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

*^{Dirección para correspondencia:}
Abstract

Ferns, which are usually colonizing different environments and their roots frequently present mycorrhization, have two adult stages in their life cycle, the sporophytic and the gametophytic phase. This paper describes the experimental mycorrhizal association betweenPteris vittataleptosporangiate fern and a strain ofGlomus intraradicesduring 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 ofin vitrocultures ofG. intraradicesalong the fern life cycle with those found in nature. For this, mature spores were obtained from fertileP. vittatafronds growing in walls of Buenos Aires city, Argentina. Roots were stained and observed under the light microscope for arbuscular mycorrhizal colonization. Approximately, 75 fern spores were cultured in each pot filled with a sterile substrate andG. 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 colonization dynamics each sample was observed under optical and confocal microscope after staining. Gametophyte was classified asAdiantumtype. Male and female gametangia were limited to the lower face, mycorrhizal colonization 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 embrionary foot. It was colonized by external mycelium. When the first root was developed soil inoculum colonizedde 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 determined growth andParis-type colonization, while, sporophyte roots have undetermined growth and Arum-type colonization. The structures foundin vitrocultures 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.

Resumen

Los helechos presentan dos etapas en su ciclo de vida, una fase esporofítica y una gametofítica. Estos por lo general pueden colonizar diferentes ambientes y frecuentemente presentan raíces micorrizadas. Este estudio describe la asociación experimental entrePteris vittata, un helecho leptosporangiado y una cepa de Glomus intraradicesdurante el ciclo de vida del helecho, desde la germinación de las esporas hasta el desarrollo del esporofito maduro. El objetivo de este estudio fue comparar los patrones de colonización deG. intraradicesa lo largo de todo el ciclo de vida del helecho con los tipos encontrados en la naturaleza. Las esporas maduras fueron obtenidas de frondes fértiles deP. vittataque crecen sobre las paredes de la ciudad de Buenos Aires, Argentina. Las raíces se tiñeron y fueron observadas bajo microscopio óptico para el estudio de la colonización micorrízica. Aproximadamente 75 esporas de helecho se cultivaron en macetas con un sustrato estéril y con un inóculo deG. intraradices(N° 51.331 BAFC) en la superficie. Después de la germinación, se tomaronmuestras cada 15 días hasta que se completó el ciclo de vida del helecho. Con el fin de determinar la dinámica de la colonización, cada muestra se observó con el microscopio óptico y el microscopio de confocal luego de la tinción correspondiente. El gametofito fue clasificado como del tipo “Adiantum”. Los gametangios femeninos y masculinos se desarrollaron en la cara inferior del mismo. La micorrización comenzó cuando los gametangios estaban ya diferenciados y la colonización se produjo a través de los rizoides. Las esporas y las vesículas no se encontraron en esta fase del ciclo. La micorrizacion tipoParisse observó sobre la línea de la nervadura central. El pie del esporofito fue colonizado por el micelio externo. Cuando la raíz se desarrolló, se colonizó “de novo”, y se observó una colonización de tipoArum. Este estudio demuestra que el tipo de colonización está determinado por la estructura del helecho y no por el hongo. Tanto el gametofito como el pie del embrión tienen crecimiento definido y colonización tipoParis, mientras que las raíces del esporofito presentan un crecimiento indeterminado y una colonización tipoArum. Las estructuras que se encontraron bajo cultivo coinciden con las que se encontraron en condiciones naturales.

Palabras clave: Pteris vittata, Glomus intraradices,Pteridophyta-gametofito, esporofito, colonización tipoArum, colonización tipo Paris.

Over 90% of terrestrial plant groups have some type of symbiosis with soil fungi, and/or some mycorrhizal form. During the Devonic period, the first plants with roots appeared on land, the ferns belonging to Pteridophyta, Filicales groups, still exist today. They are widely distributed, particularly in tropical environments, and many of them have roots colonized by arbuscular mycorrhizal fungi (AM) (Brundrett 2002). Fern species with fine roots and long absorbent hairs sometimes limit mycorrhizal colonization. This facultative association is considered to be a feature of more evolved ferns (Fernandez et al. 2010). Cairney (2000) and Brundrett (2002) suggested that mycorrhizal symbiosis probably enabled plants to colonize land, conferring advantages such as increased fitness and resistance to drought or pathogenic microorganisms under certain conditions (Smith & Read 1997, Daniellet al. 1999, Read 1999).

