Optimal conditions of in vitro mycelial growth of Lentinus strigosus, an edible mushroom isolated in the Brazilian Amazon

Abstract

The protocols of in vitro cultivation described in the literature for mushrooms are usually correlated with temperate climate
habitat, but it is necessary to study protocols for species of tropical climates. In this article, we collected, isolated,
and evaluated the conditions of in vitro mycelial growth of Lentinus strigosus and correlated these with the characteristics of its habitat. These results indicate, as optimal conditions of in vitro mycelial
growth for L. strigosus, the use of 35°C for incubation, initial pH from 5 to 7, without illumination, Sabouraud dextrose agar medium, and agitation
for culture in liquid medium.

Full text

NOTE
Mycoscience (2008) 49:215–219 © The Mycological Society of Japan and Springer 2008
DOI 10.1007/s10267-007-0404-2
R. Vargas-Isla · N.K. Ishikawa (*)
Coordenação de Pesquisas em Tecnologia de Alimentos, Instituto
Nacional de Pesquisas da Amazônia, Av. André Araújo, 2936,
Manaus, Amazonas – CEP: 69060-020, Brazil
Tel. +55-92-3643-1890; Fax +55-92-3643-1846
e-mail: noemia@inpa.gov.br
Ruby Vargas-Isla · Noemia Kazue Ishikawa
Optimal conditions of in vitro mycelial growth of
Lentinus strigosus
, an
edible mushroom isolated in the Brazilian Amazon
1990; Boa 2004). The potential of the diversity of mush-
rooms in the tropical ecosystems has been much discussed,
but little explored scientifi cally (Hawksworth 2001; Mueller
and Schmit 2007; Mueller et al. 2007).
The genus Lentinus Fr. (Polyporaceae tribe Lentineae
Fayod) has a worldwide distribution but with species occur-
ring more abundantly in tropical regions. The basidiocarps
are often leathery to almost woody in consistency and are
therefore more persistent than typical members of the
Agaricales. It is because of their persistent nature that they
frequently represent the dominant group of agaricoid fungi
in the tropical rainforests, being resistant to adverse periods
of drought (Pegler 1975). Ethnomycology studies have
identifi ed different species of edible mushrooms of the
Lentinus genus consumed by indigenous groups such as the
Yanomami in the Brazilian Amazon: Lentinus crinitus (L.)
Fr., L. velutinus Fr., L. glabratus Mont., L. cubensis Berk.
and M.A. Curtis, L. strigosus (Schwein.) Fr. (Fidalgo and
Prance 1976; Fidalgo and Hirata 1979; Prance 1984). In
addition, the Uitoto natives of the area of Araracuara in
the Colombian Amazon consume L. strigosus, L. concavus
(Berk.) Corner, L. crinitus, and L. scleropus (Pers.) Fr.
(Vasco 2002). Chang and Mao (1995) related that this
species can be cultivated. We tasted this mushroom sautéed
with margarine and some salt and thought it had a good
fl avor, a high umami taste, and was slightly fi brous.
Most of the cultivated mushrooms originated in coun-
tries of temperate climate, and the protocols of in vitro
cultivation described in the literature are correlated with
the habitat of these species. As environmental conditions in
the tropics are different, however, it is necessary to study
protocols for the tropical climate species. As commented by
Mswaka and Magan (1999), the optimum temperature for
growth of most wood-decay fungi from temperate regions
is between 25°C and 30°C. Very few if any detailed studies
have been carried out on the temperature profi les of tropi-
cal wood-decay fungi. In Brazil, the species of cultivated
edible mushrooms are mainly originally of Europe and
Asia, as A. bisporus, L. edodes, and Pleurotus spp. Mush-
room cultivation is more developed in the south and
southeast of Brazil where the subtropical climate is more
Received: June 28, 2007 / Accepted: November 30, 2007
Abstract The protocols of in vitro cultivation described in
the literature for mushrooms are usually correlated with
temperate climate habitat, but it is necessary to study pro-
tocols for species of tropical climates. In this article, we
collected, isolated, and evaluated the conditions of in vitro
mycelial growth of Lentinus strigosus and correlated these
with the characteristics of its habitat. These results indicate,
as optimal conditions of in vitro mycelial growth for L.
strigosus, the use of 35°C for incubation, initial pH from 5
to 7, without illumination, Sabouraud dextrose agar medium,
and agitation for culture in liquid medium.
