OVERDUE FINES ARE 25¢ PER DAY PER ITEM Roturn to Book drop to remove this checkout from your record. THE USE OF SEROLOGY AND SEMI-SELECTIVE MEDIA AS AIDS IN THE DETECTION OF XANTHOMONAS BEAN BLIGHT BACTERIA By Gustavo E. Trujillo A DISSERTATION Submitted to Michigan State University in partia] fulfiliment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and PTant Pathoiogy 1979 ABSTRACT THE USE OF SEROLOGY AND SEMI-SELECTIVE MEDIA AS AIDS IN THE DETECTION OF XANTHOMONAS BEAN BLIGHT BACTERIA By Gustavo E. Trujillo Seed-borne common (Xanthomonas phaseoli, 32) and fuscous (X: phaseoli var. fuscans, pr) bacterial blights continue to affect dry bean production in many areas of the U.S., Canada and Latin America. While planting of pathogen-free seed remains effective for control of the Xanthomonas diseases, techniques for detection of internal seed contamination by blight bacteria are often time- consuming (seedling injection) or require large bacterial popula- tions (serology). A combined serological and semi-selective media technique for the detection of internally-borne §p_and [pf in dry bean seed was developed. Antisera of §p_and 1p: at titres of l:2000—lz5000 were pro- duced in rabbits by intravenous injfection of formalin-killed cells suspended in buffered saline (l09 cells/ml). Injections were made at 0 (0.l ml), 4 (0.3 ml), 8 (0.5 ml), ll (1.0 ml), and l4 (2.0 ml) days; sera were collected 7, l4, and 2l days after the last injec- tion. Gustavo E. Trujillo Agar gel double diffusion tests were found more reliable than micro- and tube-agglutinin tests in serological studies of {p and 13:. Live cells of Agrobacterium, Bacillus, and Corynebacterium sp. did not react in agar gel double diffusion tests; when steamed cells (60 min. at l00°C) of other bacterial isolates (Erwinia or Pseudomonas) were tested, the reaction was Xanthomonas specific. In agar double diffusion tests, §p_and [pf antisera reacted positively to steamed cells of 20/20 §p_and 29/29 52f isolates, but did not react to steamed cells of 19 internal bacterial contaminants obtained from surface-sterilized bean seeds. In absorption tests it was shown that {p and KB: possess species-specific heat stable antigens. 52 and Kai concentrations near l07 cells/ml were sufficient to be detected in agar gel double diffusion tests. A semi-selective media (SSM), highly selective for Xanthomonas, was developed and contained: l.0 gm yeast extract, 25 mg cycloheximide, 2 mg nitrofurantoin, l mg nalidixic acid, and .05 mg gentamicin in 1000 ml .0l M phosphate buffer pH 7.2. Utilizing gijlO, resistant to 50 ppm rifampin, and selec- tive plating on media with and without rifampin, we obtained maximum lijlO and minimum bacterial contaminant populations by incubating Rl0-infected seed (l infected seed:4 non-infected seed) in the SSM. SSM was inhibitory to all of the Gram positive bacterial idolates tested, including different isolates of Corynebacterium and Bacillus sp., and inhibited the growth of most of the Gram negative bacteria tested. Gustavo E. Trujillo The Michigan Department of Agriculture (MDA) test for internal blight contamination of bean seed currently involves: (1) surface sterilization of l.9 kg seed for l0 minutes in 2.6% NAOcl; (2) rinsing in sterile H20; (3) incubation of seed for 18-24 hours in sterile H20 containing 10 gm/litre yeast extract; and (4) injfection of a sample of liquid surrounding seed into primary leaf node of young kidney bean seedlings. Samples of l ml of the surrounding liquid obtained from the MDA were individually incubated in 25 ml SSM for 24-36 hours in a rotary shaker. Bacteria were then sedimented by l5 minutes centri- fugation at 5000 x g, resuspended in l ml buffered saline, steamed 60 min. at l00°C, and tested serologically (SSMS). Sixty-one of sixty-five bean seed samples found to carry internal blight contami- nation in the Michigan Department of Agriculture test reacted posi- tively in the serological test. Thirty-seven of the ninety-nine navy bean samples obtained from the MDA showed positive results for internal blight contami- nation with the SSMS procedure. Of these 37 the MDA seedling injection detected blight in only 25. The SSMS also consistently detected Xanthomonas blight in other plant tissues infected with blight (stems and leaves). Pseudomonas phaseolicola and Bé: syringae were found to be internally seed-borne in navy (pea) beans. To my father, Juan, and my friend, Salomon ii ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Dr. A. w. Saettler who provided counsel, guidance and encouragement throughout the entire investigation, and during the preparation of this manuscript. Appreciation is likewise extended to Drs. w. M. Adams, E. J. K105 and w. J. Hooker who served as members of my guidance committee and who reviewed this manuscript. The Facultad de Agronomia and the Consejo de Desarrollo Cientifico y Humanistico, Universidad Central de Venezuela Venezuela) provided financial assistance to the author during this graduate program. LIST OF LIST OF GENERAL Chapter I. II. TABLE OF CONTENTS TABLES . FIGURES INTRODUCTION . SEROLOGY OF XANTHOMONAS PHASEOLI (BEAN COMMON BACTERIAL BLIGHT) AND XANTHOMONAS PHASEOLI VAR. FUSCANS (BEAN FUSCOUS BACTERIAL BLIGHT) Literature Review Objectives . . . Materials and Methods . Antisera Preparation Serological Techniques . Results . . Titres of Different Antisera to Xanthomonas Using the Tube and Microagglutinin Tests Antisera Titres as Determined by Immunodiffusion in Agar Plates . . . The Effect of Antigen Concentration on Band Formation in Agar Gel Diffusion Tests . Reactions of Xanthomonas Antisera Against Live and Steamed Cells of Various Bacteria Agar Gel Double Diffusion Tests of Xanthomonas Antisera Against Whole and Steamed Cell Antigens . . Agar Double Diffusion Test of Xanthomonas Anti-o sera Against Cell Free Steamed Supernant as Antigen . . . . . . . Discussion DEVELOPMENT OF A LIQUID MEDIUM SELECTION FOR XANTHOMONAS PHASEOLI AND XANTHOMONAS PHASEOLI VAR. m Introduction . Objective . iv Page vii ix l2 l3 T3 19 l9 22 25 25 31 34 48 55 55 55 Chapter Page Materials and Methods . . . . . . . . 57 Bacterial Storage and Culture . . . . . . 57 Selection of a Basal Medium . . . . 58 Preparation of Bean Seed Flour Containing [prlO . . . . . . . . 58 Experiments with Liquid Media . . . . . . 59 Experiments Using Antimicrobial Discs . . . . 59 Origin of Isolates . . . . . . . 60 Methods of Antibiotic Preparation . . . . . 60 Results . . 6l The Effect of Yeast Extract Concentration on the Detection of Internal Xanthomonas phaseoli Contamination of Navy Bean Seeds . . 6l Sensitivity of Various Plant Pathogenic and Non- Pathogenic Bacteria to Several Antibiotics in Bioassay Tests . . 63 Growth of Several Xanthomonas Bacterial Blight Isolates in LiquidTMedia with Various Antibiotics . . 63 The Effect of Antibiotics and Antibiotic Combi-o nations on Populations of §p_R10 and Bacterial Contaminants . . 74 Minimal Number of Bacterial Cells Detected by SSM . . 77 Internal Bacterial Contaminants of Navy Bean Seeds . . . . . . . . . . . . . 77 Discussion . . . . . . . . . . . . . . 79 III. A LABORATORY TEST FOR THE DETECTION OF XANTHOMONAS PHASEOLI AND XANTHOMONAS PHASEOLI VAR. FUSCANS IN BEAN SEED. . . . . . . . 86 Introduction . . . . . . . . . . . . . . 86 Objective . . . . . . . . . . . . . 90 Materials and Methods . . . . . . . . . . . 90 Samples . . . . . . . . . . . . . 90 Serological Test . . . . . . . . . . . 92 Seedling Injection . . . . . . . . . . 92 Results . . . . 93 Michigan Department of Agriculture Liquid Samples . 93 Michigan Department of Agriculture Seed Samples. 95 MDA Stem Sections . . . lOl Stems Suspected of Being Infected with Blight Bacteria from Greenhouse Experiments . . lOl Leaves from Plants Suspected of Being Infected with Blight Bacteria . . . . . . . . lOl Chapter Page Dried Material . . . . . . 102 Stored Seeds Infected with Blight . . . . . 102 Discussion . . . . . . . . . . 102 APPENDICES . . . . . . . . . . . . . . . . . ll6 vi Table 10. ll. l2. LIST OF TABLES Titres of Different Xanthomonas Antisera as Determined by Agglutinin Tests with Homdlogous Antigen . Titres of Different Xanthomonas Antisera as Determined by Microagglutination Tests with Homologous Antigen Agar Gel Double Diffusion Tests of Different Dilutions of Xanthomonas Antisera Against the Homologous Antigen . . . . . . . . Agar Gel Double Diffusion Tests of Xanthomonas Anti- sera with the Homologous Antigen . Agar Gel Double Diffusion Analysis of Xanthomonas Antisera Tested Against Various Concentrations of Homologous Antigen Agar Gel Double Diffusion Analysis of Different Xanthomonas Antisera Against Different Concentrations of the Homologous Antigen . . Reactions of Several Plant Pathogenic Bacteria in Agglutination Tests Against Xanthomonas Antisera Reactions of Several Plant Pathogenic Bacteria in Agglutination Tests Against Xanthomonas Antisera Cross Agglutination Reactions of Various Xanthomonas Phaseoli (12) and Xanthomonas Phaseoli var. Fuscans (fipj) Isolates Against Xanthomonas Antisera . Reactions of Different Plant Pathogenic and Non- Pathogenic Bacteria to Xanthomonas Antisera in Agar Gel Double Diffusion Tests . . . . . Reactions of Different Plant Pathogenic and Non- Pathogenic Bacteria to Xanthomonas Antisera in Agar Gel Double Diffusion Tests . . . . . Reactions of Various Bacterial Isolates Against Xanthomonas Antisera in Agar Bel Double Diffusion Tests . . . . . . . vii Page 20 2l 23 24 26 27 3O 32 33 35 36 38 Table 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. Tube Agglutinin Absorption Tests with Different Xanthomonas Antisera . . . . . . The Effect of Media Composition on the Detection of Internal Xanthomonas phaseoli Contamination in Navy Bean Seed in Agar Gel Double Diffusion Tests The Effect of Media Composition on the Detection of Internal Xanthomonas phaseoli var. Fuscans Contamina- tion in Navy Bean Seed in Microagglutinin Tests The Effects of Media Composition on the