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

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

Host-specificity of symbiotic mycorrhizal fungi for enhancing seed germination,protocorm formation and seedling development of over-collected medicinal orchid, <Emphasis Type="Italic">Dendrobium devonianum</Emphasis>

All orchids maintain an obligate relationship with mycorrhizal symbionts during seed germination. In most cases, germination-enhancing fungi have been isolated from roots of mature plants for conservation and cultivation purposes. To understand the germination biology of Dendrobium devonianum, an over-collected medicinal orchid, the seeds of D. devonianum were inoculated with a fungal strain (FDd1) isolated from naturally occurring protocorms of D. devonianum and two other germination-enhancing fungal strains (FDaI7 and FCb4) from D. aphyllum and Cymbidium mannii, respectively. The fungal strain was isolated from five protocorms of D. devonianum and identified as a species of the genus Epulorhiza. In germination trials, treatments with all of the three fungal strains showed a significant promoting effect on seed germination and protocorm formation, compared with the control treatment (no inoculation). However, FDd1 fungal strain showed the greatest effectiveness followed by FDaI7 and FCb4. For all inoculation and control treatments, seeds developed to protocorms regardless of the presence of illumination, whereas protocorms did not develop to seedlings unless illumination was provided. The results of our manipulative experiments confirmed the hypothesis that mycorrhizae associated with orchid seedlings are highly host-specific, and the degree of specificity may be life stagespecific under in vitro conditions. The specific mycorrhizal symbionts from protocorms can enhance restoration efforts and the conservation of orchids such as D. devonianum.     

Specificity and preference of mycorrhizal associations in two species of the genus Dendrobium (Orchidaceae)

Dendrobium is a large genus of tropical epiphytic orchids. Some members of this genus are in danger of extinction across China. To investigate orchid mycorrhizal associations of the genus Dendrobium, plants from two Dendrobium species (Dendrobium officinale and Dendrobium fimbriatum) were collected from two habitats in Guangxi Province, China, and clone libraries were constructed to identify the mycorrhizal fungi of individual plants. A low and high degree of specificity was observed in D. officinale and D. fimbriatum, respectively. Phylogenetic analysis revealed that the majority of Dendrobium mycorrhizal fungi are members of the Tulasnellaceae, but, in some plants, members of the Ceratobasidiaceae and Pluteaceae were also found. In D. officinale, individual plants associated with more than three fungi simultaneously, and, in some cases, associations with five fungi at the same time. One fungus was shared by individual plants of D. officinale collected from the two habitats. In D. fimbriatum, only one fungal partner was found in each population, and this fungus differed between populations. The two species of Dendrobium sampled from the same habitat did not share any fungal taxa. These results provide valuable information for conservation of these orchid species.     

In situ seed baiting techniques in <Emphasis Type="Italic">Dendrobium officinale</Emphasis> Kimuraet Migo and <Emphasis Type="Italic">Dendrobium nobile</Emphasis> Lindl. : the endangered Chinese endemic <Emphasis Type="Italic">Dendrobium</Emphasis> (Orchidaceae)

The new in situ seed baiting method using seed packets to assess germination of orchid species within soil provides a means of locating, collecting, and identifying specific fungi that are involved in the lifecycle of orchids in the wild and investigating their relationship with the orchids under natural field conditions. Two isolates (SHH44, SHH53) originating from the seedlings found in the seeds packets in situ were demonstrated to support seed germination and seedling development (in vitro) of two endangered Chinese endemic herbs, Dendrobium officinale and Dendrobium nobile (Orchidaceae). Advanced protocorm development (Stage 3 and greater) in this study of these species of Dendrobium only occurred under the 12/12 h L/D photo-period indicated that illumination may play an important role in seedling recruitment of terrestrial orchid species in their natural environment. The information provided in this study may prove invaluable in the conservation of D. officinale and D. nobile and congener species in China, especially given their rare status.     

