Virulence of Hymenoscyphus albidus and H. fraxineus on Fraxinus excelsior and F. pennsylvanica

European ash (Fraxinus excelsior) is currently battling an onslaught of ash dieback, a disease emerging in the greater part of its native area, brought about by the introduction of the ascomycete Hymenoscyphus fraxineus (= Hymenoscyphus pseudoalbidus). The closely-related fungus Hymenoscyphus albidus, which is indigenous to Europe, is non-pathogenic when in contact with F. excelsior, but could pose a potential risk to exotic Fraxinus species. The North American green ash (Fraxinus pennsylvanica) is planted widely throughout Europe and regenerates naturally within this environment but little is known about the susceptibility of this species to ash dieback. We performed wound inoculations with both fungi (nine strains of H. fraxineus and three strains of H. albidus) on rachises and stems of F. excelsior and F. pennsylvanica under field conditions in Southern Poland. Necrosis formation was evaluated after two months on the rachises and after 12 months on the stems. After inoculation of H. albidus, only small lesions (of up to 1.3 cm in length) developed on the F. excelsior and F. pennsylvanica rachises, but with no significant distinction from the controls. Hymenoscyphus albidus did not cause necrotic lesions on the stems of either Fraxinus species. In contrast, H. fraxineus induced necroses on all inoculated rachises of both ash species with mean lengths of 8.4 cm (F. excelsior) and 1.9 cm (F. pennsylvanica). Necroses also developed on all of the inoculated F. excelsior stems (mean length 18.0 cm), whereas on F. pennsylvanica such lesions only occurred on about 5% of the stems (mean length 1.9 cm). The differences between strains were negligible. No necroses were observed on the control plants. Reisolations of H. albidus were only successful in around 8–11% of the cases, while H. fraxineus was reisolated from 50–70% of the inoculated organs showing necrotic lesions. None of the Hymenoscyphus species were isolated from the control plants. Our data confirm H. fraxineus’ high virulence with regards to F. excelsior and demonstrate a low virulence in relation to F. pennsylvanica under field conditions in Poland. Hymenoscyphus albidus did not express any perceivable pathogenicity on both host species.     

Demography of persistent juveniles in Fraxinus excelsior

Seedling emergence, survival, growth, and juvenile development of Fraxinus excelsior were followed over 10 yr in permanent plots m a dense Alnus-Fraxinus woodland in the outer archipelago of Stockholm, Sweden Number of germinated seedlings varied between years, due to variation in total seed crop but was also affected by weather conditions Seedlings and juveniles showed high survivorship 39 2% of the emerged seedlings of the two oldest cohorts were alive in the 9th growth season Mortality was somewhat raised during the first two years, but was age-independent from the third year The density of the field-layer and climatic factors were the most important determinants of mortality The juveniles produced 1 2 pairs of leaves per year and had an annual height increment of 7 1 mm, on average, from their second growth season The ontogenetic development, e g the increase m leaf complexity, was correspondingly slow and included regressions to more simple leaves
Fraxinus excelsior shows a regenerative strategy involving persistent juveniles The divergence between this and previous studies may be due to the lack of rabbit grazing     

Dormancy in Fruits of Fraxinus excelsior L.

The germination requirements of Fraxinus excelsior, the Europeanash, were determined as a preliminary stage to an investigationof the growth-substance content of dormant and germinating fruits.The embryo grows in size within the imbibed fruit before germinationcan take place and this growth is delayed through the firstwinter by the presence of an indehiscent pericarp which is impermeableto oxygen. Even when fully grown, the embryo is dormant andcompletes its chilling requirement during the second winter,after which a normal seedling can be produced.     

Success in the demographic expansion of Fraxinus excelsior L.

