Seed Collection
Mature seed capsules of P. concreta (Jacq.) Garay & H.R. Sweet were collected from a native population in the Northern tropical Andes of Ecuador on 23 August 2018. Plants with closed mature capsules were located along the roadside to Chical (0º48'36.5"N 78º12'55.30"W, 1370 m.a.s.l.), Carchi Province, Ecuador. Upon return to the lab, capsules were place in a sealed container containing CaSO4 desiccant at 22±2 ºC. After 24 hours, seeds were removed from the capsules and stored in glass vials over CaSO4 for shipping to the laboratory located at the University of Florida, Gainesville, Florida, USA. Seeds were stored at -10 ºC in the dark at 0% relative humidity until used in the experiments. Two vials of seeds were maintained at 22±2 ºC in the dark for 16 weeks before transferred to storage conditions.
Seed Viability Testing
Seed viability tests using triphenyl tetrazolium chloride (TTC) were performed in seeds before being transfer to storage cold conditions and after 7 weeks in storage. Approximately 5 mg of seed contained in 1.5 ml microtube was preconditioned in a 10% sucrose solution for 24 hours at 22±2 ºC. Solutions were removed and seeds were washed twice with deionized distilled (dd) water. A 1% TTC solution was added and the microtubes were incubated in the dark at 36 ºC for 24 hours. The TTC solution was removed and replaced with dd water before samples were stored at 4 ºC until their evaluation of seed viability using a light microscopy. Seeds with rose-red stained embryos were considered viable, while those with white embryos were recorded as non-viable (Hosomi et al. 2011; Hosomi et al. 2012).
Seed Sterilant Screening
To optimized seed sterilization procedures, sodium hypochlorite (NaOCl) and calcium hypochlorite [Ca(OCl)2] solutions were used to test their individual effectiveness as orchid seed surface sterilants. Seeds surface sterilization was evaluated using a 0.3% or 0.5% sodium hypochlorite/ethanol solution or 2% calcium hypochlorite in a sterile 1.5 ml sterile microtube for 1, 3 and 5 minutes. The 0.3 % and 0.5% sodium hypochlorite solutions were prepared by combining 250 or 416 µl 6.0% sodium hypochlorite (Clorox bleach), 250 µl 100% ethanol and 4,500 or 4,334 ml sterile dd water. The calcium hypochlorite solution was prepared by dissolving 307 mg Ca(OCl)2 (65% available chlorine) in 10 ml sterile dd water. This gave a final solution providing 2% available chlorine. The solution was filtered through sterile white filter paper (Whatman ®, Qualitative 1, Cat# 1001-125) immediately before use. After sterilization, solutions were removed with a micropipette by placing the sterile pipette tip into the bottom of the microtube. Seeds were then rinsed three times for 1 min. each with sterile dd water.
After sterilization, three 60 µl aliquots of the sterile seed solution were pipetted and each was placed on a separated section into a Petri dish (100x15mm) containing 30 ml ½ X Strength Murashige & Skoog (½ MS; PhytoTechnology Laboratories, Shawnee Mission, KS, USA Cat# M524, Murashige and Skoog 1962). Seeds then were distributed uniformly using a sterile inoculating loop. Four Petri dishes per treatment were sealed with one layer of sealing film (PhytoTechnology Laboratories, Shawnee Mission, KS, USA Cat# A003) and incubated at 23 ºC under a 12h-Light /12h-Dark photoperiod for 3 weeks. Light was provided by cool-white florescent tubes (General Electric F20T12/CW) at 35 µM m-2 s-1 (PAR) in a Percival I-35LL growth chamber (Percival Scientific, Boone, IA).
Asymbiotic Seed Germination
Four asymbiotic orchid seed germination media: 1) Knudson-C (KND-C; PhytoTechnology Laboratories, Shawnee Mission, KS, USA Cat# K400, Knudson 1946 ); 2) Vacin and Went Modified Orchid Medium (VW; PhytoTechnology Laboratories, Shawnee Mission, KS, Cat# V891, Vacin and Went 1949 ); 3) ½ X Strength Murashige & Skoog (½ MS; PhytoTechnology Laboratories, Shawnee Mission, KS, Cat# M524, Murashige and Skoog 1962); and 4) PhytoTechnology Orchid Seed Sowing Medium (Cat# P723) were examined for their effectiveness in facilitating seed germination and seedling development of P. concreta. To standardize the concentrations of 2% sucrose, 0.8% agar and 0.1% activated charcoal, all basal media were modified as follows: 20 g/L sucrose was added to 1/2 MS, 8 g/L TC agar (PhytoTechnology Laboratories, Shawnee Mission, KS, USA Cat# A175) to KND-C and ½ MX, 1 g/L TC agar to VW, and 1 g/L activated charcoal to KND-C, ½ MS, and VW. KND-C and ½ MS was adjusted to pH 5.8. All media were sterilized by autoclaving at 117.7 kPa and 121 ºC for 40 min.
