The role of anaerobic fungi in fundamental biogeochemical cycles in the deep biosphere

A major part of the biologic activity on Earth is hidden underneath our feet in an environment coined the deep biosphere which stretches several kilometers down into the bedrock. The knowledge about life in this vast energy-poor deep system is, however, extremely scarce, particularly for micro-eukaryotes such as fungi, as most studies have focused on prokaryotes. Recent findings suggest that anaerobic fungi indeed thrive at great depth in fractures and cavities of igneous rocks in both the oceanic and the continental crust. Here we discuss the potential importance of fungi in the deep biosphere, in particular their involvement in fundamental biogeochemical processes such as symbiotic relationships with prokaryotes that may have significant importance for the overall energy cycling within this vast subsurface realm. Due to severe oligotrophy, the prokaryotic metabolism at great depth in the crust is very slow and dominantly autotrophic and thus dependent on e.g. hydrogen gas, but the abiotic production of this gas is thought to be insufficient to fuel the deep autotrophic biosphere. Anaerobic fungi are heterotrophs that produce hydrogen gas in their metabolism and have therefore been put forward as a hypothetical provider of this substrate to the prokaryotes. Recent in situ findings of fungi and isotopic signatures within co-genetic sulfide minerals formed from bacterial sulfate reduction in the deep continental biosphere indeed seem to confirm the fungi-prokaryote hypothesis. This suggests that fungi play a fundamental biogeochemical role in the deep biosphere.     

A Fungal-Prokaryotic Consortium at the Basalt-Zeolite Interface in Subseafloor Igneous Crust

We have after half a century of coordinated scientific drilling gained insight into Earth´s largest microbial habitat, the subseafloor igneous crust, but still lack substantial understanding regarding its abundance, diversity and ecology. Here we describe a fossilized microbial consortium of prokaryotes and fungi at the basalt-zeolite interface of fractured subseafloor basalts from a depth of 240 m below seafloor (mbsf). The microbial consortium and its relationship with the surrounding physical environment are revealed by synchrotron-based X-ray tomographic microscopy (SRXTM), environmental scanning electron microscopy (ESEM), and Raman spectroscopy. The base of the consortium is represented by microstromatolites—remains of bacterial communities that oxidized reduced iron directly from the basalt. The microstromatolites and the surrounding basalt were overlaid by fungal cells and hyphae. The consortium was overgrown by hydrothermally formed zeolites but remained alive and active during this event. After its formation, fungal hyphae bored in the zeolite, producing millimetre-long tunnels through the mineral substrate. The dissolution could either serve to extract metals like Ca, Na and K essential for fungal growth and metabolism, or be a response to environmental stress owing to the mineral overgrowth. Our results show how microbial life may be maintained in a nutrient-poor and extreme environment by close ecological interplay and reveal an effective strategy for nutrient extraction from minerals. The prokaryotic portion of the consortium served as a carbon source for the eukaryotic portion. Such an approach may be a prerequisite for prokaryotic-eukaryotic colonisation of, and persistence in, subseafloor igneous crust.     

Ecological Inferences from a deep screening of the Complex Bacterial Consortia associated with the coral,Porites astreoides

The functional role of the bacterial organisms in the reef ecosystem and their contribution to the coral well‐being remain largely unclear. The first step in addressing this gap of knowledge relies on in‐depth characterization of the coral microbial community and its changes in diversity across coral species, space and time. In this study, we focused on the exploration of microbial community assemblages associated with an ecologically important Caribbean scleractinian coral, Porites astreoides, using Illumina high‐throughput sequencing of the V5 fragment of 16S rRNA gene. We collected data from a large set of biological replicates, allowing us to detect patterns of geographical structure and resolve co‐occurrence patterns using network analyses. The taxonomic analysis of the resolved diversity showed consistent and dominant presence of two OTUs affiliated with the order Oceanospirillales, which corroborates a specific pattern of bacterial association emerging for this coral species and for many other corals within the genus Porites. We argue that this specific association might indicate a symbiotic association with the adult coral partner. Furthermore, we identified a highly diverse rare bacterial ‘biosphere’ (725 OTUs) also living along with the dominant bacterial symbionts, but the assemblage of this biosphere is significantly structured along the geographical scale. We further discuss that some of these rare bacterial members show significant association with other members of the community reflecting the complexity of the networked consortia within the coral holobiont.     

