The Productivity of Genotypes of Hericium erinaceus (Bull.: Fr.) Pers. on Wheat Straw and Poplar Sawdust Based Growing Media

The genotypes of Hericium erinaceus He-101, He-102, He-104, He-105, He-107, He-108, He109, and growing media made of wheat straw+20% wheat bran (GM1) and poplar sawdust+20% wheat bran (GM2) were studied for spawn run, harvestings, numbers, yield, biological efficiency, cropping period, and average fruit body weight. The spawn run of He-108 was completed within a minimum of 24 and 26 days on GM 1&2, respectively, however, early harvesting of He-101 was recorded within 42 (GM1) and 43 days (GM2) after spawning. He104 resulted highest 205.7 g yield /1.5 kg GM 1 (31.65% BE) with 11 highest average numbers of mushroom /1.5 kg GM1 and 19.22 g highest average fruit body weight with maximum 5 harvestings and 94 days of total cropping period. The performance of GM1 was found superior to GM 2. The GM1 led to the shortest duration of spawn run (27 days), the highest number of harvestings (3), the highest number of fruits (7/bag), the highest yield (115 g/bag), and the highest BE (17.66%) on paired ‘t’ test. While the short-duration crop of 66 days was recorded in GM2 with an incomplete spawn run. Keyword: Hericium erinaceus, Wheat straw, Poplar sawdust, Genotype, Yield, BE. Research Article Cite this article: Sholyavei, Sh., & Mishra, S. K. (2020). The Productivity of Genotypes of Hericium erinaceus (Bull.: Fr.) Pers. on wheat Straw and Poplar Sawdust Based Growing Media, Ind. J. Pure App. Biosci. 8(6), 396-405. doi: http://dx.doi.org/10.18782/2582-2845.8454 Sholyavei and Mishra Ind. J. Pure App. Biosci. (2020) 8(6), 396-405 ISSN: 2582 – 2845 Copyright © Nov.-Dec., 2020; IJPAB 397 The fruit body is composed of numerous bioactive compounds like phytosterol; βsitosterols, ergosterol (Rahman et al., 2014), polysaccharides; β-D glucans (Moldavan et al., 2007), phenol analogous; hercenons, and benzyl alcohol derivatives hericenones (Mizuno, 1999; & Ma et al., 2010), diterpenoid derivatives; erinacine (Lee et al., 2014; & Ma et al., 2010). The only recent breakthrough of its first cultivation in India was in Directorate Mushroom Research (DMR), Solan, Himachal Pradesh. The present research work on Hericium was initiated in Uttarakhand State, India after receiving seven genotypes from DMR, Solan. They were tested in the natural climate of winter season in the Tarai region of Uttarakhand State on the growing media made of wheat straw and poplar sawdust for the spawn run, harvesting, cropping period, number, yield biological efficiency, and average fruit body weight. Though, H. erinaceus was cultivated earlier on various substrates, such as wheat straw, paddy straw, sawdust, cottonseed hulls, sugarcane bagasse, corncobs, supplemented with wheat bran (Oei, 1996). But during the present investigation wheat straw and poplar sawdust are selected as they are available in plenty in the Tarai region of Uttarakhand State. MATERIALS AND METHODS Genotypes The pure culture of seven genotypes of Hericium erinaceus (He-101, He-102, He-104, He-105, He-107, He-108, and He-109) was obtained from the DMR, Solan, and maintained on Potato Dextrose Agar (PDA) medium to produce master spawn and subsequent commercial spawn. Master and commercial spawn were prepared from boiled unbroken wheat grains with which 2% CaSO4 and 0.5% CaCO3 were thoroughly mixed (Gupta & Sharma, 1994). CaSO4 and CaCO3 mixed wheat grains were filled into thermostatic glass bottles of 250 ml capacity @100g/ bottle. The bottles were plugged with non-absorbent cotton and a piece of butter paper. Ready bottles were autoclaved at 20 lbs for 2 hours. The autoclaved bottles were allowed to cool in an aseptic place followed by inoculation with a pure culture of all 7 genotypes of Hericium erinaceus. The ready bottles were incubated at 25 o C for about 25 days. The same steps were adopted for the production of commercial spawn using autoclavable polypropylene bags of 1 kg capacity instead of glass bottles. The prepared bags were inoculated from master spawn. The inoculated bags of commercial spawn were incubated at 25 o C and get ready for spawning within 25 days after inoculation. Growing medium The growing medium 1 (GM1) and 2 (GM 2) were prepared from fresh wheat straw and poplar sawdust, respectively with which wheat bran was added for supporting firm mycelial growth of Hericium erinaceus. In preparation of growing