Internal Transcribed Spacer Ribosomal DNA Research Articles

Three New Fungal Leaf Spot Diseases of Spinach in the United States and the Evaluation of Fungicide Efficacy for Disease Management.

Leaf spot diseases of spinach, caused by Colletotrichum spinaciae, has become a major production constraint in several production areas, including Texas, in recent years. Leaf spot symptoms were observed in several fields in Texas in 2016 and 2017, with typical anthracnose-like symptoms and leaves with small, circular, and sunken lesions that appeared similar to injury from windblown sand. The lesions were plated on potato dextrose agar, from which fungal cultures were recovered. The fungi were identified based on morphology and sequence analysis of the introns of glutamate synthetase and glyceraldehyde-3-phosphate dehydrogenase (for isolates determined to be Colletotrichum spp.) and the internal transcribed spacer ribosomal DNA (for isolates determined to be Myrothecium spp.). Based on foliar symptoms, fungal colony and spore morphology, pathogenicity tests of fungal isolates on the spinach cultivar 'Viroflay', and DNA sequence analysis of the isolates, the symptoms on spinach leaves for two sets of samples were caused by Colletotrichum coccodes and Colletotrichum truncatum, and leaf spots resembling damage from windblown sand were caused by Myrothecium verrucaria. This is the first report of spinach leaf spot diseases caused by C. coccodes, C. truncatum, and M. verrucaria in the United States. C. coccodes and C. truncatum caused severe symptoms on the spinach cultivar 'Viroflay', whereas M. verrucaria caused symptoms of intermediate severity. Fungicide efficacy tests demonstrated that chlorothalonil, mancozeb, pyraclostrobin, fluxapyroxad + pyraclostrobin, and penthiopyrad were completely effective at preventing leaf spots caused by any of these pathogens when applied 24 h before inoculation of 'Viroflay' plants in greenhouse trials.

Molecular identification and antifungal susceptibility among clinical isolates of dermatophytes in Shiraz, Iran (2017-2019).

Dermatophytosis is a common superficial mycotic infection affecting individual's quality of life worldwide. The present study aimed to perform species-level identification and evaluate the antifungal susceptibility patterns of dermatophytes isolated in Shiraz, Iran. This cross-sectional study was conducted on clinical samples collected during 2017-2019 from 307 patients suspected of having dermatophytosis.The isolates were identified by direct microscopy, culture and internal transcribed spacer ribosomal DNA sequencing, and their antifungal susceptibility patterns were determined by the microdilution method. Among 307 patients, dermatophytosis was diagnosed by microscopy in 190 (61.8%) subjects and confirmed in 130 (42.3%) cases by both microscopy and culture. It was found out tinea pedis was the most common clinical manifestation, and Trichophyton mentagrophytes was the most prevalent species (28.4%), followed by T tonsurans (23.8%), Microsporum canis (11.5%), T interdigitale (10%), T verrucosum (6.9%), T rubrum (6.9%), T benhamiae (4.6%), T violaceum (3%), T simii (3%), Epidermophyton floccosum (0.7%) and M ferrugineum (0.7%). Moreover, it was revealed that luliconazole with a geometric mean (GM) minimum inhibitory concentration (MIC) of 0.03μgml-1 was the most effective agent against all tested isolates. Regardless of species, 30% of isolates responded to high MICs of griseofulvin (MIC90 >2μgml-1 ). The increasing prevalence of nonindigenous species of T simii, T benhamiae and M ferrugineum in Shiraz, Iran, was a notable finding. In addition, infections due to zoophilic species showed an increasing trend. These epidemiological data, along with antifungal susceptibility patterns, mayhave implications for clinicaldecision-makingand successful treatment.

New Insights Into the Skin Microbial Communities and Skin Aging.

