agronomy

Article Phylogenetic Analyses of Isolated from Nodules of Lupinus angustifolius in Northern Tunisia Reveal Devosia sp. as a New Microsymbiont of Lupin

Abdelhakim Msaddak 1,2, Luis Rey 3 , Juan Imperial 2, José Manuel Palacios 3 , Mohamed Mars 1 and José J. Pueyo 2,*

1 Laboratory of Biodiversity and Valorization of Arid Areas Bioresources, BVBAA, Faculty of Sciences, University of Gabès, Erriadh 6072, Tunisia; [email protected] (A.M.); [email protected] (M.M.) 2 Institute of Agricultural Sciences, ICA-CSIC, 28006 Madrid, Spain; [email protected] 3 Centre for Plant Biotechnology and Genomics, CBGP, UPM-INIA, Pozuelo de Alarcón, 28223 Madrid, Spain; [email protected] (L.R.); [email protected] (J.M.P.) * Correspondence: [email protected]

Abstract: Thirty-two bacterial isolates were obtained from root nodules of Lupinus angustifolius growing in Northern Tunisia. Phylogenetic analyses based on recA and gyrB partial gene sequences grouped the strains into six clusters: four clusters belonged to the Bradyrhizobium (22 isolates), one to Microvirga (8 isolates) and one to Devosia (2 isolates), a genus that has not been previously reported to nodulate lupin. Representative strains of each group were further characterized. Multi-   Locus Sequence Analysis (MLSA) based on recA and glnII gene sequences separated the strains within the genus Bradyrhizobium into four divergent clusters related to B. canariense, B. liaoningense, Citation: Msaddak, A.; Rey, L.; B. lupini, and B. algeriense, respectively. The latter might constitute a new Bradyrhizobium species. Imperial, J.; Palacios, J.M.; Mars, M.; The strains in the Microvirga cluster showed high identity with M. tunisiensis. The Devosia isolates Pueyo, J.J. Phylogenetic Analyses of Rhizobia Isolated from Nodules of might also represent a new species within this genus. An additional phylogenetic analysis based Lupinus angustifolius in Northern on the symbiotic gene nodC affiliated the strains to symbiovars genistearum, mediterranense, and Tunisia Reveal Devosia sp. as a New to a possibly new symbiovar. These results altogether contributed to the existing knowledge on Microsymbiont of Lupin Species. the genetic diversity of lupin-nodulating microsymbionts and revealed a likely new, fast-growing, Agronomy 2021, 11, 1510. https:// salt-tolerant rhizobial species within the genus Devosia as a potentially useful inoculant in agricultural doi.org/10.3390/agronomy11081510 practices or landscape restoration.

Academic Editor: Alwyn Williams Keywords: Lupinus angustifolius; lupin; rhizobium; Devosia; Bradyrhizobium; Microvirga; microsym- biont; biodiversity; microbiota; Tunisia Received: 16 June 2021 Accepted: 26 July 2021 Published: 29 July 2021 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in The Leguminosae or Fabaceae family includes more than 19,700 species, many of which published maps and institutional affil- are agronomically important [1]. The Genisteae tribe is presently considered to include iations. 618 species in 25 genera [2]. Lupinus is considered the largest Genisteae genus in terms of species number with around 270 species [3,4]. This genus may be divided into two groups, the first of which is centered in the Mediterranean Basin and the Canary Islands [5,6] and comprises 13 Old World species with smooth and rough seeds [3,7]. The second group includes the New World species [8]. The New World species number ranges from Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 200 to 400 located in the Americas. Lupins are able to colonize very different environ- This article is an open access article ments and diverse ecosystems [9], including marginal impoverished or contaminated distributed under the terms and soils [10]. L. angustifolius, commonly named narrow leaf lupin and blue lupin, grows wild conditions of the Creative Commons in agricultural lands as well as in the wetlands of coastal plains in the Northern region Attribution (CC BY) license (https:// of Tunisia. creativecommons.org/licenses/by/ Lupins, partly due to their effective nitrogen-fixing symbiosis with different soil 4.0/). (collectively called rhizobia), have been grown since antiquity as an important

Agronomy 2021, 11, 1510. https://doi.org/10.3390/agronomy11081510 https://www.mdpi.com/journal/agronomy Agronomy 2021, 11, 1510 2 of 17

pulse crop and as a green manure. Lupinus species have been studied from various perspectives as indicators of phosphorus and nitrogen in soils poor in nutrients, because of their dual efficacy in fixing atmospheric nitrogen and solubilizing soil phosphates [11], phytoremediation of polluted soils [12,13] and as medicinal plants [14].The high protein content of lupin seeds is similar to that of soybean, and lupin cultivation has been suggested as an alternative to soybean in Europe [15], as new sources are needed to meet the increasing demand for plant protein [16]. Rhizobia belong to the Alpha and Betaproteobacteria [17]. Legume-rhizobium sym- bioses provide a net input of nitrogen into the ecosystems. The great diversity of lupin species reflects a vast diversity of their nodulating rhizobia. Lupinus species are mainly nodulated by slow-growing bacteria that belong to the genus Bradyrhizobium. The major species are B. canariense, B. japonicum [18–21], B. elkanii [22], and B. valentinum [23]. Other fast-growing rhizobia species, such as Ochrobactrum lupini [24], Mesorhizobium loti [25], Phylobacterium trifolii [26] and Microvirga ssp. [27–29] can also effectively nodulate dif- ferent Lupinus spp. Recently, several microsymbionts isolated from L. albus growing in Tunisian soils were identified as species within the genera Agrobacterium, Rhizobium and Neorhizobium [30]. The genus Devosia was defined is a result of the reclassification of Pseudomonas riboflavin as Devosia riboflavina [31]. Devosia bacteria are aerobic, Gram-negative, rod-shaped or oval and they do not form spores. Twenty-one validated species have been identified within the genus (http://www.bacterio.net/devosia.html, accessed on 16 July 2020). Some Devosia species have been reported to nodulate legumes [32–34]. We have previously reported that L. angustifolius from Tunisia was nodulated by new lineages within the Bradyrhizobium and Microvirga genera [35], but, to the best of our knowledge, there are no reports on Devosia symbiotic nitrogen-fixing bacteria that nodulate Lupinus species. The main objective of the present paper was to further investigate and characterize the diversity of the rhizobia that nodulate L. angustifolius growing in different sites in Northern Tunisia.

