墨兰共生真菌一新种的分离、培养、鉴定及其生物活性

在对产于云南西双版纳的热带兰花菌根进行研究时,发现一分离自墨兰的菌株,菌丝纯白色,具典型的锁状连合,对其子实体的鉴定表明该菌株代表着一新分类单位——兰小菇Mycena orchidicola Fan et Guo sp.nov.同时,对该新种在兰科种子萌发中的作用作了初步探讨,结果表明该菌可侵入兰科种子,促进种子萌发。     

兰科菌根研究综述

兰科菌根是一种内生菌根,主要寄生于兰科（Orchidaceae）植物的种子及根系上。对兰科菌根真菌的分类及真菌资源多样性、兰科菌根的形态和菌根对兰科植物的效应等最新研究进展进行了综述。目前研究已知,感染兰科植物根部并能与之共生的真菌绝大多数属于担子菌门（Basidiomycota）和半知菌门（Deuteromycotha）,也有部分属于子囊菌门（Ascomycota）;兰科菌根的形成可分为两种情况：一是对兰科植物种子的侵染;二是对成长新根的侵染。菌根真菌对兰科植物的种子萌发及植株生长发育均有一定影响。     

Tansley Review No. 110.

S UMMARY 367
I. I NTRODUCTION 367
II. N UMBER 368
III. S IZE 379
IV. A IR SPACE IN THE SEEDS 381
V. F LOATATION AND DISPERSAL 383
1. Air 383
(a) Physical considerations 383
(b) Dispersal 387
(c) Birds 415
2. Water 416
(a) Physical considerations 416
(b) Dispersal 416
VI. C ONCLUSIONS 417
Acknowledgements 417
References 418
Orchid seeds are very small, extremely light and produced in great numbers. Most range in length from c . 0.05 to 6.0 mm, with the difference between the longest and shortest known seeds in the family being 120-fold. The 'widest' seed at 0.9 mm is 90-fold wider than the 'thinnest' one, which measures 0.01 mm (because orchid seeds are tubular or balloon-like, 'wide' and 'thin' actually refer to diameter). Known seed weights extend from 0.31 lg to 24 μg (a 78-fold difference). Recorded numbers of seeds per fruit are as high as 4000000 and as low as 20–50 (80000–200000-fold difference). Testae are usually transparent, with outer cell walls that may be smooth or reticulated. Ultrasonic treatments enhance germination, which suggests that the testae can be restrictive. Embryos are even smaller: their volume is substantially smaller than that of the testa. As a result, orchid seeds have large internal air spaces that render them balloon-like. They can float in the air for long periods, a property that facilitates long-distance dispersal. The difficult-to-wet outer surfaces of the testa and large internal air spaces enable the seeds to float on water for prolonged periods. This facilitates distribution through tree effluates and/or small run-off rivulets that may follow rains. Due to their size and characteristics, orchid seeds may also be transported in and on land animals and birds (in fur, feathers or hair, mud on feet, and perhaps also following ingestion).     

Formation of endomycorrhizas by an achlorophyllous orchid,Erythrorchis ochobiensis, andAuricularia polytricha

To test the mycorrhizal function of heterobasidiomycetous fungi on achlorophyllous orchids and to examine the symbiotic fungal range of a myco-heterotrophic orchid,Erythrorchis ochobiensis, synthetic cultures of the orchid seed were carried out withAuricularia polytricha isolates from Japan and Mexico. After three and a half mo of incubation, 57.0–70.7% of seeds germinated but none of them showed further growth. When cultured on peat moss at 25°C, the germination rate was 8.7% in the presence of Mexican isolate and 18.0% in the presence of Japanese isolate. Some germinated seeds developed into protocorms, and several seeds incubated with the Mexican isolate developed into plantlets after 5 mo. Pelotons were observed in the cells of protocorms and roots. The results indicated that some heterobasidiomycetous fungi could form endomycorrhizas with a myco-heterotrophic orchid. The results also showed that the symbiont ofE. ochobiensis extends, at least experimentally, to Heterobasidiomycetes. The variances of germination rate and seedling growth were suggested to be affected by the difference of isolates and culture conditions.     

