Species Of Labyrinthula Research Articles

Standard ecological and molecular research methods and techniques for Labyrinthula spp.

Labyrinthula are unicellular protists occupying diverse spatial and functional niches, including various roles in host and ecological function, fatty acid production, pandemic marine disease and saprobic decomposition. Labyrinthula species span tropical and temperate climates and have been isolated from each marine coastal ecosystem tested. Our understanding of primary cellular and molecular functions of Labyrinthula has substantially progressed through a combination of increased global investments, research interest and technological advances. Recent advances in molecular techniques provide a toolkit for advancing ecological questions in marine infectious disease in seagrass meadows around the world. Here we provide a comprehensive review of relevant ecological and molecular techniques used in long-term research and the progression of Labyrinthula scholarship. Our aims in preparing this review are to: 1) share, compare and advance global Labyrinthula protocols, 2) increase accessibility to robust methodology to encourage the uptake of Labyrinthula-based questions into marine studies of molecular and ecological qualities of Labyrinthula and 3) encourage uptake of robust Labyrinthula-based questions into coastal marine studies, while also encouraging international collaborative networks across multiple fields. Lastly, we discuss gaps in the over 100 years of Labyrinthula research and opportunities for expanding research on this model marine organism.

First report of Labyrinthula zosterae (Labyrinthulomycetes) as the causal pathogen of wasting disease in the seagrass Zostera marina in Korea.

Zostera marina L. plants have been seriously impacted by wasting disease along the Atlantic coasts of North America and Europe since the 1930s (Muehlstein 1989). Sudden declines in the population sizes of Zostera marina affect primary and secondary producers of different trophic levels in blue carbon ecosystems (Gleason et al. 2013). Muehlstein et al. (1991) first identified Labyrinthula zosterae (Labyrinthulomycetes) as the pathogen causing wasting disease in Zostera marina. However, there have been no reports of wasting disease pathogens affecting seagrass in Korea. In this study, we collected leaves of Z. marina showing symptoms of wasting disease in the southern region of South Korea (Dongdaeman, Namhae, Gyeongnam Province) during field monitoring (from April to September 2013). The pathogens of wasting disease, Labyrinthula zosterae has been isolated from the infected leaves of Z. marina and established as a culture strain (Supplementary Figure 1). Samples of Z. marina and L. zosterae were deposited at the Fisheries Seed and Breeding Research Institute (previous Seaweed Research Center, National Institute of Fisheries Science, South Korea). Microscopic examination of the infected leaf tissues revealed fusiform or spindle-shaped vegetative Labyrinthula cells (4-5 × 15-20 μm). These were similar in size and shape to those previously described for Labyrinthula species. The fusiform cells were cultured in 1% serum seawater agar medium, and they formed colonies and showed gliding motility along a network of hyaline slime filaments. To validate the pathogenicity, re-inoculation tests by L. zosterae were performed with the isolated strains in accordance with Koch's postulates. Healthy leaves of Z. marina collected from the field were used in the re-inoculation tests and were cultured at 15°C under white fluorescent irradiation of approximately 20 μmol·photons·m-2·s-1 and a 12:12-h light:dark cycle (Supplementary Figure 1). Labyrinthula zosterae re-isolated from artificially infected leaves of Z. marina was confirmed by DNA sequence similarity analysis. Total genomic DNA from the infected leaf cells and the culture strains was extracted using the QIAamp DNA Stool Mini Kit (Qiagen, Germany). Internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA were determined to identify Labyrinthula species. L. zosterae-specific primers (Lz2forward (5'- CTAAGACTAAACGAGGCGAAAGCCTAC-3') and Lz2reverse (5'-AGGTTTACAAAACACACTCGTCCACA-3') in Bergmann et al. (2011)) were used to confirm the infection of L. zosterae in the leaves from the field samples and the re-inoculation test samples. Next, PCR products were cloned using a pLUG-Prime® TA-cloning Vector (iNtRON Biotechnology, Korea) and commercially sequenced (SolGent, Korea). The ITS sequence of Korean L. zosterae (accession number MW357748) showed high sequence similarity (99.3-100%) with that of L. zosterae deposited in GenBank (National Center for Biotechnology Information) from BLAST searches. These findings confirm that this is the first report of L. zosterae as the causal pathogen of wasting disease in Z. marina in Korea.