Does aspartic acid racemization constrain the depth limit of the subsurface biosphere?

Previous studies of the subsurface biosphere have deduced average cellular doubling times of hundreds to thousands of years based upon geochemical models. We have directly constrained the in situ average cellular protein turnover or doubling times for metabolically active micro‐organisms based on cellular amino acid abundances, D/L values of cellular aspartic acid, and the in vivo aspartic acid racemization rate. Application of this method to planktonic microbial communities collected from deep fractures in South Africa yielded maximum cellular amino acid turnover times of ~89 years for 1 km depth and 27 °C and 1–2 years for 3 km depth and 54 °C. The latter turnover times are much shorter than previously estimated cellular turnover times based upon geochemical arguments. The aspartic acid racemization rate at higher temperatures yields cellular protein doubling times that are consistent with the survival times of hyperthermophilic strains and predicts that at temperatures of 85 °C, cells must replace proteins every couple of days to maintain enzymatic activity. Such a high maintenance requirement may be the principal limit on the abundance of living micro‐organisms in the deep, hot subsurface biosphere, as well as a potential limit on their activity. The measurement of the D/L of aspartic acid in biological samples is a potentially powerful tool for deep, fractured continental and oceanic crustal settings where geochemical models of carbon turnover times are poorly constrained. Experimental observations on the racemization rates of aspartic acid in living thermophiles and hyperthermophiles could test this hypothesis. The development of corrections for cell wall peptides and spores will be required, however, to improve the accuracy of these estimates for environmental samples.     

Life's unity and flexibility: the ecological link.

The small size, ubiquity, metabolic versatility and flexibility, and genetic plasticity (horizontal transfer) of microbes allow them to tolerate and quickly adapt to unfavorable and/or changing environmental conditions. Prokaryotes are endowed with sophisticated cellular envelopes that contain molecules not found elsewhere in the biological world. Although prokaryotic cells lack the organelles that characterize their eukaryotic counterparts, their interiors are surprisingly complex. Prokaryotes sense their environment and respond as individual cells to specific environmental challenges; but prokaryotes also act cooperatively, displaying communal activities. In many microbial ecosystems, the functionally active unit is not a single species or population (clonal descendence of the same bacterium) but a consortium of two or more types of cells living in close symbiotic association. Only recently have we become aware that microbes are the basis for the functioning of the biosphere. Thus, we are at a unique time in the history of science, in which the interaction of technological advances and the exponential growth in our knowledge of the present microbial diversity will lead to significant advances not only in microbiology but also in biology and other sciences in general.     

Organisms of deep sea hydrothermal vents as a source for studying adaptation and evolution

It has been postulated that life originated in a similar environment to those of deep sea hydrothermal vents. These environments are located along volcanic ridges and are characterized by extreme conditions such as unique physical properties (temperature, pressure), chemical toxicity, and absence of photosynthesis. However, numerous living organisms have been discovered in these hostile environments, including a variety of microorganisms and many animal species which live in intimate and complex symbioses with sulfo-oxidizing and methanotrophic bacteria. Recent proteomic analyses of the endosymbiont ofRiftia pachyptila and genome sequences of some free living and symbiotic bacteria have provided complementary information about the potential metabolic and genomic capacities of these organisms. The evolution of these adaptive strategies is connected with different mechanisms of genetic adaptation including horizontal gene transfer and . various structural and functional mutations. Therefore, the organisms in this environment are good models for studying the evolution of prokaryotes and eukaryotes as well as different aspects of the biology of adaptation. This review describes some current research concerning metabolic and plausible genetic adaptations of organisms in a deep sea environment, usingRiftia pachyptila as model.     

