1/5,..«154135 2 1t V1,». .2. J. ; =1 . , This is to certify that the dissertation entitled Induction of Resistance in Potato Tuber Tissue to Control Fusarium Sambucinum presented by Julie A. Greyerbiehl has been accepted towards fulfillment of the requirements for Ph.D. degree in Botany and Plant Pathology W Major professor Date ‘Lf' Vita-«12,00 2" MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY Michigan State University 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 FEM? 300615 6/01 c:/C|RC/DateDue.p65-p.15 INDUCTION OF RESISTANCE IN POTATO TUBER TISSUE TO CONTROL FUSARIUM SAMBUCINUM By Julie A. Greyerbiehl A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements For the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 2002 ABSTRACT Induction of Resistance in Potato Tuber Tissue to Control Fusarium sambucinum By Julie A. Greyerbiehl Fusarium sambucinum is one of the causal agents of Fusarium dry rot in potato tubers. Although thiabendazole has been the primary control of Fusarium dry rot, development of resistance to this fungicide has required that new control strategies be developed. Induced resistance may be one new approach to controlling dry rot. Potato tuber tissue was treated with several materials known to induce resistance in other plants. These included: salicylic acid, salicylic acid analogues, non-protein amino acids, chitosan, fatty acids and homogenate of Phytophthora infestans mycelium. Only treatments of potato tuber tissue with chitosan and homogenate of Phytophthora infestans mycelium (MH) reduced infection by F. sambucinum. However, the tuber tissue response was visually different between the MH and chitosan treatments and chitosan was slightly more effective at reducing infection. Protein analysis revealed both chitosan and MH induced a number of putative plant defense mechanisms. The pathogenesis-related protein chitinase was locally and systemically induced in tubers in response to both treatments. [3-1.3- glucanase was locally induced by both treatments and systemically induced in response to MH. Treatment with MH elicited a greater increase in chitinase and [3-1.3-glucanase activity than treatment with chitosan. Local induction of the oxidative enzymes peroxidase and polyphenol oxidase (PPO) was also observed in response to both treatments. Steroid glycoalkaloid accumulation, which has been shown to inhibit F. sambucinum growth and spore germination, was suppressed by both chitosan and MH treatments. Cytological examination of of chitosan-treated tissues with autofluorescence and histochemical staining revealed accumulation of non-soluble phenolic compounds in the cell wall areas of tuber tissue, suggesting cell wall modifications had occurred in response to chitosan treatment. Additionally, lacmoid staining revealed that chitosan treated tissue developed a barrier of callose two to three layers from the treated surface. Induction of resistance by chitosan and P. infestans mycelium homogenate is probably a result of multiple plant defense mechanisms (pathogenesis-related proteins, oxidative enzymes, accumulation of phenols, cell wall changes and callose deposition) working simultaneously to stop the ingression of F. sambucinum. Induced resistance is therefore a promising alternative to fungicides for the control of Fusarium dry rot and because it controls F. sambucinum using multiple defense mechanisms it may be a more stable form of resistance than fungicides. ACKNOWLEDGEMENTS I would like to thank Dr. Ray Hammerschmidt for his guidance and support in the completion of my Ph.D. and also in life. I would also like to thank my committee members Dr. Karen Klomparens, Dr. David Douches, and Dr. Frances Trail, as well as, the many colleagues and friends at MSU who have helped support me in this endeavor. Finally, I would like to thank my parents Stanley and Judith Greyerbiehl for their unending support and my husband William Chu for his patience and encouragement. iv TABLE OF CONTENTS ABSRACT ........................................................................................ ii ACKNOWLEDGEMENTS ...................................................................... iv TABLE OF CONTENTS ......................................................................... v LIST OF TABLES ............................................................................... vii LIST OF FIGURES ............................................................................ viii INTRODUCTION ................................................................................. 1 Fusarium sambucinum the Causal Agent of Potato Dry Rot ....... 2 Induced or Acquired Resistance ........................................... 3 Acquired Resistance in Potato Tuber Tissue ........................... 4 Defense Mechanisms ......................................................... 5 Elicitors and Plant Resistance Activators ............................... 15 References ...................................................................... 16 CHAPTER 1: Evaluation of Potential Inducing Agents of Acquired Resistance in Potato Tuber Tissue Against Fusarium sambucinum ........... 24 Introduction .................................................................... 25 Materials and Methods ...................................................... 26 Results .......................................................................... 30 Discussion ..................................................................... 40 References ..................................................................... 48 CHAPTER 2: The Effect of Chitosan and Phytophthora infestans Mycelium Homogenate on the Biochemistry of Potato Tuber Tissue 55 Introduction .................................................................... 56 Materials and Methods ...................................................... 56 Results .......................................................................... 64 Discussion ..................................................................... 80 References ..................................................................... 87 CHAPTER 3: Histochemistry and Autofluoresence Microscopy of Chitosan Treated Tuber Tissue ....................................................... 91 Introduction .................................................................... 92 Material and Methods ....................................................... 93 Results .......................................................................... 95 Discussion .................................................................... 101 References ................................................................... 104 CONCLUSIONS AND FUTURE RESEARCH ......................................... 106 vi LIST OF TABLES CHAPTER 1: Evaluation of Potential Inducing Agents of Acquired Resistance 1-1 1-10 1-11 in Potato Tuber Tissue Against Fusarium sambucinum Effect of salicylic acid (SA) on the ability of Fusarium sambucinum to infect potato tuber tissue .............................. 33 Effect of ASM on the ability of Fusarium sambucinum to infect potato tuber tissue ........................................................... 34 Effect of INA on the ability of Fusarium sambucinum to infect potato tuber tissue ........................................................... 34 Effect of non-protein amino acids on the ability of Fusarium sambucinum to infect potato tuber tissue .............................. 34 Effect of Auexin Corp. chemical elicitors on the ability of Fusarium sambucinum to infect potato tuber tissue ................. 35 Effect of homogenate of Phytophthora infestans mycelium (MH) on the ability of Fusarium sambucinum to infect potato tuber tissue ............................................................................ 35 Effect of Arachadonic acid (AA) on the ability of Fusarium sambucinum to infect potato tuber tissue .............................. 36 Effect of Linolenic acid (LNA) on the ability of Fusarium sambucinum to infect potato tuber tissue .............................. 36 Effect of Linoleic acid (LA) on the ability of Fusarium sambucinum to infect potato tuber tissue .............................. 37 Effect of chitosan treatment on the ability of Fusarium sambucinum to infect potato tuber tissue .............................. 37 Effect of combining elicitors on the ability of Fusarium sambucinum to infect potato tuber tissue .............................. 38 CONCLUSIONS AND FUTURE RESEARCH C-1 Review of the effect of chitosan and MH on plant defense mechanisms .................................................................. 106 vii LIST OF FIGURES INTRODUCTION 1 Major steroid glycoalkaloids found in potato tuber tissue 7 2 The major potato sesquiterpenoid phytoalexins ........................ 8 3 Common phenolic compounds found in potato ........................ 9 CHAPTER 1: Evaluation of Potential Inducing Agents of Acquired Resistance in Potato Tuber Tissue Against Fusarium sambucinum 1-1 1-2 Visual comparison of tuber slices 3 days after treatment with 0.25, 0.5, or 1.0 mg/ml MH or 0.5 or 1.0 mg/ml chitosan .......... 38 Comparison of the effect of chitosan treatment on the depth of infection by F. sambucinum in potato tuber slices made from cultivars Snowden and Atlantic ........................................... 39 CHAPTER 2: The Effect of Chitosan and Phytophthora' infestans Mycelium Homogenate on the Biochemistry of Potato Tuber Tissue 2-1 2-2 2-3 2-4 2-5 Diagram of tuber cylinders used for depth studies .................. 58 Time course of the radial growth of F. sambucinum grown in petri plates containing water agar and water agar supplemented with 0.5 or 1.0 mg/ml chitosan ........................................... 65 Chitinase activity in tuber tissue 2 and 3 days after treatment with water (control) and Phytophthora infestans mycelium homogenate (MH) ............................................................ 66 Chitinase activity in tuber tissue 2 and 3 days after treatment with water (control) and chitosan ......................................... 66 Comparison of chitinase activity in tuber tissue treated with water (control), MH, and chitosan 2 days (A) and 3 days (B) after treatment ................................................................. 67 Time course of chitinase activity in tuber tissue treated with water (control) and 1.0 mg/ml chitosan ................................. 67 viii 2-7 2-8 2-9 2-10 2-11 2-12 2-13 2-14 2-15 2-16 2-17 2-18 2-19 Glucanase activity in tuber tissue 2 and 3 days after treatment with water (control), MH, and chitosan ................................. 68 Peroxidase activity in tuber tissue 2 days after treatment with water (control), MH, and chitosan ....................................... 69 Peroxidase activity in tuber tissue 3 days after treatment with water (control), MH, and chitosan ....................................... 70 Time course of peroxidase activity in tuber tissue treated with water (control) and chitosan .............................................. 70 Seven-day time course of peroxidase activity in tuber tissue after water (control) and chitosan treatment .......................... 71 Spectrophotometric analysis of peroxidase activity in tuber tissue after water (control) and chitosan treatment .................. 71 Polyphenoloxidase activity in tuber tissue 2 and 3 days after with treatment with water (control) and chitosan ..................... 72 Steroid glycoalkaloid levels in tuber tissue 2 and 3 days after water (control), MH, and chitosan treatment .......................... 73 Depth of infection by F. sambucinum in layers of tuber tissue taken in 0.5 centimeter increments from the treated surface of tuber cylinders ................................................................ 74 Chitinase activity in layers of tuber tissue taken in 0.5 centimeter increments from the treated surface of tuber cylinders ........................................................................ 75 Chitinase activity in layers of tuber tissue taken in 0.5 centimeter increments from the treated surface of tuber cylinders ........................................................................ 76 Glucanase activity in layers of tuber tissue taken in 0.5 centimeter increments from the treated surface of tuber cylinders ........................................................................ 77 Peroxidase activity in layers of tuber tissue taken in 0.5 centimeter increments from the treated surface of tuber cylinders ........................................................................ 78 ix 2-20 2-21 2-22 CHAPTER 3: 3-1 3-2 3-4 3-5 3-6 3-7 Polyphenoloxidase activity in layers of tuber tissue taken in 0.5 centimeter increments from the treated surface of tuber cylinders ........................................................................ 79 Glycoalkaloid levels in layers of tuber tissue taken in 0.5 centimeter increments from the treated surface of tuber cylinders ........................................................................ 80 Steroid glycoalkaloid and sesquiterpenoid biosynthetic pathway. 86 Histochemistry and Autofluoresence Microscopy of Chitosan Treated Tuber Tissue Fluorescence micrograph of longitudinal sections of potato tuber discs 24 hours after treatment with water (control) or chitosan ......................................................................... 96 Fluorescence micrograph of longitudinal sections of potato tuber discs 48 hours after treatment with water (control) or chitosan ......................................................................... 96 Fluorescence micrograph of longitudinal sections of potato tuber discs 72 hours after treatment with water (control) or chitosan ......................................................................... 97 Depth of autofluorescence in longitudinal sections of potato tuber discs 24, 48, and 72 hours after treatment with water (control) or chitosan ......................................................... 97 Micrograph of longitudinal sections of potato tuber discs stained with toluidine blue 0 12, 24,48, and 72 hours after treatment with water (control) or chitosan ............................. 98 Depth of blue-green color caused by toluidine blue 0 staining 48 and 72 hours after treatment with water (control) or chitosan ........................................................................ 99 Micrographs of longitudinal sections of potato tuber discs stained with lacmoid and fluorescent micrographs of the same sections 72 hours after treatment with water (control) or chitosan .................................................................... 100 INTRODUCTION INTRODUCTION Fusarium sambucinum the Causal Agent of Potato Dry Rot Fusarium dry rot is one of the most devastating post-harvest diseases of potato. It is estimated that an average of 6-10% of the potato crop is lost to dry rot annually, with reports of losses as high as 60% (Carnegie et al, 1990; Theron, 1991 ). The major causal agent of Fusarium dry rot is Fusarium sambucinum (teleomorph Gibberella pulicafis) (USDA, 1978). In addition to causing dry rot of tubers F. sambucinum can also cause seed decay and foliage wilt (Hooker, 1981) F. sambucinum produces chlamydospores, microspores, and macrospores. The microspores and macrospores provide continual airborne inoculum, while the chlamydospores can persist in the soil for years (Hooker, 1981 ). The telcmorph, G. pulicaris, produces ascospores contained in a perithecium. F. sambucinum can not penetrate the periderm of potato tubers, consequently, infection of potato tubers can only occur through wounds or breaks in the periderm (Hooker, 1981). Potato tubers, therefore, are not infected in the soil, but are only infected post-harvest through wounds or cracks caused by the harvesting process. Tuber infection begins by the growth of hyphae intercellularly and eventually spreads intracellularly subsequent to cell death. As infection progresses hollow cavities form inside the tuber, which cause the periderm to become sunken and wrinkled (Hooker, 1981). A limited number of potato cultivars and breeding clones exhibit resistance to F. sambucinum and none of the cultivars grown in the United States are resistant (Leach 8 Webb, 1981). F. sambucinum has been controlled by cultural practices, such as, limiting wounding during harvest, transit, and storage; and storing tubers in conditions optimal for wound healing (Secor and Gudmestad, 1999). The disease has been controlled primarily by post-harvest application of the fungicide thiabendazole. However, F. sambucinum resistance to thiabendazole is now wide spread (Desjardins, 1995 and Desjardins et al, 1993). Since thiabendazole is the only fungicide approved for post-harvest application on potato tubers (Powelson et al, 1993) finding an alternative method of controlling this disease is of the utmost importance. Induced resistance may offer an alternative. Induced or Acquired Resistance Induced or acquired resistance is a form of resistance in which a susceptible plant becomes resistant after treatment with an inducing agent (Hammerschmidt and Becker, 1997; Hammerschmidt and Kuc, 1995). Acquired resistance is characterized by: a) the induced tissue responding quicker to pathogen attack through the rapid induction of plant defenses; b) increased resistance to wide range of pathogens; 0) a long lasting effect of the inducing agent; often lasting weeks or months; d) a delay between the treatment with the inducing agent and full expression of resistance; e) a local and systemic accumulation of pathogenesis-related (PR) proteins; f) typically induced by agents or pathogens that cause necrotic lesions (Hammerschmidt, 1999; Lucas, 1999). The response may be localized or systemic (Hammerschmidt and Dam, 1997; Hammerschmidt and Becker, 1997). Local acquired resistance (LAR) is confined to a few cells around the area exposed to the inducing agent. Systemic acquired resistance (SAR) increases resistance throughout the plant and appears to be mediated by salicylic acid (Hammerschmidt and Smith-Becker, 1999; Sticher et al, 1997). Acquired resistance can be induced in response to infection or chemical treatment. Activation of acquired resistance by means of infection is generally induced by necrosis caused by pathogenesis (Hammerschmidt, 1997). Induction of acquired resistance has also been associated with the hypersensitive response (HR). The HR is a localized rapid cell death and subsequent necrosis caused by an avirulent pathogen or elicitor. Cell death is coupled with the disruption of the host cell membranes and accumulation of oxidized phenolic compounds. The HR has been correlated with the Induction of putative defense mechanisms associated with acquired resistance (Goodman and Novacky, 1994). Acquired Resistance In Potato Tuber Tissue Acquired resistance has been previously demonstrated in potato tuber tissue. Treatment of tuber tissue with an incompatible race of Phytophthora infestans protected the tissue against subsequent infection by a compatible race of P. infestans and Fusarium caerulem (Miiller and Borger, 1940). A lipoglycoprotein complex (LGP-complex) extracted from P. infestans was shown to induce a resistance response against P. infestans (Chalova et al, 1977). The LGP- complex induced local resistance at high concentrations (100 jig/ml) and systemic resistance at low concentrations (5-10 pl/ml) (Ozeretskovskaya, 1995). Arachidonic acid (AA) and eicosapentaenoic acid (EPA), components of the LOP-complex, were shown to be the primary agents responsible for induction of resistance against P. infestans. AA and EPA ability to induce resistance was enhanced by glucans extracted from P. infestans. which on their own could not induce resistance (Chalova et al, 1989; Preisig and Kuc, 1985). Extracts from the nonpathogenic fungus Fusarium culmomm Sacc, chitosan, and fucosyl- containing oligosaccharides were also able to induce resistance against P. infestans (Yurganova et al, 1989; Vasyukova et al, 2000; ll’inskaya et al, 1997). Induced resistance has also been demonstrated against bacterial pathogens in potato. Treatment with oligogalacturonides and Phytophthora sojae cell wall hydrolysate decreased infection by Erwinia carotovore spp. atroseptica (Dutton et al, 1997). The ability to induce disease resistance in potato tuber tissue suggests that acquired resistance may be used in place of or in combination with