Internal Transcribed Spacer Ribosomal DNA Research Articles

Development of PCR-Multiplex Assays for Identification of the Herpotrichiellaceae Family and Agents Causing Chromoblastomycosis.

The Herpotrichiellaceae family is an important group of dematiaceous filamentous fungi, associated with a variety of pathogenic fungal species causing chromoblastomycosis (CBM) and phaeohyphomycosis (PHM), both with polymorphic clinical manifestations and worldwide incidence. Currently, the identification of this family and determination of the causative agent is challenging due to the subjectivity of morphological identification methods, necessitating the use of molecular techniques to complement diagnosis. In this context, genetic sequencing of the Internal Transcribed Spacer (ITS) has become the norm due to a lack of alternative molecular tools for identifying these agents. Therefore, this study aimed to develop PCR-Multiplex methodologies to address this gap. Sequences from the ITS and Large Subunit (LSU) of ribosomal DNA were used, and after manual curation and in vitro analyses, primers were synthesized for the identification of the targets. The primers were optimized and validated in vitro, resulting in two PCR-Multiplex methodologies: one for identifying the Herpotrichiellaceae family and the bantiana clade, and another for determining the species Fonsecaea pedrosoi and Fonsecaea monophora. Ultimately, the assays developed in this study aim to complement other identification approaches for these agents, reducing the need for sequencing, improving the management of these infections, and enhancing the accuracy of epidemiological information.

Clinical spectrum, phenotypic and molecular characterization, and antifungal susceptibility of an emerging human pathogen, Acrophialophora, from India.

Acrophialophora is implicated in superficial and invasive infections, especially in immunosuppressed individuals. The present study was undertaken to provide clinical, microbiological, phylogenetic, and antifungal susceptibility testing (AFST) profile of Acrophialophora isolated from India. All the isolates identified as Acrophialophora species at the National Culture Collection for Pathogenic Fungi, Chandigarh, India were revived. Phenotypic and molecular characterization was performed, followed by temperature studies, scanning electron microscopy (SEM), and AFST. We also performed systematic review of all the cases of Acrophialophora species reported till date. A total of nine isolates identified as Acrophialophora species were identified by molecular method as A. fusispora (n=8) and A. levis (n=1), from brain abscess (n=4), respiratory tract (n=3), and corneal scraping (n=2). All patients but two had predisposing factors/co-morbidities. Acrophialophora was identified as mere colonizer in one. Temperature studies and SEM divulged variation between both species. Sequencing of the internal transcribed spacer ribosomal DNA and beta-tubulin loci could distinguish species, while the LSU ribosomal DNA locus could not. AFST showed the lowest minimum inhibitory concentrations (MICs) for triazoles and the highest for echinocandins. Systematic literature review revealed 16 cases (11 studies), with ocular infections, pulmonary and central nervous system infections, and A. fusispora was common species. All the patients except three responded well. High MICs were noted for fluconazole, micafungin, and caspofungin. This is the first study delineating clinical, phenotypic, and genotypic characteristics of Acrophialophora species from India. The study highlights microscopic differences between both species and emphasizes the role of molecular methods in precise identification. Triazoles appear to be the most effective antifungals for managing patients.

Bioprospecting <i>Saccharomyces cerevisiae</i> in fruits from Amazonian region for beer brewing

<abstract> <p>This research aimed to isolate <italic>Saccharomyces cerevisiae</italic> strains from Amazonian fruits for potential utilization in beer production. Yeast strains were derived from the spontaneous fermentation of Arazá (<italic>Eugenia stipitata</italic> MacVaught), cocoa (<italic>Theobroma cacao</italic> L.), and cupuassu (<italic>Theobroma grandiflorum</italic> Wild. Ex Spreng. Schum) fruits. Identification of the isolated strains was achieved through biochemical assays and ITS rDNA (Internal Transcribed Spacer ribosomal DNA), sequencing, with emphasis on determining their affiliation to <italic>S. cerevisiae</italic> and assessing phylogenetic ties. Out of the 76 colonies isolated from the fruit fermentations, seven were distinctly identified as <italic>S. cerevisiae</italic>. Phylogenetic assessments unveiled significant parallels between regional isolates and commercial strains. Notably, beer brewed with the <italic>S. cerevisiae</italic> AR 03 isolate exhibits physical-chemical attributes characteristics similar to those found in American ale commercial beers. These findings underscore the untapped potential of leveraging Amazonian yeasts in brewing, a step forward for the region's burgeoning bioeconomy.</p> </abstract>

