Plant material and isolation of symbiotic fungi
Seeds of B. striata ‘Murasakishikibu’ collected five months after self-pollination of plants purchased from a garden store were used in this study. The B. striata strain ‘Murasakishikibu’ was originally selected as a specific flower-color variant from a habitat in Miyazaki Prefecture, Japan, and has been maintained for more than 20 years by gardeners.
The symbiotic fungus was isolated from roots of Pecteilis radiata (Thunb.) Raf. collected with owner’s permission on Aug. 3, 2003 at private land in Himeji, Hyogo Prefecture, Japan. The habitat of this orchid was a rough wetland, where the place had been maintained as a paddy field until 10 years ago. Mycobiont of this orchid was isolated according to the method of Warcup and Talbot [24] with slight modifications as follows. The surface of the root was washed with tap water and sterilized by immersion in 70% ethanol for 30 s and in sodium hypochlorite solution containing 1% available chlorine for 30 s. The surface-sterilized root was then cut into small pieces approximately 10 mm long. The pieces were placed into a Petri dish (9 cm diameter) with 1 ml sterilized distilled water and crushed with a sterilized glass rod to disperse the intracellular hyphal coils (pelotons). Autoclaved modified Czapek Dox agar (0.5 g sucrose, 0.33 g NaNO3, 0.2 g KH 2 PO4, 0.1 g MgSO4 · 7H 2 O, 0.1 g KCl, 0.1 g yeast extract, 15 g agar, 1 l distilled water) was cooled to 45 °C and poured into the Petri dishes (~20 ml per dish). The dishes were mixed well before solidification to disperse the pelotons throughout the medium. The plates were incubated at 25.0 ± 0.5 °C in the dark for 3 d. Fungal colonies growing from the pelotons were isolated using a sterilized scalpel and cultivated on potato dextrose agar (PDA, Difco, Franklin, New Jersey, USA) medium. One of the fungal isolates, HR1-1, was used for symbiotic germination in this study.
Phylogenetic analysis
DNA was extracted from the isolated fungus using PrepMan Ultra Reagent (Applied Biosystems, Foster City, California, USA) according to the manufacturer’s instructions. The ITS of rDNA was amplified from the extracted DNA by PCR with the primers ITS1-OF/ITS4-OF [25] using TaKaRa Ex Taq Hot Start Version (Takara Bio, Otsu, Japan). The PCR mixture contained 5 μl template DNA, 0.75 units Taq polymerase, 0.25 μmol/l each primer, 200 μmol/l each dNTP, and 3 μl of the supplied PCR buffer in a total volume of 30 μl. The amplification of the ITS region was performed on a PC-818S Program Temp Control System (Astec, Fukuoka, Japan) as follows: initial denaturation at 94 °C for 2 min followed by 35 cycles of 94 °C for 20 s, 55 °C for 30 s, and 72 °C for 1 min and a final elongation step at 72 °C for 5 min. PCR products were cloned using the pGEM-T Easy Vector System I (Promega, Tokyo, Japan), and plasmid DNAs were extracted from the cloned products using MagExtractor Plasmid (TOYOBO). The plasmid inserts were sequenced using the dye terminator method with sequencing primers T7 and SP6. All sequences were subjected to BLAST searches [26], and the related sequences were downloaded from the DDBJ/EMBL/GenBank nucleotide sequence database. Sequence alignment was performed using the CLUSTAL W program [27]. For phylogenetic analyses, neighbor-joining analysis [28] was performed with MEGA version 5 [29] with bootstrap analysis of 1000 replications [30]. Evolutionary distances were estimated using γ–distributed rates. The phylogenetic tree was drawn with TreeView software [31].
