Rhizobia nodulating African Acacia spp. and Sesbania sesban trees in southern Ethiopian soils are metabolically and genomically diverse

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1 Soil Biology & Biochemistry 36 (2004) Rhizobia nodulating African Acacia spp. and Sesbania sesban trees in southern Ethiopian soils are metabolically and genomically diverse Endalkachew Wolde-meskel a, *, Zewdu Terefework b, Kristina Lindström b,åsa Frostegård a a Department of Chemistry, Biotechnology and Food Science, Agricultural University of Norway, P.O. Box 5003, N-1432 Ås, Norway b Department of Applied Chemistry and Microbiology, University of Helsinki, Biocenter 1, Fin-0014 Helsinki, Finland Received 26 March 2004; received in revised form 10 May 2004; accepted 18 May 2004 Abstract The diversity of 110 rhizobial strains isolated from Acacia abyssinica, A. seyal, A. tortilis, Faidherbia albida, Sesbania sesban, Phaseolus vulgaris, and Vigna unguiculata grown in soils across diverse agro-ecological zones in southern Ethiopia was assessed using the Biologe system and amplified fragment length polymorphism (AFLP) fingerprinting technique. By cluster analysis of the metabolic and genomic fingerprints, the test strains were grouped into 13 Biolog and 11 AFLP clusters. Twenty-two strains in the Biolog method and 15 strains in the AFLP analysis were linked to eight and four reference species, respectively, out of the 28 included in the study. Most of the test strains (more than 80% of 110) were not related to any of the reference species by both methods. Forty-six test strains (42% of 110) were grouped into seven corresponding Biolog and AFLP clusters, suggesting that these groups represented the same strains, or in some cases clonal descendants of the same organisms. In contrast to the strains from S. sesban, isolates from Acacia spp. were represented in several Biolog and AFLP clusters indicating the promiscuous nature of the latter and widespread occurrence of compatible rhizobia in most of the soil sampling locations. The results showed that indigenous rhizobia nodulating native woody species in Ethiopian soils constituted metabolically and genomically diverse groups that are not linked to reference species. q 2004 Elsevier Ltd. All rights reserved. Keywords: African Acacia spp.; Amplified fragment length polymorphism (AFLP); Biolog; Ethiopia; Rhizobial diversity; Sesbania sesban 1. Introduction Woody legumes of different species are abundant in sub-saharan Africa, where they grow in a range of vegetation types, from dry lowland scrubs to moist highland forests. In Ethiopia, like in many countries in the region, it is a common practice to leave trees growing either in crop fields or on the field boundaries. These trees, which are often termed multipurpose trees (MPTs), provide several products such as timber, firewood, fodder, fruits etc. Often, the MPTs are leguminous and due to their ability to form nodules and fix nitrogen in symbiosis with rhizobia, they play an important role in the nitrogen budget * Corresponding author. Permanent address: Department of Plant Production and Dryland farming, Awassa College of Agriculture, Debub University, P.O. Box 5, Awassa, Ethiopia. address: endame@ikbm.nlh.no (E. Wolde-meskel). and are used to restore or increase soil fertility in degraded soils. Early reports indicate that tree rhizobia belong to either the fast-growing Rhizobium or the slow-growing Bradyrhizobium (Allen and Allen, 1981; Dreyfus and Dommergues, 1981). However, recent developments in molecular methods and examination of strains isolated from previously unexplored biogeographical regions revealed a hitherto unknown diversity in rhizobia and led to the description of new genera and species (Zakhia and de Lajudie, 2001). In West Africa, diverse groups of rhizobial strains were isolated from Acacia spp. and Sesbania spp. (de Lajudie et al., 1994, 1998). In a numerical taxonomy study, Zhang et al. (1991) reported a high diversity in physiological and biochemical properties of 60 strains isolated from tree legumes growing in Sudan and Kenya. Later, Nick et al. (1999) described two new species (Sinorhizobium kostiense and S. arboris) among these and resolved that the collection comprised strains, which belong to more than five different /$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi: /j.soilbio

2 2014 E. Wolde-meskel et al. / Soil Biology & Biochemistry 36 (2004) species (including Mesorhizobium plurifarium, S. terangae and S. saheli) and one yet undesignated Sinorhizobium group. Similarly, McInroy et al. (1999) and Odee et al. (1997, 2002) reported a high degree of phenotypic and genotypic diversity in a relatively small number of samples studied from East Africa (Sudan and Kenya). On these bases, Odee et al. (2002) suggested that this sub-saharan region could possibly be a centre of rhizobial diversity. In fact, the region has also been designated as a centre of floral and faunal diversity (World Conservation Monitoring Centre, 2000). However, lack of information on the composition and diversity of natural populations of rhizobia harbored in Ethiopian soils formed a gap in East Africa. Ethiopia is considered to be the centre of origin for many leguminous crop plants, including pea (Hawkes, 1983), chickpea (Cousin, 1997), lentils (Singh, 1997) and clover (Raven and Polhill, 1981). There is thus reason to believe that Ethiopian soils harbor diverse rhizobial strains, which form symbiotic relationships with herbaceous and woody legumes. The