Isolation and identification of endophytic and mycorrhizal fungi from seeds and roots of <Emphasis Type="Italic">Dendrobium</Emphasis> (Orchidaceae)

The seed germination of orchids under natural conditions requires association with mycorrhizal fungi. Dendrobium nobile and Dendrobium chrysanthum are threatened orchid species in China where they are considered medicinal plants. For conservation and application of Dendrobium using symbiosis technology, we isolated culturable endophytic and mycorrhizal fungi colonized in the protocorms and adult roots of two species plants and identified them by morphological and molecular analyses (5.8S and nrLSU). Of the 127 endophytic fungi isolated, 11 Rhizoctonia-like strains were identified as Tulasnellales (three strains from protocorms of D. nobile), Sebacinales (three strains from roots of D. nobile and two strains from protocorms of D. chrysanthum) and Cantharellales (three strains from roots of D. nobile), respectively. In addition, species of Xylaria, Fusarium, Trichoderma, Colletotrichum, Pestalotiopsis, and Phomopsis were the predominant non-mycorrhizal fungi isolated, and their probable ecological roles in the Dendrobium plants are discussed. These fungal resources will be of great importance for the large-scale cultivation of Dendrobium plants using symbiotic germination technology and for the screening of bioactive metabolites from them in the future.     

<Emphasis Type="Italic">Mycena</Emphasis> sp., a mycorrhizal fungus of the orchid <Emphasis Type="Italic">Dendrobium officinale</Emphasis>

An endophytic fungus, F-23, was isolated from the roots of Dendrobium officinale Kimura et Migo, an endangered Chinese medicinal plant. The sequence of the ITS region indicated that the isolate belongs to the genus Mycena. After 4 months of inoculation, the root systems of D. officinale that were inoculated with F-23 fungus were much larger than the control’s root systems. We also observed that the hyphae of F-23 penetrated the epidermal cells within the host’s roots and spread from cell to cell. A large number of pelotons existed in the root cortical cells of D. officinale inoculated with F-23 fungus. Intracellular hyphae crossing through the host walls were also observed using SEM (scanning electron microscopy). In contrast, light microscopy and SEM showed that the transverse sections of the roots of control plants remained uncolonized. Therefore, the F-23 fungus can form mycorrhizal associations with the roots of its host plant, D. officinale, and enhance the growth of seedlings and roots. In brief, Mycena sp. was identified and shown to be a mycorrhizal fungus of the epiphytic orchid, D. officinale. This might be of potential use to the mass cultivation of D. officinale under artificial conditions.     

Comparative <Emphasis Type="Italic">in vitro</Emphasis> germination ecology of <Emphasis Type="Italic">Calopogon tuberosus</Emphasis> var. <Emphasis Type="Italic">tuberosus</Emphasis> (Orchidaceae) across its geographic range

Seed responses to temperature are often essential to the study of germination ecology, but the ecological role of temperature in orchid seed germination remains uncertain. The response of orchid seeds to cold stratification have been studied, but the exact physiological role remains unclear. No studies exist that compare the effects of either cold stratification or temperature on germination among distant populations of the same species. In two separate experiments, the role of temperature (25, 22/11, 27/15, 29/19, 33/24°C) and chilling at 10°C on in vitro seed germination were investigated using distant populations of Calopogon tuberosus var. tuberosus. Cooler temperatures promoted germination of Michigan seeds; warmer temperatures promoted germination of South Carolina and north central Florida seeds. South Florida seed germination was highest under both warm and cool temperatures. More advanced seedling development generally occurred at higher temperatures with the exception of south Florida seedlings, in which the warmest temperature suppressed development. Fluctuating diurnal temperatures were more beneficial for germination compared to constant temperatures. Cold stratification had a positive effect on germination among all populations, but South Carolina seeds required the longest chilling treatments to obtain maximum germination. Results from the cold stratification experiment indicate that a physiological dormancy is present, but the degree of dormancy varies across the species range. The variable responses among populations may indicate ecotypic differentiation.     