Detection of Internal Xanthomonas phaseoli var. Fuscans Contamina- tion in Navy Bean Seed in Microagglutinin Tests Sensitivity of Various Bacteria to Several Antibiotics in Bioassay Tests Growth of Six Xanthomonas Bacterial Blight Isolates in Liquid Media Supplemented with Various Antibiotics Growth of Xanthomonas Blight Isolates and Internal Bean Seed Contaminants in Liquid Media Supplemented with Various Antibiotics . . . . . . Growth of Internal Bean Seed Bacterial Contaminants in Liquid Media Supplemented with Various Antibiotics Growth of Internal Bean Seed Bacterial Contaminants in Liquid Media Supplemented with Various Antibiotics Populations of 52:10 and Bacterial Contaminants in Liquid Media Supplemented with Various Antibiotics Populations of §p_R10 and Bacterial Contaminants in Liquid Media Supplemented with Various Antibiotics Growth of Various Plant Pathogenic and Non- Pathogenic Bacteria in Liquid Media with and Without Added Antibiotics . . . . . . The Use of Serology, Semi-Selective Media, and Seed- ling Injection for the Detection of Xanthomonas Blight Bacteria in Bean Seed . Detection of Internal Bacterial Blight Contamination in Bean Seed Lots by Several Procedures . viii Page 39 62 64 65 68 69 71 72 73 78 94 96 Figure 10. LIST OF FIGURES Serologic reactions of 5. phaseoli var. fuscans antisera at different dilutions with homologous antigen . . Serologic reactions of 1, phaseoli var. fuscans antisera at different dilutions with homologous antigen . . . . . . . . . . Serologic reactions of 5, haseoli ll antisera (central well) with live celis 48 hours old of dif- ferent Xanthomonas isolates adjusted at 1010 cells/ml. Serologic reactions of 5, phaseoli var. fuscans anti- sera (central well) with 11ve ce 5 48 hour old of different bacterial isolates adjusted at 10 0 cells/ ml . . . . . . . . . . Serologic reactions of x. phaseoli (15) antisera (central well) with steamed cells of different bac- terial isolates adjusted at 1010 cells/ml Serologic reaction of L. phaseoli (15) antisera (central well) with steamed cells of different bac- terial isolates adjusted at 1010 cells/ ml Serologic reaction of A. phaseoli (11) antisera with different concentrations of the homologous (steamed) antigen . . . . . Serologic reaction of L. phaseoli (15) antisera with different concentrations of the homologous steamed antigen . . . . . . . . Radial immuno difusion reactions of x. phaseoli (11) antisera with several pathogenic and non-pathogenic bacteria . . . . . . . . . . . . Radial immodifusion reactions of 5, phaseoli (15) antisera incorporated into agar medium at a final dilution of l:30 . . . ix Page 28 28 40 4O 42 42 44 44 46 46 Figure Page ll. Serologic reactions of X. phaseoli var. fuscans (l6) (central well) with the antigen from one blighted seed added to 250 seed samples . . . . . . . . 66 12. Symptoms of internal seed infections by Xanthomonas blight bacteria . . . . . . . . . . . . . 103 13. Seeds from the same sample . . . . . . . . . 103 14. Symptoms in red kidney bean plants after injection of Xanthomonas blight bacteria . . . . . . . . . 105 15. Red kidney bean plants injected with isolate 59 (PS. 5 rin ae) . . . . . . . 107 16. Red kidney bean plant injected with isolate 59 (P5. phaseolicola) . . . . . . . . . 107 17. Typical symptoms of Rs. Phaseolicola . . . . . . 109 GENERAL INTRODUCTION Common blight caused by Xanthomonas phaseoli (E. F. Smith) Dowson (12) and fuscous blight caused by Xanthomonas phaseoli var. fuscans (Burkh.) Starr and Burkh. (12:) are bacterial diseases of major importance to dry bean (Phaseolus vulgoris L.) production. §p_and 5 j are considered two of the most important seed-borne diseases of dry edible and green beans in many production areas throughout the world (15, 22) and have been reported from Australia (2, 11), Russia (5), Yugoslavia (19), Michigan, U.S.A. (l) and Venezuela (14). The pathogens are seed-borne, both internally and externally (16, 22) and are capable of being transmitted long distances with seed. X f can survive in seeds for three years at 20-35°C and seed transmission is the primary means for dissemination of these bac- terial diseases. Much research has been directed toward maintaining bean seed stocks free of §p_and.§pf contamination and developing methods to detect seed-borne infection (21). Disease control is based on a seed certification program to maintain clean seed stocks. The Michigan seed certification program is administered by the Michigan Crop Improvement Association under authority delegated to it by the Michigan Department of Agriculture (12). Copeland gt_gl, (4) have described the process of producing Michigan certified bean seed from breeder and foundation seed stocks. The first step in certified seed production is to plant foundation seed supplied by the Michigan Foundation Seed Association. Such seed is usually grown in the semi-arid or arid west where conditions are unfavorable for seed-borne bacterial diseases. Seeds from fields which pass visual inspection for the presence of blight symptoms, and which show no contamination in laboratory tests for §p_and.§pf, can be sold as certified. Numerous assay methods have been developed or adapted for detecting the presence of seed-borne §p_and lpf_and other bacterial diseases. Direct plating of seed in agar has been used (7, 20). The cotyledon method (13, 18) has been used for the detection of fig, glycinea in soybean seeds (13). Another method used is the phage plaque count (8, 9). Schuster has used a leaf water-soaking method (17). Serology has been used by several workers (6, 10), and, in Michigan, the Michigan Department of Agriculture Bean Seed Testing Program uses a laboratory blight test developed by Saettler (16). There is no doubt that such tests have been successful in reducing seed infection by the bacterial pathogens; nevertheless, outbreaks of common and fuscous blights persist and some fields are rejected annually for certification. This suggests that present methods for assaying seed are not entirely satisfactory for detect- ing internal blight contamination of bean seed. Objectives Production and characterization of antisera to Xanthomonas phaseoli and 5, phaseoli var. fuscans. Development of a semi-selective medium for §p_and.§pf. To determine the possible utility of combined serologi- cal and semi-selective media techniques for the detection of Xanthomonas blight bacteria in seeds and other plant tissues. REFERENCES-~GENERAL INTRODUCTION Andersen, A. L. 1951. Observations on bean diseases in Michigan during 1949-1950. U.S. Bur. Plant Indus. Soils and Agr. Eng., Plant Dis. Rep 35:89-90. Anonymous. 1947. New plant diseases. Agr. Gaz. N. S. Wales. 58:94. Basu, P. K. and V. R. Nallen. 1967. Factors affecting viru- lence and pigment production of Xanthomonas phaseoli var. fuscans. Can. J. Bot. 45:2367-2374. Copeland, L. 0., M. N. Adams, and D. C. Bell. 1975. An improved seed programme for maintaining disease-free seed on field bean (Phaseolus vulgaris). Seed Sci. and Technol. 3:719-724. Galachyan, R. 1936. Bacteriosis of bean, their noxiousness, distribution and ways of infecting. Lenin Acad. Agr. Sci. Inst. Plant Protect., Sum. Sci. Res. Norw. Inst. Plant Protect. 1935:513-515. Guthrie, J. H., D. M. Huber, and H. S. Fenwick. 1965. Serological Detection of Halo Blight. Plant Dis. Rep. 4(3297-299. Hoitink, H. A. J., D. J. Hagedorn, and E. McCoy. 1968. Survival, Transmission and Taxonomy of Pseudomonas syringae van Hall, the causal organism of bacterial brown spot of bean (Phaseolus vulgaris L.). Can. J. Microbiol. 14:437-441. Katznelson, H. and M. 0. Sutton. 1951. A rapid phage plaque count method for the detection of bacteria as applied to the demonstration of internally-borne infection seed. J. Bact. 61:689-701. Katznelson, H., M. 0. Sutton, and S. T. Vayley. 1954. The use of bacteriophage of Xanthomonas phaseoli in detecting infection in beans with observation on its growth and morphology. Can. J. Microbiol. 1:22-29. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. Lovrekovich. L. and Z. Klement. 1963. A practical method to demonstrate the bacterial infection of bean seeds. Acta. Agronomica Hung. 12:83-88. Magee, C. J. 1930. Bacterial blight of beans. Agric. Gaz. N. S. Hales 41:229-231. M.S.U. Coop. Ext. Service. 1976. Seed certification in Michigan. Extension Bulletin E-1019, No. 112, 6p. Parashar, R. D. and C. Leben. 1972. Detection of Pseudomonas glycinea in soybean seed lots. Phytopathology 62:1075- 1077. Pontis Videla, R. E. 1959. La quemazon bacteriana de la caraota. Ministerio de Agricultura y Cria. Instituto Nacional de Agricultura. Maracay. Vzla. Bolletin 9:5-6. Saettler, A. w. 1971. Seedling injection as an aid in identi- fying bean blight bacteria. Plant Dis. Reptr. 55:703-706. Saettler, A. w. and S. K. Perry.' 1972. Seed transmitted bacterial diseases in Michigan navy (pea) beans. Phaseolus vulgaris. Plant Dis. Rep. 56:378-381. Schuster, M. L. 1955. A method for testing resistance of beans to bacterial blight. Phytopathology 45:519-520. Skekhawat, P. S. and B. P. Chakravath. 1979. Comparison of agar plate and cotyledon methods for the detection of Xanthomonas vesicatoria in chili seeds. Phytopath Z., 94:80-83. Tesic, Z. P. 1949. Bacterium phaseoli. Ann. Fac. Agron. Belgrade 2:103-113. Nallen, V. R. and M. 0. Sutton. 1965. Xanthomonas phaseoli var. fuscans (Burkh.) Dowson in white-seeded dwarf bean seed stocks. Ann. Appl. Biol. 60:305-312. Weller, David M. 1978. Ecology of Xanthomonas phaseoli and Xanthomonas phaseoli var. fuscans in navy (peET'beans (Phaseolus vulgaris L.). Ph.D. thesis, Michigan State University, East Lansing, MI. 137 pp. Zaumeyer, w. J. and H. R. Thomas. 1957. A monographic study of bean diseases and method for their control. U.S.D.A. Tech. Bul. 868. 