75 The role of feeding in growth and photosynthesis of myrionecta rubra

The diversity of Scenedesmus and Scenedesmus -like taxa from Itasca State Park, Minnesota was assessed using light microscopy and molecular techniques. Thirty isolates from various ponds and lakes in Itasca State Park were examined. Light microscopy showed many similarities in morphology among isolates, but PCR-RFLP analysis of the ribosomal ITS region from these isolates revealed twenty different types. A previous study from Itasca State Park using only light microscopy found only six taxa of Scenedesmus ; however, our results suggest that there is much greater diversity than previously suspected. DNA sequences of the 5.8S ribosomal subunit and the ITS-2 region from our isolates are presently being determined and will be used to assess this diversity in greater detail.     

Observations on the diversity and ecology of freshwater Nannochloropsis (Eustigmatophyceae), with descriptions of new taxa

The genus Nannochloropsis is well known from the marine environment but has only recently been reported from fresh and brackish waters. A single species, N. limnetica, was first documented from shallow lakes in Germany, where it produced spring blooms. A second unnamed isolate from a river in the United States has been characterized by sequence analysis and light microscopy. All of the Nannochloropsis species that have been described, both marine and freshwater, are small spheres with essentially no distinguishing morphological characteristics. Therefore, they must be characterized using molecular techniques. We have cultured numerous isolates of Nannochloropsis from a series of lakes on the James River in the Arrowwood National Wildlife Refuge, North Dakota, USA, and 1 isolate from a pond in Itasca State Park, Minnesota, USA. The diversity among these isolates was determined by light microscopy and DNA sequence analysis. Seven distinct haplotypes of Nannochloropsis were found, one of which possesses 18S rDNA and rbcL sequences identical to those of N. limnetica from Europe. The 6 new haplotypes vary in rbcL sequences and some are morophologically distinct from each other and from N. limnetica. These types are described as the new taxa N. limnetica var. globosa, N. limnetica var. irregularis, N. limnetica var. dystrophica, and N. limnetica var. gutta. All of the Nannochloropsis isolates from Arrowwood and Itasca were cultured only from samples taken during cold-water periods. These results suggest that Nannochloropsis species may be better adapted to cold water conditions, including temperatures near 0 degrees C and ice cover.     

Fungal endophyte communities in four Rhizophoraceae mangrove species on the south coast of China

This study investigated the taxonomic identities and diversity of fungal endophytes isolated from four Rhizophoraceae mangrove plant species, Ceriops tagal, Rhizophora apiculata, R. stylosa and Bruguiera sexangula var. rhynchopetala, using a combination of morphological and molecular approaches. Two hundred ninety-five isolates were classified into 38 taxa by morphological characteristics. The representative 38 isolates from each taxa were selected for further molecular identification using nuclear ribosomal DNA sequences, including both the internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene region. The 38 representative endophytes were identified to various taxonomic levels. These results suggest that Pestalotiopsis and Phomopsis were the most frequent endophytes in the four host species. Some of the endophytes exhibit host and tissue specificity. The colonization frequencies of endophytic fungi in the stems of the four host plants are evidently higher than in the roots. The four Rhizophoraceae mangrove species have low similarities of endophyte communities.     

Characterization of a Chromobacterium haemolyticum population from a natural tropical lake

Aim: To study genetic diversity of Chromobacterium haemolyticum isolates recovered from a natural tropical lake. Methods and Results: A set of 31 isolates were recovered from a bacterial freshwater community by conventional plating methods and subjected to genetic and phenotypic characterization. The 16S ribosomal RNA (rRNA) gene phylogeny revealed that the isolates were related most closely with C. haemolyticum. In addition to the molecular data, our isolates exhibited strong β‐haemolytic activity, were nonviolacein producers and utilized i‐inositol, d ‐mannitol and d ‐sorbitol in contrast with the other known chromobacteria. Evaluation of the genetic diversity in the 16S rRNA gene, tRNA intergenic spacers (tDNA) and 16S‐23S internal transcribed spacers (ITS) unveiled different levels of genetic heterogeneity in the population, which were also observed with repetitive extragenic palindromic (rep)‐PCR genomic fingerprinting using the BOX‐AR1 primer. tDNA‐ and ITS‐PCR analyses were partially congruent with the 16S rRNA gene phylogeny. The isolates exhibited high resistance to β‐lactamic antibiotics. Conclusion: The population genetic heterogeneity was revealed by 16S rRNA gene sequence, ITS and BOX‐PCR analysis. Significance and Impact of the Study: This study provides for the first time an insight into the genetic diversity of phylogenetically close isolates to C. haemolyticum species.     