Although it is well-known that human skin aging is accompanied by an alteration in the skin microbiota, we know little about how the composition of these changes during the course of aging and the effects of age-related skin microbes on aging. Using 16S ribosomal DNA and internal transcribed spacer ribosomal DNA sequencing to profile the microbiomes of 160 skin samples from two anatomical sites, the cheek and the abdomen, on 80 individuals of varying ages, we developed age-related microbiota profiles for both intrinsic skin aging and photoaging to provide an improved understanding of the age-dependent variation in skin microbial composition. According to the landscape, the microbial composition in the Children group was significantly different from that in the other age groups. Further correlation analysis with clinical parameters and functional prediction in each group revealed that high enrichment of nine microbial communities (i.e., Cyanobacteria, Staphylococcus, Cutibacterium, Lactobacillus, Corynebacterium, Streptococcus, Neisseria, Candida, and Malassezia) and 18 pathways (such as biosynthesis of antibiotics) potentially affected skin aging, implying that skin microbiomes may perform key functions in skin aging by regulating the immune response, resistance to ultraviolet light, and biosynthesis and metabolism of age-related substances. Our work re-establishes that skin microbiomes play an important regulatory role in the aging process and opens a new approach for targeted microbial therapy for skin aging.

Diaporthe rosiphthora sp. nov.: Yet another rose dieback fungus

The cultivated rose (Rosa × hybrida) is the most important plant species in the cut flower industry worldwide. In Brazil, rose plantations are said to be frequently attacked by a dieback disease of uncertain etiology. A severe rose dieback outbreak was detected and closely observed in Viçosa (state of Minas Gerais, Brazil). The disease was particularly severe on the cultivar ‘Gran Gala’. A fungus with typical characteristics of the asexual state of Diaporthe spp. was regularly associated to necrotic tissues and isolated in pure culture. Morphology of the fungus was examined and described and DNA was extracted for molecular studies. Phylogenetic analysis inferred from combined dataset of the internal transcribed spacer ribosomal DNA (ITS), partial calmodulin gene (CAL) and partial translation elongation factor 1-α (TEF1) along with morphological characterization, indicated that the fungus causing dieback disease of Rosa × hybrida is a new species of Diaporthe. This is described herein as Diaporthe rosiphthora sp. nov. Its pathogenicity to Rosa × hybrida was evaluated and inoculations on additional selected rose cultivars indicated that, besides ‘Grand Gala’, another important commercial cultivar (‘Saltinho’) is susceptible. Cultivars ‘Greta’ and ‘Príncipe Negro’, were also susceptible, though less so than ‘Grand Gala’ and ‘Saltinho’. Diaporthe rosiphthora is a novel addition to the numerous fungi associated rose dieback.

Prevalence and control implications of bovine trypanosomes in endemic areas of northern Uganda.

African animal trypanosomiasis (AAT), a disease complex caused by tsetse fly-transmitted Trypanosoma brucei brucei, T. congolense savannah ITS, and T. vivax, continues to inflict heavy losses to the animal industry in terms of decreased livestock production and productivity. Live bait technology and chemotherapy have been used as a control strategy in northern Uganda since 2006 with minimal success. Here, we report the results of a cross-sectional study carried out in Lango subregion, Uganda, to assess the species prevalence of bovine trypanosome in cattle using the internal transcribed spacer (ITS) of trypanosome ribosomal DNA (rDNA). Blood samples were collected from 1090 cattle by ear vein puncture and screened using a single pair of primers designed to amplify ITS ribosomal DNA (rDNA). Our results indicate an overall prevalence of 40.18% (438/1090, 95% CI 30.82-54.51). T. vivax constituted 32.66% (356/1090), T. congolense 2.39% (26/1090), T. brucei 1.28% (14/1090), T. godfreyi 0.09%(1/1090), T. brucei and T. congolense 0.36% (4/1090), T. brucei and T. vivax 1.47% (16/1090), T. vivax and T. congolense 1.65% (18/1090), T. vivax and T. simiae 0.18% (2/1090), and T. vivax and T. godfreyi 0.09% (1/1090) of infections. Over 91.7% of infections involved single species, while 9.5% were mixed infections. Over 90.2% (37/41) of the mixed infections involved T. vivax as one of the species, while 53.7% (22/41) involved T. congolense. The high prevalence of AAT and the continued presence of T. brucei raise public health concerns because of the zoonotic implications. An integrated approach that involves mass treatment of cattle, vector, and animal movement control should be adopted to reduce the risk of both AAT and HAT.