Fusarium equiseti as the pathogen causing stem rot disease of red-fleshed dragon fruit (Hylocereus polyrhizus) in India

Dragon fruit (red skin and red fleshed, Hylocereus polyrhizus), newly introduced cactus plant in Mizoram, India, exhibited stem rot (35.38%) disease in established orchards during 2017–2018. Recorded symptoms included small red-brown spots encircled with yellowish halos. The size of the spots gradually enlarged and later the tissues turned completely watery and rotten. Fusarium species were consistently isolated from the stem rot tissues of red-fleshed dragon fruit. The pathogen associated with the stem rot disease was identified as Fusarium equiseti based on the morphological characters (production of abundant macroconidia, single-celled microconidia, and solitary and intercalary chlamydospores) and multigene phylogenetic analysis of internal transcribed spacer of ribosomal DNA (ITS-rDNA), translation elongation factor 1α (TEF-1) and beta-tubulin (TUB2). The phylogenetic trees showed that all isolates belonged to the F. equiseti, a member of Fusarium incarnatum-equiseti species complex (FIESC). Four isolates were tested for pathogenicity that produced the symptoms of stem rots (up to 57.64% severity) at 15 days after inoculation. Koch's postulate was further established by re-isolating the F. equiseti from artificially infected plants. To our best knowledge, this is the first report of F. equiseti as a pathogen causing the stem rot of dragon fruit (red-fleshed) in India.

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.

IDphy: An International Online Resource for Molecular and Morphological Identification of Phytophthora.

Phytophthora, with 203 species, is a genus of high importance in agriculture worldwide. Here, we present the online resource "IDphy", developed to facilitate the correct identification of species of Phytophthora using the type specimens from the original descriptions wherever possible. IDphy emphasizes species of high economic impact and regulatory concern for the United States. IDphy presents an interactive Lucid key and a tabular key for 161 culturable species described as of May 2018, including 141 ex-types and 20 well-authenticated specimens. IDphy contains standard operating procedures for morphological and molecular characterization, as well as a glossary, image gallery, and numerous links. Each of the 161 factsheets includes access to nomenclature and morphological and molecular features, including sequences of the internal transcribed spacer ribosomal DNA, cytochrome C oxidase subunit I (barcoding genes), YPT1, β-tubulin, elongation factor 1a, L10, heat shock protein 90, and other genes. IDphy contains an innovative in silico BLAST and phylogenetic sequence analysis using NCBI. The IDphy mobile app, released in August 2021 (free for Android or iOS), allows users to take the Lucid key into the laboratory. IDphy is the first online identification tool based on the ex-types implemented for plant pathogens. In this article, we also include information for 21 new species and one hybrid described after the publication of IDphy, the status of the specimens of the types and ex-types at international herbaria and culture collections, and the status of genomes at the GenBank (currently 153 genome assemblies which correspond to 42 described species, including 16 ex-types). The effectiveness of the IDphy online resource and the content of this article could inspire other researchers to develop additional identification tools for other important groups of plant pathogens.

Molecular characterization of Peronospora variabilis isolates infecting Chenopodium quinoa and Chenopodium album in Spain.