ITS-RFLP fingerprinting and molecular marker for detection of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">ciceris</Emphasis>

Genetic diversity of 11 representative isolates of Fusarium oxysporum f.sp. ciceris causing chickpea wilt was determined through internal transcribed spacer (ITS) region of the ribosomal DNA-restriction fragment length polymorphism (ITS-RFLP). ITS1+5.8s+ITS2 regions of the isolates were amplified with a set of primers ITS1 and ITS4 and amplified products were digested with 4 restriction enzymes (AluI, MboI, RsaI, MseI). Six different kinds of ITS-RFLP patterns were obtained. The ITS region of these isolates was sequenced and deposited to NCBI GeneBank. The nucleotide sequence homology of ITS region grouped the isolates into 5 categories. Primers were designed with sequence information using Primer 3 software. F. oxysporum f.sp. ciceris specific markers (FOC F2 and FOC R2) based on ITS region were developed for the first time for detection of the pathogen. The markers produced an amplicon of 292 bp; they were validated against the isolates of the pathogen collected from different locations of India.     

Molecular diversity of fungi from ericoid mycorrhizal roots

In order to investigate the diversity of fungal endophytes in ericoid mycorrhizal roots, about 150 mycelia were isolated from surface-sterilized roots of 10 plants of Calluna vulgaris. Each mycelium was reinoculated to C. vulgaris seedlings under axenic conditions, and the phenotype of the plant-fungus association assessed by light and electron microscopy. Many isolates that were able in vitro to produce typical ericoid mycorrhizae did not form reproductive structures under our culture conditions, whereas others could be identified as belonging to the species Oidiodendron maius. Morphological and molecular analysis of the fungal isolates showed that the root system of a single plant of C. vulgaris is a complex mosaic of several populations of mycorrhizal and non mycorrhizal fungi. PCR-RFLP techniques, used to investigate the mycorrhizal endophytes, revealed up to four groups of fungi with different PCR-RFLP patterns of the ITS ribosomal region from a single plant. Some of the mycorrhizal fungi sharing the same PCR-RFLP pattern showed high degree of genetic polymorphism when analysed with the more sensitive RAPD technique; this technique may prove a useful tool to trace the spread of individual mycorrhizal mycelia, as it has allowed us to identify isolates with identical RAPD fingerprints on different plants.     

Genetic characterization of Mortierella alpina by sequencing the 18S‐28S ribosomal gene internal transcribed spacer region

Aims: The aim of this study was to establish a genetic background of Mortierella alpina, which is a cosmopolitan, soil‐inhabiting Zygomycete that also has important biotechnology potential. Methods and Results: A total of 44 18S‐28S ribosomal gene internal transcribed spacer (ITS1‐5·8S rDNA‐ITS2 DNA) regions of M. alpina from three diverse locations (Far East Asia, North America and West‐Central Europe) were sequenced and investigated. The sequences between M. alpina and the three closely related species (Mortierella macrocystis, Mortierella gamsii and Mortierella humilis) showed 74–84% sequence identity. When a phylogenetic tree was constructed with a neighbour‐joining algorithm, four clades of M. alpina isolates were clearly distinct with high bootstrap values. In addition, isolates from the West‐Central Europe were found to have the highest gene and nucleotide diversities. Conclusions: The ITS region was a suitable tool for distinguishing M. alpina from other closely related species. The region also provided information of the diversity of M. alpina. Significance and Impact of the Study: The study established a genetic background of M. alpina for identification and the diversity of M. alpina provided information for further isolation and screening of the fungus.     

