Feeding by Scolytus bark beetles to test for differently susceptible elm varieties

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.     

Metabolic distinction of Ulmus minor xylem tissues after inoculation with Ophiostoma novo-ulmi

Dutch elm disease (DED) is the most devastating and widespread disease of elms. The pathogen, Ophiostoma novo-ulmi, spreads systemically causing xylem vessels blocking and cavitation, and ultimately resulting in the development of a wilt syndrome. Twig samples from susceptible and resistant Ulmus minor trees were harvested at 0, 5, 15, 30, 60, and 120 days post-inoculation (dpi) with O. novo-ulmi. Fourier transform-infrared (FT-IR) spectroscopy, in tandem with chemometrics, was used to monitor changes in wood chemistry as consequence of infection. Principal component analysis distinguished between spectra from inoculated and control elms, and from susceptible- and resistant-inoculated elms. By 30 dpi, infected xylem showed reduced relative levels of carbohydrates and enhanced relative levels of phenolic compounds, probably due to the degradation of cell wall polysaccharides by fungal enzymes and the synthesis of host defence compounds. On 15 dpi, samples from resistant-inoculated elms showed higher levels of starch than samples from susceptible-inoculated elms, suggesting that availability of starch reserves could affect the tree's capacity for defensive responses. The results showed the power of FT-IR spectroscopy for analysing changes in the major components of elm xylem as consequence of infection by DED, and its potential for detecting metabolic profiles related to host resistance.     

2004 SIVB Congress Symposium Proceeding: Biotechnological progress in dealing with dutch elm disease

Summary Applications of biotechnological tools, including genetic modification aimed at combating Dutch elm disease, are described. In vitro shoot cultures of Ulmus procera (English elm) SR4, U. glabra, U. americana, and U. parvifolia have been established and used as source material in genetic transfer experiments. Biolistic transformation of U. procera leaf material with cauliflower mosaic virus 35S-promoted constructs resulted in transient gusA (β-glucuronidase) expression. Subsequently, regenerant U. procera have been obtained following transformation of stem pieces with wild-type tumor-inducing or root-inducing plasmid-harboring Agrobacterium strains. Genetically modified elms expressing gusA, gfp (green fluorescent protein), and nptII (neomycin phosphotransferase II) genes have been produced using disarmed vectors. Regenerant English elms have been produced following transformation with anti-fungal genes, transferred to soil, and are currently being tested for their ability to resist the Dutch elm disease fungus Ophiostoma novo-ulmi. Future prospects for fighting this fungal wilt using biotechnological tools are discussed.     

Experiments with MBC derivatives for the control of Dutch elm disease

Preparations of methyl benzimidazol-2-ylcarbamate (MBC) hydrochloride stabilized with HC1, MBC nitrate stabilized with lactic acid and MBC bisul-phate stabilized with KHS0₄ were made from technical MBC. As it was the least phytotoxic to elm shoots, the hydrochloride was selected for field experiments on the control of Dutch elm disease using 6 m elms. In protectant experiments using 1.51 of 0.25 or 0.5 % MBC per tree, only five of eighteen injected trees became infected while all the control trees were severely diseased. In curative experiments, injection with 0.25 % MBC 2 and 4 wk after inoculation with Ceratocystis ulmi kept symptoms to < 1 and 5% respectively, compared with 76% in untreated inoculated trees. MBC at 0.5 % Save similar results.     

Use of culture filtrates of Ceratocystis ulmi as a bioassay to screen for disease tolerant Ulmus americana

Cullus cultures of elm (Ulmus americana L.) derived from Dutch elm disease susceptible, intermediate-resistant, and resistant genotypes were exposed to the culture filtrates of three patogenic isolates of Ceratocystis ulmi, the causal agent of Dutch elm disease. Callus fresh weights, cell viability, and reactions of stem cuttings were determined after exposure to various concentrations of the filtrates. Calli from the susceptible elm failed to increase in fresh weight and lost viability after exposure to media containing culture filtrate. Calli from the resistant and the intermediate-resistant elms exhibited growth rates and maintained viability similar to controls not exposed to culture filtrate. Stem cuttings of the susceptible elm wilted after exposure to the culture filtrate. The symptoms were similar to wilt seen with the disease. Cuttings from the resistant elm had no disease symptoms whereas, the intermediate elm had some leaf chlorosis. Callus screening may thus be useful for selection of Ulmus germplasm for Dutch elm disease resistance.     

