Experimental Design and Observations

2. REVIEW OF LITERATURE

The genus Lentinus Fr. belongs to class Agaricomycetes, order Polyporales

and family Polyporaceae (Kirk et al., 2008). Its name Lentinus Fr. has been derived

from the Latin words “Lent”, meaning “Pliable”, and “Inus”, meaning “resembling”

(http://en.wikipedia.org/wiki/Lentinus). It is characterized by xeromorphic tough

carpophores having gills with serrated margins (Pegler, 1977 and Singer, 1986). The

fungi belonging to Lentinus Fr. are typically lignicolous and have a widespread

distribution, especially in subtropical regions (Pegler, 1977). Presently, 40 species

are recognized in this genus throughout the world (Kirk et al., 2008). Some of the

prominent contributions from India on the systematics of this mushroom are by

Manimohan et al., 2004, Kumar and Manimohan, 2005). In India the genus is

represented by 37 species (Bilgrami et al., 1979, 1991, Purkayastha and Chandra,

1985, Sarbhoy et al., 1996, Jamaluddin et al., 2004, Manimohan et al., 2004 and

Natarajan et al., 2005).

Some of the well known species of this genus are Lentinus squarrosulus

(Mont.) Singer, L. cladopus Lév, L. lepideus (Buxb.) Fr., L. tuber-regium (Fr.) Fr.,

L. sajor caju (Fr.) Fr., L. crinitus (L.) Fr., L. tigrinus (Bull.) Fr., L. polychorus Lév.,

L. conatus Berk., L. strigosus Fr., L. kauffmanii A. H. Smith, etc. Most of the

physiological, biological and cultivation work has been done on Lentinus edodes

(Berk.) Singer. Now this mushroom has been shifted to family Marasmiaceae under

new genus Lentinula Earle as Lentinula edodes (Berk.) Pegler (Kirk et al., 2008).

Lentinus squarrosulus (Mont.) Singer is one of the important species of this

genus on which the present work has been undertaken. It is vernacularly known as

‘Tifa’ in Nigeria (Oso, 1975) and ‘Hed Khon Khao’ in Thailand (Petcharat, 1995). It

44

Experimental Design and Observations

is an edible mushroom which is widely distributed throughout Thailand and is

normally encountered under dense vegetation as well as in open habitats and thus

exposed to large temperature variations (Castillo et al., 2004). It is also very

common in the southern part of Nigeria and has been highly recommended for

commercialization (Oso, 1975, Akpaja et al., 2003, Okhuoya, 1997). Neda and Doi

(1998) reported its widespread presence throughout equatorial Africa, South-East

Asia, the Pacific island and Australia. In India its presence in the wild has been

documented from South by (Montagne, 1842, Natarajan and Manjula, 1978 and

Manimohan et al., 2004) as well as from the North (Personal communication).

The proximate and amino acid composition as well as mineral content of L.

squarrosulus (Mont.) Singer has been worked out by number of workers including

Alofe (1985), Fasidi and Kadiri (1991), Aletor (1995) and Nwanze and Adamu

(2004). In case of L. squarrosulus (Mont.) Singer Nwanze et al. (2006) reported

22.82 % crude protein, 7.64% crude fiber, 7.25 % ash, 27.6 % moisture, 97.25 % dry

weight, 6.29 % crude fat and 60.65 % soluble carbohydrates. Royse et al. (1990)

while working on the proximate composition of L. squarrosulus reported the

mushroom to be rich in protein, sugar, lipid, amino acid, vitamins B, C, D and

minerals. Gbolagade et al. (2006) worked out the proximate composition of L.

subnudus Berk. and documented 90.3 % moisture, 9.7 % dry matter content, 8.9 %,

soluble sugar, 4.5 % lipid, 10.7 % of glycogen, 5.1 % protein content, 6.5 % fiber

and 7.1 % ash in this mushroom. Natarajan and Manjula (1978) estimated 1.00 %

proteins, 0.55 % lipids and 1.38 % carbohydrates per 100 gm fresh weight of fruit

bodies.

Physical, physiological, biochemical and cultivation aspects of some species

of genus Lentinus Fr. namely L. squarrosulus (Mont.) Singer, L. connatus Berk., L.

