Variation in mycorrhizal performance in the epiphytic orchid <Emphasis Type="Italic">Tolumnia variegata in vitro</Emphasis>: the potential for natural selection

Symbiotic seed germination is a critical stage in orchid life histories. Natural selection may act to favor plants that efficiently use mycorrhizal fungi. However, the necessary conditions for natural selection – variation, heritability, and differences in fitness – have not been demonstrated for either orchid or fungus. With the epiphytic orchid Tolumnia variegata as a model system, we ask the following questions: (1) Do seeds from different individuals in a population differ in germination and seedling development in the presence of the same fungi? (2) Do different mycorrhizal fungi (Ceratobasidium spp.) differ in ability to stimulate seed germination and growth in T. variegata? And (3) are the Ceratobasidium isolates that best induce seed germination and seedling development more closely related to each other than to isolates that are less effective? We performed symbiotic seed germination experiments in vitro. The experiments were done using mycorrhizal fungi isolated from T. variegata; relationships among the fungi were inferred from nuclear ribosomal ITS sequences. We found significant variation for both symbiotic germination and seedling growth among biparental seed crops obtained from a population of T. variegata plants. Differences among Ceratobasidium fungi in seed germination were significant. The fungi that induced highest seed germination and seedling development belonged to two of four clades of Ceratobasidium. The two experiments show that there is potential for natural selection to act on orchid–fungus relationships. Given that orchids vary in performance, and that mycorrhizal fungi are not geographically distributed homogeneously, mycorrhizae may affect population size, distribution and evolution of orchids.     

Asymbiotic seed germination,in vitro seedling development,and greenhouse acclimatization of the threatened terrestrial orchid <Emphasis Type="Italic">Bletia purpurea</Emphasis>

Procedures for asymbiotic seed germination and seedling acclimatization were developed for Bletia purpurea, a threatened North America native terrestrial orchid. Six asymbiotic orchid seed germination media (Knudson C, PhytoTechnology Orchid Seed Sowing Medium, Malmgren Modified Terrestrial Orchid Medium, Vacin &; Went Modified Orchid Medium, ½-strengh Murashige &; Skoog, and BM-1 Terrestrial Orchid Medium) were examined for their effectiveness in promoting seed germination and protocorm development of B. purpurea in either a 0/24 h or 16/8 h L/D photoperiod. Germination occurred regardless of medium or photoperiod treatment. However, advanced seedling development (Stage 6) only occurred on Vacin &; Went Modified Orchid Medium in the 16/8 h L/D photoperiod. Further effects of photoperiod on in vitro seedling development were also examined. Shoot length, leaf width, root number and length, and fresh weight and dry weight in the 16/8 h L/D photoperiod were all significantly different when compared to the 8/16 h and 12/12 L/D photoperiods. In vitro seedlings were readily acclimatized to greenhouse conditions. Seedlings showed high survival all potting media. Seedlings acclimatized in Fafard Mix 4 potting medium developed significantly longer roots. Corm formation occurred regardless of potting media used.     

Ultrastructural studies and molecular characterization of root-associated fungi of $$\textit{}$$ (D. Don) Szlach.: a threatened and medicinally important taxon

Crepidium acuminatum (Orchidaceae) is a threatened medicinal orchid that grows under shady and moist forest floor where light remains for a very short period of time. Mycorrhizal association is known to be essential for seed germination and seedling establishment in a majority of orchids. Identification of fungi that form mycorrhizae with orchids is of crucial importance for orchid conservation. We used both morphological as well as molecular approaches to study this plant–fungal interaction. Scanning electron microscopy showed that fungi grow and proliferate in the middle layers of the cortex. Also, spiral-root hairs were found along with root hairs, which is an unusual observation. Spiral-root hairs provide more surface area for fluid absorption and entrance of colonizers. Further, total root genomic DNA was isolated and fungal internal-transcribed spacer (ITS) regions were polymerase chain reaction (PCR)-amplified using specific primer combinations ITS1F/ITS4 and ITS1/ITS4tul. ITS sequences were obtained and analysed to know the closest sequence matche in the GenBank using BLASTn hosted by NLM-NCBI. Subject sequences were identified to be belonging to three main genera, namely, Tulasnella, Aspergillus and Penicillium. Results indicate that mycorrhizal association is necessary for the growth and development of the plant. In addition, this symbiosis influences the distribution and rarity of this medicinally valuable taxon. Specific fungal partners may lead to an enhanced seed germination rate and increased efficiency of nutrient exchange between both the partners. Hence, knowledge of mycorrhizal fungi is essential for future in vitro germination and seedling establishment programmes, because they rely on fungi for germination. Identification of mycorrhizal fungi can be used for orchid propagation and conservation programmes.     

