Ophiostoma novo-ulmi sp. nov., causative agent of current Dutch elm disease pandemics

The aggressive subgroup of the Dutch elm disease pathogen Ophiostoma ulmi (Buism.) Nannf. syn. Ceratocystis ulmi (Buism.) Moreau is named as a new species, O. novo-ulmi, and is thereby separated from the old non-aggressive subgroup, which is retained as O. ulmi. O. novo-ulmi differs from O. ulmi in colony morphology, growth rate, optimum temperature for growth, perithecial neck length, pathogenicity to elm, bark colonising ability, cerato-ulmin protein production, synnemetal and protoperithecial production, mating type frequency, protein and isozyme polymorphisms, mitochondrial DNA and nuclear DNA polymorphisms, and mitochondrial DNA size. In addition, a strong unidirectional fertility barrier operates between the two species, while their hybrids show remarkable variation, poor fitness, and many are infertile. These aspects are summarised. New information on perithecial dimensions is presented. O. ulmi is redefined and a neotype designated. The status of the Eurasian and North American races of O. novo-ulmi is currently under investigation.Abbreviations EAN Eurasian race - NAN North American race     

Accumulation of mansonones E and F in seedlings of Ulmus americana in response to inoculation with Ophiostoma ulmi

Summary The accumulation of mansonones E and F was investigated in Ulmus americana L. seedlings 5 weeks after inoculation with three aggressive and three non-aggressive isolates of Ophiostoma ulmi (Buism.) Nannf. The three non-aggressive isolates stimulated significantly more mansonone E and F accumulation than the three aggressive isolates of O. ulmi. Mansonone induction also varied within both the aggressive and the non-aggressive groups. Aggressive and non-aggressive isolates were recovered in equal frequencies from the inoculation wounds, whereas the aggressive isolates were recovered more frequently than the non-aggressive isolates 15 cm and 25 cm up the seedlings' stem. Vascular browning in the outer xylem of the seedlings correlated with mansonone E and F accumulation. Mansonone accumulation in U. americana seedlings is therefore associated with vascular browning and a reduction in fungal spread.     

Electrophoretic karyotypes of the elm tree pathogen Ophiostoma ulmi (sensu lato)

Pulsed field gel electrophoresis using OFAGE, TAFE, and CHEF systems has been used to more fully characterize karyotypic variation within the two closely related fungal species of Ophiostoma ulmi sensu lato. Twelve wild-type and laboratory strains, representing the less agressive species O. ulmi and both of the biotypes of the more aggressive species O. novo-ulmi were studied and their karyotypes determined. Depending on the strain, a minimum of four to a minimum of eight chromosomal DNA bands were present that fall into three distinct size classes, with one exception. Strain CESSI6K (O. novo-ulmi, North American aggressive subgroup) contains a unique chromosomal DNA band which comigrated near a Saccharomyces cerevisiae chromosome of 0.95 Mb. This unique band was the smallest O. ulmi s. l. chromosomal DNA observed. Seven of the twelve strains shared a common chromosomal DNA banding pattern, whereas each of the other five had a unique karyotype. There was no correlation between chromosome profile and species, as some O. novo-ulmi and O. ulmi strains shared common electrophoretic karyotypes.     

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.     

Electrophoretic karyotypes of the elm tree pathogen Ophiostoma ulmi (sensu lato)

Pulsed field gel electrophoresis using OFAGE, TAFE, and CHEF systems has been used to more fully characterize karyotypic variation within the two closely related fungal species of Ophiostoma ulmi sensu lato. Twelve wild-type and laboratory strains, representing the less agressive species O. ulmi and both of the biotypes of the more aggressive species O. novo-ulmi were studied and their karyotypes determined. Depending on the strain, a minimum of four to a minimum of eight chromosomal DNA bands were present that fall into three distinct size classes, with one exception. Strain CESSI6K (O. novo-ulmi, North American aggressive subgroup) contains a unique chromosomal DNA band which comigrated near a Saccharomyces cerevisiae chromosome of 0.95 Mb. This unique band was the smallest O. ulmi s. l. chromosomal DNA observed. Seven of the twelve strains shared a common chromosomal DNA banding pattern, whereas each of the other five had a unique karyotype. There was no correlation between chromosome profile and species, as some O. novo-ulmi and O. ulmi strains shared common electrophoretic karyotypes.     