45

Experimental Design and Observations

edodes (Berk.) Singer, L. sajor-caju (Fr.) Fr., L. strigosus Fr., and L. subnudus Berk.

have been worked out by number of workers including Ishikawa (1967), Gbolagade

et al. (2006), Suayan Zenaida (1980), Singh et al. (1990), Inglet et al. (2006), Kaur

and Lakhanpal (1995, 1999), Hiroe and Ikuda (1960) and Wuyep et al. (2003).

While evaluating media, Gbolagade et al. (2006) reported that Potato

dextrose agar and Yellow corn agar stimulated the best mycelial extension in case of

L. subnudus Berk. Singh et al. (1990) observed that the mushroom L. sajor-caju

(Fr.) Fr. mycelium attained maximum radial growth on Potato dextrose agar and

Sabouraud’s agar amongst the solid media and Malt extract broth gave best mycelial

yield on dry weight basis amongst liquid media. Singh et al. (1990) while working

on L. connatus Berk. reported that the mushroom grew well on Malt extract and

Sabouraud’s agar media. Amongst the liquid media L. connatus Berk. gave best

mycelial yield on dry weight basis in Soybean extract broth

( Singh et al., 1990). Nawanze et al. (2005) studied culture of L. squarrosulus

(Mont.) Singer at the hyphal level in submerged liquid cultures as well as at the

carpophore level on various different media. Natarajan and Manjula (1978) observed

best vegetative growth of L. squarrosulus (Mont.) Singer on Malt extract.

Hydrogen ion concentration of the culture medium is an important parameter

for cultivation of mushrooms. Gbolagade et al. (2006) reported L. subnudus Berk.

grew best within pH range of 5.0 - 8.0 with optimum pH at 5.5. Iinglet et al. (2006)

evaluated that pH range of 4.0 - 6.0 favours maximum mycelial growth rate in L.

edodes (Berk.) Singer. The optimum pH for mycelial growth in Lentinula edodes

(Berk.) Pegler was evaluated at 3.0 to 3.5 while for production of antibacterial

substances best pH has been worked out at 4.5 (Hassegawa et al., 2005). Suayan

46

Experimental Design and Observations

Zenaida (1980) demonstrated that L. sajor-caju (Fr.) Fr. gave best mycelial growth

at pH 6.0.

Temperature has considerable impact on the growth of the mycelium.

Vargas-Isla and Ishikawa (2008) while investigating the temperature requirement of

L. strigosus Fr. documented maximum vegetative growth at 350C. Khan et al. (1991)

obtained best vegetative growth of L. edodes (Berk.) Pegler at the temperature

ranging from 20 - 300C. The vegetative growth has been reported to decline at

temperature above 300C and below 200C. Gbolagade et al. (2006) worked out the

temperature range of 15 - 400C for stimulating the mycelial growth in case of L.

subnudus Berk. Suayan Zenaida (1980) found the maximum mycelial growth and

fruiting of L. sajor-caju (Fr.) Fr. at 300C temperature.

Light, though inhibits the mycelial growth, however, it is necessary for

fruiting. Vargas-Isla and Ishikawa (2008) indicated that L. strigosus Fr. show best

mycelial growth without illumination. Suayan Zenaida (1980) reported that two

minute exposures to UV radiation twice daily enhanced mycelial growth of L. sajor-

caju (Fr.) Fr.

Carbon is an essential element for mycelial growth. Ishikawa (1967) reported

3 - 5% concentration of certain monosaccharides, oligosaccharides and

polysaccharides in liquid culture satisfactory for mycelial growth of L. edodes

(Berk.) Singer. Kaur and Lakhanpal (1995) documented dextrose as the most

suitable carbon source for mycelial growth of L. edodes (Berk.) Singer. Alofe

(1985) found glucose to be a good carbon source for L. squarrosulus (Mont.)

Singer. Gbolagade et al. (2006) observed that mycelial growth in case of L.

subnudus Berk. was supported by fructose, followed by glucose and myo-inositol in

decreasing order. The best mycelial growth of L. sajor-caju (Fr.) Fr. was obtained in

47

Experimental Design and Observations

25% rice bran decoction agar (Suayan Zenaida, 1980). Nwanze et al. (2005)

reported optimum production of fungal biomass in L. squarrosulus in liquid culture

by using glucose and butter as carbon and lipid sources, respectively.

