Micología Aplicada International

ISSN: 1534-2581

dcarrera@colpos.mx

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. 25-30

Colegio de Postgraduados

Puebla, México

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25Ganoderma lucidum cultivated in Mexico

micol. apl. int., 27(2), 2015, pp. 25-30

Micologia aplicada international, 27(2), 2015, pp. 25-30© 2015, Berkeley, CA, U.S.A.www.micaplint.com

Cultivation of ganoderMa luciduM on agriCultural by-produCts in MexiCo*

t. bernabé-gonzález1, 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. E-mail: teobernaglez@hotmail.com

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 70-72 days. The mushroom yield varied from 40.9 to 47.9 g per 0.586 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.

1 Corresponding author.

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Micol. apl. int., 27(2), 2015, pp. 25-30

INTRODUCTION

The medicinal mushroom Ganoderma lucidum (Curtis) P. Karst. has long been esteemed for its functional and medicinal properties10. 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 level6,7. 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, Mexico7. 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 28-29 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 (CaSO4). 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

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micol. apl. int., 27(2), 2015, pp. 25-30

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 (25.3-27.8 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 percentage9.

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-145 80.5 ± 5.0 a High Cottony CP-405 75.5 ± 2.7 a High CottonyPeanut shell CP-145 80.5 ± 2.1 a High Cottony CP-405 79.5 ± 3.7 a High CottonyCoffee pulp CP-145 36.2 ± 3.2 c High Cottony CP-405 48.8 ± 4.3 cd High CottonyCoconut husk CP-145 55.8 ± 7.9 b Low Hairy CP-405 58.7 ± 3.0 bc Low HairyCorn-cob CP-145 78.7 ± 3.3 a High Cottony CP-405 68.5 ± 5.5 ab High CottonyRice straw CP-145 36.2 ± 6.3 c Low Hairy CP-405 43.2 ± 3.4 d Medium HairyBean stubble CP-145 19.0 ± 10.0 d Low Cottony CP-405 36.7 ± 8.9 d Medium HairyMaize ear husk CP-145 71.7 ± 7.3 a Low Hairy CP-405 46.3 ± 16.0 cd Low HairyBean pod CP-145 78.7 ± 4.6 a High Cottony CP-405 81.0 ± 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.

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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

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.

A B

C D

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micol. apl. int., 27(2), 2015, pp. 25-30

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-145 586 10 17-18 70 40.9 ± 4.5 6.9 ± 2.7 a

CP-405 586 10 18-19 72 47.9 ± 4.2 8.2 ± 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.

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 cultivation5. Cultivation of G. lucidum on agricultural by-products. The substrate was colonized by the mushroom mycelium in 10 days. Mushroom primordia appeared after 17-19 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 31-38 x 45-65 mm for strain CP-145, while 27-55 x 47-78 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

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3. Jaramillo, C., N. Rodríguez and S. T. Chang. 2010. Simple methodology for the cultivation of the medicinal mushroom Ganoderma lucidum in Colombian coffee farms. Pp. 397-405. In: Hacia un Desarrollo Sostenible del Sistema de Producción-Consumo de

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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.

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6. Martínez-Carrera, D., N. Curvetto, M. Sobal, P. Morales & V. M. Mora (Eds.). 2010. 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. 2010. Los hongos comestibles y medicinales en México: recursos genéticos, biotecnología, y desarrollo del sistema de producción-consumo. Pp. 91-108. 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. 2009. Tea waste as a supplement for the cultivation of Ganoderma lucidum. World Journal of Microbiology and Biotechnology 25: 611-618.

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Fig. 2. Basidiocarps of Ganoderma lucidum cultivated on a selected substrate containing diverse agricultural by-products. A: strain CP-145. B: Strain CP-405.

A B