Quinoa is an expanding crop in Southern Spain. Downy mildew, caused by Peronospora variabilis, is the most important quinoa disease in Spain and worldwide. In Spain, this disease has also been observed oin the weed Chenopodium album. The objectives of this study were to unravel the origin of the P. variabilis isolates currently infecting quinoa in Southern Spain and to study their genetic diversity. We hypothesized that P. variabilis isolates infecting quinoa in Spain could have been introduced through the seeds of the quinoa varieties currently grown in the country, or, alternatively, that these isolates are endemic isolates, originally infecting C. album, that jumped to quinoa. In order to test these hypotheses, we sequenced the ITS, cox1 and cox2 regions of 33 P. variabilis isolates infecting C. quinoa and C. album in Southern Spain and analysed their phylogenetic relationship with isolates present in other countries infecting Chenopodium spp. cox1 gene sequences from all the Spanish P. variabilis isolates were identical and exhibited 9 SNPs compared to a single P. variabilis cox1 sequence found at GenBank. Phylogenetic analyses based on ITS rDNA region were not suitable to differentiate isolates according to their geographical origin or host. Cox2 sequences from P. variabilis Spanish isolates collected from C. quinoa and C. album were all identical and had a distinctive SNP in the last of four polymorphic sites that distinguished Spanish isolates from isolates from other countries. These results suggest that P. variabilis infecting quinoa in Southern Spain could be native isolates infecting originally C. album.

Acquired fluconazole resistance and genetic clustering in Diutina (Candida) catenulata from clinical samples

ObjectivesDiutina (Candida) catenulata is an ascomycetous yeast isolated from environmental sources and animals, occasionally infecting humans. The aim of this study is to shed light on the in vitro antifungal susceptibility and genetic diversity of this opportunistic yeast. MethodsForty-five D. catenulata strains isolated from various sources (including human and environmental sources) and originating from nine countries were included. Species identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and confirmed via internal transcribed spacer ribosomal DNA barcoding. In vitro antifungal susceptibility was determined for seven systemic antifungals via the gradient strip method after 48 hours of incubation at 35°C using Etest® (Biomérieux) or Liofilchem® strips. Isolates exhibiting fluconazole minimal inhibitory concentrations (MICs) of ≥8 μg/mL were investigated for mutations in the ERG11 gene. A novel microsatellite genotyping scheme consisting of four markers was developed to assess genetic diversity. ResultsMIC ranges for amphotericin B, caspofungin, micafungin, isavuconazole, and posaconazole were 0.19–1 μg/mL, 0.094–0.5 μg/mL, 0.012–0.064 μg/mL, 0.003–0.047 μg/mL, and 0.006–0.032 μg/mL, respectively. By comparison, a broad range of MICs was noted for fluconazole (0.75 to >256 μg/mL) and voriconazole (0.012–0.38 mg/L), the higher values being observed among clinical strains. The Y132F amino acid substitution, associated with azole resistance in various Candida species (C. albicans, C. tropicalis, C. parapsilosis, and C. orthopsilosis), was the main substitution identified. Although microsatellite typing showed extensive genetic diversity, most strains with high fluconazole MICs clustered together, suggesting human-to-human transmission or a common source of contamination. DiscussionThe high rate of acquired fluconazole resistance among clinical isolates of D. catenulata is of concern. In this study, we highlight a link between the genetic diversity of D. catenulata and its antifungal resistance patterns, suggesting possible clonal transmission of resistant isolates.

A fast and accurate method for specific detection and quantification of the bloom-forming microalgae Karlodinium veneficum in the marine environment.

Karlodinium veneficum is a toxic benthic globally distributed dinoflagellate which has direct impacts on human health and the environment. Early and accurate detection of this harmful algal bloom-forming species could be useful for potential risks monitoring and management. In the present work, a real-time PCR targeting the internal transcribed spacer ribosomal DNA region for the specific detection and absolute quantification of K. veneficum was designed. Then, the assay conditions were adjusted and validated. The developed qPCR was highly specific for the target species and displayed no cross-reactivity with closely related dinoflagellates and/or other microalgal species commonly distributed along the Tunisian coast. Its lowest detection limit was 5 rDNA copies per reaction, which is often considered satisfying. qPCR assay enumeration accuracy was evaluated using artificially inoculated environmental samples. The comparison of the cell abundance estimates obtained by qPCR assay with the theoretical estimates showed no statistically significant difference across a range of concentrations. We suggest that the qPCR approach developed in the present study may be a valuable tool to investigate the distribution and seasonal dynamics of K. veneficum in marine environments.

