Synthetic Tetraploid of Oncidium crispum Lodd. (Orchidaceae).

In ornamental plants, artificial polyploidization has enabled the creation of new cultivars. Due to their high commercial value in the international flower market and their ornamental characteristics, such as the shape, size, color, and durability of their flower, orchids have received great attention in studies of artificial polyploidization. Here we described the protocol used for polyploid induction in Oncidium crispum, an epiphyte species native of southeastern Brazil, of great ornamental interest and widely sold in flower shops. The species stands out for having inflorescence with large flowers, brown in color with yellow spots. In addition, O. crispum has great potential for use in genetic improvement programs since the species is widely used in interspecific crosses. Closed capsules containing mature O. crispum seeds were subjected to running sterilized water for 10 min and then to a 1.5% sodium hypochlorite solution for 10 min. Small portions of seeds were introduced into 50 mL of water-soluble fertilizer with macro- and micronutrients (B>M) plus 0.7% agar. Explants originating from seeds previously in vitro germinated were submitted to 0.05% and 0.1% of colchicine for 4 days and 8 days. Flow cytometry and chromosome counts confirmed that the protocol successfully produced synthetic polyploid plants.

PaWOX3 and PaWOX3B Regulate Flower Number and The Lip Symmetry of Phalaenopsis.

The standout characteristic of the orchid perianth is the transformation of the upper median petal into a distinctively formed lip, which gives orchid flowers their typically zygomorphic symmetry and makes them the most popular ornamental plants worldwide. To study orchid flower development, two WUSCHEL-related homeobox (WOX) genes, PaWOX3 and PaWOX3B, were identified in Phalaenopsis. PaWOX3 and PaWOX3B mRNAs accumulate abundantly during early reproductive development and perianths of young buds, significantly decrease in mature flower and absent in vegetative leaves and roots. PaWOX3 and PaWOX3B virus-induced gene silencing (VIGS) knockdown in Phalaenopsis significantly reduces floral bud numbers, suggesting that PaWOX3/PaWOX3B may be involved in flower initiation. Transgenic Arabidopsis ectopically expressing repressor forms of PaWOX3/PaWOX3B and their Oncidium orthologue, OnPRS, exhibit lateral organ development defects, implicating these genes likely have function in regulating growth and differentiation for lateral organs. Neither PaWOX3, PaWOX3B single nor PaWOX3/PaWOX3B double VIGS Phalaenopsis altered the flower morphology. Interestingly, double silencing of PaWOX3 or PaWOX3B with OAGL6-2, which controlled the identity/formation of lips, altered the symmetry of 'BigLip' produced in OAGL6-2 VIGS. This result indicated that the levels of PaWOX3/PaWOX3B are still sufficient to maintain the symmetry for the OAGL6-2 VIGS 'BigLip'. However, the symmetry of the OAGL6-2 VIGS 'BigLip' can not be maintained once the expression of PaWOX3 or PaWOX3B is further reduced. Thus, in addition to control lip identity, this study further found that OAGL6-2 could cooperate with functionally redundant PaWOX3/PaWOX3B in maintaining the symmetric axis of lip.

Evidence of introduced honeybees (Apis mellifera) as pollen wasters in orchid pollination.

Biological invasions threaten global biodiversity, altering landscapes, ecosystems, and mutualistic relationships like pollination. Orchids are one of the most threatened plant families, yet the impact of invasive bees on their reproduction remains poorly understood. We conduct a global literature survey on the incidence of invasive honeybees (Apis mellifera) on orchid pollination, followed by a study case on Australian orchids. Our literature survey shows that Apis mellifera is the primary alien bee visiting orchids worldwide. However, in most cases, introduced honeybees do not deposit orchid pollen. We also test the extent to which introduced honeybees affect orchid pollination using Diuris brumalis and D. magnifica. Diuris brumalis shows higher fruit set and pollination in habitats with both native and invasive bees compared to habitats with only introduced bees. Male and female reproductive success in D. magnifica increases with native bee abundance, while conversely pollinator efficiency decreases with honeybee abundance and rises with habitat size. Our results suggest that introduced honeybees are likely involved in pollen removal but do not effectively deposit orchid pollen, acting as pollen wasters. However, Apis mellifera may still contribute to pollination of Diuris where native bees no longer exist. Given the global occurrence of introduced honeybees, we warn that certain orchids may suffer from pollen depletion by these invaders, especially in altered habitats with compromised pollination communities.

