Micropropagation of common ash (Fraxinus excelsior)

Embryos extracted from dried seeds of common ash (Fraxinus excelsior), were germinated on growth regulator-free culture medium. Cotyledonary nodes from these seedlings were placed onto Murashige and Skoog, Woody Plant or Driver and Kuniyuki culture media with 22.2 or 44.4 M benzyladenine, on which they developed into shoot cultures following the outgrowth of axillary buds. With Murashige and Skoog medium, cultures often died. With Woody Plant Medium, survival of the cultures was considerably improved, but large amounts of callus were produced at the cut ends of the explants, and new axillary shoots had long internodes and small leaves. With Driver and Kuniyuki medium, both survival and callus formation were much improved, and the shoots produced were of high quality. Proliferation of axillary shoots was obtained from both shoot tip and nodal explants placed onto Driver and Kuniyuki medium with 22.2 M benzyladenine. Adventitious root formation was best with shoots inserted into half-strength Woody Plant Medium containing 2.45, 4.9 or 9.8 M indolebutyric acid. All of the rooted plantlets tested have successfully established in soil.     

Shoot initiation in the leaflet axils of compound leaves from micropropagated shoots of juvenile and mature common ash (Fraxinus excelsior L.)

Micropropagation of a mature ash tree has been achieved forthe first time. The main obstacle encountered was contaminationof the initial explants with microorganisms. However, once apparentlysterile shoots had been obtained, shoot proliferation was achievedmost effectively by culturing nodes on Driver and Kuniyuki walnutmedium containing 22.2 µM benzyladenine. After severalsubcultures, a species of Bacillus appeared with the matureculture line, but it did not affect shoot or root developmentadversely. With successive subculturing, shoots of the matureash clone became progressively easier to root. Pinnately compoundleaf explants from micropropagated shoots of two seedlings andthe mature tree, placed on to Murashige and Skoog-based culturemedia supplemented with 4.4µM-phenyl-N-1,2,3-thiadiazol-5-ylurea,produced shoot buds. Most buds developed from the rachis atthe points of attachment of the leaflets. When transferred toDriver and Kuniyuki walnut medium, buds from one of the seedlinglines and from the mature tree, elongated into shoots, and weresubsequently transferred to media for micropropagation and rooting.Shoot initiation is discussed in relation to possible occurrenceof vestigial meristems in the axils of leaflets and the partialshoot theory of leaf structure. Key words: Forestry, leaf structure, partial shoot theory, thidiazuron, tree     

Underwater germination potential of common ash seed (Fraxinus excelsior L.) originating from flooded and non-flooded sites

Oxygen availability is one of the main factors affecting seed germination capacity. On floodplains, oxygen supply is seasonally restricted because of flooding events. Seed ecotypes are able to cope with total submergence to different extents, depending on the species and its position in a given environmental gradient. This investigation tested three provenances of common ash (Fraxinus excelsior L.), one from a lowland site and two from upland sites, for germination capacity and germination velocity under water. The final germination capacity of all provenances was unaffected, but the germination velocity was decreased for all provenances. The adaptational significance of these results is discussed.     

Phyllactinia fraxinicola,another Asian fungal pathogen on Fraxinus excelsior (common ash) introduced to Europe?

Since the mid-nineties Phyllactinia fraxini has become frequent in Germany. This species has hitherto been characterized by having straight conidiophore foot cells. However, we found that recent collections from Germany have conidiophores with sinuated and twisted foot cells. So far sinuated foot cells were only known from the related P. fraxinicola, another species with Eastern Asian origin. We thus hypothesized that recent collections from Germany belong to P. fraxinicola which might have been introduced to Europe. Using morphological and molecular rDNA data we found that no introduction took place and that there is only P. fraxini in Germany.     

