Rapid sourdough yeast identification using panfungal PCR combined with high resolution melting analysis.

The microbial composition of the sourdough starter affects the sourdough bread properties. Therefore, it is crucial to find a tool for rapid, time-saving, and economical identification of the sourdough microbiota. We focused on the rapid identification of sourdough yeasts. We designed a panfungal real time-PCR targeting the ITS2 region (ITS-amplicon) and a fragment of D1/D2 region of 26S rRNA gene (U-amplicon) and used high resolution melting analysis (HRM) for subsequent species identification. The sensitivity and specificity of our method were tested on the reference yeast cultures. We obtained divergent melting peaks (Tm). The further analysis of melt curves suggests the possibility to discriminate yeasts on the genus- and some on species-specific level in the mixed sample. The applicability of this method in routine practice was evaluated on nine sourdough samples. Revealed melt curves of U-amplicons were predominantly characteristic of the sourdough. The evaluation of the Tm and the shape of the melt curve was used to assess the sourdough yeasts. Additionally, using the HRM-PCR method the contamination with the ergot fungus DNA was revealed. Our data showed HRM-PCR is a simple, rapid, and inexpensive tool useful in identifying sourdough yeasts.

Intestinal mycobiome associated with diagnosis of inflammatory bowel disease based on tissue biopsies.

Analysis of mycobiome from formalin-fixed, paraffin-embedded (FFPE) biopsies should preferentially detect only fungi which are actually present in the intestine wall, in contrast to stool samples, which are limited by the diet composition. Next generation sequencing provides the advantage of analyzing many species from a single sample. Consequently, canonical correspondence analysis divided fungal genera present in FFPE intestinal tissues into three well-defined experimental groups (negative controls - NC, Crohn's disease - CD, ulcerative colitis - UC). Simultaneously, the analysis showed that particular fungal genera are associated with these experimental groups and several fungal genera occurred in all experimental groups equally. Our results also showed a noticeable increase of Ascomycota proportion from NC, through CD to UC. Fungal genera Malassezia, Cladosporium and Toninia occurred in all experimental groups assuming that they are common components of the intestinal mycobiome. Other fungal genera found only in the NC experimental group were non-pathogenic and might bring some benefits. In contrast, CD and UC samples were characterized by an accumulation of genera with inhibitive effects on growth of other fungal genera and the presence of opportunistic pathogens. Furthermore, a decrease in the fungal genus Malassezia in inflammatory tissues was observed; Specifically, the UC experimental group showed a connection between the presence of Candida and seven time's lower amounts of Malassezia (compared to amounts found in NC). The CD experimental group was characterized by the simultaneous presence of Engyodontium album with Lecanicillium, and indicates a possible pathogenic effect of Ramularia in disease development. LAY SUMMARY: Mycobiome analysis of formalin-fixed and paraffin-embedded biopsies may highlight actual fungal genera composition in the intestinal wall. Interestingly, experimental groups of Crohn's disease and ulcerative colitis clearly differed by structure of their mycobiomes.

Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) as a potential mycoparasite on Sphaerotheca fuliginea (Ascomycotina: Erysiphales).

Hyphomycete Paecilomyces fumosoroseus that is well known as saprophytic and entomopatogenic fungus was investigated for its mycoparasitism on the cucumber powdery mildew pathogen. Mycoparasitism was documented by using standard bioassay and SEM. Effects of mycoparasitism were evaluated in three types of experiments. Paecilomyces fumosoroseus was applied in the form of graded suspensions into a colony of powdery mildew on a leaf segment. Interaction between both fungi was observed as the percentage of colonized area vs. experimental time. In the second experiment, young cucumber plants were sprayed with a suspension of Paecilomyces fumosoroseus 24 h before inoculation of Sphaerotheca fuliginea. Pre-treatment with P. fumosoroseus reduced development and spreading of powdery mildew infection significantly 15 days post-inoculation in contrast to pre-treatments with sulfur fungicide and distilled water. The development of pure culture powdery mildew under determined experimental conditions was observed and compared with treated variants. In the third experiment, mildewed plants were treated with a suspension of P. fumosoroseus. The control treatments with sulfur fungicide and distilled water were tested. Effects of P. fumosoroseus on the dispersion of powdery mildew during a 21-day period were observed. P. fumosoroseus suppressed the development and spread of cucumber powdery mildew significantly during the time of the experiment. The mechanical and physical damages and disruptions of vegetative and fruiting structures of powdery mildew were recorded under light microscopy and S.E.M. Results were concluded in pursuance to differences between the natural behaviour and development of S. fuliginea on cucumber plants treated with P. fumosoroseus and non-treated plants.