A METHOD FOR EVALUATING ERGOSTEROL CONTENT IN WOOD-DECAY FUNGI

Ergosterol is responsible for important functions in the fungal plasma membrane. The infl uence of fungitoxic agents on membrane ergosterol content is one of the most important mechanisms of antifungal action and its knowledge allows the generation of products that associate active compounds of diff erent mechanisms, consequently improving the eff ectiveness of wood preservatives. Therefore, this study optimized a method for quantifying ergosterol in wood-decay fungi. The white-rot species selected were Ganoderma applanatum and Trametes versicolor, while the brown-rot were Gloeophyllum trabeum and Lentinus lepideus. Mycelial discs of each species were transferred to Petri dishes containing a cellophane-covered potatodextrose-agar medium. Mycelia of each fungus were collected, weighed, and transferred to test tubes with 5 mL of 25% alcoholic potassium hydroxide. The tubes were vortexed for 5 min, subjected to ultrasound for 5 min, incubated at 85 °C for 4 h, followed by the addition of 2 mL of sterile distilled water and 5 mL of n-heptane and subsequent ultrasound shaking for 2 min. The n-heptane layer was analyzed by UV spectrophotometry between 230 and 300 ηm. The blank sample only contained n-heptane. The mycelia wet weight of the fungi ranged from 0.061 to 0.296 g. Ergosterol content was 0.007% for Lentinus lepideus and 0.004% for the other species. The absorbance was higher than the ones observed in the blank for all samples. The adapted method was effi cient for ergosterol extraction.


INTRODUCTION
Diff erent biological agents can attack wood-based products, including wood-decay fungi (Stangerlin et al., 2013). These organisms can cause serious damage to wooden structures, which may result in considerable economic and resource losses (Cheng et al., 2008). These fungi belong to the class Basidiomycetes and are classifi ed as white and brown rots (Oliveira et al., 2005). The white-rot group is characterized by the capacity to degrade lignin, hemicellulose, and cellulose, while the brown-rot fungi are characterized by the degradation of wood polysaccharides (Martínez et al., 2005).
To circumvent the losses caused by wood-decay fungi on wood products, preservatives are usually used (Mendes et al., 2014;Vivian et al., 2015). However, some preservatives have high toxicity, therefore, research is necessary to obtain alternative fungitoxic agents that are eff ective in combating wood rot (Brand et al., 2006;Xie et al., 2017).
Ergosterol is the major sterol in the fungal membrane (Hu et al., 2017) and responsible for important growth functions (Mota et al., 2012). Among these functions are its contribution to membrane fl uidity and integrity in addition to supporting the normal operation of membrane-bound enzymes (Lupetti et al., 2002). Ergosterol is produced by wood-decay fungi, both in white-rot and brown-rot fungi (Presley and Schilling, 2017;Xie et al., 2017).
The eff ect of a product on ergosterol content in plasma membrane is a known mechanism of antifungal action (Tian et al., 2012;Kedia et al., 2014;Avanço et al., 2017). Determining a method for ergosterol evaluation is necessary in order to confi rm the mechanism of antifungal action of antifungal agents in wood-decay fungi. Knowledge of fungitoxic compound mechanisms allows the generation of products that associate active compounds of diff erent mechanisms that are more eff ective as wood preservatives.
Considering that ergosterol content in wooddecay fungi vary between white-rot and brown-rot ones (Niemenmaa et al., 2008), the possibility that the evaluation of this sterol can be used to study mechanism of antifungal action, and that the extraction methods proposed by Arthington-Skaggs et al. (1999) and Tian et al. (2012) were not eff ective for this group of fungi, the present study optimized a method for ergosterol evaluation in wood-rotting fungi using the white-rot fungi Ganoderma applanatum and Trametes versicolor and the brown-rot fungi Gloeophyllum trabeum and Lentinus lepideus as models.

Fungal growth in PDA with and without cellophane membrane
Both the white-rot fungi (G. applanatum and T. versicolor) and brown-rot fungi (G. trabeum and L. lepideus) were provided by the Forest Products Laboratory (Laboratório de Produtos Florestais) situated at Universidade de Brasília, DF, Brazil. The fungal strains were deposited at the Herbarium of the Biology Department, UFSM, Brazil. Ganoderma applanatum (SMDB 17,748), T. versicolor (SMDB 17,749), G. trabeum (SMDB 17,751), and L. lepideus (SMDB 17,750) were initially cultivated in 9-cm Petri dishes containing potato-dextrose-agar (PDA). Two treatments were used: PDA with and without a cellophane membrane (Bento et al., 2014) in order to confi rm whether the membrane infl uences mycelial growth. Mycelial discs (11 mm of diameter) of each fungus species were transferred (in three repetitions) to the Petri dishes which were then sealed with parafi lm and kept in a Biochemical Oxygen Demand (BOD) chamber at 25 °C (± 1) at light/dark cycle of 12/12 h. The fi rst evaluation was done on the 7 th day after the beginning of the experiment through two measurements diametrically opposite to each other. The test was considered fi nished when the fungal growth of each dish completely covered the medium (Badawy and Abdelgaleil, 2014). Data normality and homogeneity of variances were confi rmed, one-way ANOVA performed, and the averages were compared by Tukey's test using SigmaPlot 11.0 software.

