Small Oogonia Research Articles

Phylogeny and Pathogenicity of Phytophthora Species Associated with Artichoke Crown and Root Rot and Description of Phytophthora marrasii sp. nov.

Field surveys conducted on nine farms over a 2-year period showed the widespread presence of Phytophthora-related diseases on globe artichoke plants in the main growing area in Sardinia (Italy). Characteristic symptoms included wilting and necrosis of the outermost leaves and dark brown discoloration of stem tissues, as well as root rot. A total of 18 Phytophthora colonies belonging to three species were isolated and characterized. Based on morphological features and ITS sequence data, Phytophthora isolates were identified as P. crassamura (eight isolates) and P. cactorum (four isolates). Six isolates could not be assigned to any formally described species of Phytophthora and are therefore described here as Phytophthora marrasii sp. nov. The ITS phylogeny places P. marrasii in a terminal clade basal to the sister taxa (P. foliorum, P. hibernalis, P. lateralis, and P. ramorum) of the clade 8c. In particular, P. marrasii is phylogenetically related to P. foliorum, a species from which it differs in 62 nucleotides in the ITS region. At the same time, it can easily be distinguished morphologically from P. foliorum mainly because of the low minimum temperature for growth, the bigger and persistent non-papillate sporangia, and smaller oogonia. Pathogenicity tests confirmed that all three Phytophthora species are pathogenic on globe artichokes, which represent a new host for these pathogens.

Annual gametogenesis and reproductive effort of the limpet Cellana grata (Gould, 1859) (Gastropoda: Nacellidae) in a rocky intertidal beach at Ulleungdo Island off the east coast of Korea

Widely distributed from the northern coast of Vietnam to the northern Japan, the limpet Cellana grata (Gould, 1859) occurs commonly on the south and east coasts of Korea. Despite their wide distribution range, few studies have investigated the annual gametogenesis and reproductive effort of C. grata. In an attempt to understand the reproductive physiology of the limpet, we investigated the annual gametogenesis and reproductive effort of C. grata from Ulleungdo Island off the east coast of Korea. Histology revealed that the gonial mitosis commenced in January, as the female exhibited small oogonia (10–40 μm) in the follicle. From March to June, the oocyte size increased dramatically, and fully mature eggs (110–170 μm in diameter) appeared in early summer. First spawning males and females were observed in July, as the surface seawater temperature (SST) reached 22.1°C. The spawning male and females could be observed until the end of December. Gonad somatic index (GSI), a ratio of gonad mass to the total tissue weight, of the male ranged from 0.6 (April) to 17.9 (July), while the female GSI varied from 1.0 (February) to 18.3 (July). GSI of male and female declined rapidly from July to August, suggesting that the major purse of the spawning at the study site was between July and August. Our study suggested that the commercial catch of C. grata during July and August must be suspended at Ulleungdo Island, in order to protect the spawning limpets, which enhances C. grata recruitment and the population.

울릉도 남해역에 서식하는 키조개의 연중 번식주기에 관한 연구

Gametogenesis of the comb pen shell, Atrina pectinata (Linnaeus, 1767) (Bivalvia: Pinnidae) on the southern coast of Ulleungdo Island, Korea was assessed monthly (November 2013 to October 2014) using histology. Gametogenesis commenced in January when the surface water temperature was 12.6℃ and pen shells evidenced an early development phase with small oogonia from January to April, although few females exhibited ripe eggs in their follicular epithelium. In April, the oocyte diameter increased rapidly, and fully mature eggs appeared in May. First spawning males and females were observed in June as the surface water temperature reached 19.3℃ and July (23.2℃) respectively. The spawning activity continued until the end of September. Histology indicated that the spawning peak of the females in Ulleungdo Island was July to August. During October to January, most of the pen shells were in spent and resting stages. Our data suggested that A. pectinata is a summer spawner, and their annual gametogenesis is closely associated with the seasonal variation in the surface water temperature. The present study is the first provided fundamental information on the life history of A. pectinata in Ulleungdo Island, and this can be put to good use in the management of this pen shell in the study area.

