CULTIVATING MUSHROOMS on SMALL FARMS

CULTIVATING MUSHROOMS
on SMALL FARMS
Commonly Cultivated
Agaricus Mushrooms
􀂐Agaricus augustus Prince Button Mushroom
􀂐Agaricus bisporus Italian Agaricus,
“portobello” Portabella, Portabellini
􀂐Agaricus bitorquis Warm-Weather Button
􀂐Agaricus bisporus White Button Mushroom,
(=A. brunnescens) Cremini
Limited Cultivation
Edible Fungi
􀂐Morel Morchella esculenta
􀂐Black Morel Morchella angusticeps
􀂐Summer White Truffle Tuber aestivum
􀂐White Italian Truffle Tuber magnatum
􀂐Black Perigord Truffle Tuber melanosporum
􀂐Chanterelles Cantharellus spp.
􀂐Maize Mushroom Ustilago maydis
Culture Parameters
or Conditions to Monitor
􀁽 Relative Humidity:
􀁽 Air T:
􀁽Duration of Stage:
􀁽CO2:
􀁽Fresh Air Exchanges:
􀁽Light:
􀁽Intervals:Semi-Controlled
Conditions
􀁽Temperature of substrate: pocket thermometers
(metal 1-inch dial and 5-inch stem)
􀁽 Air temperature and relative humidity: battery
operated LCD digital thermometer/hygrometer
(Thermo-Hygro ®), which also shows
temperature maximums and minimums.
􀁽Relative Humidity: time-set semi-automated
misting system (Raindial – Irritrol Systems ®
Model RD 600 Ext).
􀁽PVC pipes: along the production areas on the
ceiling, with Turbo-Flo ® Misters, extremely low
flow (1/2 gph) and very small droplet size.ABSTRACT: Interspecific hybridization studies were carried out between P/eurotus sajor-caju, P. sapidus
andP. cornucopiae for obtaining better quality strains. The resultant hybrids were categorised on phenotypic
basis into two categories. In first group, hybrids obtained were similar in appearance, yield and colour etc.
with either of their parents. The second group hybrids had blending of characters of their parents and
possessing the yield potential more or less similar to them. A specific hybrid (hybrid no. 3) obtained with
mating between P. sajor-caju and P. cornucopiae, in which the shape and size of the sporophore was similar
to P. sajor-caju and white colour was resembling with P. cornucopiae. The total average yield of this hybrid
was found to be 23.3 per cent more than the parent.
Keywords: Pleurotus, oyster, interspecific hybridization, mating, desirable characters
Species of Pleurotus can grow on a number of
substrates and a wide range of temperature. But
inspite of easy method of cultivation its production
is very less in comparison to button mushroom in
India. Pleurotus sajor-caju is the most preferred
species suiting to Indian conditions, having 22-
28°Ctemperature range for its vegetative as well
as reproductive growth. Agaricus bisporus, the
commonEuropean white button mushroom requires
more or less the same temperature requirement
for its vegetative growth i.e. 22-25°C but prefers
16-18°Cfor its fruiting. In northern plains of India,
growing season for both the mushrooms is more
or less same, i.e. November to March for Agaricus
bisporus and October to April for Pleurotus sajorcaju.
As the button mushroom is preferred than
the oyster mushroom due to socio-economic
reason,the growers like to grow button mushroom
during this season. In 1996-97, total mushroom
production in India was 40,000 tons, of which
Agaricus contributed 90 percent of the total
production,whereas Pleurotus and Volvariella have
only 10 percent share (Dhar, 1997). During rest of
the year from May to September any of the two
mushrooms (Agaricus and Pleurotus) can not be
grown under natural conditions as the temperature
during this period ranges between 28-46°C in
northern plains. Therefore, there is a need to find
out a strain which can be grown under natural
conditions at a higher temperature and may have
other desirable characters like white colour,
sporelessness, better texture, taste and also high
yielding capability. These qualities are not found in
existing species of Pleurotus. The only exception
is P. fossulatus, which is still not cultivated
commercially and brought from wild for
consumption.
