«By ZAHI KANAAN-ATALLAH A dissertation submitted in partial fulfillment of the requirement of the degree of DOCTOR OF PHILOSOPHY WASHINGTON STATE ...»
DEVELOPMENT AND GENETIC DIVERSITY OF SCLEROTINIA
SCLEROTIORUM ON POTATO IN THE COLUMBIA BASIN
A dissertation submitted in partial fulfillment of the requirement of the degree of
DOCTOR OF PHILOSOPHY
WASHINGTON STATE UNIVERSITYDepartment of Plant Pathology MAY 2003
To the Faculty of Washington State University:
The members of the Committee appointed to examine the dissertation of Zahi Kanaan- Atallah find it satisfactory and recommend that it be accepted Chair _____________________
ACKNOWLEDGMENTI am grateful to Dennis Johnson for taking me under his wing and providing me with the ability to push my curiosity to the utmost. He also extended his arm to help me in every way he could and presented relentless and unequivocal support.
Without Dr. Xianming Chen’s material support and great clarifications, the section on the population biology of S. sclerotiorum would not have seen the light. I am thankful for his willingness to allow me to use his equipment and solicit the help of his former technician, David Christian, whom I thank as well.
My gratitude extends also to Dr. Frank Dugan and Dr. Gary Grove who discussed various topics with me repeatedly and steered my work to this end. They also provided me with food for thought during my preliminary exam, even though I still have not figured out the answers to some of their questions.
I would also like to thank Drs. T. Peever and J. Rogers whose several comments and answers to my questions made better sense of the chaos of the learning process.
I acknowledge and thank the Washington State Potato Commission for supporting my research by all necessary means. I also am grateful to the following collaborators for the isolates they provided: Drs. L Kohn, J. Steadman, M. Cubeta, T. Miller, L. Dutoit and Mr. Gary Pelter.
Thanks also go to Tom Cummings for his willingness to help me constantly; and to my dear friend Lyndon “the dream” Porter for those great scientific, religious and sport discussions we had over three years. I also thank Lyndon for the butchery he did to my name, in the name of humor.
DEVELOPMENT OF WHITE MOLD AND GENETIC DIVERSITY OF SCLEROTINIA
SCLEROTIORUM IN POTATO IN THE COLUMBIA BASINAbstract By Zahi Kanaan-Atallah, Ph.D.
Washington State University May 2003 Chair: Dennis A. Johnson Sclerotinia sclerotiorum (Lib.) de Bary is a cosmopolitan, homothallic and necrotrophic ascomycetous fungus dispersed by airborne ascospores and soilborne sclerotia. It causes disease in over 400 host species. A significant correlation between potato yield losses and stem rot incidence has not been observed, while 1-3 fungicide applications are made to manage the disease. In the Pacific Northwest, iprodione, dichloran, quintozene and fluazinam are registered for stem rot control; labels recommend fungicide applications be made prior to row closure.
Reduced control could be caused by mistimed applications, inadequate tissue coverage or a selection for fungicide resistant isolates.
Ascospores were captured and disease incidence was monitored in ten potato fields in the Columbia Basin to determine when infections occur. Peak ascospore release corresponded with initial full bloom (7 to 10 days after row closure) and disease onset occurred 10-14 days following row closure. Airborne S. sclerotiorum ascospores impacted potato blossoms attached to plants. Blossoms fell to the ground and onto stems, senesced and were colonized by the fungus. Potato vines became infected after dropping to the ground and coming in contact with the fungus. Flower removal and fungicide applications at initial full bloom reduced disease incidence, in comparison to untreated controls. These observations indicate that fungicide label recommendations need to be based on initial full bloom, instead of row closure, to provide
efficacies of iprodione, dichloran or fluazinam in greenhouse and in-vitro trials; quintozene failed to provide effective protection on inoculated stems.
