Molecular and phenotypic description of the widespread root symbiont Acephala applanata gen. et sp. nov., formerly known as dark-septate endophyte type 1

Acephala applanata gen. et sp. nov. is described. A. applanata is a dark-septate endophyte (DSE) of conifer roots and belongs to the Phialocephala fortinii species complex. Several genetic markers, including isozymes, inter-simple-sequence-repeat (ISSR) fingerprints, single-copy restriction fragment length polymorphisms (RFLP) and sequences of the internal transcribed spacers (ITS), let us unambiguously separate isolates of A. applanata from isolates of P. fortinii s.l. and other dark-septate endophytes. Alleles at four RFLP loci and two fixed nucleotides in the ITS region were diagnostic for A. applanata. One of the fixed nucleotides resulted in the addition of an Afa I restriction site. PCR amplification with primers prITS4 and the newly developed primer PF-ITS_F (ACT CTG AAT GTT AGT GAT GTC TGA GT) and restriction digestion with Afa I yielded three fragments (203 bp, 117 bp, 56 bp) in A. applanata but only two (260 bp and 117 bp) in P. fortinii s.l. Population differentiation (GST) between A. applanata and other cryptic species of P fortinii was pronounced, and the index of association (IA) did not deviate significantly from zero, showing that recombination occurs or had occurred in A. applanata. Although isolates of A. applanata never were observed to sporulate, it can be distinguished morphologically from P fortinii s.l. by the scarcity of aerial mycelium, significantly slower growth and denser mycelium on cellophane overlaid on water agar. These phenotypic characteristics, combined with diagnostic RFLP alleles and/or PCR-RFLP of the ITS fragment with the fixed Afa I restriction site, unequivocally allow identification of A. applanata.     

A new PCR-RFLP-based method for an easier systematic affiliation of European water frogs

We describe a non‐invasive, PCR‐RFLP‐based method that allows reliable determination of the European water frog species Pelophylax lessonae and Pelophylax ridibundus and the hybrid form Pelophylax esculentus. Maximum‐likelihood analysis of ITS2 sequences revealed two robust monophyletic clades corresponding to water frogs of the P. lessonae and P. ridibundus groups. Three restriction enzymes (KpnI, HaeII, and SmaI) were used to digest three conserved ITS2 domains. Taxonomic identification was unambiguous; the three restriction enzymes gave the same results. A French reference sample was identified using allozyme electrophoresis. Our PCR‐RFLP method confirmed circa 83% of identification of the allozyme method. We conclude that the difference between identifications was caused by introgression.     

Relationships Among Group IV Phytophthora Species Inferred by Restriction Analysis of the ITS2 Region

The polymerase chain reaction (PCR) was used to amplify the ITS2 region of nuclear ribosomal DNA from six Phytophthora species which comprise taxonomic Group IV. Digestion of the ca. 600 bp PCR product with restriction enzymes Alu I, Dra I, Hha I, Hinf I, Msp I, and Taq I revealed variation which allowed relationships among the species to be assessed. P. infestans , P. mirabilis and P. phaseoli were indistinguishable from one another with all enzymes tested. With Alu I and Taq I. P. ilicis , P. colocasiae . and P. hibernalis each showed unique banding patterns different from the common banding pattern shared by P. infestans . P. mirabilis . and P. hibernalis . Dra I allowed differentiation of P. ilicis and P. colocasiae from P. infestans , P. mirabilis , P. phaseoli , and P. hibernalis . all of which shared a common banding pattern. Hha I allowed differentiation of P. colocasiae and P. hibernalis from P. infestans, P. mirabilis, P. phaseoli , and P. ilicis . Hinf I allowed differentiation of P. ilicis and P. hibernalis , (each of which showed a unique banding pattern) from P. infestans, P. mirabilis, P. phaseoli , and P. colocasiae . Msp I allowed differentiation of P. hibernalis from the other five species. Species groupings determined by restriction analysis of ITS2 were consistent with those based on morphological criteria. These results show that restriction analysis of PCR-amplified TS2 regions can be useful as an adjunct to morphological criteria in Phytophthora species identification.     

