Zuzana Sýkorová

Communities of arbuscular mycorrhizal fungi in arable soils are not necessarily low in diversity

Communities of arbuscular mycorrhizal fungi (AMF) in five agricultural field sites of different management intensities were studied. Variable regions of the ribosomal RNA genes were used to detect and identify AMF directly within colonized roots. Roots from a continuous maize monoculture showed low AMF diversity, in agreement with previous reports on molecular diversity of AMF in agricultural soils. In contrast, a substantially higher diversity of AMF was found throughout the long term ‘DOK’ field experiment, where organic and conventional agricultural practices have been compared side by side since 1978. In this experiment, a 7‐year crop rotation is performed under lower levels of inorganic fertilizer input and chemical pest control. These results are in good agreement with analyses of the spore community previously conducted in these field sites. In a third site, an organically managed leek field with soil of very high phosphate content reflecting the highly intensive conventional field history and intensive tillage, we detected a low‐diversity community comparable to the maize monoculture. In addition to fungi from Glomus group A, which have previously been reported to dominate arable soils, we regularly found members of the genera Scutellospora, Paraglomus and Acaulospora. The genus Acaulospora was shown to occur more frequently early in the growing season, suggesting that the life history strategy of AMF may influence the active community at a given time. These data show that the diversity of AMF is not always low in arable soils. Furthermore, low‐input agriculture involving crop rotation may provide better conditions to preserve AMF diversity, by preventing the selection for the few AMF taxa tolerating high nutrient levels.

Sebacinales Everywhere: Previously Overlooked Ubiquitous Fungal Endophytes

Inconspicuous basidiomycetes from the order Sebacinales are known to be involved in a puzzling variety of mutualistic plant-fungal symbioses (mycorrhizae), which presumably involve transport of mineral nutrients. Recently a few members of this fungal order not fitting this definition and commonly referred to as ‘endophytes’ have raised considerable interest by their ability to enhance plant growth and to increase resistance of their host plants against abiotic stress factors and fungal pathogens. Using DNA-based detection and electron microscopy, we show that Sebacinales are not only extremely versatile in their mycorrhizal associations, but are also almost universally present as symptomless endophytes. They occurred in field specimens of bryophytes, pteridophytes and all families of herbaceous angiosperms we investigated, including liverworts, wheat, maize, and the non-mycorrhizal model plant Arabidopsis thaliana. They were present in all habitats we studied on four continents. We even detected these fungi in herbarium specimens originating from pioneering field trips to North Africa in the 1830s/40s. No geographical or host patterns were detected. Our data suggest that the multitude of mycorrhizal interactions in Sebacinales may have arisen from an ancestral endophytic habit by specialization. Considering their proven beneficial influence on plant growth and their ubiquity, endophytic Sebacinales may be a previously unrecognized universal hidden force in plant ecosystems.

Acaulospora alpina, a new arbuscular mycorrhizal fungal species characteristic for high mountainous and alpine regions of the Swiss Alps

Acaulospora alpina sp. nov. forms small (65–85 μm diam), dark yellow to orange-brown spores laterally on the neck of hyaline to subhyaline sporiferous saccules. The spores have a three-layered outer spore wall, a bi-layered middle wall and a three-layered inner wall. The surface of the second layer of the outer spore wall is ornamented, having regular, circular pits (1.5–2 μm diam) that are as deep as wide and truncated conical. A “beaded” wall layer as found in most other Acaulospora spp. is lacking. The spore morphology of A. alpina resembles that of A. paulinae but can be differentiated easily by the unique ornamentation with the characteristic pits and by the spore color. A key is presented summarizing the morphological differences among Acaulospora species with an ornamented outer spore wall. Partial DNA sequences of the ITS1, 5.8S subunit and ITS2 regions of ribosomal DNA show that A. alpina and A. paulinae are not closely related. Acaulospora lacunosa, which has similar color but has generally bigger spores, also has distinct rDNA sequences. Acaulospora alpina is a characteristic member of the arbuscular mycorrhizal fungal communities in soils with pH 3.5–6.5 in grasslands of the Swiss Alps at altitudes between 1800 and 2700 m above sea level. It is less frequent at 1300–1800 m above sea level, and it so far has not been found in the Alps below 1300 m or in the lowlands of Switzerland.

