Fungal Culture Collection Research Articles

Axenic Culture and DNA Barcode Identification of Wood Decay Fungi from the Maltese Islands

Wood-decaying fungi are important study subjects for their ecological role as well as for their biotechnological applications. They break down lignin, cellulose, and hemicelluloses using enzymes that modify the chemical structure of these complex macromolecules. Due to their ability to degrade wood, these fungi can create structural damage to wooden structures and to trees, especially those with very low level of fitness. Previous studies on wood decay fungi in the Maltese Islands are limited to records and checklists described by a handful of authors. The aim of this study was to provide a comprehensive description of wood decay fungal diversity in the Maltese Islands including an updated checklist based on DNA barcoding, as well as to establish the first wood-decay fungal culture collection at the Biology Department Seed Bank of the University of Malta. Several surveys were carried out during the rainy season along wooded areas of the Maltese Islands as well as in historical gardens. Isolates were identified using macro- and micro-morphological features, dichotomous keys, as well as molecular data. Basidiomes were recorded growing on 14 different host plant species, 11 axenic cultures have been made and 9 species of wood decay fungi have been conclusively identified by DNA barcoding. The collection of the axenic isolates includes one of Aurificaria cf. euphoria, three of Ganoderma resinaceum sl., two of Laetiporus sulphureus, one of Inonotus sp., one of Inonotus rickii anamorph, one of Inocutis tamaricis, one of Stereum hirsutum, and one of Pleurotus eryngii. However, the mycelium of Coriolopsis gallica, though collected and identified, could not be isolated.

An Example of the Conservation of Wood Decay Fungi: The New Research Culture Collection of Corticioid and Polyporoid Strains of the University of Salamanca (Spain)

Over the last decade, fungal conservation has become an increasingly important topic, especially for species tied to forest ecosystems. Among these, wood decay fungi are a group of interesting species from ecological and applicative points of view. Culture collections represent an important tool for the conservation of species and research material. The aim of this study was to establish the first research culture collection of wood decay fungal strains, mainly corticioid and polyporoid species, at Salamanca University (Spain). From two areas of the Iberian Peninsula, a total of 120 basidiomata were collected and morphologically identified. From these, 55 strains were successfully isolated in pure culture and their identity was confirmed by DNA molecular analysis. The average growth rate of each strain was recorded and mycelium characteristics, such as colony morphology and microscopic features, were described. Notable strains in the collection included: (1) Botryobasidium asperulum and Phlebia rufa for taxonomical studies; (2) Hericium erinaceus, Grifola frondosa and Pleurotus species for medicinal properties; (3) Irpex lacteus, Phanerochaete sordida and Trametes versicolor for their degradation capabilities; (4) Stereum gausapatum and Stereum hirsutum for their applicative and enzymatic potential. The new fungal strain culture collection represents a valuable tool for the ex situ conservation of Mediterranean wood decay fungi.

Isolation, Identification, Chemical and Biological Synthesis of silver nanoparticles andits Antimicrobial Activity

Abstract: Extracellular biosynthesis of silver nanoparticles (AgNPs) by Aspergillus Fumigatus isolated from Soil was reported in the present study. The biosynthesis of AgNPs was monitored by ultraviolet-visible spectroscopy and its antimicrobial activity, Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC). The isolated fungus was identified by observing morphological characteristics and Lactophenol cotton blue staining and confirmed as Aspergillus fumigatus from the report given by National Fungal Culture Collection of India (NFCCI). Based on the study, it is concluded that the AgNP’s which is produced by biological methods is showing the antimicrobial activity

First report of wilt disease in cashew (Anacardium occidentale L.) caused by Fusarium decemcellulare in Kerala, India

