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Hybridization and the resulting introgression can drive the success of invasive species via the rapid acquisition of adaptive traits. The Dutch elm disease pandemics in the past 100 years were caused by three fungal lineages with permeable reproductive barriers: Ophiostoma ulmi, Ophiostoma novo-ulmi subspecies novo-ulmi and Ophiostoma novo-ulmi subspecies americana. Using whole-genome sequences and growth phenotyping of a worldwide collection of isolates, we show that introgression has been the main driver of genomic diversity and that it impacted fitness-related traits. Introgressions contain genes involved in host–pathogen interactions and reproduction. Introgressed isolates have enhanced growth rate at high temperature and produce different necrosis sizes on an in vivo model for pathogenicity. In addition, lineages diverge in many pathogenicity-associated genes and exhibit differential mycelial growth in the presence of a proxy of a host defence compound, implying an important role of host trees in the molecular and functional differentiation of these pathogens. Genome analysis of fungi responsible for Dutch elm disease shows that introgression has contributed to genomic diversity and has impacted fitness-related traits in these pathogens.

Dutch elm disease (DED) is a vascular wilt disease caused by the pathogens Ophiostoma ulmi and Ophiostoma novo-ulmi with multiple ecological phases including pathogenic (xylem), saprotrophic (bark) and vector (beetle flight and beetle feeding wound) phases. Due to the two DED pandemics during the twentieth century the use of elms in landscape and forest restoration has declined significantly. However new initiatives for elm breeding and restoration are now underway in Europe and North America. Here we discuss complexities in the DED ‘system’ that can lead to unintended consequences during elm breeding and some of the wider options for obtaining durability or ‘field resistance’ in released material, including (1) the phenotypic plasticity of disease levels in resistant cultivars infected by O. novo-ulmi; (2) shortcomings in test methods when selecting for resistance; (3) the implications of rapid evolutionary changes in current O. novo-ulmi populations for the choice of pathogen inoculum when screening; (4) the possibility of using active resistance to the pathogen in the beetle feeding wound, and low attractiveness of elm cultivars to feeding beetles, in addition to resistance in the xylem; (5) the risk that genes from susceptible and exotic elms be introgressed into resistant cultivars; (6) risks posed by unintentional changes in the host microbiome; and (7) the biosecurity risks posed by resistant elm deployment. In addition, attention needs to be paid to the disease pressures within which resistant elms will be released. In the future, biotechnology may further enhance our understanding of the various resistance processes in elms and our potential to deploy trees with highly durable resistance in elm restoration. Hopefully the different elm resistance processes will prove to be largely under durable, additive, multigenic control. Elm breeding programmes cannot afford to get into the host–pathogen arms races that characterise some agricultural host–pathogen systems.

Humankind owes much to trees, given their major role in sequestering carbon and providing oxygen, sugars and much of the energy on which ourselves and terrestrial ecosystems depend. Trees and forests are important culturally, economically, environmentally and socially. And yet, despite this, trees throughout the world are currently facing an increasing number of serious challenges. On a global scale, Curtis et al. (2018) report most forest loss is due to commodity driven deforestation through permanent conversion to nonforest land uses including agriculture (e.g. palm oil production), energy production and mining. The other main drivers of global forest loss, that might instead be considered less permanent and associated with subsequent regrowth, include forestry, shifting agriculture and wildfire. Additional threats to forests include those posed by climate change, and invasive biotic agents such as insect pests and pathogens. Most new tree disease outbreaks are due to introduction events, with a potential pathogen introduced from their endemic centres of origin (where they generally cause little or no disease on their plant host due to long-term coevolution) into a new geographic location, in which a naive host has not previously been exposed and can thus be highly susceptible. The incidence of such 'new encounter' diseases is increasing at an unprecedented rate due to globalisation with increased international trade in plants and travel, a scenario potentially exacerbated by a changing climate better suited to establishment of a pathogen once introduced. Identification of the centres of origin of fungal pathogens can be important for several reasons. First, given that the original host-fungus interaction will have typically stabilised over long periods of time, such geographic regions could be useful sources of host genetic resistance. Moreover, the longer time periods involved will have likely resulted in greater genetic diversity accruing in such endemic populations. More diverse pathogen populations have greater evolutionary potential, with increased genetic variation available for response to environmental change. This could enable host tolerance to be overcome, unexpected 'jumps' onto new hosts, increased risk of fungicide resistance, and better adaptability to changing environmental conditions (e.g. temperature). Thus, strategies to reduce introduction of additional genetic variation from source to sink regions can reduce tree health threats. In this article, such introduction events are considered in the context of three devastating tree diseases, namely ash dieback, Dutch Elm Disease (DED) and Dothistroma Needle Blight (DNB, mainly on pine). On all these tree hosts, multiple closely-related fungal species have now been associated with each of these different diseases. Such related species are often morphologically very similar or even indistinguishable by eye, and consequently this can result in taxonomic confusion and species misidentification, leading to delayed diagnosis of the true causal agent of a given disease outbreak. Research into such related species is important as they might pose very different plant health threats that require distinct disease management strategies. These differences might relate to pathogenicity, geographic distribution, host range, effectiveness of host resistance, sensitivity to fungicides, temperature optima, reproductive strategy and so on. Furthermore, when related fungal species come into physical contact with each other after a long period of separation, for instance via an introduction event, various outcomes are possible including: (1) replacement (and possible extinction) of one species by the other; (2) coexistence of the species; or (3) cross-species hybridisation. The remainder of this article focuses, using three major tree disease case histories, on how fundamental research on pathogen biology can provide new insights into the genetic structure of related pathogen populations that can be usefully applied to reduce the threat to tree health posed by invasive fungal species.

