Detection of Chalara fraxinea from tissue of Fraxinus excelsior using species‐specific ITS primers

Chalara fraxinea (teleomorph: Hymenoscyphus albidus) is known as a serious pathogen of Fraxinus excelsior, causing massive dieback of trees in Europe. The fungus is able to cause latent infections, and has been previously detected as an endophyte in asymptomatic tissues. Chalara fraxinea is a slow grower in culture, and is thus likely to be overgrown by faster growing fungi whenever pure culture isolations are being attempted. This study reports species‐specific ITS primers allowing fast and reliable detection of the pathogen directly from infected tissues of F. excelsior.     

Natural infection of Fraxinus excelsior seeds by Chalara fraxinea

The occurrence of Chalara fraxinea, the fungus responsible for dieback of European ash (Fraxinus excelsior), was investigated in the current and previous seed years collected from symptomatic trees in Latvia and Sweden using molecular techniques (DNA extraction, ITS‐PCR, Sanger sequencing). Molecular analysis of seeds revealed the presence of 30 different fungal taxa. Chalara fraxinea was detected in 8.3% of seeds tested from the current year originating from Latvia. The presence of C. fraxinea in seeds of F. excelsior is of great concern to phytosanitary protection authorities in countries outside the current zone of infestation.     

First record of Chalara fraxinea in Finland and genetic variation among isolates sampled from Åland,mainland Finland,Estonia and Latvia

We have isolated and confirmed the identification of ash dieback fungus (Chalara fraxinea, teleomorph Hymenoscyphus albidus) for the first time in Finland. In a preliminary analysis, considerable amount of genetic variation was detected among 20 Finnish (Åland and mainland Finland), one Latvian and 11 Estonian isolates, analysed by random amplified microsatellite (RAMS) markers.     

The teleomorph of Chalara fraxinea,the causal agent of ash dieback

Ash dieback, caused by the pathogen Chalara fraxinea, is an emerging lethal disease of Fraxinus excelsior, threatening the host species in large parts of Europe. The ascomycete Hymenoscyphus albidus (Helotiaceae, Helotiales) was identified as the teleomorph of C. fraxinea by culturing from ascospores, morphological comparison and nuclear ribosomal internal transcribed spacer (ITS) sequencing.     

Temperature effect on Chalara fraxinea: heat treatment of saplings as a possible disease control method

Ash dieback is an emerging disease caused by the fungus Chalara fraxinea that severely affects Fraxinus excelsior and F. angustifolia stands in Europe. Previous studies have shown that this pathogen prefers temperatures around 20°C, while its growth in pure cultures at 30°C proved to be very limited. The purpose of this study was to determine the effects of temperature on the development and growth of C. fraxinea in pure cultures and in plant tissues, as well as to test the heat tolerance of F. excelsior saplings. The sensitivity of fungus to heat in ash tissues was higher than in pure cultures. Low isolation success rate from diseased ash tissue after a five‐hour hot water treatment at 36°C and the relatively high survival rate of ash saplings after hot water treatments at 36°C and 40°C indicate possibilities for the development of a C. fraxinea eradication method in ash saplings. Field monitoring showed that in hot weather periods, thermal conditions inside the ash tissues can be extreme enough to markedly decrease the viability of C. fraxinea in infected plant tissues.     

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.     

Occurrence and pathogenicity of fungi in necrotic and non-symptomatic shoots of declining common ash (<Emphasis Type="Italic">Fraxinus excelsior</Emphasis>) in Sweden

Currently, massive dieback of Fraxinus excelsior is observed in countries of eastern, northern and central Europe, and the reasons for it are unclear. The aims of the present work were (a) to study fungal communities in declining F. excelsior crowns; (b) to clarify role of fungi in the decline. Shoots from symptomatic crowns were collected in four localities in central Sweden, and distributed into the following categories: (a) visually healthy; (b) initial necroses; (c) advanced necroses; (c) dead tops. The most frequently isolated fungi were Gibberella avenacea, Alternaria alternata, Epicoccum nigrum, Botryosphaeria stevensii, Valsa sp., Lewia sp., Aureobasidium pullulans and Phomopsis sp., and these taxa were consistently found in shoots of all four symptomatic categories. Forty-eight taxa of other fungi were isolated, and fungal diversity was not exhausted by the sampling effort. The same taxa of fungi were dominant in F. excelsior shoots of different symptomatic categories, and moderate to high similarity of fungal communities was observed in shoots despite the symptoms. Forty-four isolates from 24 fungal taxa were used for artificial inoculations of 277 1-year-old F. excelsior seedlings in bare root nursery. After 2 years, only four fungi caused symptomatic necroses of bark and cambium: A. alternata, E. nigrum, Chalara fraxinea and Phomopsis sp. The most pathogenic was C. fraxinea, inducing symptoms on 50% of inoculated trees, while three other fungi caused necroses on 3–17% of inoculated trees. Infection biology of C. fraxinea and environmental factors determining susceptibility of F. excelsior to decline deserve future investigations.     

