2.1. Origin of Cultures

The culture of P. eryngii M 2600 strain was acquired from Mycelia nv. (https://www.mycelia.be/en/strain-list/m-2600-pleurotus-eryngii), maintained on potato dextrose agar (PDA) media slants, and stored at 2°C–4°C until used. P. eryngii IBK 2032 and 2033 strains were isolated from carpophores collected in the wild in the Dnepropetrovsk and Kharkov regions of Ukraine. Strain 2032 was collected from decaying leaf litter near the base of Eryngium campestre L. and 2033 near the thicket of Ferula communis L. The strains were identified following the description of Zerova et al. [9], and deposited in the IBK Mushroom Culture Collection [10,11]. The cultures were activated and grown on PDA (Difco Cat#: 213400). They were cultured onto new PDA medium plates (90 mm) and incubated for 10 days at 22°C–24°? before use to inoculate sterile grain material to obtain spawn.

2.2. Mycelia Spawn

Spawn was made with barley, wheat, rape, flax, and chalk (CaCO₃) combined in the ratio 60:30:8:1:1. Precooked barley and wheat and presoaked rapeseeds, flax, and chalk were mixed and loaded into polypropylene bags of size 570 × 470 mm, PP75/BEU6/X47-57 (https://saco2.com/shop/zipper-filter/autoclavable-pp-123-c%CB%9A-253-f%CB%9A/pp75beu6x47-57/), and sterilized at 128°C and 1.8 atm for 180 minutes. Upon cooling, the sterile grain mixture was inoculated with the mother spawn (0.5% w/w), and sealed and incubated at 24°C ± 1°C for 8 days before thorough mixing to achieve uniformity. After 11 ± 1 days, the spawn was ready and it was stored in a refrigerator (2°C ± 1°C) until used.

2.3. Substrates

The cultivation substrate was composed of alder (Alnus glutinosa (L.) Gaertn) sawdust. The moisture content in the sawdust, wheat bran, and chalk was 10%, 7%, and 5%, respectively. The substrate materials (sawdust, wheat bran, and chalk) were combined in the ratio of 18:17:1, respectively, and the water content was adjusted to 65% [12]. The substrate was packed into polypropylene bags (580 × 490 mm), PP75/BEU6/X47-57 (from Sac02, Belgium). The bags (average weight 3,256 ± 18 g) were sterilized at 121°C ± 1°? and 15 pounds per square inch for 120 minutes. The average weight of the substrate bags after sterilization was 3,249 ± 7 g. The substrate bags were inoculated with spawn (5% w/w), sealed under aseptic conditions, and incubated at 24°C ± 2°C and relative humidity 68% ± 2%.

2.4. Fruiting and Yield Characteristics

At the initiation of fruiting, i.e., when pinheads appeared, the substrate bags were weighed again, and the weight loss in each bag was recorded. The bags were randomly distributed in a grow room with conditions set at temperature 16°C ± 2°C, relative humidity 95% ± 3%, and CO₂ 1,250 ± 150 ppm. At maturity, each bag was opened at the top and the bag’s sides were rolled down to expose about 30% of the total surface area. In the first 5 days of fruiting, illumination was maintained at 150 lux for 8 hours per day, after which daylight (not more than 40 lux) was sufficient until harvest. Data for the following parameters were collected: 1. the number of days from inoculation to pinning, 2. the number of days from inoculation to harvest, 3. the total weight of FBs from each bag, and 4. basidiocarp morphological indexes (BMI) for each mushroom [weight (g), height (mm), stipe diameter (mm), and cap diameter (mm)]. BMI was measured with 25 FBs (five FBs collected from five random bags) and was measured for each strain during each repeat experiment (75 total FBs in the three repeat experiments). The biological efficacy (BE) was calculated using the following formula: the weight of freshly picked mushrooms/substrate weight (dry matter) 100% [12].

2.5. Substrate Analysis

Since the experiment was repeated over three seasons, the substrates were analyzed. Moisture content in the substrate was determined as the difference in weight after drying a 10 g quantity of the substrate to constant weight at 102°C ± 2°C (usually 6–8 hours). The ash content was determined by weighing 3 g of dry milled substrate material into ceramic crucibles of known weights and put in the oven (550°C ± 10°C) for 3 hours and cooled in a desiccator. The leftover material in the crucible was weighed to obtain ash content [13]. Total nitrogen was determined by the Kjeldahl method [14]. The C/N ratio was determined with the formula C/N = 0.52 (100−a)/N, where a is ash content (%), 0.52 is carbon content coefficient [15,16], and N is total nitrogen content (%).

Statistical analysis on the data obtained was carried out on the above parameters with Microsoft Office Excel 2016 MSO (16.0.4266.1001) kod 00339-10000-00000-AA963 and QI Macros superstructure. Statistics on BMI were evaluated with STATISTICA 13.

