Establishment of conidial fusion in the asexual fungus Verticillium dahliae as a useful system for the study of non-sexual genetic interactions.

Cell-to-cell fusion is a fundamental biological process across the tree of life. In filamentous fungi, somatic fusion (or anastomosis) is required for the normal development of their syncytial hyphal networks, and it can initiate non-sexual genetic exchange processes, such as horizontal genetic transfer and the parasexual cycle. Although these could be important drivers of the evolution of asexual fungi, this remains a largely unexplored possibility due to the lack of suitable resources for their study in these puzzling organisms. We thus aimed at the characterization of cell fusion in the important asexual fungus Verticillium dahliae via Conidial Anastomosis Tubes (CATs), which can be useful for the analysis of parasexuality. We optimized appropriate procedures for their highly reproducible quantification and live-cell imaging, which were used to characterize their physiology and cell biology, and to start elucidating their underlying genetic machinery. Formation of CATs was shown to depend on growth conditions and require functional Fus3 and Slt2 MAP kinases, as well as the NADPH oxidase NoxA, whereas the GPCR Ste2 and the mating-type protein MAT1-2-1 were dispensable. We show that nuclei and other organelles can migrate through CATs, which often leads to the formation of transient dikaryons. Their nuclei have possible windows of opportunity for genetic interaction before degradation of one by a presumably homeostatic mechanism. We establish here CAT-mediated fusion in V. dahliae as an experimentally convenient system for the cytological analysis of fungal non-sexual genetic interactions. We expect that it will facilitate the dissection of sexual alternatives in asexual fungi.

Sarocladium and Acremonium infections: New faces of an old opportunistic fungus.

The genera Acremonium and Sarocladium comprise a high diversity of morphologically and genetically related fungi generally found in the environment, although a few species, mainly Sarocladium kiliense and Acremonium egyptiacum, can also be involved in many human infections. Clinical management of opportunistic infections caused by these fungi is very complex, since their correct identification is unreliable, and they generally show poor antifungal response. More than 300 clinical cases involving a broad range of Acremonium/Sarocladium infections have so far been published, and with this review we aim to compile and provide a detailed overview of the current knowledge on Acremonium/Sarocladium human infections in terms of presentation, diagnosis, treatments and prognoses. We also aim to summarise and discuss the data currently available on their antifungal susceptibility, emphasising the promising results obtained with voriconazole as well as their impact in terms of animal infections.

Metabolic engineering of Acremonium chrysogenum for improving cephalosporin C production independent of methionine stimulation.

BACKGROUND: Cephalosporin C (CPC) produced by Acremonium chrysogenum is one of the most important drugs for treatment of bacterial infectious diseases. As the major stimulant, methionine is widely used in the industrial production of CPC. In this study, we found methionine stimulated CPC production through enhancing the accumulation of endogenous S-adenosylmethionine (SAM). To overcome the methionine dependent stimulation of CPC production, the methionine cycle of A. chrysogenum was reconstructed by metabolic engineering. RESULTS: Three engineered strains were obtained by overexpressing the SAM synthetase gene AcsamS and the cystathionine-γ-lyase gene mecB, and disrupting a SAM dependent methyltransferase gene Acppm1, respectively. Overexpression of AcsamS resulted in fourfold increase of CPC production which reached to 129.7 µg/mL. Disruption of Acppm1 also increased CPC production (up to 135.5 µg/mL) through enhancing the accumulation of intracellular SAM. Finally, an optimum recombinant strain (Acppm1DM-mecBOE) was constructed through overexpressing mecB in the Acppm1 disruption mutant. In this strain, CPC production reached to the maximum value (142.7 µg/mL) which was 5.5-fold of the wild-type level and its improvement was totally independent of methionine stimulation. CONCLUSIONS: In this study, we constructed a recombinant strain in which the improvement of CPC production was totally independent of methionine stimulation. This work provides an economic route for improving CPC production in A. chrysogenum through metabolic engineering.

A super-infection in the cornea caused by Stemphylium, Acremonium, and α-Streptococcus.

