Ribosomal DNA ITS Research Articles

MARKER REGIONS OF THE GENOME FOR MOLECULAR DIAGNOSTICS OF OPISTHORCHIIDAE FAMILY

Infectious diseases caused by representatives of the Opisthorhidae family rank 8th in terms of health concern in the world among all food parasites. According to FAO data, studies of representatives of the Opisthorhidae family rank first and range from 0.66 to 88.7%. When performing a PCR analysis, a molecular marker plays an important role. In our studies, ribosomal genome clusters and mitochondrial genes were used as molecular markers. In eukaryotic organisms, ribosomal genes are presented in the form of clusters located in groups on different chromosomes. Each cluster includes three ribosomal genes (18S, 5.8S and 28S), separated by spacer regions. The ribosomal gene cluster consists of a transcribed region, internal transcribed spacers (ITS - Internal Transcribed Spacer) and flanking external transcribed spacers (ETS - Eternal Transcribed Spacer) The ITS noncoding sequence differs greatly among closely related organisms and is therefore used to classify parasites at the genus, species, and subspecies level. The ribosomal nuclear gene 5.8S forms the complete transcribed internal spacer region together with the molecular markers ITS1 and ITS2. This region of the genome is used in species studies of 19 families of digenetic flukes. Mitochondrial genes are also one of the most commonly used mtDNA gene fragments in the molecular identification of the family Opisthorchiidae. The advantage of using COX in research is the high degree of mutation, which makes it possible to differentiate closely related species The use of these genome regions makes it possible to quickly and accurately identify pathogens of the family Opishorchiidae, even for such individuals with practically indistinguishable morphological stages of the life cycle, such as parasite eggs and capsules in fish muscles. Therefore, in our studies, primers were used for the nuclear regions of ribosomal DNA ITS1, ITS2 and mitochondrial genes COX1, COX3.

MARKER REGIONS OF THE GENOME FOR MOLECULAR DIAGNOSTICS OF OPISTHORCHIIDAE FAMILY

Infectious diseases caused by representatives of the Opisthorhidae family rank 8th in terms of health concern in the world among all food parasites. According to FAO data, studies of representatives of the Opisthorhidae family rank first and range from 0.66 to 88.7%. When performing a PCR analysis, a molecular marker plays an important role. In our studies, ribosomal genome clusters and mitochondrial genes were used as molecular markers. In eukaryotic organisms, ribosomal genes are presented in the form of clusters located in groups on different chromosomes. Each cluster includes three ribosomal genes (18S, 5.8S and 28S), separated by spacer regions. The ribosomal gene cluster consists of a transcribed region, internal transcribed spacers (ITS - Internal Transcribed Spacer) and flanking external transcribed spacers (ETS - Eternal Transcribed Spacer) The ITS noncoding sequence differs greatly among closely related organisms and is therefore used to classify parasites at the genus, species, and subspecies level. The ribosomal nuclear gene 5.8S forms the complete transcribed internal spacer region together with the molecular markers ITS1 and ITS2. This region of the genome is used in species studies of 19 families of digenetic flukes. Mitochondrial genes are also one of the most commonly used mtDNA gene fragments in the molecular identification of the family Opisthorchiidae. The advantage of using COX in research is the high degree of mutation, which makes it possible to differentiate closely related species The use of these genome regions makes it possible to quickly and accurately identify pathogens of the family Opishorchiidae, even for such individuals with practically indistinguishable morphological stages of the life cycle, such as parasite eggs and capsules in fish muscles. Therefore, in our studies, primers were used for the nuclear regions of ribosomal DNA ITS1, ITS2 and mitochondrial genes COX1, COX3.

