison with the growth [optical density (OD)] of Ochrobactrum sp. Pv2Z2 bean plants inoculated with the Pv2Z2 may be attributed to its physiological phenomena. Moreover, the ability to produce N-fixing ability. AHL will help in rhizosphere colonization and might give this The Pv2Z2 strain was isolated from the soil of Zanzibar, strain a competitive advantage under field conditions. To date, Tanzania, where crop productivity is usually low due to soil no member of genus Ochrobactrum has been reported to acidity and moisture stress. Cell and colony morphology contain AHL production activity, although it does have the features, biochemical reactions and the antibiotic resistance activity to produce quorum quenching lactonase enzymes. pattern showed a maximum relatedness with genus This Ochrobactrum sp. was isolated from potato rhizosphere Ochrobactrum. The 16S rRNA sequence analysis also sup- and produces lactonase enzyme which is able to disrupt the ported the genus affiliation. The strain exhibited maximum signals of potato pathogen Erwinia carotovora (Jafra et al. similarities (99 %) with Ochrobactrum anthropi strains CLM6 2006). Following separation by RP-TLC, the crude AHL (AJ867292), CLM5 (AJ867291) and SAIII104 (AJ867290). extract showed the presence of 3O-C7-HSL and 3OH-C7- All of these strains showing maximum similarities to HSL, but the exact mass and nature of these compounds will Ochrobactrum strain Pv2Z2 are isolates from the wheat rhi- remain questionable unless confirmed through detailed mass zosphere (Lebuhn et al. 2000). Although phylogenetic analy- spectrometry analysis. sis grouped Pv2Z2 in the same phylogenetic lineage in which The biodegradation study is the most disparate of the the type strain of O. anthropi was present, the nearest phylo- investigations since it does not directly address plant–microbe genetic relative was the nodulating strain O. cytisi rather than interaction but was undertaken because the root exudates the clinical isolate or any pathogenic strain (Fig. 1), suggesting contain a number of phenolic compounds and bacteria contain the non-pathogenic nature of the strain Pv2Z2 which was the ability to utilize these compounds as an energy source. further validated by the RAPD and TP-RAPD assays This ability might give the bacterium an ecological advantage (Fig. 2). Moreover, the plant inoculation data obtained in this in the rhizosphere, which (due to its nutrient richness) works and other studies (Faisal and Hasnain 2004; Chakraborty et al. as “microbial hot-spot” for diverse plant–plant, plant–microbe 2009;Riazetal. 2010) show that O. anthropi or and microbe–microbe interactions. The strain proved itself a O. intermedium can be safely used as PGPR. candidate of choice for the biodegradation of phenol- The production of IAA, solubilization of inorganic contaminated soils. The degradation of phenol by phosphate/zinc and the promotion of growth of common bean Ochrobactrum species (Lechner et al. 1995;El-Sayed etal. plants in the pot experiment as well as the significant increase 2003) has been reported to be fourfold higher than that of in N uptake by plants suggest that the strain can be a potential other reported phenol-degrading bacteria. The known phenol- PGPR candidate for field application. The production of AHL degrading Ochrobactrum species include both those isolated was found to be a novel trait that had not earlier been reported from activated sludge and contaminated industrial environ- for the members of this genus. N2 fixation, phosphate solubi- ments or the plant-associated O. tritici NBRC 102585, lization and biocontrol activities of bacteria are known to be O. grignonense NBRC 102586, and O. lupini NBRC controlled by the AHL signal molecules. The varied charac- 102587 (Yamada et al. 2008). Phenol degradation by nodule ters of this strain suggest that its quorum sensing signals might bacteria, such as Rhizobium leguminosarum bv trifolli, has be involved in all of these processes as well as in many other been reported (Parke et al. 1991), as has the degradation of Ann Microbiol (2014) 64:1797–1806 1805 Amer MM (2008) Monitoring of Variation among Faba Bean Rhizobium brominated phenols by plant-associated Ochrobactrum sp. Isolates: 2. Biodegradation of Herbicide, 3(3,4 Dichlorophenyl) -1- TB01, O. tritici NBRC102585, O. grignonense NBRC methoxy-1-methylurea. Aust J Basic Appl Sci 2(3):540–548 102586 and O. lupini NBRC 102587 (Yamada et al. 2008). An D, Danhorn T, Fuqua C, Parsek MR (2006) Quorum sensing and These studies show that this phenol-degradation phenomenon motility mediate interactions between Pseudomonas aeruginosa and Agrobacterium tumefaciens in biofilm cocultures. Proc Natl Acad is wide-spread in the genus. The Pv2Z2 strain was, however, Sci USA 103(10):3828–3833 not able to grow in the medium containing 4-nitrophenol and Antunes LC, Ferreira RB (2009) Intercellular communication in bacteria. 2-(5-bromo-2-pyridylazo)-5-diethyl amino phenol. One pos- Crit Rev Microbiol 35(2):69–80 sible explanation for the failure of PV2Z2 to grown on Anwar S, Liaquat F, Khan QM, Khalid ZM, Iqbal S (2009) Biodegradation of chlorpyrifos and its hydrolysis product 3,5,6- nitrophenol-supplemented medium might be the unfavourable trichloro-2-pyridinol by Bacillus pumilus strain C2A1. J Hazard conditions, as p-nitrophenol degradation has been shown to be Mater 168(1):400–405 accelerated at alkaline pH (8–10) and following the addition Asad S, Malik KA, Hafeez FY (1991) Competition between inoculated of organic N (i.e. yeast extract and peptone; Qiu et al. 2007). and indigenous Rhizobium/Bradyrhizobium spp. strains for nodula- tion of grain and fodder legumes in Pakistan. Biol Fertil Soils 12: The ability of plant-associated bacteria to degrade aromatic 107–111 compounds allows these microorganisms to utilize multiple Beringer JE (1974) R factor transfer in Rhizobium leguminosarum. J Gen energy sources, thereby improving their ecological success in Microbiol 84:188–198 the rhizosphere. Bhattacharyya PN, Jha DK (2012) Plant growth-promoting rhizobacteria (PGPR): emergence in agriculture. World J Microbiol Biotechnol Ochrobactrum species are widespread and frequent inhab- 28:1327–1350 itants of the rhizosphere and other environmental habitats. The Cavalcante VA, Döbereiner J (1988) A new acid-tolerant nitrogen fixing results presented here have implications for using bacterium associated with sugarcane. Plant Soil 108:23–31 Ochrobactrum strain Pv2Z2 as biofertilizer and in biodegra- Chakraborty U, Chakraborty BN, Basnet M, Chakraborty AP (2009) Evaluation of Ochrobactrum anthropi TRS-2 and its talc based dation. The strain Pv2Z2 was isolated from the root nodule of formulation for enhancement of growth of tea plants and manage- common bean; it is genetically different from clinical/ ment of brown root rot disease. J Appl Microbiol 107:625–634 pathogenic O. anthropi/intermedium isolates and has positive Chen WM, Chang JS, Wu CH, Chang SC (2004) Characterization of effect on plant growth. Therefore, its use as plant and/or soil phenol and trichloroethene degradation by the rhizobium Ralstonia taiwanensis. Res Microbiol 155(8):672–680 inoculum is not expected to cause any human pathogenic Compant S, Duffy B, Nowak J, Clement C, Barka EA (2005) Use of plant consequences. In this study, we report for the first time a growth-promoting bacteria for biocontrol of plant diseases: princi- new strain of Ochrobactrum sp. Pv2Z2 isolated from the ples, mechanisms of action, and future prospects. Appl Environ common bean nodules with the ability to produce AHL Microbiol 71(9):4951–4959 Czajkowski R, Krzyżanowska D, Karczewska J, Atkinson S, Przysowa J, molecules. Lojkowska E, Williams P, Jafra S (2011) Inactivation of AHLs by Ochrobactrum sp. A44 depends on the activity of a novel class of AHL acylase. Environ Microbiol Rep 3(1):59–68 Acknowledgements The authors are grateful to Dr. Allan J. Dowine El-Sayed W, Ibrahim MK, Abu-Shady M, El-Beih F, Ohmura N, Saiki H, (JIC. Norwich, UK) for providing reference and indicator strains for the Ando A (2003) Isolation and identification of a novel strain of the AHL assay and to Dr. Erko Stackebrandt (DSMZ, Germany) for provid- genus