IJA-2016_1.qxp_Hrev_master 07/04/16 10:31 Pagina 35
Italian Journal of Agronomy 2016; volume 11:704
Molecular identification and artificial cultivation of a wild isolate
of oyster mushroom in Albania
Jordan Merkuri,1 Stefania Mirela Mang,2 Ippolito Camele,2 Magdalena Cara,1
Gian Luigi Rana2
1Department of Plant Protection, Agricultural University of Tirana, Tirana, Albania;
2School of Agricultural, Forestry, Food and Environmental Sciences, University of Basilicata,
Basidiomata of a wild mushroom macroscopically recognised as
Although cultivation of Pleurotus ostreatus
(Jacq.: Fr.) Kum., as
were observed on an oak trunk in a mixed wood of
reported by Delmas (1989), was initially attempted in Germany at
northern Albania. Pure cultures of the fungus were then obtained on
beginning of last century using pieces or wheels of broadleaf wood as
potato-dextrose-agar medium. Molecular analyses of genomic DNA of
a growth substrate, its large spread on straw compost in the world
the fungus confirmed its identification. The rDNA ITS region
occurred through ‘70 years of the last century thanks to the initialworks of Vessey (1969) and Ferri (1972). Ferri et al.
report, in 2007,
nucleotide sequence of the studied Pleurotacea
matched at 99% those
China as the major producer of P. ostreatus
basidiomata with about 2.3
of two P. ostreatus
strains already present in NCBI GenBank database.
ton milions and Italy, with about 30,000 tons as the first producer in
The rDNA ITS nucelotide sequences of two pure cultures of the
Europe. Pileus of P. ostreatus
, as well as that of Pleurotus eryngii
Albanian P. ostreatus
were deposited in EMBL database under the
Fr.) Quél., contains lovastatin, a statin drug used for lowering choles-
accession numbers LN849458 and LN849459. One of the fungus iso-
terol ematic level and prevention of cardiovascular diseases (Gunde-
lates was subsequently cultivated under protected and semi-natural
Cimerman, 1999; Wasser and Weis, 1999). Furthermore, the cellular
conditions. Productivity and biological efficiency of the Albanian P.
wall of both Pleurotus
species contains some particular carbohydrates
ranged from about 10% to 16% and from 33 to 53.33%, respec-
which seem useful to trigger human defences against viral, bacterial
tively. This seems to be the first report on the artificial cultivation of P.
and fungal diseases. Some basidiomata of a mushroom closely resem-
in Albania and could have, in the next future, a high econom-
bling P. ostreatus
(Ferri, 1985; Ferri et al
., 2007) were found growing on
ic impact on development and diffusion of this important edible mush-
an oak trunk in a mixed wood of the Derven forest located near
room over the country.
Rreshen town (Tirana province) in autumn 2009. Because noPleurotus
species was so far artificially and intensively cultivated inAlbania, it seemed worthwhile, first of all to identify by molecularmethods the mushroom and successively to attempt its artificial culti-vation in the same country.
Correspondence: Ippolito Camele, School of Agricultural, Forestry, Food andEnvironmental Sciences, University of Basilicata, viale dell'Ateneo Lucano10, 85100 Potenza, Italy.
Materials and methods
E-mail: [email protected]
Mushroom isolation and storage
Key words: Cultivation, molecular analysis; polymerase chain reaction;Pleurotus ostreatus
; substrate pasteurisation.
Isolation of dikaryotic mycelium of the fungus was accomplished in
axenic conditions at Plant Protection Laboratory belonging to the
Funding: this work was supported by the Agency of Research Technology and
Department of Plant Protection, Faculty of Agriculture and
Innovation in Albania.
Environment, Agricultural University of Tirana at the end of October
agar added of 300-ppm/L streptomycin (PDA +
Received for publication: 30 June 2015.
s) was used as the first isolation medium. Small fungus flesh pieces,
Revision received: 23 October 2015.
Accepted for publication: 24 October 2015.
axenically picked-up from different wild basidiomata, specifically fromthe internal zone between stem and pileus, were singly placed into 10
Copyright J. Merkuri et al., 2016
cm diameter Petri dishes containing the above-mentioned medium.