On Neotropical Fuscoporia with strigose pileus surface: Redescription and phylogenetic study of Polyporus sarcites and a new species Fuscoporia dollingeri (Hymenochaetaceae, Basidiomycota)

Specimens of poroid Hymenochaetaceae with uniquely strigose pileus surfaces were collected and studied morphologically and phylogenetically (using as markers ITS and nrLSU ribosomal DNA). Detailed morphological examination showed that the specimens belong to two distinct species of Fuscoporia. Fuscoporia sarcites comb. nov., which is proposed and recorded for the first time in Guatemala, Honduras, and Venezuela, and the newly described Fuscoporia dollingeri sp. nov., which was collected several times in Florida (USA). Morphological and ecological data of these species are compared to other similar species, and an identification key of Neotropical Fuscoporia is provided.

Molecular diagnosis of echinococcosis in patients based on frozen paraffin tissue samples and fixed formalin and hydatid cysts isolated from livestock in a slaughterhouse.

Various genotypes of Echinococcus granulosus have been studied in high-disease-risk areas and identified as causative agents of cystic echinococcosis (CE). This study was performed to examine and identify the molecular hydatid cyst in the dissected human specimens in paraffin tissue, and the dissected animal cyst was characterized using the DNA polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer 1 (ITS1). To determine the molecular properties of E. granulosus, 20 hydatid cyst samples (including 6 sheep samples, 9 camel samples, and 10 human paraffin samples) were collected from Zahedan and Zabol cities. After DNA extraction, molecular PCR was performed, and RFLP was evaluated. In this study, the Taq1 endonuclease cleavage enzyme was used. The patterns of DNA bands found in the isolates from human CE and animal bladder cysts were the same, as indicated by the results of ribosomal DNA-ITS1 amplification from E. granulosus. Two nested primer pairs were used. The rough size of the enhanced ITS1 piece was 444 and 391 base pairs (bp), individually. After cutting the PCR product with the Taq1 enzyme, the patterns of the fragments revealed that the samples had two identical RFLP patterns. The aftereffects of this study showed that the parasite genotypes confined to sheep, camels, and people had hereditary changes. The transcendent type of E. granulosus sensu lato in the area is E. granulosus sensu stricto, which featured the meaning of the sheep/canine cycle in human transmission. Albeit the band profile in the camel is now and again like the sheep strain, RLFP can be recognized utilizing the PCR strategy, and two differentiating band profiles using the chemical were found in this review.

Antifungal Potential of Cladosporium sp. (Endophytic fungi) Associated with Olea europaea L. Leaves

In the leaves of Olea europaea L. Olive trees an endophytic fungus was discovered. Cladosporium sp. was identified to be the fungus based on its morphological characteristics and nuclear ribosomal DNA ITS sequence analysis and was registered in NCBI as the Cladosporium genus has been registered under the number (0P939922.1) The species was not specified, and it was considered of unknown species after comparing it to global isolates. In comparison to olive leaf extract, Cladosporium sp. including total flavonoid, total phenolic, total terpenoid, and total saponins, Which were 121.9%, 198.1%, 89.13%, and 29.87 % respectively compared to its content in olive leaf extract, which was 61.54 %, 67.88 % , 17.1%, and 20.19% respectively. The Cladosporium sp. extract inhibited the growth of 27 isolates belonging to different species of candida which were Candida albicans , C. lypolitica , C. tropicalis , C. sphaerica , C. krusei , C. guilliermondii , C. parapsilosis , C. norvegicus , C. glabrata , and C. kefyr , the inhibition effects increased with increasing concentration to reach the highest level to suppress fungal growth when concentrated 30 mg/ml. This proves the antifungal potential of endophytic fungi in the future.