Ochrobactrum with phenol-degrading activity. J Biosci ing the DNA of O. anthropi and other type strains of the genus. The Bioeng 96(3):310–312 research work was partially supported by IDB-BIRCEN Project on “Use Faisal M, Hasnain S (2004) Comparative study of Cr(VI) uptake and of Biofertilizers for Increasing Sustainable Crop Production in Develop- reduction in industrial effluent by Ochrobactrum intermedium and ing Countries: Establishment of a Biofertilizer Resource Center Brevibacterium sp. Biotechnol Lett 26:1623–1628 (BIRCEN) at NIBGE” (2000–2005). Feng YS, Lee CM (2009) The potential of the acetonitrile biodegradation by Mesorhizobium sp. F28. J Hazard Mater 164(2–3):646–650 Frassinetti S, Setti L, Corti A, Farrinelli P, Montevecchi P, Vallini G (1998) Biodegradation of dibenzothiophene by a nodulating isolate of Rhizobium meliloti. Can J Microbiol 44(3):289–297 References Garnett TP, Conn VM, Kaiser BN (2009) Root based approaches to improving nitrogen use efficiency in plants. Plant Cell Environ 32: 1272–1283 Abbas A, Adams C, Scully N, Glennon J, O'Gara F (2007) A role for Gordon SA, Weber RP (1951) Colorimetric estimation of indole acetic TonB1 in biofilm formation and quorum sensing in Pseudomonas acid. Plant Physiol 26:192–195 aeruginosa. FEMS Microbiol Lett 274(2):269–278 Greenburg AE, Trussell RL, Clesceri LS (1985) Standard menthods for Adesemoye AO, Torbert HA, Kloepper JW (2009) Plant Growth- the examination of water and waste water, 16th edn. American Promoting Rhizobacteria allow reduced application rates of chemi- Public Health Association, Washington, DC cal fertilizers. Microbiol Ecol 58:921–929 Hafeez FY, Naeem F, Naeem R, Zaidi AH, Malik KA (2005) Symbiotic Afzal M, Iqbal S, Rauf S, Khalid ZM (2007) Characteristics of phenol effectiveness and bacteriocin production by Rhizobium biodegradation in saline solutions by monocultures of Pseudomonas leguminosarum bv. Viciae isolated from agriculture soils in aeruginosa and Pseudomonas pseudomallei. J Hazard Mater Faisalabad. Environ Exp Bot 54:142–147 149(1):60–66 Hafeez FY, Yasmin S, Airan D, Rahman M, Zafar Y, Malik KA (2006) Agrawal A, Pandey RS, Sharma B (2010) Water pollution with special Plant growth-promoting bacteria as biofertilizer. Agron Sustain Dev reference to pesticide contamination in India. J Water Res Protect 2: 26:143–150 432–448 1806 Ann Microbiol (2014) 64:1797–1806 Imran A, Hafeez FY, Fruhling A, Schumann P, Malik KA, Stackebrandt E Seo JS, Keum YS, Harada RM, Li QX (2007) Isolation and characterization (2010) Ochrobactrum ciceri sp. nov., isolated from nodules of Cicer of bacteria capable of degrading polycyclic aromatic hydrocarbons arietinum. Int J Syst Evol Microbiol 60(7):1548–1553 (PAHs) and organophosphorus pesticides from PAH-contaminated Jafra S, Przysowa J, Czajkowski R, Michta A, Garbeva P, van der Wolf soil in Hilo, Hawaii. J Agric Food Chem 55(14):5383–5389 JM (2006) Detection and characterization of bacteria from the potato Shah NH, Hafeez FY, Asad S, Hussain A, Malik KA (1995) Isolation and rhizosphere degrading N-acyl-homoserine lactone. Can J Microbiol characterization of indigenous Rhizobium leguminosarum bv viciae 52(10):1006–10015 nodulating Lens culinaris Medik. from four Pakistani soils. In: Kantar F, Hafeez FY, Shivakumar BG, Sundaram SP, Tejera NA, Aslam Malik KA, Nasim A, Khalid AM (eds) Biotechnology for sustain- A, Bano A, Raja P (2007) Chickpea: Rhizobium management and able development. NIBGE, Faisalabad, pp 211–219 nitrogen fixation. In: Yadav SS, Redden R, Chen W, and Sharma B Smejkal CW, Seymour FA, Burton SK, Lappin-Scott HM (2003) (eds) Chickpea breeding and management. Cormwell Press, Characterisation of bacterial cultures enriched on the Trowbridge, pp 179–192 chlorophenoxyalkanoic acid herbicides 4-(2,4-dichlorophenoxy) Lebuhn M, Achouak W, Schloter M, Berge O, Meier H, Barakat M, butyric acid and 4-(4-chloro-2-methylphenoxy) butyric acid. J Ind Hartmann A, Heulin T (2000) Taxonomic characterization of Microbiol Biotechnol 30(9):561–567 Ochrobactrum sp. isolates from soil samples and wheat roots, and Somasegaran P, Hoben HJ (1994) Handbook for Rhizobia Methods in description of Ochrobactrum tritici sp. nov. and Ochrobactrum Legume–Rhizobium Technology. Springer, Heidelberg grignonense sp. nov. Int J Syst Evol Microbiol 50:2207–2223 Steinshamn H, Thuen E, Bleken MA, Brenoe UT, Ekerholt G, Yri C Lechner U, Baumbach R, Becker D, Kitunen V, Auling G, Salkinoja- (2004) Utilization of nitrogen (N) and phosphorus (P) in an organic Salonen M (1995) Degradation of 4-chloro-2-methylphenol by an dairy farming system in Norway. Agric Ecosyst Environ 104:509– activated sludge isolate and its taxonomic description. 522 Biodegradation 6(2):83–92 Taminiau B, Daykin M, Swift S, Boschiroli ML, Tibor A, Lestrate P, Lugtenberg B, Kamilova F (2009) Plant-growth-promoting rhizobacteria. Bolle XD, O'Callaghan D, Williams P, Letesson JJ (2002) Annu Rev Microbiol 63:541–556 Identification of a quorum-sensing signal molecule in the facultative Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A labora- intracellular pathogen Brucella melitensis. Infect Immun 70(6): tory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor 3004–3011 McLean RJ, Pierson LS 3rd, Fuqua C (2004) A simple screening protocol Tan ZY, Xu XD, Wang ET, Gao JL, Martinez-Romero E, Chen WX for the identification of quorum signal antagonists. J Microbiol (1997) Phylogenetic and genetic relationships of Mesorhizobium Methods 58(3):351–360 tianshanense and related rhizobia. Int J Syst Bacteriol 47(3):874– Mirza BS, Mirza MS, Bano A, Malik KA (2007) Inoculation of chickpea 879 with Rhizobium isolates from roots and nodules and phytohormone Tien TM, Gaskins MH, Hubbell DH (1979) Plant Ggrowth substances producing Enterobacter strains. Aust J Exp Agric 47:1008–1015 produced by Azospirillum brasilense and their effect on the growth Murphy J, Riley JP (1962) A modified single solution method for the of pearlmillet(Pennisetum americanum L.). Appl Environ determination of phosphate in natural waters. Anal Chim Acta 27: Microbiol 37(5):1016–1024 31–36 Tilman D, Cassman KG, Matson PA, Naylor R, Polasky S (2002) Parke D, Rynne F, Glenn A (1991) Regulation of phenolic catabolism in Agricultural sustainability and intensive production practices. Rhizobium leguminosarum biovar trifolii. J Bacteriol 173(17):5546– Nature 418:671–677 5550 Vincent JM, Humphrey B (1970) Taxonomically significant group anti- Pikovskaia RI (1948) Metabolism of phosphorous in soil in connection with gens in Rhizobium. J Gen Microbiol 63(3):379–382 vital activity of some microbial species. Microbiology 17:362–370 Wei G, Yu J, Zhu Y, Chen W, Wang L (2008) Characterization of phenol Qiu X, Zhonga Q, Lia M, Baia W, Li B (2007) Biodegradation of P- degradation by Rhizobium sp. CCNWTB 701 isolated from nitrophenol by methyl parathion-degrading Ochrobactrum sp. B2. Astragalus chrysopterus in mining tailing region. J Hazard Mater Int Biodeterior Biodegrad 59:297–301 151(1):111–117 Radwan SS, Dashti N, El-Nemr I, Khanafer M (2007) Hydrocarbon Williams JGK, Kubelik AR, Livak KJ, Rafalski JA (1990) DNA poly- utilization by nodule bacteria and plant growth-promoting morphisms amplified by arbitrary primers are useful as genetic rhizobacteria. Int J Phytoremed 9(6):475–486 markers. Nucleic Acids Res 18:6531–6535 Riaz S, Faisal M, Hasnain S (2010) Cicer arietinum growth promotion by Yamada T, Takahama Y, Yamada Y (2008) Biodegradation of 2,4,6- Ochrobactrum intermedium and Bacillus cereus in the presence of tribromophenol by Ochrobactrum sp. strain TB01. Biosci CrCl and K CrO . Ann Microbiol 60:729–733 Biotechnol Biochem 72(5):1264–1271 3 2 4 Sakthivel N, Gnanamanickam SS (1986) Toxicity of Pseudomonas Yoshida S, Forno DA, Cock JH, Gomez KA (1976) Laboratory manual fluorescens towards rice sheath rot pathogen Acrocylindrium oryzae. for physiological studies of rice, 3rd edn. The International Rice Curr Sci 5:106–107 Research Institute, Manila http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Ochrobactrum sp. Pv2Z2 exhibits multiple traits of plant growth promotion, biodegradation and N-acyl-homoserine-lactone quorum sensing

Download PDF
10 pages

Loading next page...
