Latent infections of <Emphasis Type="Italic">Fomes fomentarius</Emphasis> in the xylem of European beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis>)

Fungal colonisation originating from endophytic thalli in wood of healthy European beech trees (Fagus sylvatica) was studied. Fungi were isolated from wood immediately after felling and after incubation for 8, 16 and 24 weeks under two different drying regimes. Two media were used to isolate fungi: malt extract agar with and without thiabendazol. Thiabendazol was added to inhibit non-basidiomycetes. The two drying regimes had no influence on the species composition of the recovered mycobiota and the frequency of isolation of these species. Mycelia of basidiomycetes except Coniophora puteana emerged only from wood samples inoculated onto malt extract agar containing thiabendazol. Only a few isolates were obtained from freshly cut wood, but a great number of isolates was recovered already after eight weeks of wood incubation. Four taxa accounted for 88 % of the total number of isolates: Hypoxylon fragiforme, Trichoderma spp., and the basidio-mycetes Coniophora puteana and Fomes fomentarius. The latter had not been considered an endophyte before. The isolates of F. fomentarius were made exclusively from the stem and some large diameter branches, which are the locations of its basidiocarps on dying trees. Every isolate of F. fomentarius was genetically different as revealed by tests for somatic compatibility. Advantages of the presumed endophytic strategy of F. fomentarius are discussed. Microscopic investigations showed a great number of hyphae within the cell lumina of vessels and a distinct wood degradation already after eight weeks of wood incubation.     

Variation of cytosine methylation patterns in European beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.)

The role of epigenetic phenomena in plant adaptation is becoming widely recognized and the potential of epigenetics for forestry practice has been demonstrated as well. In this study, methylation-sensitive amplification polymorphism (MSAP) markers were investigated in 20 European beech (Fagus sylvatica L.) provenances that cover most of Europe and were planted in two climatically contrasting provenance trial plots. Correlations of cytosine-methylation patterns at five loci and overall DNA methylation with climatic conditions of the sites of population origin and budburst phenology were detected, suggesting that methylation at particular loci was influenced by the weather or photoperiod during embryogenesis or even earlier. Alternation of methylation patterns may also have been caused by genetic mutation. Frequencies of methylation patterns at three loci differed between the two trial locations, indicating that a climatically induced change of methylation during the ontogeny occurs as well. The results suggest that the rules for collection, transfer, and use of forest reproductive materials should also consider epigenetic effects.     

Geographic distribution of chloroplast variation in Italian populations of beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.)

The distribution of chloroplast DNA (cpDNA) variation in Italian beech (Fagus sylvatica L.) populations was studied using PCR-RFLP and microsatellite markers. In total, 67 populations were analysed, and 14 haplotypes were identified by combining the two marker types. A remarkable subdivision of cpDNA diversity in Italian beech was found, as indicated by a high level of genetic differentiation (G_st=0.855). The highest level of total haplotype diversity (h_t=0.822) was estimated for southern Italian populations. The highest number of haplotypes was found in the central-southern region of the peninsula. The nested clade analysis provided evidence for past fragmentation events that may have been occurred during the Quaternary glaciations and had a major role in defining the genetic structure of the central-southern Italian beech populations. Only one haplotype apparently spread towards the north of Italy along the Apennine chain and reached the Italian slope of the western part of the Alps (Maritime Alps, Liguria). All haplotypes found along the Apennines remained trapped in the Italian peninsula. Southern and central Italy represent hotspots of haplotype diversity for Italian beech.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by O. Savolainen     

Proteins and polyamines during dormancy breaking of European beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.) seeds

