Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Sat, 15 Dec 2018 09:17:02 Description of Xenorhabdus khoisanae sp. nov., the symbiont of the entomopathogenic nematode Steinernema khoisanae Tiarin Ferreira, 1 Carol A. van Reenen, 2 Akihito Endo, 2 Cathrin Spro ¨ er, 3 Antoinette P. Malan 1 and Leon M. T. Dicks 2 Correspondence Leon M. T. Dicks LMTD@sun.ac.za 1 Department of Conservation Ecology and Entomology, University of Stellenbosch, Private Bag X1, 7602 Matieland, South Africa 2 Department of Microbiology, University of Stellenbosch, Private Bag X1, 7602 Matieland, South Africa 3 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffenstrasse 7B, 38124 Braunschweig, Germany Bacterial strain SF87 T , and additional strains SF80, SF362 and 106-C, isolated from the nematode Steinernema khoisanae, are non-bioluminescent Gram-reaction-negative bacteria that share many of the carbohydrate fermentation reactions recorded for the type strains of recognized Xenorhabdus species. Based on 16S rRNA gene sequence data, strain SF87 T is shown to be closely related (98 % similarity) to Xenorhabdus hominickii DSM 17903 T . Nucleotide sequences of strain SF87 obtained from the recA, dnaN, gltX, gyrB and infB genes showed 96–97 % similarity with Xenorhabdus miraniensis DSM 17902 T . However, strain SF87 shares only 52.7 % DNA–DNA relatedness with the type strain of X. miraniensis, confirming that it belongs to a different species. Strains SF87 T , SF80, SF362 and 106-C are phenotypically similar to X. miraniensis and X. beddingii, except that they do not produce acid from aesculin. These strains are thus considered to represent a novel species of the genus Xenorhabdus, for which the name Xenorhabdus khoisanae sp. nov. is proposed. The type strain is SF87 T (5DSM 25463 T 5ATCC BAA-2406 T ). Xenorhabdus species are bound in obligate alliance of mutual benefit with specific species of entomopathogenic nematodes of the family Steinernematidae (Thomas & Poinar, 1979). Thus far, 65 Steinernema species have been described worldwide, of which Steinernema khoisanae (Nguyen et al., 2006) was isolated in the Western Cape province of South Africa. Thomas & Poinar (1979) were the first to describe symbiotic bacteria in Steinernema as members of the genus Xenorhabdus. Since then, 23 species in the genus Xenorhabdus have been described as listed in Fig. 1 and Table 1. In this paper, we describe a novel symbiont, Xenorhabdus khoisanae sp. nov., associated with the entomopathogenic nematode S. khoisanae collected in South Africa. Bacterial strains SF87 T , SF80, SF362 and 106-C were isolated from different S. khoisanae nematode populations, as previously described (Malan et al., 2006, 2011). Cultures were obtained indirectly from the nematodes by sampling the haemocoel of wax moth larvae (Galleria mellonella L.), which was plated onto nutrient agar (Biolab Diagnostics), supplemented with 0.004 % (w/v) triphenyltetrazolium chloride and 0.025 % (w/v) bromothymol blue (NBTA) according to the procedure described by Akhurst (1980). Plates were incubated at 26 u C for 72 h and blue–green colonies were randomly selected for identification and further characterization. Isolates of Xenorhabdus species were also routinely cultured in trypticase soy broth (TSB; Beckton Dickinson) and Luria broth (LB; Biolab), and stored in 40 % (v/v) glycerol at 280 u C. Xenorhabdus miraniensis DSM 17902 T , Xenorhabdus hominickii DSM 17903 T and Bacillus subtilis subsp. subtilis DSM 10 T were obtained from the DSMZ. Escherichia coli transformants were cultured in LB containing 100 mg ampicillin ml 21 . Bacillus subtilis subsp. subtilis DSM 10 T was grown in nutrient broth (NB; Biolab). Cell size measurements were taken by means of a Leica DM2000 research microscope (Leica Microsystems) equipped The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, dnaN, gltX, gyrB, recA and infB gene sequences of Xenorhabdus khoisanae sp. nov. SF87 T are HQ142625, AB685733–AB685736 and JX623984, respectively. The GenBank/EMBL/DDBJ accession num- bers for the 16S rRNA, recA, dnaN, gltX, gyrB and infB gene sequences of X. khoisanae sp. nov. SF 80, 106-C and SF362 are JX623966– JX623983, respectively. Seven supplementary figures are available with the online version of this paper. International Journal of Systematic and Evolutionary Microbiology (2013), 63, 3220–3224 DOI 10.1099/ijs.0.049049-0 3220 049049 G 2013 IUMS Printed in Great Britain