Ribosomal Internal Transcribed Spacer Research Articles

Conventional and molecular diagnostics in onychomycosis-part2 : Molecular identification of causative dermatophytes by polymerase chain reaction and sequence analysis of the internal transcribed spacer region of ribosomal DNA

Dermatophyte identification using traditional methods such as optics-based direct fluorescence microscopy and culture is nowadays supplemented by molecular biological methods. The validity of dermatophyte DNA detection with direct uniplex-polymerase chain reaction-enzyme immunoassay (PCR-EIA) in nail samples was proven by sequence analysis of the ribosomal internal transcribed spacer (ITS) region. A total of 108dermatophytes, isolated from patients with onychomycosis, were positive for Trichophyton rubrum (TR) and Trichophyton interdigitale (TI) in culture and/or uniplex-PCR-EIA. Conventional methods for dermatophyte identification were complemented by direct uniplex-PCR-EIA and sequence analysis of the ribosomal ITS region (18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA). Of 108 patients (average age62, median age 73), 56showed cultural growth with 31of them being identified as TR and23 as TI. There was high agreement with the sequence analysis. Surprisingly, the pathogen of asingle nail sample was identified as T.quinckeanum (formerly T.mentagrophytes sensu stricto), arare zoophilic dermatophyte in Germany. Asingle TI strain turned out to be amisidentified T.tonsurans based on the sequence analysis. In all, 34of the 52specimens lacking cultural growth were detected by PCR as TR, and 18specimens could be identified as TI. The results of dermatophyte identification of culture-negative nail samples were also in agreement with the results of sequence analysis. Molecular biological methods are well applicable, and they show high reliability for direct dermatophyte identification in nail samples without prior cultivation. Especially for nail samples without cultural growth, PCR-based dermatophyte identification was highly specific and sensitive.

Molecular prevalence of Enterocytozoon bieneusi in stray cats of İzmir, Türkiye

The phylum Microsporidia contains obligate single celled parasites that can infect many vertebrate hosts including humans. Enterocytozoon bieneusi is considered as the most diagnosed species in humans. E. bieneusi has also been detected in many animals such as cats, dogs and cattle. Among these animals, cats are carriers of type D and IV which are the most common human pathogenic genotypes of E. bieneusi. In Türkiye, the prevalence of E. bieneusi in stray cats is not well known. Therefore, in this study, the molecular prevalence of E. bieneusi in stray cats (n = 339) was determined by Real-Time PCR targeting ribosomal DNA ITS (internal transcribed spacer) region of E. bieneusi. Initially, the analytical sensitivity of Real-Time PCR was determined by a plasmid control and then E. bieneusi DNA was investigated in fecal samples of stray cats. The results showed that the analytical sensitivity of Real-Time PCR targeting ITS region of E. bieneusi was ≤1 copy plasmid/reaction. Analysis of fecal samples revealed that the molecular prevalence of E. bieneusi was 50.15% (170/339). Overall, these results showed that the Real-Time PCR successfully detected E. bieneusi in cat’s fecal samples and stray cats can be an important source for transmission of E. bieneusi to humans and other animals.

Astragalicola ephedrae sp. nov., isolated from the stem of Ephedra gerardiana in Ladakh, India.

An endophytic fungus designated as EIT4T (MCC 9756T) was isolated from the asymptomatic stem tissue of Ephedra gerardiana collected from the Kargil district of Ladakh Union territory, India. Phylogenetic analysis based on concatenated nuclear ribosomal ITS (internal transcribed spacer) and LSU (large ribosomal subunit) sequence datasets revealed its placement within the genus Astragalicola. However, it formed a separate clade exhibiting strong bootstrap support value (80%). The highest nrITS sequence similarity between EIT4T and species of Astragalicola was 95.19% (A. vasilyevae) and 94.26% (A. amorpha), while nrLSU sequence similarity was 99.27% (A. amorpha). Morphologically, EIT4T differs from the other species of Astragalicola in having larger sub-globose to pyriform conidiomata, smaller and mostly unbranched conidiophores, and polymorphic translucent conidia with two terminal guttules. Based on combined cultural, micromorphological, molecular, and phylogenetic analyses, EIT4T represents a novel species in the genus Astragalicola proposed here as Astragalicola ephedrae sp. nov. Detailed description and illustrations of the novel species are provided. The type strain is EIT4T (= MCC 9756T = MN29T).