Taxonomy and phylogeny of some geophilic Umbelliferae - Apioideae genera based on anatomical, morphological and molecular data

The paper provides an overview of the concordance between molecular (nuclear ribosomal DNA ITS) and morphological data in the group of geophilic Umbelliferae of Middle Asia, which includes the genera Elwendia, Elaeosticta, Hyalolaena, Galagania, Oedibasis, Mogoltavia and Gongylotaxis. In general, a good consistency of data of different types can be observed. At the same time, at some levels of the taxonomic hierarchy, the consistency between morphological and molecular data looks much higher than at others. The identification of the group of geophilic Umbelliferae agrees much better with molecular data than the boundaries between genera within it and the proposed intrageneric groupings. Some observed cases of inconsistency, such as the position of Hyalolaena melanorrhiza among the species of the genus Elaeosticta and the polyphyly of the genera Hyalolaena and Oedibasis, are rather difficult to explain from a morphological point of view, only from a biogeography.

Isolation and Characterization of Novel Huperzine-Producing Endophytic Fungi from Lycopodiaceae Species.

Huperzine A (HupA) is an important drug for treating Alzheimer's disease (AD) and is primarily extracted from the Huperzia serrata (Lycopodiaceae). Failures in the chemical synthesis of Hup and in vitro culture have put H. serrata in danger of extinction, and there is a need for an extensive investigation of Hup from alternative perspectives. The aim of this study is to identify endophytic fungi that produce high Hup or simultaneously produce many types of Hup and have high genetic stability derived from other Lycopodiaceae species as a source of materials for natural Hup production. In this work, Hup-producing endophytic fungi were isolated from three species: Lycopodium clavatum, Phlegmariurus squarrosus, and P. phlegmaria. Of these, L. clavatum and P. squarrosus were confirmed as novel sources of Hup-producing fungi. Based on morphological characteristics and nuclear ribosomal DNA ITS sequences, four endophytic fungi Colletotrichum siamense THG1-17, Epicoccum sorghinum THG01-18, Phoma sp. TKH3-2, and Phyllosticta sp. THG2-27 were firstly isolated from these Lycopodiaceae plants, which were capable of simultaneously producing both HupA and HupB, as evidenced by high-performance liquid chromatography analysis. The four strains showed stability in Hup yield over 50 generations of culture with an in vitro storage period of 3 months. These isolated fungi will provide a new source of materials for further research to develop drugs containing HupA as well as HupB for AD treatment in the future.

Ganoderma gibbosum newly recorded from Pakistan

Ganoderma gibbosum is a new record for Pakistan, identified by morphological and molecular data. DNA sequence analysis of ribosomal 5.8S rRNA gene including the flanking nuclear ribosomal DNA-ITS regions confirmed the Pakistani specimens as G. gibbosum. Morphologically, the specimens are characterized by their non-laccate sessile basidiomata, truncate, short fine minute inter walled pillars, elongated basidiospores (7.5– 10.2 × 4.6–5.9 μm), and greyish to greyish-brown dull pileal surface.

Antagonistic activity of Trichoderma asperellum against Fusarium species, chemical profile and their efficacy for management of Fusarium-root rot disease in dry bean.

Diseases caused by Fusarium pathogens lead to significant yield losses on many economically important crops. The purpose of this study was to evaluate the antagonistic capability and chemical profile of the bioagent Trichoderma asperellum against several Fusarium strains. The efficacy of this strain in reducing Fusarium-root rot disease in dry bean was also examined. The T. asperellum strain was identified based on sequencing the internal transcribed spacer (ITS) and tef1 gen regions of ribosomal DNA. Dual cultural assay demonstrated their antagonistic activity against the studied Fusarium strains due to the probable combination of competition, mycoparasitism and antibiosis. This strain was positive for cellulase, chitinase and protease activity. The crude extracts of T. asperellum significantly suppressed the growth of the tested Fusarium strains with inhibition zone values ranging from 7.3 to 19.7 mm and minimum inhibitory concentration (MIC) values ranging from 0.15 to 1.42 mg mL-1 . The gas chromatography-mass spectrometry (GC-MS) analysis of cell free supernatant and mycelial biomass of T. asperellum showed the presence of 27 and 21 compounds, respectively. The main compounds responsible for the bioactivity were butylated hydroxytoluene, hexadecanoic acid, 9-octadecenoic acid, ergosterol and hexadecanoic acid, ethyl ester. Trichoderma asperellum significantly increased plant emergence and reduced root rot caused by Fusarium solani in dry bean grown under glasshouse and field trials. Further, plant biomass and dry bean yield were higher in T. asperellum-treated plants than in control plants. Trichoderma asperellum was highly effective, through various mechanisms, against Fusarium strains especially F. solani which causes root rot in dry bean. © 2023 Society of Chemical Industry.