Chloroplast DNA variation and postglacial recolonization of common ash (Fraxinus excelsior L.) in Europe

We used chloroplast polymerase chain reaction-restriction-fragment length polymorphism (PCR-RFLP) and chloroplast microsatellites to assess the structure of genetic variation and postglacial history across the entire natural range of the common ash (Fraxinus excelsior L.), a broad-leaved wind-pollinated and wind-dispersed European forest tree. A low level of polymorphism was observed, with only 12 haplotypes at four polymorphic microsatellites in 201 populations, and two PCR-RFLP haplotypes in a subset of 62 populations. The clear geographical pattern displayed by the five most common haplotypes was in agreement with glacial refugia for ash being located in Iberia, Italy, the eastern Alps and the Balkan Peninsula, as had been suggested from fossil pollen data. A low chloroplast DNA mutation rate, a low effective population size in glacial refugia related to ash's life history traits, as well as features of postglacial expansion were put forward to explain the low level of polymorphism. Differentiation among populations was high (GST= 0.89), reflecting poor mixing among recolonizing lineages. Therefore, the responsible factor for the highly homogeneous genetic pattern previously identified at nuclear microsatellites throughout western and central Europe (Heuertz et al. 2004) must have been efficient postglacial pollen flow. Further comparison of variation patterns at both marker systems revealed that nuclear microsatellites identified complex differentiation patterns in south-eastern Europe which remained undetected with chloroplast microsatellites. The results suggest that data from different markers should be combined in order to capture the most important genetic patterns in a species.     

Assessment of genetic structure within and among Bulgarian populations of the common ash (Fraxinus excelsior L.)

We analysed genetic variation within and between populations of the common ash from Bulgaria in order to extract biological information useful in the context of conservation management of eastern European genetic resources of noble hardwood species. A total of 321 trees from three regions of Bulgaria were typed at six highly polymorphic microsatellite loci. Analysis of within-population inbreeding suggests an upper boundary value of 2.7% for the selfing rate. Significant spatial genetic structure consistent with models of isolation by distance was detected within four out of 10 populations as well as among populations. Estimates of neighbourhood size in the range 38-126 individual trees were obtained based on spatial genetic structure analyses at either the intrapopulation or interpopulation level. Differentiation among populations explained only about 8.7% of total genetic diversity. These results are discussed in comparison with data from social broad-leaved species such as oak and beech.     

Nitrogen storage and remobilization in ash (Fraxinus excelsior) under field and laboratory conditions

 Storage and remobilization of nitrogen (N) were studied in ash trees (Fraxinus excelsior) under both field and greenhouse conditions. Experiments in the greenhouse providing ¹⁵N labelled fertilizer to the trees showed that the major quantity of N remobilized during subsequent spring was from the roots, and only a small amount from the stem. This corresponded with a loss of soluble N (proteins and low-molecular-weight compounds) from both roots and stem. On the two field sites, which differed in water availability, there was a decrease in bark N content during leaf growth, but on the dry site net N export from the bark was sustained throughout the whole vegetation period. Remobilized N was derived from soluble proteins and low-molecular-weight compounds on the moist site, which was demonstrated by the seasonal dynamics of a 56 kDa polypeptide in bark and wood. On the dry site, lower contents of soluble proteins were associated with smaller amounts of N remobilized compared to the moist site. Uptake studies of ¹⁵N labelled fertilizer indicated a higher contribution of current uptake to leaf N increment during spring at the dry site compared to the moist site. Differential N availability during the season had a decisive effect on the nitrogen storage dynamics at the two sites. Thus the influence of current N supply on N remobilization and storage as found in the greenhouse-grown plants could be verified under field conditions. Received: 28 July 1995 / Accepted: 17 July 1996     

Spatio-temporal dynamics of endophyte diversity in the canopy of European ash (Fraxinus excelsior)

Leaf-inhabiting endophytic fungi of Fraxinus excelsior growing in a floodplain forest were isolated during 2008 to investigate vertical community structure, species richness and seasonal variation. The analysis of 848 fungal endophytes from 213 leaves resulted in 50 different species. In the understorey, infection density and species richness were higher than in the crowns of mature trees throughout the whole vegetation period. Within tree crowns, sun-exposed leaves of the top canopy exhibited the lowest infection rates. Most species were rare or absent in spring and in the light crowns and frequent in autumn and the understorey. However, some species, especially the two most frequent, Alternaria infectoria and A. alternata, deviated from these patterns. Young leaves were nearly free of endophytes. Apparently, the subsequent infection and establishment of fungi strongly depend on microclimatic parameters and leaf characters, which create highly variable spatial and temporal colonisation patterns within an individual tree.     