255pp. CHAPTER I SEROLOGY OF XANTHOMONAS PHASEOLI (BEAN COMMON BACTERIAL BLIGHT) AND XANTHOMONAS PHASEOLI VAR. FUSCOUS (BEAN FUSCOUS BACTERIAL BLIGHT) Literature Review The genus Xanthomonas was proposed in 1939 by Dawson (9) and and was defined as follows: “Xanthomonas, n.g., non-sporing rod- shaped bacteria, gram negative, motile by means of one polar flagel- lum (rarely two present) or non-motile, yellow in the (mass) of nutrient agar and on potato, on which abundant slimy growth is formed. Most species digest starch and produce acid in lactose. None produce acid in salicin; they are all pathogenic for plants, and there seems no doubt that these organisms form a definitive group." On the basis of morphology and biochemical properties, the individual species have few distinguishing features (3). In 1962 Dye (10) performed a comparative study of 209 phyto- pathogenic Xanthomonas species comprising 57 recognized species, using numerous standard identification methods. He concluded that they formed a remarkably uniform group which could easily be dis- tinguished from some other yellow pgimented organisms. Dye and Le11iot described the genus Xanthomonas in the 1974 Bergey Manual of Determinative Bacteriology (4). Up until this time, serological work with Xanthomonas species was scattered and incomplete (11). St. John-Brooks, Nain and Rhodes in 1925 (38), working with 33 strains received from Dr. Erwin Smith and using tube agglutina- tion tests, found that of three strains of Xanthomonas campestris, two appeared to act alike, while one was unique in its agglutinative reactions in ten antisera prepared with bacterial plant pathogens, and that two strains of Xanthomonas malvacearum showed identical cross reactions. They also observed a close relationship between .1. campestris and 5, malvacearum and between L. phaseoli, 1. phaseoli var. sojense, 5, pelargoni and 5, vitians. In 1927 Sharp (36) produced sera against Corynebacterium flaccumfaciens, x, phaseoli and E: phaseoli var. sojense, with which these bacteria could be differentiated in tube agg1utination tests with live cells as antigen. Link and Link in 1928 (21) found that the agglutination tests could differentiate Xanthomonas malvacearum from 5. campestris, x, phaseoli, 5, $1531, 1. cucurbitae and 5, prgnj, These workers found that direct agglutination tests were of little use in dis- tinguishing between 5, malvacearum and 1, phaseoli sojense. They concluded that not all of the yellow organisms tested form a single serological group. In 1929 Link, Edgecombe, and Godkin (20) utilized agglutinin absorption as well as agglutination tests. They stated that sero- logical studies apparently gave promise in grouping and classifying at least some of the closely related species of phytopathogenic bacteria. In 1931, William and Glass (42) and Horgan (17) independently established the serological homogeneity of Xanthomonas malvacearum by agglutinin absorption tests. In 1940, McNew and Braun (27) and Braun and McNew (5) con- cluded that 5, stewartii was not serologically homogenous; we know now that 3, stewartii corresponds to the genus Erwinia (3). In 1947, Elrod and Braun reported serological studies in the genus Xanthomonas (13, 14, 15). They found that when phyto- bacteria were grown in sugar-rich media for 72 hours at room temper- ature, the bulk of the growing mass was composed of extracellular mucoid material and that the mucoid material interfered with anti- genic patterns. Mucoid material extracted with warm saline and harvested according to the method of Morgan and Beckwith (28) yielded a large quantity of polysaccharide. In agglutinin tests the semi-purified polysaccharide acted as a common component giving strong reactions at high serum concentrations. They stated that the common mucoid material, produced by many bacteria, was respons- ible for the cross reaction obtained by earlier workers in the field. Elimination of the mucoid exudate resulted in cellular antigens which, on cross agglutination, reacted more specifically than gummy suspensions. They defined five immunological groups: "vascularum," "phaseoli," "campestris," "translucens," and "pruni." The X: phaseoli group contained X: phaseoli, 3, phaseoli var. fuscans, 5, geranii, x, pelargoni, and l: malvacearum. The phaseoli block showed strong antigenic ties to one another and to 5, campestris and 5, barbarbareae. The "fuscans" bacteria agglutinated in mahy "vascularum" antiserum but the reciprocal case was not true. They stated that members of the Xanthomonas phaseoli group had common group components and absorption of any of the individual antisera by a heterologous organism (of the same group) removed all group components and left specific factors. The next report on serology of Xanthomonas appeared in 1959, when Mushin g§_gl. (31) mentioned the use of slide and tube aggluti- nation and agglutinin absorption tests. "0" antisera were prepared with bacteria grown 24 hours on carbohydrate-free medium (meat infusion or brain heart agar); both steamed and Roschka's antigens (alcohol-acetone treated suspensions) were used for antisera pro- duction. Mucoid was removed from Xanthomonas cultures using trypsin digestion. "H" antisera (flagella) were prepared by passing bacteria several times through semi-solid (0.4%) agar in Craigie tubes until active motility was induced. Preliminary agglutination tests were performed with steamed, Roschka's, alcoholized, and autoclaved bacterial suspensions as antigens. The first two antigens proved to be the most sensitive. Antisera to Xanthomonas phaseoli reacted non-specifically with 5, campestris, 5, carotae, 5, vesicatoria, 5, incanae, 5, juglandis, and 5, albineans. . Until 1959, then, the only information relative to the serology of Xanthomonas was that various species cross agglutinate and that some species agglutinate in some sera prepared against 10 other bacterial genera. 0nly short notes and abstracts on serologi- cal techniques applied to the genus Xanthomonas have been presented in recent years. In 1964, Morton (30) detected Xanthomonas vesicatoria in leaves of Capsicum frutescens by bentonite flocculation. The same author (29) suggested the use of antibodies conjugated with fluores- cent iso-thiocyanate and ultraviolet examination to detect 5, vesicatoria on pepper leaf sap. In 1970, R. Charudatan and R. E. Stall (7) used agar gel double diffusion tests and examined 72 isolates of 5, vesicatoria from pepper and tomato against antisera prepared with sonicated cells; two serotypes were described based on the presence or absence of specific precipitin bands. In 1971, Orellana and Weber (33), working with 5, cyamopsidis races 0 and l and using immunodiffusion analysis, found that the two races differed antigenically. In 1972, Saaltink et a1. (35) prepared antisear to 5, hyacinthi and 5, gummisudans and used a microdroplet agglutination method under liquid paraffin, described by van Slogteren (37). In the same year Namekata and Oliveira (32) reported a comparative gel diffusion and immuno-electrophoresis study of 5. giggi_and a bacterium causing a canker disease on Mexican lime; the authors used bacterial extracts obtained by heating suspensions for 45-60 minutes at 100°C. Saalting and others (35) present procedures for serological identification of bacteria in tissues of leaves or bulb scales of 11 infected hyacinths. 5, hyacinthi was identified with the micro- droplet agglutination method (37). Erwinia and Corynebacterium species have been identified using agar gel double diffusion (18, 41). Recently, a new bacterial disease attacking onions was iden- tified serologically as being caused by a Xanthomonas sp. (1). A thorough literature search, including a computer search in 1977 and 1978, disclosed no additional work on the relationship between 5, phaseoli and 5, phaseoli var. fuscans or any other Xanthomonas nomen species. However, there are a number of papers on similar other genera, in particular the genus Pseudomonas. Pertinent papers include: in 1969 Lucas and Grogan (24, 25) reported on serological variation and identification of Pseudomonas lachrymans and other Pseudomonas nomen species. They used gel diffusion tests with antigens from different plant pathogenic Pseudomonas and found that all shared common antigens but each possessed at least one specific antigen. Common antigen bands were eliminated by heating antigen preparations one hour at 100°C. The same workers also found that specific antigens from smooth serotypes I and II of Pseudomonas lachrymans and smooth isolates of other nomen species could be extracted with trichlor- acetic acid or phenol water; antigens of rough isolates could not be extracted with these procedures. Antigens from smooth isolates were serologically active, heat stable with properties similar to "0" antigens of other Gram negative bacteria. Otta and English in 1970 (34) divided 450 isolates of fig, syringae into ten distinct serotypes based on the reaction of their 12 heat stable antigens in gel diffusion tests. Rough and smooth isolates possessed serologically identical heat-stable antigens. In the same year, Taylor (39) showed that the heat labile antigen possessed by 35, phaseolicola was common to 12 other plant pathogenic Pseudomonas species. The heat stable antigen was more specific and was detected only in Eé, morspronorum and £5, primilae, neither of which occur on bean. Coleno g§_gl. (8) in 1971 studied several phytopathogenic Pseudomonas by the complement fixing technique and showed the existence of several antigenic determinants of which some were common, and others were specific. The specificity could be used in taxonomy or detection of seed-borne bacteria (with the pos- sibility of estimating contamination rates). In 1975, Tunstall and Gowland (40) found that three different antigens were associated with the cell walls of Pseudomonas species: (1) a highly specific antigen of high molecular weight conposed of protein or lipoprotein in nature as an envelope around the cell; (2) another antigen common to all Pseudomonas sp. was studied and was situated below the first antigen, and composed of polysaccharide or lipopolysaccharide in nature; and (3) a heat-labile antigen which was common to all Pseudomonas sp. and consisted of the mucopeptide of the cell wall, and which was dependent on both the carbohydrate and polypeptide components of the macro-molecule. Objectives The objectives of this study were threefold: (l) the pro- duction and characterization of antisera to Xanthomonas phaseoli 13 and 5, phaseoli var. fuscans using 0 antigen (steamed) and H antigen (formalized); (2) to determine the specificity of the antisera obtained against Xanthomonas phaseoli and 5, phaseoli var. fuscans; and (3) to examine the use of microagglutination and agar gel double diffusion techniques for studying antisera to Xanthomonas phaseoli and 5, phaseoli var. fuscans. Materials and Methods Antisera Preparation Antisera were prepared against formalized and steamed cells of several isolates each of Xanthomonas phaseoli (bean common blight) and Xanthomonas phaseoli var. fuscans (bean foscous blight). Formalized cells.