A new species of Siphoderina Manter, 1934 (Digenea: Cryptogonimidae) infecting the Dory Snapper Lutjanus fulviflamma (Teleostei: Lutjanidae) from the east coast of South Africa

Parasitological assessment of marine fishes at Sodwana Bay in the iSimangaliso Marine Protected Area on the east coast of South Africa revealed a new species of cryptogonimid trematode infecting the pyloric caeca of the Dory Snapper, Lutjanus fulviflamma (Forsskål) (Lutjanidae). The new species is morphologically consistent with the concept of the large genus Siphoderina Manter, 1934; its phylogenetic position within this genus was validated through molecular sequencing of the ITS2 and partial 28S ribosomal DNA sub-regions. We name this species Siphoderina nanan. sp. and comment on the current state of understanding for this genus of cryptogonimids.

First report of the wattle rust pathogen, Uromycladium acaciae (Raveneliaceae, Pucciniales) in Ethiopia

Abstract Australian Acacia species are among the most important trees planted for wood and pulp production in several African countries, including Ethiopia. In 2020, symptoms of a serious shoot and leaf rust disease were observed on black wattle (Acacia mearnsii De Wild.) trees across the three main wattle growing regions of Ethiopia. The aim of this study was to describe the disease and identify its causal agent based on morphological characteristics as well as DNA sequence data for the ITS and LSU regions of ribosomal DNA. Here we report for the first time, the presence of the wattle rust pathogen, Uromycladium acaciae, in Ethiopia.

Plastome evolution in the genus Sium (Apiaceae, Oenantheae) inferred from phylogenomic and comparative analyses

BackgroundSium L. (Apiaceae) is a small genus distributed primarily in Eurasia, with one species also occurring in North America. Recently, its circumscription has been revised to include 10 species, however, the phylogenetic relationships within its two inclusive clades were poorly supported or collapsed in previous studies based on nuclear ribosomal DNA ITS or cpDNA sequences. To identify molecular markers suitable for future intraspecific phylogeographic and population genetic studies, and to evaluate the efficacy of plastome in resolving the phylogenetic relationships of the genus, the complete chloroplast (cp) genomes of six Sium species were sequenced.ResultsThe Sium plastomes exhibited typical quadripartite structures of Apiaceae and most other higher plant plastid DNAs, and were relatively conserved in their size (153,029–155,006 bp), gene arrangement and content (with 114 unique genes). A total of 61–67 SSRs, along with 12 highly divergent regions (trnQ, trnG-atpA, trnE-trnT, rps4-trnT, accD-psbI, rpl16, ycf1-ndhF, ndhF-rpl32, rpl32-trnL, ndhE-ndhG, ycf1a and ycf1b) were discovered in the plastomes. No significant IR length variation was detected showing that plastome evolution was conserved within this genus. Phylogenomic analysis based on whole chloroplast genome sequences produced a highly resolved phylogenetic tree, in which the monophyly of Sium, as well as the sister relationship of its two inclusive clades were strongly supported.ConclusionsThe plastome sequences could greatly improve phylogenetic resolution, and will provide genomic resources and potential markers useful for future studies of the genus.

Accidental cultivation of the European truffle Tuber brumale in North American truffle orchards.

