Mixotrophy in orchids: insights from a comparative study of green individuals and nonphotosynthetic individuals of Cephalanthera damasonium

Some green orchids obtain carbon (C) from their mycorrhizal fungi and photosynthesis. This mixotrophy may represent an evolutionary step towards mycoheterotrophic plants fully feeding on fungal C. Here, we report on nonphotosynthetic individuals (albinos) of the green Cephalanthera damasonium that likely represent another evolutionary step. Albino and green individuals from a French population were compared for morphology and fertility, photosynthetic abilities, fungal partners (using microscopy and molecular tools), and nutrient sources (as characterized by 15N and 13C abundances). Albinos did not differ significantly from green individuals in morphology and fertility, but tended to be smaller. They harboured similar fungi, with Thelephoraceae and Cortinariaceae as mycorrhizal partners and few rhizoctonias. Albinos were nonphotosynthetic, fully mycoheterotrophic. Green individuals carried out photosynthesis at compensation point and received almost 50% of their C from fungi. Orchid fungi also colonized surrounding tree roots, likely to be the ultimate C source. Transition to mycoheterotrophy may require several simultaneous adaptations; albinos, by lacking some of them, may have reduced ecological success. This may limit the appearance of cheaters in mycorrhizal networks.     

Comparison of green and albino individuals of the partially mycoheterotrophic orchid Epipactis helleborine on molecular identities of mycorrhizal fungi,nutritional modes and gene expression in mycorrhizal roots

Some green orchids obtain carbon from their mycorrhizal fungi, as well as from photosynthesis. These partially mycoheterotrophic orchids sometimes produce fully achlorophyllous, leaf‐bearing (albino) variants. Comparing green and albino individuals of these orchids will help to uncover the molecular mechanisms associated with mycoheterotrophy. We compared green and albino Epipactis helleborine by molecular barcoding of mycorrhizal fungi, nutrient sources based on ¹⁵N and ¹³C abundances and gene expression in their mycorrhizae by RNA‐seq and cDNA de novo assembly. Molecular identification of mycorrhizal fungi showed that green and albino E. helleborine harboured similar mycobionts, mainly Wilcoxina. Stable isotope analyses indicated that albino E. helleborine plants were fully mycoheterotrophic, whereas green individuals were partially mycoheterotrophic. Gene expression analyses showed that genes involved in antioxidant metabolism were upregulated in the albino variants, which indicates that these plants experience greater oxidative stress than the green variants, possibly due to a more frequent lysis of intracellular pelotons. It was also found that some genes involved in the transport of some metabolites, including carbon sources from plant to fungus, are higher in albino than in green variants. This result may indicate a bidirectional carbon flow even in the mycoheterotrophic symbiosis. The genes related to mycorrhizal symbiosis in autotrophic orchids and arbuscular mycorrhizal plants were also upregulated in the albino variants, indicating the existence of common molecular mechanisms among the different mycorrhizal types.     

Comparative study of nutritional mode and mycorrhizal fungi in green and albino variants of Goodyera velutina,an orchid mainly utilizing saprotrophic rhizoctonia

The majority of chlorophyllous orchids form mycorrhizal associations with so‐called rhizoctonia fungi, a phylogenetically heterogeneous assemblage of predominantly saprotrophic fungi in Ceratobasidiaceae, Tulasnellaceae, and Serendipitaceae. It is still a matter of debate whether adult orchids mainly associated with rhizoctonia species are partially mycoheterotrophic. Here, we investigated the nutritional modes of green and albino variants of Goodyera velutina, an orchid species considered to be mainly associated with Ceratobasidium spp., by measuring their ¹³C and ¹⁵N abundances, and by molecular barcoding of their mycorrhizal fungi. Molecular analysis revealed that both green and albino variants of G. velutina harbored a similar range of mycobionts, mainly saprotrophic Ceratobasidium spp., Tulasnella spp., and ectomycorrhizal Russula spp. In addition, stable isotope analysis revealed that albino variants were significantly enriched in ¹³C but not so greatly in ¹⁵N, suggesting that saprotrophic Ceratobasidium spp. and Tulasnella spp. are their main carbon source. However, in green variants, ¹³C levels were depleted and those of ¹⁵N were indistinguishable from the co‐occurring autotrophic plants. Therefore, we concluded that the albino G. velutina variants are fully mycoheterotrophic plants whose C derives mainly from saprotrophic rhizoctonia, while the green G. velutina variants are mainly autotrophic plants, at least at our study site, in spite of their additional associations with ectomycorrhizal fungi. This is the first report demonstrating that adult nonphotosynthetic albino variants can obtain their nutrition mainly from nonectomycorrhizal rhizoctonia.     

