Oospore Wall Research Articles

Taxonomy, morphology and phylogenetic placement of a new species Nitella pluripapillifera sp. nov. (Charales, Charophyta) from Japan

ABSTRACT We examined the morphology and taxonomy of specimens of the genus Nitella, collected from the main island of Okinawa, Japan. Based on their vegetative and reproductive characteristics, the samples were most similar to Nitella tumulosa, as the two species have a large monoicous thallus without mucus, an abbreviated two-celled dactyl and papillate oospore walls. Nevertheless, the two species can be differentiated based on variations in the overall appearance of thalli and the size of oogonia. Moreover, scanning electron microscopy observations revealed the detailed structures of oospores of specimens collected from Okinawa, and their oospore characteristics were clearly distinguishable from those of N. tumulosa and other related taxa. Additionally, molecular phylogenetic analyses demonstrated that the specimens collected from the main island of Okinawa were phylogenetically separated from a clade comprising specimens of N. tumulosa. Therefore, we propose a new species from the specimen collected from the main island of Okinawa as, Nitella pluripapillifera sp. nov.

Climate acts as an environmental filter to plant pathogens.

Climate shapes the distribution of plant-associated microbes such as mycorrhizal and endophytic fungi. However, the role of climate in plant pathogen community assembly is less understood. Here, we explored the role of climate in the assembly of Phytophthora communities at >250 sites along a latitudinal gradient from Spain to northern Sweden and an altitudinal gradient from the Spanish Pyrenees to lowland areas. Communities were detected by ITS sequencing of river filtrates. Mediation analysis supported the role of climate in the biogeography of Phytophthora and ruled out other environmental factors such as geography or tree diversity. Comparisons of functional and species diversity showed that environmental filtering dominated over competitive exclusion in Europe. Temperature and precipitation acted as environmental filters at different extremes of the gradients. In northern regions, winter temperatures acted as an environmental filter on Phytophthora community assembly, selecting species adapted to survive low minimum temperatures. In southern latitudes, a hot dry climate was the main environmental filter, resulting in communities dominated by drought-tolerant Phytophthora species with thick oospore walls, a high optimum temperature for growth, and a high maximum temperature limit for growth. By taking a community ecology approach, we show that the establishment of Phytophthora plant pathogens in Europe is mainly restricted by cold temperatures.

Plastid DNA sequences and oospore characters of some European taxa of Tolypella section Tolypella (Characeae) identify five clusters, including one new cryptic Tolypella taxon from Sardinia, but they do not coincide with current morphological descriptions.

In Europe, the genus Tolypella (Characeae) comprises four to eight Tolypella taxa in sections Rothia and Tolypella that have been distinguished by vegetative morphology and gametangial characters such as antheridial size and oospore wall ornamentation. However, morphological differentiation is difficult in some cases due to overlapping and variable vegetative features, which in many cases are difficult to observe clearly. To clarify the taxonomic status of the five European taxa of Tolypella in section Tolypella, sequence data of the plastid genes atpB, rbcL and psbC for Tolypella glomerata (Desv.) Leonh., Tolypella hispanica Allen, Tolypella nidifica (O.F. Müll.) A. Braun, Tolypella normaniana (Nordst.) Nordst. and Tolypella salina Cor. were combined with data on oospore morphology, including oospore wall ornamentation. Gene sequence data identified five distinct clusters, but they were not consistent with the morphologically identified five taxa. T. glomerata consisted of some of the samples morphologically identified as T. glomerata and seven samples of T. normaniana, while the remaining T. glomerata samples clustered with specimens of unclear affiliation (Tolypella sp.). We identified two clusters of T. hispanica within the European material: cluster T. hispanica I consisted of samples from various locations, whereas the second cluster (T. hispanica II) consisted of samples of T. hispanica from Sardinia Island. The remaining cluster consisted of all the specimens that had been determined as T. salina or T. nidifica in addition to two specimens of T. normaniana. Oospore morphology was most clearly distinguishable for T. glomerata. Oospore characteristics for all other taxa were not as informative but showed some geographical and/or environmentally influenced differences, especially for T. nidifica and T. salina. Our results suggest the need to further check the different taxonomy of Tolypella sect. Tolypella in which specimens normally identified as T. glomerata might be two different taxa, T. glomerata and an unidentified taxon; T. nidifica and T. salina are not separate taxa; T. normaniana is a diminutive variant of two different Tolypella taxa; and T. hispanica comprises two different taxa, one from the Mediterranean island Sardinia.

