mycological research 112 (2008) 1063–1068 journal homepage: www.elsevier.com/locate/mycres Does Lunularia cruciata form symbiotic relationships with either Glomus proliferum or G. intraradices? Henrique M. A. C. FONSECAa,*, Ricardo L. L. BERBARAb aCentre of Cellular Biology, Department of Biology, University of Aveiro 3810-193, Aveiro, Portugal bUniversidade Federal Rural of Rio de Janeiro, Soil Department, Serope´dica, Itaguaı´, RJ, CEP 23851-970, Brazil article info abstract Article history: The present study was undertaken to investigate whether the behaviour in vitro of Lunularia Received 17 January 2008 cruciata grown with Glomus intraradices and G. proliferum, on SRV medium with 29.2 mM Received in revised form sucrose satisfies the requirements of Koch’ postulates for mutualistic symbiosis. Hyphae 14 March 2008 emerging from mycothallus were able to grow over a two-compartment Petri dish barrier Accepted 18 March 2008 and capture and translocate phosphorus into the host liverwort. Thus, there were Corresponding Editor: increases in plant dry weight, higher AM fungi spore production, and higher plant total Paola Bonfante phosphorus content. Moreover, this colonization of L. cruciata reproduces typical symp- toms generally associated with mycorrhizae. These results showed that mycothalli of Keywords: L. cruciata have available functionalities generally associated with mycorrhizal symbiosis Arbuscular mycorrhizal fungi in higher plants; however, the energy/photosynthetic carbon requirements to maintain Glomus intraradices a mutualistic symbiosis may be a limiting factor in vivo. Features here discussed indicate Glomus proliferum that, at least in tested experimental conditions, the endophytic association of L. cruciata Lunularia cruciata with both G. intraradices and G. proliferum is a parasitic/opportunistic partnership rather Opportunistic Partnership than a mutualistic symbiosis. Parasitic ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction amongst the original colonizers of terrestrial habitats. Data from early fossil records, some dating from the Ordovician, AM are ubiquitous, underground, symbiotic associations show that these taxa appear to have remained relatively involving a wide diversity of plants and obligate symbiotic unchanged through time, hence, holding a possible key to fungi of the phylum Glomeromycota (Schu¨ bler et al. 2001). For early terrestrial diversification of land plants (Read et al. vascular plants it is commonly accepted that AM increases 2000; Renzaglia et al. 2007). Some complex thalloid liverworts host resistance to biotic and abiotic stresses, as well as stimu- (Marchantiales) are known to form mycorrhiza-like associa- lating growth by enhancing soil nutrient uptake, particularly tions with AM fungi, with the presence of structures that are inorganic phosphate (Smith & Read 1997). Concurrently, analogous to those observed in AM of vascular plants, thus plants supply the fungus with photosynthate carbon (Bago indicating possible functional similarities (Read et al. 2000; et al. 2000; Pfeffer et al. 2004). Nebel et al. 2004; Kottke & Nebel 2005; Russell & Bulman Liverworts are an ancient and extremely successful group 2005; Fonseca et al. 2006; Ligrone et al. 2007). of plants with a remarkable variety of species and forms The present work tests whether the plant–fungi associa- that are only supplanted by the flowering plants. They are tion satisfies the requirements of Koch’s postulates for mu- found in all continents and habitats, and are thought to be tualistic AM symbiosis as suggested by Read et al. (2000). * Corresponding author. E-mail address: [email protected] 0953-7562/$ – see front matter ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2008.03.008 1064 H. M. A. C. Fonseca, R. L. L. Berbara Hence, we address two questions concerning the effect of and necrotic discs, as well as those emitting only few external colonization of a liverwort plant (Lunularia cruciata) by two hyphae. Glomus fungi: (1) does the colonization of liverworts by AM fungi (G. intraradices or G. proliferum) produce within the thal- lus the typical traits of symbiosis generally observed in the Phosphorus experiment roots of mycorrhizal vascular plants? (2) Does this coloniza- tion stimulate plant and fungal growth with the concomitant Selected discs with AM fungi were normalized for phosphorus acquisition and transfer of inorganic phosphate by the content by subculturing for 3 d in SRV medium with 29.2 mM fungus? sucrose but without phosphorus. Then discs were randomly transferred into two-compartment Petri dishes as previously described by StArnaud et al. 1996 and Nielsen et al. 2002 (Fig 2A–B). Hence one disc per dish was inoculated into the Materials and methods compartment containing 15 ml solidified SRV medium with 29.2 mM sucrose but no added phosphorus (liverwort compart- Biological material and growth conditions ment). The other compartment, that was left