Mushroom samples
Five morphologically different wild mushrooms labeled Mush 1 to 5 were collected from their natural habitat in Al-Beheira Governorate and Cairo, Egypt, between January and February 2017. The mushroom samples were characterized morphologically following the methodology suggested by Largent and Stuntz [17]. The samples verified up to genus level [18] as follows: Mush 1, Bjerkandera sp.; Mush 2 and 3, Cyclocybe sp.; Mush 4, Chlorophyllum sp.; and Mush 5, Lentinus (Fig. 1). The samples were dried at room temperature to a constant weight, then preserved in a dry place until use. The macroscopic features of the collected mushroom were recorded. The specimens collected were deposited in Mycology Laboratory, Faculty of Science, Helwan University, Egypt.
DNA isolation and molecular identification
Total genomic DNA was isolated from fruit bodies according to [19]. The total isolated DNA were column purified using DNA Purification MiniSpin Kit (VIOGENE cat# PF1001) according to the manufacturer’s protocol. The purified DNA was used as a template for PCR reaction using Mytaq Red DNA polymerase master mix (BIOLINE cat # BIO-21108) according to manufacture instructions. Briefly, the reaction was containing 1× PCR red master mix buffer, 2.0 μl of 10 pm/μl of each primer; ITS1 (5′ TCCGTAGGTGAACCTGCGG 3′) and ITS4 (5′ TCCTCCGCTTATTGATATGC 3′), 1.0 μl of DNA template (~ 30 ng), 0.25 μl of MyTaq™ DNA polymerase (5 U/μl), then the total volume was adjusted to 50 μl using sterile water. The amplification reactions were performed in Thermal Cycler (Biometra, Germany) as follow: 1st cycle of 3 min at 95 °C for initial denaturation, followed by 35 cycles of 20 s at 95 °C (denaturation), 20 s at 55 °C (annealing), 30 s at 72 °C (extension), and then a final extension was carried out for 10 min at 72 °C; the reaction was held at 4 °C. The PCR products were separated on 1% agarose gel, the amplified fragments were purified using a PCR-M clean-up system (VIOGENE cat# PF1001) according to the manufacturer’s protocol, followed by sequencing with ITS1 primer at GATC Company using ABI 3730xl DNA-sequencer.
Sequencing data analysis
The obtained nucleotide sequences were aligned to the total nucleotide collection of NCBI using Basic Local Alignment Search Tool for nucleotide blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The phylogenetic tree is constructed using the UPGMA tree build method with 100 bootstrapping in Geneious 8.1.9 software [20].
Preparation of ethanol crude extracts
Dried fruiting bodies were grounded in a grinder into a fine powder before extraction. Five grams of the powdered samples were extracted with 95% ethanol (50 mL) at 30 °C at constant shaking of 150 rpm for 24 h. The extracts were centrifuged at 3000 rpm, 4 °C for 15 min to give a clear supernatant that was filtered through Whatman filter paper number 4. The clear filtrate was concentrated by evaporation under vacuum, then the crude extract was stored at 4 °C for further use [21]. The crude extracts have been evaluated for their antimicrobial activity.
Test microorganisms and growth conditions
The antimicrobial activity of extracts from the investigated mushrooms was tested against 6 different pathogenic bacterial strains and one yeast species, namely, Candida albicans ATCC1031. Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC 7853, and Staphylococcus aureus ATCC25923 were kindly provided from Pharmaceutical Control Authority, Giza, Egypt, while Proteus mirabilis, Micrococcus luteus, and Streptococcus pneumoniae were in-house isolates. The strains were routinely cultured in Nutrient agar (Diffco) medium at 37 °C. A single colony from each corresponding organism was inoculated into Nutrient broth medium and allowed to grow for 24 h at 37 °C. The grown cultures were used for the antimicrobial assay.
