ECOLOGICAL DIFFERENTIATION BETWEEN SYMPATRIC PSEUDOCRYPTIC SPECIES IN THE ESTUARINE BENTHIC DIATOM NAVICULA PHYLLEPTA (BACILLARIOPHYCEAE)1

The occurrence of cryptic and pseudocryptic species, often living in sympatry, is widespread among microalgae. This phenomenon raises important questions about niche partitioning between these closely related species. To date, however, few studies have addressed the ecological mechanisms underlying sympatry in cryptic and pseudocryptic species. As a result, we have only a limited understanding of the factors that govern their distribution along environmental gradients. Here, we used the ribosomal internal transcribed spacer (ITS), 18S rRNA gene, and the RUBISCO LSU (rbcL) chloroplast gene sequence data together with cell wall morphology to show that estuarine populations of the widespread and common benthic diatom Navicula phyllepta Kütz. consist of pseudocryptic species. Growth rate measurements in function of salinity showed that N. phyllepta strains assigned to the different species differed in their tolerance to low salinities (<5 practical salinity units, psu), which was reflected by their different (but widely overlapping) distribution in the Westerschelde estuary (the Netherlands). Multiple regression analyses of the factors determining the abundance of the different species in field samples revealed that, in addition to salinity, sediment type and ammonium concentrations were probably equally important. Our results show that N. phyllepta sensu lato comprises different species with specialized ecophysiological characteristics rather than generalists with a broad adaptability to different environmental conditions.     

Spatial Patterns of Benthic Macroinvertebrates in Intertidal Areas of a Southern European Estuary: The Tagus, Portugal

This study characterizes the composition and spatial distribution patterns of the benthic macrofauna in the intertidal mudflats of the Tagus estuary, western Portugal. A total of 68 species, more than 226,000 specimens with a total wet weight biomass of approximately 1170 g were identified in 380 sites. The species Streblospio shrubsolii, Cyathura carinata, Tharyx sp., Hydrobia ulvae and Tubificids were the most common and abundant. Scrobicularia plana strongly dominated the biomass. The invertebrate macrofauna of the Tagus estuary shows similarities to what is known from other temperate mudflats. The diversity of species, their overall abundance and the ratio of Molluscs plus Crustaceans to Polychaete species corroborate the distinctiveness between temperate and tropical mudflats and sandflats. The spatial distribution of the fauna reflects the sediment characteristics but the relationship between the environmental and the biological data is not as strong as obtained for sublittoral areas. This relationship diminishes from the sublittoral shelf to sublittoral estuarine areas, showing minimum values in this study, suggesting that such a relationship is less straightforward as natural disturbance increases. Nevertheless, a mixture of grain-size, elevation (inundation time) and particular habitats (relic oyster beds) form the best explanatory factors for the spatial distribution patterns of the intertidal benthic macrofauna of the Tagus estuary.     

Long‐term stability of entomopathogenic nematode spatial patterns in soil as measured by sentinel insects and real‐time PCR assays

Quantitative real‐time PCR (qPCR) techniques are being increasingly used to provide accurate and reliable methods to identify and quantify cryptic organisms in soil ecology. Entomopathogenic nematode (EPN) diversity in Florida is known to be extensive and our phylogenetic studies of the D2D3 and ITS regions showed the occurrence of an additional species‐complex in the Steinernema glaseri‐ group in widely separated locations of the peninsula. To address ecological studies, we developed and used qPCR assays to detect and quantify six species of EPN that are naturally distributed in Florida citrus orchards (Steinernema diaprepesi, Steinernema riobrave, Heterorhabditis indica, Heterorhabditis zealandica, Heterorhabditis floridensis and an undescribed species in the S. glaseri group) and an exotic species, S. glaseri. Species‐specific primers and TaqMan® probes were designed from the ITS rDNA region. No nonspecific amplification was observed in conventional or qPCR when the primers and probes were tested using several populations of each of the Florida species and other exotic EPN species. Standard curves were established using DNA from pure cultures. We optimised a protocol for extracting nematodes and DNA from soil samples that can detect one EPN added to nematode communities recovered by conventional extraction protocols. A survey of an 8‐ha orchard in April 2009 compared the EPN spatial patterns derived from qPCR to that obtained by baiting soil samples with Galleria mellonella larvae. The patterns were also compared to those derived from the same site in 2000–01 by repeatedly (12 sampling events) baiting soil in situ with caged larvae of the root weevil Diaprepes abbreviatus. The qPCR assay was more efficient than the Galleria baiting method for detecting the EPN species composition in population mixtures. Moreover, the spatial patterns of EPN in this orchard were remarkably stable over the course of nearly a decade. The pattern of H. zealandica detected at the site 8 years earlier was related to those derived by qPCR (P = 0.002) and from sample baiting (P = 0.02). The spatial pattern of H. indica derived from qPCR, but not that from sample baiting, was also related to the earlier pattern (P = 0.01). The qPCR assay developed here is a fast, affordable and accurate method to detect and quantify these EPN species in soil and offers great potential for studying the ecology of EPN.     

