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.     

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.     

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.     

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.     

‘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.     

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.     

Penetration of Picea sitchensis root bark by Armillaria mellea,Armillaria ostoyae and Heterobasidion annosum

Penetration of root bark tissues of Picea sitchensis by Armillaria ostoyae, Armillaria mellea and Heterobasidion annosum was examined in the absence of wounds, in superficial wounds (rhytidome tissues removed to expose the secondary phloem) and in wounds to the depth of the vascular cambium (deep wounding). Both species of Armillaria penetrated bark without prior wounding, but neither species formed rhizomorphs in this treatment. Armillaria ostoyae penetrated to 39 cell layers in depth by 48 days after inoculation of unwounded bark, whereas A. mellea penetrated 25 cell layers in the same time. Armillaria mellea penetrated superficial wounds significantly more rapidly than did A. ostoyae. Both species produced rhizomorphs within wounded host tissues. Inoculation of deep wounds with Armillaria resulted in a greater depth of bark necrosis with A. mellea than with A. ostoyae. In the absence of wounding, H. annosum failed to penetrate root bark tissues, but in both superficial and deep wounds hyphae penetrated beyond the ligno–suberized boundary zone (LSZ) by 12 days after inoculation. Where no inoculations were made, superficial or deep wounding led within 25 days to the restoration of a structurally continuous LSZ, and by day 48 the wound periderm (WP) was fully differentiated. In inoculated wounds, however, formation of the LSZ and WP was delayed or inhibited in most trees, particularly following inoculation with A. ostoyae or A. mellea. Suberization in the LSZ and WP remained diffuse and discontinuous 48 days after inoculation. Moreover, the presence of WP did not prevent further penetration of the tissues by the pathogens. Variations between trees in the depth of pathogen penetration were noted, possibly indicating differing susceptibilities of individual host genotypes. The possible host factors involved in resistance to penetration of root bark tissues by Armillaria and Heterobasidion are discussed.     

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.     

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.     

Variation in nutrient concentrations of Pinus sylvestris stems

Spatial and temporal variation of N, P, K, Ca, Mg, Mn, Zn, B and Al concentrations in Scots pine (Pinus sylvestris L.) bark and stemwood was studied in three stands at different stages of development. Concentrations of the mobile nutrients N, P and K increased in stemwood and inner bark towards the youngest tissues vertically towards the top of the stem, and horizontally from the pith to the youngest annual rings. The concentrations of Ca, Mg, Mn and Zn were greatest in the oldest tissues and decreased horizontally towards the youngest annual rings. There was a slight increase of most nutrients near the divide between sapwood and heartwood. Seasonal nutrient variation was greatest in the inner bark. The highest nutrient concentrations occurred in winter and spring. Concentrations also varied with stand age, especially in the inner bark, being lowest in the oldest stand.     

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.     

Interactions between callus cultures of Pinus sylvestris and pine fungi with different trophic properties

Fungal virulence may be studied using tissues cultures of host plants in dual cultures in vitro, enabling analyses of interactions with undifferentiated cells of their host plants. Three genotypes of Pinus sylvestris callus, initiated by somatic embryogenesis, were used for establishing dual cultures with fungi pathogenic, endophytic or saprotrophic on pine needles or shoots. Fungal growth towards the plant callus tissue differed, depending on the life strategy of the fungus. The pathogen Gremmeniella abietina proved the slowest colonizer of callus whereas the saprotrophic Phacidium lacerum was the fastest. Gremmeniella abietina partially overgrew the callus, causing extensive necrosis and death within 10 days after inoculation. Anthostomella formosa, an endophyte of pines, did not cause evident symptoms of callus degradation: after 10 days of dual culture, the callus cells remained greenish and at least 50% of cells were alive. In dual cultures Ph. lacerum, callus remained alive until the end of the experiment, maintaining a white‐creamy colour with a loose cell structure. Electrophoresis of protein extracts from the callus showed the presence of additional bands of 25–35 kDa only in host tissues challenged with the pathogen G. abietina, possibly indicating the production of pathogenesis‐related proteins. This work has shown that pine callus does not respond equally to challenge with different fungal isolates. In general, one‐third of the isolates of each fungus examined showed greater virulence compared to other isolates.     

