Temporal turnover in mycorrhizal interactions: a proof of concept with orchids.

Temporal turnover events in biotic interactions involving plants are rarely assessed, although such changes might afford a considerable acclimation potential to the plant. This could enable fairly rapid responses to short-term fluctuations in growth conditions as well as lasting responses to long-term climatic trends. Here, we present a classification of temporal turnover encompassing 11 possible scenarios. Using orchid mycorrhiza as a study model, we show that temporal changes are common, and discuss under which conditions temporal turnover of fungal symbiont is expected. We provide six research questions and identify technical challenges that we deem most important for future studies. Finally, we discuss how the same framework can be applied to other types of biotic interactions.

Partial mycoheterotrophy is common among chlorophyllous plants with Paris-type arbuscular mycorrhiza.

BACKGROUND AND AIMS: An arbuscular mycorrhiza is a mutualistic symbiosis with plants as carbon providers for fungi. However, achlorophyllous arbuscular mycorrhizal species are known to obtain carbon from fungi, i.e. they are mycoheterotrophic. These species all have the Paris type of arbuscular mycorrhiza. Recently, two chlorophyllous Paris-type species proved to be partially mycoheterotrophic. In this study, we explore the frequency of this condition and its association with Paris-type arbuscular mycorrhiza. METHODS: We searched for evidence of mycoheterotrophy in all currently published 13C, 2H and 15N stable isotope abundance patterns suited for calculations of enrichment factors, i.e. isotopic differences between neighbouring Paris- and Arum-type species. We found suitable data for 135 plant species classified into the two arbuscular mycorrhizal morphotypes. KEY RESULTS: About half of the chlorophyllous Paris-type species tested were significantly enriched in 13C and often also enriched in 2H and 15N, compared with co-occurring Arum-type species. Based on a two-source linear mixing model, the carbon gain from the fungal source ranged between 7 and 93 % with ferns > horsetails > seed plants. The seed plants represented 13 families, many without a previous record of mycoheterotrophy. The 13C-enriched chlorophyllous Paris-type species were exclusively herbaceous perennials, with a majority of them thriving on shady forest ground. CONCLUSIONS: Significant carbon acquisition from fungi appears quite common and widespread among Paris-type species, this arbuscular mycorrhizal morphotype probably being a pre-condition for developing varying degrees of mycoheterotrophy.

Germination and seedling establishment in orchids: a complex of requirements.

BACKGROUND: Seedling recruitment is essential to the sustainability of any plant population. Due to the minute nature of seeds and early-stage seedlings, orchid germination in situ was for a long time practically impossible to observe, creating an obstacle towards understanding seedling site requirements and fluctuations in orchid populations. The introduction of seed packet techniques for sowing and retrieval in natural sites has brought with it important insights, but many aspects of orchid seed and germination biology remain largely unexplored. KEY CONSIDERATIONS: The germination niche for orchids is extremely complex, because it is defined by requirements not only for seed lodging and germination, but also for presence of a fungal host and its substrate. A mycobiont that the seedling can parasitize is considered an essential element, and a great diversity of Basidiomycota and Ascomycota have now been identified for their role in orchid seed germination, with fungi identifiable as imperfect Rhizoctonia species predominating. Specificity patterns vary from orchid species employing a single fungal lineage to species associating individually with a limited selection of distantly related fungi. A suitable organic carbon source for the mycobiont constitutes another key requirement. Orchid germination also relies on factors that generally influence the success of plant seeds, both abiotic, such as light/shade, moisture, substrate chemistry and texture, and biotic, such as competitors and antagonists. Complexity is furthermore increased when these factors influence seeds/seedling, fungi and fungal substrate differentially. CONCLUSIONS: A better understanding of germination and seedling establishment is needed for conservation of orchid populations. Due to the obligate association with a mycobiont, the germination niches in orchid species are extremely complex and varied. Microsites suitable for germination can be small and transient, and direct observation is difficult. An experimental approach using several levels of environmental manipulation/control is recommended.

Composition of Cypripedium calceolus (Orchidaceae) seeds analyzed by attenuated total reflectance IR spectroscopy: in search of understanding longevity in the ground.

PREMISE OF THE STUDY: Orchid seeds are minute and covered with a thin coat, yet they often have a long life after dispersal. They are notorious for low and irregular germination, in nature as well as in vitro. Since orchids are often rare species of conservational and commercial interest, reproduction by seeds is an important concern. The purpose of this study was to learn more about the resilience of these highly specialized seeds and stimulatory processes toward germination. • METHODS: We studied testa and embryos of Cypripedium calceolus to identify natural components in intact seeds and the impact of 7 yr in soil in its natural habitat. We also analyzed the effects of Ca(OCl)2, used technically to enhance germination for cultivation in vitro. For the first time with this kind of plant material, we used attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, an ideal method for minute sample sizes and surface selectivity. Thus, we could link treatments with changes in seed surface chemistry. • KEY RESULTS: A lignin-like polymer is an essential testa component that undergoes degradation by soil or hypochlorite processes. In both cases, we found a build-up of CaCO3 on the testa, which could interact with lignin to enhance germination. Very minor changes occurred in embryo reserve nutrient content after a long sojourn underground, which supports their continued viability. • CONCLUSIONS: We suggest that degradation of lignin and enrichment of the testa surface with CaCO3 are important stimulants of germination both in the habitat and during laboratory sowing.

