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Molecular Identification of Some Wild Nigerian Mushrooms Using Internal Adeniyi et al. AMB Expr (2018) 8:148 https://doi.org/10.1186/s13568-018-0661-9 ORIGINAL ARTICLE Open Access Molecular identifcation of some wild Nigerian mushrooms using internal transcribed spacer: polymerase chain reaction Mobolaji Adeniyi1,2, Yinka Titilawo3,4,5*, Anthonia Oluduro1, Olu Odeyemi1, Motebang Nakin4 and Anthony Ifeanyi Okoh5 Abstract Identifcation of fungal species based on morphological characteristics is tedious, complex, prone to errors, and thus cannot be completely relied upon. In this study, internal transcribed spacers (ITS 1 and 4)—polymerase chain reaction was employed to amplify DNA of 19 mushroom isolates collected at Environmental Pollution Science and Technology farm, Ilesa, Southwest Nigeria. The PCR amplifcation of ITS1 and 4 of the mushrooms isolates yielded approximately 850 bp. Amplicons obtained were sequenced and identifed using BLASTn in the NCBI. The BLASTn results revealed that Termitomyces aurantiacus (3), Tricholoma matsutake (8), Tricholoma robustum (2), P. ostreatus (4), Schizophyllum commune (1) and Pleurotus pulmonarius (1) were fully represented. Only Tricholoma matsutake (KT273371), Pleurotus pulmonarius (KY962469) and Tricholoma matsutake (AF438605) had 100% similarity with reference strain. However, the phylogenetic analysis of the isolates showed low genetic relatedness with reference strains. This study revealed the novelty of the mushroom strains and thus advocating the need for strict conservation measures and further investiga- tions on their potential benefts to mankind. Keywords: Mushrooms, ENPOST, Molecular identifcation, Food security, Nigeria Introduction Generally, macrofungi are regarded as important Mushrooms are regarded as visible fungi with distinc- bioresource because of their diverse ecological, nutri- tive carpophores (basidiocarps or fruiting bodies) which tional, health and medicinal benefts (Odeyemi et al. represent the reproductive stage in the life cycle of 2014). Tey are decomposers of dead organic matters Basidiomycetes and some Ascomycetes (Das 2010). Mor- especially those containing lignin and cellulose, and also phologically, they are classifed as pufballs, stinkhorns, aid nutrient recycling in the environment (Nilsson et al. bracket and gilled fungi, and may either be edible, ined- 2011). In addition, they are rich in protein, crude fbre, ible or poisonous (Bates 2006). Tey are usually abundant vitamins and minerals, and possess medicinal benefts during the wet season in most countries of the world and including anticholesterol, antitumor, antimicrobial, anti- have been found thriving on diferent substrates (Rog- viral, antineoplastic, antimutagenic, antioxidant, antilipi- ers 1994; Straatsma et al. 2001; LaRochelle and Berkes demic, antidiabetic antihyperglycaemic, antihypotensive, 2003; Gbolagade et al. 2006; Djelloul and Samraoui 2011; antiparasitic, anti-infammatory hepatoprotective, hypo- Dwivedi et al. 2012; Pushpa et al. 2012; Andrew et al. cholesterolemic, immunodulatory and anti-ageing prop- 2013; Vyas et al. 2014; Rashid et al. 2016; Rumainul and erties (Borchers et al. 2008; Rathee et al. 2011; Patel et al. Aminuzzaman 2016). 2012; Duru and Çayan 2015). Accurate taxonomy is essential for exploitation of the numerous advantages an organism ofers (Odeyemi et al. *Correspondence: [email protected] 2014). Before now, traditional method of mushroom 3 Department of Biology/Microbiology/Biotechnology, Faculty of Science, Federal University, Ndufu-Alike, Ikwo, Nigeria identifcation is based on the size, shape and margin of Full list of author information is available at the end of the article carpophore, length, diameter, location and texture of © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Adeniyi et al. AMB Expr (2018) 8:148 Page 2 of 9 stipe, the spore-bearing surface, habitat, habit, season their taxonomy using genetic approach greatly limits they appear, spore prints, use of coloured photographs, optimal exploitation for diverse human benefts. To the and any other unique feature present on the fruiting body best of our knowledge, this is the frst of its kind in the (FAO 2006; Odeyemi and Adeniyi 2015). However, muta- region and it is in the light of this we aimed at investi- tion resulting in genetic changes and which may not be gating the variability of some wild mushrooms collected expressed, and the indistinctive nature of mushroom at the ENPOST farm, Ilesa, Southwest Nigeria using ITS mycelial forms remains a setback with taxonomy using regions. morphological characteristics (Lian et al. 2008; Appiah