Gymnadenia winkeliana-A New Orchid Species to Romanian Flora.

A novel species, Gymnadenia winkeliana, has been identified in the Bucegi Natural Park ROSCI0013, located in the Southern Carpathians of Central Romania. Two moderately sized populations of Gymnadenia winkeliana, totalling 120-140 individuals, were discovered inhabiting the alpine grasslands of the park, situated 2.000 m above sea level. To describe this newly found population as comprehensively as possible, 44 vegetative and floral organs/organ parts were directly studied and measured from living plants. Special attention was focused on the characteristics that proved to have taxonomic significance, particularly those involving distinctive details in the morphology of the leaves, perianth, labellum and gynostemium. A total of 223 characteristics were analysed encompassing the morphology of every organ of the plant, cytology and breeding system. Furthermore, comprehensive taxonomic treatment and description, accompanied by colour photographs illustrating the holotype, are provided. Voucher specimens were deposited at the Herbarium of the University of Agriculture and Veterinary Medicine, Bucharest (USAMVB Herbarium barcode: 40102, NEA); Gymnadenia winkeliana, a (micro)endemic species, is characterized as a putative allogamous, facultatively apomict that significantly differs from other Gymnadenia R.Br. species found in Romania. Notably, it distinguishes itself through its smaller habitus (reaching heights of up to 8-10 cm), its two-coloured, rounded/hemispherical inflorescence displaying a gradient of pink hues in an acropetal fashion (ranging from whitish-pink at the base to vivid-pink at the topmost flowers), and its limited distribution in high-altitude areas, encompassing approximately 8-10 km2 in the central area of the Bucegi Natural Park. This species has been under observation since 2005, with observed population numbers showing a significant increase over time, from ca. 50-55 (counted at the time of its discovery) to 120-140 individuals (counted in June 2023). Additionally, comprehensive information regarding the habitat, ecology, phenology and IUCN conservation assessments of Gymnadenia winkeliana are provided, including maps illustrating its distribution.

Evaluation of the anticancer and antibacterial activities of moscatilin.

Orchids (Dendrobium sp.) have been the subject of extensive research due to their ubiquitous pharmacological, antimicrobial, and anticancer properties. Moscatilin is a bibenzyl secondary metabolite enriched in orchids that exhibits anticancer and antimicrobial properties through mechanisms that have not yet been fully elucidated. The current study aimed to assess the in vitro anticancer and antibacterial potential of moscatilin. The in vitro anti-proliferative effects of moscatilin against breast cancer-MCF-7 and liver-HepG2 cells were assessed using the dimethylthiazol-diphenyltetrazolium bromide assay. Selected six pro-apoptotic (caspase-3, 8, 9, p53, p21 & Bax) and two anti-apoptotic (Bcl-xL & Bcl-2) gene markers were assessed via qPCR and tested antibacterial activity against various bacterial strains using disc diffusion and broth dilution methods. Moscatilin decreased the cellular viabilities of HepG2 and MCF-7 cancer cells, with anti-proliferation rates of 66 % (IC50 51 ± 5.18 µM) and 58 % (IC50 57 ± 4.18 µM), respectively. This effect was selectively observed in cancer cells, and the impact of moscatilin on non-cancerous MCF-12 cells was marginal. Moreover, moscatilin-treated cells exhibited higher mRNA levels of caspase-3,8, 9, Bax, p53, and p21, whereas lower levels of Bcl-2 and Bcl-xL, two anti-apoptotic markers, were observed. Furthermore, moscatilin exhibited varying degrees of antibacterial activity against the bacterial strains investigated. Notably, the highest antibacterial potentials were observed against Staphylococcus epidermidis and Klebsiella pneumonia, while the lowest inhibitory activity was observed in Escherichia coli and Pseudomonas aeruginosa. Overall, these findings demonstrated that moscatilin exerts potent anticancer effects via apoptosis and has antimicrobial properties against Gram-negative and Gram-positive bacteria that are clinically relevant. These findings highlight the potential of moscatilin as a natural therapeutic candidate for the treatment of cancer and clinically important bacterial pathogens.

