Micología Aplicada International ISSN: Colegio de Postgraduados México

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1 Micología Aplicada International ISSN: Colegio de Postgraduados México Bernabé-González, T.; Cayetano-Catarino., M.; Bernabé-Villanueva, G.; Romero-Flores, A.; Ángel-Ríos, M.D.; Pérez-Salgado, J. Cultivation of ganoderma lucidum on agricultural by-products in Mexico Micología Aplicada International, vol. 27, núm. 2, julio, 2015, pp Colegio de Postgraduados Puebla, México Available in: How to cite Complete issue More information about this article Journal's homepage in redalyc.org Scientific Information System Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Non-profit academic project, developed under the open access initiative

2 Micologia Ganoderma Aplicada lucidum International, cultivated 27(2), in Mexico 2015, pp , Berkeley, CA, U.S.A. Cultivation of Ganoderma lucidum on agricultural by-products in Mexico* T. Bernabé-González 1, M. Cayetano-Catarino, G. Bernabé-Villanueva, A. Romero-Flores, M. D. Ángel-Ríos and J. Pérez-Salgado Universidad Autónoma de Guerrero, Cuerpo Académico de Agroproducción, Unidad Académica de Ciencias Naturales, ex-rancho Shalako, Petaquillas, Guerrero, México. Accepted for publication: July 24, 2015 ABSTRACT Most basic and applied studies on Ganoderma lucidum have used strains from Southeast Asia. In this work, we studied Mexican genetic resources of G. lucidum from the central region of the country. Strains CP-145 and CP-405 were characterized on nine agricultural by-products in petri dishes. The best mycelial growth and colonization were recorded on bean pod (Phaseolus vulgaris), maize stem (Zea mays), and corn-cob. Several agricultural products, including bean pod, maize stem, and coffee pulp (Coffea arabica) were mixed, supplemented with wheat bran, and used as substrate for the cultivation of G. lucidum. Basidiocarps were harvested after days. The mushroom yield varied from 40.9 to 47.9 g per kg dry substrate weight, showing biological efficiencies ranging from 6.9% to 8.2% for strains CP-145 and CP-405, respectively. Native strains of G. lucidum can be cultivated using local agricultural by-products as substrate. Key words: Agricultural by-products, Ganoderma lucidum, Mexican genetic resources, mushroom cultivation. * Project funded partially by the Research Directorate from the Autonomous University of Guerrero, Mexico, in order to support the Academic Body of Agro-production, UAGRO-CA-139. Micol. 1 Corresponding Apl. Int., author. 27(2), 2015, pp

3 26 T. Bernabé-González et al. INTRODUCTION The medicinal mushroom Ganoderma lucidum (Curtis) P. Karst. has long been esteemed for its functional and medicinal properties 10. There is accordingly a growing demand for G. lucidum and its products at the international market. Most strains of G. lucidum studied, so far, come from Southeast Asia. They have been cultivated on a variety of sawdust substrates (beech, Carpinus, oak, poplar) mixed with diverse supplements (bran, sugar cane bagasse, tea residues, straw, coffee by-products) 1,2,3,4,8. In Mexico, there is a great diversity of Ganoderma genetic resources and several species have been identified at the molecular level 6,7. In the present study, we studied two native strains of G. lucidum cultivated on several agricultural by-products. MATERIALS AND METHODS Strains. The strains CP-145 and CP- 405 of G. lucidum were studied, which are deposited at the Genetic Resources Unit of Edible, Functional and Medicinal Mushrooms (CREGENHCFM), College of Postgraduates (CP), Campus Puebla, Mexico 7. Strains were isolated from wood substrates at the central region of Mexico. They were maintained and subcultured on malt extract agar medium (MEA, Bioxon), and incubated at C for 15 days. Mycelial growth on substrates in petri dishes. The following dry substrates were studied: rice straw (Oryza sativa L.), maize stem (Zea mays L.), coconut husk (Cocos nucifera L.), coffee pulp (Coffea arabica L.), peanut shell (Arachis hypogaea L.), bean pod (Phaseolus vulgaris L.), maize ear husk (Zea mays L.), corn-cob (Zea mays L.), and bean stubble (Phaseolus vulgaris L.). Each substrate was cut into pieces of ca. 3-5 mm, and its moisture content adjusted to 80-85%. The wet substrate (9 g) was placed in a petri dish (90 x 15 mm), and sterilized at 121 C for 15 min. After cooling, three replicates of each sterilized substrate in petri dishes were inoculated with a MEA agar disc (7 mm diameter) from strains CP-145 and CP-405 of G. lucidum, and incubated at 27±1 C in the dark. Mycelial colony diameter (mm) was measured daily for 10 days, and the macroscopic colony morphology was recorded. Cultivation of G. lucidum on agricultural by-products. After substrate selection in the laboratory, the strains CP-145 and CP-405 of G. lucidum were cultivated in a mushroom growing room. The substrate was prepared as follows (10 kg, dry weight; proportion): 4.4 (44.4%), maize stem; 2.2 (22.2%), bean pod; 2.2 (22.2%), coffee pulp; 1.1 (11.1%), wheat bran; 0.031, calcium hydroxide [Ca (OH) 2 ]; and 0.084, calcium sulfate (CaSO 4 ). The substrate was well mixed, adjusted to 69% moisture content, placed into polypropylene bags (40 x 60 cm; 2 kg wet substrate per bag) with cotton and gauze stoppers at the top for allowing gas exchange, and sterilized at 121 C for 1.5 h. The preparation of spawn was carried out as follows: 200 g of sorghum kernels (60% moisture content) were placed into polypropylene bags, sterilized at 121 C for 1 h, cooled, inoculated with a MEA agar disc (7 mm diameter) from strains CP-145 and CP-405 of G. lucidum, and incubated at 27±1 C in the dark until completely colonized by the mushroom mycelium. Sterilized substrate bags previously prepared were spawned with uniform mixing under aseptic conditions at a rate of ca. 200 g inoculum from each strain studied. Five replicate bags were done for each strain. Inoculated bags were placed

