Marine Oomycetes Research Articles

Antifungal Potential of Yellow Bur Head Limnocharis flava (Buchenau, 1868) Against Pathogenic Oomycete, Lagenidium thermophilum

Fungal infection by marine oomycetes is the main problem that hinders crustaceanproduction. Therefore, a study to find an alternative fungal treatment that is safer thanchemical treatment is currently needed. One of the potential sources of antifungalproperties is macrophytes. Limnocharis flava, known as yellow bur head, was foundto have antimicrobial properties. Thus, this research was conducted to determinethe potential of L. flava extract as an antifungal agent against the marine oomycetes Lagenidium thermophilum IPMB 1801. In this study, ethanol and methanol solvents were used to extract L. flava. The results showed that the methanol extraction yield of L. flavais higher (7.03 g, 35.16%) compared to ethanol extract (3.26 g, 16.26%). The antifungal screening test was conducted using the disc diffusion method. Ethanol and methanol extract of L. flava had antifungal activities against the hyphal growth of L. thermophilum.Continuation from the screening test, the minimum inhibitory concentration for both ethanol and methanol extracts was determined to be at 100 mg/ml respectively. These findings suggest that L. flava has the potential to become an antifungal treatment forthe control of fungal infections in the crustacean industry.

Multiple Viral Infections Detected in Phytophthora condilina by Total and Small RNA Sequencing.

Marine oomycetes have recently been shown to be concurrently infected by (−)ssRNA viruses of the order Bunyavirales. In this work, even higher virus variability was found in a single isolate of Phytophthora condilina, a recently described member of Phytophthora phylogenetic Clade 6a, which was isolated from brackish estuarine waters in southern Portugal. Using total and small RNA-seq the full RdRp of 13 different potential novel bunya-like viruses and two complete toti-like viruses were detected. All these viruses were successfully confirmed by reverse transcription polymerase chain reaction (RT-PCR) using total RNA as template, but complementarily one of the toti-like and five of the bunya-like viruses were confirmed when dsRNA was purified for RT-PCR. In our study, total RNA-seq was by far more efficient for de novo assembling of the virus sequencing but small RNA-seq showed higher read numbers for most viruses. Two main populations of small RNAs (21 nts and 25 nts-long) were identified, which were in accordance with other Phytophthora species. To the best of our knowledge, this is the first study using small RNA sequencing to identify viruses in Phytophthora spp.

Cox2 community barcoding at Prince Edward Island reveals long-distance dispersal of a downy mildew species and potentially marine members of the Saprolegniaceae

Marine oomycetes are highly diverse, globally distributed, and play key roles in marine food webs as decomposers, food source, and parasites. Despite their potential importance in global ocean ecosystems, marine oomycetes are comparatively little studied. Here, we tested if the primer pair cox2F_Hud and cox2-RC4, which is already well-established for phylogenetic investigations of terrestrial oomycetes, can also be used for high-throughput community barcoding. Community barcoding of a plankton sample from Brudenell River (Prince Edward Island, Canada), revealed six distinct oomycete OTU clusters. Two of these clusters corresponded to members of the Peronosporaceae—one could be assigned to Peronospora verna, an obligate biotrophic pathogen of the terrestrial plant Veronica serpyllifolia and related species, the other was closely related to Globisporangium rostratum. While the detection of the former in the sample is likely due to long-distance dispersal from the island, the latter might be a bona fide marine species, as several cultivable species of the Peronosporaceae are known to withstand high salt concentrations. Two OTU lineages could be assigned to the Saprolegniaceae. While these might represent marine species of the otherwise terrestrial genus, it is also conceivable that they were introduced on detritus from the island. Two additional OTU clusters were grouped with the early-diverging oomycete lineages but could not be assigned to a specific family. This reflects the current underrepresentation of cox2 sequence data which will hopefully improve with the increasing interest in marine oomycetes.

Antifungal Prospect of Bacillus cereus Postbiotic on Crustacean Pathogen, Lagenidium thermophilum.

