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Root rot is a major constraint on field pea ( Pisum sativum L.) production. Species of Phytophthora can cause seed rot, pre- and post-emergence damping-off, and stem and root rot in many legume crops, but have not been reported in association with pea root rot in Canada. Following a survey of pea fields in Alberta, Canada, in 2015, root tissue with typical symptoms of root rot was plated onto a growth medium, and one isolate was identified as a putative Phytophthora spp. based on its colony characteristics. The isolate was homothallic, producing smooth, thick-walled oospores and both paragynous and amphigynous antheridia on lima bean agar. The sporangia were ovoid or elliptical, non-papillate, and had internal proliferations. Based on these criteria, the isolate was tentatively identified as Phytophthora sansomeana . Sequencing of the internal transcribed spacer (ITS) and Cox II regions revealed 100% identity with the ITS and Cox II sequences of P. sansomeana available in GenBank, confirming this identification. A pathogenicity test indicated that the isolate was pathogenic and caused pre-emergence damping-off, root discoloration, and root rot with brown to dark brown lesions on pea cultivars. This is the first report of P. sansomeana causing root rot in pea in North America.

Charophytes, as a group of algae inhabiting waters characterized by varied fertility levels, represent highly critical attributes which are important for ecosystem services. One of the popular methods adopted for lake restoration is chemical inactivation of phosphates using iron coagulants. This paper presents the findings of laboratory experiments on the effects of iron sulphates on the growth and production of oospores in the charophyte Chara hispida L. In the course of the investigations, responses of this species to three dosages (Low, Medium, High) of iron (III) sulphate corresponding to the Fe concentrations of: 5.4, 10.8 and 21.6 g m−3 Fe were analysed. The results demonstrated there was a decrease in the speed of growth of the major axis and reduced production of oospores on the one hand (differences were statistically significant only between High vs. Control), and stimulation of the development and growth of side branches of the first order on the other. Several factors that cause disorders in oospore growth and production were encountered, with a wide spectrum of physico-chemical changes in the water, which resulted in the charophyte thallus being coated by a brown colloid film (limiting access of light) and lowered pH level. The study showed that the use of iron coagulants for lake restoration poses a threat to the development (growth) of charophytes.

Lychnothamnus barbatus (Meyen) Leonhardi is a rare, endangered charophyte species considered to be a reliable indicator of low water fertility, with its fossilized gyrogonites (calcified oospores) being applied as a biomarker of past oligotrophic conditions. This paper presents a case study from a lake in which L. barbatus occurs abundantly under eutrophic conditions (Lake Kuźnickie, central-western Poland). The species overwinters in this lake in the form of extensive meadows along a wide depth gradient and, in addition, produces fully developed gyrogonites. The occurrence of L. barbatus in Lake Kuźnickie was investigated at 1 m depth intervals at nine permanent transects in November 2015 and in April 2016. Spring thalli were observed microscopically to determine whether L. barbatus overwintered (with new outgrowths on old thalli) or grew anew in spring (from oospores). The species occurred at almost 50% of the surveyed sites in both sampling periods. At most sites L. barbatus formed compact carpets in which it was the only species or co-occurred with the charophyte Nitellopsis obtusa or the angiosperm Ceratophyllum demersum . L. barbatus occurred along a wide depth gradient: 1.5–6.5 m deep in autumn and 2–6.5 m after the winter period. The species coverages in April 2016 were comparable to those observed in November 2015 (Mann-Whitney U -test, P > 0.05). The thalli collected in April were well preserved after the winter, with numerous spring outgrowths. In addition, fully developed gyrogonites were found in April 2016. This study indicates wider fertility tolerance of L. barbatus and its ability to overwinter under eutrophic conditions.

This work proposes the use of a biomaterial sodium alginate for microencapsulating stonewort oospores. The microencapsulation method has been adopted and optimized to improve research work carried out on the oospores. A detailed protocol for the production of microcapsules with stonewort oospores has been presented, with particular attention paid to the critical stages and factors interfering with the efficient production process. The advantages and disadvantages of this method have also been indicated. Microcapsules with oospores can be successfully used in various laboratory (observation of the germination phases) and field experiments (reintroduction of rare species of stonewort). Alginate microcapsule may consist of water taken directly from the habitat of the stonewort, a medium, as well as specific additives (antibiotics or fungicides). The hardness of the encasement is regulated by the concentration of the sodium alginate. The optimum alginate concentration for oospores is between 2 and 3%. Encasements produced with less than 2% sodium alginate concentration do not form circular microcapsules during gelation. On the other hand, concentration >3% may limit the oospores' ability to germinate (higher density of the microcapsule). In such a dense environment, the germinating primary cells may encounter difficulties in outgrowing the microcapsule. It has been shown that alginate encasement creates an effective protective layer for the oospores inside it. The oospore is protected from mechanical damage as well as from direct exposure to the external environment; especially from pathogens and other microorganisms that can inhibit its germination and limit the growth of the protonemal and primary rhizoidal cell. The main application of oospore microencapsulation using sodium alginate is facilitating further mechanical manipulations of oospores, which by nature are very small structures (max. length 1 mm), while a microcapsule with an oospore embedded inside it can have a diameter from 3 to 5 mm (as required).

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.