Raman microspectrometry sulfur detection and characterization in the marine ectosymbiotic nematode Eubostrichus dianae (Desmodoridae, Stilbonematidae)

Background information. Marine nematodes belonging to the Stilbonematidae (Desmodoridae) family are described as living in obligatory association with sulfur‐oxidizing chemoautotrophic ectosymbionts. The symbiotic bacteria carrying out this chemosynthesis should contain elemental sulfur in periplasmic granules as sulfur granules of chemoautotrophic endosymbionts described in various marine invertebrates. Results. Based on TEM (transmission electron microscopy) analyses, extracellular bacteria surrounding Eubostrichus dianae possess these spherical periplasmic granules. Few investigative techniques can be used to identify elemental sulfur, S₈, such as EDXS (energy dispersive X‐ray spectroscopy) and EELS (electron energy loss spectroscopy), which are associated with cryo‐fixation of the sample to avoid sulfur loss. These techniques are time consuming, expensive and require technical skills. Raman microspectrometry applied to the analysis of E. dianae allowed us to detect elemental sulfur, S₈, and confirmed the location of these sulfur clusters in the bacterial coat. In the same way, Raman spectrometry was positively applied to the endosymbiotic bivalve Codakia orbicularis, suggesting that this technique can be used to characterize sulfur in ecto‐ as well as in endo‐symbiotic sulfur‐oxidizing bacteria. Conclusions. As Raman spectrometry can be used on living organisms (without preliminary fixation) without sample damage and preserving the molecular structure of the sulfur (denatured during chemical fixation), it represents a very well‐adapted investigative tool for biologists. This technique therefore permits us to detect quickly and easily (in a few seconds and on entire living animals) the presence of sulfur compounds in the symbiotic nematode.     

Geomicrobiology of a Weathering Crust from an Impact Crater and a Hypothesis for its Formation

Understanding the role of microbe-mineral interactions in rock weathering is vital to an understanding of nutrient availability to the biosphere and, in so far as weathering influences carbon dioxide drawdown, climate control. We studied a weathering crust on a resurge tsunami deposit (Loftarstone) from the ～ 455 Ma old Lockne impact crater, central Sweden with an integrated approach using XRD, electron microprobe analysis, SEM-EDS and GCMS analysis of organics. The lichens and fungal hyphae network preferentially weather the chlorite in the Loftarstone compared to feldspars and quartz. We demonstrate, using a fungal isolate (identified by ITS sequencing), that biologically induced dissolution of the calcite component produces cavities which increase the surface area of interaction between the biota and the rock substrate. The weathering crust exfoliates from the rock surface in sheets, which we attribute to the dissolution of the calcite matrix. We present a hypothesis for the crust development. As well as providing insights into weathering on substrates derived from a diversity of high-energy geological disturbances, such as impact events and tsunamis, the weathering crust provides a model system to understand weathering processes in other common lithologies with mixed mineralogies at small spatial scales, including many sedimentary rocks. This work reveals how each different clast plays a unique part in the weathering process, leading to a well-defined weathering sequence.     

Symbiotic lactobacilli stimulate gut epithelial proliferation via Nox‐mediated generation of reactive oxygen species

The resident prokaryotic microbiota of the metazoan gut elicits profound effects on the growth and development of the intestine. However, the molecular mechanisms of symbiotic prokaryotic–eukaryotic cross‐talk in the gut are largely unknown. It is increasingly recognized that physiologically generated reactive oxygen species (ROS) function as signalling secondary messengers that influence cellular proliferation and differentiation in a variety of biological systems. Here, we report that commensal bacteria, particularly members of the genus Lactobacillus, can stimulate NADPH oxidase 1 (Nox1)‐dependent ROS generation and consequent cellular proliferation in intestinal stem cells upon initial ingestion into the murine or Drosophila intestine. Our data identify and highlight a highly conserved mechanism that symbiotic microorganisms utilize in eukaryotic growth and development. Additionally, the work suggests that specific redox‐mediated functions may be assigned to specific bacterial taxa and may contribute to the identification of microbes with probiotic potential.     