2. Materials and Methods 2.1. Sampling Sites, Isolation of Bacterial Strains and Culture Conditions Effective nodules were collected from L. angustifolius roots growing wild or in soil collected from six different locations in Northern Tunisia: Mraissa, Takelsa, Borj Hfaied, Hammamet, Sejnane and Tabarka. The ecological and climatic characterization of the sampling sites is indicated in Table1. Nodules were surface-sterilized with 95% ethanol for 1 min, 25% sodium hypochlorite for 3 min, then rinsed several with sterile distilled water; they were individually crushed on sterile plates, and a loopful of nodule material was streaked onto yeast-mannitol agar (YMA) [36]. Plates were then incubated aerobically at 28 ◦C and colonies could be visualized after 4 to 10 days. Single colonies were streaked on fresh YMA plates. Pure isolates were maintained on YMA plates at 4 ◦C for temporary preservation, or stored in 20% (w/v) glycerol suspensions at −80 ◦C. The isolates used in this study are listed in Table1.

2.2. Nodulation and Cross-Inoculation Test The ability of all purified isolates to nodulate their original host was assessed. The isolates were also tested for nodulation on L. luteus, L. micranthus, L. mariae-josephae, Retama sphaerocarpa, Vigna unguiculata and Glycine max. Seed germination and plant inoculation were carried out as previously described [37]. Plants were grown under bacteriologically controlled conditions in a greenhouse for 3 to 8 weeks, depending on the legume host, with a 16.0/8.0 h light/dark photoperiod at 25/23 ◦C, and they were watered with sterile Jensen’s liquid medium once a week. Three uninoculated Leonard jars per legume were included as negative controls. Nodules were examined for number and inside color. Three replicates of each strain were used in the nodulation tests. Agronomy 2021, 11, 1510 3 of 17

Table 1. Designations and geographical origins of L. angustifolius endosymbionts in Northern Tunisia.

Number Isolate Isolate Soil Bioclimatic Soil Site of Environment Code Code Number pH Stage Texture Isolates Takelsa 5 LanT 1, 3, 5, 6, 7 7.5 Area with orange trees semi-arid superior sandy Mraissa 5 LanM 4, 5, 6, 7, 9 8.0 Area planted with cereals semi-arid superior sandy Borj 2 LanB 5, 6 8.5 Area with olive trees semi-arid superior sandy Hfaiedh Hammamet 3 LanH 1, 2, 5 9.0 Semiurban area, next to the sea semi-arid superior sandy Sejnane 2 LanS 2, 5 7.5 Semiurban area humid clay 3, 4, 5, 6, 8, 9, 11, Tabarka 15 LanTb 12, 14, 15, 16, 17, 6.5 Area planted with cereals humid sandy 18, 19, 20

2.3. DNA Isolation, PCR Amplifications and Sequencing Bacterial DNA was obtained by the alkaline lysis method [38]. PCR was performed in a 25 µL volume containing DNA (5–10 ng), 2.5 µL of 10× PCR buffer containing magnesium chloride (Roche Applied Science), 10 µM of each primer, 10 mM of each dNTP, 1 µL DMSO and 1 U of Taq DNA polymerase (Roche Applied Science, Mannheim, Germany). PCR amplicons of genes coding for DNA recombination and repair protein RecA (recA), DNA gyrase subunit B (gyrB), 16S rRNA (rrs), chaperone protein DnaK (dnaK), glutamine synthetase 2 (glnII), and N-acetylglucosaminyl transferase (nodC) were obtained using previously described primers and conditions [37,39]. PCR amplifications of recA, 16S rRNA, glnII and nodC genes were performed and amplicons were sequenced as previously reported [37]. nifH was amplified by using the appropriate primers with PCR conditions previously described [34]. glnII appeared to be absent in the genomes of Microvirga spp., as it could not be retrieved from data banks or from any sequenced Microvirga genomes. Hence, dnaK was used, and the phylogenetic tree for this group of strains was assembled using gyrB and dnaK concatenated sequences. Amplification products were purified from the unincorporated primers and (deoxynu- cleosidetriphosphates (dNTPs) with the NucleoSpin® Extract II (Macherey-Nagel, Dueren, Germany) or by gel electrophoresis followed by band purification with the same kit, if necessary. Sequencing was performed externally at Secugen (Madrid, Spain) and STAB Vida (Lisbon, Portugal).

2.4. Phenotypic Characterization Cell morphology and size after growth on YMA agar plates at 28 ◦C for 10 days were visualized. The growth temperature range (20–40 ◦C) was tested on YMA plates at pH 7.0 for 10 days. pH tolerance was determined over the pH range 4.0–12.0 at one unit intervals. The resistance to various antibiotics (ampicillin: 100 µg/mL, gentamicin: 30 µg/mL, tetracycline: 5 µg/mL, spectinomycin: 50 µg/mL, kanamycin: 50 µg/mL, streptomycin: 10 µg/mL and chloramphenicol: 10µg/mL), as well as salt tolerance (0–5%, w/v), were assessed on YMA plates at pH = 7.0 after 10 days at 28 ◦C. All phenotypic analyses were performed in triplicate.

2.5. Phylogenetic Analyses We used a polyphasic approach including genetic analyses by sequencing of house- keeping (rrs, recA, glnII, gyrB and dnaK) and symbiotic (nodC and nifH) genes. Sequences were compared with those in GenBank using BLASTN (http://www.ncbi.nlm.nih.gov/ blast, accessed on 6 March 2021) and the EzBiocloud Database (https://www.ezbiocloud. net/, accessed on 6 March 2021), and aligned with CLUSTALW. Phylogenetic and molecular evolutionary analyses were conducted using the MEGA7.0 software [40]. The maximum likelihood (ML) and neighbor-joining (NJ) statistical methods Agronomy 2021, 11, 1510 4 of 17

and the Kimura two-parameter model were used for gene and for multilocus sequence analysis (MLSA) (combined sequences of glnII and recA; gyrB and dnaK). ML and NJ anal- yses provided similar results. NJ phylogenetic trees are shown in Figures. Phylogenetic trees were bootstrapped with 1000 bootstrap replications. Accession numbers are shown in the tree figures and were obtained after deposit in the Genbank sequence database.

