Updates Ontropical Mushrooms 2018

Updates on Tropical Mushrooms.
Basic and Applied

Research
José E. Sánchez, Gerardo Mata and Daniel J. Royse
Editors
EE
635.8
U6
Updates on Tropical Mushrooms. Basic and applied research / José E. Sánchez, Gerardo Mata
and Daniel J. Royse, editors.- San Cristóbal de Las Casas, Chiapas, México: El Colegio de la
Frontera Sur, 2018.
227 p. : photographs, illustrations, maps, portraits; 22.7X17 cm.
ISBN: 978-607-8429-60-8
It includes bibliography
1. Tropical mushrooms, 2. Agaricus, 3. Edible mushrooms, 4. Medicinal mushrooms, 5.

Mushroom cultivation, 6. Biotechnology, 7 Mexico, 8. China, 9. Guatemala, 10 Cuba, I. Sánchez,
José E. (editor), II. Mata Gerardo (editor). III. Royse, Daniel J. (editor).
1st. Edition, 2018
The content of this book was subjected to

a process of blind peer reviewing according to
rules established by the Editorial Committee of
El Colegio de la Frontera Sur.
DR © El Colegio de la Frontera Sur

www.ecosur.mx
El Colegio de la Frontera Sur
Carretera Panamericana y Periférico Sur s/n
Barrio de María Auxiliadora
CP 29290
San Cristóbal de Las Casas, Chiapas, México
Photographies of the front page: clockwise, Agaricus subrufescens (D.C. Zied), Agaricus
martinicensis (C. Angelini),. Lentinula boryana (G. Mata), Lepista nuda ( M . C . B r a n ) ,
Agaricus trisulphuratus (P. Callac), Sparassis latifolia (Lu Ma), L. boryana (G. Mata), S.
latifolia (Lu Ma). At the center, A. subrufescens (G. Mata)
No part of this book may be reproduced or transmitted in any form by any means without written
permission from the editors.
Printed and made in Mexico

Acknowledgments
Printing of this book was supported financially by Fondos Mixtos Conacyt through
the Project FOMIX-13149 “Design, construction, equipment and startup of a state
center for innovation and technology transfer for the development of coffee
growing in Chiapas, Mexico” and through the Project MT-11063 of Ecosur “Social
and environmental innovation in coffee growing areas for reducing vulnerability”.
REFEREES LIST
The editors are grateful to the following scientists, researchers, and Professors for the critical peer
review of each of the chapters in this book:
O. P. Ahlawat ICAR-Directorate of Mushroom Research Solan, India

Edgardo Albertó Instituto Tecnológico de Chascomús Argentina
René Andrade El Colegio de la Frontera Sur Tapachula, Chiapas, México
John A. Buswell Shanghai Academy of Agricultural Sciences
China/England
Cristina Burrola Aguilar Universidad Autónoma del Estado de México, México
Reina L. Camacho Inst. de Ciencias Agrícolas Universidad de Baja
California, México
Santiago Chacón Instituto de Ecología, Xalapa, Veracruz, México
Ofer Danai MIGAL Galilee Research Institute and Tel-Hai Academic
College Israel
Francisco Gea Alegría Centro de Estudios sobre el Champiñón, La Rioja, España
Hermilo Leal Lara Facultad de Química, UNAM. Mexico City, Mexico
Dan Levanon MIGAL Galilee Research Institute and Tel-Hai Academic
College Israel
Adolfo Paz Facultad de Veterinaria Universidad de Santiago de
Compostela, España
Marian Petre Faculty of Sciences. University of Pitesti, Romania
Antonios Philippoussis Institute of Technology of Agricultural Products (ITAP)
Likovrisi Athens, Greece
Jean Michel Savoie INRA MycSA (Mycologie et sécurité des aliments),
Villenave d’Ornon, France
Sigfrido Sierra Galván Facultad de Ciencias, UNAM, Mexico City, Mexico
Manjit Singh Mushroom Adviser, Government of Punjab, India
V.K. Singh Department of Biotechnology Faculty of Sciences, V.B.S.
Purvanchal University, Jaunpur, India
Gustavo Valencia Laboratorio de Cultivos Celulares. UPIBI, IPN, Mexico
City, Mexico
List of Contributors
Acosta-Urdapilleta Ma. de Lourdes. Laboratorio de Micología, Centro de Investigaciones

Biológicas. Universidad Autónoma del Estado de Morelos. Avenida Universidad 1001, Colonia
Chamilpa, Cuernavaca Morelos. México. C.P. 62209 < [email protected]>
Aguilar-Marcelino Liliana. Centro Nacional de Investigación Disciplinaria en Parasitología
Veterinaria, INIFAP. Jiutepec, Mor. Mexico. <[email protected]>
Andrade René H. El Colegio de la Frontera Sur. Apdo. Postal 36. Tapachula, Chiapas, México.
30700. <[email protected]>
Batista Pedro L. Center of Toxicology and Biomedicine (TOXIMED), Medical University of
Santiago de Cuba, CP 90 400, Cuba
Beltrán Yaixa. Center for Studies on Industrial Biotechnology (CEBI), Universidad de Oriente.
Santiago de Cuba 5, CP 90 500, Cuba
Bermúdez Rosa C. Center for Studies on Industrial Biotechnology (CEBI), Universidad de
Oriente. Santiago de Cuba 5, CP 90 500, Cuba
Bidart Maíra. Laboratory for Microbiology, Parasitology and Hygiene, Faculty of
Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Belgium
Bran María del Carmen. Departamento de Microbiología, Facultad de Ciencias Químicas y
Farmacia, Universidad de San Carlos, Guatemala. Edificio T-12, 2º nivel. Ciudad universitaria,
Zona 12, 01012, Ciudad de Guatemala, Guatemala, Centroamérica y Programa Universitario de
Investigación en Desarrollo Industrial, Dirección General de Investigación, Universidad de San
Carlos Guatemala, Edificio S-11, 3er nivel. Ciudad Universitaria, zona 12, 01012, Ciudad de
Guatemala, Guatemala, Centroamérica. <[email protected]>
Cáceres Roberto. Departamento de Microbiología, Facultad de Ciencias Químicas y Farmacia,
Universidad de San Carlos, Guatemala. Edificio T-12, 2º nivel. Ciudad universitaria, Zona 12,
01012, Ciudad de Guatemala, Guatemala, Centroamérica y Programa Universitario de
Guatemala, Guatemala, Centroamérica
Callac Philippe. INRA, MycSA (Mycologie et sécurité des aliments), CS20032, 33882,
Villenave d’Ornon, France <[email protected]>.
Cappello García Silvia. Universidad Juárez Autónoma de Tabasco. Carretera Villahermosa-
Cárdenas km 0.5, entronque a Bosques de Saloya, Villahermosa, Tabasco. Tel: 01 993 3 54 43
08, C. P. 85039. <[email protected]>
Carreño Ruiz Santa Dolores. Universidad Juárez Autónoma de Tabasco. Carretera
Villahermosa-Cárdenas km 0.5, entronque a Bosques de Saloya, Villahermosa, Tabasco. Tel: 01
993 3 54 43 08, C. P. 85039
Chen Jie. Instituto de Ecología, A.C., CP 91070, Xalapa, Veracruz, Mexico
Chen Mingjie Academy of Agricultural Sciences. 1000 Jinqi Road, Shanghai 201403, China
Chen Yong-hui. Edible-fungi Industry Adiminstration Gutian, Standardization Working Group
on Tremella of Standardization Administration of China, Ningde, Fujian, 352200, China
Cos Paul. Laboratory for Microbiology, Parasitology and Hygiene, Faculty of Pharmaceutical,
Biomedical and Veterinary Sciences, University of Antwerp, Belgium
Cuevas-Padilla Edgar J. Centro Nacional de Investigación Disciplinaria en Parasitología
Veterinaria, INIFAP. Jiutepec, Mor. Mexico
Cunha Zied Diego. Universidade Estadual Paulista (UNESP), Câmpus Experimental de Dracena,
Rod. Cmte João Ribeiro de Barros, km 651, Bairro das Antas, 17900-000, Dracena, SP, Brazil.
< [email protected]>
Díaz-Godínez Gerardo. Laboratorio de Biotecnología, Centro de Investigación en Ciencias
Biológicas, Universidad Autónoma de Tlaxcala. Mexico.
Diez José C. Department of Biochemistry and Molecular Biology, University of Alcalá de
Henares, Madrid, Spain
Gaitán Hernández Rigoberto. Instituto de Ecología, A.C. Carretera antigua a Coatepec 351. El
Haya CP 91070, Xalapa, Vereacruz, México.
García Nora. Center for Studies on Industrial Biotechnology (CEBI), Universidad de Oriente.
Santiago de Cuba 5, CP 90 500, Cuba
González-Cortázar Manasés. Centro de Investigación Biomédica del Sur, IMSS. Xochitepec,
Mexico
Gonzáalez Ferdiner. Departamento de Microbiología, Facultad de Ciencias Químicas y
Farmacia, Universidad de San Carlos, Guatemala. Edificio T-12, 2º nivel. Ciudad universitaria,
Zona 12, 01012, Ciudad de Guatemala, Guatemala, Centroamérica
G o n z á l e z G a r d u ñ o R o b e r t o . Universidad Autónoma Chapingo (Unidad Regional
Universitaria Sursureste). Teapa, Tabasco, Mexico
Gurriarán Natalia. Departamento de Microbiología, Facultad de Ciencias Químicas y Farmacia,
Universidad de San Carlos, Guatemala. Edificio T-12, 2º nivel. Ciudad universitaria, Zona 12,
01012, Ciudad de Guatemala, Guatemala, Centroamérica y Programa Universitario de
Guatemala, Guatemala, Centroamérica
He Huanging. Guangdong Academy of Agricultural Sciences. Wushan Road, Guangzhou
510640, China
Hernández Edgar. Department of Biochemistry and Molecular Biology, Dalhousie University,
Halifax, Canada
Hernández-Velázquez Victor M. Centro de Investigación en Biotecnología, UAEM (Morelos),
Cuernavaca, México
Huicochea-Medina Magaly. Centro Nacional de Investigación Disciplinaria en Parasitología
Veterinaria, INIFAP. Jiutepec, Mor. México.
Jiang Xiaoling. Institute of Edible Fungi, National and Local Joint Engineering Research Center
for Breeding & Cultivation of Featured Edible Fungi, Fujian Academy of Agricultural Sciences,
No 10, Lane 95, Qianheng Road 10, Jin’an district, Fuzhou City, 350014, P.R.China
Ke Binrong. Institute of Edible Fungi, Fujian Academy of Agricultural Science, Fuzhou 350014,
China
Lebeque Yamila. Center for Studies on Industrial Biotechnology (CEBI), Universidad de
Lin Yanquan, Institute of Edible Fungi, National and Local Joint Engineering Research Center
No 10, Lane 95, Qianheng Road 10, Jin’an district, Fuzhou City, 350014, P.R. China
Llauradó Gabriel. Center for Studies on Industrial Biotechnology (CEBI), Universidad de
López-Arellano Ma. Eugenia. Centro Nacional de Investigación Disciplinaria en Parasitología
Veterinaria, INIFAP. Jiutepec, Mor. México
Lu Ma, Institute of Edible Fungi, National and Local Joint Engineering Research Center for
Breeding & Cultivation of Featured Edible Fungi, Fujian Academy of Agricultural Sciences, No
10, Lane 95, Qianheng Road 10, Jin’an district, Fuzhou City, 350014, P.R. China.
<[email protected]>
Lu Zhenghui. Institute of Edible Fungi, Fujian Academy of Agricultural Science, Fuzhou
350014, China. <[email protected]>
Mata Gerardo. Instituto de Ecología, A. C. carretera antigua a Coatepec 351, El Haya, Xalapa,
Veracruz, C.P. 91070. Tel: 01 228 8 42 18 30. <[email protected]>
Medel Rosario. Instituto de Investigaciones Forestales, Universidad Veracruzana, Xalapa,
Veracruz, México. <[email protected]>
Mendoza-de-Gives Pedro. Centro Nacional de Investigación Disciplinaria en Parasitología
Veterinaria, INIFAP, Jiutepec, Mor. México
Morales Osberth. Departamento de Microbiología, Facultad de Ciencias Químicas y Farmacia.
Universidad de San Carlos de Guatemala. Edificio T-12, 2º nivel. Ciudad Universitaria. Zona
12, 01012, Ciudad de Guatemala, Guatemala, Centroamérica. <[email protected]>
Moreno Lilia. El Colegio de la Frontera Sur. Apdo. Postal 36. Tapachula, Chiapas, México.
Morris Humberto J. Center for Studies on Industrial Biotechnology (CEBI), Universidad de
Oriente. Santiago de Cuba 5, CP 90 500, Cuba. <[email protected]>
Moukha Serge. Laboratory of Toxicology and Applied Hygiene/ INRA, Bordeaux University-
Campus de Carreire, 33076 Bordeaux Cedex, France
Palestina Naara. Instituto de Investigaciones Forestales, Universidad Veracruzana, Xalapa,
Veracruz, México
Pardo-Giménez Arturo.. Centro de Investigación, Experimentación y Servicios del Champiñón
(CIES), C/ Peñicas, s/n, Apartado 63. 16220 Quintanar del Rey, Cuenca, Spain
Parra Sánchez Luis A. Avenida Padre Claret n° 7, 5° G, 09400 Aranda de Duero, Burgos, Spain
Peng Dongxiang. Edible-fungi Industry Adiminstration Gutian, Standardization Working Group
on Tremella of Standardization Administration of China, Ningde, Fujian, 352200, China
Perraud-Gaime Isabelle. Mediterranean Institute of Biodiversity and Ecology, UMR CNRS
IRD 7263, Aix Marseille University - Campus de l'Etoile, 13397 Marseille Cedex 20, France
Royse Daniel J. Professor Emeritus, Department of Plant Pathology and Environmental
Microbiology, 115 Buckhout Laboratory, The Pennsylvania State University, University Park,
PA 16802 USA <[email protected]>
Salmones Dulce. Red de Manejo Biotecnológico de Recursos. Instituto de Ecología, A. C.
Carretera antigua a Coatepec 351, Xalapa, Ver., México. <[email protected]>
Sánchez José E. El Colegio de la Frontera Sur. Apdo. Postal 36. Tapachula, Chiapas, México.
Savoie Jean-Michel. INRA, Unité de Recherche 1264, MycSA, 71, Avenue Edouard Bourlaux,
CS20032, 33882 Villenave d’Ornon, Cedex, France
Tan Qi. Shanghai Academy of Agricultural Sciences. 1000 Jinqi Road, Shanghai 201403, China.
<[email protected]>
Téllez Téllez Maura. Laboratorio de Micología, Centro de Investigaciones Biológicas.
Universidad Autónoma del Estado de Morelos. Avenida Universidad 1001, Colonia Chamilpa,
Cuernavaca Morelos. México. C.P. 62209. <[email protected]>
Wang Zesheng. Institute of Edible Fungi, Fujian Academy of Agricultural Science, Fuzhou
350014, China
Xiao Donglai, Institute of Edible Fungi, National and Local Joint Engineering Research Center
Yang Chi, Institute of Edible Fungi, National and Local Joint Engineering Research Center for
Breeding & Cultivation of Featured Edible Fungi, Fujian Academy of Agricultural Sciences, No
10, Lane 95, Qianheng Road 10, Jin’an district, Fuzhou City, 350014, P.R.China
Ying Zhenghe, Institute of Edible Fungi, National and Local Joint Engineering Research Center
Yu Meilian Academy of Agricultural Sciences. 1000 Jinqi Road, Shanghai 201403, China
Yu Xinmin. Edible-fungi Industry Adiminstration Gutian, Standardization Working Group on
Tremella of Standardization Administration of China, Ningde, Fujian, 352200, China
Zamilpa-Álvarez Alejandro. Centro de Investigación Biomédica del Sur, IMSS. Xochitepec,
Mexico
CONTENTS
Prologue 13
Introduction
1 IMPORTANCE AND POTENTIAL OF TROPICAL MUSHROOMS 17
Daniel J. Royse
Biodiversity and Taxonomy

2 TROPICAL SPECIES OF Agaricus Philippe Callac, Jie Chen 25
3 AN OVERVIEW OF TROPICAL AND SUBTROPICAL SPECIES OF 39
Agaricus IN MEXICO Rosario Medel, Naara Palestina, Gerardo Mata, Luis A.
Parra Sánchez
Cultivation
4 DEVELOPMENT OF THE STRAW MUSHROOM SECTOR IN 51
CHINA Qi Tan, Mingjie Chen, Meilian Yu, Huanqing He
5 ENHANCED PRODUCTION OF THE MEDICINAL MUSHROOM 59
Agaricus subrufescens PECK Diego Cunha Zied, Arturo Pardo-Giménez
6 ARTIFICIAL CULTIVATION OF THE MEDICINAL MUSHROOM 71
Sparassis latifolia Yanquan Lin, Lu Ma, Donglai Xiao, Chi Yang, Zhenghe
Ying, Xiaoling Jiang
7 EFFECT OF SOAK TIME AFTER HARVEST ON NUTRITIONAL 87
QUALITY OF Tremella fuciformis DRY PRODUCT Lu Zhenghui, Ke
Binrong, Peng Dongxiang, Chen Yong-hui, Yu Xinmin, Wang Zesheng
8 FRUIT BODY PRODUCTION OF Schizophyllum commune Silvia 95
Cappello García, Santa Dolores Carreño Ruiz, Rigoberto Gaitán
Hernández
9 BASIDIOME PRODUCTION OF GUATEMALAN STRAINS OF 105
Lepista nuda María del Carmen Bran, Osberth Morales, Roberto Cáceres,
Natalia Gurriarán, Ferdiner González
10 SHIITAKE CULTIVATION ON STRAW: AN ALTERNATIVE FOR 115
SUBTROPICAL REGIONS Gerardo Mata, Jean-Michel Savoie
Biotechnological applications
11 Auricularia spp.: EDIBLE MUSHROOM WITH BROAD 137
BIOTECHNOLOGICAL PROPERTIES José E Sánchez, René H.
Andrade, Lilia Moreno
12 ANTIOXIDANT ACTIVITY AND CHEMICAL COMPOSITION OF 151
Grifola frondosa. Ma de Lourdes Acosta-Urdapilleta, Gerardo Díaz-
Godínez, Maura Téllez Téllez
13 IMMUNOMODULATING AND ANTITUMOR PROPERTIES OF 159
Pleurotus sp. IN CUBA Humberto J. Morris, Gabriel Llauradó, Yaixa
Beltrán, Yamila Lebeque, Pedro L. Batista, Serge Moukha, Isabelle
Perraud-Gaime, Nora García, Rosa C. Bermúdez, Maíra Bidart, Paul Cos,
Edgar Hernández, José C. Diez
14 CULTIVATION BIOTECHNOLOGY FOR Volvariella spp. IN 181
MEXICO: ADVANCES, CHALLENGES AND PERSPECTIVES
Dulce Salmones
15 A Pleurotus spp. HYDROALCOHOLIC FRACTION POSSESS A 193
POTENT in vitro OVICIDAL ACTIVITY AGAINST THE SHEEP
PARASITIC NEMATODE Haemonchus contortus Edgar J. Cuevas-
Padilla, Liliana Aguilar-Marcelino, José E. Sánchez, Manasés González-
Cortázar, Alejandro Zamilpa-Álvarez, Magaly Huicochea-Medina, Ma.
Eugenia López-Arellano, Pedro Mendoza-de-Gives Pedro, Víctor M.
Hernández-Velázquez
Perspectives 215
Appendix 217
1 Mushrooms mentioned in this book
2 Tropical species of Agaricus. Philippe Callac and Jie Chen
PROLOGUE
This book contains a selection of work presented at the International Symposium on Tropical
Mushrooms, organized by El Colegio de la Frontera Sur and Instituto de Ecología, in Tapachula,
Chiapas, during 1-2 December 2016.
The Tropics is a very large area of the planet Earth with abundant and surprising biodiversity;
however, it remains poorly understood. One group of organisms present in the Tropics, that have
been little studied are Macromycetes. It is this lack of information, the desire to highlight them,
and to show that these organisms should and deserve to be further studied, what led to the
holding of this event and later, to this book.
The aim of the symposium was to bring together specialists in the field and to draw attention to
some of the research on tropical macromycetes made today. Furthermore, we sought to help
motivate researchers, students, professionals and other individuals involved, to increase interest,
and to redouble efforts to confront the enormous -Herculean- task of research, knowledge and
study remaining to be done.
From this point of view, the symposium was a great success, because researchers from around the
world assembled and presented their findings and experiences on the topic. Our level of
knowledge for several mushrooms was documented and also it was clearly appreciated that more
research needs to be done. Macromycetes are generally organisms with great potential for
humanity as food, medicine and for biotechnological applications. This has been amply
demonstrated by the few examples worldwide already studied and exploited today. Certainly,
there is in tropical macromycetes an additional interest in conducting research in a poorly
explored field that is necessary for better development and benefit of mankind.
This book brings together a selection of the work presented at the symposium as a reliable record
of what was discussed there. It is intended not only for the interest of those involved, but above
all, for those who could not join us for the event.
We are especially grateful for the contributions of our collaborators for their willingness to share
their knowledge and experience to complete this document. We also give special recognition and
appreciation to all reviewers of the different chapters. Their disinterested and timely
collaboration, their comments and suggestions were a very important and enriching input that led
to the observed academic level. Last but not least, we would like to thank all persons who
contributed to making this publication a reality, namely Laura López, Carla Quiroga, Margarita I.
Hernández, Ana M. Galindo, Fabiola Roque, Graciela Ocampo and René H. Andrade.
The editors
INTRODUCTION
Updates on Tropical Mushrooms. Basic and Applied Research. Sánchez JE, Mata G, Royse DJ. (eds)
1. IMPORTANCE AND POTENTIAL OF TROPICAL MUSHROOMS
Daniel J. Royse
Professor Emeritus, Department of Plant Pathology and Environmental Microbiology, 115 Buckhout
Laboratory, The Pennsylvania State University, University Park, PA 16802 USA, Email: [email protected]
ABSTRACT
The region between the Tropic of Cancer in the Northern Hemisphere (23.4 N) and the Tropic of Capricorn in the
Southern Hemisphere (23.4 S) is defined as the tropics. The tropics encompass the largest range of climates and
habitats on earth. Most of the ecological conditions present in the world can be found at climatic convergences of
altitude and latitude in the tropics. Thus, significant areas of biodiversity are found in rainforests, dry deciduous
forests, spiny forests, desert and other habitat types. Fungi have significant functions in ecosystems and are found in
all kinds of environments. The many ecological and functional attributes of fungi are reflected in their great variety
of forms and physiological and biochemical properties. Recent estimates of the number of fungi that exist on the
earth are as many as 5.1 million species. It is estimated that fungi outnumber plants by at least 6 to 1. The
significance of fungi for mankind has a long history. Fungi have been exploited in fermentation processes for
thousands of years and mushrooms are known to possess pharmaceutical properties that are of major benefit to
human health. It is thought that medicinal mushrooms and fungi produce over 125 medicinal functions. In the
future, it is expected that additional medicinal substances and drugs may be found in, as yet, unidentified tropical
mushrooms. Finally, an increasing number of edible and medicinal mushrooms of tropical origin are being
commercially cultivated for food, dietary supplements and for medicine.
Keywords: edible mushrooms, macromycetes, mycology, ethnomycology, biodiversity.
TROPICS DEFINED
The tropics are defined as the region between the Tropic of Cancer in the Northern Hemisphere (23.4 N)
and the Tropic of Capricorn in the Southern Hemisphere (23.4 S). The tropics cover an immense area and
encompass the largest range of climates and habitats on Earth (J.M. Moncalvo 1997).
CLIMATE AND CLIMATE SYSTEMS
Climate has been defined as the statistics of weather over a 30-year interval. Several systems have been
developed over the years to classify climates into similar regimes. One of the most popular systems is the
Koppen classification that divides climates into five primary types labeled A through E as follows: A =
tropical, B = dry, C = mild mid-latitude, D = cold mid-latitude, and E = polar. These five primary types
can be further divided into secondary types such as rainforest, monsoon, tropical savanna, humid
subtropical, etc. The Koppen Classification System recognizes a total of 29 climate types.
17
ECOLOGICAL CONDITIONS AND CLIMATE
Most of the ecological conditions present in the world can be found at climatic convergences of altitude
and latitude in the tropics. These convergences have substantial ecological impacts on the types and
populations of organisms found there. Thus, significant areas of biodiversity are found in rainforests, dry
deciduous forests, spiny forests, desert and other habitat types.
BIODIVERSITY
Biodiversity encompasses species diversity, genetic diversity and ecosystem diversity. Fungi have
significant functions in ecosystems and are found in all kinds of environments. The many ecological and
functional attributes of fungi are reflected in their great variety of forms and physiological and
biochemical properties. It is estimated that fungi outnumber plants by at least 6 to 1 (Blackwell 2011).
Recent estimates of the number of fungi that exist on the earth are as many as 5.1 million species
(Blackwell 2011). Only about 2 to 3% of these species are known. The genera of wild edible fungi found
in tropical and subtropical climates are broadly similar to those found in the mycota of temperate regions
(Lincoff, 2010). However, species diversity is much greater in tropical regions compared to temperate
regions.
As rainforests in the tropics continue to disappear (currently at the rate of about 2,400 hectares per h)
biodiversity is reduced due to extinctions. Without sufficient biodiversity on earth, survival of humankind
is threatened. As the human population continues to increase, enormous pressure is placed on the planet
and many species are heading toward extinction. Habitat destruction, the spread of invasive species,
pollution and climate change are major threats to human survival.
As a way to help prevent extinction of species, the International Union for the Conservation of Nature and
Natural Resources (IUCN) was established in 1948 and is now working in over 160 countries. This
organization has had several successful interventions for reversing habitat loss and restoring ecosystems
and is vital for protecting the natural resources we need to survive. IUCN’s species Red List is a critical
indicator of the health of the world’s biodiversity. Assessments for the Red List of vertebrates,
invertebrates, plants and fungi are shown in Table 1. A total of about 80,000 species have been assessed
(as of 2015) but this is only half of the goal of 160,000 species by 2020. Currently, fungi are highly
underrepresented (only 34 out of a goal of 14,500 have been assessed) so much more work is needed in
this area. Only one cultivated species (Pleurotus nebrodensis) that is found in the wild in Sicily, Greece
and China (Zervakis et al. 2014) is considered critically threatened.
Table 1. Goal and current number of species assessed for the Red List worldwide by the International
Union for the Conservation of Nature and Natural Resources (IUCN) for vertebrates, invertebrates, plants
and fungi (2015).
Category Goal Species Assessed
Vertebrates 61,635 41,517
Invertebrates 45,344 17,516
Plants 38,521 20,755
Fungi 14,500 34
18
HEALTH OF FORESTS
Tropical rainforests provide over 40% of the world’s oxygen supply. Rainfall is critical to the health of
plants, animals and fungi of the rainforest. It is generally considered that tropical mushrooms may help
bring rainfall to forests. Each mushroom may release up to 30,000 spores/sec that can be carried in air
currents as high as 85 km in altitude. Millions of tons of spores are released each year that may act as
nuclei for the formation of raindrops in clouds. This may promote rainfall in places like the tropics that
have relatively high populations of mushrooms and other fungi. Thus, spores are considered vital to
productivity of tropical forests (Milliken 2015).
Mycorrhizal plants are also vitally important for the health of rainforests. Soils in over two-thirds of the
world’s rainforests are acidic and very low in minerals and nutrients. Fortunately, uptake of these scarce
nutrients and minerals is facilitated by a unique relationship between the roots of vascular plants and
fungi, i.e., mycorrhizae. Plants benefit from this relationship through the availability of additional water,
minerals and nutrients supplied by the fungi while fungi benefit by additional access to carbohydrates,
vital for mycelial growth, provided by the plant’s roots.
ETHNOMYCOLOGY - MUSHROOMS AND HUMANKIND
Wild Mushrooms
Ethnomycology is the study of people and fungi. Wild mushrooms have been gathered for food and
income for many decades in various counties. During the last 30 to 40 years, research has substantially
increased our knowledge of local traditions in Africa, Asia and Mesoamerica (Mexico and Guatemala)
(Boa 2004). These studies have made a distinction between mycophilia and mycophobia. In mycophillic
societies, fungi are esteemed and there is a long tradition of popular use. Mycophobic cultures have a
minor regard for mushrooms and they are often actively feared (Boa 2004). China, Japan and the Koreas
are examples of mycophyllic societies while India, Pakistan and other former British colonies such as
Australia and the U.S.A. are usually considered mycophobic. Traditions vary within countries, too. For
example, Hispanic Mexico is generally considered mycophobic while native peoples in Mexico are
considered mycophilic (Lincoff 2010). Another example is Africa, where most of the continent is
mycophobic with some scattered peoples in West and East Africa considered mycophilic. Variable
traditions also exist in Tanzania (Boa 2004).
Wild mushrooms are collected and used as food and to generate income in over 80 countries. There is a
relatively large diversity of different types (1,000-plus species) recorded for these purposes. Some of the
wild mushrooms are exported but most are used for subsistence. In 2013, wild mushrooms represented
about 8% of the component value of the world mushroom industry (Fig 1) (Royse et al. 2017).
19
Edible (54%)
Medicinal (38%)
Wild (8%)
Total = $63 billion
Figure 1. Component value of the world mushroom industry in 2013 showing wild mushrooms
accounting for approximately 8% or $5.04 billion of the total value ($63 billion).
Medicinal mushrooms
Medicinal mushrooms are routinely used in traditional Chinese medicine and wild medicinal mushrooms
are also used in Mexico and many other countries. It is thought that medicinal mushrooms and fungi
produce over 125 medicinal functions (Chang and Wasser 2012). These include antitumor,
immunomodulating, antioxidant, radical scavenging, cardiovascular, anti-hypercholesterolemia, antiviral,
antibacterial, antiparasitic antifungal, hepatoprotective, detoxification and anti-diabetic. Many, if not all
basidiomycetes contain biologically active polysaccharides in fruitbodies and cultured mycelium and
broth (Chang and Wasser 2012). Component value of medicinal mushrooms for the world in 2013 was
estimated at $23.9 billion or about 38% of the total value (Fig. 1) (Royse et al. 2017).
One medicinal component of mushrooms that has received considerable attention in recent years is
ergothioneine (ERGO) (Dubost et al. 2006). This compound functions as an intra-mitochondrial
antioxidant that acts to interrupt the vicious cycle of oxidative stress, cell injury and inflammation. ERGO
is made in relatively few organisms with fungi as one of the main sources (Kalaras et al. 2017). Humans
acquire ERGO through diet and it accumulates in erythrocytes, bone marrow, liver, kidneys, seminal fluid,
and the eyes. Humans have a specific transporter gene (OCTN1) so it is thought that ERGO plays an
important role as a “cytoprotectant” in human health (Grundemann 2012). Some scientists have proposed
that ERGO be elevated to vitamin status (Paul and Synder 2010).
It is estimated that nearly 90% of human diseases known to medical science can be treated with
prescription drugs derived from nature (Torrence 2013). In the future, it is expected that additional
medicinal substances and drugs may be found in, as yet, unidentified tropical mushrooms and in those that
20
are already known. The potential of these fungi for improving human health is enormous and their
potential impact is incalculable.
MUSROOM SAFETY
Some mushrooms, harvested from the wild, are known to be poisonous. Fungal poisoning is a relatively
rare event and is related to local habits, economic factors and lifestyle. Among thousands of known
mushroom species, fewer than a hundred are toxic. For example, Amanita phalloides and several other
members of the genus Amanita, as well as Conocybe, Galerina and Lepiota contain amatoxins that can
cause severe gastroenteritis and hepatic necrosis. Ingestion of Psilocybe semilanceata (liberty cap),
containing a potent halluciogen, may cause anxiety, panic reactions, and peripheral sympathomemetic
symptoms (Fenney et al. 2014). Another toxin, contained in Cortinarius speciosissimus, may cause
permanent kidney failure. Most mushroom poisonings occur due to ingestion of a toxic mushroom
misidentified as an edible one. It is always best to have the wild mushroom inspected by a certified expert
or mycologist prior to consumption (Schenk-Jaeger et al. 2012).
SUMMARY
The tropics cover an immense area and encompass the largest range of climates and habitats on earth.
Fungi outnumber plants by at least 6 to 1 and a very small percentage of the 5.1 million species of fungi
on the earth are probably known. Tropical mushrooms are invaluable today – serving as a source of food
and income and helping to maintain the health of forests and of humans. It is expected they will be even
more valuable to humankind in the future.
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Chang ST, Wasser SP (2012). The role of culinary-medicinal mushrooms on human welfare with a pyramid
model for human health. Int. J. Med. Mush. 14(2):95-134.
Dubost NJ, Beelman R, Peterson D, Royse DJ (2006). Identification and quantification of ergothioneine in
cultivated mushrooms by liquid chromatography-mass spectroscopy. Int. J. Med. Mush. 8:215–22.
Feeney MJ et al. 2014. Mushrooms and health summit proceedings. J. Nutrition 144:1128S-1136S.
Grundemann D (2012) The ergothioneine transporter controls and indicates ergothioneine activity—a
review. Prev. Med. 54:S71–4.
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ergothioneine and glutathione. Food Chemistry 233:429-433.
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Mushrooms. Quarry Books, Beverly, MA.
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2016).
Moncalvo JM (1997) Evaluation of fungal biological diversity in the tropics: systematics perspectives. Pages 1-
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Paul BD, Snyder SH (2010) The unusual amino acid L-ergothioneine is a physiologic cytoprotectant. Cell Death
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Edible and Medicinal Mushrooms: Technology and Applications, John Wiley & Sons, NY.
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poisoning: A study on circumstances of exposure and patterns of toxicity. European J. Internal Med.
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Torrence PF (2013) Owed to nature: medicines from tropical forests.
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Biol. 118:814-834.
22
Biodiversity and Taxonomy
Updates on Tropical Mushrooms. Basic and Applied Res earch. Sánchez JE, Mata G, Royse DJ. (eds)
2. TROPICAL SPECIES OF AGARICUS
Philippe Callac 1 and Jie Chen2

1
INRA, MycSA (Mycologie et sécurité des aliments), CS20032, 33882, Villenave d’Ornon, France.
([email protected])
2
Instituto de Ecología, A.C., CP 91070, Xalapa, Veracruz, México
ABSTRACT
Agaricus is a large genus of mushrooms including species of nutritional and medicinal interest. With research
programs developed over the last decade, a larger diversity of Agaricus came to light. The present review focuses on
tropical species of Agaricus and their classification, including a discussion of the definition of tropical species, a
brief h istory of the taxonomic system of classification since 2000, an updated presentation of the revised system
including its recent emendation, and the distribution of tropical species in the subgenera and sections of the current
system. Tropical species of interest and methods to identify tropical species of Agaricus are also introduced. The new
classification system should facilitate studies of tropical Agaricus; however, mo re investigations in unexp lored areas
are necessary to complete the classification system and to clarify the evolutionary history of the genus in which
climate and geography seem to have been the main factors of diversificat ion.
Keywords: classification, climate, fungal diversity, subgenus, systematics, taxonomy
THE GENUS AGARICUS
The genus Agaricus (Agaricales, Basidiomycota) is characterized by a stipe separable from the pileus
(heterogeneous context) provided with one or several annuli and free lamellae that produce brown
basidiospores. Its distribution range extends to all continents except Antarctica (Parra 2008, Zhao et al.
2011). Species of Agaricus are saprobic and grow in forest, grassland, dune, or any place with decaying
organic matter (Karunarathna et al. 2016). Zhao et al. (2011) counted 386 recognized species including
183 that were tropical. With 170 new species described from 2011 to September 2018, the number of
species recognized today exceeds 500 (Chen et al. 2017, Karunarathna et al. 2016, Kerrigan 2016). In
addition, many putative new species have not yet been named and species diversity remains poorly known
in many regions. Indeed, 185 new species that were proposed and included in phylogenetic analyses since
2000 (see list of species in Appendix 2, at the end of the book) are heterogeneously distributed as follows:
--- 55% (102) were described from Asia, mostly from China and Thailand (Ariyawansa et al. 2015, Chen
et al. 2012, 2015, 2017, Dai et al. 2016, Gui et al. 2015, He & Zhao 2015, He et al. 2017, 2018a,b, Hyde
et al. 2017, Karunarathna et al. 2014, Li et al. 2014, 2016, Liu et al. 2015, Thongklang et al. 2014a, 2016,
Wang et al. 2015, Zhang et al. 2017, Zhao et al. 2012a, b, 2016, Zhou et al. 2016), and some from India,
Iran, and Pakistan (Bashir et al. 2018, Chen et al. 2016c, Kaur et al. 2016, Mahdizadeh et al. 2018).
--- 25% (47) were from the Americas, mainly from North America (Callac & Mata 2004, Kerrigan 2016,
Kerrigan et al. 2008), and some from the Caribbean and South America (Albertó et al. 2000, de Meijer
2008, Drewinski et al. 2017, Parra et al. 2018).
--- 14% (26) were from Europe (Blanco-Dios 2001, Callac & Guinberteau 2005, Kerrigan et al. 2008,
Lanconelli 2002, Mahdizadeh et al. 2018, Mua et al. 2017, Parra 2013, Parra & Arrilaga 2002, Parra &
Caballero 2017, Parra et al. 2011, 2014, 2015),
--- 5% (9) were from Oceania (Geml et al. 2007, Lebel 2013, Lebel & Syme 2012),
--- 1% (1) was from Africa (Hama et al. 2010).
25
We excluded A. dilutibrunneus R.L. Zhao (Zhao et al. 2016) from this count because it is a doubtful
species of which the sequences of different genes seem mutually incompatible (Mahdizadeh et al. 2018).
Among the 185 new species proposed since 2000, A. heinemannii Albertó & G. Moreno (Albertó et al.
2000), A. pachydermus T. Lebel (Lebel & Syme 2012), A. patialensis M. Kaur & Harw. Kaur (Kaur et al.
2016), and A. stijvei de Meijer (Meijer 2008) are the only species for which sequence data are not
available.
Species of Agaricus have both nutritional and medicinal interest. Certain edible species are tasty and
frequently collected in the wild for consumption, such as A. augustus Fr. and A. campestris L. : Fr., but the
latter has never been successfully domesticated (Grigson 1975). In contrast, A. bisporus (J.E. Lange)
Imbach (the button mushroom) is not encountered frequently in the wild but has been domesticated and
remains the species that is the most cultivated worldwide (Chang 1999, Savoie et al. 2013). Some other
species are cultivated to a lesser extent, such as A. bitorquis (Quél.) Sacc. and A. arvensis Schaeff.
Agaricus subrufescens Peck (the almond mushroom) is cultivated primarily for medicinal use. Despite
their ecological, nutritional, medicinal, and economical potential interest, species of Agaricus remain little
documented in certain countries, particularly in the tropics.
Breeding work, developed mainly for A. bisporus, requires genetic resources and knowledge of its
reproductive biology and genetics. For this purpose, a large collection of germplasm is currently available
(Callac et al. 2002, Savoie et al. 2013); the three main types of life cycles (heterothallism,
pseudohomothallism, or homothallism) are present in this species (Callac et al. 2003, Savoie et al. 2013)
and complete genomes and genetic maps are available (Foulongne-Oriol et al. 2010, Morin et al. 2012). A
wide diversity of germplasm, life cycles, and a genetic map are also documented in A. subrufescens
(Foulongne-Oriol et al. 2016, Rocha de Brito et al. 2016, Thongklang et al. 2014b).
We focus, with the present review, on the tropical species of Agaricus and their classification. Despite
efforts of some mycologists regarding molecular delimitation of species, we are a long way from fully
clarifying the classification of Agaricus. The state-of-the-art of classification that we detail here most
likely will be considerably improved in the coming years. This new classification was required to
accommodate the numerous clades of tropical species evidenced in Zhao et al. (2011).
WHAT DOES IT MEAN TROPICAL SPECIES OF AGARICUS?
Our definition of tropical Agaricus is empirical and neither absolute nor definitive. Among several
hundred samples that were collected during ten years in north Thailand (including highlands), none of the
species known in Europe was detected except in two specific cases: (1) A. subrufescens, which is one of
the rare cosmopolitan species of Agaricus (Wisitrassameewong et al. 2012) and (2) A. endoxanthus Berk.
& Broome, which is a tropical species relatively frequently introduced in tropical greenhouses in
temperate areas (Chen et al. 2016c, Zhao et al. 2012a). Interestingly, 500 km to the north, in the Yunnan
Province of China, it is possible to find both ‘temperate species’ including certain known in Europe such
as A. abruptibulbus Peck and ‘tropical species’ known in north Thailand such as A. flocculosipes R.L.
Zhao, Desjardin, Guinb. & K.D. Hyde (Zhao et al. 2016). This boundary between south China and north
Thailand that temperate species cannot cross corresponds to the climatic boundary between humid
subtropical (Cfa or Cwa) and tropical (A) climate areas according to the Köppen-Geiger climate
classification (Peel et al. 2007). We have simply defined the tropical species as the species native from the
tropical climate regions.
Are such species exclusively tropical? It is generally the case but there are exceptions as A. endoxanthus
and A. subrufescens cited above. The latter species is cosmopolitan as, to a lesser extent, A. bisporus
26
reported from D. R. Congo (Heinemann 1956) and possibly A. bitorquis. There are also some tropical
species such as A. flocculosipes that extends to neighboring subtropical climatic areas. Possibly, some
species discovered first in subtropical regions could be in fact tropical species colonizing in subtropical
areas. In other respects, Agaricus species in relatively arid/hot climate (including hot Mediterranean or hot
temperate) are poorly known because the fruiting periods are often short and unpredictable.
Tropical and non-tropical species are generally grouped in distinct clades that diverged a long time ago
(Zhao et al. 2016). This suggests that adaptation to a different climate is not a frequent event. For
temperate species, fruiting at high temperature (i.e. 25°C or more) is not an optimal condition because the
hot season is not sufficiently rainy for optimal reproductive fitness. The rare cosmopolitan species of
Agaricus belong to clades that are not strictly tropical or temperate. Their ancestral condition is unknown.
Investigations of genetics of fruiting ability at high temperature in A. bisporus are in progress (Foulongne-
Oriol et al. 2014, Navarro et al. 2014). Current cultivars of this species are unable to fruit at 25°C.
However, fruiting tests revealed that the percentage of wild isolates able to fruit in cultivation at 25°C was
100% with a good yield for the population of A. bisporus var. burnettii Kerrigan & Callac distributed in
the Sonoran Desert of California. On the contrary, it varies, on average, from 35% to 78% with a lower
yield among different populations of A. bisporus var. bisporus from temperate regions of Europe and
North America (Largeteau et al. 2011). These data suggest that temperate populations of this species
retain an evolutionary potential to adapt to a hot climate. Unfortunately, it was not possible to study the
adaptation to tropical climate in absence of an isolate from the only known population in D. R. Congo.
However, we suspect that optimal fruiting temperature is not the single criterion for adaptation to tropical
or temperate climates.
We also use the terms of ‘tropical section’ or ‘tropical subgenus’ to indicate that most species belonging to
the corresponding clades are known from tropical climate regions and none are known from temperate
climate regions. We could not use a strict definition because there are always some species that can be
found in neighboring subtropical regions (as in the palaeotropical A. sect. Brunneopicti) or even hot
temperate climate (as in the neotropical A. subg. Minoriopsis), but never in typical temperate regions. In
other respects, the terms palaeotropical and neotropical are used in the strict sense at any taxonomic rank.
They indicate that all specimens of the taxon are exclusively known today from Africa or Asia, or from
the Americas, respectively. The geographical status of palaeo- or neotropical clades may change in the
future with reports of new specimens or species.
A BRIEF HISTORY OF THE CLASSIFICATION SINCE 2000
Until the year 2000, taxonomic classification did not reflect phylogeny of the species, mainly because few
morphological characters were available and their evolutionary histories were completely unknown. For
example, species were grouped in sections according to their discoloration (pink, red, yellow, or none)
when the mushroom is rubbed or cut. However, this criterion of flesh discoloration was overemphasized
because it was subsequently shown that unrelated sections (Arvenses and Xanthodermatei) are both
characterized by yellow discoloration. Morphology of the annulus, macrochemical reactions and
organoleptic characters seems more reliable but in quasi-absence of knowledge on their function and their
adaptive role, and more generally on the ecology and physiology of the species it remains very difficult to
apprehend their evolutionary history. However, before the development of molecular tools, some
mycologists began to emphasize other criteria such as the structure of the annulus and others suspected
that some secotioid genera should be included in Agaricus hypothesizing that their gasteroid appearance
would result from adaptation to dryness. Despite the attempts of mycologist Paul Heinemann to propose
new subgenera and sections for tropical species, many of them were classified in sections based on
temperate species.
27
From 2000 to 2010 classification continued to progress. Phylogenetic reconstruction of sections Bivelares
(including the tasty edible A. bisporus) and Xanthodermatei (including toxic species such as A.
xanthodermus) were performed based on rDNA-ITS sequences (Challen et al. 2003, Kerrigan et al. 2005,
2008). Analyses using ITS and LSU sequence data (Geml et al. 2004, Mitchell & Bresinsky 1999) also
indicated that these two sections were unexpectedly closely related to each other while they exhibit
different flesh discolorations (pink and yellow, respectively). At that time, eight temperate sections were
considered: Agaricus, Arvenses, Bivelares, Chitonioides, Minores, Sanguinolenti, Spissicaules and
Xanthodermatei. Although sections Xanthodermatei and Arvenses exhibit a yellow discoloration, they are
not phylogenetically related. Several secotioid species were included in the genus and 13 new species
were described. Structure of the annulus (superous vs. inferous; simple vs. double or two layered,
microscopic features), odor, and cross reaction of Schäffer (aniline × nitrogen acid) became major criteria
of classification.
Monographs using molecular data for the first time, were completed on European species (Parra 2008,
2013) and North American species (Kerrigan, 2016). Concomitantly, species from Asia (mainly Thailand
and China) were studied through a collaboration between European and Asian teams (mainly P. Callac at
INRA-MyscSA in France and K. D. Hyde at MFLU in Thailand). Following field work of R-L Zhao that
began in 2005 in Thailand, these studies were supported in 2010 by an Integration Research Grant from
the European Distributed Institute of Taxonomy (EDIT). They included European specimens of reference
and many tropical species, mainly from Thailand and to a lesser extent from Africa and the Caribbean
thanks to collaboration with the University of Lille, France (R. Courtecuisse, LIP Herbarium), the
National Botanic Garden of Belgium (O. Raspé and A. De Kesel, BR Herbarium) and L. A. Parra (private
herbaria LAPAG, LAPAM and LAPAF; Avda. Padre Claret n°7, 5°G, 09400 Aranda de Duero, Burgos,
Spain). From 2011 to September 2018, 170 new species were described, some subsections and sections
were proposed (Parra 2013, Kerrigan 2016), numerous tropical clades were delineated (Zhao et al. 2011),
a revised system of classification was proposed (Zhao et al. 2016) and emended (Chen et al. 2017, Parra et
al. 2018, He et al. 2018a). Although all these studies contributed more or less directly to the revision of
the classification, we will focus here on the first work that included tropical species in phylogenetic
analyses (Zhao et al. 2011) and on the studies supporting tropical subgenera or sections (Chen et al. 2015,
2017, Parra et al. 2018, Zhao et al. 2016, He et al. 2018a).
WHY WAS A REVISED CLASSIFICATION REQUIRED?
There were three main reasons for revising the classification. The first reason was the small number of
subdivisions of the genus. Indeed, eight sections were not sufficient for a number of species that already
exceeded 500 and will continue to increase. The second reason was that three of the traditional sections
(Sanguinolenti, Spissicaules, and Xanthodermatei) were polyphyletic and required splitting into several
sections:
--- three sections (Bohusia, Nigrobrunescentes, and Sanguinolenti) for A. sect. Sanguinolenti (Parra 2008,
Parra et al. 2014, 2015, Peterson et al. 2000, Zhao et al. 2011, 2016),
--- three sections (Rarolentes, Spissicaules, Subrutilescentes) for A. sect. Spissicaules (Kerrigan 2016,
Zhao et al. 2011, 2016)
--- two sections (Hondenses, Xanthodermatei) for A. sect. Xanthodermatei (Kerrigan 2016, Kerrigan et al.
2005, Thongklang et al. 2014a, Zhao et al. 2011, 2016)
With these five new sections, the number of sections increased from eight to 13 for species distributed in
temperate climate regions.
The third reason for revising the classification was to accommodate unclassified tropical clades described
by Zhao et al. (2011). In this study, there were seven strongly supported tropical clades (TRI to TRVII)
28
and four lesser supported tropical clades (TRa to TRd). In the revised system of classification proposed by
Zhao et al. (2016) and subsequently emended (Chen et al. 2017, Parra et al. 2018, He et al. 2018a), one
tropical subgenus and 11 tropical sections were retained. Correspondence between the clades observed by
Zhao et al. (2011) in their phylogenetic tree, based only on ITS sequence data and the 6 subgenera and 24
sections of the revised system based on multi-gene phylogenetic analyses with dated trees (Zhao et al.
2016, Chen et al. 2017, He et al. 2018a) and taxonomic emendations (Parra et al. 2018), are shown in
Table 1.
Table 1. Distribution of tropical species in the current revised system of classification *.