media 1, 70 kg of fresh dry wheat straw was soaked in a water tank overnight followed by sun-drying on a pre-disinfested cemented platform for 4 hrs. Moist wheat straw was thoroughly mixed with 14 kg wheat bran and packed in autoclavable polypropylene bags @ 1.5 kg/ bag followed by plugging them with thermostatic polypropylene ring, non-absorbent cotton, and butter paper. Ready bags were autoclaved at 20 lbs for 2 hours. The next day, the autoclaved bags were inoculated with the commercial spawn @ 90 g /bag. Inoculated bags were incubated at 25 o C for spawn run. Growing media 2 was made of 117 kg dry poplar sawdust in which 23 kg wheat bran was added. Total 140 kg dry mixture was getting moist with 70 liters of fresh tap water. The moist mixture was heaped overnight to have uniform moisture. Very next day, the process of bagging, autoclaving, inoculation and incubation was done similar to those of GM1. The inoculated bags of GM1 and GM2 were tagged with date of inoculation, rate of spawning, name of genotype, number of bags, and number of replication. The details of GM1and GM2 with their treatments and replications are mentioned in table 1. Sholyavei and Mishra Ind. J. Pure App. Biosci. (2020) 8(6), 396-405 ISSN: 2582 – 2845 Copyright © Nov.-Dec., 2020; IJPAB 398 Table 1: Calculations of GM1 and GM2 Activity Wheat straw Sawdust Quantity of dry substrate (A) 70 kg 117 kg Quantity of dry wheat bran (B) 14 kg 23 kg Total dry matter C (A+B) 84 kg 140 kg Addition of water in the dry matter C (D) 126 liter 70 liter Total weight of wet substrate E (C+D) 210 kg 210 kg Weight of one bag 1.5 kg 1.5 kg Number of genotypes 7 7 Number of replications 5 5 Number of bags per replication 4 4 Total number of bags 7X 5X4 = 140 7X 5X4 = 140 Spawning rate with commercial spawn (6% on a wet weight basis of GM) 90 g 90 g After completion of the spawn run, polythene bags were removed completely from the logs of GM1 but polypropylene bags of GM2 were folded upside down to half of the substrate level and punched holes at the bottom. The process of cultivation of Hericium erinaceus was started in October. In this month the temperature reached down to about 25 o C which is an ideal temperature for spawn run. As temperature constantly decreased, it would have been favorable for the development of fruiting of Hericium erinaceus and found most favorable from November to December (15-20 o C). The observations on the yield were recorded at the three ranges of temperatures above 20 o C, 20-15 o C, and 1512 o C. The crop room was kept in dark. A regular spray of water was provided in the morning to maintain 9095% RH during the cropping. Usually, 4-5 days were taken for primordia formation but sometimes it extended 7 days. After 8-10 days of primordia formation, harvesting was done. The crop was maintained until the mushrooms stopped coming out of the bags. At most 4 harvestings were obtained. Data of yield (g) and the number of fruits per bag were recorded for all the genotypes grown on both the media. Physical properties of GM 1 and 2 Wheat straw and sawdust were characterized by their particle size, bulk density, particle density, and porosity. The particle size of wheat straw was measured by Vernier Caliper. However, the particle size of sawdust was measured under a microscope with the help of the ocular and stage micrometer at 40X. The bulk and particle density of both the substrates were calculated using the method as suggested by Blake and Hartge, 1986. In which two cylindrical beakers of 1000ml capacity were filled up with wheat straw and sawdust separately to the half of the beaker with the gentle press at each step of filling. The respective cylindrical beakers are dried in a hot air oven at 100°C for 6 hours and weight. The dry substrates were water up to 800 ml in each cylinder and heat gently to remove entrapped air inside the media then make the volume up to 1000 ml and weight. The bulk density, particle density, and porosity were calculated by the following formula and concerned data are mentioned in table 2. Bulk density g/cm 3 (BD) = Particle density g/cm 3 (PD) =


INTRODUCTION
Hericium erinaceus (Bull.: Fr.) Pers. is an edible and medicinal mushroom and has been used for centuries in traditional Chinese medicine and Japanese cuisine. It is a member of toothed fungi and ecologically behaves like white-rot fungi of several hardwood trees such as Beech, Oak, Poplar, and Pine trees.