This review examines the ecological traits and ecophysiological characteristics of the common ash (Fraxinus excelsior L.) that help to explain the current spread of ash communities in Europe. As a consequence of anthropogenic disturbance, the common ash has experienced new environmental conditions and the frequency of the species has fluctuated over space and time. Owing to its ubiquity, over the last few decades the common ash has been greatly favoured in areas abandoned by agriculture and also along impounded rivers. F. excelsior is a mesophilic species that usually thrives on alluvial soils and can also survive the strong water deficit on hillslopes. Drought tolerance in ash trees is related to stomatal regulation, a decrease in osmotic potential and also an increase in the elastic modulus ). Malic acid plays a central role in drought tolerance, first as one of the major solutes involved in osmotic adjustment, and second as an effector for stomatal regulation through a drought-induced increase in its xylem concentration.     

Differentiation of circular vessels in isolated segments of Fraxinus excelsior

The differentiation of circular vessels was studied in stem segments which had passed winter dormancy and were stimulated by application of auxin in vitro. The circular vessels differentiated near the basal end of internodes. The prevailing form of circular vessels was a pair of cells connected by two perforations. Two-cell vessels never had only one perforation. The occurrence of two perforations strongly supports the view that auxin flux is a stimulus for vessel differentiation and not the very presence or concentration of auxin; the circular vessels developed as a results of a circular flux. In general, the circular vessels appeared in contact with rays. Their formation was delayed in comparison with that of normal vessels.     

Maternal inheritance of a chloroplast microsatellite marker in controlled hybrids between Fraxinus excelsior and Fraxinus angustifolia

Restriction fragment length polymorphism, polymerase chain reaction-restriction fragment length polymorphism and simple sequence repeat (SSR or microsatellites) analyses were performed to detect chloroplast DNA polymorphisms between two ash species, Fraxinus excelsior and F. angustifolia. Only one SSR locus was found to be polymorphic, confirming the very close relatedness of these species. Inheritance of this marker was studied in hybrids obtained from controlled crosses between the two tree species. Results indicated, for the first time in Oleaceae, that chloroplasts are maternally inherited. This chloroplast SSR marker is now used concomitantly with nuclear markers to analyse ash populations in sympatric areas.     

Germination uniformity of Fraxinus excelsior controlled by seed water content during cold treatment

The seeds of Fraxinus excelsior L. are dormant after harvest, since they need a period of chilling for germination. Moist treatment at 20°C for 2–3 months followed by stratification at 4°C for 7 months breaks dormancy. We observed that germination occurred during stratification and was spread over a period of 3 months. Germination at low temperature was temporarily inhibited by a moderate reduction of the seed water content initiated after the third month of stratification. This allowed the afterripening process to continue.
The following procedure was developed to suppress dormancy and to induce uniform germination:

• 1. 

  Imbibition of the seeds and moist treatment at 20°C for 2–3 months;
• 2. 

  stratification for 3 months;
• 3. 

  treatment at low temperature and low water potential for at least 4 months, this treatment should not exceed 6 months;
• 4. 

  complete rehydration of the seeds at 16°C.

     

Seasonal Variation in the Prevalence of Sand Flies Infected with Leishmania donovani

Background

Visceral Leishmaniasis (VL) is a life threatening neglected infectious disease in the Indian subcontinent, transmitted by the bite of female sand flies. Estimation of the infectivity in the vector population, collected in different seasons, may be useful to better understanding the transmission dynamics of VL as well as to plan vector control measures.

Methodology

We collected sand flies from highly endemic regions of Bihar state, India for one year over three seasons. The species of the sand flies were confirmed by species-specific PCR-RFLP. Leishmania donovani infection was investigated in 1397 female Phlebotomus argentipes using PCR, targeting the Leishmania specific minicircle of the kDNA region. Further, the parasitic load in the infected sand flies was measured using quantitative PCR.

Conclusion

Though sand flies were most abundant in the rainy season, the highest rate of infection was detected in the winter season with 2.84% sand flies infected followed by the summer and rainy seasons respectively. This study can help in vector elimination programmes and to reduce disease transmission.     