Revised system Major clades
Study Subgenus Section (Zhao et al. 2011)
Zhao et al. Flocculenti Closely related to TRa
2016 Brunneopicti TRI
Trisulphurati TRb (in part)
He et al. Crassispori TRb (in part)
2018a Cymbiformes TRb (in part)
Rubricosi TRc
Bivelares Bivelares
Pseudochitonia Chitonioides Chitonioides
Xanthodermatei Xanthodermatei (in part)
Hondenses Xanthodermatei (in part)
Sanguinolenti Sanguinolenti I
Bohusia Sanguinolenti II
Nigrobrunnescentes Sanguinolenti III
Agaricus Agaricus Agaricus
Amoeni TRIII
Spissicaules Rarolentes Spissicaules (in part)
Spissicaules Spissicaules (in part)
Subrutilescentes Spissicaules (in part)
Flavoagaricus Arvenses Arvenses
Chen et al. Minoriopsis Minoriopsis TRd (in part)
2017 Kerrigania TRII
Parra et al. Leucocarpi
2018 Minores Pantropicales
Minores TRV, TRVI, TRVII, Minores
Unclassified TRIV (A. deserticola)
*Sections are indicated in red, bold red or highlighted when the corresponding clade includes a
proportion of tropical species reaching 10-20%, reaching or exceed ing 40-50%, or of 100%,
respectively. High lighting in green, blue, or yellow, ind icates that the clade exclusively includes to our
knowledge either palaeotropical, neotropical species, or both palaeo - and neotropical species,
respectively.
29
Figure 1. a. Agaricus niveogranulatus, LD2012148 (A. sect. Brunneopicti), b. Agaricus suthepensis,

LD2012100 (A. sect. Amoeni), c. Agaricus trisulphuratus complex, LD2012176 (A. sect. Trisulphurati), d.
Agaricus variicystis, LD201228 (A. sect. Crassispori).
DISTRIBUTION OF TROPICAL TAXA IN THE REVISED SYSTEM OF CLASSIFICATION
Zhao et al. (2016) proposed a revision of the taxonomic system of classification in the genus Agaricus
considering stem age as a criterion for standardizing taxonomic ranks (Avise & Johns 1999). In their
Maximum Clade Credibility tree of Agaricus based on ITS, LSU, tef1α, and rpb2 genes, strongly
supported clades that diverged about 30-33 million years ago (Ma) or about 18-26 Ma were ranked as
subgenera or sections, respectively. Zhao et al. (2016) proposed five subgenera, then a sixth subgenus was
added by Chen et al. (2017). After a taxonomic emendation (Parra et al. 2018) and a recent contribution
(He et al. 2018a), the current system includes 24 sections (Table 1; note that the species listed in
Appendix 2 are grouped by section).
30
Eleven of the 24 sections are tropical and their history is briefly presented below with species illustrated in
Figs. 1 and 2 for eight of them.
--- Two sections (Brunneopicti and Trisulphurati) have been previously proposed by Heinemann (1956).
However, the current concept of A. sect. Brunneopicti, that corresponds to clade TRI, is much broader
than its original circumscription (Chen et al. 2015, Karunarathna et al. 2014, Zhao et al. 2016). This
section is illustrated in Fig. 1a with A. niveogranulatus Linda J. Chen, R.L. Zhao, Callac & K.D. Hyde
showing the pileus and stipe base covered with granulose or punctiform squamules. In A. sect.
Trisulphurati, sporocarps of the species of the A. trisulphuratus Berk. complex exhibit an unusual orange
color (Fig. 1c). These two sections are palaeotropical and belong to A. subg. Pseudochitonia.
--- Four sections (Amoeni, Crassispori, Flocculenti, and Rubricosi) were recently proposed by Zhao et al.
(2016). Agaricus sect. Amoeni is a pantropical section in A. subg. Spissicaules. Agaricus suthepensis
Linda J. Chen, K.D. Hyde & R.L. Zhao, illustrated in Fig. 1b, should be sequenced for a reliable
identification. The three remaining sections are in A. subg. Pseudochitonia. Agaricus sect. Rubricosi,
which is pantropical, is illustrated (Fig. 2c) with a specimen from Martinique of the species A. fiardianus
Callac & L.A. Parra (≡ A. magnivelaris Pegler; Mahdizadeh et al. 2018). This species is better
characterized by its narrow spores than by a large annulus according the notes of J.-P. Fiard. The two
remaining sections (Crassispori, and Flocculenti) are palaeotropical and are illustrated by A. variicystis
Linda J. Chen, K.D. Hyde & R.L. Zhao (Fig. 1d) characterized by lageniform cheilocystidia, and A.
erectosquamosus Linda J. Chen, K.D. Hyde & R.L. Zhao (Fig. 2a) characterized by erect, dark brown
squamules on the pileus, respectively.
--- Two sections (Leucocarpi and Pantropicales) belong to A. subg. Minores. Agaricus sect. Leucocarpi
was recently proposed by Chen et al. (2017) for a single Asian species, A. leucocarpus Linda J. Chen,
Callac, R.L. Zhao & K.D. Hyde (Fig. 2b). This species exhibits a Schäffer positive reaction (Parra et al.
2018) contrarily to what was indicated in its original description. In a recent study, at least one unnamed
species from the Caribbean clusters with A. leucocarpus in A. sect. Leucocarpi (Ortiz-Santana et al. 2018),
which therefore is a pantropical section. Agaricus sect. Pantropicales, which appeared as ‘section 1’ in the
phylogenetic study in Chen et al. (2017), was proposed by Parra et al. (2018) and also includes species
described from Asia and the Caribbean.
--- Two sections (Minoriopsis and Kerrigania) were recently proposed by Parra et al. (2018). In fact, these
two neotropical sections already appeared in Chen et al. (2017) as two sister clades representing putative
sections in A. subg. Minoriopsis. Agaricus subg. Minoriopsis was previously considered by Zhao et al.
(2016) as being Agaricus sect. Laeticolores because it includes species such as A. rufoaurantiacus
Heinem. that Heinemann (1961) placed in this section. However, the ITS sequence of the type specimen
of A. laeticulus Callac, L.A. Parra, Linda J. Chen & Raspé (≡ A. laeticolor Heinem. & Gooss.-Font.),
which is the type species of A. sect. Laeticolores, was recently obtained and phylogenetic analyses
revealed that it does not belong to this clade but to A. sect. Minores. In addition, Chen et al. (2017)
estimated the stem age of this clade at 31.02 Ma. Therefore, Chen et al. (2017) proposed A. subg.
Minoriopsis and designated A. martinicensis Pegler (Fig. 2d) as the type species of this new neotropical
subgenus.
--- The remaining section (Cymbiformes) was recently proposed by He et al. 2018a for a single species
from Thailand, A. angusticystidiatus M.Q. He, Desjardin, K.D. Hyde & R.L. Zhao. This section is
palaeotropical like its two closely related sections (Trisulphurati and Crassispori).
The 13 remaining sections include temperate species. In these sections, tropical species are unevenly
distributed and often grouped into distinct subclades. According to our counts based on current available
data, the proportion of tropical species is greater than 50% in A. sect. Minores and A. sect. Rarolentes,
achieving around 40% in A. sect. Xanthodermatei, lower than 20% in two subgenera and three sections (A.
31
subg. Agaricus, A. subg. Flavoagaricus, A. sect. Nigrobrunnescentes, A. sect. Spissicaules, A. sect.

Subrutilescentes), and close to zero in the five remaining sections.
The clade TRIV, that includes A. deserticola G. Moreno, Esqueda & Lizárraga (Zhao et al., 2011) and a
few species, such as A. martineziensis Heinem. were not included in the recent revision of the system.
Therefore, the classification of these species is presently not resolved as this is also the case for a few
incertae sedis in Zhao et al (2016).
Fig. 2. a. Agaricus erectosquamosus, LD2012165 (A. sect. Flocculenti), b. Agaricus leucocarpus, SCK089
(A. sect. Leucocarpi), c. Agaricus fiardianus, F2389 (A. sect. Rubricosi), d. Agaricus martinicensis,
LAPAM43 (A. sect. Minoriopsis). Photo credit: b. Samantha C. Karunarathna, c. Jean-Pierre Fiard (LIP
herbarium), d. Claudio Angelini.
SPECIES OF INTEREST
Agaricus sect. Arvenses is now ranked as A. subg. Flavoagaricus. Certain species of this subgenus such as
A. subrufescens, A. arvensis, and A. fissuratus are cultivated and are of nutritional or medicinal interest.
The three species collected in Thailand (A. subrufescens, A. flocculosipes, and A. subtilipes Thongklang,
Linda J. Chen, Callac & K.D. Hyde) fruited on standard compost substrate (Thongklang et al. 2014b,
2016, Zhao et al. 2012b). Cultivated sporocarps of Agaricus flocculosipes (Fig. 3) were tasty, while A.
subtilipes was not consumed and thus, its edibility remains unknown. The life cycle of a specimen of A.
subrufescens was studied and hybridizations with French and Brazilian specimens were performed
32
(Thongklang et al. 2014b). Parental and hybrid strains were subsequently analyzed for their production of
blazeispirol A, a spiro-triterpenoid conferring a cholesterol-lowering activity to the mycelium of A.
subrufescens. Two other species, that are in the same ‘tropical’ clade as A. flocculosipes and A. subtilipes
within A. subg. Flavoagaricus, have not been formally described but are potentially interesting since their
samples were collected from marketplaces in Africa by Thoen (Gui et al. 2015, Thongklang et al. 2016).
Two species of A. sect. Brunneopicti, A. bingensis Heinem. and A. subsaharianus L.A. Parra, Hama & De
Kesel, are also consumed by local people according to Pegler (1977) and Hama et al. (2010), respectively.
Fig. 3. First cultivation of Agaricus flocculosipes, isolate CA917, 20 November 2011 at INRA-MycSA
(France) with PhD students of the Center of Excellence in Fungal Research, Mae Fah Luang University
(MFLU, Thailand). a. Culture trays with, from left to right, Guinberteau J., Coldefy C., Thongk lang N.
(MFLU), Callac P., Chen J. (MFLU), Sysouphanthong P. (MFLU), b. and c. sporocarps.
HOW TO IDENTIFY TROPICAL AGARICUS?
As for any specimen of Agaricus, during fieldwork, it is necessary to note the odor, the discoloration of
the sporocarp when rubbed or longitudinally cut, the structure and ornamentation of the annulus, and to
test the Schäffer reaction on fresh tissue or as soon as possible. For tropical species, a clear and strongly
positive Schäffer reaction indicates with high confidence that the specimens belong to A. subg. Minores,
A. subg. Minoriopsis or A. subg. Flavoagaricus. The distinction between the tropical sections as well as
diversity within these sections is based on recent data and it is still difficult to identify many species
33
without ITS sequence data even in the A. sect. Brunneopicti that was recently reconstructed (Chen et al.
2015). ITS sequences are generally sufficient to identify a species when a highly similar sequence of a
specimen of reference is available in GenBank. Generally, ‘highly similar’ means that there is no more
than one difference between the sequences. However, we do not count heteromorphisms as true
differences (for example one sample has two peaks, C and T, at a given position of the chromatogram and
the reference sample has only T at this position) which might reflect intraspecific allelic polymorphism.
The best specimen of reference is of course a type, if possible the holotype. For most species described
since 2000, the ITS sequence of their holotype is available in GenBank; those species newly described
between 2000 and June 2015 are listed in Callac (2015). For species proposed before 2000, it is necessary
to sequence herbarium specimens and this has already been completed for some tropical species. Species
of Agaricus from tropical and humid subtropical regions of Asia were recently listed (Karunarathna et al.
2016). There are some exceptions to the rule above. More than one difference may be observed between
ITS sequences of different collections within some species. Two differences are observed in some variable
species or even more in cosmopolitan species such as A. subrufescens (Chen et al. 2016a, 2016b) and A.
endoxanthus (Chen et al. 2016c). In contrast, two species that diverged recently from each other could
share identical ITS sequences as this is the case for A. gemellatus Kerrigan, L.A. Parra, Cappelli & Weholt
and the secotioid species A. inapertus Vellinga (Kerrigan 2016). Such examples indicate that
morphological descriptions remain useful. In addition, a biological circumscription of the species of
interest remains crucial to characterize available germplasm and develop breeding programs.
CONCLUSIONS
The number of species, sections and subgenera has recently increased and, in the new system of
classification, tropical species are placed more accurately. Despite recent advances in taxonomy and
phylogeny, enormous taxonomic work remains. Species diversity in Africa, South America, and Australia
remains poorly known. More investigations in all these continents may allow clarification of the
evolutionary history of this genus in which climate and geography seem to have been the main factors of
diversification. The new system of classification should facilitate studies of Agaricus in the tropics and,
reciprocally, such studies in various regions should allow to further improve the system of classification.
ACKNOWLEDGMENTS
The authors are grateful to L. A. Parra for his suggestions, to R. L. Zhao and K. D. Hyde who initiated the
project in Asia, and to the contributors who collected, provided, or studied tropical Agaricus with us since
2010: C. Angelini, G. Barroso, H. Bashir, C. Billette, R. Courtecuisse, M. Foulongne-Oriol, A. Guelly, Y.
Gui, J. Guinberteau, S. C. Karunarathna, R. W. Kerrigan, A. De Kesel, A. N. Khalid, G. Mata, V.
Mahdizadeh, M. Moinard, R. Nawaz, B. Ortiz-Santana, L. A. Parra, T. Qasim, S. Rapior, O. Raspé, V.
Sabaratnam, N. Safaie, P. Sysouphanthong, N. Thongklang, K. Wisitrassameewong, and J. Xu.
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Parra LA, Muñoz G, Callac P (2014) Agaricus caballeroi sp. nov., una nueva especie de la sección
Nigrobrunnescentes recolectada en España. Micol. Veg. Medit. 29:21-38.
Parra LA, W isman J, Guinberteau J, Wilhelm M , Weholt Ø, Musumeci E, Callac P, Geml J. (2015) Agaricus
collegarum and Agaricus masoalensis, two new taxa of the section Nigrobrunnescentes collected in Europe.
Micol. Veget. Medit. 30:3-26.
Parra LA, Angelin i C, Ortiz-Santana B, Mata G, Billette C, Rojo C, Chen J, Callac P (2018) The genus Agaricus in
the Caribbean. Nine new taxa mostly based on collections fro m the Do minican Republic. Phytotaxa 345:219-271.
Peel M C, Finlayson BL, McMahon TA (2007) Updated world map of the Köppen -Geiger climate classificat ion.
Hydrol. Earth Syst. Sci. 4:439-73.
Pegler DN (1977) A preliminary Agaric flora of east Africa. Kew Bull., Addit. Ser. 6:1– 615.
Peterson KR, Desjard in ED, Hemmes DE (2000) Agaricales of Hawaiian Islands. 6. Agaricaceae L. Agaricaceae:
Agaricus and Melanophyllum. Sydowia 52:204-257.
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subrufescens. Appl. Microbiol. Biotechnol. 100:781-796.
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Mycoscience 4:239-250.
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reveals a new group in section Xanthodermatei. Mycologia 106:1220-1232.
Thongklang N, Sysouphanthong P, Hyde KD, Mo inard M, Hoang E, Rodriguez A, Kerrigan RW, Foulongne –Oriol
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(Agaricaceae) fro m Xinjiang Province (Western China). Phytotaxa 202:185-197.
Wisitrassameewong, K, Karunarathna, SC, Thongklang N, Zhao RL, Callac P, Chukeat irote E, Bahkali AH, Hyde
KD (2012) Agaricus subrufescens: new records to Thailand. Chiang Mai J. Sci. 39:281-291.
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Phytotaxa 257:99-121.
38
3. AN OVERVIEW OF TROPICAL AND SUBTROPICAL SPECIES OF Agaricus IN

MEXICO
1
Rosario Medel, 1 Naara Palestina, 2 Gerardo Mata, 3 Luis A. Parra Sánchez
1
Instituto de Investigaciones Forestales, Universidad Veracruzana, Xalapa, Veracruz, Mexico.
2
Instituto de Ecología A.C., Xalapa, Veracruz, Mexico.
3
Avenida Padre Claret n° 7, 5° G, 09400 Aranda de Duero, Burgos, Spain.
ABSTRACT
The genus Agaricus comprises around 400 species, most of which are described in temperate zones, with a smaller
number found in tropical zones. Due to phenotypic plasticity of the genus, many Agaricus species are difficult to
identify using morphological characters, so recent studies have included molecular characters for identificat ion.
While there have been some studies in tropical zones of Central America, South America and the Caribbean, there is
litt le knowledge of the diversity of this genus in tropical and subtropical zones of Mexico. The last review of the
genus for Mexico was conducted in 2011. This study analyzed species of tropical and subtropical zones in Mexico
and included up to 29 species of Agaricus that are currently known in 12 states. The vegetation type, where most
(15) species of Agaricus have been found, is tropical montane cloud forest. This is followed by nine species of
Agaricus found in the deciduous and sub-evergreen tropics. Advances and perspectives in the study of the genus
Agaricus in tropical and subtropical zones of Mexico are presented.
Keywords: cloud forest, diversity, edible fungi, tropical rain forest, trends.
INTRODUCTION
The genus Agaricus L. includes saprobic basidiomycetes fungi, featuring some highly valued edible
species (A. bisporus) and others with medicinal properties (A. subrufescens). The genus is widely
distributed and is present on all continents, except Antarctica. To date, 434 species have been described
worldwide (Karunarathna et al. 2016). However, the most extensive studies, with keys for identification
and descriptions, focus mainly on species from the temperate zones of Europe (Cappelli 1984, Parra 2008,
2013). Over the years, various studies have been published pertaining to tropical regions of America
(Heinemann 1961, 1962a, 1962b, 1962c, 1993, Murrill 1918, 1942, 1945, 1946, Rick 1906, 1919, 1920,
1930, 1939, 1961), Africa (Heinemann 1956, 1961, Pegler 1966, 1968, 1969, 1977), Asia (Heinemann
1980, Pegler 1986) and Oceania (Heinemann 1982). In addit ion, the extensive study of Kerrigan (2016),
relating to the genus Agaricus of North America, was published recently.
It is known that tropical regions host a wide diversity of fungi, greater than that found in temperate zones
(Blackwell 2011). Furthermore, even with the few studies conducted in tropical zones, it is known that
nearly half of the total number of species recorded in the genus comes from these regions (Karunarathna et
al. 2016). This suggests that there is insufficient knowledge of the species of this genus that inhabit
tropical and subtropical zones.
PHYLOGENETIC RELATIONSHIPS
One of the greatest impediments to classify these species is the recognition of sections within the genus
Agaricus, based on morphological and macrochemical characters that have varied according to the
taxonomic criteria of each author. The study of Zhao et al. (2011) proposed a new scheme of division into
sections based on molecular characters of the genus and including species from tropical zones of Asia
(Thailand), Africa and America. These authors found that up to a third of the studied species did not fit
39
into the eight classic sections in modern studies (Parra 2008, 2013) and proposed three new clades.
Subsequently, studies in tropical regions of Asia and Oceania (Chen et al. 2012, 2015, Gui et al. 2015,
Zhao et al. 2016) recorded new species and included phylogenetic proposals that placed the species into
new tropical clades. Recently, Zhao et al. (2016) proposed a new classification of the genus that was
divided into five subgenera and 20 sections, including some previously proposed tropical clades. As a
result, infrageneric categories are gradually being clarified.
ETHNOMYCOLOGICAL IMPORTANCE IN MEXICO
In Mexico, species of the genus Agaricus have been used since pre-Hispanic times for food and medicine.
While it is true that Herrera and Guzmán (1961) commented on the difficulty of conducting a historic
analysis of edible fungi in this country, the fact that many species of fungi (including th ose of the genus
Agaricus) have common or popular names and are sold in markets in some parts of the Mexican Republic.
This indicates familiarity with and use of these species by consumers of mushrooms in Mexico, who go to
buy them using popular names such as “hongo de Sanjuan”, “Sanjuanero de llano”, “llanero”, “champiñón
de campo”, “hongo blanco”, “pípila” and “mazayel”, among others (Fig. 1). A study of the main edible
fungi of Mexico by Herrera and Guzmán (1961) was published 55 years ago and describes seven edible
species sold in various markets of Mexico City and Valle de Mexico, such as the Amecameca, and the
markets of the Distrito Federal, among others. Since then, several general studies have been conducted in
order to provide knowledge of this theme (Estrada-Martínez et al. 2009, Galván et al. 1998, Garibay-
Orijel et al. 2006, Guzmán 1981, 1984, 1994, 1995, 1997, Herrera and Guzmán 1961, Pérez-Moreno et al.
2008, Velandia et al. 2008).
Figure 1. Sale of mushrooms in the market of San José in Xalapa, Veracru z.
THE GENUS AGARICUS IN MEXICO
The floristic composition of Mexican forests that includes species of both Neotropical and Nearctic zones,
allows existence of diverse vegetation types that feature a biodiversity that is particular to each one of
these forests, above all in the tropical and subtropical regions. These forests, that cover less than 30% of
40
the country, favor the presence of different species of fungi, especially those that present dark spores, such
as the genera Agrocybe, Agaricus, Bolbitius, Coprinus, Cortinarius, Galerina and Inocybe, among others
(Guzmán-Dávalos 2002).
While some studies are related to the genus Agaricus, most are focused on species that inhabit subtropical
and temperate zones of Mexico (Gutierrez-Ruiz and Cifuentes 1990, Montoya-Bello et al. 1987, Pérez-
Silva et al. 2011). Other studies are basically general inventories that register collections from different
zones. The most complete studies are those of Guzmán (1983), who describes A. benzodorus, A.
endoxanthus, A. denisii, A. purpurellus, A. singeri and A. trinitatensis of the Yucatán Peninsula. In one of
the pioneering studies on tropical species of this genus, Gutiérrez-Ruiz and Cifuentes (1990) describe
species of various vegetation types including those of the tropical montane cloud forest (A. comtulus, A.
essettei, A. impudicus, A. phaeolepidotus, A. moelleri, A. semotus, A. benesii, A. xanthodermus and A.
xantholepis), tropical low deciduous forest (A. moelleri), tropical sub-evergreen forest (A. fuscofibrillosus)
and tropical deciduous forest (A. semotus). Finally, Medel et al. (2015) describe six species of tropical
montane cloud forest (A. augustus, A. comtulus, A. sylvicola, A. sylvaticus and A. xanthodermus) and
tropical deciduous forest (A. phaeolepidotus). Only A. yucatanensis is excluded from the list, due to the
fact that the type specimen of this species deposited in the Farlow herbarium (FH, Harvard University)
was reviewed by the first author of this paper who concluded that it did not correspond to a species of the
genus Agaricus.
The last review of the genus for Mexico was conducted by Mata et al. (2011), who highlighted 32 species
distributed across 25 states of the country. In that study, the authors mention diversity of species in
tropical and subtropical zones of Mexico could be significant due to the fact that, in these regions, the
genus has been insufficiently studied. Since the date of that review, only two new studies have been
published (Medel et al. 2015, Mata et al. 2016) and progress in the knowledge of this genus, therefore,
remains moderate.
This study includes a bibliographic review of all studies published in Mexico from 1896 to 2016. Fifty-
five species have been recorded to date for temperate, tropical and subtropical forests. In this chapter, we
discuss only the species known to inhabit tropical zones.
Table 1 lists 29 species of the genus Agaricus corresponding to records for tropical and subtropical forests.
These are ordered into nine categories or vegetation types (Fig. 2). In order to group types of vegetation,
equivalences were established with vegetation types according to Rzedoswki (1978). In cases where types
such as tropical forest or tropical vegetation are mentioned these were conserved. Such denominations
cannot have equivalents since they constitute very wide categories. The tropical montane cloud forest has
the most records, with 11 species. In tropical forests (deciduous, evergreen and sub-evergreen) there are
nine species and in the xerophylous scrub there are only two records. Analysis per vegetation type
corroborates, at least quantitatively, the hypothesis that a non-determined number of new records will be
found in that vegetation type, even including new species. This is supported by composition of the floristic
kingdoms and provinces of Mexico (Rzedowski 1978) where the Neotropical kingdom coincides exactly
with the location of states with the highest number of records of Agaricus species in tropical and
subtropical zones (Fig. 3).
41
Table 1. Agaricus species recorded from tropical areas in Mexico.*

Species States Vegetation Edi bility References
A. arvensis Ver., Mor. Tsv, Cf, Tv (+) 8, 13, 21
Gro., Edo. Méx., Tamps.,
A. augustus Cf (+) 3, 6, 12, 15
Ver.
A. benesii Gro. CF (+) 7
A. benzodorus Q. Roo Tv (*) 9
A. bitorquis w. l. Tsv (+) 8
Chis., Hgo., Jal., M ich., Tsv, Ats, Cf, Thf, 2, 8, 10, 14, 20,
A. campestris (+)
Oax., Ver. Tdf, Tdfp 21, 22
A. comtulus Gro. Cf (+) 7
A. aff. dennisii Q. Roo Tv (*) 9
A. deserticola Son. Thf (*) 17
A. endoxanthus Q. Roo Tv (-) 9
A. essettei Son. Tdfp (+) 18
A. fuscofibrillosus Gro. Tef (+) 7
Chis., Gro ., Hgo., M ich.,
A. impudicus Tef (+) 4, 7
Ver.
A. martineziensis w. l. Tsv (+) 8
A. moelleri (como A.
Gro., Mor., Oax., Ver. Cf, Tdfp (-) 3, 7
praeclaresquamosus)
A. osecanus Mor. Tsv (+) 16
A. phaeolepidotus Gro., Edo. Méx., Mor., Ver. Tdf, Cf (-) 7, 15
A. placomyces Mich., Tamps., Ver. Ats, Cf (-) 1, 4, 14
A. purpurellus Q. Roo Tv (+) 9
A. semotus Gro., Mor. Tf, Cf (+) 7
A. singeri Q. Roo Tv (+) 9
A. subrutilescens Mich. Cf (+) 14
Chis., Hgo., Jal., M ich.,
A. sylvaticus Cf, Tdf (+) 14, 19
Mor., Oax., Tamps., Ver.
A. sylvicola Hgo., M ich., Oax. Cf (+) 5, 21, 22
A. trinitatensis Q. Roo Tv (*) 9
A. variegatus Jal. Tf (+) 11
A. volvatulus Ver. Cf (-) 1
Gro., Jal., Q. Roo,Tamps.,
A. xanthodermus Tdf, Cf, Tf, Tv (-) 6, 7, 9, 15, 18
Ver.
A. xantholepis Gro., Hgo. Cf (*) 7
*Symbols: States: Ch is=Ch iapas, Gro =Guerrero, Hgo=Hidalgo, Jal=Jalisco, Edo. Méx=Estado de México,
Mich=Michoacán, Mor=Morelos, Oax=Oaxaca, w.l.=without locality, Son=Sonora, Tamps=Tamaulipas,
Ver=Veracruz. Vegetati on: Thf=thorn forest, Cf=cloud forest, Tf=tropical forest, Tdf=tropical deciduous forest,
Tef= tropical evergreen forest, Tdfp=tropical deciduous forest (partly), Ats=arid tropical scrub, Tv=tropical
vegetation, Tsv=tropical and subtropical zone. Edi bility: (+)=edible, ( -)=to xic, (*)=unknown. References:
1=Chacón and Guzmán 1997, 2=Chanona-Gó mez et al. 2007, 3=Cifuentes et al. 1993, 4=Cifuentes et al. 1990,
5=Frutis and Guzmán 1983, 6=García-Jiménez and Valenzuela 2005, 7=Gutiérrez-Ru iz and Cifuentes 1990,
8=Gu zmán 1977, 9=Guzmán 1983, 10=Gu zmán and García-Saucedo 1973, 11=Gu zmán-Dávalos et al. 1983,
12=Herrera and Guzmán 1961, 13=López et al. 1985, 14=Mapes et al. 1981, 15=Medel et al. 2015, 15=Mora and
Gu zmán 1983, 16=Moreno et al. 2007, 17=Pérez-Silva et al. 2006, 18=Portugal et al. 1985, 19=Salvador and
Gu zmán-Dávalos 1988, 20=Varela and Cifuentes 1979, 21=Welden and Gu zmán 1978.
42
Cf
8
15 Thf
3 Tsv
2
Tdfp
4 Ats
2
2
3 5 Tdf
Tef
Tf
Tv
Figure 2. Records of species of Agaricus by vegetation type. Thf=thorn forest, Cf=cloud forest, Tf=tropical forest,
Tdf=tropical deciduous forest, Tef= tropical evergreen forest, Tdfp=tropical deciduous forest (partly), Ats=arid
tropical scrub, Tv=tropical vegetation, Tsv=tropical and subtropical zone.
Figure. 3. Graphical representation of the Holarctic and Neotropical kin gdoms in Mexico (Fro m Rzedowski 1978).
Geographic distributions of the species found are represented in 12 states of Mexico (Fig. 4), with
Guerrero, Veracruz and Quintana Roo those that have the highest number of records (10, 10, 7 records,
respectively). The fewest records are presented in Estado de México and Sonora (2 records each). This
43
does not mean that these states do not have diversity of species, but rather that there has been little
exploration. Furthermore, there are states (e.g., Estado de México) that have more species associated with
temperate forests. Of the 29 species, at least 12 have been mentioned in studies of Central and South
America and that confirms some of these may be more American than European in distribution.
10
9
8
7
6
5
4 No. species
3
2
1
0
Figure 4. Records of tropical species by state.
PERSPECTIVES OF STUDIES IN TROPICAL AND SUBTROPICAL AREAS OF MEXICO
Following analysis of information obtained from the last update of knowledge of this genus in Mexico,
some published studies have suggested a few guidelines for study of the genus Agaricus (Medel et al.
2015). These suggested that scarcity of studies of this genus is significant and that there is still a lack of
literature to enable identification of species collected in tropical and subtropical zones. It is very important
to document macro and micro morphological characters, important microchemical reactions (i.e., Schäffer
and 5% KOH), odor and change of color in the context and surface of the pileus and stipe, as well as
presence of structures such as rhizomorphs, in order to complete a more certain identification. Mata et al.
(2016), in a study of wild Agaricus bisporus in Mexico, emphasized the need to support identifications
with molecular characters, since this is a genus that is sensitive to environmental change and therefore the
morphology can change within the same species to an extent that there would be variations in number of
spores per basidium.
Finally, study of the genus Agaricus from the point of view of diversity is interesting per se. However,
various species produce compounds of medical interest, including Agaricus subrufescens, the “almond
mushroom” from which compounds with anti-cancer activity have been obtained (Wisitrassameewong et
al. 2012).
Study of this genus is ongoing in Mexico and the tropical zones of that country are reservoirs of new or
previously unknown species. Knowledge from these studies may lead to additional commercial
44
opportunities for both the agricultural and pharmaceutical industries.
ACKNOWLEDGMENTS
The authors thank authorities of the Universidad Veracruzana and the Dirección General de
Investigaciones for their support. Thanks go to the Instituto de Ecología, A.C. for the facilities provided
for conducting this study. The second author is grateful to CONACyT for a scholarship awarded for
Masters studies. We thank Emilia Belingheri for her support in various aspects of the work and Juan Lara
Carmona of the XAL herbarium for providing facilities for consulting material of this collection. Finally,
we thank Keith MacMillan for English translation of this work.
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47
Cultivation
4. DEVELOPMENT OF THE STRAW MUSHROOM SECTOR IN CHINA
Qi Tan1, Mingjie Chen1, Meilian Yu1 and Huanqing He2
1
Shanghai Academy of Agricultural Sciences. 1000 Jinqi Road, Shanghai 201403, China
2
Guangdong Academy of Agricultural Sciences. Wushan Road, Guangzhou 510640, China
ABSTRACT
This paper provides an historical overview of straw mushroom (Volvariella volvacea) cultivation, development of the
straw mushroom sector and progress in research on the straw mushroom in China. Cultivation of the straw
mushroom originated in southern China more than 300 years ago. Its cultivation method was introduced to Malaysia
in 1930s and soon cultivation of the straw mushroom was carried out in Southeast Asia and North Africa. Cultivation
methods of the straw mushroom are among the simplest of all mushroom species and its production was the third
largest in the world in 1986. However, in the past three decades, development of the straw mushroom sector in China
was very slow, mainly due to its low biological conversion efficiency and short shelf-life. It is anticipated that the
industrialized and large-scale production of straw mushroom soon will replace the traditional cultivation methods in
China in response to growing demand in the Chinese market.
Keywords: mushroom production, straw mushroom, mushroom cultivation, edible mushroom, fungal technology
INTRODUCTION
The straw mushroom [Volvariella volvacea (Bull.) Singer] is a species of edible mushroom belonging to
kingdom fungi, division Basidiomycota, class Agaricomycetes, order Agaricales, family Pluteaceae, genus
Volvariella. It is an important grass-rotting fungus growing on fiber wastes such as rice straw, cotton and
hemp. The straw mushroom prefers high temperature and high humidity and therefore, it is traditionally an
edible mushroom of the tropics and subtropics. The straw mushroom has high nutritional and medicinal
values since it is rich in proteins, vitamin C and contains 18 amino acids. China is the largest producer of
the straw mushroom in the world and the main producing areas of this mushroom in China are provinces
in southern China and southwest China, including Guangdong, Guangxi, Sichuan, Fujian, Hunan and
Jiangxi.
HISTORY OF STRAW MUSHROOM CULTIVATION
Cultivation of the straw mushroom originated in southern China, with a history of more than 300 years. As
early as the Ming Dynasty (1368-1644), there was a description of straw mushroom cultivation in a book
51
called “Book of Planting Trees” written by Yu Zongben. Records of straw mushroom cultivation also
appeared in county annals or annals of local governments in southern China in the Qing Dynasty
(1644-1912), such as “Annals of Guangdong” compiled by Ruan Yuan in 1822, “Annals of Yingde County:
Products” complied by Huang Peirong in 1844, “Annals of Ningde County” in Fujian Province, and
“Annals of Shaozhou Prefecture” complied by Lin Shuxun in 1874. In those documents, the straw
mushroom was clearly defined as a kind of wild edible mushroom that originally grew on decaying straw
in southern China. Monks in the Nanhua Temple in Guangdong were the first consumers of straw
mushrooms who collected them in the wild and then started to use rice straw to cultivate them in rice
fields (Chang 1978, He et al. 2010).
According to studies by Malaysian scholar JA Baker and Thai scholar K Jalaricharana, cultivation
methods of the straw mushroom were brought to Malaysia by overseas Chinese people in 1932. Soon
afterwards, cultivation of straw mushrooms quickly spread to Southeast Asia and North Africa. Therefore,
globally, the straw mushroom is known as the Chinese mushroom (He et al. 2010).
Before 1960s, a traditional method of “piling up straw and pouring manure” had been adopted in artificial
cultivation of straw mushrooms. Outdoor cultivation of straw mushrooms that uses raw material piling up
and cultivation had low and unstable yield, with only around 7% biological conversion efficiency. Along
with development of modern biology, genetics, microbiology and environmental engineering, researchers
in China, led by Professor Chang Shuting and Professor Deng Shuqun, assisted the rapid development of
straw mushroom production, through continuous accumulation of technologies and research achievement.
In the 1960s, a field cultivation method and a simple structure room cultivation method were developed
for the first time in China by Guangdong Institute of Microbiology. The Institute introduced the concept of
pure culture spawn and selected the first batch of straw mushroom strains, V20 and V23. These strains are
still the main cultivars of straw mushrooms throughout the country. In the 1970s, Professor Chang Shuting
of the Chinese University of Hong Kong made the first successful production of straw mushrooms using
cotton waste. Along with improvement of strains and cultivation facilities, biological conversion
efficiency increased to 20 to 35% and development of straw mushroom production increased rapidly. In
the 1980s, bed cultivation in thermally-insulated and/or brick rooms was adopted in Guangdong, Jiangsu,
Zhejiang, and Shanghai, improving the production cycle. In the late 1980s and early 1990s, bag cultivation
of the straw mushroom was adopted and resulted in more efficient and stable yield. He Huanqing and his
team at Guangdong Academy of Agricultural Sciences, experimented with Chinese herbal residue to
cultivate straw mushrooms and this proved a success. Thus, a new source of raw material for straw
mushroom cultivation became available and this method was widely adopted in Guangdong Province (He
et al. 2010).
Compost for straw mushroom cultivation undergoes a development process from first fermentation
(composting) to secondary fermentation (composting and pasteurization) and then to indoor pasteurization.
When using rice straw as the main raw material and using secondary fermentation and a bed-type
cultivation model, in general, the biological conversion efficiency is 10 to 20%. This is 2 to 3 times higher
52
than that of outdoor “piling up” cultivation. Straw mushrooms may be cropped 18 to 20 times in one year
when using cotton seed hulls and/or waste cotton as the main raw materials and using pasteurization
treatment method for indoor cultivation. So far, various cultivation modes of straw mushrooms have been
established in various geographic regions in China. In recent years, Jiangsu Jiangnan Biotech Co., Ltd., as
the representative of some enterprises in China, started the industrialized production of straw mushrooms.
ISSUES IN DEVELOPMENT OF THE STRAW MUSHROOM INDUSTRY
The cultivation method of the straw mushroom is the simplest among methods of all edible mushrooms.
The straw mushroom requires the shortest time for fruiting, compared with other edible mushrooms. Raw
materials for straw mushroom cultivation are abundant. Total output of straw mushrooms reached 38,000
tons in 1979 and its production scale was third highest in the world relative to button mushroom and
shiitake in 1986. However, in the past 3 decades, development of the straw mushroom industry was very
slow, while the industries of other mushroom species have been developing very rapidly. In 2014, China’s
total output of mushrooms reached 32.7 million tons, while the output of straw mushrooms was only
250,000 tons, accounting for 0.8% of total output. The main reasons for this result include biological
characteristics of straw mushrooms and the influence of several factors in the process of production and
distribution (He et al. 2010).
Strain
As the most important and most effective method for edible fungi breeding, cross-breeding has made an
outstanding contribution to rapid development of China’s edible mushroom industry and to helping China
take the lead position in the world. However, breeders are not able to distinguish between homokaryotic
and heterokaryotic mycelium or between parents and hybrids, by morphological characters, due to the fact
that straw mushroom is regarded as a kind of primary homothallic fungus and its hypha has
multinucleate cells and no clamp connections. Therefore, progress in straw mushroom breeding has been
very slow. Very few researchers work on straw mushroom breeding and efforts so far have not resulted in
improved strains (Zhao et al. 2015a).
Growth characteristics
The straw mushroom prefers high temperature and high humidity so it can grow well only when air
temperature is above 25℃ and humidity is above 80%. In addition, its fruiting temperature is more than
35℃. These growth characteristics greatly limit large-scale cultivation and popularization.
Biological conversion efficiency
Compared with other major cultivated edible mushrooms, the straw mushroom's biological conversion
53
efficiency is still low. For instance, its biological conversion efficiency of natural straw substrate is less
than 10%. Its biological conversion efficiency tops out at about 40%, although waste cotton, cottonseed
hulls, and other materials have added to the efficiency in some cases. In comparison, the biological
conversion efficiencies of oyster mushrooms (Pleurotus spp.), button mushroom (Agaricus bisporus),
shiitake (Lentinula edodes), black ear mushroom (Auricularia auricula), and enoki (Fammulina velutipes)
are all above 80% (He et al. 2010).
Poor storage durability
Pilei of straw mushrooms can continue to stretch and open after harvesting fruit bodies, which lower their
quality and commodity value. Under conditions of conventional low temperature storage, straw mushroom
fruit bodies become soft and even liquefy or decay, meaning straw mushrooms have a very short shelf life.
Its poor storage durability is one of the main reasons that limit development of the straw mushroom
industry.
PROGRESS IN STRAW MUSHROOM RESEARCH
Having recognized the above-mentioned factors limiting the development of the straw mushroom industry,
researchers in China have been making great efforts and attempts to overcome problems limiting straw
mushroom production and its industry.
Using modern molecular biology to improve breeding efficiency
Researchers at Shanghai Academy of Agricultural Sciences and Fujian Agriculture and Forestry
University have completed whole-genome sequencing of the straw mushroom. Bao Dapeng (2013)
conducted genome sequencing of Volvariella volvacea V23 by Roche 454 GS FLX and Illumina
Solexa ． The genome is 35.7 Mb in size and encodes 11,084 proteins based on bioinformatics
prediction．Among the total predicted, 5,516 genes have been assigned either a definitive or tentative
function (Bao et al. 2013). Comparative analysis found that there is a mating-type system in straw
mushrooms that is similar to the two polar mating-type system in Pholiota nameko. Zou (2012) concluded
that Volvariella volvacea is a two polar mating type mushroom after using conventional pairwise mating
method and combining with morphological marker of colony and SV molecular marker to verify the
paring result. Xue Chengqin et al. established specific molecular markers on the basis of straw mushroom
A factor, and thus, established molecular marker-assisted breeding technology system of straw mushroom
(Xue et al. 2013). Compared with the conventional cross-breeding method of straw mushroom, molecular
marker-assisted breeding technology system can effectively improve the probability of mating success and
obtain fertile stains, because many hybrids do not produce fruit bodies in conventional cross-breeding.
Therefore, application of molecular marker-assisted breeding technology system can greatly improve the
efficiency of cross-breeding (Xiong et al. 2014, Zhao et al. 2015).
54
Studies on molecular biology of the straw mushroom are mainly focused on application of a variety of
molecular markers in strain identification. Through identification of gene library of straw mushroom,
Chen Mingjie et al. (1996) found that the straw mushroom genome has a high percentage of repetitive
sequences. Among these repetitive sequences, the moderately repetitive sequences are unique in different
interspecific and intraspecific strains, so they can be used to construct molecular markers (Chen et al.
1996). The highly repetitive sequences may be more conservative DNA sequences that have important
values in the study of evolution and classification of fungi. Fu Junsheng et al. (2010) screened hereditary
differences in bands of single-spore strain of parents by SRAP, then, converted hereditary difference bands
to SCAR markers to identify hybrids (Fu et al. 2010). Chen et al. (2011) identified straw mushroom
hybrids using RAPD technique that made use of the phenomenon that parents’ specific bands are always
present in their offspring.
Use of modern molecular biology to improve biological conversion efficiency of the straw mushroom
Straw mushrooms can produce a variety of enzymes that degrade cellulose to glucose, including β-1,
4-endoglucanase, β-glycosidase, and cellobiohydrolase. In recent years, study of the isolation, cloning,
and gene transfer of a cellulase gene has become a hot topic of research on molecular biology of straw
mushrooms in the world. Based on accomplishment of whole-genome sequencing project of Volvariella
volvacea and establishment of the genome-wide framework map of Volvariella volvacea, Wang Menglan
et al. (2014) found the genes encoding hemicellulase degrading enzymes from whole-genome by
bioinformatics analysis. By comparing structures of hemicellulase degrading enzymes of straw mushroom
of some grass-rotting fungi and wood-rotting fungi and by analyzing the level of gene expression and
enzyme activity, researchers attempted to identify differential genes between straw mushroom and other
edible mushrooms and to explore reasons for low biological conversion efficiency of straw mushroom. It
is speculated that xylanase may be a crucial enzyme in straw mushroom hemicellulase, and xynII may be
an important gene in the process of degrading hemicellulose (Wang et al. 2014, Zhao et al. 2015)
Bao et al. (2013) found 11 laccase isozyme genes (lac genes) and 4 manganese peroxidase genes in straw
mushrooms, but did not find lignin peroxidase genes. Lignin peroxidase genes, that are important for
lignin degradation, are absent in straw mushroom genome so this may be associated with straw
mushroom’s poor ability to utilize lignin. Reports associated with ligninolytic enzymes in straw
mushroom are relatively few and mainly focused on laccase that has the largest amount in straw
mushroom, especially on laccase gene cloning and expression (Wu et al. 2014). In the study by Zhu
(2013), 11 laccase isozymes genes were found in straw mushroom genome and laccase gene expression
was detected at the pinhead, button, egg, elongation and mature stage of straw mushroom. The results
show that laccase gene transcription can occur at all stages, but expression levels at different stages are
dissimilar. It is speculated that those lac genes may play a key role in morphogenesis of straw mushroom
fruit bodies (Zhu et al. 2013).
55
Improving product quality and freshness-keeping technology of the straw mushroom
Initially, many studies were focused on low temperature storage of the straw mushroom while a number of
studies were directed toward cultivation management and strain domestication. For instance, plastic
tunnels, arch sheds, ground sheds and other protected cultivation measures are used to conduct spore
domestication at low temperature and induced domestication by cultivation in spring season. But these
measures can only play a mitigation role when temperatures change and the low temperature tolerance of
straw mushroom strains is not stable. Shelf life of straw mushrooms is still a problem, although some
measures such as 60 Co-γ-irradiation, method of charging air and exhausting oxygen and modified
atmosphere storage can extend the shelf life of straw mushroom to 74 hours (Lu et al. 2013).
Rapid development of modern molecular biology and its applications in various domains of biology has
brought new approaches and ideas for theoretical studies on straw mushrooms and for genetic
improvement of strains. Chinese researchers also conducted studies on straw mushroom autolysis at low
temperature. However, the fundamental cause and role of autolysis at low temperature remains unknown.
By focusing on protease, proteins and nucleic acids, through comparative analysis of mRNA differential
display technique and protein spectrum (Chen et al. 2001), Chen et al. (1998) screened and cloned a low
temperature response gene. Then they analyzed the change of gene expression at low temperatures,
conducted expression validation after obtaining low temperature mutant straw mushroom strain and
preliminarily established a low temperature response model on straw mushroom V23 strain. Low
temperature can damage cell membranes of V23 strain resulting in autolysis leading to death. Synthesis of
lipids and other components on the plasma membrane is accelerated in a low temperature mutant strain,
thus improving cell membrane fluidity and allowing the mutant stain to survive longer at low temperature
(Sun et al. 2005, Sun et al. 2012, Jiang et al. 2014). Guo et al. (2005) cloned and isolated freezing stress
induced expression gene of straw mushroom by cDNA-AFLP technology and conducted sequencing and
structure analysis of DNA sequence.
With the development of genetic engineering, it was found that the time of low temperature storage of
straw mushroom can be effectively extended by transgenic manipulation. Guo et al. (2005) used a gene
gun to transfer an antifreeze protein gene into straw mushroom and obtained transgenic straw mushroom
by hygromycin resistance selection. Results of a low temperature stress experiment show that the
transgenic mushrooms have a strong tolerance to low temperature and this tolerance is stable over
generations (Guo et al. 2005). Based on these experiments and findings, using antifreeze protein gene afp
as target gene and selectable marker gene for straw mushroom transformation, Wang et al. (2010)
screened transformants at 0℃ and obtained cold resistant transgenic straw mushrooms without selectable
markers. Therefore, transgenic manipulation is one of the ideal approaches to develop new strains of straw
mushroom that are suitable for low temperature storage (Wang et al. 2010).
56
DEVELOPMENT PROSPECT OF INDUSTRIALIZED CULTIVATION OF STRAW

MUSHROOMS
With economic development and improvement of living standards in China, consumers are demanding
fresh, healthy and safe agricultural products. Thus, market demand for edible mushrooms is also changing.
In some rural areas, the edible mushroom industry is playing an important role in helping farmers alleviate
poverty and generate more income. The development of the edible mushroom industry is supported by the
Chinese Central Government and local governments in a bid to build new rural areas in China and realize
the sustainable development of agriculture and the rural economy.
The straw mushroom is native to regions with high temperature and high humidity in southern China and
it needs very strict conditions of temperature and humidity for storage or its nutrients will be lost quickly.
Therefore, if transporting straw mushrooms from southern China to northern China, the nutritional value
of straw mushrooms will decrease and costs will be increased. The supply of fresh straw mushrooms is
limited to certain regions, mostly in southern and southwest China, while the demand for fresh straw
mushrooms in northern China is very strong and the market potential is very large. In order to satisfy new
market demand and enhance market competitiveness of products, upgrading current production modes and
adopting industrialized, large-scale production is the direction of development of the straw mushroom
industry in China. In recent years, some Chinese enterprises have already started industrialized production
of straw mushrooms. It is expected that industrialized and large-scale production of straw mushrooms will
replace traditional cultivation methods in China, in efforts to satisfy growing demand in the Chinese
market.
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5 ENHANCED PRODUCTION OF THE MEDICINAL MUSHROOM

