2.1. Mushroom Strains Source

The wild V. volvacea fruiting bodies [Figure 1] growing on the decaying banana leaves [Figure 1] were collected from La Clementina (1°44’18.6”S, 79°19’45.3”W), Vinces (1°34’44.0”S, 79°44’13.9”W), and Montalvo (1°48’29.6”S, 79°17’13.4”W) in Ecuador. The collected fruiting bodies were tissue cultured using potato dextrose agar (PDA) as culture medium [1]. Successful cultures were used as source of mycelial inoculant in the optimization study.

Figure 1: Wild fruiting body of V. volvacea taken from decaying banana leaves collected in Ecuador [Click here to view]

2.2. Influence of Culture Media and pH

The procedures on the evaluation of culture media and pH, as described by Kalaw et al. [8], were followed with minor modification. Three dehydrated mycological culture media, namely, Sabouraud dextrose agar (SDA), potato dextrose agar (PDA), and malt extract agar (MEA) were evaluated in this study. Media were prepared following the product instructions, adjusted to pH 6, dispensed into 500 mL capacity flask plugged with cotton, covered with aluminum foil, and sterilized at 15 psi, 121°C for 30 min. The sterilized media were pour-plated, solidified, and inoculated with 10 mm diameter mycelia discs from the 7-day-old culture. Each treatment was replicated 3 times. Assay plates were incubated at 30°C. The diameter of mycelial growth was measured every 24 h until the full mycelial ramification. To evaluate the effect of pH, the identified best medium was adjusted to varying levels of pH using 1 M NaOH and HCl. Sterilization of pH media, inoculation, and incubation was the same as described in the preceding section.

2.3. Influence of Physical Factors

The best medium with optimum pH was used as basal medium in the evaluation of the influence of aeration, illumination, and temperature. To evaluate the aeration requirements, inoculated plates were sealed with parafilm while other plates remained unsealed. In lighted condition, the inoculated plates were exposed to artificial white light (322.92 lumens/m2), while the other plates were maintained in total dark by covering with black paper, both at 30°C. For temperature, culture plates were incubated in required aeration and illumination conditions, and at different temperature conditions: 9°C, 26–28°C, and 32°C. Mycelial growth diameters were measured. All evaluations were done in triplicate.

2.4. Statistical Analysis

Analysis of variance was employed to analyze the data. Comparison among treatment means was done using Tukey’s HSD at 0.05 significance level. T-test was used for the comparison of means with only two treatments.

3.1. Effect of Culture Media and pH

In nature, mushrooms are commonly growing on lignocellulosic substrates such as fallen logs, dead trunks and branches of trees, pile of leaves, stumps, on soil, and other agro-industrial waste. Hence, mushrooms require suitable nutrients for their efficient growth. In mushroom production, the success of fruiting body development is dependent on the good quality mycelial culture. Thus, the present work evaluated the growth of mycelia of the three V. volvacea strains on the three culture media as source of nutrients. Interestingly, the three strains exceedingly favored SDA followed by PDA [Figure 2]. In contrast, MEA recorded the lowest mycelial growth diameter. Tukey’s HSD analysis revealed the significant difference of the three culture media. Looking at the nutritional components of the three culture media evaluated. SDA has higher amounts of mycological peptone (10 g/L) than malt extract agar (3 g/L) as source of nitrogenous compounds. Both SDA and PDA contain dextrose as a major carbon source. The higher amount of peptone and the presence of dextrose as ingredients of SDA make this medium superior than the other media used. The present study suggests that the three V. volvacea strains have greater preference for the combination of peptone and dextrose in the medium. This finding conforms with the study of Dulay et al. [9] who reported that Sabouraud dextrose broth produced the highest biomass yield of Schizophyllum commune, Ganoderma lucidum, V. volvacea, and Pleurotus cystidiosus in submerged culture. However, in the evaluation of mycelial growth using indigenous culture media, the two strains of V. volvacea (Rang-ayan and CLSU) showed the maximum mycelial growth on potato sucrose gulaman (PSG) and corn grit gulaman (CGG) [8]. In addition, Ukoima et al. [10] demonstrated that the highest growth of V. volvacea mycelia was observed on palm fiber culture medium. Accordingly, aside from the commercially available dehydrated culture media, indigenous culture media derived from natural sources could also be used as suitable media for the excellent mycelial growth of V. volvacea, which are dependent on their components, their nutrient concentration, and preparation of the medium.