Preliminary data indicated that the presence of AM fungi at both the sporophyte and the gametophyte stage, stimulates fern growth (Turnauet al.2005).P. vittatasporophytes grown in controlled conditions showed an increase in fresh and dry weight shoots when grown in contaminated soil (Trottaet al.2006, Leunget al.2006). In natural conditions (Zhiwei 2000) from 12 different species ofPteris (P. vittataincluded) found sporophytic mycorrhization only inP. setulosocostulata.

P. vittatais a widely distributed fern in Buenos Aires city, often growing on the substrate that fills hollows in damp walls. Under these stressful conditions, it was observed that the sporophyte was always colonized by arbuscular mycorrhizal fungi.

P. vittatahas a chlorophyllous gametophyte of limited growth, the most vulnerable phase of the life cycle (Zhanget al.2008). The appearance of AM fungiGlomus intraradicesat the gametophyte phase may significantly shorten the period when the small plants are especially susceptible to drought, allowing them to adapt better to the environment (Boullard 1957, 1979, Pirozynski & Malloch 1975).

There are two main morphological types of arbuscular mycorrhizas (AM), theArum-type and the Paris-type and a continuum between them. They are responsible of the P plant nutrition (van Aarleet al. 2005). In theArum-type, fungi form intercellular hyphae between the cortical cells and intracellular arbuscules within them. TheParis-type is characterized by extensive intracellular hyphal coils and arbusculate coils in the root cortex. In theParis-type the intercellular phase of colonization is almost absent. With very few exceptions, members of single plant species formed only one type of colonization. It is often accepted that AM morphology is controlled by plant identity (Smith & Smith 1997). The aim of this study is to determine the relationship between the life cycle ofPteris vittataand the colonization of the arbuscular mycorrhizal fungiG. intraradices.

Materials and methods

Collection of plant material:Approximately 100 specimens ofP. vittataL. at different stages of sporophyte development were collected from hollows filled of saline substrate on damp walls of Buenos Aires city, and analysed for root colonization. Sampled sporophytes ranged in height from few millimetres up to 6cm. A number of 20 samples were attached to the gametophyte.

Cultivation of plant material:Spores were obtained from fertileP. vittatafronds and kept in dry, covered containers until they were used. Thirty 50mL pots were filled with a sterile mixture of perlite:peat:soil 5:2:1V/V, and 500mg ofG. intraradicesinoculum placed on the surface of each pot, containing approximately 75 fern spores on top.

The mycorrhizal strain used in this experiment is a pure culture identified asG. intraradices, originated from a pastureland from Buenos Aires province in Argentina, grown in association with white clover (Trifolium repens). Spores have been preserved as herbarium material (BAFC N° 51.331). The cultura was replicated, and part of it kept in our collection under the name Strain GB1 (Banco de GlomeromycotaIn vitro(BGIV)http://www.bgiv.com.ar/strains/glomus-intraradices/gb1).

Pots were watered to field capacity and kept in a humid chamber (relative humidity100%) at 27-30°C and a regime of 16 hours light/8 hours darkness. As from germination, samples were taken every 15 days until the fern cycle was complete, in order to determine colonization dynamics.

Microscopy analyses:The cultured and field material was cleared and stained using the method of Phillips & Hayman (1970) as follows: fresh roots were heated in a 10% KOH solution at 90ºC for 15 minutes, washed in tap water and immersed in 20 vol (H₂O₂) for 10 minutes until bleached. Then, they were rinsed in tap water to remove H₂O₂, acidified in 0.1N HCl, and stained with 0.05% Trypan Blue solution for 20 minutes at 90ºC. Dye excess was removed in clear 85% lactic acid. Root segments were mounted on slides in 85% Lactic acid. Observation and microphotography were assessed using a Nikon Opthot-2 microscope fitted with a digital Coolpix 950 camera.

For a more accurate observation of root colonization, confocal microscopy was used. Root samples were fixed at least 12 hours in 50% ethanol. Roots were cleared by heating them in 5% KOH (w/v) at 90ºC, roots were washed with tap water, and then acidified with 0.1N HCl for 5-10 minutes. Roots were stained with 0.01% acid fuchsin (w/v) in solution of acid-glycerin-water (875mL lactic acid, 63mL glycerin, 63mL water) for one hour at 55ºC. Dye excess was removed in 100% glycerin. Observations and microphotography were conducted using an Olympus FV300 confocal scanning HeNe green laser microscope and with excitation at 543nm (Argon laser). The lenses used included Zeiss UplanFI 20x/0.5 and UplanApo 40x/1.0 water immersion objectives. Images were captured and processed using Photoshop v 5.5 software Adobe Systems, San Jose, CA, USA (Petersonet al. 2004).