Diel,semi‐lunar and seasonal patterns in the fish community of an intertidal zone of the Yangtze estuary

The structure and temporal variations of the fish community in the intertidal estuarine zone of shallow mud areas have been poorly studied in China. This paper analyses the diel, semi‐lunar and seasonal patterns of fish assemblages in the Yangtze estuary in 2006. Fish were collected by consecutive day and night samplings using tide‐stow‐nets deployed parallel to each other in three stations. A total of 56 fish species belonging to 21 families was caught during the study period. The family Cyprinidae dominated with 25 species. Freshwater fish species were the important dominant commercial fishery species and well represented with five species (sharpbelly Hemiculter bleekeri, goldfish Carassius auratus, bream Parabramis pekinensis, likely‐bream Pseudobrama simony, and glossy yellow catfish Pelteobagrus nitidus) in the three stations. Juvenile fishes dominated the fish community, comprising 93.9% in station 1 and 96.6% in station 2 of the total abundance. The number of fish species in day tides was slightly lower than those in night tides in spring and summer, but the opposite in other seasons. In neap tides, the numbers and abundance of fish species were both lower than those in the spring tides. Fish abundance was lowest in winter, increasing during spring and summer (March–September) in both stations 1 and 2, with obviously large fluctuations in each season. The pattern of habitat selection of fishes could effectively decrease the food competition of intraspecies or interspecies and favour the growth and nursing of young fishes. These findings indicate that the intertidal zones in the estuary may serve as important nursery areas for fish communities.     

Investigating the ecology and evolution of cryptic marine nematode species through quantitative real-time PCR of the ribosomal ITS region

The presence of morphologically similar but genetically distinct species has impacted biogeographical and ecological paradigms. In marine sediments, free‐living nematodes form one of the most abundant and diverse faunal groups. Inferring the importance of nematode diversity for ecosystem functioning requires species‐level identification, which is hampered by the lack of easily observable diagnostic characters and the presence of cryptic species. New techniques are urgently needed to adequately study the ecology and evolution of cryptic species. The aim of the present study was to evaluate the potential of a quantitative real‐time PCR (qPCR) assay using the internal transcribed spacer (ITS) region of the ribosomal DNA to detect and quantify cryptic species of the R. (P.) marina complex. All primer pairs proved to be highly specific, and each primer pair was able to detect a single juvenile in a pool of 100 nematodes. C_t values were significantly different between developmental stages for all species except for PmIII. Despite differences between developmental stages, a strong correlation was observed between the amount of extracted DNA and the number of nematodes present. Relative and absolute quantification estimates were comparable and resulted in strong positive correlations between the qPCR estimate and the actual number of nematodes present in the samples. The qPCR assay developed here provides the ability to quickly identify and quantify cryptic nematode species and will facilitate their study in laboratory and field settings.     