兰科石斛属植物菌根真菌研究进展

石斛属(Dendrobium)隶属于兰科(Orchidaceae)树兰亚科(Epidendroideae)石斛兰族(Dendrobiinae),是兰科最大的属之一,终生附生于树上或岩石上。石斛属很多种类具有很高的药用价值与观赏价值。由于人为过度采挖和野生生境的破坏,使得野生石斛资源濒临灭绝。石斛属植物为典型的兰科菌根植物,在自然条件下需要与真菌共生,才能完成生活史。菌根真菌对于石斛属植物的种子萌发和植株生长具有重要的作用。对石斛属植物菌根的形成、菌根真菌的作用、菌根真菌多样性及菌根技术在石斛属植物中的应用做了评述,并对今后的研究内容和重点提出了一些思路。     

Endophytic fungi assemblages from 10 <Emphasis Type="Italic">Dendrobium</Emphasis> medicinal plants (Orchidaceae)

Dendrobium is the largest genus of tropical epiphytic orchid, some of which are traditional Chinese medicinal plants. The therapeutic components varied significantly among species. Endophytic microbes (fungi) hidden in medicinal plants may play an important effect on the overall quality of herb. Investigation of fungal composition in host plants is the first step toward elucidating the relationship endophyte-therapeutic content of herbal medicine. In this study, 401 culturable fungal endophytes were isolated and identified from 10 species of medicinal Dendrobium based on morphological and molecular techniques. The results showed that endophytic fungi from Dendrobium plants exhibited high biodiversity (37 genera, about 80 species). Acremonium, Alternaria, Ampelomyces, Bionectria, Cladosporium, Colletotrichum, Fusarium, Verticillium and Xylaria were the dominant fungal endophytes. Tropical epiphytic orchids appear to vary in degree of host specificity in their endophytic fungi.     

Mycorrhizal compatibility and symbiotic reproduction of Gavilea australis,an endangered terrestrial orchid from south Patagonia

Gavilea australis is a terrestrial orchid endemic from insular south Argentina and Chile. Meeting aspects of mycorrhizal fungi identity and compatibility in this orchid species is essential for propagation and conservation purposes. These knowledge represent also a first approach to elucidate the mycorrhizal specificity of this species. In order to evaluate both the mycorrhizal compatibility and the symbiotic seed germination of G. australis, we isolated and identified its root endophytic fungal strains as well as those from two sympatric species: Gavilea lutea and Codonorchis lessonii. In addition, we tested two other strains isolated from allopatric terrestrial orchid species from central Argentina. All fungal strains formed coilings and pelotons inside protocorms and promoted, at varying degrees, seed germination, and protocorm development until seedlings had two to three leaves. These results suggest a low mycorrhizal specificity of G. australis and contribute to a better knowledge of the biology of this orchid as well as of other sympatric Patagonian orchid species, all of them currently under serious risk of extinction.     

干旱-复水对两种石斛属植物叶水势的影响

石斛属植物多附着在其他植物体或岩石上,水分获取困难,其特殊的水分利用策略是其生存和发展的重要保证.为弄清石斛属植物对干旱胁迫的适应能力和机制,该文选用3年生金钗石斛和铁皮石斛,通过盆栽控水进行干旱胁迫和复水处理,探讨在不同干旱历时和干旱后复水条件下两种石斛的叶水势变化情况.结果表明:随着干旱时间的延长,两种石斛叶水势均...     

