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The present review discusses the phylogenomic diversity of root nitrogen-fixing bacteria associated to wild legumes under North African soils. The genus Ensifer is a dominant rhizobium lineage nodulating the majority of the wild legumes, followed by the genus Rhizobium and Mesorhizobium. In addition, to the known rhizobial genera, two new Microvirga and Phyllobacterium genera were described as real nodulating and nitrogen-fixing microsymbiotes from Lupinus spp. The promising rhizobia related to nitrogen fixation efficiency in association with some legumes are shared. Phylogenetic studies are contributing greatly to our knowledge of relationships on both sides of the plant-bacteria nodulation symbiosis. Multiple origins of nodulation (perhaps even within the legume family) appear likely. However, all nodulating flowering plants are more closely related than previously suspected, suggesting that the predisposition to nodulate might have arisen only once. The origins of nodulation, and the extent to which developmental programs are conserved in nodules, remain unclear, but an improved understanding of the relationships between nodulin genes is providing some clues.
Laboratory of Biodiversity and Valorization of Arid Areas Bioresources (BVBAA), Faculty of Sciences of Gabes, Erriadh, Zrig, Tunisia
Mohamed Ali BenAbderrahim
Arid and Oases Cropping Laboratory, Arid Area Institute, Tunisia
Mohamed Mars
Laboratory of Biodiversity and Valorization of Arid Areas Bioresources (BVBAA), Faculty of Sciences of Gabes, Erriadh, Zrig, Tunisia
*Address all correspondence to: rejili_mokhtar@yahoo.fr
1. Introduction
Africa has a vast array of indigenous legumes, ranging from large rain forest trees to small annual herbs [1]. However, in recent years, there has been a tendency in agriculture and forestry to use exotic species for crops and wood. As has been pointed out several times over nearly 30 years, most recently [2], by the US National Academy of Sciences, this ignores the potential of the native species, which are arguably better adapted to their environment. For this review, the nodulated indigenous legume genera in Northwestern Africa with known uses have been selected to illustrate the problems and potential for their better exploitation.
The wild legume flora in Northwestern Africa is rich, with great specific and infraspecific diversity [3]. The overgrazing and expansion of agriculture has gradually led to the regression and extinction of many pastoral and forage species. In addition, desertification causes disturbance of plant-microbe symbioses, which are a critical ecological factor in helping further plant growth in degraded ecosystems [4]. In this context, the establishment of indigenous pastoral legume species associated with their appropriate symbiotic bacterial partners may be of increased value for success in soil fertility restoration. Biological N2 fixation (BNF) is the major way for N input into desert ecosystems. Rhizobium-legume symbioses represent the major mechanism of BNF in arid lands, compared with the N2-fixing heterotrophs and associative bacteria [5, 6] and actinorhizal plants [7, 8]. Deficiency in mineral N often limits plant growth, and so symbiotic relationships have evolved between plants and a variety of N2-fixing organisms [9]. The symbiotically fixed N2 by the association between rhizobium species and the legumes represents a renewable source of N for agriculture. Values estimated for various legume crops and pasture species are often impressive [10]. In addition to crop legumes, the nodulated wild (herb and tree) legumes have potential for nitrogen fixation and reforestation and to control soil erosion [11]. It has been reported that a novel, suitable wild legume-rhizobia associations are useful in providing a vegetational cover in degraded lands [12].
Considering the major ecological importance of many wild legumes such as Retamasp., Acaciasp., Lotussp., Lupinussp., Medicagosp., etc. in Northwest Africa by their important role in soil fertility maintenance, coverage, and dune stabilization, the present chapter proposes to review the phylogenomic diversity of root nitrogen-fixing symbiont population nodulating Northwestern African wild legumes listed in the bibliography, some of which are common and play important ecological and pastoral roles, but others are rare and endangered. As well as the host legumes, the nodule endosymbionts also vary widely in Africa and include newly described members of both α and β branches of the Proteobacteria, now often referred to as α- or β-rhizobia, even though they do not have “rhizobium” as part of their generic names [13].
Therefore, understanding the nature of indigenous populations of rhizobia-nodulating wild legumes is of considerable agricultural significance. It is also of interest to identify a wider variety of bacterial strains in a bid to define new strains for the production of inoculants for smallholder farms.