Evaluation of in vitro alpha-glucosidase inhibitory, antimicrobial, and cytotoxic activities of secondary metabolites from the endophytic fungus, Nigrospora sphaerica, isolated from Helianthus annuus

PurposeThis study aimed to evaluate alpha-glucosidase inhibition and antimicrobial activity as well as cytotoxic activity of extracts from the endophytic fungus, Nigrospora sp., isolated from leaves of Helianthus annuus, which is widely cultivated for food and used as a medicinal plant.MethodsThe fungus (TSU-CS003) was identified based on internal transcribed spacer ribosomal DNA sequences and fungal biomass, and fermentation broth was subjected to extraction by solvents (hexane and ethyl acetate). All extracts were tested for their antimicrobial activity, alpha-glucosidase inhibition, and cytotoxicity activity. In addition, the active extract was analyzed by using gas chromatography mass spectrometry (GC-MS)ResultsTSU-CS003 was identified as Nigrospora sphaerica. The fermentation broth extract (BE) showed strong antimicrobial activity against Staphylococcus aureus and methicillin-resistant S. aureus (Gram-positive bacteria) with minimum inhibitory concentration (MIC) values in the range of 16–32 μg/mL and a few yeasts with MIC values ranging from 64 to 128 μg/mL, especially Talaromyces marneffei with an MIC value of 4 μg/mL. The effects of BE were observed by SEM. The results showed that this extract affected the cell morphology of T. marneffei. The half-maximal inhibitory concentration (IC50) of BE from alpha-glucosidase inhibition was recorded as 17.25 μg/mL and also showed significant cytotoxicity against A549 human cancer cell lines with an IC50 value of 22.41 μg/mL. Furthermore, BE was analyzed by using GC-MS and divided into three main compounds, including 5-pentyldihydrofuran-2(3H)-one, (Z)-methyl 4-(isobutyryloxy)but-3-enoate, and 2-phenylacetic acid.ConclusionThis was the first report of the endophytic fungus N. sphaerica from H. annuus. It is a potential source of active metabolites, which gave the strong antifungal activity, antioxidant activity, and cytotoxicity to A549 cancer cell lines.

<p><strong><em>Alternaria</em></strong><strong> <em>ershadii</em> <em>sp. nov</em>. a new species isolated from wheat black head mold in Iran</strong></p>

In the continuation of studies on Alternaria species from wheat plants with black head mold symptoms, 10 isolates with typical characteristics of the section Pseudoalternaria were isolated and studied by a combined approach based on morphological characteristics and phylogenetic analyses using sequence data sets of the internal transcribed spacer of ribosomal DNA (ITS-rDNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and plasma membrane adenosine triphosphatase (ATPase) genes. These isolates represented a new and previously undescribed species in this section which is illustrated and described here as Alternaria ershadii sp. nov. Also based on our phylogenetic analyses, it was revealed that Alternaria brassicifolii, which was recently described from Brassica rapa subsp. pekinensis in Korea and placed in this section, formed a well separated monotypic lineage distinct from the other members in the section Pseudoalternaria. Comparisons of three-dimensional sporulation patterns and conidial characteristics supported the molecular results. So, at present, Alternaria section Pseudoalternaria contains only seven species viz. Alternaria altcampina, A. arrhenatheri, A. ershadii, A. inflata, A. kordkuyana, A. parvicaespitosa and A. rosae. Morphological comparisons of Alternaria ershadii with other species in the section Pseudoalternaria and with Alternaria brassicifolii is also provided.

Cryptic speciation in Protoceratium reticulatum (Dinophyceae): Evidence from morphological, molecular and ecophysiological data