Mechanisms Involved in Biological Control of Dutch Elm Disease

An effective suppression of Dutch elm disease symptom development was observed in elms resistant to the non-aggressive strain of Ophiostoma ulmi, but not to the aggressive strain, after a preventive treatment by this non-aggressive strain. Anequally effective isolate of Verticillium dabliae suppressed symptom development even in the very susceptible field elm (Ulmus carpinifolia). Induction of resistance is proposed for the major mechanism explaining these and earlier experiments. The primary stimuli initiating a resistance reaction are unknown, but the intermediates of the univalent reduction of oxygen may play a role. In an experiment the activities of two groups of enzymes involved in the catalytical scavenging of these radicals, superoxide dismutase and peroxidase, were monitored, but no correlation with induction of resistance was observed. Resistant elms showed the highest activities, probably reflecting a more effective defence against radicals than susceptible ones.     

Distribution and survival of overwintering adults of the Dutch elm disease vector, Hylurgopinus rufipes (Coleoptera: Scolytidae), in American elm trees in Manitoba

Abstract 1 The native elm bark beetle, Hylurgopinus rufipes, is the principal vector of Dutch elm disease in Manitoba, Saskatchewan and North Dakota, and disease management measures include applying residual insecticides to the lower part of elm tree trunks where the adult beetles overwinter. 2 In American elm trees in southern Manitoba, we counted entrance holes produced by beetles then felled and dissected trees to determine numbers of tunnels and numbers and survival of overwintering beetles. 3 Densities of entrance holes, tunnels and beetles followed a logistic relationship with tree trunk diameter; densities were near zero at diameters < 10 cm and reached a site‐specific asymptote at diameters > 20 cm. 4 Asymptotic densities of holes, tunnels and beetles in samples from 55 to 190 cm above the ground were, respectively, 22%, 22% and 0.7% of those within 25 cm of the ground. 5 Within the height range 0–190 cm, the proportion of living beetles declined steeply with increasing height. 6 Average density of holes at height 0–25 cm estimated from a sample of several trees of diameter ≥15 cm could be used to predict the asymptotic maximum density of overwintering beetles in the site; predictions of beetle densities for individual trees were not reliable.     

Elm defence against herbivores and pathogens: morphological,chemical and molecular regulation aspects

Elms (Ulmus spp.) have long been appreciated for their environmental tolerance, landscape and ornamental value, and the quality of their wood. Although elm trees are extremely hardy against abiotic stresses such as wind and pollution, they are susceptible to attacks of biotic stressors. Over 100 phytopathogens and invertebrate pests are associated with elms: fungi, bacteria and insects like beetles and moths, and to a lesser extent aphids, mites, viruses and nematodes. While the biology of the pathogen and insect vector of the Dutch elm disease has been intensively studied, less attention has been paid so far to the defence mechanisms of elms to other biotic stressors. This review highlights knowledge of direct and indirect elm defences against biotic stressors focusing on morphological, chemical and gene regulation aspects. First, we report how morphological defence mechanisms via barrier formation and vessel occlusion prevent colonisation and spread of wood- and bark-inhabiting fungi and bacteria. Second, we outline how secondary metabolites such as terpenoids (volatile terpenoids, mansonones and triterpenoids) and phenolics (lignans, coumarins, flavonoids) in leaves and bark are involved in constitutive and induced chemical defence mechanisms of elms. Third, we address knowledge on how the molecular regulation of elm defence is orchestrated through the interaction of a huge variety of stress- and defence-related genes. We conclude by pointing to the gaps of knowledge on the chemical and molecular mechanisms of elm defence against pest insects and diseases. An in-depth understanding of defence mechanisms of elms will support the development of sustainable integrated management of pests and diseases attacking elms.     

The pathogen causing Dutch elm disease makes host trees attract insect vectors

Dutch elm disease is caused by the fungal pathogen Ophiostoma novo-ulmi which is transmitted by the native elm bark beetle, Hylurgopinus rufipes. We have found that four semiochemicals (the monoterpene (-)-beta-pinene and the sesquiterpenes (-)-alpha-cubebene, (+)-spiroaxa-5,7-diene and (+)-delta-cadinene) from diseased American elms, Ulmus americana, synergistically attract H. rufipes, and that sesquiterpene emission is upregulated in elm trees inoculated with O. novo-ulmi. The fungus thus manipulates host trees to enhance their apparency to foraging beetles, a strategy that increases the probability of transportation of the pathogen to new hosts.     

