Article

Specific Features of Mycorrhizal Symbiosis Formation in the Ontogeny of Orchids of the Temperate Zone

Authors:
  • Botanical Garden of Ural Branch of Russian Academy of Science, Ekaterinburg
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Abstract

The study of relationships between symbiotic fungi and germinating seeds of the Orchidaceae showed that the effect of a fungus on germination depends on the presence of seed dormancy and its pattern. The specificity of symbiosis varies widely. The development of symbiotic sprouts in situand in vitro differs only in the rate, whereas the morphogenesis of asymbiotic sprouts is characterized by several anomalies.

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... Rhizoctonia repens Bernard (= Epulorhiza repens Moore), an anamorph of Tulasnella calospora (Boudier) Juel. has been isolated from the roots of Malaxis brachypoda (Kulikov and Filippov 2001). The symbiotic specificity of M. brachypoda appears to be high. ...
... The symbiotic specificity of M. brachypoda appears to be high. Kulikov and Filippov (2001) observed normal development of sprouts of M. brachypoda only with its natural symbionts. Seed germination and sprout growth did not occur when inoculated with root isolates from other orchid species. ...
... Особую актуальность для разработки эффективных мер по сохранению локальных популяций редких видов орхидей представляет изучение их консортивных связей с почвенными микроорганизмами. К настоящему времени доказана положительная роль микоризы в адаптации орхидных к малоплодородным субстратам, почвам с избыточным количеством металлов и другим неблагоприятным эдафическим условиям (Jurkiewicz et al., 2001;Kulikov, Filippov, 2001;Rasmussen, 2002;Smith, Read, 2008). ...
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The article presents data obtained in the study of bacteria isolated from the rhizosphere of the orchid Epipactis atrorubens (Hoffm.) Besser. Analysis was carried out to compare some morphological, physiological, and biochemical characteristics of plant rhizobacteria growing on serpentine rocks in two biotopes: in the natural forest community (control habitat) and on the asbestos mine dump (the Sverdlovsk region, Middle Urals). An assessment of the plant growth promoting (PGP) activity of the isolated strains did not show significant differences in the ability of rhizobacteria to synthesize indol-3-acetic acid (IAA) and solubilize phosphates between the study sites. However, the proportion of isolates capable of nitrogen fixation was higher in the rhizosphere of E. atrorubens growing on the dump compared to the control habitat. The tolerance of isolates to heavy metals was assessed by the maximum metal concentration (400, 600, and 1000 mg/L, respectively, for Ni, Cu,-298-Olga V. Voropaeva, Galina G. Borisova… Plant Growth Promoting Activity and Metal Tolerance of Bacteria Isolated… and Zn) at which bacterial growth was observed. Rhizobacteria from the dump were found to be more resistant to elevated concentrations of metals compared to their counterparts from the natural forest community. The molecular genetic analysis of isolates with the highest PGP-activity (IAA >1.0 mg/L; PO 4 3->50.0 mg/L) revealed that most of the E. atrorubens rhizobacteria in both habitats belonged to the genera Buttiauxella and Pseudomonas. In model experiments, the PGP ability of four selected strains was tested on zinnia seeds. Seed inoculation with Pseudomonas sp. and Buttiauxella sp. did not have any significant effect on their germination; however, Buttiauxella sp. contributed to the increase in the length of seedlings compared with the control (by 25 %, on average). It has been suggested that the selected isolates of E. atrorubens rhizobacteria, due to their growth promoting activity and metal tolerance, can facilitate naturalization of the orchid in an industrially disturbed area.
... The number of young D. traunsteineri individuals was positively correlated with the seed yield of the previous year, as well as with the precipitation amount and the August temperature of the previous vegetative season. The importance of moisture at the time of seed dispersal to the ground was also indicated for other orchid species (Kulikov & Filippov, 2001;Scott & Carey, 2002). ...
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Due to specific biological features, high ornamental value and low resistance to anthropogenic factors, Orchidaceae species belong to the most vulnerable plants. To successfully protect their populations, comprehensive investigations of orchid biology and ecology are necessary. Long-term population studies are the most valuable. The paper presents data of population studies of Dactylorhiza traunsteineri, an orchid species listed in the Red Data Book of the Russian Federation. The field investigations have been conducted in the Komi Republic where the orchid species is located at the northeastern limit of its range. The seasonal development of D. traunsteineri lasts from May to August. We found the weather factors (air temperature, precipitations) impact features of small and big life cycles of this orchid species. The size of plant individuals is influenced by weather conditions of both the current and previous vegetative season. The number of generative (flowering) individuals per population had a positive correlation with the air temperature and humidity in August of the previous vegetative season. The fruit set of D. traunsteineri is high (50.4%). This parameter is negatively correlated with the air temperature at the flowering period, while it is positively correlated with precipitation values. The seed number per capsule (4090 seeds in average) was higher than it is known for other Dactylorhiza species in the Komi Republic. The real seed production is associated with the moisture content level during the vegetative season. The seed production of D. traunsteineri was high, from 88 000 to 199 000 seeds per 1 m2 in different study years. The presence of juvenile individuals (3.5–9.4%) over all study years indicates a successful seed reproduction in this population. This parameter was positively correlated with precipitation, air temperature in August, and seed production at the previous vegetative season.