Fungal diversity driven by bark features affects phorophyte preference in epiphytic orchids from southern China.

Epiphytic orchids exhibit varying degrees of phorophyte tree specificity. We performed a pilot study to investigate why epiphytic orchids prefer or avoid certain trees. We selected two orchid species, Panisea uniflora and Bulbophyllum odoratissimum co-occurring in a forest habitat in southern China, where they showed a specific association with Quercus yiwuensis and Pistacia weinmannifolia trees, respectively. We analysed a number of environmental factors potentially influencing the relationship between orchids and trees. Difference in bark features, such as water holding capacity and pH were recorded between Q. yiwuensis and P. weinmannifolia, which could influence both orchid seed germination and fungal diversity on the two phorophytes. Morphological and molecular culture-based methods, combined with metabarcoding analyses, were used to assess fungal communities associated with studied orchids and trees. A total of 162 fungal species in 74 genera were isolated from bark samples. Only two genera, Acremonium and Verticillium, were shared by the two phorophyte species. Metabarcoding analysis confirmed the presence of significantly different fungal communities on the investigated tree and orchid species, with considerable similarity between each orchid species and its host tree, suggesting that the orchid-host tree association is influenced by the fungal communities of the host tree bark.

Plagiotropism and auxin in Abies nordmanniana.

Main branches of Abies nordmanniana Spach. were examined through their first growth season from subapical buds around the leader bud to fully expanded shoots. Plagiotropism was evident in branch orientation, which was almost horizontal, as well as in the orientation of buds developing on the branches. Auxin transport capacity was predominantly basipetal (> 90%) and consistently higher in the middle part of the branch than in the distal and proximal ends. Auxin transport capacity was higher on the dorsal side of the branch during the short initial hyponastic growth phase, but the difference disappeared when the branch became horizontal. No dorsal-ventral differences could be detected in young horizontal branches in concentrations of indole acetic acid, cytokinins, gibberellins or abscisic acid. Branch orientation was unaffected by decapitation of the leader apex or by decapitation and replacement with exogenous auxin. However, decapitation resulted in a less plagiotropic bud arrangement on the branches, and auxin application to the leader bud scar counteracted this effect. Thus, a signal originating in the stem seems to be involved in regulating branch bud positioning, whereas the horizontal branch orientation must rely on a different mechanism, presumably autonomic within the branch.

Phenology of roots and mycorrhiza in orchid species differing in phototrophic strategy.

• The mycorrhiza of orchids represents an energy source that may replace or supplement photosynthesis. Dependency on mycotrophy in adult life stages would thus be expected to be inversely related to the prevalence of phototrophic structures. • The phenology of underground parts and mycorrhizal infection were monitored in five terrestrial species differing in leaf phenology (and thus in phototrophic strategy): Goodyera pubescens (evergreen), Tipularia discolor (wintergreen), Galearis spectabilis and Liparis lilifolia (summergreen) and Corallorhiza odontorhiza (chlorophyll deficient), growing sympatrically in a North American deciduous forest. • Mycorrhizal infection was extensive in T. discolor roots and C. odontorhiza rhizomes. Only the proximal part of roots was infected in G. pubescens, and mycorrhizal colonisation was patchy in roots and tubers of G. spectabilis and localized in the rhizome in L. lilifolia. Mycotrophic roots were long-lived (1.5-3 yrs) determinate structures and mycorrhizal infection reached maximum intensity 2-6 months after development. Mycotrophy appeared to be active all year round in mature organs. • The phenology of mycotrophic roots and patterns of mycorrhizal infection were not related to the leafy season. The hypothesis that phototrophic and mycotrophic nutrition alternate through the seasons could not be confirmed.

Ontogeny in terminal buds of Abies nordmanniana (Pinaceae) characterized by ubiquitin.

Meristematic activity in the bud meristem of Abies nordmanniana was visualized by ubiquitin immunohistochemical localization from before bud break and throughout shoot expansion. Ubiquitin was detected in meristematic cells either in the cytosol or nucleus, or both, depending on tissue type and developmental stage. During winter dormancy, ubiquitin was only observed in the protodermal/hypodermal layers, but at bud break in mid May, the signal expanded to the entire shoot tip. At the end of May, a clear zonation in ubiquitin localization appeared that lasted about one month. Throughout this period, ubiquitin was barely detectable in a central group of cells that might indicate an organizing center with stem cells. At the end of June, coinciding with the transition from scale leaf to needle primordia production, ubiquitin again was more prevalent in the peripheral cell layers. During shoot expansion, a strong ubiquitin signal developed in the axil of all needles. Most of these signals later disappeared, except for those few axils where buds actually developed. A strong ubiquitin signal was also observed in cells lining the young resin ducts. Our data showed that ubiquitin may be used as a marker for metabolic activity associated with seasonal development in the apical meristem.

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.