Specific fluorescent antiglobulins for the detection and identification of blastomyces dermatitidis yeast-phase cells

Summary Two lots of rabbit anti-Blastomyces dermatitidis globulins were conjugated with fluorescein isothiocyanate. These reagents stained brightly elements of the yeast and mycelial phases of 10 strains ofB. dermatitidis. In addition, the labeled antibodies cross-reacted with elements of the yeast and mycelial phases of 7 strains ofHistoplasma capsulatum and cells of numerous other heterologous fungi. Adsorption of one lot of labeled antibodies twice with yeast cells ofH. capsulatum and once with elements ofGeotrichum candidum rendered the conjugate specific for the yeast phase ofB. dermatitidis. Three adsorptions with yeast cells ofH. capsulatum followed by a single adsorption with elements ofG. candidum rendered the second conjugate specific for yeast-phase cells ofB. dermatitidis. The specific reagents did not react with the mycelial phase of this fungus.     

Mechanisms Involved in Biological Control of Dutch Elm Disease

An effective suppression of Dutch elm disease symptom development was observed in elms resistant to the non-aggressive strain of Ophiostoma ulmi, but not to the aggressive strain, after a preventive treatment by this non-aggressive strain. Anequally effective isolate of Verticillium dabliae suppressed symptom development even in the very susceptible field elm (Ulmus carpinifolia). Induction of resistance is proposed for the major mechanism explaining these and earlier experiments. The primary stimuli initiating a resistance reaction are unknown, but the intermediates of the univalent reduction of oxygen may play a role. In an experiment the activities of two groups of enzymes involved in the catalytical scavenging of these radicals, superoxide dismutase and peroxidase, were monitored, but no correlation with induction of resistance was observed. Resistant elms showed the highest activities, probably reflecting a more effective defence against radicals than susceptible ones.     

Rapid Evolutionary Changes in a Globally Invading Fungal Pathogen (Dutch Elm Disease)

Two enormously destructive pandemics of Dutch elm disease occurred in the 20th century, resulting in the death of a majority of mature elms across much of the northern hemisphere. The first pandemic, caused by Ophiostoma ulmi, occurred as this pathogen spread across Europe, North America and Southwest and Central Asia during the 1920s–1940s. The current pandemic is caused by another Ophiostoma species, O. novo-ulmi. Since the 1940s, O. novo-ulmi has been spreading into the regions previously affected by O. ulmi. It has also spread as two distinct subspecies, termed subsp. americana and subsp. novo-ulmi. This sequence of events has resulted in competitive interactions between these previously geographically isolated pathogens. This article summarizes the biological properties of the Dutch elm disease pathogens and their history of spread. It reviews the remarkable series of genetic events that have occurred during their migrations; including the emergence of genetic clones, the spread of deleterious fungal viruses within the pathogen clones, and the rapid and continuing evolution of O. novo-ulmi via horizontal gene flow. The wider role of horizontal gene flow in the evolutionary potential of migratory plant pathogens is discussed.     

Protoplasts of systemic dimorphic fungal pathogens: Histoplasma capsulatum and blastomyces dermatitidis

Summary Protoplasts have been released fromH. capsulatum in the mycelial and yeast phases and from the mycelial and incompletly converted yeast phase ofB. dermatitidis by the enzymatic action of snail digestive juice. There is great variation in the mode of protoplast formation not only between species but between the two morphological forms, particularly inH. capsulatum These studies were supported in part by grants from the American Thoracic Socicty and the United States Public Health Service National Institute of Allergy and Infectious Diseases (1-R1-AI-7520-01).     

Breeding ofl-threonine hyper-producer ofEscherichia coli W

Summary l-Threonine hyper-producing mutants were obtained fromEscherichia coli W strain KY-8366, by reducingl-threonine degradation activity and enhancingl-threonine biosynthetic activity. Anl-threonine degradation reaction test using resting cells of KY-8366 suggested that the main pathway ofl-threonine degradation by KY-8366 is via glycine. A strain with reducedl-threonine degradation activity was obtained among those mutants that could not utilizel-threonine as sole nitrogen source. Rifampicin-resistant mutants andl-lysine plus methionine-insentitive mutants were isolated. These mutants showed enhanced aspartokinase levels and accumulated morel-threonine than the parental strains. Mutant H-4290 accumulated 58 g/l ofl-threonine.     

Phenolic metabolites of Ceratocystis ulmi

The C₁₀ acid 2,4-dihydroxy-6-(1-hydroxyacetonyl) benzoic acid, together with the 6-acetonyl- and 6-pyruvyl-analogues, has been identified as a metabolic product of Ceratocystis ulmi, the causative agent of Dutch elm disease. In a comparison of aggressive “fluffy” and non-aggressive “waxy” strains of C. ulmi, the C₁₀ acids were produced more rapidly and in greater yield by the former group.     