BACKGROUND: Polymicrobial keratitis with fungus and bacteria can lead to blindness and is challenging to treat. Here, we introduce a case of fungal keratitis caused by two different strains in addition to definite bacterial super-infection caused by an α-Streptococcus sp., and describe the importance of microscopic examination. CASE PRESENTATION: A 74-year-old woman, who had a past history of infection with leprosy, presented with conjunctival hyperaemia, pain, and corneal opacity in her right eye. Under the presumptive diagnosis of infectious keratitis, corneal scrapings were stained by various reagents and inoculated on several agar plates. Microscopic findings of the scrapings revealed fungi and a small number of Gram-positive cocci. Multiple anti-fungal therapies with levofloxacin ophthalmic solution were administered. Although empiric treatment was initially effective, keratitis recurred 10 days after its initiation. Repeated corneal scraping revealed an abundance of Gram-positive chain cocci and a small amount of fungi, resulting in the switching of an antibiotic medication from levofloxacin to moxifloxacin and cefmenoxime. Keratitis resolved gradually after the conversion. Stemphylium sp., Acremonium sp., and α-Streptococcus sp. were simultaneously isolated from the corneal scrapings. CONCLUSIONS: To the best of our knowledge, this is the first case of fungal keratitis caused by Stemphylium sp., and also the first case of super-infection in the cornea caused by two different fungi and one bacterium. Microscopic examination of the corneal scrapings was beneficial in rapid decision of changing to appropriate drug according to the dominancy of pathogenicity.

Identification of Cephalosporium stripe resistance quantitative trait loci in two recombinant inbred line populations of winter wheat.

KEY MESSAGE: Identification of genome regions linked to Cephalosporium stripe resistance across two populations on chromosome 3BS, 4BS, 5AL, C5BL. Results were compared to a similar previous study. Cephalosporium stripe is a vascular wilt disease of winter wheat (Triticum aestivum L.) caused by the soil-borne fungus Cephalosporium gramineum Nisikado & Ikata. In the USA it is known to be a recurring disease when susceptible cultivars are grown in the wheat-growing region of Midwest and Pacific Northwest. There is no complete resistance in commercial wheat cultivars, although the use of moderately resistant cultivars reduces the disease severity and the amount of inoculum in subsequent seasons. The goal of this study was to detect and to compare chromosomal regions for resistance to Cephalosporium stripe in two winter wheat populations. Field inoculation was performed and Cephalosporium stripe severity was visually scored as percent of prematurely ripening heads (whiteheads) per plot. 'Tubbs'/'NSA-98-0995' and 'Einstein'/'Tubbs', each comprising a cross of a resistant and a susceptible cultivar, with population sizes of 271 and 259 F (5:6) recombinant inbred lines, respectively, were genotyped and phenotyped across four environments. In the quantitative trait loci (QTL) analysis, six and nine QTL were found, explaining in total, around 30 and 50 % of the phenotypic variation in 'Tubbs'/'NSA-98-0995' and 'Einstein'/'Tubbs', respectively. The QTL with the largest effect from both 'NSA-98-0995' and 'Einstein' was on chromosome 5AL.1 and linked to marker gwm291. Several QTL with smaller effects were identified in both populations on chromosomes 5AL, 6BS, and 3BS, along with other QTL identified in just one population. These results indicate that resistance to Cephalosporium stripe in both mapping populations was of a quantitative nature.

[Onychomycoses due to molds]. / Les onychomycoses à moisissures.

