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The ongoing global climate change has aroused interest in the spring phenology of woody species in boreal and temperate regions that require cold temperatures to break bud dormancy. Most research on this topic has been concentrated on fruit species, rather than forest trees. We studied the effect of chilling duration in vitro and ex vitro on the timing of dormancy release in European ash plants. In vitro chilling (at 4–5ºC) of rooted ash plants delayed their subsequent growth when planted out in the greenhouse, and the length of delay correlated with chilling duration. The fastest growth was observed after 5–7 weeks of cold treatment, whereas 13 weeks reduced the plant viability. The impact of cold treatment for 300–3000 h was assessed on one-year-old ash plantlets. Subsequently, clonal micropropagation of European ash on a dihydroquercetin (taxifolin)-containing medium significantly reduced the requirement for chill temperatures: the plantlets came out of endodormancy after having accumulated 500 chill hours, whereas control (unchilled) plantlets required more than 1000 h. The budburst period was also significantly shorter in chilled plants. We suppose that the effect might have been due to the antioxidant activity of the flavonoid dihydroquercetin. The thermal time requirement for budburst in European ash was well described as an inverse exponential function of the number of chill hours at ≤ 5 °C. Late-flowering and photoperiod-insensitive European ash is a good object for studying the impact of external and internal factors on the mechanisms behind the induction and release of dormancy. [ABSTRACT FROM AUTHOR]

Plant diseases are mostly multicausal with several factors influencing the health status of affected hosts. Common ash (Fraxinus excelsior), a significant tree species of European forests, is currently mostly endangered by ash dieback, caused by the invasive fungus Hymenoscyphus fraxineus. However, contributing factors, including pathogenic viruses, are poorly understood. Here, we report the results of a virus screening conducted on selected special stands of F. excelsior. Over three consecutive years, ash trees from different origins were tested, including leaf material from mature seed trees, young trees and ash seedlings from the natural regeneration. Using RT-PCR, we screened for five viruses, including the generalist species ArMV (Nepovirus arabis) and CLRV (Nepovirus avii), as well as newly discovered viruses in ash, including the emaravirus ASaV (Emaravirus fraxini), the idaeovirus PrLBaV (Idaeovirus ligustri), and cytorhabdoviruses. The results revealed a high virus diversity in common ash. An association of ASaV detection with specific leaf symptoms, including shoestring, chlorotic ringspots, and vein yellowing, was documented. An analyses of relevant gene products of cytorhabdoviruses obtained from ashes of different sites revealed sequence diversities and two distinct phylogenetic groups present in ash populations. Signatures of novel viruses from different families have been identified by high-throughput sequencing. Together, our results provide insights into the virus diversity and distribution of viruses in ash and expand our knowledge about the virome of this endangered tree species. [ABSTRACT FROM AUTHOR]

Abstract Objective The endophytic mycobiome is present in all studied plant compartments, including fruits and seeds. Here, we studied the mycobiome of seed wings as they are transferred with seeds in common ash and tested whether the mycobiome differs among trees. To achieve this, we used ITS1-based amplicon sequencing and two genotypes of F. excelsior as a model to compare the mycobiome of mother trees and their wings. Results We compared the mycobiome of 57 seed wings to the seed stalks (57) collected from two genotypes of F. excelsior using three ramets of each genotype. Alpha diversity indices (ACE, Fisher and Observed OTUs) suggested a higher richness of the mycobiome associated with the seed wing than the seed stalk within each genotype. However, there were neither significant differences in Shannon diversity between the mycobiomes from the two tissue types nor the two genotypes. PERMANOVA revealed significant differences in the mycobiome composition between tissue types (P

Objective: The endophytic mycobiome is present in all studied plant compartments, including fruits and seeds. Here, we studied the mycobiome of seed wings as they are transferred with seeds in common ash and tested whether the mycobiome differs among trees. To achieve this, we used ITS1-based amplicon sequencing and two genotypes of F. excelsior as a model to compare the mycobiome of mother trees and their wings. Results: We compared the mycobiome of 57 seed wings to the seed stalks (57) collected from two genotypes of F. excelsior using three ramets of each genotype. Alpha diversity indices (ACE, Fisher and Observed OTUs) suggested a higher richness of the mycobiome associated with the seed wing than the seed stalk within each genotype. However, there were neither significant differences in Shannon diversity between the mycobiomes from the two tissue types nor the two genotypes. PERMANOVA revealed significant differences in the mycobiome composition between tissue types (P < 0.001). It also showed a significant difference between seed wings (P = 0.04), but not between seed stalks of the two genotypes. Our results suggest that Fraxinus excelsior mother trees disperse different sets of mycobiomes with their seed wings, which may be important for germination and seedling establishment—especially in the light of ash dieback. [ABSTRACT FROM AUTHOR]

The increase in introduced insect pests and pathogens due to anthropogenic environmental changes has become a major concern for tree species worldwide. Common ash (Fraxinus excelsior L.) is one of such species facing a significant threat from the invasive fungal pathogen Hymenoscyphus fraxineus. Some studies have indicated that the susceptibility of ash to the pathogen is genetically determined, providing some hope for accelerated breeding programs that are aimed at increasing the resistance of ash populations. To address this challenge, we used a genomic selection strategy to identify potential genetic markers that are associated with resistance to the pathogen causing ash dieback. Through genome‐wide association studies (GWAS) of 300 common ash individuals from 30 populations across Poland (ddRAD, dataset A), we identified six significant SNP loci with a p‐value ≤1 × 10−4 associated with health status. To further evaluate the effectiveness of GWAS markers in predicting health status, we considered two genomic prediction scenarios. Firstly, we conducted cross‐validation on dataset A. Secondly, we trained markers on dataset A and tested them on dataset B, which involved whole‐genome sequencing of 20 individuals from two populations. Genomic prediction analysis revealed that the top SNPs identified via GWAS exhibited notably higher prediction accuracies compared to randomly selected SNPs, particularly with a larger number of SNPs. Cross‐validation analyses using dataset A showcased high genomic prediction accuracy, predicting tree health status with over 90% accuracy across the top SNP sets ranging from 500 to 10,000 SNPs from the GWAS datasets. However, no significant results emerged for health status when the model trained on dataset A was tested on dataset B. Our findings illuminate potential genetic markers associated with resistance to ash dieback, offering support for future breeding programs in Poland aimed at combating ash dieback and bolstering conservation efforts for this invaluable tree species. [ABSTRACT FROM AUTHOR]

