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Daphne Sudden Death Syndrome (DSDS) : pathogen identification, characterization and screening for disease… Noshad, David 2007

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Daphne Sudden Death Syndrome (DSDS): Pathogen Identification, Characterization and Screening For Disease Resistance  by  David Noshad B.Sc. (Hons), University of Tabriz (Azerbaijan)  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in  THE FACULTY OF GRADUATE STUDIES (Plant Science)  THE UNIVERSITY OF BRITISH COLUMBIA December 2007  © David Noshad  ABSTRACT  Daphne is a widely dispersed genus with large variation in morphology, native habitats,  and use. Unfortunately, broader acceptance of Daphne in the ornamental trade has been limited due to Daphne Sudden Death Syndrome (DSDS), a disease that kills the plant without warning. The results of this research identified Thielaviopsis basicola (Berk. et Br.) Ferr. as the causal agent for this disease. Pure cultures of the pathogen were developed and used in a germplasm screen.  To evaluate Daphne germplasm in vitro, species-specific protocols were developed that alleviated two common problems in Daphne micropropagation, browning and hyperhydricity. Optimizing the concentrations of both PGRs and charcoal was able to control these problems. Selected species were evaluated for resistance against Thielavipsis basicola in both, in vivo and in vitro, conditions. The results of both  methods displayed a strong correlation and indicated significant differences among the taxa. However, there were differences in disease progression rates. Typically, the in vitro challenge produced a comparable level of disease as the in vivo challenge but in two to three weeks less time. Across both screening methods, the most resistant species evaluated were D. tangutica and D. retusa, while D. cneroum was the most susceptible.  Based on ITS sequences, phylogenetic relationships among selected Daphne species were established and associated with their resistance against T basicola. The phylogeny indicated that Daphne is possibly a monophyletic group. However, placement of D.  ii  genkwa remained problematic. The analysis of ITS sequences data resulted in a parsimony consensus tree with two well-supported major clades and one Glade with less support. In general, the evolutionary tree for resistance, inferred from the phylogenetic data and the results of the screening project, indicate that resistance is a derived character and that plants recently evolved this ability.  iii  ^  TABLE OF CONTENTS  ABSTRACT ^  ii  TABLE OF CONTENTS ^  iv  LIST OF TABLES ^  vii  LIST OF FIGURES^  ix  ABBREVIATIONS ^ PREFACE  ^xi  ACKNOWLEDGEMENT ^  xii  DEDICATION ^  xiii  CO-AUTHORSHIP STATEMENT ^  xiv  CHAPTER ONE: INTRODUCTION ^ I.I  DAPHNE IN THE PLANT KINGDOM ^  I  .1.1 The Family Thymelaeaceae ^  1  .1.2 The Genus Daphne L ^ 1.2 PLANT BREEDING AND CROP IMPROVEMENT ^ ^1 .2. ^Overview of plant improvement strategies ^  1 .2.2^Breeding of Ornamental Plants ^ 1 .23^Breeding for Important Traits in Horticultural Crops ^ 1.3 PLANT FUNGAL DISEASES ^  ^1 .3.1^Disease Development ^ .3.2 Thielaviopsis basieola; a Fungal Root Pathogen ^ 1.4 DAPHNE DISEASES ^  ^1 .4.1^Overview ^ 1 .4.2 Daphne Sudden Death Syndrome ^  3 4 5 8 10 13 14 17 21 21 21  1.5 OBJECTIVES ^  22  1.6 REFERENCES ^  24  CHAPTER TWO: FIRST REPORT OF THIELAVIOPSIS BASICOLA ON DAPHNE L.^33 2.1 INTRODUCTION ^ 2.2 MATERIAL AND METHODS ^  38  2.2.1^Fungi isolation, inoculation and evaluation procedures ^ 2.2.2 Daphne production ^  40  2.3 RESULTS ^  42  2.4 REFERENCES ^  44  CHAPTER THREE: IN VITRO PROPAGATION OF SEVEN DAPHNE L. SPECIES ^ 51 3.1 INTRODUCTION ^  51  3.2 MATERIALS AND METHODS ^ 3.2.1^Plant material and growing conditions ^  53 53  3.2.2^Establishment phase ^  53  3.2.3^Multiplication phase ^  54  3.2.4^Elongation and rooting phases ^ 3.2.5^Experimental design and analyses ^  56  3.3 RESULTS ^  55 56  3.3.1^Establishment phase ^  56  iv  3.3.2^Multiplication phase ^ 3.3.3^Rooting and acclimation ^ 3.4 DISCUSSION ^ 3.7 REFERENCES ^  57 58 59 61  CHAPTER FOUR: EVALUATION OF DAPHNE GERMPLASM FOR RESISTANCE TO DAPHNE SUDDEN DEATH SYNDROME (DSDS) CAUSED BY THE SOIL-BORNE PATHOGEN 76 THIELAVIOPSIS BASICOLA ^ 4.1 INTRODUCTION  ^  4.2 MATERIALS AND METHODS ^  4.2.1^Plant material ^ 4.2.2 Pathogen culture and suspension preparation ^ 4.2.3^In vivo challenge^ 4.2.4^In vitro challenge ^ 4.2.5^Disease assessment ^ 4.2.6 Data analyses ^ 4.3 RESULTS ^ 4.3.1^In vivo screen ^ 4.3.2^In vitro screen^ 4.3.3 Assay comparison ^ 4.4 DISCUSSION ^  4.5 REFERENCES ^  76 81 81 82  82 83 83 84 85 85  85 86 86 90  CHAPTER FIVE: COMPARATIVE EVOLUTIONARY ANALYSIS OF RDNA ITS SEQUENCES OF SELECTED DAPHNE SPECIES WITI I REFERENCE TO RESISTANCE TO 102 THIELAVIOPSIS BASICOLA ^ -  5.1 INTRODUCTION  ^  5.2 MATERIAL AND METHODS^  5.2.1^Plant materials ^ 5.2.2 DNA extraction and sequence data ^ 5.2.3 Phylogenetic reconstruction ^ 5.3 RESULTS ^ 5.3.1 DNA matrix features and sequence divergence ^ 5.3.2 Phylogenetic results ^ 5.4 DISCUSSION ^ 5.5 REFERENCES ^ CHAPTER SIX: CONCLUSIONS ^ 6.1 CoNcLusioNs AND FUTURE DIRECTIONS 6.2 REFERENCES ^  ^  102 106 I06 107 107 108 108 108 109 112 121 121 125  APPENDIX I: PRELIMINARY EXPERIMENTS ON INOCULATION PROTOCOL DEVELPMENT ^ 126 INTRODUCTION ^ MATERIAL AND METHODS ^  RESULTS ^  126 126 128  APPENDIX 2: EXPERIMENTS ON PGR STABILITY FOLLOWING AUTOCLAVING ^ INTRODUCTION ^ MATERIALS AND METHODS ^  RESULTS ^ APPENDIX 3:  129 129 129 129  PRELIMINARY EXPERIMENTS ON IN VITRO PROPAGATION MEDIA COMPOSITION ^ 131 1 NTRODUCIION  131 131 131 131 1333  ^  MATERIAL AND METHODS ^ I. Plant material ^ IL General procedures ^  RESULTS ^ REFERENCES ^  1344  vi  LIST OF TABLES Table 3.1. Explant survivorship (%) among five Daphne species following a 4 wk establishment phase on one of five basal media without PGR supplements; mean ± SD  ^33  Table 3.2. Shoot explant multiplication rates following 8 weeks of culture on basal media supplemented with a single cytokinin and no auxin; mean ± SD ^  34  Table 3.3. Shoot explant multiplication rates following 8 weeks of culture on basal media supplemented with a combination of cytokinins and auxins; mean ± SD ^  35  Table 3.4. Daphne rooting frequency (%) after 8 weeks of culture on media incorporating PGRs  36  (traditional method) ; mean ± SD. ^  Table 3.5. Daphne species rooting frequency (%) after 8 weeks of culture on 2 layered media incorporating auxins in one of the basal medium layers (no PGRs in the other basal medium layer); mean ± SD. ^  .........37  Table 3.6. Rooting frequency after 8 weeks of culture on basal medium following treatment with auxin solution dip treatment; mean ± SD  ^38  Table 4.1. Daphne taxa used in the Thielaviopsis basicola resistance bioassay with region of nativity or origin and mean plant disease index (PDI) values eight weeks following inoculation ^  59  vii  Table 5.1. Daphne taxa used in the Thielaviopsis basicola resistance bioassay with region of nativity or origin and mean plant disease index (PDI) values ^  .78  Table 5.2. ITS sequence characteristics of the selected species ^  ..79  viii  LIST OF FIGURES  Figure 2.1. Infection of D. cneorum by Thielavopsis basicola  Figure 3.1. Multiplication stage of Daphne species  Figure 3.2. Root formation from Daphne microshoots  . 16  ^ 39  ^ .40  Figure 4.1. Bioassay containers used for Daphne taxa challenged with Thielaviopsis basicola ^  61  Figure 4.2. Comparison of the in vitro with in vivo assays using seven Daphne taxa and Thielaviopsis basicola  .62  Figure 5.1. The consensus parsimony tree which is a strict consensus of the most parsimonious trees ^  80  Figure 5.2. The evolutionary tree of resistance for the selected Daphne species ^81  ix  Abbreviations  BA: 6- benzyl aminopurine Dl: disease incidence DS: disease severity dsRNA: double stranded RNA DSDS: Daphne Sudden Death Syndrome IAA: indole-3 acetic acid IBA: indole 3- butyric acid NAA: a-naphthalene acetic acid MS: Murashige and Skoog medium PDI: plant disease index PGR: plant growth regulator TDZ: thidiazuron WPM: woody plant medium  PREFACE  `To see a world in a grain of sand And a heaven in a wild flower, Hold infinity in the palm of your hand And eternity in an hour.' William Blake  xi  ACKNOWLEDGEMENT  Many people contributed significantly to this project. My research supervisor and four committee members guided this study from its beginning to its final completion, and my gratitude for their valuable input and support is profound. I take this opportunity to thank Andrew Riseman for his great help. Without his help, this study could not have been accomplished. I also thank Zamir Punja, Murray lsman, Brian Ellis and Quentin Cronk for providing critical feedback and guidance on my project.  Many thanks to Vippen Jushi and her colleagues, at the BC Ministry of Agriculture, for helping in the pathogen identification; Douglas Justice, Ron Rollo, and their colleagues at the UBC Botanical Garden, for helping me with the plant material.  Finally, my deepest appreciation goes to my family and friends. Special thanks to Eva Buschmann, Saber Miresmaili, Peter and Tanya Kalyniak, Isidro Ojeda, Nyssa Temmel, Sarah Martz, Yasmin Akhtar, Trish Osterberg and Eduardo Jorel for their help and support.  xii  DEDICATION  Tor my mother, the symhof of lave, and  -  myfither; w/io wantearta see tllic di zy Girt conaizt.. -  CO-AUTHORSHIP STATEMENT  Modified versions of Chapters 2 and 4 are manuscripts accepted for publication (Chapter 2 is published; Chapter 4 is in press). I designed the project in consultation with Andrew Riseman and Zamir Punja, and I conducted all laboratory work, data analyses and wrote the manuscripts. A. Riseman provided insights and contributed to the writing.  Chapter 3 is a draft manuscript submitted for publication. I designed the project in consultation with Andrew Riseman, and I conducted all laboratory work, data analyses and wrote the manuscript. A. Riseman provided insights and contributed to the writing.  Chapter 5 is a draft manuscript that will be submitted for publication. I designed the project in consultation with Andrew Riseman and Quentin Cronk, and I conducted all laboratory work, data analyses and wrote the manuscript. A. Riseman provided insights and contributed to the writing.  xiv  CHAPTER ONE  INTRODUCTION  1.1 Daphne in the Plant Kingdom  1.1.1 The Family Thymelaeaceae Daphne L. is a genus within the family Thymelaeaceae Juss. (syns: Aquilariaceae,  Daphnaceae, Gonystylaceae, Phaleriaceae, Tepuianthaceae). The family is comprised of 44 genera, approximately 500 species (Watson and Dallwitz, 2007), and has a cosmopolitan distribution with concentrations in tropical Africa, Southeast Asia and Australasia (Watson and Dallwitz, 2007). The plant forms found in this family are mostly trees and shrubs, with a few vines and herbaceous types known. The family is within subclass Dicotyledonae and order Malvales. A few genera within Thymelaeaceae have economic importance as cultivated ornamental shrubs (e.g., Daphne, Dais L., Dirca L. (leatherwood), Pimelea Banks ex Gaertn. (rice flower) Wikstroemia L.), and for paper production (e.g., Thymelaea Mill.) (Watson, 2007; Flora of China, 2005; Soltis and Soltis, 2000). It has also been reported that many species in the family have bioactive compounds e.g. Daphne genkwa (Zhou, 1991) and have been used as medicinal plants in Chinese and other Asian cultures. Studies on the chemical profiles of these plants have identified several groups of active compounds including Ilavonoids, lignans and diterpenes (Zhou, 1991).  The phylogenetic relationships among the genera in Thymelaeaceae have been under extensive discussion recently with various authors proposing a relationship between this family and Euphorbiales, Malvales, and/or Gutiferales (Crawford, 1995; Van der Bank and Fay, 2002). More recently, Thymelaeaceae was investigated by parsimony analysis of 41 rbcL (a chloroplast gene) nucleotide sequences, including 27 genera and several outgroup  taxa. These results supported the Malvalean placement (Alverson and Baum, 1998). In a second study, Soltis and Soltis (2000) used plastid trnL intron sequence analysis that produced highly congruent results to the first rbcL results. Therefore, Malvalean placement of this family has been confirmed by both rbcL (Alverson and Baum, 1998) and trnL (Alverson and Baum, 1998; Bayer and Fay, 1999; Soltis and Soltis, 2000) sequence  data. However, within Malvales, the position of Thymelaeaceae remains unresolved because of some characteristic traits of these plants including: plant forms that are mostly shrubs, lianas or herbs (rarely), and non-succulent; leaves that are small to medium-sized, alternate, opposite, or whorled; internal phloem that is present in nearly all genera with secondary thickening developing mostly from a conventional cambial ring. Using both classical and modern data, the accepted systematic placement of Thymelaeaceae is as follows (Watson and Dallwitz, 2007; Flora of China, 2005):  Kingdom: Plantae Division: Magnoliophyta Class: Magnoliopsida Order: Malvales Family: Thymelaeaceae  2  1.1.2 The Genus Daphne L. The genus Daphne is comprised of 95 species distributed primarily in Europe and Asia, with a few species also endemic to Africa and Australia (Flora of China, 2005). Botanically, the petals, which in many familiar flowers form an inner whorl, have all but disappeared in the genus. However, in a few species, the petals are present and represented by a small disk inside the base of the calyx-tube. Within this tube are eight stamens arranged in two whorls of four at different levels. The ovary, which sits at the base, has a short (often absent) style and a fairly obvious (capitate) stigma (Halda, 2001; Mathew et al, 2000).  Based in part on its wide distribution, Daphne contains significant variation in morphology, native habitats, and use. Important uses include pharmaceutical, ethnobotanical, and ornamental applications (Zhou, 1991; Brickell and White, 2000a; Mathew, 2000). In horticulture, several species have been commercially introduced because of their many desirable characteristics including attractive foliage, plant habit, flower color and most of all, pleasant fragrance. Propagation of Daphne in the horticulture trade can be from seed or cuttings. However, seeds are genetically variable adding undesirable variation to a production (or evaluation) system. Vegetative propagation is more desirable, but traditional cuttings are typically very difficult to root (Brickell and White, 2000b; Matthew, 2000; Chen etal, 1995; Dixon, 1994). For example, D. cneorum (Rose daphne, or garland flower), a very fragrant species with good commercial potential, is one of the most popular species in this genus. It forms a low  3  shrub that, during April and May, bears clusters of fragrant rose-pink flowers (Brickell and White, 2000b). However, it seldom produces seed in cultivation and has to be increased by division, grafting, or cuttings. Unfortunately, rooting percentages experienced by commercial propagators are often between 5% - 20% (Riseman, personal communication). However, as with other crops, the development of in vitro propagation protocols can allow for the conservation and production of uniform, disease-free plants (Cohen, 1977; Li and White, 1998; Marks and Simpson, 2000; Sediva, 2002).  1.2 Plant Breeding and Crop Improvement  Prior to the 20th century, plant breeding was largely an art with little or no knowledge of genetic principles (Debener, 1999). However, since the rediscovery of Menders seminal work and the application of genetic principles, plant breeding has made enormous gains over the last century. Plant breeders have made significant progress in developing high yielding cultivars, cultivars that can be grown in less favorable soils/environments, and cultivars that are resistant to a variety of pests and diseases. The development of cultivars with disease resistance is one of the major goals for plant breeders due to its direct (e.g., greater yields) and indirect (e.g., reduced pesticide use) importance (Stuber etal, 1999; Fehr, 1987). In general, disease resistance can be thought of as either narrow (i.e., vertical, qualitative), where one or a few major genes condition resistance, or broad (i.e., horizontal, quantitative), where many genes, each with a small contribution, affect resistance (Kozumplinki etal, 2004; Agrios, 2005). Each form has its own advantages and disadvantages. For example, identification and use of vertical resistance is thought of as being easier and more straightforward to manipulate than horizontal resistance.  4  However, vertical resistance is often specific to only one or a few races of a particular disease while horizontal resistance can be more broadly effective. One basic but critical disadvantage of vertical resistance compared to horizontal resistance is its often short effective lifespan due to pathogen evolution.  1.2.1 Overview of plant improvement strategies In general, plant improvement is defined as the process of identifying and selecting plants with desirable traits and using them for the production of more plants with favorable traits (Simmonds and Smart, 1999). The production of these plants can be by sexual or asexual systems. Depending on which system is used, various plant improvement strategies are available.  1.2.1.1 Sexual Reproduction and Plant Breeding Sexual reproduction is the primary system by which crops are improved allowing for the recombination of favorable characters into superior individuals. It involves knowledge of the reproductive biology of the crop of interest as well as knowledge of the techniques and procedures required to efficiently identify and recombine the traits of interest. Over the next section, I will review the basic biology of sexual reproduction and how plant breeders work within this context to develop superior germplasm.  Sexual reproduction occurs in the flowers with flower form having significant implications for the plant breeder. Flowers have four major parts: calyx (sepals), corolla (petals), androecium (stamens) and gynaecium (carpel) (Neil, 2006). A flower having all  5  of these organs is called complete while a flower lacking one or more parts is incomplete. The stamen and carpel are the essential parts of a flower as these organs produce the male and female gametes. A flower that has both of these organs is called perfect while a flower that lacks one of these organs is refered to as imperfect. By definition, perfect flowers are bisexual while imperfect flowers are unisexual (i.e., either pistillate and staminate). Species that bear imperfect flowers on the same plant are called monoecious. However if the staminate and pistillate flowers occur on different individuals, the species is known as dioecious. The mode of reproduction and the ease with which it can be modified by plant breeders largely depends upon these differences, especially with respect to the presence and location of the stamens and pistils (Neal et al, 2006; Neil, 2006).  The morphological features of flowers influence both natural and human directed pollination (i.e., the process of transferring pollen grains from the anther to the stigma). When flower form and development favor pollination within a single flower, the crop is referred to as self-pollinated or an inbreeder. On the other hand, when flower form and development inhibit pollination within a flower and instead, promote inter-flower pollination, the crop is referred to as cross- pollinated or an outcrosser. The impacts of mode of reproduction to the plant breeder are many and are very influential factors in plant breeding and crop improvement practices (Niel, 2006; Neal et al, 2006). However, two of the most important impacts include its effect on the genetic structure of the breeding population and the associated ability to tolerate inbreeding. First, populations of a naturally self-pollinated crop will increase homozygosity across generations while a  6  cross-pollinated crop will naturally increase the level of heterozygosity across generations. Second, a self-pollinated crop is normally tolerant of inbreeding while a cross-pollinated crop is typically intolerant of inbreeding often suffering from inbreeding depression after a few generations of self-pollination. In addition to these impacts, the mode of reproduction can be exploited by the plant breeder to help them better manage and manipulate the genetics of the population. For example, a self-pollinated crop breeder can easily increase homozygosity and inbred line development by simply allowing pollination to occur naturally. Following an initial cross-pollination generation (i.e., labor intensive step), 6-8 generations of natural self-pollination will produce a functional level of homozygosity and sufficient uniformity for commercialization.  1.2.1.2 Asexual/Clonal Reproduction  Although sexual reproduction is the primary way plant breeders manipulate genetic variation and create new cultivars, it is not the only way. Asexual or clonal reproduction is a valuable tool that both allows for mass production of individual genotypes but also as an important source of genetic variation. In many crops where sexual reproduction and/or the level of variation is problematic for cultivar development (e.g., low fecundity, inbreeding depression, low genetic diversity), clonal reproduction can be of value. Specifically, clonal reproduction can allow plant breeders to exploit naturally occurring somatic mutations (e.g., sports), somatic mutations induced by chemical or radiation treatments, or somaclonal variation induced via tissue culture technologies. Even for crops that form adequate quantities of seed for commercial production (e.g., diploid seed  7  geraniums), asexual propagation may be of value for specific applications (e.g., bacterial/viral indexing) or germplasm types (e.g., tetraploid geraniums)  1.2.2^Breeding of Ornamental Plants 1.2.2.1 Overview Many cultures around the world use flowers and ornamental plants to decorate and celebrate major events, symbols of beauty and respect, and objects of art. When this practice began, most ornamental plants were wild collected and used directly as they occurred in nature. As time progressed to the Victorian era, gardening and ornamental plants reached its pinnacle in terms of social status. Great effort was extended to collect the most unusual and unique specimens. At the same time, gardeners started to generate non-naturally occurring forms for use in this budding industry. Works of art from this period provide us with a rich accounting of the numerous flowers and cultivars common at that time, most of which were either natural mutants clonally propagated or the result of 'happenstance' breeding. The vast majority of plants grown at this time were ones that could be easily propagated either by seed (e.g., annuals) or by asexual propagation (e.g., cutting, divisions, bulblets, etc). To help support this growing interest in gardening, commercial horticulture firms formed and became a very powerful industry. For example the Dutch bulb industry, which started its work by introducing tulip bulbs imported from today's Turkey, is currently providing 93% of the world's bulbs with over nine billion bulbs sold per year (Perry, personal communications,). Over time, many seed and vegetativly propagated cultivars have been preserved as important sources of germplasm and are now known as heirloom or heritage cultivars. In the modern  8  ornamental plant breeding industry, tools involving molecular biology are commonly used for the identification and selection of improved cultivars (Harrera, 1992). In addition, modern ornamental plant breeders often rely on modern greenhouses and nurseries to help maintain environmental conditions at optimum levels thereby allowing for maximum growth potential to be observed among breeding selections.  