' ”T: “A « quzv‘r O .. n K .V- t. 4:» w-( a 4 *u o “: Ev‘ l‘.l « ':"s“‘ ,.r [raz- «..‘| “‘53:. m ., ,,- 1 -V 14,1. J‘af‘v.‘ I“: «.- ‘Z' 'L 'n.“ éfiufifi .R. .3 «flu 35“.?“ ~ --r1-‘-‘:':., v mm 1 is n.'.1,f", ." v».., . r “3:3. .. ‘3“. I." .1” ...‘ , .1:L§._,'.. n! , u, .. . ~« ' H. L y“... . 5-,..2- .12. I m t x . , 3. 'n .. " "v .-.i, m. .-..._ . 354.4. , . ‘ ‘ >- 3‘ 2_ . .‘ ' -~- . . t' . , :77}. - >~'~' - .‘ ””1221. ' . . . a. “41:, » -~. 2.. ¢>ao "w' tuff" m'.'.:...-—. 0.- vol.- ".3 '1'.“ /‘ . 3:1: :4; , 2 79:". U Lif"i(_-.r?x' , y" ‘5;ng ":1 _.',_, llllllllllllllllllll lllllllllllllllllllll 300876 4858 This is to certify that the thesis entitled Factors Influencing the Resistance of the Potato to Common Scab presented by Karen E. Ludlam has been accepted towards fulfillment of the requirements for M.Sc. degree in Plant Breeding & Genetics >J<>a4~ Major professor Date May 8, 1991 0.7639 MS U is an Affirmative Action/Equal Opportunity Institution LIBRARY Mlchlgan State Unlverslty PLACE IN RETURN BOX to remov- thlo checkout from your record. TO AVOID FINES Mum on or before dds duo. DATE DUE DATE DUE DATE DUE W» IL JL V—ll ll MSU I. An Affirmative ActiorVEqual Opportunlty Institution cmms-m FACTORS INFLUENCING THE RESISTANCE OF THE POTATO TO COMMON SCAB BY Karen Ludlam A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Crop and Soil Science 1991 ABSTRACT FACTORS INFLUENCING THE RESISTANCE OF THE POTATO TO COMMON SCAB by Karen Ludlam Three basic questions about the resistance of potato cultivars (Solanum tuberosum L.) and diploid potato lines to the common scab disease (Streptomyces spp.) were addressed. 1) Six methods for scoring resistance of potato lines were‘ assessed. The methods compared average scores per pot verses high scores per pot; cover score ratings vs. lesion type ratings vs. a combined rating of cover score and lesion type. Results indicated that an average combined rating score per pot is the best method for scoring scab resistance. 2) The effect of six strains of the scab organism on potato lines was evaluated. There were significant interactions between varieties and strains, and the cover score and lesion type were effected by these interactions independently of one another. 3) Soluble peroxidase isozymes and acidic and total soluble peroxidase activity levels were compared among potato lines. There was no correlation between isozyme patterns or activity levels and scab resistance. ACKNOWLEDGMENTS I would like thank my advisors, Dr. Dave Douches and Dr. Ray Hammerschmidt for their time and effort on this project. I would also like to thank Dr. Amy Iezzoni for valuable input into the final draft of this thesis. I thank all of the people who helped me to complete this research: Chien-An Liu, Katherine Hicks, Donna Kells, Theresa Wood, Dick Kitchen, Dick Chase, George Silva, Cara Wallace,' Yihua Zeng, Frank Spooner, and many, many more. Thanks to my parents, family, and friends for their support. And a very special thanks to Stan Hokanson for all of his time, patience, advise, love, and support. ii TABLE OF CONTENTS Page LISTOFTABLES...0.0.0....0000000.000 ........ .0.......... v LIST OF FIGURES 0 . 0 0 0 0 0 0 0 . 0 0 0 0 . 0 . . . 0 . 00000 . . . 0 0 . 0 . . 0 . 0 0 0 . . Viii INTRODUCTION 0 . . . . . 0 0 0 0 . 0 0 . . . 0 0 . 0 0 0 0 0 0 0 . . 0 . 0 0 . . 0 . . . . 0 0 0 . 0 . 1 LITERATURE REVIEW. . . 0 O . 0 . . . . 0 0 0 0 . 0 0 0 0 0 . 0 . 0 0 0 0 . 0 0 0 . 0 0 0 0 0 0 . 3 LITEMTURECITED.....000000.0.......0000...0.0......0.00. 12 CHAPTER I METHODS FOR SCORING SCAB RESISTANCE Abstract................................................. 18 Introduction............................................. 19 Materials and Methods.................................... 20 Results.................................................. 28 Discussion............................................... 45 LiteratureCited...0.0.00...0.00000.00.0000..000.00000000 53 CHAPTER II THE EFFECT OF STRAINS OF THE SCAB ORGANISM ON THE POTATO Abstract....................... ......... ................. 56 Introduction............................................. 56 MaterialsandMethods. ...... ........ 57 Results......... ..... ...... .................. ............ 59 Discussion............................................... 65 iii LiteratureCited....0....0..00..000..O0.00..0000..0.0000. 71 CHAPTER III EVALUATION OF PEROXIDASE IN RELATION TO POTATO SCAB RESISTANCE Page AbstractOOOOOO0.00.000.00.0000...OOOOOOOOOOOOOOOOOOOOOOOO 74 IntrOduCtiono O O O O I O I O I O O O O O 0 O O O O O O O O O O O O O O O I O 0 O O O O O O O O O O O 74 Materials and HethOdSO O O O O O O O O O O O 0 O O O O O 0 O O O O O O O O O O O O O O O O O 77 ResultSOOOOOOOOOOOOOO...OOOOOOOOIOOOOOOOOOOOOO0.0.0.0.... 79 DiSCUSSionooooooooooooooooooooooooo00000000000000.0000... 86 LiteratureCited......................................... 88 APPENDIX EXTRACTION OF CHLOROGENIC ACID FROM TUBER PERIDERM Materials and Methods.......... ....... 9O LiteratureCited...... ...... 91 iv LIST OF TABLES Table CHAPTER I 1 Cultivars (4x) and diploid lines (2x) evaluated for scab resistance...... ..... ................... 2 Numerical rating of % surface cover, lesion type and combined rating for each tuber and the class of resistance (R, MR, MS, S)‘ designated by the number ratings................................... 3 Classifications, rankings, significant differences and coefficients of variation for cultivars evaluated in the summer 1989 according to the highest scab per pot... ...... .................... 4 Classifications, rankings, significant differences and coefficients of variation for cultivars evaluated in the summer 1989 according to the average scab per pot............................. 5 Classifications, rankings, significant differences and coefficients of variation for cultivars evaluated in the spring 1990 according to the highest scab per pot............................. 6 Classifications, rankings, significant differences and coefficients of variation for cultivars evaluated in the spring 1990 according to the average scab per pot........ ...... ............... 7 Correlation coefficients among the different scoring systems for the cultivar greenhouse and field evaluations... ............. ............ 8 Classifications, rankings, significant differences and coefficients of variation for cultivars evaluated in the field according to the highest scab per pot............ ..... .................... Page 22 25 29 30 31 32 33 35 Table Page 9 Classifications, rankings, significant differences and coefficients of variation for cultivars evaluated in the field according to the average scab per pot..................................... 36 10 Classifications, rankings, significant differences and coefficients of variation for diploid lines evaluated in the summer 1989 according to the highest scab per pot.............................. 38 11 Classifications, rankings, significant differences and coefficients of variation for diploid lines evaluated in the summer 1989 according to the average scab per pot..... ......... ................ 39 12 Classifications, rankings, significant differences and coefficients of variation for diploid lines evaluated in the winter 1990 according to the highest scab per pot.............................. 40 13 Classifications, rankings, significant differences and coefficients of variation for diploid lines evaluated in the winter 1990 according to the average scab per pot.............................. 41 14 Classifications, rankings, significant differences and coefficients of variation for diploid lines evaluated in the spring 1990 according to the highest scab per pot.............................. 42 15 Classifications, rankings, significant differences and coefficients of variation for diploid lines evaluated in the spring 1990 according to the average scab per pot.............................. 43 16 Correlation coefficients among the different scoring systems for the diploid greenhouse evaluations. ..... 0.0.0.0. 00000 .O........0.0. 000000 44 CHAPTER II 1 Potato cultivars (4x), diploid lines (2x), and strains of the organism (Streptomyces) used to evaluatescabinfectionOlO0.000000IOOOOOOOOIOOOOO. 58 2 Tabular and calculated F values from analysis of variance of cultivars and diploid lines and strains of the organism........................... 60 vi Table Page 3 Classification‘ according to a combined rating of seven potato cultivars and six diploid lines infected with six strains of the scab organism.... 61 4 Type of lesion1 produced on seven potato cultivars and six diploid lines infected with six strains of the scab organism................................. 63 5 Surface coverage (%V of seven potato cultivars and six diploid lines infected with six strains of thescaborganism....0.0..000..0.....0..0....0.0.. 64 CHAPTER III 1 Cultivars and diploid lines used to evaluate peroxidase isozymes and total peroxidase activity. 78 2 Total soluble peroxidase activity (Am/min/mg protein) of eighteen tetraploid and diploid lines averaged over three replications.................. 85 3 Total soluble peroxidase activity (Am/min/mg protein) of six round white potato varieties averaged over seven.replications.................. 85 vii LIST OF FIGURES Figure Page CHAPTER I 1 Tubers representing the classes of surface cover and their corresponding numerical ratings (left to right): (2) 1-5% ; (3) 5-25% ; (4) > 25%....... 26 2 Tubers representing classes of lesion types and their corresponding numerical ratings (clockwise from bottom): (2) superficial; (3) slightly pitted; (4) deep pitted; (4) deep pitted.......... 26 CHAPTER III 1 The phenylpropanoid pathway leading to the synthesis of the aromatic domains of suberin and of chlorogenic acid.............................. 76 2 Starch gel electrophoresis isozyme patterns of thirteen round white tetraploid lines, from left to right: Hindenberg(R); Burbank(R); Ontario(R); Superior(R); Atlantic (8); Onaway(S); Spartan Pearl(S); ND860-2(S); Monona(S); Katahdin(S); M8716-15(S); NDD277-2(S); Y245-7(S). R=Resistant, S=Susceptible....................... 8O 3 Starch gel electrophoresis isozyme patterns of two yellow flesh tetraploids, three russets, and six diploids, from left to right: Michigold(S); Saginaw Gold(S); Lemhi Russet(R); Nooksack(R); Russet Burbank(R); 84SlO(R); 84SD22(R); W5337.3 (S); W5295.7(S); P100-2(S); cuss-4(3). R=Resistant, S=Susceptible....................... 81 4 PAGE isozyme patterns of siz round white potato varities, left to right: Atlantic(S); Superior (R); Ontario(R); Spartan Pearl(S); Onaway(S); N0860-2(S). R=Resistant, S=Susceptible ............. .......... 82 viii Figure Page 5 PAGE isozyme patterns of six diploid lines and two tetraploids, left to right: W5337.3(S); 84810 (R); 84SDZZ(R); P100-2(S); DM56-4(S); Y245-7(S); Lemhi Russet(R). R=Resistant, S=Susceptible.. .......... ........... 83 ix INTRODUCTION Common Scab Disease of the potato (Solanum tuberosum L.) , caused by the soil-borne bacterium. Streptomyces scabies (Thaxter) , is a major problem in the production of high quality potatoes. The organism infects the apical end of growing tubers and induces the formation of corky lesions on the tuber surface. The lesions may vary in appearance from raised on the surface to deeply pitted, depending on the' cultivar and the strain of the organism. When scab lesions cover greater than five percent of the tuber surface, those potatoes are rejected from fresh.market. Potatoes with deep- pitted scab are eliminated from the processing markets. A number of cultural practices help reduce the severity of the disease, but there has been no consensus on how to completely control it. The most effective means of controlling the disease is to grow scab resistant cultivars. Therefore, ‘most. U.S. potato {breeding’ programs ‘take scab resistance into account. Although the disease has been studied for many years, the genetic nature of resistance is not known. Studies on the inheritance of scab resistance in S. tuberosum are further complicated by a basic lack of understanding of the organism and its ecology, as well as the 2 physiology and molecular or biochemical basis of the host/pathogen interaction. In anticipation of future studies on the inheritance of scab resistance, the research presented in this thesis will address some critical preliminary questions about 1) the methods for scoring scab, 2) the interaction between varieties of potatoes and the strains of the scab organism, 3) biochemical factors involved in host resistance, and 4) levels of resistance in some diploid lines. LITERATURE REVIEW WM Potato varieties are known to vary in their susceptibility to infection by S. scabies (Stuart, 1914; Walker et al., 1938; Blodgett and Stevenson, 1946; McKee, 1958; 1963) . Various studies have been done to determine the mode of inheritance of resistance. Leach et al.