Pathogenic marine fungi, Lagenidium thermophilum is known causative agent in the crustacean industry. Current disinfection practice in hatchery has risks and negative impacts which prompts suitable substitute to synthetic antifungal agents. Thus, this study was conducted to evaluate the antifungal potential of postbiotic from four potential probiotics towards marine oomycetes, L. thermophilum IPMB 1401. The screening test showed that the Lactobacillus plantarum GS12 and Bacillus cereus GS15 postbiotics were positive for antifungal activity on L. thermophilum IPMB 1401. These two bacterial extracts have minimum inhibitory concentration （MIC） at 50%. The toxicity assay on MIC level of the postbiotic revealed that the cumulative mortality of brine shrimp nauplii exposed to B. cereus postbiotic was significantly lower compared to L. plantarum GS12 postbiotic and formalin. This indicates a high potential of B. cereus GS15 as a prospect for alternative control method for fungal infections in the crustacean culture industry.

Marine Oomycetes of the Genus Halophytophthora Harbor Viruses Related to Bunyaviruses.

We investigated the incidence of RNA viruses in a collection of Halophytophthora spp. from estuarine ecosystems in southern Portugal. The first approach to detect the presence of viruses was based on the occurrence of dsRNA, typically considered as a viral molecule in plants and fungi. Two dsRNA-banding patterns (∼7 and 9 kb) were observed in seven of 73 Halophytophthora isolates tested (9.6%). Consequently, two dsRNA-hosting isolates were chosen to perform stranded RNA sequencing for de novo virus sequence assembly. A total of eight putative novel virus species with genomic affinities to members of the order Bunyavirales were detected and their full-length RdRp gene characterized by RACE. Based on the direct partial amplification of their RdRp gene by RT-PCR multiple viral infections occur in both isolates selected. Likewise, the screening of those viruses in the whole collection of Halophytophthora isolates showed that their occurrence is limited to one single Halophytophthora species. To our knowledge, this is the first report demonstrating the presence of negative (−) ssRNA viruses in marine oomycetes.

The oomycete Lagenisma coscinodisci hijacks host alkaloid synthesis during infection of a marine diatom

Flagellated oomycetes frequently infect unicellular algae, thus limiting their proliferation. Here we show that the marine oomycete Lagenisma coscinodisci rewires the metabolome of the bloom-forming diatom Coscinodiscus granii, thereby promoting infection success. The algal alkaloids β-carboline and 2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylic acid are induced during infection. Single-cell profiling with AP-MALDI-MS and confocal laser scanning microscopy reveals that algal carbolines accumulate in the reproductive form of the parasite. The compounds arrest the algal cell division, increase the infection rate and induce plasmolysis in the host. Our results indicate that the oomycete manipulates the host metabolome to support its own multiplication.

Growth and fatty acid profiles of Halophytophthora vesicula and Salispina spinosa from Philippine mangrove leaves.

Studies on marine-sourced fatty acids have gathered significant interest recently as an important component of aquaculture feeds and of biofuel production. Of the organisms capable of producing fatty acids, marine oomycetes are promising model organisms. One group of marine oomycetes are the Halophytophthora spp. which is known to have an important role in leaf decomposition, thereby changing the plant debris into exudates which are usable to consumers in the mangrove ecosystems. This study reports the three mangrove oomycetes isolated from Philippine mangrove forests, identified herein as Halophytophthora vesicula AK1YB2 (Aklan), H. vesicula PQ1YB3 (Quezon) and Salispina spinosa ST1YB3 (Davao del Norte). These isolates were subjected to growth analyses using varying incubation parameters (salinity level and pH), and for fatty acid production. Results revealed the presence of different fatty acids such as Arachidonic acid, Linoleic acid and Vaccenic acid when grown on V8S and PYGS media. This study is the first observation of fatty acids from S. spinosa and H. vesicula from the Philippines. SIGNIFICANCE AND IMPACT OF THE STUDY: Tropical Philippines straddling west of the Pacific Ocean and East of South China Sea is rich in marine and estuarine oomycetes. These micro-organisms, hitherto poorly known and unstudied in the country, play an important role in the nutritive cycle of the mangrove ecosystem. Due to the increasing demand for an alternative source of fatty acids, species of Oomycetes isolated from select mangrove forests in Luzon, Visayas and Mindanao were analysed for their fatty acid contents. Prospects for industrially-important fatty acids make these Oomycetes all-important to study in applied microbiology in the Philippine setting where these structurally simple micro-organisms abound.

