Dutch elm disease pathogen transmission by the banded elm bark beetle Scolytus schevyrewi

Dutch Elm Disease (DED) is a vascular wilt disease of Ulmus species (elms) incited in North America primarily by the exotic fungus Ophiostoma novo‐ulmi. The pathogen is transmitted via root grafts and elm bark beetle vectors, including the native North American elm bark beetle, Hylurgopinus rufipes and the exotic smaller European elm bark beetle, Scolytus multistriatus. The banded elm bark beetle, Scolytus schevyrewi, is an exotic Asian bark beetle that is now apparently the dominant elm bark beetle in the Rocky Mountain region of the USA. It is not known if S. schevyrewi will have an equivalent vector competence or if management recommendations need to be updated. Thus the study objectives were to: (i) determine the type and size of wounds made by adult S. schevyrewi on branches of Ulmus americana and (ii) determine if adult S. schevyrewi can transfer the pathogen to American elms during maturation feeding. To determine the DED vectoring capability of S. schevyrewi, newly emerged adults were infested with spores of Ophiostoma novo‐ulmi and then placed with either in‐vivo or in‐vitro branches of American elm trees. The inoculation of trees via feeding wounds was successful 30% of the time for in‐vivo trials and 33% for in‐vitro trials. Although the infection rate of DED has declined in Colorado over the past 10 years, the disease is still present in urban elms. While it appears that S. schevyrewi is another vector of the DED pathogens, it appears that S. schevyrewi is no more efficient than S. multistriatus. Thus, management programs that remove elm bark beetle breeding sites, rapidly remove DED‐infected elms and include the planting of DED‐resistant elms should continue to be effective management tactics.     

Evaluation of Dutch elm disease susceptibility and pathogenicity of Ophiostoma ulmi using micropropagated elm shoots

Elm (Ulmus sp.) shoots from in vitro micropropagated plants were tested with Ophiostoma ulmi culture filtrates, at different dilutions to determine the susceptibility of different host clones to Dutch elm disease (DED). ‘Commelin’, a susceptible Dutch hybrid, showed a high wilting index but that of American elm was higher. Although U. pumila × japonica (Sapporo gold 2) was the most tolerant to spore inoculation, its wilting index was as high as that of ‘Commelin’. The absorption capacity and wilting index of the elm shoots showed consistent correlation to isolate effectiveness (as determined by inoculation experiments), to production of toxin (cerato-ulmin) and to filtrate dilution. This method could be used to assess the effectiveness of the different O. ulmi isolates and for the detection of elm genotypes that are highly susceptible to Dutch elm disease.     

Starch‐like exopolysaccharide produced by the filamentous fungi Ophiostoma ulmi and O. novo‐ulmi

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.     

Fungitoxic properties of an antibiotic against dutch elm disease

A formulation containing solubilized Nystatin called Ceratocide, is fungitoxic to Ceratocystis ulmi (Buis.) Moreau as shown in laboratory studies. In controlled inoculations studies in eight-year-old elm saplings, Ceratocide was shown to have prophylactic utility in Dutch elm disease control when injected 2, 4 or 10 days before a challenge inoculation with C. ulmi. Therapeutic control was not obtained with a single injection of Ceratocide. Most infected trees succumbed to Dutch elm disease either during the same year of treatment or a year later.     

Detection of differential changes in lignin composition of elm xylem tissues inoculated with Ophiostoma novo‐ulmi using Fourier transform‐infrared spectroscopy

Differences in elm susceptibility against Ophiostoma novo‐ulmi have been related with differences in timing and degree of the tree defence responses. In this study, we used Fourier transform‐infrared spectroscopy, coupled with chemometric methods, to detect progressive changes in the metabolic profile of Ulmus minor and U. minor × U. pumila xylem tissues after inoculation with O. novo‐ulmi. Differences between control and inoculated trees were detected at 30 and 60 days post‐inoculation (dpi) in U. minor, and at 15, 30 and 60 dpi in U. minor × U. pumila. These differences were related with increased levels of lignin in the xylem tissues, suggesting an earlier defence response to the infection in the hybrids.     

