Effects of plant-symbiotic relationships on the living soil microbial community and microbial necromass in a long-term agro-ecosystem.

We examined the impact of arbuscular mycorrhizal fungi and rhizobia on the living microbial community and microbial necromass under different long-term fertilization treatments at the long-term Static Fertilization Experiment Bad Lauchstädt (Germany). Phospholipid fatty acids (PLFA) and amino sugars plus muramic acid, were used as biomarkers for soil microbial bio- and necromass, respectively, and analyzed from six treatments imposed on two crop rotations, varying only in the inclusion/non-inclusion of a legume. Treatments included: two levels of only farmyard manure (FYM), only mineral fertilizer (NPK), the combined application of both fertilizer types and a non-fertilized control. PLFA profiles differed clearly between the investigated crop rotations and were significantly related to labile C, mineral N, and soil pH. This emphasizes the role of carbon, and of mycorrhizal and rhizobial symbioses, as driver for changes in the microbial community composition due to effects on the living conditions in soil. We found some evidence that legume associated symbiosis with arbuscular mycorrhizal fungi and rhizobia act as a buffer, reducing the impact of varying inputs of mineral nutrients on the decomposer community. While our results support former findings that living microbial populations vary within short-term periods and are reflective of a given crop grown in a given year, soil necromass composition indicates longer term changes across the two crop rotation types, mainly shaped by fertilizer related effects on the community composition and C turnover. However, there was some evidence that specifically the presence of a legume, affects the soil necromass composition not only over the whole crop rotation but even in the short-term.

Endogenous rhythmic growth in oak trees is regulated by internal clocks rather than resource availability.

Common oak trees display endogenous rhythmic growth with alternating shoot and root flushes. To explore the mechanisms involved, microcuttings of the Quercus robur L. clone DF159 were used for (13)C/(15)N labelling in combination with RNA sequencing (RNASeq) transcript profiling of shoots and roots. The effect of plant internal resource availability on the rhythmic growth of the cuttings was tested through inoculation with the ectomycorrhizal fungus Piloderma croceum. Shoot and root flushes were related to parallel shifts in above- and below-ground C and, to a lesser extent, N allocation. Increased plant internal resource availability by P. croceum inoculation with enhanced plant growth affected neither the rhythmic growth nor the associated resource allocation patterns. Two shifts in transcript abundance were identified during root and shoot growth cessation, and most concerned genes were down-regulated. Inoculation with P. croceum suppressed these transcript shifts in roots, but not in shoots. To identify core processes governing the rhythmic growth, functions [Gene Ontology (GO) terms] of the genes differentially expressed during the growth cessation in both leaves and roots of non-inoculated plants and leaves of P. croceum-inoculated plants were examined. Besides genes related to resource acquisition and cell development, which might reflect rather than trigger rhythmic growth, genes involved in signalling and/or regulated by the circadian clock were identified. The results indicate that rhythmic growth involves dramatic oscillations in plant metabolism and gene regulation between below- and above-ground parts. Ectomycorrhizal symbiosis may play a previously unsuspected role in smoothing these oscillations without modifying the rhythmic growth pattern.

Genome Sequence of the Mycorrhiza Helper Bacterium Streptomyces sp. Strain AcH 505.

A draft genome sequence of Streptomyces sp. strain AcH 505 is presented here. The genome encodes 22 secondary metabolite gene clusters and a large arsenal of secreted proteins, and their comparative and functional analyses will help to advance our knowledge of symbiotic interactions and fungal and plant biomass degradation.

Draft Genome Sequence of Streptomyces sp. Strain 150FB, a Mushroom Mycoparasite Antagonist.

Streptomyces sp. strain 150FB, isolated from the cap surface of a bolete mushroom, inhibits the growth of the mycoparasitic Sepedonium species. Functional annotation of the strain 150FB draft genome identified 22 putative secondary metabolite biosynthetic gene clusters and genes encoding secreted proteins, which may contribute to the inhibition of the mycoparasite.

Community structure of arbuscular mycorrhizal fungi associated to Veronica rechingeri at the Anguran zinc and lead mining region.

Root colonization and diversity of arbuscular mycorrhizal fungi (AMF) were analyzed in Veronica rechingeri growing in heavy metal (HM) and non-polluted soils of the Anguran Zn and Pb mining region (Iran). Three species could be separated morphologically, while phylogenetic analyses after PCR amplification of the ITS region followed by RFLP and sequencing revealed seven different AMF sequence types all within the genus Glomus. Rarefaction analysis confirmed exhaustive molecular characterization of the AMF diversity present within root samples. Increasing heavy metal contamination between the sites studied was accompanied by a decrease in AMF spore numbers, mycorrhizal colonization parameters and the number of AMF sequence types colonizing the roots. Some AMF sequence types were only found at sites with the highest and lowest soil HM contents, respectively.

