Clive M. Brasier

Rapid Evolution of Introduced Plant Pathogens via Interspecific Hybridization

P lant disease epidemics resulting from introduction of exotic fungal pathogens are a well-known phenom- enon. Limited resistance in the host and excessive aggres- siveness in the pathogen (reflecting their lack of prior co- evolution) can result in an explosive outbreak of disease. Introduction events also present a window of evolutionary op- portunity for the pathogen. In its endemic location, a plant pathogen tends to be subject to routine selection constraints, favoring maintenance of a relatively stable, if fluctuating, population structure over time. When introduced into a new environment, it will often be subject to novel or episodic se- lection, reflecting sudden exposure to new biotic and abiotic influences, such as a new host population, new vectors, new competitors, or a different climate. These influences provide the potential for rapid evolution (Brasier 1995). Falling within the category of episodic selection is the sud- den contact that can occur between closely related but pre- viously geographically isolated pathogens as a result of in- troductions. Theoretically, this process presents an opportunity for rapid emergence of new or modified pathogens via interspecific gene flow (Brasier 1995). Until recently, this phenomenon has been little studied. Now Dutch elm disease, a major ecological accident of the 20th century (Heybroek 1993), is providing remarkable insights into rapid evolution of a plant pathogen outside its en- demic environment. This article describes the migratory events and unusual genetic events that have occurred in this fungus and other new examples of rapid pathogen evo- lution via interspecific gene flow, and discusses some of the wider environmental, evolutionary, and quarantine impli- cations. The Dutch elm disease pathogens Elm trees (Ulmus) are confined mainly to the temperate re- gions of the Northern Hemisphere. China and Japan have a total of about 25 elm species, while Eurasia, North Amer- ica, and the Himalayas each have about five or six species. Dutch elm disease (so called because the early seminal re- search was in The Netherlands (Holmes and Heybroek 1990)) is the elms main enemy. It is a wilt disease, caused by ascomycete fungi of the genus Ophiostoma, that spreads within the trees vascular system. The pathogens are trans- mitted from diseased to healthy elms by elm bark beetles of the genus Scolytus (Fransen 1935, Webber and Brasier 1984).

Phytophthora alni sp. nov. and its variants: designation of emerging heteroploid hybrid pathogens spreading on Alnus trees

In 1993 a destructive new Phytophthora pathogen of riparian Alnus trees was discovered in the UK and subsequently shown to be present in other parts of Europe. The new Phytophthora comprised a group of emergent heteroploid hybrids, probably between P. cambivora and a species related to P. fragariae. These included a common, near tetraploid standard hybrid, the presumptive allopolyploid; and four scarcer major variant types with chromosome numbers intermediate between diploid and tetraploid, named the Swedish, Dutch, German and UK variants. The standard hybrid type is formally designated here as Phytophthora alni subsp. alni. The Swedish variant is designated as P. alni subsp. uniformis; and the Dutch, German and UK variants collectively as P. alni subsp. multiformis. The properties of the Dutch, German and UK variants within subsp. multiformis are informally described. The problems of designating emergent species hybrids under the International Code of Botanical Nomenclature and the reasons for the taxonomic choices made are discussed.

Phytophthora kernoviae sp. nov., an invasive pathogen causing bleeding stem lesions on forest trees and foliar necrosis of ornamentals in the UK

A new Phytophthora pathogen of trees and shrubs, previously informally designated Phytophthora taxon C, is formally named here as P. kernoviae. P. kernoviae was discovered in late 2003 during surveys of woodlands in Cornwall, south-west England, for the presence of another invasive pathogen, P. ramorum. P. kernoviae is self-fertile (homothallic), having plerotic oogonia, often with distinctly tapered stalks and amphigynous antheridia. It produces papillate sporangia, sometimes markedly asymmetric with medium length pedicels. Its optimum temperature for growth is ca 18 degrees C and upper limit ca 26 degrees. Currently, P. kernoviae is especially noted for causing bleeding stem lesions on mature Fagus sylvatica and foliar and stem necrosis of Rhododendron ponticum. P. kernoviae is the latest of several invasive tree Phytophthoras recently identified in the UK. Its geographical origins and the possible plant health risk it poses are discussed.

Plant pathology: Sudden larch death

An aggressive and unpredictable fungal pathogen is devastating larch plantations in Britain. Its remarkably broad host range, and the possibility of further geographical spread, give heightened cause for concern.

The rise of the hybrid fungi.

Hybrids between fungal pathogens of plants seem to be arising with increasing frequency. The most recently described example is a cross between two species of rust fungus that can attack poplar trees bred for commercial purposes.

Taxonomy of 'Phytophthora palmivora' on cocoa.