1.2.2.2 Daphne Breeding and Improvement During the last 25-30 years, there has been a surge of activity among horticulturists to produce new and superior hybrids through classical breeding methods. However, sexual hybridization within and among species of Daphne has been either unsuccessful (i.e., no fruit), or the hybrids produced were of low fertility or sterile (Brickell and White, 2000b). Because of this difficulty in reliably producing seeds/hybrid progeny and the need for increased uniformity during production, Daphne plants are primarily propagated for commercial (or research) use via asexual propagation. Unfortunately, Daphne cuttings are typically very difficult to root (Brickell and White, 2000a; Matthew etal, 2000; Chen etal, 1995; Dixon, 1994). Typical rooting percentages of the most common daphne in the horticulture trade, D. cneorum, are below 30% and are limited to a three month window (i.e., May-July) during the year. In addition, Daphne stock plants have been observed to decline over time yielding fewer and fewer cuttings. Therefore, the development of micropropagation protocols is seen as a way to address these limitations by allowing for the year-round production of genetically uniform, disease-free plants for use (Cohen, 1977; Li and White, 1998; Marks and Simpson, 2000; Sediva, 2002).  9  1.2.3^Breeding for Important Traits in Horticultural Crops 1.2.3.1 Overview When horticulture firms first started to breed ornamental crops, the most important characters identified included fragrance, plant form and morphology, and leaf and flower colour, all associated with the aesthetics of the plant. For many years, these remained the primary traits of interest. For example, initial rose breeding efforts were directed at increasing the range of colors, plant forms, and flower size (Debener, 1999). However, more recently, breeding for resistance to environmental stressors, both biotic and abiotic has become a priority for many plant breeding programs (Agrios, 2005; Ashraf and Harris, 2005). Specifically, traits of modern interest include fungal/bacterial disease resistance, insect resistance, and drought and cold tolerance; all non-aesthetic traits but important for sustainable production. Breeding disease resistant cultivars has received more attention in recent years and has been greatly helped by advances in genetics and other related sciences. With the heightened awareness of the environment and sustainability, the incorporation of these traits is vital to the future of this industry and the success of many crops (Agrios, 2005; Neil, 2006).  1.2.3.2 Breeding for resistance to abiotic and biotic stressors Plant stress experienced by plants can be divided into two groups: abiotic and biotic. Abiotic stressors are derived from interactions between the plant and its physical environment while biotic stressors involve the interaction between two organisms, the plant and the pest, (Yeo and Flowers, 1989; Ashraf and Harris, 2005). Abiotic stressors include the harmful effects of salinity, drought, metal toxicity, nutrient deficiency and/or  10  temperature extremes. For example, salinity stress occurs when there is an excessive amount of soluble salts in the soil that negatively affect the physiology of plant. It can be quantified as electrical conductivity (EC), exchangeable sodium percentage (ESP), or sodium adsorption ratio (SAR) (Yeo, 1989; Ashraf and Harris, 2005). In addition, abiotic stressors can include air pollution, wind, shade, or any shortage in an essential resource that affects plant growth e.g., gaseous or light sources. In some cases, such as the supply of water, either too little (drought) or too much (flooding) can cause stress on the plant (Ashraf and Harris, 2005). In addition, abiotic and biotic stressors can interact magnifying their individual effects. For example, plants suffering from nutrient deficiency are generally more susceptible to pathogens (Ashraf and Harris, 2005).  1.2.3.2.1 Breeding for resistance to abiotic stressors Plant resistance to abiotic stressors in nature evolved through stress avoidance or stress tolerance mechanisms. Avoidance enables the plant to avoid exposure to the stressor by exclusion or minimizing the damage caused. Avoidance mechanisms are diverse and depend on the type of stress. They can take place in the whole plant, an individual organ, or at the cellular level. Stress tolerance refers to the ability of the plant to maintain normal function in the presence of the stressor or the damage caused by a stressor (Ashraf and Harris, 2005). In the recent years, many cultivars of plants resistant to individual stressors have been developed through traditional plant breeding, asexual selection, or other more technological methods e.g., recombinant DNA (Yeo, 1989; Ashraf and Harris, 2005).  11  1.2.3.2.2 Breeding for resistance to biotic stressors Plants are constantly being challenged by pathogens and a wide range of control strategies (e.g., chemical control, environmental manipulation) have been developed to control them. Although these methods are somewhat efficient in controlling the negative impacts of these pathogens in the short term, they do not lead to long-term sustainability. For example, reliance on fungicides/pesticides is expensive, environmentally damaging, and potentially hazardous for human health. On the other hand, developing cultivars with host-plant resistance to these pests can provide a long-lasting and sustainable solution. While the incorporation of genetic resistance into plants is a lengthy and expensive process, it is deemed relatively inexpensive compared to the costs associated with not pursuing this goal (Neal et al, 2006).  Many authors have suggested that the differences between horizental and vertical resistance are artificial and question if horizontal resistance truly exists (Agrios, 2005). Future research will examine this issue and will hopefully elucidate the situation more clearly. In the meantime, discussions on this topic as related to the mechanisms of disease resistance and the genetic control of resistance continue (Agrios, 2005; Neal et al, 2006; Johnson, 1978).  Development of pathogen resistant cultivars requires a thorough understanding of the evolutionary interrelationships between plants and their pathogens. The co-evolution of host and pathogen can not be understood without knowledge relating to the pathogen including its pathogenecity (i.e. the ability the pathogen to display virulence and induce  12  disease on a given host). The efficiency of developing resistant cultivars depends on this understanding and the exploitation of the interactions between host and pathogen (Neal etal, 2006; Agrios, 2005). In contrast to breeding for yield or other horticultural traits, the development of cultivars with pathogen resistance is more complex because it involves the interaction between two dynamic organisms (Neal etal, 2006; Agrios, 2005). The process of developing these resistant types typically involves a type of germplasm screen that allows for the identification of genotypes possessing resistance against a specific pathogen.  1.3 Plant Fungal Diseases  The majority of the 100,000 described fungi, are saprophytic and not pathogens (Agrios, 2005). However, approximately 10% are considered plant pathogens and may cause disease on many plant species (Agrios, 2005). Fungal pathogens, based on their life style, are categorized into two major groups: obligate and non-obligate. Obligate fungi only grow and complete their life-cycle on living hosts while non-obligate pathogens need a living host for only part of their life cycle, completing the remainder of their life cycle on dead material. The capacity of pathogen to incite disease among members of a host species is called 'pathogenicity' and when it is able to induce symptoms of disease, the pathogen is termed 'virulent'. The term `avirulent', often used to describe lack of pathogenicity of a certain isolate, suggests an internal failure of a pathogen to be able to induce disease on the host (Agrios, 2005; Johnson, 1978).  13  1.3.1 Disease Development 1.3.1.1 Process of infection and disease progression Three basic factors are involved in the development of disease; the pathogen, the host plant, and the environment surrounding these two interacting organisms. If any one of these factors is lacking or not promotive of disease (e.g., proper conditions for disease development), disease cannot develop. Every disease needs a specific range of environmental conditions (e.g., favorable temperature, proper humidity) to develop (Agrios, 2005). In addition to this three-way interaction, the genetic interaction between host plant and pathogen plays a crucial role in disease development. The concept of `parallel evolution' for the compatibility between the host and pathogen genomes, first introduced in the mid 1950's (Flor, 1955), is generally accepted by most plant pathologists today (Agrios, 2005). In support of this concept, it has been shown that there is a balance between pathogen virulence and host plant resistance (Agrios, 2005). The concept of 'gene-for-gene' (Flor, 1955) describes the interaction between a pathogen and host where a balance exists between a pathogen's gene required for virulence and a host's gene responsible for resistance. Based on this theory, for every virulent gene in a pathogen's genome, there is a corresponding resistance gene in the host (Flor, 1955; Agrios, 2005). Generally, these genes are active during different stages of disease development (Agrios, 2005; Johnson, 1978; Flor, 1955).  The process of disease development involves many individual events based on the interaction between pathogen and host plant. The major events in this process are:  14  inoculation, penetration, infection, invasion (colonization), growth, multiplication, and spread of the pathogen (Agrios, 2005). Inoculation, the initial contact between plant and pathogen, may involve two steps i.e. attachment and germination (Osherov and May, 2001; Agrios, 2005). However, for some pathogens, additional steps are present and include specific attachment processes (adhesion/ bond) and/or thigmotropism (surface sensing). Initial adhesion of the spore to the plant cuticle is commonly based on hydrophobic interactions. Once securely attached, the spore germinates and produces a germ tube for penetration of the plant's cuticle. Usually, spore germination is affected by the amount of nutrients (e.g., sugars, potassium, calcium and amino acids) exuded or cutin monomers from the plant (Osherov and May, 2001; Agrios, 2005). Pathogens most often penetrate the plant surface through natural openings such as stomata or through wounds. In addition, some pathogens (e.g., rust fungi, Uromyces sp.) can sense appropriate points of entry on the plant (Osherov and May, 2001; Agrios, 2005). Other pathogens do not use existing openings but instead, penetrate plant surfaces through direct action. These pathogens can penetrate through a plant surface by using either physical (e.g., appressorium) or enzymatic (i.e., cell wall degrading enzymes including cellulase, pectinase and cutinase) forces. However, successful penetration does not always lead to infection. Successful infection relies on the pathogen's contact with susceptible cells and tissues of the host. During infection, fungal pathogens enter plant cells in order to derive nutrition. As nutrients are made available, the pathogen grows and multiplies within these tissues leading to more widespread invasion of the plant (Osherov and May, 2001; Agrios, 2005).  15  One of the fundamental problems associated with controlling fungal pathogens is their ability to easily infect neighboring plants. Fungi use a range of vectors to gain access to new hosts including water (e.g., overhead irrigation), air flow (e.g., horizontal air flow fans), insects, and/or humans (e.g., contaminated secateurs) (Agrios, 2005). In addition, infection can occur on different plant parts/organs with each pathogen specialized for a specific host. For example, some root pathogens (e.g., Fusarium spp., Phytophtora spp. Thielaviopsis basicola) penetrate the plant surface through lateral roots and degrade root tissues using specific enzymes (Agrios, 2005; Rothrock, 1992; Hood and Shew, 1997a).  1.3.1.2 Plant Defense Systems Most plants are resistant to most of the pathogens found in their environment although each plant may be attacked by many pathogens every day (Osherov and May, 2001; Agrios, 2005). While these plants will suffer to some extent from these pathogens, many survive and live for long periods (Agrios, 2005). The failUre of most pathogens to infect plants other than their host indicates that plants can generally defend themselves against pathogens (Agrios, 2005). In these plants, defense against pathogen attack can be based on either physical or biochemical barriers. The physical or structural defense system works as a barrier against pathogen intrusion while the biochemical system can generate compounds toxic to the pathogen or able to prevent its growth. These systems can he either pre-existing or induced, with different plants using various combinations based on their interactions with their pathogen (Agrios, 2005). The first line of defense for many plants is their pre-existing structural defense system that may include waxes, a thick cuticle layer, and/or cell walls. Plants may have a pre-existing biochemical defense  16  system, such as the use of phenolic compounds, which can inhibit pathogen growth and multiplication (Agrios, 2005). While these primary defense systems are essential, in many cases of disease, pathogens can easily overcome these pre-existing barriers to reach the internal tissues. To augment the pre-existing defense systems, many plants are able to induce a secondary defense system after detection of a pathogen. These systems are typically more targeted and intense defense strategies against the pathogen. Some induced defense systems are structural and include cytoplasmic defense reaction, cell wall defense reactions, and hypersensitive defense reactions (Agrios, 2005). In cytoplasmic defense reactions, the cytoplasm is modified into a granular and dense matrix thereby inhibiting fungal growth. Cell wall defense reactions may include a thickening of the cell wall in the areas around sites of penetration (Agrios, 2005). This reaction can lead to the death of invaded cells i.e. a necrotic or hypersensitive defense reaction. In addition, specific biochemicals (e.g., phytoalexins; very toxic antimicrobial compounds) can be produced following infection as part of an induced defense system. In all induced defense mechanisms, early recognition of the pathogen by the host plays a vital role in successful defense.  1.3.2 Thielaviopsis basicola; a Fungal Root Pathogen  1.3.2.1 Overview  Some plant pathogens are specialized for life in the soil and have special abilities to infect plants via their root systems (Hood and Shew, 1997a; Agrios, 2005). Typically, symptoms induced by these pathogens first appear on the roots as water soaked regions  17  that later discolor and become necrotic. Root pathogens are mostly non-obligate parasites that can live and grow in the soil as without a host using organic material (e.g., dead plant tissue) available in the soil. These fungi typically prefer soils with high moisture content and a high atmospheric relative humidity. Many of the most aggressive, and economically important, plant pathogens are among this group and include Fusarium spp., Rhizoctonia spp., Phytophthora spp., and Thielaviopsis basicola (Agrios, 2005; Hood and Shew, I997b).  1.3.2.2 Thielaviopsis basicola: Taxonomy and Pathogenicity Thielaviopsis hasicola (Berk & Br.) Ferr. (synanamorph Chalara elegans, Nag Raj and Kendrick; Torula hasicola Berk. & Broome; Trichocladiu#n hasicola (Berk. & Broome) J.W. Carmich.) is a widespread root fungal pathogen found in both agricultural and nonagricultural soils (Trojak-Goluch and Berbec, 2005; Hood and Shew, 1996). A wide range of economically important plants are infected by T. hasicola including carrot (Daucus carota L.) (Punja et al, 1992), cotton (Gossypium hirsutum L.) (Wheeler et al, 1999), and tobacco (Nicotiana tahacum L.) (Hood and Shew, 1996 & 1997b). Infection by T hasicola commonly results in a form of black root rot disease, whole plant stunting, and delayed maturity, all of which reduce yield and crop quality. This pathogen is generally considered a facultative parasite, recently described (Walker etal, 1999; Hood and Shew, 1997b) as a hemibiotrophic pathogen. This designation refers to T. basicola's ability to survive for long periods of time outside of a living host by saprophytic consumption of soil organic matter (Hood and Shew, 1997b; Punja et al, 1993). The fungus produces two spore types, phialospores (endoconidia) and  18  chlamydospores (aleuriospores). The chlamydospore is characterized by a thick pigmented wall and divisions into compartments or segments, each of which is able to germinate. The phialospores are hyaline, not septate, and have a rectangular shape. The taxonomic classification of T. basicola is as follows (Nag Raj and Kendrick, 1975):  Kingdom: Fungi Division: Eumycota Subdivision: Deuteromycotina (Fungi Imperfecti) Class: Moniliales Genus: Chalara Species: Thielaviopsis basicola (syn. Chalara aeons)  The literature indicates that variability in virulence, due to both external and internal factors, exists within T basicola. For example, T. basicola is reported to he most aggressive during the growing season when soil temperatures are 24°C (Walker et al, 1999) and soil water content is high (Rothrock, 1992; Walker et al, 1999). Bottacin et al. (1994) described variation in virulence among several isolates of T. basicola and related this to their genetic composition. Specifically, they described the existence of dsRNAs in some, but not all, isolates of T basicola (Bottacin et al., 1994) and related this to changes in the level of virulence (Park et al, 2005; Punja et al, 1992; Geldenhius et al, 2004). In support, it has been shown that strains without dsRNA were less pathogenic than strains with one dsRNA strand and that these were more pathogenic than strains with more than one dsRNA strand (Punja, 1995). As a result, the authors have concluded that the  19  presence of this particular dsRNA in T. basicola increases the pathogenicity of this fungus (Punja et al, 1992; Park et al, 2005). Most recently, Park et al (2005) examined infection of T basicola by two totivirus-like dsRNAs. A full-length cDNA clone was developed from the 5.3 kb dsRNA element present. Sequence analysis revealed that it contained three large putative open reading frames (ORFs). These results indicate that the presence of putative virus-like particles in the cytoplasm, which were similar in both shape and size to viruses in the Totiviridae, increase the virulence in certain isolates of T basicola (Punja, 1995; Park et al, 2005).  Observations on the interactions of T basicola with other organisms show that T basicola can be sensitive to antagonism (Howell, 2003, Flood and Shew, 1997b). For  example, Pseudomonas spp. populations in the rhizosphere of tobacco roots produced biocontrol compounds (e.g., hydrogen cyanide 1HCN1 suppressive to T. basicola (Troxlert and Berling, 1997). The antagonistic activity of Trichodertna spp. against many pathogens, including T basicola, have also been reported. However, biocontrol using Trichoderma against T basicola has not yet been approved for commercial applications (Howell, 2003). In another study, a synergism between the root-knot nematode, Meloidogyne incognita (Kofoid & White), and T basicola, was identified. When occurring together, these pests greatly facilitate the development of black root rot in cotton; a seedling disease that is characterized by severe rotting of the cortex of young cotton hypocotyls and roots (Walker etal, 1999).  20  1.4 Daphne Diseases 1.4.1 Overview Daphne species are well-known because of their delightful scent and attractive flowers.  However, many problems have been associated with growing these plants by both commercial growers and Daphne enthusiasts. Reports indicate that one of the major limitations to Daphne's survival in cultivation is susceptibility to fungal root pathogens that cause root rot and chlorosis (Linderman and Toussoun, 1967). Previously, pathology reports have identified several pathogens as possible causal agents including Fusarium spp. (Pataky, 1988), Phytophthora cactorum (Lebert& Cohn) J. Schroeter (Linderman and Zeitoun, 1977; Anonymous-USDA, 1960), P. nicotiana var. parasitica (Breda de Haan) Tucker (Tompkins, 1951), and Pythium spp. (Grand, 1985), as well as several unidentified fungal pathogens. In addition to fungal pathogens, many viral diseases have been reported in Daphne. These viruses have been categorized into three groups based on mode of transmission: aphid-borne viruses, nematode-borne viruses, and contact spread viruses (Moran, 1995; Halda, 2001). Although many of these viruses remain unknown, several that have been confirmed include Alfalfa Mosaic Virus, Cucumber Mosaic Virus, Daphne Virus S and Y, Arabis Mosaic Virus, Tobacco Ringspot Virus, Daphne Latent Ringspot Virus, and Carnation Mottle Virus (Moran, 1995; Halda, 2001).  1.4.2 Daphne Sudden Death Syndrome A disease, refered to as 'Daphne Sudden Death Syndrome' (DSDS) or 'Mad Daphne Disease' by growers and gardening enthusiasts, kills the plant, as the name suggests, very quickly. The symptoms are to some extent different from those previously reported on  21  Daphne. In this case, diseased plants developed black lesions on their roots and died  within 2-3 weeks after the first appearance of foliar symptoms (i.e., leaf chlorosis). Once foliar symptoms are seen, plant death is imminent. The key symptoms of this disease are the brown to black necrotic lesions on the roots, leaf chlorosis leading to abscission, stunting, and whole plant collapse.  