2. Genetics and functional genomics of legume nodulation
The interaction between rhizobia and legumes in root nodules is an essential element in sustainable agriculture, as this symbiotic association is able to enhance biological fixation of atmospheric nitrogen (N2) and is also a paradigm in plant-microbe signaling [14, 15, 16]. The knowledge of the whole genome would allow the specific features of each rhizobium to be identified. The prominent feature of this group of bacteria is their molecular dialog with plant hosts, an interaction that is enabled by the presence of a series of symbiotic genes encoding for the synthesis and export of signals triggering organogenetic and physiological responses in the plant [17, 18]. In recent years, significant progress has been made in resolving the complex exchange of signals responsible for nodulation through genome assembly, mutational and expression analysis, and proteome characterization of legumes [14, 19, 20] and rhizobia [15, 21, 22, 23].
3. Phylogenomic of wild legume root nitrogen-fixing symbionts
The known diversity of rhizobia increases annually and is the subject of several reviews, the most recent and comprehensive being that of [24]. It is not our intention to revisit this subject nor the genetic basis of nodulation [25, 26], the horizontal transfer of symbiosis-related genes [23], or the symbiovar concept [27] but instead to attempt to link, where possible, rhizobial genotypes with their geographical locations and/or legume tribes/genera. At the time of writing, rhizobia consist of a diverse range of genera in the Alphaproteobacterial and Betaproteobacterial classes and are termed “alpha-rhizobia” and “beta-rhizobia,” respectively (Figure 1).
Figure 1.
Phylogenetic tree showing the relationships of currently described genera and species of alpha- and beta-rhizobia, based on aligned sequences of the 16S rRNA gene (1341-bp internal region) (adapted from [28]).
3.1 The genus Bradyrhizobium(Bradyrhizobiaceae)
The Bradyrhizobiumgenus was described by Jordan in 1982 [29]. It currently consists of nine rhizobia species.
For the Loteaetribe, previous studies found that Lotus palustrisand L. purpureusspecies from Algeria were nodulated by Bradyrhizobium lupini, and L. pedunculatusby B. japonicum[30]. However, L. creticusssp. maritimusis nodulated by both [30]. At Tunisia, L. roudaireimicrosymbiont is closely related to B. japonicum[31]. For the Acacieaetribe, two studies reported that rhizobial strains associated to the Acacia saligna, an Australian introduced species, belonged to the genus Bradyrhizobiumgenus under Algerian and Moroccan soils [32, 33, 34]. For the Genisteaetribe, it has been noticed that Bradyrhizobiumis the dominant genus of symbiotic nitrogen-fixing bacteria associated with Retamaspecies in North Africa: Retama monosperma, R. raetam, and R. sphaerocarpa(Algeria: [35, 36]; Morocco: [37]). Recently, the novel B. retamaespecies, in which groups with B. elkaniiand B. pachyrhiziand related B. lablabiand B. jicamaetype strains are included in Bradyrhizobiumgroup II [38], has been isolated from R. sphaerocarpaand R. monospermain Morocco [37]. For the genus Cytisus, two studies reported that Cytisus villosusis nodulated by B. cytisisp. nov. and B. rifensesp. nov. in Morocco [39, 40] and by genetically diverse Bradyrhizobiumstrains in Algeria belonging to B. japonicumand B. canarienseand to new lineage within the Bradyrhizobiumgenus [41]. Fifty-two strains isolated from root nodules of the Moroccan shrubby legume Cytisus trifloruswere genetically characterized, and results showed that it is nodulated by Bradyrhizobiumstrains, with 99% homology with Bradyrhizobiumgenosp. AD [42]. For the genus Lupinus, some endosymbiotic bacteria of L. luteus and L. micranthusfrom Tunisia and Algeria belonged to B. lupini, B. canariense, B. valentinum, B. cytisi/B. rifense, B. japonicum, B. elkanii, and B. retamae[43, 44, 45].
3.2 The genus Mesorhizobium(Phyllobactericeae)
The genus Mesorhizobiumwas described by Jarvis et al. [46]. Several Rhizobiumspecies were transferred to this genus. It currently consists of 21 rhizobia species.