Soybean [Glycine max (L.) Merr.] has great potential as an alternative crop in western Canada. Root rot is a common constraint in soybean production, and the occurrence of root rot was documented in all 29 fields surveyed in southern Alberta in 2014. Phytophthora spp. cause seed rot, pre- and post-emergence damping-off, and stem and root rot wherever soybean is produced, but it has not been previously reported in association with soybean in Alberta. Following a survey of soybean fields in 2014, root tissue with typical symptoms of root rot was plated onto a selective growth medium, and two isolates were designated as putative Phytophthora spp. based on their colony characteristics. These two isolates were homothallic on lima bean agar, producing smooth, thick-walled oospores and mostly paragynous with some amphigynous antheridia. Based on these criteria, the two isolates were identified as Phytophthora sojae Kaufmann & Gerdemann. Sequencing of the internal transcribed spacer (ITS) region revealed 100% identity with ITS sequences of P. sojae available in GenBank. A pathogenicity test revealed that both isolates of P. sojae were able to cause root rot on soybean and produce typical chocolate brown stem lesions just above the base of the plants. The two isolates were classified as races 4 and 28 of P. sojae based on their virulence on a set of soybean differential cultivars. Race 28 was more virulent than race 4 on the soybean cultivars ‘XBP27005’ and ‘XBP29002’. Regression analysis showed the positive linear relationships of the log of inoculum density with disease severity, and negative linear relationships with seedling emergence, plant height, and plant biomass. This is the first report of Phytophthora spp. causing root rot in soybean in Alberta.

Phytophthora sojae is a widely distributed, destructive oomycete plant pathogen that has been developed as a model for oomycete biology. Given the important but limited reports on the comparison of the sexual and asexual stages in Phytophthora, we performed a large-scale quantitative proteomics study on two key asexual life stages of P. sojae-the mycelium and cyst-as well as on the oospore, which is a key sexual stage. Over 29,631 peptides from 4688 proteins were analyzed. Briefly, 445 proteins, 624 proteins, and 579 proteins were defined as differentially quantified proteins in cyst vs mycelium, oospore vs cyst, and oospore vs mycelium comparisons, respectively (|log2 fold change| 1 and P 0.05). Compared to the mycelium and oospore, fatty acid and nitrogen metabolism were specifically induced in cysts. In oospores, the up-regulated proteins focused on RNA transport and protein processing in endoplasmic reticulum, indicating translation, folding, and the secretion of core cellular or stage-specific proteins active in oospores, which might be used for oospore germination. The data presented expand our knowledge of pathways specifically linked to asexual and sexual stages of this pathogen. BIOLOGICAL SIGNIFICANCE: The sexual spores (oospores) in oomycetes have thick cell walls and can survive in the soil for years, thus providing a primary source and allowing the reinfection of their host plant in subsequent growing seasons. However, the proteomic study on oospores remains very limited as they are generally considered to be dormant. In the present study, we successfully isolated oospores, and performed a large-scale comparative quantitative proteomics study on this key sexual stage and two representative asexual stages of P. sojae. The results provide an improved understanding of P. sojae biology and suggest potential metabolic targets for disease control at the three different developmental stages in oomycetes.

International audience; Charophytes (Charales) are multicellular algae growing in fresh- to hypersaline waters and recorded as fossils by their oospores (formed by resistant organic walls) and gyrogonites (formed by intracellular calcification of the spiral cells surrounding the oospore). Preservation of oospores is mostly restricted to Late Pleistocene–Holocene sediments, whereas calcareous gyrogonites provide the fossil record of charophytes since the Late Silurian (425 Ma) and constitute the only link between living and fossil charophytes. Oospores and gyrogonites represent successive developmental stages occurring after fertilisation of the oogonia; however, gyrogonites do not develop in the extant genera Nitella and Tolypella section Tolypella of Tribe Nitelleae, and in most species of the subgenus Charopsis. For Tribe Chareae and Sphaerochara (Tolypella section Rothia), gyrogonites display more useful characters than the oospores, allowing easy identification of the extant genera Nitellopsis, Lychnothamnus, Lamprothamnium, Sphaerochara and Chara subgenus Chara. Determination to species level, and in particular the study of plastic species, needs a statistical approach. Extant oospores/gyrogonites can be used as modern analogues to infer palaeohydrological conditions and record past biodiversity. The ecological requirements of the living species provide important information for the restoration/conservation of wetlands and their management, and contribute to the reconstruction of Quaternary palaeoenvironments. The aim of the present paper is to highlight the importance of the gyrogonite, especially when studying extant charophytes, the application of both gyrogonites and oospores to palaeoenvironmental reconstructions, and to provide an accurate terminology and determination criteria for their study.

Oospores of charophytes allow species delineation in palaeoecology, but are rarely used for determination of recent taxa. Especially in the genus Chara there is no generally accepted catalogue of oospore characters and the existing determination keys do not include brackish water material. Parameters with discriminatory power by statistical analysis are required to exploit the potential of oospore morphometry for species identification. Taking into consideration the conflicting species concepts in use for charophytes, we propose to continue building a database providing information about the parent plant of each oospore, including geographical coordinates and morphological details. This database is used here for the first time to determine which oospore parameters discriminate brackish water charophyte taxa from Germany. The results are discussed in the context of the recent debate of the systematic status of these taxa. In relation to Chara baltica var. baltica (Hartman) Bruzelius 1824, Chara baltica var. liljebladii (Wallm.) R.D.W. 1853 and Chara intermedia A. Braun 1859, our results indicate that C. baltica var. liljebladii is within the variability range of C. baltica var. baltica . On the other hand, C. intermedia and C. baltica var. liljebladii appear better separated in the statistical analyses, but still these two taxa cannot be clearly segregated except for ecological characters as, e.g. habitat salinity and incrustation. The article also presents a method for reliable discrimination between oospores of Chara aspera Willd. 1809 and Chara canescens Desv. & Loisel. In Loisel. 1810.

a b s t r a c t Plant macro remains of more than 40 samples from 13 lakes of the Trans-Urals have been radiocarbon dated. Sediment samples from one lake (Lake FS, Russia, oblast Tscheljabinsk; 50◦-54◦ N, 59◦-62◦ E) con- tained oospores of Characeae which were transferred to water containers to test their viability. Oospores of Nitella mucronata (A. Braun) Miquel (Characeae) germinated and completed their lifecycle coming from a sediment depth of more than 50 cm. Radiocarbon datings derived from seeds of the same depth of the lake sediments imply an age over 300 years (339 ± 33 uncal. BP (1469-1641 cal. AD) and 386 ± 36 uncal. BP (1440-1633 cal. AD). Results indicate the potentials of oospores to survive under favourable conditions for much longer time as previously suggested. © 2014 Elsevier B.V. All rights reserved.