Ophiostoma spp. (Ophiostomatales, Ascomycota) are well-known fungi associated with bark and ambrosia beetles (Curculionidae: Scolytinae, Platypodinae). Fungi in the Ophiostomatales include serious tree pathogens as well as agents of timber blue-stain. Although these fungi have been extensively studied in the northern hemisphere, very little is known regarding their occurrence on hardwoods in Europe. The aims of the present study were to identify and characterize new Ophiostoma spp. associated with bark and ambrosia beetles infesting hardwoods in Norway and Poland, and to resolve phylogenetic relationships of Ophiostoma spp. related to the Norwegian and Polish isolates, using multigene phylogenetic analyses. Results obtained from five gene regions (ITS, LSU, β-tubulin, calmodulin, translation elongation factor 1-α) revealed four new Ophiostoma spp. These include Ophiostoma hylesinum sp. nov., O. signatum sp. nov., and O. villosum sp. nov. that phylogenetically are positioned within the Ophiostoma ulmi complex. The other new species, Ophiostoma pseudokarelicum sp. nov. reside along with Ophiostoma karelicum in a discrete, well-supported phylogenetic group in Ophiostoma s. stricto. The results of this study clearly show that the diversity and ecology of Ophiostoma spp. on hardwoods in Europe is poorly understood and that further studies are required to enrich our knowledge about these fungi.

Several inhibitors of polyamine biosynthesis reduced mycelial growth of the fungus Ophiostoma ulmi (Buism.) Nannf. cultured on malt extract-agar medium. Growth inhibition was particularly evident when expressed in terms of fresh weight rather than colony diameter. Of the different drugs tested, the most effective was difluoromethylarginine (DFMA), while difluoromethylornithine (DFMO) and methylglyoxal(bis)guanyl-hydrazone (MGBG) plus cyclohexylamine (CHA) reduced growth only by 75 and 62% respectively. The growth inhibition by DFMO + DFMA, measured as colony diameter, was apparently reversed by putrescine (PUT), but not when expressed in terms of fresh weight. Spermidine (SPD) was the most abundant polyamine in control cultures, followed by PUT and spermine (SPM). PUT was no longer detectable 8 d after inoculation. On day 10, DFMO and DFMA, alone and in combination, caused a significant reduction in cellular SPD concentrations, while exogenously supplied PUT restored the levels of this polyamine to control values. MGBG + CHA caused a conspicuous accumulation of PUT and an approximately 50% reduction in SPD titres. DFMA, alone or in combination with DFMO and with or without PUT, led to increased cellular levels of SPM. The latter polyamine, but not PUT or SPD, strongly retarded growth when added to the growth medium. As suggested by the effectiveness of DFMA in inhibiting growth, arginine decarboxylase activity was shown to be prevalent over ornithine decarboxylase activity in this fungus.

Dutch elm disease (DED) is a vascular wilt disease of elms caused by ascomycetous fungi of the genus Ophiostoma. The initial DED pandemic was caused by Ophiostoma ulmi, a relatively weak pathogen when compared to Ophiostoma novo- ulmi, the causal agent of the current pandemic. The latter has been spreading as two distinct subspecies: O. novo-ulmi ssp. novo-ulmi and O. novo-ulmi ssp. americana. Weak prezygotic barriers to hybridization have resulted in the emergence of ssp. novo-ulmi × ssp. americana hybrids in subspecies overlap zones. In this study, samples of host species Ulmus laevis, Ulmus minor and Ulmus glabra from across Croatia were analysed for DED prevalence and pathogen distribution and characterization. Dutch elm disease was confirmed in all three elm species, but the prevalence of the infection was approximately four times higher in U. minor and U. glabra than in U. laevis. Ophiostoma novo-ulmi was identified as the only cause of DED, while O. ulmi was not detected, indicating complete replacement of O. ulmi by O. novo-ulmi in Croatia. PCR–RFLP analysis and sequencing of the cu and col1 genes indicate the presence of both O. novo-ulmi subspecies as well as their hybrids at investigated sites, often in high frequency. The results provide insight into distribution and population structure of Dutch elm disease pathogen in Croatia, as well as an assessment of virulence for selected isolates, which could be utilized for making informed management decisions regarding DED and elm conservation in Croatian forests.