Based on preliminary sterilant screening, seed sterilization was performed using a filtered solution of 2% calcium hypochlorite for 1 min. followed by three 1-min. rinses with sterile dd water. After sterilization, three 60 µl aliquots containing between 60-80 sterile seeds each, were pipetted and distributed uniformly using a sterile inoculating into Petri dishes (100x15mm) containing 30 ml of each of the four asymbiotic seed germination media. Petri dishes were sealed with one layer of sealing film and incubated under both 0/24 h and 12/12 h Light/Dark photoperiod at 23 ºC. The complete darkness treatment was achieved by wrapping stacked Petri dishes in heavy duty aluminum foil.
Plates were first inspected for seed germination after 1-week culture. At 3 weeks Petri dishes from the 0/24 h photoperiod were unwrapped from the aluminum foil and placed under a 12/12 h Light/Dark photoperiod for the rest of the experiment. Seed germination and seedling developmental stages were assessed on a scale of 0-6 (Table 1). Germination and seedling growth and development were recorded every 2 weeks after the first week using a stereomicroscope.
Seedling Development Experiment
Survival and further development of P. concreta seedlings were evaluated using three media: 1) P723; 2) PhytoTechnology Orchid Maintenance/Replace Medium (Cat# P748); and 3) PhytoTechnology Orchid Multiplication Medium (Cat# P793). After 16 weeks on P723, 12 Stage 6 seedlings were transferred into each of four Phytatray™ II (Sigma-Aldrich, Cat# P-5929) vessels containing 125 ml of each medium. Vessels were sealed with one layer of sealing film and incubated under a 12h-Light /12h-Dark photoperiod at 23 ºC. Percent survival and seedling length were recorded after 25 weeks.
Symbiotic Seed Germination
Isolation of putative mycobionts
Isolation of root endophytes from root cortical tissue was performed at IDgen, a commercial molecular diagnostic laboratory, located in Quito, Ecuador. Standard methods described by Zettler et al. (2003) were applied. Briefly, roots from young orchids or seedlings, when available, were collected in the field and transported to the laboratory. Roots were rinsed with sterile distilled water, then surface sterilized with a 0.4% sodium hypochlorite/ethanol solution, followed by three 1-minute rinses with sterile distilled water. This solution was prepared by combining 8 ml 5% sodium hypochlorite (Clorox bleach), 5 ml 98% ethanol and 87.0 ml sterile distilled water.
Roots were cut into 1 cm long segments starting at the tip. Each segment was placed into a separate sterile Petri dish containing 200 µl sterile distilled water. Root segments were macerated using sterile scalpel and forceps to separate and break open the cortical cells containing fungal pelotons. Warm Fungal Isolation Medium (FIM) (Clements et al. 1986) containing 10ml/L streptomycin sulfate antibiotic, was slowly added to the dish. To uniformly distribute the macerated tissue, dishes were gently swirled before solidification of the medium. Using a dissection microscope, plates were observed after 24 hours for active hyphal growth emerging from the pelotons. Individual pelotons with hyphal tips were subcultured using a sterile scalpel into ½ Potato Dextrose Agar (PDA, PhytoTechnology Laboratories, Shawnee Mission, KS, USA Cat# P772) and incubated for 24-72 hours at 22 ± 2 ºC. Hyphal tips or pelotons were then transferred to fresh ½ PDA solidified on a 45º angle in glass culture tubes. Glass tubes were sealed with a layer of sealing film and incubated at 22 ± 2 ºC for at least 48 hours and then stored at 4 ºC until their transportation to the laboratory located at the University of Florida, Gainesville, Florida, USA.
Fungal strains with morphological features that have been previously assigned to the form-genus Rhizoctonia (Currah et al. 1997) were subcultured on ½ PDA for further identification to the genus level using Sanger sequencing of the ribosomal DNA internal transcribed spacer (ITS) sequences (Otero et al. 2002). Subcultures of putative mycorrhizal fungi were transferred to fresh ½ PDA plates every 4 months and maintained at 10 ºC in the dark.