Current status of the Dutch elm disease pathogen populations affecting Ulmus minor in Greece

One hundred and eight Ophiostoma ulmi sensu lato isolates were collected from field elm trees with symptoms in 14 Prefectures of Greece. The purpose of this study was to assign Greek isolates to species and afterwards subspecies of the DED fungi and to analyse the genetic variability within the Greek populations of these pathogens. Isolates were compared with six reference strains belonging to O. ulmi and the two subspecies of O. novo‐ulmi. The structure of the population has been analysed by means of morpho‐physiological features (growth rates, colony morphology and fertility responses) and by DNA sequencing and PCR‐RFLP amplification of the cerato‐ulmin (cu) and the colony type (col1) gene regions. According to fertility tests, both subspecies of O. novo‐ulmi were detected in Greece, but none of the isolates collected was identified as O. ulmi. O. novo‐ulmi ssp. novo‐ulmi occurred more frequently than ssp. americana (73 and 35 isolates, respectively) and their ranges overlapped. All isolates that behaved as ssp. novo‐ulmi in the fertility tests had the cu, as well as the col1 profile of ssp. novo‐ulmi. Surprisingly, all isolates that behaved as O. novo‐ulmi ssp. americana in the fertility test had the cu, as well as the col1 (with one exception) profile of O. novo‐ulmi ssp. novo‐ulmi. A possible explanation for this inconsistency could be the occurrence of hybridization between the two subspecies in Greece.     

Effects of Ophiostoma ulmi and Ophiostoma novo-ulmi culture filtrates on elm cultures from genotypes with different susceptibility to Dutch elm disease

Cell cultures of callus tissue cultures obtained from four elm genotypes (Ulmus minor; Ulmus minor×Ulmus pumila; [Ulmus carpinifolia×Ulmus glabra] × [Ulmus wallichiana×Ulmus glabra]; and Ulmus pumila), either susceptible or resistant to Dutch elm disease (DED) were exposed to culture filtrates of Ophiostoma ulmi and Ophiostoma novo-ulmi. Elm cells were largely affected by crude culture filtrate incorporated into the media. However, the correlation between ‘in vivo’ cell resistance and growth in the presence of culture filtrate was poor: the effects of fungal media components were greater than that exerted by fungal exotoxins. Therefore, it is concluded that these ‘in vitro’ assays cannot be used to evaluate resistance sources to DED in elms, or to assess specific pathogenicity of fungal isolates.     

‘Morfeo’ Elm: a new variety resistant to Dutch elm disease

Dutch elm disease (DED) has spread through Europe and North America since the beginning of the twentieth century. In response, several independent genetic improvement programmes for breeding DED‐resistant elms have been established on both sides of the Atlantic. Assimilating a wide range of resistant clones of different parentage should help ensure survival of the elm in the event of mutation of the pathogen. The Italian elm breeding programme began in the late 1970s and has recently raised a new variety by crossing a specimen of U. chenmoui with the Dutch hybrid clone ‘405’. This new release, named ‘Morfeo’, is extremely resistant to DED and has an attractive form and foliage. It is also fast‐growing, tolerant of drought and soils waterlogged in winter, therefore proving well adapted to the climates of both north‐western Europe and the Mediterranean. Following trials in England, ‘Morfeo’ is considered of potential importance in the conservation of several invertebrates endangered by the consequences of DED.     

American elm cultivars: Variation in compartmentalization of infection by Ophiostoma novo‐ulmi and its effects on hydraulic conductivity

Five cultivars and two populations of wild‐type seedlings of American elm (Ulmus americana), 3 and 4 years old, were examined for differences in their abilities to compartmentalize and resist infection by artificially inoculating with Ophiostoma novo‐ulmi. Morphological characteristics of tree defence, often referred to as the compartmentalization of decay in trees model, were used as a conceptual framework, with particular emphasis on the limiting of tangential spread of infection within the xylem and barriers that limit spread outwards to cells formed after infection. To investigate the change in functional xylem over time, 3‐year‐old trees were assessed at multiple time points following inoculation for hydraulic conductivity. Three and four‐year‐old cut trees were placed in 0.1% w/v safranin O for 18 to 24 hr to indicate functional xylem. Transverse sections of the stained stems were used to calculate per cent of sap‐conducting xylem area and the per cent of circumference conducting of first formed cells and later formed cells. At each collection time, trees were assessed for disease severity on a 1–12 scale, based on the percentage of permanent wilt in the crown. There was considerable variation between cultivars in disease severity and their capacity to localize and resist infection. “Prairie Expedition,” which had the lowest disease severity rating in 2015 and the second lowest in 2016, consistently limited the spread of infection into newly formed xylem and had functional xylem around the entire circumference of the stem at 90 days post‐inoculation. “Valley Forge” in 2016 had the lowest overall disease severity rating and was the only cultivar to consistently limit the tangential spread of infection within extant xylem. This research identifies key characteristics that some cultivars have to resist and limit infection and provides new information that can be used in disease screening programmes to evaluate other cultivars and older plant material.     