Fraxinus excelsior L. (ash) is a key forest tree species challenged by Hymenoscyphus fraxineus (T. Kowalski) Baral, Queloz, Hosoya, the causal agent of ash dieback. The goals of this study were (I) to assess the presence, spatial distribution, and incidence of H. fraxineus in the inner-alpine valleys of northwestern Italy, along with the severity of ash dieback; (II) to model the probability of infection by H. fraxineus based on environmental variables; (III) to reconstruct the direction of provenance of the front of invasion of the pathogen; and (IV) to test whether H. fraxineus has replaced the native relative Hymenoscyphus albidus (Gillet) W. Phillips, a saprobe of ash litter. By combining phytosanitary monitoring and samplings in 20 forest stands, laboratory analyses, and statistical modelling, this study showed that H. fraxineus was present in 65% of stands with an average incidence of 27%, reaching peaks of 80%. Rainfalls were the most relevant drivers of the probability of infection by H. fraxineus, rising up to 80% with the increased precipitation in April and July. Other drivers included elevation, maximal temperatures, latitude, and longitude. The front of invasion likely moved from Italy and/or Switzerland, rather than from France, while the replacement of H. albidus is uncertain. [ABSTRACT FROM AUTHOR]

This study describes the seasonal changes in cell-to-cell transport in three selected angiosperm tree species, Acer pseudoplatanus (maple), Fraxinus excelsior (ash), and Populus tremula × tremuloides (poplar), with an emphasis on the living wood component, xylem parenchyma cells (XPCs). We performed anatomical studies, dye loading through the vascular system, measurements of non-structural carbohydrate content, immunocytochemistry, inhibitory assays and quantitative real-time PCR to analyse the transport mechanisms and seasonal variations in wood. The abundance of membrane dye in wood varied seasonally along with seasonally changing tree phenology, cambial activity, and non-structural carbohydrate content. Moreover, dyes internalized in vessel-associated cells and 'trapped' in the endomembrane system are transported farther between other XPCs via plasmodesmata. Finally, various transport mechanisms based on clathrin-mediated and clathrin-independent endocytosis, and membrane transporters, operate in wood, and their involvement is species and/or season dependent. Our study highlights the importance of XPCs in seasonally changing cell-to-cell transport in both ring-porous (ash) and diffuse-porous (maple, poplar) tree species, and demonstrates the involvement of both endocytosis and plasmodesmata in intercellular communication in angiosperm wood. [ABSTRACT FROM AUTHOR]

Plants are especially useful as biological indicators to assess air pollution and the effects of urbanisation. The aim of this study was to investigate tree species' sensitivity to air pollution using the Air Pollution Tolerance Index (APTI), the dust content (PM10) of leaves, and the heavy metal concentration of leaves. Sampling sites were in a city park (urban area), a location on the edge of the city (suburban area), and a forested location (rural area) along an urbanisation gradient in the city of Vienna in Austria. Leaf samples were collected from Acer platanoides, Fraxinus excelsior, and Quercus robur tree species. The APTI was determined by measuring the content of relative water ascorbic acid and chlorophyll, and the pH of leaf extracts. We found significant difference among species in their relative water content, pH, and content of ascorbic acid and chlorophyll Additionally, we found significant differences in chlorophyll content and pH based on sampling site. The highest chlorophyll content was found in A. platanoides. There was a significant difference among species in Al, Ba, Cr, Ni, and Zn concentrations in each stage of urbanisation. Based on APTI values, the studied species were sensitive indicators of air pollution; thus, they are useful bioindicator species, and they are suitable for air pollution monitoring. We demonstrated the effects of urbanisation with these bioindicator species with elevated PM10, Al, Ba, Cu, Fe, Sr, and Zn concentrations in urban area samples. [ABSTRACT FROM AUTHOR]

Abstract The increase in introduced insect pests and pathogens due to anthropogenic environmental changes has become a major concern for tree species worldwide. Common ash (Fraxinus excelsior L.) is one of such species facing a significant threat from the invasive fungal pathogen Hymenoscyphus fraxineus. Some studies have indicated that the susceptibility of ash to the pathogen is genetically determined, providing some hope for accelerated breeding programs that are aimed at increasing the resistance of ash populations. To address this challenge, we used a genomic selection strategy to identify potential genetic markers that are associated with resistance to the pathogen causing ash dieback. Through genome‐wide association studies (GWAS) of 300 common ash individuals from 30 populations across Poland (ddRAD, dataset A), we identified six significant SNP loci with a p‐value ≤1 × 10−4 associated with health status. To further evaluate the effectiveness of GWAS markers in predicting health status, we considered two genomic prediction scenarios. Firstly, we conducted cross‐validation on dataset A. Secondly, we trained markers on dataset A and tested them on dataset B, which involved whole‐genome sequencing of 20 individuals from two populations. Genomic prediction analysis revealed that the top SNPs identified via GWAS exhibited notably higher prediction accuracies compared to randomly selected SNPs, particularly with a larger number of SNPs. Cross‐validation analyses using dataset A showcased high genomic prediction accuracy, predicting tree health status with over 90% accuracy across the top SNP sets ranging from 500 to 10,000 SNPs from the GWAS datasets. However, no significant results emerged for health status when the model trained on dataset A was tested on dataset B. Our findings illuminate potential genetic markers associated with resistance to ash dieback, offering support for future breeding programs in Poland aimed at combating ash dieback and bolstering conservation efforts for this invaluable tree species.

