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Rhizobia Indigenous to the Okavango Region in Sub-Saharan Africa: Diversity, Adaptations, and Host Specificity Jann L. Grönemeyer, Ajinkya Kulkarni, Dirk Berkelmann, Thomas Hurek, Barbara Reinhold-Hurek Department of Microbe-Plant Interactions, Faculty of Biology and Chemistry, Center for Biomolecular Interactions Bremen (CBIB), University of Bremen, Bremen, Germany The rhizobial community indigenous to the Okavango region has not yet been characterized. The isolation of indigenous rhizo- bia can provide a basis for the formulation of a rhizobial inoculant. Moreover, their identification and characterization contrib- ute to the general understanding of species distribution and ecology. Isolates were obtained from nodules of local varieties of the pulses cowpea, Bambara groundnut, peanut, hyacinth bean, and common bean. Ninety-one of them were identified by BOX re- petitive element PCR (BOX-PCR) and sequence analyses of the 16S-23S rRNA internally transcribed spacer (ITS) and the recA, glnII, rpoB, and nifH genes. A striking geographical distribution was observed. Bradyrhizobium pachyrhizi dominated at sam- pling sites in Angola which were characterized by acid soils and a semihumid climate. Isolates from the semiarid sampling sites in Namibia were more diverse, with most of them being related to Bradyrhizobium yuanmingense and Bradyrhizobium daqin- gense. Host plant specificity was observed only for hyacinth bean, which was nodulated by rhizobia presumably representing yet-undescribed species. Furthermore, the isolates were characterized with respect to their adaptation to high temperatures, drought, and local host plants. The adaptation experiments revealed that the Namibian isolates shared an exceptionally high temperature tolerance, but none of the isolates showed considerable adaptation to drought. Moreover, the isolates’ performance on different local hosts showed variable results, with most Namibian isolates inducing better nodulation on peanut and hyacinth bean than the Angolan strains. The local predominance of distinct genotypes implies that indigenous strains may exhibit a better performance in inoculant formulations. T he catchment area of the Okavango River extends across parts of the three sub-Saharan African countries of Angola, Na- mibia, and Botswana and is mainly characterized by smallholder and subsistence farming. Since cultivation techniques remain un- derdeveloped, e.g., in terms of irrigation and the application of agrochemicals or rhizobial inoculants, local farmers are con- fronted with low yields and decreasing soil fertilities (1). Pulses like cowpea (Vigna unguiculata), Bambara groundnut (Vigna sub- terranea), peanut (Arachis hypogaea), hyacinth bean (Lablab pur- pureus), and common bean (Phaseolus vulgaris) are cultivated and occasionally intercropped, but natural nodulation rates are often low (2), presumably due to a seasonal reduction of the rhizobial population caused by drought and heat. Thus, nitrogen input via symbiotic N 2 fixation might be very limited. Field-grown cowpea plants in Ghana have been reported to fix more than 200 kg N/ha (89% of plant N) (3). In contrast, values of only 4 to 29 kg N-fixed/ha (15 to 56% of plant N) were observed in semiarid southwestern Zimbabwe (4). In such areas, the applica- tion of a rhizobial inoculant may thus hold the potential to in- crease plant nutrition and soil fertility. Many effective rhizobial strains have been identified and are available. However, it has been observed that rhizobial strains often perform poorly under condi- tions dissimilar to their original habitat (5–7) and that their effec- tiveness depends on environmental factors such as soil tempera- ture (5, 6, 8), soil pH (9), soil texture (7), and host plant variety (10), an issue which might be especially relevant for the Okavango region with its harsh climate, heterogeneous soils, and local plant varieties. In this regard, Law et al. (7) already reported that a prominent inoculant strain had no effect on cowpea and peanut grown in Botswana. Furthermore, soybean inoculant application increased seed yields at only one out of three locations in South Africa (9). Hence, inoculant formulations consisting of effective indigenous rhizobia being adapted to the local conditions may exhibit a better performance. Most grain legume species grown in the Okavango region are known to be almost exclusively nodulated by a wide range of Bra- dyrhizobium spp. which were originally grouped as heterogeneous “cowpea miscellaneous rhizobia” according to, and solely based on, their host range. A more precise classification to discrete spe- cies is hampered by the exceptional conservation of the 16S rRNA gene sequence, routinely used as a marker for species discrimina- tion, in the genus Bradyrhizobium (e.g., see references 11 and 12). Thus, for a long time the genus consisted of only three described species: Bradyrhizobium japonicum (13), already described in 1896 by Kirchner; Bradyrhizobium elkanii (14); and Bradyrhizo- bium liaoningense (15). Alternative markers such as the 16S-23S rRNA intergenic spacer (internally transcribed spacer [ITS]) (11, 12, 16, 17) and the multilocus sequence analysis (MLSA) ap- proach to bradyrhizobia (e.g., see references 18 to 20) allowed uncovering of a high species diversity inside the genus Bradyrhi- zobium. It currently consists of 18 validly published species (LPSN database [21]). Studies on soybean-nodulating bradyrhizobia already revealed a clear species geographical distribution related to soil tempera- Received 21 July 2014 Accepted 10 September 2014 Published ahead of print 19 September 2014 Editor: C. R. Lovell Address correspondence to Barbara Reinhold-Hurek, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AEM.02417-14. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.02417-14 7244 aem.asm.org Applied and Environmental Microbiology p. 7244 –7257 December 2014 Volume 80 Number 23 on October 1, 2018 by guest http://aem.asm.org/ Downloaded from

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Page 1: Rhizobia Indigenous to the Okavango Region in Sub …aem.asm.org/content/80/23/7244.full.pdf · ture (8), soil pH (22), and climate (22–24). Furthermore, even though it is well

Rhizobia Indigenous to the Okavango Region in Sub-Saharan Africa:Diversity, Adaptations, and Host Specificity

Jann L. Grönemeyer, Ajinkya Kulkarni, Dirk Berkelmann, Thomas Hurek, Barbara Reinhold-Hurek

Department of Microbe-Plant Interactions, Faculty of Biology and Chemistry, Center for Biomolecular Interactions Bremen (CBIB), University of Bremen, Bremen, Germany