Orchid seed removal by ants in Neotropical ant‐gardens

• Most plants that inhabit ant‐gardens (AGs) are cultivated by the ants. Some orchids occur in AGs; however, it is not known whether their seeds are dispersed by AG ants because most orchid seeds are tiny and dispersed by wind.
• We performed in situ seed removal experiments, in which we simultaneously provided Azteca gnava ants with seeds of three AG orchid species and three other AG epiphyte species (Bromeliaceae, Cactaceae and Gesneriaceae), as well as the non‐AG orchid Catasetum integerrimum.
• The seeds most removed were those of the bromeliad Aechmea tillandsioides and the gesneriad Codonanthe uleana, while seeds of AG orchids Coryanthes picturata, Epidendrum flexuosum and Epidendrum pachyrachis were less removed. The non‐AG orchid was not removed. Removal values were positively correlated with the frequency of the AG epiphytes in the AGs, and seeds of AG orchids were larger than those of non‐AG orchids, which should favour myrmecochory.
• Our data show that Azt. gnava ants discriminate and preferentially remove seeds of the AG epiphytes. We report for the first time the removal of AG orchid seeds by AG ants in Neotropical AGs.

     

兰科植物种子共生萌发真菌多样性及共生萌发机制研究进展

自然环境下,兰科植物种子细小无胚乳,需要和适宜的真菌共生才能萌发,因而与真菌有天然的共生关系。自身繁殖率低加之近年来栖息地环境破坏导致兰科植物资源更加濒危,而通过筛选适合的真菌进行种子的共生萌发可以有效地实现兰科植物的种质保育及濒危种类野生居群的生态恢复。本文对地生型、附生型以及腐生型等兰科植物已发现的萌发真菌的多样性进行了系统地梳理,发现担子菌门的胶膜菌科、角担菌科以及蜡壳耳目真菌为已报道共生萌发真菌的主要类群;同时对兰科植物种子的共生萌发机制,包括形态学机制、营养机制和分子机制等方面的相关研究进行了归纳论述,但是当前关于兰科植物和真菌互作机制方面的研究还相对较少,许多问题需要进一步明确。本文对共生萌发真菌在兰科植物保育和繁育中的应用以及共生萌发机制的研究等方面具有一定的参考价值。     

Orchid-associated bacteria produce indole-3-acetic acid,promote seed germination,and increase their microbial yield in response to exogenous auxin

Germination of orchid seeds is a complex process. In this paper we focus on interactions between the host-plant and its bacterial partners via indole-3-acetic acid (IAA). Originally isolated from the roots of the epiphytic orchid Dendrobium moschatum, the strains of Rhizobium, Microbacterium, Sphingomonas, and Mycobacterium genera were among the most active IAA producers. Addition of exogenous tryptophan significantly enhanced auxin formation both in mineral and complex media. The presence of IAA and indole-3-acetaldehyde was confirmed by HPLC. Indole-3-pyruvic and indole-3-lactic acids were also detected in supernatants of culture filtrates of Sphingomonas sp., Rhizobium sp., and Microbacterium sp., while indole-3-acetamide was identified only in Mycobacterium sp. Some concentration- and strain-dependent effects of exogenous IAA on bacterial development were also established. Treatment of the cultures with 10 and 100 μg/ml of auxin resulted in an increase in microbial yield. None of the investigated strains was able to utilize IAA as a source of carbon and energy. Furthermore, inoculation of D. moschatum seeds with Sphingomonas sp. and Mycobacterium sp. resulted in considerable enhancement of orchid seeds germination. This growth-promoting activity was observed in the absence of any plant growth stimulators or mycorrhizal fungi, usually required for orchid germination.     

Absence of Recruitment Limitation in Restored Dune Slacks Suggests That Manual Seed Introduction Can Be a Successful Practice for Restoring Orchid Populations

Answering the question whether or not to introduce seeds of target species in restored habitats depends on the relative importance of dispersal and recruitment limitation. Especially in orchid species, recruitment limitation is expected to be important because of their dependence on mycorrhiza for germination. Using a large seed introduction experiment we investigated the relative importance of dispersal versus recruitment limitation in the failure of three rare orchid species to colonize restored habitat patches. For all species, seeds developed successfully into protocorms in both occupied and unoccupied habitats, and no significant differences in the number of protocorms per seed packet were found between occupied and unoccupied habitats. These results show that orchid species do not necessarily suffer from recruitment limitation when introduced in restored habitats, and that manual introduction of orchid seeds can be a successful and necessary ecological restoration practice.     