Molecular biodiversity and population structure in common ash (Fraxinus excelsior L.) in Britain: implications for conservation

Current forestry policy promotes the use of local seed for new plantings, on the assumption that local material may be better adapted to local conditions. However, landscape‐scale genetic studies which are necessary to underpin conservation and breeding strategies are often lacking. We investigated molecular diversity in common ash (Fraxinus excelsior L.) sampled from 42 British and six French sites with microsatellites. Chloroplast haplotype H04 was the most common and widespread in Britain, although rare and localized individuals with H02 and H09 were also detected. In addition, three new chloroplast haplotypes were identified, and these were rare and highly localized. In terms of nuclear microsatellite markers, allelic richness differed between sites and decreased in an east to west direction. Differentiation between sites was often very low (mean F_ST 0.025), indicating few differences between the majority of sites. There was a clear excess of homozygotes (mean H_O 0.669, mean H_E 0.818) and a relatively high F_IS (mean 0.182), suggests a consistent level of inbreeding or a widespread Wahlund effect in many F. excelsior sites. Gene pool ancestry analysis suggested that the majority of British F. excelsior belongs to a single meta‐population which covers mainland western and central Europe. Three northern and western sites diverged markedly from the dominant population, and may represent remnants of two late potential Ice Age refugia in northern Britain. The data provide new information which will aid development of appropriate conservation policies for ash and other wind pollinated tree species.     

Micropropagation of hornbeam (Carpinus betulus L.) and ash (Fraxinus excelsior L.)

Shoot multiplication of hornbeam was stimulated onWPM, QL andDKW medium supplemented with a low concentration of BAP or BPA (0.1-0.2 mg I -1) andIBA (0.1 mg I -1). Low concentration of thidiazuron promoted axillary bud formation, higher concentration inhibited shoot elongation. Microshoots were rooted onWPM supplemented with a low auxin concentration (IBA or NAA 0.2-0.5 mg I -1). High rooting percentages were obtained. Shoot proliferation of ash was stimulated on MS andDKW medium supplemented withBAP orBPA (2.0-5.0 mg I -1) andIBA (0.1 mg I -1). Root formation was promoted onWPM containing a low auxin concentration. Rooted plantlets were transplanted into soil and after hardening off the micropropagated trees were planted in the field. The planted trees grew normally without showing signs of abnormality.     

Changes in ecosystem carbon stocks in a grassland ash (Fraxinus excelsior) afforestation chronosequence in Ireland

Aims Government policy in Ireland is to increase the national forest cover from the current 10% to 18% of the total land area by 2020. This represents a major land use change that is expected to impact on the national carbon (C) stocks. While the C stocks of ecosystem biomass and soils of Irish grasslands and coniferous forests have been quantified, little work has been done to assess the impact of broadleaf afforestation on C stocks.Methods In this study, we sampled a chronosequence of ash (Fraxinus excelsior) forests aged 12, 20, 27, 40 and 47 years on brown earth soils. A grassland site, representative of the pre-afforestation land use, was sampled as a control.Important findings Our results show that there was a significant decline (P < 0.05) in the carbon density of the soil (0–30cm) following afforestation from the grassland (90.2 Mg C ha^-1) to the 27-year-old forest (66.7 Mg C ha^-1). Subsequently, the forest soils switched from being a C source to a C sink and began to sequester C to 71.3 Mg C ha^-1 at the 47-year-old forest. We found the amount of C stored in the above- and belowground biomass increased with age of the forest stands and offset the amount of C lost from the soil. The amount of C stored in the above- and belowground biomass increased on average by 1.83 Mg C ha^-1 year^-1. The increased storage of C in the biomass led to an increase in the total ecosystem C, from 90.2 Mg C ha^-1 at the grassland site to 162.6 Mg C ha^-1 at the 47-year-old forest. On a national scale, projected rates of ash afforestation to the year 2020 may cause a loss of 290 752 Mg C from the soil compared to 2 525 936 Mg C sequestered into the tree biomass. The effects of harvesting and reforestation may further modify the development of ecosystem C stocks over an entire ash rotation.     