3.1. Substrate Composition

The substrate analysis data from the three repeat tests did not show significant differences in the parameters of moisture contents (Table 1).

Wheat bran-supplemented sawdust substrate was used for this study to compare our results with reports in the literature about the cultivation of P. eryngii (Wanzenbock et al., 2017). The substrate compositions in different seasons had a C/N ratio in the range 45–47, which is optimum for P. eryngii cultivation [17]. Philippoussis et al. [18] noted a positive correlation (r² = 0.98) between the C/N ratio (within the range of 32.7–59.3) and mushroom yield in P. eryngii. The C/N ratio of 46.86 ± 1.11 found in our substrate is suitable for the cultivation of other Pleurotus species on sawdust substrate [18,19].

3.2. Colonization and Mushroom FB Development

The fastest strain (2032) reached pinning 37.3 ± 0.9 days after inoculation, followed by 2033 (43.2 ± 1.2), and the last was 2600 (57.4 ± 4.8 days). Strains 2032, 2033, and 2600 reached the harvesting stage 49.2 ± 6.1, 53.1 ± 3.2, and 68 ± 3.4 days after inoculation, respectively (Fig. 1).

Single-factor statistical analysis (ANOVA) on the number of days to reach pinning and first harvest indicated no significant difference within the repeat cultivation in each strain, but a significant difference between the three strains tested (p < 0.01). Moonmoon et al. [20] used sawdust substrate and reported 27–30 and 39–52 days to pinning and harvest, respectively, during the cultivation of P. eryngii. However, their substrate composition (sawdust, wheat bran, and rice husk) and the strains were different from those used in our experiment. Szarvas and Gyorfi [21] grew P. eryngii isolates using the substrate from birch sawdust supplemented with wheat bran and soybean, and they reported about 60 days to attain the harvesting stage.

3.3. Yield and BE

In general, the maximum average total FB yield from each bag was 565.9 ± 12.3 g in strain 2032, and the least was 312.2 ± 7.8 g in strain 2600 (Table 2). The FB yield in each strain during the three seasons of cultivation indicated slight variations that were not significantly different (p >0.05), except in the Oct–Dec 2018 cultivation in strain 2032. The FB yield, which was 603.5 ± 9.2, was significantly different from 568.4 ± 12.6 and 525.8 ± 15.2 obtained during the Feb–Apr 2018 and Jan–March 2019 cultivation seasons, respectively, in strain 2032. However, statistical analyses indicate significant differences (p = 0.02) in total FB yield among the three strains tested during each season (Table 2).

Similarly, statistical analyses (ANOVA) on BE values indicated significant differences between strains (p = 0.04). The highest BE was in strain 2032 (47.5% ± 6.6%), followed by strain 2033 (32.7% ± 3.9%), and the least was in 2600 (25.1% ± 3.2%). The BE values for 2032 and 2033 were 25% and 5% higher than strain 2600 in this experiment, respectively. The BE in all the strains tested were higher than the 20.3 BE reported by Wanzenbock et al. The BE of 2032 and 2033 strains was similar to the report of Sardar et al. [22], who obtained BE values of 35.47 ± 0.76 when P. eryngii was cultivated on a substrate containing sawdust, rice, and wheat straw. The results from strains 2032 and 2033 were lower than those reported by Xie et al. [23], who used different agriculture wastes to obtain BE as high as 36.8% and 52.4%. Nevertheless, higher yield and BE from local isolates 2032 and 2033 could be due to these isolates’ unique genetic backgrounds. Furthermore, evolution/adaptation to prevailing ecogeographic conditions in Ukraine could be responsible for the superior performance of strains 2032 and 2033.

Table 1: Chemical and physical properties of substrate used in different seasons. [Click here to view]

Figure 1: Time to primordia/pinning and FB development in P. eryngii. [Click here to view]

Table 2: Average FB yield (g/bag ± standard error) from three seasons of cultivation of P. eryngii. [Click here to view]

Figure 2: Shapes of FBs from different strains of P. eryngii: ?) strain 2600, b) strain 2033, ?) strain 2032, and d) FB diameter. [Click here to view]

3.4. Morphological Characteristics

Representative FBs from strains 2600, 2033, and 2032 are shown in Figure 2a, b, and c, respectively. The morphological parameters of FB weight, height, stipe, and cap diameter that were measured indicated significant differences in test strains’ FBs (Table 3).