fungicides to control diseases such as F. sambucinum. Defense Mechanisms It Is believed that acquired resistance involves the induction of or the ability to rapidly induce upon infection a number of putative defense mechanisms. These mechanisms may include phytoalexins, cell wall alterations, activated oxygen, oxidative enzymes, and PR proteins. Phytoalexins Phytoalexins are low molecular weight, antimicrobial compounds that are synthesized and accumulate in the plant after exposure to microorganisms (Paxton, 1981). Phytoalexins are believed to contribute to defense because their accumulation has long been correlated with cessation of pathogen development. In addition, fungal detoxification of phytoalexins has been correlated with an increase in vimlence, mutants expressing increased levels of phytoalexins have been demonstrated to be more resistant to pathogens, and chemical inhibitors of phytoalexins have been shown to increase susceptibility (Hammerschmidt, 1999). Phytoalexins present in potato tuber tissue include steroid glycoalkaloids and sesquiterpenes (Kuc, 1982). Steroid glycoalkaloids (SGA) are antimicrobial compounds produced by many species in the Solanaceae family, including potato (Kuc, 1982). The major SGA found in potato tuber tissue are a-solanine and a-chaconine (Figure 1). SGA are highly toxic to humans and can cause potatoes to have a bitter taste (Valkonen et al, 1996). Therefore, the concentration of SGA in new potato cultivars has been limited to a maximum level of 20 mg per 100 g fresh weight (Sinden and Webb, 1972). SGA are found in high concentration in potato peel and at wound sites of tuber tissue. SGA biosynthesis is induced by wounding and is inhibited by a number of pathogens and non-pathogens, as well as, preparations of P. infestans cell walls, AA and EPA (lshizaka and Tomiyama, 1972; Kuc, 1982; Tjamos and Kuc, 1982). SGA are believed to have a role in plant defense against pathogens. SGA have been demonstrated to have antifungal activity that synergistically increases when SGA are combined (Kuc, 1968; Fewell and Roddick, 1997; Allen and Fewell et al, 1994; Fewell and Roddick, 1993). Additionally, light induced increases of SGA in tuber tissue were correlated with resistance to the tuber rot pathogens Fusarium sulphureum and Fusarium solani var. coeruleum (Percival et al, 1998). SGA has been shown to inhibit the growth and spore germination of F. sambucinum in vitro (Zeng, 1993). Despite the fact that SGA accumulation is inhibited by F. sambucinum infection, (Zeng, 1993) it is believed that SGA may play a role in defense against F. sambucinum. Inhibition of SGA accumulation has been reported to be lower in potatoes resistant to F. sambucinum than susceptible potatoes (Corsini and Pavek, 1980). Furthermore, increased SGA accumulation, which occurs during wounding, may initially play a role in slowing infection by F. sambucinum. a-Solanine (R = B-solatriosyl) a-Chaconine (R = B-chacotriosyl) RO Figure 1: Major steroid glycoalkaloids found in potato tuber tissue. The major potato sesquiterpenoid phytoalexins are lubmin and rhisitin (Figure 2). Sesquiterpenes accumulate in response to infection, cell wall preparations of P. infestans. AA, and EPA (Lisker and Kuc, 1977; Henfling et al, 1980; Bostock et al, 1981). However, F. sambucinum can tolerate high levels of rhisitin and can detoxify lubmin (Desjardins and Gardner, 1989; Desjardins et al, 1989). Therefore, here sesquiterpenes are probably not involved in tuber defense against F. sambucinum. CHO Rhisitin Lubmin Figure 2: The major potato sesquiterpenoid phytoalexins. Phenolic compounds (Figure 3) rapidly accumulate upon infection and are thought to slow or halt infection through direct antimicrobial activity, oxidation into toxic quinones, or esterification to cell wall materials (Nicholson and Hammerschmidt, 1992). The phenolic compound, chlorogenic acid has also been reported to accumulate upon wounding and has been correlated with resistance to potato tuber scab. However, chlorogenic acid is a relatively non- toxic compound therefore, resistance is believed to be due to the oxidation of chlorogenic acid to a more toxic compound(s) upon infection (Johnson and Schaal, 1952 and 1959). Chlorogenic acid has also been shown to accumulate in potato tuber tissue after infection by P. infestans (Smith and Rubery, 1981; Friend et al, 1973). However, the accumulation of chlorogenic acid has been reported to be more rapid in some susceptible cultivars than in resistant cultivars (Henderson and Friend, 1979). The accumulation of chlorogenic acid in susceptible cultivars may represent activation of the phenylpropanoid pathway and an increase in the biosynthesis of phenolic compounds due to tissue damage rather than a specific response to infection. The slower accumulation of chlorogenic acid in resistant cultivars may indicate that phenols are being shunted from chlorogenic acid biosynthesis to the production of phenols that are more important in plant defense. ll . .. fi Caffeic acid (R = OH) . Ferulic acid (R = OCHa) Chlorogenic acid : p-Coumaric acid (R = H) HO 2': H Figure 3: Common phenolic compounds found in potato. Cell Wall Modifications The first barrier encountered by pathogens is the cell wall and most pathogens can easily penetrate the cell wall (Schafer, 1994). Thus, rapid modifications in the wall may prevent the pathogen from further invading the plant tissue. Cell wall modifications may prevent infection by: a) increasing the mechanical strength of the wall preventing penetration by the pathogen; b) decreasing the susceptibility of the wall to cell wall degrading enzymes; c) providing a barrier to nutrient flow, thus starving the pathogen and d) providing a barrier to toxins or enzymes produced by the pathogen (Ride, 1978). In many cases cell wall modifications act as an initial defense blocking the pathogen until other defenses are induced. In other cases lignin deposition may actually contain the pathogen by the Iignification of the pathogen cell wall surrounding the pathogen or by the deposition of lignin around the penetrating pathogen (Ride, 1978). In potato tuber tissue, the periderm protects tuber tissue against invading pathogens, and wounding of the periderm allows infection by a number of pathogens to occur (O'Brien and Leach, 1983; Hooker,1981). As the wounded tissue ages, wound periderm is deposited. The deposition of wound periderm correlates with a decrease in susceptibility of infection, demonstrating the importance of the periderm in tuber tissue defense (Nolte et al, 1987; Hooker, 1981; Miiller, 1957; Smith and Smart, 1955). The cell walls of the periderm consist partly of suberin, which is composed of a phenolic and lipid matrix with waxes embedded into the matrix (Kolattukudy, 1984). Lulai and Corsini (1998) showed that deposition of both the phenolic and lipid component of the suberin was necessary for resistance to F. sambucinum during potato tuber wound healing. The waxes in the periderm provide the major barrier of water vapor diffusion from the host (Soliday et 31,1979). It has been demonstrated that an increase in water vapor diffusion across this layer 10 increases susceptibility to F. sambucinum, implicating the suberin-associated waxes with plant defense (Vander Zaag et al, 1984). In addition to suberin, the periderm contains a lignin-like material, which also appears to be important in plant defense. Deposition of the lignin-like material occurred more rapidly in response to incompatible race of P. infestans than to susceptible strain (Hammerschmidt, 1984). Furthermore, tuber tissue treated with a- aminooxyacetic acid, an inhibitor of phenylalanine ammonia Iyase, showed a decrease in lignin deposition and was then susceptible to the non-pathogen Cladosporuim cucmerinum (Hammerschmidt, 1984). Deposition of lignin, hydroxyproline-rich glycoproteins, and callose may also contribute to the restriction of pathogens attempting to invade potato tuber tissue. Activated Oxygen Production of the activated oxygen species (hydrogen peroxide, hydroxyl radicals and superoxide) are associated with the plant’s resistance response (Wojtaszek, 1997). In particular, increases in activated oxygen species are associated with the HR (Goodman and Novacki, 1994). Active oxygen species are believed to function in resistance by crosslinking cell wall polymers, via antimicrobial activity, and as a local signal for induction of defense related genes (Hammerschmidt and Schultz, 1996). Superoxide has been shown to increases in potato tuber tissue in response to incompatible races of P. infestans, but not in response to compatible races (Doke, 1983). Hyphal cell wall components of P. infestans, including AA, were also shown to increase superoxide in potato tuber tissue (ll’inskaya et al.1999; Doke, 1983). Diffusates from AA treated tuber 11 tissue inhibited P. infestans growth. If superoxide dismutase, which inhibits generation of superoxide, was added to the diffusates P. infestans growth was not inhibited (ll’inskaya et al, 1999). Superoxide, therefore, appears to have a role in resistance. Oxidative Enzymes Induction of resistance is often accompanied by activation or synthesis of the oxidative enzymes peroxidase, polyphenoloxidase, and lipoxygenase (Goodman and Novacky, 1994). Peroxidase may have a role in defense by producing toxic levels of hydrogen peroxide and phenolic free radicals (Peng and Kuc, 1992; Nicholson and Hammerschmidt, 1992). Peroxidase is also involved in the polymerization of phenolic precursors producing lignin, suberin, and hydroxyproline-rich glycoprotein crossing linking of the cell wall (Nicholson and Hammerschmidt, 1992; Espelie and Kolattukudy, 1985; Espelie et al, 1986). Polyphenoloxidase may be involved in defense by interacting with phenolic compounds producing toxic quinones from phenolic compounds (Appel, 1993). Lipoxygenase (LOX) may contribute to plant defense by forming toxic lipid oxidation products and by the formation of lipid signal molecules (Ricker and Bostock, 1994; Croft et al, 1993; Farmer, 1994). AA has been reported to increase levels of LOX in potato tuber tissue (ll’inskaya et al, 2000; Bostock et al, 1992). AA induced increases in LOX activity correlated with an increase in P. infestans resistance. If AA treated tuber tissue was treated with salicylhydroxamic acid, an inhibitor of LOX, resistance to P. infestans was 12 inhibited, suggesting that LOX plays a role in tuber tissue resistance response (ll’inskaya et al, 2000). Pathogenesis-related Proteins Pathogenesis-related (PR) proteins are defined as proteins coded for by the host plant and induced by pathogen, nematode, insect and herbivore attack or comparable condition including wounding and chemical agents that mimic the effect of pathogen infection or induce the host response (van Loon, 1999). PR proteins are divided into 11 families, named PR1 through PR11. PR1 proteins are the most abundant and therefore, are often used as markers of acquired resistance. The function of PR1 proteins is not known, however, they have been implicated in virus and fungal resistance (Buchel and Linthorst, 1999). Constitutive high-level expression of PR1a reduced infection by Peronospora tabacina and Phytophthora parasitica (Alexander et al, 1993). Additionally, PR1 proteins isolated from tomato infected with P. infestans or virus infected tobacco have been shown to have fungicidal activity against P. infestans in both in vitro and in vivo assays (Niderman et al, 1995). PR2 proteins are 8-1.3-glucanases that cleave 1,3-8-D-glucosidic linkages in 8-1.3-glucans (Leubner-Metzger and Meins, 1999). Many fungal cell walls are partly composed of 8-1.3-glucan (Gooday, 1994). It has been proposed that [3- 1,3-glucanases may be directly involved in fungal resistance by inhibiting fungal growth through the degradation of fungal cell walls. The direct action of 8-1.3- glucanases has been supported by in vitro studies that have shown [3-1.3- glucanases to have antifungal activity against a number of fungal pathogens. [3- 13 1,3-glucanases may also be involved indirectly in fungal resistance by producing 8-glucan by fungal cell wall degradation. 8-glucans have been reported to elicit resistance against fungal and viral pathogens by inducing plant defenses (Koop et al, 1989 and Bhandal and Paxton, 1991). Further evidence for the role of 8- 1,3-glucanase in resistance has been provided by transgenic expression of PR2 proteins. Expression of 8-1.3-glucanases regulated by a constitutive promoter has been reported to reduce infection by fungal pathogens in numerous hosts (Leubner-Metzger and Meins, 1999). PR3, PR4, PR8, and PR11 proteins are chitinases. Chitinases are able to hydrolyze 8-1 ,4-linkages between N-acetylglucosamine residues of chitin (Neuhaus, 1999). Many fungal cell walls are composed of chitin (Gooday, 1994). It has been proposed that chitinases may be involved directly in defense by breaking down fungal cell walls and indirectly involved by producing elicitors that induce other plant defenses. The role of chitinases in resistance has been supported by their antifungal activity in vitro and by reports of increased resistance to a number of pathogens in transgenic plants constitutively expressing chitinases (Neuhaus, 1999). Combinations of chitinase and 8-1 ,3-glucanase may increase resistance to pathogens. A combination of chitinase and 8-1.3-glucanase synergistically increases antifungal activity in vitro compared to their activity alone. Additionally, simultaneous constitutive expression of a 8-1.3-glucanase protein and chitinase protein synergistically increased fungal resistance (Leubner-Metzger and Meins, 1999; Neuhaus, 1999). 14 PR5 proteins are similar to thaumatin in sequence and therefore have been called thaumatin-like proteins. PR5 proteins are believed to be involved in plant defense due to their antifungal activity (Velazhahan et al, 1999). The remaining PR proteins roles in pathogen resistance have been studied less extensively. PR6 proteins are proteinase inhibitors involved in defense against herbivores. There is also some evidence that PR6 proteins are involved in resistance against pathogens (Heitz et al, 1999). PR9 proteins are a specific group of peroxidases in tobacco involved in lignin synthesis. PR10 proteins have sequence similarity to ribonucleases (van Loon, 1999). Elicitors and Plant Resistance Activators Elicitors are compounds that are able to induce resistance in plants to subsequent infection by a pathogen or induce typical defense responses in a plant (Lyon et al, 1995). Elicitors must meet the following criteria to be considered an inducer of plant defense: a) the elicitor must not have antimicrobial activity or metabolites derived from the elicitor must not have antimicrobial activity; b) the elicitor must induce defense mechanisms that resemble the response of the host to an incompatible interaction and c) the elicitor should make the plant resistant to pathogen(s) (Kessmann et al, 1994). There are two types of elicitors, biotic and abiotic elicitors. Biotic elicitors include carbohydrates, phenols, salicylic acid, jasmonic acid, and amino acids. Abiotic elicitors include synthetic chemicals such as 2,6-dichloroisonicotinic acid (INA) and acibenzolar-S-methyl (ASM), which are believed to act as salicylic acid 15 analogues. They also include inorganic compounds such as phosphate salts, potassium phosphate salts, silicates, and oxalic acid (Lyon et al, 1995). Elicitor mediated induction of resistance has potential to control diseases. The use of elicitors to control F. sambucinum will be focus of this thesis. REFERENCES Alexander 0., Goodman R.M., Gut-Rella M., Glascock C., Weymann K., Friedrich L., Maddox D., Ahl-Goy P., Luntz T., Ward E., and Ryals J., 1993. Increased tolerance to two oomycete pathogens in transgenic tobacco expressing pathogenesis-related protein 13. Proceedings of the National Academy of Science USA 90:7327-7331. Allen E. and Kuc J., 1968. a-Solanine and a-chaconine as fungitoxic compounds in extracts if Irish potato tubers. Phytopathology 58:776-781. Appel H.M., 1993. Phenolics in ecological interactions: the importance of oxidation. Journal of Chemical Ecology 19:1521-1552. Bhandal LS. and Paxton JD, 1991. Phytoalexins biosynthesis induced by the fungal glucan polytran l in soybean, pea, and sweet pepper tissue. Journal of Agriculture and Food Chemistry 39:2156-2157. Bostock R.M., Kuc J., and Laine RA, 1981. Eicosapentaenoic and arachidonic acids from Phytophthora infestans elicit fungitoxic sesquiterpenes in potato. Science 212:67-69. Bostock R.M., Yamamoto H., Choi D., Ricker K.E., and Ward BL, 1992. Rapid stimulation of 5-lipoxygenase activity in potato by the fungal elicitor arachidonic acid. Plant Physiology 100:1448-1456. Buchel AS and Linthorst H.J.M., 1999. PR-1: A group of plant proteins induced upon pathogen infection. In: Datta SK. and Muthukrishnan S. (eds). Pathogenesis-related Proteins in Plants. CRC Press: New York. p. 21 ~47. Carnegie S.F., Ruthven A.D., Lindsay DA, and Hall TD, 1990. Effects of fungicides applied to seed potato tubers at harvest or after grading on fungal storage diseases and plant development. Annals of Applied Biology 116:61-72. 16 Chalova L.l., Avdyushko S.A., Chalenko G.l., Karavaeva K.A., and Ozeretskovskaya O.L., 1989. Arachidonic and eicosapentaenoic acid - long- action systemic immunizers of the potato. Doklady Akademii Nauk SSSR, 305:1262-1265. Chalova L.l., Baramidze V.G., Yurganova L.A., Yu T., Ozeretskovskaya O.L., and Metlitskii L.V., 1977. Isolation and characteristics of inductors of defense responses in potato from cytoplasm of Phytopthora infestans. Doklady Akademii Nauk SSSR, 235:1215-1216. Corsini D.L. and Pavek J.J., 1980. Phenylalanine ammonia lyase activity and fungitoxic metabolites produced by potato cultivars in response to Fusarium tuber rot. Physiological Plant Pathology 16:63-72. Croft K.P.C., Juttner F ., and Slusarenko A.J., 1993. Volatile products of the lipoxygenase pathway evolved from Phaseolus vulgaris (L.) leaves inoculated with Pseudomonas syringae pv phaseolicola. Plant Physiology 101 :13-24. Desjardins A.E., 1995. Population structure of Gibberella pulicaris (anamorph Fusarium sambucinum) from potato tuber dry rot in North America and Europe. American Potato Journal 72:145-156. Desjardins A.E., Christ-Hamed E.A., McCormick S.P., and Secor GA, 1993. Population structure and genetic analysis of field resistance to thiabendazole in Gibberella pulicaris from potato tubers. Phytopathology 83:164-170. Desjardins AB. and Gardner H.W., 1989. Genetic analysis in Gibberella pulicaris: rishitin tolerance, rishitin metabolism, and virulence of potato tubers. Molecular Plant-Microbe Interactions 2:26-34. Desjardins A.E., Gardner H.W., and Plattner RD, 1989. Detoxification of the potato phytoalexin lubimin by Gibberella pulicaris. Phytochemistry 28:431-437. Doke N., 1983. Involvement of superoxide anion generation in the hypersensitive response of potato tuber tissues to infection with an incompatible race of Phytophthora infestans and to the hyphal wall components. Physiological Plant Pathology 23:345-357. Dutton M.V., Rigby NM, and MacDougall A.J., 1997. Induced resistance to Erwinia carotovora ssp. atroseptica, through the treatment of surface wounds of potato tubers with elicitors. Journal of Phytopathology 145:163-169. Espelie K.E. and Kolattukudy, 1985. Purification and characterization of an abscisic acid-inducible peroxidase associated with suberization in potato (Solanum tuberosum). Archival Biochemistry and Biophysics 240:539. 