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Made available in DSpace on 2022-05-01T09:30:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-02-01 During the winter, there is a high occurrence of downy mildew on lettuce caused by Bremia lactucae. This oomycete shows variability in virulence, so understanding the genetic structure of the pathogen population becomes essential for obtaining resistant cultivars. Thus, the objective of this study was to determine sexual compatibility in Brazilian populations of B. lactucae and investigate the occurrence of sexual reproduction of the pathogen on lettuce (Lactuca sativa) and prickly lettuce (Lactuca serriola). Leaf samples were collected in 33 municipalities in seven Brazilian states. The virulence structure of the populations was monitored using the EU-C sextet code. B. lactucae populations from the states of São Paulo, Paraná, Rio de Janeiro, and Rio Grande do Sul shared six of the 15 virulence factors evaluated. Twenty-five virulence phenotypes (v-phenotypes) were found, with the sextet codes 31-00-02, 31-16-02, 31-24-02, and 31-01-02 being more frequent. The predominance of some v- phenotypes indicates that clonal reproduction is still the main form of B. lactucae propagation. The genes and resistance factors of the cultivars Argelès (Dm38), Balesta, and Bartoli are recommended as suitable sources of lettuce resistance in Brazil. Natural occurrence of oospores was detected in most sampled locations, in lettuce and prickly lettuce plants. Virulence variability of Brazilian isolates is the result of the pathogen's ability to reproduce both sexually and asexually, with a prevalence of homothallic isolates; although the majority were of the predominant B2 mating type, there was a high incidence of predominant B1 in addition to B1 = B2. Department of Agricultural Production Sciences São Paulo State University (Unesp) School of Agricultural and Veterinarian Sciences, SP Department of Plant Pathology Gulf Coast Research and Education Center University of Florida Research Department – Lettuce Breeding Agristar do Brasil, SP Department of Botany Faculty of Science Palacký University in Olomouc Department of Agricultural Production Sciences São Paulo State University (Unesp) School of Agricultural and Veterinarian Sciences, SP

Impatiens downy mildew is caused by Plasmopara obducens, a pathogen known in the United States for over a hundred years, but newly attacking ornamental Impatiens walleriana in production and in the landscape. Little is known about the life cycle of P. obducens; thus, in this study an attempt was made to determine whether the pathogen is homothallic or heterothallic. Fourteen single-sporangium isolates and three single-zoospore isolates were used in single and dual inoculations of stem tissue to see whether the pathogen was homothallic or heterothallic; all isolates tested were able to produce oospores when inoculated singly, suggesting homothally. Cold treatment at 0 C for at least 1 mo induced oospores to germinate and produce primary sporangia. Inoculation of plant tissue with germinating oospores resulted in infection. Other incubation temperatures (-10, 10, and 20 C) did not induce germination, but fluctuating temperatures (between -10 and 0 C, or 0 and 10 C) induced some germination. Spores incubated at -10 C had significantly thicker walls than spores incubated at other temperatures. Evidence suggests that oospores can serve as an overwintering stage.

Potato crop losses can be substantial when conditions for late blight (Phytophthora infestans) development and spread are favourable. In this study, drivers of differences between the P. infestans population structures in Latvia and Lithuania, two neighbouring countries with similar potato-growing traditions, were investigated. Genotypes of P. infestans and population genetic diversity were analysed using a 12-plex simple sequence repeat (SSR) marker assay. High genetic diversity was demonstrated in both populations, with population diversity being higher in Latvia. It would appear that local populations established from soilborne oospores early in the season are well adapted to the conditions in the region. However, somewhat greater spread and survival of local clones was detected in Lithuania, suggesting that potato cropping there is more vulnerable to clonal invasion than in Latvia. For effective disease management, current strategies should be adjusted according to the specific pathogen populations in the region, considering the reproduction and survival of the pathogen. Potato growers should implement late blight preventive measures such as longer field rotation to prevent oospore infections, especially in Latvia, and should use more disease resistant cultivars and high-quality seed potatoes.

Sexual reproduction is an essential stage of the oomycete life cycle. However, the functions of critical regulators in this biological process remain unclear due to a lack of genome editing technologies and functional genomic studies in oomycetes. The notorious oomycete pathogen Pythium ultimum is responsible for a variety of diseases in a broad range of plant species. In this study, we revealed the mechanism through which PuM90, a stage-specific Puf family RNA-binding protein, regulates oospore formation in P. ultimum. We developed the first CRISPR/Cas9 system-mediated gene knockout and in situ complementation methods for Pythium. PuM90-knockout mutants were significantly defective in oospore formation, with empty oogonia or oospores larger in size with thinner oospore walls compared with the wild type. A tripartite recognition motif (TRM) in the Puf domain of PuM90 could specifically bind to a UGUACAUA motif in the mRNA 3′ untranslated region (UTR) of PuFLP, which encodes a flavodoxin-like protein, and thereby repress PuFLP mRNA level to facilitate oospore formation. Phenotypes similar to PuM90-knockout mutants were observed with overexpression of PuFLP, mutation of key amino acids in the TRM of PuM90, or mutation of the 3′-UTR binding site in PuFLP. The results demonstrated that a specific interaction of the RNA-binding protein PuM90 with the 3′-UTR of PuFLP mRNA at the post-transcriptional regulation level is critical for the sexual reproduction of P. ultimum., Author summary Oomycetes are a class of eukaryotic microorganisms with life cycles and growth habits similar to filamentous fungi, but are not true fungi. Although sexual reproduction, which produce oospores, is an essential stage of life cycle, the functions of critical regulators in this biological process remain unclear due to a lack of genome editing technologies and functional genomic studies in oomycetes. In this study, we developed the first CRISPR/Cas9 system-mediated gene knockout and in situ complementation methods for Pythium ultimum, a notorious oomycete pathogen that is responsible for a variety of diseases in a broad range of plant species. We further identified the Puf family RNA-binding protein PuM90 and the flavodoxin-like protein PuFLP as major functional factors involved in P. ultimum oospore formation. We proposed a new model that PuM90 acts as a stage-specific post-transcriptional regulator by specifically binding to the 3′-UTR of PuFLP and then repressing PuFLP mRNA level. This study describes new technologies and data that will help to elucidate sexual reproduction and post-transcriptional regulation in oomycetes.