A fast and simple polymerase chain reaction method has been developed for detection of Phytophthora infestans oospores, the causal agent of Late blight of Potato in soil. The method involves the disruption of oospores by grinding dry soil, using abrasive properties, in the presence of glass powder and skimmed milk powder within a short time. The latter prevents loss of DNA by adsorption to soil particles or by degradation and reduces the co-extraction of PCR inhibitors with the DNA. After phenol/chloroform extraction; the DNA is suitable for direct PCR amplification without a precipitation step. This amplification leads to detection of pathogen in infested soils before planting of crop. The real-time PCR assay we describe is highly sensitive and specific, and has several advantages over conventional PCR assays used for P. infestans detection to confirm positive inoculum level in potato seeds and elsewhere. With increasing amounts of standard DNA templates, the respective threshold cycle (Ct) values were determined and a linear relationship was established between these Ct values and the logarithm of initial template amounts. The method is rapid, cost efficient, and when combined with suitable internal controls can be applied to the detection and quantification of P. infestans oospores on a large-scale basis.

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 charophyte flora of the Hamilton Group consists of both vegetative and reproductive organs. The external structure of Sycidium is elucidated by the discovery of antheridial shields at the surface of the utricle containing the oospore. The oldest representative of the genus, the Ludlowian (Upper Silurian) Sycidium siluricum (Ishchenko and Ishchenko) which presents the same complex structure, is thus featured as the oldest bisexual plant. Unknown in North America prior to the Lower Carboniferous, the discovery of Sycidium sp. 1 in the Givetian Hamilton Group provides precise evidence of the distribution of Sycidiaceae during the Middle Devonian. Besides Sycidium, the charophyte assemblage includes gyrogonites and oospores, as well as thallus remains; on the whole, the three orders of Charophyta are represented. The fine grey shales of the Plattekill Formation suggest an aquatic, non-detrital habitat, which was certainly suitable to charophyte vegetation. The preservation of delicate structures reflects an absence of transport during fossilization.

Late blight, caused by Phytophthora infestans, is one of the most devastating diseases found in potato and tomato crops worldwide. In Colombia it also attacks other important crops: cape gooseberry and tree tomato. The knowledge of the pathogen population is determinant to effectively design control strategies.To determine the physiological and molecular characteristics of a set of Colombian P. infestans isolates.Strains isolated from Cundinamarca and Boyacá were examined for the level of resistance to mefenoxam and cymoxanil. Virulence was tested for all strains and crosses between A1 mating type, from different hosts, and the Colombian A2 mating type were tested for the production and viability of oospores in different substrates. Additionally, the molecular diversity of the avirulence gene Avr3a, the β-tubulin gene, and two single copy genes showing RxLR motif, was assessed.We found all levels of mefenoxam sensitivity, with 48% of the strains resistant. A high diversity of races was detected and the population was genetically clonal. Colombian strains had the possibility of sexual reproduction.These results will help in optimizing the use of fungicides and deployment of resistance as control strategies and will contribute to broader studies on diversity of this pathogen.

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

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.

The biological control activity of Pythium oligandrum against black scurf of potato caused by Rhizoctonia solani AG-3 was evaluated in field experiments after treatment of potato seed tubers with P. oligandrum. Seed tubers infected with black scurf sclerotia were dipped for a few seconds in a suspension of 103, 104 or 105 mL−1 P. oligandrum oospores and were then air-dried. Each level of P. oligandrum-treatment significantly reduced the disease rates of stolon at a level similar to that achieved by chemical control. When P. oligandrum populations adherent to the surface of seed tubers were determined, oospore counts on tubers treated with 104 or 105 oospores mL−1 were about 540/cm2 or about 22,000/cm2 just after dipping and decreased to about 170/cm2 or 2900/cm2 after a 3-week incubation, respectively. Confocal laser scanning microscopic observation with an immuno-enzymatic staining procedure showed that P. oligandrum hyphae had colonized the sclerotia and established close contact by coiling around the R. solani hyphae present on the surface of seed tubers, in a manner similar to that observed in the dual-culture test. Quantification of R. solani DNA by PCR indicated that the R. solani population was reduced on the seed tubers treated with P. oligandrum compared to untreated tubers. Furthermore, the ability of P. oligandrum to induce resistance against black scurf was determined using a potato tuber disk assay. Treatment of tuber disks with the cell wall protein fraction of P. oligandrum enhanced the expression of defense-related genes such as 3-deoxy- d -arabino-heptulosonate 7-phosphate synthase, lipoxygenase and basic PR-6 genes, and reduced disease severity upon challenge with R. solani compared with untreated controls. These results suggest that biocontrol mechanisms employed by P. oligandrum against black scurf involve both mycoparasitism and induced resistance.

Background: Oospores are the most persistent propagules of Phytophthora capsici. The viability of oospores is determined by staining methods such as the tetrazolium bromide (MTT) test. Objectives: The aim was to assess the MTT test and a plasmolysis method for their utility in determining viability of oospores of P. capsici. Methods: For either procedure the reactions of non-treated (viable) and lethally heat-treated (nonviable) oospores were assessed and compared with different matings of isolates. Results: The plasmolysis method provided lower estimates of oospore viability relative to MTT in almost all cases. Viability of autoclaved oospores determined by MTT ranged from 26 to 35%, while in the plasmolysis method, the false positive rate was null. Data of non-treated (viable) oospores determined by both techniques showed a significant positive correlation (r = 0.998, P = 0.0001). Viability assessments in the plasmolysis method of heated oospores in sterile water at 52 ◦C (0-2-4-20 hours) showed viability values of 81-4-0.7-0% while MTT values were 78-15.3-9-5.7% respectively. They both exhibited different values for the other tested mating (57.7-1-1-0% and 50-7.3-7.7-9% respectively). Data of autoclaved, heattreated (52 ◦C), and non-treated oospores determined by both techniques showed a significant positive correlation (r = 0.946, P < 0.0001). However, MTT always overestimated viability rates when values were near zero. Conclusions: MTT staining was non-objective, unstable and with a high rate of false positives. In contrast, reliable results were obtained using the plasmolysis method. MTT should be used in combination with other techniques such as the plasmolysis method for determining viability of P. capsici oospores.