Cyst‐motile stage relationship and molecular phylogeny of a new freshwater dinoflagellate Gymnodinium plasticum from Plastic Lake,Canada

The dinophyceaen genus Gymnodinium was established with the freshwater species G. fuscum as type. According to Thessen et al. (2012), there are 268 species, with the majority marine species. In recently published molecular phylogenies based on ribosomal DNA sequences, Gymnodinium is polyphyletic. Here, a new freshwater Gymnodinium species, G. plasticum, is described from Plastic Lake, Ontario, Canada. Two strains were established by incubating single cysts, and their morphology was examined with light microscopy and scanning electron microscopy. The cyst had a rounded epicyst and hypocyst with a wide cingulum and smooth surface. Vegetative cells were characterized by an elongated nucleus running vertically and a deep sulcal intrusion. The apical structure complex was horseshoe‐shaped and consisted of two pronounced ridges with a deep internal groove, encircling 80% of the apex. Small subunit ribosomal DNA (SSU rDNA), large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer (ITS) sequences were obtained from cultured strains. Molecular phylogeny based on concatenated SSU, LSU and ITS sequences supports the monophyly of the Gymnodiniales sensu stricto clade but our results suggest that many Gymnodinium species might need reclassification. Gymnodinium plasticum is closest to Dissodinium pseudolunula in our phylogeny but distant from the type species G. fuscum, as are the other gymnodiniacean taxa.     

44 Diversity of the picoplankter choricystis (trebouxiophyceae,chlorophyta) from minnesota and north dakota lakes

Choricystis (Trebouxiphyceae, Chlorophyta) is considered a very common member of the freshwater picoplankton. We have established a culture collection from Itasca State Park (ISP), Minnesota, and Arrowwood National Wildlife Refuge (ANWR), North Dakota, that includes many Choricystis isolates. We examined 109 Choricystis isolates from ISP and 19 isolates from ANWR using PCR‐RFLP of the rbcL gene. Twelve types for ISP and 7 types forANWR were distinguished by this technique. Sequence analysis revealed additional diversity within some of the RFLP types. Moreover, none of the Choricystis isolates from ANWR possessed rbcL sequences identical to any isolate from ISP. Phylogenetic analysis of the sequence data revealed at least 2 major lineages. These results indicate that Choricystis is much more diverse than previously thought. This material is based upon work supported by the National Science Foundation under Grant Nos. DEB‐0128953, DBI‐0070387 and MCB     

Phylogenetic analysis of Dunaliella (Chlorophyta) emphasizing new benthic and supralittoral isolates from Great Salt Lake

Dunaliella, a commercially important chlorophyte, is globally distributed in saline habitats. Morphological species have not been definitively reconciled with phylogenetic analyses. Considerable genetic diversity continues to be discovered in new isolates, especially from soil and benthic habitats. Twenty‐nine new isolates from Great Salt Lake, Utah, many from benthic or supralittoral habitats, were phylogenetically analyzed using ITS1+5.8S+ITS2 in comparison to a broad sampling of available sequences. A few new isolates align in one branch of a bifurcated monophyletic Dunaliella salina clade and several cluster within monophyletic D. viridis. Several others align with relatively few unnamed strains from other locations, comprising a diverse clade that may represent two or more new species. The overall Dunaliella clade is relatively robust, but the nearest outgroups are ambiguously placed with extremely long branches. About half of the isolates, all from benthic or supralittoral habitats, have been persistently sarcinoid in liquid media since isolation. This trait is spread across the Dunaliella phylogeny. The morphology of two sarcinoid strains was documented with light microscopy, revealing an extensive glycocalyx. Clumping behavior of unicellular and sarcinoid strains was unaffected by presence or absence of Mg²⁺ or Ca²⁺, addition of lectin‐inhibiting monosaccharides, or water‐soluble factors from morphologically opposite strains. Results from this investigation have significantly expanded our current understanding of Dunaliella diversity, but it seems likely that much remains to be discovered with additional sampling.     