Ecological perspectives on female and male reproductive success with competition in two Serapias species.

BACKGROUND AND AIMS: The deceptive strategies by which orchids are pollinated and how these are capable of attracting pollinators remain understudied with regard to their implications for plant fitness. Despite their ecological importance, limited investigations have been conducted on sexual deception and shelter mimicry in orchid species, making this a compelling avenue in orchid biology research. To expand the knowledge of these reproductive mechanisms, we studied the pollination of Serapias lingua and S. parviflora in co-occurring and isolated sites in the Balearic Islands (Spain), further accentuated by the presence of a hybrid, indicating shared pollinators. METHODS: We employed bagging and hand pollination experiments to examine the reproductive biology of the two species. Furthermore, we evaluated the influence of phenotypical and ecological factors on reproductive success, including biometric measurements, reproductive performance and neighbourhood diversity (co-flowering and pollinator communities). KEY RESULTS: Reproductive mechanisms between these two orchid species exhibit substantial disparities. Serapias lingua relies primarily on insect-mediated pollination, while S. parviflora demonstrates self-reproduction capacity. Although events of open pollination are rare, hybridization occurs predominantly when S. lingua is the pollen donor. Fruit set in S. parviflora was positively correlated with plant height, while in S. lingua it was negatively associated with flower size. The coexistence of the two species positively affected pollinium removal in S. parviflora, but did not exert an influence on reproductive traits in S. lingua. Overall, biometric parameters were diminished in isolated compared with co-occurring sites. At the community level, the increased diversity of co-flowering species in the vicinity exhibited an inhibitory effect on pollinium removal in S. parviflora. CONCLUSIONS: Under a context of pollinator loss or phenological mismatch between pollinator presence and flowering, the selfing capacity of S. parviflora would guarantee reproduction whereas S. lingua survival would be compromised. Furthers studies are needed to assess the effects of phenotypical and ecological factors on reproductive success of S. lingua in pollinator-decline scenarios.

Photosynthate transfer from an autotrophic orchid to conspecific heterotrophic protocorms through a common mycorrhizal network.

The minute 'dust seeds' of some terrestrial orchids preferentially germinate and develop as mycoheterotrophic protocorms near conspecific adult plants. Here we test the hypothesis that mycorrhizal mycelial connections provide a direct pathway for transfer of recent photosynthate from conspecific green orchids to achlorophyllous protocorms. Mycelial networks of Ceratobasidium cornigerum connecting green Dactylorhiza fuchsii plants with developing achlorophyllous protocorms of the same species were established on oatmeal or water agar before the shoots of green plants were exposed to 14CO2. After incubation for 48 h, the pattern of distribution of fixed carbon was visualised in intact entire autotrophic/protocorm systems using digital autoradiography and quantified in protocorms by liquid scintillation counting. Both methods of analysis revealed accumulation of 14C above background levels in protocorms, confirming that autotrophic plants supply carbon to juveniles via common mycorrhizal networks. Despite some accumulation of plant-fixed carbon in the fungal mycelium grown on oatmeal agar, a greater amount of carbon was transferred to protocorms growing on water agar, indicating that the polarity of transfer may be influenced by sink strength. We suggest this transfer pathway may contribute significantly to the pattern and processes determining localised orchid establishment in nature, and that 'parental nurture' via common mycelial networks may be involved in these processes.