Seventy-five FBs were collected per strain and analyzed for variations in individual FB weight. In strain 2600, 54 out of 75 FBs had weights that ranged between 25 and 45 g, indicating a tight peak shown in Figure 3. However, strains 2032 and 2033 gave FBs with higher weights and a wider spread of FB weight across the spectrum. For strains 2032 and 2033, the distribution of values in samples had significant right-sided asymmetry, and descriptive statistical analysis indicated coefficients of 1.73 and 1.31, respectively. The estimation of FBs’ weight in strain 2600 showed the smallest dispersion of results from average and a lower sample asymmetry coefficient of 1.09.

Table 3: Morphological characteristics in FBs (± standard error) of P. eryngii. [Click here to view]

Figure 3: Variations and distribution of FB weight (g) among P. eryngii strains. [Click here to view]

The average height of FBs in strain 2600 was 11 and 14 mm higher than results from strains 2032 and 2033, respectively (Fig. 4). The maximum FB height was 127 mm in strain 2600, the minimum was 40 mm in strain 2033, and the FBs from strains 2032 and 2033 did not exceed 100 mm. The samples’ distribution based on FBs’ height was more uniform and indicated that the asymmetry coefficient did not exceed 0.3 in strains 2032 and 2600; it was below 0.2 in strain 2033.

The largest FB cap diameter (59.7 ± 2.0 mm) was in 2033, and the least (46.4 ± 1.4 mm) was in strain 2600. The highest variation in pileus diameter was found in strain 2033, ranging from 22 to 115 mm. The least cap diameter variations were between 22 and 75 mm in strain 2600 (Fig. 5). Most of the FBs of strains 2032 and 2033 had a cap diameter within the range of 45–65 mm, while those of strain 2600 were within a 30–50 mm diameter. The average FB cap diameter of strain 2600 was 12 and 13 mm less than those of strains 2032 and 2033. The cap diameter range values in strain 2600 were significantly (p < 0.01) different from those in strains 2032 and 2033. The range of variations in cap diameter in all strains tested is in line with the earlier report of Ha et al. [24], who recorded a 59–64.9 mm diameter in the cultivation of P. eryngii. Strain 2032 had the least stipe diameter (15 mm) and the highest was 61 mm. The stipe diameter of FBs from strain 2600 was from 18 to 50 mm and was on average 4 and 6 mm less than FBs from strains 2032 and 2033, respectively (Fig. 6). Strains 2600, 2032, and 2033 have morphological characteristics comparable to earlier reports of stipe diameters 27–32 mm and cap diameter 53–82 mm [20]. However, the variations in FB morphological parameters during three repeat cultivations could account for the predictability of occurrence in mushrooms’ morphological features in terms of weight, height, cap, and stipe, which are very important in commercial cultivation.

The analysis of the FBs’ morphological parameters determined that commercial strain 2600 had significant differences in all morphological characteristics, FB weight and height, cap, and stipe diameter (p < 0.01), in comparison to local isolates IBK 2032 and 2033. The FBs of strain 2600 had an elongated cylindrical shape compared to the FBs from isolates 2032 and 2033, which showed substantially larger stipe diameters toward the base of the FB.

Figure 4: Variations and distribution of FB height (mm) among P. eryngii strains. [Click here to view]

Figure 5: Variations and distribution of FB cap diameter (mm) among P. eryngii strains. [Click here to view]

Figure 6: Variations and distribution of FB stipe diameter (mm) among strains of P. eryngii. [Click here to view]

In the morphological characteristics studied, most samples from strains 2032 and 2033 had similar features and characteristics that were, in most cases, different from 2600. About 48% of individual FB weight was between 40 and 80 g, 64% individual FB from both strains had height between 50 and 70 mm, 55% (2032) and 72% (2033) had a cap diameter between 50 and 70 mm, and 82% (2032) and 64% (2033) had a stipe diameter between 20 and 40 mm. In M 2600, 58% FB weighed between 20 and 60 g, 40% between 70 and 90 mm in height, 67% between 20 and 40 mm in cap diameter, and 50% between 40 and 60 mm in stipe diameter. Figs. 3–6 represent low dispersion from average values obtained. The fact that the same substrate was used for cultivation and the fruiting was carried out in the same climate-controlled environment may indicate that the differences between 2600 and the two local strains, 2032 and 2033, are most likely due to inherent genetic traits in the strains tested. This line of thought is supported further by the fact that strains 2032 and 2033 that were collected from the same locality showed no difference in most characteristics studied.

The results obtained from morphological studies could be useful for designing the package’s size to reduce mechanical damage to the delicate FB of the P. eryngii mushrooms from strains 2032 and 2033 under commercial cultivation. Such an approach will help to increase the shelf life of mushrooms when they are sold fresh. Furthermore, the wide variation in FB morphological characteristics of strains 2032 and 2033 suggests that making different FB size products, e.g., small, medium, and large FB packages, would benefit marketers of consumer mushroom products.