Ulmus americana (American elm) was an important urban tree in North America prior to the introduction of the Dutch elm disease (Ophiostoma ulmi and O. novo-ulmi) pathogen in 1930. Subsequently, urban and community forests were devastated by the loss of the large canopies. Disease tolerant elms (Ulmus spp.) of various species were later developed; however, consumer acceptance of these trees was slow. The goal of the National Elm Trial was to evaluate newer cultivars of elm across the United States. Trees were monitored for survival and growth for 7 to 10 years at 16 different locations. 'Morton' elm had >90% survival, while 13 cultivars averaged 70 to 90%, and five cultivars ranged from 25 to 69% survival. Trunk diameter growth by location ranged from 0.5 cm year-1 at Colorado to more than 2.0 cm year-1 at Iowa. By taxa, trunk diameter growth ranged from a low of 'JFS Bieberich' elm (0.7 cm year-1) to a high of 'New Horizon' elm (1.7 cm year-1). The fastest height growth was found with the American elm cultivars 'New Harmony' and 'Princeton' (0.63 and 0.61 m year-1, respectively).

The Dutch elm disease (DED) pathogens, Ophiostoma ulmi (Buisman) Nannf. and the more aggressive Ophiostoma novo-ulmi Brasier, have decimated European elm populations in the last 100 years. Today, the number of tolerant elm varieties available on the market is limited, partly due to the long breeding cycles and expensive facilities they require. Developing a low-cost technique to allow early screening of elm tolerance based on simple morphological and/or biochemical traits would considerably boost elm breeding and research. Within this general aim, we developed an in vitro plant culture system to (i) characterize stress responses to O. novo-ulmi-root inoculation in two Ulmus minor Mill. clones of contrasting susceptibility level to DED (termed ‘tolerant’ and ‘susceptible’) and (ii) compare the upward dispersal rate of the pathogen in the two clones. Constitutive xylem anatomy was similar in both clones, indicating that differences in plant responses to the pathogen are not attributable to anatomical factors (e.g., conduit size). Susceptible plantlets suffered a significant delay in apical growth and a decrease in chlorophyll content at 21 days post-inoculation (dpi). The rate of pathogen dispersal from roots to aerial tissues was similar in both clones. However, the tolerant clone showed a marked increase in lipid peroxidation at 1 dpi, while the susceptible clone showed enhanced values of lipid peroxidation during most of the experimental period (1–21 dpi). Despite wide stem colonization by the pathogen, the tolerant clone effectively regulated the oxidative stress levels and showed remarkable resilience to inoculation. These results extend current knowledge on elm defense mechanisms, and the proposed in vitro plant culture system emerges as a promising early screening method for tolerance to improve elm breeding.

Dutch elm disease (DED), caused by the fungi Ophiostoma ulmi and O. novo-ulmi, has reduced elm populations severely in Europe and North America. Breeding programmes are in action to find less susceptible elm varieties suitable for re-establishing elm stands. Bark beetles, mainly Scolytus spp., are the only known natural vectors of DED. During twig feeding, beetles transfer Ophiostoma spores to healthy elms. Thus, less palatable elms should run a lower risk of DED infections. In feeding preference bioassays, we offered twigs from elms exhibiting different degree of susceptibility to O. novo-ulmi, together with non-host trees to Scolytus beetles. Scolytus multistriatus preferred wych elm, Ulmus glabra, to 100% in two-choice tests, whereas S. laevis did not discriminate between a tolerant and a susceptible variety of field elm, U. minor. We suggest that the feeding assay is useful as a low-tech method in breeding programmes for evaluating the suitability of promising elm genotypes to vector insects. (Less)

Dutch elm disease (DED) is caused by the dimorphic fungi Ophiostoma ulmi, Ophiostoma novo-ulmi, and Ophiostoma himal-ulmi. A cell population density-dependent phenomenon related to quorum sensing was previously shown to affect the reversible transition from yeast-like to mycelial growth in liquid shake cultures of O. novo-ulmi NRRL 6404. Since the response to external stimuli often varies among DED fungal strains, we evaluated the effect of inoculum size on 8 strains of the 3 species of DED agents by determining the proportion of yeast and mycelium produced at different spore inoculum concentrations in defined liquid shake medium. The results show that not all DED fungi strains respond similarly to inoculum size effect, since variations were observed among strains. It is thus possible that the different strains belonging to phylogenetically close species use different signalling molecules or molecular signalling pathways to regulate their growth mode via quorum-sensing mechanisms.