Earlier, attempts have been made for producing
quality strains of Pleurotus. In P. ostreatus,
heterokaryons were developed by Eugenio and
Anderson (1968) and Eger et al., (1976). In P.
sajor-caju, P. florida and P. sapidus, hybridization
were carried out by Bahukhandi and Munjal (1990),
Ghosh and Chakravarty (1991), Thakur and
Bhandal (1993). The hybrids obtained by these
workers did not show much improved characters
and hence lacked popularity and importance among
mushroom growers. Therefore, the studies were
carried out on interspecific hybridization among
the selected three species of oyster mushrooms
namely, P. sajor-caju, P. sapidus and P.
cornucopiae due to their popularity in different
parts of the world, to develop a suitable strain with
desired characters.
62 Indian Phytopathology
MATERIALS AND METHODS
The type cultures of P. sajor-caju, P. sapidus
and P. cornucopiae were obtained from Indian
Type Culture Collection (ITCC), Division of Plant
Pathology, Indian Agricultural Research Institute,
New Delhi vide Acc. Nos. 1725, 2575 and 3312,
respectively and sub-cultured at regular intervals.
Spawn was made on wheat grains and the
sporophores were obtained by standard cultivation
method (Bahukhandi et aI., 1989). The spore prints
were taken from healthy mature sporophores, which
served as spore stock for further study (Watling
1970).
Spore suspension was made by taking small
loop of spores from the spore print and suspended
in sterile distilled water. This solution was diluted
to 10-4 dilutions, where spore concentration remains
4-5 spores per low power microscopic field (10X).
One loop of this solution was streaked on plain
agar in petri plates (pre-demarcated lines) and
incubated at 25-27DC. The germinating spores
were isolated and picked up by a needle! spatula
and placed in P.D.A. slants. A total of 26 single
spore isolates of P. ·sajor=caju, 23 of P. sapidus
and 19 of P. cornucopiae were obtained. These
monokaryans were grown in mediated petri plates
and the hybridization between the three species
was done randomly. Three millimeter diameter
bits were cut out from seven days old monokaryotic
culture and the two monokaryotic cultures were
placed at two opposite ends in petri plates
containing PDA. The single spore isolates were
kept for interspecific crossing in the following way
(Table 1).
[Vol. 55( 1) • 2002]
1. P.sajor-caju x P.sapidus = 26x23=598 pairings
2. P.sajor-caju x P.comucopiae = 26x19=494
pairings
3. P.sapidus x P.comucopiae = 23x19=437
pairings
In this way a total of 1519 number of pairings
were obtained. These paired isolates were
incubated at 25-27DC for 7-10 days for mating. A
small inoculum was taken from the meeting point
of the paired monokaryons if any. The spawn was
made by the standard method and cultivation was
done by the method described earlier (Bahukhandi
et a/., 1989). The sporophore characters like colour,
size, yield and temperature requirement for
cultivation were recorded in different hybrids and
compared with their parents. Hybrids were grown
on chopped paddy straw in small poly bags and
were categorized in 9 groups based on size,
shape, colour and yield etc. From each group one
type sample having higher yield, early fruiting and
light-colour was selected and were used for further
cultivation studies. Hybrid nos. 1-3 were as a
result of mating between P. sajor-caju x
P.comucopiae, 4-6, were between P.sajor-caju x
P.sapidus and 7-9 were between P. sapidus x
P.cornucopiae. Cultivation studies were carried
out in polybags having capacity of one kg. dry
straw. Time taken for substrate colonization,
cropping period and total yield obtained in 4 flushes
was recorded.
The non-mated pairs in all the three crossing
(P.sajor-caju x P. sapidus, P.sajor-caju x P.
cornucopiae and P. sapidus x P. comucopiae)
were cateqorized into 3 classes namely a, band
c according to their hypha I growth pattern.