Optimization of cultural conditions for Labyrinthula species isolated from mangrove leaves

Labyrinthulids which are marine osmoheterotrophic, straminipilan protists under Labyrinthulomycetes, thrive in brackish waters and mangrove-rich areas. Despite their long history and widespread occurrence in marine and terrestrial environments, this group of organisms is less studied compared to their closely-related organisms such as thraustochytrids, and so far less is known about their optimum growth conditions in laboratory setups. Although the labyrinthulid group is important ecologically, our understanding of their true culturable diversity, isolation, and long-term viability remains poorly documented. Thus, three labyrinthulid strains (AK1, DV1, and DV2) were studied here for culture optimization, mode of growth and prolonged cell viability (at least 14 d) by using customized agar media. Optimal temperature (35°C) and pH conditions (pH 6.0-7.0) were best for their growth specifically on media with 6% glucose concentration and 60% salinity (parts per thousand). Broth medium with carabao grass extract was suitable in keeping the isolates viable for more than 14 d but their growth in extract-based media was not significantly different from those grown on base medium, Glucose-Yeast Extract-Peptone agar (GPYA). Although it is not well understood what defense mechanism this organism needs to provide them longer viability in the environment, this study gives insights on their preferred culturable conditions.

Functional, Phylogenetic and Host-Geographic Signatures of Labyrinthula spp. Provide for Putative Species Delimitation and a Global-Scale View of Seagrass Wasting Disease

Seagrass meadows form ecologically and economically valuable coastal habitat on every continental margin except the Antarctic, but their areal extent is declining by approximately 2–5 % per year. Seagrass wasting disease is a contributing factor in these declines, with the protist Labyrinthula identified as the etiologic agent. To help elucidate the role of Labyrinthula spp. in global seagrass declines, we surveyed roughly one fourth of all seagrass species to identify Labyrinthula diversity at the strain and/or species level, combining results from culturing methods and two common nuclear DNA markers: the ITS and 18S regions of the ribosomal RNA gene complex. After assaying a subset of the resulting isolates (of which 170 were newly sequenced), we produced a cladogenic context for putative seagrass-pathogenic versus non-pathogenic Labyrinthula while also defining host and geographic ranges. Assays also suggest that pathogenicity is consistently high (when present; and, even when comparing susceptibility of US East- versus West Coast Zostera marina hosts) while virulence is variable, that some isolate-host combinations have the potential for host cross-infection, and that several modes of transmission can be effective. Taken together, these data provide additional means for delimiting putative species of Labyrinthula, suggesting at least five seagrass-pathogenic and perhaps ten or more non-pathogenic marine “species”, yielding a working definition for ecologists and epidemiologists attempting to reconcile the sundry data related to seagrass wasting disease.

Labyrinthula species associated with turfgrasses in Arizona and New Mexico

Labyrinthula terrestris, the causal agent of rapid blight disease in cool-season turfgrasses, is the first Labyrinthula species found to be associated with higher plants in terrestrial ecosystems. An initial study suggested a single common genotype associated with turfgrass in the US, but a later study revealed genetic diversity in this pathogen. In the present study, genetic diversity as well as differences in pathogenicity and morphology was determined in Labyrinthula spp. from turfgrasses in New Mexico and Arizona where rapid blight had not previously been identified. In total, 96 Labyrinthula isolates from New Mexico and four from Arizona were sequenced in 2011 and 2012. A phylogenetic approach based on 18S and ITS-LSU of ribosomal DNA was used for genotypic diversity, and pathogenicity and microscopy were used for phenotypic diversity. A maximum parsimony analysis revealed four genetic groups including two previously described Labyrinthula groups, Laby10 (L. terrestris) and Laby31. Laby10 was the most predominant with 70 % of isolates followed by Laby31 with 24 %. Two new groups, Laby879 and Laby2020, contained only five and one isolate, respectively. An association between genotypes and phenotypes was observed. Laby10 and Laby31 were highly virulent as well as larger in cell size (14.9 and 18.1 μm, respectively), while Laby879 was less virulent with an average cell size of 8.5 μm. Laby2020 was the smallest (0.8 μm) and non-pathogenic. Results showed a prevalence of pathogenic and non-pathogenic Labyrinthula spp. in turfgrasses in New Mexico and the presence of the Laby31 group in the US for the first time.

Potential roles of Labyrinthula spp. in global seagrass population declines

Abstract Overwhelming evidence suggests that seagrass ecosystems are declining around the world. Pathogens from the genus Labyrinthula have repeatedly been found to cause disease in a variety of seagrass species. For example, the ‘wasting disease’ of Zostera marina has been attributed to Labyrinthula infection. Although poorly characterized taxonomically, species of Labyrinthula are very common in marine ecosystems, virulence of genotypes/phylotypes is known to be variable, and highly virulent species are able to cause ecologically significant diseases of protists, plants and animals. Here, the pathosystem model is applied to host–parasite relationships in seagrass ecosystems. Known physical and biological stressors of seagrass are reviewed. Finally, we make the case that it is time to expand research on this poorly studied microorganism in order to quantify the role of disease in seagrass populations world-wide.