Phylogenetic analysis of black piper (Piper spp.) population collected in different locations of Viet Nam based on the ITSU1-4 gene region

The internal transcribed spacer (ITS) of nuclear ribosomal DNA is one of the most commonly used DNA markers in plant phylogenetic and DNA barcoding analyses and it has been recommended as a core plant DNA barcode. To compare and find out the genetic diversity difference, some individuals Peper were collected in different localities in Vietnam when using the ITS of nuclear ribosomal DNA. The ITS gene region from the nuclear genomes was tested for suitability as DNA barcoding regions of thirty-nine Peper individuals. Universal primers were used and sequenced products were analyzed using the Maximum Likelihood method and Tamura-Nei model in the MEGA X program. We did not observe high variability in intraspecific distance within the ITSu1-4 gene region between individuals ranging from 0.000 to 0.155. The size of the gene region has fluctuated from 667 to 685 bp between different individuals with the percentage (G + C) contained in the ITSu1-4 gene region which ranged from 54.776% to 60.805%, mean = 60.174%. The values of Fu’s Fs, D, Fu and Li’s D* and F* were negative as well (Fs = -0.209, D = -1.824; p < 0.05, D* = -1.205; not significant, p > 0.10 and F* = -1.699; not significant, 0.10 > p > 0.05) indicating an excess of recently derived haplotypes and suggesting that either population expansion or background selection has occurred. The value of Strobeck’s S is high (S = 0.684). The results of evolutionary relationships of taxa obtained 3 groups with the highest value of Fst are shown in the pairs of groups II and III (Fst = 0.151) and the lowest is in groups II and I (Fst = 0.015). All of the new sequences have been deposited in GeneBank under the following accession numbers MZ636718 to MZ636756. This database is an important resource for researchers working on Species of Peper in Vietnam and also provides a tool to create ITSu1-4 databases for any given taxonomy.

Efficacy and Mechanism of Thymol/KGM/LG Edible Coating Solution on Inhibition of Mucor circinelloides Isolated From Okra.

With the increasing demand and quality requirement for the natural nutritious food in modern society, okra has attracted much attention because of its high nutritional value and remarkable functionality. However, the occurrence of postharvest diseases of fresh okra severely limited the application and the value of okra. Therefore, in this study, the dominant pathogens causing postharvest diseases such as soft rot were isolated from naturally decaying okra. It was identified as Mucor circinelloides by its morphological characteristics and standard internal transcribed spacer ribosomal DNA sequence. Furthermore, the biological characteristics of M. circinelloides were studied, and the inhibitory effect of thymol/KGM/LG (TKL) edible coating solution on M. circinelloides and its possible mechanism was discussed. In addition, TKL edible coating solution had a dose-dependent inhibitory effect on M. circinelloides, with a 50% inhibitory concentration (EC50) of 113.55 mg/L. The TKL edible coating solution at 960 mg/L of thymol completely inhibited mycelial growth and spore germination of M. circinelloides. The results showed that the best carbon source of M. circinelloides was maltose, the best nitrogen source was beef extract and potassium nitrate, the best pH was 6, the best temperature was 28°C, the best NaCl concentration was 0.5%, and the light was conducive to the growth of M. circinelloides. It was also observed by scanning electron microscope (SEM) that TKL was more likely to destroy the cell wall integrity of M. circinelloides, inhibit spore morphology and change mycelium structure. Meanwhile, the activity of chitinase (CHI), an enzyme related to cell wall synthesis of M. circinelloides, was significantly decreased after being treated by TKL with thymol at 100 mg/L (TKL100). The content of Malondialdehyde (MDA) in M. circinelloides decreased significantly from 12 h to 48 h, which may cause oxidative damage to the cell membrane. The activity polygalacturonase (PG), pectin methylgalacturonase (PMG), and cellulase (Cx) of M. circinelloides decreased significantly. Therefore, the results showed that TKL had a good bacteriostatic effect on okra soft rot pathogen, and the main bacteriostatic mechanism might be the damage of cell membrane, degradation of the cell wall, inhibition of metabolic activities, and reduction of metabolites, which is helpful to further understand the inhibitory effect of TKL on okra soft rot pathogen and its mechanism.

