Exploring the diversity, bioactivity of endophytes, and metabolome in Synsepalum dulcificum.

Synsepalum dulcificum exhibits high edible and medicinal value; however, there have been no reports on the exploration of its endophyte resources. Here, we conducted analyses encompassing plant metabolomics, microbial diversity, and the biological activities of endophytic metabolites in S. dulcificum. High-throughput sequencing identified 4,913 endophytic fungal amplicon sequence variants (ASVs) and 1,703 endophytic bacterial ASVs from the roots, stems, leaves, flowers, and fruits of S. dulcificum. Fungi were classified into 5 phyla, 24 classes, 75 orders, 170 families, and 313 genera, while bacteria belonged to 21 phyla, 47 classes, 93 orders, 145 families, and 232 genera. Furthermore, there were significant differences in the composition and content of metabolites in different tissues of S. dulcificum. Spearman's correlation analysis of the differential metabolites and endophytes revealed that the community composition of the endophytes correlated with plant-rich metabolites. The internal transcribed spacer sequences of 105 isolates were determined, and phylogenetic analyses revealed that these fungi were distributed into three phyla (Ascomycota, Basidiomycota, and Mucoromycota) and 20 genera. Moreover, 16S rDNA sequencing of 46 bacteria revealed they were distributed in 16 genera in three phyla: Actinobacteria, Proteobacteria, and Firmicutes. The antimicrobial activities (filter paper method) and antioxidant activity (DPPH and ABTS assays) of crude extracts obtained from 68 fungal and 20 bacterial strains cultured in different media were evaluated. Additionally, the α-glucosidase inhibitory activity of the fungal extracts was examined. The results showed that 88.6% of the strains exhibited antimicrobial activity, 55.7% exhibited antioxidant activity, and 85% of the fungi exhibited α-glucosidase inhibitory activity. The research suggested that the endophytes of S. dulcificum are highly diverse and have the potential to produce bioactive metabolites, providing abundant species resources for developing antibiotics, antioxidants and hypoglycemic drugs.

Haplotype-resolved chromosomal-level genome assembly of Buzhaye (Microcos paniculata).

Microcos paniculata is a shrub used traditionally as folk medicine and to make herbal teas. Previous research into this species has mainly focused on its chemical composition and medicinal value. However, the lack of a reference genome limits the study of the molecular mechanisms of active compounds in this species. Here, we assembled a haplotype-resolved chromosome-level genome of M. paniculata based on PacBio HiFi and Hi-C data. The assembly contains two haploid genomes with sizes 399.43 Mb and 393.10 Mb, with contig N50 lengths of 43.44 Mb and 30.17 Mb, respectively. About 99.93% of the assembled sequences could be anchored to 18 pseudo-chromosomes. Additionally, a total of 482 Mb repeat sequences were identified, accounting for 60.76% of the genome. A total of 49,439 protein-coding genes were identified, of which 48,979 (99%) were functionally annotated. This haplotype-resolved chromosome-level assembly and annotation of M. paniculata will serve as a valuable resource for investigating the biosynthesis and genetic basis of active compounds in this species, as well as advancing evolutionary phylogenomic studies in Malvales.

Comparative chloroplast genomics of three species of Bulbophyllum section Cirrhopetalum (Orchidaceae), with an emphasis on the description of a new species from Eastern Himalaya.

Background: Chloroplast (cp) genomes are useful and informative molecular markers used for species determination and phylogenetic analysis. Bulbophyllum is one of the most taxonomically complex taxa in Orchidaceae. However, the genome characteristics of Bulbophyllum are poorly understood. Methods: Based on comparative morphological and genomic analysis, a new species Bulbophyllum pilopetalum from eastern Himalaya belonging to section Cirrhopetalum is described and illustrated. This study used chloroplast genomic sequences and ribosomal DNA (nrDNA) analysis to distinguish the new Bulbophyllum species and determine its phylogenetic position. An additional phylogenetic analysis was conducted using 74 coding sequences from 15 complete chloroplast genomes from the genus Bulbophyllum, as well as nrDNA sequences and two chloroplast DNA sequences from 33 Bulbophyllun species. Results: The new species is morphologically similar to B. pingnanense, B. albociliatum, and B. brevipedunculatum in vegetative and floral morphology, but it can be distinguished by its ovate-triangle dorsal sepal without a marginal ciliate. The chloroplast genome of the new Bulbophyllum species is 151,148 bp in length, and includes a pair of inverted repeats (IRs) of 25,833 bp, a large single-copy region (LSC) of 86,138 bp, and a small single-copy region (SSC) of 13,300 bp. The chloroplast genome includes 108 unique genes encoding 75 proteins, 30 tRNAs, and four rRNAs. Compared with the cp genomes of its two most closely-related species, B. pingnanense and B. albociliatum, this chloroplast genome exhibited great interspecific divergence and contained several Indels that were specific to the new species. The plastid tree showed that B. pilopetalum is most closely-related to B. pingnanense. The phylogenetic tree based on combined nrDNA and chloroplast DNA sequences indicated that section Cirrhopetalum was monophyletic and B. pilopetalum was a member of this section. Discussion: The taxonomic status of the new species is strongly supported by cp genome data. Our study highlights the importance of using the complete cp genome to identify species, elucidate the taxonomy, and reconstruct the phylogeny of plant groups with complicated taxonomic problems.

