Veronaea Botryosa Research Articles

Evaluation of three inactive vaccines against Veronaea botryosa infection in white sturgeon (Acipenser transmontanus)

Veronaea botryosa is the etiological agent of a systemic phaeohyphomycosis known as “fluid belly” in white sturgeon (Acipenser transmontanus). Fluid belly is a critical disease affecting sturgeon aquaculture and the caviar industry for which there are no commercially available vaccines or approved antifungal treatments to manage outbreaks. The primary aim of this study was to investigate the effect of a V. botryosa [conidia], a V. botryosa [mold], and a Saccharomyces cerevisiae [yeast] formalin-killed vaccine on sturgeon immune responses to fungal challenge. Immunization consisted of an initial intracoelomic injection with one of the three treatment preparations, followed by a vaccine booster four weeks later by the same route and dose. Experimental challenge by intramuscular injection with a virulent V. botryosa conidia suspension followed after another four weeks. Non-challenged control fish received injections of PBS. The inactivated vaccines proved safe for white sturgeon fingerlings. Sturgeon immunized with either V. botryosa [mold] or S. cerevisiae [yeast] exhibited a significantly different pro-inflammatory response upon challenge with V. botryosa compared to non-immunized fish. Challenged fish developed clinical signs similar to those reported during natural outbreaks of fluid belly. Positive control treatments (those not immunized but challenged with V. botryosa) experienced the highest mortality; however, survival curves were similar amongst all treatments (p < 0.05). Furthermore, the S. cerevisiae [yeast] vaccine resulted in comparatively lower fungal persistence and fewer lesions following histological analysis. Further efforts evaluating the potential of Saccharomyces spp. as a vaccine candidate against fluid belly are warranted.

Disseminated phaeohyphomycosis in a beluga sturgeon (Huso huso)

Sturgeon aquaculture is an emerging worldwide industry both for human consumption as well as for conservation. Several transmissible diseases limit this industry including mycosis by dematiaceous fungi such as Veronaea botryosa. This study reports the first diagnosis of phaeohyphomycosis in a beluga sturgeon (Huso huso) in Italy.

In vitro susceptibility testing for the emerging pathogenic mould Veronaea botryosa and pharmacokinetic parameters of natamycin in white sturgeon (Acipenser transmontanus).

Systemic phaeohyphomycosis caused by the dematiaceous mould Veronaea botryosa is an important emergent disease affecting captive sturgeons (Acipenser spp.). The disease, colloquially known as "fluid belly," causes morbidity and mortality in adult animals resulting in significant economic losses to the aquaculture industry. Advancements in therapeutic and prophylactic protocols have been partially hampered by the lack of basic protocols to grow and manipulate the fungus in the laboratory. In this study, microbroth kinetic protocols were established to analyse V.botryosa growth in seven nutrient media at different temperatures. Generated area under the curve (AUC) indicates that potato flake dextrose broth (PFD-B) and Sabouraud dextrose broth (SD-B) incubated at 25°C provided the greatest growth. The generated protocol was then used to test the susceptibility of V.botryosa isolates to natamycin, a macrolide polyene antifungal agent used as a food preservative. SD-B and RPMI with l-glutamine (+RPMI-B) containing different concentrations of natamycin were inoculated with V.botryosa conidia and the generated growth curves were compared using cubic smoothing spline model. The non-inhibitory concentration and minimal inhibitory concentration (MIC; decrease of AUC by 90% compared with control) were determined to be <1μg/mL and 16 μg/mL of natamycin in SD-B media. To gain an understanding of the tissue distribution of natamycin in white sturgeon, pharmacokinetics was tested. Based on pharmacokinetic parameters determined in this study and targeting a blood concentration >16 μg/mL for 24 h, an intravenous dose >1g/kg would be needed, making the use of this drug unrealistic. The information presented in this study can be used to investigate susceptibility of pathogenic fungus to antimicrobials and disinfectants as well as support future therapeutic protocols against emerging fungal diseases like fluid belly.

Glucose and Lactate Reference Intervals for White Sturgeon and Evaluation of Two Point-of-Care Devices in Sturgeon Infected with Veronaea botryosa.

