6/11/03 Input from the Forest Pathology committee on 3 fungal forest pathogens. From Louis Bernier - Forest Pathology: Ophiostoma novo-ulmi as a candidate for microbial genome sequencing 1) General description of the organism The ascomycete Ophiostoma novo-ulmi belongs to a large and complex group of species collectively known as Ophiostomatoid fungi. These include pathogens as well as saprobes and are globally responsible for serious diseases of various broadleaved and coniferous tree species, cause economic losses to lumber exporters when they stain wood, and are considered significant quarantine organisms and invasive pests. For its part, O. novo-ulmi is the main agent of the vascular wilt known as Dutch elm disease (DED) and is responsible for the pandemic which has been decimating elm populations in North America and Europe since the 1960's. Understanding the biology (including both parasitic and saprophytic phases) of this highly aggressive organism is basic to developing control methods involving manipulation of O. novo-ulmi, its insect vectors (elm bark beetles) or its hosts (species in the genera Ulmus and Zelkova). 2) Significance Because of the dramatic impact of DED on elm populations worldwide, several laboratories in North America and Europe are investigating various aspects of parasitic fitness in O. novo-ulmi. Information gained on O. novo-ulmi would also be useful for investigations on phylogenetically close, but less-studied, Ophiostomatoid fungi, including several ubiquitous sapstaining species. Moreover, comparative DNA sequence analysis of 18SrDNA- and chitin synthase A genes have shown that O. novo-ulmi is closely related to Sporothrix schenckii, the etiological agent of sporotrichosis which causes cutaneous infections in humans and animals. Therefore, while O. novo-ulmi is poorly characterized when compared with "model fungi" such as Saccharomyces cerevisiae and the filamentous molds Neurospora crassa and Aspergillus nidulans, it is nevertheless a genetically tractable organism (see below) whose study could lead to findings with potential application in both plant and animal host-pathogen systems. 3) Genomic facts Ophiostoma novo-ulmi possesses several attributes which make it attractive for genetic studies. These include the occurrence of haploid yeast and mycelial phases which are both easily grown in vitro, the ability to carry out laboratory crosses by pairing sexually compatible strains, and the relative ease with which mutants can be obtained by conventional chemical and physical mutagenesis. Protocols are also available for efficient transformation, insertional mutagenesis (including REMI), and electrophoretic karyotyping. While natural populations of O. novo-ulmi tend to be rather uniform genetically, several laboratory mutants are available, including a growing number of genetically tagged mutants. Genomic banks have been constructed in cosmid vectors. The size of the O. novo-ulmi nuclear genome is evaluated at 30-40 Mb. A genetic map of O. novo-ulmi containing so far 172 loci (157 RAPD loci and 15 coding sequences) on 10 linkage groups representing 6 chromosomes has been obtained, whereas a few genes involved in parasitic fitness have been localized and, in some instances, cloned and further analyzed through gene disruption assays. Study of genes involved in pathogenicity is facilitated by the possibility of carrying out artificial inoculations on elm saplings and calli under controlled environmental conditions. In 2001, the first genomics-based research on an ophiostomatoid fungus was initiated by four university research laboratories. This project, funded for three years by the Natural Sciences and Engineering Research Council of Canada (NSERC), involves: 1) The construction of complementary DNA (cDNA) libraries from O. novo-ulmi; 2) The obtention of Expressed Sequence Tags (ESTs) by single-pass, partial sequencing of clones from these cDNA libraries; 3) The study of differential gene expression, first by Real-Time PCR and, eventually, by EST microarrays; 4) The design of studies on the function of genes of interest that display a strong pattern of differential expression, using both gene disruption- and gene over-expression approaches; 5) The