Species separation in <Emphasis Type="Italic">Curvularia</Emphasis> “geniculata” group inferred from <Emphasis Type="Italic">Brn1</Emphasis> gene sequences

 Brn1, a reductase gene involved in the melanin biosynthetic pathway, was adopted for species delimitation among members in the “geniculata” group of Curvularia species and proved to be useful for this purpose. Phylogenetic trees of these fungal members were constructed from nucleotide sequences of this region. The so-called geniculata group of Curvularia was separated into several clusters. The conidial morphology of the members in each cluster is closely similar but clearly different among discrete clusters. The phylogenetic groups almost concurred with the morphological grouping. Thus, the synonymous treatment of Curvularia affinis, C. fallax, and C. senegalensis to C. geniculata in a previous study was supported. The isolates with warping hilum conidia were clearly different from C. geniculata and separated into two clusters. C. geniculata ATCC 6671 made an independent cluster situated near these clusters. The protuberant hilum species were located separately in the phylogenetic trees. For sound taxonomic treatment of these isolates, we should accumulate more information and retain our species determination for them. Received: September 26, 2002 / Accepted: March 12, 2003     

Effect of polymer differences on fungal penetration of hydrogel lenses

Summary Two chemically distinct types of hydrogel lenses, vifilcon A and bufilcon A, each with a water content of 55%, were challenged in a balanced salts solution withAspergillus fumigatus, Cladosporium cladosporioides, Curvularia lunata andFusarium solani. The lenses were cleaned, disinfected and stained after varying periods of incubation and examined with light microscopy and scanning electron microscopy. For three of the four fungi, the bufilcon A lens was more susceptible to fungal attack than the vifilcon A lens.Curv. lunata produced the greatest number of penetration pegs within 72 h for both lens types. Etching of lens surfaces was observed withC. cladosporioides. In general, the susceptibility of a hydrogel lens to penetration with a fungus appeared to vary with the species of fungus and the chemical composition of the lens.     

Violent Spore Release in Some Fungi Imperfecti

Violent spore discharge in the Hyphomycetes Alternaria tenuis,Memnoniella supsimplex, Curvularia lunata, C. geniculata, Corynesporacassiicola, and Zygophiala jamaicensis occurs after transferfrom a humid to a dry atmosphere. Mechanisms of discharge aresuggested.     

TWO DIFFERENT SPECIES OF EUGLENA,E. GENICULATA AND E. MYXOCYLINDRACEA (EUGLENOPHYCEAE), ARE VIRTUALLY GENETICALLY AND MORPHOLOGICALLY IDENTICAL1

We investigated the similarity of a single Euglena myxocylindracea strain, isolated originally by Bold and MacEntee, to several Euglena geniculata strains on both morphological and DNA levels. We found the three DNA stretches, consisting of fragments coding for the parts of cytoplasmic and chloroplast small subunit rRNA, and the internal transcribed spacer (ITS2) of cytoplasmic rDNA, with the combined length of 4332 nucleotides, are identical in E. myxocylindracea and E. geniculata, strain SAG 1224‐4b. Morphological differences between E. myxocylindracea and any E. geniculata strain examined were well within the range of E. geniculata variability as well. The only difference behind the distinction of E. myxocylindracea from E. geniculata is the presence of the second chloroplast in the latter. However, we were able to induce the appearance of the second chloroplast in the cells of E. myxocylindracea and its disappearance in the cells of E. geniculata by changing the composition of the culture media. We therefore conclude that E. myxocylindracea Bold and MacEntee should be regarded as an environmental form of E. geniculata Dujardin. For the first time the morphology of E. geniculata chloroplasts was shown as revealed by confocal laser microscopy.     

Eumycetoma caused by Curvularia lunata in a dog

Curvularia lunata was cultured from black granules found in granulomatous tumefactions excised from the subcutis of a three year old Medium Schnauzer dog. Draining sinuses were present in some of the tumefactions. Accordingly the diagnosis of eumycotic mycetoma was made. This diagnosis was confirmed by histopathological examination. During the four years following the first surgical intervention, several more similar tumefactions were excised on three different occasions. The dog died of chronic renal failure at the age of 8 years. There was no bone involvement or visceral diffusion of the fungus. The granules were examined by scanning electron microscopy. Immunoglobulins in the dog's serum, assessed by a qualitative test, proved to be equal to immunoglobulins in the serum of a control dog. Precipitating antibodies against C. lunata were not found. The dog was treated for 150 days with itraconazole. In spite of good initial results, recurrence of the fungal lesions were observed after the treatment's interruption. Further treatment with itraconazole for 45 days proved ineffective. No side effects of the drug were observed. This is, to the best of our knowledge, the first case in which C. lunata is identified as the causative agent of an animal eumycetoma.     

