lll3l|lllllllzllfllljllllljfllfllllllllflllfll LIBRARY Michigan Sun: U . . This is to certify that the thesis entitled EFFECT OF HOST GENOTYPE 0N MULTIPLICATION, DISTRIBUTION AND SURVIVAL OF BEAN COMMON BLIGHT BACTERIA (XANTHOMONAS PHASEOLI) presented by Claudio R. Cafati has been accepted towards fulfillment of the requirements for Ph.D. dpgnmin Plant Pathology Major professor Date JU] 19 1979 0-7 539 U'JERDIFE FIRES ARE. 25C. T’ER DAY PEI; TERM ‘54.; vim. Return to Book drop to remove x. .‘fig; this ';.i.eck;311£ from your record. r' I‘. ”-I 's «m ';?¥lfI-I :1 .fl: . W x “'2'. _ .uaw‘m — ——— EFFECT OF HOST GENOTYPE ON MULTIPLICATION, DISTRIBUTION AND SURVIVAL OF BEAN COMMON BLIGHT BACTERIA (XANTHOMONAS PHASEOLI) BY Claudio R. Cafati A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1979 ABSTRACT EFFECT OF HOST GENOTYPE ON MULTIPLICATION, DISTRIBUTION AND SURVIVAL OF BEAN COMMON BLIGHT BACTERIA (XANTHOMONAS PHASEOLI) BY Claudio R. Cafati Naturally—occurring rifampin—resistant mutants of common blight bacteria (Xanthomonas phaseoli, RlS-l) and of fuscous blight bacteria (g, phaseoli var fuscans, R17) have been used as an antibiotic selective system in epidemiological studies. A wide range of Phaseolus germplasm.was examined for their reaction to Xp and for their ability to support population build-ups of blight bacteria. Results indicate that most of the germplasm sources being utilized in bean breeding programs throughout the world may be potential "symptomless carriers" of the common blight pathogen. Multiplication and distribution patterns of Xp (RlS-l) in resistant (Tepary beans -— E, acutifolius - P597 and Arizona-Buff), moderately-resistant (MSU-513l9 and G.N. Valley), and common blight susceptible (Seafarer and Tuscola) bean genotypes, were Claudio R. Cafati studied in 1977 and 1978. While bacterial grown patterns were similar in and on leaves of moderately-resistant and susceptible cultivars, maximum bacterial populations were generally lower in the former, particularly during the reproductive stage of plant development. High RlS—l populations were detected in non- inoculated, symptom-free leaves of both susceptible and moderately- resistant genotypes. g, phaseoli populations in and on pods of susceptible and moderately-resistant genotypes were initially similar, but approximately eight days after inoculation, populations on the moderately-resistant genotypes reached stationary phase and most bacteria were epiphytic. In Tepary bean (Arizona-Buff), bacterial populations remained at relatively low levels in both leaves and pods, although detectable levels of R15-l occurred 20 days after inoculation; however, no disease symptoms developed. In the resistant Tepary genotype, bacteria were detected consistent- ly only in inoculated primary leaves. In moderately-resistant G.N. Valley, bacteria were recovered from primary, first, second and 3rd + 4th trifoliolate leaves and also from stems. In susceptible Tuscola, bacteria were isolated from primary, first, second, 3rd + 4th, 5th and 6th trifoliolate leaves; and from stem.and roots. High bacterial populations and symptoms were detected first in the older leaves and later in the younger leaves, from the primary to the 5th trifoliolate leaves in the susceptible, from.the primary to the 2nd trifoliolate leaves in the moderately—resistant. No symptoms and very low densities of bacteria were detected in inoculated and lst trifoliolate leaves of resistant Tepary. Tepary bean continues to Claudio R. Cafati be the best source of blight resistance presently available. Results of greenhouse and field studies indicate that leaves of susceptible and resistant bean genotypes and non-host plants may support epiphytic multiplication of Xp and that the bacteria may possess a resident phase in its life cycle. Xp and pr survived and retained