Table 1. Number and type of mated and non-mated pairs and hybrids obtained after mating three species of
Pleurotus
Species of Total Total Segregation of Total non Morphological
Pleurotus Pairs mating Hybrids (fertile) mating pairs variations (sterile),
obtained pairs A B C a b c
P.sajor-caju x P. sapidOs 598 92 26 ~5) 21 506 425 58 23
P.sajor-caju x P. cornucopiae 494 80 23 39 18 414 360 42 12
P. sapidus xO P cornucopiae 437 90 28 40 22 347 308 23 16
A-Hybrids with characters of first-parent, B-Hybrids with blending of characters of both the parents, C-Hybrids with
characters of second parent, a-Simple non mated pairs, b-Neutral, c-Inhibitory
[Vol. 55(1) : 2002]
RESULTS AND DISCUSSION
It was observed that the single spore
isolates obtained in the three species of Pleurotus
have some peculiar features, by which they were
differentiated from dikaryotic mycelium, (i) The
single spore isolates were having slower growth
and in some cases the growth was limited to
inoculum. (ii) Absence of clamps. (iii) They did not
produce fruiting bodies, during cultivation. Although
some primordial formation were seen in some
monokaryons but they were abortive and did not
grow further. Some of the isolates were not able to
colonize the substrate (Fig. 1).
During the mating process between
monokaryons, four types of reactions were seen
in petriplates on the basis of the nature of the
growth of hyphae. As a result of first reaction
mating occurred and fertile progeny was produced
(A, Band C, Table 1). In this process the hyphae
developing from both the oppositely placed inocula
were anastomosed and showed clear difference in
appearance between individual parents and mating
hyphae, when tested for fertility, they were found
to be fertile (Fig. 2).
The non-mated pairs belonging to remaining
three categories have some special morphological
characters (a,b and c, Table 1). In first category
(a), the hyphae of the monos porous pairs were
intermingled and gave an idea that these have
Indian Phytopathology 63
mated, but after testing they did not produce fruit
bodies and were found sterile. Most of the non
mated pairs belonging to this category (425, 360and
308 respectively in the 3 crosses). In the second
category the hyphae of both the paired monos pores
grew but did not mate and remain separated. This
reaction of the non mated isolates was neutral (b)
and their number in each crossing were 58, 42
and 23 respectively. In the third category, a zone
of inhibition was developed between paired isolates
and their growth occurred in different direction to
each. This reaction was termed as inhibitory (c)
and the number of pairs in this category were
lowest among all the three crosses (23, 12 and 16
respectively).
In fertile isolates (first category) dikaryotization,
clamp connection and sporophore formation were
observed. In three sets of possible crosses the
sterile:fertile ratios were recorded as 506:92, 414:80
and 347:90, respectively in P. sajar-caju x P.
sapidus, P. sajar-caju x P. carnucapiae and P.
sapidus x P.carnucapiae. The hybrids obtained in
the present study in all the above pairings showed
characters corresponding to their parents. There
was hardly any difference between hybrids and
their parents in their vegetative growth characters.
The sporophores in all the above combinations
were categorized in two types. Some of the hybrids
were similar to their either of the parents and
some of them had blending of characters from
Fig.1. Mycelial growth of a dikaryon (A) and monokaryon (B) of Pleurotus sajor- caju
64 Indian Phytopathology [Vol. 55(1) : 2002)
Fig.2. Paired single spore isolates showing mating (A) and non-mating (B) in Pleurotus sajor-caju and P.comucopiae
both the parents. In first set of mating (P. sajorcaju
x P. sapidus) 26 hybrids (sample hybrid-4)
were phenotypically similar to P. sajor-caju (Fig.
3A) and 21 (sample hybrid-5) were similar to P.
sapidus, (Table 1, Fig. 3C). Remaining 45 hybrids
(sample hybrid-6) were possessing blending of
characters between the two parent (Fig. 3B). The
similarities were considered in appearance, colour,
size and texture of fruiting bodies and temperature
requirements for cultivation of the hybrids, etc.
The hybrids had blending of characters having
colour of sporocarp like P. sapidus and size like P.
sajor-caju but there was no other specific character
and the yield was also similar to their parents
(Table 2 shows sample hybrids selected for
cultivation as 4, 5, 6 nos.)
In the crosses between P. sapidus x P.
cornucopiae, more or less same results were
obtained and the resultant number of three types
of hybrids were 28:40:22 (Table 1, Fig 3). The
hybrids did not show any specific character and
were similar in all the respects including yield, with
their parents (Table 2 shows sample hybrids
selected for cultivation as 7,8,9 respectively).