First report of fusarium wilt disease caused by Fusarium equiseti on grafted watermelon in Korea.

In Korea, most of the grafted watermelons are a fusion of bottle gourd (Lagenaria siceraria) as a rootstock and watermelon (Citrullus lanatus) as a scionstock (Lee et al., 2010). Currently, we have collected several samples from grafted watermelon displaying symptoms of yellowing, withered and wilting leaves. When the symptomatic stem was excised, browning vascular tissues were observed due to the colonization of fungal pathogen. From the samples obtained, 25 fungal isolates were identified as species of Fusarium. Among 25 isolates, 18 were identified as Fusarium oxysporum, four as Fusarium solani, and three as Fusarium equiseti (F. equiseti) . Initial assessment showed that one of the F. equiseti isolates (NIHHS 16-126) was highly virulent to rootstock. Interestingly, this is the first time F. equiseti has been identified pathogenic to grafted watermelon. NIHHS 16-126 isolate was collected from watermelon cultivation field around Buyeo-gun (36.25951°N, 126.92044°E) county. Disease incident was estimated to infect approximately 10% of the watermelon plants cultivated in this area. NIHHS 16-126 isolate was examined to confirm its identity. On potato dextrose agar, colonies appeared yellowish-brown while the aerial mycelium was whitish to peach in color. Macroconidia were relatively long (20.21 - 51.13 × 2.30 - 4.5 μm, n=50), comprise of 3-6 septa, curvature shape and its conidiophores were with monophialides. However, microconidia formation was not observed. These morphological characteristics resemble F. equiseti characters as described by Hyun (2019). For molecular identification, an internal transcribed spacer of ribosomal DNA (ITS-rDNA), elongation factor-1α (EF-1α), and beta-tubulin (β-tub) genes were sequenced using primer pairs of ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R (Glass and Donaldson 1995), and Bt2a/Bt2b (Carbone and Kohn 1999). BLASTN analysis revealed that ITS-rDNA (LC648248), EF-1α (LC648250), and β-tub (LC648249) sequences were 99-100% identical to F. equiseti reference sequences (KF515650, KF747331, and KF747330) infected Avicennia marina in China (Lu 2014). Phylogenetic analysis of concatenated ITS-rDNA, EF-1α and β-tub sequences showed that this isolate clustered in the same clade as F. equiseti, confirming its identity as F. equiseti. For the inoculation, roots of 12-days-old seedlings (watermelon and bottle gourd, n=10 each) were dipped in the conidia suspension (1x106 conidia/µL) for 30 min. Inoculated seedlings were planted in the soil before being transferred to the greenhouse (temperature; 30°C, daylight; 14 hours). Control plants were inoculated with sterile water. Results showed that after 21 days post-inoculation, all inoculated bottle gourd seedlings (n=10) wilted and eventually died. In contrast, none of the inoculated watermelons or control seedlings were affected. Re-isolation of three fungal isolates (infected root) showed that their morphology and gene markers sequence were identical to the original isolates thus fulfilled Koch's postulates. Bottle gourd is the most preferred rootstock for grafted watermelons among Korean farmers due to its ability to resist Fusarium spp. infection. Therefore, the identification of F. equiseti as a fungal that is pathogenic to rootstock is crucial information to manage fusarium wilt disease among grafted watermelon. To our knowledge, this is the first report confirming F. equiseti infection in grafted watermelon plants in Korea.

Phylogeny and Ecology of Trebouxia Photobionts From Bolivian Lichens.

