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Sclerotia Production: A Way Ahead to Morchella Cultivation

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 Recent Advances in
Mushroom Cultivation
Technology
and Its Application
Volume - 1

Chief Editor
Dr. Shweta Kulshreshtha
Associate Professor, Amity University, Jaipur, Rajasthan, India
Co Editor
Ukaogo, Prince Onyedinma
Lecturer, Analytical/Environmental Unit, Department of Pure and Industrial
Chemistry, Abia State University, Uturu, Nigeria
Dr. Siddhant
Assistant Professor, Department of Botany, Durgesh Nandini Degree
College, Purabazar, Faizabad, Uttar Pradesh, India

Bright Sky Publications

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Published By: Bright Sky Publications

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Chief Editor: Dr. Shweta Kulshreshtha

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Publication Year: 2021
Pages: 126
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Price: ` 736/-
 Contents

Chapters Page No.

1. Mushroom and Its Choice of Substrates 01-13
(Nwaru C.E and Iwuagwu M.O)

2. Mushroom Cultivation: A Sustainable Solution for the

Management of Agriculture Crop Residues 15-26
(Nanje Gowda N.A, Chennappa Gurikar and Lokesh A.C)

3. Disinfection Methods for Mushroom Substrate Preparation 27-48

(Nanje Gowda N.A, Chennappa Gurikar and Hanumantharaju KN)

4. Mushroom Pests and Disease Management 49-66

(Sunanda Mandal)

5. Edible Mushrooms as Nutraceuticals: Boon or Bane 67-86

(Baby Sharma and Shruti Mathur)

6. Disease Management of Insect and Pest in Mushroom

Cultivation 87-106
(Dr. Monalisa Kulshrestha)

7. Sclerotia Production: A Way Ahead to Morchella Cultivation 107-126

(Siddhant, Shweta Kulshreshtha, PO Ukaogo, Vigi Chaudhary and Mahesh
Kumar)
 Chapter - 7
Sclerotia Production: A Way Ahead to Morchella
Cultivation
Siddhant, Shweta Kulshreshtha, PO Ukaogo, Vigi Chaudhary and Mahesh Kumar

Abstract
Morchella, the true morels, belonging to Helvellaceae family of class
Ascomycetes, are amongst the most highly priced fungi in the world. Their
artificial production is still a challenge, even though patents for their
cultivation do exist. The tissue of Morchella sp. was transferred aseptically
to Potato Dextrose Agar medium (peeled, sliced and boiled potato, 200 g;
dextrose, 20 g; agar, 20 g L-1) to grow hyphae. The mycelium showed
fastest growth as compared to other edible mushrooms. It covered entire area
of Petri plate (90 mm) within 4-5 days with the growth rate of 18-
22.5mm/day. A unique growth pattern i.e., vertically oriented mycelia were
observed. Brown coloured pigmentation in the culture was also observed
during the study. The basal media for spawn (wheat grains; Glucose, 1%,
CaCO3, 2%; CaSO4, 1.5% and MgSO4, 1%) was aseptically inoculated with
the mushroom culture. The spawn substrate was colonized by mushroom
mycelium in 7-8 days. The sclerotia were formed in unused (old) spawn.
Wheat straw was used as a substrate for mushroom cultivation. It was
supplemented with wheat bran, 20%, Glucose (1%) and MgSO4 (1%). It
showed prolific growth when it was seeded by mushroom spawn using jar
method. Once substrate was fully covered with mushroom mycelium, casing
was applied. Sclerotia were successfully obtained after 14 days of incubation
in our experiment both in the substrate and casing soil but failed to give rise
to fruiting primordial. Further research is going on to domesticate this
species in our country.
Keywords: ascomycetes, jar method, Morchella, sclerotium
Introduction
Morchella, the true morels belonging to Helvellaceae family of class
Ascomycetes, are amongst the most highly priced fungi in the world (Pegler
2003) because of their medicinal and nutritional qualities. The average