To characterize the molecular mechanisms underlying life-stage transitions in Phytophthora infestans, we initiated a chemical genetics approach by screening for a stage-specific inhibitor of morphological development from microbial culture extracts prepared mostly from actinomycetes from soil in Japan. Of the more than 700 extracts, one consistently inhibited Ph. infestans cyst germination. Purification and identification of the active compound by ESI–MS, 1H-NMR, and 13C-NMR identified β-rubromycin as the inhibitor of cyst germination (IC50 = 19.8 μg/L); β-rubromycin did not inhibit growth on rye media, sporangium formation, zoospore release, cyst formation, or appressorium formation in Ph. infestans. Further analyses revealed that β-rubromycin inhibited the germination of cysts and oospores in Pythium aphanidermatum. A chemical genetic approach revealed that β-rubromycin stimulated the expression of RIO kinase-like gene (PITG_04584) by 60-fold in Ph. infestans. Genetic analyses revealed that PITG_04584, which lacks close non-oomycete relatives, was involved in zoosporogenesis, cyst germination, and appressorium formation in Ph. infestans. These data imply that further functional analyses of PITG_04584 may contribute to new methods to suppress diseases caused by oomycetes.

The downy mildew pathogen, Pseudoperonospora humuli, forms oospores abundantly in diseased hop tissue. Diverse monosporangial isolates of P. humuli derived from samples collected in Japan, Germany and the USA readily formed oospores within hop leaves when inoculated singly, suggesting homothallism. Single zoospore isolates also readily formed oospores within hop leaves, further supporting the homothallic nature of this oomycete. The majority of oospores were deemed viable based on cytoplasm characteristics and plasmolysis assays. However, disease symptoms failed to develop when hop leaves were inoculated with newly formed oospores, even when oospore conditioning was attempted with treatment with potassium permanganate or β-glucuronidase/arylsulphatase, brief exposure to freezing temperature, or passage through an earthworm. Oospores derived from a monosporangial isolate of P. humuli that overwintered outdoors in infested leaves buried in soil also failed to cause downy mildew. Pseudoperonospora humuli is homothallic and oospores of the organism appear to require as yet unknown conditions to stimulate their germination and/or infection.

The Phytophthora genus is composed, mainly, of plant pathogens. This genus belongs to the Oomycete class, also known as “pseudo-fungi”, within the Chromista Kingdom. Phytophthora spp. is highlighted due to the signifcant plant diseases that they cause, which represents some of the most economically and cultural losses, such as European chestnut ink disease, which is caused by P. cinnamomi. Currently, there have been four genome assemblies placed at the National Center for Biotechnology Information (NCBI), although the progress to understand and elucidate the pathogenic process of P. cinnamomi by its genome is progressing slowly. In this review paper, we aim to report and discuss the recent fndings related to P. cinnamomi and its genomic information. Our research is based on paper databases that reported probable functions to P. cinnamomi proteins using sequence alignments, bioinformatics, and biotechnology approaches. Some of these proteins studied have functions that are proposed to be involved in the asexual sporulation and zoosporogenesis leading to the host colonization and consequently associated with pathogenicity. Some remarkable genes and proteins discussed here are related to oospore development, inhibition of sporangium formation and cleavage, inhibition of fagellar assembly, blockage of cyst germination and hyphal extension, and bioflm proteins. Lastly, we report some biotechnological approaches using biological control, studies with genome sequencing of P. cinnamomi resistant plants, and gene silencing through RNA interference (iRNA). The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO UID/AGR/00690/2019. info:eu-repo/semantics/publishedVersion

International audience; Aphanomyces euteiches is a polyphagous, homothallic soilborne pathogen producing asexual (zoospores) and sexual (oospores) spores. Even if oospores are essential for disease development and survival, to date, no study has focused on the production rates of oospores or the quality of the offspring produced by oospores. In this study, a nonabrasive oospore extraction method from infected roots of leguminous species (pea, faba bean and vetch) was developed. This methodology includes steps of grinding and filtration. The quality of oospores (viable, dormant and dead) was assessed with tetrazolium bromide staining, and germination of oospores was tested using exudates of peas, faba bean and vetch. The average yield of the extraction method was approximately 21%. Staining revealed some differences between strains and between leguminous species. The germination percentage of oospores extracted from pea, faba bean and vetch was 25%, 62% and 70%, respectively, and a significant difference was observed according to the origin of A. euteiches‐inoculated strains. Application of exudates seems to stimulate the germination of oospores (2% for the control, 18% for pea exudates and 1% for vetch exudates). Differences observed between A. euteiches strains and leguminous species indicate that more knowledge concerning the biology of oospores is needed. This will help to better estimate evolution process of the pathogen and manage resistance and crop successions.

Sexual and asexual reproduction are two key processes in the pathogenic cycle of many filamentous pathogens. However in Peronophythora litchii, the causal pathogen for the litchi downy blight disease, critical regulator(s) of sexual or asexual differentiation has not been elucidated. In this study, we cloned a gene named PlM90 from P. litchii, which encodes a putative Puf RNA-binding protein. We found that PlM90 was highly expressed during asexual development, and much higher than that during sexual development, while relatively lower during cyst germination and plant infection. By polyethylene glycol (PEG)-mediated protoplast transformation, we generated three PlM90-silenced transformants and found a severely impaired ability in sexual spore production and a delay in stages of zoospore release and encystment. However, the pathogenicity of P. litchii was not affected by PlM90-silencing. Therefore we conclude that PlM90 specifically regulates the sexual and asexual differentiation of P. litchii.

Syringomycin E (SRE) is a cyclic lipodepsinonapeptide with potent antifungal activity and is produced by certain strains of Pseudomonas syringae pv. syringae. In this study, its potential as an organic-compatible agrofungicide and vegetable seed treatment against the soilborne pathogen Pythium ultimum var. ultimum was examined. A variant of P. syringae pv. syringae strain B301D with enhanced SRE-producing capabilities was isolated and grown in a bioreactor with SRE yields averaging 50 mg/l in 40 h. SRE was extracted and purified through a large-scale chromatography system using organic-compatible processes and reagents. The minimum concentrations of the purified product required to inhibit 50 and 90% of P. ultimum oospore germination were determined as 31.3 and 250 μg/ml, respectively. Drench treatment of cucumber seeds in P. ultimum-infested potting medium (500 oospores/g) with 50 μg/ml SRE or water with no SRE resulted in 90.2 ± 4.5% and 65.7 ± 4.6% germination rates, respectively. Seed coating with 0.03% (w/w) SRE allowed 65.7 ± 4.6% seedlings to germinate on naturally infested soil while 100.0 ± 0.0% of non-coated seeds were unable to germinate due to Pythium infection. Organic-compatible and scalably produced SRE is potentially a novel organic fungicide seed protectant.

In the spring of 2007, a serious disease on amaranth was noticed in several farms in the major amaranth production area in central Taiwan. Abundant oospores were found in the disease tissues. A species of Phytophthora was consistently isolated from disease tissues. The organism formed abundant oospores with smooth walls and with amphigynous antheridia in single culture. Sporangia were partially deciduous with short- to medium-length pedicels. Morphological characteristics of this organism did not match any reported Phytophthora species, and the organism was named Phytophthora amaranthi. Pathogenicity tests and molecular characterization confirmed the identity of the organism as a new pathogen of amaranth and a new species of Phytophthora.