Morphologically it has a long spiny hymenophore that looks like a white snowball (Conde et al., 1972) with no distinct cap and stipe. The hot water extract of mushroom is considered a healthy drink and strengthens immunity and neurological health (Kolotushkina et al., 2003).
The fruit body is composed of numerous bioactive compounds like phytosterol; βsitosterols, ergosterol (Rahman et al., 2014), polysaccharides; β-D glucans (Moldavan et al., 2007), phenol analogous; hercenons, and benzyl alcohol derivatives hericenones (Mizuno, 1999;& Ma et al., 2010), diterpenoid derivatives; erinacine (Lee et al., 2014;& Ma et al., 2010). The only recent breakthrough of its first cultivation in India was in Directorate Mushroom Research (DMR), Solan, Himachal Pradesh. The present research work on Hericium was initiated in Uttarakhand State, India after receiving seven genotypes from DMR, Solan. They were tested in the natural climate of winter season in the Tarai region of Uttarakhand State on the growing media made of wheat straw and poplar sawdust for the spawn run, harvesting, cropping period, number, yield biological efficiency, and average fruit body weight. Though, H. erinaceus was cultivated earlier on various substrates, such as wheat straw, paddy straw, sawdust, cottonseed hulls, sugarcane bagasse, corncobs, supplemented with wheat bran (Oei, 1996). But during the present investigation wheat straw and poplar sawdust are selected as they are available in plenty in the Tarai region of Uttarakhand State.

MATERIALS AND METHODS Genotypes
The pure culture of seven genotypes of Hericium erinaceus  was obtained from the DMR, Solan, and maintained on Potato Dextrose Agar (PDA) medium to produce master spawn and subsequent commercial spawn.
Master and commercial spawn were prepared from boiled unbroken wheat grains with which 2% CaSO 4 and 0.5% CaCO 3 were thoroughly mixed (Gupta & Sharma, 1994). CaSO 4 and CaCO 3 mixed wheat grains were filled into thermostatic glass bottles of 250 ml capacity @100g/ bottle. The bottles were plugged with non-absorbent cotton and a piece of butter paper. Ready bottles were autoclaved at 20 lbs for 2 hours. The autoclaved bottles were allowed to cool in an aseptic place followed by inoculation with a pure culture of all 7 genotypes of Hericium erinaceus. The ready bottles were incubated at 25 o C for about 25 days. The same steps were adopted for the production of commercial spawn using autoclavable polypropylene bags of 1 kg capacity instead of glass bottles. The prepared bags were inoculated from master spawn. The inoculated bags of commercial spawn were incubated at 25 o C and get ready for spawning within 25 days after inoculation.

Growing medium
The growing medium 1 (GM1) and 2 (GM 2) were prepared from fresh wheat straw and poplar sawdust, respectively with which wheat bran was added for supporting firm mycelial growth of Hericium erinaceus. In preparation of growing media 1, 70 kg of fresh dry wheat straw was soaked in a water tank overnight followed by sun-drying on a pre-disinfested cemented platform for 4 hrs. Moist wheat straw was thoroughly mixed with 14 kg wheat bran and packed in autoclavable polypropylene bags @ 1.5 kg/ bag followed by plugging them with thermostatic polypropylene ring, non-absorbent cotton, and butter paper. Ready bags were autoclaved at 20 lbs for 2 hours. The next day, the autoclaved bags were inoculated with the commercial spawn @ 90 g /bag. Inoculated bags were incubated at 25 o C for spawn run.
