Hindawi Publishing Corporation International Journal of Microbiology Volume 2013, Article ID 831308, 6 pages http://dx.doi.org/10.1155/2013/831308 Research Article Agromyces arachidis sp. nov. Isolated from a Peanut (Arachis hypogaea) Crop Field Chandandeep Kaur, Anil Kumar Pinnaka, Nitin Kumar Singh, Monu Bala, and Shanmugam Mayilraj Microbial Type Culture Collection and Gene Bank (MTCC), CSIR Institute of Microbial Technology (IMTECH), Sector 39-A, Chandigarh 160 036, India Correspondence should be addressed to Shanmugam Mayilraj; mayil@imtech.res.in Received 1 August 2013; Accepted 25 September 2013 Academic Editor: Carla Pruzzo Copyright © 2013 Chandandeep Kaur et al. his is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A Gram-positive, yellowish bacterium strain AK-1T was isolated from soil sample collected from peanut (Arachis hypogaea) crop ield and studied by using a polyphasic approach. he organism had morphological and chemotaxonomic properties consistent with its classiication in the genus Agromyces. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain AK-1T was closely related to Agromyces aurantiacus (98.6%) followed by Agromyces soli (98.3%), Agromyces tropicus (97.6%), Agromyces ulmi (97.3%), Agromyces lavus (97.2%), and Agromyces italicus (97.0%), whereas the sequence similarity values with respect to the other Agromyces species with validly published names were between 95.3 and 96.7%. However, the DNA-DNA hybridization values obtained between strain AK-1T and other related strains were well below the threshold that is required for the proposal of a novel species. he DNA G + C content of the strain is 71.8 mol%. he above data in combination with the phenotypic distinctiveness of AK-1T clearly indicate that the strain represents a novel species, for which the name Agromyces arachidis sp. nov. is proposed. he type strain is AK-1T (=MTCC 10524T = JCM 19251T ). 1. Introduction he genus Agromyces was irst proposed by Gledhill and Casida Jr [1] and later on emended by Zgurskaya et al. [2]. At present, the genus Agromyces comprises 24 species with validly published names (http://www.bacterio.net/a/ agromyces.html), and all these species have been isolated from diferent environmental sources: soils from fertile meadows, rhizosphere, and plants to rock art paintings [1– 18]. In the present study, bacterial strain AK-1T , isolated from soil sample, is described and subjected to the polyphasic taxonomy. 16S rRNA gene sequence comparison revealed that the isolate is Agromyces-like organism. he aim of the present study is to determine the exact taxonomic position of the isolate. 2. Materials and Methods Strain AK-1T was isolated from a soil sample collected from peanut (Arachis hypogaea) crop ield, Srikakulam, Andhra Pradesh, India (18∘ 14� N latitude 83∘ 58� E longitude), by the dilution-plate technique on tryptic soy agar medium (TSA; HiMedia) and maintained as glycerol stocks at −70 ∘ C. he reference strains A. aurantiacus (MTCC 11069T ), A. soli (MTCC 11074T ), A. tropicus (MTCC 11075T ), A. ulmi (MTCC 10783T ), A. lavus (MTCC 11103T ), and A. italicus (MTCC 10784T ) were obtained from the Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Chandigarh, India. Colony and cell morphologies were studied according to standard methods [19]. he Gram reaction was determined using the HiMedia Gram staining kit according to the manufacturer’s instructions. Physiological tests like growth at diferent temperatures ranging from 10 to 55∘ C and NaCl concentrations (1–15%) were performed by growing the strain on TSA supplemented with diferent concentrations of NaCl. he pH range (5.0–12.0) and the optimum pH for growth were examined as described by Xu et al. [20] using TSB as basal medium. For anaerobiosis, the cultures 2 International Journal of Microbiology Table 1: Diferential characteristics that diferentiate strain AK-1T along with the closest species AK-1T (MTCC 10524T ), A. aurantiacus (MTCC 11069T ), A. soli (MTCC 11074T ), A. tropicus (MTCC 11075T ), A. ulmi (MTCC 10783T ), A. lavus (MTCC 11103T ), and A. italicus (MTCC 10784T ). AK-1T (MTCC 10524T ) A. aurantiacus (MTCC 11069T ) A. soli (MTCC 11074T ) A. tropicus (MTCC 11075T ) A. ulmi (MTCC 10783T ) A. lavus (MTCC 11103T ) A. italicus (MTCC 10784T ) 37∘ C 42∘ C 2% NaCl 5% NaCl pH 5.0 pH 10.0 pH 12.0 Starch hydrolysis Casein hydrolysis Urease Catalase Acid production from carbohydrates + − − − + − − + − − − + − − − − + − + − + + + + + + − + + − − − + + − + − + + + + − − + − − − − + − − − − − − + − − − + − − + + − + + − + + + + + − − − − Salicin Mannitol Melibiose Galactose Arabinose Cellobiose Sucrose Xylose Inositol Rhamnose Lactose Trehalose Mannose Maltose Rainose Sensitivity to antibiotics (�g/disc) − − − − + + − + − − + + − − − + − − − − − + − − − + + + − + + − − + − + + − − + − + + + + + + + + + + + + − + + + + + + + + − + − + + + − + − + + + + − + − − + − − + + − − − + − − + − − + − + + − − + − − + + + Nitrofurantoin (300) Norloxacin (10) Polymyxin B (300) Kanamycin (30) Colistin (10) Methicillin (5) Oxacillin (5) Gentamycin (10) Trimethoprim (5) Oxytetracycline (30) Cefoxitin (30) Biochemical tests using VITEK 2GP card Arginine dihydrolase 1 S R S S R R R S S S S R S S S R S S R S S S R R R R R R R S R R R S R S S S S S S S S S S R S S R R R S S S R S S S S S S S S S S S S R R R S S S S S R S − − − − − − + Characteristics Growth at International Journal of Microbiology 3 Table 1: Continued. T Characteristics Leucine arylamidase �-Glucosidase L-Proline arylamidase �-Galactosidase Alanine arylamidase Tyrosine arylamidase L-Lactate alkalinization Salicin Quinone type Total lipid pattern DNA G + C mol% AK-1 (MTCC 10524T ) A. aurantiacus (MTCC 11069T ) A. soli (MTCC 11074T ) A. tropicus (MTCC 11075T ) A. ulmi (MTCC 10783T ) A. lavus (MTCC 11103T ) A. italicus (MTCC 10784T ) + + + − + − − − MK12 11, 13 DPG, PG 71.8 + + + + + + − − + + + − + + − − + + + + + − − − + − − − − − − − + − + − + + − − + + + − + + + + MK12, 13 MH12 MK12 M12, 11, 10 MK12 MK12, 13 DPG, PG 72.8 DPG, PG 73.4 DPG, PG 72.7 DPG, PG 72.0 DPG, PG 70.9 DPG, PG 70.8 All the strains were positive at pH 8.0 and 9.0, at temperatures 25∘ C and 30∘ C, and acid production from fructose; negative at 12∘ C, 10%, 15% NaCl, dulcitol, inositol, sorbitol, adonitol, citrate, methyl-red, Voges-Proskauer, indole, nitrate, and gelatin liquefaction. All the strains are negative for the following biochemical tests using VITEK 2-GP card: D-Amygdalin, phosphatidylinositol phospholipase C, D-xylose, �-galactosidase, Ala-Phe-Pro-arylamidase, cyclodextrin, L-aspartate arylamidase, �-galactopyranosidase, �-mannosidase, phosphatase, �-glucuronidase, L-pyrrolldonyl arylamidase, D-sorbitol, urease, polymyxin B resistance, D-galactose, D-ribose, lactose, N-acetyl-D-glucosamine, D-maltose, bacitracin resistance, novobiocin resistance, growth in 6.5% NaCl, D-mannitol, D-mannose, methyl-�-D-glucopyranoside, pullulan, D-rainose, O/129 resistance (comp. vibrio.), sucrose, D-trehalose, arginine dihydrolase 2 and optochin resistance. All the strains were sensitive to triple sulphas, kanamycin, sulfonamide, novobiocin, ampicillin, and rifampicin. S: sensitive; R: resistance. were streaked on TSA plates and placed in an anaerobic jar (MART), which was evacuated and lushed with Anoxomat unit (MART) using anaerobic gas mixture consisting of nitrogen (85%), carbon dioxide (10%), and hydrogen (5%). Plates were incubated at 30∘ C for 5 days. Catalase activity, citrate utilization (using Simmons’ citrate agar), and urea hydrolysis were determined as described by Cowan and Steel [21]. he hydrolysis of casein, gelatin, Tween 80, tyrosine, starch and indole, methyl red test, Voges-Proskauer test, and oxidase activity were assessed as described by Smibert and Krieg [22]. Nitrate reduction was tested as described by Lányi [23]. VITEK 2-GP cards were used as per the instructions of the manufacturer (bioMérieux). Acid production from various sugars was tested on minimal medium by using the method described by Smith et al. [24]. For cellular fatty acid analysis, the strains were grown on TSA medium at 30 ∘ C for 36 h; fatty acids were saponiied, methylated, and extracted using the standard protocol of MIDI (Sherlock Microbial Identiication System, version 4.0). he fatty acids were analysed by GC (Hewlett Packard 6890) and identiied by using the TSBA50 database of the Microbial Identiication System as described by Sasser [25] and Pandey