A search for microscopic fungi with directed hydroxylase activity for the synthesis of steroid drugs

The hydroxylase activities of new strains such as Curvularia lunata, C. geniculata, C. eragrostidis, C. prasadii, Ulocladium botrytis, Alternaria tenuis, and Fusarium oxysporum toward three steroid substrates, namely, androstenedione (AD), cortexolone (S), and dehydroepiandrosterone acetate (DAA), were characterized. The 9α-hydroxylase activity of C. lunata 1011 cells against S to form 9α-hydroxy-S was shown for the first time. It was found that C. geniculata 837 and F. oxysporum 11dn1 strains can hydroxylate substrates to form pharmacologically promising 7α-hydroxysteroids. C. geniculata 837 cells selectively hydroxylate AD, resulting in 7α-hydroxytestosterone, whereas F. oxysporum 11dn1 leads to the transformation of DAA to 7α-hydroxydehydroepiandrosterone.     

The effect of prednisolone trimethylacetate on the pathogenicity of dermatophilus congolensis to white mice

Albino mice were successfully infected intravenously, subcutaneously, intraperitoneally and on the intact and scarified skin with Dermatophilus congolensis. Various gross lesions were observed. Large subcutaneous nodules were produced in mice that were injected subcutaneously. Lesions involving the internal organs were obtained in mice that had been injected intravenously and intraperitoneally in addition to microabsessation and scab formation on the tails of those that were injected intravenously. The application of D. congolensis on scarified skin resulted in scab formation covering the whole area while its application on non-scarified skin produced isolated lesions. The organism was isolated in pure culture from exudate, pus and scab material obtained from lesions. Severe histopathological changes were noted in sections prepared from the lesions. Premedication of mice with prednisolone trimethylacetate increased the severity of the experimentally induced lesions.     

Protection induced in BALB/c mice by the high-molecular-mass (hMM) fraction of <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis>

Paracoccidioidomycosis (PCM) is a granulomatous disease caused by a dimorphic fungus, Paracoccidioides brasiliensis. The present study investigated the protective activity of the P. brasiliensis high-molecular-mass (hMM) fraction (~380 kDa) in experimental murine PCM. In the first step, lymphocyte proliferation and production of IFNγ (but not IL-4) were observed in “in vitro” spleen cells (from female BALB/c mice infected (i.v.) with P. brasiliensis) that were stimulated with hMM fractions. In the second step, female BALB/c mice were previously immunized (s.c.) with hMM fraction (25 μg/protein = F-25 and 50 μg/protein = F-50), and the colony-forming units (CFU) of the lung and spleen, the histopathological characteristics of the granulomatous lesions, and plasmatic gp43 soluble antigens and anti-hMM IgG levels were analyzed at 28 and 56 days after infection. The lung and liver CFU were lower in mice previously immunized with the hMM fraction (P < 0.05). The granulomatous lesions revealed a greater degree of compaction and organization, with no dissemination of the fungus to other organs. Lower soluble antigen levels (P < 0.05) and higher IgG anti-hMM fraction (P < 0.05) were observed in immunized groups. The results for CFU, histopathology and antigenemia suggest that the hMM fraction has a protective effect in experimental paracoccidioidomycosis in BALB/c mice.     

Culture Conditions Affect Colonization of Watermelon by Colletotrichum Orbiculare

Area of lesions produced by Colletotrichum orbiculare on watermelon (Citrullus lanatus) seedlings was not affected by amount of culture medium on which the fungus was grown, and decreased as length of time the fungus was in culture increased. Internal integrity of C. orbiculare spores appeared degraded, and this was coincident with decreases in lesion area.     

Micosis cutánea por Curvularia pallescens en un paciente trasplantado pulmonar: primer caso descrito en España

BackgroundCurvularia is a filamentous dematiaceous fungus increasingly recognized as a pathogen in immunocompromised patients. The most common clinical entities associated with this fungus are allergic sinusitis, cutaneous infection and keratitis. In this article, a report on the first clinical case of Curvularia pallescens cutaneous infection in Spain and its treatment is described.Case reportA 68 year-old man with a history of lung transplantation presented to Dermatology Unit due to a skin lesion in the knee that had been evolving for 6 months. A skin biopsy was performed for its study. In the histopathological study, an intense and non-specific inflammatory reaction in the dermis was observed, and with Grocott stain and periodic acid Schiff abundant septate hyphae and spores were found in the dermis. The culture of the sample revealed a filamentous fungus whose microscopic examination allowed to identify the genus as Curvularia. Using MALDI-TOF mass spectrometry and molecular identification, the fungus was finally identified as Curvularia pallescens. The patient underwent surgical resection of the lesion and was treated with posaconazole, evolving favorably.ConclusionsThe species of Curvularia should be considered causal agents of fungal skin infections in immunosuppressed patients. This clinical case, which showed good clinical response after surgical resection and treatment with posaconazole, is the first described in Spain due to this species.     