Ramularia coleosporii,a Hyperparasite on Coleosporium plumeriae in India

During a survey of fungal plant diseases, parasitized uredinial pustules of Coleosporium plumeriae were observed in Umiam, Meghalaya, India. Microscopic examination was conducted using light and scanning electron microscopy which revealed the occurrence of the hyperparasitic hyphomycete Ramularia coleosporii. Molecular characterization using the ITS1‐5.8S‐ITS2 region of nrDNA also confirmed our results. This is the first record of R. coleosporii on C. plumeriae from India.     

SymPortal: A novel analytical framework and platform for coral algal symbiont next‐generation sequencing ITS2 profiling

We present SymPortal (SymPortal.org), a novel analytical framework and platform for genetically resolving the algal symbionts of reef corals using next‐generation sequencing (NGS) data of the ITS2 rDNA. Although the ITS2 marker is widely used to genetically characterize taxa within the family Symbiodiniaceae (formerly the genus Symbiodinium), the multicopy nature of the marker complicates its use. Commonly, the intragenomic diversity resultant from this multicopy nature is collapsed by analytical approaches, thereby focusing on only the most abundant sequences. In contrast, SymPortal employs logic to identify within‐sample informative intragenomic sequences, which we have termed ‘defining intragenomic variants' (DIVs), to identify ITS2‐type profiles representative of putative Symbiodiniaceae taxa. By making use of this intragenomic ITS2 diversity, SymPortal is able to resolve genetic delineations using the ITS2 marker at a level that was previously only possible by using additional genetic markers. We demonstrate this by comparing this novel approach to the most commonly used alternative approach for NGS ITS2 data, the 97% similarity clustering to operational taxonomic units (OTUs). The SymPortal platform accepts NGS raw sequencing data as input to provide an easy‐to‐use, standardization‐enforced, and community‐driven framework that integrates with a database to gain resolving power with increased use. We consider that SymPortal, in conjunction with ongoing large‐scale sampling and sequencing efforts, should play an instrumental role in making future sampling efforts more comparable and in maximizing their efficacy in working towards the classification of the global Symbiodiniaceae diversity.     

First report of the potentially toxic marine diatom Pseudo‐nitzschia simulans (Bacillariophyceae) from the East Australian Current

Certain species of the marine diatom genus Pseudo‐nitzschia are responsible for the production of the domoic acid (DA), a neurotoxin that can bioaccumulate in the food chain and cause amnesic shellfish poisoning (ASP) in animals and humans. This study extends our knowledge by reporting on the first observation of the potentially toxic species Pseudo‐nitzschia simulans from this region. One clonal strain of P. simulans was isolated from the East Australian Current and characterized using light and transmission electron microscopy, and phylogenetic analyses based on regions of the internal transcribed spacer (ITS) and the D1–D3 region of the large subunit (LSU) of the nuclear‐encoded ribosomal deoxyribonucleic acid (rDNA), as well as examined for DA production as measured by liquid chromatography–mass spectrometry. Although this strain was non‐toxic under the defined growth conditions, the results unambiguously confirmed that this isolate is the potentially toxic species P. simulans – the first report of this species from the Southern Hemisphere.     

A wide diversity of previously undetected free‐living relatives of diplomonads isolated from marine/saline habitats

Over the last 15 years classical culturing and environmental PCR techniques have revealed a modest number of genuinely new major lineages of protists; however, some new groups have greatly influenced our understanding of eukaryote evolution. We used culturing techniques to examine the diversity of free‐living protists that are relatives of diplomonads and retortamonads, a group of evolutionary and parasitological importance. Until recently, a single organism, Carpediemonas membranifera, was the only representative of this region of the tree. We report 18 new isolates of Carpediemonas‐like organisms (CLOs) from anoxic marine sediments. Only one is a previously cultured species. Eleven isolates are conspecific and were classified within a new genus, Kipferlia n. gen. The remaining isolates include representatives of three other lineages that likely represent additional undescribed genera (at least). Small‐subunit ribosomal RNA gene phylogenies show that CLOs form a cloud of six major clades basal to the diplomonad‐retortamonad grouping (i.e. each of the six CLO clades is potentially as phylogenetically distinct as diplomonads and retortamonads). CLOs will be valuable for tracing the evolution of diplomonad cellular features, for example, their extremely reduced mitochondrial organelles. It is striking that the majority of CLO diversity was undetected by previous light microscopy surveys and environmental PCR studies, even though they inhabit a commonly sampled environment. There is no reason to assume this is a unique situation – it is likely that undersampling at the level of major lineages is still widespread for protists.     

