Introduction

Orchid plants have unique a nutritional mode through mycorrhizal associations (Leake 2004;Smith and Read 2008). Several terrestrial orchids are nutritionally dependent on their mycorrhizal fungi partners (Dearnaley 2007) and are sustained by forming associations with fungi that are co-associated with the roots of neighboring autotrophs.

In Korea, approximately 70% of the native orchid plants are grown in Jeju island (Lee and Choe 2006). Among them, Cymbidium goeringii (C. goeringii) and Cymbidium kanran (C. kanran) are very important species as natural genetic resources to be conserved and as floriculture crops. C. goeringii is known as the spring orchid in Korea and has been cultivated for a long time owing to the variation of its leaves and flowers. In terms of nutrient uptake, it has a mixotrophic mode which shifts to autotrophy with the development of photosynthetic organs (Zhao et al. 2014). It is also enlisted in the Korea Red List (KORED) and categorized as Least Concern. C. kanran has an attractive scent to its flowers and a mixotrophic nutritional mode. It was designated as a Korean National Monument, number 191, in 1967 and is enlisted in KORED categorized as Critically Endangered.

Orchid roots infections spread from one cell to another through fungal hyphae and fungal pelotons (Rasmussen 2002). The hyphae grow into the orchid tissues and form coiled structures called pelotons in the cortical cells of host root tissues. Endophytic fungi were reported as fungal colonization in the healthy tissue and it were able to produce secondary metabolites and associated with plants without any disease or damage exhibited by the host (Zhang et al. 2006). These fungi produce hydrolytic enzymes which break down macromolecules for utilizing nutrients (Arditti 1992). Endophytic fungi infections do not cause morphological changes or form fruit bodies in the roots; besides, they can survive for many years in the soil in the absence of a host (Marx 1975).

Previous studies have reported the identification of orchid mycorrhizal fungi (OMF) from several orchids native to Korea; they are, the study of OMF from two species of Cypripedium (Sim et al. 2010) and a study of several indigenous orchids including Oreochis, Cymbidium, Gymnadenia and Cephalanthera by Youm et al. (2011). These studies revealed that Tulasnella was identified as a general symbiont. Furthermore, there are studies reporting OMF identification from Spiranthes, Calanthe, Bletilla and Pogonia (Youm et al. 2012), and from Epipactis thunbergii (Han et al. 2013). These reports identified Sebacina as well as Tulasnella as OMF. Besides, Russula was reported in Cymbidium kanran (Hong et al. 2015). Ectomycorrhiza could be identified in addition to Tulasnella (Okayama et al. 2012). Using two species of Cephalanthera, different OMF were investigated such as Russulaceae, Sebacinaceae, and Thelephoraceae (Sakamoto et al. 2015). Investigation of five species of Cymbidium revealed that population diversity of mycorrhizal fungi could change according to the growth stage (Ogura-Tsujita et al. 2012).

In this study, microscopic observation was performed to find out the existing symbiotic fungi and their pelotons in root cortical tissues of sampled Cymbidium. Molecular identification was performed to identify the OMF. Producing elementary but fundamental data about Cymbidium’s nutritionally helpful endophytic mycorrhizal fungi is important to help endangered native orchids survive in-situ and in ex-situ conservation programs. We hope this study will improve the understanding of the interaction between native Cymbidium species and their mycorrhizal symbionts, and the knowledge obtained will be useful to investigate specific inter-dependence between orchid plants and mycorrhizal fungi during symbiotic growth.

Materials and Methods

Plant source and preparation for root microscopy observation

Two species, C. goeringii and C. kanran were collected in March, 2015 in Jeju-do, Korea for scientific purposes without environmental disturbances (Table 1). The root surfaces were sterilized in 50% ethanol, 2% sodium hypochlorite (NaOCl) and rinsed with sterilized distilled water (SDW). Prepared root samples were subjected to a modified staining method based on Koske and Gemma (1989). Root tissues were treated with 2.5% KOH (Duchefa, Netherland) at 121°C for 10 min, and rinsed with SDW. Softened root tissues were treated with 1% HCl at room temperature for 24 h. The fixed root tissues were stained with 0.05% trypan blue (MP Biomedicals, USA) at 121°C for 10 min. The stained root tissues were placed in acidic glycerol (500 mL Glycerol, 450 mL SDW, 50 mL 1% HCl) for a quarter of a day. The stained root tissues were cut with a sterilized blade and the root pieces were put on a slide and covered with a cover glass. To observe the colonized endophytes and pelotons in the root cells, microscopic examination was performed (Nikon E100, Japan).