Results

Experimental life cycle ofP. vittatashowed that spores germinated after eight days and they gave rise to a few cell filaments, and a rhizoid able to differentiate. Gametophyte development (Fig. 1A) was of Adiantum-type (Nayar & Kaur 1969, Martinez 2010), characterized by a meristematic apical cell that divided laterally giving rise to new cells, which formed a notch. The apical cell divided transversally into further meristematic cells, which began to divide rapidly on another plane, producing a central zone made up of several cell layers with rhizoids and two wings. Male and female gametangia were limited to the lower face and to the ventral surface below the notch, mainly between the rhizoids (Fig. 1D). About 75% of the gametophytes examined were colonized byG. intraradices, of sporophytes arising from embryo development.

The first stages of prothallus development lack mycorrhizal colonization even when rhizoids have developed (Fig. 1A). Gametophyte colonization only began when gametangia were differentiated (Fig. 1D). Gametangial differentiation was separated in the time (protandric gametophyte). Colonization took place through the rhizoids (Fig. 1B-F), on which an appresoriumformed and penetrated the cell. On reaching the basal cells of the midrib, the hypha grew forming coils and spread through cells (Paris-type), taking up ¾ of the midrib (Fig. 1B, D). The arbuscules, being very small, grew from these coils (Fig. 1C, E, F). There was little, if any, intercellular growth. Arbuscules were ephemeral, and in most cells we observed amorphous, blue-stained material. Spores and vesicles were not found in this phase of the cycle. Gametophyte wings and growth ápex were uncolonized, probably because the wings were unstratified.

When the embryo developed, the foot that penetrated the gametophyte also showedParis-type colonization (Fig. 2A). This colonization did not originate from the colonized gametophyte cells, but rather from external mycelium (Fig. 2B), as zones remain separated by uncolonized cells, the foot had determinate growth and enabled the young sporophyte to obtain nutrients from the gametophyte (Fig. 2B).

Right from the start, there wasArum-type colonization of the first sporophyte root (Fig. 2D). Penetration points, intercellular arbuscules and intercellular hyphae could be seen (Fig. 2C-E). There was no colonization through the gametophyte. The sporophyte was colonized by inoculum in the medium (Fig. 1F).

The substrate accompanying the samples was analyzed, resulting in a salt imbalanced substrate with the following characteristics: pH 7.6 (1:2.5 H₂O), C.E. dS/m 3.59, C_t(W. Black) 24.98g/kg, Nt (Kjeldahl) 1.92g/kg, P (K y B) 9.3mg/kg, CIC (Ac. NH₄ pH7 μDest.) 18.4cmolc/kg, Ca²⁺ (Ac. NH₄pH7 A.A) 30.5cmolc/kg, Mg²⁺(Ac NH₄ pH7 A.A) 1.7cmolc/kg, Na⁺ (Ac NH₄pH7 E/A.A) 4.2 cmolc/kg, K⁺(Ac. NH4 pH7 E/A.A) 1.7 cmolc/kg.

Most gametophytes ofP. vittatathat developed in hollows in damp walls were mycothallic (Fig. 3A). Fern gametophytes and roots of sporophytes were strongly colonized (Fig. 3A, E). Thin fungal mycelium with extraradical hyphae of 0.8-1.3μm, hyphae developing within the gametophyte often forming complex coils (Paris-type arbuscules) filling the gametophyte cells (Fig. 3A), strongly stained in trypan blue, were visible either in the basal part of the rhizoids or within cells of theAdiantum-type gametophyte, where the rhizoids were initiated. The mycelium was also observed within the elongated part of the rhizoids (Fig. 3B). The fungus was spreading from one cell to another without the development of the intercelular phase (Fig. 3A). The colonization Arum-type of sporophyte roots was observed by the mycelium originating from the extraradical hyphal net (Fig. 3D-F). Vesicles of 3-7μm diameter were found in root cortical cells. (Fig. 3E-F).