3. Results 3.1. Isolation and Symbiotic Efficiency of L. angustifolius-Nodulating Bacteria from Northern Tunisia A total of 32 rhizobial bacteria were isolated from L. angustifolius nodules growing naturally in six different areas in Northern Tunisia (Table1). These isolates were tested for re-nodulation and efficient nitrogen-fixing activity by re-inoculating L. angustifolius seedlings and growing the plants under bacteriologically axenic conditions. All isolates were able to nodulate their original host, as corroborated by the formation of nodules that were pink inside, which is indicative of active nodules. The nodule number ranged from 3 to 10 per plant. Indirect nitrogen fixation effectiveness could be assessed by the dark green color of the leaves compared to uninoculated yellowish control plant leaves, and by visual observation of pink-red color due to the leghemoglobin presence inside the nodules.

3.2. Phylogenetic Analysis Based on recA and gyrB Genes The recA gene was amplified and sequenced for all isolates. The sequences were compared with those available in GenBank. As shown in the recA phylogenetic tree topology (Figure1a), L. angustifolius strains are grouped in six different clades, four within the Bradyrhizobium genus (22 strains) and one with Devosia (2 strains). A sixth group, based on gyrB gene phylogeny, formed by Microvirga (8 strains) is shown in Figure1b. The bradyrhizobia subgroups contained 17 strains affiliated to B. lupini, 2 strains to B. canariense, 1 to B. liaoningense (86%) and 2 strains to B. algeriense. At least one representative isolate of each pattern was selected for further phylogenetic analyses. In order to define a more phylogenetically robust position of the strains, an analysis based on the concatenation of relevant housekeeping genes was performed. recA and glnII were used for Bradyrhizobium strains as they have been proven to be appropriate in phylogenetic studies of this genus [23,37]. glnII could not identified in any Microvirga strains draft genome sequences. Thus, dnaK was used instead, and the phylogenetic tree for this group of strains was made using gyrB and dnaK concatenated sequences. The trees again distributed L. agustifolius nodulating strains in six distinct groups, four (I, II, III and IV) contained all the bradyrhizobia, one (V) grouped the Devosia strain and one (VI) comprised the Microvirga strains (Figure2a,b). Strains LanT1, LanTb3, LanM5 and LanTb18 clustered with B. lupini USDA3051.LanS2 and LanTb17 grouped with B. canariense BTA-1 and B. liaoningense LMG18230, respectively. LanH1 occupied a position between B. valentinum LmjM3 and B. algeriense RST89. LanTb5 was located in a taxonomic position separate from the Bradyrhizobium spp., which suggested that it might constitute a new species under a different genus. The phylogenetic tree based on the concatenated gyrB and dnaK sequences (Figure2b) revealed that strains in Group VI (LanM6, LanTb9 and LanH5) grouped with M. tunisiensis LmiM8T with almost 100% identity. Agronomy 2021, 11, 1510 5 of 17 Agronomy 2021, 11, x FOR PEER REVIEW 5 of 18

B. lupini USDA 3051 (KM114866) LanT6 (MT861031) LanT5 (MT861030) LanT3 (MT861029) LanT1 (MT861028) LanM9 (MT861025) LanM7 (MT861024) LanM5 (MT861023) 99 LanM4 (MT861022) Group I LanTb14 (MT861018) LanTb6 (MT861017) LanTb19 (MT861014) LanTb18 (MT861013) LanTb16 (MT861012) LanTb15 (MT861011) LanTb12 (MT861010) LanTb3 (MT861009) LanT7 (MT861032) B. canariense BTA-1(AY591553) LanS2 (MT861026) Group II 100 LanS5 (MT861027) B. rifense CTAW71(GU001585) B. cytisi CTAW11(GU001575) B. ganzhouense RITF806(JX277144) B. amphicarpaeae 39S1MB (CP029426) B. betae LMG 21987(AB353734) B. diazoefficiens USDA 110 (BA000040) B. japonicum (AM168341) B. arachidis CCBAU 051107(HM107233) B. ottawaense OO99 (HQ587287) B. forestalis INPA54B (PGVG01000004) B. yuanmingense CCBAU 10071(AY591566) B. liaoningense LMG 18230 (AY591564) Group III 85 LanTb17 (MT861019) B. shewense ERR11 (NZ FMAI01000022) B. centrolobii BR 10245 (NZ LUUB01000078) (a) B. iriomotense EK05(AB300996) B. sacchari BR10280 (KX065095) B. manausense BR 3351(KF785992) B. kavangense 14-3 (KM378399) B. vignae7-2 (KM378374) B. daqingense CCBAU 15774(HQ231270) B. huanghuaihaiense CCBAU 23303(HQ231595) 98 B. jicamae LMG 24556(HM047133) B. paxllaeri LMTR21(JX943617) B. lablabi CCBAU 23086 (GU433522) B. icense LMTR 13(JX943615) B. retamae Ro19(KC247094) B. valentinum LmjM3(JX518589) B. algeriense RST89 (FJ264927) LanH1 (MT861020) Group IV 98 LanH2 (MT861021) 89 B. brasilense UFLA03-321 (KT793142) B. pachyrhizi LMG 24246(HM047130) B. macuxiense BR 10303 (NZ LNCU01000014) B. elkanii USDA 76(AM168342) B. ferriligni CCBAU 51502(KJ818112) 100 B. mercantei SEMIA 6399 (NZ MKFI01000001) B. erythrophlei CCBAU 53325(KF114669) B. viridifuturi SEMIA 690 (KR149140) B. embrapense SEMIA 6208 (HQ634899) B. tropiciagri SEMIA 6148 (NZ LFLZ01000039) B. oligotrophicum LMG 10732(JQ619231) B. denitrificans LMG 8443(FM253196) 96 LanTb4 (MT861015) Group V LanTb5 (MT861016) Devosia insulae DS-56 (NZ LAJE02000162) Devosia elaeis S37 DIN R1 (NZ LVVY01000067) Devosia submarina JCM 18935 (NZ PYWB01000003) 84 IFO13584 (NZ JQGC01000010) Devosia soli GH2-10 NODE 14 (NZ LAJG01000014)

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Figure 1. Cont.