Amplification of rDNA ITS region for molecular identification of mycorrhizal fungi

The surface-sterilized root was cut into 0.5 cm pieces with a sterilized blade. Root tissues were immediately used for genomic DNA extraction. Total genomic DNA of the individual orchid root tissues was extracted using the DNeasy Plant mini kit (Qiagen, USA). For each sample, 100 mg of root tissues were placed into 1.5 mL microcentrifuge tubes and were treated as recommended by the manufacturer’s procedure. Concentration of the extracted DNA was measured using the ND 2000 spectrophotometer (Nanodrop Technologies, USA) by determining the absorbance at 260 nm. Polymerase chain reaction (PCR) was performed to amplify the rDNA internal transcribed region (ITS) region of the orchid mycorrhizal fungi. The reaction was performed in a volume of 30 μL containing 20 ng of genomic DNA, 3 μL of 10 × PCR buffer (100 mM Tris-HCl (pH 8.3), 500 mM KCl and 15 mM MgCl2), 1 μL of 2.5 mM dNTP mixture, 0.2 μL of 1U Taq polymerase (Takara, Japan), and 1 μL of 10 pM ITS1-OF and ITS4-OF primers (Taylor and McComick, 2008). The PCR mixtures were p re-denaturated at 96°C f or 2 m in. Pre-denaturated PCR mixtures were denatured at 94°C for 30 sec, annealed at 62°C for 40 sec, elongated at 72°C for 1 min, and 35 cycles were repeated. Final extension time was at 72°C for 10 min. Amplicons were examined by 1% of agarose gel electrophoresis, then stained with EtBr and visualized on the UV transilluminator.

The amplified fungal rDNA ITS sequence which was derived from the root tissues was ligated into the pGEM-T easy vector (Promega, USA) and ligation was performed. The ligation mixture was mixed with competent DH5α Escherichia coli cells (RBC, Taiwan). Luria-Bertani (LB) broth (BD, USA) was added into the cell mixture and incubated for 1 h with shaking at 37°C and 2,000 rpm. For the selection of correct clones, the cultured cells were spread on a LB agar plate containing ampicillin, isopropyl-1-thio-β-D-galactopyranoside, and X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) and incubated at 37°C for 16 h. White colonies were selected and recombinant plasmids were separated by alkaline lysis method (Sambrook et al. 1989) from the cultured cell. The nucleotide sequence of the selected DNA clones was determined (Macrogen, Korea) and analyzed by using the BLAST program at the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/).

Phylogeny construction for comparison among other orchid fungal isolates

To estimate the genetic relationships between the resulting sequences and other reference sequences isolated from diverse orchid species, phylogeny analysis was conducted. Determined ITS nucleotide sequences of fungal symbionts from Cymbidium root samples were analyzed by using the BLAST program (NCBI). The sequences analyzed were compared to rDNA ITS sequences in the GenBank database and compared sequences were aligned in the Molecular Evolutionary Genetics Analysis (MEGA) software, version 6.0 (Tamura et al. 2013), with the ClustalW alignment tool. The phylogenetic tree was constructed using the Unweighted Pair Group Method using Arithmetic average (UPGMA) method. The optimal tree with the sum of branch length = 1.552972 was obtained. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are reported as the number of base differences per site. The analysis involved 20 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 434 positions in the final dataset.

Results and Discussion

Observation of the symbiotic state of mycorrhizal fungi in Cymbidium roots

To observe the symbiotic states and the hyphae of fungi in the orchid cortical tissues, roots were stained using trypan blue and observed through a light microscope. The microscopic observation identified that the hyphae grew into the cortical root tissues of the orchid plants, and formed coiled structures called pelotons (Fig. 1). The portions of stained blue were seen to be a typical construct of the pelotons derived from symbiotic fungal hyphae in cortical tissues. Morphological characteristics of pelotons appeared to be differentiated for each species. Cylindrical shaped cells filled with compact coiled pelotons situated in the center of the root cortical cells were seen in C. goeringii, whereas in C. kanran, the spherical shaped-peloton was observed (Fig. 1A and 1B). In this study, mycorrhizal fungi appeared to be in symbiotic states in the root cells of the native Cymbidium species.

