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/lp/springer-journals/characterization-of-as-iii-oxidizing-achromobacter-sp-strain-n2-0BF96QcLdi
- Publisher
- Springer Journals
- Copyright
- Copyright © 2018 by Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan
- Subject
- Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
- ISSN
- 1590-4261
- eISSN
- 1869-2044
- DOI
- 10.1007/s13213-018-1338-y
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- See Article on Publisher Site
Abstract
Achromobacter sp. strain N2 was isolated from a pyrite-cinder-contaminated soil and presented plant growth promoting traits (ACC deaminase activity, production of indole-3-acetic and jasmonic acids, siderophores secretion, and phosphate solubilization) and arsenic transformation abilities. Achromobacter sp. strain N2 was resistant to different metals and metalloids, including arsenate (100 mM) and arsenite (5 mM). The strain was resistant to ionic stressors (i.e., arsenate and NaCl), whereas bacterial −1 growth was impaired by osmotic stress. Strain N2 was able to oxidize 1.0 mmol L of arsenite to arsenate in 72 h. This evidence was supported by the retrieval of an arsenite oxidase AioA gene highly homologous to arsenite oxidases of Achromobacter and Alcaligenes species. Rice seeds of Oryza sativa (var. Loto) were bio-primed with ACCD-induced and non-induced cells in order to evaluate the effect of inoculation on rice seedlings growth and arsenic uptake. The bacterization with ACCD-induced cells significantly improved seed germination and seedling heights if compared with the seeds inoculated with non-induced cells and non-primed seeds. Enhanced arsenic uptake was evidenced in the presence of ACCD-induced cells, suggesting a role of ACCD activity on the mitigation of the toxicity of arsenic accumulated by the plant. This kind of responses should be taken into account when proposing PGP strains for improving plant growth in arsenic-rich soils. . . . . Keywords Arsenic Arsenite oxidase Plantgrowthpromotion ACC deaminase Rice Introduction carboxylic acid deaminase (ACCD) leads to a direct stimula- tion of plant growth due to the catabolism of molecules related Environmental stresses such as contaminants, drought and to the stress hormone ethylene (Glick et al. 1998). Under salinity are some of the limiting factors in crop production metal-stress conditions, IAA and ethylene are released due to their effect on plant functioning. Soil bacteria having resulting in an increased uptake of metal ions (Dell’Amico plant growth promoting (PGP) characteristics might be envis- et al. 2005). However, the knowledge on plants hormone pro- aged as enhancers for plant resistance to abiotic and biotic duction by bacteria under stress conditions is still very limited. (phytopathogens) stresses. Besides biological nitrogen fixa- In rice fields, arsenic (As) contamination represents a pub- tion, important direct PGP mechanisms are: release of bacte- lic health issue, due to cultivation under flooded conditions rial siderophores that supply iron or phosphate to the plant with As-contaminated water in many parts of the world (Zhao (Burd et al. 2000; Abou-Shanab et al. 2003; Madhaiyan et al. 2010). Such agronomic scheme increments As mobility et al. 2004), synthesis of phytohormons, such as indole-3- in soil solution resulting in higher As concentration in rice acetic acid (IAA) and jasmonic acid (JA), and degradation grains with respect to dry rice (Spanu et al. 2012). In such of stress-related molecules. The expression in many plant- anoxic conditions, the predominant As species is arsenite associated bacteria of the enzyme 1-aminocyclopropane-1- [As(III)], more mobile and toxic than arsenate [As(V)]. Italy is the first rice producer in Europe. Here, the cultivation of rice under flooded conditions in soils with natural As levels (i.e., −1 * Lucia Cavalca tot As < 20 mg kgdw ) (Mandal et al. 2002) leads to an lucia.cavalca@unimi.it average metalloid concentration in grains that exceeds the European limits (Commission regulation (EU) 2015/1006) Department of Food, Environmental and Nutritional −1 of 100 μgkg for baby food production (Meharg et