Morphology and Molecular Phylogeny of Genus Oedogonium (Oedogoniales, Chlorophyta) from China.

Oedogoniales comprises the three genera Oedogonium, Oedocladium, and Bulbochaete, which include more than 600 described species. The classification of Oedogoniaceae is currently based on morphology, and the complicated morphological characteristics make species identification difficult, with the limited molecular data also restricting the phylogenetic analysis. In the present study, we collected 47 Oedogonium specimens from China and sequenced 18S rDNA, ITS2, ITS (ITS1 + 5.8S + ITS2), and rbcL sequences to conduct phylogenetic analyses. We selected nine morphological characteristics, most of which were considered important in traditional systematics, for comparison with the molecular phylogeny results. All the topologies based on different datasets showed similar results; Oedogonium was a paraphyletic group, and Oedocladium and Bulbochaete clustered with Oedogonium. The morphological characteristics matching the phylogenetic results showed that the types of sexual differentiation, characteristics of the oogonium (including shape, types of aperture, and ornamentation of oospore wall), division types of antheridial, and number of sperm of each antheridial, which are considered the most important morphological characteristics in traditional taxonomy of Oedogonium, did not form monophyletic lineages respectively, indicating that traditional systematics may not reflect the real phylogeny of the genus Oedogonium. In addition, a new taxonomical classification of the genus Oedogonium was presented according to the shapes of basal cells, which matched well with the phylogenetic topologies. In addition, we propose to divide the genus Oedogonium into two sections, section Globosum and section Elongatum, representing the species with spherical or sub-hemispherical basal cells and elongated basal cells, respectively.

PHYTO-THREATS: Addressing Threats to UK Forests and Woodlands from Phytophthora; Identifying Risks of Spread in Trade and Methods for Mitigation

The multidisciplinary ‘Phyto-threats’ project was initiated in 2016 to address the increasing risks to UK forest and woodland ecosystems from trade-disseminated Phytophthora. A major component of this project was to examine the risk of Phytophthora spread through nursery and trade practices. Close to 4000 water and root samples were collected from plant nurseries located across the UK over a three-year period. Approximately half of the samples tested positive for Phytophthora DNA using a metabarcoding approach with 63 Phytophthora species identified across nurseries, including quarantine-regulated pathogens and species not previously reported in the UK. Phytophthora diversity within nurseries was linked to high-risk management practices such as use of open rather than closed water sources. Analyses of global Phytophthora risks identified biological traits and trade pathways that explained global spread and host range, and which may be of value for horizon-scanning. Phytophthoras having a higher oospore wall index and faster growth rates had wider host ranges, whereas cold-tolerant species had broader geographic and latitudinal ranges. Annual workshops revealed how stakeholder and sector ‘appetite’ for nursery accreditation increased over three years, although an exploratory cost-benefit analysis indicated that the predicted benefits of introducing best practice expected by nurseries outweigh their costs only when a wider range of pests and diseases (for example, Xylella) is considered. However, scenario analyses demonstrated the significant potential carbon costs to society from the introduction and spread of a new tree-infecting Phytophthora: Thus, the overall net benefit to society from nurseries adopting best practice could be substantial.

Specific interaction of an RNA-binding protein with the 3'-UTR of its target mRNA is critical to oomycete sexual reproduction.

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.