Similar to carbon, nitrogen is also essential element for the growth of

mycelium. Ishikawa (1967) reported best mycelial growth of L. edodes (Berk.)

Singer with peptone and very poor growth with nitrate and nitrites. Kaur and

Lakhanpal (1995) reported the maximum mycelial growth of L. edodes (Berk.)

Singer in peptone. Suayan Zenaida (1980) reported that 0.0001% concentration of

ammonium nitrate induced slight mycelial growth in L. sajor-caju (Fr.) Fr.

Gbolagade et al. (2006) evaluated yeast extract giving the maximum mycelial dry

weight in case of L. subnudus Berk. and least dry weight with L-glutamic acid as a

nitrogen source. Addition of adenine or cytosine to the medium has been reported to

stimulate mycelial growth of L. edodes (Berk.) Singer (Ishikawa, 1967).

Jonathan and Fasidi (2001) evaluated Biotin as the most utilizable vitamin

for L. subnudus Berk. followed by Thiamine and Folic acid, respectively. Hiroe and

Ikuda (1960), Ishikawa (1967) and Kaur and Lakhanpal (1995) reported the role of

Thiamine in stimulating the mycelial growth of L. edodes (Berk.) Singer. Ray and

Samajpati (1997) worked out the requirement of Thiamine for the best mycelial

growth in case of L. squarrosulus (Mont.) Singer and L. polychorus Lév. Suayan

Zenaida (1980) worked out the concentration of Pyridoxine and Biotin for fruiting of

L. sajor-caju (Fr.) Fr.

Jonathan and Fasidi (2001) reported that 1.0 ppm of 2, 4 - D stimulated the

optimum mycelial growth of L. subnudus Berk. followed by 0.1 ppm of Gibberellic

acid. Kaur and Lakhanpal (1995) reported the enhanced mycelial growth of L.

edodes (Berk.) Singer with Gibberellic acid.

48

Experimental Design and Observations

Jonathan and Fasidi (2001) reported Mg, K and Ca supporting good mycelial

growth in L. subnudus Berk. Ishikawa (1967) found that trace elements Mn, Fe, Zn,

Cu and Mg were essential for good mycelial growth of L. edodes (Berk.) Singer.

Wuyep et al. (2003) documented the importance of metal ions Mn2+ and Ca2+ in

stimulating the mycelial growth in case of L. squarrosulus (Mont.) Singer as

compared to Mg2+ and K+ ions which do not stimulate the vegetative growth of

mycelia in this mushroom.

The cultivation of L. edodes (Berk.) Singer, now Lentinula edodes (Berk.)

Pegler, on wooden logs is the oldest technique. Saw dust of various hardwood tree

species have been used for the production of shiitake by different workers (Ito and

Imai, 1925, Farr, 1983, Leatham, 1982, San Antonio, 1981, Kuo and Kuo, 1983,

Harris, 1986 and Singer and Harris, 1987). The earliest commercial technique for

cultivation of Lentinus edodes (Berk.) Singer on supplemented saw dust was

developed about 20 years ago in Japan, Taiwan and China (Ishikawa, 1967, Ando,

1974, Han et al. 1981 and Song, 1983). Royse (1996) reported a composition of saw

dust (80 %), wheat bran (10 %) and millet (10 %) for successful production of L.

edodes (Berk.) Singer. Wooden logs of Chlorophora excelsa have been reported to

support excellent fruiting in case of L. subnudus Berk. followed by logs of Spondias

mombin (Kadiri and Arzai , 2004). In case of L. sajor-caju Fr., Suayan Zenaida

(1980) reported fruiting in Dextrose bottle and Erlenmeyer flask which apparently

provided the aeration needed for initiation and differentiation of fruiting.

Oghenekero et al. (2009) while working with L. squarrosulus (Mont.) Singer

showed highest fruiting in Brachystegia nigerica saw dust supplemented with 1%

CaCO3, 1 % sugar and 20 % wheat bran. Kadiri and Arzai (2004) reported that bed

logs treated with GA and IAA are best in enhancing mushroom yield in case of L.