In the past few years, new phylogenetic lineages in Trebouxia were detected as a result of molecular approaches. These studies included symbiont selectivity in lichen communities, transects along altitudinal gradients at local and global scales and the photobiont diversity in local populations of lichen-forming fungal species. In most of these studies, phylogenetic and haplotype analyses based on the internal transcribed spacer (ITS) locus have continuously allowed the recognition of new monophyletic lineages, which suggests that still numerous undiscovered Trebouxia lineages can be hidden in lichens from unexplored areas, especially in the tropics. Here, we estimated the biodiversity of photobionts in Bolivian Andean vegetation and assessed their specificity. About 403 lichen samples representing 42 genera, e.g., Haematomma, Heterodermia, Hypotrachyna, Lecanora, Lepra, Leucodermia, Parmotrema, Pertusaria, Polyblastidium, and Usnea, containing Trebouxia photobionts, were analyzed. ITS ribosomal DNA (rDNA) and rbcL markers were used. We obtained Trebouxia sequences from Bolivian samples belonging to already described clades A, C, I, and S. Thirty-nine Trebouxia lineages were distinguished within these clades, while 16 were new. To reveal the structure of the community of Bolivian photobionts and their relationships with mycobionts, the comparative effects of climate, altitude, geographical distances, substrate, and habitat type, as well as functional traits of lichens such as growth forms, propagation mode and secondary metabolites, were analyzed. Furthermore, new Bolivian records were included in analysis on a global scale. In our study, the mycobiont genus or even species are the most important factors correlated with photobiont identity. Moreover, we revealed that the community of Bolivian photobionts is shaped by altitude.

Molecular characterization and study of phylogeny of some commercially important Vandaceous orchids (Orchidaceae) based on the internal transcribed spacer (ITS)

Habitat destruction, indiscriminate and rampant collection of Vandaceous orchids for their ornamental and medicinal values have resulted many of the important species pushed to threatened category and to struggle for their survival in nature. Illegal trade of this commercially important group of orchids continues as these orchids are similar in shape, size and difficult to identify and classify, especially when they are not in flowering stage. To address this issue of authentic identification, molecular barcode can be a useful tool for rapid identification even from vegetative tissues which will help in preventing indiscriminate collection of the species. The present study was carried out to test the discriminatory power of Internal Transcribed Spacer (IT1 and IT2) region in barcoding and to ascertain their phylogenetic relationship among these 31 species (80 individuals) from 15 genera of Vandaceous orchids. The sequences were aligned using ClustalW and genetic distances were computed using MEGA 7.0. Data analyses suggest that ITS of nuclear ribosomal DNA is a reliable marker which can be used as an efficient barcode to identify. The species resolution by three methods stood at 95.52% by genetic distance, 79.40% by BLAST analysis and 95.52% by phylogenetic tree building method and also analyze their phylogenetic relationships among Vandaceous orchids of Nagaland.

Description of Scutylenchus quercinus n. sp. (Nematoda: Tylenchomorpha) from Asalem forests, north Iran

Summary A new species of Scutylenchus was recovered from soil collected in the rhizosphere of forest trees in natural Asalem forests, Gilan province, north Iran. The new species is described and illustrated based upon morphological and molecular data. Scutylenchus quercinus n. sp. is mainly characterised by having four lines in the lateral fields, thus delimiting it from currently known species of the genus, which have six lines. It is further characterised by 605-709 μm long females having a slightly offset cephalic region with five or six annuli, stylet 19-21 μm long with conus ca 50% of its total length and three slightly posteriorly sloping knobs, vulva in a cavity, with epiptygma, vagina vera squarish in lateral view, female tail subcylindrical with smooth end, and males with 26-30 μm long spicules. The new species was morphologically compared with eight similar species. The molecular phylogenetic relationships of the new species with other relevant species and genera were reconstructed using partial small, large subunit and internal transcribed spacer ribosomal DNA (SSU, LSU D2-D3 and ITS1 rDNA) sequences and the resulting topologies were discussed.