Improved methods for axenic culture of Labyrinthula terrestris, causal agent of rapid blight of turfgrasses

The genus Labyrinthula is a group of unicellular microorganisms with spindle-shaped cells that move in an ectoplasmic network. Most Labyrinthula species are saprotrophic and found in coastal marine or estuarine habitats; however, exceptions exist, such as Labyrinthula terrestris , a terrestrial plant pathogen that causes rapid blight on cool-season turfgrasses. Labyrinthula spp. can be grown in culture, which facilitates studies on their biology and pathology. However, axenic culture of L.terrestris has always been challenging. We modified the most commonly used Labyrinthula growth medium, serum seawater agar (SSA), and designed 2 media for improved pure culture, modified SSA (MSSA) and grass extract SSA (GESSA). A comparative assessment of these 2 media and basic SSA was made to measure the growth responses of 18 L.terrestris isolates. Results indicate that the average colony area was greatest on GESSA followed by MSSA, while cultures lived longest on MSSA followed by GESSA. We also suggest an improved long-term culture technique to maintain viable L.terrestris isolates for at least 2 years.

Widespread occurrence of endophytic Labyrinthula spp. in northern European eelgrass Zostera marina beds

Seagrasses worldwide are commonly infected by endophytic protists of the genus Labyrinthula. To date, the nature of interaction of endophyte and host is not well understood. In eelgrass (Zostera marina) Labyrinthula zosterae may become virulent (pathogenic) and lead to the loss of entire sea grass beds. One of the best known examples of any marine epidemic were outbreaks of the ´wasting disease´ on both sides of the Atlantic in the 1930s, but smaller infestations have been reported until recently. Up to now, detection of infection by Labyrinthula was based on the wasting index, i.e. the relative area of leaf lesions or microscopy, while genetic data are virtually absent. We characterized a ~1400 base pair portion of the 18S small subunit rDNA in L. zosterae isolates (N=41) from six northern European sites and one southern location (Adriatic Sea) in order to assess identity and potential diversity of endophytic protists. Because there are indications that low salinity impedes Labyrinthula growth, sampling sites included a wide range of salinities from 5-34 psu. A search against the non-redundant GENBANK data base revealed that most isolates are 99% similar to the only L. zosterae 18S sequence available from the data base at all but the Finish site (salinity values 5-7 psu). At the latter site, a different Labyrinthula species occurred, which was also found in fully marine Wadden Sea cultures. A third species was detected in Skagerrak, south-western Baltic and North Sea samples (20-25 psu). We conclude that L. zosterae is widespread among northern European eelgrass sites across wide ranges of salinity.

Genetic diversity of Labyrinthula terrestris, a newly emergent plant pathogen, and the discovery of new Labyrinthulid organisms

Labyrinthuloid organisms are thought almost exclusively to be only associated with marine environments. However in 1995, a disease of turfgrass suddenly appeared that was eventually determined to be caused by a new Labyrinthula species ( Labyrinthula terrestris). The disease is primarily thought to be caused by the use of elevated salinity irrigation water, making it a unique example of an emergent plant disease potentially induced by human activity. Our objective was to examine diversity of L. terrestris from broadly distributed isolates using AFLP, sequence analysis of two rDNA loci (SSU & LSU-ITS), and pathogenicity tests since previous research on a limited number of isolates found no variability based in ITS and SSU. In contrast to previous work, 18 unique genotypes were found out of a total of 29 analyzed based on AFLP. Sequence variability was only found in a single pathogenic isolate (Laby 31) that was isolated from the United Kingdom. The divergence based on AFLP and sequence analysis suggests that this isolate is a distinct species but closely related to the other L. terrestris isolates examined. Two putatively new nonpathogenic Labyrinthulid species were also found (Laby 13 & 32). Our results suggest that these organisms may be widely distributed in terrestrial environments based on the diversity found in this study and may have long been associated with terrestrial plants. Our results also suggest that more Labyrinthulid organisms may potentially emerge as new plant pathogens in the future if salinification of agricultural systems continues to increases worldwide.

Labyrinthula terrestris sp. nov., a new pathogen of turf grass

A species of Labyrinthula that causes ‘rapid blight’ and death of turfgrass has been isolated and studied. We name this new species Labyrinthula terrestris and briefly summarize morphological characteristics and growth patterns of this pathogen of turfgrass.

Labyrinthula terrestris sp. nov., a new pathogen of turf grass

A species of Labyrinthula that causes 'rapid blight' and death of turfgrass has been isolated and studied. We name this new species Labyrinthula terrestris and briefly summarize morphological characteristics and growth patterns of this pathogen of turfgrass.