Canadian and southeastern US populations of S. sclerotiorum have been previously described as clonal, with a few genotypes composing the majority of populations. Analyses of isolates from the Columbia Basin and other U.S. areas using microsatellites revealed high genotypic variability, but genotypes were not segregated by host or geographic location. The discovery of a 25% rate of outcrossing in the Columbia Basin, in addition to gene flow explain in large part the high genotypic variability observed. Furthermore, no correlations were found between genotypes and mycelial compatibility groups, reduced sensitivity to fungicides, and response to various temperatures or aggressiveness.
LIST OF TABLES
LIST OF FIGURES
1. Development of Sclerotinia stem rot in potato fields in south central Washington Abstract
Materials and Methods
2. High genetic variability, phenotypic uniformity and outcrossing in populations of Sclerotinia sclerotiorum in the Columbia Basin of Washington Abstract
Materials and Methods
1. Locations of studied fields
2. Incidence of stem rot in fungicide-treated or blossom removed potato plots
3. Incidence of stem rot in a field treated with various fungicides before row closure............ 29
4. Mean number of lesions in greenhouse fungicide-treated and inoculated potato plants...... 29 Chapter 2.
1. List of potato isolates of S. sclerotiorum collected from the Columbia Basin
2. List of non-potato S. sclerotiorum isolates
3. Fungicides used to identify sensitivity of S. sclerotiorum
4. List of microsatellite primers showing polymorphisms
5. Pairwise FST comparisons among S. sclerotiorum isolates from the Columbia Basin and other North American regions
1. Mean number of ascospores captured in Pasco and Basin City, WA in 2002
2. Means of Sclerotinia stem rot incidence on two dates
3. Disease progress curves of Sclerotinia stem rot in commercial fields
1. Colonies from S. sclerotiorum ascospores captured on top of plant canopy
2. Histogram of frequencies of the 20 MCGs including more than one individual out of a population of 167 potato isolates of S. sclerotiorum
My work is dedicated to the two people who sowed and grew the seeds of curiosity in me, my parents: Daad and Nabih. They believed in me and steered me toward safer shores.
I also dedicate my dissertation to my country: The land of Cedars, biblical Lebanon. Many forget that it gave the world its alphabet, Jesus’ first miracle and more recently the mind behind “The Prophet”, Jibran Khalil Jibran.
Say not, "I have found the truth," but rather, "I have found a truth."
The two chapters included in this dissertation have been prepared for submission to professional journals. Chapter one will be submitted to Plant Disease and chapter two will be submitted to Phytopathology. Citations and references in those two chapters are formatted according to the guidelines of the respective journals and are listed at the end of each chapter before the accompanying tables and figures. Citations included in the general introduction section follow the guidelines of WSU graduate school and are found at the end of the dissertation.
Cultivated potatoes (Solanum tuberosum spp. tuberosum L.) are plagued by a variety of fungal diseases of economic importance. Stem rot (aka white mold) caused by Sclerotinia sclerotiorum (Lib.) de Bary occurs in temperate potato growing areas and was reported to have caused the destruction of entire crops in Ireland during the 19th century (Partyka and Mai, 1962).
Symptoms of stem rot start as soaked/watery lesions on main and secondary stems that turn into severely rotted areas girdling infected stems and devouring the inner pith, thus causing the death of the canopy. Infected stems turn brownish, become soft and as they dry out acquire a bleached and papery appearance. A whitish mycelium is frequently observed on infected parts hence the name white mold is used to describe the symptoms on legume plants (chiefly bean and soybean).
In potato the nature of the symptoms and their restriction to stems are in conformity with the name stem rot. Stems are hollowed by the fungus and are filled with black and hardened sclerotia, which allow the fungus to survive adverse conditions. With the degradation of stems on the soil surface or by plowing, sclerotia are liberated and after conditioning can either undergo a sexual cycle and produce apothecia, or germinate directly to infect plant tissues in the immediate vicinity.