ON THE NATURE OF INTRA- AND INTERSPECIFIC INCOMPATIBILITY IN ULMUS

Efforts to introduce Dutch Elm Disease resistance into the American elm (Ulmus americana L.) through breeding with Asian elms has been hampered by sexual incompatibility. Controlled pollinations of Ulmus americana and the Siberian elm (Ulmus pumila L.) were studied in detail to gain insight into the nature of this incompatibility. Microscopic observations revealed that germination and early pollen tube growth were inhibited on the stigmatic surface following both intra- and interspecific incompatible pollinations. Both qualitative and quantitative differences in pollen inhibition on the stigmatic surface indicated that the inhibition may involve the action of an inhibitory substance. Detailed observations on callose deposition indicated that this β-1,3 glucose polymer may implement the inhibition.     

Breeding populations of the Hobby Falco subbuteo on farmland in the southern Midlands of England

Site occupancy, dispersion, density and nest sites of Hobbies Falco subbuteo were studied in two areas of farmland in the English southern Midlands between 1975 and 1983. Nesting ranges were identified within which Hobby nests were regularly found but which contained only one pair of birds in any one year. Breeding was not proved in all nesting ranges in every year. Within nesting ranges, birds sometimes used the same nest in consecutive years but nests were also commonly found more than 1 km apart in consecutive years. The mean nearest-known-neighbour distance between simultaneously occupied breeding sites was 4 6 km (range 20-9.6 km) and the minimum estimated densities of Hobbies in the two study areas were 3.8 and 4.8 nesting ranges/100 km. Small inter-nest distances, less than 30 km, were recorded only in the area with the lowest density. Hobbies nested in old Crow Corvus corone nests in a wide range of tree species but prior to the onset of Dutch elm disease, elms Ulmus spp. were probably used almost exclusively in one of the areas. It is concluded that substantial numbers of Hobbies nest on farmland north of the River Thames, outside what has been traditionally regarded as the main range of the species, and that previous published estimates of the size of the British population have been far too low.     

Rapid Evolutionary Changes in a Globally Invading Fungal Pathogen (Dutch Elm Disease)

Two enormously destructive pandemics of Dutch elm disease occurred in the 20th century, resulting in the death of a majority of mature elms across much of the northern hemisphere. The first pandemic, caused by Ophiostoma ulmi, occurred as this pathogen spread across Europe, North America and Southwest and Central Asia during the 1920s–1940s. The current pandemic is caused by another Ophiostoma species, O. novo-ulmi. Since the 1940s, O. novo-ulmi has been spreading into the regions previously affected by O. ulmi. It has also spread as two distinct subspecies, termed subsp. americana and subsp. novo-ulmi. This sequence of events has resulted in competitive interactions between these previously geographically isolated pathogens. This article summarizes the biological properties of the Dutch elm disease pathogens and their history of spread. It reviews the remarkable series of genetic events that have occurred during their migrations; including the emergence of genetic clones, the spread of deleterious fungal viruses within the pathogen clones, and the rapid and continuing evolution of O. novo-ulmi via horizontal gene flow. The wider role of horizontal gene flow in the evolutionary potential of migratory plant pathogens is discussed.     

Optimizing Seed Water Content: Relevance to Storage Stability and Molecular Mobility

This research was conducted to determine the optimum moisture content (MC) that gave maximum longevity to seeds. Three species were used to represent seeds with different dry matter reserves, which gives them different sorption properties: maize (Zea mays L.), elm (Ulmus pumila L.) and safflower (Carthamus tinctorius L.). The seeds of elm, safflower, and maize embryos with MC ranging from 0.00–0.15 g H₂O/g dry weight (DW) were stored at 35 °C for different periods of time. The results showed that the optimum MC for seed and embryo storage varied between species (0.057 g H₂O/g DW for maize embryos, 0.045 g H₂O/g DW for elm, and 0.02 g H₂O/g DW for safflower). Drying below this optimum MC increased the aging rate and there were detrimental effects of drying. The relative humidity corresponding to optimum MC in embryos of maize, elms and safflower was about 15%, 12% and 7% respectively, according to the lipid composition of the embryos. The data provided confirmatory evidence that molecular mobility (ΔAzz) in elms, maize and safflower embryos was compatible with the optimum moisture content.     