Agaricus subrufescens PECK
Diego Cunha Zied1 and Arturo Pardo-Giménez2

1
Universidade Estadual Paulista (UNESP), Faculdade de Ciências Agrárias e Tecnológicas (FCAT),
Câmpus de Dracena, Rod. Cmte João Ribeiro de Barros, km 651, Bairro das Antas, 17900-000, Dracena,
SP, Brazil.
2
Centro de Investigación, Experimentación y Servicios del Champiñón (CIES), C/ Peñicas, s/n, Apartado
63. 16220 Quintanar del Rey, Cuenca, Spain.
ABSTRACT
Agaricus subrufescens Peck [A. blazei (Murrill) ss. Heinemann] has been studied by researchers in several countries
(Brazil, USA, China, Taiwan, etc.) primarily because of its medicinal and pharmacological properties. Cell walls of
the fungus contain polysaccharides called β-glucans that have structural and other functions. Several studies
highlight the importance of A. subrufescens medicinal properties. It was traditionally used to treat many common
diseases like atherosclerosis, hepatitis, hyperlipidemia, diabetes, dermatitis and cancer. Beneficial properties of A.
subrufescens include tumor growth reduction, immune modulatory activities, immune stimulatory effects,
antimicrobial and antiviral activities and anti-allergy effects. In this work, various aspects of production of A.
subrufescens are presented. Production of this edible medicinal mushroom has generated a notable interest
worldwide in the last few years, becoming increasingly popular and as a result has expanded into other countries
(Spain, France, South Korea, etc.). This is mainly due to its high international market price that is related not only to
the remarkable medicinal properties contributed by high content of bioactive compounds but also to the culinary
value added by its slightly nutty pleasant aroma.
Keywords: Agaricus blazei, medicinal mushrooms, production, compost, casing
INTRODUCTION
In recent years, production of the mushroom Agaricus subrufescens Peck has aroused increasing interest
around the world, achieving great popularity. Its medicinal and culinary properties make it possible to
foresee a rapid expansion of production around the world.
It is frequently referred to in the literature as Agaricus blazei (Murrill) ss. Heinemann or Agaricus
brasiliensis Wasser although these names are presented, not without some controversy, as incorrectly
applied or illegitimate (Kerrigan 2005, Wisitrassameewong et al. 2012). In Brazil, A. subrufescens is
commonly known as “Cogumelo do Sol®”, “Cogumelo Medicinal”, “Cogumelo de Piedade” “Cogumelo
de Deus”, “Portobello de almendra” or simply as “Blazei”, while in the USA, it is known as the “Almond
mushroom” and as “Royal Sun Agaricus”, in Japan as “Himematsutake”, “Agarikusutake” and
“Kawarihiratake”, in Spain as “Champiñón del Sol” and in China as “Ji Rong Song” (Kopytowski Filho et
al. 2006, Firenzuoli et al. 2007; Moukha et al. 2011).
The tradition and history of this mushroom in Brazil strongly represent an important mycological
relationship between growers and merchandizers. "Blazei" is associated with mushrooms harvested in the
city of Piedade (Fig. 1) in the 1960s and was originally collected by Mr. Furumoto. A detailed description
of the history of A. blazei in Brazil can be found in the chapter published by Zied et al. (2012).
59
A B
Figure 1. A) City of Piedade, region of Mata Atlantida (it is possible to visualize some specific growing
houses for the production of A. subrufescens), B) First field crops exposed to environmental conditions
that possibly gave the popular name of Sun Mushroom (substrate covered with soil ready for production).
Currently, only a few countries produce A. subrufescens on a commercial scale. Its production is now
well-established in Brazil, Japan, China and Korea (Gregori et al. 2008). It has generated a notable interest
worldwide in the last few years becoming increasingly popular and as a result expanding into many
countries. This is mainly due to its high international market price in comparison to other mushrooms that
is related not only to the remarkable medicinal properties contributed by high content of bioactive
compounds but also to the culinary value added by its slightly nutty, pleasant aroma (Largeteau et al.
2011).
Besides fresh fruit bodies, this mushroom is mainly processed in a dried and powdered form, in capsules
or pills or as an extract. Furthermore, it has been used as additive ingredients in cosmetic products
(Stamets 2000, Wisitrassameewong et al. 2012).
Several studies recently reviewed by Wisitrassameewong et al. (2012) highlight the importance of A.
subrufescens medicinal properties. It was traditionally used to treat many common diseases like
atherosclerosis, hepatitis, hyperlipidemia, diabetes, dermatitis and cancer (Firenzouli et al. 2008). Among
the beneficial properties from A. subrufescens that have been reported are tumor growth reduction,
immune modulatory activities, immune stimulatory effects, antimicrobial and antiviral activities and anti-
allergy effects (Wisitrassameewong et al. 2012).
In fact, in Brazil a methodology was never established regarding cultivation practices to consistently
produce relatively high levels of β-glucans in the basidiomas until the publication of Zied et al. (2014).
The authors clearly detailed the influence of strain, compost formulation, type of casing layer and
environment of production on the total amount of β-glucans found in the dehydrated basidiomas.
Zied et al. (2014) characterized all stages of production according to the amounts of β-glucans in the
basidiomas and the biotechnological behavior in its production. They concluded that the major
contributors to differences in β-glucan content were as follows (in descending order): 1) strain (35.8%), 2)
casing layer (34.5%), 3) environment of production (15.7%) and 4) type of compost (9.9%). On the other
hand, variations in yield were mainly affected by the environment of production (82.1%), followed by
strain (81.3%), casing layer (49.1%) and type of compost (15.2%). Camelini et al. (2005) also conducted
research that showed the amount and structural characterization of β-glucans in basidiomas varied with
different maturation stages. The amount of (1,3)-β-glucans in the mature stage was higher than in the
immature stage.
60
PRODUCTION PROCESS
The processes and techniques previously established for the production of Agaricus bisporus (Lange)
Imbach have been adopted for A. subrufescens cultivation (Martos et al. 2017). Parameters, such as the
conditions of growing in the different stages of the cycle (temperature, relative humidity, carbon dioxide
concentration, light exposure), materials, composting processes, moisture content and different aspects of
casing layers and fruit-formation should be examined to increase yields and adapt the crop to specific
conditions native to various countries.
In order to improve production of this crop and to provide guidance to growers, the following
considerations and practical aspects should be considered for commercial production.
SPAWN
Strains used for production of A. subrufescens in Brazil are marketed as varieties collected indigenously
that were selected through anthropization and adaptation to the cultivation conditions of the growers (type
and formulation of the compost and local environmental conditions). The consequences are substantial
variability in yield, a long growing cycle and a lack of control over specific growth characteristics of the
strains.
Production of spawn on grain (wheat or rye) is carried out similarly to A. bisporus. Figure 2 outlines the
necessary steps for production of inoculum i.e, sub-culturing and production of mother spawn in Petri
dishes using culture medium and production of grain spawn and spawn in bottles and high-density
polyethylene plastic bags using cereal grains.
COMPOST
Production of compost is carried out in two phases, similar to the method developed for A. bisporus,
although the compost obtained should have a lower total nitrogen content (1.15-1.45%), providing a
higher C/N ratio (25-27:1 final of Phase II). In Brazil, the traditional process of composting has been
widely practiced by growers (Fig. 3) following the steps of: pre-wetting (4-7 days), fermentation
(formation of the windrow 2 m wide x 2 m high, with intervals of turning every 2-3 days), pasteurization
(58±2°C) and physical, chemical and biological conditioning (47±2°C) (Eira, 2003). Although the
bibliographic references report a wide range of materials, characteristics for the growth substrates and C:N
values higher than those commonly used for the production of A. bisporus are recommended. Several
strains evaluated have shown good adaptation to production on different composts commercialized for
mushroom growing by several composting plants in Spain (Pardo-Giménez et al. 2014a).
As for compost load densities, a range between 60 and 70 kg m-2 may be considered suitable.
Application of commercial supplements to compost at the time of spawning may provide increases in
biological efficiency.
61
62
Figure 2. Sequence of procedures used for preparation of matrix and spawn and potential yield from a
single basidioma fragment, where in approximately 70 days a quantity of mycelium capable of inoculating
321,600 kg substrate is produced.
A B
Figure 3. A: Phase I composting, wood structure used to make the windrows (2 m wide by 2 m high),
bales of Brachiaria sp. and perforated grid on the floor where compost is heaped above for air circulation.
B: General view of the pasteurization tunnel (Phase II), a wooden platform where air circulates from the
bottom up through the compost. It is also possible to see in the photo the air outlet, which aims to
eliminate excess ammonia and reduce the temperature at time of spawning.
PRODUCTION OF MUSHROOMS
Production of the Sun mushroom in Brazil is carried out by farmers at the family level and by mid-level
mushroom growers. The number of mushrooms produced monthly at the family level may reach an
average of up to 60 kg of dry mushrooms. Above this value, professionalized production has been defined
with sectors of composting, production and mushroom processing areas, where the average production is
around 150 kg of dried mushrooms per month.
Usually, facilities used in the production process have a low technological level, do not have adequate
thermal insulation and do not usually have automated equipment. Thus, it is difficult to induce
programmed fruit-formation.
Producers have been investing recently in facilities with a more controlled environment (Fig. 4) to allow
production during all times of the year. Production in open fields (Fig. 1B) has not been carried out in
Brazil since the beginning of the 2000s.
Cultivation stages are the same as those for Agaricus bisporus: Spawning and filling, spawn run, casing,
pinning, fruit-maturation and harvesting, emptying, cleaning and disinfection. The remarkable feature is
that the environmental conditions in each of these stages for A. subrufescens differ substantially from
those applied for A. bisporus. During cultivation, optimum temperature for mycelial growth is around
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28±2 °C, while for development of basidiomata values between 24 and 27°C are recommended. Also,
high relative humidity and a high level of fresh air are required in the growing room. The growing cycle
lasts an average of 120 days (period considered after addition of the casing layer) that allows the producer
to perform three annual cycles per growing room.
A B
Figure 4. A: Modern production buildings with environmental control in the mountainous region of Mata
Atlantica. B: Internal view of a growing room that has thermal insulation and wooden benches containing
3 levels. Each room is 25 m long, 7 m wide and 3 m high.
CASING
A B
Figure 5. Production using casing soils of various textures. A: Mixture of loam soil with silt clay soil,
3:1V/V. B: Silt clay soil only.
Application of a casing layer on compost colonized with mycelium is an essential operation in commercial
production of both A. bisporus and A. subrufescens. This layer is where the change from vegetative to
reproductive growth occurs. In the case of A. bisporus, numerous materials have been used for this
purpose, with different types of peat moss being the most widely used in the world, mainly due to its
exceptional structural and water retention properties (Yeo and Hayes, 1979). In the case of A.
subrufescens, materials used are based, in most cases, by availability in producing countries. Casings are
64
usually based on mineral soils (different textures, Fig. 5) and different types of local peat, although we can
find other materials such as charcoal, sawdust, sand, vermiculite, pine bark and coconut fiber pith, among
others (Silva et al. 2007, Siqueira et al. 2009, Cavalcante et al. 2008, Colauto et al. 2010, Zied et al. 2010,
Zied 2011, Zied et al. 2011).
Our experience indicates that peat-based casings (Fig. 6) are preferable to those based on mineral soil
(Pardo-Giménez et al. 2014b). This is justified if we consider that, in the course of the growing cycle,
there is a high degree of evaporation associated with high ambient temperature, high rate of ventilation
required (CO2 concentrations between 650 and 700 ppm) and long cycle duration (4 to 5 flushes are
harvested at 12 to 14-day intervals). The ruffling operation, performed 5 days after application of casing
on compost, provides a significant increase in total yield.
Figure 6. Use of Dutch Commercial Casing in production of A. subrufescens. This casing has a greater
water holding capacity, high porosity and it provides a reduction in the irrigation program when compared
to soil-based casing.
BASIDIOME FORMATION
Regarding induction of fruit formation, two different conditions can be initially proposed, applicable for
successive flushes of mushrooms. A rapid induction that we might call "aggressive", adapted from
Kopytowski Filho and Minhoni (2007) and a slow one, adapted from Kopytowski Filho et al. (2008), is
less demanding a priori regarding the cooling capacity of the air conditioning system (Fig. 7 and 8). A
third possibility, later evaluated in order to reduce the energy cost associated with the significant decrease
in temperature required for induction, is what we termed moderately slow induction, reducing the
amplitude of the applied temperature range (Fig. 9).
Although the results, in terms of biological efficiency, do not differ significantly between different
induction conditions, the production pattern in terms of temporal distribution of the same is substantially
modified. Thus, rapid induction allows greater control over the crop by concentrating production of each
flush in fewer days. Intervals of flushes are between 12 and 14 days in this case. Slow inductions, on the
other hand, provide a greater number of harvest days that may be beneficial in some cases in terms of
manpower needs.
65
Figure 7. Control of internal and external temperatures of compost during induction of fruit-body
formation by fast cooling. Reduction of compost temperature from 28 to 20°C is accomplished in one day
and is maintained for 3 days at 20°C.
Figure 8. Control of internal and external temperatures of compost during induction of fruit formation by
slow cooling. Reduction of compost temperature from 28 to 20°C occurs over four days and then is held
for 1 day at 20°C.
66
Figure 9. Control of internal and external temperatures of compost during induction of fruit formation by
moderate cooling. Reduction of compost temperature from 28 to 22°C occurs over three days and then is
held for 3 days at 22°C.
YIELD OF MUSHROOMS
Biological efficiency reported in the consulted literature is in a range of 13.1 to 60.4 kg dt-1, with an
average value around 35 kg dt-1. According to Silva et al. (2007), in Brazil it is considered that
productivity, expressed as dry matter of mushrooms with respect to the fresh weight of compost, must be
at least 1% for the crop to be economically viable. Translated to biological efficiency, it should exceed the
threshold of 25 kg dt-1 compost. Our experience shows that, under climatically controlled conditions, and
depending on different factors associated with the production process (compost and casing type, schedule
of operations, watering program, induction of fruit formation conditions, etc.), biological efficiency values
of 50 to 70 kg dt-1 may be obtained.
After harvest, excess soil must be removed from the base of the stipe, then basidiomata are washed, cut
and dehydrated (Fig. 10). A. blazei mushrooms are usually commercialized dehydrated, whole or crushed
(flour, tablets, capsules, tea bags, etc.) and also combined with other foods (coffee, chocolate, honey, etc.).
It should be noted that there are standards of commercialization for the export of mushrooms. Detailed
information on the subject may be found in Zied et al. (2017).
67
A B
Figure 10. A: Freshly harvested mushrooms with residue of the casing layer and fragments of mycelium at
the base of the stipe. B: Washed mushrooms cut in half showing brown color absent. C: Mushrooms in
trays that are placed in a dehydrator. D: Mushroom dehydration room; each dehydrator (covered by
aluminium door) has an electric resistance heating system and forced ventilation and has a capacity of 100
kg of mushrooms per day. E: Dehydrated mushrooms sliced in half (light color and without signs of
oxidation, standard for export) and powder and capsules, ready to be marketed.
FINAL CONSIDERATIONS
Requirements regarding necessary growing conditions and particularly, high ambient temperature, make
production of A. subrufescens an alternative for consideration by growers in summer months. Limited
technological capacity of some cultivation facilities along with high energy costs, limits production
significantly in summer season. These same facilities could be used by producers to carry out a crop cycle
of A. subrufescens in those months. Production technology and substrate processing and crop cycle
management for A. bisporus is mostly applied to A. subrufescens, so growers should have few problems
switching to this crop. In addition, conditions of commercialization of this mushroom, mainly dehydrated,
avoid the limitation that supposes a short shelf life that generally is encountered with fresh mushrooms.
This allows staggered commercialization throughout the year.
68
ACKNOWLEDGMENTS
We would like to thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP-
2015/15306 and 2015/24788-1) in Brazil and the Diputación Provincial de Cuenca and the Consejería de
Agricultura, Medio Ambiente y Desarrollo Rural of Castilla-La Mancha in Spain.
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6. ARTIFICIAL CULTIVATION OF THE MEDICINAL MUSHROOM Sparassis latifolia
Yanquan Lin, Lu Ma, Donglai Xiao, Chi Yang, Zhenghe Ying, Xiaoling Jiang
Institute of Edible Fungi, National and Local Joint Engineering Research Center for Breeding &
Cultivation of Featured Edible Fungi, Fujian Academy of Agricultural Sciences, No 10, Lane 95,
Qianheng Road 10, Jin’an district, Fuzhou City, 350014, P.R.China
ABSTRACT
Sparassis latifolia is an edible and medicinal mushroom that is cultivated in East Asia. It contains important
bioactive molecules that have several biological effects including antiangiogenic activity, tumor-suppressing effect,
etc. This review presents a comprehensive summary of the genomics, proteomics, biological characteristics,
nutritional physiology, breeding and artificial cultivation of S. latifolia.
Keywords: Sparassis latifolia, genomics and proteomics, intrinsic and extrinsic factors, industrial cultivation
INTRODUCTION
Sparassis latifolia (also known as hanabiratake in Japanese or cauliflower mushroom in English) is an

edible and medicinal mushroom that is cultivated in Japan, Korea and China. Taxonomy of S. latifolia is
as follows: Fungi, Basidiomycota, Agaricomycetes, Polyporales, Sparassidacea, Sparassis (Li et al. 2010).
The color of S. latifolia can range from white to creamy white and its unique shape and appearance can be
described as similar to a large petal or a head of cauliflower, from which it has been given its popular
name (Figure 1). S. latifolia is a brown-rot fungus that grows on stumps of coniferous trees (pine or larch)
during the summer and autumn seasons and is widely distributed throughout the Northern Temperate Zone
(Kimura 2013, Kim et al. 2013). It is a rare species of edible and medicinal mushroom and produces
highly nutritious basidiocarps that are more precious than Cordyceps sinensis, Morchella esculenta and
Truffle.
S. latifolia contains a remarkably high concentration of β-glucan as measured by an enzyme method of the
Japan Food Research Laboratories (Ohno et al. 2000). β-glucan content from stipes and petals of S.
latifolia is over 40% with its content of stipe significantly higher than that of the petal (Lian et al. 2014).
In addition, β-glucan from the basidiocarp of S. latifolia has many biological and pharmacologic activities
including antiangiogenic activity (Harada and Ohno 2008, Yamamoto et al. 2009), tumor-suppressing
effect (Ohno et al. 2000, Hasegawa et al. 2004), etc. Polysaccharide fractions have been prepared from
cultivated S. latifolia and its major structural units and biological activities of the extracts have also been
examined (Tada et al. 2007).
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Figure 1. Fruit body of Sparassis latifolia
Because of the nutritional and medicinal value given above, S. latifolia has attracted interest of researchers
throughout the world and, as a result, consumer demand has increased. However, the production of S.
latifolia is still very low and industrial cultivation techniques of S. latifolia have been limited to a few
commercial farms because of its slow mycelial growth into cultivation substrate. In addition, control of
environmental conditions is also required for primordium formation and growth of fruit bodies, which is
hardly achievable on general commercial farms (Kim et al. 2013). Up to now, there are only three farms
that have artificially cultivated this mushroom in China so the total fresh production of S. latifolia is just
over 2.5 tons/d (Figure 2). Our previous study has shown that S. latifolia requires about 120 days to
complete its crop cycle from inoculation to harvest, which is longer than Flammulina velutipes,
Hypsizigus marmoreus and Pleurotus eryngii cultivation (Lin et al. 2012). S. latifolia yield is a complex
trait and the mushroom’s survival and multiplication are related to a number of factors including intrinsic
and extrinsic factors and their interactive effects. Intrinsic factors include carbon and nitrogen sources,
minerals, pH, type of mushroom spawn, substrate composition, moisture, ratio of carbon to nitrogen,
particle size, etc. Extrinsic factors include temperature, humidity, luminosity and air composition of the
surrounding substrate, such as concentration of oxygen and carbon dioxide.
Considering nutritional characteristics and growth conditions, domestication of S. latifolia has been a
difficult endeavor for a long time. The Institute of Edible Fungi, Fujian Academy of Agricultural
Sciences (FAAS) has focused on S. latifolia breeding, biological characteristics, nutritional physiology,
submerged fermentation and commercial cultivation aspects since 2004. In this review, we provide a
comprehensive insight into the above-mentioned factors of S. latifolia with the intent to provide
knowledge to people for improving yield and quality of S. latifolia.
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Figure 2. Industrial cultivation of S. latifolia in China
GENOMICS AND PROTEOMICS OF S. latifolia
Genome features
We sequenced the whole genome of S. latifolia and further assembled it into a 48.13 Mb draft genome.
The S. latifolia genome is similar in size to the genomes of several other species from the order
Polyporales, including Trametes versicolor (44.79Mb), Wolfiporia cocos (50.48 Mb) (Floudas et al. 2012),
Phanerochaete carnosa (46.29 Mb) (Suzuki et al. 2012) and Polyporus brumalis (45.72Mb)
(http://genome.jgi.doe.gov/Polbr1/Polbr1.info.html). We further mapped the genome to Eukaryotic
Clusters of Orthologs (KOG), Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes
(KEGG) databases to further characterize the predicted proteins.
Proteomes
We investigated protein expression at different developmental stages of S. latifolia using Isobaric Tags for
Relative and Absolute Quantification (iTRAQ)-coupled 2D LC-MS/MS (Two-Dimensional Liquid
Chromatography Tandem Mass Spectrometry). A total of 2305 reliable proteins were identified using
Q-Exactive mass spectrometry and ProteinPilot search engines and 2219 of these proteins had a
quantitative dimension. Of quantitatively different proteins, 104 were significantly up-regulated and 142
down-regulated at the early fruiting stage (80 days following inoculation) and 155 were significantly
up-regulated and 460 down-regulated at the fruiting stage (115 days following inoculation), compared
with the primordium stage (60 days following inoculation), respectively. Data from gene ontology (GO)
molecular functional analysis revealed that differentially expressed proteins were mainly involved in
catalytic activity, protein binding and hydrolase activity (Figure 3). Significantly enriched KEGG
pathways included those related to carbon metabolism, biosynthesis of amino acids, ribosome pathways
73
and glycolysis/gluconeogenesis (Figure 4). Furthermore, the numbers of differentially expressed proteins
related to signal transmission and transcription factors were 27 and 7, respectively.
Figure 3. GO analysis of proteins expressed during the early fruiting and fruiting stages of S. latifolia.
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Figure 4. Functional category of proteins differentially expressed during the early fruiting and fruiting
stages of S. latifolia.
Figure 5. KEGG pathways of proteins differentially expressed during the early fruiting and fruiting stages
of S. latifolia.
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The Molecular mechanism of fruiting development
We obtained the mRNA sequence of S. latifolia aegerolysin-like gene namely latifolysin by transcriptome
sequencing and analyzed it using bioinformatics methods. mRNA and protein expression were detected by
Real-time quantitative PCR and Isobaric Tags for Relative and Absolute Quantification (iTRAQ) coupled
two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS), respectively.
Latifolysin gene contained a 399 bp ORF that was predicted to encode a 133-ammino acid protein. The
deduced latifolysin contained the structure domain of aegerolysin family, showing 40% identity to
aegerolysin (Figure 6 and Figure 7). Fungal aegerolysin family proteins were inconsistently distributed
among species. Phylogenetic analysis showed that latifolysin protein was clustered with basidimycete
group and closely related to Volvariella volvacea (Figure 8). Quantitative real-time PCR and proteomics
analysis revealed that the gene and protein of latifolysin had the highest expression level in the early
fruiting stage and particularly higher than the mycelium stage (Figure 9). This study would be useful for
understanding the developmental molecular mechanism of S. latifolia.
Figure 6. Cluster analysis of predicted amino acid sequences of latifolysin.
Figure 7. Predicted tertiary structures of hemolysin from four mushrooms.
76
Figure 8. Phylogenetic analysis of some aegerolysins in fungi (Vibrio as outgroup).

Ⅰ: Basidiomycota; Ⅱ: Ascomycota
Figure 9. Differential expression of latifolysin gene at various developmental stages.
77
FACTORS AFFECTING S. latifolia GROWTH
Intrinsic factors
Carbon source
Carbohydrates are major components of the cytoskeleton, important nutritional requirements for growth
and development (Xiao et al. 2006) and some of the most important required nutrients for S. latifolia.
According to Shim et al. (1998), among 19 carbon sources, 16 carbon sources were favorable to the
mycelial growth of Sparassis crispa. Except for salicin, adonitol and trehalose, mycelial growth of S.
crispa was most favorable on culture media supplemented with maltose, followed by arabinose and
mannitol. Huang et al. (2007b) found that highest growth rate and most luxuriant mycelia were observed
when sticky rice powder served as the major carbon source. Vigorous growth was recorded on maltose,
followed by glucose or sucrose, while no mycelial growth was observed when methylcellulose served as
the carbon source. Our previous study showed that 27 g/L glucose was suitable for mycelium growth (Lin
et al. 2011). Wang et al. (2012) also confirmed that starch was the main factor impacting the dry weight of
mycelium. Farooq and Alfred Chioza (2014) reported that fructose followed by glucose supported the best
mycelial growth while the most unsuitable carbon source was galactose (no mycelial growth). In liquid
culture, You et al. (2006) found that starch and maltose were appropriate carbon sources for mycelial
growth in submerged fermentation. Productivity of S. crispa mycelia reached its maximum at an initial
glucose concentration of 30 g/L (Kurosumi et al. 2006). Our previous study showed that glucose was
favorable for formation of small mycelial pellets and maximum biomass production was 3.53 g/L.
Highest biomass (4.07 g/L) in medium was achieved when wheat ﬂour was added in liquid culture,
followed by glutinous rice ﬂour and corn starch. These findings suggest that maltose, glucose and starchy
carbon sources were suitable for mycelial growth of S. latifolia.
Nitrogen source
Shim et al. (1998) reported that glycine stimulated mycelial growth of S. crispa on culture media, but
there was no mycelial growth on culture media that were supplemented with nitrogen sources such as
methionine, glutamine, urea, histidine, ammonium acetate, calcium nitrate, sodium nitrate and potassium
nitrate. You et al. (2006) showed that corn flour and bran were optimum nitrogen sources. Accord to Lin et
al. (2007), S. crispa grew the best with culture media containing peptone, followed by vitriol ammonium
and urea. PDA with 3 g/L peptone was suitable for mother-culture media. Huang et al. (2007b) also found
that peptone, yeast extract or soybean meal was a favored nitrogen source. Farooq and Alfred Chioza
(2014) found the most favorable nitrogen source was glycine followed by alanine and this is in agreement
with findings of a previous study (Shim et al. 1998). In conclusion, organic nitrogen was more suitable for
mycelial growth compared with inorganic nitrogen.
pH
Shim et al. (1998) reported that mycelial growth of S. crispa was most favorable at pH 4, whereas there
was no mycelial growth at pH 8 and pH 9. Cheong et al. (2008) related that optimal pH for mycelial
78
growth was 6.0. According to Huang et al. (2007b), S. crispa mycelia grew over the full range of pH
values tested (4.0-7.5), but the most suitable pH was 5.25 (highest mycelial growth rate). Slightly acidic to
neutral initial agar media pH is most favorable for mycelial growth of S. crispa and PDA medium with
initial pH of 6 resulted in highest mycelial growth among pH levels tested (Farooq and Alfred Chioza,
2014).
Substrate
Mushrooms can be classified into three categories by their tropic pattern: saprophytes, parasites or
mycorrhizae. S. latifolia grows parasitically on stumps or roots of conifer trees during summer and autumn
seasons (Kim et al. 2013). Lee et al. (2004) found that highest mycelial growth of S. crispa was recorded
on larch sawdust. When S. crispa DUM-04 was cultured on a medium of larch sawdust + pine sawdust,
formation of its basidiocarps was more outstanding on media containing larch/pine sawdust than those of
only larch sawdust. Ryu et al. (2009) considered that Larix kaempferi was suitable for S. crispa cultivation
including mycelial growth period, harvest period and mushroom production, respectively. Park et al. (2011)
recommended that sawdust-based medium with larch for the cultivation of cauliflower mushroom be
prepared with 0.76 g/cm3 in medium density and excluding particles less than 1 mm. Researchers at FAAS
have focused on S. latifolia artificial cultivation techniques since 2004 and successfully cultivated this
mushroom in 2005. PDPA was the optimum mother culture medium of S. crispa while culture media from
Mango and Pinus massoniana were suitable for S. crispa growth. Rice bran and corn powder were
suitable complementary culture materials (Lin et al. 2005).
In conclusion, the most suitable substrate for mycelial growth and fruit body formation of S. latifolia is a
mixture of larch sawdust and pine sawdust such as Abies holophylla (Ryu et al. 2009), L. kaempferi (Ryu
et al. 2009), Pinus koraiensis (Oh et al. 2009), Pinus densiflora (Lee et al. 2004), Larix gmelini (Rupr.)
Kuzenneva, Larix olgensis A. Henry and Pinus yunnanensis Franch (Liu 1986).
Extrinsic factors
Temperature
S. latifolia requires low temperatures in order to form fruit bodies. The range of temperature for mycelial
growth of S. latifolia is 10～30℃ and the optimal temperature for S. latifolia mycelial growth is 23～
24℃. S. latifolia mycelia stop growing above 30℃ and serious mycelial damage occurs at 30℃.
Mycelium dies above 40℃ (Huang et al. 2007b). The optimal temperature for primordium formation was
20～21℃ (Huang et al. 2007a).
Humidity
Water is important for S. latifolia growth and production. Nutrients should be dissolved in water in order
to be absorbed by the mycelium. The suitable moisture content of substrate is 60%～65% for vegetative
growth of S. latifolia. Optimum humidity range of the incubation room is 85%～90% while during
basidiocarp formation it is 90%～95%. (Huang et al. 2007a).
79
Air composition
S. latifolia is an aerobic fungus. Well-circulated fresh air and more frequent ventilation during the
reproductive phase are important. According to our previous study, S. latifolia was very sensitive to carbon
dioxide during primordium formation (Lin et al. 2007) and carbon dioxide levels were maintained below
0.03% (Zhu and He 2008).
Luminosity
Light is the direct or indirect source of energy for S. latifolia growth. Mycelium can grow in darkness
without light, but light is required for basidiospore formation. S. latifolia needs much more light than other
mushrooms and our previous study showed that fruit body formation was possible when the light was at
500～800 Lux.
ARTIFICIAL CULTIVATION
Selection of strains with high yield and good quality
Quality strains result in high yield and good quality mushrooms. Six strains of S. crispa were tested for
their suitability for mycelial growth and primordium formation on various sawdust media in bottles. Strain
DUM-04 showed good performance for fruit body formation. Agronomic characters of the fruit bodies
were (average): 7.2 cm in height, 10 cm in diameter and the dry weight was 7 g (Lee et al. 2004). Ryu et
al. (2009) investigated cultivation characteristics of 12 stains on L. kaempferi sawdust medium. Strain
KFRI 700 showed the highest yield (163 g from 380 g sawdust media) with 103 days in the growth period.
Features of the fruit body were: 14.9 cm in width and 8.5 cm in height.
Our team has artificially cultivated S. latifolia since 2005. We have investigated agronomic characters
including biological characteristics, nutritional physiology, undesired microorganism resistance capacity
and productivity. One strain, certified by the Variety Certification Committee of Fujian Province in April
2013, was formally registered as ‘Min Xiu No.1’. This was China’s first self-owned intellectual property
variety. It was suitable for commercial cultivation with high biological efficiency and yield and harvest
time at 150～200 g/bag and 120 d, respectively. Fresh S. latifolia fruit bodies had good shelf life and were
hard and brittle with golden yellow color after drying.
Spawn making
In China, researchers tried to domesticate this mushroom early in the 1980s (Liu 1986), but little progress
was made on artificial cultivation. FAAS has focused on S. latifolia artificial cultivation techniques since
2004 and has developed a culture medium (PDPA) for mycelium growth that contains 20% potato, 2.1%
dextrose, 0.32% fish peptone and 2% agar (Lin et al. 2011). For S. latifolia, sawdust spawn is widely used
80
for its industrial cultivation because it is easy to manufacture without specific fermentation equipment.
Mango and Pinus massoniana sawdust were suitable for S. latifolia mycelial growth (Lin et al. 2005). S.
latifolia sawdust spawn was made using glass bottles (750 ml) containing P. massoniana sawdust (70%),
corn flour (28%) and calcium carbonate (2%). After inoculation with mycelial plugs from PDPA slants,
spawn was incubated at 25ºC in the dark until the substrate was completely colonized. According to our
previous study, liquid spawn led to rapid mycelial growth on sterile pine sawdust substrate compared to
traditional sawdust spawn (Ma et al. 2016). Further work on primordium development and fruit body
formation by inoculating with liquid spawn is under investigation in our laboratory.
Substrate composition, spawn run and fructification
Korean scientists first artificially cultivated S. latifolia in 2004. Lee et al. (2004) found highest mycelial
growth on larch sawdust, but larch + pine sawdust was more effective in forming fruit bodies than larch
sawdust. Culture media included: L. kaempferi (60%), P. densiflora (20%), wheat bran (15%), and glucose
(5%). According to Ryu et al. (2009), S. crispa strain KFRI 700 had the highest yield at 163 g from 380 g
L. kaempferi sawdust media. Substrate used to cultivate S. latifolia contained pine sawdust (76%), wheat
bran (18%), corn flour (2%), sucrose (2%) and calcium superphosphate (2%). The dry ingredients were
thoroughly mixed and then tap water was added to reach a moisture content of 65%. A bag-filling machine
was used to fill and compact substrate into polyethylene bags (17 cm in diameter and 36 cm long), a single,
vertical hole was made in the center of the compacted substrate for the spawn inoculation and aeration.
The bags were enclosed with plastic rings and vent caps, autoclaved at 122℃ for 2.5 h, then bags were
cooled down to room temperature. The bags were inoculated with sawdust spawn (15±2 g) into the
vertical hole of the substrate. Inoculated bags were incubated at 24±1℃ under dark conditions. After the
substrate was completely colonized by the mycelia, the bags were then transferred into the fruiting
chamber where the environmental conditions were maintained at 20～21℃, and 85 %～90% relative
humidity, with a 10 h illumination. When primordial formed, the necks and covers were removed from the
bags. Only a single flush of fruit bodies was harvested.
Cultivation
Until now, there are only few farms in Japan, Korea and China where this edible and medicinal mushroom
is cultivated on an industrial scale. In the 1980s, researchers in Japan began to isolate wild strains of S.
latifolia and initiated efforts to domesticate this mushroom including suitable culture media selection,
identifying environment conditions for primordium formation, etc. In 1993, S. latifolia was successfully
artificially cultivated. Bottle cultivation was realized in 1996. After 2000, artificially cultivated S. latifolia
captured consumer interest in the Japan mushroom market. At present, there are some companies that
cultivate this mushroom, including UNITIKA (Kwon et al. 2009, Yao et al. 2008, Yamamoto and Kimura
2010, Yamamoto et al. 2009, Yoshikawa et al. 2010, Yamamoto and Kimura 2013), Minahealth (Ohno et
al. 2000, Harada et al. 2002a, b), etc., but cultivation techniques were not known to the public.
81
In order to shorten harvest time and improve yield performance, biological and cultivation characteristics
of S. latifolia have been studied systematically by our research group. Huang et al. (2007a) and Jia et al.
(2010) also reported a cultivation technique for S. crispa. Liu et al. (2010) found that S. crispa mycelial
growth was rapid on pine sawdust after high temperature and pressure treatment and this process is now
used for industrial cultivation.
Figure 10. Commercial cultivation factories of S. latifolia in China
FUTURE PERSPECTIVES
S. latifolia is an edible and medicinal mushroom that has been cultivated in recent years. The survival and
multiplication of S. latifolia is related to a number of factors that may act individually or have interactive
effects. A combination of these factors may have a complicated effect on production of S. latifolia. With
intensive studies on its medicinal value, a number of new related products were successfully produced. In
our country, research on S. latifolia developed late but is progressing rapidly mostly related to breeding,
biological characterization, artificial cultivation, extraction of bioactive compounds, etc. Although we
have made substantial progress additional work remains. Collection, conservation and domestication of
germplasm resources of S. latifolia should receive more attention in the future. Breeding high yield,
good quality and disease-resistant cultivars of S. latifolia that are suitable for industrial cultivation are
crucial. And finally, bioactive compound extraction and its pharmacological action should also be
emphasized.
ACKNOWLEDGEMENTS
This work was partially funded by the Special Fund for Scientific Research in the Public Interest of Fujian
Province (2014R1020-1, 2014R1020-4) and by the Science and Technology Innovations Program at
Fujian Academy of Agricultural Science (STIT-I-0309).
82
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7. EFFECT OF SOAK TIME AFTER HARVEST ON NUTRITIONAL QUALITY OF

Tremella fuciformis DRY PRODUCT
LU Zhenghui1, KE Binrong1, PENG Dongxiang2, CHEN Yong-hui2, YU Xinmin2, WANG Zesheng1
1
Institute of Edible Fungi, Fujian Academy of Agricultural Science, Fuzhou 350014, China;
2
Edible-fungi Industry Adiminstration Gutian, Standardization Working Group on Tremella of
Standardization Administration of China, Ningde, Fujian, 352200, China
ABSTRACT
This work examined the influence of soak time prior to drying Tremella fuciformis on sensory quality and nutrient
composition. Soak time caused changes of pH of soak water, affecting color and tightness of the dry product. Soak time
was linearly related to dry matter and ash content while changes of crude protein, crude fiber, crude fat and crude
polysaccharide were less affected by soak time.
Keywords: Tremella fuciformis, soak time, nutritional quality, dry product
INTRODUCTION
Tremella fuciformis is an important edible fungus and is one of the historical treasures of China (Huang
2000). Wild T. fuciformis is widely distributed in China, but it is difficult to find specimens. At present, the
vast majority of T. fuciformis is cultivated on sterilized substrate contained in bags (Peng et al. 2005). Gutian
County of Fujian Province is the main producing area of T. fuciformis with an annual output of 30,000 tons,
accounting for more than 90% of the national production of T. fuciformis. Products are exported to 27
countries and regions including Europe, United States and Southeast Asia. Because fresh fruit bodies of T.
fuciformis do not have a long shelf life, they are usually stored and sold as dry product (Huang and Zheng
2009).
Before drying, freshly-picked T. fuciformis are soaked in water to remove debris and improve consumer
appeal (Wang and Yang 2007). There is no uniform standard for soak time for T. fuciformis and soak water
waste is a concern for local environments. Therefore, we began this work as commissioned by the
Standardization Administration of China to examine the influence of soak time during pre-processing of T.
fuciformis on sensory quality and nutrient composition. We sought to: 1) better understand the effect of soak
time on quality, 2) assess the potential effect of discharge water on the environment and 3) develop relevant
standards to guide production and processing of this mushroom.
87
MATERIALS AND METHODS
Materials
Fresh T. fuciformis fruit bodies produced in Gutian county were used as raw materials. Research was
conducted at Yancang Edible Fungus Professional Cooperatives in Daqiao, Gutian county.
Test design
Fresh cut fruit bodies (40 kg) of T. fuciformis from the same producer were used for soak treatments (Table
1). Mushrooms for all treatments were completely immersed (soaked) in potable water in the same size
containers. After soaking, mushrooms were placed on bamboo mats. Samples (0.5 kg) of fruiting bodies
were randomly selected from each treatment after drying and evaluated for sensory quality and other
parameters.
Table 1. Soak times for five treatments of Tremella fuciformis before drying.
Trt. No Soak time (min)a

1 Not soaked
2 5
3 30
4 60
5 120
a
Excess water was removed after soaking
Determination of indicators and methods
pH of soak water
After soaking, pH value of the water (23º C) was measured and 500 ml of soak water samples were taken.
pH was measured again after standing 24 h and 5 days.
Sensory quality evaluation

After drying, shape, cleanliness and color of fruit bodies were estimated by sensory evaluation while
diameter, height and other indicators were recorded.
Determination of nutrient composition

Analyses were made for moisture, crude protein, crude fat, crude fiber, crude polysaccharide, ash, colloidal
protein, etc.
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Figure 1. Fruit bodies of Tremella fuciformis Figure 2. Fruit bodies of Tremella fuciformis
after soaking and cleaning. after drying with hot air.
RESULTS AND ANALYSIS
Changes of pH value of water after soaking of Tremella fuciformis
The pH values of water collected after soaking tended to decrease with increasing soak time and increasing
time of incubation. The pH value of Treatment 2 (5 min soak) was 6.7 while the pH value of water soak
treatment of 30 min - 120 min decreased to 5.4 - 5.7 (Table 2). pH value of treatment 2 was 6.7, 6.0 and 5.2,
immediately after soaking, at 24 h and 5 days, respectively. The pH value of water samples for Treatments 3
to 5 reduced by 0.2 - 0.4 with slightly acid water smell. After the water samples were kept in the
environment at 25°C for 5 days, the pH value of treatment 2 samples decreased from 6.0 to 5.2 and the water
had a slightly acid odor. The pH value of the water samples of treatments 3 - 5 reduced to 4.2 - 5.0 and the
water was malodorous. Odor ranged from normal water smell to weak acidity for Treatment 2 over the
5-day period (Table 2).
The influence of soak time on color and shape of Tremella fuciformis
Length of soaking time had a substantial influence on color and shape of T. fuciformis (Table 3). The color of
non-soaked treatment 1 was the most yellow. The longer the fruit body was soaked the lighter the color.
After 120 min soak time, dry fruit body color change was difficult to distinguish.
The shape of fruit bodies of treatment 1 was compact and tight. As soak time increased, the shape of the
whole fruit body (treatments 2-5) became looser and the spread was larger. After 60 min soak time, fruit
body shape tended to stabilize. Soak time did not appear to have much effect on the average height of the
whole fruit body.
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Table 2. pH value and “odor” of water after different soak times (not soaked or soak times)
Treatment No. Standing water time after soaking fruitbodies
(soak time) (pH/odor)
Water immediately after Water after standing for Water after standing for
soaking 24 h 5 days
1 (not soaked) - - -
2 (5 min) 6.7 6.0 5.2
Odor normal Odor normal Odor slightly acid
3 (30 min) 5.7 5.5 5.0
Odor normal Odor slightly acid Odor sour
4 (60 min) 5.6 5.2 4.4
5 (120 min) 5.4 5.3 4.5
Table 3. Influence of soak time on color and shape of T. fuciformis

Fresh
The average The average Dry weight
No. weight Color1 Tightness2
diameter（cm)3 height（cm)3 （kg）
（kg）
1 4 Yellow 1 Tightness - 5 10.8 5.8 1.30
2 4 Yellow-2 Tightness - 4 12.9 6.0 1.28
3 4 Yellow-3 Tightness -3 13.5 6.0 1.30
4 4 Yellow-4 Tightness - 2 13.6 5.6 1.28
5 4 Yellow-5 Tightness -1 13.4 6.0 1.25
Note：
1
Yellow 1-5, where 1= darker yellow and 5= lighter yellow.
2
Tightness 1-5, where 1=slightly tight and 5=tightest
3
Average measurement of 10 random samples each.
Influence of different soaking time on nutritional components of Tremella fuciformis
The comparison of nutrients as affected by soak time is presented in Table 4. It is observed there that the
water content of each treatment of dry product of T. fuciformis was less than 15%, according to the national
standards, they meet the water content requirements of dry products. Also, the nutrient contents after T.
fuciformis drying is presented at different water soaking times (Table 5).
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Table 4. Comparison of nutritional components of Tremella fuciformis as affected by soak time

Crude
Moisture Crude Crude Crude
No. Ash% polysaccharide Collagen
% protein % fiber % fat%
%
1 11.8 7.9 8.8 2.4 1.8 9.0 not detected
2 11.1 7.8 10.0 2.2 2.6 6.6 not detected
3 13.8 6.8 10.1 2.6 2.0 10.0 not detected
4 13.7 5.8 10.2 2.6 2.3 7.8 not detected
5 12.4 6.2 10.0 2.3 2.2 9.7 not detected
Table 5. Nutrient content of dried Tremella fuciformis after different soaking time
Unit: kg
Crude
water Crude Crude Crude
No. Ash% polysacch Collagen
content % protein % fiber % fat%
aride %
1 1.147 0.091 0.101 0.028 0.021 0.103 0
2 1.138 0.089 0.113 0.025 0.03 0.075 0
3 1.121 0.076 0.113 0.029 0.022 0.112 0
4 1.105 0.064 0.113 0.029 0.025 0.086 0
5 1.095 0.068 0.11 0.025 0.024 0.106 0
1.16
y = -0.0137x + 1.1623
1.15
R² = 0.9895
1.14
1.13
Dry matter（kg）
1.12
1.11
1.1
1.09
1.08
1.07
1.06
1 2 3 4 5
Treatment
Fig.3 Column chart of dry matter after different treatment
91
1.16
1.15
1.14 y = -0.0137x + 1.1623
R² = 0.9895
1.13
1.12
Ash（kg）
1.11
1.1
1.09
1.08
1.07
1.06
1 2 3 4 5
Treatment
Fig.4 Column chart of ash after different treatment
As soak time increased, total weight and ash of dry products decreased - this trend was linear (Table 4, Figs
3-4). The longer T. fuciformis absorbed water the more substances were lost. Highest loss of dry substances
and ash occurred in Treatment 5. Although there were differences in aspects of crude protein, crude fiber,
crude fat and crude polysaccharide, depending on treatment, no apparent linear changes were observed.
Therefore, we concluded that the changes of crude protein, crude fiber, crude fat and crude polysaccharide
were not related to soak time.
DISCUSSION
As soak time increased, the pH of soak water gradually decreased. The reason may be that nutrient
substances of T. fuciformis fruit bodies were dissolved into the water. These substances would provide
nutrients for bacterial growth and reproduction and thus, cause water rancidity. Therefore, freshness of soak
water should be monitored in real-time and changed in time to reduce microbial growth. Soak time
significantly affected the sensory quality of T. fuciformis, such as color and shape, and appears to cause the
loss of the nutrient substances. Therefore, soak time of T. fuciformis should be well controlled under
practical operation, to avoid a drop in nutritional value (Huang et al. 2009).
Changes in contents of crude protein, crude fiber, crude fat and crude polysaccharide were slightly affected
by length of soak time. Protein components are just part of the fruit body and free state protein components
were not lost. Soak water used in these experiments was well water with normal temperature. Therefore,
degeneration, decomposition and precipitation of protein components were minimized. Crude fiber is the
main portion of the cell wall of the fruit body and since it is not soluble, it remained intact. Most of
polysaccharides were not dissolvable, so the soaking time would not cause the loss of polysaccharides. Most
fungal polysaccharides are insoluble in water so soak time does not affect polysaccharides content (Chen
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2011). Polysaccharide content of T. fuciformis was not affected by soak time.
After cooking, the soup of T. fuciformis is “sticky” while the fruit body is gelatinous. Many consumers
believe T. fuciformis is rich in gelatin, which is considered to be collagen. Collagen is nutritional
complement for anti-aging and exists in the animal body. Hydroxyproline is the characteristic amino acids
of collagen. Collagen was not detected in the 5 samples tested. There are two possibilities. Firstly, there is no
collagen in the fruit body of T. fuciformis. Secondly the content of collagen of the fruit body of T. fuciformis
is quite low and can’t be detected (Huang et al. 2010, Liu et al. 2006). The minimum detection volume was
0.1mg/100 g. The materials we studied show that the fruit body of T. fuciformis doesn’t contain this kind of
protein. Therefore, it is presumed that T. fuciformis does not contain collagen. The gelatin of the fruit body
of T. fuciformis is a kind of mucopolysaccharide that is rich in glucose, trehalose, acid pentose, mannitol, etc.
Mucopolysaccharide is the essential substance of the cell cytomembrane, similar in structure to
hyaluronic acid with excellent moisturizing properties (Yan et al. 2006).
It is assumed that the length of soak time mainly depends on consumer and market demand of the shape,
looseness and color of T. fuciformis. Meanwhile, it is necessary to correctly guide sales of T. fuciformis in
order to make sure quality and nutrition are considered first followed by appearance.
ACKNOWLEDGMENTS
This research was supported by the Standardization Working Group on Tremella of Standardization
Administration of China. The authors are grateful to Yancang Edible Fungus Professional Cooperatives in
Daqiao town of Gutian county.
REFERENCES
Chen G (2011) Functional characteristics and application of Tremella polysaccharides. China Food
Additives (4):141-144.
Huang JL, Huang Y, Zheng BD (2009) Study on Optimization of Combined Hot-air and Microwave
Vacuum Drying Technology for Tremella fuciformis. Agricultural Science Bulletin. 25(22):88-91.
Huang NL (2000) China Tremella fuciformis production. China Agricultural Press 1-187.
Huang Y, Huang JL, Zheng BD (2010) Microwave vacuum drying properties and kinetics model of white
fungus. Transactions of the CSAE 4:362-367.
Huang Y, Zheng BD (2009) Study on Optimization of Microwave Vacuum Drying Technology for Tremella
fuciformis. Chinese Agricultural Science Bulletin 25(20):82-89.
Liu PX, Gao XR, Xu WQ, Zhou ZW, Shen X (2006) Antioxidation activities of polysaccharides extracted
from Tremella fuciformis Berk. Chinese Journal of Biochemical Pharmaceutics. 26(3):169-170.
Peng WH, Wang Y, Huang ZQ, Gan BC(2005) The present situation of Tremella fuciformis research and
93
problems existed in china. Acta Edulis Fungi 12(1):51-56.