White Sturgeon Acipenser transmontanus are cultured for human consumption as well as for conservation purposes. In this study, two commercially available portable devices for measuring glucose and lactate were compared to a benchtop analyzer and blood reference intervals were generated using heparin plasma collected from 43 healthy White Sturgeon yearlings. The generated normal ranges were used to compare plasma values collected from Veronaea botryosa-infected White Sturgeon at 10, 20, and 30 d postchallenge (dpc). In the 43 healthy yearlings, significantly different glucose and lactate values were obtained when comparing the portable devices to the benchtop analyzer. In the portable devices, blood glucose showed a consistent bias of 12.3 mg/dL and blood lactate showed a proportional bias. The detected blood glucose values in infected sturgeon were significantly different from those in noninfected controls when using the benchtop analyzer at 20 and 30 dpc and when using the portable device at 20 dpc. However, blood glucose in infected individuals and controls was within the reference interval on either device. No significant difference was noted for lactate measurements in infected sturgeon. The results of this study indicate that portable devices for blood glucose and blood lactate evaluation in White Sturgeon may be useful in a setting where relative values are an acceptable approximation of absolute values.

Expression of immune markers of white sturgeon (Acipenser transmontanus) during Veronaea botryosa infection at different temperatures

Systemic phaeohyphomycosis caused by Veronaea botryosa is one of the most important emergent diseases to affect sturgeon aquaculture in North America. White sturgeon (Acipenser transmontanus) cultured at temperatures above 15 °C are at higher risk of severe disseminated disease and higher mortalities. Despite this, little is known regarding disease pathogenesis and the immune response to infection. The objective of this study was to investigate the acute (2 days post-challenge [dpc]) and chronic (32 dpc) response of white sturgeon at 13 °C and 18 °C challenged with V. botryosa via intramuscular injection, using gene expression analysis of a diverse array of soluble immune and inflammatory mediators. Significantly greater amounts of irf8 (p < 0.05) and tfg-β (p < 0.05) genes were detected in gills of exposed fish at 18 °C when compared to those at 13 °C 32 dpc. Transcript levels of haptoglobin, serotransferrin, serum amyloid, cathelicidin, tnf-α, and il-17 were significantly increased in splenic tissues of challenged fish maintained at 18 °C late in infection (p < 0.05). However, only haptoglobin and serotransferrin transcript abundance were significantly greater in exposed fish when compared to controls 32dpc. Moreover, haptoglobin transcripts at this time point were significantly greater in exposed fish at 18 °C when compared to those challenged at 13 °C. Fewer differences were detected in fish kept at 13 °C. In agreement with transcript quantification, western blot assessment of haptoglobin showed increased levels in the challenged fish maintained at 18 °C.

Analysis of the white sturgeon (Acipenser transmontanus) immune response during immunostimulation and Veronaea botryosa infection

Systemic phaeohyphomycosis caused by Veronaea botryosa is regarded as an important emerging mycotic disease of sturgeon aquaculture. However, no vaccines or treatments are currently available. The effects of dietary β-glucan supplementation on resistance to V. botryosa infection was examined in controlled challenges by exposing immunostimulated and control fish to ~7.25 × 105 fungal spores/fish via intra-muscular injection. Six weeks post-challenge, cumulative mortality was determined and antibodies to acute phase-proteins (APP) were used to quantify the conserved APP peptides in the serum of challenged and control fish using Western blot. Transcript levels for all tested pro-inflammatory cytokines, APP, and regulatory cytokines in the spleen were similar amongst treatments at the end of the three-week feeding period. However, significantly higher survival occurred in fingerlings fed 0.3% β-glucans compared to non-immunostimulated fish groups (p < 0.05) six weeks post-challenge. A strong proinflammatory response was detected in exposed treatment groups, and greater survival at 6 weeks was associated with higher transcript abundance of Il-17 in fish fed β-glucans. Findings support the important role of this cytokine in response to fungal infection.

Quantitative PCR for detection and quantification of Veronaea botryosa in fish and environmental samples.

Systemic phaeohyphomycosis, aka 'fluid belly', is one of the most important emergent diseases in sturgeon Acipenser spp. aquaculture. The etiologic agent is the saprobic, dematiaceous fungus Veronaea botryosa. Effective vaccines and chemotherapeutic treatments are currently unavailable. Additionally, the fungus is a slow-growing organism, taking from 10-15 d for colonies to be observed in agar media. To this end, a specific quantitative PCR (qPCR) targeting the V. botryosa β-tubulin gene was developed and validated. The specificity of the assay to V. botryosa was initially confirmed in silico and in vivo against common fungal fish pathogens, including closely related members of the order Chaetothyriales (Exophiala spp.) and other black pigmented fungi (Alternaria spp. and Cladosporium spp.), as well as tissues from uninfected sturgeon. The assay possessed high clinical specificity (100%) and clinical sensitivity (74%) in detecting V. botryosa DNA in splenic tissues from laboratory-infected sturgeon. Using V. botryosa genomic DNA as a template, the limit of detection was equivalent to 10 conidia, and the method was found suitable for the detection of fungal DNA in fresh and formalin-fixed tissues. In addition, the presence of non-target DNA from white sturgeon did not influence assay sensitivity. The developed qPCR assay is a sensitive, specific, and rapid diagnostic method for the detection and quantification of V. botryosa DNA from white sturgeon tissues.