17 Espelie K.E., Franceschi V.R. and Kolattukudy PE, 1986. lmmunocytochemical localization and time course of appearance of an anionic peroxidase associated with suberization in would-healing potato tuber tissue. Plant Physiology 87:487. Farmer E.E., 1994. Fatty acid signalling in plants and their associated microorganisms. Plant Molecular Biology 26:1423-1437. Fewell AM. and Roddick J.G., 1993. Interactive antifungal activity of the glycoalkaloids a-solanine and a-chaconine. Phytochemistry 33:323-328. Fewell AM. and Roddick J.G., 1997. Potato glycoalkaloid impairment of fungal development. Mycological Research 101:597-603. Fewell A.M., Roddick J.G., and Weissenberg M., 1994. Interactions between the glycoalkaloids solasonine and solamargine in relation to inhibition of fungal growth. Phytochemistry 37:1007-101 1. Friend J., Reynolds 83., and Aveyard MA, 1973. Phenylalanine ammonia- Iyase, chlorogenic acid and lignin in potato tuber tissue inoculated with Phytophthora infestans. Physiological Plant Pathology 32495-507. Gooday G.W., 1994. Cell walls. In: Gow N.A.R. and Gadd GM. (eds). The Growing Fungus. Chapman and Hall: London. p. 43-62. Goodman RN. and Novacky A.J., 1994. The Hypersensitive Reaction in Plants to Pathogens. APS Press, St. Paul. Hammerschmidt R., 1984. Rapid deposition of lignin in potato tuber tissue as a response to fungi non-pathogenic on potato. Physiological Plant Pathology 24:33-42. Hammerschmidt R., 1997. Local and systemic defensive responses to infection. Stress-Inducible Processes in Higher Eukaryotic Cells. Plenum Press, New York. Hammerschmidt R., 1999. Induced disease resistance: how do induced plants stop pathogens? Physiological and Molecular Plant Pathology 55:77-84. Hammerschmidt R., 1999. Phytoalexins: What have we learned after 60 years? Annual Reviews of Phytopathology 37:285-306. Hammerschmidt R. and Becker J.S., 1997. Acquired resistance to disease in plants. Horticultural Reviews 18:247-289. 18 Hammerschmidt R. and Dam E.K., 1997. Induced resistance to disease. In: Recheigl NA. and Recheigl J.E. (eds.), Environmentally Safe Approaches to Plant Disease Control. CRC Lewis Publishers, Boca Raton. p. 177-199. Hammerschmidt R. and Kuc J. (eds.), 1995. Induced Resistance to Disease in Plants. Kluwer, Dordrecht. Hammerschmidt R. and Schultz J.C., 1996. Multiple defenses and signals in plant defense against pathogens and herbivores. In: Romeo (ed) Phytochemical Diversity and Redundancy in Ecological Interactions. Plenum Press, New York. Hammerschmidt R. and Smith-Becker J.A., 1999. The role of salicylic acid in disease resistance. In: Agrawal A.A, Tuzun S., and Bent E. (eds), Induced Plant Defenses Against Pathogens and Herbivores. APS Press, St. Paul. p. 1-17. Heitz T., Geoffroy P., Fritig B., and Legrand M., 1999. The PR-6 family: proteinase inhibitors in plant-microbe and plant-insect interactions. In: Datta SK. and Muthukrishnan S. (eds). Pathogenesis-related Proteins in Plants. CRC Press: New York. p. 131-155. Henderson SJ. and Friend J., 1979. Increase in PAL and lignin-like compounds as race-specific resistance responses of potato tubers to Phytophthora infestans. Phytopathology Z. 94:323-34. Henfling J.W.D.M., Bostock RM. and Kuc J., 1980. Cell walls of Phytophthora infestans contain an elicitor of terpene accumulation in potato tubers. Phytopathology 70:772-776. Hooker W.J., 1981. Compendium of potato diseases. American Phytopathological Society. p. 58-60. ll'iskaya L.l., Chalenko G.l., Ozeretskovskaya O.L., and Aver'yanov AA, 1999. Induction of superoxide radical and peroxidase in a potato Phytophthora infestans system by arachidonic acid. Microbiology 68:183-188. ll’inskaya L.I., Perekhod E.A., Chalenko G.l., Gerasimova N.G., Ozeretskovskaya O.L., Piskarev V.E., Borovskaya, A.A., Yamskov IA, and Babakov A.V., 1997. FucosyI-containing oligosaccharides and potato resistance to Phytophthora infection. Russian Journal of Plant Physiology 44:771-776. ll’inskaya L.l., Perekhod E.A., Chalenko G.l., Gerasimova N.G., Romanenko E.N., Zinov'eva S.V., and Ozeretskovskaya O.L., 2000. Lipoxygenase activity in plants with induced resistance to disease. Russian Journal of Plant Physiology 47:449-455. 19 Ishizaka N. and Tomiyama K., 1972. Effect of wounding or infection by Phytophthora infestans on the contents of terpenoids in potato tubers. Plant and Cell Physiology 1 3:1 053-1063. Johnson G. and Schaal L., 1952. Relation of chlorogenic acid to scab resistance in potatoes. Science 115:627-629. Johnson G. and Schall L., 1955. The inhibitory effect of phenolic compounds on growth of Streptomyces scabies as related to the mechanism of scab resistance. Phytopathology 45:626-628. Kessmann H., Staub T., Hofmann C., Maetzke T., Herog J., Ward E., Uknes S., and Ryals J., 1994. Induction of systemic acquired resistance in plants by chemicals. Annual Review of Phytopathology 32:439- 459. Kolattukudy, PE, 1984. Biochemistry and function of cutin and suberin. Canadian Journal of Botany 62:2918-2933. Koop M., Rouster J., Fritig B., Darvill A., and Albersheim P., 1989. A fungal glucan preparation protects Nicotianae against infection by viruses. Plant Physiology 90:208-216. Kuc J., 1982. Phytoalexins from the Solanaceae. In: Bailey J.A. and Mansfield J.W. (eds), Phytoalexins. p. 81 -1 05. Leach S. and Webb R., 1981. Resistance of selected potato cultivars and clones to Fusarium dry rot. Phytopathology 71:623-629. Leubner-Metzger G. and Meins F., 1999. Functions and regulation of plant 8- 1,3-glucanases (PR-2). In: Datta SK. and Muthukrishnan S. (eds). Pathogenesis-related Proteins in Plants. CRC Press: New York. p. 49-76. Lisker N. and Kuc J., 1977. Elicitors of terpenoid accumulation in potato tuber slices. Physiology and Biochemistry 67:1356-1359. Lucas J.A., 1999. Plant immunization: from myth to SAR. Pesticide Science 55:193-196. Lulai EC. and Corsini D.L., 1998. Differential deposition of suberin phenolic and aliphatic domains and their roles in resistance to infection during potato tuber (Solanum tuberosum L.) wound-healing. Physiological and Molecular Plant Pathology 53:209-222. 20 Lyon G.D., Reglinski T., and Newton AC, 1995. Novel disease control compounds: the potential to ‘immunize’ plants against infection. Plant Pathology 44:407-427. Muller K.O., 1957. Wound healing of the potato tuber in relation of infection by Phytophthora infestans (Mont) de Bary. Austrian Journal of Biological Science 10:189-196. Mtiller, K.O. and Borger H., 1940. Experimentelle Untersuchungen uber die Phytophtora-Resistanz der Kartoffel. Arbeiten Biologischen Aanst. Reichsanstalt. 23:189. Neuhaus J., 1999. Plant Chitinases (PR-3, PR-4, PR-8, PR—11). In: Datta SK. and Muthukrishnan S. (eds). Pathogenesis-related Proteins in Plants. CRC Press: New York. p. 77-105. Nicholson R.L. and Hammerschmidt R., 1992. Phenolic compounds and their role in disease resistance. Annual Review of Phytopathology 30:369-389. Niderrnan T., Genetet l., Bmyére T., Gees R., Stintzi A., Legrand M., Fritig B., and Mdsinger E., 1995. Pathogenesis-related PR-1 proteins are antifungal. Plant Physiology 108:17-27. Nolte P., Secor GA, and Gudmestad N.C.,1987. Wound healing, decay and chemical treatment of cut potato tuber tissue. American Potato Journal 64:1-9. O'Brien V.J. and Leach 8.8., 1983. Investigations into the mode of resistance of potato tubers to Fusarium roseum ‘sambucinum'. American Potato Journal 60:227-233. Ozeretskovskaya O.L., 1995. Induced resistance in the Solanaceae. In: Hammerschmidt R., Kuc J. (eds), Induced Resistance to Disease in Plants. Kluwer, Dordrecht. p. 31-62. Paxton JD, 1981. Phytoalexins — A working redefinition. Phytopathology Z. 101:106-109. Peng M. and Kuc J. 1992. Peroxidase-generated hydrogen peroxide as a source of antifungal activity in vitro and on tobacco leaf disks. Phytopathology 82:696- 699. Percival G.C., Karim MS, and Dixon GR, 1998. Influences of light-enhanced glycoalkaloids on resistance of potato tubers to Fusarium sulphureum and Fusarium solani var. coemleum. Plant Pathology 47:665-670 21 Powelson M.L., Johnson KB, and Rowe RC, 1993. Management of diseases caused by soilbome pathogens. In: Rowe, R.C. (ed). Potato Health Management. American Phytopathological Society Press: St. Paul, MN. Preisig CL. and Kuc J.A., 1985. Arachidonic acid-related elicitors of the hypersensitive response in potato and enhancement of their activities by glucans from Phytophthora infestans (Mont) deBary. Archives of Biochemistry and Biophysics 236:379-389. Ricker K.E. and Bostock RM, 1994. Eicosanoids in the Phytophthora infestans- potato interaction: lipoxygenase metabolism of arachidonic acid and biological activities of selected lipoxygenase products. Physiological and Molecular Plant Pathology 44:65-80. Ride JP, 1978. The role of cell wall alteration in resistance to fungi. Annals of Applied Biology 89:302-6. Schafer W., 1994. Molecular mechanisms of fungal pathogenicity to plants. Annual Reviews of Phytopathology 32:461-477. Secor GA. and Gudmestad NC, 1999. Managing fungal diseases of potato. Canadian Journal of Plant Pathology 21:213-221. Sinden S.L. and Webb RE, 1972. Effect of variety and location on the glycoalkaloid content of potatoes. American Potato Journal 49:334-338. Smith BC. and Rubery P.H., 1981. The effects of infection by Phytophthora infestans on the control of phenylpropanoid metabolism in wounded potato tissue. Planta 151 :535-540. Smith W.L. and Smart HF, 1955. Relation of soft rot development to protective barriers in Irish potato slices. Phytopathology 45:649-654. Soliday C.L., Kolattukudy PE, and Davis R.W., 1979. Chemical and ultrastructural evidence that waxes associated with the suberin polymer constitute the major diffusion barrier to water vapor in potato tuber (Solanum tuberosum L.). Planta 146:607-614. Sticher L., Mauch-Mani B., and Métraux JP, 1997. Systemic acquired resistance. Annual Reviews Phytopathology 35:235-270. Theron DJ, 1991. Prediction of potato dry rot on the presence of Fusarium in soil adhering to tubers at harvest. Plant Disease 75:126-129. 22 Tjamos E. C. and Kuc J. A., 1982. Inhibition of steroid glycoalkaloid accumulation by arachidonic and eicosapentaenoic acids in potato. Science 217:542-543. USDA, 1978. Market diseases of potatoes. p. 19-23. Valkonen P.T., Keskitalo M., Vasara T., and Pietilla T., 1996. Potato glycoalkaloids: a burden or a blessing? Critical Reviews in Plant Science 15:1- 20. Vander Zaag R., Chase R.W., and Hammerschmidt R., 1984. The effect of chlorine wash treatments on potato suberization. American Potato Journal 61:540-541. van Loon LC, 1999. Occurrence and properties of plant pathogenesis-related proteins. In: Datta SK. and Muthukrishnan S. (eds). Pathogenesis-related Proteins in Plants. CRC Press: New York. p. 1-19. Vasyukova N.l., Chalenko G.l., Gerasimova N.G., Perekhod E.A., Ozeretskovskaya O.L., ll’ina A.V., Varlamov VP, and Albulov AL, 2000. Chitin and chitosan derivatives as elicitors of potato resistance to late blight. Applied Biochemistry and Microbiology 36:372-376. Velazhahan R., Datta S.K., and Muthukrishnan S., 1999. The PR-5 family: thaumatin-like proteins. In: Datta SK. and Muthukrishnan S. (eds). Pathogenesis-related Proteins in Plants. CRC Press: New York. p. 107-129. Wojtaszek P., 1997. Oxidative burst: an early plant response to pathogen infection. Biochemistry Journal 322:681-692. Yurganova L.A., Nogaideli D.E., Chalenko G.l., Chalova LL, and Ozeretskovskaya O.L., 1989. Inducing activity of Fusarium culmorum Sacc. Prikladnaya Biokhimiya i Mikrobiologiya 25:805-810. Zeng Y., 1993. Biochemical and physiological aspects of the plant host response in the Fusarium dry rot disease of potato. Ph.D. Dissertation, Michigan State University, East Lansing, Michigan USA. 23 CHAPTER 1 Evaluation of Potential Inducing Agents of Acquired Resistance In Potato Tuber Tissue Against Fusarium sambucinum 24 INTRODUCTION Acquired resistance is a type of resistance in which a susceptible plant becomes resistant after treatment with an inducing agent (Hammerschmidt and Becker, 1997; Hammerschmidt and Kuc, 1995). It has been demonstrated in over 25 plant species from at least six different families (T uzun and Kuc, 1991). Inducing agents include a wide range of biotic elicitors including carbohydrates, phenols, salicylic acid, jasmonic acid, and amino acids; and abiotic elicitors including synthetic chemicals and inorganic compounds (Lyon et al, 1995). Acquired resistance has been proposed as an alternative to pesticides for the control of disease. It has a number of advantages over pesticide use: a) activators of acquired resistance are safer for the environment and animal health b) protection can be systemic c) protection is long lasting d) protection is against a broad spectrum of pathogens including bacteria and viruses to which there are no pesticide controls and e) multiple plant defenses are induced making acquired resistance more stable than pesticides which is directed at a single metabolic function of the pathogen (Kuc, 1995). F. sambucinum, the causal agent of dry rot in potato tubers, has developed resistance to thiabendazole, (Desjardins, 1995; Desjardins et al, 1993) the only approved fungicide for post-harvest application on potato tubers (Powelson et al, 1993). Additionally, most potato cultivars and breeding lines have little or no resistance to F. sambucinum (Leach and Webb, 1981). Acquired resistance is a promising method of controlling post harvest dry rot of potato tubers. Acquired resistance has been demonstrated in potato tuber tissue. Treatment of tuber 25 tissue with an incompatible race of Phytophthora infestans, glycoprotein extracts from P. infestans cell walls, arachidonic acid, eicosapentaenoic acid, extracts from Fusarium caerulem and Fusarium culmorum Sacc, chitosan, and fucosyl- containing oligosaccharides protected tissue against P. infestans (Vasyukova et al, 2000; ll’inskaya et al, 1997; Ozeretskovskaya, 1995; Chalova et al, 1989; Yurganova et al, 1989; Preisig and Kuc, 1985; Chalova et al, 1977; Miiller and Borger, 1940). Additionally, potato tuber tissue was protected against Erwinia carotovora spp. atroseptica infection when treated with oligogalacturonides and Phytophthora sojae cell wall hydrolysate (Dutton et al, 1997) and against Fusarium caemlem when treated with an incompatible race of P. infestans (Miiller and Borger, 1940). The goal of this study was to find an activator of acquired resistance in potato tubers that would protect against F. sambucinum. MATERIALS AND METHODS Preparation of Potato Tuber Disks Tubers from potato (Solanum tuberosum L.) cultivar Snowden, which were stored at 4°C for up to 6 months, were used in all experiments. Procedure for preparation of potato tuber disks was modified from Hammerschmidt (1984). Tubers were allowed to warm to room temperature overnight. They were then washed and surface sterilized by soaking in a 5% solution of Clorox for 5-10 minutes. Just prior to making the tuber disks, tubers were dipped in 95% ethanol and flamed to ensure surface sterilization. The apical and stolen ends of the 26 tubers were excised and a 2.0 cm core borer was pressed through the cut end producing a cylinder of tuber tissue from the pith medullary tissue. The cylinder was then sliced into 0.5 cm thick slices. The disks were rinsed three times in sterile water and placed in sterile petri plates lined with Whatman #1 filter paper. Fungal Cultures Fusarium sambucinum isolate RN1 was grown on potato dextrose agar at 23°C in the dark. The cultures were transferred every 7-10 days. Preparation of Elicitors Salicylic Acid Salicylic acid sodium salt was purchased from Sigma and prepared at concentrations of 1, 5, and 10 mM by dissolving in distilled water. Non-protein Amino Acids L-, D-, and DL-a-aminobutyric acid (AABA); 8-aminobutyric acid (BABA); and y-aminobutyric acid (GABA) were purchased from Sigma Chemical Company, St. Louis, MO, USA. The compounds were prepared at concentration of 3000 ppm by dissolving them in distilled water. Commercial Chemicals 2,6—dichloro-isonicotinic acid (INA, CGA 279202) and acibenzolar-S-methyl (ASM, CGA 245704) were obtained from Novartis Crop Protection, Greensboro, North Carolina, USA. INA was prepared at concentrations 10, 25, and 50 ul/ml by dissolving it in distilled water. ASM was prepared at concentrations between 27 0.1 and 100 ppm by dissolving it in distilled water. The ASM was formulated with 50% active ingredient; concentrations were prepared based on the concentration of active ingredient. AXP and GCC were obtained from Auexin Corporation, Lansing, Michigan, USA. The materials were prepared at concentrations between 300 and 3000 ppm by dissolving them in distilled water. Phytophthora infestans Mycelium Homogenate Isolate 97-2 of P. infestans was obtained from Dr. William Kirk, Michigan State University. It was grown for approximately one month on Iima bean agar at 23°C in the dark. Mycelium was peeled from the surface of the lima bean agar, autoclaved, lyophilized, and ground with a pestle and mortar into a fine powder. Mycelium homogenate (MH) were prepared at concentrations of 0.25, 0.5, and 1.0 mglml by suspending the powdered mycelium in sterile water by sonication. Fatty Acids Arachidonic acid, linolenic acid, and linoleic acid were purchased from Sigma Chemical Company, St. Louis, MO, USA. The fatty acids were prepared at concentrations between 1x10'5 and 50 ul/ml by homogening the fatty acids in distilled water using a Polytron homogenizer. Chitosan Chitosan preparation was based on a procedure from Benhamou and Thériault (1992). Crab-shell chitosan, purchased from Sigma Chemical Company, St. Louis, MO, USA was washed by homogenizing the chitosan in distilled water using a Polytron homogenizer. Centrifuging the homogenate at 28 10,000 rpm for 10 minutes pelleted the homogenate. The pellet was than air- dried. Twenty grams of dried chitosan were then dissolved in 1000 ml of 0.25 N HCl and the insoluble material was removed by centrifugation. The chitosan was precipitated by adjusting the pH to 9.8 using 2.5 N NaOH. The precipitate was collected by centrifugation. washed three times with distilled water, and lyophilized. The Iyophilized chitosan was stored at 4°C. A stock solution was prepared from the washed chitosan by dissolving it in 0.25 N HCI. The pH was then adjusted to 5.5-6.0 using 2.0 N NaOH. The solution was then adjusted with distilled water to a concentration of 4.0 mg chitosan/ml. The stock solution was sterilized by autoclaving and stored at 4°C. The stock solution was adjusted to the concentrations of 0.5 and 1.0 mglml using sterile distilled water just prior to use. Identification of Elicitors that Induce Resistance to Fusarium sambucinum The surfaces of the tuber slices were treated with 200 pl of sterile water, as a control, or with 200 pl of the elicitor solutions. The treatment was applied within 1 hour of slicing and was spread evenly across the surface with a glass rod. The slices were incubated for 1, 2, or 3 days after treatment at room temperature in the dark. The tuber slices were then challenge with F. sambucinum by placing in the center of each tuber slice a 5.0 mm plug of F. sambucinum. Plugs were taken from the margin of hyphal growth of 3 or 4 day old cultures. Three or 4 days after challenge each slice was cut in half and the depth and width of infection was measured. Each treatment consisted of 15 slices. Data were 29 analyzed for significance by one-way analysis of variance, followed by Tukey’s significant difference test (a=0.05), using GraphPad lnStat software. Comparison of Cultivars Potato tuber slices (Solanum tuberosum L.), cultivars Snowden and Atlantic, were treated with 1.0 mg chitosan/ml as described in the procedures above. Three days after treatment the slices were challenged with F. sambucinum and depth of infection was measured 4 days after challenge. RESULTS Effect of Salicylic Acid Salicylic acid (SA), applied at 1.0, 5.0 and 10.0 mM, did not cause any visible effect on the appearance of treated tuber tissue. SA also did not have an effect on infection by F. sambucinum when the treated tuber slices were challenged 2 or 3 days after treatment (Table 1-1). Effect of Salicylic Acid Functional Analogues Acibenzolar-S-methyl (ASM, CGA 245704) applied at 100, 50, 25, 0.5, 0.1 ppm had no effect on disease when the treated tuber slices were challenged at 1, 2, or 3 days after treatment (Table 1-2). Tuber tissue treated with 2,6-dichloro-isonicotinic acid (INA, CGA 41396) applied at 50, 25, and 10 mglml, tended to be more susceptible to F. 30 sambucinum when the treated tissue slices were challenged 2 or 3 days after treatment; however, the increase was not significantly over the control (Table 1- 3). Effect of Non-Protein Amino Acids D-, L-, and DL-a-aminobutyric acid (AABA) applied at 3000 ppm, slightly reduced infection in potato tuber tissue challenged 1, 2, or 3 days after treatment. However, the reduction was not significantly different than the control. 