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Studies on the Molecular Identification, Biological Characteristics and Indoor Cultivation of Morchella Spp

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Studies on the Molecular Identification, Biological Characteristics and Indoor Cultivation of Morchella Spp The Pennsylvania State University The Graduate School Department of Plant Pathology and Environmental Microbiology STUDIES ON THE MOLECULAR IDENTIFICATION, BIOLOGICAL CHARACTERISTICS AND INDOOR CULTIVATION OF MORCHELLA SPP. A Thesis in Plant Pathology by Siyi Ge © 2019 Siyi Ge Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science August 2019 The thesis of Siyi Ge was reviewed and approved* by the following: John A. Pecchia Associate Professor of Plant Pathology and Environmental Microbiology Thesis Advisor David M. Beyer Professor of Plant Pathology and Environmental Microbiology Gretchen A. Kuldau Associate Professor of Plant Pathology and Environmental Microbiology Michael A. Fidanza Professor of Plant and Soil Sciences Carolee T. Bull Professor of Plant Pathology and Environmental Microbiology Head of the Department of Plant Pathology and Environmental Microbiology *Signatures are on file in the Graduate School iii ABSTRACT True morel (Morchella) is a genus of edible fungi in the order Pezizales, Ascomycota, well known for its desirable flavor. However, most morels are collected in the wild and the short fruiting season makes it one of the most prized edible fungi in the world. Recently, morels have been cultivated in China to meet the growing demand of edible morels. The purpose of my thesis is to study the biological characteristics of Morchella and provide information to improve on the success of future Morchella cultivation. The Morchella cultural collection in the Spawn Lab at the Pennsylvania State University is reidentified. The isolates in the collection were identified decades ago based on morphology and older species concepts. As a result, many species seem to be misidentified. Here, the Morchella isolates were reidentified based on molecular phylogenetic analysis of the nuc rDNA internal transcribed spacer (ITS1- 5.8D-ITS4) sequences. Based on the cultivation history, geographic origin and characteristic recorded fruiting patterns, four Morchella isolates (WC 833 M. rufobrunnea, China 1 M. importuna, WC 374 M. americana and WC 766 M. exuberans) were selected to study the growth characteristics and used for indoor cultivation cropping trials. Mycelial growth is the first stage in the life cycle of Morchella, and Morchella fruit bodies develop from pseudosclerotia, which are masses of mycelia acting as a resting stage. Satisfactory mycelial growth and pseodosclerotia production are both thought to be required for successful morel cultivation. The effects of pH, light and temperature on mycelial growth and pseudosclerotia formation were described for four selected Morchella isolates. PH and light had a significant effect on mycelial growth for three of the Morchella isolates, with no affect observed for M. exuberans. Light significantly affected the pseudosclerotia formation for M. rubobrunnea and M. importuna, while pH had no effect. With the exception of M. exuberans, which obtained the most mycelial growth at 30C, mycelial growth and pseudosclerotia formation were maximized at 20C. M. americana and M. exuberans did not produce any pseudosclerotium under any of the tested conditions. Conditions resulting in both the most mycelial growth and pseudosclerotia production in vitro were pH6, darkness (0 lux), and 20 to 25C for M. rufobrunnea, pH5 to 6, darkness (0 lux), and 20 to 25C for M. iv importuna. The condition preference for mycelial growth of M. americana was found to be pH5 to 7, darkness (0 lux) to low light intensity (400 lux), and 20C. Mycelia of M. exuberans achieved the fastest mycelial growth in media with a pH value of 5, 25 to 30C, and light intensity did not influence its mycelial growth. In addition, two indoor morel cultivation experiments were conducted in the Mushroom Research Center (MRC). The results indicated that the outdoor morel cultivation techniques, currently being followed in China, are feasible for indoor cultivation, even though many questions remain. Mature fruit bodies of M. rufobrunnea were successfully obtained from two substrates: soil + straw and soil + sawdust in experiment 1 but not in experiment 2, even though many primordia were formed in experiment 2. M. rufobrunnea and M. importuna appear to be the most promising candidates of morel cultivation based on the results from both biological growth studies and the indoor cultivation experiments. These studies can contribute knowledge to the field of Morchella cultivation by providing information on environmental factors that contribute to desirable mycelial growth and pseudosclerotia formation, which are both thought to be required for successful morel cultivation. These results will certainly contribute to our understanding of the required growing parameters needed for successful Morchella indoor cultivation. Key Words: Morchella, morel cultivation, environmental factors, identification v TABLE OF CONTENTS LIST OF FIGURES ................................................................................................................. VIII LIST OF TABLES ....................................................................................................................... X ACKNOWLEDGEMENT ........................................................................................................ XII CHAPTER 1 LITERATURE REVIEW .................................................................................... 1 Economic importance of morels ............................................................................................................ 1 Taxonomy of Morchella .......................................................................................................................... 1 Global distribution pattern of Morchella ............................................................................................. 3 Trophic mode of Morchella .................................................................................................................... 4 Life cycle of Morchella............................................................................................................................ 5 Life cycle .............................................................................................................................................. 5 Pseudosclerotia of Morchella ............................................................................................................... 8 Factors that affect pseudosclerotia production ................................................................................... 10 Mitospore ........................................................................................................................................... 13 Etiology of Morchella ............................................................................................................................ 14 Carbon source ..................................................................................................................................... 14 Nitrogen sources ................................................................................................................................. 15 Mineral salts ....................................................................................................................................... 15 Temperature ....................................................................................................................................... 16 Humidity ............................................................................................................................................ 16 pH ....................................................................................................................................................... 17 Light ................................................................................................................................................... 17 Oxygen and carbon dioxide ................................................................................................................ 17 Morel cultivation ................................................................................................................................... 18 History and progress of morel cultivation .......................................................................................... 18 Morel cultivation in China ................................................................................................................. 21 Issues and perspectives ......................................................................................................................... 27 Spawn quality ..................................................................................................................................... 27 Reduction of soil nutrients ................................................................................................................. 28 Discussion .......................................................................................................................................... 28 CHAPTER 2 IDENTIFICATION OF MORCHELLA CULTURAL COLLECTION ....... 32 Abstract ................................................................................................................................................. 32 Introduction .......................................................................................................................................... 32 Material and Methods .........................................................................................................................
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