Influence of a Ceratocystis ulmi Toxin on Water Relations of Elm (Ulmus americana)

Water-soluble glycopeptides isolated from cultures of Ceratocystis ulmi have been reported to be toxins involved in Dutch elm disease. The influence of the glycopeptides on the water relations of Ulmus americana seedlings was tested by placing cut stems in glycopeptide preparations. After 4 hours in 200 micrograms per milliliter toxin the stem conductance of the seedlings was reduced by 79% and the leaf water potential was reduced by 3 bars to that at which the seedlings wilted, the stomata closed, and transpiration decreased. Decrease in stem conductance as the mode of action of the toxin was further confirmed by forcing toxin through the stem and petiole of elm and measuring the effects on stem conductance. High molecular weight dextrans were found to mimic the action of toxin on stem and petiole conductance, and their ability to do so was found to be correlated with their molecular weight. As low as 4 micrograms of toxin or dextrans were found to measurably decrease the stem and petiole conductance of elms. Disruption of the water-conducting system of elms and other plants by small quantities of high molecular weight compounds may be a factor in diseases with wilting symptoms.     

Micropropagation of mature wych elm (<Emphasis Type="Italic">Ulmus glabra</Emphasis> Huds.)

Explants of mature vigorous donor trees of wych elm (Ulmus glabra Huds.) that had not been previously exposed to Dutch elm disease were investigated for the influence of phytohormones and media on shoot multiplication rates and organogenic capacity. The regenerates were micropropagated from cultures that originated from 15-year-old progeny of plus trees. Two plus trees aged over 70 years showed recalcitrant responses. Thidiazuron in combination with 6-benzylaminopurine (BAP) induced a significantly higher number of shoots per explant than the most optimal BAP treatment (5.88 vs. 3.05 shoots). Woody plant medium and Dubovský minimal medium had no significant effects on shoot formation and multiplication rates. All plantlets raised in vitro were phenotypically normal and successfully hardened to ex vitro conditions. Two experimental field plots with 3-year-old in vitro-propagated trees were established.Abbreviations DED: Dutch elm disease - BAP: 6-Benzylaminopurine - IBA: Indole-3-butyric acid - TDZ: Thidiazuron - WPM: Woody Plant Medium - DM: Dubovský Minimal Medium Communicated by D. Bartels     

Exogenous phenol increase resistance of Ulmus minor to Dutch elm disease through formation of suberin-like compounds on xylem tissues

The survival of some elms to Dutch elm disease (DED) epidemics could be related with the application of disinfectant products based on simple phenols. To test this hypothesis, the protective effect of different phenolic treatments in Ulmus minor trees was evaluated through inoculations with Ophiostoma novo-ulmi, the current DED pathogen. During spring 2004 and spring 2005, 4-year-old elms were: (i) watered with a 0.02% solution of the phenolic fraction of phenolic oil, (ii) watered with a 0.02 and 0.2% solution of a phenol–cresol mixture, and (iii) trunk injected with a 0.2% solution of phenol–cresol mixture. In May, trees were artificially inoculated with O. novo-ulmi. At the end of the 2004 and 2005 vegetative periods, phenol-treated trees showed significantly lower wilting values than control trees. One week of bud break delay was observed in trees watered with the 0.2% solution of phenol–cresol mixture. Fourier transform-infrared spectroscopy and electrospray ionisation mass spectrometry evidenced enhanced levels of suberin-like compounds in phenol-treated trees with respect to non-treated trees. The deposition of suberin in xylem tissues, as a response to phenol treatments, might be considered as one of the mechanisms of resistance of elms to O. novo-ulmi.     