... Van de generatieve voortplanting is niet duidelijk hoe succesvol deze is. Net als andere orchideeën leeft Veenmosorchis in symbiose met een mycorrhizaschimmel; het lijkt er op dat deze symbiose in het geval van de Veenmosorchis algemenere mycorrhizaschimmels betreft (Kulikov & Filippov, 2001;Illyés et al., 2012). Veenmosorchis komt zowel in het holocene deel als in het pleistocene deel van Nederland voor op venige bodems, en hoofdzakelijk in door veenmos gedomineerde vegetaties (Kreutz & Dekker, 2000). ...
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A closer look on the habitat conditions of Bog Orchid in The Netherlands The Bog orchid (Hammarbya paludosa) is a small, inconspicuous orchid occurring in peatlands. The Bog orchid has experienced a strong decline during the last decades. The species occurs in wet habitats with Sphagnum mosses, in the gradient from acid to slightly alkaline conditions. Detailed measurements of the habitat of the species were however absent. In this article, results of a study on the habitat characteristics of twelve locations in The Netherlands are presented. The species was mainly found in peatland areas, but also in sandy areas with seepage of groundwater. The orchid was found in open vegetation dominated by Sphagnum species. The habitats showed slightly buffered conditions already at 10 cm below the soil surface. Due to the influence of surface water or seepage, the water table remained close to the surface even in summer. Nutrient concentrations, in particular of nitrogen, are very low. The management of the populations should be aimed at the preservation of the horizontal and vertical pH-gradient, a high groundwater table, an open vegetation structure as well as the creation of new habitats. Furthermore, a low nitrogen deposition seems indispensable. Measurements on seasonal fluctuations in pore water quality may reveal the mechanisms responsible for the pH-gradients and for the very low nutrient availability of the habitat of the Bog orchid. Major knowledge gaps exist on subjects such as mycorrhizal-plant symbiosis and interactions with habitat conditions, dispersal limitations, genetic diversity and population dynamics.
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In nature all orchids develop a mutualistic association with specialized group of fungi called orchid mycorrhiza. In natural environment, orchids are exclusively reliant on mycorrhizal fungi for seed germination, establishment, growth and development. In the early developmental stages, all orchids are mycoheterotrophic, and in most cases the association subsists throughout their lifespan. The significance of orchid mycorrhizal fungi (OMF) for seed germination, nourishment, growth promotion, and survival has been established more than a century ago and tremendous advancements were made in recent decades. Till date, understanding the mode of interaction between orchids and mycorrhizal fungi and determining the architecture of interaction networks are major challenges in ecology and evolutionary biology. The development of molecular techniques has empowered us to unscramble the fungal diversity comprised in the symbiosis and has facilitated us to overcome the difficulties associated with the conventional identification of OMF and orchid-fungal interactions. In addition, the application of modern biotechnological approaches resolved many controversial issues of OMF. Recently, OMF are used for biological hardening and growth promotion of in vitro raised seedlings, early flowering and quality improvement of flowers, and diseases and pests management. The aim of this communication is to update and summarize orchid mycorrhizal research including isolation, culture, and characterization of OMF, understanding the mechanisms of orchid-fungus symbiosis, and application of OMF in seed germination, growth promotion, and survival of asymbiotically raised seedlings and conservation of orchids. This appraisal will convey valuable insights to orchidologists engaged in biology, propagation, and conservation of orchids.