Onychomycoses represent about 30% of superficial mycosis that are encountered in Dermatology consults. Fungi such as dermatophytes, which are mainly found on the feet nails, cause nearly 50% of these onychopathies. Yeasts are predominantly present on hands, whereas non-dermatophytic moulds are very seldom involved in both foot and hand nails infections. According to literature, these moulds are responsible for 2 to 17% of onychomycoses. Nevertheless, we have to differentiate between onychomycoses due to pseudodermatophytes such as Neoscytalidium (ex-Scytalidium) and Onychocola canadensis, which present a high affinity for keratin, and onychomycoses due to filamentous fungi such as Aspergillus, Fusarium, Scopulariopsis, Acremonium... These saprophytic moulds are indeed most of the time considered as colonizers rather than real pathogens agents. Mycology and histopathology laboratories play an important role. They allow to identify the species that is involved in nail infection, but also to confirm parasitism by the fungus in the infected nails. Indeed, before attributing any pathogenic role to non-dermatophytic moulds, it is essential to precisely evaluate their pathogenicity through samples and accurate mycological and/or histological analysis. The treatment of onychomycoses due to non-dermatophytic moulds is difficult, as there is today no consensus. The choice of an antifungal agent will first depend on the species that is involved in the infection, but also on the severity of nail lesions and on the patient himself. In most cases, the onychomycosis will be cured with chemical or mechanical removing of the infected tissues, followed by a local antifungal treatment. In some cases, a systemic therapy will be discussed.

Experimental murine acremoniosis: an emerging opportunistic human infection.

Acremonium is an emerging fungal pathogen causing severe infections. We evaluated the virulence of three clinically relevant species within the genus, i.e., Acremonium kiliense (currently Sarocladium kiliense), Acremonium sclerotigenum-A. egyptiacum complex and Acremonium implicatum in a murine model of disseminated infection. Both immunocompetent and immunosuppresssed mice were infected with two inocula concentrations (2 × 10(6) and 2 × 10(8) conidia/animal) of two strains of each species. Tissue burden, mortality rate, histopathology and levels of (1→3)-ß-D-glucan were used as virulence markers. None of the species of Acremonium tested was able to cause infection in immunocompetent mice. Conversely, severe infections were produced in immunocompromised mice, the spleen being the most affected organ. In general, the virulence of the Acremonium species tested was low, S. kiliense being the most virulent species.

Evaluation of the efficacies of Amphotericin B, Posaconazole, Voriconazole, and Anidulafungin in a murine disseminated infection by the emerging opportunistic Fungus Sarocladium (Acremonium) kiliense.

We evaluated and compared the efficacies of different antifungal drugs against Sarocladium kiliense (formerly Acremonium kiliense), a clinically relevant opportunistic fungus, in a murine model of systemic infection. Three clinical strains of this fungus were tested, and the therapy administered was as follows: posaconazole at 20 mg/kg of body weight (twice daily), voriconazole at 40 mg/kg, anidulafungin at 10 mg/kg, or amphotericin B at 0.8 mg/kg. The efficacy was evaluated by prolonged animal survival, tissue burden reduction, and (1→3)-ß-d-glucan serum levels. In general, the four antifungal drugs showed high MICs and poor in vitro activity. The efficacy of the different treatments was only modest, since survival rates were never higher than 40% and no drug was able to reduce fungal load in all the organs for the three strains tested. Posaconazole, in spite of its high MICs (≥16 µg/ml), showed the highest efficacy. The (1→3)-ß-d-glucan serum levels were equally reduced by all drugs evaluated.

An unusual cluster of Acremonium kiliense fungaemias in a haematopoietic cell transplantation unit.

The genus Acremonium (formerly known as Cephalosporium) is a large polyphyletic fungal genus that comprises approximately 150 phylogenetically distant species, commonly isolated from the environment. Clinical cases concern mostly superficial infections after traumatic inoculation, but there are reports of opportunistic invasive infections in immunocompromised patients. Acremonium kiliense has been described as a cause of mycetoma, keratitis, endophthalmitis, endocarditis, and continuous ambulatory peritoneal dialysis-associated peritonitis. We describe an unusual cluster of possible catheter-related bloodstream infections due to this pathogen in patients who underwent haematopoietic cell transplantation.

