CAUSAL AGENT, BIOLOGY AND MANAGEMENT OF THE LEAF AND STEM DISEASE OF BOXWOOD {BUXUS SPP.) A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by FANG SHI In partial fulfillment of requirements for the degree of Master of Science May, 2011 ©Fang Shi, 2011 Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington OttawaONK1A0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-82801-4 Our file Notre reference ISBN: 978-0-494-82801-4 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. 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While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. 1+1 Canada ABSTRACT CAUSAL AGENT, BIOLOGY AND MANAGEMENT OF THE LEAF AND STEM DISEASE OF BOXWOOD (BUXUS SPR) Fang Shi Advisor: University of Guelph, 2011 Professor T. Hsiang An outbreak of a boxwood disease was observed in Southern Ontario nurseries in 2008, but appears to have been present in Ontario for at least 10 years. From 2008 to 2010, over fungal 312 isolates were obtained from diseased samples. Eight major fungal morphotypes associated with the blight were identified with morphological and molecular techniques. Among them, only Volutella buxi successfully satisfied Koch's postulates, which confirmed it is the causal agent of boxwood blight. Wounds may be the major penetration points for V. buxi since non-wounded inoculated tissues did not become diseased. The Buxus cultivar 'Green Gem' was the most susceptible compared to 'Green Velvet', 'Green Mound', or 'Green Mountain'. ISSR markers revealed low genetic variation of V. buxi between different cultivars and locations indicating asexual reproduction and probably a recent origin in Canada. Six fungicidal treatments showed strong preventive activity and some curative activity against Volutella blight. ACKNOWLEDGEMENTS I really appreciate my advisor Dr. Tom Hsiang providing me this opportunity to obtain an M.Sc degree. With his knowledge, patient and encourage, I have learned how to do research in science. I would like to thank to my advisory committee member Dr. Allen Xue for his advice. I would like to give my thanks to Dr. George Barron for his suggestions during the study and kindly offering his plant disease specimens. I would also like to thank to Dr. Janice Elmhirst for sending me plant samples from B.C. and sharing her experience and knowledge. I would like to thank to everyone in Dr. Hsiang's lab for their help with my research and sharing their professional and life experiences. Special thanks to Dr. Lynn Tian, Linda He and Karla De la Cerda for their technical support and helping me get through hard times. I would like to thank to Jennifer Llewellyn and everyone in the nursery industries. Without them, I would not be able to be involved in such an interesting and challenging project. I would like to give my thanks to my family members and friends. Without their spiritual support, I would not be able to accomplish what I have done, many thanks for their endless support and unconditional love. i TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii LIST OF TABLES vi LIST OF FIGURES viii LIST OF APPENDICES x LIST OF ABBREVIATIONS AND ACRONYMS xi Chapter One: Literature Review 1 1.1 Introduction 1 1.2 Major Boxwood Diseases Caused by Fungi 3 1.2.1 Fungi associated with boxwood 3 1.2.2 Volutella leaf and stem blight 5 1.2.2.1 Taxonomy 5 1.2.2.2 Morphology 6 1.2.2.3 Disease history and description 9 1.2.3 Macrophoma leaf spot 11 1.2.4 Phytophthora root rot 11 1.2.5 Cylindrocladium leaf spot 12 1.3 Control Methods for Boxwood Diseases 13 1.3.1 Cultural methods 13 1.3.2 Chemical methods 15 1.4 Hypotheses and Objectives 15 Chapter Two: Causal Agent of A Disease of Boxwood 20 2.1 Introduction 20 2.1.1 Morphological methods of identification 20 2.1.2 