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.     

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.     

Relative effectiveness of Scolytus scolytus,S. multistriatus and S. kirschi as vectors of Dutch elm disease

Three scolytid species consistently associated with Dutch elm disease were assessed and compared for their effectiveness as vectors of Ophiostoma ulmi. Assessments were based on the number of beetles within a sample carrying any detectable O. ulmi, the number of O. ulmi spores carried by individual beetles and the number of discrete gallery systems colonised by O. ulmi.     

Physiological and biochemical differences among Ulmus minor genotypes showing a gradient of resistance to Dutch elm disease

Dutch elm disease (DED) spread across Europe and North America in the 20th century killing most natural elm populations. Today, breeding programmes aim at identifying, propagating and studying elm clones resistant to DED. Here, we have compared the physiology and biochemistry of six genotypes of Ulmus minor of variable DED resistance. Leaf gas exchange, water potential, stem hydraulic conductivity and biochemical status were studied in 5‐year‐old trees of AB‐AM2.4, M‐DV2.3, M‐DV2 × M‐CC1.5 and M‐DV1 and 6‐year‐old trees of VA‐AP38 and BU‐FL7 before and after inoculation with Ophiostoma novo‐ulmi. Leaf water potential and net photosynthesis rates declined, while the percentage loss of hydraulic conductivity (PLC) increased after the inoculation in susceptible trees. By the 21st day, leaf predawn and midday water potential, stomatal conductance to water vapour and net photosynthesis rates were lower, and PLC was higher in trees of susceptible (S) genotypes inoculated with the pathogen than in control trees inoculated with water, whereas no significant treatment effect was observed on these variables in the resistant (R) genotypes. Fourier transform infrared spectroscopy analyses revealed a different biochemical profile for branches of R and S clones. R clones showed higher absorption peaks that could be assigned to phenolic compounds, saturated hydrocarbons, cellulose and hemicellulose than S clones. The differences were more marked at the end of the experiment than at the beginning, suggesting that R and S clones responded differently to the inevitable wounding from inoculation and repeated sampling over the experimental course. We hypothesize that a weak activation of the defence system in response to experimental wounding can contribute to the susceptibility of some genotypes to O. novo‐ulmi. In turn, the decline in shoot hydraulic conductivity and leaf carbon uptake caused by the infection further exacerbates tree susceptibility to the fungus.     

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.     

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.     

‘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.     

Variation among Italian and French elm clones in their response to Ophiostoma novo‐ulmi inoculation

In 1995 a clone bank of elms from across Italy was created at Antella (Florence, I) under a European Union project. In 1998 an experimental planting of some clones from this source was established in Antella together with material from a similar clone bank in France and two reference clones: Commelin (relatively susceptible) and Lobel (relatively resistant). In May 2001, the elms were inoculated with Ophiostoma novo‐ulmi and disease development was assessed throughout that summer and the next. Significant differences in susceptibility were found between taxonomic groups, with putative natural hybrids between Ulmus minor and Ulmus pumila being the most resistant and Ulmus glabra the most susceptible. Significant differences were also found within U. minor, some clones being as resistant as Lobel. For U. minor, a statistically significant relation was found with latitude, southerly clones showing less disease than more northerly ones. Relationships were also found between disease severity and times to bud burst, these relationships applying both to the different taxonomic groups and within U. minor. Relationships between growth increment and disease severity were inconsistent. These results are discussed in relation to the intrinsic genetically‐controlled differences that exist between elms and to differences in susceptibility that occur as the growing season proceeds and as a consequence of environmental influences.     

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.     

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.     

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.     

Ophiostoma ulmi metabolites and elm cell membrane permeability. Possible use in early tests of resistance

Electrolyte leakage from leaf discs of three Ulmus species when challenged with cultural filtrate of an NAN aggressive isolate of Ophiostoma ulmi paralleled their susceptibility to the disease in the field. But the correlation was not working with some hybrids derived from the common field elm showing intermediate resistance. The possible explanation will be checked in further tests which are in progress to assess the possibility of using this technique in early screening for resistance.     

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.     

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.     

A scanning electron microscope study of cell wall degradation in elm wood by aggressive and non-aggressive isolates of Ophiostoma ulmi

Degradation of cell walls in elm wood tissue (Ulmus americand) by aggressive and non-aggressive isolates of Ophiostoma ulmi was studied by scanning electron microscopy. Xylem vessel walls were much more affected after infection with the aggressive isolate than after infection with the non-aggressive one.     

Cellulolytic activity and virulence of Ophiostoma ulmi and O. novo‐ulmi isolates

The cellulolytic activity (exoglucanase, endoglucanase and β‐glucosidase) of Ophiostoma ulmi (four isolates), O. novo‐ulmi (19 isolates) and ‘fast‐waxy’ (five isolates) was determined in growth media containing carboxymethylcellulose (CMC) and cellulose powder. Differences in enzyme activities were observed among isolates, irrespective of the species and substrate used. Inoculation experiments on Ulmus minor with randomly selected isolates of O. ulmi (two isolates), O. novo‐ulmi (five isolates) and ‘fast‐waxy’ (two isolates) were also performed. Disease was assessed as the percentage of leaves showing yellowing and browning. Ophiostoma novo‐ulmi and ‘fast‐waxy’ isolates exhibited a great variability in their capacities to cause the disease. In the presence of CMC, a significant correlation between the activity of exoglucanase and β‐glucosidase in vitro and virulence was found.     

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.     

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.     

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.     

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.