A review on natural phenylbutanoid attractants: Occurrence, distribution, and role in nature, especially in relation to Dacini fruit fly behavior and pollination.

The natural occurrence, distribution (within a plant) and roles of four phenylbutanoid compounds (anisyl acetone, cue-lure, raspberry ketone and zingerone) are elucidated for the Asia-Pacific and Oceania regions. These phenylbutanoids may act individually or in combination to attract true fruit fly males belonging to a tribe Dacini of subfamily Dacinae (Diptera: Tepritidae). Of special interest are the mutualistic interactions between the Dacini fruit fly males and the tropical daciniphilous (attracting exclusively Dacini fruit flies) orchids - leading to cross pollination for the orchids and enchanced mating success for the flies. When offered to male flies, anisyl acetone and cue-lure are generally converted to raspberry ketone. Upon consumption, raspberry ketone and zingerone are individually sequestered in the male rectal (pheromonal) gland unchanged. Attracted male flies readily imbibe the phenylbutanoid(s) in the floral synomone to compliment the endogenously synthesized male sex pheromonal components - to enhance attraction of conspecific females during courtship as well as attract conspecific males to form 'leks'. The phenylbutanoid(s) may also act as an allomone to deter vertebrate predators, especially geckos, besides possessing antimicrobial and antioxidant activities. Cue-lure, raspberry ketone and zingerone are important attractants/lures used in pest surveillance and mass trapping under the integrated pest management (IPM) program against quarantine Dacini fruit fly pest species, particularly Bactrocera tryoni and Zeugodacus cucurbitae.

Morphological and Ultrastructural Features of Selected Epidendroideae Pollen Dispersal Units and New Insights into Their Chemical Nature.

Orchidaceae display enormous diversity in their flower morphology, which is particularly evident in their pollen dispersal units (pollinia, pollinaria). The packaging of pollen by elastoviscin leads to a great diversity of these morphologically and structurally complex pollen units. Despite being one of the most diverse angiosperm families, the available palynological data on orchids remain limited and sometimes contradicting. This study provides new insights into the pollen morphology and ultrastructure of five orchid species from the subfamily Epidendroideae, using combined light, scanning electron, and transmission electron microscopy. The aim was to compare the morphology and ultrastructure of pollen dispersal units and to elucidate the chemical nature of the pollen wall layers and of elastoviscin. Our combined light and electron microscopy investigation demonstrated the presence of six tetrad types even within a single pollinium, which is unique for orchids. The application of different staining methods confirmed the assumed lipidic nature of elastoviscin and the differences in its contrast and ultrastructure suggest a mixture of sticky materials with dissimilar chemical compositions. This study affirmed that sporopollenin is mostly restricted to the outer pollen grains of peripheral tetrads in compact and sectile pollinia, while inner tetrads exhibit highly reduced non-sporopollenin pollen walls.

What are the drivers of female success in food-deceptive orchids?

A large suite of floral signals, and environmental and biotic characteristics influence the behavior of pollinators, affecting the female success of food-deceptive orchids. In this study, we examined the many factors shaping the reproductive output of three orchid taxa: Dactylorhiza majalis, D. incarnata var. incarnata, and D. fuchsii. We applied a statistical model to correlate female success (number of fruit sets) with individual characteristics (plant and inflorescence height, number of flowers, and spur length), number of pollinaria removed, flowering time, and density of floral units of co-flowering rewarding plants. Our findings suggested that the broad spectrum of variations in Dactylorhiza's morphological traits, floral display, and flowering phenology within different environmental contexts has a significant impact on their reproductive success. The number of fruits increased with an increase in the number of pollinaria removed in the studied Dactylorhiza taxa. In contrast, a higher number of flowers per inflorescence and higher inflorescences in relation to individual height always decreased fruit set. We observed that low number of co-flowering rewarding plants in populations could affect the Dactylorhiza reproductive output as magnets and competitor plants. The synchronization of flowering, or lack thereof, between Dactylorhiza and rewarding plants can limit reproductive success. This demonstrates that the food deception strategy is multidirectional, and reproductive output can vary considerably both spatially and temporally within the context of this strategy.

