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.     

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.     

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.     

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.     

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.

Evolving strategies for biological control of weeds with plant pathogens

Current biological control strategies involve the use of exotic or native biocontrol agents, proven to be host-specific and lethal. This is a successful field of endeavor, attractive lo a large cadre of entomologists and a few plant pathologists. The main limitation is that each weed species must be attacked by a separate host-specific agent and these organisms are not always lethal. Only by using a radical paradigm shift can we reach a new strategy of biocontrol. In this strategy, lethal, broad host-range pathogens are genetically modified to permit their safe release. Either they are rendered host-specific or they are given a chemical dependency that prevents their spread or long-term survival. This genetic-manipulative approach offers numerous and diverse scenarios for biocontrol. Host-specific promoters or toxins, host-dependency by multiple auxotrophy, or mutants dependent on specific environmental conditions are all approaches that lend themselves to genetic modification. Such self-delimiting pathogens may have the advantage of repeated marketability, environmental safely and multiple target weeds.     

The growth of aggressive and non-aggressive strains of Ophiostoma ulmi in susceptible and resistant elms,a scanning electron microscopical study

Different wood preparation techniques for a SEM study of the growth ofOphiostoma ulmi (Buisman) Nannf. in elm tissue were compared. Critical point drying appeared to be the most suitable method. All observations were made 10–14 cm above the site of inoculation. In the resistant elm,Ulmus hollandica cl. 390, inoculated with a non-aggressive strain ofO. ulmi, no hyphae could be found until 5 days after inoculation. However, whenU. hollandica cl. 390 was inoculated with the aggressive strain, the fungus could already be observed after 3 days. In the susceptible elm,U. hollandica cl. Belgica, whether inoculated with an aggressive or non-aggressive strain, hyphae were found within 2 days after inoculation. The fungus appears only to penetrate the vessel pits and has at no time been seen to penetrate the cell wall directly.Samenvatting Verschillende houtpreparatie-technieken voor bestudering van de groei vanOphiostoma ulmi in iepeweefsel met behulp van de SEM werden vergeleken. Alle waarnemingen werden 10–14 cm boven de plaats van inoculatie gedaan. Critical point drying bleek de meest bruikbare methode. In de resistente iep,Ulmus hollandica kloon 390, geïnoculeerd met een niet-agressieve stam vanO. ulmi werden eerst 5 dagen na inoculatie hyfen gevonden in tegenstelling tot de vatbare iep,U. hollandica kloon Belgica waarin zowel na inoculatie met de agressieve als met de niet-agressieve stam hyfen konden worden gevonden binnen 2 dagen. AlsU. hollandica kloon 390 geïnoculeerd werd met de agressieve stam kon de schimmel reeds na 3 dagen worden gevonden. Binnendringen van de schimmel door de vaatwand werd waargenomen, maar alleen via de stippels.     

Studies on the biological control of plant pathogens in Finland1

Studies on biological control of plant pathogens in Finland were started by Pohjakallio in the 1950s, concentrating especially on the microbes parasitizing fungal sclerotia. At the turn of the 70s, Kallio studied the protective effect of Finnish isolates of Phlebia gigantea against the most important forest pathogen Heterobasidion annosum with positive results. The preparation was put on the market. Later in the 1970s, the observation by Tahvonen that Finnish light-coloured peat had an inhibitory effect on the infection of plants by some seed and soil-borne fungal pathogens led to further investigations. Of the microbes isolated from peat, Streptomyces spp. proved the most effective antagonists in both in vitro and in vivo tests. Studies are in progress with S. griseoviridis and several other fungal and bacterial antagonists as potential biocontrol agents against some seed and soil-borne pathogens and storage diseases.     

Production in vitro of phytotoxic compounds by non-aggressive and aggressive isolates of Ophiostoma ulmi, the Dutch elm disease pathogen

Development in shake culture of two non-aggressive and two aggressive isolates of the Dutch elm disease fungus, Ophiostoma ulmi, was monitored in a time course study. The time sequences for the production of total biomass, spores, polysaccharides and phytotoxic compounds were followed, but none of them showed a clear correlation with aggressiveness. The same parameters were examined for nine non-aggressive and 11 aggressive isolates after 10 days growth. Of two phytotoxic compounds, a glycopeptide and the protein cerato-ulmin, only the latter was generally produced in larger quantities by aggressive isolates. However, variability in production and a number of exceptions to the trend made cerato-ulmin production an uncertain criterion for assessing the aggressiveness of an individual isolate.Immunofluorescence of sections of infected elms treated with an anti-glycopeptide serum showed intense fluorescence only in the cell walls of the fungus. Apparently the glycopeptide is abundantly present in or on fungal cell walls.The roles of the different compounds in relation to Dutch elm disease symptom development are discussed.