1.5 Objectives  Although there is a great amount of passion directed towards Daphne species and their cultivation, very few peer-reviewed publications exist that address their pathology, phylogeny, or crop improvement techniques (e.g., breeding and biotechnology). This research addresses several fundamental questions as they relate to Daphne disease and the development of improved cultivars. The main objectives of this research were:  1) To identify the causal agent(s) of Daphne Sudden Death Syndrome (DSDS) affecting Daphne cultivars grown in British Columbia;  2) To develop efficient in vitro micropropagation protocols for selected Daphne species;  3) To develop a robust germplasm screen to identify variation in taxon x pathogen interactions;  22  4) To develop a phylogeny for selected Daphne species based on DNA sequence information and associate DSDS resistance to Glade structure.  23  1.6 References  Agrios GN (2005) Plant Pathology. 5th ed. Academic Press, Elsevier Science and Technology books, NY.  Alverson WS and Baum D (1998) Circumscription of the Malvales and relationships to other Rosidae. Amer. J. Rot. 85, 876-877.  Anonymous (1960) Index of plant diseases in the United States, U.S. Department of Agriculture. Handbook No.165. p. 531. Washington DC.  Ashral M and Harris P (2005) Abiotic stresses : plant resistance through breeding and molecular approaches. The Haworth Press, Inc., Binghamton, NY.  Bayer C and Fay M (1999) Support for an expanded concept of Malvaceae within recircumscribed order Malvales: a combined analysis of plastid atpB and rbeL DNA sequences. Bot. J. Linn. Soc. 129, 267-303.  Bottacin AM, Levesque CA and Punja ZK (1994) Characterization of dsRNA in Chalara elegans and effects on growth and virulence. Phytopathology. 84, 303 -312.  Brickell CD and White R (2000a) A quartet of New Daphnes. The New Plantsman. March, 6-18. The Royal Horticultural Society, London.  24  Brickell CD and White R (2000b) Further trio of new Daphne hybrids. The New Plantsman. December, 236-248. The Royal Horticultural Society, London.  Chen DZ, Liu T and Li Y (1995) Studies on rapid clonal propagation of Daphne odora in vitro culture. Acta Hort. 404, 15-20.  Cohen D (1977) Thermotherapy and meristern-tip culture of some ornamental plants. Acta Hort. 78, 381-387.  Crawford DJ (1995). Plant Molecular Systematics, Macromolecular Approaches, Wileylnterscience publication, New York, NY.  Dixon RA (1994) Plant Cell Culture, A Practical Approach. Oxford University Press. Oxford [England]; New York: IRL Press at Oxford University Press, c1994.  Debcner T (1999) Genetic analysis of horticulturally important morphological and physiological characters in diploid roses. Gartenbauwissenschaft. 64 (1) 14-20.  Fehr WR (1987) Principles of cultivar development. Macmillan Publishing Co. New York.  25  Flor H (1955) Host-parasite interactions in flax rust-its genetics and other implications. Phytopathology. 45, 680-685.  Flora of China (2005) Vol. 13 [Clusiaceue through Araliaceaej. Science Press, Beijing, and Missouri Botanical Garden Press, St. Louis.  Geldenhius M, Roux J, Wingfield M and Wingfield B (2004) Development of polymorphic markers for the root pathogen Thielaviopsis basicola using ISSR-PCR. Mol. Eco. Notes. 4, 547-550.  Grand LF (1985) North Carolina Plant Disease Index. North Carolina Agri. Res. Ser. Tech. Bul. 240, 1-157.  HaIda JJ (2001) The Genus Daphne. Eva Kucerova- Sen Dobre Publikaca, Prague, Czeck Republic.  Harrera J (1992) Systematics and evolution flower variation and breeding systems in the Cistaceae. Plant Syst. Evol. 179, 245-255.  Hood ME and Shew HD (1996) Pathogenesis of Thielaviopsis basicola on a susceptible and a resistant cultivar of burley tobacco. Phytopathology. 86, 38-44.  26  Hood ME and Shew HD (1997a) Initial cellular interactions between Thielaviopsis basicola and tobacco root hairs. Phytopathology. 87, 228-235.  Hood ME and Shew HD (1997b) Reassement of the role of saprophytic activity in the ecology of Thielaviopsis basicola. Phytopathology. 87, 1214- 1219.  Howell C (2003) Mechanisms employed by Trichoderma species in the biological control of plant diseases: The history and evolution of current concepts. Plant Disease. 87 ( 1), 410.  Johnson R (1978) Practical breeding for durable resistance diseases in self-pollinating cereals. Euphyt. 27, 529-540.  Kozumplinki V, Barlei M and Barkie I (2004) Plant breeding and environment. Agri. Consp. Sci. 69, 67-75.  Li 11 and White D (1998) Biological control of Botrytis, Phytophtora and Pythium by Bacillus subtilis of micropropagated plants. Plant Cell, Tis. Org . Cult. 52, 109-112.  Linderman RG and Zeitoun F (1977) Phytophthora causing root rot and wilt of nurserygrown native Western azalea and other ornamental plants. Plant Dis. Rep. Vol.16, No.12.  27  Linderman RG and Toussoun TA (1967) Behavior of chlamydospores and endoconidia of Thielaviopsis basicola in nonsterilized soil. Phytopathology. 57, 729-731.  Marks T and Simpson S (2000) Interaction of explant type and indol-3-butyric acid during rooting in vitro in a range of difficult and easy to root woody plants. Plant Cell Tiss. Org . Cult. 62, 65-74.  Mathew B, Brickell C and White R (2000). The smaller Daphnes. The Proceeding of the Conference " Daphne 2000 " April 2000. The Royal Horticultural Society, London.  Moran J (1995) Daphne viral diseases. Agri. Notes 175, 1-3, State of Victoria, Department of Primary Industeries, Melbourne, Australia.  Nag Raj T and Kendrick B (1975) A Monograph of Chalara and Allied Genera. Wilfred Laurier Universiiy Press. Waterloo, Ontario, Canada.  Neal C, Stoskopf D, Tomes T and Christie B (2006) Plant Breeding: Theory and practice. Westview Press. Reprint. Jodhpur, Scientific.  Neil A (2006) Introduction to Flower Breeding & Genetics; Issues, challenges, and opportunities for the 21st Century. Springer, St. Paul, MN, USA.  28  Osherov N and May GS (2001) The molecular mechanisms of conidial germination. Microbiol. Lett. 199 153-160.  Park Y, James D and Punja ZK (2005) Co-infection by two distinct totivirus-like doublestranded RNA elements in Chalara elegans (Thielaviopsis basicola). Virus Res. 109, 7185.  Pataky NR (1988) Report on Plant Disease. RPD No. 650, Department of Crop Sciences, University of Illinois at Urbana-Champaign.  Punja ZK (1993) Influence of culture conditions on mycelial growth and phialospore production and germination in Chalara elegans. Can. J. Bot. 71, 447-456.  Punja ZK (1995) influence of double-stranded RNAs on growth, sporulation, pathogenicity, and survival of Chalara elegans. Can. J. Bot. 73, 1001-1009.  Punja ZK, Chittaranjan, S and Gaye, M.M (1992) Development of black root rot caused by Chalara elegans on fresh market carrots. Can. J. Plant Path. 14, 299-309.  Rothrock CS (1992) Influence of soil temperature, water, and texture on Thielaviopsis basicola and black root rot of cotton. Phytopathology.82, 1202-1206.  29  Sediva J (2002) Using micropropagation for Daphne conservation (Daphne cneorum L.) in the Czech Republic. Acta Pruhoniciana. 2002, No.73, 52-59.  Simmonds N and Smart J (2000) Principles of crop improvement. Second Edition. University of Southampton press, UK.  Soltis DE and Soltis P S (2000) Angiosperm phylogeny inferred from 18s rDNA, rbcL, and atpB sequences. Bot. J. Linn. Soc.133, 381-461.  Stuber C, Polacco M and Senior L (1999) Synergy of empirical breeding, marker-assisted selection, and genomics to increase crop yield potential. Crop Sci. 39, 1571-1583.  Tompkins CM (1951) Stem and root rot of Daphne odora caused by Phytophthora parashica. Phytopathology. 41, 654-656.  Trojak-Goluch A and Berbec A (2007) Meiosis and fertility in interspecific hybrids of Nicotiana tabacum L., N. glauca Grah. and their derivatives. Plant Breeding 126, 201-  206.  30  Troxler J and berling C (1997) Interactions between the biocontrol agent Pseudomonas Iluorescens CHAO and Thielaviopsis basicola in tobacco roots observed by immunolluorescence microscopy. Plant Path. 46, 62-71.  Van der Bank M and Fay M F (2002) Molecular phylogeny of Thytnelacciceac with particular reference to African and Australian genera. Taxon. 51, 329-339.  Walker N, Kirkpatrick T, and Rothrock C (1999) Effect of temperature on and histopathology of the interaction between Meloidogyne incognita and Thielaviopsis basicola on cotton. Phytopathology. 89 613-617.  Watson L and Dallwitz MJ (2007) The families of flowering plants: descriptions, illustrations, identification, and information retrieval. Delta press, Albany, Australia.  Wheeler TA, Gannaway JR and Keating K (1999) Identification of resistance to Thielaviopsis basicola in diploid cotton. Plant Disease. 83, 831-833.  Yeo AR and Flowers TJ (1989) Selection for physiological characters-examples from breeding for salt tolerance. Plants under stress biochemistry, physiology and ecology and their application to plant improvement. Cambridge University Press.  31  Zhou B (1991) Some progress on the chemistry of natural bioactive terpenoids from Chinese medicinal plants. Memorias do instituto Oswaldo Cruz. 86 (suppl. 2) 219-226.  32  CHAPTER TWO  First report of Thielaviopsis basicola on Daphne L.  2.1 Introduction  Rose daphne or garland flower (Daphne cneorum L.) is one of the most popular perennial flower species among discriminating ornamental plant growers. Daphne's appeal is based on many desirable characteristics, including its attractive foliage, variable plant habits and flower colors, but most of all, its sweet fragrance or perfume. However, due to problems reported by commercial growers and homeowners, daphne has acquired a poor reputation for long-term performance. Reports indicate that one of the major limitations to daphne's survival in cultivation is susceptibility to fungal root pathogens.  Previously, reports have identified Fusariurn sp. (Pataky, 1988), Phytophthora cactorum (Lebert& Cohn) J. Schroeter (Linderman and Zeitoun, 1977), P. nicotiana var. parasitica (Breda de Haan) Tucker (Tompkins, 1951), and Pythium ,vp. (Grand, 1985), as well as several unidentified fungal pathogens, as possible causal agents of this problem in  A version of this chapter has been published.  Noshad D, Punja ZK, Riser Ilan A, 2006. First report of Thielaviopsis basicola on Daphne -  L. Canadian Journal of Plant Pathology 28, 310-312. 33  different regions of the USA. In 2001, symptoms of an undescribed daphne disease were reported in Vancouver, British Columbia. Typical symptoms were somewhat inconsistent from those in previous pathology reports on daphne in that these plants all had black lesions on the roots and died within 2 weeks following appearance of the first foliar symptoms. This disease, coined 'Daphne Sudden Death Syndrome' (DSDS) or `Mad Daphne Disease' by gardening enthusiasts, kills plants, as the names suggests, quickly following the first foliar symptoms. Evaluation of DSDS indicates the following progression of symptoms: (i) brown to black necrotic lesions on the roots, (ii) leaf chlorosis leading to abscission, (iii) whole plant stunting, and (iv) stem collapse and plant death.  The sypotoms described are consistent with infection by Thielaviopsis basicola (Berk. & Br.) Ferraris (syn. Chalara elegans Nag Raj et Kendrick). In Canada, this pathogen causes the disease 'black root-rot' on crops such as carrot (Daucus carom L.) (Punja et al., 1992) and tobacco (Nicotiana tabacum L.) (Gayed 1972; Stover 1950a, 1950b) while also found on several ornamental species such as poinsettia (Euphorbia pulcherrima  (Willd. ex Klotzsch) Graham) and petunia (Petunia hybrida Vilm.) (Punja et al., 1992). In addition, it has been reported to parasitize important agricultural crops including cotton (Gossypium spp.), beans, (Phaseolus vulgaris L.), pansy (Viola tricolor L.), and peanuts (Arachis hypogaea L.) (Hood and Shew, 1997a).  Thielaviopsis basicola is a widespread root fungal pathogen found in both agricultural and nonagricultural soils (Trojak-Goluch and Berbec, 2005; Hood and Shew, 1996). A  34  wide range of economically important plants are infected by T basicola including carrot (Daucus carota L.) (Punja etal, 1992), cotton (Gossypium hirsutum L.) (Wheeler etal, 1999), and tobacco (Nicotiana tabacum L.) (Hood and Shew, 1997h & 1996). Infection by T. basicola commonly results in a form of black root rot disease, whole plant stunting, and delayed maturity, all of which reduce yield and crop quality. This pathogen is generally considered a facultative parasite recently, has been described (Walker etal, 1999; Hood and Shew, 1997a&b) as a hemibiotrophic pathogen. This designation refers to T basicola's ability to survive for long periods of time outside of a living host by saprophytic consumption of soil organic matter (Hood and Shew, 1997b; Punja etal, 1993). The fungus produces two spore types, phialospores (endoconidia) and chlamydospores (aleuriospores). The chlamydospore is characterized by a thick pigmented wall and divisions into compartments or segments, each of which is able to germinate. The phialospores are hyaline, not septate, and have a rectangular shape. The taxonomic classification of T basicola is as follows (Nag Raj and Kendrick, 1975):  Kingdom: Fungi Division: Eumycota Subdivision: Deuteromycotina (Fungi Imperfecti) Class: Moniliales Genus: Chalara Species: Thielaviopsis basicola (syn. Chalara elegans)  35  The literature indicates that variability in virulence, due to both external and internal factors, exists within T. hasicola. For example, T. basicola is reported to be most aggressive during the growing season when soil temperatures arc 5 24°C (Walker etal, 1999) and soil water content is high (Rothrock, 1992; Walker et al, 1999). Bottacin et al. (1994) described variation in virulence among several isolates of T. hasicola and related this to their genetic composition. Specifically, they described the existence of dsRNAs in some, but not all, isolates of T basicola (Bottacin et al., 1994) and related this to changes in the level of virulence (Park etal, 2005; Punja etal, 1995; Geldenhius etal, 2004). In support, it has been shown that strains without dsRNA were less pathogenic than strains with one dsRNA strand and that these were more pathogenic than strains with more than one dsRNA strand (Punja, 1995). As a result, the authors have concluded that the presence of this particular dsRNA in T. basicola increases the pathogenicity of this fungus (Punja, 1995; Park etal, 2005). Most recently, Park et al (2005) examined infection of T. hasicola by two totivirus-like dsRNAs. A full-length cDNA clone was developed from the 5.3 kb dsRNA element present. Sequence analysis revealed that it contained three large putative open reading frames (ORFs). These results indicate that the presence of putative virus-like particles in the cytoplasm, which were similar in both shape and size to viruses in the Totiviridae, increase the virulence in certain isolates of T hasicola (Punja, 1995; Park etal, 2005; Wattimena, 2001).  Observations on the interactions of T. basicola with other organisms show that T hasicola can be sensitive to antagonism (Howell, 2003, Hood and Shew, 1997b). For example, Pseudomonas spp. populations in the rhizosphere of tobacco roots produced  36  biocontrol compounds (e.g., hydrogen cyanide (HCN)) suppressive to T. basicola (Troxlert and berling, 1997). The antagonistic activity of Trichoderma spp. against many pathogens, including T. basicola, have also been reported (Howell etal, 2003). However, biocontrol using Trichoderma against T. basicola has not yet been approved for commercial applications (Howell etal, 2003). In another study, a synergism between the root-knot nematode, Meloidogyne incognita (Kofoid & White), and T. basicola, was identified. When occurring together, these pests greatly facilitate the development of black root rot in cotton; a seedling disease that is characterized by severe rotting of the cortex of young cotton hypocotyls and roots (Walker etal, 1999). Evaluating genetic variation in T basicola using ISSR-PCR, seven of fourteen primer pairs resulted in the amplification of single polymorphic fragment indicating quantifiable variation is present among populations (Geldenhuis etal., 2004). Continued use of these primers will enable further molecular characterization of this important pathogen resulting in an enhanced understanding of its population structure (Geldenhuis et al, 2004).  Little is known about the factors (e.g., cultural conditions, host plant genetics) that affect DSDS development beyond anecdotal observations and practices. For example, during nursery production of daphne cultivars, many producers apply prophylactic fungicide treatments to help ensure crop health. While this is a relatively common practice, no published literature was found that directly addresses the efficacy of this practice to control DSDS. In addition, even if effective at controlling DSDS, reliance on fungicides is unsustainable and undesirable. One alternative to this practice is the development of disease resistant daphne cultivars. This strategy is typically more desirable because it can  37  be highly effective in reducing disease, is environmentally benign, and usually entails little or no additional expense to producers (Crute, 1996; Dahlberga, 2001; Diaz-Perez 1995; Reeleder, 1999). However, it typically requires a long time horizon to achieve. To date, native host plant resistance to T basicola has been identified during germplasm screens as part of various crop improvement or breeding programs. For example, Nicotiana glauca Graham was identified as resistant to T. basicola and subsequently  incorporated into a tobacco (N. tabacum) breeding program (Trojak-Goluch, 2005). In another germplasm screen, Gossypium arboreum L. PI 1415 was found to be resistant to T basicola and subsequently incorporated into a diploid cotton breeding program  (Wheeler, 1999; Shankara, 1999).  Based on these data, I designed experiments to identify the casual agent of DSDS for subsequent use in a germplasm screen of Daphne species.  2.2 Material and Methods  2.2.1 Fungi isolation, inoculation and evaluation procedures To identify the causal agent of DSDS, tissue samples were collected from diseased and healthy plants (paired samples of diseased and healthy plants acquired from individual nurseries throughout the greater Vancouver region) of Daphne cneorum 'Ruby Glow' which included roots of various diameters, discoloration, and degree of degradation. Special attention was paid to sampling tissue from the margins surrounding necrotic lesions. Samples were cut into 2-4 mm lengths and surface-disinfested with 10% bleach (5.25% sodium hypochlorite) for 5 min, and rinsed in water. These samples were  38  subsequently cultured on both general media (e.g., potato dextrose agar and corn meal agar) as well as media specific for an individual groups of fungi; e.g., cornmeal for detection of Phytophthora (Ferguson, 1999), Komada's medium for detection of Fusarium (Komada, 1975), V8 ® (Campbell Soup Co. Camden, NJ) for detection of Phytophthora or Thielaviopsis basicola, and carrot sections for specific detection of Thielaviopsis basicola (Tsao, 1970; Punja etal, 1992). After initial growth, all fungi were subsampled 3-5 times to eliminate bacterial contamination.  Following subculturing, single spore colonies were produced for each isolate. For each individual isolate, a spore suspension was prepared by gently washing the surface of 3week-old colonies (abraded with a fine brush) with deionized water and vortexing the wash solution for 30 sec. The resulting suspension was twice filtered through four layers of cheesecloth to remove agar, hyphae, and other debris. The suspensions were calibrated with a haemocytometer and adjusted with deionized water to obtain a final concentration of lx10 6I spores prior to inoculation. The concentration of spores chosen was based on preliminary experiments that determined the optimum concentration for effective inoculation (Appendix 1).  Following pure culture production and inoculum concentration determinations, isolate pathogenicities were tested by applying 5 ml of the conidial suspension to healthy roots of both 2-year old nursery-grown and rooted in vitro-produced plantlets of D. cneorum. Distilled water alone was applied to the control plants. Following inoculation, nurserygrown plants were kept under ambient conditions (24°C/14°C day/night averages over  39  the experimental period) while in vitro plantlets were maintained at 24 °C with a 16 h photoperiod. This temperature was chosen after observation of several isolates' growth rates under laboratory conditions. At this temperature, all isolates grew equally well with comparable vigor. The experiment was repeated over three different seasons of the year.  In these experiments, a completely randomized design was used with three replications. Each replication consisted of 15 plants (i.e., 45 plants for each fungal isolate) with two plants serving as controls. Descriptive statistics, analysis of variance, correlation, and Tukey's honest significant differences (HSD) test were generated using SPSS 11.5 software (SPSS Inc. Statistical Package for the Social Sciences, Chicago, US).  Weekly, data on disease progression were collected using the following 0-5 rating: 0= healthy plant, no symptoms; 1= less than five lesions on lateral roots, no lesions on tap root, no foliar symptoms; 2= greater than five lesions on lateral roots, less than five lesions on tap root, no foliar symptoms; 3= most lateral roots with lesions and some necrosis, greater than five lesions on tap root, five to ten chlorotic leaves; 4= most lateral roots necrotic, greater than five lesions on tap root, most leaves chlorotic with some leaf abscission; 5= plant is dead (Fig 2.1).  2.2.2 Daphne production Rooted plants of Daphne cneorum were produced in July and August from terminal cuttings (50-100 mm in length) with the flower buds and lower leaves removed. Cuttings were made with a single shallow cut and soaked in an anti-fungal solution (Physan 20, 40  Maril Products Inc.) for 60 seconds. The cuttings were allowed to dry momentarily before being dipped in 0.4% IBA powder (Stim Root #2, Plant Products. Co. Ltd.) and then placed in 6 cm pots filled with a course rooting medium (10 parts propagation grade perlite, 8 parts peat, 6 parts granite grit #2, 1 part pumice (double screened to remove fine particles), dolomite lime 65AG at 900 g m -3 , and Micromax (trace elements; Scotts International Co., Geldermalsen, The Netherlands) at 400 g m -3 . The flats were placed under intermittent mist with bottom heat set at 22°C. Rooted cuttings were transferred to a polyhouse in October where they were allowed to go dormant but kept frost free. They were repotted in May into 12 cm pots filled with a well-drained medium (8 parts peat, 8 parts Turface MVP (Profile Products LLC, Buffalo Grove, IL 60089), 6 parts granite grit #2, 4 parts screened and pasteurized soil, 1 part pumice, dolomite lime 65AG at 670 g m 3  , Micromax micronutrients at 540 g m , Osmocote (Scotts Miracle-Gro Co., Marysville, OH) 18-6-12 at 2150 g m -3 , and Psi Matric (TerraLink Horticulture Inc., Abbotsford, BC) wetting agent. All stock plants were groWn under shade cloth during the summer months and moved to a heated polyhouse during the winter months to prevent frost damage. Fertilization regiment included yearly top-dressing with Osmocote 18-6-12 at 5 g 1 -I gal pot. Fungicides were not used during stock production because very little disease pressure was present and I did not want to risk cross-contamination affecting the in vivo assay.  