et al. [26]. Freeze-dried cells for other chemotaxonomic analyses were prepared following growth of the strains in tryptic soy broth for 4 days at 30∘ C. he peptidoglycan structure was determined by using a hydrolysate of puriied cell walls, according to Schleifer [27]. he diagnostic amino acids were separated by single dimensional ascending TLC as described by Schleifer and Kandler [28], with the modiication that TLC on cellulose sheets (Merck 5577) was used instead of paper chromatography. Polar lipids and menaquinones were extracted and analysed by using the methods described by Minnikin et al. [29] and Kroppenstedt [30]. Genomic DNA extraction, ampliication, and sequencing were performed as described previously by Mayilraj et al. [31]. he complete sequence of the 16S rRNA gene was aligned with those of representative related taxa using the EzTaxon server (http://www.eztaxon.org/) [32]. he 16S rRNA gene sequence of AK-1T and the representative of closely related species were retrieved from the EzTaxon server and aligned using MEGA version 5.0 [32]. Phylogenetic trees were constructed using the neighbour-joining as well as maximum parsimony algorithms and maximum likelihood algorithms. Bootstrap analysis was performed to assess the conidence limits of the branching. DNA-DNA hybridization was performed by the membrane ilter method [33]. he G+C content of the genomic DNA was determined spectrophotometrically (Lambda 35; Perkin Elmer) using the thermal denaturation method [34]. 3. Results and Discussion Detailed phenotypic properties that diferentiate strain AK-1T from closely related species of the genus Agromyces are summarized in Table 1. Most of the chemotaxonomic properties, including the fatty acid composition, were typical of members of the genus Agromyces. he major menaquinone detected for the strain AK-1T is MK-12 (54.13%), while MK11 (14.08%) and MK-13 (31.77%) are the other minor components; major fatty acids are anteiso-C15:0 , anteisoC17:0 , iso-C15:0 , and iso-C16:0 (Table 2); cell wall diagnostic amino acid is 2,4-diaminobutyric acid. Major lipids are diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), two unknown phospholipids, and one unknown glycolipid (Figure 2). he almost complete 16S rRNA gene sequence 4 International Journal of Microbiology Strain AK-1T (FN868445) Agromyces tropicus CM9-9T (AB454378) Agromyces soli MJ21T (GQ241325) 71 79 Agromyces lavus CPCC 202695T (FJ529717) Agromyces ulmi XIL01T (AY427830) Agromyces luteolus IFO 16235T (AB023356) 91 95 72 77 82 98 Agromyces bauzanensis BZ41T (FJ972171) Agromyces neolithicus 23-23T (AY507128) Agromyces humatus CD5T (AY618216) Agromyces italicus CD1T (AY618215) Agromyces lapidis CD5T (AY618217) Agromyces allii UMS-62T (DQ673873) 99 Agromyces terreus DS-10 T (EF363711) Agromyces salentinus 20-5T (AY507129) Agromyces rhizospherae IFO 16236T (AB023357) Agromyces ramosus DSM 43045T (X77447) Agromyces subbeticus Z33T (AY737778) Agromyces albus VKM Ac-1800 T (AF503917) Agromyces fucosus VKM Ac-1345T (AY158025) Agromyces subsp. cerinus DSM 8595T (X77448) Agromyces cerinus subsp. nitratus ATCC 51763T (AY277619) Agromyces atrinae P27T (FJ607310) Leifsonia lichenia 2SbT (AB278552) 0.005 Figure 1: Phylogenetic neighbour-joining tree based on 16S rRNA gene sequences (1442 bases) showing the relationship between Agromyces arachidis AK-1T and related members of the genus Agromyces. Leifsonia lichenia 2SbT (AB278552) was used as an outgroup. Bootstrap values (expressed as percentages of 1000 replications) greater than 70% are given at nodes. Filled circles indicate that corresponding nodes were also recovered in the tree generated with maximum parsimony and maximum likelihood algorithms. Bar, 0.005% sequence variation. GenBank accession numbers are given in parentheses. Table 2: Percentage of total cellular fatty acids from strains AK-1T (MTCC 10524T ), A. aurantiacus (MTCC 11069T ), A. soli (MTCC 11074T ), A. tropicus (MTCC 11075T ), A. ulmi (MTCC 10783T ), A. lavus (MTCC 11103T ), and A. italicus (MTCC 10784T ). Type of fatty acids AK-1T (MTCC 10524T ) A. aurantiacus (MTCC 11069T ) A. soli (MTCC 11074T ) iso-C14:0 iso-C15:0 anteiso C15:0 iso-C16:0 C16:0 iso-C17:0 anteiso C 17:0 C18:0 C18:3 �6c iso-C19:0 0.8 9.4 47.7 11.3 0.9 3.3 21.86 tr ND ND 0.7 7.7 32.7 11.4 2.8 3.0 31.6 0.6 0.7 tr 0.6 6.1 39.5 18.1 0.6 1.8 31.2 tr ND tr A. tropicus A. ulmi (MTCC A. lavus A. italicus (MTCC 11075T ) 10783T ) (MTCC 11103T ) (MTCC 10784T ) 3.0 3.6 31.9 32.7 0.5 3.3 20.6 tr ND ND 2.5 15.5 58.3 1.8 2.5 3.0 1.6 1.5 1.3 tr 1.4 2.8 41.5 23.6 0.5 0.7 23.7 tr 0.7 1.0 0.6 6.3 40.2 18.6 0.7 1.7 29.2 tr tr ND Data from the present study. Fatty acids amounting to <0.5% of the total fatty acids in all strains are not shown or shown as tr: traces. ND: not detected. of strain AK-1T (1442 bases) was determined. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain AK-1T was closely related to A. aurantiacus (98.6%) followed by A. soli (98.3%), A. tropicus (97.6%), A. ulmi (97.3%), A. lavus (97.2%), and A. italicus (97.0%). he similarities with respect to the type strains of the remaining species of the genus were signiicantly lower (95.3–96.7%). he 16S rRNA gene sequence-based phylogenetic analysis revealed that strain AK-1T forms a separate branch within the lineage that includes A. aurantiacus, A. soli, A. tropicus, A. ulmi, and A. lavus (Figure 1); this was also evident in the phylogenetic tree constructed using maximum parsimony and maximum likelihood algorithms (shown as closed circles at the nodes in Figure 1) where the strain was recovered as a separate clade. International Journal of Microbiology PL DPG PG GL PL 5 (54.1%), while MK-13 (31.7%) and MK-11 (14.0%) are the other components. he predominant fatty acids are anteiso-C15:0 , anteiso-C17:0 , iso-C15:0 , and iso-C16:0 . he diagnostic diamino acid in cell wall hydrolyzate is 2,4-diaminobutyric acid. he DNA G + C content of the strain is 71.8 mol%. he type strain, AK-1T (=MTCC 10524T = JCM 19251T ), was isolated from a soil sample collected from peanut (Arachis hypogaea) crop ield, Srikakulam, Andhra Pradesh, India. Acknowledgments he authors thank Mr. Malkit Singh for his excellent technical assistance. his work was supported by the Council of Scientiic and Industrial Research (CSIR Network Project NWP006), Government of India. his is IMTECH communication no. 056/2013. he GenBank accession number for the 16s rRNA gene sequence of Agromyces arachidis strain is AK-1T FN868445. Figure 2: Two-dimensional thin layer chromatograms of the total lipids of strain AK-1T , detected with molybdophosphoric acid (5% w/v) in absolute ethanol. Phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), unknown phospholipid (PL), and unknown glycolipids (GL). he DNA-DNA hybridization values for strain AK-1T with the closely related species were less than 56.2%, which is well below the 70% threshold value recommended for the delineation of bacterial species [35]. he levels of DNA-DNA relatedness between strain AK-1T and other Agromyces species were not determined, since it has been shown that organisms with more than 3% 16S rRNA gene sequence dissimilar belong to diferent genomic species [36]. On the basis of the polyphasic data presented previously, strain AK-1T should be placed in the genus Agromyces within a novel species, for which we propose the name Agromyces arachidis sp. nov. 3.1. Description of Agromyces arachidis sp. nov. Agromyces arachidis sp. nov. (a.ra� chi. dis. N. L. n. Arachis-idis, a botanical generic name; N. L. gen. n. arachidis, of Arachis, isolated from a peanut (Arachis hypogaea) crop ield). he cells are Gram-positive, strictly aerobic, nonspore forming, and occurring in straight or curved rods. Colonies are yellowish, opaque, convex, entire and 1-2 mm in diameter on tryptic soy agar medium, and capable of growing from 25∘ C to 37∘ C, with optimum for growth at 30∘ C and a pH range from 6.0 to 10.0; they can tolerate up to 1.0% NaCl. Strain AK-1T shows positive reaction for hydrolysis of starch and negative for casein hydrolysis, urease production, MR-VP reaction, hydrogen sulphide production, and nitrate reduction. Acid is produced from arabinose, xylose, inulin, and lactose; it is negative for salicin, mannitol, melibiose, galactose, sucrose, rhamnose, trehalose, mannose, maltose, and rainose. Other detailed characteristics features are mentioned in Table 1. 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