Evolutionary Relationships of the Dutch Elm Disease Fungus Ophiostoma novo-ulmi to Other Ophiostoma Species Investigated by Restriction Fragment Length Polymorphism Analysis of the rDNA Region

Restriction fragment length polymorphisms (RFLPs) in the ribosomal RNA gene (rDNA) region were used to assess relationships between the Dutch elm disease fungi Ophiostoma novo-ulmi and Ophiostoma ulmi , the recently described Himalayan Dutch elm disease pathogen, Ophiostoma himal-ulmi , the morphologically similar sapstain fungi, Ophiostoma piceae and Ophiostoma quercus , and several Ophiostoma species from hardwood trees, including Ophiostoma stenoceras and Ophiostoma proliferum . A distance matrix and cluster analysis indicated that the rDNA region of O. himal-ulmi is more closely related to those of O. novo-ulmi and O. ulmi than to those of O. piceae and O. quercus and is more distantly related to O. stenoceras and the other Ophiostoma species, which formed a separate clade. The rDNA region of O. quercus was found to be at least as closely related to that of O. novo-ulmi and O. ulmi as it is to that of O. piceae . The implications of these results for the evolution of the Dutch elm disease fungi are discussed.     

Transgenic American elm shows reduced Dutch elm disease symptoms and normal mycorrhizal colonization

The American elm (Ulmus americana L.) was once one of the most common urban trees in eastern North America until Dutch-elm disease (DED), caused by the fungus Ophiostoma novo-ulmi, eliminated most of the mature trees. To enhance DED resistance, Agrobacterium was used to transform American elm with a transgene encoding the synthetic antimicrobial peptide ESF39A, driven by a vascular promoter from American chestnut. Four unique, single-copy transgenic lines were produced and regenerated into whole plants. These lines showed less wilting and significantly less sapwood staining than non-transformed controls after O. novo-ulmi inoculation. Preliminary observations indicated that mycorrhizal colonization was not significantly different between transgenic and wild-type trees. Although the trees tested were too young to ensure stable resistance was achieved, these results indicate that transgenes encoding antimicrobial peptides reduce DED symptoms and therefore hold promise for enhancing pathogen resistance in American elm.     

Use of insertional mutagenesis to tag putative parasitic fitness genes in the Dutch elm disease fungus Ophiostoma novo-ulmi subsp. novo-ulmi

We used insertional mutagenesis to produce genetically tagged mutants of the Dutch elm disease fungus Ophiostoma novo-ulmi subsp. novo-ulmi. We first optimized transformation of O. novo-ulmi protoplasts by the restriction enzyme mediated integration method. A concentration of 80 U of HindIII with 108 fungal protoplasts and 5 mug of plasmid DNA was the most efficient for generating a high number of O. novo-ulmi mutants carrying a single insertion in their genome. Mycelium- and yeast-like growth kinetics of 24 O. novo-ulmi mutants were evaluated in vitro. Flanking sequences were successfully recovered in 8% of the transformants analyzed. Some mutant phenotypes appeared to result from gene disruption events, whereas others likely involved modifications of noncoding regions. Several nuclear loci that control vegetative growth and could potentially impact parasitic fitness were successfully tagged.     

Stress-induced mobility of OPHIO1 and OPHIO2, DNA transposons of the Dutch elm disease fungi

The mobility of transposable elements (TEs) can contribute to genome plasticity, under- or over-expression of genes and ectopic recombination. The data collected in this study provide evidence of stress-induced mobility of OPHIO1 and OPHIO2 transposons, recently detected in Ophiostoma ulmi and O. novo-ulmi, the causal agents of Dutch elm disease (DED). The analyses of OPHIO UTRs and TIRs indicated the presence of two potential binding site motifs and a heat shock protein (hsp) promoter which could be involved in the mobility of OPHIO1 following a heat shock stress. The exact position of the hsp promoter was determined by 5' RACE PCR. After confirmation of the expression by RT-PCR of both OPHIO1 and OPHIO2 transposases in the absence of stress factors, we tested two experimental procedures to induce mobility of OPHIO TEs: (1) an exogenous (cloned) copy of OPHIO1 was introduced into the O. novo-ulmi subsp. americana strain W2 (OPHIO1 free strain) to give mutant strain W2:OPHIO1. After exposure of W2:OPHIO1 to a 55 degrees C heat shock treatment, some of the survivors showed signs of incomplete transposition (excision without reinsertion) of OPHIO1. (2) The O. novo-ulmi subsp. novo-ulmi strain AST27, introgressed from O. ulmi and carrying a distinct endogenous copy of OPHIO2 (OPHIO2-int.), was subjected to a series of abiotic stress treatments. Although a promoter sequence could not be identified, both exposures to UV light and to a 4 degrees C cold treatment caused perfect excision of OPHIO2-int. In contrast to OPHIO1, heat shock stress did not induce OPHIO2-int. mobility. Taken together, these results allow us to hypothesize a potential interspecific invasion of OPHIO transposons due to their mobility in Ophiostoma spp.     