Internal Transcribed Spacer (ITS) Region as Molecular Marker for Parasite Identification in Aquaculture: A Review

Molecular technologies may be quicker and more accurate than the culture, serology, and histology procedures that have historically been employed to identify fish parasites. By using molecular diagnostic methods, parasites can be found in asymptomatic fish, allowing disease outbreaks to be stopped. This review discusses the use of Internal Transcribed Spacer (ITS) markers to detect parasite in aquaculture and their potential applications are discussed. It was found that it is a more reliable marker and species identification in combination with other DNA markers. ITS region are highly polymorphic non - coding region with sufficient taxonomic units. Thus, sequences can be separated at the species level. though ribosomal ITS as a universal barcode marker for parasite identification is still hampered by few limitations, the ITS will remain as the key choice for parasite identification. It remains the most important option for parasite identification. Many studies have been carried out using ITS markers for parasites identification such as cestode, nematodes, Pseudoterranova spp., Anisakis type I, Diplostomum, Gyrodactylids Hysterothylacium sp, Glochidia larvae. This identification of parasites are taken from the species Rainbow trout (Oncorhynchus mykiss), Argentinesea bass, (Acanthistius patachonicus), and other samples from various places.

Comparison of two strains of the edible cyanobacteria Arthrospira: Biochemical characterization and antioxidant properties

Arthrospira is a cyanobacteria broadly cultivated worldwide due to its high nutritional value, metabolites with therapeutic properties, non-toxic character and easy culture. The genus Arthrospira has been historically confused with the genus Spirulina (with potential toxicity) therefore, an accurate taxonomic identification of strains for food applications, is essential. In this study we compared two strains of Arthrospira with remarkable morphological differences, preliminary classified as Arthrospira maxima (CCM-UdeC 040) and Arthrospira platensis (CCM-UdeC 136). Despite of their morphological differences, we confirmed that both strains belong to the same genetic species, according to their ribosomal ITS (internal transcribed spacer) sequences. We also compared the biochemical composition of both strains, including analysis of antioxidant properties of their aqueous protein extracts, both crude, as well as, hydrolyzed with commercial proteases. Biotechnological potential of these strains as raw material for nutraceutical and functional food markets is discussed. Strain CCM-UdeC 040 showed the highest biotechnological potential so further studies should be conducted for optimizing its culture conditions.

First Report of Dieback Caused by Neofusicoccum batangarum in Cashew in Guinea-Bissau.