(1938) made a number of crosses to study the inheritance of scab resistance. ‘ They found that mean scab ratings of progenies between resistant x resistant parents were more resistant than the progeny from all other types of crosses. Progenies from crosses with susceptible x susceptible parents had the highest scab susceptibility. Krantz and Eide (1941) had suggested that inheritance to common scab in potato is tetrasomic and that the differences observed in reaction to scab were controlled by one locus (fig) . Five breeding types, nulliplex (sq g _s_g s9) , simplex (5.: so so s9.) . duplex (S2 .82 s2 s_c) . triplex (S9 82 5.9 3.2) . and quadraplex (Sq x 52 E) were postulated to explain variation in scab reaction. In a similar study, Lauer and Eide (1963) reported that clonal resistance of parents was directly reflected in the resistance of their progenies. They 4 explained the inheritance of resistance to common scab on a single gene basis with dominance and the duplex genotype (59 fig 59 fig) necessary for effective clonal resistance. Bloomquist (1963) also suggested a single dominant gene as the basis for resistance to common scab from analysis at the tetraploid level. Genetic studies at the diploid level are less complex than at the tetraploid level of the cultivated species S. tuberosum. Cipar and Lawrence (1971) isolated haploids from some tetraploid cultivars and tested them in the greenhouse for resistance to common scab. They found that the' distribution of haploids generated from the resistant variety Hindenberg was skewed strongly toward high levels of resistance to scab. In contrast, the haploids from Avon, a cultivar with moderate levels of resistance, were predominantly susceptible. They suggested that scab resistance of these haploids is controlled by more than one locus, yet the mode of inheritance is relatively simple, i.e., probably involves no more than one or two other loci. Resistance to common scab has been found in several diploid Solanum species (2n=24) . Reddick (1939) reported scab resistance in the 24-chromosome species 5. chacoense, S. caldasii var glabrescens, S. comersonii, and S. jamesii. Hougas and Ross (1954), in a survey of resistance in species, also reported resistance to scab in other Solanum species. Rudorf (1958) was able to confirm that some species of the 5 Commersonianna series, especially S. chacoense, demonstrate good resistance. Dionne and Lawrence (1961) suggested transferring resistance from diploid species of the series Commersonianna to the early maturing diploid Group Phureja. In crosses between S. chacoense and Group Phureja, segregation in the progeny of scab reaction from susceptible to resistant was obtained. They suggested that transferring resistance to Solanum tuberosum at the diploid level should be possible by crossing the S. chacoense-Group Phureja hybrids (2n=24) with haploids (2n=24) of TUberosum (2n=48). Alum (1972), following the evaluation of haploid-Phureja hybrids, concluded that there are two major loci contributing to scab resistance. At one locus (Sc1) the presence of one dominant allele confers resistance at either the diploid or tetraploid level of ploidy. In contrast, another locus (Sc2) conditions scab resistance only when homozygous recessive. e s of aluat'o Resistance to scab infection has traditionally been tested in fields where the scab organism is known to frequently occur. Standard procedures for field tests of varietal resistance have been described by Walker et a1. (1938), Stevenson et a1. (1942), and Leach et al. (1938). Hillard and Burr (1926), Schaal (1944), Taylor and Decker 6 (1947), and others have described methods for growing plants in field soil artificially contaminated with S. scabies. However, environmental conditions, such as soil pH and soil moisture levels are known to influence scab development (Terman.et al., 1948; Lapwood, 1966; Lapwood.and.Hering, 1968, 1970; Adams and Lapwood, 1978). Also, because infection only occurs on growing tubers (Fellows, 1926; Hooker and Page, 1960), an interaction between environmental conditions, especially soil moisture, and genotypic differences in dates of tuber initiation may confound varietal evaluations in the field (Wiersema, 1974). For these reasons, more reliable” tests for resistance have been developed for the greenhouse (Taylor and Decker, 1947; Hooker, 1950; Dykstra, 1956; McKee, 1958). Many greenhouse methods suggest growing potatoes in pots filled with naturally infested soil from the field. These greenhouse tests have been modified and improved to include artificially inoculated soil in pots rather than naturally infested soil (Bjor and Roer, 1980; Gunn et al., 1933). In order to make quantitative comparisons of scab resistance among varieties, a numerical system for scoring infection is necessary. Walker et a1. (1938) simply placed tubers into classes according to»general appearance.such.as 1) clean, 2) slightly scabbed, 3) moderately scabbed, or 4) severely scabbed. More commonly, numerical ratings for scab evaluation have been based on either the proportion of the 7 tuber surface affected (cover) (Darling, 1937; Clark et al., 1938; Large, 1955) or on the most severe type of scab lesion observed (highest scab) (Leach et al, 1938; Schaal, 1944; Lauer and Eide, 1963). A number of papers have considered both methods ("cover“ and ”highest scab") for rating scab resistance (Emilsson and Gustafsson, 1953; McKee, 1958). Bjor and Roer (1980) suggested that the use of both variables in a compound index should give a better estimate of resistance than either index alone. Stevenson et al. (1942) and Leach et al. (1938) both suggested that there is a correlation between lesion and area‘ affected. In general, they observed that the more superficial the lesion type, the smaller' the area affected» iMost investigators consider the type of lesion to be a more important criterion for judging the scab resistance of a potato variety (Emilsson and Gustafsson, 1953). Dowley(1972) compared highest scab and cover score and found that the coefficient of variation in.cover is higher than highest scab. He suggested the high variation is due to low scab incidence considering that under field conditions a high proportion of the plots are classified as "free from scab". yariatign in the pathogen In addition to climatic conditions, the population of S. scabies in the soil and the virulence of the strain of the organism should also be considered as factors which may 8 influence scab development and therefore evaluation. Numerous strains of the scab producing organism have been isolated and have been found to differ in morphology, physiology and pathogenicity (Drechsler, 1919; Waksam, 1919; Schaal, 1944; Taylor and Decker, 1947). Some studies have concluded that there is a relationship between different strains of the organism and specific lesion types (Millard and Burr, 1926; Schaal, 1940). De Bruyn (1939) concluded that potato varieties are not equally susceptible to different strains of the organism and that different varieties react differently to the same strain. Leach et al. (1939) indicated that the’ lesion is actually the result of an interaction between the pathogen and the host tissue and may be influenced as much by the host as by the pathogen. In these studies, it is suggested that infection in the field occurring on varieties that were previously classified as resistant might be due to different races of the pathogen. Variation in scab resistance at different locations has been reported (Stevenson and Clark, 1942; Darling, 1937). Thomas (1947), in studying several races on several cultivars in the greenhouse, concluded that a definite specificity for resistance and virulence exists between host and pathogen. On the other hand, Afanasiev (1937) and Emilsson and Gustafsson (1953) found no relationship between strains of Streptomyces and scab type. Varietal differences were found to be the major factor in determining pustule type. Stevenson 9 et al. (1942) showed that there were significant differences between potato varieties in their reactions to scab infection, and that comparative differences were fairly constant for the twenty—two varieties tested in four widely separated plots. This indicated that physiological forms of S. scabies did not play a very important part in these tests. McKee (1958) and Bjor and Roer (1980) found differences in several isolates of S. scabies in their ability to infect tubers; however, no host-pathogen interaction was reported. This suggests that varieties resistant in one area would be resistant in other areas. Although most researchers agree that physiological forms of the organism exist and show' a ‘wide range of pathogenicity, opinions differ on the occurrence of strains causing specific types of lesions and of strains especially virulent towards specific varieties. Nature of resistance in the potato A number of theories have been advanced to explain resistance. According to Sanford (1926), there seems to be a critical period of susceptibility during tuber growth. Fellows (1926) also found that infection appeared to be coincidental with a particular stage of growth. Richardson (1952) indicated that scab infection in the field takes place almost entirely on actively growing tubers, not on mature tubers or newly formed tubers. In addition, a number of studies have been done on the association of cellular 10 structure of the epidermis and lenticels with scab resistance (Lutman, 1919; Stuart, 1914; Darling et al., 1935; Longree, 1931). Darling (1937) concluded that the organism enters the tuber through the lenticels. Thus the amount of scab lesions covering the tuber surface may be a function of the number of lenticels infected. Woodcock (1952), however, did not find a correlation between the number of lenticels of varieties and scab resistance. The relationship of periderm development to resistance has also been considered. Histological studies (Jones, 1931; Labruyere, 1971) have suggested resistance is associated with' the effectiveness of the periderm to underlie lesions and wall them off from the tuber. Penetration of the actinomycete stimulates the tuber tissue to form a wound barrier a few cells below the surface. If this stops further invasion, a "superficial" scab, with little or no disruption of the tuber skin, is formed. If the barrier is destroyed, a second and third barrier may form before penetration is stopped, resulting in increasingly severe lesion types (Jellis, 1977). It has been known for many years that potato cultivars differ in the rate at which wounds in the tubers heal (McGee et al., 1985). Darling (1937) suggested resistance is associated with the time of suberization of the periderm. Cooper et al. (1954) proposed resistance is closely associated with the nature of the development of the periderm during tuberization. 11 Johnson and Schaal (1952, 1957) suggested there is a strong correlation between the amount of chlorogenic acid (CGA) in the periderm of the tuber and scab resistance. They reported. tubers ‘with. a :relatively’ high. chlorogenic acid content to be notably resistant, and susceptibility increased as the quantity of CGA decreased. Schaal and Johnson (1955) also studied the inhibitory effect of phenolic compounds on the growth of S. scabies as related to the mechanism of scab resistance. They offered two suggestions as to the effect of CGA: 1) It lowers the pH of the cells thereby creating an unfavorable medium for the growth of the bacteria; or 2) The acid itself or its quinone may be directly or indirectly involved in the formation of the suberized periderm. However, in detailed studies, Emilsson (1953) and Wolffgang and Hoffman (1959) were unable to show any relationship between scab resistance in field tests and high chlorogenic acid content. Holm and Adams (1960) investigated both chlorogenic acid and phenol oxidase content in potato cultivars and their relationship to resistance to common scab and found no correlation. LITERATURE CITED Adams, M.J. and D.H. Lapwood. 1978. Studies on the lenticel development surface microflora and infection by common scab (Streptomyces scabies) of potato tubers growing in wet and dry soils. Ann Appl Biol 90:336-343. Afanasiev, H.M. 1937. Comparative physiology of Actinomyces in relation to potato scab. Nebr Agr Exp Sta Res Bul 92. Alum, z. 1972. Inheritance of scab resistance in 24-chromosome potatoes. Ph.D. Diss. University of Wisconsin. Bjor, T. and L. Roer. 1980. Testing the resistance of potato varieties to common scab. Potato Res 23:33-47. Blodgett, F.M. and F.J. Stevenson. 1946. The new’ scab- resistant potatoes, Ontario, Seneca, and cayuga. Amer Potato J 23:315-329. Bloomquist, A.W. 1963. A quantitative genetic analysis of resistance to common scab (Streptomyces scabies Thaxt.) in the Irish potato (Solanum tuberosum L.). Ph.D. Diss. University of Minnesota. Cipar, M.S. and C.H. Lawrence. 1971. Scab resistance of haploids from two Solanum tuberosum cultivars. Amer Potato J 49:117-119. Clark, C.F., F.R. Stevenson. and L.A. Schaal. 1938. The inheritance of scab resistance in certain crosses and selfed lines of the potato. Phytopath 28:878-890. Cooper, D.C., G.W. Stokes and G.H. Rieman. 1954. Periderm development of the potato tuber and its relationship to scab resistance. Amer Potato J 31:58-66. Darling, H.M. 1937. A study of scab resistance in the potato. J Agr Res 54:305-317. Darling, H.M., J.G. Leach and F.A. Krantz. 1935. Scab resistance in potato seedlings. Phytopath 25:13-14. 12 13 De Bruyn, H.L.G. 1939. Onderzoekingen over enkele Actinomycetec welke Aardappelschurft verwekken. Tijdschr Plantenziekt 45:131-154. Dionne, L.A. and C.H. Lawrence. 1961. Early scab resistant derivatives of Solanum chacoense x Solanum pbureja. Amer Potato J 38:6-8. Dowley, L.J. 1972. Reliability of methods of assessing the degree of tuber attack by common scab of potatoes. Potato Res 15:263-265. Dreschler, C. 1919. Morphology of the genus Actinomyces. Bot Gaz 67:65-83, 147-168. Dykstra, T.P. 1956. A new method for screening first-year potato seedlings for scab resistance. Phytopath 25: 13-14. Emilsson, B. 1953. The relation between content of chlorogenic acid and scab resistance in potato varieties. Acta Agr Scand 3:328-333. Emilsson, B. and N. Gustafsson. 