Differential gene expression in brain tissues of aggressive and non-aggressive dogs

Background  

Canine behavioural problems, in particular aggression, are important reasons for euthanasia of otherwise healthy dogs. Aggressive behaviour in dogs also represents an animal welfare problem and a public threat. Elucidating the genetic background of adverse behaviour can provide valuable information to breeding programs and aid the development of drugs aimed at treating undesirable behaviour. With the intentions of identifying gene-specific expression in particular brain parts and comparing brains of aggressive and non-aggressive dogs, we studied amygdala, frontal cortex, hypothalamus and parietal cortex, as these tissues are reported to be involved in emotional reactions, including aggression. Based on quantitative real-time PCR (qRT-PCR) in 20 brains, obtained from 11 dogs euthanised because of aggressive behaviour and nine non-aggressive dogs, we studied expression of nine genes identified in an initial screening by subtraction hybridisation.     

Additional studies of histoplasmin formation

Summary Culture filtrates of 20 strains ofHistoplasma capsulatum were studied to determine the effect of certain growth conditions on histoplasmin formation. The presence of histoplasmin was denoted by an antigenic titer of 1:4 or higher with the complement fixation test.The data indicated that, in addition to verifying that the strain used affected histoplasmin formation, the morphological condition of the inoculum was extremely important. It was found that most strains which converted readily to the yeast phase at 37° C produced histoplasmin poorly. Tests with different volumes of media also showed that 500 ml volumes of culture media produced histoplasmin with higher titers than 3 liter volumes when cultured at 25° C for six months.Some additional histoplasmin could be liberated by sonification of the mycelial pad from culture filtrates which contained histoplasmin. A few strains produced high titer histoplasmin by the shake method if incubated for three months, but they had low titers after only six weeks.Complement fixation tests with sera from proven cases of histoplasmosis indicated that histoplasmin from a single strain ofH. capsulatum can give identical results with those obtained with histoplasmin from a pool ofH. capsulatum strains if H and M antigen components are present.     

Antigenic relationships between Aracoccidioides loboi and other pathogenic fungi determined by immunofluorescence

Summary The fluorescent antibody technique was used to study antigenic relationships betweenParacoccidioides loboi and other pathogenic fungi. The findings suggest thatP. loboi is more closely related antigenically to certainP. brasiliensis strains than to others and that it has antigens in common with the yeast form ofHistoplasma capsulatum, H. duboisii, Blastomyces dermatitidis, Candida albicans and also the mycelial form ofCoccidioides immitis. Serum globulins from 3 cases of keloidal blastomycosis were labelled with fluorescein isothiocyanate. These conjugates showed slight or no reactivity withP. loboi, the yeast forms ofP. brasiliensis, H. capsulatum, H. duboisii andB. dermatitidis, However, they stained brightlyC. albicans, serotypes A and B, the tissue form ofC. immitis and the yeast form ofSporotrichum schenckii. Adsorption of these reagents withC. albicans eliminated all staining except that forS. schenckii. These patients had no history of clinical sporotrichosis.Deceased. Last address: Fundacão Gonçalo Moniz, Salvador, Bahia, Brazil. Requests for reprints should be sent to Dr.William Kaplan.Dr.Miranda is in private practice in Rio de Janeiro, Brazil.     

rtpA, a gene encoding a bacterial two-component sensor kinase, determines pathogenic traits ofPseudomonas tolaasii, the causal agent of brown blotch disease of a cultivated mushroom,Pleurotus ostreatus

Pseudomonas tolaasii strain PT814 produces extracellular toxins, tolaasins, and a volatile toxin, tovsin, that are responsible for the induction of brown blotch and rotting, respectively, in a cultivated mushroom,Pleurotus ostreatus. Insertions of single transposon mini-Tn5Km 1 into the chromosome ofP. tolaasii strain PT814 generated mutants that are pleiotropically defective in tolaasin and protease production, and altered in colony morphology. The mutants, however, produce tovsin at the level of wild-type. Variants phenotypically similar to the pleiotropic mutants ofP. tolaasii strain PT814 spontaneously occurred inP. tolaasii strain S8501 at 22–30°C in vitro. The occurrence of variants was significantly reduced in the presence of extracts ofP ostreatus or at a temperature of 15–20°C. ThertpA gene (rtpA=regulator gene of tolaasin production and other pleiotropic traits) isolated from aP. tolaasii strain PT814 gene library restored the wild-type phenotype in both the mini-Tn5km 1 insertion and spontaneous mutants. mini-Tn5km 1 insertions were also located in the allele ofrtpA. Nucleotide sequencing of thertpA DNA revealed an open reading frame of 2,751 bp predicted to encode a protein consisting of 917 amino acid residues with a molecular mass of 100.6 kDa and displaying the conserved amino acid sequence of both sensor, and receiver domains of “bacterial two-component regulators”. The data suggest that the machinery responding to environmental stimuli is essential for the pathogenic interaction ofP. tolaasii with the mushroom.     