Cashew (Anacardium occidentale L.) is a cash crop with a highly significant economic importance in West Africa, particularly in Guinea-Bissau (Monteiro et al. 2015, 2017). In October 2018, dieback-like symptoms such as wilt and necrosis of apical shoots were observed in 10 % of the cashew trees grown in a 100 plant-orchard in Bolama Island at Bijagós archipelago, Guinea-Bissau. Six symptomatic apical shoots from individual plants were collected for fungal isolation and identification. Tissue pieces (3 × 2 mm) from healthy to diseased margins were surface sterilized with 1 % sodium hypochlorite, washed twice with sterilized water, placed on potato dextrose agar (PDA, Difco® Laboratories) supplemented with potassium thiocyanate (50 µg/ml), and incubated at 24 ± 1 °C in the dark for 7 days. Four fungal colonies were isolated (67 %) and purified through hyphal tips removal, displaying rapid growth rate, and aerial mycelia that initially was white, turning later to dark greenish on PDA. Pycnidia produced on 1.5 % water agar and sterilized pine needles (± 25ºC; near-UV light) were solitary, covered by mycelium, obpyriform to ampulliform (152.5 ± 41.6 × 135.2 ± 30.8 μm, n = 30). Conidia were unicellular, hyaline, smooth, fusoid to ovoid, thin-walled, measuring 16.21 ± 1.52 × 5.84 ± 0.66 μm (n = 50, L/W 2.8). Such morphological features are characteristic of Neofusicoccum spp. (Phillips et al. 2013). For molecular identification, genomic DNA was extracted from a representative isolate GB160 and partial regions of ribosomal internal transcriber spacer (ITS) (ITS1/ITS4; White et al. 1990), translation elongation factor 1-α (EF1-α) (EF1-688F/EF1-1251R; Alves et al. 2008) and β-tubulin (β-tub) (Bt2a/Bt2b; Glass and Donaldson 1995) genes were amplified as previously described, respectively, with BSA (50 mg/ml). Amplicons were sequenced and deposited in GenBank (ITS, MN952993; EF1-α, MN952204; β-tub, MN952208). BLAST analysis of ITS, EF1-α and β-tub gene sequences showed 100 % identity with Neofusicoccum batangarum reference strain CBS124923 (FJ900608, FJ900654, FJ900635, respectively). Maximum-likelihood and Bayesian Inference analyses from the concatenated dataset placed GB160 isolate within the N. batangarum cluster (BS = 72 %; PP = 0.95). For pathogenicity assessment, 3-month-old cashew "Caju di Terra" plants (n = 8) grown in a greenhouse under controlled conditions were inoculated following a randomized block design as described by Lima et al. (2013). Briefly, 3 mm diam. stem tissue bark was removed and replaced with a 3 mm diameter PDA plug retrieved from the colony margin. Inoculation wound was covered with sterilized wet cotton and sealed with parafilm. Eight control plants were only treated with PDA plugs and the wound covered and sealed as described. After 15 days, all inoculated plants displayed similar symptoms to those observed in the field, and vascular lesions (10.8 ± 4.0 cm), whereas control plants remained symptomless. Koch's postulates were fulfilled by successful re-isolation of the pathogen from all inoculated stems and identification by morphology and gene sequencing. N. batangarum was identified associated with Anacardium spp. in Brazil (Netto et al. 2017) and recently reported as causing grapevine dieback in Brazil (Rêgo et al. 2020). To our knowledge, this is the first report of N. batangarum causing cashew dieback in Guinea-Bissau and West Africa. Occurrence of this disease may represent a significant impact for cashew production since this crop is the major agricultural commodity in Guinea-Bissau.

Genetic variation and demographic history analysis of Pestalotiopsis, Pseudopestalotiopsis, and Neopestalotiopsis fungi associated with tea (Camellia sinensis) inferred from the internal transcribed spacer region of the nuclear ribosomal DNA

AbstractTo elucidate the demographic history within and among the genera Pseudopestalotiopsis, Neopestalotiopsis, and Pestalotiopsis and their genetic diversity, we studied internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA (nrDNA) with no saturation effects in 172 Pestalotiopsis‐like fungal accessions isolated from Camellia sinensis planted in nine provinces in China (64 from Yunnan, 4 from Guangxi, 2 from Guizhou, 42 from Jiangxi, 3 from Sichuan, 3 from Zhejiang, 1 from Henan, 18 from Fujian, 12 from Chongqing, and 18 from other provinces) and in Japan (1), France (1), Turkey (1), and Thailand (2). Among these, 44 Pestalotiopsis‐like isolates from Yunnan in this study formed a clade in phylogenetic trees with Pestalotiopsis accessions in GenBank, despite their cultural characteristics on potato dextrose agar displaying no genus specificity. Based on estimated sequence polymorphism parameters of average pairwise differences (K), haplotype diversity (Hd), nucleotide diversity (Pi), θ (per site) from η, and recombination event rate (Rm), we identified moderate genetic variation in these three genera. Molecular evolution analysis with neutrality tests and mismatch distribution demonstrated that Pseudopestalotiopsis and Pestalotiopsis ribosomal ITS sequences evolved under a neutral model. However, the recent demographic expansion of the three genera promoted geographical isolation but showed no clear geographical structure. Pairwise genetic divergences between haplotypes and a median‐joining network revealed Yunnan or Jiangxi as a possible centre of origin, and that Pseudopestalotiopsis and Neopestalotiopsis may derive from Pestalotiopsis.