PeNAC67-PeKAN2-PeSCL23 and B-class MADS-box transcription factors synergistically regulate the specialization process from petal to lip in Phalaenopsis equestris.

The molecular basis of orchid flower development involves a specific regulatory program in which MADS-box transcription factors play a central role. The recent 'perianth code' model hypothesizes that two types of higher-order heterotetrameric complexes, namely SP complex and L complex, play pivotal roles in the orchid perianth organ formation. Therefore, we explored their roles and searched for other components of the regulatory network.Through the combined analysis for transposase-accessible chromatin with high-throughput sequencing and RNA sequencing of the lip-like petal and lip from Phalaenopsis equestris var.trilip, transcription factor-(TF) genes involved in lip development were revealed. PeNAC67 encoding a NAC-type TF and PeSCL23 encoding a GRAS-type TF were differentially expressed between the lip-like petal and the lip. PeNAC67 interacted with and stabilized PeMADS3, which positively regulated the development of lip-like petal to lip. PeSCL23 and PeNAC67 competitively bound with PeKAN2 and positively regulated the development of lip-like petal to petal by affecting the level of PeMADS3. PeKAN2 as an important TF that interacts with PeMADS3 and PeMADS9 can promote lip development. These results extend the 'perianth code' model and shed light on the complex regulation of orchid flower development.

Distinct orchid mycorrhizal fungal communities among co-occurring Vanilla species in Costa Rica: root substrate and population-based segregation.

Despite being the second largest family of flowering plants, orchids represent community structure variation in plant-microbial associations, contributes to niche partitioning in metacommunity assemblages. Yet, mycorrhizal communities and interactions remain unknown for orchids that are highly specialized or even obligated in their associations with their mycorrhizal partners. In this study, we sought to compare orchid mycorrhizal fungal (OMF) communities of three co-occurring hemiepiphytic Vanilla species (V. hartii, V. pompona, and V. trigonocarpa) in tropical forests of Costa Rica by addressing the identity of their OMF communities across species, root types, and populations, using high-throughput sequencing. Sequencing the nuclear ribosomal internal transcribed spacer (nrITS) yielded 299 fungal Operational Taxonomic Units (OTUs) from 193 root samples. We showed distinct segregation in the putative OMF (pOMF) communities of the three coexisting Vanilla hosts. We also found that mycorrhizal communities associated with the rare V. hartii varied among populations. Furthermore, we identified Tulasnellaceae and Ceratobasidiaceae as dominant pOMF families in terrestrial roots of the three Vanilla species. In contrast, the epiphytic roots were mainly dominated by OTUs belonging to the Atractiellales and Serendipitaceae. Furthermore, the pOMF communities differed significantly across populations of the widespread V. trigonocarpa and showed patterns of distance decay in similarity. This is the first report of different pOMF communities detected in roots of wild co-occurring Vanilla species using high-throughput sequencing, which provides evidence that three coexisting Vanilla species and their root types exhibited pOMF niche partitioning, and that the rare and widespread Vanilla hosts displayed diverse mycorrhizal preferences.

Partial mycoheterotrophy in the leafless orchid Eulophia zollingeri specialized on wood-decaying fungi.

