FEB 29 1999 .‘vj‘. OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return to ream charge from circulation recur POPULATION TRENDS AND SEED TRANSMISSION OF PSEUDOMONAS PHASEOLICOLA IN SUSCEPTIBLE AND TOLERANT BEAN GENOTYPES By Sara J. Stadt A THESIS Submitted to Michigan State University in partial fquiIIment of the requirements for the degree of MASTER OF SCIENCE Botany and Piant Pathology 1980 ABSTRACT POPULATION TRENDS AND SEED TRANSMISSION OF PSEUDOMONAS PHASEOLICOLA IN SUSCEPTIBLE AND TOLERANT BEAN GENOTYPES by Sara J. Stadt Growth of Pseudomonas phaseolicola (Pp), cause of bean halo blight, was studied in susceptible Charlevoix and tolerant Montcalm and Seafarer field bean cultivars inoculated with rifampin resistant isolate, £p_R13. Total populations were determined on inoculated first, and noninoculated third and fifth trifoliolate leaves; surface populations were determined on third trifoliolates. ‘32 R13 grew exponentially and remained at high stationary phase levels on all leaves of Charlevoix. Although maximum bacterial populations were lower in Montcalm and Seafarer, Pp did spread to noninoculated third and fifth trifoliolates. A higher percentage of the total p0pulation was surface-borne on third trifoliolates of Charlevoix than on Montcalm and Seafarer. The fact that halo blight bacteria were detected on symptomless leaves of Montcalm and Seafarer cultivars suggests that these cultivars may serve as symptomless carriers. Internal seed infection by EB Rl3 was directly related to susceptibility of the cultivar to leaf infection. ACKNOWLEDGMENTS I thank Dr. A. w. Saettler for the opportunity to learn and study. I appreciated his advice and continual optimism during the course of my research. I also wish to thank my guidance committee members, Dr. J. L. Lockwood and Dr. E. Klos for their suggestions and critical evaluation of this manuscript. I express special thanks to Terry McCoy-Trese for the long uncomplaining hours of field and technical assistance, and to Scott P. Eisensmith for his generous and valuable assistance in computer analysis and plotting of the data. I am grateful to my parents, Mr. and Mrs. H. L. Beltman, and to my in-laws, Mr. and Mrs. Martin Stadt, for their encouragement and enthusiasm toward my work. I thank Mark for his intelligent perspective and love during my research. ii TABLE OF CONTENTS Page GENERAL INTRODUCTION AND LITERATURE REVIEW ................... 1 Section I. ISOLATION AND CHARACTERIZATION OF RIFAMPIN- RESISTANT MUTANTS OF PSEUDOMONAS PHASEOLICOLA ........ 12 MATERIALS AND METHODS ................................. 13 Isolation and Storage of Rifampin-Resistant Mutants ... 13 Physiological Tests ................................... 13 Pathogenicity Tests ................................... 13 Growth Rates of Rifampin-Resistant R13 and Wild Type Isolates in Bean Leaves ............................. 14 Growth Rates of Rifampin-Resistant R13 and Wild Type Isolates in Liquid Culture .......................... 15 RESULTS ............................................... l7 Physiological and Virulence Tests ..................... 17 In Vivo and In Vitro Growth Rates of the Wild Type and Pp_Rl3 .......................................... 17 DISCUSSION ............................................ 23 II. POPULATION TRENDS 0F PSEUDOMONAS PHASEOLICOLA R13 IN SUSCEPTIBLE AND TOLERANT BEAN GENOTYPES ............... 27 MATERIALS AND METHODS ................................. 28 Field Study 1978 ...................................... 28 Field Design ........................................ 28 Bacterial Isolate and Inoculation ................... 28 Determination of Bacterial Populations .............. 29 Disease Evaluation .................................. 30 Field Study 1979 ...................................... 30 Field Design ........................................ 3O Bacterial Isolate and Inoculation ................... 31 Determination of Bacterial Populations .............. 31 iii Section Page Disease Evaluation .................................. 32 Statistical Analysis ................................ 32 RESULTS ............................................... 33 Field Study 1978 ...................................... 33 Field Study 1979 ...................................... 42 DISCUSSION ............................................ 55 III. TRANSMISSION OF PSEUDOMONAS PHASEOLICOLA R13 IN SEED OF SUSCEPTIBLE AND TOLERANT BEAN—BENOTVPES ............ 59 MATERIALS AND METHODS ................................. 60 Seed Study 1978 ....................................... 60 Seed Study I979 ....................................... 60 RESULTS ............................................... 62 DISCUSSION ............................................ 66 iv Table LIST OF TABLES Page Disease severity in leaves of greenhouse-grown Charlevoix kidney bean plants inoculated with rifampin-resistant and wild type Pseudomonas phaseolicola isolates at 103 cells/ml .................. 18 Disease severity in leaves of greenhouse-grown Charlevoix kidney bean plants inoculated with rifampin-resistant and wild type Pseudomonas phaseolicola isolates at 106 cells/ml ................. 19 Disease severity in leaves of greenhouse-grown Charlevoix kidney bean plants inoculated with rifampin-resistant wild type Pseudomonas phaseolicola isolates at 104 cells/ml ................. 20 Halo blight lesions on first, third, and fifth trifoliolate leaves of susceptible (Charlevoix) and tolerant (Montcalm and Seafarer) field bean cultivars during the growing season ............................. 41 Total populations of Pseudomonas phaseolicola (R13 mutant) associated with first trifoliate leaves of susceptible (Charlevoix) and tolerant (Montcalm and Seafarer) bean cultivars .......................... 