Licensee PAGEPress, Italy
Pure cultures of the mushroom under study were obtained by succes-
Italian Journal of Agronomy 2016; 11:704
sive transfers on PDA of periferal small portions of the fungal colonies
grown on PDA + s (Cara et al
This article is distributed under the terms of the Creative Commons
Several pure culture replicates were kept in vials at 4°C on oat-
Attribution Noncommercial License (by-nc 4.0) which permits any noncom-
meal-agar and then stored in the fungal collection of School of
mercial use, distribution, and reproduction in any medium, provided the orig-
Agricultural, Forestry, Food and Environmental Sciences (University of
inal author(s) and source are credited.
Basilicata, Potenza, Italy).
[Italian Journal of Agronomy 2016; 11:704] [page 35]
IJA-2016_1.qxp_Hrev_master 07/04/16 10:31 Pagina 36
DNA extraction, polymerase chain reaction amplifica-
initial pH of 6.5. Substrate pasteurisation was performed at 70-72°C for
tion and molecular identification
12 h, after a pre-heating phase with hot water at 60°C for 2 h accordingto Muez-Orobia (1993). Substrate was then kept at 50-52°C for 36 h and
Genomic DNA (gDNA) was extracted from 400 mg mycelium aliquots
gradually cooled to 25°C in the successive 12 h. Sterile wheat kernels
of ten single pure cultures with the commercial DNeasy Plant Mini
well invaded by mycelium of the selected strain were prepared as
DNA extraction kit (Qiagen, Hilden, Germany), following manufactur-
spawn at the Department of Plant Protection, Faculty of Agriculture and
er's instructions. Concentration of the gDNA was determined using a
Environment, Agricultural University of Tirana. Inoculation was accom-
plished in axenic conditions thoroughly mixing 15-20 g of spawn to
Wilmington, DE, USA) and, then, adjusted to 50-100 ng/mL. DNA sam-
each 10 kg substrate portions which were then packed into single plas-
ples were stored at 20 and 80°C for short and long-term use, respec-
tic bags furnished, on their surface, of 15 holes, each having 2 cm in
diameter. Incubation phase lasted about 25 days and was performed at
The gDNA was amplified using the universal primer pair ITS4/ITS5
28-30°C directly in cultivation greenhouses or tunnels shadowed at
(White et al.,
1990). Sterile distilled water was used as a negative con-
75% with polyethylene black net. Four cultivation tests were performed:
trol. Polymerase chain reaction (PCR) was performed with an automat-
three under protected conditions and one in a woody habitat. More pre-
ed thermal cycler (Model ‘One personal') of EuroClone S.p.A. (Pero,Milan, Italy) in a 50 mL reaction volume containing 125 mM of the four
cisely, the first three were carried out with 200 substrate bags/each
dNTPs, 0.5 mM of each primer, 1 U of DyNAzyme EXT DNA polymerase,
trial, in the agricultural farms Irakli Shkoza in Divjaka territory
1x polymerase buffer, 4 mL of template DNA (40-50 ng) and water. More
(Lushnja province), Mirela Muca (Tirana) and Ilir Beshi (Tirana) from
specifically, amplification was carried out according to the following
October to December 2010 whereas the fourth was performed employ-
protocol: an initial heating at 94°C for 3 min, followed by 35 cycles at
ing sixty substrate bags, in the farm of Xhevdet Mancaku (Ndroq dis-
94°C for 30 s, 50°C for 30 s and 72°C for 1 min and a final extension at
trict-Tirana) from February to March 2011 (Figure 1). In this last culti-
72°C for 10 min. After electrophoresis, carried out with a 1 Kb DNA
vation test, the substrate bags were transferred, after incubation, in a
Ladder (Invitrogen, Thermo Fischer Scientific, Rodano, Milan, Italy) in
hidden woody location, characterised by abundance of Crataegus
1.2% agarose gel in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8),
as a shrub, were there hunged to branches of Quercus robur
five mL of the amplified products were visualised under a UV transillu-
Mill. at 120-170 cm from soil surface and never
minator and stained with ethidium bromide (0.5 mg mL–1). Finally,
sprayed with tap water because of the rainy season. Cultivation envi-
amplicons were purified using a QIAquick Gel Extraction Kit (Qiagen)
ronments of the first three farms were vice versa
equipped with auto-
and directly sequenced by BMR Genomics (Padua, Italy). The resulting
matic sprinklers that guaranteed repeated atomised water sprays dur-
sequences were compared with those available in NCBI GenBank for P.
