Characterization of Sporophores, Spore Prints, Spines, Basidia, and Basidiospores of Seven Genotypes of Hericium erinaceus (Bull.: Fr.) Pers

In the present investigation, the morphology, dry matter, water content, fragrance of dry powder, the dimension of sporophores, basidia, and basidiospores, and length of spines of genotypes He101, He-102, He-104, He-105, He-107, He-108, and He-109 of Hericium erinaceus were studied. The above genotypes were broadly categorized into clumping type (single clumping typeHe-108 and multiple clumping typeHe-101, He-104, He-107, He-109) and branching type (thick branching typeHe-105 and diffuse branching type-He-102). The highest 29.99 % dry matter was calculated with He-102 along with 70.01% water content. The dry powder of He-101, He104, He-105, He-108, He-109 fragranced like vanilla essence. The cooked mushroom of even all genotypes rather gave lobster-like taste. However, He-102 and He-107 produced unpleasant raw lichen and mixed spicy-vanilla type fragrances, respectively. The dimension of each of sporophores, basidia, and basidiospores was found identical and measured 4.1-6.2 x 3.8-5.7 cm, 27-45x 5.20-7.51 μm, and 5.20-6.9 x 4.20-5.80 μm, respectively. However, the genotypes were characterized with the longest 2.08 cm (He-108), medium 1.05-1.52 cm (He-101, He-104, He107, and He-109), and short 0.32-0.52 cm (He-102, He-105) spines. The spore prints of the genotypes were white and varied in shape, texture, and thickness.


INTRODUCTION
Hericium erinaceus (Bull.: Fr.) Pers. is a mostly saprophyte but rarely act as a week wood degrading fungal pathogen of many deciduous trees of Quercus sp., Fagus sp., Acer sp., Juglans sp., Ulmus sp. etc. (Stamets, 1993;Pegler, 2003& Fora et al., 2009) and indeterminately grows on them. The sporophores of Hericium are varied in dimension from 5 to 30 cm and attached latterly on the substratum strongly with the round or subglobose, protruding, unbranched (Kuo, 2016) and rudimentary stipe.

MATERIAL AND METHODS Genotype and growing medium
The pure culture of seven genotypes of Hericium erinaceus  was obtained from the Directorate Mushroom Research, Solan, and maintained on Potato Dextrose Agar (PDA) medium. The pure culture of all genotypes was used in the preparation of master spawn and subsequent commercial spawn with the standard procedure described by Gupta and Sharma, 1994. A total of 210 kg growing medium was also prepared from 70 kg of fresh dry wheat straw and 14 kg wheat bran. The dry wheat straw was soaked in water overnight followed by sun-drying for 4 hrs on a pre-disinfested cemented platform with 4% formalin. Wet wheat straw was thoroughly mixed with 14 kg wheat bran and packed in polypropylene bags @ 1.5 kg/ bag followed by autoclaving (at 20 lbs for 2 hours), inoculation (with 90 g commercial spawn /bag), and incubation (at 25 o C for spawn run). Every genotype of Hericium eranceus was maintained with 5 replications. Each replication contained 4 bags. The inoculated bags of growing medium were tagged with date of inoculation, rate of spawning, name of genotype, number of bags, and number of replication. After completion of the spawn run, polyethylene bags were removed completely from the logs of the growing medium for pinhead initiation. The 8-10 days matured sporophores were selected for morphological and micrometrical study including their dry matter content, fragrance and taste. The process of spawn run was started in October around 25 o C. However, the production of sporophores was led by December at 15-20 o C. During production, a regular spray of water was provided every morning to maintain 90-95% RH in the production room. Morphology and micrometry of genotypes Five uniform samples of 8-10 days matured freshly harvested sporophores of each He-101,  were selected for morphological characters. Selected sporophores were split longitudinally with the help of a sharp knife and characterized the stipes (length, surface, flesh, basal end, a distal end, and branching pattern of stipe) and spines (location and length). The branches of stipe were studied similar to Sokół, et al. 2015. The arrangement of the spines formed a distinct shape/appearance of the sporophores. The texture of the sporophores was felt while holding the sporophores on the palm and recorded whether they had a wet fleshy feel, wet stemy thick feel or dry rough light feel. The dry and water content of the 100 g fresh sporophores with 3 replications of each genotype were calculated after drying them in a tray dryer at 45°C for 48 hours. The dry matter of sporophores was calculated using the formula, (%) dry matter of sporophores = (Dry weight of sporophores (g) /100 g fresh weight of sporophores) x100. However, the (%) moisture content of sporophores was measured with the formula, 100-(%) dry content of sporophores was calculated. The dried sporophores of all genotypes were powdered using Wiley grinder and confirmed their fragrance based on smell perception and memory-based comparison. The measurement of sporophores was taken using Vernier Calliper from the lower plane of the sporophores where the main stipe is over. Since the shape of sporophores was ovoid or irregular so, an average of two or more lengths was measured at longer, shorter, and intermediate horizontal axis. The spore prints of all genotypes were also obtained. The selected sporophores of each genotype were attached to the holder in an invert position just a few cm above the piece of black paper on which spore mass was shaded. The setting was kept undisturbed for 12 hrs in an aseptic place for a successful spore print.
The micrometry of basidia and basidiospores of all genotypes of Hericium erinaceus was done with a compound microscope (on which Magcam DC 14 camera was attached) and computer-based Magnus Pro Image Analysis Software. Fifteen observations of each genotype for both basidia and spore micrometry were taken. In the micrometry of basidia, the selected matured spines were embedded in a piece of potato and cut into possible thinnest transverse (TS) sections with the help of a sharp blade in presence of distilled water. The possible thinnest TS of the spine was placed on the glass slides and stained with a drop of cotton blue. The prepared sections were covered with coverslips. The micrometry of basidiospores was done using 8-10 days matured sporophores. The selected sporophores of each genotype were plucked from the logs and kept for 12 hrs directly onto the clean glass slides in an aseptic place for spore shedding. The coverslips were mounted on the slide followed by the drop of water and finally covered the area of shaded spores. The prepared slides of basidia and basidiospores were observed using Magnus Pro Image Analysis Software. The respective data of the experiments were statistically analyzed using one factorial CRD of Web-Based Agricultural Statistics Software Package (WASP 2.0) at www.ccari.res.in/wasp 2.0/index.php.