Taxon-specific oligonucleotide primers for detection of Glomus etunicatum

The 5.8S subunit and flanking internal transcribed spacer (ITS) regions in nuclear ribosomal DNA (rDNA) from spores of Glomus etunicatum MD107, MD127, TN101, and FL329 were amplified by polymerase chain reaction (PCR) using ITS1Kpn and ITS4Pst as primers. The amplification products (597, 599, 598, and 613 bp, respectively) were cloned and sequenced. The similarity among ITS region sequences from MD107, MD127, and TN101 was 99%, whereas the sequence similarity between the ITS regions of these three DNAs and that from FL329 was 91%. The 5.8S rDNA sequences of all four G. etunicatum isolates were identical. In contrast, major dissimilarities in the corresponding rDNA sequence regions of other glomalean taxa were observed. Oligonucleotide sequences unique to G. etunicatum were tested for their specificity in PCR amplification of genomic DNA from spores of 55 isolates comprising 29 glomalean fungi: 18 isolates of G. etunicatum, five G. intraradices, three G. claroideum, 16 other Glomus isolates, and 11 other glomalean taxa from each of four other genera. The G. etunicatum isolates were from a broad range of geographic regions and soils. The oligonucleotide pair GETU1:GETU2 primed specific amplification of an oligonucleotide sequence (approximately 400 bp) present in all G. etunicatum. This primer pair did not prime PCR when template consisted of DNA from any of the other glomalean fungi or any of the non-mycorrhizal controls, including roots of corn (Zea mays). In addition, the pair successfully detected G. etunicatum in nested PCR using a primary PCR product amplified from highly diluted extracts of colonized corn roots using modified ITS1:ITS4 primers. In the phylogenetic analysis of Glomus 5.8S and ITS2 rDNA region sequences, which included 500 bootstrap data sets, confidence in the G. etunicatum branch was very strong (90%) and clearly independent of G. claroideum and G. intraradices, to which it is very closely related. Accepted: 15 October 2000     

Genetic Diversity, Aggressiveness and Metalaxyl Sensitivity of Pythium spinosum Infecting Cucumber in Oman

A total of 24 isolates of Pythium spinosum from cucumber obtained from five regions in Oman were characterized for genetic diversity using amplified fragment length polymorphism (AFLP) fingerprinting and three isolates from the Netherlands, South Africa and Japan were included for comparison. Isolates from Oman were also characterized for aggressiveness on cucumber seedlings and sensitivity to metalaxyl. Identity of all isolates was confirmed using sequences of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA), which showed more than 99% nucleotide similarity among all isolates. Using six primer‐pair combinations, AFLP fingerprinting resolved 295 AFLP markers of which 193 were polymorphic among isolates from other countries and only six were polymorphic among isolates of P. spinosum from Oman. Seven different AFLP phenotypes of P. spinosum were recovered in Oman; two of them were found to contain over 79% of isolates and one was recovered from all regions in Oman. Phenotypes from Oman showed very high (≥99%) levels of genetic similarity to each other compared to moderate (mean =53%) levels of genetic similarity with phenotypes from other countries. In addition, all isolates from Oman were found to be highly sensitive to metalaxyl and all were aggressive on cucumber seedlings at 25°C. The high genetic similarity among phenotypes of P. spinosum in Oman as well as recovering two major clones across regions may suggest that P. spinosum has been recently introduced in Oman via a common source.     