8- aminobutyric acid (BABA), and y- aminobutyric acid (GABA) applied at 3000 ppm did not significantly decrease infection by F. sambucinum. In fact, BABA appeared to make the tuber tissue more susceptible to infection (Table 1-4). Effect of Commercial Formulations of Non-Protein Amino Acids AXP applied at 300 and 3000 ppm did not significantly reduce infection by F. sambucinum when challenged 1, 2, or 3 days after treatment. However, AXP did appear to slightly reduce infection. This reduction was greater at 3000 ppm than 300 ppm (Table 1-5). GCC applied at 1000 ppm did not reduce infection when challenged 1, 2, or 3 days after treatment. However GCC applied at 3000 ppm reduced infection when challenged at all three time points. This reduction was significantly different than the control when challenged 1 and 3 days after infection (Table 1- 2). 31 Effect of Phytophthora infestans Mycelium Homogenate P. infestans mycelium homogenate (MH), applied at 0.25, 0.5 and 1.0 mglml, significantly decreased the depth of infection by F. sambucinum when treated tissues were challenge inoculated 2 or 3 days after treatment (Table 1-6). Effect of Fatty Acids Arachidonic acid (AA), applied at 50, 25, 10, 5, and 1 uI/ml, slightly decreased infection by F. sambucinum when treated tuber tissue was challenged at 2 or 3 days after treatment. However, the decrease was not significantly different than controls. AA applied at concentration lower the 1 ppm did not decrease infection and in some cases appeared to make the tuber tissue more susceptible (Table 1- 7). Linolenic acid (LNA), applied at 2.0, 1.0, 0.5, and 0.1 uI/ml, did not decrease infection by F. sambucinum when the treated tissue were challenged at 2 or 3 days after treatment (Table 1-8). Linoleic acid (LA), applied at 10, 5, and 1 pl/ml, significantly decreased infection by F. sambucinum when challenged at 2 or 3 days after treatment in some trial. However, other trials showed no reduction in disease. LA applied at concentration less than 1.0 pllml consistently had less infection. However, the decrease was not significantly different than controls (Table 1-9). Tuber tissue treated with AA, LNA and LA exhibited browning and cell death similar to a hypersensitive response. 32 Effect of Chitosan Chitosan applied at 1.0 and 0.5 mglml significantly decrease both the width and depth of infection by F. sambucinum when the tuber slices were challenged at 2 or 3 days after treatment. When challenged 1 day after treatment, infection was reduced, but not significantly compared to the control (Table 1-10). Effect of Combining Elicitors ASM at concentrations of 0.5 and 0.1 ppm in combination with 3000 ppm GGC had no significant effect on F. sambucinum infection when the treated tuber tissue was challenged 2 or 3 days after treatment (Table 1-11). Chitosan at concentrations of 1.0 and 0.5 mglml in combination with 3000 ppm BABA decreased F. sambucinum infection. However, disease reduction was only significantly different then controls when challenged 3 days after treatment with 1.0 mglml chitosan and 3000 ppm ASM (Table 1-11). SA at concentrations of 1.0 and 10.0 mM in combination with 1000 ppm BABA decrease disease, however, it was only significantly decreased when challenged 3 days after treatment with 1.0 mM SA and 1000 ppm BABA (Table 1-11). TABLE 1-1: Effect of salicylic acid (SA) on the ability of Fusarium sambucinum to infect potato tuber tissue 2 Days 3 Days Treatment Control Treatment Control Treatment 1 mM SA 2.9 2.7 2.7 2.2 5 mM SA 2.9 2.6 2.7 3.0 10 mM SA 2.9 3.0 2.7 3.1 Tuber slices were treated with 1, 5, and 10 mM of SA and challenged with F. sambucinum 2 and 3 days after treatment. Depth of infection (mm) was measured 4 days after challenge. The depth of infection in tuber slices treated with 1, 5, and 10mM SA was not significantly different (P<0.05) from controls. 33 TABLE 1-2: Effect of ASM on the ability of Fusarium sambucinum to infect potato tuber tissue 1 Day 2 DaLs 3 Days Treatment Control Treatment Control Treatment Control Treatment 0.1 ppm ASM 3.5 3.5 3.8 3.6 2.5 2.4 0.5 ppm ASM 3.5 3.7 3.8 3.7 2.5 1.9 25 ppm ASM 2.7 3.4 3.1 3.4 3.5 3.7 50 ppm ASM 2.7 3.1 3.1 3.2 3.5 3.2 100 ppm ASM 2.7 3.6 3.1 3.6 3.5 3.6 Tuber slices were treated with 0.1, 0.5, 25, 50, and 100 mM of ASM and challenged with F. sambucinum 1, 2 and 3 days after treatment. Depth of infection (mm) was measured 4 days after challenge. The depth of infection in tuber slices treated with ASM was not significantly different (P<0.05) from controls. TABLE 1-3: Effect of INA on the ability of Fusarium sambucinum to infect potato tuberfissue 2 Days 3 Days Treatment Control Treatment Control Treatment 10 ul/ml INA 2.3 3.1 2.8 3.2 25 ul/ml INA 2.3 3.1 2.8 2.6 50 pl/ml INA 2.3 2.8 2.8 3.1 Tuber slices were treated with 10, 25, and 50 ul/ml of INA and challenged with F. sambucinum 2 and 3 days after treatment. Depth of infection (mm) was measured 4 days after challenge. The depth of infection in tuber slices treated with INA was not significantly different (P<0.05) from controls. TABLE 1-4: Effect of non-protein amino acids on the ability of Fusarium sambucinum to infect potato tuber tissue Treatment 1 Ba 2 Days 3 Days (3000 ppm) Control Treatment Control Treatment Control Treatment L-AABA 4.4 4.2 4.0 3.0 4.1 3.6 D-AABA 4.4 4.0 4.0 3.7 4.1 3.3 DL-AABA 4.4 4.3 4.0 3.4 4.1 3.9 BABA 3.3 3.6 3.0 2.9 2.7 3.1 GABA 3.3 2.8 3.0 2.9 2.7 2.5 Tuber slices were treated with 3000 ppm of L-, D-, and DL-AABA, BABA and GABA and challenged with F. sambucinum 1, 2 and 3 days after treatment. Depth of infection (mm) was measured 4 days after challenge. BABA and GABA were tested separately from the AABA isomers. The depth of infection in tuber slices treated with L-, D-, and DL-AABA; BABA; GABA was not significantly different (P<0.05) from controls. 34 TABLE 1-5: Effect of Auexin Corp. chemical elicitors on the ability of Fusarium sambucinum to infect potato tuber tissue 1 Day 2 Dafi 3 Days Treatment Control Treated Control Treated Control Treated 300 ppm AXP 2.8 2.3 3.1 2.6 2.5 1.9 3000 ppm AXP 2.8 2.5 3.1 2.5 2.5 1.7 1000 ppm GGC 4.7 4.1 3.9 4.1 4.1 4.2 3000 ppm GGC 4.7 3.8* 3.9 3.7 4.1 3.1* Tuber slices were treated with 300 and 3000 ppm AXP and 1000 and 3000 ppm GGC. The tuber slices were then challenged with F. sambucinum 1, 2 and 3 days after treatment. Depth of infection (mm) was measured 4 days after challenge. AXP and GGC were tested in separate experiments. Asterisks (*) represent a significant difference (P<0.05) from controls. TABLE 1-6: Effect of homogenate of Phytophthora infestans mycelium (MH) on the ability of Fusarium sambucinum to infect potato tuber tissue 2 Day 3 Days Treatment Width Depth Width Depth C°""°' 12.5:11 3.93 11.83 3.38 0.25 mg MH/ml 13.68 2.9b 13._,a,b 22b 0.5 mg MH/ml 122a 2.3b 9.633 15b 1'0 "'9 MH’m' 13.2a 2.3b 12.13 1.5b Tuber slices were treated with 0.25, 0.5, and 1.0 mg MH/ml and challenged with F. sambucinum 2 and 3 days after treatment. Depth of infection (mm) was measured 4 days after challenge. Superscripts indicate significant difference (P< 0.05) between control and treatments. 35 TABLE 1-7: Effect of Arachadonic acid (AA) on the ability of Fusarium sambucinum to infect potato tuber tissue 2 Days Treatment Control Treatment Control Treatment 50 ul/ml AA 2.1 1.6 2.1 1.6 25 ul/ml AA 2.1 1.5 2.1 1.5 10 ill/ml AA 2.1 1.6 2.1 1.5 5.0 uI/ml AA 2.1 1.6 2.1 1.6 1.0 ul/ml AA 2.1 1.8 2.1 1.8 0.1 ullml AA 2.4 3.3 2.5 2.4 0.03 ul/ml AA 2.4 3.3 2.5 2.3 0.01 ul/ml AA 2.4 3.3 2.5 2.4 0.003 ul/ml AA 2.4 3.0 2.5 2.2 0.001 uI/ml AA 2.4 2.6 2.5 2.3 0.0003 uI/ml AA 3.7 3.5 3.2 3.2 0.0001 pl/ml AA 3.7 4.1 3.2 3.3 0.00003 1.1le AA 3.7 3.6 3.2 3.1 0.00001 ul/ml AA 3.7 3.7 3.2 3.3 Tuber slices were treated with AA at concentrations ranging from 0.00001 ule to 50 III/ml. The tuber slices were challenged with F. sambucinum 2 and 3 days after treatment. Depth of infection (mm) was measured 4 days after challenge. The depth of infection in tuber slices treated with AA was not significantly different (P<0. 05) from controls. TABLE 1-8: Effect of Linolenic acid (LNA) on the ability of Fusarium sambucinum to infect potato tuber tissue 2 Days 3 Days Treatment Control Treatment Control Treatment 2.0 ul/ml LNA 2.2 2.2 2.1 3.0 1.0 ullml LNA 2.2 2.2 2.1 3.2 0.5 ul/ml LNA 2.2 1.9 2.1 2.4 0.1 pllml LNA 2.2 1.9 2.1 1.8 Tuber slices were treated with 2.0, 1.0, 0.5, and 0.1 pI/ml LNA and challenged with F. sambucinum 2 and 3 days after treatment. Depth of infection (mm) was measured 4 days after challenge. The depth of infection in tuber slices treated with LNA was not significantly different (P<0.05) from controls. 36 TABLE 1-9: Effect of Linoleic acid (LA) on the ability of Fusarium sambucinum to infect potato tuber tissue 2 Days 3 Days Treatment Control Treatment Control Treatment 10 ul/ml LA 2.8 2.7 2.6 3.4 10 uI/ml LA 3.3 0.6* 3.8 1.0* 10 ul/ml LA 3.4 1.2* 2.6 0.5* 5.0 ul/ml LA 2.8 3.0 2.6 3.7 1.0 ul/ml LA 2.8 3.6 2.6 2.5 1.0 ul/ml LA 3.3 0.4* 3.8 0.1* 1.0 11le LA 3.4 2.6 2.6 2.7 0.8 ul/ml LA 3.1 2.5 3.8 3.2 0.6 III/"TI LA 3.1 2.7 3.8 3.7 0.4 tLI/mI LA 3.1 2.7 3.8 3.4 0.2 til/ml LA 3.1 2.8 3.8 3.3 0.1 11le LA 3.3 2.4 3.8 2.1* 0.1 pllmI LA 3.4 3.0 2.6 2.7 Tuber slices were treated with LA at concentrations ranging from 0.1 pI/ml to 10 ullml. The tuber slices were challenged with F. sambucinum 2 and 3 days after treatment. Depth of infection (mm) was measured 4 days after challenge. Asterisks (*) represent significant difference (P< 0.05) from controls. TABLE 1-10: Effect of chitosan treatment on the ability of Fusarium sambucinum to infect potato tuber tissue 1 Day 2 Days 3 Days Treatment Width Depth Width Depth Width Depth 8 a a a C°"t"°' 12.7:11 4.36' 12.9 4.0 11.5 4.2 - b b b b 0'5 "‘g’m' Ch'msa“ 11.3a 3.5a 6.3 2.0 5.6 2.3 1.0mglmlCh1tosan 1238 4.0 7.6 2.3” 3,5 1,5 Tuber slices were treated with 0.5 and 1.0 mglml chitosan and challenged with F. sambucinum 1, 2 and 3 days after treatment. Depth of infection (mm) was measured 4 days after challenge. Superscripts represent significant differences (P<0.05) between control and treatments. 37 TABLE 1-11: Effect of combining elicitors on the ability of Fusarium sambucinum to infect potato tuber tissue 2 Days 3 Days Treatment Control Treated Control Treated 0.1 ppm ASM/ 3000ppm GGC 3.3 3.5 3.6 4.0 0.5 ppm ASM/3000 ppm GGC 3.3 3.1 3.6 2.9 0.5 mglml Chitosan/3000ppm BABA 2.8 2.9 3.8 3.0 1.0 mglml Chitosan/3000ppm BABA 2.8 2.3 3.8 2.7* 1 mM Salicylic acid/1000ppm BABA 2. 8 2.4 3.1 2. 5 10 mM Salicylic acid/1000ppm BABA 2. 8 2. 5 3.1 2. 0* Tuber slices were treated with combinations of ASM and GGC, chitosan and BABA, and SA and BABA. The tuber slices were challenged with F. sambucinum 2 and 3 days after treatment. Depth of infection (mm) was measured 4 days after challenge. Asterisks (*) represent treatments that were significantly different (P<0.05) from the control. Visual Comparison of Tuber Tissue Response to MH and Chitosan Tuber tissue treated with MH exhibited browning and cell death similar to a hypersensitive response while tuber tissue treated with chitosan did not display a visual response to treatment (Figure 1-1). O O —8 O o .3 g 9'01 92: pg '01 o " 6‘3 6‘3 3 33 z3 i are as Ia IQ Is :1 =3 3 a a Figure 1-1: Visual comparison of tuber slices 3 days after treatment with 0.25, 0.5, or 1.0 mglml MH or 0.5 or 1.0 mg/ml chitosan. Images in this dissertation are presented in color. 38 Comparison of Cultivars Tuber slices made from Atlantic tubers appeared to be more susceptible to F. sambucinum infection than Snowden tuber slices, however, the difference was not significant (Figure 1-2). Treatment of the Snowden and Atlantic tuber slices with chitosan made them significantly less susceptible to F. sambucinum than the controls (Figure 1-2). Chitosan treated Atlantic slices were less susceptible than chitosan treated Snowden slices, however the difference was not significant (Figure 1-2). These results may indicate Atlantic tuber tissue is more inducible than Snowden tuber tissue. ° 1 5 ‘ Snowden E3 Atlantic Depth (mm) 00 h N 41 ......... .......... ................... ......... .......... ................... ......... .......... ......... .......... ................. ........... 0- ~ . s Control 1.0 mglml Chitosan Figure 1-2: Comparison of the effect of chitosan treatment on the depth of infection by F. sambucinum in potato tuber slices made from cultivars Snowden and Atlantic. Bars indicate standard deviation. 39 DISCUSSION Salicylic acid (SA) appears to have a role in plant resistance by inducing plant defense responses (Hammerschmidt and Becker, 1999). The amount of endogenous SA in potato plants has been correlated with resistance to pathogens (Coquoz et al, 1995). However, potato plants transformed with the nahG gene, which encodes for salicylate hydroxylase (an enzyme that converts SA to catechol), were not more susceptible to P. infestans, indicating that basal levels of SA are not responsible for resistance (Yu et al, 1997). Exogenous treatment of potato plants with SA has been reported to cause local induction of PR-1, (Coquoz et al, 1995) and to decrease susceptibility to P. infestans in cultivars resistant to P. infestans (Quintanilla and Brishammar, 1998). SA treatment of seed tubers from cultivars resistant to P. infestans has also been shown to increase resistance of the plants to P. infestans (Quintanilla and Brishammar, 1998). However, SA also increased susceptibility in cultivars susceptible to P. infestans (Quintanilla and Brishammar, 1998). SA has been implicated in systemic acquired resistance (SAR) based on the observations that SA levels systemically increase at the onset of SAR (Ryals et al, 1996). Accumulation of SA throughout the plant is believed to be responsible for the systemic induction of PR proteins observed in SAR (Hammerschmidt and Smith-Becker, 1999; Klessig and Malamy, 1994). Initially it was believed that SA was the systemically translocated signal for SAR. However, it is more likely that systemic increases in SA are induced by a translocated signal that has yet to be 40 identified (Hammerschmidt and Smith-Becker, 1999) and, to a lesser degree, by the movement of SA through the phloem (Klessig and Malamy, 1994). Treatment of potato plants with AA induced systemic resistance to P. infestans and Alteman'a solani, even though SA accumulation and PR-1 expression was only at the site of AA treatment (Coquoz et al,1994; Coquoz et al, 1995). Infection with P. infestans also caused only local induction of SA (Coquoz et al, 1994), but systemic protection against P. infestans (Cohen et al, 1991). As a result, SA appears not to have a role in SAR in potato plants when induced by fatty acids. Induction of SAR by fatty acids may instead be due to oxidation of fatty acids leading to production of jasmonic acid (JA), which in turn triggers induction of resistance. However, AA did not induce SAR in potato plants expressing the nahG gene, suggesting SA is necessary for SAR expression (Yu et al, 1997). It has been suggested that increases in SA are not observed in potato because there is an increase in sensitivity to SA instead of an increase in SA (Yu et al, 1997). There has been very little research done on the accumulation or affect of SA in potato tuber tissue and it is uncertain whether tuber tissue responds in the same manner as the foliage. SA has been shown to stimulate production of ethylene in tuber tissue (Liang et al, 1997). Ethylene has been reported to induce some PR proteins and enhance cell wall modifications (Sticher et al, 1997). AA and EPA are able to induce resistance to P. infestans in potato tuber tissue; however, exogenous application of acetyl salicylic acid has been shown to inhibit induction of phytoalexins and HR cell death caused by AA and EPA 41 treatment (Lee and Currier, 1996). SA has been reported to inhibit JA biosynthesis (Farmer and Ryan, 1992) and may block 3 JA induced ISR pathway. SA’s role in potato induced resistance, as well as, its ability to induce resistance to P. infestans in some potato cultivars, locally induce PR proteins, and stimulate production of ethylene made it a candidate as an activator of acquired resistance against F. sambucinum. However, application of SA to potato tuber tissue did not induce resistance against F. sambucinum (Table 1-1). Several problems have previously arisen with the use of SA as an activator of acquired resistance: 3) SA is not translocated when applied exogenously, therefore, only treated tissue is resistance; b) SA is not always taken up by the plant tissue; and c) SA can be phytotoxic and the range between SA efficiency as an activator and its phytotoxicity is narrow (Kessmann at al, 1994). In this experiment the tuber tissue was evenly treated with SA and there were no apparent symptoms of phytotoxicity. The SA treatment may not have induced resistance because SA was not taken up the tuber tissue, the concentration of SA applied was not suitable to activate a response, or SA is not an activator of acquired resistance in potato tuber tissue. INA and ASM are functional analogs of SA (Hammerschmidt and Dam, 1997). They are structurally different from SA, but they induce SAR and activate the same plant defenses as SA (Kessmann et al, 1994; Tally of al, 1999). There is evidence that INA and ASM work down stream of SA in the SAR signal transduction pathway (Kessmann et al, 1994; Tally et al, 1999). ASM and INA 42 have both been shown to induce resistance in wide range of crops against viruses, bacteria, and fungi (Tally et al, 1999; Kessman et al, 1994). However, neither of these compounds induced resistance to potato tuber tissue against F. sambucinum (Table 1-2 and Table 1-3). SA, INA, and ASM are not promising elicitors for the control of F. sambucinum. Amino acids have been considered as a potential method of controlling disease for years, because certain amino acids or metabolites of amino acids have antimicrobial activity, the ability to affect the development and pathogenicity of fungi, and the ability to increase resistance of plant tissue to pathogens (van Andel, 1966). The ability of the non-protein amino acids AABA, BABA, and GABA to induce resistance has been extensively studied. BABA has been shown to increase resistance to viruses, fungal pathogens, and nematods. BABA elicited protection against pathogens in potato (van Andel, 1966), tomato (Cohen, 1993), tobacco (Cohen, 1994), pea (Papavizas, 1964), pepper (Sunwoo et al, 1996), and cucurbits (van Andel, 1966). AABA has been shown to increase resistance to fungal pathogens in tobacco (Cohen, 1994), tomato (Cohen, 1993), pepper (Sunwoo et al, 1996) pea and apple (van Andel, 1966) and GABA has been shown to increase resistance to P. infestans in tomato (Cohen, 1993). BABA appears to have a broader affect than AABA and GABA, and BABA has been shown to be more effective than AABA and GABA at inducing resistance in tomato against Phytophthora infestans, pepper against Phytophthora capsici, and tobacco against Peronospore tabacina (Cohen, 1993; Sunwoo et al, 1996 and Cohen, 1994). AABA, BABA, and GABA were shown to induce PR—proteins 43 (Cohen, 1994; Cohen et al,1994; Eyal et al, 1992; Lotan and Fluhr, 1990; Asselin et al, 1985) and AABA and BABA caused ethylene evolution (Cohen, 1994; Lotan and Fluhr, 1990), which has been reported to induce PR proteins and cause cell wall alterations (Sticher et al, 1997). However AABA, BABA, and GABA did not significantly reduce infection by F. sambucinum in potato tuber tissue (T able 1-4). AXP and GGC are commercial formulations of non-protein amino acids. AXP also had no affect on tuber tissue resistance to F. sambucinum (Table 1-5). However, GGC significantly decreased F. sambucinum infection. Why GGC caused an increase in resistance and not the purified non-protein amino acids is unknown. It may be a result of other substances in the formulation rather than the proteins, a synergistic affect of compounds in the formulation, or the concentration of the amino acids. * BABA was tested in combination with SA. SA has been shown to enhance the inducing activity of some elicitors. BABA in combination with SA significantly reduced infection by F. sambucinum after 3 days (Table 1-11), indicating that SA may enhance the activity of BABA. GGC was tested in combination with ASM, however, there was no significant decrease in disease (Table 1-11). Non-protein amino acids do not seem to be promising elicitors of resistance in tuber tissue against F. sambucinum. Even though GCC and BABA in combination with SA decreased infection, the level of disease reduction is not great enough for effective control of the disease. Extracts from P. infestans mycelium have long been studied as elicitors of resistance in potato tuber tissue against P. infestans. Extracts or homogenates of P. infestans mycelium have been reported to increase accumulation of