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.     

Studies on insects and mites causing galls on the leaves of common ash <Emphasis Type="Italic">Fraxinus excelsior</Emphasis> L. in the Ojców National Park in Poland

The occurrence frequency of galls caused by insects and mites on the leaves of Fraxinus excelsior L. was investigated, based on leaf samples collected in the Ojców National Park in 2000?–?2001. The species composition of galls was identical in two consecutive years. Galls were caused by 5 species a total of 2881 galls. The most numerous species was Dasineura fraxinea (Kieffer) in both years.Index of occurrence frequency and Agrell's index of species co-existence have been calculated. This index reached the highest values in case of Dasineura fraxinea and Vasates fraxini (Nalepa) in 2000 as well as D. fraxinea and Psyllopsis fraxini (L.) in 2001.     

Isolation,identification and necrotic activity of viridiol from Chalara fraxinea,the fungus responsible for dieback of ash

Investigation of secondary metabolite production of Chalara fraxinea, the fungus responsible for dieback of ash, led to the isolation of the phytotoxin viridiol and the fungistatic compound viridin. Viridiol showed necrotic activity towards ash‐seedlings.     

A molecular toolkit for population genetic investigations of the ash dieback pathogen Hymenoscyphus pseudoalbidus

The recently described ascomycete fungus Hymenoscyphus pseudoalbidus (anamorph: Chalara fraxinea) causes the current dieback of ash (Fraxinus excelsior) in large parts of Europe. The origin of this species and its relation to the native cryptic species Hymenoscyphus albidus are still enigmatic. The spatiotemporal pattern of the epidemic is typical for an introduced invasive species. However, the presence of two cryptic species indicates that hybridization or mutation might have been involved in driving speciation in this case. In this study, we present a set of 18 polymorphic microsatellite markers to study these processes in more detail on a population genetic level. Markers were designed such that they can be amplified in three individual multiplex PCRs and analysed in two fragment analysis runs. We thoroughly tested the marker set for pairwise linkage among loci, selective neutrality and Mendelian inheritance. Additionally, the markers were applied on two large collections of isolates derived from study sites in Germany. Population genetic calculations suggested a low yet significant level of differentiation, a large genotypic diversity and a limited genetic diversity within populations. Furthermore, we present additional data concerning the phylogenetic relation between H. albidus and H. pseudoalbidus, which seems to be more distantly related to each other than expected previously.     

Mycological and histological investigations of Fraxinus excelsior nursery saplings naturally infected by Chalara fraxinea

Ash dieback is an emerging disease of Fraxinus excelsior in Germany. To date, economical damage is significant in nurseries, which also contribute towards spread of the disease, but damage to forests is increasing. The study presents the results of mycological and histological investigations on three hundred 3‐year‐old nursery ash saplings. The infection rate by the causative pathogen was determined for bark, outer and inner xylem, the pith and also separately for the above‐ground portion and root system of the plants. The invasion and colonization strategy of the fungus in the woody stem was examined. In addition, the presence of soil‐borne Oomycetes as possible primary or accompanying causal organisms was investigated. The results verify the dominant role of Chalara fraxinea as a causal agent of ash dieback and rule out the role of Oomycetes in the disease process. We conclude that C. fraxinea is not primarily endophytic in nature and spreads very effectively in the central stem tissues, which enables colonization of the woody stem in all three dimensions. Infections arising in the upper part of plants can thus spread extensively to lower parts.     

Hymenoscyphus albidus is not associated with an anamorphic stage and displays slower growth than Hymenoscyphus pseudoalbidus on agar media

Examination of isolates of Hymenoscyphus albidus from France revealed that this fungus does not form an anamorphic stage in culture. The lack of an asexual stage in this fungus is a conspicuous morphological difference to the ash dieback pathogen Hymenoscyphus pseudoalbidus, which is associated with its Chalara fraxinea anamorphic state. In growth studies on malt extract agar (MEA) and MEA amended with ash leaflets (ash leaf malt extract agar, AMEA) at 20°C, isolates of H. albidus grew slower than those of H. pseudoalbidus. On AMEA, the growth of cultures of both species was greatly enhanced.     