Studies of Mycorrhizal Fungi of Chinese Orchids and Their Role in Orchid Conservation in China—A Review

China has over 1,200 species of native orchids in nearly 173 genera. About one fourth of native species are of horticultural merit. Some species are of Chinese medicinal value. In fact, the demand on orchid species with high Chinese medicinal values such as Gastrodia elata, Dendrobium offcinale, along with demands on species of cultural importance, such as those in the genus of Cymbidium, is a major factor causing wild populations to diminish and in some cases, drive wild populations to the brink of extinction. These market demands have also driven studies on the role of mycorrhizal fungi in orchid seed germination, seedling and adult growth, and reproduction. Most of these mycorrhizal studies of Chinese orchids, however, are published in Chinese, some in medical journals, and thus overlooked by the mainstream orchid mycorrhizal publications. Yet some of these studies contained interesting discoveries on the nature of the mycorrhizal relationships between orchids and fungi. We present a review of some of these neglected publications. The most important discovery comes from the mycorrhizal studies on G. elata, in which the researchers concluded that those fungi species required to stimulate seed germination are different from those that facilitate the growth of G. elata beyond seedling stages. In addition, presence of the mycorrhizal fungi associated with vegetative growth of post-seedling G. elata hindered the germination of seeds. These phenomena were unreported prior to these studies. Furthermore, orchid mycorrhizal studies in China differ from the mainstream orchid studies in that many epiphytic species (in the genus of Dendrobium, as medicinal herbs) were investigated as well as terrestrial orchids (mostly in the genus Cymbidium, as traditional horticultural species). The different responses between epiphytic and terrestrial orchid seeds to fungi derived from roots suggest that epiphytic orchids may have a more general mycorrhizal relationship with fungi than do terrestrial orchid species during the seed germination stage. To date, orchid mycorrhizal research in China has had a strongly commercial purpose. We suggest that this continuing research on orchid mycorrhizal relationships are a solid foundation for further research that includes more rare and endangered taxa, and more in-situ studies to assist conservation and restoration of the endangered orchids. Knowledge on the identities and roles of mycorrhizal fungi of orchids holds one of the keys to successful restoration and sustainable use of Chinese orchids.     

Asymbiotic germination of immature seeds,plantlet development and <Emphasis Type="Italic">ex vitro</Emphasis> establishment of plants of <Emphasis Type="Italic">Dendrobium tosaense</Emphasis> makino—A medicinally improrant orchid

Summary Shi-hu (Dendrobium spp. or Dendrobii Herba) is one of the important traditional Chinese medicines. The commercially available crude drug in the traditional medicine market is composed mainly of three species: Dendrobium tosaense, D. nobile, and D. moniliforme. An efficient method of propagation has been developed via asymbiotic germination of seeds in vitro for the medicinally important D. tosaense. Seeds from capsules of D. tosaense collected 8–14 wk after artificial pollination germinated after being cultured on full-strength or half-strength Murashige and Skoog (MS) medium devoid of plant growth regulators and with 3% sucrose. Germination of seeds varied with the medium type and seed maturity. Germinated seedlings after transfer to MS medium with 1.5% sucrose and 8% banana homogenate or potato juice or coconut water and 20 wk of incubation developed into healthy plantlets. Well-developed plantlets were transplanted to moss or moss and tree fern or tree fern as substrates in plastic trays and transferred to a greenhouse for hardening. All plants survived, attained maturity, and developed normal flower and capsule after one and a half years. This protocol of successful plant regeneration by asymbiotic seed germination should permit rapid propagation and conservation of this medicinally important Dendrobium species.     

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

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

Fungi associated with terrestrial orchid mycorrhizas,seeds and protocorms

The identity and ecological role of fungi in the mycorrhizal roots of 25 species of mature terrestrial orchids and in 17 species of field incubated orchid seedlings were examined. Isolates of symbiotic fungi from mature orchid mycorrhizas were basidiomycetes primarily in the generaCeratorhiza, Epulorhiza andMoniliopsis; a few unidentified taxa with clamped hyphae were also recovered. More than one taxon of peloton-forming fungus was often observed in the cleared and stained mycorrhizas. AlthoughCeratorhiza andEpulorhiza strains were isolated from the developing protocorms, pelotons of clamped hyphae were often presents in the cleared protocorms of several orchid species. These basidiomycetes are difficult to isolate and may be symbionts of ectotrophic plants. The higher proportion of endophytes bearing clamp connections in developing seeds than in the mycorrhizas is attributed to differences in the nutritional requirements of the fully mycotrophic protocorms and partially autotrophic plants. Most isolates ofCeratorhiza differed enzymatically fromEpulorhiza in producing polyphenol oxidases. Dual cultures with thirteen orchid isolates and five non-orchid hosts showed that some taxa can form harmless associations with non-orchid hosts. It is suggested that most terrestrial orchid mycorrhizas are relatively non-specific and that the mycobionts can be saprophytes, parasites or mycorrhizal associates of other plants.     