Caracterización morfológica de Acremonium sp. asociado a Neonectria fuckeliana en Pinus radiata en Chile / Morphological characterization of Acremonium-like asociated with Neonectria fuckeliana in Pinus radiata in Chile

En este estudio se realizaron los primeros aislamientos del estado asexual del patógeno Neonectria fuckeliana asociado a cancros o ®revirado del pino¼ en plantaciones de Pinus radiata. Se caracterizaron morfológicamente las cepas del sinanamorfo semejante a Verticillium (Acremonium), obtenidas en cultivo in vitro a partir de peritecios. El material para los aislamientos consistió en trozos de corteza de P. radiata con presencia de peritecios, colectados en Toltén, región de La Araucanía, lugar donde se realizó el primer reporte de N. fuckeliana en Chile. Se utilizaron diez cepas del semejante a Acremonium para la caracterización morfológica, mediciones de estructuras fúngicas, ritmo de crecimiento in vitro y morfología de las colonias. Las colonias presentaron un micelio flocoso y ralo de bordes blanquecinos e irregulares, destacándose tres tipos de colonias, blancas, naranjo oscura y naranjo claro. Taxonómicamente, las cepas coinciden con las estructuras mencionadas en la literatura, caracterizándose por la presencia de glioconidios. Las fialides midieron entre 7 – 78,4 x 1,4 - 4,9 ìm. Los conidios, de formas ovoides y algunas bicelulares, midieron entre 4,2 - 8,4 x 2,6 - 3,5 ìm. El ritmo de crecimiento in vitro fue lento, completando su desarrollo a los 19 días con un promedio de 71 +/- 0,3 mm de diámetro, a una tasa de crecimiento diario de 3,8 mm. Los resultados obtenidos hacen necesario futuros estudios de carácter molecular para analizar la variabilidad genética poblacional que puede presentarse en Chile.

First Neonectria fuckeliana isolates in Chile. Strains of Acremonium obtained from in vitro peritecia culture were morphologically characterized. The samples were collected in Toltén, La Araucanía region, were Neonectria fuckeliana was first reported in Chile. The material used for the isolation came from pieces of Pinus radiata bark with peritecia. Ten Acremonium-like strains were used for characterization of fungal structures, in vitro growth and strains morphology. The colonies were a floccose mycelium and thin edges whitish and irregular, varying color highlighting three types of strains, white, dark orange and pale orange. Taxonomically, the strains match the structures referred in the literature, characterized by the presence of gliospores. The phialides dimensions ranged from 7 to 7.8 ìm long and 1.4 to 4.9 ìm wide. The conidio of ovoid shapes and some bicelular measured between 4.2 to 8.4 ìm in length and width 2.6 and 3.5 ìm. In vitro growth rates were slow, the complete development take19 days with a daily growth average of 71 +/-3 mm in diameter, at a rate of 3.8 mm. It is necessary future molecular studies to analyze the population genetic variation that may occur in Chile.

Quantitative trait loci analysis for resistance to Cephalosporium stripe, a vascular wilt disease of wheat.

Cephalosporium stripe, caused by Cephalosporium gramineum, can cause severe loss of wheat (Triticum aestivum L.) yield and grain quality and can be an important factor limiting adoption of conservation tillage practices. Selecting for resistance to Cephalosporium stripe is problematic; however, as optimum conditions for disease do not occur annually under natural conditions, inoculum levels can be spatially heterogeneous, and little is known about the inheritance of resistance. A population of 268 recombinant inbred lines (RILs) derived from a cross between two wheat cultivars was characterized using field screening and molecular markers to investigate the inheritance of resistance to Cephalosporium stripe. Whiteheads (sterile heads caused by pathogen infection) were measured on each RIL in three field environments under artificially inoculated conditions. A linkage map for this population was created based on 204 SSR and DArT markers. A total of 36 linkage groups were resolved, representing portions of all chromosomes except for chromosome 1D, which lacked a sufficient number of polymorphic markers. Quantitative trait locus (QTL) analysis identified seven regions associated with resistance to Cephalosporium stripe, with approximately equal additive effects. Four QTL derived from the more susceptible parent (Brundage) and three came from the more resistant parent (Coda), but the cumulative, additive effect of QTL from Coda was greater than that of Brundage. Additivity of QTL effects was confirmed through regression analysis and demonstrates the advantage of accumulating multiple QTL alleles to achieve high levels of resistance.

