Bacteria associated with the roots of epiphytic orchids

This work is the first to report the isolation and identification of bacteria colonizing the roots of tropical epiphytic orchids Acampe papillosa (Lindl.) Lindl. and Dendrobium moschatum (Buch.-Ham.) Swartz. and bacteria inhabiting inner layers of the aerial and substrate roots of A. papillosa. We showed by the example of this epiphyte that associative bacteria are present in large amounts on the aerial but not substrate roots. We isolated and identified bacteria from the substrate roots of D. moschatum and from its growth substrate (pine bark). The structure of the intercellular matrix of the associative bacteria was studied.     

海南霸王岭不同森林类型附生兰科植物的多样性和分布

附生兰科植物是热带林附生植物的主要类群之一,对于维持热带林生态系统的物种多样性及生态功能具有重要的作用。以海南岛霸王岭国家级自然保护区内的6种热带原始林类型(热带季雨林、低地雨林、热带针叶林、山地雨林、山地常绿林及山顶矮林)中的附生兰科植物为研究对象,通过样带调查(每个森林类型设置12个10m×50m的样带,记录每个样带内胸径(DBH)≥5cm的树木及藤本上附生兰科植物的物种名称、株数及附生位置)分析了附生兰科植物的物种多样性、附生位置及其在不同森林类型中的分布规律。结果表明:1)3.6hm2森林调查样带内共记录到附生兰科植物9634株,分属于26属60种;2)除趋势对应分析(DCA)结果表明,6种森林类型中的附生兰科植物可分成5组(其中,山地常绿林与山顶矮林内的附生兰科植物归为一组);3)分布海拔范围相临近的森林类型的附生兰科植物具有较高的相似性,山地常绿林和山顶矮林附生兰科植物的相似性最高(88.9%);4)6种森林类型中,较高海拔的3种森林类型(山地雨林、山地常绿林和山顶矮林)中,附生兰科植物的丰富度和多度均显著高于其在较低海拔的3种森林类型(热带季雨林、低地雨林和热带针叶林),其中,附生兰科植物在山地常绿林内的丰富度和多度均最高;5)热带季雨林、低地雨林、热带针叶林及山地雨林内,宿主冠区附生兰科植物的多度均高于干区;山地常绿林内两者之间无显著差异;而山顶矮林干区的附生兰科植物的多度高于冠区;6)调查木上附生兰科植物的发生率在高海拔森林类型均高于其在低海拔森林类型,各森林类型内附生兰科植物的多度及物种丰富度与宿主胸径均存在显著正相关关系。     

Auxin production by bacteria associated with orchid roots

Bacteria associated with the roots of greenhouse tropical orchids were shown to produce indole-3-acetic acid (IAA) and to excrete it into the culture liquid. The presence and activity of IAA were demonstrated colorimetrically, by thin-layer chromatography, and by biotests. The associated bacteria varied in their ability to excrete indole compounds (1–28 µg/ml nutrient broth). Addition of tryptophan to the growth medium enhanced phytohormone production. Upon addition of 200 µg/ml tryptophan, the bacteria isolated from Dendrobium moschatum roots (Sphingomonas sp. 18, Microbacterium sp. 23, Mycobacterium sp. 1, Bacillus sp. 3, and Rhizobium sp. 5) produced 50.2, 53.1, 92.9, 37.6, and 60.4 µg IAA/ml, respectively, while the bacteria isolated from Acampe papillosa roots (Sphingomonas sp. 42, Rhodococcus sp. 37, Cellulomonas sp. 23, Pseudomonas sp. 24, and Micrococcus luteus) produced 69.4, 49.6, 53.9, 31.0, and 39.2 µg IAA/ml. Auxin production depended on cultivation conditions and on the growth phase of the bacterial cultures. Treatment of kidney bean cuttings with bacterial culture liquid promoted formation of a root brush with a location height 7.4- to 13.4-fold greater than the one in the control samples. The ability of IAA-producing associated bacteria to act as stimulants of the host plant root development is discussed.Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 55–62.Original Russian Text Copyright © 2005 by Tsavkelova, Cherdyntseva, Netrusov.     

