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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,
Potenza, Italy
Basidiomata of a wild mushroom macroscopically recognised as Although cultivation of Pleurotus ostreatus (Jacq.: Fr.) Kum., as Pleurotus ostreatus 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 (D.C.: 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 ostreatus 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- ostreatus 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] Non 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
2009. potato-dextrose-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., 2012). 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- spectrophotometer 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 spp.
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 L. or five mL of the amplified products were visualised under a UV transillu- Carpinus orientalis 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. ostreatus using the BLAST software (Altschul et al., 1997). 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., 1981): 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) 12-day intervals.
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 perfectly 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., 1999).
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.
ostreatus 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., 2012). 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 basid- iomata had a light grey colour (Figure 3) and their flesh consistencywas tender. Performance of the Albanian isolate of P. ostreatus in cultivation 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
Trichoderma 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, i.e., 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 isolates even- Pleurotus sajor- caju. Eur. J. Appl. Microbiol. Biotechnol. 12:58-62.
tually characterised by a more pleasant, intense taste and a higher Delmas J, 1989. Les champignons et leur culture. Flammarion ed., basidiomata yield in artificial cultivation conditions or to improve the Paris, France.
substrate composition. 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- Ferri F, 1985. I funghi - micologia, isolamento, coltivazione. Edagricole, ter mushroom, for producing them in some pioneer intensive cultiva- Bologna, Italy.
tions with the goal to commercialise a Pleurotus species undoubtedly, Ferri F, Zjalic S, Reverberi M, Fabbri AA, Fanelli C, 2007. I funghi.
for taste and flesh tenacity, known to be superior to the oyster mush- Coltivazione e proprietà medicinali. Edagricole, Bologna, Italy.
room even if Albanian P. ostreatus basidiomata artificially produced in Gunde-Cimerman N, 1999. Medicinal value of the genus Pleurotus (Fr.) this work, when cooked, resulted tender and tasteful. P. Karst. (Agaricales S.R., Basidiomycetes). Int. J. Med. Mushrooms Considering that the fresh pro capite mushroom consumption per year in Albania is very low (400-500 g) especially in comparison to Italy Muez-Orobia MA, 1993. Bases para o cultivo de Pleurotus. pp 129-137 (about 1 kg) and other countries (Germany, Belgium, United States of in Jornadas técnicas del champiñ n y otros hongos comestibles em America) in which it reaches 2-2.5 kg and results of a preliminary mar- Castilla-La Mancha, I, Anais Cuenca. Consejera de Agricultura y ket investigation (Merkuri and Cara, unpublished results), it seemsadvisable to promote a major production of fresh basidiomata of P. ostreatus and popularise knowledge regarding artificial cultivation of P. Papetti C, Consiglio G, Simonini G, 1999. Atlante fotografico dei funghi eryngii in Albania, in the next future. d'Italia. Vol. 1. AMB Ed., Trento, Italy.
It is matter of fact that the oyster mushroom so far encountered the Phillips R, 1985. Mushroom and other fungi of Great Britain and consumer's and restaurator's favour in the country and request of its Europe. Istituto Geografico De Agostini Ed., Novara, Italy.
fresh basidiomata is high and remains mostly unsatisfied. Vessey E, 1969. Culture industrielle des champignons sylvestres en Hongrie. Rev. Myc. 34:7-108. Wasser SP, Weis AL, 1999. Medicinal properties of substances occurring in higher Basidiomycetes mushrooms: current perspectives (Review). Int. J. Med. Mushrooms 1:31-62. White TJ, Bruns T, Lee S, Taylor JW, 1990. Amplification and direct Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, sequencing of fungal ribosomal RNA genes for phylogenetics. In: Lipman DJ, 1997. Gapped BLAST and PSIBLAST: a new generation M.A. Innis, D.H. Gelfand, J.J. Sninsky, T.J. White (Eds.), PCR proto- of protein database search programs. Nuc. Ac. Res. 25:3389-402.
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