A rare temperate terrestrial orchid selects similar Tulasnella taxa in ex situ and in situ environments

Mycorrhizal symbiosis in orchids is unique in that fungal presence is considered a requirement for germination as well as for further development. Additionally, orchid fungal associations can exhibit high specificity in nature. Yet, an important ecological question remains unanswered: ‘With which orchid mycorrhizal fungi (OMF) do un-inoculated orchid seedlings form symbiosis when cultured ex situ?’ Simultaneously, it is asserted that orchid conservation efforts involving ex situ plant culture should exclusively utilize natural symbionts of the respective orchid taxa. We present a first comparison of OMF communities within the roots of asymbiotically cultured plants of the rare orchid Platanthera chapmanii grown ex situ (ES), and those occurring naturally in situ (IS). Nuclear ribosomal internal transcribed spacer (nrITS) barcoding region was used to identify peloton forming OMF from roots collected between 2012 and 2014 from both growing environments. Our 114 sequences clustered into 11 operational taxonomic units (OTUs) belonging to four closely related clades of the fungal family Tulasnellaceae. Shannon–Wiener (H) and Simpson diversity (D) indices were similar (p = 0.81 for both) for ES and IS OMF communities. Beta diversity comparisons also showed similarity between ES and IS treatments based on weighted (p = 0.10) and unweighted (p = 0.20) Bray–Curtis dissimilarity matrices. Bayesian and Maximum Likelihood (ML) phylograms clustered ES and IS derived fungal OTUs into the same clades. Our data suggest that P. chapmanii: (1) forms symbiosis with taxonomically similar fungi in ex situ culture and in its native soil, and (2) exhibits a narrow phylogenetic breadth of mycorrhizal fungal OTUs within the Tulasnellaceae.     

Host-specificity of symbiotic mycorrhizal fungi for enhancing seed germination,protocorm formation and seedling development of over-collected medicinal orchid, <Emphasis Type="Italic">Dendrobium devonianum</Emphasis>

All orchids maintain an obligate relationship with mycorrhizal symbionts during seed germination. In most cases, germination-enhancing fungi have been isolated from roots of mature plants for conservation and cultivation purposes. To understand the germination biology of Dendrobium devonianum, an over-collected medicinal orchid, the seeds of D. devonianum were inoculated with a fungal strain (FDd1) isolated from naturally occurring protocorms of D. devonianum and two other germination-enhancing fungal strains (FDaI7 and FCb4) from D. aphyllum and Cymbidium mannii, respectively. The fungal strain was isolated from five protocorms of D. devonianum and identified as a species of the genus Epulorhiza. In germination trials, treatments with all of the three fungal strains showed a significant promoting effect on seed germination and protocorm formation, compared with the control treatment (no inoculation). However, FDd1 fungal strain showed the greatest effectiveness followed by FDaI7 and FCb4. For all inoculation and control treatments, seeds developed to protocorms regardless of the presence of illumination, whereas protocorms did not develop to seedlings unless illumination was provided. The results of our manipulative experiments confirmed the hypothesis that mycorrhizae associated with orchid seedlings are highly host-specific, and the degree of specificity may be life stagespecific under in vitro conditions. The specific mycorrhizal symbionts from protocorms can enhance restoration efforts and the conservation of orchids such as D. devonianum.     

