Mycorrhizal association in gametophytes and sporophytes of the fern Pteris vittata (pteridaceae) with Glomus intraradices.
Preliminary data indicated that the presence of AM fungi at both the sporophyte and the gametophyte stage, stimulates fern growth (Turnau et al. 2005). P. vittata sporophytes grown in controlled conditions showed an increase in fresh and dry weight shoots when grown in contaminated soil (Trotta et al. 2006, Leung et al. 2006). In natural conditions (Zhiwei 2000) from 12 different species of Pteris (P. vittata included) found sporophytic mycorrhization only in P. setulosocostulata.
P. vittata is 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. vittata has a chlorophyllous gametophyte of limited growth, the most vulnerable phase of the life cycle (Zhang et al. 2008). The appearance of AM fungi Glomus intraradices at 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), the Arum-type and the Paris-type and a continuum between them. They are responsible of the P plant nutrition (van Aarle et al. 2005). In the Arumtype, fungi form intercellular hyphae between the cortical cells and intracellular arbuscules within them. The Paris-type is characterized by extensive intracellular hyphal coils and arbusculate coils in the root cortex. In the Paris-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 of Pteris vittata and the colonization of the arbuscular mycorrhizal fungi G. intraradices.
MATERIALS AND METHODS
Collection of plant material: Approximately 100 specimens of P. vittata L. 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 fertile P. vittata fronds 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 of G. intraradices inoculum 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 as G. 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 No. 51.331). The culture was replicated, and part of it kept in our collection under the name Strain GB1 (Banco de Glomeromycota In vitro (BGIV) http://www. bgiv.com.ar/strains/glomus-intraradices/gb1).
Pots were watered to field capacity and kept in a humid chamber (relative humidity 100%) at 27-30[degrees]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 90[degrees]C for 15 minutes, washed in tap water and immersed in 20 vol ([H.sub.2][O.sub.2]) for 10 minutes until bleached. Then, they were rinsed in tap water to remove [H.sub.2][O.sub.2], acidified in 0.1N HCl, and stained with 0.05% Trypan Blue solution for 20 minutes at 90[degrees]C. 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 90[degrees]C, 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 55[degrees]C. 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 (Peterson et al. 2004).
Experimental life cycle of P. vittata showed 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 by G. 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 appresorium formed 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 3/4 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 apex were uncolonized, probably because the wings were unstratified.
When the embryo developed, the foot that penetrated the gametophyte also showed Paris-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 was Arum-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 [H.sub.2]O), C.E. dS/m 3.59, [C.sub.t](W. Black) 24.98g/kg, Nt (Kjeldahl) 1.92g/kg, P (K y B) 9.3mg/kg, CIC (Ac. N[H.sub.4] pH7 [mu]Dest.) 18.4cmolc/kg, [Ca.sup.2+] (Ac. N[H.sub.4] pH7 A.A) 30.5cmolc/kg, [Mg.sup.2+] (Ac N[H.sub.4] pH7 A. A) 1.7cmolc/kg, [Na.sup.+] (Ac N[H.sub.4] pH7 E/A.A) 4.2 cmolc/kg, [K.sup.+] (Ac. NH4 pH7 E/A.A) 1.7 cmolc/kg.
[FIGURE 1 OMITTED]
Most gametophytes of P. vittata that 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 0.8-1.3 [micro]m, 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 the Adiantum-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 intercellular 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 3-7 [micro]m diameter were found in root cortical cells. (Fig. 3E-F).
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
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 intracellular hyphae, arbuscules and vesicles (Fig. 3C-F) similar to the ones found in the sporophytes cultured with G. intraradices.
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 in Pteridophyte sporophytes, particularly in P. vittata roots (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. vittata is growing under highly stressful conditions (wild P. vittata) or under low experimental soil fertility (cultured P. vittata) (Hajiboland et 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 pusilla grown in natural environments were colonized by arbuscular mycorrhiza when they were found in places where nutrients were unavailable (Cooper 1976, Swatzell et 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 by Glomus intraradices, leading to the notion that sporophytes could be colonized by other mycorrhizal fungus.
Our observation showed Paris-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-Jaramillo et 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 (Dopp 1950), which is a gibberellin-like compound (Wynne et al. 1998, Banks 1999). This change in the phytohormonal balance (Shaul-Keinan et 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. In P. vittata the embryo foot established a close association with the gametophyte from which it takes nutrients for its growth, but was not colonized by the intracellular 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 in Gleichenia bifida (Schmid & Oberwinkler 1995). We report the simultaneous development of colonization units of Paris-type and Arum-type mycorrhizae with the same fungus in different stages of the life cycle concluding that the morphology of arbuscular mycorrhizae, (Arum versus Paris-types), is solely under the control of P. vittata genome during its life cycle.
We wish to thank to UBA, CONICET and ANPCYT for financial support. The authors acknowledge especially to Monica Ponce from Instituto Darwinion, Monica Palacios-Rios from INECOL (Instituto de Ecologia, A.C.) Xalapa, Veracruz, Mexico, Romina Giacometti from CONICET and Roberto Fernandez IFYBIME (Instituto de Fisiologia y Biologia Molecular) for his technical assistance in confocal microscopy.
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Alicia E. Martinez (1,3), Viviana Chiocchio (2,3), Lo Tai Em (1), Maria A. Rodriguez (1,3) & Alicia M. Godeas (1,3)
(1.) Departamento de Biodiversidad y Biologia Experimental. Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Av. Int. Guiraldes s/N. Pabellon II. Ciudad Universitaria. 1428, Buenos Aires, Argentina; email@example.com, firstname.lastname@example.org, email@example.com, firstname.lastname@example.org
(2.) Catedra de Microbiologia Agricola y Ambiental. Facultad de Agronomia. Universidad de Buenos Aires - Av. San Martin 4453. 1417. Buenos Aires. Argentina; email@example.com
Received 09-V-2011. Corrected 10-XI-2011. Accepted 12-XII-2011.
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|Title Annotation:||articulo en ingles; pteris vitatta, glomus intraradices, gametofito, colonizacion|
|Author:||Martinez, Alicia E.; Chiocchio, Viviana; Em, Lo Tai; Rodriguez, Maria A.; Godeas, Alicia M.|
|Publication:||Revista de Biologia Tropical|
|Date:||Jun 1, 2012|
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