4 Ganoderma lucidum cultivated in Mexico 27 on shelves for incubation in the mushroom growing room. The roof had two rows of transparent plastic sheets, which allowed indirect daylight (about 12 h) during the cultivation cycle. After one month, the top of each bag was removed to promote fruiting, keeping about 70% relative humidity and a suitable temperature range ( C) in the growing room. Basidiocarps were harvested when mature. The biological efficiency (BE) was determined dividing the fresh mushroom yield (fresh weight) by the dry weight of substrate at spawning, and expressed as a percentage 9. Statistical analysis. All experiments followed a completely randomized design. Data were subjected to analysis of variance, and average values were compared using the multiple range test of Tukey (α = 0.05). RESULTS AND DISCUSSION Mycelial growth on substrates in petri dishes. Colony morphology after ten days of mycelial growth of Ganoderma lucidum on diverse agricultural by-products is shown in Table 1 and Fig. 1. Faster growth rate of Table 1. Colony morphology of two strains of Ganoderma lucidum after ten days of mycelial growth on substrates studied in petri dishes. Substrate Strain Colony diameter (mm)* Density Texture Maize stem CP ± 5.0 a High Cottony CP ± 2.7 a High Cottony Peanut shell CP ± 2.1 a High Cottony CP ± 3.7 a High Cottony Coffee pulp CP ± 3.2 c High Cottony CP ± 4.3 cd High Cottony Coconut husk CP ± 7.9 b Low Hairy CP ± 3.0 bc Low Hairy Corn-cob CP ± 3.3 a High Cottony CP ± 5.5 ab High Cottony Rice straw CP ± 6.3 c Low Hairy CP ± 3.4 d Medium Hairy Bean stubble CP ± 10.0 d Low Cottony CP ± 8.9 d Medium Hairy Maize ear husk CP ± 7.3 a Low Hairy CP ± 16.0 cd Low Hairy Bean pod CP ± 4.6 a High Cottony CP ± 4.3 a High Cottony * Different letters at the same column indicate significant differences between mean values according to the multiple range test of Tukey with α= 0.05.

5 28 T. Bernabé-González et al. A B C D Fig. 1. Mycelial growth of Ganoderma lucidum on substrates studied. A: Strain CP-405 growing on bean pod. B: Strain CP-145 growing on maize stem. C: Strain CP-405 growing on peanut shell. D: Strain CP-145 growing on coffee pulp. strain CP-145 was recorded on peanut shell and maize stem, followed by corn-cob and bean pod. All showed mycelial colonies of high density and cottony texture. In a similar manner, strain CP-405 grew faster on bean pod, followed by peanut shell and maize stem, showing high-density and cottony mycelial colonies. The strain CP-145 grew slowly on coffee pulp, rice straw and bean stubble. Slow growth by the strain CP-405