额尔齐斯河鱼类双身虫科一种属新记录

研究报道了2009年4月至2013年5月采自新疆北部额尔齐斯河(中国段)东方欧鳊(Abramis brama orientalis Berg, 1949)鳃上的1种双身虫科(Diplozoidae Palombi, 1949)、双身虫亚科(Diplozoinae Palombi, 1949)、双身虫属(Diplozoon Nordmann, 1832)的奇异双身虫(Diplozoon paradoxum Nordmann, 1832)。所获标本与Nordmann (1832)的原始描述基本一致, 仅各部分度量等方面略有差异, 经比较鉴定, 为我国一新记录种。同时, 双身虫属在我国也尚属首次发现, 为我国一新记录属。研究对奇异双身虫的主要形态特征进行了描述并绘制了全图和特征图, 测定其ITS-2 rDNA序列并与GenBank同亚科虫种进行比对, 确定其与欧洲已发现的奇异双身虫相似度达到99.5%, 并对该寄生虫的地理分布特点进行了探讨。       

Differentiation ofMelampsora rust species on willows in Japan using PCR-RFLP analysis of ITS regions of ribosomal DNA

To develop a reliable method for identifyingMelampsora species parasitic on willows in Japan, we differentiated 10Melampsora species by PCR-RFLP analysis. Internal transcribed, spacer (ITS) regions, including 5.8S ribosomal DNA, of 63 collections of 10Melampsora species and 4 collections of unidentified species were amplified by PCR. The fragments from the 67 collections varied in size (approximately 880 bp, 860 bp and 840 bp). The restriction sites in the amplified DNA fragments were mapped after the RFLP analysis using four restriction enzymes,Dra I,EcoRI,SspI andTaqI. All the collections were divided into 11 RFLP types. In the 6 species,M. caprearum, M. epiphylla, M. kamikotica, M. larici-urbaniana, M. microsora andM. yezoensis, the RFLP type was species-specific. The RFLP type ofM. chelidonii-pierotii andM. coleosporioides was identical. The collections ofM. epitea were separated into three RFLP types. One of these three types was identical with the type ofM. humilis. It is suggested that the PCR-RFLP analysis of ITS regions is a useful and reliable method for species identification ofMelampsora. Contribution No. 131, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Molecular identification of house dust mites and storage mites

Mites are known causes of allergic diseases. Currently, identification of mites based on morphology is difficult if only one mite is isolated from a (dust) sample, or when only one gender is found, or when the specimen is not intact especially with the loss of the legs. The purpose of this study was to use polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) of the ITS2 gene, to complement the morphological data for the identification of mites to the species level. For this, six species were cultured: Dermatophagoides pteronyssinus, D. farinae, Blomia tropicalis, Tyrophagus putrescentiae, Aleuroglyphus ovatus and Glycycometus malaysiensis. Genomic DNA of the mites was extracted, quantified, amplified and digested individually with restriction enzymes. Hinf I and Ple I differentiated the restriction patterns of D. pteronyssinus and D. farinae. Bfa I and Alu I enzymes differentiated B. tropicalis and G. malaysiensis. Ple I enzyme was useful for the differentiation between T. putrescentiae and A. ovatus. Bfa I was useful for the differentiation of G. malaysiensis from the rest of the species. In conclusion, different species of mites can be differentiated using PCR–RFLP of ITS2 region. With the established PCR–RFLP method in this study, identification of these mites to the species level is possible even if complete and intact adult specimens of both sexes are not available. As no study to date has reported PCR–RFLP method for the identification of domestic mites, the established method should be validated for the identification of other species of mites that were not included in this study.     

Comparison of PCR-RFLP analysis of the ITS region with morphological criteria of various strains of Dunaliella