Growing media 2 was made of 117 kg dry poplar sawdust in which 23 kg wheat bran was added. Total 140 kg dry mixture was getting moist with 70 liters of fresh tap water. The moist mixture was heaped overnight to have uniform moisture. Very next day, the process of bagging, autoclaving, inoculation and incubation was done similar to those of GM1. The inoculated bags of GM1 and GM2 were tagged with date of inoculation, rate of spawning, name of genotype, number of bags, and number of replication. The details of GM1and GM2 with their treatments and replications are mentioned in table 1. After completion of the spawn run, polythene bags were removed completely from the logs of GM1 but polypropylene bags of GM2 were folded upside down to half of the substrate level and punched holes at the bottom. The process of cultivation of Hericium erinaceus was started in October. In this month the temperature reached down to about 25 o C which is an ideal temperature for spawn run. As temperature constantly decreased, it would have been favorable for the development of fruiting of Hericium erinaceus and found most favorable from November to December (15-20 o C). The observations on the yield were recorded at the three ranges of temperatures above 20 o C, 20-15 o C, and 15-12 o C. The crop room was kept in dark. A regular spray of water was provided in the morning to maintain 90-95% RH during the cropping. Usually, 4-5 days were taken for primordia formation but sometimes it extended 7 days. After 8-10 days of primordia formation, harvesting was done. The crop was maintained until the mushrooms stopped coming out of the bags. At most 4 harvestings were obtained. Data of yield (g) and the number of fruits per bag were recorded for all the genotypes grown on both the media. Physical properties of GM 1 and 2 Wheat straw and sawdust were characterized by their particle size, bulk density, particle density, and porosity. The particle size of wheat straw was measured by Vernier Caliper. However, the particle size of sawdust was measured under a microscope with the help of the ocular and stage micrometer at 40X. The bulk and particle density of both the substrates were calculated using the method as suggested by Blake and Hartge, 1986. In which two cylindrical beakers of 1000ml capacity were filled up with wheat straw and sawdust separately to the half of the beaker with the gentle press at each step of filling. The respective cylindrical beakers are dried in a hot air oven at 100℃ for 6 hours and weight. The dry substrates were water up to 800 ml in each cylinder and heat gently to remove entrapped air inside the media then make the volume up to 1000 ml and weight. The bulk density, particle density, and porosity were calculated by the following formula and concerned data are mentioned in table 2.

Statistical analysis
The experimental data were recorded on the duration of spawn run, a total number of harvestings, cropping period (days), number, yield (g), biological efficiency (% BE), and average fruit body weight (g), of the genotypes of Hericium erinaceus. The respective data were statistically analyzed using Web-Based Agricultural Statistics Software Package (WASP 2.0) at www.ccari.res.in/wasp 2.0/index.php. One factorial RBD was used in all crop room experiments Standard deviation (SD) and critical difference (CD) were also calculated at a 5% level of significance. A paired T-test was used to compare the GM1 and GM2 by using the means of their respective treatments.

RESULTS AND DISCUSSION Spawn run
The mycelium of genotype He-107, He-108, He-109, and He-108 of Hericium erinaceus was spread fast and colonized entire growing medium 1 (wheat straw + 20% wheat bran of dry weight of wheat straw) and growing medium 2 (sawdust + 20% wheat bran of dry weight of sawdust) within 24-25 and 26 days, respectively (Table 3 and photo 1). The genotype He-104 was grown very slow on GM 1 and 2 both to be colonized within 29 and 35 days after inoculation. Growing medium made of sunflower seed hull 8 parts and wheat bran 2 parts was also colonized by Hericium erinaceus within 25 days (Figlas et al., 2007). Hassan (2007) observed that the growth of mycelium of Hericium erinaceus was slow on the oak sawdust and wheat straw-based growing medium and colonized them completely within 40 and 38 days at a 2% spawning rate (wet weight basis of growing medium). However, in the present study, the 6% spawning rate (on a wet weight basis of growing medium) was successfully reduced the time of spawn run in both the growing medium. As per the paired 't' test the growing medium 1 and growing medium 2 has a statistically significant difference for the duration of spawn run at a 5% level of significance (table 7).

Harvesting and cropping period
In the growing medium 1 and 2, the first harvest of He-101 and 104 was done at 42, 43 days, and 43, 45days after spawning, respectively (table 4). However, the remaining genotypes were differed statistically and taken a comparatively long duration for the first harvest ranged from 51-55 days in GM1 and 47-57 days in GM2. Hassan (2007) took 1 st harvest of Hericium erinaceus at 14 and 20 days after completion of spawn run in wheat straw and sawdust based growing medium, respectively.