BackgroundThe incidence of wilt disease of cashew (Anacardium occidentale L.) caused by Fusarium sp. has recently emerged in two-year-old cashew trees in an experimental field at the Regional Agricultural Research Station, Pilicode, in the Kasaragod district of Kerala, India. The disease was characterized initially by chlorosis of foliage and shoots, followed by necrosis and wilting, with internal symptoms including vascular necrosis, eventually resulting in complete plant mortality.MethodsA fungal pathogen was isolated repeatedly from the infected vascular tissues onto potato dextrose agar medium. Symptomology, cultural, morphological and molecular studies were performed to characterize the pathogen causing the disease.ResultsIn culture, the colony of fungus was white, later turning pink. The fungus produced both macro and micro conidia in culture. Molecular characterization revealed the identity of the isolates as Fusarium decemcellulare Brick. Pathogenicity of the isolate was confirmed in cashew seedlings, thereby demonstrating Koch’s postulates.ConclusionTo our knowledge, this is the first record of wilt disease of cashew caused by F. decemcellulare in India. The culture of the fungus has been deposited with the National Fungal Culture Collection of India at Agharkar Research Institute, Pune, India with accession number NFCCI 4801, and sequence of the fungus was deposited at GenBank of NCBI with accession number OP942472.

350. Clinical impact of positive fungal blood cultures for diagnosis and treatment of fungal infections.

Abstract Background Fungal blood cultures are usually ordered when sepsis secondary to disseminated fungal infection is suspected. We aimed to analyze whether positive fungal blood cultures had an added clinical impact over other conventional microbiological tests. Methods We performed a retrospective study of all patients for whom fungal blood cultures were performed for any indication at our institution from June 2018 to March 2022. We reviewed informatics database and medical records to assess the microbiological and clinical impact of positive fungal blood cultures during the initial admission. Assessment of clinical impact was analyzed using a list of predefined positive, negative, and no impact scenarios (Table 1) based by the treating team’s decisions. Results In total, 4447 fungal blood cultures were performed in 3648 admissions during our study period. The overall positivity rate of fungal blood cultures was 6.4% (n=284), of which only 130 (2.9%) were positive for fungi. The most common isolated fungi were Candida spp. (71, 55%), followed by Histoplasma spp. (16, 12%), Cryptococcus spp. (12, 9%), and unidentified mold (9, 7%) (Table 1). The median time to positivity was 106 hours (IQR 79-177). Only 21 (16%) fungal blood cultures resulted in a change in management. Positive fungal blood cultures for fungi led to a positive clinical impact in 17 (13%), negative clinical impact in 4 (3%), and no clinical impact in 109 (84%). Most fungal blood cultures confirmed other conventional microbiological diagnosis (71, 55%) or result was not acted upon (26, 20%). Organisms where fungal blood culture had a positive impact included C. albicans (n=4), and C. parapsilosis (n=3), while Cladosporium spp. (n=2) was the most frequent fungi with a negative clinical impact. Isolated fungi that had no clinical impact because of other confirmatory tests included Candida spp. (n=57), Histoplasma spp. (n=13) and Cryptococcus neoformans (n=10). Table 1.Microbiological characteristics and clinical impact of fungal blood cultures.Abbreviations: h hours; IQR, interquartile range.*Time to positivity was defined as the time from the fungal blood culture collection until growth and preliminary identification of a fungal organism on staining by the microbiology laboratory. **Time to identification was defined as the time from the fungal blood culture collection until identification of the fungal organism by the microbiology laboratory. Conclusion Most of positive fungal blood cultures for fungi resulted in no immediate clinical impact due to other microbiological tests available sooner or isolates thought to be non-clinically significant. Further studies should evaluate the long-term impact of fungemia and identify stewardship interventions to optimize clinical utility of fungal blood cultures. Disclosures All Authors: No reported disclosures.

First Report of Anthracnose Caused by Colletotrichum acutatum on Olive Fruits in Pakistan.