Wang CY, Yang WB (2007) A Study on the Technique of Reversible Hot Air Drying for Tremella
Fuciformis. Acta Agriculturae Universitatis Jiangxiensis 29(1):158-163.
Yan J, Guo XQ, Wu XY (2006) Research on the Ability to Scavenge Free Radical of Tremella
Polysaccharide. Journal of Chengdu University (Natural Science) 25(1):35-38.
94
8 FRUIT BODY PRODUCTION OF Schizophyllum commune
Silvia Cappello García1, Santa Dolores Carreño Ruiz1 and Rigoberto Gaitán Hernández2
1
Universidad Juárez Autónoma de Tabasco. Carretera Villahermosa-Cárdenas km 0.5, entronque a
Bosques de Saloya, Villahermosa, Tabasco, Mexico <[email protected]>
2
Instituto de Ecología, A. C. carretera antigua a Coatepec 351, El Haya, C.P. 91070, Xalapa, Veracruz,
Mexico
ABSTRACT
Cultivation of tropical edible mushrooms has generated an enormous and growing interest because it
represents a new alternative for various sectors to produce beneficial food products using environmentally
friendly biotechnologies. In particular, the academic sector has prompted development of research to
diversify means and techniques of cultivation while also integrating various aspects such as traditional
knowledge and use of local genetic resources. In this chapter, data on biology and consumption of
Schizophyllum commune Fr. in tropical regions are presented in addition to observations on cultivation of
this fungus under semi-controlled conditions on several agricultural by-products from Tabasco, México.
Keywords: edible mushrooms, basidiomycetes, mushroom cultivation, fungal technology
INTRODUCTION
In recent years, efforts have increased in Latin America to integrally understand diversity of macroscopic
fungi in different environments. Edible and medicinal mushrooms have been catalogued in micro-floristic
studies and their sustainable cultivation on agricultural by-products, which presents multiple benefits for
both humans and the environment. However, in spite of being a relatively accessible activity with a
growing tendency, knowledge on fungi cultivation in tropical areas has been generated to a lesser extent as
a result of multiple factors.
Since substantial research exists on edible mushrooms and their cultivation in temperate climates but not
tropical climates, one strategy has been to develop sustainable production of commercial strains with the
capacity to produce fruit bodies at an average ambient temperature of 30 °C. This strategy was based on
experiments and improvements in the laboratory as well as in conditioned cultivation chambers, in
addition to considering other technical aspects. In this way, greater knowledge on fungi production,
including appropriate conditions and culture practices, has been generated. Even so, the majority of
commercial species optimally produce fruit bodies at an average temperature of 20 °C (Marshall and Nair
2009).
Another cultivation strategy has focused on the use of available genetic resources in the region, employing
native strains that are adapted to conditions of a tropical climate in addition to developing various
cultivation processes and techniques that include substrate management and that also consider the
economic and cultural characteristics of local populations. With this strategy, traditional knowledge has
formed a fundamental part of the development of cultivation techniques and related research on this theme
has undoubtedly increased the growth opportunities for mushroom cultivation.
In Mexico, several fungal species are cultivated using imported strains, such as button [Agaricus bisporus
(J.E. Lange) Imbach] and oyster mushrooms [Pleurotus ostreatus (Jacq. ex Fr.) Kumm] that are
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merchandized in supermarkets, street markets and small fruit and vegetable shops, mainly in the central
and southern regions of the country. However, at least 371 wild species of edible mushrooms have been
identified (Garibay and Ruan 2014). At least one dozen of these may be distinguished for their important
ties to local and indigenous cultures and are currently widely accepted and consumed amongst rural
populations of temperate and tropical zones. Given the biology of these species as facultative saprophytes,
they are ideal candidates for experimentation in order to examine their cultivation potential. Amongst
these tropical edible species, many fungi belonging to the genus Pleurotus, Hydnopolyporus, Lentinus,
Auricularia, Polyporus and Schizophyllum are found (Moreno-Fuentes 2014).
Especially, in the state of Tabasco, Auricularia delicata (Fr.) Henn, A. polytricha (Mont.) Sacc., Pleurotus
djamor (Rumph. ex Fr) Boedijn and Schizophyllum commune are traditionally eaten, mainly in the
municipalities of Macuspana and Teapa. The frequent sale and cultural importance of S. commune have
also been highlighted. In this regard, research on the management of strains of these species and their
production is of great importance, with the goal of developing a technical guide to facilitate production of
these fungi for diverse sectors of the population.
At the Universidad Juárez Autónoma de Tabasco (UJAT), studies have been carried out since 2012 to
obtain and characterize native strains of the aforementioned species. Comparative tests of mycelial
growth were performed in vitro using various substrates, such as banana leaves (Musa paradisiaca), cacao
shells (Theobroma cacao), coconut fibre (Cocos nucifera) and cedar sawdust (Cedrela odorata) (Carreño-
Ruiz et al. 2014).
Schizophyllum commune is a species, that due to its medicinal and gastronomic properties, as well as its
resistance to high temperatures has generated an interest in the continuity of these studies realized in the
UJAT to optimize its production. One of the advantages of S. commune is its cartilaginous texture, which
compared with other species of fleshy edible fungi, is more resistant to decomposition in tropical climates.
In this chapter, experiences in cultivating this species using agricultural by-products of the region are
described.
BIOLOGY AND CULTIVATION OF Schizophyllum commune
Several studies have shown that S. commune has a wide distribution around the world, except in
Antarctica (Chang and Lui 1969, Adejoye et al. 2007) and grows throughout the year (Degreef et al.
1997). This fungus is found on trunks of fallen trees, on recently cut branches, on dead wood and on more
than 300 live plant hosts (Vázquez-Mendoza 2012). It develops in areas covered with vegetation or in
grasslands, can survive during the dry season or on wood exposed to sun (Mata 1999) and leads to the
white rotting of wood. It has been found growing in numerous types of vegetation like tropical and
subtropical forests, tropical montane cloud forests, Quercus, Pinus and Abies forests, thorn forests,
xerophytic scrub, grasslands, coastal and urban vegetation and fruit crops (Olivo-Aranda and Herrera
1994). In the state of Tabasco, it has been found on banana pseudostems, fallen wood of ‘palo mulato’
(Bursera simaruba), cacao shells and coconut fibre, amongst other substrates.
This species is characterised by a flabelliform or fan-shaped fruit body of 7 to 25 mm in width and 10 to

30 mm from the base toward the outer edge (Figure 1 A). It presents sessile to substipitate pilei with
irregular edges that are leathery in texture. The pileus is greyish white (identified with the key N10M00C00
according to the Color Guide of Küppers 1996) and the upper surface is hairy. The hymenium presents
longitudinally split gills (lamella) (Figure 1 B) that are light brown in color (N00A10M00-Kuppers 1996)
(Carreño- Ruiz et al. 2014).
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Other morphological forms of the species also are found. For example, sessile spatulate or with
psuedostipitates or with semi-circular and connate forms. Varying sizes and colors are also found,
depending on the state of development of basidiocarps, availability of nutrients and environmental
conditions.
Schizophyllum commune has long generated human interest and is recognised by various ethnic groups
and common names in distinct languages. Initial records have shown that this fungus was both eaten and
used as a type of chewing gum (Cooke 1961).
More recently, several research studies were carried out highlighting the consumption of this species in
countries such as Mexico (Olivo-Aranda and Herrera 1994), India (Longvah and Deosthale 1998), Japan
(Paulí 2000), Nigeria (Adejoye et al. 2007), Guatemala (Bran-González et al. 2009), the Philippines
(Reyes et al. 2013), Malaysia, Thailand (Mirfat 2014) and Argentina (Figlas et al. 2014). Related research
studies have been initiated to obtain native strains, to examine mycelial growth and to evaluate the fruiting
process of this fungus. In addition, Boa (2005) highlights the consumption of this species in other
countries such as Benin, China, Ethiopia, Ghana, Hong Kong, Madagascar, Malawi, Peru, the Central
African Republic, Laos and Zambia, where, in addition to being considered a food resource, it has also
been used in traditional medicine.
A B
Figure 1. A) Flabelliform basidiocarps of Schizophyllum commune (photo by Mario Eduardo Sosa).

B) Lamellae of the hymenium in detail (photo by Víctor Herman Gómez García).
In Mexico, S. commune is reported in nearly all states. However, its consumption has mainly been
recorded in states of the south-eastern region, such as Veracruz (where it is known as ‘chiquite’ or ‘hongo
de chaca’), Puebla (‘oreja de ratón’), Oaxaca (‘hongo blanco’), Chiapas (‘xikin che’, ‘afumaditos’,
‘cascarilla de madera’, ‘pajarito’, ‘sulte’ and ‘tehuitzhanacame’), Tabasco (‘oreja de palo’, ‘cusuche’,
‘muca’ and ‘orejita criolla’) and Quintana Roo (‘oreja de palo’) (Guzmán 1990, Guzmán 2003, Ruan-Soto
et al. 2004, Ruan-Soto et al. 2006, Ruan-Soto et al. 2009, Ruan-Soto and Cifuentes-Blanco 2011,
Vázquez-Mendoza 2012).
In the coastal plains of the Gulf of Mexico, this species is frequently sold and is also abundant in some
traditional markets in Oaxaca and Tabasco. Sellers are largely indigenous women who commercialise
either small sacks or larger quantities, demonstrating the wide presence and consumption of this species in
the southern region of the country (Ruan-Soto et al. 2006).
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In regard to its preparation, in Teapa, Tabasco, S. commune is used to make several traditional dishes,
such as the so-called ‘mone’ (Ruan-Soto et al. 2006, Ruan-Soto and Cifuentes Blanco 2011), amongst
others. In the Northern Sierra of Puebla, this species is used in soups or as a slight stimulant (Vázquez-
Mendoza 2012).
Generally, the sale of S. commune occurs during the rainy season. Both sellers and consumers have
demonstrated preference for larger fruit bodies of white color, although the diverse morphologies and
variations of this species are also sold in markets or collected for self-consumption (Figure 2).
A B
Figure 2. A) Sale of S. commune along with other regional products in the Diana Córdova de Balboa market in
Teapa, Tabasco. B) Presentation of S. commune along with suggested ingredients for its preparation (photos by Santa
Dolores Carreño Ruiz).
With respect to the nutritional properties of S. commune, studies have found a protein content of 16% to
27% (Aletor 1995, Longvah and Deosthale 1998), a low-fat content of 2% (20 g/kg in dry weight) and
abundant oleic and linoleic fatty acids. The fungus also contains 34% of amino acids essential to the
human diet (Longvah and Deosthale 1998).
In addition, S. commune possess medicinal properties, such as antibiotic, antitumor, antioxidant and
anticarcinogen properties, highlighting its pharmacological importance. This fungus produces
immunostimulant polysaccharides, schizophyllan or sonifilan ((1 3)-β-D-glucans with ramifications (1 6)-
β-D-glucosyl) that are widely used for treatment of cervical cancer, mainly in Asian countries (Ooi and
Liu 2000, Chang and Miles 2004). In traditional Chinese medicine, S. commune is used in the form of
infusions to treat leucorrhoea (Ying et al. 1987, Chang and Miles 2004, Hobbs 2005, Adejoye et al. 2007,
Calonge 2011, Vázquez-Mendoza 2012) and has also been used for regulating blood pressure (Boa 2005).
With respect to its cultivation, S. commune has been little studied at the international level for production
purposes (Chang and Miles 2004). To date, methods to produce its fruit bodies were developed in
Guatemala by Bran-González et al. (2009), who obtained basidiocarps of this species on various
substrates, including pine shavings, dry coffee pulp, maize cobs and stalks, and oat and wheat flours. This
98
latter study established parameters for their optimal growth and development in this region and also
experimentally studied different parameters in the production of fruit bodies.
Meanwhile, Figlas et al. (2014) evaluated growth of S. commune on sunflower seed residues from the oil
industry in Argentina, suggesting that discarded sunflower shells may be used as a nutrition source and as
a substrate for the cultivation of this species on synthetic trunks. This technique, in addition to substrate
supplementation with wheat bran, was shown to significantly improve basidiocarp production.
Important and complementary studies exist on mycelial growth of S. commune on various culture media
and substrates at average temperatures ranging from 26 to 30°C. These studies contain valuable references
for the adequate isolation, conservation and management of the strains (Adejoye et al. 2007, Bran-
González et al. 2009, Teoh and Don 2012, Figlas et al. 2014, Carreño-Ruiz et al. 2014).
CULTIVATION OF Schizophyllum commune IN TABASCO
In 2012, a collection of edible macroscopic mushrooms was started in the Tropical Mycology Laboratory
of the UJAT (Cepario Cappello-García-CCG). To date, twelve strains of different species have been
collected with five corresponding to the genus Schizophyllum. Several of these strains were deposited in
the mushroom collection of the Instituto de Ecología, A.C. INECOL. Xalapa, México (Cepario de Hongos
comestibles IE) (World Data Centre for Microorganisms) (Figure 3).
Figure 3. General aspect of the culture Schyzophyllum commune strain CCG003 on PDA medium (photograph by
Santa Dolores Carreño Ruiz).
For fruiting experiments of S. commune, mainly cacao shells and coconut fibre were used. These
substrates are collected fresh and then cleaned and sun dried for approximately five days. Afterwards, the
cacao shells are broken into pieces of 2 to 3 cm2 and the coconut fibres into strips of 5 cm in length. Then,
the substrates are hydrated for 12 h. Excess water was subsequently drained until the approximate
moisture content of 70% was reached, and 0.1% lime and gypsum were added. A thermal treatment was
performed by immersing the substrate, contained in a metal recipient, in water at 80 °C for 1 h. Before
inoculation, the substrate was drained and cooled.
99
The cultivation of S. commune was performed in a small greenhouse under semicontrolled conditions
using a system of hanging bags (Figure 4 A) in which plastic, transparent bags of 35 x 45 cm were hung
after manually being filled with alternating layers of the primary inoculum and substrate until reaching 1
kg (wet weight). Primary inoculum was grown on popcorn kernels (Zea mays L. var. everta).
The incubation period was under conditions of darkness during 16 days at ambient temperature. On the
following day of inoculation, 35 perforations were made with dissecting needles and radially distributed to
favor oxygenation. Initially, mycelium aggregates (joint of hyphae) appeared at the zones of perforation
after approximately 12 days of incubation (Figure 4 B). When the mycelium had covered the substrate,
bags were exposed to light in order to stimulate formation of primordia. And to allow for adequate
development of fruit bodies, in this period cuts were then made in the form of an ‘X’ on the plastic bag
and daily watering was performed to maintain a relative humidity of 75% to 85%. After maturation, fruit
bodies were harvested (Figure 4 C).
Figure 4. A) Incubation of S. commune cultures under conditions of darkness using a system of hanging bags. B)
mycelium aggregates during the incubation period and C) formation of primordia on the substrate (coconut fibre)
(photographs by Santa Dolores Carreño Ruiz).
Biological efficiency, yield and rate of production (Gaitán-Hernández et al. 2006) were obtained.
Likewise, periods of incubation and appearance of fruit bodies were recorded, including variables such as
temperature, total harvest and size and fresh weight of fruit bodies. The above data were based on a single
crop.
Production of fresh fruit bodies of S. commune on cacao shells and coconut fibres encompasses a cycle of
approximately 47 days from time of inoculation. The appearance of primordia occurs two days after
exposing the samples to light. Four days after, the harvest period is initiated for fungi grown on cacao
shells, whilst the harvest of fungi grown on coconut fibres can vary by one day. Similarly, duration of the
fruiting period is approximately 23 days.
These time periods are similar to those reported by other authors such as Bran-González et al. (2009) who
reported that the cultivation of S. commune on maize stalks and corncobs (1:1) had a duration of 28 to 48
days, including the time lapsed from inoculation to the harvest of basidiocarps. Figlas et al. (2014)
recorded a cultivation cycle of 36 days and a reduction to 31 days using supplemented sunflower seeds.
100
With respect to biological efficiency of substrates containing cacao shells and coconut fibres, values of
12.35% and 7.7% were obtained, respectively, which were slightly higher than the biological efficiency
(5.5%) reported by Bran-González et al. (2009) for a Guatemalan strain of S. commune cultivated on
maize stalks and cobs (1:1). Figlas et al. (2014) cited a higher value of biological efficiency (48.3%)
using supplemented sunflower seeds in studies carried out in Argentina.
With respect to morphology of fruit bodies, flabelliform pilei have been observed (Figure 5 A) in addition
to connate formations (Figure 5 B), with an average size of 9 to 40 mm and 15 to 50 mm in diameter,
respectively. Larger sizes have been recorded for basidiocarps harvested on cacao shells.
During the harvest period, temperatures of 28 to 40°C were reported. Greater production was obtained
with temperatures surpassing 30°C, demonstrating the resistance of S. commune to relatively high
temperatures.
Contamination by bacteria or other fungi has not proven a problem. Although it is indispensable to
disinfect the substrate to maintain aseptic conditions during culture, this species shows resistance to other
microorganisms, which could be related to its antibacterial and antifungal properties. Studies on
production of metabolites by S. commune have confirmed the effective medicinal properties of this fungus
(Mirfat et al. 2014). However, it is essential to maintain daily cleaning of the greenhouse or production
area.
Other important factors for development of fruit bodies are moisture content of the substrate and the
environment, given that high temperatures can cause a decrease in their ability to retain moisture. Even so,
S. commune can adapt to relatively dry environments and can resume growth and development when
humidity conditions become more favourable. Cooke (1961) and Vellinga (2013) have confirmed that,
after loss of humidity, the fruiting bodies of this fungus are able to return to their initial conditions
following a recovery in humidity levels.
A B
Figure 5. Shape patterns of pilei in Schizophyllum commune cultivation. A) Flabelliform pilei and B) connate pilei on
cacao shells (photographs by Santa Dolores Carreño Ruiz).
PERSPECTIVES AND RECOMMENDATIONS
In Tabasco, the quantity of residues generated by the most intensive agricultural activities is estimated at
approximately 16,000 t per year. Cacao and coconut production generate approximately 4,000 t of
101
residues in the form of fibrous shells (SIAP, 2016). Currently, these latter substrates are not reused and
therefore could represent a valuable resource for S. commune production, as fruiting bodies occur
naturally on these substrates.
Several aspects of S. commune production require further exploration. Evaluation of other substrates in
addition to those previously mentioned, both alone or as a mixture, and the effects of fermentation
processes should also be investigated. Despite the documented information on cultivation of this
mushroom, the role of incubation systems and the appropriate conditions for cultivation rooms constitute
other fundamental topics of study in tropical regions. In this sense, the optimisation of production and, in
particular, the production of certain fruit body morphologies represents other challenges to address.
Currently, at the UJAT, considerable interest lies in evaluating cultivation of this species under distinct
production schemes, for example, in systems exposed to the environment that utilise other substrates such
as sugarcane bagasse (Saccharum officinarum) (Figure 6 A) or different types of wood shavings (Figure 6
B).
This experience may be added to efforts to incentivise cultivation of S. commune in tropical regions.
Culture techniques should be improved to achieve greater biological efficiency and to develop additional
aspects of production. Production capacity of this species under semi-controlled conditions as well as its
resistance to high temperatures may be highlighted as important qualities.
Furthermore, the improvement of culture techniques is indispensable in order to maintain the accessibility
and viability of S. commune production for interested growers and to promote its consumption in the rural
sector as a strategy for mitigating the lack of highly nutritional foods. In addition, developed culture
systems can have multiple benefits for rural environments, such as, for example, the use of agricultural by-
products.
A special point of interest is to keep studying this fungus, since there have been some reports indicating
that under certain conditions it can be considered as pathogen for humans, causing mainly infections (Rihs
et al. 1996, Kamei et al. 1999).
A B
Figure 6. A) Fruit bodies of S. commune on sugarcane bagasse and B) wood shavings (photographs by Silvia
Cappello García).
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ACKNOWLEDGMENTS
This study was carried out with the support of financing from FOMIX-CONACYT for the projects
‘Diversity and conservation of macro and microscopic saprophytic fungi in several environments of Agua
Blanca State Park, Macuspana, Tabasco’ (‘Diversidad y conservación de los hongos macro y
microscópicos saprobios de algunos ambientes del Parque Estatal Agua Blanca, Macuspana, Tabasco’)
and ‘Evaluation of the production of edible mushrooms at a pilot scale’ (‘Evaluación de la producción de
hongos comestibles a nivel de planta piloto’).
Thanks are extended to the population and mushroom sellers of Teapa, Tabasco, who provided invaluable
information for the development of this research. Likewise, the support of the academic sector and of
students who have directly or indirectly contributed toward this project are appreciated.
REFERENCES
Adejoye OD, Adebayo-Tayo BC, Ogunjobi AA, Afolabi OO (2007) Physicochemical studies in Schizophyllum
commune (Fries) a Nigerian edible fungus. World Applied Sciencies Journal, 2: 73-76.
Aletor VA (1995) Compositional studies on edible tropical species of mushrooms. Food Chemistry, 54: 264-268.
Boa E (2005) Los hongos comestibles silvestres. Roma: Food and Agriculture Organization.
Bran-González MC, Morales-Esquivel OI, Flores-Arzú RE, Cáceres-Staackmann RA (2009) Caracterización y
producción de cuerpos fructíferos de cepas nativas del hongo comestible Asam (Schizophyllum commune Fr.).
Universidad de San Carlos, Guatemala.
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9. BASIDIOME PRODUCTION OF GUATEMALAN STRAINS OF Lepista nuda
María del Carmen Bran1,2*, Osberth Morales1, Roberto Cáceres1,2, Natalia Gurriarán1,2, Ferdiner González1
1
Departamento de Microbiología, Facultad de Ciencias Químicas y Farmacia, Universidad de San Carlos
de Guatemala. Edificio T-12, 2º. Nivel, Ciudad Universitaria zona 12, 01012, Ciudad de Guatemala,
Guatemala, Centroamérica. *<[email protected]>
2
Programa Universitario de Investigación en Desarrollo Industrial, Dirección General de Investigación,
Universidad de San Carlos de Guatemala, Edificio S-11, 3º. nivel, Ciudad Universitaria zona 12, 01012,
Ciudad de Guatemala, Guatemala, Centroamérica.
ABSTRACT
This study determined biological efficiency and pileus diameter of basidiomes of five native strains of Lepista nuda
on three substrates. Highest biological efficiency was 57.1% from a substrate formulated with 75% wheat straw and
25% rice husk (strain 21.10). Strains 17.01 and 50.09 fruited only on compost used for Agaricus cultivation. Largest
pileus diameter (6.19 cm) was obtained for strain 21.10.
Key words: edible mushrooms, substrate, biological efficiency.
INTRODUCTION
Lepista nuda (Bull.) Cooke is consumed in several countries of Europe, America and Australia (Stott et al.
1996). This mushroom has good potential for production using the A. bisporus cultivation system because
both species show similar patterns of growth (Danai et al. 2008). The origin of L. nuda cultivation is in
Europe, but there are complications in production due to a slow and prolonged life cycle (Castro et al.
2014). Several studies were conducted in Australia, Israel and Spain that evaluated production of fruit
bodies on different substrates. However, yield was relatively low and further experimental studies were
recommended before attempting its commercial cultivation (Stott et al. 1996, Danai et al. 2008, Castro et
al. 2014).
In Guatemala, L. nuda is used as food by the Maya-Kaqchikel people in the highlands of Chimaltenango
(Morales 2001, Bran et al. 2003a, Bran et al. 2003b, Morales et al. 2010) (Figure 1). Due to the saprobic
nature of this species, it can be cultivated on wastes that are generated from agricultural activities in the
country. Several native strains have been evaluated on various culture media and temperatures. In
addition, production of spawn on various grains and the development of primordia on two substrates have
been accomplished. Given the progress that has been made for the cultivation of Guatemalan strains, it is
desirable to evaluate production of fruit bodies (Bran et al. 2011, Bran et al. 2015).
The objective of this study was to determine biological efficiency (BE) and pileus diameter of basidiomes
(fruit bodies) of Guatemalan strains of L. nuda produced on various substrates. Our work aims to: 1)
develop an appropriate technology for cultivation of L. nuda in Guatemala, 2) serve as a preliminary step
of cultivation technology transfer to rural communities, 3) provide the possibility of an alternative food, 4)
assist in economic development of the country, and 5) serve as a bioprospecting model of fungal diversity.
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Figure 1. L. nuda in Tecpán-Guatemala, Chimaltenango, Guatemala. Left: Wild fruit bodies on leaf litter
in Quercus-dominated forest. Right: Mushrooms for sale at 94 Km Interamerican highway, including L.
nuda, Amanita garabitoana, Chroogomphus jamaicensis and Ramaria sp.
L. nuda strains
All strains were deposited in the Saprobe and Mycorrhizal Fungi Strain Collection, at Departamento de
Microbiología, Facultad de Ciencias Químicas y Farmacia, Universidad de San Carlos de Guatemala. The
code and source of the strains are as follows: 17.01 (Tecpán-Guatemala, Chimaltenango), 54.02 (94 Km
Interamerican Highway, Tecpán-Guatemala, Chimaltenango), 50.09 (San Juan Comalapa,
Chimaltenango), 4.10 (San Jorge Muxbal, Santa Catarina Pinula, Guatemala) and 21.10 (Tecpán-
Guatemala, Chimaltenango municipal market).
Biomass production of L. nuda strains
Pure cultures of different strains were prepared on malt extract agar and incubated at 26°C in the dark for
30 days. Subculturing was done on potato dextrose agar and mycelium was incubated at 26 °C for 15 to
20 days, according to the procedure recommended by Quimio & Chang (1990) and Bran et al. (2011).
Spawn
Spawn was prepared on wheat grains, which were soaked for 24 hours until they reached about 43%
moisture and then boiled for 20 minutes. Subsequently, 200 g were placed in polypropylene bags with
CaCO3 (1% w/w) and sterilized (1 h, 121 °C). Five fragments of mycelium (1.0 cm2) of each of the L.
nuda strains were inoculated on the grains and incubated at 26°C in the dark, until growth of the mycelium
was observed (Quimio & Chang, 1990; Bran et al. 2015).
Substrates for basidiome production
Cultivation of L. nuda was studied using polypropylene bags in units of 1 kg of compost that typically is
used for cultivation of Agaricus (substrate 1, S1), wheat straw supplemented with either 25% rice bran
(substrate 2, S2) or 5% soy flour (substrate 3, S3) (substrates 2 and 3 were sterilized, 2 h, 121°C) (Stott et
al.1996, Sierra-Fernández et al. 2002, Bran et al. 2011).
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Determination of moisture, dry weight and C:N ratio of the substrates
Five samples of the substrates were randomly taken. The moisture percentage was determined using a
moisture analyzer MB35 (OHAUS®), then the dry weight average (g) was calculated for the substrates.
The C:N ratio of the substrates was determined by Unit of Quimica de Suelos, Facultad de Agronomía,
Universidad de San Carlos de Guatemala, and obtained the following results: Substrate 1 (20.6), Substrate
2 (48.2), Substrate 3 (40.8) and wheat straw (67.8).
Incubation and fructification
The procedure was based on methods proposed by Guinberteau et al. (1989), Stott et al. (1996), Sierra-
Fernández et al. (2002) and Bran et al. (2012). Substrates were inoculated with 200 g of spawn of the L.
nuda strains and incubated at 22 to 24°C until the mycelium completely colonized the substrates. Ten
replicates per substrate and L. nuda strains were prepared. When the mycelium colonized substrate S1, it
was overlaid with approximately 5 cm of sterilized and neutralized peat (Canadian Sphagnum Peat
Moss®). A thermal shock (8°C) was performed to induce production of fruit bodies. Bags were then
moved to a rustically crafted culture room. Environmental parameters recorded during production time
were temperature (18 to 22°C) and humidity (75 to 85%). Substrates were irrigated by spraying with fresh
water three times a day.
Quantification of fruit body production
Fruiting time was defined as number of days from substrate inoculation to first harvest. Fruit bodies
produced were collected, weighed (g) and counted for one flush. Also, pileus diameter was measured and
basidiomes were classified into group 1 (G1) <2 cm, group 2 (G2) 2-4 cm and (G3) > 4 cm. Percentage
biological efficiency (BE) was determined using the formula as follows: BE = Biomass (fresh weight) x
100/Substrate weight (dry basis) (Chang & Miles 2004).
Statistical analysis
An analysis of variance (ANOVA) was performed by a SPSS®19 statistical program for BE, fruiting time
and pileus diameter groups. Means were separated using Duncan's multiple comparison test with α= 0.05
(Nurosis 2011).
RESULTS
Lepista nuda strains 17.01, 50.09 and 21.10 fruiting on substrate S1, however only strain 21.10 produced
fruit bodies on substrates S2 and S3. Strain 4.10 colonized the substrates but did not produce fruit bodies
and the strain 54.02 did not colonize the substrates. In general, all strains fructified in less time in substrate
S1. However, strain 21.10 had the shortest fruiting time and was statistically significant with respect to the
others (p<0.05). Strains 17.01 and 50.09 did not show statistical difference in this same substrate (p>0.05).
The fruiting time in substrates S2 and S3 in strain 21.10 was statistically significant (p=0.003) and showed
the longest fruiting times (Table 1, Figure 2).
Highest BE was obtained by strain 21.10 in substrate S2, which was significantly different with respect to
the other strains and substrates evaluated (p<0.05). BEs for this same strain in substrates S1 and S3 were
not significantly differently (p = 0.508). There was no significant difference in BEs from strains 17.01 and
50.09 on substrate S1 (p> 0.05). Strain 50.09 had the lowest BE in substrate S1 (Table 1).
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Table 1. Biological efficiency for L. nuda strains cultivated on three substrates.

Strain Substrate1 Fruiting time2,3 BE (%)3
21.10 S1 121.80 ± 3.96 a3 32.80 ± 08.90 b4
S2 186.40 ± 1.82 c 57.06 ± 26.34 a
S3 196.60 ± 1.82 d 18.27 ± 18.08 b, c
17.01 S1 137.60 ± 2.61 b 09.01 ± 02.46 c

S2 0 0
S3 0 0
50.09 S1 137.40 ± 6.58 b 02.08 ± 00.79 c

S2 0 0
S3 0 0
1
Compost used for cultivation of Agaricus (S1), wheat straw supplemented with 25% rice bran (S2), wheat straw
supplemented with 5% soy flour (S3). 2Days from substrate inoculation to first harvest. 3Mean ± standard deviation.
4
Different letters in the same column indicate statistically significant difference according to Duncan's multiple
comparison test (α = 0.05).
A B
C D
Figure 2. Basidiome production of L. nuda strains. A-C. Strain 21.10. A) Gregarious fruit bodies on
substrate S1. B) Caespitose fruit bodies on substrate S2. C) Fruit bodies harvested from substrate S3. D)
Gregarious fruit bodies on substrate S1 (strain 17.01).
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The overall effect of strains on BE showed that strain 21.10 was statistically significant with respect to the
others (p<0.05). There was no significant difference for BE for strains 17.01 and 50.09 (Figure 3).
Substrate was not a significant factor for BE for substrates S1 and S3 (Figure 4).
Figure 3. BE of L. nuda strains on evaluated substrates. Bars represent the BE mean obtained from three
substrates. Error bars indicate ± 95% confidence interval. Different letters indicate statistically significant
difference according to Duncan's multiple comparison test (α = 0.05).
Figure 4. General effect of the substrates on the EB of the L. nuda strains. The bars represent the EB mean
obtained in the substrates evaluated. The error bars indicate ± the standard deviation from the mean to
95% confidence interval. Different letters indicate statistically significant difference, according to
Duncan's multiple comparison test (α = 0.05).
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Table 2. Pileus diameters (cm) of L. nuda strains produced in three substrates.

Strain Substrate1 G12,3 G22,3 G32,3 Total3
21.10 S1 1.37 ± 0.23 2.90 ± 0.43 6.19 ± 0.61 3.91 ± 2.30 a4
S2 1.32 ± 0.04 2.75 ± 0.19 5.47 ± 0.44 2.61 ± 1.65 b
S3 1.31 ± 0.15 2.13 ± 1.22 3.36 ± 3.15 2.34 ± 1.48 b
17.01 S1 1.37 ± 0.15 2.37 ± 01.34 04.84 ± 0.36 2.64 ± 1.39 b

S2 0 0 0 0
S3 0 0 0 0
50.09 S1 0.73 ± 0.69 2.58 ± 1.45 4.41 ± 2.57 2.95 ± 2.08 b

S2 0 0 0 0
S3 0 0 0 0
1
Compost used for cultivation of Agaricus (S1), wheat straw supplemented with 25% rice bran (S2),
wheat straw supplemented with 5% soy flour (S3). 2Pileus diameter groups: G1 (<2 cm), G2 (2 to 4
cm) and G3 (> 4 cm). 3Mean ± standard deviation. 4Different letters in the column indicate
statistically significant difference, according to Duncan's multiple comparison test (α = 0.05).
Strains 17.01, 50.09 and 21.10, produced fruit bodies with pileus diameter in three classification groups
(G1, G2 and G3) on substrate S1. Only strain 21.10 produced fruit bodies of the three categories on
substrates S2 and S3. Strain 21.10 produced the largest pileus diameter in substrate S1, which was
statistically significant with respect to the others (p<0.05). Pileus diameters obtained by this same strain in
substrates S2 and S3, as well as by the strains 17.01 and 50.09 in the substrate S1 no showed statistically
significant difference (p>0.05) (Table 2, Figure 5).
Figure 5. Classification of pileus diameter produced by L. nuda strains on three substrates: Compost used for
cultivation of Agaricus (S1), wheat straw supplemented with 25% rice bran (S2), wheat straw supplemented with 5%
110
soy flour (S3). Bars represent ranges of minimum and maximum values of pileus diameter. Horizontal lines indicate
groups of classification of pileus diameter groups: G1 (<2 cm), G2 (2 to 4 cm) and G3 (> 4 cm).
DISCUSSION
Literature indicates that cultivation of L. nuda is a long-time process, since that strains colonize substrates
between 25 to 60 days and fruiting occurs between 24 and 52 weeks (168 to 364 days). In addition, using
mycelium inoculated in beds of horse dung and straw, fruit bodies were produced after the appearance of
mycelial rhizomorphs between 7 and 14 months (Stamets & Chilton 1983). Other authors report that
fruiting time on compost used for cultivation of Agaricus lasted 7 to 10 weeks (49-70 days), producing
one or two harvests with an interval of 14 to 20 days (Sierra-Fernandez et al. 2002).
In this study, the mycelium of L. nuda produced the first crop between 121-196 days in substrate S1
(compost used for cultivation of Agaricus). This period is longer than that reported by Sierra-Fernandez et
al. (2002), but is within the period reported by Stamets & Chilton (1993). In the case of this substrate,
fruiting time was shorter than that observed in substrates S2 and S3. This may be due to the C:N ratio of
20.6, which is very close to the optimal ratio (15-16) for development of Agaricus (Vedder 1996).
Fruiting time observed for L. nuda strain 21.10 in substrates S2 and S3, consisting of wheat straw
supplemented with 25% rice bran or 5% soybean meal respectively, coincides with the period reported by
Stamets & Chilton (1983). The delay in colonization by L. nuda strain 21.10 of substrates S2 and S3 (186-
196 days) compared to substrate S1, may be due to the high initial carbon nitrogen (C:N) ratio (48.2 and
40.8, respectively). Microorganisms, in general, require a C:N ratio of 20 to 30 to initiate the degradation
process (Roy et al. 1981). Wheat straw only substrate (C:N ratio 67.8), therefore, would need to be
supplemented to increase nitrogen levels for degradation by this fungus.
Highest BE (57.06%, strain 21.10) was obtained on the substrate composed of wheat straw supplemented
with 25% rice bran (S2). Strains 17.01 and 50.09 did not fructify on substrates S2 and S3 (wheat straw
supplemented with 5% soybean meal or with 5% soy flour, respectively). Because use of supplemented
wheat straw has not been reported for L. nuda cultivation, it is not possible to compare our results with
others. Stott et al. (1996) used compost prepared for A. bisporus supplemented with 10% cereal straw and
found that hyphal growth increased, but they were unable to obtain fruit bodies.
BE obtained from commercial mushroom compost (substrate S1) was lower than that from substrate S2,
however, strains 21.10, 17.01 and 50.09 all fructified there. This is understandable, since this substrate has
been recommended for the cultivation of L. nuda (Guinberteau et al. 1989, Stott et al. 1996, Vedder 1996,
Sierra-Fernandez et al. 2002, Danai et al. 2008). BE (32.8%) obtained in this study in substrate S1 was
higher compared to 5% yield (based on wet substrate) obtained by Danai et al. (2008). Unfortunately, in
the publication of Guinberteau et al. (1989), it was impossible to determine BE and, in another case, it was
not possible to obtain fruit bodies (Stott et al. 1996).
Other studies have reported that L. nuda strains also fructify on supplemented oak or alder sawdust, as
well as willow, poplar and maple (Stamets & Chilton 1983) or olive leaves (Castro et al. 2014). We
suggest evaluation of other substrates to determine fruiting capacity of Guatemalan L. nuda strains.
Another measure of productivity of L. nuda strains was pileus diameter. In this study, pileus diameters
obtained were significantly larger in substrate S1 with strain 21.10. All pileus diameters were smaller than
maximum diameters (14-15 cm) observed in nature (Bigelow & Smith 1969, Bran et al. 2003a).
111
From a commercial point of view, each species of edible mushrooms possesses some standard diameter
for merchandizing, for example, around 15 cm is optimum for the cultivated mushroom A. bisporus
(Malloch 1976). Although L. nuda cultivation is recent and not widespread, the ideal pileus diameter for
commercialization has not been established. However, in the wild, L. nuda in Guatemala reaches 10.5 to
14 cm diameter (Bran et al. 2003a) whereas, in the United States it can measure between 4 to 15 cm
(Bigelow & Smith 1969).
Finally, the fact that strain 54.02 did not colonize the substrates and strain 4.10 did not produce fruit
bodies, may be because these strains did not adapt to substrates evaluated. Another possibility may be that
they lost the capacity of colonization and fruiting due to multiple transfers in laboratory media as has been
reported for most fungal strains (Labarére & Bois 2001).
The results of this study demonstrated that it is possible to obtain a high BE of Guatemalan L. nuda strain
21.10 in the substrate consisting of wheat straw supplemented with 25% rice bran. This combination of
strain and substrate may have potential for commercial cultivation of this species. This substrate is
already used in the production of other mushrooms such as Pleurotus spp. and A. cylindracea (Chang &
Miles 2004, Bran et al. 2014).
ACKNOWLEDGMENTS
This work was supported by Dirección General de Investigación and Facultad de Ciencias Químicas y
Farmacia, Universidad de San Carlos de Guatemala (research project 4.8.63.6.61). The autors are gratefull
to the Unit of Química de Suelos, Facultad de Agronomía, Universidad de San Carlos de Guatemala for
performing the chemical analyzes. We thank Agrícola Grotto and Finca La Suiza for facilitating the
substrates. We also appreciate the efforts of Liuba Cabrera and Ricardo Figueroa for technical and
administrative assistance. The authors express their sincere gratitude to anonymous reviewers for critical
review of the manuscript.
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UAEM-UPAEP-IMINAP. Puebla. 437-464.
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10. SHIITAKE CULTIVATION ON STRAW: AN ALTERNATIVE FOR

SUBTROPICAL REGIONS
Gerardo Mata 1 and Jean-Michel Savoie 2

1
Instituto de Ecología, A.C., Red Manejo Biotecnológico de Recursos, Carretera antigua a Coetepec 351,
El Haya, C.P. 91070, Xalapa, Veracruz, Mexico. <[email protected]>
2
INRA, Unité de Recherche 1264, MycSA, 71, Avenue Edouard Bourlaux, CS20032, 33882 Villenave
d’Ornon, Cedex, France
ABSTRACT
The traditional cultivation of shiitake (Lentinula edodes) on oak logs has been replaced in part by cultivation in
plastic bags with sterilized sawdust since this method offers a higher biological efficiency and a shorter cultivation
cycle. However, the sterilization process has a high initial installation cost and consumes more energy. The
feasibility of cultivating shiitake in pasteurized straw offers a very important alternative for subtropical regions since
straw is generally an easily accessible substrate. Pasteurized straw (of different cereals: wheat, barley, etc.) has
become, in recent years, a suitable substrate for the cultivation of shiitake since some strains adapted to this substrate
have been selected. The selection of efficient strains is fundamental because a non-sterile substrate is used. An
update of the cultivation of shiitake on straw and an analysis of its possibilities of adaptation in subtropical climates
is presented.
INTRODUCTION
The edible mushroom Lentinula edodes (Berk. & Mont.) Pegler, is known as shiitake in Japan as well as
xiang-gu in China (Chen 2005). It is also popularly known as oak mushroom and is one of the best-known
and most studied mushrooms. Until the 1980s, the main world producer of shiitake was Japan, using the
traditional technique of cultivation in logs. However, China is currently the world's leading producer of
shiitake with more than 95% of the total produced, and this species has become the most cultivated
worldwide with 22% of the total, about 7.48 x 106 tons, displacing the white button mushroom (Agaricus
bisporus) that has been relegated to the fourth position with 15% of the total (Royse et al. 2017). The
mushroom is estimated to have been cultivated for the first time in China between 1,000 and 1,100 AD on
wood logs (Chang 1993). Lentinula edodes in China is an important part of gastronomy, art and culture,
which is why there are often diverse manifestations alluding to this species (Figure 1).
According to Chang (2001), the shiitake cultivation system can be divided into 6 different stages that
involve changes in the technologies used: 1) cutting method that was developed about 1,000 years ago, 2)
wood log method, based on a mushroom spawn inoculation system invented by Japanese growers in 1928,
3) plastic bag method using small plastic bags with sawdust substrate, developed in Taiwan in the early
1970s, 4) brick- or pressed-cake method introduced in Shanghai in 1979, 5) synthetic log method,
developed in 1986 in Fujian Province. This innovative method has allowed China to position itself as the
world's leading producer of shiitake, and 6) small rush/plastic shed method that is derived from the
synthetic log method, developed and adapted in Biyang County in Henan Province. The bags are much
bigger and are laid on shelves of multiple layers within the shed.
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Figure 1. Jade crafts showing wild shiitake specimens.
Since the decade of 1990, the traditional system of culture has been gradually replaced by a more modern
method that uses plastic bags with lignocellulosic substrates (Oei 2003, Chang and Miles 2004, Chen
2005). For the cultivation of shiitake, hardwoods are traditionally used (Chang and Miles 1989, Kozak and
Krawczyk 1993, Sobata and Nall 1994), but as mentioned previously, a more efficient and faster system
has focused on using a substrate with enriched sawdust that is placed in plastic bags (Przybylowicz and
Donoghue 1988, Chen 2005) (Figure 2). This method has potential for relatively high yields in a short
time and in relatively small spaces. Different alternative substrates have also been tested that are generally
abundant locally and at an affordable price (Chen 2005). In addition to China, shiitake is produced in
different countries: Japan, Korea, USA, Mexico, Brazil, France, Spain, and others, but China produces
more than 95% of worldwide production (Royse et al. 2017). In Mexico, the production of L. edodes has
been tested on wood chips from different trees such as Carpinus, Bursera, Alnus, Quercus, Eliocarpus and
Jacaranda, among others (Mata et al. 1990, Morales and Martínez-Carrera 1991, Morales et al. 1991,
Curiel Pérez et al. 2012, Manero Colín et al. 2012, Martínez-Guerrero et al. 2012). Tests have also been
conducted on agroindustrial wastes such as coffee pulp, sugarcane bagasse and vineyard residues (Mata
and Gaitán-Hernández 1992, 1994, Salmones et al. 1999, Gaitán-Hernandez et al. 2006).
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Figure 2. Shiitake cultivation on sawdust substrate. a: polypropylene bags with eucalyptus substrate during
incubation process, b: shiitake substrate bags during the formation of the dark layer at the end of
incubation, c: culture on logs in a green house.
CULTIVATION ON WHEAT STRAW
Among the most important advantages of growing edible mushrooms in plastic bags is the possibility of
using very different types of substrates. However, such substrates must be rich in their content of
cellulose and lignin. In tropical and subtropical regions, diversity of substrates is abundant and often easily
accessible and inexpensive. Many of these substrates can be adapted relatively easily to shiitake
cultivation through simple processes such as soaking and fermentation. Various substrates have shown
potential for cultivation of shiitake such as coffee husk, coffee pulp, spent coffee grounds, sugarcane
bagasse, corncobs, millet straw, wheat straw, tea leaves, peanut hulls, cotton seed hulls, sunflower seed
hulls, dried grass powder, water hyacinth, etc. (MushWorld 2005).
Most of the agricultural by-products used for shiitake cultivation are subjected to autoclave sterilization
treatment in plastic bags. This system, although very effective, is quite expensive for its implementation in
tropical and subtropical regions. In order to reduce production costs, a method has been developed to use
steam-pasteurized cereal straw as a substrate for shiitake cultivation (Delpech and Olivier 1991). This
process requires a rigorous selection of strains, as well as production of fast growing inoculum that helps
to limit growth of antagonistic molds (Mata et al. 1998, 2001, 2002, Savoie et al. 2000, Gaitán-Hernández
et al. 2014). In Mexico, progress has been made on cultivation of shiitake on pasteurized cereal straw
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(Gaitán-Hernández and Mata 2004, Gaitán-Hernández et al. 2006, Peralta Márquez and Frutis Molina
2010). However, this technology has not been implemented commercially. Work in Mexico on this
species has not only focused on its cultivation on alternative substrates such as those already mentioned,
but research has also been carried out with the objective of selecting strains that adapt to the different
substrates tested. Also, research trials have been initiated with antagonistic fungi, hybridization, inoculum
improvement and culture conditions to increase yields (Mata and Guzmán 1989, Savoie et al. 2001, Mata
et al. 2002, Ramírez-Carrillo and Leal-Lara 2002, Mata and Savoie 2005a,b, Gaitán-Hernández et al.
2014).
Wheat straw preparation
Both the composition of substrate materials and heat treatments have substantial effects on substrate
selectivity. A selective substrate allows growth of shiitake mycelium but inhibits growth of competitors.
To prepare a substrate, wheat straw is shredded into pieces from 4 to 6 cm in length for easy handling
during pasteurization and bagging. Crushing straw might be beneficial. It is soaked in water for 6 to 12
hours at room temperature, drained and then mixed with 2 to 10% (dry weight) gypsum. Several
supplements containing materials that are lacking in wheat straw itself are added to provide sufficient
nutrients for shiitake mycelium. As a nitrogen and oligoelements source, soybean flour added at 4 kg per
ton can increase yield by 30%, but soybean flour is more likely to lower substrate selectivity (Figure 3).
Generally, substrate selectivity decreases by addition of components rich in nitrogen or oligoelements.