Although the absence of normal leaves is often considered a sign of full heterotrophy, some plants remain at least partially autotrophic despite their leafless habit. Leafless orchids with green stems and capsules probably represent a late evolutionary stage toward full mycoheterotrophy and serve as valuable models for understanding the pathways leading to this nutritional strategy. In this study, based on molecular barcoding and isotopic analysis, we explored the physiological ecology of the leafless orchid Eulophia zollingeri, which displays green coloration, particularly during its fruiting phase. Although previous studies had shown that E. zollingeri, in its adult stage, is associated with Psathyrellaceae fungi and exhibits high 13C isotope signatures similar to fully mycoheterotrophic orchids, it remained uncertain whether this symbiotic relationship is consistent throughout the orchid's entire life cycle and whether the orchid relies exclusively on mycoheterotrophy for its nutrition during the fruiting season. Our study has demonstrated that E. zollingeri maintains a specialized symbiotic relationship with Psathyrellaceae fungi throughout all life stages. However, isotopic analysis and chlorophyll data have shown that the orchid also engages in photosynthesis to meet its carbon needs, particularly during the fruiting stage. This research constitutes the first discovery of partial mycoheterotrophy in leafless orchids associated with saprotrophic non-rhizoctonia fungi.

Identification of Viruses Infecting Phalaenopsis Orchids Using Nanopore Sequencing and Development of an RT-RPA-CRISPR/Cas12a for Rapid Visual Detection of Nerine Latent Virus.

Phalaenopsis orchids are one of the most popular ornamental plants. More than thirty orchid viruses have been reported, and virus-infected Phalaenopsis orchids significantly lose their commercial value. Therefore, the development of improved viral disease detection methods could be useful for quality control in orchid cultivation. In this study, we first utilized the MinION, a portable sequencing device based on Oxford Nanopore Technologies (ONT) to rapidly detect plant viruses in Phalaenopsis orchids. Nanopore sequencing revealed the presence of three plant viruses in Phalaenopsis orchids: odontoglossum ringspot virus, cymbidium mosaic virus, and nerine latent virus (NeLV). Furthermore, for the first time, we detected NeLV infection in Phalaenopsis orchids using nanopore sequencing and developed the reverse transcription-recombinase polymerase amplification (RT-RPA)-CRISPR/Cas12a method for rapid, instrument-flexible, and accurate diagnosis. The developed RT-RPA-CRISPR/Cas12a technique can confirm NeLV infection in less than 20 min and exhibits no cross-reactivity with other viruses. To determine the sensitivity of RT-RPA-CRISPR/Cas12a for NeLV, we compared it with RT-PCR using serially diluted transcripts and found a detection limit of 10 zg/µL, which is approximately 1000-fold more sensitive. Taken together, the ONT platform offers an efficient strategy for monitoring plant viral pathogens, and the RT-RPA-CRISPR/Cas12a method has great potential as a useful tool for the rapid and sensitive diagnosis of NeLV.

Identification and Analysis of PEPC Gene Family Reveals Functional Diversification in Orchidaceae and the Regulation of Bacterial-Type PEPC.

Phosphoenolpyruvate carboxylase (PEPC) gene family plays a crucial role in both plant growth and response to abiotic stress. Approximately half of the Orchidaceae species are estimated to perform CAM pathway, and the availability of sequenced orchid genomes makes them ideal subjects for investigating the PEPC gene family in CAM plants. In this study, a total of 33 PEPC genes were identified across 15 orchids. Specifically, one PEPC gene was found in Cymbidium goeringii and Platanthera guangdongensis; two in Apostasia shenzhenica, Dendrobium chrysotoxum, D. huoshanense, Gastrodia elata, G. menghaiensis, Phalaenopsis aphrodite, Ph. equestris, and Pl. zijinensis; three in C. ensifolium, C. sinense, D. catenatum, D. nobile, and Vanilla planifolia. These PEPC genes were categorized into four subgroups, namely PEPC-i, PEPC-ii, and PEPC-iii (PTPC), and PEPC-iv (BTPC), supported by the comprehensive analyses of their physicochemical properties, motif, and gene structures. Remarkably, PEPC-iv contained a heretofore unreported orchid PEPC gene, identified as VpPEPC4. Differences in the number of PEPC homolog genes among these species were attributed to segmental duplication, whole-genome duplication (WGD), or gene loss events. Cis-elements identified in promoter regions were predominantly associated with light responsiveness, and circadian-related elements were observed in each PEPC-i and PEPC-ii gene. The expression levels of recruited BTPC, VpPEPC4, exhibited a lower expression level than other VpPEPCs in the tested tissues. The expression analyses and RT-qPCR results revealed diverse expression patterns in orchid PEPC genes. Duplicated genes exhibited distinct expression patterns, suggesting functional divergence. This study offered a comprehensive analysis to unveil the evolution and function of PEPC genes in Orchidaceae.