Anthracnose symptoms on olive (Olea europaea) fruits cv. "Gamlick" were found in farmer orchards in Chakwal, Punjab, Pakistan (32° N and 72° E), with an average prevalence of 59%. Fruit symptoms start as thin, black, sunken lesions with a watery appearance that grow in diameter and coalesce into a large sunken soft zone. Lesions on mature fruit become noticeable in 5 to 6 days after infection, if temperatures are favorable (28°C). On the fruit lesion, orange conidial masses in dispersed or concentric circle arrangement can appear. Fragments (5 mm) were taken from the margins of fruit lesions and surface-sterilized with 70% ethanol (1 min) and 1% NaClO (2 min), cleaned with sterile purified water, blotted dry, and plated on potato dextrose agar (PDA) in Petri dishes. The petri plates were incubated at 27°C. A fungus was consistently isolated, and thirty-five isolates were characterized. Aerial mycelia from olive isolates Colonies were compact, initially white or cream white, then grey, and eventually dark grey, with conidium masses forming in the middle. Mycelium is branched, septate, and hyaline. Conidia are hyaline, aseptate, fusiform, or often cylindrical, with obtuse apices and tapering bases. Their mean size was 8.5µm in length and 3.0 µm in width. Based on morphological features, the fungus was tentatively identified as Colletotrichum acutatum (Agosteo G.E., 2010). The identification was confirmed by amplification and sequencing of a representative isolate's internal transcribed region (ITS), Beta- tubulin region (TUB2), Actin region (ACT), and Glyceraldehyde 3-phosphate dehydrogenase region (GAPDH) with the primers ITS1/ITS4 (Gardes & Bruns 1993), TUB4/TUB5 (Woudenberg et al. 2009), ACT1/ACT3 (Carbone & Kohn 1999) and GDF1/GDR1 (Guerber et al. 2003). BLAST analysis revealed 100% identity for ITS, GAPDH and ACT and 99% identity for TUB, between the sequences of the olive fruit isolate (GenBank Accessions MW647502, MZ436968, MZ714412 and MW810331, respectively) and sequences of C. acutatum reference isolates (GenBank Accessions GO613492, KF975660.1, MT274752.1 and MH547616 respectively). Phylogenetic analysis based on ITS, GAPDH and TUB regions of the olive fruit isolates and reference isolates of various Colletotrichum species using the MEGA X software program confirmed the isolate from olive was C. acutatum. The fungal isolate was deposited as a living culture in the Barani Agricultural Research Institute's fungal culture collection center (BACA.9381). Pathogenicity tests were conducted with this isolate by placing a 20 µl drop of a conidial suspension (3 × 107conidia ml-1) on five healthy olive cv. Gemlik fruits. As a control, five non-inoculated olive fruits were used. Fruits were placed at a temperature of 27°C with artificial light and a photoperiod of 12 hours. Anthracnose symptoms developed only on inoculated fruits after seven days of inoculation. The fungus was re-isolated from symptomatic fruits, and its identity was confirmed through morphological characteristics, thus verifying Koch's postulates. To the best of our knowledge, this is the first report of C. acutatum infecting olive fruits in Chakwal region of Pakistan.

Change in dominant genotype of Microsporum canis, a causative fungus of zoonotic dermatophytosis, in Japan over the past 40 years

Multilocus microsatellite typing was performed on 124 strains of Microsporum canis, which is the most prevalent causative fungus of zoonotic dermatophytosis, isolated in Japan between 1974 and 1981. The strains had been enclosed in glass ampoules by freeze dried process. Genotypes were detected by capillary electrophoresis targeted on six microsatellite regions, and 19 genotypes were found among these 124 strains. The most frequent genotype, which had been reported as genotype L in a previous study, comprised 56 of the 124 strains (45.2%), although genotype L was reported to comprise only six of 165 strains (3.6%) isolated between 2010 and 2017. A decrease in the prevalence of genotype L may have led to the retreat of M. canis infection in 1996 to 2006. On the other hand, genotype A, the second most predominant genotype in the aforementioned studies of strains isolated between 2010 and 2017, comprised just four of 124 strains (3.2%) in the present study. Thus, these studies reveal that prevalences of some major genotypes have changed over the last 40 years. Genotype consistency of strains was proven in all of 12 familial cases, each of which was infected with a single genotype. We emphasize the importance of fungal culture collection for further studies with new techniques in the future.