45 Total and surface populations of Pseudomonas phaseolicola (R13 mutant) in and on third trifoliolate leaves of susceptible (Charlevoix) and tolerant (Montcalm and Seafarer) bean cultivars ................. 48 Total populations of Pseudomonas phaseolicola (R13 mutant) associated with fifthltrifoliolate leaves of susceptible (Charlevoix) and tolerant (Montcalm and Seafarer) bean cultivars .............................. 52 Progression of halo blight in leaves of susceptible (Charlevoix) and tolerant (Montcalm and Seafarer) beans cultivars during the growing season ............. 53 Table Page 9. Incidence of internal seed infection by Pseudomonas phaseolicola R13 in pods harvested from field grown beans ................................................ 64 lO. Incidence of internal seed infection by Pseudomonas phaseolicola R13 in pods harvested from field grown beans .......................................... 65 vi Figure LIST OF FIGURES Growth of rifampin-resistant R13 and Wild Type isolates of Pseudomonas phaseolicola in primary leaves of greenhouse-grown Charlevoix kidney bean plants ............................................... mutant associated with first trifoliolate leaves of susceptible (Charlevoix) and tolerant (Montcalm Total populations of Pseudomonas phaseolicola (R13 and Seafarer) bean cultivars ......................... Total populations of Pseudomonas phaseolicola (R13 mutant) associated with third trifoliolate leaves of susceptible (Charlevoix) and tolerant (Montcalm and Seafarer) bean cultivars ......................... Total populations of Pseudomonas phaseolicola (R13 mutant) associated with fifth trifoliolate leaves of susceptible (Charlevoix) and tolerant (Montcalm and Seafarer) bean cultivars ......................... Total populations of Pseudomonas phaseolicola (R13 mutant) associated with first trifoliolate leaves of susceptible (Charlevoix) and tolerant (Montcalm and Seafarer) bean cultivars ......................... Total and surface populations of Pseudomonas phaseolicola (R13 mutant) in and on the third trifoliolate leaves of susceptible (Charlevoix) and tolerant (Montcalm and Seafarer) bean cultivars ...... Total populations of Pseudomonas phaseolicola (R13 mutant) associated with fifth trifoliolate leaves of susceptible (Charlevoix) and tolerant (Montcalm and Seafarer) bean cultivars ................ g, ............ vii Page 21 34 36 38 43 46 SO GENERAL INTRODUCTION AND LITERATURE REVIEW Halo blight of bean (Phaseolus vulgaris L.), caused by the bacterium Pseudomonas phaseolicola (Burk.) Dows., occurs in many bean growing areas throughout the world (57). The disease is especially destructive on snap beans, and on colored bean types such as dark and light red kidney, cranberry, pinto, and yelloweye; the colored bean types are important to bean production in Latin America, and of increasing value to Michigan's bean industry (32). Halo blight remains an important economic concern due to periodic epidemics and resultant yield losses (6,7), and the annual rejection of numerous bean fields for certification. Halo blight was first described by Burkholder in 1926 (3). Due to morphological and cultural similarities to Phytomonas medicaginis (bacterial disease of alfalfa), the organism from bean was treated as a variety and given the name Phytomonas medicaginis var. phaseolicola n. var. (3). About the same time Florence Hedges isolated and described a bacterium from the kudzu vine (Pueraria thunbergiana) similar to Phytomonas medicaginis. The bacterium was tentatively named Bacterium puerariae Hedges (17). After thorough comparison of the kudzu and bean organisms, Hedges established that the two were identical (l8). Phytomonas medicaginis var. phaseolicola, having priority, was adopted; Bacterium puerariae became a synonym (18). Although Pseudomonas phaseolicola is now the accepted name, Young et al. (56) have proposed that Pseudomonas phaseolicola be designated as a pathovar of the closely related species Pseudomonas syringag. If accepted, 3. phaseolicola would become Pseudomonas syringag pv. phaseolicola. Pseudomonas phaseolicola is a small (0.7 to 1.2 u, by 1.5 to 3.0 u), gram negative, obligately aerobic rod (2). The bacteria is motile by means of multitrichous flagella. Motile bacteria caused increased disease as compared to non-motile mutants, evidence that flagella are important for leaf invasion and subsequent disease development (37). Link and Hull (30) reported the occurrence of rough and smooth colony types in Pseudomonas phaseolicola. Later, Adam and Pugsley (1) found the smooth colony more virulent than the larger, wavy margined, rough colony type. Also smooth colony types were lysed by a bacteriophage obtained from diseased bean seed while the rough colony type was not lysed. A somatic antigenic component in the smooth colony type was hypothesized as a possible site for bacteriophage attachment (1). A nonhost-specific toxin produced PY.E- phaseolicola is responsible for the distinct halo and chlorotic symptoms character- istic of the disease (21). Recently, Mitchell isolated and characterized the toxin as (N-phosphosulphamyl)ornithylalanylhomo- arginine; its trivial name is designated phaseolotoxin (34). When applied to bean leaves, the compound causes the accumulation of ornithine, which occurs in diseased tissue (41), as well as symptoms resembling halo blight. Important diagnostic tests which aid in distinguishing P.phaseolicola, a plant pathogen, from other fluorescent saprophytic Pseudomonads (29) include: (1) presence of arginine dihydrolase (50), (2) presence of oxidase activity (27), and (3) production of hypersensitivity on tobacco (26). Disease reactions in injected bean seedlings are useful in distinguishing E, phaseolicola from P, syringae (43). Pseudomonas phaseolicola enters the plant through natural openings such as stomata or hydathodes and through wounds (57). Water congestion facilitates the infection process (37). Subsequent symptom expression and disease development are dependent on bacterial strain (24,45), temperature (4,14,38,46), cultivar (23), plant age (l4,36,38), and nutritional status of the host (38). Generally, small, angular, watersoaked areas appear on leaves approximately three to five days after infection (36). On susceptible varieties, lesions enlarge, coalesce, and a distinct yellow-green halo is pro- duced at temperatures ranging from 12°C to 20°C (14), thus the general characterization of halo blight as a cool temperature disease. At higher temperatures (28°C to 32°C), Goss found increased numbers of infection per leaf, from which E, phaseolicola could be isolated, but no halo was visible (l4). Burkholder (4) also noted numerous small angular spots late in the season, but no halo was evident. It is of note that these symptoms are also associated with physiological resistance to E. phaseolicola (23) and to certain strains of the bacterium (24,45). Newly emerged trifoliolate leaves appear most susceptible to halo blight, becoming stunted, deformed, and chlorotic (l4,36,38,57). Pseudomonas phaseolicola may move through the vascular tissue particularly the xylem (3.36.57), resulting in reddish longi- tudinal lesions on stems and petioles. Viscous drops of cream- colored bacterial ooze are often associated with stem lesions (57). Lesions on green pods are usually dark green, with a greasy appear- ance. The lesions may remain olive-green as the pod ripens, and are often surrounded by a reddish brown border (57). A slight crust of dried bacterial ooze can be found on pod lesions later in the season. Bacteria may move through pod lesions and developing seed coats infecting seed internally through the vascular system of the suture, entering seed via the funiculus (57). Dark colored bean seeds may become wrinkled and shriveled while beans with light colored seed may become discolored (57). External infestation occurs during threshing when infested dust and plant material adhere to the seed coat (15,16). Infected seeds are responsible for most outbreaks of halo blight with reports of as few as a dozen seeds per acre capable of causing an epidemic (51). Rain and overhead irrigation facilitate plant to plant spread of halo blight bacteria with the general direction of spread determined by the prevailing winds (25.28.30.31, 51). Although some disease control has been achieved by the use of fungicide (35,39,40), antibiotic (20,33,47,49,52), and copper sprays (ll,44,49,52), use of resistant varieties and disease free seed produced in semi-arid regions, are the most practical and effective measures of control. Varietal resistance to halo blight was recognized by Burkholder and Zaleski (5) and Higgins (19) soon after the disease was characterized. Jensen and G055 (23) classified the following as symptoms of physiological resistance: small, rusty colored necrotic lesions on pods, inconspicuous necrotic lesions on leaves, and the absence of systemic infection. In vivo growth of P. phaseolicola in leaves of resistant and susceptible greenhouse-grown bean cultivars has been examined by several workers (12,36,42,46). Although bacteria were found to multiply in resistant hosts, final population levels were lower than in susceptible cultivars. Omer and Wood (36) also found that halo blight bacteria moved systemically for a greater distance in a susceptible than in a resistant host. More recently Katherman et a1. (25) demonstrated a reduced ability of resistant as compared to susceptible varieties to serve as secondary inoculum sources under field conditions. There are no previous reports relative to population trends of halo blight bacteria in tissue of resistant and susceptible beans in the field. Isolation of plant pathogenic bacteria from the ubiquitous field microflora has in general been difficult and a major obstacle to epidemiological studies. The introduction of antibiotic markers into plant pathogenic bacteria has improved field recovery and has facilitated basic studies of bacterial epidemiology (8,9,10, l3,22,48,53,55). The use of rifampin-resistant mutants was tested (54) and effectively used by Weller and Saettler (53,55) in field studies of the epidemiology of bean common and fuscous bacterial blights. Populations were monitored from seedling to reproductive stage of plant development over the course of a growing season. Cafati (8), and Cafati and Saettler (9) further elaborated on the study, identifying weeds as possible alternate hosts, and following population levels in resistant and susceptible hosts. A similar rifampin selection system would be useful in studies of bean halo blight epidemiology. Since the main strategy of halo blight control involves the production and planting of disease free seed, the possibility of seed transmission in resistant cultivars needs to be determined; no data are available. Simulation of a disease epidemic in the field with a rifampin-resistant isolate 0f.EE and subsequent testing of possible seed infection, would prove useful in obtaining quantitative information on seed transmission of blight in resistant cultivars. The purpose of this study was to: (1) develop and character- ize a rifampin-resistant mutant of P. phaseolicola, (2) determine populations of Pp on field-grown resistant and susceptible sultivars, and the symptomology associated with the disease, (3) study the role of resistant and susceptible cultivars in seed transmission of the bacteria, and (4) determine whether halo blight is transmitted in seed contained in symptom free pods. IO. LITERATURE CITED Adam, D. B., and A. T. Pugsley. 