서 론
Monotropoideae is a subfamily of the Ericaceae, and most of the species in this subfamily are achlorophyllous, and thus, heterotrophic plants [1]. These plants are not able to fix carbon by themselves because they have very low amounts of chlorophyll-related pigments [2]. They obtain fixed carbon from photosynthetic plants through mycorrhizal hyphae; plants exhibiting this relationship are referred to as mycoheterotrophic plants [3, 4]. The mycorrhizal relationship between Monotropoideae plants and fungi is referred to as monotropoid mycorrhiza. Such relationships are associated only with certain genera of fungi, including M. uniflora, M. uniflora, Russula, and Tricholoma, which also form ectomycorrhizas with sur- rounding photosynthetic plants [5-8]. In particular, Lactarius and Russula are well known as relatively dominant ectomycorrhizal fungi in forest ecosystems [9, 10].
Structures of monotropoid mycorrhizas have been stu- died in species of several Monotropoideae plants [11-13]. The mycorrhizal structure has typical ectomycorrhizal characteristics, including a hyphal mantle covering the roots and Hartig nets between cortex cells of the roots. In addition, fungal hyphae penetrate and produce fungal pegs inside the epidermal cells of plant roots, which is the characteristic structure of monotropoid mycorrhizas. Fungal pegs have been known as structures that trans- locate photosynthetic carbon compounds from the fungi to photosynthetic plants.
Monotropoideae consists of 15 species belonging to 10 genera [1], and many species of Monotropoideae are en- dangered because of habitat destruction. It is important to conduct studies on Monotropoideae to protect the de- clining populations of these plants, as well as to provide important clues regarding translocation of nutrients through the hyphal networks [14-16].
Monotropa spp. are distributed in the northern hemi- sphere. In particular, M. uniflora are often found in sha- ded and wet areas of old forests in Korea. The mycor- rhizal status and fungal symbionts of M. uniflora have not yet been determined in Korea. The purpose of this study was to investigate the symbiotic fungi in the roots of M. uniflora collected from a forest in Korea.
A total of seven individual M. uniflora were collected from a mixed forest in Gangwon-do, Korea (37º43'29" N, 128º35'53" W). The roots along with the soil were collec- ted and taken to the laboratory. These were then washed under running tap water to remove the attached soil par- ticles. The root balls were observed under a stereomicro- scope (Olympus SZX9, Olympus, Japan). Root tips were removed and sectioned using a microtome (CM1850, Leica, Germany) to examine the morphological charac- teristics of monotropoid mycorrhizas.
To examine the molecular characteristics of the fungi, the individual root tips were removed and washed seve- ral times with sterile water, then, the genomic DNA was extracted. The fungal specific primers ITS1F and ITS4 were used for PCR to amplify the internal transcribed spacer (ITS) region of fungal rDNA [17]. Subsequently, about 650 bp of amplified products was confirmed from 1.5% agarose gel electrophoresis and sequenced at Mac- rogen (Seoul, Korea). Sequences were compared with si- milar sequences obtained using BLAST in NCBI; further, Bayesian phylogenetic analysis was performed using MrBayes [18].
Morphological characteristics of monotropoid mycor- rhizas of M. uniflora were examined under a microscope. The roots of all seven plants were colonized by mycor- rhizal fungi, forming root balls (Fig. 1A). The mycor- rhizal root tips were short and unbranched, and the shape of mycorrhizal tips observed in the roots was identical in all the individual plants. In cross-sections of the root tips, a thick hyphal mantle covered the roots (Fig. 1B), and cystidia were observed outside the mantle (Fig. 1C). In particular, the structure of the mantle covered with the cystidia is the typical characteristic of mycorrhiza between Russula spp. and M. uniflora, which has been supported by several studies, including the results of this study [19-21]. Moreover, fungal pegs were observed in- side the epidermal cell of the mycorrhizal root tips (Fig. 1D), which is another typical characteristics of monotro- poid mycorrhizas, and form the location of nutrient ex- change between the plants and fungi.
Fig. 1. Structure of monotropoid mycorrhizas of Monotropa uniflora collected from this study. A, fungal root tips of M. uniflora; B, thick mantle structure of the root tip; C, numerous cystidia on the root surface; D, fungal peg (arrows). Scale bars: 10 µm.
Sequence analysis of the fungal ITS region amplified from the root tips of M. uniflora showed that all of the samples collected in this study were Russula sp. (Fig. 2). This indicates that a single species of fungus formed mycorrhizal relationship with the M. uniflora collected in this study. The result of BLAST search showed that Russula heterophylla was the most similar with the sequence from this study but the similarity was not enough to identify these fungi in species level (94~97%). Therefore, additional analyses are needed to confirm the phylogene- tic identity of this fungus.
Fig. 2. Bayesian phylogenetic tree constructed using ITS sequences of the fungi associated with the Monotropa uniflora. Lactarius oomsisiensis was used as an outgroup. Sequences from this study are in bold.
In general, it has been reported that ectomycorrhizal fungi have symbiotic relationships with various host plants [3]. However, there seems to be strong specificity with fungi among mycoheterotrophic plants. Strong specificity of Russula species with M. uniflora has been reported in studies from Japan and China, which are geographically close to the sample sites of this study [20-22].
In previous studies, it was shown that Monotropa spe- cies are able to form symbiotic relationships with differ- ent species of Russulaceae depending on the area, even though they showed strong specificity with fungi at the family level [5, 21, 22]. Therefore, the symbiotic relation- ships between M. uniflora and fungi have a geographical mosaic appearance. This could be because of coevolution between plants and fungi in different ecological environ- ments; however, strong preference of Monotropa to Rus- sulaceae suggests the presence of specific recognition pro- cess between the plants and fungi.
Russula species form ectomycorrhizal relationships with various host plants in forests. In general, it is known that the mutualistic relationship has relatively weak host spe- cificity; however, host specificity develops when one of the two interacting organisms receives relatively more benefits. Therefore, mycoheterotrophic plants such as M. uniflora not only provide important evidence regarding nutrient transfer through the mycorrhizal hyphal network in soil [15] but also provide important insights into understanding the coevolution between plants and fungi [16]. In addition, determining the diversity of mycorrhi- zal fungi associated with Monotropoideae plants provides an important basis to understand the evolutionary paths of mycoheterotrophy in Ericaceae and Orchidaceae [23].