Morphological and physiological studies-chromosome type, colony morphology and growth rate on carrot agar, cocoa pod lesion characteristics, morphology and size of sporangia, sporangial pedicels, chlamydospores and sex organs, compatibility type, growth on a synthetic medium, response to Trichoderma, and temperature relations-were made with c. 950 Phytophthora isolates from cocoa (Theobroma cacao L.) attributed to P. palmivora (Butl.) Butl. The survey covered isolates from all the major cocoa growing areas of the world, and included isolates studied by C. H. Gadd and S. F. Ashby in the 1920s. The majority of the isolates could be assigned to one of three distinct forms, termed S, L and MF4. The S-type is attributed here to P. palmivora, which is redefined. Both L and MF4 are considered to be distinct species of Phytophthora. The L-type could not be identified with any known species and is described here as P. megakarya sp.nov. P. palmivora occurred world-wide on cocoa, whereas P. megakarya was obtained only from West Africa and MF4 only from Central and South America and the West Indies. Isolates attributed to P. palmivora from other hosts were also examined. Some isolates from rubber, coconut and durian were P. palmivora (S-type). Isolates from pepper comprised a group closely resembling MF4. Isolates from coconut and rubber originally attributed to P. palmivora by S. F. Ashby, C. H. Gadd and E. M. Blackwell comprised a further group, and were also attributed here to P. palmivora, yet were somewhat different morphologically from the S-type on cocoa. It is not clear which of these two types is authentic P. palmivora. The significance of these results, and the value of the various diagnostic criteria used, is discussed.

Multiple new phenotypic taxa from trees and riparian ecosystems in Phytophthora gonapodyides-P. megasperma ITS Clade 6, which tend to be high-temperature tolerant and either inbreeding or sterile

Phytophthora isolates associated with Phytophthora major ITS Clade 6 were grouped into 11 phenotypic taxa. These comprised the described morphospecies P. gonapodyides, P. megasperma s. str. and P. humicola; four previously identified but so far undescribed taxa, informally designated here P. sp. O-group, P. sp. Apple-cherry, P. taxon Pgchlamydo, and P. taxon Walnut; and four previously unknown taxa, designated P. taxon Oaksoil, P. taxon Raspberry, P. taxon Forestsoil, and P. taxon Riversoil. With the exception of P. gonapodyides, each phenotypic taxon represented an unique ITS lineage. Two isolates morphologically identical to P. gonapodyides comprised a separate lineage and probably represent another taxon, designated here P. taxon Salixsoil, P. humicola, P. sp. O-group, P. sp. Apple-cherry and P. taxon Walnut grouped together as subclade I. Within subclade II, P. taxon Oaksoil, P. taxon Raspberry, P. taxon Forestsoil, P. taxon Riversoil and P. taxon Pgchlamydo formed a cluster of closely related but phenotypically distinct lineages basal to P. gonapodyides and P. megasperma, P. taxon Salixsoil being the most basal member. The taxonomy, adaptation and breeding systems of Clade 6 taxa are discussed. They show a strong association with forests and riparian ecosystems, only a limited association with agriculture and an ability to tolerate high temperatures. Also, in contrast to most other Phytophthora clades, Clade 6 taxa are predominantly sterile or inbreeding in culture. Only one taxon, P. sp. O-group, appears classically A1/A2 heterothallic.

The taxonomic structure of Phytophthora megasperma: evidence for emerging biological species groups.

Nomenclatural uncertainty surrounds P. megasperma as various authors, working with limited groups of isolates, offer their interpretations of this species based on pathology, morphology, or cytology. We compared 93 isolates, including many described by others, for classical morphological features, growth behaviour and appearance, electrophoretic pattern of total proteins, chromosome number and nuclear DNA content. Nine distinct sub-groups were distinguished. While most groups could be distinguished by each of the criteria, protein electrophoresis was the most sensitive. The groups included: ALF, pathogenic to alfalfa, n = 12–15; SOY, pathogenic to soybean, n = 12–15; CLO, pathogenic to clover, n = 11–15; DF, pathogenic to Douglas fir, n = 17–24; AC, isolated from rosaceous fruit trees; and BHR, a major group obtained from a broad range of hosts. The last two groups, distinguished primarily by protein pattern, comprised at least four karyotypes: KI, n = 12–17; KII, n = 15–23; KIII, n = 22–28; and KIV, n = 26–34. All four karyotypes occur within the BHR protein group, suggesting a polyploid series within a closely related genotype. Two broad lines of evolution are hypothesized, a legume line comprising ALF, SOY, CLO, and perhaps DF isolates, and a Broad Host Range line of AC and BHR isolates. Sub-groups within each line may represent emerging biological species, isolated by host specificity or karyotype. Taxonomic designation for the various groups must await confirmation of the hypothesis by demonstration of the extent of barriers to gene flow between the groups.

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.

Designation of the EAN and NAN races of Ophiostoma novo-ulmi as subspecies

The two subpopulations of the Dutch elm disease pathogen Ophiostoma novo-ulmi , previously known as the Eurasian (EAN) and North American (NAN) races, are redesignated as subspecies novo-ulmi and americana. In addition to their partial reproductive isolation, wide range of physiological and molecular differences and different geographic ranges, the two subspecies can be discriminated by their perithecial form and dimensions. These perithecial differences are described, using perithecia produced in multiple intra-subspecies crosses. Perithecia of subsp. novo-ulmi have an average neck length of ca 450 μm, base width of ca 103 μn and neck length:base width ratio ca 4.4. Perithecia of subsp. americana have an average neck length of ca 295 μn, base width ca 116 μm and neck length:base width ratio ca 2.6. The average shape and dimensions of subsp. americana perithecia is similar to that of O. ulmi. The average perithecial form of subsp. novo-ulmi , as well as of O. himal-ulmi , is rather distinctive. The current known geographical distribution of subspecies novo-ulmi and americana , based on > 6500 samples, is presented.