Ergosterol content analysis in the plasma membrane of the wood-decay fungi
The white-rot fungi G. applanatum and T. versicolor and the brown-rot G. trabeum and L. lepideus were fi rst cultivated in Petri dishes containing PDA, for fi fteen days, in a BOD chamber at 25 °C (± 1) at a light/dark cycle of 12/12 h. Then, mycelial discs (11-mm diameter) of each fungal species were transferred to Petri dishes containing cellophane membrane-covered PDA (Bento et al., 2014). Afterwards, the dishes were sealed and moved to a BOD chamber in which they remained for 5 days. All treatments were done with three repetitions.
An adaptation of the method by Arthington-Skaggs et al. (1999) and Tian et al. (2012) was developed for mycelial ergosterol extraction. After the incubation period, mycelia from each wood-decay fungus were harvested and their wet weight determined. Fungal mycelia along with 5 ml of 25% alcoholic potassium hydroxide were transferred to test tubes and vortex mixed for 5 min (Velp Scientifi ca, Vortex Mixer, Wizard X). The mixed samples were transferred to ultrasound (Unique Ultrasonic Clean, model Ultra Cleaner 1450A with heating) and remained there for 5 min. After ultrasound treatment, the test tubes were incubated at 85 °C for 4 h. Then, 2 ml of sterile distilled water and 5 ml of n-heptane were added to the test tubes, which remained in an ultrasound bath for 2 min. The solution layers of each fungus were separated into funnels for 1 h at room temperature. The n-heptane layer of each tube was collected and analyzed by UV spectrophotometry (Biospetro sp-220) between 230 and 300 ηm. The absorbance at 282 ηm characterized the presence of ergosterol in n-heptane layer, while the reading obtained at 230 and 282 ηm corresponded to the late sterol intermediate 24 (28) dehydroergosterol (Tian et al., 2012). Blank samples containing only n-heptane were also analyzed by UV spectrophotometry at the same wavelengths.

RESULTS
After 15 days, all Petri dishes containing PDA with and without cellophane membrane were completely covered by wood-decay fungi. No signifi cant diff erences were found in mycelial growth in PDA among G. applanatum, T. versicolor, G. trabeum, and L. lepideus with and without the cellophane membrane (Table 1).
In order to demonstrate the effi ciency of the ergosterol extraction method, the results are presented in two diff erent manners: the percentage of ergosterol obtained for each fungus and the UV spectrophotometric profi les of this sterol for each species (Figure 2). For all samples, the absorbance values between 230 and 300 ηm wavelengths were higher than the blank, indicating the presence of ergosterol.

DISCUSSION
The cellophane membrane was fi rst used to determine growth of wood-decay fungi in a study by Bento et al. (2014) in order to obtain a nutrient medium free of mycelium. The aforementioned authors determined the activity of antioxidant enzymes of fi lamentous fungi in the presence of plant extracts and only evaluated the white-rot species Trametes villosa and Pycnoporus sanguineus. In some previously described methods, mycelia were washed in sterile distilled water after removing the culture medium (Arthington-Skaggs et al., 1999;Tian et al., 2012;Kedia et al., 2014). The use of cellophane membranes make the removal of the fungal mycelia possible without the need for washing.  This alternative prevents the washing water from infl uencing mycelial wet weight.
The percentages of ergosterol vary depending on the fungal species (Barajas-Aceves et al., 2002). The readings of absorbance values between 230 and 300 ηm wavelengths were previously used as an indication of ergosterol presence in diff erent studies (Breivik and Owades, 1957;Tian et al., 2012;Kedia et al., 2014). Several studies on ergosterol in wood-decay fungi are found in the literature (Gao et al., 1993;Barajas-Aceves et al., 2002;Eikenes et al., 2005;Niemenmaa et al., 2008;Chedgy et al., 2009;Song et al., 2012), although no information was reported regarding the extraction procedure used in this study. Nevertheless, a similar method was already used for extracting ergosterol from yeasts, such as Saccharomyces cerevisiae (Breivik and Owades, 1957), Candida albicans (Arthington-Skaggs et al., 1999), and the fi lamentous fungus Aspergillus fl avus (Tian et al., 2012;Kedia et al., 2014). Since the previously described methods were not eff ective for ergosterol extraction of white and brown-rot fungi, the standardization of a new method was necessary.
In addition to the use of cellophane membrane, other steps were added and changes applied to the previously described methods (Tian et al., 2012;Kedia et al., 2014) in order to extract ergosterol from the selected species. After adding 25% alcoholic potassium hydroxide to the test tubes containing mycelia, the literature recommends a vortex mix for 2 min (Tian et al., 2012;Kedia et al., 2014). In our method, the time of vortexing was extended to 5 min and a period of 5 min in ultrasound was added. After the addition of 2 ml sterile distilled water and 5 ml n-heptane to the test tubes, the previous methods described a vortex mix for 2 min (Tian et al., 2012;Kedia et al., 2014). In present study, we opted to transfer the test tubes to ultrasound for 2 min.

CONCLUSION
Our study describes a method to quantify ergosterol in the plasma membrane of wood-decay fungi. The use of cellophane membrane did not interfere in the mycelial growth of the white-rot species G. applanatum and T. versicolor, or the brown-rot species G. trabeum and L. lepideus. The adaptation and additional steps added to the methods already described in the literature were effi cient for ergosterol extraction of wood-rot fungi and their quantitative evaluation.