'3’: ,1.» - «:1 3i u:. . 231nm? , , mr: 3?..qu 4‘... \. 9.1. ; u .v 1 $.33... «Minx. .43:;.,:3 1.3. “$5... , ”in aw “gm. 2.. 1%.“. 2!. was"; 2 gri‘é- 3751? H8) This is to certify that the thesis entitled RESISTANCE MECHANISMS IN POTATO TO PHYTOPHTHORA INFESTANS, POTATO LATE BLIGHT presented by AMY BOLINE READER PETERSON DUNFEE has been accepted towards fulfillment of the requirements for the Master of degree in Plant Pathology Science Major Professor's Signature ii Minot/L Zoo 4(— Date MSU is an Afiirmative Action/Equal Opportunity Institution LlBRARY *y MiCthan State L‘flflversity 4 PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/01 c:/CIRC/DateDuerp65-p. 1 5 RESISTANCE MECHANISMS IN POTATO TO PH Y T OPH T HORA INFEST ANS, POTATO LATE BLIGHT By Amy Boline Reader Peterson Dunfee A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Plant Pathology 2004 ABSTRACT RESISTANCE MECHANISMS IN POTATO TO PH Y T OPHT HORA INFEST ANS, POTATO LATE BLIGHT By Amy Boline Reader Peterson Dunfee Late blight (Phytophthora infestans (Mont.) De Bary), is the most important and devastating disease of potato worldwide. Currently, no marketable, resistant cultivars exist, thus potato breeders depend on the investigation of resistance mechanisms to incorporate such characteristics into their program. This research was undertaken to screen several characteristics in the potato-P. infestans response, that have been correlated with resistance in other plant-pathogen interactions. Varieties from Michigan State University’s potato breeding program were screened and no correlations were found between late blight development and stomatal density or conductance. The same varieties were evaluated for differences in glucanase, peroxidase and polyphenol oxidase activity. Only polyphenol oxidase activity (both induced and constitutive) was correlated with resistance to P. infestans. MSEl49-SY was used to create a set of transgenic plants sense and antisense for two genes involved in the production of phytoalexins. The constructs were evaluated for terpenoid variations in foliage and tuber tissue, and for evident deleterious effects of genetic engineering. The results of this research are a small part of an effort to identify and provide potato resistance mechanisms and markers to breeders who can then offer quality, late blight resistant varieties to growers. DEDICATION It makes me smile and even laugh a little when giving a sincere and heartfelt dedication of this thesis to my friend, Ana Luiza Resseguie Barnes. Ana, my roommate and closest fi'iend here at Michigan State University, earned her degree in zoology from 1996—2000. Throughout our whole experience she would always teasingly say to me “Who cares about plants, when you can study animals!” Even though I would try to persuade her that plants are interesting too, I know that I never gave her a good enough answer! Ana and I shared and laughed so much here at MSU that it only seems natural to dedicate this thesis to her and everything that she stands for. She was one person who made this a wonderful place for me to be and I am forever thankful to her. I am lucky to have all of our great stories and experiences from here. There are just so many. I really miss her and all of her pizzazz, but with such an unbelievable person I have discovered that our friendship can always endure. iii ACKNOWLEDGEMENTS I would graciously like to thank my committee members, Dr. Willie Kirk and Dr. Dave Douches for their readily accessible help and good humor throughout this project. My major professor, Dr. Ray Hammerschmidt, deserves more than a special thanks for all of the assistance, guidance and especially the opportunities that he has given to me since the time that I first set foot in his introductory plant biology class six years ago as a college freshman. My parents are also thankful for all of these things! More than I can ever begin to acknowledge, I have acquired amazing friends from the Hammerschmidt Lab who have been the true highlight of this whole experience. I want to thank Elise Poole Hollister, my favorite tuber technologist; Dr. Luis Velasquez, my swimming buddy; and Dr. Andrea daRocha, my sergeant mom, for their continuous support and unexplainable, lifetime friendships. Last, but certainly not least, I would like to thank my husband Jeff, my mom and dad, and my sisters Eliza and Carrie for being the very best family ever and for always being supportive of my academic work. I would also like to acknowledge my Grandma and Grandpa Reader who have always encouraged education above all else. iv TABLE OF CONTENTS LIST OF TABLES ................................................................................. vii LIST OF FIGURES .............................................................................. viii CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW The cultivated potato and Potato Late Blight: an Introduction ........................ 2 Potato Late Blight: Management strategies .............................................. 6 Potato Late Blight: General resistance responses ...................................... 15 The production of antimicrobial compounds (PR-proteins) .......................... 21 The production of antimicrobial compounds (phytoalexins) ......................... 25 Potato Late Blight: Resistance through breeding and genetic engineering ....... 26 OBJECTIVE OF THIS THESIS .................................................................. 28 LITERATURE CITED ............................................................................. 30 CHAPTER 2 RESISTANCE AND SUSCEPTIBILITY TO PH Y T OPHTHORA INFESTANS IN ADVANCED BREEDING LINES INTRODUCTION .................................................................................. 48 MATERIALS AND METHODS ................................................................. 53 Plant material ............................................................................... 53 Phytophthora infestans isolates, maintenance and inoculations ..................... 54 Field inoculations and disease assessment .............................................. 55 Screening for physical resistance mechanisms ......................................... 56 Screening for biochemical resistance mechanisms .................................... 57 RESULTS ............................................................................................ 61 Field disease assessment results .......................................................... 61 Screening of physical resistance mechanisms .......................................... 67 Screening of biochemical resistance mechanisms ..................................... 73 DISCUSSION ....................................................................................... 86 LITERATURE CITED ............................................................................. 91 CHAPTER 3 EVALUATION OF TERPENOIDS AND THE EFFECTS OF ENGINEERING POTATOES USING SENSE AND ANT ISENSE CONSTRUCTS OF STVS3 AND STVSS, GENES INVOLVED IN PHYTOALEXIN PRODUCTION INTRODUCTION .................................................................................. 97 MATERIALS AND METHODS ................................................................ 103 Transgenic plants ......................................................................... 103 P. infestans cultures ....................................................................... 107 Inoculation and disease assessment of transgenic potato foliage .................. 108 Inoculation procedures and terpenoid extractions from potato tuber tissue ...... 108 Inoculation procedures and terpenoid extractions from potato leaf tissue ....... 110 Enzymatic analysis of potato leaf tissue ............................................... 112 RESULTS .......................................................................................... 113 Disease assessment of transgenic plants ................................................ 113 Terpenoid extractions from potato leaf and tuber tissue ............................. 118 Screening for non-target effects of transforming plants ............................. 128 DISCUSSION ...................................................................................... 123 LITERATURE CITED ........................................................................... 126 SUMMARY ........................................................................................ 129 APPENDIX ......................................................................................... 131 vi LIST OF TABLES 1. Resistance and susceptibility potato leaf tissue of cultivars tested in the field as determined by disease assessment in the field, 2001. vii LIST OF FIGURES . A comparison of the average Relative Area Under the Disease Progress Curve (RAUDPC) values that were calculated for the advanced breeding lines that were included in field trials from 2000 and 2001 at Muck Soils Research F arms; Bath, MI. . A comparison of the average Relative Area Under the Disease Progress Curve (RAUDPC) values for the advanced breeding lines tested in the field in the year 2001. Their designated resistance, moderate resistance or susceptibility was determined, based on significant differences in their RAUDPC values. . Disease progression in the field 2001. Disease was assessed in the potato foliage every 3 to 4 days between 0 and 72 days after planting of the advanced breeding lines. Disease progression was evaluated in order to calculate the RAUDPC values for each tested cultivar at the end of the growing season. These values were based on the percentage of infected foliar tissue. . Average stomatal densities of the abaxial leaflet surface from greenhouse-grown advanced breeding lines. Cultivars Atlantic and Snowden differed significantly from the others, yet there was no correlation with resistance or susceptibility. . Average stomatal densities of adaxial leaflet surface from greenhouse-grown advanced breeding lines. Stomatal density varied between the different cultivars yet there was no correlation with resistance or susceptibility. . Comparison of stomatal conductance in advanced breeding lines as measured by diffusive resistance in the field, 2000. It was found that there was no correlation between the disease assessment of any of the varieties and their stomatal conductance. Glucanase activity evaluated from six to eight-week-old greenhouse-grown plants of which the leaves were collected and inoculated in the lab with water or Phytophthora infestans. There was not a significant difference in glucanase activity between the water and P. infestans treatments. Glucanase activity was only significantly induced with P. infestans inoculations in MSG274-3. . Glucanase activity evaluated from mature field-gown plants of which the leaves were collected before disease was present in the field (Muck Soils Research Farm; Bath, MI.) and inoculated in the lab with water or Phytophthora infestans. It was found that glucanase was induced in the leaflets that had been treated with the pathogen, but there was no significant difference in glucanase concentration between the specific varieties. viii 9. Glucanase activity evaluated in leaf tissue collected 64 days after planting that were infected with Phytophthora infestans in the field, 2001 at Muck Soils Research Farm; Bath, MI. Glucanase concentration was not significant between the different varieties. 10. Peroxidase activity evaluated from six to eight-week-old greenhouse-grown plants of which the leaves were collected and inoculated in the lab with water or Phytophthora infestans. There was a significant difference between some of the tested varieties both constitutively and induced there was not a significant different between the water and P. infestans treatments. Peroxidase activity in this trial was not correlated with the evaluated resistance or susceptibility responses. 11. Peroxidase activity evaluated from mature, field-gown plants of which the leaves were collected before disease was present in the field (Muck Soils Research Farm; Bath, MI.) and inoculated in the lab with water or Phytophthora infestans. There is no significant difference between varieties. There was no correlation between peroxidase activity and susceptibility or resistance. 12. Peroxidase activity evaluated in plants that were infected with Phytophthora infestans in the field, 2001 at Muck Soils Research Farm; Bath, MI. There was no significant difference between peroxidase activity between the different varieties. There was no correlation between peroxidase activity and the foliar disease assessment. 13. Polyphenol oxidase activity evaluated fi'om six to eight-week-old greenhouse- grown plants of which the leaves were collected and inoculated in the lab with water or Phytophthora infestans. A Pearson Product Moment Correlation revealed that there was a correlation between the polyphenol oxidase activity of these varieties, before and after inoculation, with the disease assessment from the field. 14. Polyphenol oxidase activity evaluated fiom mature field-gown plants of which the leaves were collected before disease was present in the field (Muck Soils Research Farm; Bath, MI.) and inoculated in the lab with water or Phytophthora infestans. There was no difference in PPO activity between the varieties and there was no correlation with disease assessed in the field. 15. Polyphenol oxidase activity evaluated in plants that were infected with Phytophthora infestans in the field, 2001 at Muck Soils Research Farms; Bath, MI. There was not a significant difference in PPO activity amongst the different varieties nor was there a correlation between PPO activity and disease assessed in the field. ix l6. Simplified general pathway of isoprenoid biosynthesis (Voet and Voet 1990). 17. Simplified pathway of the branching point between phytoalexin and steroid alkaloid synthesis. Two genes, STVS3 and STVSS, encode proteins for the enzymes involved in the branching point between famesyl pyrophosphate and phytoalexin production. 18. Construction of transgenic potato plants using variety E149-5Y (Kelly Zarka, Michigan State University). 19. Phytophthora infestans disease assessment expressed as RAUDPC (Max. = 1.00) for cv. El49-5Y and its transgenic constructs. The bars within each construct represent the different lines. 20. Average RAUDPC values for the different transgenic constructs when inoculated with Phytophthora infestans compared to the untransformed, control E149-5Y. 21. Average glucanase activity of STVS transformed plants with water or Phytophthora infestans inoculations compared to the untransformed plant, E149- 5Y. 22. Average peroxidase activity of STVS transformed plants with water and Phytophthora infestans inoculation compared to the untransformed plant, E149- 5Y. 23. Average polyphenol oxidase activity of STVS transformed plants with water and Phytophthora infestans inoculation compared to the untransformed plant, E149- 5Y. CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW GENERAL INTRODUCTION The cultivated potato and Potato Late Blight: an Introduction The botanical genus Solanum is extremely extensive, comprising some 2,000 known species (Rowe 1993). Only eight of these species are cultivated for food, and Solanum tuberosum is the only species grown in quantity worldwide. The cultivated potato is thought to have originated in the Andean highlands of South America and has been a valuable crop for more than 8,000 years. Wild potatoes, many of which can freely interbreed, can be found in a range of ploidy levels from the common diploid (2n = 2x = 24) to tetraploid (including S. tuberosum) and hexaploid with most functioning as autopolyploids and only a few as allopolyploids. The potato was introduced into Europe around 1570 and into the United States around 1621 (Rowe 1993). The potato is nutritious in terms of both protein and vitamin C, and provides more yield per unit area than the major grain crops. These factors combined have made it a desirable crop for many people including those with little money or little land (Btu'ton 1989). Potatoes are fourth in world production behind wheat, maize and rice, reaching over 250 million tons in annual production, (Rowe 1993). The potato continues to serve as a crop of major significance in human nutrition, thus making it absolutely necessary to ensure plant health, economic return for growers, and to ultimately maintain crop quality. The largest problem facing potato growers globally is potato late blight caused by Phytophthora infestans (Mont) DeBary. Evidence indicates that Phytophthora infestans probably originated along with the cultivated potato in the Andean highlands of South America, and consequently, this is where the most genetic sources of resistance to late blight can be found. In the 1840’s this potato disease first appeared in Europe and North America causing the potato foliage to blacken and the tubers to rot. In regions such as Ireland, where both environmental and social conditions favored dependence on the potato, the outcome was devastating. Potato late blight completely destroyed the potato crops as well as the lives of many people. As a result of this epidemic, over one million people died in Ireland and another one and a half million people emigrated (Burton 1989). Phytophthora infestans, the casual agent of potato late blight, is a member of the family Oomycota (Agrios, 1997). Oomycetes have little taxonomic relation to filamentous fungi, but are more closely related to golden—brown algae and heterokont algae in Stramenophiles of the Kingdom Protista. P. infestans is broadly distinct from most other Phytophthora species. P. infestans is a coenocytic oomycete with rare cross walls. It can reproduce asexually by sporangia that are ellipsoid to lemon shaped with a small pedicel. The zoospores of P. infestans have two flagella, one forward-directed tinsel type and a backward-directed whiplash type. In culture, the mycelium of P. infestans is white and fluffy and the colony is somewhat slow growing, yet the growth- rates can vary dramatically amongst isolates. P. infestans does not produce chlamydospores. Potato late blight continues to cause immense losses in crop yield everywhere potatoes are grown. Controlling P. infestans costs potato growers hundreds of dollars per acre because control relies mainly on the intensive application of fungicides. The combined costs of crop losses and pesticide applications have been estimated at $3 billion annually (Mackay 1996). Because potatoes are propagated vegetatively from tubers which can carry pathogens and pests, these problems follow potatoes wherever they are grown. The disease cycle of P. infestans has been well described. P. infestans is heterothallic and produces three different spore types. P. infestans produces sporangia and biflagellate, motile zoospores asexually in 5 to 7 day cycles under ideal conditions (Kirk 1996). Persistent, sexual oospores are produced when compatible strains of the opposite mating types, A1 and A2, interact (Galindo and Gallegly 1960). In addition to having different mating types, P. infestans also has different genotypes which creates additional variation within this pathogen. Losses due to potato late blight in North America have become even more devastating in recent years due to the introduction of the A2 mating type (Spielrnan et a1. 