Preliminary studies on establishment of genetic transformation system in loblolly pine

A transformation system was established for loblolly pine (Pinus taeda L.) mature zygotic embryos usingAgrobacterium tumefaciens. The gene coding for the β-glucuronidase (GUS) gene was introduced into loblolly pine tissues and its transient expression was detected with histochemical staining. The influences of different genotypes.Agrobacterum concentrations. and cocultivation time on GUS expression and kanamycin resistant callus and shoot regeneration were investigated. The results showed that the highest GUS expression frequency (16.3%) and shoot regeneration frequency (78%) were obtained from genotype 9–1003 withAgrobacterium concentration decreased 9 times and cocultivation time of 56 hours respectively. GUS expression was obtained in all genotypes tested. The successful expression of the GUS gene in different genotypes suggested that it will be a useful transformation system for loblolly pine. (Responsible Editor: Chai Ruihai)     

The importance of wild plant species as potential inoculum reservoirs of white root rot disease

Rosellinia necatrix is a soilborne plant pathogenic fungus belonging to the phylum Ascomycota. The fungus is distributed widely in temperate zones and often causes huge economic losses on numerous crop plants. To determine the potential for wild plants to serve as inoculum reservoirs of white root rot disease, eight woody and two herbaceous plant species belonging to nine families were inoculated with two R. necatrix isolates. The species used were Actinidia polygama, Broussonetia kazinoki, Camellia sinensis, Castanea crenata, Hydrangea serrata, Magnolia obovata, Pteridium aquilinum, Petasites japonicus, Quercus serrata and Rosa multiflora, all common flora in the cool temperate forests of Japan. The mortality of plants varied between species, independent of the effect of fungal isolates. Although some of the plants survived fungal infection, all plant species tested were more or less susceptible, having rotting lesions on the inner bark and cortex. For the woody plants, most of the mycelial strands in the diseased tissues became plumose mycelia, growing compressed between the outer and inner barks. However, mycelia found in diseased tissues of the two herbaceous plants were not in the form of plumose mycelia. All plant species harboured mycelial strands on the surface of their root systems and underground main stem, and the amount of the mycelial strands did not correlate with the mortality of the plants. Even if plants exhibited no external symptoms, some of them harboured abundant mycelial strands. In conclusion, wild plants can serve as alternative host plants for R. necatrix and function as persistent inoculum reservoirs, irrespective of their apparent health status. Thus, assessments of such latent host plants must be considered in the course of managing white root rot.     

Ophiostomatoid fungi associated with root‐feeding bark beetles on Scots pine in Poland

Ophiostomatoid fungi are known to be associated with various species of bark beetles. However, information about fungal associates of root‐feeding bark beetles in Europe is still fragmentary. For this reason, the fungal associates of Hylastes ater, H. opacus and Hylurgus ligniperda on Pinus sylvestris were isolated and identified. A total of 743 fungal isolates were collected and separated into 10 morphological groups. Analyses of ITS rDNA and partial β‐tubulin gene sequences confirmed that these groups represented distinct species. The 10 species included a total of 13 associations between fungi and bark beetles that had not been recorded previously. All of the bark beetles examined were frequently associated with ophiostomatoid fungi. The fungal diversity and relative abundance of species were very similar in the three species of root‐feeding bark beetles. The most commonly encountered associates of these beetles were Grosmannia radiaticola, Leptographium lundbergii, L. procerum and L. truncatum. Insect infestation data furthermore suggest that Hylastes spp. and Hg. ligniperda are also important vectors of the fungal pathogen Sphaeropsis sapinea.     