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LanTb20 (MT800905) LanB5 (MT800900) LanTb11 (MT800904) LanTb9 (MT800903) 100 LanTb8 (MT800899) Group VI LanM6 (MT800898) LanB6 (MT800901) 71 LanH5 (MT800902) Microvirga tunisiensis LmiM8 (KX784068) 98 Microvirga ossetica V5/3M (KX576560) 93 Microvirga zambiensis WSM3693 (JF428160) Microvirga arabica SV2184P (KT832845) Microvirga lotononidis WSM3557 (JF428159) (b) 100 100 Microvirga pakistanensis NCCP-1258 (LC085516) DSM14364 (JF428162) Microvirga flocculans ATCC BAA-817 TFB (JF428161) Microvirga vignae BR3299 (JX504836) Lut6 (JF428163) Microvirga makkahensis SV1470 (KT832844) 99 Methylobacterium extorquens DM4 (FP103042) Methylobacterium populi BJ001(CP001029) 83 Methylobacterium radiotolerans JCM 2831(CP001001) 100 Methylobacterium oryzae CBMB20(CP003811) Bradyrhizobium japonicum USDA 6 (AP012206)

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FigureFigure 1. Neighbor-joining 1. Neighbor-joining phylogenetic phylogenetic trees trees based based on on housekeeping housekeepingrecA recA ((aa)) andand gyrB (b)) gene gene sequences sequences of of Tunisian Tunisian L.L. angustifoliusangustifolius-nodulating-nodulating isolates isolates in this in this study study and and reference reference strains. strains. Black Black diamonds diamonds (N) ( indicate◆) indicate Tunisian Tunisian strains. strains. Bootstrap Boot- valuesstrap (above values 70%) (above using 70%) 1000 using replicates 1000 replicates are indicated are indicated at branching at branching points. points. Accession Accession numbers numbers are are shown shown in in brackets. brack- Bars:ets. 0.1 Bars: and 0.050.1 and estimated 0.05 estimated substitutions, substitutions, respectively. respectively.

3.3. 16SIn rRNA order Gene to define Phylogeny a more phylogenetically robust position of the strains, an analysis based on the concatenation of relevant housekeeping genes was performed. recA and glnII Phylogenetic analysis of the nearly full length sequences of 16S rRNA gene of 11 repre- were used for Bradyrhizobium strains as they have been proven to be appropriate in phy- sentativelogenetic strains studies showed of this genus five large [23,37] groups. glnII could (Figure not3). identified Groups in I andany Microvirga II of the analysis strains baseddraft on genome housekeeping sequences. genes Thus, form dnaK a singlewas used group instead, when and analyzed the phylogenetic based on tree the forrrs thisgene. Thisgroup group of containedstrains was five made strains using (LanTb18, gyrB and dnaK LanM5, concatenated LanS2, LanT1, sequences. and LanTb3) The trees grouped again withdistributedB. canariense L. agustifolBTA-1ius and nodulatingB. lupini USDAstrains in 3051. six distinct The rest groups, of the groupsfour (I, wereII, III similarlyand IV) distributed.contained LanTb17all the bradyrhizobia grouped with, oneB. (V) liaoningense grouped LMG18230.the Devosia strain Strain and LanH1 one occupied(VI) com- a separateprised positionthe Microvirga in the strains extra-slow (Figure bradyrhizobial 2a,b). clade formed by B. valentinum LmjM3, B. pachyrhiziStrainsPAC48 LanT1, and B. lablabiLanTb3,CCBAU LanM5 23086, and and LanTb18 might constitute clustered a new with species B. lupini in the BradyrhizobiumUSDA3051.LanS2genus. and Three LanTb17 fast growing grouped strains with (LanM6,B. canariense LanH5 BTA and-1 LanTb9) and B. liaoningense were identi- calLMG18230, to Microvirga respectively. tunisiensis LmiM8LanH1 ,alsooccupied isolated a position in Northern between Tunisia B. valentinum from L. micranthusLmjM3 and [B.37 ]. Thealgeriense 16S rRNA RST89. sequence LanTb5 of LanTb5 was located shared in 99% a taxonomic identity withpositionDevosia separatesp. Dc2a-9 from the isolated Bradyrhi- from algal-bacterialzobium spp., consortiawhich suggested (https://www.ncbi.nlm.nih.gov/nuccore/300068687 that it might constitute a new species under a different, accessed ge- on 10nus. March The 2021). phylogenetic Altogether, tree the based phylogenetic on the concatenated analyses revealed gyrB and several dnaK sequencesBradyrhizobium (Figure, Mi- crovirga2b) revealedand Devosia that strainsspecies, in whichGroup had VI (LanM6, not been LanTb9 previously and described,LanH5) grouped as microsymbionts with M. tu- of L.nisiensis angustifolius LmiM8. T with almost 100% identity.

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79 LanM5 (MT861023.MT800917) LanT1 (MT861028.MT800920) 100 LanTb18 (MT861013.MT800916) Group I LanTb3 (MT861009.MT800915) 60 B lupini USDA 3051 (KM114866.KM114862) 99 B canariense BTA-1(AY591553.AY386765) LanS2 (MT861026.MT800919) Group II B ferriligni CCBAU 51502(KJ818112.KJ818099) 55 56 B cytisi CTAW11(GU001575.GU001594) 90 B rifense CTAW71(GU001585.GU001604) B icense LMTR 13(JX943615.KF896175) B iriomotense EK05(AB300996.AB300995)

68 B betae LMG 21987(AB353734.AB353733) 57 B. japonicum USDA6 (AM168341.NC 004463) B ganzhouense RITF806(JX277144.JX277110) B stylosanthis BR 446 (KU724163.KU724148) LanTb17 (MT861019.MT800918) Group III 67 B liaoningense LMG 18230(AY591564.AY386775) B yuanmingense CCBAU 10071(AY591566.AY386780) B arachidis CCBAU 051107(HM107233.HM107251) 99 B ottawaense OO99(HQ587287.HQ587750) (a) B daqingense CCBAU 15774(HQ231270.KF962690) 56 99 B americanum CMVU44 (KC247141.KX012942) B manausense BR 3351(KF785992.KF785986) B elkanii USDA 76(AM168342.AY599117) B mercantei SEMIA 6399 (KX690615.KX690621) 57 99 98 B erythrophlei CCBAU 53325(KF114669.KF114693) B tropiciagri SEMIA 6148 (FJ391168.FJ391048) B namibiense 5-10 (KM378377.KM378440) B huanghuaihaiense CCBAU 23303(HQ231595.HQ231639)

90 B valentinum LmjM3(JX518589.JX518575) 97 56 B algeriense RST89 (FJ264927.FJ264924) LanH1(MT861020.MT800921) Group IV B retamae Ro19(KC247094.KC247108) 54 B lablabi CCBAU 23086(GU433522.GU433498) 94 97 B paxllaeri LMTR21(JX943617.KF896169) B denitrificans LMG 8443(FM253196.HM047121) 100 B oligotrophicum LMG 10732(JQ619231.JQ619233) LanTb5 (MT861016.MT800922) Group V Phyllobacterium sophorae CCBAU 03429 (KM396596.KM396513) Devosia soli GH2-10 (NZ LAJG01000014.LAJG01000021)

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Figure 2. Cont.

