Pathogenicity of fungi associated with ash dieback symptoms of one‐year‐old Fraxinus excelsior in Montenegro

In addition to Hymenoscyphus fraxineus, two fungi identified as Diaporthe eres aff. and Fusarium sambucinum aff. were also isolated from necrotic bark lesions on declining one‐year‐old Fraxinus excelsior in a forest stand in Montenegro. To examine their involvement in ash decline, a pathogenicity test was performed using under bark inoculations on one‐year‐old Fraxinus excelsior. Hymenoscyphus fraxineus was included as comparison. All three fungal species proved highly pathogenic towards one‐year‐old seedlings although lesion sizes differed significantly between the different species. Hymenoscyphus fraxineus was most aggressive, followed by F. sambucinum aff., while D. eres aff. caused the smallest lesions. This study demonstrates for the first time the ability of isolates in the D. eres and F. sambucinum species complexes to cause decline on one‐year‐old common ash seedlings.     

Genetic variability and divergence among Italian populations of common ash (Fraxinus excelsior L.)

The level of genetic variation throughout the Italian range of common ash (Fraxinus excelsior L.) was estimated using six microsatellite markers. High levels of allelic diversity was detected. The levels of expected heterozygosity for each of the populations ranged from 0.726 to 0.871, with an average of 0.798, and indicated that populations have a high level of genetic variation. A general and significant homozygote excess was found at most loci in all populations, with an overall mean F _IS of 0.284. Possible explanations for such situations are discussed. Only 4.9% of the total diversity was attributable to differentiation among populations. Although divergence among pedo-climatic regions explained only a small part of the variance it was possible to observe some partial clustering of populations belonging to the same regions. The contribution of the results in relation to the definition of the most appropriate strategies to collect forest reproductive material is discussed.     

A review of black heart of ash (Fraxinus excelsior L.)

Black heart is a non-fungal stain of ash (Fraxinus excelsiorL.) which results in a price penalty compared with white wood.The formation of black heart varies within individual trees,between trees and on different sites; the evidence availablesuggests that it can be present in up to 45 per cent of treesat age 60. Once formed the proportion of cross-sectional areaaffected increases with age. Previous work has suggested thatblack heart has no effect on timber properties or strength butits occurrence has been linked with high soil moisture. Howeverthese studies were conducted in the 1950s and need to be confirmedusing more rigorous research methods. Scope exists for innovativemarketing of black heartwood to emphasize that it is a naturalfeature and can be visually pleasing; this may lead to betterreturns for the grower.     

Differences in susceptibility to ash dieback in Czech provenances of Fraxinus excelsior

The impact of ash dieback caused by Hymenoscyphus fraxineus on 17 provenances of Fraxinus excelsior and one provenance of Fraxinus angustifolia was studied in an extensive field trial established in the Czech Republic prior to the H. fraxineus invasion in 1999. A difference in the level of resistance to ash dieback between the species was found: F. angustifolia was significantly less affected by the disease than F. excelsior. Moreover, particular provenances of F. excelsior showed important differences in the level of resistance to H. fraxineus. A relationship between the impact of ash dieback and altitude was also discovered – the provenances from altitudes above 600 m a.s.l. were less affected by the pathogen than were the provenances from lower areas. No difference in the impact of the disease among provenances of F. excelsior from different ecotopes (ravine, calcareous ravine and alluvial) was found. Substantial among‐tree variability in resistance to H. fraxineus was observed throughout the trial – promising genotypes (with crown defoliation up to 5%) were identified in all 18 tested provenances. In regard to this finding, it appears that the main source of resistance to the pathogen is probably at the individual genotype level in the trial. A secondary but massive attack by Hylesinus fraxini was identified in the trees that had been greatly damaged by ash dieback, and the beetle caused their health to deteriorate significantly. A significant negative effect of the presence of collar necroses caused by H. fraxineus and browse damage was also identified.     

