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.     

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.     

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.     

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     

Biological control of Dutch elm disease by Pseudomonas species

Antagonism of a selected group of bacteria against Ophiostoma (=Ceratocystis) ulmi, the Dutch elm disease pathogen, was determined on agar media. Four promising bacterial isolates, all fluorescent Pseudomonads, were used for field experiments and for further tests on in vitro antagonism against several fungi. It was shown that only slight differences existed in antagonism against different O. ulmi isolates. Also Ceratocystis fagacearum and C. fimbriata were inhibited similarly to O. ulmi. In field experiments bacteria were applied to elm trees by low pressure injection or by injection with a specially developed ‘gouge-pistol’. Elms treated only with bacteria remained healthy throughout two growing seasons. Elms inoculated with O. ulmi developed severe Dutch elm disease symptoms. Trees, inoculated first with O. ulmi and treated subsequently with bacteria also developed severe Dutch elm disease symptoms. Trees treated with bacteria first and inoculated subsequently with O. ulmi showed significantly fewer symptoms, especially those where treatments were carried out with the gouge-pistol.     

<Emphasis Type="Italic">Ophiostoma tsotsi</Emphasis> sp. nov., A Wound-infesting Fungus of Hardwood Trees in Africa

Polymorphic sequence-characterised marker assays from a recent diversity study on the Ascomycete fungus Ophiostoma quercus reported that some isolates from Africa were genetically distinct from O. quercus. In the present study, these African isolates were compared with authentic O. quercus isolates by evaluating morphological characters, growth in culture, mating compatibility and DNA sequence data. The isolates from Africa were morphologically similar to O. quercus, presenting Pesotum and Sporothrix synanamorphs in culture. Phylogenetic analyses of the ribosomal internal transcribed spacer regions 1 and 2, β-tubulin and translation elongation factor 1-α gene regions confirmed that the African group represents a distinct species within the hardwood lineage of the O. piceae complex, closely related to O. ulmi and O. himal-ulmi. Mating studies between O. quercus and the African isolates showed that isolates mated predominantly with those of their own group, although there were rare cases of fertile crosses between the groups. Isolates residing in the African lineage are described here as a new species, O. tsotsi sp. nov.     

Molecular diversity of porcine and human isolates of Pasteurella multocida

Aims: To examine the variability among Pasteurella multocida strains isolated from pigs (nasal, tonsil and lung specimens) and humans in France. Methods and Results: The genetic diversity of 117 French isolates of P. multocida, obtained from pigs (n = 101) and humans (n = 16) and three reference strains, was evaluated by pulsed‐field gel electrophoresis (PFGE) after macrorestriction with ApaI. Sixty‐four patterns were detected. The genetic relationships revealed five clusters (Aa1, Aa2, Aa3, Ab and B). The pig isolates obtained from pneumonic lungs and nasal cavities were clustered in groups Ab and Aa1, respectively (P < 0·05). Up to four different PFGE patterns were detected in the same farm. Isolates producing dermonecrotic toxins were clustered only in group Aa1, suggesting that the toxigenic isolates were more genetically homogenous than the others. Conversely, cluster Aa3 was significantly associated with human isolates even if the human isolates are spread over most of the clusters. Conclusions: Pasteurella multocida strains were genetically diverse, but pig and human isolates were significantly clustered in distinct phylogenetic groups. Significance and Impact of the Study: The discrimination index was >0·95 in both populations of human and pig isolates. Therefore, ApaI‐PFGE seems to be a useful tool for epidemiological tracing of P. multocida infections.     

High genetic diversity of Vibrio cholerae in the European lake Neusiedler See is associated with intensive recombination in the reed habitat and the long‐distance transfer of strains

Coastal marine Vibrio cholerae populations usually exhibit high genetic diversity. To assess the genetic diversity of abundant V. cholerae non‐O1/non‐O139 populations in the Central European lake Neusiedler See, we performed a phylogenetic analysis based on recA, toxR, gyrB and pyrH loci sequenced for 472 strains. The strains were isolated from three ecologically different habitats in a lake that is a hot‐spot of migrating birds and an important bathing water. We also analyzed 76 environmental and human V. cholerae non‐O1/non‐O139 isolates from Austria and other European countries and added sequences of seven genome‐sequenced strains. Phylogenetic analysis showed that the lake supports a unique endemic diversity of V. cholerae that is particularly rich in the reed stand. Phylogenetic trees revealed that many V. cholerae isolates from European countries were genetically related to the strains present in the lake belonging to statistically supported monophyletic clades. We hypothesize that the observed phenomena can be explained by the high degree of genetic recombination that is particularly intensive in the reed stand, acting along with the long distance transfer of strains most probably via birds and/or humans. Thus, the Neusiedler See may serve as a bioreactor for the appearance of new strains with new (pathogenic) properties.     