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Microvirga tunisiensis LmiM8 (KX784068.KX784053) Microvirga tunisiensis LmiE10 (KX784069.KX784054) 100 Lan M6 (MT800898.MT787163) Group VI Lan Tb9 (MT800903.MT787168) Lan H5 (MT800902.MT787167) Microvirga tunisiensis LluTb3 (KY445771.KY445769) 93 Microvirga arabica SV2184P (KT832845.KT832847) Microvirga ossetica V5/3M (KX576560.KX576556) 86 Microvirga zambiensis WSM3693 (JF428160.JF428153) Microvirga lotononidis WSM3557 (JF428159.JF428152) 57 99 Microvirga pakistanensis NCCP-1258 (LC085516.LC085514) (b) Microvirga lupini Lut6 (JF428163.JF428156) 99 Microvirga flocculans ATCC BAA-817 TFB (JF428161.JF428154) 55 Microvirga vignae BR3299 (JX504836.JX504820) 99 Microvirga makkahensis SV1470 (KT832844.KT832846) Microvirga subterranea DSM14364 (JF428162.JF428155) Methylobacterium nodulans ORS 2060(CP001349.CP001349) 100 Methylobacterium extorquens DM4 (FP103042.FP103042) 91 Methylobacterium populi BJ001(CP001029.CP001029) 84 Methylobacterium radiotolerans JCM 2831(CP001001.CP001001) 100 Methylobacterium oryzae CBMB20(CP003811.CP003811) Bradyrhizobium japonicum USDA 6 (AP012206.BA000040)

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FigureFigure 2. Neighbor-joining 2. Neighbor-joining phylogenetic phylogenetic trees trees of Tunisian of TunisianL. angustifolius L. angustifolius(Lan) (Lan) microsymbionts microsymbionts and reference and reference strains strains basedbased on concatenated on concatenated gene gene sequences. sequences. (a) Phylogeny(a) Phylogeny of the of theBradyrhizobium Bradyrhizobiumisolates isolates based based on onrecA recAand andglnII glnIIsequences. sequences. (b) (b) PhylogenyPhylogenyof of the theMicrovirga Microvirgaisolates isolates based based on gyrBon gyrBand anddnaK dnaKsequences. sequences. Filled Filled diamonds: diamonds: Tunisian Tunisian Lan isolatesLan isolates obtained obtained in thisin work. this work. Bootstrap Bootstrap values values calculated calculated for 1000 for 1000 replications replications and thoseand those greater greater than than 50% are50% indicated are indicated at the at internodes. the internodes. GenBankGenBank accession accession numbers numbers are shown are shown in brackets. in brackets.

3.4. NodC3.3. 16S Gene rRNA Phylogeny Gene Phylogeny PartialPhylogeneticnodC sequences analysis were of obtainedthe nearly from full onlylength six sequences bradyrhizobial of 16S strains. rRNA gene Five wereof 11 rep- in groupsresentative I and IIstrains defined showed in Figure five1 andlarge lined groups up with(Figure symbiovar 3). Groupsgenistearum I and II formedof the analysis by strainsbased able on to housekeeping nodulate legumes genes in form the Genisteae a single tribe,group such when as analyzedB. lupini, basedB. rifense on, theB. cytisi rrs gene. and B.This canariense group containedtype strains five (Figure strains4). (LanTb18, LanH1, in LanM5, group IV LanS2, of Figure LanT1,1, might and LanTb3 correspond) grouped to awith new symbiovarB. canariense designated BTA-1 and new B. lupini sv. A, USDA which 3051. is distant The rest from of the other groupsBradyrhizobium were similarly symbiovarsdistributed. and closeLanTb17 to symbiovar grouped withretamae B. ,liaoningense defined with LMG18230. strains nodulating Strain LanH1Retama occupiedspp., a and L.separate mariae-josephae position[ 41in]. thenodC extrasequences-slow bradyrhizobial from Microvirga cladestrains formed (LanM6, by B. LanTb9 valentinum LanH5) LmjM3, wereB. identical pachyrhizi to thatPAC48 of Microvirga and B. lablabi tunisiensis CCBAULmiM8 23086, belonging and might to theconstitute new mediterraneense a new species in symbiovarthe Bradyrhizobium (Figure4)[ 42genus]. The. Three LmiM8 fast straingrowing was strains an effective (LanM6, endosymbiont LanH5 and LanTb9) isolated were fromidenticalL. micranthus. to Microvirga tunisiensis LmiM8,also isolated in Northern Tunisia from L. micran- thus [37]. The 16S rRNA sequence of LanTb5 shared 99% identity with Devosia sp. Dc2a-9 isolated from algal-bacterial consortia (https://www.ncbi.nlm.nih.gov/nuccore/300068687, accessed on 10 March 2021). Alto- gether, the phylogenetic analyses revealed several Bradyrhizobium, Microvirga and Devosia species, which had not been previously described, as microsymbionts of L. angustifolius.