The rhizobial community indigenous to the Okavango region has not yet been characterized. The isolation of indigenous rhizo-bia can provide a basis for the formulation of a rhizobial inoculant. Moreover, their identification and characterization contrib-ute to the general understanding of species distribution and ecology. Isolates were obtained from nodules of local varieties of thepulses cowpea, Bambara groundnut, peanut, hyacinth bean, and common bean. Ninety-one of them were identified by BOX re-petitive element PCR (BOX-PCR) and sequence analyses of the 16S-23S rRNA internally transcribed spacer (ITS) and the recA,glnII, rpoB, and nifH genes. A striking geographical distribution was observed. Bradyrhizobium pachyrhizi dominated at sam-pling sites in Angola which were characterized by acid soils and a semihumid climate. Isolates from the semiarid sampling sitesin Namibia were more diverse, with most of them being related to Bradyrhizobium yuanmingense and Bradyrhizobium daqin-gense. Host plant specificity was observed only for hyacinth bean, which was nodulated by rhizobia presumably representingyet-undescribed species. Furthermore, the isolates were characterized with respect to their adaptation to high temperatures,drought, and local host plants. The adaptation experiments revealed that the Namibian isolates shared an exceptionally hightemperature tolerance, but none of the isolates showed considerable adaptation to drought. Moreover, the isolates’ performanceon different local hosts showed variable results, with most Namibian isolates inducing better nodulation on peanut and hyacinthbean than the Angolan strains. The local predominance of distinct genotypes implies that indigenous strains may exhibit a betterperformance in inoculant formulations.

The catchment area of the Okavango River extends across partsof the three sub-Saharan African countries of Angola, Na-

mibia, and Botswana and is mainly characterized by smallholderand subsistence farming. Since cultivation techniques remain un-derdeveloped, e.g., in terms of irrigation and the application ofagrochemicals or rhizobial inoculants, local farmers are con-fronted with low yields and decreasing soil fertilities (1). Pulseslike cowpea (Vigna unguiculata), Bambara groundnut (Vigna sub-terranea), peanut (Arachis hypogaea), hyacinth bean (Lablab pur-pureus), and common bean (Phaseolus vulgaris) are cultivated andoccasionally intercropped, but natural nodulation rates are oftenlow (2), presumably due to a seasonal reduction of the rhizobialpopulation caused by drought and heat. Thus, nitrogen input viasymbiotic N2 fixation might be very limited.

Field-grown cowpea plants in Ghana have been reported to fixmore than 200 kg N/ha (89% of plant N) (3). In contrast, values ofonly 4 to 29 kg N-fixed/ha (15 to 56% of plant N) were observed insemiarid southwestern Zimbabwe (4). In such areas, the applica-tion of a rhizobial inoculant may thus hold the potential to in-crease plant nutrition and soil fertility. Many effective rhizobialstrains have been identified and are available. However, it has beenobserved that rhizobial strains often perform poorly under condi-tions dissimilar to their original habitat (5–7) and that their effec-tiveness depends on environmental factors such as soil tempera-ture (5, 6, 8), soil pH (9), soil texture (7), and host plant variety(10), an issue which might be especially relevant for the Okavangoregion with its harsh climate, heterogeneous soils, and local plantvarieties. In this regard, Law et al. (7) already reported that aprominent inoculant strain had no effect on cowpea and peanutgrown in Botswana. Furthermore, soybean inoculant applicationincreased seed yields at only one out of three locations in SouthAfrica (9). Hence, inoculant formulations consisting of effective

indigenous rhizobia being adapted to the local conditions mayexhibit a better performance.

Most grain legume species grown in the Okavango region areknown to be almost exclusively nodulated by a wide range of Bra-dyrhizobium spp. which were originally grouped as heterogeneous“cowpea miscellaneous rhizobia” according to, and solely basedon, their host range. A more precise classification to discrete spe-cies is hampered by the exceptional conservation of the 16S rRNAgene sequence, routinely used as a marker for species discrimina-tion, in the genus Bradyrhizobium (e.g., see references 11 and 12).Thus, for a long time the genus consisted of only three describedspecies: Bradyrhizobium japonicum (13), already described in1896 by Kirchner; Bradyrhizobium elkanii (14); and Bradyrhizo-bium liaoningense (15). Alternative markers such as the 16S-23SrRNA intergenic spacer (internally transcribed spacer [ITS]) (11,12, 16, 17) and the multilocus sequence analysis (MLSA) ap-proach to bradyrhizobia (e.g., see references 18 to 20) alloweduncovering of a high species diversity inside the genus Bradyrhi-zobium. It currently consists of 18 validly published species (LPSNdatabase [21]).

Studies on soybean-nodulating bradyrhizobia already revealeda clear species geographical distribution related to soil tempera-

Received 21 July 2014 Accepted 10 September 2014

Published ahead of print 19 September 2014

Editor: C. R. Lovell

Address correspondence to Barbara Reinhold-Hurek, [email protected].

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.02417-14.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.02417-14

7244 aem.asm.org Applied and Environmental Microbiology p. 7244 –7257 December 2014 Volume 80 Number 23

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Page 2: Rhizobia Indigenous to the Okavango Region in Sub …aem.asm.org/content/80/23/7244.full.pdf · ture (8), soil pH (22), and climate (22–24). Furthermore, even though it is well

ture (8), soil pH (22), and climate (22–24). Furthermore, eventhough it is well established that the host plant compatibility of arhizobial strain is generally dependent on the quality of its symlocus (19, 25, 26), chromosomal backgrounds can also have adecisive influence on the selection by the host plant (27–29). Thus,the assignment of a bradyrhizobial strain to a distinct species cangenerate information on adaptational properties, which in turnaccount for a strain’s suitability to serve as an inoculant at a par-ticular site.

Here, we report on the distribution and adaptation of rhizobiain the Okavango region and the identification of strains that holdthe potential to be used for the inoculation of locally grown pulses.

MATERIALS AND METHODSSampling and isolation of rhizobia from root nodules. Root noduleswere collected from the grain legumes cowpea (Vigna unguiculata), Bam-bara groundnut (V. subterranea), peanut (Arachis hypogaea), hyacinthbean (Lablab purpureus), and common bean (Phaseolus vulgaris). Theywere stored in 2-ml glass vials with silica gel desiccant at room tempera-ture during the field trip and at 4°C upon arrival in Bremen, Germany.The surveyed plants were growing in different soils, mainly on small-holder farms in the Namibian Mashare and the Angolan Cusseque areas(Fig. 1 and Table 1). Sampling dates and sites and sampling and isolationprocedures on modified arabinose gluconate (MAG) (30) medium aregiven in the work of Grönemeyer et al. (2). The isolates were designatedconsidering the individual host plant or soil sample (first number), therespective nodule (second number), and an isolate number (third num-ber). In cases where only two numbers are given, they correspond to theindividual host plant and the isolate number. For detailed information onsoil properties and climate, see the work of Gröngröft et al. and of Weber(31, 32). Soils were transported to Germany in plastic bags to preventdrying and under cooling (around 4°C). For trapping of rhizobia, a tea-spoon of the respective soil sample was used to inoculate cowpea andBambara groundnut seedlings which were grown aseptically in Magentajars containing vermiculite supplemented with half-concentrated N-freeJensen’s medium (33) as described for the cross-inoculation experiments.Root nodules were taken 3 weeks postinoculation.