classical phenotypic characterisation of rhizobia has often been the first method employed when classifying unknown strains of rhizobia (Lindström and Lehtomäki, 1988; Zhang et al., 1991; Wolde-meskel et al., 2004). Though this method provides valuable information, it is laborious and time consuming. The Biologe system (Biolog, Hayward, CA, USA), which is based on metabolic fingerprinting by determination of C source utilization, offered a less tedious alternative for large scale screening of isolates. It has been used to characterize and classify bacteria and bacterial communities in a wide range of environments, including marine, agricultural and nonagricultural soils, and sludge (Rüger and Krambeck, 1994; van Heerden et al., 2001; McCaig et al., 2001). The Biolog method has, for examle; been reported to be the best out of five typing methods for differentiating fluorescent pseudomonads (Dawson et al., 2002). It has also been applied to characterize members of Rhizobiaceae (Dupuy et al., 1994; McInroy et al., 1999). Amplified fragment length polymorphism (AFLP) is a high-resolution genomic fingerprinting method (Vos et al., 1995; Blears et al., 1998). It has been used to study diversity at species and strain level in several groups of bacteria. Due to its high discriminatory power, it has been used as identification tool (Coenye et al., 1999) or for typing purposes (Thyssen et al., 2000; Jiang et al., 2000). AFLP has also been applied to study genetic diversity of R. galegae (Terefework et al., 2001) and bradyrhzobia (Willems et al., 2000, 2001), and for initial grouping of unclassified Rhizobium strains (Gao et al., 2001). This study aims to analyse, using Biolog and AFLP fingerprinting methods, the diversity of 110 rhizobium strains isolated from root nodules of African Acacia spp., Sesbania sesban and herbaceous trap host species grown in soils from diverse agro-ecological zones in southern Ethiopia. We constructed a Biolog user-database of 23 rhizobia and five Agrobacterium reference species, based on which the metabolic diversity of our 110 unnamed test strains was examined. In addition, classification of the reference species into Biolog metabolic fingerprint groups was compared to phylogenetic classification of the species (based on 16S rrna gene analysis) in the literature. 2. Materials and methods 2.1. Rhizobial strains The test strains and reference species used in this study are listed in Tables 1 and 2, respectively. Twenty-three rhizobial and five agrobacterial species were included as reference in the study. The 110 unnamed test strains were isolated from seedlings of three African Acacia spp. (Acacia abyssinica, A. seyal and A. tortilis) and a number of their provenances, Faidherbia albida, S. sesban and herbaceous trap host species (Phaseolus vulgaris and Vigna unguiculata) grown in soil samples collected from diverse ecoclimatic zones as previously described (Wolde-meskel et al., 2004) Biolog metabolic profiling In the Biologe system (Biolog Inc., Hayward, CA, USA) where the identification of bacterial strains is based on their utilization pattern of 95 C sources, the limited number of environmental bacteria in the commercially available MicroLog database is a major drawback. For example, the database (Version 4.0) for Gram-negative (GN) bacteria contains only four Agrobacterium spp. and Sinorhizobium meliloti. Therefore, a user-database was created from the 23 reference rhizobia and five agrobacterium species (Table 2) with which the C utilization patterns of our unknown test strains were compared for identification. Prior to transfer into the microplates, rhizobia were grown on YMA and plated on freshly prepared R2A agar medium (McInroy et al., 1999) (yeast extract 0.5 g, proteose peptone 0.5 g, casamino acids 0.5 g, glucose 0.5 g, soluble starch 0.5 g, sodium pyruvate 0.3 g, K 2 HPO g, MgSO 4 $7H 2 O 0.05 g, noble agar 15 g in 1 l distilled H 2 O and a ph of 7.2). The inocula were prepared by suspending cells from plates in 0.5% saline solution. The density was adjusted to the turbidity level of 52% transmittance recommended by the Biolog plate manufacturer (Biolog GN2 MicroPlatee instructions). All 96 wells on a Biolog GN microplate were filled with 150 ml of the cell suspension and incubated at 25 8C. The colour density in each well was recorded spectrophotometrically after 6, 24, 48 and 72 h using an automated plate reader (Emax precision microplate reader, USA) at 590 nm absorbance. Each of the reference strains was assayed in four replications and the profiles obtained were used to construct a user-database. Initially, some test strains were assayed twice. However, C utilization patterns of the duplicates were found to be consistently