<p><strong><em>Urocystis piptatheri</em></strong><strong> sp. nov. (Urocystaceae), a new species of smut fungus on <em>Piptatherum laterale</em> (Poaceae) from Pakistan</strong></p>

A new species of smut fungi, Urocystis piptatheri is described from Kumrat Valley, Pakistan based on morphological characters and molecular phylogenetic analysis of nuclear ribosomal DNA comprising the internal transcribed spacer (ITS) regions along with large subunit (LSU). The new species is characterized by the large size of its spore balls, size and number of central spores, spore wall thickness, sori in leaves and by the host plant species (Piptatherum laterale). Phylogenetic analysis of nuclear ribosomal ITS and LSU nucleotide sequences confirmed the novelty of Urocystis piptatheri. A key for the identification of Urocystis species on Pipthatherum spp. is provided.

DNA detection of Gyrodactylus spp. in skin mucus of Nile tilapia Oreochromis niloticus

Monogeneans Gyrodactylus von Nordmann 1832, cause outbreaks of gyrodactylosis in aquaculture settings worldwide. Detection of Gyrodactylus spp. is based on the morphological identification of isolated parasites after fish necropsy. Contributing to the diagnosis of gyrodactylosis, in this study, a non-destructive PCR assay was standardized; the PCR was first performed using genomic DNA of Gyrodactylus spp. isolated from the surface of the Nile tilapia Oreochromis niloticus (Linnaeus 1758), and subsequently tested with mucus samples of infected and uninfected Nile tilapia fish. The primers (Ekgyro1) were designed from the ribosomal Internal Transcriber Spacer (ITS) RNA region (ITS1, 5.8S and ITS2 rRNA gene) of Gyrodactylus cichlidarum Paperna 1968. The positive control group included the DNA of 30 monogeneans Gyrodactylus spp. The heterologous control group included 75 monogeneans Cichlidogyrus Paperna 1960, 75 protozoans Ichthyophthirius multifiliis Fouquet 1876 and 75 Trichodina Ehrenberg 1830. PCR products of each parasite and from the external mucus samples (described as P and M respectively), were sequenced. The average DNA concentration of the ectoparasites was of 13.5 ng/μl. The PCR test had an analytical sensitivity of 0.0039 ng μl-1 of DNA of Gyrodactylus spp. No cross-reactions were observed with the heterologous group. The sensitivity and specificity of the PCR test were of 100% either with genomic DNA or with DNA from mucus samples. Six DNA consensus sequences with sizes ranging from 568 bp to 571 bp were obtained and the BLAST analysis matched with DNA sequences of G. cichlidarum.

Molecular Identification of Human Sporotrichosis in Korea

Background: Sporotrichosis is a common deep mycosis caused by the Sporothrix schenckii complex. Until 2016, no molecular studies had been conducted on these fungi, and all the included strains were reported as S. schenckii. However, investigations conducted in northeast China, Japan, and India revealed that S. globosa was the most prevalent Sporothrix species, whereas S. schenckii sensu stricto was reported very rarely. Objective: To investigate the accurate prevalent causative species of sporotrichosis among strains reported as S.schenckii in Korea. Methods: We isolated strains of Sporothrix spp. Prevalent in Korea from fungus collection centers or private collections and reviewed the available literature on molecular studies of strains from this region. We found five S. schenckii (1998-2016) and three S. globosa (2016-2018) strains. Ribosomal DNA internal transcribed spacer (ITS) sequences of these strains were compared with those of the S. schenckii complex strains. Results: The ribosomal ITS sequences of the eight strains were 100% identical with that of S. globose. No S. schenckii sensu stricto was found. In addition, a study on the molecular analysis of Korean S. schenckii published by Ishizaki et al. (2004) demonstrated that the eight strains were of the mitochondrial subtype group B (S. globosa). Thus, all the 16 strains examined within the Korean S. schenckii complex were determined to be S. globosa. Conclusion: In summary, S. globosa is the causative species within the tested Korean sporotrichosis cases reported between 1998 and 2018. Based on our analyses, S. globosa, and not S. schenckii, may be the predominant species in Korea.