Acremonium species: a review of the etiological agents of emerging hyalohyphomycosis.

Unusual fungal agents that exist environmentally as saprophytes can often lead to opportunistic infections. Hyalohyphomycosis is a group of fungal infections caused by fungi characterized by hyaline septate hyphae and can infect both immunocompetent as well as immunocompromised patients. Many a times it becomes difficult to distinguish a pathogenic and a contaminant fungus, because many such agents can assume clinical significance depending on circumstances. Subcutaneous and invasive fungal infection due to the emerging hyalohyphomycotic fungus, Acremonium, has drawn the attention of clinicians and microbiologists, as a potential pathogen in patients with and without underlying risk factors. Generally considered to be minimally invasive in the past, genus Acremonium has been responsible for eumycotic mycetomas and focal infections in otherwise healthy individuals. It has also been increasingly implicated in systemic fungal diseases. The management with different antifungals in various clinical situations has been very conflicting and hence needs to be carefully evaluated. This overview is an endeavor to consolidate the available clinical infections due to Acremonium and the recommendations on treatment.

A case of colonization of a prosthetic mitral valve by Acremonium strictum

Se describe un caso de colonización por el hongo Acremonium strictum W. Gams en un paciente varón trasla implantación de una válvula mitral protésica. El paciente falleció a causa de fallo multiorgánico. La identificacióndel hongo se realizó morfológicamente y se confirmó mediante análisis de las secuencias de laregión ITS. Los estudios moleculares demuestran que A. strictum constituye un complejo de especies. Elpresente caso enfatiza el alto riesgo potencial de infección fúngica para los pacientes con prótesis de válvulascardíacas(AU)

A case of colonization of a prosthetic mitral valve in a 73-year-old Spanish male by the fungus Acremoniumstrictum W. Gams is described. The valve was replaced due to paravalvular leak and severe insufficiencyand the patient died of multiorgan failure. The identity of the fungus was determined by morphologicalstudies and it was confirmed by the analysis of the ITS region sequence analysis. Molecular studies seem todemonstrate that A. strictum is a species complex. The case emphasizes the potential high risk of fungalinfection for patients with prosthetic valves(AU)

Mycoparasitism of Acremonium strictum BCP on Botrytis cinerea, the gray mold pathogen.

A fungal strain BCP, which parasitizes Botrytis cinerea gray mold pathogen, was isolated and identified as Acremonium strictum. BCP strain overgrew the colonies of B. cinerea and caused severe lysis of the host hyphae. Frequent penetration and hyphal growth of A. strictum BCP inside the mycelia of B. cinerea were observed under light microscopy. In addition, some morphological abnormalities such as granulation and vacuolation of the cytoplasm were observed in mycelia and spores of B. cinerea. In dual culture test, A. strictum BCP strongly inhibited the mycelial growth of several plant pathogenic fungi as well as B. cinerea. To our knowledge, this is the first report on mycoparasitism of Acremonium species on B. cinerea.

Onicomicosis por Acremonium Kiliense / Onychomycosis by Acremonium Kiliense

El rol de los hongos filamentosos hilalinos no dermatofitos, como agentes causales de onicomicosis, ha sido documentado en las últimas tres décadas por diferentes autores y son considerados oportunistas emergentes. El objetivo de este trabajo es presentar un caso de onicomicosis producido por Acremonium kiliense, cuya evolución fue estudiada. Se presentó al laboratorio de micología un paciente de 18 años de edad de sexo femenino con traumatismo en la uña del primer dedo del pie derecho. Se tomaron muestras seriadas de la lesión para la observación microscópica directa con OHK 40% p/v y para cultivos en Agar Sabouraud, Agar Lactrimel y DTM (medio selectivo para dermatofitos) y con las colonias recuperadas se efectuaron microcultivos. Se implementó tratamiento con ciclopirox laca al 8% combinado al principio con Itraconazol vía oral (100 mg/día), con buena evolución. Es importante tener en cuenta a estos hongos oportunistas como agentes etiológicos potenciales de micosis ungueales, en especial en pacientes inmunodeprimidos, ya que la afección ungueal puede ser la puerta de entrada para la diseminación de la infección.