Persistence of Veronaea botryosa in marine and freshwater microcosms and susceptibility evaluation to three disinfectants

Veronaea botryosa is a dematiaceous fungus and the causative agent of systemic mycosis known as “fluid belly’, one of the most important emergent diseases in sturgeon aquaculture. A better understanding of the pathogenesis of the disease requires knowledge of environmental conditions that promote colonization, infection and disease. In this study, both traditional cultured-based methods and qPCR assays were used to evaluate V. botryosa persistence in water microcosms at two different temperatures and salinities, as different environmental factors can predispose poikilotherm animals to infection. Results indicate that V. botryosa persisted significantly greater in fresh than marine water microcosms (p-values < .05). However, fungus persisted similarly at 15 °C and 25 °C at both salinities over 60 days without a host. Furthermore, due to the lack of effective preventive and therapeutic methods to combat fungal infections, proper disinfection of aquaculture equipment and facilities is needed. To this end, the susceptibility of V. botryosa to three frequently used disinfectants in aquatic facilities was investigated to provide efficient biocide concentrations against V. botryosa. The results show that a dose of at least 75 mg/L iodine for 5 min, 390 mg/L household bleach for 5 min, and 3.75% hydrogen peroxide for 30 min are effective against V. botryosa and they are applicable in biosecurity protocols for captive sturgeon.

Experimental Infection of Mice with Veronaea botryosa as a Model for Human Phaeohyphomycosis.

Veronaea botryosa is a ubiquitous, dematiaceous mold capable of causing cutaneous and subcutaneous lesions in humans. In the last decade, V. botryosa has been associated with emergent systemic fungal infections in aquatic animals, including cultured sturgeon (Acipenser spp.), captive amphibians, and wild reptiles. Recently, repetitive extragenic palindromic PCR (rep-PCR) fingerprinting has demonstrated intraspecific variability among V. botryosa isolates from different clinically affected hosts and geographic regions. However, little is known regarding the pathogenic potential of the different genetic clades, and no mammalian model currently exists to investigate V. botryosa phaeohyphomycosis. In this study, we inoculated immunocompetent heterozygotic (nu/+) and immunodeficient homozygotic (nu/nu) Hsd:Athymic Nude-Fox1nu mice subcutaneously or through orogastric gavage with 1 of 3 representative V. botryosa strains that had been recovered from white sturgeon (Acipenser transmontanus), green sea turtle (Chelonia mydas), and human hosts and typed by using rep-PCR analysis. Daily mortality and morbidity were recorded, and dissemination of the fungus was investigated through culture of splenic samples and histologic analysis of the injection site, regional lymph nodes, salivary gland, spleen, liver, mesenteric lymph node, and gastrointestinal tract. No differences in survival, fungal burden, or dissemination were observed between fungal strains, routes of inoculation, or host immune status. Fungal infection was observed after subcutaneous inoculation only, was localized to the inoculation site, and was identified in both nu/nu and nu/+ mice. Fungal strain variability was not associated with virulence in a murine model of infection, and this novel mouse model of V. botryosa phaeohyphomycosis recapitulates the human clinical condition.

Introducing Aculeata aquatica gen. et sp. nov., Minimelanolocus thailandensis sp. nov. and Thysanorea aquatica sp. nov. (Herpotrichiellaceae, Chaetothyriales) from freshwater in northern Thailand

Aculeata gen. nov., Minimelanolocus thailandensis sp. nov., Thysanorea aquatica sp. nov. and Veronaea botryosa are described and illustrated from submerged wood collected from a freshwater habitat in Thailand. Morphological examination and phylogenetic analyses support the classification of the four isolates in Herpotrichiellaceae. Their relationships with other morphologically similar taxa are discussed. Veronaea botryosa is reported for the first time from a freshwater habitat.