AgronomyAgronomy2021 ,201121,, 1510 11, x FOR PEER REVIEW 9 of9 of18 17

LanT1 (MT779767) Bradyrhizobium lupini USDA 3051 (KM114861) 86 LanTb3 (MT779771) LanS2 (MT779766) Group I and II 85 LanM5 (MT779765) LanTb18 (MT779772) Bradyrhizobium canariense BTA-1 (AJ558025) Bradyrhizobium ottawaense OO99 (JN186270) Bradyrhizobium japonicum USDA6(AB231927) Bradyrhizobium yuanmingense CCBAU 45321 (HM107161) Bradyrhizobium liaoningense LMG 18230(AJ250813) LanTb17 (MT779769) Group III 74 Bradyrhizobium americanum CMVU44 16S (NR 149803) Bradyrhizobium daqingense CCBAU 15774 (HQ231274) Bradyrhizobium centrosematis A9 (NR 149804) Bradyrhizobium diazoefficiens USDA110 (NR 074322) Bradyrhizobium betae PL7HG1(NR 029104) Bradyrhizobium manausense BR 3351 (HQ641226) Bradyrhizobium rifense CTAW71(NR 116361) 97 Bradyrhizobium cytisi CTAW11 (NR 116360) Bradyrhizobium stylosanthis BR 446 (NR 151930) 94 Bradyrhizobium iriomotense EK05 (NR 112671) 100 Bradyrhizobium denitrificans LMG 8443 (X66025) 96 Bradyrhizobium oligotrophicum LMG 10732 (JQ619230) Bradyrhizobium retamae Ro19(NR 118548) Bradyrhizobium algeriense RST89 (FJ546419) 100 Bradyrhizobium jicamae PAC68(AY624134) Bradyrhizobium erythrophlei CCBAU 53325 (KF114645) Bradyrhizobium mercantei SEMIA 6399 (NZ MKFI01000007) Bradyrhizobium valentinum LmjM3 (JX514883) 95 99 Bradyrhizobium elkanii USDA 76 (HQ233240) Bradyrhizobium pachyrhizi PAC48(AY624135) LanH1 (MT779770) Group IV Bradyrhizobium lablabi CCBAU 23086(NR 117513) Bradyrhizobium namibiense 5-10 (KX661401) Bradyrhizobium icense LMTR 13 (KF896156) Microvirga subterranea DSM14364T(FR733708) Microvirga aerophila 5420S-12 16S (GQ421848) Microvirga zambiensis WSM3693 (HM362433) 100 Microvirga aerilata 5420S-16 (GQ421849) Microvirga lotononidis WSM3557 (HM362432) 86 Microvirga lupini Lut6 (EF191408) Microvirga vignae BR3299 (JX504804) Microvirga tunisiensis Lmi M8 LanTb9 (MT779773) Group VI 100 LanM6 (MT779777) LanH5 (MT779778) Devosia insulae DS-56 (NR 044036) Devosia ginsengisoli (AB271045) 100 LanTb5 (MT779768) Group V 99 Devosia sp. DC2a-9 (AB552853) Rothia mucilaginosa (AY244786)

0.02

Figure 3. Neighbor-joining phylogenetic tree based on 16S rRNA sequences of Tunisian isolates and reference strains. Black diamonds (N) indicate Tunisian strains. Bootstrap values using 1000 replicates are indicated at branching points. Bar 0.02 estimated substitutions.

Agronomy 2021Agronomy, 11, 1510 2021, 11, x FOR PEER REVIEW 11 of 18 10 of 17

M loti MAFF303099 (BA000012) S meliloti 1021(GQ507335) R leguminosarum 3841(AM236084) S fredii USDA 205(GU994072) R tropici (AY664626) B ingae BR 10250 (KF927054) B americanum CMVU44 (KC247130) 99 B iriomotense EK05(AB301000) 100 B manausense BR 3351 (KF786002) LanH5 (MT800913)

100 LanTb9 (MT800912) sv. mediterraneense Lan M6 (MT800914) Microvirga tunisiensis LmiM8 (KX272803) M temperatum (JN991136) Microvirga lupini Lut6(AZYE00000000.1/AZYE01) B jicamae PAC68(AB573869) LanH1 (MT800909) new sv. A Microvirga sp. BR3296 (KC768862)

100 B lablabi CCBAU 23086(GU433565) B paxllaeri LMTR 21(KF896160) B icense LMTR 13 (KF896159) 100 B retamae Ro19(KC247112) 99 Bradyrhizobium sp. RST89 (FJ348666) sv. retamae 84 B valentinum LmjM3(JX514897) B neotropicale BR 10247 (KJ661727) B japonicum USDA 6 (NZ AXAV01000039) B ottawaense OO99 (HQ587980) 100 B huanghuaihaiense CCBAU 23303(HQ231507) B diazoefficiens USDA110 (BA000040) 71 99 B daqingense CCBAU 15774 (HQ231326) B liaoningense CCBAU 33113(FJ418727) 98 82 B yuanmingense NBRC 100594(AB354633) B arachidis CCBAU 051107 (HM107267) 78 B vignae 7-2 (KT362339) 89 90 B kavangense 14-3 (KT033402) B pachyrhizi PAC48 (AB573868) 99 B elkanii USDA 76(HQ233221) 100 99 B ferriligni CCBAU 51502 (KJ818109) B tropiciagri SEMIA 6148 (KP234520) B embrapense SEMIA 6208 (KP234521) 100 100 B viridifuturi CMVU02 (KC247131) B erythrophlei CCBAU 53325 (KF114576) B cytisi CTAW11 (EU597844) 78 B rifense CTAW71(EU597853) B lupini USDA 3051 (KM114864) 100 LanTb3 (MT800906) 99 LanM5 (MT800910) sv. genistearum LanTb18 (MT800911) 93 LanS2 (MT800908) B canariense. BTA-1 (AJ560653) LanT1 (MT800907) B ganzhouense RITF806(JX292035) A caulinodans ORS571 (AP009384)

0.05

Figure 4. Neighbor-joining phylogenetic tree based on nodC sequences (620 bp) of Tunisian L. angustifolius endosymbionts and reference strains: Tunisian Lan (N) isolates obtained in this work. Bootstrap values calculated for 1000 replications and those greater than 70% are indicated at the internodes. Accession numbers from GenBank and NCBI databases are shown in brackets. Bar: 0.05 estimated substitutions. M., Mesorhizobium; B., S., Sinorhizobium, Bradyrhizobium, R., Rhizobium. Agronomy 2021, 11, 1510 11 of 17

Amplification of nodC and nodA could not be obtained for strains LanTb17 and LanTb5. nifH gene sequencing affiliated LanTb17 (MZ343378) to Bradyrhizobium japon- icum CCBAU43129 with 97% identity. LanTb5 (MZ369158) nifH gene was aligned to Rhizobium tropici CIAT 899 with 78% identity. These results might indicate that these novel L. angustifolius microsymbionts might have acquired their nif genes from phylogenetically distant sources through lateral transfer.

3.5. Host Range of Rhizobial Strains Isolated from L. angustifolius Nodules The symbiotic specificity of L. angustifolius-nodulating rhizobia was investigated in plant cross-inoculation assays under axenic conditions. The selected strains were tested for nodulation with their original host and six additional legumes (L. luteus, L. micranthus, L. mariae-josephae, Retama sphaerocarpa, Vigna unguiculata and Glycine max) (Table2). All tested strains were able to nodulate L. angustifolius, L. luteus and L. micranthus,with the exception of LanTb17, which only nodulated its original host (Table2). LanT1, LanM5, LanTb3, LanTb18 and LanS2 strains formed white nodules with V. unguiculata. Microvirga sp. strains induced the formation of oversized non-effective nodules in L. mariae josephae roots. LanS2, Tb3, M5 and LanTb5 were unable to form root nodules in L. mariae josephae roots. LanH1 affiliated to extra-slow-growing Bradyrhizobium species, such as B. valentinum,which was originally isolated from L. mariae josephae, and formed effective nodules with L. mariae josephae. All tested strains were unable to form nodules on soybean, but formed white nodules on R. sphaerocarpa roots (Table2). Overall, the nodulation analyses revealed that L. angustifolius endosymbionts present a diverse host range and symbiotic specificity.

