Symbiosis between Dendrobium catenatum protocorms and Serendipita indica involves the plant hypoxia response pathway.

Mycorrhizae are ubiquitous symbioses established between fungi and plant roots. Orchids, in particular, require compatible mycorrhizal fungi for seed germination and protocorm development. Unlike arbuscular mycorrhizal fungi, which have wide host ranges, orchid mycorrhizal fungi are often highly specific to their host orchids. However, the molecular mechanism of orchid mycorrhizal symbiosis is largely unknown compared to that of arbuscular mycorrhizal and rhizobial symbiosis. Here, we report that an endophytic Sebacinales fungus, Serendipita indica, promotes seed germination and the development of protocorms into plantlets in several epiphytic Epidendroideae orchid species (6 species in 2 genera), including Dendrobium catenatum, a critically endangered orchid with high medicinal value. Although plant-pathogen interaction and high meristematic activity can induce the hypoxic response in plants, it has been unclear whether interactions with beneficial fungi, especially mycorrhizal ones, also involve the hypoxic response. By studying the symbiotic relationship between D. catenatum and S. indica, we determined that hypoxia-responsive genes, such as those encoding alcohol dehydrogenase (ADH), are highly induced in symbiotic D. catenatum protocorms. In situ hybridization assay indicated that the ADH gene is predominantly expressed in the basal mycorrhizal region of symbiotic protocorms. Additionally, the ADH inhibitors puerarin and 4-methylpyrazole both decreased S. indica colonization in D. catenatum protocorms. Thus, our study reveals that S. indica is widely compatible with orchids and that ADH and its related hypoxia-responsive pathway are involved in establishing successful symbiotic relationships in germinating orchids.

What role does the seed coat play during symbiotic seed germination in orchids: an experimental approach with Dendrobium officinale.

BACKGROUND: Orchids require specific mycorrhizal associations for seed germination. During symbiotic germination, the seed coat is the first point of fungal attachment, and whether the seed coat plays a role in the identification of compatible and incompatible fungi is unclear. Here, we compared the effects of compatible and incompatible fungi on seed germination, protocorm formation, seedling development, and colonization patterns in Dendrobium officinale; additionally, two experimental approaches, seeds pretreated with NaClO to change the permeability of the seed coat and fungi incubated with in vitro-produced protocorms, were used to assess the role of seed coat played during symbiotic seed germination. RESULTS: The two compatible fungi, Tulasnella sp. TPYD-2 and Serendipita indica PI could quickly promote D. officinale seed germination to the seedling stage. Sixty-two days after incubation, 67.8 ± 5.23% of seeds developed into seedlings with two leaves in the PI treatment, which was significantly higher than that in the TPYD-2 treatment (37.1 ± 3.55%), and massive pelotons formed inside the basal cells of the protocorm or seedlings in both compatible fungi treatments. In contrast, the incompatible fungus Tulasnella sp. FDd1 did not promote seed germination up to seedlings at 62 days after incubation, and only a few pelotons were occasionally observed inside the protocorms. NaClO seed pretreatment improved seed germination under all three fungal treatments but did not improve seed colonization or promote seedling formation by incompatible fungi. Without the seed coat barrier, the colonization of in vitro-produced protocorms by TPYD-2 and PI was slowed, postponing protocorm development and seedling formation compared to those in intact seeds incubated with the same fungi. Moreover, the incompatible fungus FDd1 was still unable to colonize in vitro-produced protocorms and promote seedling formation. CONCLUSIONS: Compatible fungi could quickly promote seed germination up to the seedling stage accompanied by hyphal colonization of seeds and formation of many pelotons inside cells, while incompatible fungi could not continuously colonize seeds and form enough protocorms to support D. officinale seedling development. The improvement of seed germination by seed pretreatment may result from improving the seed coat hydrophilicity and permeability, but seed pretreatment cannot change the compatibility of a fungus with an orchid. Without a seed coat, the incompatible fungus FDd1 still cannot colonize in vitro-produced protocorms or support seedling development. These results suggest that seed coats are not involved in symbiotic germination in D. officinale.