1991; Drenth et a1. 1993). Prior to this time, only the A1 mating type was present in North American fields. Using DNA-fingerprinting to identify isolates, US-8, a newly introduced isolate that has the A2 mating type, has been shown to produce more late blight infection on inoculated tubers than was seen on tubers infected with the previously dominant US-l genotype, an Al mating type (Marshall and Stevenson 1996). It has also been shown that US-8 isolates produce faster visible rot on tubers (Lambert and Currier 1997; Kirk et al. 2001) and are resistant to the fungicide metalaxyl, which had been the predominant chemical used for late blight control prior to this time. In the new, dominant strains, the absence of genetic markers which were associated with the previously important US-l clonal lineage, indicates that the new strains have probably not arisen via sexual reproduction involving the US-l lineage (Fry and Goodwin 1997). Instead, it is thought that the re—emergence of late blight around the world in the past couple decades, is due to the recent migration of exotic strains of P. infestans. The oospores of P. infestans can survive in the soil for up to four years in the absence of a host (Drenth et a1. 1995; Turkensteen et a1. 2000) which is the longest survival period of all the infective propagules of this pathogen (Erwin and Ribeiro 1996). However, the actual role of oospores in late blight epidemiology is poorly understood (Andrivon 1995). Direct evidence of oospore production in the field has been scarce. Oospores have been shown to over winter in the northeastern region of North America (Medina et a1. 1999) and there has also been indirect evidence for sexual recombination in the form of probable recombinant genotypes found in some Canadian populations (Goodwin et a1. 1995; Punja et a1. 1998). However, only a few oospores in Canada have actually been recovered (Chycoski and Punja 1996; Peters et a1. 1998). Although the epidemiological function of oospores is poorly understood, the asexual spores including sporangia and zoospores are of great concern. P. infestans spreads prolifically in the field by asexual sporangia and asexual, motile, wall-less zoospores which are released under cool, wet conditions. Upon encystrnent on the potato leaf surface, the zoospores retract or sometimes shed their flagella, and attach to the host surface by secreting an adhesive material. The cysts begin germination by producing a germ tube that differentiates into an appressorium at the apex. Likewise, sporangia can directly cause infection by germinating to produce germ tubes and appressoria. The papillate sporangium of P. infestans is readily detached and is well designed for a splash droplet mode of dispersal (Coffey and Gees 1991). The appressorium penetrates the epidermal cell wall with the production of a penetration peg while the encysted zoospore and the major part of the germ tube becomes devoid of cytoplasm (Shimony and Friend 1975). Once inside the host cell, the pathogen produces an “infection vesicle” (Shimony and Friend 1975) which gives rise to secondary hyphae and then grows into the mesophyll cells or intercellular spaces. Growth into the mesophyll cells has been termed transcellular (Hohl and Stossel 1975) and is different fiom intracellular growth, the usual mode of development which results in the production of haustoria (Hohl and Stossel 1975; Shimony and Friend 1975). Haustoria are specialized intracellular structures that are enveloped by the host protoplast. Such an intimate relationship between the pathogen and the host cytoplasm suggests that this is a highly evolved relationship. P. infestans may be conceived as having evolved towards an increasing dependence on its host (Coffey and Gees 1991). It is possible that it began as a necrotroph and progressively developed the mechanisms to become a hemibiotrophic organism. Thus, it is not beneficial for P. infestans to kill the host cells immediately. Potato Late Blight: Management strategies Potato late blight can affect potato foliage and tubers at any stage of growth and can easily lead to 100% losses (Fry 1994). One major problem is the fact that there are currently no late blight resistant potato cultivars that meet commercial standards in the United States (Kirk et a1. 2001). Following the re-emergence of late blight as a global concern in the late 1980’s and early 1990’s, intense research efforts regarding Phytophthora infestans have led to disease management programs that integrate both cultural practices and chemical treatments. Chemical Controls More chemicals are applied to the potato than to any other food crop. The cost of simply attempting to protect potato crops from late blight in the United States alone is estimated at $155 million annually (Sender 2000). In order to reduce the amount of fungicides applied to potato, several strategies can be used. These strategies include using fungicides with less active ingredients, reducing application rates and implementing longer application intervals (Kirk et a1. 2001). Typical fungicide application programs use 5 to 7 day spray intervals, depending on environmental conditions and grower preference (Kirk et a1. 2001). However, most growers are skeptical of cutting back on fungicide applications because they are faced with the fear of losing their entire crop. The occurrence of metalaxyl-resistant strains of P. infestans in the United States was first reported in 1992 (Deahl et al. 1993). Metalaxyl is an acylalanine, phenylamide fungicide that was effective against sensitive strains of P. infestans. It had an immediate effect on epidemic progress due to the suppression of lesion expansion and the prevention of latent infections when applied prior to establishment of late blight in the crop (Fry et a1. 1979). In sensitive strains of the pathogen, metalaxyl can also suppress established epidemics by reducing sporulation and preventing sporangium germination (Bruck et a1. 1980). Within the last ten years, severe late blight epidemics caused by metalaxyl- resistant strains of P. infestans, have created a demand for new, effective, systemic fungicides. Researchers continue to try to identify a strategy that would be helpful to growers even after infection has already taken place in the field. However, the primary strategy currently used most by growers, is to use all firngicides in a protectant manner in hopes of preventing infection. Recently tested fungicides such as Tattoo C (propamocarb hydrochloride), Curzate M8 (cymoxanil) and Acrobat MZ (dimethomorph) have not been shown to be as effective against the US-8 clonal lineage, as metalaxyl was against the metalaxyl-sensitive US-l clonal lineage investigated in previous experiments (Fry et a1. 1979; Mayton et a1. 2001). Numerous fungicides are being tested against new isolates of P. infestans including Cyazofamid (Ranman), a fungicide in the cyanoimidazole class which has been shown to exhibit strong firngicidal activity against Oomycetes (Mitani et a1. 2001). It has even been shown to be effective against metalaxyl- resistant isolates of P. infestans. At low concentrations cyazofamid is reported to inhibit all stages of the life cycle of P. infestans, including zoospore motility and release, cytospore germination, oospore formation and mycelial growth (Mitani et a1. 2001). It is known that zoospore motility and release are affected by a inhibition of energy supply which suggests that the mode of action of cyazofamid may be due to the impairment of this system. Product development for late blight control is focused on preventing the transmission of disease at seed cutting (Kirk et al. 1999). Seed health is of utmost importance to commercial potato growers. While seed treatments are sometimes recommended for protection against early- season late blight, this does not appear to be an easy answer. The emergence of late blight infected plants is still a risk when using seed treatments due to the fact that slower sprout emergence rates caused by treatments may still enable late blight to be carried into the new crop and initiate infection (Kirk et al. 1999). In order to maximize the efficiency of chemical treatments, many disease- forecasting models have been being developed (Gudmestad et a1. 1995). Forecasting models incorporate plant nutrition, age, day-length and the presence or absence of other pathogens and pests, which are all factors known to affect late blight development. The aim of these late blight forecasting programs is to improve the timing of fungicide applications relative to the onset of disease development and to improve the overall efficiency of fungicide applications. The continual development of these programs, in addition to the discovery of effective fungicides, will additionally help contribute to more successfirl late blight control. Cultural Control The increasing occurrence of devastation worldwide caused by late blight and decreased fungicide efficiency has forced the consideration of all potential management tools in controlling P. infestans. Many of these strategies ultimately attempt to reduce or prevent the introduction of primary inoculum into the field, reduce inoculum survival, reduce infection rates, and generally try to establish conditions that are unfavorable to P. infestans proliferation. There are very specific measures that can be taken to reduce the presence of primary inoculum. Because P. infestans mainly over winters within infected, living potato tissue, the elimination of infected tubers, transplants, cull piles and volunteer potatoes is essential. Permitting tubers to freeze during the over wintering period is one of the most economical means for management in many North American production areas. Likewise, it is recommended that growers always use certified seed. Burying, composting, mechanical cultivation and herbicide treatments are all also possible strategies for controlling volunteers and cull piles. It was estimated by Van Der Zaag (1956) that one infected plant per square kilometer (245 acres) is sufficient to create an epidemic. A wide range of cultural management strategies have been investigated. Studies by Garrett and Mundt (2000), demonstrated the effects of potato cultivar diversity in the field. It was found that the Area Under Disease Progress Curve (AUDPC) for foliar late blight was reduced in host-diversity plots when compared to uniform plots. However, it is not practical for growers to simply implement a multi-variety strategy when the potato plants grow and mature at different rates and produce tubers of varying quality and end- use. With all of the variables and uncertainties, it is wise for growers to instigate an Integrated Pest Management (1PM) strategy which integrates multiple disease management practices. These strategies include; 1) optimizing disease control both ecologically and economically, 2) using multiple tactics to enhance stable potato production and 3) keeping disease damage below injurious levels while minimizing the risks to humans, animals and the environment. The time of planting, soil type, plant density, seed placement depth, irrigation practices, planting direction, and even canopy architecture may influence disease development. Many factors must be taken into consideration when trying to control P. infestans. lO Resistant Cultivars Another way to achieve quality potato production through management is the use and development of potato cultivars that are resistant to late blight. Due to recent changes in the population structure of Phytophthora infestans, resistance to this pathogen and the production of desirable processing quality has become the focus of potato breeders globally. However, this issue of resistance is extremely complicated due to the complex nature of both the host and the pathogen and there are currently no late blight resistant varieties that meet market standards. The development of resistant varieties may be one of the most promising aspects of potato late blight management. Solanum tuberosum supsp. tuberosum, the European cultivated potato, was derived from a narrow genetic base. Consequently, it lacks genes for adequate resistance to many pests and pathogens, which quickly developed into a significant problem when the potato became a major food crop and began to demand continuous genetic improvement. Potato breeding is a difficult task due to many factors including cytoplasmic and nuclear sterilities, tetrasomic inheritance, and inbreeding depression (Douches et a1. 1996). Wild species, which sometimes serve as sources of resistance, must first be hybridized with S. tuberosum. To compensate for the lack of genetic diversity, desirable genes fiom wild and primitive cultivated potato species are introgressed. The development of new varieties became essential after the devastating European late blight epidemic of the 1840’s. From 1851 to 1910 it is estimated that 380 new potato varieties were developed or introduced in The United States and many more elsewhere (Rowe 1993). In 1905, crosses between Solanum tuberosum and Solanum demissum, a Mexican hexaploid, led to hybrid ll resistance (Ross 1986). However, this resistance was very short lived These initial efforts to develop late blight resistant cultivars emphasized vertical, or specific resistance, which is conferred by major resistance genes (R genes) (Flor 1955), usually from wild Mexican species. It has been suggested that pyramiding R genes may confer an additive, quantitative, resistance effect (Pedersen and Leath 1988). Correlations between Rgenes and higher resistance levels, has been tested based on a comparison of plants known to contain R genes with genetically unrelated plants that are free of the R genes (Darsow et a1. 1987). These authors concluded that the R genes were associated with an increase in quantitative resistance and hypothesized that this was due to linkage. In cases such as the early crosses between Solanum tuberosum and a Mexican hexaploid which led to short-lived, hybrid resistance (Ross 1986), vertical resistance had been achieved. Vertical resistance is the specific resistance of a plant to some races of a pathogen while remaining susceptible to other races of the same pathogen. Vertical resistance is always controlled by one or a few genes and is sometimes referred to as monogenic or oligogenic resistance (Agrios 1997). Breeders found commercial potato varieties with the desirable dominant resistant R genes which could confer complete resistance in the absence of the specific races of P. infestans that could successfully infect them. It has been generalized that in race-specific interactions the dominant host genes for resistance are matched by complimentary, dominant, avirulence genes in the pathogen. This concept was first illustrated by Flor’s analysis of the flax-Melampsora lini interaction, which demonstrated that for each gene conditioning resistance in the host, there is a corresponding gene in the pathogen controlling pathogenicity (Flor 1955). It is not surprising that there is more recent genetic analysis of the segregation of virulence in 12 P. infestans that reveals a more complex design (Spielman et a1. 1989). Still, the pressure of the pathogen is more than the hosts can compete with, and vertical resistance is usually quite short-lived. The emphasis of breeders is now on horizontal resistance, which is a more general, non-specific resistance to a range of races within the same pathogen. It is controlled by many genes and is also referred to as partial, polygenic or mutigenic resistance (Agrios 1997). It has been found that the specific components that contribute towards reduced disease severity vary among and within species (Canizares and Forbes 1995; Colon et a1. 1995). Some of the cultivars that have been developed are accepted for fresh market, but most of them have a long growing season and many do not have agronomic quality for the industry. One difficulty in breeding for horizontal resistance is that it is hard to maintain in crossing and backcrossing which is often necessary to recover commercially acceptable tubers (Black 1970). Many different potato characteristics must be considered including the development of cultivars with other attributes such as the “cold chippers” which do not accumulate reducing sugars at cooler storage temperatures (Chase 1995). The continued isolation and characterization of potato R genes and the corresponding avirulence genes from P. infestans is leading to a better understanding of the mechanisms involved in the induction of the defense responses. Still, very little is known about the molecular basis underlying pathogenicity of this diploid pathogen. Cultivar specificity caused by P. infestans in which the pathogen loci interact with nine of the 11 known resistance genes in potato has been studied using F1, F2 and backcross populations (Al-kherb et a1. 