Table 2. Legume host-range analysis of representative rhizobial strains isolated from L. angustifolius nodules.

Host Plant L. angusti- L. L. mariae- Retama Vigna L. luteus Glycine max folius micranthus josephae sphaerocarpa unguiculata Strain PG LanT1 I + + + +W +W +W - LanM5 I + + + - +W +W - LanTb3 I + + + - +W +W - LanTb18 I + + + +W +W +W - LanS2 II + + + - +W +W - LanTb17 III + ------LanH1 IV + + + + + - - LanTb5 V + + + - +W - - LanM6 VI + + + +W +W - - LanH5 VI + + + +W +W - - LanTb9 VI + + + +W +W - - PG: Phylogenetic groups identified in Figure1. Color of nodules: (+) red, (+W) white, (-) no nodules.

3.6. Phenotypic Analyses of Narrow-Leaf Lupin-Nodulating Strains The rhizobial isolates presented a broad biodiversity in their growth characteristics. Ten isolates could be classified as fast-growing (Devosia and Microvirga) and twenty-two of them as slow-growing rhizobia (Bradyrhizobium). The phenotypic traits of the 11rep- resentative strains are summarized in Table3. All strains were able to grow in optimal growth conditions (28 ◦C, pH 6.8 and <0.01% NaCl). Cells were rod shaped, Gram neg- ative and aerobic. The Bradyrhizobium isolates were slow growers with mean generation times >15 h in YMB. Colonies produced in YMA were translucent, gummy and circular. They presented convex smooth margins, and their sizes were 2–3 mm in diameter after 6–7 days in YMA medium at 28 ◦C. LmiH1 was an extra-slow grower (generation time in YMB > 30 h) that produced punctiform, non-mucoid colonies (<1 mm) after 10 to 11 days Agronomy 2021, 11, 1510 12 of 17

in YMA plates. Fast growers showed mean generation times of 4–7 h in TY. They also grew well on YMA plates. After 8 days at 28 ◦C, Microvirga colonies were 2–3 mm in diameter, convex, smooth, mucilaginous pink, and circular. The effect of temperature, salinity, pH and some antibiotics, used to quantify and qualify stress tolerance, showed a degree of het- erogeneity (Table3). All the Microvirga strains grew well between 20 and 37 ◦C, at pH 4–12, and on media containing 0.1; 0.5 and 1% salt. These strains were very sensitive to all antibiotics used (kanamycin (50 µg/mL), ampicillin (100 µg/mL), gentamicin (30 µg/mL), spectinomycin (50 µg/mL), tetracycline (5 µg/mL), streptomycin (10 µg/mL). The Bradyrhi- zobium strains grew at temperatures between 20 and 28 ◦C, at pH range 4 to 11 and on media containing 0.1% salt, with only strains LanS2, LanTb17 and LanH1 growing at temperatures between 20 and 37 ◦C. These strains were sensitive to kanamycin, gentamicin, spectinomycin, and streptomycin and were less resistant to ampicillin and tetracycline, except for strain LanTb17, which showed a good resistance to kanamycin, ampicillin and chloramphenicol. The Devosia LanTb5 strain grew well between 20 and 28 ◦C, at pH 4–11, and tolerated 1% salt added to the medium. This strain was sensitive to ampicillin, gentam- icin, tetracycline, and chloramphenicol. LanTb5 could grow in presence of spectinomycin, kanamycin and streptomycin.

Table 3. Phenotypic characteristics of representative strains isolated from L. angustifolius growing in Northern Tunisia.

Salt Tolerance GT T Range pH Antibiotics Resistance Strain PG (NaCl %) (h) (◦C) Range 0.1 0.5 1 2 Amp Gen Tet Spe Kan Str Chl LanT1 I 10 20–28 4–11 + - - - + - + - - - + LanM5 I 9 20–28 4–11 + - - - + - + - - - + LanTb3 I 9.5 20–28 4–11 + - - - + - + - - - + LanTb18 I 8.7 20–28 4–11 + - - - + - + - - - + LanS2 II 9.3 20–37 4–11 + +/------/+ - - - -/+ LanTb17 III 8 20–37 4–12 + +/- - - + - + -/+ + - + LanH1 IV 18 20–37 4–11 + +/------/+ - - - -/+ LanTb5 V 4 20–28 4–11 + + +/- - - - - + + + - LanM6 VI 4.6 20–37 4–12 + + +/------LanH5 VI 4.6 20–37 4–12 + + +/------LanTb9 VI 4.6 20–37 4–12 + + +/------PG: Phylogenetic groups identified in Figure1. GT: Generation Time-, no growth; +/-, weak growth (10–30% compared to the control in YMA); +, good growth (similar to the control) Amp: ampicillin (100 µg/mL); Gen: gentamicin (30 µg/mL); Tet: tetracycline (5 µg/mL); Spe: spectinomycin (50 µg/mL); Kan: kanamycin (50 µg/mL), Str: streptomycin (10 µg/mL).and Chl: chloramphenicol (10 µg/mL). Values represent the average of two experiments with three replicates each time.

4. Discussion In this study, the isolation, identification and phenotypic characterization of rhizobial bacteria isolated from effective root nodules of L. angustifolius plants growing in Northern Tunisia is reported. All isolated strains were effective endosymbionts of their original host, as evidenced by the formation of red–pink nodules and the leaves’ dark green color. Nodule numbers ranged from 3 to 10 per plant Phylogenetic analyses revealed high genetic diversity levels among the L. angustifolius nodule isolates. Previous reports also described considerable genetic diversity among rhizobia that nodulate Lupinus by using diverse methodological approaches, such as M13 random amplified polymorphic DNA-PCR (RAPD-PCR) [20,21,23]. High diversity was shown by phylogenetic, symbiotic and phenotypic analyses. Some rhizobia assigned to the Bradyrhizobium genus were slow-growing bacteria, while some isolates were fast- growing and were assigned to the Microvirga and, unexpectedly, for the first time to our knowledge, to the Devosia genus. It is not unusual that legumes nodulated by slow-growing Agronomy 2021, 11, 1510 13 of 17