A long-read amplicon approach to scaling up the metabarcoding of lichen herbarium specimens.

Reference sequence databases are critical to the accurate detection and identification of fungi in the environment. As repositories of large numbers of well-curated specimens, herbaria and fungal culture collections have the material resources to generate sequence data for large number of taxa, and could therefore allow filling taxonomic gaps often present in reference sequence databases. Financial resources to do that are however often lacking, so that recent efforts have focused on decreasing sequencing cost by increasing the number of multiplexed samples per sequencing run while maintaining high sequence quality. Following a previous study that aimed at decreasing sequencing cost for lichen specimens by generating fungal ITS barcodes for 96 specimens using PacBio amplicon sequencing, we present a method that further decreases lichen specimen metabarcoding costs. A total of 384 mixed DNA extracts obtained from lichen herbarium specimens, mostly from the four genera Buellia, Catillaria, Endocarpon and Parmotrema, were used to generate new fungal ITS sequences using a Sequel I sequencing platform and the PacBio M13 barcoded primers. The average success rate across all taxa was high (86.5%), with particularly high rates for the crustose saxicolous taxa (Buellia, Catillaria and others; 93.3%) and the terricolous squamulose taxa (Endocarpon and others; 96.5%). On the other hand, the success rate for the foliose genus Parmotrema was lower (60.4%). With this taxon sampling, greater specimen age did not appear to impact sequencing success. In fact, the 1966–1980 collection date category showed the highest success rate (97.3%). Compared to the previous study, the abundance-based sequence denoising method showed some limitations, but the cost of generating ITS barcodes was further decreased thanks to the higher multiplexing level. In addition to contributing new ITS barcodes for specimens of four interesting lichen genera, this study further highlights the potential and challenges of using new sequencing technologies on collection specimens to generate DNA sequences for reference databases.

English

Infectious diseases, including skin infections, have always been treated by plants and other natural products since ancient times. This study investigated the antimicrobial activity of nine medicinal plant species from Gonsé Forest and Bangreweogo Park, Burkina Faso on seven microbial strains involved in skin infections. Sixty-eight organic and aqueous extracts from leaves, stem and root barks of the nine plants were tested on five bacterial and two fungal American Type Culture Collection (ATCC) strains using the disk diffusion and microdilution methods. Twenty-eight extracts (41.17%) from eight of the plants differently inhibited bacteria and fungi, the exception being Wissadula amplissima specie. Gram-positive Cocci (GPC) were the most sensitive to the extracts. Eleven extracts had a Minimum Inhibitory Concentration (MIC) lower than 1 mg/ml (MIC < 1 mg/ml). The extracts of Opilia celtidifolia gave the largest zones of inhibition ranging from 21.67 to 24.33 mm at 100 to 300 mg/ml on GPC and Candida albicans. Its MIC varied from 0.03 to 0.06 mg/ml on Streptococcus pyogenes, and from 0.07 to 0.24 mg/ml on Streptococcus agalactiae. Ethanolic extracts (80 and 96%) of Lawsonia inermis had MIC ranging from 0.29 to 9.37 mg/ml on the sensitive strains. The variable inhibitory activity displayed by the plants confirmed in part their traditional use in the treatment of some bacterial and fungal skin infections. Key words: Infectious diseases, inhibitory activity, plant extracts, minimal bactericidal concentration (MBC), minimum inhibitory concentration (MIC), skin infections.

Fungus-bacterium associations are widespread in fungal cultures isolated from a semi-arid natural grassland in Germany.

We report on a study that aimed at establishing a large soil-fungal culture collection spanning a wide taxonomic diversity and systematically screening the collection for bacterial associations. Fungal cultures were isolated from soil samples obtained from a natural grassland in eastern Germany and bacterial associations were assessed by PCR-amplification and sequencing of bacterial 16S rRNA. In addition, intraspecies genetic diversities of a subset of the isolated species were estimated by double-digest restriction associated DNA sequencing. A total of 688 fungal cultures, representing at least 106 fungal species from 36 different families, were obtained and even though clonal isolates were identified in almost all fungal species subjected to ddRAD-seq, relatively high genetic diversities could be observed in some of the isolated species. A total of 69% of the fungal isolates in our collection were found to be associated with bacteria and the most commonly identified bacterial genera were Pelomonas, Enterobacter and Burkholderia. Our results indicate that bacterial associations commonly occur in soil fungi, even if antibiotics are being applied during the isolation process, and provide a basis for the use of our culture collection in ecological experiments that want to acknowledge the importance of intraspecies genetic diversity.

