Applying a Modified Metabarcoding Approach for the Sequencing of Macrofungal Specimens from Fungarium Collections

Premise: Fungaria are a largely untapped source for understanding fungal biodiversity. The effort and cost in producing DNA barcode sequence data for large numbers of fungal specimens can be prohibitive. This study applies a modified metabarcoding approach that provides a labor and cost-effective solution for sequencing the fungal DNA barcode from hundreds of specimens at once. Methods: A two-step PCR approach uses nested barcoded primers to nrITS2 sequence data. We applied this to 766 macrofungal specimens that represent a broad taxonomic sampling of the Dikarya, of which 382 Lactarius specimens are used to identify molecular operational taxonomic units (MOTUs) through a phylogenetic approach. Scripts in Python and R were used to organize sequence data and execute packages CutAdapt and DADA2 were used for primer removal and assessing sequence quality. Sequences were compared to NCBI and UNITE databases and Sanger-produced sequences. Results: Specimen taxonomic identities from nrITS2 sequence data are >90% accurate across all specimens sampled. Phylogenetic analysis of Lactarius sequences identified 20 MOTUs. Discussion: The results demonstrate the capacity of these methods to produce nrITS2 sequences from large numbers of fungarium specimens. This provides an opportunity to more effectively use fungarium collections in advancing fungal diversity identification and documentation.

Fungal DNA extraction for Nanopore sequencing v1

This protocol is intended for extraction of high molecular weight DNA from fungal samples.

Isolation and Identification of Dermatophytes from Collegiate Runners

Competitive runners experience various risk factors that render them more susceptible to superficial cutaneous fungal infections, including the use of occlusive footwear, shared locker rooms, submission of feet to constant maceration, trauma, sweating, and having depressed immune function. The goal of this work was to assess the prevalence of athlete’s foot fungi in cross country runners at St. John Fisher College. Toe webs of 16 collegiate runners were sampled and volunteers surveyed about their shoe habits, foot hygiene, and average miles run per week. Lack of tinea pedis-causing fungi in asymptomatic cross- country runners shifted the study to investigate the identities of fungi morphologically similar to athlete’s foot and look for correlations with volunteers’ running habits and hygiene. Thirty-five distinct fungal cultures were isolated and compared to a known Trichophyton rubrum strain both microscopically and macroscopically. Four samples were preliminarily identified as tinea pedis-causing fungi and sequenced to confirm molecular identification. Fungal DNA was isolated, purified, and PCR amplified using primers for the internal transcribed spacer region, D1/D2 region of the 28S subunit, and β-Tubulin gene. Three of the four isolates were identified as Fusarium equiseti, a soil-borne plant pathogen with rare human pathogenicity reported. The fourth isolate was Beauveria bassiana, a common soil-borne pathogen that can infect immunocompromised individuals. Correct dermatophytic identification and understanding of the interplay between species is important to provide correct treatment, prevent spread among athletes and within facilities, and determine how opportunistic pathogens might play a role in people with immune suppressed function, which includes runners.

The compositional turnover of grapevine-associated plant pathogenic fungal communities are greater among intraindividual microhabitats and terroirs than among healthy and Esca-diseased plants

Grapevine trunk diseases (GTD) are a major threat to the wine industry, causing yield loss and dieback of grapevines. While the increasing damage caused by GTDs in recent decades have spurred several studies on grapevine-associated pathogenic fungi, key questions about the emergence and severity of GTDs remain unanswered, including possible differences in plant pathogenic fungal communities in asymptomatic and symptomatic grapevines. We generated fungal DNA metabarcoding data from soil, bark, and perennial wood samples from asymptomatic and symptomatic grapevines sampled in three terroirs. We observed larger compositional differences in plant pathogenic fungi among different plants parts within grapevine plants than among individual grapevines. This is driven by the dominance of GTD-associated fungi in perennial wood and non-GTD pathogens in soil, as well as by the lack of significant differences among asymptomatic and Esca symptomatic grapevines. These results suggest that fungi generally associated with Esca disease belong to the core grapevine microbiome and likely are commensal endophytes and/or latent saprotrophs, some of which can act as opportunistic pathogens on stressed plants. In addition, we found significant compositional differences among sampling sites, particularly in soil, which suggest a certain influence of local edaphic and mesclimatic factors on plant pathogenic fungal communities. Furthermore, the observed differences among terroirs in plant pathogenic fungal communities in grapevine woody parts indicate that environmental factors likely are important for the development of Esca disease and further studies are needed to investigate the abiotic conditions on fungal compositional dynamics in Esca-affected plants.