sesquiterpene phytoalexins (Henfling et al, 1980) and induce resistance to P. infestans (Chalova et al, 1977). P. infestans mycelium homogenate (MH) significantly increased the resistance of tuber tissue to F. sambucinum as shown by decreasing the degree of infection by as much as 50% (Table 1-6). It is unlikely that induction of sesquiterpene phytoalexin accumulation is responsible for the increase in resistance since F. sambucinum can detoxify lubimin (Desjardins et al, 1989) and with stand high concentrations of rishitin (Desjardins and Gardner, 1989). However, MH may induce other putative plant defense mechanisms, which could cause the tuber tissue to be more resistant to F. sambucinum. Further studies demonstrated that the fatty acids arachidonic acid (AA) and eicosapentaenoic acid (EPA) from P. infestans mycelium were the most active elicitors of sesquiterpene phytoalexin accumulation (Bostock et al, 1981) and resistance to P. infestans in potato tuber tissue (Chalova at al, 1989). Additionally, the fatty acids AA and EPA, as well as, linoleic acid (LA) and linolenic acid (LNA) from P. infestans mycelium were able to protect the foliage of potato plants against P. infestans (Cohen et al, 1991). AA and LNA were unable to induce resistance in potato tuber tissue to F. sambucinum (Table 1-7 and Table 1-8). LA did increase resistance, however, the results were very inconsistent between replications (Table 1-9). It appears 45 that component(s) other than these fatty acids are responsible for the tuber tissue resistance induced by MH. It has been reported that a 8-1,3-8-1,6 glucan from P. infestans mycelium homogenate can also induce sesquiterpene phytoalexin and resistance to P. infestans (Chalova et al, 1976). Additionally, glucans have been shown to increase the effect of fatty acids (Bostock et al, 1982; Maniara et al, 1984; Preisig and Kuc, 1985). The increased resistance produced by the MH may be a result of untested fatty acids (EPA and eicosatrienoic acid), glucans or an additive affect between glucans and fatty acids. If the active component(s) of the MH can be determined they may be a successful elicitor of resistance against F. sambucinum in potato tuber tissue. Chitosan, a polymer of 8-1,4-Iinked glucosamine, has also been extensively studied as an elicitor of resistance. Chitosan has been shown to decrease susceptibility in celery (Bell et al, 1998), bean (Pospieszny et al, 1991), pea (Hadwiger and Beckman, 1980; Pospieszny et al, 1991), carrot (Chaeh et al, 1997), potato (Vasyukova et al, 2000), tomato (Pospieszny et al, 1991; Benhamou et al, 1992; Benhamou et al, 1994), wheat (Raddy et al, 1999), peppers (Kim et al, 1997), tobacco (Pospieszny et al, 1991) and fruit (Ghaouth et al, 1992; Du et al, 1997; Fajardo et al, 1998; Reddy et al, 2000) against viruses, bacteria and fungal pathogens. Reduced susceptibility may be due to chitosan’s reported antifungal activity (Allen and Hadwiger, 1979; Stdssel and Leuba, 1984) or by induction of plant defense mechanisms. Chitosan has been shown to induce a number of putative plant defense mechanisms including accumulation of phytoalexins (Kendra and Hadwiger, 1984; Kneer et al, 1999), deposition of 46 phenolic material (Benhamou et al, 1994; Reddy et al, 1999; Stange and McDonald, 1999), induction of PR-proteins (Wilson et al, 1994; Fajardo et al, 1998), production of hydrogen peroxide (Orozco-Cardenas and Ryan, 1999), accumulation of jasmonic acid (Doares et al, 1995), and increase phenylalanine ammonia-lyase activity (Peltonen et al, 1997). Treatment of potato tuber slices with chitosan significantly decreased infection by F. sambucinum (Table 1-10). Increased resistance did not occur until two days after treatment (Table 1-10) indicating the increase in resistance was not due to antifungal activity of chitosan. Therefore, it appears that chitosan is inducing a response in the potato tuber tissue that is able to decrease infection. Chitosan was also tested in combination with BABA and BABA was shown to reduce the effectiveness of chitosan to increase resistance in the tissue (Table 1- 11). ' ' Chitosan and MH were both effective at reducing infection by F. sambucinum and may be potential methods of controlling dry rot of potato tubers. However, the responses to the treatments were noticeably different. MH caused cell death and browning while chitosan treated tuber tissue did not exhibit cell death or browning (Figure 1-1). This may be an indication of difference between the plants response to MH and chitosan. The effectiveness of elicitors to induce resistance has been shown to vary between cultivars (Quintanilla and Brishammar, 1998). Tuber tissue from potato cultivar Atlantic is a more susceptible than Snowden to F. sambucinum infection (Figure 1-2). However, when treated with chitosan the Atlantic tuber tissue 47 becomes more resistant than the Snowden tuber tissue (Figure 1-2). Chitosan is therefore very promising method of controlling F. sambucinum in cultivars that are more susceptible to dry rot. REFERENCES Allan CR. and Hadwiger LA, 1979. The fungicidal effect of chitosan on fungi of varying cell wall composition. Experimental Mycology 32285-287. Asselin A., Grenier J., and Cbté F., 1985. Light-influenced extracellular accumulation of b (pathogenesis-related) proteins in Nicotiana green tissue induced by various chemicals or prolonged floating on water. Canadian Journal Botany 63:1276-1283. Bell A.A., Hubbard J.C., and Liu L., 1998. Effects of chitin and chitosan on the incidence and severity of fusarium yellows of celery. Plant Disease 82:322-328. Benhamou N., Lafontanine P.J., Nicole M., 1994. Induction of systemic resistance to Fusarium crown and root-rot in tomato plants by seed treatment with chitosan. Phytopathology 84:1432-1444. Benhamou N., and Thériault G., 1992. Treatment with chitosan enhances resistance of tomato plants to the crown and root rot pathogen Fusarium oxyspomm f. sp. Radicis-chopersici. Physiological and Molecular Plant Pathology 41:33-52. Bostock R.M., Kuc J., and Laine RA, 1981. Eicosapentaenoic and arachidonic acids from Phytophthora infestans elicit fungitoxic sesquiterpenes in potato. Science 212:67-69. Bostock R.M., Laine RA and Kuc J.A., 1982. Factors affecting the elicitation of sesquiterpenoid phytoalexin accumulation by eicosapentaenoic and arachidonic acids in potato. Plant Physiology 70:1417-1424. Chalova L.I., Avdyushko S.A., Chalenko G.l., Karavaeva K.A., and Ozeretskovskaya O.L., 1989. Arachidonic and eicosapentaenoic acid - long- action systemic immunizers of the potato. Doklady Akademii Nauk SSSR, 305:1262-1265. Chalova L.I., Baramidze V.G., Yurganova L.A., Yu T., Ozeretskovskaya O.L., and Metlitskii L.V., 1977. Isolation and characteristics of inductors of defense 48 responses in potato from cytoplasm of Phytophthora infestans. Doklady Akademii Nauk SSSR, 235:1215-1216. Chalova L.I., Ozeretskovskaya O.L., Yurganova L.A., Baramidze V.G., Protsendo M.A., D’yakov Y.T., and Metlitskii L.V., 1976. Metabolites of phytopathogenic fungi as inductors of defense responses in plants. Doklady Akademii Nauk SSSR 230:722-725. Cheah L.H., Page 8.8.0., and Shepherd R., 1997. Chitosan coating for inhibition of Sclerotinia rot of carrots. New Zealand Journal of Crop and Horticultural Science 25:89-92. Cohen Y., 1993. Local and systemic control of Phytophthora infestans in tomato plants by DL-3-amino-n-butanoic acids. Phytopathology 84:55-59. Cohen Y., 1994. 3-aminobutyric acid induces systemic resistance against Peronospore tabacina. Physiological and Molecular Plant Pathology 44:273-288. Cohen Y., Gisi U., and Mosinger E., 1991. Systemic resistance of potato plants against Phytophthora infestans induced by unsaturated fatty acids. Physiological and Molecular Plant Pathology 38:255-263. Cohen Y., Niderrnan T., Mosinger E., and Fluhr R., 1994. 8-Aminobutyric acid induces the accumulation of pathogenesis related proteins in tomato (Lycopersicon esculentum L.) and resistance to late blight infection by Phytophthora infestans. Plant Physiology 104:59-66. Coquoz J.L., Buchala A.J., and Meuwly P., 1994. Salicylic acid: substance produced upon eliciting potato leaves with arachidonic acid? Acta Horticulturae 381 :379-382. Coquoz J.L., Buchala A.J., Meuwly P.H., Métraux JP, 1995. Arachidonic acid induces local but not systemic synthesis of salicylic acid and confers systemic resistance in potato plants to Phytophthora infestans and Alteman'a solani. Phytopathology 85: 1 21 9-1 224. Desjardins A.E., 1995. Population stmcture of Gibberella pulicaris (anamorph Fusarium sambucinum) from potato tuber dry rot in North America and Europe. American Potato Journal 72:145-156. Desjardins A.E., Christ-Hamed E.A., McCormick S.P., and Secor GA, 1993. Population structure and genetic analysis of field resistance to thiabendazole in Gibberella pulicaris from potato tubers. Phytopathology 83:164-170. 49 Desjardins A.E. and Gardner H.W., 1989. Genetic analysis in Gibberella pulicaris: rishitin tolerance, rishitin metabolism, and virulence of potato tubers. Molecular Plant-Microbe Interactions 2:26-34. Desjardins A.E., Gardner H.W., and Plattner RD, 1989. Detoxification of the potato phytoalexin lubimin by Gibberella pulicaris. Phytochemistry 28:431-437. Doares S.H., Syrovets T., Weller E.W., and Ryan CA, 1995. Oligogalcturonides and chitosan activate plant defensive genes through the octadecanoid pathway. Proceedings of the National Academy of Science USA. 92:4095-4098. Du J.M., Gemma H., and lwahori S., 1997. Effects of chitosan coatings on the storage of peach, Japanese pear, and kiwifmit. Journal of the Japanese Society for Horticultural Science 66:15-22. Dutton M.V., Rigby NM, and MacDougalI A.J., 1997. Induced resistance to Erwinia carotovora ssp. atroseptica, through the treatment of surface wounds of potato tubers with elicitors. Journal of Phytopathology 145:163-169. Eyal Y., Sagee O., and Fluhr R., 1992. Dark-induced accumulation of a basic pathogenesis-related (PR-1) transcript and a light requirement for its induction by ethylene. Plant Molecular Biology 19:589-599. Fajardo J.E., McCoIlum T.G., McDonald RE, and Mayer R.T., 1998. Differential induction of proteins in orange flavedo by biologically based elicitors and challenged by Penicillium digitatum Sacc. Biological Control 13:143-151. Farmer E.E. and Ryan CA, 1992. Octadecanoid precursors of jasmonic acid activate the synthesis of wound-inducible proteinase inhibitors. Plant Cell 4:129- 134. Ghaouth A.E., Arul J., Grenier J., and Asselin A., 1992. Antifungal activity of chitosan on two postharvest pathogens of strawberry fruits. Phytopathology 82:398-402. Hadwiger LA, and Beckman J.M., 1980. Chitosan as a component of pea- Fusan'um solani interactions. Plant Physiology 66:205-211. Hammerschmidt R., 1984. Rapid deposition of lignin in potato tuber tissue as a response to fungi non-pathogenic on potato. Physiological Plant Pathology 24:33-42. Hammerschmidt R. and Kuc J. (eds), 1995. Induced Resistance to Disease in Plants. Kluwer, Dordrecht. 50 Hammerschmidt R. and Schultz J.C., 1996. Multiple defenses and signals in plant defense against pathogens and herbivores. In: Romeo (ed) Phytochemical Diversity and Redundancy in Ecological Interactions. Plenum Press, New York. Hammerschmidt R. and Becker J.S., 1997. Acquired resistance to disease in plants. Horticultural reviews 18:247-289. Hammerschmidt R. and Dam E.K., 1997. Induced resistance to disease. In: Recheigl NA and Rechcigl J.E. (eds.), Environmentally Safe Approaches to Plant Disease Control. CRC Lewis Publishers, Boca Raton. p. 177-199. Hammerschmidt R. and Smith-Becker J.A., 1999. The role of salicylic acid in disease resistance. In: Agrawal A.A, Tuzun S., and Bent E. (eds.), Induced Plant Defenses Against Pathogens and Herbivores. APS Press, St. Paul. p. 1-17. Hengling J.W.D.M., Bostock RM, and Kuc J., 1980. Cell walls of Phytophthora infestans contains an elicitor of terpene accumulation in potato tubers. Phytopathology 70:772-776. ll’inskaya L.I., Perekhod E.A., Chalenko G.l., Gerasimova N.G., Ozeretskovskaya O.L., Piskarev V.E., Borovskaya, A.A., Yamskov IA, and Babakov A.V., 1997. Fucosyl-containing oligosaccharides and potato resistance to Phytophthora infection. Russian Journal of Plant Physiology 44:771-776. Kendra 0F. and Hadwiger LA, 1984. Characterization of the smallest chitosan oligomer that is maximally antifungal to Fusarium solani and elicits pisatin formation in Pisum sativum. Experimental mycology 8:276-281. Kessmann H., Staub T., Hofmann C., Maetzke T., Herzog J., Ward E., Uknes S., and Ryals J., 1994. Induction of systemic acquired disease resistance in plants by chemical. Annual Review of Phytopathology 32:439-459. Kim K.D., Named 3., and Musson G., 1997. Control of Phytophthora root and crown rot of bell pepper with composts and soil amendments in the greenhouse. Applied soil Ecology 5:169-179. Kneer R., Poulev A.A., Olesinski A., and Raskin I., 1999. Characterization of the elicitor-induced biosynthesis and secretion of genistein from roots of Lupinus Iuteus L. Journal of Experimental Botany 50:1553-1559. Kuc J., 1995. Induced systemic resistance - an overview. In: Hammerschmidt R., Kuc J. (eds), Induced Resistance to Disease in Plants. Kluwer, Dordrecht. p. 1 69-1 75. 51 Leach SS. and Webb RE, 1981. Resistance of selected potato cultivars and clones to Fusarium roseum, Fusarium solani dry rot breeding for disease resistance. Phytopathology 71:623-629. Lee M. and Currier W.W., 1996. Suppression of the hypersensitive response in potato by acetyl salicylic acid. Biochemical and Biophysical Research Communications 222:309-31 1. Liang W., Wen J., and Liang H., 1997. Stimulation of ethylene production in aged potato tuber slices by salicylic acid. Phytochemistry 44:221-223. Lotan T., and Fluhr R., 1990. Xylanase, a novel elicitor of pathogenesis-related proteins in tobacco, uses a non-ethylene pathway for induction. Plant Physiology 93:811-817. Lyon G.D., Reglinski T., and Newton AC, 1995. Novel disease control compounds: the potential to ‘immunize’ plants against infection. Plant Pathology 44:407-427. Maniara G., Laine R., and Kuc J., 1984. Oligosaccharides from Phytophthora infestans enhance the elicitation of sesquiterpenoid stress metabolites by arachidonic acid in potato. Physiological Plant Pathology 24:177-186. MIJIler, K.O. and Borger H., 1940. Experimentelle Untersuchungen uber die Phytophtora-Resistanz der Kartoffel. Arbeiten Biologischen Aanst. Reichsanstalt. 23:189. Orozco-Cardenas M. and Ryan CA, 1999. Hydrogen peroxide is generated systemically in plant leaves by wounding and systemin via the octadecanoid pathway. Proceedings of the National Academy of Sciences of the United States of America 96:6553-6557. Ozeretskovskaya O. L. 1995. Induced resistance in the Solanaceae. In: Hammerschmidt R., Kuc J. (eds), Induced Resistance to Disease in Plants. Kluwer, Dordrecht. p. 31-62. Papavizas 6.0., 1964. Greenhouse control of Aphanomyces root rot of peas with aminobutyric acid and methylaspartic acid. Plant Disease Reporter 48:537- 541. Peltonen S., Mannonen L., and Karjalainen R., 1997. Elicitor-induced changes of phenylalanine ammonia-lyase activity in barley cell suspension cultures. Plant Cell Tissue and Organ Culture 55:185-193. Pospieszny H., Chirkov s., and Atabekov J., 1991. Induction of antiviral resistance in plants by chitosan. Plant Science 79:63-68. 52 Powelson M.L., Johnson KB, and Rowe RC, 1993. Managementof diseases caused by soilbome pathogens. In: Rowe, R.C. (ed). Potato Health Management. American Phytopathological Society Press: St. Paul, MN. Preisig CL and Kuc J.A., 1985. Arachidonic acid-related elicitors of the hypersensitive response in potato and enhancement of their activities by glucans from Phytophthora infestans (Mont) deBary. Archives of Biochemistry and Biophysics 236:379-389. Quintanilla P. and Brishammar s., 1998. Systemic induced resistance to late blight in potato by treatment with salicylic acid and Phytophthora crytogea. Potato Research 41:135-142. Reddy M.V.B., Arul J., Angers P., and Couture L., 1999. Chitosan treatment of wheat seeds induces resistance to Fusarium graminearum and improves seed quality. Journal of Agricultural and Food Chemistry 47:1208-1216. Reddy M.V.B., Belkacemi K., Corcuff R., Castaigne F ., and Arul J., 2000. Effect of pre-harvest chitosan sprays on post-harvest infection by Botrytis cinerea and quality of strawberry fruit. Postharvest Biology and Technology 20:39-51. Stange RR. and McDonald R.E., 1999. A simple and rapid method for determination of lignin in plant tissues - its usefulness in elicitor screening and comparison to the thioglycolic acid method. Postharvest Biology and Technology 1 5:185—193. Ryals J.A., Neuenschwander U.H., Willits M.G., Molina A., Steiner H., and Hunt MD, 1996. Systemic acquired resistance. The Plant Cell 8:1809-1819. Sticher L., Mauch-Mani B., and Métraux JP, 1997. Systemic acquired resistance. Annual Reviews of Phytopathology 35:235-270. Stbssel P., and Leuba J.L., 1984. Effect of chitosan, chitin, and some aminosugars on growth of various soilbome phytopathogenic fungi. Phytopathology Zeitschrift 1 11:82-90. Sunwoo, J.Y., Lee Y.K., and Hwang B.K., 1996. Induced resistance against Phytophthora capsici in pepper plants in response to DL-8-amino-n-butyric acid. European Journal of Plant Pathology 102:663-670. Tally A., Oostendrop M., Lawton K., Staub T., and Bassi B., 1999. Commercial development of elicitors of induced resistance to pathogens. In: Agrawal A.A., Tuzun S., and Bent E. (eds), Induced Plant Defenses Against Pathogens and Herbivores. APS Press, St. Paul. p. 357-369. 53 Tuzun S. and Kuc J., 1991. Plant immunization: An alternative to pesticides for control of plant diseases in the greenhouse and field. In: Bay-Petersen J. (ed.), Biological Control of Plant Diseases, FFTC Book Series No. 42, Taiwan, p. 30- 40. Van Andel OM, 1966. Amino acids and plant diseases. Annual Review of Phytopathology 4:349-368. Vasyukova N.l., Chalenko G.l., Gerasimova N.G., Perekhod E.A., Ozeretskovskaya O.L., ll’ina A.V., Varlamov VP, and Albulov AL, 2000. Chitin and chitosan derivatives as elicitors of potato resistance to late blight. Applied Biochemistry and Microbiology 36:372-376. Wilson C.L., Ghoauth A.E., Chalutz E., Droby 8., Stevens 0., Lu J.Y., Khan V., and Arul J., 1994. Potential of induced resistance to control postharvest diseases of fruits and vegetables. Plant Disease 78:837-844. Yu 0., Liu Y., Fan 8., Klessig BF, and Chen Z., 1997. Is the high basal level of salicylic acid important for disease resistance in potato? Plant Physiology 1 15:343-349. Yurganova L.A., Nogaideli D.E., Chalenko G.l., Chalova LL, and Ozeretskovskaya O.L., 1989. Inducing activity of Fusarium culmorum Sacc. Prikladnaya Biokhimiya i Mikrobiologiya 25:805-810. 54 CHAPTER 2 The Effect of Chitosan and Phytophthora infestans Mycelium Homogenate on the Biochemistry of Potato Tuber Tissue 55 INTRODUCTION The criterion used to establish if a material is a “resistance inducer" states that the material must not be antimicrobial or be converted by the plant to an antimicrobial compound. In addition, it must be able to activate plant defense mechanisms such as pathogenesis-related proteins, which are often used as a marker for the induction of resistance, as well as oxidative enzymes, and antimicrobial secondary metabolites (Hammerschmidt and Dann, 1997). These changes must alter the ability of the pathogen to infect and colonize the plant. The goal of this study was to determine if chitosan and Phytophthora infestans mycelium homogenate (MH) are “resistance inducers” and to determine which plant defense mechanisms are involved in the resistance response induced by these two treatments. The antifungal activity of chitosan against F. sambucinum and its potential role in reducing infection were evaluated. Induction of chitinase, 8-1,3-glucanse, peroxidase, and polyphenoloxidase were examined as well as the effect chitosan and MH treatments had on steroid glycoalkaloid accumulation. MATERIALS AND METHODS Assessment of Chitosan’s Antifungal Activity Water agar containing 0, 0.5, and 1.0 mglml chitosan was prepared in 9 cm polystyrene Petri dishes as previously described a mycelium plug (5.0 mm) of F. sambucinum taken from the outer edge of growth was placed in the center of 56 each plate and incubated in the dark at 23°C. Six plates were used for each chitosan concentration. The diameter of the radial growth of F. sambucinum was measured at 2, 4, 6, 8, 10, and 30 days. Spore Germination Tuber discs were prepared and treated as described in chapter 1. Seventy- two hours after treatment with 1.0 mglml chitosan or water, the tuber discs were inoculated with a 200 pl suspension of Fusarium sambucinum spores at a concentration of 1X10‘. The discs were sliced longitudinally into 2-4 cell layer sections 1, 3, 5, 8, and 12 hours after inoculation using a Hooker microtome. The sections were immersed in a solution of alcoholic lactophenol cotton-blue and heated until boiling. The sections were left in the alcoholic lactophenol blue solution for 48 hours. The sections were then washed with water and destained in a saturated solution of chloral hydrate (Keeling and Banttari, 1975). The sections were mounted in 50% glycerol and examined using light microscopy. Potato Tuber Tissue Preparation To study the local response of potato tuber tissue to chitosan and P. infestans mycelium homogenate (MH) treatment, potato tuber slices were prepared and treatments were applied as described in chapter 1. To study the systemic response of tuber tissue to chitosan and MH treatment, potatoes were prepared as described in chapter 1. Three centimeter long cylinders of potato tuber tissue were made from the center of tubers using a 2 cm 57 cork borer. The cylinders were rinsed with sterile water and stood on end in sterile petri dishes. The top surface of the cylinders were treated with 0.5 ml of 0.5 and 1.0 mg/ml chitosan; 0.25, 0.5, and 1.0 mglml MH; or water. After 2 or 3 days the cylinders were sliced into six 0.5 cm slices and changes in the potato tissue was studied for each 0.5 cm depth. 