Ultrastructural study of ectomycorrhizas on Pinus caribaea Morelet . var . hondurensis Barr . & Golf . seedlings

(Ultrastructural study of ectomycorrhizas of Pinus caribaea Morelet. var. hondurensis Barr. & Golf. seedlings). The ultrastructure of ectomycorrhizas formed between Pinus caribaea var. hondurensis inoculated with Pisolithus tinctorius (Pers.) Coker & Couch and Telephora terrestris (Ehrenb.) Fr. was analyzed just before the transplant of these seedlings to the field to ascertain if fungi are established in the roots. Ectomycorrhizal fungi formed a well-developed compact mantle in lateral roots. Vacuoles, nuclei and dolipore septa were observed in mantle hyphae and numerous nuclei, endoplasmatic reticulum and polymorphic mitochondria were frequently located in the cytoplasm of Hartig net hyphae adjacent to plant cortical cells. Highly vacuolated cortical cells contained droplets of electron-dense material, nucleus and some organelles were observed in a narrow region of peripheral cytoplasm. The ectomycorrhizas of P. caribaea var. hondurensis exhibited typical ultrastructural characteristics of a compatible and physiological active association.


Introduction
Ectomycorrhiza is the symbiotic association between roots of some woody plants and fungus, which belong to the Basidiomycota, Ascomycota and Glomales (Zygomycota), characterized by the presence of hyphae between root cortical cells producing a netlike structure called the Hartig net (Sylvia et al. 1997).In this association carbon flows to the fungus, which absorb and translocate nutrients from the soil to the host plant (Sylvia et al. 1997).The ultrastructure of many ectomycorrhizal forest tree species has been widely studied since the early works of Foster & Marks (1966;1967).Electron microscopy is a useful tool for cytochemical localization (Rincon et al. 2001) and imunolocalization (Tagu et al. 2001) allowing the researches to compare the ultrastructure of different types of ectomycorrhizas.
The importance of ectomycorrhizal fungi in forestry was first revealed when attempts to establish exotic pine forests were enhanced when transplanted seedlings were first colonized by ectomycorrhizal fungi (Daniel et al. 1982).It is now a common practice to inoculate nursery seedlings in reforestation programs.In the Southern plains of Anzoategui and Monagas States (Venezuela) in a large forestation area belong to CVG (Corporación Venezolana de Guayana) PROFORCA (Productos Forestales de Oriente Compania Anonima), seedlings of Caribbean pine (Pinus caribaea var.hondurensis) are inoculated with the ectomycorrhizal fungi Pisolithus tinctorius and Thelephora terrestris to improve the effectiveness on seedlings establishment.For more than 30 years this region of Venezuela has been planted with Caribbean pine and until December 2000, pine comprised an extension of 615,000ha, which represents the largest tropical area with a monospecific forestal plantation (Cedeno et al. 2001).The purpose of the present study is to examine the ultrastructure of P. caribaea var.hondurensis ectomycorrhizas just before the transplant of these seedlings to the field to ascertain if fungi are established in the roots.Electron microscopy -The root samples collected in El Merey were post-fixed at the Universidade Estadual Paulista, Rio Claro, SP, Brazil.For scanning electron microscopy (SEM) the samples were rinsed in cacodylate buffer, dehydrated in ethanol, and critical point dried in a Balzers CPD 030 instrument using CO 2 as the transition fluid.Dried specimens were sputtercoated with gold in a Balzers SCD 050 sputter coater, mounted on stubs and examined with a Jeol P-15 SEM.For transmission electron microscopy (TEM), samples were rinsed in cacodylate buffer, post-fixed for 1h in osmium tetroxide (2%) in 0.05M of the same buffer, dehydrated in a graded acetone series, and embedded in Spurr resin.Thick sections (3.0 to 5.0nm) were stained with 1.5% methylene blue and 1.5% azure II for light microscopy.Ultrathin sections were cut with a glass knife using a Porter-Blum MT2-B ultramicrotome, picked up on copper grids, stained for 10 min with ethanolic uranyl acetate and 5 min with aqueous lead citrate, and observed with a Zeiss EM900 and a Philips CM100 TEMs at 80kV.

Materials and methods
Different developmental regions of the ectomycorrhiza were identified using conventional light microscopy and ultrastructural observations were restricted to the active mycorrhiza infection zone.Approximately 34 root segments were analyzed by electron microscopy.

