First report on the occurrence of the uncultivated cluster 2 Frankia microsymbionts in soil outside the native actinorhizal host range area

1 1 1 2 IMEN NOUIOUI , IMED SBISSI , FATEN GHODHBANE-GTARI , KAWTAR FIKRI BENBRAHIM , PHILIPPE NORMAND3 and MAHER GTARI1,* 1Laboratoire Microorganismes et Biomolécules Actives, Université de Tunis El Manar (FST) et Université de Carthage (INSAT) 2092, Tunis, Tunisia 2Laboratoire de Biotechnologie Microbienne, Faculté des Sciences et Technologies de Fès Université Sidi Mohammed Ben Abdellah, P. O. Box 2202, Fès, Maroc. 3Ecologie Microbienne, Centre National de la Recherche Scientifique UMR 5557, Université Lyon I, Université Lyon, Villeurbanne 69622 cedex, France

*Corresponding author (Email, [email protected])

The occurrence of uncultivated Frankia was evaluated in Tunisian soils by a -trapping assay using myrtifolia seedlings. Despite the lack of this compatible host plant for more than two centuries, soil-borne Frankia cells were detected in one sampled soil as shown by the development of nodules on 2-year-old seedlings. Based on glnA sequences, Tunisian trapped Frankia strains belong to the uncultivated cluster 2 strains that associate with other Coriaria species and also with Ceanothus, Datisca and Rosaceae actinorhizal species. This is the first report on the occurrence of Frankia cluster 2 strains in soils from areas lacking compatible host plant groups.

[Nouioui I, Sbissi I, Ghodhbane-Gtari F, Benbrahim KF, Normand P and Gtari M 2013 First report on the occurrence of the uncultivated cluster 2 Frankia microsymbionts in soil outside the native actinorhizal host range area. J. Biosci. 38 695–698] DOI 10.1007/s12038-013-9366-z

Coriaria myrtifolia, a shrub with myrtle-like , occurs in et al. 2004), one of which has recently had its genome sequenced northern Mediterranean coastal , Southern France, Italian starting from symbiotic tissues (Persson et al. 2011). Cluster 2 Apennine mountains and the Balearic islands (Montserrat 1958) Frankia are also characterized by a narrow geographic distribu- as well as in Northern Africa from western Morocco to the tion and are highly dependent on their respective host for Algerian Kabylia (Good 1930; Gtari and Dawson 2011). In their distribution in soil. To date, no studies have been made on Tunisia, C. myrtifolia has never been mentioned except for a the distribution of these strains in soils from areas that lack single record in a hedge of Tunis City dating from more than a compatible host plants. In this study Coriaria myrtifolia was century. The plant was thus considered among the disappeared used in plant-trapping assay to evaluate whether the disappear- species in Tunisia (Alapetite 1979). Natural nodulation of this ance of this host plant from Tunisian soils resulted in the absence actinorhizal species in Morocco has been confirmed and the of compatible Frankia in soil. Soil samples were collected microsymbionts were assigned to Frankia cluster 2 (Nouioui et beneath Alnus glutinosa, Casuarina glauca and Elaeagnus al. 2011), a group comprising un-isolated strains from various angustifolia plants, respectively, in Tamra (37°3′ N, 9°7′ E, host plants belonging to Rosaceae, Datiscaceae, Coriariaceae and 150 m a.s.l.), Zerniza (37°9′ N, 9°7′ E, 60 m a.s.l.) and Ghar Rhamnaceae (Normand et al. 1996). Coriaria-infecting Frankia Elmelh (37°10′ N, 10°11′ E, 10 m a.s.l.) stands. Coriaria strains lack variety and are undistinguishable from other un- myrtifolia seeds kindly provided by A Ennabli (Université Sidi isolated cluster 2 strains in root nodules of Ceanothus, Datisca, Mohamed Ben Abdellah, Fès, Morocco) were surface sterilized and members of the actinorhizal Rosaceae (Benson and Clawson by treating with concentrated sulphuric acid for 5 min, rinsed 2000). This group of Frankia strains has never been isolated in several times with sterile distilled water and germinated in pure culture despite many attempts by several investigators and watered sterile vermiculite. After germination, three seedlings is, therefore, considered an uncultivated microsymbiont (Benson were placed in plastic pots containing 2 kg of each sampled soil

Keywords. Coriaria myrtifolia; Frankia; soil; Tunisia http://www.ias.ac.in/jbiosci J. Biosci. 38(4), November 2013, 695–698, * Indian Academy of Sciences 695

Published online: 1 October 2013 696 Imen Nouioui et al.