First Report on the Annual Gametogenesis ofHeteromacoma irus(Hanley, 1845) in a Rocky Intertidal Area, Northern Jeju Island, Korea

The annual reproductive cycle of the Indo-Pacific tellinid clam Heteromacoma irus (Hanley, 1845) was investigated, using histology, for the first time in this study. The sex ratio of H. irus collected from a rocky intertidal area on the northern coast of Jeju Island, Korea, was found to be 1:1, indicating that H. irus is gonochoric. In January, most clams were in the resting phase, although a small number of females showed small oogonia on their follicular epithelium. From February to May, oocyte diameter increased rapidly, and fully mature eggs (44.8–56.8 μm) appeared in May. The first spawning males and females were observed in July, when the water temperature reached 18.0°C, and spawning activity continued until the end of August. During September and December, most clams were sexually in the resting stage. H. irus is considered to be a summer spawner, and annual gametogenesis was closely associated with the seasonal variation of the surface water temperature. Despite warm water temperatures in Jeju, the resting phase of female and male clams at the study site was prolonged from October to March, suggesting that food availability at this site could be poor and may act as a limiting factor in gametogenesis.

Isolation of oogonia from ovaries of the sea urchin Strongylocentrotus nudus

The presence of oogonia in the ovaries of adult females is typical in species with a broadcast spawning reproductive strategy, including invertebrates and lower vertebrates. In sea urchins, difficulties in the study of oogonia arise from the small number of these cells and the lack of specific markers for their identification. Therefore, more reliable methods are needed for identifying and manipulating oogonial cells in quantities sufficient for experimentation. Homologs of the DEAD-box RNA helicase vasa expressed in germline cells have been proposed for use as markers to detect germline cells in diverse species. We have developed a method for the isolation of sea urchin oogonia by using immunocytochemistry with vasa antibodies, together with reverse transcription and the polymerase chain reaction to detect the expression of Sp-vasa and Sp-nanos2 homologs and a morphological approach to identify germline cells in sea urchin ovaries and cell fractions isolated from the ovarian germinal epithelium. This method has allowed us to obtain 15%-18% of small oogonia with 70%-75% purity from the total amount of isolated germ cells. Our findings represent the first methodological basis for obtaining cell populations containing sea urchin oogonia; this method might be useful as a tool for further investigations of the early stages of sea urchin oogenesis.

First Report of Phytophthora Rot on Alders Caused by Phytophthora alni subsp. alni in Spain.

Phytophthora alni, a soil- and waterborne pathogen, causes aggressive root and collar rot on riparian alder populations (1,2,4). The disease has been described from several European countries with a destructive impact in Great Britain (1,2). All European alder species and the red alder (Alnus rubra) are highly susceptible. P. alni has multiple variants that have been placed in three subspecies: P. alni subsp. alni, P. alni subsp. uniformis, and P. alni subsp. multiformis (1). In July 2009, a survey of symptoms of Phytophthora rot from A. glutinosa at 20 riparian stands along the Avia River in Galicia (northwest Spain) was conducted. Affected trees showed symptoms of Phytophthora rot including abnormally small, sparse, and yellowish foliage, dieback in the canopy, necroses of the inner bark and cambium, and bleeding cankers on the trunks (2,4). Phytophthora spp. were baited from saturated rhizosphere soil and watercourses using oak leaflets (4). Roots and tissue from fresh active inner bark lesions were transferred to selective medium V8-PARPH agar (4) and incubated for 7 days at 22°C in the dark. A Phytophthora sp. was isolated, transferred to carrot agar (CA), and incubated in the dark. Colonies were appressed, often irregular in outline, and with limited aerial mycelium (1). Growth on CA occurred from 4 to 31°C with optimum growth at 23 to 25°C. Chlamydospores were not observed. Ellipsoid, nonpapillate, noncaducous sporangia had a length/breadth average ratio of 1.4. Nesting and extended internal proliferation occurred. Oogonia, antheridia, and oospores were abundantly produced in a single culture. Oogonia with tapered stalks were spherical (mature oogonia 38 to 50 μm in diameter) and some had ornamented walls or bullate protuberances (1,2). Antheridia were large, amphigynous, and predominantly two-celled (23 to 37 × 16 to 23 μm). Oospores were plerotic. Distorted comma-shaped or smaller oogonia and aborted oospores were observed (1). Amplification of DNA was accomplished by using sequence-characterized amplification region-PCR primers (3). The amplicon sizes obtained were identical to P. alni subsp. alni (3). Internal transcribed spacer (ITS)-DNA and nadh1 mitochondrial gene were also amplified. DNA sequences of ITS and mt-DNA regions were deposited in GenBank (Nos. GU108602 and GU108603). Comparison of the sequences showed 100% homology with P. alni subsp. alni (GenBank Nos. FJ746679 and DQ202490). P. alni subsp. alni was recovered from trees at 3 of 20 riparian alder stands with symptoms. Pathogenicity of one representative isolate was confirmed by inoculating 10 3-year-old A. glutinosa seedlings grown in pots. One shallow cut was made into the bark at the collar level. A colonized agar plug, from the margin of an actively growing colony of P. alni subsp. alni, was inserted beneath the flap that was sealed with Parafilm. Five controls seedlings received only sterile CA agar plugs. Plants were incubated at 24°C and 95% humidity for 30 days. On inoculated plants, necroses progressed bidirectionally from the wound, and dead leaves and wilting of shoots were observed. P. alni subsp. alni was recovered from inoculated seedlings, but not from controls. To our knowledge, this is the first report of Phytophthora rot on alder caused by P. alni subsp. alni in Spain.