The studies were carried out on Fagus sylvatica seeds during stratification and their germination. After imbibition beechnuts were subjected to cold (3 °C — temperature which breaks dormancy) or warm (15 °C — temperature unable to break dormancy) stratification and alternatively were treated with polyamine synthesis inhibitors: canavanine and DFMO (difluoromethylornithine). After cold stratification in embryo axes we found (using 2-D electrophoresis) about 150 new proteins absent in dry seeds. Exogenous spermidine increased the protein synthesis, percent of germinated seeds and accelerated breaking of dormancy. In contrast, canavanine and DFMO decreased dynamic of protein synthesis, quantity of proteins probably synthesised de novo, and percent of germinated seeds. The maximum of polyamine content in embryo axes during cold stratification preceded such the maximum during warm stratification. Irrespective of the influence of PAs and inhibitors of PA synthesis, the comparison of electrophoregrams and autoradiograms showed that different groups synthesised de novo appeared after different periods of cold stratification. Probably the part of this protein is associated with Fagus sylvatica seeds dormancy breaking.     

Optimized nonradioisotopic amplified fragment length polymorphism method for European beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.)

The amplified fragment length polymorphism (AFLP) technique has increasingly been used for the study of forest tree species. A nonradioisotopic AFLP method was optimized for European beech (Fagus sylvatica L.) and found reproducible. However, type ofTaq DNA polymerase and choice of primers must be considered for a consistent AFLP pattern.     

Fine-root trait plasticity of beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis>) and spruce (<Emphasis Type="Italic">Picea abies</Emphasis>) forests on two contrasting soils

Aim

The fine roots of trees may show plastic responses to their resource environment. Several, contrasting hypotheses exist on this plasticity, but empirical evidence for these hypotheses is scattered. This study aims to enhance our understanding of tree root plasticity by examining intra-specific variation in fine-root mass and morphology, fine-root growth and decomposition, and associated mycorrhizal interactions in beech (Fagus sylvatica L.) and spruce (Picea abies (L.) Karst.) forests on soils that differ in resource availability.

Methods

We measured the mass and morphological traits of fine roots (i.e. ≤ 2 mm diameter) sampled to 50 cm depth. Fine-root growth was measured with ingrowth cores, and fine-root decomposition with litter bags. Mycorrhizal fungal biomass was determined using ingrowth mesh bags.

Results

Both tree species showed more than three times higher fine-root mass, and a ten-fold higher fine-root growth rate on sand than on clay, but no or marginal differences in overall fine-root morphology. Within the fine-root category however, beech stands had relatively more root length of their finest roots on clay than on sand. In the spruce stands, ectomycorrhizal mycelium biomass was larger on sand than on clay.

Conclusions

In temperate beech and spruce forests, fine-root mass and mycorrhizal fungal biomass, rather than fine-root morphology, are changed to ensure uptake under different soil resource conditions. Yet enhancing our mechanistic understanding of fine-root trait plasticity and how it affects tree growth requires more attention to fine-root dynamics, the functional diversity within the fine-roots, and mycorrhizal symbiosis as an important belowground uptake strategy.

     

Seasonal activity of non-specific acid and alkaline phosphatases in beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis>) leaves

The activities of acid and alkaline phosphatases along with phosphorus content in leaves of European beech (Fagus sylvatica L.) were studied for a period from April to October. The phosphorus content of beech leaves was highest in April, at the beginning of the vegetation period; from May to October it was twofold lower than in April. Acid phosphatase activity (per unit fresh weight) in leaves collected from the middle part of the crown decreased significantly in May and July compared to the enzyme activity in April. In both the low and middle parts of the crown, the acid phosphatase activity had a peak in August, and thereafter decreased in September and October. No correlations between acid phosphatase activity and phosphorus concentrations were found. Alkaline phosphatase activity was very low and in some cases near the detection limit during the whole observation period.     

