Genetic evidence for natural hybridization between the Dutch elm disease pathogens Ophiostoma novo-ulmi ssp. novo-ulmi and O. novo-ulmi ssp. americana

The Dutch elm disease pathogen Ophiostoma novo-ulmi is separated into subspecies novo-ulmi , formerly known as the Eurasian (EAN) race, and subspecies americana , formerly known as the North American (NAN) race. Both subspecies occur in Europe, and hybrids between them are suspected to have emerged in parts of Europe where their ranges overlap. Authenticated isolates of both subspecies were examined in order to determine whether fixed mutations occur in the cerato-ulmin ( cu ) and in the colony type gene ( col1 ). One and six mutations were detected between subspp. novo-ulmi and americana in cu and col1 , respectively. The mutation in cu and one mutation in col1 proved to be located within restriction sites, and were used for PCR-RFLP. This method provides a quick and reliable diagnostic method to differentiate the two subspecies. Seven isolates of O. novo-ulmi from Austria were suspected to be hybrids between subspp. novo-ulmi and americana , and were tested by PCR-RFLP of their cu and col1 genes. Two of the suspected hybrids from Austria (isolates AT73 and AT146) had the cu PCR-RFLP profile of ssp. americana and the col1 PCR-RFLP profile of ssp. novo-ulmi . Furthermore, a ssp. novo-ulmi tester isolate from Poland (P150) showed a similar hybrid pattern. This is the first evidence revealing recombination between two genes and thus hybridization between the two subspecies of O. novo-ulmi in nature.     

Monoclonal antibodies to specific components of the Dutch elm disease pathogen Ophiostoma ulmi

The potential of polyclonal antisera and monoclonal antibodies to differentiate the EAN and NAN aggressive subgroups of Ophiostoma ulmi was explored. Polyclonal antisera, when tested by ELISA, cross-reacted widely with unrelated species and failed to distinguish between the two aggressive subgroups but small quantitative differences were found, particularly between antigens secreted overnight, by EAN and NAN germlings. Monoclonal antibodies were raised in mice against mycelial homogenates. From two fusions, 33 cell lines were raised that secreted antibodies positive for O. ulmi. Approximately one third were non-specific; 11 were specific either to species or subspecies. Two cell lines differentiated mycelial antigens of the aggressive isolates of O. ulmi from those of the non-aggressive subgroup, but not antigens from surface washings. Only quantitative differences were detected between the EAN and NAN aggressive subgroups. Almost all the monoclonal antibodies and antiserum recognized antigens present in surface washings of cultures on solid medium, in cell-free extracts of mycelial homogenates, in cell-free culture fluids, and in substances secreted overnight by germinating spores. Specific detection of such molecules promises to provide a highly sensitive mechanism for studying early pathogen/host plant interactions. Most of the monoclonal antibodies appeared to have potential diagnostic value; they gave readings twofold to tenfold higher with extracts from diseased than from healthy tissue. However, one cell line that secreted antibodies specific to O. ulmi cross-reacted strongly with extracts of healthy tissue.     

Analysis of Spanish populations of Ophiostoma ulmi and O. novo-ulmi using phenotypic characteristics and RAPD markers

One hundred and six isolates of the Dutch elm disease (DED) fungi Ophiostoma ulmi and Ophiostoma novo-ulmi were collected from elm trees with symptoms in 15 regions of Spain. Isolates were compared with eight reference strains belonging to O. ulmi and the two subspecies of O. novo-ulmi. The purpose of this study was to assign Spanish isolates to species and subspecies of the DED fungi and to analyse the genetic variability within the Spanish populations of these pathogens. Isolates were examined for their growth rates, colony morphologies and fertility responses and by using random amplified polymorphic DNA (RAPD) markers. Six isolates were identified as O. ulmi , 16 as O. novo-ulmi ssp. novo-ulmi and 78 as O. novo-ulmi ssp. americana . Nei's and Shannon's diversity indices and the upgma dendrogram from RAPD profiles indicated a high level of variation among isolates, probably reflecting post-epidemic status of DED in Spain. Although most isolates were separated into three major clusters representing the three taxa of DED fungi in the RAPD analysis, two isolates from central Spain clustered between O. novo-ulmi ssp. americana and O. novo-ulmi ssp. novo-ulmi , and four isolates from Mallorca clustered between O. ulmi and the group representing O. novo-ulmi ssp. novo-ulmi . Mating tests conducted with these isolates revealed a variety of fertility responses. The novel combinations between molecular profile and fertility reaction suggest that three isolates from Mallorca may be interspecific hybrids of the DED fungi.     

