In memory of Achim Trebst (1929-2017): a pioneer of photosynthesis research.

The life and work of Achim Trebst (1929-2017) was dedicated to photosynthesis, involving a wide span of seminal contributions which cumulated in more than five decades of active research: Major topics include the separation of light and dark phases in photosynthesis, the elucidation of photosynthesis by the use of inhibitors, the identification of the three-dimensional structure of photosystem II and its degradation, and an explanation of singlet oxygen formation. For this tribute, which has been initiated by Govindjee, twenty-two personal tributes by former coworkers, scientific friends, and his family have been compiled and combined with an introduction tracing the different stages of Achim Trebst's scientific life.

Divergent biology of facultative heavy metal plants.

Among heavy metal plants (the metallophytes), facultative species can live both in soils contaminated by an excess of heavy metals and in non-affected sites. In contrast, obligate metallophytes are restricted to polluted areas. Metallophytes offer a fascinating biology, due to the fact that species have developed different strategies to cope with the adverse conditions of heavy metal soils. The literature distinguishes between hyperaccumulating, accumulating, tolerant and excluding metallophytes, but the borderline between these categories is blurred. Due to the fact that heavy metal soils are dry, nutrient limited and are not uniform but have a patchy distribution in many instances, drought-tolerant or low nutrient demanding species are often regarded as metallophytes in the literature. In only a few cases, the concentrations of heavy metals in soils are so toxic that only a few specifically adapted plants, the genuine metallophytes, can cope with these adverse soil conditions. Current molecular biological studies focus on the genetically amenable and hyperaccumulating Arabidopsis halleri and Noccaea (Thlaspi) caerulescens of the Brassicaceae. Armeria maritima ssp. halleri utilizes glands for the excretion of heavy metals and is, therefore, a heavy metal excluder. The two endemic zinc violets of Western Europe, Viola lutea ssp. calaminaria of the Aachen-Liège area and Viola lutea ssp. westfalica of the Pb-Cu-ditch of Blankenrode, Eastern Westphalia, as well as Viola tricolor ecotypes of Eastern Europe, keep their cells free of excess heavy metals by arbuscular mycorrhizal fungi which bind heavy metals. The Caryophyllaceae, Silene vulgaris f. humilis and Minuartia verna, apparently discard leaves when overloaded with heavy metals. All Central European metallophytes have close relatives that grow in areas outside of heavy metal soils, mainly in the Alps, and have, therefore, been considered as relicts of the glacial epoch in the past. However, the current literature favours the idea that hyperaccumulation of heavy metals serves plants as deterrent against attack by feeding animals (termed elemental defense hypothesis). The capability to hyperaccumulate heavy metals in A. halleri and N. caerulescens is achieved by duplications and alterations of the cis-regulatory properties of genes coding for heavy metal transporting/excreting proteins. Several metallophytes have developed ecotypes with a varying content of such heavy metal transporters as an adaption to the specific toxicity of a heavy metal site.

Albanian violets of the section Melanium, their morphological variability, genetic similarity and their adaptations to serpentine or chalk soils.

Violets of the section Melanium from Albanian serpentine and chalk soils were examined for their taxonomic affiliations, their ability to accumulate heavy metals and their colonization by arbuscular mycorrhizal fungi (AMF). The sequence analysis of the ITS1-5.8S rDNA-ITS2 region showed that all the sampled six Albanian violets grouped between Viola lutea and Viola arvensis, but not with Viola tricolor. The fine resolution of the ITS sequences was not sufficient for a further delimitation of the Albanian violets within the V. lutea-V. arvensis clade. Therefore, the Albanian violets were classified by a set of morphological characters. Viola albanica, Viola dukadjinica and Viola raunsiensis from serpentine soils as well as Viola aetolica from a chalk meadow were unambiguously identified, whereas the samples of Viola macedonica showed high morphological variability. All the violets, in both roots and shoots contained less than or similar levels of heavy metals as their harboring soils, indicating that they were heavy metal excluders. All the violets were strongly colonized by AMF with the remarkable exception of V. albanica. This violet lived as a scree creeper in shallow serpentine soil where the concentration of heavy metals was high but those of P, K and N were scarce.

Mycorrhizal symbiosis effects on growth of chalk false-brome (Brachypodium pinnatum) are dependent on the environmental light regime.