From the field material collected, the samples presenting gametophyte (n=20), showed a Paris-type colonization occurring through the rhizoids in contact with the inoculum (Fig. 3A, B). A large amount of extra-radical mycelium can be seen penetrating root hairs in the sporophytes, forming intercellular and intracelular hyphae, arbuscules and vesicles (Fig. 3C-F) similar to the ones found in the sporophytes cultured withG. intraradices.

Discussion

Mycorrhizal dependence of a plant species is one of its constitutive features, allowing plant classification as facultative or obligately mycotrophic. Facultative mycotrophs are those that can grow without mycorrhizae in relatively fertile natural soils. Previous studies, found a low mycorrhization level inPteridophytesporophytes, particularly inP. vittataroots (Zhiwei 2000). On the contrary our results showed a high level of fern roots colonization, supporting the previous hypothesis that the amount of available nutrients in the substrate regulates this process (Janos 1993, 2007, Brundrett 2002), leading to the notion that symbiosis may be necessary when P. vittatais growing under highly stressful conditions (wildP. vittata) or under low experimental soil fertility (cultured P. vittata) (Hajibolandet al. 2010). This symbiotic relationship between plant and fungi allowed completing its life cycle in different substrates, enabling the estimation of the colonization at all stages of the fern life cycle. To our knowledge, this is the first time the experimental AMF development in fern sporophytes and gametophytes under culture is reported.

Both gametophytes and sporophytes can form symbioses with the same Glomeromycota fungi that are common symbionts of phanerogamic plants. Previous studies showed that gametophytes of different genera as:Pteridium, Histiopteris, Todea, Cyathea, Asplenium, Blechnum and Schizaea pusillagrown in natural environments were colonized by arbuscular mycorrhiza when they were found in placeswhere nutrients were unavailable (Cooper 1976, Swatzellet al. 1996). Although, as it was shown in this study, the development of the symbiosis between the gametophytes and the Glomalean fungi is not obligatory, we observed that the sporophyte was usually colonized byGlomus intraradices, leading to the notion that sporophytes could be colonized by other mycorrhizal fungus.

Our observation showedParis-type colonization in cells of the gametophyte and sporophyte foot, while the mycelium never colonized the sporophyte through the junction between the gametophyte and the sporophyte, in line with Turnau (2005) and Reyes-Jaramilloet al.(2008) reports. Previous studies found hyphal coils in the gametophyte, near the penetration point considering this structure as an adaptation of the fungus to the limited growth of the gametophyte and the embryo foot (Schmid & Oberwinkler 1995). Gametophyte colonization is restricted to the basal zone of the midrib. Fungus colonization always occurs through the rhizoids after male gametangia differentiation. Previous experiments have shown that the gametophytes synthesize antheridiogen (Döpp 1950), which is a gibberellin-like compound (Wynneet al.1998, Banks 1999). This change in the phytohormonal balance (Shaul-Keinanet al. 2002), enabling the hypha to penetrate the rhizoid and colonize the gametophyte, suggests a biochemical regulation of the mycorrhizal colonization.

Reyes-Jaramillo et al.(2008) found sporophyte root does not become colonized from the gametophyte; this finding is in agreement with our observations. InP. vittatathe embryo foot established a close association with the gametophyte from which it takes nutrients for its growth, but was not colonized by the intracelular mycelium growing in the gametophyte. A plant tissue with several layers of uncolonized cells separated structures: embryo foot/gametophyte. Sporophyte colonization took place after development of root hairs. There were intercellular hyphae in the subepidermic tissue, parallel to the root surface, which formed intracellular branches that gave rise to arbuscules (Arum-type). This type of colonization has also been found inGleichenia bifida(Schmid & Oberwinkler 1995). We report the simultaneous development of colonization units ofParis-type andArum-type mycorrhizae with the same fungus in different stages of the life cycle concluding that the morphology of arbuscular mycorrhizae, (ArumversusParis-types), is solely under the control ofP. vittata genome during its life cycle.

Acknowledments

We wish to thank to UBA, CONICET and ANPCYT for financial support. The authors acknowledge especially to Mónica Ponce from Instituto Darwinion, Mónica Palacios-Rios from INECOL (Instituto de Ecología, A.C.) Xalapa, Veracruz, México, Romina Giacometti from CONICET and Roberto Fernandez IFYBIME (Instituto de Fisiología y Biología Molecular) for his technical assistance in confocal microscopy.

References

Banks, J.A. 1999. Gametophyte development in ferns. Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 50:

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