Stress-induced mobility of OPHIO1 and OPHIO2, DNA transposons of the Dutch elm disease fungi.

The mobility of transposable elements (TEs) can contribute to genome plasticity, under- or over-expression of genes and ectopic recombination. The data collected in this study provide evidence of stress-induced mobility of OPHIO1 and OPHIO2 transposons, recently detected in Ophiostoma ulmi and O. novo-ulmi, the causal agents of Dutch elm disease (DED). The analyses of OPHIO UTRs and TIRs indicated the presence of two potential binding site motifs and a heat shock protein (hsp) promoter which could be involved in the mobility of OPHIO1 following a heat shock stress. The exact position of the hsp promoter was determined by 5' RACE PCR. After confirmation of the expression by RT-PCR of both OPHIO1 and OPHIO2 transposases in the absence of stress factors, we tested two experimental procedures to induce mobility of OPHIO TEs: (1) an exogenous (cloned) copy of OPHIO1 was introduced into the O. novo-ulmi subsp. americana strain W2 (OPHIO1 free strain) to give mutant strain W2:OPHIO1. After exposure of W2:OPHIO1 to a 55 degrees C heat shock treatment, some of the survivors showed signs of incomplete transposition (excision without reinsertion) of OPHIO1. (2) The O. novo-ulmi subsp. novo-ulmi strain AST27, introgressed from O. ulmi and carrying a distinct endogenous copy of OPHIO2 (OPHIO2-int.), was subjected to a series of abiotic stress treatments. Although a promoter sequence could not be identified, both exposures to UV light and to a 4 degrees C cold treatment caused perfect excision of OPHIO2-int. In contrast to OPHIO1, heat shock stress did not induce OPHIO2-int. mobility. Taken together, these results allow us to hypothesize a potential interspecific invasion of OPHIO transposons due to their mobility in Ophiostoma spp.     

Characterization of three DNA transposons in the Dutch elm disease fungi and evidence of repeat-induced point (RIP) mutations

Transposable elements (TEs) are fundamental components of eukaryotic genomes and can contribute in various ways to genome plasticity and evolution. We describe here the first three DNA transposons in the Dutch elm disease (DED) pathogens Ophiostoma ulmi and O. novo-ulmi, named OPHIO1, OPHIO2 and OPHIO3. We demonstrate that OPHIO transposons, which show high homology to Fot1/pogo TEs within the Tc1/mariner superfamily, have different distribution patterns and specificity in the DED fungi and that interspecific hybrids could act as genetic bridges for transmission of TEs between closely related fungal species. OPHIO3 was found to have undergone repeat-induced point mutations (RIP). We have also developed a complementary method to Margolin's ratios based on the computation of cumulative transition scores (CTS) in order to visualize rapidly RIP signatures on individual DNA strands of OPHIO transposons and TEs found in other ascomycete fungi.     

The pathogen causing Dutch elm disease makes host trees attract insect vectors

Dutch elm disease is caused by the fungal pathogen Ophiostoma novo-ulmi which is transmitted by the native elm bark beetle, Hylurgopinus rufipes. We have found that four semiochemicals (the monoterpene (-)-beta-pinene and the sesquiterpenes (-)-alpha-cubebene, (+)-spiroaxa-5,7-diene and (+)-delta-cadinene) from diseased American elms, Ulmus americana, synergistically attract H. rufipes, and that sesquiterpene emission is upregulated in elm trees inoculated with O. novo-ulmi. The fungus thus manipulates host trees to enhance their apparency to foraging beetles, a strategy that increases the probability of transportation of the pathogen to new hosts.     