Nectar-mediated avian pollination in Cattleya (Orchidaceae: Laeliinae).

Orchids are notable for the variety of rewards produced for their pollinators. Nevertheless, many species are rewardless and pollinated by food-deceptive strategies. This is the case for Cattleya, a Neotropical genus of more than 100 species supposedly pollinated by food-deception. Here, I studied a member of the clade Sophronitis (Cattleya cernua) in forest areas of southeastern Brazil. The study included analyses of floral morphology, light microscopy of secretory tissues, histochemistry of flower resources, and measurements of nectar volume and concentration. Data on pollinators and mechanisms of pollination were collected in the field by means of film records, while breeding systems were studied using experimental treatments applied to cultivated specimens. The flower traits of Cattleya cernua show strong adaptation to avian pollination. Flowers produce 1-7 µl of a moderately concentrated nectar (17-50%). A secretory tissue at the distal portion of the cuniculus is responsible for nectar secretion. Secreted nectar is stored in the nectar chamber. Pollinaria deposition on the apex of bird beaks allows both short- and long-billed birds to pollinate the flowers. Plants from the studied population were fully self-compatible but pollinator-dependent. Pollinator frequency was higher than in food-deceptive Cattleya. As far as is known, this is the first record of nectar production in Cattleya. The discovery of a reward-producing species (C. cernua) within a genus entirely pollinated by food-deception strategies (Cattleya) provides clues and new insights into understanding the diversification of Laeliinae across the Neotropics.

Antioxidant and L-asparaginase activities of culturable endophytic fungi from ornamental Dendrobium orchids.

This study reports the antioxidant potential and L-asparaginase production of culturable fungal endophytes from Dendrobium orchids in Malaysia. Twenty-nine isolates were screened using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay to determine their free radical scavenging activities and antioxidant capacity (IC50 and AEAC). L-asparaginase production of fungal endophytes was detected by the qualitative plate assay, and the enzyme activities estimated via the Nesslerization method. All 29 endophytic isolates exhibited various degrees of radical scavenging activities (35.37%-77.23%), with Fusarium fujikuroi (D1) identified as having the highest antioxidant capacity (IC50 6.097 mg/mL) and the highest AEAC value (11.55  mg/g). For L-asparaginase production, the majority of the isolates (89.66%) showed positive results, especially among the culturable species of Fusarium, Trichoderma, and Daldinia. Most Fusarium spp. were able to produce L-asparaginase (80.77%), but the highest L-asparaginase activity was detected in Daldinia eschscholtzii (D14) with 2.128 units/mL. Results from this study highlighted the potential of endophytic fungi from medicinal orchids (Dendrobium sp.) as natural sources of bioactive compounds to be developed into novel antioxidants and anticancer drugs.

Insights to Phenylalanine Ammonia Lyase (PAL) and Secondary Metabolism in Orchids: An in silico Approach.