3 E. Wolde-meskel et al. / Soil Biology & Biochemistry 36 (2004) Table 1 Rhizobium test strains used and their Biolog and AFLP groups Strains a Host species (provenance) Geographic origin b Biolog AFLP AC 10a1 A. seyal (Alemaya) Nazret 1A 4 AC 38b2 A. abyssinica Akaki 1A 6B AC 38b1 A. abyssinica Akaki 1A 6B AC 20b A. tortilis (Arba-minch) Akaki 1A 5 AC 08e A. seyal (Alemaya) Debrezeit 1A 5 AC 38d1 A. abyssinica Akaki 1B 5 AC 38d2 A. abyssinica Akaki 1B 5 AC 10d A. seyal (Alemaya) Nazret 1B 5 AC 16c A. seyal (Surupa) Nazret 1B 7 AC 16a A. seyal (Surupa) Nazret 1B 7 AC 22d A. tortilis (Arba-minch) Awassa-RFC 1C 7 AC 27c A. tortilis (Meki) Debrezeit 1C 7 AC 27e1 A. tortilis (Meki) Debrezeit 1C 7 AC 40a A. abyssinica Debrezeit 1D 7 AC 27a2 A. tortilis (Meki) Debrezeit 1D 7 AC 27a3 A. tortilis (Meki) Debrezeit 1D 7 AC 27a1 A. tortilis (Meki) Debrezeit 1D 7 AC 27d2 A. tortilis (Meki) Debrezeit 1D 7 AC 16b1 A. seyal (Surupa) Nazret 1D 7 AC 21c1 A. tortilis (Arba-minch) Nazret 1D 6B AC 22c A. tortilis (Arba-minch) Awassa-RFC 1E 6B AC 22e A. tortilis (Arba-minch) Awassa-RFC 1E 6B AC 25c A. tortilis (Mega) Awassa-RFC 1E 6B AC 25b A. tortilis (Mega) Awassa-RFC 1E 6B AC 25a A. tortilis (Mega) Awassa-RFC 1E 6B AC 17b A. seyal (Surupa) Awassa-RFC 2A 12 AC 50a S. sesban Debrezeit 2A 8 AC 50e S. sesban Debrezeit 2A 8 AC 14d A. seyal (Awassa) Awassa-ACA 2B 4 AC 07b1 A. seyal (Akaki) Awassa-RFC 2B 12 AC 07b2 A. seyal (Akaki) Awassa-RFC 2B 12 AC 07c A. seyal (Akaki) Awassa-RFC 2B 12 AC 17d A. seyal (Surupa) Awassa-RFC 2B 12 AC 19e A. tortilis (Abergele) Awassa-RFC 2B 12 AC 25d A. tortilis (Mega) Awassa-RFC 2B 12 AC 25e A. tortilis (Mega) Awassa-RFC 2B 12 AC 28a A. tortilis (Meki) Awassa-RFC 2B 12 AC 28d1 A. tortilis (Meki) Awassa-RFC 2B 12 AC 01d2 A. seyal (Abergele) Debrezeit 2C 6A AC 01d1 A. seyal (Abergele) Debrezeit 2C 6A AC 01c1 A. seyal (Abergele) Debrezeit 2C 6A AC 01c2 A. seyal (Abergele) Debrezeit 2C 6A AC 01e A. seyal (Abergele) Debrezeit 2C 6A AC 01b A. seyal (Abergele) Debrezeit 2C 6A AC 17e2 A. seyal (Surupa) Awassa-RFC 3 6B AC 17e1 A. seyal (Surupa) Awassa-RFC 3 6B AC 19a A. tortilis (Abergele) Awassa-RFC 3 6B AC 07e A. seyal (Akaki) Awassa-RFC 3 7 AC 07a A. seyal (Akaki) Awassa-RFC 3 7 AC 17a A. seyal (Surupa) Awassa-RFC 3 7 AC 17c A. seyal (Surupa) Awassa-RFC 3 7 AC 19d A. tortilis (Abergele) Awassa-RFC 3 7 AC 08a2 A. seyal (Alemaya) Debrezeit 3 7 AC 27d1 A. tortilis (Meki) Debrezeit 4 7 AC 16b2 A. seyal (Surupa) Nazret 4 7 AC 24d1 A. tortilis (Mega) Debrezeit 5 5 AC 24d2 A. tortilis (Mega) Debrezeit 5 5 AC 61a V. unguiculata Debrezeit 6A 1A AC 61d V. unguiculata Debrezeit 6A 1A AC 73b1 P. vulgaris Debrezeit 6A 1A AC 73b2 P. vulgaris Debrezeit 6A 1A (continued on next page)

4 2016 E. Wolde-meskel et al. / Soil Biology & Biochemistry 36 (2004) Table 1 (continued) Strains a Host species (provenance) Geographic origin b Biolog AFLP AC 73e1 P. vulgaris Debrezeit 6A 1A AC 73e2 P. vulgaris Debrezeit 6A 1A AC 51a1 S. sesban Nazret 6A 1A AC 51a2 S. sesban Nazret 6A 1A AC 51c S. sesban Nazret 6A 1A AC 51e S. sesban Nazret 6A 1A AC 50b S. sesban Debrezeit 6B 1B AC 50c S. sesban Debrezeit 6B 1B AC 50d S. sesban Debrezeit 6B 1B AC 73c P. vulgaris Debrezeit 6B 1B AC 73d P. vulgaris Debrezeit 6B 1B AC 42c A. abyssinica Awassa-RFC 7 8 AC 11c A. seyal (Alemaya) Awassa-ACA 8 U c AC 11a A. seyal (Alemaya) Awassa-ACA 8 U AC 13b A. seyal (Awassa) Debrezeit 8 U AC 28c2 A. tortilis (Meki) Awassa-RFC 9 4 AC 39e2 A. abyssinica Chofa 9 10 AC 39e1 A. abyssinica Chofa 9 10 AC 21a2 A. tortilis (Arba-minch) Nazret 9 8 AC 21c2 A. tortilis (Arba-minch) Nazret 9 16 AC 11d A. seyal (Alemaya) Awassa-ACA 12 3 AC 52e S. sesban Awassa-ACA 12 3 AC 52d S. sesban Awassa-ACA 12 3 AC 52c S. sesban Awassa-ACA 12 3 AC 52a2 S. sesban Awassa-ACA 12 3 AC 52a1 S. sesban Awassa-ACA 12 3 AC 52b S. sesban Awassa-ACA 12 3 AC 47b S. sesban Arba-minch 13 2 AC 47d S. sesban Arba-minch 13 2 AC 47a S. sesban Arba-minch 13 2 AC 39d A. abyssinica Chofa 14 4 AC 39a A. abyssinica Chofa AC 29c F. albida (Arba-minch) Awassa-RFC 16 4 AC 70c P. vulgaris Akaki 16 1B AC 62a V. unguiculata Dilla 16 1B AC 64b V. unguiculata Nazret 16 4 AC 64a V. unguiculata Nazret 16 4 AC 65c V. unguiculata Nazret 16 4 AC 64c V. unguiculata Nazret 16 5 AC 14c A. seyal (Awassa) Awassa-ACA U 5 AC 04d A. seyal (Abergele) Awassa-RFC U 2 AC 26e A. tortilis (Meki) Akaki U 1B AC 56b V. unguiculata Akaki U 1B AC 47c2 S. sesban Arba-minch U 7 AC 72a P. vulgaris Chofa U 4 AC 39c1 A. abyssinica Chofa U 10 AC 08a1 A. seyal (Alemaya) Debrezeit U 7 AC 18a A. tortilis (Abergele) Debrezeit U 7 AC 10a2 A. seyal (Alemaya) Nazret U 4 a Source of all AC strains is Wolde-meskel et al. (2004); AC, Awassa College culture collections. b Soil sampling locations in southern Ethiopia from which strains were isolated; ACA, Awassa College of Agriculture campus; RFC, Research and Farm Centre of the Awassa College of Agriculture. c U, strains unclustered on the dendrogram. similar and thus all the remaining test strains were assayed only once. The Biolog metabolic fingerprint data were used for cluster analysis and to construct dendrograms using Bionumerics software program, version 2.0 (Applied Maths, Kortrijk, Belgium). The resemblance between pairs of strains was calculated using the Pearson correlation coefficient and presented as dendrograms by unweighted pair-group method with arithmetic average (UPGMA) DNA isolation and AFLP fingerprinting Total genomic DNA was isolated according to Boom et al. (1990) with slight modifications (Terefework et al.,