6 Ganoderma lucidum cultivated in Mexico 29 was recorded on maize ear husk, rice straw, and bean stubble. Although growth rate of strains studied on coffee pulp was slow, their mycelial density and vigour was high confirming that such substrate is suitable for mushroom cultivation 5. Cultivation of G. lucidum on agricultural by-products. The substrate was colonized by the mushroom mycelium in 10 days. Mushroom primordia appeared after days, depending on the strain. Mature basidiocarps of strain CP-145 were harvested after 70 days, showing a mushroom yield of 40.9 g and a BE of 6.9%. A mushroom yield of 47.9 g and a BE of 8.2% were recorded for strain CP-405, 72 days after spawning (Table 2). Basidiocarps of each strain studied are shown in Fig. 2. They developed normal morphology, and their pileus length and width were x mm for strain CP-145, while x mm for strain CP-405. These results showed that native genetic resources of G. lucidum can be cultivated using diverse substrates available locally. ACKNOWLEDGEMENTS The authors thank Prof. Dr. Daniel Martínez-Carrera, College of Postgraduates, Campus Puebla, Mexico, for providing the strains CP-145 and CP-405 of Ganoderma lucidum. They are also grateful to Dr. Ascencio Villegas Arrizon ( ), Past President of the Autonomous University of Guerrero (UAG), Mexico, as well as to Dr. Arturo Contreras Gómez, Head of the Academic Unit of Natural Sciences, UAG, for their support to achieve this study. LITERATURE CITED 1. Ćilerdžić, J., J. Vukojević, M. Stajić, T. Stanojković and J. Glamočlija Biological activity of Ganoderma lucidum basidiocarps cultivated on alternative and commercial substrate. Journal of Ethnopharmacology 155: Erkel, E. I The effect of different substrate mediums on yield of Ganoderma lucidum (Fr.) Karst. Journal of Food, Agriculture and Environment 7 (3-4): Jaramillo, C., N. Rodríguez and S. T. Chang Simple methodology for the cultivation of the medicinal mushroom Ganoderma lucidum in Colombian coffee farms. Pp In: Hacia un Desarrollo Sostenible del Sistema de Producción-Consumo de Table 2. Mushroom yield and biological efficiency of two strains of Ganoderma lucidum cultivated on a selected substrate containing maize stem, bean pod, and coffee pulp, supplemented with wheat bran. Strain Substrate Mycelial Appearance of Total cycle until Yield Biological dry weight colonization primordia harvesting (g) efficiency* (g) (days) (days) (days) (%) CP ± ± 2.7 a CP ± ± 1.4 a * Different letters at the same column indicate significant differences between mean values according to the multiple range test of Tukey with α= 0.05.

7 30 T. Bernabé-González et al. A B Fig. 2. Basidiocarps of Ganoderma lucidum cultivated on a selected substrate containing diverse agricultural by-products. A: strain CP-145. B: Strain CP-405. los Hongos Comestibles y Medicinales en Latinoamérica: Avances y Perspectivas en el Siglo XXI. Eds. D. Martínez-Carrera, N. Curvetto, M. Sobal, P. Morales and V. M. Mora. Red Latinoamericana de Hongos Comestibles y Medicinales-COLPOS- UNS-CONACYT-AMC-UAEM-UPAEP- IMINAP, Puebla, Mexico. 4. Manavalan, T., A. Manavalan, K. P. Thangavelu and K. Heese Secretome analysis of Ganoderma lucidum cultivated in sugarcane bagasse. Journal of Proteomics 77: Martínez-Carrera, D., A. Aguilar, W. Martínez, M. Bonilla, P. Morales and M. Sobal Commercial production and marketing of edible mushrooms cultivated on coffee pulp in Mexico. Chapter 45, pp In: Coffee Biotechnology and Quality. Eds. T. Sera, C. Soccol, A. Pandey and S. Roussos. Kluwer Academic Publishers, Dordrecht. 6. Martínez-Carrera, D., N. Curvetto, M. Sobal, P. Morales & V. M. Mora (Eds.) Hacia un Desarrollo Sostenible del Sistema de Producción-Consumo de los Hongos Comestibles y Medicinales en Latinoamérica: Avances y Perspectivas en el Siglo XXI. Red Latinoamericana de Hongos Comestibles y Medicinales-COLPOS- UNS-CONACYT-AMC-UAEM-UPAEP- IMINAP, Puebla. 648 pp. 7. Morales, P., M. Sobal, M. Bonilla, W. Martínez, P. Ramírez-Carrasco, I. Tello, T. Spezzia, N. Lira, R. De lima, S. Villa, E. Montiel and D. Martínez Carrera Los hongos comestibles y medicinales en México: recursos genéticos, biotecnología, y desarrollo del sistema de producción-consumo. Pp In: Hacia un Desarrollo Sostenible del Sistema de Producción-Consumo de los Hongos Comestibles y Medicinales en Latinoamérica: Avances y Perspectivas en el Siglo XXI. Eds. D. Martínez-Carrera, N. Curvetto, M. Sobal, P. Morales and V. Mora. Red Latinoamericana de Hongos Comestibles y Medicinales-COLPOS- UNS-CONACYT-AMC-UAEM-UPAEP- IMINAP, Puebla, México. 8. Peksen, A. and G. Yakupoglu Tea waste as a supplement for the cultivation of Ganoderma lucidum. World Journal of Microbiology and Biotechnology 25: Tschierpe, H. J. and K. Hartmann A comparison of different growing methods. Mushroom Journal 60: Wasser, S. P Reishi or Ling Zhi (Ganoderma lucidum). Pp.: In: Encyclopedia of Dietary Supplements. Marcel Dekker, Inc., New York.