Identification,characterization, and expression of the <Emphasis Type="Italic">SWEET</Emphasis> gene family in <Emphasis Type="Italic">Phalaenopsis equestris</Emphasis> and <Emphasis Type="Italic">Dendrobium officinale</Emphasis>

Sugars are important molecules that function not only as primary metabolites, but also as nutrients and signal molecules in plants. The sugar transport protein genes family SWEET has been recently identified. The availability of the Dendrobium officinale and Phalaenopsis equestris genome sequences offered the opportunity to study the SWEET gene family in this two orchid species. We identified 22 and 16 putative SWEET genes, respectively, in the genomes of D. officinale and P. equestris using comprehensive bioinformatics analysis. Based on phylogenetic comparisons with SWEET proteins from Arabidopsis and rice, the DoSWEET and PeSWEET proteins could be divided into four clades; among these, clade II specifically lacked PeSWEETs and clade IV specifically lacked DoSWEETs, and there were orthologs present between D. officinale and P. equestris. Protein sequence alignments suggest that there is a predicted serine phosphorylation site in each of the highly conserved MtN3/saliva domain regions. Gene expression analysis in four tissues showed that three PeSWEET genes were most highly expressed in the flower, leaf, stem, and root, suggesting that these genes might play important roles in growth and development in P. equestris. Analysis of gene expression in different floral organs showed that five PeSWEET genes were highly expressed in the column (gynostemium), implying their possible involvement in reproductive development in this species. The expression patterns of seven PeSWEETs in response to different abiotic stresses showed that three genes were upregulated significantly in response to high temperature and two genes were differently expressed at low temperature. The results of this study lay the foundation for further functional analysis of SWEET genes in orchids.     

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.     

Fruit harvesting time and corresponding morphological changes of seed integuments influence in vitro seed germination of <Emphasis Type="Italic">Dendrobium nobile</Emphasis> Lindl.

In vitro asymbiotic seed germination of Dendrobium nobile varied significantly with fruit harvesting time and growth medium used for culturing seeds. Seeds harvested 129 days after pollination (DAP) possessing globular shaped embryos and a discontinuous cuticle layer showed a substantially greater germination on P668 medium. Alternatively, immature seeds harvested 96 and 116 DAP displayed a significantly lower germination response on various growth media. Most of the ovules at 96 DAP are in archesporial and megaspore mother cell stages, whereas the majority of ovules are mature and fertilized at 116 DAP. Mature seeds harvested 158 DAP also germinated at a higher frequency at Stage 5 (emergence of the first leaf) after 8 weeks of culture on different growth media indicating the absence of testa imposed dormancy in this endangered epiphytic orchid.     

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.     

Antimicrobial Activity and Biodiversity of Endophytic Fungi in <Emphasis Type="Italic">Dendrobium</Emphasis><Emphasis Type="Italic">devonianum</Emphasis> and <Emphasis Type="Italic">Dendrobium thyrsiflorum</Emphasis> from Vietman

Endophytic fungi are rich in orchids and have great impacts on their host plants. 53 endophytes (30 isolates from Dendrobium devonianum and 23 endophytic fungi from D. thyrsiflorum) were isolated, respectively, from roots and stems of Dendrobium species. All the fungi were identified by way of morphological and/or molecular biological methods. 30 endophytic fungi in D. devonianum were categorized into 11 taxa and 23 fungal endophytes in D. thyrsiflorum were grouped into 11 genera, respectively. Fusarium was the dominant species of the two Dendrobium species in common. Antimicrobial activity of ethanol extract of fermentation broth of these fungi was explored using agar diffusion test. 10 endophytic fungi in D. devonianum and 11 in D. thyrsiflorum exhibited antimicrobial activity against at least one pathogenic bacterium or fungus among 6 pathogenic microbes (Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus). Out of the fungal endophytes isolated from D. devonianum and D. thyrsiflorum, Phoma displayed strong inhibitory activity (inhibition zones in diameter >20 mm) against pathogens. Epicoccum nigrum from D. thyrsiflorum exhibited antibacterial activity even stronger than ampicillin sodium. Fusarium isolated from the two Dendrobium species was effective against the pathogenic bacterial as well as fungal pathogens. The study reinforced the assumption that endophytic fungi isolated from different Dendrobium species could be of potential antibacterial or antifungal resource.     

