Host-Dependent Symbiotic Efficiency of Rhizobium Leguminosarum Bv
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Antonie van Leeuwenhoek 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-Wro´bel . Michał Kalita . Mykhaylo Chernetskyy . Kamil Deryło . Marek Tcho´rzewski . Anna Skorupska Received: 22 May 2017 / Accepted: 29 July 2017 Ó The Author(s) 2017. This article is an open access publication Abstract Trifolium rubens L., commonly known as pratense, T. repens and T. resupinatum. We deter- the red feather clover, is capable of symbiotic mined that Rlt strains formed mostly inefficient interactions with rhizobia. Up to now, no specific symbiosis with their native host plant T. rubens and symbionts of T. rubens and their symbiotic compat- weakly effective (sub-optimal) symbiosis with T. ibility with Trifolium spp. have been described. We repens and T. pratense. The same Rlt strains were characterized the genomic diversity of T. rubens fully compatible in the symbiosis with T. resupinatum. symbionts by analyses of plasmid profiles and BOX– T. rubens did not exhibit strict selectivity in regard to PCR. The phylogeny of T. rubens isolates was inferred the symbionts and rhizobia closely related to Rhizo- based on the nucleotide sequences of 16S rRNA and bium grahamii, Rhizobium galegae and Agrobac- two core genes (atpD, recA). The nodC phylogeny terium radiobacter, which did not nodulate Trifolium allowed classification of rhizobia nodulating T. rubens spp., were found amongst T. rubens nodule isolates. as Rhizobium leguminosarum symbiovar trifolii (Rlt). The symbiotic efficiency of the Rlt isolates was Keywords Trifolium rubens Á Rhizobia Á Symbiosis Á determined on four clover species: T. rubens, T. MLSA Electronic supplementary material The online version of this article (doi:10.1007/s10482-017-0922-7) contains supple- mentary material, which is available to authorized users. Introduction M. Marek-Kozaczuk (&) Á S. Wdowiak-Wro´bel Á In their symbiotic association with legume plants, M. Kalita Á A. Skorupska rhizobia have the potential to fix nitrogen in amounts Department of Genetics and Microbiology, Maria Curie- Skłodowska University, Akademicka 19, 20-033 Lublin, sufficient to reduce the dependence of plants on Poland nitrogen fertilizers (Herridge 2008). They are dis- e-mail: [email protected] tributed worldwide in many types of soil where they can be found as free-living organisms or symbionts of M. Chernetskyy The Botanic Garden of Maria Curie-Skłodowska leguminous plants. Rhizobium-legume symbiosis University, Sławinkowska 3, 20-810 Lublin, Poland plays a critical role in sustainable agriculture, because it reduces the need for nitrogen fertilizer while K. Deryło Á M. Tcho´rzewski ensuring efficient protein-rich production. Rhizobia Department of Molecular Biology, Maria Curie- Skłodowska University, Akademicka 19, 20-033 Lublin, attach to the root hairs of plants, invade plant tissues, Poland and colonize the cells, forming nodules where they 123 Antonie van Leeuwenhoek differentiate into nitrogen-fixing bacteroids. The Rhi- truncatula is mediated by a large family of legume zobium-legume symbiosis is specific and depends on nodule-specific cysteine-rich (NCR) peptides trans- the exchange of signal molecules, such as flavonoids, ported to symbiosomes, which have antimicrobial secreted by plants, which induce expression of bacte- activity in vitro and have a critical role in bacteroid rial nodulation (nod) genes via interaction with the development and persistence in vivo (Haag et al. 2011; NodD regulatory protein (Perret et al. 2000; Jones Van De Velde et al. 2010; Kondorosi et al. 2013). et al. 2007; Oldroyd and Downie 2008; Wang et al. The genome of Rhizobium leguminosarum is large 2012). The Nod proteins synthesize lipochitin and complex, consisting of a chromosome and a oligosaccharides (Nod factors) recognized by host variable number of large plasmids (Young et al. 2006; plant receptors and, in response, tubular structures Mazur et al. 2011; Kumar et al. 2015). Symbiotic called infection threads are formed where bacteria functions are encoded by genes located in symbiotic proliferate and are released into plant nodule cells plasmids (pSym) (Perret et al. 2000; Young et al. forming symbiosomes. In these structures, bacteria 2006; Mazur et al. 2011, 2013). The plasmids differentiate into nitrogen-fixing bacteroids and turn constitute a pool of accessory genetic information N2 into ammonia, which is assimilated by the legume and contribute to the plasticity and dynamic state of host (Heidstra and Bisseling 1996; Gage and Margolin the genome commonly observed among members of 2000). In the indeterminate nodules formed by gale- the Rhizobiaceae family (Palacios and Newton 2005). goid