Introduction

Mushrooms are fleshy, spore bearing reproductive structures of fungi. For a long time, wild edible mushrooms have played an important role as a human food. However, empirical methods for their cultivation are relatively recent (Martinez-Carrera, 2000). The consumption of mushrooms probably occurred during prehistory, in the hunting and gathering period. Unlike plants, mushrooms could not be cultivated at first and were collected for a long period of time. Even today, relatively few species of mushrooms can be cultivated compared to the number of edible species. Mushrooms were thought to be special and supernatural in origin as 4600 years ago, the Egyptians believed mushrooms to be the plant immortal; the Pharaohs decreed that only they could eat mushrooms (Goldstein and Goldstein, 2010). The Romans thought mushrooms were the food of the Gods (Valverde et al., 2015). Many people collect mushrooms for the purpose of consumption, but lots of myths and false concepts still survive today (Rogers, 2015).

V. volvacea is an edible mushroom of tropics and subtropics. The optimum temperature and moisture for the growth of this mushroom are 35 °C and 57-60%, respectively. It can be cultivated in North Indian plains from July to September and in peninsular India from March to November. However, in the hilly areas during the November to July months artificial heating is necessary to raise the bed temperature but in the plains, artificial heating can be minimized by the incorporation of Melia azadirachta and Tamarindus indicus leaves in alternate layers (Singh et al., 2011).

Mushrooms grow on natural or semi synthetic compost and absorb nutrients for their survival. The maintenance and revival of pure culture mycelium with magnificent quality is the first critical stage towards the success of spawn preparation (Kumar et al., 2018). To maintain any microorganism in artificial conditions, the former has to be cultured on a suitable nutrient medium. All microbes require a set of conditions under which they can grow and sporulate best and culture medium is the major factor influencing fungal cultivation (Dhingra and Sinclair, 2014). Much literature is not available on this aspect of V. volvacea, however there are few reports on the cultural studies of Volvariella species (Gupta et al.,1970;Ukoima et al., 2009;Tudses, 2016 and Kapoor,2004). Keeping in view the importance of cultural studies in the cultivation process of a particular mushroom and paucity of literature on this aspect of V. volvacea,   the present investigations were conducted with an objective to study cultural parameters of different strains of V. volvacea.

Materials and Methods

2.1.  Screening of liquid medium

Five different nutrient media viz., malt extract broth, potato dextrose broth, oat meal broth, Czapek’s dox broth, sweet potato dextrose broth were evaluated for mycelial growth of different strains of V. volvacea. A 5 mm bit of different strains of V. volvacea (DMR-484, DMR-463, DMR-819, DMR-820) was taken with the help of sterilized cork borer from the pure culture plate and inoculated in respective broth (150 ml capacity Erlenmeyer’s flasks). After inoculation, the flasks were incubated in a BOD incubator at 30 °C. Each treatments were replicated thrice and the data were recorded after 7, 14 and 21 days of the inoculation in terms of dry mycelial weight (mg). For the calculation of fungal biomass in liquid media, mycelial mats of the test fungus were filtered through Whatmann no. 1 filter paper disc having 11 cm diameter and dried at 50 °C overnight. The dry weight of the fungus was calculated by using the following formulae:

Dry weight=(weight of filter paper+mycelium)–(weight of filter paper)

2.2.  Screening of solid media

Five different solid media viz., malt extract agar (MEA), potato dextrose agar (PDA), oatmeal agar (OMA), Czapek’s dox agar (CA) and sweet potato dextrose agar (SDA) were evaluated for mycelial growth of different strains of V. volvacea. A 5 mm bit of test fungus was taken with the help of sterilized cork borer from the pure culture plate and inoculated in the centre of petriplate and then incubated in BOD at 30°C. Each treatments were replicated thrice and data were recorded in terms of average diametric growth (mm) and type of growth (fluffy/ strandy/ sparse) after 48, 72 and 96 h of incubation. Based on these studies best nutrient medium was selected for further experiments.