 
/lp/springer-journals/ochrobactrum-sp-pv2z2-exhibits-multiple-traits-of-plant-growth-y0Im8gTxc5

References (59)

Publisher
Springer Journals
Copyright
Copyright © 2014 by Springer-Verlag Berlin Heidelberg and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
eISSN
1869-2044
DOI
10.1007/s13213-014-0824-0
Publisher site
See Article on Publisher Site

Abstract

Ann Microbiol (2014) 64:1797–1806 DOI 10.1007/s13213-014-0824-0 ORIGINAL ARTICLE Ochrobactrum sp. Pv2Z2 exhibits multiple traits of plant growth promotion, biodegradation and N-acyl-homoserine-lactone quorum sensing Asma Imran & Marryam Jumma Abdulla Saadalla & Sami-Ullah Khan & Muhammad Sajjad Mirza & Kauser Abdulla Malik & Fauzia Yusuf Hafeez Received: 21 November 2013 /Accepted: 22 January 2014 /Published online: 19 February 2014 Springer-Verlag Berlin Heidelberg and the University of Milan 2014 Abstract Rhizosphere bacteria play a vital role in plant solubilized zinc sulphate and zinc oxide in the LG1 medium. growth, pathogen control, biodegradation and rhizosphere The production of AHLs (e.g. 3O-C7-HSL, 3OH-C7-HSL) signaling. A motile, rod-shaped bacterium, Pv2Z2, isolated was detected with biosensor strains CV026 using reverse from the nodules of the common bean grown in Tanzanian soil phase thin layer chromatography. The bacterium was able to was characterized using a polyphasic approach. The traits grow in minimal salt medium supplemented with 100 mg/L assessed included the production of indole-3-acetic acid and each of phenol, 2-bromophenol, 2,4-diamino phenol hydro- N-acyl homoserine lactone (AHL) molecules, solubilization chloride, 3,4-dimethoxy benzyl alcohol and 4-methoxy ben- of insoluble phosphate and zinc compounds and biodegrada- zyl alcohol. Phenol degradation was recorded up to a level of tion of a number of toxic compounds. The 16S rRNA se- 94 % within 12 days. Inoculation of common bean plants quence of Pv2Z2 (EU399793) showed 99 % homology to resulted in a significant increase in plant height, fresh/dry Ochrobactrum anthropi isolates (Accession no. AJ867292, weight and nitrogen uptake as compared to non-inoculated AJ867291, AJ867290) from soil samples of wheat root. Phy- plants. The data suggest that the plant growth-promoting and logenetic analysis showed relatedness to nodulating strain biodegradation potential of this bacterium may be exploited Ochrobactrum cytisi rather than to the clinical/pathogenic type on a large scale. The capacity to produce AHL molecules by strain of O. anthropi. Moreover, it showed unique fingerprints members of the Ochrobactrum genus has not been previously in the randomly amplified polymorphic DNA (RAPD) and reported and needs to be explored in detail. two primers-RAPD assays which were different from those of the pathogenic type strain of O. anthropi. The bacterium . . . Keywords Legumes Ochrobactrum Biodegradation -1 produced 6.68 μg/mL indoleacetic acid in the presence of Phosphate solubilization AHLs -1 tryptophan, released 25.7 μg/mL phosphorus from inorganic tri-calcium phosphate in the Pikoviskaya’smediumand Introduction : : : : A. Imran (*) M. J. A. Saadalla S.<U. Khan M. S. Mirza K. A. Malik F. Y. Hafeez (*) Although nitrogen (N) and phosphorus (P) are vital nutrients National Institute for Biotechnology and Genetic Engineering, for plant growth and development, only 30–50 % of exoge- P.O. Box 577, Jhang Road, Faisalabad, Pakistan nously applied N and 10–45 % of P are taken up by plants e-mail: asmaaslam2001@yahoo.com e-mail: fauzia_y@yahoo.com (Adesemoye et al. 2009; Garnett et al. 2009). The rest of the fertilizer fraction becomes unavailable either due to leaching M. J. A. Saadalla or nutrient run-off. In addition, long-term use of synthetic Ministry of Agriculture, Natural Resources, fertilizers negatively affects the overall biogeochemical cycles Environment and Co-operatives, Zanzibar, Tanzania and ecosystem (Steinshamn et al. 2004) and has become a K. A. Malik global environmental concern (Tilman et al. 2002). An alter- FC College University, Lahore, Pakistan native to the use of chemical fertilizers is the application of biological fertilizer technology. This technology not only F. Y. Hafeez COMSTAS Institute of Information Technology, Islamabad, Pakistan benefits plants in terms of growth and yield but improves soil 1798 Ann Microbiol (2014) 64:1797–1806 health and the environment, reduces reliance on chemical and soybean to trap the indigenous rhizobia specific to these fertilizers and provides economic benefits. hosts. After screening a number of isolates, we identified one Biological fertilizers composed of plant growth-promoting nodule isolate having the ability to produce AHL compounds rhizobacteria (PGPR) are often described as having multiple and multiple traits of plant growth promotion and biodegra- traits of plant growth promotion. They convert atmospheric N dation. The isolate, when characterized in detail, showed great to ammonia, thereby making N available to plants, transform promise for use in the detoxification of hazardous chemicals unavailable phosphate or zinc compounds to plant-available and as a biofertilizer. The AHL quorum-sensing system might fractions, produce different growth hormones and amino be involved in regulating its function in the rhizosphere and acids, produce signal compounds, such as N-acyl homoserine environment. lactone (AHL) molecules, to interact with the host and coor- dinate their activities in the rhizosphere, exhibit 1- aminocyclopropane-1-carboxylate-deaminase activity to mit- Materials and methods igate the stress-induced negative impacts and produce differ- ent antifungal compounds to combat pathogen attack Strain isolation (Compant et al. 2005). In addition to the biotic stress caused by pathogen attack, The strain was isolated from soil collected from the Maruhubi plant growth and yield are seriously affected by abiotic stress- region of Tanzania, which was a sandy-loam soil having a pH -1 es, such as salinity, drought, frost and the presence of toxic of 5.69, electrical conductivity of 10.2 dS/m ,11%Ccon- compounds in water or soil. Each day huge amounts of tent, 2.2 % organic matter content, 0.08 % total N content, 10 organic compounds are released as industrial by-products, mg/kg P, 0.72 mg/100 g calcium, 0.48/100 g magnesium, 0.50 pesticides or agrochemicals into the environment, of which mg/100 g potassium and a C/N ratio of 13. The last crop phenolic compounds are very hazardous (Afzal et al. 2007). grown in the soil was common bean. Serial eightfold dilutions Microbes found in natural waters and soils have an amazing, were prepared from 1 g of soil (Somasegaran and Hoben wide-ranging ability to utilize many of these toxic compounds 1994), and 1 mL of each dilution was applied to the base of as carbon (C) and energy sources and have a great potential for the root tip of 3-day-old seedlings of common bean (var. use in large-scale bioremediation programs. Biodegradation Lyamungu 90). The plants were irrigated with N-free ability has been reported in many soil bacteria, particularly Hoagland solution and observed for nodule formation. Nod- PGPR [e.g. Rhizobium sp. (Amer 2008; Wei et al. 2008), ules were detached from the root, flame sterilized, crushed on R. leguminosarum bv. Trifolli (Parke et al. 1991), R. meliloti a sterile glass surface and streaked onto YEM-Congo red (Frassinetti et al. 1998), Ralstonia taiwanensis (Chen et al. plates as described by Shah et al. (1995) to isolate the bacteria. 