Therefore, it is recommended to either add only a small quantity of them, or to sterilize the substrate if a
large amount of nitrogen rich components are added. Other supplements improve competitiveness of
shiitake against competing molds such as Trichoderma sp. Peat, sawdust or other lignin derivatives not
only have absorbing properties but also contain phenolic compounds that most competitors cannot degrade
easily. Shiitake can degrade these lignin components with oxidative enzymes, so the shiitake mycelium
has almost exclusive access to these supplements (Mata et al. 2001, Savoie et al. 2000). Phenolic
compounds may also contribute to the active defense of L. edodes. When mycelia of shiitake and
Trichoderma are confronted on wheat straw substrate, shiitake mycelium forms a barrier with dense
mycelium, initially white that then turns dark brown. In this dark zone, shiitake produces a quantity of
enzymes from the group of laccases that serve to defend themselves from Trichoderma. (Figure 4). For
instance, when 10% peat moss was added to a wheat substrate, the contamination rate by Trichoderma
was reduced by 50% and mushroom yield increased by 30% (Mata et al. 1998).
Heat treatment with steam
Wheat straw must be pasteurized in order to kill possible competitor microorganisms as well as insects in
the straw. Another goal of pasteurization is to propagate thermophilic microorganisms that will improve
substrate selectivity by immobilizing readily available nutrients to competitors and by producing toxic or
inhibitory molecules to limit rapid growth of competitors. The substrate mixture is placed in containers or
directly in special rooms for pasteurization with steam at 65 ºC for 12 to 24 hours and then cooled to room
temperature (Figure 5). Water content of substrates after pasteurization must be about 70% (Mata et al.
1998). During the pasteurization process, it is very important to encourage vapor recirculation in order that
the temperature across the substrate is homogeneous. The most efficient way to perform this operation is
to use motors that allow steam to move from the bottom to the top of the pasteurization room. If possible,
the steam recirculation system should have an air filter to favor the cooling of the substrate at the end of
the process. It is very important to maintain the temperature during the pasteurization process at 65°C. If
the temperature increases too much there will be a decrease in the population of thermophilic organisms
and if the substrate is spawned in non-sterile conditions it is highly susceptible to contamination.
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However, if treatment of the substrate is adequate, a selective substrate will be obtained that will favor
mycelial development of shiitake (Mata et al. 1998).
Figure 3. Preparation of wheat straw for shiitake cultivation. a: chopped, fragmented straw, b:
fermentation of substrate in a covered area, c: addition of supplements for small-scale cultivation.
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Figure 4. Confrontation of mycelia of shiitake and Trichoderma. a: formation of a mycelial barrier

between shiitake (right) and Trichoderma (left), b: mycelium of shiitake forming a dark zone with high
laccase production (top = mycelium of L. edodes, bottom = Trichoderma harzianum).
Figure 5. Different systems for straw pasteurization with steam. a: single-door pasteurization room for
bulk substrate, b: pasteurization system type "box", c: pasteurization system of double door (outdoor-
indoor), with rollers, for substrate in containers, d: single door pasteurization system for substrate in
containers, e: detail of the motor for steam recirculation and air filter, f: metallic container for fermenting
or pasteurizing straw.
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Spawning substrate and incubation
After cooling, pasteurized substrates are mixed with spawn in a clean environment. Aseptic conditions are
not necessary because the substrate is not sterilized. In order to improve competitiveness of shiitake
during the first days after spawning, mycelium in spawn must be vigorous, adapted to components of the
substrate and able to colonize all particles. For these reasons, Delpech and Olivier (1991) recommended
limiting use of supplementation in wheat straw substrates to prevent growth of bacteria and molds. Spawn
must be mixed with sterilized or pasteurized straw at 5% (w/w) and the mixture should be placed in plastic
bags that are lightly perforated or equipped with a microporous filter.
Selection of genotypes appropriate for chemical and structural properties of chosen substrates and thermal
treatments are critically important to ensure good production of fruiting bodies in the shortest time
possible. Due to relatively few shiitake strains that are well adapted for growth on pasteurized wheat straw
(Levanon et al. 1993, Mata and Savoie 1998, Mata et al. 2002), it is recommended that growers using
wheat straw choose a shiitake strain with high competitiveness. However, it was demonstrated that use of
supplemented spawn considerably reduces substrate contamination (Mata et al. 1998, Savoie et al. 2000).
Selection of good spawn adapted to cultivation substrates, as well as to thermal treatments, is critically
important to ensure a high production of mushrooms in the shortest time possible. Grains of different
seeds have been used as substrate to produce shiitake spawn. Millet was tested with the addition of
different supplements (Mata et al. 1998, Mata et al. 2002, Gaitán et al. 2014) and also to increase yield of
shiitake by millet supplementation of wood chip substrate (Royse 1996). Sorghum seeds have been also
tested with very good results (Salmones et al. 1999, Peralta Márquez and Frutis Molina 2010, Martínez-
Guerrero et al. 2012). Preparation of nutritionally-supplemented spawn as well as preadaptation of the
mycelium to final components of the culture substrate have allowed for a considerable reduction in
contamination during the first growth stages (Savoie et al. 2000).
Incubation is one of the most important phases for shiitake cultivation on alternative substrates because of
the competition between shiitake and competitor molds that occurs during the first weeks. The initial rate
of substrate colonization by antagonistic fungi is an important factor of the competitive interaction. If
shiitake rejects an attack by the mold at this stage, no other problem is encountered. Some strains of
shiitake are able to reject mold attacks under temperature and nutritive conditions favorable to them
(Badham 1991) if their mycelium has colonized enough space before contacting competitor fungi (Savoie
et al. 1998). Incubation must be carried out at 25 ºC ± 2 with a 12-hour light and 12-hour dark cycle that is
recommended at least for shiitake cultivation on wheat straw for 1–2 months depending on the strain. At
the end of the incubation period, the entire surface of the substrate turns brown, indicating that mycelium
is ready for fructification (Przybylowicz and Donoghue 1988, Donoghue and Dennison 1996). Generally,
transparent bags are used and are placed on shelves inside the incubation rooms to promote mycelial
growth. Various bag sizes are used to contain 3 to 30 kg of substrate. Sometimes shiitake growers prefer
to use pre-perforated bags because if the substrate has been properly prepared there should be no
contamination problems. Many producers use the same incubation rooms to induce and obtain the
mushrooms but some producers prefer to have specific rooms to carry out only incubation of the
inoculated bags (Figure 6).
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Figure 6. Incubation of shiitake on wheat straw contained in bags. a: sample of shiitake in pre-perforated
plastic bags, b: shiitake substrate contained in polypropylene bag with filter, c: bags with substrate on
wooden shelves in an incubation/production room, d: bags with substrate on metal shelves in an
incubation room, e: samples of shiitake (without plastic bag) hanging on a shelf at the end of the
incubation period. Substrate has turned brown indicating that the mycelium is ready for fruiting.
Shiitake production on pasteurized wheat straw
After the incubation period, plastic bags are removed and substrate blocks are sprinkled with cold water.
Room temperature must be adjusted to 17 to 19 ºC. A relative humidity of 90% and a cycle of 12 hours
light/12 hours dark are necessary to encourage mushroom development. After obtaining the first harvest,
blocks can be rehydrated to induce a second flush by soaking them in water for 12 hours (Gaitán-
Hernández and Mata 2004). Although it is not a general rule, soaking substrate is a method that is
frequently used since mycelial growth compacts the substrate and hinders hydration of the samples. On
the other hand, the dark external layer, also known as pseudosclerotium or pellicle, protects the samples
from dehydration but at the same time hinders absorption of environmental humidity and causes samples
to float during soaking.
Under commercial production conditions, when large blocks of supplemented and pasteurized straw are
used (10-16 kg), mushrooms may be harvested for 12 to 16 weeks and biological efficiency reaches 50 to
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100%. It is difficult to make comparisons between the results of different investigations since a large
number of substrates, strains and particular conditions have been studied in shiitake cultivation. Table 1
shows a non-exhaustive comparison of some substrates used in the cultivation of shiitake comparatively
with wheat straw. It should be taken into account that even when the same strains or substrates are used,
biological efficiency can vary with the culture conditions used, such as temperature and humidity in each
experiment.
Table 1. Biological efficiency obtained for cultivation of shiitake on wheat straw, with various heat
treatments and spawn types, compared with other substrates.
Substrate Heat Spawn Biological Reference
treatment efficiency (%)
Wheat straw Pst C 15.9 Delpech and Olivier 1991
C 59.2 Mata and Savoie 1998
S 59.0 Mata et al. 1998
S 116.0 Savoie et al. 2000
S 84.2 Peralta Márquez and Frutis Molina 2010
Phw S 55.6 Gaitán-Hernández and Mata 2004
S 188.3 Gaitán-Hernández et al. 2014
St C 75.2 Philippoussis et al. 2007
C 66.8 Sharma et al. 2013
Paddy straw St C 50.1 Puri 2012
Coffee residues St C 88.6 Leifa et al. 1999
C 64.3 Mata and Gaitán-Hernández 1994
C 90.0 Fan and Soccol 2005
Sunflower seeds St C 108.0 Curvetto et al. 2005
hulls
Vineyard pruning St C 93.3 Gaitán-Hernández et al. 2006
Corn cobs St C 80.6 Philippoussis et al. 2007
Sugar cane bagasse St S 133.4 Salmones et al. 1999
Sawdust Stst C 25.0 Thevasingh et al. 2005
St C 60.0 Fan et al. 2005b
C 79.0 Sobal et al. 2010
C 168.0 Curiel Pérez et al. 2012
C 70.4 Manero Colín et al. 2012
C 103.0 Martínez-Guerrero et al. 2012
Pst C 80.4 Royse and Sánchez 2007
Pst = pasteurization with steam, Phw = pasteurization in hot water, St = sterilization, Stst = sterilization with steam,
C = conventional, S = supplemented.
Generally, the first flush of mushrooms obtained from wheat straw substrate is the most abundant with 50-
70% of the total harvest, depending on strain and growing conditions (Mata et al. 1998, Gaitán-Hernández
et al. 2014). Mushrooms should be harvested when they are turgid and before the pileus extends fully
(Figure 7). Harvest may be done by hand, grasping the mushrooms at the base and turning them slightly so
that they can be removed without physical damage. Some strains of shiitake produce mushrooms with
many scales while others produce smoother fruitbodies. Through control of ventilation and humidity, final
morphology of harvested mushrooms may be influenced. In several Asian countries, the "flower shiitake"
presents a cracked pileus and is very appreciated. The flower pattern is not a characteristic of a particular
genotype or genetically inherent trait, but rather a morphological, flower-like cracking pattern on the
upper surface of the cap. This white cracking pattern is produced by different growth rates between the
surface and the inner tissue of the cap due to drastic diurnal fluctuations of temperature and humidity.
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Flower shiitake production requires high humidity and temperature in daytime and lower humidity and
temperature during nighttime (Fan et al. 2005a) (Figure 8a).
Figure 7. Production of shiitake on wheat straw. a: formation of primordia, b-d: mushrooms ready to
harvest.
Perhaps one of the biggest advantages of shiitake cultivation is that a major part of its market is directed to
dried product. Many people prefer fresh shiitake as it combines very well when cooked with vegetables.
However, dried shiitake has a particular aroma that has placed it in the preferences of consumers in the
international market. Although there are different commercial presentations of shiitake that include fresh,
canned, dried and canned products such as brine, many producers prefer to dry shiitake and store it and
then sell larger volumes (Figure 8).
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Figure 8. Commercial presentation of shiitake mushrooms. a: fresh “flower mushrooms”, b: fresh

mushrooms with special packaging, c: slices of dried mushrooms, d: whole dried mushrooms.
The purpose of the drying process is, in addition to dehydrating the product for its preservation, to
maintain its appearance and color as well as enhancing its aroma. Drying may be done in a traditional way
by exposing the product to the sun or using dryers in a chamber with recirculation of hot air. Size of the
drying chamber varies depending on the production scale. The drying chamber should be maintained at 40
to 50 ºC for 24 hours. Dried shiitake should be cooled one hour before termination of the drying process
and then should be placed into polyethylene bags, sealed and kept in a dry, cool and dark place. Shiitake
produced by this method have better quality including better hygienic conditions and brighter color
compared to sun-dried mushrooms (Fan et al. 2005b) (Figure 9).
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Figure 9. Electric dryer for shiitake. a: metallic cabinet, b: extraction fan, c: wooden trays for placement of
mushrooms.
The growing shiitake market has prompted producers to offer new shiitake-based products that are
appealing to the consumer and that quickly position themselves in new niches within the market. In
addition to health care products such as extracts or tisanes, one may now find a variety of snacks produced
with shiitake and beers and different preserves (Figure 10).
Figure 10. Various products based on shiitake. a: shiitake and enokitake (Flammulina velutipes) brine, b:
snacks based on shiitake, c: dry shiitake cooked and seasoned, d: shiitake beer.
126
Aroma characteristics of shiitake produced on wheat straw
Aroma is one of the most important characteristics of shiitake. Due to its organoleptic properties, L.
edodes was introduced into the French market as the “fragrant mushroom” (champignon parfumé) (Olivier
et al. 1991) where it is used in gourmet dishes. Dried shiitake possesses a fragrance that is not found in
fresh mushrooms and it is related to the presence of lenthionine plus some other sulfur-based odor
compounds and alcohols. In addition to lenthionine, substances such as 1,2,4-trithiolane and 1,2,4,6-
tetrathiepane play an important role in the odor of dried shiitake (Hiraide et al. 2004, Hiraide 2006).
Volatile compounds of shiitake produced in logs or in synthetic logs have been widely studied and
identified as complex metabolites (Wu and Wang 2000) that may be increased by adding cysteine (or
methionine), glutamic acid or other amino acids to the medium (Hiraide 2006, Hiraide et al. 2010).
It is important to note that volatile compounds of fresh shiitake vary depending on the substrate used for
its cultivation (Mata et al., 2014). In this way, perhaps, specialists can differentiate a mushroom grown on
an oak wood-based substrate from a substrate that has been produced on wheat straw or some other cereal.
According to Mata et al. (2014), when shiitake was cultivated on 4 different substrates, 8 volatile
compounds were obtained from the basidiocarps with differences depending on the substrates (Table 2).
The most abundant compound (3-octanone) was present in shiitake cultivated on all substrates evaluated.
The substrate based on oak wood showed a greater diversity in volatile compounds and the only one in
which the mushrooms produced 2-pentylfuran and limonene. With this evidence it is expected that in
addition to the aroma, some of the medicinal compounds that have been recognized in shiitake, would be
affected when the fungus is produced on different substrates. For these reasons, it is expected that
medicinal properties would also vary depending on substrate. Consequently, shiitake grown in cereal
straw, does not have the same aroma and its medicinal properties could be different when it is cultivated
on oak wood. However, more studies are needed to understand the effect of substrates on the production
of various shiitake metabolites.
Table 2. Main volatile compounds identified in shiitake cultivated on various substrates.

Compound Substrates Percentage Aroma
1 2 3 4
3-octanone X X X X 71.4 - 97.8 Sweet fruity
1-octen-3-ol X X 9.9 - 18.9 Mushroom, butter, resinous
Benzaldehyde X X 0.6 - 4.2 Bitter almond
Benzeneacetaldehyde X X 0.8 - 5.8 Fruity
2-penten-1-ol X X 2.2 - 9.3 Mushroom-like odor
1,2,4-trithiolane X 1.2 Egg and garlic
2-pentylfuran X 11.8 Butter, cooked rice
Limonene X 6.9 Citric
Substrates: 1= Barley straw 84 % - oak powder 16 %, 2= sugarcane bagasse 100%, 3= Beech tree litter 84 % - oak
powder 16 %, 4= oak sawdust 84 % - oak powder 16 %. Modified from Mata et al. 2014.
Some pests and diseases encountered during shiitake cultivation
Like all cultivated mushrooms, production of shiitake may be seriously affected by the development of
some pests and diseases. With cultivation of shiitake on logs, more than 150 species of fungi are known
competitors (Lou 1981). For outdoor shiitake crops, it is practically impossible to control spore dispersal
of wood destroyers since they are transported by air. According to damage they may produce during the
crop cycle, these fungi can be classified into three categories: a) disease-causing fungi, i.e., they are
capable of attacking and killing shiitake mycelium, b) competitor fungi, i.e., they do not attack shiitake
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mycelium but decrease the amount of nutrients available for its growth and development, and c) weed
fungi, i.e., they are not usually a problem but sometimes can act as competitors (Przybylowicz and
Donoghue 1988). In addition to fungi, some insects and other pests may also reduce production of shiitake
on logs. Insects that might damage cultivated shiitake by affecting logs or fruiting bodies include termites,
beetles (Families Cerambycidae, Scolytidae, Erotylidae), moths, flies and springtails. Slugs and snails are
probably the most commonly encountered animal pests of shiitake and they cause serious damage by
feeding directly on mushroom caps. Some mammals also like to eat shiitake fruit bodies (deer, mice,
squirrels, rabbits and pigs) (Bak and Kwon 2005).
Fortunately, for cultivation of shiitake in bags, diseases and pests are limited when compared to those
found during log cultivation. In most cases, contamination that occurs in bags with cereal straw is due to
fungi of the genus Trichoderma (Fan et al. 2005c). Trichoderma, the asexual stage of the genus Hypocrea
(Ascomycetes), is common in natural habitats containing organic matter (Samuels 1996). Trichoderma
harzianum Rifai, which has a rapid mycelial growth rate, is the cause of green mold, a disorder that affects
cultivated mushrooms such as Lentinula edodes. Shiitake resists Trichoderma spp. attack by producing
high quantities of laccases (p-phenol oxidase, EC 1.10.3.2) in barrages that are characterized by brown
line formation in contact zones of the mycelia (Savoie and Mata 1999) (Figure 11, see also Figure 4).
Laccase production is also induced by extracellular metabolites produced by T. harzianum (Savoie et al.
1998). Extracellular laccase production by shiitake is a defensive metabolite produced during interaction
with T. harzianum. This characteristic of shiitake allows stimulation of the production of some enzymes
that are considered important at the beginning of mycelial growth. To stimulate production of laccase,
phenolic compounds may be added to grain used in spawn preparation. This increases the probability of
resisting antagonistic fungi in early stages of mycelial development during incubation (Mata et al. 1998).
On the other hand, antagonistic relationships between shiitake and molds depend enormously on the
composition and thermic treatment of the final production substrate. For this reason, it is very important to
properly perform pasteurization of substrate and not exceed the recommended temperature (65 ºC) to
favor development of microorganisms that inhibit the growth of Trichoderma (Mata et al. 1998).
Supplementation of substrate is recommended, but when a steam pasteurization method is followed,
supplementation should be light and preferably by adding elements that help inhibit growth of antagonist
molds. After first harvest of mushrooms, it is necessary to check substrate blocks frequently, as
Trichoderma spots may begin to appear over time (Figure 11).
A substantial part of the success for control of Trichoderma has to do with good practices carried out
during cultivation of shiitake. Strict hygiene must be maintained in all facilities including personnel access
control. During spawning, access to facilities should be restricted to a minimum. If, despite all precautions
taken, there is contamination by Trichoderma, a rapid evaluation should be made to consider whether it is
necessary to eliminate all contaminated samples. Contamination of substrate with bacteria may also be a
major problem. In addition to affecting mycelial growth, bacteria often attack shiitake fructifications,
producing deformations and greatly decreasing production. Bacterial attacks can also produce spots on the
hymenium that diminish quality of the product. Samples with severe bacterial attacks should be eliminated
quickly. Sometimes, deformations of basidiocarps occur due to lack of ventilation in the cultivation room,
to excess moisture or to virus infection (Figure 11). During cultivation of shiitake in bags, some pests
may also appear that are attracted by the odor of fructifications. The most frequent pests encountered
include mites, springtails, slugs, and small rodents (Fan et al. 2005c). It is also common to find ladybugs.
In most cases, these pests can be controlled relatively easily through specific traps.
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Figure 11. Contamination encountered on shiitake substrate and fruit bodies. a-b: shiitake substrate
contaminated with Trichoderma mold, c: Trichoderma contaminating a shiitake block substrate during
second flush, d-e: shiitake fruit bodies contaminated with bacteria, f-g: deformed shiitake fruit bodies.
THE AMERICAN SHIITAKE
Among the species that have a promising future for their cultivation is the so-called American shiitake,
Lentinula boryana (Berk. & Mont.) Pegler (Figure 12). This species is an edible neotropical fungus that
grows in subtropical and tropical forests from the southeastern United States, Mexico, the Caribbean,
Central America to north of South America (Guzmán 1972, Mata and Guzmán 1991, Guzmán et al. 1997).
Although in Mexico it is not one of the most popular species in traditional markets, it is frequently found
for sale in the center of the state of Veracruz where it is known as "hongo de encino” (oak mushroom),
"hongo de palo” (stick mushroom), "cuerudo" (leatherly) (Mata and Guzmán 1989) while in Chiapas it
129
receives the indigenous name of "nla" (Guzmán 1997). Currently there are no data on its commercial
production and only a few studies have been carried out on the feasibility of its cultivation on different
substrates (Mata and Guzmán 1993, Soto-Velazco et al. 1995, Mata et al. 2001, Salmones and Gutiérrez-
Lecuona 2008). However, despite morphological similarities between the two species, it has been shown
that these are two completely separate and genetically distinct species, they are not interfertile and
although they belong to the same genus they are two phylogenetically separated species (Mata and
Guzmán 1989, Guzmán et al. 1997, Nicholson et al. 1997). Besides their macroscopic similarities, both
species have similar nutritional properties, suggesting the possibility of cultivating L. boryana using
technology developed for cultivation of L. edodes. This would suggest that the American shiitake could be
produced in large quantities. However, with this species it is necessary to carry out an intense work
regarding the selection of strains.
Figure12. Lentinula boryana, the American shiitake, cultivated on wheat straw.
On the other hand, aroma of a species is a key feature that can help to identify and create a “personality”
when trying to introduce the mushroom commercially. Differences in the aroma between L. boryana and
L. edodes allow to distinguish them with relative ease (Mata et al. 2014). The American species, L.
boryana, is consumed in Mexico fresh and is appreciated for its natural flavor and there is no record of
any process for preservation and stimulation of the production of aroma. Despite the morphological
similarity of both species, aromatic differences may provide a sufficient basis for believing that the
introduction of L. boryana could help to diversify the supply of edible species for human consumption.
The medicinal properties of L. boryana and its comparison with L. edodes remains unknown, but one
might expect that the American species possesses similar properties to those of shiitake.
PERSPECTIVES
Worldwide, more than 500 species of wild mushrooms are known to possess medicinal properties, such as
anti-inflammatory, antioxidant, antihypertensive, anti-cholesterol, antiviral and immune system stimulants
(Pérez-Moreno et al. 2010). Shiitake contains β-glucans and may be beneficial for human health. It has
been used for treatment of cancer, hypertension and high cholesterol levels (Bak et al. 2014). Edible
mushrooms are currently appreciated not only for their culinary value but also for the benefits they may
provide to human health. Consumers are increasingly interested and informed of the benefits they may
130
obtain through food and, as a result, a whole new market has been created for so-called "functional" foods.
A functional food provides health benefits beyond the basic nutritional requirements, that is, it contains
ingredients that have proven to help improve human health (Soler-Rivas and Reglero 2012).
Nutraceuticals are products of pure fungi or partially refined, from fruiting bodies, mycelium, or from the
culture medium filtered after mycelial growth in submerged culture. These products have nutritional
properties that promote health and they are usually consumed in the form of capsules or tablets as a
dietary supplement (Chang and Buswell 1996, Wasser et al. 2000, Trigos and Suárez Medellín 2010).
Edible mushroom cultivation is not only an ancestral agricultural practice, but an activity that currently
requires technological and scientific knowledge. Mushroom production from agricultural wastes for both
food and medicine help maintain an ecological balance that is sustainable. Edible fungi will undoubtedly,
due to their health benefits, be one of the most important elements in the diet of humans in the near future.
The cultivation of shiitake in tropical and subtropical regions using straw from various cereals represents
an alternative that should be considered in the near future. However, it is worth noting that research is
needed to select strains with a high production capacity at temperatures close to 25 ºC. The effects of
imminent global warming are becoming more and more noticeable in the temperate zones and the increase
of temperature reduces favorable zones for crops of many mushroom species. Deepening knowledge of
tropical and subtropical species of edible mushrooms and their management is essential for economical
production of these crops. Due to its nutritional characteristics and relative ease of production, shiitake is
destined to be a very important food in the near future.
ACKNOWLEDGMENTS
We thank the authorities of the Instituto of Ecología, A.C. and the CONACYT of Mexico as well as the
Institut National de la Recherche of France for the facilities provided to carry out this work.
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Biotechnological Applications
11. Auricularia spp.: EDIBLE MUSHROOMS WITH BROAD BIOTECHNOLOGICAL

PROPERTIES
José E Sánchez, Lilia Moreno and René H. Andrade

El Colegio de la Frontera Sur. Km 2.5 Carretera al Antiguo Aeropuerto
Tapachula, Chiapas, 30700, Mexico
ABSTRACT
An account is made of the importance, technology of cultivation and advances in medicinal and
biotechnological applications of the edible species of Auricularia. This genus represents the third most
cultivated mushroom worldwide and is the oldest cultivated by humans. It is a tropical and subtropical fungus
that grows frequently in places with average temperatures between 18 and 27°C. It is grown mainly in
Southeast Asia. As a formula for cultivation in different parts of the world, various sawdust and wheat straw
raw materials used either alone or mixed with bran are reported, as well as supplemented corncobs.
Antioxidant and medicinal qualities are mentioned in the treatment of heart problems, diabetes and certain
types of cancer. From a biotechnological point of view, this genus has broad expectations of application for
bioremediation of recalcitrant and emerging pollutants.
Keywords: wood ear mushroom, tropical mushrooms, medicinal uses, antioxidants
INTRODUCTION
Auricularia auricula-judae (≡A. auricula) is the first species of edible mushroom cultivated by
humankind. There are reports on its cultivation in China, from about 300 BC using logs (Cheng and Tu
1978, Quimio 2002). Its cultivation in plastic bags is more recent, with this method developed in Taiwan
in the early 1970's. Auricularia auricula-judae and A. nigricans (≡A. polytricha) (Chang and Quimio
1982) are the two most cultivated species, although A. fuscosuccinea is also cultivated to a lesser extent in
China (Quimio 2002). As a whole, these species now account for around 6,264 x 103 t of Auricularia
in China only, reference year 2013 (Figure 1, Chang 2005, Li 2002, Zhang et al. 2014.). It should be
noted that cultivation of mushrooms of this genus is mainly in Southeast Asia, especially China,
which produces about 90% of the total, in addition to Taiwan, Thailand, the Philippines, Indonesia
and Malaysia. With current world production, the genus Auricularia occupies third place in
production, only after Lentinula edodes and Pleurotus spp. and more than Agaricus bisporus, the
fourth most cultivated species (Royse 2014, Zhang et al. 2014, Royse et al. 2017).
Outside of Southeast Asia, cultivation of Auricularia is practically nonexistent, although some cases of
small-scale production have been reported in certain places in America and even Africa (Apetorgbor et al.
2005, Lou Hsu Pers Comm, Sawyer 2000, Ibáñez et al. 2009). The consumption of Auricularia occurs
mainly in producing countries, although not exclusively. Thus, it is consumed in Europe and America,
mainly within Asian communities and in specialized restaurants. In several regions, wild basidiomes of
some species of this genus are consumed in a traditional way by the local population (Ruan-Soto et al.
2004, 2009, Robles et al. 2007).
137
6000
Production (Thousand tons)

5000
4000
3000
2000
1000
0
1980 1985 1990 1995 2000 2005 2010 2015
Time (years)
Figure 1. Production of Auricularia spp. in China (thousand tons).

Source: Chang 2005, Li 2002, Zhang et al. 2014.
Substantial production and consumption of Auricularia, its ease of cultivation and the absence of
cultivators in large regions outside of Southeast Asia, make it an excellent alternative for production and
diversification. In Latin America, for example, there are growers in regions with several years' experience
cultivating Pleurotus spp. In some of these regions, there are signs of saturation of the local market, and
this likely will result in a decline in the price of oyster mushrooms with a collapse of some growers.
Initiating production of an edible mushroom other than the one already grown is a good option that opens
up new markets for the producer and helps maintain stability of prices.
An overview of cultivation of Auricularia spp., a common genus in the tropics and of growing interest, is
presented here. It is detailed in some cases, with data obtained at El Colegio de la Frontera Sur (Ecosur),
with species found in the state of Chiapas, Mexico and a culture of A. fuscosuccinea. Also, an overview of
Chinese wood ear mushroom production is presented.
Auricularia spp.
Index Fungorum (2016) indicates that historically, from about 529,712 online incomes, 173 records have
been named within the genus Auricularia. Of these, 55 are not identified to species, 43 belong to other
genera and 75 names represent the 32 species so far recognized within the genus.
For the state of Chiapas, Mexico, A. auricula-judae, A. fuscosuccinea and A. nigricans (≡ A. polytricha)
species are very frequent and grow mainly during the rainy season. These mushrooms grow on branches
and stumps of plants such as coffee (Coffea arabica), mango (Mangifera indica), rubber (Hevea
brasiliensis), primavera (Tabebuia donnell-smithii), laurel (Ficus benjamina L.), almond (Prunus dulcis),
capulín (P. salicifolia), Inga spp., and Yucca spp, from May to December on the Mexican side of the
Pacific and from June to February on the slope of the Gulf of Mexico.
Some sites within the state of Chiapas where specimens of this genus have been collected are shown in
Table 1 and Figure 2. The range of sites in terms of heights above sea level and temperatures varies
138
widely, ranging from 100 to 150 m (Huehuetán with average temperature of 27°C and extremes minimum
and maximum of 23 to 31°C) and 1800 to 1950 m (Chiquihuites, with average temperature of 18°C and a
range of 13 to 23°C). Under the same conditions it is possible to find A. delicata, although less frequently.
Auricularia spp. also grow on cedar trunks (Cedrela mexicana), coconut (Cocus nucifera), cocoa
(Theobroma cacao), Bursera simaruba, chile (Capsicum spp.) and Mexican lemon, a variety of Citrus
limon (Sánchez Vázquez et al. 1995). The four mentioned mushroom species are used for food by some
settlers of diverse localities of the entity. A. mesenterica is another species that also grows in the region. It
fructifies after the rainy season on branches and trunks and has a leathery consistency, therefore, it is not
as desirable as the other species. In coffee plantations of Soconusco, it is common to find five species: A.
auricula, A. delicata, A. mesenterica, A. fuscosuccinea and A. nigricans (≡ polytricha) (Andrade et al.
1996).
Table 1. Some municipalities where specimens of Auricularia spp. have been collected in Chiapas,
Mexico with average, minimum and maximum temperatures during the year and height above sea
level (m).
Municipality Average temperature Temperature range Altitude above sea
(°C) (ºC) level (m)
Cacahoatán 26 23-28 450-1700
Chiquihuites 18 13-23 1800-1950
Huehuetán 27 23-31 100-150
Salto de agua 26 20-33 200
Salvador Urbina 26 22-28 500
Tapachula 26.5 23-30 150-1200
Tila 23 18-31 1000
Tuxtla chico 26 22-29 120-450
Unión Juárez 21 17-24 800-1300
Yajalón 22 19-28 800-1100
139
Figure 2. Areas where specimens of Auricularia spp. were collected in Chiapas, México.
In the Mycological Collection ECO-TA-HO- of ECOSUR, there are specimens of the five species
mentioned above (Andrade and Pérez 2013). Table 2 presents a synopsis of the specimens in said
collection and Table 3 shows the distribution of the specimens collected throughout the year in those
municipalities.
Table 2. Specimens of Auricularia of the state of Chiapas present in the mycological collection of Ecosur
ECO-TA HO and its frequency.
Number of specimens Municipalities Localities
A auricula 45 9 30
A. delicata 80 6 28
A. fuscosuccinea 176 12 77
A. mesenterica 59 11 35
A. nigricans 222 14 76
Table 3. Species of Auricularia found during the year in municipalities of the state of Chiapas, Mexico.*
Municipality Jan Feb Mar Apr May Jun Jul Aug Sept Oct Nov Dec
Acapetahua M
A-D- A-D- A-F- F-N-
Cacahoatán N F F-N F-N A-D-N N M A-N
Heuhuetán A N-M N M
Mazatán F F-N N-M
Motozintla F-N N F-N A-F
140
Ocozocuautla N N A-N M
Salto de Agua F F-N N F-M
Suchiate F N-M
A-D- A-D-
D-F- D-F- D-F- A-D-F- F-N- D-F- F-N- N-
Tapachula M N-M N-M N-M N N-M M N-M M M
Tila F-N D
A-F-
Tumbalá N
A-D-
A-F- F-N-
Tuxtla Chico N N F-N M F-M N-M M
F-N-
Tuzantán F-N M N-M M N-M
A-D- A-D- A-D-F- A-D- D-F- A-F-
Unión Juárez N F-N F-N F-N N F-N N N
F-
F-N- D-F- N-
Yajalón M N-M M
*
Species of Auricularia: A=auricula D= delicata F= fuscosuccinea N= nigricans M= mesenterica
CULTIVATION OF Auricularia spp.
Cultivation methods of Auricularia spp. is much like that used for other edible white rot mushrooms, such
as Pleurotus spp. and Lentinula edodes. Practically, the three most popular species of this genus (A.
delicata, A. polytricha and A. fuscosuccinea) can be cultivated with the same culture method, under
similar environmental conditions (Wong 1993, 2016). Oei (2005), however, indicates that A. polytricha is
the most appropriate species for cultivation in tropical regions where temperatures are usually higher and
A. auricula-judae can only be grown in cooler areas.
The most favorable conditions reported for mycelial growth of A. polytricha include mycelial growth on
malt extract agar, a temperature of 25-30°C and pH of 7 (Khan et al. 1991). In the Philippines, cultivation
of Auricularia spp. is on a composted (five days) mixture of sawdust (78%), rice bran (20%), sugar (1%)
and calcium carbonate (1%) with 65% moisture (Vilela and Silverio 1982, Oei 2005). This mixture is
autoclaved for 1.5 h and then cooled for spawning. Also reported is the cultivation of A. polytricha on
wheat straw only and supplemented with rice bran. In the latter case, a biological efficiency of 87.7 g
mushrooms/100 dry substrate was obtained (Bhandal and Mehta 1989, Sharma and Jandaik 1992, Bisko
and Bilay 1995). Auricularia polytricha was successfully cultivated on sawdust of Acacia arabica mixed
with cotton waste (Khan et al. 1991, Irawati et al. 2012). Three types of tropical hardwoods (Falcataria
moluccana, Shorea sp. and Tectona grandis) were used for the cultivation of this species with the first
species giving higher yield. Information on the cultivation of A. auricula on sterile mixtures of sawdust
with wheat bran is also available on the internet. Some of these websites are listed at the end of this
chapter.
In Mexico, the cultivation of A. fuscosuccinea at a pilot plant level was accomplished on a mixture of
sterile corncobs supplemented with Leucaena or tamarind leaves (Calvo Bado et al. 1995, 1996,
Castillejos Puón et al. 1996). Morales et al. (2000) showed that the diameter of the substrate directly
influences the production of basidiomata and production is better in substrate blocks with a diameter of 6-
141
9 cm (Figure 3). Recently it was possible to cultivate this species using a self-heating pasteurization
method for substrate containing corncobs and on blends of corncobs and sawdust (Morales and Sánchez
2017.) A BE of 22.2% was obtained, which is considered low. Some research is under way using
supplements to see if yield may be improved. We were able to cultivate this mushroom on sterile Pangola
grass Digitaria decumbens, but not on this grass pasteurized by self-heating.
110
100
Biological Efficiency (%)
90
80
70 ECS-203
R² = 1 ECS-0210
60
50 R² = 0.9521
40
0 5 10 15 20
Bag Diameter (cm)
Figure 3. Effect of diameter of the substrate (corncobs) on production of basidiomes of two strains of A.
fuscosuccinea. Source: Morales et al. (2000).
Figures 4 and 5 show some aspects of the cultivation of Auricularia spp. in China. This includes
appearance of basidiomes, presence of insects and post-harvest management. In general, species of
Auricularia are not marketed fresh, but dried. Drying is done in the sun (Figure 5d), although it is more
advisable to do it in the shade (Oei 2005). Figure 6 shows some aspects of the cultivation process of A.
fuscosuccinea at a laboratory scale in Chiapas, Mexico.
Chemical composition of A. fuscosuccinea
Chemical composition (as determined in the Laboratory of Bromatology of Ecosur) of A. fuscosuccinea

cultivated on corncobs is shown in Tables 4 and 5. Protein content (13.5%) is lower than that of other
cultivated mushrooms, such as the button mushroom (29-45%), or the oyster mushroom (17-25%) and it is
rich in crude fiber (5.8%) relative to the button mushroom (1.9%). Carbohydrate content (62.4%) is high
compared to the range observed for other edible mushrooms (38 to 70%). Also, potassium content is
relatively high (Lelley 2017).
142
a) b)
c) d)
Figure 4. Various aspects of the cultivation process of Auricularia spp. in China a) basidiomes of A.
polytricha before harvest, b and c) presence of larvae in developing basidiomes, and d) quality control of
harvested mushrooms.
a) b)
143
c) d)
Figure 5. Processing Auricularia spp. fruitbodies in China: a) disinfection treatment of harvested
basidiomes, b) postharvest treatment chain: draining and slicing of basidiomes, c) sliced A. auricula
basidiomes, and d) sun drying of sliced basidiomes.
a)
b)
Figure 6. Basidiomes of A. fuscosuccinea

cultivated on corncobs in Chiapas Mexico a) on
the substrate (corncob), b) freshly harvested, c)
dry. Photographies, a & b) Kary G. Trujillo c)
René H. Andrade.
c)
144
Table 4. Proximal analysis of A. fuscosuccinea cultivated on corncob (g/100g).

Moisture Crude Crude Fat Carbohydrates Reducing Ash Energetic
Protein Fiber sugars value
(Kjoule)
7.9 13.5 5.8 13.5 62.4 10.3 4.7 1780.5
1 Kjoule=0.239 kcal.
Source: Bromatology Laboratory. Ecosur
Table 5. Mineral content of Auricularia fuscosuccinea cultivated on corncobs.

Calcium Magnesium Potassium Sodium Iron Copper Zinc Manganese
(g/100 g) (g/100 g) (g/100 g) (g/100 g) (g/100 (mg/kg) (mg/kg) (mg/kg)
g)
0.072 0.110 1.108 0.005 0.005 Less than 0.005g/100g
Source: Bromatology Laboratory, ECOSUR
MEDICINAL USES
Various medicinal properties are attributed to this genus, including antibiotic, anti-inflammatory, anti-
diabetic, etc. Tang et al. (2010) reported that A. auricula and A. polytricha are used in China and other
parts of Asia to treat various health conditions, such as hemorrhoids, hemoptysis, angina, diarrhea, and
gastrointestinal ailments. More recently, several species of this genus are used to prevent clotting, stroke,
heart attack, and to lower cholesterol and triglycerides. They also have antioxidant and useful properties
for treatment of diabetes and certain types of cancer. Polysaccharides of Auricularia auricula-judae are
known to provide myocardial protection benefits by improving superoxide dismutase (SOD) activity and
reducing lipid peroxidation in the heart (Ye et al. 2010). Other authors have examined the ability of
ethanolic extracts and polysaccharides of species of this genus for activity as antioxidants, antibiotics,
hypoglycemics, hypolipidemics or hepatoprotectives (Yuan et al. 1998, Mau et al. 2001, Yang et al. 2011,
Zeng et al. 2012, Cai et al. 2015, Reza et al. 2015, Fang et al. 2016). Antioxidant, hypoglycemic activities
and anti-lipid accumulation in the liver have also been found (Yang et al. 2002, Sun et al. 2010, Chiu et
al. 2014).
BIOTECHNOLOGICAL USES
Auricularia fuscosuccinea possesses an important ligninolytic capacity due to the presence of the enzymes
laccase and phenol oxidase (Figure 7, Montoya et al. 2014, Yanez-Montalvo et al. 2015, 2016). These
ligninolytic properties appear responsible for important capacities for recalcitrant substances degradation,
such as the insecticide endosulfan and the growth on agroindustrial polluted effluents (Nieto López y
Sánchez Vázquez 1997). In fact, Escobar et al. (2002) demonstrated that this species is able to degrade
100 mg/kg endosulfan in eight days of liquid culture. On the other hand, Yánez Montalvo et al. (2016)
demonstrated that the crude extract from a liquid culture of A. fuscosuccinea is able to degrade 97% of this
insecticide in a solution of 35 ppm in 4 days of contact, suggesting the presence and action of extracellular
enzymes. Furthermore, this species is able to decolorize the bright blue R dye R Remazol (RBBR,
Machado et al. 2005) and recent studies have demonstrated the ability of this species to degrade 100% of
the emerging metamizole sodium pollutant in just six hours of exposure to a culture extract of this fungus
(Mayorga et al. 2017).
145
200 10
180 9
160 8
Enzimatic Activity (U/ml)
140 7
Protein (μg/ml)
120 6
100 5
80 4
60 3
40 2
20 1
0 0
1 2 3 4 5 6 7
Days
Figure 7. Production of laccase (▲), phenol oxidase (●) and protein concentration of cell free supernatant
(gray bars) of liquid culture of A. fuscosuccinea ECS-0210, in flasks (per L) of culture medium (2 g
glucose, 5 g yeast extract), during 7 days at 110 rpm and 26-28°C. Values are means of three replicates
with standard deviation (Yánez-Montalvo 2014 and Yánez-Montalvo et al. 2015).
PERSPECTIVES
Although the first edible mushroom cultivated worldwide, about 2300 years ago, belongs to the genus
Auricularia, this genus is not one of the most studied or known today. Other mushrooms, that were grown
at a later date, but that now have significant production worldwide, such as A. bisporus, L. edodes and P.
ostreatus have been studied in more detail. It is well known, for example, that the basidiomes of
Auricularia spp. are very sensitive to and affected by insect attack, mainly of the larval state. However,
there are no reports or studies carried out on this subject in cultivation. With other cultivated mushrooms,
there is much more research on pest management. However, with Auricularia spp., much work remains in
order to provide more knowledge to better control this fundamental aspect of the crop and reduce current
costs in post-harvest treatments.
Information on medical and biotechnological qualities of Auricularia spp. is rudimentary. The important
findings of the nature of its polysaccharides and their enzymes suggest that there is still much potential in
this genus of edible fungus.
ACKNOWLEDGMENTS
The authors thank Ma. Guadalupe Pérez Escobar for chemical analysis made on A. fuscosuccinea samples.
146
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Websites of related interest to Auricularia spp.:
http://unicornbags.com/cultivation/auau.shtml. Visited on 6 September 2018.

http://www.amanitacesarea.com/auricularia.html
http://herbolaria.altervista.org/plantas/oreja-judas.html.
http://www.sonremedioscaseros.com/oreja-de-judas/
http://www.tusplantasmedicinales.com/oreja-de-judas/
http://www.davidmoore.org.uk/Sec04_07.htm
http://revistaciencias.univalle.edu.co/volumenes/vol_15/ABolanos.pdf
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12. ANTIOXIDANT ACTIVITY AND CHEMICAL COMPOSITION OF Grifola frondosa
Ma de Lourdes Acosta-Urdapilleta1, Gerardo Díaz-Godínez2, Maura Téllez Téllez1*

1
Laboratorio de Micología, Centro de Investigaciones Biológicas. Universidad Autónoma del Estado de
Morelos. Avenida Universidad 1001, Colonia Chamilpa, Cuernavaca Morelos. México. C.P. 62209.
2
Laboratorio de Biotecnología, Centro de Investigación en Ciencias Biológicas, Universidad Autónoma de
Tlaxcala. *[email protected]
ABSTRACT
Grifola frondosa is marketed in China, Japan and other Asian countries as a medicinal and edible fungus. Several
bioactive properties have been reported. This mushroom also has considerable levels of protein and its content may
vary depending on culture conditions and the type of substrate used. In this study, we worked with dry and crushed
fruit bodies of Grifola frondosa (HEMIM-43) cultivated on supplemented oak sawdust. Its proximal chemical
composition, polyphenol content and inhibitory concentration (IC 50) of an aqueous extract with ABTS and DPPH
radicals were determined. Fruit bodies contained 28.9% protein, 2.4% ash, 6.8% ethereal extract, 44.8% nitrogen free
extract and 17% fiber. The content of polyphenols was 0.26 mg GAE/g and antioxidant activity of ABTS and DPPH
radicals was positively correlated with extract concentration. It is possible that fruit bodies contained substances that
act as donors of hydrogen and react with free radicals to turn them into stable products and to complete the chain
reaction.
Keywords: Grifola frondosa, chemical composition, polyphenols, radical DPPH, radical ABTS.
INTRODUCTION
Grifola frondosa (Dickson: Fries), also called maitake and hen of the woods (Stamets 1993), often
develops as a heavy mass (groups can weigh many kilos) on the base of stumps and on roots of oaks,
elms, persimmons and other trees. Like many other fungi, optimal maitake growth conditions exist within
a limited range of temperature, humidity and other environmental factors. Maitake can be found in
temperate forests of northern Asia, Europe and eastern North America (Mayell 2001). An increase in
forage crop areas and civilian development have combined to limit availability of maitake in the wild.
Cultivation of maitake is of recent development, only in the last three decades, so producers have been
able to pass from the dependence on wild maitake to that of cultivated maitake. Maitake is often grown on
substrate formulated with a combination of sawdust/bran/soybeans (in a proportion 80:10:10) (Mizuno et
and Zhuang 1995) contained in bottles or bags. Japanese commercial cultivation, mainly for food, began
in 1981 with 325 tons (Takama et al. 1981), and grew to 1,500 tons in 1985, 8,000 tons in 1991 and
almost 10,000 tons in 1993. Commercial production of maitake worldwide is now more than 40,000 tons
(Mayell 2001). Rendón-Ramírez et al. (2012) reported the production of G. frondosa on a mix of maize
straw and maize oat, where they obtained fruit bodies with typical characteristics of the species and a
biological efficiency of 19.9% at 120 days of incubation. In the last two decades, maitake was also grown
for use as a dietary supplement. It may be the most versatile and promising medicinal supplement,
although currently less well known than shiitake (Lentinula edodes) and reishi (Ganoderma lucidum). The
nutritional content of mushrooms is considered to be of high quality, not only for their proteins, but also
because they contain biologically active compounds considered secondary metabolites that play an
important role in the structure and survival of the fungus. There are several reports that edible fungi
produce a large number of these compounds, including phenolic compounds that offer a health benefit and
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provide protection against chronic degenerative diseases. Therefore, these fungi are traditionally
considered a fundamental part of healthy diets and additionally have been processed for nutraceuticals
(Chang and Miles 2004).
Diseases caused by poor eating habits, excessive consumption of alcohol and smoking may shorten the
human life span due to oxidative stress (Sohal 2002). Although reactive oxygen and nitrogen species play
an important role in the maintenance of health, their excess leads to situations of oxidative stress that are
detrimental (Alves et al. 2010). The fungi have components that influence antioxidant activity and include
phenolic compounds, tocopherols, ascorbic acid, polysaccharides, terpenoids and polysaccharide-protein
complexes (Kosanić et al. 2012). Recently, several bioactive properties of this fungus have been explored,
attracting considerable attention around the world. Fruit bodies and mycelium grown in liquid culture
contain antitumor polysaccharides that have been identified as glucans (β-1,6 and β-1,3) (Masuda et al.
2009). Research efforts have focused mainly on therapeutic effects and methods of cultivation of this
edible fungus, so there is relatively less information available on its antioxidant properties. Therefore, the
aim of this work was to determine the antioxidant activity of aqueous extracts of fruit bodies of G.
frondosa.
Drying of material
Fruit bodies of G. frondosa were dried immediately after harvest. Drying was performed via an airstream
at 20 to 24 °C in darkness to a constant weight. They were then macerated, sieved and stored in the dark
until used (Salas et al. 2003).
Chemical analysis
Official methods of Association of Official Analytical Chemists (AOAC) were used for determination of
moisture, crude protein, ash, ethereal extract, nitrogen-free extract and fiber (AOAC 1990).
Preparation of aqueous extract
Aqueous extract of fruit bodies was prepared with 0.5 g of sample in 10 mL of water with heat treatment
(at 95 °C for 5 min). Subsequently, the supernatant was recovered by centrifugation (8000 x g/15 min).
Determination of total polyphenols
Total polyphenols were measured according to the method of Singleton et al. (1999). The sample (0.5 ml)
was added to 4.5 ml of distilled water and mixed with 0.2 ml of Folin-Ciocalteu reagent, 0.5 ml of
saturated Na2CO3 solution and 4.3 mL of distilled water. The reaction mixtures were incubated for 60 min
in the dark at room temperature. Absorbances were measured at 725 nm. Total phenolic content was
expressed in mg gallic acid equivalents per g of dry sample (mg GAE/g).
Evaluation of antioxidant activity
Scavenging activity of DPPH radical

DPPH radical scavenging activity was determined according to Moraes-de-Souza et al. (2008) with some
modifications. The reaction mixture consisted of 0.5 ml of extract, 3 ml of methanol, and 0.3 ml of 0.5
mM DPPH radical solution in methanol. After incubation for 45 min, absorbance was determined in a
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spectrophotometer at 517 nm. The antioxidant activity (% inhibition) was calculated by Eq. 1,
% Inhibition = [(Acontrol–Asample)/Acontrol] × 100
Where Acontrol is the absorbance of the negative control at the moment of solution preparation and
Asample is the absorbance of the sample after 45 min.
Scavenging activity of ABTS radical

ABTS radical scavenging activity was determined according to Re et al. (1999) with some modifications.
ABTS was dissolved in water to a 7 mM concentration. The ABTS radical cation (ABTS•+) was produced
by reacting ABTS stock solution with 2.45 mM potassium persulfate (final concentration) and incubating
the mixture in the dark at room temperature for 12 to 16 hours before use. The ABTS•+ solution was
diluted with water to an absorbance of 0.70 (± 0.02) at 734 nm. The reaction mixture consisted of 0.07 ml
of extract and 3 ml of the ABTS radical. After incubation for 6 min, absorbance was determined in a
spectrophotometer at 734 nm. The antioxidant activity was calculated by Eq. 1, where Acontrol is
absorbance of the negative control at the moment of solution preparation and Asample is absorbance of
the sample after 6 min.
The IC50 values were calculated from the graph that represents the concentration of the sample required
to scavenge 50% of the ABTS or DPPH free radicals. The IC50 is often used to express the amount or
concentration of extracts needed to scavenge 50% of the free radicals. ABTS and DPPH were expressed as
mg GAE/L.
RESULTS AND DISCUSSION
Dry extract of fruiting bodies from G. frondosa contained 28.9% protein, 44.9% nitrogen free extract,
6.8% ethereal extract and 17% fiber (Table 1).
Table 1. Chemical composition of fruit bodies of Grifola frondosa