Resistance of Cucurbita spp. germplasm tothe fungus Macrophomina phaseolina

The objective of this study was to evaluate genotypes of Cucurbita moschata and Cucurbita maxima for resistance to the fungus Macrophomina phaseolina (Taissi) Goid. The experiment used 17 accessions of Cucurbita moschata Duchesne (ABTOU 805 F1, ABO 168, ABPUN 206 F4, ABPUN 201 F6, ABTOU 805 F4, ABPUN 206 F6, ABPUN 206 F2, ABPUN 206 F1, ROCA DE PAI, ABTOU 805 F4, P114-1, P160-2, P11-2, P114-6, P131-21, P114-02 and P97-1), 20 accessions of Cucurbita maxima Duchesne (ABPUN-213, ABCRN-304, ABTOU-802-F4, ABAPO-002, ABAPO-005, JCCM-2014, ABO-156, ABPUN-212, ABCRN-315, ABTOU-802 F1, ABAPO-007, JERIMUM LU, ABAPO-024, ABPUN-211, RGV 2011.1 01, ABCRN-306, ABBAR-101, ABCRN-302, ABTOU-802 F3 and JCCA) preserved in the Cucurbit Germplasm Collection of the Federal Rural University of the Semi-Arid Region (UFERSA). Four commercial controls were also used, two for each species. In both studies, three isolates of M . phaseolina were inoculated, which are kept at the Fungal Culture Collection of UFERSA: MM1531 (GenBank: MN136199), ME249 and ME250. Plants received inoculum of the pathogen in pieces of colonized toothpick and were evaluated for incidence and severity of the disease. Both species showed resistant plants, but with varied frequencies for the accessions. Considering the species, the frequencies observed for C. moschata were higher than those observed for C. maxima.

IMPACT OF CORONA DISCHARGE ON ASPERGILLUS NIGER SPORES AND CYATHUS OLLA MYCELIUM GROWTH

The article presents an investigation of the influence of the corona discharge with needle-ring electrode configuration on the efficiency of Aspergillus niger spore germination and Cyathus olla mycelium development. Current-voltage characteristics were measured for different distances between the needle and the metal ring. The plot of the dependence of conductance on voltage was built and the corona discharge breakthrough voltage was determined to be (2.7±0.1) kV. The treatment of Aspergillus niger spores was conducted at different development stages: at the germination stage (corona discharge treatment 1 day after the seeding) and dormant state (corona discharge treatment on the day of seeding). The growth rate of Cyathus olla mycelium in samples treated by corona discharge and in control group samples was investigated. The influence of the corona discharge on the potato dextrose agar growth medium before the seeding of Cyathus olla was studied. The pure cultures were obtained from the Culture Collection of Fungi at Kyiv University (FCKU) at the «Institute of Biology and Medicine» Educational and Scientific Centre of the Taras Shevchenko National University of Kyiv.

Aspergillus sydowii: Genome Analysis and Characterization of Two Heterologous Expressed, Non-redundant Xylanases.

