Original article Curing of a non-symbiotic plasmid of the Rhizobium tropici strain CIAT 899 affected nodule occupancy and competitiveness of the bacteria in symbiosis with common beans Elan Ferreira Barreto, Rosângela Straliotto, José Ivo Baldani * Embrapa Agrobiologia, BR 465, Km 07, 23851-970 Seropédica, RJ, Brazil article info Article history: Received 29 August 2011 Received in revised form 2 December 2011 Accepted 7 December 2011 Available online 22 December 2011 Handling editor: Kristina Lindström Keywords: Nitrogen fixation Cryptic plasmids Phaseolus vulgaris Symbiosis Plasmid loss abstract Functional analyses of rhizobial plasmids have concentrated mainly on symbiotic plasmids (pSym) that carry genes required for nitrogen fixation and nodule formation. However, information on the other plasmids, termed cryptic or non-symbiotic, is still scarce. In this work, a collection of sequential cured derivatives with different combinations of plasmids was generated from the Rhizobium tropici strain CIAT 899 to study their functions in symbiosis with common bean plants. PCR analysis of the nif genes from all cured derivatives indicated that plasmid b is the symbiotic plasmid (pSym) while a and c are the cryptic plasmids. This genotype was confirmed by the presence of nodules in common bean plants inoculated with derivatives containing plasmid b. However, when plasmid a was missing in the deriv- ative containing the pSym, nodules were formed but in lower numbers and were smaller in size. The derivative cured of plasmid a formed nodules with a large quantity of starch and crystals and showed a significant decrease in nitrogenase activity. In addition, the presence of bacteria in the intercellular spaces of the nodules was observed. A co-inoculation experiment involving both the wild-type CIAT 899 strain and derivative CIAT 899a suggested that the cryptic plasmid a contains genes involved in the competition for nodulation of Phaseolus vulgaris. Ó 2011 Elsevier Masson SAS. All rights reserved. 1. Introduction The genus Rhizobium is known to carry 1 e 10 plasmids (ranging from 100 to 300 MDa in size) which could represent up to 40% of the total bacterial genome [1,2]. Most of the genes involved in the nodulation (nod) and nitrogen fixation (nif and fix) processes are located on the symbiotic plasmid or pSym [3]. However, rhizobial strains also carry other plasmids, which are termed cryptic, and although these are not essential for a complete symbiosis but they do perform other beneficial functions [4e6]. Indeed, their high number, stability and molecular weight suggests that cryptic plasmids play a role in ecology or physiology of Rhizobium since a high energetic cost is necessary for their maintenance within the cells [7]. Notwithstanding the potential role(s) of the cryptic plas- mids, curing of pSym plasmids in rhizobia that nodulate common beans, clover and alfalfa has led to a complete loss of the nitrogen fixation and nodulation ability of the strains [8]. Many studies focusing on cryptic plasmids have shown that, in addition to genes involved in survival in soil and tolerance to high temperature [9,10], these plasmids also carry genes coding for the symbiotic process [6,11,12]. Moënne-Loccoz and Weaver [13] showed that curing of cryptic plasmids in the Rhizobium legumi- nosarum bv. trifolii strain W14-2 affected the rhizosphere coloni- zation of clover when co-inoculated with the wild-type strain. Other studies with plasmid-cured derivatives of R. leguminosarum bv. viciae [11], Rhizobium etli [14] and Sinorhizobium fredii [15] strains also showed that the genes involved in the competitive- ness of the strains are located in non-symbiotic plasmids. Soto and co-authors [16] showed that a gene related to nodule efficiency and competitiveness in Sinorhizobium meliloti was located within the non-symbiotic megaplasmid. Furthermore, loss of plasmid b (non- symbiotic) in the R. etli strain CFN 42 affected nodule formation due to the absence of LPS genes [6]. This plasmid also harbors genes such as rmrA and rmrB that seemed to be involved in phytoalexin detoxification which once mutagenized resulted in the formation of 40% less nodules than the wild-type strain [17]. Lower nodulation was also obtained with a derivative strain cured of non-symbiotic plasmids a (strain W14-2) and b (strain W8-7) from R. leg. bv. tri- folii that carry genes involved in cell motility and LPS synthesis * Corresponding author. Tel.: þ5521 3441 1555; fax: þ5521 2682 1230. E-mail addresses: elanbarreto@yahoo.com.br (E.F. Barreto), straliot@cnpab. embrapa.br (R. Straliotto), ibaldani@cnpab.embrapa.br (J.I. Baldani). Contents lists available at SciVerse ScienceDirect European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi 1164-5563/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejsobi.2011.12.003 European Journal of Soil Biology 50 (2012) 91e96