A phylogeographical study of the toxic benthic dinoflagellate genus Ostreopsis Schmidt

Aim Ostreopsis is a benthic and epiphytic dinoflagellate producing potent toxins widespread in tropical and warm temperate coastal areas world‐wide. We tested the hypothesis that as it is benthic, it would show distinct biogeographical patterns in comparison with planktonic species. Here, we analyse sequence variability in ribosomal DNA markers to provide the first phylogeographical study of this toxic benthic dinoflagellate. Location Mediterranean Sea, Atlantic Ocean, Pacific Ocean. Methods Ribosomal DNA sequence data from partial nuclear LSU (D1/D2 domains) and 5.8S genes and non‐coding internal transcribed spacer (ITS) regions were obtained from 82 isolates of Ostreopsis species, collected at 26 localities throughout the world. Molecular sequence data were analysed using maximum parsimony, maximum likelihood and Bayesian methods for phylogenetic inference. A statistical parsimony network was obtained based on concatenated LSU and 5.8S rDNA–ITS region sequences of the Mediterranean/Atlantic Ostreopsis cf. ovata isolates to infer haplotype distribution over their geographical range. Light epifluorescence microscopy analyses were performed on cultured and field Ostreopsis material for taxonomic identification, while laboratory experiments for encystment induction were carried out on selected O. cf. ovata isolates. Toxin assays of Ostreopsis species isolates were carried out using the haemolytic‐based method. Results Analyses based on single and concatenated ribosomal genes gave substantially similar results. The rDNA phylogeny revealed different clades corresponding to different species within the genus Ostreopsis. In the species O. cf. ovata, different genetic lineages were correlated with macrogeographical distribution. A network of haplotypes inferred from the Atlantic and Mediterranean isolates of O. cf. ovata revealed that these two areas might host a single panmictic population. The Atlantic/Mediterranean population of O. cf. ovata was differentiated considerably from the Indo‐Pacific populations. Other species of Ostreopsis were found, but they turned out to be restricted to just one of the two main warm‐water oceanic basins, the Mediterranean/Atlantic and the Indo‐Pacific. Main conclusions Ostreopsis cf. ovata was found to be widely dispersed throughout the coastal areas of tropical and some warm temperate seas. In the Atlantic/Mediterranean region it may constitute a panmictic population that is highly distinct from Indo‐Pacific populations. Ostreopsis cf. siamensis was found only in the Mediterranean Sea, and strains identified as Ostreopsis lenticularis and Ostreopsis labens were found only in the Indo‐Pacific region.     

Characterization of Thielaviopsis paradoxa Isolates from Oil Palms in Colombia,Ecuador and Brazil

Ceratocystis paradoxa (Anamorph: Thielaviopsis paradoxa) is parasitic on a range of economic and food crops and is the cause of dry basal rot, a limiting disease in oil palm. The objective of this study was to determinate the pathogenic and genetic diversity of Thielaviopsis isolates from oil palms in Colombia, Ecuador and Brazil. A total of 164 strains of Thielaviopsis paradoxa were characterized using pathogenicity tests, random amplified polymorphic DNA (RAPD) markers and PCR sequencing of the internal transcribed spacer (ITS) region of 5.8 S ribosomal DNA. Oil palm seedlings were inoculated by injecting the base of stems in the seedling stage with a fungal suspension and severity scores of disease reactions were evaluated. PCR amplification of the ITS region resulted in a 590 base pair (bp) product. Digestion of the PCR product with two restriction enzymes produced three restriction patterns, which according to ITS sequences could be classified as T. paradoxa. Six RAPD primers gave polymorphic bands in T. paradoxa. Population structure analyses of the RAPD data suggested that most of the isolates obtained in this study belonged to a single population. The genetic diversity of the isolates from South America was intermediate, and therefore, T. paradoxa is likely to be predominantly clonal compared with Ceratocystis species. Sporadic sexual reproduction may occur for T. paradoxa but is secondary to clonal reproduction. Data on pathogen diversity will provide information on breeding strategies and population structures.