Zur Verbreitung und Schädlichkeit der Gallmücken (Cecidomyiidae,Diptera) an Waldbäumen in Mitteleuropa

Zusammenfassung In 3 Staaten Mitteleuropas, Tschechien, Slowakei und Österreich wurde die Vorkommensfrequenz von Gallmückenarten, die sich an Waldbäumen entwickeln, an mehr als 1200 Fundorten untersucht. Insgesamt wurden 44 Gallmückenarten an 20 Wirtspflanzen als schädlich erkannt (Tab. 1). Alle festgestellten Gallmückenarten wurden nach ihrer Gesamtvorkommensfrequenz bewertet (Tab. 2). Die Gallmückenarten können erhebliche Schäden verursachen, wenn ihre Larven wichtige Pflanzenorgane, z. B. Blütenknospen oder junge Blattknospen, befallen, oder eine wesentliche Verminderung der Assimilationsfläche verursachen. Unter den 44 Gallmückenarten von Waldbäumen treten nur 8 Arten als wirkliche Schädlinge auf:Contarinia fagi an jungen Blattknospen vonFagus sylvatica, Contarinia quercina an jungen Blattknospen vonQuercus robur undQ. petraea (beide Arten vornehmlich in Forstgärten),Thecodiplosis brachyntera an Nadeln vonPinus sylvestris undP. mugo, Harrisomyia vitrina undDrisina glutinosa an Blättern vonAcer pseudoplatanus, Dasineura fraxini undD. fraxinea an Fiederblättchen vonFraxinus excelsior sowieMikiola fagi an Blättern vonFagus sylvatica.

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On the occurrence and harmfulness of gall midges (Cecidomyiidae, Diptera) on forest trees

The occurrence of 44 gall midge species developing on 20 forest trees was investigated at more than 1200 localities in the territory of 3 countries in Central Europe, viz. in Czech Republic (670 localities), in Slovak Republic (340 localities) and in Austria (230 localities) (see fig. 1 and table 1). The evaluation of frequency of gall midges in the territory of Central Europe (table 2) showed that the most abundant gall midge species cannot be always the most serious pests of forest trees. Gall midge species change into pests if their larvae destroy important plant organs (e. g. flower or leaf buds), or ential considerable reducing of assimilation surface, mainly together with the significantly increasing of their occurrence. The following gall midge species have been evaluated as serious pests of forest trees in Central Europe:Contarinia fagi onFagus sylvatica, Contarinia quercina onQuercus robur andQ. petraea (mainly in nurseries),Thecodiplosis brachyntera onPinus mugo andP. sylvestris, Harrisomyia vitrina andDrisina glutinosa onAcer pseudoplatanus, Dasineura fraxini andD. fraxinea onFraxinus excelsior, andMikiola fagi onFagus sylvatica. The fig. 2 gives the relative occurrence (dominance) of gall midge species in 3 Central-European countries.

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Mit 2 Abbildungen und 2 Tabellen     

Cryptic speciation in Hymenoscyphus albidus

Ash dieback caused by the mitosporic ascomycete Chalara fraxinea is a novel disease of major concern affecting Fraxinus excelsior and Fraxinus angustifolia in large parts of Europe. Recently, its teleomorph was detected and assigned to Hymenoscyphus albidus, which has been known from Europe since 1851. In this study, we present molecular evidence for the existence of two morphologically very similar taxa, H. albidus, which is lectotypified and Hymenoscyphus pseudoalbidus sp. nov. Differences were found between the species in the loci calmodulin, translation elongation factor 1‐α and the internal transcribed spacers of the rDNA genes, and strong differentiation was obtained with ISSR markers. It is likely that H. albidus is a non‐pathogenic species, whereas H. pseudoalbidus is a virulent species causing ash dieback. Genotyping herbarium specimens showed that H. pseudoalbidus has been present in Switzerland for at least 30 years prior to the outbreak of the epidemic.     

Efficacy of fungicides on Hymenoscyphus fraxineus and their potential for control of ash dieback in forest nurseries

Twenty‐six commercial formulations of fungicides at six concentrations were evaluated in vitro for their efficacy on mycelial growth of Hymenoscyphus fraxineus (anamorph Chalara fraxinea). The results are presented as EC₅₀, EC₉₀ and minimal inhibitory concentration values; the comparisons with the recommended application concentrations showed that 10 of the 26 fungicides were highly effective in their ability to inhibit the mycelial growth of the pathogen. The eight most effective fungicides identified based on multiple comparisons analysis were azoxystrobin, bitertanol, captan (in two‐component preparation with trifloxystrobin), difenoconazole, kresoxim‐methyl, spiroxamine (in multicomponent preparation with tebuconazole and triadimenol), tebuconazole and trifloxystrobin. Azoxystrobin, difenoconazole, kresoxim‐methyl, mancozeb, myclobutanil, pyrimethanil, tebuconazole and trifloxystrobin were selected to verify their effectiveness in in planta tests. During two field tests in nurseries, it was found out that tebuconazole (triazole), trifloxystrobin and kresoxim‐methyl (strobilurins) and mancozeb (dithiocarbamate) were significantly effective against H. fraxineus. The best results were gained with tebuconazole, when mean percentage of diseased saplings was 16.3%, whereas in the control, it was 63.6%. The combination and alternation of fungicides from the triazole, strobilurine and dithiocarbamate chemical groups should be an effective tool for protecting ash saplings in forest nurseries. Moreover, exact timing of the treatment reflecting rainfall and development of first symptoms in the foliage will be also important.     