Temporal development of ash dieback symptoms and spatial distribution of collar rots in a provenance trial of Fraxinus excelsior

European ash (Fraxinus excelsior) is threatened by Hymenoscyphus pseudoalbidus, the agent of ash dieback disease. Beside ordinary ash dieback symptoms, collar rots have been reported on declining ashes as an additional problem of increasing severity. Since 2009, ash dieback has been surveyed annually in a provenance trial on four different sites in southwest Germany (Metzler et al. in Ger J For Res 183:168–180, 2012). The trial was established in 2005. Data of tree growth, ash dieback symptoms and collar rot prevalence were collected by surveying the trial in summer 2012 and compared with previously published data of the trial. Evaluations revealed a continuous and considerable increase in dieback severity since 2009. The results suggest that the infection process has not come to a standstill yet. Up to 2012, 6 % of the trees remained symptomless, whereas mortality added up to 9 %. There were significant differences in ash dieback severity between the investigated provenances. Collar rot prevalence ranged from 19 to 59 % between study sites. Moreover, high spatial dependency of collar rot prevalence could be detected within sites. Collar rots were more abundant on trees of severe ash dieback intensity, but could also be detected on 15 % of otherwise healthy trees. Mycelium from collar rots could be identified by means of RFLP analyses and sequencing of the ITS region as most likely belonging to Armillaria gallica. The possible roles of Armillaria spp. and H. pseudoalbidus in collar rot formation are discussed.     

Chalara fraxinea sp. nov. associated with dieback of ash (Fraxinus excelsior) in Poland

A new species of the hyphomycete genus Chalara is described. It has been frequently isolated from stems and branches and sometimes also from roots of wilting and dying Fraxinus excelsior in Poland. It differs from previously described species of Chalara by its small, short cylindrical phialoconidia extruded in chains or in slimy droplets, morphological features of the phialophores and by colony characteristics. It is non‐tolerant to cycloheximide.     

Diminished vessel diameter as a possible factor in the decline of European ash (Fraxinus excelsior L.)

? The aim of this work was to examine the anatomy and functioning of secondary xylem in stems in relation to the decline of European ash (Fraxinus excelsior L.).

? We tested the hypothesis that declining trees show changes in the structure of wood, which result in impaired water transport.

? Anatomical analyses were carried out on wood samples (comprising all annual rings formed during the 30 years life of the analysed trees) collected at breast height from the main stem of healthy, weakened and dead ash trees. The width of annual wood increments, the diameter and density of earlywood vessels were measured and the theoretical hydraulic conductivity index through the secondary xylem calculated by application of Hagen-Poisseuille’s formula.

? Anatomical characteristics changed both with the age of trees and in response to unfavorable factors. The largest vessels were observed in healthy trees, which implied that they had the highest hydraulic conductivity index, whereas trees considered to be in decline produce smaller vessels and hence had reduced conductivity.

     

Die Esche(Fraxinus excelsior L.) — ein Baum des Leipziger Auwaldes?

Zusammenfassung Die historische Analyse der Waldentwicklung des Leipziger Auwaldes ergab, da?Fraxinus excelsior bis Mitte des 19. Jahrhunderts nicht in der Baumschicht vorkam. Ihre Einbringung erfolgte durch forstliche Ma?nahmen beginnend am Ende des 18. Jahrhunderts und wurde ab 1870 verst?rkt. Heute ist die Esche die h?ufigste Baumart im Leipziger Auwald. Weitere Beispiele zeigen, da? der Eschenanbau keine lokale Erscheinung darstellt, sondern auch in anderen europ?ischen Gebieten zur gleichen Zeit erfolgte.      

Wood-inhabiting fungi in stems of Fraxinus excelsior in declining ash stands of northern Lithuania,with particular reference to Armillaria cepistipes

Abstract

Stem bases of 210 Fraxinus excelsior trees of three different health categories were sampled by the means of an increment borer in declining ash stands in northern Lithuania. From this number, 15 sound-looking, 132 declining and 63 dead trees from three discrete plots yielded 352 isolates, representing 75 fungal species. In addition, mycelial fans and rhizomorphs typical of Armillaria spp. from 205 and 20 trees, respectively, were sampled and subjected to fungal isolations. Species richness was similar in trees from each health category, but community structures differed, indicating that species composition of wood-inhabiting fungi in stems changes along with the changes in tree health condition. Armillaria cepistipes was the most common species (86 isolates from 210 wood samples, or 41.0%), isolated more frequently and consistently than any other potential tree pathogen. It also showed abundant occurrence on a majority of trees in the form of mycelial fans and rhizomorphs, from which 64 and 14 isolates of the fungus were obtained, respectively. The population structure of A. cepistipes revealed the presence of 53–93 genets per hectare, some of which extended up to 30–55?m.     