A prerequisite for the transition toward a biobased economy is the identification and development of efficient enzymes for the usage of renewable resources as raw material. Therefore, different xylanolytic enzymes are important for efficient enzymatic hydrolysis of xylan-heteropolymers. A powerful tool to overcome the limited enzymatic toolbox lies in exhausting the potential of unexplored habitats. By screening a Vietnamese fungal culture collection of 295 undiscovered fungal isolates, 12 highly active xylan degraders were identified. One of the best xylanase producing strains proved to be an Aspergillus sydowii strain from shrimp shell (Fsh102), showing a specific activity of 0.6 U/mg. Illumina dye sequencing was used to identify our Fsh102 strain and determine differences to the A. sydowii CBS 593.65 reference strain. With activity based in-gel zymography and subsequent mass spectrometric identification, three potential proteins responsible for xylan degradation were identified. Two of these proteins were cloned from the cDNA and, furthermore, expressed heterologously in Escherichia coli and characterized. Both xylanases, were entirely different from each other, including glycoside hydrolases (GH) families, folds, substrate specificity, and inhibition patterns. The specific enzyme activity applying 0.1% birch xylan of both purified enzymes were determined with 181.1 ± 37.8 or 121.5 ± 10.9 U/mg for xylanase I and xylanase II, respectively. Xylanase I belongs to the GH11 family, while xylanase II is member of the GH10 family. Both enzymes showed typical endo-xylanase activity, the main products of xylanase I are xylobiose, xylotriose, and xylohexose, while xylobiose, xylotriose, and xylopentose are formed by xylanase II. Additionally, xylanase II showed remarkable activity toward xylotriose. Xylanase I is stable when stored up to 30°C and pH value of 9, while xylanase II started to lose significant activity stored at pH 9 after exceeding 3 days of storage. Xylanase II displayed about 40% activity when stored at 50°C for 24 h. The enzymes are tolerant toward mesophilic temperatures, while acting in a broad pH range. With site directed mutagenesis, the active site residues in both enzymes were confirmed. The presented activity and stability justify the classification of both xylanases as highly interesting for further development.

Curvularia eragrostidis, a new threat to large cardamom (Amomum subulatum Roxb.) causing leaf blight in Sikkim

Large cardamom (Amomum subulatum Roxb.) is now affected by several diseases caused by both viruses and fungi. At present, leaf blight is considered a major threat to cardamom cultivation in Sikkim. During the past two decades, cultivation of the crop in this region has dropped by almost 60%. Hence, to quantify the severity of leaf blight damage and identification of the causal organism for the disease, a survey was conducted from May to August 2017 in different large cardamom growing regions of Sikkim. During this survey, a typical symptom of leaf blight was observed on cardamom leaves in many locations. The leaves with blights were collected, surface sterilized, and inoculated on potato dextrose agar (PDA). The pathogen was isolated as pure culture, and on the basis of morphological and microscopic characteristics, the fungus was identified species of Curvularia. Molecular characterization of the fungal isolate with ITS-rDNA partial gene amplification using universal primers (ITS4 and ITS5), showed 100% similarity with Curvularia eragrostidis (family: Pleosporaceae). The fungal isolate and nucleotide sequence was deposited in National Fungal Culture Collection of India (NFCCI), Pune and NCBI with accession numbers NFCCI 4541 and MN710527, respectively. This is the first report on the occurrence of C. eragrostidis pathogen causing leaf blight of large cardamom grown in Sikkim.

Fungi, bacteria and oomycota opportunistically isolated from the seagrass, Zostera marina.

Fungi in the marine environment are often neglected as a research topic, despite that fungi having critical roles on land as decomposers, pathogens or endophytes. Here we used culture-dependent methods to survey the fungi associated with the seagrass, Zostera marina, also obtaining bacteria and oomycete isolates in the process. A total of 108 fungi, 40 bacteria and 2 oomycetes were isolated. These isolates were then taxonomically identified using a combination of molecular and phylogenetic methods. The majority of the fungal isolates were classified as belonging to the classes Eurotiomycetes, Dothideomycetes, and Sordariomycetes. Most fungal isolates were habitat generalists like Penicillium sp. and Cladosporium sp., but we also cultured a diverse set of rare taxa including possible habitat specialists like Colletotrichum sp. which may preferentially associate with Z. marina leaf tissue. Although the bulk of bacterial isolates were identified as being from known ubiquitous marine lineages, we also obtained several Actinomycetes isolates and a Phyllobacterium sp. We identified two oomycetes, another understudied group of marine microbial eukaryotes, as Halophytophthora sp. which may be opportunistic pathogens or saprophytes of Z. marina. Overall, this study generates a culture collection of fungi which adds to knowledge of Z. marina associated fungi and highlights a need for more investigation into the functional and evolutionary roles of microbial eukaryotes associated with seagrasses.