Frost periods and beginning of the ash (Fraxinus excelsior L.) pollen season in Basel (Switzerland)

In order to be able to forecast the beginning of the ash pollen season, several meteorological factors have been considered. During these research activities it appeared that the ash pollen release is closely linked to frost periods happening during the months of February and March. For 24 years over a period of 29 consecutive years, a daily concentration of 30 ash pollen grains/m³/day has been reached when the cumulated mean daily temperatures starting on the last day when the absolute minimum temperature is lower or equal to −2 °C amounts on average to 214 °C – admitting a variation of plus or minus 42 °C. As for the five remaining years, rain is three times responsible for the delay of the pollen release. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ethylenediurea (EDU) affects the growth of ozone-sensitive and tolerant ash (Fraxinus excelsior) trees under ambient O3 conditions

Adult ash trees (Fraxinus excelsior L.), known to be sensitive or tolerant to ozone, determined by presence or absence of foliar symptoms in previous years, were treated with ethylenediurea (EDU) at 450 ppm by gravitational trunk infusion over the 2005 growing season (32.5 ppm h AOT40). Tree and shoot growth were recorded in May and September. Leaf area, ectomycorrhizal infection, and leaf and fine root biomass were determined in September. EDU enhanced shoot length and diameter, and the number and area of leaves, in both O3-sensitive and tolerant trees. However, no EDU effects were recorded at the fine root and tree level. Therefore, a potential for EDU protection against O3-caused growth losses of forest trees should be evaluated during longer-term experiments.     

Genetic variability of Chalara fraxinea, dieback cause of European ash (Fraxinus excelsior L.)

A total of 159 colonies of Chalara fraxinea were isolated between 2005 and 2006 from dying trees of European ash (Fraxinus excelsior L.) aged between 3 and 10 years. They derived from five regions of Poland differing by geographic location and climatic conditions. On the basis of 90 RAMS markers, pathogen intra- and inter-population variability, as well as its dependency on geographic distance and climatic conditions in the regions of strain origin, was analysed. The applied measures of intrapopulation genetic variability (genetic distance, Nei’s unbiased diversity, Shannon’s Information Index and percentage of polymorphic loci) allowed for differentiation of two strain groups: the first deriving from lowlands and the second from uplands and mountainous areas. Strains in lowlands were characterised by smaller number of markers, smaller number of polymorphic loci and smaller intrapopulation genetic variability. Positive and statistically significant correlation was shown between variability of isolates and elevation of regions above sea level. Pair-wise genetic distances between groups of isolates (Nei’s unbiased genetic distance) from particular regions were not significantly correlated with the corresponding geographic distances. On the basis of AMOVA, it was shown that 85% of variability was within-region differences and 2% between-region differences, whereas differences between lowlands and uplands were 13%. Principal Components Analysis (PCA) for the investigated regions confirmed the results from Nei’s genetic distance matrix.     

Cumulative temperatures for prediction of the beginning of ash (Fraxinus excelsior L.) pollen season

The aim of this study is the analysis of the meteorological conditions leading to the release of ash pollen in order to predict the beginning of the pollen season in Zurich, Switzerland. For 12 years over a period of 14 consecutive years, it appears that ash pollen release begins when the cumulated mean daily temperatures starting on January 1 reach 220.7°C, admitting a variation of ±30°C that more or less corresponds to 3 consecutive days at 10°C.     

Nuclear microsatellites reveal contrasting patterns of genetic structure between western and southeastern European populations of the common ash (Fraxinus excelsior L.)

To determine extant patterns of population genetic structure in common ash and gain insight into postglacial recolonization processes, we applied multilocus-based Bayesian approaches to data from 36 European populations genotyped at five nuclear microsatellite loci. We identified two contrasting patterns in terms of population genetic structure: (1) a large area from the British Isles to Lithuania throughout central Europe constituted effectively a single deme, whereas (2) strong genetic differentiation occurred over short distances in Sweden and southeastern Europe. Concomitant geographical variation was observed in estimates of allelic richness and genetic diversity, which were lowest in populations from southeastern Europe, that is, in regions close to putative ice age refuges, but high in western and central Europe, that is, in more recently recolonized areas. We suggest that in southeastern Europe, restricted postglacial gene flow caused by a rapid expansion of refuge populations in a mountainous topography is responsible for the observed strong genetic structure. In contrast, admixture of previously differentiated gene pools and high gene flow at the onset of postglacial recolonization of western and central Europe would have homogenized the genetic structure and raised the levels of genetic diversity above values in the refuges.     

Die Esche (Fraxinus excelsior) auf den Bergen des Wienerwaldes

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