CARBOHYDRATE PHYSIOLOGY OF ORCHID SEEDLINGS. III. HYDROLYSIS OF MALTOOLIGOSACCHARIDES BY PHALAENOPSIS (ORCHIDACEAE) SEEDLINGS

Germinating seeds and developing seedlings of Phalaenopsis Habsburg and Phalaenopsis Ruth Burton × (Phalaenopsis Abendrot × Phalaenopsis Abendrot) can utilize glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose as carbon sources. Fresh weight decreased significantly with increased polymerization from glucose through maltoheptaose. Seedling survival declined on higher molecular weight sugars reaching levels which were significantly different from those on glucose. Sugar uptake increased moderately with increasing molecular weight of oligomers. The maltooligosaccharides used in these experiments are hydrolyzed by the orchid seedlings and of the sugars which can support good growth glucose, but not maltose accumulate in culture media. As a result, media which supported seedlings contained substantial levels of glucose, the starting sugars, and decreasing amounts of the next shorter oligomers. This suggests enzymatic endwise hydrolysis of these maltooligosaccharides. Similar results were obtained with Phalaenopsis seedlings produced from seeds which were germinated on sugar-free medium and transferred to a solution containing the same oligomers. Sugars in media which did not support seedlings were not hydrolyzed.     

MORPHOMETRY OF ORCHID SEEDS. II. NATIVE CALIFORNIA AND RELATED SPECIES OF CALYPSO,CEPHALANTHERA, CORALLORHIZA AND EPIPACTIS

A study of Calypso, Cephalanthera, Corallorhiza and Epipactis seeds suggests that morphometric analysis can be useful as a taxonomic tool and an indicator of developmental patterns.     

齿瓣石斛的原生境播种及其株丛生长特性研究

兰科（Orchidaceae）植物是植物保护中的“旗舰”类群,其种子细小粉末状,缺乏萌发所需的营养物质,研究种子萌发和幼苗生长特性对于兰科植物保育、资源可持续利用和产业化等具有重要意义。本研究以附生齿瓣石斛（Dendrobium devonianum Paxt.）为对象,建立原生境播种方法,研究其生活史周期和不同附主树种齿瓣石斛株丛生长特性。结果显示,采取稀牛粪＋种子（或米汤＋种子）的原生境播种方法,齿瓣石斛种子可大量萌发生长形成幼苗;种子萌发当年生长缓慢,完成由种子到种子的生活史周期需3~5年或更长;不同附主树种的齿瓣石斛株丛生长及结果情况存在差异,4年生假鳞茎在不同附主树种上的茎节数指标为：李树 > 梨树 > 桃树 > 披针叶楠;茎粗指标为：披针叶楠 > 梨树 > 桃树 > 李树;茎长指标为：梨树 > 披针叶楠 > 李树 > 桃树;结实率指标为：梨树 > 桃树 > 李树 > 披针叶楠,其中,梨树上的齿瓣石斛株丛自然结果率达24.86%。该研究为附生石斛的原生态栽培和回归保育提供了一条新途径。     

The effects of smoke derivatives on in vitro seed germination and development of the leopard orchid Ansellia africana

Plant‐derived smoke and smoke‐isolated compounds stimulate germination in seeds from over 80 genera. It has also been reported that smoke affects overall plant vigour and has a stimulatory effect on pollen growth. The effect of smoke on orchid seeds, however, has not been assessed. In South Africa, orchid seeds from several genera may be exposed to smoke when they are released from their seedpods. It is therefore possible that smoke may affect their germination and growth. Therefore, the effects of smoke [applied as smoke‐water (SW)] and two smoke‐derived compounds, karrikinolide (KAR₁) and trimethylbutenolide (TMB), were investigated on the germination and growth of orchid seeds in vitro. The effect of SW, KAR₁ and TMB were investigated on the endangered epiphytic orchid, Ansellia africana, which is indigenous to tropical areas of Africa. Smoke‐water, KAR₁ and TMB were infused in half‐strength MS medium. The number of germinated seeds and number of seeds and protocorm bodies to reach predetermined developmental stages were recorded on a weekly basis using a dissecting microscope for a 13‐week period. Infusing SW 1:250 (v:v) into half‐strength MS medium significantly increased the germination rate index (GRI) and the development rate index (DRI) of the A. africana seeds. All the SW treatments significantly increased the number of large protocorm bodies at the final stage of development. Infusing KAR₁ into the growing medium had no significant effect on germination or development of the seeds. The TMB treatment, however, significantly reduced the GRI and DRI of A. africana seeds.     