The harvesting frequency of mushroom fruits of all genotypes of Hericium erinaceus was studied under natural conditions. It was invariably found that the first harvest of Hericium erinaceus crop of all genotypes was late due to higher temperature recorded around 25 o C in the crop room at the beginning of November, and then in the coming months, the temperature was reduced gradually by12 o C. As the temperature was down by 20-15 o C, the proceeding harvestings have been achieved comparatively in less time. After each harvesting generally 4-5 days were taken for new primordia formation. Re-lowering of temperature extends the time of new primordia formation by 7 days for 3 rd and 4 th flush, and the next 8-10 days were taken for their development to attain the right stage of the harvesting. The fruits of genotype He-104 and 105 harvested a maximum of 4 times at different intervals from GM1 within a maximum cropping period of 94 and 91 days, respectively. However, in GM 2 average number harvestings were limited by 1-2 only among all genotypes within 63-71 days of average cropping period (table 5).

Number and yield of mushroom fruits
In wheat straw-based growing medium1, the genotype He-104, and He-107 produced maximum10 numbers of fresh mushroom. However, these genotypes were differed in their yields and BE. Maximum 205.70 g (31.65% BE) yield of He-104 was recorded/1.5 kg wet substrate with highest 19.22 g average fruit body weight. This genotype also gave uniform fruiting in all the bags, with bigger fruit size. The yields and BE and average fruitbody weight of He-107 and He-109 were statistically at par but inferior to the genotype He-104 (table 6). The remaining genotypes He-105, He-101, He-108, and He-102 were found poor in the yield, BE and average fruit body weight. Hassan (2007) observed the highest 140g (39.4% BE) -157 g (43.5% BE) of Hericium erinaceus /kg wheat straw. Figlas et al. (2007) obtained 37.2% BE in sunflower seed hull with 20% wheat bran. Even all the genotypes of present research grown on the same climatic conditions showed different BE ranged from 5.14% to 31.65%. It might be due to their different inherent quality characters. Kirchhoff (1996) also found 14 heterokaryotic strains of Hericium erinaceus showed a large variation in the yield and quality of the sporophores ranging from 5%-33% on the wet weight of the substrate.
Sawdust based growing medium 2 resulted in fairly fewer numbers, yield, BE, and average fruit body weight among genotypes (Table 6), nevertheless, a maximum of 3 fruits of genotype He-101 were harvested from 1.5 kg wet substrate and highest 42.22 g (5.63% BE) yield and 15.81 g average fruit body weight of genotype He-108 were recorded from 1.5 kg wet substrate. In contrast to the above results, Hassan (2007) recorded the highest yield and highest BE of Hericium erinaceus in the sawdust based growing media. Ehlers and Schnitzler (2000) had grown six strains of Hericium erinaceus on fine beech sawdust with 20% wheat bran and obtained a 254.3g yield of fresh mushroom/kg substrate. Ko et al. (2005) obtained 31±2% BE of Hericium erinaceus in the oak sawdust substrate with 20% rice bran. Similarly, Eisenhut and Fritz (1995) recorded a 29.3% average BE of Hericium erinaceus in the substrate of sawdust. Siwulski et al. (2009) obtained BE of three H. erinaceus strains cultivated on beech sawdust substrates ranging from 20.1-28.4%.
The comparative effect of growing medium 1 and 2 was also studied. It was found that GM1 was superseded GM2 for spawn run, harvestings, cropping period, number of fruits, yield, and their BE. However, the cropping period was long (83 days) with GM1 than that of 66 days cropping of GM2 (table 7). Further, both the GMs were statistically at par with their respective duration of the first harvest and average fruit body weight. It was observed that mycelia of all tested genotypes couldn't penetrate deep in the GM2 bags during spawn run and restricted superficially. The fine sawdust of GM2 might be also made the logs compact and impermeable of air in comparison to the wheat straw of GM1. Consequently, the sawdust logs resulted in poor numbers of mushrooms and their yield. The bags of GM2 were rarely formed fruit after the first harvest. According to Figlas et al. 2007 higher mycelial growth rate of Hericium erinaceus (3.3 mm/day) was observed with large sunflower seed hull size (12x 4 mm) with the irrespective  3.21% NS = Not significant; S = Significant