2004, Bradyrhizobium japonicum (Radwan et al. 2007), The plates were incubated at 28±2 °C for 3–4 days until the Sinorhizobium (Seo et al. 2007), Mesorhizobium (Feng and appearance of separate colonies. Lee 2009)]. Bacterial activities (either beneficial or pathogenic) are Biochemical characterization mainly controlled by different signal molecules which are produced by bacteria to regulate the expression of specific Colony morphology, size, color, shape, gum production and genes and to interact with the environment. The AHLs are growth pattern were recorded after 24 hofgrowthonYeast signal compounds that are produced specifically by Gram- Extract Mannitol (YEM) and Luria–Bertani(LB)agarplatesat negative bacteria and are involved in signaling processes such 28±2 °C as described by Somasegaran and Hoben (1994). Cell as antibiotic production and conjugal plasmid transfer. This size and motility was observed by light microscopy. Acid/alkali kind of signaling has been identified in Pseudomonas, production was tested on LB-agar plates containing 0.025 % Agrobacterium and Rhizobium (An et al. 2006; Abbas et al. (w/v) bromothymol blue as pH indicator. The Gram reaction 2007; Antunes and Ferreira 2009). The members of family was performed as described by Vincent and Humphrey (1970). Brucellaceae are known to produce signal molecules Amino-peptidase and cytochrome oxidase tests were per- (Taminiau et al. 2002), but to date this activity has not been formed by using commercially available strips (Merck, Darm- reported for the genus Ochrobactrum. Quorum-quenching stadt, Germany). Catalase production was checked by placing a activity, however, has been reported for one member of this drop of H O onto the bacterial colony on a glass slide. Resis- 2 2 genus (Jafra et al. 2006; Czajkowski et al. 2011). tance to the antibiotics chloromphenicol (30 μg), erythromycin The aim of this study was to isolate the nodulating bacteria (15 μg), aztreonam (30 μg), carbenicillin (100 μg), ampicillin that can be used for inoculum production for common bean, (10 μg), gentamycin (10 μg), amikacin (30 μg), streptomycin cowpeas and soybean in Zanzibar, Tanzania. For that purpose, (10 μg), doxicycline (30 μg), neomycin (30 μg) and soil was collected from different regions of Zanzibar, Tanza- trimethoprim/sulfamethoxazole (25 μg) was determined on nia, and soil inoculum was applied to common bean, cowpea antibiotic sulphonamide sensitivity-test agar (Merck) plates Ann Microbiol (2014) 64:1797–1806 1799 using commercially available discs (Bioanalyse®, Ankara, Tur- Solubilization of tri-calcium phosphate, zinc oxide and zinc key). The utilization of different carbon sources and enzymatic sulphate reactions were performed using the QTS-24 kit (DESTO, Ka- rachi, Pakistan) following the manufacturer‘s protocol. The Aliquots (10 μL) of Pv2Z2, after overnight growth in LB capacity to utilize mannitol, maltose, fructose, sucrose, D-glu- medium, were spot inoculated onto Pikovskaia’sagar cose, levulose, sorbitol, myo-inositol, glycerol and yeast extract (Pikovskaia 1948) containing tri-calcium phosphate as the insol- as carbon sources was determined as described by uble P source and onto LGI medium (Cavalcante and Somasegaran and Hoben (1994). Döbereiner 1988) supplemented with 0.1 % zinc oxide and zinc sulphate. The plates were incubated at 28±2 °C for 10–14 days and examined for the formation of a clear zone around the Molecular characterization bacterial growth spot daily. The appearance of a clear zone was considered to be a positive indication for phosphate and zinc Total genomic DNA of the bacterium was isolated by the solubilization activities. P solubilization was quantified using the alkaline lysis method (Maniatis et al. 1982) and used to phosphomolybdate blue colour method on a spectrophotometer amplify the 16S rRNA gene with primers P1/P6 (Tan et al. (λ=882 nm) as described by Murphy and Riley (1962) 1997). The PCR assay was carried out in a thermal cycler (Eppendorf, Hamburg, Germany) as described by Imran Antifungal activity et al. (2010). The amplified PCR product was purified using the QIA Quick PCR Purification kit (Qiagen, Hilden, Antifungal activity was tested using a dual-culture assay as Germany), ligated in TA cloning vector pTZ57R/T described by Sakthivel and Gnanamanickam (1986). A drop (Fermentas, Thermo Fisher Scientific, Waltham, MA) and of the exponentially grown bacterial culture (approx. 20 μL) cloned in Escherichia coli strain DH5α as described by was spotted onto potato dextrose agar (PDA) plates close to the Maniatis et al. (1982). Cloned PCR products were se- walls of the petri dish on both sides and the plate left in laminar quenced commercially from Macrogen (Seoul, Korea). The air flow cabinet until dry. A 6-mm agar disk of each of three gene sequence was compared with others in the GenBank fungal species, namely, Fusarium solani, F. oxysporum and database using the NCBІ BLASTn. Multiple sequence align- F. moniliforme (obtained from the lab collection), were sepa- ments were performed by ClustalX, and phylogeny was rately placed at the centre of PDA plates. The plates were determined by neighbor-joining method. Random amplified incubated at 28±2 °C for 5 days to measure the inhibition of polymorphic DNA (RAPD) analysis was performed as de- radial fungal growth between fungal and bacterial colonies. scribed by Williams et al. (1990) using primers OPC-13 and Percentage inhibition was determined by the reduction in fungal OPC-15 (Operon Technologies, Alameda, CA). Two primers growth compared to control (un-inoculated bacterial strain). (TP)-RAPD was performed as described by Imran et al. (2010). The DNA of the O. anthropi type strain LMG3331 The AHL assay and detection by reverse-phase thin-layer and other type strains of the genus obtained from DSMZ chromatography (German Collection of Microorganisms and Cell Cultures) were used as reference. The Pv2Z2 isolate was streaked onto the center of a Tryptone Yeast extract (TY) (Beringer 1974) agar plate and grown overnight at 28 °C to achieve appreciable growth. The indica- Bioassays for plant growth-promoting activities tor strain Chromobacterium violaceum CV026 (obtained from John Innes Centre, Norwich, UK) was grown separately in LB Production of indole-3-acetic acid broth at 30 °C with constant shaking overnight up to an OD of 10 CFU/mL. AHLs were detected by the plate overlay assay The production of indole-3-acetic acid (IAA) was determined as described by McLean et al. (2004). The reference strain A34 using the colorimetric method of Gordon and Weber (1951). (obtained from the John Innes Centre) was used as the positive Quantitative estimation was done by growing Pv2Z2 in LB- control. A34 is a Rhizobium leguminosarum 8401 containing broth supplemented with 100 mg/L tryptophan as IAA pre- pRLIJ1. Plates were incubated at 30±2 °C overnight, and cursor for 5 days. IAA was extracted with an equal volume of purple pigmentation produced by the indicator strain on the ethyl acetate as described by Tien et al. (1979) and finally plates was considered to indicate AHL production. collected in ethanol. A 100-μL sample of the extract was AHLs were extracted from the culture supernatant analysed on high-performance liquid chromatograph with an equal volume of ethyl acetate (acidified with equipped with Turbochrom software (Perkin Elmer Life Sci- 0.1 % v/v glacial acetic acid).Water residues were re- ence, Boston, MA) and on a C-18 column at a flow rate of moved by treating the extract with anhydrous Na SO . 