DIVERSITY OF ENDOPHYTIC FUNGI ASSOCIATED WITH FRUITS AND LEAVES OF TAMARIND (Tamarindusindica L.) BASED O N ITS RIBOSOMAL DNA SEQUENCES

Plant-associated microbes are among the important natural resources that abundantly exist in natural environment such as endophytic fungi. The studies on endophytic fungi in medicinal plants have allowed the discovery of numerous fungi species and their hidden potentials. Therefore, this study focused on the isolation and identification of endophytic fungi from several plant parts of tamarind such as leaves and fruits. A total of 69 fungal cultures were successfully isolated and identified into 33 distinct species from 14 genera based on morphological characteristics and internal transcribed spacer (ITS) sequence analysis using a Maximum Likelihood method. There were six different species obtained from the genus Colletotrichum (C. aenigma, C. brevisporum, C. cobbittiense, C. fructicola, C. gloeosporioides and C. siamense), and Diaporthe (D. arecae, D. ceratozamiae, D. phaseolorum, D. pseudomangiferae, D. pseudooculi and D. pseudophoenicicola), four species of Aspergillus (A. aculeatus, A. carbonarius, A. flavus and A. tubingensis), three species of Curvularia (C. aeria , C. geniculata and C. lunata) and Nigrospora (N. lacticolonia, N. oryzae and N. sphaerica), two species of Lasiodiplodia (L. pseudotheobromae and L. theobromae) and Penicillium (P. rolfsii and P. verruculosum). Other fungal species that were also identified are Botryosphaeria mamane, Fusarium solani, Perenniporia tephropora, Phyllosticta fallopiae, Sarcostroma bisetulatum, Trichoderma asperellum and Xylaria feejeensis. The isolates were grouped into different clades according to their class. In conclusion, endophytic fungi isolated from tamarind plant are diverse and provides an insight into species diversity of endophytic fungi in tamarind.

COX1 gene and ITS-2 region: A comparative study of molecular diagnosis of Parabronema skrjabini in camels (Camelus dromedaries), Al-Najaf Province, Iraq

The current study was contributed to the analysis of the nucleotide sequence pattern of the nucleotide sequence of the tissue DNA isolates based on the Internal Transcribed Spacer ribosomal DNA (ITS2rDNA) gene and Cytochrome c oxidase subunit 1 mitochondrial DNA (COX1 mtDNA) using the traditional polymerase chain reaction (150 samples of abomasum collected directly from camel carcasses after working in Al-Najaf slaughterhouse from 1/10/2019 to 1/2/2020). ITS2rDNA were well suited for the prepared primer with size783 bp and identical ratio ranged 81.17-99.73% of the same species, as indicated a high similarity of the isolates taken from two-humped camels in China or less related to Parabronema skrjabini in sheep and goat. In addition, the study identified the number of the mutations within the four COX1 gene and ITS-2 region, which were the most conservative region of the host's species, supporting the concept of host specificity with Parabronema skrjabini. The COX1 gene and ITS-2 region applied to confirm the diagnosis using a universal primer, as it included eight isolates with a size of 689 bp, identical values were ranged from 84.99-98.02% depending on the multiple sequence alignment and showed an increase in the substitution level among isolates at an upper taxonomic level. Studying of the COX1 gene and ITS-2 region in Parabronema skrjabini demonstrated a significant relation in the cluster and an early common ancestor with isolates of the two-humped camel (China). As for the COX1 gene and ITS-2 region, the phylogenetic relationship supported the ribosomal gene results, especially with Habronema muscae or related species such as Habronema majus, Dirofilaria repens, Dipetalonema evansi, Setaria tundra, Cercopithifilaria sp towards the root node. Therefore, considering COX1 gene and ITS-2 region as an ideal tool in determining the phylogenetic history of the sequence maps, but less conservative mode than the ITS2 ribosome gene based to a taxonomic species level.

Isolation and molecular characterization of clinical and environmental dematiaceous fungi and relatives from Iran.