1934. 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Nature (Londonlil78: 703. LeBaron, M., G. McMaster, and J. Guthrie. 1976. Spread of halo blight with sprinkle irrigation. U. of Idaho Coop. Ext. Ser. Agr. Exp. Sta. Bull. No. 359, 2pp. Lelliott, R. A., E. Billing, and A. C. Hayward. 1966. A determinative scheme for the fluorescent plant pathogenic Pseudomonads. The Journal of Applied Bacteriology 29(3): 470-489. Link, G.K.K., and K. L. Hull. 1927. Smoothness and roughness and spontaneous agglutination of Bacterium citri, Baterium medicaginis var. phaseolicola, Bacterium phaseoli so'ense, and Bacterium tumefaciens. Botanical Gazette 83: 412. Menzies, J. D. 1954. Effect of sprinkler irrigation in an arid climate on the spread of bacterial diseases of bean. Phytopathology 44: 553-556. McGill, J. A. 1980. 1980 Michigan-Mexican Bean Contract signed. Michigan Dry Bean Digest 4(3): 2-3. Mitchell, J. W., W. J. Zaumeyer, and W. S. Preston Jr. 1954. Absorption and translocation of streptomycin by bean plants and its effect on the halo and common blight organisms. Phytopathology 44: 25-30. Mitchell, R. E. 1976. Isolation and structure of a chlorosis- inducing toxin of Pseudomonas phaseolicola. Phytochemistry 15: 1941-1947. Morris, H. E., and M. M. Afanasiev. 1954. Control of bacterial halo blight on garden beans in Montana in 1952 and 1953. Phytopathology 44: 499. (Abstract). Omer, M.E.H., and R.K.S. Wood. 1969. Growth of Pseudomonas phaseolicola in susceptible and in resistant bean plants. Ann. Appl. Biol. 63: 103-116. Panopoulos, N. J., and M. N. Schroth. 1974. Role of flagellar motility in the invasion of bean leaves by Pseudomonas phaseolicola. Phytopathology 64: 1389-1397. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 10 Patel, P. N., and J. C. Walker. 1963. Relations of air temperature and age and nutrition of the host to the development of halo and common bacterial blights of bean. Phytopathology 53: 407-411. Reid, W. D. 1945. Control of halo-blight and anthracnose of beans. New Zealand Journal of Science and Technology 27: 90-93. Reid, W. D. 1948. Control of halo blight of beans. New Zealand Journal of Science and Technology 30: 45-48. Rudolph, K., and M. A. Stahmann. 1966. The accumulation of L. -ornithine in halo-blight infected bean plants (Phaseolus vulgaris L.), induced by the toxin of the pathogen Pseudomgnas phaseolicola (Burk.) Dowson. Phytopathol. Z. 57: 29- 6. Russel, P. E. 1977. Observations on the in vivo growth and symptom production of Pseudomonas phaseolicola on Phaseolus vulgaris. Journal of Appliengacteriology 43: 167-170. Saettler, A. W. 1971. Seedling injection as an aid in identifying bean blight bacteria. Plant Dis. Reptr. 55: 703-706. Saettler, A. W., and H. S. Potter. 1967. Chemical control of bacterial blights of dry field beans in Michigan by foliage sprays applied by ground and air equipment. Plant Dis. Reptr. 51: 622-625. Schroth, M. N., V. B. Vitanza, and D. C. Hildebrand. 1971. Pathogenic and nutritional variation in the halo blight group of fluorescent Pseudomonads of bean. Phytopathology 61: 852-857. Skoog, H. A. 1952. Studies on host-parasite relations of bean varieties resistant and susceptible to Pseudomonas phaseolicola and toxin production by the parasite. Phytopathology 42: 475. (Abstract). Smith, W. L. 1949. Seed treatment with streptomycin for the control of bacterial blight of beans. The Journal of the Colorado-Wyoming Academy of Science 4: 49. Stall, R. E., and A. A. Cook. 1966. Multiplication of Xanthomonas vesicatoria and lesion development in resistant and susceptible pepper. Phytopathology 56: 1152-1154. 49. 50. 51. 52. 53. 54. 55. 56. 57. 11 Taylor, J. D. 1972. Field studies on halo-blight of beans (Pseudomonas phaseolicola) and its control by foliar sprays. Ann. Appl. Biol. 70: 191-197. Thornley, M. J. 1960. The differentiation of Pseudomonas from other gram-negative bacteria on the basis of arginine metabolism. Journal of Applied Bacteriology 23: 37-52. Walker, J. C., and P. N. Patel. 1963. Splash dispersal and wind as factors in epidemiology of halo blight of bean. Phytopathology 54: 140-141. Walters, M. J., and G. H. Starr. 1952. Bacterial diseases of beans in Wyoming. Wyoming Agricultural Exp. Station Bulletin. No. 319, 12pp. Weller, D. M. 1978. Ecology of Xanthomonas phaseoli and Xanthomonas phaseoli var. fuscans in navy (pea) beans (Phaseolus vulgaris L.). Pfi.D. Thesis, Michigan State Univ., East Lansing, 137pp. Weller, D. M., and A. W. Saettler. 1978. Rifampin-resistant Xanthomonas phaseoli var. fuscans and Xanthomonas phaseoli: Tools for field study of bean Blight bacteria. Phytopathology 68: 778-781. Weller, D. M., and A. W. Saettler. 1980. Colonization and distribution of Xanthomonas phaseoli and Xanthomonas phaseoli var. fuscans in field-grown beans. Phytopathology 70: 500-506. Young, J. M., D. W. Dye, J. F. Bradbury, C. G. Panagopoulos, and C. F. Robbs. 1978. A proposed nomenclature and classification for plant pathogenic bacteria. N. Z. Journal of Agricultural Research 21: 155-177. Zaumeyer, W. J., and H. R. Thomas. 1957. A monographic study of bean diseases and methods for their control. USDA Agric. Tech. Bull. No. 868. 