Dynamics ofPanonychus ulmi andTyphlodromus pyri: factors contributing to persistence

We addressed the question of persistence of predator and prey in a biological control system by examining temporal patterns ofPanonychus ulmi (Koch) and its predator,Typhlodromus pyri Scheuten at two geographic locations and at two spatial scales. At the scale of an orchard, bothP. ulmi andT. pyri were persistent over the time frame of 6 years. At the scale of an individual tree,T. pyri appeared to be more persistent than its prey,P. ulmi. We used a simulation model of single populations ofP. ulmi andT. pyri to determine which of several aspects of the biology of the two species could contribute to such a pattern. Spatial incongruity between predator and prey was essential for persistence of both species. The generalist food habit ofT. pyri probably contributes to the persistence ofT. pyri on individual trees, and may cause occasional extinction ofP. ulmi at this spatial scale. The presence of alternate food is likely an essential element for successful biological control in this system. Cannibalism byT. pyri results in higher prey densities, that is, it is detrimental to the biological control ofP. ulmi, but has no effect on the relative persistence of the two species.     

Fungicide inhibition of aflatoxins,diacetoxyscirpenol and zearalenone production

The effect of fungicides on the production of aflatoxin byAspergillus flavus IMI 89717, diacetoxyscirpenol and zearalenone byFusarium graminearum was studied. In a yeast extract - sucrose medium, dicloran, iprodione and vinclozolin fungicides significantly inhibited mycelial growth ofA. flavus at 250 ppm and significantly decreased aflatoxin production at 100, 250 and 500 ppm, respectively. In potato — dextrose broth, these fungicides diminished the mycelial growth ofF. graminearum and production of diacetoxyscirpenol and zearalenone at 100 ppm. Sensitivity of toxigenic mycelia to fungicides increased approximately five-fold in a yeast extract-starch medium with an appreciable reduction in sugar uptake andα-amylase activity.     

Isolation of auxotrophic mutants of the methylotrophic yeastCandida boidinii and determination of its ploidy

Auxotrophic mutations in the methylotrophic yeast strainCandida boidinii 11Bh were induced by different mutagens and their combinations (nitrosoguanidine, UV light, HNO₂+UV). Majority of the mutants obtained carried defects in histidine, arginine, proline and/or adenine biosynthetic pathways. His^- mutants were distributed into four complementation groups using the protoplasts fusion technique. Ploidy determination ofCandida boidinii 11Bh was performed by measuring its DNA content and by following its survival after chemical mutagens treatment. The DNA content of this strain was found to be similar to that of aSaccharomyces cerevisiae diploid strain. Also the kinetics of survival ofCandida boidinii cells indicate thatCandida boidinii 11Bh is a diploid.     

Developmental times and oviposition rates of predatory mitesTyphlodromus pyri andAmblyseius andersoni (Acari: Phytoseiidae) reared on different foods

The developmental times and the reproduction of two resistant Italian strains ofTyphlodromus pyri Scheuten andAmblyseius andersoni (Chant) were studied in the laboratory by rearing them on the spider mitesPanonychus ulmi (Koch) andEotetranychus carpini (Oud.), on the eriophyidColomerus vitis (Pgst.) and on pollen ofMesembryanthemum criniflorum. The response ofT. pyri andA. andersoni females to a spider mite supply (P. ulmi orE. carpini) of 4, 8 and 16 adult female prey per female predator per day was also studied.Development ofT. pyri onE. carpini andC. vitis required a shorter period than onM. criniflorum pollen, while intermediate values were recorded forP. ulmi. When the highest number of prey was offered, the influence of different foods on oviposition rates ofT. pyri was not significant. An increase in spider mite supply favoured a shorter pre-oviposition period and higher oviposition rates.Development ofA. andersoni was faster on pollen than on spider mites, while intermediate values were found concerningC. vitis. Differences statistically significant were recorded for development onP. ulmi andC. vitis. Colomerus vitis proved to be the more suitable food in terms of oviposition. The oviposition rate decreased when feeding uponP. ulmi, but reached intermediate values onE. carpini andM. criniflorum. Increasing spider mite densities caused shorter pre-oviposition times and higher oviposition rates. Using a given number ofE. carpini females, rather than those ofP. ulmi, resulted in higher oviposition rates and shorter pre-oviposition times.For both predators, the results suggest a higher intrinsic rate of population increase onE. carpini orC. vitis than onP. ulmi.The research was supported by a grant from Regione Veneto (Lotta biologica ed integrata nel controllo di insetti ed acari dannosi).The general lines of the research have been planned by C. Duso. The authors contributed equally to the experimental work.