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This account presents information on all aspects of the biology of Hammarbya paludosa (L.) Kuntze (bog orchid, bog adder's‐mouth orchid) (Malaxis paludosa (L.) Sw., Ophrys paludosa L.), that are relevant to understanding its ecological characteristics and behaviour. The main topics are presented within the standard framework of the Biological Flora of Britain and Ireland: distribution, habitat, communities, responses to biotic factors, responses to environment, structure and physiology, phenology, floral and seed characters, herbivores and disease, history and conservation. Hammarbya paludosa is a small bisexual perennial forb. It occurs in open habitats in bogs, mires and heaths as well as semi‐shaded open woodland and, in some areas, in shaded coniferous forest. It is found in a few locations in the southern England (the New Forest, Dorset, Devon and Cornwall), in Cumbria and Northumberland, and in western counties in Wales. It is rare in Ireland but wide spread but very local in Scotland. H. paludosa has a Holarctic circumboreal range. Ninety per cent of known locations are recorded in Northern Europe and the British Isles. Hammarbya paludosa occurs in Sphagnum, other mosses and bare peat, overlying a variety of superficial deposits and sedimentary, metamorphic and igneous rocks. The pH of the rooting substrates ranges from acidic to neutral and slightly alkaline. Its nutrient status is oligotrophic, although occasionally mesotrophic, and very rarely eutrophic. It grows well in conditions of consistent lateral flow of water in the substratum. Hammarbya paludosa is visited by small insects of the order Diptera. Two species of gnats are recorded as orchid pollinators: Phronia digitata and Sciara thomae. It produces a small amount of nectar but is often considered as non‐rewarding. The seeds are dust‐like, dispersed by wind and water and the species has a short‐term seed bank. Tiny bulbils (propagules) are formed on the leaf margins. They are dispersed by water. Hammarbya paludosa has declined in Britain since the late 19th century primarily due to drainage of its mire habitats in lowland regions of southern and eastern England and Wales. The decline slowed down by the 2000s, and new locations have been found in Scotland and Ireland.
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Orchidaceae is one of the largest flowering plant families of the plant kingdom. The habitats of orchids are highly diverse, ranging from tree bark and damp forest floors to rock crevices, sandy dunes and semi-arid deserts. The diversity of endophytes (internal symbiotic fungi) associated with orchids is enormous. Most studies of endophytic fungi from orchids in the past have focused on mycorrhizal endophytes (internal symbiotic fungi associated with plant roots). There has, however, been an increasing trend to study nonmycorrhizal endophytes from orchids because of their physiological roles and their potential as sources of novel bioactive compounds. This review discusses the methods used in the isolation and identification of endophytic fungi from orchids, their diversity and host-specificity, their significance in orchid conservation and cultivation, and their potential application in the discovery of bioactive compounds.
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The diversity and host specificity of endophytic and Rhizoctonia-like fungi were investigated in orchids from the Aegean and Mediterranean regions. Endophytic fungi from various Aegean and Mediterranean orchids (Anacamptis pyramidalis (L.) L.C.M.Richard, Orchis sancta L., Ophrys fusca Link., and Serapias vomeracea subsp. orientalis Greuter) were isolated and identified partially. Surface sterilisation of roots and tubers was carried out in laminar airflow under aseptic conditions. Several modified methods for the isolation of symbiotic fungi from orchid roots and tubers were used. Most of the orchid endophytes isolated was found to be Fusarium Link ex Fr. spp. A total of 47 isolates, having genus characterisations as 44 (94%) isolates belonging to the genus Fusarium, 2 (4%) isolates belonging to the Rhizoctonia DC. ex Fr.-like fungi, and 1 (2%) isolate belonging to the genus Papulaspora Preuss, were found from the orchid root and tubers. Endophytic Fusarium spp. were isolated from Aegean and Mediterranean orchids Anacamptis pyramidalis, Orchis sancta, Ophrys fusca, and Serapias vomeracea subsp. orientalis. Rhizoctonia-like fungi were only isolated from Orchis sancta whereas Papulaspora sp. was only isolated from Anacamptis pyramidalis.
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Pure cultures of endophytic fungi were obtained from the mycorrhizae of some native Alberta orchids (Amerorchis rotundifolia, Calypso bulbosa, Coeloglossum viride, Corallorhiza maculata, Platanthera dilatata, P. hyperborea, P. obtusata). Isolates of Rhizoctonia constituted the largest group of endophytes, but few could be identified to species. One of these strains was identified as Rhizoctonia repens, a species reported to be a ubiquitous and common mycorrhizal fungus of orchids. Another strain produced the teleomorph stage and was identified as Ceratobasidium obscurum. Rhizoctonia anaticula Currah, sp.nov. is described based on five isolates bearing monilioid cells linked by prominent, narrow connections. A second group of isolates consisted of sterile, greenish black to grey fungi. After prolonged incubation, eight of these isolates formed conidia. Two isolates produced phialoconidia and were identified as Phialocephala fortinii. Five isolates, demonstrating sympodial development of conidia in the apical region of swollen or unswollen hyaline conidiogenous cells, are disposed in Leptodontidium orchidicola Sigler & Currah, sp.nov. The remaining conidial isolate was identified as Trichocladium opacum. Two isolates, resembling Rhizoctonia in cultural features, sporulated and are described as Trichosporiella multisporum Sigler & Currah, sp.nov.
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A modified technique for the symbiotic germination of terrestrial orchids has been used to propagate 23 species of European orchids in vitro. Of these, 14 have been grown on to the seedling stage of development. So far, tubers have formed on three of these and four have been transferred to a soil-based compost.