2.3.  Effect of different pH level on growth of V. volvacea

To see the effect of different pH levels on the mycelial growth of different strains of V. volvacea, the best nutrient medium selected in 3.4.2 was adjusted to different pH levels viz., 5.0, 6.0, 7.0, 8.0, and 9.0. Then each of the media with different pH levels was poured in petriplates and after solidifying of the media a culture bit of 5 mm was placed on each of the petriplates. These petriplates were then incubated at 30°C for 48 hrs to record the data. Each treatments were replicated thrice and the data were recorded in terms of average diametric growth (mm) and type of growth (fluffy/ strandy/ sparse) after 48, 72 and 96 h of inoculation.

2.4.  Effect of different temperature regimes

To study the effect of different temperature regimes, petriplates containing MEA as a basal medium along with culture bit of 5 mm diameter of the test fungus were subjected to different temperatures viz., 25, 30, 35, 40 and 45 °C in different incubators for 48 h. Each treatments were replicated thrice and data were recorded in terms of average diametric growth (mm) and type of growth (fluffy/ strand/ sparse) after 48, 72 and 96 h of inoculation.

Results and Discussion

3.1.  Screening of liquid media

Data presented in Table 1 clearly depict that on an average malt extract broth was recorded to support significantly maximum (2625.42 mg) dry weight of the test fungus followed by potato dextrose broth (1667.22 mg) and sweet potato dextrose broth (604.17 mg) while,

Table 1: Effect of different liquid media on the growth of different strains of V. volvacea

average minimum (246.94 mg) dry weight of the fungus was observed in Czapek’s dox broth followed by oat meal broth (400.83 mg), irrespective of the different strains used and days after inoculation. As far as the growth behaviours of different strains of V. volvacea was concerned, strain DMR-484 exhibited significantly maximum (1780.67 mg) average dry weight of the fungus followed by strain DMR-463 (1124.00 mg) while, average minimum dry weight of the fungus was recorded in strain DMR-820 (713.00 mg) followed by the strain DMR-819 (818.00 mg), irrespective of different media used and time after inoculation. Irrespective of nutrient media and different strains of V. volvacea under investigations, significantly maximum average dry weight (2231.25 mg) was recorded after 21 days of inoculation followed by 14 (764.17 mg) and 7days of the inoculation (331.33 mg). The body of table reveals that strain DMR-484 exhibited significantly maximum average dry weight (8250.00 mg) in malt extract broth after 21 days of inoculation. However, minimum (53.33 mg) average dry weight was recorded in strain DMR-820 grown in sweet potato dextrose broth after 7 days of inoculation. Rest of the strains exhibited intermediate range of average dry weight in different media under studies after 7, 14 and 21 days of the inoculation except strain DMR-819 and DMR-820 grown in Czapek’s dox broth, where no growth was recorded even after 21 days of incubation.

3.2.  Screening of solid media

It is evident from the data presented in Table 2 that on an average malt extract agar medium was observed to support the maximum average diametric growth (38.92 mm) of the fungus significantly followed by sweet potato dextrose agar (36.14 mm) and oat meal agar (33.64 mm) while, the minimum average diametric growth (26.86 mm) was recorded in Czapek’s dox agar followed by potato dextrose agar (28.69 mm), irrespective of different strains and time of inoculation under study. Irrespective of different media and hours of incubation, strain DMR-484 was observed to exhibit maximum average diametric growth (41.38 mm) followed significantly by DMR-463 (33.40 mm), DMR-820 (29.04 mm) and DMR-819 (27.58 mm). As far as time of incubation was concerned, the maximum average diametric growth (50.63 mm) was recorded after 96 h of incubation while, the minimum average diametric growth (16.42 mm) was recorded after 48 h of inoculation.

Table 2: Effect of different solid media on the growth of different strains of V. volvacea

The growth of the test fungus depicted significant increase after each duration of incubation. The body of the table reveals that significantly maximum average diametric growth (79.00 mm) was recorded in the strain DMR-484 grown in malt extract agar after 96 h of inoculation while, minimum average diametric growth (9.00 mm) was recorded in strain DMR-819 after 48 h of incubation grown in Czapek’s dox agar. Rest of the strains exhibited intermediate range of average diametric growth grown on various solid media after different hours of incubation.