Detection of Chalara fraxinea in common ash (Fraxinus excelsior) using real time PCR

A real time PCR assay was developed for the detection of Chalara fraxinea in common ash. PCR primers and Taqman probes, based on the internal transcribed spacer region of the multi‐copy gene rDNA, were tested for specificity and sensitivity. The primers amplified an 81 bp fragment for C. fraxinea but did not amplify DNA from other Chalara species or from other fungi isolated from ash, whether pathogenic or saprophytic. The limit of detection was 5 pg of genomic DNA per PCR. Moreover, naturally‐infected samples were correctly diagnosed. A procedure for DNA extraction from woody tissues using an electric drill yielded DNA of an appropriate quality for real time PCR. This molecular method could be useful for routine analysis of this emergent pathogen and for epidemiological studies.     

The effects of propagation environment on the rooting of leafy cuttings of ash (Fraxinus excelsior L.), sycamore (Acer pseudoplatanus L.), and sweet chestnut (Castanea sativa Mill.)

The effect of propagation environment on the rooting of field collected leafy cuttings of ash (Fraxinus excelsior, sycamore (Acer pseudoplatanus) and sweet chestnut (Castanea sativa) was investigated in two experiments. In the first experiment, rooting of all three species in mist enclosed inside a polythene tent was greater than 50%, while rooting of ash cuttings was reduced from 63% in enclosed mist to only 16% in open mist. Rooting in open mist, enclosed mist, and contact polythene was studied in a second experiment. Sycamore cuttings rooted equally well in the two mist systems with an average rooting of 78%. In contrast cuttings of both ash and sweet chestnut rooted most under the enclosed mist system, at 64 and 46% respectively. Rooting of ash cuttings was again depressed in the open mist system where less than 30% of cuttings rooted. This reduction was related to an increase in the percentage of cuttings which remained alive without rooting, while showing proliferation of callus at the cutting base. The percentage of sweet chestnut cuttings which callused without rooting was also much greater in the open mist system than in enclosed mist or contact polythene.     

Genetic parameter estimates for growth and form traits in common ash (Fraxinus excelsior, L.) in a breeding seedling orchard at Little Wittenham in England

Data from a trial of 36 families at Little Wittenham, North Oxford was used to assess growth traits and stem form in common ash (Fraxinus excelsior, L.). The trial followed a design described by Barnes (Silv Genet 44(2–3):81–88, 1995) as the Breeding Seedling Orchard (BSO). The objectives of the study were to determine variation in growth and form traits and estimate genetic parameters of families. Assessments conducted at 3–8 years for height, diameter at breast height (dbh), straightness and forking showed significant differences (P < 0.05) among families with Guys (19), Settrington (34), Overbury (23) and Grimthorpe (36) being the best performers while Coleford (3 and 5) Gwynedd (27) and Bacheiddon (32) were the least performers. At the overall level, the additive genetic coefficient of variation (CV_A) ranged between 10.2 and 12.7% for height and 1.5 and 2.1% for dbh. Height had a high genetic correlation (r_g = 0.85) with dbh whilst forking had low correlation (r_g = 0.25) with height. Estimates of heritability decreased with age from  = 0.62 at 3 years to  = 0.30 at 8 years for height while values for dbh decreased from  = 0.48 at 4 years to  = 0.27 at 8 years. Some genetic gains are possible despite the low genetic variation among families; height had the greatest potential of genetic gain (12.5% at 8 years). Results suggest that early selection of superior families of Fraxinus excelsior appears to be feasible and it could be possible to achieve simultaneous improvement in both growth and form.     

Fungal colonization patterns in necrotic rootstocks and stem bases of dieback‐affected Fraxinus excelsior L.

European ash (Fraxinus excelsior) trees currently face the major threat of ash dieback caused by an invasive fungus, Hymenoscyphus fraxineus. Collar rots in F. excelsior have been increasingly associated with infections by this pathogen. However, the aetiology of the collar rots is still unclear and remains heavily debated. In contrast to most studies of this kind, entire rootstocks of four diseased ash trees were dug out to examine necrotic tissues in these rootstocks and stem bases in detail and to sample necrotic wood for fungal isolation. With the aid of morphological and molecular identification techniques, five to twelve fungal taxa were detected per tree. Members of the Nectriaceae family and Botryosphaeria stevensii, the causal agent of stem and branch cankers on many tree species, were frequently isolated from outer xylem. In contrast, H. fraxineus was the dominating species in interior wood layers. Microsatellite genotyping of 77 H. fraxineus isolates helped to identify up to six different genotypes per tree. The role of H. fraxineus and other isolated fungi in the aetiology of ash collar rots are discussed.     

