Growing Medicinal Mushroom Lentinus Tigrinus

A preliminary study on cultivation of Iranian wild- growing medicinal mushroom Lentinus tigrinus

Shadi Shahtahmasebi1, Hamid R. Pourianfar1*, Sharareh Rezaeian1, Javad Janpoor1

1 Industrial Fungi Biotechnology Research Department, Research Institute for Industrial Biotechnology, Academic Centre for Education, Culture and Research (ACECR)-Khorasan Razavi Province Branch, Mashhad, Iran.

Corresponding author: Hamid R. Pourianfar

ABSTRACT: Wild mushrooms have been known to provide a significant source of nutritional and medicinal bioactive compounds. However, the production of fruiting bodies or mycelia in a standardized quality remains a challenge in utilization of wild mushrooms as drugs, nutraceuticals or other purposes. The present work, for the first time, aimed to evaluate mycelium growth characteristics and cultivate an ITS-identified Iranian wild strain of Lentinus tigrinus using agro-waste substrates locally available in the country. The findings indicated that the radial growth of mycelia on MEGA media was significantly greater with a higher density than that on PDA or MEA media (p < 0.05). The wild strain was then demonstrated to successfully produce normal mushrooms at a biological efficiency of 56.66% in sawdust substrates enriched with wheat bran. Therefore, the findings of this study may facilitate further investigations which require fruiting bodies or fresh mycelia in order to evaluate biological activities of wild strains of L. tigrinus.

Keywords: Lentinus tigrinus, Iranian wild mushrooms, substrates, domestication, growth characteristics

INTRODUCTION

An increase in the world demand of healthy foods has made functional foods an important part of lifestyle that also includes a balanced diet and physical activity (Das et al., 2016). In addition to well-known sources such as animals and plants (Das et al., 2016), wild mushrooms could also be one of the best sources for production of functional foods. Wild mushrooms possess nutritional as well as bioactive compounds that are effective in improving human health (Singh et al., 2014). Thus, there is an increasing interest in research into nutritional and medicinal properties of wild mushrooms, as compared to limited cultivated mushroom strains (Oyetayo, 2011). One of the most important challenges in utilization of wild mushrooms as drugs, nutraceuticals or other purposes is the continuous production of fruiting bodies in a standardized quality (Lindequist et al., 2005). Accordingly, since 2012 our research team in Iran has made efforts to collect and molecularly authenticate wild populations of mushrooms across the country. These efforts were followed by studies on cultivation and growth characteristics of the wild mushrooms in lignocellulosic substrates locally available (Ghanbari et al., 2015; Masoumi et al., 2015; Rezaeian et al., 2017; Rezaeian et al., 2016) and their medicinal properties (Tajalli et al., 2015, Soltanian et al., 2016). The fungus Lentinus tigrinus is also among medicinal mushrooms collected and molecularly identified (using ITS sequence analysis) by us in 2012. L. tigrinus is a white-rot fungus belonging to Polyporaceae family, which could be found in fallen logs in forest along with the onset of rainy season and increased humidity (Dulay et al., 2013). The leathery flesh and strong aroma and taste of the fruit bodies have made it a pleasant mushroom (Lechner and Albertó, 2007). While the cap of L. tigrinus is 1 to 3 cm in width with small brown scales on its surface and a white spore print, the mushroom has a stem with 2–4 cm long and 2–5 mm wide. In addition to North American, North and Western Europe, Eastern Asia, Turkey and Pakistan, L. tigrinus is also native to various regions in Iran. To the best of our knowledge, no study has reported cultivation of Iranian wild-growing L. tigrinus Intl J Farm & Alli Sci. Vol., 6 (6): 149-153, 2017

using locally available substrates so far,. This research, thus, aimed to evaluate mycelium growth characteristics and cultivate wild L. tigrinus under conditions of commercial production.

Materials and Methods

2.1. Strain preparation A wild strain of L. tigrinus utilized in this study has previously been collected from the Northeastern Iran during the year 2012. This strain was identified using morphological characteristics and Internal Transcribed Spacer (ITS) analysis, designated as Lt004 code.

2.2. Mycelial culture in solid medium Various culture media were employed to attain the best mycelial growth of L. tigrinus, including PDA (potato dextrose agar), MEA (Malt extract agar), and MEGA (Malt extract glucose agar). All the media were sterilized for 20 minutes at 121℃ and then aseptically poured into 8-cm petri dishes. The mycelial cultures were then incubated in the dark for 10 days at 25℃, after which the mycelial growth, density and color of the colonies were evaluated as reported elsewhere (Masoumi et al., 2015).

2.3. Submerged cultures of mycelia Liquid cultures were prepared using MEG broth media (as MEGA was selected to be the best solid media). Each 40-mL liquid culture was kept at 25°C, 140 rpm for 8 days. 2.4. Spawn production A mixture of boiled wheat grains supplemented with 0.18% w/v CaCO3 and 1% w/v CaSO4 in 20× 45 cm polypropylene bags was prepared. The bags were then autoclaved at 121 °C, 15 psi pressure for two hours. Then two pieces of mycelial disc (10 mm) from a 7-day old pure culture of L. tigrinus was used to inoculate the sterilized spawn bags, incubated at 25 ± 2°C until the grains were fully colonized by the mycelia.