Two natural cerato-ulmin (CU)-deficient mutants of Ophiostoma novo-ulmi: one has an introgressed O. ulmi cu gene, the other has an O. novo-ulmi cu gene with a mutation in an intron splice consensus sequence

The nucleotide sequences of the cerato-ulmin ( cu ) genes of two naturally occurring pathogenic CU-deficient mutants, PG470 and MAFf8, of the Dutch elm disease fungus, Ophiostoma novo-ulmi , were determined. The PG470  cu gene sequence was identical to that of CU-secreting isolates of O. novo-ulmi , except for a G to A mutation in the GT splice consensus sequence at the start of intron 1, suggesting that the CU deficiency was due to a splicing defect in the premRNA. In contrast, the MAFf8  cu gene showed a 99.1% sequence identity with cu genes of O. ulmi isolates, but only 92.8% sequence identity with cu genes of CU-secreting isolates of O. novo-ulmi , and in a dendrogram clustered with cu gene sequences of O. ulmi isolates with 100% bootstrap support. Restriction fragment length polymorphisms of the ribosomal RNA region, random amplified polymorphic DNA markers, and many biological properties of MAFf8, including pathogenicity, were typical of O. novo-ulmi . It is therefore likely that the cu gene of MAFf8 has been introgressed from O. ulmi , probably as a result of rare hybrid formation between O. ulmi and O. novo-ulmi , followed by backcrossing of the hybrid with O. novo-ulmi . The presence of an O. ulmi -like cu gene in MAFf8 is consistent with its CU deficiency, since the O. ulmi cu gene is known to be poorly expressed and O. ulmi isolates secrete little or no CU in culture.     

Correlation of resistance and H2O2 production in Ulmus pumila and Ulmus campestris cell suspension cultures inoculated with Ophiostoma novo-ulmi

The Dutch elm disease (DED) pathogen Ophiostoma novo-ulmi Buissm. elicited the production of H₂O₂ in cell suspension cultures of the resistant species Ulmus pumila L. This response was not observed in suspensions of the susceptible elm U. campestris Mill. H₂O₂ production started after a lag time of 30–40 min following inoculation, peaked between 4 and 6 h and lasted up to 24 h. Treatment of the suspensions with exogenously added H₂O₂ did not cause accumulation of the sesquiterpene phytoalexins mansonones nor of the coumarin scopoletin. Spore germination and growth of O. novo-ulmi were significantly delayed with different amounts of H₂O₂ (0.1–1 m M ). These results suggest that H₂O₂ production is an inducible defence response which may contribute to DED resistance by delaying the growth of the pathogen at the earliest stages of infection. Whether H₂O₂ is involved in other elm defence responses to the pathogen is presently unknown, but its production seems to be an independent event from phytoalexin formation.     

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.     

Sequence analysis of the chitin synthase A gene of the Dutch elm pathogen Ophiostoma novo-ulmi indicates a close association with the human pathogen Sporothrix schenckii.

Degenerate oligonucleotide primers were designed according to conserved regions of the chitin synthase gene family and used to amplify a 621 basepair (bp) fragment from genomic DNA of Ophiostoma novo-ulmi, the causal agent of Dutch elm disease. The amplification product was used as a hybridization probe to screen a library of genomic DNA sequences and to retrieve a full-length chitin synthase gene (chsA). The putative coding region of the gene was 2619 bp long, lacked introns, and encoded a polypeptide of 873 amino acids. Based on the similarity of the predicted amino acid sequence to the full-length chsC gene of Aspergillus nidulans and chsA gene of Ampelomyces quisqualis, the O. novo-ulmi chsA was classified as a Class I chitin synthase. The phylogenies constructed, according to a subregion of all available chitin synthases, showed that O. novo-ulmi consistently clustered most closely with the human pathogen Sporothrix schenckii, recently classified as a member of the mitosporic Ophiostomataceae. Disruption of the chsA gene locus had no obvious effects on the growth or morphology of the fungus.     

