ORCHID PHYTOALEXINS. II. ISOLATION AND CHARACTERIZATION OF POSSIBLE STEROL COMPANIONS

Four sterols have been isolated from extracts of Cymbidium pseudobulbs infected with Rhizoctonia repens M 32. One of them, ergosterol peroxide, is most probably an artifact of extraction. The other three, sitosterol, stigmasterol, and campesterol, occur in a 70:25:5 ratio. Appearance of phytoalexin(s) in pseudobulb extracts coincides with increase of sterol production. This raises the question whether Cymbidium phytoalexins are related, biosynthetically or structurally, to sterols. Since the same three sterols occur (free or conjugated) in Cattleya and Arundina, but in different ratios to each other than in Cymbidium, they may be of value in chemotaxonomy.     

基于MaxEnt模型预测中国兰属植物的分布格局及主导气候因子(附表)

兰属(Cymbidium)中，除了兔耳兰C.lancifolium以外的所有种均被列为国家重点保护野生植物。为探究其在未来气候条件下的潜在分布格局，该研究基于兰属植物已知的分布点和19个气候因子，利用最大熵(MaxEnt)模型和地理信息系统(ArcGIS)模拟兰属以及其中20种兰属植物在9种不同气候情景(当代以及未来2030s、2050s、2070s和2090s 4个时间段各两种温室气体排放情景)下的潜在分布格局。结果表明：(1)最干旱季降水量(Bio17)、年降水量(Bio12)和温度季节性变化(Bio4)是影响兰属植物地理分布格局的主导气候因子。(2)不同兰属植物在未来情景下的适生区表现出不同的变化趋势，并且影响其分布的主导气候因子也有所不同。其中，冬凤兰(C.dayanum)等8个物种的适生区面积整体呈扩张趋势，而西藏虎头兰(C.tracyanum)等12个物种的适生区面积整体则呈缩减趋势。该研究结果为兰属植物就地保护与迁地保护提供了重要参考，对兰属等濒危野生植物的保护具有积极意义。     

Identification of Fusarium proliferatum causing leaf spots on Cymbidium orchids in Taiwan

Previous research indicated that black and yellow leaf spots on Cymbidium, Ondontioda, Dendrobium and Cattleya could be caused by Fusarium proliferatum worldwide. However, the agent causing leaf spot on Cymbidium spp. plants is still obscure in Taiwan. Thirty‐five F. fujikuroi species complex (FFSC)‐like isolates were collected from Cymbidium leaf spot from different greenhouses in Taiwan. All isolates were identified as F. proliferatum based on morphological characteristics and molecular analysis. Sequence of translation elongation factor 1‐alpha gene showed 99%–100% homology with F. proliferatum. In addition, two assay techniques using either detached leaves or seedlings were used to evaluate the pathogenicity and host range of the isolates and consequently their effects on Cymbidium and other orchid plants. Pathogenicity assays revealed that all isolates induced black and necrotic spots on detached leaves of Cymbidium, showing 9.4%–29.5% severity on seedlings of Cymbidium. Results of host specificity tests on detached leaves of different plants indicated that the F. proliferatum isolates collected from Cymbidium plants caused severe black spots on Oncidium, Cymbidium, Dendrobium and Cattleya plants. The symptoms on Phalaenopsis plants were relatively mild. Results of host specificity tests on plant seedlings indicated that the F. proliferatum isolates of Cymbidium origin were also pathogenic to Oncidium, Cymbidium and Dendrobium, but not to Cattleya and Phalaenopsis. Phylogenetic analysis of the translation elongation factor (TEF) gene among all fungal isolates using maximum parsimony (MP) and Bayesian inference (BI) methods revealed that the isolates of F. proliferatum from Cymbidium spp. could be separated from other FFSC‐like species with high phylogenetic support.     