In central Europe, the endangered Rosalia longicorn Rosalia alpina (L.) has rarely been observed to develop in the wood of elms Ulmus sp.; however, this beetle has recently expanded into habitats formed by elms. The factors driving these changes are unknown in detail, but a potential one is the increasing amount of breeding material, namely, dead elm wood. Dutch elm disease, caused by the fungi Ophiostoma ulmi and O. novo-ulmi, by killing off elm trees, has created a supply of dead wood. The aim of this work was to assess the potential role of Dutch elm disease in the formation of habitats suitable for the endangered Rosalia longicorn. The health of four elm stands (Carpathians, SE Poland) was analysed and the pathogen responsible for elm death were determined by DNA sequence comparison. O. novo-ulmi — the causative agent of Dutch elm disease — was isolated from weakened and dying trees at all studied localities. In total, 30% of the elm trees were dead, 34% showed signs of dieback or were moribu...

During the last decades, the development of ever more powerful genetic, molecular and omic approaches has provided plant pathologists with a wide array of experimental tools for elucidating the intricacies of plant-pathogen interactions and proposing new control strategies. In the case of the Dutch elm disease (DED) pathosystem, these tools have been applied for advancing knowledge of the host (Ulmus spp.) and the causal agents (Ophiostoma ulmi, O. novo-ulmi and O. himal-ulmi). Genetic and molecular analyses have led to the identification, cloning and characterization of a few genes that contribute to parasitic fitness in the pathogens. Quantitative PCR and high-throughput methods, such as expressed sequence tag analysis, have been used for measuring gene expression and identifying subsets of elm genes that are differentially expressed in the presence of O. novo-ulmi. These analyses have also helped identify genes that were differentially expressed in DED fungi grown under defined experimental conditions. Until recently, however, functional analysis of the DED fungi was hampered by the lack of protocols for efficient gene knockout and by the unavailability of a full genome sequence. While the selective inactivation of Ophiostoma genes by insertional mutagenesis remains a challenge, an alternative approach based on RNA interference is now available for down-regulating the expression of targeted genes. In 2013, the genome sequences of O. ulmi and O. novo-ulmi were publicly released. The ongoing annotation of these genomes should spark a new wave of interest in the DED pathosystem, as it should lead to the formal identification of genes modulating parasitic fitness. A better understanding of DED, however, also requires that omic approaches are applied to the study of the other biotic components of this pathosystem.

Bark beetles of the genus Scolytus Geoffroy are the main vectors of the fungus Ophiostoma ulmi s.l., which causes the Dutch elm disease. The large and small elm bark beetles - S. scolytus (F.) and S. multistriatus (Marsham), respectively - are the most common and important species spreading the pathogen worldwide. The success of the pathogen-insect interactions is mainly due to the characteristic reproductive behavior of the elm bark beetles, which, however, largely depends on the occurrence of infected trees. During feeding activity on elm twigs, callow adults carrying pathogen conidia on their bodies contaminate healthy trees and facilitate pathogen development and movement within the wood vessels. Infected trees become then suitable for insect breeding in the stem bark. This well-known mutualistic association has devastating consequences for elm survival. Although much is known about insect-pathogen interactions and transmission mechanisms, many topics still deserve additional attention, as, for example, beetle systematic based on new molecular tools and morphological characters; selection of European elm clones based on disease avoidance; consequences of global warming on life-history of the three organisms (fungus-insect-tree) involved in the pathosystem; new problems resulting from the rapid increase of international trade among continents, leading to the accidental introduction of new vector species or new pathogen species or races, or to the introduction of new highly susceptible elm species in gardens and public parks. A holistic approach to tackle the problem is highly recommended, taking into account how these organisms interact with each other and the environment, and how their interactions could be modified in order to face one of the most destructive diseases ever known in plant pathology.

Ophiostomatoid fungi are vectored by their bark-beetle associates and colonize different host tree species. To survive and proliferate in the host, they have evolved mechanisms for detoxification and elimination of host defence compounds, efficient nutrient sequestration, and, in pathogenic species, virulence towards plants. Here, we assembled a draft genome of the spruce pathogen Ophiostoma bicolor. For our comparative and phylogenetic analyses, we mined the genomes of closely related species (Ophiostoma piceae, Ophiostoma ulmi, Ophiostoma novo-ulmi, and Grosmannia clavigera). Our aim was to acquire a genomic and evolutionary perspective of gene families important in host colonization. Genome comparisons showed that both the nuclear and mitochondrial genomes in our assembly were largely complete. Our O. bicolor 25.3 Mbp draft genome had 10 018 predicted genes, 6041 proteins with gene ontology (GO) annotation, 269 carbohydrate-active enzymes (CAZymes), 559 peptidases and inhibitors, and 1373 genes likely involved in pathogen-host interactions. Phylogenetic analyses of selected protein families revealed core sets of cytochrome P450 genes, ABC transporters and backbone genes involved in secondary metabolite (SM) biosynthesis (polyketide synthases (PKS) and non-ribosomal synthases), and species-specific gene losses and duplications. Phylogenetic analyses of protein families of interest provided insight into evolutionary adaptations to host biochemistry in ophiostomatoid fungi.