Molecular identification of orchid mycorrhizal fungi through the rDNA sequence analysis

One fungal sequence of rDNA ITS was obtained from each individual Cymbidium, and used for sequence comparison. OMF sequences obtained from C. goeringii were determined to be 673 bp nucleotides (MF101371), and those from C. kanran were 788 bp nucleotides (MF101372). Based on the nucleotide BLAST result, OMF sequences obtained from C. goeringii in this study corresponded with the highest similarity of 98.3% to those of Tulasnellaceae isolated from C. goeringii from Japan. OMF sequences obtained from C. kanran root tissues corresponded with 90.4% similarity to those of Russula sp. obtained from Gymnadenia conopsea from Germany.

The resulting of rDNA ITS sequences were analyzed by the UPGMA cluster analysis method, and are represented as a phylogenetic tree in Fig. 2. For comparison, nucleotide sequences of OMF isolates obtained from other orchid species were used. According to the phylogenetic tree, the OMF ITS sequence from C. goeringii root tissues was clustered with other Tulasnella species, and that from C. kanran root tissues was clustered with other Russula species. A total of 20 fungal isolate samples, including one outgroup species ‘Cenococcum from unknown orchid’, were divided into two main groups (Fig. 2). The outgroup species was positioned at the base of the tree, distinctively differentiated from the main groups.

As one of the most common fungal partners in orchids, Tulasnellaceae is the main mycorrhizal fungi associated with the Cymbidiums (Rasmussen 2002). In the review of Ogura-Tsujita et al. (2012), Tulasnellaceae had been isolated from roots of autotrophic Cymbidium species (Warcup 1981;Lee 2002;Athipunyakom et al. 2004;Nontachaiyapoom et al. 2010). The OMF derived from C. goeringii was the closest to Tulasnella in the Tulasnellaceae family according to the nucleotide blast result and the phylogeny analysis. Therefore, isolated OMF from C. goeringii in this study appeared be included in Tulasnella, in agreement with the previous studies. In Korea, studies on the identification of similar OMF were carried out in C. goeringii and C. kanran. (Youm et al. 2011, 2012;Hong et al. 2015). When analyzing two different species of Cypripedium collected in Gyeonggi province in Korea (Sim et al. 2010), Tulasnella was found to be present in the roots, showing above 88% nucleotide sequnece similarity in the ITS region among the same species collected from the same spot. Youm et al. (2011, 2012) reported that cultured fungi isolated from C. goeringii root showed 99% similarity with Tulasnella calospora (AB369940) and 87% similarity with Sebacina vermifera (EU625994). Hong et al. (2015) reported that predominant species sampled from C. kanran were Russulaceae, followed by Mortierellaceae and Sebacinaceae, based on mycorrhizae analysis. We compared these Russulaceae with the sequences obtained from C. kanran in this study, and the sequence similarity ranged from 76.9% to 82.5% (data not shown). Despite all samples being collected from Jeju in Korea, these sequences did not seem to be high related. However, Russula obtained in this study clustered with other Russula isolates (Fig. 2).

The myco-heterotroph plants were associated with Russulaceae family (Leake 2004;Ma et al. 2015). Russulaceae is one of the ectomycorrhizal fungi, which are enriched with nitrogen and carbon associated with autotrophs (Leake 2004). In this study, OMF derived from C. kanran could be classified as a member of Russula species based on the results obtained in the nucleotide sequence and phylogeny analysis. Two species in this study, C. goeringii and C. kanran had mixotrophic nutritional modes, but the ITS sequences of symbionts in each species were identified as different fungi, though they were habituated closely. The results obtained by Okayama et al. (2012) were similar to those in this study; they reported that most mycoheterotrophic orchids were associated with diverse ectomycorrhizal fungi, including those from the family Russulaceae. So, the results of this study also accounted for the conclusion that the OMF involved in the nutritional mode was not the only factor determined by the host.

Results of this study would be useful for further studies such as OMF identification by pure culture and could help in the proper germination of diverse native orchids in Korea and contribute to large-scaled micropropagation. OMF isolates obtained in this study will contribute to the better understanding of their diversity and host-specificity, considering conservation and cultivation. Additionally, our next step would be the study of OMF to validate the functionally expressed genes related to orchid mycorrhizal association.

Conclusion

Terrestrial orchids are nutritionally dependent on their mycorrhizal fungi partners and are sustained by forming associations with fungi that are co-associated with the roots of neighboring autotrophs. To investigate the relationships between the resulting ITS sequences in this study and other reference sequences, phylogenetic tree analysis was performed and their taxonomic positions confirmed. Based on the BLAST results, sequences obtained from C. goeringii were revealed to have the highest homology with Tulasnella (98%), and those from C. kanran root tissues showed 96% homology with Russula subrubescens.