AMF (arbuscular mycorrhizal fungi) colonization of the grass chalk false-brome (Brachypodium pinnatum (L.) P. B.) was studied in selected habitats under spatially different light regimes: (a) shade condition under oak trees, (b) half shade in a shrubby area and (c) full-sun conditions on unshaded grassland. This study assessed the variations in AMF colonization of the grass dependent on the light supply in field habitats. Soil, root and shoot samples were collected four times during the vegetation period (in June, July, September and October). Root colonization, root and shoot biomass as well as soil water content were determined. The highest rate of AMF colonization was detected in June under half-sun and full-sun conditions, where about 50% of the roots were colonized. The average amount of arbuscules was less than 20% in the roots at the three sites, with the highest number of arbuscules in June, under half-sun and full-sun conditions, however, not under the trees. Overall, best mycorrhizal colonization occurred during summer, and its rate decreased in autumn. This tendency inversely correlated with the amount of precipitation, and thus with the water content of soils. The high colonization rate of the examined root samples, and also its seasonal fluctuation, might reflect the importance of the symbiosis where inorganic nutrients and water are the growth-limiting factors. The marginal AMF colonization of chalk false-brome under shade conditions indicates that plants do not use AMF under all stress conditions. When low light limits photosynthesis and thus growth of the plants, they dispense with the colonization of AMF in order to save the expenditure of organic carbon.

Molecular identification of the microbial diversity in two sequencing batch reactors with activated sludge.

The diversity of the microbial community was identified in two lab-scale, ideally mixed sequencing batch reactors which were run for 115 days. One of the reactors was intermittently aerated (2 h aerobically/2 h anaerobically) whereas the other was consistently aerated. The amount of biomass as dry matter, the degradation of organic carbon determined by chemical oxygen demand and nitrogen-degradation activity were followed over the operation of the two reactors and did not show significant differences between the two approaches at the end of the experiment. At this point, the composition of the microbial community was determined by a terminal restriction fragment length polymorphism approach using multiple restriction enzymes by which organisms were retrieved to the lowest taxonomic level. The microbial composition was then significantly different. The species richness was at least five-fold higher in the intermittently aerated reactor than in the permanently kept aerobic approach which is in line with the observation that ecosystem disturbances result in higher diversity.

Nitrogen fixation and hydrogen metabolism in cyanobacteria.

This review summarizes recent aspects of (di)nitrogen fixation and (di)hydrogen metabolism, with emphasis on cyanobacteria. These organisms possess several types of the enzyme complexes catalyzing N(2) fixation and/or H(2) formation or oxidation, namely, two Mo nitrogenases, a V nitrogenase, and two hydrogenases. The two cyanobacterial Ni hydrogenases are differentiated as either uptake or bidirectional hydrogenases. The different forms of both the nitrogenases and hydrogenases are encoded by different sets of genes, and their organization on the chromosome can vary from one cyanobacterium to another. Factors regulating the expression of these genes are emerging from recent studies. New ideas on the potential physiological and ecological roles of nitrogenases and hydrogenases are presented. There is a renewed interest in exploiting cyanobacteria in solar energy conversion programs to generate H(2) as a source of combustible energy. To enhance the rates of H(2) production, the emphasis perhaps needs not to be on more efficient hydrogenases and nitrogenases or on the transfer of foreign enzymes into cyanobacteria. A likely better strategy is to exploit the use of radiant solar energy by the photosynthetic electron transport system to enhance the rates of H(2) formation and so improve the chances of utilizing cyanobacteria as a source for the generation of clean energy.

Unicellular cyanobacteria with a new mode of life: the lack of photosynthetic oxygen evolution allows nitrogen fixation to proceed.

Some unicellular N(2)-fixing cyanobacteria have recently been found to lack a functional photosystem II of photosynthesis. Such organisms, provisionally termed UCYN-A, of the oceanic picoplanktion are major contributors to the global marine N-input by N(2)-fixation. Since their photosystem II is inactive, they can perform N(2)-fixation during the day. UCYN-A organisms cannot be cultivated as yet. Their genomic analysis indicates that they lack genes coding for enzymes of the Calvin cycle, the tricarboxylic acid cycle and for the biosynthesis of several amino acids. The carbon source in the ocean that allows them to thrive in such high abundance has not been identified. Their genomic analysis implies that they metabolize organic carbon by a new mode of life. These unicellular N(2)-fixing cyanobacteria of the oceanic picoplankton are evolutionarily related to spheroid bodies present in diatoms of the family Epithemiaceae, such as Rhopalodia gibba. More recently, spheroid bodies were ultimately proven to be related to cyanobacteria and to express nitrogenase. They have been reported to be completely inactive in all photosynthetic reactions despite the presence of thylakoids. Sequence data show that R. gibba and its spheroid bodies are an evolutionarily young symbiosis that might serve as a model system to unravel early events in the evolution of chloroplasts. The cell metabolism of UCYN-A and the spheroid bodies may be related to that of the acetate photoassimilating green alga Chlamydobotrys.