Mock communities highlight the diversity of host‐associated eukaryotes

Host‐associated microbes are ubiquitous. Every multicellular eukaryote, and even many unicellular eukaryotes (protists), hosts a diverse community of microbes. High‐throughput sequencing (HTS) tools have illuminated the vast diversity of host‐associated microbes and shown that they have widespread influence on host biology, ecology and evolution (McFall‐Ngai et al. 2013 ). Bacteria receive most of the attention, but protists are also important components of microbial communities associated with humans (Parfrey et al. 2011 ) and other hosts. As HTS tools are increasingly used to study eukaryotes, the presence of numerous and diverse host‐associated eukaryotes is emerging as a common theme across ecosystems. Indeed, HTS studies demonstrate that host‐associated lineages account for between 2 and 12% of overall eukaryotic sequences detected in soil, marine and freshwater data sets, with much higher relative abundances observed in some samples (Ramirez et al. 2014 ; Simon et al. 2015 ; de Vargas et al. 2015 ). Previous studies in soil detected large numbers of predominantly parasitic lineages such as Apicomplexa, but did not delve into their origin [e.g. (Ramirez et al. 2014 )]. In this issue of Molecular Ecology, Geisen et al. ( 2015 ) use mock communities to show that many of the eukaryotic organisms detected by environmental sequencing in soils are potentially associated with animal hosts rather than free‐living. By isolating the host‐associated fraction of soil microbial communities, Geisen and colleagues help explain the surprisingly high diversity of parasitic eukaryotic lineages often detected in soil/terrestrial studies using high‐throughput sequencing (HTS) and reinforce the ubiquity of these host‐associated microbes. It is clear that we can no longer assume that organisms detected in bulk environmental sequencing are free‐living, but instead need to design studies that specifically enumerate the diversity and function of host‐associated eukaryotes. Doing so will allow the field to determine the role host‐associated eukaryotes play in soils and other environments and to evaluate hypotheses on assembly of host‐associated communities, disease ecology and more.     

Fine structure of the host-fungus interface in orchid mycorrhiza

Summary Electron microscopy of protocorms of Dactylorhiza purpurella infected with a symbiotic Rhizoctonia sp. showed that the intracellular hyphae examined did not penetrate the plasmalemma of the host cell. Walls of hyphae within cells bore many hemispherical protuberances over which the host plasmalemma was closely pressed. we estimate that these protuberances would increase the area of contact between hyphae and host plasmalemma by about 15%. They were not found on hyphae growing on agar. Except for these protuberances, and some vesicles or tubules which invaginated the fungus plasmalemma, no other structures were seen which could be suggested to be adaptations to transport across the living fungus-host interface.     

Middle Jurassic stromatactis mud-mound in the Pieniny Klippen Belt (Western Carpathians)