In the third set of crossing between P. sajorcaju
x P. cornucopiae, the phenotypic ratio of
hybrids was 23:39:18 (Table 1, Fig. 3D,E). All
hybrids performed more or less similar results as
in first two cases (Table 2 shows sample hybrids
A, C and D selected for cultivation as 1, 2, 3
respectively). The authors took only sample hybrids
from each groups obtained after hybridization
among the three species numbered as 1, 2, 3, 4,
5, 6, 7, 8 and 9 as mentioned in the text. It has
been observed that total yield in all the hybrids
Table 2. Comparison in yield, spawn running and
total cropping period of the selected hybrids
and their parer.ts
Parents and Time for Cropping Yield
Hybrids substrate period after for 4
colonization colonization flushes
(days) (days) (g)
Parents
Pleurotus sajor-caju16 38 900
P. cornucopiae 17 42 800
P. sapidus 16 40 840
Hybrids
Hy. 1 16 35 950
Hy. 2 17 35 850
Hy.3* 15 31 1080
Hy. 4 17 36 836
Hy. 5 17 35 786
Hy. 6 17 36 860
Hy. 7 18 37 840
Hy. 8 18 38 800
Hy. 9 17 38 850
*Hybrid NO.3 deposited vide I.T.C.C. Ace. No. 3828
in Indian Type Culture Collection
[Vol. 55(1) : 2002]
was more or less similar, however hybrid no. 3
(Fig. 3E) as a result of mating between P. sajorcaju
and P. corn ucopia e, took lesser time in
subtrate colonization and higher yield in shorter
cropping period than rest of the isolates (Table 2).
The fruit bodies of hybrid no. 3 were of whitish
colour, which is an additional advantage over
others as people prefer white-coloured mushrooms.
It was also found to produce fruit bodies upto
32°C.
Fig.3. P. sajor-caju, P. sapidus and P. cornucopiae
and inter specific hybrids developed by crossing
among them. A. Hybrids resembling to P. sajorcaju,
B. Hybrids having blending of characters of
P. sajor-caju and P. sapidus, C. Hybrids
resembling to P. sapidus, D. Hybrids having
blending of characters of P. sajor-caju and P.
cornucopiae, E. Hybrids no. 3 recommended for
cultivation
The data reveals that the hybrid no. 3 is better
yielder than the others and maybe recommended
for cultivation to the mushroom growers. The culture
of hybrid nO.3 was deposited in Indian Type Culture
Collection, Division of Mycology and Plant
Pathology, Indian Agricultural Research Institute,
New Delhi vide Acc. No. 3828 (Table 2).
Pleurotus is heterothallic and tetrapolar
(Anderson et a/., 1973, Bahukhandi and Munjal
Indian Phytopathology 65
1990, Thakur and Bhandal 1993). A single
basidiocarp produces four types of basidiospores
with two different mating factors termed as 'A' and
'B'. Dikaryon is produced by the basidiospores
having different incompatibility factors, which can
be detected on the basis of presence of clamps
and conjugate nuclear division (Raper, 1966).
Roxon and Jong (1977) described four mating
types of Pleurotus sajor-caju as A 1B1, A2B2,
A1B2 and A2B1. Kaufert (1936) reported four
types of reactions, when he mated monokaryons
of P. corticatus and observed mated or compatible,
neutral, antagonistic and inhibitory reactions. In
the present study, however the paired monokaryons
resulted in three types of colonies, which may be
termed as mated, non mated, neutral and
antagonistic. This combination may be compared
with the results of Kaufert (1936). It is also reported
that interstock hybrids gave better yield than
intrastock hybrids in Pleurotus species (Jandaik
1987, Rajarathnam and Bano 1987, Thakur and
Bhandal 1993). During this study it has been
observed that the resultant hybrids produced more
or less similar yield as their parents but the hybrid
produced by crossing between P. sajor-caju and
P. cornucopiae, resulted 23.3 per cent more yield
as compared to his parents, which is in agreement
with that of earlier workers. Like Schizophyllum
and Coprinus, in Pleurotus also, among four
basidiospores one type can mate only with one of
the remaining three basidiospores, therefore the
sterile:fertile ratio is 75:25. It is only possible to get
a clear picture of sterile:fertile ratio, when the
pairing will be maximum (Eger 1974, Liang and
Chang 1989, Toyomasu and Mori 1989).