Antimycotic Activity of Seaweed Extracts（ Caulerpa lentillifera and Eucheuma cottonii） against Two Genera of Marine Oomycetes, Lagenidium spp. and Haliphthoros spp.

Fungal infection mostly caused by marine oomycetes had hindered crustacean production thus searching for natural and safe treatment is currently needed. Thus, this study was conducted to investigate the antimycotic effect of different seaweed extract against marine oomycetes (Lagenidium spp. and Haliphthoros spp) . Two seaweeds species (Eucheuma cottonii and Caulerpa lentillifera) were extracted using ethanol, methanol and water. Each extracts was tested on four fungi strains of marine oomycetes species for minimum inhibitory concentration (MIC) and fungicidal activities. C. lentillifera ethanol extract showed the highest antifungal effect where it can inhibit three from four fungal strains. Meanwhile, E. cottonii ethanol extract has lowest MIC (500 ppm) and inhibit L. thermophilum IPMB 1401 and H. sabahensis IPMB 1402 hyphal growths. Antimycotic effect on zoospores production shows reduction in production after 12 h immersion for three marine oomycetes species. Seaweed extracts toxicity on Artemia sp. showed approximately 5% mortality at 12 h immersion. It is suggested that 12 h immersion of seaweed extract is a suitable treatment for marine oomycetes in aquaculture. This study does not only show potential alternative control method for crab larvae health management, it may also contribute to the sustainable development and food security of aquaculture industry.

Marine Oomycetes (Halophytophthora and Salispina): A Potential Source of Fatty Acids with Cytotoxic Activity Against Breast Adenocarcinoma Cells (MCF7).

Marine oomycetes are ubiquitous, fungus-like eukaryotes known to produce fatty acids with potential anticancer activity. The long chain omega-3 and omega-6 fatty acids are currently popular and considered as safe when used as nutraceuticals in cancer treatment. In this study, crude fatty acids from three marine oomycetes, Halophytophthora spp. (T12GP1 and T12YBP2) and Salispina hoi (USTCMS 1611), were explored for their cytotoxic and apoptotic potentials against human breast adenocarcinoma cells (MCF7) and normal human dermal fibroblasts (HDFn). Extracts from mycelia mats consisted of diverse saturated, monounsaturated, and polyunsaturated fatty acids such as linoleic, α-linolenic, γ-linolenic, eicosatrienoic and eicosapentaenoic acids. The crude fatty acids from all three oomycetes in in vitro assays for cytotoxicity showed no toxicity (30% toxicity values) on HDFn cells. On MCF7 cells, however, IC50 values of 23.44, 15.63, and 26.15 µg/mL were obtained with extracts from Halophytophthora T12GP1 and T12YBP2 and S. hoi, respectively. Treated MCF7 cells exhibited deformed cell membrane in MTT assay and also aggregation of DNA and disruption of nuclear membrane aggregation in nuclear staining; further, green signals indicative of apoptosis was recorded in caspase 3/7 assay.

Novel Widespread Marine Oomycetes Parasitising Diatoms, Including the Toxic Genus Pseudo-nitzschia: Genetic, Morphological, and Ecological Characterisation.

Parasites are key drivers of phytoplankton bloom dynamics and related aquatic ecosystem processes. Yet, the dearth of morphological and molecular information hinders the assessment of their diversity and ecological role. Using single-cell techniques, we characterise morphologically and molecularly, intracellular parasitoids infecting four potentially toxin-producing Pseudo-nitzschia and one Melosira species on the North Atlantic coast. These sequences define two, morphologically indistinguishable clades within the phylum Oomycota, related to the genera of algal parasites Anisolpidium and Olpidiopsis and the diatom parasitoid species Miracula helgolandica. Our morphological data are insufficient to attribute either clade to the still unsequenced genus Ectrogella; hence it is proposed to name the clades OOM_1 and OOM_2. A screening of global databases of the barcode regions V4 and V9 of the 18S rDNA demonstrate the presence of these parasitoids beyond the North Atlantic coastal region. During a biweekly metabarcoding survey (Concarneau Bay, France), reads associated with one sequenced parasitoid coincided with the decline of Cerataulina pelagica bloom, whilst the other parasitoids co-occurred at low abundance with Pseudo-nitzschia. Our data highlight a complex and unexplored diversity of the oomycete parasitoids of diatoms and calls for the investigation of their phenology, evolution, and potential contribution in controlling their host spatial-temporal dynamics.