Summary A stromatactis mud-mound has been found near Slavnické Podhorie in the Czorsztyn Unit of the Pieniny Klippen Belt (Western Carpathians, Slovakia). Its stratigraphic range is Bathonian to Callovian and it is one of the youngest known true stromatactis mud-mounds. The complete shape the mound is not visible since the klippe is a tectonic block encompassed by younger Cretaceous marls. The matrix is micritic to pelmicritic mudstone, wackestone to packstone with pelecypods, brachiopods, ammonites, and crinoids. An important component of the mound is stromatactis cavities that occur as low as the underlying Bajocian-Bathonian crinoidal limestones. The stromatactis cavities are filled by radiaxial fibrous calcite (RFC) as well as in some places by internal sediment and, finally, by clear blocky calcite. Some cavities remain open with empty voids in the centres. In some stromatactis cavities, tests of cavedwelling ostracodsPokornyopsis sp. were found, surrounded by the latest stages of the RFC. This indicates that stromatactis cavities formed an open network enabling migration of the ostracods and their larvae over a period of time. Except in the case of the stromatactis cavities, there are numerous examples of seeming recrystallizationsensu Black (1952) and Ross et al. (1975) and Bathurst (1977). The radiaxial fibrous calcite encloses patches of matrix and isolated allochems. The RFC crystals are oriented perpendicularly to the substrate whether it is a cavity wall or enclosed allochems. This means that the RFC crystals could not grow from the centre of the cavity outward as postulated by Ross et al. (1975). There are also numerous “floating” isolated allochems, which are much smaller than the surrounding RFC crystals. The explanation involving three-dimensional interconnection of allochems seems to be unlikely. In the discussed mud-mound there is a conflict between apparently empty cavities that had to exist in the sediment and seeming “recrystallization” related to the same RFC that forms the initial void filling. The authors favor an alternative explanation of the “recrystallization”. We presume that the allochems served as nucleation points on which the crystals started to grow. Obviously, the allochems and the micritic patches were different from the surrounding material. RFC crystals (either short-or long-bladed) of the “recrystallization” spar grew at the expense of decaying microbial mucillages. The mucus can enclose peloids, allochems, or whole micritic patches that “floated” in the cavity and served as nucleation sites for the RFC crystals. The entire mud-mound represents a microbially bound autochthonous micritic mass; the stromatactis and stromatactis-like cavities originated where purer mucillage patches occurred, giving rise to open spaces. Such features as the morphological variety of stromatactis fabrics, the pervasive penetration of the sparry calcite into matrix, and the enclosure of the “floated” allochems and mudstone patches by sparry calcite, seem to provide support for the presence of mucus aggregates within the mound body. The mucus might be related to protozoans rather than to sponges or other well organized metazoan organisms. Occurrence of the stromatactis cavities in the underlying Bajocian-Bathonian crinoidal limestones support the inference on biological origin of the stromatactis fabrics. The alternative inorganic models of stromatactis origin (e.g., internal erosion or water-escape) are hardly applicable to the sediment formed by crinoidal skeletal detritus.     

Cryptic Sex in Symbiodinium (Alveolata,Dinoflagellata) is Supported by an Inventory of Meiotic Genes

Symbiodinium encompasses a diverse clade of dinoflagellates that are ecologically important as symbionts of corals and other marine organisms. Despite decades of study, cytological evidence of sex (karyogamy and meiosis) has not been demonstrated in Symbiodinium, although molecular population genetic patterns support the occurrence of sexual recombination. Here, we provide additional support for sex in Symbiodinium by uncovering six meiosis‐specific and 25 meiosis‐related genes in three published genomes. Cryptic sex may be occurring in Symbiodinium's seldom‐seen free‐living state while being inactive in the symbiotic state.     

Peptidoglycan in obligate intracellular bacteria

Peptidoglycan is the predominant stress‐bearing structure in the cell envelope of most bacteria, and also a potent stimulator of the eukaryotic immune system. Obligate intracellular bacteria replicate exclusively within the interior of living cells, an osmotically protected niche. Under these conditions peptidoglycan is not necessarily needed to maintain the integrity of the bacterial cell. Moreover, the presence of peptidoglycan puts bacteria at risk of detection and destruction by host peptidoglycan recognition factors and downstream effectors. This has resulted in a selective pressure and opportunity to reduce the levels of peptidoglycan. In this review we have analysed the occurrence of genes involved in peptidoglycan metabolism across the major obligate intracellular bacterial species. From this comparative analysis, we have identified a group of predicted ‘peptidoglycan‐intermediate’ organisms that includes the Chlamydiae, Orientia tsutsugamushi, Wolbachia and Anaplasma marginale. This grouping is likely to reflect biological differences in their infection cycle compared with peptidoglycan‐negative obligate intracellular bacteria such as Ehrlichia and Anaplasma phagocytophilum, as well as obligate intracellular bacteria with classical peptidoglycan such as Coxiella, Buchnera and members of the Rickettsia genus. The signature gene set of the peptidoglycan‐intermediate group reveals insights into minimal enzymatic requirements for building a peptidoglycan‐like sacculus and/or division septum.     