The potential role of arbuscular mycorrhizal fungi in protecting endangered plants and habitats.

Ecosystems worldwide are threatened with the extinction of plants and, at the same time, invasion by new species. Plant invasiveness and loss of species can be caused by similar but opposing pressures on the community structures. Arbuscular mycorrhizal fungi (AMF) can have multiple positive effects on plant growth, productivity, health, and stress relief. Many endangered species live in symbiosis with AMF. However, the list of the International Union for Conservation of Nature and Natural Resources (IUCN Red List of Threatened Species) indicates that the mycorrhizal status of most of the threatened species has not been assessed. Rare plants often occur in specialized and also endangered habitats and might utilize specialized or unique AMF. The specificity of any endangered plant to its AMF population has not been investigated. Because most of the current AMF isolates that are available colonize a broad range of plant species, selected inocula could be used to promote growth of endangered plants before the proper and more effective indigenous AMF are characterized. Application of AMF in field sites to protect endangered plants is hardly feasible due to the complexity of plant community structures and the large amount of fungal inocula needed. Endangered plants could, however, be grown as greenhouse cultures together with appropriate fungi, and, at the relevant developmental stage, they could be re-planted into native sites to prevent extinction and to preserve plant community ecology.

Biodiversity of arbuscular mycorrhizal fungi in roots and soils of two salt marshes.

The occurrence of arbuscular mycorrhizal fungi (AMF) was assessed by both morphological and molecular criteria in two salt marshes: (i) a NaCl site of the island Terschelling, Atlantic Coast, the Netherlands and (ii) a K(2)CO(3) marsh at Schreyahn, Northern Germany. The overall biodiversity of AMF, based on sequence analysis, was comparably low in roots at both sites. However, the morphological spore analyses from soil samples of both sites exhibited a higher AMF biodiversity. Glomus geosporum was the only fungus of the Glomerales that was detected both as spores in soil samples and in roots of the AMF-colonized salt plants Aster tripolium and Puccinellia sp. at both saline sites and on all sampling dates (one exception). In roots, sequences of Glomus intraradices prevailed, but this fungus could not be identified unambiguously from DNA of soil spores. Likewise, Glomus sp. uncultured, only deposited as sequence in the database, was widely detected by DNA sequencing in root samples. All attempts to obtain the corresponding sequences from spores isolated from soil samples failed consistently. A small sized Archaeospora sp. was detected, either/or by morphological and molecular analyses, in roots or soil spores, in dead AMF spores or orobatid mites. The study noted inconsistencies between morphological characterization and identification by DNA sequencing of the 5.8S rDNA-ITS2 region or part of the 18S rDNA gene. The distribution of AMF unlikely followed the salt gradient at both sites, in contrast to the zone formation of plant species. Zygotes of the alga Vaucheria erythrospora (Xanthophyceae) were retrieved and should not be misidentified with AMF spores.

Maximizing hydrogen production by cyanobacteria.

When incubated anaerobically, in the light, in the presence of C2H2 and high concentrations of H2, both Mo-grown Anabaena variabilis and either Mo- or V-grown Anabaena azotica produce large amounts of H2 in addition to the H2 initially added. In contrast, C2H2-reduction is diminished under these conditions. The additional H2-production mainly originates from nitrogenase with the V-enzyme being more effective than the Mo-protein. This enhanced H2-production in the presence of added H2 and C2H2 should be of interest in approaches to commercially exploit solar energy conversion by cyanobacterial photosynthesis for the generation of molecular hydrogen as a clean energy source.

Drought, but not salinity, determines the apparent effectiveness of halophytes colonized by arbuscular mycorrhizal fungi.

