Elsevier

Mycological Research

Volume 112, Issue 9, September 2008, Pages 1063-1068
Mycological Research

Does Lunularia cruciata form symbiotic relationships with either Glomus proliferum or G. intraradices?

https://doi.org/10.1016/j.mycres.2008.03.008Get rights and content

Abstract

The present study was undertaken to investigate whether the behaviour in vitro of Lunularia cruciata grown with Glomus intraradices and G. proliferum, on SRV medium with 29.2 mm sucrose satisfies the requirements of Koch' postulates for mutualistic symbiosis. Hyphae emerging from mycothallus were able to grow over a two-compartment Petri dish barrier and capture and translocate phosphorus into the host liverwort. Thus, there were increases in plant dry weight, higher AM fungi spore production, and higher plant total phosphorus content. Moreover, this colonization of L. cruciata reproduces typical symptoms generally associated with mycorrhizae. These results showed that mycothalli of L. cruciata have available functionalities generally associated with mycorrhizal symbiosis in higher plants; however, the energy/photosynthetic carbon requirements to maintain a mutualistic symbiosis may be a limiting factor in vivo. Features here discussed indicate that, at least in tested experimental conditions, the endophytic association of L. cruciata with both G. intraradices and G. proliferum is a parasitic/opportunistic partnership rather than a mutualistic symbiosis.

Introduction

AM are ubiquitous, underground, symbiotic associations involving a wide diversity of plants and obligate symbiotic fungi of the phylum Glomeromycota (Schüβler et al. 2001). For vascular plants it is commonly accepted that AM increases host resistance to biotic and abiotic stresses, as well as stimulating growth by enhancing soil nutrient uptake, particularly inorganic phosphate (Smith & Read 1997). Concurrently, plants supply the fungus with photosynthate carbon (Bago et al., 2000, Pfeffer et al., 2004).

Liverworts are an ancient and extremely successful group of plants with a remarkable variety of species and forms that are only supplanted by the flowering plants. They are found in all continents and habitats, and are thought to be amongst the original colonizers of terrestrial habitats. Data from early fossil records, some dating from the Ordovician, show that these taxa appear to have remained relatively unchanged through time, hence, holding a possible key to early terrestrial diversification of land plants (Read et al., 2000, Renzaglia et al., 2007). Some complex thalloid liverworts (Marchantiales) are known to form mycorrhiza-like associations with AM fungi, with the presence of structures that are analogous to those observed in AM of vascular plants, thus indicating possible functional similarities (Read et al., 2000, Nebel et al., 2004, Kottke and Nebel, 2005, Russell and Bulman, 2005, Fonseca et al., 2006, Ligrone et al., 2007).

The present work tests whether the plant–fungi association satisfies the requirements of Koch's postulates for mutualistic AM symbiosis as suggested by Read et al. (2000). Hence, we address two questions concerning the effect of colonization of a liverwort plant (Lunularia cruciata) by two 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 roots of mycorrhizal vascular plants? (2) Does this colonization stimulate plant and fungal growth with the concomitant acquisition and transfer of inorganic phosphate by the fungus?

Section snippets

Biological material and growth conditions

Glomus intraradices (MUCL 43204) and Glomus proliferum (MUCL 41827), acquired from GINCO (Mycotheque de l'Université Catholique de Louvain, Laboratoire de Mycologie, Belgique), were multiplied and maintained, since 2003, in monoxenic cultures of Lunularia cruciata. Throughout the experiments plants and fungi were kept at 25 °C with a 10/14 h light/dark photoperiod in a Sanyo MLR–350H chamber. The light had an average intensity of 68.3 ± 6.4 μmol s−1 m−2 measured at eight different positions per shelf

LM

To observe fungal distribution within Lunularia cruciata, pieces of mycothalli and adjacent medium were cropped from cultures, fixed in Bouin's fluid and cleared with 10 % KOH, at 80 °C for 20 min. Samples were washed in distilled water, acidified in 1 n HCl before being dehydrated and embedded in paraffin wax. Sections of ca 10 μm were cut with a microtome (Leitz model 1512), mounted on microscope slides, and stained overnight in 0.05 % trypan blue (Phillips & Hayman 1970). Images were digitally

Acquisition of phosphorus by the hyphae crossing the PB and its effect on Lunularia cruciata

Only Glomus intraradices and G. proliferum were able to cross over the dual compartment PB (Fig 2), thus allowing the plant to access the medium in the phosphorus compartment during 72 ± 5 and 67 ± 4 d, respectively, for G. intraradices and G. proliferum. Mycothallic plants with hyphae crossing the PB into the phosphorus compartment showed significantly higher biomass than those with fungus restricted to the liverwort compartment (Table 1), i.e. when restricted to a medium without added phosphorus

Discussion

For most land plants, sensu tracheophytes, it is widely accepted that AM fungi are obligate symbiotic organisms able to assist their hosts in the acquisition of inorganic nutrients, such as phosphates (Smith & Read 1997). The plant in turn supplies the fungi with carbohydrates required to fulfil its life-cycle (Pfeffer et al., 1998, Pfeffer et al., 1999, Bago et al., 2000). With non-vascular plants little information is available for a consensus on the symbiotic behaviour of liverworts and AM

Acknowledgements

We thank Lourdes Pereira and Manuel Santos for the use of their Laboratory facilities. We acknowledge the financial support of Fundação para a Ciência e a Tecnologia (SFRH/BSAB/740/2007), Portugal. R.L.L.B. has a fellowship grant from Inter-American Institute for Global Change Research (IAI) CRN II/14 which is supported by the US National Science Foundation (Grant GEO-04523250).

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