Smelling the tree and the forest: elm background odours affect egg parasitoid orientation to herbivore induced terpenoids

Parasitic insects use herbivore induced plant volatiles as signals for host location. However, their responses to these volatiles in the background of natural habitat odours need further evaluation for developing successful biological control strategies. Field elms (Ulmus minor Miller (Ulmaceae)) release a blend of volatiles in response to oviposition of the elm leaf beetle, Xanthogaleruca luteola Müller (Coleoptera: Chrysomelidae), a major urban and forest pest in the USA and Australia. This induced blend attracts the beneficial egg parasitoid Oomyzus gallerucae Fonscolombe (Hymenoptera: Eulophidae). Our olfactory assays showed that an odorous background of non-attractive host plant volatiles from feeding damaged elms or (Z)-3-hexenyl acetate masks the attractive effect of the host-induced (E)-β-caryophyllene to O. gallerucae. Quantitative GC–MS analyses revealed decreased concentrations of (Z)-3-hexenyl acetate accompanied by highly increased concentrations of sesquiterpenes in oviposition and feeding treated elms compared to undamaged elms. This finding hints to how the parasitoid might distinguish between different odorous backgrounds. It is corroborated by the outcome of our field study in natural elm stands, where the egg parasitoid parasitized more host egg masses due to an artificially induced blend of elm terpenoids.     

Establishment and characterization of American elm cell suspension cultures

Cell suspension cultures of Dutch elm disease (DED)-tolerant and DED-susceptible American elms clones have been established and characterized as prerequisites for contrasts of cellular responses to pathogen-derived elicitors. Characteristics of cultured elm cell growth were monitored by A₇₀₀ and media conductivity. Combined cell growth data for all experiments within a genotype showed relatively low variances and between-genotype contrasts during repeated passages showed no significant differences. Subculturing exponentially growing cells at 8–14 day intervals, within readily measured parameters of media conductivity (4.95–4.2 mmhos) and cell concentration (≥ 1.4 A₇₀₀), consistently resulted in repeatable profiles of elm cell growth and minimized lag phase. Culture cells were essentially homogeneous after 5 subculture passages and their overall appearance was stable. We conclude that the described procedure resulted in consistent cultures suitable for elicitor treatment experiments. This revised version was published online in June 2006 with corrections to the Cover Date.     

Conservation of genetic diversity in slippery elm (<Emphasis Type="Italic">Ulmus rubra</Emphasis>) in Wisconsin despite the devastating impact of Dutch elm disease

Forest pest epidemics are responsible for many population declines reported in forest trees. While forest tree populations tend to be genetically diverse, in principle mortality resulting from disease could diminish that genetic diversity and alter the genetic structure of the remnant populations with consequences for the ability of a species to adapt to changing environments. Slippery elm (Ulmus rubra Muhl.) is a long-lived, wind-pollinated forest tree with a native range covering essentially all of eastern North America. Dutch elm disease (DED) caused by an introduced fungal pathogen (Ophiostoma ulmi) devastated North American elm populations, including slippery elm, beginning in the 1930s. Estimates of the numbers of elms lost to DED are unknown but range into the hundreds of millions of trees given their former abundance. In this study, the genotypes of 77 herbarium specimens collected between 1890 and 2004 in Wisconsin, and of 100 slippery elm trees from five wild Wisconsin populations, were characterized using 13 microsatellite loci. Levels of genetic diversity were compared between the herbarium specimens collected pre- and post-DED spread in Wisconsin. In addition, the levels of genetic diversity and degree of genetic differentiation were quantified in the five wild populations. The allelic diversity and expected levels of heterozygosity were similar between the pre- and post-DED herbarium specimens. The five wild populations were only slightly differentiated and no genetic bottleneck was detected for any population. At least in Wisconsin, slippery elm apparently has maintained levels of genetic diversity that could facilitate adaptation to future climatic and environmental changes.     

Chemical changes in Ulmus minor xylem tissue after salicylic acid or carvacrol treatments are associated with enhanced resistance to Ophiostoma novo-ulmi

Application of endogenous plant hormone salicylic acid (SA) or essential oil component carvacrol (CA) in elms enhances tree resistance to the Dutch elm disease pathogen, although the effect of these compounds on tree metabolism is unknown. The chemical changes induced by SA or CA treatments in Ulmus minor were studied through gas chromatography–mass spectrometry (GC–MS) analysis of xylem tissues. Treatments consisted of fortnightly irrigating seedlings with water, SA or CA at 600 mg L⁻¹. The chemical composition of the xylem tissues sampled from treated trees was significantly altered depending on the treatment type. SA treatment induced an accumulation of the sinapyl alcohol, a precursor of lignin and other phenylpropanoid-derived products. CA treatment induced an accumulation of the methyl esters of palmitic, linoleic and stearic acids. Both treatments resulted in early bud burst and SA significantly reduced sapwood radial growth, possibly as a consequence of a trade-off between tree growth and tree defence. The enhanced resistance provided by these treatments is discussed.