2.5. Substrates Two different formulations of substrates (S1 and S2) were evaluated. S1 and S2 contained wheat straw and sawdust, respectively, while both substrates were enriched with wheat bran. The substrates were then soaked in water for 24h to adjust the moisture content as much as 65-70%. To each substrate, 1% lime and 3% gypsum (on dry weight basis) were also added. The substrates were filled in polypropylene bags and autoclaved at 15 psi, 121°C for 120 minutes. 2.6. Vegetative growth of mycelia in substrates After cooling, the substrates were inoculated with 5% (wet weight) of spawn of L. tigrinus. The temperature and humidity conditions were set to 25ºC and 80-90%, respectively for 12-14 days until the spawn run was completed.

2.7. Cropping After the completion of spawn run in the substrates, the bags were placed at 18oC with a humidity of 60–70% in order to stimulate primordia formation. Further, the bags containing primordia were moved to a cropping room with light exposure in which temperature and humidity were maintained at 18-20oC and 85%, respectively. After appearance of primordia, several small cuts were made in bags and harvesting was performed for 10-14 day, during which various growth parameters including yield (weight of fresh mushrooms), biological efficiency, period from inoculation until appearance of first primordia, and total number of mushrooms were measured and recorded. Biological efficiency was determined using the following formula: Biological efficiency (%) = [weight of fresh basidiocarps/weight of dry substrate] × 100.

Results and Discussion

3.1. The growth characteristics of mycelia in solid and liquid cultures The findings revealed that the texture of mycelia was normal and cottony in all of the tested media. However, the growth of mycelia on MEGA media was significantly faster with a higher density than that on PDA or MEA (p < 0.05). The mycelia fully colonized the 8-cm petridish containing MEGA after eight days of incubation at 25°C (Fig. 1). The quantitative growth data showed that the radial growth rates of L. tigrinus on MEGA, MEA, and PDA were 7.45, 6.62, and 5.60 mm/day, respectively. MEG also showed to be the optimum media for liquid culture of L. tigrinus, where 1.62 grams dry mycelia were obtained after 8 days of incubation at 25°C. 3.2. The effect of substrates on agronomic characteristics of L. tigrinus

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The effects of substrate composition on mycelial growth of wild L. tigrinus were also investigated. The results showed that L. tigrinus significantly grew slower in S1 as compared to S2 (7.2 mm/day) (p ≤ 0.05). Accordingly, mycelial colonization in S2 was found to be completed within 12-14 days, while it took 20-25 days in S1. The primordia of L. tigrinus appeared 19-22 days after inoculation in S2 (Fig. 2). Also, 4-6 fruiting bodies per each bag were harvested during fructification in S2 (p ≤ 0.05) (Fig. 2). The mushrooms harvested in S1 also displayed some types of deformation in their shape. The results also showed that the best yield of L. tigrinus was obtained with substrate S2 with an average BE of 56.66 % which was statistically greater than that of S1 (p ≤ 0.05). Thus, the wild strain of L. tigrinus was successfully cultivated in S2 at a semi-commercials scale in the pilot plant (Fig. 3). This study is the first report on domestication of Iranian wild-growing L. tigrinus in locally available low-cost substrates. The findings indicated that the wild strain successfully produced normal mushrooms at a biological efficiency of 56.66% in sawdust enriched with wheat bran. Very few studies from other countries have reported cultivation of L. tigrinus, including rice straw-sawdust enriched with rice bran or rice grits in Philippine (De Leon et al., 2013), sawdust + rice straw substrate formulation in Philippine (Dulay et al., 2012), and sawdust with different supplements in Argentina (Lechner and Albertó, 2007). Medicinal properties of L. tigrinus have also been the subject of limited studies (Garcia et al., 2014; Xu et al., 2012; Dulay et al., 2015). Therefore, the findings of this study may facilitate further investigations on biological activity of wild strains of L. tigrinus.

Acknowledgments This study was conducted in Industrial Fungi Biotechnology Research Department, ACECR-Khorasan Razavi Province Branch, Iran, funded by an ACECR-Khorasan Razavi Province Branch approved research project (an internal grant to Shadi Shahtamsebi in the year 2016-2017).

Figure 1 – The high mycelial growth rate of L. tigrinus (Lt004) in MEGA (Malt extract glucose agar) after eight days of incubation at 25°C. The texture of mycelia was found to be normal and cottony.

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Figure 2– Appearance of primorida of L. tigrinus (Lt004) in sawdust enriched with wheat bran 19-22 days after inoculation (left). The fruiting bodies grew in cluster (middle) and growth characteristics were measurement by using a caliper (right).

Figure 3. Cultivation of L. tigrinus in semi-commercial scale in the pilot plant of Industrial Fungi Biotechnology Research Department, ACECR-Khorasan Razavi Province Branch, Mashhad, Iran in 2017.

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