DNA barcoding of Cymbidium by genome skimming: Call for next-generation nuclear barcodes

Cymbidium is an orchid genus that has undergone rapid radiation and has high ornamental, economic, ecological and cultural importance, but its classification based on morphology is controversial. The plastid genome (plastome), as an extension of plant standard DNA barcodes, has been widely used as a potential molecular marker for identifying recently diverged species or complicated plant groups. In this study, we newly generated 237 plastomes of 50 species (at least two individuals per species) by genome skimming, covering 71.4% of members of the genus Cymbidium. Sequence-based analyses (barcoding gaps and automatic barcode gap discovery) and tree-based analyses (maximum likelihood, Bayesian inference and multirate Poisson tree processes model) were conducted for species identification of Cymbidium. Our work provides a comprehensive DNA barcode reference library for Cymbidium species identification. The results show that compared with standard DNA barcodes (rbcL + matK) as well as the plastid trnH-psbA, the species identification rate of the plastome increased moderately from 58% to 68%. At the same time, we propose an optimized identification strategy for Cymbidium species. The plastome cannot completely resolve the species identification of Cymbidium, the main reasons being incomplete lineage sorting, artificial cultivation, natural hybridization and chloroplast capture. To further explore the potential use of nuclear data in identifying species, the Skmer method was adopted and the identification rate increased to 72%. It appears that nuclear genome data have a vital role in species identification and are expected to be used as next-generation nuclear barcodes.     

Effects of permanent magnetic fields on in vitro growth of Cymbidium and Spathiphyllum shoots

Magnetic fields affect biological systems. However, this is the first study on the effects of permanent magnetic fields (MFs) on the micropropagation of two ornamental plants, Spathiphyllum cv. i.e ‘Merry’ and Cymbidium Music Hour ‘Maria’. Cymbidium and Spathiphyllum shoots cultured in the ‘Miracle Pack’? culture system were exposed to MFs of different intensities, polarities, and duration of exposure. The results show that by increasing intensity from 5 × 10⁻⁶ Tesla (T) as the geo-magnetic field to 0.1, 0.15, and 0.2 T negatively influenced height and fresh mass of roots of Cymbidium plants (except for 0.1 T–S and 0.2 T–N treatments), but had no significant effect on other plantlet parameters. Long-term exposure (1, 2, or 3 mo) of Cymbidium shoots to 0.15 T–MFs negatively influenced plant height, positively affected the number of leaves (with the exception of 0.15 T–S—1 mo), and had no clear effect on other parameters compared to the control. MFs (0.1, 0.15, and 0.2 T), regardless of their polarity, increased chlorophyll content (SPAD value) and the number of leaves, but slightly decreased the dry mass of Spathiphyllum shoots. Different exposure duration to 0.15 T (i.e., 2, 4, or 8 wk) had no significant influence on Spathiphyllum plantlet development other than increasing the SPAD value. These two ornamentals could serve as model systems to study plant development, space production, yield maximization, and the development of new morphotypes essential for the floricultural market.     

Organic Nutrients in the Media for Propagation of Cymbidium in vitro

Protocorms of Cymbidium (Orchidaceae) were grown on media containing different organic nutrients. Of the sugars tested sucrose was better than maltose, glucose and fructose, and sucrose had an optimum concentration of 3 to 4 %. D-Mannose was significantly less effective than the other sugars. The amino acid mixtures casamino acids (casein hydrolysate) and tryptone increased growth while yeast extract was inhibitory and malt extract without effect. Optimal concentrations were 2 to 3 g · l^-1 casamino acids and 3 to 4 g · l^-1 tryptone. It was to some extent possible to substitute the amino acid mixtures with a single amino acid (glutamine at 300 mg · l^-1). Arginine was inhibitory and asparagine was without any effect. Vitamins proved to be unnecessary although there was a tendency towards increased growth with nicotinic acid and meso-inositol. Purines and pyrimidines were added to the medium but with no effect. Liquid endosperm from coconuts (10 to 15%) increased growth while the liquid endosperm from Aesculus hippocastanum was inhibitory. On the basis of these results a revised medium is proposed for the in vitro propagation of Cymbidium.     