78. Non-Invasive Prediction of Invasive Fungal Infection by Plasma-Based Microbial Cell-Free DNA Next-Generation Sequencing (mcfDNA NGS) in Pediatric Patients with Relapsed or Refractory Leukemia

Background Diagnosis of invasive fungal infections (IFIs), a life-threatening complication of cancer therapy or hematopoietic cell transplantation (HCT) can be challenging, and IFI has poor outcomes. Prediction or early non-invasive diagnosis of IFI in high-risk hosts before onset of symptoms could reduce morbidity and mortality. Because non-invasive plasma mcfDNA NGS can detect invasive fungal infections, and may predict bloodstream infections in immunocompromised patients, we hypothesized that mcfDNA NGS might also predict invasive fungal infection before clinical presentation. Methods In a prospective study, serial remnant plasma samples were collected from pediatric patients undergoing treatment for relapsed or refractory leukemia. IFI events were classified according to EORTC criteria by 2 independent experts, and episodes empirically treated for suspected IFI, but not meeting ‘possible’ criteria were classified as ‘suspected’. All samples collected within 30 days before clinical diagnosis of non-fungemic IFI were tested for fungal DNA by mcfDNA NGS using a research-use only assay by Karius, Inc. optimized for fungi; because of overlapping clinical syndromes, non-fungal DNA was not considered in this study. Results There were 15 episodes of suspected IFI in 14 participants with ≥1 sample available from either diagnostic (within 1 day of diagnosis) or predictive (2 to 30 days prior to diagnosis) periods (5 “suspected”, and 4 probable and 6 proven by EORTC definitions). Of 10 probable or proven IFIs, 6 (60%) had a relevant fungal pathogen identified mcfDNA NGS at diagnosis. In each of these cases the fungal DNA was also detectable prior to clinical onset of IFI (Range 2 to 41 days; Figure 1). In an additional case, manual review of sequence data identified the fungal DNA at diagnosis and during the prior month. Of 5 “suspected” IFI episodes, all were determined by expert review as not representing fungal infection; fungal DNA was identified by mcfDNA NGS in 2/54 (3.7%) of samples from these episodes. Table 1. Characteristics of Invasive Fungal Infections Conclusion mcfDNA NGS can identify fungal pathogen DNA before clinical onset of IFI, so might predict IFI in immunocompromised hosts, and may help differentiate fungal infection from other etiologies of lung nodules or infiltrates. Disclosures Joshua Wolf, MBBS, PhD, FRACP, Karius Inc. (Research Grant or Support) Joshua Wolf, MBBS, PhD, FRACP, Nothing to disclose Radha Duttagupta, PhD, Karius inc (Employee) Lily Blair, PhD, Karius Inc. (Employee) Asim A. Ahmed, MD, Karius, Inc. (Employee)

Influence of DNA extraction kits on the fungal DNA metabarcoding for freshwater environmental DNA

Background: Environmental DNA (eDNA) metabarcoding is a rapidly expanding technique for efficient biodiversity monitoring, especially of animals. Recently, the usefulness of aquatic eDNA in monitoring the diversity of both terrestrial and aquatic fungi has been suggested. In eDNA studies, different experimental factors, such as DNA extraction kits or methods, can affect the subsequent analyses and the results of DNA metabarcoding. However, few methodological studies have been carried out on eDNA of fungi, and little is known about how experimental procedures can affect the results of biodiversity analysis. In this study, we focused on the effect of the DNA extraction method on fungal DNA metabarcoding using freshwater samples obtained from rivers and lakes. Methods: DNA was extracted from freshwater samples using the DNeasy PowerSoil kit, which is mainly used to extract microbial DNA from soil, and the DNeasy Blood & Tissue kit, which is commonly used for eDNA studies on animals. We then compared PCR inhibition and fungal DNA metabarcoding results [i.e., operational taxonomic unit (OTU) number and composition] of the extracted samples. Results: No PCR inhibition was detected in any of the samples, and no significant differences in the number of OTUs and OTU compositions were detected between the samples processed using different kits. These results indicate that both DNA extraction kits may provide similar diversity results for the river and lake samples evaluated in this study. Therefore, it may be possible to evaluate the diversity of fungi using a unified experimental method, even with samples obtained for diversity studies on other taxa such as those of animals.