The halophytes Plantago maritima, Aster tripolium, Artemisia santonicum, Puccinellia limosa, Festuca pseudovina and Lepidium crassifolium from two different saline soils of the Hungarian steppe were examined for colonization by arbuscular mycorrhizal fungi (AMF). The salt aster (A. tripolium) and the sea plantain (P. maritima) were examined more thoroughly by recording root colonization parameters, the salt content in the soil and monthly precipitations in 2001 and 2002. Mycorrhizal colonization was maximal in late spring to early summer and had a second peak later in the autumn. Arbuscule formation and overall mycorrhizal colonization appeared to be inversely correlated with the intensity of rainfall at the investigated sites. The results suggest that, in addition to seasonality, drought may play an important role in governing mycorrhizal activity in saline habitats. In greenhouse experiments, conditions in which AMF could overcome the inhibitory effects of sodium chloride on establishing plant-mycorrhizal symbiosis were not met.

Prokaryotic life in a potash-polluted marsh with emphasis on N-metabolizing microorganisms.

Prokaryotic life along the salt gradient of the potash marsh resulting from mining waste at Schreyahn, Northern Germany, was screened for the distribution of total prokaryote (assessed by the 16S rRNA gene) and of N2-fixing (nifH gene), denitrifying (nosZ) and nitrifying (amoA) microorganisms. Information on prokaryotes was retrieved from the different soil sites (a) by culturing in conventional media, (b) by isolating the DNA, amplifying the target genes by PCR followed by sequencing, (c) by employing the recently developed computer program (TReFID [Rösch, C., Bothe, H., 2005. Improved assessment of denitrifying, N2-fixing, and total-community bacteria by terminal restriction fragment length polymorphism analysis using multiple restriction enzymes. Applied and Environmental Microbiology 71, 2026-2035]) based on tRFLP data. New sequences were obtained as well as ones that were almost identical to those found at far distant locations. Whereas the distribution of plants strictly follows the salt gradient, this is apparently not the case with prokaryotes. Bacteria of hypersaline areas coexist with salt-non-tolerant species. The recently developed TReFID program is successfully applied to characterize a prokaryote community structure.

Arbuscular mycorrhiza and heavy metal tolerance.

Arbuscular mycorrhizal fungi (AMF) have repeatedly been demonstrated to alleviate heavy metal stress of plants. The current manuscript summarizes results obtained to date on the colonization of plants by AMF in heavy metal soils, the depositions of heavy metals in plant and fungal structures and the potential to use AMF-plant combinations in phytoremediation, with emphasis on pennycresses (Thlaspi ssp.). The focus of this manuscript is to describe and discuss studies on the expression of genes in plants and fungi under heavy metal stress. The summary is followed by data on differential gene expression in extraradical mycelia (ERM) of in vitro cultured Glomus intraradices Sy167 supplemented with different heavy metals (Cd, Cu or Zn). The expression of several genes encoding proteins potentially involved in heavy metal tolerance varied in their response to different heavy metals. Such proteins included a Zn transporter, a metallothionein, a 90 kD heat shock protein and a glutathione S-transferase (all assignments of protein function are putative). Studies on the expression of the selected genes were also performed with roots of Medicago truncatula grown in either a natural, Zn-rich heavy metal "Breinigerberg" soil or in a non-polluted soil supplemented with 100 microM ZnSO(4). The transcript levels of the genes analyzed were enhanced up to eight fold in roots grown in the heavy metal-containing soils. The data obtained demonstrate the heavy metal-dependent expression of different AMF genes in the intra- and extraradical mycelium. The distinct induction of genes coding for proteins possibly involved in the alleviation of damage caused by reactive oxygen species (a 90 kD heat shock protein and a glutathione S-transferase) might indicate that heavy metal-derived oxidative stress is the primary concern of the fungal partner in the symbiosis.

The rice field cyanobacteria Anabaena azotica and Anabaena sp. CH1 express vanadium-dependent nitrogenase.

