Saprochaete clavata invasive infection: characterization, antifungal susceptibility, and biofilm evaluation of a rare yeast isolated in Brazil

ABSTRACT Rare emerging pathogens such as Saprochaete clavata are associated with invasive fungal diseases, high morbidity, mortality, rapidly fatal infections, and outbreaks. However, little is known about S. clavata infections, epidemiology, risk factors, treatment, biofilms, and disease outcomes. The objective of this study was to describe a new case of severe S. clavata infection in a patient diagnosed at a referral children's hospital in Brazil, including antifungal minimal inhibitory concentration, S. clavata biofilm characterization, and molecular characterization. The S. clavata isolated from an immunocompromised 11-year-old male patient was characterized using MALDI-TOF, Gram staining, scanning electron microscopy (SEM), and next generation sequencing (NGS) of genomic DNA. Biofilm production was also evaluated in parallel with determining minimal inhibitory concentration (MIC) and biofilm sensitivity to antifungal treatment. We observed small to medium, whitish, farinose, dry, filamentous margin colonies, yeast-like cells with bacillary features, and biofilm formation. The MALDI-TOF system yielded a score of ≥ 2,000, while NGS confirmed S. clavata presence at the nucleotide level. The MIC values (in mg L-1) for tested drugs were as follows: fluconazole = 2, voriconazole ≤ 2, caspofungin ≥ 8, micafungin = 2, amphotericin B = 4, flucytosine ≤ 1, and anidulafungin = 1. Amphotericin B can be active against S. clavata biofilm and the fungus can be susceptible to new azoles. These findings were helpful for understanding the development of novel treatments for S. clavata-induced disease, including combined therapy for biofilm-associated infections.

Caracterización de Fusarium spp. asociado con la pudrición basal de la cebolla de rama / Characterization of Fusarium spp. associated with the basal rot of the green onion

RESUMEN El departamento de Nariño ocupa el cuarto lugar como productor de cebolla de rama, a nivel nacional. En los últimos años, su producción y área de cultivo se han reducido por múltiples limitantes, destacándose, como la más importante, la susceptibilidad al ataque de hongos causantes de pudriciones radicales, los cuales, perjudican el sistema productivo y la rentabilidad. El objetivo del estudio fue caracterizar morfológica y molecularmente las poblaciones de Fusarium, asociados a la enfermedad de pudrición basal de cebolla de rama. Para ello, en los municipios de Pasto, Potosi y Buesaco, se colectaron plantas con síntomas de pudrición basal, acompañada de necrosis de raíces y ablandamiento de tejido. En el laboratorio de Sanidad Vegetal de la Universidad de Nariño, se sembraron tejidos en medio PDA y, a continuación, se purificaron los aislamientos para su posterior caracterización morfológica y molecular. El estudio morfológico, se realizó usando claves taxonómicas para el género Fusarium y la caracterización molecular con cebadores específicos para el género Fusarium y mediante secuenciación. Finalmente, se realizó un análisis filogenético de la variabilidad intraespecífica. Los resultados de la caracterización morfológica y molecular corroboran la presencia de dos especies dentro del género asociadas a esta patología, F. oxysporum f sp. cepae y F. solani. Los análisis filogenéticos muestran alta variabilidad intraespecífica entre los aislamientos de F. oxysporum y F. solani, formando dos complejos Fusarium oxysporum (FOSC) y Fusarium solani (FSSC), manifestando que estas especies no parten de un ancestro común.

ABSTRACT The department of Nariño occupies the fourth place as producer of green onion nationwide. In recent years, its production and cultivation area has been reduced by multiple limitations, highlighting as the most important, the susceptibility to the attack of fungi causing radical problems, which harm the productive system and profitability. This study was carried out in order to morphologically and molecularly characterizes Fusarium populations associated with green onion basal rot disease. For this, in Pasto, Potosi and Buesaco municipalities, plants were collected with basal rot symptoms, tissue deterioration and root necrosis. In the Plant Health laboratory of the University of Nariño, tissues were planted in PDA medium and subsequently the isolates were purified for further morphological and molecular characterization. The morphological study was carried out using taxonomic keys for the genus Fusarium and molecular characterization with specific primers for the genus Fusarium, and by sequencing. Finally, a phylogenetic analysis of the intraspecific variability was carried out. Morphological and molecular characterization results corroborate the presence of two species within the genus associated with this pathology, F. oxusporum f sp. cepae and F. solani. Phylogenetic analyzes show high intraspecific variability between the isolates of F. oxysporum and F. solani, forming two Fusarium oxysporum (FOSC) complexes and Fusarium solani (FSSC), evidencing that these species do not start from a common ancestor.

Optimization of Fungal Enzyme Production by Trichoderma harzianum KUC1716 through Surfactant-Induced Morphological Changes

The morphological optimization of Trichoderma harzianum was carried out using several surfactants to achieve increased cellulase production. Addition of the surfactants to the culture medium successfully modified the fungal morphology from an aggregated form to a dispersed form. Optimization of the fungal morphology increased cellulase activity up to 177%. The morphologically optimized conditions enhanced the accessibility of the fungus to substrates and thus promoted cellulase production.