Asymbiotic seed germination and in vitro seedling development of <Emphasis Type="Italic">Cyrtopodium punctatum</Emphasis>: a propagation protocol for an endangered Florida native orchid

Cyrtopodium punctatum Lindley is an endangered epiphytic orchid restricted in the United States to southern Florida. Due to its ornamental value, the species was extensively collected from the wild during the past 100 years. Today, only a few plants remain in protected areas. As part of a conservation plan for the species, procedures for asymbiotic seed germination were developed. Five asymbiotic orchid seed germination media (PhytoTechnology Orchid Seed Sowing Medium, Knudson C, Malmgren Modified Terrestrial Orchid Medium, Vacin &; Went Modified Orchid Medium, and ½-strengh Murashige &; Skoog) were examined for their effectiveness in promoting seed germination and protocorm development under a 16/8 h L/D photoperiod and dark (0/24 h L/D). The influence of photoperiod on growth and development was also examined. Seeds were germinated under a 16/8 h, 12/12 h, 8/16 h L/D photoperiod, at 25 ± 3°C and allowed to develop in vitro for 10 weeks. After 10 weeks, developing seedlings were transferred to Sigma Phytatrays and returned to their assigned photoperiod treatments for continued seedling development for an additional 15 weeks. Highest germination occurred in 0/24 h L/D on PhytoTechnology Orchid Seed Sowing Medium and seedlings displayed more advanced development when cultured under 16/8 h L/D photoperiod after 15 weeks in Phytatrays. Thirty-five week old seedlings potted in coconut husk growing medium exhibited 90% survival following 5 weeks acclimatization to greenhouse conditions. This asymbiotic seed germination protocol for C. punctatum will facilitate future reintroduction projects involving this endangered species.     

Impacts of Host Trees and Sowing Conditions on Germination Success and a Simple Ex Situ Approach to Generate Symbiotic Seedlings of a Rare Epiphytic Orchid Endemic to Hainan Island,China

For species relying on seeds for population regeneration, knowledge on seed germination behaviors in relation to environmental factors is critical in designing species recovery strategy. Dendrobium sinense is an orchid endemic to Hainan Island of China and listed as Endangered by the IUCN Redlist. It reproduces primarily via seeds in its natural habitat. However, how germination is impacted by major environmental factors is poorly known. This study aimed to examine germination success of D. sinense seeds using two approaches, i.e. in situ and ex situ, using host tree barks as germination media. The latter was intended to generate symbiotic seedlings in a simple and economic approach which could be used for reintroduction efforts. In addition, three factors of in situ symbiotic seed germination success, including different sowing time, location (distance from an adult plant), and host tree were investigated. Our results showed that seeds sown ex situ and in situ in July had the highest rates of germination. Seed germination was significantly higher ex situ using bark as medium than in situ. Seeds sown directly on Rhododendron moulmainense, the most common host tree, with naturally occurring conspecific orchids had the highest rate of germination. In contrast, ex situ seeds sown on the bark of Cyclobalanopsis blakeii, a non-host species, had the highest rate of seed germination. In situ a positive correlation was found between the seed germination rate and the distance of the seeds from the adult D. sinense. Based on these results, it is likely seedling recruitments are determined by host tree species, the presence of and the distance from an adult conspecific orchid, which imply the importance of the mycorrhizal fungi, which were not reported here. This study provided important information on the optimal environmental conditions for population augmentation and reintroduction, which can be used as part of the species recovery strategy.     

Do novel weapons that degrade mycorrhizal mutualisms promote species invasion?