The growth characteristics of the different strains of V. volvacea varied differently in various liquid as well as solid media under study. The present investigations reveal malt extract broth to be the best nutrient medium for the growth of all four strains of V. volvacea while, Czapek’s dox broth was least supportive for the growth of test fungus. These results are in conformity with the findings of Gupta (2015) who reported that malt extract broth supported maximum mycelial growth while, Czapek’s dox broth supported the minimum mycelial growth of V. volvacea both in liquid and solid phase.

Among five different solid media studied, malt extract agar was found to support the maximum mycelial growth followed by sweet potato dextrose agar. These results are in accordance with the findings of Nasim et al. (2001) who reported that malt extract agar supported the maximum mycelial growth of V. volvacea and potato dextrose agar was found to support least mycelial growth. Further the present findings are in conformity with the results of Nie et al. (2016) who reported that malt extract agar supported faster mycelial growth of V. volvacea than potato dextrose agar. On the contrary, Kumar et al. (2016b) reported potato dextrose agar to support the maximum mycelial growth followed by malt extract agar.

3.3.  Evaluation of different pH regimes

It is clear from the Table 3 that maximum average diametric growth (30.75 mm) was recorded at pH 7.0 significantly followed by pH level 8.0 (25.25 mm) which was statistically at par with the growth at pH 9.0 (25.11 mm) and pH 6.0 (24.19 mm) while, significantly minimum average diametric growth (13.75 mm) was recorded at pH 5.0, irrespective of different strains and hours of incubation. Evaluating the performance of different strains of the test fungus, strain DMR-484 was observed to exhibit maximum average diametric growth (29.64 mm) of fungus significantly followed by strain DMR-463 (24.42 mm) while, minimum average diametric growth (20.27 mm) was recorded in DMR-819 which was statistically at par with DMR-820 (20.91 mm),

Table 3: Effect of different pH levels on mycelial growth of different strains of V. volvacea

irrespective of the different pH level and hours of incubation. As far as the average diametric growth after different intervals is concerned, significantly maximum average diametric growth (37.70 mm) was recorded after 96 h of incubation while, minimum average diametric growth (9.58 mm) was recorded after 48 h of incubation, irrespective of the different pH levels and strains under evaluation. It was interesting to note that on an average, all the strains grew faster between 48 to 72 h of incubation while, after 72 to 96 h of incubation the increase in mycelial growth was comparatively lesser. The body of table reveals that average diametric growth was significantly maximum (62.00 mm) in strain DMR-484 grown at pH 7.0 after 96 h of incubation while, minimum (4.67 mm) growth was recorded in strain DMR-819 grown at pH 5.0 being statistically at par with rest of the three strains grown at same pH level; DMR-463, DMR-819, DMR-820 grown at pH 6.0; DMR-819, DMR-820 grown at pH 8.0 and DMR-819, DMR-820 grown at pH 9.0 after 48 h of incubation. Intermediate range of average growth of different strains was recorded when grown at different pH levels after 42, 72 and 96 h of incubation. It was interesting to note that at pH level 5.0, strain DMR-819 did not exhibit significant increase in growth from 72 to 96 h of incubation.

In the present investigations, pH 7.0 was found to be the best pH for the mycelial growth of V. volvacea as it supported the maximum growth of the test fungus followed by pH 8.0. The present findings regarding the optimum pH levels are in accordance with the findings of Kumar et al. (2016a) who reported that the best pH is 7.0 for the growth of V. volvacea however, Kapoor (2004) reported that pH between 6.0 and 7.0 was more suitable for the growth of straw mushroom. On the contrary, Ahlawat and Tewari (2007) reported pH 9.0 to be the best supportive pH for the growth of V. volvacea.