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To develop an effective propagation method, a symbiotic culture was attempted in nine species of Japanese terrestrial orchids. Seeds of three species of Goodyera (Goodyera biflora var. macrantha, G. foliosa var. laevis, and G. hachijonesis var. matsumurana), when inoculated non-symbiotically or with fungi from the binucleate Rhizoctonia fungus group among the 20 fungus strains tested germinated with virtually the same TTC activity level as embryos. Those on a symbiotic culture with binucleate Rhizoctonia grew markedly well compared with seeds on a non-symbiotic culture. The low germination rates in six of the species in the TTC test results are attributed to the unsuitability of the test culture conditions. There were numerous effective fungal strains with 5 species other than Cypripedium macranthos var. speciosum, which developed a symbiotic relationShip with only one of the fungal isolates tested. Among the 3 species (Aorchis cyclochila, Dactylorhiza aristata, and Gymnadenia camtschatica) the binucleate Rhizoctonia fungal group would be suitable for symbiotic germination and subsequent growth, whereas the 2 species Amitostigma kinoshitae and Ponerorchis graminifolia var. graminifolia, responded better to R. repens.
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Seeds of Dactylorhiza majalis (Rchb. f.) Hunt & Summerhayes were sown in vitro with a compatible fungus. A mycorrhiza was established after infection through the rhizoids of the germinating seedling. Infection through the suspensor did occur, but these hyphae did not form pelotons, and embryo cells in contact with the hyphae were filled with a tannin-like substance. Germination, and establishment of mycorrhiza, took about 14 days in vitro. From day 9 the protein reserves were hydrolysed, the protein vacuoles coalesced and starch accumulated in plastids. Certain epidermal cells developed nuclei about eight times original volume and produced rhizoids which emerged from day 11. After infection the hyphae formed pelotons in central cells with enlarged nuclei (16–64 times original volume). The intracellular hyphae developed close contacts with the hypertrophied host nuclei. Collapsed pelotons could be observed in cells from day 12, one day after infection. Meristematic activity in the uninfected chalazal end of the seedling began on day 12. On day 28 the first vascular tissue started to develop and on day 35 the beginning of a leafy shoot could be detected.
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A method is described by which seeds of terrestrial orchids are sown and retrieved in the field under almost natural conditions. For the first time it is possible to conduct a quantitative study of orchid germination in situ and observe seasonal growth and mortality of seedlings. The technique has also enabled us to investigate the relation between the site where the seeds are sown, the availability of an appropriate fungus to infect the seeds, and seedling establishment in the soil. Five local species were studied. Corallorhiza odontorhiza, Goodyera pubescens, and Galearis spectabilis all began to germinate in May-June, after 23-30 weeks in the soil. These species differed in their dependency on infection at germination time, but none of the seedlings developed beyond the point of rupturing the testa except when infected. Seeds of Liparis lilifolia and Tipularia discolor did not germinate within the first 12 months of the experiment. The implications and potential uses of this field sowing technique for further studies and for other kinds of minute seeds are discussed.
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Some terrestrial orchid species, including Epipactis palustris (L.) Crantz, are considered extremely difficult to germinate and cultivate in vitro. Observations of orchids germinating in nature are very few, and the timing and requirements for seedling establishment are unknown for most species. Seeds of E. palustris were incubated in vitro with an appropriate fungus, but germination was poor unless several other conditions were also met: scarification of the testa in Ca(OCL)2, an initial incubation for several weeks at 27°C, and a subsequent cold stratification for 8–12 weeks at 4–8°C, With these pretreatments, germnation responses exceeded 50% after incubation for 4 weeks at 20°C. Healthy protocorms with normal organ development were only produced by symbiotic culture following this lengthy seed preparation. The findings suggest that under natural conditions the seeds need some after-ripening, and the testa needs to be partially decomposed before germination. The requirement for chilling suggests that germination of seeds in situ occurs in spring.
Mykorrhizen der Orchideen, Die Orchidee
  • E Dijk
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Keimungsfördernde Faktoren bei schwerkeimenden europäischen Orchideen: 1. Bodenpilze und Agarbedeckung, Die Orchidee
  • M Weinert
Orchideenmykorrhiza: Symbiotische Anzucht Einiger Dactylorhiza-Arten
  • H Beyrle
  • F Penningsfeld
  • B Hock
Mikosimbiotrofizm kak forma konsortivnykh svyazei v rastitel'nom pokrove Sovetskogo Soyuza (Mycosymbiotrophism as a Form of Consortism in the Plant Cover of the Soviet Union
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Factors Affecting Symbiotic Germination of Orchid Seed
  • J H Warcup
On the Methods of Reproduction in Orchids of the Temperate Zone Cultivated in vitro
  • P V Kulikov
  • E G Filippov