255pp. SECTION I ISOLATION AND CHARACTERIZATION OF RIFAMPIN-RESISTANT MUTANTS 0F PSEUDOMONAS PHASEOLICOLA 12 MATERIALS AND METHODS Isolation and Storage of Rifampin- Resistant Mutants Naturally occurring rifampin-resistant mutants of wild-type (WT) isolate Shiels #2 of Pseudomonas phaseolicola were obtained by spreading approximately 109 cells onto the surface of modified King's Medium B (KMB: 15 ml glycerol, 20 g peptone, 3 g MgSO4'7H20, 2 g KZHP04, 15 g agar, in 1000 m1 of distilled water) amended with 50 ug/ml or 100 ug/ml rifampin (15,16). Colonies exhibiting pheno- typic characteristics similar to WT were selected for further characterization. Colonies were subcultured twice on modified KMB and stored in 40% v/v aqueous glycerol at -10°C. Physiological Tests Four rifampin-resistant mutants of P. phaseolicola, R12, R13, R15, and R100 were isolated and then compared to the wild type in the following tests: (1) fluorescence on modified KMB (7), (2) oxidase activity (9), (3) production of hypersensitivity in tobacco (8), (4) arginine dihydrolase (14), and utilization of carbohydrates (1). Pathogenicity Tests Virulence of the four rifampin-resistant mutants was com- pared to that of the WT by the seedling injection technique (11). 13 14 Plants of bean cv. Charlevoix were grown in 15 cm diameter clay pots containing standard greenhouse soil mix; plants were watered alternately with tap water and Rapid-Gro (commercial fertilizer, N = 23%, P 0 = 19%, K 0 = 17%) at 2.45 g/liter of H 2 5 2 2 was supplemented with twelve hours of fluorescent lighting. Pots O. Daylight were arranged in a randomized block design. TWenty-four hour old cultures of fi.phaseolicola WT and mutants were adjusted to 108, 106, and 104, cells/ml with distilled water and injected into the primary leaf and cotyledon node of fifteen-day-old seedlings. Each injection was replicated three times with two seedlings per replication. Plant response was rated at six days after inoculation, and at two- day intervals thereafter over a fifteen day period using the follow- ing scale: 0 = no symptoms, 1 = slight marginal chlorisis and wilting, 2 = advanced chlorosis along leaf veins, 3 = both primary leaves collapsed, newly emerged trifoliolate leaves chlorotic, 4 = complete stem and leaf collapse. Data were analyzed as a split plot design with isolates as the whole plot factor and time the subplot factor. Significant differences between treatments was estimated using least significant ranges (L.S.R.) obtained from Tukey's w-procedure (13). Growth Rates of Rifampin-Resistant R13 and Wild Type Isolates in Bean Leaves The growth rates of rifampin-resistant R13 and WT isolates were compared in primary leaves of greenhouse-grown susceptible Charlevoix kidney beans. Plants were grown as previously described. Fully expanded primary leaves on fourteen-day-old plants were 15 lightly sprayed to run-off with bacterial suspensions containing 5 x 105 cells/ml. Plants with the same treatment were grouped on the greenhouse bench to prevent cross contamination. Each sample consisted of three randomly selected primary leaves (average leaf area = 100 cm2) with three replications per sample. Leaves were homogenized in a Waring blender with 100 m1 of 0.01 M PO4 buffer, pH 7.2. The slurry was serially diluted and aliquots were distributed over the surface of modified KMB (for WT and R13 bacteria) or KMBR supplemented with 50 ug/ml of cycloheximide (for R13 bacteria). Samples were taken at l,2,4,6,8, and 12 days after inoculation. Bacterial numbers were expressed as logarithm of colony forming units/100 cm2 (average leaf area). Leaf area was measured by a'Li Cor area meter (Model 3000, Lambda Instruments Corp.) using leaf tracings on paper. Data were analyzed as a split plot design with bacteria-media combinations as the whole plot factor and time the subplot factor. Significant differences between treatments were estimated using least significant ranges (L.S.R.) obtained from TUkey's‘w-procedure. Growth Rates of Rifampin-Resistant R13 and Willeype Isolates in Liquid Culture Doubling times of R13 and WT isolates of_£. phaseolicola were compared in nutrient broth (NB: 3 9 beef extract, 5 g peptone per 1000 m1 of distilled water). Bacterial suspensions (5 m1) of R13 and WT isolates at 1.2 x 108 cells/ml were individually added to 50 m1 of NB in Bellco side arm flasks. Flasks were shaker incubated, and, at one hour intervals, samples were plated on modified KMB and 16 percent transmission determined in a Bausch and Lamb Spectronic 20 spectrophotometer. Colonies were counted after four days incubation at 21°C. RESULTS Physiological and Virulence Tests The four rifampin-resistant mutants and the WT isolates of P, phaseolicola gave identical responses in all of the physiological tests. R13 and R15 were as virulent as the WT in the seedling injection test at all three inoculum concentrations. Mutant R12 at 4 10 cells/ml was significantly less virulent than the WT at 14 days after inoculation. No differences in virulence between the WT and R12 were found at 108 or 106 cells/ml. Isolate R100 was signifi- cantly less virulent than the WT and the other mutants in a number of cases (Tables 1, 2, and 3). In Vivo and In Vitro Growth Rates of the Wild Type and Pp R13 In vivo growth rates of fig R13 and the WT were similar over the twelve day sampling time (Figure 1). In addition there was no significant difference in the plating efficiencies of fig R13 on KMBR and modified KMB. 