Decomposers of the Diatom Lawn and Reticulosphaera

Summary: “Lawns” of the pennate diatom Amphiprora spec. can be decomposed by three osmotrophs: two filamentous bacteria and a species of Labyrinthula . The appearance of the diatoms after extracellular digestion is different in all cases. Osmotrophs may also have a deleterious effect on the cell bodies of the plasmodial protist Reticulosphaera spec., at least when they phagocytose the diatoms (heterotrophic phase). When food becomes exhausted and the reticulopodia are withdrawn, they are protected (autotrophic phase). It is assumed that the degenerative appearance of many plastids in Reticulosphaera socialis is caused by the bacteria found in the cytoplasm.

The host – pathogen interaction in the wasting disease of eelgrass, Zostera marina

A marine epidemic of wasting disease decimated populations of eelgrass, Zostera marina L., in the early 1930s. Labyrinthula, a marine slime mold was the suspected pathogen, although the cause was never clearly determined. Presently, a recurrence of wasting disease of Z. marina was documented in populations along the coasts of North America and Europe. A pathogenic species of Labyrinthula, described as Labyrinthula zosterae Porter et Muehlstein, was identified as the primary microorganism causing the present wasting disease. Of all the microorganisms tested in laboratory disease tests, only L. zosterae caused disease symptoms. Direct microscopic observations revealed that Labyrinthula cells were found most frequently associated with marginal areas of disease symptoms and appeared to move rapidly through the tissue, directly penetrating cell walls. The ectoplasmic network that surrounds Labyrinthula cells appeared to have an important role in the enzymatic degradation of plant cell walls and presumably a role in the destruction of cytoplasmic contents of the plant cells. Direct contact of diseased leaves with healthy leaves was the mechanism of disease spread from plant to plant. Key words: Labyrinthula, Zostera marina, eelgrass wasting disease.

Ribosomal RNA molecular weights and the phylogeny of Labyrinthula

Ribosomal RNA molecular weights were determined for several species of Labyrinthula, members of the Thraustochytriaceae, and other lower fungi in an attempt to clarify phylogenetic relations among these groups of organisms. The molecular weights of both 18 S and 25 S ribosomal RNA components were calculated from their relative electrophoretic mobility in polyacrylamide gels. The ribosomal RNA molecular weights of the Labyrinthula spp. were lighter than any other fungi thus far examined, averaging 1.22 × 106 and 0.69 × 106 for the heavy and light components, respectively. The ribosomal RNA molecular weights of the thraustochytrids, including Labyrinthuloides spp., are distinctly heavier than those of Labyrinthula spp., averaging 1.32 × 106 and 0.71 × 106. Those of Lagenidium sp. and Didymium iridis are heavier still. The results suggest that Labyrinthula is not closely related to the thraustochytrids, the Oomycetes, or the Myxomycetes, and that the thraustochytrids have appropriately been removed from the Oomycetes.

Structure of Labyrinthula sp. Zoospores*

SYNOPSIS. A species of Labyrinthula closely resembling L. algeriensis was isolated from marsh grass, Spartina alterniflora. Zoosporulation was obtained in ∼50% of the cultures, which were grown on a modified Vishniac medium, when yeasts were used as food organisms; when the temperature was ∼22 C; when thiamine (0.2 mg/l, biotin (1 μg/l, Ba (1 μg/l, and cholesterol (5 mg/l) were present; and when the cells were grown on a semisolid medium (0.6% agar) rather than higher concentrations of agar. The motile cells were similar to biflagellated fungal zoospores in that they were pyriform and laterally biflagellated. The anterior flagellum (13‐15 μ long) possessed what appeared to be 2 rows of mastigonemes and was uniform in diameter while the posterior flagellum (6‐10 μ) lacked mastigonemes and was tapered distally into a whiplash. The planonts are apparently not isogametes since planogametic copulation could not be induced and since synaptinemal complexes were not observed in sectioned presporangia.

Labyrinthula minuta sp. nov.

SUMMARY: A new marine species of Labyrinthula is described for which the name L. minuta is proposed because of its diminutive proportions. This species has been isolated repeatedly from Ulva lactuca and other algae collected from the Atlantic Ocean in the region of Woods Hole, Massachusetts. The vegetative stage, as in other species of Labyrinthula, consists of independent cells interconnected by threads of slime which form a net-like structure called the net-plasmodium. L. minuta differs from all previously described species of this genus in the size, shape, and movement of its cells, in its method of cell division, its colonial morphology and by the formation in aged cultures of multinucleate bodies which resemble miniature plasmodia. No fructification has yet been observed. This species has been grown in pure reproducible laboratory culture on a serum + sea water medium for a period of five years.