The phylogenetic relationship and non‐specific symbiotic habit of mycorrhiza fungi from a terrestrial orchid (Cymbidium)

To investigate the specificity of the symbiotic relationship between Cymbidium plants and their mycorrhiza fungi, thirty mycorrhiza fungi were isolated from roots of six terrestrial Cymbidium species. The internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) were amplified by polymerase chain reaction (PCR) with universal fungal primers ITS1/ITS4. All fungal strains isolated from natural roots of orchids were inoculated into the rhizomes of in vitro Cymbidium goeringii. Phylogenetic analysis indicated fungal isolates of different cluster could be obtained from a special terrestrial Cymbidium species. Observation of light microscope and scanning electron microscope showed that fungi entered the cortical tissue by destroying cell wall of epidermal cells, where they formed hyphal knots in the cortical cells and were digested gradually. A large number of small protuberances were visible on cross sections of the rhizome. There was no strict inter‐species specificity between the isolated mycorrhiza fungi and terrestrial Cymbidium.     

The occurrence of crassulacean acid metabolism in Cymbidium (Orchidaceae) and its ecological and evolutionary implications

Crassulacean acid metabolism (CAM) is one of the photosynthetic pathways regarded as adaptations to water stress in land plants. Little is known about correlations among the level of CAM activity, environment of habitat, life form, and phylogenetic relationship of a plant group from an evolutionary perspective. We examined these relationships in 18 species of Cymbidium (Orchidaceae) because the genus shows distinctive diversification of habitats and life forms. The photosynthetic type was classed into three categories, strong CAM, weak CAM, and C₃ on the basis of CAM activity. CAM expression in Cymbidium was confined to the epiphytic and lithophytic species. Especially, all of these species from tropical to subtropical rainforest exhibited CAM activity. On the other hand, the terrestrial species always exhibited C₃ metabolism irrespective of their varied habitats. Regarding the evolution of photosynthetic characters, weak CAM was the ancestral state in Cymbidium and strong CAM and C₃ metabolism occurred subsequently. The evolution of strong CAM likely enabled Cymbidium to extend to exposed sites in tropical lowland where marked water stress exists. Further, different levels of CAM activity characterized each species and such potential plasticity of CAM may realize the radiation of Cymbidium into sites with different environmental conditions.     

The Effects of Cymbidium Mosaic Virus and Odontoglossum Ringspot Virus on the Growth of Cymbidium Orchids

Two cultivars of Cymbidium orchid were mechanically inoculated with Odontoglossum ringspot virus (ORSV) and Cymbidium mosaic virus (CyMV), individually and in combination. ORSV was found to have an infection rate of 70% (as determined by ELISA), but seldom induced easily discernable leaf symptoms. CyMV had an infection rate of only 20%, but infected plants invariably produced a pronounced leaf mosaic either with or without necrotic streaks. Both viruses were found to reduce plant growth, the effects of CyMV being more severe than those of ORSV.     

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.     

Further Observations on the Effects of Cymbidium Mosaic Virus and Odontoglossum Ringspot Virus on the Growth of Cymbidium Orchids

Two cultivars of Cymbidium orchid were mechanically inoculated with Odontoglossum ringspot virus (ORSV) and Cymbidium mosaic virus (CyMV), individually and in combination, one year after transfer from in vitro culture to the glasshouse. Plant growth and disease symptoms were monitored over the following 4 years. Plants infected with CyMV showed severe mosaic symptoms with necrotic streaks and the virus was easily detectable by ELISA throughout the experiment. Plants infected with ORSV alone showed no obvious symptoms, and by the end of the experiment the virus could not be detected in the new growth using ELISA. Both viruses reduced plant growth, the effect of CyMV being more severe than that of ORSV.     

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.     

The effect of fungicides on survival of the crayfish plague fungus,Aphanomyces astaci,Oomycetes, growing on fish scales

An antifungal factor isolated from extracts of Cymbidium (Orchidaceae) roots and infected pseudobulbs was identified as monolinolein.     