For subtribe Astragalinae(Coluteinae Clade), Guerrouj et al. [37] reported that rhizobial symbiont of Astragalus gombiformisin Eastern Morocco is closely related to M. camelthorni. A polyphasic approach analysis indicated that bacterial strains isolated from the pasture legume Biserrula pelecinusgrowing in Morocco belong to the genus Mesorhizobium. At Tunisia, Mahdhi et al. [47] showed that five strains isolated from Astragalus corrugatuswere phylogenetically related to M. temperatumand to Mesorhizobiumsp. From the tribe Galegeae(subtribe Coluteinae), Ourarhi et al. [48] reported that Colutea arborescensis nodulated by diverse rhizobia in Eastern Morocco, among them, the genus Mesorhizobium. For the Loteaetribe, M. alhagias well as M. temperatumwere isolated, at Tunisia, from Lotus creticus[49, 50, 51]. Zakhia et al. [31] reported that Lotus argenteusmicrosymbiotes are closely related to M. mediterraneumin the infra-arid zone of Tunisia. Roba et al. [52] reported that M. delmotiiand M. prunaredenseare two new rhizobial species nodulating Anthyllis vulnerariagrowing on Tunisian soils. From the Acacieaetribe, Boukhatem et al. [33] reported that rhizobial strains associated to the Acacia saligna, an Australian introduced species, to A. ehrenbergianaand F. albidabelonged to M. mediterraneumunder Algerian soils. From the Genisteaetribe, the genetic diversity of Genista saharaemicrosymbionts in the Algerian Sahara reported that they belonged to M. camelthorni[53]. For the Mimoseaetribe, root-nodulating bacteria associated to Prosopis farctagrowing in the arid regions of Tunisia were assigned to the genus Mesorhizobium[54]. From the Hedysareaetribe, Zakhia et al. [31] reported that one strain isolated from Ebenus pinnataroot nodules is closely related to M. ciceriin the infra-arid zone of Tunisia.
3.3 The genus Rhizobium(Rhizobiaceae)
The genus Rhizobiumwas the first named (from Latin meaning “root living”), and for many years this was a “catch all” genus for all rhizobia. Some species were later moved in to new genera based on phylogenetic analyses [55]. It currently consists of 49 rhizobial species.
For Galegaetribe, Zakhia et al. [31] reported that rhizobial symbionts of Astragalus gombiformis, A. armatus, and A. cruciatusare closely related to Rhizobium mongolense, R. leguminosarum, and R. galegae, in the infra-arid zone of Tunisia. From Genisteaetribe, it was shown that strains from Tunisia nodulating Argyrolobium uniflorumare closely affiliated to R. giardinii, Calicotome villosato R. mongolense, and Genista microcephalato R. mongolenseand R. leguminosarum[31]. Mahdi et al. [56, 57, 58] reported that strains nodulating Genista saharaeand Retama retamare members of the genus Rhizobium. Nonetheless, there are reports indicating that members of the genus Rhizobiumnodulate Adenocarpus decorticansand Cytisus arboreusat Morocco [59]. For the Loteaetribe, R. leguminosarumand R. mongolensewere isolated, at Tunisia, from Anthyllis henoniana, R. leguminosarumfrom Coronilla scorpioides, and R. mongolensefrom Lotus creticus[31]. Rejili et al. [51] reported that Lotus creticusmicrosymbiotes are closely related to R. huautlensein the arid areas of Tunisia. Bacterial strains isolated from root nodules of Scorpiurus muricatussampled from different regions of western Algeria are affiliated to R. vignae, R. radiobacter, and R. leguminosarum[60]. For the Trifolieaetribe, R. galegaespecies was isolated, in Tunisia and Algeria, from Medicago marimaand M. truncatula[31]. In Algeria, Merabet et al. [61] reported that Medicago ciliarisand M. polymorphaare nodulated by Rhizobiumsp. Similarly, genetic diversity of rhizobia from annual Medicago orbicularisshowed that they are affiliated to Rhizobium tropici[62]. For the Vicieaetribe, R. leguminosarumspecies was isolated from Lathyrus numidicus[31]. Mahdhi et al. [63] reported that Vicia sativaisolates from Tunisia had 16S rDNA type identical to that of the reference R. leguminosarum. From Acacieae, Boukhatem et al. [33] reported that bacteria-nodulating Acacia salignaand A. seyalunder Algerian soils are affiliated to the R. tropiciclade and R. sullaeclade, respectively. On the other hand, the same study mentioned that five bacterial isolates, all from A. saligna, formed a separate clade in the vicinity of the R. galegae-R. huautlense-R. loessensebranch [33]. The same authors showed that the R. leguminosarumreference strain was represented by five A. karrooisolates and five A. seyalisolates [33]. At Tunisia, the genetic diversity of root nodule bacteria associated to Hedysarum coronarium(sulla), from Hedysareaetribe, showed that they are closely related to R. sullae[64]. Similarly, Ezzakkioui et al. [65] indicated that the strains from the Moroccan Hedysarum flexuosumlegume had 99.75–100% identity with R. sullae.