Effects of a Dark Septate Fungal Endophyte on the Growth and Physiological Response of Seedlings to Drought in an Epiphytic Orchid.

Dark septate endophytes (DSE) are a group of facultative biotrophic root-colonizing fungi that live within a plant for a part of their life cycle without causing any apparent, overt negative effects. These fungi have been found in >600 different plant species, including orchids. Although the precise ecological functions of dark septate fungal endophytes are not yet well understood, there is increasing evidence that they enhance host growth and nutrient acquisition, and improve the plant's ability to tolerate biotic and abiotic stresses. In this research, we tested the effects of a DSE isolated from the roots of the epiphytic orchid Coelogyne viscosa on the growth and drought tolerance of orchid seedlings. Our results showed that addition of DSE inoculum significantly enhanced biomass of seedlings and increased the activities of drought resistance related enzymes and the accumulation of osmoregulatory substances. These results suggest that DSE can fulfill important ecological functions in stressful environments and potentially play an important role in the life cycle of epiphytic orchids.

Diversity of Cantharellus (Cantharellales, Basidiomycota) in China with Description of Some New Species and New Records.

Cantharellus is a well-known genus of edible mushrooms, belonging to the family Hydnaceae in the class Agaricomycetes. In this study, a phylogenetic overview of Cantharellus subg. Cinnabarinus and C. subg. Parvocantharellus in China is carried out with the description of four new species. Species description are based on morphological characters of basidiomata and phylogenetic analyses of multi-locus dataset of 28S + tef1 + rpb2. Among the new species, two species, C. chrysanthus and C. sinocinnabarinus, belong to C. subg. Cinnabarinus and two new species, C. convexus and C. neopersicinus, belong to C. subg. Parvocantharellus. Species delimitation characters of the new taxa are compared with closely related species. In addition, three new records of Cantharellus are reported for China: C. albovenosus and C. citrinus of subg. Cinnabarinus and C. koreanus of subg. Parvocantharellus. A key to the species of subg. Cinnabarinus in China was provided.

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.

Successful reintroduction releases pressure on China's orchid species.

Orchids have suffered dramatic declines in China and elsewhere in the world and several species are at the brink of extinction. Recent developments in orchid reintroduction programs could help establish new populations in natural habitats and release the current pressure on China's most threatened orchid species.

Chloroplast genomic diversity in Bulbophyllum section Macrocaulia (Orchidaceae, Epidendroideae, Malaxideae): Insights into species divergence and adaptive evolution.

Bulbophyllum is the largest genus in Orchidaceae with a pantropical distribution. Due to highly significant diversifications, it is considered to be one of the most taxonomically and phylogenetically complex taxa. The diversification pattern and evolutionary adaptation of chloroplast genomes are poorly understood in this species-rich genus, and suitable molecular markers are necessary for species determination and phylogenetic analysis. A natural Asian section Macrocaulia was selected to estimate the interspecific divergence of chloroplast genomes in this study. Here, we sequenced the complete chloroplast genome of four Bulbophyllum species, including three species from section Macrocaulia. The four chloroplast genomes had a typical quadripartite structure with a genome size ranged from 156,182 to 158,524 bp. The chloroplast genomes included 113 unique genes encoding 79 proteins, 30 tRNAs and 4 rRNAs. Comparison of the four chloroplast genomes showed that the three species from section Macrocaulia had similar structure and gene contents, and shared a number of indels, which mainly contribute to its monophyly. In addition, interspecific divergence level was also great. Several exclusive indels and polymorphism SSR loci might be used for taxonomical identification and determining interspecific polymorphisms. A total of 20 intergenic regions and three coding genes of the most variable hotspot regions were proposed as candidate effective molecular markers for future phylogenetic relationships at different taxonomical levels and species divergence in Bulbophyllum. All of chloroplast genes in four Bulbophyllum species were under purifying selection, while 13 sites within six genes exhibited site-specific selection. A whole chloroplast genome phylogenetic analysis based on Maximum Likelihood, Bayesian and Parsimony methods all supported the monophyly of section Macrocaulia and the genus of Bulbophyllum. Our findings provide valuable molecular markers to use in accurately identifying species, clarifying taxonomy, and resolving the phylogeny and evolution of the genus Bulbophyllum. The molecular markers developed in this study will also contribute to further research of conservation of Bulbophyllum species.