The genus Dunaliella comprises 28 species defined primarily by morphological and physiological criteria, which vary considerably depending on growth conditions. Concomitantly, the taxonomic status of various species is uncertain. To confirm the taxonomic identity and to better understand the relationship within Dunaliella, seven taxa ( D. salina, D. bardawil, D. tertiolecta, D. parva, D. viridis, D. lateralis, D. peircei) were compared using RFLP analysis of the nuclear rDNA repeats, specifically the internal transcribed spacer regions, including the 5.8S rRNA gene. Volvox aureus was used as an outgroup. A single ITS PCR amplification product was obtained for each taxon. An ITS fragment of ca. 640 bp was present in all the taxa within the subgenus Dunaliella, except for D. salina CCMP 1303 (ca. 540 bp) and D. lateralis (subgenus Pascheria) (ca. 600 bp). A cluster analysis based on the presence or absence of bands generated by digestion of the PCR product with 8 restriction endonucleases (DpnI, HhaI, EcoRI, PvuII, TaqI, HaeIII, MspI, StyI) revealed no correlation between the genetic relationship inferred from the ITS-RFLP data and the morpho-physiological attributes used for taxonomy. In addition, differences in morphology, physiology and in the length and restriction fragment patterns of the ITS region of D. salina CCMP 1303 suggest that this strain does not belong to Dunaliella. This revised version was published online in August 2006 with corrections to the Cover Date.     

Polymorphism in the internal transcribed spacer (ITS) region of the ribosomal DNA among different Fusarium species

Fusarium species causing wilt diseases in different plants were characterised by comparing nonpathogenic and different pathogenic species using rDNA RFLP analysis. The ITS (internal transcribed spacer) region of 12 isolates belonging to the section Elegans, Laseola, Mortiella, Discolor, Gibbosum, Lateritium and Sporotrichiella were amplified by universal ITS primers (ITS-1 and ITS-4) using polymerase chain reaction (PCR). Amplified products, which ranged from 522 to 565 bp were obtained from all 12 Fusarium isolates. The amplified products were digested with seven restriction enzymes, and restriction fragment length polymorphism (RFLP) patterns were analysed. A dendrogram derived from PCR-RFLP analysis of the rDNA region divided the Fusarium isolates into three major groups. Assessment of molecular variability based on rDNA RFLP clearly indicated that Fusarium species are heterogeneous and most of the forma speciales have close evolutionary relationships.     

Restriction fragment length polymorphism catalog for molecular identification of Japanese Tetranychus spider mites (Acari: Tetranychidae)

Species identification is a basic issue in biosecurity. Polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) is a useful molecular diagnostic tool for species identification. However, the lack of transferability of data has been a serious shortcoming of this method. A RFLP catalog, i.e., a graph of PCR-RFLP patterns expected from sequence data, was devised as a tool to facilitate PCR-RFLP data sharing among laboratories. Twelve species of Tetranychus spider mites have been recorded in Japan to date. In this study, we analyzed DNA sequences of the internal transcribed spacer (ITS) region in nuclear ribosomal DNA of 11 Tetranychus species. For the species identification using PCR-RFLP, we chose six candidates from 131 restriction endonucleases and developed an RFLP catalog of all known Japanese Tetranychus species except Tetranychus neocaledonicus André. The RFLP catalog revealed that most Tetranychus species had diagnostic restriction fragments. The RFLP catalog is transferable and simple molecular diagnostic tool, and it has the ability to add more species and newly found intraspecific variations. Therefore, we believe that the RFLP catalog will contribute to biosecurity as a practical diagnostic tool for species identification of spider mites.     

Species-specific polymorphisms in transcribed ribosomal DNA of fivePythium species

Twenty-five isolates representing fivePythium species collected from diverse hosts and geographic origins were evaluated using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis. DNA regions coding for the small-subunit ribosomal RNA (SrDNA) and the internal transcribed spacer (ITS) were amplified and analyzed by restriction enzyme digestion. The amplified SrDNA was about 1800 bp long and uniform in length among the five species. However, restriction digestion revealed three polymorphic groups. They areP. arrhenomanes andP. graminicola,P. irregulare andP. spinosum, andP. ultimum. The amplified-ITS region showed three different lengths which corresponded to the three polymorphic groups of SrDNA. Each length variant of the ITS showed distinct banding patterns after restriction enzyme digestion. In addition,P. irregulare andP. spinosum each showed distinct banding patterns after digestion with enzymesHinfI andMboI. Physical maps of the restriction sites in the SrDNA and the ITS were determined. Length variation occurred primarily in the spacer between the SrDNA and 5.8 S rDNA; although, it also was detected in the ITS-2 region. Little intraspecific variation was observed in the SrDNA and ITS, and species could be reliably distinguished by RFLP analysis of the amplified rDNA regions. Data presented do not support the maintenance ofP. arrhenomanes andP. graminicola as distinct species. Results indicate that PCR-RFLP can be used as a simple and speedy taxonomical tool for ecological studies ofPythium species.     