Key words Basidiomycetes · High temperature · Lentinus
strigosus · Thermophiles fungi
There are more than 200 genera of macrofungi that contain
species of use to people, mostly because of their edible
properties. These are distinguished between those recorded
as simply “edible” and those that are actually eaten (“food”).
To include all edible species as “food” would greatly over-
state the number of species consumed by people around the
world (Boa 2004). There are nearly 100 species of fungi that
can be cultivated (Boa 2004). Commercial markets are
dominated by Agaricus bisporus (J.E. Lange) Pilát, Pleuro-
tus spp. (Fr.) P. Kumm., Lentinula edodes (Berk.) Pegler,
Volvariella volvacea (Bull.) Singer, Flammulina velutipes
(Curtis) Singer, and Pholiota nameko (T. Ito) S. Ito and S.
Imai (Kendrick 2000; Sánchez 2004). On the other hand,
the wild mushrooms that are still not cultivated commer-
cially have great ethnomycological importance because
they constitute a much appreciated food (Herrera and Ulloa

216
appropriate for the production of these species. In the north
region of tropical climate, the production costs for these
species are very high, making cultivation at the highest
scales not feasible. Therefore, we began research on domes-
tication and cultivation of species adapted to the tropical
climate using as base agroforestry residues of the Amazo-
nian to reduce the costs of production.
The domestication of naturally occurring edible fungi in
Brazil will allow their production and commercial exploita-
tion so that the country would have a national product
adapted to our climate and atmosphere, competing on the
external market (Maki and Paccola-Meirelles 2002).
In the present article, we collected, isolated, and evalu-
ated the conditions of in vitro mycelial growth of Lentinus
strigosus and correlated the results with the characteristics
of the habitat of this mushroom.
The basidiocarps of L. strigosus were collected in lignico-
lous substratum on the campus III of the Instituto Nacional
de Pesquisas da Amazônia (INPA) (3°5′31.6″ S, 59°59′36.4″
W), Manaus–AM, Brazil. The mycelium was isolated with
inoculation of fragments of the basidiocarp context in
potato dextrose agar (PDA; Acumedia) culture medium,
incubated at 25°C, without light. Part of the collection was
made exsiccate, to be deposited in the Herbarium of INPA.
The species was identifi ed with the help of available litera-
ture (Stankovicová 1973; Pegler 1975, 1983).
The mycelial growth of the L. strigosus isolate was evalu-
ated at temperatures of 25°, 30°, 35°, 40°, and 45°C, on PDA
medium. Each Petri dish (90 mm diameter) was inoculated
with one disk (10 mm diameter) of the mycelia freshly
grown on PDA plates and cultured for 5 days at 25°C. The
mycelial growth was evaluated by measuring the diameter
of the colony and the mycelial dry weight on the 5th and on
the 6th day, respectively. The two common measures of
growth rate are (1) increase in radius of colony over time
and (2) increase in dry mass of colony over time. The fi rst
has the advantage that sequential records can be obtained
from each colony. The second is a more absolute measure-
ment, but can be performed only once for each colony
(Kendrick 2000). In this study, we adopted a methodology
that allowed evaluation of the colony diameter and dry
weight, cultivated in solid medium. For evaluation of myce-
lial dry weight, after the growth of the colony in the respec-
tive culture media, the Petri dish was placed in a microwave
oven for 20 s to melt the medium. Soon after, the mycelium
was separated from the medium for fi ltration and washed
with distilled water, at an approximate temperature of 60°C.