2 4 0.5 mL/min. The extract was filtered, concentrated in a rotary evaporator 1800 Ann Microbiol (2014) 64:1797–1806 (Rota vapor R-210; Buchi Corp, New Castle, DE) and re- of indicator strain CV026, and AHLs were identified as purple confirmed by reverse plate assay as described by McLean spots on the off-white background. et al. (2004). The AHL extract (10 μL) was separated on glass-backed C18 reverse-phase thin-layer chromatography Plant inoculation assay (RP-TLC) plates (Merck) in methanol/water (60:40 v/v) along with the AHL extract of A34 as reference. The TLC plate was The Pv2Z2 strain, which had been grown in yeast-extract- developed with the overlay of an exponentially grown culture mannitol (YEM) medium to a concentration of up to 10 cells/ Table 1 Morphological and biochemical characters of Ochrobactrum sp. Pv2Z2 Cultural/Morphological characters Growth on Colony morphology on LB agar Circular, Nutrient agar + Low convex, entire LB agar + Smooth, shining YEM agar + Optimum growth temperature on LB, NA, YEM agar 28°C Colony color on LB agar Light-Yellow Colony color on Nutrient agar Off-white to creamy Pigment production on LB, NA, YEM & TY agar No Cell shape in Nutrient agar Small rods Gum production on LB, NA, TY & YEM agar + Cell motility in LB and Nutrient broth + Biochemical characters Reaction/test PV Z Reaction/test PV Z 2 2 2 2 Cytochrome oxidase + Catalase + Aminopeptidase + Nitrate reduction + β-galactosidase production + Production of acid from Glucose + Sodium citrate utilization UI Production of acid from Maltose + Sodium malonate fermentation - Production of acid from Sucrose - Lysine decarboxylase - Production of acid from Mannitol + Arginindihydrolase - Production of acid from Arabinose + Ornithine decarboxylase - Production of acid from Rhamnose + H S production - Production of acid from Sorbitol + Urea hydrolysis - Production of acid from Inositol + Tryptophanedeaminase + Production of acid from Adonitol - Indole production - Production of acid from Melibiose + Acetion formation + Production of acid from Raffinose - (Voges-Proskauer test) Gelatin hydrolysis - Utilization of carbon source Mannitol +++ Yeast extract ++ Maltose ++ Myo-inositol +++ Sucrose +++ Sorbitol +++ Fructose + Levulose +++ D-Glucose - Glycerol +++ Resistant to antibiotic Erythromycin (15 μg) + Kanamycin (10 μg) + Ampicillin (100 μg) + Spectinomycin(50 μg) + Aztreonam (30 μg) + Chloramphenicol (30 μg) + Carbenicillin (100 μg) + Gentamycin (10 μg) - Streptomycin (10 μg) - Neomycin (10 μg) - LB=Luria-Bertani; YEM=Yeast Extract Mannitol, TY=Trypton Yeast medium. - shows the reaction/test is negative + shows that reaction is positive +++/++ shows that growth is sufficiently good on agar plates containing respective carbon source UI reaction/test cannot be categorized as positive or negative Ann Microbiol (2014) 64:1797–1806 1801 mL, was inoculated (1 mL inoculum) onto the base of the yellow colonies in TY and LB medium. Colonies are root tips of common bean (var. Lyamungu 90) in growth generally convex and shiny, with smooth margins, and pouches (Asad et al. 1991). The plants were harvested after produce high amounts of gum. It is an acid-producing 30 days, and the effectiveness of nodules was determined by strain and showed complete resistance to chloromphenicol the acetylene reduction assay as described by Hafeez et al. (30 μg), erythromycin (15 μg), aztreonam (30 μg) and (2005). Total N content of the plants was determined by the carbenicillin (100 μg), but was highly sensitive to ampi- micro-Kjeldahal method (Yoshida et al. 1976) on a Kjeldahal cillin (10 μg), gentamycin (10 μg), amikacin (30 μg), system (Rapid Kjeldahl System; Labconco Corp., Kansas streptomycin (10 μg), doxicycline (30 μg), neomycin City, MO), and the results were statistically analysed using (30 μg) and trimethoprim/ sulfamethoxazole (25 μg). the MSTAT Version C statistical package. Significance at the The almost full-length 16S rRNA (1,461 bp) fragment of 5 % level was tested by New Duncan’s Multiple Range. PV2Z2 showed its maximum homology (99 %) to Ochrobactrum anthropi strain CLM6 (AJ867292). The nucle- otide accession number for strain PV2Z2 is EU399793. Full- Biodegradation potential length 16S rRNA gene sequences were obtained from the NCBI (www.ncbi.nlm.nih.gov) site and were aligned with A 1 mL sample of bacterial culture, after overnight growth in LB medium, was inoculated into 100 mL of minimal salts PV2Z2 to generate a phylogenetic tree. This phylogenetic -1 tree (Fig. 1) showed that the strain falls in the same cluster medium (MSM) supplemented with 100 mg L each of the phenol, 2-bromo-phenol, 4-nitrophenol, 2, 4-diamino phenol with the type strains of O. anthropi LMG3331 , O. lupini T T HCL, 3, 4-dimethoxy benzyl alcohol, 1, 3-dimethoxy benzene LUP21 and O. cytisi ESC1 and that it is more related to and 2(5-bromo-2-pyridylazo)-5-diethylamino phenol. Cultures O. cytisi from an evolutionary point of view than to O. were grown with constant shaking for 12 days at 28 °C, and growth was monitored by measuring the optical density of the culture at λ with a CamSpec M350 double-beam UV- visible spectrophotometer (Spectronic Camspec, Leeds, UK) using non-inoculated MSM medium as blank. Bacte- rial growth was quantified by counting the colony-forming units (CFU) as described (Somasegaran and Hoben 1994). The cells after growth in phenol-supplemented MSM were centrifuged, and the residual phenol concentration in the cell-free supernatant was estimated at λ as described (Greenburg et al. 1985) based upon the reaction between phenol and 4-amino antipyrine in the presence of ferricya- nide under alkaline conditions. Percentage degradation was calculated by the formula %phenoldegradation ¼½ ðÞ C −C =C 100 f 0 0 where C is the final concentration of phenol in medium, and C is the initial concentration of phenol in the medium. Results Physicochemical characters and identification of Pv2Z2 The detailed physicochemical characters of the bacterial strain Pv2Z2are giveninTable 1. Based on the test results, cells of the strain Pv2Z2 are Gram negative (ami- Fig. 1 Neighbour-joining tree, based on 16S rRNA gene sequences nopeptidase positive) and aerobic (catalase and oxidase showing the phylogenetic position of strain Pv2Z2 among recognized members of the genus Ochrobactrum and other related species of the positive), and they appear as highly motile, short rods family Brucellaceae. Bootstrap values of >50 %, based on 1,000 replica- commonly observed as single cells under the light micro- tions, are shown at branch points. The tree was rooted with scope. Growth occurs on LB, YEM, nutrient agar (NA) Pseudochrobactrum asaccharolyticum CCUG 46016 (accession no. and TY media within 10–12 hat28°C, resultinginlight- AM180485). Bar 0.01 substitutions per nucleotide position 1802 Ann Microbiol (2014) 64:1797–1806 that strain Pv2Z2 produces the AHLs 3O-C7-HSL and 