al. Sciences—DeFENS, Università degli Studi di Milano, Via Celoria 2, 2009). Bacteria play a crucial role in As geochemical cycling 20133 Milan, Italy 296 Ann Microbiol (2018) 68:295–304 through microbial transformation processes, including reduc- was cultured first in 1/10 TSB medium until mid-exponential tion, oxidation, and methylation (Cavalca et al. 2013). phase and 5% (v/v) of inoculum was transferred in DF medi- Recently, the role of As(III) oxidizing rhizobacteria in lower- um containing 3.0 mM ACC (DF_ACC, Sigma–Aldrich) in- ing As content in rice plant and in the relief of As toxicity has stead of (NH ) SO as N source (Grichko et al. 2000). The 4 2 4 been revealed (Yang et al. 2015). The presence of As(III) culture was incubated at 30 °C on a rotary shaker at 180 rpm oxidizing bacteria on rice roots iron plaques has been found for 48 h. The ability to utilize ACC was verified by inoculat- to be correlated with As content in the plant. Since As(V) is ing the strain in control tubes containing DF medium without bound to iron minerals, As(III) oxidizing bacteria were active- any N source, and incubating the tubes in the above- ly catalyzing As transformation and greatly influencing metal mentioned conditions for 10 days. uptake by rice (Hu et al. 2015). Siderophore secretion was determined as described by In this context, the aim of this study was to characterize Schwyn and Neilands (1987) using blue agar plates contain- PGP traits and As transformation abilities of a rhizosphere ing Chrome azurol S dye (CAS, Sigma–Aldrich). Orange bacterial strain isolated from a pyrite-cinders contaminated halos around the colonies after 5 days incubation at 30 °C soil and to evaluate the effect of As on PGP characteristics. on blue agar were indicative of siderophore secretion. The influence of strain inoculation was evaluated in relation to The mineral P-solubilizing ability of the strain was assayed the growth of rice seedling and As sensitivity. on agar plates containing insoluble Ca (PO ) according to 3 4 2 Goldstein and Liu (1987). Strain N2 was streaked on agar −1 medium (pH 7.2) containing glucose 10 g L ,NH Cl −1 −1 . −1 Materials and methods 5g L ,NaCl1 gL ,MgSO 7H O1gL ,and 4 2 −1 Ca (PO ) 5g L . The plates were incubated at 30 °C for 3 4 2 Bacterial strain N2 5 days. The development of a clear zone around the colonies was indicative of P-solubilizing activity. The bacterial strain N2 was isolated from sunflower Proteolytic activity was tested by inoculation of the strain −1 (Helianthus annuus, L.) rhizosphere grown in an As pyrite- into skim milk agar medium containing 5 g L pancreatic −1 −1 cinder polluted soil (Torviscosa, Italy). The strain was identi- digest of casein, 2.5 g L yeast extract, 1 g L glucose, 15 g −1 fied by 16S rRNA nucleotide sequence analysis and preserved agar, and 100 ml L of 7% skim milk solution. A clear zone in glycerol stocks at − 80 °C. Prior to use, the strain was grown around the cells on plates incubated at 30 °C for 3 days indi- to mid-exponential phase in 1/10 Tryptic Soy Broth (TSB) cated positive proteolytic activity (Smibert and Krieg 1994). medium at 30 °C with shaking. Chitinase activity was tested by streaking the strain on M9 −1 chitin agar medium containing 1.62 g L nutrient broth, −1 −1 −1 Determination of plant growth promotion traits 0.5gL NaCl, 8 g L colloidal chitin and 15 g L agar (Kunz and Chapman 1981). The formation of clear halos The isolate was qualitatively tested for its ability to produce 3- around the bacterial growth after 7 days incubation at 30 °C indoleacetic acid (IAA), abscisic acid (ABA) and jasmonic indicated positive chitinase activity (Sahoo et al. 1999). acid (JA), growth on 1-aminocyclopropane-1-carboxylic Biocontrol activity of strain N2 against Botrytis cinerea (ACC) as the sole source of nitrogen, siderophores secretion, ss177v and B. cinerea ss140t was determined by inoculating phosphate mineralization, protein and chitin hydrolyzation, fungal mycelium and strain N2 on plates containing CYA and as biocontrol agent. medium (Barka et al. 2002). After 7 days of incubation at IAA production was estimated according to Glickmann 30 °C, the growth of mycelium was measured and compared and Dessaux (1995). The strain was cultured for 4 days at to that obtained without bacterial inoculation (control). 