Epidemiology and Clinical Management of Fusarium keratitis in the Netherlands, 2005-2016.

Introduction: Recognizing fungal keratitis based on the clinical presentation is challenging. Topical therapy may be initiated with antibacterial agents and corticosteroids, thus delaying the fungal diagnosis. As a consequence, the fungal infection may progress ultimately leading to more severe infection and blindness. We noticed an increase of fungal keratitis cases in the Netherlands, especially caused by Fusarium species, which prompted us to conduct a retrospective cohort study, aiming to describe the epidemiology, clinical management, and outcome.Materials and Methods: As fungi are commonly sent to the Dutch mycology reference laboratory for identification and in vitro susceptibility testing, the fungal culture collection was searched for Fusarium isolates from corneal scrapings, corneal swabs, and from contact lens (CL) fluid, between 2005 and 2016. All Fusarium isolates had been identified up to species level through sequencing of the ITS1-5.8S-ITS2 region of the rDNA and TEF1 gene. Antifungal susceptibility testing was performed according to the EUCAST microbroth dilution reference method. Antifungal agents tested included amphotericin B, voriconazole, and natamycin. In addition, susceptibility to the antisepticum chlorhexidine was tested. Ophthalmologists were approached to provide demographic and clinical data of patients identified through a positive culture.Results: Between 2005 and 2016, 89 cases of Fusarium keratitis from 16 different hospitals were identified. The number of cases of Fusarium keratitis showed a significant increase over time (R2 = 0.9199), with one case in the first 5 years (2005–2009) and multiple cases from 2010 and onwards. The male to female ratio was 1:3 (p = 0.014). Voriconazole was the most frequently used antifungal agent, but treatment strategies differed greatly between cases including five patients that were treated with chlorhexidine 0.02% monotherapy. Keratitis management was not successful in 27 (30%) patients, with 20 (22%) patients requiring corneal transplantation and seven (8%) requiring enucleation or evisceration. The mean visual acuity (VA) was moderately impaired with a logMAR of 0.8 (95% CI 0.6–1, Snellen equivalent 0.16) at the time of Fusarium culture. Final average VA was within the range of normal vision [logMAR 0.2 (95% CI 0.1–0.3), Snellen equivalent 0.63]. CL wear was reported in 92.9% of patients with Fusarium keratitis. The time between start of symptoms and diagnosis of fungal keratitis was significantly longer in patients with poor outcome as opposed to those with (partially) restored vision; 22 vs. 15 days, respectively (mean, p = 0.024). Enucleation/evisceration occurred in patients with delayed fungal diagnosis of more than 14 days after initial presentation of symptoms. The most frequently isolated species was F. oxysporum (24.7%) followed by F. solani sensu stricto (18%) and F. petroliphilum (9%). The lowest MICs were obtained with amphotericin B followed by natamycin, voriconazole, and chlorhexidine.Conclusion: Although Fusarium keratitis remains a rare complication of CL wear, we found a significant increase of cases in the Netherlands. The course of infection may be severe and fungal diagnosis was often delayed. Antifungal treatment strategies varied widely and the treatment failure rate was high, requiring transplantation or even enucleation. Our study underscores the need for systematic surveillance of fungal keratitis and a consensus management protocol.