Page | 109
morels are sold at a price of Rs. 5000 per Kg (Prasad et al., 2002) that makes
a good income generating source for livelihood (Semwal et al., 2014, Singh
and Rawat 2000). These were easily recognizable of all the edible mushroom
species because of their distinctive shape and appearance. In India, majority
of Morchella species are reported primarily in Northern Western Himalayan
regions (Bhatt et al., 2016, Ghosh and Pathak 1962, Hennings 1901, Kaul
1981, Khan et al., 2009, Kumar and Sharma 2010, Lakhanpal and Shad
1986, Manikandan et al., 2011, Negi 2006, Singh and Verma 2000a, Singh et
al., 2004, Sohi et al., 1965, Theissen 1911, Vishwakarma et al., 2011,
Waraitch 1976). However, occasional reports on collection of Morchella sp.
have also been documented from rest of the country (Acharya et al., 2005,
Bhattacharya and Baruah 1953, Ghurde and Wakode 1981, Kaviyasaran et
al., 2006, Moinudheen 2019, Paliwal et al., 2013, Purkayastha and Chandra
1985, Rai et al., 1999, Wakode 1983). All the true morels are known to
edible (Arora 1986) with excellent flavor and taste. They are rich in proteins,
minerals (Gençcelep et al., 2009) and vitamins viz., thiamine, riboflavin,
niacin, pantothenic acid, pyridoxine and cyanocobalamine (Samajpati 1979).
They contain 44.56-57.99% carbohydrate, 17.32-25.65% crude proteins,
9.34-15.33% crude fibre, and 6.45-10.87% ash content with the mean value
of 49.56%, 21.54%, 11.83% and 8.44%, respectively (Odoh et al., 2017).
Morels fruit bodies have same nutritive value as their mycelia have (Hayes
and Hadded 1976). They have also been used in traditional medicine for
centuries, due to their health-related benefits (Tietel and Masaphy 2018).
They contain a wide range of pharmacological properties which includes
antioxidant, antitumor, antimicrobial and anti-inflammatory property, it also
acts as an immune-stimulant due to the presence of various active
constituents (Ajmal et al., 2015). It may be used as purgative, laxative, body
tonic, emollient and used for stomach problems, heal the wound and for
general weakness (Raman et al., 2018). The fruiting period of Morchella is
very short. Generally, they appear in the nature during the spring, i.e. March
and April (Lakhanpal et al., 2010) while some species can be grown in dry
regions all year-round. Furthermore, the accumulation of heavy metals in the
naturally grown ascocarps was reported (Daniel-Umeri et al., 2015, Isidak et
al., 2004, Shavit, 2008, Shavit & Shavit 2010). The distinctive culinary
flavour, nutritive and medicinal values, rare occurrence, the short fruiting
season, high price, and the heavy metal accumulation of wild morels have
led to the need to establish a biotechnological method to grow mushrooms
under controlled conditions. Morchella produces sclerotia in their life cycle.
The sclerotia represent an intermediate stage between mycelial growth and
fructification. These are hard-surfaced resting bodies composed of