undisturbed, contained 15 ml of the same medium with 61.5 mgKH2PO4 Glomus intraradices (MUCL 43204) and Glomus proliferum (MUCL (phosphorus compartment). A 2 Â 2 factorial design was set 41827), acquired from GINCO (Mycotheque de l’Universite´ up, with the fungal treatment consisting of monoxenic L. cru- Catholique de Louvain, Laboratoire de Mycologie, Belgique), ciata with G. intraradices or G. proliferum. The phosphorus treat- were multiplied and maintained, since 2003, in monoxenic ment consisted of two sets of Petri dishes: in the first set only cultures of Lunularia cruciata. Throughout the experiments the external mycelium was allowed to grow over the plastic plants and fungi were kept at 25 C with a 10/14 h light/dark barrier (PB) into the phosphorus compartment. At the end of photoperiod in a Sanyo MLR–350H chamber. The light had the experiment the distances between the plants and PB an average intensity of 68.3 Æ 6.4 mmol sÀ1 mÀ2 measured at were at least twice the length of rhizoids (Fig 2A–B); hence no eight different positions per shelf with a Li-COR photometer contact was observed between the liverworts and PB. In the (Model Li-250) equipped with a spherical sensor (Fonseca second set of dishes the hyphae were prevented from estab- et al. 2006). lishing contact with the phosphorus compartment by cutting with a scalpel. There were six replicate Petri dishes per treat- ment, each containing one plant colonised by AM fungus. Inocula preparation Sucrose experiment All inoculated thalli were single thallus discs of 10.7 Æ 1.7 mm2 diam (Fig 1A) made using 3.27 mm diam cork-borers on Lunu- Selected discs with and without fungi were first cultured for laria cruciata with and without Glomus intraradices or G. prolif- 3 d in SRV medium with 7.3 mM sucrose in order to normalize erum grown for 60 d on SRV (Fonseca et al. 2006) with the inocula for sucrose content. Then they were randomly 29.2 mM sucrose; discs were cultured, before selection, on transferred to 90 mm diam (one compartment) plastic Petri fresh medium for 7 d in order to choose discs that were homo- dishes containing 30 ml SRV with different concentrations of geneously green and, when colonized by the fungus, had sucrose (Fig 1A). Hence, a 3 Â 3 factorial design was set up, strong extra-mycelium production, hence excluding uneven with fungal treatment consisting of Lunularia cruciata with Fig 1 – Lunularia cruciata culture with and without AM fungi (Glomus intraradices or G. proliferum) in SRV medium with 29.2 mM sucrose. (A) Disc of thallus at the beginning of the experiment, after 10 d pre-culture (see method above); (B) Culture of L. cruciata with G. intraradices after 100 d culture showing half a Petri dish (90 mm diam) with the plant and extensive external hyphae and spores. (C) As in (B) but with G. proliferum; (D) 100-d-old culture of axenic L. cruciata. Bar [ 10 mm. Does Lunularia cruciata form symbiotic relationship 1065 acidified in 1 N HCl before being dehydrated and embedded in paraffin wax. Sections of ca 10 mm were cut with a micro- tome (Leitz model 1512), mounted on microscope slides, and stained overnight in 0.05 % trypan blue (Phillips & Hayman 1970). Images were digitally acquired with a Carl Zeiss Axio- cam HR apparatus. Data collection Plant biomass was estimated as dry weight after oven-drying (60 C) to a constant weight. The number of spores and hy- phal length were measured under a stereomicroscope with Fig 2 – Lunularia cruciata cultured with (A) Glomus intraradi- a6Â 6 square hairline graticule of 20.25 mm2 regularly ces and (B) G. proliferum for 100 d in SRV medium with placed at 0.5 cm intervals over the surface of inverted Petri 29.2 mM sucrose showing hyphae transposing the divider dishes and following the method by McGonigle et al. (1990). into the phosphorus compartment. On the right detail views Analyses of plant phosphorus content was performed using of G. proliferum external mycelium and spores accumulated whole dry plants ashed in a muffle furnace (stages: 200 C near the crossing point (arrows) over the divider into the at 700 ChÀ1; 300 Cat70ChÀ1; 600 C at 700 ChÀ1; 600 C phosphorus compartment. (C) Sideway view from the over a period of 6.7 h) and dissolved in 10 ml of 1 M HCl. phosphorus compartment. (D) Above view of a section of Aliquots of supernatant were mixed with ammonium molyb- both compartments near the edge of the dish. (LP), liver- date, ammonium vanadate, and 1 M HCl in a 1:2:2:17 mixture wort compartment, without added phosphorus; (DP), (v/v/v/v). After a 30 min delay to allow colour development, phosphorus compartment medium with added 61.5 mg the absorbance at 440 nm was measured using a Shimadzu [ [ KH2PO4. Bar (A–B) 10 mm; (C–D) 1 mm. UV-120-02 spectrophotometer against a reagent blank. Data were evaluated for significance (P < 0.05) using multivariate analysis of variance (ANOVA/MANOVA) and post hoc Scheffe´’s test with Statistica software for Windows v4.5 Glomus intraradices; L.