Figure 2: Mycelial growth diameter of the three strains of V. volvacea on the different culture media at pH 6 in 4 days of incubation. MEA: Malt extract agar, PDA: Potato dextrose agar; SDA, Sabouraud dextrose agar. Means with the same letters of superscript are not significantly different according to Tukey’s HSD (P < 0.05) [Click here to view]

The growth performance of mycelia at varying pH levels was also determined. Figure 3 shows the growth of mycelia of the three V. volvacea strains on SDA at varying pH levels. It can be seen that the three strains could grow from pH of 5.5–8.0, but with optimum pH at 6.5–7.5 for La Clementina and Vinces and at 6.5–7.0 for Montalvo. Similarly, Rang-ayan and CLSU strains of V. volvacea showed favorable mycelial growth at a wide range of pH with very thick mycelia at pH 7.5–8.0 [8]. In liquid culture, the optimum pH of V. volvacea was at pH 6.0 [9]. However, other species of Volvariella such as V. esculenta could produce good quality mycelia at pH 6 [11], and V. speciosa mycelia could grow at pH 3–9 [12]. Therefore, optimum pH of the genus Volvariella is species and strain dependent.

Figure 3: Mycelial growth diameter of the three strains of V. volvacea on Sabouraud dextrose agar at varying pH levels in 4 days of incubation [Click here to view]

3.2. Effect of Physical Factors

Aside from the nutritional requirements, environmental factors such as aeration, illumination, and temperature are also essential for efficient mushroom growth. Hence, the effects of environmental factors on the growth of mycelia of the three V. volvacea strains were studied. In aeration, the mycelial growths of the three strains in the two aeration conditions were not significantly varied [Figure 4]. The results suggest that the mycelial growths of the three strains are not influenced by aeration. They could grow either in sealed and unsealed conditions. This response is in contrast with the observation of Reyes et al. [13] that V. volvacea favorably respond in sealed conditions. Likewise, the mycelia of S. commune, Lentinus sajor-caju, and Lentinus tigrinus showed luxuriant growth when incubated in either sealed and unsealed conditions [14-16].

Figure 4: Mycelial growth diameter of the three strains of V. volvacea on Sabouraud dextrose agar at pH 6.5 incubated in sealed and unsealed conditions [Click here to view]

The influence of illumination on the growth of mycelia of V. volvacea strains is shown in Figure 5. Mycelia of La Clementina and Montalvo strains favorably grew in lighted condition, whereas Vinces strain exceedingly favored dark condition. However, T-test revealed that the two conditions were significantly different in Vinces and Montalvo strains only. These results clearly indicate that the three strains have varying responses to illumination. Kalaw et al. [8] reported the excellent mycelial performance of two strains of V. volvacea in both lighted and dark conditions. However, Reyes et al. [13] confirmed that light has positive effect on the growth of V. volvacea mycelia.

Figure 5: Mycelial growth diameter of the three strains of V. volvacea on Sabouraud dextrose agar at pH 6.5 incubated in lighted and dark conditions. Asterisks indicate significant difference of the two conditions using t-test [Click here to view]

Temperature is another important physical factor that influenced the mycelial growth. In general, fungi can be classified as temperate, semi-temperate, or tropical depending on their optimum temperature for mycelial growth [17]. The mycelial growth of the three mushroom strains as affected by temperature is presented in Figure 6. Noticeably, the maximum mycelial growth of the three strains was achieved when incubated at 26–28°C and 32°C. The mycelial growths of the three strains of V. volvacea on SDA at pH 6.5, incubated in the optimal physical conditions, are shown in Figure 7. In contrast, no growth of mycelia was observed when incubated at 9°C. The results suggest that the three strains of V. volvacea are tropical mushrooms. Since the Philippines is a tropical country, cultivation of these exotic strains would not require sophisticated temperature-controlled facility. The fruiting body production technology established for the Philippine strains of V. volvacea could be utilized for the production of these new strains from Ecuador.

Figure 6: Mycelial growth diameter of the three strains of V. volvacea on Sabouraud dextrose agar at pH 6.5 incubated in three temperature conditions. Means with the same letters of superscript are not significantly different according to Tukey’s HSD (P < 0.05). [Click here to view]

Figure 7: Mycelial growth of the three strains of V. volvacea; (a) La Clementina, (b) Vinces, and (c) Montalvo on Sabouraud dextrose agar at pH 6.5 incubated in the optimal physical conditions for growth [Click here to view]