1953. Scab resistance in potato varieties. Acta Agr Scand 3:33-53. Fellows, H. 1926. Relation to growth in the potato tuber to the potato-scab disease. J Agr Res 32:757-781. Gunn, R.E., G.J. Jellis, P.J. Webb, and N.C. Sterling. 1983. Comparison of three methods for assessing varietal differences in resistance to common scab disease (Strep Streptomyces scabies) of potato. Potato Res 26: 173-178. Holm, E.T. and A.P. Adams. 1960. An investigation of the relationship of the chlorogenic acid and phenol oxidase content of a potato variety and its resistance to common scab. Enzymologia 22:245-250. Hooker, W.J. 1950. A technique for observing tuber enlargement and scab development in potatoes. Phytopath 40:390-391. Hooker, W.J. and O.T. Page. 1960. Relation of potato tuber growth and skin maturity to infection by common scab, Streptomyces scabies. Amer Potato J 37:414. Hougas, R.W. and R.W. Ross. 1954. The use of foreign introductions in the breeding of U.S. potato varieties. Amer Potato J 31:369 (Abstract). Jellis, G.J. 1977. The relative importance of host and environment in determining the incidence and severity of lesions of common scab (Streptomyces scabies) on potato. Potato Res 20:295-301. 14 Johnson, G. and IHA, Schaal. 1952. Relation of chlorogenic acid to scab resistance in potatoes. Science 115:627-629. Johnson, G. and L.A. Schaal. 1957. Chlorogenic acid and other orthodihydricphenols in scab-resistant Russet Burbank and scab-susceptible Triumph. potato tubers of different maturities. Phytopath 47:253-255. Jones, A.P. 1931. The histogeny of potato scab. Ann Appl Biol 18:313. Krantz, F.A. and C.J. Eide. 1941. Inheritance of reaction to common scab in the potato. J Agr Res 63:219-231. Labruyere, R.E. 1971. Common scab and its control in seed potato crops. Ag Res Rep 767:71pp. Lapwood, D.H. 1966. The effects of soil moisture at the time potato tubers are forming on the incidence of common scab (Streptomyces scabies). Ann Appl Biol 58:447-456. Lapwood, D.H. and T.F. Hering. 1968. Infection of potato tubers by common scab (Streptomyces scabies) during brief periods when soil is drying. Eur Potato J 11:177-187. Lapwood, D.H. and T.F. Hering. 1970. Soil moisture and the infection of young potato tubers by Streptomyces scabies (common scab). Potato Res 13:296-304. Large, E.C. 1955. Survey of common scab of potatoes in Great Britian, 1952 and 1953. Plant Path 4:1-8. Lauer, F.I. and.C.J. Eide. 1963. EvaluationLOf parental clones of potato for resistance to common scab by the "highest scab" method. Eur Potato J 6:35-44. Leach, J.G., F.A. Krantz, P. Decker, and.H. Mattson. 1938. The measurement and inheritance of scab resistance in selfed and hybrid progenies of potatoes. J Agr Res 56:843-853. Leach, J.G., P. Decker, and H. Becker. 1939. Pathogenic races of Actinomyces scabies in relation to scab resistance. Phytopath 29:204-209. Longree, K. 1931. Untersuschungen uber die ursache des verschiedenen verhaltens der kartoffelsorten gegen schorf. Arb Biol Reichaust Land-u Forstw 19:285-336. Lutman, B.F. 1919. Resistance of potato tubers to scab. Vt.Agr Exp Sta Bul 215. 15 McGee, E., M.C. Jarvis, and H.J. Duncan. 1985. Wound healing in. potato ‘tuber tissue. 2. ‘Varietal and anatomical variation. Potato Res 28:101-108. McKee, R.K. 1958. Assessment of resistance of potato varieties to common scab. Eur Potato J 1:65-80. McKee, R.E. 1963. Scab resistance of potato varieties. Plt Path 12:106-109. Millard, W.A. and S. Burr. 1926. A study of twenty-four strains of Actinomyces and their relation to types of common scab of potato. Ann Appl Biol 13:580-644. Reddick, D. 1939. Scab immunity. Amer Potato J 16:71-76. Richardson, J.K. 1952. The influence of tuber development on scab infection in Katahdin potatoes. Phytopath 42:297- 298. Rudorf, W. 1958. The significance of wild species for potato' breeding. Eur Potato J 1:10-20. Sanford, G.B. 1926. Some factors affecting the pathogenicity of Actinomyces scabies. Phytopath 16:525-547. Schaal, L.A. 1940. Cultural variation and physiologic specialization of Actinomyces scabies. Phytopath 30:21 (Abst.). Schaal, L.A. 1944. Variation and physiologic specialization in the common scab fungus (Actinomyces scabies). J Agr Res 69:169-186. Schaal, L.A. and G. Johnson. 1955. The inhibitory effect of phenolic compounds on the growth.of Streptomyces scabies as related to the mechanism of scab resistance. Phytopath 45:626-628. Stevenson, P.J., L.A. Schaal, C.F. Clark, R.V..Akeley, and.co- operators. 1942. Potato scab gardens in the United States. Phytopath 32:965-971. Stuart, W. 1914. Disease resistance of potatoes. Vt Agr Exp Sta Bul 179:145-183. Taylor, C.F. and P. Decker. 1947. A correlation between pathogenicity and cultural characteristics in the genus Actinomyces. Phytopath 37:49-58. 16 Terman, G.L., F.H. Steinmetz, and A. Hawkins. 1948. Effects of certain soil conditions and treatments upon potato yields and the development and control of potato scab. Maine Agr Exp Sta Bul 463. Thomas, W.D. 1947. Growth and variation of six physiologic races of Actinomyces scabies on different culture media. Phytopath 37:49-58. Waksam, S.A. 1919. Cultural studies of species of Actinomyces. Soil Sci 8:71-207. Walker, J.C., R.H. Larson, and A.R. Albert. 1938. Studies of resistance to potato scab in Wisconsin. Amer Potato J 15:246-252. Wiersema, H.T. 1974. Testing for resistance to common scab. Potato Res 17:356-357. Wolffgang, H. von and G.M. Hoffman. 1959. Die bedeuting der' chlorogensauer als resistanzefaktor. Zuchter 29:335-339. Woodcock, E.F. 1952. The correlation between the number of lenticels on potato tubers and susceptibility to the potato scab organism (Streptomyces scabies). Pap Mich Acad Sci 38:109-111. Chapter I: Methods for Scoring Scab Resistance 17 ABSTRACT Six methods were examined for scoring resistance of the potato to common scab disease (Streptomyces scabies). Eighteen cultivars were evaluated in the greenhouse and eight cultivars were evaluated in the field. Seven diploid lines were also included in the analysis. Tubers were scored for resistance according to 1) the highest percent tuber surface covered with scab in a pot; 2) the highest (most severe) lesion type on a tuber in a pot; 3) the highest combined‘ rating'of percent surface cover and most severe lesion.type of a tuber in a pot; 4) the average percent surface covered with scab of all the tubers in.a pot; 5) the average lesion type of all the tubers in a pot; 6) the average combined rating of all the tubers in a pot. There were significant differences between the cultivars for all six of the scoring systems in the greenhouse, but only for the average scoring systems in the field, and only for some of the scoring systems among the diploid potato lines. The highest scoring systems tended to place the cultivars and diploid lines into the susceptible classes in the greenhouse and in the field. In the greenhouse, the average combined rating placed the cultivars into the expected classes of resistance. 18 19 INTRODUCTION The most effective means of controlling the common scab disease of potato is to grow resistant varieties. Thus, for variety development, it is essential to be able to score potential varieties for scab resistance efficiently and accurately. Unfortunately, scab resistance is inherently difficult to evaluate because the symptoms are variable, and breeders are tempted to evaluate scab in a rather subjective manner. However, in order to determine the genetic basis of scab resistance, it is necessary to use a system for scoring resistance that is consistent and will divide infected progeny ' into distinct classes. Many methods for scoring scab resistance have been employed through the years. There are two common methods. One is to score tubers based on the percent surface area of the tuber that is covered with scab lesions (Darling, 1937; Clark et al., 1938; Large, 1955). The other is to score tubers based on the type of lesion observed on the tuber (Leach et al, 1938; Schaal, 1944; Lauer and Eide, 1963). There does not seem.to be a consensus on whether a cover score rating or a lesion type rating is better to quantify and compare resistance among varieties. Most studies that have attempted to critically compare these different methods have been concerned with field evaluation of a large number of lines, where environment and the need for ease and speed of evaluation are factors 20 (Stevenson. et al., 1942; Emilsson. and. Gustafsson, 1953; Dowley, 1972) . In the greenhouse, at least two studies regarding the criteria for the scoring of scab resistance have been conducted. McKee (1958) classified varieties on the proportion of surface affected by scab and on the amount of damage. Bjor and Roer (1980) considered percent surface covered and lesion type as criteria for scab assessment in a greenhouse study of scab resistance. It is apparent that an efficient and consistent means of scoring tubers for scab resistance in the greenhouse as well as the field is necessary. In this study, cultivars and' diploid lines were evaluated in the greenhouse according to cover score and the type of lesion, and these criteria for scoring tubers were compared. A combined rating (cover score x lesion type) was also considered. Average scores per pot were compared with.a highest scab score per pot. ‘Use of these same criteria for evaluation in the field were also considered. Because the resistance levels of the cultivars have been characterized in the past, it will be possible to compare the scoring systems according to which systems place the cultivars into the expected classes of resistance. MATERIALS AND METHODS Cultivars were evaluated twice in the greenhouse, in the summer of 1989 and in the winter of 1990. The diploid lines were evaluated three times in the greenhouse: 1) Summer of 21 1989; 2) Winter of 1990; and 3) Spring of 1990. The cultivar and diploid lines evaluated are listed in Table 1. Cultivars of known scab resistance were chosen for this analysis, although.the levels of resistance among the diploid lineS‘were not known at the time of screening. The strain of Streptomyces used in this study, DP(z), was one known to cause deep-pitted lesions on susceptible tubers. The organism was maintained at 22°C on a yeast malt extract (YME) agar. Cultures would typically sporulate 7-10 days after transfer to fresh agar. Sporulating’ cultures 'were blended. with ‘water and a' measured amount was then hand-mixed separately for each pot into a premixed potting soil (Baccto). The amount of inoculum was approximately 0.4 to 0.5 petri dish culture per 8 inch.pot and 0.3 petri dish culture per 5 inch pot. Cultivars were grown in 8 inch clay pots and diploids were grown in 5 inch clay pots. Each evaluation was conducted using a randomized complete block design with three blocks. In the summer 1989 experiments, a low moisture level was maintained by watering into aluminum trays placed underneath the pots. In the winter and spring 1990 experiments, a low moisture level was :maintained. by less frequent ‘watering directly to the pot based on individual inspection. The tubers were harvested for evaluation at maturity (14 weeks). Tubers under two centimeters in diameter for 22 Table 1. Cultivars (4x) and diploid lines (2x) evaluated for scab resistance. Clones Ploidy Species‘ 19892 19902 OH Field “— Burbank 4x S. tuberosum X X Russet 4x 8. tuberosum X X Burbank Superior 4x 8. tuberosum X X X X Atlantic 4x 8. tuberosum X X X X Lemhi 4x 3. tuberosum X X X X Russet Saginaw 4x S. tuberosum X X X X Gold Spartan 4x S. tuberosum X X X X Pearl Onaway 4x S. tuberosum X X X X ND860-2 4x 5. tuberoswm X X X X Monona 4x 8. tuberosum X X X Y245.7 4x S. tuberosum X X X Nooksack 4x S. tuberosum X X X Xatahdin 4x S. tuberosum X X X Ontario 4x 3. tuberosum X X X X MS716-15 4x 5. tuberosum X X X Michigold 4x 8. tuberosum X X X NDD277-2 4x S. tuberosum X X Hindenburgfi 4x S. tuberosum X X Continued. 23 Table 1. Continued. Clones Ploidy Species1 19892 19902 GB: Field BSSDSS 2x S. tuberosum x X X S. bertbaltii 84810 2x 8. phureja X X X 84SD22 2x S. tuberosum x X X X S. chacoense DM56-4 2x S. phureja X X X W5295.7 2x S. phureja x X X X S. tuberosum PlOO-2 2x S. tuberosum x X X X S. tarijense W5337.3 2x S. phureja x X X S. tuberosum 'Huaman and Ross, 1985. 