Anabaena azotica FACHB-118 and Anabaena sp. CH1, heterocystous cyanobacteria isolated from Chinese and Taiwanese rice fields, expressed vanadium-containing nitrogenase when under molybdenum deficiency. This is the second direct observation of an alternative nitrogenase in cyanobacteria. The vanadium nitrogenase-specific genes vnfDG are fused and clustered in a phylogenetic tree next to the corresponding genes of Methanosarcina. The expression of vnfH in cells cultured in Mo-free medium and of nifH in Mo-grown cells was shown for the first time by sequencing cDNA derived from cultures of A. azotica and Anabaena sp. CH1. The vnfH sequences clustered with that of Anabaena variabilis. The vnf genes were strongly transcribed only in cultures grown either in Mo-free medium, or in W-containing medium, but also weakly in Mo-containing medium. NifH was transcribed in all media. On-line measurements of acetylene reduction by Mo-free A. azotica cultures demonstrated that the V-nitrogenase was active. Ethane was formed continuously at a rate of 2.1% of that of ethylene. Acetylene reduction of cultures grown either with or without Mo had a high temperature optimum of 42.5 degrees C. The uptake hydrogenase gene hupL was expressed in Mo-free medium concomitantly with vnfDG in A. azotica, Anabaena sp. CH1, and A. variabilis.

The bacterium Paenibacillus validus stimulates growth of the arbuscular mycorrhizal fungus Glomus intraradices up to the formation of fertile spores.

Two isolates of Paenibacillus validus (DSM ID617 and ID618) stimulated growth of the arbuscular mycorrhizal fungus Glomus intraradices Sy167 up to the formation of fertile spores, which recolonize carrot roots. Thus, the fungus was capable of completing its life cycle in the absence of plant roots, but relied instead on the simultaneous growth of bacteria. The supernatant of a mixed batch culture of the two P. validus isolates contained raffinose and another, unidentified trisaccharide. Among the oligosaccharides tested, raffinose was most effective in stimulating hyphal mass formation on plates but could not promote growth to produce fertile spores. A suppressive subtractive hybridization library followed by reverse Northern analyses indicated that several genes with products involved in signal transduction are differentially expressed in G. intraradices SY 167 when grown in coculture with P. validus (DSM 3037). The present investigation, while likely representing a significant step forward in understanding the arbuscular mycorrhizal fungus symbioses, also confirms that its optimal establishing and functioning might rely on many, as yet unidentified factors.

Differential gene expressions in arbuscular mycorrhizal-colonized tomato grown under heavy metal stress.

When tomato was grown in either "Breinigerberg" soil, which has a high content of Zn and of other heavy metals or in non-polluted soil enriched with up to 1 mM CdCl2, plants colonized with the arbuscular mycorrhizal fungus (AMF) Glomus intraradices grew distinctly better than non-mycorrhizal controls. An analysis of differential mRNA transcript formations was performed on several plant genes coding for products potentially involved in heavy metal tolerance. Northern blot analyses indicated that the mRNA from either roots or leaves was not differentially expressed in the case of LePCS1 (coding for phytochelatin synthase), Lemt1, Lemt3 and Lemt4 (for metallothioneins) or LeNramp2 (for a broad range heavy metal transporter) in both mycorrhizal and non-mycorrhizal plants, grown either with or without heavy metals. In contrast, Lemt2 was strongly expressed only in non-AMF-colonized roots, and only after growth in the Breinigerberg soil or in the presence of high CdCl2-concentrations. AMF colonization distinctly reduced the level of Lemt2 transcripts. This was also the case for the root specific LeNramp1 transporter, however, only after growth in the Breinigerberg soil, but not under Cd-stress. Likewise, the levels of LeNramp3 transcripts were reduced by the AMF colonization in roots, but not in leaves. Quantitative Real-Time RT-PCR-experiments performed with Lemt2, LeNramp1 and LeNramp3 largely corroborated the Northern analysis data. In situ hybridization experiments with Lemt2 and LeNramp1 showed that both genes were strongly expressed throughout the plant cells in non-colonized roots, whereas colonized roots revealed only few signals restricted to some parenchyma cells. All the data suggest that the transcript levels of some, but not all genes of the Nramp or mt family are elevated under heavy metal stress. AMF colonization results in a down-regulation of these genes, presumably due to the fact that the content of heavy metals is lower in mycorrhizal than in non-colonized roots. A suppression subtractive hybridization (SSH) Library from hyphae of the AMF G. intraradices grown in high versus low Zn++ provided none of the genes which were down-regulated at the plant side (mt or Nramp genes). In contrast, several gene sequences coding for enzymes potentially catalysing the detoxification of reactive oxygen species were found. Thus the fungal cells in the symbiosis may primarily have to cope with the heavy metal-induced oxidative stress.

Improved assessment of denitrifying, N2-fixing, and total-community bacteria by terminal restriction fragment length polymorphism analysis using multiple restriction enzymes.