20 mm Diameter 0.5 ml of Treatment Applied Layer 1 ——> Layer 2 ——> 5 mm Layers Figure 2-1: Diagram of tuber cylinders used for depth studies. The surface of the cylinder was treated with chitosan or MH. Two to three days after treatment the cylinders were sliced into 0.5 cm layers. Each layer was evaluated for increased resistance to F. sambucinum and biochemical changes. Determination of Systemic Protection Against Fusarium sambucinum Each 0.5 cm slice prepared from the tuber cylinders were inoculated with F. sambucinum and disease progression was measured following the protocol described in chapter 1. 58 Protein Extraction and Quantification The upper 0.5 mm of tissue from five potato slices was removed using a vegetable peeler. The tissue was quartered and ground with a pestle and mortar in 3.0 ml of 0.2 M borate buffer (pH 8.8) with glass beads and polyvinylpyrrolidone to bind phenolic compounds. The ground tissue was filtered through a double layer of Miracloth. The filtered extract was centrifuged at 10,000 rpm for 10 minutes at 4°C. The supernatant was frozen until needed. Protein concentrations of the extracts were determined using the Bio-Rad Protein Assay, purchased from Bio-Rad Hercules, California. The Bio-Rad standard assay procedure was used with bovine semm albumin as the protein standard. Native Polyacrylamlde Gel Electrophoresls (PAGE) Polyacrylamide separating gels (7.5%, 1.5mm) were prepared by mixing 4.85 ml deionized water, 2.5 ml 1.5 M Tris-HCI (pH 8.8), 2.5 ml 30% acrlyamidelbis (29:1 ), 50 pl 10% ammonium persulfate. The solution was degassed for 10 minutes, then 5 pl of TEMED was added and the gel was poured. The stacking gel (4.0 %) was prepared by mixing 6.1 ml of deionized water, 0.5 M Tris-HCI (pH 6.8), 1.33 ml 30% acrylamide/bis, and 50 pl of 10% ammonium persulfate. The solution was degassed for 10 minutes, then 10 pl of TEMED was added and the gel was poured. The upper tank buffer was prepared by mixing 5.16 g Tris base, 3.48 g glycine and 1 liter of water. The lower tank buffer was prepared by mixing 14.5 g Tris base, 60 ml of 1 N HCI. and diluting to 1 liter with water. A Mini- 59 Protean II or III electrophoresis system from Bio-Rad was used to run the gels. The gels were run at 50 V for approximately 4 hours. Sample Preparation for PAGE Protein extracts were mixed at a 1:1 (v/v) ratio with sample buffer. The sample buffer consisted of 5.4 ml of deionized water, 1.0 ml 0.5 M Tris-HCl (pH 6.8), 0.8 ml glycerol, and 0.8 ml 0.05% (wlv) bromophenol blue. An equal concentration of protein was loaded for each sample based on the Bio-Rad protein assay. PAGE Assay for Peroxidase Activity PAGE was performed as described above. Peroxidase stain was prepared by dissolving 50 mg of 3-amino-9-ethyl carbazole (AEC) in 2 ml dimethyl foramide. The AEC solution was slowly added to 200 ml 0.2 M sodium acetate buffer (pH 5.0) then 200 pl of 30% hydrogen peroxide was added. The gels were incubated at room temperature in the peroxidase stain until bands developed (Graham et al, 1965). The gels were then rinsed in water and photographed. Spectrophotometric Assay for Peroxidase Peroxidase activity was determined spectrophotometrically using 0.25% guaiacol and 0.3 % hydrogen peroxide (30%) in 0.1 M sodium phosphate buffer (pH 6) as a substrate. Crude protein extract was added to 1 ml of substrate solution and the absorbance was taken every 30 seconds for 4 minutes at 470 60 nm using a Cary 50 Bio UV-visible spectrophotometer. The slope of the absorbance verses time was used as a relative measure of peroxidase activity. Activity was based on protein concentration of the crude protein extract and three replicates were done for each sample and averaged. PAGE Assay for Polyphenoloxidase Activity PAGE was preformed as described above. Gels were then stained in a solution of L-8-3,4 dihydroxyphenylalanine (L-Dopa) for 1 hour or until brown bands appeared. The L-Dopa solution contained 4 mg of L-Dopa per milliliter of 0.1 M phosphate buffer pH 6.0 (Gomes and Ledward, 1996). PAGE Assay for Chitinase Activity PAGE was preformed as described above except 100 pl of a 1% stock solution of glycol chitin was added to the separating gel. Glycol chitin was obtained by acetylation of glycol chitosan (T rudel and Asselin, 1989). Gels were then incubated at 37°C in 1 M sodium acetate buffer (pH 5.0) for 2 hours. Following incubation gels were stained in a solution of 0.02 g fluorescent brightener 28 in 200 ml of 500mM Tris-HCI (pH 8.9) at room temperature for 5 minutes. The gels were removed from the stain and incubated in distilled water over night to destain the gel. Clear zones signifying chitinase activity were visualized under UV light. Activity could be compared by the size of the lytic zone. 61 Extraction and Quantification of Steroid Glycoalkalolds in Potato Tissue Steroid glycoalkaloids were extracted according to Allen and Kuc (1968). Steroid glycoalkaloids were extracted from the upper 0.5 mm of 20 tuber slices for each treatment. The tissue was removed using a vegetable peeler. quartered and homogenized for 30 seconds in 100 ml of 10 chloroform: 9 methanol: 1 acetic acid (CAM). The homogenate was stored at room temperature for 24 hours then were filtered through Whatman #1 filter paper. The filtrate was concentrated to near dryness using a rotary-evaporator. 15 ml of 2% acetic acid was added to the concentrated filtrate and it was centrifuged at 12,000 rpm for 10 minutes to remove any insoluble material. The supernatant was washed with chloroform two times by adding 10 ml of chloroform, vortexing, centrifuging at 12,000 for 10 minutes, and discarding the chloroform layer. After washing the aqueous layer was adjusted to pH 10 using ammonium hydroxide. The solution was then heated at 80° C for 30 minutes, cooled to room temperature, and stored overnight at 4° C. The solution was centrifuged at 12,000 rpm for 10 minutes and the supernatant was poured off and the pellet was dried in a desiccator over NaOH pellets for 2-3 days. The pellet was resuspended in 3 ml of 5% acetic acid by sonication. Concentration of SGA was determined spectrophotometrically (Cadle et al,1978). One-half ml of SGA extract was mixed with 1.5 ml of 85% phosphoric acid by vortexing. One ml of p-formaldehyde reagent (0.2 grams of p- formaldehyde dissolved in 15 ml of deionized water was combined with 85 ml of 85% phosphoric acid to make p-formaldehyde reagent) was added by vortexing 62 and the mixture was incubated at 60° C for 10 minutes. The mixture was allowed to cool to room temperature and absorbency was read at 600 nm using a Cary 50 Bio Liv-visible spectrophotometer. Each sample was done in triplicate and the average absorbency was calculated. The concentration of SGA was determined using a standard curve produced using a-solanine as a SGA standard. Spectrophotometric Assay for 8-1,3-glucanase Activity B-1,3-glucanase activity was assayed using AZCL-pachyman (obtained from Megazyme) as a substrate. AZCL-pachyman substrate was prepared by adding 0.1 g of AZCL-pachyman to 6 ml water. To assay B-1,3-glucanase activity, 0.3 ml of 10 mM potassium acetate buffer (pH 5.0) was added to 0.1 ml of crude protein extract, spun briefly to mix, and equilibrated in 30° C water bath for 3-4 minutes. Substrate (0.2 ml) was added and allowed to incubate 10 minutes after which 0.7 ml of 20% (wlv) Tris was added and the solution was vortexed to stop the reaction. The mixture was keep at room temperature for 3-4 minutes then spun at 12,400 rpm for 2 minutes to precipitate unreacted substrate. Absorbency of supernatant was determined at 595 nm using a Cary 50 Bio UV-visible spectrophotometer. Each assay was done in triplicate and the average absorbency was used as a relative measure of B-1,3—glucanase activity. 63 Statistical Analysis Data was analyzed for significance by one-way analysis of variance, followed by Tukey’s significant difference test (a=0.05), using GraphPad lnStat software. RESULTS Antifungal Activity of Chitosan At a concentration of 0.5 mglml, chitosan significantly reduced radial growth of F. sambucinum after 4 days growth (Figure 2-2). Mycelial growth was also more sparse than in water controls (data not shown). At a concentration of 1.0 mglml, chitosan completely inhibited F. sambucinum growth (Figure 2-2). Spore Germination The germination of F. sambucinum spores on the surface of chitosan treated tuber discs and water treated tuber discs were compared. Spore germination was observed 3 hours after the tubers were inoculated with the spore suspension. There was no visible difference in the spore germination on the water treated disks verses chitosan treated discs. 64 9.0 ~ 8.0 - 7.0 - 6.0 ~ 5.0 ~ 4.0 4 3.0 - 2.0 ~ 1.0 — i 0.0 - l Radius of Growth (cm) slit—20 z sfiea p sfieo g sliea 9 sfieo 0L sfieo 09 0 Control +0.5 mglml Chitosan +1.0 mglml Chitosan Figure 2-2: Time course of the radial growth of F. sambucinum grown in petri plates containing water agar and water agar supplemented with 0.5 or 1.0 mglml chitosan. Local Effect of Chitosan and P. infestans Mycelium Homogenate Treatment of Tuber Tissue on the Activity of PR-Proteins Chitinase activity increased in tuber tissue after P. infestans mycelium homogenate (MH) treatment at 0.25, 0.5 and 1.0 mglml and chitosan treatment at 0.5 and 1.0 mglml (Figure 2-3 and 2-4). There was a marked increase in chitinase activity as the concentration of MH increased (Figure 2-3), however, there was no discernible difference in chitinase activity as the concentration of chitosan was increased (Figure 24). MH treatment induced a greater increase in chitinase activity than chitosan at all concentrations and time points tested (Figure 2-5). Chitinase activity occurred lower in the gel as activity increased (Figure 2-3). This appears to be due to the concentration of chitinase and is not due to induction of a different chitinase isozyme. 65 | = Initial C = Control T1 = 0.25 mg/ml MH T2 = 0.5 mg/ml MH T3 = 1.0 mg/ml MH INC T1 T2 T3 C T1 T2 T3 -———»_ (T‘ . ,;-.§ Tiff: 5 it!“ ‘ - ,— '1?‘ .' 2 Days 3 Days Figure 2-3: Chitinase activity in tuber tissue 2 and 3 days after treatment with water (control) and Phytophthora infestans mycelium homogenate (MH). initial indicates chitinase levels at time of treatment (0 days). TZCT1 l = Initial C = Control T1 = 0.5 mg/ml Chitosan T2 = 1.0 mglml Chitosan 2 Days 3 Days Figure 2-4: Chitinase activity in tuber tissue 2 and 3 days after treatment with water (control) and chitosan. Initial indicates chitinase levels at time of treatment (0 days). Further examination of the affect of chitosan treatment revealed that chitinase activity increased in response to chitosan within 2 hours of treatment and continued to increase through at least 12 hours and was still elevated at 72 hours after treatment (Figure 2—4 and 2-6). 66 I = Initial C = Control T1 = 0.25 mglml MH T2 = 0.5 mglml MH T3 = 1.0 mglml MH T4 = 0.5 mglml Chitosan T5 = 1.0 mglml Chitosan Figure 2-5: Comparison of chitinase activity in tuber tissue treated with water (control), MH, and chitosan 2 days (A) and 3 days (B) after treatment. Initial indicates chitinase levels at time of treatment (0 days). .' ,. .s , .3.” a: 9 w M * m ;.,~».i§ «.-..-2 ; ' 4 . 2 Hours 4 Hours 6 Hours . 8 Hours 10 Hours 12 Hours ' I = Initial C = Control T1 = 1.0 mglml Chitosan Figure 2-6: Time course of chitinase activity in tuber tissue treated with water (control) and 1.0 mglml chitosan. Initial indicates chitinase levels at time of treatment (0 days). Glucanase activity increased in tuber tissue treated with 0.25, 0.5, and 1.0 mglml MH and 0.5 and 1.0 mg/ml chitosan (Figure 2-7). Increases in glucanase activity were observed 2 and 3 days after treatment with MH. Three day levels were slightly higher than 2 day levels (Figure 2-7). Chitosan treatments did not 67 significantly increase glucanase activity until 3 days after treatment (Figure 2-7). MH treatments increased glucanase activity significantly more than chitosan at 2 and 3 days after treatment (Figure 2-7). El Initial I Control I0.25 mg/ml MH [11110.5 mg/ml MH [$11.0 mg/ml MH I0.5 mglml Chitosan $1.0 mglml Chitosan Absorbance \. § § §l a. §I 0) 2 Days Days Figure 2-7: Glucanase activity in tuber tissue 2 and 3 days after treatment with water (control), MH, and chitosan. Initial indicates glucanase levels at time of treatment (0 days). The letters indicate significant differences (P< 0.05) between treatments. Local Effect of Chitosan and P. infestans Mycelium Homogenate Treatment of Tuber Tissue on the Activity of Oxidative Enzymes Peroxidase activity was increased by treatment with 0.25, 0.5, and 1.0 mglml MH and 0.5 and 1.0 mglml chitosan (Figure 2-8 and 2-9). Increased peroxidase activity was observed 2 and 3 days after treatment with MH (Figure 2-8 and 2-9). Chitosan treatment induced peroxidase activity within 12 hours of treatment and peroxidase activity continued to increase for at least 7 days after treatment (Figure 2-10 and 2-11). 68 There was no detectable difference in induction of peroxidase activity as the concentration of MH increased (Figure 2-8 and 2-9). At two days after treatment there was no detectible difference in the level of peroxidase activity in tissue treated with 0.5 mglml and 1.0 mglml chitosan (Figure 2-8 and 2-12). At three days after treatment, spectrophotometric analysis of total peroxidase activity indicated there was slightly more peroxidase activity in tissue treated with 0.5 mglml chitosan than 1.0 mg/ml chitosan (Figure 2-12). However, analysis of peroxidase activity by gel electrophoresis did not show a difference between the treatments (Figure 2-9). MH induced higher levels of peroxidase activity than chitosan, and MH and chitosan induced different isozymes of peroxidase (Figure 2-8 and 2-9). Chitosan induced an isozyme pattern similar to that induced by wounding (Figure 2-8, 2-9, 2-10, and 2-11). MH induced some isozymes that were not induced by wounding and inhibited induction of other isozymes normally induced by wounding (Figure 2-8 and 2-9). I CT1 T2 T3 T4 T5 I = Initial C = Control T1= 0.25 mg/ml MH T2 = 0.5 mg/ml MH T3 = 1.0 mglml MH T4 = 0.5 mg/ml Chitosan T5 = 1.0 mg/ml Chitosan Figure 2-8: Peroxidase activity in tuber tissue 2 days after treatment with water (control), MH, and chitosan. Initial indicates peroxidase levels at time of treatment (0 days). 69 | C T1 T2 T3 T4 T5 I = Initial C = Control T1= 0.25 mglml MH T2 = 0.5 mglml MH T3 = 1.0 mglml MH T4 = 0.5 mglml Chitosan T5 = 1.0 mglml Chitosan Figure 2-9: Peroxidase activity in tuber tissue 3 days after treatment with water (control), MH, and chitosan. Initial indicates peroxidase levels at time of treatment (0 days). CTC Hours After Treatment l = Initial C = Control T = 1.0 mglml Chitosan Figure 2-10: Time course of peroxidase activity in tuber tissue treated with water (control) and chitosan. Initial indicates peroxidase levels at time of treatment (0 days). 70 |CT1T2CT1T2 ”1334.. '§); yc‘”M I. C T1 T2 C T1T2 9.1117 C T1 T2 C T1 T2 .T'. 0 1 Day 2 Days 3 Days 4 Days 5 Days 6 Days 7 Days I = Initial C = Control T1 = 0.5 mglml Chitosan T2 = 1.0 mglml Chitosan Figure 2-11: Seven-day time course of peroxidase activity in tuber tissue after water (control) and chitosan treatment. Initial indicates peroxidase levels at time of treatment (0 days). 0.14 - 0.12 ~ 8 b C to 0.1 A b E g 0.08 _ EIControl E l 0.5 mglml Chitosan g9) 0'06 ‘ [11111.0 mg/mlChitosan § 0.04 — O 0.02 a 0 Initial (0.0013) 3 Days Figure 2-12: Spectrophotometric analysis of peroxidase activity in tuber tissue after water (control) and chitosan treatment. Initial indicates peroxidase levels at time of treatment (0 days). The letters indicate significant differences (P< 0.05) between treatments. 71 Polyphenoloxidase activity increased after treatment with 0.5 and 1.0 mglml chitosan (Figure 2-13). Increased activity was observed 2 and 3 days after treatment (Figure 2-13). T1 T2 I = Initial (0 Days) ‘ C = Control T1 = 0.5 mg/ml Chitosan T2 = 1.0 mglml Chitosan 2 Days 3 Days Figure 2-13: Polyphenoloxidase activity in tuber tissue 2 and 3 days after with treatment with water (control) and chitosan. Initial indicates polyphenoloxidase levels at time of treatment (0 days). Local Effect of Chitosan and P. infestans Mycelium Homogenate Treatment of Tuber Tissue on Steroid Glycoalkaloid Levels Wound induced steroid glycoalkaloid (SGA) accumulation was suppressed in response to treatment with MH at concentrations of 0.25, 0.5 and 1.0 mg/ml and by treatment with chitosan at concentrations of 0.5 and 1.0 mg/ml (Figure 2-14). When tuber discs are prepared wounding occurs causing an increase in SGAs over the initial level (Figure 2-14). However, this increase was suppressed by MH and chitosan treatment (Figure 2-14). Levels were still above initial SGA levels, however significantly less than controls (Figure 2-14). Decreases were observed at 2 and 3 days after treatment and were greater at 3 days (Figure 2- 72 14). MH reduced SGA accumulation more than treatment with chitosan at all tested concentrations and both time points (Figure 2-14). 250 200 - i a d DControl :5 150 C c [1110.25 mglml MH g c 130.5 mglml MH @1004 d I1.0mg/ml MH .1: I05 mglml Chitosan 31.0 mglml Chitosan =- 50 . ‘1" :-:-.:: _--..- ;. - -Initial (40.7) 0 . 2 Days 3 Days Figure 2-14: Steroid glycoalkaloid levels in tuber tissue 2 and 3 days after water (control), MH, and chitosan treatment. Initial indicates steroid glycoalkaloid levels at time of treatment (0 days). The letters indicate significant differences (P< 0.05) between treatments. Systemic Effect of Chitosan and P. infestans Mycelium Homogenate Treatment of Tuber Tissue on F. sambucinum Infection Tuber tissue treated with 1.0 mglml chitosan and challenged three days after treatment with F. sambucinum had a significant decrease in the depth of infection by F. sambucinum 5 days after challenge in layer 2, 0.5 cm from the treated surface, and layer 3, 1.0 cm from the treated surface (Figure 2-15). MH at 0.25, 0.5 and 1.0 mglml and chitosan at 0.5 mglml did not reduce infection systemically (Figure 2-15). 