Page | 110
aggregated mycelium-large cells with thick wall, that act as nutrient
reservoir and therefore, helps the survival of mushroomsin the adverse
natural conditions and formation of fruiting bodies (Leonard and Volk
1992). Because of their complex life cycle, these delicacies are not
artificially cultivated, and the successful cultivation of morels remains a rare
and difficult task. Although, there are some reports on Morchella cultivation
(Longley et al., 2019, Masaphy 2010, Ower 1982, Ower et al., 1986), but
stable production remains difficult because its mechanisms of fruiting body
formation are unclear (Hao et al., 2019). The authors have reported the
occurrence of Morchella sp. in Ayodhya during December to January of
2014-2015 (Siddhant et al., 2014, 2016, 2019). The incidence of Morchella
in this region makes it the suitable place for seasonal cultivation in the same
way as cultivation of button mushroom is made, therefore, an attempt has
been made to cultivate it in this part of the country.
Materials and Methods
The specimens of Morchella sp. were collected during December 04 to
27, 2014 from district Faizabad (officially Ayodhya) which is one of the 75
districts of Uttar Pradesh state in northern India and lies between the
parallels of 26047’N to 26078’N latitude and 82.080E to 82.13’E longitude
having an average elevation of 97 meters above the sea level (Siddhant et al.,
2014).
Culture
The pure culture of Morchella was prepared on Potato Dextrose Agar
medium (peeled, sliced, and boiled potato, 200 g; dextrose, 20 g; agar, 20 g;
DW, L-1) by following tissue culture method (FAO 1990). To obtain tissue
culture, ascocarp was cut longitudinally and tissue from stipe region were
aseptically removed and placed on culture medium and incubate at 20°C for
5 days. It was maintained on same medium by serial subculture method
(FAO 1990).
Spawn preparation
Wheat grains (Triticum aestivum) were used as a spawn substrate which
was purchased from the seed market of Faizabad. These were cleaned
manually to remove inert matter, stubble, and debris. The grains were water
soaked overnight. On the following day, these were washed again and boiled
with water for 10-15 minutes until they cooked but not broken. The excess
water was drained off and boiled grains were allowed to cool. It was then
thoroughly mixed with 1% Glucose (w/w), 2% CaCO3 (w/w), 1.5% CaSO4
(w/w) and 1% MgSO4 (w/w). The mixture was then packed in heat resistant

Page | 111
polythene bags to two-third of their capacities and autoclaved at 15 lbs for
60 min. Immediately after sterilization, bags were shaken well to dislodge
the grains and cooled overnight. Next day, these were inoculated with fungal
discs under aseptic conditions and incubated at 20 °C until complete
colonization. During this period, bags were moved daily, so that fungal
mycelia may uniformly be mixed and cover around the grains. Spawn was
ready to use after the Morchella mycelia completely colonized the grains.
Substrate preparation
Wheat straw was used as a substrate for mushroom cultivation. It was
soaked overnight in tap water. After soaking excess water was drained off to
maintain appropriate moisture content which was supplemented with wheat
bran (20%), glucose (1%) and MgSO4 (1%; dissolve in water and spray).
The prepared substrate was filled in the jar. After the jar (empty bottle) has
been filled, substrate was pressed manually and a vertical hole is made
through this compact mixture which permits even distribution of mushroom
spawn to the bottom. The Jar was autoclaved at 15 lbs pressure for 60
minutes (Tewari & Panday 1988).
Preparation of casing soil
Farmyard soil and cow dung manure (1:1) were used as casing material,
which were collected from the village Madrahiya. These were sieved to
avoid foreign particles and then properly mixed to a uniform texture. The pH
of mixture was adjusted at 7.0-7.5 by addition of CaCO3. It was then
sterilized by with 2% formaldehyde solution and covered with the polythene
sheet for next 72 hours. After this period, the mixture was uncovered to
remove the extra trace of formaldehyde. It was then spread, collected and
stored for use (Biswas et al., 2012).
Method of cultivation
The jar method was used in this experiment (Ower et al., 1986). The
column spawning was done with the spawn of Morchella sp. at the rate of
15% w/w on dry weight basis under aseptic conditions. Once substrate was
colonized by mushroom mycelium, the mouth of jar was opened to apply the
casing soil. Watering was done at regular interval to maintain moisture.
Growth rate
The culture medium was inoculated by placing fungal disk from the end
of colony and then incubated at 18-22 ºC. Three replicates of each plate were
used and then the growth rate was calculated as follows:
Growth rate (mm/day) = x/y