Asymbiotic seed germination of Cymbidium bicolor Lindl. (Orchidaceae) and the influence of mycorrhizal fungus on seedling development

An in vitro plant regeneration protocol was successfully established for Cymbidium bicolor an epiphytic orchid by culturing seeds from green pods. Immature seeds were germinated on four basal media viz., Murashige and Skoog (MS) medium, Knudson C (KC) orchid medium, Knudson C modified Morel (KCM) medium and Lindemann orchid (LO) medium. Seed germination and protocorm development was significantly higher in LO medium (96.6 %) followed by KCM (89 %), MS (77.5 %) and KC (62.7 %) media after 56 days. For multiple shoot induction the protocorms were transferred to B5 medium supplemented with cytokinin. Among the various cytokinins tested, BAP (4.42 μM) induced maximum (27.59) number of multiple shoots per explant. IBA was effective in inducing healthy roots. Tissue-cultured protocorms and seedlings of C. bicolor were inoculated with AC-01 fungal strain (Moniliopsis sp.) isolated from the mycorrizal roots of an epiphytic orchid Aerides crispum. Mycorrhizal fungi significantly enhanced number of roots, root length and shoot number.     

四川黄龙沟森林植被中兰科植物群落优势种种间联结和相关分析

四川黄龙沟内分布着十分丰富的兰科植物(19属30余种),并且部分种类在沟内形成优势群落。目前尚不清楚在群落中这些兰科植物种之间作用关系。运用种间关联分析和相关分析对黄龙沟森林植被兰科植物群落中的24个优势种的种间关系进行了研究。两种分析方法得到的结果相近,表明黄龙沟优势兰科植物可以分为两组。一组包括无苞杓兰(Cypripedium bardolphianum)、黄花杓兰(C. flavum)、二叶红门兰(Orchis diantha)、广布红门兰(Orchis chusua)、少花虾脊兰(Calanthe delavayi)和西藏杓兰(C. tibeticum),主要分布在光线充足但又具有一定遮荫条件的环境中;另一组包括筒距兰(Tipularia szechuanica)、沼兰(Malaxis monophyllos)、珊瑚兰(Corallorhiza trifida)、小斑叶兰(Goodyera repens)、布袋兰(Calypso bulbosa)、小花舌唇兰(Platanthera minutiflora)和小叶对叶兰(Listera smithii),它们主要分布于荫蔽的环境中。这些兰科植物在组内大多呈现显著的正相关关系,组间大多呈现显著的负相关关系,说明黄龙沟兰科植物在资源利用方式上可能产生了分化。     

ORCHID MYCORRHIZA: VITAMIN PRODUCTION AND REQUIREMENTS BY THE SYMBIONTS

A Rhizoctonia species isolated from Cymbidium has been cultured successfully on a defined medium consisting of minerals, sugar, thiamine, and folic acid. Thiamine can be replaced by its thiazole component, which is probably produced by germinating orchids. The fungus apparently produces the pyrimidine moiety of thiamine, a compound which may enhance growth of certain orchid seedlings. Niacin is also provided by the fungus. Para-aminobenzoic acid, a constituent of folic acid, produced and released by orchid seeds, can satisfy the vitamin requirement of the fungus. These findings point to the possibility that orchids and their fungi may have coevolved with respect to vitamin requirements. The data also suggest that exchanges of vitamins or their components between orchids and endophytes are important aspects of the symbiotic relationship.     