Evaluating Verticillium dahliae for biological control of Ophiostoma novo-ulmi in Ulmus minor

The ability of isolate Vd-48 of Verticillium dahliae to induce resistance against subsequent challenge with Ophiostoma novo-ulmi was examined in Ulmus minor . In the first experiment, conditioning inoculation of 5-year-old elm trees (2–3 m in height) with Vd-48 15 days prior to challenge inoculation with O. novo-ulmi significantly reduced wilting ( P  ≤ 0·05) compared with trees not conditioned with Vd-48. However, in another experiment on 6-year-old trees (2–3·5 m in height), no protection was achieved when the length of time between conditioning and challenge inoculations was 45 or 60 days. In a further experiment, inoculations with Vd-48 alone resulted in severe wilting in 22 out of 118 trees (6–10 years old and 4–7 m in height). Across the 2 years of this last trial, nine trees showed massive feeding wounds made by Scolytus sp., while O. novo-ulmi strains were isolated from six trees. Vd-48 provided a variable prophylactic effect against O. novo-ulmi in U. minor . Major difficulties with this approach to control Dutch elm disease are discussed.     

Influence of the d2 factor on survival and infection by the Dutch elm disease pathogen Ophiostoma ulmi

A particular form of mycovirus, the d² factor, which attacks the elm pathogen Ophiostoma ulmi, was found to be extremely deleterious to this fungus both in vitro and in vivo. Most significantly, d²-infected conidia were poor at persisting in feeding grooves, the usual infection court of the pathogen, and also showed a 50-fold reduction in successful xylem infection compared with their healthy counterparts. In addition, transmission of the d² factor from diseased to healthy isolates of O. ulmi was found to take place in feeding grooves, indicating that the fungus exists in a mycelial phase prior to invading xylem vessels.     

Pseudomonas for biological control of Dutch elm disease. III. Field trials at various locations in the Netherlands

The prophylactic effect in elm of one treatment with aPseudomonas isolate was monitored in two types of field trials. In one type only natural Dutch elm disease infections were monitored and hence large numbers of trees were necessary due to the low incidence of natural occurring infections. In the other type trees were artificially infected.The large-scale field trials in which only natural infections were monitored, were based on expected annual losses due to Dutch elm disease of approximately 2%. As a result of the Dutch sanitation program, which was based on the prompt removal of every weakened or diseased elm, the actual losses were generally threefold lower. Dutch elm disease incidence was 22–45% lower in the trees treated with aPseudomonas isolate in the year of treatment and the year after. The results of the biocontrol treatment were negatively influenced because on several locations trees were felled that showed initial signs of Dutch elm disease, which probably would have disappeared during the season.The advantage of artificial infections withOphiostoma ulmi was a reproducable development of symptoms and the possibility to maintain diseased trees, at least till the first signs of elm bark beetle breeding. For Commelin elms an increase in symptoms was observed with increasingO. ulmi dose till 3000 conidia per tree; the standard 500 000 conidia used for most experiments was well above this critical value. No decrease in effectiveness of the bacterial pre-treatment was observed with increasingO. ulmi inoculum. Different bacterial treatments suggested that injections at a smaller interval (i.e. more injections per tree) may result in a better prophylactic effect, but the significance of the correlation remained doubtful. A comparison of several elm species and clones showed the importance of the host tree. Prophylaxis as a result of one bacterial treatment was shown repeatedly in Commelin elms; the numbers of trees showing symptoms by the end of the second year were 10 to 85% lower in the bacteria-treated groups in comparison with the controls. Also in one experiment with Belgica elms prophylaxis was observed, resulting in a 84% decrease in the number of trees showing symptoms by the end of the second year after the prophylactic treatment followed by inoculation withO. ulmi. In Vegeta symptom development was only less severe and in field elms (Ulmus carpinifolia) some prophylactic effect was observed in one experiment, but no effect in two others.Samenvatting De mogelijke bescherming tegen de iepeziekte, verkregen door injectie van de boom met bacteriën van het geslachtPseudomonas, werd gemeten in twee soorten experimenten. In het ene soort werden natuurlijke infecties gemeten, waardoor grote proefgroepen nodig waren. In het andere soort werden de iepen kunstmatig geinfecteerd.De grootschalige veldexperimenten waarbij natuurlijke iepeziekte-infecties werden gemeten, waren gebaseerd op een verwachte jaarlijkse uitval van 2%. Als gevolg van de landelijke bestrijdingscampagne bleken de verliezen slechts ongeveer een derde hiervan te zijn. Er kwam minder iepeziekte voor in de metPseudomonas geïnjecteerde bomen in het jaar van injectie en in het jaar daarna. Een storende invloed op de resultaten had het effect dat ook met bacteriën geïnjecteerde bomen soms beginnende symptomen vertonen na infectie metOphiostoma ulmi, symptomen die in de loop van het seizoen soms weer verdwijnen. Als gevolg van de bestrijdingscampagne werden zulke bomen toch geveld.Het voordeel van kunstmatige infecties metO. ulmi was een voorspelbaar verloop van de symptoomontwikkeling en de mogelijkheid om zieke bomen te laten staan tot er iepespintkevers in kwamen. In Commelin iepen bleken de symptomen toe te nemen met een tot 3000 conidiën per boom toenemende dosisO. ulmi. De gebruikelijke 500000 conidiën die in de meeste experimenten werden gebruikt lagen ver boven deze kritische waarde. Er werd geen effect van een toenemende dosisO. ulmi op de effectiviteit van een bacteriebehandeling waargenomen.Uit variaties in de diverse bacteriebehandelingen kwam naar voren dat injecties met een kleinere tussenruimte (dus meer injecties per boom) mogelijk het effect verbeterden, maar de significantie van deze correlatie bleef twijfelachtig.Vergelijken van diverse iepen toont dat soort en kloon type een belangrijke rol speelt bij deze bestrijdingsmethode. Bescherming tegen de iepeziekte als gevolg van een bacteriebehandeling werd diverse malen aangetoond in Commelin iepen; het aantal bomen met iepeziekte-symptomen was aan het eind van het tweede seizoen in de met bacteriën behandelde groepen 10 tot 85 % lager dan in de controlegroepen. Ook in een experiment met Belgica iepen werd een goede bescherming gemeten. In Vegeta werd slechts een verminderde symptoomontwikkeling gemeten en in veldiepen (U. carpinifolia) werd enige bescherming gevonden in één experiment, maar geen effect in twee andere.     