Rapid Identification of Seven Waterborne Exophiala Species by RCA DNA Padlock Probes

The black yeast genus Exophiala includes numerous potential opportunistic species that potentially cause systematic and disseminated infections in immunocompetent individuals. Species causing systemic disease have ability to grow at 37–40 °C, while others consistently lack thermotolerance and are involved in diseases of cold-blooded, waterborne vertebrates and occasionally invertebrates. We explain a fast and sensitive assay for recognition and identification of waterborne Exophiala species without sequencing. The ITS rDNA region of seven Exophiala species (E. equina, E. salmonis, E. opportunistica, E. pisciphila, E. aquamarina, E. angulospora and E. castellanii) along with the close relative Veronaea botryosa was sequenced and aligned for the design of specific padlock probes for the detection of characteristic single-nucleotide polymorphisms. The assay demonstrated to successfully amplify DNA of target fungi, allowing detection at the species level. Amplification products were visualized on 1% agarose gels to confirm specificity of probe–template binding. Amounts of reagents were reduced to prevent the generation of false positive results. The simplicity, tenderness, robustness and low expenses provide padlock probe assay (RCA) a definite place as a very practical method among isothermal approaches for DNA diagnostics.

Effects of temperature on Veronaea botryosa infections in white sturgeon Acipenser transmontanus and fungal induced cytotoxicity of fish cell lines

Veronaea botryosa is a melanized mold and cause of systemic fungal infections in cultured sturgeon (Acipenser spp.). Mortality in adult female sturgeon caused by this emergent pathogen results in significant economic losses for the caviar industry. Little is known regarding environmental conditions conducive to V. botryosa infection. This study evaluated the effect of temperature on V. botryosa infectivity and dissemination following intramuscular injection challenge of white sturgeon maintained at 13 or 18 °C for 40 days. Daily mortality was recorded and persistence of the fungus in the livers of moribund and surviving fish was investigated using culture and histopathological analysis. Fish maintained at 18 °C developed systemic phaeohyphomycosis and had significantly greater mortality than controls and fish maintained at 13 °C (p < 0.05). Challenged fish, regardless of temperature, exhibited lesions in multiple organs. However, muscle lesions, angioinvasion, and systemic dissemination were more severe and widespread in fish challenged at the higher temperature. In vitro cytotoxicity of V. botryosa was evaluated in white sturgeon skin (WSSK-1) and epithelioma papulosum cyprini (EPC) cell lines inoculated at spore:cell ratios of 1:10, 1:1 and 10:1, then incubated 15, 20 and 25 °C. Cytotoxicity, as indicated by quantifying the release of lactate dehydrogenase into culture supernatants, increased with increasing spore dose and incubation temperature in both fish cell lines. Findings suggest that temperature significantly influences the development of systemic V. botryosa infection in white sturgeon and that WSSK-1 and EPC cells are suitable in vitro models for the study of host–pathogen interactions between V. botryosa and fish epithelial cells.

Diversity of Veronaea botryosa from different hosts and evaluation of laboratory challenge models for phaeohyphomycosis in Acipenser transmontanus.

Veronaea botryosa has been identified as a pathogen of cultured white sturgeon Acipenser transmontanus. In 2015, samples from 19 white sturgeon were received for diagnosis, of which 14 cultured positive for V. botryosa. Intraspecific variability among V. botryosa isolates from different clinically affected hosts and geographic regions was investigated using repetitive extragenic palindromic PCR fingerprinting (rep-PCR). The rep-PCR profiles of 16 V. botryosa isolates from a human, sea turtles, and cultured fish were distinct from those of other phaeoid fungi belonging to the genera Cladophialophora and Exophiala. To gain a better understanding of the pathogenesis of V. botryosa mycosis, 5 laboratory challenge methods were evaluated in white sturgeon fingerlings. Intramuscular (IM) and intracoelomic (IC) injection challenges produced cumulative mortalities of 13.3% (8/60) and 3.3% (2/60), respectively, and V. botryosa was recovered from 100% (10/10) of dead fingerlings. Affected fish exhibited abnormal orientation and/or failure to maintain neutral buoyancy, emaciation, coelomic distension, exophthalmos, cutaneous erythema, and ulcerated skin. After 6 wk, surviving fish were euthanized, and samples of liver were taken for mycological evaluation. Viable fungus was detected in 90% and 100% of fish surviving IM and IC challenge, respectively. No V. botryosa-associated mortality was detected in other groups challenged by immersion, immersion with abrasion, or orally. Both IM and IC challenge routes appear suitable for the induction of V. botryosa infection in white sturgeon and can serve as models for the study of disease pathogenesis associated with this emergent pathogen.