In vitro antimicrobial activity assay of mushroom’ extracts
Antimicrobial assay of mushroom extracts was verified using the well diffusion method [22]. The inoculum size of each test organism was adjusted to a concentration of 1.5 × 108 CFU/mL by comparing with 0.5 McFarland standards. Bacteria and yeast seeded plates were prepared by inoculating 100 μl suspension of each tested culture into nutrient agar media. Wells were made on the agar surface with a 6-mm cork borer. The extracts were dissolved in sterile distilled water to a final concentration of 10 μg/ml. A hundred microliters of extracts were poured independently into the well using a sterile syringe. The seeded plates were refrigerated for 8 h at 4 °C to allow extract diffusion into agar then, were incubated at 37 ± 2 °C for 24 h. The plates were observed for the inhibition zone formation around the wells.
The inhibition zone was calculated by measuring the diameter of the inhibition zone around the well (in millimeters) including the well diameter in three independent replicas. The measurements were taken in three different fixed directions for each plate and the average values for the three independent replicas were tabulated. Gentamycin (10 μg/disk) was used as a standard reference antibiotic.
Chemical composition of the crude extract of Cyclocybe cylindracea and Agrocybe aegerita fruiting bodies
Even identified differentially, Mush 2 and 3 show 100% similarity based on the sequenced ITS-rDNA fragment that argues for the same species for both samples; however, dissimilar antimicrobial activities were detected. Therefore, both samples were used to evaluate their chemical compositions by measuring the total soluble sugars, proteins, and phenolic contents.
Total soluble sugars
As described by Umbreit et al., total sugars were estimated using the anthrone technique [23]. Six milliliters anthrone solution (2 g anthrone /L H2SO4 of 95 %) was added to 3 ml mushroom extract and the mixture was kept in a boiling water bath for 3 min. The formed color was measured at 620 nm by a spectrophotometer after cooling. The calibration curve was constructed with different concentrations of glucose as a standard. Total soluble sugar content of the sample was determined as glucose equivalent and expressed as milligrams per gram of the extract.
Total soluble proteins
Total soluble proteins were determined according to Lowry et al., [24]. One milliliter of each mushroom extract was mixed with 5 ml of freshly prepared solution of 2% sodium carbonate, 4% sodium hydroxide, and 0.5% copper sulfate in 1% sodium tartrate. The mixture was incubated at room temperature for 10 min before the addition of 0.5 ml Folin and made up to 10 ml. After 30 min, the optical density of the mixture was measured at 750 nm. The calibration curve was constructed with different concentrations of albumin bovine serum as a standard. The total soluble proteins were calculated using albumin bovine serum equivalent and expressed as milligrams per gram of the extract.
Total phenolic contents
Using Folin-Ciocalteu reagent and gallic acid as a standard, total phenolic contents were determined according to Kujala et al., (2000) [25]. Half a millimeter of each mushroom’s extract was mixed well with 2.5 mL of 1:10 ethanol diluted Folin-Ciocalteu’s reagent, 2 mL of 7.5% Na2CO3. After 15 min incubation at room temperature, the absorbance of mixtures was recorded by spectrophotometer at 765 nm. Different concentrations of gallic acid as standard have been prepared to construct the calibration curve. The total phenolic contents were calculated as gallic acid equivalent (GAE) and expressed as milligram per gram of the extract (mg GAE/g sample extract).
HPLC analysis
According to Roberts et al., (2018) [26] HPLC was carried out at the National Research center, Cairo, Egypt using Agilent 1260 series with a C18 column (4.6 mm × 250 mm i.d., 5 μm) at 35 °C to identify and quantify the phenolics and related compounds present in Bjerkandera adusta extract as it displayed the highest antimicrobial activity [26]. The mobile phase consisted of water (A) and acetonitrile (B) at a flow rate of 1 ml/min. The multi-wavelength detector was monitored at 280 nm. The sample injection volume was 10 μl. For the quantitative analysis of phenolic compounds, a calibration curve was obtained by injection of different concentration of gallic acid, chlorogenic acid, catechin, caffeine, syringic acid, rutin, ellagic acid, coumaric acid, vanillin, ferulic acid, naringenin, propyl gallate, 4′,7-dihydroxyisoflavone, quercetin, and cinnamic acid standards.
Statistical analysis
All analyses were performed in triplicate. The data were recorded as means ± SD (standard deviation). The statistical (SPSS version 17.0) package program was used to perform one way ANOVA. P ≤ 0.05 is considered statistically significant.