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Mycelial Growth of Native Strains of Neolentinus Ponderosus and N Institute of Chemical and Biological Research - Faculty of Chemical Sciences and Pharmacy – San Carlos de Guatemala University Mycelial growth of native strains of Neolentinus ponderosus and N. lepideus at different pH and their Pinus spp. Wood degradation Claudia Rangel del Valle, Diego de León Ruíz, Osberth Morales Esquivel, María del Carmen Bran González Microbiology Department, Faculty of Chemical Sciences and Pharmacy,San Carlos de Guatemala University [email protected] Received: May 31st. 2017 Approved: July 02nd. 2018 Abstract Neolentinus ponderosus and N. lepideus are two saprophytic fungi species used traditionally in Huehuetenango and Totonicapán, Guatemala. The degradative capacity of both species confers them potential for fruiting bodies production. This study evaluated the mycelial growth of two native strains of N. ponderosus and N. lepideus in malt extract agar (EMA) at different pH and the degradation of wood from two pine species in rot chambers during 12 months. pH 7.0 was the most appropriate for the mycelial growth of N. ponderosus and for N. lepideus were 5.0 and 5.6. The colonies of both strains showed fruity odor, velvety texture, regular to irregular edge, white color, with or without diffusible pigment, hyphae with 1-5 μm width, chlamydospores and clamp connections. Wood from Pinus tecunumanii and P. ayacahuite exhibit weight-loss percentages between 8.76 ± 5.58 and 12.07 ± 5.66, with N. ponderosus 145.2003 and N. lepideus 90.2002, respectively. In both cases reached the early stage of brown-rot decay. These results could be useful for future research that evaluate the fruiting bodies production in logs for food and commercial purposes. Keywords: edible fungi culture, brown-rot decay, fruiting bodies, saprophytic fungi. Revista Científica|Vol.28 No.1|Publication year 2018|ISSN 2070-8246 Institute of Chemical and Biological Research - Faculty of Chemical Sciences and Pharmacy – San Carlos de Guatemala University Introduction In Guatemala there is a great variety of The cultivation of edible mushrooms is a edible fungi and the use of 83 species biotechnological process that that are consumed in 48 towns of 20 contributes to recycle lignocellulosic departments of the country has currently agricultural and forest residues, since been documented (Morales, Bran, & the mushrooms are consumed as food Cáceres, 2010). Within this great variety for humans and the degraded of species, N. ponderosus (Fr.) substrates can be used in several ways Redhead & Ginns and N. lepideus (OK (Sánchez, 2004). There are 7,000 Mill.) Redhead & Ginns stand out, which species of fungi that are considered are very popular for their consumption edible in the world but only 200 have and commercialization in the been studied for cultivation on an departments of Huehuetenango and experimental basis and about ten have Totonicapán (Bran , Morales, Cáceres, been produced on an industrial scale: & Flores, 2003a, 2003b). Agaricus bisporus (JE Lange) Imbach, Auricularia spp., Flammulina Velutipes ( The natural habitat of the Neolentinus Curtis) Singer, Grifola frondosa (Dicks.) genus is the wood of Pinus spp, where it Gray, Hypsizygus marmoreus (Peck) causes brown rot (Pegler, 1893; HIM. Bigelow, Lentinula edodes (Berk.) Redhead & Ginns, 1985). In Guatemala Pegler, Pholiota nameko (T. Itô) S. Ito N. ponderosus has been found on wood and S. Imai, Pleurotus spp., Tremella of P. tecunumanii Eguiluz & Perry, while fuciformis Berk. and Volvariella volvacea N. lepideus develops on P. ayacahuite (Bull.) Singer (Chang & Miles, 2004). Ehren., Therefore, it is considered that both species are saprobia in nature and New species of edible fungi have been with the possibility of being grown in successfully cultivated in recent years, forest waste (Bran et al., 2003a). Agrocybe cylindracea (DC.) Maire, Therefore, in 2007 several native strains Favolus tenuiculus P. Beauv., Hericium of these species were isolated and erinaceus (Bull.) Pers., Lepista nuda studies on in vitro growth were also (Bull.) Cooke , Falo indusiatus Vent, carried out with different culture media Pleurotus albidus (Berk.) Pegler, P. and different temperatures; as well as citrinopileatus Singer and Stropharia the production of inoculum and fruit rugosoannulata Farl. ex Murrill (Chang bodies with different disinfection and Miles, 2004 are the ones that are treatments, in order to achieve the outstanding ; Omarini, Lechner and production of fruit bodies on Albertó, 2009; Lechner and Albertó, lignocellulosic waste generated in the 2011; Bran, Cáceres, Gurriarán, country (Bran, Morales, Flores, Morales and Flores, 2014). For this Cáceres, & Blanco, 2007). reason that currently many studies have Because there are no studies about focused on the search for new species mycelial growth under different culture of wild edible fungi, to study their conditions, this study evaluated the cultivation and thus be able to expand behavior of two native strains (N. the number of species available for ponderosus and N. lepideus) in the EMA human consumption (Omarini et al., medium