Morphological characters of the sporophores of Hericium erinaceus
The sporophores of He-101,  genotypes were matured within an average of 8-10 days from the date of their appearance on the growing media. The dimension of sporophores of all genotypes was statistically identical (table 3). Although, He-104 had the biggest sporophore of 6.2 X 5.7cm followed by the 5.8 X 5.0 cm dimension of He-109. The smallest sporophore of He-105 was measured by 4.1 X 3.8 cm. However, 5-9 cm cap diameter of Hericium erinaceus was recorded by Hassan, 2007. The length of spines of all genotypes was measured differently. He-108 had the longest spines of 2.08 cm length followed by He-101 (length 1.52 cm). Branched type sporophores had very short bristle-like spines (0.32-0.52), while clumping type had long spines. As the sporophores were grown indeterminately, the length of spines was increased by 3-4 cm. Ouali et al. (2020) described the fruit body of Hericium erinaceus consisting of dangling spines of 2 cm long and 2mm in diameter. Similarly, Ginns (1985) noted 1-5cm long and 2-3mm diameters of geotropic dangling spines of Hericium erinaceus on its lower face.
The sporophores of He-101, He-102, He-104, He-105, He-107, He-108, and He-109 genotypes of Hericium erinaceus were broadly divided into two groups; 1) clumping type and 2) branch type ( fig. 1). The clumping type was further divided into a single clumping type (He-108) and multiple clumping type . Both the clumping type sporophores were similar in many characters. Branch type sporophores were divided into thick branching type (He-105) and diffuse branching type (He-102) which differed in many characters as shown in table 1. These three groups of sporophores were differentiated on the appearance, texture, dry matter, moisture, fragrance, and taste of sporophores, length, surface, flesh, basal end, distal end, and branching pattern of stipe and location, and length spines. (table1). Ginns (1985) and Harrison (1973) stated that species in the genus Hericium are distinguished macroscopically by the presence of branched or unbranched spines on which spines/tooth of various lengths are attached. Aggregation of spines formed single and multiple clumps of fruit bodies. Conde and Wolf (1972) defined Hericium erinaceus as large, snow-white, fleshy sporophores. Sometimes the fruit body of Hericium erinaceus appeared as nodular or ovoid basidioma with compact tuft-shaped, white-cream with a lateral short stipe attached on the trunk (Ouali et al., 2020). Some species of Hericium were described with the sporophores having thick primary branches originating from a single hard base. These primary branches were further divided into many progressive branches and formed multitudes of ramuli of short spines (Hallenberg, 1983& Hallenberg et al., 2013. The spore print of all genotypes having single, multiple, thick, and diffuse branching pattern of sporophores was varied in the pattern, thickness of spore mass, and shape. The basidiospores were formed externally on the tuberculate spines and at maturity, they were released in form of white spore mass. The spore print of He-102 was insignificant in appearance due to very little in the release of spores on the black surface (Fig.1).  Fig. 1: Types of sporophores (a, c, e, g) and spore print (b, d, f, h) The dry powder of He-101, He-104, He-105, He-108, He-109 was sensed vanilla type fragrance. However, He-102 produced unpleasant raw lichen fragrance and He-107 was known for mixed spicy vanilla fragrance (table 2). Ahmed et al. 2008 advocated the citrus-floral and musky delicate flavor of Hericium species. The cooked Hericium reportedly tastes as delicious to someone as lobster cooked in butter (https://www.firstnature.com/fungi/hericium-erinaceus.php). Thongbai, 2015 found out the seafood type taste of Hericium reminiscent of crab or lobster with fleshy, tough, and watery texture. Several aromatic compounds such as hericerin A, isohericenone J, isoericerin, hericerin, N-dephenylethyl isohericerin, hericenone J, 4-(3′,7′-dimethyl-2′,6′-octadienyl)-2-formyl-3hydroxy-5-methoxybenzylalcohol, erinacene D, esorcinols, erinacerins, and hericenols were reported earlier (Chaiyasut & Sivamaruthi, 2017) probably responsible for the fragrance and taste of Hericium. The sporophores of all tested genotypes of Hericium were composed of different dry matter and moisture of their sporophores. Out of 100 g fresh weight of sporophores, a maximum of 29.99 g dry matter was obtained with He-102. However, a minimum of 6.25 g dry matter was recorded in the case of He-108. The moisture content among the genotypes was varied from 89.10 to 93.75% except for He-102 in which 70.01% moisture content was determined. Chaiyasut and Sivamaruthi, 2017 reported 4% water content from the dry matter of Hericium erinaceus. The water content of the sporophores was varied from 73 to 91% (Friedman, 2015).