Symbiotic and asymbiotic germination
The seeds were surface sterilized in sodium hypochlorite with 1% available chlorine concentration containing 0.05% Tween 80 for 2 min and rinsed with sterilized water. Approximately 50 sterilized seeds were placed into plates containing either 20 ml original (1×), double (2×), or quadruple (4×) strength of oatmeal agar medium (2.5 g, 5.0 g, or 10.0 g, respectively, of oatmeal agar [Difco, Franklin, New Jersey, USA], 6.5 g agar, 1 l distilled water, pH 5.5) with symbiotic fungus which is precultured on 1× oatmeal agar medium for a week at 25 °C for symbiotic germination or 20 ml Hyponex agar medium (3.0 g Hyponex [6.5–6-19] [Hyponex Japan, Osaka, Japan], 2.0 g peptone, 30 g sucrose, 10 g agar, 1 l distilled water, pH 5.5) for asymbiotic germination. The germination experiments were conducted at 25 °C in the dark, and several randomly chosen protocorms were collected every seven days for four weeks. At each protocorm collection, images of the protocorms were taken under an SZX16 stereomicroscope (Olympus, Tokyo, Japan).
Protocorm growth measurements
The length and width of the collected protocorms were measured using the following procedure with Image J software version 1.47 (http://imagej.nih.gov/ij) as shown in Additional file 1: (1) a straight line (broken line) was drawn from the basal end to the apical end of the protocorm to measure the length (L), (2) a straight line (solid line) was drawn through both ends of the swollen embryo, and (3) a straight line (dotted line) was drawn perpendicular to the solid line at the most swollen site to measure the width (W). Three protocorms were measured at each sampling time point, and each experiment was repeated five times.
After rinsing the protocorms with distilled water, 10 protocorms were placed in a single ∅5 × 19 mm tin capsule (Ludi Swiss AG, Switzerland) and dried for 1 week at 60 °C. The dry weights were then measured using a microbalance (Mettler Toledo, Columbus, OH). Three independent germination experiments were performed for the dry weight measurements.
Quantitative evaluation of seed germination
The number of germinated seeds on oatmeal agar medium or Hyponex agar medium was counted under an SZX16 stereomicroscope (Olympus, Tokyo, Japan). At least 50 seeds were observed for each germination method, in which germination was defined as the emergence of a rhizoid or shoot. Three independent germination experiments were performed for the measurements of the germination rates.
Ink staining of hyphal coils in protocorm
The germinated symbiotic protocorms were stored in FAA solution at 4 °C for subsequent quantitative evaluation of fungal colonization. The FAA-fixed protocorms were rinsed with distilled water through a 40-μm cell strainer (Corning, NY, USA) and autoclaved at 121 °C for 20 min in 10% (w/v) KOH solution. The autoclaved protocorms were neutralized in 2% (v/v) HCl for 5 min, transferred to 10% (v/v) ink dye solution (10% Pelikan 4001 Brilliant Black and 3% acetic acid), heated at 95 °C for 30 min, and soaked in 100% lactic acid (Nakarai tesque Inc., Kyoto, Japan) at 4 °C before microscopic observation.
Quantitative evaluation of fungal colonization
The stained protocorms were processed as shown in Additional file 2. The testa of the stained protocorm was removed using a dissecting needle under an SZX16 stereomicroscope (Olympus, Tokyo, Japan), and the protocorm was transferred to a glass slide. The protocorm was covered with a cover glass and crushed using the end of the grip of a dissecting needle. The number of symbiotic cells was counted under a BX53 light microscope (Olympus, Tokyo, Japan) in at least 10 different protocorms at each sampling time. Three independent germination experiments were performed for this quantitative evaluation.
Staining hyphae on oatmeal agar medium
Hyphae of the symbiotic fungus on oatmeal agar medium were stained for two hours in a solution of 0.05% Trypan blue in lactic acid at room temperature. The hyphae were washed three times with distilled water. Stained hyphae were observed under an SZX16 stereomicroscope (Olympus, Tokyo, Japan). Five replicate plates were prepared for each concentration of oatmeal medium (1×, 2×, and 4×).