In this work, the total content of phenolics, flavonoids, and phenolic acids in the leaves and bark extracts of Fraxinus ornus L. and Fraxinus excelsior L. was determined. In addition, the identification and quantification of phenolic acids was performed using the HPLC-DAD technique. Samples were tested for antioxidant activity using the ABTS and DPPH methods. Eight extract samples were prepared by Soxhlet and ultrasound extraction using 70 % ethanol as solvent. The content of phenolic compounds ranged from 7.59 for F. ornus to 88.93 mg GAE/g for F. excelsior in the bark extracts obtained by Soxhlet extraction. The highest content of total flavonoids in F. ornus and F. excelsior was found in the leaves extracts obtained by ultrasound extraction, which was 5.68 and 1.74 mg GAE/g extract, respectively. The results also showed that the highest content of total phenolic acids was found in F. ornus (105.33 mg CAE/g) and F. excelsior (97.97 mg CAE/g) in the bark extract obtained by Soxhlet extraction. The highest content of gallic acid (112.96 ± 1.32 mg g–1 extracts) and chlorogenic acid (246.94 ± 0.82 mg g–1 extracts) was found in the F. ornus bark extract obtained by ultrasound extraction. As for antioxidant activity, the bark extract obtained by Soxhlet extraction of F. ornus showed the best antioxidant activity by ABTS method with IC50 value of 0.062 mg ml–1. The results of the DPPH method show that the leaves extract obtained by Soxhlet extraction had the best antioxidant activity for the F. ornus sample, with an IC50 value of 0.23 mg ml–1.

In light of the increase of environmental pollution, we tested the effect of cadmium (Cd) and lead (Pb) soil contamination on ash dieback. The experiment included the inoculation of Hymenoscyphus fraxineus on Fraxinus excelsior and Fraxinus angustifolia seedlings growing on unpolluted soil, soil contaminated with cadmium (Cd), and soil contaminated with lead (Pb). At the end of the experiment, 173 days after soil contamination and 50 days since inoculation, all F. excelsior and F. angustifolia seedlings inoculated with H. fraxineus showed ash dieback symptoms in comparison to their control groups. However, both F. excelsior and F. angustifolia seedlings grown on contaminated soil had significantly increased necrotic lesions in comparison to the seedlings grown on uncontaminated soil. Our results showed for the first time that cadmium (Cd) and lead (Pb) soil contamination can significantly contribute to ash dieback and increase damage to F. excelsior and F. angustifolia seedlings.

Plant diseases are mostly multicausal with several factors influencing the health status of affected hosts. Common ash (Fraxinus excelsior), a significant tree species of European forests, is currently mostly endangered by ash dieback, caused by the invasive fungus Hymenoscyphus fraxineus. However, contributing factors, including pathogenic viruses, are poorly understood. Here, we report the results of a virus screening conducted on selected special stands of F. excelsior. Over three consecutive years, ash trees from different origins were tested, including leaf material from mature seed trees, young trees and ash seedlings from the natural regeneration. Using RT-PCR, we screened for five viruses, including the generalist species ArMV (Nepovirus arabis) and CLRV (Nepovirus avii), as well as newly discovered viruses in ash, including the emaravirus ASaV (Emaravirus fraxini), the idaeovirus PrLBaV (Idaeovirus ligustri), and cytorhabdoviruses. The results revealed a high virus diversity in common ash. An association of ASaV detection with specific leaf symptoms, including shoestring, chlorotic ringspots, and vein yellowing, was documented. An analyses of relevant gene products of cytorhabdoviruses obtained from ashes of different sites revealed sequence diversities and two distinct phylogenetic groups present in ash populations. Signatures of novel viruses from different families have been identified by high-throughput sequencing. Together, our results provide insights into the virus diversity and distribution of viruses in ash and expand our knowledge about the virome of this endangered tree species.

In this work, the total content of phenolics, flavonoids, and phenolic acids in the leaves and bark extracts of Fraxinus ornus L. and Fraxinus excelsior L. was determined. In addition, the identification and quantification of phenolic acids was performed using the HPLC-DAD technique. Samples were tested for antioxidant activity using the ABTS and DPPH methods. Eight extract samples were prepared by Soxhlet and ultrasound extraction using 70 % ethanol as solvent. The content of phenolic compounds ranged from 7.59 for F. ornus to 88.93 mg GAE/g for F. excelsior in the bark extracts obtained by Soxhlet extraction. The highest content of total flavonoids in F. ornus and F. excelsior was found in the leaves extracts obtained by ultrasound extraction, which was 5.68 and 1.74 mg GAE/g extract, respectively. The results also showed that the highest content of total phenolic acids was found in F. ornus (105.33 mg CAE/g) and F. excelsior (97.97 mg CAE/g) in the bark extract obtained by Soxhlet extraction. The highest content of gallic acid (112.96 ± 1.32 mg g−1 extracts) and chlorogenic acid (246.94 ± 0.82 mg g−1 extracts) was found in the F. ornus bark extract obtained by ultrasound extraction. As for antioxidant activity, the bark extract obtained by Soxhlet extraction of F. ornus showed the best antioxidant activity by ABTS method with IC50 value of 0.062 mg ml−1. The results of the DPPH method show that the leaves extract obtained by Soxhlet extraction had the best antioxidant activity for the F. ornus sample, with an IC50 value of 0.23 mg ml−1. [ABSTRACT FROM AUTHOR]

Leaf litter decomposition is a major component of nutrient cycling which depends on the quality and quantity of the leaf material. Ash trees (Fraxinus excelsior, decay time ∼ 0.4 years) are declining throughout Europe due to a fungal pathogen (Hymenoscyphus fraxineus), which is likely to alter biochemical cycling across the continent. The ecological impact of losing species with fast decomposing leaves is not well quantified. In this study we examine how decomposition of three leaf species with varying decomposition rates including ash, sycamore (Acer pseudoplatanus, decay time ∼ 1.4 years), and beech (Fagus sylvatica, decay time ∼ 6.8 years) differ in habitats with and without ash as the dominant overstorey species. Ten plots (40 m × 40 m) were set up in five locations representing ash dominated and non-ash dominated habitats. In each plot mesh bags (30 cm × 30 cm, 0.5 mm aperture) with a single leaf species (5 g) were used to include (large holes added) and exclude macrofauna invertebrates (with a focus on decomposer organisms such as earthworms, millipedes, and woodlice). The mesh bags were installed in October 2020 and retrieved without replacement at exponential intervals after 6, 12, 24 and 48 weeks. Total leaf mass loss was highest in the ash dominated habitat (ash dominated: 88.5%, non-ash dominated: 66.5%) where macrofauna were the main contributor (macrofauna: 96%, microorganisms/mesofauna: 4%). The difference between macrofauna vs microorganisms and mesofauna was less pronounced in the non-ash dominated habitat (macrofauna: 68%, microorganisms/mesofauna: 31%). Our results suggest that if ash dominated habitats are replaced by species such as sycamore, beech, and oak, the role of macrofauna decomposers will be reduced and leaf litter decomposition rates will decrease by 25%. These results provide important insights for future ash dieback management decisions.