This preparation was placed in an oven at 105°C until con-
stant in weight.
The effect of agitation of the liquid culture was evaluated
by measuring L. strigosus mycelial growth, using potato
dextrose broth (PDB): an infusion of 200 g potato, 20 g
glucose, and distilled water to complete the volume of
1000 ml (pH = 6.0), sterilized at 121°C for 15 min. Erlen-
meyer fl asks (250 ml) containing 100 ml PDB were inocu-
lated with fi ve disks of the mycelia. For the agitation
treatment, the fl asks were placed in a table agitator
(TECNAL TE-140) at 75 rpm. As control, other fl asks were
maintained in static condition. Both treatments were incu-
bated in an atmosphere conditioned to 25° ± 2°C and natural
illumination. After 15 days of mycelial growth, the myce-
lium was separated for fi ltration. The obtained mass was
placed in an oven at 105°C until constant in weight. To
measure the effect of initial culture media pH, Erlenmeyer
fl asks (250 ml) containing 100 ml PDB with respective pH
(4, 5, 6, and 7) were inoculated with fi ve disks of mycelia.
The pH was adjusted with HCl and NaOH solutions before
sterilization. Flasks were incubated at 35°C and with agita-
tion, the optimal conditions observed in the previous exper-
iments. In this experiment the dry weight was obtained after
10 days of mycelium growth.
The L. strigosus mycelial growth was evaluated in solid
culture media: PDA (Acumedia), malt extract and peptone
agar (MEPA) [3% malt extract (Becton Dickinson); 0.3%
soy peptone (Acumedia); 1.5% agar (Becton Dickinson)],
Sabouraud dextrose agar (SDA) (Becton Dickinson), V8
medium (V8) [200 ml V8 juice (Campbell’s); 1.5% agar,
800 ml H2O], and minimum culture medium (MM) (Pon-
tecorvo et al. 1953). The fl asks with the respective culture
media were sterilized at 121°C for 15 min, and the media
were poured into Petri dishes (90 mm diameter) and inocu-
lated with one disk of the mycelia. This experiment was also
incubated at 35°C. Mycelial growth was evaluated by mea-
suring the colony diameter on the 5th day. The dry weight
of the mycelium was evaluated according to the methodol-
ogy described in the evaluation of mycelial dry weight in
solid medium.
To evaluate the effect of the infl uence of light, Petri
dishes with PDA medium were inoculated with one disk of
the mycelia and incubated at 35°C for 24 h light (1250 lx).
Light emission spectra were taken by Illuminance meter
IM-5 (Topcon, Japan). Dark treatments were carried out in
a box made lightproof with a dark cloth. Dark-grown cul-
tures were viewed and scored for development only after
the termination of the experiments. The mycelial growth
was evaluated by measuring the diameter of the colony on
the 5th day, and mycelial dry weight was measured using
the temperature evaluation procedure. To evaluate the
mycelial growth in liquid medium, fi ve disks of the mycelia
were placed in Erlenmeyer fl asks (250 ml) containing 100 ml
PDB medium and incubated at 35°C and 75 rpm for 10
days. Light treatments were 24 h light; dark treatments
were carried out in fl asks covered with aluminum foil. After
this period, the mycelium was separated by fi ltration, obtain-
ing the dry weight.
The experiments were tested in fi ve replications and two
repetitions. For statistical evaluation, the experiments were
submitted to analysis of variance (ANOVA) and the aver-
ages were compared by the Tukey test at the 5% level of
signifi cance.