The Waiting Room Hypothesis revisited by orchids: were orchid mycorrhizal fungi recruited among root endophytes?

BACKGROUND: As in most land plants, the roots of orchids (Orchidaceae) associate with soil fungi. Recent studies have highlighted the diversity of the fungal partners involved, mostly within Basidiomycotas. The association with a polyphyletic group of fungi collectively called rhizoctonias (Ceratobasidiaceae, Tulasnellaceae and Serendipitaceae) is the most frequent. Yet, several orchid species target other fungal taxa that differ from rhizoctonias by their phylogenetic position and/or ecological traits related to their nutrition out of the orchid roots (e.g. soil saprobic or ectomycorrhizal fungi). We offer an evolutionary framework for these symbiotic associations. SCOPE: Our view is based on the 'Waiting Room Hypothesis', an evolutionary scenario stating that mycorrhizal fungi of land flora were recruited from ancestors that initially colonized roots as endophytes. Endophytes biotrophically colonize tissues in a diffuse way, contrasting with mycorrhizae by the absence of morphological differentiation and of contribution to the plant's nutrition. The association with rhizoctonias is probably the ancestral symbiosis that persists in most extant orchids, while during orchid evolution numerous secondary transitions occurred to other fungal taxa. We suggest that both the rhizoctonia partners and the secondarily acquired ones are from fungal taxa that have broad endophytic ability, as exemplified in non-orchid roots. We review evidence that endophytism in non-orchid plants is the current ecology of many rhizoctonias, which suggests that their ancestors may have been endophytic in orchid ancestors. This also applies to the non-rhizoctonia fungi that were secondarily recruited by several orchid lineages as mycorrhizal partners. Indeed, from our review of the published literature, they are often detected, probably as endophytes, in extant rhizoctonia-associated orchids. CONCLUSION: The orchid family offers one of the best documented examples of the 'Waiting Room Hypothesis': their mycorrhizal symbioses support the idea that extant mycorrhizal fungi have been recruited among endophytic fungi that colonized orchid ancestors.

Low Specificity but Dissimilar Mycorrhizal Communities Associating with Roots May Contribute to the Spatial Pattern of Four Co-Occurring Habenaria (Orchidaceae) Species.

Fungi with orchid roots have been increasingly proven to play important roles in orchid growth, spatial distribution, and coexistence of natural communities. Here, we used 454 amplicon pyrosequencing with two different primer combinations to investigate the spatial variations in the community of OMF and endophytic fungi associates within the roots of four co-occurring Habenaria species. The results showed that all investigated Habenaria species were generalists and the different fungi communities may contribute to the spatial separation of the four Habenaria species. Firstly, the fungal OTUs identified in the roots of the four species overlapped but their presence differed amongst species and numerous distinct OMF families were unique to each species. Second, NMDS clustering showed samples clustered together based on associated species and PERMANOVA analyses indicated that fungi communities in the roots differed significantly between the Habenaria species, both for all endophytic fungi communities and for OMF communities. Third, the network structure of epiphytic fungi was highly specialized and modular but demonstrated lowly connected and anti-nested properties. However, it calls for more soil nutrition and soil fungal communities' studies to elucidate the contribution of habitat-specific adaptations in general and mycorrhizal divergence.

A comparative study on the reproductive success of two rewarding Habenaria species (Orchidaceae) occurring in roadside verge habitats.