Reactivation of Vessel Production in Ash (Fraxinus excelsior L.) Trees

Stages of earlywood vessel development have been compared withstages of bud and shoot growth throughout 12-year-old treesof ash (Fraxinus excelsior L.). Reactivation of vessel productionwas not simultaneous throughout the tree. There was evidencethat vessel expansion progressed basipetally down branches andacropetally up the main stem. The earliest expanding vesselswere found scattered around the circumference of main stem andbranches about 3 weeks before the emergence of foliage leavesfrom the buds. Other vessels expanded later between the earlierones so the whole of the first earlywood layer was expandingby 1.5 weeks before leaf emergence; this is suggested as a convenientstage to use as a baseline for a model of wood production inash. Vessel maturation progressed basipetally down the mainstem as well as branches, the first mature (presumably functional)vessels appearing in the upper stem shortly before leaf emergence.Mature vessels were not found in the lower part of the mainstem until after the beginning of rapid leaf expansion afterbudbreak, contrary to a previous assumption that functionalearlywood vessels are of necessity produced before leaf expansionin ring-porous trees. Patterns of vessel expansion are comparedbetween the ring-porous ash and the diffuse-porous sycamore;these data suggest that expansion of earlywood vessels beganat the same time in relation to budbreak in the two species,but the location of the first vessel expansion differed. ash, cambial reactivation, Fraxinus excelsior L., ring-porous hardwood, vessel expansion, vessel maturation     

Establishment of Fagus sylvatica and Fraxinus excelsior in an old-growth beech forest

Abstract. Distribution of tree seedlings, forest architecture, light conditions, ground vegetation and humus conditions were studied in a 45 m × 100 m area including multiple gaps in an old-growth beech forest. Gaps were created after some beech trees had been felled in severe storms in February 1990. A group of adult ash trees is found near the study site. The data were analyzed by Correspondence Analysis. Young seedlings (< 4 yr), of both Fraxinus (a sun species) and Fagus (a shade species), were most abundant under the crown of beech trees in semi-shade conditions, and where beech litter did not accumulate. Differences in the dissemination of Fraxinus and Fagus explained differences in the establishment of the two species. In contrast, older seedlings of beech established before the storms were more numerous in the gaps, suggesting a change in the ecological requirements of beech seedlings in the course of time.     

The replacement of Alnus glutinosa by Fraxinus excelsior during succession related to regenerative differences

Mechanisms of the replacement of one species by another during a primary succession were studied in the deciduous woodland of the outer archipelago of Stockholm Sweden where there are only two principal tree species Alnus glutmosa (alder) was most abundant close to the sea shore while the abundance of Fraxinus excelsion (ash) increased with increasing elevation above sea level which due to the isostatic land upheaval corresponds to increasing age of the site Regeneration of the two species followed in permanent plots, was different Alder did not regenerate from seeds in the woodland No seedling survived 3 yr even under conditions of thin eanopy and removed field-layer. and most died within the first year In contrast survival of ash was 35% after 10 yr Annual height increments of the ash juveniles were < 8 mm under a closed eanopy but growth of the juveniles increased rapidly when a gap was opened out by a tree-fall Thus ash in eontrast to alder possesses characters advantageous for regeneration in gaps which may explain the successive replacement of alder by ash in this woodland     

Estimating Coextinction Risks from Epidemic Tree Death: Affiliate Lichen Communities among Diseased Host Tree Populations of Fraxinus excelsior