pathogenicity after two years storage in the laboratory in dry infected tissue samples of susceptible and resistant plant species; however, neither isolates were detected in similar samples maintained on or buried in field soil at three different Michigan localities for the period November 1977 to June 1978. It is unlikely that leaf debris plays an important role in between-season survival of these pathogens under environmental conditions present in Michigan. Pods of resistant and susceptible bean genotypes inoculated by scratching the dorsal suture with the needle of s syringe containing the bacterial suspension developed different disease reactions; however, seeds with and without disease symptoms of both susceptible and resistant genotypes, carried internal blight infection. The data suggest that tests to detect seed borne bacterial blight should be a component in certified, blight-free bean seed production programs of all dry bean cultivars. Ultrastructural evidence suggests that attachment of Xp occurs in the intercellular spaces of leaves of blight-resistant Tepary bean (Arizona-Buff). To my wife and daughters To my parents ii ACKNOWLEDGMENTS I would like to express my sincerest gratitude and appreciation to my major professor, Dr. Alfred W. Saettler. I am indebted to him for his guidance, interest, and friendship during the period of this study and throughout my graduate program at Michigan State University. I also thank the other members of my graduate committee, Drs. Alan L. Jones, Albert H. Ellingboe, and James M. Tiedje for their helpful suggestions. To all my laboratory mates, for their cooperation I wish to express my gratitude. My sincere appreciation is also extended to the Instituto de Investigaciones Agropecuarias, Chile, for allowing me to continue my graduate education. To the Rockefeller Foundation, the fellowship is gratefully acknowledged. iii PART II. TABLE OF CONTENTS LIST OF TABLES. . . . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . . . . GENERAL INTRODUCTION AND LITERATURE REVIEW . . . LITERATURE CITED . . . . . . . . . . . . . . . . PART I. PRELIMINARY STUDIES .'. . . . . . . . . 1.1 Isolation rifampin-reistant mutants of Xanthomonas phaseoli (Xp) and Xanthomonas phaseoli var. fuscans (pr). . . . . . 1.2 Epiphytic growth of Xp on susceptible and resistant plant tissue . . . . . . . . . 1.3 Population trends of Xanthomonas phaseoli (xp) in bean germplasm as related to disease reaction Literature Cited . . . . . . . . . . . . . . (XANTHOMONAS PHASEOLI) . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . Host Genotypes . . . . . . . . . . . . . Bacterial Isolate . . . . . . . . . . . Experimental Plots . . . . . . . . . . . Inoculation Technique . . . . . . . . Isolation Procedures . . . . . . . . . . Evaluation of Disease Reaction . . . . . Statistical Analysis . . . . . . . . . RESULTS . . . . . . . . . . . . . . . . . . EFFECT OF HOST GENOTYPE ON MULTIPLICATION AND DISTRIBUTION OF BEAN COMMON BLIGHT BACTERIA Multiplication of Xp in and on Leaves and Pods Resistant, Moderately—Resistant and Susceptible Bean Genotypes . . . . . . . . . . . . . iv Page vi ix 11 16 16 18 21 35 38 38 41 41 41 42 42 42 43 44 45 45 Leaf populations, 1977 . . . . . . . . . . . . . . Pod populations, 1977 . . . . . . . . . . . . . . Leaf populations, 1978 . . . . . . . . . . . . . . Pod populations, 1978 . . . . . . . . . . . . . . Multiplication, Movement and Distribution of Xp in Resistant, Moderately-Resistant and Susceptible Bean Genotypes . . . . . . . . . . . . . . . .q. . . . . . DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . PART III. SURVIVAL AND TRANSMISSION OF BEAN BLIGHT BACTERIA (XANTHOMONAS PHASEOLI AND £3 PHASEOLI VAR. FUSCANS) IN TISSUES OF SUSCEPTIBLE AND RESISTANT PLANT SPECIES . . . . . . . . . . . . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . Bacterial Isolates . . . . . . . . . . . . . . . . . . Inoculation Techniques . . . . . . . . . . . . . . . . Determination of Bacterial Populations . . . . . . . . Survival in Leaf Tissues . . . . . . . . . . . . . . . Secondary Spread . . . . . . . . . . . . . . . . . . . Seed Transmission . . . . . . . . . . . . . . . . . . I. Greenhouse study . . . . . . . . . . . . . . . II. Field study . . . . . . . . . . . . . . . . . RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . Multiplication of Xp (RlS-l) in Leaves of Beans and Non—Host Species . . . . . . . . . . . . . . . . . . . Secondary Spread of Xp from Resistant to Susceptible Bean Genotypes . . . . . . . . . . . . . . . . . . . . Reciprocal Secondary Spread of Xp Between Susceptible Bean Cultivar to weeds . . . . . . . . . . . . . . . . Survival of Xp (R15-l) and pr (R17) in Dry Tissues of Susceptible and Resistant Bean Genotypes and Non—Host Species . . . . . . . . . . . . . . . . . . . Survival and Seed Transmission of Xp (RlS—l) in Seed of Resistant and Susceptible Bean Genotypes . . . DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . APPENDIX C O O O O O O 0 O O O O O O O O O I O O I I O O O O 0 Preliminary Ultrastructural Evidence for Immobilization of Xanthomonas phaseoli in Tepary Bean (Phaseolus acutifolius) Leaves . . . . . . . . . . . . . . . . . . . Tepary bean (resistant) . . . . . . . . . . . . . . Tuscola bean (susceptible) . . . . . . . . . . . . . Literature Cited . . . . . . . . . . . . . . . . . . . . . Page 45 49 56 6O 64 68 74 78 78 82 82 82 83 83 84 85 85 86 88 88 88 91 91 98 107 111 114 114 116 116 124 Table LIST OF TABLES Page PART I. PRELIMINARY STUDIES Relative population levels of Xanthomonas phaseoli (R15-l) on several host and non-host species as determined by leaf-prints on YCA-rifampin media. . . . . . 20 Germplasm used a sources of disease-, insect-, and nematode—resistance by the major Bean Breeding Programs . . . . . . . . . . . . . . . . . . . . . . . . . 23 Reactions of various disease-, insect-, and nematode-resistant bean germplasm to Xanthomonas phaseoli . . . . . . . . . . . . . . . . . . . . . . . . . 26 Population trends of Xanthomonas phaseoli (R15—l mutant) in trifoliolate leaves of resistant (45-1 and 29-BK, Phaseolus coccineus lines) and susceptible (Searfarer, P, vulgaris) bean genotypes . . . . . . . . . 28 Population trends of Xanthomonas phaseoli (RlS-l mutant) in trifoliolate leaves of resistant (Tepary bean, P. acutifolius, and PI 207262) and susceptible (Seafarer) bean genotypes . . . . . . . . . . 29 Population levels of Xanthomonas phaseoli (R15-l mutant) in plants of resistant (Tepary), moderately- resistant (MSU-51319), and susceptible (Seafarer) bean genotypes . . . . . . . . . . . . . . . . . . . . . . 31 Recovery of Xanthomonas phaseoli (RlS-l mutant) 21 days after inoculation of primary leaves in resistant and susceptible bean genotypes . . . . . . . . . 33 PART II. EFFECT OF HOST GENOTYPE ON MULTIPLICATION AND DISTRIBUTION OF BEAN COMMON BLIGHT BACTERIA (XANTHOMONAS PHASEOLI) Population trends in Xanthomonas phaseoli (R15-1 mutant) in and on trifoliolate leaves of moderately- resistant (MSU-51319) and susceptible (Seafarer) bean genotypes . . . . . . . . . . . . . . . . . . . . . . 