--Cells were obtained by inoculating the surface of YCA plates (10 gm yeast extract, 2.5 gm CaC03, 20 gm of agar in 1,000 m1 of distilled water), with the desired isolate of Xanthomonas. The agar cultures were incubated at room temperature (23°C) for 24-48 hours. The growth then was removed with formal saline. The cell suspension was allowed to stand at room temperature for 48 hours to kill the cells but maintain the flagella intact (Formalin = 40% v/v formaldehyde in psysiological saline. Formol- saline = .6% v/v Formalinin Physiological saline. Saline = 8.5 gm NaCl in 1,000 ml distilled water). Cells were then centifugated twice in a Sorvall centrifuge SS-l set at 30 for 20 minutes and resuspended in saline. The cells 9 were adjusted to a density of 1 x 10 cells/m1 using McFarland nephelometer tube no. 3 (2). 14 Five ml volumes of the suspension were aseptically stored in sterile serum bottles; 2-3 drops were removed and transferred to YCA plates to check for sterility. Steamed cells.--The somatic antigens were prepared by remov- ing the 24-48 hr. growth from YCA plates with saline. Cell suspensions were washed twice by centrifuging as in Formalized Cells and resuspended in a buffer saline. Cell suspensions standardized to 109 cells/ml were steamed at 100°C for one hour. This treatment killed the cells as well as destroyed the flagella (19). Volumes of 5 ml cell suspension were stored in sterile serum bottles and checked for sterility as before. The antigens prepared in Formalized Cells and Steamed Cells were stored in 4°C and used when needed. Immunization of rabbits.--Young female rabbits were injected at 0 (0.1 ml), 4 (0.3 m1), 8 (0.5 ml), 11 (1.0 ml) and 14 (2.0 ml) days; blood for sera was collected 7, l4, and 21 days after the last injection (6). The marginal ear vein was used for injection. The first injection of the series was always made as near the tip of the ear as possible; succeeding injections were made closer to the animal's head, so the scar tissue would not interfere with the injections. Rabbits were bled for normal sera, prior to the first injec- tion; titre of the normal sera were determined using standard tube agglutinin tests. 15 The marginal ear vein was used for bleeding. The vein is located at the outer edge of the dorsal side of the ear. One ear was used for bleeding and the other one for injections. The first bleeding was made from a site near the middle of the ear; succeeding punctures were made distal to the head. Antisera collection.--Blood was collected in a sterile, dry glass test tube and allowed to stand undisturbed until a firm clot formed. The clot was carefully detached from the inside surface of the container using a sterile wooden applicator. The sample was left at room temperature for 3 hours and placed in a refrigerator over- night. The fluid serum was carefully removed, centrifuged 2,000 x g for 5 minutes to remove cellular components and stored at -5°C in serum bottles. Serological Techniques Agglutination.--The clumping of particulate antigen--in our case bacterial cells, mediated by antibodies (agglutinin). Aggluti- nation is the specific combination of agglutinin with its homologous antigen or a closely related one, followed by aggregation of the particles and antibody (6, 12). a. General Procedure 1. 12 x 75 mm test tubes were placed in a rack. 2. 0.8 m1 of saline was added to tube 1 and 0.5 to the remaining tubes. 3. 0.2 ml of serum was added to tube 1. 16 4. 0.5 m1 from tube 1 was transferred and mixed with tube 2; 0.5 ml from tube 2 was transferred and mixed into tube 3; continuing until contents of the next to the last tube had been mixed; 0.5 ml was then discarded from the next to the last tube. 5. 0.5 ml of antigen (adjusted to 1 x 108 cells/ml) was added to all tubes and mixed. The last tube contained no serum and represented the control. 6. The tubes were incubated 24-48 hr. in a water bath at 37°C. b. Agglutination Reactions Each tube was examined for settled aggregates of cells and the cell patterns on the bottom of the titration tubes were compared with the pattern of the cells in the control tube. Tubes were marked 4+, 3+, 2+, 1+ or negative, depending on the amount of agglutinated cells on the bottom of the tube relative to the control. Microagglutination.--0ne disadvantage of the tube agglutinin test is the requirement for large volumes of antigen and antiserum (12). On the other hand, the use of a grid titration system in the microagglutination test would utilize only 0.2 m1 total volume of antiserum and antigen. a. Procedure A square plastic petri dish (9 cm square) was divided in a checkerboard pattern with wax pencil; each check 17 was 1.5 cm square. A small drop of the serial dilution of tne antisera was placed in each square. A drop of 8 cells/ml was placed over each antigen adjusted at 10 drop of the dilution. A check consisting of a drop of saline plus a drop of antigen was always used. The petri dishes were incubated at room temperature and were carefully sealed with two layers of parafilm paper. The reaction was read after two hours and again in the morning using obliquely-transmitted light in a stereoscopic microscope. Gel diffusion tests.--These tests utilize the precipitation of antigens and antibodies in a gel medium rather than a fluid medium. Lines of precipitation will form when specific antigen and antibody come together in equivalent proportions by diffusing through the agar media. a. Ouchterlony (Immunodiffusion in agar plates) The media contained 8.5 gm, purified agar, 10 ml of solution containing 1% w/v Orange G, 30 ml of a solution containing 10 mg/ml NaN3, and 1000 m1 of buffered saline. NaN3 was added to prevent bacterial and fungal contami- nation (16); Orange G was added to increase contrast during photography (18). Petri dishes of 9 cm diameter were filled with 20 m1 of melted medium, and 2 serological tests were routinely performed in each plate. Each serological test 18 consisted of one central hole of 5 mm diameter sur- rounded by 6 holes of 5 mm diameter. Normally, the central hole contained the antisera and the surrounding holes contained the antigens. All plates were maintained inside plastic bags to avoid loss of humidity, and readings were taken every two days. Radial immunodiffusion.--This procedure employs the precipi- tation of antigen-antibody complex in a gel for quantification of one of the reacting components (19). Antibody is incorporated into a buffered agar medium, and the soluble antigen is placed in wells cut in the medium. Antigen diffuses Out from the well and precipi- tates with the antibody in the medium in a radial pattern. The diameter of the ring of precipitation directly reflects the concen- tration of antigen in the well. a. Procedure 1. 1.7 gm of purufied agar (Difco Co.); 100 m1 Borate buffer (pH 8.4); 6 m1 of NaN3 solution containing 10 mg/ml. The media was melted at 100°C and equili- brated to 55°c in water bath. 2. Antisera were diluted 1:15 or 1:20 with borate buffer and maintained at 55°C in a water bath. Equal volumes (10 ml) of (1) and (2) were mixed in a petri dish and allowed to cool. Equally spaced wells (6) of 5 mm diameter were cut in each petri plate. 19 In each petri plate two of the wells were filled with reference antigens, the remaining wells were filled with test antigens. Rings were measured one week later. Preparation of the absorption-antisera.--Agglutinin- absorption is used to prepare “typing serum" which contain the anti- bodies desired to identify (by agg1utination or precipitation) only those major determinants as desired (19). a. Procedure 1. 1 ml of the desired antiserum was diluted 1:10 with saline and mixed with 1 ml of packed cells of the selected antigen in a'12 x 75 mm test tube. 2. Tubes were incubated for 4 to 5 hours in 45°C water bath and then refrigerated overnight. 3. Contents of the tube were centrifuged at 2000 x g, the supernant was carefully removed to a clean test tube. Efficiency of the absorption was determined by a standard agglutination test against the homologous antigen, and the procedures in (l) and (2) were repeated as necessary. Results litres of Different Antisera to Xanthomonas Using the Tube and Microagglutinin Tests Antisera titres obtained when formalized cells (H antigen) were used as antigen were equal to or greater than titres obtained when steamed cells (0 antigen) were used (Tables 1, 2). Titres 20 .mczouo FFme cowgmcwuapmmm cows: pm mcmmwucm mcp co cowuzfiwu ummummcm mg» acmmmcnmc mums a .m.o u 5 $0 zuwmcmc Fmowpno cm op umumzmum mP—wu cosmmpm mucmmmcamc :mmwucmmz acmmwuc< umcmamca :mmv#:< maomo—oeo: esp we Any zuwmcmo _mowuao mcmmwpc< n.gmmwuc< maomopoeo: mo meowuwcucmucoo maowco> umcwmm< vmpmmh mcwmwuc< mmcoeogpcmx eo memzpm=< cowmzmmwo mpnaoo Pow cmm<11.m m4m