The basidiocarps (Fig. 1) presented pileus 4–7 cm diam-
eter, convex, subinfundibuliform to infundibuliform, or
laterally attached and spathulate; surface pale, whitish to
pallid ochraceous, more brownish toward the center, at fi rst
often with violaceous or purplish tints especially toward the
margin, densely villose to hispid-tomentose; margin curved,
thin, undulate; context thick at the disk, white. Lamellae
deeply decurrent, white to ochraceous buff, or with

217
violaceous tints especially on the edge, 1–2 mm wide,
crowded, with lamellulae of four lengths. Stipe excentric to
lateral, more rarely central, short, cylindrical or with sub-
bulbous base; colorless surface with the pileus but not vio-
laceous, tomentoso, with fi brillose hairs extending to the
apex, these characteristics led us to identify this mushroom
as L. strigosus, considering the descriptions by Pegler (1983).
L. strigosus is encountered under dense vegetation as well
as in semiopen habitat and is exposed to large temperature
variations (Castillo et al. 2004). The isolate used in this
work was collected in November 2006 in open habitats on
lignicolous substratum.
The effects of temperature on L. strigosus grown on
PDA over a range of temperatures from 25° to 45°C are
shown in Fig. 2. Statistical analysis of the data of the diam-
eter of the colonies showed that 35°C and 40°C were the
best temperatures for mycelial growth, not differing at 5%
signifi cance (Fig. 2A). However, the results of the mycelial
mass dry weight showed a signifi cant difference between
35°C and 40°C (Fig. 2B); this measure of mycelial growth
indicated that the temperature of 35°C provided a colony
with larger mass. This experiment demonstrated the advan-
tage of evaluating colony dry mass in solid medium for
determining the size and the density of the colony without
the need of cultivation in liquid medium. Mswaka and
Magan (1999) studied basidiomycetes and divided them
into three groups based on their optimum and maximum
temperatures for growth: (1) low-temperature group with
optimal temperatures lying between 25°C and 30°C and no
growth above 37°C; (2) intermediate group with optimal
temperature range of 30°–37°C with no growth at 45°C; and
(3) high-temperature group with growth in the range 37°–
40°C and growth ceasing at 55°C. Kendrick (2000) defi ned
thermophile fungi with minima temperature above 20°C,
maxima above 50°C, and optima between 35°C and 50°C.
Once there was growth to 45°C, although it was smaller, the
colony reached 2.5 cm diameter in 5 days at 45°C; thus, we
considered this mushroom to be of the high-temperature
group or a mushroom thermophile. This result is easily
understood, because when measuring the internal tempera-
ture of the log where the mushrooms were collected, a
Fig. 1. Basidiocarps of Lentinus strigosus collected in lignicolous substratum. A Young state. B Mature state of basidiocarps
Fig. 2. Lentinus strigosus mycelial growth on potato dextrose agar
incubated at different temperatures. Means with the same letter (s) are
not signifi cantly different (P < 0.05) by the Tukey test. Average of fi ve
replications. A Diameter of colony (cm) on the 5th day. B Colony dry
weight (mg/plate) on the 6th day
temperature from 35° ± 3°C was usually registered at
midday, which explains the correlation with the optimum
temperature obtained in the laboratory. Castillo et al. (2004)
studied correlation between the in vitro growth response to
temperature and the habitat of some lignicolous fungi from
Papua New Guinea coastal forests and obtained similar
results for L. strigosus.
For aerobes, aeration is one of the most important culti-
vation conditions. Usually, wood-rotting basidiomycetes

218
grow only on wood where the moisture content is not
greater than 60%. Therefore, they are more sensitive to
oxygen defi ciency than the fungi imperfecti (Emelyanova
2005). Furthermore, biomass production, the intensity of
aeration, and agitation conditions also infl uenced the form
of the mycelium growth (diffused fi lamentous mycelium or
agglomerated mycelium: pellets) and the form of the pellets
(Emelyanova 2005). These aspects were observed in this
experiment (Table 1); the treatment with agitation pre-
sented greater mycelial growth compared with stationary
condition. Agglomerated mycelium was observed in the
treatment with agitation. There was no signifi cant differ-
ence (P < 0.05) between pH 5, 6, and 7; however, pH 4
presented the lowest mycelial growth (Table 2).