In recent decades the vitality and physical stability of European ash trees in Germany have been reduced by European ash dieback, especially when associated with stem collar necroses and rots. This study was carried out to investigate the composition of the fungal communities associated with stem collar necroses. Filamentous fungi were isolated from 58 ash trees out of nine forest stands in northern, eastern, and central Germany. Obtained isolates were identified to a genus or species level by means of morphological and molecular analyses. In total 162 morphotypes including endophytic, saprotrophic, and pathogenic fungi were isolated. For 33 species found no prior reports from Fraxinus excelsior were recognised, including Cryptostroma corticale and Diplodia sapinea. None of the identified species were found at all studied sites, though Diplodia fraxini was the most common fungus with regard to frequency within all isolates, occurring at seven sample sites. This species is followed by Hymenoscyphus fraxineus, Armillaria spp., Neonectria punicea, Diaporthe cf. eres, Fusarium cf. lateritium, and Paracucurbitaria sp. in order of frequency within all isolates. The aforementioned species are characterised and analysed in respect to their occurrence in stem collar necroses and at sample sites. The influence of site conditions on the fungal composition was described for five intensively sampled sites with a minimum of five studied trees (Schwansee, Rhüden, Berggießhübel, Satrup, and Schlangen). The sampling site of Schlangen was further subdivided into four subplots with different positions in the terrain. In the remaining four extensive sample sites, either one or two trees, respectively, were sampled and analysed (Oranienbaumer Heide, Woltershausen, Wolfenbüttel, and Neuhege). Over all sample sites, fungal communities of symptomatic stem tissue are similar concerning the most frequent fungi, but vary greatly according to singularly isolated fungi. [ABSTRACT FROM AUTHOR]

Inflammation is the first physiological defence mechanism against external and internal stimuli. The prolonged or inappropriate response of the immune system may lead to the persistent inflammatory response that can potentially become a basis for chronic diseases e.g., asthma, type II diabetes or cancer. An important role in the alleviation of inflammatory processes, as an adjunct to traditional pharmacological therapy, is attributed to phytotherapy, especially to raw materials with a long tradition of use, e.g., ash leaves. Despite their long-term use in phytotherapy, the specific mechanisms of action have not been confirmed in a sufficient number of biological or clinical studies. The aim of the study is a detailed phytochemical analysis of infusion and its fractions, isolation of pure compounds from the leaves of Fraxinus excelsior and evaluation of their effect on the secretion of anti-inflammatory cytokines (TNF-α, IL-6) and IL-10 receptor expression in an in vitro model of monocyte/macrophage cells isolated from peripheral blood. Methods: Phytochemical analysis was carried out by the UHPLC-DAD-ESI-MS/MS method. Monocytes/macrophages were isolated from human peripheral blood using density gradient centrifugation on Pancoll. After 24 h incubation with tested fractions/subfractions and pure compounds, cells or their supernatants were studied, respectively, on IL-10 receptor expression by flow cytometry and IL-6, TNF-α, IL-1β secretion by the ELISA test. Results were presented with respect to Lipopolysaccharide (LPS) control and positive control with dexamethasone. Results: The infusion, 20% and 50% methanolic fractions and their subfractions, as well as their dominating compounds, e.g., ligstroside, formoside and oleoacteoside isolated from the leaves, show the ability to increase the IL-10 receptor expression on the surface of monocyte/macrophage cells, stimulated by LPS, and to decrease the secretion of pro-inflammatory cytokines, e.g., TNF-α, IL-6. [ABSTRACT FROM AUTHOR]

Objectives: Fraxinus excelsior L. (FE) is traditionally used to treat inflammatory and pain disorders. This study aimed to identify the constituents of FE leaves and evaluate the effects of its n-hexane (FEH), ethyl acetate (FEE), methanol (FEM) extracts and constituents on the viability of THP-1 cells and their ability to release pro-inflammatory cytokines. Methods: THP-1 cell viability was assessed using an MTT assay. The immunomodulatory activity was evaluated by measuring tumour necrosis factor-alpha (TNF-α) and interleukin 12 (IL-12) released by lipopolysaccharide-stimulated THP-1 cells using enzyme-linked immunosorbent assays. Key findings: Triterpenes, tyrosol esters, alkanes, phytyl and steryl esters, pinocembrin and bis(2-ethylhexyl)phthalate were isolated from FE. The tyrosol esters showed no significant effect on THP-1 cell viability. FEH, FEE, FEM, and pinocembrin, ursolic acid, oleanolic acid had IC50 values of 56.9, 39.9, 124.7 µg/ml and 178.6, 61.5 and 199.8 µM, respectively. FE extracts, ursolic acid, oleanolic acid and pinocembrin significantly reduced TNF-α/IL-12 levels. The tyrosol esters did not significantly affect TNF-α/IL-12 production. Conclusions: FE was able to reduce pro-inflammatory cytokine production indicating a mechanistic focus in its use for inflammation and pain. Further investigations are warranted to unravel the mode of action of the tested constituents and discover other potentially active compounds in FE extracts. [ABSTRACT FROM AUTHOR]