带叶兜兰种子原地共生萌发及有效菌根真菌的分离与鉴定

为获得带叶兜兰(Paphiopedilum hirsutissimum)种子萌发的共生真菌,采用原地共生萌发技术获得了2株自然萌发的小幼苗,并分离和筛选出了有效的种子萌发共生菌——瘤菌根菌(Epulorhiza sp.)。为验证分离菌株对带叶兜兰种子萌发的有效性,将Phs34号菌株与带叶兜兰种子在灭菌后的原生境基质上进行室内共生萌发试验,结果表明,经过6周的培养,对照组没有观察到种子的萌发;接菌的种子胚明显膨大,突破种皮,形成原球茎,平均萌发率为(58.35±3.41)%。这表明分离得到的瘤菌根菌能促进带叶兜兰的种子萌发。     

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

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

硬叶兰种子的迁地共生萌发及有效共生真菌的分离和鉴定

兰科植物的种子原地和迁地共生萌发技术是近年发展起来的开展兰科植物种子和共生真菌研究的有效方法。该研究对兰属(Cymbidium)附生植物硬叶兰(C. mannii)开展了种子的迁地共生萌发研究, 试图获得其种子萌发的有效真菌。利用硬叶兰成年植株根部周围的树皮、苔藓、枯枝落叶、腐殖质等作为培养基质, 进行种子的共生培养。在培养133天后, 成功地获得了处于不同阶段的已萌发种子、原球茎和幼苗, 并从原球茎中分离得到一种瘤菌根菌属(Epulorhiza)真菌。用所分离到的FCb4菌株和一种从兜唇石斛(Dendrobium aphyllum)分离到的胶膜菌属(Tulasnella) FDaI7菌株和硬叶兰种子在燕麦琼脂培养基上进行共生萌发, 设置不接菌作为对照处理, 以检验FCb4菌株对硬叶兰种子萌发的有效性。经过58天的培养, 不接菌的对照处理中种子没有萌发, 接种FCb4和FDaI7菌株的处理都有很高的种子萌发率, 两种接菌处理在不同光照条件下的种子萌发率均无显著性差异。但暗培养条件下, 种子萌发形成原球茎后, 表现出生长停滞的趋势, 仅有很少的原球茎继续生长达到幼苗阶段, 说明原球茎发育后期与幼苗发育阶段需要光照。在光照条件下, 接种FCb4菌株处理中达到幼苗阶段种子的比例为(25.67 ± 9.27)%, 显著高于接种FDaI7菌株处理的(3.04 ± 2.27)% (W = 56, p = 0.026, Mann-Whitney U-test), 表明此研究中分离到的瘤菌根菌属真菌能有效地促使硬叶兰种子萌发并生长发育到幼苗阶段。     

Isolation and Identification of Endophytic Fungi in Roots of Nine <Emphasis Type="Italic">Holcoglossum</Emphasis> Plants (Orchidaceae) Collected from Yunnan,Guangxi, and Hainan Provinces of China