Samples were collected under the research and collection permits1569/2011, 1635/2011, 1757/2012, and 1780/2013 and export permits83786, 90409, and 90990.

BOX repetitive element PCR (BOX-PCR) and amplification of the16S-23S rRNA ITS, nifH, and housekeeping genes. The templates for allPCR applications were crude extracts. A portion of a bacterial colony wassuspended in 10 �l Lyse and Go PCR reagent (Thermo Fisher ScientificInc., Rockford, IL, USA), which was then subjected to thermal cyclingaccording to the manufacturer’s instructions to release genomic DNA.

The suspension was briefly centrifuged, and 2 �l of the supernatant servedas a template in one PCR mixture.

PCR mixtures were composed of 2.5 U MolTaq DNA polymerase(Molzym, Bremen, Germany), 5 �l of the respective 10� PCR buffer, 50�M (each) deoxynucleoside triphosphate (dNTP), 0.5 �M (each) primer,and 2 �l template in a total volume of 50 �l. The 16S-23S rRNA gene ITSwas amplified using the primer pair FGPS1490/FGPS132= under the cy-cling conditions described by Laguerre et al. (34) with increased annealingtemperature at 58°C. Partial amplification of glnII, recA, and rpoB frag-ments was achieved using the primer pairs and cycling parameters re-ported previously (19, 23). Amplification of nifH fragments was con-ducted with primers FGPH19/PolR (35) under the cycling conditionsdescribed by Demba-Diallo et al. (36).

For BOX-PCR, the reaction mix of 50 �l consisted of 2.5 U DreamTaqDNA polymerase (Thermo Fisher Scientific), 5 �l of the respective 10�PCR buffer, 100 �M (each) dNTP, 2 �M primer BOXA1R (37), and 2 �lof template. DNA amplification was carried out in a Biometra TProfes-sional Thermocycler (Biometra, Göttingen, Germany) conducting 35 cy-cles under the cycling conditions described previously (37). PCR productswere separated by horizontal gel electrophoresis on 1.5% (wt/vol) agarosegels in Tris-acetate-EDTA (TAE) buffer for 1.5 h at 90 V. Ethidium bro-mide-treated gels were scanned with a Typhoon FLA 9500 scanner (GEHealthcare Life Sciences).

Sequencing and phylogenetic analysis. Amplification products wereeither purified using the QIAquick PCR purification kit (Qiagen, Hilden,Germany) or custom purified before Sanger sequencing (LGC Genomics,Berlin, Germany). Sequencing primers were the same forward primerspreviously used for PCR amplification, except for nifH (primer PolF)(35).

Multiple sequence alignments were generated using either ClustalWversion 5.1 (38) or MUSCLE (39) incorporated in the MEGA 5.2 software(40). For ITS sequences, ClustalW was used and the gap open/extensionpenalties were set to 20/6.66 for the pairwise and 50/6.66 for the multiplealignment, respectively. The alignment was visually inspected and manu-ally edited. Positions containing gaps were deleted in the phylogeneticanalysis, and distances were calculated from the number of differences assuggested by Willems et al. (12). For the analysis of protein-coding se-quences, the MUSCLE aligner was used under default settings. Thealigned housekeeping gene fragment sequences were manually trimmedto remove overhangs and analyzed individually as well as in a concate-nated fashion. The best-fit models of evolution were determined usingMODELTEST (41) integrated in MEGA5 software, and the substitutionmodel with the lowest Bayesian information criterion (BIC) (42) scorewas chosen for a maximum likelihood-based phylogenetic analysis. Thereliability of the tree topology was estimated by conducting a bootstraptest (43) with 500 pseudoreplicates.

Tests for temperature tolerance and osmotolerance of isolates. Theisolates’ maximum growth temperature was surveyed on MAG agar slants(at 28.5, 35.5, and 38.5°C � 0.5°C each) as well as in liquid cultures.Precultures grown at 28°C in MAG medium were adjusted to an opticaldensity at 600 nm (OD600) of 0.2, and 25 �l was used for inoculation of25-ml test cultures. Test cultures were grown on a rotary shaker (200 rpm)at 35.5°C (�0.5°C) or in an agitating water bath at higher temperatures(38, 40, and 42°C). Cultures were inspected for visible growth within 7days of incubation. To survey the isolates’ drought tolerance, MAG me-dium was supplemented with various amounts of polyethylene glycol(PEG) 6000 (Rotipuran; Carl Roth, Karlsruhe, Germany) (15, 20, 25, and30% [wt/vol]), and cultures were grown on a rotary shaker with 200 rpmat 28°C. Bacterial density was monitored at OD600 over a period of 3weeks.

Cross-inoculation experiments. The isolates were tested for theirability to nodulate cowpea variety Lutembwe as well as local varieties ofcowpea, Bambara groundnut, peanut, hyacinth bean, and common bean.Hand-sorted beans free of visible damage and of uniform size were surfacesterilized by rinsing them in 70% ethanol for 10 s followed by three wash-

FIG 1 Okavango region and core sampling sites.

Rhizobial Community Indigenous to the Okavango Region

December 2014 Volume 80 Number 23 aem.asm.org 7245

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Page 3: Rhizobia Indigenous to the Okavango Region in Sub …aem.asm.org/content/80/23/7244.full.pdf · ture (8), soil pH (22), and climate (22–24). Furthermore, even though it is well

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Rhizobial Community Indigenous to the Okavango Region