1995). In most cases, specificity appeared to be determined 13 by single loci with dominant alleles for avirulence (Spielman et a1. 1989; Spiehnan et al. 1990; Al-kherb et a1. 1995). However, there is of course conflicting evidence. In some isolates, avirulence against potato genes R2 and R4 was dominant (Al-kherb et a1. 1995), while others appeared recessive (J udelson et a1. 1995). It has also been suggested that there is linkage of some avirulent loci, which has not been described in fungi (Al-kherb et a1. 1995). When breeding for resistance to potato late blight, most emphasis is given to late blight resistance in the foliage. It is conunonly thought that high levels of foliar resistance may lead to a reduction in the amount of inoculum capable of infecting tubers, which therefore would automatically reduce tuber blight incidence (Toxopeus 1958; Wastie 1991). To date, there is very little known about the way in which newly established strains, which are extremely aggressive on potato foliage, interact with potato tubers Bjor and Mulelid (1991) gave evidence supporting their hypothesis that the adaptation of specific isolates to tubers of potato cultivars might lead to the erosion of tuber blight resistance. As most popular cultivars are susceptible to late blight, the use of resistant varieties remains a highly desirable goal that has yet to be achieved. However, breeders have developed varieties with partial resistance and guidelines have been established for adjusting fungicide doses based on cultivar resistance (Halseth 1987; Kirk et al. 2001). Still, the widespread use of resistant varieties depends on the future development of cultivars that are acceptable to the market, which requires extensive investigation of this potato-pathogen interaction. 14 Potato Late Blight: General resistance responses Again and again researchers have made detailed studies involving the potato-P. infestans interaction and tirelessly work to define the characteristics that comprise horizontal resistance. The host range specificity (all host plants being Solanaceae) of P. infestans, suggests that there are specific recognition mechanisms between the pathogen and the host that involve the exchange of signals (Pieterse et al. 1992). Recognition of P. infestans by the host plant may lead to the induction of critical plant defense responses, while pathogen contact with the host may also induce responses that are required for pathogenesis. Therefore, it is likely that such interactions induce the expression of many genes in both the host plant and the pathogen. One type of resistant reaction is characterized by rapid host cell death, localized browning of tissue, a shift in terpenoid metabolism resulting fiom the accumulation of fungitoxic sesquiterpenoids, and finally the inhibition of the pathogen (Preisi g and Kuc 1987). In a susceptible reaction, the same events typically take place, but not until after the pathogen has proliferated and spread through the tissue. It has become a major objective to discover potato characteristics that directly correspond to this generalized late blight resistance response. Plants are equipped with an array of mechanisms for resistance to pathogens. This includes preformed barriers to hypersensitive response and production of antimicrobial compounds. Regardless of all the efforts and studies that have gone into this area, there is still much unknown. Tuber infection typically takes place through natural openings such as buds, lenticels, cracks or wounds. The cytological resistance and active responses of potato 15 tubers to late blight can be attributed to three major components (Pathak and Clarke 1987). The periderrn, composed of several layers of phellem cells is the first barrier to infection. The second tuber defense barrier is composed of the outer cortex layers, which can retard lesion expansion and block hyphal growth. The third defense layer is the medulla, the storage tissue of the tuber. Here the tuber reduces hyphal growth and expansion. Based on observations by Flier et a1 (2001), it was concluded that it is actually quite unlikely that the gene-for-gene pathosystem in potato is responsible for differences in tuber infection (Black et al. 1953; Malcohnson and Black 1966). The cell walls and cuticle may be important structural barriers to P. infestans infection in the foliage. It has been suggested that the new important genotype, US-8, preferentially infects through stomatal openings. Thus, the presence of fewer stomata on the abaxial or adaxial leaf surface may also reduce infection potential. It seems likely that topographic signals and chemical attractants together trigger the differentiation and orientation of the infection structures. Early evidence suggests that meSOphyll cells respond differently in compatible and incompatible interactions in addition to differences in timing and the cytological appearance of the responses between cultivars and organs (Coffey and Wilson 1983). Cuypers and Hahlbrock, (1988) investigated compatible and incompatible interactions in one potato cultivar to two P. infestans races, and also explored the non-host response. It was found that the compatible, incompatible and non-host (Phytophthora megasperma f.sp. glycinea) spores all rapidly germinated and penetrated the host cultivar within 1 to 2 hours (Cuypers and Hahlbrock 1988). Large differences were seen between the different interactions during the next intermediate stages of infection. The non-host hyphae reached the palisade l6 parenchyma quickly but went no further. P. infestans hyphae reached the palisade parenchyma approximately two hours later. A hypersensitive response was rarely observed in the compatible interaction while it appeared strong in the incompatible interaction. It was also found that subsequent sporulation occurred only in the compatible interaction (Cuypers and Hahlbrock 1988). These data support an apparent role of mesophyll cells in potato resistance. This study also indicated that there is additional variation in resistance to be explained by plant genotype, leaf age and experimental or environmental conditions. An active defense that is thought to be important in the resistance response of potato is the hypersensitive response (HR). It has been repeatedly shown that incompatible potato-P. infestans interactions result in a rapid plant cell death, known as hypersensitive response, upon penetration of the epidermal cell. HR is credited with preventing the spread of the pathogen which ultimately results in a resistance phenotype (Ferris 1955; Hohl and Stossel 1975; Wilson and Coffey 1980; Gees and Hohl 1988). In compatible interactions, the pathogen spreads throughout the host tissue causing infection while HR is not induced, slowly induced or induced to a lesser extent. Studies by Freytag et al. (1994), demonstrated that hypersensitive cell death was only ever observed when the pathogen had formed haustoria. In this case, the cytoplasm and nucleus conglomerate around the intracellular fungal structure, which is followed by a sudden collapse of the whole conglomerate and an instantaneous collapse of the fungal haustorium (Freytag et al. 1994). The quantitative nature of P. infestans resistance is described as the competition between mycelial growth and the HR of invaded cells (Umaerus 1969; Tomiyarna 1983). 17 In studies by Vleeshowers et al (2000b), it was shown that fully resistant, wild, Solanum species and non-hosts displayed HR within 22 hours of P. infestans inoculation, while plants with partial resistance induced HR between 16 and 46 hours and also displayed larger lesions. The sites of HR are consistently the center for transcriptional activation of a large variety of plant defense genes in neighboring cells (Kombrink and Schmelzer 2001). Subsequently, the biosynthesis of protective secondary metabolites and inhibitory proteins near the site of infection are thought to be important in containing the pathogen. To complicate matters, there are different forms of cell death and mechanisms leading to HR. It has been determined that many small molecules are important for the establishment of HR cell death, yet the direct links between their production and disease resistance remains to be understood. It is believed that signal transduction pathways leading to HR in fully resistant plants, are triggered by pathogen elicitors that recognize specific receptors encoded by plant R- genes (Hammond-Kosack and Jones 1997). Partial resistance in the plant may be induced by nonspecific elicitors produced by all races of the pathogen (Agrios 1997). Karnoun et al., (1999) suggested that multiple pathogen elicitors interact with R-gene receptors from a range of plants to confer non-host resistance. Studies have investigated a range of potential components such as the role of glucans in host-parasite specificity as a suppressor of HR (Doke and Tomiyama 1980), as well as the involvement of cytoplasmic aggregation and actin filaments in signaling transduction for defense (Furuse et al. 1999). The formation of papillae and cell wall appositions seem to be common features of both susceptible and resistant interactions 18 (Wilson and Coffey 1980; Cuypers and Hahlbrock 1988). However, studies have also demonstrated that resistance responses in tuber tissue appears to be correlated with more rapid papillae formation and encasements (Allen and Friend 1983; Hachler and Hohl 1984) Researchers have also found a reversibility response in highly resistant clones during the early stages of HR (V leeshouwers, van Dooijeweert et al. 2000). Mesophyll cells adjacent to HR cells sometimes showed the ability to restore their normal appearance in all of the studied Solanum clones. Thus, a cell that actively responds to pathogen attack is not necessarily committed to HR. An earlier study using video microscopy examined the potato-P. infestans interaction in living potato leaf cells and reported that each individual host cell exhibited a behavior that appeared to be directly linked to the growth of the pathogen (Freytag et al. 1994). It was observed that cytoplasmic rearrangement took place directly at the site of pathogen penetration and that if the pathogen stopped at this stage, the host cell would restore its normal cytoplasmic activity. It was concluded that the cells of the potato leaf perceive the pathogen and signals defense responses during or after perforation of the anticlinal cell wall by a penetration peg (F reytag et al. 1994). The germination of the cyst and appressorial formation did not trigger responses in the underlying cells. This evidence complimented reports that demonstrated that the mere penetration of the cell wall did not seem to trigger defense responses and that there are no immediate initial differences between penetration by a compatible or incompatible race (Gees and Hohl 1988). The first response upon attack by an incompatible race of P. infestans is thought to be the immediate translocation of the cytoplasm and nucleus to the penetration site. 19 This phenomenon called cytoplasmic aggregation, may actually be a defense reaction of the plant and has been reported in potato tissue infected with P. infestans (Kitazawa 1973). To further investigate the function of this process, cytoplasmic aggregation has been suppressed using cytochalasin D, an inhibitor of actin polymerization and the resulting effect on defense responses was examined. (Takemoto et al. 1999). When cytoplasmic aggregation was inhibited, there was a delay in phenylalanine ammonia lyase (PAL) mRNA induction, a decrease in the accumulation of PR-proteins, as well as a partial suppression of cell death. This suggests that cytoplasmic aggregation is essential for the timely induction of other defense reactions (Takemoto et al. 1999). Such inhibition has permitted non-pathogens to penetrate the host plant (Kobayashi et al. 1997). Cytoplasmic aggregation may be a non-specific response because it has also been induced by mechanical stimuli (Laclaire 1989; Hush and Overall 1992; Gus-Mayer et al. 1998). Studies have also indicated that cytoplasmic aggregation alone is not enough to induce resistance in a susceptible host to P. infestans (Gross et al. 1993). This reaction in the plant may assist other defense reactions yet more information is needed regarding the involved compounds in order to better understand the function of this process. Kramer et a1 (1997) analyzed protein synthesis at four distinct developmental stages of P. infestans infection including hyphae, cysts, germinating cysts and appressoria. Changes in protein synthesis occurred during cyst germination and germ tube development. Appressorium formation, the most important infection structure, displayed three unique proteins that may be related to infection and may play a key role in pathogenicity. This data indicates the complexity of gene activation and repression 20 beginning with cyst germination. Protein synthesis is required for cyst germination of P. infestans as it can be inhibited by cyclohexarnide (Clark et al. 1978). Hypersensitive response (HR) has been associated with the induction of plant genes encoding structural defense proteins with roles in pathogen confinement, secondary metabolism enzymes such as those of antibiotic biosynthesis, as well as pathogenesis related-proteins (PR-proteins) (Stintzi et al. 1993). The production of antimicrobial compounds (PR-proteins) Pathogenesis-related (PR) proteins are a group of structurally diverse plant proteins that exist in plants cells constitutively, but are produced in much greater concentrations following pathogen attack or stress. PR-proteins were first detected in tobacco leaves expressing HR to tobacco mosaic virus (TMV) (Van Loon and Van Karnmen 1970). It was found in leaves that the regions of highly induced defense responses exhibited bright blue fluorescence under UV light representing derivatives of the phenylpropanoid pathway (Fritig et al. 1976; Legrand et al. 1976; Legrand 1983). A few days after infection, PR—proteins may account for 10% of the soluble proteins found in leaves (Van Loon 1982; Jarnet et al. 1985; Van Icon 1985; Jamet and Fritig 1986; Pierpoint 1986; Van Loon et al. 1987). Many of the PR-proteins have been classified based on characteristics, including the 3-1 ,3-glucanases and peroxidases which are included in the group PR1 (Agrios 1997). The only PR-proten that has been described to have inhibitory activity towards P. infestans is osmotin, isolated from tobacco and tomato (Woloshuk et al. 1991). While phenylpropanoid metabolites are produced locally, the production of PR-proteins is induced in uninoculated and uninfected parts of partially - 21 infected plants. Thus, the uninoculated parts of the plant seem to develop an increased state of resistance known as Systemic Acquired Resistance (SAR) (Ye et al. 1989). Because PR-proteins are induced in association with the onset of SAR, they may have a role in its efficiency. However, a causal relationship has still to be shown. It is thought that PR-proteins may actually serve as markers for signals involved in SAR. PR-proteins are also of great interest because they have been found to be produced constitutively in interspecific hybrids which exhibit high levels of resistance to viruses (Ahl and Gianinazzi 1982). Vleeshouwers et al. (2000) provided results which suggested that constitutive expression of PR—genes may contribute to non-specific resistance to P. infestans (V leeshouwers, Van Dooijeweert et al. 2000). PR-proteins have been shown to be induced by chemical treatments (White 1979; Ahl and Gianinazzi 1982; Asselin et a1. 1985; De Tapia et al. 1986; Dumas et al. 1987; Nasser et al. 1990; Van de Rhee et al. 1990; Ward et al. 1991; Yalpani et al. 1991; Malamy and Klessig 1992; Uknes et al. 1992; B01 et al. 1996), air pollutants (Ernst et al. 1992; Didierjean et al. 1993), phytohorrnones (Vanloon 1983; Memelink et al. 1990; Hughes and Dickerson 1991) and even osmotic stress (King et al. 1986; Grosset et al. 1990). Some PR-proteins are developmentally regulated in healthy plants (Fraser 1981; Felix and Meins 1986; Shinshi et al. 1987; Lotan et a1. 1989; Castresana et al. 1990; Neale et a1. 