BACKGROUND: Most orchid species have been shown to be severely pollination limited, and the factors affecting reproductive success have been widely studied. However, the factors determining the reproductive success vary from species to species. Habenaria species typically produce nectar but exhibit variable fruit set and reproductive success among species. Here, we investigated the influence of the flowering plant density, inflorescence size, breeding system, and pollinator behaviour on the reproductive success of two rewarding Habenaria species. RESULTS: Our observations indicated that Habenaria limprichtii and H. petelotii co-occur in roadside verge habitats and present overlapping flowering periods. Both species were pollination limited, although H. limprichtii produced more fruits than H. petelotii under natural conditions during the 3-year investigation. H. petelotii individuals formed distinct patches along roadsides, while nearly all H. limprichtii individuals clustered together. The bigger floral display and higher nectar sugar concentration in H. limprichtii resulted in increased attraction and visits from pollinators. Three species of effective moths pollinated for H. limprichtii, while Thinopteryx delectans (Geometridae) was the exclusive pollinator of H. petelotii. The percentage of viable seeds was significantly lower for hand geitonogamy than for hand cross-pollination in both species. However, H. limprichtii may often be geitonogamously pollinated based on the behaviours of the pollinators and viable embryo assessment. CONCLUSIONS: In anthropogenic interference habitats, the behaviours and abundance of pollinators influence the fruit set of the two studied species. The different pollinator assemblages in H. limprichtii can alleviate pollinator specificity and ensure reproductive success, whereas the more viable embryos of natural fruit seeds in H. petelotii suggested reducing geitonogamy by pollinators in the field. Our results indicate that a quantity-quality trade-off must occur between species with different breeding strategies so that they can fully exploit the existing given resources.

Transitions between the Terrestrial and Epiphytic Habit Drove the Evolution of Seed-Aerodynamic Traits in Orchids.

Orchids are globally distributed, a feature often attributed to their tiny dustlike seeds. They were ancestrally terrestrial but in the Eocene expanded into tree canopies, with some lineages later returning to the ground, providing an evolutionarily replicated system. Because seeds are released closer to the ground in terrestrial species than in epiphytic ones, seed traits in terrestrials may have been under selective pressure to increase seed dispersal efficiency. In this study, we test the expectations that seed airspace-a trait known to increase seed flotation time in the air-is (i) larger in terrestrial lineages and (ii) has increased following secondary returns to a terrestrial habit. We quantified and scored 20 seed traits in 121 species and carried out phylogenetically informed analyses. Results strongly support both expectations, suggesting that aerodynamic traits even in dust seeds are under selection to increase dispersal ability, following shifts in average release heights correlated with changes in habit.

Are fungi from adult orchid roots the best symbionts at germination? A case study.

We studied mycobionts from advanced seedlings and adult mycorrhizal roots of the terrestrial orchid Arundina graminifolia. Fungi were isolated, identified by ITS sequencing, and tested for their impact on seed germination, protocorm formation, and development of advanced seedlings (emergence of first leaf) in vitro. Among the six fungal species isolated, four were not standard orchid mycorrhizal fungi (Fusarium solani, Cylindrocarpon sp., Acremonium sp., and Phlebiopsis flavidoalba) and did not support germination beyond imbibition and greening of the seeds during a span of 35 days. Over the same time, one Tulasnella species isolated from adult mycorrhiza allowed protocorm formation but not further development. However, another Tulasnella species isolated from advanced seedlings facilitated development to the advanced seedling stage. Our results support (i) the inability of occasional orchid root colonizers to support late seed germination, and (ii) the growing literature showing that fungal associates can change over orchid development. Functionally, we show that mycorrhizal taxa isolated from advanced seedlings can be more efficient than those from adults in supporting germination in some species, leading to recommendations for ex situ orchid conservation.

[Effects of different endophytic fungi on seedling growth of Dendrobium devonianum].

To obtain seedling growth-promoting fungi is a key step in restoration-friendly cultivation of medicinal Dendrobium species, since there are a large number of functionally-unknown endophytic fungi in the roots of Dendrobium plants.In this study, six functionally-unknown endophytic fungal strains were isolated from roots of D.devonianum using single peleton isolation technology, and used in inoculation experiments to test their effectiveness for seedling growth in D.devonianum.After 90 days of inoculation, comparing with the control treatment, FDdS-1, FDdS-2 and FDdS-4 showed strong pathogenic or fatal effects on seedlings; while, FDdS-12, FDdS-9 and FDdS-5 had different effects on seedling growth.FDdS-5 had significant promoting effects on height, fresh and dry weight, stem diameter and root numbers, while FDdS-9 only had significant promoting effect on seedling height, and FDdS-12 had a negative effect on seedling growth.According to the anatomical features of the inoculated roots, FDdS-5 fungi could infect the velamina of seedlings and the existence of symbiosis pelotons in the cortex cells, suggesting that FDdS-5 is a mycorrhiza fungi of D.devonianum.FDdS-5 and FDdS-9 were identified as Sebacina vermifera and Sebacina sp.by molecular technologies.By using FDdS-5 in the restoration-friendly cultivation of D.devonianum, it could effectively promote seedling growth and shorten the seedling growth periods.The results will aid in reintroduction and cultivation of D.devonianum.