Paleoproteomics of Mesozoic Dinosaurs and Other Mesozoic Fossils

Molecular studies have contributed greatly to our understanding of evolutionary processes that act upon virtually every aspect of living organisms. However, these studies are limited with regard to extinct organisms, particularly those from the Mesozoic because fossils pose unique challenges to molecular workflows, and because prevailing wisdom suggests no endogenous molecular components can persist into deep time. Here, the power and potential of a molecular approach to Mesozoic fossils is discussed. Molecular methods that have been applied to Mesozoic fossils—including iconic, non‐avian dinosaurs— and the challenges inherent in such analyses, are compared and evaluated. Taphonomic processes resulting in the transition of living organisms from the biosphere into the fossil record are reviewed, and the possible effects of taphonomic alteration on downstream analyses that can be problematic for very old material (e.g., molecular modifications, limitations of on comparative databases) are addressed. Molecular studies applied to ancient remains are placed in historical context, and past and current studies are evaluated with respect to producing phylogenetically and/or evolutionarily significant data. Finally, some criteria for assessing the presence of endogenous biomolecules in very ancient fossil remains are suggested as a starting framework for such studies.     

Actin dynamics in Phytophthora infestans; rapidly reorganizing cables and immobile,long‐lived plaques

The actin cytoskeleton is a dynamic but well‐organized intracellular framework that is essential for proper functioning of eukaryotic cells. Here, we use the actin binding peptide Lifeact to investigate the in vivo actin cytoskeleton dynamics in the oomycete plant pathogen Phytophthora infestans. Lifeact–eGFP labelled thick and thin actin bundles and actin filament plaques allowing visualization of actin dynamics. All actin structures in the hyphae were cortically localized. In growing hyphae actin filament cables were axially oriented in the sub‐apical region whereas in the extreme apex in growing hyphae, waves of fine F‐actin polymerization were observed. Upon growth termination, actin filament plaques appeared in the hyphal tip. The distance between a hyphal tip and the first actin filament plaque correlated strongly with hyphal growth velocity. The actin filament plaques were nearly immobile with average lifetimes exceeding 1 h, relatively long when compared to the lifetime of actin patches known in other eukaryotes. Plaque assembly required ～30 s while disassembly was accomplished in ～10 s. Remarkably, plaque disassembly was not accompanied with internalization and the formation of endocytic vesicles. These findings suggest that the functions of actin plaques in oomycetes differ from those of actin patches present in other organisms.     

Morpho‐anatomical and molecular characterization of the mycorrhizas of European Polygala species

