Tulasnella calospora (UAMH 9824) retains its effectiveness at facilitating orchid symbiotic germination in vitro after two decades of subculturing

Background The technique of symbiotic germination—using mycorrhizal fungi to propagate orchids from seed in vitro—has been used as one method to cultivate orchids in North America and abroad for > 30 years. A long-held assumption is that mycorrhizal fungi used for this purpose lose their effectiveness at germinating seeds over time with repeated subculturing. Results We provide evidence for the lingering efficacy of one particular strain of Tulasnella calospora (266; UAMH 9824) to stimulate seed germination exemplified by the North American terrestrial orchid, Spiranthes cernua, as a case study. This fungus was originally acquired from roots from Spiranthes brevilabris in 1999 and sub-cultured during the two decades since. Seeds inoculated with the fungus in vitro developed to an advanced protocorm stage after 16 days, and leaf elongation was pronounced after 42 days. In a pilot study, seedlings co-cultured with Tulasnella calospora 266 were deflasked after 331 days and later transferred to soil under greenhouse conditions where they eventually initiated anthesis. During the course of two decades, seeds of 39 orchid species, cultivars and hybrids spanning 21 genera, germinated in vitro co-cultured with Tulasnella calospora 266. These orchids included temperate terrestrials and tropical epiphytes alike. Conclusions The sustained effectiveness of this fungus is noteworthy because it argues against the concept of mycorrhizal fungi losing their symbiotic capability through prolonged subculturing. This study serves as an example of why in situ habitat preservation is essential for the conservation of orchids as a source of potentially useful mycorrhizal fungi.

Protocorm-Supporting Fungi Are Retained in Roots of Mature Tipularia discolor Orchids as Mycorrhizal Fungal Diversity Increases

Mycorrhizal fungi are critical to understanding the distribution patterns of many plants, but they are especially important for orchids. Some orchids may change the mycorrhizal fungi they use through their lives, either in response to changes in abiotic or biotic conditions, or as a result of ontogenetic changes that alter the orchid’s need for fungal nutrition. The temperate terrestrial orchid Tipularia discolor germinates only on decomposing wood, but often persists well after the wood has completely decomposed and has been incorporated into the soil. We used PCR and Sanger sequencing to ask: (1) Do mature T. discolor retain protocorm fungi or are protocorm and adult mycorrhizal fungi mutually exclusive? (2) Are protocorm fungi limited to areas with decomposing wood? (3) Does the abundance of protocorm fungi in the substrate differ between decomposing wood and bare soil? We found that T. discolor retained protocorm fungi into maturity, regardless of whether they were growing in persistent decomposing wood or soil. Protocorm fungi were not restricted to decomposing wood but were more common and abundant in it. We conclude that the mycorrhizal fungi associated with T. discolor change during the ontogeny of individuals. These results highlight the importance of assessing protocorm fungi, in addition to mycorrhizal fungi associating with adult orchids, to understand the conditions needed for orchid germination, growth, and reproduction.

Terrestrial Orchid Diversity in Sharavati River Valley of Shimoga District Karnataka (India)

Sharavati is a river which originates and flows entirely within the state of Karnataka in India. Sharavati river valley is the one of the most beautiful valley in the Western Ghats. Extensive field surveys during 2018-2020, revealed the occurrence of 25 species of orchids under 13 genera in river basin region in Shimoga district. Habenaria with 9 species is the largest genus in the river basin region, followed byNervilia with 3 species;Malaxis, Peristylus are 2 species each and the remaining genera by a single species each. The habit, distribution, phenology and population status of terrestrial orchid species is presented in this paper. Efforts have also been initiated for their conservation at Sharavati river valley region in Shimoga district Karnataka.

Influence of scarification method on seed germination of the terrestrial orchid Anacamptis laxiflora (Lam.)