Phenotypic and molecular analysis of Mexican Metarhizium anisopliae strains

Metarhizium anisopliae is an anamorphic fungus widely studied due to its use as a biological control agent. In Mexico, it is mainly used as an integrated management strategy to control populations of spittlebugs (Hemiptera: Cercopidae) that affect sugarcane fields and cattle pastures. In this study, 13 strains isolated from Aeneolamia spp. (Hemiptera: Cercopidae) from the main areas of sugarcane production of Mexico and 11 reference strains of Metarhizium from different insect hosts and geographic origins were evaluated based on their conidial dimensions, thermotolerance at 15, 25, 30, and 35 oC, random amplification of polymorphic DNA (RAPD) patterns using a combined analysis with 14 different oligonucleotides and diversity estimators, and a phylogenetic analysis with the ribosomal RNA internal transcribed spacer (ITS) region. All strains used in this study showed typical morphological characteristics corresponding to M. anisopliae and were able to grow at 25 and 30 oC with restricted growth at low (15 oC) and high temperatures (35 oC). Polymorphism analysis clustered all strains from Aeneolamia sp. in one welldefined group with low variability among them. Phylogenetic relationships based on nuclear ribosomal ITS region sequences recovered a subclade within M. anisopliae formed by 10 of the Mexican strains.

Sequence analysis and molecular evolution of Tunisian date palm cultivars (Phoenix dactylifera L.) based on the internal transcribed spacers (ITSs) region of the nuclear ribosomal DNA

Sequences of the internal transcribed spacers (ITSs) of nuclear ribosomal DNA (nrDNA) were studied in 15 Tunisian date palm accessions (Phoenix dactylifera L.) in order to establish genetic relationships among date palm cultivars and to elucidate its molecular evolution. The length size of the ribosomal intergenic spacer ranged from 441 bp and 445 bp. Variation of GC contents has been scored as 45.6–46.5% in the ITS region. The overall transition/transversion bias R is 4.375 indicating that the transitions are more frequent than transversions at this region. The aligned Sequences allowed us to identify 4 haplotypes among the 15 examined cultivars. Intraspecific variation has been occurred in sequences of the internal transcribed spacers (ITSs) of date palm. The haplotypic and nucleotide diversities appeared low. Phylogenetic trees generated using distance (Neighbor-joining) and maximum parsimony (MP) methods supported significantly the organization of the tested accessions that are ordered independently of the tree’s sex and the chlorotype classification. This gives evidence of an unstructured geographic pattern among the studied cultivars. Molecular evolution analysis demonstrates that the ribosomal intergenic spacer ITS evolved under a strictly neutral model. A stable population of Tunisian date palm seems to be retained within the studied genotypes. Our results indicate that the diversity detected among closely related genotypes supported strongly the efficiency of ITS sequences for establishing relationships between cultivars and determining the evolutionary history of this species.

Aureobasidium mangrovei sp. nov., an ascomycetous species recovered from Hara protected forests in the Persian Gulf, Iran

A new ascomycetous black yeast-like species was recovered from healthy plant (Avicennia marina) of Hara protected mangrove forests at Qeshm Island, Iran. Morphological, physiological analysis as well as a molecular analysis of the internal transcribed spacer (ITS) and partial large ribosomal subunit (D1/D2 domains) confirmed the placement of this strain in the genus Aureobasidium and based on considerable sequence divergence, distinguishable cardinal growth temperatures and salt tolerance a new species Aureobasidium mangrovei sp. nov. is proposed. However, the type strain micro-morphologically is not clearly distinguishable from other members of the genus. The type strain, Aureobasidium mangrovei was preserved in a metabolically inactive state at the Iranian Biological Resource Centre, Tehran, Iran as IBRC-M 30265T and the ex-type culture is deposited in the CBS yeast collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands as CBS 142205T. The GenBank accession numbers for the nucleotide sequences of the large subunit ribosomal DNA and ITS region are KY089084 and KY089085, respectively. The MycoBank number of the new species is MB 823444.