The mycorrhizas of 12 species of Polygala (Polygalaceae), including herbs, subshrubs and one shrub, collected from Germany, Mallorca (Spain) and Malta, were investigated by morpho‐anatomical and molecular methods. Aseptate hyphae, arbuscules and vesicles indicate an arbuscular mycorrhiza in all species examined. Hyphal spread in Polygala is predominantly, but not exclusively, intracellular and comprises three characteristic stages of colonization: (i) intracellular, linear hyphal growth in a cascading manner after penetration towards the penultimate parenchyma layer (layer 2), (ii) initially linear hyphal growth in the cells of layer 2 from where hyphal branches repeatedly penetrate the anatomically distinct innermost parenchyma layer (layer 1), forming arbuscule‐like structures therein which are subject to degeneration, (iii) more branches from the linear hyphae in layer 2 develop, but coil and make contact to the layer outside layer 2 (layer 3) in which arbuscule‐like structures similar to those in layer 1 form and degenerate. This general colonization pattern differs in details between the species, and critical comparisons, in particular between the woody P. myrtifolia, the herbaceous Polygala spp. and the mycoheterotrophic Epirixanthes spp. (Polygalaceae) suggest an evolutionary shift of mycorrhizal features within the family towards an optimization of plant benefit through the fungus. Based on the molecular marker 18S rDNA mycorrhizal fungi detected in roots of Polygala spp. are largely restricted to five clades of Glomeraceae 1 (Glomus Group A). This result rejects the hypothesis of a strict symbiotic specificity in Polygalaceae but may stimulate a discussion on functionally compatible groups of fungi.     

Re‐Evaluation of the EUK516 Probe for the Domain Eukarya Results in a Suitable Probe for the Detection of Kinetoplastids,an Important Group of Parasitic and Free‐Living Flagellates

ABSTRACT. Two frequently used universal eukaryote probes, EUK1209 and EUK516, are not consistent with one branch of the eukaryotic phylogenetic tree, the Kinetoplastida, which has undergone rapid evolution of their small subunit rRNA gene. Kinetoplastids include medically important parasitic organisms (e.g. Trypanosoma, Leishmania) and free‐living flagellates that occur in all aquatic environments and in soils (e.g. Bodo, Neobodo, Rhynchomonas). A modified probe presented here as KIN516, now based on the kinetoplastid sequence, provides a strong signal with Neobodo designis, Leishmania donovani, and Trypanosoma cruzi using the catalyzed reporter deposition protocol. EUK516 and KIN516 function as competitor probes, thereby greatly increasing discriminatory power when used in combination. The probe pair was tested in field samples collected in a freshwater pond in Norfolk, the mesohaline Elizabeth River, Norfolk, Virginia, and a tropical lagoon in Belize. The combined probes bound to 58–84% of organisms identified as eukaryotic based on having large DAPI‐stained nuclei. The contribution of kinetoplastids to total eukaryotes (positive signal of EUK516+KIN516) was much higher in marine samples (ca. 17%) than in either the freshwater or brackish water sites (<0.2%).     

Succinate dehydrogenase activity of external and internal hyphae of a vesicular-arbuscular mycorrhizal fungus,Glomus mosseae (Nicol. & Gerd.) Gerdmann and Trappe,during mycorrhizal colonization of roots of leek (Allium porrum L.), as revealed by in situ histochemical staining

The succinate dehydrogenase (SDH) activity of hyphae of the vesicular-arbuscular (VA) mycorrhizal fungus Glomus mosseae (Nicol. & Gerd.) Gerdmann and Trappe, in symbiotic association with leek (Allium porrum L.) roots, was investigated by histochemical staining in situ. Leek seedlings were transplanted to sand culture and inoculated with spores of G. mosseae placed just below the base of the stem. At intervals (14, 25, 35 and 60 days) after transplanting, the growth medium of seedlings was flooded with nitro blue tetrazolium chloride solution, thereby displacing the nutrient solution. This allowed sites of SDH activity of external and internal fungal structures of the mycorrhizas to be stained without physically disturbing the symbiotic system. After counterstaining harvested roots and mycelium with acid fuchsin, it was possible to differentiate clearly metabolically active and inactive regions of the fungus. The lengths of external hyphae and infected root both increased nearly exponentially, and were in constant proportion (1.4 m hyphae per cm of infected root) for up to 60 days. The percentage length of external hyphae with SDH activity remained almost constant (80%). In each infected length of root there was a gradation of SDH activity from inactive distal (older) hyphae to uniformly active proximal (younger) hyphae. These findings are discussed in relation to the symbiotic activity of the mycobiont.Deceased