Simultaneous identification of Grapholita molesta Busck and Grapholita dimorpha Komai by PCR‐RFLP

Although several molecular diagnostic techniques are available for the identification of the apple‐feeding pests Grapholita molesta Busck and Grapholita dimorpha Komai, these pests are severely affecting apple orchards in Korea. These two pests may be misidentified or the available molecular diagnostic techniques may not facilitate the simultaneous identification of the morphological features of both species. In this study, we developed a multiplex assay for these two species using the polymerase chain reaction – restriction fragment length polymorphism (PCR‐RFLP) method. Sixty‐two specimens were collected from apples presumed infested with moth larvae and from pheromone traps from 2013 to 2014. Both species were identified morphologically, and a partial region of the cytochrome b gene was sequenced to design primers for PCR‐RFLP. Digestion profiles of G. molesta and G. dimorpha, using the Sau3A1 restriction enzyme, were characterized using three DNA fragments each for G. molesta (363 bp, 91 bp and 31 bp) and G. dimorpha (220 bp, 234 bp and 31 bp). The RFLP assay developed for both species in this study was more efficient and accurate than other currently used diagnostic assays and would be helpful to identify field‐collected specimens for pest control research.     

Mite species identification in the production of allergenic extracts for clinical use and in environmental samples by ribosomal DNA amplification

The identification of allergy‐causing mites is conventionally based on morphological characters. However, molecular taxonomy using ribosomal DNA (rDNA) may be particularly useful in the analysis of mite cultures and purified mite fractions in the production of allergenic extracts. Full‐length internal transcribed spacers (ITS1 and ITS2) were obtained from Dermatophagoides farinae, Dermatophagoides pteronyssinus, Dermatophagoides microceras and Euroglyphus maynei (Astigmata: Pyroglyphidae), Glycyphagus domesticus and Lepidoglyphus destructor (Astigmata: Glycyphagidae), Tyrophagus fanetzhangorum, Tyrophagus putrescentiae, Tyrophagus longior, Tyrophagus neiswanderi, Acarus farris and Acarus siro (Astigmata: Acaridae), and Blomia tropicalis (Astigmata: Echymopodidae), using mite‐specific primers. Polymerase chain reaction (PCR) products were digested with HpaII and RsaI restriction enzymes in order to produce species‐specific PCR restricted fragment length polymorphism (RFLP) profiles. A semi‐nested re‐amplification step was introduced before the RFLP in order to apply the method to environmental samples. Results demonstrate that rDNA sequences can be used for the unambiguous identification of mite species. The PCR–RFLP system allows the identification of species in purified mite fractions when the availability of intact adult mite bodies for morphological identification is limited. This reliable and straightforward PCR–RFLP system and the rDNA sequences obtained can be of use in the identification of allergy‐causing mite species.     

Identifying freshwater mussels (Unionoida) and parasitic glochidia larvae from host fish gills: a molecular key to the North and Central European species

Freshwater mussels (order Unionoida) represent one of the most severely endangered groups of animals due to habitat destruction, introduction of nonnative species, and loss of host fishes, which their larvae (glochidia) are obligate parasites on. Conservation efforts such as habitat restoration or restocking of host populations are currently hampered by difficulties in unionoid species identification by morphological means. Here we present the first complete molecular identification key for all seven indigenous North and Central European unionoid species and the nonnative Sinanodonta woodiana, facilitating quick, low-cost, and reliable identification of adult and larval specimens. Application of this restriction fragment length polymorphisms (RFLP) key resulted in 100% accurate assignment of 90 adult specimens from across the region by digestion of partial ITS-1 (where ITS is internal transcribed spacer) polymerase chain reaction (PCR) products in two to four single digestions with five restriction endonucleases. In addition, we provide protocols for quick and reliable extraction and amplification of larval mussel DNA from complete host fish gill arches. Our results indicate that this new method can be applied on infection rates as low as three glochidia per gill arch and enables, for the first time, comprehensive, large-scale assessments of the relative importance of different host species for given unionoid populations.     