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ings in sterile distilled water and immersion in 2.5% sodium hypochloritefor 6 min. Seeds were again washed five times in sterile distilled water,followed by soaking in water for at least 2 h. To ensure that the seeds werethoroughly surface sterilized, randomly chosen beans were placed onMAG agar plates and inspected for microbial growth within 3 days. Sur-face-disinfected seeds were allowed to pregerminate on water agar (1.5%[wt/vol]) in the dark at 28°C for up to 4 days (depending on bean), plantedinto Magenta jars (Magenta Corp., Chicago, IL, USA) containing sterilewashed vermiculite supplemented with 100 ml of 0.5� Jensen’s medium(33), and inoculated with 109 cells of the respective isolate in 1 ml of a 1%(wt/vol) sucrose solution. Each isolate was assayed at least in duplicate,with uninoculated control plants. The plants were maintained under con-trolled conditions with day/night (11.5-h/12.5-h) temperatures of 28°C/25°C and lighting at 16.4 klx. The humidity was set to 60%, and plantswere watered once a week with sterile distilled water. After 3 (cowpea,Bambara groundnut, and hyacinth bean) to 4 (peanut and common bean)weeks of growth, the presence and appearance of root nodules were con-trolled. To test for nitrogenase activity, an acetylene reduction assay(ARA) on whole roots was carried out in sealed 80-ml glass tubes with10% (vol/vol) acetylene in the headspace. After 4 h of incubation in thedark at 28°C, a 1-ml gas sample was transferred to 3-ml Exetainers (LabcoLtd., Lampeter, Wales, United Kingdom) and stored until gas chromato-graphic quantification of ethylene.

Nucleotide sequence accession numbers. Sequences were submittedto GenBank (accession no. KM378245 to KM378555).

RESULTSIsolation of root nodule rhizobia from the Okavango region. Atotal of 91 putative rhizobia (60 from Namibia, 31 from Angola)were isolated from 86 individual root nodules of various pulses(Table 1). The 75 isolates from nodules collected in the fields werecomplemented by 16 isolates obtained from trapping from soilsamples. Among others, the trapping experiment added some An-golan rhizobia trapped by Bambara groundnut, which was notgrown on fields sampled in Angola.

As expected, the majority of the isolates (75/78) obtained fromcowpea, Bambara groundnut, peanut, and hyacinth bean wereslow growing, with single colonies becoming visible after 5 to 7days of incubation on MAG agar plates, indicating that they arebradyrhizobia. However, from two nodules (sampled from plants54 and 62), only fast-growing isolates with colony morphologytypical for Rhizobium sp. were obtained (isolates 54 3-1 and 621-1), while for another nodule from plant 54, some fast-growingcolonies (which turned out to be genotypically identical with 543-1) were observed on plates dominantly populated by slow-growing isolate 54 1-1. From common bean, most strains (7/13)were fast growing, with single colonies appearing after 1 to 2 daysof growth as expected, but slow growers were isolated also.

All isolates were initially screened for the presence of the nifHgene, and a PCR amplification product with the expected size ofabout 450 bp was observed for all of the 91 isolates except forisolate 22 2-1. As colony morphology was similar to that of bra-dyrhizobia, this strain was included in the subsequent analysis.

Grouping of isolates by BOX-PCR fingerprinting. In order toidentify similar isolates and to select representatives for furtherstudies, the BOX-PCR fingerprinting technique was applied. Theresulting patterns were highly reproducible and were rather influ-enced by PCR parameters (e.g., the thermal cycler used) than bythe PCR template (data not shown). The patterns of slow-growingisolates originating from the same individual nodule were alwaysidentical (Fig. 2), thus indicating their clonality. Most of the othervery similar patterns belonged to isolates from the same individual

plant. However, in some rare cases identical to very similar pat-terns were observed for isolates obtained from different individualplants (34 1-1 and 35 3-3; 30 2-1 and 31 1-1) as well as fromdifferent plant species (54 2-1 and 58 2-1) at different samplingsites (10-2 and 28 2-1). These results indicated that some rhizobialgenotypes are competitive in nodulation of several host plants atvarious locations. In contrast, the isolation of different genotypesfrom nodules of the same plant was rare, indicating that the indi-vidual plants were preferentially nodulated by a group of veryclosely related rhizobia at a distinct location.

The fast-growing isolates and the isolates obtained from thetrapping experiment were analyzed separately (data not shown).Fifty-six slow-growing and six fast-growing isolates, representingeither a distinct BOX-PCR pattern or an individual plant, wereselected for the subsequent analyses.

Phylogenetic classification of rhizobia. A first classificationwas based on sequence comparisons of the 16S-23S rRNA geneITS region. As expected, the ITS regions varied in length (approx-imately 800 to 1,000 bp), resulting in sections with large gaps inthe alignment. Phylogenetic trees computed with and without in-clusion of gaps led to similar but slightly different topologies (datanot shown). As Willems et al. (12) observed a better agreementwith their results from DNA-DNA hybridizations when gaps inthe ITS alignment were not penalized, we adopted this approach.GenBank accession numbers of reference sequences are listed inthe supplemental material (see Table S1 in the supplemental ma-terial). The resulting phylogenetic tree was based on 634 positionsand separated (100% and 98% bootstrap support) the B. japoni-cum well from the B. elkanii lineage (20, 23), the latter also show-ing good bootstrap support of the subclades (Fig. 3). The isolateswere highly diverse, forming several distinct clusters distributedover both major clades. As most of these clusters were composedof isolates obtained from different plant species and often evenshared identical ITS sequences, the plant origin had only a minorinfluence on the relatedness of the isolates. Exceptions were therhizobia obtained from hyacinth bean.

Interestingly, a geographic distribution was observed, with allAngolan rhizobia apart from isolate 45 1-4 clustering in proximityto Bradyrhizobium pachyrhiziT on the B. elkanii branch. For mostNamibian isolates, the ITS sequence had highest similarity to theones of Bradyrhizobium daqingenseT, Bradyrhizobium yuanmin-genseT, or Bradyrhizobium iriomotenseT located on the B. japoni-cum branch. The remaining Namibian isolates showed remark-ably low relatedness to any bradyrhizobial species type strain,suggesting that they represent novel species. This was especiallyevident for the isolates 26 3-1 and 18C 2-1 early branching fromthe B. elkanii lineage as well as for isolates 5-10 and 30 3-2.

The isolates were grouped by their sequence identity to facilitatecomparisons with the phylogeny of concatenated housekeepinggenes and to select representatives for the adaptation experiments.The genospecies threshold of 95.5% ITS sequence identity proposedpreviously (44) was not chosen because higher similarities amongdifferent species were observed in our sequence comparisons: Brady-rhizobium betaeT shared 96.7% and 96.0% of its ungapped ITSsequence with Bradyrhizobium canarienseT and BradyrhizobiumdiazoefficiensT, respectively. Furthermore, some other pairs of typestrains showed similarities slightly above 95.5%, which were B.canarienseT and Bradyrhizobium cytisiT (95.9%), B. canarienseT

and B. diazoefficiensT (95.9%), B. betaeT and B. liaoningenseT

(95.6%), B. cytisiT and B. japonicumT (95.9%), Bradyrhizobium

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lablabiT and Bradyrhizobium jicamaeT (95.7%), and B. yuanmin-genseT and Bradyrhizobium huanghuaihaienseT (95.6%), respec-tively. Therefore, the threshold was set to 97%, resulting in 15distinct clusters with �98% sequence identity among the isolateswithin one cluster. Isolates from clusters IV and XIII showed highsequence similarities to B. yuanmingenseT (96.8 to 97.1%) and B.pachyrhiziT (98.4 to 98.7%), respectively, strongly supporting theidea that they are members of the corresponding species. In con-trast, the sequence identity to the closest related species type strainwas rather low for clusters IX (88.7 to 88.9%) and XI (91.7%).