At least 10% of the world’s tree species are threatened with extinction and pathogens are increasingly implicated in tree threats. Coextinction and threats to affiliates as a consequence of the loss or decline of their host trees is a poorly understood phenomenon. Ash dieback is an emerging infectious disease causing severe dieback of common ash Fraxinus excelsior throughout Europe. We utilized available empirical data on affiliate epiphytic lichen diversity (174 species and 17,800 observations) among 20 ash dieback infected host tree populations of F. excelsior on the island Gotland in the Baltic Sea, Sweden. From this, we used structured scenario projections scaled with empirical data of ash dieback disease to generate probabilistic models for estimating local and regional lichen coextinction risks. Average coextinction probabilities (Ā) were 0.38 (95% CI ±0.09) for lichens occurring on F. excelsior and 0.14 (95% CI ±0.03) when considering lichen persistence on all tree species. Ā was strongly linked to local disease incidence levels and generally increasing with lichen host specificity to F. excelsior and decreasing population size. Coextinctions reduced affiliate community viability, with significant local reductions in species richness and shifts in lichen species composition. Affiliates were projected to become locally extirpated before their hosts, illuminating the need to also consider host tree declines. Traditionally managed open wooded meadows had the highest incidence of ash dieback disease and significantly higher proportions of affiliate species projected to go extinct, compared with unmanaged closed forests and semi-open grazed sites. Most cothreatened species were not previously red-listed, which suggest that tree epidemics cause many unforeseen threats to species. Our analysis shows that epidemic tree deaths represent an insidious, mostly overlooked, threat to sessile affiliate communities in forested environments. Current conservation and management strategies must account for secondary extinctions associated with epidemic tree death.     

Defining the twig fungal communities of Fraxinus species and Fraxinus excelsior genotypes with differences in susceptibility to ash dieback

Ash dieback disease (caused by Hymenoscyphus fraxineus) has affected European ash species (Fraxinus spp.) in recent decades. However, some Asian and American species of Fraxinus and certain genotypes of Fraxinus excelsior are less affected by the disease. We used ITS1-metabacoding to explore the drivers influencing diversity and composition of the twig fungal communities of Fraxinus species and F. excelsior genotypes. Our results revealed that fungi in the classes Eurotiomycetes and Dothideomycetes were among the most prevalent taxa in both Fraxinus species and F. excelsior genotypes. The diversity of the fungal communities differed significantly among Fraxinus species and could be explained by seed origin. Neither host genotype nor season had a significant effect on the community diversity of F. excelsior genotypes. On the other hand, the composition of twig fungal communities differed significantly among host species and among F. excelsior genotypes, and in F. excelsior there was also a significant effect of season on the composition of the fungal community. We did not find a clear effect of ash dieback susceptibility on either diversity or composition of fungal communities in twigs of Fraxinus species, although the effect was significant on the composition of fungal communities among F. excelsior genotypes. Our results demonstrated differences in fungal communities among species of Fraxinus and of F. excelsior genotypes, suggesting specific relationship between individual host genotypes and endophytic fungi.     

Analysis of the diurnal change in osmotic potential in leaves of Fraxinus excelsior L.

The daily cycle of the transpiration rate, stomatal conductance,water potential, and the concentration of the main osmoticumidentified in ash leaves, malate and mannitol, were monitoredin a field on the Isere river plain. On sunny days, the stomatalconductance tends to remain close to its maximun value allowinga high transpiration rate and diurnal variations in leaf waterpotential, _w, which may fall as low as –2 MPa at solarnoon. These variations of _w are closely correlated with changesin malate, mannitol and the concentration of the well-knownosmoticum K⁺, which agree with the involvement of an osmoticadjustment to counteract the evaporative demand during daylighthours. How malate, mannitol and K⁺ contribute to the osmoticadjustment was analysed subsequently by comparing the solutepotential _s, evaluated by the Boyle-Van't Hoff relation, tothe osmotic potential measured by thermocouple psychrometry.These experiments have led us to suspect some errors in themeasurement of , presumably due to experimental artefacts andthe ability of Ca ²⁺ , present in high levels in leaves, toform chelates with malate once the cells have been decompartmentedby freezing and thawing. Since significant changes of Ca²⁺ alsooccurred during the diurnal variations of _w, the possible mechanismsby which Ca²⁺ may be implicated in controlling the water statusof the tree are discussed. Key words: Fraxinus excelsior L, osmotic adjustment, thermocouple psychrometry, malate, calcium     