73 3.5 a A 3 0 a a a a E ' 1 a a a a E b a V 2.5 ' a a c b 3% 2.0 7 BControl 3:3 I05 mglml MH “3 1.5 2 11111.0 mglml Chitosan 5 1.0 O. 0 D 0.5 - 0.0 -J I'— I— I" I— I— 3 2‘2 2‘2 2 53 ‘3 2 9: Q 2 N w A U! 05 Figure 2-15: Depth of infection by F. sambucinum in layers of tuber tissue taken in 0.5 centimeter increments from the treated surface of tuber cylinders. Cylinders of tuber tissue were prepared and the surface was immediately treated with water (control), MH, and chitosan. Tissue layers were made and challenged with F. sambucinum 3 days after treatment. Depth of infection was measured 5 days after challenge. The letters represent significant differences (P<0.05) between treatments. Systemic Effect of Chitosan and P. infestans Mycelium Homogenate Treatment of Tuber Tissue on the Activity of PR-Proteins Chitinase activity was increased at least 2.5 cm from the treated surface, when treated with 0.25, 0.5, and 1.0 mg/ml MH (Figure 2-16) and at least 3.5 cm from the treated surface 3 days after treatment with 0.5 and 1.0 mglml chitosan (Figure 2-16 and 2-17). Increases in activity decreased as distance from the treated surface increased (Figure 2-16 and 2-17). Increases were higher at all depths in tissue treated with MH and were greater at higher concentrations of MH (Figure 2-16). Glucanase activity significantly increased 1.0 cm from the treated surface 3 days after treatment with 0.25, 0.5 and 1.0 mg/ml MH (Figure 2-18). MH 74 treatment increased glucanase 1.5 - 2.0 cm, however, the increases were not always significantly different than the control (Figure 2-18). Chitosan treatment at 0.5 and 1.0 mglml did not cause a significant systemic increase in glucanase activity (Figure 2-18). C = Control T1 = 0.25 mglml MH T2 = 0.5 mg/ml MH T3 = 1.0 mglml MH T4 = 0.5 mglml Chitosan T5 = 1.0 mglml Chitosan Figure 2-16: Chitinase activity in layers of tuber tissue taken in 0.5 centimeter increments from the treated surface of tuber cylinders. Chitinase activity was assessed 3 days after treatment of the surface of the tuber tissue cylinders with water (control), MH, or chitosan. 75 U“ 2.; Laver1 .. . , Laver2 : . _'~-‘-$‘ 5;: .“ T7975“: Layer 3 Laver4 Layer 5 Laver6 5*” “ ' 2.4.5 Layer 7 Laver8 I = Initial C = Control T1 = 0.5 mglml Chitosan T2 = 1.0 mglml Chitosan Figure 2-17: Chitinase activity in layers of tuber tissue taken in 0.5 centimeter increments from the treated surface of tuber cylinders. Chitinase activity was assessed 3 days after treatment of the surface of the tuber tissue cylinders with water (control) or chitosan. 76 Absorbance C' s § \ x x \ § § Layer 3 Layer 4 Layer 5 Layer 6 ElControl I0.25 mglml MH I0.5 mglml MH [11111.0 mglml MH I0.5 mg/ml Chitosan I1.0 mglml Chitosan Figure 2-18: Glucanase activity in layers of tuber tissue taken in 0.5 centimeter increments from the treated surface of tuber cylinders. Glucanase activity was assessed 3 days after treatment of the surface of the tuber tissue cylinders with water (control), MH, or chitosan. The letters represent significant differences (P< 0.05) between treatments. Systemic Effect of Chitosan and P. infestans Mycelium Homogenate Treatment of Tuber Tissue on the Activity of Oxidative Enzymes After 3 days peroxidase activity increased 2.5 cm from the treated surface, when treated with 0.25, 0.5 and 1.0 mg/ml MH and 0.5 and 1.0 mg/ml chitosan (Figure 2-19). However, increases were not consistent and were not always significantly different (Figure 2-19). Polyphenoloxidase activity increased 0.5 cm below the tissue surface 3 days after treatment with 0.5 and 1.0 mg/ml chitosan (Figure 2-20). 77 0.35 Change in Absorbancy Layer 2 Layer 3 Layer 4 Layer 5 Layer 6 121 Control I 0.25 mglml MH 0.5 mg/ml MH 11111.0 mg/ml MH 0.5 mg/mlChitosan1.0 mglml Chitosan Figure 2-19: Peroxidase activity in layers of tuber tissue taken in 0.5 centimeter increments from the treated surface of tuber cylinders. Peroxidase activity was assessed 3 days after treatment of the surface of the tuber tissue cylinders with water (control), MH, or chitosan. The letters represent significant differences (P< 0.05) between treatments. 78 .9. .I‘... IE... Layer 2 C = Control T1 = 0.5 mg/ml Chitosan T2 = 1.0 mglml Chitosan Layer 3 Layer 4 Layer 5 Layer 6 Figure 2-20: Polyphenoloxidase activity in layers of tuber tissue taken in 0.5 centimeter increments from the treated surface of tuber cylinders. Polyphenoloxidase activity was assessed 3 days after treatment of the surface of the tuber tissue cylinders with water (control) or chitosan. 79 Systemic Effect of Chitosan and P. infestans Mycelium Homogenate Treatment of Tuber Tissue on Steroid Glycoalkaloid Levels Steroid glycoalkaloids (SGA) did not significantly decrease below the treated surface of the tuber tissue 3 days after treatment with 0.5 and 1.0 mglml chitosan (Figure 2-21). 100 ~vv. 7.77.7.7 ,, W-,- micrograms/gram fresh weight 01 O Layer 2 Layer 3 Layer 4 Layer 5 Layer 6 Layer 7 Layer 8 El Initial I Control [1]] 0.5 mglml Chitosan 11511.0 mglml Chitosan Figure 2-21: Glycoalkaloid levels in layers of tuber tissue taken in 0.5 centimeter increments from the treated surface of tuber cylinders. Glycoalkaloid levels were assessed 3 days after treatment of the surface of the tuber tissue cylinders with water (control) or chitosan. DISCUSSION Chitosan has been reported to inhibit growth of a number of fungal pathogens including various Fusarium species (Allen and Hadwiger, 1979; Stdssel and Leuba, 1984). This research has shown that chitosan also Inhibited growth of F. 80 sambucinum in vitro at concentrations of 0.5 and 1.0 mglml (Figure 2-2). It is unlikely that the antifungal activity of chitosan accounts for the increased resistance to F. sambucinum observed in potato tuber tissue treated with chitosan. Increases in resistance were not observed until 2 days after chitosan treatment (Table 1-10), suggesting resistance is not caused by the direct affect of chitosan, but more likely an induced plant response. Additionally, F. sambucinum spore germination on tuber tissue was not affected by chitosan treatment supporting a role of plant defense mechanisms in F. sambucinum resistance. However, these results do not eliminate any role for the antifungal activity of chitosan in the increased resistance observed after chitosan treatment. Chitosan and the P. infestans mycelium homogenate (MH) treatment locally activate a number of puntative defense mechanisms including the pathogenesis- related proteins chitinase and B-1,3—glucanase and the oxidative enzymes peroxidase and polyphenoloxidase (Figure 2-3, 2-4, 2-7, 2-8, 2-9, 2-12, and 2- 13). Additionally, steroid glycoalkaloid levels were suppressed in treated tissue (Figure 2-14). Chitinase and B-1,3—glucanase are induced in potato upon pathogen attack suggesting they may have a role in defense against pathogens (Kombrink et al, 1988; Godoy et al, 1996). The cell walls of the hyphae of Fusarium species are typically composed of [3-1,3-glucan and chitin (Gooday, 1994). B-1,3—glucanase and chitinase may therefore directly inhibit growth and infection by F. sambucinum by breaking down the cell walls of F. sambucinum hyphae. It has also been suggested that 0-1 ,3—glucanase and chitinase may release elicitors 81 from fungal cell walls that can induce plant defense reactions (Mauch and Staehelin, 1989; Bowles, 1990; Boller, 1995). Chitinase activity increased within two hours (Figure 2-6) of treatment. However, resistance was not observed until 2 days after treatment. This suggests that if chitinase has a role in defense against F. sambucinum it may not be a direct role. Additionally, higher levels of chitinase activity occurred when the tuber tissue was treated with MH verses chitosan (Figure 2-5). However, MH conferred less resistance to F. sambucinum than chitosan. The size of chitosan oligomers has been shown to affect the ability of chitosan to induce defense responses (Kendra and Hadwiger, 1984; Doares et al, 1995). Potentially, the size of chitin fragments produced by chitinase may also affect induction of plant defenses. MH, which induces a higher level of chitinase, may produce smaller chitin fragments that might be less effective at inducing plant defense mechanisms, thus explaining why chitosan treatments are more effective at controlling F. sambucinum than MH. 1 Peroxidase has previously been reported to increase in potato tuber tissue in response to F. sambucinum infection suggesting it may have a role in potato tuber defense against F. sambucinum (Zeng, 1993). A specific role of peroxidase in plant defense against pathogens has not been demonstrated (Chittoor et al, 1999). However, peroxidase activity has been correlated with a number of puntative plant defense mechanisms including Iignification and suberization of cell walls, cross-linking of hydroxyproline-rich glycoproteins in cell walls, and production of toxic phenolic free radicals (Chittoor et al, 1999). In potato tuber tissue, peroxidase has been associated with suberization (Espelie et 82 al, 1986; Espelie and Kolattukudy, 1985). The formation of suberin and wound periderm has been reported to be an important defense mechanism of potato tuber tissue against F. sambucinum (O’Brien and Leach, 1983). Treatment with arachidonic acid, a component of the MH has previously been reported to stimulate peroxidase activity and increase accumulation of lignin (Bostock and Schaeffer, 1986). Induction of resistance caused by MH and chitosan may in part be caused by increased suberization and wound healing caused by increased peroxidase activity. It is also possible that increases in peroxidase activity caused by MH and chitosan treatments may produce toxic levels of phenolic free radicals inhibiting F. sambucinum growth. Peroxidase activity increased within 12 hours of chitosan treatment and continued to increase for at least 7 days (Figure 2-10 and 2-11). The timing and duration of increased peroxidase activity would allow for accumulation of phenolic free radicals as well as changes in the structure of the cell wall before induction of resistance was observed two days after treatment (Hammerschmidt, 1984). Similar to the PR-protein response, peroxidase activity also increased more in MH treated tissue than in tissue treated with chitosan (Figure 2-8 and 2-9). However, the chitosan treated tissue was more resistant to F. sambucinum infection. This indicates that other defense responses are at least partially responsible for the increase in resistance. However, higher levels of peroxidase may not always lead to increased resistance because the substrate of peroxidase may be the limiting factor in peroxidase induced defense responses and not the peroxidase enzyme. On the other hand, chitosan appears to 83 enhance the wound response while MH may induce isozymes similar to those induced during the HR (Figure 2-8 and 2-9). Therefore, peroxidase isozymes induced by MH may have a greater role in plant defense against F. sambucinum than those induced by chitosan. The oxidative enzyme polyphenoloxidase increases upon infection by pathogens and therefore has been implicated in host defense, however, the role of polyphenoloxidase in defense in unclear (Thygesen et al, 1994; Mayer and Harel, 1979). Polyphenoloxidase catalyzes the conversions of phenols into quinones (Vaughn et al, 1988). The quinones can react with proteins and are often more toxic to pathogens then the unoxidized phenols used to produce them (Hammerschmidt and Schultz, 1996). Increased polyphenoloxidase activity may increase quinones levels and contribute to resistance against F. sambucinum by inhibiting growth. I Steroid glycoalkaloids (SGA) accumulate in response to wounding of potato tuber tissue and are generally considered a defense mechanism against herbivores (Kuc, 1984: Valknonen et al, 1996). SGA may also be important in defense against pathogens (Valkonen et al, 1996; Kuc, 1984). The SGAs a- solanine and a-chaconine were reported to inhibit F. sambucinum spore germination and growth (Zeng, 1993). SGAs were reported to be suppressed upon infection by F. sambucinum (Zeng, 1993) and in response to P. infestans and the fatty acid components of P. infestans mycelium arachidonic acid and eicosapentaenoic acid (lshizaka and Tomiyama, 1972; Tjamos and Kuc, 1982). This research showed SGA was suppressed in response to chitosan treatment 84 and by an even larger degree by MH treatment (Figure 2-14). It is unlikely that SGA contributes to the resistance response since levels in the MH and chitosan treated tissue were substantially lower than the control tissue, which was not resistant to F. sambucinum. Steroid glycoalkaloid and sesquiterpenoid biosynthesis share a common biosynthetic pathway until famesyl pyrophosphate at which point they diverge (Figure 2-22). Suppression of steroid glycoalkaloids is often associated with increases in the sesquiterpenoid phytoalexins (Kuc, 1984). However, sesquiterpenoid phytoalexins are probably not involved in resistance of potato tuber tissue to F. sambucinum because F. sambucinum has the ability to detoxify and tolerate high levels of the sesquiterpenoid phytoalexins rishitin and lubimin (Desjardins and Gardner, 1989; Desjardins et al, 1989). Chitosan and MH treatments triggered systemic changes in addition to the localized changes. Chitosan treated tuber tissue showed increased resistance 0.5 cm below the treated surface (Figure 2-15) and exhibited an increase in chitinase throughout the entire 3.5 cm depth of the tuber tissue cylinder (Figure 2-17) and polyphenoloxidase 0.5 cm from the treated surface (Figure 2-20). MH treated tissue also exhibited an increase in chitinase throughout the entire 2.5 cm depth of the tuber tissue cylinder (Figure 2-16) as well as an increase in 8-1 ,3- glucanase 1.0 cm from the treated surface (Figure 2-18), however, there was no increase in resistance below the treated surface (Figure 2-15). The ability of chitosan to induce resistance below the surface may account for the higher resistance levels obtained by chitosan treatment verses MH treatment. Hyphae 85 that penetrate the surface tissue of chitosan treated tissue may encounter mechanisms that would prevent further penetration. Steroid Alkaloids l l I l l \ —+ C / STVS OPP Vetispiradie ne Farnesyl PPi I Ow I... HO «— + .. no HO Rishitin Lubimin Figure 2-22: Steroid glycoalkaloid and sesquiterpenoid biosynthetic pathway. The local and systemic responses of the tuber tissue to MH and chitosan treatments are clearly unique based on the differences in the induction of local and systemic resistance to F. sambucinum and induction of punatitive plant defense mechanisms. Both treatments give protection against F. sambucinum. 86 However, each treatment induces different defense responses and different levels of resistance. Treatment with MH and chitosan are promising methods of controlling F. sambucinum. These treatments may also prove effective against other potato tuber diseases. Both treatments induce several putative defense mechanisms both locally and systemically, which could protect the tuber against diseases that infect through the outer surface as well as those that infect the interior of the tuber. It is not likely that resistance is due to just one of these defense mechanisms, but a number of defense mechanisms working simultaneously to stop the pathogen. The multi-defense nature of this approach of controlling F. sambucinum will make it more difficult for the fungus to over come control of the disease as has occurred with current pesticide control methods and should allow for protection against a broad spectrum of pathogens. REFERENCES Allan CR. and Hadwiger LA, 1979. The fungicidal effect of chitosan on fungi of varying cell wall composition. Experimental Mycology 3:285—287. Allen E.H. and Kuc J., 1968. a-Solanine and a-chaconine as fungitoxic compounds in extracts of Irish potato tubers. Phytopathology 58:776-781. Boller T., 1995. Chemoperception of microbial signals in plant cells. Annual Review of Plant Physiology and Plant Molecular Biology 46:189. Bostock RM. and Schaeffer DA, 1986. Comparison of elicitor activities of arachidonic acid, fatty acids and glucans from Phytophthora infestans in hypersensitivity expression in potato tuber. Physiological and Molecular Plant Pathology 29:349-360. Bowles, JD, 1990. Defense-related proteins in higher plants. Annual Review of Biochemistry 59:873. 87 Cadle L.S., Stelzig D.A., Harper K.L., and Young R.J., 1978. Thin-layer chromatography system for identification and quantification of potato tuber glycoalkaloids. Journal of Agriculture and Food Chemistry 26:1453-1454. Chittoor J.M., Leach J.E., and White F.F., 1999. Induction of peroxidase during defense against pathogens. In: Datta SK. and Muthukrishnan S. (eds.) Pathogenesis-Related Proteins in Plants. CRC Press: Boca Raton. p. 171-193. Desjardins A.E. and Gardner H.W., 1989. Genetic analysis in Gibberella pulicaris: rishitin tolerance, rishitin metabolism, and virulence on potato tubers. Molecular Plant-Microbe Interactions 2:26-34. Desjardins A.E., Gardner H.W., and Plattner RD, 1989. Detoxification of the potato phytoalexin lubimin by Gibberella pulicaris. Phytochemistry 28:431-437. Doares S.H., Syrovets T., Weiler E.W. and Ryan CA, 1995. Oligogalacturonides and chitosan activate plant defensive genes through the octadecanoid pathway. Proceedings of the National Academy of Science USA. 92:4095-4098. Espelie K.E. and Kolattukudy PE, 1985. Purification and characterization of an abscisic acid-inducible anionic peroxidase associated with suberization in potato (Solanum tuberosum). Archives of Biochemistry and Biophysics 240:539-545. Espelie K.E., Franceschi V.R., and Kolattukudy PE, 1986. lmmunocytochemical localization and time course of appearance of an anionic peroxidase associated with suberization in wound-healing potato tuber tissue. Plant Physiology 81:487-492. Godoy A.V., Olivieri PP, and Casalongue CA, 1996. Induction of hydrolytic enzymes and antifungal in vitro activity in S. tuberosum subsp. andigena after infection with Fusan'um solani f.sp. eumartii. Potato Research 39:259-266. Gomes M.R.A. and Ledward DA, 1996. Effect of high-pressure treatment on the activity of some polyphenoloxidases. Food Chemistry 5621-5. Gooday G.W., 1994. Cell walls. In: Gow N.A.R. and Gadd G.M. (eds.), The Growing Fungus. Chapman 8. Hall: London. p. 43-62. Graham R.C., Lundholm U., Kamovsky M.J., 1965. Cytochemlcal determination of peroxidase activity with 3-amino-9-ethylcarbazole. Journal of Histochemistry and Cytochemistry 13:150-152. 88 Hammerschmidt R., 1984. Rapid deposition of lignin in potato tuber tissue as a response to fungi non-pathogenic on potato. Physiological Plant Pathology 24:33-42. Hammerschmidt R. and Schultz J.C., 1996. Multiple defenses and signals in plant defense against pathogens and herbivores. In: Romeo (ed) Phytochemical Diversity and Redundancy in Ecological Interactions. Plenum Press: New York. p. 121-154. Ishizaka N. and Tomiyama K., 1972. Effect of wounding or infection by Phytophthora infestans on the content of terpenoids in potato tubers. Plant and Cell Physiology 13:1053-1063. Keeling B.L. and Banttari E.E., 1975. Factors associated with the resistance of barley to Helminthospon’um teres (net blotch). Phytopathology 65:464-467. Kendra DP. and Hadwiger LA, 1984. Characterization of the smallest chitosan oligomer that is maximally antifungal to Fusarium solani and elicits pisatin formation in Pisum sativum. Experimental Mycology 8:276-281. Kombrink E., Schrdder M., and Hahlbrock K., 1988. Several “pathogenesis- related proteins in potato are 1,3-8-glucanases and chitinases. Proceedings of the National Academy of Science USA. 85:782-786. Kuc J., 1984. Steroid glycoalkaloids and related compounds as potato quality factors. American Potato Journal 61 :123—139. Mauch F. and Staehelin LA, 1989. Functional implications of the subcellular localization of ethylene-induced chitinase and 8-1,3-glucanase in bean leaves. Plant Cell 1:447. Mayer AM. and Harel E., 1979. Polyphenol oxidase in plants. Phytochemistry 1 8: 1 93-21 5. O’Brien V.J. and Leach 8.8., 1983. Investigations into the mode of resistance of potato tubers to Fusarium Roseum ‘Sambucinum’. American Potato Journal 60:227-233. Stdssel P. and Leuba J.L., 1984. Effect of chitosan, chitin and some aminosugars on growth of various soilbome phytopatholgenic fungi. Phytopathology Zeitschrift. 111:82-90. Thygesen P.W., Dry LB, and Robinson S.P., 1994. Polyphenol oxidase in potato tubers. In: Belknap W.R., Vayda M.E., and Park W.D. (eds) The Molecular and Cellular Biology of the Potato. Second edition. CAB lntemational: Wallingford, UK. p. 151-159. 