Phytophthora root and collar rot of mature Fraxinus excelsior in forest stands in Poland and Denmark

In recent years, Common ash (Fraxinus excelsior) throughout Europe has been severely impacted by a leaf and twig dieback caused by the hyphomycete Chalara fraxinea. The reasons for its current devastating outbreak, however, still remain unclear. Here, we report the presence of four Phytophthora taxa in declining ash stands in Poland and Denmark. Phytophthora cactorum, Phytophthora plurivora, Phytophthora taxon salixsoil and Phytophthora gonapodyides were isolated from rhizosphere soil samples and necrotic bark lesions on stems and roots of mature declining ash trees in four stands. The first three species proved to be aggressive to abscised roots, twigs and leaves of F. excelsior in inoculation experiments. Soil infestation tests also confirmed their pathogenicity towards fine and feeder roots of ash seedlings. Our results provide first evidence for an involvement of Phytophthora species as a contributing factor in current decline phenomena of F. excelsior across Europe. Specifically, they may act as a predisposing factor for trees subsequently infected by C. fraxinea. Phytophthora species from ash stands also proved to be aggressive towards a wide range of tree and shrub species commonly associated with F. excelsior in mixed stands. Although damage varied considerably depending on the Phytophthora species/isolate–host plant combination, these results show that many woody species may be a potential source for survival and inoculum build‐up of soilborne Phytophthora spp. in ash stands and forest ecosystems in general.     

Genetic variation in dieback resistance: growth and survival of Fraxinus excelsior under the influence of Hymenoscyphus pseudoalbidus

The invasive fungal pathogen, Hymenoscyphus pseudoalbidus V. Queloz, has decimated stands of Fraxinus excelsior L. over most of the species' natural distribution area. We assessed crown damage from 2009 to 2014 (at ages 8, 11 and 13) in 43 open-pollinated ash families planted in north-eastern Zealand, Denmark, and confirmed the presence of substantial genetic variation in ash dieback susceptibility. The average crown damage increased in the trial from 61% in 2009 to 66% in 2012 and 72% in 2014, while the estimated heritability was 0.42 in both 2009 and 2012 but increased to 0.53 in 2014. Genetic correlation between assessments was 0.88 between 2009 and 2012 and 0.91 between 2009 and 2014, suggesting fairly good possibilities for early selection of superior genotypes in the presence of high infection levels in the trial. The level of crown damage had strong negative effect on growth and survival. Only 34% of the trees with high levels of damage in 2009 were still alive in 2014, emphasising that high susceptibility is associated with low fitness.     

Effects of benomyl and captan on growth and mycorrhizal colonization of Sitka‐spruce (Picea sitchensis) and ash (Fraxinus excelsior) in Irish nursery soil

The effects of two fungicides (benomyl and captan ‐ at recommended doses and up to three soil drenches) on root development and mycorrhizal colonization of Sitka‐spruce (Picea sitchensis) and ash (Fraxinus excelsior) were examined after 20 weeks growth, under environmentally controlled conditions, in soil from an Irish tree nursery. Although four mycorrhizal types have been found on Sitka‐spruce at the nursery, only one ectomycorrhizal type (Piceirhiza horti‐inflata) was identified on the short roots in this study. An inoculant (Vaminoc: MicroBio Ltd, Hemel Hempstead, UK) was used to inoculate ash and 20‐week‐old seedlings had a higher arbuscular mycorrhizal (AM) colonization in comparison with uninoculated controls. Multiple applications (2–3) of benomyl reduced the length of root and shoot and shoot dry mass of Sitka‐spruce, whereas in ash, it only depressed root length. Benomyl decreased the numbers of ectomycorrhizas of Sitka‐spruce and arbuscular mycorrhizal colonization of Vaminoc‐inoculated ash. A single application of captan stimulated root length and ectomycorrhizal colonization of Sitka‐spruce and root dry mass in ash compared with uninoculated controls. Applications of captan reduced arbuscular mycorrhizal colonization of Vaminoc‐inoculated ash to levels near to those of uninoculated controls. Of the two fungicides used, benomyl had the most deleterious effect on root length and mycorrhizal colonization of Sitka‐spruce and ash.