ing basidioma differentiation and growth phases.
using the BLAST software (Altschul et al
Each cultivation cycle lasted about 60-65 days and 70-75 days under
protected and semi-natural conditions, respectively. Visual inspections
Cultivation and productivity
of the substrate bags put in cultivation were done each 2-3 days to ver-
Preliminary artificial cultivation tests showed that all the pure cul-
ify the eventual occurrence of insect attacks and/or appearance of alter-
tures of Pleurotus
species under study grew and produced in a satisfac-
ations caused by fungal or bacterial parasites. Basidiomata harvesting
tory mode and without significant differences on a pasteurised stan-
was operated, just before sporulation, at 15-20 days intervals, during
dard substrate wetted at 70% with water (w:w) and composed by 2-3-
each cultivation cycle, mainly three times in the first conditions and
cm-long pieces of wheat straw (50%) and shattered corn stems and
four times in the second. Successive, minor and economically not
bracts (50%) (Ferri et al
., 2007). Therefore, only one of them was then
important sporophore harvests were not registered. Basidiomata were
employed in cultivation tests on the above substrate, added of 10% soy
commonly picked up when their cap diameter at maximum measured
flour, 2.5% ammonium nitrate and 5% CaCO3 powder and adjusted to an
Figure 1. An internal view of a cultivation tunnel (A). Substrate bags hunged to branches of Carpinus orientalis in the Ndroq forest (B).
[page 36] [Italian Journal of Agronomy 2016; 11:704]
IJA-2016_1.qxp_Hrev_master 07/04/16 10:31 Pagina 37
Yield and biological efficiency
shown in semi-natural conditions with an average FBW/substrate bagof 1.5-1.8 kg. Finally, the FBAW ranged from 54.3 to 67.8 g.
Fresh basidioma average weight (FBAW), expressed in grams, was
calculated on a representative sample of 400 sporofores randomly col-lected among those altogether harvested during the four cultivation tri-als. Biological efficiency was calculated using the following formula
(Chang et al.
The xylophilous mushroom, found in Mirdita forestry territory in
BE = FWB/DWC × 100 (1)
Albania and identified as P. ostreatus
on the basis of morphological andmolecular analyses, was successfully cultivated for the first time in the
FWB = fresh weight of basidiomata; and
The highest BE value, i.e
., 53.33%, was achieved in Mancaku farm.
DWC = dry weight of compost or substrate.
This very probably happened thanks to the almost complete absence of
Productivity (P) expressed in percentage, was determined according to
insect infestations, Spring season incoming and consequent prolonged
the following equation:
basidiomata production in forestry conditions. In fact, in the abovelocation, it was possible to operate four harvests in about 75 days with
P = FWB/FWS × 100 (2)
Results here presented, if compared with those regarding the opti-
where:FWB = fresh weight of basidiomata; and FWS = fresh weight of substrate.
Morphology of the fungus
Wild basidiomata of the Albanian isolates of Pleurotus
matched morphological features of P. ostreatus
well described in severalmycological treatises and electronic resources (see at: www.indexfun-gorum.org) (Phillips, 1985; Papetti et al
Isolation of the studied fungus was successful and fifteen pure cul-
tures were obtained. Ten of them were selected for further investiga-tions. Morphology of their colonies on PDA was identical to that of P.
