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Mycorrhizal morphology of Monotropastrum humile collected from six different forests in central Japan

     Laboratory of Forest Pathology and Mycology, Department of Sustainable Resource Science, Faculty of Bioresource, Mie University, Mie, 514–8507, Japan

Akiyoshi Yamada

     Department of Biosciences and Biotechnology, Faculty of Agriculture, Shinshu University, Minami Minowa 8304, Nagano, 399–4598, Japan

	ABSTRACT

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A survey of the nonphotosynthetic plant Monotropastrum humile was conducted to determine its mycorrhizal status and characterize the fungal structures observed. Thirteen populations and 40 individuals were collected from six forest types, including coniferous and broadleaf trees, in central Japan. The nearly spherical root system of M. humile intertwines with the root systems of neighboring trees, and individual roots were branched up to third-order structure, forming monopodial-pinnate or monopodial-pyramidal morphologies. In addition to the formation of a fungal mantle and Hartig net in association with the epidermis, fungal penetration pegs consistently were observed around and within the epidermal cells. These structures indicate that the mycorrhizal status of M. humile is of the monotropoid type.

Key words: ecology, microscopic survey, Monotropoideae, monotropoid mycorrhizae, root architecture

	INTRODUCTION

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The nonphotosynthetic plant, Monotropastrum humile (D. Don) H. Hara, is a well-known species of the Monotropoideae (Hara 1941, Wallace 1975, 1987, Wu 1978) and is distributed in East Asia from the Himalaya to Japan (Kitamura et al 1975). The shoot of this plant always produces a single flower that appears in Japan between Mar and Aug, coinciding with rising temperatures (Tsukaya 1998). Like all other members of the Monotropoideae, M. humile is nonphotosynthetic and must depend on exogenous organic carbon resources for its development. Plants that depend on living fungi for nutrition are called myco-heterotrophic (Leake 1994).

Since the 19th century the genus Monotropa, a close relative of Monotropastrum, has been observed to have root-associated fungi (Kamienski 1881, in Rayner 1927). The unique mycorrhizal association of the Monotropoideae has been classified as being of the monotropoid type (Duddridge and Read 1982, Smith and Read 1997). Since monotropoid mycorrhizae are normally ensheathed with fungal mycelium, their appearance is similar to that of ectomycorrhizae. In fact, monotropoid mycorrhizal fungi have been shown to form ectomycorrhizae on neighboring autotrophic plants based on studies of histology (Lutz and Sjolund 1973, Duddridge and Read 1982, Robertson and Robertson 1982, Castellano and Trappe 1985), nutritional physiology (Björkman 1960, Vreeland et al 1981) and molecular biology (Cullings et al 1996, Kretzer et al 2000, Bidartondo and Bruns 2001, 2002, Young et al 2002). Monotropastrum humile, together with Cheilotheca malayana and Monotropa uniflora, belongs to the Russulaceae-specialized clade of the Monotropoideae (Bidartondo and Bruns 2001, Bidartondo pers comm). However, the protrusion of a single terminate hypha into root epidermal cells, i.e., fungal penetration pegs, is found only in monotropoid mycorrhizae (Smith and Read 1997).

Few studies on Monotropastrum humile mycorrhizae have been carried out. Kasuya et al (1995) observed that the root system of M. humile formed monotropoid mycorrhizae. However, the fungal pegs illustrated in that study differed in size when compared to those described in other members of the Monotropoideae (Lutz and Sjolund 1973, Duddridge and Read 1982, Robertson and Robertson 1982). Moreover, the mycorrhizal observation by Kasuya et al (1995) of M. humile was conducted from a single Fagus crenata forest, although M. humile is distributed in a wide range of forest vegetation including Fagaceae and Pinaceae. The present study describes the root architecture and mycorrhizal structure of M. humile collected from several forests that differ in geography and vegetation. The objective was to clarify whether there are features of the mycorrhizal association that are unique to this plant species or a particular habitat.

	MATERIALS AND METHODS

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Populations of Monotropastrum humile, which consisted of more than several individual flowering axes of the plant growing at six different forests, were sampled during the flowering period from May to Aug 2000 in central Japan (Table I). The distance and altitude among the forests varied from 80 km to 420 km and from 50 m to 1800 m, respectively. According to the developmental index of Duddridge and Read (1982), the plants sampled were at developmental stages 3 or 4. Each sample of M. humile was taken to the laboratory and kept at 4 C for less than 2 wk until further root examinations.

	RESULTS

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Within a population of Monotropastrum humile, the floral axes arose from compact spherical root systems intertwined with the roots of neighboring plants (Fig. 1a). Roots of surrounding woody plants occasionally were found within the root system of Monotropastrum plants (Fig. 1b). When root tips were excised from the root system, it was found that they were up to third-order branching and formed monopodial-pinnate or monopodial-pyramidal morphologies (Fig. 1c). The roots varied from light yellowish brown to dark brown, depending on age.