Translocation of threatened plants as a conservation measure in China.

We assessed the current status of plant conservation translocation efforts in China, a topic poorly reported in recent scientific literature. We identified 222 conservation translocation cases involving 154 species, of these 87 were Chinese endemic species and 101 (78%) were listed as threatened on the Chinese Species Red List. We categorized the life form of each species and, when possible, determined for each case the translocation type, propagule source, propagule type, and survival and reproductive parameters. A surprisingly large proportion (26%) of the conservation translocations in China were conservation introductions, largely implemented in response to large-scale habitat destruction caused by the Three-Gorge Dam and another hydropower project. Documentation and management of the translocations varied greatly. Less than half the cases had plant survival records. Statistical analyses showed that survival percentages were significantly correlated with plant life form and the type of planting materials. Thirty percent of the cases had records on whether or not individuals flowered or fruited. Results of information theoretic model selection indicated that plant life form, translocation type, propagule type, propagule source, and time since planting significantly influenced the likelihood of flowering and fruiting on the project level. We suggest that the scientific-based application of species conservation translocations should be promoted as part of a commitment to species recovery management. In addition, we recommend that the common practice of within and out of range introductions in nature reserves to be regulated more carefully due to its potential ecological risks. We recommend the establishment of a national office and database to coordinate conservation translocations in China. Our review effort is timely considering the need for a comprehensive national guideline for the newly announced nation-wide conservation program on species with extremely small populations, which is expected to stimulate conservation translocations for many species in the near future.

Orchid conservation in the biodiversity hotspot of southwestern China.

Xishuangbanna is on the northern margins of tropical Asia in southwestern China and has the largest area of tropical forest remaining in the country. It is in the Indo-Burma hotspot and contains 16% of China's vascular flora in <0.2% of the country's total area (19,690 km(2) ). Rapid expansion of monoculture crops in the last 20 years, particularly rubber, threatens this region's exceptional biodiversity. To understand the effects of land-use change and collection on orchid species diversity and determine protection priorities, we conducted systematic field surveys, observed markets, interviewed orchid collectors, and then determined the conservation status of all orchids. We identified 426 orchid species in 115 genera in Xishuangbanna: 31% of all orchid species that occur in China. Species richness was highest at 1000-1200 m elevation. Three orchid species were assessed as possibly extinct in the wild, 15 as critically endangered, 82 as endangered, 124 as vulnerable, 186 as least concern, and 16 as data deficient. Declines over 20 years in harvested species suggested over-collection was the major threat, and utility value (i.e., medicinal or ornamental value) was significantly related to endangerment. Expansion of rubber tree plantations was less of a threat to orchids than to other taxa because only 75 orchid species (17.6%) occurred below the 1000-m-elevation ceiling for rubber cultivation, and most of these (46) occurred in nature reserves. However, climate change is projected to lift this ceiling to around 1300 m by 2050, and the limited area at higher elevations reduces the potential for upslope range expansion. The Xishuangbanna Tropical Botanical Garden is committed to achieving zero plant extinctions in Xishuangbanna, and orchids are a high priority. Appropriate in and ex situ conservation strategies, including new protected areas and seed banking, have been developed for every threatened orchid species and are being implemented.

In situ seed baiting to isolate germination-enhancing fungi for an epiphytic orchid, Dendrobium aphyllum (Orchidaceae).