LEAD PHYTOEXTRACTION BY WHEAT IN RESPONSE TO THE EDTA APPLICATION METHOD

Lead solubilization in soil and accumulation by spring wheat (Triticum aestivum L.) was studied in response to the ethylenediaminetetraacetic acid (EDTA) application method. In this study, 4 mmol EDTA kg^?1 was applied using two application methods (a single dose and split doses) either alone or in combination with elemental sulfur. Results indicate that the application of EDTA in four equal splits at 1 mmol kg^?1 during the growth period resulted in significantly higher shoot dry matter than its application at 4 mmol kg^?1 at once 10 d before harvesting the wheat crop at the bolting stage. EDTA applied in split doses resulted in less lead (Pb) solubilization as compared with the single-dose application. The split application also significantly increased the shoot Pb concentration and Pb accumulation by wheat shoots as compared with the single-dose application. Despite its lesser effect on Pb solubilization, the EDTA application in split doses substantially increased Pb accumulation; thus, it is expected to minimize the risk of groundwater contamination.     

In vitro growth and single-leaf photosynthetic response of Cymbidium plantlets to super-elevated CO2 under cold cathode fluorescent lamps

To examine the effectiveness of super-elevated (10,000 μmol mol⁻¹) CO₂ enrichment under cold cathode fluorescent lamps (CCFL) for the clonal propagation of Cymbidium, plantlets were cultured on modified Vacin and Went (VW) medium under 0, 3,000 and 10,000 μmol mol⁻¹ CO₂ enrichment and two levels of photosynthetic photon flux density (PPFD, 45 and 75 μmol m⁻² s⁻¹). Under high PPFD, 10,000 μmol mol⁻¹ CO₂ increased root dry weight and promoted shoot growth. In addition, a decrease in photosynthetic capacity and chlorosis at leaf tips were observed. Rubisco activity and stomatal conductance of these plantlets were lower than those of plantlets at 3,000 μmol mol⁻¹ CO₂ under high PPFD, which had a higher photosynthetic capacity. On the other hand, plantlets on Kyoto medium grown in 10,000 μmol mol⁻¹ CO₂ under high PPFD had a higher photosynthetic rate than those on modified VW medium; no chlorosis was observed. Furthermore, growth of plantlets, in particular the roots, was remarkably enhanced. This result indicates that a negative response to super-elevated CO₂ under high PPFD could be improved by altering medium components. Super-elevated CO₂ enrichment of in vitro-cultured Cymbidium could positively affect the efficiency and quality of commercial production of clonal orchid plantlets.     

Agrobacterium-mediated transformation of protocorm-like bodies in Cymbidium

Genetically transformed plants of Cymbidium were regenerated after cocultivating protocorm-like bodies (PLB) with Agrobacterium tumefaciens strain EHA101 (pIG121Hm) that harbored genes for β-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII). PLB of three genotypes maintained in liquid new Dogashima medium (NDM), were subjected to transformation experiments. The PLB inoculated with Agrobacterium produced secondary PLB, 4 weeks after transfer onto 2.5 g L⁻¹ gellan gum-solidified NDM containing 10 g L⁻¹ sucrose, 20 mg L⁻¹ hygromycin and 40 mg L⁻¹ meropenem. Transformation efficiency was affected by genotype and the presence of acetosyringone during cocultivation. The highest transformation efficiency was obtained when PLB from the genotype L4 were infected and cocultivated with Agrobacterium on medium containing 100 μM acetosyringone. Transformation of the hygromycin-resistant plantlets regenerated from different sites of inoculated PLB was confirmed by histochemical GUS assay, PCR analysis and Southern blot hybridization.     

Use of VIRAZOLE® to eradicate odontoglossum ringspot virus from in vitro cultures of Cymbidium Sw.