The role of non-dermatophytic hyaline filamentous fungi as agents of onychomycosis has been documented in the last three decades by different authors, who have considered these fungi emerging opportunists. A 18-year-old female patient with trauma to the first toenail of the right foot came to the mycology laboratory. Serial samples of the lesion were collected for direct microscopic observation using 40% (w/v) potassium hydroxide (KOH) and for culture in Sabouraud agar, Lactrimel agar and DTM (dermatophyte selective medium). Microcultures were grown from the colonies recovered. Treatment with 8% ciclopirox nail lacquer, combined at the beginning with oral administration of itraconazole (100 mg a day), was implemented with good evolution. It is important to consider these opportunist fungi as potential etiological agents for nail mycosis, specially in immunosuppressed patients, since nail disease can be the entry point for the spread of infections.

Human hyalohyphomycoses: a review of human infections due to Acremonium spp., Paecilomyces spp., Penicillium spp., and Scopulariopsis spp.

Human infections due to opportunistic molds are on the rise. This is due to recent advances in medical technology that have led to increased numbers of patients who are immunosuppressed, receiving broad-spectrum antibiotics, or have indwelling medical devices. In this article, human infections caused by four hyalohyphomycoses, Acremonium spp., Paecilomyces spp., Penicillium spp., and Scopulariopsis spp., will be reviewed. Specific areas of focus will include the epidemiology, mycology, clinical presentations, and treatment options for each of these four hyaline molds.

A simple thin-layer chromatographic method for the detection of ergovaline in leaf sheaths of tall fescue (Festuca arundinacea) infected with Neotyphodium coenophialum.

A relatively simple and inexpensive thin-layer chromatographic (TLC) method is described for the detection and semiquantitative measurement of ergovaline in leaf sheaths of tall fescue (Festuca arundinacea). Samples were finely ground and extracted with methanol. The extracts were filtered and the methanol was evaporated. The aqueous residue was extracted with hexane, followed by chloroform at pH 9. The chloroform extract was concentrated and further purified on a preparative silica gel TLC plate, developed with toluene/ethyl acetate/acetonitrile (50:10:40). The ergovaline band was scraped and eluted with methanol. The eluant was concentrated and an aliquot was applied to a silica gel TLC plate. The plate was developed successively with chloroform/acetone/acetic acid (90:10:5) and chloroform/ethanol (9:1). Ergovaline was visualized with p-dimethylaminobenzaldehyde and sulfuric acid. Semiquantitation of ergovaline was achieved by comparison with a known standard of ergotamine, which was shown to have the same Rf as ergovaline in this system. Spike recovery of ergotamine averaged 60%, with a limit of detection of 200 microg/kg of dry tall fescue leaf sheaths. The method was applied to 15 tall fescue samples with varying degrees of fungal infection, and ergovaline was identified in all contaminated samples with endophyte infection above 15%. Thin-layer chromatography may be also applicable for tall fescue seed, where the ergovaline content is usually higher and the amount of interfering pigments is much lower.

Terpendole M, a novel indole-diterpenoid isolated from Lolium perenne infected with the endophytic fungus Neotyphodium lolii.

Terpendole M (1), a novel indole-diterpenoid, was isolated from perennial ryegrass (Lolium perenne) infected with the endophytic fungus Neotyphodium lolii. It was identified as 14alpha-hydroxyterpendole C by NMR and mass spectral techniques. The known indole-diterpenoids paspaline (3) and 13-desoxypaxilline (4) were also isolated from perennial ryegrass for the first time. Terpendole M was less tremorgenic than terpendole C (2) in a standard mouse bioassay. These findings provide clues to the biogenesis of the lolitrem neurotoxins, and information on the structure-activity relationships within the indole-diterpenoids.