DEVELOPMENT OF SEMI‐QUANTITATIVE PCR ASSAYS FOR THE DETECTION AND ENUMERATION OF GAMBIERDISCUS SPECIES (GONYAULACALES,DINOPHYCEAE)1

Ciguatera fish poisoning (CFP) is a serious health problem in tropical regions and is caused by the bioaccumulation of lipophilic toxins produced by dinoflagellates in the genus Gambierdiscus. Gambierdiscus species are morphologically similar and are difficult to distinguish from one another even when using scanning electron microscopy. Improved identification and detection methods that are sensitive and rapid are needed to identify toxic species and investigate potential distribution and abundance patterns in relation to incidences of CFP. This study presents the first species‐specific, semi‐quantitative polymerase chain reaction (qPCR) assays that can be used to address these questions. These assays are specific for five Gambierdiscus species and one undescribed ribotype. The assays utilized a SYBR green format and targeted unique sequences found within the SSU, ITS, and the D1/D3 LSU ribosomal domains. Standard curves were constructed using known concentrations of cultured cells and 10‐fold serial dilutions of rDNA PCR amplicons containing the target sequence for each specific assay. Assay sensitivity and accuracy were tested using DNA extracts purified from known concentrations of multiple Gambierdiscus species. The qPCR assays were used to assess Gambierdiscus species diversity and abundance in samples collected from nearshore areas adjacent to Ft. Pierce and Jupiter, Florida USA. The results indicated that the practical limit of detection for each assay was 10 cells per sample. Most interestingly, the qPCR analysis revealed that as many as four species of Gambierdiscus were present in a single macrophyte sample.     

Genetic delineation between and within the widespread coccolithophore morpho‐species Emiliania huxleyi and Gephyrocapsa oceanica (Haptophyta)

Emiliania huxleyi and Gephyrocapsa oceanica are abundant coccolithophore morpho‐species that play key roles in ocean carbon cycling due to their importance as both primary producers and cal‐cifiers. Global change processes such as ocean acidification impact these key calcifying species. The physiology of E. huxleyi, a developing model species, has been widely studied, but its genetic delineation from G. oceanica remains unclear due to a lack of resolution in classical genetic markers. Using nuclear (18S rDNA and 28S rDNA), mitochondrial (cox1, cox2, cox3, rpl16, and dam), and plastidial (16S rDNA, rbcL, tufA, and petA) DNA markers from 99 E. huxleyi and 44 G. oceanica strains, we conducted a multigene/multistrain survey to compare the suitability of different markers for resolving phylogenetic patterns within and between these two morpho‐species. The nuclear genes tested did not provide sufficient resolution to discriminate between the two morpho‐species that diverged only 291Kya. Typical patterns of incomplete lineage sorting were generated in phylogenetic analyses using plastidial genes. In contrast, full morpho‐species delineation was achieved with mitochondrial markers and common intra‐morpho‐species phylogenetic patterns were observed despite differing rates of DNA substitution. Mitochondrial genes are thus promising barcodes for distinguishing these coccolithophore morpho‐species, in particular in the context of environmental monitoring.     

RECOGNIZING DINOFLAGELLATE SPECIES USING ITS rDNA SEQUENCES1

Dinoflagellate taxonomy is based primarily on morphology and morphometric data that can be difficult to obtain. In contrast, molecular data can be rapidly and cost‐effectively acquired, which has led to a rapid accumulation of sequence data in GenBank. Currently there are no systematic criteria for utilizing taxonomically unassigned sequence data to identify putative species that could in turn serve as a basis for testable hypotheses concerning the taxonomy, diversity, distribution, and toxicity of these organisms. The goal of this research was to evaluate whether simple, uncorrected genetic distances (p) calculated using ITS1/5.8S/ITS2 (ITS region) rDNA sequences could be used to develop criteria for recognizing putative species before formal morphological evaluation and classification. The current analysis used sequences from 81 dinoflagellate species belonging to 14 genera. For this diverse assemblage of dinoflagellate species, the within‐species genetic distances between ITS region copies (p=0.000–0.021 substitutions per site) were consistently less than those observed between species (p=0.042–0.580). Our results indicate that a between‐species uncorrected genetic distance of p≥0.04 could be used to delineate most free‐living dinoflagellate species. Recently evolved species, however, may have ITS p values <0.04 and would require more extensive morphological and genetic analyses to resolve. For most species, the sequence of the dominant ITS region allele has the potential to serve as a unique species‐specific “DNA barcode” that could be used for the rapid identification of dinoflagellates in field and laboratory studies.     