Seasonal changes in wood formation of Ulmus pumila and U. minor and its relation with Dutch elm disease

Elms containing narrow and scattered vessels have been reported to be more resistant to Ophiostoma novo-ulmi (Dutch elm disease pathogen) than elms with large and contiguous vessels. However, recent measurements in Ulmus pumila and U. minor showed a contrary trend. The pin method was applied to 4-yr-old branches of eight clones planted in Madrid. During 2002, radial growth increments and vessel diameters were measured monthly, and beetle trapping was undertaken weekly. U. minor formed larger vessels at the beginning of the season, coinciding with a peak of captured beetles, but, up to June 15, vessels were larger for U. pumila. The number of vessels per group, the transversal area per vessel group, and the mean theoretical hydraulic conductances were significantly higher for U. minor on most dates. Researchers should take into consideration the seasonal changes in vessel size. The results highlight that seasonal variation of vessel diameters and hydraulic parameters, in combination with beetle abundance, are the main factors that could explain the different susceptibility of both elm species to O. novo-ulmi.     

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.     

Selective acquisition of novel mating type and vegetative incompatibility genes via interspecies gene transfer in the globally invading eukaryote Ophiostoma novo-ulmi

The Dutch elm disease fungus Ophiostoma novo-ulmi, which has destroyed billions of elm trees worldwide, originally invaded Europe as a series of clonal populations with a single mating type (MAT-2) and a single vegetative incompatibility (vic) type. The populations then rapidly became diverse with the appearance of the MAT-1 type and many vegetative incompatibility types. Here, we have investigated the mechanism using isolates from sites in Portugal at which the rapid evolution of O. novo-ulmi populations from clonality to heterogeneity was well established. We show by genetic mapping of vic and MAT loci with AFLP markers and by sequence analysis of MAT loci that this diversification was due to selective acquisition by O. novo-ulmi of the MAT-1 and vic loci from another species, Ophiostoma ulmi. A global survey showed that interspecies transfer of the MAT-1 locus occurred on many occasions as O. novo-ulmi spread across the world. We discuss the possibility that fixation of the MAT-1 and vic loci occurred in response to spread of deleterious viruses in the originally clonal populations. The process demonstrates the potential of interspecies gene transfer for facilitating rapid adaptation of invasive organisms to a new environment.     

Conservation of genetic diversity in slippery elm (<Emphasis Type="Italic">Ulmus rubra</Emphasis>) in Wisconsin despite the devastating impact of Dutch elm disease

Forest pest epidemics are responsible for many population declines reported in forest trees. While forest tree populations tend to be genetically diverse, in principle mortality resulting from disease could diminish that genetic diversity and alter the genetic structure of the remnant populations with consequences for the ability of a species to adapt to changing environments. Slippery elm (Ulmus rubra Muhl.) is a long-lived, wind-pollinated forest tree with a native range covering essentially all of eastern North America. Dutch elm disease (DED) caused by an introduced fungal pathogen (Ophiostoma ulmi) devastated North American elm populations, including slippery elm, beginning in the 1930s. Estimates of the numbers of elms lost to DED are unknown but range into the hundreds of millions of trees given their former abundance. In this study, the genotypes of 77 herbarium specimens collected between 1890 and 2004 in Wisconsin, and of 100 slippery elm trees from five wild Wisconsin populations, were characterized using 13 microsatellite loci. Levels of genetic diversity were compared between the herbarium specimens collected pre- and post-DED spread in Wisconsin. In addition, the levels of genetic diversity and degree of genetic differentiation were quantified in the five wild populations. The allelic diversity and expected levels of heterozygosity were similar between the pre- and post-DED herbarium specimens. The five wild populations were only slightly differentiated and no genetic bottleneck was detected for any population. At least in Wisconsin, slippery elm apparently has maintained levels of genetic diversity that could facilitate adaptation to future climatic and environmental changes.     

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.     