In vitro Inhibitory Effects of Two Bornean Medicinal Wild Gingers against Pathogenic Lagenidium thermophilum Infected Mud Crab Scylla tranquebarica.

The antifungal activity of two Bornean medicinal wild gingers Plagiostachys megacarpa and Zingiber phillippsiae were examined against Lagenidium thermophilum. The most active extract was P. megacarpa at concentration of 320 µg/mL inhibiting both hyphal growth and zoospore production of L. thermophilum in 24 h. Toxicity tests were conducted using mud crab (Scylla tranquebarica) larva. Bath treatment of P. megacarpa at concentrations of 320 and 640 µg/mL for 24 h were highly effective against hyphae and zoospores of the strain and it is non-toxic to mud crab larva. Therefore, crude extracts P. megacarpa may be used as alternative treatment for marine Oomycete infection of mud crab.

Filamentous brown algae infected by the marine, holocarpic oomycete Eurychasma dicksonii

The important role of the cytoskeletal scaffold is increasingly recognized in host-pathogen interactions. The cytoskeleton potentially functions as a weapon for both the plants defending themselves against fungal or oomycete parasites, and for the pathogens trying to overcome the resisting barrier of the plants. This concept, however, had not been investigated in marine algae so far. We are opening this scientific chapter with our study on the functional implications of the cytoskeleton in 3 filamentous brown algal species infected by the marine oomycete Eurychasma dicksonii. Our observations suggest that the cytoskeleton is involved in host defense responses and in fundamental developmental stages of E. dicksonii in its algal host.

Cytoskeleton organisation during the infection of three brown algal species, Ectocarpus siliculosus, Ectocarpus crouaniorum and Pylaiella littoralis, by the intracellular marine oomycete Eurychasma dicksonii.

Oomycete diseases in seaweeds are probably widespread and of significant ecological and economic impact, but overall still poorly understood. This study investigates the organisation of the cytoskeleton during infection of three brown algal species, Pylaiella littoralis, Ectocarpus siliculosus, and Ectocarpus crouaniorum, by the basal marine oomycete Eurychasma dicksonii. Immunofluorescence staining of tubulin revealed how the development of this intracellular biotrophic pathogen impacts on microtubule (MT) organisation of its algal host. The host MT cytoskeleton remains normal and organised by the centrosome until very late stages of the infection. Additionally, the organisation of the parasite's cytoskeleton was examined. During mitosis of the E.dicksonii nucleus the MT focal point (microtubule organisation centre, MTOC, putative centrosome) duplicates and each daughter MTOC migrates to opposite poles of the nucleus. This similarity in MT organisation between the host and pathogen reflects the relatively close phylogenetic relationship between oomycetes and brown algae. Moreover, actin labelling with rhodamine-phalloidin in E.dicksonii revealed typical images of actin dots connected by fine actin filament bundles in the cortical cytoplasm. The functional and phylogenetic implications of our observations are discussed.

Host–parasite interactions and host species susceptibility of the marine oomycete parasite, Olpidiopsis sp., from Korea that infects red algae