Prior to inoculations, nursery production containers were modified to contain a clear panel behind a lightproof 'door' to allow for direct observation of the infection process without further disturbance to the root system. All procedures were the same for control plants  41  except for the application of distilled water instead of the spore suspension. To allow for uniform conditions following inoculation, plants were transferred to a greenhouse and grown under natural light at 24 +1 °C and a relative humidity between 70 and 80%.  2.3 Results  From diseased plants, the following fungi were isolated: Fusarium roseum (Snyder & Hansen), Fusarium oxysporum (Snyder & Hansen), Trichoderma sp. (Persoon ex Gray), Aspergillus sp. (Micheli ex Link) and Thielaviopsis basicola (Berk. et Br.) Ferr. However, only Thielaviopsis basicola was isolated from all diseased plants but was absent from healthy plants. Identification was based on the presence of characteristic chlamydospores in root tissues. As mentioned earlier, T basicola forms two spore types: hyaline cylindrical phialospores (endoconidia) and thick-walled pigmented chlamydospores (aleuriospores). As Nag Raj and Kendrick (1975) explain, both spore types are used as the basis for taxonomic identification of this species. Regardless of nursery- vs in vitro-based inoculations, all plants inoculated with T basicola, developed stunted and chlorotic shoots with the roots displaying black lesions containing the characteristic spores of the fungus (Punja et al. 1992). These symptoms are consistent with those reported for DSDS. However, neither these symptoms, nor any other symptoms, were induced by any of the other fungi isolated from Daphne diseased tissue.  Comparing the nursery-inoculated plants to the in vitro inoculated plants four weeks postinoculation, all nursery plants developed symptoms consistent with DSDS (average rating 3.5), while all other plants, either inoculated with the other isolates or clear water, 42  remained symptomless and healthy. In addition, in vitro inoculated plants displayed the same pattern of disease occurrence as expressed on the nursery-grown plants. However, T. basicola induced symptoms in significantly less time (< 2 weeks) on these plantlets than on the nursery-grown plants. Based on this observation, I conclude the temperature used during inoculation and disease evaluation (i.e., 24 C) demonstrates that this isolate of T basicola can be an aggressive virulent pathogen to Daphne at this temperature. Furthermore, the higher temperature used during the in vitro evaluation, despite being higher than the optimum temperature reported for T hasicola, facilitated disease progression. The difference between my results and prior reports on temperature affects on T basicola may be related to the fact that temperature guides for T. basicola are based on field, not laboratory, conditions. Field measured temperatures are typically heterogeneous with significant day/night fluctuations while laboratory conditions are significantly more consistent and stable. Following Koch's postulates, T. basicola was successfully re-isolated and re-identified from all plants expressing symptoms. Although my use of only one isolate of T hasicola (i.e., the most aggressive and robust isolate identified) imposes limitations on the breath of conclusions that can be made, (e.g., can not compare pathogenicity of other races on Daphne),1 am able to conclude with confidence that T. basicola is the causal agent of Daphne Sudden Death Syndrome. This conclusion is based on the facts that only T basicola was recovered from all diseased Daphne plants collected and that it was the only fungal isolate recovered able to induce DSDS.  43  2.4 References  Bottacin AM, Levesque CA and Punja ZK (1994) Characterization of dsRNA in Chalara elegans and effects on growth and virulence. Phytopathology. 84, 303 -312.  Crute IR and Pink DA (1996) Genetics and utilization of pathogen resistance in plants. The Plant Cell 8, 1747-1755.  Dahlberga JA and Bandyopadhyay R (2001) Evaluation of sorghum germplasm used in US breeding programmes for sources of sugary disease resistance. Plant Pathology. 50, 681-689.  Diaz-Perez J (1995) Acclimatization and subsequent gas exchange, water relations, survival and growth of microcultured apple plantlets after transplanting them in soil. Physiologia Plantarum 95, 225-229.  Ferguson AJ (1999) Detecting multiple species of Phytophthora in container mixes from ornamental crop nurseries. Plant Disease. 83(12), 1129-1136.  Gayed SK (1972) Host range and persistence of Thielaviopsis basicola in tobacco soil. Can. J. Plant Sci. 52, 869-873.  44  Geldenhius M, Roux J, Wingfield M and Wingfield B (2004) Development of polymorphic markers for the root pathogen Thielaviopsis basicola using ISSR-PCR. Mol. Eco. Notes. 4, 547-550.  Grand LF (1985) North Carolina Plant Disease Index. North Carolina Agri. Res. Ser. Tech. Bul. 240, 1-157.  Hood ME and Shew I-1D (1996) Pathogenesis of Thielaviopsis basicola on a susceptible and a resistant cultivar of burley tobacco. Phytopathology. 86, 38-44.  Hood ME and Shew HD (1997a) Initial cellular interactions between Thielaviopsis basicola and tobacco root hairs. Phytopathology. 87, 228-235.  Hood ME and Shew HD (1997b) Reassessment of the role of saprophyti activity in the ecology of Thielaviopsis basicola. Phytopathology. 87, 1214-1219.  Howell C (2003) Mechanisms employed by Trichoderma species in the biological control of plant diseases: The history and evolution of current concepts. Plant Disease. 87, 4-10.  Komada I 1 (1975) Development of a selective medium for quantitative isolation of -  Fusarium oxysporum from natural soils. Rev. Plant Prot. Res. 8, 114-124.  45  Linderman RG and Zeitoun F (1977) Phytophthora causing root rot and wilt of nurserygrown native western azalea and other ornamental plants. Plant Dis. Rep. 16(12), 2-3.  Mauk PA and Hine RB (1988) Infection, colonization of Gossypium hirsuturn and G. barbaden.ve and development of black root rot caused by Thielaviopsis basicola.  Phytopathology. 78,1662-1667.  Nag Raj TR and Kendrick B (1975) A monograph of Chalara and allied genera. Wilfred Laurier University Press, Waterloo, Ont.  Pany D (1990) Plant Pathology in Agriculture. Cambridge University Press, Cambridge.  Park Y, James D and Punja ZK (2005) Co-infection by two distinct totivirus-like doublestranded RNA elements in Chalara elegans (Thielaviopsis bavicola). Virus Research. .  109,71-85.  Pataky NR (1988) Report on Plant Disease. RPD No. 650, Department of Crop Sciences, University of Illinois at Urbana-Champaign.  Punja ZK Chittaranjan S and Gaye MM (1992) Development of black root rot caused by Chalara elegans on fresh market carrots. Can. J. Plant Pathology. 14,  299-309.  46  Punja ZK (1993) Influence of culture conditions on mycelial growth and phialospore production and germination in Chalara elegans. Can. J. Bot. 71, 447-456.  Punja ZK (1995) Influence of double-stranded RNAs on growth, sporulation, pathogenicity, and sumival of Chalara elegans. Can. J. Bot. 73, 1001-1009.  Reeleder R (1999) Septoria leaf spot of Stevia rebaudiana in Canada and methods for screening for resistance. J. of Phytopathology. 147, 605-613.  Rothrock CS (1992) Influence of soil temperature, water, and texture on Thielaviopsis basicola and black root rot of cotton. Phytopathology.82, 1202-1206.  Stover RH (1950a) The black root-rot disease of tobacco. I. Studies on the causal organism Thielaviopsis basicola. Can. J. Res. Sect. C Bot. Sci. 28, 445-470.  Stover RH (1950b) The black root-rot disease of tobacco. II. Physiologic specialization of Thielaviopsis basicola on Nicotiana tabacum. Can. J. Res. Sect. C Bot. Sci. 28, 726-738.  Tompkins CM (1951) Stem and root rot of Daphne odora caused by Phytophthora parasitica. Phytopathology. 41, 654-656.  47  Trojak-Goluch A and Berbec A (2007) Meiosis and fertility in interspecific hybrids of Nicotiana tahacum L., N. glauca Grah. and their derivatives. Plant Breeding 126, 201206.  Troxler J and berling C (1997) Interactions between the biocontrol agent Pseudomonas fluorescens CHAO and Thielaviopsis basicola in tobacco roots observed by immunolluorescence microscopy. Plant Pathology. 46, 62-71.  Tsao PH (1970) Selective media for isolation of pathogenic fungi. Ann. Rev. Phytopathology. 8, 157-186.  Walker N, Kirkpatrick T, and Rothrock C (1999) Effect of temperature on and histopathology of the interaction between Meloidogyne incognita and Thielaviopsis basicola on cotton. Phytopathology. 89 (8) 613-617.  Wattimena S (2001) Studies of factors affecting virulence of Chalara Elegans on Bean (Phaseolus Vulgaris L.). MSc. Thesis. Simon Fraser University.  Wheeler TA, Gannaway JR and Keating K (1999) Identification of resistance to Thielaviopsis basicola in diploid cotton. Plant Disease. 83, 831-833.  48  Yarwood CE (1981) Occurrence of Chalara elegans. Mycology. 73, 524-530.  49  Figure 2.1. Infection of D. cneorum 'Ruby Glow' by Thielavopsis basicola: (A) Stages of disease progression from left to right- healthy shoot (rating of 0), lesions on fine roots; leaf abscission, fine root necrosis (rating of 4); plant death (rating of 5); (B) Roots showing lesions and tissue necrosis, (circle indicates typical region for sample collection), scale bar= 0.33 mm; (C) T basicola chlamydospores in root tissue, scale bar= 50p.m; (D) T. basicola chlamydospores in culture, scale bar= 25[1.m.  50  CHAPTER THREE  IN VITRO PROPAGATION OF SEVEN DAPHNE L. SPECIES  3.1 INTRODUCTION The genus Daphne L. (Thymelaeaceae) is comprised of 95 recognized species (Flora of China, 2005) distributed primarily in Europe and Asia with a few species endemic to Africa and Australia. This genus of flowering plants includes deciduous, semi-evergreen and evergreen shrubs. Many species are spring flowering and have deliciously fragrant flowers that range in color from white to purple with a few having greenish-yellow or yellow flowers. Plant habits range from low growing prostrate forms, only a few inches high, to large shrubs. Several species have been commercially propagated because of their desirable horticultural characteristics, including attractive foliage, plant habit, flower color and most of all, pleasant fragrance. Specifically, D. cneroum L. (rose daphne or garland flower) has become one of the most popular perennial flowering shrubs among discriminating ornamental plant growers (Brickell and White, 2000a; Halda, 2001). Commercial propagation of Daphne is typically from seed or vegetative cutting. However, seeds are genetically variable adding undesirable variation to a production system.  A version of this chapter has been submitted for publication. Noshad I), Riseman A. In vitro propagation of Seven Daphne L. Species  51  Vegetative propagation is more desirable but root induction is very difficult for many Daphne taxa (Brickell and White, 2000a; Mathew et al, 2000). In addition to rooting difficulties, commercial production of some species was in decline due to the build-up of internal pathogens within the stock plants (Noshad, 2006; Green etal, 1992; Hartmann and Preece, 1990; Havel and Kolar, 1983). However, these species were successfully 'refreshed' by producing new stock plants through tissue culture techniques, thereby eliminating most of the internal pathogens (Jayasankar etal, 2001; Chen etal, 1995). Although in vitro techniques successfully generated disease-free plants in a relatively short time period (Bajaj, 1996), Daphne species remain very difficult to establish and manipulate in culture. Part of the  problem appears to be associated with significant species-specific culture requirements and the presence of polyphenol oxidases in Daphne tissue that cause explant browning and death during establishment (Marks and Simpson, 2000; Green etal, 1992; Cohen, 1977). Therefore, the development of species-specific in vitro protocols is needed and will allow for the production of uniform, disease-free plants for commercial and experimental use (Krogstrup and Norgaard, 2001; Bennett and Davis, 1986). Rooting and in vivo establishment of micropropagated Daphne has also proven difficult for several species (Tricoli etal, 1985). Therefore, the objective of this study was to develop efficient establishment, proliferation, and rooting protocols for the in vitro propagation of selected Daphne species. The present work established protocols for the micropropagation of seven species of Daphne.  52  3.2 MATERIALS AND METHODS 3.2.1 Plant material and growing conditions  Seven Daphne species, collected by the UBC Botanical Garden, Vancouver, BC, Canada, were used in this research and included D. cneorum L., D. caucasica Pall., D. retusa Hemsl., D. giraldii Nitsche, D. jasminea Sibth.& Sm., D. laureola L. and D. tangutica Maxim.  Apical shoot tips, 2-5 cm in length, 3-5 mm in diameter, and bearing 1-3 nodes were collected during the summer and fall seasons from 4-6 year old container-grown plants. Collected shoots were stripped of all leaves and rinsed under running tap water for 15 min. Under aseptic conditions, shoots were then surface sanitized by treatment with 70% ETON for 30 seconds followed by treatment with 0.5% sodium hypochlorite solution containing 0.5 ml 1 -1 Tween-20 (Sigma Chemical, St. Louis, MO) and gently stirred for 10 min. After sanitation, shoots were rinsed three times with sterile distilled water for 5 min each and placed individually in culture vessels. Cultures were maintained at 25°C under 16 h photoperiod with irradiance intensity of 350 µW cm -2 supplied by cool white fluorescent lamps. Following this disinfestations procedure, culture contamination rates were below 10%.  3.2.2 Establishment phase  Individual nodal explants 1-2 cm long were cut aseptically and positioned vertically in individual 25 x 150 mm test tubes containing 25 ml of medium. Several base media were evaluated and included MS (Murashige and Skoog, 1962), WPM (Woody Plant Medium; McCown and Lloyd, 1983), B5 (Gamborg ctal, 1968), LS (Linsmaier & Skoog, 1965), and SH (Schenk and Hildebrandt, 1972). All base media were supplemented with 20 gl  53  -I  sucrose,  and 5.6 g 1 1 high gel strength agar (Sigma-Aldrich, St. Louis, MO). The pH of the various media was adjusted based on published protocols (e.g., MS media was adjusted to 5.8, WPM media to 5.2) before autoclaving for 15 min at 121°C. Experiments designed to determine the effects of autoclaving on PGR activities found no significant reduction in activity following these sterilization procedures (Appendix 2). Explants were maintained on establishment media for four weeks prior to subculture on multiplication media.  3.2.3 Multiplication phase Following a four week establishment period, shoots were sub-cultured into 150 mm baby food jars (Sigma-Aldrich, St. Louis, MO) containing 25 ml multiplication media. Shoots were harvested from stock cultures, cut into nodal explants 3-5 cm long and place vertically into media containing either MS or WPM basal salts, and 5.6 g I I agar (Sigma-Aldrich, St. Louis, MO). Based on explant growth during the establishment phase, D. cneoruin and D. jasminea were sub-cultured on WPM based media while the other species were sub-cultured on MS based media (Table 3.1). Various concentrations of 6- benzyl aminopurine (BA), kinetin (KIN), and thidiazuron (TDZ) were used alone or in conjunction with one of three concentrations of indole-3 acetic acid (IAA), indole 3- butyric acid (IBA), or a-naphthalene acetic acid (NAA) (Tables 3.2 and 3.3). The experiment was conducted in a randomized complete block design with three replications. Shoot proliferation data were based on the number of 'usable' shoots (>1 mm long) produced per explant after 8 wks in culture (Table 3.2).  54  3.2.4 Elongation and rooting phases  Following multiplication, new shoots were used in elongation and rooting trials. For shoot elongation, shoots 1 cm long with intact apices and 2-4 leaves were cultured on either MS or WPM basal medium supplemented with 1^charcoal. After four weeks, the longest shoots (4-6 cm) were transferred to individual test tubes with 20 ml rooting media consisting of standard MS or WPM salts, vitamins (nicotinic acid 0.5 ^pyridoxine-HCI 0.5 g thiamine-HCI 1 g I -I , and glycine 2 g1 -1 ), 20 g I -I sucrose, and 5.6 g I agar. in addition, shoots were exposed to various combinations of PGRs through one of three methods as follows (individual PGR treatments used are detailed in Tables 3.4, 3.5 and 3.6): 1) conventional method: PGRs were added to the medium before autoclaving and were  homogeneous within the container (Table 3.4); 2) two layer method: two media layered with the upper basal medium containing PGR(s) and -  the lower basal medium with charcoal but without PGR(s) (Table 3.5); 3) PGR dip method: shoots were directly dipped into a PGR solution before culture on basal medium sans PGR(s). For this method, all PGRs were filter sterilized before use. Shoot bases (lower 2-3 cm) were dipped for 5, 15, or 30 mins in 1 mM solution of an individual PGR or dipped for 15 min in a combination of PGRs (Table 3.6). Shoots were then transferred directly to basal medium without PGRs. Rooting, expressed as the percentage of shoots producing root initials >5 mm in length, was recorded after 8 weeks. Preliminary experiments were conducted to evaluate the effects of changes in component concentrations of the base media (Appendix 3). I found all alterations to the published media compositions tested inferior for Daphne rooting.  55  3.2.5 Experimental design and analyses  All experiments used a complete randomized design with three replications and 15 shoots per replication (i.e. 45 shoots). Data were recorded as number of multiplied shoots after 8 weeks, and percentage of shoots rooted after another 8 weeks. The percentage data were subjected to arcsin transformation before ANOVA analysis. Descriptive statistics, analysis of variance, Tukey's honest significant differences (HSD) test, and other analysis results were generated using SPSS® 11.5 software (SPSS Inc. Statistical Package for the Social Sciences, Chicago, US).  3.3 RESULTS 3.3.1 Establishment phase  All primary explants harvested from stock plants were contamination-free 2 weeks after establishment. In addition, the inclusion of activated charcoal was effective in reducing the negative effects of phenolic compounds produced by these initial explants. Significant medium effects were observed for species survivorship after two weeks with MS-based media most appropriate for five species (D. caucasica, D. giraldii, D. 'aureola, D. retusa, and D. tangutica) and WPM-based media most appropriate for the remaining two species (D. cneorum and D. jasminea) (Table 3.1). The remaining three media were ineffective in  supporting growth of any Daphne species evaluated. Axillary meristems from all species initiated growth after 2 weeks of culture in a species-most-favorable medium (Table 3.1) and grew to 10-15 mm after 4 weeks.  56  3.3.2 Multiplication phase Significant effects were observed for axillary shoot production by species and media (Tables 3.2 and 3.3; Fig 3.1). Continuing to follow the medium preference observed earlier, D. caucasica, D. giraldii, D. retusa, D. laureola and D. tangutica displayed the greatest  proliferation on MS-based media, while D. jasminea and D. cneorum displayed the greatest proliferation on WPM-based media. When apical and lateral explants were compared, no significant differences in multiplication capacity were detected.  In addition to base media effects, significant effects on multiplication rate were observed for PGR supplement. When neither cytokinin nor auxin were incorporated into media, shoots grew with strong apical dominance without axillary shoot proliferation. When a cytokinin was incorporated without auxin, the BA treatments produced the greatest multiplication rates with increasing response observed with increasing concentrations up to 2 mg I I (Table 3.2). With the incorporation of one cytokinin, D. retusa produced the greatest proliferation rate with an average of 1.56+0.5 shoots per explant. KIN treatments also induced greater response with increasing concentrations, but the overall rates remained below those of BA. Shoots induced on BA-containing media were usually of normal appearance and, in the moderate concentration ranges, did not cause any hyperhydricity. The TDZ treatments were less effective in higher concentrations than the other two cytokinins tested with a general trend of decreasing response as concentration increased. In addition, TDZ induced a very high frequency of hyperhydricity.  57  When a cytokinin was used in combination with an auxin, increases in multiplication rates were observed. Incorporation of NAA, regardless of cytokinin or species, greatly increased multiplication rates over those of IAA, IBA or the use of the cytokinin alone (Table 3.3). Among species, D. tangutica produced the greatest proliferation rate with an average of 3.6+0.2 shoots per explant (Table 3.2). D. caucasica, D. giraldii, and D. laureola produced the greatest number of shoots per explant when cultured on medium supplemented with 2 mg F1 BA+ 0.01 mg NAA while D. retusa and D. tangutica responded best to medium supplemented with 1 mg 1 -1 BA + 0.01 mg NAA. However, the remaining two species, D. cneorum and D. jasminea, produced their greatest respective multiplication rates when two  cytokinins (0.5 mg 1 1 BA and 0.001 mg 1 1 TDZ) were combined with 0.01 mg NAA (Table 3). In general, BA was more efficient for inducing shoot formation than other cytokinins. Also, combinations of cytokinins and auxin resulted in a higher number of shoots with the combination of BA and NAA inducing very strong shoot proliferation (Table 3.3).  3.3.3 Rooting and acclimation Root initiation and development required approximately 60 days (Fig 3.2) with significant treatment effects on the frequency of rooting observed (Table 3.4, 5 and 6). Root development was induced by the addition of auxin, either NAA and/or IBA, for all species tested with variable rates. No rooting occurred in the absence of auxins. Generally, the inclusion of IBA or NAA resulted in a higher number of roots than IAA. Treatments with only NAA resulted in the formation of thicker roots than roots induced by IBA. The twolayer media and dipping techniques were superior in inducing rooting than the traditional medium with homogeneous PGR incorporation (Table 3.5 and 6). Regardless of species,  58  both techniques produced rooting frequencies of >85% in some individual PGR combinations (Table 3.5 and 6). However, explants in the two-layer media often produced callus and/or adventitious roots from callus, while no such growth was observed with the dipping method. Therefore, the dipping technique appeared to be the most efficient technique for use with these selected species (Table 3.6). Rooted shoots were successfully acclimatized in a fog chamber and survived transfer to the greenhouse at >85%.  