Component Wet basis Dry basis
g/100g g/100g
Moisture 3.35 ± 0.15 0
Ash 2.29 ± 0.51 2.37
Crude protein 27.90 ± 0.20 28.87
Nitrogen-free extract (Carbohydrates) 43.38 ± 1.87 44.88
Ethereal extract (Fats) 6.55 ± 1.15 6.78
Fiber* 16.53* 17.10
* Obtained by weight difference.
In general, in terms of the amount of crude protein, the fungi are in a lower range than animal meat, but
far above most other foods (Chang and Wasser 2012). In ancient times, edible wild mushrooms were
popularly known in Central Europe as the "meat of the poor" because of the quality and quantity of
proteins and amino acids they contain (Kalac 2013). The protein content of the fungus is considered high,
ranging from 10 to 44% of dry weight (Longvah and Deosthale 1998). Petrovska (2001) analyzed the
protein content of 47 species, obtaining an average of 22.8%. Uzun et al. (2009) studied 30 species of
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fungi and obtained average protein values of 24.9%. Some authors have detected protein values of 54%
and 59% in Cantharellus cibarius and Lepista nuda, respectively (Barros et al. 2008).
The ethereal extract content of fungi is relatively low, with values reported between 1% and 8% on a dry
basis, although it may vary between species or between strains of the same species depending on several
factors (Barros et al. 2008). Some species of the genus Cordyceps contain 10.06% fat, while others such
as Lentinula edodes, Tremella fuciformis, Clitocybe maxima, Trametes versicolor, Auricularia
mesenterica and Auricularia polytricha are below 1.5% (Ulziijargal and Mau 2011). The ratio of
unsaturated to saturated fatty acids in fungi may range from 4.3 to 12.7 in wild fungi (Liu et al. 2012) and
0.7 to 4.5 in cultured fungi (Zhang and Ran 2005). Oleic acid and linoleic acid are the most prevalent,
constituting two-thirds of all fatty acids that have been identified in fungi, followed by saturated palmitic
acid (Kalac 2009).
Carbohydrates (simple sugars as monosaccharides, oligosaccharides and construction polysaccharides as
glucans) vary depending on species and they constitute approximately half of the dry matter with their
average values between 35 and 70 g/100 g on dry basis. The major sugars in fungi are mannitol and
trehalose with mean values of 28.9 g/kg and 39.2 g/kg (dry wt basis), respectively (Kalac 2009). Fiber is a
complex mixture of carbohydrates such as: chitin, mannans and glucans with proteins, waxes, saponins or
phytosterols and are not digestible by humans. Edible fungi represent an important source of fiber: 4.5 to
54.5 g/100 g of dry matter (Manzi et al. 2004) with mean values of 40 to 90 g/kg for soluble fiber and 220
to 300 g/kg for insoluble fiber (Kalac 2009).
The polyphenol content of Grifola frondosa extract was 10.4± 0.001 mg GAE/ml. Polyphenols are organic
compounds whose basic structure is characterized by containing at least one aromatic ring with one or
more linked hydroxyl groups. These can range from simple molecules (phenolic acids, flavonoids or
phenylpropanoids) to highly polymerized compounds (tannins, lignins or melanins). The phenolic content
of fungi is determined by the presence, but not exclusive, of phenolic acids, as other phenolic compounds
are also present depending on the species analyzed. Arbaayah and Kalsom (2013) found that phenolic
content differed between species, indicating that each species has its own pathway in the metabolism of
phenolic compounds. Exposure of living cells to various sources of radicals such as sunlight and
chemicals may cause organisms to develop their protective systems with both enzymatic and non-
enzymatic reactions. Most of the antioxidant properties present in fungi are mainly in the form of phenolic
acids and flavonoids, followed by tocopherols, ascorbic acid and carotenoids (Ferreira et al. 2009). It is
suggested that production of phenolic compounds in fungi provides defensive mechanisms for free
radicals and reactive species of certain chemicals. In a study of Pleurotus eryngii and P. ostreatus, total
phenol content was 0.03 mg/g and 0.09 mg/g dry weight (Kim et al. 2008), respectively, while in another
P. ostreatus strain total phenol content was 0.71 mg/g dry weight (Jayakumar et al. 2009). Mau et al.
(2004) obtained 1.59 mg/g phenols using the Folin-Ciocalteu reagent method from the mycelium of G.
frondosa and Smith et al. (2015) reported 2.31 mg GAE/g. In both studies they established phenols as the
main antioxidant component found in the methanolic extracts of the mycelium. Flavonoids represent the
lowest levels of phenolic compounds found in fungi (Barros et al. 2008). In fact, there are few sources that
prove their presence in scarce, and very specific, species (Iwashina 2000, Ribeiro et al. 2007).
The percentage of inhibition of both radicals increased in relation to concentration, i.e., the IC50 of the
aqueous extract with the DDPH radical was 17.1 mg GAE/L and with ABTS was 101.3 mg GAE/L. Yeh
et al. (2011) reported a higher percentage of DPPH radical in water extracts than in ethanolic extracts of
G. frondosa. Percentages of inhibition were in descending order of cold water (62.6%-59.6%)> hot water
(53.1%-50.8%)> ethanol (17.1%-18.6%). Inhibition of both aqueous extracts and ethanolic extract may be
due to the presence of soluble polysaccharides. Percentage of inhibition of aqueous extracts was higher in
cold water compared to hot water. Another study also found that the percentage inhibition of cold-water
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extract was higher than that of warm-water extract of Pleurotus citrinopileatus fruit bodies (Lee et al.
2007).
Fungi with these type compounds are already available in commercial formulas with proven antioxidant
activity, as in the case of powdered preparations of two of the most famous and studied: Agaricus blazei
and L. edodes (Carneiro et al. 2013). So, the fungi are considered as possible dietary supplements or
functional foods against chronic diseases related to oxidative stress. However, when administering them in
the diet, it should be kept in mind that during their cooking or processing, their antioxidant activity may be
affected (Barros et al. 2007). In this work, extracts were heat-treated for 5 min and still maintained
antioxidant activity, so it is a good alternative to use as a component of certain enriched foods in which
some thermal processes are performed.
ACKNOWLEDGMENTS
We thank the Biological Research Center of the Autonomous University of the State of Morelos and the
Biotechnology Laboratory of the Autonomous University of Tlaxcala.
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13. IMMUNOMODULATING AND ANTITUMOR PROPERTIES OF Pleurotus

sp. IN CUBA
Humberto J. Morris1, Gabriel Llauradó1, Yaixa Beltrán1, Yamila Lebeque1, Pedro L. Batista2,
Serge Moukha3, Isabelle Perraud-Gaime4, Nora García1, Rosa C. Bermúdez1, Maíra Bidart5, Paul Cos5,
Edgar Hernández6, José C. Diez7
1
Center for Studies on Industrial Biotechnology (CEBI), Universidad de Oriente. Santiago de Cuba 5,
CP 90 500, Cuba. <[email protected]>
2
Center of Toxicology and Biomedicine (TOXIMED), Medical University of Santiago de Cuba,
CP 90 400, Cuba
3
Laboratory of Toxicology and Applied Hygiene/ INRA, Bordeaux University-Campus de Carreire, 33076
Bordeaux Cedex, France.
4
Mediterranean Institute of Biodiversity and Ecology, UMR CNRS IRD 7263, Aix Marseille University -
Campus de l'Etoile, 13397 Marseille Cedex 20, France
5
Laboratory for Microbiology, Parasitology and Hygiene, Faculty of Pharmaceutical, Biomedical and
Veterinary Sciences, University of Antwerp, Belgium
6
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Canada
7
Department of Biochemistry and Molecular Biology, University of Alcalá de Henares, Madrid, Spain
ABSTRACT
In this chapter, we report on the effects of Pleurotus (oyster mushroom) extracts (Myc-E and FB-E) and powders
(FB-P) on immunodeficient BALB/c mice. The anti-proliferative effect on NB4 human leukemia cells was measured
by flow-cytometry. In addition, antioxidant activity was investigated by scavenging of DPPH and ABTS radicals,
reducing power and inhibition of lipid peroxidation. Pleurotus mycelial extract (Myc-E) exerted a protective effect in
both cyclophosphamide and whole-body irradiated mice in terms of bone marrow cellularity, white blood cell counts
and enhancement of the monocyte-macrophage system. Cyclophosphamide treated mice also showed a stimulant
effect on cell immune response when administered with fruiting body powder (FB-P). Fruit body-derived extract
(FB-E) stimulated immunonutritional recovery of malnourished mice after activation of gut-associated lymphoid
tissues. Myc-E reduced viability of NB4 leukemia cells, particularly at a concentration of 200 μg/ml, by arresting
cells in the G2/M phase. At 10 mg/ml, FB-E showed scavenging effects for DPPH and ABTS radicals (90.4% and
80%, respectively) and inhibited lipid peroxidation (51.2%), whereas at 5 mg/ml manifested a reducing power of
0.438. Pleurotus derived-products could be considered good candidates for developing nutraceuticals and innovative
myco-therapeutics, as judged by their immunomodulating/antitumor and antioxidant effects.
Keywords: antioxidant, antitumor, immunomodulating, mushrooms, myco-therapeutics, nutraceuticals, Pleurotus
INTRODUCTION
Today, the well-being of humankind faces unprecedented challenges involving inadequate regional food
supplies, deficiency in new insight into healthy eating, diminishing quality of health, and increasing
environmental deterioration (Chang and Wasser 2012). In this context, mushrooms are emerging as a vital
component of the human diet and have become attractive as a functional food and as a source of drugs and
nutraceuticals (Ferreira et al. 2009, Patel et al. 2012, Gomes Corrêa et al. 2016, Morris et al. 2017a).
Fruit bodies as well as mushroom mycelia have a broad range of bioactive properties. Mushrooms are
thought to exert approximately 130 pharmacological functions such as antitumor, immunomodulatory,
antigenotoxic, antioxidant, anti-inflammatory, hypocholesterolemic, antihypertensive,
antiplatelet‐aggregating, antihyperglycemic, antimicrobial, and antiviral activities (Lindequist 2013,
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Paterson and Lima 2014, Wasser 2014, Prasad et al. 2015). These pharmacological effects have been
demonstrated for many traditionally used mushrooms, including species from the genera Ganoderma,
Lentinula, Agaricus, Auricularia, Flammulina, Grifola, Hericium, Pleurotus, Trametes (Coriolus),
Schizophyllum, Lactarius, Phellinus, Cordyceps, Inonotus, Inocybe, Tremella, and Russula (Roupas et al.
2012, Vikineswary and Chang 2013, Valverde et al. 2015).
Several ongoing research projects are directed toward the promotion of mushrooms as a new generation of
“biotherapeutics” (Pereira et al. 2012, Patel and Goyal 2012). Mycotherapy comprises the use of
mushroom-derived extracts and bioactive compounds for their utilization as functional products or drugs
with the ability of promoting health. As part of cancer research, mycotherapy is a relatively new and
promissory field as a source of agents with immunomodulating and antitumor properties (Popovic et al.
2013, Peña-Luna et al. 2016). The bioactive molecules comprise high molecular weight compounds,
mainly polysaccharides, and low molecular weight secondary metabolites (de Silva et al. 2013). Given
that only about 10% of mushroom biodiversity has been studied so far, and few of them have been
characterized with regard to health benefits, it is likely that new active compounds will be discovered in
the future (Hawksworth 2012). Particularly in tropical areas, 22 to 55% (in some cases up to 73%) of
mushroom species have not yet been described (Bass and Richards 2011).
Although Pleurotus (oyster mushroom) is the second most important mushroom of culinary value (Royse
2014), there has been an upsurge in Pleurotus research activities in the last two decades in view of its
biopotentialities. This genus includes some of the most popular Basidiomycetes edible mushrooms which
cultivation has increased greatly throughout the world during the last few decades (Sánchez and Royse
2002, Gomes Corrêa et al. 2016). Its popularity has been expanded due to its vigorous growth on a variety
of agroforestry substrates and for the production of a high nutritional value-food containing biologically
active compounds with therapeutic effects (Carrasco-González et al. 2017).
Pleurotus species have been recognized as mushrooms with dual functions to humans both as food and
medicine (Khan and Tania 2012, Patel et al. 2012). Recent studies on various Pleurotus species have
shown a number of the pharmacological activities mentioned above (Gregori et al. 2007, Deepalakshmi
and Mirunalini, 2014, Beltrán et al. 2015, Fu et al. 2016, Sun et al. 2017). In particular, Pleurotus spp.,
are distinguished as important natural resources for immunotherapy, in view of the content of several
bioactive compounds able to augment or complement a desired immune response defined as ‘host defense
potentiators’ (HDPs) (El Enshasy et al. 2012, Morris et al. 2015, Pérez-Martínez et al. 2015).
Such bioactive compounds include polysaccharopeptides, polysaccharide-proteins, functional proteins,

glucans, proteoglycans and many others. Most of these bioactive compounds follow the
immunomodulatory pathway mechanism of mushroom β-glucans by stimulating activities for both innate
and adaptive immune systems (El Enshasy et al. 2013, Facchini et al. 2014). They activate innate immune
system components such as natural killer (NK) cells, neutrophils, and macrophages, and stimulate
cytokines expression and secretion. These cytokines in turn activate adaptive immunity through the
promotion of B cells for antibodies production and stimulation of T-cell differentiation to T helper (Th1
and Th2) cells, which mediate cell and humoral immunities, respectively (Morris et al. 2007, Ike et al.
2012, Oloke and Adebayo 2015, Tanaka et al. 2016).
Both fruit bodies and mycelia of Pleurotus spp. have been studied in search of effector molecules
(Kyakulaga et al. 2013, Morris et al. 2012, 2017b). In the opinion of Chang (2001), mycelial products are
the “wave of the future” because they ensure standardized quality and year‐round production. Thus,
submerged liquid fermentation can provide more uniform and reproducible biomass and may afford
valuable medicinal products. However, fruit bodies obtained under good manufacturing practice (GMP)
can also be used in the formulation of consistent and safe mushroom products such as functional foods,
nutraceuticals and biologically active compounds (Morris et al. 2014).
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Much research work has been reported for various extracts and isolated compounds, particularly
polysaccharides, and efforts to find new immunomodulators are ongoing (El Enshasy et al. 2013).
Therefore, the study of synergy exerted by the vast structural diversity of biomolecules, found in
Pleurotus crude extracts, powders and other preparations on immune responses, deserves special attention.
Better insight into the different roles of multiple active compounds and mechanisms underlying their
biological action will accelerate commercial production of pharmaceuticals for therapeutic applications
(Figure 1).
In Cuba, implementation of technologies for cultivation of Pleurotus spp. on agricultural substrates, in

addition to food generation for human consumption, has opened new research activities towards
mushroom immunoceuticals. These immunomodulating therapeutic agents can be used in the management
of some immunocompromised patients suffering from various diseases, like cancer, HIV/AIDS, liver
cirrhosis, acute respiratory failure and recent bone marrow transplants.
The present chapter gives, from an experimental perspective, an updated comprehensive account of some
medicinal properties of Pleurotus sp. involved in anti-cancer mechanism, such as immunomodulating,
anti-proliferative and antioxidant, exerted by extracts and powder preparations obtained from both
mycelium and fruit bodies. These results are significant in that they provide a framework for further
exploration of the use of Pleurotus bioactive preparations for immunotherapy as new life-saving drugs.
Mushroom material
Pleurotus sp. strain CCEBI-3024 is deposited at the Culture Collection of the Center for Studies on
Industrial Biotechnology (CEBI, Cuba). The strain was maintained on slants with solid medium of potato
dextrose agar (PDA) incubated at 5 °C.
Preparation of Pleurotus-derived products
Pleurotus sp. cultivation was performed by solid-state fermentation of mushroom spawn on pasteurized
coffee pulp used as substrate in plastic bags of 2 kg (30-40 cm) (Bermúdez et al. 2001). Fruit bodies were
harvested, sliced into small pieces and dried at 45 °C for 24 h. Dried material was milled and the resulting
powder was preserved away from light and humidity in plastic bags for further use (FB-P). The powder
contained 55% (w/w) carbohydrate, 20% (w/w) protein and 4% (w/w) lipids.
Cold water extracts of Pleurotus fruit bodies (FB-E) were produced by exhaustively washing the
carpophores with distilled water and then slicing them into 1 cm2 pieces. They were weighed and 5 ml of
distilled water was added per g of mushroom tissue. The extraction was made at 20 °C with continuous
stirring at 100 rpm for three hours and final extracts were collected by centrifugation and filtration.
Extracts were stored at -20 °C and freeze dried. They were mainly composed of 43% carbohydrate and
35% protein.
The preparation of Pleurotus mycelium hot-water extract (Myc-E) began with inoculation of mycelium in
YPG medium (yeast-peptone-glucose) contained in Erlenmeyer flasks. The flasks were incubated at 27 °C
with continuous stirring at 100 rpm for 15 days. After the submerged fermentation was carried out,
mycelia were collected by centrifugation at 4,000 rpm and washed twice with distilled water. Isolated
mycelia, suspended in 200 g (wet weight)/l of distilled water, were extracted with boiling water for 10 h
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and the final extracts were collected by centrifugation and filtration. Extracts were stored at -20 °C and
freeze-dried. The major components of Myc-E were carbohydrate (76.8%) and protein (12%).
Figure 1. Protocol for developing nutraceuticals and effector molecules with biological activity from
Pleurotus sp. mushrooms in Cuba.
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Laboratory animals
BALB/c mice purchased from the National Center for Production of Laboratory Animals (CENPALAB,
Havana) were used. Experiments were carried out under conventional sanitary conditions and animals
were maintained at controlled temperature and humidity throughout the investigation ensuring the optimal
interval for the specie. The administration of products was made daily in the morning between 9 to 10 am.
The research was approved by the institutional Ethical Committee (University of Oriente) and was
performed in accordance with Cuban legislation and the National Research Council Guidelines for Care
and Use of Laboratory Animals.
Effect of intraperitoneal (i.p.) administration of Pleurotus mycelium hot-water extract (Myc-E) to

cyclophosphamide-treated or whole-body irradiated mice
Cyclophosphamide (CY)-treated mice.

Fifteen male mice (20-25 g) were divided into two groups. Myc-E was administered intraperitoneally (i.p.)
at 100 mg/kg for 7 days to ten Balb/c mice and cyclophosphamide (CY) USP 23 for injection, obtained
from JSLYP (China), at 100 mg/kg was given i.p. on the fifth day. The control group, comprised of five
mice, was injected i.p. with physiological saline. On the eighth day, blood was collected from the orbital
vein and animals were then bled to death.
Whole-body irradiated mice

Male mice were randomly allocated into two groups (n= 10) for eventual whole-body irradiation with a
60
Co source Theratron teletherapy unit (Siemens, Erlanger, Germany) in the Oncological Hospital
“Conrado Benítez” (Santiago de Cuba, Cuba) at a dose rate of 0.43 Gy/min for 20 min (date of exposure
designated as day 0). For the analyses of effects of the mushroom-derived materials, one group of mice
was administered with the extract intraperitoneally (i.p.) at a dose of 100 mg/kg in a volume of 0.2 ml on
days -10 to -6 and -2 to +1 with respect to the irradiation. Mice in the control group (n=10) were injected
with saline solution in place of the extract; non-irradiated mice were used as negative controls. All mice
were euthanized 24 h after the final administration of extract or saline, and tissues/blood were isolated for
analyses.
In both experiments, blood specimens were analyzed for white blood cell count. Femoral bone marrow
cells were withdrawn with Hanks’ solution and counted with a Neubauer chamber (Germany). Spleen cell
suspensions were prepared by gently teasing the tissue with ice-cold Hanks’ solution and passing it
through antiseptic gauze (Johnson & Johnson Medical, TX, USA) and counted with a Neubauer chamber.
Peritoneal exudate cells were collected from the peritoneal cavity of mice by washing with Hanks’
solution and also counted.
Functional activity of the monocyte-macrophage system of each host was evaluated using a carbon
clearance test. The clearance rate of carbon was expressed as the ratio of absorbance for samples from
Myc-E treated (or saline-control) mice with respect to values from immunocompetent mice injected with
carbon particles (i.e., received no test substances) (see Morris et al. 2008 for details).
Effect of oral administration of Pleurotus sp. fruit bodies powder (FB-P) on cell immune response of
cyclophosphamide treated mice
The 20 to 25 g male BALB/c mice were fed a standard diet and acidified water ad libitum. Fifteen mice
were divided into three groups (n= 5). The two experimental groups were treated intraperitoneally (i.p)
with cyclophosphamide (CY) USP 23 for injection obtained from JSLYP (China) at 100 mg/kg on day 0.
FB-P was administered by oral route (1,000 mg/kg) for 7 days to the ‘CY/ FB-P¨ group, whereas
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physiological saline solution was administered to the ‘CY/ Saline’ group in a similar schedule. Non-
treated mice were used as controls in the experiment.
The effect of FB-P on cell-mediated immunity was determined by the delayed-type hypersensitivity
(DTH) reaction (Kim et al. 1998). Mice were immunized by an intradermal (i.d.) injection of 50 l of 5
mg/ml BSA emulsified in Complete Freund Adjuvant (CFA; Sigma, St. Louis, MO, USA) at two sites on
the abdomen. Eight days after immunization, mice were re-challenged by injection of 20 l of 5 mg/ml
BSA into one rear foot pad, while the other received a comparable volume of phosphate buffered saline
(PBS). Measurements of foot pad swelling were taken at 24, 48 and 72 h after challenge by use of a
micrometer (Mitutoyo, Tokyo, Japan). The magnitude of the DTH response was determined as differences
in foot pad thickness between the antigen and PBS-injected foot pads. A similar immunization protocol
was applied to control animals. Histological studies were made with samples taken from the antigen-
injection sites with the hematoxylin-eosin staining and observations performed under an optical
microscope (100x).
Popliteal lymph nodes (right and left) of antigen sensitized and re-challenged animals were removed and
washed with PBS pH 7.4. Excess moisture was discarded with a filter paper and the lymph nodes were
immediately weighed separately with an electronic analytical balance (Sartorius). The mass index was
expressed as the relation between weights of the popliteal node belonging to BSA-injected foot pad with
respect to that of PBS-injected pad (Descotes 2006).
Effect of oral administration of Pleurotus sp. fruit bodies cold water-extract (FB-E) to malnourished
BALB/c mice
Female BALB/c mice, weighing 20 g, were housed individually at 23 °C with a 12-hour/12-hour

light/dark cycle. Thirty mice that were starved for 3 days and had free access to salted water were studied.
After this time, blood was collected from the orbital vein of 10 mice and the animals were killed (M
group). The others were re-fed ad libitum for 8 days with commercial pelleted diet (M-CD group) or with
the commercial diet and the Pleurotus fruiting bodies cold water-extract (FB-E) administered orally at a
dose of 100 mg/kg of body weight per day (M/FB-E group). A control group of 10 mice was fed with
commercial diet throughout the study.
After the small intestine was collected, the segment correspondent to jejunum was rinsed thoroughly with
ice-cold saline solution, opened, and blotted dry. Mucosa was scraped with a glass slide and weighed
separately. Jejunal mucosa was homogenized with ice-cold phosphate-buffered saline with a pH of 6.0
(1:3 w/v). Total protein and DNA were estimated by the methods of Lowry et al. (1951) and Burton
(1956), respectively.
Functional activity of the monocyte-macrophage system was evaluated as described previously for
cyclophosphamide-treated or whole-body irradiated treated with Myc-E.
Humoral immune response was evaluated through an immunization protocol with sheep red blood cells
(SRBC) as antigen. Three groups, comprised of five mice, were designed: M-DC, M/FB-E and control as
described above. After the starvation (day 0) mice were injected intraperitoneally (i.p.) with 0.2 ml of a
25% SRBC saline solution. After 7 days from the first injection, blood samples of 50 μl were drawn from
the orbital plexus to measure antibody titres by a haemagglutination (HA) reaction. Reciprocal serum
dilution, which just gave agglutination, was considered the titre. At this time, mice received the second
immunization and on day 14, antibody titres were determined.
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Cytometric analysis of cell viability of human acute promyelocytic leukemia (NB4) cells
Cells were maintained in RPMI medium (Gibco-Life Technologies) with 10% fetal bovine serum (FBS),
1% penicillin-streptomycin and 0.02 mg/ml gentamycin. Cells were cultured at 37°C in a humidified air
atmosphere with 5% CO2. Cell viability of NB4 cells was determined by flow cytometry by measuring the
level of impermeability to propidium iodide (PI). Briefly, 5×105 cells were seeded in a 12-well culture
plate (Falcon, Becton Dickinson, NJ, USA) 24 h prior to treatments. Then, NB4 cells were incubated for
24 h with Myc-E extract at doses of 100 and 200 μg/ml; cells incubated with culture medium alone served
as controls. After incubation, cells were collected and washed in PBS and centrifuged at 1200 rpm for 5
min. The cells were resuspended in 500 μl of PBS and stained with PI to a final concentration of 50 μg/ml
and analyzed in a FACS caliber flow cytometer (Becton Dickinson, San José, CA, USA). Data analysis
was performed using WinMDI 2.8 software (Trotter 2011).
For analysis of cell-cycle distribution of NB4 cells, 5×106 treated cells were centrifuged at 1500 rpm for 3
min. Then, they were resuspended in 500 μl PBS containing 0.1% Nonidet P-40 and 0.5 mg/ml DNAse-
free RNAse A. Cell suspensions were incubated at room temperature for 30 min in order to extract low
molecular weight DNA from cell nuclei. Remnant DNA in cells was stained with 50 μg/ml PI and
immediately measured in the cytometer. Cell-cycle progress was expressed as the percentage of cells in
G0/G1, S, and G2/M phases. Histograms of untreated cells were used to define positions of different phases
of the cell cycle.
Assays for in vitro antioxidant activity with FB-E extract
DPPH assay
Radical scavenging ability (RSA) of mushroom extract against 1,1-diphenyl-2-picryl-hydrazyl (DPPH,
Sigma-Aldrich) was measured according to Cheung et al. (2003) using spectrophotometry. FB-E (1 ml) at
10 mg/ml was mixed with 0.5 ml of 0.1 mM DPPH ethanolic solution. Then, the mixture was shaken
vigorously and incubated at 25°C for 1 h in the dark. The absorbance of the sample was measured at 520
nm (VIS-723G spectrophotometer, Beijing Rayleigh Analytical Instrument Corporation) and the
scavenging ability against DPPH radicals was calculated as a percentage of DPPH discoloration using the
equation: % RSA = (ADPPH – AS)/ADPPH x 100, where AS is the absorbance in the solution when the
sample extract has been added, and ADPPH is the absorbance of DPPH solution. L-ascorbic acid was used
as a standard.
ABTS assay
Scavenging effect on 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS, Sigma-Aldrich)
radicals was measured according to Choi et al. (2006). ABTS radicals were generated by mixing 7 mM of
the ABTS stock solution and 2.45 mM of potassium persulfate (Sigma-Aldrich) in distilled water, and
storing this mixture overnight at room temperature in the dark. The mixture (10 ml) was diluted with 840
ml of distilled water. Pleurotus extract (50 µl) at 10 mg/ml was added to 3 ml of ABTS solution and after
90 min, the absorbance was measured at 414 nm (VIS-723G spectrophotometer, Beijing Rayleigh
Analytical Instrument Corporation). The scavenging ability against ABTS radicals was calculated using
the equation: % RSA = (AABTS – AS)/AABTS x 100, where AS is the absorbance in the solution when the
sample extract has been added, and AABTS is the absorbance of ABTS solution. L-ascorbic acid was used
as a standard.
Reducing power
The reducing power was determined according to the method of Lee et al. (2007). Mushroom extract FB-
E (2.5 ml) at 5 mg/ml was mixed with 2.5 ml of 10 g/l potassium ferricyanide (Sigma-Aldrich) and the
mixture was incubated at 50°C for 20 min. Then 2.5 ml of 100 g/l trichloroacetic acid (Merck) was added,
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and the mixture was centrifuged at 2 000 g for 10 min. A sample of the supernatant (5 ml) was mixed with
5 ml of distilled water and 1 ml of 1 g/l FeCl3 (Merck), and the absorbance was measured at 700 nm (VIS-
723G spectrophotometer, Beijing Rayleigh Analytical Instrument Corporation). Butylated
hydroxyltoluene (BHT) was used as a standard.
Determination of inhibitory activity on lipid peroxidation.

A reaction mixture containing 8 ml of a suspension of 20% sheep erythrocytes, 7.52 ml of physiological
saline, 80 l of 0.5 mol/l FeCl3, 0.4 ml of ascorbic acid (0.5 mM) and 1 ml of FB-E (10 mg/ml) was
incubated at 37 oC in a water bath for 120 min. The lipid peroxide formed was estimated by measuring
thiobarbituric acid reacting substances (TBARS) with some modifications (Okhawa et al. 1979). For this,
2 ml of the incubation mixture was treated with 1 ml of trichloroacetic acid at 10%, the samples were
shaken in a vortex for 1 min and centrifuged at 6 000 g for 15 min. Then, 2 ml of the supernatant was
transferred to test tubes with 2.5 ml of 0.335% thiobarbituric acid (TBA) and the reaction tube was kept in
a water bath at 100oC for 1 h. After cooling, the absorbance was measured at 532 nm. The percentage of
inhibition of lipid peroxidation was determined by comparing the results of the test compounds (treated
with mushroom extract) with those of control (not treated with the mushroom extract). The percentage of
lipid peroxide-scavenging ability of the extract was calculated by the formula described in DPPH and
ABTS radicals scavenging effect.
Statistical analysis
The results were expressed as mean ± standard deviation (SD). One-way analysis of variance and post hoc
Tukey’s tests or Kruskal-Wallis rank test followed by the Student-Newman-Keuls test was applied to
determine the significance of differences between treatments. The Student’s t-test or Mann-Whitney´s U-
test was used to compare the two means in the experiments related to the effects of extracts in
cyclophosphamide-treated or whole-body irradiated mice. Differences at p< 0.05 were accepted as
significant. The software Statgraphics Plus v. 5.1 (Statistical Graphics Corporation, 1994-2001) was used
in the analysis.
RESULTS AND DISCUSSION
Cancer is a worldwide disease that is causing serious damage to human health. How to conquer cancer is
one of the most important research topics in medicine. The immune system is the human’s ultimate
defense against infectious diseases, tumors, and cancer growth (El Enshasy and Hatti-Kaul 2013).
Minimal toxicity and potent biopharmacological activities of mushroom metabolites has received
increasing attention in cancer therapy. Recently, numerous reports have been published on preclinical
studies and clinical trials related to the functionality and bioactive properties of Pleurotus mushrooms and
their nutraceutical derivatives, including immune modulatory and antitumor effects (Pérez-Martínez et al.
2015, Xu et al. 2016, Carrasco-González et al. 2017).
Although in our study polysaccharides appear to be the most important bioactive component in Pleurotus-
derived preparations with respect to immunomodulation, the presence in varying amounts of different
secondary metabolites could lead to a synergy for immune enhancing activity. Results of mycochemical
tests showed that both Pleurotus fruit bodies and mycelial extracts contain alkaloids, phenolic compounds
like flavonoids and tannins, reducing sugars and amino acids (Morris et al. 2014). Moreover, fungal
immunomodulatory proteins purified from medicinal mushrooms, comprise a group of novel proteins that
possess immunomodulatory properties and have a strong potential of being applied to food or
pharmaceutical products for commercial development (Ou et al. 2009). Differences in biosynthesis
patterns of cell molecular components in distinct stages of the vital cycle would explain the dissimilarities
in biochemical composition of fruit bodies and mycelial extracts.
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Immunomodulating effects of Myc-E in cyclophosphamide-treated or whole-body irradiated mice
Chemotherapy and radiotherapy in cancer treatment contribute to further depression of the immune
system. Use of immunomodulating therapeutic agents can help to minimize these problems and efforts to
find new immunomodulators are on-going (Zhuang 2009). For that reason, we studied the effects of
intraperitoneal administration of Pleurotus mycelium hot-water extract (Myc-E) to cyclophosphamide-
treated or whole-body irradiated mice.
Cyclophosphamide (CY) is currently one of the most widely prescribed alkylating agents in cancer
chemotherapy; however, CY treatment often results in potent immunosuppressive and cytotoxic effects
(Morris et al. 2003). Immunosuppression caused by CY and other anticancer drugs significantly
complicates the course of cancer chemotherapy and worsens the condition of patients (Hou et al. 2007).
As expected, cyclophosphamide severely impaired mice hematopoietic tissue, but Myc-E was found to
have an active protective effect. Myc-E increased bone marrow cellularity and white blood cell counts
compared to the CY-saline control group (p< 0.05) (Figure 2) and no significant increase was observed in
spleen cellularity. On the other hand, the radioprotective effect exerted by mycelium Myc-E was evident
by increases in bone marrow cellularity, leukocyte counts and in spleen cellularity (p< 0.05), compared
with the irradiated control group (Figure 2). Stimulation of production of white blood cells by bone
marrow in an immunosuppressed animal model has been classified as an immunomodulatory effect
(Gupta et al. 2010).
Figure 2. Effects of Pleurotus sp. mycelial extract (Myc-E) on haemopoiesis of cyclophosphamide-treated

or irradiated BALB/c mice. Values shown are means (SE) of each group (n= 10). Different letters
indicate significant differences among groups (Kruskal–Wallis, Student–Newman–Keuls, p< 0.05).
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Since macrophages have been suggested to play important roles in immunological surveillance, we
studied the influence of administration of Myc-E on the number of peritoneal exudate cells and phagocytic
activity of macrophages (Table 1). In the CY-treated or irradiated mice, immunosuppression manifests as
a markedly decrease in the numbers and phagocytic activities of macrophages. Myc-E at 100 mg/kg
remarkably increased the number of peritoneal exudate cells compared with saline control groups in both
immunosuppression models (p< 0.05). In the study to evaluate effects of the extract on in vivo phagocytic
activity by measuring carbon clearance in peripheral blood (as an index of the phagocytic activity of liver
and spleen), a low ratio was deemed to correspond to a high clearance of carbon from the blood. Data
show that treatment with Myc-E extract potentiated activity of the host monocyte-macrophage system
(relative to that in the CY or irradiated saline treated mice) (Table 1).
Table 1. Effects of Pleurotus sp. mycelial extract (Myc-E) on the number of peritoneal exudate cells and
macrophage phagocytic activity of cyclophosphamide-treated or irradiated BALB/c mice.
Experimental groups Number of peritoneal exudate Macrophage phagocytic activity

cells (x 106/mouse) (Absorbance ratio at 5 min)
Myc-E/ irradiated mice 4.61 ± 1.43 a 1.62 ± 0.12 b
Irradiated-saline control 1.82 ± 0.65 b 2.01 ± 0.31 a
Myc-E/ CY treated mice 4.9 ± 1.4 a 1.67 ± 0.11 b
CY-saline control 2.9 ± 0.1 b 1.98 ± 0.02 a
Non-immunosuppressed control 3.41 ± 0.57 a -
Values are means ± SE, n= 10. Different letters indicate significant differences, Student’s t-test, p< 0.05)
For number of peritoneal exudate cells, comparisons were made with respect to non-immunosuppressed
mice and for phagocytic activity between both irradiated or CY-treated animals.
(-) The value was used in estimation of absorbance ratios.
These results were in keeping with the finding of another study wherein water-soluble fractions of P.
ostreatus mycelium exerted modulating effects on macrophage activation in vitro, as reflected in enhanced
glucose consumption and acid phosphatase activity by treated cells (Morris et al. 2007).
Immunomodulatory effects of Myc-E in in vitro systems have been investigated (murine macrophages
RAW 264.7). The extract can induce functional activation of macrophages by inducing nitric oxide (NO)
release and increasing mRNA expression of inducible nitric oxide synthase (iNOS) (Llauradó et al. 2016).
These results confirm the vital role of Myc-E in triggering immune responses.
Noted increases in macrophage activation might be related to binding of one or more extract components
to receptors found on macrophage surface such as glucan receptors (e.g. dectin-1). Polysaccharides appear
to be the most important component with respect to antitumor effect and on average, 1.5% of Myc-E
dried mass consists of -1,3-1,6-glucans (Morris et al. 2014).
Pillai and Uma Devi (2013) reported an increase in the survival index (66%) and an improvement in
hepatic function and haematological parameters in bone marrow of irradiated mice (6 and 8 Gy) treated
with Ganoderma preparations. These findings agree with those of a clinical trial wherein patients with
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different types of cancer (hepatic, lung, gastric, colorectal and nasopharyngeal) who were undergoing
chemotherapy or radiotherapy received a nutritional supplement containing polysaccharides extracted
from six different mushrooms (Novaes and Fortes 2005).
Reversing the function of immune suppressed cells may improve efficacy of cancer therapy (Cui et al.
2015). Hence, Myc-E may be a candidate therapeutic agent with chemo- and radioprotective activity for
hematopoiesis damage, particularly to cells involved in immune function. Although other studies
described the radioprotector effect on the immune system of mushroom products (Guggenheim et al.
2014), application of Myc-E (from Pleurotus mycelium) in immune suppression research appears to be
new as reflected in the literature. These data could be complemented by further experiments, possibly
investigating also cytotoxicity exerted by the extract on tumor cell lines, as shown later in this chapter.
Immunoenhancing activity of FB-P on cyclophosphamide treated mice
In vivo efficacy of Pleurotus preparations in immunological effector cells that have a key function against
tumor growth under immunosuppression, is poorly understood. Most of the in vivo results come from
studies with a polysaccharide injection, rather than oral administration (Wang et al. 2014).
Polysaccharides enriched-extracts that elicit effects in vitro, or by injection, may be ineffective or may
exhibit different effects when taken orally (Boh et al. 2007). Evidence of the effectiveness of oral
mushroom preparations against immune challenges must be ultimately demonstrated in animals.
Moreover, well-characterized formulations must be developed based on a complete understanding of the
immunomodulatory effects and specific applications of oral myco-products. Thus, dried and powdered
Pleurotus mushroom (FB-P) could become an attractive alternative for the development of functional
foods and nutraceuticals preparations.
In the present section, activation of FB-P on cell immune response was evaluated in vivo on
cyclophosphamide treated mice. Results indicated that none of the FB-P-orally treated mice died and their
body weights showed no significant change during the course of the experiment (p 0.05, data not shown).
Mice supplemented with Pleurotus powder (FB-P) showed a higher delayed-type hypersensitivity (DTH)
response, as judged by the increase of foot pad swelling compared to the saline control group, particularly
at 48 and 72 h after antigen re-challenge (p 0,05; Table 2). DTH response mounted at these times by the
CY-FB-P group was similar to that of control mice. Reconstitution of DTH response reflected induction of
CD4+ Th1 cells and the activation of macrophages by cytokines: tumor necrosis factor alpha (TNF-) and
gamma interferon (IFN-) (Kim et al. 1998, Murphy and Weaver 2016). Mononuclear cells were shown
infiltrating antigen-injection sites in CY/FB-P treated mice (Figure inserted in Table 2). In addition, DTH
reconstitution was associated with the increase observed in the mass index of popliteal lymph nodes of
FB-P supplemented animals (p 0.05; Table 2). In sum, results evidenced that FB-P reversed CY-induced
damage on adaptive cell immunity and promoted proliferation of T cells and macrophages.
It has been demonstrated that a polysaccharide of P. citrinopileatus (PCPS) has the capacity to activate
human dendritic cells (DCs) via multiple pathways, leading to secretion of pro-inflammatory cytokines
TNF, IL-1, IL-6 and IL-12, as well as anti-inflammatory cytokine IL-10 (Minato et al. 2016). As is
known, activated macrophages, NK cells, cytotoxic T cells and their secretory products, such as tumor
necrosis factor, reactive nitrogen and oxygen intermediates and interleukins are involved in
immunomodulatory responses (Wang et al. 2015).
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Table 2. Effect of oral administration of FB-P on cell immune response (DTH response) of
cyclophosphamide treated BALB/c mice.
Experimen Foot pad thickness (mm) Mass index of CY/ FB-P 48 h
tal groups popliteal lymph nodes (H/E x100)
24 h 48 h 72 h
a a a
Control 0.48 ± 0.07 0.46 ± 0.04 0.38 ± 0.07 -
b a a
CY/ FB-P 0.14 ± 0.05 0.43 ± 0.02 0.29 ± 0.05 1.87 ± 0.27*
CY/ Saline 0.11 ± 0.06b 0.39 ± 0.01b 0.12 ± 0.04b 1.34 ± 0.15
All values are expressed as the arithmetic mean ± S.D. of five mice. Different letters indicate significant
differences among groups (Kruskal-Wallis, Student-Newman-Keuls, p< 0.05). The (*) reflects significant
differences between the two groups in the Mann-Whitney test (p 0.05).
Mononuclear cells infiltrating antigen-injection sites in CY/FB-P are shown in the inserted figure
(hematoxylin-eosin staining, 100x).
Oral administration of P. nebrodensis polysaccharide (PN-S) to CY-immunosuppressed mice increased

thymic and splenic indices and promoted proliferation of T lymphocyte, B lymphocyte and macrophages.
PN-S also enhanced activity of natural killer cells and increased immunoglobulin M (IgM) and
immunoglobulin G (IgG) levels in serum. PN-S also increased levels of interleukin-6 (IL-6), tumor
necrosis factor-α (TNF-α), interferon-γ (INF-γ) and nitric oxide (NO) in splenocytes. These results suggest
that PN-S treatment enhances immune function of immunosuppressed mice. This study may provide a
basis for application of this fungus in adjacent immunopotentiating therapy against cancer and in
treatment of chemotherapy- induced immunosuppression (Cui et al. 2015).
Mariga et al. (2014) reported that P. eryngii fruit bodies powder is an active antitumor agent with
immunomodulatory activity, where, it targets lysosomes of cancerous cells concomitantly stimulating
macrophage-mediated immune response. Based on current data, we demonstrated that Pleurotus sp.
mushrooms might be of potential benefit in anticancer-drug induced immunosuppression. Our findings
suggest that oral administration of Pleurotus sp. powder would stimulate the immune system after their
absorption in the gastrointestinal tract and the activation of gut-associated lymphoid tissues, thus
integrating different elements of immune function (see results on the animal model of malnutrition).
Immunonutritional restoration effects of FB-E on malnourished BALB/c mice
In protein-energy malnutrition (PEM), there are reductions in nutrient absorption and depression of the
immune system in gut-associated lymphoid tissues (Schaible and Kaufmann 2007). Functionality and
integrity of the small intestine also deteriorated. Administration of FB-E to malnourished mice during the
refeeding period provided beneficial effects and improved functional alterations in the intestinal tract (p<
0.05) (Table 3). The increase of mucosal weight in association with high values of protein and DNA
content suggests a higher rate of protein biosynthesis and could be related to the recovery of
gastrointestinal tract function. Some components present in Pleurotus mushrooms could re-establish
intestinal architecture after oral consumption.
In general, enteral nutrition is considered the first method of feeding in critical patients (Botrán and
López-Herce 2011) and mushroom substances might be potential candidates for use in immunonutritional
diets. Some studies with mushrooms reported the biological potential of polysaccharides at the intestinal
level by means of stimulation of gut-associated lymphoid tissues and intestinal macrophages (Bouike et al.
2011).
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Table 3. Effect of oral administration of FB-E on immunonutritional restoration of malnourished BALB/c

mice with respect to gut mucosa and humoral immune response.
Gut mucosa parameters Antibodies titres against
Experimental
SRBC (day 14)
groups Protein (g/ 10 cm) DNA (g/ 10 cm)
Control 352 ± 78b 59 ± 16b
Malnourished 108 ± 38c 45 ± 20b

mice (M)
M/ CD 277 ± 76b 68 ± 6b
M/ FB-E 576 ± 87a 98 ± 21a
Results are expressed per ten centimeters of intestine. All values are the arithmetic mean ± SD of 10 mice.
Different letters indicate significant differences among groups (Kruskal-Wallis, Student- Neuman-Keuls,
p< 0.05). Subscript legend: (a) (b)  (c).
Oral administration of FB-E at a dose of 100 mg/kg stimulated phagocytic activity in comparison with the
standard diet group. The ratio of carbon clearance at 5 min was lower in FB-E group than in the M-CD
group (1.4  0.1 vs. 1.9  0.1) (p< 0.05); a low ratio was deemed to correspond to a high clearance of
carbon from the blood. Nevertheless, FB-E does not increase spleen weight and splenic cell counts (p<
0.05) (data not shown). Augmentation of phagocytic activity may be owing to activation of phagocytes
and not by an increase in the number of total phagocytes. Effects on the intestinal tract linked to
macrophage activation might be influenced by a partial absorption of bio-compounds from Pleurotus sp.,
or by stimulation of gut-associated lymphoid tissues. Nevertheless, the mechanism(s) of action of several
orally administered bio-substances from mushrooms is still unclear. Among various myco-chemicals, it
has been suggested that only fragments of polysaccharides partially hydrolyzed or degraded after ingestion
might bind to gut epithelia and exert localized and/or systemic effects on the immune system or the
mechanisms could be mediated via a non-specific intestinal absorption (Wasser et al. 2014). Although
most of the bio-components in FB-E could be implicated as immunomodulatory agents, more evidence is
required to link observed actions to any of the identified bio-components.
Although cell-mediated immunity is severely affected in PEM, atrophy of lymphoid tissues leads to a
decrease of circulating and splenic B cell numbers (Lykke et al. 2013). However, the role of humoral
immune response in malnourished mice is not well documented. Antibody production by B cells after 14
days of immunization with sheep red blood cells (SRBC) was significantly higher compared with mice
refed with commercial diet (p< 0.05) (Figure inserted in Table 3). Anti-SRBC antibodies (directed against
a T-dependent antigen) titres might also suggest stimulation of cellular immunity. Other studies indicated
that the humoral response might respond to malnutrition, depending on malnutrition type (e.g. acute vs.
chronic, protein malnutrition vs. energy restriction) (Neyestani et al. 2004).
The term immunonutrition was introduced as emergent subject in the last few years (Zapatera et al. 2015).
In this context, FB-E could be used to develop specific enteral formulations with potential applications in
immunotherapy and as immunonutritional support during recovery of metabolic and immunological
disorders associated with malnutrition. This study is a contribution to the knowledge of the
immunonutritional properties of Pleurotus mushrooms and suggests its prospective use in
immunocompromised people with special nutritional requirements.
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Antiproliferative effects of Myc-E on human acute promyelocytic leukemia (NB4) cells
Since research has tended to focus on the dietary value of species of the genus Pleurotus, there is
relatively little information pertaining to their anticancer mechanisms, particularly in mycelia-derived
products. Treatment with Pleurotus mycelial extract (Myc-E) reduced viability of NB4 human leukemia
cells, particularly at a concentration of 200 μg/ml, to 82% relative to control nontreated cells (p< 0.05)
(data not shown).
Our results presented in Figure 3 indicated that the cytotoxic effect of a hot water extract from Pleurotus
sp. mycelia on NB4 cells is related to its ability to arrest the cell cycle. Independent of concentration, the
extract lowered leukemic cells in the G0/G1 phase compared to untreated cells (p< 0.05), but had no
significant effect on the S population. Overall, Pleurotus sp. mycelial extract significantly increased the
number of NB4 cells in G2/M phase (15.82% and 18.35% for cells treated, respectively, with 100 and 200
μg/ml, vs 8.78% in control cells, p< 0.05). Thus, Pleurotus sp. extract arrested NB4 cells in the G2/M
phase supporting a cell-cycle dependent anticancer mechanism.
While a water-soluble non-starch polysaccharide extracted from mycelium (EDP) of P. tuber-regium