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.

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.

A dynamic model for Plasmopara viticola primary infections on grapevine was elaborated according to a mechanistic approach. Development of the sexual stage of the pathogen was split into different state variables, in which changes from one state to another were regulated by rates depending on environmental conditions. The conceptual model was based on the definition of a primary inoculum season, a seasonal oospore (inoculum) dose, and its division into many coeval cohorts. Each cohort progresses along the primary infection cycle (production and survival of sporangia, release, survival and dispersal of zoospores, infection, appearance of disease symptoms) simultaneously, with a time step of one hour. The model was evaluated over a 12-year period (1995–2006) in Emilia-Romagna (Northern Italy) by comparing simulations with actual observations of the first seasonal symptoms of downy mildew in 43 vineyards. This data set was not used in model building and represents different environmental conditions, with both early and late primary infections. The model showed high sensitivity, specificity and accuracy. The achieved probabilities that an oospore cohort produce or not an infection were 0.994 and 0.999, respectively, while there was practically no probability that an oospore cohort produce infection when infection is not predicted by the model. Because of a small probability (

Germination and dormancy-breaking requirements have been investigated of Chara vulgaris , L. oospores harvested from surface sediments and from the plants of two lakes, Schumacher and Little Pearl in the locality of Timmins, northern Ontario. Oospores exhibited contrasting germination rates of 4–5% for fresh and 28–45% for sedimentary oospores. Dormancy of sedimentary oospores was readily broken by after-ripening for 60 days at 4 °C followed by exposure to white or red light, or by gibberellic acid (GA) in light (49–74% germination), whereas fresh oospores were less responsive (12–22% germination) to such treatments. Treatment of oospores by stratification or with GA or exposure to red light caused the induction of α-amylase activity. Abscisic acid reduced the effect of stratification and inhibited the GA-enhanced germination of sedimentary oospores, providing evidence of the participation of a phytochrome mechanism and hormonal regulation in the breaking of oospore dormancy. These results indicate that C. vulgaris oospores exhibit primary dormancy when first released from the parent plant, which is broken by exposure to low temperature, followed by secondary dormancy which is broken by a further period of stratification and exposure to light. Oospores from both lakes responded similarly to the dormancy-breaking treatments, but their overall viabilities were significantly different.

Factors governing the germination of Chara vulgaris L. and Nitella flexilis L. were investigated in a series of three experiments. Temperature, light regimes, drying, and changes in oxidation–reduction potential are postulated to be possible triggers working in isolation or in combination on dormant oospores originating in sediments with different physical/chemical characteristics. In Experiment 1, none of the N. flexilis oospores inoculated into Petri plates and culture tubes containing either agar or water and then exposed to ‘normal’ 12 h daylight 12 h dark germinated when the redox of the media remained relatively constant over the 20-day observation period, while all of the oospores in a tube in which the redox of the agar declined to below 200 mV, germinated. Similarly no germination occurred in the dark without a decrease in redox. When redox fell to below 200 mV, 11% of the inoculated oospores germinated. In Experiment 2, N. flexilis and C. vulgaris oospores were subjected to cold pre-treatments and inoculated into either water or agar in both plates and tubes. Of the N. flexilis oospores 53% germinated in the dark, and 31% under normal lighting. Of the C. vulgaris , 3% germinated in the dark and 53% under normal lighting. The stimulation of germination with the decrease in redox in the media did not re-occur. Experiment 3 compared the germination of C. vulgaris oospores from four ponds contaminated by mining wastes and from a comparatively clean wetland. The oospores were again subjected (or not) to cold pre-treatments, and were drawn from both freshly concentrated sediments and air-dried sediments that had been held in storage for either 97 or 376 days. Notably, a consistent reduction in oospore viability occurred in oospores from a particular mine pond, contaminated by nickel tailings.

The potential for field soils to cause Aphanomyces root rot of pea (Pisum sativum) was estimated for a large number of samples from commercial pea fields over a period of 5 years, using a greenhouse bioassay. The aim of the research project was to gain a mechanistic understanding of soil suppressiveness to the disease. Regression analysis showed that of the measured soil variables (Ca, Mg, K, P, pH), soil Ca concentrations had the strongest (negative) correlation with disease prevalence, and also a significant negative correlation with disease severity in samples with confirmed presence of the disease. Greenhouse bioassays using a set of non-infested soils inoculated with artificially produced oospore inoculum of the casual organism Aphanomyces euteiches, showed a similar negative correlation between soil Ca content and disease severity. Disease severity was not consistently affected by soil sterilisation, but was lowered by the addition of two different Ca salts. In contrast, addition of sodium bicarbonate to two soils lowered the content of water-soluble Ca in the soils and increased disease severity. Studies of cultures of A. euteiches exposed to varying Ca concentrations in vitro showed that zoospore production was inhibited at submillimolar concentrations, while mycelial growth was stimulated or unaffected. We conclude that free Ca is a major variable controlling the degree of soil suppressiveness against A. euteiches, and that inhibition of zoospore production from oospores is a possible mechanism.