Micrometry of basidia and basidiospores of genotypes of Hericum erinaceus
During the microscopic study, club type basidia and globose to sub-globose type spores were found (Fig 3). The dimension of basidia was found almost identical among all genotypes. Although, the He-102 had the longest and slender type basidia with length and 45µm length X 5.20 µm width (table 3). The widest basidia of He-107 was measured at approximately 7.51µm. Statistical analysis of the spore dimension was also done and found with no significant difference. The majority of basidiospores were dark thick walled at periphery and sub globose / mango /pear shaped. The papillae (remaining part of the sterigmata) were also appeared on the basidiospores by which they probably attached with the basidium However, the colour of the spore print of basidiospores of all genotypes was recorded as white. The basidiospores of He-102 were measured longest and widest with the dimension of 6.90 X 5.80µm (table  3). Harrison (1973) and Kuo (2016) found about 25-40 × 5-7μm dimension of basidia and 5.5-6.8 × 4.5-5.6μm dimension of the short ellipsoid to subglobose white coloured basidiospores. According to Ouali et al. (2020), the basidiospores (5-7µm X 4-5µm) are white in mass, short ellipsoid, little visible ornamentation with finely roughened, amyloidal.    CONCLUSION Among all genotypes of Hericium erinaceus, the dimension of each of sporophores, basidia, basidiospores were found identical. However, the length of spines of all genotypes was varied from0.32 to1.52 cm. The variation in the length of spines and branching of stipes was collectively responsible to develop different shapes and textures of the sporophores. Thus, these sporophores were grouped into clumping (single and multiple clumping type) and branching type (thick and diffuse branching). The dry matter of the sporophores was varied from 6.25 to 9.66% except for He-102 in which a maximum of 29.99% dry matter was calculated. The fragrance of the sporophores was also studied and found that the dry powder of He-101, He-104, He-105, He-108, He-109 were sensed vanilla type fragrance. However, He-102 produced an unpleasant raw lichen fragrance and He-107 was known for a mixed spicy vanilla fragrance. The taste of butter cooked sporophores of all genotypes was felt like cooked lobster except He-102 and He-105. The white coloured spore prints with different pattern and thickness were noticed except the spore print of He-102.