Phylogenetic study of the endemic species Oxytropis almaatensis (Fabaceae) based on nuclear ribosomal DNA ITS sequences

BackgroundOxytropis almaatensis Bajt. is a rare, narrow endemic species of the Trans-Ili Alatau mountains in Kazakhstan. Up to now, no studies regarding the taxonomy and variation of key morphological traits of O. almaatensis were undertaken. The purpose of this analysis was to evaluate phenotypic variation of O. almaatensis and assess the position of the species within the genus based on nucleotide sequences of the nuclear ribosomal DNA internal transcribed spacer (ITS) region.ResultsTwo populations of O. almaatensis were collected in neighboring gorges of the Trans-Ili Alatau Mountains. The ITS sequences from the samples of two populations of O. almaatensis were identical. The phylogenetic analysis indicated that O. almaatensis is within Oxytropis genetically close to O. glabra as these species formed a separate subclade. The phenotypic variation of populations was assessed using nine morphological traits and compared to descriptions of O. glabra. The range of variation for the traits between two populations was established. A clear morphological difference of O. almaatensis and O. glabra was found in peduncle length to leaf length ratio. This was in O. almaatensis 1.56, while in O. glabra, it was 1.0.ConclusionsThe study provides the first phenotypic description and phylogenetic placement of the rare endemic species O. almaatensis. The morphological traits in two O. almaatensis populations showed a high level of phenotypic variability. Although clearly different from O. glabra, the ITS phylogeny grouped these species in a subclade within the genus.

Amanita glarea, a new species in section Vaginatae from Pakistan

A new species, Amanita glarea, is described and illustrated from Pakistan. Molecular phylogenetic data derived from internal transcribed spacer (ITS) and nuclear ribosomal large subunit (LSU) regions along with morphological characters make it distinct from other currently known Amanita species. Amanita glarea is characterized by a grayish brown, slightly umbonate, pileus having universal veil remnants and striate margins, a long ringless stipe with a saccate volva bearing yellowish brown patches on the verrucose surface, inamyloid globose to subglobose basidiospores, and a filamentous pileipellis with some inflated round to elongated cells. Phylogenetic analysis of nuclear ribosomal ITS and LSU nucleotide sequences resolve A. glarea with other taxa in Amanita section Vaginatae.

Phylogenetic congruence of ribosomal operon and plastid gene sequences for the Characeae with an emphasis on Tolypella (Characeae, Charophyceae)

:To clarify species diversity of Tolypella (Characeae, Charophyceae) and the relationship between Tolypella and other genera in Characeae, sequence data from the nuclear ribosomal operon were assembled for 18 individuals of Tolypella. These represented nine named species and two potentially new species from Australia, as well as 13 other taxa of Characeae. Phylogenetic analyses were performed on ribosomal gene sequences (18S, 5.8S and 28S) for Characeae and on ribosomal gene and internal transcribed spacer (ITS) sequences (ribosomal genes + ITS1 + ITS2) for Tolypella. Analyses were also performed on the ribosomal gene and ITS sequences combined with plastid gene sequences (atpB, psbC and rbcL). Ribosomal gene sequences alone were conservative and their phylogenetic analyses resulted in topologies similar to a previously published plastid gene-based study but with less support and resolution. In contrast, ITS sequences showed greater variability and their addition resulted in greater resolution among some species relationships of Tolypella. The addition of plastid sequences also resulted in greater support and increased resolution in some lineages but not all.