determination of the chromosomal location of the genes represented by ESTs, in order to build a consensus genetic map for Ophiostomatoid fungi; 6) The production of a saturated genetic map using both coding and non-coding segregating loci. As of May 2003, 2000 clones from an O. novo-ulmi yeast-phase expression library had been sequenced and had yielded over 1200 authentic sequences which generated ca 620 BLASTX hits. Differential analysis of genes expressed during vegetative growth, reproduction (asexual and sexual) and pathogenesis is under way using subtractive hybridization approaches. The Ophiostoma EST database is expected to be online within a few weeks and will be available for consultation by public users. 4) Research community information The following four investigators are currently involved in the Ophiostoma EST sequencing project described above: Louis Bernier, U. Laval, Quebec, QC (CAN), 418-656-7655, Louis.bernier@rsvs.ulaval.ca; Colette Breuil, U. British Columbia, Vancouver, BC (CAN), 604-822-9738, breuil@interchange.ubc.ca; Will E. Hintz, U. Victoria, Victoria, BC (CAN), 250-721-7104, whintz@uvic.ca; Paul A. Horgen, U. of Toronto at Mississauga, Mississauga, ON (CAN), 905 828 5424, phorgen@utm.utoronto.ca. Additional potential collaborators would include the following reasearchers working on ophiostomatoid fungi: Clive M. Brasier, Forest Research (UK); Richard C. Hamelin, Canadian Forest Service, Quebec (CAN); Tom C. Harrington, Iowa State U., (USA); Georg Hausner, U. Manitoba (CAN); Martin Hubbes, U. Toronto (CAN); Aniello Scala, U. Firenze (ITAL); Michael Wingfield, U. Pretoria, (SA). In addition to the above, researchers working on elm genetics would also be interested by advances on Ophiostoma genomics. From Ned B. Klopfenstein - Forest Pathology Armillaria ostoyae: A candidate for genomic sequencing The genus Armillaria has a worldwide distribution and affects an extremely wide range of broad-leaved and coniferous woody hosts. Ecological roles of Armillaria species range from aggressive pathogenicity that causes root and butt rot, to beneficial saprophytic and symbiotic relationships. In many forests, damage from pathogenic Armillaria, either directly or through predisposition to insect attack, accounts for a greater loss in forest productivity than that from any other forest pathogen. In such situations, Armillaria spp. appear to be the primary force for forest succession and other ecological processes. In many forest ecosystems, Armillaria spp. represent an extremely large microbial biomass. Genets (vegetative diploid clones) of Armillaria can extend up to 4.1 km (890 ha), and appear to have occupied some sites for more than 2,400 years. Armillaria spp. possess a bifactoral mating system, and somatic compatibility reactions among diploid strains have been well studied and are of ongoing interest. Antibiotics produced by Armillaria spp. have potential use by the pharmaceutical industry and in research projects on biological control. Catabolic enzymes from these fungi are being examined for roles in wood decomposition and bioremediation. Also, bioluminescence occurs in Armillaria but is poorly understood. Because Armillaria spp. are the subject of worldwide interest from diverse perspectives, a member of this genus is a strong candidate for genomic sequencing. Furthermore, Armillaria spp. are easily maintained in culture as haploids or stable diploids. Mating is routinely achieved in vitro, and in vitro fruiting has been achieved at several laboratories. Genomic sequencing of an Armillaria sp., especially A. ostoyae, would contribute greatly to international collaborative research efforts involving diverse government agencies, academic institutions, and private industry. A. ostoyae genets (e.g., RMRS C53, P1404) form basidiocarps in culture, which has resulted in the production of hundreds of basidospore-derived, haploid lines that have been characterized as to mating type and are being examined for vegetative compatibility traits. Efforts are underway to produce an AFLP-based genomic map. Nuclear DNA content of A. ostoyae is known, and is ca. 0.065 pg (55 Mb) per haploid genome. Armillaria ostoyae is a primary research focus at several laboratories. The