The phenylalanine ammonia lyase (PAL) catalyses the first step of phenylpropanoid metabolic pathway which leads to the biosynthesis of a diverse group of secondary metabolites. Orchids serve as a rich source of metabolites and the availability of genome or transcriptome for selected orchid species provides an opportunity to analyse the PAL genes in orchids. In the present study, 21 PAL genes were characterized using bioinformatics tools in nine orchid species (Apostasia shenzhenica, Cypripedium formosanum, Dendrobium catenatum, Phalaenopsis aphrodite, Phalaenopsis bellina, Phalaenopsis equestris, Phalaenopsis lueddemanniana, Phalaenopsis modesta and Phalaenopsis schilleriana). Multiple sequence alignment confirmed the presence of PAL-specific conserved domains (N-terminal, MIO, core, shielding and C-terminal domain). All these proteins were predicted to be hydrophobic in nature and to have cytoplasmic localisation. Structural modelling depicted the presence of alpha helices, extended strands, beta turns and random coils in their structure. Ala-Ser-Gly triad known for substrate binding and catalysis of MIO-domain was found to be completely conserved in all the proteins. Phylogenetic study showed that the PALs of pteridophytes, gymnosperms and angiosperms clustered together in separate clades. Expression profiling showed tissue-specific expression for all the 21 PAL genes in the various reproductive and vegetative tissues which suggested their diverse role in growth and development. This study provides insights to the molecular characterization of PAL genes which may help in developing biotechnological strategies to enhance the synthesis of phenylpropanoids in orchids and other heterologous systems for pharmaceutical applications.

Characters evolution of Encyclia (Laeliinae-Orchidaceae) reveals a complex pattern not phylogenetically determined: insights from macro- and micromorphology.

Encyclia is the second-largest genus in the neotropical subtribe Laeliinae (Orchidaceae) and has more than 150 species, which are characterized by fairly consistent flower morphology. Its taxonomy and species boundaries, however, seem to be still under debate. In the present study, we first examined the lip micromorphology of 61 species of Encyclia sensu stricto. We correlated our results with external flower morphology and phylogenetic analyses performed on a combined dataset that included both nuclear (ITS, Xdh, PhyC) and plastid markers (ycf1, rpl32, and trnL-trnF). Phylogenetic reconstruction showed that Encyclia sensu stricto species form a coherent, monophyletic group. However, it is difficult to determine the relationships between the different groups within one larger clade. The groups all form distinct lineages that evolved from a common ancestor. The UPGMA cluster analysis for the seven qualitative micromorphological features clearly divides the genus into two main groups, the larger of which is further subdivided into two subgroups. None of these, however, overlap with any of the phylogeographic units distinguished in previously published papers or in presented article. It is worth noting that the groups resulting from the UPGMA analysis cannot be defined by macromorphological features. The pattern of similarities between species, taking into account both macro- and micromorphological features, is eminently mosaic in nature, and only a multifaceted approach can explain this enigmatic group.

Characterization and identification of antimicrobial compounds from endophytic Fusarium incarnatum isolated from Cymbidium orchids.

This study characterized and identified the antimicrobial compounds from an endophytic fungus (Fusarium incarnatum (C4)) isolated from the orchid, Cymbidium sp. Chromatographic techniques were employed to separate the bioactive compounds from the crude extracts of F. incarnatum (C4). Following bio-guided fractionation, two fractionated extracts (fractions 1 and 2) of F. incarnatum (C4) exhibited antibacterial and antifungal activities against Bacillus cereus (MIC: 0.156 mg/mL) and Ganoderma boninense (MIC: 0.3125 mg/mL), respectively. The active fractions were discovered to comprise of a variety of bioactive compounds with pharmacological importance (alkaloids, flavonoids, phenolic compounds, terpenoids, peptides and fatty acids). Liquid chromatography mass-spectrometry (LCMS) analysis detected the presence of antibacterial (kanzonol N, rifaximin, linoleic acid (d4), cannabisativine, docosanedioic acid, and stearamide) and antifungal components (3-methyl-quinolin-2-ol, prothiocarb, kanzonol N, peganine, 5Z-tridecene, and tetronasin) in fractions 1 and 2, respectively, which may have contributed to the antimicrobial effects. Findings from this study highlighted the important potential of fungal endophytes from medicinal hosts as producers of antimicrobials and antibiotics.