3OH- C7-HSL. Plant inoculation assay The inoculation of common bean with Pv2Z2 resulted in increased plant weight, plant height and N uptake as compared to the non-inoculated plants (Fig. 5). Biodegradation assays The strain Pv2Z2 was able to grow on MSM containing Fig. 2 Representative random amplified polymorphic DNA profile of 100 mg/L of each of the phenols 2-bromophenol, 2,4- strain Pv2Z2 and some type strains of genus Ochrobactrum generated with primer OPC-15. Lanes: M DNA marker (1 kb/100 bp), 1 O. ciceri, 2 diamino phenol hydrochloride, 3,4-dimethoxy benzyl alco- T T O. tritici LAIII106, 3O. tritici ScII24 , 4 O. lupini LUP-21 , 5 O. oryzae hol and 4-methoxy benzyl alcohol, demonstrating that it was T T MTCC4195 , 6, 7 O. gallinifaecis ISO196 , 8O. intermedium DSM T T able to utilize these substrates as carbon sources, forming 17986 , 9 O. anthropi DSM 6882 , 10 Ochrobactrum sp. PV Z 2 2 populations of >10 cells per millilitre (Table 2). Maximum and minimum growth was observed in phenol (8.88 CFU) and 3,4-dimethoxy benzyl alcohol (7.53 CFU), respectively. anthropi. The RAPD results (Fig. 2) further validated the Strain Pv2Z2 was unable to grow in MSM containing 4- genetic differences between strains PV2Z2 and O. anthropi nitrophenol and 2 (5-bromo-2-pyridylazo)-5-diethyl amino LMG3331 . phenol. Within 12 days, it degraded 94 % of 100 mg/L phenol (Fig. 6). Bioassay for beneficial plant traits Discussion After 7 days of incubation a clear halo zone around the bacterial growth spot was clearly observed, showing the sol- The beneficial effects of the rhizosphere microbiome have ubilization of zinc oxide (Fig. 3a) and inorganic phosphate been known about for some time, but the wide-spread use of (Fig. 3b). The quantification data of these and other beneficial microbes as a crop supplement and bioremediation has in- planttraitsare giveninTable 2. No antagonistic activity creased in recent years due to the deleterious effects of chem- was observed against Fusarium solani, F. oxysporum and ical fertilizers/pesticides on the environment and non-target F. moniliforme. organisms, the increasing costs of chemical fertilizers and the AHL assay and RP-TLC massive increase in environmental pollution and contaminat- ed water due to rapid industrialization (Agrawal et al. 2010). The production of a violet colour in the TY/LB media detected Beneficial bacteria generally include members of the genera with the biosensor strain after overnight incubation demon- Bacillus, Pseudomonas, Azospirillum, Azotobacter, Serratia, strated the production of AHL molecules in the medium Enterobacter, Arthrobacter, Burkholderia and Ochrobactrum (Fig. 3c). These AHLs after extraction with ethyl acetate were (Lugtenberg and Kamilova 2009; Bhattacharyya and Jha analysed by RP-TLC, and the chromatogram (Fig. 4)showed 2012). These bacteria have been reported to act as plant Fig. 3 Solubilization of zinc oxide (a), tri-calcium phosphate (b) and the production of N-acyl homoserine lactone (AHL) molecules (c)by Ochrobactrum sp. Pv2Z2 in vitro Ann Microbiol (2014) 64:1797–1806 1803 Table 2 Plant growth promoting and biodegradation potential of strain PV2Z2 Beneficial plant traits Growth in the MSM-containing substrate Trait/character Values Substrate/compound Colony-forming units −1 Indole-3-acetic acid production 6.68 μgmL Phenol 8.88 Solubilization of inorganic phosphate 25.7 μg mL 2-Bromophenol 8.17 Production of AHL signal molecule + 2,4-diaminophenol dihydrochloride 8.25 Solubilization of zinc oxide + 4-Nitrophenol 0 Solubilization of zinc sulphate + 2 (5-Bromo-2-pyridylazo)-5-diethyl amino phenol 0 Antagonistic activity - 3,4-Dimethoxybenzyl alcohol 7.53 4-Methoxybenzyl alcohol 7.82 MSM, Minimal salts medium; AHL, N-acyl homoserine lactone -, The reaction/test is negative; +, the reaction/test is positive growth-promoting factors (Hafeez et al. 2006;Kantar et al. biodegradation or bioremediation (Smejkal et al. 2003;Afzal 2007; Mirza et al. 2007; Bhattacharyya and Jha 2012)and in et al. 2007; Anwar et al. 2009). In the study reported here we describe the taxonomic affiliation of a common bean nodule isolate Pv2Z2 and dem- onstrate its beneficial plant traits and biodegradation traits as well as its likely contribution in promoting plant growth. The strain was found to be metabolically diverse, to utilize a number of carbon sources and substrates, to be resistant to different antibiotics and to show the presence of different enzymes. These characteristics show the ecological fitness of the strain in a range of environments. Strain Pv2Z2 was isolated from sterilized nodules, indicating its endophytic nature. Its colony morphology was similar to that of other rhizobia, and nodulation assays were performed to confirm its nodulation ability. The Ochrobactrum strain Pv2Z2 induced nodulation in plant inoculation assay on common bean and -1 - showed ARA activity (3.34 μmol of ethylene g h 1). How- ever we were unable to confirm the identity of the endophyte to our inoculated strain using in situ hybridization or other molecular techniques. The improved N contents in common Fig. 5 Effect of inoculation with Ochrobactrum sp. Pv2Z2 on common -1 bean in growth pouches. Nitrogen uptake is given in units of mg g shoot Fig. 4 Thin-layer chromatogram of AHL extracts of Ochrobactrum sp. dry weight. The results are the average of three replicates. Un-inoculated Pv2Z2 compared with reference strain Rhizobium leguminosarum (A34). control contained sterile water. The concentration of the N+control was HSL Homoserine lactone 1 M potassium nitrate 1804 Ann Microbiol (2014) 64:1797–1806 Fig. 6 Degradation of phenol in comparison with the growth [optical density (OD)] of Ochrobactrum sp. Pv2Z2 bean plants inoculated with the Pv2Z2 may be attributed to its physiological phenomena. Moreover, the ability to produce N-fixing ability. AHL will help in rhizosphere colonization and might give this The Pv2Z2 strain was isolated from the soil of Zanzibar, strain a competitive advantage under field conditions. To date, Tanzania, where crop productivity is usually low due to soil no member of genus Ochrobactrum has been reported to acidity and moisture stress. Cell and colony morphology contain AHL production activity, although it does have the features, biochemical reactions and the antibiotic resistance activity to produce quorum quenching lactonase enzymes. pattern showed a maximum relatedness with genus This Ochrobactrum sp. was isolated from potato rhizosphere Ochrobactrum. The 16S rRNA sequence analysis also sup- and produces lactonase enzyme which is able to disrupt the ported the genus affiliation. The strain exhibited maximum signals of potato pathogen Erwinia carotovora (Jafra et al. similarities (99 %) with Ochrobactrum anthropi strains CLM6 2006). Following separation by RP-TLC, the crude AHL (AJ867292), CLM5 (AJ867291) and SAIII104 (AJ867290). extract showed the presence of 3O-C7-HSL and 3OH-C7- All of these strains showing maximum similarities to HSL, but the exact mass and nature of these compounds will Ochrobactrum strain Pv2Z2 are isolates from the wheat rhi- remain questionable unless confirmed through detailed mass zosphere (Lebuhn et al. 2000). Although phylogenetic analy- spectrometry analysis. sis grouped Pv2Z2 in the same phylogenetic lineage in which The biodegradation study is the most disparate of the the type strain of O. anthropi was present, the nearest phylo- investigations since it does not directly address plant–microbe genetic relative was the nodulating strain O. cytisi rather than interaction but was undertaken because the root exudates the clinical isolate or any pathogenic strain (Fig. 1), suggesting contain a number of phenolic compounds and bacteria contain the non-pathogenic nature of the strain Pv2Z2 which was the ability to utilize these compounds as an energy source. further validated by the RAPD and TP-RAPD assays This ability might give the bacterium an ecological advantage (Fig. 2). Moreover, the plant inoculation data obtained in this in the rhizosphere, which (due to its nutrient richness) works and other studies (Faisal and Hasnain 2004; Chakraborty et al. as “microbial hot-spot” for diverse plant–plant, plant–microbe 2009;Riazetal. 