Please cite this article in press as: J. Puławska, et al., Rhizobium nepotum sp. nov. isolated from tumors on different plant species, Syst. Appl. Microbiol. (2012), doi:10.1016/j.syapm.2012.03.001 ARTICLE IN PRESS G Model SYAPM-25455; No. of Pages 6 Systematic and Applied Microbiology xxx (2012) xxx–xxx Contents lists available at SciVerse ScienceDirect Systematic and Applied Microbiology jo u rn al homepage: www.elsevier.de/syapm Rhizobium nepotum sp. nov. isolated from tumors on different plant species  Joanna Puławska a,∗ , Anne Willems b , Sofie E. De Meyer b , Sandor Süle c a Research Institute of Horticulture, ul. Pomologiczna 18, 96-100 Skierniewice, Poland b Laboratory of Microbiology, Dept. Biochemistry and Microbiology, Fac. Science, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium c Plant Protection Institute of Hungarian Academy of Sciences, Herman Otto Str. 15, 1022 Budapest, Hungary a r t i c l e i n f o Article history: Received 26 October 2011 Received in revised form 23 February 2012 Accepted 2 March 2012 Keywords: Agrobacterium sp. Crown gall DNA–DNA hybridization FAME MLSA RAPD a b s t r a c t Five Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from galls on different plant species in Hungary: strain 39/7 T from Prunus cerasifera Myrobalan, strain 0 from grapevine var. Ezerjó, strain 7/1 from raspberry var. Findus and in Poland, strain C3.4.1 from Colt rootstock (Prunus avium × Prunus pseudocerasus) and strain CP17.2.2 from Prunus avium. Only one of these iso- lates, strain 0, is able to cause crown gall on different plant species. On the basis of 16S rRNA gene sequence similarity, the strains cluster together and belong to the genus Rhizobium and their closest relative is Rhizobium radiobacter (99.1%). Phylogenetic analysis of the novel strains using housekeep- ing genes atpD, glnA, gyrB, recA and rpoB revealed their distinct position separate from other known Rhizobium species and confirmed their relation to Rhizobium radiobacter. The major cellular fatty acids are 18:1 w7c, 16:0, 16:0 3OH, summed feature 2 (comprising 12:0 aldehyde, 16:1 iso I and/or 14:0 3OH) and summed feature 3 (comprising 16:1 w7c and/or 15 iso 2OH). DNA–DNA hybridization of strain 39/7 T with the type strain of R. radiobacter LMG 140 T revealed 45% DNA–DNA hybridization. Phenotypic and physiological properties differentiate the novel isolates from other closely related species. On the basis of the results obtained, the five isolates are considered to represent a novel species of the genus Rhizobium, for which the name Rhizobium nepotum sp. nov. (type strain 39/7 T = LMG 26435 T = CFBP 7436 T ) is proposed. © 2012 Elsevier GmbH. All rights reserved. Crown gall is one of the most serious plant diseases that can affect over 800 plant species. It is caused by tumorigenic bacteria classified earlier in the genus Agrobacterium [28] and since 2001 in the revised genus Rhizobium [29], although this taxonomic revision has been contested [3]. Recently, the International Committee on Systematics of Prokaryotes, Subcommittee on the taxonomy of Agrobacterium and Rhizobium published minutes of its latest  Note: The GenBank/EMBL/DDBJ accession numbers for the partial 16S rRNA gene sequences of strains 0, 7/1, 39/7 T , C3.4.1, CP17.2.2 are: FR870229–FR870233, respectively. Accession numbers for the partial glnA gene sequences of strains 0, 7/1, 39/7 T , C3.4.1, CP17.2.2, R. pusense NRCPB10 T are: FR870240–FR870245, respec- tively. Accession numbers for the partial gyrB gene sequences of strains 0, 7/1, 39/7 T , C3.4.1, CP17.2.2, R. pusense NRCPB10 T are: FR870234–FR870239, respectively. Accession numbers for the partial rpoB gene sequences of strains 0, 7/1, 39/7 T , C3.4.1, CP17.2.2, R. pusense NRCPB10 T are: FR871199–FR871204, respectively. Accession numbers for the partial atpD gene sequences of strains 0, 7/1, 39/7 T , C3.4.1, CP17.2.2, R. skierniewicense Ch11 T are: HE646679–HE646684, respectively. Accession num- bers for the partial recA gene sequences of strains 0, 7/1, 39/7 T , C3.4.1, CP17.2.2, R. skierniewicense Ch11 T are: HE646685–HE646690, respectively. ∗ Corresponding author at: Research Institute of Horticulture, Pomology Division, ul. Pomologiczna 18, 96-100 Skierniewice, Poland. Tel.: +48 46 8345366; fax: +48 46 8345375. E-mail address: joanna.pulawska@inhort.pl (J. Puławska). meeting where status of genera Agrobacterium and Rhizobium was debated and the possibility of a re-definition of the genus Agrobac- terium considered [6]. However, a formal proposal with a definition or the new borders of Agrobacterium (and clarification of the posi- tion of related Rhizobium species) is still lacking and we therefore retain the Rhizobium nomenclature here. At present, pathogenic bacteria can be found in six Rhizobium species: Rhizobium radiobacter, Rhizobium rhizogenes, Rhizobium rubi, Rhizobium vitis, Rhizobium larrymoorei and Rhizobium skierniewicense [19,27,29]. However, bacteria belonging to R. radiobacter (also called Agrobac- terium biovar 1) were previously found to be more heterogeneous. Exhaustive investigations have shown that this species/biovar includes at least nine genomospecies [1,2,15,16]. Possibly, also Rhizobium pusense – a newly described species based on one non-pathogenic strain isolated from rhizosphere of chickpea [14] – may in future be found to harbour tumorigenic bacteria. On the basis of recA gene (Fig. S1) and gyrB gene (Puławska, unpublished data) analyses, it clusters together with members of genomovar G2 of the former Agrobacterium biovar 1 species complex. Using com- parative genomics, Lassalle and co-workers [5] recently showed that genomovar G8 represents a species and suggested the name Agrobacterium fabrum. However, no formal species description was proposed yet and thus the new species name has not been validly 0723-2020/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2012.03.001