1990; Cote et al. 1991; Leung 1992; Richard et al. 1992). In potato plants, resistance can be induced by treatrrrent with hyphal wall components, unsaturated fatty acids and jasmonic acid resulting in enhanced resistance to P. infestans (Cohen et al. 1991; Cohen et al. 1997; Takemoto et al. 1997). Resistance against P. infestans has also been investigated in tomato. Resistance was induced by pre- 22 treatment with a chemical inducer (3-aminobutyric acid) or by a pre-inoculation with tobacco necrosis virus (TNV) in tomato when subsequently infected with P. infestans (J eun and Buchenauer 2001). In the same study, the role of PR-proteins was investigated and it was found that they accumulated systemically under both of the resistance inducing treatments. The PR-protein AP24 was detected in the pathogen cell walls after invasion, as well as in the space between the cell walls and the invaginated plasma (papillae) in leaves. Leaves expressing SAR demonstrated the densest accumulation of AP24. Therefore, it is not surprising that HR is accompanied by PR-protein production and it is valid to analyze a possible role of PR-proteins in resistance responses. Plant encoded enzymes, such as 6-1, 3-glucanases, are capable of hydrolyzing fungal cell walls and may serve as an inducible antimicrobial defense system in plants (Tonon et al. 2001). A major component of the P. infestans cell walls is 0-1 ,3-glucan. Therefore, 0-], 3- glucanases can potentially degrade the walls of this pathogen. Kombrink et al. (1988) identified two PR—proteins with 0-1, 3-glucanase activity in the intercellular spaces of potato leaves. This process would most likely serve as a defense response based on studies by Andreu et al., (1998) which reported that glucans from virulent strains of the pathogen inhibited phytoalexin accumulation which may also be a plant defense response. It has been repeatedly suggested (Garas et al. 1979; Doke and Tomiyama 1980; Currier 1981) that susceptibility results from the suppression and/or delay of the production or activation of resistance reactions by glucans released from compatible races of the pathogen. Soybean 6-1 ,3-endoglucanase cDNA was overexpressed in tobacco plants and these plants showed a positive correlation between glucanase activity and disease (Y oshikawa et al. 1993). This data also supports the 23 possibility that disease-resistant transgenic plants may be developed by expressing genes encoding nonfungitoxic proteins (Kuc 1995). Peroxidase is an enzyme that is required for the final polymerization of phenolic derivates into lignin. It has been found that peroxidase activity increases in potato following F usarium sambucinum infection (Ray and Hammerschmidt 1998). It is known that peroxidase is also implicated in the process of suberization (Mohan and Kolattukudy 1990). Potatoes produce several types of peroxidases with roles that are yet to be determined. Several investigators have found a positive correlation between peroxidase activity in potato foliage and resistance to P. infestans (Kammermann 1951; Kedar and Kammermann 1959). However, Sakai and Tomiyama (1964) found no correlation between peroxidase activity and blight resistance in the early stages of growth. Polyphenol oxidase is located in plasmids and catalyzes the conversion of monophenols to 0- diphenols and 0- dihydroxyphenols to o— quinones. The quinone products can then polymerize and/or react with amino acid groups of cellular proteins, resulting in black or brown pigment deposits (Thygesen et al. 1995). It is widely accepted that polyphenol oxidase may be involved in defense against pathogens (Mayer 1987; Steffens et al. 1994). Evidence has been provided suggesting that polyphenol oxidase activity in leaves is highest in developing tissue with expression declining as development proceeds (Cary et al. 1992; Hunt et al. 1993; Dry and Robinson 1994; Boss et al. 1995; Thygesen et a1. 1995). In tubers, polyphenol oxidase activity remained high throughout tuber development and growth (Thygesen et al. 1995). This maintenance and the localization of the highest levels of polyphenol oxidase at the exterior of the tuber, suggests some role in protection from infection. 24 The production of antimicrobial compounds (phytoalexins) In 1940, Muller and Borger suggested that a prior infection of potato tuber tissue with an incompatible race of P. infestans, induced resistance to a subsequent challenge with a compatible race. Muller and his coworkers hypothesized that toxic compounds formed after infection were a component of this resistance response. This idea gave rise to the phytoalexin theory. A combination of experiments years later by Muller demonstrated that bean pod tissues infected with Sclerotinia fructicola and Phytophthora infestans produced fungistatic substances (Muller 1958). During this same time, Kuc demonstrated that potato tuber tissue infected with incompatible pathogens produced antimicrobial compounds (Kuc 1957). Phytoalexins are defined as low molecular weight antimicrobial compounds that are produced by plants in response to infection or stress (Kuc 1995). While it may seem like a simple idea to induce resistance in plants by inducing phytoalexin accumulation, the whole picture is much more complicated. The number of compounds that are capable of eliciting phytoalexin production is both immense and extremely diverse (Kuc 1995). For example, over 200 compounds, microorganisms and physiological stresses are capable of eliciting the phytoalexin pisatin in pea, phaseollin and kievitone in green bean and glyceollins in soybean (Kuc 1991). Some elicitors, such as chitosan are even antifungal (Kendra and Hadwiger 1984). Scientists have found that firngicides, temperatures and ultraviolet radiation can also elicit phytoalexins (Hadwiger and Schwochaw 1971; Mercier et al. 1993). It is thought that P. infestans species produce three types of elicitors; glucans, glycoproteins and unsaturated fatty acids (de Wit 1986). Bostock et al., (1981) reported 25 that arachidonic and eicosapentaenoic acid from cell wall preparations of P. infestans were effective resistance inducers. Subsequently, Bostock et al. (1982) and Maniara et al. (1984) found that the elicitor activity of these acids were extremely enhanced by the addition ofB—1,3 and B- 3,6-glucans. Thus, inducing these elicitors to magnify phytoalexin production is complex. An alternative to directly applying these compounds or their elicitors in the manner that pesticides are applied to a plant, which may be toxic or washed away, is the use of gene expression to alter phytoalexin accumulation. Still, the role of phytoalexins in plant resistance remains to be elucidated. The suppression of phytoalexin accumulation has been well documented in determining susceptibility or resistance (Ouchi and Oku 1982; Ziegler and Pontzen 1982; Kessmann and Barz 1986; Doke et al. 1987; Preisig and Kuc 1987; Yamada 1989). However, Stolle et al., (1988) found that it was unlikely that phytoalexin accumulation was the cause of restricted Pernospora tabacina colonization in immunized tobacco expressing induced resistance as both the total phytoalexin concentration and the fraction of the leaf area showing symptoms was about five times higher in the control leaves than in the immunized leaves. Potato Late Blight: Resistance through breeding and genetic engineering Genetic engineering methods allow potato breeders to introduce genes into the potato that are outside conventional, sexual hybridization techniques. These effects are intended to compliment traditional breeding efforts. There is some evidence that raises the possibility of the induction of resistance against fungal pathogens by inserting foreign genes coding for phytoalexins (Hain et al. 26 1993). Grapevine synthesizes the stilbene-type phytoalexin resveratrol following pathogen attack. Stilbene biosynthesis only specifically requires the presence of stilbene synthase which is isolated from grapevine and transferred into tobacco. It was reported that these regenerated tobacco plants were actually more resistant to infection by Botrytis cinerea. Such methods seem to be usefirl tools in creating resistance. Genetic engineering has been investigated in aspects of potato breeding as well. In 1995, resistance to Erwinia carotovora was conferred by Wu et al. by expressing the peroxide-generating glucose oxidase gene. However, in order to firlly utilize such methods, much research is needed in order to correctly direct these techniques for obtaining resistance. 27 OBJECTIVE OF THIS THESIS North American agriculture continues to undergo many changes that require new approaches to crop health management. Potato production is no exception. Studying the interactions between the potato plant and the correlating pests and diseases allows researchers to contribute to the overall goal of a comprehensive strategy for potato health management. This management includes the production of quality potatoes that are in line with environmental and legal guidelines while yielding a reasonable profit for growers. Even though there is a large pool of resistance sources available in wild Solanum species, very little is known about the mechanisms that characterize resistance. In fact, very little is known about the disease reactions of North American cultivars to the US-8 genotype of Phytophthora infestans. In order to achieve successful potato breeding focusing on resistance to P. infestans the defense responses in potato must be more intensely evaluated. A better understanding of what makes certain potato plants resistance to late blight may allow breeders to selectively incorporate these characteristics into their program. Complimented by good cultural practices, growers would be able to reduce their investment in costly chemical controls. Due to the immense costs for growers to attempt to control late blight, there is a heavy pressure for researchers to explore the natural resistance mechanisms in potato. This requires a better understanding of the underlying resistance mechanisms and their cytological effects. The objective of this thesis is to contribute to this effort. 28 What are the mechanisms in potato that contribute to late blight resistance? An attempt to identify potato characteristics, that may be involved in potato resistance against P. infestans, can be made by the assessment of potato varieties that are used as advanced breeding lines in breeding programs for late blight resistance. The general resistance characteristics to be evaluated include stomatal density and conductance. Additionally, biochemical processes that may be involved in the resistance responses between the advanced breeding lines and P. infestans will be investigated using B—1,3- glucanase assays, peroxidase assays and polyphenol oxidase assays. Additionally, to further explore possible mechanisms of resistance, potato variety E149-5Y has been genetically altered in the phytoalexin pathway for use as a tool. These plants allow for the introductory investigation of the role of antimicrobial compounds. Further tests will be conducted to examine any adverse effects of this genetic modification. 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Naturwissenschaflen 80: 417-420. Ziegler, E., and R. Pontzen. 1982. Specific inhibition of glucan elicited glyceollin accumulation in soybeans by an extracellular mannan glycoprotein of Phytophthora megasperma f sp glycinea. Physiological Plant Pathology 20: 321- 331. 46 CHAPTER 2: RESISTANCE AND SUSCEPTIBILIT Y TO PHY T OPHT HORA INFESTANS IN ADVANCED BREEDING LINES 47 INTRODUCTION Potato late blight, caused by the oomycete, Phytophthora infestans (Mont) De Bary, is the most serious disease affecting potato crops worldwide. Infection can be initiated on any part of the potato plant, including the tubers. F oliar infections may appear as water-soaked lesions that rapidly enlarge into irregularly shaped regions of necrosis under cool, moist condition. The white mycelium that is characteristic of P. infestans can be observed on the under side of the leaf. A brownish-purple coloration on the surface of infected tubers can cause rot and discoloration in the flesh. Late blight infected tubers are often invaded by secondary pathogens and saprophytes in storage. Currently, growers combine cultural practices such as the elimination of cull piles, frequent field scouting, and the aggressive application of protectant fungicides to try to prevent infection. Prior to 1992, the fungicide metalaxyl was used to prevent the spread of late blight once the pathogen had been identified in the field. However, the introduction of the metalaxyl resistant US-8 genotype of P. infestans into North America has eliminated this chemical as a management tool (Fry and Goodwin 1997). This forces growers and researchers alike to also seriously consider other means of control. Presently, there are no commercially acceptable late blight resistant potato cultivars. Potato breeders are working to try to supply growers with late blight resistant varieties that also possess the demanded market characteristics. This includes high yield, early maturity, smooth flesh, high specific gravity and an overall attractive appearance. This is a serious challenge considering that the sources of resistance come fi'om wild 48 Solanum species which lack virtually all of the characteristics that are demanded by the market. Extensive testing is being done including experiments conducted at Michigan State University (MSU) which have identified foreign cultivars and advanced breeding lines (ABL) that are not fully resistance to potato late blight, but are less susceptible in the absence of fungicides when compared to the widely used, important cultivars (e. g. Snowden, Atlantic, and Russet Burbank) (Douches et al. 1997; Douches et al. 1997; Douches et a1. 1998; Douches et al. 1999). It is the hope of potato breeders that different sources of resistance can be combined to develop cultivars with durable resistance. Therefore, the potato characteristics that may directly or indirectly be involved in resistance to late blight are being sought While potato varieties are being screened for their resistance or susceptibility to late blight, it has become crucial to investigate potato characteristics that may be contributing to this response. Various potato cultivars have been examined in their specific interactions with P. infestans, due to the fact that resistance or susceptibility may be determined at this initial interaction between the pathogen and the host. Leaves appear to be the primary site of infection for P. infestans (Hohl and Stossel 1975; Shimony and Friend 1975; Coffey and Wilson 1983). It has been suggested that the preferential penetration site on potato leaves is the anticlinal wall of epidermal cells, immediately adjacent to stomata guard cells (Wilson and Coffey 1980; Cuypers and Hahlbrock 1988; Gees and Hohl 1988). Topographic features, such as stomatal density, may actually play an important role in the induction of appressorium formation of P. infestans (Kramer et al. 1997). There is evidence that P. infestans senses 49 the groove above anticlinal cell walls on the leaf surface and orients the growth of its germ tube tip towards epidermal cells immediately adjacent to stomatal guard cells (Kramer et al. 1997). It is possible that variations in the cytology of leaf topographic features may play a role in resistance to P. infestans. Another possible component of potato resistance to late blight may be the production of B-l,3-glucanases by the plant to potentially break down glucans in the cell walls of P. infestans. One component of potato resistance to P. infestans is the ability of the tissue to inhibit penetration by the pathogen. It is generally thought that it is the initial stages of infection by the pathogen that are inhibited by highly resistant varieties, rather than subsequent growth or sporulation (Clarke and Kassim 1977). In both woody and herbaceous plants, the process of lignification is important in the compressive strength and stiffiress that it adds to the cell walls. Lignin deposition has a role in water transport but has also been correlated with various types of injury and attack by pathogens. This phenolic polymer increases the resistance of cell walls to mechanical penetration and reduces the diffusion of enzymes and toxins into the plant (Raven et al. 1999). The deposition of lignin has been associated with increased resistance to infection in several plants (Brown 1989). The process of lignification occurs through a series of enzymatic steps, starting with a phenylalanine ammonia-lyase catalyzed reaction to produce lignin precursors, and terminating with a process that requires either hydrogen peroxide and a cell-wall-bound peroxidase (Gross et al. 1977) or oxygen and laccase (Driouich et al. 1992; Savidge and Udagamarandeniya 1992; Sterjiades et al. 1992; Bao et al. 1993; Omalley et al. 1993) in order to polymerize the C6 — C3 monolignols into lignin (Hahlbrock 1979). 50 A third possible component of potato resistance involves using simple assays that are available for detecting the activity of the cell-bound enzyme peroxidase, which provides a possible indication of lignin production. The role of peroxidase and its involvement in plant resistance may provide potato breeders valuable information. It has been shown that toxins from the culture medium of Phytophthora infestans increased phenylalanine ammonia-lyase activity and induced lignification in potatoes (Henderson and Friend 1979). It was additionally reported that lignin in the cell walls of tuber slices was involved in the resistance response of potato tubers to non-pathogenic fungi (Harnmerschmidt 1984). In other host plant interactions, Martinez et al. (1996) indicated that bacterial infection of resistant cotton plants greatly enhanced the activity of two constitutive isoperoxidases suggesting that the stimulation of peroxidase may be associated with resistance (Martinez et al. 1996). The possible correlation between peroxidase activity, lignin production and plant resistance makes this an important enzyme for evaluation. The enzyme-catalyzed browning reactions in potatoes may also have a role in resistance. These reactions are involved in the oxidation of phenolic compounds by the enzyme tyrosinase (polyphenol oxidase, PPO) to quinones, followed by the transformation of quinones into dark pigments (Friedman 1997). Phenolic compounds accumulating in healthy tissue next to infected tissue is thought to have a role in resistance. Gregory et al. (1986) supplied evidence that glandular, trichome- bearing leaves of wild potato species such as Solanum barthaulti Hawkes, conferred resistance to foliage-feeding pests by causing insect entrapment. It was shown that S. tuberosum 51 leaves contain lower amounts of polyphenol oxidase and do not have this type of resistance, but may still have another role in resistance. Numerous studies suggest that PPO may selectively be involved in host-plant resistance. Further work should better define the contribution of PPO in the overall resistance reaction and its interaction with other defense compounds. It is known that potatoes contain polyphenolic compounds such as chlorogenic acid (5-0—caffeoquuinic acid). Chlorogenic acid constitutes about 90% of the total polyphenolic content of potatoes (Malmberg and Theander 1985; Mondy and Gosselin 1988; Dao and Friedman 1992; Ramarnurthy et al. 1992). In addition to possibly being responsible for the discoloration of boiled or steamed potatoes following exposure to air (Swain 1962) and for enzymatic browning (Schwimmer 1981; Hurrell and Finot 1984), chlorogenic acid may be involved in the defenses of potatoes against insects and phytopathogens (Deshpande et a1. 