Molecular identification of mycorrhizal fungi in Neuwiedia veratrifolia (Orchidaceae)

We here apply a previously described method for identification of single peloton orchid mycorrhiza to a key orchid group and extend the usefulness in the heterobasidiomycetes of an existing fungal database for identification of mycorrhizal fungi. We amplified and sequenced mitochondrial ribosomal large subunit DNA from fungi in roots of Neuwiedia veratrifolia (Orchidaceae), a member of the small subfamily Apostasioideae that is sister to the remainder of Orchidaceae, and used the extended database to identify the mycorrhizal fungi. Sequences from fungi cultured from Neuwiedia roots and from direct peloton amplifications were analyzed cladistically with sequences determined from reference fungal collections and published sequences. The fungi from Neuwiedia are referred to the heterobasidiomycetous orders Tulasnellales and Ceratobasidiales, indicating that apostasioids utilize the same fungi as other photosynthetic orchids. The majority of Neuwiedia mycobionts came together in a clade with Tulasnella species, but some were most closely related to Thanatephorus. In some cases members of these two clades were isolated from the same orchid plant, providing another example of multiple mycobionts occurring in a single plant.     

Allozyme Diversity in Populations of Cymbidium goeringii (Orchidaceae)

Abstract: Using 14 allozyme loci, we investigated levels of genetic diversity within populations, and degree of genetic divergence among 24 populations of Cymbidium goeringii (Orchidaceae) in Korea and Japan. Cymbidium goeringii maintains high levels of genetic diversity both at population (mean expected heterozygosity, H _e = 0.238) and species levels (0.260). Means of H _e found in 24 populations were not significantly different from each other. About 90 % of the total variation in the species is common to all populations (mean G _ST = 0.108). No unique allele was found in any population. The indirect estimate of gene flow based on the mean G _ST was high ( Nm = 2.06). Nei's genetic identities for pairs of populations had high values (mean = 0.974 [SD = 0.013]). The Mantel-Z test showed a significant correlation between genetic distance and geographic distance. However, the mean G _ST value between 17 populations in Korea and seven Japanese populations was relatively low (0.029), even though the land connection between the southern Korean peninsula and southern Japanese archipelagos has not existed since the middle Pleistocene. Large numbers of small seeds of C. goeringii might travel long distances by wind from populations to populations both in Korea and Japan, increasing genetic diversity within populations and maintaining low genetic differentiation among populations.     

Pollination biology of Cymbidium goeringii (Orchidaceae) in China

The genus Cymbidium comprises three subgenera with ca. 50 species. Interactions between pollinators and plants have been studied in the two subgenera Cymbidium and Cyperorchis, but only a few studies in the subgenus Jensoa have been reported. Here we report on the reproductive characteristics of C. goeringii (in sub-genus Jensoa) in three populations in the southwest of Hunan Province, China, during the winter from December 2005 to March 2006. Floral phenological and morphological features, behavior of visitors, the breeding system, and fruit sets under natural conditions were studied. The flowers of C. goeringii were strongly fragrant, but did not present any rewards to the visitors. The flowering period of C. goeringii in the studied populations lasted about 40 days, and most flowers (about 60%) opened within 30 days. Flowers opened immediately when temperature increased obviously and reached to about 16 ℃. The flowering time of the pollinated flowers, unpollinated flow-ers and the flowers with pollinarium removed were similar. Two bees of Apidae, Apis cerana cerana (honeybee) and Anthophora melanognatha, were observed visiting flowers of C. goeringii, but only the honeybees performed as pollinators. The honeybees mostly visited the orchid flowers at 10:00–17:00 on sunny days with temperature above 10 ℃. A total of only thirteen visits were observed during 20 days of observation, indicating pollinators were rare. Honeybees directly landed on the upper surface of the labellum and inserted their heads into the flowers between column and labellum, while the hind legs trod on the surface of the curved downward mid-lobe of label-lum. When a honeybee landed on the labellum of a flower, the labellum moved up and down slightly. After the honeybee entered the flower further, its head might touch the foot of the column. At this time, the body of the honeybee was parallel with the upper surface of the labellum. Then the honeybee used its front legs to scratch on the callus ridges on the upper surface of the labellum, and its hind legs hooked the edges of the side lobe of label-lum, trying to exit forcibly from the flower. During exiting process, sometimes the honeybee’s body was arched owing to tension. In this case, the surface of the scutellum came into close contact with the viscidium and then pollinaria, together with the anther cap, were removed. When this honeybee visited next flower, the pollinaria would be adhered to the stigma when it arched its body during the exiting process. Three plant species flowered synchronously with C. goeringii in the studied areas, but their flowers were different with C. goeringii in color or shape. Because C. goeringii is rewardless to pollinators, the flowers probably attract visitors by olfactory stimu-lus. Breeding system experiments showed no spontaneous autogamy and pollination success relying on pollinators in C. goeringii. Artificial pollination resulted in 90% fruit set by induced autogamy, 100% by pollinating within clone, and 100% by xenogamous pollination, respectively. These results indicate that C. goeringii is highly self-compatible and the fruit production is pollen limited. Because pollinators are essential for fruit set of C. goeringii in natural habitats, protection of wild honeybee populations or apiculture is likely a simple but effective strategy to maintain the orchid populations.     