Rationalizing molecular analysis of field-collected roots for assessing diversity of arbuscular mycorrhizal fungi: to pool, or not to pool, that is the question.

For rationalizing molecular analysis of field-collected roots in diversity studies on arbuscular mycorrhiza, we compared three different approaches. After DNA extraction from 50 root samples of Plantago lanceolata grown on monoculture plots at a former arable field site, (1) DNAs were amplified separately by nested PCR and each amplicon was cloned separately; (2) DNAs were amplified separately by nested PCR, 1 mul of each amplicon was pooled, and a single cloning was made from the resulting amplicons mix; and (3) DNAs were pooled and the single amplicon derived from the nested PCR was cloned. Based on these three different methods, 109 nuclear ribosomal internal transcribed spacer sequences were obtained. Methods 1 and 2 enabled the detection of almost similar levels of arbuscular mycorrhizal fungal diversity. However, method 1 was expensive and time-consuming as much more cloning had to be done. Method 3 was completely biased by preferential amplification of nontarget organisms, which were only detected in low frequencies by the other methods.

Oribatid mites as potential vectors for soil microfungi: study of mite-associated fungal species.

The ability of soil-living oribatid mites to disperse fungal propagules on their bodies was investigated. Classical plating methods were applied to cultivate these fungi and to study their morphology. Molecular markers were used for further determination. The nuclear ribosomal large subunit and the nuclear ribosomal internal transcribed spacer of DNA extracts of the cultured fungi as well as total DNA extracts of the mites themselves, also containing fungal DNA, were amplified and sequenced. Based on phylogenetic analysis, a total of 31 fungal species from major fungal groups were found to be associated with oribatid mites, indicating that mites do not selectively disperse specific species or species groups. The detected taxa were mainly saprobiontic, cosmopolitan (e.g., Alternaria tenuissima), but also parasitic fungi (Beauveria bassiana) for whose dispersal oribatid mites might play an important role. In contrast, no mycorrhizal fungi were detected in association with oribatid mites, indicating that their propagules are dispersed in a different way. In addition, fungi that are known to be a preferred food for oribatid mites such as the Dematiacea were not detected in high numbers. Results of this study point to the potential of oribatid mites to disperse fungal taxa in soil and indicate that co-evolutionary patterns between oribatid mites and their associated fungi might be rare or even missing in most cases, since we only detected ubiquitous taxa attached to the mites.

Diversity of arbuscular mycorrhizal fungi in grassland spontaneously developed on area polluted by a fertilizer plant.

Mycorrhizal colonization and diversity of arbuscular mycorrhizal fungi (AMF) were analyzed in a calcareous grassland with residual phosphate contamination 10 years after the closure of a pollutant fertilizer plant in Thuringia (Germany). AMF were detected in 21 of 22 plant species analyzed. Mean mycorrhization levels reached up to 74.5% root length colonized. AMF diversity was analyzed based on 104 sequences of the internal transcribed spacer (ITS) of the ribosomal DNA. Phylogenetic analyses revealed a total of 6 species all belonging to the genus Glomus. There was no overlap between species detected as active mycorrhizas on roots (2 taxa) or as spores (4 taxa). Compared to the regional context, the diversity of AMF at our field site was reduced, which may reflect a residual disturbance effect. However, none of the detected species was exclusive to the polluted site as they are commonly found in the region.

Recent advances in exploring physiology and biodiversity of ectomycorrhizas highlight the functioning of these symbioses in ecosystems.

Ectomycorrhizas, the dominating mycorrhizal symbiosis in boreal, temperate and some tropical forests, are formed by 5000-6000 species of the asco- and basidiomycetes. This high diversity of fungal partners allows optimal foraging and mobilisation of various nitrogen and phosphorus forms from organic soil layers. In this review, two approaches to study the functioning of this multitude of symbiotic associations are presented. On selected culture models, physiological and molecular investigations have shown that the supply of hexoses has a key function in controlling the plant-fungus interaction via partner-specific regulation of gene expression. Environmental factors which affect fungal carbon supply, such as increased nitrogen availability, also affect mycorrhiza formation. Based on such laboratory results, the adaptative capability of ectomycorrhizas to changing field conditions is discussed. The second approach consists of analysing the distribution of mycorrhizas in ecosystem compartments and to relate distribution patterns to variations of ecological factors. Recent advances in identification of fungal partners in ectomycorrhizas by analysing the internal transcribed spacer of ribosomal DNA are presented, which can help to resolve sampling problems in field studies. The limits of the laboratory and the field approaches are discussed. Despite some problems, this combined approach is the most promising. Direct investigation of gene expression, which has been introduced for soil bacteria, will be difficult in the case of mycorrhizal fungi which constitute organisms with functionally varying structures.