A homothallic Phytophthora species was found to be consistently associated with a rot of mature fruits of two local cultivars of olive (Olea europaea) in Calabria, southern Italy. The phylogenetic analysis of sequences of the ITS1‐5.8S‐ITS2 region and cox1 gene enabled its identification as a new species of clade 2, with a basal position compared to previously described subclades. The new species is described formally with the epithet Phytophthora oleae, referring to the natural matrix from which it was isolated. A unique combination of molecular and morphological characters clearly separates P. oleae from other already described Phytophthora species. This new species produced semipapillate, occasionally bipapillate, persistent sporangia on simple sympodially branching sporangiophores as well as globose and smooth‐walled oogonia, paragynous antheridia and spherical, plerotic oospores. The pathogenicity of P. oleae was confirmed in inoculation trials on fruits of three olive cultivars, including the two local cultivars from which the pathogen had been isolated.

Sexual reproduction remains an understudied feature of oomycete biology. To expand our knowledge of this process, we used RNA-seq and quantitative proteomics to examine matings in Phytophthora infestans. Exhibiting significant changes in mRNA abundance in three matings between different A1 and A2 strains compared to nonmating controls were 1170 genes, most being mating-induced. Rising by >10-fold in at least one cross were 455 genes, and 182 in all three crosses. Most genes had elevated expression in a self-fertile strain. Many mating-induced genes were associated with cell wall biosynthesis, which may relate to forming the thick-walled sexual spore (oospore). Several gene families were induced during mating including one encoding histidine, serine, and tyrosine-rich putative wall proteins, and another encoding prolyl hydroxylases which may strengthen the extracellular matrix. The sizes of these families vary >10-fold between Phytophthora species and one exhibits concerted evolution, highlighting two features of genome dynamics within the genus. Proteomic analyses of mature oospores and nonmating hyphae using isobaric tags for quantification identified 835 shared proteins, with 5% showing >2-fold changes in abundance between the tissues. Enriched in oospores were β-glucanases potentially involved in digesting the oospore wall during germination. Despite being dormant, oospores contained a mostly normal complement of proteins required for core cellular functions. The RNA-seq data generated here and in prior studies were used to identify new housekeeping controls for gene expression studies that are more stable than existing normalization standards. We also observed >2-fold variation in the fraction of polyA+ RNA between life stages, which should be considered when quantifying transcripts and may also be relevant to understanding translational control during development.

The genus Chlamydomyzium is a little studied holocarpic oomycete parasite of nematodes of uncertain phylogenetic and taxonomic position. A new holocarpic species, Chlamydomyzium dictyuchoides, is described which has usually refractile cytoplasm and a dictyuchoid pattern of spore release. This new species infects bacteriotrophic rhabditid nematodes and was isolated from diverse geographical locations. Infection was initiated by zoospore encystment on the host surface and direct penetration of the cuticle. A sparsely branched, constricted, refractile thallus was formed which eventually occupied almost the entire host body cavity, often accompanied by complete dissolution of the host cuticle. Walled primary cysts formed throughout the thallus and each cyst released a single zoospore via an individual exit papillum, leaving a characteristic dictyuchoid wall net behind. At later stages of infection some thalli formed thick-walled stellate resting spores in uniseriate rows. Resting spore formation appeared to be parthenogenetic and was not accompanied by the formation of antheridial compartments. These spores had ooplast-like vacuoles and thick multi-layered walls, both of which suggest they were oospores. The maximum likelihood tree of sequences of the small ribosomal subunit (SSU) gene placed this new isolate in a clade before the main saprolegnialean and peronosporalean lines diverge. A second undescribed Chlamydomyzium sp., which has direct spore release forms a paraphyletic clade, close to C. dictyuchoides and Sapromyces. The fine structure of other documented Chlamydomyzium species was compared, including an undescribed (but sequenced) isolate, SL02, from Japan, Chlamydomyzium anomalum and Chlamydomyzium oviparasiticum. Chlamydomyzium as currently constituted is a paraphyletic genus that is part of a group of phylogenetically problematic early diverging clades that lie close to both the Leptomitales and Rhipidiales.

The microscopic examination of Phytophthora cinnamomi in plant tissues is often difficult as structures such as hyphae, chlamydospores and oospores are frequently indistinguishable from those of other fungi and oomycetes, with histological stains not enabling species differentiation. This lack of staining specificity makes the localization of P. cinnamomi hyphae and reproductive structures within plant tissues difficult, especially in woody tissues. This study demonstrates that with the use of a species-specific fluorescently labelled DNA probe, P. cinnamomi can be specifically detected and visualized directly using fluorescent in situ hybridization (FISH) without damage to plant or pathogen cell integrity or the need for subculturing. This approach provides a new application for FISH with potential use in the detailed study of plant–pathogen interactions in plants.

The mycoparasitic oomycete Pythium oligandrum is homothallic, producing an abundance of thick-walled spiny oospores in culture. After mining a cDNA sequence dataset, we identified a family of genes that code for small tyrosine rich (Pythium oligandrum small tyrosine rich (PoStr)) proteins. Sequence analysis identified similarity between the PoStr proteins and putative glycine-rich cell wall proteins from the related plant pathogenic oomycete Pythium ultimum, and mating-induced genes from the oomycete Phytophthora infestans. Expression analysis showed that PoStr transcripts accumulate during oospore production in culture and immunolocalisation indicates the presence of these proteins in oogonial and oospore cell walls. PoStr protein abundance correlated positively with production of oogonia as determined by antibiotic-mediated oogonia suppression. To further characterise the role of PoStr proteins in P. oligandrum oospore production, we silenced this gene family using homology-dependent gene silencing. This represents the first characterisation of genes using gene silencing in a Pythium species. Oospores from silenced strains displayed major ultrastructural changes and were sensitive to degradative enzyme treatment. Oogonia of silenced strains either appeared to be arrested at the mature oosphere stage of development or in around 40 % of the structures, showed a complete suppression of oospore formation. Suppressed oogonia were highly vacuolated and the oogonium wall was thickened by a new inner wall layer. Our data suggest PoStr proteins are probably integral structural components of the normal oospore cell wall and play a key role in oospore formation. QC 20130524

The biology of late blight of potato and tomato, caused by Phytophthora infestans, changed when sexual reproduction by the pathogen became possible in many parts of the world, including Europe. In northern Europe, especially Scandinavia, there is increasing evidence that the pathogen is reproducing sexually on a regular basis, although in other regions further south or to the west it appears to reproduce primarily in a clonal manner. The presence of both mating types, the production of viable oospores, and observations of fields with soilborne sources of inoculum are consistent with sexual reproduction. Studies with different marker systems have revealed a population structure without any dominating clonal lineages in Scandinavia, and that is most easily explained by sexual reproduction. Phytophthora infestans recovered from the soil can also be linked to parental genotypes using likelihood-based methods when codominant markers are used. A synthesis of all the available data points to a second centre of sexual reproduction in northern Europe.

