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.     

Effects of induced water stress on leaf trichome density and foliar nutrients of three elm (Ulmus) species: implications for resistance to the elm leaf beetle

Seedlings of three elm species with variable susceptibility to the elm leaf beetle (Pyrrhalta luteola Müller) (Coleoptera: Chrysomelidae) were subjected to three water stress treatments (no stress, low stress, and high stress) in a greenhouse experiment. The species tested were Ulmus pumila L. (Siberian elm = highly susceptible), U. parvifolia Jacq. (Chinese elm = resistant), and U. americana L. (American elm = intermediate). The seedlings were analyzed for changes in the levels of selected host traits (trichome density, foliar concentration of nitrogen [N], phosphorus [P], potassium [K], calcium [Ca], magnesium [Mg], iron [Fe], and manganese [Mn]), some of which had previously been implicated in resistance to the elm leaf beetle. Density of leaf abaxial surface trichomes (simple, bulbous, and total trichomes) and foliar Fe and Mg concentrations increased significantly in the highly susceptible Siberian elms under water stress. In contrast, stress reduced trichome density in the moderately susceptible American elms, but it had no effect on levels of foliar mineral nutrients. The stress treatments had no influence on host traits in the resistant Chinese elms. The results suggest that environmental stress can alter plant traits that are likely involved in determining resistance of elms to the elm leaf beetle.     

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.     

Inheritance of pathogenicity and cultural characters in Ceratocystis ulmi: hybridization of aggressive and non-aggressive strains

When British isolates of Ceratocystis ulmi were surveyed for compatibility type, both A- and B-types were found in the non-aggressive strain, but only the B-type in the aggressive strain. Single ascospore progeny from crosses between compatible aggressive and non-aggressive isolates showed a near-normal growth rate distribution, with a mean lying between the parents. Many grew either faster than the aggressive or slower than the non-aggressive parent. The progeny were highly variable in culture morphology and could not be classified in terms of the parental types. When inoculated into English elm they showed a marked skewness towards low pathogenicity. None approached the aggressive strain in pathogenicity. It is concluded that the above characters are under polygenic control, and that the aggressive strain could not arise from the non-aggressive by a simple mutation. The results suggest that the two strains may be reproductively isolated.     

Development of the Dutch elm disease epidemic in southern England, 1971-6

The current epidemic of Dutch elm disease was studied by recording the fate of individual hedgerow elms (Ulmus procera) in five plots in the West Midlands, and by analysing data from successive Forestry Commission surveys of non-woodland elms in 234 plots in southern England. Ninty-five percent of the individual trees died between May 1972 and September 1975. The average infection rate (r) was found to be 1 -35 during the period when the proportion of disease, x, increased from 0–16 to 0–42. In the plots of the main survey the average infection rate was 0–65 and the cumulative loss increased from 6 to 62% between 1971 and 1976, with little evidence that the course of the epidemic was influenced by variations in the weather from year to year. These infection rates are as high as those recorded in Dutch elm disease epidemics elsewhere in the world. The infection rate in English elm was higher than in either the wych elm or the heterogeneous ‘smooth-leaved elm’. The study of English elm in four geographical areas of southern Britain showed that there was an initial drop in infection rate until x = 0–12, when a steady infection rate obtained in all four areas, ranging from 0–56 in the Midlands to 0–76 in the south-east. It is concluded that the epidemic is likely to continue at a high rate until most non-woodland elm have died. Most trees which survive are likely to be smooth-leaved elm in East Anglia. Few communities in southern England have been able to practice vigorous sanitation control programmes, but data from two, in East Sussex and Brighton, are analysed and the effect on disease progress discussed.     

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.     

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.     

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.     

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.     

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.     