Pollen are an important food source for numerous insects and may be used as natural markers in ecological studies. However, to make inferences about the movement rates of insects based on their gut contents, information on pollen digestion rates is needed. Here, we assessed how the consumption and digestion rates of pollen ingested by Chrysoperla carnea Stephens (Neuroptera: Chrysopidae) adults are influenced by plant species, temperature, and sex. We offered pollen of two insect‐pollinated plants – Acer pseudoplatanus L. (Sapindaceae) and Helianthus annuus L. (Asteraceae) – and two wind‐pollinated plants – Fraxinus excelsior L. (Oleaceae) and Quercus rubra L. (Fagaceae) – differing in size and protein content, to adult lacewings at two temperatures, 20 and 25 °C. After feeding, lacewings were allowed to digest pollen for up to 14 days, sampled at 10 time intervals. At each of these intervals, lacewings were frozen and the internal pollen were obtained through acetolysis and quantified under a light microscope. The number of pollen grains decreased exponentially over time and declined faster for Acer than for the other three plant species. The half‐life and the time at which 95% of the pollen grains were digested were lower for Acer than for the other plant species. Lacewings consumed more pollen grains from Acer and Quercus than from Fraxinus and Helianthus. Male lacewings consumed 30% fewer pollen grains than females, but without differences in their digestion rates. Our results show that lacewings consumed higher amounts of high‐protein pollen (Acer and Quercus) and that digestion rates differed among plant species, which could be linked to their structural characteristics. The variable digestion rates of pollen grains may influence the study of lacewing diet composition. Studies that make inferences about the pollen diet or movement ecology of lacewings by analysing their gut contents should account for species‐specific pollen digestion rates. [ABSTRACT FROM AUTHOR]

European ash dieback caused by the alien, invasive ascomycete species Hymenoscyphus fraxineus currently represents, along with its side effects, the greatest threat to common ash (Fraxinus excelsior) trees in Europe. The disease is widely distributed in Germany and present in all regions where common ash is growing. In order to study European ash dieback in Germany and to develop management strategies to conserve common ash as a forest tree species, the concerted, interdisciplinary research project FraxForFuture was initiated. FraxForFuture consist of an association of five sub-networks: FraxConnect, FraxMon, FraxGen, FraxPath, and FraxSilva. In total, 27 individual projects conduct research on the epidemiology of the disease, the pathogen and the preservation of ash, including various control, breeding, and silvicultural strategies. The main goals of the FraxForFuture network and the sub-networks as well as essential information about the network of common research plots are provided. [ABSTRACT FROM AUTHOR]

Urbanization impacts plant-herbivore interactions, which are crucial for ecosystem functions such as carbon sequestration and nutrient cycling. While some studies have reported reductions in insect herbivory in urban areas (relative to rural or natural forests), this trend is not consistent and the underlying causes for such variation remain unclear. We conducted a continental-scale study on insect herbivory along urbanization gradients for three European tree species: Quercus robur, Tilia cordata, and Fraxinus excelsior, and further investigated their biotic and abiotic correlates to get at mechanisms. To this end, we quantified insect leaf herbivory and foliar secondary metabolites (phenolics, terpenoids, alkaloids) for 176 trees across eight European cities. Additionally, we collected data on microclimate (air temperature) and soil characteristics (pH, carbon, nutrients) to test for abiotic correlates of urbanization effects directly or indirectly (through changes in plant secondary chemistry) linked to herbivory. Our results showed that urbanization was negatively associated with herbivory for Q. robur and F. excelsior , but not for T. cordata. In addition, urbanization was positively associated with secondary metabolite concentrations, but only for Q. robur. Urbanization was positively associated with air temperature for Q. robur and F. excelsior , and negatively with soil nutrients (magnesium) in the case of F. excelsior , but these abiotic variables were not associated with herbivory. Contrary to expectations, we found no evidence for indirect effects of abiotic factors via plant defences on herbivory for either Q. robur or F. excelsior. Additional biotic or abiotic drivers must therefore be accounted for to explain observed urbanization gradients in herbivory and their interspecific variation. In more urbanized areas, insect leaf herbivory was lower for Quercus robur and Fraxinus excelsior but not for Tilia cordata , while leaf secondary metabolites were higher for Q. robur but not for F. excelsior and T. cordata. [Display omitted] • The association between the urbanization degree and herbivory varied among the tree species. • Urbanization is positively associated with leaf secondary metabolite concentrations in Q. robur. • Abiotic proxies of urbanization showed no direct or indirect association with herbivory in either species. [ABSTRACT FROM AUTHOR]

Ash dieback caused by the invasive fungus Hymenoscyphus fraxineus leads to massive mortality among common ash (Fraxinus excelsior) in Europe. To select tolerant genotypes, inoculation experiments are frequently conducted with isolates of the fungus. The aim of this study was to improve the inoculation methodology for evaluating susceptibility of ash genotypes to ash dieback through (i) testing the virulence of different isolates of Hymenoscyphus fraxineus and (ii) investigating temporal lesion development using three widely applied cultivars: Altena, Atlas and Westhof's Glorie. First, an experiment was conducted with a repeated measure design in which virulence of five different isolates was tested. Second, a subset of two isolates that induced the longest downward lesion length in experiment one was used in combination with a slightly adjusted inoculum protocol. Significant differences were found between isolates for downward lesion length, but a significant interaction effect of isolates and cultivars was absent. Also, the inoculation position within the stem affected lesion length; the largest lesions were found on the highest inoculation position within the stem. Furthermore, we found that cryopreserved isolates can remain virulent over years. The timing of inoculation at the end of the growing season was effective as large lesions already occurred during winter dormancy. For future inoculation studies, we propose to use: (i) isolates that induce large lesions, as less virulent isolates induced not only shorter but also fewer lesions; (ii) a similar inoculation position to better compare inoculations within and between experiments; (iii) cryopreserved isolates for testing over years; (iv) mycelial suspensions for inoculum preparation to cover wood chips more evenly; (v) reference clone—like the studied cultivars—to standardize research outcomes between years and research groups. [ABSTRACT FROM AUTHOR]

A large proportion of the western marginal range for common ash (Fraxinus excelsior L.) is located on the island of Ireland. However, the molecular diversity of common ash in Ireland has only been studied in a limited number of populations and using mainly non-standard chloroplast and nuclear simple sequence repeat (SSR) markers. This has prevented direct comparisons with studies on the rest of the species' range across Europe. Here, four chloroplast and six nuclear SSR markers were used to infer the genetic diversity from 347 trees sampled across 20 populations. Results confirmed that, like Britain, Ireland is dominated by one main haplotype (H04) which originates from an Iberian glacial refugium. The occurrence of a second, rarer haplotype (H13) that also occurs as a rare haplotype in Britain but nowhere else, suggests at least some post-glacial recolonisation from the east. Chloroplast allelic richness was similar to Norway, which constitutes the species' northern marginal range, but lower than in Britain and the European average. Nuclear allelic richness was also comparable with Norway, but Irish common ash differed in a complete absence of sub-population structure and geographic variability at both the chloroplast and nuclear level. Analysis of nuclear genetic structure indicated that common ash in Ireland mainly comprises one genetic group which is likely part of a single, western European meta-population. However, a less frequent genetic cluster is hypothesised to represent a mix of non-native alleles from imported plantation ash. Finally, conservation recommendations and the consequences of a uniform and low genetic diversity are discussed in the context of ash dieback disease, which was present in all populations sampled here. [ABSTRACT FROM AUTHOR]