PCR-RFLP of ITS rDNA for the rapid identification of Penicillium subgenus Biverticillium species

RFLP of ITS rDNA is proposed as a useful tool for molecular identification of the most common species of biverticillate penicillia. 60 isolates were analysed representing 13 species and 21 unique sequences were produced. The combination of five restriction enzymes was successful in separating 12 species. However, the variety Penicillium purpurogenum var. rubrisclerotium remained indistinguishable from Penicillium funiculosum. P. funiculosum appeared as the most confused species, being mis-identified with Penicillium miniolutum and Penicillium pinophilum, which were originally part of the species, and with P. purpurogenum perhaps because of the common production of red pigment. Penicillium variabile was difficult to investigate as introns were found on half of the isolates. Penicillium piceum, Penicillium rugulosum, Penicillium loliense, Penicillium erythromellis and P. purpurogenum were homogeneous from molecular and morphological positions and corresponded to a well circumscribed taxon. Furthermore, intraspecific variability was evidenced within P. pinophilum and P. funiculosum. The ex-type isolate of P. funiculosum produced a unique pattern. The method is sensitive, rapid and inexpensive and can be used for isolate identification of the biverticillate species. It is recommended particularly when many isolates have to be authentificated prior to analysis for phylogenetic assessment or population genetics.     

The use of the polymerase chain reaction and restriction fragment length polymorphism technique associated with the classical morphology for characterization of Lymnaea columella, L. viatrix, and L. diaphana (Mollusca: Lymnaeidae)

The specific identification of Lymnaeid snails is based on a comparison of morphological characters of the shell, radula, renal and reproductive organs. However, the identification is complicated by dissection process, intra and interspecific similarity and variability of morphological characters. In the present study, polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) techniques targeted to the first and second internal transcribed spacers (ITS1 and ITS2) rDNA and to the mitochondrial 16S ribosomal gene (16S rDNAmt) were used to differentiate the species Lymnaea columella, L. viatrix, and L. diaphana from some localities of Brazil, Argentina, and Uruguay as well as to verify whether the molecular results corroborates the classical morphological method.PCR-RFLP analysis of the ITS1, ITS2, and 16S using 12 restriction enzymes revealed characteristic patterns for L. columella and L. diaphana which were concordant with the classical morphology. On the other hand, for L. viatrix populations a number of 1 to 6 profiles were generated while morphology provided the species pattern results.     

Role of fimbriae as antigens and intestinal colonization factors of Salmonella serovars

The ascomycete Pulvinula constellatio was found to form competing ectomycorrhizal relationships with plants that had been inoculated with Tuber spp. and other edible ectomycorrhizal fungi. In order to be able to distinguish P. constellatio mycorrhizae from those of the Tuber spp., we determined the morphological and bio-molecular characteristics of P. constellatio. The complete sequence of the ITS regions was determined, in order to select specific primers. The ITS region was also studied using restriction fragment length polymorphism analyses with several restriction enzymes that allowed an unambiguous identification of the species.     

GENETIC DIVERGENCE CORRELATES WITH MORPHOLOGICAL AND ECOLOGICAL SUBDIVISION IN THE DEEP-WATER ELK KELP, PELAGOPHYCUS PORRA (PHAEOPHYCEAE)

Pelagophycus porra (Leman) Setchell has a narrow distribution confined to deep water from the Channel Islands off the southern California coast to central Baja California, Mexico. Distinct morphotypes are consistently correlated with distinctive habitats, that is, windward exposures characterized by strong water motion and rocky substrates, and sheltered areas with soft substrates found on the lee sides of the islands. We tested the hypothesis that morphologically and ecologically distinct forms reflect genetically distinct stands. Individuals representing populations from three islands and the mainland were compared using RFLP analyses of the nuclear rDNA internal transcribed spacers (ITS1 and ITS2), chloroplast trn L (UAA) intron sequences, and random amplified polymorphic DNA (RAPDs). No variation was found in a survey of 20 restriction sites of ITS1 (ca. 320 base pair [bp]) and ITS2 (ca. 360 bp) among individuals from six populations. Likewise, comparisons of trn L intron (241 bp) sequences among nine individuals from seven populations were identical with the exception of a CATAGT insert in two adjacent stands. A RAPD analysis of 24 individuals from nine populations (4 windward and 5 leeward) using 16 primers generated 166 bands. Thirty-eight percent of the bands did not vary, 16% were unique to a given individual, and 46% were variable. Neighbor joining analysis produced a well-resolved tree with moderately high bootstrap support in which windward and leeward populations were easily distinguished. The lack of divergence in both the fast evolving nuclear rDNA-ITS and the chloroplast trn L intron does not support the morphotypes as different species. However, the compartmentalized differentiation shown in the RAPD data clearly points to isolation. This, and previous ecological studies that demonstrate habitat specificity suggest that leeward stands probably comprise a species in statu nascendi.     