Genetic variation in dieback resistance in Fraxinus excelsior confirmed by progeny inoculation assay

Ash dieback caused by the pathogenic fungus Hymenoscyphus fraxineus [previously known as H. pseudoalbidus (sexual stage) and Chalara fraxinea (asexual stage)] is a widespread problem in Europe. Here, we assess crown damage from natural infection and necrosis development following artificial controlled inoculations on full‐sib and half‐sib progeny from Danish Fraxinus excelsior clones with contrasting and well‐characterized levels of susceptibility to the disease. The inoculation assay was performed on a total of 123 offspring, and necrosis development monitored over two years. The offspring from low susceptible mother clones developed smaller necroses when compared to offspring from susceptible clones. Their crown damage due to natural infections was also significantly less. The correlation coefficient between average crown damages of mother clones and the average of their progeny was 0.85 (natural infections), while the correlation between crown damage of mother clones and the average necrosis development in their progeny after controlled inoculation was 0.73. The correlation between resistance of parent trees and crown damage/necrosis development on their offspring confirms the presence of heritable resistance and indicates that a bioassay based on controlled inoculations has the potential of becoming a fast and cost‐effective tool for estimation of dieback susceptibility in breeding programmes for resistance in ash trees.     

Durability evaluation of particleboards by accelerated aging tests

Summary Specimens of four commercially produced particleboards were exposed to various accelerated aging treatments: 1) 6-cycle ASTM D-1037 exposure, 2) 6-cycle WCAMA exposure, 3) BS 5669 test, 4) cyclic soak-dry test, and 5) cyclic boil-dry test. Thickness swelling (TS) and internal bond strength (IB) were measured after each step of each cycle. Phenol and phenol (face)/isocyanate (core)bonded boards are more durable than urea and melamine urea bonded boards. The effect of treatment 1) is most severe on TS and IB retention, followed in order by treatments 2), 5), 4) and 3). When the value of springback reached about 35%, IB became equal to zero.     

Genetic diversity of the myrtle rust pathogen (Austropuccinia psidii) in the Americas and Hawaii: Global implications for invasive threat assessments

Since the myrtle rust pathogen (Austropuccinia psidii) was first reported (as Puccinia psidii) in Brazil on guava (Psidium guajava) in 1884, it has been found infecting diverse myrtaceous species. Because A. psidii has recently spread rapidly worldwide with an extensive host range, genetic and genotypic diversities were evaluated within and among A. psidii populations in its putative native range and other areas of myrtle rust emergence in the Americas and Hawaii. Microsatellite markers revealed several unique multilocus genotypes (MLGs), which grouped isolates into nine distinct genetic clusters [C1–C9 comprising C1: from diverse hosts from Costa Rica, Jamaica, Mexico, Puerto Rico, and USA‐Hawaii, and USA‐California; C2: from eucalypts (Eucalyptus spp.) in Brazil/Uruguay and rose apple (Syzygium jambos) in Brazil; C3: from eucalypts in Brazil; C4: from diverse hosts in USA‐Florida; C5: from Java plum (Syzygium cumini) in Brazil; C6: from guava and Brazilian guava (Psidium guineense) in Brazil; C7: from pitanga (Eugenia uniflora) in Brazil; C8: from allspice (Pimenta dioica) in Jamaica and sweet flower (Myrrhinium atropurpureum) in Uruguay; C9: from jabuticaba (Myrciaria cauliflora) in Brazil]. The C1 cluster, which included a single MLG infecting diverse host in many geographic regions, and the closely related C4 cluster are considered as a “Pandemic biotype,” associated with myrtle rust emergence in Central America, the Caribbean, USA‐Florida, USA‐Hawaii, Australia, China‐Hainan, New Caledonia, Indonesia and Colombia. Based on 19 bioclimatic variables and documented occurrences of A. psidii contrasted with reduced sets of specific genetic clusters (subnetworks, considered as biotypes), maximum entropy bioclimatic modelling was used to predict geographic locations with suitable climate for A. psidii which are at risk from invasion. The genetic diversity of A. psidii throughout the Americas and Hawaii demonstrates the importance of recognizing biotypes when assessing the invasive threats posed by A. psidii around the globe.