Wide geographical and ecological distribution of nitrogen and carbon gains from fungi in pyroloids and monotropoids (Ericaceae) and in orchids

* Stable isotope abundance analyses recently revealed that some European green orchids and pyroloids (Ericaceae) are partially myco-heterotrophic, exploiting mycorrhizal fungi for organic carbon and nitrogen. Here we investigate related species to assess their nutritional mode across various forest and climate types in Germany and California. * C- and N-isotope signatures of five green pyroloids, three green orchids and several obligate myco-heterotrophic species (including the putatively fully myco-heterotrophic Pyrola aphylla) were analysed to quantify the green plants' nutrient gain from their fungal partners and to investigate the constancy of enrichment in (13)C and (15)N of fully myco-heterotrophic plants from diverse taxa and locations relative to neighbouring autotrophic plants. * All green pyroloid and one orchid species showed significant (15)N enrichment, confirming incorporation of fungi-derived N compounds while heterotrophic C gain was detected only under low irradiance in Orthilia secunda. Pyrola aphylla had an isotope signature equivalent to those of fully myco-heterotrophic plants. * It is demonstrated that primarily N gain from mycorrhizal fungi occurred in all taxonomic groups investigated across a wide range of geographical and ecological contexts. The (13)C and (15)N enrichment of obligate myco-heterotrophic plants relative to accompanying autotrophic plants turned out as a fairly constant parameter.     

Application of in vitro methods to study carbon uptake and transport by AM fungi

Just as multi-compartmented root chambers have advantages over standard plastic pots for the study of nutrient uptake by arbuscular mycorrhizal [AM] fungi in soil, so the split-plate in vitro system has advantages over the standard dual culture system for the study of the physiology of AM fungi. We used the split-plate culture system of Ri T-DNA transformed Daucus carota L. roots and Glomus intraradices Schenck & Smith, in which only the fungus has access to the distal compartment, to study the ability of germ tubes and extraradical and intraradical hyphae to take up ¹³C-labeled substrates. Labeled substrates were added to one side of the plate divider and plates were incubated for 8 weeks while the fungus proliferated on the side from which the root was excluded. Tissues then were recovered from the plate and examined via NMR spectroscopy. Results showed that the morphological phases of the fungus differed in their ability to take up these substrates, most notably that intraradical hyphae take up hexose while extraradical hyphae cannot. In addition, NMR studies indicated that intraradical hyphae actively synthesized lipids while extraradical hyphae did not. These data show that eventual axenic culture of AM fungi is more than a matter of finding the proper substrate for growth. Genetic regulation must be overcome to make extraradical hyphae behave like intraradical hyphae in terms of C uptake and metabolism. This revised version was published online in June 2006 with corrections to the Cover Date.     

