Genetic diversity and phylogeny of indigenous rhizobia nodulating faba bean (Vicia faba L.) in Greece

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Abstract

The genetic diversity and phylogeny of fast-growing rhizobia isolated from root nodules of Vicia faba grown in different geographical regions of Greece were assessed. Although Rhizobium leguminosarum sv. viciae is the most common symbiont of Vicia spp. in European soils, there is no available information on native rhizobia nodulating faba bean in Greece. Seventy bacterial strains were isolated and grouped into sixteen distinct profiles based on BOX-PCR fingerprinting. The phylogenetic affiliation was further defined by sequence analysis of the rrs and multilocus sequence analysis (MLSA) of three housekeeping genes (recA, atpD and gyrB). Fifty-eight isolates were affiliated with recently described genospecies gsF-2, represented by R. laguerreae FB206T, whereas six isolates were closely related to gsB and two isolates might belong to gsA. Two isolates assigned to R. hidalgonense and another two non-nodulating strains could not be assigned to any validly defined species and possibly belong to a new rhizobial lineage. Interestingly, R. laguerreae strains were commonly found at all sampling sites, suggesting that they could be the main symbionts of faba beans in Greek soils. According to the phylogenies of two symbiosis-related genes (nodC and nifH), all nodulating isolates belonged to symbiovar (sv.) viciae harboring four distinct nodC gene haplotypes and they were grouped into two clades together with strains assigned to R. laguerreae and genospecies of R. leguminosarum isolated from other countries and continents. This is the first report that R. hidalgonense strains belong to sv. viciae. No correlation was observed between the nodC haplotypes, geographic origin and chromosomal background of the isolates in the study.

Introduction

Nitrogen is one of the most limiting nutrients for plant growth, and nitrogen fertilizers have major costs in crop production. Biological nitrogen fixation (BNF) is a key component in sustainable agriculture, since it represents an economic environmentally friendly and renewable nitrogen resource for crops [37]. BNF is a natural process for the conversion of atmospheric N2 to ammonia (NH3) and it can be performed by free-living, associative, endophytic and symbiotic rhizobacteria. The latter are collectively called rhizobia and they form a symbiotic relationship with legumes in which the plant host provides the rhizobia with nutrients, while rhizobia provide the host with fixed atmospheric dinitrogen in the form of ammonia. Notably, estimates of N fixed annually in agricultural systems have shown that symbiotic BNF has the greatest annual input of fixed N (∼60% of the total BNF inputs) into agricultural systems worldwide [19], [44]. Estimates of N2 fixation in experimental and farmers’ fields have shown that faba bean and lupin (Lupinus spp.) possess the highest capacity for N2 fixation, followed by soybean and groundnut, winter and summer pulses and common bean [44]. Therefore, the utilization of N2-fixing leguminous crops represents a sustainable way to increase BNF input in agrosystems.

In symbiosis, nitrogen fixation takes place in specialized root organs called nodules that are formed by a highly specific process defined as nodulation. Nodulation specificity is genetically determined by both symbiotic partners. Due to this specificity, a subset of rhizobia can establish symbiosis with certain legumes. Based on the range of rhizobia capable of nodulating a particular legume host, some legumes are considered to be restrictive hosts, since they form nodules with a few rhizobial strains, whereas others are more promiscuous hosts and are nodulated by a wide range of rhizobia [45].

Faba bean is considered to be a restrictive legume host and commonly establishes effective nitrogen fixation symbiosis with genospecies within Rhizobium leguminosarum sensu lato (henceforth abbreviated to Rl) that are distributed worldwide [6], [26]. Previous studies have shown the existence of seven distinct genospecies (gsA, gsB, gsC, gsD, gsE, gsF-1 and gsF-2) within the Rhizobium species complex that are not symbiovar specific [6], [26]. Similarly, a number of genospecies that nodulate Phaseolus vulgaris have been defined and some of them are equivalent to those found in Vicia spp. and Trifolium spp. [26], [64]. Besides faba bean, some Rl genospecies also effectively nodulate cultivated and wild legumes of the tribe Vicieae, which comprises the genera Vicia, Pisum, Lens and Lathyrus [2], [24], [29], [45]. Despite the fact that wild Vicia species are more promiscuous in their interaction with microsymbionts than the crop legumes, Rl is the predominant species in the nodules of wild species [25], [27], [29], [33], [42], [61], [27], [29], [33], [42], [61]

The symbiovar (sv.) viciae is the sole symbiovar able to establish effective N2-fixing symbiosis with V. faba [2], even though it could be present in different chromosomal backgrounds [3], [13], [25], [29], [33], [38], [41], [56], [61], [62], [66], [67], [81]. Previous reports have shown that faba bean also establishes effective nitrogen fixation symbiosis with R. etli in China, Peru and Egypt [56], [57], [61], R. fabae in China [9], [62], R. laguerreae in Algeria, Peru, Spain, and Tunisia [5], [6], [55], [56], R. mesosinicum in China [74], R. anhuiense, R. sophorae and R. vallis in China [9], [82], the unidentified lineages Rhizobium sp. I and II in China [9], [74], and Sinorhizobium/Ensifer meliloti in Egypt [57].

In European soils, Rl is ubiquitous in arable soils and has been isolated from faba bean in Spain, France, Germany, Italy, Serbia, Sweden, and the Czech Republic [6], [28], [38], [41], [42], [46], [49], [52], [53], [63], [69], [79]. Rl isolates have also been found in other crops and wild legumes of the tribe Vicieae, such as Pisum sativum, Lens culinaris, Lathyrus sativus and Vicia spp. in the UK, Germany, Belgium, Italy, France, Serbia, Czech Republic, Poland, Spain and Sweden [3], [4], [6], [13], [23], [26], [38], [41], [47], [48], [51], [54], [68], [69], [71].