Cladosiphon umezakii sp. nov. (Ectocarpales, Phaeophyceae) from Japan

The new species Cladosiphon umezakii Ajisaka (Ectocarpales, Phaeophyceae) is described from Japan based on morphology and DNA sequences. The species resembles Cladosiphon okamuranus Tokida in its gross morphology; somewhat slimy, cylindrical, multiaxial and sympodial erect thallus, arising from a small disc‐shaped holdfast, and branching once to twice. However, C. umezakii has considerably longer assimilatory filaments (up to 840 μm long, composed of up to 90 cells) than any known taxa of the genus. The species is a winter to spring annual, growing on lower intertidal to subtidal rocks of more or less exposed sites on the north‐eastern coast of Kyushu and on both the Pacific and the Sea of Japan coasts of Honshu. Specimens from the Sea of Japan coast had both unilocular and plurilocular zoidangia, whereas those from Kyushu and from the Pacific had only unilocular zoidangia. Unilocular zoidangia were formed on the basal part of assimilatory filaments, and plurilocular ones were transformed from the distal part of assimilatory filaments. DNA sequences of the Rubisco‐spacer (rbc‐spacer) region and the nuclear rDNA ITS region (ITS1, 5.8S and ITS2) supported the distinctness of the species.     

Sequencing type material resolves the identity and distribution of the generitype Lithophyllum incrustans,and related European species L. hibernicum and L. bathyporum (Corallinales,Rhodophyta)

DNA sequences from type material in the nongeniculate coralline genus Lithophyllum were used to unambiguously link some European species names to field‐collected specimens, thus providing a great advance over morpho‐anatomical identifi‐cation. In particular, sequence comparisons of rbcL, COI and psbA genes from field‐collected specimens allowed the following conclusion: the generitype species, L. incrustans, occurs mostly as subtidal rhodoliths and crusts on both Atlantic and Mediterranean coasts, and not as the common, NE Atlantic, epilithic, intertidal crust reported in the literature. The heterotypic type material of L. hibernicum was narrowed to one rhodolith belonging in Lithophyllum. As well as occurring as a subtidal rhodolith, L. hibernicum is a common, epilithic and epizoic crust in the intertidal zone from Ireland south to Mediterranean France. A set of four features distinguished L. incrustans from L. hibernicum, including epithallial cell diameter, pore canal shape of sporangial conceptacles and sporangium height and diameter. An rbcL sequence of the lectotype of Lithophyllum bathyporum, which was recently proposed to accommodate Atlantic intertidal collections of L. incrustans, corresponded to a distinct taxon hitherto known only from Brittany as the subtidal, bisporangial, lectotype, but also occurs intertidally in Atlantic Spain. Specimens from Ireland and France morpho‐anatomically identified as L. fasciculatum and a specimen from Cornwall likewise identified as L. duckerae were resolved as L. incrustans and L. hibernicum, respectively.     

CRYPTIC DIVERSITY AND PHYLOGENETIC RELATIONSHIPS WITHIN THE MASTOCARPUS PAPILLATUS SPECIES COMPLEX (RHODOPHYTA,PHYLLOPHORACEAE)1

Mastocarpus papillatus (C. Agardh) Kütz. is a common intertidal red alga occurring along the west coast of North America from Baja California to Alaska. Sequencing of both the chloroplast‐encoded rbcL gene and the nuclear ribosomal internal transcribed spacer (ITS) regions of ～200 specimens from California to Alaska revealed that M. papillatus is actually a complex of at least five species. All five species have high bootstrap support in phylogenetic analyses of both genetic regions, and in the case of the ITS marker, the species also have distinctive patterns of indels. Three of the species are localized in the mid‐ to upper intertidal, whereas two of the species occur in the low intertidal. The species also have different geographic ranges that overlap in the Vancouver Island area of British Columbia. No distinctive, reliable morphological differences were observed among the species. Although a variety of names are available for species in the complex, it is not yet clear which name goes with which species. As part of the survey, I also sequenced other species of Mastocarpus in the northeast Pacific region, and I provide new distribution records for M. jardinii ( J. Agardh) J. A. West and for a nonpapillate and probably undescribed species of Mastocarpus.     