Integrative Taxonomic, Ecological and Genotyping Study of Charophyte Populations from the Egyptian Western-Desert Oases and Sinai Peninsula.

Present-day information available on the charophyte macroalgae in Egypt, including their phylogenetic affinities, remains largely incomplete. In this study, nine charophyte populations were collected from different aquatic biotopes across the Egyptian Western-Desert Oases and Sinai Peninsula. All populations were investigated using an integrative polyphasic approach including phylogenetic analyses inferred from the chloroplast-encoded gene (rbcL) and the internal transcribed spacer (ITS1) regions, in parallel with morphotaxonomic assignment, ultrastructure of the oospore walls, and autecology. The specimens identified belonged to the genera Chara, Nitella, and Tolypella, with predominance of the first genus to which five species were assigned though they presented some interesting aberrant taxonomic features: C. aspera, C. contraria, C. globata, C. tomentosa, and C. vulgaris. Based on our integrative study, the globally rare species C. globata was reported for the second time for the whole African continent. The genus Nitella was only represented by N. flagellifera, and based on the available literature, it is a new record for North Africa. Noteworthy, an interesting Tolypella sp., morphologically very similar to T. glomerata, was collected and characterized and finally designated with the working name ‘Tolypella sp. PBA–1704 from a desert, freshwater wetland’, mainly based on its concatenated rbcL+ITS1 phylogenetic position. This study not only improved our understanding on the diversity, biogeography and autecological preferences of charophytes in Egypt, but it also broadened our knowledge on this vulnerable algal group in North Africa, emphasizing the need of more in-depth research work in the future, particularly in the less–impacted desert habitats.

Evolutionary trait-based approaches for predicting future global impacts of plant pathogens in the genus Phytophthora.

Plant pathogens are introduced to new geographical regions ever more frequently as global connectivity increases. Predicting the threat they pose to plant health can be difficult without in-depth knowledge of behaviour, distribution and spread. Here, we evaluate the potential for using biological traits and phylogeny to predict global threats from emerging pathogens.We use a species-level trait database and phylogeny for 179 Phytophthora species: oomycete pathogens impacting natural, agricultural, horticultural and forestry settings. We compile host and distribution reports for Phytophthora species across 178 countries and evaluate the power of traits, phylogeny and time since description (reflecting species-level knowledge) to explain and predict their international transport, maximum latitude and host breadth using Bayesian phylogenetic generalised linear mixed models.In the best-performing models, traits, phylogeny and time since description together explained up to 90%, 97% and 87% of variance in number of countries reached, latitudinal limits and host range, respectively. Traits and phylogeny together explained up to 26%, 41% and 34% of variance in the number of countries reached, maximum latitude and host plant families affected, respectively, but time since description had the strongest effect.Root-attacking species were reported in more countries, and on more host plant families than foliar-attacking species. Host generalist pathogens had thicker-walled resting structures (stress-tolerant oospores) and faster growth rates at their optima. Cold-tolerant species are reported in more countries and at higher latitudes, though more accurate interspecific empirical data are needed to confirm this finding. Policy implications. We evaluate the potential of an evolutionary trait-based framework to support horizon-scanning approaches for identifying pathogens with greater potential for global-scale impacts. Potential future threats from Phytophthora include Phytophthora x heterohybrida, P. lactucae, P. glovera, P. x incrassata, P. amnicola and P. aquimorbida, which are recently described, possibly under-reported species, with similar traits and/or phylogenetic proximity to other high-impact species. Priority traits to measure for emerging species may be thermal minima, oospore wall index and growth rate at optimum temperature. Trait-based horizon-scanning approaches would benefit from the development of international and cross-sectoral collaborations to deliver centralised databases incorporating pathogen distributions, traits and phylogeny.