Dutch elm disease (DED) is a fungal disease that causes high mortality of elms. DED and its vector beetles are widely present in most of the countries in the Northern Hemisphere, but they are not known to be present in Finland. DED is a major risk to plant health in Finland. DED and its vectors are moderately likely to enter Finland by natural spread aided by hitchhiking, because they are present in areas close to Finland. Entry via other pathways is much less likely, mainly due to the low volume of trade of untreated wood and plants for planting. DED and its vectors could likely establish in the southern parts of the country, since they currently occur in similar climatic conditions in other countries. DED could cause massive environmental damage as natural elm groves are critically endangered habitats in Finland. The economic consequences to the owners of mature elms could also be significant. Eradication or containment of DED could be possible if strict measures were taken as the patchy distribution of elms would limit the spread of the disease. The most important source of uncertainty in this assessment is the lack of information regarding the amount of elm in fuel wood, wood waste and wood chips imported to Finland.

Aims: For Ophiostoma (Ceratocystis) ulmi, the ability to undergo morphological change is a crucial factor for its virulence. To gain an understanding of quorum-sensing activity in O. ulmi as it relates to yeast-mycelium dimorphism control, this study examines the effects of branched-chain amino acids as well as their fusel alcohols and fusel acids as quorum sensing molecules. Methods and Results: In a defined medium containing glucose, proline and salts, O. ulmi grew as yeasts when the culture was inoculated with a high density of spores (2 × 107 CFU ml−1) and as mycelia when inoculated with a low spore density (4 × 105 CFU ml−1). The cultures displaying yeast morphology secreted a quorum-sensing factor that shifted the morphology from mycelia to yeast. This quorum-sensing molecule was lipophilic and extractable by organic solvents from the spent medium. Using GC/MS analysis, it was determined that the major compound in the extract was 2-methyl-1-butanol. A similar effect was observed when the branched-chain amino acids (fusel alcohol precursors) were used as the nitrogen source. E, E-farnesol had no effect on the morphology of O. ulmi. Conclusions: Addition of the branched-chain amino acids or one of the compounds detected in the spent medium, 2-methyl-1-butanol or 4-hydroxyphenylacetic acid, or methylvaleric acid, decreased germ tube formation by more than 50%, thus demonstrating a quorum sensing molecule behaviour in O. ulmi cultures. Significance and impact of the study: This study presents advances in the investigation of dimorphism in O. ulmi, complementing the existing scientific basis, for studying, understanding and controlling this phenomenon.

Conventionally hybrid elm clones obtained within breeding programs for Dutch elm disease (DED) resistance were selected to meet requirements for use as ornamentals. However, it has been long and commonly observed that these clones may show hybrid vigour and enhanced growth. Nowadays DED resistant hybrid elm clones, which have been released to the market or are under evaluation for an upcoming release, are numerous enough to be considered for timber production or short rotation coppice (SRC). But experimental testing of the growth performances of these clones in different environments is still lacking. In this paper, growth and stability of performance of 24 DED resistant hybrid elm clones planted at three experimental sites with contrasting environmental conditions in Italy were studied. Height and diameter were measured yearly from 2001 to 2009, and the mean yearly increments after plant establishment were calculated. The study revealed a general good growth performance of the majority of the clones with mean height increments above 1 m/year, and an excellent growth performance of some genotypes. Analysis of variance showed significant effects of clone, site and clone × site interaction, for both height and diameter increments. Stability analysis of diameter and height increments was performed by using two parametric (CV% and W2) and two non-parametric (Huhn's Si(1) and Si(2)) indexes. According to all indexes, two clones showed superior and stable growth. These clones may be suitable for planting in a range of environments. In addition, several other clones had high growth in general or at a particular site. The results support our belief that these elm clones could be successfully used for timber and biomass production, and provide new knowledge for an informed choice of the most suitable genotypes.

Molecular mechanisms underlying the interaction leading to Dutch elm disease were studied in vitro using Ulmus americana L. callus culture inoculated with budding cells of the fungal pathogen Ophiostoma novo-ulmi (Brasier). An interaction cDNA library employing suppression subtractive hybridization was constructed from infected elm callus tissue 72 h post-inoculation. Five hundred and thirty-five expressed sequence tags, mostly from the host, were grouped into 314 unisequences and distributed into functional categories. After differential screening, 53 U. americana unisequences were considered upregulated during the interaction. The expression profiles at six time points of a subset of 18 elm transcripts were analyzed in more detail by quantitative reverse-transcriptase polymerase chain reaction. Upregulated sequences included transcripts with sequence similarity to genes coding for different classes of pathogenesis-related proteins and enzymes belonging to different branches of the phenylpropanoid pathway. The possible association with compartmentalization-related compounds and phytoalexin production is discussed. This study provides, for the first time, snapshots of molecular mechanisms involved in the compatible interaction between U. americana and O. novo-ulmi. The interaction library dataset also represents a valuable genomic resource for the highly appreciated urban tree U. americana.