Orchid Reintroduction Based on Seed Germination-Promoting Mycorrhizal Fungi Derived From Protocorms or Seedlings.

Orchids are among the most endangered in the plant kingdom. Lack of endosperm in their seeds renders orchids to depend on nutrients provided by orchid mycorrhizal fungi (OMF) for seed germination and seedling formation in the wild. OMF that parasitize in germination seeds is an essential element for orchid seedling formation, which can also help orchid reintroduction. Considering the limitations of the previous orchid reintroduction technology based on seed germination-promoting OMF (sgOMF) sourced from orchid roots, an innovative approach is proposed here in which orchid seeds are directly co-sown with sgOMF carrying ecological specificity from protocorms/seedlings. Based on this principle, an integrative and practical procedure concerning related ecological factors is further raised for re-constructing long-term and self-sustained orchid populations. We believe that this new approach will benefit the reintroduction of endangered orchids in nature.

The genus Paraconiothyrium: species concepts, biological functions, and secondary metabolites.

The genus Paraconiothyrium has worldwide distribution with diverse host habitats and exhibits potential utilisation as biocontrol agent, bioreactor and antibiotic producer. In this review, we firstly comprehensively summarise the current taxonomic status of Paraconiothyrium species, including their category names, morphological features, habitats, and multigene phylogenetic relationships. Some Paraconiothyrium species possess vital biological functions and potential applications in medicine, agriculture, industry, and environmental protection. A total of 147 secondary metabolites have been reported so far from Paraconiothyrium, among which 95 are novel. This paper serves to provide an overview of their diverse structures with chemical classification and biological activities. To date, 27 species of Paraconiothyrium have been documented; however, only seven have been investigated for their secondary metabolites or biological functions. Our review is expected to draw more attention to this genus for providing a taxonomic reference, discovering extensive biological functions, and searching in-depth for new bioactive natural products.

Complete mitochondrial genome of the edible Basidiomycete mushroom Thelephora aurantiotincta (Aphyllophorales: Thelephoraceae) from China.

The complete mitochondrial genome of Thelephora aurantiotincta, an edible Basidiomycete mushroom species with ecological and economic value is reported in this study. The whole genome is a circular molecule 50,672 bp in length and encodes 42 genes as follows: 15 protein-coding genes, two rRNA genes and 25 tRNA genes. The A, T, C, G contents in the genome are 35.60%, 35.31%, 13.89%, and 15.20%, respectively. Phylogenetic analysis revealed a close relationship between T. aurantiotincta and T. ganbajun. This is the first complete mitochondrial genome for T. aurantiotincta that will be useful for providing basic genetic information for this important species.

Extracellular Enzyme Activities and Carbon/Nitrogen Utilization in Mycorrhizal Fungi Isolated From Epiphytic and Terrestrial Orchids.

Fungi employ extracellular enzymes to initiate the degradation of organic macromolecules into smaller units and to acquire the nutrients for their growth. As such, these enzymes represent important functional components in terrestrial ecosystems. While it is well-known that the regulation and efficiency of extracellular enzymes to degrade organic macromolecules and nutrient-acquisition patterns strongly differ between major fungal groups, less is known about variation in enzymatic activity and carbon/nitrogen preference in mycorrhizal fungi. In this research, we investigated variation in extracellular enzyme activities and carbon/nitrogen preferences in orchid mycorrhizal fungi (OMF). Previous research has shown that the mycorrhizal fungi associating with terrestrial orchids often differ from those associating with epiphytic orchids, but whether extracellular enzyme activities and carbon/nitrogen preference differ between growth forms remains largely unknown. To fill this gap, we compared the activities of five extracellular enzymes [cellulase, xylanase, lignin peroxidase, laccase, and superoxide dismutase (SOD)] between fungi isolated from epiphytic and terrestrial orchids. In total, 24 fungal strains belonging to Tulasnellaceae were investigated. Cellulase and xylanase activities were significantly higher in fungi isolated from terrestrial orchids (0.050 ± 0.006 U/ml and 0.531 ± 0.071 U/ml, respectively) than those from epiphytic orchids (0.043 ± 0.003 U/ml and 0.295 ± 0.067 U/ml, respectively), while SOD activity was significantly higher in OMF from epiphytic orchids (5.663 ± 0.164 U/ml) than those from terrestrial orchids (3.780 ± 0.180 U/ml). Carboxymethyl cellulose was more efficiently used by fungi from terrestrial orchids, while starch and arginine were more suitable for fungi from epiphytic orchids. Overall, the results of this study show that extracellular enzyme activities and to a lesser extent carbon/nitrogen preferences differ between fungi isolated from terrestrial and epiphytic orchids and may indicate functional differentiation and ecological adaptation of OMF to local growth conditions.