Leaf trait dissimilarities between Dutch elm hybrids with a contrasting tolerance to Dutch elm disease

Background and Aims

Previous studies have shown that Ophiostoma novo-ulmi, the causative agent of Dutch elm disease (DED), is able to colonize remote areas in infected plants of Ulmus such as the leaf midrib and secondary veins. The objective of this study was to compare the performances in leaf traits between two Dutch elm hybrids ‘Groeneveld’ and ‘Dodoens’ which possess a contrasting tolerance to DED. Trait linkages were also tested with leaf mass per area (LMA) and with the reduced Young''s modulus of elasticity (MOE) as a result of structural, developmental or functional linkages.

Methods

Measurements and comparisons were made of leaf growth traits, primary xylem density components, gas exchange variables and chlorophyll a fluorescence yields between mature plants of ‘Groeneveld’ and ‘Dodoens’ grown under field conditions. A recently developed atomic force microscopy technique, PeakForce quantitative nanomechanical mapping, was used to reveal nanomechanical properties of the cell walls of tracheary elements such as MOE, adhesion and dissipation.

Key Results

‘Dodoens’ had significantly higher values for LMA, leaf tissue thickness variables, tracheary element lumen area (A), relative hydraulic conductivity (RC), gas exchange variables and chlorophyll a fluorescence yields. ‘Groeneveld’ had stiffer cell walls of tracheary elements, and higher values for water-use efficiency and leaf water potential. Leaves with a large carbon and nutrient investment in LMA tended to have a greater leaf thickness and a higher net photosynthetic rate, but LMA was independent of RC. Significant linkages were also found between the MOE and some vascular traits such as RC, A and the number of tracheary elements per unit area.

Conclusions

Strong dissimilarities in leaf trait performances were observed between the examined Dutch elm hybrids. Both hybrids were clearly separated from each other in the multivariate leaf trait space. Leaf growth, vascular and gas exchange traits in the infected plants of ‘Dodoens’ were unaffected by the DED fungus. ‘Dodoens’ proved to be a valuable elm germplasm for further breeding strategies.     

Control of yeast-mycelium dimorphism in vitro in Dutch elm disease fungi by manipulation of specific external stimuli

Dutch elm disease (DED) fungi exhibit yeast-mycelium dimorphism both in planta and in vitro. However, previously published data on the transition between these two growth forms in vitro were mostly obtained from a single strain. We examined the effect of six factors on yeast-mycelium dimorphism in vitro in ten strains of Ophiostoma ulmi, Ophiostoma novo-ulmi and Ophiostoma himal-ulmi. Nitrogen sources, calcium, and yeast extract, altogether with inhibitors of phosphodiesterase (caffeine) and dioxygenases (propyl gallate and salicylic acid) were tested in defined culture media. Morphological response to manipulation of several of these factors varied according to the strain of Ophiostoma being analysed. Responses ranged from no statistical differences in morphological transitions to stimulation or reversion of yeast-mycelium dimorphism with the treatments that were tested. These results suggest that different mechanisms and pathways operate in the control of the yeast-mycelium transition in DED pathogens. Oxylipins could be involved in the yeast-to-mycelium transition, since the addition of a dioxygenase inhibitor, salicylic acid, reduced mycelium production in all strains that were tested.     

Sap flow-based quantitative indication of progression of Dutch elm disease after inoculation with Ophiostoma novo-ulmi

Key message

The rate of progression of Dutch elm disease can be continuously and quantitatively estimated from sap flow measurements.

Abstract

Response of sap flow to inoculation with Ophiostoma novo-ulmi, a causal agent which causes vascular mycosis called Dutch elm disease, was studied in a field experiment comprised of 4-year-old wych elm trees (Ulmus glabra). Sap flow was measured on inoculated trees using the trunk heat balance method with external heating (EMS 62, Czech Republic) throughout the experiment. The first detectable symptoms of reduction in sap flow occurred 6 days after inoculation and all inoculated trees died within 16 days. Our experiment confirmed the ability of O. novo-ulmi to quickly kill young elm trees. The disease progressed faster than in previous experiments utilizing O. ulmi. To the best of our knowledge, this is the first experiment using sap flow measurements on trees inoculated by O. novo-ulmi. The trunk heat balance sap flow method is an effective non-invasive tool for continuous quantitative monitoring of the progression of vascular tree diseases, and show increased potential for field and greenhouse studies on changes in xylem hydraulic conductivity in a wide range of broadleaved and coniferous tree species.