Non-native plants often dominate novel habitats where they did not co-evolve with the local species. The novel weapons hypothesis suggests that non-native plants bring competitive traits against which native species have not adapted defenses. Novel weapons may directly affect plant competitors by inhibiting germination or growth, or indirectly by attacking competitor plant mutualists (degraded mutualisms hypothesis). Japanese knotweed (Fallopia japonica) and European buckthorn (Rhamnus cathartica) are widespread plant invaders that produce potent secondary compounds that negatively impact plant competitors. We tested whether their impacts were consistent with a direct effect on the tree seedlings (novel weapons) or an indirect attack via degradation of seedling mutualists (degraded mutualism). We compared recruitment and performance using three Ulmus congeners and three Betula congeners treated with allelopathic root macerations from allopatric and sympatric ranges. Moreover, given that the allelopathic species would be less likely to degrade their own fungal symbiont types, we used arbuscular mycorrhizal (AMF) and ectomycorrhizal (ECM) tree species to investigate the effects of F. japonica (no mycorrhizal association) and Rhamnus cathartica (ECM association) on the different fungal types. We also investigated the effects of F. japonica and R. cathartica exudates on AMF root colonization. Our results suggest that the allelopathic plant exudates impact seedlings directly by inhibiting germination and indirectly by degrading fungal mutualists. Novel weapons inhibited allopatric seedling germination but sympatric species were unaffected. However, seedling survivorship and growth appeared more dependent on mycorrhizal fungi, and mycorrhizal fungi were inhibited by allopatric species. These results suggest that novel weapons promote plant invasion by directly inhibiting allopatric competitor germination and indirectly by inhibiting mutualist fungi necessary for growth and survival.     

Arbutoid mycorrhizas of the genus <Emphasis Type="Italic">Cortinarius</Emphasis> from Costa Rica

Arbutoid mycorrhizas of Comarostaphylis arbutoides (Arbutoidea, Ericaceae) from neotropical montane forests are rarely described. To date, only mycorrhizal associations with the fungal species Leccinum monticola, Leotia lubrica and Sebacina sp. are known from literature. The genus Cortinarius is one of the most species-rich ectomycorrhizal taxa with over 2000 assumed species. In this study, two sites in the Cordillera de Talamanca of Costa Rica were sampled, where Com. arbutoides is endemic and grows together with Quercus costaricensis. Using a combined method of rDNA sequence analysis and morphotyping, 33 sampled mycorrhizal systems of Cortinarius were assigned to the subgenera Dermocybe, Phlegmacium and Telamonia. Specific plant primers were used to identify the host plant. Here, we present the phylogenetic data of all found Cortinarii and describe four of the arbutoid mycorrhizal systems morphologically and anatomically.     

Mechanisms of influence of invasive grass litter on germination and growth of coexisting species in California

In grasslands, litter has been recognized as an important factor promoting grass persistence and the suppression of forbs. The invasive European annual grass Bromus diandrus (ripgut brome) is widespread throughout California, where it produces a persistent and thick litter layer. The native grass, Stipa pulchra, is also common in some grassland settings and can also produce persistent litter, yet it is typically associated with more forbs. Very little is known about the mechanisms through which these two common grass species influence seedling establishment of both exotic invasive and native herbs. Here, we evaluated the effect of B. diandrus and S. pulchra litter on seedling establishment of two invasive (the grass B. diandrus and the forb Centaurea melitensis) and two native (the grass S. pulchra, and the forb Clarkia purpurea) herbaceous plants in a greenhouse setting. Our results showed that B. diandrus litter cover hindered seedling establishment of the four species tested, but that the degree and mechanism of inhibition was dependent on which species was tested, life form (e.g. monocot/dicot) and seed size. Seedling emergence of the two forb species was more vulnerable to litter cover than either grass species and both forbs had smaller seed size. After germination, only seedling biomass of B. diandrus itself was reduced by litter (both B. diandrus and S. pulchra). We found no significant effects of leachate of either grass species on seedling emergence of any species, while a high concentration of B. diandrus leachates inhibited root growth of all species including B. diandrus seedlings. Stipa pulchra litter leachates did not affect S. pulchra or C. melitensis seedlings although it did suppress B. diandrus and C. purpurea seedling growth. Our findings provide direct experimental evidence for the mechanism of effect of litter on these coexisting invasive and native species. Such evidence helps advance our understanding of role of B. diandrus and S. pulchra litter in California grassland.     