We investigated the distribution of two charophyte species, Chara fibrosa var. fibrosa (A. Br.) and Nitella hyalina (DC.) Ag., in Myall Lake, a shallow lake in New South Wales, Australia, in an attempt to elucidate the factors causing their distribution patterns. The field study was carried out from July 2003 to May 2005 and charophytes were sampled together with bottom sediments at 20 sampling locations in the lake on 13 occasions. Charophyte biomass (0–321 g DW m−2) displayed an optimum curve with depth and maximum biomass occurred between 1 and 2.5 m depth. In deeper water, shoots were longer (i.e., around 30 cm at 1 m depth to 60–90 cm between 2 and 4 m depth). Oospore and antheridia densities were higher in shallower water with a maximum around 80 cm. Plants growing in shallow depths had shorter internodes implying a short life cycle of shoots, and nodal spacing was relatively regular in contrast to its deep water counterparts although spacing tended to increase at locations farther from the apex. The present study also reports that there is an apparent decline in sexual propagule production rates with increasing water depth, further highlighting the different morphological and reproductive acclimations of charophytes in shallow and deep water.

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.

In this study, we investigated the growth responses of a native, non-vascular charophyte (Chara globularis var. globularis Thuill.) and an invasive vascular angiosperm (Egeria densa Planch.) on three contrasting New Zealand lake sediment types (fine silt from beneath charophyte beds, sand from an exposed shoreline and low density organic-rich substrate from beneath Ceratophyllum demersum L. beds) under low and high light conditions (6 and 15% ambient). In a shallow outdoor trough, the growth responses of transplanted C. globularis and E. densa plants were examined over a 12-month period and the germination response of C. globularis oospores over a 5-month period. Under low light, growth of both species, but particularly C. globularis, was poor and no significant differences were observed between sediment types. Under high light, growth of C. globularis plants was strongly inhibited (by 96 and 49%) on the sand and C. demersum sediments relative to the charophyte sediment (320 g DM m−2 y−1). However, germination and short-term (

Pythium attrantheridium sp. nov. is a new species isolated from cavity spot lesions of carrots as well as apple and cherry seedlings from various locations widely distributed in Canada and the USA. This fungus is closely related to the heterothallic P. intermedium, but is distinguished by: (1) unique molecular characteristics; (2) unique morphological characteristics; and (3) mating incompatibility with P. intermedium. The ITS region of the nuclear rDNA of all strains of P. attrantheridium studied is different from that of all other known Pythium spp. The oogonia attract a large number of antheridia when compatible mating types contact each other. The positive mating type produces zoospores unlike those of P. intermedium. Thus, biological, morphological and molecular data support the recognition of a new species.

Charophyte establishment from oospores in lake sediment was measured under nine light regimes (range −2 d −1 ) in two short-term experiments (13–20 weeks). Natural light was modified by inorganic turbidity in Experiment 1 and controlled artificial light was used in Experiment 2. Germling response was dominated by Chara australis Brown or Chara globularis Thuill. Germling number was not significantly affected by the light regimes and germination occurred under −2 s −1 (‘Dark’ treatment). A three-fold range in PAR (1.7−5.2 mol photons m −2 d −1 ) had less influence on the final germling number than pre-drying sediment, which increased germlings ca. 1.5- to 3-fold. Drying lake sediment could be used as a management tool to stimulate charophyte germination. Light influenced germling biomass, with negligible gain at ≤0.4 mol photons PAR m −2 d −1 and biomass accrual with light ≥1.7 mol photons PAR m −2 d −1 . Germination at an irradiance insufficient to sustain germling growth suggests that this may be an important loss for oospore banks under unfavourable light conditions and might be an adaptation to germination within the soil profile. The results of these experiments indicate that for these species, light is required for establishment rather than germination.

Analysis of 18S rDNA sequence and morphological data indicate that Achlya s. lat. is polyphyletic. Aplanopsis spinosa and Achlya species with subcentric oospores and laterally applied antheridia are transferred to Newbya gen. nov. The relationship of Calyptralegnia and Aplanes androgynus to Newbya is discussed. Molecular data support morphologically based conclusions concerning the relatedness of Pythiopsis and Aplanopsis terrestris. Achlya truncatiformis nom. nov., is provided for material previously identified as Achlya recurva. The following new combinations within Newbya are made: N. pascuicola (type species), N. apiculata, N. braziliensis, N. curvicollis, N. megasperma, N. oblongata, N. oblongata var. gigantica, N. oligacantha, N. polyandra, N. recurva, N. spinosa, and N. stellata combs nov.

Charophyte oospore banks have been studied in different habitat types, whereas only one detailed study of bulbil banks has been recorded from the literature. The density of oospore banks generally appears to be very high, dominating the propagule bank in many wetlands. Oospore banks are characterised by high spatial variability within and between the sites. Bulbils and oospores are not uniformly distributed with sediment depth, oospores being generally more numerous in the top few centimetres. Animal activities can mix the sediment, bury propagules and/or homogenise their distribution. Burial deeper than 2 cm limits oospore germination, but burial to a depth of 5 mm can improve their germination capacities. Oospore banks are highly variable between years. They are generally perennial, cumulating several generations of oospores, and buffering the risk of reproductive failure with time. The limited germination rates recorded for oospores from the bank could be related to this perennial strategy, whereas up to 100% of freshly harvested oospores may germinate. Germination rates and patterns and the conditions required for germination vary greatly from one study to another as a result of the large range of species and habitats investigated. Certain water birds consume charophytes and could be efficient dispersal agents for oospores. Various abiotic factors can affect the charophyte biomass and therefore the oospore production which then in turn could affect the propagule bank. The oospore bank density appears to influence charophyte abundance in the field, but above a threshold, its abundance is not related to the number of oospores in the soil. Below a threshold of density, charophytes may be unable to establish dense stands of vegetation.