USDA Forest Service, Rocky Mountain Research Station Laboratory in Moscow, Idaho has the following research scientists working on A. ostoyae and other Armillaria spp.: Geral I. McDonald (208-883-2343; gimcdonald@fs.fed.us), Ned B. Klopfenstein (208-883-2310; nklopfenstein@fs.fed.us), Mee-Sook Kim (208-883-2362; mkim@fs.fed.us), and Paul Zambino (208-883-2334; zambino@fs.fed.us). The Moscow laboratory has an archive that contains over 5,000 isolates of Armillaria along with associated collection information, and associated basidiospore-derived lines. Related research efforts include studies of th e genetic diversity of A. ostoyae, the phylogeny of North American Armillaria spp., intraspecific and interspecific hybridization within Armillaria spp., population structure of Armillaria spp. in the Pacific Northwest as influenced by environmental factors, factors influencing fruiting, culture reactions related to somatic compatibility and mating, and other topics. The Moscow laboratory has a number of well-characterized genets of Armillaria, especially A. ostoyae, that would be ideal candidates for genomic sequencing. Other research contacts include C.G. "Terry" Shaw (USDA Forest Service, Vegetation Management and Protection Research, Washington, DC; 703-605-5261; cgshaw@fs.fed.us); Jean-Pierre Bérubé (Natural Resources Canada, Quebec City, Canada; 418-648-5899; jpberube@nrcan.gc.ca); James B. Anderson (University of Toronto, Toronto, ON, Canada: 905-828-5362; jandero@utm.utoronto.ca); Phil Cannon BOISE® Corp., Boise, ID: 208-384-6522; PhilCannon@BC.com); David Rizzo (University of California, Davis, CA; 530-754-9255; dmrizzo@ucdavis.edu); Duncan Morrison (Natural Resources Canada, Victoria, BC, Canada; 250-363-0642; dmorriso@nrcan.gc.ca); Catherine Parks (USDA Forest Service, Lagrande, OR; 541-962-6531; cparks01@fs.fed.us); Paul Hessburg (USDA Forest Service, Wenatchee, WA; 509-664-2709; 509-662-4315; phessburg@fs.fed.us); and James Worrall (USDA Forest Service, Gunnison, CO; 970-641-0471; 970-642-4424; jworrall@fs.fed.us). From Paul Zambino - Forest Pathology Cronartium ribicola as a Candidate for Genomic Sequencing Cronartium species are obligately parasitic rust fungi that are important pathogens of pine, on which they produce persistent galls or cankers. Blister rust, the disease caused by the pathogen C. ribicola, affects five-needle pines in Asia, and more recently, North American five-needle pines in Europe and North America. C. ribicola not only has tremendous importance on five-needle pines and commercial Ribes; it is also of critical importance to the post-introduction functioning of five-needle pine ecosystems across wide expanses of eastern, midwestern, and western North America. All five-needle pines in North America that grow under climatic conditions favoring rust infection have been severely decimated following C. ribicola's introduction into North America at the beginning of the twentieth century. For example, in the Inland Northwest, western white pine (Pinus monticola) has been reduced to 5% of its historical range. The loss has not only crippled the timber industry but has also altered species composition and ecosystem processes so that entire ecosystems are now at risk. Besides the importance of white pine blister rust to North American forestry, C. ribicola presents an ideal model system for a microbial genomic sequencing project because of the following genetic pathways that could be examined within a single pathosystem: 1) The comparative genetic basis could be determined for race-specific and non-race-specific, host-parasite interactions for both the pycnial / aecial hosts (five-needle pines hosts in which infections are perennial but also progress from a haploid to a dikaryotic condition every growing season) and uredinial / telial hosts (Ribes species hosts in which infections are dikaryotic and limited to the current growing season); 2) The genetic basis for aggressive vs. non-aggressive growth habit in native vs. introduced pathosystems can be studied; 3) The genetic basis for environmental adaptation could be determined; and 4) Sequencing of C. ribicola, coupled with examination of sister species, could reveal genes and signal transduction pathways that