The cosmopolitan, potentially toxic dinoflagellate Protoceratium reticulatum possesses a fossilizable cyst stage which is an important paleoenvironmental indicator. Slight differences in the internal transcribed spacer ribosomal DNA (ITS rDNA) sequences of P. reticulatum have been reported, and both the motile stage and cyst morphology of P. reticulatum display phenotypic plasticity, but how these morpho-molecular variations are related with ecophysiological preferences is unknown. Here, 55 single cysts or cells were isolated from localities in the Northern (Arctic to subtropics) and Southern Hemispheres (Chile and New Zealand), and in total 34 strains were established. Cysts and/or cells were examined with light microscopy and/or scanning electron microscopy. Large subunit ribosomal DNA (LSU rDNA) and/or ITS rDNA sequences were obtained for all strains/isolates. All strains/isolates of P. reticulatum shared identical LSU sequences except for one strain from the Mediterranean Sea that differs in one position, however ITS rDNA sequences displayed differences at eight positions. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference based on ITS rDNA sequences. The results showed that P. reticulatum comprises at least three ribotypes (designated as A, B, and C). Ribotype A included strains from the Arctic and temperate areas, ribotype B included strains from temperate regions only, and ribotype C included strains from the subtropical and temperate areas. The average ratios of process length to cyst diameter of P. reticulatum ranged from 15% in ribotype A, 22% in ribotype B and 17% in ribotype C but cyst size could overlap. Theca morphology was indistinguishable among ribotypes. The ITS-2 secondary structures of ribotype A displayed one CBC (compensatory change on two sides of a helix pairing) compared to ribotypes B and C. Growth response of one strain from each ribotype to various temperatures was examined. The strains of ribotypes A, B and C exhibited optimum growth at 15 °C, 20 °C and 20–25 °C, respectively, thus corresponding to cold, moderate and warm ecotypes. The profiles of yessotoxins (YTXs) were examined for 25 strains using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The parent compound yessotoxin (YTX) was produced by strains of ribotypes A and B, but not by ribotype C strains, which only produced the structural variant homoyessotoxin (homoYTX). Our results support the notion that there is significant intra-specific variability in Protoceratium reticulatum and the biogeography of the different ribotypes is consistent with specific ecological preferences.

Morphology, ultrastructure and molecular phylogeny of cyst-producing Caladoa arcachonensis gen. et sp. nov. (Peridiniales, Dinophyceae) from France and Indonesia

ABSTRACTThe dinoflagellate order Peridiniales encompasses several well circumscribed families. However, the family level of some genera, such as Bysmatrum and Vulcanodinium, has remained elusive for many years. Four Peridinium-like strains were established from the Atlantic coast of France and North Sulawesi, Indonesia through cyst germination or isolation of single cells. The cyst-theca relationship was established on specimens from the French Atlantic. Their morphologies were examined using light, scanning and transmission electron microscopy. The cells were characterized by a much larger epitheca relative to the hypotheca, a large anterior sulcal (Sa) plate deeply intruding the epitheca and a small first anterior intercalary plate. The plate formula was identified as Po, cp, X, 4′, 3a, 7′′, 6C, 5S, 5′′′, 2′′′′, shared by Apocalathium, Chimonodinium, Fusiperidinium and Scrippsiella of the family Thoracosphaeraceae but the configuration of Sa plate and anterior intercalary plates is different. Transmission electron microscopy showed that the eyespot was located within a chloroplast comprising two rows of lipid globules and thus belongs to type A. All four strains were classified within a new genus Caladoa as C. arcachonensis gen. et sp. nov. Small subunit ribosomal DNA (SSU rDNA), partial large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer ribosomal DNA (ITS rDNA) sequences were obtained from all strains. Genetic distance based on ITS rDNA sequences between French and Indonesian strains reached 0.17, suggesting cryptic speciation in C. arcachonensis. The maximum likelihood and Bayesian inference analysis based on concatenated data from SSU and LSU rDNA sequences revealed that Caladoa is monophyletic and closest to Bysmatrum. Our results supported that Caladoa and Bysmatrum are members of the order Peridiniales but their family level remains to be determined. Our results also support that Vulcanodinium is closest to the family Peridiniaceae.

Scrippsiella masanensis sp. nov. (Thoracosphaerales, Dinophyceae), a phototrophic dinoflagellate from the coastal waters of southern Korea