To better support this classification, the evolutionary history ofpartial sequences of the glnII, recA, and rpoB genes was analyzedindividually and in a concatenated fashion. The proportion of theindividual genes in the 1,625-bp-long concatenated sequences was505 bp for glnII, 381 bp for recA, and 739 bp for rpoB. The best-fitmodel of evolution evaluated by MODELTEST was the generaltime reversible model with a gamma-distributed rate heterogene-ity and invariant sites (GTR �G �I). The calculated phylogenetictree corroborated geographic distribution of the isolates, as theclustering appeared to be very similar to the one obtained fromITS sequence analysis (Fig. 4). Some inconsistencies could befound in early branching inside the B. japonicum lineage, mostprobably deriving from the fact that the rapidly evolving ITS lacks

resolution at higher taxonomic ranks. One conspicuous differencewas the separation of isolates 2-13 and 22 2-1 from previous clus-ter III, with B. liaoningenseT showing a close relationship to isolate22 2-1. While the divergence of 2-13 and 22 2-1 was observed foreach individual housekeeping gene (see Fig. S1 to S3 in the sup-plemental material), the shift of B. liaoningenseT was mainly basedon rpoB phylogeny (Fig. S3).

The range of sequence variation of concatenated sequences wassimilar to the one observed for the ITS sequences, but in several cases,the resolution was slightly increased, discriminating isolates withidentical ITS sequences. The sequence identity between species typestrains was up to 96.3% (B. lablabiT and B. jicamaeT); thus, theisolates were grouped by 97% sequence identity. The resulting 14groups were almost identical to the ITS-based ones, giving goodsupport in the overall classification of the isolates. Exceptions werethe change of isolate 2-13 to cluster I and merging of ITS clustersXIII to XV. One additional cluster resulted from isolate 36 1-1 notoccurring in ITS phylogeny. For 36 1-1, the ITS PCR yielded twoproducts slightly different in size which were thus not assigned inFig. 3, indicating that this isolate is one of the rare Bradyrhizobiumspecies which exhibit more than one rRNA operon (44). Thus,assignment based on housekeeping genes may be more reliable.

Additionally, the relatedness of the isolates’ symbiotic islands

FIG 2 BOX-PCR fingerprints of isolates from nodules. Solid and dashed lines indicate identical or very similar genotypes, respectively.

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FIG 3 Neighbor-joining phylogram inferred from ITS sequences of bradyrhizobial isolates and species type strains. Rhodopseudomonas palustrisT was includedas an outgroup strain. A bootstrap value is indicated when a given node appeared in �50% of 500 pseudoreplicates. The following symbols indicate the plantorigin of the isolates: �, Namibian cowpea; Œ, Namibian Bambara groundnut; �, Namibian peanut; }, Namibian hyacinth bean; p, Angolan cowpea; o,Angolan Bambara groundnut; �, Angolan common bean. Isolates labeled with “(soil)” were trapped from soil samples. Roman numerals cluster isolates with�97% sequence identity. The scale bar indicates the number of base differences per sequence.

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FIG 4 Maximum likelihood phylogram inferred from glnII-recA-rpoB sequence concatemers based on the GTR �G �I model. Rhodopseudomonas palustrisBisB5 was included as an outgroup strain. A bootstrap value is indicated when a given node appeared in �50% of 500 pseudoreplicates. The following symbolsindicate the plant origin of the isolates: �, Namibian cowpea; Œ, Namibian Bambara groundnut; �, Namibian peanut; }, Namibian hyacinth bean;p, Angolancowpea;o, Angolan Bambara groundnut; �, Angolan common bean. Isolates labeled with “(soil)” were trapped from soil samples. Roman numerals clusterisolates with �97% sequence identity. The scale bar indicates the number of substitutions per site.

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was investigated. As several studies reported a congruent phylog-eny among nodulation genes and nifH (45–47), the latter wasregarded to be representative. The final alignment consisted of 286nucleotides, and the Tamura 3-parameter model with gamma-distributed evolutionary rates among sites (T92 �G) was the best-fit model according to MODELTEST analysis. The resulting phy-logenetic tree showed a slightly differing topology from theprevious ones (Fig. 5), which might be due to some strain se-quences not included in both alignments or to lateral gene transferof nifH. Three main clusters were observed, the first one (A) in-cluding most Namibian isolates, the second one (B) exclusivelyharboring Angolan isolates, and a third one (C) containing theNamibian isolates from the previous B. elkanii branch which weremainly obtained from the newly introduced hyacinth bean.

Adaptation to temperature and water stress. The rhizobiawere subjected to high growth temperatures and water stress toevaluate their adaptation to the harsh Okavango climatic condi-tions. Nine bradyrhizobial species type strains, which showed thehighest degree of relatedness to the isolates in the phylogeneticanalyses, were included as references. Furthermore, stress toler-ance of the indigenous rhizobia was compared to that of threeUSDA strains (U.S. Department of Agriculture, Beltsville, MD,USA), which are recommended for the inoculation of legumes insub-Saharan Africa (Table 1).