et al. 2017). Materials and methods Modern molecular technique reduces the challenges Description of study area of inconspicuous nature, inconsistent morphology and Environmental Pollution Science and Technology indiscrimination among fungal species often associated (ENPOST) farm, Ilesa, Southwest Nigeria is located with traditional method of nomenclature (Blackwell between (Latitude 4°42′30″E to 4°42′45″E longitude et al. 2006; Nilsson et al. 2011). Tis procedure employs 7°36′55″N to 7°37′10″N). Te farm which spans about 10 the use of genetic marker identifable to a specifc region hectares was established primarily to address the chal- of either the coding or non-coding portion of the fun- lenges of environmental pollution, food insecurity and gal genome, usually the nuclear encoded ribosomal agroforestry/biodiversity destruction and provide oppor- RNA (rRNA). Tese genes present in multiple copies tunities for research on natural resources. and contain conserved coding (small subunit—SSU and large subunit—LSU) as well as variable non-coding parts Sample collection and presumptive identifcation (internal transcribed spacers—ITS) are highly conserved Nineteen mushroom samples were randomly collected within a species (Cui et al. 2011). In addition, they are at ENPOST farm, Ilesa, Southwest Nigeria. Te mush- suitable for developing genetic probes to classify fungi rooms were from decaying woods and leaves, soil debris and also reveal their phylogenetic relationship (Cui et al. and termite mounds from diferent locations within 2011; Siddiquee et al. 2015). the farm over a 2 year sampling regime (April 2014 and In China, PCR-based approach was employed to iden- March 2016). Samples were harvested from their respec- tify Boletus edulis and Verpa bohemica using rDNA ITS tive substrate, placed in diferent labelled sterile paper sequences (Lian et al. 2008; Anand and Chowdhry 2013). bags and immediately taken to the laboratory for further In addition, eight wild mushrooms belonging to the gen- analysis. Te samples were kept in the refrigerator (4 °C) era Amanita, Astraceus, Termitomyces and Volvariella before analysis. Presumptive identifcation was based on were characterized in the region (Das et al. 2013). Simi- the morphology of the mushrooms previously described lar identifcation was performed on Boletus edulis, B. (Rogers 1994; Nwordu et al. 2013). aestivalis, B. luridus, Amanita muscaria and Hebeloma radicosum in Italy (Iotti et al. 2005) and Tricholoma Tissue culture of mushroom fruiting bodies giganteum and Calocybe indica isolates in Bangalore Fruiting bodies collected from the wild were gently (Pushpa et al. 2012). Likewise, Agaricus bisporus white cleaned with water, the undercap surface (gills) cut into and brown varieties, Pleurotus sajor-caju, Pleurotus eryn- small sizes (1 cm2) and surface sterilized in 70% ethanol gii, Lentinula edodes and Flammulina velutipes were for 30 min. Te surface sterilized mushroom pieces were identifed in Malaysia (Avin et al. 2014) and Amanita sp. aseptically placed on sterile potato dextrose agar (PDA) in India (Zhang et al. 2004). plates and incubated for 7–10 days. Sub-culturing of In the black African countries, studies on mushrooms mushroom mycelia was done and pure isolates were pre- taxonomy using the molecular technique have not been served on PDA slant at 4 °C until when needed. widely investigated. In Kenya, Ojwang (2012) charac- terized seventy-one Pleurotus species collected from Extraction and PCR amplifcation of genomic DNA the wild whereas in Nigeria, literatures revealed that Total genomic DNA was extracted from a 5–7 day old Oyetayo (2009, 2014), Bankole and Adekunle (2012) and mycelial mat using the ZR Fungal/Bacterial DNA kit™ Awala and Oyetayo (2015) have identifed mushrooms. (Zymo Research, USA) following the manufacturer Te samples were collected from Ekiti, Lagos, Ondo and instructions. Briefy, 100 mg wet weight of mushroom Oyo states of the Southwestern Nigeria and character- mycelium suspended in 200 µl phosphate bufer solu- ized using the internal transcribed spacer—polymer- tion bufer was added to a ZR BashingBead ™ lysis tube ase chain reaction ITS-PCR). Diversity of mushrooms and vortexed at 10,000 rpm for 5 min. Tereafter, the ZR abounds at ENPOST farm, Ilesa, Osun State, Southwest BashingBead ™ lysis tube was centrifuged at 1000 rpm Nigeria. However, paucity of information regarding for 1 min, 400 µl of the supernatant transferred into a Adeniyi et al. AMB Expr (2018) 8:148 Page 3 of 9 Zymo-Spin™ IV spin flter in a collection tube and cen- Editor (Hall 2004). After this, the resulting consen- trifuged
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