Identification of RAPD markers linked to a black leaf spot resistance gene in Chinese elm

Black leaf spot (Stegophora ulmea) is a common foliage disease on Chinese (Ulmus parvifolia) and Siberian elms (U. pumila), two species which have been widely used as sources of Dutch-elm disease-resistance genes for interspecific elm hybrids. A dominant gene controlling resistance to black leaf spot was identified in a population derived from self-pollination of a single U. parvifolia tree. Using RAPD markers, in combination with bulked segregant analysis, we have identified three markers linked to this resistance gene. A survey of Chinese-elm hybrids revealed that the same gene is likely to confer a high level of resistance to black leaf spot in interspecific elm hybrids, although other genetic factors may also be involved in the determination of a disease phenotype.     

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.     

Phenylalanine ammonia-lyase activity in suspension cultures of Ulmus pumila and U. campestris treated with spores of Ceratocystis ulmi

Summary Cell suspension cultures of a Ceratocystis ulmi-resistant (Ulmus pumila) and a -susceptible elm (U.campestris) were established from leaf callus tissue. Treatment of cultures with spores of C.ulmi induced a large increase in the activity of phenylalanine ammonialyase, only in the cells of the resistant species U.pumila with a maximum after 24 h. Inoculated U.pumila cells also excreted a red unidentified chemical into the culture medium. Neither responses were induced in inoculated U.campestris cultures. The results are discussed in relation to the development of the elm cell culture system as a model for studying the differential biochemical mechanisms of disease resistance in elms.     

Response of the elm leaf beetle to host plants induced by oviposition and feeding: the infestation rate matters

We investigated by olfactometry and feeding‐ and oviposition‐choice‐tests how the highly specialised elm leaf beetle, Xanthogaleruca luteola Müller (Coleoptera: Chrysomelidae), responds to conspecifically induced defences in the field elm Ulmus minor Miller (Ulmaceae). While egg deposition of the beetle induced elms to release volatiles attractive to the egg parasitoid Oomyzus gallerucae Fonscolombe (Hymenoptera: Eulophidae), feeding alone did not. In the present study, females of the elm leaf beetle showed preferences for the odours of twigs induced by low egg deposition and feeding over odours from uninfested twigs. In contrast, heavy infestation rendered elm odours less attractive to the beetles. Feeding and oviposition bioassays revealed an oviposition preference for leaves from uninfested twigs when compared to locally infested leaves. However, beetles preferred to feed upon systemically induced leaves compared to uninfested ones. The different preferences of the elm leaf beetle during host plant approach might be explained by a strategy that accounts for both gaining access to high quality nutrition and avoiding competition or parasitism.     

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.     

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.     

Transgenic tobacco plants expressing the coat protein of cucumber mosaic virus show different virus resistance

Transgenic tobacco (Nicotiana tabacum cv. Xanthi-nc) plants were regenerated after cocultivation of leaf explants withAgrobacterium tumefaciens strain LBA4404 harboring a plasmid that contained the coat protein (CP) gene of cucumber mosaic virus (CMV-As). PCR and Southern blot analyses revealed that the CMV CP gene was successfully introduced into the genomic DNA of the transgenic tobacco plants. Transgenic plants (CP⁺) expressing CP were obtained and used for screening the virus resistance. They could be categorized into three types after inoculation with the virus: virus-resistant, delay of symptom development, and susceptible type. Most of the CP⁺ transgenic tobacco plants failed to develop symptoms or showed systemic symptom development delayed for 5 to 42 days as compared to those of nontransgenic control plants after challenged with the same virus. However, some CP⁺ transgenic plants were highly susceptible after inoculation with the virus. Our results suggest that the CP-mediated viral resistance is readily applicable to CMV disease in other crops.     

Resistance to Dutch Elm Disease Reduces Presence of Xylem Endophytic Fungi in Elms (Ulmus spp.)