Ten Phytophthora infestans isolates were analysed for maximal growth rate (μmax), sporulation capacity (K), ploidy and the ability to produce infective F1 progeny when combined in different crosses on tomato leaflets. Ploidy was determined using three different methods: simple sequence repeats (SSR, microsatellites), allele quantification at F382Y in the RNA polymerase, and flow cytometry. Eight out of 10 isolates were classified as diploid, the other two as triploid. There was no correlation between ploidy, growth rate and sporulation capacity although the latter was rather low for triploid isolates. Ten crosses were investigated in tomato leaflets (five 2n × 2n; four 2n × 3n; one 3n × 3n). Oospore production was observed in all crosses independent of parental ploidy. Germination and pathogenicity of oospores were investigated by measuring the number of infection sites (pathogenic F1 progeny) over a time period of 72 days. All crosses containing a triploid parent produced many fewer F1 isolates than 2n × 2n crosses. The generation of pathogenic F1 progeny isolates was best for 2n × 2n crosses. A total of 319 F1 offspring were produced and analysed for mating type and ploidy; a subset was tested for ploidy with all three methods. For 2n × 2n crosses, the majority of offspring were diploid, whereas in crosses containing one or two triploid parents, diploid, triploid and trisomic offspring were observed. The results suggest that less pathogenic F1 progeny isolates are produced if at least one parent is triploid.

Pythium polare sp. nov. is a new heterothallic oomycete species isolated from fresh water and moss from various locations in both the Arctic and Antarctic. This water mould is able to infect stems and leaves of Sanionia moss (Sanionia uncinata). Pythium polare causes brown discolouration in in vitro inoculation tests at 5 °C after 5 weeks of inoculation. It is characterized by globose sporangia with various lengths of discharge tubes releasing zoospores and aplerotic oospores with usually one to five antheridia. The sexual structures are only produced in a dual culture of antheridial and oogonial isolates. Phylogenetic analysis, based on ITS sequencing, places all isolated strains of P. polare in a unique new clade, hence it is considered a novel species. Pythium canariense and Pythium violae are the most closely related species of P. polare based both on morphology and the phylogenetic analysis.

Oospore formation of Pseudoperonospora cubensis was investigated in 10 Chinese locations: Mohe, Harbin, Changchun, Shenyang, Beijing, Liaocheng, Yinchuan, Xining, Yangling and Haikou. Oospores were observed in all but Haikou. Oospore viability was monitored from 10 January to 10 July 2009, in Harbin. Percentages of activated oospores increased from 10 April with a peak in late May (14.0% on 25 May 2009), and then decreased. This is in accordance with the usual time of downy mildew appearance in Harbin, 20–30 May. Inoculation tests using the oospores overwintered in Harbin, whether in the greenhouse or outdoors, showed that these were viable, with a disease occurrence of 26.6–95.0%. Oospores overwintering locally could be primary infection sources of downy mildew in cool temperate northern China.

A sensitive real-time polymerase chain reaction (PCR) assay was developed for the quantification of Phytophthora infestans, the cause of foliar and tuber late blight in potato. A primer pair (PinfTQF/PinfTQR) and a fluorogenic probe (PinfTQPR) were designed to perform a quantitative assay for the detection of P. infestans in leaves, tubers and soils. The assay was shown to be specific to P. infestans and the very closely taxonomically related non-potato pathogen species P. mirabilis, P. phaseoli and P. ipomoea, but did not detect the potato pathogens P. erythroseptica and P. nicotianae. The assay was able to reliably detect P. infestans DNA at 100 fg per reaction and was effective in quantifying P. infestans in infected leaf tissue from 24 h after inoculation and also in infected symptomless tubers and diseased tubers. Attempts to detect oospores of P. infestans in naturally and artificially infested soil samples are described and compared with baiting tests and previous literature. It was not possible to detect oospores in soil samples due to problems with DNA extraction from the oospores themselves. However, the assay was shown to detect even very low levels of asexual inoculum (sporangia and mycelium) in soil. This work assembles all the necessary features of a quantitative P. infestans assay, which have previously been somewhat disparate: the sensitivity, specificity and quantitation are fully validated, the assay is shown to work in common applications in leaf and tuber tissue and the problems with P. infestans oospore detection are explored and tested experimentally.

White tip, caused by Phytophthora porri, is a devastating disease in the autumn and winter production of leek (Allium porrum) in Europe. This study investigated the disease cycle of P.porri in laboratory and field conditions. Oospores readily germinated in the presence of non-sterile soil extract at any temperature between 4 and 22 degrees C, with the formation of sporangia which released zoospores. The zoospores survived at least 7 weeks in water at a temperature range of 0 till 24 degrees C. Microscopic examinations revealed that zoospores encysted and germinated on the leek leaf surface and hyphae entered the leaf directly through stomata or by penetrating via appressoria. Oospores were formed in the leaves within 6 days, while sporangia were not produced. By monitoring disease progress in fields with a different cropping history of leek, it could be deduced that P.porri survives in soil for up to 4 years. Disease progress during three consecutive years was correlated with average daily rainfall in the infection period. Disease incidence on leek was reduced when rain splash was excluded by growing the plants in an open hoop greenhouse. Based on these findings, we propose a disease cycle for P.porri in which oospores germinate in puddles, and zoospores reach the leaves by rain splash and survive in water in the leaf axils, from where they infect the plant by direct penetration or via stomata. When conditions become unfavourable, oospores are produced in the leaves which again reach the soil when leaves decay. Secondary spread of the disease by sporangia does not seem to be important.