Presence and accumulation of fungitoxic substances against Ceratocystis ulmi in Ulmus americana: possible relation to induced resistance1

This paper reports the differential accumulation of inhibitory compounds in American elm seedlings after inoculation with a non-aggressive and an aggressive strain of C, ulmi.     

Dutch elm disease has currently a low incidence on wych elm in Norway

A rapid increase in the frequency of Dutch elm disease (DED), a wilting disease of elm trees caused by bark‐beetle vectored fungi, was observed in the early 1990s on several wych elm stands around Oslofjord, southern Norway. To examine the current status of the disease and its impacts on elm population, disease frequency and size distribution of elms were recorded at four locations. Northern parts of Lier, a municipality most affected by DED in Norway 15 years ago, showed in the survey season 4% disease frequency, whereas 13.8% of trees were dead, the dead trees having accumulated over several years in the unmanaged stands. In southern parts of the municipality the mean disease and death percentages were 1.9 and 2.4%. Compatible with their low disease incidence in early 1990s, the other two areas now examined, municipality of Larvik and district of Grenland, showed comparably low frequency of DED. Northern part of Lier showed significantly higher overall density of elm trees per hectare than the other examined areas, and also the small elms below 5 cm in d.b.h. were most frequent in this region. In contrast, the density of large trees was lower in northern Lier than in the other examined areas. These data suggest that regeneration of the tree is not prohibited owing to the disease but that the large trees have been locally reduced in frequency as a result of DED. The superior general density of elm trees in northern Lier, owing to the exceptionally rich soil in the warm southern slopes of the region,> may have contributed to the rapid increase of DED in the area 15 years ago and to the subsequent settlement of the disease outbreak as a chronic stage.     

Genotype × environment interaction and growth stability of several elm clones resistant to Dutch elm disease

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 (Hühn's S_i⁽¹⁾ and S_i⁽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.     

Effect of Betula pendula clone and leaf age on Melampsoridium betulinum rust infection in a field trial

Ten 5‐year‐old Betula pendula clones were studied for their rust resistance in the field. The trees were treated by inoculating 10 leaves on a shoot with Melampsoridium betulinum urediniospore suspension or spraying the control leaves with water. The birch clones differed significantly in their resistance to M. betulinum leaf rust fungus and the clones also varied in their responses to the local rust strain and the inoculated rust strains. However, natural rust infections and inoculation treatment were positively correlated. The older leaves had fewer infections than the younger ones on the tip of the shoot in the control trees, but in the inoculation treatment no significant correlation was found between the leaf ages and rust infection. The factors behind the different leaf susceptibilities are discussed.     

Induced resistance of Norway spruce,variation of phenolic compounds and their effects on fungal pathogens

Three clones of Norway spruce (Picea abies) were studied for their response to mass‐inoculation with the blue‐stain fungus Ceratocystis polonica. The effect of different pretreatments (fungal inoculation and wounding) before mass‐inoculation was investigated for their possible role in an acquired resistance reaction. Pretreated trees showed enhanced resistance to the subsequent mass‐inoculation relative to control trees that received no pretreatment. Furthermore, the fungal colonization of inoculated trees was less than that of wounded trees. The phenolic content of the bark, analysed by RP‐HPLC, was compared in trees receiving different treatments. Trees inoculated with C. polonica had higher average concentration of (+)‐catechin, taxifolin and trans‐resveratrol than wounded trees. Both inoculated and wounded trees had higher average concentrations of these compounds than control trees. The effect of the phenolic extract of Norway spruce bark on the growth of the root rot fungus Heterobasidion annosum and the blue‐stain fungi C. polonica and Ophiostoma penicillatum were investigated in vitro. Heterobasidion annosum was not negatively affected, and the extracts had fungistatic effects on the blue‐stain fungi. The growth of O. penicillatum was more inhibited than the growth of the more aggressive C. polonica.     

First report on the Dutch elm disease pathogen Ophiostoma novo‐ulmi from Latvia

During 2017, Dutch elm disease was investigated on 67 Ulmus glabra and 29 Ulmus laevis samples from 28 sites in Latvia. The presence of Ophiostoma novo‐ulmi was detected in 61.2% of the samples from U. glabra and in 34.5% of the samples from U. laevis. In 11% of cases, hybrids between subsp. novo‐ulmi and subsp. americana were identified, particularly in the sites in the southern part of Latvia. This study presents the first verified record of Ophiostoma novo‐ulmi and its subspecies in Latvia.     