Page | 112
Where,
X: Represents the diameter of the petri-dish (mm).
y: The time required for the fungal growth to reach the edge of the plate
(day). (Chechan et al., 2017).
Results
Cultural characteristics of Morchella on PDA media
The Morchella fruit bodies, obtained from District Ayodhya were used
for culture preparation (Fig.1). The large tissue from stipe region (10 mm)
was aseptically transferred on PDA media. The mycelia development began
on the tissue of Morchella as well as culture media within 24 hours after
inoculation which covered the whole area (90 mm) of the Petri plate in 4-5
days with the growth rate of 18-22.5mm/day. The colour of mycelia was
white when it was developed on the tissue of Morchella but on the culture
media it showed light brown colour. During this study, brown colour
pigmentation was also observed in the culture media. A very different
pattern was experienced during the mycelial run on the culture media which
generally do not happen with other mushroom species such as Pleurotus and
Agaricus. Initially mycelium was non-aerial during first 24 hours but in next
24 hours aerial mycelia were developed vertically to media surface and
formed a fluffy appearance, occupied the entire volume of the Petri plate. It
was observed that sclerotial formation did not initiate until mycelial growth
filled the Petri plate. White colour cell aggregations of pin head size were
developed after 12 days from the date of inoculation which were irregularly
dispersed on the media surface. Soon, after maturity (16-18 days) it
converted in to brown coloured sclerotia.
Growth characteristics of mycelia in spawn substrate
When supplemented wheat grains were inoculated with the fungal disc
of Morchella, extensive mycelial growth was observed within 24 hours in
spawn substrate. However, it was not as dense as in other edible mushrooms.
Despite of thick and light brown colour of mycelia, mycelial run was
difficult to visualize in first 24 hours. It was identified by the sizable lump
formation. Sooner, it was visualized due to its cottony appearance. The
mycelium took 7-8 days to cover the spawn substrate. Once spawn was
prepared, it was used to inoculate growing media. The orange pigmentation
was observed in the unused (old) spawn which became brownish on
maturity. The sclerotial formation was observed initially only on the surface
of grains, soon grains were entirely replaced by brown mass (sclerotia) (Fig.
2).

Page | 113
 Fig 2: Morchella sclerotia
Fig 1: Morchella sporocarp
formation in old spawn

Evaluation of mycelia colonization and sclerotia production on substrate

Wheat straw supplemented with wheat bran (20%), Glucose (1%) and
MgSO4 (1%) was utilized as a substrate for sclerotia production/Morchella
cultivation. After inoculation, the mycelia took 7 days for complete
colonization of the substrate. However, as compared to grains, mycelial
growth was not as dense as on the lignocellulosic (wheat straw) substrate.
Once beds were fully covered with mushroom mycelium, casing was
applied. Both the substrate and casing layer showed mycelial growth and
sclerotial formation with the size of 2-4 mm. The orange coloured sclerotia
were formed mostly in the substrate, filled in upper half of the bottle after 14
days of inoculation. In our experiment, these sclerotia were failed to give rise
to fruiting primordia. Overall, the formation of sclerotia, in our investigation,
shows a big move towards Morchella cultivation.
Discussion
The Morchella fruit bodies were earlier reported by authors (Siddhant et
al., 2014, 2016 & 2019) from District Ayodhya (Figure. 1). Because the
tissue size influences the mycelia density of mushroom (Liu et al., 2016b),
the large tissue from stipe region (10 mm) was aseptically transferred on
PDA media. As Ower (1982) stated that both the stipe tissue cultures and
ascospore cultures were effective for obtaining vegetative hyphae, we prefer
tissue culture over spore culture method on PDA media. This media has
already been proved best for the mycelia growth of Morchella species.
Morchella tibelica, M. conica, M. deliciosa and M. esculenta showed best
mycelia growth on PDA media with the colony diameter of 7.0 cm, 8.5 cm,
6.5 cm and 9.0 cm, respectively (Lakhanpal et al., 2010). Our finding is very
close to Papadaki et al., (2019) who determined a growth rate of 13.1 -22.2
mm/day for Morchella on PDA media. In comparison to other edible
mushrooms (Zervakis et al., 2001), like Pleurotus pulmonarius, (6.6