春兰(兰科)传粉生物学的研究

为探讨兰属Cymbidium建兰亚属subgen.Jensoa植物与传粉者之间的关系,对湖南省西南部野生春兰Cymbidiumgoeringii的传粉生物学进行了研究.结果表明春兰花期近40天,多数花(60%左右)在30天内开放.春兰的花开放与气温有直接的关系,当温度明显升高时,春兰的花会大量开放;春兰的花唇瓣3裂,中裂片强烈外弯成90°至180°不等,且少数花的中裂片上有紫褐色斑点;花能够散发出浓郁的香气,距离花1m左右就可以闻到香气.春兰唯一的传粉者为中华蜜蜂Apis cerana cerana,主要在晴天1000-1700气温较高的时间段活动.中华蜜蜂访花时,直接落在唇盘上,后腿蹬在唇瓣中裂片上.头部直接进入花内.当中华蜜蜂头部接近合蕊柱基部时便不能继续进入,开始挣扎退出.退出过程中有时中胸会随着挣扎而拱起,接触到合蕊柱上部,蹭到粘盘(viscidium).当中华蜜蜂中胸背部蹭到粘盘后,花粉块连同药帽就粘到蜜蜂的背部被一起带出.中华蜜蜂访花后,如果没有带出花粉块,则继续访花或者飞离居群;访花后若带出花粉块,则会立即飞离该居群.黑颚条蜂Anthophora melanognatha只是春兰的访问者.春兰不会为传粉者提供花蜜、花粉等报酬,也没有模仿同期开花植物花的形态特征,因而可能是通过其强烈的香味来吸引传粉者.自然条件下,春兰的结实率仅为6.67%.人工异花授粉、自花授粉、同克隆内授粉的结实率分别为100%、90%和100%,表明春兰是高度自交亲和的.而套袋后不采取措施、去雄的花均不结实,表明春兰不存在自动的自花授粉、无融合生殖,因而,春兰必须通过一定的媒介传粉才能繁衍后代.     

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.     

菌根真菌与兰科植物氮营养关系的研究进展

兰科植物是典型的菌根植物。兰菌根是兰科植物根与真菌形成的菌根共生体。兰菌根真菌的营养来源影响宿主植物的生活方式和营养水平。氮是植物生长的主要限制因子。兰科植物具有富集氮的特征, 其组织和器官的氮含量通常高于同生境中的其他植物。该文综述了兰菌根真菌类别、兰科植物氮营养特征和兰菌根的氮转移机制等的研究进展, 以期为兰科植物资源的保护、再生及可持续利用的相关研究提供参考和借鉴。     

Isolation and identification of mycorrhizal fungi associating with an achlorophyllous plant, Epipogium roseum (Orchidaceae)

The identity of mycorrhizal fungi associated with the achlorophyllous orchid Epipogium roseum was investigated by DNA analysis. The fungi were isolated from each coiled hypha (peloton), and the ITS region of nuclear rDNA was sequenced. Phylogenetic analysis based on the neighbor-joining method showed that all the isolates clustered with fungi belonging to Psathyrella or Coprinus in Coprinaceae. Those fungi are known as saprobes, using dead organic materials for a nutritive source. Large colonies of this orchid were frequently found around tree stumps or fallen logs. In such colonies, these decaying wood materials would be used as a large and persistent carbon source for the growth of this orchid. This is the first report of Coprinaceae as an orchid mycorrhizal fungi.     

Pollination-Induced Transcriptome and Phylogenetic Analysis in Cymbidium tortisepalum (Orchidaceae)

Russian Journal of Plant Physiology - Orchids comprise a group of ecologically and evolutionarily significant plants, and the Orchidaceae is one of the most abundant angiosperm families. Yet little...     