Taxonomy and phylogeny of Nitella (Charophyceae, Characeae) from Brazil with emphasis on the midwest and southeast regions

The genus Nitella is the most species-rich within the Charales. Brazilian studies on the genus are relatively scarce and consist of floristic surveys, lacking modern and more precise information. This investigation applied scanning electron microscopy to analyze the oospore wall and molecular data associated with traditional morphological characters to analyze forty-two populations of Nitella from the midwest and southeast regions of Brazil. Forty-two new sequences of rbcL, twelve of ITS1 and twenty-three of ITS2 were generated for the five species recognized in this study: Nitella acuminata A. Braun ex Wallman, Nitella axillaris A. Braun, Nitella elegans B. P. Pal, Nitella flagellifera J. Groves & G. O. Allen and Nitella microcarpa A. Braun.. Phylogenetic analyses of sequences of these three markers were congruent in that they grouped our species with others from different countries to form five clades. Our results on ultrastrucure of the oospore wall were consistent with previous studies for the same species from other regions of the world. The data reinforced the conclusion that the use of ornamentation of oospore wall may be extremely useful for the construction of a natural system for Characeae at section level. Molecular evidence, reinforced by morphological data, for the Brazilian material analyzed suggests that Nitella subglomerata A. Braun and Nitella gollmeriana A. Braun could be synonymys of Nitella acuminata; and Nitella axilliformis K. Imahori appears to be the same as Nitella axillaris. However, no formal proposition was made considering that type specimens were not analyzed and these observations were based on a relatively small number of samples strictly from Brazil. We showed that even among geographically distant populations, such as from other continents, of some Nitella species, the degree of identity among DNA sequences was high.

Transcriptomic and proteomic analysis reveals wall-associated and glucan-degrading proteins with potential roles in Phytophthora infestans sexual spore development.

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.

Taxonomical analysis of closely related species of Chara L. section Hartmania (Streptophyta: Charales) based on morphological and molecular data

In this study, five charophyte species belonging to the genus Chara L. section Hartmania, collected in Poland, were examined to determine their taxonomic status. Oospore dimensions of all investigated species differed significantly in their length, width and number of striae. However, no clear differences were found in the case of the oospore wall ornamentation that could be used for clear differentiation of the studied species. Detailed SEM images showed that the ornamentation on the oospore wall is not species specific, and also does not allow for differentiation of the species. Moreover, molecular phylogenetic analyses based on the matK and atpB DNA sequences reveal that the five studied species of section Hartmania are closely related and exhibit low genetic variation. Both types of analyses, based on oospore morphology and DNA barcoding, suggest a close phylogenetic relationship among the investigated species. The low levels of variability between species suggest that they had a relatively recent common ancestor, and probably exhibit a still incomplete, speciation process.

Taxonomy and phylogeny of Chara (Charophyceae, Characeae) from Brazil with emphasis on the midwest and southeast regions

South American studies on the genus Chara are relatively scarce, most consisting of floristic surveys and using only traditional morphological characters. This study is a first approach to the systematics of the genus Chara applying modern techniques (DNA sequences and oospore SEM analyses) in addition to the alpha-taxonomy investigations that have been conducted in Brazil. Twelve populations of Chara were analyzed from the midwest and southeast regions of Brazil. Sequences of three molecular markers were applied to infer phylogenies. The ultrastructure of the oospore wall and currently used morphological characters were analyzed for Chara populations. Maximum likelihood and Bayesian analyses of sequences of rbcL, ITS2, and matK were congruent in that they grouped the species in six clades, each representing one species: Chara braunii C.C. Gmelin, C. foliolosa C.L.Willdenow, C. guairensis R.Bicudo, C. haitensis M.P.J.F. Turpin, C. hydropitys H. Reichenbach and C. rusbyana M. Howe. Morphological characters, including ultrastructure of oospore wall, provided good evidences to characterize each species. Molecular data supported the recent view that some traditional infra-generic taxa (e.g. subgenus Charopsis and subsection Willdenowia) are not supported in phylogenetic analyses, whereas some species (e.g. C. foliolosa, C. haitensis, C. hydropitys and C. rusbyana previously considered as varieties and forms of C. zeylanica) were consistently distinguished in the analyses for the three molecular markers.