Diversity of mitochondrial large subunit rDNA haplotypes of Glomus intraradices in two agricultural field experiments and two semi-natural grasslands

Glomus intraradices, an arbuscular mycorrhizal fungus (AMF), is frequently found in a surprisingly wide range of ecosystems all over the world. It is used as model organism for AMF and its genome is being sequenced. Despite the ecological importance of AMF, little has been known about their population structure, because no adequate molecular markers have been available. In the present study we analyse for the first time the intraspecific genetic structure of an AMF directly from colonized roots in the field. A recently developed PCR‐RFLP approach for the mitochondrial rRNA large subunit gene (mtLSU) of these obligate symbionts was used and complemented by sequencing and primers specific for a particularly frequent mtLSU haplotype. We analysed root samples from two agricultural field experiments in Switzerland and two semi‐natural grasslands in France and Switzerland. RFLP type composition of G. intraradices (phylogroup GLOM A‐1) differed strongly between agricultural and semi‐natural sites and the G. intraradices populations of the two agricultural sites were significantly differentiated. RFLP type richness was higher in the agricultural sites compared with the grasslands. Detailed sequence analyses which resolved multiple sequence haplotypes within some RFLP types even revealed that there was no overlap of haplotypes among any of the study sites except between the two grasslands. Our results demonstrate a surprisingly high differentiation among semi‐natural and agricultural field sites for G. intraradices. These findings will have major implications on our views of processes of adaptation and specialization in these plant/fungus associations.

Ericaceous dwarf shrubs affect ectomycorrhizal fungal community of the invasive Pinus strobus and native Pinus sylvestris in a pot experiment

This study aimed to elucidate the relationship between ericaceous understorey shrubs and the diversity and abundance of ectomycorrhizal fungi (EcMF) associated with the invasive Pinus strobus and native Pinus sylvestris. Seedlings of both pines were grown in mesocosms and subjected to three treatments simulating different forest microhabitats: (a) grown in isolation and grown with (b) Vaccinium myrtillus or (c) Vaccinium vitis-idaea. Ericaceous plants did not act as a species pool of pine mycobionts and inhibited the ability of the potentially shared species Meliniomyces bicolor to form ectomycorrhizae. Similarly, Ericaceae significantly reduced the formation of Thelephora terrestris ectomycorrhizae in P. sylvestris. EcMF species composition in the mesocosms was strongly affected by both the host species and the presence of an ericaceous neighbour. When grown in isolation, P. strobus root tips were predominantly colonised by Wilcoxina mikolae, whereas those of P. sylvestris were more commonly colonised by Suillus and Rhizopogon spp. Interestingly, these differences were less evident (Suillus + Rhizopogon spp.) or absent (W. mikolae) when the pines were grown with Ericaceae. P. strobus exclusively associated with Rhizopogon salebrosus s.l., suggesting the presence of host specificity at the intrageneric level. Ericaceous plants had a positive effect on colonisation of P. strobus root tips by R. salebrosus s.l. This study demonstrates that the interaction of selective factors such as host species and presence of ericaceous plants may affect the realised niche of the ectomycorrhizal fungi.

Surprising spectra of root‐associated fungi in submerged aquatic plants

Similarly to plants from terrestrial ecosystems, aquatic species harbour wide spectra of root-associated fungi (RAF). However, comparably less is known about fungal diversity in submerged roots. We assessed the incidence and diversity of RAF in submerged aquatic plants using microscopy, culture-dependent and culture-independent techniques. We studied RAF of five submerged isoetid species collected in four oligotrophic freshwater lakes in Norway. Levels of dark septate endophytes (DSE) colonization differed among the lakes and were positively related to the organic matter content and negatively related to pH. In total, we identified 41 fungal OTUs using culture-dependent and culture-independent techniques, belonging to Mucoromycotina, Chytridiomycota, Glomeromycota, Ascomycota as well as Basidiomycota. Sequences corresponding to aquatic hyphomycetes (e.g. Nectria lugdunensis, Tetracladium furcatum and Varicosporium elodeae) were obtained. Eight arbuscular mycorrhizal taxa belonging to the orders Archaeosporales, Diversisporales and Glomerales were also detected. However, the vast majority of the fungal species detected (e.g. Ceratobasidium sp., Cryptosporiopsis rhizophila, Leptodontidium orchidicola, and Tuber sp.) have previously been known only from roots of terrestrial plants. The abundance and phylogenetic distribution of mycorrhizal as well as nonmycorrhizal fungi in the roots of submerged plants have reshaped our views on the fungal diversity in aquatic environment.