30 °C in flasks containing 20 mL of Dworkin and Foster All tests were performed in triplicate and repeated three (DF) mineral medium (Dworkin and Foster 1958) supple- times. All solutions were prepared with MilliQ water, steril- −1 mented with 0.5 g L of L-tryptophan (Sigma-Aldrich). ized by membrane filtration (0.22 μm pore size, Millipore, After incubation, 1 mL of cell suspension was transferred into Merck), and stored at 4 °C in the dark. All chemicals were a tube and mixed vigorously with 2 mL of Salkowski’sre- of the highest purity available. agent and left at room temperature for 20 min. Development of pink color indicated IAA production. JA and ABA produc- Metal resistance tion was determined by Ultra performance liquid chromatog- raphy—tandem mass spectrometry (UPLC-MS/MS, Waters, The strain was tested for its resistance to antimony (Sb) as Milford, MA) after growing strain N2 for 72 h at 30 °C in SbCl , As(III) as (NaAsO ), As(V) as (Na HAsO x7H O), 3 2 2 4 2 yeast mannitol medium (YEM). cadmium (Cd) as CdCl , copper (Cu) as CuCl , chromium 2 2 The ability to use ACC as nitrogen source is a consequence (Cr) as K Cr O , nickel (Ni) as NiCl , and zinc (Zn) as 2 2 7 2 of the enzymatic activity of ACCD. To test this trait, strain N2 ZnSO ,(Sigma–Aldrich, St. Louis, MO, USA). 4 Ann Microbiol (2018) 68:295–304 297 The growth of strain N2 was determined in liquid Tris Arsenic transformation of strain N2 mineral medium (TMM) at low phosphate content −1 (0.12gL of Na HPO ) to avoid metal precipitation The ability of strain N2 to oxidize As(III) or to reduce As(V) 2 4 (Sadouk and Mergeay 1993) supplemented with a range of was tested both in growing cells or and resting cells. concentrations of the different metals, added separately. Cells grown to mid-exponential phase (growing cells) were Gluconate (0.6% w/v) was used as the carbon source in inoculated into three flasks containing 20 mL of TMMG with −1 TMM medium (TMMG) and the pH was adjusted to 7.0. either 1 mmol L As(V) or As(III) to obtain an initial Triplicate 100 mL flasks were inoculated with 1 mL of an OD of about 0.05. In a parallel experiment, As transfor- 600nm overnight culture of the strain grown on TMMG. Flasks were mation capability was tested as ACCD-induced and incubated for 5 days at 30 °C and the growth was measured as uninduced growing cells by inoculating cells grown to mid- optical density at 600 nm (OD ). exponential phase in DF and DF_ACC with either 600nm −1 0.05 mmol L As(V) or As(III). Control flasks of TMMG, Osmotic and ion toxicity tests DF, or DF_ACC without inoculum were incubated to check abiotic transformation of As. Flasks without As were inocu- To differentiate osmotic from ion toxicity effects, experiments lated to compare the growth of the microorganisms in the were conducted under an osmotic potential Ψ = − 1.5 MPa absence of As(V) or As(III). Three replicates per treatment using three different sources. To achieve this osmotic poten- were performed. All flasks were incubated at 30 °C under −1 −1 tial, 175 mmol L of sodium As(V), 400 mmol L of NaCl shaking at 150 rpm. After 72 h of incubation, aliquots of and 26% (w/v) polyethylene glycol 6000 (PEG 6000) were controls and cell suspensions were sampled to measure cell separately added to Luria Bertani (LB) medium (Sosa et al. growth by OD and to determine total As, As(V), and 600 nm 2005). Cells of N2 strain were grown at 30 °C with 150 rpm As(III) by Inductively Coupled Plasma Mass Spectrometry shaking for 24 h and inoculated in triplicate in the media. (ICP-MS, Agilent Technologies, USA) analysis as described Bacterial growth was measured at successive incubation times below. (0–30 h) after inoculation using a spectrophotometer at As(III) oxidation and As(V) reduction ability of strain N2 546 nm (Forchetti et al. 2007). The ability of N2 strain to was also tested as As(III)-induced or uninduced resting cells. tolerate different osmotic stresses was compared by calculat- A pre-culture of N2 cells was established in TMMG in pres- ing a resistance index (RI). The RI was defined as the ratio of ence or in the absence of As for 24 h at 30 °C under shaking at the exponential growth rate in the medium with stress to that 150 rpm in the dark. After growth, cells were centrifuged at in the control medium (Huang et al. 2010). The closer RI was 12,857 g at 20 °C for 15 min. The cell pellet was washed three −1 to 1, the smaller was the stress agent toxicity. times with 500 mmol L CaSO solution and resuspended in the same medium. This cell suspension was inoculated in −1 Determination of stress-related phytohormons 60 mL of 500 mmol L CaSO solution supplemented with −1 0.1 mmol L As(III), in order to obtain a final cell optical The production of stress-related phytohormons IAA, JA, and density OD of 0.35, corresponding to about 10 cell 600nm −1 ABA was determined in YEM in the absence and in the pres- mL . ence of As(III) and As(V). In parallel experiments, the strain Resting cell experiment was carried out in triplicate for −1 N2 was inoculated in YEM with L-tryptophan 0.5 g L 48 h under shaking at 150 rpm in the dark at 30 °C. At the (YEMT) to check whether the strain utilized L-tryptophan as end of both growing and resting cells experiments, 10 mL of precursor to produce IAA. Aliquots of a mid-exponential cell suspensions were collected, centrifuged and syringe- phase culture of strain N2 grown in YEM for 72 h were used filtered through 0.22 μm nitrocellulose membranes. Total to inoculate 60 mL of YEM or YEMT (final cell number 10 As, As(III), and As(V) were determined by ICP-MS analysis −1 cells mL ) in the presence and in the absence of As(III) as described below. −1 −1 (3 mmol L ) and As(V) (50 mmol/ L ), separately supple- mented. Three replicates were used for each growth condition. Rice germination tests The flasks were incubated at 30 °C under shaking at 150 rpm for 72 h. Bacterial suspensions were collected and centrifuged Germination tests of rice (Oryza sativa L. var. Loto) seeds at 8000 rpm at 4 °C for 15 min. From each supernatant, 50 mL were conducted in triplicate in 120 mm ∅ Petri dishes con- were acidified with 0.25 mL of 12 M HCl and then extracted taining filter paper moistened with 15 mL of CaSO solution with ethylacetate (2 × 70 mL). The organic phase was dried (Meharg and Hartley-Whitaker 2002) either supplemented under N and the residue dissolved in 0.5 mL of methanol with increasing concentrations of As(III) (0.05, 0.1, and (Merck, Darmstadt, Germany). The solution was centrifuged 0.5 mM) or without As. and 10 μL were injected in an UPLC-MS/MS spectrometer Prior to inoculation with Achromobacter sp. N2, rice seeds and analyzed as described below. were surface-sterilized according to the procedure from 298 Ann Microbiol (2018) 68:295–304 Pandey et al. (2011). Rice seeds were rinsed in 1.5% sodium transition. The source temperature was 130 °C, the hypochlorite solution for 15 min and then washed three times desolvating temperature was 350 °C, and argon was used at −3 with sterile deionized water. Seed sterility was verified by 2.0 × 10 mbar to improve fragmentation in the collision cell. incubating 10 seeds onto LB agar plates at 30 °C for 10 days. Masslinx 4.0 acquired data with Quan-Optimize option for Bacterial cell suspension was prepared by growing strain N2 fragmentation study. The fragmentation transitions for the in 200 mL DF_ACC medium. Cells were harvested by cen- multiple reaction monitoring (MRM) was (m/z) 263→153 − + trifugation (11,000×g for 30 min) and suspended in for ABA, (m/z) 209→59 for ABA, (m/z) 176→130 for 3- −1 500 mmol L CaSO until final OD of 0.35 was IAA, with a dwell time of 0.2 s. 4 600nm 8 −1 achieved (corresponding to about 10 CFU mL ). For inoc- The primary stock solutions of IAA, JA, and ABA −1 ulation, sterile seeds were soaked in 40 mL bacterial suspen- (0.1 mg mL , Sigma-Aldrich) were prepared in methanol sion and gently stirred in the dark at room temperature for 2 h, and diluted to give working solutions in the range of 0.5– −1 after which they were removed from the suspension by using 50 ng mL . All stock solutions and the working solutions sterile pliers. Twenty inoculated seeds were added to three were stored at – 80 °C and − 20 °C, respectively. Petri dishes with or without 0.1 mM As(III), whereas addition of uninoculated seeds was used as control. Petri dishes were ICP-MS incubated at 28 °C per 10 days (3 days in the dark and 7 days in the light) according to Pandey et al. (2011). After 10 days of Arsenic species in the samples were determined by ICP-MS incubation, the percentage of germination, the root length, analysis according to Kim et al. (2007). Specifically, total As seedling dry weight (7 days at 100 °C), and height were was determined in 5 mL of the sample previously acidified measured. with HNO to achieve a final concentration of 2% (v/v). Arsenic content of seedlings was determined in seedling 3 Inorganic As forms were determined in 5 mL of samples biomass by digesting samples (0.5 g) in a mixture of concen- passed through a WATERS Sep-Pak®Plus Acell Plus QMA trated HNO and HClO (4:1, v/v). After digestion, the vol- 3 4 cartridge (Waters). As(V) was retained in the cartridge while ume of each sample was adjusted to 20 mL using deionized allowing As(III) to pass through and collected. The cartridge water. Arsenic content was determined by ICP-MS as speci- was then washed with 0.16 M HNO to extract As(V) from it. fied below. 3 Total As, As(III), and As(V) contents were determined by ICP-MS. Standards of As for concentrations ranging from 0 −1 Analytical methods to 1 mg L were prepared from sodium As(III) NaAsO (Sigma Aldrich, USA) solution. UPLC-MS/MS IAA, ABA, and JA were determined by UPLC-MS/MS. Molecular methods Chromatographic system consisted of an UPLC mod. Acquity (Waters, Milford, MA) coupled to a triple quadrupole Genomic DNA was isolated from strain N2 using the mass spectrometer mod. Quattromicro (Waters). A 1.7 μmC Microbial DNA Extraction Kit (Mo Bio Laboratories Inc., BEH column (150 × 2.1 mm, Waters) was used for separation Carlsbad, CA, USA) according to the manufacturer’sproto- at a flow-rate of 0.5 mL/min. The column was maintained at col. The yield and quality of DNA were analyzed by agarose 50 °C and the separation was performed by means of a linear gel electrophoresis. gradient elution (eluent A, 0.05% acetic acid; eluent B, 0.05% PCR amplification of 16S rRNA and of As genes was acetic acid in acetonitrile, Merck, Darmstadt, Germany). The performed in a final volume of 25 μL containing 10 ng of gradient was as follows: 20 to 60% B in 3 min, and then 60% genomic DNA, 1.5 U of Taq polymerase, 0.4 μM of each B for 1 min. The capillary voltage was set to 3 kV, and the primer, 0.2 mM of dNTPs, 1.75 mM MgCl , and 1× PCR cone voltage and the collision energy was specific for each buffer. All reagents were obtained from Invitrogen. Table 1 Primer pairs used to Target Primer Sequence (5’-3’)Reference amplify As-transforming genes from Achromobacter sp. N2 arsC P52f AGCCAAATGGCAGAAGC-3 Bachate et al. (2009) P323r GCTGGRTCRTCAAATCCCCA arsB darsB1F TGTGGAACATCGTCTGGAAYGCNAC Achour et al. (2007) darsB1R CAGGCCGTACACCACCAGRTACATNCC aioA aoxBM1-2F CCACTTCTGCATCGTGGGNTGYGGNTA Quèmèneur et al. (2008) aoxBM3-2R TGTCGTTGCCCCAGATGADNCCYTTYTC Ann Microbiol (2018) 68:295–304 299 Table 2 Plant growth promotion traits, biocontrol properties and metal An universal primer pair for bacterial 16S rRNA gene was resistance of Achromobacter sp. N2 used: P27f and P1495r (Weisburg et al. 1991). DNA amplifi- cation conditions were initial denaturation at 95 °C for 5 min, Characteristic N2 35 cycles of 95 °C for 1 min, 55 °C for 40 s, 72 °C for 1 min PGP traits 40 s followed by a final extension step at 72 °C for 10 min. Indole acetic acid production + Genes for As(V) reductase (arsC), As(III) efflux pump (arsB) Jasmonic acid production + and As(III) oxidase (aioA) were amplified using the primer Abscissic acid production – pairs listed in Table 1. Growth on ACC + PCR reactions were performed on a T-Gradient Biometra Siderophores production + apparatus (Germany). PCR products were checked on 2% (w/ Mineral-P solubilization + v) agarose gel containing 0.01% (v/v) GelRed™ (Biotium, Proteoliytic activity + CA, USA) and visualized using the GelDoc image analyzer Chitinase activity – system (Biorad, CA, USA). Biocontrol activity Sequence