3.4 DISCUSSION Daphne has been categorized as a "difficult-to-root" group, both with conventional  propagation via cuttings and following micropropagation (Brickell and white, 2000b; Marks and Simpson, 2000). In addition to rooting difficulties, browning of tissue and hyperhydricity (or vitrification) are reported to be problematic during in vitro propagation of several Daphne species (Chen and Li, 1995; Wang and Tang, 1994; Cohen, 1977). My research is the first systematic attempt to optimize the protocols for in vitro propagation and root induction of a number of Daphne species. My results mostly support previous research on the micropropagation of one species, D. cneorum (Mala etal, 2004; Marks and Simpsons, 2000) having produced similar results. However, new optimizations of these protocols are now established as well as the addition of species-specific protocols for an additional six species.  In general, species responded differently to media, individual PGRs, combinations of PGRs, rooting technique, and required species-specific protocol development. With species-specific protocols optimized, successful multiplication was achieved without the common problems of  59  browning or hyperhydricity. Adding activated charcoal to the media was very helpful for the control of browning in all species. Hyperhydricity was observed more often in some treatments than in others. Reports suggest hyperhydricity could result from the type and concentration of cytokinins, gelling agent, or medium used (Preece and Compton, 1991; Escobar and Villalobos, 1986; Krulik, 1980). My cultures usually became vitrified on media with high concentrations of cytokinins, particularly with TDZ. High TDZ concentrations have been shown to induce hyperhydricity in several other plant species e.g. Populus tremula (Vinocur et al., 2000; Clayton and Hubstenberger, 1995; Huetteman and Preece, 1993; Garton etal, 1981) and support my findings. This cytokinin-like compound has been used for microprogation of many woody species (Zhang et al., 2001; Clayton and Hubstenberger, 1995; Mohamed-Yasseen etal, 1994; Drew etal, 1993), but has not been widely tested for members of the genus Daphne. Based on TDZ concentrations comparable to those used by other authors for shoot induction (Mulwa and Bhalla, 2000; Martinez-Vazquez and Rubluo, 1989; McComb, 1985; Tricoli etal, 1985), I do not recommend TDZ use in Daphne micropropagation. This investigation also revealed that auxin supplementation was required for in vitro rooting. However, species still responded slowly (approximately 7-9 weeks) to the various rooting treatments.  60  3.7 REFERENCES Bajaj YPS (1996) Biotechnology in Agriculture and Forestry Trees IV. 35, 20-290. SpringerVerlag, Berlin.  Bennett CK and Davis J (1986) In vitro propagation of Quercus schumerida seedling. HortSci. 21, 1045-1047.  Brickell CD and White R (2000a) A quartet of New Daphnes. The New Plantsman. March, 618. The Royal Horticultural Society.  Brickell CD and White R (2000b) Further trio of new Daphne hybrids. The New Plantsman. December, 236-248. The Royal Horticultural Society.  Chen DZ, Liu T and Li Y (1995) Studies on rapid clonal propagation of Daphne odora in vitro culture. Acta Hort. 404, 15-20.  Clayton PW and Hubstenberger JF (1990) Micropropagation of members of the Cactaceae Subtribe Cactinae. J. Am. Soc. Hort. Sci. 115, 337-343.  Cohen D (1977) Thermotherapy and meristem-tip culture of some ornamental plants. Acta Hort. 78, 381-388.  61  Drew RA, McComb JA and Considine JA (1993) Rhizogenesis and root growth of Carica papaya L. in vitro in relation to auxin sensitive phases and use of riboflavin. Plant Cell Tiss.  Org . Cult. 33, 1-7  Escobar H and Villalobos V (1986) Opuntia micropropagation by axillary proliferation. Plant Cell Tiss. Org . Cult. 7, 269-277.  Flora of China Vol. 13 (Clusiaceae through Araliaceae). (2005) Science Press, Beijing, and Missouri Botanical Garden Press, St. Louis.  Gamborg, OL, Miller, RA and Ojima, K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res. 50, 151-158  Garton S, Hosier MA and Read PE (1981) In vitro propagation of Alnus glutinosa Gaertn. Hort Sci. 16, 758-759  Green MJ, Godkin SE and Monette PL (1992) Use of in vitro cultures of Daphne cneorum L. for the western detection of daphne virus X. J. Enviro. Hort. 10(3), 153-155.  Halda JJ (2001) The Genus Daphne. Eva Kucerova- Sen Dobre Publikaca, Prague, Czeck Republic.  62  Hartmann HT and Kester DE (1990) Plant propagation: principles and practices. Englewood Cliffs, NJ: Prentice Hall.  Havel L and Kolar Z (1983) Microexplant isolation from Cactaceae. Plant Cell Tiss. Org . Cult. 2, 349-353.  Huetteman CA and Preece JE (1993) Thidiazuron: a potent cytokinin for woody plant tissue culture. Plant Cell Tiss. Org . Cult. 33, 105-119.  Jayasankar S, Vanaman M, Li Z and Gray DJ (2001) Direct seeding of grapevine somatic embryos and regeneration of plants. In Vitro Cell & Dev. Biol. - Plant 37: 476-479(4)  Krogstrup P and Norgaard J V (1991) Micropropagation of Psiadia coronopus (Lam.) Benth, a threatened endemic species from the island of Rodrigues. Plant Cell, Tiss. Org . Cult. 27, 227-230.  Krulik G (1980) Tissue culture of succulent plants. Nat. Cact. Succ. J. 35,14-17.  Linsmaier E and Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant. 18, 100-127.  Mala J and V. BylinsI4 (2004) Micropropagation of endangered species Daphne cneorum. Biol. Plant. 48 (4), 633-636.  63  Marks T and Simpson S (2000) Interaction of explant type and indol-3-butyric acid during rooting in vitro in a range of difficult and easy to root woody plants. Plant Cell, Tiss. Org . Cult. 62, 65-74.  Martinez-Vazquez 0 and Rubluo A (1989) In vitro mass propagation of the near-extinct Mammillaria sanangelensis Sanchez-Mejorada. J. Hort. Sci. 64, 99-105.  Mathew B, Brickell C and White R (2000) The smaller Daphnes. The Proceeding of the Conference " Daphne 2000 " April 2000. The Royal Horticultural Society, London.  McComb JA (1985) Micropropagation of the rare species Stylidium coroniforme and other Stylidium species. Plant Cell Tiss. Org . Cult. 4, 151-158.  McCown BH and Lloyd GB (1983) A survey of the response of Rhododendron to in vitro culture. Plant Cell Tiss. Org . Cult. 2, 75-85  Mohamed-Yasseen Y (1994) Micropropagation of pitaya (Hylocereus undatus Britt. et Rose). HortSci. 29, 559-560.  Mulwa RMS and Bhalla PL (2000) In vitro shoot multiplication of Macadamia tetraphylla L. Johnson. J. Hortic. Sci. Biotechnol. 75, 1-5.  64  Murashige T and Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15,473-497.  Noshad D, Punja ZK and Riseman A (2006) First report of Thielaviopsis hasicola on Daphne cneorum. Can. J. Plant Pathol, 28(3), 310-312.  Preece JE and Compton ME (1991) Problems with explant exudation in micropropagation. In: Bajaj YPS (ed) Biotechnology in Agriculture and Forestry, 17,168-169. High-tech and Micropropagation 1. Springer-Verlag, Berlin.  Schenk RU and Hildebrandt AC (1972) Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can. J. Bot. 50,199-204.  Tricoli DM, Maynard CA and Drew AP (1985) Tissue culture propagation of mature trees of Prunus seritona Ehrh. I. Establishment, multiplication and rooting of in vitro. For. Sci. 31, 201-208.  Vinocur B, Carmi T, Altman A and Ziv M (2000) Enhanced bud regeneration in aspen (Populus tremula L.) roots cultured in liquid media. Plant Cell Rep.19, 1146-1154.  Wang Q and Tang H (1994) Phenol induced browning and establishment of shoot-tip explants of fuji apple and jinhua pear cultured in vitro. J. Hort. Sci. 69,833-839.  65  Zhang CH, Chen D, Elliott MC and Slater A (2001) Thiazuron-induced organogenesis and somatic embryogenesis in sugar beet (Beta vulgaris L.). In Vitro Cell. Dev. Biol.—Plant 37, 305-310.  66  Table 3.1. Explant survivorship (%) among Daphne species following a 4 wk establishment phase on one of five basal media without PGR supplements; mean percent ± SD.  Species  MS  WPM  85  SH  LS  D. caucasica  82+2.1  2.5+1.5  0  2.5+1.6  0  D. cneorum  5+0.3  85+3.8  0  0  0  D. giraldii  87+3.8  5+2.4  5+1.3  0  0  D. jasmi ilea  7+1.6  92+2.1  0  0  0  D. laureola  75+0.8  2.5+0.6  0  0  0  D. retu.va  92+3.8  2.5+0.8  0  0  0  D. tangutica  92+2.6  5+1.3  0  0  2.5+1.1  67  Table 3.2. Shoot explant multiplication rates following 8 weeks of culture on basal media supplemented with a single cytokinin and no auxin; mean ± SD.  Treatment Cvloki loin Supplement  Species D. caucasica  D. cneorum  D. girokli  D. jasminea  D. !aureola  D. reins('  D. langulica  No cytokinin  0.33+0.0  0.06+0.4  0.13+0.0  0.20+0.3  0.20+0.0  0.46+0.2  0.48+0.1  0.1 mg 1 - ' BA  0.80+0.1  0.08+0.3  0.31+0.1  0.20+0.4  0.26+0.1  1.02+0.8  0.93+0.3  0.5 mg  r' BA  1.51+0.3  0.28+0.2  1.75+0.3  0.44+0.0  0.82+0.5  2.22+1.2  2.08+0.8  I mg I - ' BA  1.77+0.5  0.53+0.1  2.04+1.1  0.60+0.3  1.64+1.1  2.13+0.8  7.02+0.5  r' BA  2.46+1.1  0.66+0.3  2.71+0.8  0.73+0.2  1.95+0.8  2.55+1.4  2.60+1.3  4 ing 1' BA  1.82+0.3  0.33+0.2  2.28+1.3  0.40+0.1  1.77+1.5  7.53+0.3  2.33+0.9  IA KIN  0.46+0.2  0.06+0.1  0.46+0.6  0.13+0.3  0.20+0.1  0.53+0.1  0.46+0.1  0.5 mg 1 1 KIN  0.62+0.3  0.06+0.0  0.55+0.3  0.20+0.1  0.60+0.4  0.80+0.6  0.60+0.5  1 mg 1 1 KIN  1.22+0.8  0.20+0.4  1.66+1.1  0.40+0.6  0.86+0.3  1.13+0.7  1.17+0.8  2 mg  r' KIN r' KIN 0.4)001 mg rfrrnz  1.73+0.0  0.28+0.3  1.40+0.8  0.42+0.3  1.77+0.5  2.20+1.1  1.71+0.7  4 mg  1.82+0.3  0.20+0.1  1.42+0.5  0.33+0.2  1.68+1.2  1.97+0.3  1.86+1.1  0.91+0.5  0.13+0.0  0.73+0.3  0.26+0.1  0.86+0.6  1.33+0.2  1.26+0.0  0.001 mg 1 1 TD7.  1.91+0.3  0.42+0.1  1.33+0.7  0.73+0.5  1.66+0.9  1.55+1.1  1.57+0.2  1.33+0.7  0.42+0.2  1.22+0.5  0.66+0.3  1.73+0.5  1.88+1.2  1.53+0.5  1.06+0.8  0.22+0.3  1.08±0.8  0.33+0.2  1.42+1.1  1.86+0.8  0.95+0.6  0.84+0.1  0.13+0.2  0.86+0.3  0.06+0.1  0.55+0.8  0.86+0.5  0.93+0.8  1.28+0.3  0.25+0.1  1.24+0.4  038+0.1  1.12+0.3  1.56+0.5  1.4+0.4  2 mg  0.1 mg  rfrrnz 0.1 lug 1 Inz 0.2 mg rbrnz 11.01 mg  -  Average  68  Table 3.3. Shoot explant multiplication rates following 8 weeks of culture on basal media supplemented with a combination of cytokinins and auxins; (P< 0.05). Species  Supplement  I mg 1 - ' BA + 0.001 mg 1 -1 IAA  1 mg 1 -1 BA + 0.01 mg 1 -I IAA I mg 1 -1 BA + 0.1 mg 1 -1 IAA 2 mg 1 -1 BA + 0.001 mg l i IAA 2 mg 1 -1 BA + 0.01 mg F' IAA 2 mg 1 - BA + 0.1 mg I - ' IAA I mg I BA + 0.001 mg 1 -1 NAA I mg 1 - BA + 0.01 mg 1 -I NAA I mg 1 - BA + 0.1 mg 1"' NAA 2 mg 1 - BA + 0.001 mg 1 - ' NAA 2 mg,1 - BA + 0.01 mgl -I NAA 2 mg 1 - BA + 0.1 mg 1 -1 NAA I mg 1 -^BA + 0.001 mg 1 -1 IBA I mg 1 -^BA + 0.01 mg 1 -1 IBA I mg 1 -^BA + 0.1 mg 1 -1 IBA 2 mg F ' BA + 0.001 mg I - ' IBA 2 mg 1 -^BA + 0.01 mg I - ' IBA 2 mg F' BA +0.1 mg 1 - ' IBA 2 mg 1 -^KIN + 0.01 mg 1 - ' IAA 2 mg F' KIN + 0.01 mg 1 - ' NAA 2 mg F' KIN + 0.01 mg 1 - ' IBA 4 nig 1 -^KIN + 0.01 mg 1 - ' IAA 4 mg 1 - ' KIN + 0.01 mg 1 - ' IAA 4 mg 1 - KIN + 0.01 mg 1 -i IAA 0.001 mg I ' TDZ + 0.01 mg 1 -1 IAA 0.001 mg 1 - ' TDZ + 0.01 mg F' NAA 0.001 mg 1 -i TDZ + 0.01 mg 1 -1 IBA 0.001 mg 1 - ' TDZ + 0.1 mg 1 -1 IAA 0.001 mg F' TDZ + 0.1 mg I - ' NAA 0.001 mg F I TDZ + 0.01 mg 1 -1 IBA -  D. caucasica  D. cneorum  D. jaSnlinea  D. laureola  D. retusa  giroldn  D.  D.  1.9+0.3 1.9+0.9 2.2+0.6 1.6+0.5 1.91+0.0 2.20+0.3 4.00+0.8 5.33+0.7 3.73+0.8 4.66+0.5 5.64+0.7 4.31+0.8 2.75+0.0 3.71+0.9 3.06+0.6 2.93+0.7 3.93+0.9 3.37+0.6 1.95+0.3 3.06+0.4 2.24+0.2 1.86+0.5 2.66+0.8 2.08+0.7 2.84+0.9 3 - 57 0.5 3.13+0.2 2.11+0.8 2.91+0.9 2.55+0.2  1.46+0.6 2.13+0.0 2.06+0.2 1.71+0.3 2.13+0.8 2.26+0.7 3.20+0.9 5.06+0.6 4.57+0.8 3.40+0.0 5.13+0.4 4.66+0.3 2.60+0.9 3.04+0.3 2.75+0.7 2.66+0.0 3.13+0.1 2.86+0.7 1.33+0.0 2.73+0.8 1.77+0.3 2.17+0.2 2.60+0.4 2.35+0.3 1.86+0.2 2.62+0.3 2.33+0.0 2.17+0.3 2.42+0.3 2.08+0.3  1.80+0.8 2.42+0.3 2.15+0.3 1.93+0.1 2.75+0.2 2.66+0.4 4.33+0.3 4.91+0.8 4.37+0.2 4.64+0.3 5.06+1.1 4.77+0.3 2.93+0.0 3.64+0.3 3.40+0.1 2.86+0.6 3.66+0.2 3.26+0.0 2.84+0.3 3.06+0.1 2.75+0.3 2.20+0.5 2.64+0.3 2.40+0.4 2.06+0.6 2.75+0.3 2.91+0.7 2.26+0.4 2.40+0.2 2.33+0.7  1.55+0.3 2.40+0.0 2.00+0.0 2.13+0.6 2.26+0.2 I .93±0.5 5.35+0.3 6.93+0.7 5.28+0.3 6.13+0.9 6.46+0.2 5.53+0.2 3.66+0.3 3.91+0.4 3.20+0.6 3.57+0.3 3.84+0.3 3.80+0.0 1.68+0.3 2.57+0.3 1.93+0.0 2.13+0.4 2.60+0.4 2.40+0.3 2.33+0.5 2.86+0.7 2.55+0.1 2.26+0.4 2.55+0.2 2.33+0.3  1.46+0.5 2.08+0.3 2.15+0.3 1.33+0.0 2.26+0.4 2.53+0.6 4.06+0.1 5.17+0.8 3.64+0.5 4.80+0.2 5.46+0.5 4.73+0.4 2.75+0.4 3.91+0.8 3.46+0.2 2.97+0.6 4.06+0.9 3.86+0.4 1.88+0.2 2.93+0.2 2.28+0.4 1.93+0.3 2.73+0.3 2.15+0.7 2.91+0.4 3.71+0.5 3.17+0.3 2.28+0.2 3.13+0.4 2.86+0.0  1.93+0.4 2.26+0.2 2.35+0.0 2.06+0.4 2.86+0.7 2.75+0.3 5.31+0.3 6.48+0.2 4.60+0.5 5.00+0.6 5.46+0.6 4.66+0.9 3.08+0.4 4.06+0.5 3.97+0.3 3.00+0.1 4.15+0.3 4.22+0.3 2.55+0.2 2.64+0.2 2.66+0.1 2.13+0.0 2.95+0.3 2.53+0.0 2.93+0.2 3.20+0.6 3.00+0.2 2.55+05 3.00+0.7 2.53+0.4  2.13+0.1 2.33+0.0 2.40+0.3 2.00+0.2 2.84+0.2 2.80+0.2 5.35+0.4 7.73+0.8 4.48+0.3 5.13+0.0 5.66+0.0 4.55+0.3 3.13+0.0 4.28+0.3 3.93+0.0 3.06+0.0 4.24+0.3 3.84+0.3 2.40+0.0 3.13+0.0 2.86+0.0 2.08+0.3 3.04+0.3 2.33+0.2 3.00+0.3 3.26+0.4 2.93+0.1 2.13+0.3 2.86+0.0 2.51+0.7  4.93+0.8  4.60+0.0  4.13+0.9  4.82+0.6  3.80+0.3  4.28+0.4  4.84+0.3  4.53+0.7  4.35+0.3  4.15+0.4  4.46+0.8  3.64+0.7  4.68+0.8  4.42+0.1  tangtuica  0.5 mg1 -1 BA+0.001 mg r' I'm + 0.01 mg I - '^IAA 0.5 mg,1 -1 BA+0.001 mgl -I TDZ + 0.01 mg I - ' IBA 0.5 mg1 -1 I3A+0.001 mg 1 - ' TDZ + 0.01 mg 1 -1 NAA 0.5 mgr s BA+0.01 mg 1 -1 TDZ + 0.01 mg 1 -1 NAA  5.57+0.7  5.37+0.3  4.33+0.5  7.84+0.6  4.04+0.8  5.46+1.1  6.86+0.8  5.26+0.6  5.26+0.6  4.40+0.6  5.46+0.9  4.15+0.7  4.88+0.9  5.13+0.6  A verage  3.24+0.6  2.96+0.3  3.2+0.4  3.55+0.5  3.18+0.4  3.5+0.3  3.6+0.2  408.9  1045.7  4137.7  F(49, 100) ( P< 0.05 )  ,  69  2871.2  6528.5  7788.9  9755.5  Table3.4. Daphne rooting frequency (%) after 8 weeks of culture on media incorporating PGRs  Species  Supplement/Modification D. caucasica  D. encomia  D. giraldi  D. jasminea  D. !aureola  D. retusa  D. tartgutica  0+0  0+0  0+0  0+0  No PGR Supplement  0 +0  0+0  0+0  1/2 strength salts  0 +0  6.67+0.24  0+0  15.56+2.4  6.67+1.667  6.67+0.8  6.67+1.6  1/2 sucrose  0 +0  0+0  0+0  6.67+0.00  0+0  0+0  0+0  0.001 mg 1 1 IAA  0 +0  3.33+0  0+0  8.89+3.8  11.11+3.8  0+0  0+0  0.01 mg 1' 1 IAA  13.33+6.6  6.67+3.8  17.78+3.1  26.67+2.4  20+1.1  0+0  0+0  0.1 mg r' IAA  15.56+3.8  6.67+0  22.22+3.84  40+3.8  33.33+6.6  11.11+3.8  20.00+2.4  0.5 mg 1 1 IAA  2.22+3.8  17.78+3.8  31.11+1.05  26.67+1.2  13.33+0.8  17.78+3.8  20.00+3.8  0.01 mg r' IAA+ 1 mg 1 4 Charcoal  13.33+-6.6  22.22+0.6  13.33+1.67  46.67+2.8  20+2.4  26.67+0.8  13.33+1.1  0.001 mg r' NAA  42.22+3.8  28.89+6.6  40+2.0  37.78+3.8  41.11+4.6  26.67+1.8  35.56+4.3  0.01 mg r' NAA  37.78+3.8  33.33+3.8  43.33+2.3  48.89+3.8  40.00+0  33.33+3.8  33.33+1.6  0.1 mg r' NAA  24.44+3.8  15.56+3.8  28.89+0  41+1.5  34.44+3.8  38.25+4.1  43.44+2.4  0.5 mg r' NAA  31.11+3.8  13.33+6.6  26.67+1.3  26.67+1.2  33.33+2.4  22.22+3.8  26.67+3.8  0.01 mg 1 4 NAA+ 1 mg r' Charcoal  46.67+0.6  33.33+0  44.44+3.8  46.67+3.8  46.67+3.8  46.67+2.6  51.11+2.1  0.001 mg r' IBA  31.11+3.8  8.89+3.8  40+ 2.4  37.78+2.7  44.44+3.8  33.33+0.8  26.67+3.8  0.01 mg I -I IBA  35.56+3.4  31.11+3.8  33.33+1.4  48.89+3.8  33.33+2.3  40.00+4.1  26.67+1.6  0.1 mg 1'' IBA  17.78+3.8  22.2+1.4  24.44+3.8  46.67+2.1  33.33+6.6  20.00+1.6  17.78+2.4  20.00+0.6  8.89+3.8  13.33+1.5  40.00+1.6  13.33+1.5  40.00+3.8  26.67+2.1  3556+3.8  24.44+0  35.56+3.8  44.4+3.6  35.56+3.8  31.11+2.6  22.22+1.6  r' NAA+ 0.1 mM1 -1 1AA  42.22+3.8  37.78+3.8  40+ 2.1  46.67+3.6  35.56+3.8  33.33+3.1  40.00+3.8  0.1 m1111 ' NAA+ 0.1 mM 1 4 IBA  53.33+6.6  41.44+3.8  40+3.8  76.14+ 2.4  42.18+2.6  46.67+2.4  53.33+2.6  0.1 mM 1 1 IAA+ 0.1 mM !IBA  22.22+3.8  28.89+2.4  24.44+3.8  40.23+1.3  28.89+3.8  22.22+3.8  13.33+1.1  1 mM r' IBA+ 1 mM r' NAA  42.22+3.8  35.56+3.8  44.44+2.4  78.00+3.8  39.88+3.8  40.00+2.8  51.11+3.8  Average  23.94+1.1  20.24+0.8  25.6±13  37.76+2.6  27.56+3-8  24.36+2.1  23.99+1.6  31.8  66.2  443  31.1  29.6  0.5 mg I A IBA 0.01 mg 1 4 IAA+ 1 mg 0.1 mM  r' Charcoal  -  F value (21,44)  0.05 =  373  39.7  70  ^ ^ ^ ^  .  0 0  cG bq  01 O O  a)  E  a) CC con  ^rz:i 0  ^,  a a cs0 (1)  cu  .2Le  00  a)  ccs  O  al  a  O  ^ci  rn  0  cu sa. ass  (1.-s in fn  cd  1111.1  Q  O  4.1  1  •  oo fn  -F 1  \O \.0 +1  cp  s.0  00 -1-1 "1/N crl  ',el  c-,1  11---  0\  4.--1 en  00 +1  00  Ir---  +1  ,-,  +i  kr1 en  oo en  7  ,.17 '.0 +1  .6  00 en +1 VD  0\  en  00 en  +1  +1 en  en  en  CT\ +1 ■ID  +1  +1 O  oo^Q1^00^0.^oo^oo en  +I^+I^+1^-I- 1^-I- 1^1^+1  tri^"1-^  en  ▪  00  +1  en  '17  r-tti  CN  -1-1 00 11,1.1  o^en^"1 ^ +I N^fn^00 r-^.O^en^  0  -  fn^\O^\CD^00^en^fn^N^11,1 1-1^-1-1^-1-1^+1^+1 +1^ CO^ +1^+1^1-1 00 \O^•0^71-  00^00^■0  ..0^00^00^en^-Cr^Cl^en^en^  a";^r--.^ca^,..0.^ca^0,^WI^r--^ri If)  -1oo^oo^O. vD^o0 en^Ch^ co^'Cr^en^en^.—. 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'" at .,.,.^= 4,^;--  76^4.  <  . . . ,^__,^__, _.E. . . ___,E .:::^-^.:-Ill  , :,  '..^<  cco^E 4i^MI  72  00 00  r)  00  (NI  nl  73  Figure 3.1. Multiplication stage of Daphne species  74  Figure 3.2. Root formation of Daphne microshoots after 8 weeks on WPM media.  75  CHAPTER FOUR EVALUATION OF DAPHNE GERMPLASM FOR RESISTANCE TO DAPHNE SUDDEN DEATH SYNDROME (DSDS) CAUSED BY THE SOIL-BORNE PATHOGEN THIELAVIOPSIS BASICOLA  4.1 Introduction The genus Daphne L. (Thymelaeaceae Juss.) is comprised of approximately 95 species (Flora of China, 2005) distributed through Africa, Northern and Southern Europe, the Middle East, Asia and regions of Oceania. Of these species, several have been commercialized because of their many desirable horticultural characteristics including attractive foliage, plant habit, flower color, and most of all, pleasant fragrance. Specifically, D. cneroum L. (Rose daphne or garland flower) has become one of the most popular perennial flowering shrubs among ornamental plant growers. However, the genus has acquired a poor reputation because of poor long-term performance of this and other Daphne introductions. One of the major limitations to daphne's survival in cultivation is Daphne Sudden Death Syndrome (DSDS), a disease caused by the fungal root pathogen Thielaviopsis basicola (Berk. & Br.) Ferraris (syn. Chalara elegans Nag Raj et Kendrick) (Noshad et al. 2006).  I A version of this chapter has been accepted for publication in Hortscience. Noshad D, Punka ZK, Riseman A. Evaluation of Daphne gennplasm for resistance to Daphne Sudden Death Syndrome caused by the soil-borne pathogen  76  Thielaviopsis hasicola  This disease kills plants quickly, as the name suggests, following the first foliar symptoms. Observations on DSDS-infected plants indicate the following progression of symptoms: (i) brown to black necrotic lesions on the roots, (ii) leaf chlorosis leading to abscission, (iii) whole plant stunting, and (iv) stem collapse and plant death (Noshad et al, 2006).  Thielaviopsis basicola is a widespread root fungal pathogen found in both agricultural and nonagricultural soils (Anderson and Welacky, 1988; Trojak-Goluch and Berbec, 2005; Hood and Shew, 1996). A wide range of economically important plants are infected by T basicola including carrot (Daucus carom L.) (Punja etal, 1992), cotton (Gossypium hirsutum L.) (Wheeler etal, 1999), and tobacco (Nicotiana tabacum L.) (Hood and Shew, 1997b & 1996). Infection by T basicola commonly results in a form of black root rot disease, whole plant stunting, and delayed maturity, all of which reduce yield and crop quality. This pathogen is generally considered a facultative parasite, recently described (Reddy, 1989; Walker etal, 1999; Hood and Shew, 1997a&b) as a hemibiotrophic pathogen. This designation refers to T basicola's ability to survive for long periods of time outside of a living host by saprophytic consumption of soil organic matter (Hood and Shew, 1997b; Punja etal, 1993; Shew and Meyer, 1992). The fungus produces two spore types, phialospores (endoconidia) and chlamydospores (aleuriospores). The chlamydospore is characterized by a thick pigmented wall and divisions into compartments or segments, each of which is able to germinate. The phialospores are hyaline, not septate, and have a rectangular shape. The taxonomic classification of T. basicola is as follows (Nag Raj and Kendrick, 1975):  Kingdom: Fungi  77  Division: Eumycota Subdivision: Deuteromycotina (Fungi Imperfecti) Class: Moniliales Genus: Chalara Species: Thielaviopsis basicola (syn. Chalara elegans)  The literature indicates that variability in virulence, due to both external and internal factors, exists within T basicola. For example, T. basicola is reported to be most aggressive during the growing season when soil temperatures are 24°C (Walker etal, 1999) and soil water content is high (Rothrock, 1992; Walker et al, 1999). Bottacin et al. (1994) described variation in virulence among several isolates of T basicola and related this to their genetic composition. Specifically, they described the existence of dsRNAs in some, but not all, isolates of T basicola (Bottacin et al., 1994) and related this to changes in the level of virulence (Park etal, 2005; Punja etal, 1995; Geldenhius etal, 2004). In support, it has been shown that strains without dsRNA were less pathogenic than strains with one dsRNA strand and that these were more pathogenic than strains with more than one dsRNA strand (Punja, 1995). As a result, the authors have concluded that the presence of this particular dsRNA in basicola increases the pathogenicity of this fungus (Punja, 1995; Park etal, 2005). Most recently, Park et al (2005) examined infection of T. basicola by two totivirus-like dsRNAs. A full-length cDNA clone was developed from the 5.3 kb dsRNA element present. Sequence analysis revealed that it contained three large putative open reading frames (ORFs). These results indicate that the presence of putative virus-like particles in the cytoplasm, which were  78  similar in both shape and size to viruses in the Totiviridae, increase the virulence in certain isolates of T basicola (Punja, 1995; Park etal, 2005; Wattimena, 2001).  Observations on the interactions of T basicola with other organisms show that T. basicola can be sensitive to antagonism (Howell, 2003, Hood and Shew, 1997b). For example, Pseudomonas spp. populations in the rhizosphere of tobacco roots produced biocontrol  compounds (e.g., hydrogen cyanide (HCN)) suppressive to T. basicola (Troxlert and berling, 1997). The antagonistic activity of Trichoderma spp. against many pathogens, including T. basicola, have also been reported (Howell etal, 2003). However, biocontrol using Trichoderma against T. basicola has not yet been approved for commercial applications  (Howell etal, 2003). In another study, a synergism between the root-knot nematode, Meloidogyne incognita (Kofoid & White), and T basicola, was identified. When occurring  together, these pests greatly facilitate the development of black root rot in cotton; a seedling disease that is characterized by severe rotting of the cortex of young cotton hypocotyls and roots (Walker etal, 1999). Evaluating genetic variation in T basicola using 1SSR-PCR, seven of fourteen primer pairs resulted in the amplification of single polymorphic fragment indicating quantifiable variation is present among populations (Geldenhuis etal., 2004). Continued use of these primers will enable further molecular characterization of this important pathogen resulting in an enhanced understanding of its population structure (Geldenhuis et al, 2004).  