described in details in a previous work on the same Albanian
Figure 2. Result of gel electrophoresis of polymerase chain reac-
mushroom (Cara et al
tion amplification products obtained from Pleurotus ostreatus
with ITS4/ITS5 primers set. Lane M = 1 Kb DNA ladder
DNA extraction, polymerase chain reaction amplifica-
(Invitrogen); Lanes: 1-9 = ITS amplicons of 9 different Albanian
P. ostreatus gDNA samples; 10 = negative control.
tion and molecular identification
Genomic DNA extraction was successfully performed for all the
selected pure cultures of the studied mushroom. The extracted gDNAconcentration ranged from 250 to 350 ng/mL. PCR, performed with theITS4/ITS5 primer pair, produced visible amplicons around 600 bp
(Figure 2) for all samples tested. No amplification products were foundin negative controls. Two oyster mushroom ITS nucleotide sequencesobtained in this study were deposited into EMBL database archiveunder the accession numbers LN849458 and LN849459. Albanian rDNAITS nucleotide sequences were very similar and closely related (simi-larity coefficient=99%) to those of the P. ostreatus
strains S041 andS042 from China, present in NCBI GenBank with the accession num-bers AY540324 and AY540325.
Cultivation and productivity
Pileus cuticle of the Albanian artificially produced P. ostreatus
iomata had a light grey colour (Figure 3) and their flesh consistencywas tender.
Performance of the Albanian isolate of P. ostreatus
tests was satisfactory. In fact, its P and BE ranged from 10 to 16% andfrom 33 to 53.33%, respectively (Table 1). Each single substrate bagproduced a minimum of 0.5 kg and a maximum of 3 kg of fresh basid-iomata depending on the occurrence of massive or weak insect and/or
Figure 3. Close up of the gregarious basidiomata growth of the
spp. attacks, respectively. The best performance was
Albanian Pleurotus ostreatus.
[Italian Journal of Agronomy 2016; 11:704] [page 37]
IJA-2016_1.qxp_Hrev_master 07/04/16 10:31 Pagina 38
Table 1. Productivity and biological efficiency of the Albanian Pleurotus ostreatus in cultivation tests carried under protected and semi-natural
(Xhevdet Mancaku) conditions.
Farm Dry substrate weight Wet substrate weight Total basidiomata fresh yield Productivity Biological efficiency
(q) (q) (q) (q) (%)
Irakli Shkoza 6.00 20.00 1.98 9.90 33.00
Mirela Muca 6.00 20.00 2.00 10.00 33.33
Ilir Beshi 6.00 20.00 2.53 12.65 42.16
Xhevdet Mancaku 1.80 6.00 0.96 16.00 53.33
mal Italian P values for the same Pleurotacea
., 20-25% (Ferri et al
via "in vitro" cultivation of edible mushrooms Pleurotus spp. pp.
2007), are encouraging and along with the relatively low cost of the
346-351 in Proc. Intern. Conf. "Towards future sustainable develop-
mushroom on market and its high versatility in kitchen, would suggest
ment", Nov. 16-17, Shkodër, Albania. University of Shkodra, Luigj
the opportunity of continuing these preliminary studies.
Gurakuqi, Shkodër, Albania.
All future efforts should be planned to either investigate the natural
Chang ST, Lau OW, Cho KY, 1981. The cultivation and nutritive value of
presence in other Albanian areas of different P. ostreatus
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Delmas J, 1989. Les champignons et leur culture. Flammarion ed.,
basidiomata yield in artificial cultivation conditions or to improve the
Ferri F, 1972. Prove di coltivazione di alcuni isolamenti di Pleurotus
Interesting could be also to extend these studies to search Albanian
ostreatus Quél. Micol. Ital. 1:11-8.
basidiomata of P. eryngii
, mushroom commonly known as the king oys-
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, for producing them in some pioneer intensive cultiva-
tions with the goal to commercialise a Pleurotus
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for taste and flesh tenacity, known to be superior to the oyster mush-
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room even if Albanian P. ostreatus
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[page 38] [Italian Journal of Agronomy 2016; 11:704]
EASTERN PURPLE 1986). The Delaware used an infusion of coneflower root for gonorrhea and found it to be highly effective. The purple coneflower was the only native prairie Echinacea purpurea (L.) plant popularized as a medicine by folk practitioners and doctors. It was used extensively as a folk remedy (Kindscher 1992). Purple coneflower root was used
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