Chemical constituents and their antioxidant activities from the leaves of Synsepalum dulcificum.

Synsepalum dulcifificum is an evergreen shrub native of tropical West Africa. It is very effective against a variety of diseases. However, the phytochemical investigation on this plant is limited till now. In our current study, seventeen compounds were isolated and identified from the ethyl acetate extract of the leaves. All of the compounds were obtained from S. dulcifificum for the first time. Compounds 4 and 9 were isolated from natural source for the second time. Moreover, the antioxidant activities of compounds 1, 4, 5, 9-11, and 15 were evaluated firstly. Compounds 6, 7, 13, and 14 exhibited significant antioxidant activity in DPPH and ABTS + assays, suggesting their potential application for an antioxidant drug.

Co-Cultures of Mycorrhizal Fungi Do Not Increase Germination and Seedling Development in the Epiphytic Orchid Dendrobium nobile.

Orchids are highly dependent on mycorrhizal fungi for seed germination and subsequent growth to a seedling as they provide essential carbon, water, and mineral nutrients to developing seeds. Although there is mounting evidence that orchid seeds are often colonized by multiple fungi simultaneously, most in vitro germination experiments focus on mycorrhizal monocultures and little is known about how mycorrhizal assemblages affect seed germination and growth of seedlings. In this study, we compared the effects of mycorrhizal monocultures and co-cultures on seed germination and seedling growth of the epiphytic orchid Dendrobium nobile. In situ baiting was used to isolate mycorrhizal fungi from protocorms for germination experiments. Germination experiments were conducted under two light regimes for 90 days. In total, five fungal strains were isolated from protocorms of D. nobile, indicating that the species was not highly specific to its fungal partners. Four strains (JC-01, JC-02, JC-04, and JC-05) belonged to the Serendipitaceae and one (JC-03) to the Tulasnellaceae. In vitro germination experiments showed that germination percentages were higher under light-dark conditions than under complete dark conditions, supporting previous findings that light facilitates germination in epiphytic orchids. While all strains were able to induce protocorm formation and growth into the seedling stage, large differences between fungal strains were observed. Co-cultures did not result in significantly higher seed germination percentages and seedling development than monocultures. Taken together, these results demonstrate that effects of fungal assemblages are not predictable from those of component species, and that more work is needed to better understand the role of fungal assemblages determining seed germination and subsequent growth under natural conditions.

Fungi isolated from host protocorms accelerate symbiotic seed germination in an endangered orchid species (Dendrobium chrysotoxum) from southern China.

To ensure long-term survival of epiphytic orchids through active reintroduction, more research on critical life cycle stages such as seed germination and seedling establishment are needed. In this study, we used in vitro germination experiments to investigate the role of mycorrhizal fungi in determining seed germination and growth in the endangered epiphytic orchid species, Dendrobium chrysotoxum. Symbiotic seed germination experiments were conducted for 90 days under different light conditions with fungal strains isolated from protocorms of D. chrysotoxum and three sister species. Molecular analyses showed that five strains belonged to the typical orchid mycorrhizal family Tulasnellaceae, whereas the other two strains belonged to the Sebacinaceae and the genus Coprinellus. Fungal inoculation, light conditions, and their interaction had a significant effect on protocorm formation and seedling development. Three fungal isolates, including two from D. chrysotoxum and one from D. catenatum, significantly stimulated protocorm formation and seedling development under light conditions. However, fungi isolated from host protocorms (GC-14 and GC-15) produced the highest number of seedlings after 50 days (49.5 ± 8.5%, 51.3 ± 9.0%, respectively), while the fungus isolated from D. catenatum protocorms produced the maximum number of seedlings only after 90 days (48.7 ± 16.1%). To conclude, this study has shown that light conditions and the identity of fungi had a strong effect on in vitro seed germination and seedling formation in an epiphytic orchid, with fungi isolated from host protocorms leading to accelerated germination and seedling formation. Therefore, fungal source should be taken into account when using seeds and compatible fungi for seedling propagation and in situ reintroduction.