In several studies of oak decline in Europe, one semi-papillate (Phytophthora psychrophila sp. nov.) and two non-papillate homothallic Phytophthora species (P. europaea and P. uliginosa spp. nov.) were isolated, together with other Phytophthora species, from rhizosphere soil samples which could not be assigned to existing taxa. P. psychrophila differs from other semi-papillate species of Waterhouse's morphological Group IV, like P. ilicis and P. hibernalis, by its uniform, dome-shaped and cottonwool-like colony growth pattern on V8 juice agar and malt extract agar, the occurrence of sympodially branched primary hyphae, the high variation in size and shape of the sporangia, shorter pedicels, lower optimum temperature for growth, and ITS sequences. P. europaea is distinguished from related non-papillate Group V and VI species, namely P. fragariae, P. cambivora, and the ‘alder phytophthora’, by producing oogonia with tapered bases, irregular walls and exclusively paragynous antheridia, its cardinal temperatures for growth, and ITS sequences. P. uliginosa differs from related Group V and VI species by its large oogonia with exclusively paragynous antheridia, the predominant occurrence of ellipsoid sporangia with markedly wide exit pores, its slow growth, low cardinal temperatures, its colony growth patterns, and ITS sequences. P. uliginosa is separated from P. europaea by its larger oogonia without tapering bases, lower cardinal temperatures and growth rates, different colony growth patterns, and greater aggressiveness on Q. robur.

Evidence that the biometric characters of the oogonial unit of Pythium are of significant taxonomic value, allowing for a good species separation, has been presented by other workers, based on 40 species, each represented by fairly few isolates. Since broad intraspecific variation and interspecific overlapping of biometric parameters was observed in isolates of four Pythium species from Denmark, it was suspected that a such wide variability would weaken the conclusions obtained by previous biometric approaches. Species to be studied should be represented by higher numbers of isolates to reflect intraspecific variation. In the present biometric study 49 isolates were used to test the robustness of the biometric approach and to examine the taxonomic value of biometric characters from a practical perspective. Good species separation was obtained despite intraspecific variation. The biometric parameter set for the oogonium, consisting of oogonial, oospore and ooplast diameters, supplemented with the thickness of the oospore wall or the protoplast diameter and with derived indices of volume or linear ratios of the measured parameters were all of taxonomic value for the separation of species, as shown from canonical variate plots after linear discriminant analysis. More detailed information on species separation, isolate-species or isolate—isolate associations could be obtained by separation based on the Mahalanobis distance function in the linear discriminant analysis. Both the derived volume and linear indices contributed significantly to species separation and appeared to be equally satisfactory. The substrates used for oogonium production may influence the biometric parameters to an extent which may seriously affect the results of the biometric approach to taxonomy. These results stress the importance of standardizing substrates in morphological taxonomy and identification work.

A total of 81 isolates of Phytophthora infestans (Mont.) de Bary were recovered from late blight infected samples collected from different areas of Uganda in 1998/1999. They were analyzed for their resistance to metalaxyl fungicide, mating types, and cross infection between potato and tomato hosts. Sensitivity to metalaxyl was determined by growing isolates on 10% V8 medium amended with 0, 5 and 100 μg/ml metalaxyl. Overall 44.4% of the isolates tested were resistant to metalaxyl, 23.5% were intermediate and 31.2% were sensitive. Mating type was determined on 80 isolates using an A1 (1724) isolate and by growing the same isolates in pure culture (selfing). Fifty percent of the isolates produced oospores by matings and selfings, 22.5% by mating only and 10% by selfing only; 18% did not produce oospores in the two tests. Twenty seven of the P. infestans isolates from potato-infected tomato. The majority of these isolates were highly resistant to metalaxyl and produced oospores by either mating or selfing or both.

The analysis of random amplified microsatellite (RAMS) markers of Phytophthora cactorum from strawberry showed that leather rot of strawberry fruits and crown rot are not caused by genetically different strains of this species. UPGMA-analysis showed that the populations of P. cactorum on strawberry differ between North America and Europe and that no clear genetic separation between isolates from different plants can be made among these populations. Slight morphological variation exists in the diameters of oogonia and oospores among the isolates but is insufficient for the identification of genetic groups or host specificity of P. cactorum isolates. Pathogenicity experiments proved that strains show a tendency towards host specialization.

Using a membrane filter system, oospores of Pythium aphanidermatum were exposed for 80 d to soil of pH 6.9 at 0, −1 or −10 kPa, and 25°C or to soil (−1 kPa) of pH 5 or 8 at 15, 25 or 35°C. Carbon loss and pathogenicity of oospores were recorded during the 80-d period. Oospores lost most endogenous carbon to soil of pH 6.9 at −1 kPa or to soil of pH 8 at 35°C. These soil conditions induced a rapid decline of pathogenicity of oospores for cucumber seedlings. Carbon loss was negatively correlated with the pathogenicity of oospores to cucumbers. In contrast, oospores lost least carbon to soil of pH 6.9 at −10 kPa, or to soil at 15°C regardless of pH. Pathogenicity of oospores to cucumbers under these conditions did not decline, even after 80 d.

Dimensions of oospores such as length, width, fossa-breadth, length-to-width ratio and number of striae were considered for morphological identification of the members of Charales. Oospores were collected from natural stands of three species, viz. Chara braunii , C. corallina and C. wallichii in ponds in West Bengal (India). Oospore dimensions (length, width and fossa-breadth) correlated well: though the fossa breadth–length relation of C. braunii showed lower correlation coefficient ( r 2 = 0.076, p r 2 ≥ 0.23, p p C. braunii and C. corallina has similar length to width ratios, C. wallichii and C. corallina were similar in width, whereas the species differed in breadth and length in the order C. braunii , C. wallichii , and C. corallina .