5 E. Wolde-meskel et al. / Soil Biology & Biochemistry 36 (2004) Table 2 Rhizobium reference species used and their Biolog and AFLP groups Reference species HAMBI a strain no. Other strain designation a Biolog AFLP Sinorhizobium saheli 215 T ORS 609 T 1A 9 S. arboris 1552 T LMG T 2A 9 S. medicae 1838 T 2A 9 S. terangae 220 T ORS 1009 T 2A 11 S. meliloti 2148 T LMG 6133 T 2A 9 Rhizobium huautlense 2409 T 6B 16 Mesorhizobium huakuii 1674 T LMG T 7 16 A. rubi 1812 T LMG 156 T 7 8 Allorhizobium undicola LMG T 8 9 R. hainanense 1930 T 10 9 R. tropici 1163 T CIAT 899 T 10 U Agrobacterium rhizogenes 1816 T LMG 150 T 10 U A. radiobacter 1814 T LMG 140 T A. tumefaciens 1811 T LMG 187 T R. gallicum 2326 T R. leguminosarum 12 T LMG T 15 1A R. etli 1727 T CFN 42 T U 11 R. galegae 540 T LMG 624 T U U R. giardinii 2323 T U U R. mongolense 234 T USDA 1844 T U 15 S. kostiense 1489 T LMG T U 9 M. ciceri 1750 T LMG14989 T U 14 M. loti 1129 T LMG 6125 T U 14 M. mediterraneum 2096 T U U Azorhizobium caulinodans 216 T ORS 571 T U U Bradyrhizobium elkanii LMG 6164 U 15 B. japonicum 2314 T USDA 6 T U U A. vitis 1815 T LMG 257 T U 13 T Type strain. a CFN, Centro de Investigación sobre Fijación de Nitrógeno, Universidad Nacional Autónoma de México, Cuernavaca, Mexico; LMG, Collection of Bacteria of the Laboratorium voor Microbiologie, Ghent, Belgium; HAMBI, Culture collection of the Division of Microbiology, University of Helsinki, Finland; ORS, I.R.D./ORSTOM Collection, Institut de Recherche Pour le Développement, BP 1386, Dakar, Senegal; USDA, US Department of Agriculture, Beltsville, MD. 2001) where diatomaceous earth or Celite analytical filter aid (BDH Laboratory Supplies, Poole BH15 1TD, UK) was used as DNA-binding solid support. The AFLP procedure was as described by Vos et al. (1995) with some modifications (Gao et al., 2001) where the digestion and ligation were combined in a single step. A 20 ml reaction mixture containing 0.5 mg of genomic DNA, 10 U of EcoRI, 10 U of MseI, 10 U T4 DNA-ligase, 10! DNA-ligase reaction buffer, 5 mm of EcoRI and 10 mm MseI adapters were digested in a PTC-200 Peltier Termal Cycler (MJ Research, Watertown, MA). The temperature profile for restriction ligation was 37 8C for 2 h followed by a 0.1 8C s K1 decrease to 16 8C in 30 min and finally heating at 70 8C for 20 min. Five microlitres of the restricted tagged (with specific adapters) fragments was used as a template in 20 ml of reaction mixture for selective PCR amplification by MseI-gc (5 0 -GAT GAG TCC TGA GTA AGC-3 0 ) and EcoRI-gag (5 0 -GAC TGC GTA CCA ATT CGA ATT CGA GAGK3 0 ) selective primers (underlined bases at 3 0 end are selective). The amplification procedures were: cycle 1, heating for 2 min at each of 50, 55, 60, 65 and 70 8C, and then for 30 s at 95 8C, 30 s at C, and 90 s at 72 8C. Cycles 2 30, the last three steps and a final extension at 72 8C for 3 min. After the PCR reaction, an equal volume (2.5 ml) of loading buffer (98% formamide, 10 mm EDTA [ph 8.0], 0.1% xylene cyanol) was added to the reaction mixtures and pgem DNA marker. Prior to loading and electrophoresis, the samples were heated for 3 min at 95 8C and then rapidly cooled on ice. The amplified fragments were separated in 5% denaturing polyacrylamide gel, as described by Jenssen et al. (1996), with some modification using 1!TTT buffer instead of 1!TBE buffer (Gao et al., 2001). Sample aliquots of 3 ml were loaded in the gel. Electrophoresis was performed at 500 V for 10 min until the samples entered the gel and then the voltage was increased to 1500 V for 1.5 h using a sequencing gel apparatus with 1!TTT as a running buffer. After electrophoresis, the gels were stained using silver staining method and dried overnight at room temperature. The dried gels were scanned using HP DeskJet II scanner and the AFLP fingerprints were analyzed by the GelCompar 4.1 program (applied Maths BVBA, Kortijk, Belgium), and dendrograms were constructed using the Pearson correlation

6 2018 E. Wolde-meskel et al. / Soil Biology & Biochemistry 36 (2004) coefficient of similarity calculations and the average linkage (UPGMA) method. the reference species into different clusters. This was used for further characterization and analysis of the test strains. 3. Results 3.1. Duration of incubation and reproducibility of Biolog fingerprinting Colour development was weak in microplate wells at 6 h of incubation. The intensity of colour development and the number of coloured wells increased after 6 h of incubation. On comparison of cluster analyses of C utilization patterns and dendrograms generated (not shown) at 6, 24, 48 and 72 h of incubations for the reference strains, the 24 h incubation gave consistent pattern and best separation of 3.2. Biolog fingerprints of reference strains Cluster analysis of C utilization patterns of the reference species revealed three main clusters, clusters 1 3, on the dendrogram (Fig. 1). Cluster 1 contained 20 out of 28 reference species included in this study. This cluster further subdivided into several sub-clusters (designated 1A, 1B, etc.) and separate positions, i.e. non-clustered species (S. kostiense and R. etli). Cluster 2 consisted of five reference species, which further divided into two distinct sub-clusters; one contained R. leguminosarum, R. gallicum and R. mongolense and the other composed of R. galegae and M. loti. Species within the latter sub-cluster were only Fig. 1. Relationships between reference rhizobial strains based on the Biolog sole C source utilization. Clusters 1 3 represent groups of strains with similar C profiles at the level shown on the scale; A, B, C, etc. represent groups of strains more related to each other within the main clusters and indicated as 1A, 1B, 1C, etc. in the text. The dendrogram was constructed by the UPGMA method in the Bionumerics program.