Tuber brumale is a European edible truffle species that is often viewed as a contaminant in truffle orchards, as it visually resembles more valuable black truffles such as T. melanosporum, but differs in aroma and flavor and sells for a much lower price. Although T. brumale is not native to or intentionally cultivated in North America, it was reported to have been accidently introduced into British Columbia in 2014 and North Carolina in 2020. However, in winter of 2021, various truffle orchards in eastern North America produced truffles that differed from the anticipated harvest of T. melanosporum. Molecular analysis of these specimens confirmed T. brumale truffle fruiting bodies from ten orchards distributed across six eastern USA states. Phylogenetic analysis of nuclear ribosomal ITS and 28S DNA sequences indicated that all samples belong to the T. brumale A1 haplogroup, the genetic subgroup of T. brumale that is more common in western Europe. This pattern of widespread fruiting of T. brumale in North American truffle orchards is likely the result of T. brumale being introduced in the initial inoculation of trees used as hosts in T. melanosporum truffle cultivation. We review other examples of introduced non-target truffle species and strategies for limiting their impact on truffle cultivation.

The Occurrence of the Strongylid Nematodes, Kalicephalus viparae viparae (Nematoda: Diaphanocephalidae), in Viper Snakes, Macrovipera lebetina (Reptilia: Viperidae), Southwestern Iran.

ancylostomatid Kalicephalus spp. is the common parasitic intestinal nematode of reptiles. West-Asian blunt-nosed viper is a venomous snake found in extensive areas of Iran. From June to September 2017, two dead viper snakes were referred to a parasitology laboratory and examined for intestinal parasites. Several white elongated roundworms were collected and fixed to identify under light and scanning electron microscopes (SEM) based on morphological and molecular characteristics. For the molecular survey, some parts of the identified worms were extracted and the ITS of nuclear ribosomal DNA (rDNA) was amplified by polymerase chain reaction (PCR). Five roundworms were found in one snake and three worms with similar morphological characteristics in another one. All the collected female hookworms were taxonomically identified as Kalicephalus viperae viperae. The SEM findings showed the head was small and had three dorsal, ventral, and middle circumoral papillae with a spike-like process on the median papilla of K. viperae. Moreover, the buccal capsule was bivalvular and included two lateral valves consisting of several chitonid pieces. The tail of the female worm was slim and long with a blunt end and had a terminal spike at its end. In the molecular survey, the ITS of rDNA amplified at about 850 bp was identified as K. viperae. The ITS gene rDNA phylogeny analysis of the K. viperae sequence showed that the isolated species had high similarity to Ancylostoma species from around the world and is close to Ancylostoma braziliense with 88% discrepancies in the phylogenetic tree. The morphological characteristics and a large part of K. viperea viperea rDNA nucleotide sequence were reported in viper snakes for the first time in the world and in Iran.

Molecular characterization of Fasciola spp. from ruminants in Duhok province using the ITS1 ribosomal DNA marker

This study aimed to characterize and identify the genotypes of Fasciola spp. isolated from sheep, goats, and cattle in Duhok province based on the ITS1 region of rDNA. About 54 adult Fasciola flukes were individually isolated from the livers of naturally infected ruminants. After morphological identification, the genomic DNA of 54 isolated Fasciola spp. was successfully extracted, and the ITS1segment (518 bp) of rDNA was amplified. The amplicons were confirmed by gel electrophoresis and yielded mono cleared bands. Five amplicons from these samples (2 sheep, 2 cattle, and 1 goat) were selected for sequencing and then compared with NCBI-GenBank sequences for genotyping and phylogenetic analysis. Sequencing analysis and the BLAST results revealed that 3/5 of the resultant sequences were F. hepatica and 2/5 were F. gigantica. The ITS1 sequences were submitted to NCBI-GenBank with accession numbers: OM920533, OM920534, OM948733, OM948683, and OM918714. Alignment analysis of the current study and GenBank ITS1 sequences showed the presence of nucleotide variations between F. hepatica and F. gigantica species (interspecific), which were enough to separate them. At the same time, they were not observed within the same species of Fasciola (intraspecific). The pairwise identity percentage of intraspecific and interspecific Fasciola isolates was 100% and 99.2-99.6%, respectively. Phylogenetic analysis of the ITS1 sequences demonstrated that the Fasciola isolates of this study were clustered into two clades (hepatica and gigantica clades). The present study concluded that both Fasciola spp. (F. hepatica and F. gigantica) existed among the infected ruminants in Duhok province and are closely related to intraspecific Fasciola isolates from different countries in the Middle East, Asia, Europe, and Africa.