Summary The intact yeast phase spores of Ophiostoma ulmi and O. novo-ulmi isolates were analysed by matrix-assisted-laser-desorption/ionization time-of-flight/time-of-flight mass spectrometry. The results clearly showed that isolates of O. ulmi and O. novo-ulmi have different mass peak profiles. The specific mass in m/z may vary slightly. One of the peaks from m/z 7623 to 7630 dominated (in high intensity) mass spectra for all isolates of O. novo-ulmi tested in this study. This specific peak is believed to be CU (cerato-ulmi) protein. However, no specific m/z peak can be found to distinguish between the isolates of ssp. novo-ulmi and ssp. americana. On the contrary, all isolates of O. ulmi tested possessed one of a dominate peak at m/z 7337 to 7342, but none around m/z 7626 as shown in O. novo-ulmi isolates.

European elms (Ulmus glabra Huds., Ulmus laevis Pall. and Ulmus minor Mill.) have been severely damaged and are still endangered by an alien hypervirulent pathogen, Ophiostoma ulmi s. l., agent of the Dutch elm disease (DED). Consequently, several ex situ clone collections have been established throughout Europe for elm breeding and conservation. In this paper we summarise the studies carried out within the EU project RESGEN CT96-78, which launched the EU-coordinated evaluation of these collections. Our aim was to analyse the variation in timing of bud burst and to acquire basic knowledge on the environmental control of this adaptive trait in European elms, under the hypothesis that DED susceptibility is related to spring phenology. The variation in the bud burst date of European elms among collections and years was explained by a phenological model assuming that the thermal time required for bud burst decreases exponentially with increasing chilling during winter down to a level where chilling requirement is fully met. According to the fitted curves, European elms have low dormancy and short chilling requirement for dormancy release. Although no simulation was performed, on the basis of the model applied we can hypothesise that under climate warming elms would flush earlier in most of Europe. The bud burst date was directly related to latitude and elevation in U. minor. The order of bud burst of clones from different origins was stable among years. The observed geographic trends were largely determined by differences in chilling requirements that increased with latitude and elevation. Susceptibility to DED varied greatly within U. minor and was directly correlated with geographic origin and bud burst date, southern and early flushing clones showing the least symptoms. Our results suggest that early flushing represents a mechanism of disease avoidance owing to asynchrony between host’s susceptible period and time of natural infection by DED vectors (Scolytus). This escape mechanism, combined with true resistance and avoidance owing to unattractiveness for the vector’s feeding, might be exploited for breeding DED-resistant clones of indigenous species.

The mobility of transposable elements (TEs) can contribute to genome plasticity, under- or over-expression of genes and ectopic recombination. The data collected in this study provide evidence of stress-induced mobility of OPHIO1 and OPHIO2 transposons, recently detected in Ophiostoma ulmi and O. novo-ulmi, the causal agents of Dutch elm disease (DED). The analyses of OPHIO UTRs and TIRs indicated the presence of two potential binding site motifs and a heat shock protein (hsp) promoter which could be involved in the mobility of OPHIO1 following a heat shock stress. The exact position of the hsp promoter was determined by 5' RACE PCR. After confirmation of the expression by RT-PCR of both OPHIO1 and OPHIO2 transposases in the absence of stress factors, we tested two experimental procedures to induce mobility of OPHIO TEs: (1) an exogenous (cloned) copy of OPHIO1 was introduced into the O. novo-ulmi subsp. americana strain W2 (OPHIO1 free strain) to give mutant strain W2:OPHIO1. After exposure of W2:OPHIO1 to a 55 degrees C heat shock treatment, some of the survivors showed signs of incomplete transposition (excision without reinsertion) of OPHIO1. (2) The O. novo-ulmi subsp. novo-ulmi strain AST27, introgressed from O. ulmi and carrying a distinct endogenous copy of OPHIO2 (OPHIO2-int.), was subjected to a series of abiotic stress treatments. Although a promoter sequence could not be identified, both exposures to UV light and to a 4 degrees C cold treatment caused perfect excision of OPHIO2-int. In contrast to OPHIO1, heat shock stress did not induce OPHIO2-int. mobility. Taken together, these results allow us to hypothesize a potential interspecific invasion of OPHIO transposons due to their mobility in Ophiostoma spp.

