TAXONOMIC DESCRIPTION Araújo et al., Int J Syst Evol Microbiol 2017;67:2236–2241 DOI 10.1099/ijsem.0.001932

Bradyrhizobium cajani sp. nov. isolated from nodules of Cajanus cajan

Juan Araújo,1† Jose David Flores-Felix, 2† Jose M. Igual,3,4 Alvaro Peix,3,4 Fernando Gonzalez-Andr es, 5 Cesar Antonio Díaz-Alcantara 1 and Encarna Velazquez 2,4,*

Abstract Two slow-growing strains, AMBPC1010T and AMBPC1011, were isolated from nodules of Cajanus cajan in the Dominican Republic. 16S rRNA gene analysis placed these strains within the genus , being phylogenetically equidistant to several species of this genus. Analysis of the recA and atpD genes showed that the strains isolated belong to a cluster containing the strains Bradyrhizobium ottawaense OO99T, ‘Bradyrhizobium americanum’ CMVU44 and Bradyrhizobium daqingense CCBAU 15774T, and presented similarity values lower than 96 % for both genes with respect to the strains nodulating C. cajan. DNA–DNA hybridization analysis showed averages of 36, 40 and 39 % relatedness with respect to the representative strains of Bradyrhizobium ottawaense, ‘Bradyrhizobium americanum’ and Bradyrhizobium daqingense, respectively. Phenotypic characteristics also differed from those of the most closely related species of the genus Bradyrhizobium. Therefore, based on the data obtained in this study, we propose to classify the strains AMBPC1010T (=LMG 29967T=CECT 9227T) and AMBPC1011 into a novel species named Bradyrhizobium cajani sp. nov.

The genus Bradyrhizobium comprises Gram-stain-negative, ability to reinfect their host was confirmed according to the aerobic showing slow growth in media containing method of Araújo et al. [8]. mannitol as a carbon source [1]. This genus currently con- Strains AMBPC1010T and AMBPC1011 were subjected to tains more than 30 species, some of them recently isolated randon amplification of polymorphic DNA (RAPD) finger- – from nodules of American tropical legumes [2 5]. The printing as was previously described [10] using the M13 seeds of some of these legumes have great interest as human primer (5¢-GAGGGTGGCGGTTCT-3¢) and Go Taq G2 foods, particularly for developing countries, such as those Green Master Mix (Promega). The results showed that the from Cajanus cajan L., which establishes nitrogen-fixing two strains have different RAPD profiles between them and symbiosis with strains of the genus Bradyrhizobium [6–8]. with respect to the most closely related type strains (Fig. S1, available in the online Supplementary Material). In this work, we characterized two strains isolated from C. cajan nodules in the Dominican Republic and from the Amplification and sequencing of the complete 16S rRNA results obtained we propose a novel species with the name gene of the newly isolated strains was carried out according Bradyrhizobium cajani sp. nov. to the method of Rivas et al. [11], that of the recA and glnII T genes was performed according to the method of Vinuesa The strains, named AMBPC1010 and AMBPC1011, were et al. [12] and that of the nodC gene was carried out accord- isolated from two effective root nodules (pink colour) of ing to the method of Velazquez et al. [13]. The sequences plants from C. cajan L. cultivated at the site named ‘Monte obtained were compared with those from GenBank using Monito’ located in Azúa province in the Dominican Repub- the BLASTN program [14]. Sequences were aligned using the lic. The isolation was performed on YMA plates following CLUSTAL X software [15]. The distances were calculated the classic methodology described by Vincent [9], and the according to Kimura’s two-parameter model [16].

Author affiliations: 1Facultad de Ciencias Agronómicas y Veterinarias, Universidad Autónoma de Santo Domingo, Santo Domingo, Dominican Republic; 2Departamento de Microbiología y Genetica and Instituto Hispanoluso de Investigaciones Agrarias (CIALE), Universidad de Salamanca, Sala