Little is known about the factors (e.g., cultural conditions, host plant genetics) that affect DSDS development beyond anecdotal observations and practices. For example, during  79  nursery production of daphne cultivars, many producers apply prophylactic fungicide treatments to help ensure crop health. While this is a relatively common practice, no published literature was found that directly addresses the efficacy of this practice to control DSDS. In addition, even if effective at controlling DSDS, reliance on fungicides is unsustainable and undesirable. One alternative to this practice is the development of disease resistant daphne cultivars. This strategy is typically more desirable because it can be highly effective in reducing disease, is environmentally benign, and usually entails little or no additional expense to producers (Crute, 1996; Dahlherga, 2001; Diaz-Pdrez 1995; Reeleder, 1999). However, it typically requires a long time horizon to achieve. To date, native host plant resistance to T. basicola has been identified during germplasm screens as part of various crop improvement or breeding programs. For example, Nicotiana glauca Graham was identified as resistant to T. basicola and subsequently incorporated into a tobacco (N. tabacum) breeding program (Trojak-Goluch and Berbec, 2005). In another germplasm  screen, Gossypium arborettm L. PI 1415 was found to be resistant to T basicola and subsequently incorporated into a diploid cotton breeding program (Berbec, 1976; Wheeler, 1999; Shankara, 1999). Based on these reports, evaluating Daphne germplasm for resistance to DSDS, via controlled screens, is a reasonable approach to identify host plant resistance to this pathogen. Therefore, the objectives of this study were: I) to develop an efficient method for evaluating resistance of Daphne taxa to T basicola; 2) to compare in vivo and in vitro methods for their efficiency in identifying resistance of selected Daphne taxa; 3) to develop a useful disease progression index (DPI) for use in taxa evaluations; and 4) to rank Daphne germplasm for resistance to 7. basicola.  80  4.2 Materials and methods  4.2.1 Plant material Thirty-two species and cultivars of Daphne were collected and maintained at the UBC Botanical Garden and Center for Plant Research, Vancouver, BC, Canada. All 32 taxa were included in an in vivo challenge while a subset of seven species was used in an in vitro challenge (Table 4.1). Container-grown stock plants were used to supply tissue for both in vitro tissue culture establishment and traditional vegetative propagation. Rooted plants were produced in July and August from terminal cuttings (50-100 mm in length) with the flower buds and lower leaves removed. Cuttings were made with a single shallow cut and soaked in an anti-fungal solution (Physan 20, Maril Products Inc., Tustin, CA) for 60 seconds. The cuttings were allowed to dry momentarily before being dipped in 0.4% IBA powder (Stim Root #2, Plant Products. Co. Ltd., Brampton, Ontario) and then placed in 6 cm pots filled with a course rooting medium (10 parts propagation grade perlite, 8 parts peat, 6 parts granite grit #2, 1 part pumice (double screened to remove fine particles), dolomite lime 65AG at 900 g m , and Micromax (trace elements) at 400 g m _3 ). The flats were placed under intermittent mist with bottom heat set at 22°C. Rooted cuttings were transferred to a polyhouse in October where they were allowed to go dormant but kept frost free. They were repotted in May into 12 cm pots filled with a well-drained medium (8 parts peat, 8 parts Turface MVP, 6 parts granite grit #2, 4 parts screened and pasteurized soil, 1 part pumice, dolomite lime 65AG at 670 g m -3 , Micromax micronutrients at 540 g m -3 , Osmocote 18-6-12 at 2150 g m -3 , and Psi Matric wetting agent). All stock plants were grown under shade cloth during the summer months and moved to a heated polyhouse during the winter months to prevent frost damage. Fertilization regiment included yearly top-dressing with Osmocote 18-6-12 at 5 g 1  81  gal pot. Fungicides were not used during stock production because we had very little disease pressure and we did not want to risk cross-contamination affecting the in vivo assay.  4.2.2 Pathogen culture and suspension preparation  A single aggressive pathogenic isolate of T. basicola was cultured from diseased daphne plants and used throughout this study (Noshad et al., 2006). A suspension of endoconidia was prepared by gently washing the surface of 3-week-old colonies with deionized water and vortexing the wash solution for 30s. The resulting suspension was twice filtered through four layers of cheesecloth to remove agar, hyphae, and chlamydospores. The spore suspension was calibrated with a haemocytometer and adjusted with deionized water to obtain a final I  concentration of endoconidia of l x 10 6 ml prior to inoculation. A preliminary experiment was conducted to determine the optimum concentration of pathogen spores for effective inoculation. The results of this experiment indicated that a concentration of I x10 6 ml l induces infection within a reasonable time (Appendix 1).  4.2.3 ►z viva challenge  The conidial suspension (5 ml) was topically applied to healthy roots of 2-year old nurserygrown plants. Production containers were modified to contain a clear panel behind a lightproof 'door' to allow for direct observation of the infection process without further disturbance to the root system (Fig 4.1). All procedures were the same for control plants except for the application of distilled water instead of the spore suspension. To allow for uniform conditions following inoculation, plants were transferred to a greenhouse and grown under natural light at 25 +1 °C and a relative humidity between 70 and 80%.  82  4.2.4 In vitro challenge  Seven of the 32 taxa were selected for inclusion in an in vitro challenge (Fig 4.2). Clean cultures of these taxa were established and axillary shoot proliferation obtained from nodal explants cultured on either MS (Murashig and Skoog, 1962) or WPM (Woody Plant Medium; McCown and Lloyd, 1983) supplemented with plant growth regulators (i.e., 2 mg 1 -I BA + 0.01 mg I-1 NAA), recommended minerals and 5.6 g I agar based on these two major  protocols (Murashig and Skoog, 1962; McCown and Lloyd, 1983). All the in vitro plant cultures were maintained at 24°C with a 16/8 h photoperiod supplied by cool white fluorescent lamps delivering 3501LIW cm -2 . Axillary shoots were rooted by subculturing on WPM/MS medium with vitamins (nicotinic acid 0.5 g F 1 , pyridoxine-HCI 0.5 g 1 -1 , thiamineHC1^, and glycine 2 g 1 1 ), 20 g 1 -1 sucrose, and 5.6 g 1 -1 agar following dipping in plant growth regulators filter sterilized solution i.e. IBA (10 mM) + NAA (I mM). Following six to eight weeks, shoots were rooted and ready for inoculation. Using a I ml syringe, I ml of the conidial suspension was injected next to a root segment while still embedded in the culture medium (Fig 4.1). All control plants were treated equally to the test plants except for injection of distilled water instead of the conidial suspension (Svabova, 2005; Jarausch, 1999).  4.2.5 Disease assessment  For both methods, observations on disease progression following inoculation were made weekly over eight weeks. The following disease progression rating (DPR) was developed: 0 = healthy plant, no symptoms; 1 = less than five lesions on lateral roots, no lesions on tap  83  root, no foliar symptoms; 2 = greater than five lesions on lateral roots, less than five lesions on tap root, no foliar symptoms; 3 = most lateral roots with lesions and some necrosis, greater than five lesions on tap root, five to ten chlorotic leaves; 4 = most lateral roots necrotic, greater than five lesions on tap root, most leaves chlorotic with some leaf abscission; 5 = plant is dead. A plant disease index (PDI) was developed based on both disease incidence (DI) and disease severity (DS) as follows:  PDI = DS/L X DI/N X 100  where DS= disease severity (i.e., average DPR value among diseased plants); L= the number of DPR disease categories; DI= disease incidence (i.e., number of diseased plants); and N= total number of plants. This approach allows for greater resolution of absolute taxon susceptibility by combining both relative disease progression within individuals with the absolute occurrence of the disease within a taxon. Therefore, based on this assessment, a completely susceptible taxon would score 100 while a completely resistant taxon would score 0.  4.2.6 Data analyses Both methods used a completely randomized design with three replications. Each replicate consisted of 24 plants per taxon with two plants serving as controls. Descriptive statistics, analysis of variance, correlation, and Tukey's honest significant differences (HSD) test were generated using SPSS 11.5 software (SPSS Inc. Statistical Package for the Social Sciences, Chicago, US).  84  4.3 Results  4.3.1 In vivo screen Significant differences (P<0.05) among taxa were observed for PDI values of T. basicola infection. Eight weeks following inoculation, PDI values ranged from 64.6 for D. cneorum to 0 for D. tangutica and D. retusa (Table 4.1). For all other taxa, PDI values ranged between these extremes and with varying levels of chlorosis, leaf abscission, and stunting observed. Typical disease progression in susceptible taxa can be generalized as follows: 1-3 weeks post-inoculation- brown to black necrotic lesions on the roots; 4-6 weeks post inoculation- leaf chlorosis leading to abscission; and 6-8 weeks post-inoculation- whole plant stunting, stem collapse and plant death. However, for highly resistant taxa, no visible infection or discoloration developed at any time.  4.3.2 In vitro screen Similar to the in vivo challenge, significant differences between the susceptible and resistant taxa evaluated were identified for T basicola development. As in the in vivo screen, D. cneorum was the most susceptible to T basicola infection (PDI = 72.2) while D. tangutica  and D. retusa were the most resistant (PDI = 3.3 and 3.6, respectively) (Fig 4.2). For the five remaining taxa, PDI ranged between these extremes and with varying levels of chlorosis, leaf abscission, and stunting observed (Fig 4.2).  85  4.3.3 Assay comparison For the seven taxa included in both assays, strong similarities were present for overall taxa performance while a significant difference in the timing of disease progression was present. A high correlation coefficient (R=0.87) was calculated between these systems for PDI values, indicating both systems were comparable in evaluating disease susceptibility among these seven taxa. In addition, the rank order of taxa based on DPI values for the two methods were identical, further supporting these methods as equal in evaluating disease susceptibility. However, the in vitro system produced results in significantly less time. On average, three fewer weeks were required to reach the same level of disease progression as compared to the  in vivo system.  4.4 Discussion The identification and incorporation of host-plant resistance into susceptible plants is an often sought-after goal for many breeding programs. It has been successfully achieved using both conventional breeding as well as biotechnological methods. However, despite significant differences between these two approaches, both rely on a robust germplasm screen to differentiate germplasm performance following pathogen exposure. A screen incorporated into a traditional breeding program often involves the evaluation of progeny derived from hybridizations between resistant and susceptible parents or the evaluation of related taxa if resistance was not present in the most advanced gene pool (Punja, 2001; Agrios 2005; Iglesias, 2000; Daryonol 2005). This approach has been successfully used to transfer T basicola resistance between related species. In independent breeding programs, T.  basicola resistant tobacco genotypes were developed based on germplasm screens of related  86  Nicotiana species (Bai et al. 1996; Palakarcheva 1995; Trojak-Goluch and Berbec 2005).  Specifically, the use of a robust germplasm screen allowed researchers to identify T. basicola resistance in Nicotiana debneyi (Bai et al. 1996; Wilkinson, 1991) and to further conclude it was conditioned by a single dominant gene. Once transferred to a susceptible genetic background, this gene conferred the same degree of resistance as found in the original N. debneyi accession (Legg, 1981; Palakarcheva 1995, Bai et al. 1996; Keller, 1999). In a Gossypium germplasm screen designed to identify T. basicola resistance, significant  variation was observed among taxa, with the strongest resistance identified in Gossypium arboreum. This resistance was then successfully transferred to commercial cotton cultivars  (Walker 1999; Weeler et al, 1999; Rothrock, 1992). Development of a robust T. basicola germplasm screen is valuable in not only identifying taxon-specific variation for pathogen resistance but also for further evaluation of the plant's genetic structure. Among the 32 Daphne taxa evaluated, significant differences were present for resistance to T. basicola  under both in vivo and in vitro challenges. Of the 32 taxa, D. langulica and D. retusa displayed the greatest resistance and remained symptom-free during the in vivo challenges, while displaying only mild symptoms in the in vitro challenges. At the other extreme, D. cneroum was clearly the most susceptible taxon in both screens and became fully diseased  followed by plant death in the shortest amount of time. The observed range in disease resistance among Daphne taxa indicates my challenge was effective and identifies D. tangutica and D. retusa as potential sources for resistance.  The inoculation and screening methods were proven robust in terms of the isolate's pathogenicity, disease characterization, consistency over time, and in its ability to  87  differentiate taxa. Based on the one isolate used, overall pathenogenicity was adequate in allowing assessment of disease incidence and severity (i.e., root and foliar symptoms) on all taxa. Also, the concentration of conidia used was sufficient to cause disease but not to overwhelm the defense mechanism(s) and prevent taxa differences from being displayed. Finally, both screening methods produced comparable and consistent results over an 11month period despite the in vivo challenge being conducted in an outdoor polyhouse exposed to seasonal fluctuations. These observations validate our screen methodology and support its continued use in identifying genetic variation among Daphne species for resistance to DSDS.  The results of the in vitro and in vivo experiments indicate a strong correlation between these two assay methods. However, there were differences in disease progression rates between them. Typically, the in vitro challenge produced a comparable level of disease as the in vivo challenge but in two to three weeks less time. Differences in disease development rates between plants produced from tissue culture and traditional propagation have been reported and may be based on anatomical (e.g., root structure), biological (e.g., adaptation mechanisms with other organisms) or physiological (e.g., difference in biochemical compounds) differences (Diaz-Perez, 1995). In addition, there was a small temperature difference between my two assay environments with the in vitro assay conducted at a higher temperature than the previously reported optimum temperature for T. basicola (Walker et al., 1999). Based on 100% infection of D. cneorum, the most susceptible taxon, and rebust growth of the pathogen, l conclude this higher temperature is still within the effective range needed for infection.  88  One limitation of this research was the use of a single isolate of T basicola. The use of a single isolate limits the breath of conclusions that can be made. Various isolates of T. basicola, from different regions, may have genomic differences that could affect my assay  results. Resistance to one isolate cannot be assumed to extend to all races of this pathogen. It is possible that some of the Daphne taxa could react differently to other races. These other races could have different virulent genes (based on gene-for-gene theory) that interact with another type of resistance gene in Daphne. However, despite this limitation, for the one aggressive isolate used, significant taxa differences were observed for resistance.  In future studies, additional races of T basicola should be collected and used in the germplasrn screen to increase our knowledge about how these two organisms interact. This research may reveal important information on the pathogen's infection process and the various factors that influence both successful infection and successful defense.  89  4.5 References  Adams PB and GC Papavizas (1969) Survival of root-infecting fungi in soil. Phytopathology. 59, 135-138.  Agrios GN (2005) Plant Pathology. 5th ed. Academic Press, Elsevier Science and Technology books, NY.  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Phytopathology. 84, 303-312.  90  Crute IR and Pink DA (1996) Genetics and utilization of pathogen resistance in plants. The Plant Cell 8, 1747-1755.  Dahlberga JA and Bandyopadhyay R (2001) Evaluation of sorghum germplasm used in US breeding programmes for sources of sugary disease resistance. Plant Pathol. 50, 681-689.  Daryonol BS and Natsuaki KT (2005) Screening for resistance to Kyuri green mottle mosaic virus in various melons. Plant Breeding 124, 487-490.  Diaz-Perez J (1995) Acclimatization and subsequent gas exchange, water relations, survival and growth of microcultured apple plantlets after transplanting them in soil. Physiologia Plantarum 95, 225-229.  Flora of China Vol. 13 (Clusiaceae through Araliaceae). (2005) Science Press, Beijing, and Missouri Botanical Garden Press, St. Louis.  Gayed SK (1972) Host range and persistence of Thielaviopsis basicola in tobacco soil. Can. J. Plant Sci. 52, 869-873.  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Nag Raj TR and Kendrick B (1975) A monograph of Chalara and allied genera. Wilfred Laurier University Press, Waterloo, Ontario.  Noshad D, Punja ZK and Riseman A (2006) First report of Thielaviopsis basicola on Daphne cneorum. Can. J. Plant Pathol. 28, 310-312.  Palakarcheva M (1995) Transfer of disease resistance genes by interspecific hybridization of wild growing Nicotiana species in Nicotiana tabacum. J. Genet. and Breeding 94, 99-105.  Papavizas GC (1968) Survival of root-infecting fungi in soil. Phytopathology. 58, 421-428.  93  Park Y, James D and Punja ZK (2005) Co-infection by two distinct totivirus-like doublestranded RNA elements in Chalara elegans (Thielaviopsis basicola). Virus Research. 109, 71-85.  Punja ZK (2001) Genetic engineering of plants to enhance resistance to fungal pathogens, a review of progress and future prospects. Can. J. Plant Pathol. 23, 216-235.  Punja ZK (1995) Influence of double-stranded RNAs on growth, sporulation, pathogenicity, and survival of Chalara elegans. Can. J. Bot. 73, 1001-1009.  Punja ZK, Chittaranjan S and Gaye MM (1992) Development of black root rot caused by Chalara elegans on fresh market carrots. Can. J. of Plant Pathol. 14, 299-309.  Punja ZK and Sun LI (1999) Morphological and molecular characterization of Chalara elegans (Thielaviopsis basicola), cause of black root rot on diverse plant species. Can. J. Bot. 77, 1801-1812.  Reddy MS and Patrick ZA (1989) Effects of host, nonhost, and fallow soil on populations of Thielaviopsis basicola and severity of black root rot. Can. J. Plant Pathol. 11, 68-74.  Rec.leder R (1999) Septoria leaf spot of Stevia rebaudiana in Canada and methods for screening for resistance. J. Phytopathology. 147, 605-613.  94  Rothrock CS (1992) Influence of soil temperature, water, and texture on Thielaviopsis basicola and black root rot on cotton. Phytopathology. 82, 1202-1206.  Schipper B (1970) Survival of endoconidia of Thielaviopsis basicola in soil. Netherlands J. Plant Pathol. 76, 206-211.  Shankara M and Cowling WA (1999) Screening for resistance to Diaporthe toxica in lupins by estimation of phomopsins and glucoseamine in individual plants. Plant Pathol. 48, 320324.  Shew HD and Meyer JR (1992) Thielaviopsisln: L. L. Singleton, J.D. Mihail, and C. M. Rush, eds. Methods for Research on Soilborne Phytopathogenic Fungi. Amer. Phytopathology, P. 171-174. Soc., St. Paul, MN.  Stover RH (1950a) The black root rot disease of tobacco. I. Studies on the causal organism Thielaviopsis basicola. Can. J. of Bot. 28, 445-470.  Stover RH (1950b) Effect of nutrition on growth and chlamydospore formation in brown and gray cultures of Thielaviopsis basicola. Can. J. of Bot. 34, 459-472.  Svabova L (2005) In vitro selection for improved plant resistance to toxin-producing pathogens. Phytopathology. 153, 52-64.  95  Tabachnik M, DeVay JE, Garber RH and Wakeman RJ (1979) Influence of soil inoculum concentrations on host range and disease reactions caused by isolates of Thielaviopsis basicola and comparison of soil assay methods. Phytopathology. 69, 974-977.  Trojak-Goluch A and Berbec A (2005) Meiosis and fertility in interspecific hybrids of Nicotiana tabacum L., N. glauca Grah. and their derivatives. Plant Breeding 126, 201-206.  Troxler J and berling C (1997) Interactions between the biocontrol agent Pseudomonas fluorescens CHAO and Thielaviopsis hasicola in tobacco roots observed by immunofluorescence microscopy. Plant Pathology. 46, 62-71.  Walker NR, Kirkpatrick TL and Rothrock CS (1999) Effect of temperature on and histopathology of the interaction between Meloidogyne incognita and Thielaviopsis hasicola on cotton. Phytopathology. 89:613-617.  Wattimena S (2001) Studies of factors affecting virulence of Chalara Elegans on Bean (Phaseolus Vulgaris L.). MSc. Thesis. Simon Fraser University.  Wheeler MH, Stipanovic RD (1979) Melanin biosynthesis in Thielaviopsis basicola. Expt. Mycol. 3, 340-350.  96  Wheeler TA, Gannaway JR and Keating K (1999) Identification of resistance to Thielaviopsis basicola in diploid cotton. Plant Disease. 83, 831-833.  Wick RL and Moore LD (1983) Histopathology of root disease incited by Thielaviopsis basicola in Ilex crenata. Phytopathology. 73, 561-564.  Wilkinson CA and Rufty RC (1991) Inheritance of partial resistance to black root rot in burley tobacco. Plant Disease. 75, 889-892.  Yarwood CE (1981) Occurrence of Chalara elegans. Mycol. 