ABSTRACTA phototrophic dinoflagellate belonging to the genus Scrippsiella was isolated from a water sample collected from Masan Bay, Korea. Using a clonal culture of this dinoflagellate, its morphology was analysed with light, scanning and transmission electron microscopy and small subunit (SSU), large subunit (LSU), and internal transcribed spacer ribosomal DNA (ITS rDNA) sequences were studied. The dinoflagellate plate formula was Po, x, 4ʹ, 3a, 7″, 6c (5c + t), 5s, 5ʹ″, and 2″″, consistent with Scrippsiella. The dinoflagellate had a rounded epitheca, unlike most Scrippsiella species, which have a conical epitheca. Furthermore, it had a narrow, symmetrical, and pentagonal 1ʹ plate, unlike other Scrippsiella species. In addition, it had three intercalary plates of different sizes. This species had noncalcareous cysts, unlike most Scrippsiella species, which have calcareous cysts. The LSU rDNA sequence of the dinoflagellate was about 2% different from Scrippsiella enormis, the closest Scrippsiella species, and the ITS rDNA sequence was about 8% different from S. enormis. The sequence was identical to an unidentified Scrippsiella strain from Chinese waters (SSND14) and 0.55% different from an unidentified Scrippsiella strain from Norwegian waters (GeoB 277). On the basis of its unique morphological and molecular features, this species was named Scrippsiella masanensis sp. nov.

Talaromyces verruculosus tannase production, characterization and application in fruit juices detannification

Abstract A potential tannase producing fungus S3-07 was isolated from the tannin-rich soil sample and screened as better tannase producer with 38 ± 3 mm diameter of hydrolysis zone formation. It was identified as T. verruculosus (accession number KX863699) on the basis of the internal transcribed spacer ribosomal DNA (ITS-rDNA) sequence analysis. Tannase was produced with 8.22 ± 0.35 Unit per gram dry substrate (U/gds) activity in the unoptimized condition through solid state fermentation (SSF) using Babul (Acacia nilotica) bark as a solid substrate. Central composite design (CCD) was used to maximize the production and achieved 3.91 fold increments (32.18 ± 1.31 U/gds). Ammonium sulfate precipitation with 70% saturation and DEAE fractionation obtained 13.289 specific activity, 47.489% yield and 6.896 purification fold. SDS-PAGE and zymographic analysis showed two bands of tannase with ∼75 kDa and 58 kDa pH optima of tannase was measured at 8.00, whereas pH stability was recorded between 4.0 and 8.0. Tannase had temperature optima at 60 °C, however, stability was observed between 30 and 60 °C. In presence of ethanol, SDS, Mn2+ ions and gallic acid increased tannase activity, whereas ethyl acetate, tween 20, Ba2+ ion and urea inhibited maximum activity. Most of tannin reduction (93.27%) was measured in case of orange juice after 15 min incubation and 89.36% reduction in pomegranate juice after 30 min, whereas aonla juice was measured with 75.49% tannin reduction after 45 min incubation.

An amended description of two Gyrodactylus species (Platyhelminthes: Monogenea) parasitizing Antarctic Notothenioid fish.

Species identification based on the morphometry of opisthaptoral hard parts, in combination with internal transcribed spacer ribosomal DNA (ITS rDNA) region sequences, confirmed the presence of four viviparous Gyrodactylus von Nordman, 1832 (Plathyhelminthes, Monogenea) species on Nototheniid fish from the Prince Gustav Channel (Weddell Sea, Antarctica). Gyrodactylus antarcticus Gusev, 1967 was found mostly on Trematomus newnesi Boulenger (93 specimens) but also on T. bernacchii Boulenger (one specimen), the latter representing a new host record for this species. Gyrodactylus byrdi Hargis & Dillon, 1968 and G. coriicepsi Rokicka, Lumme & Ziętara, 2009 were recorded on their type hosts, T. newnesi and Notothenia coriiceps Richardson, respectively. Gyrodactylus wilkesi Hargis & Dillon, 1968 was found mostly on the fins of T. bernacchii (29 specimens), but also on T. hansoni Boulenger (one specimen) and T. newnesi (three specimens). The finding of G. wilkesi on T. newnesi represents a new host record. The low number of Gyrodactylus specimens may indicate an accidental infection. The occurence of all four Gyrodactylus species in the Prince Gustav Channel represents a new locality record. According to phylogentic methods, the newly redescribed monogeneans belong to the Antarctic lineage, forming a sister group to North American and European marine Gyrodactylus species, and consist of two species groups, one comprising G. coriicepsi and G. nudifronsi Rokicka, Lumme & Ziętara, 2009, and the other G. anarcticus and G. wilkesi.

Efficacy and Mechanism of Cinnamon Essential Oil on Inhibition of Colletotrichum acutatum Isolated From 'Hongyang' Kiwifruit.