The tolerance of ash trees against the pathogen Hymenoscyphus fraxineus seems to be associated with the occurrence of specific microbial taxa on leaves. A group of bacterial isolates, primarily identified on tolerant trees, was investigated with regard to their taxonomic classification and their potential to suppress the ash dieback pathogen. Examination of OGRI values revealed a separate species position. A phylogenomic analysis, based on orthologous and marker genes, indicated a separate genus position along with the species Achromobacter aestuarii. Furthermore, analysis of the ratio of average nucleotide identities and genome alignment fractions demonstrated genomic dissimilarities typically observed for inter-genera comparisons within this family. As a result of these investigations, the strains are considered to represent a separate species within a new genus, for which the name Schauerella fraxinea gen. nov., sp. nov. is proposed, with the type strain B3P038T (=LMG 33092 T = DSM 115926 T). Additionally, a reclassification of the species Achromobacter aestuarii as Schauerella aestuarii comb. nov. is proposed. In a co-cultivation assay, the strains were able to inhibit the growth of a H. fraxineus strain. Accordingly, a functional analysis of the genome of S. fraxinea B3P038T revealed genes mediating the production of antifungal substances. This potential, combined with the prevalent presence in the phyllosphere of tolerant ash trees, makes this group interesting for an inoculation experiment with the aim of controlling the pathogen in an integrative approach. For future field trials, a strain-specific qPCR system was developed to establish an efficient method for monitoring the inoculation success. [ABSTRACT FROM AUTHOR]

Inflammation is the first physiological defence mechanism against external and internal stimuli. The prolonged or inappropriate response of the immune system may lead to the persistent inflammatory response that can potentially become a basis for chronic diseases e.g., asthma, type II diabetes or cancer. An important role in the alleviation of inflammatory processes, as an adjunct to traditional pharmacological therapy, is attributed to phytotherapy, especially to raw materials with a long tradition of use, e.g., ash leaves. Despite their long-term use in phytotherapy, the specific mechanisms of action have not been confirmed in a sufficient number of biological or clinical studies. The aim of the study is a detailed phytochemical analysis of infusion and its fractions, isolation of pure compounds from the leaves of Fraxinus excelsior and evaluation of their effect on the secretion of anti-inflammatory cytokines (TNF-α, IL-6) and IL-10 receptor expression in an in vitro model of monocyte/macrophage cells isolated from peripheral blood. Methods: Phytochemical analysis was carried out by the UHPLC-DAD-ESI-MS/MS method. Monocytes/macrophages were isolated from human peripheral blood using density gradient centrifugation on Pancoll. After 24 h incubation with tested fractions/subfractions and pure compounds, cells or their supernatants were studied, respectively, on IL-10 receptor expression by flow cytometry and IL-6, TNF-α, IL-1β secretion by the ELISA test. Results were presented with respect to Lipopolysaccharide (LPS) control and positive control with dexamethasone. Results: The infusion, 20% and 50% methanolic fractions and their subfractions, as well as their dominating compounds, e.g., ligstroside, formoside and oleoacteoside isolated from the leaves, show the ability to increase the IL-10 receptor expression on the surface of monocyte/macrophage cells, stimulated by LPS, and to decrease the secretion of pro-inflammatory cytokines, e.g., TNF-α, IL-6.

Agrilus planipennis (Coleoptera: Buprestidae) is the most serious invasive pest of ash trees (Fraxinus spp., Oleaceae) in the world. It has not yet reached the range of olive tree plantations, however it has been shown to complete its life cycle in this host tree. This pest native to East Asia was first found in Europe in Moscow in 2003 and has been spreading ever since. The aims of this study were to determine if the southern border of the range has already reached the Caucasus and to assess the potential range in this region based on host availability and heat availability. In 2021, we surveyed ash trees south to the known range in Russia. We did not find the pest in six cities in the North Caucasus region but found it in the city of Azov close to the Caucasus. Analysis of information about 550 localities of ash trees in the Caucasus showed that Fraxinus spp. is common throughout the region. The calculation of annual growing degree days base 10 °C indicated that the places where Fraxinus spp. grow above 1500 m are too cold for A. planipennis and therefore could potentially become a refuge for ash trees. The spread of A. planipennis to the Caucasus is of concern. First, the forest ecosystems of this region are endangered, since European ash Fraxinus excelsior is much more common there than in the currently infested regions in Russia. Second, the Russian Caucasus can become the gateway to Georgia, Armenia, Azerbaijan, Turkey and other countries of the Middle East and Southern Europe. We propose using Fraxinus angustifolia, F. ornus and Olea europaea growing in the Caucasus and adjacent regions as sentinel trees for the monitoring and pest risk assessment of these tree species. [ABSTRACT FROM AUTHOR]

European ash (Fraxinus excelsior) is highly affected by the pathogenic fungus Hymenoscyphus fraxineus in all of Europe. Increases in plant’s secondary metabolite (SM) production is often linked tol enhanced resistance to stress, both biotic and abiotic. Moreover, plant-associated bacteria have been shown to enhance SM production in inoculated plants. Thus, our hypothesis is that bacteria may boost ash SM production, hence priming the tree’s metabolism and facilitating higher levels of resilience to H. fraxineus. We tested three different ash genotypes and used Paenibacillus sp. and Pseudomonas sp. for inoculation in vitro. Total phenol (TPC), total flavonoid (TFC) and carotenoid contents were measured, as well as the chlorophyll a/b ratio and morphometric growth parameters, in a two-stage trial, whereby seedlings were inoculated with the bacteria during the first stage and with H. fraxineus during the second stage. While the tested bacteria did not positively affect the morphometric growth parameters of ash seedlings, they had a statistically significant effect on TPC, TFC, the chlorophyll a/b ratio and carotenoid content in both stages, thus confirming our hypothesis. Specifically, in ash genotype 64, both bacteria elicited an increase in carotenoid content, TPC and TFC during both stages. Additionally, Pseudomonas sp. inoculated seedlings demonstrated an increase in phenolics after infection with the fungus in both genotypes 64 and 87. Our results indicate that next to genetic selection of the most resilient planting material for ash reforestation, plant-associated bacteria could also be used to boost ash SM production. [ABSTRACT FROM AUTHOR]

