Host-dependent symbiotic efficiency of Rhizobium leguminosarum bv. trifolii strains isolated from nodules of Trifolium rubens

Antonie van Leeuwenhoek (2017) 110:1729–1744 DOI 10.1007/s10482-017-0922-7 ORIGINAL PAPER Host-dependent symbiotic efficiency of Rhizobium leguminosarum bv. trifolii strains isolated from nodules of Trifolium rubens Monika Marek-Kozaczuk . Sylwia Wdowiak-Wróbel . Michał Kalita . Mykhaylo Chernetskyy . Kamil Deryło . Marek Tchórzewski . Anna Skorupska Received: 22 May 2017 / Accepted: 29 July 2017 / Published online: 8 August 2017 Ó The Author(s) 2017. This article is an open access publication Abstract Trifolium rubens L., commonly known as the red feather clover, is capable of symbiotic interactions with rhizobia. Up to now, no specific symbionts of T. rubens and their symbiotic compatibility with Trifolium spp. have been described. We characterized the genomic diversity of T. rubens symbionts by analyses of plasmid profiles and BOX– PCR. The phylogeny of T. rubens isolates was inferred based on the nucleotide sequences of 16S rRNA and two core genes (atpD, recA). The nodC phylogeny allowed classification of rhizobia nodulating T. rubens as Rhizobium leguminosarum symbiovar trifolii (Rlt). The symbiotic efficiency of the Rlt isolates was determined on four clover species: T. rubens, T. Electronic supplementary material The online version of this article (doi:10.1007/s10482-017-0922-7) contains supplementary material, which is available to authorized users. M. Marek-Kozaczuk (&)
S. Wdowiak-Wróbel
M. Kalita
A. Skorupska Department of Genetics and Microbiology, Maria CurieSkłodowska University, Akademicka 19, 20-033 Lublin, Poland e-mail: M. Chernetskyy The Botanic Garden of Maria Curie-Skłodowska University, Sławinkowska 3, 20-810 Lublin, Poland K. Deryło
M. Tchórzewski Department of Molecular Biology, Maria CurieSkłodowska University, Akademicka 19, 20-033 Lublin, Poland pratense, T. repens and T. resupinatum. We determined that Rlt strains formed mostly inefficient symbiosis with their native host plant T. rubens and weakly effective (sub-optimal) symbiosis with T. repens and T. pratense. The same Rlt strains were fully compatible in the symbiosis with T. resupinatum. T. rubens did not exhibit strict selectivity in regard to the symbionts and rhizobia closely related to Rhizobium grahamii, Rhizobium galegae and Agrobacterium radiobacter, which did not nodulate Trifolium spp., were found amongst T. rubens nodule isolates. Keywords MLSA Trifolium rubens
Rhizobia
Symbiosis
Introduction In their symbiotic association with legume plants, rhizobia have the potential to fix nitrogen in amounts sufficient to reduce the dependence of plants on nitrogen fertilizers (Herridge 2008). They are distributed worldwide in many types of soil where they can be found as free-living organisms or symbionts of leguminous plants. Rhizobium-legume symbiosis plays a critical role in sustainable agriculture, because it reduces the need for nitrogen fertilizer while ensuring efficient protein-rich production. Rhizobia attach to the root hairs of plants, invade plant tissues, and colonize the cells, forming nodules where they 123 1730 differentiate into nitrogen-fixing bacteroids. The Rhizobium-legume symbiosis is specific and depends on the exchange of signal molecules, such as flavonoids, secreted by plants, which induce expression of bacterial nodulation (nod) genes via interaction with the NodD regulatory protein (Perret et al. 2000; Jones et al. 2007; Oldroyd and Downie 2008; Wang et al. 2012). The Nod proteins synthesize lipochitin oligosaccharides (Nod factors) recognized by host plant receptors and, in response, tubular structures called infection threads are formed where bacteria proliferate and are released into plant nodule cells forming symbiosomes. In these structures, bacteria differentiate into nitrogen-fixing bacteroids and turn N2 into ammonia, which is assimilated by the legume host (Heidstra and Bisseling 1996; Gage and Margolin 2000). In the indeterminate nodules formed by galegoid plants, five developmental zones are distinguished: the apical meristem functioning during nodule development (I), the invasion zone (II) into which infection threads release rhizobia, the interzone (II–III), the nitrogen-fixing zone (III), the senescence zone (IV), and the saprophytic zone (V) in older nodules (Vasse et al. 1990; Timmers et al. 2000). In the saprophytic zone, bacteroids degenerate and nonnitrogen fixing, undifferentiated rhizobia are released from infection threads, which increase the rhizobial population in the rhizosphere after nodule senescence (Timmers et al. 2000; Wielbo et al. 2010a, b). Thus, the nitrogen-fixing nodule is an organ where reciprocal benefits for both partners occur in different regions of the nodules. Although the legume-Rhizobium symbiosis is beneficial to the host, the nitrogenfixation efficiency significantly varies between different plant-Rhizobium interactions and the molecular mechanisms of strain-specific nitrogen fixation are largely unknown (Schumpp and Deakin 2010; Wang et al. 2012). Plants play a significant role in the control of later stages of symbiosis, such as bacteroid differentiation inside nodules of some galegoid (IRLC) plants (Medicago, Trifolium, Vicia, Pisum, Astragalus) and endoreduplication of bacterial genomes forming bacteroids (Mergaert et al. 2006; Haag et al. 2013). At this stage, bacteroids exhibit decreased cytoplasmic membrane integrity and undergo terminal differentiation in relation to their free-living form while maintaining the metabolic activity required for nitrogen fixation and nutrient exchange with the host plant. Bacteroid differentiation studied in Medicago 123 Antonie van Leeuwenhoek (2017) 110:1729–1744 truncatula is mediated by a large family of legume nodule-specific cysteine-rich (NCR) peptides transported to symbiosomes, which have antimicrobial activity in vitro and have a critical role in bacteroid development and persistence in vivo (Haag et al. 2011; Van De Velde et al. 2010; Kondorosi et al. 2013). The genome of Rhizobium leguminosarum is large and complex, consisting of a chromosome and a variable number of large plasmids (Young et al. 2006; Mazur et al. 2011; Kumar et al. 2015). Symbiotic functions are encoded by genes located in symbiotic plasmids (pSym) (Perret et al. 2000; Young et al. 2006; Mazur et al. 2011, 2013). The plasmids constitute a pool of accessory genetic information and contribute to the plasticity and dynamic state of the genome commonly observed among members of the Rhizobiaceae family (Palacios and Newton 2005). The host range of R. leguminosarum (Rl) species varies; R. leguminosarum bv. viciae (Rlv) is able to induce efficient symbiosis with legumes belonging to the genera Pisum, Vicia, Lathyrus, and Lens forming several species and biovars (symbiovars) (Laguerre et al. 2003; Alvarez-Martinez et al. 2009; RamirezBahena et al. 2009; Rogel et al. 2011; Rashid et al. 2015). The development of effective symbiotic associations of Rlv with the large group (...truncated)