Widespread mycorrhizal specificity correlates to mycorrhizal function in the neotropical, epiphytic orchid Ionopsis utricularioides (Orchidaceae)

Tropical orchids constitute the greater part of orchid diversity, but little is known about their obligate mycorrhizal relationships. The specificity of these interactions and associated fungal distributions could influence orchid distributions and diversity. We investigated the mycorrhizal specificity of the tropical epiphytic orchid Ionopsis utricularioides across an extensive geographical range. DNA ITS sequence variation was surveyed in both plants and mycorrhizal fungi. Phylogeographic relationships were estimated for the mycorrhizal fungi. Orchid functional outcomes were determined through in vitro seed germination and seedling growth with a broad phylogenetic representation of fungi. Most fungal isolates derived from one clade of Ceratobasidium (anamorphs assignable to Ceratorhiza), with 78% within a narrower phylogenetic group, clade B. No correlation was found between the distributions of orchid and fungal genotypes. All fungal isolates significantly enhanced seed germination, while fungi in clade B significantly enhanced seedling growth. These results show that I. utricularioides associates with a phylogenetically narrow, effective fungal clade over a broad distribution. This preference for a widespread mycorrhizae may partly explain the ample distribution and abundance of I. utricularioides and contrasts with local mycorrhizal diversification seen in some nonphotosynthetic orchids. Enhanced orchid function with a particular fungal subclade suggests mycorrhizal specificity can increase orchid fitness.     

兔耳兰食源性欺骗传粉的研究

兰科植物具有精巧、多样化的花部结构以及高度多样的吸引传粉者方式。作者对广西雅长兰科植物自治区级保护区内的一个兔耳兰(Cymbidium lancifolium)居群进行了连续2年的观察和研究。观察发现兔耳兰唯一的传粉者为膜翅目蜜蜂科的中华蜜蜂(Apis cerana cerana)。中华蜜蜂一般直接落在唇瓣外弯的中裂片上, 然后调整身体的方向, 进入花中, 当发现花中无蜜液等回报时, 借助于后足的蹬力退出花朵。在退出的过程中, 花粉块连同药帽会通过粘盘粘附在中华蜜蜂的胸部。中华蜜蜂在花内的停留时间为8–71 s, 平均18.3 s (N = 11)。根据观察我们推测兔耳兰可能是通过其唇瓣上无规则的紫栗色小斑点(假蜜导)来吸引中华蜜蜂为其传粉, 属于食源性欺骗方式。在传粉过程中兔耳兰的药帽与花粉团和粘盘一起粘在中华蜜蜂背部。药帽的存在能够阻止下一朵被拜访的花实现雌性功能。兔耳兰药帽高度(0.154 ± 0.032 cm) (N = 10)加上传粉昆虫胸高(2005年为0.37 ± 0.03 cm (N = 10), 2006年0.35 ± 0.04 cm (N = 7))大于传粉通道入口的高度(0.29 ± 0.04 cm) (N = 21), 支持兔耳兰可能通过药帽来减少同株异花授粉现象的推测。2005和2006年该兔耳兰居群的自然繁殖成功率分别为21.13%和21.28%。繁育系统实验证明兔耳兰是高度自交亲和物种, 自交和异交的繁殖成功率没有显著性差异, 表明该种在结实过程中未显示近交衰退。兔耳兰不存在无融合生殖和自花授粉的现象, 其结实依赖传粉者。TTC法检测结果显示兔耳兰种子活力达85.78%(N = 11), 可见种子活力不是制约兔耳兰种子萌发的主要原因。因此传粉者的密度和访问频率可能是影响兔耳兰结实的重要因素, 并最终影响兔耳兰种群的维持和扩张。     

Development of polymorphic microsatellite markers for Cymbidium goeringii (Orchidaceae)

? Premise of the study: Microsatellite markers were developed in Cymbidium goeringii to investigate its genetic diversity and population genetic structure. ? Methods and Results: From a microsatellite-enriched genomic library, 21 novel polymorphic microsatellites were isolated. The polymorphic patterns were verified in four populations (East Korea, West Korea, China, and Japan). The number of alleles per locus ranged from 11 to 29 with a mean of 20.29. The observed and expected heterozygosities varied from 0.272 to 0.799 and from 0.461 to 0.911, respectively. ? Conclusions: These microsatellite markers will be useful tools for understanding genetic variation and population ecogenetic structure in C. goeringii.     

Mycoheterotrophy evolved from mixotrophic ancestors: evidence in Cymbidium (Orchidaceae)

Background and Aims

Nutritional changes associated with the evolution of achlorophyllous, mycoheterotrophic plants have not previously been inferred with robust phylogenetic hypotheses. Variations in heterotrophy in accordance with the evolution of leaflessness were examined using a chlorophyllous–achlorophyllous species pair in Cymbidium (Orchidaceae), within a well studied phylogenetic background.

Methods

To estimate the level of mycoheterotrophy in chlorophyllous and achlorophyllous Cymbidium, natural ¹³C and ¹⁵N contents (a proxy for the level of heterotrophy) were measured in four Cymbidium species and co-existing autotrophic and mycoheterotrophic plants and ectomycorrhizal fungi from two Japanese sites.