Associative cyanobacteria isolated from the roots of epiphytic orchids

Associative cyanobacteria were isolated from the rhizoplane and velamen of the aerial roots of the epiphytic orchids Acampe papillosa, Phalaenopsis amabilis, and Dendrobium moschatum and from the substrate roots of Acampe papillosa and Dendrobium moschatum. Cyanobacteria were isolated on complete and nitrogen-free variants of BG-11 medium. On all media and in all samples, cyanobacteria of the genus Nostoc predominated. Nostoc, Anabaena, and Calothrix were isolated from the surface of the A. papillosa aerial roots, whereas the isolates from the substrate roots were Nostoc, Oscillatoria, and representatives of the LPP-group (Lyngbia, Phormidium, and Plectonema, incapable of nitrogen fixation). On the D. moschatum substrate roots, Nostoc and LPP-group representatives were also found, as well as Fischerella. On the aerial roots of P. amabilis and D. phalaenopsis grown in a greenhouse simulating the climate of moist tropical forest, cyanobacteria were represented by Nostoc, LPP-group, and Scytonema in the D. phalaenopsis and by Nostoc, Scytonema, Calothrix, Spirulina, Oscillatoria, and the LPP-group in P. amabilis. For D. moschatum, the spectra of cyanobacteria populating the substrate root zhizophane and the substrate (pine bark) were compared. In the parenchyma of the aerial roots of P. amabilis, fungal hyphae and/or their half-degraded remains were detected, which testifies to the presence of mycorrhizal fungi this plant. This phenomenon is attributed to the presence of a sheath formed by cyanobacteria and serving as a substrate for fungi.     

Localization of associative cyanobacteria on the roots of epiphytic orchids

This work is the first study of the localization of phototrophic microorganisms in the rhizoplane and velamen of epiphytic orchids, namely on the aerial and substrate roots of Acampe papillosa and Dendrobium moschatum and on the aerial roots of the Phalaenopsis amabilis and Dendrobium phalaenopsis. The composition of the bacterial community on the plant roots depended on the conditions of plant growth. Under conditions simulating climate of moist tropical forests, the aerial roots proved to be populated with phototrophic microorganisms among which cyanobacteria predominated. Interlaced fungal hyphae and filamentous cyanobacteria formed a sheath on the surface of aerial roots. The nitrogen-fixing capacity of the sheath of aerial roots was studied by the example of P. amabilis.     

西双版纳片段化石灰岩森林附生兰科植物多样性研究

西双版纳石灰岩地区拥有丰富的兰科植物资源,但近年来随着橡胶树的大面积种植,使得该地区很多石灰岩森林呈片段化。为了解片段化石灰岩森林中附生兰科植物多样性状况以及片段化对附生兰科植物的影响,该研究选取了生境片段化的青岩寨和曼纳览,以及连续生境的绿石林和巴卡新寨等4个样地的29个样方进行多样性调查和对比研究。结果表明:4个石灰岩森林样共记录到附生兰科植物34属76种1 528株(丛)。通过对坡向、坡度、海拔、地形、郁闭度和森林类型等6个环境因子与附生兰科植物丰富度进行CCA分析,发现石灰岩地区附生兰科植物的分布主要受海拔和森林类型2个因素的影响,在海拔较高的青岩寨和巴卡新寨附生兰科植物物种丰富度高于低海拔的绿石林和曼纳览,而片段化对附生兰科植物的物种多样性和多度均无显著影响,这可能与片段化的历史较短有关。虽然目前来看生境片段化对石灰岩地区的附生兰科植物多样性无显著影响,但生境脆弱的石灰岩森林植被的保护对于兰科植物多样性保护则更具重要性。     

The photosynthetic pathway of the roots of twelve epiphytic orchids with CAM leaves