2Cultivars or diploids were included for evaluation in the experiment or experiments of this year. 24 cultivars were not evaluated. Diploid tubers were generally very small (less than one centimeter in diameter) and any size tuber was evaluated. Each pot was treated as one replicate. There were six systems for scoring the potato lines in this study: 1) Highest percent tuber surface covered with scab per pot; 2) Highest (most severe) lesion type per pot; 3) Highest combined rating per pot; 4) Average percent tuber surface covered with scab per pot; 5) Average lesion type per pot; 6) Average combined rating per pot. Every tuber in a pot was given a numerical rating from one to four (Table 2) according to the percent surface area covered with scab (Figure 1) and then according to the predominant type of lesion present on the tuber (Figure 2). For each tuber, the numerical ratings for cover and type were then multiplied together to form a combined rating of scab, also shown in Table 2. The six systems (listed above) for scoring the tubers were based on the scores of the individual tubers from each pot. Among all the tubers in one pot, the three highest scab ratings per pot were determined (highest % surface cover, highest lesion type, and highest combined rating per pot). The scores of the individual tubers were then added together and the total was divided by the number of tubers in the pot to determine the three average ratings per pot. The mean 25 Table 2. Numerical rating of % surface cover, lesion type and combined rating for each tuber and the class of resistance (R, MR, MS, S)l designated by the numerical ratings. Class of Numerical % Surface Lesion Resistance Rating cover Type R 1 No Scab No Scab MR 2 1-5 % Surface MS 3 5-25 % Slightly Pitted S 4 > 25 % Deep Pitted Combined Rating R 1 No Scab MR 4 1-5 % , Surface Scab MR 6 1-5 % , Slightly Pitted -OR- 5-25 % , Surface Scab MS 8 1-5 % , Deep Pitted -OR- > 25 % , Slightly Pitted MS 9 5-25 % , Slightly Pitted S 12 5-25 % , Deep Pitted -OR- > 25 % , Slightly Pitted S 16 > 25 % , Deep Pitted 'R-Resistant, MR=Moderately SsSusceptible Resistant, MS=Moderately Susceptible, 26 Figure 1. Tubers representing the classes of surface cover and their corresponding numerical ratings (left to right): (2) 1-5% ; (3) 5-25% ; (4) >25%. Figure 2. Tubers representing classes of lesion types and their corresponding numerical ratings (clockwise from bottom): (2) superficial; (3) slightly pitted; (4) deep pitted; (4) deep pitted. 27 numerical rating across replications was used to place the lines into resistant, moderately resistant, moderately susceptible, or susceptible classes (Table 2). Three replications (ten tubers from each plot) of eight cultivars (Table 1) were similarly evaluated from the field in one year to make a comparison of field evaluation and greenhouse evaluation. The same DP(z) strain of Streptomyces was used to artificially infest the field with the organism at the time of planting. Significant differences between lines, rankings of lines, and the coefficients of variation were compared between the. six scoring systems. Correlation coefficients were also calculated among the scoring systems. All statistical analysis was performed using the Michigan State university' MSTATC program. Analysis of variance was used to test for significant differences among means. Duncan‘s Multiple Range Test was used to determine ranking. Cultivars and diploid lines were placed into the classes of resistance according to the following divisions of numerical scores: For the % surface cover and for the lesion type scoring systems, 0 - 1 was resistant (R), >1 - 2 was moderately resistant (MR), >2 - 3 was moderately susceptible (MS), and >3 - 4 was susceptible (S); For the combined rating scoring systems, 0 - <4 was resistant (R), 4 - <8 was moderately resistant (MR), 8 - <12 was moderately susceptible (MS), and 12 - 16 was susceptible (S) . 28 RESULTS Outliers Classifications of cultivars, significant differences, and.the coefficients of variation (C.V.) for the three highest scab scoring systems in the summer 1989 experiment are shown in Table 3. The results for the three average scab scoring systems in the summer 1989 experiment are shown in Table 4. Similarly, results from.the spring of 1990 are shown.in.Tables 5 and 6. The cultivars are ranked from most resistant to most susceptible according to Duncan's Multiple Range test. Correlation coefficients among the different scoring systems. are shown in Table 7. The incidence of scab varied significantly between the summer 1989 and winter 1990 experiments for all methods of scoring. Disease was less severe in the summer 1989 experiment than in the spring 1990 experiment. There were significant differences (p=0.01) between the cultivars for all of the scoring systems both years. The coefficient of variation was lower for the three highest scab ratings than the three average scab ratings in.both.years. 'The coefficient of variation was higher when the combined rating was considered than when either the percent coverage or type of lesion were considered alone in both years. For all of the scoring systems, according to Duncan's Multiple Range Test, the order in ‘which the cultivars are ranked from :most resistant to most susceptible was similar. 29 Table 3. Classifications, rankings, significant differences and coefficients of variation for cultivars evaluated in the summer 1989 according to the highest scab per pot. RESISTANT MODERATELY MODERATELY SUSCEPTIBLE SCORING RESISTANT SUSCEPTIBLE SYSTEM HIGHEST Burbank Superior Onaway Atlantic PERCENT Nooksack R. Burbank M8716-15 SURFACE Lemhi Russet Ontario Monona COVER 2245-7 Saginaw Gold Spartan Pearl Michigold ** Katahdin ND860—2 C.V.= 8.60% H P HIGHEST Nooksack Burbank Ontario N0860-2 LESION Lemhi Russet Superior Katahdin Spartan Pearl TYPE Michigold 2245-7 R. Burbank Onaway Atlantic ** Ms7l6-15 Monona C.V.- Saginaw Gold 10.61% HIGHEST Burbank Ontario Onaway R. Burbank COMBINED Nooksack Atlantic RATING Lemhi Russet Xatahdin Superior Michigold MS716-15 Monona Saginaw Gold ** 2245-7 Spartan Pearl C.V.= N0860-2 18.53% "Significant differences between the cultivars within the scoring system at p20.01. 30 Table 4. Classifications, rankings, significant differences and coefficients of variation for cultivars evaluated in the summer 1989 according to the average scab per pot. RESISTANT MODERATELY MODERATELY SUSCEPTIBLB SCORING RESISTANT SUSCEPTIBLE SYSTEM . AVERAGE Nooksack Burbank R. Burbank Monona PERCENT Lemhi Russet Superior Atlantic Saginaw Gold SURFACE Ontario Spartan Pearl COVER Onaway Michigold M8716-15 Xatahdin Y245-7 N0860-2 ** C.V.- 21.24% " AVERAGE Burbank Ontario R. Burbank. N0860-2 LESION Nooksack Superior Atlantic Spartan Pearl TYPE Lemhi Russet Onaway 2245-7 Xatahdin Michigold Saginaw Gold ** Monona MS716-15 C.V.8 23.61% :3 — AVERAGE Burbank Superior Onaway Monona COMBINED Nooksack Ontario Atlantic Xatahdin RATING Lemhi Russet R. Burbank M8716-15 Michigold Saginaw Gold Spartan Pearl ND860-2 2245-7 ** C.V.- 31.40% "Significant differences between the cultivars within the rating system.at p-0.01. 31 Table 5. Classifications, rankings, significant differences and coefficients of variation for cultivars evaluated in the spring 1990 according to the highest scab per pot. RESISTANT MODERATELY MODERATELY SUSCEPTIBLE SCORING RESISTANT SUSCEPTIBLE SYSTEM HIGHEST Lemhi Russet Hindenburg Ontario PERCENT Superior Onaway SURFACE Nooksack ND860-2 COVER M8716-15 Michigold Monona Saginaw Gold NDD277-2 ** Spartan Pearl Xatahdin C.V.- Atlantic 15.09% Y245-7 HIGHEST Lemhi Russet Nooksack Ontario Atlantic LESION Hindenburg Onaway Superior TYPE ND860-2 Y245-7 Michigold Monona M8716-15 ** Spartan Pearl NDD277-2 C.V.= Saginaw Gold 14.72% .J Katahdin HIGHEST Lemhi Russet Hindenburg Superior Onaway COMBINED Nooksack Ontario Atlantic RATING N0860-2 Y24S-7 Michigold Monona Ms7l6-15 ** Spartan Pearl NDDZ77-2 C.V.- Saginaw Gold 18.50% Katahdin ”Significant differences between cultivars within the p-0.0l. rating system at Table 6. Classifications, 32 rankings, significant differences and coefficients of variation for cultivars evaluated in the spring 1990 according to the average scab per pot. RESISTANT MODERATELY MODERATELY SUSCEPTIBLE SCORING RESISTANT SUSCEPTIBLE SYSTEM AVERAGE Lemhi Russet Hindenburg Nooksack Michigold PERCENT Superior Atlantic Spartan Pearl SURFACE Ontario Katahdin COVER Saginaw Gold ND860-2 Onaway NDD277-2 Y245-7 MS7l6-15 ** Monona C.V.3 21.24% _‘ AVERAGE Lemhi Russet Hindenburg Ontario Saginaw Gold LESION Nooksack Atlantic Michigold TYPE Superior Spartan Pearl. Xatahdin Onaway Y245-7 ND860—2 MS716-15 Monona ** NDD277-2 C.V.- 23.61% m AVERAGE Lemhi Russet Nooksack Atlantic Saginaw Gold COMBINED Hindenburg Superior Onaway Michigold RATING Ontario Spartan Pearl. Xatahdin Y245-7 ND860-2 MS716-15 ND0277-2 ** Monona C.V.= 25.47% “Significant differences between cultivars within the scoring system at p-o.01. 33 Table 7. Correlation coefficients among the different scoring systems for the cultivar greenhouse and field evaluations. Correlations: AVG % - High % Avg Type-High Avg Combined- Type High Combined Summer, 1989 .901 .887 .928 Spring, 1990 .780 .806 .822 Field .806 .705 .885 High % - High High % - High High Type-High Type Combined Combined Summer, 1989 .902 .950 .969 Spring, 1990 .773 .892 .954 Field .655 .841 .886 Avg % - Avg Avg % - Avg Avg Type - Avg Type Combined Combined Summer, 1989 .910 .963 .914 Spring, 1990 .908 .955 .978 Field .888 .949 .950 34 However, classification as resistant, moderately resistant, moderately susceptible, and susceptible according to the numerical ratings was different between the different scoring systems. There was a significant correlation between all of the scoring systems that were compared. £121.51 The classifications and rankings, significant differences and. coefficients of ‘variation for the. different scoring systems of the eight cultivars in the field are shown in Tables 8 and 9. The correlation coefficients are shown in Table 7. In the field, the highest scab methods of rating (Table 8) did not determine significant differences among the means of the cultivars, but the average methods of rating (Table 9) did. There was a significant difference at p=0.05 for the average percent coverage, and at p=0.01 for average type and average combined ratings. The order in which the cultivars were ranked was similar for the different scoring systems. IHowever, the average rating methods (Table 9) tended to classify most of the cultivars as moderately resistant or resistant, while the highest scab ratings (Table 8) divided the cultivars into moderately resistant, moderately susceptible, and susceptible classes. The coefficient of variation is relatively high for all of the scoring systems in the field. All of the scoring systems were significantly correlated. 35 Table 8. Classifications, rankings, significant differences and coefficients of variation for cultivars evaluated in the field according to the highest scab per pot. RESISTANT MODERATELY MODERATELY SUSCEPTIBLE SCORING RESISTANT SUSCEPTIBLE SYSTEM 1 HIGHEST Lemhi Russet Saginaw Gold PERCENT Superior Atlantic SURFACE Ontario Spartan Pearl COVER Onaway ND860-2 n.s. C.V.- 28.72 HIGHEST Lemhi Russet Saginaw Gold Spartan Pearl ' LESION Ontario Onaway ND860-2 TYPE Atlantic Superior n.s. C.V.- 31.08% HIGHEST Lemhi Russet Atlantic Saginaw Gold COMBINED Ontario Onaway Spartan Pearl RATING Superior ND860-2 n.s. Cov.‘ 46.02% n.s. system. no significant differences between cultivars within the scoring 36 Table 9. Classifications, rankings, significant differences and coefficients of variation for cultivars evaluated in the field according to the average scab per pot. RESISTANT MODERATELY MODERATELY SUSCEPTIBLE SCORING RESISTANT SUSCEPTIBLE SYSTEM AVERAGE Lemhi Russet ND860-2 PERCENT Superior SURFACE Ontario COVER Onaway Saginaw Gold * Spartan Pearl C.V.- Atlantic 21.49% AVERAGE Lemhi Russet ND860-2 LESION Ontario TYPE Superior Spartan Pearl ** Saginaw Gold Atlantic C.V.- Onaway 21.68% AVERAGE Lemhi Onaway ND860-2 COMBINED Russet Saginaw Gold RATING Ontario Spartan Superior Pearl Atlantic ** c.v.- 39.82% 'significant differences between cultivars within the scoring system at 80.05. 2significant differences between cultivars within the scoring system at p-o.01. 37 111219.112 The classifications and rankings, significant differences, and coefficients of variation for the diploid lines evaluated in the summer of 1989, winter of 1990, and spring of 1990 are shown in Tables 10 and 11, 12 and 13, and 14 and 15, respectively. The correlation coefficients among the different scoring systems for these three experiments are shown in Table 16. Like the cultivar evaluation, less severe disease was observed in the summer 1989 experiment than in the winter or spring, 1990 experiments. In fact, in the latter two experiments, the diploid lines were only classified as moderately susceptible or susceptible. Among the diploids, there were fewer significant differences in the winter and spring, 1990 experiments. The general order in which the diploid lineS'were ranked was.different from.one experiment to the next. There were also differences between the different scoring systems in the order in which the tubers were ranked in the winter and spring, 1990 experiments. The combined rating generally had a higher coefficient of variation than the cover ratings or type of lesions ratings in all three experiments. The coefficients of variation were lower for the highest scab rating systems than the average scab rating systems in the winter and spring 1990 experiments, as was the case for the cultivars. This was not true, however, in the summer 1989 evaluation of theidiploid lines. ‘The correlation 38 Table 10. Classifications, rankings, significant differences and coefficients of variation for diploid lines evaluated in the summer 1989 according to the highest scab per pot. RESISTANT MODERATELY MODERATELY SUSCEPTIBLE SCORING RESISTANT SUSCEPTIBLE SYSTEM HIGHEST 84810 W5295.7 DM56-4 PERCENT 84SD22 BSSDSS SURFACE FIDO-2 COVER ** C.V.- 17.678 HIGHEST 84810 W5295.7 PIOO-Z LESION 84SD22 BSSDSS TYPE DM56-4 * c0v.. 20.59‘ HIGHEST 84810 W5295.7 DM56-4 COMBINED 84SD22 BSSDSS RATING FIDO-2 fit C.V.' 29.878 'significant differences between diploids within the scoring system at 0.05. 2:ignificant differences between diploids within the scoring system at p=0.01. 39 Table 11. Classifications, rankings, significant differences and coefficients of variation for diploid lines evaluated in the summer 1989 according to the average scab per pot. RESISTANT MODERATELY MODERATELY SUSCEPTIBLE SCORING RESISTANT SUSCEPTIBLE SYSTEM 1 AVERAGE 84810 858D55 9100-2 PERCENT 84SD22 DM56-4 SURFACE ws295.7 COVER it c.v.