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Weller and Saettler (16) demonstrated the specific value of rifampin-resistant mutants in epidemiological studies of bean common blight bacteria (Xanthomonas phaseoli), and attributed the success of their system to the wide spectrum of antibacterial activity and high toxicity of rifampin. That resistance to an antibiotic changes cell physiology or structure of the bacterial organism is known. Russel found variations in streptomycin-resistant Pseudomonas phaseolicola mutants (10). Com- pared to the wild type parent, mutants exhibited changes in symptom production, lower population levels within the plant, and loss of the ability to produce toxin (10). Reversions back to parental type also occurred after passage of the pathogen through the plant. In their initial screening and characterization of rifampin-resistant Kg mutants, Weller and Saettler (16) found mutants with reduced virulence. Therefore, careful characterization and screening are important prior to using a mutant to model the wild type pathogen in epidemiological studies. Our data with several £p_rifampin-resistant mutants indicate that R13 adequately models the wild type. Mutant R13 and the wild type isolates responded identically in the biochemical tests 23 24 conducted. Also, when injected into susceptible bean seedlings, mutant R13 tested as virulent as the wild type at three levels of inoculum concentration. Rate and severity of wilting, chlorosis, and systemic infection were identical in R13 and the wild type. Such results suggested that R13 would accurately model the wild type in disease development under natural conditions. Several of the other mutants were less virulent than the wild type, indicating that evaluation of virulence is important. In vivo growth of R13 and the wild type were similar over the course of a twelve day sampling period, suggesting that multipli- cation of R13 within the plant is similar to that of the wild type. Such data also imply that population buildups in the field will model a natural halo blight infection. Similar plating efficiencies of R13 on the two media (KMB, KMBR) imply that reversion of the mutant after passage through the plant is probably insignificant. Adequate recovery of R13 from the field throughout the entire growing season is probably assured. Growth rate of R13 in broth culture was slightly slower than the wild type. However, differences in multiplication under arti- ficial conditions is probably insignificant. Since R13 grew in vivo at a rate identical to that of the wild type, it is felt that R13 will simulate the wild type under field conditions. 10. 11. LITERATURE CITED Buchanan, R. E., and N. E. Gibbons, eds. 1974. Bergey's Manual of Determinative Bacteriology, 8th ed. Williams and Wilkins, Baltimore, 1246pp. Cafati, C. R. 1979. Effect of host genotype on multiplication, distribution and survival of bean common blight bacteria (Xanthomonas phaseoli), Ph.D. Thesis, Michigan State Univ., East Lansing,2124pp. Cafati, C. R., and A. W. Saettler. 1980. Role of nonhost species as alternate inoculum sources of Xanthomonas phaseoli, Plant Disease 64: 194-196. Cunfer, B. M., N. W. Schaad, and D. D. Morey. 1978. Halo blight of rye; multiplicity of symptoms under field condi- tions. Phytopathology 68: 1545-1548. Gardner, J. M., and C. J. Kado. 1973. Evidence of systemic movement of Erwinia rubrifaciens in Persian walnuts by use of double-antibiotic markers. *Phytopathology 63: 1085-1086. Hsieh, S.P.Y., I. W. Buddenhagen, and H. E. Kauffman. 1974. An improved method for detecting the presence of Xanthomonas oryzae in rice seed. Phytopathology 64: 273-274. King, E. 0., M.K.W. Ward, and D. E. Raney. 1954. Two simple media for the demonstration of pyocyanin and fluorescin. J. Lab. Clin. Med. 44: 301-307. Klement, Z. 1963. Rapid detection of the patho enicity of phytopathogenic pseudomonads. Nature (London? 199: 299-300. Kovacs, N. 1956. Identification of Pseudomonas pyocyanea by the oxidase reaction. Nature (LondOn) 178: 703. Russel, P. E. 1975. Variation in the virulence of some streptomycin resistant mutants of Pseudomonas phaseolicola. J. App. Bact. 39: 175-180. Saettler, A. W. 1971. Seedling injection as an aid in identifying bean blight bacteria. Plant Dis. Reptr. 55: 703-706. 25 12. 13. 14. 15. 16. 17. 26 Stall, R. E., and A. A. Cook. 1966. Multiplication of Xanthomonas vesicatoria and lesion development in resistant and susceptible pepper. Phytopathology 56: 1152-1154. Steel, G. 0., and J. H. Torrie. 1960. Principles and Procedures of Statistics. New York: McGraw-Hill Book Company, Inc., 481pp. Thornley, M. J. 1960. The differentiation of Pseudomonas from other gram negative bacteria on the basis of arginine metabolism. Journal of Applied Bacteriology 23: 37-52. Weller, D. M. 1978. Ecology of Xanthomonas phaseoli and Xanthomonas phaseoli var. fuscans in navy (pea) beans (Phaseolus vulgaris L.), Ph.D. Thesis, Michigan State Univ., EastTLansing, 37pp. Weller, D. M., and A. W. Saettler. 1978. Rifampin-resistant Xanthomonas phaseoli var. fuscans and Xanthomonas phaseoli: Tools for fiéldistudy of bean blight bacteria. Phytopathology 68: 778-781. Weller, D. M., and A. W. Saettler. 1980. Colonization and distribution of Xanthomonas phaseoli and Xanthomonas haseoli var. fuscans in field grown beans. Phytopathology 70: 500-506. SECTION II POPULATION TRENDS OF PSEUDOMONAS PHASEOLICOLA R13 IN SUSCEPTIBLE AND TOLERANT BEAN GENOTYPES 27 MATERIALS AND METHODS Experiments were conducted under field conditions at the Botany and Plant Pathology Research Farm, Michigan State University, East Lansing, Michigan, during the summers of 1978 and 1979. Field Study 1978 Field Design Certified disease-free seed of Charlevoix (susceptible dark red kidney 3. vulgaris), Montcalm (tolerant dark red kidney,_fl. vulgaris), and Seafarer (tolerant navy, P. vulgaris) cultivars were hand planted on July 10, 1978. Plots consisted of four rows each of 4.9 meters in length with 50 cm between rows; seed spacing was about 9 cm in the row. One row of tolerant Tuscola separated each plot, and plots were replicated three times. Bacterial Isolate and Inoculation Rifampin-resistant mutant