The photosynthetic pathway of the roots (both the white velamentous main portions and the green, nonvelamentous tips) was investigated in twelve taxa (natural species and intergeneric hybrid cultivars) of epiphytic orchids having CAM leaves. All organs contained chlorophyll, and the a/b ratios indicate that the organs, especially the roots, are likely shade-adapted. Stable carbon isotope ratios of the tissues were near −15‰ for all organs, a value typical of obligate (constitutive) CAM plants. Values for root tissues were slightly lower (more negative) than those of the leaves. The presence of CAM in the leaves of these orchids did not ensure that their roots performed CAM photosynthesis. Further work is needed to address the questions raised in this study and to determine if the photosynthetic roots of these taxa are capable of assimilating atmospheric CO₂.     

Pollination ecology of four epiphytic orchids of New Zealand

BACKGROUND AND AIMS: In New Zealand epiphytic orchids are represented by four genera and eight species. The genera Earina (three species) and Winika (one species) are the most conspicuous and widespread. These are likely to be some of the southernmost distributed genera of epiphytic orchids in the world. METHODS: To identify the pollination strategies that have evolved in these orchids, hand-pollination treatments were done and floral visitors were observed in several wild populations at two areas of southern North Island (approx. 40 degrees S). Pollen:ovule ratio and osmophores were also studied and the total carbohydrate content of the nectar produced by each species was measured. KEY RESULTS: Earina autumnalis and Earina mucronata are self-compatible, whereas Earina aestivalis and Winika cunninghamii appear to be partially self-incompatible. All four orchids are incapable of autonomous selfing and therefore completely dependent on pollinators to set fruits. Floral visitors observed in the genus Earina belong to Diptera, Coleoptera and Hymenoptera and to Diptera and Hymenoptera in W. cunninghamii. CONCLUSIONS: Contrary to many epiphytic orchids in the tropics, the orchid-pollinator relationship in these orchids is unspecialized and flowers are visited by a wide range of insects. Putative pollinators are flies of the families Bibionidae, Calliphoridae, Syrphidae and Tachinidae. All four orchids display anthecological adaptations to a myophilous pollination system such as simple flowers, well-exposed reproductive structures, easily accessed nectar and high pollen : ovule ratios.     

Bacteria associated with orchid roots and microbial production of auxin

Associative bacteria of terrestrial (Paphiopedilum appletonianum) and epiphytic (Pholidota articulata) tropical orchids were investigated. Microbial community of epiphytic plant differed from that of the terrestrial one. Streptomyces, Bacillus, Pseudomonas, Burkholderia, Erwinia and Nocardia strains populated Paphiopedilum roots, whereas Pseudomonas, Flavobacterium, Stenotrophomonas, Pantoea, Chryseobacterium, Bacillus, Agrobacterium, Erwinia, Burkholderia and Paracoccus strains colonized Pholidota roots. Endophytic bacteria populations were represented with less diversity: Streptomyces, Bacillus, Erwinia and Pseudomonas genera were isolated from P. appletonianum, and Pseudomonas, Bacillus, and Flavobacterium genera were isolated from Ph. articulata. Microorganisms produced indole-3-acetic acid (IAA). Variations in its biosynthesis among the strains of the same genus were also observed. The highest auxin level was detected during the stationary growth phase. Biological activity of microbial IAA was proved by treatment of kidney bean cuttings with bacterial supernatants, revealing considerable stimulation of root formation and growth.     

Endophytic diversity of hanging velamen roots in the epiphytic orchid Acampe praemorsa

ABSTRACT

Background

The ecological functions of the aerial hanging roots (velamen roots) in orchids are not yet fully understood.     