Introduction Elms are important trees in urban environments and coastal areas due to their resistance to harsh conditions such as wind, salt, flood, and narrow root space, as well as to their attractive architecture. However, a devastating disease, the so-called Dutch elm disease (DED), appeared in Europe during the 1920s and in North America by 1930. Sudden wilting and dying of the leaves and branches was caused by Ophiostoma ulmi during the first pandemic from 1920s to 1940s, while an even more aggressive strain O. novo-ulmi is responsible for the current pandemic. Elm bark beetles, mainly Scolytus scolytus and S. multistriatus, transmit the disease by breeding in weakened and dead elms (Scheffer et al, 2008). Approximately 100 years after its first introduction into Europe, biological control of Dutch elm disease is an effective component of an integrated control strategy for this disease. Conidiospores of Verticillium albo-atrum isolate WCS850 (active ingredient of Dutch Trig®) enhance the natural defence mechanism of elms after being injected in the xylem tissue of tree, where they germinate and induces resistance (Scheffer, 1990). The isolate was proven to be non-pathogenic for all the 19 tree species that had been tested (http://www.dutchtrig.com/home)

The parasitoids of the elm bark beetlesScolytus ensifer, S. kirschi, S. multistriatus, S. pygmaeus andPteleobius vittatus were studied in ten localities in Serbia. Eleven species of parasitoids ofS. ensifer, 10 ofS. kirschi, 13 ofS. multistriatus, 12 ofS. pygmaeus, and four ofP. vittatus were found. The most abundant wereEcphylus silesiacus, Dendrosoter protuberans, Cheiropachus quadrum andRhaphitelus maculatus. Acrocormus semifasciatus, Cerocephala eccoptogastri, Mesopolobus typographi andDoryctes pomarius were found in some samples.

Summary This paper describes the chemical and biochemical properties of exopolysaccharides (EPS) produced by Ophiostoma ulmi and O. novo-ulmi isolates, the Dutch elm disease (DED) fungi. Some of EPS have been considered as pathogenicity factor in the DED complex. The selected isolates grow well and produce EPS in a medium containing various types of carbon and nitrogen sources. EPS obtained from potato dextrose broth (PDB) medium appeared to be opaque, firm and stained purple blue with iodine-potassium iodide solution, whereas those from yeast extract (YE) medium were less opaque, jelly-like and remained unchanged in iodine solution. The selected fungal isolates produced much higher molecular weight EPS from the medium containing YE than from PDB. The results of this study suggest that high molecular weight compounds produced by O. ulmi (W9) and O. novo-ulmi (R136) are not involved in DED pathogenesis. Spectrometric analysis of acid-digested EPS obtained from PDB and YE revealed the presence of a monomer similar to glucose used as a standard. Thin layer chromatography indicated that glucan-1,4-α-glucosidase (glucoamylase) only hydrolyses EPS from PDB media and releases glucose. The results strongly indicate that isolates of O. ulmi and O. novo-ulmi produce starch-like EPS from PDB medium. The EPS obtained from YE medium lacked this characteristic. The biological significance and the potential use of these EPS are discussed.

Species of the genus Geosmithia are associated with insect species, mainly bark beetles. On Ulmus spp., the same beetles are also vectors of Ophiostoma ulmi s.l., the agent of Dutch elm disease (DED), a worldwide elm disease. Aim of this paper is to characterise Geosmithia species associated with elms and/or elm beetles in Europe. Seventy-two strains representative of all morphological taxonomic units were used to build a phylogenetic tree based on ITS, β-tubulin and elongation factor 1-α gene regions. On the basis of molecular and morpho-physiological traits, seven taxonomic entities were identified. In addition to the species previously known our results assigned strains previously identified as Geosmithia pallida to two separate taxa: Geosmithia sp. 2 and Geosmithia sp. 5. Two new species, Geosmithia omnicola and Geosmithia ulmacea, are described. Two strains were assigned to the partially described species Geosmithia sp. 20. Geosmithia species living on Ulmus do not discriminate between elm species, but between different environments. The association between Ulmus and Geosmithia is common, stable, and seems to be related to specific vectors. The relationship between Geosmithia and Ophiostoma would deserve further investigation, as these fungi share the same vectors and habitat for a significant part of their life cycles.

Elms, especially Ulmus minor and U. americana, are carrying out a hard battle against Dutch elm disease (DED). This vascular wilt disease, caused by Ophiostoma ulmi and O. novo-ulmi, appeared in the twentieth century and killed millions of elms across North America and Europe. Elm breeding and conservation programmes have identified a reduced number of DED tolerant genotypes. In this study, three U. minor genotypes with contrasted levels of tolerance to DED were exposed to several biotic and abiotic stresses in order to (i) obtain a de novo assembled transcriptome of U. minor using 454 pyrosequencing, (ii) perform a functional annotation of the assembled transcriptome, (iii) identify genes potentially involved in the molecular response to environmental stress, and (iv) develop gene-based markers to support breeding programmes. A total of 58,429 putative unigenes were identified after assembly and filtering of the transcriptome. 32,152 of these unigenes showed homology with proteins identified in the genome from the most common plant model species. Well-known family proteins and transcription factors involved in abiotic, biotic or both stresses were identified after functional annotation. A total of 30,693 polymorphisms were identified in 7,125 isotigs, a large number of them corresponding to single nucleotide polymorphisms (SNPs; 27,359). In a subset randomly selected for validation, 87% of the SNPs were confirmed. The material generated may be valuable for future Ulmus gene expression, population genomics and association genetics studies, especially taking into account the scarce molecular information available for this genus and the great impact that DED has on elm populations. © 2015 Perdiguero, Venturas, Cervera, Gil and Collada.

