Mycorrhizal association in gametophytes and sporophytes of the fern Pteris vittata (Pteridaceae) with Glomus intraradices Alicia E. Martinez1,3, Viviana Chiocchio2,3, Lo Tai Em1, María A. Rodriguez1,3 & Alicia M. Godeas1,3 1. Departamento de Biodiversidad y Biología Experimental. Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Av. Int. Güiraldes s/N. Pabellón II. Ciudad Universitaria. 1428, Buenos Aires, Argentina; [email protected], [email protected], [email protected], [email protected] 2. Cátedra de Microbiología Agrícola y Ambiental. Facultad de Agronomía. Universidad de Buenos Aires - Av. San Martín 4453. 1417. Buenos Aires. Argentina; [email protected] 3. INBA - CONICET Received 09-V-2011. Corrected 10-XI-2011. Accepted 12-XII-2011. Abstract: Ferns, which are usually colonizing different environments and their roots frequently present mycor- rhization, have two adult stages in their life cycle, the sporophytic and the gametophytic phase. This paper describes the experimental mycorrhizal association between Pteris vittata leptosporangiate fern and a strain of Glomus intraradices during the life cycle of the fern, from spore germination to the development of a mature sporophyte. The aim of this study was to compare the colonization pattern of in vitro cultures of G. intraradices along the fern life cycle with those found in nature. For this, mature spores were obtained from fertile P. vittata fronds growing in walls of Buenos Aires city, Argentina. Roots were stained and observed under the light micro- scope for arbuscular mycorrhizal colonization. Approximately, 75 fern spores were cultured in each pot filled with a sterile substrate and G. intraradices (BAFC N° 51.331) as inoculum on the surface. After germination took place, samples were taken every 15 days until the fern cycle was completed. In order to determine coloni- zation dynamics each sample was observed under optical and confocal microscope after staining. Gametophyte was classified as Adiantum type. Male and female gametangia were limited to the lower face, mycorrhizal colo- nization started when they were differentiated and took place through the rhizoids. Spores and vesicles were not found in this cycle stage. Paris-type mycorrhizal colonization was established in the midrib and in the embrion- ary foot. It was colonized by external mycelium. When the first root was developed soil inoculum colonized de novo this structure and Arum-type colonization was observed. This study proves that the type of colonization is determined by the structure of the host, not by the fungus. Both the gametophyte and embryo foot have deter- mined growth and Paris-type colonization, while, sporophyte roots have undetermined growth and Arum-type colonization. The structures found in vitro cultures were highly similar to those found under natural conditions. Rev. Biol. Trop. 60 (2): 857-865. Epub 2012 June 01. Key words: Pteris vittata, Glomus intraradices, Pteridophyta-gametophyte-sporophyte, Arum colonization, Paris colonization. Over 90% of terrestrial plant groups have colonized by arbuscular mycorrhizal fungi some type of symbiosis with soil fungi, and/or (AM) (Brundrett 2002). Fern species with fine some mycorrhizal form. During the Devonic roots and long absorbent hairs sometimes limit period, the first plants with roots appeared mycorrhizal colonization. This facultative asso- on land, the ferns belonging to Pteridophy- ciation is considered to be a feature of more ta, Filicales groups, still exist today. They evolved ferns (Fernandez et al. 2010). Cairney are widely distributed, particularly in tropical (2000) and Brundrett (2002) suggested that environments, and many of them have roots mycorrhizal symbiosis probably enabled plants Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 60 (2): 857-865, June 2012 857 to colonize land, conferring advantages such the life cycle of Pteris vittata and the colo- as increased fitness and resistance to drought nization of the arbuscular mycorrhizal fungi or pathogenic microorganisms under certain G. intraradices. conditions (Smith & Read 1997, Daniell et al. 1999, Read 1999). MATERIALS AND METHODS Preliminary data indicated that the pres- ence of AM fungi at both the sporophyte and Collection of plant material: Approxi- the gametophyte stage, stimulates fern growth mately 100 specimens of P. vittata L. at dif- (Turnau et al. 2005). P. vittata sporophytes ferent stages of sporophyte development were grown in controlled conditions showed an collected from hollows filled of saline substrate increase in fresh and dry weight shoots when on damp walls of Buenos Aires city, and ana- grown in contaminated soil (Trotta et al. 2006, lysed for root colonization. Sampled sporo- Leung et al. 2006). In natural conditions (Zhi- phytes ranged in height from few millimetres wei 2000) from 12 different species of Pteris up to 6cm. A number of 20 samples were (P. vittata included) found sporophytic mycor- attached to the gametophyte. rhization only