ww .eemwue wpeemeem am ++++ ++++ ++++ ++++ ++++ ++++ mp mm“ ++++ ++++ ++++ ++++ ++++ ++++ Pp wpeemeem AM ++++ ++++ ++++ ++++ ++++ ++++ weemce em aw ++++ ++++ ++++ ++++ ++++ ++++ .x ++++ ++++ ++++ ++++ ++++ ++++ mweumeeseu meeeseepeex ++ ++ +++ ++ +++ ++ mom e weew Pemmeem .m ++ ++ +++ ++ ++ ++ Pom eweewpeemeen .me + ++ + ++ + + memewcxm 2mm ++ ++ ++ ++ ++ ++ eeewexpm amm ++ ++ ++ ++ + + meeemecezpw meeeeeeeeme + + ++ + + ++ epeewecee am + + ++ + + + eewumemecpe .cm> eee>epecee am + + ++ + + + ece>eP>Ee ewewzem + + + + + + emeeeemweowe «w + + + ++ ++ + em we meewpeeem11.n mem wweemeem am ++++ ++++ ++++ ++++ ++++ ++++ m—.mm ++++ +++ ++++ ++++ ++++ ++++ FF wpeemeem mm +++ +++ ++++ +++ +++ ++++ weemcepem AM +++ +++ +++ ++++ +++ +++ mweeewmen .x +++ +++ ++++ ++++ ++++ ++++ mwcemeasee meeeseepeex + ++ + + ++ + mom eewuepeemeee. mm + ++ + ++ ++ + Pom epeewpeemmmm amm 1 1 + 1 1 + memewexwi.me + 1 + 1 + + eeewexpm .mm. + 1 + + 1 + meeemeee:_w meeeEeezeme - - - - - + eweewece; em 1 1 1 1 + + mewmmemecee .ce> eee>eeecee am 1 1 1 1 + + ece>epxse ewewzcm 1 1 1 1 1 1 emeeeemweowe am 1 1 1 1 1 + em we meeweeeem11.w u4m wweemeem meeeEeeeeex eee ammv wweemeem,meee5eeueex maewce> we meeweeeem eeweeewezpmm< mmeLU11.m mem<|>< aAntigen consisted of steamed cells adjusted to about 1010 cells/m1. b- = no band formation; + = 1 or 2 bands; ++ = 2 or 3 bands. 37 were tested against: Pseudomonas fluorescens, fig. syringae, fig, phaseolicola, Erwinia herbicola. Agrobacterium flaccumfaciens, g, fascians and 5, megaterium (Table 12) in similar tests. Absorption Tests Antisera prepared against whole cells of 5915 did not cross react with the following bacteria after absorption with 511516 or of 5251085: 5, juglandis, 5, Qelargoni, 5, phaseoli var. fuscans (2 isolates), Bacillus megaterium, g, herbicola. Agrobacterium tumefaciens, fig. fluorescens, fig, syringae and fig, phaseolicola. The antisera did react with 3 isolates of 5. Ehaseoli (Table 13). After absorption, final antisera titres were reduced from 2560 to 320. Antisera prepared against whole cells of 52116 did not cross-react with the following bacteria after absorption with 5211 or 5215: 5, juglandis, 5, Eelargoni, 5, phaseoli (isolates 11 and 15), 5, mggaterium, g, herbicola. A, tumefaciens, g, michiganense, g, fascians, fig. fluorescens, fig. syringae or_fig. Phaseolicola. However, the absorbed antisera did react only to 595 isolate 1085 and to the homologous isolate 5gj16. Absorption reduced titres from 2560 to 320. Antisera prepared against whole cells of 5215 or 53516 reacted only with the Xanthomonas group after absorption of the antisera with fig, phaseolicola; absorption reduced antisera titres from 2560 to 640. 38 .ueemece meeee N so P n + meewpeELew eeee n 1e .eewee~wpweeem ceuwww eee .u°oo_ em .ewe oe mewEeeum .eewwem cewwze ew eewmeeemsmee .mxoooo.m we .ewe mp mmceepze Fewceeeee we eewuemzwwcueee 8e eeeweeee ewuweweeee n eemwue.flflmmmmmm am mp wweemmea meeeeeeeeex 1 1 1 1 1 meeemeeeeww meeeEeeeeme + + + + + + + + + + + + + + + 1 1 1 1 1 epeeweeee eweszN 1 1 1 1 1 emceeemweewe am 1 1 1 1 1 emme meeweew530eepw am 1 1 1 1 1 meewemew Ezwcepeeeeeameu 1 1 1 1 e1 Eewceeemme mepwweem 2mm 2% 88% :mm 2% 83 e88 88:8 mFPeu eeEemem mF—eo eerFeEcee umewemq eeceeece ecemwue< .mpmew eewmewwwa epezeo Few cem< ew ecemwee< meeeeeeueex emewem< wepepemH wewceueem meewce> we meewueemm11.NF memez ew eewueewEeeeeO wweemeem.meeeeeepeex FeeceeeH we eeweeeeee wee ee eewewmeeeee eweez we ueewwm eew11.ew memwe>ew eeeewceexu .Aepeeemeee eewwae z wO.O we eeeww eee ew eewewxeeeweze we OE va eeeweeeee ewees wememe 1 - - - - 1 - - + + o 3 - - - - - - - - + + e to 1 1 1 1 + + + + + + _ O.m 1 1 1 w + + + + + + F O.F 1 1 1 w + + + + + + P —.O 1 1 1 w + + + + + + _ o.o xumcu ompmup ommmup owmpu— ovoup ONmup oopup ownp oeup ONHP ummnmwmm ~\Em vmvu< eewuawwO ecemwue< we ceeeOz ueecexm umee> n1 e.memew ewewuawmmeecewz ew eeem eeem >>ez ew eewpeewEepeeO meeemze .ee> wweememm,meee5eeeeex weeceeeH we eeweeeeeo we» ee eewpwmeeeeu eweez we ueewwu eew11.m_ NOOwe>ew peeeweeexm .Aepeeemeee eewwee z FO.O we eepww eee ew eewewxeeepeze we Os va eeewepeee eweee Pememe - 1 - - - _ - .. + + e 04.. - - .. - - - - + + 0 re - - 1 + + + + + + F 9,1. 1 1 + + + + + + + w O.w 1 1 + + + + + + + w F.O - - + + + + + + + e 0.0 ON_mu_ OemNu_ eeN_ F eee _ ONm P eee F eee, O8”. ONHP eemnmwmm w\ee eeeee eeequPwO eeemwpe< we eeesaz aoeeaxm umee> e.memew ewewOOPOOeeeewz ew eeem eeem 8>ez ew eewueewEOHeeO meeemze .ee> wweememm,meeeseeueex weeeeeeH we eeweeepeO eee ee eewpwmeOEeO eweez we mueewwu eew11.ep meme appeweeeEEOOe .mN.O n e we eepmanee eewmeeemem weweeueee we w.O euwz eeeeem eee: meuepe e . N_ o Ne S 8 8e 2 8:93.53 2 e e e e 3 2: 58528 8 8 8 S 8 8e. 8 88:.er 8 8 2 8 N 8e m £88885 8 NN o , 8 8 8e. 8 5828858528 m 8 8 8 2 8 8e 8 588852... 8 e o 2 NF 8... 8 E23528 w N O O_ Ow Oe Ow ewezseeea< eewpeeweeee meeemeeee—w epeeweeee emeeeemweews aneeeemee eewueeu eweeweewee< 88 + em 85888.. g 821588 a -888 a 1eeNeeeO epmeweO< eeume» eee: eeeN eewpweweeH we “say eeuesewO .mpmew xemmeewm ew mewuewewee< Feee>em ee eweeeeem msewee> we 8¢w>wuwmeem11.np m4meemee OP we eOeee>e eee mw eOFe> eeee .eewueeaeew eexeem we meeee we eeewe memepw eee we Ase ONO n ev OO eee we eemmeeexe mw eezeeO e .m.O u e ee eeemewee eewmeeemem weweeeeee eee we we P.O sew: eeeewseeew eee .ewueweweee eeeeeweew eee epwz eeueeEewOOOm we; Aeeeeemeee eewwze N.N :e z p0.0 we eepww eee ew eewewxeeepuxe we OE mN .ueeeuxe Heme» EO O.wv eewuwmeeEee eweee wememe e e O o e 80. me. me. m_. eN. eeweaeaeeeew e e e 0 8o. e_. NN. ewaeEeeemw e e e e o e e o FN. e_NEewwe e e o o o e we. eN. m eoeee_=m ewaee°uaeeem O O O O m—. Np. ON. eN. eN. 8N. eweewee>ez OF. N_. mN. NN. eN. eN. eweueeeeweeewz e o o e e m_. MN. 8N. ewUNEeez O. mO. Ow. NN. mN. 8N. ewee ewxwewpez O O O O mp. NN. ON. ON. ewwpwewepez O O O O NO. NP. PN. NN. ON. ewewEeeeeO o o 0 mo. op. e_. FN. 8N. eeawemeQEeeo_ee O.m O.e O.N O.N m. P. mO. NO. PO. O ewuewewue< A._e\OOV ewuewewee< we meewueeueeeeeu eeeeeweeH um eeezeeO a.mawoo_e_ee< mzawea> eee: eeeeeEeeeeam eweaz eeaewe ew meee_ame eemw_m _eweeeuem meeaeaeeeex xwm we eezaee1-.ee memew 7O Twelve antibiotics were evaluated relative to their effects on various seed borne contaminants and on several Xanthomonas iso- lates in liquid media. Concentrations of antibiotics that were used were those that were shown to be inhibitory to contaminants in pre- vious studies but not to Xanthomonas phaseoli or to Xanthomonas ghaseoli var. fuscans (Table 19). 0f the contaminants tested, Gram positive C4 and C4 were both effectively inhibited by Chloramphenicol, nalidixic acid, nitrofurantoin, penicillin, and streptomycin sulphate. Nalidixic acid at 1.0 ug/ml inhibited six of the seven contaminants tested. Penicillin, rifampin and terramycin, however, were inhibitory to the two Xanthomonas control isolates. Antibiotics showing good activity against seed borne contaminants were then eval- uated relative to their effect on internal bean seed microflora of disease-free seeds incubated in liquid media (Table 20). All of the antibiotics used were able to significantly reduce bacterial contaminants in the liquid media. Gentamicin at 0.05 ug/ml, methi- cillin at 0.5 ug/ml, nalidixic acid at 0.5 and 1.0 ug/ml, and nitrofurantoin at 2.0 ug/ml provided the greatest degree of control of the growth of internal bean seed bacterial contaminants. Combinations of various antibiotics at effective concentra- tions as determined in previous studies were then evaluated as to their inhibitory effect upon the growth of internal bean seed bacterial contaminants in liquid media (Table 21). The following antibiotic combinations gave statistically more significant control of the bacterial contaminants: nalidixic 71 .eewpeeseew eexeem mezee Om eepwe memeww eee we A.Ee ONO u ev OO eee we eemmeeexe mw euzeeO .eewueewwwpeeew peeewEeueeu eew eweew eeme .meewee>eemee eeeep we eOeee>e eew mw ezwe> eeee e .Ee ONO we epOeewe>Oz we m.O n e ea eepmamue eewmeeemsm _eweeueee eee we FE F.O new: eeeeweeeew .ewuewewuee eeeeeweew eee eew: eeueeEePOOOm we: Aeueeemeee eewwze N.N :O z F0.0 we eepww eee ew eOwwaeeeFOAO we OE ON .eeeepxe pmeea EO O.wv eewewmeeEee ewOeE wememe me. me. OP. OO. Om. ON. Nm. Om. Om. N_. PO. OOweaeaeeeew OP. OO. OO. NO. ON. Om. w8. me. O ON. _. eeaeeOeeew me. O_. OF. mO. NO. mm. m_. O NO. O OO. O OOOEOPOO Owaeeaeaeeem OO. OO. OO. O OO. em. Om. Om. OO. O NO. O_OeOwwO O_. OO. OO. NO. ON. em. Oe. NO. O ON. O.O e_e\OO ew__waweea O_. OF. Oe. NM. NM. _m. NO. Om. Om. O_. F. OOOOOOOOOZ O_. me. me. we. ON. OF. Om. OP. m_. ON. O.O ewaeeeeewaeewz OF. OP. me. me. Om. _m. Nm. em. Om. OO. OO. Oeaeeaez NF. OP. OF. OO. NO. ON. O_. NO. O OO. O.N OOOO waweweez N_. O_. Ne. NO. ON. ON. Om. PO. Om. ON. _. Oweeeaweeez Ne. Ne. OP. OO. O Om. NO. me. MO. O OO. ewOOEOOOOO O_. O_. Ne. me. N_. mm. Om. ON. O NN. F. _Oeweeeaeeea_ee Oe. O_. N_. me. mm. mm. Om. me. me. NN. Oeaz Omwmm memm _Omm O_O O_O eee OOO OO OO _O _e\OO OOOO OOOOOOOOOO imepcwucou e.eewewemH Peweeeeem O.mO_OOwOwOO< OOOweOO new; OOOOOEOFOOOO Oweez Owner; ew emueeewEeeeeO eeem eeem PeeeeeeH eee meuewema ueOwwm meeeEeeueex we epzeeO11.O_ Oem .4. . ee OeewEeueO eeez mpeeewEOpeee Peweeueem .Ava eweeE e>weeewem eweEewwe eew: eeewEeeuee eee: mewmm .OE eee Opmwmm OOP x N Oeweweueee esepw eeee we OE ON eew: eeeepeeeew .meweewewuee eeueeweew eee eew: eeeeeEeweezm we: Aeeeeemeee eewwse N.N :O z PO.O we eepww eee ew eewwaeeewexe we OE ON .peeeuxe emeea EO OPFV eewuwmeeEee OweeE womeme o888. NO, x O.O OO_ x O.N O.O APO\OO O Ow_ewo_eea eeOONm. mow x O.w OOF x O.P O.N eweueeezweepwz OOOONO. NOP x 0.0 NO, x 0.0 O.” ewe< ewxwewwez OONNON. NO_ x 0.0 OOP x m.m O. ewe< ewxwewwez ee OOO. New x N.O OOF x O.N O. ewwwweweeez eeeONN. OOP x N.O OOF x N.m OO. ewewEOpeeO OONOm. OOF x O.N OOP x m.m w. weeweeeOEeeeweO 5e OOO. NOP x P.N OFOF x O.m eeez .OOOO\Hmm New .OOOO Fe\OO Owoawewoee eewueepeeeeeu .M Ow eweem meewpeewweem eew: OeueeEeweeam Oweez eweewe ew mpeeewEeueeO O.moeoaeewoe< meoeee> eeweeooem OOO O.NNm.wO OOOOOO_OOOO--.NN memew 76 TABLE 23.