7 E. Wolde-meskel et al. / Soil Biology & Biochemistry 36 (2004) loosely linked at a similarity level of 60%. Cluster 3 comprised the two slow growing Bradyrhizobium species and Azorhizobium caulinodans for which the dendrogram suggested distant relatedness to the rest of the reference species found in Clusters 1 and Biolog fingerprints of test strains A Biolog user-database of the 28 reference strains was created to identify test strains (Table 1) using the MicroLog Software Program version 4.0 (Biolog Inc., Hayward, CA, USA). However, the software program was able to name only few test strains with O0.5 similarity index (SIM) value, the lowest acceptable degree of certainty specified by the manufacturer. The bulk of the strains (91%) had SIM values below The quantitative Biolog fingerprint data (absorbance record at 590 nm) derived from the automatic reading was clustered (using UPGMA) and a dendrogram was generated (Fig. 2). At a similarity level of 77%, 16 main clusters (designated Biolog 1 16) and 23 ungrouped strains were identified. Out of the 16 clusters, 13 contained test strains while three clusters (Biolog 10, 11, 15) comprised only reference strains. Of the 13 clusters, Biolog 1, 2 and 6 comprised 59 out of 110 test strains included in this study. Each of these was divided into a number of distinct subclusters, out of which only one sub-cluster was linked to reference species. Biolog 1 contained 25 strains isolated from seedlings of three Acacia spp. (A. abyssinica, A. seyal and A. tortilis) grown in soils from four locations. Out of five distinct subclusters in this cluster (designated 1A 1E), sub-cluster 1A was loosely linked to a reference strain, S. saheli. Each of sub-cluster 1B and 1C contained strains isolated at two different locations. Except one strain, sub-cluster 1D contained strains isolated at one specific location (Debrezeit). All strains in sub-cluster 1E were isolated from A. seyal seedlings grown in soil from one site (Awassa-RFC). Biolog 2 included 19 strains and was subdivided into three distinct sub-clusters, 2A, 2B and 2C. Sub-cluster 2A consisted of three strains, two of which were isolated from S. sesban and one from A. seyal. These were linked to three Sinorhizobium reference species (S. arbores, S. terangae and S. medicae). All strains in sub-cluster 2B, except one, were isolated from seedlings of A. seyal and A. tortilis grown in one site-soil (Awassa-RFC). Sub-cluster 2C contained strains isolated only from A. seyal seedlings grown in another soil (Debrezeit). Sub-clusters 2B and 2C formed tight clusters which were not linked to reference species. Biolog 6 comprised 15 strains from S. sesban and the herbaceous species (V. unguiculata and P. vulgaris), and separated into two distinct sub-clusters, 6A and 6B. The first contained a tightly clustered group (similarity of O96%) to which no reference species was linked while 6B was loosely connected to R. huautlense. All strains in sub-cluster 6B were isolated from one soil (Debrezeit). Biolog 7 9 which consisted of one, three and five strains, respectively, were isolated from different Acacia sp. grown in soils obtained from five different locations. The clusters were loosely connected to Allorhizobium undicola, Mesorhizobium huakuii, and M. loti, respectively. Biolog 3 5, and 16 each consisted of two to nine strains. Altogether these clusters contained 32 test strains (29% of 110), which were not linked to any of the reference species included in this study. All but one strain in cluster 3 was isolated from one site-soil (Awassa-RFC) and one host species (A. seyal). Clusters 4, 5, and 14 contained strains from A. seyal and A. tortilis seedlings grown in soils from four locations (Akaki, Awassa-RFC, Debrezeit and Nazret). Clusters 12 and 13 contained tightly clustered groups of strains, which were isolated from only S. sesban seedlings grown in two different soils (Awassa-ACA and Arbaminch). Biolog 16 consisted of all the slow growing strains in this experiment, isolated from F. albida, V. unguiculata and P. vulgaris grown in four different soils. Thirteen reference strains (A. caulinodans, A. vitis, B. elkani, B. japonicum, M. ciceri, M. mediterraneum, M. loti, R. etli, R. galegae, R. giardinii, R. mongolense, S. kostiense and S. meliloti) and 10 test strains (AC 26e, AC 8a1, AC 18a, AC 14c, AC 10a2, AC 39c2, AC 4d, AC 56b, AC 47c2, and AC 72a) were not clustered, i.e. formed separate positions on the dendrogram AFLP fingerprinting Cluster analysis of the AFLP patterns of 110 test strains and 28 reference species revealed 16 clusters (designated AFLP 1 16) on the dendrogram at a similarity level of 60% (Fig. 3). The unnamed test strains occupied 11 of these clusters (AFLP 1 8, 10, 12 and 16) while only reference species occupied the remaining five clusters (AFLP 9, 11, 13 15). Eight clusters out of the 11 (2 7, 10 and 12) contained 92 test strains, which were not linked to any reference species in this study. The other three clusters [clusters 1 (sub-cluster 1A), 8 and 16] contained 15 strains loosely linked to one or two of four reference species (R. leguminosarum, A. rubi, M. huakuii and R. huautlense). Six reference species (R. galegae, R. tropici, R. giardinii, A. rhizogenes, A. caulinodans, B. japonicum) and three test strains (AC 11a, AC 11c, AC 13b) occupied separate positions on the dendrogram, i.e. did not cluster with any of the other strains. In some cases there was a good agreement between AFLP and Biolog grouping of the test strains. Thus, 46 strains in seven Biolog clusters/sub-clusters (6A, 6B, 13, 12, 2C, 1E, and 2B) were grouped into seven corresponding AFLP clusters/sub-clusters (1A, 1B, 2, 3, 6A, 6B and 12) (Table 1). The strains in each of these corresponding Biolog/ AFLP clusters or sub-clusters were isolated mostly from one host species grown in specific soils. For example, all strains