2010) show that O. anthropi or and microbe–microbe interactions. The strain proved itself a O. intermedium can be safely used as PGPR. candidate of choice for the biodegradation of phenol- The production of IAA, solubilization of inorganic contaminated soils. The degradation of phenol by phosphate/zinc and the promotion of growth of common bean Ochrobactrum species (Lechner et al. 1995;El-Sayed etal. plants in the pot experiment as well as the significant increase 2003) has been reported to be fourfold higher than that of in N uptake by plants suggest that the strain can be a potential other reported phenol-degrading bacteria. The known phenol- PGPR candidate for field application. The production of AHL degrading Ochrobactrum species include both those isolated was found to be a novel trait that had not earlier been reported from activated sludge and contaminated industrial environ- for the members of this genus. N2 fixation, phosphate solubi- ments or the plant-associated O. tritici NBRC 102585, lization and biocontrol activities of bacteria are known to be O. grignonense NBRC 102586, and O. lupini NBRC controlled by the AHL signal molecules. The varied charac- 102587 (Yamada et al. 2008). Phenol degradation by nodule ters of this strain suggest that its quorum sensing signals might bacteria, such as Rhizobium leguminosarum bv trifolli, has be involved in all of these processes as well as in many other been reported (Parke et al. 1991), as has the degradation of Ann Microbiol (2014) 64:1797–1806 1805 Amer MM (2008) Monitoring of Variation among Faba Bean Rhizobium brominated phenols by plant-associated Ochrobactrum sp. Isolates: 2. Biodegradation of Herbicide, 3(3,4 Dichlorophenyl) -1- TB01, O. tritici NBRC102585, O. grignonense NBRC methoxy-1-methylurea. Aust J Basic Appl Sci 2(3):540–548 102586 and O. lupini NBRC 102587 (Yamada et al. 2008). An D, Danhorn T, Fuqua C, Parsek MR (2006) Quorum sensing and These studies show that this phenol-degradation phenomenon motility mediate interactions between Pseudomonas aeruginosa and Agrobacterium tumefaciens in biofilm cocultures. Proc Natl Acad is wide-spread in the genus. The Pv2Z2 strain was, however, Sci USA 103(10):3828–3833 not able to grow in the medium containing 4-nitrophenol and Antunes LC, Ferreira RB (2009) Intercellular communication in bacteria. 2-(5-bromo-2-pyridylazo)-5-diethyl amino phenol. One pos- Crit Rev Microbiol 35(2):69–80 sible explanation for the failure of PV2Z2 to grown on Anwar S, Liaquat F, Khan QM, Khalid ZM, Iqbal S (2009) Biodegradation of chlorpyrifos and its hydrolysis product 3,5,6- nitrophenol-supplemented medium might be the unfavourable trichloro-2-pyridinol by Bacillus pumilus strain C2A1. J Hazard conditions, as p-nitrophenol degradation has been shown to be Mater 168(1):400–405 accelerated at alkaline pH (8–10) and following the addition Asad S, Malik KA, Hafeez FY (1991) Competition between inoculated of organic N (i.e. yeast extract and peptone; Qiu et al. 2007). and indigenous Rhizobium/Bradyrhizobium spp. strains for nodula- tion of grain and fodder legumes in Pakistan. Biol Fertil Soils 12: The ability of plant-associated bacteria to degrade aromatic 107–111 compounds allows these microorganisms to utilize multiple Beringer JE (1974) R factor transfer in Rhizobium leguminosarum. J Gen energy sources, thereby improving their ecological success in Microbiol 84:188–198 the rhizosphere. Bhattacharyya PN, Jha DK (2012) Plant growth-promoting rhizobacteria (PGPR): emergence in agriculture. World J Microbiol Biotechnol Ochrobactrum species are widespread and frequent inhab- 28:1327–1350 itants of the rhizosphere and other environmental habitats. The Cavalcante VA, Döbereiner J (1988) A new acid-tolerant nitrogen fixing results presented here have implications for using bacterium associated with sugarcane. Plant Soil 108:23–31 Ochrobactrum strain Pv2Z2 as biofertilizer and in biodegra- Chakraborty U, Chakraborty BN, Basnet M, Chakraborty AP (2009) Evaluation of Ochrobactrum anthropi TRS-2 and its talc based dation. The strain Pv2Z2 was isolated from the root nodule of formulation for enhancement of growth of tea plants and manage- common bean; it is genetically different from clinical/ ment of brown root rot disease. J Appl Microbiol 107:625–634 pathogenic O. anthropi/intermedium isolates and has positive Chen WM, Chang JS, Wu CH, Chang SC (2004) Characterization of effect on plant growth. Therefore, its use as plant and/or soil phenol and trichloroethene degradation by the rhizobium Ralstonia taiwanensis. Res Microbiol 155(8):672–680 inoculum is not expected to cause any human pathogenic Compant S, Duffy B, Nowak J, Clement C, Barka EA (2005) Use of plant consequences. In this study, we report for the first time a growth-promoting bacteria for biocontrol of plant diseases: princi- new strain of Ochrobactrum sp. Pv2Z2 isolated from the ples, mechanisms of action, and future prospects. Appl Environ common bean nodules with the ability to produce AHL Microbiol 71(9):4951–4959 Czajkowski R, Krzyżanowska D, Karczewska J, Atkinson S, Przysowa J, molecules. Lojkowska E, Williams P, Jafra S (2011) Inactivation of AHLs by Ochrobactrum sp. A44 depends on the activity of a novel class of AHL acylase. Environ Microbiol Rep 3(1):59–68 Acknowledgements The authors are grateful to Dr. Allan J. Dowine El-Sayed W, Ibrahim MK, Abu-Shady M, El-Beih F, Ohmura N, Saiki H, (JIC. Norwich, UK) for providing reference and indicator strains for the Ando A (2003) Isolation and identification of a novel strain of the AHL assay and to Dr. Erko Stackebrandt (DSMZ, Germany) for provid- genus Ochrobactrum with phenol-degrading activity. J Biosci ing the DNA of O. anthropi and other type strains of the genus. The Bioeng 96(3):310–312 research work was partially supported by IDB-BIRCEN Project on “Use Faisal M, Hasnain S (2004) Comparative study of Cr(VI) uptake and of Biofertilizers for Increasing Sustainable Crop Production in Develop- reduction in industrial effluent by Ochrobactrum intermedium and ing Countries: Establishment of a Biofertilizer Resource Center Brevibacterium sp. Biotechnol Lett 26:1623–1628 (BIRCEN) at NIBGE” (2000–2005). Feng YS, Lee CM (2009) The potential of the acetonitrile biodegradation by Mesorhizobium sp. F28. J Hazard Mater 164(2–3):646–650 Frassinetti S, Setti L, Corti A, Farrinelli P, Montevecchi P, Vallini G (1998) Biodegradation