Phytophthora kernoviae exhibits comparable epidemiology to Phytophthora ramorum in invaded UK woodlands. Because both pathogens have an overlapping geographic range in the UK and often concurrently invade the same site, it is speculated that P. kernoviae may also invade North American (NA) forests threatened by P. ramorum, the cause of Sudden Oak Death. This paper addresses the susceptibility of select NA plants to P. kernoviae, including measures of disease incidence and severity on wounded and unwounded foliage. The potential for pathogen transmission and survival was investigated by assessing sporangia and oospore production in infected tissues. Detached leaves of Rhododendron macrophyllum, Rhododendron occidentale and Umbellularia californica, and excised roots of U. californica and R. occidentale were inoculated with P. kernoviae and percent lesion area was determined after 6 days. Leaves were then surface sterilized and misted to stimulate sporulation and after 24 h sporangia production was assessed. The incidence of symptomless infections and sporulation were recorded. All NA native plants tested were susceptible to P. kernoviae and supported sporangia production; roots of U. californica and R. occidentale were both susceptible to P. kernoviae and supported sporangia production. Oospore production was also observed in U. californica roots. The results highlight the vulnerability of select NA native plants to infection by P. kernoviae, suggest that symptomless infections may thwart pathogen detection, and underscore the importance of implementing a proactive and adaptive biosecurity plan.

Two novel homothallic species of Phytophthora causing dieback of Kwongan vegetation in south-west Western Australia are described here as Phytophthora arenaria sp. nov. andPhytophthora constricta sp. nov. DNA sequencing of the ITS rDNA and cox1 gene confirmed that P. arenaria and P. constricta are unique species residing in ITS clades 4 and 9, respectively. Phytophthora arenaria has been isolated from vegetation occurring on the northern sandplains which are warmer and drier than the southern sandplains from which P. constricta has been predominantly isolated, and both species appear morphologically and physiologically well adapted to the ecosystems in which they occur. Both species have been associated mainly with dead and dying Banksia species and the pathogenicity of both P. arenaria and P. constricta to Banksia attenuata was confirmed in this study. The combination of unique DNA sequences, including considerable variation in cox1 sequence data, thick oospore walls and physiological characteristics that appear to be adaptations favouring survival in the harsh Kwongan ecosystem suggest that these species may be endemic to Western Australia.

The dynamics of a late blight epidemic and sexual reproduction in Phytophthora infestans were studied in an experimental field in mid-Sweden. The field was inoculated with six isolates of P. infestans taken from another potato field where sexual reproduction of the pathogen was suspected. Three weeks after inoculation single-lesion leaflets were sampled and the resulting isolates characterized using microsatellites (SSRs) and mating type as markers. Among the 151 isolates analysed, the inoculum genotypes constituted more than 80% of the genotypes found, with three other genotypes making up the remainder. The following year, P. infestans obtained from soil samples taken from this field were analysed, and six novel genotypes were identified. Genotypes from the previous summer’s population were not detected. Analysis of the genotypes recovered was consistent with them being recombinants, with the previous summer’s population acting as parents. These findings are consistent with the hypothesis that oospores produced during a summer epidemic in Sweden can overwinter and cause infection the next year.

Limited information is known on the basic biology of the recently described Phytophthora kernoviae that produces homothallic oospores. In this study, different P. kernoviae isolates were used to investigate oospore maturity, germination, and infection. All isolates produced oospores in V8 broth at 20°C in the dark by 6d. Oospores also formed at 10 and 15°C, but did not form at 25 and 28°C. Continuous light inhibited oospore production of some isolates but had no negative effect on others. Maturation time of the oospores, as noted by germination and staining with tetrazolium bromide, was not much different among the isolates between 2 and 14 weeks. Oospore germination was optimal at 18 and 20°C, and did not occur at 5, 25, and 30°C. Oospore germination under continuous light was higher than in the dark, but individual isolates showed variable results. Rhododendron leaf disks inoculated with oospores and maintained in the dark at 20°C were necrotic after 1 week, while those kept under continuous light did not develop necrosis. The percentage of leaf disks infected with P. kernoviae was lower in the leaves exposed to continuous light (40%) compared to those kept in the dark (100%).

Oospores of Phytophthora ramorum were produced from intraspecific pairings between a European A1 and European or American A2 strains. Their viability was evaluated through colouration with tetrazolium bromide. The distribution of oospores in the different classes of colouration was similar to that found in other Phytophthora species (homothallic and heterothallic): most of the oospores stained purple, which corresponds to spores in dormancy. In order to produce single-oospore cultures, a method was developed to separate oospores from mycelium and chlamydospores. Germination of oospores was observed in 110, 250, 350 and 500-d-old cultures at a low proportion. Microsatellite marker analyses on oospore progenies revealed that the oospores resulted from hybridisation. More than 50 oospore progenies were characterised in terms of mating type, aggressiveness on Rhododendron leaves, and growth rate on two different media. The results are discussed in the context of pest risk analysis.

An experimental field infested with Phytophthora fragariae var. fragariae (Pff) and used for strawberry red core fungicide and cultivar resistance trials until 1981 was surveyed for the presence of inoculum of the pathogen 11 and 12 years later. Alpine strawberries, highly susceptible to all races of Pff, were grown from true seed and planted as a bait crop on a 0·5 m-spaced grid. Rapid and widespread red core infection was observed, which provided good evidence that oospores had survived in soil for this extended period. Site elevation and the distribution of red core infected plants showed a strong correlation, with a higher frequency of infected and dead plants in the lowest areas of the field. The race designation of 18 recovered isolates were determined and AFLP fingerprint patterns of some of these and their single-spore derivatives were analysed. The isolates differed little in race type, and the majority were genetically identical at 433 AFLP loci. Races used to inoculate the site in the 1970s were recovered. The fingerprints of the single variant isolate matched that of an isolation made by Hickman in the 1950s, originally used to inoculate the site. Clearly Pff is a very stable and long-lived pathogen able to retain its genetic integrity and lie dormant in soil for many years, ensuring its survival between epidemiologically favourable conditions which occur erratically.

Chara altaica A. Braun (Charales, Charophyceae), a monoecious species of the section Desvauxia, was newly found in Japan and studied by light and scanning electron microscopy (SEM) as well as gene sequence analysis of the large subunit of Rubisco (rbcL). Our first SEM observations of C. altaica oospores revealed that small papillae were scattered on the fossa wall, an SEM oospore wall ornamentation that is essentially different from that of the monoecious species C. (sect. Desvauxia) evoluta T. F. Allen. Phylogenetic analysis demonstrated that C. altaica and 10 European samples of the dioecious species C. (sect. Desvauxia) canescens Desvaux & Loiseleur-Deslongchamps formed a robust monophyletic group, in which the rbcL gene sequence from C. altaica is identical to those from 4 parthenogenetic samples, but different from 6 other bisexual and parthenogenetic samples of C. canescens.