Porphyra farms in Korea occasionally suffer from Olpidiopsis infection. As Porphyra farming proceeds from October to March, this obligatory biotrophic parasite may need an alternative host to survive during other months of the year. To find a possible alternative summer host, we collected algae from Wando, Korea, where extensive Porphyra plantations are located, and discovered an oomycete assignable to the genus Olpidiopsis from Heterosiphonia pulchra. Host susceptibility tests showed that this oomycete could also infect Heterosiphonia japonica, Dasya sp., Dasysiphonia chejuensis, and also blades of Porphyra tenera. The minimum incubation time for this Olpidiopsis sp. to infect its hosts was approximately 4 h. Zoosporangia matured in 2 days and biflagellate zoospores were released. Free zoospores remained infective in seawater for up to 7 days. The infection of Olpidiopsis sp. to H. japonica was cell-type specific and extended rhizoid-like apical cells of determinate branches were preferentially infected. FITC-conjugated lectin staining showed specific binding of concanavalin A (ConA) to extended rhizoid-like apical cells. Attachment of Olpidiopsis sp. zoospores to the host cells was inhibited by α-mannosidase. Monosaccharide inhibition experiments showed that d(+)-mannose, complementary to the lectin ConA, could also block the infection, suggesting a lectin–carbohydrate interaction during host–parasite recognition.

Taxonomy, molecular phylogeny, and ultrastructural morphology of Olpidiopsis porphyrae sp. nov. ( Oomycetes, straminipiles), a unicellular obligate endoparasite of Bangia and Porphyra spp. ( Bangiales, Rhodophyta)

Olpidiopsis porphyrae sp. nov., a marine oomycete endoparasite that infects the commercially cultivated red alga Porphyra yezoensis, is described and its phylogenetic position based on molecular data and ultrastructural morphology is discussed. O. porphyrae infects the host Porphyra by means of encysted zoospores. Spherical-shaped holocarpic thalli develop within the cytoplasm of its algal host, which produce monoplanetic, subapically biflagellate zoospores. The characteristic features of this isolate are the ellipsoidal, unicellular thallus and simple holocarpic zoosporangial development, which show morphological similarity with the genus Olpidiopsis. Laboratory infection experiments with a wide range of green, brown, and red algae revealed that O. porphyrae infects several stages of the bangialean red algae (the genera Bangia and Porphyra). Molecular phylogenetic analyses inferred from both SSU rRNA and cox2 genes showed O. porphyrae branched before the main saprolegnian and peronosporalean lineages within the monophyletic oomycete clade, indicating its phylogenetic separation from them. A single or double K-body-like organelle, which contains tubular inclusions, is found located to one side of the zoospore nucleus and shows similarities to homologous organelles previously described in O. saprolegniae. The ultrastructural morphology of O. porphyrae with zoospore initials containing K-bodies and tubular mitochondrial cristae is characteristic of oomycetes. Group I intron-like multiple insertions were found in the SSU rRNA gene of O. porphyrae. This is the first report of SSU group I introns in the class Oomycetes.

Molecular phylogeny of an unidentified Haliphthoros-like marine oomycete and Haliphthoros milfordensis inferred from nuclear-encoded small- and large-subunit rRNA genes and mitochondrial-encoded cox2 gene

The SSU rRNA, LSU rRNA, and cox2 genes of an unidentified Haliphthoros-like marine oomycete (NJM0034) and Haliphthoros milfordensis (NJM0131) were sequenced, and their phylogenetic relationships are analyzed and discussed. All phylogenetic trees showed that NJM0034 and NJM0131 were branched before separation of the two main saprolegnian and peronosporalean clades. These data suggest that the clear phylogenetic separation of those marine oomycete endoparasites from the two main oomycete clades. Excepting the LSU rRNA gene tree, NJM0034 and Haliphthoros spp. did not form a monophyletic group. On the other hand, H. milfordensis NJM0131 clustered with H. philippinensis SANK 15178, not with H. milfordensis NJM9434 in the cox2 amino acid sequence (COII) tree. This result strongly suggests that a taxonomic reinvestigation of the genus Haliphthoros should be considered.

Molecular phylogeny of an unidentified <i>Haliphthoros</i>-like marine oomycete and <i>Haliphthoros milfordensis</i> inferred from nuclear-encoded small- and large-subunit rRNA genes and mitochondrial-encoded cox2 gene

The SSU rRNA, LSU rRNA, and cox2 genes of an unidentified Haliphthoros-like marine oomycete (NJM0034) and Haliphthoros milfordensis (NJM0131) were sequenced, and their phylogenetic relationships are analyzed and discussed. All phylogenetic trees showed that NJM0034 and NJM0131 were branched before separation of the two main saprolegnian and peronosporalean clades. These data suggest that the clear phylogenetic separation of those marine oomycete endoparasites from the two main oomycete clades. Excepting the LSU rRNA gene tree, NJM0034 and Haliphthoros spp. did not form a monophyletic group. On the other hand, H. milfordensis NJM0131 clustered with H. philippinensis SANK 15178, not with H. milfordensis NJM9434 in the cox2 amino acid sequence (COII) tree. This result strongly suggests that a taxonomic reinvestigation of the genus Haliphthoros should be considered.