caused G2/M arrest in HL-60 cells by lowering Cdk1 expression, its fruiting-body analog (HWE) caused S
arrest in HL-60 cells by a depletion of Cdk2 and an increase in cyclin E expression (Wong et al. 2007).
This was in contrast to a previous study of a β-glucan obtained from Poria cocos mycelium that was found
to inhibit proliferation of MCF-7 cancer cells by G1 arrest and apoptotic induction via down regulating
anti-apoptotic protein Bcl-2 (Zhang et al. 2006).
On the other hand, the effect of several extracts of various polarities obtained from fruiting bodies of
Pleurotus sp. was tested on apparent growth of different cell lines (U937, N2A and Caco2 tumoral cells
compared to Vero cells). In vitro growth activity of cells with aqueous (CW-P at 4°C and HW-P at
100°C), methanol (MetOH-P) or dichloromethane (DM-E) extracts were estimated through mitochondrial
activity using an MTT (a Tetrazolium salt) Test and by Neutral Red Uptake assays. Inhibition of cell
respiration and cell uptake was observed with CW-P extracts while HW-P and MetOH-P extracts were
less efficient. Compared to HW-P, substance(s) responsible for this effect in CW-P appeared thermo-
labile. However, cell proliferation was shown in U937 with intermediate dilutions of HW-P. N2A was
specifically sensitive to inhibition by MetOH-P extract (Llauradó et al. 2014). The mechanisms by which
they drive benefits remain obscure, while the effective doses and their safety need to be evaluated.
Dietary supplements of edible Pleurotus spp. rich in fungal polysaccharides is associated with anticancer
health benefits. Tong et al. (2009) isolated a novel water-soluble polysaccharide (POPS-1) from fruit
bodies of P. ostreatus by hot water extraction. Cytotoxicity assay showed that POPS-1 presented
significantly higher antitumor activity against Hela tumor cells in vitro, in a dose-dependent manner, and
exhibited significantly lower cytotoxicity to human embryo kidney 293T cells than Hela tumor cells.
These results suggest that P. ostreatus water-soluble preparations may be considered as potential
candidates for developing new low toxicity antitumor agents.
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Figure 3. Cell-cycle analysis of human leukemia NB4 cells treated with Pleurotus sp. mycelial extract
Myc-E for 24 h at concentrations of 100 and 200 μg/ml. Values represent the mean ± SE of three
independent experiments. Significantly differences in Tukey’s test (p< 0.05) were shown with different
letters compared to control cells. Non-significant differences were found in the S-phase of cell-
cycle.
In vitro antioxidant activities
Free radicals are known to be a major cause of various chronic and degenerative diseases, including aging,
coronary heart disease, inflammation, stroke, diabetes mellitus and cancer. Antioxidant compounds play
an important role in preventing and curing chronic inflammation, atherosclerosis, cancer and
cardiovascular disorders (Sun et al. 2017). The antioxidant potential of a compound could be attributed to
its various characteristics; the most important of these is the ability to scavenge and reduce free radicals,
to chelate transition metal ions and to inhibit lipid peroxidation, among others (Rajeshwar et al. 2005).
The antioxidant effects displayed by edible mushrooms, in addition to their immunomodulating properties
represent an important contribution to their antitumor activities (Patel and Goyal 2012). Antioxidant
properties of Pleurotus spp. were of both enzymatical and non-enzymatical nature (Khatun et al. 2015).
Until now, research has tended to focus on the dietary significance of edible mushrooms; however, there is
relatively little information relating to antioxidant activity and possible use of such mushrooms to
neutralize oxidative stress (Jayakumar et al. 2011).
Antioxidant activities of FB-E determined using four in vitro assays are presented in Table 4. DPPH is a
stable free radical that shows maximum absorbance at 517 nm in methanol; when DPPH encounters a
proton-donating substance such as an antioxidant, the radical is scavenged and absorbance is reduced
(Kohen and Nyska 2002). As shown in Table 4, the DPPH radical scavenging activity of the aqueous
extract obtained from Pleurotus fruit bodies was 90.4%, lower than values achieved with ascorbic acid
used as control. In previous studies, a DPPH radical scavenging ability of 96% was reported with a
mycelial extract (Morris et al. 2017b). Scavenging effects of extracts from several specialty and
commercial mushrooms on DPPH radicals augmented with increased extract concentrations. Thus, DPPH
radical scavenging activity varied from 9% (P. nebrodensis) to 57% (P. cystidiosus). Moreover, DPPH
scavenging action for P. citrinopileatus and some other fungi significantly improved with gradual
elevation of sample concentration from 0.5 to 9.0 mg/ml (Asatiani et al. 2010).
173
Table 4. In vitro antioxidant activity of a water extract of fruit bodies of Pleurotus sp (FB-E).
Sample Scavenging of DPPH Scavenging of ABTS Reducing power Inhibition of
radicals (%) radicals (%) (A700 nm) lipid
peroxidation
(%)
Pleurotus extract 90.4  0.8 80  0.9 0.438  0.034 51.2  4.8
(FB-E) (10 mg/ml) (10 mg/ml) (5 mg/ml) (10 mg/ml)
Control 96.3  0.6* 98  0.2* 0.700  0.018* 92  1.4*
(ascorbic acid) (ascorbic acid) (BHT) (ascorbic acid)
Results are showed as means ± standard deviation of three replicates; means with an (*) differ when
compared with the Mann–Whitney test (p< 0.05).
With the ABTS radical scavenging test, we can measure activity of both hydrophilic and lipophilic
compounds; therefore, it is useful in the simultaneous study of several natural ingredients (Kuskoski et al.
2005). In this study, ABTS radical scavenging activity of FB-E mycelium at 10 mg/ml was of 80%, higher
than a mycelial extract with a value of 55% (Morris et al. 2017b). On the other hand, hydro-alcoholic
extracts of Grifola gargal showed an ABTS radical scavenging ability of 90.9-93.3 ascorbic acid
equivalents (mg of ascorbic acid per l of sample) (De Bruijn et al. 2009). This method also has been used
in evaluation of antioxidant activity of neutral polysaccharides from Auricularia auricula and their
homologues sulfated in concentrations of 0.2-10 mg/ml, without significant differences (Zhang et al.
2011).
Moreover, it has been discussed that the diphenylpropane structure of flavonoids and the aromatic ring
structure of phenolics, such as aromatic oxy phenol acids, might contribute to free radical scavenging
ability of these compounds (De Bruijn et al. 2008).
Reducing capability of a compound may serve as a significant indicator of its potential antioxidant activity
and the efficacy of certain antioxidants is known to be associated with their reducing power (Lü et al.
2010). In the present study, reducing power of a 5 mg/mL concentration of mushroom extract was 0.438,
which was relatively lower than that of BHT (p 0.05) (Table 4). The reducing power of medicinal
mushrooms might be due to their hydrogen-donating ability (Jayakumar et al. 2011). Possibly, medicinal
mushrooms contain high amounts of reductones that could react with radicals to stabilize and terminate
radical chain reactions. Reducing power of the ethanolic extract of P. ostreatus fruit bodies steadily
increased in direct proportion to increasing concentration of the extract. Reducing power of a 10 mg/ml
concentration of mushroom extract was 1.367 and was relatively higher than that of BHT (1.192)
(Jayakumar et al. 2009). Further, ethanolic extract from fruit bodies of Pleurotus citrinopileatus was
reported to exhibit reducing power of 1.05 at 10 mg/ml (Lee et al. 2007).
Lipid peroxidation (LPO), a process induced by free radicals, leads to oxidative deterioration of
polyunsaturated lipids, inactivates cellular components and therein plays a key role in oxidative stress in
biological systems (Niki 2010). Hence, inhibitory activity of mushroom extract on LPO was evaluated.
Both mushroom extract and ascorbic acid standard inhibited lipid peroxidation (Table 4). At a
concentration of 10 mg/ml, mushroom extract effected 51.2% inhibition of LPO activity and the ascorbic
acid standard effected 92%. At least until now, there is no data available for comparison of our results
obtained with FB-E extract on lipid peroxidation in the in vitro erythrocyte membrane model estimated by
TBARS. Erythrocytes are excellent subjects for studies of biological effects of free radicals, since they are
structurally simple, are continuously exposed to high oxygen tensions, the membrane lipids are composed
partly of polyunsaturated fatty acid side chains which are vulnerable to peroxidation, and they have
antioxidant enzyme systems (Konyalioglu et al. 2005).
174
In vitro antioxidant properties exhibited by Pleurotus mushroom preparation may be due to presence of
antioxidant mycochemicals, like polyphenols. In addition, antioxidant activities (chelating ability of
ferrous ion, inhibition of LPO and reducing power) found in polysaccharide extracts from widely used
mushrooms Ganoderma applanatum, Ganoderma lucidum, Lentinus edodes, Trametes versicolor and
Pleurotus eryngii does not negate the possible contribution of β-1,3-1,6-glucans polysaccharides to the
antioxidant effect (Kozarski et al. 2012, Fu et al. 2016).
In our experiment of LPO inhibition, butylated hydroxyltoluene (BHT) was used as a standard. Butylated
hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are amongst the most commonly used
synthetic antioxidants that are used in fats and oily foods to prevent oxidative deterioration. However,
BHA and BHT have restricted use in foods as they are suspected carcinogens and may cause liver damage
(Botterweck et al. 2000). This may explain why there is currently much research on the application of
antioxidants from natural products. Although BHT and ascorbic acid, substances used as standards in our
work, have significant antioxidant activity, they are additives and are used or are present in mg levels in
foods. Pleurotus powder could be used in gram levels as functional food or nutraceutical, thus providing
health protection to help humans reduce oxidative damage daily.
Research carried out this way, showed that polysaccharides of P. tuber-regium (Fr.) Sing. had strong
antioxidant potency and might be exploited as effective natural antioxidant to alleviate oxidative stress.
The antioxidant activities of two homogeneous polysaccharides, water-extracted polysaccharide (W-PTR)
and alkali-extracted polysaccharide (A-PTR) were evaluated. Results indicated that W-PTR was stronger
than A-PTR in superoxide scavenging activity, while A-PTR was stronger than W-PTR in the scavenging
activities to hydroxyl, DPPH , inhibition effects on liver lipid peroxidation, liver mitochondria swelling,
and red blood cell (RBC) hemolysis (Wu et al. 2014).
In the link between antioxidant and antitumor activities, Ren et al. (2015) isolated the polysaccharide
(PAP) from fruit bodies of P. abalonus and evaluated their antiproliferative activity in human colorectal
carcinoma LoVo cells. PAP exerted a high antioxidant activity in vitro and a dose-dependent
antiproliferative effect against LoVo cancer cells. Flow cytometry analysis demonstrated that PAP
exhibited a stimulatory effect on apoptosis of LoVo cells and induced cell-cycle arrest at the S phase. PAP
also increased generation of intracellular Radical Oxygen Species (ROS), critical mediators in PAP-
induced cell growth inhibition. These findings suggested that PAP may serve as a potential novel dietary
agent for human colon cancer chemoprevention.
Additionally, Pleurotus eryngii residue polysaccharides obtained by ultrafiltration showed in vitro

antioxidant properties and cytotoxicities with CPPS-1 the strongest activity. Expression of tumor
suppressor p53 and apoptosis activator Bax were up-regulated by CPPS-1 fraction while the expression of
Bcl-2 was down-regulated. The results suggested that antitumor activity of CPPS-1 may be related to its
capability of inducing apoptosis via activation of mitochondria apoptosis pathway (Ma et al. 2016).
CONCLUSIONS
Taken together, these observations indicate that Pleurotus sp. extracts (Myc-E and FB-E) and powders
(FB-P) possess bioactivities involved in antitumor mechanism including immunostimulatory,
antiproliferative and antioxidant toward free radicals. Both fruit bodies and mycelial preparations were
well tolerated and can be used in formulation of consistent and safe mushroom products. Thus, Pleurotus
sp., a common edible and medicinal mushroom, exerted health promoting benefits to maintain good health
by activating the immune system for a multitude of defensive functions. Pleurotus sp. may be developed
as a functional food and potential myco-therapeutic agent for human diseases, especially for enhancing
175
anticancer and immune responses. It appears that more studies are necessary to explore the complete
structural characteristics of preparations tested, structure–activity relationship and molecular signaling
pathways of their antitumor activity. As a result, future research may be oriented in that direction.
ACKNOWLEDGMENTS
The authors would like to thank the Cuban Ministry of Science, Technology and Environment (Territorial
Project 9072 of the Program for Development of Health Products, CITMA). The authors also thank the
“Biotechnologie de Pleurotus sp. à Cuba et diversification de sa Culture pour des Applications
Environementales et Nutraceutique” (FSP Coopération Scientifique Franco-Cubaine) project and the IRD
under the Programme Jeunes Equipes AIRD (JEAI). This work also was supported by the Belgian
Development Cooperation through VLIR-UOS (Flemish Interuniversity Council-University Cooperation
for Development) in the context of the Institutional University Cooperation Program with University of
Oriente.
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14. CULTIVATION BIOTECHNOLOGY FOR Volvariella spp. IN MEXICO:

ADVANCES, CHALLENGES AND PERSPECTIVES
Dulce Salmones
Red de Manejo Biotecnológico de Recursos. Instituto de Ecología, A. C.
Carretera antigua a Coatepec 351, Xalapa, Ver., México
[email protected]
ABSTRACT
Edible fungi are a traditional food that has been eaten in Mexico since precolonial times and are currently an
emerging agrofood industry supported mainly by production and commercialization of species of temperate climates.
Considering the extensive biological and cultural diversity of the country, as well as the notable area of the country
that has warm weather throughout most of the year, it is desirable to diversify with other species of commercial
interest that can be grown under these conditions. Among fungi currently cultivated worldwide, Volvariella spp. are
a productive alternative for tropical and subtropical regions, ideal for growing in rural areas since they only require a
relatively small investment. Even though species such as V. volvacea and V. bombycina are collected and eaten by
people in various regions of the country, development of biotechnology for their cultivation in Mexico has been slow
and limited to laboratory experiments, the storage of genetic material and field trials to adapt their growth and
development on different lignocellulose materials. This study summarizes results obtained to date and provides a
prospective of the potential for producing these species at the regional level, based on existing genetic, agricultural
and environmental resources.
Keywords: edible mushrooms, tropical species, paddy straw mushroom, cultivation prospects
INTRODUCTION
Mexico is one of the most important cultural and biological reservoirs of edible wild mushrooms in the
world. This is mainly the result of the peculiar biological diversity, geological and geographic conditions,
as well as the ancestral cultural history of the country (Pérez-Moreno et al. 2010) that have survived to the
present day through texts, archeological relicts and traditional knowledge handed down from generation to
generation (Garibay-Orijel et al. 2010). According to Boa (2004), in Mexico at least 303 species of fungi
are used traditionally, the majority for culinary purposes.
In contrast, cultivation of edible fungi in Mexico dates back to only the 1930s and the first fifty years were
focused on producing the white button mushroom, Agaricus bisporus (J.E. Lange) Imbach. It was only
starting in the 1980s, in parallel with the consolidation of the mushroom industry in temperate-cold
regions, that experimentation with species in tropical regions was presented as a novel feasible alternative,
based on the fact that most of the commercial species grew wild in the country, especially those of the
genera Pleurotus (Fr.) P. Kumm. and Volvariella Speg. (Guzmán et al. 1993). Additionally, academic
groups interested in the study of wild germplasm and the implementation of techniques for growing
commercially known species were formed (Martínez-Carrera 2000, Mata et al. 2016, Sánchez et al. 2016).
Early studies focused on temperate species in the genus Pleurotus, but continued with species for which
reproduction was potentially viable in tropical and subtropical regions of the country, such as Auricularia
(Bull.) spp., Cookeina sulcipes (Berk.) Kunt., Pleurotus djamor (Rumph. ex Fr.) Boedijn and Volvariella
spp. (Guzmán et al. 1993, Sánchez Vázquez et al. 1995a, 1995b).
Several edible species belonging to the genus Volvariella are grown commercially in southeastern Asia
(Miles & Chang 2004). Volvariella volvacea (Bull.) Sing. is one of the most extensively cultivated
mushrooms in tropical and sub-tropical regions and requires relatively high temperatures (28–35 °C) for
vegetative growth and fruiting (Chen et al. 2003). It is widely used in traditional oriental cuisine and is
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reported to possess anti-tumor, immunosuppressant and immunomodulatory effects (Buswell & Chen
2005, Mathew et al. 2008). It is known as a rich source of protein, fiber (chitin), vitamins (large amount of
vitamin C, and also all water-soluble vitamins including riboflavin, biotin and thiamine), fat (5.7%),
carbohydrates (56.8%), amino acids (all essential amino acids: alanine, arginine, glycine, serine etc.),
unsaturated fatty acids, essential minerals (potassium, sodium and phosphorus) and has low calorific
values (Chang 1980a, Ahlawat et al. 2016). The innumerable medicinal properties of V. volvacea along
with its phytochemical properties are evidence of its value as a medicinal mushroom (Mathew et al.
2008). Annual production of V. volvacea has increased in recent years due to a higher demand for health
foods. In 2010, Chinese production of Volvariella was 330,000 tons, accounting for 80% of the total world
production of this genus. Although V. volvacea has been cultivated for 300 years, multiple problems
associated with the practice has greatly restricted development of the industry (Bao et al. 2013).
Volvariella mushrooms are also known as “paddy straw or warm mushrooms”. They are fast growing and
under favorable production conditions, the total crop cycle is completed within 4-5 weeks. This mushroom
can use a wide range of cellulosic materials and the C:N ratio needed is 40 to 60, quite high in comparison
to other cultivated mushrooms (Biswas 2014). It can be grown quite quickly on non-composted substrates
such as paddy straw and cotton waste (Miles & Chang 2004, Ahlawat 2011). In addition to these
substrates, V. volvacea grows on water hyacinth, palm oil bunch wastes, pericarp wastes, banana leaves,
and cotton waste (Chang 1980b, Belewu & Belewu 2005). Various grains (wheat, sorghum) and raw
substrates (sawdust, rice bran, paddy husk etc.) have been used to produce spawn of Volvariella species
(Chang 1978).
Paddy straw mushrooms have been traditionally cultivated on beds in the open field or using wooden
frames (Chang 1993). In some regions, the bed is made of non-composted and unpasteurized bundles of
rice straw exposed directly to the sun and covered with plastic sheets (Reyes 2000). The yield on beds is
unstable and irregular, because the mycelium is exposed to microbial contamination and fluctuating
environmental conditions. In recent times, development of indoor cultivation in growing houses under a
controlled environment incorporated composting and pasteurization of substrates. Additionally, use of
cotton waste in place of rice straw resulted in a significant increase of biological efficiency and more
stable production but, it is still lower than other cultivated species (Kurtzman & Yung 1982, Chang 1993).
Volvariella basidiomes are commercially classified based on their stage of development: 1) primordia, 2)
button, and 3) adult (Chang & Yau 1971). Each stage has different morphological and anatomical
characteristics, with the button phase the most highly valued in the market since adult specimens
deteriorate rapidly owing to polyphenol oxidative activity (Cho et al. 1982).
GENERAL CHARACTERISTICS OF Volvariella spp. AND THEIR DISTRIBUTION IN MEXICO

Volvariella Speg. is a cosmopolitan genus belonging to the Pluteaceae family in the Agaricales order and
Basidiomycotina class (Singer 1986). It is characterized by basidiocarps initially covered with a veil that
breaks during development, leaving as a remnant a membranous saccate volva at the base of the stipe. The
spore print is pink to brownish pink. The context (“meat”) is spongy, white to whitish, with a pleasant
mushroom smell. At the microscopic level, it has tetrasporic basidia, spores ellipsoid, rather thick-walled,
pinkish, cheilocystidia and pleurocystidia present, hymenophoral trama inverse (Li 1982, Seok et al.
2002).
Traditionally, the sexual pattern of mushrooms in the genus Volvariella has been described as primary
homothallic, so the homokaryotic mycelium that emerges from the germination of a basidiospore is able to
convert to the dikaryotic form and complete the sexual cycle without crossing (Chang & Yau 1971, Chiu
et al. 1995). However, phylogenetic data presented by Bao et al. (2013) supported the notion that V.
volvacea, like A. bisporus, is a pseudo-homothallic species. The fungi present multinucleate hyphae and
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the dikaryotic mycelia have no clamp connections (Chang & Ling 1970). Recent studies on the size of V.
volvacea’s genome indicate values of 35.7 mB and are very similar to those reported for P. ostreatus (Bao
et al. 2013).
A B
Figure 1. Basidiomes of V. volvacea collected from the wild on coffee pulp (A) and cultivated on
mushroom beds at a Chinese farm (B).
In Mexico, 13 species of Volvariella have been cited, the majority of which are considered edible, with V.
volvacea and V. bombycina (Schaeff.) Sing. the best known by rural inhabitants. These species grow wild
on a variety of decomposing lignocellulose substrates: wood, bagasse (sugar cane, henequen from agave,
tequila maguey), banana pseudostem and coffee berry pulp (Salmones et al. 1996) (Figure 1). V. volvacea
is popularly known as the “bagasse mushroom”, “sparrow-hawk’s breast” or “pink mushroom”, among
other names, while V. bombycina var. bombycina is known as “banana mushroom” or “orchard fungus”
and V. bombicina var. flaviceps is known as “yellow mushroom” or “little chicken” (Guzmán et al. 1993).
These species have been cited for the states of Baja California, Jalisco, Morelos, Oaxaca, Puebla, Sonora,
Quintana Roo, Yucatán and Veracruz (Ayala et al. 2015, Sobal Cruz et al. 2016, Pérez-Silva et al. 2006,
Salmones et al. 1988, Vázquez et al. 1989).
EXPERIMENTAL WORK WITH VOLVARIELLA IN MEXICO
Studies carried out in Mexico to date have focused on establishing conditions required for its cultivation in
vitro, with minimal in situ testing. Trials have focused on evaluating mycelial growth on agroindustrial
residues available in the country, such as: grass straw, crop stubble, corn cobs, wood shavings from
cazahuate (Ipomoea arborescens), coconut fiber, coffee pulp, pineapple crown bracts, banana leaves and
henequen bagasse (Acosta-Urdapilleta et al. 1992; Guzmán et al. 1993, Ancona & Salmones 1996,
Salmones et al. 1996).
In the laboratory, Martínez et al. (1986) grew wild Mexican and commercial Asian strains of V. volvacea
in culture medium in order to compare their macroscopic morphology and the microscopic characteristics
of their mycelia. Later, Vela and Martínez-Carrera (1989) isolated monospore cultures of the Mexican and
Asian strains and obtained fruit bodies on barley straw (Table 1), leading them to the conclusion that this
American species, formerly known as V. bakeri, was a synonym of the Asian V. volvacea, the latter being
the taxonomically correct name. In parallel, Salmones et al. (1988) compared mycelial growth of strains
of V. volvacea and V. bombycina (Schaeffer: Fr.) Sing. on various culture media and lignocellulose
materials, identifying suitable conditions for growing them in vitro and successfully developing V.
bombycina basidiomes on oat straw.
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A B E
C D
Figure 2. Volvariella mycelia growing on agro-industrial residues in the tropics. A: wheat straw, B: barley
straw, C: banana leaves, D: rice straw, E: coffee pulp with rice husk.
Table 1. Biological efficiency of strains of Volvariella volvacea on various substrates made from raw
materials found in Mexico.
Substrate Biological efficiency Reference
(%)
Barley straw 5.3-22.7 Vela & Martínez Carrera (1989)
Barley straw 14.2 Salmones & Guzmán (1994)
Henequen bagasse 10.2 Ancona & Salmones (1996)
Pineapple bracts, coffee pulp, rice 6.2-33.8 Salmones et al. (1996)
straw
Straw, banana leaves and 19-25.6 Julián Carlos & Salmones (2006)
pseudostem
Barley straw, sugar cane bagasse 46.4-64.8 Sobal Cruz et al. (2016)*
Barley straw + 10 mM 27.1-135
acetylsalicylic acid
Sugar cane bagasse + 100 µM
acetylsalicylic acid 25.5-34.2
*Study done with V. bombycina.
Volvariella spp. are highly sensitive and will not fruit at low temperatures, so Salmones and Guzmán
(1994) designed a laboratory fruiting chamber to maintain a suitable temperature range for development of
basidiomes. Using this technique, Salmones et al. (1996) fruited wild strains of V. volvacea on
agroindustrial residues as follows: coffee pulp, pineapple crown bracts and coconut fiber (Figure 2) and
obtained biological efficiency values of 6.2 to 33.8%. Recently, Sobal Cruz et al. (2016) grew V.
bombycina on barley straw and sugar cane bagasse, achieving a remarkable increase in productivity by
applying acetylsalicylic acid in concentrations of 100 µM and 10 mM (Table 1).
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At the ex situ level, the only study is that of Julián Carlos and Salmones (2006), who grew V. volvacea
under rustic conditions in a banana-growing region in eastern Mexico (Veracruz state). They used waste
from a banana harvest as the substrate (leaves and pseudostem) with biological efficiency values of 19 to
25.6 % (Table 1).
CONSERVATION OF WILD GERMPLASM
In Mexico, there are around eight fungi collections specializing in conserving species of alimentary and
medicinal interest (Salmones & Mata 2012). Of these, Instituto de Ecología, A.C. (INECOL), Colegio de
la Frontera Sur (ECOSUR) and Colegio de Postgraduados, Campus Puebla (COLPOS) curate isolates of
Volvariella though there are no data on the total number or origin of the strains in these collections (Mata
et al. 2016, Sánchez et al. 2016, Sobal Cruz et al. 2016).
The traditional method of conservation is by continuous sub-culturing on culture media or using sterilized
water (Jong 1978). The germplasm of Volvariella is characterized by rapid mycelial growth. Routine
storage at low temperatures (4 °C) causes mycelia to undergo autolysis. Low temperatures also cause fruit
bodies to become soft, liquid, and even rotten (Chang 1978), which is why they should not be refrigerated.
These two characteristics—fast growth and sensitivity to low temperatures—have adverse effects on
conservation of strains, since periodical sub-culturing can induce physical and genetic changes that
increase the risk of losing the biological material. Conservation of strains under ultracold conditions (-196
°C) is an advantageous alternative method since decreasing metabolism of the mushroom mycelium
means it can be stored for extended periods of time (years) and, consequently decreases the possibility of
physiological and genetic changes.
The Fungus Ceparium at the INECOL is one of the few institutes that has experimented with this
conservation technique for edible species and has managed to implement a technique that uses sterilized
grass seeds as a support for the mycelium (Mata et al. 2004). Following this methodology, Pérez and
Salmones (1997) carried out trials to determine freezing protocols for the genetic material of 11 strains of
Volvariella, evaluating different times of being in contact with a cryoprotectant solution of dimethyl
sulfoxide (5% v/v). The recovery rate of strains was variable depending on conditions used. In general, 60
and 90 minutes of contact with the solution were the best for achieving total recovery of samples in the
least amount of time (Figure 3).
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A 70
B
Recovered samples (%) at 48 h
60
50
40
30
20 B
10
0
Volvariella strains
Contact time with DMS 5% 30´ 60´ 90´
Figure 3. A: behavior of Volvariella strains during cryogenic storage. B: mycelia recovered after being
frozen at -196 °C.
INOCULUM SUPPLEMENTATION TRIALS
Preparation of inoculum or spawn is the basis of commercial mushroom cultivation. For Volvariella,
various lignocellulose materials are used commercially, either alone or in combination to prepare the
inoculum. Best results were obtained with cotton, coconut palm and tea crop waste, as well as rice straw
(Miles & Chang 2004). In Mexico, owing to its greater availability, grass straw was initially used as the
substrate to prepare inoculum (Guzmán et al. 1993). The use of this organic material, however, ideally
requires prior aerobic fermentation that increases the availability of the cellulose fraction. This is because
the enzyme system of Volvariella is poorly equipped to degrade the ligninolytic fraction (Cai et al. 1999).
Therefore, it necessary to have additional physical space for substrate fermentation.
One alternative for preparing inoculum of Volvariella is to use grass seeds (Quimio 2002). For this reason,
in our laboratory, we have evaluated various formulations using wheat, millet and sorghum grains alone or
mixed with carbohydrate-rich supplements (corn flour and coffee pulp) and limestone (CaCO3) (Julián
Carlos 2006). The results did not indicate a specific preference by the strains for any particular grain or
C
supplement, although some combinations such as millet with corn flour 90:10 (p/p) and sorghum with
limestone (99.5:0.5) (p/p) favored mycelial growth of Volvariella, which covered 59.52 cm2 (93%) and
50.12 cm2 (78%) of the Petri dishes’ surface after 6 days of incubation (Table 2).
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Table 2. Development of V. volvacea mycelium in formulations evaluated after six days of incubation.
Formulation* Area Petri dish pH
(cm2) area
covered
(%)
NS 9.95 15.6 6
CF 22.84 35.9 5.8
Wheat
CP 12.78 20.1 5.6
L 24.6 38.7 7.9
NS 38.35 60.3 6
CF 59.52 93.6 5.6
Millet
CP 30.49 47.9 5.6
L 3.4 5.34 8.2
NS 39.45 62.0 6
CF 34.14 53.7 5.9
Sorghum
CP 27.16 42.7 5.8
L 50.1 78.8 8.2
*NS: seeds with no supplementation, CF: seeds with corn flour (90:10), CP: with coffee pulp
(90:10) and L: with limestone, CaCO3 (99.5:0.5).
PERSPECTIVES
It is evident that mushroom diversity in the tropics is abundant and that humanity has yet to study or take
advantage of the vast majority of the species, many of which will probably go extinct before becoming
known to science. In Mexico alone, it is estimated that we have studied a mere 4.5% of our mycological
diversity (Guzmán 1998).
Professor S. T. Chang, a renowned specialist in the cultivation of tropical mushrooms, thinks that the
tropical regions of the world are suitable for developing this biotechnology, mainly because they have the
following characteristics: a) wet, warm climate and an abundant supply of agricultural waste, b)
mushrooms are relatively fast-growing organisms; some tropical mushrooms can be harvested and
consumed within 10 days after spawning, c) mushroom cultivation is labor intensive, however, this may
not be a problem in tropical regions, d) while land availability is usually a limiting factor in most types of
primary production, cultivating mushrooms requires little space because they can be stacked using trays,
and e) mushrooms have been accepted as human food from time immemorial and can immediately supply
additional protein (Chang 2007).
For the specific case of growing Volvariella in Mexico, the country satisfactorily meets the conditions
listed below: a) close to 28% of its area has a warm climate (Villers-Ruiz & Trejo-Vázquez 1998) as
recommended for the in situ reproduction of this genus, b) annually, more than 60 billion tons of organic
waste material are produced during harvest and post-harvest management of at least 20 different
agricultural crops (Valdez-Vázquez et al. 2010), and the chemical composition and physical structure of
most of these materials is suitable for mushroom production. An example of this is sugar cane, one of the
main agricultural crops in Mexico, with around 570,000 hectares designated to its cultivation. Depending
187
on the harvest method used, sugar cane cultivation produces 28 to 57 million tons of organic material that
is left in the fields (CONADESUCA 2016). Using some of this waste for growing mushrooms would
represent a sustainable way of using it that benefits the environment, c) wild strains of Volvariella have
fruited 13 days after spawning (Salmones et al. 1996) and d) rural inhabitants appreciate and eat both V.
volvacea and V. bombycina (Guzmán et al. 1993).
In Mexico, there are academic groups in research centers and universities located in tropical and
subtropical regions that specialize in mushroom cultivation. These groups could have a very important
impact by generating knowledge about techniques, substrates, varieties, control of diseases, etc.,
applicable to wild species. They also have the capacity to disseminate knowledge, through training
courses and by providing advice to growers, both from Mexico and other countries on the continent
interested in working with tropical species (Martínez-Carrera et al. 2016). Furthermore, some of these
institutions have experience in the preservation, reproduction and genetic improvement of wild germplasm
(Mata et al. 2016, Sánchez et al. 2016, Sobal Cruz et al. 2016).
Moreover, the national production of edible and medicinal mushrooms is noteworthy and accounts for
about 50% of Latin America’s entire production. It is considered the most important emerging agrofood
chain in the country, with a direct impact on food production for direct human consumption and on
producing new, high added value products with functional and medicinal properties (Martínez-Carrera et
al. 2016). Added to this consolidated production chain and the expansion in national consumption, the
strategic geographic location of Mexico has favored its leadership in this agroindustry, with a sustained
increase in exports to Latin American countries (INEGI 2016), some of which, like Peru and Guatemala,
share the ancestral tradition of eating mushrooms (Garibay-Orijel et al. 2010, Pavlich 2001). In
Guatemala, there have even been trials with the aim of cultivating Volvariella (De León et al. 1987).
In spite of notable growth in the agroindustry dedicated to cultivating edible mushrooms in Mexico, much
remains to be done in the areas of species diversification and innovation in growing techniques.
Specifically, to establish commercial cultivation of Volvariella, it is necessary to deepen our
understanding of the following aspects: a) basidiome production in greenhouse or field conditions, in
order to develop a systematic production system, b) an evaluation of production of wild germplasm, and c)
how to improve productivity of strains on residues available in the country, all in order to make the
process more economical.
ACKNOWLEDGMENTS
The author is grateful to the Institute of Ecology (INECOL) for financial support assigned for preparation of this
contribution, and Gerardo Mata for providing some photographs used in this chapter.
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15. A Pleurotus spp. HYDROALCOHOLIC FRACTION POSSESS A POTENT in vitro

OVICIDAL ACTIVITY AGAINST THE SHEEP PARASITIC NEMATODE Haemonchus
contortus
Edgar J. Cuevas-Padilla1,4, Liliana Aguilar-Marcelino1*, José E. Sánchez2, Manasés González-Cortázar3,

Alejandro Zamilpa-Álvarez3, Magaly Huicochea-Medina1,4, Ma. Eugenia López-Arellano1, Pedro
Mendoza-de-Gives1, Víctor M. Hernández-Velázquez4, Roberto González-Garduño5
1
Centro Nacional de Investigación Disciplinaria en Parasitología Veterinaria, INIFAP
2
El Colegio de la Frontera Sur
3
Centro de Investigación Biomédica del Sur, IMSS
4
Centro de Investigación en Biotecnología, UAEM (Morelos)
5
Universidad Autónoma Chapingo (Unidad Regional Universitaria Sursureste)
*<[email protected]>
ABSTRACT
Gastrointestinal parasitic nematodes cause important economic harm to animal health, seriously affecting the
livestock industry. Worldwide, economic losses from nematodiasis is reported at more than USD 10 billion a year in
anthelmintic treatments alone in 2013. In Mexico, the livestock industry has reported losses of about $8.902 million
Mexican pesos in the zootechnical potential of ruminants that interfere in the profitability of livestock farms. So far,
these diseases have been controlled by use of anthelmintic products (AH). In addition, improper use of AH
contributes to the imbalance of the environment, as well as an ecotoxicological risk to soil, plants, aquifers and
beneficial organisms. In this context, the search for alternative and sustainable complementary methods that reduce
the need for use of synthetic products of chemical origin, for example natural derivatives of edible fungi with a
nematicidal effect, is evident. The edible fungi Pleurotus spp. possesses nutraceutical and therapeutic properties
including their use as anti-parasitic. In the present investigation, in vitro effects of compounds derived from
Pleurotus spp. were assessed against various stages of the parasitic nematode Haemonchus contortus. Pleurotus
ostreatus had higher nematicidal activity than P. eryngii. The "F" fraction of hydroalcoholic extract of P. ostreatus
mycelium showed the highest nematicidal activity (25.29%) with respect to other fractions. FpMeOH and FMeOH
fractions had the highest L4 mortality rate of 84.8 and 100, respectively at 24 and 72 h. The spent mushroom
substrates (SMS) of P. djamor had biological activity against H. contortus in its egg stages and L3. Furthermore,
such activity is possibly influenced by the composition and/or the type of substrate. EHA-SA11 was lethal against L3
exsheathed with a 45% mortality at 20 mg/ml at 72 h postexposure, but did not affect nematode eggs. In contrast,
EHA-SA13 was the most effective against parasite eggs with 100% inhibition starting at 2.5 mg/ml that was
determined at CL90= 625 μg/ml. The use of a "nutraceutical" food based on SMS of edible mushroom culture
represents a potential method of prevention of nematode infections.
Keywords: ovine cattle, nematicidal mushrooms, biological control
INTRODUCTION
Gastrointestinal nematodes (GIN) are a major concern for livestock breeders worldwide, as they cause
anemia, diarrhea, weight loss and even death in young animals (von Son de Fernex et al. 2014). Among
various gastrointestinal parasitic nematodes species, H. contortus, possess haematophagia properties
(Besier et al. 2016a,b). This parasite has been found with a high prevalence in different parts of the world
with an enormous global economic impact. Treatment costs against parasites alone are more than USD 10
billion a year and do not include productivity losses (Roeber et al. 2013). In Mexico, losses of $8,902
million pesos have been reported in the zootechnical potential of ruminants, interfering with the
profitability of livestock farms (Rodríguez-Vivas et al. 2017). Nematode parasitosis of cattle and small
193
ruminants has been treated for decades with chemical anthelmintic products that possess undesirable
effects; for example, their excessive and continuous use has led to the development of a severe problem of
resistance in the parasites to these compounds. Additionally, chemical anthelmintic drugs affect beneficial
organisms such as dung beetles ie., Onthophagus landolti and Canthon indigaceus chevrolati (Basto-
Estrella et al. 2014). Therefore, it is necessary to seek alternatives to implement sustainable control
methods of these parasitic diseases. Edible fungi are considered a traditional component in the diets of
various communities nationally and globally, with important nutritional characteristics. These edible fungi
have not only been appreciated as food but have served as an important utility within traditional medicine.
They contain compounds with different therapeutic properties, such as anti-inflammatory,
antihypertensive, immuno-modulators, anti-viral, anti-microbial, anti-carcinogenic, anti-oxidant, anti-
cholesterolemic, anti-allergic, insecticidal, anti-fungal and anti-parasitic (Glamočlija et al. 2015). Species
of the edible fungus Pleurotus possess nutraceutical and nematicidal properties (Khaund and Joshi 2015,
Kirsch et al. 2016). Regarding nematicidal properties, 23 species of the genus Pleurotus are active against
nematodes (Li and Zhang 2014). In addition, some products such as proteases (André-Genier et al. 2015),
fatty acids (Pineda-Alegría et al. 2017), alkaloids, peptidic compounds, terpenes (Li et al. 2007),
condensed tannins and phenolic compounds (Ganeshpurkar et al. 2012) obtained from Pleurotus spp. have
shown antiparasitic activity (Shariat et al. 1994, Palizi et al. 2009, Del Carmen et al. 2015). Secondary
metabolites produced by Pleurotus species have nutraceutical characteristics and have been mostly
isolated from basidiomes of the fungus. In the present investigation, products derived from Pleurotus spp.
were tested in vitro against different stages of the sheep parasitic nematode, H. contortus.
Biological material
Mushrooms
Mycological material (mycelium, basidiomes and spent mushroom substrate) was provided by the
Tropical Mushrooms Laboratory of the Southern Border College (ECOSUR), Tapachula Headquarters,
Chiapas, Mexico.
Mycelium
Mycelium of Pleurotus ostreatus ECS-1123 and P. eryngii ECS-1292 were used and mycelium was
produced on an agar medium of whole wheat flour (HIT) contained in Petri dishes after 14 days’ growth
(Comans-Pérez et al. 2014).
Basidiomes
Basidiomes of P. djamor strain ECS-123 were produced following methodology of Avendaño and
Sánchez (2013).
Pleurotus djamor production substrate

Various ingredients were used to prepare substrate mixtures to promote P. djamor production. The
composition of each substrate used is shown in Table 1.
Nematode
The H. contortus strain was obtained from the INIFAP "Las Margaritas" Experimental Site and
maintained with periodic passes in experimental sheep at the Helminthology Unit of the CENID-
Veterinary Parasitology in Jiutepec, Morelos, Mexico (Campos et al. 1990).
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Table 1. Substrates for P. djamor production

Substrate Mixture 1 (g) Mixture 2 (g) Mixture 3 (g) Mixture 4 (g)
06 12-20 60-71 11-18 18-25
10 30-35 0 30-40 43-50
11 30-40 30-40 0 40-50
12 40-48 38-45 38-45 0
13 28-38 27-35 27-37 40-45
Substrates were weighed to obtain a similar starting amount in dry weight at 100 g.
Eggs and L3 larvae of H. contortus

One three-month-old male sheep that was previously infected with 350 third stage larvae (L 3) per kg/live
weight was used. After the pre-patent period (21 days), samples of fresh feces collected directly from the
rectum of the donor ovine were taken to confirm infection using the McMaster technique. Feces were
macerated with potable water until a homogeneous mixture was obtained and filtered through various
sieves of descending sizes. Eggs were recovered and purified by a gradient with 40% sucrose and
centrifugation at 3500 rpm for 5 min. The ring formed by the eggs was recovered and washed using sterile
distilled water, by centrifugation. For the production of L3 larvae, samples of feces from the sheep were
taken after the pre-patent period and a coproculture was made in a plastic container to obtain L 3. Finally,
at 7 days, L3 larvae were recovered using the Baermann technique and stored in a refrigerator at 4 °C until
used (Liébano et al. 2011).
Exsheathed of H. contortus L3 larvae
Three ml of 6% sodium hypochlorite were used to make dilutions and obtain concentrations of 3, 1.5,
0.750, 0.375, and 0.187%. This process required 7 min, then three washes were performed with distilled
water and centrifuged at 2500 rpm for 1 min each wash; the supernatant was discarded and the larvae were
recovered (Liébano et al. 2011).
Development of H. contortus L4 larvae
The fourth evolutionary stage was obtained under a controlled CO2 atmosphere, using L3 exsheathed
following methodology described by Ramírez et al. (2006).
Preparation of hydroalcoholic extracts of mycelium P. ostreatus and P. eryngii
A hydroalcoholic solution was prepared using a 70:30 ethanol-water ratio. The solution covered all fungal
material contained in an Erlenmeyer flask and was allowed to stand for 24 h. Subsequently, samples were
subjected to the distillation process using a rotavaporator and finally, the concentrated extract was dried at
high vacuum using a lyophilizer (Huicochea-Medina et al. 2015).
Chemical fractionation of a hydroalcoholic extract of the mycelium of P. ostreatus
A hydroalcoholic extract (3 g) of mycelium of P. ostreatus was weighed and adsorbed onto 7 g of normal
phase silica and a glass column was packed with 60 g of normal phase silica (Durst and Gokel 2007). The
elution was started with a dichloromethane system (100%) and 10% volume-to-volume increments of
methanol were made, ending with a dichloromethane-methanol system (50:50 v/v) and ending with
methanol (100%) washing (Huicochea-Medina et al. 2015).
195
Purification of fraction "F" from P. ostreatus mycelium
Purification was performed by high performance liquid chromatography (HPLC). Fraction "F" extract (3
mg) was weighed and diluted in 1 ml of methanol HPLC to have a concentration of 3 mg/ml. From this
solution, 1 ml was taken and the sample injected into the chromatograph. Previously, equipment was
washed with water and methanol HPLC grade. Samples were programmed for detection at a wavelength
of 280 nm (González 2010).
Chemical fractionation of fruit bodies of P. djamor
Fruit bodies were fragmented and placed in a 40:60 water-ethanol solution. The solution covered the
fungal material in a 3:1 ratio and was allowed to soak for 24 h at room temperature. Subsequently, the
sample (hydroalcoholic extract) was filtered with a gauze and cotton system, concentrated in a rotary
evaporator and dried under high vacuum using a freeze-dryer. Dry material was fractionated by the open
column chromatography technique using dichloromethane-methanol systems. Fractions evaluated were
obtained in a 1:1 dichloromethane-methanol system. Two fractions were recovered: one-part from the
sediment and another part remained soluble. They were denominated "FpMeOH" and "FMeOH".
Obtaining hydroalcoholic extracts from spent mushroom substrate
The dry weight of each of five spent mushroom substrates (SMS) previously described in Table 1, were
individually placed in 1 liter flasks containing a hydroalcoholic solution in a ratio of 70:30, ethanol:water.
A volumetric ratio of 3:1, hydroalcoholic solution:SMS was used. Samples were macerated at room
temperature for 24 h protected from light. Biomass was removed by filtration through sterile cotton gauze
and each of the filtrates were collected in 1 liter flasks. Filtrates were concentrated by removal of solvent
by vacuum distillation in a rotary evaporator at 49 °C temperature and 80 rpm. Extracts were obtained by
lyophilization of the concentrated macerates. Five hydroalcoholic extracts of depleted substrate (EHA-SA)
were obtained and were named as follows: EHA-SA06, EHA-SA10, EHA-SA11, EHA-SA12 and EHA-
SA13.
Experimental designs
Bioassays were carried out in 96-well microtiter plates as described below:
In vitro comparison of hydroalcoholic extracts and mycelial fractions of P. ostreatus and P. eryngii
against L3 of H. contortus
In each well, 80 μl of hydroalcoholic extract of P. ostreatus and P. eryngii or fractions denominated: B, D,

E, F and G of mycelium of P. ostreatus were placed at a concentration of 400, 200, 100, 50, 25 ml and 20
μl of the aqueous suspension containing 200 L3 of H. contortus sheathed and exsheathed, leaving a final
volume of 100 μl. Two control groups were included: positive control containing distilled water and a
negative control with commercial ivermectin [10 mg/ml], with four replicates per treatment and three
post-challenge readings: 24, 48, and 72 h. Data were analyzed using the SAS GLM procedure (SAS,
2004). Adjusted mortality was calculated according to the formula: % nematicidal effectiveness = average
of the control-average of the treated group/average of the control group * 100 (Eguale and Giday 2009).
Subsequently, mortality was transformed to a square root and then to a sinus arc to homogenize the
variance and obtain an approximation to a normal distribution. A 2x5x3 factorial design was used where
the main effects were: hydroalcoholic extracts obtained from P. ostreatus and P. eryngii, five
concentrations (400, 200, 100, 50 and 25) and 3 times (24, 48 and 72 h). Duncan's test (Steel and Torrie
1988) was used to separate means according to the following model: Yijkl = μ + Fi + Dj + Tk + F * Tik + D
* Tjk + F * Dij + D * Et * T ijk + εijkl Where: Yijkl = response variable (adjusted mortality). Μ = population
196
mean. Fi = fixed effect of extracts, Dj = fixed effect of j-th concentration (j = 400, 200, 100, 50 and 25). Tk
= fixed effect of k-th reading time (k = 24, 48, 72). (F * T) ik = effect of extract and time. (D * T) jk =
effect of the jth concentration and time. F * Dij = combined effect of extract and concentration. F * D * T
ijk = effect of extract, concentration and time. εijkl ~NI (0, 2e). A second 5x5x3 factorial design was used
where the effects were: fractions obtained from P. ostreatus (B, D, E, F and G), 5 concentrations (400,
200, 100, 50 and 25) and 3 times (24, 48 and 72h), the same statistical model described above was used.
In vitro comparison of two methanolic fractions (FpMeOH and FMeOH) of basidiomes of P. djamor
against L4 larvae of H. contortus
100 L4 and a final volume of 100 μl were placed in each well. Concentrations (via serial dilutions) for the
fraction that precipitated (FpMeOH) were: 0.3125, 0.625, 1.25, 2.5, 5, 10, 20, 40 and 60 mg/ml, whereas
for the fraction that was soluble (FMeOH) were: 0.3125, 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 mg/ml.
A negative control (enriched Hanks medium) and a positive control (Ivermectin 5 mg/ml) were used.
Plates were then covered with foil and incubated at 28 ±1 °C in a CO2 incubator. Readings were
performed at 24, 48 and 72 h post-confrontation. For statistical analysis, a completely randomized design
was used (Buzatti et al. 2015). An analysis of variance and a comparison of means by Tukey's method
(α=0.05) were performed. SAS (2004) software was used. Adjusted mortality was calculated according to
the formula: % nematicidal effectiveness = mean of the control group - mean of the treated group/mean of
the control group * 100.
In vitro evaluation of five hydroalcoholic extracts of depleted substrates of P. djamor against eggs
and L3 larvae of H. contortus
A bioassay was performed for each of the five hydroalcoholic extracts from different P. djamor-SMS
named: EHA-SA (06, 10, 11, 12 and 13) by placing six concentrations (0.5, 1.25, 2.5, 5, 10 and 20 mg/ml)
dissolved in sterile distilled water and their respective controls, where 1% commercial ivermectin was
used as a positive control in egg inhibition tests and mortality of H. contortus L3 larvae. Four replicates
(n=4) of each treatment were used by placing 50 μl of treatment at concentrations of: 1, 2.5, 5, 10, 20 and
40 mg/ml to obtain previously described working concentrations and 50 μl of an aqueous suspension
containing 100 eggs and 200 L3 of H. contortus sheathed. The final volume was 100 μl. Inoculated
microplates were incubated at 29 °C and protected from light until they were read (48 and 72 h for eggs
and 24, 48 and 72 h for L3 sheathed) where aliquots were observed corresponding to each replicate of
treatments in a 40X light field microscope. H. contortus eggs and/or larvae present in each of the
treatments were quantified to obtain percent inhibition of hatching of each replicate as well as live larvae
and dead larvae confirmed by physical stimulation. Subsequently, percentages of the four replicates were
used to obtain percent inhibition and corrected mortality rate that was determined based on the Schneider-
Orelli formula (1972): % inhibition of corrected eggs = group mean mean-control group / mean control
group mean * 100. Data analysis was performed individually for each of the five EHA-SAs (06, 10, 11, 12
and 13) from the data weighted with the mean inhibition percentage of the corresponding negative control.
We used a variance analysis of treatments in a completely randomized design and means were compared
according to Tukey's test at a level of significance of 0.05. The statistical model used is as follows:
Where:
= Variable response (Inhibition at 48,72 h; mortality at 24, 48 y 72 h)
= Overall average
= Effect of treatment (EHA-SA06, EHA-SA10, EHA-SA11, EHA-SA12, EHA-SA13)
= Random error
197
RESULTS
In vitro comparison of hydroalcoholic extracts and mycelial fractions of P. ostreatus and P. eryngii
against L3 of H. contortus
Results of nematicidal efficacy of the hydroalcoholic extract of P. ostreatus and P. eryngii against H.
contortus L3 sheathed at five different concentrations are presented in Table 2. Highest mortality rate of P.
ostreatus was 23.3 at a concentration of 100 μg/ml at 72 h and for P. eryngii of 16.6% at 24 h at 200
μg/ml.
Table 2. In vitro interaction of Haemonchus contortus infective larvae with a hydroalcoholic extract of Pleurotus
ostreatus.
Treatment Concentration Time (h) X, SD Mortality (%)
24 0.92  0.38 11.78a

25 μg/ml 48 1.20  0.31 12.20a
72 0.75  0.23 13.51a
24 0.98  0.25 10.51a
50 μg/ml 48 1.15  0.42 11.59a
72 1.07  0.23 17.69b
Pleurotus ostreatus 24 0.77  0.41 8.96a
100 μg/ml 48 1.15  0.12 12.13a
ECS-1123 72 1.57  0.42 23.33b
24 0.95  0.31 9.57a
200 μg/ml 48 1.17  0.57 10.70a
72 1.17  0.28 16.54b
24 1.17  0.20 11.3a
400 μg/ml 48 1.07  0.53 12.72a
72 1.52  0.63 21.28b
24 0.10  0 1.28
Distilled water 48 0.07  0.05 0.64
Controls 72 0.02  0.05 0.45
24 9.05  0.93 100
Ivermectin (10 mg/ml) 48 9.10  1.49 100
72 6.07  0.71 100
X = Average; SD = Standard Deviation. Values followed by a different letter indicate statistical differences (P<0.05).
Results of nematicidal effectiveness of fractions against H. contortus L3 sheathed are shown in Tables 3-7.
Fraction "F" had the highest mortality rate of 25.2% at 72 h with a concentration of 400 μg/ml, while the
lowest percentage was from fraction "D" with 1.64.
Table 3. In vitro interaction of infective larvae of Haemonchus contortus with the fraction “B” of the hydroalcoholic
extract of the mycelium of Pleurotus ostreatus.
24 0.47  0.29 5.13ab
Fraction B 25 μg/ml 48 0.58  0.26 5.36ab
72 0.52  0.15 6.34ab
198
24 0.58  0.22 4.96ab

50 μg/ml 48 0.50  0.45 4.48ab
72 0.42  0.12 4.22b
24 0.85  0.43 8.19a
100 μg/ml 48 0.47  0.17 4.43ab
72 0.75  0.28 8.19a
24 0.55  0.17 6.94ab
200 μg/ml 48 0.62  0.28 6.02ab
72 0.67  0.33 6.63ab
24 0.70  0.21 5.49ab
400 μg/ml 48 0.62  0.12 5.68ab
72 0.67  0.27 7.54ab
24 0.05  0.06 0.68
72 0.05  0.05 0.62
Controls
24 9.48  0.82 100
Ivermectin (10 mg/ml) 48 10.65  0.9 100
72 8.02  0.87 100
Average (X) and standard deviation (SD) were calculated from three repetitions each. Values followed by a different letter indicate statistical
differences (P<0.05) among concentrations and time.
Table 4. In vitro interaction of infective larvae of Haemonchus contortus with fraction “D” of a hydroalcoholic
24 0.33  0.13 2.68b
25 μg/ml 48 0.50  0.16 5.31b
72 0.15  0.13 3.59b
24 0.20  0.15 1.64b
50 μg/ml 48 0.40  0.08 3.5b
72 0.25  0.13 5.18b
24 0.37  0.28 2.88b
Fraction D 100 μg/ml 48 0.62  0.45 5.85ab
72 0.12  0.12 2.09b
24 0.53  0.15 4.17b
200 μg/ml 48 1.32  0.92 9.61a
72 0.32  0.17 3.97b
24 0.80  0.80 5.83b
400 μg/ml 48 0.35  0.19 3.53b
72 0.30  0.18 3.64b
24 0.02  0.05 0.23
Distilled water 48 0 0
72 0 0
Controls
24 15.6  4.43 100
Ivermectin (10
48 12.2  0.59 100
mg/ml)
72 10.42  1.00 100
Average (X) and standard deviation (SD) were calculated from three repetitions each. Values followed by a different letter
indicate statistical differences (P<0.05) among concentrations and time.
199
Table 5. In vitro interaction of infective larvae of Haemonchus contortus with the fraction “E” of a hydroalcoholic
24 0.30  0.08 6.06c
25 μg/ml 48 0.45  0.30 7.03bc
72 0.30  0.18 6.09c
24 0.50  0.52 6.82bc
50 μg/ml 48 0.63  0.39 9.12bc
72 0.70  0.11 10.04ab
24 0.72  0.26 7.02bc
Fraction E 100 μg/ml 48 0.43  0.26 5.33c
72 1.02  0.28 13.09a
24 0.43  0.17 4.67c
200 μg/ml 48 0.43  0.28 5.28c
72 0.80  0.62 7.74bc
24 0.75  0.17 6.71bc
400 μg/ml 48 0.55  0.19 5.80c
72 0.60  0.27 6.52c
24 0.05  0.06 0.68
72 0.05  0.05 0.62
Controls
24 9.48  0.82 100
Ivermectin (10 mg/ml) 48 10.65  0.90 100
72 8.02  0.08 100
Table 6. In vitro interaction of infective larvae of Haemonchus contortus with fraction “F” of a hydroalcoholic
extract of the mycelium of Pleurotus ostreatus
24 1.62  0.27 10.19cd
25 μg/ml 48 1.65  0.05 10.1cd
72 2.15  0.28 16.2b
24 1.55  0.66 9.24cd
50 μg/ml 48 2.12  0.39 11.89c
72 1.80  0.40 14.12b
24 1.90  0.70 10.97cd
Fraction F 100 μg/ml 48 2.82  0.12 13.51bc
72 2.37  0.54 16.73b
24 1.62  0.35 11.02cd
200 μg/ml 48 2.55  0.42 12.37c
72 1.97  0.22 14.93b
24 1.10  0.21 7.24d
400 μg/ml 48 2.92  0.65 16.34b
72 3.27  0.35 25.29a
24 0.20  0 1.25
72 0.10  0.08 0.74
Controls
24 11.10  1.73 100
Ivermectin (10 mg/ml) 48 12.83  3.20 100
72 12.72  1.04 100
200
Table 7. In vitro interaction of infective larvae of Haemonchus contortus with fraction “G” of a hydroalcoholic
extract of mycelium of Pleurotus ostreatus
24 1.28  0.13 10.28ab
25 μg/ml 48 1.65  0.20 9.42ab
72 1.00  0.40 7.73b
24 1.50  0.26 10.90ab
50 μg/ml 48 1.95  0.33 11.90ab
72 1.30  0.21 9.50ab
24 1.50  0.39 9.42ab
Fraction G 100 μg/ml 48 1.70  0.54 9.97ab
72 1.60  0.21 9.22ab
24 1.50  0.26 11.10ab
200 μg/ml 48 1.35  0.34 8.01b
72 1.50  0.21 9.78ab
24 1.47  0.33 10.2ab
400 μg/ml 48 1.63  0.53 9.54ab
72 1.75  0.46 11.51a
24 0.20  0 1.25
72 0.10  0.08 0.74
Controls
24 11.10  1.73 100
Ivermectin (10 mg/ml) 48 12.83  3.20 100
72 12.72  1.04 100
Chemical fractionation of mycelium of P. ostreatus yielded 12 fractions and were named A-G as shown in
Table 8.
201
Table 8. Fractionation of hydroalcoholic extract of mycelium of Pleurotus ostreatus.