R13, of P.phaseolicola, similar in virulence to the Shiels #2 parental wild type, was used through- out these studies. Inoculum was prepared by rinsing bacterial growth from 24-hour cultures on KMB plates into sterile distilled water. Bacterial suspension were adjusted to approximately 6 x 108 cells/ml as determined by standard turbidimetric and dilution plate techniques. Plants were inoculated when twenty-two days old 28 29 by gently spraying the upper and lower leaf surfaces of expanded primary and first trifoliolate leaves using a knapsack sprayer. No watersoaking of leaf tissue was observed. Determination of Bacterial Populations Total bacterial populations were determined on first, third, and fifth trifoliolate leaves of each genotype. Each leaf was assayed for the presence of the mutant for a period of approximately sixteen to twenty days after emergence of the leaf. A sample of seven leaves (twenty-one leaflets) was randomly selected from each cultivar and ground three minutes with 0.01 M P04 buffer pH 7.2 in a 2 of leaf Waring Blendor. Buffer was added at a rate of 1 ml per 6 cm tissue. Serial dilutions were prepared and plated on KMBR; samples were replicated three times for each bean genotype. Bacterial colonies were counted after four to five days incubation at 21°C. Bacterial numbers were converted to logarithm and expressed as logarithm number of colony formint units per 30 cm2 of leaf tissue (average size of one leaflet). Leaf area was measured on a Li Cor area meter (Model 3000, Lambda Instruments Corp.) using leaf tracings on paper. Stem tissues were sampled to determine internal levels of EE.R13- Stem pieces from the first through third node were excised from twelve plants of each genotype; and surface sterilized in 2.5% sodium hypochlorite for three minutes (three replications per geno- type). All stem pieces from one genotype were collectively rinsed in running distilled water for one minute, rinsed in sterile 3O distilled water for one minute, and ground in 200 ml of .01 M PO4 buffer, pH 7.2, for three minutes. Serial dilutions were prepared and samples plated on KMBR plates. Bacterial numbers were expressed as CFU per 2.3 g fresh weight (average fresh weight of one cut stem piece). Disease Evaluation Disease symptoms in the three bean genotypes were evaluated by counting the number of lesions per leaflet on the first, third and fifth trifoliolate leaves. Lesion counts were made twelve times during the growing season. Leaf areas were measured on a Li Cor area meter using leaf tracings on paper and lesion numbers were expressed as number per 30 cm2 leaf area (average leaflet area). Leaflets with over 100 lesions were counted as 100. Twenty-one plants (63 leaflets) were sampled at each date for each trifoliolate leaf. Field Study 1979 Field Design Disease-free seed of Charlevoix, Montcalm, and Seafarer cultivars were hand planted in a randomized block design, with three replications of each block. Six rows of 2.8 m length, of each cultivar were planted in each block. Bean cultivars within each block were separated by two rows of Tuscola; blocks were isolated by a 1.5 m buffer space. 31 Bacterial Isolate and Inoculation The bacterial isolate and inoculation procedures were similar to those described for 1978 except that (l) inoculum was prepared in sterile PO4 buffer, (2) the inoculum contained approximately 7 1.5 x 10 cells/ml and (3) plants were twenty-seven days old when inoculated. Determination of Bacterial P6pulations Total bacterial populations were determined as previously described. In addition numbers of surface (external) bacteria were determined on the third trifoliolate leaf. To determine surface populations, seven leaves were shaken for two minutes with 150 m1 P04 buffer in sterile flasks. Serial dilutions of the rinse solution were made and plated on KMBR. The rinsed leaves were subsequently homogenized as described previously for determination of internal bacteria. Total bacterial populations were the sum of surface plus internal populations. At the flowering stage of plant development blossoms were assayed for the presence of R13. Twenty freshly-opened blossoms were removed from each cultivar and homogenized in 50 ml of P04 buffer in a Waring Blendor. Serial dilutions were prepared and plated on KMBR; the procedure was replicated three times for each genotype. Bacterial numbers were expressed as CFU per .135 g fresh weight of blossoms (average weight of one blossom). Surface populations of fig R13 on mature pods were determined by rinsing twenty-one pods of each genotype in 150 ml of P04 buffer 32 pH 7.2 for two minutes, preparing serial dilutions of the rinse and plating on KMBR. Populations were expressed as CFU per 8 g fresh weight of pod tissue. Disease Evaluation Disease reactions incited by Pp R13 in the three bean geno- types were determined periodically on the first, third, and fifth trifoliolate leaves throughout the growing season. Individual leaflets obtained from twenty-one plants were rated for percent leaflet infection at thirteen times according to the following scale: 1 = 1-4% infection, few watersoaked or hypersensitive spots; 5-9%, lesions enlarged, halo; 3 = 10-19% leaflet infection; 20-29% leaflet infection; 5 = 30-39%; 6 = 40-49%; 7 - 50-59%; co 5 N II 60-69%; 9 = 70-79%; 10 = 80-89%; 11 = 90-99%; and 12 = 100%. Isolations were made from leaves of Montcalm and Seafarer cvs. which exhibited atypical symptoms. Lesions were aseptically excised, rinsed in sterile distilled water, and ground in 1 m1 of sterile PO4 buffer in a sterile mortar. Streaks for single colonies were made on KMBR. Statistical Analysis Bacterial populations in the three bean genotypes were analyzed