Orchid conservation efforts, using seeds and species-specific fungi that support seed germination, require the isolation, identification, and germination enhancement testing of symbiotic fungi. However, few studies have focused on developing such techniques for the epiphytes that constitute the majority of orchids. In this study, conducted in Xishuangbanna Tropical Botanical Garden, Yunnan, China, we used seeds of Dendrobium aphyllum, a locally endangered and medicinally valuable epiphytic orchid, to attract germination promoting fungi. Of the two fungi isolated from seed baiting, Tulasnella spp. and Trichoderma spp., Tulasnella, enhanced seed germination by 13.6 %, protocorm formation by 85.7 %, and seedling development by 45.2 % (all P < 0.0001). Epulorhiza, another seed germination promoting fungi isolated from Cymbidium mannii, also enhanced seed germination (6.5 %; P < 0.05) and protocorm formation (20.3 %; P < 0.0001), but Trichoderma suppressed seed germination by 26.4 % (P < 0.0001). Tulasnella was the only treatment that produced seedlings. Light increased seed imbibition, protocorm formation, and two-leaved seed development of Tulasnella inoculated seeds (P < 0.0001). Because the germination stage success was not dependent on fungi, we recommend that Tulasnella be introduced for facilitating D. aphyllum seed germination at the protocorm formation stage and that light be provided for increasing germination as well as further seedling development. Our findings suggest that in situ seed baiting can be used to isolate seed germination-enhancing fungi for the development of seedling production for conservation and reintroduction efforts of epiphytic orchids such as D. aphyllum.

[Effects of different fungi on symbiotic seed germination of two Dendrobium species].

The epiphytic orchid, Dendrobium aphyllum and D. devonianum are used as traditional Chinese medicine, and became locally endangered in recent years because of over-collection. We test the effect of inoculations of endophytic fungi FDaI7 (Tulasnella sp.), FDd1 (Epulorhiza sp. ) and FCb4 (Epulorhiza sp.), which isolated from D. aphyllum, D. denonianum and Cymbidium mannii, respectively, on artificial substrate in these two Dendrobium species. In the symbiotic germination experiment, FDaI7 and FDd1 were effective for protocorm formation and seedling development of D. aphyllum and D. denonianum separately. After 60 days, 14.46% of the D. aphyllum seeds grown to protocorms and 12.07% developed to seedlings inoculated only with FDaI7, while contrasted with 0 when inoculated the other two isolates and non-inoculation treatment. However, in D. denonianum, seeds only grown to protocorms and developed to seedlings when inoculated with FDd1, the percentages were 44.36% and 42.91% distinguishingly. High specificity was shown in symbiotic germination on artificial substrate of Dendrobium. Protocorms could further develop to seedlings within or without light when inoculated the compatible fungi. However, light condition (12/12 h Light/Dark) produced the normal seedlings, while dark condition (0/24 h L/D) produced the abnormal seedlings. These may suggest that the development of young seedlings require light based on the effective symbiotic fungi. These findings will aid in seedling production of simulation-forestry ecology cultivation, conservation and reintroduction of Dendrobium.

Floral mechanisms promote pollination success and reduce the incidence of self-pollination in a fly-pollinated self-incompatible orchid.

Among flowering plants, self-incompatibility is considered the most efficient system for avoiding self-fertilization. However, many self-incompatible plants have also evolved floral mechanisms to reduce sexual conflict. In China, some studies of Bulbophyllum have been reported to be self-incompatible and no fruit sets. However, we have observed relatively high fruit sets in Bulbophyllum funingense. Therefore, we speculated that if B. funingense is also self-incompatible, and it might present a floral mechanism to avoid sexual conflict. Natural fruit sets, pollinia removal and deposition rates were determined and breeding system was tested in a hand-pollination experiment. The pollination process and visiting frequency of pollinators and their behavior after escape from access were observed and recorded. Floral traits associated with pollination and pollinator size were measured. B. funingense was completely self-incompatible, the fruit sets of cross-pollination in 2 years were all more than 70%, and the natural fruit sets for 2 years were 1.70 ± 4.31% and 6.63 ± 5.29%, respectively. B. funingense did not produce strong odor or nectar, but produced a kind of secretions from its labellum that attracted flies. Calliphora vicina (Calliphoridae) was its only effective pollinator. When C. vicina licked the secretions, they were stuck in the access for a long time. Thus, when they escaped from access, they almost always flew quickly away from the inflorescence removing pollinia most of the times. In B. funingense, a floral mechanism improves pollinia transfer efficiency, reduces pollinia waste, promotes pollination success, reduces the incidence of self-pollination, and avoids sexual conflict to a certain extent.