In this study, one of the dominant pathogens, which caused postharvest diseases such as anthracnose, was isolated from decayed ‘Hongyang’ kiwifruit. It was identified as Colletotrichum acutatum by its morphological characteristics and standard internal transcribed spacer ribosomal DNA sequence. Further, the efficacy and possible mechanism of cinnamon essential oil on inhibition of C. acutatum were investigated. Results showed that C. acutatum was dose-dependently inhibited by cinnamon essential oil. Meanwhile, the mycelial growth and spore germination of C. acutatum were completely inhibited at the concentrations of 0.200 μL/mL and 0.175 μL/mL (v/v), respectively. Indeed, both minimal inhibitory and minimum fungicidal concentrations of cinnamon essential oil were measured as 0.200 μL/mL. Additionally, the possible antifungal mechanism of cinnamon essential oil on C. acutatum was demonstrated. Results showed that the cinnamon essential oil could destroy the cell membrane integrity of C. acutatum, and the structure of cell membrane was changed. Indeed, the cell cytoplasm including soluble protein, sugar, and nucleic acid was released, which significantly changed the extracellular conductivity. Results suggested that the cinnamon essential oil exerted great potential to be used as a natural and efficient preservative for kiwifruit postharvest storage, which were helpful for the better understanding of the efficacy and mechanism of cinnamon essential oil on inhibition of pathogens isolated from decayed ‘Hongyang’ kiwifruit.

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.

Development of Real-Time and Conventional PCR Assays for Identifying Stubby Root Nematode Paratrichodorus allius.

Paratrichodorus allius is an important pest on many crops, particularly on potato due to its ability to transmit Tobacco rattle virus causing corky ringspot disease on tubers. Detection and identification of P. allius are important for effective disease management. In this study, a rapid and reliable molecular diagnosis of this nematode targeting internal transcribed spacer ribosomal DNA was established. The specificity of the designed primers was evaluated using 29 nematode species and results showed that a single amplicon was produced from DNA of P. allius only. Detection sensitivity analysis indicated that a 9.6 × 10-4 ng of DNA template could be detected by conventional PCR and 1.92 × 10-4 ng of DNA by real-time PCR. The PCR assays amplified DNA of stubby root nematodes isolated from 18 soil samples in North Dakota and Minnesota, which were confirmed as P. allius by sequencing. Both conventional PCR and real-time PCR assays amplified target nematodes from complex nematode communities, supporting the success of this molecular diagnosis of P. allius. This is the first report of P. allius identification using the real-time PCR method and from nematode communities with other nematodes using conventional PCR. The new PCR assays provide rapid species identification and are suitable for use in diagnostic laboratories and detection of field infestations with P. allius.

Risk factors for Candida parapsilosis bloodstream infection in a neonatal intensive care unit: a case-control study

BackgroundCandida parapsilosis is increasingly responsible for invasive candidiasis in neonates. This study investigates phenotypic and genotypic features of C. parapsilosis microbial isolates and underlying clinical conditions associated with acquisition of C. parapsilosis in a neonatal intensive care unit (NICU) in Italy.MethodsIdentification of C. parapsilosis was performed by VITEK® 2 and MALDI TOF and confirmed by analysis of internal transcribed spacer ribosomal DNA sequences. Genotyping was performed by PCR fingerprinting. Antifungal susceptibility of strains was evaluated by microdilution. A case-control study was designed to identify risk factors for C. parapsilosis bloodstream infection.ResultsDuring the study period (April 2009- April 2012), C. parapsilosis was responsible for 6 umbilical catheter and 11 central catheter-associated bloodstream infection in 17 neonates in the NICU. Molecular typing identified identical fingerprinting profile in all C. parapsilosis isolates from neonates. Fifteen of 17 C. parapsilosis isolates were susceptible to all antifungal drugs, two isolates were resistant to fluconazole and intermediate susceptible to itraconazole. Low birthweight, gestational age and time to exposure to assisted ventilation were risk factors for C. parapsilosis infection in neonates in the NICU at univariate and multivariate analysis.ConclusionC. parapsilosis bloodstream infections in the NICU were caused by a single epidemic clone. Low birthweight, gestational age and time to exposure to invasive devices, with predominance of assisted ventilation, were the clinical conditions associated with C. parapsilosis bloodstream infection in the NICU.