Oospores were identified in pure culture ofPhytophthora infestansstrains of the clonal lineages US-7 and US-8 (A2 mating type), but not in the US-1 clonal lineage (A1 mating type). This self-fertility was not due to contamination, a mixture of isolates, or heterokaryosis because all of the single-zoospore isolates (n = 128) from these cultures displayed the same phenotype as the parental isolate from which they were derived. The self-fertility in US-7 and US-8 was different from self-fertility previously reported forP. infestansand otherPhytophthoraspecies because the oospores appeared in young cultures and the mycelia were not appressed to the medium. The number of oospores produced by US-7 and US-8 cultures was much lower than the number produced in response to an A1 mating type isolate. Analysis of eight oospore progeny forGlucose-6-phosphate isomeraseallozyme patterns revealed that six had genotypes different from the parental isolate.

The production of oospores in monoculture (homothallism) and sensitivity to the phenylamide fungicide metalaxyl, in its racemic mixture form of (R)- and (S)-enantiomers (Ridomil-25WP) or the latest isomeric form (R)-enantiomer (Metalaxyl-M), was tested using 11 single-spore and 14 field isolates of Peronospora parasitica . Three isolates were from Brassica napus (UK), 13 from B. juncea (India), four from B. rapa (India), one from Eruca sativa (India) and four from B. oleracea (one from the UK, two from France and one from India). All isolates were found to be homothallic, as they produced oospores in monoculture, except P006 (a UK single-spore isolate derived from B. oleracea and previously known to be insensitive to Ridomil-25WP) which was heterothallic. All isolates were found to be sensitive to the two forms of the phenylamide fungicide metalaxyl (Ridomil-25WP and Metalaxyl-M) except P006 and FP13, a French isolate derived from B. oleracea in Brittany. These isolates were insensitive to both fungicides at the three concentrations used in this study (0.5, 5.0 and 50.0 μg/ml). To our knowledge, this is the first reported case of insensitivity to metalaxyl in P. parasitica from France. This study emphasises the importance of deploying sources of host resistance with fungicides in prolonging the effectiveness of both control procedures.

The morphology of gametangia was examined in 43 pairs of isolates (mating types A1‹A2; 11 A1 and 24 A2 isolates; five isozyme}electrophoretic types) of Phytophthora cinnamomi. An amphigynous antheridium always formed with each oogonium. However, in 41 of the crosses a proportion (39 had 0‐2‐10% and two had " 30%) of oogonia also consistently had single or multiple paragynous antheridia. Single or multiple paragynous antheridia formed concurrently with amphigynous ones during the period of gametangial production in paired colonies. Where there were multiple paragynous antheridia associated with an oogonium, sometimes additional antheridia formed after fertilization or even after oospores were visible. Developmental studies showed that when meiosis in amphigynous and paragynous antheridia was simultaneous, fertilization tubes developed synchronously from both. However, cytological examination indicated that either a nucleus from an amphigynous or a paragynous antheridium fertilized the oosphere. Observations of paragynous and amphigynous, and amphigynous-only associations suggested that fertilization from either type of antheridium only occurred when meiosis in the oogonium was nearly synchronous with that of the antheridium. Asynchronous meiosis between oogonia and antheridia may contribute to failed fertilization and aborted oospore development. This appears to be the first description of paragynous antheridia in P. cinnamomi and the second observation of oogonia with both paragynous and amphigynous antheridia in a heterothallic Phytophthora species. Moreover, the development of both paragynous and amphigynous antheridia with an oogonium is rare in Phytophthora, as is the development of multiple antheridia. Antheridial variation is a characteristic to be taken into account in isolate identification. Nuclei from paragynous antheridia appear able to fertilize oospheres and therefore, have a role in sexual reproduction.

After purification by thin-layer chromatography (tlc) several fatty acids stimulated oospore formation in Phytophthora cactorum but not in Phytophthora parasitica. Palmitoleic acid was the most effective and was the only fatty acid tested that promoted oospore formation in P. parasitica. Purification of palmitoleic acid by Florisil column chromatography resulted in a two-fold increase in activity over that purified by tlc. When 0.01 to 100 ng of cholesterol was added to basal medium containing palmitoleic acid, the added cholesterol did not significantly change oospore production by P. cactorum. Oospore formation in P. cactorum was slightly stimulated by tlc-treated aliphatic hydrocarbons, or derivatives of hydrocarbons, but was strongly stimulated by geraniol and squalene. Vitamin A and vitamin A esters stimulated oospore formation in both P. cactorum and P. parasitica. tlc treatment also significantly increased the activity of cholesterol and β-sitosterol on oospore formation by P. cactorum. Both compounds did not stimulate oospore formation by P. parasitica even after tlc treatment.

Mating interactions in Phytophthora infestans, a heterothallic oomycete, are regulated by sexual compatibility loci and a system of sexual preference. The development of gametangial associations in this species were examined using matings between strains containing a β-glucuronidase (GUS) transgene and nontransformed strains. The GUS gene enabled the facile assessment of whether oospores resulted from hybridization between isolates or from self-interactions and allowed measurements of the sexual preference of strains. Depending on the strains being mated, from 5 to 99% of the total oospores formed resulted from outcrossing. In such hybrid pairings, A1 and A2 field isolates generally acted as predominant females and males, respectively. Nevertheless, genetic analyses indicated that sexual preference and compatibility type were determined by unlinked loci. The sexual preference of most strains varied depending on the relative preference of their mating partners. Both sexual preference and the frequency of outcrossing versus self-interactions varied with the position of gametangia within the mating culture.