Even though Rl is considered to be the most common symbiont of Vicia spp. worldwide, several lines of evidence have documented that Rl indigenous populations are phenotypically and genetically polymorphic and their diversity varies in different legume hosts within the tribe Vicieae and across continents [3], [6], [23], [29], [30], [31], [41], [55], [58]. Rl diversity studies have shown the existence of phenotypic and genetic differences between European Rl isolates and those from Jordan and China [6], [29], [33], [40], [41], [56], [61], [63]. In addition, several lines of evidence have demonstrated that specific Rl genotypes show preferences either for different species within the tribe Vicieae (Vicia, Pisum, Lens and Lathyrus), or for different genotypes within the same legume host species [3], [6], [8], [15], [17], [21], [23], [29], [41].

Specificity differences within Rl strains were recorded in different wild and crop species (faba bean, pea, lentils, vetch and vetchlings) grown in the same soil [17], [21], [28], [29], [31], [41]. Notably, faba beans were almost exclusively nodulated by Rl strains harboring a particular nodulation genotype (nodD type g or nodDF-2) independently of the rhizobial genomic background, while peas and vetches were more promiscuous than faba beans in the selection of nodulation genotypes, but showed preference for particular associations of genomic backgrounds and nod types [29], [41]. This nodulation genotype is present in Rl strains 248 and 3841, both isolated in the United Kingdom from nodules of V. faba and P. sativum, respectively [29], [41], [66].

Moreover, rhizobial strains isolated from nodules of V. faba and P. sativum growing in Andean and coastal regions of Peru were genetically diverse even though the environmental conditions and/or the host could be responsible for the selection of particular rhizobial genotypes [56]. Notably, distinct preferences of different V. faba genotypes for particular Rl lineages have been recorded previously [29], [61], [74]. Similarly, differences in Rl specificity have been reported among different pea lines. Rl strains found predominantly in the Middle East and Central Asia (Turkey, Israel and Afghanistan) were capable of nodulating some primitive pea lines from Afghanistan and Iran as well as modern European pea lines. However, European Rl strains isolated from cultivated pea plants were unable to nodulate primitive pea lines [34], [77], [78]. The ability of Rl strains to nodulate primitive peas was attributed to the presence of a single gene, nodX, located in the sym plasmid pRL5JI [11]. Depret et al. [14] also found that the ability of particular Rl nodulation genotypes to form nodules varied with the pea line and the plant developmental stage. In addition, strain and cultivar effects on nitrogen fixation and nodulation have been reported for different Rl strains nodulating lentils, peas and faba beans [1], [29], [68], [76]. Recent pioneering studies confirmed previous findings that differences of partner choice in faba bean- and pea-Rl associations were dependent on both legume hosts and Rl genotypes [6], [7]. Moreover, they showed that competitiveness for nodulation was not associated with BNF efficiency, and genetic determinants linked to nod or other symbiosis-related loci can be involved in the differential selection of Rl by host plants. Therefore, improvement of biological nitrogen fixation (BNF) by selecting suitable combinations of bacterial strains and legume genotypes will lead to increased productivity and reduced nitrogen (N) fertilizer use.

Therefore, the aim of this study was to isolate and characterize the rhizobial diversity found associated with field-grown faba beans originating from different Greek regions. To our knowledge, there has been no report on the genetic diversity of rhizobia nodulating V. faba in Greece, and no data are available concerning the introduction of rhizobial inoculants in the regions analyzed. Thus, all rhizobia isolated from field-grown faba bean nodules were assumed to represent members of the indigenous population. In the present study, the genetic diversity and phylogeny of root nodule bacteria isolated from different local varieties of field-grown V. faba in different locations in Greece were assessed.

Section snippets

Nodule and soil sampling

Nodules were collected from local faba bean varieties grown in seven different geographical regions of Greece, namely Athens, Crete, Epirus, Feneos, Imathia, Kythira, and Lefkada (Fig. S1). The sampling sites were located in fields without rhizobial inoculation history. The soil samples were slightly acidic to alkaline, with a pH range of 6.2–8.1.

Isolation and purification of nodules and rhizobial strains

Four nodules per plant were randomly selected from four plants of each region, and at least three isolates were retained from each nodule. A great

Isolated strains and BOX fingerprinting

A total of 70 bacterial strains were isolated from root nodules of local faba bean varieties grown in seven different regions located on the mainland (Athens, Epirus, Feneos, Imathia) and islands (Crete, Kythira, and Lefkada) of Greece. Strains were named “VF”, representing the host V. faba (VF), followed by two letters representing the region of isolation. Strains isolated from Athens, Crete, Epirus, Feneos, Imathia, Kythira, and Lefkada, were named either “AT”, or “CR”, or “EP”, or “FE”, or

Conclusions

The vast majority of isolates obtained in the present study belonged to genospecies gsF-2, represented by the type strain of R. laguerreae, while only a few isolates were related to genospecies gsA and gsB [26], [64]. A single isolate was assigned to R. hidalgonense, while another non-nodulating rhizobial isolate could not be assigned to any validly described species and it might constitute a novel rhizobial lineage. In European soils, Rl is the most common symbiont of Vicia spp., while R.

Author contributions

EE performed the experiments, APT, EE, and DS participated in collecting faba bean nodules, APT conceived and designed the experiments, analyzed the data and wrote the paper. All authors have read and approved the manuscript.

Acknowledgements

This research was funded by European Union’s Horizon 2020 research and innovation programme under grant agreement No. 727929, project “TOMRES-A novel and integrated approach to increase multiple combined stress tolerance in plants using tomato as a model” and the Hellenic Foundation for Research and Innovation (HFRI) under the HFRI PhD Fellowship grant (Fellowship Number: 957).

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