by a split plot design with bean genotypes as the whole plot factor and time as the subplot factor. Significant differences between the populations on the three genotypes were estimated using least significant ranges (L.S.R.) obtained from Tukey's w-procedure (9). RESULTS Field Study 1978,(Preliminary Studies) Populations of fig R13 were monitored in first, third, and fifth trifoliolate leaves of susceptible Charlevoix and tolerant Montcalm and Seafarer bean cultivars. Population trends of R13 in inoculated first trifoliolate leaves are shown in Figure 2. Survival of the initial inoculum was high in Charlevoix where populations increased slowly and stabilized nine days after inoculation at approximately 5 x 107 CFU/3O cm2 leaf tissue. Lower populations of £9 R13 were isolated from the tolerant cultivars and levels fluctuated between 104 and 106 CFU/3O cm2 leaf tissue. Noninoculated third trifoliolates in Charlevoix cv. were heavily colonized soon after emergence (Figure 3). Leaf tissue 2 populations of 107 CFU/30 cm were detected in the first day sample with a gradual increase to 108 CFU/3O cm2 leaf tissue over sixteen days. Tolerant cultivars supported lower population levels. Low numbers of R13 were detected on the third trifoliolate leaves of Montcalm cv. at early sampling dates. As leaves expanded the Montcalm became more heavily colonized. Noninoculated fifth trifoliolate leaves of susceptible Charlevoix were colonized upon emergence (Figure 4). High bacterial levels of about 108 cells/leaf were maintained throughout the entire 33 34 .meweuwwees eessw we memese>e ese meewe> .oewN we sewwessusw sewwe exeu e>ww ew szew uewseee ese: mewse—ee pewsewuem .mmzx se uewews use ueseeese ese: msewwswwu wewsem .seusewm mswsez e sw sewwee cos 2 wo.o sw Amwewweew wNV me>eew se>em mswuswsm we ueswsseweu ese: msewwewsses .pe\mwwee wowxo mswswewsee sewmsesmem wewseweee e swwz me>eew ewewewwewwsw wmsww use aseewss ueuseexe we meeewssm weep sezew use sees: esw mswaessm zwwsem an ueweweeesw ese: mwsews upeIAeuuezwuzwsezp .mse>www:e sees Aseseweem use Eweewsezv wsesewew use Axwe>ewsesuV ewewweeemzm we me>eew ewewewwewwsw sww: ueweweemme Awsewes mwmv eweewweemeam.meseeeusems we msewwewssee wewew--.N eszmwu 35 NN 0N _ ow _ zowwcqzoozw mmwmc w>¢o mw “w mw ma .0 .m M b b b D mmmcmcmw 4 :4cowzo: X x~o>u4m¢zu B I l 1 0'8 0'3 0'1 j 1 j 1 j I 0'8 D'L 0'9 0'9 0'? 309911 9831 zNJOE/DJU 001 36 .meweewwees eessw we meuese>e ese meswe> .UOFN we sewweneesw sewwe maeu e>ww ew szew uewssee ese: mewseweu pewsewuem .amzx se uewews use ueseeess ese: msewwewwu wewsem .seusewm mswsez e sw esmmww weep we Nae m see we p we esswe> e we ueuue sewwes cos x Po. sw Amwe—weew wNv me>ee_ se>em mswuswsm an ueswEseweu ese: msewweweses .we\mwwee wowxu oswswewsee sewmseemem wewsewees e swwz me>eew ewewewwewwsw wmsww use xseswse ueusesxe we meeewsam weep sezew use sees: esw mswaessm awwsem he uewewsuesw ese: mwsewe upenxeuuezwustezw .mse>www=e sees Aseseweem use Eweewsezv wsesewew use Axwe>ewsesuv ewswwseemsm we me>eew ewewewwewwsw uswsw swwz ueweweemme Awsewse mwmv eweewweemesmlmeseEeusems we msewwewssee wewew--.m essmws 37 zowwseauozw mmwss wwso we we we we we me P b n P I _ ow mummmcmw d zucowzoz ex x a o>w4mczo B 0'9 0'8 0'1 309911 9831 ZNOOS/Ddfl 001 O'L 0'6 38 .meweewwses eessw we memese>e ese meewe> .uewN we sewweseusw sewwe exeu e>ww ew s=ew uewsseu ese; mewseweu wewsewuem .mng se uewewe wse ueseeess ese: msewwswwu wewsem .seusewm mswsez e sw sewwss cs 2 we. sw Amwewweep FNV me>eew se>em mswuswsm we ueswsseweu ese: msewwewases .uewessm ese: mwsews sesz uemsese wes ues me>eew ewe—ewpewwsw swwww .ws\mw_eu opxm mswswewsee sewmseemsm wewsewees e sww: me>eew ewewewwewwsw wmmww use wseswss ueuseexe we meeewssm weep sezew use sees: esw mswxesem awwsem an uewewseesw ese: mwsews uweuaeuuezwuawsezw .mse>www:e seen Aseseweem use Eweewsezv wsesewew use Axwe>ewsesuv ewnwweeemem we me>ee— ewewewwewwsw swwww swwz uewewuemme wsewse mwmv eweewweememm.meseseu=ems we msewwe—sees wewewuu.¢ essmww 39 22.5.5003 mmwwc w>¢o mm mm mm mm on mm mm uN s s w L NN _ s t w t F L . . c _ 8 O .7 1mg 1111 V A! 1%. 0 9 1|. 0 mumcwcmm 4 1.1. z.._¢u._.zoz X 0 x~o>qu¢zu B 1 11 91 ($11 ){ 0 s. 3n9911 9931 zuaoe/naa 901 4O sampling period with little variation in total numbers. Pp R13 was detected on fifth trifoliolate leaves of both tolerant cultivars, however, the levels were lower and relatively stable at about 104 CFU/3O cm2 leaf tissue. Lesions were observed on the susceptible cultivar five days after inoculation (Table 4). Small watersoaked areas were observed along the lower edges of the leaves when Pp_populations were approximately 106 CFU/leaflet. Lesion development on Charlevoix increased over the growing season with severity highest on the young, later emerging leaves. Although detectable levels of bacteria were associated with leaves of tolerant cultivars, lesion development on Montcalm and Seafarer was minimal and observed only sporadically. Very few symptoms were observed on the noninoculated third and fifth trifoliolate leaves of Montcalm, and lesions in Seafarer were atypical and consisted of hypersensitive-like spots and no water soaking. The appearance of lesions in Montcalm and Seafarer did not appear to be associated with a particular population level of bacteria. Stem tissues were sampled to determine internal population levels of fig. Bacterial levels comparable to the numbers colonizing 7 the leaves were found in stem pieces of Charlevoix (1.9 x 10 CFU/ 2.3 g of stem tissue). Levels of Pp in the vascular tissue of the tolerant cultivars were lower; 1.9 x 103 Seafarer and 5.2 x 102 CFU/2.3 g of stem tissue in Montcalm. 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