Key Results

δ¹³C and δ¹⁵N values of the achlorophyllous C. macrorhizon and C. aberrans indicated that they are full mycoheterotrophs. δ¹³C and δ¹⁵N values of the chlorophyllous C. lancifolium and C. goeringii were intermediate between those of reference autotrophic and mycoheterotrophic plants; thus, they probably gain 30–50 % of their carbon resources from fungi. These data suggest that some chlorophyllous Cymbidium exhibit partial mycoheterotrophy (= mixotrophy).

Conclusions

It is demonstrated for the first time that mycoheterotrophy evolved after the establishment of mixotrophy rather than through direct shifts from autotrophy to mycoheterotrophy. This may be one of the principal patterns in the evolution of mycoheterotrophy. The results also suggest that the establishment of symbiosis with ectomycorrhizal fungi in the lineage leading to mixotrophic Cymbidium served as pre-adaptation to the evolution of the mycoheterotrophic species. Similar processes of nutritional innovations probably occurred in several independent orchid groups, allowing niche expansion and radiation in Orchidaceae, probably the largest plant family.     

Comparative vegetative anatomy and systematics of Cymbidium (Cymbidieae: Orchidaceae)

The genus Cymbidium (Orchidaceae) exhibits distinctive ecological diversification and occurs in terrestrial, epiphytic, and lithophytic life forms. One species, Cymbidium macrorhizon , lacks foliage leaves and has a strongly mycoparasitic existence. Correlation between habitat differentiation and anatomical characters was tested for 21 species of Cymbidium and its putative sister groups. Although hypostomaty characterizes the genus, C. canaliculatum shows amphistomaty. Ecological preference of this species indicates that amphistomaty is likely adapted to intensive insolation. Four types of subepidermal foliar sclerenchyma were found. Two forest floor species, C. goeringii and C. lancifolium as well as the mycoparasitic C. macrorhizon , do not have this sclerenchyma. In this genus, development of sclerenchyma is correlated with the degree of epiphytism. Palisade mesophyll evolved in Cymbidium section Cymbidium . As members of this section grow on isolated trees in tropical lowland forests or on rocks, the differentiation of palisade tissue is probably correlated with immigration to high light habitats. With the exception of C. macrorhizon , stegmata were found in leaves and stems of Cymbidium . Furthermore, a few epiphytic species have stegmata in their roots; this is a curious feature rarely found in vascular plants. Subterranean rhizomes characterize terrestrial species, while ageotropic roots are found in some epiphytic species. Cymbidium macrorhizon shows peculiar features such as degeneration of stomata, anomocytic stomata, and lack of stegmata and sclerenchyma. This set of character transformations is probably correlated with the evolution of mycoparasitic existence. © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 138 , 383–419.     

Multiple Regeneration Pathways via Thin Cell Layers in Hybrid Cymbidium (Orchidaceae)

Protocorm-like body (PLB) and subsequent shoot development in hybrid Cymbidium Twilight Moon ‘Day Light’ can be established in vitro via 3 pathways: PLBs, PLB thin cell layers (TCLs), or embryogenic callus (EC). Traditionally Cymbidium hybrids are mass-produced commercially through the neo-formation of secondary PLBs (2° PLB) from initial or primary PLBs (1° PLB) or PLB segments, or from PLB TCLs, resulting in a moderate number of 2° PLBs (average 4.46 2° PLBs/1° bisected PLB, or 1.12 2° PLBs/ PLB TCL). This study shows that EC can be induced from 1° PLBs or PLB TCLs. Thereafter, resulting 2° PLBs (average 22.1 2° PLBs/EC cluster derived from 1° PLB) form directly from the EC on the same medium or following the transfer of EC onto PGR-free medium. By flow cytometry and PCR-RAPD analysis, the cytogenetic stability of 1° PLBs, of resulting 2° PLBs and EC, and plants derived therefrom was demonstrated.     

Infection of Spathoglottis plicata (Orchidaceae) seeds by mycorrhizal fungus

Spathoglottis plicata seeds were encapsulated in 4-mm-diameter capsules of alginate-chitosan or alginate-gelatin and infected with the mycorrhizal fungus Rhizoctonia AM9. The encapsulated seeds were placed directly on Rhizoctonia culture. About 66% of the seeds encapsulated in sucrose-free chitosan-alginate established a symbiotic relationship with the mycorrhizal fungus after co-culturing for 2 weeks. The highest percentage of infection observed was about 84%. Addition of sucrose or using gelatin-alginate for encapsulation reduced the percentage of infection by about half. The growth of Rhizoctonia AM9 in sucrose-free alginate, chitosan and gelatin was found to be minimal. The advantages of germinating orchid seeds, encapsulated in sucrose-free polymers, through mycorrhizal infection is discussed. Received: 19 February 1998 / Revision received: 8 May 1998 / Accepted: 20 May 1998