Paronychia and black discoloration of a thumb nail caused by Curvularia lunata

Chronic paronychia associated with black discoloration of the left thumb nail in a 51 year old female caused by Curvularia lunata is reported for the first time. The keratolytic activity of the fungus in the nail and its complete clearance by topical clotrimazole are reported.     

Subcutaneous infection of mice with Paracoccidioides brasiliensis induces a peculiar pattern of inflammatory and immune responses

It is well established that resistance or susceptibility to Paracoccidioidis brasiliensis infection in mice is under strict host's genetic control. Mice from A/Sn strain inoculated by the ip route are resistant to fungal infection while infection induced in mice from B10.A strain results in a fatal disease. The early cellular events of infection in both strains are characterized by a marked neutrophilic infiltration that is more prominent in B10.A mice. A peculiar characteristic of the Paracoccidioides brasiliensis-mouse model is that the subcutaneous (sc) inoculations of the fungus either in resistant (A/Sn) or susceptible (B10.A) mice is self-curing and turns mice from the B10.A strain able to express typical DTH reaction to fungal antigens, as observed in A/Sn mice. Here we report the investigation on the early events of the inflammatory response induced by the inoculation of live fungus into the hind footpad of A/Sn (resistant) and B10.A (susceptible) mice. The influence of neutrophils on the inflammatory response and antibody titers or DTH response to gp43, the major fungal antigen, was also evaluated. Results showed a different course of the inflammatory response induced by fungal inoculation in A/Sn and B10.A mice. Neutrophil depletion before infection differently influenced the kinetics of the inflammatory process in both mice strains but did not modifythe fungal load in the lesions. In neutrophil depleted mice from both strains, a decrease in DTH response and an increase in total antibody titers to gp43 were observed. The significant increase in the fungal load in lesions seen in nude mice indicates that the self-limited infection evoked by fungal inoculation into the subcutaneous tissue is a T-cell dependent phenomenon. The implications of these observations in the pathogenesis of paracoccidioidomycosis are discussed.This revised version was published online in October 2005 with corrections to the Cover Date.     

Genome constitutions of Pseudoroegneriageniculata, P. geniculata ssp. scythica and P. geniculata ssp. pruinifera (Poaceae: Triticeae) revealed by genomic in situ hybridization

Genomic constitutions of three taxa of Pseudoroegneria á. L?ve, P. geniculata, P. geniculata ssp. scythica, and P. geniculata ssp. pruinifera, were characterized using genomic in situ hybridization (GISH). The results indicated that ploidy level and genomic constitution in the three Pseudoroegneria taxa studied were as follows: P. geniculata (2n = 4x = 28; genomic formula StSt ^g ), P. geniculata ssp. scythica (2n = 4x = 28; genomic formula ESt), and P. geniculata ssp. pruinifera (2n = 6x = 42; genomic formula EESt). P. geniculata ssp. scythica and P. geniculata ssp. pruinifera should be transferred to Trichopyrum á. L?ve following L?ve’s principles and treated as T. scythicum and T. pruiniferum, respectively.     

Comparative total and proportional rate of germination of Bipolaris sorokiniana and Curvularia geniculata conidia is influenced by culture age and temperature

Comparative observations of Bipolaris sorokiniana and Curvularia geniculata conidia germination as influenced by culture age and temperature showed some distinct differences, but generally established the ability of these organisms to function under similar conditions. Total germination of B. sorokiniana conidia was favored by increasing culture age from 20 to 60 days and temperature to 25°C; total conidia germination of C. geniculata was favored by increasing temperature to 25°C, but increasing culture age decreased germination. These reactions seem associated with conidia age. Maximum proportional intra-population germination of conidia of each organism also varied with culture age and temperature. At temperatures of 5°C and 15°C, amplitude of maximum proportional germination of both organisms increased as culture age was increased from 20 to 40 days and then decreased among 60-day-old cultures. At 25°C and above, amplitude of maximum proportional germination of conidia of both organisms decreased from each older culture. Progressively increasing temperature at a given culture age increased the amplitude of maximum proportional germination up to 25°C for conidia of B. sorokiniana, but generally decreased it for conidia of C. geniculata (except 20-day-old cultures). Frequency (specific 2 h interval) of maximum proportional intrapopulation germination of B. sorokiniana shifted from 6 h to 2 h in response to increasing temperature and culture age; conidia from youngest cultures of C. geniculata shifted to intervals of 4 h and 2 h in response to increasing temperature to 25°C, but among conidia from 60-day-old cultures, frequency shifted to 6 h intervals at all temperatures. Above 25°C, maximum proportional germination of C. geniculata conidia from cultures of all ages occurred at 6 h. It was concluded that the germination response of B. sorokiniana and C. geniculata conidia to temperature and culture age (and, subsequently, conidia age) are enough similar that these organisms could function in a potential ‘disease complex’ on Poa pratensis and Agrostis palustris.     