To test their ability to grow at high temperatures, rhizobia weregrown on solid (28, 35, and 38°C) as well as in liquid medium at28, 35, 38, 40, and 42°C. On solid media, higher maximal growthtemperatures were reached (Table 1), which was already observedelsewhere (48). Most type strains and all Angolan bradyrhizobiaexcept for 45 1-4 did not grow in liquid medium at 35°C, whichwas also the case for two of the USDA strains. However, for severalspecies type strains the observed temperature tolerances were in-consistent with their original description, which might be a matterof different growth media. In contrast, most Namibian isolatesproliferated at 38°C, with some even tolerating growth tempera-tures as high as 40°C. The Namibian isolates most sensitive to hightemperatures were the ones obtained from hyacinth bean belong-ing to cluster VIII and one isolate from ITS cluster I. The pheno-types were for most strains in agreement with the genotypicgrouping. To investigate the isolates’ drought tolerance, waterstress was induced by the addition of PEG 6000 to the liquidgrowth medium at final concentrations of 15, 20, 25, and 30%(wt/vol). In a pretest, the influence of PEG on growth rates withinthe first 3 days postinoculation was monitored for a selection ofisolates. In the presence of lower PEG concentrations (15%),growth kinetics were characterized by longer lag phases (or celldeath due to osmotic shock), after which doubling times ap-proached the ones observed without PEG (not shown), Thus,these conditions were not selected for differentiation. At PEG con-centrations of 20%, longer lag phases were detected and doublingtimes in the exponential phase were strongly increased. In order totest the isolates’ general ability to grow under different concentra-tions of PEG, we thus finally used extended incubation times.Rhizobium bacteria were grown for 1 week, and slow-growing Bra-dyrhizobium bacteria were surveyed over a period of 3 weeks. Al-most all bradyrhizobial cultures became stationary within 1 weekof growth at 15% PEG and 2 weeks of growth at 20% PEG, andnone of the bradyrhizobial isolates showed detectable growth athigher PEG concentrations (Table 1). As similar results were ob-tained for the species type strains, no adaptation exclusive to in-

digenous rhizobia was detected. In contrast, three isolates (bothhyacinth bean isolates forming cluster XI and isolate 45 1-4) werenot even growing at 15% PEG and therefore were highly suscep-tible to water stress. All isolates identified as Rhizobium sp. toler-ated higher PEG concentrations of up to 30%.

Cross-inoculation of groups of isolates. The isolates weretested for their ability to nodulate a range of locally grownlegumes, mostly belonging to the “cowpea miscellany” cross-in-oculation group (cowpea, Bambara groundnut, hyacinth bean,and peanut). The only exception was the common bean, belong-ing to the bean cross-inoculation group. In agreement with thecross-inoculation group concept, isolates which were identified asRhizobium sp. formed nitrogen-fixing nodules on common beanbut failed to nodulate all other plants (Table 1).

Among the bradyrhizobial isolates, three (3-2, 21 1-1, and 22 2-1)did not induce root nodules on any of the legumes tested, indicatingthat they are not symbiotic. This was especially surprising for isolates3-2 and 21 1-1, which possessed the nifH gene and were highly relatedto nodulating isolates. Furthermore, B. liaoningenseT failed to nod-ulate any legume under the selected conditions. All other brady-rhizobia induced root nodules on both Vigna species (cowpea andBambara groundnut) and were actively fixing nitrogen as deter-mined by acetylene reduction assays of nodulated roots.

Variable results were observed for hyacinth bean and peanut.Even though almost all isolates were able to nodulate hyacinthbean, remarkable variation in nodule size was observed, correlat-ing with previously defined groups. All isolates located on branchC in nifH phylogeny (groups VIII to XI) induced 10 to 30 bignodules of up to 5 mm in diameter, while for most isolates fromnifH branch A (groups II to VII), nodules appeared to be smaller(up to 3 mm in diameter). Nevertheless, the nitrogen fixationactivities determined by the ARA were similar (data not shown).In contrast, when Angolan isolates from nifH branch B (groupsXIII to XV except for isolate 46 1-1) were used for inoculation,root nodules remained tiny (mostly about 1 mm in diameter), andno nitrogen fixation could be detected with a detection limit cor-responding to 50 ppm ethylene in 80 ml of headspace. This resultwas corroborated by the observation that the plants were appar-ently suffering nitrogen shortage, exhibiting light green leaves andceasing to grow upon prolonged time (data not shown).

For peanut, the results were similar to the ones observed forthe hyacinth bean: while the bulk of isolates induced effectivenodulation, nodules did not fully develop and no nitrogen fix-ation activity was measured when peanuts were inoculatedwith Angolan isolates (see Fig. S4 in the supplemental mate-rial). Moreover, isolates from two more groups, which wereITS groups I and IX located on nifH branches A and C, respec-tively, failed to nodulate. However, moderate nodulation andsome nitrogenase activity could be detected for isolate 6-8 fromgroup I. Again, the different degrees of effectiveness could beclearly seen from various green color intensities of the leavesafter 4 weeks of growth (not shown).

Apart from the four strains mentioned above, which did notnodulate any plant, all bradyrhizobia induced whitish and irregu-larly shaped nodule-like structures on common bean, as previ-ously observed (49). The plants showed clear symptoms of Nshortage, and no N2 fixation could be detected by ARA (see Fig. S4in the supplemental material).

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FIG 5 Maximum likelihood phylogram inferred from nifH sequences based on the T92 �G model. Rhodopseudomonas palustrisT was included as an outgroupstrain. A bootstrap value is indicated when a given node appeared in �50% of 500 pseudoreplicates. The following symbols indicate the plant origin of theisolates: �, Namibian cowpea; Œ, Namibian Bambara groundnut; �, Namibian peanut; }, Namibian hyacinth bean;p, Angolan cowpea;o, Angolan Bambaragroundnut; �, Angolan common bean. Isolates labeled with “(soil)” were trapped from soil samples. The scale bar indicates the number of substitutions per site.Grouping is not based on a sequence identity threshold.

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DISCUSSION

Our study reports on the rhizobial population in the OkavangoRiver region, which is characterized by a harsh climate, heteroge-neous soils, and local pulse varieties. Since no inoculation historyis known at the sampled sites, the isolates are expected to representthe indigenous rhizobial population that is adapted to the localenvironmental conditions. Thus, our results contribute to thegeneral understanding of regional species abundance. Moreover,they provide a basis for the formulation of a rhizobial inoculantmatching the local settings at the best.

As could be expected from the widely recognized compatibilityof the sampled pulses, the spectrum of isolates comprised bothBradyrhizobium sp. and Rhizobium sp., which were mainly ob-tained from “cowpea group” pulses and common bean, respec-tively. However, occasionally Rhizobium bacteria were isolatedfrom cowpea as well as Bradyrhizobium species from commonbean. Even though other studies reported having isolated fast-growing rhizobia nodulating cowpea (50–52), all our Rhizobiumisolates failed to nodulate any plant except for the common beanand did thus not represent the actual symbionts. This might alsobe the case for Bradyrhizobium spp. obtained from common bean.However, the cross-inoculation experiments revealed that all bra-dyrhizobial isolates induced ineffective nodules on the commonbean. Even though it was not tested whether these structures werepseudonodules or contained rhizobia, the rather unusual isolationof bradyrhizobia from common bean could also be explained bythe sampling of some ineffective nodules. In rare cases, the rhizo-bia did not effectively nodulate their original host plant under theselected conditions. For some peanut isolates, it was confirmedthat they were symbionts as they nodulated cowpea and Bambaragroundnut, which might be affected by the experimental growthconditions.