1984; Sinden et al. 1988). An investigation into these possible resistance responses of potato may provide useful information to breeders and ultimately lead to late blight resistance. 52 MATERIALS AND METHODS Plant material Greenhouse plant material The potato cultivars used in this study were provided by Michigan State University’s Potato Breeding Program. Experiments were done using material grown from in vitro plantlets as well as from cuttings. In vitro plantlets were grown in sterile 50 ml glass test tubes containing 8ml MS medium (Murashige and Skoog 1962). This general propagation medium was supplemented with 30g/L sucrose, 0.17g/L dibasic sodium phosphate, 0.4g/L thiamine and 0.1 g/L myo-inositol before the pH was adjusted to 6.0 with 1N KOH. After adjustment, 8.0g/L of Bacto-Agar was added. For propagation, shoots were out just below and slightly above each node and transferred to fresh MS medium using sterile technique. The tissue cultures were maintained in a growth chamber where 75% relative humidity (RH), 25°C chamber temperature and a 16h day/8h night regime was provided. The plants were maintained and monitored in tissue culture before being transplanted into the greenhouse into pots containing Bacto potting medium (Michigan Peat Co., Houston, TX) 2-5 weeks after the original propagation. Field Plant Material In the field, the potato plants were propagated differently in the two years that the trials were carried out. Field plants were established by directly planting tubers in the 53 field (2000), which were produced at the Montcahn Research Farm, or were grown for four weeks in the greenhouse before being transferred outside (2001). The field experiments were conducted at the Michigan State University’s Muck Soils Research Station in Bath, Michigan. The soil is comprised of 90% organic muck and was plowed to a 20-cm depth in October following harvest. The soils were prepared for planting with a mechanical cultivator in May. Cultivars were planted on 28 June 2000 and 11 July 2001. Weeds were controlled by hilling and with Dual 8E at 1.13652 liters/acre at 10 days after planting, Basagran at 1.13652 liters/acre, 1.13652 liters/acre, 1.13652 liters/acre, 20 at 40 days after planting and Poast at 0.85239 literes/acre 58 days after planting. Phytophthora infestans isolates, maintenance and inoculations Phytophthora infestans isolates Pi95-7 and Pi98-1 of the US-8 genotype were obtained from W.W. Kirk, Michigan State University. Isolates were maintained at room temperature on Rye media and Rye RAN media (Caten and Jinks 1968) (see appendix). One-month-old cultures were used for inoculum preparation. The inoculum was prepared under sterile conditions by directly pouring sterile water onto the plates and releasing the hyphae and sporangia with a sterile glass rod. The inoculum was then filtered through two layers of cheesecloth and incubated in a refrigerator at 4° C for 4-5 hours to allow the release of zoospores. Inoculation: Conditions and plant material Eight to twelve weeks old greenhouse-grown plants were used for detached leaf inoculations. The potato leaves were inoculated as previously described (Vleeshouwers 54 2001). The third to fifth fully developed leaves (counted from the top) were spot- inoculated (ten spots on each of the top three leaflets) by pipetting 10p] droplets (104 spores/ml) of P. infestans or water onto the top surface. The detached leaves were placed into sterile petri dishes with water-saturated filter paper, wrapped in dark plastic bags and incubated at 15-18°C for 72 hours in a climate chamber at 100% RH. The maintenance of high humidity was essential. The petri dishes were monitored each day and sometimes required additional sprays of sterile water. Leaf discs were collected from the site of inoculation after 72 hours. Field inoculations and disease assessment The field plots were not directly inoculated but were infected with late blight following the inoculation of nearby plots with zoospores suspensions of P. infestans via sprinkler irrigation. Foliar late blight was assessed in the field every three to six days following the first symptom observation until the time of harvest or 100% infection was observed. Each plant in the field plot was individually assessed for late blight by recording the approximate overall percentage of infected tissue over time. For each plant, the relative area under the disease progress curve (RAUDPC) (Shanner 197 7) was calculated. The use of this method is considered the best estimate for a multi-cycle pathogen such as P. infestans (Fry 1978). RAUDPC = z [(T..,- T.) x (D... +D,-)/2]/ m... x 100] T = time since planting D = % area blighted foliage 55 Analysis of variance (ANOVA) was conducted using SigmaStat 2.03 and the varieties were compared with each other using Tukey’s multiple comparison test. Based on the calculated RAUDPC values, the tested varieties were classified as resistant, moderately resistant or susceptible. Those varieties which varied significantly from the most resistant variety in the study were classified as susceptible, while those varieties which differed significantly from the most susceptible variety were classified as resistant. Varieties that differed significantly from both the most resistant and most susceptible varieties were classified as moderately resistant. Screening of physical resistance mechanisms Measuring Stomatal Density The advanced breeding lines were tested for stomatal density differences in the leaves. Both the abaxial and adaxial surfaces of the leaflets were examined from field and greenhouse-grown plants. Leaflets were collected from the different varieties and leaf discs with a diameter of 1.5 cm were cut from the tissue and cleared using 3:1 ethanol and acetic acid (v/v). The clearing solution was added until no pigments were released fi'om the tissue. The leaf disks were mounted on microscope slides using glycerol. The leaf surfaces were viewed using a microscope with a 40X objective lens and the number of stomata in the field of view were counted. The field of view at 40X was measured (0.0425 mmz). Stomata were counted in the field of view on both the abaxial and adaxial surfaces. Separate samples were prepared for the two surfaces, as simply turning over the leaf disc made viewing more difficult. The greenhouse and field 56 trials were each replicated three times using six leaflets per variety, measured in three positions each. Measuring Diffusive Resistance The diffusive resistance of potato leaves from the advanced breeding varieties was measured to provide an indication of stomatal conductance. This was done using a porometer that measured C02 efflux through the leaf in centimeters per second. Plants were tested in the field on four different days. Measurements were always taken at the same time of day, 12 noon. On each day, three plants of each variety were tested at the first or second fully expanded leaf using two randomly selected leaflets. Prior to the study, a screening had been done which indicated that there was not a significant difference in resistance between the different leaflets of the same leaf. Screening of biochemical resistance mechanisms Potato leaf tissue for Enzymatic Assays Potato leaf tissue was collected from several environments for enzymatic analysis. Six to eight-week-old greenhouse-grown plants were used for controlled analysis while healthy plants from the field were also used for inoculations in the lab with P. infestans. Plants that had been exposed to P. infestans in the field were also used and analyzed for enzymatic differences. 57 Sample preparation for Enzymatic Assays Leaflets inoculated in petri dishes with water or P. infestans were used for enzymatic assays. After 72 hours of incubation, a core borer was used to collect 1-2 g of fresh leaf tissue at the sites of inoculation for one replication. Three replications per treatment were collected and the weight of the fresh tissue was recorded. Each replicate was placed into a 1.5 ml eppendorf tube and immediately immersed into liquid nitrogen. Samples were stored at -80°C until used. Small holes were melted in the top of each eppendorf tube before the samples were dried using a lypholyzer for 3-5 days. When the samples were completely dry, they were removed from the lypholyzer. The dried samples were prepared for analysis by adding 0.3m] of 10mM potassium acetate buffer, pH 5.0 per gram of leaf tissue. The buffer and samples were well homogenized by centrifirging each sample for 15-20 minutes at 10,000 g at 4°C. The samples were stored until use at -80°C. The ,B—I,3-glucanase Assay The B-1,3-glucanase assay was modified from Dann (Dann et al. 1996). The ACZL-PACHYMAN endo-l,3- B-glucanase substrate (Megazyme) was prepared at the rate of 0. 1 g substrate per 4.0 ml 10mM potassium acetate buffer pH 5.0. The substrate was best if prepared as described above in a scintillation vial just before use. The substrate was mixed continuously on a stir plate to prevent clogging. Potassium acetate buffer (0.4 ml), pH 5.0 was added to empty eppendorf tubes, then, 0.1m] of the enzyme sample was added. The tubes were vortexed for ten seconds to ensure that the buffer and sample were mixed. Additionally, a blank control containing 58 0.5m] of buffer without an enzyme sample was prepared. The mixed samples were equilibrated in a 30°C water bath for 3-4 minutes, then 0.1 ml of the B-l,3-glucanase substrate was added to all of the tubes including the control. When adding the substrate, the 2001.11 pipette tips were cut 1-2 mm from the end to keep the substrate from clotting. The reaction was run for ten minutes. During the reaction time, the samples were vortexed twice for a few seconds to keep the substrate from settling. The reactions were stopped by adding 0.7 ml Tris (20% w/v). The samples were again vortexed to completely mix the reaction and then spun at high speed for 2-4 minutes to precipitate the unreacted substrate. The samples were analyzed using a Cary SO-Bio UV-Visible spectrophotometer. A standard curve was created using known enzyme concentrations for comparison. Based on the original standard curve, the glucanase activity of each sample was determined by measuring the absorbency of each sample at 595nm. The activity was expressed as p. units glucanase/ gram fresh tissue weight. Peroxidase Assay The substrate for peroxidase analysis was created by combining 1.25 ml 0.25% guiacol, 5 ml 0.3% hydrogen peroxide and 500 ml 0.1M sodium phosphate, pH = 6.0. The substrate was stored in the refrigerator in a dark bottle and was allowed to warm to room temperature before use. The samples were analyzed using a Cary 50-Bio UV-Visible spectrophotometer. lml of the buffer was used as a blank to zero the spectrophotometer. 1 ml of the peroxidase substrate was mixed in a cuvette with 25 ul of the prepared protein extract. 59 The absorbency was measured at 470nm every 60 seconds for 10 minutes. Peroxidase activity was expressed as the change in absorbency per minute per fresh tissue weight. Polyphenol Oxidase Assay The standard assay for polyphenol oxidase (PPO) activity consisted of 10 mM L- dihydroxyphenylalanine (L-DOPA) in 10 mM phosphate buffer (pH = 7.0) measuring the increase in absorbance at 470 nm (Gomes and Ledward 1996). The tissue samples were analyzed using a Cary 50-Bio UV-Visible spectrophotometer. In a cuvette, 1 ml of the L-DOPA solution was mixed with 50 pl of protein extracted from the sample. Absorbency was measured at 480 nm at 15 second intervals for 90 seconds. The slope was calculated and polyphenol oxidase activity was expressed as the change in absorbency per minute times the flesh tissue weight. 60 RESULTS Field disease assessment results Disease assessment field 2000 The advanced breeding lines of potato used in this study and their late blight resistance characteristics (determined in 2001) are listed in Table 1. Disease was assessed in the advanced breeding lines in the field in 2000 and 2001 (Figure 1). The difference in the amount of disease that was found in the field trial in 2000 and 2001 was not significantly different (P = 0.150). The RAUDPC values from the 2000 field trial were similar to previous studies (Douches et al. 1997; Douches et al. 1998; Douches et al. 1999; Douches et al. 2000). However, RAUDPC values from 2001 were used in comparison tests throughout this study due to the fact that they provide the most recent ratings. Disease Assessment Field 2001 Disease was first found in the field plot at 44 days after planting. Based on the amount of foliar infection recorded for the remainder of the growing season, the RAUDPC values for each variety were calculated. To find if these varieties differed significantly in late blight disease progression, a one way Analysis of Variance (ANOVA) test was performed. The Mean Square (MS) values from the AN OVA, demonstrated that most of the variability in the data was due to differences between the varieties and not to differences within the varieties. The variability between the different cultivars tested in the field accounted for (100%) * [SS (between treatments)/SS(within 61 treatments)] = (100%) *[(0.328/O.435)] = 75.40% of the total variability. Fobs=13.564 >1, allowing the null hypothesis (H0 = mean values of treatments are equal) to be rejected. There was sufficient evidence to conclude that at least two or more of the ten cultivars tested in the field did not develop the same amount of infection or at the same rate. A Tukey multiple comparison test analyzed every combination and demonstrated specific significant differences between the different variety’s RAUDPC values. These results were used for cultivar classification. The tested potato varieties were classified as being resistant, moderately resistant or susceptible in the field based on the significant differences between the calculated RAUDPC values (Figure 2). Atlantic had displayed the most disease in the field while TF75-5 (wild species) demonstrated the lowest amount of disease in the field. As a result, Atlantic, Zarevo and Snowden were classified as susceptible while TF75-5, J 138K6A22, NY121, BO718-3 and AWN86514-3 were classified as resistant. Libertas and MSG274-3 were significantly different from both extremes and therefore were classified as moderately resistant (Table 1). Initially eight plants of each variety were transplanted into the field. Wet conditions that followed planting reduced the number of plants amongst some varieties. When the RAUDPC values from the 2000 and 2001 trials were compared to the RAUDPC values obtained for some of the same varieties by the MSU Potato Breeding Lab in 1999, it appears that overall the values are in line with previous results with the only real discrepancies being Zarevo and MSG274-3. In the field trials in 2000 and 2001 there were natural, severe epidemics of potato early blight, Alternaria solani, in addition to disease pressure from late blight 62 inoculations. Both Zarevo and MSG274-3 are varieties that are quite susceptible to early blight. As a result, the plants were likely weakened and displayed added disease symptoms in the field, thus increasing the RAUDPC values of these varieties higher than may have been expected. In order to calculate the RAUDPC values for each cultivar at the end of the growing season, the disease progression for each cultivar was well documented by estimating the percentage of infected tissue (Figure 3). In the 2001 trial, this began 44 days after planting and continued until 72 days after planting. When the percentage of infected tissue was plotted against the number of days after planting, the disease progression was evident. The three susceptible varieties, (Atlantic, Zarevo and Snowden) all displayed the earliest development of foliar infection and consistently had the highest levels of infection throughout the growing season. Both Atlantic and Snowden reached 100% infection. Libertas and MSG274-3, the moderately resistant varieties displayed a steady, almost linear, development of disease throughout the growing season. The five resistant varieties (AWN86514-3, BO718-3, NY121, J 138K6A22 and TF75-5) did not typically display disease development until later in the growing season. 63 cl 8 D U 0.40 (I) 3 E” 0.35 . 04 ii 0 30 . — Field Trial 2000 § ' Field Trial 2001 Q B A 0.25 ~ l: E 8 . 3 TI 0 20 4 i ii °’ ' “xi o 2 g " i J o 0 2 I: g i" a .5 v 0.15 - .l‘ a o :3 . {a i jg v 0.10 - , 3 ii 11 -‘ ‘8 U ‘ .J z... > m I .. . i i .- “’ g 0-05 ‘ g g E 8 i , -':_. S 4 :1 Ir ii in o ‘-' "1? c: on 0.00 - i; l: g E < <‘r <1 05 g: 2 1A M N If} I\ E \0 I14 E 3 8 m 0 3 g a i: 5 N Z E E’ E a g m < ._, 3 Figure 1: A comparison of the average Relative Area Under the Disease Progress Curve (RAUDPC) values that were calculated for the advanced breeding lines that were included in field trials from 2000 and 2001 at Muck Soils Research Farms; Bath, MI. 64 MSGZ74-3 .. ,. U B :1 U E 51) Susceptible E 0.35 .——i———. a 0.30 . All A Moderately 2’ Resistant e 0'25 ‘ Resistant 8 8 l: o 1 D F“ 0.20 - 3 II E s 0.15 - 1...; .A .g a :3}: “:3, "‘ g 0.10 — .1». . ‘. V. .; 4,... g 0.00 v“ .. g5 o > § N < ATLANTIC - ._ SNOWDEN - LIBERTAS T j BO718-3 NY121 Jl38K6A22 (Wild)TF75-5 E o AWN86514-3 Figure 2: A comparison of the average Relative Area Under the Disease Progress Curve (RAUDPC) values for the advanced breeding lines tested in the field in the year 2001. Their designated resistance, moderate resistance or susceptibility was determined, based on significant differences in their RAUDPC values. * Significance determined by an all pairwise Tukey multiple comparison test; 5% significance level 65 + Atlantic 100‘ —o— Zarevo + Snowden --v— Libertas 80 . + MSGZ74-3 . g —o— AWN86514-3 /, . - .