红壤中VA菌根真菌侵染力及接种效应的研究

本文报道了丘陵地区发育于第四纪红色粘土母质的马尾松林自然植被下红壤、栽种胡枝子的两种侵蚀红壤及花生-油菜轮作的耕作红壤等3种生态条件下的土壤中VA菌根真菌繁殖体数量、种类、侵染势、施用石灰石粉改土和增施磷肥对VA菌根真菌侵染的影响以及VA菌根真菌的接种效应。试验结果表明,3种生态条件下的土壤中VA菌根真菌繁殖体数量和侵染势显然不同:马尾松林自然植被下的红壤中VA菌根真菌繁殖体数量大,侵染迅速;花生-油菜轮作的耕作红壤VA菌根真菌繁殖体数量也大,但侵染缓慢,有明显的滞后期;栽种胡枝子的侵蚀红壤VA菌根真菌繁殖体数量小,侵染力很低。对侵蚀红壤与耕作红壤接种VA菌根真菌,显著提高了VA菌根的侵染率,在适当施用磷肥及施石灰石粉改土后,都能促进VA菌根侵染,从而增加了植物的吸磷量,植物生长量显著提高。     

菌根真菌提高植物抗旱性机制的研究回顾与展望

菌根真菌与全世界约97％的维管植物具有广泛的共生关系.大量研究结果显示菌根植物相比于非菌根植物对于干旱胁迫具有更高的耐受性,说明菌根真菌在植物抗旱过程中发挥着重要作用.本文对近年来国内外在菌根真菌协助植物抵御干旱作用机制方面的研究进行了归纳和总结,主要包括在干旱胁迫下菌根真菌对植物生理学特性的影响机制、菌根真菌提高植物...     

In situ management and domestication of plants in Mesoamerica

BACKGROUND AND AIMS: Ethnobotanical studies in Mexico have documented that Mesoamerican peoples practise systems of in situ management of wild and weedy vegetation directed to control availability of useful plants. In situ management includes let standing, encouraging growing and protection of individual plants of useful species during clearance of vegetation, which in some cases may involve artificial selection. The aim of this study was to review, complement and re-analyse information from three case studies which examined patterns of morphological, physiological and genetic effects of artificial selection in plant populations under in situ management in the region. METHODS: Information on wild and in situ managed populations of the herbaceous weedy plants Anoda cristata and Crotalaria pumila, the tree Leucaena esculenta subsp. esculenta and the columnar cacti Escontria chiotilla, Polaskia chichipe and Stenocereus stellatus from Central Mexico was re-analysed. Analyses compared morphology and frequency of morphological variants, germination patterns, and population genetics parameters between wild and managed in situ populations of the species studied. Species of columnar cacti are under different management intensities and their populations, including cultivated stands of P. chichipe and S. stellatus, were also compared between species. KEY RESULTS: Significant differences in morphology, germination patterns and genetic variation documented between wild, in situ managed and cultivated populations of the species studied are associated with higher frequencies of phenotypes favoured by humans in managed populations. Genetic diversity in managed populations of E. chiotilla and P. chichipe is slightly lower than in wild populations but in managed populations of S. stellatus variation was higher than in the wild. However, genetic distance between populations was generally small and influenced more by geographic distance than by management. CONCLUSIONS: Artificial selection operating on in situ managed populations of the species analysed is causing incipient domestication. This process could be acting on any of the 600-700 plant species documented to be under in situ management in Mesoamerica. In situ domestication of plants could be relevant to understand early processes of domestication and current conditions of in situ conservation of plant genetic resources.     

The role of the external mycelial network of vesicular-arbuscular mycorrhizal fungi: a study of carbon transfer between plants interconnected by a common mycelium

The transfer of ¹⁴C from Lolium perenne (the donor) to Plantago lanceolata (the receiver), mediated by vesicular-arbuscular (VA) mycorrhizal fungi, was examined when the two species were grown together or separately. The VA mycorrhizal infection led to a significant increase, relative to that in uninfected plants, in the ¹⁴C transferred from donor to receiver plants, not only when the roots of the two plants were growing in intimate mixture, but also when they were separated by a root-free zone of 2.33 cm. The majority of isotope transfer between the two plant species was along the direct pathway via VA mycelium.     