The bradyrhizobial population in the Okavango region ap-peared to be diverse, being divided into 15 groups based on un-gapped ITS sequence comparisons. While it is best to considermore than one gene for taxonomic inferences (53) with some-times-limited threshold suitability (54), bradyrhizobial strainswith more than 95.5% ungapped ITS sequence similarity usuallybelong to the same genospecies (44). However, a higher thresholdof 97% was chosen here, since the common genospecies cutoffvalue would have merged several species type strains in our se-quence comparisons. Although our analyses of physiologicalproperties were limited to maximum growth temperature, PEGtolerance, and host range, members of the same group mostlybehaved uniformly and could be clearly discriminated fromclosely related groups of isolates; whether they indeed representdifferent species needs to be elucidated in future. For example,isolates from cluster IV showed a maximal growth temperature of40°C, which was 35°C for cluster V and 38°C for cluster VI andVII, the latter two being differentiable by their susceptibility toPEG. ITS clusters XIII to XV were phenotypically similar as well,supported by being grouped together in core gene analysis. Dis-similar maximum growth temperatures of isolates 2-13 and 22 2-1supported their separation as indicated by phylogeny of house-keeping genes.

A clear, striking geographic distribution of bradyrhizobial spe-cies was uncovered. Except for one genotype (45 1-4), all Angolanbradyrhizobia were located on the B. elkanii major clade in thephylogenetic analyses, most of them being closely related to B.

pachyrhiziT. These isolates clustered together in all phylogeneticanalyses and behaved uniformly and identically to B. pachyrhiziT,indicating that they belong to the same species. Albeit promiscu-ous cowpea was grown and a high pedodiversity was observed(55), B. pachyrhizi appears to be a predominant species at thesampled sites of the Cusseque area in Angola. Reports on B. pachy-rhizi are rare, and not much is yet known about its distributionand ecology. A high proportion of B. pachyrhizi bacteria was de-tected under different land use systems in Mexican tropics usingcowpea and siratro (Macroptilium atropurpureum) as trap plants(56). The Mexican sampling sites were characterized by a warm,humid climate and acid soils (pH 4 to 6), strikingly matching thewarm, semihumid climate with an annual mean rainfall of 987mm and an annual mean temperature of 20.4°C (57) in theCusseque area. Almost all soils were also extremely acidic (pH 3.5to 4.4). Except for peaty wetland soils, they are characterized bysmall amounts of nitrogen and carbon (55). Even though the orig-inal host plant Pachyrhizus erosus (54) was usually present at thesampling sites in Mexico, these correlations indicate that B. pachy-rhizi is most competitive and can be preferentially found undersuch conditions, which is in line with its capability to grow at lowpH (pH 4.5) (54). In another report, Bradyrhizobium strainSEMIA 6099, a commercial inoculant strain in Brazil, was identi-fied as B. pachyrhizi (20). Possessing a core gene equipment wellmatching the environmental conditions, such effective B. pachy-rhizi strains might be the most promising candidates for the for-mulation of an inoculant for the Angolan Cusseque area. Interest-ingly, the B. pachyrizi isolates showed only low effectiveness in thecross-inoculation experiments on peanut, which was not culti-vated on the sampled fields.

In contrast, B. pachyrhizi was not found at the Namibian sam-pling sites in the Mashare area, which were characterized by asemiarid climate with a lower annual mean rainfall of 571 mm andhigher annual mean temperature of 22.3°C (32). Soils in theMashare area are mostly characterized by low nutrient content aswell, but higher pH variability, most being only slightly acidic (pHof approximately 4 to 6) (31). Commonly grown pulses are morediverse, with mostly cowpeas but also Bambara groundnuts andpeanuts. Concordantly, the Namibian bradyrhizobia were phylo-genetically and physiologically more diverse, with the bulk beingrelated to B. yuanmingense and B. daqingense. While B. daqingensewas described just recently (58) and information is limited, B.yuanmingense seems to be widespread in the subtropics and trop-ics and was found, e.g., in China (59), India and Mexico (23), Peru(60), Botswana (61), and South Africa (10). Moreover, the Na-mibian isolates were very diverse even within the groups, as couldbe seen in the BOX-PCR fingerprinting. The overall higher diver-sity among Namibian isolates might to some extent be explainedby a higher diversity of legume plants; however, most indigenousNamibian pulses obviously shared the same bradyrhizobial spe-cies (Table 1 or phylogenetic trees). Some influence on the nodu-lating bradyrhizobial population could be found in soil heteroge-neity and land use, but often members of one group originatedfrom different locations under different agricultural practices(Table 1). Semiarid conditions might be another relevant factoraccounting for the higher diversity. The diversity of bradyrhizo-bial nodulating populations was found to increase under water-limited conditions (62). Even though more diverse, almost all Na-mibian isolates shared the adaptational trait of being able to growat much higher temperatures than Angolan isolates and most spe-

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cies type strains. Remarkably, the only species type strains show-ing similar temperature adaptation in our tests, which were some-times inconsistent with the literature, were B. yuanmingenseT andB. daqingenseT, which might explain the frequent detection of B.yuanmingense in warm regions. The high temperature tolerance ofNamibian isolates of up to 40°C makes it a prerequisite for prom-ising inoculant strains in this region. In this regard, only inoculantstrain USDA3834, originally isolated in Nigeria, showed moderateadaptation to high temperatures and grew up to 35°C.