Identification and quantification of viable Bifidobacterium breve strain Yakult in human faeces by using strain-specific primers and propidium monoazide

Aims: To develop a quick and accurate PCR‐based method to evaluate viable Bifidobacterium breve strain Yakult (BbrY) in human faeces. Methods and Results: The number of BbrY in faeces was detected by using strain‐specific quantitative real‐time PCR (qPCR) derived from a randomly amplified polymorphic DNA analysis. And using propidium monoazide (PMA) treatment, which combined a DNA‐intercalating dye for covalently linking DNA in dead cells and photoactivation, only viable BbrY in the faeces highly and significantly correlated with the number of viable BbrY added to faecal samples within the range of 10⁵–10⁹ cells per g of faeces was enumerated. After 11 healthy subjects ingested 10·7 log CFU of BbrY daily for 10 days, 6·9 (±1·5) log CFU g^?1 [mean (±SD)] of BbrY was detected in faeces by using strain‐specific transgalactosylated oligosaccharide–carbenicillin (T‐CBPC) selective agar medium. Viable BbrY detected by qPCR with PMA treatment was 7·5 (±1·0) log cells per g and the total number (viable and dead) of BbrY detected by qPCR without PMA treatment was 8·1 (±0·8) log cells per g. Conclusions: Strain‐specific qPCR with PMA treatment evaluated viable BbrY in faeces quickly and accurately. Significance and Impact of the Study: Combination of strain‐specific qPCR and PMA treatment is useful for evaluating viable probiotics and its availability in humans.     

Assessing the potential of candidate DNA barcodes for identifying non‐flowering seed plants

In plants, matK and rbcL have been selected as core barcodes by the Consortium for the Barcode of Life (CBOL) Plant Working Group (PWG), and ITS/ITS2 and psbA‐trnH were suggested as supplementary loci. Yet, research on DNA barcoding of non‐flowering seed plants has been less extensive, and the evaluation of DNA barcodes in this division has been limited thus far. Here, we evaluated seven markers (psbA‐trnH, matK, rbcL, rpoB, rpoC1, ITS and ITS2) from non‐flowering seed plants. The usefulness of each region was assessed using four criteria: the success rate of PCR amplification, the differential intra‐ and inter‐specific divergences, the DNA barcoding gap and the ability to discriminate species. Among the seven loci tested, ITS2 produced the best results in the barcoding of non‐flowering seed plants. In addition, we compared the abilities of the five most‐recommended markers (psbA‐trnH, matK, rbcL, ITS and ITS2) to identify additional species using a large database of gymnosperms from GenBank. ITS2 remained effective for species identification in a wide range of non‐flowering seed plants: for the 1531 samples from 608 species of 80 diverse genera, ITS2 correctly authenticated 66% of them at the species level. In conclusion, the ITS2 region can serve as a useful barcode to discriminate non‐flowering seed plants, and this study will contribute valuable information for the barcoding of plant species.     

Growing coffee: Psilanthus (Rubiaceae) subsumed on the basis of molecular and morphological data; implications for the size,morphology, distribution and evolutionary history of Coffea