This study investigated the role of Peronospora arborescens oospores as primary inoculum for downy mildew of opium poppy and infection types that they may give rise to in Spain using an integrative experimental approach that combined pathogenicity tests in growth chambers and field microplots, together with molecular detection of P. arborescens infection by specific nested-PCR assays. The results demonstrated that oospores in infested soil or leaf debris were effective inoculum for ingress of the pathogen through underground plant tissues early in poppy seedling growth. This gave rise to systemic infections that reproduced the stunting, chlorotic syndrome frequently observed in affected plants in commercial fields. Additionally, infection of underground tissues of older plants by oospore inoculum could remain asymptomatic. Results also suggested that sporangia formed on infected plants are effective in producing secondary local infections that later may become systemic and either symptomatic or asymptomatic. Finally, and more importantly, those delayed symptomatic or asymptomatic systemic infections, as well as secondary local infections of capsules, can give rise to infected seeds. This research on the biology of P. arborescens on poppy plants and epidemiology of downy mildew may help to develop knowledge-based disease-management strategies of use in the protection of yields of opium poppy crops in Spain and elsewhere.

Pythium stipitatum is a slow-growing oomycete and has been isolated from soil samples and plant materials from France, Tunisia, Turkey and India. Its morphological characteristics are reminiscent of those of Pythium ramificatum, discovered in Algeria by the corresponding author. Unfortunately, the Algerian isolate was not deposited in any culture collection and ultimately got lost. Those were the days when molecular description of fungi was not a fashion; hence, no molecular characteristics of the Algerian isolates were deposited to the GenBank. Moreover, its coralloid antheridial branches made it an easy prey to be considered as synonymous to Pythium minus. Because there are no living strains of P. ramificatum, and no sequence at the GenBank, it is being treated as 'nomen invalidum' here. However, we have now isolated the same type of oomycete from four different countries and we have sufficient evidence, both molecular and morphological, to describe it as a new species, quite different from P. minus. In this article, we are giving the morphological and molecular evidence to separate it as a distinct species, P. stipitatum, belonging to the 'Clade E' of the genus Pythium. Taxonomic description of this oomycete, its comparison with related species, and the sequence of the internal transcribed spacer region of its rRNA gene, are discussed here.

A new species of Pythium isolated from soybean in Ohio is described. Pythium delawarii sp. nov. is characterized by globose internally proliferating sporangia, aplerotic oospores and diclinous antheridia that make broad lengthwise contact. Sporangia produce conspicuous papilla and germinate indirectly by producing zoospores via a vesicle and proliferate internally or the sporangia germinate directly with either one or more germ tubes. Pythium delawarii is pathogenic on soybean causing damping-off of seedlings. This oomycete can grow at 10-34 C with an optimum of 28 C. The sequence of the ITS1, 5.8S and ITS2 region of the rDNA did not match the sequence of any known Pythium species but was similar to P. citrinum, P. litorale and P. sterilum. P. delawarii can be distinguished from these three species based on the presence of aplerotic oospores and diclinous antheridia and the absence of hypogynous antheridia. Therefore biological, morphological and molecular data support the recognition of a new species.

The determination of the members of Charales (Charophyceae) is not easy. The main reason of this is a wide range of variation in vegetative characters within most of the species. Many features such as the extent of branchlet cortication, the degree of incrustation, length and number of bract cells, spine cells, stipulodes, diameter of the main axis and oospore size and color do not have universal taxonomic significance. In similar, many Nitella species characters such as: general habitus, branchlets and presence of the top segment (mucro), are helpful in determination, however they are often not developed. In this case, the features of oospore wall ornamentation revealed by light and scanning microscopy can be important taxonomic characters of the Charales. The present study shows interspecific variations of the oospore in two Nitella species from Poland which can be helpful in correct determination of both species. Some of the oospore features like number of striae and fossa width can not be used, as they appeared statistically unimportant, and cannot be used in distinguishing between both species. The presented results are the first detailed report of oospore wall ornamentation pattern from Poland for the species studied. Oospore features are presented in detail on the background of some additional tips and among them particularly: length and width of the oospore, ISI idex, width of fossa, width of striae and number of striae.

The effect of different parameters, including the type of nutrients, the quality of the gelling agent, and the genotype of the strain, were evaluated in the production of gametangia by Phytophthora ramorum in vitro . By comparing different agar sources on a carrot-based medium, a delay or a failure in the production of oospores was observed in pairings carried out on media supplemented with technical agar. In contrast, oospores were produced on other agar types, the production on media supplemented with agarose being slightly higher. The formation of gametangia was also influenced by the genotype of the strains involved in the pairing. A European A1 strain producing very few chlamydospores was found to be a better mating partner than other A1 strains. Using a carrot–agarose medium and selected genotypes, all European isolates were characterized in terms of mating type. A macroscopic experiment highlighted a particular spatial distribution of P. ramorum oospores in vitro . A method using polycarbonate membrane was evaluated to assess the selfing ability of P. ramorum .

During the past two years the integrity of Albugo candida as the only species of Albugo parasitic to Brassicaceae has been challenged. The existence of two distinct species parasitic to Brassicaceae has been confirmed, to which a third species was added. For the purpose of further exploring the diversity of the A. candida complex, eight Albugo specimens on Draba lasiocarpa, D. nemorosa, and D. verna (Brassicaceae) were morphologically and molecularly compared with other Albugo species. Based on sequence comparisons and thorough investigation of the characteristics of the oospores, especially surface ornamentation, Albugo voglmayrii sp. nov., parasitic to Draba nemorosa, is described from five specimens collected in Korea and China. It differs from the previously described species, A. candida, A. koreana, and A. lepidii, by its oospore wall ornamentation. The morphological discrepancy is supported by high genetic distances to other species of Albugo in ITS rDNA and cox2 mtDNA. Albugo specimens from D. lasiocarpa and D. verna were grouped with A. candida, revealing that two distinct species may cause white blister rust on the genus Draba. Therefore, the paradigms that: (1) there is only a single species parasitic to Brassicaceae, that (2) oospore morphology is useful only for distinguishing between largely unrelated species; and (3) in general only one species of Albugo may occur on a single host genus need to be discarded.

Leaf blight and corm rot caused by Phytophthora colocasiae are the most devastating diseases of taro. Oospores of P. colocasiae have not been considered important in the survival in natural soil because the pathogen is heterothallic and there is essentially no chance for the presence of both A1 and A2 mating types in the same host tissue. During our recent survey of the mating type distribution of P. colocasiae in Taiwan seven homothallic isolates of Phytophthora were obtained from diseased taro leaves at Tsu Chi in central Taiwan. These organisms were identified as P. colocasiae based on morphological characteristics, ITS sequence homology and pathogenicity to taro plants. The homothallic isolates of P. colocasiae segregated into A1 and A2 types in addition to the original A1A2 type during asexual reproduction and vegetative growth. The homothallic isolate and the mixture of its A1 and A2 segregants produced abundant oospores in live tissue of taro petioles on or away from soil, indicating the possibility of oospores as a survival structure and the source of genetic variation in certain areas in nature.