The avocado subgroup of Phytophthora citricola constitutes a distinct species, Phytophthora mengei sp. nov.

Isolates from avocado tree cankers have been recognized as a distinct subgroup within the P. citricola complex since 1974, both morphologically and molecularly (isozyme and amplified fragment length polymorphism [AFLP] analyses). This subgroup is formally separated from P. citricola after comparative DNA fingerprinting and sequence analyses of the ITS region, as well as by morphological examinations. This new taxon is homothallic, produces plerotic oospores with paragynous antheridia and noncaducous semipapillate sporangia. Morphologically it differs from other species of Waterhouse group III by producing many large bizarre-shaped sporangia and smaller oogonia with asymmetric capitate antheridia. It belongs to clade 2 and is phylogenetically closer to P. siskiyouensis, P. capsici and P. tropicalis than to P. citricola. P. mengei can be easily differentiated from its relatives in the same clade and other species of this morpho-group by DNA fingerprints and sequence analysis. This new taxon is named Phytophthora mengei sp. nov.

Pythium sukuiense, a new species from undisturbed natural forest in Taiwan

A new species, Pythium sukuiense, was isolated from an undisturbed natural forest in northern Taiwan. The fungus produces sporangia indistinguishable from hyphae and very small oogonia and oospores. Oogonia were smooth and terminal or intercalary and attached with a single antheridium. Oospores were aplerotic, with an average size of only 11 μm.

Intraspecific DNA polymorphisms of Pythium irregulare

Forty-seven isolates of Pythium Irregulare from different hosts and geographic origins were compared from molecular, morphological and physiological viewpoints. They were divided into four groups (I-IV) based on ITS-RFLP analysis and RAPD analysis. Groups I and II included 32 and eight isolates, respectively, collected from diverse hosts and geographic origins, and groups III and IV comprised seven isolates derived from sugar beet and sugar beet field soil. Group I had smaller oogonia and oospores than did the other three groups. In groups I and II, a significantly higher percentage of the oogonia produced multiple projections compared to groups III and IV which occasionally produced one projection. The growth rate of the four groups was similar at 5-30°C. At 33°, many isolates of group I grew rapidly but most of the isolates of other groups grew slowly, and at 35°, the former grew but the latter did not. In phylogenetic analysis based on sequences of the ITS region, four groups of P. irregulare were included in one cluster with P. sylvaticum. Groups I-II and III-IV clustered more tightly in the same branch, respectively. The genetic divergence between I-II and III-IV was higher than between each group (I-II and III-IV) and P. sylvaticum, indicating that groups I-II and III-IV may represent two different species.