Background and Aims Quantifying the Earth's forest above-ground biomass (AGB) is indispensable for effective climate action and developing forest policy. Yet, current allometric scaling models (ASMs) to estimate AGB suffer several drawbacks related to model selection and uncertainties about calibration data traceability. Terrestrial laser scanning (TLS) offers a promising non-destructive alternative. Tree volume is reconstructed from TLS point clouds with quantitative structure models (QSMs) and converted to AGB with wood basic density. Earlier studies have found overall TLS-derived forest volume estimates to be accurate, but highlighted problems for reconstructing finer branches. Our objective was to evaluate TLS for estimating tree volumes by comparison with reference volumes and volumes from ASMs. Methods We quantified the woody volume of 65 trees in Belgium (from 77 to 2800 L; Pinus sylvestris , Fagus sylvatica , Larix decidua , and Fraxinus excelsior) with QSMs and destructive reference measurements. We tested a volume expansion factor (VEF) approach by multiplying the solid and merchantable volume from QSMs by literature VEF values. Key Results Stem volume was reliably estimated with TLS. Total volume was overestimated by +21 % using original QSMs, by +9 % and –12 % using two sets of VEF-augmented QSMs, and by –7.3 % using best-available ASMs. The most accurate method differed per site, and the prediction errors for each method varied considerably between sites. Conclusions VEF-augmented QSMs were only slightly better than original QSMs for estimating tree volume for common species in temperate forests. Despite satisfying estimates with ASMs, the model choice was a large source of uncertainty, and species-specific models did not always exist. Therefore, we advocate for further improving tree volume reconstructions with QSMs, especially for fine branches, instead of collecting more ground-truth data to calibrate VEF and allometric models. Promising developments such as improved co-registration and smarter filtering approaches are ongoing to further constrain volumetric errors in TLS-derived estimates. [ABSTRACT FROM AUTHOR]

Agrilus planipennis (Coleoptera: Buprestidae) is the most serious invasive pest of ash trees (Fraxinus spp., Oleaceae) in the world. It has not yet reached the range of olive tree plantations, however it has been shown to complete its life cycle in this host tree. This pest native to East Asia was first found in Europe in Moscow in 2003 and has been spreading ever since. The aims of this study were to determine if the southern border of the range has already reached the Caucasus and to assess the potential range in this region based on host availability and heat availability. In 2021, we surveyed ash trees south to the known range in Russia. We did not find the pest in six cities in the North Caucasus region but found it in the city of Azov close to the Caucasus. Analysis of information about 550 localities of ash trees in the Caucasus showed that Fraxinus spp. is common throughout the region. The calculation of annual growing degree days base 10 °C indicated that the places where Fraxinus spp. grow above 1500 m are too cold for A. planipennis and therefore could potentially become a refuge for ash trees. The spread of A. planipennis to the Caucasus is of concern. First, the forest ecosystems of this region are endangered, since European ash Fraxinus excelsior is much more common there than in the currently infested regions in Russia. Second, the Russian Caucasus can become the gateway to Georgia, Armenia, Azerbaijan, Turkey and other countries of the Middle East and Southern Europe. We propose using Fraxinus angustifolia, F. ornus and Olea europaea growing in the Caucasus and adjacent regions as sentinel trees for the monitoring and pest risk assessment of these tree species.

Some common ash trees (Fraxinus excelsior) show tolerance towards shoot dieback caused by the invasive ascomycete Hymenoscyphus fraxineus. Leaf petioles are considered to serve as a pathogen colonization route to the shoots. We compared four common ash clones with variation in disease tolerance, and included the native host, Manchurian ash (Fraxinus mandshurica), as a reference. Tissue colonization, following rachis inoculation by H. fraxineus, was monitored by histochemical observations and a quantitative polymerase chain reaction (qPCR) assay specific to H. fraxineus. Axial spread of the pathogen towards the petiole base occurred primarily within the phloem and parenchyma, tissues rich in starch in healthy petioles. In inoculated petioles, a high content of phenolics surrounded the hyphae, presumably a host defense response. There was a relationship between field performance and susceptibility to leaf infection in three of the four studied common ash clones, i.e., good field performance was associated with a low petiole colonization level and vice versa. Low susceptibility to leaf infection may counteract leaf-to-shoot spread of the pathogen in common ash, but the limited number of clones studied warrants caution and a larger study. The Manchurian ash clone had the highest petiole colonization level, which may suggest that this native host has evolved additional mechanisms to avoid shoot infection.

Development of effective biocontrol procedures using ash endophytes to combat an ash pathogen Hymenoscyphus fraxineus would be an appropriate contribution to the ongoing effort to protect European ash stands against ash decline. In this study we investigated the biocontrol potential of two ash endophytes, Thielavia basicola and Minimidochium sp., against H. fraxineus using in vitro plant-fungus and fungus-fungus dual cultures approach in three biocontrol models. The tests aimed to determine whether the endophytes show antagonism toward Fraxinus excelsior and F. pennsylvanica, to assess the level of antagonism of the endophytes toward H. fraxineus and to identify potential secondary metabolites induced by the presence of H. fraxineus. The results that dual culture experiments modeled according to our design may be a very useful tool to precisely study biocontrol potential of fungi, i.e., without the impact of environmental factors. Such experiments also enable the selection of most resistant ash genotypes and rapid propagation, producing large numbers of pathogen-free seedlings. It should be noted, however, that both of the endophytes tested in the dual cultures strongly inhibited the growth of H. fraxineus. Their growth under the influence of callus/seedlings was also inhibited. Comparison of HPLC profiles showed that the presence of H. fraxineus in the post-culture medium induced the production of an unknown secondary metabolite in this species. Such results suggest that some of the plant–fungus combinations examined in this study may have potential to be developed as biocontrol methods, thus increasing the survivability of ash stands under natural conditions.