Efforts to introduce pathogen resistance into landscape tree species by breeding may have unintended consequences for fungal diversity. To address this issue, we compared the frequency and diversity of endophytic fungi and defensive phenolic metabolites in elm (Ulmus spp.) trees with genotypes known to differ in resistance to Dutch elm disease. Our results indicate that resistant U. minor and U. pumila genotypes exhibit a lower frequency and diversity of fungal endophytes in the xylem than susceptible U. minor genotypes. However, resistant and susceptible genotypes showed a similar frequency and diversity of endophytes in the leaves and bark. The resistant and susceptible genotypes could be discriminated on the basis of the phenolic profile of the xylem, but not on basis of phenolics in the leaves or bark. As the Dutch elm disease pathogen develops within xylem tissues, the defensive chemistry of resistant elm genotypes thus appears to be one of the factors that may limit colonization by both the pathogen and endophytes. We discuss a potential trade-off between the benefits of breeding resistance into tree species, versus concomitant losses of fungal endophytes and the ecosystem services they provide.     

Host responses and metabolic profiles of wood components in Dutch elm hybrids with a contrasting tolerance to Dutch elm disease

Background and Aims

Changes occurring in the macromolecular traits of cell wall components in elm wood following attack by Ophiostoma novo-ulmi, the causative agent of Dutch elm disease (DED), are poorly understood. The purpose of this study was to compare host responses and the metabolic profiles of wood components for two Dutch elm (Ulmus) hybrids, ‘Groeneveld’ (a susceptible clone) and ‘Dodoens’ (a tolerant clone), that have contrasting survival strategies upon infection with the current prevalent strain of DED.

Methods

Ten-year-old plants of the hybrid elms were inoculated with O. novo-ulmi ssp. americana × novo-ulmi. Measurements were made of the content of main cell wall components and extractives, lignin monomer composition, macromolecular traits of cellulose and neutral saccharide composition.

Key Results

Upon infection, medium molecular weight macromolecules of cellulose were degraded in both the susceptible and tolerant elm hybrids, resulting in the occurrence of secondary cell wall ruptures and cracks in the vessels, but rarely in the fibres. The ¹³C nuclear magnetic resonance spectra revealed that loss of crystalline and non-crystalline cellulose regions occurred in parallel. The rate of cellulose degradation was influenced by the syringyl:guaiacyl ratio in lignin. Both hybrids commonly responded to the medium molecular weight cellulose degradation with the biosynthesis of high molecular weight macromolecules of cellulose, resulting in a significant increase in values for the degree of polymerization and polydispersity. Other responses of the hybrids included an increase in lignin content, a decrease in relative proportions of d-glucose, and an increase in proportions of d-xylose. Differential responses between the hybrids were found in the syringyl:guaiacyl ratio in lignin.

Conclusions

In susceptible ‘Groeneveld’ plants, syringyl-rich lignin provided a far greater degree of protection from cellulose degradation than in ‘Dodoens’, but only guaiacyl-rich lignin in ‘Dodoens’ plants was involved in successful defence against the fungus. This finding was confirmed by the associations of vanillin and vanillic acid with the DED-tolerant ‘Dodoens’ plants in a multivariate analysis of wood traits.     

Fitness costs and morphological change of laboratory‐selected thiamethoxam resistance in the B‐type Bemisia tabaci (Hemiptera: Aleyrodidae)

Thiamethoxam has been used as a key insecticide to control the whitefly, B‐type Bemisia tabaci, for several years in China with no known cases of resistance in field populations. To evaluate the risk of resistance, a field population was collected and resistant strains were developed by exposure to thiamethoxam in the laboratory. After selection for 36 generation, a strain with 60‐fold resistance was successfully identified. Fitness analysis by constructing life tables, demonstrated that resistant B‐type whiteflies had obvious fitness disadvantages in their development and reproduction. The fitness of resistant B‐type whiteflies decreased dramatically, to only one‐half that of the susceptible strain. Some changes in the morphological characteristics of the resistant strain were observed. The lengths of first, second and third instars of the resistant strain were significantly smaller than those of the susceptible strain, and the width of the first and the fourth instars were also significantly smaller than in the susceptible strain. Our results suggest that the B‐type B. tabaci has the potential to develop high resistance to thiamethoxam, and that the resistance changed the morphology of the insects. The slow development of resistance and the lower fitness of resistant B. tabaci strains may result in a quick recovery of sensitivity when the population is no longer in contact with thiamethoxam in the field.