Long-range transport of beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.) pollen to Catalonia (north-eastern Spain)

Local and long-range transport of beech (Fagus sylvatica) pollen was analysed by using 23-year data (1983-2007) at six stations in Catalonia, Spain, and numerical simulations. Back trajectories and synoptic meteorology indicated a consistent north European provenance during beech pollen peak days. Specifically, the area from northern Italy to central Germany was the most probable source, as indicated by a source-receptor model based on back trajectories. For the event with the highest pollen levels (17 May 2004), back trajectories indicated a source in the Vosges (NE France) and the Schwarzwald (SW Germany) regions. By applying a mesoscale model (MM5) to this event, pollen transport could be further refined, allowing its entrance to Catalonia through the lower easternmost pass of the Pyrenees (the Alberes pass, 500 m a.s.l.) to be described. Hourly counts of Fagus pollen allowed the timing of pollen arrival during this episode to be matched with the model results regarding the above-mentioned passage. This study may help to interpret some results of modern beech genetic diversity and contribute to the understanding of paleopalynological records by taking long-range transport into consideration.     

Clustered root distribution in mature stands of <Emphasis Type="Italic">Fagus sylvatica</Emphasis> and <Emphasis Type="Italic">Picea abies</Emphasis>

Distribution of small roots (diameter between 2 mm and 5 mm) was studied in 19 pits with a total of 72 m² trench profile walls in pure stands of Fagus sylvatica and Picea abies. Root positions within the walls were marked and transformed into x-coordinates and y-coordinates. In a GIS-based evaluation, zones of potential influence around each root were calculated. The total potential influence produced isoline maps of relative root influence zones, thus indicating small root clustering. The questions studied were (1) whether there were marked clusters of small roots in the soil and (2) whether trees surrounding the pit (defined as tree density) correlate with the root abundance and distribution on the trench profile walls. Small roots of both species showed maximum abundance in the top 20 cm of the soil, where pronounced root clusters occurred next to areas with only low root accumulation. The area of root clusters did not differ significantly between the two stands. Weighted clumping, WC, calculated as a product of root class, and its area was used as an index of root clustering, which again did not differ between beech and spruce stands. However, evaluations on a single root level showed that beech achieved the same degree of clustering with lower number of roots. Regardless of soil properties related to root clusters, a significantly higher clustering acquired per root for beech than for spruce suggests beech to be more efficient in belowground acquisition of space. Because none of the parameters describing root clustering were correlated with tree density around the investigated soil profiles, clusters of small roots are inherently present within the tree stands.     

Climate sensitivity of radial growth in European beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.) at different aspects in southwestern Germany

The climate sensitivity of radial growth in European beech (Fagus sylvatica L.) was analyzed within a narrow valley in the Swabian Alb (southwestern Germany). We collected stem disks from three aspects (NE, NW and SW) of trees belonging to different social classes. Common climatic factors limiting growth across the valley were identified using a principal component analysis (PCA). Further, we performed hierarchical cluster analysis (HCA), redundancy analysis (RDA) and bootstrapped correlation analysis to reveal differences in chronologies and climate-growth relationships between aspect and social class. Climatic variables considered in our analyses were monthly and seasonal data on temperature and precipitation, as well as a self-calibrating Palmer drought severity index (sc-PDSI). We identified drought in the period June–August as the most prominent factor limiting growth across the valley. Dominant and co-dominant trees at the NW and SW aspects were found to be particularly drought sensitive, whereas intermediate trees were less susceptible to drought. Underlying causes of established climate–growth relationships are discussed in the context of drought susceptibility, tree-size modulation and tree physiological processes.     

Ascorbate and glutathione metabolism during development and desiccation of beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.) seeds

The ascorbate-glutathione system was studied during development and maturation of beech (Fagus sylvatica L.) seeds, the classification of which in the orthodox category is controversial. This study revealed an increase in glutathione content after acquisition of desiccation tolerance, which was more intensive in embryonic axes than in cotyledons. During seed maturation, the redox status of glutathione markedly changed toward the more reducing state, especially in cotyledons. Ascorbic acid content decreased during maturation, mostly in cotyledons. Activities of the enzymes of the ascorbate-glutathione cycle—ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2)—were markedly higher in embryonic axes than in cotyledons throughout the study period. In the course of seed maturation, the activities of these enzymes decreased. Importance of the ascorbate-glutathione cycle in desiccation tolerance of beech seeds was discussed in relation to results for typical orthodox and recalcitrant seeds of other broadleaved species.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

A RAPD,AFLP and SSR linkage map,and QTL analysis in European beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.)