Carbendazim formulations for the eradication of Dutch elm disease

Formulations containing carbendazim (40 or 80 mM) in hydrochloric acid (180 or 320 mM) were pressure-injected into elms previously inoculated with the fungus Ceratocystis ulmi (the cause of Dutch elm disease) and their performance as eradicants assessed. All formulations caused some damage to foliage but leaves regenerated. Treatment with carbendazim (80 mM) in hydrochloric acid (180 mM) resulted in four out of five trees remaining free from disease, not only immediately after treatment, but also throughout the following season. Injection of acid alone resulted in rapid death of the trees.     

Transmission of double-stranded RNA and a disease factor in Ophiostoma ulmi

A diseased isolate of Ophiostoma ulmi was found to contain 10 segments of double-stranded RNA (dsRNA) with molecular weights ranging from 2.40 x 10⁶ to 0.23 x 10⁶. In contrast seven other healthy isolates in the same vegetative compatibility group contained either no dsRNA or up to four dsRNA segments. Transmission of the disease to healthy isolates by hyphal anastomosis was accompanied by-transmission of the 10 dsRNA segments. In a genetic cross in which the diseased isolate acted as the female parent single-ascospore progeny were healthy and either contained no dsRNA or only one segment of dsRNA. When elm trees were inoculated with diseased isolates, subsequent reisolations were healthy and retained only two to seven of the dsRNA segments or were diseased and retained all 10 dsRNA segments. Following conidiogenesis a diseased isolate gave rise to single-conidial progeny which were either slow growing and diseased, like their parent, with all 10 dsRNA segments, or faster growing like healthy isolates. Some of the faster growing conidial isolates retained only two to seven of the dsRNA segments and were disease-free. However a majority of the faster growing conidial isolates retained all 10 dsRN A segments and were shown to carry the disease in a latent form. The possibility that the disease of O. ulmi is conferred by specific segments of dsRNA and the potential of d-factors for the control of Dutch elm disease are discussed.     