89 Tjamos EC. and Kuc J.A., 1982. Inhibition of steroid glycoalkaloid accumulation by arachidonic and eicosapentaenoic acids in potato. Science 217:542-543. Trudel J. and Asselin A., 1989. Detection of chitinase activity after polyacrylamide gel electrophoresis. Analytical biochemistry 178:362-366. Valkonen J.P.T., Keskitalo M., Vasara T., and Pietila, 1996. Potato glycoalkaloids: a burden or a blessing? Critical Reviews of Sciences 15:1-20. Vaughn P.F.T., Eason R., Paton J.Y., and Ritchie G.A., 1988. Polyphenol oxidase: the chloroplast enzyme with no established function. Physiologica Plantarum 72:659-665. Zeng Y., 1993. Biochemical and physiological aspects of the plant host response in the fusarium dry rot disease of potato. Ph.D. Dissertation, Michigan State University, East Lansing, Michigan USA. 90 CHAPTER 3 Histochemistry and Autofluoresence Microscopy of Chitosan Treated Tuber Tissue 91 INTRODUCTION Plant cell walls often respond to pathogen attack or elicitor treatment by modification of their cell walls. These changes in cell wall composition include lignification, esterification of phenols, and deposition of callose, and suberin (Hammerschmidt and Nicholson, 1999). They may be involved in host defense by increasing the mechanical strength of the cell wall, decreasing the susceptibility of the wall to cell wall degrading enzymes, preventing nutrient flow across the wall, and providing a barrier to toxins and enzymes produced by pathogens (Ride, 1978). Phenolic compounds often accumulate in plant cells in response to pathogen attack and to resistance inducing agents and are believed to have an important role in plant defense (Nicholson and Hammerschmidt, 1992). In some cases, increased resistance has been correlated with phenol accumulation (Shiraishi et al, 1989; Cahill et al, 1989; Ryugo et al, 1990; Lyons et al, 1993; Bostock et al, 1999). Resistance may be attributed to the accumulation of toxic phenols, which inhibit growth of the pathogen. Additionally, polymerization of phenols into the cell wall may create a physical barrier, which can prevent pathogen infection (Nicholson and Hammerschmidt, 1992). Histochemistry and autofluorescence microscopy are useful methods of observing phenol accumulation and deposition of phenolic cell wall material. This study investigated the effect of chitosan treatment on phenol accumulation and deposition of cell wall material in order to determine if phenols have a role in the increased resistance observed after chitosan treatment. 92 Callose is a polysaccaride often deposited in the cell wall in response to infection (Hammerschmidt and Nicholson, 1999). It may play a role in resistance by forming a physical barrier to pathogen ingression. Histochemical staining was also used to evaluate deposition of callose in response to chitosan treatment to elucidate the role of callose in chitosan induced resistance. MATERIALS AND METHODS Evaluation of Autofluorescence Tuber discs were prepared and treated as described in chapter 1. The discs were then sliced longitudinally into 24 cell layer thick sections 24, 48, and 72 hours after treatment using a Hooker microtome. The sections were rinsed in water and mounted in Hoyers media (Cunningham, 1972) and observed with a light microscope under UV light. Color digital photographs were taken of the treated surface of 24 sections. The depth of fluorescence was measured from the digital photos. Three measurements were taken from each photo and averaged. The data were analyzed for significance by one-way analysis of variance, followed by Tukey’s significant difference test (a=0.05), using GraphPad lnStat software. Toluidine Blue 0 Staining Tuber discs were prepared and treated as described in chapter 1. The discs were sliced longitudinally into 2-4 cell layer sections 12, 24, 48, and 72 hours 93 after treatment using a Hooker microtome. The sections were soaked overnight in 95% ethanol in order to clear the soluble phenols. The sections were then stained with 0.1% toluidine blue 0 in 0.1 M sodium phosphate buffer pH 6.5 for 2 minutes. The section were then rinsed three times with water and mounted in a 50% solution of glycerol (Hammerschmidt and Kuc, 1982). Sections were observed using a light microscope and color digital photographs were taken. Three photographs were taken from each of 12 sections. The depth of toluidine blue 0 staining were measured for section made 48 and 72 hours after chitosan treatment and analyzed for significance by one-way analysis of variance, followed by Tukey’s significant difference test (a=0.05), using GraphPad lnStat software. Lacmoid Test for Callose Tuber discs were prepared and treated as described in chapter 1. The discs were sliced longitudinally into 24 cell layer thick sections 24, 48, and 72 hours after treatment using a Hooker microtome. The sections were rinsed in water and placed in a 0.1% solution of lacmoid in 50% ethanol for 24 to 48 hours (Vance and Sherwood, 1976). The sections were rinsed in water and mounted in Hoyers medium. The sections were observed using a light microscope under bright field and UV light and color digital photographs were taken. 94 Histochemical Staining for Lignin Tuber discs were prepared and treated as described in chapter 1. The discs were sliced longitudinally into 2-4 cell layer sections 24, 48, and 72 hours after treatment using a Hooker microtome. Sections were rinsed in water and soaked in 95% ethanol overnight. Sections were then stained in a saturated aqueous solution of phloroglucinol in 20% HCl for 10 minutes, rinsed in water, and washed in 70% ethanol (Hammerschmidt and Kuc, 1982). Alternatively sections were stained in a 1% aqueous solution of KMnO4 and rinsed in water (Hepler et al, 1970). Sections were mounted in Hoyers medium and examined using a light microscope. RESULTS Autofluorescence Under UV-Light Potato tuber discs treated with 0.5 and 1.0 mglml chitosan or water were examined for autofluorescence 24, 48, and 72 hours after treatment. Compared to water treated tuber discs chitosan treated disks exhibited a higher intensity of autofluorescence and autofluorescent material occurred at a greater depth from the treated surface of the discs (Figure 3-1). The autofluorescence was concentrated in the cell wall area of both water and chitosan treated discs (Figure 3-1, 3-2 and 3-3). At the later time points the intensity and depth of fluorescence increased in tissue treated with both water and chitosan (Figure 3-2, 3-3, and 3- 95 4). By 72 hours the depth of autofluorescence in the water treated tissue begun to approach that of the chitosan treated tissue (Figure 3-4). After 48 hours the chitosan treated tissue began to exhibit a yellowish fluorescence at the tissue surface, which was not visible in the water treated tissue (Figure 3-2). This yellowish fluorescence became even more evident after 72 hours (Figure 3-3). Control 0.5 mglml Chitosan 1.0 mglml Chitosan Figure 3-1: Fluorescence micrograph of longitudinal sections of potato tuber discs 24 hours after treatment with water (control) or chitosan. Blue-green or blue fluorescence indicates phenolic deposition. 400x magnification. Control 0.5 mglml Chitosan 1.0 mglml Chitosan Figure 3—2: Fluorescence micrograph of longitudinal sections of potato tuber discs 48 hours after treatment with water (control) or chitosan. Blue-green, blue, or yellow fluorescence indicates phenolic deposition. 400x magnification. 96 Control 0.5 mg/ml Chitosan 1.0 mglml Chitosan Figure 3-3: Fluorescence micrograph of longitudinal sections of potato tuber discs 72 hours after treatment with water (control) or chitosan. Blue-green, blue, or yellow fluorescence indicates phenolic deposition. 400x magnification. _s m (D O l 1 El Control I0.5 mglml Chitosan \ 1.0 mglml Chitosan Depth of Fluoresence (microns) o-‘Nw-bo'lmfl v 11111 24 Hours 48 Hours 72 Hours Figure 3-4: Depth of autofluorescence in longitudinal sections of potato tuber discs 24, 48, and 72 hours after treatment with water (control) or chitosan. The letters represent significant differences (P< 0.05) between treatments. Toludine Blue Staining Potato tuber discs treated with 1.0 mg/ml of chitosan or water were stained with toluidine blue 0 at 12, 24,48, and 72 hours after treatment. Twelve hours after treatment with chitosan or water the cell walls were stained purple color, 97 typical of normal cell walls (Figure 3-5). After 24 hours the cell walls of the first two to three cells layers of the chitosan treated tissue stained blue-green in color; indicating deposition of phenolic compounds. The water treated tissue. however, did not stain blue-green (Figure 3-5). At 48 and 72 hours the intensity and depth of the blue-green staining increased in the chitosan treated tuber discs (Figure 3- 5 and 3-6). Also, the water treated tissue, exhibited blue-green staining, but at a much lower intensity and at a lesser depth than the chitosan treated tissue (Figure 3-5 and 3-6). Control 2 . 1.0 mglml Chitosan 12 Hours 24 Hours 48 Hours 72 Hours Figure 3-5: Micrograph of longitudinal sections of potato tuber discs stained with toluidine blue 0 12, 24,48, and 72 hours after treatment with water (control) or chitosan. Purple staining indicates typical cell wall material. Blue-green staining indicates phenolic deposition. 400x magnification. 98 \\\\\ E51 Control 1 .0mg/ml Chitosan 48 Hours 72 Hours Figure 3-6: Depth of blue-green color caused by toluidine blue 0 staining 48 and 72 hours after treatment with water (control) or chitosan. The letters indicate significant differences (P<0.05) between treatments. Lacmoid Test for Callose Potato tuber discs treated with 1.0 mglml chitosan or water were stained for callose deposition. Lacmoid staining revealed that 72 hours after treatment with chitosan, a barrier of callose was deposited 2 to 3 cells layers from the treated surface (Figure 3—7). Callose deposition was not observed in water treated tuber discs (Figure 3-7). The callose deposition corresponded with the interface between tissue exhibiting autofluorescence and tissue not exhibiting autofluorescence (Figure 3—7). 99 Callose Staining Autofluorescence Control 1.0 mg/ml Chitosan Figure 3-7: Micrographs of longitudinal sections of potato tuber discs stained with lacmoid and fluorescent micrographs of the same sections 72 hours after treatment with water (control) or chitosan. The red color of the lacmoid stain indicated the presence of callose. The blue-green, blue, and yellow fluorescence indicated deposition of phenolic compounds. 400x magnification. Histochemical Staining for Lignin Potato tuber tissue was stained for lignin deposition 24, 48, and 72 hours after treatment with water or chitosan using phloroglucinal H01 and 1% aqueous solution of KMnO4. Neither the phloroglucinal HCI stain nor the KMnO4 stain revealed lignin deposition in response to chitosan or water treatments at any of the time points. 100 DISCUSSION Treatment of tuber tissue with chitosan increased the depth and intensity of autofluorescence above levels observed in tuber tissue treated with water. This indicated that chitosan induced phenolic compound accumulation (Figure 3-1, 3- 2, 3-3, and 3-4). Previous experiments have shown a correlation between the accumulation of phenolic compounds and the slowing or halting of pathogen growth resulting in the isolation of the pathogens ability to further infect the plant tissue (Shiraishi et al, 1989; Cahill et al, 1989; Ryugo et al, 1990; Lyons et al, 1993; Bostock et al, 1999) . Therefore, increases in the concentration of phenols, as well as, increases in the area of phenol deposition observed after chitosan treatment may in part be responsible for the increase in resistance to F. sambucinum observed after chitosan treatment. Accumulation of phenols may completely inhibit F. sambucinum infection. It has also been suggested that the initial rapid accumulation of phenols slows the growth of pathogens, which allows time for the induction of other plant defenses that can completely stop invasion, by the pathogen (Mansfield, 1982 and Matem and Kneusel, 1988). Slowing the growth of F. sambucinum in chitosan treated tuber tissue may allow the tuber tissue to become suberized, which would prevent further infection by F. sambucinum (O'Brien and Leach, 1983). It has been shown to take five to seven days for tuber tissue to become suberized enough to prevent F. sambucinum from penetrating the tissue (Lulai and Corsini, 1998). Accumulation of phenolic 101 compounds may be in part responsible for slowing growth until suberization can occur. Phenolic compounds can also be polymerized into cell material (Nicholson and Hammerschmidt, 1992). Deposition of phenols in the cell wall may provide a mechanical barrier to pathogen penetration, prevent penetration of enzymes and toxins produced by the pathogen, and prevent nutrient flow to the pathogen (Ride, 1978). Additionally, phenolic free radicals, precursors to the polymerization of phenols into cell wall material, may be toxic to the pathogen (Nicholson and Hammerschmidt, 1992). Examination of autofluorescence in chitosan treated tuber tissue showed that the phenolic compounds were localized in the cell wall areas of the tuber tissue (Figure 3-1, 3-2, and 3-3). Toluidine-blue O staining also revealed the deposition of non-soluble phenols in the cell walls (Figure 3-5) thus confirming the autofluorescence results. These cytological studies provide evidence that chitosan may cause polymerization of phenols into the cell wall; indicates that cell wall changes may be in part responsible for the increase in resistance of tuber tissue observed after chitosan treatment. Phloroglucinol HCI and KMnO4 staining revealed that the phenolic material deposited in the cell wall was not lignin. However, it has been suggested that esterification of phenols to cell wall material may be involved in resistance against pathogens (Nicholson and Hammerschmidt, 1992). After 48 hours, tissue treated with chitosan begun to exhibit a yellowish fluorescence in addition to the blue-green fluorescence that had been observed previously (Figure 3-2 and 3-3). The yellowish fluorescence may also indicate 102 cell wall deposition of phenols. The localization and timing of the yellowish fluorescence corresponded with the localization and timing of toluidine-blue O staining, thus supporting the idea that the fluorescence is caused by phenols deposited in the cell wall (Figure 3-2, 3-3, and 3-5). Alternatively, the yellowish fluorescence may indicate a change in the type of phenols accumulating in the cells. It has been suggested that chlorogenic acid, which in itself is not very toxic, may be used to synthesize other phenols that can prevent pathogen infection (Friend, 1981). Such changes may affect the fluorescent properties of the accumulating phenolics. Lacmoid staining revealed that chitosan treated tissue developed a barrier of callose two to three cell layers from the surface of the treated tissue after 72 hours (Figure 3-7). Callose has also been implicated as a barrier to pathogen invasion (Hammerschmidt and Nicholson, 1999). Therefore, the callose barrier may prevent infection by F. sambucinum beyond the first two or three of cell layers. The cytological data collectively suggests that F. sambucinum infection may be slowed or halted within the first few cell layers. F. sambucinum growth may be initially inhibited by exposure to toxic phenols and/or cell wall changes caused by the deposition of phenols that can prevent F. sambucinum penetration. The callose barrier appeared to correspond with the interface between those cells accumulating phenols and those in which phenolic accumulation was not evident. The phenolic accumulation may slow hyphal penetration long enough for the 103 callose barrier to form. The callose barrier may then prevent further penetration until suberization is complete. REFERENCES Bostock R.M., Wilcox S.M., Wang G., and Adaskaveg SE, 1999. Suppression of Monilinia fmcticola cutinas production by peach fruit surface phenolic acids. Physiological and Molecular Plant Pathology 54:37-50. Cahill D., Legge N., Grant 8., and Waste 6., 1989. Cellular and histological changes induced by Phytophthora cinnamomi in a group of plant species ranging from fully susceptible to fully resistant. Phytopathology 79:417-424. Cunningham J.L., 1972. A miracle mounting fluid for permanent whole-mounts of microfungi. Mycologia 64:906-911. Friend J., 1981. Plant phenolics, lignification and plant disease. Progress in Phytochemistry 7: 1 97-261 . Hammerschmidt R. and Kuc J., 1982. Lignification as a mechanism for induced systemic resistance in cucumber. Physiological Plant Pathology 20:61-71. Hammerschmidt R. and Nicholson R.L., 1999. A survey of plant defense responses to pathogens. ln: Agrawal A.A., Tuzun s., and Bent E. (eds). Induced Plant Defenses Against Pathogens and Herbivores. p. 55-71.bb Hepler P.K., Fosket DE, and Newcomb EH, 1970. Lignification during secondary wall formation in Coleus: An electron microscopy study. American Journal of Botany 57:85-86. Lulai EC. and Corsini D.L., 1998. Differential deposition of suberin phenolic and aliphatic domains and their roles in resistance to infection during potato tuber (Solanum tuberosum L.) wound-healing. Physiological and Molecular Plant Pathology 53:209-222. Lyons P.C., Hipskind J., Vincent JR, and Nicholson R.L., 1993. Phenylpropanoid dissemination in maize resistant or susceptible to Helminthospon’um maydis. Maydica 38:175-181. Mansfield J.W., 1982. The role of phytoalexins in disease resistance. In: Bailey J.A. and Mansfield J.W. (eds). Phytoalexins. New York: Willey. p. 253-288. 104 Matern U. and Kneusel R.E., 1988. Phenolic compounds in plant disease resistance. Phytoparasitica 16:153—170. Nicholson R.L. and Hammerschmidt R., 1992. Phenolic compounds and their role in disease resistance. Annual Review of Phytopathology 30:369-389. O’Brien V.J. and Leach 8.8., 1983. Investigations into the mode of resistance of potato tubers to Fusarium roseum 'Sambucinum'. American Potato Journal 60:227-233. Ride JP, 1978. The role of cell wall alteration in resistance to fungi. Annals of Applied Biology 89:302-306. Ryugo K., Okuse 1., and Fujii Y., 1990. Correlation between fire blight resistance and phenolic levels in pears. Acta Horticulturae 273:335-338. Sherwood RT and Vance GP, 1976. Histochemistry of papillae formed in reed canarygrass leaves in response to noninfecting pathogenic fungi. Phytopathology 66:503-510. Shiraishi T., Yamaoka N., and Kunoh H., 1989. Association between increased phenylalanine ammonia-lyase activity and cinnamic acid synthesis and induction of temporary inaccessibility caused by Erysiphe graminis primary germ tube penetration of the barley leaf. Physiological and Molecular Plant Pathology 34:75-83. 105 CONCLUSIONS AND FUTURE RESEARCH Chitosan and P. infestans mycelium homogenate induced several putative defense mechanisms in potato tuber tissue including: oxidative enzymes, PR proteins, and cell wall alterations (Table C-1). Associated with the onset of these defenses was induced resistance to F. sambucinum infection. Induced resistance is therefore, a promising alternative to fungicides for the control of F usarium dry rot. The resistance observed after treatment with chitosan and P. infestans mycelium homogenate is probably a result of multiple plant defense mechanisms working simultaneously to stop the ingression of F. sambucinum; because this resistance controls F. sambucinum using several defense mechanisms it may be a more stable form of resistance than fungicides which only target one aspect of fungal development. ~ Table C-1: Review of the effect of chitosan and MH on plant defense mechanisms. PR-Proteins Oxidative Enzymes Phytoalexins Chitinase 0-1 ,3- Peroxidase Polyphenol Steroid Glucanase oxidase Glycoalkaloids Chitosan Local Local Local Local Local Decrease (2 hours) (12 hours) Systemic Systemic (0.5 cm) (2.5 cm) MH Local Local Local Not Tested Local Decrease Systemic Systemic (2.5 cm) (1.0 cm) 106 Further research questions include: 1. Determining if treatment with chitosan or P. infestans mycelium homogenate would effectively induce resistance in whole tubers and seed pieces reducing storage losses and seed decay. Evaluate the ability of chitosan and P. infestans mycelium homogenate to reduce infection in tubers and foliage by other potato pathogens and evaluate their effect on herbivory. In light of differences in the appearance of tuber tissue after chitosan and P. infestans mycelium homogenate and differences in the induction of putative defense mechanisms, it would be interesting to assess whether chitosan and P. infestans mycelium homogenate induce the same or different signally pathways. Analyze phenolic and callose deposition after treatment with P. infestans mycelium homogenate. Assess whether potato chitinase or 8-1,3-glucanase inhibit F. sambucinum growth or if they produce elicitors that may induce other defense mechanisms. 107 IIIIIIIIIIIIIIIIIIIIIIIIIIIIII lllllllllllllllllllllllllllllllllllll 293 023