Morphological and molecular phylogenetic studies show that there is a close relationship between Coffea and Psilanthus. In this study we reassess species relationships based on improved species sampling for Psilanthus, including P. melanocarpus, a species that shares morpho‐taxonomic characters of both genera. Analyses are performed using parsimony and Bayesian inference, on sequence data from four plastid regions [trnL–F intron, trnL–F IGS, rpl16 intron and accD–psa1 intergenic spacer (IGS)] and the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (ITS 1/5.8S/ITS 2). Several major lineages with geographical coherence, as identified in previous studies based on smaller and larger data sets, are supported. Our results also confirm previous studies showing that the level of sequence divergence between Coffea and Psilanthus species is negligible, particularly given the much longer branch lengths separating other genera of tribe Coffeeae. There are strong indications that neither Psilanthus nor Coffea is monophyletic. Psilanthus melanocarpus is nested with the Coffea–Psilanthus clade, which means that there is only one critical difference between Coffea and Psilanthus; the former has a long‐emergent style and the latter a short, included style. Based on these new data, in addition to other systematically informative evidence from a broad range of studies, and especially morphology, Psilanthus is subsumed into Coffea. This decision increases the number of species in Coffea from 104 to 124, extends the distribution to tropical Asia and Australasia and broadens the morphological characterization of the genus. The implications for understanding the evolutionary history of Coffea are discussed. A group of closely related species is informally named the ‘Coffea liberica alliance’. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167 , 357–377.     

Feeding ecology and seasonal diet overlap between Stellifer brasiliensis and Stellifer stellifer in a tropical estuarine ecocline

Seasonal freshwater discharge was important for defining habitat utilization by different ontogenetic phases of Stellifer brasiliensis and Stellifer stellifer along the estuarine ecocline. The middle estuary was important as a nursery and feeding ground for young‐of‐the‐year, and a feeding ground for sub‐adults and adults of both species. These species are zoobenthivorous, but during their life cycle and between different habitats and seasons, their trophic guild can change to opportunist and zooplanktivore. During the late rainy season in the lower estuary, all phases of both species, except juveniles of S. brasiliensis and adults of S. stellifer, showed a niche overlap indicating similarity in prey utilization. The diet composition was qualitatively similar, showing an evident niche overlap of intra and interspecific competition among the Stellifer spp. Although the niches of these species appeared to significantly overlap, some resource partitioning patterns were apparent. The niche overlap was significantly reduced due to the seasonal difference in habitat use and prey consumption along the ecocline of the estuary by different ontogenetic phases. The ingestion of blue nylon fragments by both species was observed and quantified.     

A quantitative real‐time PCR assay for the detection of Ramularia collo‐cygni from barley (Hordeum vulgare)

Aims: The aim of this study was to develop a real‐time quantitative PCR test to recognize and quantify the DNA levels of the increasingly important barley pathogen Ramularia collo‐cygni. Methods and Results: The method described uses specifically designed primers and a molecular beacon probe based on an internal transcribed spacer (ITS) sequence. Pathogen extracted from barley leaves could be quantified to the picogram level in both leaves showing symptoms of infection and symptomless barley leaves. Conclusions: A relationship between R. collo‐cygni DNA levels and disease symptoms was established in spring barley under natural infection conditions. Significance and Impact of the Study: To our knowledge, this is the first report of a test of this type and makes an important contribution to studies into the life cycle of this pathogen.     

River flow,zooplankton and dominant zooplanktivorous fish dynamics in a warm‐temperate South African estuary

The possible links between river flow, zooplankton abundance and the responses of zooplanktivorous fishes to physico‐chemical and food resource changes are assessed. To this end, the seasonal abundance, distribution and diet of the estuarine round‐herring Gilchristella aestuaria and Cape silverside Atherina breviceps were studied in the Kariega Estuary. Spatio‐temporal differences were determined for selected physico‐chemical variables, zooplankton abundance and zooplanktivorous fish abundance and distribution. Results indicated that, following a river flood event in winter (>30 m³ s^?1), altered physico‐chemical conditions occurred throughout the estuary and depressed zooplankton stocks. Abundance of G. aestuaria was highest in spring, with this species dominant in the upper and middle zones of the estuary, while A. breviceps was dominant in summer and preferred the middle and lower zones. The catch per unit of effort of both zooplanktivores also declined significantly following the flooding, thus suggesting that these fishes are reliant on zooplankton as a primary food source for healthy populations. Copepods dominated the stomach contents of both fish species, indicating a potential for strong interspecific competition for food, particularly in the middle reaches. Temporal differences were evident in dietary overlap between the two zooplanktivorous fish species and were correlated with river flow, zooplankton availability and fish distribution. The findings of this study emphasize the close trophic linkages between zooplankton and zooplanktivorous fishes under changing estuarine environmental conditions, particularly river flow and provide important baseline information for similar studies elsewhere in South Africa and the rest of the world.     