Fungal Metabarcoding Data for Two Grapevine Varieties (Regent and Vitis vinifera ‘Cabernet-Sauvignon’) Inoculated with Powdery Mildew (Erysiphe necator) Under Drought Conditions

Fungal Metabarcoding Data for Two Grapevine Varieties (Regent and <i>Vitis vinifera</i> ‘Cabernet-Sauvignon’) Inoculated with Powdery Mildew (<i>Erysiphe necator</i>) Under Drought Conditions

A Molecular Systematics and Taxonomy Research on Trechispora (Hydnodontaceae, Trechisporales): Concentrating on Three New Trechispora Species from East Asia.

Trechispora are an important genus of wood-inhabiting fungi that have the ability to decompose rotten wood in the forest ecosystem. In this study, we reported three new species of Trechispora: T. murina, T. odontioidea, T. olivacea from a subtropical region of Yunnan Province, China. Species descriptions were based on a combination of morphological features and phylogenetic analyses of the ITS and LSU region of nuclear ribosomal DNA. Trechispora murina is characterized by the resupinate basidiomata, grandinioid hymenial surface with a greyish tint, monomitic hyphal system and ellipsoid, thick-walled, ornamented basidiospores; T. odontioidea has an odontioid hymenial surface with cylindrical to conical, blunt aculei and subglobose to globose, colorless, slightly thick-walled, ornamented basidiospores; T. olivacea has a farinaceous hymenial surface with olivaceous tint, basidia clavate and thick-walled, ornamented, broadly ellipsoid to globose basidiospores. Sequences of the ITS and nLSU rDNA markers of the studied samples were generated, and phylogenetic analyses were performed with maximum likelihood, maximum parsimony, and Bayesian inference methods. After a series of phylogenetic analyses, the 5.8S+nLSU dataset was constructed to test the phylogenetic relationship of Trechispora with other genera of Hydnodontaceae. The ITS dataset was used to evaluate the phylogenetic relationship of the three new species with other species of Trechispora. Using ITS phylogeny, the new species T. murina was retrieved as a sister to T. bambusicola with moderate supports; T. odontioidea formed a single lineage and then grouped with T. fimbriata and T. nivea; while T. olivacea formed a monophyletic lineage with T. farinacea, T. hondurensis, and T. mollis.

Time and cost-efficient identification of Trichophyton indotineae.

During the past 5 years, an outbreak of recalcitrant dermatophytosis due to a novel Trichophyton species generally resistant to terbinafine, T.indotineae, has spread out from South Asia to many countries around the World. These isolates cannot be reliably differentiated from other Trichophyton spp. on the basis of morphological traits and the current laboratory diagnostics relies on sequencing of ribosomal DNA ITS region. In this study, we aimed to introduce two inexpensive and rapid PCR-based assays for differentiation between T.indotineae and other dermatophytes. The first introduced assay is based on PCR-restriction fragment length polymorphism (PCR-RFLP) analysis, involving the amplification of TOP2 sequences and the digestion of PCR products by Cfr13I restriction enzyme. The second assay is proposed as conventional endpoint species-specific PCR amplification of the C120-287 intergenic locus. To validate the assays, a total of 191 Trichophyton spp. and 2 Microsporum canis strains with known ITS region sequences were used. From the T.mentagrophytes / T.interdigitale species complex (TMTISC), strains with 18 different ITS genotypes were tested. The sample of TMTISC isolates included 41 T.indotineae strains. TOP2 PCR-RFLP and T.indotineae-specific PCR were positive with testing on DNA of all 41 T.indotineae isolates and two strains of T.mentagrophytes belonging to ITS Types XIII and XVI, but negative with other species and other TMTISC ITS genotypes (n=152). Therefore, the specificity of both new assays was 99%. The two developed diagnostic assays provide accurate and cost-effective means of identifying cultured T.indotineae isolates.