Figure 1. (a) Nodulated Coriaria myrtifolia plantlets on soil sampled beneath Alnus glutinosa stand; bar =1 cm. (b) Typical actinorhizal root nodules; bar = 2 mm. (c) Light micrograph of lobe cross-section showing elongated vesicle; bar = 20 μm. and were watered daily with tap water and maintained under Datisca cannabina plants present in those sites (Mirza et al. natural conditions in a garden in Tunis City in March. Plantlets 1994). The time course for obtaining root nodules in actinorhizal were examined carefully for the formation of nodules after plants depends on the inoculum, plant species and culture tech- 1, 6, 12, 18 and 24 months. While no nodulation was observed niques used, and it usually ranges from some weeks to many for all plants grown on sterile sand used as negative controls, months (Burgess and Peterson 1987; Berry and Sunell 1990). To coralloid root nodules of a size typical of those seen in Coriaria our knowledge, this may be the longest reported period in spp. (figure 1a and b) were observed after 18 months in 80% obtaining root nodulation in plant-trapping assays. This may be (n=5) of seedlings growing on soil sampled from the Tamra site, due to the relatively low predisposition of Coriaria to nodulate, where A. glutinosa is the only native species. As shown in as suggested by Mirza et al. (1994), who reported a poorer figure 1, the nodules are mature and are several months old. nodulation for Coriaria than for Datisca using the same set of Several studies showed Frankia strains to be present in soils well soils as inocula. Another explanation may be the time course outside the normal geographic range of appropriate host plants taken for the proliferation in the rhizophere of the compatible (Benecke 1969) or long after host plants have disappeared from a host plant of the soil-borne Frankia cell units (persisting in the site (Rodriguez-Barrueco 1968; Smolander and Sundman 1987; soil in the absence of host plant) to reach the Frankia cell units Wollum et al. 1968;Gtariet al. 2004). While the fact is con- threshold required to initiate nodulation. This result may be also firmable for Frankia from Alnus and Myrica (Cluster 1) and explained by various soil factors also reported to affect nodula- Elaeagnaceae (Cluster 3) host compatible strains, the situation tion in plant trapping assay (Chaia et al. 2010). The harvested remains uncertain for Frankia associated with Coriaria, Datisca, nodule lobes from Coriaria myrtifolia plantlets show the classic Ceanothus and Rosaceae (Benson et al. 2004)orCasuarina asymmetrical structure (figure 1c) as described in other Coriaria (Zimpfer et al. 1999). Data on Frankia infecting Coriaria sp. species (Canizo and Rodriguez-Barrueco 1978; Newcomb and is limited to soil samples collected from northern areas of Pankhurst 1982; Silvester and Harris 1989). Branched hyphae Pakistan shown to be able to nodulate Coriaria nepalensis and and elongated vesicles that do not occupy the centre of the

65 Uncultured Frankia sp. Cowania stansburiana (AF156760) 51 Uncultured Frankia sp. Datisca glomerata (AY644895) 98 Uncultured Frankia sp. Purshia tridentata (AY644890) 76 Uncultured Frankia sp. Chamaebatia foliolosa (AY644874) F.CmT8 F.CmT7 F.CmT3 89 F.CmT4 100 F.CmT5 91 F.CmT6 Uncultured Frankia sp. Ceanothus americanus (AF156762) Uncultured Frankia sp. Cercocarpus ledifolius (AF156763) Uncultured Frankia sp. Coriaria arborea (AF156764) Frankia sp. CcI3 (AF156756)

0.01

Figure 2. Phylogenetic tree inferred from nearly 477-bp of glnA using the neighbour-joining approach (Saitou and Nei 1987). Bootstrap values determined from 1000 replicates (Felsenstein 1985) and only values >50 are shown above branches.

