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     

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.     

Sequencing of the Dutch Elm Disease Fungus Genome Using the Roche/454 GS-FLX Titanium System in a Comparison of Multiple Genomics Core Facilities

As part of the DNA Sequencing Research Group of the Association of Biomolecular Resource Facilities, we have tested the reproducibility of the Roche/454 GS-FLX Titanium System at five core facilities. Experience with the Roche/454 system ranged from <10 to >340 sequencing runs performed. All participating sites were supplied with an aliquot of a common DNA preparation and were requested to conduct sequencing at a common loading condition. The evaluation of sequencing yield and accuracy metrics was assessed at a single site. The study was conducted using a laboratory strain of the Dutch elm disease fungus Ophiostoma novo-ulmi strain H327, an ascomycete, vegetatively haploid fungus with an estimated genome size of 30–50 Mb. We show that the Titanium System is reproducible, with some variation detected in loading conditions, sequencing yield, and homopolymer length accuracy. We demonstrate that reads shorter than the theoretical minimum length are of lower overall quality and not simply truncated reads. The O. novo-ulmi H327 genome assembly is 31.8 Mb and is comprised of eight chromosome-length linear scaffolds, a circular mitochondrial conti of 66.4 kb, and a putative 4.2-kb linear plasmid. We estimate that the nuclear genome encodes 8613 protein coding genes, and the mitochondrion encodes 15 genes and 26 tRNAs.     

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.     

Characterization of microsatellite loci in the fungus, Grosmannia clavigera, a pine pathogen associated with the mountain pine beetle

The largest forest pest epidemic in Canadian history caused by the mountain pine beetle (MPB) and its fungal associates has killed over 15 million hectares of forest. Sixty simple sequence repeat regions were identified from Grosmannia clavigera, an MPB associated fungus. Eight loci genotyped in 53 isolates from two populations in British Columbia, Canada revealed three to 10 alleles per locus and gene diversities of 0 to 0.79. All but two of these loci showed length polymorphism in Leptographium longiclavatum, a related MPB fungal associate. These microsatellites will be useful in population genetic studies of these fungi.     

Extracellular lipase production by a sapwood-staining fungus,Ophiostoma piceae

The extracellular lipase production of a sapwood-staining fungus, Ophiostoma piceae, grown in liquid media, was optimally active at pH 5.5 and 37°C. Although glucose, fructose, sucrose, starch and dextrin, as carbon sources for growth gave similar mycelial yields, which were higher than those obtained with arabinose, galactose or raffinose, the cells growing on those carbohydrates produced little extracellular lipase. However, both high biomass and lipase activity were obtained when plant oils (olive, soybean, corn, sunflower seed, sesame, cotton seed or peanut) were used as carbon sources. Among the nitrogen sources examined, Casamino acids gave the best growth, whereas (NH₄)₂SO₄ gave the best lipase production. The highest lipase productivity seen was obtained in a medium with olive oil as carbon source and a combination of (NH₄)₂SO₄and peptone as nitrogen source.The authors are with Forest Products Biotechnology, Department of Wood Science, Facully of Forestry, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada     

Pathogenicity to western larch (Larix occidentalis) of two fungi, Ophiostoma pseudotsugae and Leptographium abietinum, associated with the Douglas fir beetle (Coleoptera: Scolytidae)

1 Pole-sized, live western larch Larix occidentalis Nutt. were mass-inoculated with Ophiostoma pseudotsugae (Romb.) von Arx or Leptographium abietinum (Peck) Wingf., two blue-stain fungi associated with the Douglas fir beetle Dendroctonus pseudotsugae Hopkins, to assess their pathogenicity. 2 Inoculation with O. pseudotsugae resulted in significantly greater percentages of necrotic phloem compared with L. abietinum inoculations. 3 The percentage of occluded sapwood was significantly greater after L. abietinum inoculations compared with O. pseudotsugae inoculations. 4 Within the inoculation band, all trees had more than 60% functional sapwood 4 months after treatment. 5 The results suggest that western larch can successfully limit colonization by O. pseudotsugae and L. abietinum. 6 The inability of the fungi to thrive in live western larch may be a factor in the consistent failure of Douglas fir beetle broods in this host tree species.     

Properties and substrate specificities of an extracellular lipase purified from Ophiostoma piceae

An extracellular lipase produced by the sapstaining fungus Ophiostoma piceae 387N in a liquid medium was purified to homogeneity using ammonium sulphate and acetone fractionation, hydrophobic interaction and anion exchange chromatography. The overall purification based on lipase activity was 5200-fold with a yield of 26%. The molecular mass of the lipase was 35kDa, as determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), and 37 kDa, as measured by size exclusion chromatography. The purified enzyme was resolved as three bands at pI values of 4.3, 4.1 and 3.8 in IEF (isoelectric focusing) gels. Lipolytic stain demonstrated that all three bands were lipolytically active. The N-terminal amino acid sequence was determined asD¹-V²-S³-V⁴-T⁵-T⁶-T⁷-D⁸-I⁹-D¹⁰-A¹¹-L¹²-A¹³-F¹⁴-F¹⁵-T¹⁶-Q¹⁷-W¹⁸-A¹⁹-G²⁰ . The lipase was shown to be glycosylated, containing 10.1% carbohydrate. The lipase was stable between pH 4 and pH 8 and at temperatures below 40°C. The lipase activity had a pH optimum of approximately 5 and a temperature optimum of 30°C. The enzyme activity was not influenced by N-ethylmaleimide, -mercaptoethanol or dithiothreitol, was enhanced by Ca²⁺ or Mn²⁺, but was severely inhibited by Hg²⁺, Fe³⁺, butyric acid, caproic acid, diethyl pyrocarbonate, and diethyl p-nitrophenyl phosphate. The lipase hydrolysed mainly triglycerides, although some activity was measured on waxes and cholesteryl esters. It belongs to a group of 1 (3) positional specific lipases. It showed little activity for substrates with short chain fatty acids (C2–C6), but demonstrated high specificity for substrates with intermediate and long chain fatty acid residues (C10–C18).