«2 + BO718-3 ‘g —<>— NY121 g (:60 - + J138K6A22 ‘; ‘6 —-A— TF75-5 2 S’. .— 40 - “a a, 0 DD :3 t: 8 33 20 " Q. 0 q 40 45 50 55 60 65 70 75 Days after planting Figure 3: Disease progression in the field 2001. Disease was assessed in the potato foliage every 3 to 4 days between 0 and 72 days after planting of the advanced breeding lines. Disease progression was evaluated in order to calculate the RAUDPC values for each tested cultivar at the end of the growing season. These values were based on the percentage of infected foliar tissue. 66 Table 1: Resistance and susceptibility potato leaf tissue of cultivars tested in the field as determined by disease assessment in the field, 2001. Variety 2L0] response to potato late blight ATLANTIC susceptible ZAREVO susceptible SNOWDEN susceptible LIBERTAS moderately resistant MSGZ74-3 moderately resistant AWN86514-3 resistant 3071 8-3 resistant NY121 resistant J1 38K6A22 resistant TF75-5 (wild) resistant Screening of physical resistance mechanisms Stomatal Density of greenhouse- grown potatoes (abaxial leaf surface) Stomatal density was measured in greenhouse-grown potato foliage on both the abaxial and adaxial side of the leaf to see if there may be any correlation between a variety having a high stomatal density and a high susceptibility to late blight infection, or a low stomatal density and low susceptibility (Figure 4). The differences in the mean values among the different potato cultivars that were tested for stomatal density on the abaxial leaf surface was significant (P = < 0.001) at the 5% significance level. A Tukey pairwise multiple comparison procedure was performed and it was demonstrated that the mean densities for the cultivars Atlantic and Snowden, the two most susceptible test varieties, actually differed significantly from all the other tested varieties. However, Atlantic, the most susceptible cultivar tested, exhibited the 67 highest number of stomata while Snowden, another very susceptible variety, exhibited the lowest number of stomata. The stomatal densities of the resistant and moderately resistant cultivars did not differ significantly from each other. Thus, there was no evident correlation between abaxial stomatal density and disease in the field. 70 50- 40a ‘ffl 30_ (~. mean stomatal density .1 ’1 . 20- ‘ Br I SNOWDEN —:]-m ‘2 l— m m a A ATLANTIC . ; Figure 4: Average stomatal densities of the abaxial leaflet surface from greenhouse- grown advanced breeding lines. Cultivars Atlantic and Snowden differed significantly from the others, yet there was no correlation with resistance or susceptibility. * Significance determined by an all pairwise Tukey multiple comparison test; 5% significance level 68 Stomatal Density of greenhouse- grown potatoes (adaxial leaf surface) Stomatal density was also measured on the adaxial surface of leaves from greenhouse-grown potato plants (Figure 5). As expected, the overall number of stomata on the bottom side of the leaf was significantly higher than on the upper leaf surface. The mean stomatal densities were significantly different (P=< 0.001) at the 5% significance level and a Tukey pairwise multiple comparison test indicated such differences. It was again found that there was no evident correlation between adaxial stomatal density and the amount of disease found in the field. 69 30] 25- 20- C* mean stomatal density NY121 B l—i Z 5 [— MSG274-3 LIBERTAS J l 3 8K6A22 A Figure 5: Average stomatal densities of adaxial leaflet surface from greenhouse-grown advanced breeding lines. Stomatal density varied between the different cultivars yet there was no correlation with resistance or susceptibility. * Significance determined by an all pairwise Tukey multiple comparison test; 5% significance level Leaf Diffusive Resistance Diffusive resistance was measured in the field in 2000 in order to identify if there was any correlation between disease assessment and stomatal conductance (Figure 6). A Kruskal-Wallis one way analysis of variance on ranks was run because the data failed the equal variance test. This test showed that there was a significant difference in stomatal conductance between the various potato varieties. Then, a pairwise multiple comparison 70 procedure (using Dunn’s Method) was performed to see which varieties differed significantly from other varieties. MSG274-3 was found to have the lowest diffusive resistance of all the varieties tested. The diffusive resistance of MSG274—3 was significantly lower than all varieties tested with the exception of AWN865 14-3. A low diffusive resistance is indicative of a high stomatal conductance. The varieties Zarevo, Libertas and BO718-3 displayed the highest diffusive resistance in the study. However, in the field, these varieties were identified as susceptible, moderately resistant and resistant, respectively. A Pearson Product Moment Correlation test was done to look for a possible association between stomatal conductance and disease in the field. This test indicated that there were no significant relationships between any pairs of the variables and thus no evident correlation between stomatal conductance and late blight observed in the field. 71 3.5 3.0 - g A o 2.5 a 8 AB. 1* B E . o 2.0 4 . V?“ J o . gr i" 7 .31 . :03 1.5 " " fl ._ 3* $.51? w -l r --17. .' n g, 1.0 , ¥ ‘ . R -' 6 ,' -' Ci .‘fl'ii - ’ . 1‘ 3.5? .m l'wi 0.5 5?: V ‘ .1 :fi 5;:H 0 o is «277' 4* . U c? M ,_, F v 05 5:: In K 5 w o E a °° m < E < Figure 6: Comparison of stomatal conductance in advanced breeding lines as measured by diffusive resistance in the field, 2000. It was found that there was no correlation between the disease assessment of any of the varieties and their stomatal conductance. * Significance determined by an all pairwise Tukey multiple comparison test; 5% significance level 72 Screening of biochemical resistance mechanisms ,6-1,3-Glucanase Assay Enzyme activity was assessed in three separate sets of plants. Greenhouse-grown plants and healthy plants from the field were both inoculated in the lab and examined for differences in enzyme activity. Additionally, leaflets were collected from field-grown plants 64 days afier planting following inoculation with P. infestans. Leaf tissue from greenhouse-grown plants was collected and brought to the lab for controlled lab inoculations (Figure 7). There was not a significant difference in glucanase activity between the water and P. infestans treatments. In fact, glucanase activity was only induced with P. infestans inoculations in MSGZ74-3. A correlation test determined that there was not a correlation between constitutive or inducible glucanase activity with the corresponding disease assessment fi'om the field. Tissue from healthy, field-grown plants was collected and brought to the lab for controlled lab inoculations (Figure 8). There was a significantly higher concentration of glucanase in the leaflets that had been inoculated with P. infestans versus the water- treated leaflets (P = < 0.001). While it was found that there were significant differences in the levels of glucanase between the two treatments, there was not a significant difference in constitutive or inducible glucanase activity between the tested varieties. A correlation test determined that there was no correlation between the glucanase activity of a specific variety and the amount of disease that had been assessed in the field for that same variety. 73 250 - Water 200 - [2:1 Phytophthora infestans .5... I i=2 '8 3 .s: U) 0 vi: 3. \ "E :1 y . V 100 . 3 .2. :3 ._ as :2 :1. a 50 1 g ' '1;- § .§ . n E: , f1 g ' g i O a 1 f 0 - g, ‘i :46 ATLANTIC SNOWDEN LIBERTAS MSG274-3 AWN86514-3 80718-3 NY121 J138K6A22 TF75-5 Figure 7: Glucanase activity evaluated from six to eight-week-old greenhouse-grown plants of which the leaves were collected and inoculated in the lab with water or Phytophthora infestans. There was not a significant difference in glucanase activity between the water and P. infestans treatments. Glucanase activity was only significantly induced with P. infestans inoculations in MSG274-3. 74 Leaves were also collected in the field in 2001 after infection had already taken place (Figure 9). In this case, it was 64 days after planting. The first expanded leaf was collected and brought to the lab for a determination of glucanase activity. It was found that in this case, that there was not a significant difference (P = 0.453) in glucanase activity between the different varieties. A Pearson Product Moment Correlation was performed and it was found that there was not a significant correlation (P > 0.050) between the glucanase activity in the leaves 64 days after planting and the resistance or susceptibility of any variety in the field expressed as a RAUDPC value. 75 200 _ Water g) -r I P. infestans I '5 3 —— —— g 150 4 __ I): (i I -_ 7 “6 , l t. ' E U -- ' I . a. I ~ E 1004 " ‘1 'a . "F ' __ --1 I 1 . g I ‘1 ' a Li Ll hi 3 .1, , " .g , 3 ~ - . i Q 50 " " W K . ‘, m 1 e g I g ' t N , r? 8 a l 2;. .3 ., .. =1 0 K E U m "9 ”9 N "P E: 9 E < v 0.001). 78 —e co - Water [22:] Phytophthora infestans .—5 O\ l y—a A l u—d N 1 find O I “l a _ . ;. n.3,; 1m: 'éem.‘ "‘ "‘- . .7. 1115; “ '-.-.'. . O\ l s "' 'r "" fig 4:. 1 A ' “”1”“. -.~:.'.‘.-:.'..-.- ' ”J. firearm“? “We-.975 mm 12:-432,43; .. u N l '3 “WY... ”.1. .:c_ " Perox1dase activ1ty (change in absorbancy (470nm)/minute * gram of fresh weight) :3 00 511* 4' “fir-«H- ATLANTIC SNOWDEN LIBERTAS MSG274-3 8071 8-3 NY 121 J 138K6A22 TF75-5 AWN86514-3 Figure 10: Peroxidase activity evaluated from six to eight-week-old greenhouse-grown plants of which the leaves were collected and inoculated in the lab with water or Phytophthora infestans. There was a significant difference between some of the tested varieties both constitutively and induced. There was not a significant difference between the water and P. infestans treatments. Peroxidase activity in this trial was not correlated with the evaluated resistance or susceptibility responses. 79 go 1;) - Water a P. infestans e e- c... u 0 LE 51) J ‘3 ' E b * 6 *' E; 5 '5 g E +3 ; g. g. : m g "- 3' T“ i L v \ r- bi. E. t!- :3 ”‘4 - j 3.. 7 “I; Li 2 e *' 5* - E ‘1 o ‘7 i =' 94 v w ‘ 1 , _. >~ i ‘ L" "i Q! 3: o 2 - __ 3' F #2 .2, 5 E 1i i; ‘1 5; t5 .0 ; by. ~J l-c (I) ‘x ”v . . S e ' s: . an .5 0 - . ' ' o m m m N In °° 9 § ‘2 a. 4 05 5 N .A A: Z m N In (\ >" ‘0 LL 0 < o ~o 0 Z M H H z >— 2 53. < (I) i—l v-a <1 Figure ll: Peroxidase activity evaluated from mature, field-gown plants of which the leaves were collected before disease was present in the field (Muck Soils Research Farm; Bath, MI.) and inoculated in the lab with water or Phytophthora infestans. There is no significant difference between varieties. There was no correlation between peroxidase activity and susceptibility or resistance. 80 C 3 '5 0 It! ‘0—4 g 4 _ at _Iq 8 ‘7‘- l i ._ . ' o :3 3 "‘ ’,"v :2. ’1‘” v" :9. E r " v- -. ‘3'" g; i ii‘ ~ :1 | EA .9 4.51?" ’3 " 53' H . . x .. . A,, w . ' B E .l‘ip ,5" i A“ g i 4 Dub 2‘ “.1 ‘1 ~" "I . t . .5- ' . $ ii " at 3‘ i “" >~ . ”A: ' V" b I.“ ‘7! 1" . . ‘ P . . .. q a”; ' .1 ,3 a ' 3. "- - ‘ ' V ‘ 3 ” "1": r1 1"} 1‘ ~ " E a . . . ~ . _ .‘ ., .- $9 *5 1 4 . - 3‘92: «a. ~. a»..- in» g“ .r. ., 51-", .1" - .. ‘, ;‘. a,“ pf. ' "‘3: "I: ‘ .3“ WW .-'«.~»‘- ‘ ,_ .. (5;. W . 4“ . v 37"“; ‘ ‘_ .:,,t.- . , g) 0 l (I) ”I I2 I a (I... (IV T” o M "‘ 5 < g o 3 8 E5 ‘" H N 2 § § '3 < m H < Figure 12: Peroxidase activity evaluated in plants that were infected with Phytophthora infestans in the field, 2001 at Muck Soils Research Farm; Bath, MI. There was no significant difference between peroxidase activity between the different varieties. There was no correlation between peroxidase activity and the foliar disease assessment. 81 Polyphenol Oxidase Assay: Polyphenol oxidase (PPO) was evaluated in the same three sets of plants including; greenhouse-grown plants, healthy plants from the field and field-grown plants exposed to P. infestans. Polyphenol oxidase is involved in oxidation of phenol compounds to toxic quinones which are then transformed into dark pigments (Friedman 1997). It is thought that phenolic compounds that accumulate in healthy tissue next to infected tissues, may have a role in resistance. When PPO activity was evaluated in greenhouse-grown plants that were inoculated in the lab with water or P. infestans, a Pearson Product Moment Correlation revealed that there was a correlation between the polyphenol oxidase activity of these varieties, before and afier inoculation, with the disease assessment from the field (Figure 13). When healthy tissue from the field was collected and lab inoculations were performed, it was found that there was no difference in PPO activity between the different varieties, nor was there a correlation with the amount of disease that was assessed in the field (Figure14). There was not a significant difference between the polyphenol oxidase activity of the different varieties tested in the field following exposure to P. infestans (P = <0.001) nor was there a correlation between PPO activity and the amount of disease assessed in the field (Figure 15). 82 2:3 1%“ 1.6 3 a — Water 0 1.4 c ‘5 [2:1 P. infestans 2,3, 1.2 a 2* ‘6 3 3'3 1.0T g E 5; 0.8 - '2‘, a; "' 0.6 d “ "3% g i _ .—. C.‘ I ‘, 53$. 0-4‘ :1 " g i: 8 ‘f‘ 11 if E" l. r '8 La,_ F“ i #3 , .5 0'2 ‘ 3 3 iii 3: g ii “ a) 3r 1i j {a f i 3: g g 0.0 _ m 'I-‘ -"s e, 2 m "9 r? "9 ~ N "a [— P {E .3 z 22 51' 2 a E E M 8 e 5 E § & L1.) 00 a O a g m fl < 5. A E : Figure 13: Polyphenol oxidase activity evaluated from six to eight-week-old greenhouse- grown plants of which the leaves were collected and inoculated in the lab with water or Phytophthora infestans. A Pearson Product Moment Correlation revealed that there was a correlation between the polyphenol oxidase activity of these varieties, before and after inoculation, with the disease assessment from the field. 83 _O N 1 I. . .m.‘f‘: R“ $4 x “mm 4.....1 "‘ ,. " “w,- . .. . 2',» AREFJ. . O O I W4”; :. ,_ . 2.. O .c: 1.0 .2.” a; - Water {:0 E22! P. infestans a) d: 0.8 4 E * 1‘" - [.1 a) G ‘ . ‘3‘ 1 3' a E ‘ ~ “’ ‘ g V" 5! 3,, .. g ,7 I . ” ‘3 '3 i S In: >‘ i *7": 5:»: g g '8 5 i1 51 "~' {.35 “ °‘ E 5?? ,. 3 a “’ a? o .. E’ .1: o v ATLANTIC ZAREVO SNOWDEN LIBERTAS MSG2 74-3 AWN865 14-3 8071 8-3 NY 1 2 l J l 3 8K6A22 TF75-5 Figure 14: Polyphenol oxidase activity evaluated from mature field-gown plants of which the leaves were collected before disease was present in the field (Muck Soils Research Farm; Bath, MI.) and inoculated in the lab with water or Phytophthora infestans. There was no difference in PPO activity between the varieties and there was no correlation with disease assessed in the field. 84 E '5 1.0 3 fi 0 6:: g _ :44 a: {2‘ Sb Q .;.1 Z * IL? I‘ a .x H D .> '. -> , g g t ~ i; "1‘ 0) - ~ " Cr -. JCT m "‘ . “I u“ L. ‘*r a g :5" if » Ia ‘. : ‘ i " r ‘1 al.; -' " , . ». o o - -' . .. - .. -"I» - .- _ fl. ' .: . . M. '. - 1.4 ‘ a » at? ‘_»a. o a; - 1... I; .r‘ " I: >‘ ' . 3.2:? 1* p?” 3'32‘ "fix 1.2% g o 1% j ,_ its» -.«. ._. c: r. g; 49‘ . 1 . . O O *9. i g: ,' ’_ ‘ .' 8 . "i : Y 7 g i‘ 5" y . .I "" . , ,.f 4.513;? ‘ ~ 0 "‘5 5’ J“ 00 A ..‘ .- n. g . I ‘ o O Z V) M M m --t N In V p m < é Ea Bo>v £85585 Eocoaofl mo unmanned 38:00 ”2 PawE mEeuoahozaumom T Aouanamonaoea fixes: a: Iv «Ea—8.2 228m 6 «2:59.035: T Eggnog—ESQ 35.68 .39 a a Acumnmmonaea Efiafiofifiv Aga— tv .3— Aofinamoaqoba $5338: a 23.. 35.962 9 $8 . .52maeoéxega <8 02: 4’ <5 Ea< 100 million hectares, mostly in North America (Dunwell 2000). Transgenic crops mainly include com, soybeans and cotton. Regardless, the development of transgenic plants may at least allow researchers to better elucidate and understand the individual steps of these processes. Altering plant sesquiterpenoid production by the introduction of biosynthetic enzymes also facilitates more detailed studies of plant isoprenoid metabolism as a defense mechanism. An alternative way to investigate such pathways is through the use of sense and antisense RNA and DNA to enhance or reduce specific gene expression by targeting the gene’s mRNA rather than the gene itself. The ultimate goal remains to understand the potato’s resistance mechanisms to P. infestans infection. This may be accomplished by further investigating the role of phytoalexins and their biosynthesis using sense and antisense technology. This research was undertaken to determine if enhanced resistance to late blight could be obtained by combining natural resistance with engineered resistance conferred by two genes encoding sesquiterpenoid synthases. The genes used in this investigation, Solanum tuberosum vetispiradiene synthase 3 (STVS3) and Solanum tuberosum vetispiradiene synthase 5 (STVSS), were cloned by Dr. Michael Zook, Michigan State University. These two genes encode proteins for enzymes involved in the branching point between famesyl pyrophosphate and phytoalexin production (Figure 17). We were further interested in examining the use of sense and antisense technology by evaluating several non-related enzymes to investigate any unexpected deleterious effects of such alterations. 101 Steroid Alkaloids O\. 0 OCOCH 3 T i Phytuberin \ —-» QC // srvs 3 srvss OPP Vetispiradiene 0w lCHO HO - -— .. .0 HO Rishitin Lubim in Farnesyl PPi Figure 17: Simplified pathway of the branching point between phytoalexin and steroid alkaloid synthesis. Two genes, STVS3 and STVSS, encode proteins for the enzymes involved in the branching point between famesyl pyrophosphate and phytoalexin production. 102 .5" . A Jhnuhmifl.‘ . '. MATERIALS AND METHODS Transgenic plants: Solanum tuberosum vetispiradiene synthase genes and construct summary: The construction of pCR2.ISTVS3 (Solanum tuberosum vetispiradiene synthase 3) and pCR2.ISTVSS (Solanum tuberosum vetispiradiene synthase 5) was completed by Dr. Michael Zook, Michigan State University. The STVS3 and STVSS genes were inserted into various plant plasmid vectors and then transformed into potato plants by Kelly Zarka, Michigan State University using the following methods. The binary vector pB1121(Clontech) was used as a base vector for creating the plant transformation vectors in this experiment. pB1121 which contains the CaMV 35S promoter was modified to remove the gus gene by digesting it with Smal and EcoICRI and ligating the blunt ended fragments together. The resulting construct was called pSPUD4. pCR2. 