- 17.65% I _ AVERAGE 84810 858055 P100-2 LESION 848D22 DM56-4 TYPE ws295.7 ** c.v.- 19.86% AVERAGE 84810 w529s.7 DM56-4 P100-2 COMBINED 84SD22 essoss RATING ** c.v.- 31.82% ’significant differences between diploids within the scoring system at p=0.01. 40 Table 12. Classifications, rankings, significant differences and coefficients of variation for diploid lines evaluated in the winter 1990 according to the highest scab per pot. RESISTANT MDDERATELY MODERATELY SUSCEPTIBLE SCORING RESISTANT SUSCEPTIBLE SYSTEM _— HIGHEST P100-2 PERCENT W5295.7 SURFACE DM56-4 COVER W5337.3 848022 n.s. 84810 C.V.8 5.988 _ HIGHEST 848022 W5337.3 LESION P100-2 W5295.7 TYPE 84510 DM56-4 s C.V.- 15.258 _ HIGHEST 84SD22 COMBINED P100-2 RATING W5337.3 W5295.7 84810 * DM56-4 C.V.- 15.668 n.s. no significant differences between diploids within a scoring system. ' significant differences between diploids within a scoring system at p=0.05. 41 Table 13. Classifications, rankings, significant differences and coefficients of variation for diploid lines evaluated in the winter 1990 according to the average scab per pot. RESISTANT MDDERATELY MODERATELY SUSCEPTIBLE SCORING RESISTANT SUSCEPTIBLE SYSTEM AVERAGE 84810 848022 PERCENT P100-2 W5295.7 SURFACE W5337.3 COVER DM56-4 n.s. C.V.- 17.918 AVERAGE 84810 W5337.3 LESION 848022 DM56-4 TYPE P100-2 W5295.7 * c0v0. 18.328 AVERAGE P100-2 W5295.7 COMBINED 84810 W5337.3 RATING 848022 DM56-4 * CsVO. 24.258 n.s. no significant differences between diploids within a scoring system. ' significant differences between diploids within a scoring system at p=0.05. 42 Table 14. Classifications, rankings, significant differences and coefficients of variation for diploid lines evaluated in the spring 1990 according to the highest scab per pot. RESISTANT MODERATELY MODERATELY SUSCEPTIBLE SCORING RESISTANT SUSCEPTIBLE HIGHEST 34310 PERCENT w5295 . 7 SURFACE 93455-4 COVER 848022 W5337.3 n.s. C.V.- 8.99% 11 HIGHEST 848022 LESION P100-2 TYPE W5337.3 W5295.7 DMS6-4 * 84810 C.V.- 15.25% F HIGHEST 845022 COMBINED P100-2 RATING w5337.3 W5295.7 DM56-4 * 84810 C.V.- 17.45% n.s. no significant differences between diploids within a scoring system. ' significant differences between diploids within a scoring system at p=0.05. Table 15. 43 Classifications, rankings, significant differences and coefficients of variation for diploid lines evaluated in the spring 1990 according to the average scab per pot. RESISTANT MODERATELY MODERATELY SUSCEPTIBLE SCORING RESISTANT SUSCEPTIBLE SYSTEM s L AVERAGE P100-2 84810 PERCENT W5295.7 SURFACE DM56-4 COVER 848022 W5337.3 n. I. CsVs. 17087‘ ”L AVERAGE 848022 0M56-4 LESION P100-2 TYPE W5295.7 W5337.3 84810 * GOV.- 17.88% fifi AVERAGE P100-2 848022 COMBINED W5295.7 RATING 84810 W5337.3 0M56-4 n. I. C.V.= 26.68% n.s. no significant differences between diploids within a scoring system. significant differences between diploids within a scoring system at p=0.05. 44 Table 16. Correlation Coefficients among the different scoring systems for the diploid greenhouse evaluations. Correlations: AVG % - High % Avg Type-High Avg Combined- Type High Combined Summer, 1989 .839 .884 .910 Winter, 1990 .456 .792 .666 Spring, 1990 .523 .592 .654 High % - High High % - High High Type-High Type Combined Combined Summer, 1989 .806 .925 .933 Winter, 1990 .000 .229 .918 Spring, 1990 .059 .296 .971 Avg % - Avg Avg % - Avg Avg Type - Avg Type Combined Combined Summer, 1989 .940 .959 .986 Winter, 1990 .453 .697 .950 Spring, 1990 .739 .823 .887 45 coefficients were high among all of the scoring systems compared, except between highest percent surface cover and most severe lesion type and between highest percent surface cover and highest combined rating in the winter and spring 1990 experiments. DISCUSSION The main objective of these studies was to develop a satisfactory system for scoring scab resistance of parents and progeny to be used in future studies of the inheritance of scab resistance» These ‘results should also provide an effective means of selecting new material for breeding of scab resistant cultivars. The results of the cultivar analysis are different than those for the diploid analysis. Their results are discussed separately here. We We included an analysis of varieties in the field to determine whether the results of these different systems for scoring would be similar in the greenhouse and in the field. Obviously the infection level was much lower in the field.than in the greenhouse, resulting in mostly resistant ratings when the average score per plot was considered (Tables 8 and 9). A low level of infection in field scab evaluations has been common in our breeding program. This suggests that in the field, it would be better to score lines according to the 46 highest.rating. But, as would be expected, the coefficient of variation is higher in general in the field than it is in the greenhouse. For this reason, the evaluation of parents and progeny for inheritance studies would.be best conducted in the greenhouse, where variation due to effects of the environment is reduced and the amount of inoculum is more uniform. Studies in the greenhouse, similar to this study, have been conducted. McKee (1958) conducted a greenhouse study to evaluate scab resistance according to the proportion of the surface covered with scab and the type of scab. The cover system consisted of five categories (0-1/16, 1/16-1/8, 1/8- 1/4, 1/4-1/2, 1/2-1). The type of scab was based on an estimate of damage severity (none, very slight, slight, moderate, or severe). Bjor and Roer (1980) divided cover score into ten.categories (0, < 5%, 5-10%, 10-25%, 25-50%, 50- 75%, 75-90%, 90-95%, 95-99%, 100%) and rated lesion severity as superficial, medium deep or raised, or deep. In this study, the cover score rating according to the percent surface area covered with scab is only divided into four categories: 0%, 1-5%, 5-25%, and >25%. These four categories seemed sufficient to divide the lines into the classes of resistance, i.e., resistant, moderately resistant, moderately susceptible, and susceptible. When there are as many categories as those chosen by the above mentioned studies, it is difficult to distinguish between the classes of resistance. 47 The description of the lesion type in this study was the same as Bjor and Roer (1980), except here there is no score for raised lesion types. Raised lesions were occasionally observed in the present study, but were never a predominant lesion type and were determined to be a function of environmental conditions at the time of infection. The four descriptions of the lesion types (none, superficial, slightly pitted, and deep pitted) could easily be given a numerical rating and be placed into the four resistance classes, where none would be resistant, superficial would be moderately resistant, etc. McKee (1958) did not.consider“whether an.average rating or one rating per pot would be better, but Bjor and Roer (1980) suggested that giving one score for all the potatoes in a single pot is as reliable as scoring each tuber separately. According to our results (Table 7), average percent ratings are correlated to high percent ratings (correlation coefficients in 1989 and 1990, respectively : .901, .780), average type ratings are correlated to high type ratings (.887, .806), and average combined ratings are correlated to high.combined.ratings (.928, .822). This suggests that either average ratings or one rating per pot can be used to»determine scab resistance. However, the distribution of the cultivars among the classes of resistance when the average score is considered is more even than when the high score is considered (Tables 3 to 48 6). This is especially true in the spring 1990 analysis when there was a higher level of infection. The higher level of infection could be due to the different method of watering in the two experiments. Limited watering directly to the pot may have provided dryer soil for more severe infection than does watering normally to a container underneath the pot. Also, evaluation in different seasons may effect the soil moisture and thus the severity of the disease (Lapwood, 1966; Lapwood and Hering, 1970). This type of variation is bound to occur when scab evaluation is repeated in the future. Therefore, it may be a worthy precaution to score all of the tubers in a pot and score the average rather than score according to one high score per pot. The higher coefficient of variation when the average rating is considered may be misleading. The coefficient of variation for the highest rating system is lower because there tends to be more uniformity between replicates. That is, when a highest rating system is applied, if only one tuber in a pot has deep-pitted lesions, it is given the same rating for lesion type (a numerical rating of 4) as a pot in which all of the tubers have deep-pitted lesions. In McKee's (1958) study, mean scores for cover and damage were considered, and it was concluded that scores based on cover allowed better separation of varieties into groups than did damage scores, according to agreement with field data. 49 But, McKee did not take into account the type of lesion according to the particular appearance of the lesions, i.e., superficial or pitted, etc. And he concluded that in many of his greenhouse experiments, the incidence of infection was irregular and very low so that the majority of tubers in a pot fell in the category of 0-1/16 and were designated with a score of zero. In these cases, scores based on damage were often higher and more useful than those based on cover. Bjor and Roer (1981) concluded from analysis of variance that scores for lesion severity separated varieties at least as well as scores for surface cover. They also calculated a scab index as a product of the two assessments, similar to the combined rating in this study, and suggested that the use of both in a compound index should give a better estimate of resistance than either of them alone. In this study, scores for surface cover, lesion type, and a combined rating of the two all separated varieties equally as well and were significantly correlated with each other. This would suggest that any of these scoring systems could be used to evaluate scab :resistance among' cultivars. The coefficient of variation is higher when the combined rating is considered, but this is probably only because the range of numerical rankings is larger for this method. It should be noted, however, that there ‘were some differences in the classifications of the cultivars between the different methods. In the summer of 1989 average ratings 50 (Table 4), Ontario and Russet Burbank, two varieties that we would expect to see rated as resistant according to field tests through the years, are rated as moderately susceptible according to surface cover. When type is scored, Ontario moves to the moderately resistant class, and when the combined rating is considered, both Ontario and Russet Burbank move into the moderately resistant class. Nooksack and Ontario similarly shift classes in the spring 1990 average evaluation (Table 6) . Because the combined rating most effectively placed cultivars of known resistance levels into the expected classes, we conclude that it is the most accurate means of classifying scab resistance. For inheritance studies, it would probabLy be best to score parents and progeny according to both lesion type and surface cover to determine whether they are controlled by separate genetic factors. For the selection of new'varieties, the breeder should choose a method for scoring that would.best suit breeding goals. For example, good chipping varieties would best be selected according to lesion type, because deep pitted lesions would be unacceptable in the processing market regardless of the surface cover. However, tubers from tablestock cultivars are graded out according to surface coverage. 51 1219.19.10: The evaluation of scab resistance of wild species and diploid lines containing wild germplasm has been considered valuable in the past (Reddick, 1939; Rudorf, 1958; Dionne and Lawrence, 1961; Cipar and Lawrence, 1971) . This germplasm may offer higher levels of resistance than S. tuberosum. In order for diploid lines to be useful in transferring resistance from wild species to the cultivated potato, an accurate assessment of resistance in diploid lines is essential. Furthermore, when resistance levels have been established in these diploid lines, they may be used as parents in crosses to study the inheritance of resistance at the diploid level. Unfortunately, some problems are encountered when evaluating the diploid germplasm which are not encountered when evaluating the cultivars. Specifically, because the diploid lines have been derived from South American germplasm, they tuberize more effectively under shorter day length. They also tend to produce relatively smaller tubers than the North American cultivars. The smaller size of the tubers and the dependence on shorter day lengths must contribute to the extreme differences from experiment to experiment in this analysis. The diploid tubers harvested in the summer 1989 experiment were extremely small. Most tubers were less than one centimeter. Perhaps tubers of the lines classified as resistant in that experiment 52 were so small that they did not expand enough for the lesions to become evident. The tubers from the winter and spring 1990, when the day length was shorter, were larger in size (between one centimeter and four centimeter). In these:two experiments, no lines were classified as resistant. This may suggest that all of the six diploid lines we evaluated were susceptible. However, in the cultivar analysis, there‘were also fewer lines classified as resistant in the spring 1990 experiment than in the summer 1989. The resistance of these diploid lines needs to be further investigated. When working with diploid lines such as these, it would be advisable to evaluate the lines at least three independent times before drawing any conclusions. Also, great care should be taken to note the day lengths under which the individual diploid lines are best suited for tuberization and to note the size of the tubers upon evaluation. It may even be advisable to use a growth chamber for evaluation of the diploid lines. LITERATURE CITED Bjor, T. and L. Roer. 1980. Testing the resistance of potato varieties to common scab. Potato Res 23:33-47. Cipar, M.S. and C.H. Lawrence. 