at different pH, as well as their 2009). activity. wood degradator of P. tecunumanii and P. ayacahuite, respectively, as a preliminary step to Revista Científica|Vol.28 No.1|Publication year 2018|ISSN 2070-8246 Institute of Chemical and Biological Research - Faculty of Chemical Sciences and Pharmacy – San Carlos de Guatemala University establish if possible their cultivation and Later, 10 boxes (repetitions) of each production of fruiting bodies on logs of medium were inoculated with a 0.5 cm2 pine wood and other synthetic segment of mycelium from each of the substrates (forms that are used strains. The inoculated boxes were successfully in cultivation L. edodes), as sealed and incubated at 26 ° C for 21 well as the possible application of the days. The diameter reached by the disinfection technique of the substrates colonies was measured and recorded in by immersion in alkaline water. two perpendicular planes (x, y axes) from which the average diameter (cm) was obtained. With the data obtained, Materials and methods the Excel® program created a database Reactivation of the N. ponderosus and that included the average diameter (cm) N. lepideus strains: Two strains that are of the colonies of each of the strains in deposited in the Saprobios and the media adjusted to different pH and Mycorrhizal Fungi Cepario, of the the control medium. Department of Microbiology, School of Biological Chemistry, were used; Chambers preparation of the colonized Faculty of Chemical Sciences and wood: The procedure was carried out Pharmacy, of the University of San according to Johansen, (1949) and Carlos de Guatemala. The code and the Pérez, Pinzón and Echenique, (1977): origin of the strains are: Neolentinus The formaldehyde, alcohol, acetic acid lepideus, 90.2002 (Aldea Panquix, (FAA) fixing solution was prepared as Totonicapán) and Neolentinus follows: Ethanol 95 % (50 mL), acetic ponderosus, 145.2003 (Aldea Bulej, San acid (5 mL), formaldehyde (10 mL), and Mateo Ixtatán, Huehuetenango). They distilled water (35 mL). The wooden were seeded in Petri dishes with EMA blocks were saturated with FAA solution medium and incubated at 26 ° C for two in a Kitasato that was vacuum-stripped weeks. Subsequently, they were re- for 30 min. The FAA was replaced with seeded in the mentioned culture distilled water and they were vacuumed medium and incubated for two more for 5 min, then washed with potable weeks. water for 30 min. Sections 20 µm wide were cut from the radial face of the Diameter calculation of the colonies at blocks with a sliding microtome. The different pH level: the procedure was sections were stained with the carried out according to that Cartwright method of picro-aniline blue, recommended by Stamets (1993) and which was carried out as follows: 1% Mier, Toriello and Ulloa (2002): The aqueous Safranin (30 s), distilled water EMA culture medium (Merck®) was (30 s, twice), picro-aniline blue at steam prepared, the pH was adjusted at values (30 s), distilled water (30 s twice), 50%, of 5.0, 7.0, 9.0 and 12.0 with HCl or 70%, 95% and 100% ethanol (30 s 10% KOH and sterilized in an autoclave each), 100% ethanol (2 min, two for 15 min at 121 ° C and 1.0 Kg / cm2. changes) and xylene at 100 % (3 min, 20 mL of each were served in sterile two changes). Finally they were placed disposable polystyrene Petri dishes. In in 100% xylene and they were placed on the same way, EMA medium was slides containing a small drop of prepared without pH adjustment (5.6), Entellán® resin and then coverslips which was used as a control. were placed on them, they were Revista Científica|Vol.28 No.1|Publication year 2018|ISSN 2070-8246 Institute of Chemical and Biological Research - Faculty of Chemical Sciences and Pharmacy – San Carlos de Guatemala University pressed, labeled and left to dry. The degradation of wood, no statistical sections were studied and the analysis was performed, but the distribution of the hyphae in the wood percentage of weight loss and cells was observed. degradation by the fungus in the plant cells was described. Results: the Analysis of results: The diameter of greatest mycelial growth diameter of N. mycelial growth of each strain at the ponderosus strain 145.2003 was evaluated pH (5.0, 7.0, 9.0 and 12.0) observed at pH 7.0, which presented and the control (5.6), was statistically 8.25 (± 0.13) cm after 21 days of analyzed by means of a one-way incubation. However, this diameter did analysis of variance, with a subsequent not show a significant difference (p> test of multiple Tukey comparisons (α = 0.05) with those obtained at pH 5.0 and .05) in the SPSS 19.0® program, to pH 5.6, which corresponded to the show statistically significant differences. control. The diameters observed at pH The macroscopic and microscopic 9.0 and 12.0 were 6.81 (0.87) cm and characteristics of the colonies obtained 5.71 (± 0.43) cm, respectively, which at the evaluated pH were analyzed presented a significant difference through a cluster analysis with the between each other (p = .001) and with PAST® statistical program, through the respect to the other evaluated pH (p development of dendrograms <.05).
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