1STVS3 was digested with BamHI cutting out a 1.9kb fragment that contains the entire STVS3 gene. This fragment was ligated to pSPUD4 which was linearized by BamHI. Both orientations were obtained creating pSPUD46 (antisense) and pSPUD47 (sense). pCR2.ISTVSS was digested with XbaI and SpeI cutting out a 1.9kb fragment that contains the entire STVSS gene. This fragment was ligated to pSPUD4 which was linearized by XbaJ. Both orientations were obtained creating pSPUD48 (antisense) and pSPUD49 (sense). Additionally a construct was created to contain a tuber specific expression promoter instead of the constitutive expression of the CaMV 35S promoter. The construct 103 pS20A-G which is a pBI101.l(Clontech) binary vector modified to contain the class-I patatin promoter (W enzler et al. 1989) was modified to remove the gas gene by digesting it with SmaI and EcoICRI and ligating the blunt ended fragments together. The resulting construct was called pSPUDS 1. pCR2.ISTVS3 was digested with BamHI cutting out a 1.9kb fragment that contains the entire STVS3 gene. This fragment was ligated to pSPUDSl which was linearized by BamHI. The resulting plasmid with STVS in the sense orientation is called pSPUD53. 104 383:5 28 8322 .88 E3: >m$3m~m2 on: mangoes Bazaar? mean 353 280A 01885.: no nouogmnoo ”a magma Sum 53% VmGz._. 8:8 : a 833%.. L . 25F oauquESaSmnw e moz ._. L688. 93 Eco 83:83 EFF oasesgeum moz ._. 8.8.2.5 mafia 322.3 83283 a r Ed 28% 82 .F 738.... «new 8928 £328.: I. .r 58% 53% $02 ._. 858.55. «63% mm>2w0 8328.: L . 9:. no:._.I__._.az..mo:¢ €30? mommy «magma ml. 105 Production of Transgenic Plants The transgenic potato plants were developed by Kelly Zarka, Michigan State University, using the following methods. Plants of potato line MSEl49-5Y were micropropagated in GA-7 Magenta vessels each containing 25 m1 of modified MS basal medium (Douches et al. 1998). The plants were grown at 23-27°C in a 16 hr photoperiod under fluorescent lights (30 umol'm'z's'l) for 2 weeks. The leaves, with tip and petiole ends removed, were then cultured abaxial side down on agar solidified step I medium (Y adav and Sticklen 1995) for 2 days. The pre-cultured explants were immersed for 10 min at room temperature in an Agrobacterium tumefaciens tumefaciens strain LBA4404 (Ooms et al. 1982) containing pSPUD46,47,48,49 culture, which had been grown first in liquid Ty medium (Douches et al. 1998) at 28 °C for 2 d, diluted 50-fold, and then incubated for 7 h. Following immersion, the explants were transferred onto agar-solidified step I medium and co- cultured for 2 d at 23-27 °C. After the co-cultivation, leaf explants were rinsed with liquid step II medium (Douches et al. 1998) supplemented with 200 mg - L'l Timentin (SmithKline Beecham, Philadelphia, Penn.). The explants were then transferred to agar solidified step 11 medium, supplemented with 200 mg - L’l Timentin and 50 mg - L'1 kanamycin. The leaf explants were subcultured every week. Regenerating shoots (>5mm) from separate transformation events were excised and transferred individually into 25 X 100 mm tubes each containing 20 ml of MS medium (Douches et al., 1998) supplemented with 200 mg - L'l Timentin, and 50 mg - L’l kanamycin. 106 Obtaining and maintaining transgenic plants The transgenic potato plants used in this study were provided by Kelly Zarka, Michigan State University’s Potato Breeding Program. In vitro plantlets were grown in sterile 50 ml glass test tubes containing 8 ml of MS medium (Murashige and Skoog 1962). The propagation medium was supplemented with 30 g/L of sucrose, 0.17 g/L dibasic sodium phosphate, 0.4 g/L thiamine and 0.1 g/L Myo-Inositol before the pH was adjusted to 6.0 with 1N KOH. After adjustment, 8.0 g/L Bacto- Agar was added. For f propagation, shoots were cut just below and slightly above each node and transferred to fresh MS medium using sterile technique. Transgenic tissue cultures were maintained in E a growth chamber where 7 5% relative humidity, 25’C chamber temperature and a 16- I. hour day/8- hour night regime was provided. Approximately 2-5 weeks after propagation, the small shoots were transplanted into a secure transgenic greenhouse using clay pots containing Bacto potting medium (Michigan Peat Co., Houston, TX). The plants were watered daily and fertilized regularly. Phytophthora infestans cultures Maintaining cultures Phytophthora infestans isolates 95-7 and 98-1 of the US-8 genotype were obtained from Dr. W.W. Kirk, Michigan State University. Isolates were maintained on Rye media and Rye RAN media (see appendix) (Caten and Jinks 1968). One-month-old clean cultures were used for inoculum preparation. The inoculum was prepared under sterile conditions by directly pouring sterile water onto the plates and releasing the hyphae and sporangia with a sterile glass rod. The inoculum was then filtered through 107 two layers of cheesecloth and incubated in a refrigerator at 4° F for 4—5 hours to allow the release of zoospores. Cochliobolus carbonum was maintained on potato dextrose agar (see appendix) and spores were collected from 2- to 3- week-old cultures. The cultures were washed and scraped with sterile water and the dark spore suspensions were filtered through two layers of cheesecloth before use. Inoculation and disease assessment of transgenic potato foliage Eight to twelve weeks old greenhouse-grown transgenic plants were used for detached leaf inoculations. The potato leaves were inoculated as previously described (V leeshouwers 2001). The third to fifth fully developed leaves (counted from the top) were placed into sterile petri dishes (with water-saturated filter paper. The detached leaves were spot-inoculated (ten spots on each of the top three leaflets) by pipetting 10pl droplets (104 spores/ml) of P. infestans or water onto the top surface and incubated at 15- 18°C in a climate chamber at 100% relative humidity with a 16hour day /8 hour night photoperiod. The maintenance of high humidity was essential for adequate infection. The inoculated leaves were assessed for disease by estimating the approximate percentage of infected foliage at 48, 72, 96 and 120 hours. Inoculation procedures and terpenoid extractions from potato tuber tissue Potato tuber disks, approximately 5mm in size, were prepared as previously described (Henfling and Kuc 1979). The disks that were prepared using sterile techniques, were placed into large glass petri dishes lined with filter paper that had been 108 moistened with 3 m1 of sterile water. The tuber disks were inoculated with 200p] of water or Cochliobolus carbonum (1 x 108 spores/ml). Each treatment was applied to three tuber disks. The treated disks were observed at 24, 48 and 72 hours for disease development. Extracting terpenoids from potato tuber tissue After the potato tuber disks were incubated in petri dishes for 72 hours at room temperature, the upper 1mm layer was sliced from each disc with a potato peeler and cut into four sections. The segments were combined and weighted to make approximately 1 g samples. The tissue samples were immediately transferred to 50 ml Erlenmeyer flasks containing 20 ml of methanol. The flask was covered with parafilm and placed onto a shaker overnight at medium speed. The methanol from the samples was decanted into round-bottom flasks. The flasks were rinsed twice and shaken well with 10 ml of methanol which was then also added to the round-bottoms flasks. The samples were evaporated using a rotary evaporator. Five ml of distilled water and 5 ml of ethyl acetate were added to the residue in the flask, vortexed, and then allowed to separate. The upper organic layer was siphoned off and then transferred into a test tube. The remaining water layer was then extracted two more times with 5 ml of ethyl acetate. The extracts were combined and evaporated to dryness using a stream of nitrogen. Prolonged drying was avoided because this may cause the loss of terpenoids. Thin layer chromatography (TLC ) was performed by adding 200 pl of methanol per gram of tissue. 50 ul of sample was added onto a silica TLC plate (10 ul was added 109 at a time, 5 times). The TLC plate was run in 1:1 cyclohexanezethyl acetate (v/v) and allowed to dry. The plates were sprayed with vanillin- sulfuric acid using a chromatography sprayer (Sigma). The vanillin mixture was created by dissolving 3 g of vanillin in 100ml 95% ethanol. It was put on ice and stirred while a 0.5 ml concentrated sulfirric acid was slowly added one drop at a time. The sprayed TLC plate was heated in an oven at approximately 110°C for 5-15 minutes or until color appeared (Varns 1970). Results were recorded immediately. Inoculation procedures and terpenoid extractions from potato leaf tissue Eight to twelve weeks old greenhouse-grown plants were inoculated as previously I. described. The third to fifth firlly developed leaves (counted from the top) were placed into sterile petri dishes with water-saturated filter paper. The detached leaves were spot- inoculated (ten spots on each of the top three leaflets) by pipetting 10111 droplets (104 spores/ml) of P. infestans or water onto the top surface and incubated at 15—18°C in a climate chamber at 100% relative humidity with a 16 hour day /8 hour night photoperiod. The inoculated leaves were monitored at 24 and 48 hours before leaf disks were collected at 72 hours for extractions. Extraction of terpenoids fiom potato leaf tissue Terpenoid phytoalexins from leaf tissue were extracted according to methods previously described (Hammerschmidt and Kuc 1979; Threlfall and Whitehead 1992). Leaves of 8- to 12- week-old Solanum tuberosum L., MSE149-5Y and transformed constructs of the same cultivar were treated with water or a suspension of Phytophthora 110 infestans US-8 isolate (104 spores/ml) prepared flom 2- or 3-week-old cultures. The treated leaves were observed at 24, 48 and 72 hours for disease development. At 72 hours the leaf tissue was harvested and extracted by a modification of Keen’s facilitated diffusion technique (Keen 1978). The leaf tissue was weighed and placed in 500 ml flasks containing 40% ethanol (1 5ml/g flesh weight). The samples were vacuum infiltrated for 5 minutes before being placed on the shaker for four hours. After this time, the leaf tissue was removed and the ethanol solution was emptied into round-bottom flasks, evaporated in a rotary evaporator at 40°C down to approximately 10 ml and then transferred to 50 ml glass test tubes. Equal volumes of ethyl acetate were added to each test tube and vortexed. Fifteen minutes was allowed for separation before the ethyl acetate layer was pipeted out into round-bottom flasks and evaporated to dryness using the rotary evaporator. The samples were redissolved with methanol (1 ml/ g flesh weight) and pipeted into disposable test tubes. This flask was redissolved a total of three times before the samples were evaporated with Nitrogen to dryness. The samples were used in thin layer chromatography (TLC) by diluting each sample with methanol (20 g flesh weight/ml) and then applying one gram of flesh weight equivalent (50 ul) to a silica TLC plate. The plates were developed with cyclohexane: ethyl acetate (1:1, v/v) and then dried for one hour (Shih and Kuc 1973). The plates were then sprayed with vanillin- sulfuric acid using a chromatography sprayer (Sigma) and heated in an oven at approximately 110°C for 5-15 minutes or until color appeared (Varns 1970). Results and pictures were recorded immediately. 111 Enzymatic analysis of potato leaf tissue Sample Preparation for Enzymatic Assays Potato leaf tissue was collected flom eight to twelve-week-old greenhouse-grown plants and brought to the lab for preparation and enzymatic analysis. Transgenic leaflets that had been inoculated in petri dishes with water or P. infestans were used for enzymatic analysis. After 72 hours of incubation, a core borer was used to collect 1-2 grams of flesh leaf tissue at the sites of inoculation for one replication. Three replications per treatment were collected and the exact weight of the flesh tissue was recorded. Each replicate was placed into a 1.5m1 eppendorf tube and immediately immersed into liquid nitrogen. Samples could then be stored at -80°C until use. Small holes were melted in the top of each eppendorf tube before the samples were vacuum dried using a lypholyzer for 3-5 days. When the samples were completely dry, they were then removed flom the lypholyzer. The dried samples were prepared for analysis by adding 0.3m] of 10mM potassium acetate buffer, pH = 5.0 per gram of leaf tissue. The buffer and samples were well homogenized. Each sample was centrifuged for 15-20 minutes at 10,000 g at 4°C. The samples were stored until use at -80°C. The ,B-I,3-glucanase assay The B-l,3-glucanase assay is modified from Dann et al. 1996. The ACZL- PACHYMAN endo-1,3- B-glucanase substrate was ordered flom Megazyme and prepared at the rate of 0.1 g substrate per 4.0 ml 10mM potassium acetate buffer pH=5.0. The substrate is best prepared in a scintillation vial just before use. The substrate should be kept mixed on a stir plate. 112 I.H“Ifla"- 31'.“ .r ~ ' . it). To begin the assay, 0.4 ml of 10mM potassium acetate buffer pH= 5.0 was added to empty eppendorf tubes. Then, 0.1ml of the enzyme sample was added. The tubes were vortexed briefly to ensure that the buffer and sample were mixed. Additionally, a blank was created that contained 0.5ml of buffer without an enzyme sample. The mixed samples were equilibrated in a 30°C water bath for 3-4 minutes. Then, 0.1 ml of the B- 1,3-glucanase substrate was added to all of the tubes including the blanks. When adding the substrate, the 200p.l pipette tips were cut 1-2 mm flom the end to keep the substrate flom clotting. The reaction was run for ten minutes. During the reaction time, the samples were briefly vortexed twice to keep the substrate flom settling. After exactly ten minutes, the reaction was ceased by adding 0.7 ml Tris (20% w/v). The samples were again vortexed to completely mix and stop the reaction and then spun at high speed for 2- 4 minutes to precipitate the unreacted substrate. The samples were analyzed using a Cary 50-Bio UV-Visible spectrophotometer. A standard curve was created using known enzyme levels for comparison. Based on the original standard curve, the glucanase activity of each sample was determined by measuring the absorbency of each sample at 595nm. The activity was expressed in units, i.e. p. units of glucanase per gram flesh tissue weight. The Peroxidase assay The substrate for peroxidase analysis was created by combining 1.25 ml 0.25% guiacol, 5 ml 0.3% hydrogen peroxide and 500 m1 0.1M sodium phosphate, pH = 6.0. The substrate was stored in the refligerator in a dark bottle and was allowed to warm to room temperature before use. 113 The samples were analyzed using a Cary 50-Bio UV-Visible spectrophotometer. lml of the buffer was used as a blank to zero the spectrophotometer. One ml of the peroxidase substrate was mixed in a cuvette with 25 pl of the prepared protein extract. The absorbancy was measured at 470nm every 60 seconds for 10 minutes. Peroxidase activity was expressed as the change in absorbancy per minute per flesh tissue weight. The Polyphenol Oxidase assay The standard assay for polyphenol oxidase (PPO) activity consisted of 10 mM L- dihydroxyphenylalanine (L-DOPA) in 10 mM phosphate buffer (pH = 7.0) measuring the increase in absorbance at 470 nm. The tissue samples were analyzed using a Cary SO-Bio UV-Visible spectrophotometer. In a cuvette, 1 ml of the L-DOPA solution was mixed with 50 ul of protein extracted flom the sample. Absorbancy was measured at 480 nm at 15 second intervals for 90 seconds. The slope was calculated and polyphenol oxidase activity was expressed as the change in absorbancy per minute times the flesh tissue weight. 114 RESULTS Disease Assessment of Transgenic Potatoes For each construct that was created, several transgenic lines were produced. Using a One-Way ANOVA test, it was found that the disease assessment of the constructs E149-5Y.46, El49-5Y.47, E149-5Y.48 and E149-5Y.53, did not vary significantly within their different lines, whereas only E149-5Y.49 was significantly different (P = 0.012) amongst some of its different lines (Figure 19). E149-5Y was a construct created with the patatin promoter, meaning that it should have only been expressed in the tuber tissue. This construct showed a decreased RAUDPC value when compared to the untransformed plants. The different constructs were also tested for differences in disease assessment between the five constructs. It was found that the differences in the mean values of disease assessment among the different E149-5Y constructs was not significant (P = 0.134). Due to the fact that there was not a significant difference for most of the different lines within a construct (with the exception of E4930, the average RAUDPC values were calculated for each of the five constructs for comparison (Figure 20). An ANOVA test compared the disease assessment of the five constructs to the control, untransformed plants. The difference in the mean values was greater than would be expected by chance (P = <0.001). A Tukey All Pairwise Multiple Comparison test was performed and the disease assessment of the constructs was compared to the untransformed plants to see if their transformation had altered the resistance or susceptibility of this variety. It was discovered that the sense constructs of STVS3 and STVSS (E47 .X and E49.X) displayed 115 a reduced disease assessment when compared to the untransformed plants, whereas the antisense constructs of the same genes (E46.X and E48.X) were not significantly different flom the control. However, the sense construct of STVS3 using a patatin promoter, also displayed a significantly reduced disease assessment when compared to the control. RAUDPC (Max. = 1.00) 0.4 r 0.3 - 0.2 ' _, 0.1~ " l l nan-an 1- ae- ‘: -m§e%:mm$1- "5 r11 0.04 I II II " II I E46.X E47.X E48.X E49.X E53,X untransformed antisense sense antisense sense sense STVS3 STVS3 srvss srvss STVS3 (patatin promoter) Figure 19: Phytophthora infestans disease assessment expressed as RAUDPC (Max. = 1.00) for cv. E149-5Y and its transgenic constructs. The bars within each construct represent different lines. 