Molecular and morphological studies disentangle species complex in Euphorbia sect. Esula (Euphorbiaceae) from Iran, including two new species

Southwest Asia is one of the most important centers of diversity for leafy spurges (Euphorbia subgen. Esula), and Iran is one of the richest countries both for species and endemics in the subgenus. Section Esula, the second largest of the 21 sections of this subgenus, comprises c. 96 taxonomically and phylogenetically poorly understood species with complicated patterns of morphological variation. The Euphorbia osyridea alliance is an Iranian-centered group harboring the well-known endemic and semi-endemic species, E. buhsei, E. osyridea, and two poorly understood and yet undescribed taxa. In a phylogenetic framework based on nuclear ribosomal ITS (internal transcribed spacer) and plastid ndhF sequences, inter-simple sequence repeat (ISSR) polymorphisms were used to investigate genetic structure and genetic diversity in seven populations of the endemic and semi-endemic species of E. osyridea alliance. Phenetic analysis (principal component analysis) was used to assess morphological differentiation of the alliance. Neither nuclear nor plastid sequences provided sufficient resolution to disentangle the diversification patterns within the E. osyridea alliance. The combined evaluation of phylogenetic study, ISSR analysis, and morphological data confirmed that studied populations of E. osyridea alliance can be divided into four different groups including two new endemic species, E. khabrica sp. nov. and E. austro-iranica sp. nov. Typification, description, data on distribution and habitats, key to species as well as pertinent comments are given.

Internal Transcribed Spacer (ITS) as Dna Barcoding to Identify Fungal Species: a Review

Despite the fact that fungi are important sources of both bioactive compounds and mycotoxins, and that they are very ubiquitous in our environment, their species identification is hampered by incomplete and often unclear literature. Fungi identification is primarily based on their phenotypic and physiological characteristics. Nowadays, many molecular methods to identify fungal species have been developed. One of the methods considered as a new concept to rapidly and accurately identify unknown fungal sample is DNA Barcoding. This literature review will outline the use of DNA barcoding approach to rapidly identify fungal species and the use of ITS region that recently has been designated as primary DNA barcode for fungal kingdom. “DNA barcode” is a short, highly variable and standardized DNA region with approximately 700 nucleotides in length, which is used as a unique pattern to identify living things. Internal Transcribed Spacer (ITS) region of nuclear DNA (rDNA) has become the most sequenced region to identify fungal taxonomy at species level, and even within species. ITS region is a highly polymorphic non-coding region with enough taxonomic units. Therefore, it is able to separate sequences into species level. Even though ribosomal ITS as a universal barcode marker for fungi is still hampered by few limitations, the ITS will remain as the key choice for fungal identification. The search for alternative regions as DNA marker to improve fungal identification, especially in specific heredities, has already started.

First Report of Neoscytalidium dimidiatum Causing Stem and Fruit Canker of Hylocereus undatus in Florida