Cladosporium lebrasiae, a new fungal species isolated from milk bread rolls in France

The fungal genus Cladosporium (Cladosporiaceae, Dothideomycetes) is composed of a large number of species, which can roughly be divided into three main species complexes: Cladosporium cladosporioides, Cladosporium herbarum, and Cladosporium sphaerospermum. The aim of this study was to characterize strains isolated from contaminated milk bread rolls by phenotypic and genotypic analyses. Using multilocus data from the internal transcribed spacer ribosomal DNA (rDNA), partial translation elongation factor 1-α, actin, and beta-tubulin gene sequences along with Fourier-transform infrared (FTIR) spectroscopy and morphological observations, three isolates were identified as a new species in the C. sphaerospermum species complex. This novel species, described here as Cladosporium lebrasiae, is phylogenetically and morphologically distinct from other species in this complex.

Analysis of the relationship between ribosomal DNA ITS sequences and active components in Rhodiola plants.

Rhodiola plants are a valuable resource in traditional Chinese medicine. The objective of this study was to evaluate the correlation between ribosomal DNA internal transcribed spacer (ITS) sequences and the three active components in Rhodiola plants. For this, we determined ITS sequence polymorphisms and the concentrations of active components salidroside, tyrosol, and gallic acid in different Rhodiola species from the Tibetan Plateau. In a total of 23 Rhodiola samples, 16 different haplotypes were defined based on their ITS sequences. Analysis of the active components in these same samples revealed that salidroside was not detected in species with haplotypes H4, H5, or H10, tyrosol was not detected with haplotypes H3, H5, H7, H10, H14, or H15, and gallic acid was detected in with all haplotypes except H14 and H15. In addition, the concentrations of salidroside, tyrosol and gallic acid varied between samples with different haplotypes as well as those with the same haplotype, implying that no significant correlation exists between haplotype and salidroside, tyrosol or gallic acid concentrations. However, a statistically significant positive correlation was observed for among these three active components.

Efficacy of Chaetomium Species as Biological Control Agents against Phytophthora nicotianae Root Rot in Citrus

Thailand is one of the largest citrus producers in Southeast Asia. Pathogenic infection by Phytophthora, however, has become one of major impediments to production. This study identified a pathogenic oomycete isolated from rotted roots of pomelo (Citrus maxima) in Thailand as Phytophthora nicotianae by the internal transcribed spacer ribosomal DNA sequence analysis. Then, we examined the in vitro and in vivo effects of Chaetomium globosum, Chaetomium lucknowense, Chaetomium cupreum and their crude extracts as biological control agents in controlling this P. nicotianae strain. Represent as antagonists in biculture test, the tested Chaetomium species inhibited mycelial growth by 50~56% and parasitized the hyphae, resulting in degradation of P. nicotianae mycelia after 30 days. The crude extracts of these Chaetomium species exhibited antifungal activities against mycelial growth of P. nicotianae, with effective doses of 2.6~101.4 μg/mL. Under greenhouse conditions, application of spores and methanol extracts of these Chaetomium species to pomelo seedlings inoculated with P. nicotianae reduced root rot by 66~71% and increased plant weight by 72~85% compared to that in the control. The method of application of antagonistic spores to control the disease was simple and economical, and it may thus be applicable for large-scale, highly effective biological control of this pathogen.

Rhizopus Species from Fresh Tempeh Collected from Several Regions in Indonesia

Recent changes in taxonomy of Rhizopus, which are now heavily relying on molecular approach, create significant problem in assigning species name to particular Rhizopus strains isolated from various sources, including tempeh. The present study aims to determine 36 strains of Rhizopus from tempeh originated from 26 locations in Indonesia, using combination of molecular phylogenetic analysis based on internal transcribed spacer ribosomal DNA sequence, physiology, and morphology to species level. The results showed that most of the strains belong to R. microsporus–complex, and only one strain belongs to R. delemar. Morphological variations within R. microsporus were observed, but under current approach they were insufficient for infraspecific delimitation. The current report is an important contribution in validating the identity of Rhizopus from fresh tempeh in Indonesia.