石斛离体培养中ABA对诱导花芽形成的影响

由兰科植物铁皮石斛（Ｄｅｎｄｒｏｂｉｕｍ ｃａｎｄｉｄｕｍ Ｗａｌｌ．ｅｘ Ｌｉｎｄｌ．）种子诱导形成的愈伤组织,在光照下置于ＭＳ附加０．３ ｍ ｇ／ＬＮＡＡ 的培养基上繁殖,可以形成原球茎。将原球茎转入ＭＳ含２ ｍ ｇ／Ｌ ６－ＢＡ 和０．５ ｍ ｇ／ＬＮＡＡ 的培养基上,花芽形成频率为２７．０％ 。原球茎先在０．５ ｍ ｇ／ＬＡＢＡ的培养基上预培养１５ ｄ,再转入含２ ｍ ｇ／Ｌ６－ＢＡ 的ＭＳ培养基上培养,花芽形成频率明显提高,可达８４．４％ ,而且每株植株花的数目增加;但是在仅有ＡＢＡ 的ＭＳ培养基上培养的原球茎再生的植株未见花芽形成     

In vitro culture of Chloraea gavilu Lindl., an endemic terrestrial orchid from Chile

Orchids are the second most diverse plant family, recognized for their importance as ornamental species; this has driven research development in propagation. One of the most common culture methodologies is in vitro asymbiotic germination, in which the nutritional conditions that provide orchids with a fungal partner are emulated. Although Chile possesses more than 60 terrestrial orchid species, in vitro cultivation protocols have only been developed for Chloraea crispa. In Southern Chile, Chloraea gavilu stands out due to its floral characteristics. We evaluate different explants and cultivation conditions for C. gavilu. The best germination and development results were achieved in the MM medium +0.1% yeast extract +1% sucrose +0.454 µmol l^?1 TDZ, using immature seeds of 24–30 days after pollination, which we cultivated into seedlings in order to be acclimatized and mycorrhized. In addition, induction of protocorm-like bodies was achieved from germinated seeds, using the same culture media as in the germination and development of immature C. gavilu seeds. This resulted in the successful asymbiotic germination of immature seeds, along with the micropropagation of a terrestrial, temperate orchid. We hope to use our protocol in the commercial production of Chilean orchid species as well as to propagate threatened species.     

History of orchid propagation: a mirror of the history of biotechnology

Part I Orchid seeds are nearly microscopic in size. Because of that, many fanciful theories were proposed for the origin of orchids. Almost 400 years separate the time when orchid seeds were seen for the first time and the development of a practical asymbiotic method for their germination. The seeds were first observed and drawn during the sixteenth century. Seedlings were first described and illustrated in 1804. The association between orchid and fungi was observed as early as 1824, while the requirement for mycorrhiza for seed germination was established in 1899. An asymbiotic method for orchid seed germination was developed in 1921. After Knudson’s media B and C were formulated, orchids growing and hybridization became widespread. Hybrids which early growers may not have even imagined became possible. Part II A commonly held view is that Prof. Georges Morel is the sole discoverer of orchid micropropagation and that he was the first to culture an orchid shoot tip in 1960. In fact, the first in vitro orchid propagation was carried out by Dr. Gavino Rotor in 1949. Hans Thomale was the first to culture an orchid shoot tip in 1956. The methods used by Morel to culture his shoot tips were developed by others many years before he adapted them to orchids. This review also traces the history of several techniques, additives, and peculiarities (agitated liquid cultures, coconut water, banana pulp, a patent and what appears to be an empty claim) which are associated with orchid micropropagation. A summary of plant hormone history is also outlined because micropropagation could not have been developed without phytohormones.

A new biological function of Shiitake mushroom,Lentinula edodes, in a myco-heterotrophic orchid,Erythrorchis ochobiensis

The biological function ofLentinula edodes in a myco-heterotrophic orchid,Erythrorchis ochobiensis was examined, using one local variant each from Japan (JPN), Papua New Guinea (PNG) and New Zealand (NZ). All variants induced seed germination: PNG and NZ isolates were effective at 25°C and JPN isolate showed the highest germination rate at 30°C. Germinated seeds developed into plants and formed normal endomycorrhizas. Hence, it is concluded thatL. edodes has a perfect symbiotic potential withE. ochobiensis, though it has not been observed in the root of the orchid in the field.