analyses % inhibition of Botrytis cinerea ss 177v 20% % inhibition of Botrytis cinerea ss 140 t 17% −1 Amplified genes were sequenced with the respective As(III) oxidation (1 mmol L)100% −1 primers using the Taq Dye-Deoxy Terminator Cycle Metal resistance (mmol L ) Sequencing kit (Life Technologies Co., USA). The for- As(V) 100 ward and reverse samples were run on a 310A sequence As(III) 5 analyzer (Life Tech-nologies Co., USA). Sequences SbIII 10 were edited and aligned using MEGA software version CuII 5 6 (Tamura et al. 2007), translated into amino acid se- CrVI 1 quences and compared with the entire GenBank data- NiII 0.1 base (BlastX). Obtained amino acid sequences and ref- CdII 0.1 erence sequences were then aligned with ClustalX and ZnII 0.1 trees were built with MEGA6 using the neighbor-joining +, positive; −,negative distance method based on p-distance. A total of 1000 bootstrap replications were calculated. Sequences obtain- also resistant to ionic stress under − 1.5 MPa generated by ed in the present work were deposited to GenBank- −1 −1 400 mmol L NaCl and 175 mmol L of sodium As(V) EMBL databases under the following accession num- (Fig. 1). On the contrary, it was affected by non-ionic osmotic bers: KY344276 and KY293395. stress induced by 26% (w/v) PEG6000. The resistance index Strain N2 was identified as Achromobacter sp. according to (RI) for As(V)-induced stress was 0.7, followed by NaCl and 16S rRNA nucleotide sequence analysis (100% homology to PEG6000 with a mean RI of 0.4. KT992330). Statistical analyses Obtained data were subjected to one-way analysis of variance (ANOVA) with Bonferroni tests using SPSS Statistics for Windows, version 20.0 (IBM Corp., Armonk, NY). All anal- yses were performed at the p < 0.05 level. Results Characterization of Achromobacter sp. strain N2 Metals and metalloid resistance and plant growth promotion Fig. 1 Time course of bacterial growth of Achromobacter sp. strain N2 traits of Achromobacter sp. strain N2 are shown in Table 2. under different hyperosmotic stresses (− 1.5 MPa) (mean ± SD, n =3, Strain N2 was resistant to high concentrations of As(V) where bar is absent, SD is within the point). ○,growthinLB; ■, −1 −1 (100 mM), SbIII (10 mM), As(III) (5 mM) and CuII (5 mM) growth in LB + 400 mmol L NaCl; ♦,growth inLB+175mmolL As(V); ▲, growth in LB + 26% (w/v) PEG6000 and to CrVI, NiII, CdII, and ZnII (Table 2). The isolate was 300 Ann Microbiol (2018) 68:295–304 Achromobacter sp. strain N2 was able to produce IAA and As(V), IAA production increased to 1.85 ± 0.09 and 1.14 ± −1 JA, utilize ACC as the sole N source, secrete siderophores, 0.13 nmol mL , respectively. JA production of strain N2 was −1 solubilize phosphate, and hydrolyse proteins. The strain pro- equal to 0.17 ± 0.03 pmol mL , and it was not affected by the −1 duced 0.068 ± 0.008 nmol mL IAA without the addition of presence of As(III) and As(V), being 0.16 ± 0.02, 0.13 ± −1 −1 L-tryptophan and 0.62 ± 0.08 nmol mL with the addition of 0.01 pmol mL ,respectively. tryptophan, evidencing that the amino acid was utilized as Biocontrol activity of the strain towards B. cinerea ss 177v precursor. In the presence of 3 mM As(III) and of 50 mM and B. cinerea ss 140 t was also evidenced. Fig. 2 Phylogenetic relationships deduced amino acid sequence of method. The bar indicates 2% sequence difference. The sequence of arsenite oxidase gene aioA of Achromobacter sp. Strain N2 (in bold). Hoeflea phototrophica (ZP02167371) was used as an outgroup The evolutionary history was inferred using the Neighbor-Joining Ann Microbiol (2018) 68:295–304 301 Arsenic metabolism and As-related genes The PGP effect of Achromobacter sp. strain N2 was tested by inoculating rice seeds with ACCD-induced and ACCD- Due to the relevance of bacterial metabolism in cycling As in uninduced bacterial cells (Table 4). Although seed germina- plant rhizosphere and in influencing plant growth, the ability tion was not significantly affected by the presence of the of Achromobacter sp. strain N2 to transform inorganic As ACCD-uninduced cells, root length and seedling height of forms was further characterized. samples inoculated with ACCD-induced inoculum were