Downloaded from www.microbiologyresearch.org by IP: 93.91.26.109 On: Tue, 03 Nov 2015 16:19:28 Paenibacillus castaneae sp. nov., isolated from the phyllosphere of Castanea sativa Miller Angel Valverde, 1 Alvaro Peix, 1 Rau ´ l Rivas, 2 Encarna Vela ´ zquez, 2 Sergio Salazar, 1 Ignacio Santa-Regina, 1 Claudino Rodrı ´guez-Barrueco 1 and Jose ´ M. Igual 1 Correspondence Jose ´ M. Igual mariano.igual@irnasa.csic.es 1 Instituto de Recursos Naturales y Agrobiologı ´a de Salamanca, Consejo Superior de Investigaciones Cientı ´ficas (IRNASA-CSIC), Salamanca, Spain 2 Departamento de Microbiologı ´a y Gene ´ tica, Universidad de Salamanca, 37007 Salamanca, Spain A bacterial strain, designated Ch-32 T , was isolated from the phyllosphere of Castanea sativa in Spain. Phylogenetic analysis based on 16S rRNA gene sequences placed the isolate in the genus Paenibacillus within the same subgroup as Paenibacillus xinjiangensis and Paenibacillus glycanilyticus, with similarities of 96.3 and 96.8 %, respectively. DNA–DNA hybridization values for strain Ch-32 T with these two species were lower than 20 %. The novel isolate was a Gram- variable, motile, sporulating rod. It produced catalase and oxidase and hydrolysed cellulose, gelatin and aesculin. Acetoin and urease production, nitrate reduction and starch hydrolysis were negative. Growth was supported by many carbohydrates and organic acids as carbon sources. MK-7 was the only menaquinone detected and anteiso-C 15 : 0 , iso-C 16 : 0 and C 16 : 0 were the major fatty acids. The DNA G+C content was 46 mol%. Phylogenetic, DNA relatedness and phenotypic analyses showed that strain Ch-32 T should be classified as a novel species of the genus Paenibacillus, for which the name Paenibacillus castaneae sp. nov. is proposed; the type strain is Ch-32 T (5CECT 7279 T 5DSM 19417 T ). The sweet chestnut (Castanea sativa Miller) has been cultivated systematically throughout Europe since the Middle Ages (Conedera et al., 2004) and covers large areas in the Mediterranean basin and the UK, being used for both nut and wood production (Ferna ´ndez-Lo ´pez & Alı´a, 2003). The leaves of the sweet chestnut remain on the tree for some time after drying and this stage may be considered to be the first step in degradation processes in which polymer-degrading bacteria are involved. In the case of the phyllosphere of palm tree bracts, several of these bacteria have been found (Rivas et al., 2007), mostly belonging to the genus Paenibacillus, e.g. Paenibacillus phyllosphaerae and Paenibacillus cellulosilyticus (Rivas et al., 2005, 2006). In this study, a bacterial strain, Ch-32 T , was isolated from the phyllosphere of Castanea sativa growing in the Sierra de Francia (Salamanca, Spain) and, based on genotypic and phenotypic characterization, it should be classified as a novel species of the genus Paenibacillus. Strain Ch-32 T was isolated as follows. Chestnut leaves were immersed in 50 ml pre-chilled buffer (0.1 M potassium phosphate, pH 7.0; 0.1 % peptone) and sonicated for 10 min in an ultrasonic bath; samples (0.1 ml) were then plated on tryptic soy agar medium containing cyclohex- imide (0.15 mg ml 21 ) and incubated at 30 u C. The isolate was subcultured through two rounds of single-colony purification and subsequently stored at 270 u C in 15% glycerol. On nutrient agar, colonies of strain Ch-32 T were yellow, round, smooth and convex with approximate diameters of 1–3 mm. Strain Ch-32 T was grown in nutrient agar for 48 h at 22 u C to check for motility by phase-contrast microscopy using the hanging drop method. Gram staining was carried out by the procedure described by Doetsch (1981) after 24 h incubation at 30 u C. The flagellation type was determined by electron microscopy after 48 h incubation of strain Ch-32 T in nutrient agar at 22 uC as described previously (Rivas et al., 2005). Strain Ch-32 T was Gram-variable and motile by means of polar and subpolar flagella. Ellipsoidal subterminal spores were formed in swollen sporangia. The 16S rRNA gene of strain Ch-32 T was analysed as described by Rivas et al. (2007). The sequence obtained was compared with those from GenBank using the programs BLASTN (Altschul et al., 1990) and EZTAXON (Chun et al., 2007). Sequences were aligned using CLUSTAL_X software (Thompson et al., 1997). Distances were calculated according to Kimura’s two-parameter method (Kimura, 1980). The The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of P. castaneae Ch-32 T is EU099594. An extended phylogenetic tree constructed using the neighbour-joining method and based on 16S rRNA gene sequences of Paenibacillus castaneae Ch-32 T and related species of Paenibacillus and Cohnella is available with the online version of this paper. International Journal of Systematic and Evolutionary Microbiology (2008), 58, 2560–2564 DOI 10.1099/ijs.0.65663-0 2560 65663 G 2008 IUMS Printed in Great Britain