A database of terminal restriction fragments (tRFs) of the 16S rRNA gene was set up utilizing 13 restriction enzymes and 17,327 GenBank sequences. A computer program, termed TReFID, was developed to allow identification of any of these 17,327 sequences by means of polygons generated from the specific tRFs of each bacterium. The TReFID program complements and exceeds in its data content the Web-based phylogenetic assignment tool recently described by A. D. Kent, D. J. Smith, B. J. Benson, and E. W. Triplett (Appl. Environ. Microb. 69:6768-6766, 2003). The method to identify bacteria is different, as is the region of the 16S rRNA gene employed in the present program. For the present communication the software of the tRF profiles has also been extended to allow screening for genes coding for N2 fixation (nifH) and denitrification (nosZ) in any bacterium or environmental sample. A number of controls were performed to test the reliability of the TReFID program. Furthermore, the TReFID program has been shown to permit the analysis of the bacterial population structure of bacteria by means of their 16S rRNA, nifH, and nosZ gene content in an environmental habitat, as exemplified for a sample from a forest soil. The use of the TReFID program reveals that noncultured denitrifying and dinitrogen-fixing bacteria might play a more dominant role in soils than believed hitherto.

Bacterial life and dinitrogen fixation at a gypsum rock.

The organisms of a bluish-green layer beneath the shards of a gypsum rock were characterized by molecular techniques. The cyanobacterial consortium consisted almost exclusively of Chroococcidiopsis spp. The organisms of the shards expressed nitrogenase activity (C2H2 reduction) aerobically and in light. After a prolonged period of drought at the rock, the cells were inactive, but they resumed nitrogenase activity 2 to 3 days after the addition of water. In a suspension culture of Chroococcidiopsis sp. strain PCC7203, C2H2 reduction required microaerobic conditions and was strictly dependent on low light intensities. Sequencing of a segment of the nitrogenase reductase gene (nifH) indicated that Chroococcidiopsis possesses the alternative molybdenum nitrogenase 2, expressed in Anabaena variabilis only under reduced O2 tensions, rather than the widespread, common molybdenum nitrogenase. The shards apparently provide microsites with reduced light intensities and reduced O2 tension that allow N2 fixation to proceed in the unicellular Chroococcidiopsis at the gypsum rock, unless the activity is due to minute amounts of other, very active cyanobacteria. Phylogenetic analysis of nifH sequences tends to suggest that molybdenum nitrogenase 2 is characteristic of those unicellular or filamentous, nonheterocystous cyanobacteria fixing N2 under microaerobic conditions only.

Colonization of pennycresses (Thlaspi spp.) of the Brassicaceae by arbuscular mycorrhizal fungi.

Members of the Brassicaceae are generally believed to be non-mycorrhizal. Pennycress (Thlaspi) species of this family from diverse locations in Slovenia, Austria, Italy and Germany were examined for their colonisation by arbuscular mycorrhizal fungi (AMF). Meadow species (T. praecox, T. caerulescens and T. montanum) were sparsely but distinctly colonised, as indicated by the occurrence of intraradical hyphae, vesicles, coils, and occasionally arbuscules. Species from other locations were poorly colonised, but arbuscules were not discernible. The genus Thlaspi comprises several heavy metal hyperaccumulating species (T. caerulescens, T. goesingense, T. calaminare, T. cepaeifolium). All samples collected from heavy metal soils were at best poorly colonized. Thus the chance is small to find a "hypersystem" in phytoremediation consisting of an AM fungus which prevents the uptake of the major part of the heavy metals and of a Thlaspi species which effectively deposits the residual heavy metals inevitably taken up into its vacuoles. In two different PCR approaches, fungal DNA was amplified from most of the Thlaspi roots examined, even from those with a very low incidence of AMF colonization. Sequencing of the 28S- and 18S-rDNA PCR-products revealed that different Thlaspi field samples were colonized by Glomus intraradices and thus by a common AM fungus. However, none of the sequences obtained was identical to any other found in the present study or deposited in the databanks, which might indicate that a species continuum exists in the G. intraradices clade. An effective colonization of Thlaspi by AMF could not be established in greenhouse experiments. Although the data show that Thlaspi can be colonized by AMF, it is doubtful whether an effective symbiosis with the mutual exchange of metabolites is formed by both partners.