8 2020 E. Wolde-meskel et al. / Soil Biology & Biochemistry 36 (2004) Fig. 2. Dendrogram obtained by cluster analysis of the Biolog metabolic fingerprints of test strains and reference species. Clusters 1 16 represent groups of strains with similar C profiles at the level shown on the scale; A, B, C, etc. represent groups of strains more related to each other within the main clusters and indicated as 1A, 1B, 1C, etc. in the text. The data was clustered using UPGMA method in the Bionumerics program.

9 E. Wolde-meskel et al. / Soil Biology & Biochemistry 36 (2004) in Biolog 13/AFLP 2 and in Biolog 12/AFLP 3, except one strain, were isolated from S. sesban grown in Arbaminch and Awassa-ACA soils, respectively. Likewise, Biolog 1E/AFLP 6B contained strains from A. tortilis grown in Awassa-RFC soils while all strains in Biolog 2C/ AFLP 6A were from A. seyal in Debrezeit soil. In other cases, single Biolog clusters contained strains from several AFLP groups. For example, Biolog 1 contained strains from AFLP 4 7; Biolog 2 and 9 each contained strains from four different AFLP groups; and each of Biolog 3 and 16 contained strains from two and three AFLP clusters. However, several single AFLP clusters also contained strains from various Biolog groups. For example, AFLP 4 contained strains from five Biolog groups (Biolog 1, 2, 9, 14, and 16); AFLP 5 and 7 each contained strains from four different Biolog groups; and AFLP 1, 6, 8, and 10 each contained strains from three different Biolog groups. 4. Discussion In conjunction with the progressing research effort to improve nitrogen fixation in agroforestry systems in subsaharan Africa and to uncover the biodiversity of rhizobia in previously unexplored biogeographical regions, we have studied 110 unnamed rhizobia isolated from leguminous trees and herbaceous species in Ethiopian soils. The results demonstrated that indigenous rhizobia nodulating native woody species in soils across diverse agro-ecological zones in southern Ethiopia are metabolically and genomically diverse Biolog grouping of reference species The Biolog metabolic fingerprint data of the reference species was separately clustered and a dendrogram generated was compared with classification of the species based on the widely applied 16S rrna sequence analysis in the literature (Nick et al., 1999). Of the three major clusters on our dendrogram (Fig. 1), the deeply branched cluster, containing the Bradyrhizobium spp. and A. caulinodans bare close resemblance to the grouping of the species based on 16S rrna data. The other two Biolog clusters, which contained members of Agrobacterium, Allorhizobium, Mesorhizobium, Rhizobium and Sinorhizobium, intertwined with one another and do not accord to the anticipated relatedness supported by the 16S phylogeny. Previously, McInroy et al. (1999) reported that Biolog classification of the reference species resembled grouping based on 16S rrna data. We used more reference species (28 instead of 15) and a different version of Biolog Microlog software program (version 4.0 as opposed to 3.5), but otherwise we used the same type strains and followed the same experimental protocol as McInroy et al. (1999). However, our results do not concur with those of McInroy et al. (1999). There were anomalies in the grouping of the fast and intermediate growing Rhizobium species, clusters 1 and 2(Fig. 1). Nevertheless, though the Biolog method did not clearly reflect the taxonomic and/or phylogenetic relationships between the species in these clusters, most of them were differentiated into sub-clusters and separate (unclustered) positions Biolog grouping of test strains The Biolog method classified the test strains into 13 distinct clusters and 10 separate positions. Some of these clusters (namely Biolog 1, 2 and 6) contained discrete subclusters each composed of a closely related group of strains. Twenty-two strains out of 110 could be named because they included reference strains into their respective clusters/sub-clusters. Accordingly, four strains in sub-cluster 1A were identified as S. saheli while three strains in sub-cluster 2A were related to three Sinorhizobium species (S. arboris, S. medicae and S. terangae). In view of the specific identification of these strains (in sub-clusters 1A and 2A) to species of Sinorhizobium, all the 44 strains in Biolog 1 and 2 (main clusters) might be considered to belong to the same genus. However, the Biolog method has distinguished several distinct sub-groups within these clusters. Whether these relate to other species of Sinorhizobium (not included in this study) or constitute taxonomically different groups within the same genus remains to be resolved in further phylogenetic studies. Apart from two strains in sub-cluster 2A, all rhizobia in clusters 1 and 2 were isolated from Acacia spp. grown in soils from four different locations (Akaki, Awassa-RFC, Debrezeit and Nazret). In previous studies, the majority of rhizobia isolated from root nodules of A. senegal and Prosopis chilensis in Sudan were Sinorhizobium showing similarities to S. fredii, S. meliloti, S. terangae and S. saheli (Haukka et al., 1996). In addition, the Biolog method identified five strains in sub-cluster 6B (mostly isolated from S. sesban)as R. huautlense (a species described from a narrow genetic group of isolates from S. herbacea, Wang et al., 1998) while another five strains in cluster 9 were related to M. huakuii. Our preliminary results from PCR- RFLP studies of the 16S rrna gene for selected strains in these and other clusters (e.g. 1A, 2C, 6B, 9, 14, 16) (unpublished data) are in accordance with the Biolog groupings. Eighty-eight unnamed strains out of 110 (i.e. 80%) formed distinct Biolog clusters/sub-clusters and separate positions, which did not link to reference species on the dendrogram. This could indicate that the bulk of the test strains included in this study differed from the reference species. This was supported by lack of identification of most of the test strains by the automated Microlog software program (version 4.0) using the user-database, which we had created. The grouping of 20 out of 28 reference species into separate clusters/positions on the dendrogram also indicates the same fact. The Biolog system has not been widely used to characterize members of Rhizobiaceae, and thus there are only few