Background and Purpose: The frequency and genetic diversity of black fungi in environmental and clinical settings have not been fully studied in Iran. This study aimed to identify and evaluate intra- and inter-species DNA sequence variation and also understand the phylogenetic relationships of melanized fungi and relatives isolated from different geographical regions of Iran. Materials and Methods: In total, 111 clinical and environmental strains of dematiaceous fungi were isolated, and their internal transcribed spacer ribosomal DNA (rDNA) regions were sequenced and analyzed. Results: An inter-species nucleotide sequence diversity rate of 1 to 464 nucleotides was observed between the species. Intra-species differences were found in the strains of Alternaria alternata, Cladosporium cladosporioides, Alternaria tenuissima, Curvularia spicifera, Aureobasidium pullulans, Curvularia hawaiiensis, Neoscytalidium dimidiatum, Alternaria terricola, Alternaria chlamydospora, Didymella glomerata, and Drechslera dematioidea by 0–59, 0–22, 0–4, 0–4, 0–3, 0–2, 0–2, 0–2, 0–2, 0–1, and 0–1 nucleotide, respectively. Conclusion: The internal transcribed spacer rDNA is useful for the discrimination of several taxa of dematiaceous fungi. However, a better understanding of the taxonomy of species of Alternaria requires a larger rDNA region or a library of other gene sequences.

Taxonomy and Physiology of Oxyrrhis marina and Oxyrrhis maritima in Korean Waters

The genus Oxyrrhis is a heterotrophic dinoflagellate found in diverse marine environments. Oxyrrhis spp. have received attention owing to their ecological and industrial importance, high lipid contents, and docosahexaenoic acid formation. To the best of our knowledge, contrary to O. marina, ecophysiological characterization studies on O. maritima have not yet been reported. Therefore, we investigated the taxonomy and ecophysiology of four strains of O. marina from coastal waters and two strains of O. maritima from the littoral tidepool waters of Korea. Based on phylogenetic trees constructed using internal transcribed spacer ribosomal DNA (ITS rDNA) and SSU rDNA of dinoflagellates, the clade of all four O. marina strains was divergent from that of the two O. maritima strains. We measured the growth rates of both species at various water temperatures (10–36 °C), salinities (5–90), and light intensities (0–100 µE·m−2·s−1). The lowest (O. marina and O. maritima: 10 °C) and highest temperatures (O. marina: <35 °C, O. maritima: >35 °C) revealed that O. maritima has more tolerance to high salinity. This study provides a basis for understanding the ecophysiology of O. marina and O. maritima and their population dynamics in marine ecosystems.

Clonal Candidemia Outbreak by Candida parapsilosis Carrying Y132F in Turkey: Evolution of a Persisting Challenge.

As the second leading etiological agent of candidemia in Turkey and the cause of severe fluconazole-non-susceptible (FNS) clonal outbreaks, Candida parapsilosis emerged as a major health threat at Ege University Hospital (EUH). Evaluation of microbiological and pertinent clinical profiles of candidemia patients due to C. parapsilosis in EUH in 2019–2020. Candida parapsilosis isolates were collected from blood samples and identified by sequencing internal transcribed spacer ribosomal DNA. Antifungal susceptibility testing was performed in accordance with CLSI M60 protocol and ERG11 and HS1/HS2-FKS1 were sequenced to explore the fluconazole and echinocandin resistance, respectively. Isolates were typed using a multilocus microsatellite typing assay. Relevant clinical data were obtained for patients recruited in the current study. FNS C. parapsilosis isolates were recovered from 53% of the patients admitted to EUH in 2019–2020. Y132F was the most frequent mutation in Erg11. All patients infected with C. parapsilosis isolates carrying Y132F, who received fluconazole showed therapeutic failure and significantly had a higher mortality than those infected with other FNS and susceptible isolates (50% vs. 16.1%). All isolates carrying Y132F grouped into one major cluster and mainly recovered from patients admitted to chest diseases and pediatric surgery wards. The unprecedented increase in the number of Y132F C. parapsilosis, which corresponded with increased rates of fluconazole therapeutic failure and mortality, is worrisome and highlights the urgency for strict infection control strategies, antifungal stewardship, and environmental screening in EUH.