Non-specific symbiotic germination of <Emphasis Type="Italic">Cynorkis purpurea</Emphasis> (Thouars) Kraezl., a habitat-specific terrestrial orchid from the Central Highlands of Madagascar

Orchids, particularly terrestrial taxa, rely mostly on basidiomycete fungi in the Cantharellales and Sebacinales that trigger the process of seed germination and/or initiate the full development of the seedling. During the course of development, orchids may associate with the same fungus, or they may enlist other types of fungi for their developmental needs leading to resilience in a natural setting. This study examined in vitro seed germination and seedling developmental behavior of Cynorkis purpurea, a terrestrial orchid from the Central Highlands of Madagascar. This species is mostly restricted to gallery forests in the Itremo Massif, in moist substrate between rocks bordering streams. The main objective was to understand the influence of diverse mycorrhizal fungi on seed germination and further development of C. purpurea. The study aims to compare symbiotic versus asymbiotic germination and seedling development with seeds and fungi collected from a 13-km² area in the Itremo region. Seeds collected from the wild were sown with diverse orchid mycorrhizal fungi (OMF) spanning 12 operational taxonomic units (OTUs) in three genera (Tulasnella, Ceratobasidium, and Sebacina) acquired from different habitats. Treatments were assessed in terms of the percentage of germinated seeds and fully developed seedlings against those in asymbiotic control media treatments. Overall, OMF significantly improved seedling development within the 12-week experiment period. Sebacina as a genus was the most effective at promoting seedling development of C. purpurea, as well as having the ability to enter into successful symbiotic relationships with orchids of different life forms; this new knowledge may be especially useful for orchid conservation practiced in tropical areas like Madagascar. A Sebacina isolate from an epiphytic seedling of Polystachya concreta was the most effective at inducing rapid seedling development and was among the five that outperformed fungi isolated from roots of C. purpurea. C. purpurea was found to be a mycorrhizal generalist, despite its specific habitat preference, highlighting the complex interaction between the plant, fungi, and the environment. The potential impact on conservation strategies of understanding the requirements for orchid seed germination and development by identifying and using OMF from diverse sources is discussed in detail.     

<Emphasis Type="Italic">Pterostylis nutans</Emphasis> (Orchidaceae) has a specific association with two <Emphasis Type="Italic">Ceratobasidium</Emphasis> root-associated fungi across its range in eastern Australia

In this study, we have identified the root-associated fungi of a common species of terrestrial orchid across its range in eastern Australia. We have amplified and cloned fungal ITS DNA extracted from roots of 15 Pterostylis nutans R. Br. plants from six separate geographic localities. Sequencing and GenBank comparison demonstrated two species of Ceratobasidium fungi as the main fungal partners of the orchid. Uncommon fungal associates included homobasidiomycete species such as a Gymnomyces sp. and a Tricholoma sp., Leptodontidium orchidicola, and an unidentified soil fungus. These results demonstrate that specificity for fungal partners occurs in P. nutans and reinforces the idea that conservation measures for endangered Australian orchids must include ex situ perpetuation of fungal symbionts as well as plant material.     

Evaluation of the effect of three growth regulators in the germination of <Emphasis Type="Italic">comparettia falcata</Emphasis> seeds under <Emphasis Type="Italic">in vitro</Emphasis> conditions

Summary The effect of 3-indoleacetic acid (IAA), 6-furfurylaminopurine (kinetin), and gibberellic acid (GA₃) on germination of the orchid Comparettia falcata was evaluated in a factorial experiment (4×4×4) with Murashige and Skoog (1962) basal medium. It was established that seeds of this orchid could be maintained under aseptic conditions as long as the necessary nutrients and appropriate concentrations of growth regulators were provided. Of the three growth regulators used, IAA significantly decreased seed germination of Comparettia falcata. There was a synergistic effect in the kinetin:GA₃ combination that produced a positive response in both percentage seed germination and development of seedlings. This study describes a single medium-based protocol able to achieve more than 160000 seedlings within 21 wk, starting from a single capsule, sufficient for both large-scale propagation and in vitro conservation of this threatened orchid.     

Symbiotic seed germination and evidence for <Emphasis Type="Italic">in vitro</Emphasis> mycobiont specificity in <Emphasis Type="Italic">Spiranthes brevilabris</Emphasis> (Orchidaceae) and its implications for species-level conservation