P051 A case of recalcitrant sporotrichosis by infection of Sporothrix globosa

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectivesSporotrichosis is the leading subcutaneous mycosis caused by the Sporothrix (S.) schenckii complex. S. globosa is the causative organism of fixed sporotrichosis in Korea. The preferred regimen of cutaneous sporotrichosis is itraconazole for 3-6 months, however, there were few studies for recalcitrant sporotrichosis.MethodsIn 2018, we performed a histological examination of a patient who suffered sporotrichosis for 3 years and cultured part of the specimen. Despite various regimens for years, improvement and exacerbation were repeated, so we took another skin biopsy and cultured it in 2021. Isolates from the 2018 and 2021 lesions were identified as S. globosa by ribosomal DNA ITS sequencing (GenBank accession number: MH499862 and MH499863). The in vitro antifungal sensitivity tests were performed by broth microdilution method according to CLSI M38-A2 guidelines or Sensititre YeastOne® manufacturer's instructions. They were incubated at 30°C in a non-CO2 incubator for 7 days.ResultsIn 2018, histologically, we observed chronic inflammatory granuloma comprising lymphocytes, histiocytes, and giant cells, and several spores with periodic acid-Schiff (PAS) staining. Microscopic findings and ITS sequences of rRNA gene were identical with S. globosa. The antifungal susceptibility profile in 2018 revealed sensitive to terbinafine (0.125 μg/ml), and moderate to high MIC values for amphotericin B (2 μg/ml), itraconazole (>16 μg/ml), voriconazole (>16 μg/ml), and echinocandins (>16 μg/ml). Treatment with terbinafine, itraconazole, or amphotericin B, the skin lesions were partially improved, but were not cured. In 2021, we took another skin biopsy and culture specimen. Histopathological and mycological examination results were the same as before. The antifungal susceptibility profile revealed sensitive to itraconazole (0.5/ml), and high MIC for others. Clinically, skin lesions were not improved with the use of itraconazole 200 mg/d. Itraconazole 400 mg/d with local heating induced moderate improvement. There was no evidence of immune deficiency.ConclusionWe experienced recalcitrant sporotrichosis that did not respond to itraconazole and terbinafine, and the sensitivity of antifungal was changed. In this case, the combination treatment including local heating, saturated KI may be considered, and frequent antifungal susceptibility tests are needed.

Exploring the Species Limits in Balanophora subgenus Balania

Abstract— Balanophora subgen. Balania (Balanophoraceae), whose members differ from those of the other subgenus in having three-merous male flowers, includes B. flava, B. involucrata, B. tobiracola, the B. harlandii assemblage, and the agamospermic species B. japonica. Species limits of B. flava, B. involucrata, and the B. harlandii as currently circumscribed (B. harlandii assemblage) have long been controversial. Here, species limits in subgen. Balania are explored based on morphological characters and phylogenetic analysis using nuclear 18S and ITS ribosomal DNA sequences. Subgen. Balania was monophyletic if B. japonica is excluded. Balanophora harlandii assamblage was polyphyletic and three lineages, B. harlandii, B. kawakamii, and B. henryi, were recovered in the assemblage. Molecular and morphological divergence, distribution, and phenology provided strong support for the recognition of these lineages as distinct species, namely B. harlandii, B. kawakamii, and B. henryi. The findings also suggested that B. flava should be reduced to synonym under B. involucrata, thus supporting Hansen’s treatment of both dioecious and monoecious populations as members of a single species.