1971. Scab resistance of haploids from two Solanum tuberosum cultivars. Amer Potato J 49:117-119. Clark, C.F., F.R. Stevenson and L.A. Schaal. 1938. The inheritance of scab resistance in certain crosses and selfed lines of the potato. Phytopath 28:878-890. Darling, H.M. 1937. A study of scab:resistance in the potato. J Agr Res 54:305-317. Dionne, L.A. and C.H. Lawrence. 1961. Early scab resistant derivatives of Solanum chacoense x Solanum phureja. Amer Potato J 38:6-8. Dowley, L.J. 1972. Reliability of methods of assessing the degree of tuber attack by common scab of potatoes. Potato Res 15:263-265. Emilsson, B. and N. Gustafsson. 1953. Scab resistance in potato varieties. Acta Agr Scand 3:33-52. Hauman, z. and R.W. Ross. 1985. Updated listing of potato species names, abbreviations and taxanomic status. Am Potato J 62:629-641. Lapwood, D.H. 1966. The effects of soil moisture at the time potato tubers are forming on the incidence of common scab (Streptomyces scabies). Ann Appl Biol 58:447-456. Lapwood, D.H. and T.P. Hering. 1970. Soil moisture and the infection of young potato tubers by Streptomyces scabies (common scab). Potato Res 13:296-304. Large, E.C. 1955. Survey of common scab of potatoes in Great Britian, 1952 and 1953. Plant Path 4:1-8. Lauer, F.I. and C.J. Eide. 1963. Evaluation of parental clones of potato for resistance to common scab by the "highest scab" method. Eur Potato J 6:35-44. 53 54 Leach, J.G., F.A. Krantz, P. Decker, and H. Mattson. 1938. The measurement and inheritance of scab resistance in selfed and hybrid progenies of potatoes. J Agr Res 56:843-853. McKee, R.E. 1958. Assessment of resistance of potato varieties to common scab. Eur Potato J 1:65-80. Reddick, D. 1939. Scab immunity. Amer Potato J 16:71-76. Rudorf, W. 1958. The significance of wild species for potato breeding. Eur Potato J 1:10-20. Schaal, L.A. 1944. Variation and physiologic specialization in the common scab fungus (Actinomyces scabies). J Agr Res 69:169-186. Stevenson, P.J., L.A. Schaal, C.F. Clark, R.V. Akeley and co-operators. 1942. Potato scab gardens in the United States. Phytopath 32:49-58. Chapter II: The Effect of Strains of the Scab Organism on the Potato 55 ABSTRACT The effect of six strains of the scab forming organism (Streptomyces) on seven potato cultivars and six diploid lines was examined. Plants were grown in the greenhouse in soil inoculated with the individual strains. Mature tubers were evaluated in three ways: 1) percent surface covered with lesions; 2) type of lesion; and 3) a combined rating of percent surface covered and type of lesion. There were significant differences between strains and between potato lines for each of the three rating systems. There were also significant interactions between strains and varieties for each of the rating systems, and the cover score and lesion type were effected independently by these interactions. INTRODUCTION A number of studies have addressed the question of the effect on disease severity of different strains of the scab producing organism (Streptomyces) on potato varieties (Millard and Burr, 1926; Schall, 1940; Leach et al., 1939; Taylor and Decker, 1947; Emilsson and Gustafsson, 1953; McKee, 1958; Bjor and Roer, 1980). However, there has been no conclusion about whether different strains of the scab pathogen influence the severity of the lesion on the tuber, or even whether strains 56 57 are particularly virulent toward specific varieties. Because so little is known about the effect of different strains of the organism, it is difficult to properly classify lines of potatoes as resistant or susceptible, or to predict whether varieties resistant in one area will be resistant in other areas. Some understanding of these questions can be gained by examining whether varieties resistant to one strain of the pathogen are resistant to others. Furthermore, resistance is commonly classified in two ways: 1) according to the percent surface area of a tuber covered with scab lesions and 2) according to the severity of the lesions formed on the tuber (McKee, 1958; Dowley, 1972; Bjor and Roer, 1980). It is not known whether the effect of a particular strain is the same on the percent surface covered with lesions as it is on lesion severity. Information on the interaction between the host and the pathogen will be useful in later studies on the genetic control of resistance. For these reasons, the reaction of several cultivars and diploid lines to a number of strains of the organism in a greenhouse environment is examined in this study. MATERIALS AND METHODS Six pathogenic strains of the Streptomyces organism were used to infect seven cultivars and six diploid lines (Table 1) in a randomized complete block design with four blocks. The 58 Table 1. Potato cultivars (4x) , diploid lines (2x) and strains of the organism (Streptomyces) used to evaluate scab infection. Cultivars Diploids I Strains Lemhi Russet 84810 (S. phureja) DP(Z) Saginaw Gold 84SD22 (S. chacoense x F945 Superior 8. tuberosum) RP Atlantic W5337.3 (S. phureja x Onaway Onaway S. tuberosum) 830103 Spartan Pearl W5295.7 (S. phureja x RP(F) ND860-2 S. tuberosum) P100-2 (S. tuberosum x S. tarijense) DM56-4 (S. phureja) cultivars and diploid lines were chosen on the basis of varying resistance levels to the DP(z) strain of the organism noted in previous studies. The strains of Streptomyces were isolated from infected tubers and chosen for this study based on preliminary studies of pathogenicity (Spooner and Hammerschmidt, unpublished data). The strains were maintained and mixed into the soil as previously described (Chapter I). The inoculum level was 0.5 petri dish culture per 8 inch clay pot (cultivars), and 0.3 petri dish culture per 5 inch pot (diploids) . Pots were inspected and watered individually to maintain a IOW'moisture level which is favorable for disease development. Tubers were harvested at maturity, three months after planting. 59 Scab severity was evaluated by the average cover score, type of lesion, and combined rating per pot, as described in Chapter I. To summarize, the four classes of percent surface cover (none, 0-5%, 5-25%, >25%) and those of lesion type (none, surface, slightly pitted, deep pitted) were given a numerical rating from 1 to 4, respectively. These ratings were multiplied together for each tuber to obtain a combined rating. Within each rating system, the scores for each tuber in a pot were added together and divided by the number of tubers in the pot to obtain the average score per pot. The average numerical ratings were then used for statistical analysis. RESULTS There were significant differences in resistance between cultivars and between.diploids and significant differences in virulence between strains (Tables 3, 4, and 5). There also were significant interactions between the varieties of potato and the scab strains in all of the analyses presented here. Cultivars and diploids were statistically analyzed separately. The tabular and calculated F values from analysis of variance are shown in Table 2. There were two missing values in the cultivar analysis and fifteen missing values in the diploid analysis. According to the combined rating (Table 3), there are cultivars which appear to be consistently more resistant than 60 Table 2. Tabular and calculated F values from analysis of variance of cultivars and diploid lines and strains of the organism. fl CULTIVARS I DIPLOIDS fl FT..,(p-O.01) Fm, Fm(p-0.01) F% C.V. J COMBINED 48.30% 49.39% RATING Variety 2.96 24.26 3.25 4.45 Strain 3.17 65.26 3.25 70.26 Int. 1.86 4.10 2.02 2.99 3: . — COVER 22.92% 26.70% Variety 2.96 30.47 3.25 3.48 Strain 3.17 87.10 3.25 76.63 Int. “ 1.86 5.27 n 2.02 2.51 LESION 35.23% 27.608 TYPE Variety 2.96 11.73 3.25 4.61 Strain 3.17 35.54 3.25 47.83 Int. 1.86 1.84 2.02 2.47 (p=0.05; 1.55) 61 Table 3. Classification‘ according to a combined rating of seven potato cultivars and six diploid lines infected with six strains of the scab organism. E Strain I _Yi:f::: E r945 RP(F) _ Onawa RP 830103 DP(Z)_¥. Superior E MR N R R R us Atlantic E MS R R MR R MS I l Onaway E MS R R R N S Spartan E S N R MR N S Pearl E mesa-2 E s R MR 3 R s | Saginaw " S R R MR N S Gold E Lemhi ‘ N’ N' N’ 11 N’ N Russet E w5337.3 E S R R MS R S W5295.7 E MS R R MS N S 84810 E MR R R R N S E 848022 E MR MR R R R us P100-2 S N R R N MS mass-4 E s N R MS R s E 'N-No Scab; R=Resistant; MR=Moderately Resistant; MSsModerately Susceptible; SsSueceptible. 62 others. For example, Superior is only moderately susceptible to the DP(z) strain, but it is moderately resistant, resistant, or has no scab when inoculated with the other strains. No scab lesions were observed on Lemhi Russet when inoculated with any of the strains of the scab producing organism. .Among the diploids, 84810 and 84SD22 tend.to be the most resistant to all of the strains except the DP(z) strain. According to the combined rating (Table 3), there is a similar tendency towards aggressiveness and non-aggressiveness among the strains. Fbr example, DP(z) tends to produce a susceptible or moderately susceptible classification, as does F945. The strains RP(F) and 830103 generally produce a resistant classification or produce no scab at all. Also according to the combined rating (Table 3), there are some interactions among varieties and strains. For example, Onaway is moderately susceptible to F945, but resistant to RP(F), while Spartan Pearl is susceptible to F945 but there is no scab produced by RP(F) on Spartan Pearl. Atlantic is moderately susceptible to F945, as is Onaway, but Atlantic is moderately resistant to RP and Onaway is resistant. The varieties appear to be affected differently by the different strains. In general, lesion type (Table 4) is consistently more severe or less severe on one cultivar than it is on another. Compare ND860-2 to Spartan Pearl, for example. The lesion type is always more severe on.ND860-2. At the same time, one 63 Table 4. Type of lesion1 produced on seven potato cultivars and six diploid lines infected with six strains of the scab organism. Strain I Variety _ ______ ’945__RP__P _ _~__ “L _ Superior SP N S S 8 SP 1 Atlantic . DP S S s 8 SP Onaway SP S S S N SP Spartan SP N 8 SP N SP Pearl E anew-2 ‘ DP 5 S DP S DP Saginaw E DP 5 3 SP N SP Gold E Lemhi E N N N N N N RUIIOt E W5337.3 E SP 8 8 SP S SP 9152953: SP 3 5 SP N SP E 84810 ' S S S S N SP E 848022 E S s S S 5 SP P100-2 SP N SP SP N SP 01456-4 SP N S SP S DP lN=No Scab; SsSuperficial; SP=Slight1y Pitted; DP=Deep Pitted. 64 Table 5. Surface coverage (%)‘ of seven potato cultivars and six diploid lines infected with six strains of the scab organism. Strain 7 1 Variety : F945 RP(F) Onawa RP 830103 DP(Z) _H_@_T_u._n_W___fl_m____m____mm_“______mmfl___u_u_ Superior 0-5 N 0-5 0-5 0-5 5-25 E Atlantic E 5-25 0-5 0-5 0-5 0-5 5-25 Onaway E 5-25 0-5 0-5 0-5 N >25 E Spartan E >25 N 0-5 5-25 N 5-25 Pearl E NDB60-2 >25 0-5 0-5 5-25 0-5 >25 Saginaw E N 0-5 0-5 5-25 N 5-25 Gold I Lemhi E N N N N N N Russet E w5337.3 E >25 0-5 0—5 5-25 0-5 >25 W5295-7 5-25 0-5 0-5 5-25 N 5-25 84810 0-5 0-5 0-5 0-5 N >25 84SD22 5-25 0-5 0-5 0-5 0-5 >25 moo-2 E >25 N 0-5 0-5 N >25 BESS-4 E 5-25 N 0-5 5-25 0-5 >25 lNaNo Scab; o-sa; 5-252; >25%. 