3.4. Effect of different temperature regimes

Data presented in Table 4 clearly depict that significantly maximum average diametric growth (41.78 mm) was recorded at 30 °C followed by growth at 35 °C (39.17 mm), 40°C (27.22 mm) and 25 °C (17.44 mm), irrespective of the strains and time of incubation under study. However, no growth of any of the four strains under study was recorded at 45°C even after 96 h of incubation. Irrespective of different temperature regimes and time of incubation, significantly maximum average diametric growth (37.00 mm) was recorded in DMR-484 followed by strain DMR-463 (30.81 mm) while, minimum average diametric growth (27.83 mm) was recorded in DMR-819 which was significantly followed by strain DMR-820 (29.97 mm).

Table 4: Effect of different temperature regimes on the mycilial growth of different strains of V. volvacea

Irrespective of different temperature regimes and strains under study significantly maximum diametric growth (45.44 mm) was recorded after 96 h of incubation significantly followed by growth after 72 (29.33 mm) and 48 (19.44 mm) h of incubation. It was interesting to note that the growth of all strains was faster between 72 to 96 h of incubation as compare to growth between 48 to 72 h of incubation. Interaction of different temperature regimes, strains and time of incubation reveals that strain DMR-484 exhibited significantly maximum average diametric growth (71.33 mm) at 30 °C after 96 h of incubation while, strain DMR-463 exhibited minimum average diametric growth (10.67 mm) at 25°C after 48 h of incubation which was statistically at par with growth of rest three strains at 25 °C, strain DMR-819 at 30 °C and DMR-463 at 40 °C after 48 h of incubation as well as growth of strain DMR-463 and strain DMR-484 at 25 °C after 72 h of incubation. During the present investigations, the growth of the test fungus was best supported at a temperature of 30°C followed significantly by 35 °C. These results are in accordance with the findings of Akinyele and Adetuyi (2005) who reported 30 °C to be optimum temperature for the growth of V. volvacea. The present investigations are further supported by Kumar et al. (2016a) who reported that optimum range of temperature of V. volvacea for its best growth lies between 30-35 °C. Also, Deshpande and Tamhane (1982) reported that the best yield of V. volvacea was obtained at 27-30 °C. Chang and Steinkraus (1981) also reported a temperature of 35 °C to be optimum for the growth of V. volvacea.

3.5.  Type of mycelial growth V. volvacea strains under different cultural conditions

Under cultural studies of different strains of V. volvacea, type of growth and colour of mycelium was recorded. It is clear from the Table 5 that out of five nutrient media studied, malt extract agar medium supported the best type of growth i.e. thick strandy in all strains under investigations while, Czapek’s dox broth supported only sparse growth of different strains of V. volvacea.

Table 5: Morphological characters of different strains of V. volvacea recorded in lab conditions

However, very sparse growth was observed in case of strain DMR-463 grown in oat meal agar and sweet potato dextrose agar. As far as the growth of different strains of V. volvacea at different pH levels was concerned, very sparse growth of all the strains was observed at pH 5.0 while, thick strand mycelium was observed at pH level 7.0. However, at pH 8.0 strain DMR-484 and DMR-463 exhibited thin strandy growth and DMR-819 as well as DMR-820 exhibited sparse growth.

Conclusion

V. volvacea being a very fast growing fungus grows best at malt extract medium both in solid and liquid phase, at pH 7.0 and temperature of 30 °C. Out of four different strains under investigations of V. volvaceastrain DMR-484 grew best in all the media, pH levels and temperature regimes studied. From these results we can say that it could easily be cultivated in subtropical regions having temperature range of temperature between 30-35 °C and substrates of neutral to slightly alkaline pH.

Further Research

Further detailed studies on the biochemical properties of V. volvacea should be studied for the complete knowledge of its hidden therapeutic properties and also for improving its post-harvest life so that it can be stored for long term use.

Acknowledgment

The facilities and support provided by the Department of Plant Pathology, College of Horticulture and Forestry, Neri (Hamirpur) has been duly acknowledged.

Reference

Ahlawat, O.P., Tewari, R.P., 2007. Cultivation technology of paddy straw mushroom Volvariella volvacea. National Research Centre for Mushroom, 36.