View larger version (149K): [in this window] [in a new window]   FIG. 1. Monotropastrum humile. a. Flowering scapes of a population arising from a root ball. b. Root ball of an individual plant with a neighboring oak-root system (arrowhead). c. Root branching. Single, double and triple arrowheads indicate first-, second- and third-order roots, respectively. d–f. Light microscopy view of root in transverse section. d. Fungal penetrations into the epidermal cell (arrows). Highly branched palmate-like Hartig net (arrowheads). e, f. Projections (arrowheads) and sac-like formations (arrow) attached to the tip of a fungal penetration peg. f. Sac-like structure at the tip of fungal penetration peg. g. Light microscopy view of an epidermal cell in longitudinal section showing a star-like formation. Scale bars: a, b = 1 cm; c = 1 mm; d, f = 20 µm; e, g = 10 µm

	DISCUSSION

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Root samples of Monotropastrum humile often exhibited intracellular fungal intrusions, as well as fungal mantles and Hartig nets. Following the classification of mycorrhizal types (Smith and Read 1997), the mycorrhiza of M. humile is confirmed to be of the monotropoid type, as proposed by Kasuya et al (1995). However, the shape and size of fungal pegs observed in our study differed from what was reported by Kasuya et al (1995). We observed the fungal pegs to be considerably smaller than was reported by Kasuya et al (1995) (50 µm long and 10 µm wide at the base) and oblong rather than subulate. Furthermore, Kasuya et al (1995) did not report a connection between the pegs and the surrounding fungal mantle and Hartig net, suggesting that they are artifacts. Our study shows that the fungal peg formed on Monotropastrum humile is similar morphologically to that of other monotropoid mycorrhizal species, i.e., Monotropa hypopithys (Duddridge and Read 1982), M. uniflora (Lutz and Sjolund 1973), Pterospora andromeda and Sarcodes sanguinea (Robertson and Robertson 1982).

Using light microscopy we were not able to examine in detail the interface between plant cell walls and intruding fungal hyphae. Electron microscopy of Monotropa hypopithys roots by Duddridge and Read (1982) revealed structural differences in fungal pegs during the course of shoot development. They noted that fungal pegs were abundant at stage 2, when the shoots start to emerge above the ground. From stage 3–4, during which the shoots reached anthesis or started to seed, they found that the tip of the pegs seemed to burst and possessed membranous sac-like structures. Other ultrastructural studies on monotropoid mycorrhizae of Monotropa uniflora, P. andromeda and S. sanguinea reported the formation of membranous sacs at the tips of fungal pegs, which seemed to radiate into the cytoplasm of epidermal cells (Lutz and Sjolund 1973, Robertson and Robertson 1982). Thus the star-like formations found in our study could be a membranous sac viewed from above. We also observed projections or sac-like structures at the tip of fungal pegs in developmental stages 3 and 4. These structural changes suggest that the fungal pegs of M. humile also differentiate with the growth stage of the plants.

Björkman (1960) confirmed the transfer of ¹⁴C and ³²P from Picea or Pinus trees to nearby Monotropa plants, and he concluded that M. hypopithys shares a common mycorrhizal fungus with neighboring photosynthetic woody plants. In this study, we did not confirm the hyphal linkage between Monotropastrum plants and surrounding trees. However, root systems of the plants were simple with up to third-order branchings inferred to be less functional for the uptake of water and nutrients from the soil (Leake 1994). Moreover, the tips of the pegs either were closed or open. Thus, the morphological changes at plant-fungus interfaces might be related to the transfer of material such as exogenous carbohydrate and phosphorous so as to increase the surface area between plant cell walls and fungal hyphae (Björkman 1960, Vreeland et al 1981, Duddridge and Read 1982).

The root tips of Monotropastrum humile form monotropoid mycorrhizae irrespective of forest types. Our collections of the plants were geographically distant among sites that were categorized as evergreen coniferous, deciduous broadleaf or evergreen broad leaf forests. The dominant tree species in the forests, i.e., Abies, Castanopsis, Quercus and Tsuga, are known to be ectomycorrhizal plants (Molina et al 1992, Trappe 1962).

Sporocarps of Elaphomyces granulatus were found to be partly enclosed by the spherical root system of Monotropastrum humile in a T. diversifolia forest (Trappe 1976). Although we have not uncovered the fungal species in our collection sites, sporocarps of the genera Amanita, Lactarius and Russula commonly occurred. For the direct examination of Monotropastrum mycorrhizae, Kasuya et al (1995) did not observe fungal associates. In our study, the color of all root tips examined showed a similar brown series. This color is different from that of E. granulatus illustrated in Agerer (1987–1998). Our preliminary study of the roots of M. humile indicated differences in the hyphal arrangement on the surface layer of fungal mantles among the plants. Moreover, a recent molecular study suggests that a Russula species is involved in a mycorrhizal association with this plant (Bidartondo and Bruns 2001). The population of their plant was a part of our plant population collected from a deciduous broad leaf forest at Naka, Ibaraki Prefecture. Therefore, members of the genus Russula, which are obligate ectomycorrhizal fungi and believed to have an intermediate-to-broad host range (Molina et al 1992), could be the symbionts of our M. humile plants. The morphological classification of root tips and/or molecular analyses of both root tips and sporocarps should be further required to identify the fungal symbionts of M. humile.

	ACKNOWLEDGMENTS

 
We are grateful to M. I. Bidartondo (University of California at Berkeley) for his invaluable comments on the manuscript. We also thank Dr. S. Ito and the members of the Laboratory of Forest Pathology and Mycology, Mie University, for their support. The Dr. Kazuhiko Kasai, recently deceased, kindly provided us (A. Y.) with information of plant distribution at Mount Yatsugatake. This study was supported in part by Grant-in-Aid for Scientific Research from the Japan Ministry of Education, Science, Sports and Culture (No. 11460070).

	FOOTNOTES

 
¹ Corresponding author. E-mail: m-yosuke{at}bio.mie-u.ac.jp

Accepted for publication April 8, 2003.

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