Holcoglossum is one of the smaller genera of Orchidaceae, mainly distributed in southwest China. Some members of this genus as well as H. rupestre and H. flavescens are endemic and rare Chinese orchids. As far as we know, little work has been done concerning the relationships between the Holcoglossum plants and endophytic microorganisms. In this study, 46 culturable fungal endophytes were isolated and identified from roots of nine Holcoglossum plants collected from Yunnan, Guangxi, and Hainan provinces of China based on molecular techniques. The results showed that all strains belonged to four classes, i.e., Sordariomycetes (41.30%), Dothideomycetes (36.96%), Agaricomycetes (17.39%), Leotiomycetes (4.35%). Thirty-six strains were identified at the genus level, including Alternaria, Cladosporium, Clonostachys, Colletotrichum, Cosmospora, Cryptosporiopsis, Cylindrocarpon, Didymella, Epulorhiza (Anamorphic Tulasnella), Fusarium, Myrmecridium, Leptosphaeria, Paraconiothyrium, Phomopsis, Pyrenochaeta, and Stephanonectria. Fusarium and Epulorhiza (Anamorphic Tulasnella) were the dominant fungal endophytes. Some orchids mycorrhizal fungi as well as Tulasnella calospora and Epulorhiza sp. were found in roots. This is the first report concerning endophytic fungi from Holcoglossum plants (Orchidaceae), suggesting that endophytic fungi in Holcoglossum plants are very abundant.     

实验室条件下五唇兰菌根真菌专一性研究

利用从高原温带兰科植物菌根中获得的22个菌根真菌菌株, 对五唇兰(Doritis pulcherrima)进行了室内种子萌发、原球茎分化和组培苗回接试验, 从交叉回接的角度对附生兰科植物与菌根真菌的生理专一性进行了探讨。经过20周的共生培养, 只有编号为Cf1和Mm1的两个菌株使种子表现出种胚明显膨大的萌发迹象; 9个菌株能够促使原球茎较好地分化发育出根叶; 11个菌株处理苗的平均鲜重增长率高于对照组(156.25%), 其中Mm1的效果达到极显著水平(p = 0.01)。通过根切片显微观察, 在原球茎分化根和回接效果良好的处理苗的根皮层组织发现典型的菌丝团结构, 表明菌根体系已成功建立。温带地生兰菌根真菌对五唇兰种子萌发、原球茎发育和幼苗生长等3个重要生长阶段影响的试验显示, 五唇兰的种子和菌根真菌的共生萌发效果不佳, 而原球茎及幼株更容易与之建立良好的共生关系。同时, 也没有发现同一个真菌菌株能够对五唇兰的种子、原球茎和幼苗均产生促进作用。研究结果表明, 五唇兰的菌根真菌专一性因生理生长阶段的不同而存在差异。     

Mycorrhizal fungi of Vanilla: diversity, specificity and effects on seed germination and plant growth

Mycorrhizal fungi are essential for the germination of orchid seeds. However, the specificity of orchids for their mycorrhizal fungi and the effects of the fungi on orchid growth are controversial. Mycorrhizal fungi have been studied in some temperate and tropical, epiphytic orchids, but the symbionts of tropical, terrestrial orchids are still unknown. Here we study diversity, specificity and function of mycorrhizal fungi in Vanilla, a pantropical genus that is both terrestrial and epiphytic. Mycorrhizal roots were collected from four Vanilla species in Puerto Rico, Costa Rica and Cuba. Cultured and uncultured mycorrhizal fungi were identified by sequencing the internal transcribed spacer region of nuclear rDNA (nrITS) and part of the mitochondrial ribosomal large subunit (mtLSU), and by counting number of nuclei in hyphae. Vanilla spp. were associated with a wide range of mycorrhizal fungi: Ceratobasidium, Thanatephorus and Tulasnella. Related fungi were found in different species of Vanilla, although at different relative frequencies. Ceratobasidium was more common in roots in soil and Tulasnella was more common in roots on tree bark, but several clades of fungi included strains from both substrates. Relative frequencies of genera of mycorrhizal fungi differed significantly between cultured fungi and those detected by direct amplification. Ceratobasidium and Tulasnella were tested for effects on seed germination of Vanilla and effects on growth of Vanilla and Dendrobium plants. We found significant differences among fungi in effects on seed germination and plant growth. Effects of mycorrhizal fungi on Vanilla and Dendrobium were similar: a clade of Ceratobasidium had a consistently positive effect on plant growth and seed germination. This clade has potential use in germination and propagation of orchids. Results confirmed that a single orchid species can be associated with several mycorrhizal fungi with different functional consequences for the plant.     