3.4 The genus Ensifer (Sinorhizobium) (Rhizobiaceae)
The genera Sinorhizobiumand Ensiferwere recently recognized as forming a single phylogenetic clade [66, 67] and are now united, and all species of the genus Sinorhizobiumhave been transferred to the genus Ensifer, in line with rule 38 of the Bacteriological Code [68, 69]. The genus currently consists of 17 species.
Bacteria belonging to Ensifergenus are widely distributed in arid regions of Tunisia. From the Loteaetribe, E. melilotiand E. numidicuswere isolated, at Tunisia, from Lotus creticus[49, 50, 51, 69] and Rhizobiumsp. from Hippocrepis areolata[47]. From the Acacieaetribe, genetic characterization of rhizobial bacteria-nodulating Acacia tortilissubsp. raddiana, A. gummifera, A. cyanophylla, A. karroo, A. ehrenbergiana,and A. horridain Tunisia, Algeria, and Morocco reported that they belonged to the species E. meliloti, E. garamanticus, and E. numidicusand Ensifersp. [31, 33, 70, 71, 72, 73]. At Algeria, isolates from four different host species, namely, A. karroo, A. ehrenbergiana, A. saligna, and A. tortilis, were closely related to E. fredii, E. terangae, and E. kostiensereference strains [33]. For the Mimoseaetribe, 40 isolates associated to Prosopis farctagrowing in the arid regions of Tunisia belonged to E. meliloti, E. xinjiangense/E. fredii, and E. numidicusspecies [54]. For the Trifolieaetribe, strains nodulating different Medicagospecies in Tunisia, Algeria, and Morocco such as M. sativa, M. arborea, M. truncatula, M. ciliaris, M. laciniata, M. polymorpha, Medicago arabica, M. marima, Medicago littoralis, and M. scutellaare associated to E. meliloti, E. medicae, or E. garamanticus[31, 61, 62, 69, 74, 75, 76, 77, 78, 79]. Similarly, Ononis natrixand Trigonella maritimaare nodulated by E. meliloti[31, 63]. Nodule rhizobia of Melilotus indicusgrowing in the Algerian Sahara are affiliated to E. meliloti[78]. E. melilotiand E. numidicusstrains were isolated from the Genisteae tribe such as Argyrolobium uniflorum, Retama raetam, and Genista saharae[56, 57, 58, 69]. For Galegaetribe, Mahdhi et al. [47] reported that rhizobial symbionts of Astragalus corrugatusare closely related to E. melilotiunder Tunisian soils. From the Hedysareaetribe, Mahdhi et al. [35] reported that strains isolated from Hedysarum spinosissimumroot nodules are closely related to E. melilotiin the infra-arid zone of Tunisia.
3.5 The genus Neorhizobium(Rhizobiaceae)
The genus Neorhizobiumwas proposed by Mousavi et al. [80] as an alternative to solve the issue of grouping the members of this genus with Agrobacteriumand Rhizobiumgenera. The genetic diversity of the Algerian legume Genista saharaeisolates was assessed, and results reported that they are affiliated to Neorhizobium alkalisoli, N. galegae, and N. huautlense[53]. Several studies reported that N. galegaeis isolated from different legumes in Tunisia such as Astragalus sp.[31, 54], Argyrolobium uniflorum[31], Anthyllis henoniana[31], Lotus creticus[31, 50], Medicago marima, and M. truncatula[31]. Rejili et al. [51] reported that Lotus creticusis also nodulated by N. huautlensein the arid areas of Tunisia. For Galegae tribe, Mahdhi et al. [47] reported that rhizobial symbionts of Astragalus corrugatusare closely related to N. galegaeunder Tunisian soils.
3.6 The genus Phyllobacterium(Phyllobactericeae)
The Phyllobacteriumgenus comprises of bacteria that are well-known for their epiphytic and endophytic associations with plants [81]. Nonetheless, root-nodulating and nitrogen-fixing Phyllobacteriumwas described in Tunisia, in the nodules of genistoid legume Lupinus micranthus[44, 45]. Prior to this finding, endophytic Phyllobacteriumstrains were identified on the nodules of the Tunisian legumes Genista saharae, Lotus creticus, and L. pusillus[51, 57], but they are lacking the ability to form nodules.
