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Isolation and Molecular Identification of Laccase-Producing Saprophytic Acta Mycologica DOI: 10.5586/am.1119 ORIGINAL RESEARCH PAPER Publication history Received: 2018-05-29 Accepted: 2018-10-22 Isolation and molecular identifcation Published: 2019-06-06 of laccase-producing saprophytic/ Handling editor Andrzej Szczepkowski, Faculty of Forestry, Warsaw University of phytopathogenic mushroom-forming fungi Life Sciences – SGGW, Poland from various ecosystems in Michoacán Authors’ contributions IM performed experiments and State, Mexico wrote the article; MAS helped in collection and isolation; GVM arranged the resources and conceived the idea; MSVG Irum Mukhtar1,2*, Marina Arredondo-Santoyo2, Ma. Soledad helped in data analysis and 3 2 arranged the resources Vázquez-Garcidueñas , Gerardo Vázquez-Marrufo * 1 Mycological Research Center (MRC), College of Life Science, Fujian Agriculture and Forestry Funding University (FAFU), Fuzhou, China Funding for the publication 2 Centro Multidisciplinario de Estudios en Biotecnología, Facultad de Medicina Veterinaria for this article is obtained from Mycological Research y Zootecnia, Universidad Michoacana de San Nicolás de Hidalgo, Km 9.5 Carretera Morelia- Center, College of Life Sciences, Zinapécuaro, Col. La Palma, CP 58893 Tarímbaro, Michoacán, Mexico 3 Fujian Agriculture and Forestry División de Estudios de Posgrado, Facultad de Ciencias Médicas y Biológicas “Dr. Ignacio University, Fuzhou, 350002. Chávez”, Universidad Michoacana de San Nicolás de Hidalgo, Gral. Francisco J. Múgica s/n, Fujian, China. While Ministry Felicitas del Río, CP 58020 Morelia, Michoacán, Mexico of Foreign Afair (SRE) Mexico has provided funding for * Corresponding authors. Email: [email protected] (GVM); [email protected] (IM) postdoctoral fellowship (2013– 2014) to Dr. Irum Mukhtar. Abstract Competing interests No competing interests have Te aim of this study was isolation and molecular identifcation of laccase-producing been declared. saprophytic/phytopathogen Basidiomycetes species from different geographic regions with dominant vegetation of Pinus, Abies, and Quercus spp. in the state of Copyright notice © The Author(s) 2019. This is an Michoacán, Mexico. Soil samples and visible mycelial aggregates were collected for Open Access article distributed fungal isolations. Soil samples were processed using a soil particle washing technique, under the terms of the where a selective Ascomycetes inhibitor and guaiacol, as an indicator of saprophytic Creative Commons Attribution License, which permits Basidiomycetes growth, were used. Most of the isolates were obtained from samples redistribution, commercial and collected in Parque Nacional, José Ma. Morelos (Km 23), Charo, Michoacán, Mexico. noncommercial, provided that Based on sequence comparisons and phylogenetic analysis of internal transcribed the article is properly cited. spacer regions (ITS1-5.8S-ITS4) with respect to reference taxa, identifcation of Citation saprophytic/phytopathogen Basidiomycetes species was carried out. In total, 15 Mukhtar I, Arredondo-Santoyo isolates from 12 genera (i.e., Bjerkandera, Coriolopsis, Ganoderma, Hexagonia, Irpex, M, Vázquez-Garcidueñas MS, Vázquez-Marrufo G. Isolation Limonomyces, Psathyrella, Peniophora, Phlebia, Phlebiopsis, Trametes, and Trichaptum) and molecular identifcation of and one species from family Corticiaceae were identifed. Tis study will be useful for laccase-producing saprophytic/ further investigations on biodiversity of soil Basidiomycetes in diferent ecosystems. phytopathogenic mushroom- forming fungi from various At present, these isolates are being used in our various lab experiments and can be ecosystems in Michoacán useful in diferent industrial and bioremediation applications. State, Mexico. Acta Mycol. 2019;54(1):1119. https://doi. org/10.5586/am.1119 Keywords Basidiomycetes; guaiacol; geographic regions; saprophytic Digital signature This PDF has been certifed using digital signature with a trusted timestamp to assure its origin and integrity. A verifcation Introduction trust dialog appears on the PDF document when it is opened in a compatible PDF reader. Certifcate properties provide further details such as certifcation time In forest ecosystems, various combinations of vegetation cover consistently produce and a signing reason in case any alterations huge amounts of organic matter playing a dominant role in soil structure and fertility. made to the fnal content. If the certifcate is missing or invalid it is recommended to A very large number of microorganisms are involved in litter decomposition under verify the article on the journal website. diferent environmental conditions. Saprophytic Basidiomycetes are important and dominant recyclers of plant wastes in soil. Tis fungal group is the main producer of