O‐antigen diversity and lateral transfer of the wbe region among Vibrio splendidus isolates

The O‐antigen is a highly diverse structure expressed on the outer surface of Gram‐negative bacteria. The products responsible for O‐antigen synthesis are encoded in the wbe region, which exhibits extensive genetic diversity. While heterogeneous O‐antigens are observed within Vibrio species, characterization of these structures has been devoted almost exclusively to pathogens. Here, we investigate O‐antigen diversity among coastal marine Vibrio splendidus‐like isolates. The wbe region was first identified and characterized using the sequenced genomes of strains LGP32, 12B01 and Med222. These regions were genetically diverse, reflective of their expressed O‐antigen. Additional isolates from physically distinct habitats in Plum Island Estuary (MA, USA), including within animal hosts and on suspended particles, were further characterized based on multilocus sequence analysis (MLSA) and O‐antigen profiles. Results showed serotype diversity within an ecological setting. Among 48 isolates which were identical in three MLSA genes, 41 showed gpm genetic diversity, a gene closely linked to the wbe locus, and at least 12 expressed different O‐antigen profiles further suggesting wbe genetic diversity. Our results demonstrate O‐antigen hyper‐variability among these environmental strains and suggest that frequent lateral gene transfer generates wbe extensive diversity among V. splendidus and its close relatives.     

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.     

Gene variation and gene flow inOrchis morio (Orchidaceae) from Italy

Data are presented on genetic variation at 27 enzyme loci of the Green-Winged orchid,Orchis morio, in 18 population samples from Italy. The existence in Italy of two subspecies, i.e. subspp.morio andpicta, is not supported by allozyme data. No genetic heterogeneity was found betweenmorio-like andpicta-like samples and specimens. Moreover, morphological transition between the two forms was observed in different Italian populations. The parameters of genetic variability estimated forO. morio populations are consistent with those found among monocotyledon plants, and among those outcrossing, animal-pollinated and with wind-dispersed seeds. Genetic diversity of ItalianO. morio is mostly within populations. Correspondingly, low values of interpopulational genetic distance were found. This appears to be due to high levels of gene flow, which were estimated with different methods. The lack ofO. longicornu from Italian samples, as well as of any hybrid withO. morio (F₁, backcrossed or recombinant individuals) is demonstrated on the basis of genetic data. It is concluded that recurrent reports ofO. longicornu from Italy are due to confusion withO. morio or with otherOrchis species.     

Shotgun mass mapping of Lactobacillus species and subspecies from caries related isolates by MALDI‐MS

A taxonomical study of 90 isolates of lactobacilli isolated from soft and hard carious dentine of 70 deciduous molars is presented. The Lactobacillus strains were determined by shotgun mass mapping (SMM). This method based on MALDI‐MS analysis of Lactobacillus isolates treated with trypsin followed by database comparison against a library of mass spectra derived from 20 reference strains. The SMM method allowed to discriminate different Lactobacillus subspecies. The method was used to analyse Lactobacillus isolates of unknown identity derived from carious dentine. Application of the SMM method to isolates from hard carious dentine revealed a nearly similar distribution of L. paracasei ss paracasei (29%), L. paracasei ss tolerans (32%) and L. casei ss rhamnosus (23%) as dominant subspecies. On the other hand, samples derived from soft carious dentine showed a clear bias only to L. paracasei ss paracasei (60%), whereas L. paracasei ss tolerans (14%) and L. casei ss rhamnosus (12%) were clear minorities. Compared to existent methods, SMM has unique potential for the analysis of Lactobacillus strains on subspecies level.     

Production and Degradation of N-Acylhomoserine Lactone Quorum Sensing Signal Molecules in Bacteria Isolated from Activated Sludge

N-Acylhomoserine lactones (AHLs) function as quorum-sensing signaling molecules in many Gram-negative bacteria. We isolated a total of 672 bacterial strains from activated sludge obtained from seven sewage treatment plants in Tochigi Prefecture, Japan, and screened for AHL-producing and degrading strains. Isolates (n=107) stimulated AHL-mediated purple pigment production in AHL reporter strains Chromobacterium violaceum CV026 and VIR07. Based on their 16S rRNA gene sequences, most of these AHL-producing isolates were assigned to the genus Aeromonas, and they were divided into six groups. Isolates (n=46) degraded N-decanoyl-L-homoserine lactone (C10-HSL) within 24 h. Based on their 16S rRNA gene sequences, the most dominant AHL-degrading isolates were assigned to the genus Acinetobacter and divided into six groups. Strains Ooi24, Omo91, and Uzu81, which showed higher C10-HSL-degrading activity, showed putative AHL-acylase activity.     