European ash (Fraxinus excelsior) is highly affected by the pathogenic fungus Hymenoscyphus fraxineus in all of Europe. Increases in plant’s secondary metabolite (SM) production is often linked tol enhanced resistance to stress, both biotic and abiotic. Moreover, plant-associated bacteria have been shown to enhance SM production in inoculated plants. Thus, our hypothesis is that bacteria may boost ash SM production, hence priming the tree’s metabolism and facilitating higher levels of resilience to H. fraxineus. We tested three different ash genotypes and used Paenibacillus sp. and Pseudomonas sp. for inoculation in vitro. Total phenol (TPC), total flavonoid (TFC) and carotenoid contents were measured, as well as the chlorophyll a/b ratio and morphometric growth parameters, in a two-stage trial, whereby seedlings were inoculated with the bacteria during the first stage and with H. fraxineus during the second stage. While the tested bacteria did not positively affect the morphometric growth parameters of ash seedlings, they had a statistically significant effect on TPC, TFC, the chlorophyll a/b ratio and carotenoid content in both stages, thus confirming our hypothesis. Specifically, in ash genotype 64, both bacteria elicited an increase in carotenoid content, TPC and TFC during both stages. Additionally, Pseudomonas sp. inoculated seedlings demonstrated an increase in phenolics after infection with the fungus in both genotypes 64 and 87. Our results indicate that next to genetic selection of the most resilient planting material for ash reforestation, plant-associated bacteria could also be used to boost ash SM production.

We identified a novel virus in diseased European ash (Fraxinus excelsior) and manna ash (F. ornus) trees exhibiting chlorotic ringspots, mottle and leaf deformation such as curling and shoestring symptoms. High-throughput sequencing (HTS, Illumina RNASeq) of total RNA isolated from diseased leaf material in combination with RT-PCR-based amplification techniques and Sanger sequencing determined five complete genome segments, each encoding a single open reading frame. Sequence analyses of RNA1–RNA5 revealed a genome organization typical for emaraviruses, i.e., (i) conserved and complementary terminal 5′ and 3′ termini of each genome segment (ii) proteins showing significant homologies to the RNA-dependent RNA polymerase (RdRP) encoded by RNA1, the glycoprotein precursor (GPP) encoded by RNA2, the viral nucleocapsid protein (N, RNA3), the movement protein (MP, RNA4), and a protein of 26 kDA (P26, RNA5) highly similar to proteins of unknown function encoded by other emaraviruses. Furthermore, we identified spherical particles (double-membrane bodies, DMB) of different sizes (70–80 nm in diameter) which are typical for emaraviruses exclusively in virus-infected leaf tissue exhibiting mottle and leaf deformation. Sequence comparison and phylogenetic analyses confirmed the identified novel virus as a new member of the genus Emaravirus. We established a species-specific RT-PCR detection protocol and could associate the observed disease symptoms with the infection of the novel emaravirus in F. excelsior and F. ornus. Therefore, we propose the name ash shoestring-associated emaravirus (ASaV). Investigation of ASaV-infected sample trees originating from different locations in Switzerland, Germany, Italy and Sweden provided a wide geographical distribution of the virus in affected ash species. To our knowledge, this is the first confirmation of an emaravirus affecting ash tree species with shoestring symptoms of leaves in Europe.

The population of European ash (Fraxinus excelsior L.) is currently facing the risk of collapse, mainly due to ash dieback, a disease caused by a pathogenic fungus, Hymenoscyphus fraxineus. To facilitate studies into the molecular basis of ash dieback and design breeding strategies for a generation of resistant trees, it is necessary to develop tools enabling the study of gene function in F. excelsior. Despite this, a method for the genetic engineering of F. excelsior is still missing. Here, we report the first successful genetic transformation of F. excelsior callus and a selection process enabling the formation of stable transgenic callus lines. The protocol relies on the use of Agrobacterium tumefaciens to transform callus tissue derived from embryos of F. excelsior. In our experiments, we used the β-glucuronidase (GUS) reporter system to demonstrate the transformation of callus cells and performed RT-PCR experiments to confirm the stable expression of the transgene. Since ash dieback threatens the long-term stability of many native F. excelsior populations, we hope that the transformation techniques described in this manuscript will facilitate rapid progress in uncovering the molecular basis of the disease and the validation of gene targets previously proposed to be linked to the resistance of trees to H. fraxineus pathogenicity.

The article focuses on, In August 2021, mature Fraxinus excelsior trees and associated regeneration with typical symptoms of ash dieback were observed in the localities of Oviedo and Bulnes in the principality of Asturias in north-western Spain . The lethal disease is caused by the ascomycete fungus Hymenoscyphus fraxineus , which was introduced into Europe from Asia. Since its introduction in the mid-1990s in northeast Poland.

While assessing the filamentous fungi associated with woody tissues of stem collar rots, necroses, and lesions of European ash trees (Fraxinus excelsior) presenting symptoms of ash dieback in Germany, Cryptostroma corticale was recovered from three different ash trees. These isolated strains were the first report of C. corticale on ash and the first proof of an association of this plant pathogen with woody tissues of other tree species than Acer spp. in Germany. To test the pathogenicity of C. corticale against F. excelsior and to fulfil Koch’s postulates, inoculation tests in planta with strains isolated from Acer pseudoplatanus and F. excelsior were conducted according to Henle–Koch’s postulates in a greenhouse located in Göttingen. The pathogenicity tests were performed with apparently healthy ash saplings from June 2021 until January 2022. After three and seven months, neither necroses or lesions due to C. corticale nor disease symptoms were observed. Mostly, the inoculation wounds healed over, and C. corticale could not be re-isolated from the ash woody tissue. In an attempt to re-isolate the inoculated strains, the filamentous fungal endophytes of the ash woody tissues were isolated and identified. A total of 32 taxa of the Ascomycota were found, where the most common species was Boeremia exigua. Most frequently observed orders were Pleosporales (58.4%), followed by Sordariales (13.5%), Hypocreales (9.4%), and Diaporthales (8.7%). On average, 3.7 endophytic species were recorded on each sapling. [ABSTRACT FROM AUTHOR]