Phylogenetic Placement and Morphological Characterization of Sclerotium rolfsii (Teleomorph: Athelia rolfsii) Associated with Blight Disease of Ipomoea batatas in Korea

In this study, we aimed to characterize fungal samples from necrotic lesions on collar regions observed in different sweetpotato growing regions during 2015 and 2016 in Korea. Sclerotia appeared on the root zone soil surface, and white dense mycelia were observed. At the later stages of infection, mother roots quickly rotted, and large areas of the plants were destroyed. The disease occurrence was monitored at 45 and 84 farms, and 11.8% and 6.8% of the land areas were found to be infected in 2015 and 2016, respectively. Fungi were isolated from disease samples, and 36 strains were preserved. Based on the cultural and morphological characteristics of colonies, the isolates resembled the reference strain of Sclerotium rolfsii. Representative strains were identified as S. rolfsii (teleomorph: Athelia rolfsii) based on phylogenetic analysis of the internal transcribed spacer and large subunit genes along with morphological observations. To test the pathogenicity, sweetpotato storage roots were inoculated with different S. rolfsii strains. ‘Yulmi’ variety displayed the highest disease incidence, whereas ‘Pungwonmi’ resulted in the least. These findings suggested that morphological characteristics and molecular phylogenetic analysis were useful for identification of S. rolfsii.

Induced; Thermal Influence on Morphology of Aspergillus carbonarius During Raw Starch Digesting Amylase Production.

Aim: To investigate increased thermal influence on morphology of Aspergillus carbonarius during RSDA production. Place and Duration of Study: Microbial fermentation Unit, Department of Microbiology, Faculty of Biological sciences, University of Nigeria, between July 2009 and August 2010. Methodology: In shake flask cultures thermal influence on A. carbonarius morphology and productivity investigated. Mycelial morphology was characterised by means of image analysis using as parameters, mean diameter, roughness, circularity and compactness of pellet. Thermal effect on amylase activity, total protein, biomass concentration and pH were also investigated. Results: Shifting the temperature from 27ºC to 37ºC significantly affected the morphological parameters of the pellets, but RSDA activity was not altered. The interesting thing about the morphology is the shearing off of the hairy part of the pellet at an increased temperature and subsequent agglomeration. At 27ºC the RSDA activity increased steadily with an optimum activity of 293U/ml at 96h and subsequently decreased to 75U/ml by the end of the fermentation. At 37ºC a maximum activity of 291U/ml was achieved at 72h of fermentation but this decreased to 87U/ml at the end of fermentation. Higher biomass concentration and total protein were obtained at 37ºC. The pH dropped from an initial of 5.0 to 3.0 and 2.5 for 27ºC and 37ºC temperature conditions respectively. Conclusion: Induced thermal increase resulted to changes in pellet morphology but raw starch digesting amylase activity was not altered.

Changes in hyphal morphology due to chitosan treatment in some fungal species

In this work, changes in the hyphal morphology due to chitosan treatment in some fungal species were studied. Scanning electron microscope (SEM) observations revealed that chitosans with molar fraction of acetyl groups (F A 0.16 and 0.18) and degree of polymerization (DP 1,089 and 1,242) had a direct effect on the morphology of the chitosan-treated fungi, reflecting its potential for causing a delay in the growth of Alternaria alternata (500 µg × mL-1), Botrytis cinerea (1,000 µg × mL-1), Penicillium expansum (1,000 µg × mL-1) and Rhizopus stolonifer (500 µg × mL-1). Mycelial aggregation and structural changes such as excessive branching, swelling of the cell wall and hyphae size reduction were observed in the micrographs.

Growth rate inhibition of phytopathogenic fungi by characterized chitosans

The inhibitory effects of fifteen chitosans with different degrees of polymerization (DP) and different degrees of acetylation (F A) on the growth rates (GR) of four phytopathogenic fungi (Alternaria alternata, Botrytis cinerea, Penicillium expansum, and Rhizopus stolonifer) were examined using a 96-well microtiter plate and a microplate reader. The minimum inhibitory concentrations (MICs) of the chitosans ranged from 100 µg × mL-1 to 1,000 µg × mL-1 depending on the fungus tested and the DP and F A of the chitosan. The antifungal activity of the chitosans increased with decreasing F A. Chitosans with low F A and high DP showed the highest inhibitory activity against all four fungi. P. expansum and B. cinerea were relatively less susceptible while A. alternata and R. stolonifer were relatively more sensitive to the chitosan polymers. Scanning electron microscopy of fungi grown on culture media amended with chitosan revealed morphological changes.

Influence of buffer systems on Trichoderma reesei Rut C-30 morphology and cellulase production

The cellulase enzyme production is a key issue in the enzymatic hydrolysis of lignocellulosic materials. Since fungal morphology influences the productivity of fungal fermentations, it is of major importance to well know the fungal behavior during culture for cellulase production. In this work, the influence of medium supplementation, with different buffer systems at two different concentrations and pH conditions, on the morphology of T. reesei Rut C-30 and cellulase production, was investigated. A medium without buffer was used as control. The results suggest that fungal morphology is significantly dependent on the addition of different buffer systems to the nutrient broth. The mycelial morphology shows a clear transition from clumped to pelleted forms in cultures with variation of buffer systems and concentration. The higher filter paper activity was obtained using 100 mM succinate buffer, at pH 4.8, in the medium supplementation, corresponding to a dispersed mycelial morphology.