Phytophthora meadii may be a species hybrid

In a cytological study, some isolates of Phytophthora meadii were found to be predominantly polyploid and to show normal oospore development, while others were predominantly diploid. The diploids produced mainly small oogonia, which degenerated during meiosis with the production of many empty oogonia; but also some larger oogonia with polyploid nuclei and normal oospore development. It is therefore suggested that P. meadii is derived from a species hybrid in which meiosis is upset by the genetical imbalance; and that chromosomal doubling during vegetative growth, followed by selection, leads to the production of tetraploid forms which can undergo normal meiosis. No differences were found in the soluble protein patterns of diploid and polyploid isolates. While both A1 and A2 compatibility types were found in each ploidy group, the polyploid A2's (presumed compatibility type heterozygotes) tended to be weakly self-fertile, and one of them gave rise to a self-sterile polyploid A1 culture. This demonstrates the difficulty of maintaining the heterozygous condition during vegetative growth in a polyploid. The possible role of polyploidy in the apparent decline of the A2 type in Sri Lankan P. meadii populations is discussed.

The systematics of Phytophthora sojae and P. megasperma

Isolates of Phytophthora from alfalfa and soybean and the type isolate of Phytophthora megasperma Drechsler from hollyhock were compared morphologically, for temperature response, and pathogenically. Highly pathogenic, host-specific isolates from soybean and alfalfa, with relatively small oogonia, were identified as P. sojae Kaufmann and Gerdemann and divided into two formae speciales: f.sp. glycines and f.sp. medicaginis, respectively. Less pathogenic isolates from alfalfa with large oogonia were identified as P. megasperma and were compared favorably with Drechsler's original isolate and description. Isolates of P. sojae differed in their cardinal temperatures from those of P. megasperma. We consider P. sojae to be distinct from P. megasperma and propose that this taxon be reintroduced to the literature.

An Ultrastructural and Histochemical Study of Oogenesis in the Trichostrongylid Nematode Heligmosomoides polygyrus

Oogenesis in trichostrongylids has been examined for the first time in a light and electron microscopic investigation of Heligmosomoides polygyrus. The female reproductive tract is a single straight tube containing small oogonia (6 micron in diameter), which are arranged in a rosette pattern around a central rachis at the anterior end of the tract. Developing oocytes separate from the rachis and pass posteriorly in single file down the growth zone. Oocytes increase rapidly in volume due to the accumulation of cytoplasmic inclusion granules. These granules are of 3 types. Type 1 granules are amorphous and probably consist primarily of lipoprotein. Type 2 granules are large lipid inclusions and type 3 granules are electron-dense lipoprotein yolk bodies, which are probably used for energy reserves in the developing embryo. Histochemical studies show a more intense reaction for DNA in the nuclei of oogonia than in the nuclei of oocytes. There is a strong reaction for RNA in the nucleoli and in the cytoplasm of oogonia and oocytes. Ultrastructural studies indicate that this RNA is probably in the form of rRNA in the abundant ribosomes. Mature oocytes are cylindrical (60 X 70 micron), have a distinct nucleus with nuclear pores, and the cytoplasm is filled with inclusion granules and ribosomes but contains only small amounts of glycogen. Prior to fertilization the plasma membrane of oocytes acquires a flocculent coat. These oocytes contain 6 distinct bivalent chromosomes in diakinesis. Thus the major changes that occur in developing germ cells are 2-fold: nuclear changes that prepare the chromosomes for fertilization by initiating reduction division, and cytoplasmic changes that involve the synthesis and storage of inclusion granules.

Intraspecifïc variation in gel electrophoresis patterns of soluble mycelial proteins of Phytophthora megasperma isolated from alfalfa

The electrophoretic patterns of the buffer-soluble proteins of 26 isolates of Phytophthora megasperma f. sp. medicaginis from diseased alfalfa plants and soil were studied. On the basis of the protein patterns, the isolates were divided into two groups which correlated with cardinal temperatures for growth, pathogenicity, and oogonia size. Group 1 (seven isolates) has minimum growth at 5 °C, optimum at 25–30 °C, and maximum at 35 °C, and is a highly pathogenic pathotype with small oogonia. Group 2 (19 isolates) has minimum growth at less than 5 °C, optimum at 20 °C, and maximum at 30 °C, and is a less pathogenic pathotype with large oogonia. The electrophoresis technique was useful in identifying differences among the Phytophthora isolates.

