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

Estudo ultraestrutural de ectomicorrizas em plântulas de Pinus caribaea Morelet. var. hondurensis Barr. & Golf.

Ultrastructural study of ectomycorrhizas on Pinus caribaea Morelet. var. hondurensis Barr. & Golf. seedlings¹

Estudo ultraestrutural de ectomicorrizas em plântulas de Pinus caribaea Morelet. var. hondurensis Barr. & Golf.

Eduardo GrossI, ²; Lilian I. Thomazini Casagrande^II; Flávio Henrique Caetano^II

^IDepartamento de Biologia, Universidade Estadual de Feira de Santana, C. Postal 252-294, CEP 44031-460, Feira de Santana, BA, Brasil

^IIDepartamento de Biologia, Instituto de Biociências, Universidade Estadual Paulista, UNESP, C. Postal 199, CEP 13506-900, Rio Claro, SP, Brasil

ABSTRACT

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.

Key words: ectomycorrhiza, Pinus caribaea var. hondurensis, ultrastructure, Pisolithus tinctorius, Thelephora terrestris

RESUMO

A ultraestrutura das ectomicorrizas formadas por Pinus caribaea var. hondurensis inoculado com Pisolithus tinctorius (Pers.) Coker & Couch e Telephora terrestris (Ehrenb.) Fr. foi analisada antes do transplantio dessas mudas para o campo, com o intuito de verificar se o fungo estava estabelecido nas raízes. Os fungos ectomicorrízicos inoculados formaram um manto compacto e bem desenvolvido nas raízes laterais. Nas hifas desse manto foram observados vacúolos, núcleos e septos dolipóricos, enquanto que no citoplasma das hifas da rede de Hartig, que ficam adjacentes às células corticais, foram freqüentemente observados vários núcleos, retículo endoplasmático e mitocôndrias polimórficas. Células corticais altamente vacuolizadas, contendo gotículas de material elétron-denso, apresentaram núcleo e algumas organelas na sua estreita região citoplasmática periférica. As ectomicorrizas de P. caribaea var. hondurensis apresentaram características ultraestruturais de uma associação compatível e fisiologicamente ativa.

Palavras-chave: ectomicorriza, Pinus caribaea var. hondurensis, ultraestrutura, Pisolithus tinctorius, Thelephora terrestris

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.

Materials and methods

Biological material and location - Seedlings (160-180 days old) of Pinus caribaea Morelet. var. hondurensis Barr. & Golf., grown under natural conditions in pots containing autoclaved soil from a forest of the same Pinus variety, near the El Merey reforestation area, and inoculated with Pisolithus tinctorius (Pers.) Coker & Couch and Thelephora terrestris (Ehrenb.) Fr. were obtained from the CVG PROFORCA in El Merey (latitude 8º39'N, longitude 62º48'W, 60m high) Monagas State, Venezuela. Ectomycorrhizal roots of these seedlings were collected, washed in water and 1mm³ pieces were sectioned and fixed in Karnovsky modified solution (2.0% glutaraldehyde, 2.0% paraformaldehyde in 0.05M cacodylate buffer pH 7.2) at room temperature for at least 3 hours.

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 CO₂ as the transition fluid. Dried specimens were sputter-coated 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.

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.

Results

Apices of lateral roots branched dichotomously and occasionally acquired a coraloid appearance (Fig. 1). A compact well-developed mantle of closely-packed hyphae covered the mycorrhizal roots (Fig. 2). Branching extramatrical hyphae were present (Fig. 3), and in some roots hyphae were not evident on the root cap (Fig. 4). Observation of the outer mantle hyphae by TEM revealed a fibrillar extracellular material (Fig. 5). Fungal cytoplasm was vacuolated with sparsely distributed organelles (Fig. 5). Inner mantle and Hartig net hyphae were more cytoplasmic (Fig. 6), with numerous dolipore septa (Fig. 6; 13-14). In the mature Hartig net region hyphae penetrated between the 3^th-4^th layers of the cortical cells (data not shown) and formed a typical labyrinthine Hartig net (Fig. 6-7). The fungus penetrated the middle lamella, but there were no obvious signs of root cell wall degradation. Intercellular hyphae sometimes presented mitochondria with different morphologies (Fig. 7-8), and were multi-nucleated (Fig. 11-12), contained glycogen rosettes (Fig. 9), endoplasmatic reticulum and small vacuoles (Fig. 6-9; 11-12). Plant cortical cell cytoplasm contained a nuclei (Fig. 7, 11), mitochondria (Fig. 7-8), dictyosomes (Fig. 9-10), endoplasmatic reticulum (Fig. 7-12) and electron-dense material in the vacuole (Fig. 6-9; 11-12). Dictyosomes located close to the plant cell wall appeared to be producing vesicles (Fig. 9-10), and plant cell wall ingrowths were observed in some regions adjacent to the Hartig net (Fig. 11) near to cytoplasmic hyphae (Fig. 11-12). The septal walls both from Hartig net and the mantle hyphae are thinner than the walls of outgrowths and are continuous between the outer walls of the hyphae (Fig. 13). In higher magnification endoplasmatic reticulum was observed in association with the dolipore septum (Fig. 14).

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 coinoculation with Telephora terrestris due to the nonaseptic 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.

Acknowledgements

We are grateful to Corporación Venezolana de Guayana - Productos Forestales de Oriente Compania Anonima, for the samples of pine roots; to Núcleo de Apoio à Pesquisa em Microscopia Eletrônica Aplicada à Pesquisa Agropecuária da Escola Superior de Agricultura "Luiz de Queiroz" and Centro de Microscopia Eletrônica da Universidade Estadual Paulista, Campus de Botucatu, by electron microscopy support. We thank Dr. Lewis Melville and Dr. Larry Peterson for helpful comments and review of the manuscript.

Received: March 25, 2003. Accepted: July 11, 2003

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1

Part of the MSc dissertation of the first Author; CNPq grant

Publication Dates

• Publication in this collection
  05 Aug 2004
• Date of issue
  Mar 2004

History

• Accepted
  11 July 2003
• Received
  25 Mar 2003