Discussion
Mantle morphology is the result of the interaction between mycobiont and phycobiont genomes (Massicote et al. 1987) and the environment (Peterson & Bonfante 1994).According to Melville et al. (1988) the outer mantle morphology may depend on the rate at which additional hyphae accumulate and the extent to which the growth of outer hyphae is affected by proximity to the root.As in other ectomycorrhizae (Massicotte et al. 1986;Melville et al. 1988) the outer mantle hyphae are highly vacuolated and it is assumed that the root less affects these hyphae (Melville et al. 1988).Hyphae from  the inner mantle of P. caribaea var.hondurensis are embedded in an extracellular material, which fills the interstices (Seviour et al. 1978).This material could be composed of polysaccharides and glycoproteins (Piché et al. 1983;Massicotte et al. 1987;1990;Dexheimer et al. 1994) and might act as an adhesive (Piché et al. 1983;Alexander & Hogberg 1987).Adhesins (microbial ligands) that interact with host receptors (Jones 1994;Martin et al. 1999) are often found in fibrillar material around plant and fungal cells.Increased secretion of extracellular fibrillar polymers between hyphae and root surface could denote a compatible ectomycorrhizal association.Isolates of P. tinctorius with delayed symbiosis development do not secrete this fibrillar material (Lapeyrie et al. 1989;Lei et al. 1990).Our ultrastructural observations showed this mucilaginous bridge, which links hyphae and root cells, and a substance between the mantle hyphae in P. caribaea var.hondurensis mycorrhizas, which could denote a compatible mycorrhizal association.
Distribution of glycogen-like rosettes in the hyphae was consistent with that found by several authors (Foster & Marks 1966;1967;Jordy et al. 1998).Glycogen found in the mantle and Hartig net hyphae could be a result of seasonal variation (Duddridge & Read 1984;Genet et al. 2000) and related to the physiological state of the association.Labyrinthic branching increases the apoplastic and symplastic exchange interface between the symbionts (Peterson & Bonfante 1994) and may have diagnostic value to identify compatible mycorrhizal associations, as postulated by Nylund & Unestan (1982) and Piché et al. (1983).
The presence of cellular constituints such as nuclei, mitochondria and endoplasmatic reticulum, also indicate metabolic activity in the mantle and mainly in the Hartig net hyphae found in P. caribaea var.hondurensis.Cortical cells adjacent to the Hartig net in this studied ectomycorrhiza appear to be alive and physiologically active, since they possess nuclei, a peripheral lining of cytoplasm and organelles.Presence of these ultrastructural features in P. caribaea var.hondurensis roots may denote the health of ectomycorrhizal association.Wall ingrowths similar to those observed in Alnus crispa (Massicote et al. 1986) and in Dryas integrifolia (Melville et al. 1988) ectomycorrhizas occurred in cortical cells adjacent to the Hartig net.These transfer cell-like wall ingrowths and plasma membrane could increase the surface area for nutrient exchange between root and fungus (Allaway et al. 1985).
The ultrastructural appearance and size of the dolipore septa, which is characteristic of Basidiomycota (Moore & McAlear 1962), were very similar to that of Pisolithus tinctorius (Orlovich & Ashford 1994).When combined with other evidences such as the presence of dikariotic mycelia, and the formation of fruitbodies of Pisolithus tinctorius in the pots with seedlings of P. caribaea var.hondurensis suggest that the plant roots were colonized by this fungus in spite of co-inoculation with Telephora terrestris due to the non-aseptic conditions of our experiment.
In conclusion, this study demonstrated that the P. caribaea var.hondurensis inoculated seedlings exhibited the typical ultrastructural characteristics of a compatible and physiologically active ectomycorrhizal association, giving a general view of the ultrastructural condition of the seedlings that will be transplanted to the field.

Figures
Figures 1-4.Scanning electron micrographs of Pinus caribaea var.hondurensis ectomycorrhizas.1.A young twice dichotomized lateral root with a smooth-undulate mantle.Few hyphae can be individualized (arrows).Bar = 20µm.2. Detail of inner smooth-undulate mantle with some hyphae (h) arising from it.Bar = 10µm.3. Lateral root tip entirely surrounded by a compact mantle.Bar = 20µm.4. Lateral root apex without evidences of mantle and with root cap.Some hyphae (arrows) can be seen in other part of root.Bar = 20µm.