The genetic linkage map of European beech (Fagus sylvatica L.) that we report here is the first to our knowledge. Based on a total of 312 markers (28 RAPDs, 274 AFLPs, 10 SSRs) scored in 143 individuals from a F₁ full-sib family. Two maps (one for each parent) were constructed according to a two-way pseudo-testcross mapping strategy. In the male map 119 markers could be clustered in 11 major groups (971 cM), while in the female map 132 markers were distributed in 12 major linkage groups (844 cM). In addition, four and one minor linkage groups (doublets and triplets) were obtained for the male and female map respectively. The two maps cover about 82% and 78% of the genome. Based on the position of 15 AFLP and 2 SSR loci segregating in both parents, seven homologous linkage groups could be identified. In the same pedigree we investigated the association with genetic markers of several quantitative traits: leaf area, leaf number and shape in 2 different years, specific leaf area, leaf carbon-isotope discrimination and tree height. A composite interval-mapping approach was used to estimate the number of QTLs, the amount of variation explained by each of them, and their position on the genetic linkage maps. Eight QTLs associated with leaf traits were found that explained between 15% and 35% of the trait variation, five on the female map and three on the male map.Communicated by D. B. Neale     

Stable calcium isotope speciation and calcium oxalate production within beech tree (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.) organs

In this study, we linked Ca speciation with isotope composition in plants. To do this, we performed leachate experiments to access the soluble Ca, structurally bound Ca and insoluble Ca (i.e., water and weak acid resistant) within beech tree organs (Fagus sylvatica L.). Ca isotopic measurements were combined with infrared spectroscopy and calcium oxalate biomineralization identification. The results from our study indicate that bark and leaves are the most enriched in monohydrated calcium oxalate crystals (whewellite), which are observable in parenchyma and sclerenchyma tissues, whereas roots and wood are enriched in structurally bound Ca. Our leaching experiments also show decreasing δ^44/40Ca isotopic signatures in the order of soluble Ca > structurally bound Ca > insoluble Ca. This finding implies that because leaves degrade faster than wooden organs and because Ca linked to pectate decomposes faster than Ca linked to oxalate crystals, differential Ca isotopic signatures are expected to be observed during litter degradation.     

Genome-wide identification and evolution of <Emphasis Type="Italic">TC1</Emphasis>/<Emphasis Type="Italic">Mariner</Emphasis> in the silkworm (<Emphasis Type="Italic">Bombyx mori</Emphasis>) genome

TC1/Mariner transposons belong to class II transposable elements (TEs) that use DNA-mediated “cut and paste” mechanism to transpose, and they have been identified in almost all organisms. Although silkworm (Bombyx mori) has a large amount of TC1/Mariner elements, the genome wide information of this superfamily in the silkworm is unknown. In this study, we have identified 2670 TC1/Mariner (Bmmar) elements in the silkworm genome. All the TEs were classified into 22 families by means of fgclust, a tool of repetitive sequence classification, seven of which was first reported in this study. Phylogenetic and structure analyses based on the catalytic domain (DDxD/E) of transposase sequences indicated that all members of TC1/Mariner were grouped into five subgroups: Mariner, Tc1, maT, DD40D and DD41D/E. Of these five subgroups, maT rather than Mariner possessed most members of TC1/Mariner (51.23%) in the silkworm genome. In particular, phylogenetic analysis and structure analysis revealed that Bmmar15 (DD40D) formed a new basal subgroup of TC1/Mariner element in insects, which was referred to as bmori. Furthermore, we concluded that DD40D appeared to intermediate between mariner and Tc1. Finally, we estimated the insertion time for each copy of TC1/Mariner in the silkworm and found that most of members were dramatically amplified during a period from 0 to 1 mya. Moreover, the detailed functional data analysis showed that Bmmar1, Bmmar6 and Bmmar9 had EST evidence and intact transposases. These implied that TC1/Mariner might have potential transpositional activity. In conclusion, this study provides some new insights into the landscape, origin and evolution of TC1/Mariner in the insect genomes.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Comparative <Emphasis Type="Italic">in vitro</Emphasis> germination ecology of <Emphasis Type="Italic">Calopogon tuberosus</Emphasis> var. <Emphasis Type="Italic">tuberosus</Emphasis> (Orchidaceae) across its geographic range