Morphological and molecular differentiation of smooth‐hound sharks (Genus Mustelus,Family Triakidae) from the Gulf of California

The genus Mustelus is the most species‐rich of the widespread family Triakidae whereby its taxonomy and systematics have been historically challenging. They represent a significant fraction of the shark catches of small‐scale fisheries in the Gulf of California. In order to provide information useful for their management and conservation, the morphological and genetic distinction of the four species found in the northern Gulf of California (M. albipinnis, M. californicus, M. henlei and M. lunulatus) were analyzed. Discriminant analysis of 10 morphometric variables placed each species in a distinct and significantly different region of multivariate morpho‐space. The variables contributing most to their distinction were inter‐nostril width, mouth length, upper and lower labial furrow length, and mouth width. Restriction fragment length polymorphisms (PCR‐RFLP) of the nuclear ITS2 ribosomal DNA (rDNA) confirmed that each species represents a genetically cohesive and independent evolutionary lineage. In spite of the difficulty in differentiating these Mustelus species, a few cephalic measurements are useful to separate them. A PCR‐RFLP assay (using RsaI and MspI on ITS2 rDNA amplicons) is also proposed for the molecular differentiation of these commercially harvested smooth‐hound sharks, constituting the first molecular marker available for their identification. These data provide morphological and genetic tools that can be used to improve their management and conservation.     

Development of a Real‐time Fluorescence Loop‐mediated Isothermal Amplification Assay for Detection of Burkholderia gladioli pv. alliicola

Burkholderia gladioli pv. alliicola is a causal agent of rot on a wide range of hosts including onion and tulip. It is one of quarantine phytopathogenic bacteria in China. To reduce the economic losses associated with this pathogen, simple and rapid detection methods are needed. In this study, an efficient loop‐mediated isothermal amplification (LAMP) assay with a real‐time fluorometer was developed. The analysis of 16S‐23S rRNA intergenic transcribed spacer (ITS) sequences showed considerable variability between different Burkholderia species and B. gradioli pathovars. A set of LAMP primers was designed based on the ITS region. The sensitivity and specificity of the developed assay were evaluated at the optimal temperature of 65°C. The primers were specific for B. gladioli pv. alliicola and did not react to strains of others species and other pathovars in the species B. gladioli. The sensitivity of the real‐time LAMP assay was 1 fg DNA which was 100 times higher than that of conventional PCR. The method was verified by testing natural samples and inoculated onion seeds, and it showed effectiveness. The real‐time LAMP assay established in this study is an effective method for detection of B. gladioli pv. alliicola.     

Real-time PCR as an effective technique to assess the impact of phoresy by Paenibacillus sp. bacteria on Steinernema diaprepesi nematodes in nature

Quantitative real‐time PCR (qPCR) is a powerful tool to study species of cryptic organisms in complex food webs. This technique was recently developed to detect and quantify several species of entomopathogenic nematodes (EPNs), which are widely used for biological control of insects, and some natural enemies of EPNs such as nematophagous fungi and the phoretic bacteria Paenibacillus sp. and Paenibacillus nematophilus. A drawback to the use of primers and TaqMan probes designed for Paenibacillus sp. is that the qPCR also amplified Paenibacillus thiaminolyticus and Paenibacillus popilliae, two closely related species that are not phoretically associated with EPNs. Here, we report that the detection of Paenibacillus sp. DNA in nematode samples was two orders of magnitude greater (P < 0.001) when the bacterium was added to soil together with its EPN species‐specific host Steinernema diaprepesi than when it was added concomitantly with other EPNs or with species of bacterial‐feeding nematodes. Just 6% of samples detected trace amounts of P. thiaminolyticus and P. popilliae exposed to the same experimental conditions. Thus, although the molecular assay detects Paenibacillus spp. DNA in nonphoretic associations, the levels are essentially background compared to the detection of Paenibacillus sp. in association with its nematode host.