红壤坡地杂草群落VA菌根真菌的宿主物种调查

本研究调查了红壤坡地幼龄果园杂草群落的物种多样性以及主要物种被VA菌根真菌的侵染率和侵染强度,分析了VA菌根真菌侵染与根际土壤磷系水平的关系。研究区域幼龄果园杂草群落共有杂草96种,分属27科。对17个科的39个物种的调查发现,所调查的物种均不同程度上被VA真菌所侵染,但科与科之间存在显著差异,同一科的不同种之间也差异明显。相关分析表明,VA菌根真菌对不同毁草种 的侵染率和侵染强度与土壤磷素水平关系不甚密切,VA菌根真菌强度可能主要决定于宿主植物特性和VA菌根真菌的选择。因此,在不影响农业生产的前提下,尽可能保持杂草的多样性将有利于VA菌根真菌的生存。     

Effect of phosphate-solubilizing bacteria and vesicular-arbuscular mycorrhizal fungi on the utilization of Bayovar rock phosphate by alfalfa plants using a sand-vermiculite medium

Phosphate-solubilizing bacteria (PSB) exhibited a high efficiency to improve plant growth and nutrition in the presence of Bayovar rock phosphate when sand-vermiculive was used as a culture medium. Treatments with dual inoculum (PSB plus mycorrhiza) significantly (P≤0.05) increased alfalfa growth. Bacteria-microbial fungi interactions resulted in a greater utilization of the rock phosphate added to the rooting medium. Although Bayovar rock phosphateper se can be considered an inert substrate because it did not stimulate plant growth, metabolites released by PSB were able to transform the rock into available forms which could be utilized by alfalfa plants.Glomus fasciculatum was the most efficient mycorrhizal endophyte under the experimental conditions employed.     

Effects of arbuscular mycorrhizal fungi and soil developmental stages on herbaceous plants growing in the early stage of primary succession on Mount Fuji

A pot culture experiment was conducted to examine the effects of arbuscular mycorrhizal (AM) fungi and soil developmental stages on the growth and nutrient absorption of pioneer plants growing in the early stage of primary succession on Mt. Fuji. Four herbaceous plants, Polygonum cuspidatum (Polygonaceae), Miscanthus oligostachyus (Gramineae), Aster ageratoides var. ovatus (Compositae), and Hedysarum vicioides (Leguminosae), were grown from seed in soils collected from two different successional stages, bare ground and an herbaceous plant community. Spores of indigenous AM fungi collected from the herbaceous plant community were used as inoculum. The initial colonizer P. cuspidatum showed very low levels of AM colonization (<0.4%), whereas the average AM colonization levels of M. oligostachyus, A. ageratoides var. ovatus, and H. vicioides were within the range of 13–49%. AM fungi had positive effects on the growth and N acquisition of the leguminous species (H. vicioides) irrespective of soil developmental stages. In contrast, AM colonization did not increase the plant dry weight and N content of the non-leguminous species (P. cuspidatum, M. oligostachyus, and A. ageratoides var. ovatus) in both soil developmental stages. A positive effect of AM colonization on P content was observed in M. oligostachyus, A. ageratoides var. ovatus, and H. vicioides only in soil collected from the herbaceous plant community. P. cuspidatum showed no or a negative response to AM colonization in all cases. These results suggest that the effect of AM fungi on plant growth depends more on the plant species than soil developmental stages in the early stage of primary succession in this volcanic area.     

In silico prediction of the peroxisomal proteome in fungi,plants and animals

In an attempt to improve our abilities to predict peroxisomal proteins, we have combined machine-learning techniques for analyzing peroxisomal targeting signals (PTS1) with domain-based cross-species comparisons between eight eukaryotic genomes. Our results indicate that this combined approach has a significantly higher specificity than earlier attempts to predict peroxisomal localization, without a loss in sensitivity. This allowed us to predict 430 peroxisomal proteins that almost completely lack a localization annotation. These proteins can be grouped into 29 families covering most of the known steps in all known peroxisomal pathways. In general, plants have the highest number of predicted peroxisomal proteins, and fungi the smallest number.     

