Microbiology Discovery ISSN 2052-6180 Original Open Access Molecular detection on culture medium of Acidobacteria from Amazon soils Acácio Aparecido Navarrete 1* , Cristine Chaves Barreto 2 , Marcela Arnaldo 1 and Siu Mui Tsai 1 *Correspondence: acacionavarrete@gmail.com 1 Cell and Molecular Biology Laboratory, Center for Nuclear Energy in Agriculture CENA, University of São Paulo USP, Piracicaba, SP, Brazil. 2 Catholic University of Brasília, Graduate Program in Genomic Sciences and Biotechnology, SGAN 915, Brasília - DF, 70790-160, Brazil. Abstract Cultivation and molecular approaches were combined to recover and detect Acidobacteria from Amazon soils on culture medium. he strategy of t he cultivation procedure included the following: the use of VL55 growth medium supplemented with 0.05% xylan as the carbon source and solidiied with gellan gum; incubation under hypoxic conditions (2% O 2 [vol/vol], 2% CO 2 [vol/vol], and 96% N 2 [vol/vol]) for a relatively long period; inclusion of aluminium potassium sulphate in the growth medium; and soil dilution and plating. Of the 456 colonies recovered on the growth medium and subjected to PCR screening, three colonies belonging to Acidobacteria subdivision 1 and one colony belonging to Acidobacteria subdivision 3 were detected. he use of 16S rRNA gene based clone libraries comprising 437 clones conirmed that members of the phylum Acidobacteria grew primarily on plates on wich acidobacterial colonies were detected by PCR screening. he clones in t he libraries consisted predominantly of Proteobacteria (orders Burkholderiales and Xanthomonadales). In summary, this work reports the recovery and molecular detection on VL55 growth medium of representatives of Acidobacteria subdivisions 1 and 3 concomitantly with to other bacterial groups inhabiting pasture and soybean cropland soils from the Amazon region. Keywords: Soil microbiology, tropical rainforest soils, molecular PCR screening, Acidobacteria subdivisions 1 and 3 © 2013 Navarrete et al; licensee Herbert Publications Ltd. his is an Open Access article distributed under the terms of Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0). his permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction The existence of large phylogenetic groups of bacteria that are poorly represented by cultivated strains has been revealed by culture-independent molecular surveys. Comparative phylogenetic analyses of the DNA sequences of 16S rRNA genes have shown that bacterial groups, such as the Acidobacteria, are ubiquitous and among the most abundant in different environments, including soil (Barns et al., 1999; Hugenholtz et al., 1998; Janssen, 2006). In spite of their high abundance, however, there are few members of Acidobacteria that have been cultured to date. Of 142,153 high-quality 16S rRNA gene sequences available from bacterial isolates (Ribosomal Database Project (RDP) II v10.4; http://rdp.msu.edu/), only 141 are classified as Acidobacteria, with cultured representatives of only 5 subdivisions [1,2,3,4, and 6] of the current 26 subdivisions (Barns et al., 2007; Hugenholtz et al., 1998; Zimmermann et al., 2005). The culture-independent approach based on the direct recovery of bacterial 16S rRNA from tropical soils indicated the predominance of Acidobacteria in soils from Western Amazon (Kim et al., 2007; Jesus et al., 2009), Central Amazon (Navarrete et al., 2010), Southeastern Amazon (Navarrete et al., 2013), and the Brazilian Cerrado (Araujo et al., 2012). Among the 26 acidobacterial subdivisions classified, subdivisions [1-7, 10,11,13,16-18,22, and 25] have been detected in Amazon soils using 16S rRNA gene pyrosequencing (Navarrete et al., 2013 and Cannavan, 2012). Acidobacteria subdivisions [1,2, and 3] were reported to be dominant in soils from primary forests, soybean cropland fields, and pasture. In contrast, in the anthrosols associated with pre-Colombian settlements in the Amazonian region – Amazonian Dark Earth – Acidobacteria subdivisions [4 and 6] were dominant members within acidobacterial communities. Despite the high abundance of acidobacterial 16S rRNA gene sequences recovered from the Amazon soils, there are no cultured representatives of Acidobacteria from this tropical ecosystem. Although many laboratories are attempting to obtain pure cultures of Acidobacteria from different environments worldwide, the taxonomically described diversity within this group remains very limited. More than half of the currently characterised Acidobacteria belong to subdivision [1], and only 18/141 high-quality 16S rRNA gene sequences from bacterial isolates are classified as subdivision [3] (Ribosomal Database Project (RDP) II v10.4; http://rdp.msu.edu/). Members of Acidobacteria subdivision [1] have been isolated from diverse soils (Janssen et al., 2002; Sait et al., 2002; Joseph et al., 2003; Eichorst et al., 2007; Koch et al., 2008; Stott et al., 2008), acidic mineral environments (Kishimoto et al., 1991), the termite hindgut (Eichorst et al., 2007), Sphagnum-dominated wetlands (Pankratov et al., 2008, 2012; Pankratov and Dedysh, 2010), decaying wood (Folman et al., 2008; Valášková et al., 2009) and methanotrophic enrichment culture (Koch et al., 2008; Dedysh et al., 2012). Acidobacteria subdivision [3] representatives have been isolated from soil (Sait et al., 2002; Joseph et al., 2003;