--Populations of 525 R10 and Bacterial Contaminants in Liquid Media Supplemented with Various Antibiotics.a Concen- . Ratio . . . trat1on Ant1b1ot1c ug/ml 525_ Cont. _525/cont. * None 1.2 x 107 2 x 1010 1/1.666C Nalidixic acid 1.0 9 5 x 107 3 x 108 1/3ab Nalidixic acid 1.0 8 8 a + Gentamicin .05 1.0 x 10 l 2 x 10 5/6 Nalidixic acid 1.0 8 8 ab + Nitrofurantoin 2.0 1 4 x 10 2.9 x 10 1/2 Nalidixic acid 1.0 8 8 a + Chloramphenicol .1 2 2 x 10 3 l x 10 2/3 Nalidixic acid 1.0 7 8 ab + Gentamicin 05 9.6 x 10 2 x 10 1/2 + Chloramphenicol l Nalidixic acid 1.0 8 8 a + Gentamicin .05 3.9 x 10 3 2 x 10 10/8 + Nitrofurantoin 2.0 Nalidixic acid 1.0 8 8 b + Chloramphenicol .1 1 0 x 10 8 x 10 1/8 + Nitrofurantoin 2.0 Nalidixic acid 1.0 + Chloramphenicol .1 8 8 ab + Nitrofurantoin 2.0 3’5 x 10 8 x 10 1/2 + Gentamicin .05 aBasal media composition (1.0 gm yeast extract, 25 mg cycloheximide in one litre of 0.01 M pH 7.2 buffer phosphate) was supplemented with the indicated antibiotics, inoculated with 20 mg of bean flour containing 2 x 105 525510 per mg., 525510 were determined with rifampin selective media (32), bacterial contaminants were determined on YCA. *Values followed by any common letter are not statistically different at o = 0.01. Duncan Multiple Range Test. 77 concentrations indicated in Table 22 as being effective. Therefore, in all subsequent studies the semi—selective media (SSM) contained: nalidixic acid 1.0 ug/ml, gentamicin .05 ug/ml, and nitrofurantoin 2.0 ug/ml. The SSM was then tested against various plant pathogenic and non-pathogenic bacteria in liquid culture (Table 24). SSM was very inhibitory to Agrobacterium tumefaciens, Bacillus megaterium and to species in the genera Corynebacterium and Erwinia. SSM was somewhat effective against certain Pseudomonas species and generally ineffective when tested against several other plant pathogenic Xanthomonas species. Minimal Number of Bacterial Cells Detected by SSM A serial dilution of L. phaseoli and 3, phaseoli var. fuscans cell suspensions was prepared: one ml of each dilution was added to 125 ml flasks containing 25 m1 SSM. All dilutions led to turbidity 4 of the SSM. Turbidity at 48 hours contained 10 cells (therefore, the SSM initially contained 400 cells/ml). Internal Bacterial Contaminants of Navy Bean Seeds More than 56 internal bacterial contaminants were isolated from various lots of navy bean seeds which differed, on the basis of colony type, color, rate of growth, and Gram reaction. The con- taminants were divided into 19 representative groups and these groups were selected fOr further characterization. All of the bacterial contaminants were injected into Red Kidney bean plants 78 TABLE 24.-~Growth of Various Plant Pathogenic and Non-pathogenic Bacteria in Liquid Media with and without Added Antibiotics. Growth inb a Basic Basic Media Bacteria Media + Antibiotic Agrobacterium tumefaciens .36 0 Bacillus megaterium .46 0 Corynebacterium fasciens .40 0 Q, flaccumfaciens NE23 .24 0 C, flaccumfaciens 6887 .22 0 g, flaccumfaciens .25 0 Erwinia amylovora .21 .06 E, carotovora var. atroseptica .30 0 g, herbicola .35 0 Pseudomonas fluorescens .45 .21 .Es. glycinea .27 .19 Es, phaseolicola .26 .19 Es, syringae .29 .22 £_, syringae Y30 .23 .19 Xanthomonas campestris .19 .17 5, juglandis .20 .16 5, pelargonii .21 .18 5, pruni .18 .16 aFlasks were seeded with 0.1 ml of the bacterial suspension adjusted at h = 0.3. Growth is expressed as the 0.0. (h = 620) of the flask after 36 hours shaker incubation, each value is the average of three observations. bBasal media composition (1.0 gm yeast extract, 25 mg cyclo- heximide in one litre of 0.01 M pH 7.2 buffer phosphate), basal media plus antibiotic (basal media, 1.0 ug/ml nalidixic acid, 2.0 ug/ml nitrofurantoin, .05 ug/ml gentamicin). 79 to test for pathogenicity. None of the contaminants were pathogenic to bean. 0f the 19 contaminants characterized to genus or species, 4 were found to be Pseudomonas fluorescens on the basis of: Gram negative reaction, the production of a green fluorescent pigment on King's B medium, positive for catalase activity and oxidase activity, and negative in inducing hypersensitive reaction when infiltrated into tobacco leaves (Table 81). Isolates 3, 13 and 15 were identified as Bacillus sp. because they were Gram positive, catalase positive and possessed endospores; isolates l and 10 were identified as Erwinia sp. because they were Gram negative, with production of a yellow non-diffusible pigment and did not reduce nitrates. Discussion From the initial results an optimum shaker incubation period of 48 hours was necessary to obtain a Xanthomonas population large 4 enough to detect in agar gel double diffusion tests (from 10 cells/ ml to 107 or more cells/ml). Yeast extract concentrations of 0, .l, and 1.0 were all equally capable of supporting Xanthomonas growth to levels suffi- cient for serological detection. In 1974, Szabo (27) reported that §, silvestris strains were unresponsive to nutrient concentrations but that the contaminant population tended to grow faster with increasing nutrient concen- tration. We selected a basal medium containing 1.0 gm yeast 80 extract/litre for use in antibiotic studies primarily because of its ease of preparation and its efficiency in encouraging Xanthomonas growth to levels high enough to detect serologically. The basal medium consistently permitted the detection of Xanthomonas in seed internally infected with this pathogen. Our preliminary bioassay results with commercially available impregnated discs are in agreement with previous reports (1, 2, 15, 30), which found that the antibiotics aureomycin, carbenicillin, chloromycetin, dihydrostreptomycin, erythromycin, kanamacyn, poly- myxin and tetracycline are inhibitory to Xanthomonas, as well as to other Gram negative and Gram positive bacteria. The antibiotic concentrations selected for further detailed studies were those which did inhibit the growth of several internal bean seed contaminants (Table 19). The internal bacterial contami- nants chosen for our experiments represented the same types of microflora known to inhabit bean seed, such as Bacillus, Erwinia herbicola and Pseudomonas fluorescens (12, l6, l8, 23). The combination of nalidixic acid at 1.0 ug/ml, gentamicin at .05 ug/ml and nitrofurantoin at 2.0 ug/ml was chosen for use in the semi-selective media (SEM) because of its superior performance in increasing Xanthomonas contaminant ratios. Taylor (28) found that Pseudomonas phaseolicola were readily obtained from white bean seeds if the dry seeds were ground to a flour, dispersed in sterile water, and sampled. Although there was considerable variation in the number of bacteria present in 81 individual infected seeds, 80% of the seeds contained 1 x 105 or more bacteria. Ednie and Needham (7) used in their experiments an average number of 7.8 x 107 5, phaseoli var. fuscans cells per infected seeds. SSM is highly selective for Xanthomonas. Nalidixic acid is a relatively simple synthetic antibacterial compound used in the treatment of the genitourinary tract (8). Its mode of action is unusual since it appears to depend on selective inhibition of DNA synthesis in pathogenic micro-organisms, especially in Gram nega— tives (28). Nalidixic acid was used by Grant and Hold (10) in a selec- tive medium for Pseudomonas. The author suggested that the primary function of nalidixic acid was the inhibition of enteric bacteria and of Gram negative cocci, especially Acinetobacter. Gross and Vidaver (11) recently developed a medium selective for Corynebacterium nebraskense which included nalidixic acid, lithium chloride and Bravo 6F. The medium inhibited the growth of Corynebacterium fascians; and reduced the growth of Q, flaccumfaciens. In our studies, Corynebacterium sp. did not grow in SSM. Gentamicin is an aminoglycoside antibiotic that affects protein biosynthesis and acts in a manner similar to streptomycin; gentamicin produces higher levels of M—RNA misreading than strepto- mycin (8). Many synthesis antibacterial compounds are based on the 5-nitro-2 furfurylidene structure. One of the best known is 82 nitrofurantoin, which has a wide sprectrum of antibacterial activity covering both Gram positive and Gram negative organisms (8). Our results are in agreement with the paper of Szabo (27) who used disc bioassays and showed that nitrofurantoin was highly inhibitory to many bacterial isolates but not to Xanthomonas sp. 10. REFERENCES--CHAPTER II Alexander, H. E., G. Leidy, and N. Redman. 1949. Comparison of the action of streptomycin, polymyxin, aureomycin and chloromycetin on H. Pertussis, H. Influenzae and five enteric strains of Gram negative bacilli. J. Clin. Invest. 28:867-870. Ark, P. A. 1947. Effect of crystalline streptomycin on phytopathogenic bacteria and fungi (Abstr.). Phytopath- ology 37:842. Bailey, w. Robert and Elvyn G. Scott. 1974. Diagnostic Microbiology. The C. V. Mosby Company, St. Louis, 414p. Brock, Thomas D. 1974. Effect of antibiotics on macromolecular synthesis in biology of microorganisms. Prentice Hall, New Jersey, p. 263-2666. Crosse, J. E. and R. N. Goodman. 1973. A selective medium for and a definitive colony characteristic of Erwinia amylovora. Phytopathology 63:1425-1426. Cupples, D. and A. Kelman. 1974. Evaluation of a selective media for isolation of soft-rot bacteria from soil and plant tissue. Phytopathology 64:468-475. Ednie, A. B. and Sandra M. Needham. 1973. Laboratory test for internally-borne Xanthomonas phaseoli var. fuscans in field bean (Phaseolus vulgaris L.) seed} Proc. Association of Official Seed Analyst 63:76-82. Franklin, T. J. and G. A. Snow. 1971. Biochemistry of Anti- microbial Action. Academic Press, New York, 163p. Gilliver, K. 1946. The inhibitory action of antibiotics on plant pathogenic bacteria and fungi. Ann. Botany 10: 271-282. Grant, Michael A. and John G. Holt. 1977. Medium for the selective isolation of members of the genus Pseudomonas from natural habitats. Appl. Environ. Microbiol. 33: 1222-l224. 83 ll. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 84 Gross, D. C. and A. K. Vidaver. 