First Report of Phytophthora hedraiandra Causing Rhododendron Dieback and Root Rot of Common Beech in the Czech Republic.

From 2010 to 2012, Phytophthora isolates were obtained from brownish diffusion leaf lesions usually up to 2 to 3 cm in diameter of Rhododendron caucasicum 'Cheer,' from withered twigs of Rhododendron sp. with blackish elongated lesions up to ~5 cm in length, and from rotten feeder roots of 2-year-old, chlorotic, wilting seedlings of Fagus sylvatica collected from ornamental and forest nurseries in three areas (central and eastern Bohemia and northern Moravia) in the Czech Republic. Isolates formed chrysanthemum-like to slightly stellate, appressed colonies with sparse aerial mycelium on V8 agar (V8A) plates at 20°C after 5 days, whereas on carrot agar (CA) plates the pattern was vague with no aerial mycelium. The cardinal growth temperatures were: min. 3°C, optimum 23 to 27°C, and max. 31°C. Radial growth was 5.7 to 6.6 mm/day at 20°C on V8A. The isolates were homothallic and produced colorless, smooth-walled, spherical oogonia with an average diameter 29.9 to 33.8 μm on CA. Oospores were aplerotic (avg. 26.4 to 29.3 μm), oospore wall was hyaline and averaged 1.3 to 1.7 μm thick, oospore wall index was 0.26 to 0.32. Paragynous or occasionally amphigynous antheridia averaged 13.4 to 15.0 × 10.9 to 12.5 μm (height × width). Sporangia were caducous, papillate, globose, spherical to ovoid, with short pedicels (avg. 2.1 to 2.6 μm) and averaged 30.9 to 41.5 × 25.5 to 30.6 μm, L:B ratio was 1.2 to 1.4. Chlamydospores were not observed. Morphological characters resembled those described for P. hedraiandra (1). The isolates were deposited in the collection of phytopathogenic oomycetes of RILOG Pruhonice and given accession nos. 450.11, 531.11, and 578.12. The isolates were sequenced for nuclear rDNA ITS region and partial Cox I gene. Obtained sequences were compared with sequences present in GenBank database using BLAST. The ITS sequences of all isolates (GenBank Accession Nos. KJ567081, 82, and 83) of overall length of 792 bp were identical to that of P. hedraiandra AY707987 (1). The Cox I sequences of overall length of 880 bp (KJ567084, 85, and 86) showed 99% homology (1 bp substitution) with AY769115 (1) and 100% identity with other Cox I sequences of P. hedraiandra, i.e., JN376067 (4) and EF174432 (3). Koch's postulates were confirmed by wound-inoculating of 3-year-old rhododendron and common beech plants (10 host plants per corresponding isolate). Rhododendron leaves were gently abraded near the midrib, whereas 5-mm-diameter bark plugs were removed from the beech collars. The inoculum (5-mm-diameter V8A plug with actively growing mycelium) was placed over wounds and sealed with Parafilm. Control plants were treated in the same manner with sterile agar plugs. Plants were maintained in a greenhouse at 22°C. All inoculated plants showed disease symptoms after 10 days of incubation; the lesions were up to 2 cm in rhododendron leaves and ~1 cm in beech collars. Control plants remained healthy. The pathogen was re-isolated from all infected plants. To our knowledge, this is the first report of P. hedraiandra in the Czech Republic. Besides it, the pathogen was found in southern and western Europe (Italy, Slovenia, Spain, the Netherlands) and in the United States (2).