Despite the relatively high diversity in the overall spectrum ofisolates, none of them fell into the cluster of B. japonicum, B.diazoefficiens, B. cytisi, B. canariense, and B. liaoningense. Otherstudies already indicated that these species might be rare in sub-Saharan Africa, since Steenkamp et al. (61) detected neither B.japonicum, B. canariense, nor B. liaoningense in their survey oncowpea- and peanut-nodulating strains in Botswana and SouthAfrica. Furthermore, in a comprehensive study on rhizobia nod-ulating cowpea with nine genotypes conducted in Ghana, Bo-tswana, and South Africa, B. liaoningense was not detected and B.japonicum was found only on one variety grown in South Africa(10). In the former study, it was speculated that the absence of B.canariense and B. liaoningense in the isolate spectrum might be dueto their inability to nodulate cowpea and peanut. B. liaoningenseT

indeed failed to nodulate any of the local plant varieties tested inour cross-inoculation experiment. Also, the rather pH-neutralsoils which were sampled might be relevant for lack of B. liaonin-gense (61), since it is assumed that B. canariense and B. liaoningensetolerate acidic and prefer alkaline soils, respectively (22, 63). Thebroad range of soil pHs at our sampling sites indicates that thismight not be the only crucial factor. Besides the geographical im-pact on species abundance, an influence of host plants was ob-served only for hyacinth bean, which was, under natural condi-tions, exclusively nodulated by two bradyrhizobial species; eventhough the cross-inoculations revealed that the hyacinth bean canbe nodulated by a wide range of indigenous bradyrhizobia. Fur-thermore, these strains yielded the best nodulation rates on hya-cinth bean under laboratory conditions (data not shown) andwere only distantly related to all other bradyrhizobia. Hence, theymost probably represent yet-undescribed species being welladapted to hyacinth beans. Since the hyacinth bean was newlyintroduced to the Okavango region, one can assume that theseBradyrhizobium spp. came along with the seeds, which is in agree-ment with the high susceptibility of one of the groups (VIII) tohigh growth temperatures, while the other one (XI) showed mod-erate temperature tolerance. Moreover, their nifH sequences dis-tinguished them from those of almost all other Namibian isolates,depicting a different geographical origin of their sym locus. Espe-cially in view of the fact that most studied strains from sub-Saha-ran Africa shared relatively similar nodA sequences which are lo-cated on one clade in nodA phylogeny (61), this result isremarkable. However, uncommon nifH sequences were also ob-served for a third group of strains (IX), and the hyacinth beanisolates provoked good nodulation on all indigenous Okavangopulses, which was not the case for many other strains, especiallywith respect to peanut. As one of the two groups (VIII) could alsobe trapped from a nearby maize field soil, it may spread and/orpersist in Namibian soils for at least a short period of time. Thebetter competitiveness of these strains on hyacinth bean illustratesthe benefit of being well adapted to a respective local host plant,

which has to be taken into account when introducing exogenousstrains as inoculants.

Several of our groups behaved uniquely and separated wellfrom other species in the phylogeny, indicating that they representnovel species. Among these groups, especially the taxonomic po-sition of group IX needs more clarification. ITS sequence identityin GenBank (July 2014) was found to be maximal at 86%, and thephylogeny reconstruction resulted in an early and deep branchingfrom the B. elkanii lineage for both the ITS and the housekeepinggenes. The respective ITS sequences obviously interfered with areliable phylogeny reconstruction since removing them from theanalysis yielded a much better concerned bootstrap support (in-creased from 57 to 98). Thus, from the genotype it was not evencertain if these isolates are bradyrhizobia. However, partial 16SrRNA gene sequence identity to bradyrhizobial sequences inGenBank was 99.8%, and nifH phylogeny supports a recent lateraltransfer from bradyrhizobia. Since their physiology is typical ofthe genus Bradyrhizobium, they may represent a novel bradyrhi-zobial lineage apart from the established B. japonicum and B.elkanii lineages. The existence of more such lineages was also in-dicated in a recent study (64).

Most well-studied bradyrhizobial species which have beenknown for quite some time could be isolated from different con-tinents and climatic zones and are considered to be cosmopolitan(23). Since the geographical distribution of bradyrhizobia is facil-itated by anthropogenic long-distance dispersal via seeds and in-oculants, the statement “everything is everywhere, but, the envi-ronment selects” might be especially true for rhizobia. This wasalso observed in our study owing to the isolation of B. pachyrhizi-related strains from an African environment resembling its origi-nal isolation source in Mexico. Furthermore, the presumable in-vasion of a nonindigenous species via the introduction of hyacinthbean illustrates that altering the environmental conditions maylead to a different selection by the environment.

Many prominent species, however, were not detected in our andin other surveys on sub-Sahara African bradyrhizobia, implying thatthey are less competitive under the specific environmental condi-tions. In contrast, phylogenetic analyses and comparisons of our ITSsequences with the GenBank database indicate the presence of en-demic bradyrhizobial species: except for the isolates from groups VIIIand X and the Angolan groups clustering in proximity to B. elkaniiT

and B. pachyrhiziT, all our ITS sequences either showed low iden-tity to sequences in GenBank with 86 to 94% identity at the max-imum (VI, 93%; VII, �93%; IX, 86%; XI, �94%), or theymatched sequences obtained from other sub-Saharan countries atthe best (I, 99% Senegal; II, 99% Botswana; III, 100% Senegal; IV,100% Senegal; V, 98% Senegal). In the latter cases, the maximalidentity to non-sub-Saharan sequences was often rather low (I,94% India; II, 95% China; V, �90% India). Thus, either many ofour Namibian isolates may represent local genotypes or they be-long to genotypes widespread in, and potentially restricted to,sub-Saharan countries. Similar results were observed in a recentsurvey on Australian bradyrhizobia (64), where it was found thatmost isolates were distinct from non-Australian bradyrhizobia,presumably due to geographic isolation and plant endemicity.

The geographic isolation by the Sahara Desert may have provideda basis for allopatric speciation. Heterogeneous microclimates andedaphic factors may have resulted in an unknown, highly specializedendemic species diversity in sub-Saharan Africa even at a smallerscale. This situation is supported by the fact that almost all our Na-

Rhizobial Community Indigenous to the Okavango Region

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mibian strains share an exceptional high-temperature tolerance eventhough they can be differentiated genotypically and phenotypicallyamong themselves and other bradyrhizobial species. Recently, it wasfound that two cowpea rhizobial genotypes dominated in northernand southern Senegal, respectively, and it was hypothesized that bothrepresent novel species (62).

In conclusion, we observed a predominance of distinct geno-types which were to date solely found in sub-Saharan Africawhereat in some cases the geographic distribution may be evenmore local. In view of the assumption that “the environment se-lects,” these results imply that the indigenous bradyrhizobia arehighly adapted to exclusive environmental conditions and shouldthus be preferred for the formulation of an inoculant.

ACKNOWLEDGMENTS

This work was supported by grants by the BMBF (Federal Ministry ofEducation and Research) in the framework of the project “TFO: The fu-ture Okavango” (01LL0912G) and “SASSCAL: Southern African ScienceService Centre for Climate Change and Adaptive Land Management” (01LG 1201D) to B.R.-H. and T.H.

We thank Bruce Kasoana and Roberth Mukuya for help with collec-tion of pulses.

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