A rare fungal infection: Phaehyphomycosis due to Veronaea botryosa and review of literature

We report a rare case of phaehyphomycosis in a 71-year-old heart transplant recipient Togo native patient. Four months after the transplant, he presented painless nodules on the right heel with superficial ulceration. The polyphasic identification uncovered a rare cause of phaehyphomycose: V. botryosa. The treatment combined surgical excision of the lesions and anti-fungal therapy with posaconazole. We discussed eleven reported cases in literature since 1990.

First report of Veronaea botryosa as a causal agent of chromomycosis in frogs.

A dematiaceous hyphomycete, isolated from frogs, was determined as the possible etiologic agent of a case of systemic chromomycosis this cold-blooded animal. The fungus was identified as Veronaea botryosa on the basis of morphological features observed in histopathological examination and molecular phylogenetic evidence. Although V. botryosa is known to be distributed widely in litter and as a human pathogen, this is the first confirmed report of its involvement in a lethal infection in a cold-blooded animal, including an anuran.

New disease records for hatchery-reared sturgeon. II. Phaeohyphomycosis due to Veronaea botryosa.

A series of fungal cases in hatchery-reared juvenile and young adult Siberian sturgeon Acipenser baerii and white sturgeon A. transmontanus occurred at production facilities in Florida and California, USA, respectively. Affected fish exhibited abnormal orientation and/or buoyancy, emaciation, coelomic distension, exophthalmos, cutaneous erythema, and ulcerative skin and eye lesions. Necropsies revealed haemorrhage throughout the coelom, serosanguinous coelomic effusion and organomegaly with nodular or cystic lesions in multiple organs. Fungal hyphae were observed in 27 fish (24 A. baerii and 3 A. transmontanus) via microscopic examination of tissue wet mounts and on slides prepared from colonies grown on culture media. Histopathological examination of these infected tissues revealed extensive infiltration by melanised fungal hyphae that were recovered in culture. Phenotypic characteristics and sequencing of the fungal isolates with the use of the internal transcribed spacer region and 28S rRNA gene confirmed the aetiological agent as Veronaea botryosa. To our knowledge, this is the first documentation of V. botryosa infection in fish, although melanised fungi of the closely related genus Exophiala are well-known pathogens of freshwater and marine fishes.

Severe Disseminated Phaeohyphomycosis in an Immunocompetent Patient Caused by Veronaea botryosa

We present a severe case of disseminated phaeohyphomycosis due to Veronaea botryosa. A 32-year-old female, native from Cuautla, Morelos, Mexico, presented a chronic dermatosis which started 10 years earlier with multiple exophytic, multilobulated, soft, and pedunculated or sessile neoformations of diverse sizes from 2 to 10 cm in diameter, which became verrucose and increased in size. The patient was immunocompetent, and no hereditary or familiar precedents of importance were known. No treatment was given, and the dermatosis remained relatively stable until the patient became pregnant in 2001 and 2003. The infection then exacerbated and worsened, leading to dissemination to the extremities, trunk, and face. The initial diagnosis was chromoblastomycosis which was treated with terbinafine and itraconazole but without visible improvement. Histopathology revealed pigmented, irregular, unbranched, and septate hyphae. Veronaea botryosa was isolated (CBS 127264 = JX566723), and its identity was confirmed by sequencing the internal transcribed spacer (ITS) rDNA. Therapy with posaconazole (800 mg/day) was started showing a gradual improvement of lesions with a reduction in size and flattening of the eruptions.

Veronaea botryosa: Molecular Identification with Amplified Fragment Length Polymorphism (AFLP) and In vitro Antifungal Susceptibility

Inter- and intraspecific genomic variability of 18 isolates of Veronaea botryosa originating from clinical and environmental sources was studied using amplified fragment length polymorphism (AFLP). The species was originally described from the environment, but several severe cases of disseminated infection in apparently healthy individuals have been reported worldwide. All tested strains of V. botryosa, identified on the basis of sequencing and phenotypic and physiological criteria prior to our study, were confirmed by AFLP analysis, yielding a clear separation of V. botryosa as a rather homogeneous group from related species. In vitro antifungal susceptibility testing resulted in MIC90s across all strains in increasing order posaconazole (0.25 μg/ml), itraconazole (1 μg/ml), voriconazole (4 μg/ml), terbinafine (4 μg/ml), caspofungin (8 μg/ml), anidulafungin (8 μg/ml), isavuconazole (16 μg/ml), amphotericin B (16 μg/ml), and fluconazole (32 μg/ml). Overall, the isolates showed a uniform pattern of low MICs of itraconazole and posaconazole, but high MICs for remaining agents. The echinocandins (caspofungin and anidulafungin) had no activity against V. botryosa. There was no statistically significant difference between susceptibilities of environmental (n = 11) and clinical (n = 7) isolates of V. botryosa (P > 0.05).