A gnotobiotic culture system with oak microcuttings to study specific effects of mycobionts on plant morphology before, and in the early phase of, ectomycorrhiza formation by Paxillus involutus and Piloderma croceum.

Homogeneously developed oak (Quercus robur L.) microcuttings were challenged in a Petri-dish system with the mycobionts Piloderma croceum J. Erikss. & Hjortst. and Paxillus involutus (Batsch) Fr. Non-destructive observations over 10 wk followed by d. wt measurements at the end of the assays served to precisely characterize root and shoot development, dynamics of mycorrhizal colonization and morphological ratio. In the system, plant development, and especially root morphogenesis, had more similarities to those of stump cuttings or of older seedlings than to those of 3-month-old seedlings. Whereas Paxillus involutus displayed early mycorrhizal colonization and had no significant morphological effects on the host Piloderma croceum modified markedly the entire plant development before a delayed mycorrhiza formation. The latter mycobiont stimulated elongation and production of the lateral root system and also increased the leaf surface. However, no corresponding weight increases were noted, which was reflected by significant increase of both specific root length and specific leaf area. These differential effects are discussed in relation to data concerning carbon requirement and auxin production of the mycobionts. The developed system was shown to be highly suitable for comparative studies with diverse mycobionts on recognition and physiological balance between partners before, and in the early stage of, formation of mycorrhizas.

Characterization and identification of black alder ectomycorrhizas by PCR/RFLP analyses of the rDNA internal transcribed spacer (ITS).

The identity of black alder (Alnus glutinosa (L.) Gaertn.) ectomycorrhizas was investigated using PCR/RFLP analysis of the ITS region from 16 morphotypes sampled at a 60-yr-old black alder stand. A comparison was made with restriction patterns from sporocarps of 28 mycobionts, of which 16 originated from the same stand, the remaining 12 came from two geographically distant alder stands. Eight of the mycorrhizal types could thus be identified, whereas eight mycorrhizal types remained unidentified. The identified mycorrhizas belonged to the genera Russula, Lactarius, Naucoria and Cortinarius. Four of the identified ectomycorrhizal types had identical PCR/RFLP profiles to corresponding fruit bodies from all investigated stands with no detectable intraspecific variation, despite the geographical distance of c.300 km between the sampling locations. By contrast, intraspecific variation between sporocarps from the different locations was detected in Paxillus rubicundulus, mycorrhizas of which were not found. The diversity of fruiting alder mycobionts at the main experimental plot only partly matched the diversity observed from mycorrhizas when comparing their PCR/RFLP profiles. The results are discussed regarding sampling techniques, PCR/RFLP analyses and ecological aspects.

DNA polymorphism in morels: complete sequences of the internal transcribed spacer of genes coding for rRNA in Morchella esculenta (yellow morel) and Morchella conica (black morel).

The internal transcribed spacer (ITS) of the gene coding for rRNA was sequenced in both directions with the gene walking technique in a black morel (Morchella conica) and a yellow morel (M. esculenta) to elucidate the ITS length discrepancy between the two species groups (750-bp ITS in black morels and 1,150-bp ITS in yellow morels.

Polymorphism in morels: isozyme electrophoretic analysis.

The aim of this study was to assess whether isozyme polymorphism in different members of the Morchellaceae could be used to improve the systematics in this fungal group and to characterize intraspecific crossings between monosporal strains in Morchella esculenta. For this purpose, isozyme electrophoretic analysis of the following enzymes was performed: glutamine synthetase, NAD-glutamate dehydrogenase, NADP-glutamate dehydrogenase, aspartate aminotransferase, malate dehydrogenase, NAD-glyceraldehyde phosphate dehydrogenase, glucose phosphate isomerase, and superoxide dismutase. The analyses allowed discrimination at the inter- or intra-specific levels and could help to establish a method of identification for strains in the Morchellaceae. To a certain extent they appeared to be suitable to analyze interactions of monosporal strains of Morchella esculenta in pairing experiments. The polymorphism shown in this study was consistent with the phylogenetic relationships between the investigated strains only at the genus level.Key words: isozyme analysis, electrophoresis, Morchella sp., polymorphism.