65 strain of the organism also produces consistently more severe lesions than another (compare F945 to DP(z) in Table 4.) However, cultivars which are more susceptible to one strain than the other when the combined rating is considered do not necessarily have a more severe lesion type. For example, Superior is rated moderately resistant to F945 and moderately susceptible to DP(z) in Table 3, but both strains produce slightly pitted lesions on Superior (Table 4). INotice in Table 5, however, that the surface cover does differ between these two strains. Lesions on Superior produced by F945 only cover zero to five percent of the surface, while lesions produced by DP(z) cover five to twenty-five percent. DISCUSSION The results of this study suggest that there is an interaction between the strain of the scab producing organism and the variety of the potato. That is, a particular strain does not affect one variety of the potato in the same way it does another. Most studies prior to this have concluded that there is not an interaction between strains and varieties. However, those studies differed from this one in a number of ways. For example, McKee (1958) only examined the cover score from all the tubers of only one plant for each variety. He did not look at more than one plant and he did not consider lesion type. Bjor and Roer (1980) only compared variety reactions by a scab index (combined rating), and did not 66 consider the type of lesion or the surface cover independently. Taylor and Decker (1947) only tested different isolates on varieties of the potato known to be susceptible. The combined rating, because it includes both surface cover and lesion type, was the first criteria by which the interaction between varieties and strains was examined in this study. It is very similar to the scab index rating used by Bjor and Roer (1980) to study such interactions. It allows a general classification of the varieties as resistant, moderately resistant, moderately susceptible, or susceptible (Tables 2 and 5). In this study, there is also a classification of "No Scab" (NS), which may suggest that a variety is immune to infection by a particular scab strain. According to the combined rating, the varieties appear to be affected differently by the different strains, 1... there is an interaction. This suggests that the causal organism is as important for the classification of resistant varieties as is the host reaction. A question still remains, however, about the effect of a particular strain directly on the symptoms of scab. Previous studies did not critically examine this question, although Taylor and Decker (1947) , McKee (1958) , and Bjor and Roer (1980) suggested from general observations on lesion type that host reaction is more important to the type of scab lesion than is the strain of the organism. Our results indicate that the type of lesion is a function of an interaction between the 67 strain and the variety. A particular strain of the organism does not produce a particular lesion type on every cultivar, nor does a particular cultivar develop the same lesion type when infected with every strain of the organism. This study also suggests that there is aEdifference between the effect of an organism on the percent surface of a tuber covered with scab and the effect of that organism on the type of lesion produced. This is in contradiction to suggestions by previous results that there is a correlation between the lesion type and the percent of the tuber surface covered (Stevenson et al., 1942; Leach et al., 1938). There are two possible explanations for the results shown here. One is that there may be physiological differences between the scab strains that make them more or less able to infect a greater surface area of the tuber or to produce a certain lesion type. The other is that there may be physiological differences between the potato varieties that make them more or less able to defend themselves against the spread of the organism over a greater surface area or against the more severe lesion types. Most likely, the ability of the organism to infect and the host to defend a greater surface area is controlled separately from the ability of the organism to produce and the host to prevent a more severe lesion type. Possibly, the host defense to a greater surface area being infected is a matter of structural control. It is known that the organism infects growing tubers through the lenticels 68 (Darling, 1937). Perhaps the defense of the tuber is correlated with the number of lenticels on the tuber surface or with the size of the lenticels as the tuber is developing, although Woodcock (1952) did not find any correlation between the number of lenticels in resistant and susceptible varieties and scab resistance. The effect of the pathogen on the percent surface covered with lesions may depend on the ability of the organism to locate and enter the lenticels. Perhaps the ability of the pathogen to infect has a greater effect on the host surface area covered with lesions than the host reaction does. Host control of the type of lesion is possibly a function of the jproduction. of some biochemical inhibitor to 'the pathogen. One compound for which a correlation to resistance has been suggested is the phenolic compound, chlorogenic acid (Johnson and Schall, 1952), although its relationship to resistance has been questioned (Emilsson, 1953 ; Holm and Adams, 1960). Other possible inhibitors need to be examined. If there is a chemical compound preventing more severe lesion types, the success of the organism would depend on its ability to defend itself against this inhibitor or to prevent the inhibitor from being produced in the host. The phenomenon of interaction between host and pathogen response has been studied many times in the past (Ellingboe, 1981; McDonald, et al., 1989; Gabriel and Rolfe, 1990). Often, the interaction between the potato host and the scab 69 pathogen may be so complex that it becomes very difficult to separate the effect of the host from the effect of the pathogen. The interaction becomes like a third organism in the relationship (Gabriel and Rolfe, 1990). Understanding the interaction is further confounded by the fact that the North American cultivated potato is a tetraploid (2n=4x=48). If there is a major gene contributing to resistance in the host, as has been suggested (Krantz and Eide, 1941; Lauer and Eide, 1963), it could be present in four copies. The resistance might then be determined by a dosage effect of copies of one, two, three, or four of the resistance alleles. Such a dosage effect confounded with environmental effects may make it difficult to distinguish between a single gene or polygenic based resistance. There may be a major gene for resistance in the host, but it is likely that there are a number of minor genes also contributing to Ihost resistance. It appears that ‘true susceptibility of a particular variety to a particular strain will result in some degree of pitted lesions. However, strains such as RP(F) and 830103 will at most produce superficial lesions. Perhaps the production of superficial lesions is a hypersensitive response of the host to the organism. In that case, varieties with superficial lesions would.be.considered resistant to an organism, or that organism would be considered non-pathogenic on that variety. It also seems possible that a slightly pitted lesion type may be the 70 result of an intermediate resistance level, like the "slow rusting" phenomenon in wheat. Because Streptomyces spp., the causal organism of scab, is a saprophyte and not an obligate parasite on potato, it is difficult to support the evolution of a distinct gene-for-gene interaction between the host and the pathogen. However, the results of this study do not suggest that the host-pathogen interaction fits a model of basic compatibilty either. Basic compatibilty would require that any population of the scab organism could maintain itself as a pathogen on any member of the potato species (Gabriel and Rolfe, 1990). Yet, there are varying levels of resistance among varieties in the presence of different strains of the organism. In addition, resistance to a large surface area covered with scab and resistance to more severe lesion types appear to be under separate control. LITERATURE CITED Bjor, T. and L. Roer. 1980. Testing the resistance of potato varieties to common scab. Potato Res 23:33-47. Darling, H.M. 1937. A study of scab resistance in the potato. J Agr Res 54:305-317. Dowley, L.J. 1972. Reliability of methods of assessing the degree of tuber attack by common scab of potatoes. Potato Res 15:263-265. Ellingboe, A.H. 1981. Changing concepts in host-pathogen genetics. Ann Rev Phytopath 19:123-143. Emilsson, B. 1953. The relation between content of chlorogenic acid and scab resistance in potato varieties. Acta Agr Scand 3:328-333. Gabriel, D.W. and B.G. Rolfe. 1990. Working models of specific recognition in plant-microbe interactions. Ann Rev Phytopath 28:365-391. Johnson, G. and L.A. Schaal. 1952. The relation of chlorogenic acid to scab resistance in potatoes. Science. 115:627- 629. Krantz, F.A. and C.J. Eide. 1941. Inheritance of reaction to common scab in the potato. J Agr Res 63:219-231. Lauer, F.I. and C.J. Eide. 1963. Evaluation of parental clones of potato for resistance to common scab by the "highest scab" method. Eur Potato J 6:35-44. Leach, J.G., F.A. Krantz, P. Decker, and H. Mattson. 1938. The measurement and inheritance of scab resistance in selfed and hybrid progenies of potatoes. J Agr Res 56:843-853. Leach, J.G., P. Decker, and H. Becker. 1939. Pathogenic races of Actinomyces scabies in relation to scab resistance. Phytopath 29:204-209. McDonald, B.A., HcDermott J.M., Goodwin, S.B., Allard, R.W. 1989. The population biology of host-pathogen interactions. 27:77-94. 71 72 McKee, R.H. 1958. Assessment of resistance of potato varieties to common scab. Eur Potato J 1:65-80. Millard, W.A. and S. Burr. 1926. A study of twenty-four strains of Actinomyces and their relation to types of common scab of potato. Ann Appl Biol 13:580-644. Stevenson, P.J., L.A. Schaal, C.F. Clark, R.V. Akeley and co- operators. 1942. Potato scab gardens in the United States. Phytopath 32:965-971. Taylor, C.F. and P. Decker. 1947. A correlation between pathogenicity and cultural characteristics in the genus Actinomyces. Phytopath 37:49-58. Chapter III: Evaluation of Peroxidase in Relation to Potato Scab Resistance 73 ABSTRACT Soluble peroxidase isozymes and acidic and total soluble peroxidase activity levels were compared among eighteen tetraploid and six diploid potato lines with varying levels of scab resistance. The outermost layer of periderm was sampled from actively growing, uninfected tubers. Potato lines could be distinguished according to peroxidase isozyme patterns by starch gel and polyacrylamide gel electrophoresis. Lines could also be distinguished according to acidic and total soluble peroxidase activity levels. No correlation was found between isozyme patterns or between the levels of activity and scab resistance. INTRODUCTION In 1952, Johnson and Schaal suggested there is a correlation between chlorogenic acid (CGA) levels in the potato and resistance to the common scab disease. They offered two suggestions as to the effect of CGA: 1) It lowers the pH of the cells, thereby creating an unfavorable medium for the growth of the bacteria; or 2) The acid itself or its quinone may be directly or indirectly involved in the formation of the suberized periderm. 74 75 Later studies, however, did not find any direct relationship between resistance and high chlorogenic acid content (Emilsson, 1953; Wolffgang and Hoffman, 1959; Holm and Adam, 1960) . Furthermore, Hammerschmidt (unpublished data) tested the inhibitory effect of chlorogenic acid on the growth of the organism and found that growth was only inhibited by unnaturally high concentrations of CGA. Such evidence suggests it is unlikely that CGA is concentrated enough in the periderm cells of resistant tubers to create an unfavorable medium for growth of the organism, as Johnson and Schaal (1952) first suggested. It is also unlikely that chlorogenic acid or its quinone are directly or indirectly involved in the formation of suberized periderm at the site of infection by the bacteria. However, some precursors in the biosynthetic pathway for chlorogenic acid probably are involved in the formation of suberized periderm. Suberin is a polymer of natural and wound induced periderm. The aromatic domains of suberin are derived from the phenylpropanoid pathway; Chlorogenic acid is also a product of the phenylpropanoid pathway (Fig. 1) . Caffeic acid and p-coumaric acid are precursors in the synthesis of the aromatic domains of suberin and caffeic acid is a precursor in the synthesis of chlorogenic acid. It has often been suggested that there is a relationship between periderm development and scab resistance (Jones, 1931; Darling, 1937; Cooper et al., 1954; Labruyere, 1971) . The present study 76 proposes that the formation of suberin may be correlated with resistance to Scab, even though a high level of CGA is not. If biosynthesis of the aromatic components of suberin follows the same {pathway as that established for lignification, as suggested by Kallattukady (1984), then peroxidase is the last enzyme in the pathway. In lignin biosynthesis, peroxidase is the final enzyme catalyzing the polymerization of cinnamyl alcohols (Figure 1). This study was designed to examine the possible relationship of suberin formation to scab resistance by comparing the peroxidase isozymes and acidic and total peroxidase levels in cultivars and diploid potato lines of varying scab susceptibility. phenylalanine chlorogenic acid COOH HO 0 , E NH2 ' cinnamlc acid ”' <::>,\y/coou i ©~cocoA -flo.@/VCH2°H “flue “Id p-coumarlc acid / l-iO Hip/Vow}; o‘./\/coon \ / H \ l sussnm (Phenolic matrix) “Lignln” . \\ ///’ \\\\ 0‘:"3 Ho_@/\/ COCOA-+10% CH20“ Figure 1. The phenylpropanoid pathway leading to the synthesis of the aromatic domains of suberin and of chlorogenic acid. 77 MATERIALS AND METHODS Soluble peroxidase isozymes and total soluble peroxidase activity ‘were examined in. three replications of the 18 tetraploids and 6 diploids in. Table 1, and in 7 more replications of the six round white varieties (marked by an asterisks in Table 1). W This analysis did not include tubers under two centimeters in diameter among the tetraploids or under one centimeter among the diploids. A very thin, outermost layer of skin (approximately 0.5 mm) was peeled with a razor blade from freshly harvested, immature tubers. Immature tubers were sampled because successful infection only occurs in actively growing tubers. The skin was removed from the apical end (generally the cite of infection) of all the tubers from one pot and.collected in a vial. ‘Vials were immediately stored at -20°C until the tissue could be freeze dried. 