Mating Behaviour of Australian Isolates of Phytophthora Species. II. Phytophthora cinnamomi

Determinations were made of the patterns of oogonial occurrence in the culture medium, the numbers of oogonia produced and of aborted oogonia formed in matings between 18 A1 and 116 A2 compatability type isolates of Phytophthora cinnamomi from south-eastern and Western Australia. The majority of the A2 isolates mated freely with their A1 counterparts, but three A2 isolates mated with only some of the A1 testers and five others were sterile. Patterns of occurrence of oogonia, their quantitative production and the percentage of aborted oogonia produced were affected by the particular isolates used. Statistical analysis of the data showed that the nine populations of A2 isolates were significantly different from each other in all three parameters studied. Populations from Eden and Bemm River were markedly less fertile than those from other regions. These findings are consistent with the hypothesis that P. cinnamomi is an allochthonous organism in Australia. Oogonial size was found to vary according to position within the mating plate and according to the particular isolates used, although different geographical groups of isolates could not be dis- tinguished. Abnormally small oogonia were produced in matings involving three of the A2 isolates. The taxonomic significance of these findings is discussed.

Saprolegnia shikotsuensis sp. nov. Isolated from Kokanee Salmon Associated with Fish Saprolegniasis

An epizootic of fungus disease occurred among kokanee salmon, Oncorhynchus nerka var. adonis in Lake Shikotsu of Hokkaido Island in 1974 to 1975. Fungi infected mainly on the peduncle and caudal fin of fish. One kind of fungus was isolated from diseased fish by inoculating materials from the caudal fin onto Sabouraud dextrose agar and incubating at 15°C. Inoculation experiments using moon fish, Xyphorus maculatus showed that all the isolates were virulent (Table 2).The fungus belonged to the genus Saprolegnia according to DICK (1973) from its characteristics of the asexual reproductive stages. Characteristics of the sexual reproductive structures of diclinous antheridia and subcentric oospores placed the fungus close to S. parasitica and S. australis according to SEYMOUR (1970). Comparison in biological characteristics between the present fungus, and S. parasitica and S. australis described by SEYMOUR (1970) are summarized as follows.The zoospores of the fungus were larger in size than those of S. parasitica and S. australis. The oogonia were sparsely formed only after prolonged period of time, and in this point it resembles to S. parasitica rather than S. australis. The fungus can be distinguished from S. parasitica and S. australis by the characteristic of catenulate oogonia. The fungus has smaller oogonia than those of S. parasitica and S. australis. As regards characteristics of pits of the oogonial wall the fungus lies between S. parasitica and S. australis. Oospores per oogonium of the fungus were less in number than those of S. parasitica and S. australis. Antheridial barnches are usually present in S. parasitica and S. australis, but were often lacking in the fungus.From the above mentioned the fungus was regarded as a new species of the Saprolegnia.

A new species of Pythium from Wisconsin and Florida isolated from carrots

Pythium sulcatum n. sp. was isolated from carrots with browned and forked roots grown in muck soils from Wisconsin and Florida. It is primarily distinguished by small oogonia and oospores and by antheridia which are variable in shape and frequently adorned with single or multiple transverse furrows and folds, for which the species is named. Zoospores are rarely produced; vesicles always arise from filamentous sporangia indistinguishable from vegetative hyphae. Vegetative hyphal bodies vary in shape and are outnumbered by sexual bodies on most media. Radial growth rates of P. sulcatum on potato dextrose agar are slower than those of P. irregulare, P. paroecandrum, and P. sylvaticum over a wide temperature range, and P. sulcatum may easily be distinguished from these species on that basis.

SEX DIFFERENTIATION IN THE PACIFIC SALMON ONCORHYNCHUS KETA (WALBAUM)

The differentiation of the gonad is described in chum salmon embryos and alevins. Contrary to classical findings in teleosts, sex differentiation in the chum salmon proceeds in the male or female direction without an intermediate female phase. From an initially indifferent gonad there is a progressive development of one sex or the other. The organ forms as a fold from the splanchnic mesoderm and, at the time of first appearance, contains primordial germ cells. These enlarge to form the definitive germ cells which, after a series of divisions, form smaller oogonia or spermatogonia. Oogonia are followed by primary and secondary (growing) oocytes, the appearance of which is the criterion of sex distinction. Spermatogonia continue to multiply but do not undergo growth in the alevin. The ovary develops an open endovarial canal and is supported by a prominent mesovarium. The testis remains small and, in the alevin, develops no ducts. It is suspended by a mesorchium.