J. Biosci. 38(4), November 2013 Frankia infecting Coriaria myrtifolia in Tunisia 697 infected cells were seen as described in other Coriaria species Benecke U 1969 Symbionts of nodules in New Zealand. Plant (Mirza et al. 1994; Newcomb and Pankhurst 1982). Because Soil 30 145–149 nodulation of C. myrtifolia was obtained only on a soil collected Benson DR and Clawson ML 2000 Evolution of the beneath A. glutinosa stand and since Rodriguez-Barrueco et al. symbioses; In Prokaryotic : A model system for (1988) hypothesized on the potential effective cross-nodulation analysis of biological process (ed) EW Triplett (Wymondham, UK: Horizon Scientific Press) pp 207–224 between these two actinorhizal species, there was need for further Benson D R, Vanden Heuvel BD and Potter D 2004 Actinorhizal identification of the trapped microsymbionts. DNA was extracted symbioses: diversity and biogeography; in Plant microbiology from single lobes as described by Ritchie and Myrold (1999)and (ed) M Gillings (Oxford: BIOS Scientific Publishers Ltd.) pp an approximately 477-bp-long glnA gene was PCR-amplified 97–127 using primers DB41 (5′-TTCTTCATCCACGACCCG-3′)and Berry AM and Sunell LA 1990 The infection process and nodule DB44 (5′-GGCTTCGGCATGAAGGT-3′) (Clawson et al. development; in The biology of Frankia and actinorhizal plants 2004). The 468 bp amplicons from six individual lobes were (eds) CR Schwintzer and JD Tjepkema (San Diego: Academic double-strand sequenced using an ABI v1.1 cycle sequencing Press, Inc.) pp 61–81 kit (Applied Biosystems). Sequence similarity of glnAin Burgess D and Peterson RL 1987 Development of Alnus japonica Coriaria microsymbionts trapped in the current study root nodules after inoculation with Frankia strain HFPArI3. – (Genbank accessions: KC796472-KC796477) ranged from Can. J. Bot. 65 1647 1657 96% to 100% (nucleotide) and 99% to 100% (amino acid). Canizo A and Rodriguez-Barrueco C 1978 Nitrogen fixation by – This identity level shows them to be close relative of the Coriaria nepalensis Wall. Rev. Ecol. Biol. Sol 15 453 458 Chaia EE, Wall LG and Huss-Danell K 2010 Life in soil by the microsymbionts present in the Rosaceous Chamaebatia actinorhizal root nodule endophyte Frankia. A review. Symbiosis foliolosa (AF156758) and the Rhamnaceous Ceanothus 51 201–226 – – americanus (AF156762) with 97 98% and 99 100% nucleo- Clawson ML, Bourret A and Benson DR 2004 Assessing the tide and amino acid sequences identity, respectively (figure 2). phylogeny of Frankia actinorhizal plant nitrogen-fixing root To date, a limited diversity was observed among Frankia nodule symbioses with Frankia16S rRNA and glutamine syn- associated with Coriaria. Only two 16S rRNA gene sequences thetase gene sequences. Mol. Phylogenet. Evol. 31 131–138 have been published so far, differing by a single nucleotide Felsenstein J 1985 Confidence limits on phylogenies: an approach among 12 Coriaria arborea root nodules collected in New using the bootstrap. Evolution 39 783–791 Zealand (Clawson et al. 2004). Nick et al. (1992) also detected Good RDO 1930 The geography of the genus Coriaria. New one mismatch in 274 pb on 16S rRNA gene sequence from Phytol. 29 170–198 nodules collected from New Zealand, France and Mexico. In Gtari M and Dawson JO 2011 An overview of actinorhizal plants in – view of those results, the diversity observed here on glnA gene Africa. Funct. Plant Biol. 38 653 661 Gtari M, Brusetti L, Skander G, Mora D, Boudabous A and Daffonchio sequences is considered relatively important. Moreover, the D 2004 Isolation of Elaeagnus-compatible Frankia from soils fact that the studied Coriaria microsymbionts were trapped collected in Tunisia. FEMS Microbiol. 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