During the course of the past three years, a new disease of Pinus radiata, referred to as ‘Dano Foliar del Pino’ (DFP) has appeared in the Arauco province of Chile and subsequently spread to other areas. The disease is typified by needle infections, exudation of resin at the bases of the needle brachyblasts and, in younger trees, necrotic lesions in the cambium, which eventually girdle the branches. The disease causes the death of young seedlings and mature trees can also succumb after a few years of successive infection, probably hastened by opportunistic fungi such as Diplodia pinea. Isolations on selective medium for Phytophthora spp. led to the consistent isolation of a Phytophthora sp. from needle tissue. DNA sequence comparisons for the ITS rDNA and cox II gene regions, and morphological observation showed that this oomycete represents a previously undescribed species for which the name Phytophthora pinifolia sp. nov. is provided. This new species is characterized by unbranched sporangiophores, and non-papillate, sub-globose to ovoid sporangia that are occasionally free from the sporangiophore with medium length pedicels. Despite using a number of oospore inducing techniques, oogonia/antheridia were not observed in isolates of P. pinifolia. Pathogenicity trials with P. pinifolia showed that it is pathogenic to P. radiata and causes rapid death of the succulent apical parts of young plants. Phytophthora pinifolia is the first Phytophthora known to be associated with needles and shoots of a Pinus sp. and its aerial habit is well matched with the occurrence and symptoms of DFP in Chile.

Nuclear behaviour during gametangial- and oospore development in Phytophthora palmivora was studied by light-, fluorescence-, and electron microscopy. After contact between oogonial and anther-idial initials, the antheridial initial is penetrated by the oogonial initial. The latter expands and nuclei and cytoplasm stream into it. After the delimitation of gametangia, a number of nuclei degenerate, the rest proceed through two gametic meiotic divisions both in the antheridium and the oogonium. Three of the meiotic products from each nucleus degenerate. The oogonium is penetrated by the antheridial fertilization tube and nuclear fusion occurs. The oosphere forms, then the periplasm degenerates, and the oospore wall builds up. Upon release of zoospores diploid mycelia are produced.

Morphological features of oospores of a rarely noted species Chara braunii Gmel. were examined in a local population (fishpond near Tuplice village, Ziemia Lubuska region, mid-western Poland). The largest polar axis (LPA, length), largest equatorial diameter (LED, width), isopolarity index (ISI = LPA/LED* 100), and the number of ridges and width of fossa of 90 oospores were measured. Additionally, the oospore wall ornamentation pattern was examined by means of scanning electron microscopy (SEM). As a result, a cylindrical shape and the lack of so-called “shoulder” were found. Oospore length and width ranged between 466.8–600.1 µm and 250.1–366.7 µm, respectively, with the ISI index values between 148 and 213. The number of ridges was 8–10 and the width of fossa varied between 50.0 and 66.7 µm. The oospore width appeared to be the most changeable feature, whereas, by contrast, the number of ridges was the least variable character. Wall ornamentation can be described as tuberculate and a ribbon was present on the ridges. As a comparative material for the study performed, 9 oospores from Professor Izabela Dąmbska’s Collection of Charophytes of Poland were measured. It was evidenced that variation ranges of most of the features of Chara braunii oospores from Tuplice fishpond are similar to those of Professor Dąmbska’s herbarium materials and to data reported by authors from other countries as well. The morphology of reproductive structures does not follow the variation of thalli characteristics.

Pythium kashmirense sp. nov. was isolated from soil samples taken on the Himalayas at the height of 5300 feet in the Shivalik Hill Range of the northern Indian state of Jammu and Kashmir. The oomycete has filamentous-inflated type sporangia and its antheridial filaments form loose loops around the female gametangia, and coil around the oogonial stalks. The new species is closely related to Pythium plurisporium, Pythium periilum, Pythium inflatum, and Pythium folliculosum. All of these oomycetes have filamentous-inflated type sporangia. However, P. kashmirense has its own distinguishing characteristics which can easily differentiate it among these related species. The sequences of the internal transcribed spacer (ITS) region of its rRNA and its morphological characters are unique for the genus Pythium. Taxonomic description of this new species, its comparison with related oomycetes and the sequence of the ITS region of its rRNA, are discussed here.

Biometric measurements were performed on oospores of Chara tomentosa from Core 63-J from Lake Zeribar in Iran. The partially fossilized oospores examined did not differ significantly in dimensional measurements, number of ridges, or colour from oospores of modern specimens of C. tomentosa.

Mandipropamid is a new mandelic acid amide fungicide expressing high activity against foliar infecting oomycetes, including the grapevine downy mildew, Plasmopara viticola. Because cross-resistance with the valinamide fungicides iprovalicarb and benthiavalicarb and the cinnamic acid amide fungicides dimethomorph and flumorph was postulated, all five compounds are classified as carboxylic acid amide (CAA) fungicides. To support this classification, cross-resistance among these compounds with field isolates and the segregation of resistance in F1 and F2 progeny of P. viticola were evaluated. A bimodal distribution of sensitivity in field isolates and cross-resistance among all CAAs for the vast majority of isolates were detected. Crosses between sensitive (s) and CAA-resistant (r) isolates of opposite mating types, P1 and P2, yielded abundant oospores. All F1-progeny isolates were sensitive to CAAs (s:r segregation 1:0), whereas in F2 progeny segregation of about 9:1 (s:r) was observed suggesting that resistance to CAA fungicides is controlled by two recessive nuclear genes. Mating type segregated in a ratio P1:P2 of c. 2:1 in F1 and 1:1 in F2 progeny. In the same crosses, resistance to the phenylamide fungicide mefenoxam segregated in a ratio of c. 1:3:2 (sensitive:intermediate:resistant), reflecting the monogenic, semidominant nature of resistance. The risk of resistance in P. viticola was classified as high for phenylamide and moderate for CAA fungicides. This is the first report on the inheritance of phenotypic traits in P. viticola.

Saprolegnia bulbosa sp. nov. was isolated from floating and decaying twigs and leaves in El Gato stream, Partido de La Plata, Buenos Aires Province, Argentina. The distinctive characteristics of S. bulbosa are the product of smooth oogonia and predominantly contorted monoclinous, androgynous and diclinous antheridia. The oogonial stalks are usually bent, curved or once coiled; oospores are subcentric, (1) 2–15 (45) per oogonium and are variable in size. Taxonomical description of this new species, its comparison with related oomycetes of the genus and the nucleotide sequences of the internal transcribed region (spacers ITS1, ITS2 and the gene 5.8S) of its rRNA gene are given here.