Orchid–mycobiont specificity in the Orchidaceae was considered controversial and not well understood for many years. Differences in mycobiont specificity during germination in vitro vs in situ have lead some to consider orchid–mycobiont specificity as being generally low; however, others have suggested that specificity, especially in vitro, is surprisingly high. Mycobiont specificity may be genus or species specific. An in vitro symbiotic seed germination experiment was designed to examine mycobiont specificity of the endangered Florida terrestrial orchid Spiranthes brevilabris using mycobionts isolated from both the study species and the endemic congener Spiranthes floridana. In a screen of mycobionts, isolates Sflo-305 (99.5%), Sflo-306 (99.5%), and Sflo-308 (89.9%) (originating from S. floridana) supported higher initial (stage 1) seed germination than isolate Sbrev-266 (32.4%) (originating from S. brevilabris) after 3 wk culture. However, only isolate Sbrev-266 supported advanced germination and protocorm development to stage 5 (53.1%) after 12 wk culture. These findings suggest that S. brevilabris maintains a high degree of mycobiont specificity under in vitro symbiotic seed germination conditions. High orchid–mycobiont specificity in S. brevilabris may be indicative of the rare status of this orchid in Florida.     

<Emphasis Type="Italic">NnHSP17.5</Emphasis>, a cytosolic class II small heat shock protein gene from <Emphasis Type="Italic">Nelumbo nucifera</Emphasis>, contributes to seed germination vigor and seedling thermotolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

In plants, small heat shock proteins (sHSPs) are unusually abundant and diverse proteins involved in various abiotic stresses, but their functions in seed vigor remain to be fully explored. In this study, we report the isolation and functional characterization of a sHSP gene, NnHSP17.5, from sacred lotus (Nelumbo nucifera Gaertn.) in seed germination vigor and seedling thermotolerance. Sequence alignment and phylogenetic analysis indicate that NnHSP17.5 is a cytosolic class II sHSP, which was further supported by the cytosolic localization of the NnHSP17.5-YFP fusion protein. NnHSP17.5 was specifically expressed in seeds under normal conditions, and was strongly up-regulated in germinating seeds upon heat and oxidative stresses. Transgenic Arabidopsis seeds ectopically expressing NnHSP17.5 displayed enhanced seed germination vigor and exhibited increased superoxide dismutase activity after accelerated aging treatment. In addition, improved basal thermotolerance was also observed in the transgenic seedlings. Taken together, this work highlights the importance of a plant cytosolic class II sHSP both in seed germination vigor and seedling thermotolerance.     

Sympatric reinforcement of reproductive barriers between <Emphasis Type="Italic">Neotinea tridentata</Emphasis> and <Emphasis Type="Italic">N. ustulata</Emphasis> (Orchidaceae)

Reinforcement is the process by which selection favors traits that decrease mating between two incipient species in response to costly mating or the production of maladapted hybrids, causing the evolution of greater reproductive isolation between emerging species. I have studied a pair of orchids, Neotinea tridentata and N. ustulata, to examine the level of postmating pre- and post-zygotic isolating mechanisms that maintain these species, and the degree to which the boundary may still be permeable to gene flow. In this study, I performed pollen tube growth rate experiments and I investigated pre- and post-zygotic barriers by performing hand pollination experiments in order to evaluate fruit set, embryonate seed set and seed germination rates by intra- and interspecific crosses. Fruit set, the percentage of embryonate seeds and germinability of interspecific crosses were reduced compared to intraspecific pollinations, showing significant differences between sympatric and allopatric populations. While in allopatric populations the post-pollination isolation index ranged between 0.40 and 0.11, in sympatric populations orchid pairs showed total isolation due to post-pollination prezygotic barriers, guaranteed at the level of pollen–stigma interactions. Indeed, in sympatric populations, pollen tubes reached the ovary after 24 h in only 8 out of 45 plants; in the remaining cases, the pollen tubes did not enter the ovary, and thus no fruit set occurred. This pair of orchids is characterized by postmating pre-zygotic reproductive isolation in sympatric populations that prevents the formation of hybrids. This mechanism of speciation, starting in allopatry and triggering the reinforcement mechanisms of reproductive isolation in secondary sympatry, is the most likely explanation for the pattern of evolutionary transitions found in this pair of orchids.     