In the present study, the F1 hybrid (Hybrid No.
3) obtained by crossing between P. sajor-caju and
P. cornucopiae having blended characters but
higher yield than its parents, while other F1-hybrids
were almost similar to their parents in terms of
yield potential and other morphological features.
Further studies in this direction are required for
obtaining other desirable traits in the new hybrids.
ACKNOWLEDGEMENT
The authors are very thankful to the Head,
Division of Plant Pathology, IARI, New Delhi for
providing necessary facilities to carry out this
work.
66 Indian Phytopathology
REFERENCES
Anderson, N.A., Wang, S.S. and Schwandt, J.W.
(1973). The Pleurotus ostreatus - sapidus species
complex. Mycologia 65: 28-35
Bahukhandi D. and Munjal, R.L. (1990). Studies on
evolving high yielding strains of Pleurotus sajorcaju
through hybridization. Indian Phytopath 43(1):
70-73.
Bahukhandi, D., Munjal, R.L. and Kapoor. J.N. (1989).
Cultivation trials of Pleurotus opuntiae (Dur. and
Lev.) Sacco Current Science 58(15): 856-857.
Dhar, B.L. (1997). Mushroom Industry in India - A
view., in Advances in Mushroom Biology and
Production (Eds. Rai, Dhar and Verma). Mushroom
Society of India, Solan H.P. pp.369-378.
Eger, G. (1974). Rapid method for breeding Pleurotus
ostreatus. Mushroom Science 9: 567-573.
Eger, G., Eden,' G. and Wissing, E. (1976). Pleurotus
ostreatus, breeding potential of a new cultivated
mushroom. Theor. Appl. Genet. 47: 155-163.
Eugenio, C.P. and Anderson, N.A. (1968). The
genetics and cultivation of P. ostreatus. Mycologia
60: 627-634.
Ghosh, Nand Chakravarty, D.K. (1991). Studies on
evolving new strains of Pleurotus sajor-caju by
selective dikaryotization. Indian Mushsrooms 19:
69-72.
Jandaik, C.L. (1987). Breeding potential of Pleurotus
species in India with special reference to P. sajor-
[Vol. 55(1) : 2002]
caju: Indian Mushroom Science II, (Eds. T.N. Kaul
and B.M. Kapoor) Srinagar pp.355-360.
Kaufert, F.H. (1936). The Biology of Pleurotus corticatus
Fr. Minnesota, A.E.S. Tech. Bull. 114: 3-35.
Liang, Z.R. and Chang, S.T. (1989). A study of
intergeneric hybridization between P. sajor-caju
and Schizophyllum commune by protoplast fusion.
Mushroom Sci. 12: 125-137.
Rajarathnam, S. and Bano, Z. (1987). Pleurotus
mushroom part I.A. , Morphology, life cycle,
taxonomy, breeding and cultivation. C.R.C. critical
reviews in Food Science and Nutrition 26: 179-
185.
Raper, J.R. (1966). Genetics and sexuality of higher
fungi, New York. The Ronald Press Company,
New York. 283pp.
Roxon, J. and Jong, S.C. (1977). Sexuality of an
edible mushroom, Pleurotus sajor-caju. Mycologia
69: 203-205.
Thakur, K. and Bhandal, M.S. (1993). Monosporous
isolates and their intermating in Pleurotus sapidus
and P. sajor-caju. Mushroom Res. 2: 41-44.
Toyomasu, T. and Mori, Kan-Ichi (1989)-.
Characteristics of the fusion products obtained by
intra and interspecific protoplast fusion between
Pleurotus species. Mushroom Sci. 12(1): 151-159.
Watling, R. (1970). Spore colour. Bull. Br. Mycol. Soc.
􀁽Watering