10 2022 E. Wolde-meskel et al. / Soil Biology & Biochemistry 36 (2004) reported results in the literature. McInroy et al. (1999) using strains isolated from nine leguminous tree species grown in soils from 10 different locations, reported that Biolog method gave a matching result to grouping based on partial 16S rrna sequences. On the other hand, phenotypic and genotypic studies of a large number of bradyrhizobia isolates from F. albida demonstrated that the Biolog system had less discriminating power among the strains (Dupuy et al., 1994). This might be expected as bradyrhizobia are generally slow growing strains and less distinct phenotypically as compared to their fast-growing counterparts (Zhang et al., 1991). In our study, all the slow growing strains were grouped into one separate deeply branching cluster (Biolog 3 and 16 in Figs. 1 and 2, respectively) suggesting distant metabolic relatedness to the fast-growing rhizobia. Lack of distinct internal differentiation within these clusters in our experiment supports the results of Dupuy et al. (1994). Still, our collection embraced diverse phenotypic groups (Wolde-meskel et al., 2004), which the Biolog method was able to delineate into 13 distinct clusters and 10 separate positions on the dendrogram AFLP fingerprinting of test strains Application of the AFLP fingerprinting technique in our study distinguished 11 distinct clusters demonstrating a large genetic diversity among the test strains. Several major groups, which were not linked to reference species, were also found. Of the 110 test strains, only 15 strains (belonging to clusters 1A, 8 and 16) were loosely linked to four out of 28 reference species used (Fig. 3). Eighty-four percent of the 110 strains were grouped into eight distinct AFLP clusters and were not related to reference strains, thus further supporting the Biolog result. Each of these eight clusters consisted of groups of strains isolated either mostly from one host species at a specific site (e.g. cluster 10, 6A, 3, 2) or from several host species grown in soils obtained from a number of locations (e.g. cluster 8, 7, 5, 4) Comparative AFLP/Biolog grouping of test strains Comparison of the AFLP and Biolog groupings indicated that 42% of the test strains in seven Biolog clusters occupied seven AFLP clusters (Biolog 1E, 2B, 2C, 6A, 6B,12, and 13 corresponding to AFLP 6B, 12, 6A, 1A, 1B, 3, 2, respectively; Table 1). The clusters were also associated with geographical origin and/or hosts of isolation of the strains. While the corresponding relationship between the two methods was interesting and indicates that similarity in 3 Fig. 3. Dendrogram showing the genomic diversity of the rhizobial strains, based on AFLP fingerprints. Clusters 1 16 represent groups of strains with similar genomic profiles at the level shown on the scale. Numbers in parenthesis indicate the number of strains with the same genotype. Cluster

11 E. Wolde-meskel et al. / Soil Biology & Biochemistry 36 (2004) genomic fingerprints might imply similarity in metabolic fingerprints, it is the grouping of the same strains as one and the same entity (in both methods) that is more relevant for our purpose. In view of the metabolic and genomic similarity of the strains it is quite likely that each of these groups represented the same strains or clonal descendants of the same organisms in some cases (e.g. as in AFLP 12 and 3, Fig. 3). Interestingly, the strains isolated from S. sesban occupied four of the seven corresponding Biolog/AFLP clusters, i.e. 12/3, 13/2, 6A/1A and 6B/1B (Table 1). It is also noteworthy that three of these corresponding (Biolog/ AFLP) clusters were not linked to reference species. As compared to strains isolated from Acacia spp. which were distributed along most of the Biolog and AFLP clusters, the strains from S. sesban were restricted to these Biolog/AFLP clusters (but two strains in another cluster, Biolog 2A/AFLP 8), and each cluster represented isolates from one specific site (apart from Biolog 6A/AFLP 1A, which contained strains from two soils). This indicated the specific nature and symbiotic affinity of the host, in agreement with earlier reports by Bala et al. (2003a) and Odee et al. (2002). Previous investigations showed lack of compatible rhizobia and lack of nodulation of S. sesban in many African and all Asian and South American soils tested (Bala et al., 2003b). Though test strains from Acacia species were generally diverse and distributed along several clusters (see below), three of the seven corresponding Biolog/AFLP clusters (2C/6A, 2B/12 and 1E/6B, Table 1) were distinctly formed by strains isolated from only acacia species (only A. seyal or including A. tortilis) and each representing isolates from only one specific soil, Debrezeit or Awassa-RFC site. Like most of the Sesbania clusters, these were not linked to reference species in both