Influence of mycorrhizal fungi on seed germination and growth in terrestrial and epiphytic orchids

Epiphytes constitute over 70% of orchid diversity, but little is known about the functioning of their mycorrhizal associations. Terrestrial orchid seeds germinate symbiotically in soil and leaf litter, whereas epiphytic orchids may be exposed to relatively high light levels from an early stage of development and often produce green seeds. This suggests that seedlings of the two groups of orchids may differ in their responses to light and requirements for mycorrhiza-supplied carbon. The interactive effects of light, exogenous carbon and mycorrhizal status on germination and growth were investigated in vitro using axenic agar microcosms for one tropical epiphyte and three geophytic orchid species. The geophytic species strongly depended on their mycorrhiza for growth and this could not be substituted by exogenous sucrose, whereas the epiphytic species achieved 95% of the mycorrhizal seedling volume when supplied with exogenous sucrose in the dark. Mycorrhiza status strongly interacted with light exposure, enabling germination. Light inhibited or severely reduced growth, especially for the terrestrial orchids in the absence of mycorrhiza. For the first time, this study showed the parallel ecological importance of mycorrhizal fungi in overcoming light inhibition of seed germination and growth in both terrestrial and epiphytic orchids.     

Deceptive orchids with Meliponini as pollinators

Visitation of orchids by Meliponini (stingless bees) is confirmed only in 13Melipona, Partamona andTrigona, forXylobium andMaxillaria, with the addition ofTrigona fulviventris visitingIonopsis. Some bees evinced multiple floral visitation by carrying several stipes and viscidia from pollinaria, thus may cause seed set. None foraged pseudopollen, nor is collection of this substance by bees verified. Meliponine-visited orchids had pollinia in quartets with emplacement on the bee's scutellum, possibly devices for pollinia survival on a social bee passing through its nest. Further, orchids produced no nectar, but bees repeatedly came to flowers. A testable basis for the orchid-meliponine relationship is mimicry of rewarding resources, or bee pheromone mimicry, recently documented for some honey bees. Meliponine pheromone analogs (nerol and 2-heptanol) are here noted forMaxillaria, but lack of foraging with pheromones byMelipona suggests multiple avenues of mimicry by orchids, including alarm pheromone and carrion mimicry.     

中国兰科植物保育的现状和展望

兰科植物是植物保育中的“旗舰”类群 (flaggroup)。中国不是兰科植物种类最丰富的地区 ,但具有最复杂多样的地理分布类型以及众多的原始类群 ,因此 ,开展对中国兰科植物的研究和保育是世界兰科植物研究和保育工作中的重要组成部分。本文介绍了目前中国兰科植物研究和保育的现状 ,分析了与国际同类工作相比存在的差距 ,并对今后的发展方向提出了一些看法     

Population dynamics of epiphytic orchids in a metapopulation context

Background and Aims

Populations of many epiphytes show a patchy distribution where clusters of plants growing on individual trees are spatially separated and may thus function as metapopulations. Seed dispersal is necessary to (re)colonize unoccupied habitats, and to transfer seeds from high- to low-competition patches. Increasing dispersal distances, however, reduces local fecundity and the probability that seeds will find a safe site outside the original patch. Thus, there is a conflict between seed survival and colonization.

Methods

Populations of three epiphytic orchids were monitored over three years in a Mexican humid montane forest and analysed with spatially averaged and with spatially explicit matrix metapopulation models. In the latter, population dynamics at the scale of the subpopulations (epiphytes on individual host trees) are based on detailed stage-structured observations of transition probabilities and trees are connected by a dispersal function.

Key Results

Population growth rates differed among trees and years. While ignoring these differences, and averaging the population matrices over trees, yields negative population growth, metapopulation models predict stable or growing populations because the trees that support growing subpopulations determine the growth of the metapopulation. Stochastic models which account for the differences among years differed only marginally from deterministic models. Population growth rates were significantly lower, and extinctions of local patches more frequent in models where higher dispersal results in reduced local fecundity compared with hypothetical models where this is not the case. The difference between the two models increased with increasing mean dispersal distance. Though recolonization events increased with dispersal distance, this could not compensate the losses due to reduced local fecundity.

Conclusions

For epiphytes, metapopulation models are useful to capture processes beyond the level of the single host tree, but local processes are equally important to understand epiphyte population dynamics.     

