James M. Duncan

Phylogenetic analysis of Phytophthora species based on ITS1 and ITS2 sequences of the ribosomal RNA gene repeat

The internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal RNA gene repeat from Phytophthora species were amplified using the polymerase chain reaction and sequenced. Sequences from P. cambivora, P. cinnamomi, P. citricola, P. cryptogea, P. drechsleri, P. fragariae var. fragariae, P. fragariae var. rubi, P. megasperma var. megasperma and P. nicotianae were compared with published sequences and phylogenetic trees were produced. The resultant grouping of species generally agreed with groupings established using classical morphological criteria, primarily sporangial morphology. Amongst species with non-papillate sporangia two clades were evident, one consisting of P. fragariae, P. cambivora and P. cinnamomi and the other of P. megasperma, P. drechsleri and P. cryptogea. The latter three were placed in the tree between the non-papillate groups and the papillate and semi-papillate groups which formed three distinct clades. One group comprised P. citricola, P. citrophthora and P. capsici, another P. megakarya and P. palmivora and a third P. pseudotsugae, P. cactorum, P. idaei, P. nicotianae and P. infestans. More isolates of P. megasperma, P. drechsleri and P. cryptogea will need to be examined to settle more precisely the relationship of these species to the others. PCR amplification of ITS sequences using freeze-thawed mycelial scrapings from pure cultures growing on agar followed by digestion with restriction enzymes is a quick and easy way to compare and identify isolates without the need for laborious DNA extraction procedures. With improved technology, rapid automatic sequencing of PCR-amplified ITS regions is now possible and yields detailed information of relationships within the genus as well as allowing the design of species-specific PCR primers for diagnostic purposes.

Phytophthora quercina sp. nov., causing root rot of European oaks

In a 3 year study of oak decline in Central and Southern Europe, a papillate homothallic Phytophthora species was isolated consistently, with other Phytophthora spp., from necrotic fine roots by direct plating on to selective agar medium and from rhizosphere soil samples by baiting with leaves of Quercus robur. The morphology, physiology, RAPD banding patterns and pathogenicity against apple fruits of this Phytophthora sp. are described and compared with those of other papillate Phytophthora species from Waterhouse’s Group I, namely P. cactorum, P. clandestina, P. idaei, P. iranica, P. pseudotsugae and P. tentaculata, and semipapillate Group III P. citricola. The papillate Phytophthora isolates from oak diered from all other Group I species by their uniform, dome-shaped and cottonwool-like colony growth pattern on V8 juice agar and malt extract agar, the frequent occurrence of sympodially branched primary hyphae, a high proportion of elongated, ellipsoid or ovoid oogonia, the absence of amphigynous antheridia and RAPD banding patterns. Additionally, there was no other species in Group I with as much variation in size and shape of the sporangia or large proportion of sporangia with a curved apex, hyphal projections, lateral displacement of the papilla and lateral attachment to the sporangiophore. In pathogenicity tests with infested soil, the isolates proved to be more pathogenic to Q. robur than any other Phytophthora sp. recovered from declining oaks in Central Europe. Based on their unique combination of cultural, sporangial and gametangial morphology, pathogenicity and close association with Quercus but not other trees, the papillate Phytophthora isolates from oak are described as Phytophthora quercina sp. nov. Oak decline is a serious and frequently recurring threat to European forestry. Above-ground symptoms include dieback of branches and parts of the crown, formation of epicormic shoots, high transparency of the crown, yellowing and wilting of leaves and tarry exudates from the bark (Siwecki & Liese, 1991; Luisi, Lerario & Vannini, 1993), all symptoms indicative of water stress and poor nutrition. Roots of declining and healthy trees in 33 stands of Quercus robur L., Q. petraea (Mattuscka) Liebl., Q. cerris L., Q. pubescens Willd. and Q. ilex L. in Germany, Switzerland, Hungary, Slovenia, Italy and France were examined for the presence of Phytophthora species. For the first time in Central Europe, several Phytophthora species including P. citricola, P. cactorum, P. cambivora, P. gonapodyides and P. undulata and an unidentified species, Phytophthora sp. 2, were isolated from root and soil samples of most stands investigated. Another unknown but papillate Phytophthora species, possessing a unique combi

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.