Amino nitrogen and phenolic constituents of bark of American beech,Fagus grandifolia,and infestation by beech scale,Cryptococcus fagisuga

Concentrations of amino acids, total amino nitrogen, and phenols, ratio of phenol to amino nitrogen, and pH in the bark of American beech, Fagus grandifolia, were determined to see if these chemical constituents were correlated with susceptibility of trees to infestation by the beech scale, Cryptococcus fagisuga. The relationship of these bark constituents with scale infestation levels (none, light, and moderate), tree size (large vs. small trees), and bark shading was determined. Levels of some individual amino acids and total amino acid content in moderately infested trees were significantly higher than in uninfested trees. Large uninfested beech trees, which tend to be more readily infested by scale, had significantly higher concentrations of aspartic acid. Concentrations of phenol in the outer bark of both infested and uninfested trees were significantly higher than in the inner bark. However in bark infected by Nectria, phenol levels in the inner bark were significantly higher than in the outer bark.     

Changes in root growth and relationships between plant organs and root hydraulic traits in American elm (Ulmus americana L.) and red oak (Quercus rubra L.) seedlings due to elevated CO2 level

The relationships between plant organs and root hydrological traits are not well known and the question arises whether elevated CO2 changes these relationships. This study attempted to answer this question. A pseudo-replicated experiment was conducted with two times 24 American elm (Ulmus americana L.) and 23 and 24 red oak (Quercus rubra L.) seedlings growing in ambient CO2 (around 360 μmol·L–1) and 540 ± 7.95 μmol·L–1 CO₂ in a greenhouse. After 71 days of treatment for American elm and 77 days for red oak, 14 American elm and 12 red oak seedlings from each of the two CO2 levels were randomly selected in order to examine the flow rate of root xylem sap, root hydraulic conductance, total root hydraulic conductivity, fine root and coarse root hydraulic conductivity. All seedlings were harvested to investigate total plant biomass, stem biomass and leaf biomass, leaf area, height, basal diameter, total root biomass, coarse root biomass and fine root biomass. The following conclusions are reached: 1) plant organs respond to the elevated CO2 level earlier than hydraulic traits of roots and may gradually lead to changes in hydraulic traits; 2) plant organs have different relationships with hydraulic traits of roots and elevated CO2 changes these relationships; the changes may be of importance for plants as means to acclimatize to changing environments; 3) biomass of coarse roots increased rather more than that of fine roots; 4) Lorentzian and Caussian models are better in estimating the biomass of seedlings than single-variable models.     

Infektion und abbau der Wurzelrinde von Fichte durch Fomes annaus1

Infection and Deterioration of Spruce Root Bark by Fomes Annosus . The paper describes the mode of infection and consequent deterioration of spruce root bark by the root rot fungus, Fomes annosus using light and electron microscope observations. Spread of fungal hyphae occurs through bore holes produced by enzymatic activity. Although a definte loss of wall substance occurs, the bark tissues primarily represent merely a pathway for the fungus before it colonieses the xylem.     

The effect of bark decortication for hiwada production on xylem and phloem formation in Chamaecyparis obtusa

Of all plant materials used to cover the roofs of traditional Japanese buildings, Japanese cypress (Chamaecyparis obtusa) bark, hiwada, has the longest service life and has been used from ancient times. However, wood and bark properties after hiwada harvest have not been evaluated in detail. We studied whether decortication for hiwada production in winter affected xylem and phloem formation. Decorticated trees still preserved all inner bark and part of the outer bark, and both decorticated and control trees had similar annual ring structures at all stem heights in the xylem and phloem. In both xylem and inner bark, no significant difference in ring width at any stem height was found between annual rings before and after decortication. Thus, this study revealed that the decortication of bark for hiwada production does not affect the formation of xylem and the inner and outer bark if decortication is carried out by highly skilled workers in winter.