50 milligrams of freeze dried tissue were ground in 2 ml cold 0.2M sodium phosphate buffer (pH 8.0) with mortar and pestle, then transferred to a 1.5 ml microfuge tube and centrifuged for 10 mins (12,000X g). The supernatant was separated from the pellet and stored at -20 C to be used for polyacrylamide gel electrophoresis (PAGE). The same buffer was used to grind samples for starch gel electrophoresis. Fresh extract was soaked into 3X8mm paper wicks (Whatman 3MM) and stored overnight at -20 C. 78 Table 1. Cultivars and diploid lines used to evaluate peroxidase isozymes and total peroxidase activity. I Cultivars . Diploids Michigold Y245.7 E 84810 (S. phureja) Saginaw Gold Hindenberg 84SD22 (S. chacoense x Lemhi Russet Burbank S. tuberosum) Russet Burbank Ontario* : W5337.3 (S. phureja x Nooksack Superior* S. tuberosum) Monona Atlantic* E W5295.7 (S. phureja x NDD277-2 Onaway* E . tuberosum) Katahdin Spartan Pearl* P100-2 (S. tuberosum x M8716-15 ND860-2* S. tarijense) DM56-4 (S. phureja) *designates the round white varieties in the second study. Polyacrylamide Gel Electrophoresis Protein concentrations of periderm extracts were determined according to Bradford (1976) using bovine serum albumin (BSA) as a standard. Equal amounts of protein were loaded into the gel for each sample. An anionic (pH 9.3) discontinuous native polyacrylamide slab gel system (1.5 mm thick, 7.5 % acrylamide) was used (Keleti and Lederer, 1974). Gels were stained for peroxidase activity in a solution containing 100 mls sodium acetate buffer (pH 5.0), 20 mg 3- amino-9-ethyl carbazole dissolved in .5 ml N,N-dimethyl formamide, and 66 ul 30% H5» (modified from Smith and Hammerschmidt, 1988). Gels were rinsed after 45 minutes and stored in 5% acetic acid and 50% methanol until they were vacuum dried. 79 W Peroxidase isozymes were resolved with a Lithium borate pH 8.3 buffer system (Ashton and Braden, 1961) . General techniques are described by Quiros (1981) and the staining protocol by Vallejos (1983). W): Total peroxidase activity of periderm extracts was determined using guaiacol as a substrate (Hammerschmidt, et al. 1982). Analysis of variance was used to test for significant differences among means. RESULTS Although distinguishing peroxidase isozyme patterns could be identified for the tetraploid and.diploid lines in at least two loci from both the starch and polyacrylamide gel electrophoresis systems, there was not a correlation between banding patterns at any locus with resistance of the lines to scab infection. Figures 2 and 3 show the starch gel electrophoresis isozyme patterns for all of the 18 tetraploids and six diploids which we investigated. Figures 4 and 5 show polyacrylamide gel electrophoresis patterns for eight of the tetraploids and for the six diploids. Because equal amounts of protein for each sample were loaded on a gel in the PAGE analysis, the intensity of the bands could be compared among the potato lines“ ZMore intense 2. 80 Starch gel electrophoresis isozyme patterns of thirteen round white tetraploid lines, from left to right: Hindenberg(R); Burbank(R); Ontario(R); Superior(R); Atlantic (S); Onaway(S); Spartan Pearl(S); ND860-2 (S); Honona(S); Katahdin(S); MS716-15(S); NDD277-2(S); Y245-7(S). R=Resistant, S=Susceptible. l .3‘ ‘ ”9"” 3 :‘gnf' Starch gel electrophoresis isozyme patterns of two yellow flesh tetraploids, three russets, and six diploids, from left to right: Michigold(S); Saginaw Gold(S); Lemhi Russet(R); Nooksack(R); Russet Burbank(R); 84SlO(R); 84SD22(R); W5337.3(S); W5295.7(S); P100-2(S); DM56-4(S). R=Resistant, S=Susceptible. 82 a. It. .._. - VI..- ' arr-0" Figure 4. PAGE isozyme patterns of six round white potato varities, left to right: Atlantic(S); Superior(R); Ontario(R); Spartan Pearl(S); Onaway(S); ND860-2(S). R=Resistant, S=Susceptible. 83 t -«Ayna-v—s ,2 1’. Figure 5. PAGE isozyme patterns of six diploid lines and two tetraploids, left to right: W5337.3(S); 84SlO(R); 84SD22(R); moo-2(3); DM56-4(S); Y245-7(S); Lemhi Russet(R). R=Resistant, S=Susceptible. 84 bands would suggest higher activity of the acidic peroxidases. There were distinguishing differences between the intensity of the bands among the potato lines. For example, Superior, Atlantic and Spartan Pearl always had more intense bands, suggesting higher peroxidase activity, than Ontario, ND860-2, or Onaway (See figure 3). However, no correlation could be found betweeen band intensity and scab resistance. The total soluble peroxidase activity, measured spectrophotometrically, differed significantly (p=0.01) between lines, but again there was no correlation between total peroxidase activity and scab resistance. Correction of the total peroxidase activity by the average weight of the tuber per pot for each line did not change these results. The average total peroxidase activity of the three replications of the 18 tetraploids and six diploids are shown in Table 2. Average peroxidase activity of the seven replications of the six round white varieties are shown in Table 3. It was noted that those lines which had more intense bands on the polyacrylamide gels, indicating higher acidic peroxidase activity, also had higher total soluble peroxidase activity. The russet potatoes, which are generally resistant, did have higher peroxidase levels than most of the other lines (Table 2) . The correlation to resistance, however, broke down after more replications were examined. It was found that at least one highly susceptible round white variety (Spartan 85 Table 2. Total soluble peroxidase activity (AaJmin/mg protein) of eighteen tetraploid and diploid lines averaged over three replications. I Potato line! Afljmin/mg protein I Michigold (8) 1.895 Saginaw Gold (8) 5.910 Nooksack (R) 4.903 Russet Burbank (R) 5.670 Monona (S) 1.523 Katahdin (S) 1.763 MS716-15 (S) 2.017 NDD277-2 (S) 2.727 Hindenberg (R) 1.483 Burbank (R) 2.940 Ontario (R) 1.893 Superior (R) 2.507 Atlantic (S) 1.500 Onaway (S) 0.880 Spartan Pearl (S) 2.603 ND860-2 (8) 1.963 Y245-7 (S) 2.160 Lemhi Russet (R) 3.650 ws337.3 (S) 0.963 ws295.7 (R) 1.087 84810 (R) 0.773 84SD22 (R) 2.493 P100-2 (S) 3.503 DM56-4 (S) 0.877 Table 3. Total soluble peroxidase activity (Am/min/mg protein) of six round white potato varieties averaged over seven replications. ND360-2 (S) 2.707 Onaway (S) 2.289 Spartan Pearl (S) 7.571 Ontario (R) 1.919 Superior (R) 4.703 Atlantic (S) 3.443 86 Pearl) had much higher peroxidase activity than resistant round white varieties (Superior, Ontario) (Table 3). DISCUSSION No higher activity levels or specific peroxidase isozyme patterns were found to be associated with resistant potato varieties. Possibly, levels of peroxidase activity and/or isozyme patterns will be altered in the presence of the organism. Rapid lignification has been cited as a response to infection by Phytophthora infestans in the potato (Hammerschmidt, 1984). A similar response has been noted in cucumber to the pathogen Colletotrichum lagenarium (Dean and Kuc, 1987). Furthermore, Smith and Hammerschmidt (1988) and Hammerschmidt et al. (1982) found increases in peroxidase associated with induced resistance in cucurbits species to the pathogen Colletotrichum lagenarium. Vance et al. (1976) also found increased levels of peroxidase in reed canarygrass inoculated with Helminthosporium avenae. However, it would be difficult to examine the possible role of peroxidases regarding induced resistance of the potato tuber to the scab pathogen. The difficulty arises because the scab organism after infection is generally localized in and around the scab lesion. This would be the most desirable tissue to examine for induced peroxidase activity, but the presence of the pathogen would complicate the results. 87 If it is not possible to study the effect of the pathogen on peroxidase levels in the host, other means for examining the role of peroxidase in resistance should be considered. One alternative would be to directly examine the effect of compounds produced by the scab pathogen on peroxidase isozymes and activity levels. Unfortunately, very little is known about the mechanism by which the organism is able to produce lesions in the tuber. However, recently, Lawrence et al. (1990) described two probable phytotoxins produced by the organism, which they designated thaxtomin A and thaxtomin B. In depth studies of the potato reaction specifically to these compounds will reveal critical information on the host defense to the scab pathogen. It would be interesting to know whether these vivotoxins inhibit peroxidase activity or alter peroxidase isozyme patterns. The effect of these vivotoxins on other enzymes involved in lignification will also be worth noting. More generally, the effect of these vivotoxins on suberin should be thoroughly investigated. LITERATURE CITED Ashton, G.C. and A.W. Braden. 1961. Serum B-globulin polymorphism in mice. Austral J Biol Sci 14:248-254. Bradford, H.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye bonding. Analyt Biochem 72:248- 259. Cooper, D.C., G.W. Stokes and G.H. Rieman. 1954. Periderm development of the potato tuber and its relationship to scab resistance. Amer Potato J 31:58-66. Darling, H.M. 1937. A study of scab resistance in the potato. J Agr Res 54:305-317. Dean, R.A. and J. Kuc. 1987. Rapid lignification in response to wounding and infection as a mechanism for induced systemic protection in cucumber. Phys Mol Plt Path 31:69- 81e ' Emilsson, B. 1953. The relation between content of chlorogenic acid and scab resistance in potato varieties. Acta Agr Scand 3:328-333. Hammerschmidt, R. 1984. Rapid deposition of lignin in potato tuber tissue as a response to fungi non-pathogenic on potato. Physiol Plt Path 24:33-42. Hammerschmidt, R., E.M. Nuckles, and J. Kuc. 1982. Association of enhanced peroxidase activity with induced systemic resistance of cucumber to Colletotrichum lagenarium. Physiol Plt Path 20:73-82. Holm, E.T. and A.P. Adams. 1960. An investigation of the relationship of the chlorogenic acid and phenol oxidase content of a potato variety and its resistance to common scab. Enzymologia 22:245-250. Johnson, G. and L.A. Schaal. 1952. Relation of chlorogenic acid to scab resistance in potatoes. Science 115:627-629. Jones, A.P. 1931. The histogeny of potato scab. Ann Appl Biol 18:313. 88 89 Kallattukady, P.E. 1984. Biochemistry and function of cutin and suberin. Can J Bot 62:2918-2933. Keleti, G. and W.H. Lederer. 1974. MW Biglggig§1_figigggg§. Van-Nostrand Rheinhold Co., New York. Labruyere, R.E. 1971. Common Scab and its control in seed potato crops. Ag Res Rep 767:71pp. Lawrence, C.H., M.C. Clark, and R.R. King. 1990. Induction of common scab symptoms in aseptically cultured potato tubers by the vivotoxin, thaxtomin. Phytopath 80(7):606- 608. Quiros, C.F. 1981. Starch electrophoresis techniques used with alfalfa and other Medicago species. Can J Plt Sci 61:745- 749. Smith, J.A. and R. Hammerschmidt. 1988. Comparative study of acidic peroxidases associated with induced resistance in cucumber, muskmelon, and watermelon. Physiol Mol Plt Path 33:255-261. ‘Vallejos, C.E. 1983. Enzyme(activity'staining.,L_: Isozymes in plant genetics and breeding, Vol A (S.D. Tanksley and T.J. Orton, eds.) Elsevier, New York pp. 469-516. Vance, C.P., R.T. Sherwood, and'T.K. Kirk. 1980. Lignification as a mechanism of disease resistance. Ann Rev Phytopath 81:259-288. Wolffgang, H. von and G.M. Hoffman. 1959. Die bedeuting der chlorogensauer als resistanzefaktor. Zuchter 29:335-339. APPENDIX APPENDIX EXTRACTION OF CHLOROGENIC ACID FROM TUBER PERIDERM The outermost layer of tuber periderm from immature tubers was peeled with a razor blade and lyophilized. The peeled tissue from all of the tubers from one pot (one cultivar) was combined. Tissue was immediately frozen in vials at -20°c and later lyophilized. Chlorogenic acid was extracted from 30 mg of lyophilized tissue in.3 ml methanol at approximately 60°c for 30 minutes. The extract was transferred to an evaporation tube and the tissue was extracted a second time with 2 ml methanol at 60°c for 30 minutes. The second extract was added to the first. The tissue was extracted a third time in the same way as the second and the third extract was added to the first two. The tissue was then rinsed 2x briefly with 1 ml methanol, and each rinse was added to the extraction. The 9 ml of extraction (Slightly yellow in color) was then evaporated to dryness and redissolved in 250 microliters methanol. The extract was stored at 4°c in 1 ml vials. Preparative thin-layer chromatography was performed on 0.25mm thick 20 x 20 cm silica gel plates with precut lanes. 25-50 microliters of the redissolved extract was applied as a single band on the plate. Chlorogenic acid as a control was also applied to one lane on each plate. Plates were developed with ethyl acetate-formic 90 acid-glacial acetic acid-water (100:11:11:27, v/v) until the solvent front reached 18 cm (Friedman et al., 1989). The chlorogenic acid bands could be visualized by spraying the plates with a folic acid phenol reagent. LITERATURE CITED Friedman, M, L. Dec, and M.R. Gombmann. 1989. Ergot alkaloid and chlorogenic acid content in different varieties of morning-glory (Ipomoea spp.) seeds. J Agric Food Chem 37:708-712. 91 T RTE UNIV. LIBRQRIES EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE 3008764858 nICHIGnN s :31E2 £3