Bakalaureusetöö Metsanduse õppekaval Jalakasurm on Euroopas jalaka (Ulmus spp.) liikidel laialt levinud seenhaigus, mida põhjustavad nii Ophiostoma ulmi (Buisman) Nannf. ning nüüd ka Ophiostoma novo-ulmi Brasier., millest viimane on invasiivne ja agressiivne seenpatogeen. Bakalaureusetöö eesmärgiks oli hinnata jalakate (Ulmus glabra), künnapuude (Ulmus laevis), põldjalakate (Ulmus minor) ning jalaka hübriidide tervislikku seisundit Tallinnas ja Tihemetsas ning jalakasurma levikut neis piirkondades. Proovialade valikul lähtuti Tallinnas Sulev Järve soovitustest ja Tihemetsas kirjandusandmetest leitud informatsioonist Tihemetsa parkide kohta. Puude tervisliku seisundi hindamisel lähtuti viiest haigusklassist, millest esimesse arvati täiesti terved puud ning viimasesse viiendasse surnud puud. Harilikest jalakatest kuulusid 49,3% esimesse, 28,2% teise, 4,2% kolmandasse, 8,5% neljandasse ning 9,9% viiendasse haigusklassi. Põldjalakatest kuulusid 50,0% esimesse ja 50,0% neljandasse haigusklassi. Kõik künnapuud ning hariliku jalaka kultivar `Camperdownii´ kuulusid esimesse haigusklassi. Jalaka hübriidid kuulusid kõik teise haigusklassi. Töös kasutati välitöödel kogutud andmeid ja proove, mida laboris analüüsiti – seene universaalsete ja liigispetsiifiliste DNA praimeritega. 48-lt võrselt tehti puhaskultuure. Kolme proovi sekveneerimisel tuvastati Ophiostoma novo-ulmi ja Phoma macrostoma. Seitset puhaskultuuri, mida kontrolliti liigispetsiifiliste praimeritega PCR analüüsil andis positiivse tulemuse üks proov (DNA ID 3637). Puidust eraldatud DNA-d kontrolliti 16 juhuslikul proovil liigispetsiifiliste praimeritega PCR analüüsil patogeeni olemasolu, positiivse tulemuse andis üks proov (ID 3334). Kokku analüüsiti 26 sümptomaatilist hariliku jalaka proovi ning nendest leiti kolm tüve jalakasurma tekitajat. Dutch elm disease is a widespread fungal disease of elm (Ulmus spp) species in Europe, caused by both Ophiostoma ulmi (Buisman) Nannf. and now by Ophiostoma novo-ulmi Brasier., which is more invasive and aggressive fungal pathogen. Bachelor aim was to evaluate state of health of wych elm (Ulmus glabra), European white elm (Ulmus laevis), field elm (Ulmus minor) and elm hybrids in Tallinn and Tihemetsa and the range of Dutch elm disease in these regions. In Tallinn the selection of plots were based on recommendations of Sulev Järve and in Tihemetsa the information about Tihemetsa parks found in literature. The assessment of the healthiness of the trees was based on five disease classes, absolutely healthy trees were considered to be in the first and dead trees in the fifth class. 49,3% of wych elms belonged to the first, 28,2% to the second, 4,2% to the third, 8,5% to the fourth and 9,9% to the fifth disease class. 50% of field elms belonged to the first and 50% to the fourth disease class. All European white elms and elm cultivar `Camperdownii´ belonged to the first disease class. All elm hybrids belonged to the second disease class.All the collected data from fieldwork and samples were used in this work and the last ones were analyzed in the laboratory - with fungus universal and species-specific DNA primers. Pure cultures were made from 48 twigs. Three samples were sequenced Ophiostoma novo-ulmi and Phoma macrostoma were identified. Seven pure cultures, were checked with species-specific primers in PCR analysis, one sample (DNA ID 3637) of which gave a positive result. 16 DNA samples, extrated from wood tissue, were also analyzed with species-specific PCR primers, from which one sample gave a positive result (ID 3334). A total of 26 symptomatic wych elm samples were analyzed and three strains of Dutch elm death pathogen were found.