System: CH2Cl2-CH3OH Fraction Collected fraction Yield (g)
100% CH2Cl2 1 A ----
90:10 3 B 0.1424
5
80:20 C -----
6
7
70:30 D 1.0393
8
9
50:50 E 0.2827
10
100% CH3OH 11 F 0.5534
100% CH3OH 12 G 0.1692
Results of separation of compounds from hydroalcoholic extracts of P. ostreatus and fraction "F" are
shown in chromatograms of Figures 1-2. The hydroalcoholic extract possessed four probably "active"
compounds that were also found in fraction "F". In the latter, the compound responsible for anthelmintic
activity can be observed at minute 14 with an absorbance unit of 0.004.
Hydroalcoholic extract
of Pleurotus ostreatus
Fraction F
Figures 1 (left) and 2 (right). Chromatograms of hydroalcoholic extract and fraction "F" of mycelium of P.
ostreatus (ECS-1123) on HPLC at 280 nm with a flow of 0.9 ml/min.
202
In vitro comparison of two methanolic fractions (FpMeOH and FMeOH) of basidiomes of P. djamor
against L4 larvae of H. contortus
Highest mortality rate obtained with the FpMeOH fraction was 84.89 at a concentration of 60 mg/ml at 24
h postexposure, whereas for the FMeOH fraction it was 100% at a concentration of 160 mg/ml at 48 and
72 h post-confrontation (Table 9, 10).
Table 9. In vitro evaluation of FpMeOH fraction against Haemonchus contortus L4 larvae at various post-
confrontation times.
Treatment Mortality (%) ± SD
mg/ml 24h 48h 72h
Control 100.00 a 100.00 a 100.00 a
(Ivermectina, 5) ±0.00 ±0.00 ±0.00
a b
84.89 52.76 39.19 b
60
±10.10 ±35.48 ±7.70
b c
8.25 9.06 38.24 b
40
±4.30 ±8.08 ±6.94
b c
0.00 14.91 38.65 b
20
±6.40 ±11.96 ±5.13
b c
10.22 6.41 26.71 bc
10
±2.38 ±1.05 ±8.36
c
0.00 b 10.15 17.03 cd
5
±9.44 ±7.00 ±12.67
b c
0.00 6.96 10.00 cd
2.5
±2.29 ±2.41 ±5.83
0.00 b 0.00 c 10.00 cd
1.25
±2.74 ±2.43 ±9.88
b
3.21 5.69 c 0.65 d
0.625
±3.24 ±4.03 ±0.65
0.00 b 1.67 c 4.41 d
0.3125
±3.92 ±4.75 ±5.59
14.39 b 7.75 c 4.21 d
Hanks
±6.37 ±4.80 ±3.17
E.C.M. 30.48 109.79 48.86
R2 0.97 0.89 0.95
Same letters in the same column indicate that values do not differ statistically, according to
Tukey's test. P≤0.05. N = 4 (four wells). 10 aliquots (drops) per well.
203
Table 10. In vitro evaluation of FMeOH fraction against Haemonchus contortus L4 larvae at different
post-confrontation times.
Treatment Mortality (%) ± SD
mg/ml 24 h 48 h 72 h
Control 100.00 a 100.00 a 99.32 a
(Ivermectina, 5) ±0.00 ±0.00 ±1.28
a a
96.98 100.00 100.00 a
160
±2.44 ±0.00 ±0.00
b b
33.92 27.44 36.15 b
80
±7.57 ±13.72 ±44.07
c c
13.33 6.69 0.05 bc
40
±5.32 ±3.52 ±4.04
7.41 c 7.31 c 32.90 bc
20
±2.43 ±2.22 ±6.64
c c
3.85 11.50 28.48 bc
10
±7.10 ±5.66 ±3.82
c c
9.94 11.74 24.74 bc
5
±6.80 ±5.70 ±4.64
c c
6.98 6.89 9.30 bc
2.5
±6.75 ±5.54 ±17.42
c c
8.06 11.16 9.55 bc
1.25
±4.79 ±6.58 ±10.28
c c
7.77 8.64 0.00 c
0.625
±6.97 ±6.54 ±1.69
6.04 c 7.89 c 0.00 c
0.3125
±4.84 ±2.76 ±2.35
5.51 c 3.87 c 6.24 bc
Hanks
±2.13 ±3.17 ±3.78
E.C.M. 28.27 33.7 205.99
R2 0.97 0.97 0.87
Same letters in the same column indicate that values do not differ statistically, according to
Tukey's test. P≤0.05. N = 4 (four wells). 10 aliquots (drops) per well.
204
In vitro evaluation of five hydroalcoholic extracts of different depleted substrates of P. djamor

against eggs and L3 larvae of H. contortus
Ovicidal activity of EHA-SA06, 10, 11, 12, 13

Results obtained by comparing eggs of H. contortus with extracts (EHA-SA), showed a similar tendency
in readings carried out at 48 and 72 h. However, biological activity at 72 h was lower in all extracts
evaluated except for EHA-SA13 (Figure 3). EHA-SA11 did not show ovicidal activity despite having
shared similarity in mass composition with EHA-SA13, which had the strongest and highest activity. On
the other hand, spring chip, not present in substrate 11 and if it contained substrate 13, apparently
potentiated a biological effect in all EHAs evaluated. Since EHA-SA11, without this agroindustrial waste,
did not present significant differences compared to the negative control. EHA-SA13 showed 100%
percent inhibition with the 2.5 mg/ml concentration. In fact, of all EHAs evaluated, EHA-SA13
demonstrated strong biological activity, as shown by statistical coefficients (Table 9). On the other hand,
EHAs 06, 10 and 12 resulted in variable data with high standard deviations. With low concentrations of
EHA-SA06 (0.5 to 2.5 mg/ml), percent inhibition was above 83% at 48 h. However, the reading at 72 h
showed that biological activity of EHA-SA06 was not consistent, showing values below half the previous
reading. EHA-SA10 was similar to EHA-SA06, showing nematicidal activity at the 48 h reading from
concentrations ≥1.25 mg/ml and decreasing its activity in the reading taken at 72 h.
Figure 3. Ovicidal activity of spent mushroom substrates of Pleurotus djamor of different composition.
Percentages obtained at 48 and 72 h of EHA-SA 06, 10, 11, 12 and 13 are shown. Asterisks show
statistically significant differences (Tukey P<0.05).
In general, it is observed that data from EHA-SA06 and 10 showed an increase in the standard deviation
from the mean at 72 h, perhaps due to natural inhibition of the negative control. EHA-SA12 had ovicidal
activity above 10 mg/ml in treatments at 48 and 72 h. However, like EHA-SA06 and EHA-SA10, high
coefficients of variation of the averages resulted non-significance of its nematicidal activity by the erratic
biological response against H. contortus eggs, as well as the high rate of infertility shown in the controls.
205
It should be mentioned that data used for the analysis of variance in the SAS V9 statistical package were
not modified for normalization and thus only one treatment (EHA-SA13) with a high statistical
significance (P <0.0001) was obtained at the required levels of analysis.
Nematicidal activity of EHA-SA: 06, 10, 11, 12, 13
Lethal activity obtained in the in vitro bioassay is shown in Figure 4. A broad variability of data is shown
in the different exposure times with the highest mortality rates for EHA-SA11 obtained at 24 h. At 48 h,
fluctuations were observed between EHA-SA13, 12, 06 and 11 that depended on the concentration at
which they were evaluated. EHA-SA13 gave the highest biological effectiveness against L3: 2.5 and 5
mg/ml EHA-SA 12 was the highest. At 10 mg/ml the best was EHA-SA06 and at 20 mg/ml EHA-SA11
showed the highest nematicidal effectiveness. At 72 h of interaction, EHA-SA12 and 13 showed a very
similar tendency from the concentration of 0.625 mg/ml. However, between the concentrations of 2.5 to
10 mg/ml, EHA-SA12 presented greater activity in reference to the other extracts. Likewise, similarity of
activity between EHA-SA06 and 11 up to the concentration of 10 mg/ml is remarkable. Subsequently
EHA-SA11 presented 45% mortality against L3 larvae without sheath at 20 mg/ml.
Figure 4. Nematicidal activity of SMS of Pleurotus djamor. Percentages obtained at 24, 48 and 72 h of
EHA-SA06, 10, 11, 12 and 13 are shown. The asterisks show statistically significant differences (p<0.05).
According to previous results, mortality rates of EHA-SA11 at 24, 48 and 72 h are presented in
Table 11. The percentages presented were obtained by adjusting for mortality using the formula
of Scneider-Orelli (1972). EHA-SA11 was chosen because it showed higher mortality with
respect to 24 h exposure time and confirmed its biological activity at 48 and 72 h. Statistical
analysis showed significant difference for the concentration of 20 mg/ml at 72 h exposure.
206
Table 11. Lethal activity of HAE-SA11 against Haemonchus contortus L3.

Lethal activity (%)*
HAE-ES11 (mg/ml)
24 h 48 h 72 h
20 4.47 c,d 18.20 b 45.22 b
10 14.69 b 9.68 b 19.28 c
5 6.53 b,c,d 8.61 b 13.91 c,d
2.5 12.7 b,c 18.16 b 1.8 d,e
1.25 9.51 b,c,d 5.26 b 0.00 e
0.625 6.06 b,c,d 0.98 b 0.00 e
MSE 16.53 65.78 35.89
P value < 0.0001 < 0.0001 < 0.0001
*Adjusted percentage with the Schneider-Orelli formula (Püntener 1981). Same letters in the same column are not significantly different, Tukey
(P< 0.05).
DISCUSSION
In recent decades the study of macromycetes has increased. Extracts from edible mushrooms contain
various bio-active components that are important in human and animal health. Miles and Chang (2004)
reported that 77% of medicinal mushroom products are derived from commercially grown or
commercially harvested fruit bodies, 21% of all products are derived from fungal mycelium and about 2%
are derived of filtrates of culture media containing different nutrients. Mycelium and filtered medium are
products that are increasingly important, since they meet the requirement of greater safety, quality control
and production throughout the year (Valencia and Garín 2012). While considerable information is
available regarding human health, little is known about fungal extracts and their interactions in animals
and their pathogens. Results obtained from the evaluation of the hydroalcoholic extracts of the mycelium
of P. ostreatus against different stages of H. contortus are encouraging. Additional work is needed to
further investigate compound(s) that are responsible for this activity.
The fungus P. ostreatus possesses compounds with nematicidal activity, as reported by Kwok et al. (1992).
They identified a nemato-toxin, "2-trans-acid-decenedioic" that was evaluated against a free-living
nematode Panagrellus redivivus. Additionally, Stadler et al. (1994) identified several molecules
corresponding to S-choric acid, linoleic acid and p-anisaldehyde of the fungus P. pulmonarius that was
responsible for nematicidal activity against a bacteriophage of the nematode Caenorhabditis elegans. On
the other hand, Satou et al. (2008) demonstrated that the nematicidal mechanism of P. ostreatus against a
nematode of the Diplogastridae family is through linoleic acid peroxide, a molecule that induces reduction
of the anterior part (head) of the nematode and causes its death. In addition, Khan et al. (2014) stated that
the fungus P. eryngii possesses the ability to kill Bursaphelenchus xylophilus, a nematode that attacks pine
wood. Rojas (2013) showed that the same fungus species attacks phytopathogenic nematodes such as
Meloidogyne javanica and Heterodera schachtii through the production of nematode toxins. In the present
investigation, a compound was found in fraction "F" that could coincide with that reported by Alam et al.
(2011). He found that P. ostreatus contains several phenolic compounds such as gallic acid, chlorogenic
acid, protocatecuic acid and naringenin. Our “F” fraction was obtained with a 100% methanol system with
secondary metabolites present being very polar. Our compound was purified by high performance liquid
chromatography (HPLC) and it is inferred that this is a phenolic compound. Our work and that reported by
Arizmendi et al. (2014) shows the fungus P. ostreatus possesses bioactive compounds with nematicidal
properties both in the fruit body and mycelium. In the future, we plan to continue with the study of
Pleurotus spp. against various stages of H. contortus. In relation to in vitro comparison of the two
methanolic fractions (FpMeOH and FMeOH) of basidiomes of P. djamor against L4 of H. contortus, the
main objective of using this stage of larvae was to try to simulate the conditions of the L4 in sheep
abomasum (Schwarz et al. 2013, Gadahi et al. 2016). Results of the present study demonstrate that
FpMeOH and FMeOH fractions possess nematicidal activity. These fractions may have bioactive
207
compounds that could be used to control the H. contortus L4 stage. It would be necessary to purify and
identify compounds by means of different chromatographic and spectroscopic techniques (nuclear
magnetic resonance) to elucidate molecules responsible for nematicidal activity of P. djamor. Fungi are
one of the main generators of biologically active natural products that have a high capacity for use as
therapeutic agents for use in mammals (Li and Zhang 2014). Among these compounds more than 200
nematicidal compounds are counted. In vitro evaluation of five hydroalcoholic extracts of different P.
djamor SMS against H. contortus eggs and larvae L3 indicate the composition of the substrates for fungal
production are differential in their metabolic response.
Stadler and Sterner in (1998) evaluated nematicidal activity in response to stress inducing stimuli in the
fungus, such as physical damage to the basidiome. Similarly, there is information that the substrate
composition is important for potentiating the nematicidal activity of EHAs from basidiomes of P. djamor
(Robledo et al. 2015) and that the fungus stage is also important (Valdez 2015). Mycelium has dual
activity against H. contortus larvae and egg as shown in previous studies using P. ostreatus as a
productive species (Valdez 2015). This work suggests that the active compounds against H. contortus
eggs produced by the fungus during its development and that said compounds are stable at extraction
temperature and pH, since they remain active when subjected to the chemical maceration procedure. The
fungi appear to produce bioactive compounds that are stored in mycelial structures and degradation
compounds or extracellular compounds that are excreted into the substrate.
Mushrooms are proteinaceous and the substrate formulations may include cereal straws and various grains
that are components of animal diets (Noriega et al. 2009, Esqueda-Esquivel and Tosquy-Valle 2007, FIRA,
2015, Rinker 2017). The use of a "nutraceutical" food based on SMS of edible mushroom culture
represents a potential method of prevention of nematode infections.
CONCLUSIONS
Hydroalcoholic extracts of the mycelium of P. ostreatus and P. eryngii showed a minimal nematicidal
activity at concentrations used. However, P. ostreatus had higher nematicidal activity than P. eryngii.
The "F" fraction of hydroalcoholic extract of P. ostreatus mycelium showed the highest nematicidal
activity (25.29%) with respect to other fractions. FpMeOH and FMeOH fractions had the highest L4
mortality rate of 84.8 and 100, respectively at 24 and 72 h. SMS of P. djamor had biological activity
against H. contortus in its egg stages and L3. Furthermore, such activity is possibly influenced by the
composition and/or the type of substrate. EHA-SA11 was lethal against L3 with a 45% mortality at 20
mg/ml at 72 h postexposure, but did not affect nematode eggs. In contrast, EHA-SA13 was the most
effective against parasite eggs with 100% inhibition starting at 2.5 mg/ml that was determined at CL90=
625 μg/ml.
ACKNOWLEDGMENTS
The authors are grateful for the support provided by INIFAP through projects 9442232005 and 834432984.
We also thank Lilia Moreno Ruíz (ECOSUR) and Antonio Pineda-Alegría (CENID-PAVET, INIFAP) for
their technical support.
208
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212
PERSPECTIVES
PERSPECTIVES ON MACROMYCETES OF THE TROPICS
Since publication of the first book on tropical fungi by Chang and Quimio (1982) that was devoted to the
genera Volvariella, Auricularia and Pleurotus, there have been a few other efforts to disseminate
knowledge acquired on this important subject. These include the works of Quimio et al. (1990), Quimio
(2002) and more recently, Thakur and Thakur (2015), the latter referring to the edible fungi of India.
However, given the magnitude of the potential that tropical fungi represent, these efforts are scarce and
there is much work to be done.
As mentioned in the introductory chapter of this book, the tropics contain a great diversity of climates that
embrace different biological ecosystems. There are 17 megadiverse countries on Earth, of which 15 are
precisely located (in their entirety or in a significant part), within the tropical zones (Biodiversity 2014).
This includes regions of southern China, north-central Brazil, south-central Mexico, Colombia, most of
Africa, India, etc. Although macromycetes are not mentioned specifically, it is very likely that diversity of
these organisms in these countries and in general in tropical zones is very high and, likewise, poorly
known. Except in China, where great strides have been made to study macromycetes, efforts in other
tropical countries for development of mycology in general, and in particular for the study of
macromycetes, have been and continue to be very limited. This is reflected in the scarce knowledge and
use for humankind of tropical mycobiota.
In this book, we review cultivation techniques for some tropical species such as Agaricus subrufescens,
Sparassis latifolia, Tremella fuciformis, Schizophyllum commune, Lepista nuda, and some promising
biotechnological applications of Auricularia spp., Grifola frondosa, Pleurotus spp., and Volvariella spp.
Likewise, the existence of tropical species of Agaricus and Lentinula is highlighted. Undoubtedly, this list
is only a small sample of the great diversity of macromycetes present in the tropics. It should be noted that
there are several species that are not mentioned in this book and that are already commercially grown,
such as Dictyophora indusiata, Trametes versicolor, etc. This is a reflection of the prevailing reality about
the scarce knowledge that tropical macromycetes have. A very important effort should be made
immediately to train more mycologists in these regions that are able to study and take advantage of these
organisms.
Tropical mushrooms are just beginning to be known; however, they are already seriously threatened. In
addition to the high rates of deforestation that are prevalent in tropical countries, climate change puts the
biological diversity of the planet at high risk, especially the tropical fungi. According to the data of IUCN
(2018), of the 11,783 species considered as vulnerable, only 57 belong to the Kingdom Fungi, which
shows the lack of studies on this, especially in the tropical zones. Climate change is a serious threat to the
stability of terrestrial environments. The increase in temperature threatens to modify the landscape and the
environment, a situation that is exacerbated for organisms that depend on the microclimate that is
generated by the vegetation of the area, as is the case of fungi. The human influence on climate change is
indisputable and potentially irreversible. Today, climate change affects the health of populations around
the world. All communities will be affected, although these effects will disproportionately impact those
most vulnerable (Watts et al. 2017). Climate change will affect not only the tropical zones, but through the
increase of temperature, temperate zones will also be affected. This represents both a threat and an
opportunity for the fungal diversity of the world. It will be necessary to carry out further studies and work
on obtaining and selecting new varieties of fungi capable of resisting higher temperatures as well as those
better adapted to use of different substrates.
215
For this reason, study of tropical macromycetes is urgently needed, because they are vulnerable organisms
and because knowledge allows us to take actions and decisions that lead to their best assessment and
conservation.
Currently, most commercially grown mushrooms are sold fresh. This is very good from the culinary and
nutritional point of view. However, much remains to be explored in terms of new uses and post-harvest
management of fungi, for a better exploitation of their qualities. Surely, for nutraceutical and medicinal
uses, alternative preparations may be more appropriate and will have to be developed, such as capsules,
extracts, powders, etc. If we desire to live in a sustainable society, it is essential to review development of
natural products that are friendly to the environment. In that sense, tropical mushrooms and the products
that can be obtained from them surely will be very useful to modern production systems and will
contribute elements to a society that becomes more aware every day of their impact in the world.
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z.org/content/megadiverse-countries revised on 11/03/2017.
Chang ST, Quimio TH (1982) Tropical mushrooms. Biological Nature and cultivation methods. The Chinese
University Press. ISBN 962-201- 264-7. 493p.
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01/06/2018.
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ISBN 92-5-103026-X. 155p.
Quimio TH (2002) Tropical mushroom cultivation. National Book Store. ISBN-971-08-6201-4. 163p.
Thakur T, Thakur MP (2015) Production techniques of tropical mushrooms. Nirmal Publication (India) 140p.
Watts N et al. (2017) The Lancet countdown on health and climate change: from 25 years of inaction to a global
transformation for public health. http://www.thelancet.com/pdfs/journals/lancet/PIIS0140-6736(17)32464-9.pdf
revised on 11/01/2017.
216
APPENDIX
APPENDIX 1. LIST OF MUSHROOMS MENTIONED IN THIS BOOK
Agaricus L.
A. abruptibulbus Peck
A. arvensis Schaeff.
A. augustus Fr.
A. benesii (Pilát) Pilát
A. benzodorus Heinem. & Gooss.-Font.
A. bingensis Heinem.
A. bisporus (J.E. Lange) Imbach
A. bisporus var. burnettii Kerrigan & Callac
A. bisporus var. bisporus (J.E. Lange) Imbach
A. bitorquis (Quél.) Sacc.
A. blazei (Murrill) ss. Heinemann
A. brasiliensis Wasser, M. Diduck, Amazonas & Stamets
A. campestris L.
A. comtulus Fr.
A. dennisii Heinem.
A. deserticola G. Moreno, Esqueda & Lizárraga
A. endoxanthus Berk. & Broome
A. erectosquamosus Linda J. Chen, K.D. Hyde & R.L. Zhao
A. essettei Bon A. sylvicola (Vittad) Peck.
A. fissuratus F.H. Møller Agaricus arvensis Schaeff.
A. flocculosipes R.L. Zhao, Desjardin, GGuinb. & K.D. Hyde
A. fuscofibrillosus (F.H. Møller) Pilát
A. gemellatus Kerrigan, L.A. Parra, Cappelli & Weholt
A. heterocystis Heinem. & Gooss.-Font.
A. impudicus (Rea) Pilát
A. inapertus Vellinga Endoptychum depressum Singer & A.H. Sm.
A. laeticulus Callac, L.A. Parra, Linda J. Chen & Raspé (≡ A. laeticolor Heinem. & Gooss.-Font.)
219
A. leucocarpus Linda J. Chen, Callac, R.L. Zhao & K.D. Hyde

A. magnivelaris Peck Tricholoma magnivelare (Peck) Redhead
A. martineziensis Heinem.
A. martinicensis Pegler
A. moelleri Wasser
A. niveogranulatus Linda J. Chen, R.L. Zhao, Callac & K.D. Hyde
A. phaeolepidotus F.H. Møller
A. purpurellus F.H. Møller
A. rufoaurantiacus Heinem
A. semotus Fr.
A. singeri Heinem
A. subtilipes Thongklang, Linda J. Chen, Callac & K.D. Hyde
A. subrufescens Peck
A. subsaharianus L.A. Parra, Hama & De Kesel
A. suthepensis Linda J. Chen, K.D. Hyde & R.L. Zhao
A. sylvaticus Schaeff.
A. sylvicola (Vittad.) Peck
A. trinitatensis R.E.D. Baker & W.T. Dale
A. trisulphuratus Berk.
A. variicystis Linda J. Chen, K.D. Hyde & R.L. Zhao
A. xanthodermus Genev.
A. xantholepis F.H. Møller
A. yucatanensis Ellis & Everth.
A. sect. Brunneopicti Heinem.
A. subg. Minoriopsis Linda J. Chen, L.A. Parra, Callac, Angelini & Raspé
Agrocybe Fayod
A. cylindraceae (DC.) Maire Cyclocybe cylindraceae (DC.) Vizzini & Angelini
Amanita Pers.
Amanita phalloides (Vaill. Ex Fr.) Link
Auricularia Bull.
220
A. auricula-judae (Bull.) Quél.

≡Auricularia auricula (L.) Underw.
A. delicata (Mont. Ex Fr.) Henn.
A. fuscosuccinea (Mont.) Henn.
A. mesenterica (Dicks.) Pers.
A. nigricans Sw. Birkebak, Looney & Sánchez-García (≡A. polytricha (Mont.) Sacc.)
Bolbitius Fr.
Cantharellus cibarius Fr.
Clitocybe maxima (P. Gaertn., G. Mey., & Scherb.) P. Kumm. Clitocybe gibba (Pers.) P. Kumm.
Conocybe Fayod
Cookeina sulcipes (Berk.) Kuntze
Coprinus Pers.
Cortinarius (Pers.) Gray
Cordyceps Fr.
Cordyceps sinensis (Berk.) Sacc. Ophiocordyceps sinensis (Berk.) G.H. Sung,
J.M. Sung, Hywel-Jones & Spatafora
Cortinarius speciosissimus Kühner & Romagn. Cortinarius rubellus Cooke
Flammulina P. Karst.
Flammulina velutipes (Curtis) Singer
Galerina Earle
Ganoderma P. Karst.
G. applanatum (Pers.) Pat.
G. lucidum (Curtis) P. Karst.
Grifola Gray
G. frondosa (Dicks.) Gray
G. gargal Singer
Hericium Pers.
Hydnopolyporus D.A. Reid
Hypocrea Fr.
Hypsizigus marmoreus (Peck.) H.E. Bigelow
221
Inocybe (Fr.) Fr.

Inonotus P. Karst.
Lactarius Pers.
Lentinula Earle
Lentinula boryana (Berk. & Mont.) Pegler
Lentinula edodes(Berk.) Pegler
Lentinus Fr.
Lepiota (Pers.) Gray
Lepista nuda (Bull.) Cooke
Morchella esculenta (L.) Pers.
Phallus indusiatus Vent.
Phanerochaete carnosa (Burt) Parmasto
Phellinus Quél.
Pholiota nameko (T. Itô) S. Ito & S. Imai
Pleurotus (Fr.) P. Kumm.
P. abalonus Y.H. Han, K.M. Chen & S. Cheng P. cystidiosus O.K. Mill.
P. citrinopileatus Singer
P. cystidiosus O.K. Mill.
P. djamor (Rumph. ex Fr.) Boedijn
P. eryngii (DC.) Quél.
P. nebrodensis (Inzenga) Quél.
P. ostreatus (Jacq.) P. Kumm
P. tuber-regium (Fr.) Singer
Polyporus P. Micheli ex Adans.
Polyporus brumalis (Pers.) Fr. Lentinus brumalis (Pers.) Zmitr.
Poria cocos F.A. Wolf Wolfiporia cocos (F.A. Wolf) Ryvarden & Gilb.
Psilocybe semilanceata (Fr.) P. Kumm.
Russula Pers.
Schizophyllum Fr.
Sparassis latifolia Y.C. Dai & Zheng Wang
222
Sparassis crispa (Wulfen) Fr.

Schizophyllum commune Fr.
Trametes Fr. (Coriolus Quél.),
T. versicolor (L.) Lloyd
Tremella Pers.
T. fuciformis Berk.
Trichoderma Pers.
T. harzianum Rifai
Volvariella Speg.
V. bombycina (Schaeff.) Singer
V. bombycina var. bombycina (Schaeff.) Singer
V. bombicina var. flaviceps (Murrill) Shaffer
Volvariella volvacea (Bull.) Singer
V. bakeri (Murrill) Shaffer
Wolfiporia cocos (F.A. Wolf) Ryvarden & Gilb.
223
224
Appendix 2 – Tropical species of Agaricus. Philippe Callac and Jie Chen.

List of 185 species of Agaricus newly described since 2000 and their taxonomic placement in the 24 sections of the current system of
classification. Sections are ordered like in Table 1. Considered as a doubtful species, Agaricus dilutibrunneus R.L. Zhao is excluded
from the list.
Agaricus sect. Flocculenti Linda J. Chen, K.D. Hyde & R.L. Zhao Agaricus sect. Xanthodermatei Singer
Agaricus erectosquamosus Linda J. Chen, K.D. Hyde & R.L. Zhao Agaricus arizonicus Kerrigan
Agaricus pallidobrunneus R.L. Zhao Agaricus atrodiscus Linda J. Chen, Callac, R.L. Zhao & K.D. Hyde
Agaricus sect. Brunneopicti Heinem. Agaricus berryessae Kerrigan
Agaricus brunneosquamulosus Linda J. Chen, R.L. Zhao, K.D. Hyde & Callac Agaricus bisporiticus Nawaz, Callac, Thongklang & Khalid
Agaricus chiangmaiensis Karunarathna, Guinb. & K.D. Hyde Agaricus brunneogracilis R.L. Zhao & K.D. Hyde
Agaricus megacystidiatus Karunarathna, Guinb. & K.D. Hyde Agaricus buckmacadooi Kerrigan
Agaricus niveogranulatus Linda J. Chen, R.L. Zhao, Callac & K.D. Hyde Agaricus candussoi L.A. Parra, Angelini & Callac
Agaricus pakistanicus H. Bashir, Khalid, L.A. Parra & Callac Agaricus daliensis H.Y. Su & R.L. Zhao
Agaricus sordidocarpus Linda J. Chen, Callac & K.D. Hyde Agaricus deardorffensis Kerrigan
Agaricus sparsisquamosus H. Bashir, S. Hussain, Khalid & H. Ahmed Agaricus exilissimus Linda J. Chen, Callac, R.L. Zhao & K.D. Hyde
Agaricus subsaharianus L.A. Parra, Hama & De Kesel Agaricus flavidodiscus L.A. Parra, Angelini & Callac
Agaricus toluenolens Callac, Linda J. Chen & K.D. Hyde Agaricus freirei Blanco-Dios
Agaricus sect. Trisulphurati Heinem. Agaricus fuscopunctatus Thongklang, Linda J. Chen, Callac & K.D. Hyde
Agaricus ignicolor R.L. Zhao Agaricus gregariomyces J.L. Zhou & R.L. Zhao
Agaricus sect. Crassispori R.L. Zhao Agaricus karstomyces R.L. Zhao
Agaricus lamellidistans R.L. Zhao Agaricus kriegeri Kerrigan
Agaricus variicystis Linda J. Chen, K.D. Hyde & R.L. Zhao Agaricus laskibarii L.A. Parra & Arrillaga
Agaricus sect. Cymbiformes M.Q. He & R.L. Zhao Agaricus leptocaulis Kerrigan
Agaricus angusticystidiatus M.Q. He, Desjardin, K.D. Hyde & R.L. Zhao Agaricus malangelus Kerrigan
Agaricus sect. Rubricosi R.L. Zhao Agaricus melanocarpus R.L. Zhao
Agaricus dolichopus R.L. Zhao Agaricus moelleroides Guinb. & L.A. Parra
Agaricus kunmingensis R.L. Zhao Agaricus murinocephalus R.L. Zhao, Desjardin & K.D. Hyde
Agaricus variabilicolor R.L. Zhao Agaricus parvitigrinus Guinb. & Callac
Agaricus sect. Bivelares (Kauffman) L.A. Parra Agaricus punjabensis T. Qasim, A. Ashraf & Khalid
Agaricus agrinferus Kerrigan & Callac Agaricus sinoplacomyces Callac & R.L. Zhao
Agaricus cupressophilus Kerrigan Agaricus tephrolepidus L.A. Parra, C. Billette, Angelini, G. Mata & Callac
Agaricus qilianensis S.L. Wei, M.Z. Zhang & R.L. Zhao Agaricus tibetensis J.L. Zhou & R.L. Zhao
Agaricus sinodeliciosus Z.R. Wang & R.L. Zhao Agaricus tollocanensis Callac & G. Mata
Agaricus sinotetrasporus Y.L. Xi, M.Z. Zhang & R.L. Zhao Agaricus tytthocarpus R.L. Zhao
Agaricus sipapuensis Kerrigan Agaricus xanthodermulus Callac & Guinb
Agaricus subsubensis Kerrigan Agaricus sect. Chitonioides Romagn.
Agaricus taeniatus Sai F. Li, Shao J. Li & H.A. Wen Agaricus sect. Hondenses R.L. Zhao & L.A. Parra
Agaricus tlaxcalensis Callac & G.Mata Agaricus biannulatus Mua, L.A.Parra, Cappelli & Callac
225
Agaricus grandiomyces J.L. Zhou & R.L. Zhao Agaricus planipileus R.L. Zhao
Agaricus pusillobulbosus S.Y. Su & R.L. Zhao Agaricus sect. Subrutilescentes Kerrigan
Agaricus sect. Sanguinolenti Jul. Schäff. & F.H. Møller ex L.A. Parra Agaricus brunneopileatus Callac & R.L. Zhao
Agaricus iranicus Mahdizadeh, Safaie, M. Goltapeh, L.A. Parra & Callac Agaricus catenariocystidiosus R.C. Dai & R.L. Zhao
Agaricus cordillerensis Kerrigan Agaricus inthanonensis Linda J. Chen, K.D. Hyde & R.L. Zhao
Agaricus hupohanae Kerrigan Agaricus linzhiensis R.L. Zhao
Agaricus thujae Kerrigan Agaricus parasubrutilescens Callac & R.L. Zhao
Agaricus sect. Bohusia (L.A. Parra) L.A. Parra & R.L. Zhao Agaricus thiersii Kerrigan & Vellinga
Agaricus brunneofibrillosus Kerrigan Agaricus vinosobrunneofumidus Kerrigan
Agaricus coniferarum Guinb. & Callac Agaricus sect. Arvenses (Konrad & Maub.) Konrad & Maub.
Agaricus crassisquamosus R.L. Zhao Agaricus cruciquercorum Kerrigan
Agaricus lusitanicus Callac, L.A. Parra & A. Tancrède Agaricus didymus Kerrigan
Agaricus sect. Nigrobrunnescens K.R. Peterson, Desjardin & Hemmes Agaricus diospyros B. Ortiz, Kerrigan & Skulan
Agaricus erythrosarx T. Lebel Agaricus eburneocanus T. Lebel
Agaricus caballeroi L.A.Parra, G.Muñoz & Callac Agaricus flocculosipes R.L. Zhao, Desjardin, Guinb. & K.D. Hyde
Agaricus collegarum L.A. Parra, Wisman, Guinb., Weholt, Musumeci & Geml Agaricus gemellatus Kerrigan, L.A. Parra, Cappelli & Weholt
Agaricus desjardinii Z.R. Wang, K.D. Hyde & R.L. Zhao Agaricus guizhouensis Y. Gui, Zuo Y. Liu & K.D. Hyde
Agaricus laparrae Kerrigan Agaricus indistinctus L.A. Parra & Kerrigan
Agaricus masoalensis L.A. Parra, Wilhem & Callac Agaricus julius Kerrigan
Agaricus padanus Lancon. Agaricus longistipes Y. Gui, Zuo Y. Liu, Callac, L.A. Parra & K.D. Hyde
Agaricus sect. Agaricus L. Agaricus megalocarpus Y. Gui, Zuo Y. Liu, Callac, L.A. Parra& K.D. Hyde
Agaricus erindalensis Kerrigan Agaricus mesocarpus Kerrigan
Agaricus flavicentrus Karunarathna & K.D. Hyde Agaricus moronii Kerrigan
Agaricus gastronevadensis Kerrigan Agaricus nanaugustus Kerrigan
Agaricus griseocephalus Kerrigan Agaricus ornatipes A. Mua, M. Casula & M. Sanna
Agaricus incultorum Kerrigan Agaricus reducibulbus Kerrigan
Agaricus inilleasper T. Lebel Agaricus sandianus Kerrigan
Agaricus sect. Amoeni Callac & R.L. Zhao Agaricus subantarcticus Geml, Laursen & D.Lee Taylor
Agaricus amoenomyces R.L. Zhao Agaricus subtilipes Thongklang, Linda J. Chen, Callac & K.D. Hyde
Agaricus gratolens Pradeep & R.L. Zhao Agaricus sect. Minoriopsis Linda J. Chen, L.A. Parra, Callac, Angelini & Raspé
Agaricus suthepensis Linda J. Chen, K.D. Hyde & R.L. Zhao Agaricus argenteopurpureus L.A. Parra, Angelini & Callac
Agaricus sect. Rarolentes Kerrigan Agaricus sect. Kerrigania L.A. Parra, Angelini, B. Ortiz, Linda J. Chen &
Agaricus albosquamosus Linda J. Chen, K.D. Hyde & R.L. Zhao Callac
Agaricus butyreburneus Kerrigan, Guinb. & Callac Agaricus globocystidiatus Drewinski & M.A. Neves
Agaricus hanthanaensis Karunarathna & K.D. Hyde Agaricus porphyropos L.A. Parra, Angelini & B. Ortiz
Agaricus leucolepidotus Linda J. Chen & R.L. Zhao Agaricus sect. Leucocarpi Linda J. Chen & Callac
Agaricus sect. Spissicaules (Heinem.) Kerrigan Agaricus leucocarpus Linda J. Chen, Callac, R.L. Zhao & K.D. Hyde
Agaricus bellanniae Guinb., Kerrigan & M. Kuo Agaricus sect. Pantropicales L.A. Parra, Angelini, B. Ortiz, Linda J. Chen &
Agaricus lanipedisimilis Callac & R.L. Zhao Callac
Agaricus litoraloides R.L. Zhao Agaricus lodgeae L.A. Parra, Angelini & B. Ortiz
226
Agaricus sect. Minores (Fr.) Henn. Agaricus mangaoensis M.Q. He & R.L. Zhao.
Agaricus armandomyces M.Q. He & R.L. Zhao Agaricus marisae L.A. Parra & Callac
Agaricus arrillagarum L.A. Parra, S. Serrano & Geml Agaricus matrum L.A. Parra, A. Caball., S. Serrano, E. Fern. & Callac
Agaricus badioniveus Linda J. Chen, R.L. Zhao & K.D. Hyde Agaricus megalosporus Linda J. Chen, R.L. Zhao, Karun. & K.D. Hyde
Agaricus blatteus M.Q. He & R.L. Zhao Agaricus microviolaceus M.Q. He & R.L. Zhao
Agaricus bonisquamulosus M.Q. He & R.L. Zhao Agaricus minipurpureus M.Q. He & R.L. Zhao
Agaricus brunneolutosus Linda J. Chen, Karunarathna & K.D. Hyde Agaricus neimengguensis M.Q. He & R.L. Zhao
Agaricus callacii L.A. Parra, Iglesias, Fern. Vincente & Oyarzabal Agaricus parvibicolor Linda J. Chen, R.L. Zhao & K.D. Hyde
Agaricus campbellensis Geml, Laursen & D.Lee Taylor Agaricus parvibrunneus M.Q. He, K.D. Hyde & R.L. Zhao
Agaricus catenatus M.Q. He & R.L. Zhao Agaricus patris Linda J. Chen, Callac, K.D. Hyde & R.L. Zhao
Agaricus cerinipileus M.Q. He & R.L. Zhao Agaricus pietatis L.A. Parra & A. Caball.
Agaricus chartaceus T. Lebel Agaricus pseudominipurpureus M.Q. He, K.D. Hyde & R.L. Zhao
Agaricus coccyginus M.Q. He & R.L. Zhao Agaricus pseudopallens M.Q. He & R.L. Zhao
Agaricus colpetei T. Lebel Agaricus pseudopurpurellus M.Q. He & R.L. Zhao
Agaricus dilatostipes M.Q. He & R.L. Zhao Agaricus purpureofibrillosus Linda J. Chen, R.L. Zhao & K.D. Hyde
Agaricus edmondoi L.A. Parra, Cappelli & Callac Agaricus purpureosquameus M.Q. He & R.L. Zhao
Agaricus elongatestipes M.Q. He & R.L. Zhao Agaricus robustulus Linda J. Chen, Callac, L.A. Parra, K.D. Hyde & De Kesel
Agaricus fimbrimarginatus Linda J. Chen, Callac & K.D. Hyde Agaricus rufifibrillosus M.Q. He & R.L. Zhao
Agaricus flammicolor Linda J. Chen, Callac, R.L. Zhao & K.D. Hyde Agaricus rufipileus M.Q. He & R.L. Zhao
Agaricus flavopileatus Linda J. Chen, Karunarathna & Callac Agaricus sodalis Linda J. Chen, R.L. Zhao & K.D. Hyde
Agaricus friesianus L.A. Parra, Olariaga & Callac Agaricus stevensii Kerrigan
Agaricus fulvoaurantiacus Linda J. Chen & Karunarathna Agaricus yanzhiensis M.Q. He, K.D. Hyde & R.L. Zhao
Agaricus gemlii L.A. Parra, Arrillaga, M.Á. Ribes & Callac Unclassified species (insertae sedis and species lacking sequence data)
Agaricus gemloides M.Q. He & R.L. Zhao Agaricus evertens Kerrigan
Agaricus globosporus M.Q. He & R.L. Zhao Agaricus haematinus K.D. Hyde & R.L. Zhao
Agaricus greuteri L.A. Parra, Cappelli & Kerrigan Agaricus heinemannii Albertó & G. Moreno
Agaricus jacobi L.A. Parra, A. Caball. & Callac Agaricus nigrogracilis R.L. Zhao
Agaricus jingningensis M.Q. He & R.L. Zhao Agaricus pachydermus T. Lebel
Agaricus kerriganii L.A. Parra, B. Rodr., A. Caball., M. Martín-Calvo & Agaricus patialensis M. Kaur & Harw. Kaur
Callac Agaricus pseudolangei K.D. Hyde & R.L. Zhao
Agaricus lamelliperditus T. Lebel & M.D. Barrett Agaricus stijvei de Meijer
Agaricus luteofibrillosus M.Q. He, Linda J. Chen & R.L. Zhao Agaricus tennesseensis Kerrigan
Agaricus luteopallidus Linda J. Chen, Karunarathna, R.L. Zhao & K.D. Hyde
227
Updates on Tropical Mushrooms. Basic and Applied Research was printed on November 30, 2018
by Servicios Profesionales en Impresión (Seprim) at Siembra 1, S-5. San Simón Culhuacán,
Iztapalapa, Mexico City. Front page was designed by Fabiola Roque. Print run was 300 copies.

Updates Ontropical Mushrooms 2018

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Updates Ontropical Mushrooms 2018

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Updates on Tropical Mushrooms.

Basic and Applied

José E. Sánchez, Gerardo Mata and Daniel J. Royse

1. Tropical mushrooms, 2. Agaricus, 3. Edible mushrooms, 4. Medicinal mushrooms, 5.

1st. Edition, 2018

The content of this book was subjected to

DR © El Colegio de la Frontera Sur

Printed and made in Mexico

O. P. Ahlawat ICAR-Directorate of Mushroom Research Solan, India

Acosta-Urdapilleta Ma. de Lourdes. Laboratorio de Micología, Centro de Investigaciones

Biodiversity and Taxonomy

1. IMPORTANCE AND POTENTIAL OF TROPICAL MUSHROOMS

Keywords: edible mushrooms, macromycetes, mycology, ethnomycology, biodiversity.

CLIMATE AND CLIMATE SYSTEMS

ECOLOGICAL CONDITIONS AND CLIMATE

ETHNOMYCOLOGY - MUSHROOMS AND HUMANKIND

2. TROPICAL SPECIES OF AGARICUS

Philippe Callac 1 and Jie Chen2

Keywords: classification, climate, fungal diversity, subgenus, systematics, taxonomy

THE GENUS AGARICUS

WHAT DOES IT MEAN TROPICAL SPECIES OF AGARICUS?

A BRIEF HISTORY OF THE CLASSIFICATION SINCE 2000

WHY WAS A REVISED CLASSIFICATION REQUIRED?

Table 1. Distribution of tropical species in the current revised system of classification *.

Figure 1. a. Agaricus niveogranulatus, LD2012148 (A. sect. Brunneopicti), b. Agaricus suthepensis,

DISTRIBUTION OF TROPICAL TAXA IN THE REVISED SYSTEM OF CLASSIFICATION

subg. Agaricus, A. subg. Flavoagaricus, A. sect. Nigrobrunnescentes, A. sect. Spissicaules, A. sect.

HOW TO IDENTIFY TROPICAL AGARICUS?

3. AN OVERVIEW OF TROPICAL AND SUBTROPICAL SPECIES OF Agaricus IN

ETHNOMYCOLOGICAL IMPORTANCE IN MEXICO

Figure 1. Sale of mushrooms in the market of San José in Xalapa, Veracru z.

THE GENUS AGARICUS IN MEXICO

Table 1. Agaricus species recorded from tropical areas in Mexico.*

Figure 4. Records of tropical species by state.

PERSPECTIVES OF STUDIES IN TROPICAL AND SUBTROPICAL AREAS OF MEXICO

opportunities for both the agricultural and pharmaceutical industries.

Mexico and a case study at Ozu mba. Econ. Bot. 62 (3):425-436.

4. DEVELOPMENT OF THE STRAW MUSHROOM SECTOR IN CHINA

Qi Tan1, Mingjie Chen1, Meilian Yu1 and Huanqing He2

HISTORY OF STRAW MUSHROOM CULTIVATION

ISSUES IN DEVELOPMENT OF THE STRAW MUSHROOM INDUSTRY

Biological conversion efficiency

Poor storage durability

PROGRESS IN STRAW MUSHROOM RESEARCH

Using modern molecular biology to improve breeding efficiency

Improving product quality and freshness-keeping technology of the straw mushroom

DEVELOPMENT PROSPECT OF INDUSTRIALIZED CULTIVATION OF STRAW

5 ENHANCED PRODUCTION OF THE MEDICINAL MUSHROOM

Diego Cunha Zied1 and Arturo Pardo-Giménez2

Keywords: Agaricus blazei, medicinal mushrooms, production, compost, casing

Wisitrassameewong K, Karunarathna SC, Thongkland N, Zhao R, Callac P, Moukha S, Férandon C, Chukeatirote E,

6. ARTIFICIAL CULTIVATION OF THE MEDICINAL MUSHROOM Sparassis latifolia

Sparassis latifolia (also known as hanabiratake in Japanese or cauliflower mushroom in English) is an

Figure 1. Fruit body of Sparassis latifolia

Figure 2. Industrial cultivation of S. latifolia in China

GENOMICS AND PROTEOMICS OF S. latifolia

The Molecular mechanism of fruiting development

Figure 6. Cluster analysis of predicted amino acid sequences of latifolysin.

Figure 7. Predicted tertiary structures of hemolysin from four mushrooms.

Figure 8. Phylogenetic analysis of some aegerolysins in fungi (Vibrio as outgroup).

Figure 9. Differential expression of latifolysin gene at various developmental stages.

FACTORS AFFECTING S. latifolia GROWTH