Odontoglossum Ringspot Virus has been eradicated from Cymbidium Sw. through chemotherapy based on incorporation of ribavirin (VIRAZOLE^®) into the in vitro culture medium of protocorms. Applications of the virustatic agent for several consecutive subcultures freed protocorms of the virus. Acclimated plantlets regenerated from those protocorms are healthy as determinated by enzyme-linked immunosorbent assay (ELISA). No resurgence of virus occurred over a period of 5 years. Besides, trueness to type was total at flowering level and the batch grown was perfectly homogeneous.To secure fast and effective eradication of the virus during the consecutive subcultures of protocorms with ribavirin, three factors proved to be of prime importance: accurate isolation of new growths from initial tissues, VIRAZOLE^® concentration and frequency of transplanting in new media.     

Roles of pollination and short-chain saturated fatty acids in flower senescence

Pollination induced an immediate increase in ethylene production in Dianthus caryophyllus and Petunia hybrida. In Cymbidium, a lag of several hours was observed. In all three species, pollination induced premature flower senescence. Treatment of the stigmatic surface with aminoethoxyvinylglycine prior to pollination effectively blocked the increase in ethylene production and alleviated the detrimental effect of pollination on flower life.In all three tested species, octanoic and decanoic acids, when applied to the stigmatic surface, had no effect on ethylene production and flower life. In isolated Cymbidium lips placed with their cut base in solutions containing these fatty acids, no effects on red colouration, ethylene production, and ethylene forming enzyme activity were observed. In addition, ethylene sensitivity of isolated lips was not affected. The putative regulatory role of short-chain saturated fatty acids in (pollination-induced) flower senescence is discussed.     

Temporal and spatial regulation of anthocyanin biosynthesis provide diverse flower colour intensities and patterning in <Emphasis Type="Italic">Cymbidium</Emphasis> orchid

Main conclusion

This study confirmed pigment profiles in different colour groups, isolated key anthocyanin biosynthetic genes and established a basis to examine the regulation of colour patterning in flowers of Cymbidium orchid. Cymbidium orchid (Cymbidium hybrida) has a range of flower colours, often classified into four colour groups; pink, white, yellow and green. In this study, the biochemical and molecular basis for the different colour types was investigated, and genes involved in flavonoid/anthocyanin synthesis were identified and characterised. Pigment analysis across selected cultivars confirmed cyanidin 3-O-rutinoside and peonidin 3-O-rutinoside as the major anthocyanins detected; the flavonols quercetin and kaempferol rutinoside and robinoside were also present in petal tissue. β-carotene was the major carotenoid in the yellow cultivars, whilst pheophytins were the major chlorophyll pigments in the green cultivars. Anthocyanin pigments were important across all eight cultivars because anthocyanin accumulated in the flower labellum, even if not in the other petals/sepals. Genes encoding the flavonoid biosynthetic pathway enzymes chalcone synthase, flavonol synthase, flavonoid 3′ hydroxylase (F3′H), dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) were isolated from petal tissue of a Cymbidium cultivar. Expression of these flavonoid genes was monitored across flower bud development in each cultivar, confirming that DFR and ANS were only expressed in tissues where anthocyanin accumulated. Phylogenetic analysis suggested a cytochrome P450 sequence as that of the Cymbidium F3′H, consistent with the accumulation of di-hydroxylated anthocyanins and flavonols in flower tissue. A separate polyketide synthase, identified as a bibenzyl synthase, was isolated from petal tissue but was not associated with pigment accumulation. Our analyses show the diversity in flower colour of Cymbidium orchid derives not from different individual pigments but from subtle variations in concentration and pattern of pigment accumulation.

     

POSTPOLLINATION PHENOMENA IN ORCHID FLOWERS. V. PARTICIPATION BY THE ROSTELLUM AND GYNOSTEMIUM TIP

Removal of the rostellum following pollination does not prevent stigmatic closure in Cymbidium flowers and has a minimal effect on straightening of the gynostemium (column). However, this treatment does depress anthocyanin levels in both gynostemia and labella. Excision of the rostellum 30 or 60 min after pollination has a more pronounced effect than removal after 150 min. Stigmatic closure is not inhibited by removal of the gynostemium tip, but column swelling is reduced. These findings are discussed relative to rostellar functions and theories regarding their origin.