Page | 114
mm/day), Lentinula edodes, (4.4 mm/day), Volvariella volvacea (18.8
mm/day), Auricularia auricula-judae (6.2 mm/day), Morchella showed
remarkable higher growth rate. The higher growth rate of Morchella on PDA
medium is in agreement with several researchers (Gülerand and Arkan 2000,
Gulerand and Ozkaya 2008, Kalmişand and Kalyoncu 2008, Volk and
Leonard 1990) who reported almost same time for mycelial colonization in
Petri plates. In our investigation, the mycelial colour on the media was light
brown from the beginning. However, some researchers reported white
mycelial colonies which turned brown with age (Gulerand and Ozkaya
2009). The white coloured mycelia (primary mycelia) are attributed to the
germination of ascospores in the culture. When two genetically different
primary hyphae interact, the secondary (heterokaryotic) hyphae may arise
which is represented in culture by the formation of an aerial ridge of hyphae
with the deposition of dark pigment at the line of confluence. Under
unfavourable conditions, these heterokaryotic hyphae may form a
heterokaryotic sclerotium which, after carpogenic germination, leads to
fruiting-body formation (Volk & Leonard 1990) (Fig.3).
The ascocarp consisting of heterokaryotic mycelia, therefore, produced
dark coloured mycelium by tissue culture method. Brown colour
pigmentation in the culture media was also observed during this study. It is
reported that mycelia secrete a dark brown pigment into the media and
browning starts from the centre and proceeds to the periphery with age
(Lakhanpal et al., 2010). The vertically oriented aerial mycelia were formed
in the Petri plate instead of primary vegetative mycelia. The reason has been
described earlier. In contrast to this, Hervey et al. (1978) reported both types
of mycelium in Morchella esculenta while using ascospores culture. Güler
and Arkan, (2000) also observed the vertical growing mycelia in their study.
They further stated that the density of the vegetative mycelia, increased on
joining the aerial mycelia, which resembled flappy cotton advancing from
the surface of the

Page | 115
 Fig 3: Life cycle of Morchella (Volk & Leonard 1990)
agar plate towards the petri plate cover. The old cultures showed
sclerotia development which are irregularly scattered on the media surface. It
was possibly due to lack of nutrients in old culture that trigger the sclerotia
production. However, Singh and Verma (2000b) stated that the nutrient poor
condition is not essential for sclerotia production as they could be produced