The life cycle of Hymenoscyphus fraxineus on Manchurian ash,Fraxinus mandshurica,in Japan

Hymenoscyphus fraxineus causes a lethal disease known as “ash dieback” in the common ash, Fraxinus excelsior, in Europe. It is hypothesized that the fungus originated from East Asia. This fungus is found on the leaf litter of the Manchurian ash, Fraxinus mandshurica, in Japan and is reported to produce apothecia on pseudosclerotial plates formed mainly on decomposing rachises. However, dieback disease has not been reported in Japan, and little is known about the life cycle of H. fraxineus. This study was conducted to explore the behavior and life cycle of this fungus. It was revealed that, after infection by ascospores, H. fraxineus endophytically inhabits the living leaves of F. mandshurica. On fallen leaves, the fungus behaves saprophytically, producing apothecia on pseudosclerotial plates formed mainly on the decomposing rachises. Analysis by real-time quantitative polymerase chain reaction (qPCR) revealed that the amount of H. fraxineus DNA sharply increased in rachises, while such sharp increase of DNA was not found in leaflets.     

Historical and contemporary mating patterns in remnant populations of the forest tree Fraxinus excelsior L

Genetic variation at microsatellite markers was used to quantify genetic structure and mating behavior in a severely fragmented population of the wind-pollinated, wind-dispersed temperate tree Fraxinus excelsior in a deforested catchment in Scotland. Remnants maintain high levels of genetic diversity, comparable with those reported for continuous populations in southeastern Europe, and show low interpopulation differentiation (E = 0.080), indicating that historical gene exchange has not been limited (Nm = 3.48). We estimated from seeds collected from all trees producing fruits in three of five remnants that F. excelsior is predominantly outcrossing (t(m) = 0.971 +/- 0.028). Use of a neighborhood model approach to describe the relative contribution of local and long-distance pollen dispersal indicates that pollen gene flow into each of the three remnants is extensive (46-95%) and pollen dispersal has two components. The first is very localized and restricted to tens of meters around the mother trees. The second is a long-distance component with dispersal occurring over several kilometers. Effective dispersal distances, accounting for the distance and directionality to mother trees of sampled pollen donors, average 328 m and are greater than values reported for a continuous population. These results suggest that the opening of the landscape facilitates airborne pollen movement and may alleviate the expected detrimental genetic effects of fragmentation.     

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.     

Drought-induced increase in xylem malate and mannitol concentrations and closure of Fraxinus excelsior L. stomata

Changes in the malate and mannitol composition of ash leaf (Fraxinus excelsior L.) xylem sap were studied in response to water deficit. Xylem sap was collected by the pressure method from the petiole of leaves sampled on irrigated and non-irrigated ash seedlings. As the leaf water potential decreased from -0.3 to -3.0 MPa, there was a significant increase in malate and mannitol xylem concentrations, and a concomitant decrease in maximal stomatal conductance. The functional significance of the increased malate and mannitol concentrations was investigated by using a transpiratory bioassay with mature detached leaves which exhibited, for stomatal conductance, the typical pattern showed by expanded leaves during dark/light transitions. Supplying detached leaves with mannitol in a range of concentrations found in the xylem sap had no effect on stomatal movements, but malate, for concentrations between 0.5 and 3 mM, was effective in preventing stomatal opening. The ability of malate to inhibit stomatal opening appeared to be rather non-specific. Two structural malate analogues, citrate and aspartate or an unrelated anion, shikimate, also inhibited this process. Given the drought-induced increase in xylem malate concentrations, and the fact that the range of malate levels required to close stomata was very similar to that of the concentrations found in the xylem sap, it has been hypothesized that malate is involved in the stomatal closure of ash leaves under drying conditions.Key words: Fraxinus excelsior: L., malate, mannitol, xylem sap, stomata, water deficit.