sig- Achromobacter sp. strain N2 completely oxidized nificantly (p < 0.05) higher. This data demonstrated that the −1 1.0 mmol L of As(III) to As(V) in 72 h when incubated in growth conditions of the inoculum had an effect on the dis- chemoorganotrophic condition. The strain did not oxidize placement of the PGP activity and that PGP traits might not be As(III) in chemolitho-autotrophic conditions, indicating that visible in a plant unless induced during inoculum preparation. the oxidation was a detoxification rather than an energy gen- In order to evidence the effect of ACCD activity of erating process. As(III) was oxidized to an extent of 5.4% Achromobacter sp. strain N2 on rice under As pressure, ger- when the strain grew in DF medium, and of 20% when the mination tests were performed with rice seeds inoculated with strain grew in DF_ACC medium. As(III) oxidation did not ACCD-induced and ACCD-uninduced cells in the presence −1 occur with strain N2 resting cells. As(III) 0.1 mmol L . When in the presence of As(III), In accordance with the ability to oxidize As(III), a gene frag- ACCD-induced cells significantly promoted seed germination ment corresponding to AioA was retrieved in strain N2. The (Fig. 3) and seedling height with respect to ACCD-uninduced deduced amino acid sequence of the fragment had 99% homol- cells (Table 5). The specific As content of seedlings was de- ogy to the alpha subunit of As(III) oxidase of different termined after 10 days of incubation (Table 5). In rice inocu- Achromobacter (acc. num. AEL22195 and AOS87742) and lated with ACCD-induced cells, the total As content was sig- Alcaligenes species (acc. num. ABY19322, ABY19321, and nificantly higher than in rice inoculated with ACCD- ADF47192). Phylogenetic analysis of deduced amino acid se- uninduced cells and in the non-inoculated ones. This indicated quences (Fig. 2) indicated that the sequence of strain N2 clustered that As uptake did not impair seedling germination. together with Betaproteobacteria members of Alcaligenaceae family retrieved in different As-contaminated environments. Strain N2 was not able to reduce As(V) in any of the tested Discussion conditions and arsC and arsB genes for As(V) reductase and As(III) efflux pump, respectively, were not detected. In the present work, PGP activities and As metabolism of Achromobacter sp. strain N2 was described and its capability Effect of Achromobacter sp. N2 on rice growth to promote rice growth and As uptake was evidenced. and arsenic uptake The strain was able to oxidize As(III) in chemoorganotrophic growth conditions. This ability was strongly impaired when the The effect of Achromobacter sp. N2 on rice growth and As strain grew in the absence of a nitrogen source or in the presence uptake was determined by germination tests performed at in- of ACC as sole source of nitrogen. The presence of As stimu- creasing concentrations of As(III). lated IAA production in accordance with recent evidences Rice seedling biomass production was significantly (p< −1 (Mendoza-Hernandez et al. 2016). On the contrary, JA, signal- 0.05) affected in the presence of 0.05 and 0.1 mmol L −1 ing compound involved in plant growth, development, and re- As(III) (Table 3). As(III) 0.5 mmol L completely inhibited sponse to (a)biotic stress factors (Cuypers et al. 2011)wasnot seeds germination. Table 4 Response of rice seeds to ACCD-induced Achromobacter sp. Table 3 Response of rice seeds to different As(III) concentrations N2 cells (time of incubation 10 days) As(III) Seeds Root Seedling Seedling Condition Seeds Root Seedling Seedling −1 (mmol L ) germination length height dry weight germination length height dry weight (%) (mm) (mm) (g) (%) (mm) (mm) (g) 0 75 b 50 c 36 b 0.511 b ACCD-induced 73 ns 58 c 41 b 0.364 ns 0.05 32 a 27 b 24 a 0.179 a cells 0.1 23 a 10 a 27 a 0.114 a Uninduced cells 55 40 a 33 a 0.275 0.5 0 ––– Not inoculated 75 50 b 36 ab 0.511 seeds Data followed by the same letter in a column for each treatment do not differ significantly at p < 0.05, as determined using Bonferroni test; n.d., Data followed by the same letter in a column for each treatment do not not determinable differ significantly at p < 