Aerial roots of epiphytic orchids: the velamen radicum and its role in water and nutrient uptake

The velamen radicum, a spongy, usually multiple epidermis of the roots, which at maturity consists of dead cells, is frequently described as an important adaptation of epiphytic orchids. Yet, quantitative evidence for the alleged functions, e.g., efficient water and nutrient uptake, nutrient retention, reduction of water loss, mechanical protection, or the avoidance of overheating, is rare or missing. We tested the notion originally put forward by Went in 1940 that the velamen allows plants to capture and immobilize the first solutions arriving in a rainfall, which are the most heavily charged with nutrients. In a series of experiments, we examined whether all necessary functional characteristics are given for this scenario to be realistic under ecological conditions. First, we show that the velamen of a large number of orchid species takes up solutions within seconds, while evaporation from the velamen takes several hours. Charged ions are retained in the velamen probably due to positive and negative charges in the cell walls, while uncharged compounds are lost to the external medium. Finally, we demonstrate that nutrient uptake follows biphasic kinetics with a highly efficient, active transport system at low external concentrations. Thus, our results lend strong support to Went’s hypothesis: the velamen fulfills an important function in nutrient uptake in the epiphytic habitat. Most of the other functions outlined above still await similar experimental scrutiny.     

Strong phylogenetic congruence between Tulasnella fungi and their associated Drakaeinae orchids

The study of congruency between phylogenies of interacting species can provide a powerful approach for understanding the evolutionary history of symbiotic associations. Orchid mycorrhizal fungi can survive independently of orchids making cospeciation unlikely, leading us to predict that any congruence would arise from host-switches to closely related fungal species. The Australasian orchid subtribe Drakaeinae is an iconic group of sexually deceptive orchids that consists of approximately 66 species. In this study, we investigated the evolutionary relationships between representatives of all six Drakaeinae orchid genera (39 species) and their mycorrhizal fungi. We used an exome capture dataset to generate the first well-resolved phylogeny of the Drakaeinae genera. A total of 10 closely related Tulasnella Operational Taxonomic Units (OTUs) and previously described species were associated with the Drakaeinae orchids. Three of them were shared among orchid genera, with each genus associating with 1–6 Tulasnella lineages. Cophylogenetic analyses show Drakaeinae orchids and their Tulasnella associates exhibit significant congruence (p < 0.001) in the topology of their phylogenetic trees. An event-based method also revealed significant congruence in Drakaeinae–Tulasnella relationships, with duplications (35), losses (25), and failure to diverge (9) the most frequent events, with minimal evidence for cospeciation (1) and host-switches (2). The high number of duplications suggests that the orchids speciate independently from the fungi, and the fungal species association of the ancestral orchid species is typically maintained in the daughter species. For the Drakaeinae–Tulasnella interaction, a pattern of phylogenetic niche conservatism rather than coevolution likely explains the observed phylogenetic congruency in orchid and fungal phylogenies. Given that many orchid genera are characterized by sharing of fungal species between closely related orchid species, we predict that these findings may apply to a wide range of orchid lineages.     

In vitro flowering of orchids

Abstract

Flowering is the most elusive and fascinating of all plant developmental processes. The ability to induce flowering in vitro in orchids would reduce the relatively long juvenile phase and provide deeper insight into the physiological, genetic and molecular aspects of flowering. This review synthesizes all available studies that have been conducted on in vitro flowering of orchids with the objective of providing valuable clues as to the mechanism(s) that is possibly taking place.     