Diversity and Taxonomy of Endophytic Xylariaceous Fungi from Medicinal Plants of Dendrobium (Orchidaceae)

Dendrobium spp. are traditional Chinese medicinal plants, and the main effective ingredients (polysaccharides and alkaloids) have pharmacologic effects on gastritis infection, cancer, and anti-aging. Previously, we confirmed endophytic xylariaceous fungi as the dominant fungi in several Dendrobium species of tropical regions from China. In the present study, the diversity, taxonomy, and distribution of culturable endophytic xylariaceous fungi associated with seven medicinal species of Dendrobium (Orchidaceae) were investigated. Among the 961 endophytes newly isolated, 217 xylariaceous fungi (morphotaxa) were identified using morphological and molecular methods. The phylogenetic tree constructed using nuclear ribosomal internal transcribed spacer (ITS), large subunit of ribosomal DNA (LSU), and beta-tubulin sequences divided these anamorphic xylariaceous isolates into at least 18 operational taxonomic units (OTUs). The diversity of the endophytic xylariaceous fungi in these seven Dendrobium species was estimated using Shannon and evenness indices, with the results indicating that the dominant Xylariaceae taxa in each Dendrobium species were greatly different, though common xylariaceous fungi were found in several Dendrobium species. These findings implied that different host plants in the same habitats exhibit a preference and selectivity for their fungal partners. Using culture-dependent approaches, these xylariaceous isolates may be important sources for the future screening of new natural products and drug discovery.     

树兰原球茎内生细菌群落多样性及功能分析

【目的】通过分析树兰原球茎内生细菌群落组成、多样性特征和促生功能,探究树兰种子萌发相关的核心细菌类群及生物学功能。【方法】以树兰原球茎(树兰种子在树皮基质上共生萌发、在树叶基质上共生萌发、在MS1培养基上非共生萌发)和共生萌发基质(松树皮、腐熟树叶)共5个样本为研究材料,采用高通量测序技术分析不同萌发条件下原球茎内生细菌的16S rRNA基因多样性,比较分析细菌群落多样性和物种组成特征,通过传统的内生细菌分离方法获得共生萌发原球茎内生细菌菌株,并进行促生潜力评价。【结果】从5个研究样本中共获得2 735个可操作分类单元(operational taxonomic unit, OTU),属于41门453科876属,其中变形菌门(Proteobacteria)和放线菌门(Actinobacteria)为优势门。主坐标分析(principal coordinates analysis, PCoA)结果表明,树兰原球茎与萌发基质细菌群落结构存在差异,非共生萌发原球茎与在树皮基质上共生萌发原球茎内生细菌群落结构最为接近。功能预测表明,在树叶基质上共生萌发的原球茎内生细菌固氮功能显著高于其他萌发条件。通过分离培养,共获得内生细菌19株,分属12属16种,其中鞭毛膨胀芽孢杆菌(Tumebacillus flagellatus)、Bradyrhizobium cenepequi和人参腐殖土魏茨曼氏菌(Weizmannia ginsengihumi)为共生萌发原球茎共有种;韩国假单胞菌(Pseudomonaskoreensis)和 W. ginsengihumi兼具有溶磷、产吲哚乙酸(indole-3-acetic acid, IAA)和铁载体的潜在能力。【结论】在不同环境中萌发的树兰原球茎均有丰富的内生细菌群落定殖;从共生萌发原球茎中分离的内生细菌具有固氮、溶磷、产IAA和铁载体等促生功能。本研究为兰科植物种子萌发相关微生物资源挖掘及兰科植物与微生物互作研究提供科学依据。