Seed responses to temperature are often essential to the study of germination ecology, but the ecological role of temperature in orchid seed germination remains uncertain. The response of orchid seeds to cold stratification have been studied, but the exact physiological role remains unclear. No studies exist that compare the effects of either cold stratification or temperature on germination among distant populations of the same species. In two separate experiments, the role of temperature (25, 22/11, 27/15, 29/19, 33/24°C) and chilling at 10°C on in vitro seed germination were investigated using distant populations of Calopogon tuberosus var. tuberosus. Cooler temperatures promoted germination of Michigan seeds; warmer temperatures promoted germination of South Carolina and north central Florida seeds. South Florida seed germination was highest under both warm and cool temperatures. More advanced seedling development generally occurred at higher temperatures with the exception of south Florida seedlings, in which the warmest temperature suppressed development. Fluctuating diurnal temperatures were more beneficial for germination compared to constant temperatures. Cold stratification had a positive effect on germination among all populations, but South Carolina seeds required the longest chilling treatments to obtain maximum germination. Results from the cold stratification experiment indicate that a physiological dormancy is present, but the degree of dormancy varies across the species range. The variable responses among populations may indicate ecotypic differentiation.     

The ectomycorrhizae of <Emphasis Type="Italic">Lactarius rimosellus</Emphasis> and <Emphasis Type="Italic">Lactarius acatlanensis</Emphasis> with the endangered <Emphasis Type="Italic">Fagus grandifolia</Emphasis> var. <Emphasis Type="Italic">mexicana</Emphasis>

Gut bacterium Pantoea sp. is one of the predominant bacterial species in the larval gut of the diamondback moth, Plutella xylostella. The phenotypic characters of Pantoea sp. were investigated with BIOLOG phenotype MicroArray (PM) in this study. Totally 950 different metabolic phenotypes were tested using the PM plates 1–10. Results exhibited that Pantoea sp. was able to metabolize 37.37 % of the tested carbon sources, 91.32 % of nitrogen sources, 100 % of sulfur sources, and 98.31 % of phosphorus sources. Most informative utilization patterns for carbon sources of Pantoea sp. were organic acids and carbohydrates, and for nitrogen were various amino acids. The bacterium had 94 different biosynthetic pathways. It had a wide range of adaptabilities, and could still metabolize in osmolytes with up to 9 % sodium chloride, 6 % potassium chloride, 5 % sodium sulfate, 20 % ethylene glycol, 4 % sodium formate, 4 % urea, 5 % sodium lactate, 200 mmol/L sodium phosphate (pH 7.0), 100 mmol/L ammonium sulfate (pH 8.0), 100 mmol/L sodium nitrate, and 100 mmol/L sodium nitrite, respectively. It also exhibited active metabolism under pH values between 4.5 and 10. Pantoea sp. showed active decarboxylase activities while poor deaminase activities in the presence of various amino acids. The phenotypic characterization of Pantoea sp. increased our knowledge of the bacterium, in particular its interactions with insect hosts and the adaptability in gut environments, and showed us some possible approaches to controlling diamondback moth through decreasing Pantoea sp. density.