73, 524-530.  97  Table 4.1. Daphne taxa used in the Thielaviopsis basicola resistance bioassay with region of nativity or origin and mean plant disease index (PDI) values eight weeks following inoculation.  Taxa  Nativity/Origin  D. alpina L.  Italy  12.5 (0.92)  D. arbuscula Celak.  Czech Republic  43.3 (1.74)  D. bholua Buch-Ham. ex  Nepal  12.5 (0.82)  ghl  D. Xburkwoodii 'Carol Mackie'  Horticultural Origin  29.2 (1.32)  cdeig h  D. caucasica Pall.  Russia  9.2 (1.02)  D. circassica L.  Russia  46.6 (1.66)  D. cneorum L.  Czeck republic  64.2 (1.89)  D. collina Smith  Turkey  D. Xeschrnannii  Horticultural Origin  23.3 (1.17)  dei g hl  D. genkwa Siebold & Zucc.  China  43.3 (1.31)  allcdel  D. genkwa (Hackenberry group)  Horticultural Origin  15.0 (1.22)  D. ginidiuin L  Spain  27.5 (1.22) ' k W'  D. giraldii Nitsche  W China  15.8 (1.18) I g lu  D. jasatinea Sibth.& Sm.  Greece  13.3 (1.09) I ghl  D. kosaninii Stoj.  Bulgaria  20.8  D. laureola L.  N Africa  23.3 (1.34)  D. 'Lawrence Crocker'  Horticultural Origin  40.8 (1.80)  " "  D. longilobata Turril.  China-Yunnan  30.8 (1.06)  cdef g h  D. Xmantensiana  Horticultural Origin  57.5 (1.87)  abc  Mean PIN (SD) gh1  abcdel  hi  abale  a  51.7 (1.31) abcd  98  Ighi  (1.3) sigh' del ,Iii  ! d " ig  z  D. mezereum L.  Russia  10.0 (1.02) hi  D. mezereum (alba)  Horticultural Origin  15.0 (1.32) ghi  D. Xnapolitana  Horticultural Origin  39.2 (1.46)  D. odora Thunb.  China  31.7 (1.21) edelgh  D. oleoides Schreber  Turkey  17.5 (1.19) ighl  D. pontica L.  Russia  60.8 (1.54) ab  D. retusa Hernsl.  China  0.0 (0) .1  D. Xrollsdorlii 'Arnold Cihlarz'  Horticultural Origin  25.8 (1.54) ' 1 ' 1 ° 1  D. rossettii Gab.  Horticultural Origin b  15.8 (1.06) ighi  D. Xthauma  Horticultural Origin  34.2 (1.40)  D. tangutica Maxim.  China  0.0 (0)i  D. transcau.casica Pobed.  Turkey  19.2 (1.30) sigh'  D. 'Whilhelm Shacht'  Horticultural Origin  19.2 (1.18) 'kW'  abcdetg  hcdeigh  mean values followed by a common letter are not different (P<0.05) by Tukey's Honest  Significant Differences test.  99  Figure 4.1. Bioassay containers used for Daphne taxa challenged with Thielaviopsis basicola; A) in vivo assay pots modified with viewing panel for direct observation of diseased roots; B) in vitro assay with conidial suspension injected next to a root segment while still embedded in the culture medium.  100  Figure 4.2. Comparison of the in vitro with in vivo assays using seven Daphne taxa and Thielaviopsis  hasicola. Plant disease index (PDI) values are presented at five weeks following inoculation; Bars marked with the same letter do not differ significantly; error bars = SD.  I00 ^  ^ PDI(in vitro) PDI(in vivo)  90 Mr 70 60 50 40 30 be  20  he  10 0 Cry  t,  0.  sp. 0.  9  O  101  .  CHAPTER FIVE 1  COMPARATIVE EVOLUTIONARY ANALYSIS OF rDNA ITS SEQUENCES OF SELECTED DAPHNE SPECIES WITH REFERENCE TO RESISTANCE TO THIELAVIOPSIS BASICOLA  5.1 Introduction Plants are constantly being subjected to external stressors that require them to respond in an appropriate manner. Among these stressors, the plant's response to pathogen attack has been scrutinized intensely. A thorough understanding of how plants evolved an appropriate response to these stressors (i.e., pathogen resistance) is beneficial not only for our general understanding of how these processes work, but also for the practical application to crop improvement programs (Todd etal, 2000). Following the rediscovery of Mendel's work, plant breeders soon recognized that resistance to disease was often inherited as a single dominant or co-dominant gene (Keen, 1990).  Considerable knowledge has since accumulated on the genetic and biochemical and basis of disease resistance (Hammond-Kosack and Jones, 1996). Most recently, the tools of molecular biology coupled with phylogenetic analysis are now helping researchers to study disease resistance from an entirely new perspective, one of plant evolution.  102  Molecular sequence data have revolutionized phylogenetic analysis and our understanding of how various organisms are related. Since the late 1980s, molecular-based phylogenetic hypotheses have been proposed for nearly all groups of organisms. With reference to disease resistance, identifying a plant group's phylogenetic relationships can potentially provide insight into how often and under what conditions resistance may have evolved. Karban and Baldwin (1997) speculated that if plants native to environments with a specific endemic pathogen had more resistance than those without the pathogen, phylogenetic analyses could potentially provide insight into the evolution of disease resistance. Unfortunately, no publications relating phylogenetic placement and disease resistance have been identified.  One of the most popular sequences for phylogenetic inference at the generic and infrageneric levels in plants is the internal transcribed spacer (ITS) region of the 18S-5.8S-26S nuclear ribosomal cistron. Based on a survey of 244 molecular phylogeny papers (Alvarez and Wendel, 2003), two-thirds (66%) included ITS sequence data for comparisons at the genera level or below, and one third (34%) of all published phylogenetic hypotheses have been based exclusively on ITS sequences. The ITS region has become one of the most popular sequences to base phylogenetic inference because of its presumed advantageous properties and availability of comparison sequences (Baldwin et al., 1995). Because the 18S-26S rDNA regions reside in the nuclear genome, ITS sequences are biparentally inherited, and are thus distinguished from the cpDNA loci that are often used for phylogeny construction. Using sequences based on biparental inheritance has been shown critical in revealing past cases of reticulation, hybrid speciation, and parentage of polyploids (Baldwin et al., 1995; Kim and Jansen, 1994; Wendel et al., 1995). With the development of a universal primer set  103  for amplifying ITS sequences from most plant and fungal phyla (White et al, 1990), ITS sequence data are now more readily obtained than most nuclear-sequenced markers. In addition to these advantages, nuclear ribosomal genes are constituents of individual 18S5.8S-26S repeats of about 10 kb that are reiterated at one or more chromosomal loci per haploid complement (Baldwin et al., 1995). Because there are hundreds to thousands of nuclear rDNA repeats in plant genomes, they are more easily isolated than most low-copy nuclear loci. In angiosperms, ITS sequences vary in length from approximately 500-700 hp (Baldwin et al., 1995). Based on the ITS advantages of biparental inheritance, ease of sequence generation, and variability in sequence, I have chosen this marker to construct a phylogeny of selected Daphne taxa with special reference to resistance against Daphne Sudden Death Syndrome (DSDS) (Noshad et al., 2006a).  The genus Daphne L. (Thymelaeaceae Juss.) is comprised of approximately 95 species (Flora of China, 2005); although the number of species varies in different sources from 54-95. The genus is distributed through Africa, Northern and Southern Europe, the Middle East, Asia and regions of Oceania. There is no clearly accepted systematic treatment for this group. Over time, authors have defined several number of species and sub genera for this genus highlighting the systematic challenge this group presents (Mathew, 2000; Halda, 2001; Van der Bank, 2002). I used representative species from horticultural groupings, based on morphological traits (Mathew etal, 2000; HaIda, 2001).  Several Daphne species with horticultural merit (i.e., attractive foliage, plant habit, flower color, and fragrance) have been commercialized and introduced to consumers. Specifically,  104  D. cneroum L. (Rose daphne or garland flower) has become one of the most popular perennial flowering shrubs among discriminating ornamental plant growers. However, due to poor long-term performance of this and other daphne introductions, the genus has acquired a poor horticultural reputation. One of the major limitations to survival in cultivation is due to Daphne Sudden Death Syndrome (DSDS), a disease caused by the fungal root pathogen Thielaviopsis basicola (Berk. & Br.) Ferraris (syn. Chalara elegans Nag Raj et Kendrick) (Noshad et al. 2006a). This disease kills plants quickly, as the name suggests, following the first foliar symptoms. Observations on DSDS-infected plants indicate the following progression of symptoms: (i) brown to black necrotic lesions on the roots, (ii) leaf chlorosis leading to abscission, (iii) whole plant stunting, and (iv) stem collapse and plant death (Noshad et al, 2006b).  Thielaviopsis basicola is a fungus common in both cultivated and non-cultivated soils (King and Presley, 1942; Adams and Papavizas, 1969; Nag Raj and Kendrick, 1975; Yarwood, 1981) and is generally considered a facultative parasite with the ability to parasitize a wide range of important agricultural hosts (Bottacin et al., 1994; Anderson and Welacky, 1988; Papavizas, 1968; Reddy and Patrick, 1989). However, little is known about the factors (e.g., cultural conditions, host plant genetics) that affect DSDS development beyond anecdotal observations and practices. Previously, I identified taxon differences among 32 Daphne species and cultivars for resistance to Thielaviopsis basicola, by both in vitro and in vivo-  based methods (Noshad et al., in press). Plant reactions ranged from highly resistant, e.g., D. tangutica and D. retusa, to highly susceptible, e.g., D. cneorum.  105  The current study was guided by the following questions about the evolutionary relationships among selected Daphne taxa with reference to DSDS resistance: 1) `Can the ITS region be an informative system for the phylogenetic analyses of Daphne species?'; 2) `Is there any relationship between resistance against DSDS and the phylogenetic placement of Daphne species based on the ITS sequence analysis?'; 3) `Can a pattern be identified that places the origin of disease resistance among Daphne species?'.  5.2 Material and Methods  5.2.1 Plant materials  All plant samples were taken from living plants maintained at the UBC Botanical Garden and Centre for Plant Research (UBGCPR). For all taxa, voucher specimens were prepared and deposited in the UBGCPR herbarium while photographs of flowering specimens were taken and deposited in the UBGCPR library. Twenty Daphne taxa (i.e., ingroup taxi) used in the phylogeny construction are listed with their region of nativity or origin and mean plant disease index (PDI) values (Noshad, 2006h) (Table 5.1). To investigate the relationship between Daphne taxa, appropriate outgroup selection is critical. The outgroup taxa should be systematically close enough to the species under observation to allow sequence alignment and yet distant enough to enable unequivocal rooting of the tree. Therefore, ITS 1 and ITS2 sequences of 5 outgroup species i.e., Dias cotinifolia L., Gnidia denudata Lindi., Pimelea .spectahilis Lindi., Edgeworthia chrysantha Lindi., and 7hymelaea hirsute L. from the family  Thymeleaeceae were obtained from NCBI (National Center for Biotechnology Information; http://www.nchi.nlm.nih.gov ) and used in the analyses.  106  5.2.2 DNA extraction and sequence data Total genomic DNA was extracted from fresh samples collected from plants at UBC Botanical Garden. DNA isolation, PCR conditions, cycle sequence, and automated DNA sequencing were as specified in Moller and Cronk (1997). DNA sequence data were collected using the ABI Prism Dye Terminator Cycle Sequencing Kit (ABI PRISM. 0) and visualized using an ABI model Prism-377 DNA automated sequencer at the University of British Columbia NAPS (Nucleic Acid Protein Service). DNA was sequenced in both directions. Raw sequence data were imported into Sequencher 4.10 (Gene Codes Corporation, MI, USA), edited, and combined into a consensus sequence. Consensus sequences were imported into Se-Al® v 1.0 (Rambaut, 1996) and manually aligned using sequential pairwise comparisons with relative ease. Sequence length, alignment, and number of informative characters were determined using PAUP v. 4.0b100 (Swofford, 2000) and MacClade® (Maddison and Maddison, 2000). Summary data for these features are provided (Table 5.2).  5.2.3 Phylogenetic reconstruction Phylogenetic analyses were performed using maximum parsimony (MP). Parsimony analysis was conducted assuming unordered character states and equal character weighting. Gaps were treated as missing data and these were excluded from all analyses. Parsimony analyses were completed using PAUP® v. 4.0b10 and employed a heuristic search strategy with IOU random stepwise-addition replicates, TBR branch swapping, and MULTREES optimization. Consistency index (CI; excluding uninformative characters) and retention index (RI) were also calculated (Farris, 1989; Kluge and Farris, 1969). Branch support was  107  determined with bootstrapping (Felsenstein, 1985) using a simple addition sequence and 10,000 replicates, with all other parameters equal to those used in the MP analysis. This analysis was followed by an exhaustive run with sample frequency (i.e., number of trees saved per generation) set at 20,000. The remaining trees from this analysis were saved and a consensus tree with posterior probability values was generated. Resistance patterns were examined by mapping PDI values (i.e., degree of resistance) onto the phylogeny using MacClade® v. 4.0. Ten classes of resistance were used as character states, based on statistical analysis of PDI values (Noshad, 20066), for assessing the evolution of resistance (Fig 5.2).  5.3 Results  5.3.1 DNA matrix features and sequence divergence Alignment of internal transcribed spacer sequences of the 25 taxa analyzed (Fig 5.1) resulted in a 610-bp long data matrix; the number of gaps after alignment was 166. The G+C content ranged from 48.3-65.4%. Sequence divergence values among the 25 taxa ranged from 31.8 to 49.6% with divergence among the ingroup taxa between 0.0-30.1 %. Sequence characteristics are summarized (Table 5.2).  5.3.2 Phylogenetic results The number of variable sites (excluding indels) detected in the entire ITS region was 245 (Table 5.2). Of these sites, 176 were phylogenetically informative. Parsimony analysis of unambiguously aligned ITS sequences yielded 63 most parsimonious trees (670 steps) with uninformative and gapped characters excluded. From the 63 most parsimonious trees, the  108  strict consensus tree is shown (Fig 5.1). Bootstrap and jackknife support values were similar. The topology of all parsimonious trees was nearly identical with respect to the interrelationships among Daphne taxa. The strict consensus tree supports these selected Daphne taxa as a monophyletic group.  5.4 Discussion  The phylogenetic trees constructed give insight to the interrelationships among Daphne taxa. The most parsimonious trees suggest that Daphne is a monophyletic group (Fig 5.1). However, the placement for D. genkwa was unresolved and needs further research to more reliably place it in the phylogeny. Analysis of ITS sequence data resulted in a parsimony consensus tree with two well-supported clades and one Glade with a lower level of support. Clade I (including D. pontica, D. laureola, D. retusa. D. tangtttica and D. longilobata) had a bootstrap value of 100 and was comprised of species mostly from China but also includes species from N. Africa-middle east and Russia (Fig 5.2, Table 5.1). Clade II (including D. oleoides, D. alpina, D. kosanini and D. cneorum) had a bootstrap value of 100 and was  comprised of species mainly from Europe. Clade III (including D. giraldii, D. odora, D. bholua, D. gnidium, D. arbuscula, D. jasminea, D. caucasica, D. collina and D. circassica)  had the lowest bootstrap value (i.e., 78) and included species from around the world. In addition, there are two species (D. genkwa and D. mezereum) that are placed on separate branches from the other clades in all trees constructed (Fig 5.1). When country of origin is mapped to the consensus tree, no clear pattern emerges based on Glade structure. Based on these data and the limited taxon sample, establishment of a clear pattern for nativity is difficult. When degree of resistance for individual species is mapped onto the consensus  109  parsimony tree, no clear pattern for the evolution of resistance is apparent. However, when many random trees were constructed based on degree of resistance, all were similar in structure. This step tested the null hypothesis of no structure, and confirms that the consensus tree produced is not a result of randomness. In addition, when the average PDI values of the species within the Glade are compared (i.e., Clade 1=23, Clade 11=28, and Clade 111=27), no significant differences were identified. However, despite these close PDI averages among the clades, sufficient structure is visible to propose a hypothesis on the evolution of disease resistance in Daphne (Fig 5.2). I hypothesize that resistance is a derived character and that plants recently evolved this ability. I base this hypothesis on presence of a higher number of susceptible species than resistant species in Clades II and III (Fig 5.2). Initially, the predecessor of modern Daphne may have been 'susceptible' due to a lack of selection pressure in its region of origin. However, after diversifying and colonizing new regions, they may have been exposed to the pathogen and evolved traits related to resistance. Additional taxon sampling will help identify the extent of resistance within this genus and perhaps identify its origin.  In addition, a hypothesis can be put forward that relates susceptibly of the species currently in horticulture production and the evolution of resistance in the genus. The pathogen is reported to be native to various parts of Asia preferring colder environments (Paulin-Mahady etal, 1994; Punja and Sun, 1999; Hood and Shew, 1997; Walbot, 1985; Wendel, 1992) while most of the horticulturally relevant species are native to the more moderate climates of Western European and Mediterranean regions. Therefore, when these species were grown in Canada, it may have been the first encounter with this pathogen in an environment allowing  110  for disease development. Because these plants may not be exposed to this pathogen in their native region, they would not have evolved the necessary traits for resistance and therefore display a high level of susceptibility.  The present study has limitations that future studies should address. One major limitation was the limited number of Daphne species sampled. While all accessible taxa were included, many additional taxa are described in the literature and need to be collected and included in future work. Inclusion of these additional taxa will help researchers develop a better understanding of Daphne evolution, and their evolving interaction with the pathogen. Another limitation was the use of a single isolate of pathogen in the germplasm screen. As other races of the pathogen may affect the disease ratings calculated for each taxon, subsequent changes in tree structure may occur. Therefore, collecting and using additional races of pathogen, from around the world, would help resolve this issue.  Based on the data collected from this research, I cannot establish a reliable pattern for the evolution of disease resistance for this genus. However, the results indicate that rDNA ITS sequences can be used for phylogenetic study at the sub-generic level in Daphne. Future research should target all other Daphne taxa, both in terms of disease assessment and molecular study, in order to clearly establish a valid and robust phylogeny. Once this phylogeny is complete, a more clear understanding of how resistance has evolved in this group may be possible.  111  5.5 References  Adams PB and Papavizas GC (1969) Survival of root-infecting fungi in soil. Phytopathology. 59, 135-138.  Alvarez I and Wendel JF (2003) Ribosomal ITS sequences and plant phylogenetic inference, Mol. Phylogen. Evol. 29, 417-434.  Anderson TR and Welacky TW (1988) Populations of Thielaviopsis basicola in burley tobacco field soils and the relationship between soil inoculum concentration and severity of disease on tobacco and soybean seedlings. Can. J. Plant Pathol. 10, 246-251.  Baldwin BG, Sanderson MJ, Porter JM, Wojciechowski MF, Campbell CS and Donoghue MJ (1995) The ITS region of nuclear ribosomal DNA: A valuable source of evidence on angiosperm phylogeny. Ann. Missouri Bot. Gard. 82, 247-277.  Bottacin AM, Levesque CA, and Punja ZK (1994) Characterization of dsRNA in Chalara elegans and effects on growth and virulence. Phytopathology. 84, 303-312.  Farris JS (1989) The retention index and homoplasy excess. System. Zool. 38, 406-407.  Felsenstein J (1985) Confidence limits on phylogenies: an approach using bootstrap. Evol. 39, 783-791.  112  Halda J. J (2001) The Genus Daphne. Eva Kucerova- Sen Dobre Publikaca, Prague, Czeck Republic.  Hammond-Kosack KE, Jones JDG (1996) Disease resistance gene-dependent plant defense mechanisms. Plant Cell 8, 1773-91.  Hood ME and Shew HD (1997) Reassessment of the role of saprophytic activity in the ecology of Thielaviopsis basicola. Phytopathology. 87, 1214-1219.  Karban R and Baldwin IT (1997) Induced responses to herbivory. University of Chicago Press, Chicago.  Keen NT (1990) Gene-for-gene complementarity in plant-pathogen interactions. Ann. Rev. Genetics. 24, 447-63.  Kim KJ, Jansen RK (1994) Comparisons of phylogenetic hypothesis among different data sets in dwarf dandelions (Krigia): Additional information from internal transcribe spacer sequences of nuclear ribosomal DNA. Plant Syst. Evol. 190, 157-185.  King CJ and Presley JT (1942) A root rot of cotton caused by Thielaviopsis basicola. Phytopathology. 32, 752-761.  113  Kluge AG, Farris JS (1969) Quantitative phyletics and the evolution of anurans. Syst. Zool. 18, 1-32.  Maddison WP, Maddison DR (2000) MacClade: Analysis of phylogeny and character evolution, Version 3.08. Sinauer, Sunderland, MA.  Mathew B, Brickell C and White R (2000) The smaller Daphnes. The Proceeding of the Conference " Daphne 2000 " April 2000. The Royal Horticultural Society, London.  M011er M and Cronk QC (1997) Origin and relationships of Sainipaulia (Gesneriaceae) based on ribosomal DNA internal transcribed spacer (ITS) sequences. Amer. J. Bot. 84, 956-965.  Nag Raj TR and Kendrick B (1975) A monograph of Chalara and allied genera. Wilfred Laurier University Press, Waterloo, Ontario.  Noshad D, Punja ZK, Riseman A (2006a) First report of Thielaviopsis basicola on Daphne L.. Can. J. Plant Pathol. 28, 160-164.  Noshad D, Punja ZK and Riseman A (2006b) Screening for Resistance to Thielaviopsis basicola in Daphne spp. (2006) Can. J. Plant Pathol. 28, 310-312.  Papavizas GC (1968) Survival of root-infecting fungi in soil. Phytopathology. 58, 421-428.  114  Paulin-Mahady AE, Harrington TC and McNew D (2002) Phylogenetic and taxonomic evaluation of Chalara, Chalaropsis, and Thielaviopsis anamorphs associated with Ceratocystis. Mycology. 94: 62-72.  