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MolliBase: Overview

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Introduction

Fungi are heterotrophic organisms that form various relationships along the endosymbiont-pathogen continuum with animals and plants. Among these relationships, endophytism has been receiving increasing attention. Endophytes are fungi that live inside healthy plant tissue without causing overt symptoms or apparent injury to the host (FISHER & PETRINI, 1992; PETRINI, 1991). Endophytic fungi have been reported from various plants and plant tissues, from tree leaves and grasses (REDLIN & CARRIS, 1996) to root endophytes and other root-associated fungi (SCHULZ et al., 2006). Some fungi are endophytic in roots and others may be colonizing other root associated fungi (TEDERSOO et al., 2009; HOSOYA et al., 2011). A number of studies increasingly report Helotiales as the major components of the root associates (EGGER, 2006; TEDERSOO et al., 2009; TOJU et al., 2013). However, their taxonomy is still shrouded in mystery (e.g. VAN- DENKOORNHUYSE et al., 2002) and many reported endophytes are not identified at species level, or can be barely identified at generic or upper taxonomic levels. Part of this reason is that most of the helotialean isolates never produce apothecia in culture, or rarely pro- duce conidia, which provide useful characters for morphological identification. In these cases, DNA sequences can be used to assist with identification. Identification of isolates using sequence information stored in databases is one of the most powerful taxonomic tools, and is already applied for the identification of root associated fungi (TANABE, 2012; TANABE & TOJU, 2012; TOJU et al., 2013). However, this approach usually does not work for helotialean species, because there is still not enough coverage of data or authenticated reference sequences to estimate their taxonomic position. One of the major groups of root endophytes is the family Dermateaceae (sensu lato incl. Mollisiaceae and Ploettnerulaceae), including the genus Mollisia (Fr.) P. Karst. and its allied genera (TOJU et al., 2013).

Since many of these fungi produce only dark-colored, sterile hyphae in culture, species-level identification of cultures using a morphological approach is rarely possible. A molecular approach may not resolve species-level identifications due to the narrow coverage of this family in available databases (e. g. GenBank, UNITE, BOLD). The first author has been working on various Helotiales for more than two decades, including the collecting and culturing of a number of specimens (e.g. HOSOYA & OTANI, 1997; HOSOYA et al., 2011). Within this collection, a number of Mollisia and allied taxa are included from various substrates, primarily because these fungi are commonly found in various habitats and are easily cultured. DNA sequences were obtained from most of the resulting cultures. Using these sequences, specimens may be connected with other isolates and data obtained from other field collections or root endophyte studies. In other words, sequences based on barcoding regions, namely ITS-5.8S sequences, may be used to bridge specimens collected above ground with unidentifiable root endophyte sequences or sterile cultures (SCHOCH et al., 2012).

Although Mollisia species are not easy to identify, finding phylogenetically close groups that may represent species may be possible based on ITS sequences. The presence of specimens makes morphological characterization and identification possible. Thus, having a certain amount of isolates, together with specimens from which they were isolated, regardless of being identified or not, will provide data for the identification of these organisms with apothecial morphology in the future.

To facilitate research collaboration, we release an initial subset of sequences with corresponding morphological and collection data. The specimens are being preserved as dried specimens in TNS (Mycological Herbarium of the National Museum of Nature and Science, Tokyo, Japan). In most cases, cultures are accompanied with them, kept in agar slants or in deep frozen state. Because the resources will be expanded in the future, we release the data in the form of website designated “MolliBase”. The website provides morphological data of the specimens, metadata for the specimens and isolates (when available), and sequence data for ITS-5.8S region. Initially, 51 records are released. We welcome users to provide more data and resources for the purpose of collaborative studies and to assist in species-level identification.

Material and methods

Specimens and isolates

Collection trips has been made throughout Japan, from Okinawa to Hokkaido, but in particular the Kanto area due to convenience. The specimens were air-dried and deposited in the Mycological Herbarium of the National Museum of Nature and Science (TNS; Tokyo, Japan). In most cases, single spore isolates were obtained using Skerman’s micromanipulator (SKERMAN, 1968). The isolates were preserved on potato dextrose agar (PDA; Nissui) slants at 5 °C, and also at -80 °C in 10% glycerol in 2 ml cryovials. Part of the collection has been deposited to the Biological Resource Center, National Institute of Technology and Evaluation (NITE-BRC; Tokyo, Japan). Observations.

For microscopic examination, dried specimens were rehydrated with water and observed in crush mount in lactic acid mounting fluid with cotton blue stain (CB/LA). Line drawings were prepared using CB/LA crush mounts observed with an Olympus BX51 light microscope equipped with ×100 objective lens with the aid of an Olympus U-DA drawing tube. External views of the specimens were photographed using an Olympus SZ61 dissecting microscope equipped with a Nikon DS-L photographing system.

DNA extraction, PCR, and sequencing

Isolates were cultivated in 2 ml of 2% malt extract broth for two weeks, and the mycelia were harvested and frozen at ?80° C. About 50 mg of mycelium was mechanically lysed using a Qiagen Tissue Lyser, using ceramic beads. DNA was extracted using the Qiagen DNeasy Plant Mini Kit following the manufacturer’s instruction. To amplify internal transcribed spacers (ITS1 and ITS2) and 5.8S ribosomal region, the primer pairs ITS1 or ITS1F and ITS4 (WHITE et al.,1990) were used. Direct extraction from the apothecia was not attempted to avoid contamination. The extracted DNA samples were deposited in the Center for Molecular Biodiversity Research, National Museum of Nature and Science (Tokyo, Japan). Procedure for PCR and sequencing following HOSOYA et al. (2010).

Ascal pore against MLZ reaction

For KOH pretreatment, a drop of 3% KOH was applied to a piece of apothecia cut from the dried specimen, and left until the specimen soak the reagent. Excess KOH liquid was removed by a piece of paper filter, and Melzer's reagent (0.5 g of iodine, 1.5 g of KI, 20 g of chloral hydrate, 20 ml of distilled water, MLZ) was applied, crush mout was prepared. To examine the reaction without KOH pretreatment, MLZ was directly applied to the specimen, and crush mount was prepared. Reaction against Lugol's solution (0.5 g of iodine, 1.5 g of KI, 20 ml of distilled water, IKI) follwed the same method as MLZ without KOH pretreatment. The specimen was observed at x100 objective oil immersion lens.

KOH reaction of the apothecium

An apothecium or part of apothecium was cut from the substrate and placed on a slide glass on white paper. A drop of 3% KOH was applied directly to the specimen, and observed under dissecting microscope if the specimen has yellow exudates.

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