Quorum sensing activity in Ophiostoma ulmi: effects of fusel oils and branched chain amino acids on yeast-mycelial dimorphism

Aims: For Ophiostoma (Ceratocystis) ulmi, the ability to undergo morphological change is a crucial factor for its virulence. To gain an understanding of quorum‐sensing activity in O. ulmi as it relates to yeast‐mycelium dimorphism control, this study examines the effects of branched‐chain amino acids as well as their fusel alcohols and fusel acids as quorum sensing molecules. Methods and Results: In a defined medium containing glucose, proline and salts, O. ulmi grew as yeasts when the culture was inoculated with a high density of spores (2 × 10⁷ CFU ml^?1) and as mycelia when inoculated with a low spore density (4 × 10⁵ CFU ml^?1). The cultures displaying yeast morphology secreted a quorum‐sensing factor that shifted the morphology from mycelia to yeast. This quorum‐sensing molecule was lipophilic and extractable by organic solvents from the spent medium. Using GC/MS analysis, it was determined that the major compound in the extract was 2‐methyl‐1‐butanol. A similar effect was observed when the branched‐chain amino acids (fusel alcohol precursors) were used as the nitrogen source. E, E‐farnesol had no effect on the morphology of O. ulmi. Conclusions: Addition of the branched‐chain amino acids or one of the compounds detected in the spent medium, 2‐methyl‐1‐butanol or 4‐hydroxyphenylacetic acid, or methylvaleric acid, decreased germ tube formation by more than 50%, thus demonstrating a quorum sensing molecule behaviour in O. ulmi cultures. Significance and impact of the study: This study presents advances in the investigation of dimorphism in O. ulmi, complementing the existing scientific basis, for studying, understanding and controlling this phenomenon.     

Phylogenetic Relationships Among Colletotrichum Pathogens of Strawberry and Design of PCR Primers for their Identification

Isolates of Colletotrichum acutatum, C. fragariae and C. gloeosporioides pathogenic to strawberry plants were examined by sequence analysis of the 5.8S‐ITS region. Phylogenetic relationships among isolates of Colletotrichum are, for the most part, congruent with the molecular groups established in earlier works. 5.8S‐ITS sequence analysis showed a high level of genetic divergence within C. acutatum. Isolates of this species clustered into two very distinct clusters with further subdivision. The divergences between C. fragariae and C. gloeosporioides were too low to distinguish them as separate species. On the basis of the sequence data, specific primers were designed both to identify isolates belonging to the genus Colletotrichum, and to distinguish isolates of the species C. acutatum. The specificity of these primers was validated by testing a wide range of strawberry isolates of Colletotrichum, non‐strawberry isolates of Colletotrichum and other fungi used as controls. Although the 5.8S‐ITS sequences were not polymorphic enough to allow the construction of C. gloeosporioides‐specific primers, specific PCR amplification followed by an MvnI digestion provides a tool to specifically identify strawberry isolates of C. gloeosporioides.     

Correlation between polymerase chain reaction analysis of the histidine biosynthesis operon, randomly amplified polymorphic DNA analysis and phenotypic characterization of dairy Lactococcus isolates

A collection of 32 lactococcal strains isolated from raw milk in the Camembert RDO (registered designation of origin) area were phenotypically and genotypically characterized. As expected for environmental isolates, all strains had a Lactococcus lactis subsp. lactis phenotype. The strains were then genotypically identified by the randomly amplified polymorphic DNA (RAPD) technique, using reference strains of lactococci. Two major clusters were identified containing the two subspecies lactis and cremoris. The subspecies lactis cluster could be divided into five subgroups whereas there was a high coefficient of similarity between all strains in the subspecies cremoris cluster. This RAPD classification was then compared with that of a traditional PCR assay using L.lactis species-specific primers corresponding to part of the histidine biosynthesis operon. The two subspecies were differentiated by the size of the fragment amplified (about 200 bp longer for subspecies cremoris). Unlike preliminary phenotypic assignments, the results of PCR experiments corroborated the genotypic identification of the lactococcal strains by RAPD allowing the technique to be reconsidered on the basis of its taxonomic efficiency. Received: 14 May 1998 / Accepted: 3 September 1998     

Phytomorphological and Essential‐Oil Characterization in situ and ex situ of Wild Biotypes of Oregano Collected in the Campania Region (Southern Italy)

Oregano is an aromatic species of great interest, which spreads spontaneously over the Mediterranean area, but its genetic resources are not yet adequately developed. Moreover, the results of studies of wild strains of different origin – although quite numerous – are not always comparable, and, therefore, the influence of the environment and genotype on the variability cannot be distinguished. Hence, the plant morphology and the essential‐oil composition of three wild, white‐flowering biotypes of oregano collected in the Campania region (southern Italy) were characterized, and the effects of genotype and environment were assessed by comparison in situ and ex situ. This allowed deducing that the biotypes belonged to two different subspecies, i.e., Origanum vulgare ssp. virens and O. vulgare ssp. viridulum. The essential‐oil yield was higher for the biotype belonging to ssp. virens, and it was significantly correlated with the glandular and stomatal density. The chemical composition of the oils obtained by hydrodistillation was found to be influenced by the genotype and the conditions of plant growth.     

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.