Pink rot of potato, most commonly caused by Phytophthora erythroseptica, is a major field and post-harvest problem in southern Idaho, USA, particularly since 1998 when isolates resistant to the phenylamide fungicide metalaxyl-M (mefenoxam) were detected. Isolates of P. erythroseptica were collected from infected tubers in 2001 and 2002 from six Idaho counties and tested for resistance to metalaxyl-M on amended agar. Metalaxyl-M resistant (MR) and metalaxyl-M-sensitive (MS) isolates were identified in six counties; 160 isolates were highly resistant, seven moderately resistant and 57 sensitive to metalaxyl-M with mean EC50 values of 182, 23 and 0·5 mg L−1 ai metalaxyl-M, respectively. Mycelial growth rates and oospore production in agar were assessed for 20 MS and 20 MR isolates at 10, 15, 20, 25 and 30°C. Growth rates of MR isolates were between 2·5 and 3·1 times greater (P < 0·05) than those of MS isolates at 10, 15, 20 and 25°C, and oospore production was between 6·8 and 20·5 times greater (P < 0·0001) for MR than for MS isolates at the same temperatures. Colony growth in V8 broth at 18°C was greater for MR than MS isolates (P < 0·0032). However, zoospore production at 18°C was greater for MS than for MR isolates (P < 0·0109), and zoospore production mm−1 of colony circumference was also greater for MS than for MR isolates, 14 191 and 9959, respectively (P = 0·0109). Sexual reproduction of MR isolates in nature may be greater than MS isolates, but MS isolates may be more asexually fit based on the fitness parameters studied.

The oomycete ( Peronosporomycete ) Chlamydomyzium oviparasiticum , previously recorded as a parasite of rotifer eggs, was found infecting Rhabditis nematodes in a sample of rotting garden compost. For the first time C. oviparasiticum was cultured in liquid media, which enabled more detailed studies of zoospore behaviour and facilitated the use of confocal microscopy. Rhabditis nematodes were successfully re-infected from liquid-cultured inoculum. Light (including video) microscopy and transmission electron microscopy were used to document details of thallus development, zoospore release and resting spore morphology to enable comparison with other oomycete species. This species showed several significant saprolegnialian characters such as the ‘achlyoid’ pattern of spore formation, centrifugal cleavage and structured encystment vesicles. In contrast, spore release into a transient vesicle was a peronosporalean characteristic. The thick-walled resting spores showed relatively poor cytoplasmic preservation and had a thick multi-layered wall. It was still not possible to unequivocably decide whether these were chlamydospores or parthenogenically formed oospores. The phylogenetic significance of these observations is discussed.

A total of 54 single sporangiophore isolates of Plasmopara viticola from seven regions in four European countries were examined for mating type and genotypic characteristics, using amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR, microsatellites) markers. Grape leaf discs were co-inoculated with sporangia of randomly selected single sporangiophore isolates. Based on oospore production, four fertile isolates were selected: two were defined as P1 and two as P2 mating types. This procedure was necessary because no reference isolates were available. Among the 54 isolates, 33 belonged to the P1 and 21 to the P2 mating type. AFLP and SSR analyses allocated a distinctive genotype to almost each isolate, thus reflecting a high genetic diversity among the isolates (heterozygosity index He 0.59-0.80). AFLP clustering was observed among isolates collected from the same region, whereas no clustering occurred with SSR. This study provides defined P1 and P2 isolates which will allow future genetic investigations on the inheritance of phenotypic traits in P. viticola such as the sensitivity to fungicides.

A new species, Pythiogeton zizaniae, was isolated from diseased water bamboo (Zizania latifo- lia) in central Taiwan. The organism formed a colony with scanty mycelia and mycelial aggregates on rye- water bamboo medium. Special treatments were required for production of sporangia which were terminal, noncaducous and mostly ovoid. Chlamydo- spores were absent. The fungus was homothallic. Oogonia produced on V-8 water bamboo medium in water were mostly globose to subglobose and each was attached with a club-shaped, monoclinous antherid- ium by the base of the oogonium stalk. Oospores were plerotic and globose to subglobose. Py. zizaniae caused death of water bamboo suckers but did not infect seedlings of corn, rice, wheat, sorghum, cucumber, tomato, soybean or water spinach. It also did not affect cucumber and tomato fruit, carrot roots or potato tubers.

Isolates of Pythium graminicola and related species were differentiated using restriction fragment length polymorphism (RFLP) analyses of the internal transcribed spacer (ITS) regions of rDNA and the cytochrome c oxidase subunit II (COX II) gene. These sequences were used in subsequent phylogenetic analyses. Finally, the phylogenetic placement of species was compared to that determined from morphological characteristics. The 62 isolates tested were divided into seven groups, A–G, based on RFLP analysis of the rDNA-ITS region. In the RFLP analysis of the COX II gene, isolates were divided into groups similar to those based on ITS-RFLP. Groups A and B were each separated into two additional subgroups. Grouping of isolates based on RFLP analyses agreed with the morphological differentiation. Groups A, B, D, E, F, and G were identified as P. graminicola, P. arrhenomanes, P. aphanidermatum, P. myriotylum, P. torulosum, and P. vanterpoolii, respectively. Group C was closely related to group B based on phylogenetic analysis of the rDNA-ITS region and the COX II gene and is similar to P. arrhenomanes. Each of the other species occupied their own individual clades. Although P. arrhenomanes is morphologically similar to P. graminicola, our phylogenetic analyses revealed that it was evolutionarily distant from P. graminicola and more closely related to P. vanterpoolii. Our analysis also revealed that P. torulosum with smaller oogonia is more closely related to P. myriotylum with large oogonia than to P. vanterpoolii, which forms smaller oogonia and is morphologically similar to P. torulosum. P. aphanidermatum with large oogonia and aplerotic oospores was not related to the morphologically similar species P. myriotylum. Results suggest that P. graminicola and related species are phylogenetically distinct, and molecular analyses, in addition to morphological analyses, are necessary for the accurate taxonomic placement of species in this complex.

During the course of an investigation on the Pythiaceous oomycetes occurring in the Burgundian vineyards, some species of Pythium possessing mainly hypogynous antheridia were found. These had been classified as oomycetes belonging to the ‘‘Pythium rostratum’’ group for a long time. Three of these isolates, having similar structures and growth, are very closely related to a recently described species, Pythium bifurcatum Paul. A close look at these, however, underlines some fundamental differences with the latter. Not all of them produce zoospores but have very large sporangia. The type specimen is F-1200 (B 76a) which is a medium-slow growing saprophyte. The sequence of the ITS region of the rDNA also shows a very close relationship with P. bifurcatum .O n the basis of morphological and molecular analysis, we now describe this species as Pythium longisporangium sp. nov. Morphological features of this new species, the sequences of the ITS region of its nuclear ribosomal DNA, and its comparison with related species are discussed. 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.