Phytophthora pachypleura sp. nov., a new species causing root rot of Aucuba japonica and other ornamentals in the United Kingdom

Isolates of an unknown Phytophthora species from the ‘Phytophthora citricola complex’ have been found associated with mortality of Aucuba japonica in the UK. Based on morphological characteristics, growth–temperature relationships, sequences of five DNA regions and pathogenicity assays, the proposed novel species is described as Phytophthora pachypleura. Being homothallic with paragynous antheridia and semipapillate sporangia, P. pachypleura resembles other species in the ‘P. citricola complex’ but can be discriminated by its distinctively thick‐walled oospores with an oospore wall index of 0·71. In the phylogenetic analysis based on three nuclear (ITS, β‐tubulin, EF‐1α) and two mitochondrial (cox1, nadh1) DNA regions, P. pachypleura formed a distinct clade within the ‘P. citricola complex’ with P. citricola s. str., P. citricola E and P. acerina as its closest relatives. Phytophthora pachypleura is more aggressive to A. japonica than P. plurivora and P. multivora and has the potential to affect other ornamental species.

A taxonomic re‐evaluation reveals that Phytophthora cinnamomi and P. cinnamomi var. parvispora are separate species

SummaryBetween 2008 and 2011, severe dieback associated with root and collar rot was reported on Arbutus unedo in several sites in Sardinia, Italy. Isolations from infected tissues and rhizosphere soil samples consistently yielded a Phytophthora species. It was initially identified as Phytophthora cinnamomi var. parvispora Kröber and Marwitz by comparing morphological features with the original description and the internal transcribed spacer (ITS) sequences with those present in GenBank. A multigene phylogeny based on DNA sequence data from two nuclear (ITS and β‐tubulin) and two mitochondrial (cox1 and cox2) gene regions combined with extensive morphological and physiological properties of these isolates, including the ex‐type culture of P. cinnamomi var. parvispora, demonstrates that this taxon is unique and it is redesignated here as Phytophthora parvispora sp. nov. Although morphologically similar to P. cinnamomi, P. parvispora differs by its smaller‐sized sporangia, chlamydospores, oogonia and oospores, higher oospore wall index, single‐celled antheridia, higher minimum and maximum temperatures for growth and faster growth at optimum temperature. In the phylogeny, P. parvispora falls within Phytophthora ITS clade 7a, grouped in a well‐supported clade sister to P. cinnamomi. In pathogenicity tests, P. parvispora and P. cinnamomi were equally aggressive towards A. unedo seedlings. The possible geographic origin of P. parvispora is also discussed.

Oospore Wall Ornamentation in the Genus Tolypella (Charales, Charophyceae).

The ultrastructural features of oospore wall ornamentation in the genus Tolypella were examined using scanning electron microscopy (SEM). The taxonomic relationships among several species were discussed on the basis of oospore ultrastructure and measurements. In the case of T.glomerata and T.nidifica, our results support the status of separate species. Close relationships and transitional forms may exist between T.nidifica and T.normaniana, and not only in oospore wall ornamentation. Oospores of T.hispanica exhibited the same distinct type of reticulate oospore wall as previously reported, but our results do not support the recognition of T.hispanica as a separate species. Ultrastructure of the oospore walls of T.prolifera and T.intricata was almost identical, suggesting that these species are closely related. We therefore reject previous suggestions that morphological characteristics of oospores as observed in SEM are sufficient for identification of individual species. Although significant differences were found among oospores in individual species of Tolypella, large variation among populations, and among individuals belonging to the same population, caused substantial overlap among species.

SEM Study of Oospore Characteristics in Endemic and Endangered Balkan Charophytes

Abstract The ultrastructural features of oospore wall ornamentation in endemic and critically endangered Balkan Charophytes were examined using scanning electron microscopy (SEM). The present study shows for the first time, in this amount of detail, the interspecific variations in the oospore of endemic and rare Charophytes in the genus Chara from the Balkan Peninsula, which will assist in the correct determination and taxonomy of this genus. Oospore features are presented in detail as the background for some additional tips, including the length and width of the oospore, the fossa width, the number of striae and the length to width ratio. Based on oospore ultrastructure, the taxonomic relationships between the endemic Chara conniventi-fragilis and C. connivens, C. visianii and C. intermedia species are discussed.