Page | 116
on nutrient rich media. Even sclerotia formation was favoured when mycelia
grew from nutrient poor medium to nutrient rich medium (Faris et al., 1996).
Our results are similar to finding of Amir et al., (1993) who used split plate
method and observed the mycelia growth of Morchella toward a richer
medium and the formation of sclerotia mainly on the poor medium. The
development of sclerotia on culture media has already been reported by
several workers (Buscot 1993, Singh and Verma 2000b). Robbins and
Hervey (1959) observed sclerotia of Morchella crassipes on 2% Malt agar
medium. Mehta and Sharma (1992) also observed sclerotia formation by
different species of morels on PDA and YPDA media. Recently, Liu et al.
(2018 a, b) described the artificial formation of morel sclerotia on PDA
medium.
Agar block with active growing mycelia were transferred to grains to
produce spawn. Supplemented wheat grains have already been used as a
basal medium for spawn preparation (Liu et al., 2016a, Barnes and Wilson
1998). The enzyme amylase is reported from Morchella esculenta
(Krupodorova et al., 2014, Papinutti and Lechner, 2008). Thakur (2016)
observed amylase activity in seven different Morchella species (M.
angusticeps, M. conica, M. crassipes, M. deliciosa, M. esculenta, M. tibelica
and M. simlensis). This enzyme is associated with hydrolysis starch, the
main component of wheat grains (Shevkani et al., 2016). Because of this, the
quick mycelial colonization was observed in the wheat grain substrate. It is
supported by the finding of Alvarado-Castillo et al., (2011) which reported
prolific mycelial growth on different grains. Earlier, Brock (1951) observed
significant mycelial growth of Morchella esculenta on starch. The inoculated
wheat grains took relatively shorter time (7-8 days) for mycelial colonization
as compared to the finding of Zhang (2018) who reported 20 days for above
manifestation. The sclerotial formation was observed in the old spawn. Our
finding is similar to the Gupta (2011) who reported maximum number of
sclerotia in wheat grain substrate.
Wheat straw supplemented with wheat bran (20%), glucose (1%) and
MgSO4 (1%) was utilized as a substrate for sclerotia production/Morchella
cultivation. Various lignocellulosic substrates viz., wheat straw, groundnut
shell (Kanwal and Reddy, 2014), saw dust (He et al., 2018), rice straw
(Singh et al., 1999), pine needle (Gupta 2011), rice husk (Tan et al., 2019),
peat (Eliuzand and Goksen 2017) and vegetable wastes (Papadaki et al.,
2019) were tried either for the sclerotial development or to assist the fruiting
in Morchella. These substrates are rich in cellulose, hemicelluloses and
lignin content. To utilize these substrates, mushrooms mycelia used to

Page | 117
secrete extracellular enzymes which could depolymerize these
macromolecules into simpler forms that can be processed by mushrooms. It
is reported that Morchella sp. produces appreciable activities of
endoglucanase, β-glucosidase, laccase (Papinutti and Lechner 2008),
xylanase, laminarinase (Singh et al., 2001) that help in mycelial colonization
on lignocellulosic wastes. Our result is in conformity with Singh et al.,
(2001) who observed appreciable growth of Morel mycelia on wheat straw
substrate. Column spawning was used for the substrate inoculation.
However, Liu et al. (2018a) stated that the spawning for morel cultivation is
different from that for most mushrooms given that the morel spawn is sown
directly into the cropland or forest, which is likethe seeding of wheat crops.
Once beds were fully covered with mushroom mycelium, casing was
applied. Both the substrate and casing layer showed mycelial growth and
small sized sclerotial formation. The mycelia growth of Morchella esculenta
on soil was earlier reported by Kanwal et al. (2016). The smaller size of
sclerotia might be due to smaller size of jar. It is well known that larger jar
produces quantitatively more and greater weights of sclerotia (Volk and
Leonard 1989). The orange coloured sclerotia were formed mostly in the
substrate, filled in upper half of the bottle. However, Kanwal and Reddy
(2014) reported most of the sclerotia in the soil layer in comparison to
substrate. The time, lapse in the sclerotia formation was much shorter than
that reported in other studies, where up to 70-75 days were required for
sclerotia formation (Singh et al., 1999). In our experiment, these sclerotia
were failed to give rise to fruiting primordia. Overall, the formation of
sclerotia, in our investigation, shows a big move towards Morchella
cultivation. The significance of sclerotia in the life cycle of Morchella sp.
has already been emphasized by Ower (1982) and Ower et al. (1986). They
demonstrated that, under controlled conditions, sclerotia have a central role
in the production of fruiting bodies in Morchella. Recently, Masaphy (2010)
successfully demonstrated the fruiting of M. rufobrunnea from sclerotia in
artificial conditions. Further research is going on in our laboratory to
domesticate this species in this part of country.
Conclusion
The present study showed that sclerotia of Morchella sp. can be
achieved by the conventional method of spawn and mushroom bags
preparation.
Acknowledgements
This finding is dedicated to Late Dr. C.S. Singh, Former Associate
professor, Department of Botany, K.S. P.G. College, Ayodhya.

Page | 118
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