116 RAUDPC (Max.=l.00) 0.3 A' I ~02 1‘. I. —t <17L -’ ‘01 7” . 0.0 . El49-5Y E46.X E47.X E48.X E49.X E53.X (untransformed) Figure 20: Average RAUDPC values for the different transgenic constructs when inoculated with P. infestans compared to the untransformed, control El49-5Y. * Significance determined by an all pairwise Tukey multiple comparison test; 5% significance level 117 Terpenoid extractions from potato leaf and tuber tissue Terpenoids were extracted flom potato tuber and potato leaf tissue following a water treatment or inoculation with C. carbonum. This was done to compare constitutive and induced terpenoids in the different constructs with the untransformed plants. In the tuber tissue, C. carbonum sometimes, but not always, induced terpenoid accumulation. Additionally, there did not appear to be a visual correlation between the gene expression of the different constructs and the amount of terpenoids produced as would be expected. Unexpectedly, phytuberin was isolated from a sense STVSS construct in the leaves. Previously this phytoalexin has only been observed in tuber tissue. III—T Screening for non-target effects of transforming plants Glucanase activity was assessed in the transformed and untransformed plants as part of a screening to see if altering the expression of STVS3 or STVSS had a deleterious effect on this enzyme which may be involved in the break down of the cell walls of P. infestans (Figure 21). In the cases of water (P = 0.402) and P. infestans (P = 0.738) inoculations, there was not a statistically significant difference in glucanase activity between any of the constructs or untransformed plants. 118 10 3 (Z - Water g 8 _ 12:] P. infestans 0.) <3: 9H O s 6 l l H a on I 1 it 'E I j :3 I EL I '. a» 4 " .S I '1 :I 8 t . l .. ' § 2 - 1 ‘g 2 :2 (6 U .2 on » . O I I I E149-5Y E46.X E47.X E48.X E49.X 553x untransformed Figure 21 : Average glucanase activity of STVS transformed plants with water or P. infestans inoculations compared to the untransformed plant, MSEl49-5Y. 119 U1 — Water I P. infestans a l I l‘ A J 00 l H peroxidase activity (change in absorbency (470nm)/minute * gram of fresh weight) M —k l I E149-5Y E46.X E47.X E48.X E49.X E53.X untransformed D Figure 22: Average peroxidase activity of STVS transformed plants with water and P. infestans inoculation compared to the untransformed plant, MSEl49-5Y. Peroxidase activity was also assessed to see if the creation of these transgenic plants had caused any unexpected changes to this enzyme that is involved in lignin synthesis (Figure 22). Both the transformed and untransformed plants were inoculated with water and P. infestans. The water (P = 0.108) and P. infestans (P = 1.00) inoculations of the transformed plants did not differ in peroxidase activity when compared to the untransformed, control plant. There was again no difference in activity between any of the treatment groups. However, it was interesting that the change in 120 peroxidase that occurred with the P. infestans treatment, when compared to the water treatment, was greater than would normally be expected by chance (P = 0.009). 2.0 1.8 - - Water [:21 P. infestans 1.6 s I E 01) '5 3 .C.‘ CD 0.) It: h—I o 2.» 8 IE 5‘0 1.4 - H «II 8 g ' - I -o '5? g 1.0 r o E I :2 o o I o 00 8 4 ES“ ' >~ .. 7:5 8 0.6 a. a) g 0.4 - .8 (d .9. 0.2 s Q) “3 00 c: . I I I I I I g E149-5Y E46.X E47.X E48.X E49.X E53.X V untransformed Figure 23: Average polyphenol oxidase activity of STVS transformed plants with water and P. infestans inoculation compared to the untransformed plant, MSEl49-5Y. 121 Polyphenol oxidase activity was evaluated to see if any of the transgenic potato plants had developed any adverse effects on this enzyme when compared to normal enzyme levels in the untransformed plants (Figure 23). When the different constructs were treated with water only, there was not a significant difference in the mean values (P = 0.173). When leaves flom the same plants were inoculated with P. infestans and evaluated for polyphenol oxidase activity, a Tukey Test indicated that none of the constructs differed significantly flom the untransformed plants while there were differences between some of the transforrnants. 122 .a.“‘ \Lh'.aml I DISCUSSION It was found that there was not a significant difference between the amount of disease observed within the different lines of a given construct with the exception being El49-5Y.49 which contains the sense construct of the STVSS gene. This significance amongst the different lines within E149-5Y.49 may suggest that there were some variations in the construction of this set of transgenics. It was interesting to find that there was not any significant difference in the amount of disease that was assessed in the difl‘erent E149-5Y constructs. Although these plants differ in their expression of the STVS3 or the STVS5 genes as shown by northern blots (Kelly Zarka, Michigan State University), any significant difference in these plants was observed only when compared to the untransformed plants of this variety. The sense constructs of STVS3 and STVS5 displayed a disease assessment that was significantly lower than the untransformed plants while the antisense constructs did not differ flom the control. It was hypothesized that the sense constructs may in fact be overexpressing these STVS genes and producing more phytoalexins, thus aiding in pathogen resistance. However, the sense construct of STVS3 with the patatin promoter, also differed significantly from the untransformed plants and actually displayed the lowest disease assessment of all. Because patatin is expressed only in the tubers, it was surprising to find that there was decreased disease in the foliage. It was evident that there was a much greater accumulation of terpenoids in the potato tubers than in the foliage both before and after infection with a compatible or 123 noncompatible pathogen. The observation that there was a much greater accumulation of phytoalexins in the tubers than in the foliage is in line with other current research. In the sense construct of STVS5, when terpenoids were extracted flom the foliage, there was a compound detemrined to be phytuberin as it had produced the same characteristic color as authentic phytuberin with the vanillin- sulfuric acid reagent (V ams 1970). To our knowledge, this is the first time that phytuberin has been extracted flom ‘— $52.“ potato leaf tissue. The constructs did not differ from the untransformed plants in glucanase activity which suggested that their transformation had no effect on this enzyme. When examining peroxidase activity, there was again no deleterious effect on II ._ l peroxidase caused by the transformation. Peroxidase was significantly induced in all cases with P. infestans treatments, and may be an important enzyme in this variety’s potato-patho gen interaction. When polyphenol oxidase activity was evaluated, the constructs did not differ significantly flom the activity of the untransformed plants which again suggested that the construction of these potato plants had no deleterious effect on polyphenol oxidase activity. It is really not likely that plant resistance is simply determined by the presence or absence of genes coding for gene products that restrict the pathogen (Kuc 1995). It has been stated that rather resistance is determined by the rapidity and magnitude of such gene expressions, as well as the rapidity of the gene products. It may be concluded that genetically modified plants, which are altered to utilize natural defense mechanisms, do make excellent research tools, but are much too simplified for practical use. 124 Sensitizing the plant to rapidly respond after infection by accumulating phytoalexins, hydroxyproline-rich glycoproteins and lignin at the site of infection and producing B-l,3-glucanases and other PR-proteins and peroxidases is a practical, yet complex research goal. However, exactly which compounds are important in accumulation following infection, still requires further research. 125 TI‘J‘I‘ ‘ i . LITERATURE CITED Bostock, R. M., J. A. Kuc, and R. A. Laine. 1981. Eicosapentaenoic and arachidonic acids flom Phytophthora infestans elicit fungitoxic sesquiterpenes in the potato. Science 212: 67-69. Caten, CE, and J .L. Jinks. 1968. Spontaneous variability of single isolates of Phytophthora infestans 1. Cultural variation. Canadian Journal of Botany 46: 329- 347. Doke, N., N. A. Garas, and J. Kuc. 1979. Partial characterization and aspects of the mode of action of a hypersensitivity-inhibiting factor (hit) isolated flom Phytophthora infestans. Physiological Plant Pathology 15: 127-140. Douches, D. S., K. Westedt, K. Zarka, B. Schroeter, and E. J. Grafius. 1998. Transforrnaion of potato (Solanum tuberosum L.) with the CryV-Bt transgene to combine natural and engineered resistance mechanisms for controlling tuber moth. HortScience 33: 1053-1056. Dunwell, J. M. 2000. Transgenic approaches to crop improvement. Journal of Experimental Botany 51: 487-496. Fry, W.E., and SB. Goodwin. 1997. Re-emergency of potato and tomato late blight in the United States. Plant Disease 81: 1349-1357. Hammerschmidt, R., 1999. Phytoalexins: What have we learned after 60 years? Annual Review of Phytopathology 37: 285-306. Hammerschmidt, R., and J. Kuc. 1979. Isolation and identification of phytuberin flom Nicotiana tabacum previously infiltrated with an incompatible bacterium. Phytochemistry 18: 874-875. Henfling, J .W.D.M, and J. Kuc. 1979. A semi-micro method for quantitatioin of sesquiterpenoid stress metabolites in potato tuber tissue. Phytopathology 69: 609- 612. 126 Henfling, J .W.D.M., R.M. Bostock, and J. Kuc. 1980. Cell walls of Phytophthora infestans contain an elicitor of terpene accumulation in potato tubers. Phytopathology 70: 772-776. Keen, N. T. 1978. Phytoalexins - Efficient extraction flom leaves by a facilitated diffusion technique. Phytopathology 68: 1237-1239. Kuc, J. 1957. A biochemical study of the resistance of potato tuber tissue to attack by various fungi. Phytopathology 47:676-680. Kuc, J. 1972. Compounds accumulating in plants after infection. In Microbial Toxins, edited by S. Ajl, G. Weinbaum and S. Kadis: Academic Press 21-247. Kuc, J. 1982. Phytoalexins flom the Solanaceae. In Phytoalexins, edited by J. Bailey and J. Mansfield. New York: Wiley 81-105. Kuc, J. 1995. Phytoalexins, stress metabolism, and disease resistance in plants. Annual Review of Phytopathology 33: 275-297. Maniara, G., R. Laine, and J. Kuc. 1984. Oligosaccharides flom Phytophthora infestans enhance the elicitation of sesquiterpenoid stress metabolites by arachidonic-acid in potato. Physiological Plant Pathology 24: 177-186. Muller, K0, and Borger. 1940. Experimentelle untersuchunger uber die Phytophthora infestans- Resistenz der Kartoffel. Arb. Biol. Reichsanst. Land F orstwirtsch. 23: 189. Murashige, T., and F. Skoog. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15: 473-497. Ooms, G., P. J. J. Hooykaas, R. J. M. Vanveen, P. Vanbeelen, T. J. G. Regensburgtuink, and R. A. Schilperoort. 1982. Octopine Ti-plasmid deletion mutants of Agrobacterium tumefaciens with emphasis on the right side of the T-region. Plasmid 7: 15-29. Price, K.R., B. Howard, and D.T. Coxon. 1976. Stress metabolite production in potato tubers infected by Phytophthora infestans, F usarium avenaceum and Phoma exigue. Physiological Plant Pathology 9: 189-197. 127 3h Shih, M., and J. Kuc. 197 3. Incorporation of 14C flom acetate and mevlonate into rishitin and steroid glycoalkaloids by potato slices inoculated with Phytophthora infestans. Phytopathology 63: 826-829. Threlfall, C., and I. Whitehead. 1992. Analysis of terpenoid phytoalexins and their biosynthetic enzymes. In Molecular Plant Pathology. A Practical Approach, edited by S. J. Gurr, M. J. McPherson and D. J. Bowles. New York: Oxford University Press Inc. 63-101. Vams, J .L. 1970. Purdue University, Lafayette, Indiana. Vleeshouwers, V.G.A.A. 2001. Molcular and cellular biology of resistance to Phytophthora infestans in Solanum species. PhD, laboratory of Phytopathology, Wageningen University, Wageningen, The Netherlands. Voet, D., and J .G. Voet. 1990. Biochemistry. New York: Wiley 1223. Wenzler, H. C., G. A. Mignery, L. M. Fisher, and W. D. Park. 1989. Analysis of a chimeric class-I patatin-gus gene in transgenic potato plants hi gh-level expression in tubers and sucrose inducible expression in cultured leaf and stem explants. Plant Molecular Biology 12: 41 -50. Yadav, N. R., and M. B. Sticklen. 1995. Direct and efficient plant-regeneration flom leaf explants of Solanum tuberosum L Cv Bintje. Plant Cell Reports 14: 645-647. Zhen, W , X. Chen, H. Liang, Y. Hu, Y. Gao, and Z. Lin. 2000. Enhanced late blight resistance of transgenic potato expressing glucose oxidase under the control of pathogen-inducible promoter. Chinese Science Bulletin 45: 1982-1986. 128 SUMMARY The field trials were modified between 2000 and 2001 in order to obtain a disease assessment with the available resources each year. The field results allowed for the identification of resistance and susceptibility in potato foliage. Further seasons would reveal how trial location, cultivar availability and seed useage affected the results in addition to cultivar variability. It would be interesting to perform the same trial in a season where Alternaria solani and Botrytris cinerea were less aggressive. It is likely that signs and symptoms of both of these pathogens were mistaken for late blight symptoms in the field. A greenhouse disease assessment of intact plants was not used in this study but may provide an intermediate measurement of resistance or susceptibility. The two seasons seem to have produced results close to what would have previously been expected. This information was intended to assist breeders in combining pedigree information and agronomic quality data in order to produce cultivars with a strong, late blight resistance. However, the significance of these results would increase if a larger field trial could be done including this wide range of potato cultivars. The enzymatic assays seem to have been accurate and precise methods of determining peroxidase, glucanase and polyphenol oxidase activity in potato foliage. It seems likely that the most realistic account of these enzymes came flom the previously infected potato foliage flom the field. When greenhouse or field-grown plants were inoculated in the lab, the virulence of the Phytophthora infestans isolate was the most difficult challenge. Much effort went into obtaining or maintaining pathogen virulence and initiating infection with a single isolate. The US8 genotype of P. infestans was used 129 for greenhouse inoculations to create a more controlled experiment, yet it may have been ideal to first work with multiple isolates to ensure infection. In the controlled greenhouse experiment, polyphenol oxidase was correlated with resistance. This experiment should be repeated and compared to the updated RAUDPC values. The preliminary tests with the genetically modified potato plants provided some interesting beginning findings. Terpenoid extractions proved that these plants varied in both the quantity and type of terpenoids produced by the varying constructs. More attention should be given to the construct that produced a tuber phytoalexin in the foliage tissue. Enzymatic analysis of glucanase, peroxidase and polyphenol oxidase revealed that in this case, these enzymes had not been altered by genetic engineering. As this set of plants are used in future studies, it would be interesting to test enzymes involved in other vital plant processes as well. 130 APPENDIX Rye Media The rye media recipe was modified by Caten and Jinks, 1968. 100.0 grams of pesticide flee rye seed was washed before using by placing the seed into a beaker, covering the opening with cheesecloth secured by a rubber bands and running under water for 10-15 minutes. The washed seed was then placed into a 1000ml glass beaker with ddH20 and boiled for one hour. After boiling, the solution was filtered through two layers of cheesecloth placed over the mouth of a funnel and into a 1000ml graduated cylinder. Additional ddeO was added to the beaker and poured into the cylinder until 1000m1 was attained. 8.0 grams of sucrose and 16.0 grams of Agar were added before the solution was then autoclaved in an Erlenmeyer flask to sterilize the media. Rye RAN Media Antibiotics were sometimes added to the rye media to prevent bacterial contamination. In this case, 1.0m1 aliquots were prepared in advance and stored frozen in the dark by combining 75mg Rifamicin (or Rifamycin B), 20mg Ampicillin, 75mg Nystatin and 1.0m] DMSO. This was supplemented into the media just prior to pouring at a 0.1-0.2% final concentration. Potato Dextrose Media Several large potatoes were washed and peeled before being cut into small sections. 250 grams of potato were measured to make 1 L of medial. The raw potatoes 131 were put into approximately 1L of ddHZO and microwaved on high for 10 minutes until soft. Then the potatoes were filtered through two layers of cheesecloth in a funnel and measured out to l L in a graduated cylinder. The media was then poured into a 2 L Erlenmeyer flask and supplemented with 16 g dextrose and 16 g agar. The solution was autoclaved before use. 132 I111111111111111111 ' ' . .> .‘fi‘v‘: » A E’. __‘- . ~’ .