Molecular characterization and antifungal activity against non-dermatophyte molds causing onychomycosis

Onychomycosis is a fungal disease that caused by different types of fungi. Non-dermatophyte molds are a large saprophytic fungi group that live in nature and could affect traumatic nails. The aim of this study was to identify non-dermatophyte molds causing onychomycosis and evaluation of several antifungal activities against the isolates. The samples consisted of 50 non-dermatophyte molds isolated from patients with onychomycosis confirmed by direct and culture examination fungal. DNA was extracted, amplified, and sequenced. Disk diffusion method was used to evaluate itraconazole, fluconazole, ketoconazole, terbinafine, posaconazole, and econazole activity against the isolates. The species identified as: Aspergillus flavus 22 (44%), A. niger 12 (24%), A. fumigates, 3 (6%), A. sydowii 3 (6%), A. terreus 1 (2%), Penicillium commune 2 (4%), P. glabrum 2 (4%), P. chrysogenum, 1 (2%), Fusarium solani 3 (6%) and F. thapsinum 1 (2%). Most of the samples were sensitive to terbinafine, itraconazole, and econazole and 94% of the isolates were resistant to fluconazole. This study showed that Aspergillus species were the most common cause of non-dermatophyte mold onychomycosis and fluconazole was the most resistant antifungals. Care must be taken to choose the appropriate antifungal drug for a better cure.

Quantifying the Role of Ground Beetles for the Dispersal of Fusarium and Alternaria Fungi in Agricultural Landscapes

The spread by arthropods (zoochory) is an essential dispersal mechanism for many microorganisms, like plant pathogens. Carabid beetles are very abundant and mobile ground-dwelling insects. However, their role in the dispersal of economically relevant phytopathogens, like Fusarium and Alternaria fungi is basically unknown. We quantified the total fungal, Fusarium, and Alternaria load of carabid species collected in the transition zones between small water bodies and wheat fields by screening (i) their body surface for fungal propagules with a culture-dependent method and (ii) their entire bodies for fungal DNA with a qPCR approach. The analysis of entire bodies detects fungal DNA in all carabid beetles but Alternaria DNA in 98% of them. We found that 74% of the carabids carried fungal propagules on the body surface, of which only half (49%) carried Fusarium propagules. We identified eight Fusarium and four Alternaria species on the body surface; F. culmorum was dominant. The fungal, Fusarium and Alternaria, load differed significantly between the carabid species and was positively affected by the body size and weight of the carabids. Carabid beetles reveal a remarkable potential to disseminate different fungi. Dispersal by ground-dwelling arthropods could affect the spatial-temporal patterns of plant disease and microorganisms in general.

First report of Gibellulopsis nigrescens on peppermint in Germany

Peppermint is an important medicinal plant, and it is known for its essential oils and phenolic acids. Verticillium wilt is a vascular disease resulted from several Verticillium spp. causing significant economic losses in peppermint cultivation. In this study, the fungus Gibellulopsis nigrescens (syn. Verticillium nigrescens) was isolated from symptomless peppermint plants during the regular control of Verticillium wilt on peppermint in Germany. A pure fungal culture was prepared, and fungal DNA was extracted. Ribosomal internal transcribed spacer (ITS), beta-tubulin (TUB), and translation elongation factor 1-α (TEF1-α) were amplified, sequenced, and deposited in the GenBank. These sequences are located within the Gibellulopsis nigrescens cluster. Koch’s postulate was fulfilled, and the fungus was re-isolated from the inoculated plants. Up to our knowledge, this is the first report of Gibellulopsis nigrescens on peppermint in Germany.