Electron microscope observations of Ceratocystis ulmi-induced tylosis development in Ulmus1

Electron microscope observations of Ceratocystis ulmi-induced tylosis development in 3 species of elm were made during the first 2 weeks after artificial inoculation. Tylosis forming from both ray and vasicentric parenchyma occluded vessels adjacent to those containing the fungus conidia within one day after inoculation. The expanding protoplast forced the intact vessel-parenchyma “pit membrane” and protective layer through the pit into the vessel lumen. Cell organization during this expansion is discussed. The observations on tylosis development for the 3 elms used were identical.     

Dutch elm disease on the island of Gotland: monitoring disease vector and combat measures

The island of Gotland (Sweden) possesses the largest (more than one million trees) wild population of elms in northern Europe, which until recently was not affected by Dutch elm disease (DED). In 2005, DED was observed for the first time in the northeastern part of Gotland, and in the following years, it rapidly spread in all directions, generally following the major distribution of elms. Implementation of intensive control measures including ground surveys and geographic information system mapping of DED-diseased trees, their felling and destruction, and stump treatment with the herbicide glyphosate allowed reduction of the source of infection and probably suppression of an otherwise very rapid spread of the disease. Monitoring the flying periods of Scolytus multistriatus vector beetles between the years 2007 and 2011 showed that the abundance of trapped beetles in most cases differed significantly among individual years. Over the entire monitoring period, 55.2% of the beetles were trapped in June, 31.2% in July and 13.5% in August (difference among individual months significant at p?S. multistriatus, suggesting that the removal of trees attacked in the previous vegetation period as a combat measure should be completed before the beginning of the flying season of S. multistriatus, and that the application of the integrated DED management has the potential to arrest the development of the disease in a geographically isolated area such as Gotland.     

Coremia formation by Ceratocystis ulmi on elm stem discs

The seasonal fluctuation in the production of coremia by Ceratocystis ulmi (Buis.) was studied on elm stem discs taken from trees felled in April, June, July, September, October and November 1971 at Valcartier, Quebec. More coremia formed on stem discs of trees felled during the growing season. Except for trees felled in October and November, more coremia formed on sapwood than on heartwood. Evidence obtained during the study showed that the compounds, which trigger coremia formation, are not uniformly distributed within and between trees or throughout the period from April to November.     

Xylem vessel diameter as a factor in resistance of Ulmus minor to Ophiostoma novo‐ulmi

An investigation has been carried out to study the relationship between the susceptibility of Ulmus minor to Ophiostoma novo‐ulmi and the diameter of its xylem vessels. Vessel diameters have been analysed in seven Spanish elms of known susceptibility, taking measurements from second and third year rings in 3‐year‐old branches. In the second year rings no relationship could be observed between the vessel diameters and the susceptibility of the trees. In the third year rings the mean diameter and the percentage of vessels larger than 100 μm were found to be significantly greater for the group of elms of high susceptibility. In the third year rings the vessel transectional area (VTA) and the theoretical hydraulic conductance (THC) were calculated. The decrease in VTA and THC became increasingly noticeable in the group of high susceptibility elms as vessels of larger diameter were excluded from the calculation. Xylem vessel diameters and the proportion of large vessels were correlated with the susceptibility of U. minor to Dutch elm disease. How these properties influence the dispersal of the fungus, the sap flow and the tree's ability to compartmentalize the disease is discussed.     

In vitro antagonism of Trichoderma and naturally occurring fungi from elms against Ophiostoma novo‐ulmi

The antagonist ability of four fungal endophytes isolated from elms (Trichoderma atroviride GF13, Diaporthe eres GF2, Diplodia ulmi GF5 and Alternaria alternata GF6) and two fungi from a collection (Trichoderma harzianum and Trichoderma virens) was evaluated against six strains of Ophiostoma novo‐ulmi (1, 10, 19, 32, 43 and O‐Va), the causal agent of Dutch elm disease. Dual culture tests were established in PDA and MA culture media to test the interactions between pathogenic fungi and potential antagonist ones. The fungi showed varying levels of antagonist capacity against O. novo‐ulmi. Among them, T. atroviride and T. harzianum had the greatest inhibiting effect (around 50%). Culture filtrates and previously inoculated cellophane discs from T. harzianum and T. atroviride also caused growth inhibition of O. novo‐ulmi, with less intensity in strains 10 and 19. The control mechanisms involved in the process were competition, mycoparasitism and the production of soluble and diffusible antifungal metabolites. The new isolate T. atroviride GF13 was efficient as antagonist. It showed antagonist ability against non‐elm‐derived fungi such as Verticillium, Fusarium, Aspergillus, Rhizopus and Penicillium. These results suggest interesting application possibilities in the biological control of Dutch elm disease.