1979. A selective medium fOr isolation of Corynebacterium nebraskense from soil and plant parts. PhytopathOTogy 69:82-87. Hollis, J. P. 1951. Bacteria in healthy potato tissue. Phytopathology 51:553-557. Ivanoff, S. S. 1933. Stewart's wilt disease of corn, with emphasis on the life history of Phytomonas stewartii in relation to pathogenisis. J. Agric. Res. 47:749-770. Kao, C. I. and M. H. Heskett. 1970. Selective media for isolation of Agrobacterium, Corynebacterium, Erwinia, Pseudomonas, and Xanthomonas. Phytopathology 60:969-976. Katznelson, H. and M. 0. Sutton. 1951. Inhibition of plant pathogenic bacteria in vitro by antibiotics and quaternary ammonium compounds. Science 112:645-647. Leben, C. 1961. Micro-organisms on cucumber seedlings. Phytopathology 51:553-557. McIntyre, John L. and David C. Sands. 1977. How disease is diagnosed. Pages 35-53 jg_James G. Horsfall and Ellis 8. Cowling, eds. Plant Disease, an advanced treatise. Vol. I. How disease is managed. Academic Press, New York, 465p. Mundt, Orvin J. and Nona F. Hinkle. 1976. Bacteria within ovules and seeds. App. Env. Microbiol. 32:694-698. Nesmith, u. C. and S. F. Jenkins, Jr. 1979. A selective medium for the isolation and quantificationof Pseudomonas solanacearum from soil. Phytopathology 69:182-185. New, P. B. and A. Kerr. 1971. A selective medium for Agro- bacterium radiobacter biotype 2. J. Appl. Bacteriol. 35:233-234. Ritchie, D. F. and E. J. Klos. 1978. Differential medium for isolation of Erwinia amylovora. Plant Dis. Reptr. 62: 167-169. Sands, D. C., L. Hankin, and M. Zucker. 1972. A selective medium for pectolytic fluorescent pseudomonads. Phyto- pathology 62:998-1000. Schnathorst, William C. 1954. Bacteria and fungi in seeds and plants of certified bean varieties. Phytopathology 44: 588-592. 24. 25. 26. 27. 28. 29. 30. 31. 85 Schroth, M. N., J. P. Thompson, and D. C. Hilderbrand. 1965. Isolation of Agrobacterium tumefaciens--A radiobacter group from soil. Phytopathology 55:645-647. Shaad, N. w. and Robert Kendrick, 1975. A qualitative method for detecting Xanthomonas campestris in crucifer seed. Phytopathology 65:1034-1036. Szabo, I. M. 1974. Microbial communities in a Forest-Rendzina Ewsystem. AKADEMIAI KIADO, Budapest. 466p. Taylor, J. D. 1970. The quantitative estimation of the infec- tion of bean seed with Pseudomonas phaseolicola (Burkh.) Dowson. Ann. Appl. Biol. 66:29-36. Umbreit, u. w. 1976. Essentials of Bacterial Physiology. Burgess Publishing Company, Minnesota, 304p. Haksman, S. A., E. Bugie, and H. C. Reillei. 1944. Bacterio- static and bacteriodal properties of antibiotic substances with special reference to plant-pathogenic bacteria. Bull. Torrey. Botan. Club. 71:107-121. Wallen, V. R., M. 0. Sutton, and A. J. Skolko. 1950. The effect of actidione on the growth of certain pathogenic fungi and on the germination of pea seed. Phytopathology 40:156-160. Heller, David M. and A. N. Saettler. 1978. Rifampin-resistant Xanthomonas phaseoli var. fuscans and Xanthomonas phaseoli: Tools for field study of bean blight bacteria. Phyto- pathology 68:778-781. CHAPTER III A LABORATORY TEST FOR THE DETECTION OF XANTHOMONAS PHASEOLI AND XANTHOMONAS PHASEOLI VAR. FUSCANS IN BEAN SEED Introduction Nearly half of the United States' supply of dry edible be beans (Phaseolus vulgaris L.) is produced in the Upper Great Lakes region, particularly Michigan and New York (1). Common and fuscous bacterial blights, incited by Xanthomonas phaseoli (E. F. Sm.) Dows (KB) and Burk h. (32:) respectively, are major diseases of Michigan navy beans (2, 4, 5, 15) and, according to Zaumeyer (23) and Zaumeyer and Thomas (25), internally infected seeds are the main source of primary inoculum. In addition, surface contamination of bean seed by §p_and [pf may also be important (13, 22). Weller, in 1978 (22), showed that seeds externally contami- nated with §p_or gpf_can serve as primary inocula sources and that 4 cells per seed were required for transfer inoculum levels of 103-10 of the bacteria from seed to seedling. However, treatment of bean seed with a slurry containing streptomycin sulfate effectively elim- inates external contamination by [p and 52f. Internal seed infec- tion, therefore, is considered the important inoculum source (3, 24) for disease disemination. 86 87 "Seed certification is a program to maintain and make available to the public high quality seeds and propagating material of genetically distinct crop varieties" (4). In Michigan, seed certification includes Breeder seed, Foundation seed, and Certified seed classes. The Breeder seed of Michigan certified bean varieties has been produced and maintained in irrigated regions of Cali- fornia and Idaho where climatic conditions, rigid quar- antine and blight prevention programs minimize risk of infestation by bacterial organisms. In Idaho any field in which blight is found by the Department of Agriculture is required to be completely plowed under and destroyed (4). In the case of Pseudomonas phaseolicola infection of bean seed, a minimum acceptable level of infection has not been defined. Walker and Patel, in 1964 (21), reportedthat an infection level of 0.02% in seeds is capable of promoting a general epidemic, but Guthrie, gt_al,, in 1965 (7), reported that 0.006% is sufficient contamination to result in a complete crop loss under epiphytotic conditions. In epidemiological studies it was demonstrated that even 0.5% seed infection could cause serious outbreaks of fuscous blight (6). The most corrmon method suggested to control bacterial blights of beans is to ensure that seed used be as free of infected seed as possible (6, 15, 17, 20). Numerous assay methods have been developed or adapted to detect the presence of seed borne §p_and 53:. Direct plating of seeds on agar have been used (8, 20). Taylor described the dry grind extraction method for detecting Es, phaseolicola, causal agent of bean halo blight (18). 88 The cotyledon method (12, 16) has been used for the detec- tion of fig. glycinae in soybean seed (12), and in the detectionof Xanthomonas vesicatoria in chili seeds (16) (Qapsicum annum L. and Q, Frutescens L.). Seeds were collected from diseased plants and sown in autoclaved soil. Seedlings in the cotyledonary stage were then transferred to humidity chambers (24-30°C; RH 95-100% for 3-5 days and returned to the greenhouse; and scored for disease symptoms). Recently, Venette (19) reported a Dome test, consisting of vacuum infiltration of liquid obtained from soaked bean samples into seedlings. The seedlings were obtained from surface sterilized seeds of the same seed lot germinated on filter paper in the labor- atory. Disease symptoms appeared as water-congested spots in the left tissue after 7-10 days in the laboratory under fluorescent light. Another method used is the phage plaque count (9, 10, 17). Katznelson, in 1950 (9), detected internally borne §p_by measuring phage titres after incubation of phage with seed. Ednie and Needham (6) modified the method in 1973, and wet ground 3.5 lb. (1.6 kg) samples of surface-disinfected seed. A dilution series was prepared from a sample of the slurry produced, and an aliquot of each dilution placed on agar plates and incubated five to ten days. Xanthomonas-like colonies were then isolated, phage-typed and tested for pathogenicity. Serology has been used in some states to detect blight bacteria in bean seed. Guthrie, et al., in 1965, used serological techniques to detect Pseudomonas phaseolicola (7). They used 89 slide and tube agglutinin tests and agar gel double diffusion tests. Lovrekovich and Kelment (21) combined plating techniques and serology in the identification of bacterial infection of bean seeds: they surface sterilized the seeds, prepared a suspension, an aliquot of which was spread on nutrient agar. Colonies characteristic for blight bacteria were tested against a series of antisera in agglutinin tests. In Michigan, the Michigan Department of Agriculture Bean Seed Testing Program uses a laboratory blight test developed by Saettler in 1971 (14). A 2.21 kg sample of surface sterilized seed is incu- bated in dilute yeast extract solution (10 gm/l) for 24 hours, after which a sample of the liquid is injected into the primary leaf node of young Manitou kidney bean seedlings. It is apparent that such tests for detection of bacterial blight organisms have played an important role in reducing and often eliminating seed infection by bacterial pathogens (13), but most of them have some limitations. Serological tests, in order to be used with accuracy, must rely on two important aspects: (a) specificity of the antisera used, and (b) a given concentration of bacterial cells is necessary in order to have clear results. Tests using plating techniques and plant material are not always clear, and retesting and reisolation is time consuming. Furthermore, there is indication that the sensitivity of any seed test system would be decreased as the incidence of saprophytic bacteria increased above their normal level of occurrence (6); according to some workers this could occur with seed samples 90 harvested under adverse weather conditions wherein the pods are in contact with the soil longer than normal. In 1978-79, the seed testing program of the Michigan Depart- ment of Agriculture released the following data: of 640 bean seed lots from public sources, 34% were found to carry internal blight infection, and of 404 samples from the Michigan Crop Improvement Association, 15% were found to carry internal blight infection (w. J. Young, personal communication). Although the Michigan bean seed testing program has reduced the incidence of seed borne [p and X f, outbreaks of common and fuscous blight persist, and some fields are rejected annually for certification. It was therefore important to develop an accurate and rapid technique to detect internal Xanthomonas contamination of bean seed. Objective To determine the possible utility of combined semi-selective media and serological techniques for the detection of Xanthomonas blight bacteria in seed and other plant tissue. Materials and Methods Samples a. The Michigan