0.05, as determined using Bonferroni test 302 Ann Microbiol (2018) 68:295–304 100% Fig. 3 Plant growth-promoting ability of the strain N2 in the absence (black bars) and in the presence of As(III) 0.1 mM (grey bars) as measured in terms of 80% germination percentage. Data ns reported in the bars under different doses of As with same letter are not significantly 60% different (p < 0.05) according to Bonferroni test 40% 20% 0% ACCD-induced ACCD-uninduced Not inoculated ACCD-induced ACCD-uninduced Not inoculated cells cells seeds cells cells seeds affected. The strain was characterized by an As(III) oxidase gene grain and an increment of root As content in the presence of with high degree of amino acid conservation with known As(III) As(III) oxidizing strains. On the contrary, in a molecular ecol- oxidase of Achromobacter and Alcaligenes species. Particularly, ogy study, it was demonstrated that higher number of As(III) the high conserved motif HNRPAYNSE (Quemeneur et al. oxidase genes in rice rhizosphere corresponded to lower As 2008) was present in the amino acid sequence of strain N2. content in plant (Hu et al. 2015). With this regard, a possible Arsenic exposure significantly affected the normal growth and explanation of contrasting results could be the variety of rice development of tested rice seedlings. In accordance with previous used in these studies, as previously evidenced by Yang et al. study conducted in similar conditions (Choudhury et al. 2009), (2015). The detoxification ability of strain N2 did not relay on −1 As toxicity in rice was displayed at 0.5 mmol L As(III). The oxidation of As(III) to As(V), since resting and ACCD- rate of root length inhibition was stronger than seedling height induced cells of Achromobacter sp. strain N2 were impaired inhibition, suggesting that the increment of As has negative ef- in this ability, but more likely on IAA production and ACCD fects on the area of the seedlings directly in contact with the activity that improved seedling development. metalloid (Choudhury et al. 2009;Shrietal. 2009). Total biomass ACCD activity is recognized to protect plants against trace was confirmed to be a less sensitive parameter of As toxicity, in element toxicity when growing on contaminated soils (Glick accordance with Liu et al. (2005) and Williams et al. (2005). 2003). Plants inoculated with bacterial strains carrying ACCD The presence of Achromobacter sp. strain N2 increased As activity can regulate their ethylene levels, thus presenting a content in the seedlings. Arsenic uptake by plants in relation to more extensive root system that leads to enhanced uptake of the presence of As(III) oxidizing strains in rhizosphere is still heavy metals (Arshad et al. 2007) by modification of root debated in the literature. In a recent work, Das et al. (2016) structure (Zhang et al. 2008). In accordance with previous observed a decrement of total As content in rice straw and studies (Rahman et al. 2007; Pandey et al. 2013), seed priming −1 Table 5 Evaluation of rice growth-promotion responses to inoculation of Achromobacter sp. N2 in the presence of 0.1 mmol L As(III) As(III) Condition Seeds Root Seedling Seedling dry Specific As −1 (mmol L ) germination (%) length (mm) height (mm) weight (g) content in seedling −1 (mmol kg d.w.) 0.1 ACCD-induced cells 30 b 16 b 27 b 0.060 a 1.695 d ACCD-uninduced cells 20 a 18 b 24 a 0.106 a 1.141 c Not inoculated seeds 23 a 10 a 25 ab 0.114 a 0.698 b 0 Not inoculated seeds 75 c 50 c 36 c 0.511 a 0.009 a Data followed by the same letter in a column for each treatment do not differ significantly at p < 0.05, as determined using Bonferroni test % seed germination Ann Microbiol (2018) 68:295–304 303 Dell’Amico E, Cavalca L, Andreoni V (2005) Analysis of rhizobacterial with Achromobacter sp. strain N2 increased root growth and communities in perennial graminaceae from polluted water meadow As uptake, thus demonstrating that this PGP trait is involved soil, and screening of metal resistant, potentially plant growth- in plant relief. promoting bacteria. 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Journal
Annals of Microbiology – Springer Journals
Published: Apr 15, 2018