There are many studies of the effect of light on the
development of basidiomycetes, and this is an essential
factor for the fruit-body formation (Chang and Hayes 1978;
Leatham and Stahmann 1987; Matsumoto and Kitamoto
1987; Kitamoto et al. 1999; Sakamoto et al. 2004). Consider-
ing that the place of the collection of this mushroom was
quite well illuminated (average, 61120 lx at midday), this
suggests that light has a strong infl uence on its fructifi cation.
However, in the evaluation of the infl uence of light, in the
mycelial growth in solid medium a signifi cant difference was
not present, and in the liquid medium biomass production
was higher in the absence of light (Table 3).
As to the effect of culture medium, considering the mea-
surement of the diameter of the colony on the 5th day of
incubation, the PDA medium showed greater diameter.
However, in the evaluation of colony dry weight, the highest
biomass production was obtained in SDA medium
(Fig. 3).
Thus, we suggest the use of SDA medium with pH 5,
temperature of 35°C for incubation, without illumination,
and with agitation for cultivation in liquid medium as
optimal conditions of in vitro L. strigosus mycelial growth.
Acknowledgments The authors thank MSc. Ricardo Braga-Neto for
the description, as well as Dr. Armando López Ramírez and Dr. Eiji
Nagasawa for helpful discussions on the identifi cation of the mush-
room. They also thank Dr. José Antonio Alves Gomes and Dr. Gislene
Almeida Carvalho-Zilse for the administrative support for acquisition
of the equipment used in this study. R. Vargas-Isla thanks Fundação
de Amparo à Pesquisa do Estado do Amazonas (FAPEAM) for the
scholarship.
Table 1. Effect of agitation on Lentinus strigosus mycelial growth on
potato dextrose broth medium
Treatment Colony dry weight (mg/fl ask)
With agitation 413.4 ± 22.9a
Without agitation 286.9 ± 54.7b
Colony dry weight on the 15th day incubated at 25° ± 2°C and 75 rpm
of agitation
Means with the same superscript letter(s) are not signifi cantly different
(P < 0.05) by the Tukey test; average of fi ve replications
Table 2. Effect of initial pH of culture media on Lentinus strigosus
mycelial growth
pH Colony dry weight (mg/fl ask)
4246.5 ± 21.2b
5324.6 ± 25.9a
6283.9 ± 31.3ab
7332.8 ± 39.1a
Colony dry weight on the 10th day on potato dextrose broth medium
incubated at 35°C and 75 rpm agitation
Means with the same superscript letter(s) are not signifi cantly different
(P < 0.05) by the Tukey test; average of fi ve replications
Table 3. Effect of light on Lentinus strigosus mycelial growth on
solid and liquid potato dextrose medium
Treatment Diameter of
colony* (cm) Colony dry weight (mg/plate or fl ask)
Solid medium** Liquid medium***
With light 7.49 ± 0.749a228.9 ± 13.9a369.44 ± 1.8b
Without light 7.91 ± 0.252a203.5 ± 7.2a397.04 ± 1.1a
* Diameter of colony in solid medium on the 5th day of incubation;
** colony dry weight on the 6th day of incubation; *** colony dry
weight on the 10th day of incubation with 75 rpm of agitation; incuba-
tion at 35°C
Means with the same superscript letter(s) are not signifi cantly different
(P < 0.05) by the Tukey test; average of fi ve replications
Fig. 3. Lentinus strigosus mycelial growth on different culture media
incubated at 35°C. PDA, potato dextrose agar; MEPA, malt extract
and peptone agar; SDA, Sabouraud dextrose agar; V8, V8 juice
medium; MM, minimum medium. Means with the same letter(s) are
not signifi cantly different (P < 0.05) by the Tukey test. Average of fi ve
replications. A Diameter of colony (cm) on the 5th day. B Colony dry
weight (mg/plate) on the 6th day

219
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