Phytophthora pseudosyringae sp. nov., a new species causing root and collar rot of deciduous tree species in Europe

In several studies of oak decline in Europe, a semi-papillate homothallic Phytophthora taxon was consistently isolated, together with other Phytophthora species, from rhizosphere soil samples. It was also found associated with necrotic fine roots and stem necroses of Fagus sylvatica and Alnus glutinosa. Due to morphological and physiological similarities, the semi-papillate isolates were previously identified as P. syringae by various authors. The morphology, physiology and pathogenicity against fine roots of Quercus robur, Q. petraea and F. sylvatica, bark of A. glutinosa, leaves of Ilex aquifolium and apple fruits of this Phytophthora species are described and compared with those of related and similar Phytophthora species, namely P. ilicis, P. psychrophila, P. quercina, P. citricola and P. syringae. The phylogenetic placement on the basis of ITS and mtDNA sequence data was also examined. Isolates of this taxon produce colonies with stellate to rosaceous growth patterns and limited aerial mycelium on various agar media. Antheridia are predominantly paragynous. In water culture catenulate hyphal swellings and semi-papillate caducous sporangia, that are usually limoniform, ellipsoid or ovoid, are formed abundandly, mostly in lax or dense sympodia. This taxon is a moderately slow growing, low temperature species with optimum and maximum temperatures around 20 and 25 degrees C, respectively. Tested isolates are moderately aggressive to fine roots of oaks and beech, highly aggressive to holly leaves and apple fruits, and slightly pathogenic to alder bark. Thirteen tested isolates had an identical and distinct ITS sequence which was more similar to that of P. ilicis and P. psychrophila than any other known taxa. On the basis of their unique combination of morphological characters, colony growth patterns, cardinal temperatures for growth, growth rates, pathogenicity to oaks, beech, alder, apple and holly, their host range, and ITS and mtDNA sequences the semi-papillate caducous Phytophthora isolates from oaks, beech and alder are clearly separated from related and similar Phytophthora spp., and described as a new species, P. pseudosyringae sp. nov.

Conventional PCR and real-time quantitative PCR detection of Helminthosporium solani in soil and on potato tubers

Silver scurf is an economically important blemish disease of potato caused by the fungus Helminthosporium solani. Two sets of PCR primers, Hs1F1/Hs2R1 (outer) and Hs1NF1/Hs2NR1 (nested) were designed to unique sequences of the nuclear ribosomal internal transcribed spacer (ITS1 and ITS2) regions of H. solani. Nested PCR was used to increase the specificity and sensitivity of single round PCR. Each primer set amplified a single product of 447 bp and 371 bp respectively, with DNA from 71 European and North American isolates of H. solani, and the specificity of primers was confirmed by the absence of amplified product with DNA from other fungal and bacterial plant pathogens. A simple and rapid procedure for direct extraction of DNA from soils and potato tubers was modified and developed to yield DNA of a purity and quality suitable for PCR within 3 h. The sensitivity of PCR for the specific detection of H. solani in seeded soils was determined to be 1.5 spores g−1 of soil. H. solani was also detected by PCR in naturally infested soil and from peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan™ fluorogenic probe were designed using the original primer sequences to perform real-time quantitative (TaqMan™) PCR. The same levels of sensitivity for specific detection of H. solani in soil and tubers were obtained during first round mboxTaqMan-based PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR assay allows an accurate estimation of tuber and soil contamination by H. solani, thus providing a tool to study the ecology of the organism and to serve as a crucial component for disease risk assessments.

Isolation of Potato Genes That Are Induced During an Early Stage of the Hypersensitive Response to Phytophthora infestans

Suppression subtractive hybridization (SSH) was used to generate a cDNA library enriched for sequences induced in a late-blight-resistant potato cultivar undergoing the hypersensitive response (HR). Of 100 partial cDNA sequences submitted to international DNA and protein data bases, 42 showed similarity to 35 genes, of which 31 were from plants. Of these, 13 were previously characterized as either defense-, stress-, or senescence-associated. One sequence matched (75 to 81%) all known serine palmitoyltransferases (SPTs) at the protein level. SPT catalyzes the first committed step in the synthesis of sphingolipids, important signaling molecules involved in cell differentiation and apoptosis. Putative products of other genes identified here may play a role in programmed cell death, including protein degradation, DNA degradation, metal ion chelation, and signal transduction. cDNA-amplified fragment length polymorphism was used to confirm differential expression of sequences isolated by SSH.

