Does Lunularia Cruciata Form Symbiotic Relationships with Either Glomus Proliferum Or G

mycological research 112 (2008) 1063–1068

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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 thallus the typical traits of symbiosis generally observed in the Phosphorus experiment roots of mycorrhizal vascular plants? (2) Does this colonization 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 treatment, 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 hyphal 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. cruciata with G. proliferum; and axenic L. (Statsoft). cruciata. The sucrose treatment consisted of SRV media with two levels of added sucrose (29.2 and 7.3 mM sucrose) and SRV medium without sucrose. There were six replicate Petri Results dishes per treatment, each containing one plant. Acquisition of phosphorus by the hyphae crossing the PB and its effect on Lunularia cruciata LM Only Glomus intraradices and G. proliferum were able to cross To observe fungal distribution within Lunularia cruciata, pieces over the dual compartment PB (Fig 2), thus allowing the plant of mycothalli and adjacent medium were cropped from cul- to access the medium in the phosphorus compartment during tures, fixed in Bouin’s fluid and cleared with 10 % KOH, at 72 Æ 5 and 67 Æ 4 d, respectively, for G. intraradices and G. prolif- 80 C for 20 min. Samples were washed in distilled water, erum. Mycothallic plants with hyphae crossing the PB into the

Table 1 – Monoxenic culture of Lunularia cruciata grown for 100 d with Glomus intraradices or G. proliferum in two compartment Petri dishes each with 15 ml SRV medium and 29.2 mM sucrose Fungus Barrier Lunularia cruciata Fungus transposed Dry Total Phosphorus Number of Hyphal weight (g)a phosphorus (mg)b concentration sporesa length (mm)c À1 (mgPg D.W.)

Glomus intraradices Yes 0.084 Æ 0.015b 53.0 Æ 15.2b 623.8 Æ 106.4a 151705 Æ 78077b 92440 Æ 35278b No 0.055 Æ 0.009a 33.2 Æ 1.6a 612.8 Æ 92.7a 39879 Æ 24564a 28184 Æ 11612a G. proliferum Yes 0.076 Æ 0.006b 50.3 Æ 7b 665.0 Æ 119.9a 236155 Æ 73203b 121983 Æ 29100b No 0.054 Æ 0.01a 33.2 Æ 1.5a 634.4 Æ 111.3a 111551 Æ 34841a 84517 Æ 30190b

Added phosphorus (61.5 mgKH2PO4) was only available to the culture when the hyphae were allowed to transpose the plastic barrier from the

liverwort compartment to the phosphorus compartment. In the liverwort compartment any KH2PO4 was added. Yes, fungal allowed to cross the Petri dish plastic barrier; No, hyphae not allowed to transpose the barrier. Means (Æ S.D.) followed by the same letter, within the column, do not differ signiﬁcantly (aP < 0.03; bP < 0.02; cP < 0.008). 1066 H. M. A. C. Fonseca, R. L. L. Berbara

Table 2 – Culture of Lunularia cruciata with and without AM fungi (Glomus intraradices or G. proliferum) grown for 100 d in 30 ml SRV media with different concentrations of added sucrose Fungus Sucrose (mM) Lunularia cruciata Fungus

Dry weight (g)a Number of sporesb Hyphal length (mm)c

Glomus intraradices 0.0 0.017 Æ 0.004a 43 Æ 98a 325 Æ 423a 7.3 0.067 Æ 0.006b 2673 Æ 1415a 55726 Æ 11504a 29.2 0.121 Æ 0.01c 114697 Æ 21471b 155913 Æ 25063b G. proliferum 0.0 0.012 Æ 0.005a 0 Æ 0a 14 Æ 25a 7.3 0.069 Æ 0.01b 12870 Æ 11124a 162617 Æ 69843b 29.2 0.126 Æ 0.013c 209439 Æ 69515c 243450 Æ 49319c None 0.0 0.009 Æ 0.001a –– 7.3 0.071 Æ 0.011b –– 29.2 0.153 Æ 0.011d ––

a b c Means (ÆS.D.) followed by the same letter, within the column, do not differ signiﬁcantly at P < 0.0001, P < 0.001, P < 0.02.