Molecular methods of identification 21 2.1.3 Boxwood disease causal agents 22 2.1.4 Objectives 23 2.2 Materials and Methods 23 2.2.1 Sample collection 23 2.2.2 Media preparation 24 2.2.3 Fungal isolation from diseased boxwood tissues 24 2.2.4 Identification of fungal isolates with morphological technique 25 2.2.5 DNA extraction 26 2.2.6 Primer design and selection 27 2.2.7 PCR amplification 28 2.2.8 DNA concentration 28 2.2.9 DNA sequencing and result analysis 29 2.2.10 Koch's postulates 30 2.3 Results 31 2.3.1 Disease samples 31 2.3.2 Morphological characteristics of fungal isolates 32 2.3.3 Molecular identification of morphotypes 33 2.3.4 Volutella species comparison against the database on GenBank 34 2.3.5 Testing Koch's postulates 35 2.4 Discussion 36 Chapter Three: Biological Characteristics, Pathogenicity and Genetic Variation in Volutella buxi 48 3.1 Introduction 48 3.1.1 Biological characteristics and disease development of Volutella spp. 48 3.1.2 Boxwood cultivars 49 3.1.3 Genetic diversity of Volutella buxi 51 3.1.4 Objectives 52 3.2 Materials and Methods 53 3.2.1 Boxwood plants and fungal isolates 53 3.2.2 Growth rates of Volutella buxi isolates 53 3.2.3 Pathogenicity and resistance 54 3.2.3.1 The infection process 54 3.2.3.2 Resistance 56 3.2.4 Primer screening and PCR amplification for ISSR " 57 3.2.5 DNA extraction and ISSR data analysis 59 3.2.6 Statistical analysis 60 3.3 Results 60 3.3.1 Sample collection and fungal isolates 60 3.3.2 Temperature effects on Volutella buxi 60 3.3.3 Pathogenicity and resistance 62 3.3.4 Disease development during inoculation progress 62 3.3.5 Genetic variation in Volutella buxi 62 3.4 Discussion 63 Chapter Four: Effects of Fungicides on Volutella Blight Development 80 4.1 Introduction 80 4.1.1 Fungicides used for ornamentals in Canada 81 4.1.2 Objectives 85 4.2 Materials and Methods 85 4.2.1 Boxwood plants and fungal isolates 85 4.2.2 Fungicide selection 86 4.2.3 Fungicide sensitivity on amended agar 86 4.2.4 Fungicide test on whole plants in 25 °C room 88 4.2.5 Statistical analysis 89 4.3 Results 90 4.3.1 Boxwood samples and fungal isolates of Volutella buxi 90 4.3.2 EC50 values and threshold concentrations of fungicides 90 4.3.3 The efficacy of fungicides on boxwood plants in 25 °C room 91 4.4 Discussion 91 Chapter Five: General Discussion 102 REFERENCES 110 APPENDICES 120 V LIST OF TABLES Table 2.1 Number and frequency of morphotypes recorded based on 312 fungal isolates associated with Volutella leaf and stem blight of boxwood. Species from the eight major morphotypes were identified with morphological and molecular biological techniques. 40 Table 2.2 A list of 41 accession numbers and species names of ITS sequences collected from GenBank by searching for 'Volutella', and then used to in phylogenetic analysis. 41 Table 3.1 Boxwood plants records with collection date, location, and number of each cultivar as 3-inch potted plants or cuttings. 69 Table 3.2 Number of Volutella buxi isolates collected from Georgetown, St. Catherines and B.C. from boxwood cultivars 'Chicagoland Green', 'Green Beauty', 'Green Mound', 'Green Mountain' and 'Green Velvet'. 70 Table 3.3 Mean mycelial growth of three Volutella buxi isolates at 10, 15, 20, 25, 30 and 35 °C. A 5-mm-diameter plug from a 7-d-old culture was used to inoculate each tube and the tubes were incubated for 16 d. Each isolate by temperature combination was repeated three times. 71 Table 3.4 Mean mycelial growth during 16 d at 25 °C of 32 Volutella buxi isolates collected from Georgetown and St. Catherines. There were five replicates for each isolate. 72 Table 3.5 Detached leaves of each boxwood cultivar were inoculated with a 0.14 ml of spore suspension of Volutella buxi (106 spores/mL) and incubated at 25 °C for 5 d.