Attribution of dispersal limitation can better explain the assembly patterns of plant microbiota.

Disentangling community assembly processes is crucial for fully understanding the function of microbiota in agricultural ecosystems. However, numerous plant microbiome surveys have gradually revealed that stochastic processes dominate the assembly of the endophytic root microbiota in conflict with strong host filtering effects, which is an important issue. Resolving such conflicts or inconsistencies will not only help accurately predict the composition and structure of the root endophytic microbiota and its driving mechanisms, but also provide important guidance on the correlation between the relative importance of deterministic and stochastic processes in the assembly of the root endophytic microbiota, and crop productivity and nutritional quality. Here, we propose that the inappropriate division of dispersal limitation may be the main reason for such inconsistency, which can be resolved after the proportion of dispersal limitation is incorporated into the deterministic processes. The rationality of this adjustment under the framework of the formation of a holobiont between the microbiome and the plant host is herein explained, and a potential theoretical framework for dynamic assembly patterns of endophytic microbiota along the soil-plant continuum is proposed. Considering that the assembly of root endophytic microbiota is complicated, we suggest caution and level-by-level verification from deterministic processes to neutral components to stochastic processes when deciding on the attribution of dispersal limitation in the future to promote the expansion and application of microbiome engineering in sustainable agricultural development based on community assembly patterns.

A novel method to produce massive seedlings via symbiotic seed germination in orchids.

Orchids produce large numbers of dust-like seeds that rely heavily on orchid mycorrhizal fungi (OMFs) for germination. Using OMFs to facilitate orchid proliferation is considered an effective method for orchid conservation but still presents challenges in practice. In this study, orchid seed-fungus complexes, in which orchid seeds and fungal mycelia were embedded together to form granules, were developed as platforms to facilitate seed germination and seedling production. Overall, seedlings were produced by seed-fungus complexes for five orchid species with large variations in the percentages of seedlings produced among species/treatments. For the different fungal treatments in Dendrobium officinale, Sebacinales LQ performed much better than the other fungal strains. At 90 days after sowing, 75.8±2.6% seedlings were produced in the LQ treatment, which was significantly higher than in the Tulasnella sp. JM (22.0±3.0%) and Tulasnella sp. TPYD-2 (5.3±1.0%) treatments, as well as in the LQ and TPYD-2 cocultured treatment (40.4±3.2%), while no seedlings were formed in the Tulasnella sp. SSCDO-5 or control treatments. For the other four orchid species, only one compatible fungus for each species was used, and the percentages of seedlings in epiphytic Dendrobium devonianum (67.2±2.9%) and D. nobile (38.9±2.8%) were much higher than those in terrestrial Paphiopedilum spicerianum (2.9±1.1%) and Arundina graminifolia (6.7±2.1%) at 90 days after sowing. Adding 1% polymer water-absorbent resin to the seed-fungus complexes of D. officinale seeds with fungal strain Sebacinales LQ significantly increased seedling formation, while other additional substances showed negative effects on seedling formation. For the storage of seed-fungus complexes, it is recommended to store the seed-fungus complexes in valve bags at room temperature for a short time and at a low temperature of 4°C for no more than 30 days. As a platform for symbiotic seed germination, the seed-fungus complex can facilitate seed germination, produce seedlings and support subsequent seedling growth, and its seedling productivity depends on seed germination characteristics, seed viability, and the efficiency of fungi. Seed-fungus complexes have great potential to be used as propagules in orchid conservation.

