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.

[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.

Fungus-insect gall of Phlebopus portentosus.

Phlebopus portentosus is a popular edible wild mushroom found in the tropical Yunnan, China, and northern Thailand. In its natural habitats, a gall often has been found on some plant roots, around which fungal fruiting bodies are produced. The galls are different from common insect galls in that their cavity walls are not made from plant tissue but rather from the hyphae of P. portentosus. Therefore we have termed this phenomenon "fungus-insect gall". Thus far six root mealy bug species in the family Pseudococcidae that form fungus-insect galls with P. portentosus have been identified: Formicococcus polysperes, Geococcus satellitum, Planococcus minor, Pseudococcus cryptus, Paraputo banzigeri and Rastrococcus invadens. Fungus-insect galls were found on the roots of more than 21 plant species, including Delonix regia, Citrus maxima, Coffea arabica and Artocarpus heterophyllus. Greenhouse inoculation trials showed that fungus-insect galls were found on the roots of A. heterophyllus 1 mo after inoculation. The galls were subglobose to globose, fulvous when young and became dark brown at maturation. Each gall harbored one or more mealy bugs and had a chimney-like vent for ventilation and access to the gall. The cavity wall had three layers. Various shaped mealy bug wax deposits were found inside the wall. Fungal hyphae invaded the epidermis of plant roots and sometimes even the cortical cells during the late stage of gall development. The identity of the fungus inside the cavity was confirmed by molecular methods.

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.

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.

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.

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.

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.

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.

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.

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.

Two new solanapyrone analogues from the endophytic fungus Nigrospora sp. YB-141 of Azadirachta indica.

Two new solanapyrone analogues, solanapyrones N and O (1 and 2, resp.), and three known compounds, solanapyrone C (3), nigrosporalactone (4), and phomalactone (5), were isolated from the fermentation culture of Nigrospora sp. YB-141, an endophytic fungus isolated from Azadirachta indica A. Juss. The structures of the new compounds were elucidated on the basis of spectroscopic analysis. The antifungal activities of 1-5 towards seven phytopathogenic fungi were tested. Most of the compounds exhibited no or only weak antifungal activities.

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.

Ten-membered lactones from Phomopsis sp., an endophytic fungus of Azadirachta indica.

Four new 10-membered lactones ( 1- 4) and one known one ( 5) were isolated from the broth extract of an endophytic fungus, Phomopsis sp., obtained from the stem of Azadirachta indica. Their structures were assigned by analysis of spectroscopic data, and the structures of 1 and 4 were also confirmed by X-ray analysis. Compounds 1- 5 were tested for antifungal activity against several plant pathogens. Compound 4 demonstrated antifungal activity in the MIC value range 31.25-500 microg/mL.