Bradyrhizobium species are also efficiently nodulated by different fast-growing rhizobial species [43,44]. The housekeeping genes glnII/recA and gyrB/dnaK were used because of their proven performance as molecular markers, either individually or in combination, for assessing the evolutionary genetics of Bradyrhizobium and Microvirga species, as described in previous works [28,45]. Phylogenetic analyses based on the housekeeping genes glnII/recA and gyrB/dnaK congruently placed some tested isolates in a new strongly supported clade within the Bradyrhizobium (Group III) and Devosia (Group V) genera. The concatenated tree of the representative strain showed that four strains affiliated with B. lupini USDA3051, one strain grouped with B. canariense BTA-1, one strain clustered together with B. liaoningense LMG18230, one strain occupied a separate position between B. valentinum LmjM3 and B. algeriense RST89, and could constitute a new species within the genus Bradyrhizobium, Three strains were included in the Microvirga genus and, surprisingly, one strain was related to the genus Devosia, never described before as a lupin microsymbiont. The concatenated phylogenetic trees constructed confirmed the results obtained by the single sequence of glnII, recA, gyrB and dnaK genes. The distribution of some strains separated from the existing type species might be indicative of novel species. Nonetheless, further polyphasic studies to precisely characterize and classify them are required. The genus Devosia has been reported to establish nitrogen-fixing symbiosis with the aquatic legume Neptunia natans growing in India [34,46]. To our knowledge, no Devosia strain has previously been described as a lupin symbiont. In this study, two strains (LanTb4 and LanTb5) closely clustered with Devosia species. These isolates were clearly separated from other Devosia spp. and might constitute a new species under this genus. In previous works, Lupinus spp. has been reported to be nodulated by fast-growing bacteria affiliated to Mesorhizobium, Allorhizobium, Rhizobium and Sinorhizobium [24,27,30,47–49]. However, in general, annual lupins are nodulated by slow-growing Bradyrhizobium sp. rhizobia [21,50–52]. This is in agreement with our results, as approximately 60% of our isolates belonged to different Bradyrhizobium species. The phylogeny of the important symbiotic gene nodC is well correlated with legume host range but independent of the rhizobial taxonomic position [53–56]. The nodC gene has been used as a molecular marker related to host specificity and range, and used to define Bradyrhizobium symbiovars [41,45,57]. The nodC tree revealed that the majority Bradyrhizobium strains clearly grouped with sequences withinthe genistearum symbiovar, which has been previously described for strains that nodulate species in the Genisteae tribe, including rhizobia nodulating Cytisus, Lupinus and Retama in Europe, America and Africa [41,45,58]. The sequences of Microvirga strains LanM6 and LanH5 grouped with sequences characteristic of the mediterraneense symbiovar. This type of nodC sequence was previously described for M. tunisiensis LmiM8 nodulating L. micranthus [42]. The remaining Bradyrhizobium-type representative isolate, LanH1, might correspond to a new symbiovar, as it was placed distant from other Bradyrhizobium symbiovars defined to date. Sanger chromatograms for nifH sequences of LanTb5 strain displayed sequence ambiguities. This may be explained by the presence of more than one copy [59]. Phenotypic tests have been used to study the diversity of rhizobial bacteria and to assess their tolerance under stress conditions [60,61]. The first macroscopic test showed that the morphology of the bradyrizobial strains in the present work was similar to those previously reported [23,60]. The colonies formed by the Microvirga strains were pinkish in color, similar to those of M. lupini [28] and M. vigna [39]. pH, salinity and extreme temperatures are abiotic stresses for nodulation of agricultural legumes in Mediterranean areas. The isolates exhibited differences in their response to abiotic stress (Table3). All isolates grew at pH 4, in agreement with reports of other strains isolated from Lupinus spp. showing resistance to acid soils [62]. Most strains were also tolerant to alkaline conditions (pH 11), as previously reported for rhizobia that nodulate Prosopis juliflora growing in alkaline soil [63]. All strains were sensitive to salinity, as they were not able to grow when NaCl concentration was higher than 0.5%. Conversely, Microvirga strains were able to Agronomy 2021, 11, 1510 14 of 17

grow in with the presence of 1% sodium chloride. These results are also corroborated by those reported for Microvirga vignae [39]. Slow-growing rhizobia in the Bradyrhizobium genus have been reported to be more sensitive to salt stress than fast-growing rhizobia isolated from the same soils [61,64]. All strains in the present study grew at the optimum temperature of 28 ◦C, in contrast with a previous report that stated that the optimum temperature for M. lupini growth was 39 ◦C[28]. The effect of temperature, salinity and pH, used to qualify and quantify stresses tolerance, showed a degree of heterogeneity even when they were isolated from the same site (Table3). In conclusion, the isolates in this work showed a wide phenotypic and genotypic diversity. Significant heterogeneity among lupin endosymbionts was unraveled based on phylogenetic analysis using housekeeping and symbiotic gene sequences, and on their legume-host ranges. The study revealed the presence of rhizobia belonging to the three divergent genera Bradyrhizobium, Microvirga and Devosia. To our knowledge, this is the first report on the Alphaproteobacterial genus Devosia as a nodule-forming species in Lupinus spp. A practical aspect of the ability of lupin species to trap diverse rhizobial genotypes is that they might display important differences in their capacities to fix nitrogen depending on edaphoclimatic conditions [65,66]. The potentially new Bradyrhizobium and Devosia species reported here require further taxonomic characterization for confirmation and formal description. Finally, this work provides an array of native rhizobia that might have practical application as biofertilizers and/or inoculants with native legumes, including shrubs, in the restoration or revegetation of arid and semiarid Mediterranean areas.

Author Contributions: Conceptualization, A.M., L.R., J.I., J.M.P., M.M. and J.J.P.; methodology, A.M.; validation, A.M., L.R. and J.J.P.; formal analysis, A.M.; investigation, A.M.; resources, L.R., J.I., J.M.P., M.M. and J.J.P.; data curation, A.M.; writing—original draft preparation, A.M.; writing—review and editing, A.M., L.R., J.I., J.M.P., M.M. and J.J.P.; visualization, A.M. and J.J.P.; supervision, L.R., M.M. and J.J.P.; project administration, L.R., M.M. and J.J.P.; funding acquisition, J.M.P., M.M. and J.J.P. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Ministry of High Education and Research Development, Tunisia, and by the Agencia Estatal de Investigación, AEI, Spain, grant numbers AGL2017-88381-R and RTI2018-094985-B-100. The APC was funded in part by CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). Data Availability Statement: All sequences are available from the GenBank database. Conflicts of Interest: The authors declare no conflict of interest.

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