Growth and survival of seedlings of native plants in an impoverished and highly disturbed soil following inoculation with arbuscular mycorrhizal fungi

We investigated whether arbuscular mycorrhizas influenced growth and survival of seedlings in an extremely impoverished and highly disturbed soil. Seedlings of four plants species native to the site were either inoculated with native sporocarpic arbuscular mycorrhizal (AM) fungi or fertilised prior to transplanting, and followed over 86 weeks at the site. One treatment was also irrigated with N-rich leachate from the site. In a laboratory experiment, seedlings were fertilised with excess P for 6 weeks, and location of the P store determined. Growth and survival of AM and fertilised seedlings were similar at the site. Inoculated mycorrhizal fungi and roots appeared to extend into the surrounding soil together. P concentration in leaves of all plants was extremely low. Irrigation with leachate increased growth of seedlings. In the laboratory experiment, significantly more P was stored in roots than shoots. We suggest that successful revegetation of extremely disturbed and impoverished sites requires selection of mycorrhizal fungi and plants to suit the edaphic conditions and methods of out-planting.     

Temperature constraints on the growth and functioning of root organ cultures with arbuscular mycorrhizal fungi

In this study we investigated the effects of temperature on fungal growth and tested whether the differences in fungal growth were related to the effects of temperature on carbon movement to, or within, the fungus. Growth curves and C uptake-transfer-translocation measurements were obtained for three arbuscular mycorrhizal fungi (AMF) isolates cultured within a 6-30 degrees C temperature range. A series of experiments with a model fungal isolate, Glomus intraradices, was used to examine the effects of temperature on lipid body and 33P movement, and to investigate the role of acclimation and incubation time. Temperature effects on AMF growth were both direct and indirect because, despite clear independent root and AMF growth responses in some cases, the uptake and translocation of 13C was also affected within the temperature range tested. Root C uptake and, to a lesser extent, C translocation in the fungus, were reduced by low temperatures (< 18 degrees C). Uptake and translocation of 33P by fungal hyphae were, by contrast, similar between 10 and 25 degrees C. We conclude that temperature, between 6 and 18 degrees C, reduces AMF growth, and that C movement to the fungus is involved in this response.     

Solute accumulation and decreased photosynthesis in leaves of potato plants expressing yeast-derived invertase either in the apoplast, vacuole or cytosol

Potato (Solanum tuberosum cv. Désirée) plants expressing yeast invertase directed either to the apoplast, vacuole or cytosol were biochemically and physiologically characterised. All lines of transgenic plants showed similarities to plants growing under water stress. Transformants were retarded in growth, and accumulated hexoses and amino acids, especially proline, to levels up to 40-fold higher than those of the wild types. In all transformants rates of CO₂ assimilation and leaf conductance were reduced. From the unchanged intercellular partial pressure of CO₂ and apoplastic cis-abscisic acid (ABA) content of transformed leaves it was concluded that the reduced rate of CO₂ assimilation was not caused by a limitation in the availability of CO₂ for␣the ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco). In the transformants the amount of Rubisco protein was not reduced, but both activation state and carboxylation efficiency of photosynthesis were lowered. In vacuolar and cytosolic transformants this inhibition of Rubisco might be caused by a changed ratio of organic bound and inorganic phosphate, as indicated by a doubling of phosphorylated intermediates. But in apoplastic transformants the pattern of phosphorylated intermediates resembled that of leaves of water-stressed potato plants, although the cause of inhibition of photosynthesis was not identical. Whereas in water-stressed plants increased contents of the phytohormone ABA are supposed to mediate the adaptation to water stress, no contribution of ABA to reduction of photosynthesis could be detected in invertase transformants. Received: 29 May 1996 / Accepted: 30 December 1996