The patterns of germination of oospores of several species of charophytes were investigated to explain the distribution of charophytes in permanent and temporary wetlands in north-eastern New South Wales, Australia. Germination from seed banks and after storage was evaluated. Seed banks were pre-treated with combinations of temperature and moisture to simulate the effect of inundation in different seasons. Oospores of five species of Nitella and two species of Chara germinated from the seed banks. Nitella cristata var. ambigua, N. sonderi and N. subtilissima germinated in large numbers and the pattern and timing of emergence of these species was analysed. Nitella cristata var. ambigua , a winter germinating species, had relatively synchronous and early germination from seed banks when exposed to cues for the onset of winter. Nitella sonderi and N. subtilissima displayed extended germination from seed banks of wetlands that have variable water regimes. Drying the seed bank enhanced germination of these three species. Oospores collected from plants of four species were subject to different storage treatments and were germinated in vitro after 1, 3, 7, 9, 12 and 18 months of treatment. Oospores of Chara muelleri and C. australis germinated after storage. Nitella cristata var. ambigua germinated only in the control treatment and Nitella subtilissima did not germinate at all. Highest germination of C. muelleri (76%) occurred after oospores were subject to fluctuations in light intensity and temperature, whereas highest germination of C. australis (41%) occurred after oospores were kept wet for 4months. Small oospores (680 μm) of C. muelleri germinated sooner than large (1000 μm) oospores after 1 month of storage. Oospores of C. muelleri that were stored for a short period of time (1 month) germinated faster and more synchronously than did oospores that were stored for longer. Charophytes in these temporary wetlands reduce the risk that all offspring will die in one unfavourable season by contributing to a dormant oospore bank and by having a pattern of germination that increases the likelihood of survival. Oospore germination patterns can help explain the distribution of charophytes in space and time.

Polyclonal antibodies were produced to purified oospores of Aphanomyces euteiches. The serum primarily reacted with plant pathogens of the genus Aphanomyces. The antiserum was used to detect oospores from naturally infested soils.

Freshly harvested oospores of Pythium aphanidermatum did not germinate in the absence of an external nutrient source (1% of Pfeffer's salts solution) and germinated poorly in a nutrient medium (corn meal agar, CMA). Oospores became germinable in the Pfeffer's salts solution and showed increased germinability in CMA after incubation on a membrane-soil system. However, their germinability dropped to minimum level (6 or 11%) when the incubation period was lengthened to 90 days. During this time, their virulence to cucumber declined significantly. The linear depletion of endogenous 14C significantly and negatively correlated with the germinability and virulence of oospores. Oospores of Pythium aphanidermatum lost their endogenous carbon progressively through respiration and exudation during exposure to non-sterile soil over a period of 90 days. Evolution of 14CO2 due to respiration of oospores and soil microbes, and residual 14C in soil comprised the total 14C loss which accounted to 36% of total label. Oospore respiration was the major factor contributing to endogenous C loss (61–100% of total 14C loss), whereas residual 14C in soil and soil microbial respiration contributed 7% and 4% of total label, respectively. High respiratory activity and exudation loss of endogenous C from oospores suggests that these propagules in soil are metabolically active.

Stability of the phenotypic characteristics of growth rate, colony type, sensitivity to streptomycin and sensitivity to chloramphenicol of the conidium progeny of selfed oospore cultures of Pythium splendens was investigated. Progeny were divided into parental A type, D type for those deviated from A type but with uniform pattern and M type for those with irregular growth pattern or with dissimilar appearance on two replicate plates. Conidium progeny of A type isolate 167F and its A type oospore cultures showed the same colony type and displayed little variation in growth rate, and sensitivity to streptomycin and chloramphenicol. The D and M type oospore cultures displayed considerable variation in all phenotypic characteristics measured. Similar variation in conidium progeny following selfing was observed in isolate 167F-9, an A type oospore progeny of isolate 167F. Following sexual reproduction conidium progeny continued to vary in second and third conidium generations tested. The phenomenon of continuing variation in successive asexual generations induced by sexual reproduction may plan an important role in evolution, survival and development of pathogenicity and fungicide resistance of an organism by increasing the diversity and extent of the constitutive variation resulting from gene recombination during sexual reproduction.

Mycoparasitism by Pythium periplocum, a species with spiny oogonia, was evaluated and compared on several host fungi with parasitism by P. oligandrum and P. acanthicum on the same fungi. These species were found to be aggressive mycoparasites with a wide host range. Differential reaction among some of the hosts tested were found in relation to each mycoparasitic species. Thirty isolates of each of these species, obtained from diverse agricultural soils in California were further studied. Pythium ultimum, one of the most susceptible hosts for the three species, in an in vitro test of mycoparasite virulence, was slightly susceptible, moderately susceptible, and highly susceptible to most of the isolates of P. acanthicum, P. oligandrum and P. periplocum tested, respectively. In one test, sugar beet seedlings were protected from damping-off due to P. ultimum by co-inoculation of seedlings with P. periplocum or P. oligandrum. In the same in vitro test P. acanthicum gave no protection against P. ultimum. The degree of disease protection varied among isolates within each mycoparasitic species studied. As part of the study each mycoparasitic isolate was critically characterized by the morphology of oogonia, oospores, oogonial spines, sporangia and the rate of daily growth of mycelium at 25 °C. Pythium periplocum was readily separated from the other two species by its lobate sporangia and size of the oospore. Pythium oligandrum could be separated from P. acanthium by its acutate oogonial spine apex, longer spine and faster growth rate.

The relationships between isolates of the complex species Phytophthora megasperma from chickpea in Spain and from chickpea and lucerne in Australia were investigated. In the isolates from Spain > 90% of the sporangia had flattened apices, whereas in the Australian isolates only 5·6% of sporangia had conspicuously flattened apices, the remainder being convex. No differences were found between the sex organs of the two groups, except that oospore walls were thinner in the Spanish isolates (&- x = 2·2 ± 0·6 μm) than in the Australian collection (&- x = 3·4 ± 1·0 μm). The isolates from Spain had an optimum temperature for growth of ca 24°C and a maximum temperature range of 30–33°, whereas the Australian isolates had a higher optimum growth temperature (26°) and a higher maximum temperature range (33–35°). RAPD analysis of total DNA revealed a genetic dissimilarity of ca 80% between the two groups of isolates. The study therefore differentiated two genetically distinct groups among isolates causing chickpea root and stem rot in Spain and Australia. However, it is suggested that designation of a new taxon for the Spanish isolates should be deferred until all extant representatives of the P. megasperma complex have been fully molecularly and phylogenetically analysed.