Effects of <Emphasis Type="Italic">Prangos ferulacea</Emphasis> aqueous and hydroalcoholic extracts obtained from different organs on the regeneration of <Emphasis Type="Italic">Trifolium resupinatum</Emphasis>

Prangos ferulacea is one of the widely used, nutritional and popular fodders in livestock industry. This species is also considered as an important option in rangeland restoration and management. In this study, the comparative phytotoxic activity of aqueous and hydroalcoholic extracts obtained from different organs (flower, shoot and leaf) of P. ferulacea on proline content, seed germination and seedling growth of Trifolium resupinatum has been investigated. According to the results, the hydroalcoholic extract of P. ferulaceae flower possesses the highest total phenolic and flavonoid content and the uppermost phytotoxic effect on T. resupinatum. The extracts significantly decreased seed germination and seedling growth of T. resupinatum and increased the proline content. Our findings indicate that hydroalcoholic extract induced a stronger oxidative stress in T. resupinatum. Finally, based on the results, aqueous allelochemicals that originated from P. ferulacea played a significant role in the successful propagation and development of T. resupinatum in rehabilitated pastures. According to our results, the phytotoxicity effect of the hydroalcoholic extract was significantly higher than that of the aqueous extract. Since in nature, the allelopathic interaction between plants is closer to the aqueous method, primary evaluations of rangeland restoration using this method is suggested.     

Asymbiotic germination response to photoperiod and nutritional media in six populations of Calopogon tuberosus var. tuberosus (Orchidaceae): evidence for ecotypic differentiation

Background and Aims

Ecotypic differentiation has been explored in numerous plant species, but has been largely ignored in the Orchidaceae. Applying a specific germination protocol for widespread seed sources may be unreliable due to inherent physiological or genetic differences in localized populations. It is crucial to determine whether ecotypic differentiation exists for restoration and conservation programmes. Calopogon tuberosus var. tuberosus, a widespread terrestrial orchid of eastern North America, is a model species to explore ecotypic differences in germination requirements, as this species occupies diverse habitats spanning a wide geographical range.

Methods

Mature seeds were collected from south Florida, north central Florida, three locations in South Carolina, and the upper Michigan peninsula. Effects of three photoperiods (8/16, 12/12, 16/8 h L/D) were examined on asymbiotic in vitro seed germination and seedling development of C. tuberosus. Germination and early development was monitored for 8 weeks, while advanced development was monitored for an additional 8 weeks. In an additional experiment, asymbiotic seed germination and development was monitored for 8 weeks on six culture media (BM-1 terrestrial orchid medium, Knudson C, Malmgrem, half-strength MS, P723, and Vacin and Went). A tetrazolium test for embryo viability was performed.

Key Results

Short days promoted the highest germination among Florida populations, but few differences among photoperiods in other seed sources existed. Different media had little effect on the germination of Michigan and Florida populations, but germination of South Carolina seeds was higher on media with higher calcium and magnesium. Tetrazolium testing confirmed that South Carolina seeds exhibited low viability while viability was higher in Florida seeds. Seed germination and corm formation was rapid in Michigan seeds across all treatments. Michigan seedlings allocated more biomass to corms compared with other seed sources.

Conclusions

Rapid germination and corm formation may be a survival mechanism in response to a compressed growing season in northern populations. Ecotypic differentiation may be occurring based on seed germination and corm formation data.Key words: Asymbiotic germination, corm development, Calopogon tuberosus, ecotypic differentiation, native orchid, orchid seed germination, seedling development     

Cryopreservation of seeds and protocorms of rare temperate orchids

The efficiency of cryopreservation of seeds of five rare and endangered species of temperate orchids belonging to Platanthera and Dactylorhiza genera followed by their asymbiotic culture in vitro, as well as of in vitro cultured D. fuchsii protocorms (specific stage of orchid embryo development after release from the seed coat) was investigated. Germination rates of seeds after their exposure to liquid nitrogen were species-depended and could be either higher or lower than in the unfrozen control. There was no significant difference between growth rates of protocorms of the same species obtained from seeds collected in various Russia regions and cultured for 5 months. After vitrification, 9% of D. fuchsii protocorms with a larger diameter of 1200 μm survived cryopreservation; however, their growth was retarded for three months when compared to control protocorms.     