Punja ZK and Sun LJ (1999) Morphological and molecular characterization of Chalara aeons (Thielaviopsis basicola), cause of black root rot on diverse plant species. Can. J. Bot. 77, 1801-1812.  Rambaut A (1996) Se-Al; Sequence Alignment Editor, ver. 1.0 alpha 1. Computer software available on the WWW site: htttp://evolve.zoo.ox.ac.uk/Se-Al.html.  Reddy MS and Patrick ZA (1989) Effects of host, nonhost, and fallow soil on populations of Thielaviopsis basicola and severity of black root rot. Can. J. Plant Pathol. 11, 68-74.  Swofford DL (2000) PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods). Version 4.0 Sinauer Associates, Sunderland, Massachusetts.  Todd E, Richter L, and Pamela C (2000) The evolution of disease resistance genes. Plant Mol. Biol. 42, 195-204.  Walbot V (1985) On the life strategies of plants and animals. Trends in Genetics. 1, 165-69.  115  Wendel JF and Albert VA (1992) Phylogenetics of the cotton genus (Gos.vpium): characterstate weighted parsimony analysis of chloroplast-DNA restriction site data and its systematic and biogeographic implications. Systematic Botany 17, 115-143.  White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M., Gelfand, D., Sninsky, J., White, T. (Eds.), PCR Protocols: A Guide to Methods and Amplifications. Academic Press, San Diego, p. 315-322.  Yarwood CE (1981) Occurrence of Chalara elegans. Mycology. 73, 524-530.  116  Table 5.1. Daphne taxa used in the Thielaviopsis basicola resistance bioassay with region of nativity or origin and mean plant disease index (PDI) values. Z: PDIs marked with the same letter(s) do not differ significantly, Tukey (P<0.05).  Taxa D. alpina L. D. arbuscula Celak.  Nativity/Origin  Mean PM (SD)  Italy  12.5 (0.92) ghi  Czech Republic  43.3 (1.74) ahcdef  D. bholua Buch-Ham. ex  Nepal  12.5 (0.82) ghi  D. caucasica Pall.  Russia  9.2 (1.02) hi  D. circassica L.  Russia  46.6 (1.66) abed 's'  Czeck republic  64.2 (1.89) a 51.7 (1.31) abed  D. cneorum L. D. collina Smith  Turkey  D. genkwa Siebold & Zucc.  China  D. gnidium L  Spain  D. giraldii Nitsche  43.3 (1.31) abedet 27.5 (1.22)  dgfgh  W China  15.8 (1.18) ighi  D. jasminea Sibth.& Sm.  Greece  13.3 (1.09) i g hi  D. kosaninii Stoj.  Bulgaria  20.8 (1.3)  D. /aureola L.  N Africa  23.3 (1.34)  D. longilobata Turril.  China-Yunnan  efghi derg h '  30.8 (1.06) edergil  D. mezereum L.  Russia  10.0 (1.02) I'  D. odora Thunb.  China  31.7 (1.21) " lefg h  D. oleoides Schreber  Turkey  17.5 (1.19) ighi  D. politico L.  Russia  60.8 (1.54)  D. retusa Hemsl.  China  0.0 (0)  D. tangutica Maxim.  China  0.0 (0)-1  117  -1  ab  Table 5.2. ITS sequence characteristics.  Total number of characters  610 by  The number of characters parsimony informative  176 by  The best recovered parsimony trees  63  Length  670  CI (consistency Index)  0.712  RI (Retention Index)  0.596  Figure 5.1. The consensus parsimony tree which is a strict consensus of the most parsimonious trees. values above branches represent bootstrap values (length: 670, CI: 0.712 and RI: 0.596).  Dais cotinifolia (cotinif olia)  84  Gnidia  (denudata)  97  ^Sl, ilis ) FOgewoithia tenrysaninaj 'Pirrutjaea  D. giraldii 100  D. odora D. bholua D. gnidium  100 100  GLADE III  D. arbusculz D. jasminea D. caucasica  100  99  D . colhna  D drcassim .  D. oleoides 100  D. alpina 100  94 100  D. kosanini D. cneorum  100 100  D. tandulica D.retusa  98  E.'aureola D. pontica D. mezereum D genkoia  119  I I I  D. longilobata  100 86  I I I GLADE II  CLARE I  ^  Figure 5.2. The evolutionary tree of resistance for the selected Daphne species. Solid branches and darker points indicate lineages for which resistance is stronger. PDI (Plant Disease Index) is shown next to each species. Three major clades are shown in the tree. The placement of D. genkwa is not completely clear and.the ancestral trait is equivocal and shown by the striped pattern ( L_J ).  PDI Daphne gi  ^  13 Daphne Rol ua  ^  15.8  12.5  IN Daphne °don^31.7  I  -4® Daphne gni diurn ^27.5^I  CLADE III  Daphne arb ti:3cula.^43.3  ^41111  Nu Daphne jat;min ea.^13.3  ^dioolfil Daphne ca uc.aa jut^9.2  I  I 1.40 ^ID Daphne collina.^51.7^ I "'q^ \ ^ 46.6^I -10 Daphne ci rea_s:Eica .., 111 D aphne oleciide3^17-5^I I b2 ., --^c,1111Daphn al pina^12.5^ iv-^ I .c .^ED Daphne kosanini^20.8  GLADE II  I  \1^ Daphne eneigurn^541.2 I 31 Daphne, loffigilo Nita  4  X  4  ■ Daphne ta Ngutica  ■ Daphne rat Li »,  ^  ^  20.8  0  ^0  El Daphne laureola  ^  23.3  It Daphne pontictk^60.8 me:  ^10  ' 483 Da ph ne genkwa^43.3 -  120  I  I  I I  I  I  CLADE  CHAPTER SIX  CONCLUSIONS  6.1 Conclusions and Future Directions Daphne is a widely dispersed genus with large variation in morphology, native habitats, and  use. Despite their current ornamental, economic, pharmaceutical (Brickell and White, 2000), and ethnobotanical importance (Zhou, 1991), very little information is available about its pathology, phylogeny, or plant improvement techniques (e.g., biotechnology) (Marks and Simpson, 2000; Green etal, 1992; Cohen, 1977). In horticulture, broader acceptance of Daphne has been limited due to their susceptibility to Daphne Sudden Death Syndrome  (DSDS). The major objective of the current research was to identify the causal agent for DSDS and to study variation in resistance among a sub-set of taxa. I used both in vivo and in vitro methods to screen selected Daphne taxa for resistance against DSDS. I also constructed a limited phylogeny of the selected Daphne species using rDNA ITS sequences and mapped resistance values onto the consensus parsimony tree. My analysis of resistance among taxa and their phylogenetic relationships increased our knowledge about this genus.  In Chapter 2, my observations documented the following progression of symptoms for DSDS: 1) brown to black necrotic lesions on the roots, 2) leaf chlorosis leading to abscission, 3) whole plant stunting, and 4) stem collapse and plant death. Though this research, I identified Thielaviopsis basicola (Berk. et BR.) Ferr. as the causal agent for this disease (Noshad, 2006a). In addition, a proven protocol for producing pure cultures of T. basicola 121  has been developed. These protocols were then used in the subsequent germplasm screen (Chapter 4).  In Chapter 3, 1 established species-specific protocols for in vitro propagation of the selected species for use in the in vitro disease evaluation (Noshad, 2006b). These protocols allowed for the successful production of uniform, disease-free plants for my experimental use. The results of the in vitro propagation indicated a pattern of respond to basal media. The two small shrub species of Daphne (Daphne jasminea and Daphne cneorum) responded best to WPM base media while the larger species responded best to MS based media. A more clear pattern might be identified in the future when in vitro propagation protocols are developed for additional species.  In Chapter 4, 1 evaluated the selected Daphne species for resistance against T. basieola under both in vivo and in vitro conditions by developing a disease progression rating (DPR). A plant disease index (PDI) was then developed based on both disease incidence and disease severity. Both in vitro and in vivo methods produced similar results and displayed a strong correlation. However, there was one notable difference between them for the rate of disease progression. Typically, the in vitro challenge produced a comparable level of disease as the in vivo challenge but in two to three weeks less time. For both methods, the inoculation and  screening methods were proven robust in terms of the isolate's pathogenicity, disease characterization, consistency over time, and its ability to differentiate taxa. Based on the one isolate used, overall pathenogenicity was adequate in allowing assessment of disease incidence and severity (i.e., root and foliar symptoms) on all taxa. Also, the concentration of  122  conidia used was sufficient to cause disease but not to overwhelm the defense mechanism(s) and prevent taxa differences from being displayed. Based on the germplasm screens, significant differences among taxa were identified for resistance and ranged from highly susceptible (e.g. D. cneroum) to highly resistant (e.g. D. tangutica and D. retusa).  Finally, in Chapter 5, I constructed a limited phylogeny of selected Daphne taxa based on rDNA ITS sequences. I then associated this phylogeny with individual species' resistance against T basicola. Using established phylogenetic analysis to construct a consensus parsimony tree, Daphne appears to be a monophyletic group. However, placement of D. genkwa remains problematic and needs to be studied further. Analysis of ITS sequences  resulted in a parsimony consensus tree with two well-supported major clades and one Glade with a lower level of support. The tree for resistance evolution inferred from the phylogenetic data and the results of the germplasm screen are not sufficiently clear to make conclusions regarding the evolution of disease resistance. I prOpose that resistance is a new characteristic and these plants evolved resistance against the pathogen after colonizing new regions. This hypothesis is supported by the present of more susceptible species than resistant species among the examined group. By studying additional taxa of this genus, we may be better able to relate phylogenetic relationships among Daphne taxa with respect to the evolution of disease resistance.  Many questions remain for future research and I strongly hope that these results will he of significant assistance to those future researchers. Some of the major questions remaining include "How do other Daphne species interact with T basicola?", "What are the  123  phylogenetic relationships among the whole of Daphne?", and finally " How would other races of T hasicola react with these tested Daphne as well as other species'?". Basically, every disease is a product of interactions between three major factors, the host, the pathogen, and the environment surrounding them. In my study I tried to better understand DSDS and the major issues surrounding its basis and development. Although I was able to identify the pathogen responsible and some of its characteristics, more details are needed on its interaction with other local microorganisms, its infection and penetration methods and its interaction with resistant/susceptible plants. Since many aspects of Daphne's anatomy and physiology remain unknown, but may be related to disease resistance, future research should try to identify and characterize these underlying traits. Also, the environments in which I conducted this project were limited and did not assess disease development under a wide range of conditions. Future research should also evaluate the environmental effects on DSDS development, its influence on Daphne physiology, and its interaction with the pathogen.  124  6.2 References  Brickell CD and White R (2000) A quartet of New Daphnes. The New Plantsman. 7, 6-18. The Royal Horticultural Society, London.  Cohen D (1977) Thermotherapy and meristem-tip culture of some ornamental plants. Acta Hort. 78, 381-388.  Green MJ, Godkin SE and Monette PL (1992) Use of in vitro cultures of Daphne cneorum L. for the western detection of daphne virus X. J. Enviro. Hort. 10(3), 153-155.  Marks T and Simpson S (2000) Interaction of explant type and indo1-3-butyric acid during rooting in vitro in a range of difficult and easy to root woody plants. Plant Cell, Tiss. Org . Cult. 62, 65-74.  Noshad D, Punja ZK, Riseman A (2006a) First report of Thielaviopsis basicola on Daphne L. Can. J. Plant Pathol. 28, 160-164.  Noshad D, Punja ZK and Riseman A (2006b) Screening for Resistance to Thielaviopsis basicola in Daphne spp. (2006) Can. J. Plant Pathol. 28, 310-312.  Zhou B (1991) Some progress on the chemistry of natural bioactive terpenoids from Chinese medicinal plants. Memorias do instituto Oswaldo Cruz. 86 (suppl. 2) 219-226.  125  Appendix 1 — Preliminary Experiments on Inoculation Protocol Develpment  Introduction Preliminary experiments were conducted to determine the appropriate concentration of pathogen spores needed for effective inoculation of Daphne species. The main objective of this experiment was to determine how many spores were sufficient to induce disease in the susceptible species, D. cneorum.  Material and Methods A single-spore derived, aggressive isolate of T hasicola, isolated from diseased daphne plants, was used throughout this study. A suspension of endoconidia was prepared by gently washing the surface of 3-week-old colonies with deionized water and vortexing the wash solution for 30s. The resulting suspension was twice filtered through four layers of cheesecloth to remove agar, hyphae, and chlamydospores. Prior to inoculation, spore suspensions were calibrated with a haemocytometer and adjusted with deionized water to obtain endoconidia concentrations of lx10 4^, lx10 5^, 1x10 6^, and lx10 7  Rooted plants of Daphne cneorum were produced in July and August from terminal cuttings (50-100 mm in length) with the flower buds and lower leaves removed. Cuttings were made with a single shallow cut and soaked in an anti-fungal solution (Physan 20, Maril Products Inc.) for 60 seconds. The cuttings were allowed to dry momentarily before being dipped in 0.4% IBA powder (Slim Root #2, Plant Products. Co. Ltd.) and then placed in 6 cm pots  126  filled with a course rooting medium (10 parts propagation grade perlite, 8 parts peat, 6 parts granite grit #2, 1 part pumice (double screened to remove fine particles), dolomite lime 65AG at 900 g m -3 , and Micromax (trace elements) at 400 g m -3 ). The flats were placed under intermittent mist with bottom heat set at 22°C. Rooted cuttings were transferred to a polyhouse in October where they were allowed to go dormant but kept frost free. They were repotted in May into 12 cm pots filled with a well-drained medium (8 parts peat, 8 parts Turface MVP, 6 parts granite grit #2, 4 parts screened and pasteurized soil, 1 part pumice, 3  dolomite lime 65AG at 670 g m -3 , Micromax micronutrients at 540 g , Osmocote 18-6-12 at 2150 g n1 3 , and Psi Matric wetting agent). All stock plants were grown under shade cloth during the summer months and moved to a heated polyhouse during the winter months to prevent frost damage. Fertilization regiment included yearly top-dressing with Osmocote 186-12 at 5 g 1 gal pot. Fungicides were not used during stock production because we had very little disease pressure and we did not want to risk cross-contamination affecting the in vivo assay.  Innoculation procedures consisted of topically applying 1 ml suspension to healthy roots of 2-year old nursery-grown D. cneorum plants. Control plants were treated with sterilized distilled water. Production containers were modified to contain a clear panel behind a lightproof 'door' to allow for direct observation of the infection process without further disturbance to the root system. To allow for uniform conditions following inoculation, plants were transferred to a greenhouse and grown under natural light at 24 +1 °C and a relative humidity between 70 and 80%. In this experiment, a completely randomized design  127  used with three replications. Each replication consisted of 5 plants with two plants serving as controls.  Results The results of this preliminary experiment indicated that a minimum of Ix10 6 spores were needed for regular and reliable infection. At this concentration, 100% of the treated plants displayed symptoms within four weeks (Table 1). Concentrations below this amount failed to induce disease on 100% of the treated plants. Concentrations above lx10 6 also induced disease on 100% of the plants but within the same time period as lx 10 6 . Therefore, I used this concentration for all disease assessment trials.  Table 1- Percent diseased plants 4 weeks following inoculation. #  Spore concentration per ml  1  lx 10  2  1 x10 5  3  lx10  4  1x10 7  Diseased Plants (%)  4  6.66 46.66  6  100 100  128  Appendix 2 —Experiments on PGR Stability Following Autoclaving  Introduction  Experiments were conducted to evaluate the stability of the PGRs used in this research following autoclaving. Manufacturer specifications indicate that IAA, IBA, and KIN may have reduced activities following autoclaving while NAA is reported to be stable. Specifically, I compared shoot production on D. cnerourm following treatments with PGRs that were either autoclaved (121 C for 15 min) or filtered sterilized.  Materials and Methods  Previously established cultures of D. cneorum growing on multiplication medium (Chapter 3) yielded shoots used in these trials. For the trials, fresh multiplication media were prepared as described previously but with PGR supplements either added to the medium before autoclaving (121 C for 15 min) or added following autoclaving and filter sterilization. Each treatment had 12 subsamples. Paired t-tests were performed comparing shoot production from autoclave vs. filtered sterilized within a medium.  Results  Average number of new shoots produced 4 weeks following culture are listed in Table 1. In four of the live PGR combinations, no difference in shoot production was observed between autoclaved vs. filtered PGRs. However, in the 2 mg 1 1 BA + 0.01 mg I -1 IBA treatment, a significant difference was detected. The autoclaved treatment produced more shoots than its  129  filtered counterpart. In general, these data are similar in range and magnitude of those data detailed in Chapter 3. However, in three of the five treatments, current averages are slightly lower than those previously reported. I attribute this difference to the extended evaluation time used in Chapter 3. In the current study, the data were collected at four weeks; not the eight weeks used in Chapter 3. These four additional weeks of growth may have allowed the cultures to produce more shoots than what is currently observed. Based on these results, I conclude that under the autoclave conditions used (121 C for 15 min), no reduction in PGR activity was observed and that the procedures used (i.e., autoclaving the PGR supplements) did not affect my results or conclusions.  Table 1. Shoot production of D. cneorum after four weeks of culture on one of the listed media. PGR supplements were either autoclaved with all other medium components or added after autoclaving and filter sterilization. PGR Supplement  1 mg 1 - BA + 0.1 mg  -1  Mean Shoot  Mean Shoot  P  Chapter 3  Production (SD)  Production (SD)  Two-tailed  Means  Autoclaved  Filtered  t-test  3.3 (1.2)  3.2 (0.9)  0.72  2.2 (0.6)  4.9 (1.1)  3.5 (1.4)  0.03  3.9 (0.9)  3.1 (1.7)  3 (1.3)  0.73  5.6 (0.7)  2.7 (0.9)  2.2 (1.2)  0.17  2.7 (0.8)  1.2 (0.9)  1.4 (0.9)  0.67  2.8 (0.9)  IAA  2 mg r BA + 0.01 mg 1 ' IBA -  2 mg 1' BA + 0.01 mg  -1  NAA  4 mg F i KIN + 0.01 IAA mg 1 -1 0.001 mg 1 -i TDZ + 0.01 mg 1 ' IAA -  130  Appendix 3 - Preliminary Experiments on in vitro Propagation Media Composition Introduction  Preliminary experiments were conducted to evaluate the effects of modifying the concentrations of base medium components on growth of selected Daphne species. The specific objective was to evaluate whether changes (mostly reductions) in concentrations of the base salts, sugar (carbon source) and gelling agent (agar) affected shoot and root growth of Daphne plantlets.  Material and Methods  I. Plant material In preliminary experiments, I used the following seven species D. cneorum, D. caucasica, D. retusa, D. giraldii, D. jasminea, D. laureola and D. tangutica.  II. General procedures Apical shoot tips, 2-5 cm in length, 3-5 mm in diameter, and bearing 1-3 nodes were collected during the summer and fall seasons from 4-6 year old container-grown plants. Collected shoots were striped of all leaves and rinsed under running tap water for 15 min. Under aseptic conditions, shoots were then surface sanitized by treatment with 70% ETOH for 30 seconds followed by treatment with 0.5% sodium hypochlorite solution containing 0.5  131  ml 1 1 Tween-20 (Sigma Chemical, St. Louis, MO) and gently stirred for 10 min. After sanitation, shoots were rinsed three times with sterile distilled water for 5 min each and placed individually in culture vessels. Cultures were maintained at 25°C under 16 h photoperiod with irradiance intensity of 350 jtW cm -2 supplied by cool white fluorescent lamps.  The pH of the various media was adjusted based on published protocols (e.g., MS media was adjusted to 5.8, WPM media to 5.2) before autoclaving for 15 min at I21°C. Explants were maintained on establishment media i.e. basic MS and WPM with 20 g ['sucrose, and 5.6 gl high gel strength agar (Sigma-Aldrich, St. Louis, MO) without PGRs, for four weeks prior to subculture on multiplication media. Following a four week establishment period, shoots were sub-cultured into 150 mm baby food jars (Sigma-Aldrich, St. Louis, MO) containing 25 ml multiplication media. Basically, nodal explants cultured on either MS (Murashig and Skoog, 1962) or WPM (Woody Plant Medium; McCown and Lloyd, 1983) supplemented with plant growth regulators (i.e., 2 mg 1 1 BA + 0.01 mg 1 1 NAA), recommended minerals and 5.6 g I -1 agar with vitamins (nicotinic acid 0.5 g 1 , pyridoxine-14C1 0.5 g ^thiamineHC1 0.1 g 1 1 , glycine 2 g 1 -1 ) based on these two major protocols (Murashige and Skoog, 1  1962; McCown and Lloyd, 1983).  Following multiplication, new shoots were used in elongation and rooting trials. For shoot elongation, shoots 1 cm long with intact apices and 2-4 leaves were cultured on basic media (either MS or WPM basal medium) supplemented with 1 g 1 1 charcoal without PGRs. Following four weeks, the longest shoots (4-6 cm) were transferred to individual test tubes  132  i  with 20 ml media consisting of base MS or WPM components but modified as follows (no PGR has been used):  Media  1^Basic medium (BM)- without PGR 2^1/2 BM strength 3^1/4 BM strength 4^1/8 BM strength 5^BM + 2x sucrose (40 g1 -1 ) 6^BM+ 1/2x sucrose(10 g 1 -1 ) 7^BM+ 1/4x sucrose (5 g 1 1 ) 8^BM+ 1/8x sucrose (2.5 g 1 1 ) 9^BM+ 2x gelling agent ( 11.2 g 1 1 high gel strength agar) 10^BM+ 1/2x gelling agent ( 2.8 g [ I high gel strength agar) 11^BM+ 1/4x gelling agent ( 1.4 g 1' high gel strength agar) 12^BM+ 1/8x gelling agent ( 0.7 g 1 1 high gel strength agar) BM = Standard MS or WPM base salts and vitamins  All experiments used a complete randomized design with three replications and 15 shoots per replication (i.e., 45 shoots). Data were recorded as percentage of shoots rooted after 8 weeks.  Results  The results of these experiments indicated that none of the modifications evaluated improved shoot or root growth of any Daphne species. In addition, the most robust growth was observed from the originally published composition. Therefore, all in vitro propagation experiments used the standard base media (MS/WPM) compositions.  133  References  McCown 131-1 and Lloyd GB (1983) A survey of the response of Rhododendron to in vitro culture. Plant Cell Tiss. Org . Cult. 2, 75-85.  Murashige T and Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15, 473-497.  134  

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