腐生型兰科植物研究进展

腐生型兰科植物又称完全菌根异养型兰(Fully Mycoheterotrophic Orchids),无绿叶,不含叶绿素,不能进行光合作用制造有机物,完全依靠与其共生的真菌提供营养。近年来,腐生兰独特的生活方式引起了生物学家对其生理生态及进化问题的广泛关注,但目前仍缺乏系统深入的研究。为此,作者通过整理《中国植物志》和近年来报道的相关文献,发现分布在中国的腐生型兰科植物约有23属81种,主要集中在天麻属(Gastrodia)(24种)、鸟巢兰属(Neottia)(8种)、无叶兰属(Aphyllorchis)(6种)、山珊瑚属(Galeola)(5种)等。由于腐生兰特殊的营养需求,相比于其他营养类型的兰科植物,其菌根真菌有较大区别。腐生兰的菌根真菌主要有两大类,即外生菌根真菌(Ectomycorrhizal fungi,ECM)和非丝核菌类的腐生菌(Non-rhizoctonia SAP fungi)。该文还综述了腐生兰与其菌根真菌的专一性、营养来源以及系统进化等问题,提出了目前研究存在的问题,并对今后的研究工作进行了展望,以期为腐生兰的资源保护及再生研究提供参考。     

The chemical composition,antifungal, antioxidant and antimutagenicity properties of bioactive compounds from fungal endophytes associated with Thai orchids

Some plant‐derived bioactive compounds produced by fungal endophytes have been proven to have antimicrobial and antioxidant activities. In this study, endophytic fungi were isolated from 20 orchid samples collected in northern Thailand from 12 genera of orchids. In total, 97 isolates were isolated from the leaves (44.3%), stems (40.2%) and flowers (15.5%) of the orchid samples. The antifungal activity was investigated of the endophytic isolates against the plant pathogenic fungi. The results showed that 13 endophytic isolates provided antifungal activities against Fusarium sp., Colletotrichum sp. and Curvularia sp. The endophyte CK F05‐5, which was isolated from the flower part of Dendrobium lindleyi, was chosen for further testing because it the highest level of antifungal activity against Fusarium sp. The isolate CK F05‐5 was identified as Fusarium oxysporum on the basis of its ITS sequences of 5.8 s rRNA, and phytochemical analysis revealed the presence of coumarins. The ethyl acetate extract of CK F05‐5 was examined for its total phenolic content and antioxidant activity using Folin–Ciocalteu's reagent and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical scavenging assay, respectively. The phenolic content was 160.51 mg of GAE/g of extract, and the free radical scavenging activity was 89.61 µg/ml at the half maximal inhibitory concentration (IC₅₀). The antimutagenic potential of the ethyl acetate extract of CK F05‐5 against Trp‐P‐1 mutagenic substances was determined using the Ames test which revealed that the extract of CK F05‐5 at 10 mg/plate had the highest antimutagenic activity against Trp‐P‐1 (51.2%) and 39.6% for strains TA98 and TA100, respectively. The active compounds present in the acetate extract of CK F05‐5 were examined using GC‐MS analysis, which displayed the presence of gibepyrone A, pyrrolo [1, 2‐a] pyrazine‐1, 4‐dione, hexahydro‐3‐(2‐methylpropyl) and indoleacetic acid as major components. Based on the results, this endophytic fungus contains various bioactive components that have various biological activities. This useful information could help in producing potentially valuable and novel pharmaceutical products.     

辽宁省9种兰科植物根内与根际土壤中真菌群落结构的差异

兰科植物的生存及生长高度依赖其根中的共生真菌, 其中的菌根真菌更是对兰科植物的种子萌发与后续生长有着非常重要的作用, 研究兰科植物根中的真菌, 尤其是菌根真菌, 对兰科植物的保护有重要作用。该研究利用第二代测序技术, 对中国辽宁省境内的9种属于极小种群的兰科植物的根、根际土和根围土中的真菌群落和菌根真菌组成进行了研究。结果显示, 兰科植物根中的真菌群落和根际土、根围土中的真菌群落具有显著差异。兰科植物根中的总操作分类单元(OTU)数目远小于根际土和根围土中的总OTU数目。同时, 兰科植物根中菌根真菌的种类和丰度与根际土、根围土中菌根真菌的种类与丰度没有明显联系。FunGuild分析结果显示, 丛枝菌根真菌在根际土与根围土中的丰度非常高, 但在兰科植物的根中却数量极少。这些结果表明, 兰科植物根中的真菌群落与土壤中的真菌群落在一定程度上是相互独立的。