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
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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