plants, five developmental zones are distin- The host range of R. leguminosarum (Rl) species guished: the apical meristem functioning during varies; R. leguminosarum bv. viciae (Rlv) is able to nodule development (I), the invasion zone (II) into induce efficient symbiosis with legumes belonging to which infection threads release rhizobia, the interzone the genera Pisum, Vicia, Lathyrus, and Lens forming (II–III), the nitrogen-fixing zone (III), the senescence several species and biovars (symbiovars) (Laguerre zone (IV), and the saprophytic zone (V) in older et al. 2003; Alvarez-Martinez et al. 2009; Ramirez- nodules (Vasse et al. 1990; Timmers et al. 2000). In Bahena et al. 2009; Rogel et al. 2011; Rashid et al. the saprophytic zone, bacteroids degenerate and non- 2015). The development of effective symbiotic asso- nitrogen fixing, undifferentiated rhizobia are released ciations of Rlv with the large group of legume plants from infection threads, which increase the rhizobial indicates that both partners contain compatible deter- population in the rhizosphere after nodule senescence minants for successful nodulation and N2 fixation. In (Timmers et al. 2000; Wielbo et al. 2010a, b). Thus, contrast to Rlv, R. leguminosarum bv. trifolii (Rlt) the nitrogen-fixing nodule is an organ where recipro- symbiotic host range is confined to the clover genus cal benefits for both partners occur in different regions (Trifolium spp.) (Ramirez-Bahena et al. 2008; Reeve of the nodules. Although the legume-Rhizobium et al. 2010a, b; Kumar et al. 2015). symbiosis is beneficial to the host, the nitrogen- The clover genus Trifolium L. is a member of a large fixation efficiency significantly varies between differ- clade of legumes lacking one copy of the chloroplast— ent plant-Rhizobium interactions and the molecular inverted repeat (IRLC) and is one of the largest genera in mechanisms of strain-specific nitrogen fixation are the family consisting of approximately 255 herbaceous largely unknown (Schumpp and Deakin 2010; Wang species (Watson and Sayed-Ahmed 2000; Ellison et al. et al. 2012). Plants play a significant role in the control 2006). These are commonly cultivated forage and green of later stages of symbiosis, such as bacteroid manure crops, which grow in a great range of soils with differentiation inside nodules of some galegoid the highest species diversity in the temperate climate (IRLC) plants (Medicago, Trifolium, Vicia, Pisum, (Ellison et al. 2006). Rlt - Trifolium sp. symbiotic Astragalus) and endoreduplication of bacterial gen- associations are mostly effective; however, host-depen- omes forming bacteroids (Mergaert et al. 2006; Haag dent ineffective nodulation is evident amongst Rlt et al. 2013). At this stage, bacteroids exhibit decreased strains that nodulate clover species. Some Rlt strains cytoplasmic membrane integrity and undergo terminal form effective nodules on Trifolium repens, Trifolium differentiation in relation to their free-living form pratense,andTrifolium subterraneum, whereas other while maintaining the metabolic activity required for strains are ineffective on Trifolium subterraneum nitrogen fixation and nutrient exchange with the host (Tesfaye and Holl 1999). There are Rlt strains effective plant. Bacteroid differentiation studied in Medicago on T. subterraneum, but ineffective or partially effective 123 Antonie van Leeuwenhoek on T. repens and T. pretense (Elliot et al. 1998). In The bacteria were isolated from surface-sterilized root another case, Rlt strains are able to form effective nodules as follows: nodules were intensively washed nodules on Trifolium ambiguum (Caucasian clover) but in water and sterile distilled water and then nodules ineffective ones on T. repens, T. pratense and vice versa were sterilized by 3 min in 3% sodium chlorite and (Beauregard et al. 2004;Milleretal.2007). Melino et al. washed repeatedly in sterile distilled water. After (2012) reported sub-optimal or ineffective symbiotic surface sterilization, the nodules were crushed and association between an Rlt strain and T. subterraneum, their contents were plated on solid 79CA medium T. purpureum,andTrifolium polymorphum. These (Vincent 1970). The bacteria isolated from nodules reports show that rhizobial strains commonly found in were purified by repeatedly streaking of single agricultural soils are frequently poorly compatible with colonies and pure cultures were used in further commercially grown clover species but little is known experiments. of the cause of their ineffectivity. One of the clover species is Trifolium rubens L., DNA analyses commonly known as the red feather. This is a perennial, ornamental, xerothermic species of clover Plasmid content analyses of the nodule isolates were growing in normal