Onygenalean Fungi as Major Human and Animal Pathogens

During the last two decades, many onygenalean pathogens were discovered, redefined, or re-classified from existing taxa into clusters of micro-species, among which the original genotypes often appeared to be uncommon and exceptional. The impact of these developments on the diagnosis and treatment of fungal diseases remains to be determined in most instances. This exciting collection of invited articles provides a full flavor of ongoing changes in the knowledge about taxonomy, genetics, ecology, epidemiology, and clinical spectra of human and animal pathogens classified in the order Onygenales. Recent developments have set the stage for an ambitious translational research agenda. Diagnostic mycology laboratories now need MALDI-TOF-MS spectra, PCR probes, and other specific tools to assist them in the rapid diagnosis of new species. Similarly, an educational set of type materials of new species needs to be readily available for enhanced expertise among the wider medical mycology community. As several new species were discovered retrospectively, it is crucial to expand the re-sampling to other fungal culture collections and archived paraffin tissues. Finally, clinical and laboratory investigations are needed to get an accurate assessment of the prevalence and impact of new pathogens as the cause of major fungal diseases.

<p><strong><em>Strelitziana sarbhoyi </em></strong><strong>sp. nov.<em> </em>(Strelitzianaceae, Chaetothyriales), from North-Western Himalayas, India, described based on morphology and molecular phylogeny</strong></p>

Strelitziana sarbhoyi is established as a new species to accommodate a phylloplane fungus isolated from Mallotus philippensis collected from Kangra region of North-Western Himalayas, Himachal Pradesh. The identity of the fungus is confirmed based on the asexual-morphs, cultural characteristics and phylogenetic analyses of the internal transcribed spacer (ITS) rDNA and partial nuclear ribosomal 28S large subunit (LSU) sequence data. The placement of S. sarbhoyi in the phylogenetic tree was determined based on DNA sequences from authenticated isolates of Strelitziana. Strelitziana sarbhoyi shows nearly 94% similarity with other known species of Strelitziana. Area description is provided for the proposed taxon along with microscopic images, and a phylogenetic tree. This is probably the first report of Strelitziana from India. Holotype specimen (dried voucher culture) is deposited in the Ajrekar Mycological Herbarium (AMH), and an ex-type culture is deposited in National Fungal Culture Collection of India (NFCCI).

<p><strong><em>Kamalia indica,</em></strong><strong> a new genus and species belonging to Diaporthales (Ascomycota, Fungi) described based on morphology and molecular phylogeny</strong></p>

A fungal taxa isolated from leaf spots of Mallotus philippensis from Kangra district of North-Western Himalayan region of India is established as a new genus based on morphological characters of asexual-morphs, cultural characteristics and phylogenetic analyses of the partial nuclear ribosomal 28S large subunit (LSU) and internal transcribed spacer (ITS) rDNA sequence data. The generic placement of the genus has been determined based on DNA sequences from authenticated isolates. The present taxon has turned out to be distinct, showing nearly 90% identity with other known genera in Diaporthales based on nrDNA internal transcribed spacer region. The morphological description is provided for the new taxa and compared with the similar taxa belonging to the order Diaporthales. The culture was found to show heavy sporulation in all kind of media. The type specimen and ex type culture have been deposited in the Ajrekar Mycological Herbarium (AMH) and National Fungal Culture Collection of India (NFCCI-WDCM 932), respectively.

Morphology and phylogeny of a new species of Trichomerium from North-Western Himalayas, India

The present paper describes Trichomerium bhatii, a new species in the family Trichomeriaceae (Chaetothyriales, Ascomycota). This taxon was isolated as epiphyte from dead bark of Prunus cerasoides collected from North-Western Himalayan region of India. The identity of isolate was based on asexual-morphs, cultural characteristics and phylogenetic analyses of partial nuclear ribosomal 28S large subunit and complete internal transcribed spacer rDNA sequence data with high statistical support value. The new taxon has turned out to be distinct from other known species in the genus showing 95.26% (624/655 bp) identity with closely related T. gloeosporum MFLUCC10-0087 on the basis of rDNA internal transcribed spacer region. A brief description of asexual-morph of T. bhatii with morphological and molecular data is provided. To the best of our knowledge this is the first report on this genus from India. The type specimen is deposited in the Ajrekar Mycological Herbarium (as voucher culture) and ex type cultures are deposited in Indian Type Culture Collection (ITCC-WDCM 430), New Delhi, and National Fungal Culture Collection of India (NFCCI-WDCM 932).