Long-term tracing of Rhizophagus irregularis isolate BEG140 inoculated on Phalaris arundinacea in a coal mine spoil bank, using mitochondrial large subunit rDNA markers

During the last decade, the application of arbuscular mycorrhizal fungi (AMF) as bioenhancers has increased significantly. However, until now, it has been difficult to verify the inoculation success in terms of fungal symbiont establishment in roots of inoculated plants because specific fungal strains could not be detected within colonized roots. Using mitochondrial large subunit ribosomal DNA, we show that Rhizophagus irregularis (formerly known as Glomus intraradices) isolate BEG140 consists of two different haplotypes. We developed nested PCR assays to specifically trace each of the two haplotypes in the roots of Phalaris arundinacea from a field experiment in a spoil bank of a former coal mine, where BEG140 was used as inoculant. We revealed that despite the relatively high diversity of native R. irregularis strains, R. irregularis BEG140 survived and proliferated successfully in the field experiment and was found significantly more often in the inoculated than control plots. This work is the first one to show tracing of an inoculated AMF isolate in the roots of target plants and to verify its survival and propagation in the field. These results will have implications for basic research on the ecology of AMF at the intraspecific level as well as for commercial users of mycorrhizal inoculation.

Terminal restriction fragment length measurement errors are affected mainly by fragment length, G + C nucleotide content and secondary structure melting point

Several methods of molecular analysis of microbial diversity, including terminal restriction fragment length polymorphism (T-RFLP) analysis are based on measurement of the DNA fragment length. Significant variation between sequence-determined and measured length of restriction fragments (drift) has been observed, which can affect the efficiency of the identification of microorganisms in the analyzed communities. In the past, this variation has been attributed to varying fragment length and purine content. In this study, principal component analysis and multiple regression analysis were applied to find the contributions of those and several other fragment characteristics. We conclude that secondary structure melting point and G+C nucleotide content, besides the fragment length, contribute to the variation observed, whereas the contribution of purine content is less important. Incomplete denaturation of the sample at the start of electrophoretic separation of fragments has been excluded as a major cause of the variation observed. Our regression model explains the observed drift variation by approximately 56%, with standard deviation of the prediction equal to approximately 1.3 bp.

Sulfur uptake in the ectomycorrhizal fungus Laccaria bicolor S238N

The importance of the ectomycorrhiza symbiosis for plant acquisition of phosphorus and nitrogen is well established whereas its contribution to sulfur nutrition is only marginally understood. In a first step to investigate the role of ectomycorrhiza in plant sulfur nutrition, we characterized sulfate and glutathione uptake in Laccaria bicolor. By studying the regulation of sulfate uptake in this ectomycorrhizal fungus, we found that in contrast to bacteria, yeast, and plants, sulfate uptake in L. bicolor was not feedback-inhibited by glutathione. On the other hand, sulfate uptake was increased by sulfur starvation as in other organisms. The activity of 3′-phosphoadenosine 5′-phosphosulfate reductase, the key enzyme of the assimilatory sulfate reduction pathway in fungi, was increased by sulfur starvation and decreased after treatment with glutathione revealing an uncoupling of sulfate uptake and reduction in the presence of reduced sulfur compounds. These results support the hypothesis that L. bicolor increases sulfate supply to the plant by extended sulfate uptake and the plant provides the ectomycorrhizal fungus with reduced sulfur.

Establishment of mycorrhizal symbiosis inGentiana verna

The last lowland locality ofGentiana verna in the Czech Republic is a calcareous grassland near Rovná at Strakonice in South Bohemia. This locality was the subject of a recovery programme that included support of the remaining population by micropropagation. The plants were inoculated with arbuscular mycorrhizal fungi (AMF) after their transfer toex vitro and the effect of AMF on their establishment and survival was studied. Although the conventional method of inoculation ofG. verna using spores or colonized root segments as an inoculum source resulted in no or negligible root colonization, the transplantation of gentians to the locality Rovná was successful and the plants became colonized with AMF very rapidly in the field. Successful mycorrhization of gentians under experimental conditions occurred only via the extraradical mycelial network established by neighbouring mycorrhizal plant species (nurse plant effect). Different nurse plant species formed different morphological types of mycorrhiza when inoculated with the same fungal isolate. Gentians always had theParis type of root colonization with intracellular hyphal loops and swellings. Intercellular hyphae, arbuscules and vesicles were not observed. No evidence for a positive growth response was found inG. verna.