affect and account for observed differences in life cycles among rusts and potentially, among other groups of basidiomycete fungi. Unlike cereal rusts, which represents an artificial agro-ecosystem in which one rust host has been largely eliminated and the resistance structure of the other radically altered, both hosts of C. ribicola are experimentally tractable and the subject of genetic analysis, allowing examination of epistatic effects or "costs" of virulence to one host on the development of rust on the other host. In North America alone, dozens of Ribes species (Grossulariaceae) can function as uredinial hosts. Additionally, species of Pedicularis (Scrophulariaceae) are uredinial hosts for some Ribes-infecting strains of C. ribicola in Asia, which can allow examination of our definitions of the limits to "host" versus "non-host" resistance, and of the costs of virulence across three hosts. At least three single dominant R-genes conferring immunity to the introduced rust are known in North American pines. Virulence to two of these genes has already been identified. However, virulence appears to have an epistatic cost to the rust, as shown by inability or reduced effectiveness of virulent races to develop on some clones of Ribes species from North America. The CR gene from R. ussuriense, utilized in some commercial cultivars of R. nigrum, confers total immunity but has not as yet been overcome by virulent races. Oligogenic or multigenic forms of resistance are also known in all North American five-needle pines that have been examined, and in some Ribes species. Many species of Cronartium are heteroecious, requiring two different hosts to complete their life cycle. However, many others are autoecious and microcyclic, completing their life cycle on just the pine host. Two (some suggest three) five-needle-pine infecting Asian microcyclic species have been demonstrated through rDNA sequencing to be correlated species of C. ribicola. The high proportion of autoecious and microcyclic species in Cronartium may indicate that alternative key life history traits may originate as simple dimorphisms (polyphenisms) - an alteration in expression of key developmental pathway regulators, as has already been demonstrated for environmentally affected traits of many insects, such as parthenogenicity and male/female sex ratios. If so, elucidating the genetic mechanisms involved could be as important a discovery toward understanding fungal morphogenesis and life cycles as the discovery of homeotic genes in the model insect system of Drosophila melanogaster. A C. ribicola culture collection containing over 400 single-canker isolates from many of the hosts and ecological regions across North America, with 87 single-spore derived, pure genotype isolates is maintained at the USDA Forest Service, Rocky Mountain Research Station Laboratory in Moscow. Contacts for laboratories or individuals that could interact in a sequencing project of C. ribicola, and in the host-parasite genetics and genomics of the hosts include the following: C. ribicola: At the USDA Forest Service Rocky Mt. Research Station at Moscow, ID: Ned B. Klopfenstein (208-883-2310; nklopfenstein@fs.fed.us), Paul Zambino (208-883-2334; zambino@fs.fed.us), Bryce Richardson (208-883-3389; brichardson02@fs.fed.us), and Geral I. McDonald (208-883-2343; gimcdonald@fs.fed.us). At the USDA Forest Service Pacific Southwest Research Station, Detlev Vogler (Davis, CA, 530-758-6350; dvogler@fs.fed.us), and Bohun Kinloch (Albany, CA, 510-559-6432; bkinloch@fs.fed.us). Also, at Natural Resources Canada and the Canadian Forest Service (NRC/CFS), Richard Hamelin (Laurentian Forestry Centre, Ste. Foy, Quebec, 418-648-3693; rhamelin@exchange.cfl.forestry.ca), Rich Hunt (Pacific Forestry Centre, Victoria BC 250-363-0640; rhunt@PFC.Forestry.CA). Pine genetics/genomics: David Neale, USDA Forest Service PSW (Davis, CA,530-754-8431; dneale@fs.fed.us) and Abul Ekramadoulla (NRC/CFS Pacific Forestry Centre, Victoria, BC, 250- 363-0692; aekramoddoullah@pfc.cfs.nrcan.gc.ca) Ribes genetics/genomics: Kim Hummer, USDA ARS (Corvallis, OR, 541-750-8712; khummer@ars-grin.gov), and Rex Brennan, Scottish Crop Research Institute, Dundee, Scotland (rbrenn@scri.sari.ac.uk).