Adhesion and biofilm formation of Aureobasidium pullulans on wood analyzed by multi-modal microscopy

Aureobasidium pullulans is a highly adaptable and polymorphic black yeast belonging to the phylum Ascomycota

(Zalar et al., 2008). It is commonly found in diverse ecological niches, including soil, plant surfaces, wood, air, and

even in extreme environments such as saline waters and cold climates (Andrews et al., 2002; Botić et al., 2014;

Gunde-Cimerman et al., 2000; Wang and Pecoraro, 2021). Its metabolic versatility enables the production of

various compounds, most notably pullulan, an extracellular polysaccharide with numerous industrial applications

in the food, pharmaceutical, and biodegradable packaging industries (Prasongsuk et al., 2018). Previous studies

have shown that A. pullulans have a broad spectrum of recombinant and polyextremotolerant genes that

contribute to its remarkable resistance to environmental stress (Gostinčar et al., 2019).

To investigate the adhesion capacity of A. pullulans to wood, we analysed three strains isolated from a wooden

façade (IN-007), dried olives (IN-515), and glacial ice with sediment (IN-517) on pine wood samples. Initial

morphological assessment by light microscopy (LEICA DM 2700 M) revealed several morphological forms of

strains, including yeast-like cells, pseudohyphae, hyphae, and chlamydospores. After inoculating A. pullulans

strains onto pine wood, adhesion to the wood was assessed after washing with distilled water. Each strain was

monitored for seven days at 25 °C and 75-80% relative humidity. Optical digital microscopy (Keyence VHX-

6000) and fl uorescence microscopy (EVOS M7000, ThermoFisher Scientifi c) were used following Calcofl uor

White staining. Results showed a clear increase in fungal density and surface coverage over time.

These fi ndings confi rm the strong adhesion and biofi lm-forming ability of A. pullulans on wood surfaces paving

the way for the development of quantitative assays using specifi c fl uorescent dyes to evaluate fungal adhesion.

Furthermore, this work provides a foundation for the development of protective biologically inspired living

coatings to enhance the durability and performance of various façade materials in architectural applications.