methods and might be taxonomically different groups. Both sesbania and African acacia occupy a unique position in rhizobium legume symbiosis in that many recently described rhizobium species were isolated from root or stem nodules of these trees, including four species of Sinorhizobium (S. arbores, S. kostiense, S. saheli and S. terangae), M. plurifarium, R. huautlense, and a distinct genus Azorhizobium, containing the single species A. caulinodans (Dreyfus et al., 1988; de Lajudie et al., 1994, 1998; Wang et al., 1998; Nick et al., 1999). In our study, strains isolated from Sesbania and Acacia belonged to distinct categories, as demonstrated by matching groups in both methods and the lack of similarity (non-relatedness) to the described species included in the study. This reflects that these strains may represent different taxonomic group as yet unrecognised and warrant further phylogenetic analysis. Most of the test strains isolated from Acacia spp. (A. abyssinica, A. seyal, and A. tortilis) were represented in several Biolog and AFLP clusters/sub-clusters, for example strains from A. seyal were found in six Biolog clusters (1 4, 8, 12) and in corresponding number of AFLP clusters (3 7, 12). Likewise, strains from the other acacias were distributed over 5 7 Biolog and AFLP clusters. While this indicated the metabolic and genomic heterogeneity of the strains associated with these hosts, it also reflects the promiscuous nature of the African acacia species. However, Acacia spp. can also be specific and form symbiosis with a particular type of strains in a specific soil as demonstrated by both methods, i.e. as in Biolog2C/AFLP 6A, 2B/12 and 1E/6B (Table 1). Australian acacias were reported to be predominantly nodulated by bradyrhizobia (Lafay and Burdon, 2001). In a phenotypic study of 480 strains, mainly isolated from African acacias in Kenya, the associated natural rhizobial population constituted diverse phenotypic groups (Odee et al., 1997). Using a small sample from this core collection, it was further demonstrated by Biolog and genotypic methods, that African acacias are predominantly nodulated by various species of Mesorhizobium, Rhizobium and Sinorhizobium (McInroy et al., 1999; Odee et al., 2002). In our study, the results of both methods showed that our collection embraced metabolically and genomically diverse groups of rhizobia nodulating Acacia spp. as well as S. sesban. However, since the taxonomic positions of the different Biolog and AFLP groups (especially those not linked to the reference species) is not evident from our experiment, it is not known how the observed heterogeneity reflects on the classification of these organisms and what taxonomic groups nodulate our host species. Soils at different sites contained an array of metabolically and genomically diverse rhizobial populations. This was shown for example in the Awassa-ACA soil where we obtained seven different Biolog clusters (1 3, 7, 9, 16 and U) corresponding to six AFLP clusters (2, 4, 6 8 and 12) (Table 1). The same was true for other sites, which contained a number of strains distributed over 3 7 different Biolog and corresponding number of AFLP clusters. Since the same host species were used to trap rhizobia from all the soils, it is presumed that the extent of diversity in a given soil relates to the rhizobial affinity of the trap host species and the natural composition of compatible rhizobia resident in the soil under consideration. Our results emphasized the necessity of using a large number of native as well as introduced trap host species with varying nature of symbiotic affinities to capture the full composition and diversity of indigenous rhizobial populations. This is in agreement with previously reported results on phenotypic and genotypic studies of acacia and other tropical tree rhizobia (Odee et al., 1997, 2002; Bala et al., 2003a,b; Wolde-meskel et al., 2004). Andronov et al. (2003), using AFLP and other techniques, showed that diversity in R. galegae bv orientalis corresponds well with the host plant variation (Galega orientalis) at the hosts gene center (Caucasus region). In our experiment, A. abyssinica (native to East Africa, Ross, 1979) trapped rhizobial strains distributed over six different AFLP clusters (Table 1). Though we have no specific information concerning genetic variation among the host plants, A. abyssinica is known to be widely distributed over a range of altitudinal and climatic conditions (from semi-arid lowlands to humid highlands) in the region.

12 2024 E. Wolde-meskel et al. / Soil Biology & Biochemistry 36 (2004) Whether there exists substantial genetic variation among the host plants and that the possibility that this has played a role in shaping the diversity of associated rhizobial populations that we have observed in our study, will remain for further investigations. Acknowledgements The authors thank NUFU (Norwegian Universities Committee for Development Research and Education) for providing financial assistance to undertake the research at Agricultural University of Norway (AUN), Norway and at the University of Helsinki, Finland. E. W-m. is grateful to Lånekassen, Norway, for a PhD stipend and NorFA for providing some support while in Finland. Thanks are due to G. Kobro and E.M. Aasen, technical staff in the microbiology unit at the department of Chemistry, Biotechnology and Food Sciences at AUN, and the N-fixation research group at the Biocenter, University of Helsinki. 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