Relatedness of Group I species of Phytophthora as assessed by randomly amplified polymorphic DNA (RAPDs) and sequences of ribosomal DNA

Relatedness of Group I species P. iranica, P. clandestina, P. pseudotsugae, P. cactorum and a new species, P. idaei with specificity in pathogenicity tests to raspberry (Rubus idaeus), were examined at the molecular level using twenty random ten-mer primers to amplify total DNA (RAPDs) and by sequencing of the internal transcribed spacer regions, ITS1 and ITS2, of the ribosomal RNA gene. Cluster analysis of five of the RAPD banding patterns, separately or as a combined analysis, ranked P. idaei, P. pseudotsugae and P. cactorum more or less equally in each case. The separation between them and P. iranica and P. clandestina was much greater. Within P. cactorum, collar rot isolates from apple clustered separately from strawberry crown rot isolates, while isolates from raspberry appeared to have affinities with both clusters. Sequence analyses of ITS1 and ITS2 revealed only a few differences among isolates of P. idaei, P. pseudotsugae and P. cactorum. Their sequences were much more similar to one another than to P. infestans and in ITS1 they shared a characteristic motif, resulting from a base pair inversion, that was not present in any other Phytophthora species. The inversion occurred within a sequence that binds to a DNA probe that has been used as a genus-specific probe for all Phytophthora spp.

Detection and Quantification of Spongospora subterranea in Soil, Water and Plant Tissue Samples Using Real-Time PCR

A sensitive real-time polymerase chain reaction (PCR) assay was developed for the quantification of Spongospora subterranea, the cause of powdery scab and root galling in potato, and the vector of Potato mop top virus. A specific primer pair and a fluorogenic TaqMan® probe were designed to perform a quantitative assay for the detection of S. subterranea in soil, water and plant tissue samples. The assay was tested using DNA from cystosori, zoospores, plasmodia and zoosporangia of the pathogen. DNA was extracted directly from cystosori suspended in water and from clay soil with varying levels of added cystosori. DNA obtained from zoospores released into nutrient solution by cystosori in the presence of tomato bait plants was also tested, as was DNA from plasmodia and zoosporangia in infected tomato roots. In many cases, detection was successful even at low inoculum levels. This specific quantitative assay could therefore be a useful tool for studying the biology of S. subterranea, and for the optimisation of disease avoidance and control measures.

Actin in the oomycetous fungus Phytophthora infestans is the product of several genes.

Actin (ACT) in Phytophthora infestans is encoded by at least two genes, in contrast to unicellular and other filamentous fungi where there is a single gene. These genes (designated actA and actB) have been isolated from a genomic library of P. infestans. The complete nucleotide sequence of both genes has been determined. Unlike the actin-encoding genes (act) of other filamentous fungi, no introns are obvious in the coding region, a feature shared with the act genes of certain protists. Northern blotting and primer extension studies of the mRNA show that actA and actB are actively transcribed in mycelium, sporangia and germinating cysts but only at a low level in the case of actB. Both genes display bias in their codon usage. This is more extreme in actA. The deduced ACTB protein is strikingly similar to that of the Phytophthora megasperma actin and is more diverged from other actins than ACTA.

Identity of a Phytophthora species attacking raspberry in Europe and North America

Severe root rot of raspberry is caused in Europe and North America by a homothallic, non-papillate Phytophthora sp., which has been identified by different investigators as P. erythroseptica, P. fragariae or as a highly pathogenic variant of P. megasperma . Two collections of such highly pathogenic raspberry isolates from Europe and North America were compared with recognized specimens of the three above named species, which were originally isolated from potato, loganberry and strawberry, and a variety of perennial host plants, respectively. All such raspberry isolates, regardless of previous identification, formed an essentially homogeneous group with respect to colony and growth characteristics, the production and morphology of gametangia, the morphology and dimensions of sporangia, electrophoretic banding patterns of mycelial proteins and pathogenicity. They were readily distinguishable from recognized isolates of P. erythroseptica and P. megasperma with respect to cultural, morphological, and electrophoretic criteria. In contrast, they were very similar to isolates of P. fragariae from strawberry (and a single isolate from loganberry) although the two groups could be separated by differences in growth rate on some agar media, production of oospores in culture, and small differences in electrophoretic banding patterns, as well as in pathogenicity. It was concluded that the raspberry isolates should be assigned to P. fragariae , but that they should be given a subspecific epithet at the varietal level to distinguish them from strawberry isolates of the species i.e. P. fragariae var. rubi .