A SEM and light microscopy study of the oospore wall ornamentation in Polish charophytes (Charales, Charophyceae) - genus Chara

A SEM and light microscopy study of the oospore wall ornamentation in Polish charophytes (Charales, Charophyceae) - genus Chara

Two novel and potentially endemic species of Phytophthora associated with episodic dieback of Kwongan vegetation in the south‐west of Western Australia

Two novel homothallic species of Phytophthora causing dieback of Kwongan vegetation in south‐west Western Australia are described here as Phytophthora arenaria sp. nov. and Phytophthora 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.

First Report of Root Rot of Pedunculate Oak and Other Forest Tree Species Caused by Phytophthora plurivora in the Czech Republic.

From 2006 to 2008, several similar Phytophthora isolates were obtained from roots of mature Quercus robur and other tree species (Acer platanoides, Fraxinus excelsior, Q. rubra, and Tilia cordata) in forests and parks in several areas in the Czech Republic. The trees were characterized by chlorotic and reduced foliage, crown dieback, and reduced root hairs. Several isolates of Phytophthora were obtained from necrotic roots of these trees and identified as Phytophthora plurivora Jung & Burgess (1). Isolated colonies grown on V8A medium were radiate to slightly chrysanthemum shaped with limited aerial mycelium in the center. Optimum growth was at 25°C, minimum at 5°C and maximum at 32°C. Radial growth of colonies averaged 6.4 mm/day at 20°C. The isolates were homothallic and produced abundant smooth-walled, spherical oogonia (23.3 to 29.1 μm in diameter), oospores were nearly plerotic or plerotic (21.8 to 26.9 μm in diameter), and the oospore wall was 1.2 to 1.4 μm thick. Antheridia were usually paragynous and measured 8.4 to 12 × 6.5 to 8 μm, but amphigynous antheridia were occasionally observed. Noncaducous, semipapillate sporangia formed on simple or sympodial sporangiophores, were obpyriform, ovoid, ellipsoid or irregular in shape, and occasionally distorted with more than one apex. Sporangia dimensions were 33 to 65 × 24 to 33 μm; L/B ratio 1.2 to 1.6 (-2.1). Comparison of DNA sequences of internal transcribed spacer (ITS) regions of isolates (representative strain GenBank Accession No. FJ952382) confirmed the 100% identity of P. plurivora (1). The soil infestation test was conducted using a P. plurivora isolate acquired from roots of Q. robur and 20 3-year-old plants of Q. robur. Sterilized millet seeds colonized by pathogen with the method as described (2) were used as inoculation medium and added into sterilized peat substrate at the rate of 0.5% (vol/vol). The plants were cultivated in 5.8-liter pots in a greenhouse (20°C, 16-h/8-h photoperiod). After 4 months, the roots of all plants were washed, dried, and weighed. The root biomass of 20 infected plants was significantly reduced by approximately 25% on average compared with the control 20 plants (P < 0.05, t-test, Statistica 7.1). The pathogen was consistently reisolated from the roots of infected plants but not from control plants. Stem inoculation tests were conducted with 20 replicates in each group of 2-year-old plants of oak, maple, ash, and lime and isolates acquired from the hosts. On each seedling, a 5-mm-diameter bark plug was removed 5 cm above the collar. The inoculum (5-mm-diameter V8A agar plug with actively growing mycelium) was applied to the exposed substrate. The wounds were sealed with Parafilm. Stem necrosis developed in all cases after 1 to 2 weeks, whereas control plants remained healthy. The pathogen was successfully reisolated from necrotic stem tissues. To our knowledge, this is the first report of P. plurivora causing root rot on oak, maple, ash, and lime in the Czech Republic. On the basis of the host range and distribution of P. plurivora in the Czech Republic, it can be assumed that, as elsewhere in Europe (1), this pathogen is widespread and is a common cause of decline of many tree species.