The complete chloroplast genome sequence of Dendrobium wangliangii (Orchidaceae).

Dendrobium wangliangii is a rare orchid species with extremely small populations, endemic to China. In this study, the complete chloroplast (cp) genome sequence and the genome features of D. wangliangii were analyzed. The whole cp genome sequence of D. wangliangii is 160,052 including a large single-copy region (LSC, 87,525 bp), a small single-copy region (SSC, 18,373 bp), and a pair of repeat regions (IRs, 27,077 bp, each). The contents of four bases in cpDNA were A (30.9%), C (18.9%), G (18.3%) and T (31.9%), respectively. The total content of GC is 37.1%. The cp genome contains 129 genes, consisting of 124 unique genes (78 protein-coding genes, 38 tRNAs, and 8 rRNAs). Phylogenetic analysis showed that D. wangliangii nested with other Dendrobium spp. and was closely related to D. ellipsophyllum, D. wattii and D. longicornu.

The complete mitochondrial genome sequence of Lactarius trivialis (Russulalles, Basidiomycota).

Lactarius trivialis is a very common species and widely distributes in north temperate. In this study, a complete mitogenome of L. trivialis was assembled and annotated. The whole mitogenome of L. trivialis was a circular molecule with 42,366 bp in length, encoding 44 genes as follows: 19 coding genes, two rRNAs, and 23 tRNAs. The contents of four bases in mtDNA were A (39.65%), T (38.82%), C (11.30%), and G (21.53%), respectively. Phylogenetic analysis recovered that it is nested with other Lactarius spp. in the order Russulalles.

A novel case of autogamy and cleistogamy in Dendrobium wangliangii: A rare orchid distributed in the dry-hot valley.

Dendrobium wangliangii is an epiphytic orchid distributed in the Jinshajiang dry-hot valley in Luquan County, Yunnan Province, China. Most Dendrobium spp. typically have a low fruit set, but this orchid shows a higher fruit set under natural conditions despite the lack of effective pollinators. The pollination biology of the critically endangered D. wangliangii was investigated in this study. A fruit set rate of 33.33 ± 4.71% was observed after bagging treatment in 2017 and a high fruit set rate (65.72 ± 4.44% in 2011; 50.79 ± 5.44% in 2017) was observed under natural conditions, indicating that D. wangliangii is characterized by spontaneous self-pollination. The anther cap blocked the growing pollinium; thus, the pollinium slid down and reached the stigmatic cavity, leading to autogamous self-pollination. Specifically, 51.50% of 162 unopened flowers (total 257 flowers) of this Dendrobium species under extreme water-deficit conditions developed into fruits, suggesting the presence of cleistogamy in D. wangliangii. Here, cleistogamy may represent the primary mode of pollination for this orchid. Spontaneous self-pollination and specific cleistogamous autogamy could represent major adaptions to the drought and pollinator-scarce habitat in the Jinshajiang dry-hot valley.

Using In Situ Symbiotic Seed Germination to Restore Over-collected Medicinal Orchids in Southwest China.

Due to increasing demand for medicinal and horticultural uses, the Orchidaceae is in urgent need of innovative and novel propagation techniques that address both market demand and conservation. Traditionally, restoration techniques have been centered on ex situ asymbiotic or symbiotic seed germination techniques that are not cost-effective, have limited genetic potential and often result in low survival rates in the field. Here, we propose a novel in situ advanced restoration-friendly program for the endangered epiphytic orchid species Dendrobium devonianum, in which a series of in situ symbiotic seed germination trials base on conspecific fungal isolates were conducted at two sites in Yunnan Province, China. We found that percentage germination varied among treatments and locations; control treatments (no inoculum) did not germinate at both sites. We found that the optimal treatment, having the highest in situ seed germination rate (0.94-1.44%) with no significant variation among sites, supported a warm, moist and fixed site that allowed for light penetration. When accounting for seed density, percentage germination was highest (2.78-2.35%) at low densities and did not vary among locations for the treatment that supported optimal conditions. Similarly for the same treatment, seed germination ranged from 0.24 to 5.87% among seasons but also did vary among sites. This study reports on the cultivation and restoration of an endangered epiphytic orchid species by in situ symbiotic seed germination and is likely to have broad application to the horticulture and conservation of the Orchidaceae.

[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.