A rapid virulence assay for the Dutch elm disease fungus Ophiostoma novo‐ulmi by inoculation of apple (Malus × domestica ‘Golden Delicious’) fruits

Large‐scale virulence tests using trees or saplings are expensive, time‐consuming and require a considerable amount of space. The suitability of using ‘Golden Delicious’ apples as a rapid screen for identifying Ophiostoma novo‐ulmi transformants with reduced virulence was thus evaluated. When a collection of O. novo‐ulmi field isolates belonging to subspecies novo‐ulmi or americana was inoculated to apples, members of subsp. novo‐ulmi induced, on average, larger necrotic lesions than subsp. americana isolates. The size of the lesions on apples was not correlated with mycelial growth rate of isolates on nutrient agar. Insertional mutants from O. novo‐ulmi subsp. novo‐ulmi isolate H327 were inoculated to ‘Golden Delicious’ apples and Ulmus parvifolia × U. americana saplings in parallel experiments. Results clearly indicated that the O. novo‐ulmi transformants included several exhibiting significantly altered levels of virulence. Variability among replicates within a treatment was reduced in apple inoculation data compared to elm sapling data. Overall, the ‘Golden Delicious’ apple assay was found to be an excellent means for rapidly assessing the virulence level of O. novo‐ulmi isolates.     

Formulations for the control of dutch elm disease

Solutions of methyl benzimidazol-2-ylcarbamate (carbendazim, formerly MBC) in dilute hydrochloric, orthophosphoric or hypophosphorous acid and in dilute hydrochloric acid containing methanol (“Lignasan”) and aqueous solutions of 2-(thiazol-4-yl) benzimidazole (thiabendazole) hypophosphite, 8-hydroxyquinoline (oxine)hydro-chloride and dodecylguanidine acetate (dodine), were tested in the field for their efficacy in controlling Dutch elm disease caused by Ceratocystis ulmi. All gave some degree of control although some were phytotoxic, the benzimidazoles being the most active compounds tested. The anion and the concentration of acid present in the formulation may have some effect on retention of active material within the crown of the tree.     

China and the origins of Dutch elm disease: an appraisal

Circumstantial evidence has led to the widespread assumption that Dutch elm disease (caused by Ophiostoma ulmi ) spread to Europe and North America from China. The author's recent surveys indicate that the disease is probably absent from China, and only a relatively recent arrival in the Soviet Tien Shan mountain region on the western Chinese border. Alternative origins for the disease include introduction from the Himalayas, and recent rapid evolution within Europe from a fungus such as Ophiostoma piceae . Elucidation of the various possibilities is complicated by the need to account for the spread of at least three genetically divergent subgroups of the pathogen.     

Differences in cerato-ulmin production between the EAN, NAN and non-aggressive subgroups of Ophiostoma ulmi

A test of comparative in vitro cerato-ulmin wilt toxin production in the aggressive and non-aggressive subgroups of the Dutch elm disease pathogen Ophiostoma ulmi was carried out by turbidity and ELISA methods. Ten non-aggressive, ten EAN aggressive and ten NAN aggressive isolates were tested from a range of geographical sources. In liquid shake cultures the non-aggressive isolates produced the greatest and the NAN aggressives the least mean biomass. Despite considerable variation in cerato-ulmin production by individual isolates in three separate experiments, both the turbidity and ELISA methods showed a clear separation of the non-aggressive and aggressive subgroups. Non-aggressive isolates produced little or no cerato-ulmin (ELISA range of means 0–56.0 ng/ml) and EAN and NAN aggressive isolates moderate to high levels (EAN 1.6–89.0 × 10⁴ ng/ml and NAN 0.2–300 × 10⁴ ng/ml). In the aggressive isolates no correlation was detected between cerato-ulmin production and either biomass or pathogenicity to clonal Commelin elm. The role of cerato-ulmin in the pathogenicity of O. ulmi is discussed.     

Control of Dutch elm disease by the sterol biosynthesis inhibitors fenpropimorph and fenpropidin