S. sclerotiorum is a cosmopolitan inoperculate discomycete capable of generating cup-shaped apothecia from sclerotia. Those apothecia, usually 2-10mm in diameter, are lined with asci, which are filled with 8 hyaline ascospores (110-160µm x 6-10 µm) (Kohn, 1979). Those ascospores are forcibly ejected in puffs and are carried by wind currents. It is thought that because the sizes of apothecial stalks (stipes) are usually smaller than 5cm, sclerotia in the upper 5cm soil horizons are the only ones that germinate carpogenically (Steadman, 1983). Rainfall is known to increase disease incidence and following drought it induces carpogenic germination of
the apothecia are credited for the “puffing” of ascospores, while few ascospores were released at high relative humidity (McCartney and Lacey, 1992). Light was found to have little impact on ascospore releases. Ascospore releases usually peak around midday and plummet late in the afternoon and at night (McCartney and Lacey, 1992; Gutierrez and Shew, 1998).
The maximal extent of ascospore dissemination is still uncertain. While up to 90 percent of S.
sclerotiorum ascospores are thought to deposit in a 100 meters radius from the source, the rest were assumed to travel up to 3-4 Km (Kohli et al., 1995; Cubeta et al., 1997). Furthermore, viable ascospores were captured at 6000m in altitude (Williams and Stelfox, 1979) indicating a possible dissemination over long distances. Depending on the crop growing in a field, apothecial production and ascospore releases may be staggered over a long period, especially given that soil shading and availability of moisture are required for a carpogenic germination. Epidemics occuring early in the season were usually traced back to ascopores produced by apothecia in neighboring fields cropped to winter crops (Abawi and Grogan, 1974; Abawi et al., 1975;
Williams and Stelfox, 1979).
In potato the few available reports indicated that ascospores are responsible for disease and noted that outbreaks occur at row closure when the potato canopy has achieved row closure and the soil is totally shaded, thus providing a buffered microclimate where moisture is high with reduced air movement and solar radiation (Partika and Mai, 1962; Powelson, 2001). Thick, closed and drooping canopies of certain peanut, bean and soybean cultivars have been found to sustain more severe white mold outbreaks than cultivars with sparse or upright canopies (Schwartz et al., 1978; Boland and Hall, 1988; Butzler et al., 1998). Upright-growing and sparse canopy cultivars had minimal stem contact with S. sclerotiorum mycelium on the ground.
Alternatively, other canopies provided a favorable microclimate for the carpogenic germination
collapse under the weight of stems. High crop density, close row width and excess nitrogen fertilization were also linked with increased stem rot incidence in several crops (Natti, 1971;
Grau and Radke, 1984).
Ascospores of S. sclerotiorum necessitate an external energy source to germinate. Flowers and senescing tissues, such as leaves on the soil surface, provide them with abundant and readily available nutrients, thus allowing them to build mycelial mats which could transfer the disease to healthy stems coming in contact (Keay, 1939; Abawi and Grogan, 1974, 1975; Abawi et al., 1975, Steadman, 1983; Powelson, 2001). In the absence of such accessible energy supplies, ascospores were unable to generate disease when ascospore suspensions were sprayed onto deflowered bean plants or intact green (unwounded) lettuce and clover leaves (Keay, 1939;
Abawi and Grogan, 1974) even when in high humidity chambers. Flower contamination by S.
sclerotiorum ascospores was associated with disease incidence in bean and canola (Abawi et al., 1975; Turkington and Morrall, 1993; Lefol and Morrall, 1996), where ascospores remained inactive until blossom senescence, which was followed by a rapid colonization of blossoms and subsequent disease where humidity was favorable (Steadman, 1983; Kohli et al., 1992;
Turkington and Morrall, 1993). Moreover, fungicidal coverage of blossoms at full bloom delivered best control of white mold, as opposed to applications on stems and leave but not flowers, which had no effect on either disease incidence or severity (Natti, 1971; Abawi et al., 1975; Steadman, 1983; Morton and Hall 1989).
S. sclerotiorum is described as a homothallic fungus based on the ability of colonies produced by single ascospores or hyphal tips to produce fertile apothecia and viable ascospores without requiring the presence of a mate (Keay, 1939; Kohn, 1979). Furthermore, various attempts to force crossing between pairs of isolates in the Kohn lab (University of Toronto)