49

Experimental Design and Observations

subnudus Berk. Ray and Samajpati (1997) reported that the spraying of thiamine on

substrate gave better results for the cultivation of L. squarrosulus (Mont.) Singer.

Upadhyay and Rai (1999) reported that L. squarrosulus (Mont.) Singer is easy to

grow on wheat and paddy straw compost. Natarajan and Manjula (1978) produced

abortive fruit bodies on saw dust medium. Suayan Zenaida (1980) reported 300C as

optimum temperature and pH 6.0 for fruiting in case of L. sajor-caju Fr. Ayodele et

al. (2007) evaluated the mycelial growth and yield of L. squarrosulus (Mont.)

Singer on saw dust of different economic trees supplemented with 20 % oil palm

fruit fibers. Fasidi and Kadiri (1993), Royse (1996) and Isikhuemhen et al. (1999)

reported that supplementation of agricultural wastes stimulated both mycelial

extension, sporophore emergence and yield in case of L. subnudus Berk.. Royse and

Schisler (1986) and Han et al. (1981) reported higher yield of L. edodes (Berk.)

Singer when supplements were added to the substrates at different concentrations.

Ayodele et al. (2007) reported that the biological efficiency of L. squarrosulus

(Mont.) Singer was maximum in Mansonia altissima (4.27 %) and lowest in

Terminalia species (0.8 %) saw dust. Sharma (2004) reported good success in

cultivation of Malayasian strain of L. edodes (Berk.) Singer on wheat straw with

biological efficiency of about 45 %. Suman (2008) reported the development of fruit

bodies in lesser period of time with higher biological efficiency in L. edodes (Berk.)

Singer.

Lectins are univalent or polyvalent proteins of nonimmune origin that bind

reversibly and noncovalently to specific sugars on the apposing cells, thus

precipitating polysaccharides, glycoproteins and glycolipids bearing specific sugars

(Goldstein et al., 1980 and Singh et al., 1999). Owing to their specificity to bind

carbohydrates, they are capable of agglutinating erythrocytes, making their detection

50

Experimental Design and Observations

easy (Sharon and Lis, 1972). Lectins are known to play key roles in host defense

system (Singh et al., 2010). Mushrooms elaborate high levels of lectins, suggesting

their existence as a kind of storage protein as in plants. Mushrooms have long been

known for their nutritive and medicinal values and now represent a rich source of

lectins. Most lectins agglutinate erythrocytes of all human blood groups without any

noticeable specificity and are referred to as non specific lectins or panagglutinins.

Such lectins bind to saccharide receptors present on the surface of all erythrocytes,

irrespective of blood group determinants. Specific lectins, however, bind to

saccharide determinants on the erythrocyte surface and are known to preferentially

agglutinate erythrocytes of a particular blood type. Sometimes, the susceptibility of

erythrocytes to certain lectins increases upon mild treatment with proteolytic

enzymes (Sharon and Lis, 1972) or sialidases (Schauer, 1982). Mushroom lectins are

endowed with antiproliferative, antitumor, mitogenic, hypotensive, vasorelaxing,

haemolytic, anti-HIV1 reverse transcriptase, and immunopotentiating activities (Li

et al., 2008). No work of this type is available on L. squarrosulus (Mont.) Singer.

As today, there is a better understanding of the biology, nature and

development of many species of edible mushrooms (Isikuemhen et al. 1999,

Okhuoya et al., 2000, Kurtzman, 2000, Martinez-Carrera, 2000 and Wuyep et al.,

2003) and there is need to work out the physical and physiological parameters of the

commercially important edible species like L. squarrosulus (Mont.) Singer, in

country like India with a view to understand its requirements for domestication and

large scale production. Keeping in view the economic utility of the mushroom and

the meagre amount of work done on its domestication in India, the present study on

“Physiological and biochemical investigations for the cultivation of Lentinus

squarrosulus (Mont.) Singer” was initiated. For this purpose an indigenous culture

51

Experimental Design and Observations

of this species was raised on Potato dextrose agar from the wild collection of this

species made from Kotla Barog in Sirmour District of Himachal Pradesh.

52