Sterol biosynthesis inhibitors that inhibit the yeast-hyphae conversion inOphiostoma ulmi suppressed Dutch elm disease development in two elm clones. After curative treatment with fenpropimorph-sulphate of 27 Vegeta elms which had previously been inoculated withO. ulmi, 25 trees did not show disease symptoms by the end of the second season. All 41 control trees, inoculated withO. ulmi only, were clearly diseased. In an experiment with Commelin elms three fenpropimorph salts and thiabendazole were compared. Injection of the trees three weeks after inoculation withO. ulmi gave by the end of the second season no symptoms of Dutch elm disease in any of the trees injected with fenpropimorph-phosphate or thiabendazole, and in most trees injected with fenpropimorph-acetate or-sulphate. Similar treatments with the free base of fenpropimorph and fenpropidin-sulphate were less effective due to insufficient uptake of the fenpropimorph emulsion and phytotoxicity of fenpropidin-sulphate, respectively. Injection of fenpropimorph-sulphate or thiabendazole six weeks after inoculation withO. ulmi did not result in significant differences from the control group inoculated withO. ulmi only.Fenpropimorph-phosphate and-sulphate completely suppressed Dutch elm disease upon injection of only 7.5 or 10 g per tree (average tree diameter 28 cm). Residue analyses showed only a slow decrease in concentration of the fungicide over two growing seasons and an apparent transport into the new annual ring, other prerequisites for a possible future use for control of Dutch elm disease.Samenvatting De iepeziekte kan onderdrukt worden door sterolbiosyntheseremmers die de overgang vanOphiostoma ulmi van de gistvorm in de hyfevorm remmen. Aan het eind van het tweede seizoen na een curatieve behandeling van 27 Vegeta iepen met fenpropimorfsulfaat bleken 25 bomen geen symptomen van iepeziekte te vertonen. Alle controlebomen, die alleen metO. ulmi geïnoculeerd waren, waren duidelijk ziek. In een proef met Commelin iepen werden drie fenpropimorfzouten en thiabendazool vergeleken. De zouten werden drie weken na de inoculatie metO. ulmi geïnjecteerd. Aan het eind van het tweede seizoen vertoonden geen van de bomen die met fenpropimorffosfaat of thiabendazool geïnjecteerd waren en slechts enkele bomen die met fenpropimorfacetaat of-sulfaat geïnjecteerd waren iepeziektesymptomen. Behandelingen met fenpropimorf (vrije base) en fenpropidinsulfaat werkten minder goed door de slechte opname van de fenpropimorfemulsie en de fytotoxiciteit van fenpropidin. Injectie met fenpropimorfsulfaat of thiabendazool zes weken na inoculatie leidde niet tot significante verschillen met de controlegroep die alleen metO. ulmi geïnoculeerd was.Een dosis fenpropimorffosfaat of-sulfaat van 7.5 of 10 g per boom met een gemiddelde boomdiameter van 26 cm bleek de iepeziekte volledig te kunnen onderdrukken. Uit residue-onderzoek bleek dat de concentratie van het fungicide gedurende de twee groeiseizoenen slechts langzaam afnam en dat het middel naar de nieuwe jaarring werd getransporteerd, twee voorwaarden voor een toepassing op praktijkschaal van fenpropimorf voor de bestrijding van de iepeziekte.     

Dutch elm disease revisited: past,present and future management in Great Britain

The arrival of a new species of the fungus which causes Dutch elm disease into Great Britain in the 1960s caused widespread elm death and continues to be problematic following elm regeneration. Attempts at managing the disease have been largely unsuccessful. Forty years after the outbreak, however, researchers continue to be interested in both the underlying biology of such a severe and dramatic disease event and in the policy lessons that can be drawn from it. We develop a spatial model at a 1 km² resolution. Following parameterization to replay the historical epidemic, the model is used to explore previously proposed counterfactual management strategies. A new introduction date of late 1962 is estimated. We show that, even had there been high intervention at a national level in terms of disease management early in the epidemic, there would have been little long‐term effect on elm numbers. In Brighton, a local pocket of elm which survived the peak of the initial epidemic has been successfully managed. However, Brighton and similar locations are subject to repeated waves of the disease at a 15‐ to 20‐year intervals following regeneration and reinfection of the surrounding areas, during which much more intensive management is required.     

Linkage of the genes determining mating type and fungicide tolerance in Ophiostoma ulmi

Two allelic mutations conferring high and low levels of in vitro tolerance to the fungicide methyl benzimidazole-2-ylcarbamate (MBC) were identified in the elm pathogen Ophiostoma ulmi. Variants expressing a third and intermediate level of tolerance were also identified. The locus for fungicide tolerance was linked to the mating type locus in both the aggressive and non-aggressive strains of the fungus.     

Pseudomonas for biological control of dutch elm disease. I. Labeling,detection and identification of pseudomonas isolates injected into elms; comparison of various methods

To understand the mechanisms involved in biological control of Dutch elm disease byPseudomonas, data were needed on the distribution of the introduced bacteria within elm and on the development of the bacterial population over a period of time.As traditional biochemical identification techniques are not suitable for distinguishment between individualPseudomonas isolates, three alternative approaches were compared.