in P. setulosocostulata. P. vittata is a widely distributed fern in Cultivation of plant material: Spores Buenos Aires city, often growing on the sub- were obtained from fertile P. vittata fronds strate that fills hollows in damp walls. Under and kept in dry, covered containers until they these stressful conditions, it was observed that were used. Thirty 50mL pots were filled with the sporophyte was always colonized by arbus- a sterile mixture of perlite:peat:soil 5:2:1V/V, cular mycorrhizal fungi. and 500mg of G. intraradices inoculum placed P. vittata has a chlorophyllous gameto- on the surface of each pot, containing approxi- phyte of limited growth, the most vulnerable mately 75 fern spores on top. phase of the life cycle (Zhang et al. 2008). The The mycorrhizal strain used in this experi- appearance of AM fungi Glomus intraradices ment is a pure culture identified as G. intr- at the gametophyte phase may significantly aradices, originated from a pastureland from shorten the period when the small plants are Buenos Aires province in Argentina, grown especially susceptible to drought, allowing in association with white clover (Trifolium them to adapt better to the environment (Boul- repens). Spores have been preserved as herbar- lard 1957, 1979, Pirozynski & Malloch 1975). ium material (BAFC N° 51.331). The culture There are two main morphological types of was replicated, and part of it kept in our col- arbuscular mycorrhizas (AM), the Arum-type lection under the name Strain GB1 (Banco de and the Paris-type and a continuum between Glomeromycota In vitro (BGIV) http://www. them. They are responsible of the P plant bgiv.com.ar/strains/glomus-intraradices/gb1). nutrition (van Aarle et al. 2005). In the Arum- Pots were watered to field capacity and type, fungi form intercellular hyphae between kept in a humid chamber (relative humidity the cortical cells and intracellular arbuscules 100%) at 27-30°C and a regime of 16 hours within them. The Paris-type is characterized light/8 hours darkness. As from germination, by extensive intracellular hyphal coils and samples were taken every 15 days until the arbusculate coils in the root cortex. In the fern cycle was complete, in order to determine Paris-type the intercellular phase of coloniza- colonization dynamics. tion is almost absent. With very few exceptions, members of single plant species formed only Microscopy analyses: The cultured and one type of colonization. It is often accepted field material was cleared and stained using that AM morphology is controlled by plant the method of Phillips & Hayman (1970) as identity (Smith & Smith 1997). The aim of this follows: fresh roots were heated in a 10% KOH study is to determine the relationship between solution at 90ºC for 15 minutes, washed in tap 858 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 60 (2): 857-865, June 2012 water and immersed in 20 vol (H2O2) for 10 between the rhizoids (Fig. 1D). About 75% of minutes until bleached. Then, they were rinsed the gametophytes examined were colonized by in tap water to remove H2O2, acidified in 0.1N G. intraradices, of sporophytes arising from HCl, and stained with 0.05% Trypan Blue solu- embryo development. tion for 20 minutes at 90ºC. Dye excess was The first stages of prothallus development removed in clear 85% lactic acid. Root seg- lack mycorrhizal colonization even when rhi- ments were mounted on slides in 85% Lactic zoids have developed (Fig. 1A). Gametophyte acid. Observation and microphotography were colonization only began when gametangia were assessed using a Nikon Opthot-2 microscope differentiated (Fig. 1D). Gametangial differen- fitted with a digital Coolpix 950 camera. tiation was separated in the time (protandric For a more accurate observation of root gametophyte). Colonization took place through colonization, confocal microscopy was used. the rhizoids (Fig. 1B-F), on which an appreso- Root samples were fixed at least 12 hours in rium formed and penetrated the cell. On reach- 50% ethanol. Roots were cleared by heating ing the basal cells of the midrib, the hypha them in 5% KOH (w/v) at 90ºC, roots were grew forming coils and spread through cells washed with tap water, and then acidified with (Paris-type), taking up ¾ of the midrib (Fig. 0.1N HCl for 5-10 minutes. Roots were stained 1B, D). The arbuscules, being very small, grew with 0.01% acid fuchsin (w/v) in solution of from these coils (Fig. 1C, E, F). There was acid-glycerin-water (875mL lactic acid, 63mL little, if any, intercellular growth. Arbuscules glycerin, 63mL water) for one hour at 55ºC. were ephemeral, and in most cells we observed Dye excess was removed in 100% glycerin. amorphous, blue-stained material. Spores and Observations and microphotography were con- vesicles were not found in this phase of the ducted using an Olympus FV300 confocal cycle. Gametophyte wings and growth apex scanning HeNe green laser microscope and were uncolonized, probably because the wings with excitation at 543nm (Argon laser).