采用PCR-RFLP和RAPD对球壳孢目真菌系统学的研究

对球壳孢目真菌首次采用ＰＣＲ－ＲＦＬＰ和ＲＡＰＤ进行了系统发育研究。以ＩＴＳ１和ＩＴＳ４为引物对４属１２种２４个菌株的核糖体ＤＮＡ转录间区（ＩＴＳ）进行了ＰＣＲ扩增,４种内切酶（ＡｌｕＩ,Ｈｈａ,ＭｓｐＩ,ＴａｑＩ）酶切,结果表明：主要属的ＩＴＳ区长度不同,同属不同种相同。Ｃｏｎｉｏｔｈｙｒｉｕｍ,Ｐｈｙｌｏｓｔｉｃｔａ,Ａｓｃｏｃｈｙｔａ,ＳｅｐｔｏｒｉａＩＴＳ区长度分别为６３０,５６０,５５０,５４０ｂｐ;酶切图谱属间差别明显,属内种间基本一致,暗示传统的分种可能过细,某些种的成立还有商榷的余地,ＰＣＲ－ＲＦＬＰ对确定疑难种属的地位有重要的意义。３属８种１６个菌株的ＲＡＰＤ结果表明,种内图谱相同或图谱类型相同,而种间明显不同,揭示出过去所划分的种的确存在本质的差别,但是否达到种级的差别还有待于进一步研究     

Further studies on the molecular systematics of Biomphalaria snails from Brazil

The polymerase chain reaction and restriction fragment length polymorphism (RFLP) of the internal transcribed spacer (ITS) region of the rRNA gene, using the enzyme DdeI were used for the molecular identification of ten species and one subspecies of Brazilian Biomphalaria. Emphasis is given to the analysis of B. oligoza, B. schrammi and B. amazonica. The RFLP profiles obtained using this enzyme were highly distinctive for the majority of the species and exhibited low levels of intraspecific polymorphism among specimens from different regions of Brazil. However, B. peregrina and B. oligoza presented very similar profiles that complicated their identification at the molecular level and suggested a very close genetic similarity between the two species. Others enzymes including HaeIII, HpaII, AluI and MnlI were tested for their ability to differentiate these species. For B. amazonica three variant profiles produced with DdeI were observed. The study demonstrated that the ITS contains useful genetic markers for the identification of these snails     

Identification of internal transcribed spacer sequence motifs in truffles: a first step toward their DNA bar coding

This work presents DNA sequence motifs from the internal transcribed spacer (ITS) of the nuclear rRNA repeat unit which are useful for the identification of five European and Asiatic truffles (Tuber magnatum, T. melanosporum, T. indicum, T. aestivum, and T. mesentericum). Truffles are edible mycorrhizal ascomycetes that show similar morphological characteristics but that have distinct organoleptic and economic values. A total of 36 out of 46 ITS1 or ITS2 sequence motifs have allowed an accurate in silico distinction of the five truffles to be made (i.e., by pattern matching and/or BLAST analysis on downloaded GenBank sequences and directly against GenBank databases). The motifs considered the intraspecific genetic variability of each species, including rare haplotypes, and assigned their respective species from either the ascocarps or ectomycorrhizas. The data indicate that short ITS1 or ITS2 motifs (< or = 50 bp in size) can be considered promising tools for truffle species identification. A dot blot hybridization analysis of T. magnatum and T. melanosporum compared with other close relatives or distant lineages allowed at least one highly specific motif to be identified for each species. These results were confirmed in a blind test which included new field isolates. The current work has provided a reliable new tool for a truffle oligonucleotide bar code and identification in ecological and evolutionary studies.