lignin-degrading enzymes such as manganese peroxidase and laccase [1]. Fungal Published by Polish Botanical Society 1 of 11 Mukhtar et al. / Laccase producing saprophytic higher fungi in soil laccases are of great interest due to their higher redox potential for lignin and polyphenol degradation, potential use in many industrial applications in paper, textile, food, and pharmaceutical sectors, and in the degradation of aromatic pollutants causing envi- ronmental problems [2]. Exploring novel laccases with diferent substrate specifcities and enhanced stabilities is desirable for industrial applications, besides developing an efective and economic production medium with high yields to enhance their utility [3]. Terefore, there is a need to fnd new laccase producers from diferent geographic and environmental conditions. Te state of Michoacán is among the fve states with the greatest biodiversity in Mexico [4], partly due to its geographical location in the transition zone between the Nearctic and Neotropical regions, which generates a variety of ecosystem types. To date, studies have been performed to increase the awareness about the diversity of fungi in the state of Michoacán, with a major focus on the Basidiomycota group. Signifcant classical taxonomic work within this group has been carried out in various ecosystems of this region in the past. Discovery of novel laccase-producing fungi is important to improve sources of more active, thermostable, or acid tolerant enzymes for industrial applications. Previous studies exhibited that various ecosystems have the potential to warrant an exploration of laccase-producing fungi due to the fungal diversity and geographic position of this region. Terefore, this study was designed to complement classical taxonomic work with biotechnology by building a gene bank and an ex situ collection of long-term vegetative (mycelia) or asexual (spores) propagation structures. In this study, isolation, screening, and molecular identifcation of potential laccase-producing native saprophytic Basidiomycetes species from diferent natural forest areas were performed. Material and methods Site information and samples collection For the collection of soil samples, diferent areas were selected in the state of Michoacán, Mexico. Te sites were chosen based on their ecological characteristics and geographical location. Te information on the sampling sites appears in Tab. 1. Soil samples were collected from diferent habitats and three to four replicate plots of each habitat were sampled. Soil cores of 2.5-cm diameter were taken from 25-cm depth afer the litter layer was removed. Sample for each location was pooled from four or more cores per site. Vegetative mycelial aggregates in soil were also collected separately for direct isolation from known/unknown Basidiomycetes species. Te samples were marked with information such as collection numbers along with names, sampling location, and date of collection. Mycelial samples were wrapped in aluminum foil, brought to the laboratory, and stored in a refrigerator at 4°C for further study. Tab. 1 Sample collection sites in the state of Michoacán, Mexico. Region and location Type of vegetation Coordinates Altitude (m) Ejido La Ampliación; Atécuaro, Morelia Forest of pine and pine-oak N 19°33'59.4", W 101°07'48.2" 2,350 Parque Nacional Insurgente José María Pine-oak forest and oak N 19°39'45.7", W 101°00'19.7" 2,150 Morelos y Pavón (Km 23) forest Presa La Gachupina, Municipio de Forest of pine and cedar N 19°49'23.63", W 100°39'10.35" 2,950 Jerécuaro (Ciudad Hidalgo) Parque Nacional “Barranca del Cupa- Pine-oak forest N 19°26'16.63", W 102°06'57.22" 2,090 titzio” (PNBC), Uruapan Ichaqueo, Morelia Forest of pine, pine-oak N 19°34'11.04", W 101° 8'45.0" 2,200 and mesophyll mountain Tarímbaro Mix vegetation N 19°47'0", W 101°8'0" 1,888 © The Author(s) 2019 Published by Polish Botanical Society Acta Mycol 54(1):1119 2 of 11 Mukhtar et al. / Laccase producing saprophytic higher fungi in soil Isolation of fungal isolates with the help of guaiacol as an indicator Isolation of basidiomycetes from soil samples. Selective soil particle-washing technique [5] was employed for the isolation of Basidiomycetes from soil samples. Approximately 5–6 g of each fresh soil sample was added into 500 mL of sterile 0.1% (wt/vol) sodium pyrophosphate in a 1-L beaker. Soil solution was gently stirred for 1 h with glass rod at room temperature (25 ±2°C) to disperse soil clumps and colloids. Te soil suspension was passed through a stack of 20-cm soil sieves of 250 mm (No. 60) and 53 mm (No. 270) mesh. Particles on the 53-mm mesh were washed thoroughly, organic particles were collected and added into sterile distilled water. A 0.4 mL suspension of organic particles was spread onto petri dishes with 2% Potato Dextrose
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