of dibenzothiophene by a nodulating isolate of Rhizobium meliloti. Can J Microbiol 44(3):289–297 References Garnett TP, Conn VM, Kaiser BN (2009) Root based approaches to improving nitrogen use efficiency in plants. Plant Cell Environ 32: 1272–1283 Abbas A, Adams C, Scully N, Glennon J, O'Gara F (2007) A role for Gordon SA, Weber RP (1951) Colorimetric estimation of indole acetic TonB1 in biofilm formation and quorum sensing in Pseudomonas acid. Plant Physiol 26:192–195 aeruginosa. FEMS Microbiol Lett 274(2):269–278 Greenburg AE, Trussell RL, Clesceri LS (1985) Standard menthods for Adesemoye AO, Torbert HA, Kloepper JW (2009) Plant Growth- the examination of water and waste water, 16th edn. American Promoting Rhizobacteria allow reduced application rates of chemi- Public Health Association, Washington, DC cal fertilizers. Microbiol Ecol 58:921–929 Hafeez FY, Naeem F, Naeem R, Zaidi AH, Malik KA (2005) Symbiotic Afzal M, Iqbal S, Rauf S, Khalid ZM (2007) Characteristics of phenol effectiveness and bacteriocin production by Rhizobium biodegradation in saline solutions by monocultures of Pseudomonas leguminosarum bv. Viciae isolated from agriculture soils in aeruginosa and Pseudomonas pseudomallei. J Hazard Mater Faisalabad. Environ Exp Bot 54:142–147 149(1):60–66 Hafeez FY, Yasmin S, Airan D, Rahman M, Zafar Y, Malik KA (2006) Agrawal A, Pandey RS, Sharma B (2010) Water pollution with special Plant growth-promoting bacteria as biofertilizer. Agron Sustain Dev reference to pesticide contamination in India. J Water Res Protect 2: 26:143–150 432–448 1806 Ann Microbiol (2014) 64:1797–1806 Imran A, Hafeez FY, Fruhling A, Schumann P, Malik KA, Stackebrandt E Seo JS, Keum YS, Harada RM, Li QX (2007) Isolation and characterization (2010) Ochrobactrum ciceri sp. nov., isolated from nodules of Cicer of bacteria capable of degrading polycyclic aromatic hydrocarbons arietinum. Int J Syst Evol Microbiol 60(7):1548–1553 (PAHs) and organophosphorus pesticides from PAH-contaminated Jafra S, Przysowa J, Czajkowski R, Michta A, Garbeva P, van der Wolf soil in Hilo, Hawaii. J Agric Food Chem 55(14):5383–5389 JM (2006) Detection and characterization of bacteria from the potato Shah NH, Hafeez FY, Asad S, Hussain A, Malik KA (1995) Isolation and rhizosphere degrading N-acyl-homoserine lactone. Can J Microbiol characterization of indigenous Rhizobium leguminosarum bv viciae 52(10):1006–10015 nodulating Lens culinaris Medik. from four Pakistani soils. In: Kantar F, Hafeez FY, Shivakumar BG, Sundaram SP, Tejera NA, Aslam Malik KA, Nasim A, Khalid AM (eds) Biotechnology for sustain- A, Bano A, Raja P (2007) Chickpea: Rhizobium management and able development. NIBGE, Faisalabad, pp 211–219 nitrogen fixation. In: Yadav SS, Redden R, Chen W, and Sharma B Smejkal CW, Seymour FA, Burton SK, Lappin-Scott HM (2003) (eds) Chickpea breeding and management. Cormwell Press, Characterisation of bacterial cultures enriched on the Trowbridge, pp 179–192 chlorophenoxyalkanoic acid herbicides 4-(2,4-dichlorophenoxy) Lebuhn M, Achouak W, Schloter M, Berge O, Meier H, Barakat M, butyric acid and 4-(4-chloro-2-methylphenoxy) butyric acid. J Ind Hartmann A, Heulin T (2000) Taxonomic characterization of Microbiol Biotechnol 30(9):561–567 Ochrobactrum sp. isolates from soil samples and wheat roots, and Somasegaran P, Hoben HJ (1994) Handbook for Rhizobia Methods in description of Ochrobactrum tritici sp. nov. and Ochrobactrum Legume–Rhizobium Technology. Springer, Heidelberg grignonense sp. nov. Int J Syst Evol Microbiol 50:2207–2223 Steinshamn H, Thuen E, Bleken MA, Brenoe UT, Ekerholt G, Yri C Lechner U, Baumbach R, Becker D, Kitunen V, Auling G, Salkinoja- (2004) Utilization of nitrogen (N) and phosphorus (P) in an organic Salonen M (1995) Degradation of 4-chloro-2-methylphenol by an dairy farming system in Norway. Agric Ecosyst Environ 104:509– activated sludge isolate and its taxonomic description. 522 Biodegradation 6(2):83–92 Taminiau B, Daykin M, Swift S, Boschiroli ML, Tibor A, Lestrate P, Lugtenberg B, Kamilova F (2009) Plant-growth-promoting rhizobacteria. Bolle XD, O'Callaghan D, Williams P, Letesson JJ (2002) Annu Rev Microbiol 63:541–556 Identification of a quorum-sensing signal molecule in the facultative Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A labora- intracellular pathogen Brucella melitensis. Infect Immun 70(6): tory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor 3004–3011 McLean RJ, Pierson LS 3rd, Fuqua C (2004) A simple screening protocol Tan ZY, Xu XD, Wang ET, Gao JL, Martinez-Romero E, Chen WX for the identification of quorum signal antagonists. J Microbiol (1997) Phylogenetic and genetic relationships of Mesorhizobium Methods 58(3):351–360 tianshanense and related rhizobia. Int J Syst Bacteriol 47(3):874– Mirza BS, Mirza MS, Bano A, Malik KA (2007) Inoculation of chickpea 879 with Rhizobium isolates from roots and nodules and phytohormone Tien TM, Gaskins MH, Hubbell DH (1979) Plant Ggrowth substances producing Enterobacter strains. Aust J Exp Agric 47:1008–1015 produced by Azospirillum brasilense and their effect on the growth Murphy J, Riley JP (1962) A modified single solution method for the of pearlmillet(Pennisetum americanum L.). Appl Environ determination of phosphate in natural waters. Anal Chim Acta 27: Microbiol 37(5):1016–1024 31–36 Tilman D, Cassman KG, Matson PA, Naylor R, Polasky S (2002) Parke D, Rynne F, Glenn A (1991) Regulation of phenolic catabolism in Agricultural sustainability and intensive production practices. Rhizobium leguminosarum biovar trifolii. J Bacteriol 173(17):5546– Nature 418:671–677 5550 Vincent JM, Humphrey B (1970) Taxonomically significant group anti- Pikovskaia RI (1948) Metabolism of phosphorous in soil in connection with gens in Rhizobium. J Gen Microbiol 63(3):379–382 vital activity of some microbial species. Microbiology 17:362–370 Wei G, Yu J, Zhu Y, Chen W, Wang L (2008) Characterization of phenol Qiu X, Zhonga Q, Lia M, Baia W, Li B (2007) Biodegradation of P- degradation by Rhizobium sp. CCNWTB 701 isolated from nitrophenol by methyl parathion-degrading Ochrobactrum sp. B2. Astragalus chrysopterus in mining tailing region. J Hazard Mater Int Biodeterior Biodegrad 59:297–301 151(1):111–117 Radwan SS, Dashti N, El-Nemr I, Khanafer M (2007) Hydrocarbon Williams JGK, Kubelik AR, Livak KJ, Rafalski JA (1990) DNA poly- utilization by nodule bacteria and plant growth-promoting morphisms amplified by arbitrary primers are useful as genetic rhizobacteria. Int J Phytoremed 9(6):475–486 markers. Nucleic Acids Res 18:6531–6535 Riaz S, Faisal M, Hasnain S (2010) Cicer arietinum growth promotion by Yamada T, Takahama Y, Yamada Y (2008) Biodegradation of 2,4,6- Ochrobactrum intermedium and Bacillus cereus in the presence of tribromophenol by Ochrobactrum sp. strain TB01. Biosci CrCl and K CrO . Ann Microbiol 60:729–733 Biotechnol Biochem 72(5):1264–1271 3 2 4 Sakthivel N, Gnanamanickam SS (1986) Toxicity of Pseudomonas Yoshida S, Forno DA, Cock JH, Gomez KA (1976) Laboratory manual fluorescens towards rice sheath rot pathogen Acrocylindrium oryzae. for physiological studies of rice, 3rd edn. The International Rice Curr Sci 5:106–107 Research Institute, Manila

Journal

Annals of Microbiology – Springer Journals

Published: Feb 19, 2014

There are no references for this article.