phosphorus compartment showed significantly higher the area where the hyphae crossed the PB (Fig 2C–D). Average biomass than those with fungus restricted to the liverwort external hyphae length was only observed to be statistically compartment (Table 1), i.e. when restricted to a medium with- significant for G. proliferum (P < 0.008). out added phosphorus plants showed an average reduction in dry weight of about 35 and 30 %, respectively, for Lunularia cruciata with G. intraradices and with G. proliferum. These Effect of added sucrose results suggest net transference of phosphorus from one compartment to another via AM hyphae with a direct impact Under the present culture conditions added sucrose was on plant growth. When hyphae crossed the PB and acceded shown to have a significant effect on Lunularia cruciata growth, to the phosphorus compartment the plants showed average independently of the presence or absence of AM fungi or increments in total phosphorus content of 20 mg and tested fungal species (Table 2). Higher added levels of sucrose À1 17 mgg D.W.(P < 0.02), respectively, for L. cruciata with G. resulted in higher plant dry weights (P < 0.0001). However, on intraradices and with G. proliferum (Table 1). However, liverwort comparison of monoxenic (Fig 1B–C) with axenic (Fig 1D) L. phosphorus concentration at the end of the experiment (100 d) cruciata for plants grown with 29.2 mM sucrose, monoxenic À1 did not change significantly (640 Æ 100 mgPg D.W.) whatever plant dry weight showed a significant decrease of 21 % the amount of total phosphorus available to the plant. The (P < 0.0003) and 18 % (P < 0.0003), respectively, for G. intraradi- increase availability of phosphorus and other nutrients from ces and G. proliferum (Table 2). the phosphorus compartment resulted in significant (P < 0.03) The amount of added sucrose also significantly influenced differences in the average number of spores produced by the the production of fungal spores and external hyphae length AM fungi when compared with mycothallus restricted only to (Table 2). The behaviour of mycothalli cultured on 29.2 mM the liverwort compartment (Table 1). Concurrently, an increase sucrose showed patterns (Fig 1B–C) that were in accordance agglomeration of spore numbers/clusters was observed near with those previously described by Fonseca et al. (2006).

Fig 3 – LM of trypan blue-stained samples of Lunularia cruciata cultured for 100 d with Glomus proliferum in SRV medium with 29.2 mM sucrose. (A) Anatomic section of mycothallus showing (1) the lower epidermis with rhizoids; (2) thallus’ midrib parenchyma with high concentration of arbuscules, vesicles and oil cells; (3) the photosynthetic layer under a detached (4) upper epidermis. Bar [ 200 mm. (B) Detail of a zone of thallus’ midrib parenchyma with high concentration of (5) oil cells containing collapse arbuscules. (6) Vesicle. Bar [ 50 mm. (Inset) Shows mycothallus’ arbuscular cell. Bar [ 20 mm. Does Lunularia cruciata form symbiotic relationship 1067

Structures analogous to those observed for AM of vascular photosynthate carbon to maintain monoxenic cultures of plants were present within the mycothallus of L. cruciata AM fungi with L. cruciata. The fungi required the hydrolysis (Fig 3). of added sucrose to grow, populate the cultures, and produce new spores. Moreover, when axenic cultures of L. cruciata were compared with mycothallic cultures it was clear that the pres- Discussion ence of the fungi was costly for plant growth. This suggests that in vivo and in natural environment conditions major nu- For most land plants, sensu tracheophytes, it is widely tritional parameters, such as plant sugar status and the ability accepted that AM fungi are obligate symbiotic organisms to produce enough photosynthate carbon will be of para- able to assist their hosts in the acquisition of inorganic nutri- mount importance in controlling the dynamics of symbiosis ents, such as phosphates (Smith & Read 1997). The plant in (Schwab et al. 1991; Koide & Schreiner 1992; Landis & Fraser turn supplies the fungi with carbohydrates required to fulfil 2008). From the observations in this study, and from those of its life-cycle (Pfeffer et al. 1998, 1999; Bago et al. 2000). With Fonseca et al. (2006), we may conclude that although these non-vascular plants little information is available for a con- colonizations have apparently available functionalities gener- sensus on the symbiotic behaviour of liverworts and AM fungi. ally associated with mycorrhizal symbiosis in vascular plants, To investigate the nature of the association between Lunularia the energetic constraints and the nutritional requirements of cruciata and two AM fungi (Glomus intraradices and G. prolif- liverworts and AM fungi in natural conditions may render erum) we followed an approach suggested by Read et al. these interactions in most cases more parasitic/opportunistic (2000). These authors indicated the need to determine partnerships than a mutualistic symbiosis behaviour. whether the requirements of Koch’s postulate for obligate symbionts were met. The first requirement (Read et al. 2000) was already answered favourably through data presented by Fonseca et al. (2006). Anatomic traits, such as inner paren- Acknowledgements chyma cells within the mycothallus harbouring inter- and intracellular mycelium, which produce arbuscules and vesicles, We thank Lourdes Pereira and Manuel Santos for the use of were observed in L. cruciata colonized by G. proliferum (Fig 3B). their Laboratory facilities. We acknowledge the financial sup- These typical AM features, present in the cultures of both port of Fundaçaõ para a Cieˆncia e a Tecnologia (SFRH/BSAB/ fungi species, were also described for other liverworts (e.g. 740/2007), Portugal. R.L.L.B. has a fellowship grant from In- Nebel et al. 2004; Russell & Bulman 2005). ter-American Institute for Global Change Research (IAI) CRN To address the second Koch’s requirement, two-compart- II/14 which is supported by the US National Science Founda- ment cultures (StArnaud et al. 1996; Nielsen et al. 2002) were tion (Grant GEO-04523250). constructed where plants were prevented to access added phosphorus by a Petri dish PB. In the liverwort compartment references plants only had available residual phosphorus brought with inoculated discs of mycothallus and from some medium components, e.g. Phytagel. As the amount of phosphorus Bago B, Pfeffer PE, Shachar-Hill Y, 2000. Carbon metabolism and present in the liverwort compartment was known to restrict transport in arbuscular mycorrhizas. 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