A rare complication of ear piercing: a case of subcutaneous phaeohyphomycosis caused byVeronaea botryosain China

We present the third case of phaeohyphomycosis caused by Veronaea botryosa in China and the tenth case worldwide. A 16-year-old Chinese girl developed crusted, verrucous lesions, initially on the left ear and later on the left buttock, within 2-5 months of receiving an ear piercing. Histopathological examination of biopsy specimens confirmed diagnosis of subcutaneous phaeohyphomycosis. Microscopic examination of the colonies recovered in culture from a portion of the biopsy specimen resulted in the identification of Veronaea botryosa based primarily on the presence of two-celled, brownish pigmented, cylindrical conidia produced sympodially from erect conidiogenous cells. The lesions significantly improved with daily oral treatment with itraconazole 400 mg and adjuvant thermotherapy for 6 months. A maintenance therapy with low dose itraconazole was prescribed in order to achieve clinical and mycological cure. A two-year follow-up didn't reveal any recurrence of infection. Our case is the first report of V. botryosa infection associated with a cosmetic procedure, which suggests that skin piercing could precipitate V. botryosa or other dematiaceous, as well as opportunistic fungal infections.

Cutaneous phaeohyphomycosis caused by Veronaea botryosa observed as sclerotic cells in tissue

A 65‐year‐old Japanese female farmer had an erythematous, slightly scaly, indurated plaque on the dorsum of the left wrist of more than 3 years’ duration. She had a history of hepatitis C. Her medical history and family history were otherwise unremarkable. The lesion had begun as a small region of erythema on the dorsum of the left wrist 3 years previously and had gradually enlarged. The patient had seen a local physician and had been treated with topical steroids, topical antifungals, and topical vitamin D3 agents without improvement.Initial laboratory studies revealed slightly decreased numbers of white blood cells and thrombocytes, decreased albumin levels, and elevated liver enzymes, reflecting chronic hepatitis C. Clinical examination of the lesion revealed a well‐demarcated, erythematous, slightly scaly, indurated plaque of 22 mm × 28 mm in size on the dorsum of the left wrist (Fig. 1). Superficial lymph nodes were nonpalpable. Direct microscopy revealed aggregates of thick‐walled spores, considered to be sclerotic cells, from which budding brown hyphae emanated (Fig. 2a).A well‐demarcated, erythematous, slightly scaly, indurated plaque of 22 mm × 28 mm in size on the dorsum of the left wristimage(a) Microphotograph of scales in KOH reveals aggregates of thick‐walled, brown sclerotic cells and budding brown hyphae (×400). (b) Conidia are bicellular, gourd‐shaped at maturity and pale brown. They are sympodially produced by darker conidiogenous cells (slide culture, ×400)imageCutaneous biopsy specimens revealed a chronic granulomatous inflammation infiltrated with lymphocytes, histiocytes, neutrophils, and multinucleated giant cells. Within the microabscess and within one of the giant cells were large, brown, thick‐walled cells, which were identified as sclerotic cells, and brown septate hyphae.Culture on a Sabouraud dextrose agar slant at 26 °C yielded villous, green‐tinged, black–gray colonies, with a central button‐like protuberance. By 28 days, these colonies had reached 4.8 cm in diameter. A slide culture showed pale brown septate hyphae bearing lateral and terminal conidiophores. Conidiophores were erect, straight, or flexuous, unbranched or occasionally loosely branched, smooth‐walled, septate, and brown. Conidiogenous cells were terminal or lateral and cylindrical in the apical part with numerous dark brown scars fading away to the apex. Conidia were sympodial and bicellular, with rounded apices and truncated bases (Fig. 2b). On the basis of these morphologic features, we identified the mold as Veronaea botryosa. Surgical excision of the lesion was performed, resulting in complete healing.