Akinyele, B.J., Adetuyi, F.C., 2005. Effect of agro wastes, pH and temperature variation on the growth of Volvariella volvacea. African Journal of Biotechnology 4(12), 1390–1395.

Chang, S.T., Steinkraus, K.H., 1981. Ligocellulytic enzymes produced by Volvariella volvacea the edible straw mushroom. Applied Environmental Microbiology 43(2), 440–446.

Deshpande, A.G., Tamhane, D.V., 1982. Mushrooms- Volvariella volvacea, improvement of yield. Indian Food Packer 36, 56–61.

Dhingra, O.D., Sinclair, J.B., 2014. In: Basic plant pathology methods. CRC Press, 448.

Goldstein, M.C., Goldstein, M.A., 2010. In: Healthy foods : fact versus fiction. Greenwood publishers, 310.

Gupta, G.K., Bajaj, B.S., Suryanarayan, D., 1970. Studies on the cultivation of paddy straw mushroom Volvariella volvacea and Volvariella diplasia. Indian Phytopathology 23, 615–620.

Gupta, N., 2015. In vitro cultural, nutritional and biochemical characterization of mycelia of Volvariella volvacea: An indigenous mushroom from Odisha. Applied Science Reports 12(1), 20–26.

Kapoor, J.N., 2004.  Mushroom cultivation. Indian Council of Agricultural Research, New Delhi, 89.

Kumar, N.K., Krishnamoorthy, A.S. Akannon, A.K. Amirthan, D. 2016a. Influence of temperature and pH on mycelial growth and chlamydospore production of paddy straw mushroom (Volvariella volvacea) (Bull.Ex.Fr.) Sing. The Journal of Research ANGRAU 44(1&2), 1–7.

Kumar, P., Kumar, K., Kumar, S., 2016b. Evaluation of media and substrate for spawn production of paddy straw mushroom (Volvariella volvacea). Research in Environment and Life Sciences 9(4), 446–447.

Kumar, S., Kumar, A., Chand, G., Akhtar, M.N., Kumar, T., 2018. Optimization of mycelia growth parameters for Pleurotus florida and Pleurotus sajor-caju. International Journal of Current and Applied Sciences 7, 4818-4823.

Kumar, S., Kumar, A., Chand, G., Akhtar, M.N., Kumar, T., 2018. Optimization of mycelia growth parameters for Pleurotus florida and Pleurotus sajor-caju. International Journal of Current and Applied Sciences 7, 4818–4823.

Martinez-Carrera, D., 2000. Mushroom biotechnology in tropical America. International Journal of Mushroom Sciences 3, 9–20.

Nasim, G., Malik, S.H., Bajwa, R., Afzal, M., Mian, S.W., 2001. Effect of three different culture media on mycelial growth of oyster and Chinese mushrooms. Online Journal of Biological Sciences 1(12), 1130–1133.

Nie, H.J., Mohamad, A., Joyce, Y.S.M., Muhamad, N.M., 2016. Optimisation of Volvariellavolvacea spawn production for high mycelia growth. R&D Seminar 2016: Research and Development Seminar 2016, Malaysia.

Rogers, R.D., 2015. The 19 myths of medicinal mushrooms. Medicinal Mycology 8(2), 12–15.

Singh, M., Vijay, B., Kamal, S., Wakchaure, G.C., 2011. Mushrooms: cultivation, marketing and consumption. Directorate of Mushroom Research, 12.

Tudses, N., 2016. Isolation and mycelial growth of mushrooms on different yam-based culture media. Journal of Applied Biology & Biotechnology 4(5), 33–36.

Ukoima, H.N., Ogbonnaya, L.O., Arikpo, G.E., Ikpe, F.N., 2009. Cultural studies of mycelia of Volvariella volvacea, Pleurotus tuber-regium and Pleurotus sajor-caju on different culture media. Pakistan Journal of Nutrition 8(7), 1052–1054.

Valverde, M.E., Perez, T.H., Lopez, O.P., 2015. Edible mushrooms: Improving human health and promoting quality life. International Journal of Microbiology, 367–387.