Department of Agriculture (MDA) test for internal blight contamination of bean seed currently involves: (1) surface sterilization of 2.2 kg seed for ten minutes in 2.6% NaOCl; (2) rinsing in sterile water; (3) incubating fOr 24 hours in a liquid media containing about 10 gm/l of yeast extract, and 91 (4) injfection of a sample of liquid surrounding the seeds into the primary leaf node of young kidney bean seedlings. One ml of the surrounding liquid from 175 seed samples obtained through the MDA were individually incubated in 25 m1 of the semi-selective media (SSM) (1.0 gm yeast extract, 25 mg cyclo- heximide, 2 mg nitrofurantoin, 1 mg nalidixic acid, 0.05 mg genta- micin in 1,000 ml of 0.01 M phosphate buffer pH 7.2). After 48 hours shaker incubation, bacteria were sedimented by 15 minutes centrifugation at 5,000 x g, resuspended in 1 ml buffer saline, steamed 60 minutes at 100°c, and tested using agar gel double diffusion. The results of these serological tests were then com- pared with the MDA test for internal blight detection. b. Navy bean samples from the MDA were tested. Eighty gms of navy bean seed lots (400 seeds) were weighed, surface- sterilized two minutes with 2.6% NaOCl (1:1 dilution of commercial bleach), rinsed twice with double distilled water, and divided into two parts. Samples were taken, each of approximately 40 gms and separately placed in flasks containing 120 m1 SSM. After 48 hours shaking, bacteria from 30 ml of the SSM were sedimented by 15 minutes centrifugation at 500 x g, and resuspended in 2 ml of buffer saline. One portion of the bacterial suspension was used for seedling injection, and the other was steamed 60 minutes at 100°C and tested serologically as before, in agar double diffusion tests (SSMS). c. Stem Samples: A number of stems suspected of being infected with bacterial blight (§p_or EDI) were received from the 92 MDA. A number of other stem sections from injfected plants in our greenhouse, suspected of being infected with bacterial blight (lg or 13:), were taken five weeks after plant infection. Sections of the stems at the injection point were excised, rinsed with distilled water, and placed in 25 ml of SEM, following the same steps as in (b). d. Leaf Material: When §p_and [pf_symptoms were atypical or were doubtful, three leaves were taken from the plants, rinsed with distilled water, cut in pieces with sterilized scissors, and treated as in (a). e. Stored material and seeds: Dry infected tissue samples and seed were kept in the refrigerator or stored at room temperature for different periods of time (six months to 30 years) and tested for blight following the same procedure as in (a). Most of this material had been collected by Dr. A. Saettler. Serological Test Agar gel double diffusion tests were used in all these experiments. Two antisera were consistently employed; one prepared against Xanthomonas phaseoli isolate 11 or 15, and one prepared against Xanthomonas phaseoli var. fuscans isolate 16 or 1085. Seedling Injection Disease-free seed of Manitou light red kidney bean were planted 2 cms deep in sterile soil plus vermiculite (1:1) in clay pots (3 plants per pot). Plants were grown in the greenhouse and watered alternately as needed with deionized water and Rapid Gro 93 (l tspn/gal). When ten to fourteen days old, plants were injected at the primary leaf node with bacterial suspensin using a hypodermic needle. Plants were maintained in the greenhouse at least six weeks after injection. When symptoms were unclear or were doubtful, isolations were attempted with (a) selective enrichment, (b) dilution plate methods, and (c) plant injection. Plants were considered infected with blight when at least one plant showed the typical disease symptoms. Results Michigan Department of Agriculture Liquid Samples Ninety of the 1975 liquid samples obtained from the MDA Plant Diagnostic Laboratory were found to contain §p_or‘§pj_when tested with the combined semi-selective media plus agar gel double diffusion technique (SSMS) (Table 25). MDA tests reported 65 of the 175 samples containing internal blight infection. Of these 65, 61 were found to carry internal blight contamination using the combined selective enrichment media- serology technique. Therefore, the MDA testing procedure did not detect blight in 29 samples, which were found positive for blight with the combined technique. 94 .mcwsmmam on Lowgn cowuumhcw mcwpummm com umma mm: co_mcmam:m megmpumn ms» mo mpasmm m .Lm>mzo; ”Amy cw mm umummgu ccm .mcwpmm cmmmzn cw cmucmamam mm: .u°¢ um umgopm mecmume pcmpa vmuum$cw umwgu upo cwwzugaomm .mcwsmmum op cocpa cowuumncw mcwpummm com com: mm; cormcmamzm megmpumn asp mo m—asmm m .cm>mzo; mmcomma mm umummgu can Loam: um_p_pmwu mpwgmum saw: ummcwg mcwz AmsopaEAm acmwpn Pmquau uzonuwz gznv usmwpa saw: umpomncw mucmpq soc» mm>mm4u .mcwsmmum ow Lowca cowuumncw mcwpummm com cow: was commcmnmam mecmpumn any we mFaEmm m .Lm>mzo; mmcommn mm umpmmgu vcm Loam: umppwummc mpwgmum gum: umm:_g mgmz mucms_gmaxm mmaoscmmcm soc; mucosogpcmx saw: umuuo>cw mcwmn yo umuumamam msmumu .Amv cw mm xmopogmm new 2mm cng cmummu new .gmumz umppwpmwu mpwgmum zuvz ummcwg .wmumg mgmz mwcwuuma usmwpn saw: umuum$cw mzwmn we umuumamzm mampmn .mcowmcwnmam mecmpumn mg» no umsgowgma wng mummp cowmzmmwc mpnzou gmm< .UOOOF pm mmuzcms on Low qumwpm ucm .m:_pmmucw$$=a FE P cw umccmamammg .cowpmpamwgpcmu za cmucmswumm mcmz mwgmuuma cowumnzucv gmxmnm we mgao; we gwum< .zmm FE mm :? umumpa mm: «mop usmwpn <92 mg» cw umm: mpasmm comm soc; uwzcwp m:_u:=osgam as» we Fe Pm op o o_ op op mumwgo mm op um om o¢ cmm>mm4 mm 0 mm mm ow umsmpm mm 0 mm mm mm nmsmum _o om APpumpwm-wsam .xmo_0Eam co mm: ash--.mm msmw4 . :wmwpc< .mpmmp eowmzccwa mpnzoo Paw me< cw mcmmwyc< mmcoeozucmx pm:_mm< mmumFomH Ammw wpommmmm.mm:oeo;pcmx mo meowuummmii.F< m4m_6 - cmmwpe< .uaaemucoo--.P< m4m<~ 120 .cmumm» «o: u a: mmccmn N so F u + “mason m Lo N u ++ ”mucus mcos :0 v u +++n ..e\mppmu o_op ozone cu umomahca emmwuewa - emm_ue< 11-111111 I‘lIII I‘lill‘ IIIITI 1 I11 I 11.411111 1‘. .mumm» :owm:»w_o mpnzoo Pow gmm< cw mgmmmuc< mucososucmx umcwmm< mmucpomH Awnxv memomae .gm> w—ommmcm.mmcosogucmx mo mcomuummmun.N< m4m

ws - camwp=< .cmzcwucoonu.m< m4mmz mo mucmcwEmucou mccom vmmm msowgm> we mcowpumwm cowumcmszmm< mmocu--.m< mum<~ 123 TABLE A4.--Reactions of Various Bacterial Contaminants Isolated Internally from Navy Bean Seeds Against Xanthomonas Antisera in Agar Gel Double Diffusion Tests. Antisera Prepared Against Formalized Cells Steamed Cells Isolate Used 1pf16 1915 5211 5pf16 5215 1211 c1 -b - - - - - C2a - - - - - - C3 - - - - - - 4) C4 - - - - - - 5) cs - - - - - - 6) C6 - - - - - - 7) C7a - - - - - - 8) C8b - - - - - - 9) C9 - - - - - - 10) C10 - - - - - - 11) C11 - - - - - - 12) C12 - - - - - - 13) 013 - - - - - - 14) C14 - - - - - - 15) C15 - - - - - - 16) C16 - - - - - - 17) C17 - - - - - - . 18) C18 - - - - - - 19) C19 - - - - - - 20) 12:16 + + + + + 2]) £21] + + + + + + N VVV aAntigen steamed cells adjusted to an optical density of b- = no reaction; + = band formation. APPENDIX B 124 125 .couuuzcmg mmumcu_c new me< mmocuxmu ammo» co zpzocm .mwmapocu»; socmum ”uwuozucou mew: mummy pmcouuwuum .mwcwzgm on o» umuumamsm mopmpomw suwzn .cowuummc m>wummmc u . “cowaummc w>muwmoa u +6 35.5 - - - .. - :2 a» + 8 mucosousmma . - u i + smote - m MMCOEOUDUWQ I N H H + EmeU I N - - - u . Emmgu . o AEmeuV . - u u . mumcz - co cowuommm consaz 6623mmgm mmcoam Foams; acmpcmmm Auw>wuwm co mocmo copou Sago acme? ioucm lemmgma»: -mwgoapu acopou -smucoo m.mummm :mmm tmeELmum mumeam EOLL. vmamFOmH macmcwEmucou megwuumm .._.o muwumewuumswsu mzowLm>ll._.m m.._m<._. 126 - i - u . mars: . m_ Amcocpmv - - - - - 32:; - M: 32.3 - - - - - 32E - : mucosouamma . + u u u + Eumgu . op mappwomm + - u u . Emmcu + mp ate a u - - . zoppmz + up m=__womm + u u u . Emmco + m_ u n u n . zeppm» . N— mmcosouzmmm - + u - u + Emmcu u PP mwcmzcm . i n - . zomex u op macmo unmmmca pump cum :owuumhcm ouumnoh :o .m zx co cowuommm coaszz uwsawmca mmcoam mmmuwxo Focus; acupummm Auw>wuwm co mucmu Lopou anew acme? -oucm -cmmcmaxz -mmcoapm Acopou -Emucou .umzcwucounu.Pm m4m mammcsp mzpommmcm *+ «+ «4+ + «+ cmccam umFLmum .Lm> mamcwouou mapommmcm . i + - + . xcmz .cm> .xms mcquFw . - + u + - mmmzou xumpm . u + u . mmpmz umuumpmm .Lm> E=>_pmm Ezmwm + + + + + + mpoumzh .Lm> + + + + + + cmcmmmmm .cm> + + + + + + zepwcmz .cm> .4 mwcmmpz> mzpommmsm NR No mm AmumpomP .;6wzv nmmmcwmzm mpou__ommm;a mumpomu mumpomH mumpomH mm mpouupommmgm mucosouammm a mmcosouzmmm mmcosouammm m.muou vmmm cmmm x>mz soc; umcwmuno mmumpomu mecmpmumm chm>mm mam mmmumam mmcoeouzmmm mo mmcmm umoxuu._o mumwumc_ELmumo mo Pascmz m.>mmcmm= Eogm cowumscomcHn .aa Npm .mmoeuupmm .xcmaeou mcwx_w3 ucm mEme—wz mgh .mwgmuomn quwums mo cowpmu -wmwpcmuv cow mummy Pmuwsmsuowm .oump .cwuummumz .m cmma "om mcwugouum umscowcmn mummhm ouumaop cw + + + + + + Nu_>wuum=mmema»= mowmpzm ammomu»; . - - - n . mo cowpuauocm . u u i u . pump mmmumxo + + + + + + cwpmpmo . n n - n - cucmum mo mummpocuxx . i u - n . cowumu_mwcuwcmo . u u u - . mmmpocuzcwu mcwcwmc< ng co + + + + + + ucmsmma mpawmzmwvo . u n u n - cumum Emma mm mm mm Amumpomw .sumzv ammmcwczm mpoumpommmsm mumpomH mumpomH mumpomH mz Eogm umcwmuno mmumpomfi chm>mm ccm mmwummm mmcosouammm mo mowpmwcmuomcmsu Pmuwsmcoowm ucm pmu_mopommagmiu.No mumwumcpsgmumo mo szcmz m.>mmgmm= Eocm mpmon .am Npm .mcosmupmm ..ou mcwxpuz ucm msmuppwz msh .mwcmuoma _mo_ums mo :o_umow$wucmuu mg» com mummu qumsmcuomm .mNmP .cmucmmumz .m :mma no» acmugouum umsgowcma mummhm 133 + + + + + mmopax + + + + + mmoguzm - - i - . cwumpmm . i 1 u . mmocsmsm - - u - - mmocwmwma . u n u + mmoccmz - - u u + Fouwccmz - - i u . mmoppmz - u - - . mmouumu + + + + + Fogmu>Fo + + + + + mmouzpo + + + + + mmouumpmw + + + + vmpmonmg no: mmouuzgm + + n + + mmocwnmc< NR mm mm mpoowpommmsm ammmcwczm mumpomH mumpomH mumpomfi A.mmcosogmmm mmcosouzmmm m.muou ummm :mmm asmz Eocm umcwmuno mmpmpomH mecmuumm chm>mm ucm mmwommm mmcoEocsmmm Fmgm>mm an muczomeou :oacmu mo cowumNNPwusnu.mu m4m