Differential cytotoxicity and mycotoxin content among isolates of Fusarium moniliforme

Curvularia lunata was found causing (disseminated phaeohyphomycosis among a group of Nezara viridula (Insecta:Heteroptera) parasitizing vegetable crop Vigna unguiculata. Dark lesions were seen on pronotum and abdominal sterna. Experimental lesions were produced by applying 0.1 ml of 6.2 × 10⁸cfu/ml^–1 on abdominal sterna. Histopathology revealed that almost all internal organs and tissues showed extensive damage. It is interesting to note that C. lunata exhibited predeliction for chitinous tissues and elicited cellular immune response by granulocytes (phagocytosis). This is the first report of phaeohyphomycosis of an insect, extending the disease to invertebrates.A promising research career of one of the authors (Mrs. Vinita Dubey) was cut short by untimely death. This paper is dedicated to her memory.     

Survival of Curvularia lunata var. aeria in soil

Spore suspensions from 7 day old cultures of Curvularia lunata (Wakker) Boedijn var. aeria (Batista, Lima and Vasconcelos) M.B. Ellis were added to soil samples originally devoid of Curvularia propagules. The test fungus disappeared after six weeks of inoculation irrespective of soil amendments, like the application of green manure, urea, superphosphate, or soil cropped to Sorghum (dura) or cotton seedlings.     

Short communication: Protoplast formation from the thermophilic fungus Malbranchea sulfurea,using the thermostable chitinase and laminarinase of Paecilomyces varioti

Two thermostable enzymes produced by the thermophilic fungus Paecilomyces varioti, a chitinase and laminarinase, were used to isolate protoplasts of a thermophilic fungus, Malbranchea sulfurea. The frequency of protoplast regeneration observed (35%) was considerably higher than that obtained using commercial lytic enzymes.     

Experimental histoplasmosis capsulati in athymic nude mice

Granuloma formation in nude (nu/nu) mice and their heterozygous littermates (nu/+ mice) against Histoplasma capsulatum var. capsulatum infection was studied.A culture of H. capsulatum var. capsulatum, isolated from a granuloma in the nasal cavity of a Japanese patient, was used in this experiment. Sixteen specific-pathogen-free male nu/nu and 32 nu/+ mice were used in this study.The nu/+ mice were divided into two groups. Sixteen nu/+ mice in one group and 16 nu/nu mice were inoculated intraperitoneally with 10⁶ yeast cells of the fungus, those in the other group of nu/+ mice were inoculated intravenously with the same number of the yeast cells. Two mice out of each group were sacrificed 2, 3, 7, 11, 14, 18, 25 and 30 days after inoculation, and each of their organs was examined histopathologically. In addition, pieces of these tissues were cultured on Sabouraud's dextrose agar slants.In the nu/+ mice inoculated intraperitoneally, although the fungus was recovered from the spleen, kidney and lymph nodes during the initial course of the infection, lesions were not detected in their histopathological sections. In the nu/+ mice inoculated intravenously, colonies were recovered from all of the organs examined, other than the brain and thymus, 7 days after inoculation.Histopathologically, a few microfoci consisting chiefly of mononuclear cells with or without yeast cells were found in the liver sections 4 days after inoculation. Seven and 11 days after inoculation the number of lesions had increased. They had large accumulations of mononuclear cells. From day 14 on, almost all of the yeast cells had lost most of their staining affinity or were destroyed in the granuloma. From day 25 on, the granulomatous lesions changed gradually to fibrous tissue.In the nu/nu mice the fungus was readily recovered from the spleen, liver, kidney and lymph nodes. Histopathologically, a few microfoci consisting of mononuclear cells were present in the liver sections 4 days after inoculation. That is to say, during the initial course of infection granulomas were formed. In the liver, from day 7 on, the lesions were large and their number increased. However, there was a definite difference between the nu/nu and nu/+ mice. In the former, the yeast cells were not killed, and they continued to multiply within the granulomas. These granulomas were never transformed into fibrous tissue.     

Paracoccidioidomycosis in nude mice: Presence of filamentous forms of the fungus

Congenitally athymic nude mice (nu/nu) and their phenotypically normal littermates (nu/+) were intraperitoneally infected with yeast cells of a strain of Paracoccidioides brasiliensis. The nude mice developed a severe and generalized infection with an intense parasitism of several organs, accompanied by a low-grade of tissue reaction. The lesions were characterized by abundant yeast-like cells of the fungus, and in some animals, numerous hyphal forms could be well visualized. In control animals, infection was moderate, almost exclusively restricted to the area of inoculation, and the lesions presented few parasites surrounded by an inflammatory response. Filamentous forms of the fungus were never encountered in these animals.