HomePlant DiseaseVol. 100, No. 7First Report of Neoscytalidium dimidiatum Causing Stem and Fruit Canker of Hylocereus undatus in Florida PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Neoscytalidium dimidiatum Causing Stem and Fruit Canker of Hylocereus undatus in FloridaG. Sanahuja, P. Lopez, and A. J. PalmateerG. SanahujaSearch for more papers by this author, P. LopezSearch for more papers by this author, and A. J. PalmateerSearch for more papers by this authorAffiliationsAuthors and Affiliations G. Sanahuja P. Lopez A. J. Palmateer , Tropical Research and Education Center, University of Florida, Homestead, 33031. Published Online:31 Mar 2016https://doi.org/10.1094/PDIS-11-15-1319-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Pitaya, commonly known as dragon fruit, is a cactus widely grown throughout south Florida for its edible fruit. Two species of pitaya are produced in south Florida: Hylocereus undatus has fruit with white flesh and scarlet colored skin and H. polyrhizus has red flesh with scarlet colored skin. In July 2015, a number of fruit samples of Hylocereus undatus submitted to the Florida Extension Plant Diagnostic Clinic in Homestead, FL, were discovered to have small cankers that appeared as sunken orangish brown spots. Dark gray to black aerial mycelium grew after surface-disinfected lesions were plated on acidified potato dextrose agar (APDA) and incubated at 25°C for 7 days. Colonies produced abundant conidia in arthric chains that were dark brown, cylindrical to round with zero to one-septa and averaged 9.29 ± 1.56 μm × 5.93 ± 0.66 μm (Crous et al. 2006). Genomic DNA was extracted from mycelium and partial 18S ribosomal RNA gene, complete internal transcribed spacer (ITS) 1, 5.8S ribosomal RNA gene, ITS 2, and partial 28S ribosomal RNA gene was amplified with the primers ITS1 and ITS4 and the PCR product was sequenced. The resulting sequence was deposited in GenBank (Accession No. KT803701). Based on morphological characteristics and the ITS sequence region, the pathogen was identified as Neoscytalidium dimidiatum. A BLAST search exhibited 97% nucleotide identity with an isolate of Neoscytalidium dimidiatum that caused pitaya canker disease in China (Accession No. JX524168). To further support identification, phylogenetic analysis by the maximum likelihood method (Tamura-Nei model) was performed (MEGA 6.06) and our isolate clustered with other N. dimidiatum isolates with 74% support (1000 replicates). Pathogenicity tests were conducted inoculating healthy stem and fruit of both species (H. undatus and H. polyrhizus) with 2 mm agar plugs from a 5-day old APDA culture. Two noninoculated controls were included; one with 2-mm agar plugs and one without. Five fruit and five stems were used per treatment and the experiment was repeated. Inoculated fruit and stems were placed in a greenhouse where temperature ranged from 25 to 32°C. Seven days post inoculation, both stem and fruit tissue developed small circular sunken light-brown spots. Neoscytalidium dimidiatum was isolated from all symptomatic tissue confirming Koch’s postulates. No symptoms developed on the noninoculated fruit and stem controls. This disease has been reported in Taiwan, Malaysia, and China (Chuang et al. 2012; Mohd et al. 2013; Lan and He 2012), but this is the first report of N. dimidiatum causing stem and fruit canker on Hylocereus undatus in Florida. It is important to note that Hylocereus polyrhizus is also susceptible to N. dimidiatum. Disease incidence in several local fields has been as high as 70% and appears to be more severe on fruit than stems. Very few fungicides are currently labeled for use on pitaya in Florida, so this disease has been a serious issue for pitaya growers in south Florida.

DNA barcoding of East Asian Amentotaxus (Taxaceae): Potential new species and implications for conservation

AbstractDNA barcoding is a useful tool for species identification using standardized genomic DNA fragments. The genus Amentotaxus, consisting of five or six species, is confined to South China, Northeast India, Laos, and Vietnam. All species have been assessed as globally or nationally threatened. However, there is uncertainty about the number of species involved, especially in the border areas of southern China, northern Vietnam, and Laos. We selected five DNA barcodes (rbcL, matK, trnH‐psbA, trnL‐F, and internal transcribed spacer (ITS)) to evaluate their discrimination ability in this genus, and to investigate the current taxonomy of Amentotaxus. Our results indicate that all the selected barcoding regions showed a high level of universality for PCR and sequencing. When six species are recognized, the nuclear ribosomal DNA region ITS and the chloroplast DNA region trnL‐F used on their own provided the highest identification success (60%). Two barcode combinations that included either ITS or trnL‐F had the same species discrimination ability. Combinations using additional barcodes did not improve the species identification success. When only five species are recognized, with A. hatuyenensis T. H. Nguyen treated as a synonym of A. yunnanensis H. L. Li, the discrimination rate rises to 100%. Our results also indicate that recent collections from Yunnan province, China, Lao Cai province, Vietnam, and Laos may represent a potential new species. The findings from this study will be very useful for the formulation of appropriate conservation strategies for threatened Amentotaxus species in national and trans‐boundary regions.