<Emphasis Type="Italic">In vitro</Emphasis> germination and axillary shoot propagation of <Emphasis Type="Italic">Centaurea zeybekii</Emphasis>

In vitro culture is an important aid for ex situ conservation of rare, endemic or threatened plants. In this work, we establish an efficient method for the seed germination, seedling development, and axillary shoot propagation of Centaurea zeybekii Wagenitz. The seeds, collected from a wild population, were surface sterilised and cultured on various in vitro germination media. The effects of photoperiod and temperature on seed germination were also investigated. Germinations were obtained after 6 weeks in culture and the radicle emergence was evaluated as a main indicator. A high frequency of germination was obtained on distilled water supplemented with vitamines and 1 mg/L GA₃. Although the seed germination frequencies were not affected by photoperiod, the highest germination frequency was obtained at 24 ± 2°C. A high frequency of axillary shoot proliferation was produced on MS medium supplemented with 1 mg/L BA. Then, the axillary shoots were separated and transferred to MS medium with or without plant growth regulators for rooting. Rhizogenezis was promoted after 6 weeks only in MS and 1/2 MS media containing 0.5 mg/L IBA. The rooting process was very slow and the percentage of shoot rooting was also very low (15%). The present study not only enables reinforcement of wild plant populations using ex situ growth of individuals, but it also helps to large number of aseptic seedling to use it in clonaly micropropagation studies.     

Diverse non-mycorrhizal fungal endophytes inhabiting an epiphytic,medicinal orchid (<Emphasis Type="Italic">Dendrobium nobile</Emphasis>): estimation and characterization

Although the terrestrial and temperate orchids–fungal biology have been largely explored, knowledge of tropical epiphytic orchids–fungus relationships, especially on the ecological roles imparted by non-mycorrhizal fungal endophytes, is less known. Exploitation of the endophytic fungal mycobiota residing in epiphytic orchid plants may be of great importance to further elucidate the fungal ecology in this special habitat as well as developing new approaches for orchid conversations. The composition of fungal endophytes associated with leaves, stems and roots of an epiphytic orchid (Dendrobium nobile), a famous Chinese traditional medicinal plant, was investigated. Microscopic imaging, culture-dependant method and molecular phylogeny were used to estimate their entity and diversity. Totally, there were 172 isolates, at least 14 fungal genera and 33 different morphospecies recovered from 288 samples. Ascomycetes, coelomycetes and hyphomycetes were three major fungal groups. There were higher overall colonization and isolation rates of endophytic fungi from leaves than from other tissues. Guignardia mangiferae was the dominant fungal species within leaves; while the endophytic Xylariaceae were frequently observed in all plant tissues; Colletotrichum, Phomopsis and Fusarium were also frequently observed. Phylogenetic analysis based on ITS gene revealed the high diversity of Xylariacea fungi and relatively diverse of non-Xylariacea fungi. Some potentially promising beneficial fungi such as Clonostachys rosea and Trichoderma chlorosporum were found in roots. This is the first report concerning above-ground and below-ground endophytic fungi community of an epiphytic medicinal orchid, suggesting the ubiquitous distribution of non-mycorrhizal fungal endophytes in orchid plants together with heterogeneity and tissue specificity of the endophyte assemblage. Possible physiological functions played by these fungal endophytes and their potential applications are also discussed briefly. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Seed size and seed germination in the Mediterranean fire-prone shrub <Emphasis Type="Italic">Cistus </Emphasis><Emphasis Type="Italic">ladanifer</Emphasis>

The germination response of different sized seeds from individuals of a Mediterranean fire-prone shrub (Cistus ladanifer) was investigated in relation to pre-germination heating. A control (no heating), a low temperature during a short exposure time (50°C during 5 min), a high temperature during a short exposure time (100°C during 5 min) and a high temperature during a long exposure time (100°C during 15 min) were applied to seeds from different individual plants with different mean seed weight. These pre-germination treatments resemble natural germination scenarios for the studied species, absence of fire, low intensity pasture fire, typical Mediterranean shrub fire, and severe fire with high fuel load. Mean seed weight only showed a marginally significant positive correlation with the proportion of germinated seeds whatever the pre-germination treatment. These results suggest that seed dormancy is unrelated to seed size and that under the experimental conditions used in this study, the effect of seed size on seed germination is low. Nevertheless, larger seeds could be favoured in natural conditions, especially under the high competition scenario which arise after wildfires. Control seeds showed a negative correlation between seed size and germination velocity suggesting that lighter seeds could take advantage from early germination in recruitment events in the absence of wildfires. Nevertheless, even the lower pre-germination heating treatment turns this correlation in not significant, suggesting a strong selection pressure (unrelated to seed size) for early germination after fire events. In our study, different sized seeds of C. ladanifer seem to perform better under different germination scenarios suggesting that seed size variation could be maintained by the alternation of recruitments without wildfires and recruitments after wildfire events.