Compartments of roots and mature leaves are key hubs in the connectivity of tea-plant mycobiomes and are influenced by environmental factors and host age.

Understanding the tripartite consortium of crop, mycobiome, and environment is necessary to advance smart farming. Owing to their life cycle of hundreds of years, tea plants are excellent models for studying these entwined relationships; however, observations on this globally important cash crop with numerous health benefits are still rudimentary. Here, the fungal taxa along the soil-tea plant continuum in tea gardens of different ages in famous high-quality tea-growing regions in China were characterized using DNA metabarcoding. Using machine learning, we dissected the spatiotemporal distribution, co-occurrence patterns, assembly, and their associations in different compartments of tea-plant mycobiomes, and further explored how these potential interactions were driven by environmental factors and tree age, and how they influenced the market prices of tea. The results revealed that Compartment niche differentiation was the key driving force behind variation in the tea-plant mycobiome. The mycobiome of roots had the highest specific proportion and convergence and almost did not overlap with the soil. The enrichment ratio of developing leaves to root mycobiome increased with increasing tree age, while mature leaves showed the highest value in the Laobanzhang (LBZ) tea garden with top market prices and displayed the strongest depletion effect on mycobiome association along the soil-tea plant continuum. The balance between determinism and stochasticity in the assembly process was co-driven by compartment niches and life cycle variation. Fungal guild analysis showed that altitude indirectly affected market prices of tea by mediating the abundance of the plant pathogen. The relative importance of plant pathogen and ectomycorrhizae could be used to assess the age of tea. Biomarkers were mainly distributed in soil compartments, and Clavulinopsis miyabeana, Mortierella longata, and Saitozyma sp. may affect the spatiotemporal dynamics of tea-plant mycobiomes and their ecosystem services. Soil properties (mainly total potassium) and tree age indirectly affected the developing leaves via positively influencing the mycobiome of mature leaves. In contrast, the climate directly and significantly drove the mycobiome composition of the developing leaves. Moreover, the proportion of negative correlations in the co-occurrence network positively regulated tea-plant mycobiome assembly, which significantly affected the market prices of tea in the structural equation model with network complexity as hub. These findings indicate that mycobiome signatures play pivotal roles in the adaptive evolution and fungal disease control of tea plants and can help develop better agricultural practices that focus on both plant health and financial profits, and provide a new strategy for assessing tea quality grade and age.

Rain pollination provides reproductive assurance in a deceptive orchid.

BACKGROUND AND AIMS: Abiotic pollination by wind or water is well established in flowering plants. In some species pollination by rain splashes, a condition known as ombrophily, has been proposed as a floral strategy. However, evidence for this type of abiotic pollination has remained controversial and many reported cases have subsequently been shown to be false. This study investigates ombrophily in the deceptive orchid Acampe rigida to determine the mechanism by which this species is able to maintain high fecundity, despite flowering during the rainy season in south-west China when pollinators are scarce. METHODS: The floral mechanisms promoting rain pollination in A. rigida were observed and described in detail. Controlled pollination experiments and observations of floral visitors were conducted. A field experiment using rain shelters at 14 sites in Guangxi, south-west China, evaluated the contribution of rain pollination to fruit-set. KEY RESULTS: During rainfall, raindrops physically flicked away the anther cap exposing the pollinarium. Raindrops then caused pollinia to be ejected upwards with the strap-like stipe pulling them back and causing them to fall into the stigmatic cavity, resulting in self-pollination. Neither flower nor pollen function were damaged by water. Although A. rigida is self-compatible, it is incapable of autonomous self-pollination without the assistance of rain splashes. The results of the rain-sheltering experiment indicated that rain pollination contributed substantially to increasing fruit-set, although there was variation among sites in the intensity of this effect. CONCLUSIONS: A. rigida flowers during the rainy season, when pollinators are scarce, and ombrophily functions to provide reproductive assurance without compromising opportunities for outcrossing.