3.7 The genus Microvirga(Methylobacteriaceae)
The genus Microvirgawhich comprises soil and water saprophytes was included in the alphaproteobacterial lineage of root-nodule bacteria only in 2012, although the first symbiotic strains were detected in nodules of Lupinus texensis[82, 83, 84]. Recently, Microvirgastrains were only isolated from L. micranthusand L. luteusin Tunisia, belonging to the Genisteaetribe [44, 45].
Table 1 shows the root nodule symbionts from Northwestern African wild legumes.
Subfamily tribe
Genus
Species
Symbiont
Geographic origin
Mimosoideae
Acacieae
Acacia
A. cyanophylla
E. meliloti, E. fredii, Ensifersp.
Tunisia, Morocco
A. gummifera
E. meliloti, E. garamanticus, E. numidicus, Ensifersp.
Tunisia, Morocco
A. horrida
E. meliloti, E. garamanticus, E. numidicus, Ensifersp.
Tunisia, Morocco
A. tortilis raddiana
E. meliloti, E. garamanticus, E. numidicus, Ensifersp.
The root nodule symbiosis established between legumes and rhizobia is an exquisite biological interaction responsible for fixing a significant amount of nitrogen in terrestrial ecosystems. The success of this interaction depends on the recognition of the right partner by the plant within the richest microbial ecosystems on Earth, the soil. Recent metagenomic studies of the soil biome have revealed its complexity, which includes microorganisms that affect plant fitness and growth in a beneficial, harmful, or neutral manner. In this complex scenario, understanding the molecular mechanisms by which legumes recognize and discriminate rhizobia from pathogens, but also between distinct rhizobia species and strains that differ in their symbiotic performance, is a considerable challenge.
By symbiotic efficiency and properties, strains isolated from wild legumes varied in their symbiosis effectiveness with their host plant of origin. A great diversity among and within isolates was reported by many authors. This symbiotic diversity within and between isolates growing in diverse geographical areas was also defined by Tinick and Hadobas [85] for other legume plants. All strains were capable of nodulation. Mahdhi et al. [55, 86] reported that two Retama raetamisolates RB3 and RM4 (Rhizobium) gave the highest nodule numbers per plant, 26 (±2.053) and 27 (±0.997), respectively. The effective strain LAC765 (Ensifer) was isolated from Lotus creticuswith a 91.46 (±0.01%) dry biomass of the TN control [50]. The dry matter of the aerial part is considered a criterion for assessing the efficiency of a given strain; a highly significant correlation between these two parameters has been reported. Results related to symbiotic efficiency showed that among 45 tested isolates, 20 isolates are highly efficient (relative effectiveness ≥70%), 20 isolates are partially effective (60% ≤ relative effectiveness <70%), and 5 isolates are inefficient (relative effectiveness <60%). The strain GN29 isolated from Genista saharae, affiliated to Rhizobiumgenus, is considered inefficient (relative effectiveness = 32.29%). Among the 20 isolates considered highly efficient, 5 isolates were isolated from Retama retam, five from Lotussp., 4 from Genista saharae, 3 from Vicia sativa, 2 from Argyrolobium uniflorumand 2 from Trigonella maritima. From the 20 highly efficient isolates, 13 isolates belong taxonomically to Ensifersp., 6 to Rhizobiumsp., and one to Mesorhizobiumsp.
The Mediterranean basin is a hotspot place of legume diversity and the center of diversification of many of them. Our review contributes to enlarge our knowledge on the LNB-legume symbioses. We evidenced the biodiversity among bacteria-nodulating wild legumes in Northwestern Africa and unknown associations were found. Several groups may represent new genospecies to be further characterized to assess their taxonomical status. This work thus opens further interesting perspectives and makes new models available for evolutionary studies and for understanding mechanisms involved in nitrogen-fixing symbiosis.
The authors declare that they have no conflict of interest.
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Written By
Mokhtar Rejili, Mohamed Ali BenAbderrahim and Mohamed Mars
Submitted: November 24th, 2018Reviewed: May 27th, 2019Published: September 16th, 2019
Open access peer-reviewed chapter