Mycorrhizal associations and reproductive isolation in three closely related Orchis species

Background and Aims

The maintenance of species boundaries in sympatric populations of closely related species requires some kind of reproductive isolation that limits gene flow among species and/or prevents the production of viable progeny. Because in orchids mycorrhizal fungi are needed for seed germination and subsequent seedling establishment, orchid–mycorrhizal associations may be involved in acting as a post-mating barrier.

Methods

We investigated the strength of post-mating barriers up to the seed germination stage acting between three closely related Orchis species (Orchis anthropophora, O. militaris and O. purpurea) and studied the role of mycorrhizal fungi in hybridization by burying seed packets of pure and hybrid seeds. After retrieval and assessment of seed germination, the fungi associating with protocorms originating from hybrid and pure seeds were determined and compared with those associating with adult individuals using DNA array technology.

Results

Whereas pre-zygotic post-mating barriers were rather weak in most crosses, post-zygotic post-mating barriers were stronger, particularly when O. purpurea was crossed with O. anthropophora. Germination trials in the field showed that seed germination percentages of hybrid seeds were in most cases lower than those originating from pure crosses. In all species pair combinations, total post-mating reproductive isolation was asymmetric. Protocorms associated with a smaller range of fungal symbionts than adult plants, but there was considerable overlap in mycorrhizal associations between protocorms and their respective parents.

Conclusions

Our results suggest that mycorrhizal associations contribute little to reproductive isolation. Pre-mating barriers are probably the main factors determining hybridization rates between the investigated species.     

Diversity of fungi associated with roots of <Emphasis Type="Italic">Calanthe</Emphasis> orchid species in Korea

While symbiotic fungi play a key role in the growth of endangered Calanthe orchid species, the relationship between fungal diversity and Calanthe species remains unclear. Here, we surveyed root associated fungal diversity of six Calanthe orchid species by sequencing the internal transcribed spacer (ITS) region using 454 pyrosequencing. Our results revealed that Paraboeremia and Coprinopsis are dominant fungal genera among Calanthe species. In terms of overall relative abundance, Paraboeremia was the most common fungal genus associated with Calanthe roots, followed by Coprinopsis. Overall fungal diversity showed a significant degree of variation depending on both location and Calanthe species. In terms of number of different fungal genera detected within Calanthe species, C. discolor had the most diverse fungal community, with 10 fungal genera detected. This study will contribute toward a better understanding of those fungi that are required for successful cultivation and conservation of Korean Calanthe species.     

Variation in nutrient-acquisition patterns by mycorrhizal fungi of rare and common orchids explains diversification in a global biodiversity hotspot

Background and Aims

Many terrestrial orchids have an obligate requirement for mycorrhizal associations to provide nutritional support from germination to establishment. This study will investigate the ability of orchid mycorrhizal fungi (OMF) to utilize a variety of nutrient sources in the nutrient-impoverished (low organic) soils of the Southwest Australian Floristic Region (SWAFR) in order to effectively compete, survive and sustain the orchid host.

Methods

Mycorrhizal fungi representing key OMF genera were isolated from three common and widespread species: Pterostylis recurva, Caladenia flava and Diuris corymbosa, and one rare and restricted species: Drakaea elastica. The accessibility of specific nutrients was assessed by comparing growth including dry biomass of OMF in vitro on basal CN MMN liquid media.

Key Results

Each of the OMF accessed and effectively utilized a wide variety of nutrient compounds, including carbon (C) sources, inorganic and organic nitrogen (N) and inorganic and organic phosphorus (P). The nutrient compounds utilized varied between the genera of OMF, most notably sources of N.

Conclusions

These results suggest that OMF can differentiate between niches (micro-niche specialization) in a constrained, highly resource-limited environment such as the SWAFR. Phosphorus is the most limited macronutrient in SWAFR soils and the ability to access phytate by OMF indicates a characterizing functional capacity of OMF from the SWAFR. Furthermore, compared with OMF isolated from the rare D. elastica, OMF associating with the common P. recurva produced far greater biomass over a wider variety of nutritional sources. This suggests a broader tolerance for habitat variation providing more opportunities for the common orchid for recruitment and establishment at a site.