17th Australian Nitrogen Fixation Conference 2014 Proceedings

Adelaide, Australia

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Gupta, V.V.S.R. Unkovich, M. Kaiser, B.N.

Example citation Pipai, R. 2014. Biological nitrogen fixation by legume cover in oil palm plantations’, in the 17th Australian Nitrogen Fixation proceedings, ed. Gupta V.V.S.R., Unkovich, M. and Kaiser, B. N. ASNF, Adelaide, pp. 19-21

ISBN 978-0-86396-000-0

th 17 Australian Nitrogen Fixation Conference, Adelaide 2014 2 Nodulation in the endemic Australian Papilionoideae tribes Mirbelieae and Bossiaeeae

Julie K. Ardley1, Euan K. James2, Sofie E. De Meyer1, Janet I. Sprent3 and John G. Howieson1

1Centre for Rhizobium Studies, Murdoch University, Murdoch WA 6150, Australia 2The James Hutton Institute, Invergowrie, Dundee, UK 3College of Life Sciences, University of Dundee, Dundee, UK

Key Words Legumes, nodule structure, Bradyrhizobium, biogeography

Introduction The closely related papilionoid tribes Mirbelieae and Bossiaeeae are a large (ca. 750 ) group of endemic Australasian legumes. Typically they are ericoid shrubs that are conspicuous understorey members of sclerophyll communities growing on poor soils of the southwest and southeast temperate biomes (Crisp et al. 2004). The centre of diversity is in Southwest Australia, a biodiversity hotspot, unglaciated since the Permian (ca. 250 Mya) and dominated by old landscapes with nutrient-deficient soils (Hopper and Gioia 2004). The crown clade age for the mirbelioids and their sister tribe Hypocalypteae is estimated to be ca. 55 Mya, suggesting that mirbelioids evolved in isolation, shortly after Australia separated from the Antarctic continent, having lost contact with other Gondwanan land masses (Sprent et al. 2013). Molecular-dated phylogenies indicate that the group radiated rapidly during the Mid-Cenozoic (ca. 25–10 Mya) period of climatic cooling and drying (Crisp et al. 2004). In common with most papilionoid legumes, Mirbelieae and Bossiaeeae species form nitrogen-fixing associations with rhizobia, usually with strains of Bradyrhizobium (Lafay and Burdon 1998; Stępkowski et al. 2012). Nodule morphology and structure has not yet been studied. This character trait has previously been found to be a useful phylogenetic and taxonomic marker (Sprent et al. 2013). Phylogenetic analyses place the mirbelioid legumes between the Dalbergioid and Indigoferoid clades, two groups that have quite different nodule structures (Sprent et al. 2013). This study aimed to characterise rhizobial microsymbionts of diverse mirbelioid species collected from sites across southwest Australia and to determine the mirbelioid nodule morphology and structure.

Methods Fieldwork, glasshouse work and microscopy Fieldwork was carried out during early spring. Nodules were collected and desiccated in situ or halved and fixed in glutaraldehyde. Soil samples from each site were used in glasshouse pot trials with trap hosts. Seeds supplied by Nindethana Seeds were either scarified or treated with hot water before surface sterilisation, germination and growth in glasshouse pots, using an axenic sand culture system. Selected nodules were excised from harvested plants, fixed and prepared for nodule sectioning and light microscopy, or were used for isolation of rhizobia.

Isolation of bacteria, DNA extraction, gene sequencing and phylogenetic analysis Desiccated nodules were re-imbibed in distilled water. Fresh or rehydrated nodules were surface sterilised and rhizobia subsequently isolated and routinely subcultured on ½ Lupin Agar (½ LA). Rhizobial isolates were authenticated on legume hosts in axenic glasshouse pots. Genomic DNA of isolates was prepared and PCR amplifications and sequencing and analysis of the 16S rRNA gene were performed.

Results and Discussion Rhizobial isolates were obtained from nodulated host legumes at all sites (Table 1). In total, 171 rhizobial isolates were collected from 18 mirbelioid species. The soil pH at most sites was mildly acidic and all isolates were slow-growing, phylogenetically diverse strains of Bradyrhizobium, which accords with bradyrhizobial adaptation to low pH soils (Zhang et al. 2011). There were no strong patterns of host or geographic associations. This held true also for Viminaria juncifolia, a monotypic species which is adapted to seasonally waterlogged habitats and forms both cluster roots and mycorrhizas (de Campos et al. 2013). In all cases, nodules of mirbelioid legumes were indeterminate, with a persistent meristem and central tissue containing both infected and uninfected cells (Figure 1).

th 17 Australian Nitrogen Fixation Conference, Adelaide 2014 54 Table 1. Southwest Australian sites and mirbelioid hosts from which rhizobial isolates were obtained Site ID Legume host Latitude Longitude pH Bg Bo, Ca (T), Cc, Cd, Crh (T), Gok, Gok (T)Gom (T), Vj (T) 32 10' S 116 2' E 6.2 Wh Bo, Cre, Go, Gase (T) 33 49' S 115 23' E 6.2 Me1 Bl (T), Ca (T), Gok (T), Vj 33 34' S 115 5' E 7.2 Me2 Bo, Crh, Gase, Gok 33 35' S 115 05' E 6.3 Den1 Ca (T), Cc (T), Crh (T), Gac, Go (T), Eum 34 57' S 117 17' E 6.1 Den4 Vj 34 47' S 117 27' E 6.0 TC1,2 B, Ca, Ca (T), Cc (T), Crh (T), Gasp, Gok (T) 34 36' S 117 52' E 6.2 117 50-52' TC3,4 Cg, Gase, Gasp, Gop 34 35' S E 6.2 Be = , Bl = , Bo = Bossiaea ornata, Ca = Chorizema aciculare, Cc = Chorizema cordatum, Cd = Chorizema dicksonii, Cg = Chorizema glycinifolium, Cre = Chorizema reticulatum, Crh = Chorizema rhombeum, Eum = Eutaxia myrtifolia, Gac = , Gase = Gastrolobium sericeum, Gasp = , Gok = Gompholobium knightianum, Gom = Gompholobium marginatum, Go = Gompholobium ovatum, Gop = Gompholobium polymorphum, Vj = Viminaria juncifolia, (T) = trap host

1 mm 0.5 mm a b

Figure 1. Nodulation in mirbelioid legumes: a) Indeterminate nodules of Chorizema cordatum; b) nodule section of Gastrolobium sericeum: the central tissue contains both infected (blue) and uninfected cells.

Conclusion Mirbelioid legumes are able to nodulate and fix N2 with diverse strains of Bradyrhizobium, indicating that selection pressure has not favoured the development of host specificity.

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