Microsatellite markers for Duperrea pavettifolia (Rubiaceae).

PREMISE OF THE STUDY: The development of microsatellite primers for Duperrea pavettifolia will be the foundation for mating system analysis and conservation research. METHODS AND RESULTS: Nineteen microsatellite markers were developed and characterized in two wild populations by using the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol. Polymorphisms were evaluated in 24 individuals from two natural populations. Eleven of these primers generated polymorphic loci. CONCLUSIONS: These microsatellite markers will be useful in future investigations into the population genetics and mating system of D. pavettifolia.

Compatible and Incompatible Mycorrhizal Fungi With Seeds of Dendrobium Species: The Colonization Process and Effects of Coculture on Germination and Seedling Development.

Orchids highly rely on mycorrhizal fungi for seed germination, and compatible fungi could effectively promote germination up to seedlings, while incompatible fungi may stimulate germination but do not support subsequent seedling development. In this study, we compared the fungal colonization process among two compatible and two incompatible fungi during seed germination of Dendrobium officinale. The two compatible fungi, i.e., Tulasnella SSCDO-5 and Sebacinales LQ, originally from different habitats, could persistently colonize seeds and form a large number of pelotons continuously in the basal cells, and both fungi promoted seed germination up to seedling with relative effectiveness. In contrast, the two incompatible fungi, i.e., Tulasnella FDd1 and Tulasnella AgP-1, could not persistently colonize seeds. No pelotons in the FDd1 treatment and only a few pelotons in the AgP-1 treatment were observed; moreover, no seedlings were developed at 120 days after incubation in either incompatible fungal treatment. The pattern of fungal hyphae colonizing seeds was well-matched with the morphological differentiation of seed germination and seedling development. In the fungal cocultural experiments, for both orchids of D. officinale and Dendrobium devonianum, cocultures had slightly negative effects on seed germination, protocorm formation, and seedling formation compared with the monocultures with compatible fungus. These results provide us with a better understanding of orchid mycorrhizal interactions; therefore, for orchid conservation based on symbiotic seed germination, it is recommended that a single, compatible, and ecological/habitat-specific fungus can be utilized for seed germination.

Seed viability testing for research and conservation of epiphytic and terrestrial orchids.

BACKGROUND: Seed viability testing is essential in plant conservation and research. Seed viability testing determines the success of ex-situ conservation efforts, such as seed banking but commonly testing protocols of orchids lack consistency and accuracy, therefore, there is a need to select an appropriate and reliable viability test, especially when conducting comparative studies. Here, we evaluated the suitability of three seed viability tests, Evans blue test (EB), Fluorescein diacetate test (FDA) and Tetrazolium test (TTC), with and without sterilization, on seeds of 20 orchid species, which included five epiphytes and fifteen terrestrials, using both fresh seeds and seeds stored at - 18 ºC for 6 to 8 years. RESULTS: We found that sterilization and lifeform of seeds affected seed viability across all tests but the storage time was not an influential factor. Sterilization negatively affected seed viability under EB and FDA test conditions but increased the detection of viable seeds in the TTC test in both epiphytic and terrestrial species. The EB test, when administered without sterilization provided the highest viability results. Being non-enzymatic unlike TTC and FDA tests, as expected, the EB test was the most reliable with similar results between sterilized and not sterilized seeds for most epiphytic and terrestrial species as well as when compared between groups. CONCLUSIONS: The lifeform of the species and seed sterilization prior to testing are important influential factors in orchid seed viability testing. Since EB test was found to be reliable we recommend the EB test for seed viability assessment in orchids rather than the less reliable but commonly used TTC test, or the FDA test, which require more expensive and sophisticated instrumentation. Since storage time was not an influential factor in orchid seed viability testing, the recommendations of this study can be used for both fresh as well as long-term stored orchid seeds. This is helpful for research and especially for conservation measures such as seed banking. However, due to the species specificity of the bio-physiology of orchids, we call for comprehensive viability test assessment in the hyper diverse orchid family to be extended to a greater number of species to facilitate efficient conservation and research.