A critical step during in vitro sexual propagation of terrestrial orchids is the treatment of the microscopic seeds with a disinfecting solution that kills bacteria and fungi attached to the seeds. This treatment is necessary to prevent infection of the culture vessels. At the same time, the treatment serves to scarify the seeds, a process that disrupts seed dormancy and initiates germination. The literature is inconclusive with respect to the proper combination of disinfecting solution strength and treatment duration. Both factors should be adapted to each species to guarantee minimal infection rate without damaging the embryo. This research aims to compare three disinfection/scarification methods for seeds of Anacamptis laxiflora (Lam.): (i) soaking in 0.5% NaClO, (ii) soaking in 0.5% NaClO, then centrifugation, and (iii) presoaking the seeds in sucrose solution, then soaking in 0.5% NaClO. The seeds were soaked in the disinfecting solution for 5 to 85 min. Following scarification, the seeds were sown in modified Malmgren nutrient medium. Infected and germinated vessels were counted at 41 and 189 d after sowing. We found that the longer the chemical treatment, the lower the infection rate, and the higher the germination rate. There was no significant difference in germination rate between the NaClO and the NaClO-plus-centrifugation method; in fact, the slight savings in disinfection time effected by centrifugation were more than offset by the added complexity of the method. Moreover, we found that centrifugation significantly delays germination. The sucrose presoak-plus-NaClO method was superior to plain NaClO, as the sucrose stimulates the germination of microbial spores on the surface of the seeds, making them easier to kill. Perhaps seeds with thicker testa as well as whole immature capsules could benefit even more from the pretreatment in sucrose solution.

Morphological and Genetic Diversities of Habenaria radiata (Orchidaceae) in the Kinki Area, Japan

Floral organs have evolved from leaves for reproduction, and the morphological analyses help to understand the plant diversity and evolution. Habenaria radiata (syn. Pecteilis radiata) is a terrestrial orchid living in wetlands in Japan, Russia, South Korea, and China. The habitats of this plant in Japan have been reduced because of environmental destruction and overexploitation, and thus it is on the Red List of Japan as a Near Threatened species. One of the three petals of the H. radiata flower is called a lip or labellum, which resembles a flying white bird, egret, or white heron, with its proposed function being to attract pollinators. To understand the diversity of H. radiata plants in different areas, we examined the lip morphology and phylogeny of populations from eight habitats in the Kinki area, Japan. The complex shapes of the lips were quantified and presented as a radar chart, enabling characterization of the morphological difference among populations. Phylogenetic analysis with microsatellite markers that we generated showed the variation of genetic diversity among populations, suggesting the different degrees of inbreeding, outbreeding, and vegetative propagation. Our approach offers a basic method to characterize the morphological and genetic diversity in natural populations.

Seed analysis of Dichromanthus aurantiacus, terrestrial orchid from Toluca valley, México

Objective: The objective of this work was to analyze the viability and germination of Dichromanthus aurantiacus seeds, a terrestrial orchid from Toluca valley, México. Design/methodology/approach: The size and color were evaluated. Two methods determined the viability: 1) the tetrazolium test (imbibition for 24 hours in the water, 2 hours in calcium hypochlorite (CaCOCl2), and drops of Tween-80). 2) the asymbiotic seed germination by in vitro culture (imbibition for 24 hours in the water, and the concentration of MS medium plus natural extracts). Results: The seeds of this specie showed approximately 0.2 mm long and 0.05 mm wide; they possess an embryo and a brownish testa. There were significant differences (P<0.05) between the treatments finding a positive effect with the tetrazolium test, achieving up to 91.4% viability. In the in vitro germination, the imbibition of the seeds favored contamination. The concentration of MS and the addition of natural extract presented significant differences (P<0.05), the 50% MS plus 10% of coconut water showed up to 92.8% of germination at 60 days. Study limitations/implications: The results are preliminary of a long-term experiment. Findings / Conclusions: The seeds of Dichromanthus aurantiacus showed brown testa and an oval embryo with dimensions of 0.2 mm long and 0.05 mm wide. The tetrazolium test’s viability showed 91.4% viability when they were soaked in sodium hypochlorite solution (CaCOCl2) for two hours, 24 hours soaking in tetrazolium solution (1%) plus two drops of Tween-80. The asymbiotic in vitro culture showed up to 92.8% germination in 60 days using MS medium at 50% enriched with 10% coconut water