A marine oomycete Atkinsiella panulirata sp. nov. from philozoma of spiny lobster, Panulirus japonicus

A marine oomycete fungus was isolated from philozoma of the spiny lobster,Panulirus japonicus. The fungus exhibited slow growth, occasionally submerged, with a creamy white, raised moist colony. Hyphae were stout, arranged in radiating pattern, irregularly branched, 10–22 µm diam, occasionally separated by cross walls into subthalli. Thalli occasionally consisted of swollen features. Sporangia formed from the subthalli had 1–3 wavy or partly coiled discharge tubes at the terminal or subterminal area. Zoospores were pyriform or reniform, biflagellate, isokont and diplanetic. Encysted spores germinated as a hairlike filament with a globular enlarged tip in sterilized synthetic seawater, and directly as stout initial hypha in PYGS broth. This fungus was identified as belonging to the genusAtkinsiella and was designatedAtkinsiella panulirata sp. nov. The optimal temperature for the fungus was 25°C. Hyphal growth occurred in both sterilized synthetic seawater and NaCl in the range of 1.5–6.0% and 2.0–4.0%, respectively. The fungus could utilize all carbohydrates and derivatives tested as the carbon sources. From in vitro tests of five fungicides against the fungus malachite green was found to be the most effective.

Distribution and experimental responses to substrate of marine oomycetes ( Halophytophthora spp.) in mangrove ecosystems

Two marine oomycetes, Halophytophthora vesicula and Halophytophthora spinosa , were predominantly frequent (up to 100% and 80% respectively), on newly submerged leaves of red mangrove ( Rhizophora mangle ) near or within stands of mangroves on cays surrounding Exuma Sound, Bahama Islands. Outside of the mangrove tidal channels, H. vesicula (but not H. spinosa ) was most frequent (47–93%) on newly submerged leaves placed near naturally submerged, decaying leaves. Within a tidal channel in an extensive stand of mangroves (15 km 2 ), H. vesicula was more frequent (67–93%) than H. spinosa (7–40%). H. vesicula may compete better than H. spinosa for new substrate when there is a large zoospore source nearby, and vice versa when zoospores are rare and must travel several metres to new substrate. H. vesicula and H. spinosa selectively colonized mangrove leaves (45–100% frequency) as opposed to mangrove twigs (0–45%), other leaves (0–5%), or cellulose pads (0–5%). Protection of leaves from turbulent diffusion, but not from zoospore-swimming contact, reduced frequencies of both H. vesicula and H. spinosa by ca 75–100%, suggesting that chemotaxis toward leaves is, at most, of only very localized significance.

Autumn distribution of marine Pythiaceae across a mangrove–saltmarsh boundary

Zoosporic fungi of the Pythiaceae, especially Halophytophthora vesicula, were present at high frequency (≥ 90%) on autumn-shed leaves both north and south of a mangrove–saltmarsh boundary (Florida–Georgia, U.S.A.). Submerged, decaying leaves of mangroves (three species), other trees (including conifers), shrubs, and vines bore H. vesicula. Halophytophthora bahamensis was also found north of the mangrove belt but at frequencies ≤ 10%. Halophytophthora kandeliae was found only within the mangrove environment. Leaves of intertidal grasses (Spartina and Juncus) yielded low frequencies (≤ 20%) of Halophytophthora spp., but Pythium grandisporangium exhibited frequencies of up to 40–43% on leaves of trees and of Spartina alterniflora. Key words: marine oomycetes, mangrove, saltmarsh, Avicennia germinans, Halophytophthora vesicula, Pythium grandisporangium.