Symbiotic seed culture
Fungal strains, isolated from different native orchid species from Ecuador and Florida, USA, were used to examine their ability to enhance seed germination and seedling development of P. concreta. Symbiotic treatments consisted of one fungal culture provided by Dr. Lawrence W. Zettler (Illinois College, Jacksonville, IL): Tulasnella calospora strain (University of Alberta Microfungus Herbarium UAMH 9824) isolated from a primary lateral root of Spiranthes brevilabris in Levy County, Florida, USA in 1999 (Stewart et al 2003), four fungal strains isolated from roots of plants collected from native orchids species from Ecuador: two Tulasnella sp. strains isolated one from Lepanthes acarina (EC16-LPA2T [UAMH12450]) and the other from Campylocentrum sp. (EC17-CM4T [UAMH 12451]), and two Ceratobasidium strains isolated from Ericina pumilla (EC18-ER2C) and Macroclinium sp. ( EC18-MC4C [UAMH12454]). Few pelotons were observed in cortical tissue of roots of P. concreta collected from juvenile or adult plants. Unfortunately, no putative mycorrhizal fungi could be isolated therein.
Based on the asymbiotic media screen responses, P723 was included as the asymbiotic treatment control. A second control treatment consisted of the symbiotic medium with seeds but without fungal inoculation (Fig. 9d). Oatmeal Agar (OMA; 2.5 g/L oats flour, 7 g/L agar (PhytoTechnology Laboratories, Shawnee Mission, KS, USA Cat# A175)) was used as the symbiotic seed germination medium. A triangle-shaped cellophane membrane (BIO-RAD, Cat# 1650922) with one black filter paper strip (Thomas Scientific, USA Cat# 4740C10) on each side were placed on the surface of the symbiotic medium after it solidified (Fig. 9d). Medium was sterilized by autoclaving at 117.7 kPa at 121 ºC for 40 min. Approximately 30 ml of cooled but molten sterile OMA was dispensed in each (100x15 mm) plastic Petri dish. Seeds were removed from cold storage and maintained at 22±2 ºC for at least 4 hours before sterilization. Seed sterilization was performed using the same procedure described for the asymbiotic seed germination experiment. After sterilization, 60 µl aliquots containing 60-80 seeds were placed on the surface of each 1 cm X 4 cm black sterile filter paper.
Fungal inoculation was performed by cutting a 1X1X0.5 cm3 ½ PDA block containing active growing fungal mycelia harvested from the edges of a week-old culture in the case of Tulasnella and 2 day-cultures of Ceratobasidium (Fig. 10). The PDA block was placed into each Petri dish on the center of the membrane (Fig. 9e). Petri dishes were sealed with one layer of sealing film and incubated under in both 0/24 h and 12/12 h Light/Dark photoperiods at 23 ºC. Plates were examined for evidence of germination after 5 days. Germination and seedling development stages (Table 1) were recorded every 5 days until day 20, and then weekly, thereafter until 9 weeks. Roots from seedlings produced on the symbiotic medium were finely sliced and stained using an aqueous 0.5% Toluidine Blue Staining Solution to evaluate the presence of fungal pelotons in the cortical tissue.
Experimental Design and Statistical Analysis
Treatment effects were evaluated using a completely randomized experimental design. Each factor or combination of factors consisted of 7 replicates with 3 sub-replicates. The experimental unit consisted of a Petri dish containing 30 ml culture medium with three areas or three filter paper strips with ca. the same number of seeds on the surface. These areas were considered as sub-replicates. All experiments were repeated once. Numbers of seeds in each developmental stage (Table 1) were recorded at each data collection time point. Percentages of individuals in each stage were calculated for each treatment by dividing the number of seeds in each stage by the total number of viable seeds in each sub replicate. Percent data were arcsine transformed to normalize variation prior to analysis. Data were analyzed using general linear model procedures with a multifactor Analysis of Variance (ANOVA). Mean separation was assessed using Tukey’s Methods at α = 0.05 significant level. Statistical Analysis were completed with JMP Pro15 (SAS Institute Inc.).
The time-to-event data, in this case time from sowing to germination (Stage 2), were analyzed using the survival analysis package in R studio 3.2.5. Survival analysis is a statistical method that deals with time to events. It has many important applications in clinical research and epidemiology as well as other disciplines. It is believed that time-to-event analysis provides the most appropriated method to analyze germination, because the analysis is based on the distribution of germination times of individuals seeds rather than on cumulative germination (McNair et al. 2012). A Kaplan-Meier estimator was used to estimate the survival functions with 95% confidence intervals. Log-rank or Stratified log-rank test were performed to evaluate differences between survival curves at the α = 0.05 significant level and multiple pair wise comparisons were performed using log-rank test with a Bonferroni correction to adjust p-values at α=0.05 significance level.