48 vi Table Population trends of Xanthomonas phaseoli (RlS-l mutant) in and on trifoliolate leaves of resistant (P597, 2. acutifolius) and susceptible (Seafarer) bean genotypes. . . . . . . . . . . . . . . . . . . . . . Population trends of Xanthomonas phaseoli (RlS-l mutant) in and on pods of moderately-resistant (MSU-513l9) and susceptible (Seafarer) bean genotypes . . . . . . . . . . . . . . . . . . . . . . . Population trends of Xanthomonas phaseoli (RlS-l mutant) in and on trifoliolate leaves of highly resistant (Tepary), moderately-resistant (G.N. Valley) and susceptible (Tuscola) bean genotypes . . . . Population trends of Xanthomonas phaseoli (RlS-l mutant) in and on pods of highly resistant (Tepary Arizona-Buff), moderately-resistant (G.N. Valley) and susceptible (Tuscola) bean genotypes . . . Pepulation levels of Xanthomonas phaseoli (RIS-l mutant) in resistant (Tepary Arizona-Buff), moderately-resistant (G.N. Valley) and susceptible (Tuscola) bean genotypes . . . . . . . . . . Recovery of Xanthomonas phaseoli (R15-1 mutant) from various plant parts following inoculation of the primary leaves of resistant (Tepary), moderately-resistant (G.N. Valley) and susceptible (Tuscola) bean genotypes ._. . . . . . . . Page 52 55 59 63 66 67 PART III. SURVIVAL AND TRANSMISSION OF BEAN BLIGHT BACTERIA (XANTHOMONAS PHASEOLI AND E, PHASEOLI VAR. FUSCANS) IN TISSUES OF SUSCEPTIBLE AND RESISTANT PLANT SPECIES Population trends of Xanthomonas phaseoli (R15-l mutant) in leaves of different plant species . . . . . . Secondary spread of Xanthomonas phaseoli (RlS-l mutant) from inoculated resistant (MSU-513l9) to non-inoculated susceptible (Tuscola) bean genotypes . . . . . . . . . . Reciprocal secondary spread of Xanthomonas phaseoli (RlS-l mutant) between susceptible bean genotype (Tuscola) to Chenopodium alba ("lambsquarters") and Amaranthus retroflexus ("pig weed") under field condi tions 0 O O O O O C O O O O O O O O O O O O O O O 0 Population levels of RlS-l (Xanthomonas phaseoli) and R17 OE. phaseoli var fuscans) in dry leaf tissue of greenhouse-grown plants . . . . . . . . . . . . . . vii 89 9O 92 93 Table Page Population levels of RIB-l (Xanthomonas phaseoli) and R17 (g, phaseoli var'fuscans) in dry leaf tissue of field-grown bean genotypes . . . . . . . . . . . . . . 96 Recovery of pr (R17 mutant) from dry infected leaf tissue of different bean genotypes stored at room temperature . . . . . . . . . . . . . . . . . . . . 97 Recovery of Xp (RIB-1 mutant) from seeds harvested from symptomless pods on plants of three bean genotypes inoculated at different stages of plant development . . . . . . . . . . . . . . . . . . . . . . . 99 Incidence of blight bacteria in seed harvested from greenhouse grown plants inoculated with g. phaseoli . . . 105 Incidence of blight bacteria in seed harvested from field grown plants inoculated with g. phaseoli . . . . . 106 viii Figure LIST OF FIGURES Page PART I. PRELIMINARY STUDIES Population trends of Xanthomonas phaseoli (RlS-l in and on trifoliolate leaves of moderately- resistant (MSU-51319) and susceptible (Seafarerl bean genotypes. Twenty-three day old plants (3rd and 4th trifoliolate leaves) were inoculated to run-off with a 1.0xlO7 cells/ml suspension of RlS-l at day 0. values are average of three replications. . . . . . . . . . . . . . . . . . . . . . . 46 Population trends of Xanthomonas phaseoli (RlS—l mutant) in and on trifoliolate leaves of resistant (P597, P, acutifolius) and susceptible (Seafarer) bean genotypes. Thirty-six day—old plants (3rd and 4th trifoliolate leaves) were inoculated to run-off with a 1.0xlO7 cells/m1 suspension of RlS—l at day 0. Values are average of three replications. . . . . . . . . . . . . . . . . . . . . . . 50 Population trends of Xanthomonas phaseoli (RlS—l mutant) in and on pods of moderately—resistant (MSU-51319) and susceptible (Seafarer) bean genotypes. Pods (flat-pod stage) were inoculated by gentle spraying to run-off with.a 1.0x107 cells/ml suspension of R15-l at day 0. Values are average of three replications . . . . . . . . . . . . . . . . . . . 53 Population trends of Xanthomonas phaseoli (RlS-l mutant) in and on trifoliolate leaves of resistant (Tepary, Arizona-Buff), moderately-resistant (G.N. Valley) and susceptible (Tuscola) bean ‘genotypes. Twenty-five day-old plants Can and 3rd trifoliolate leaves) were inoculated to run-off with a 1.0xlO7 cells/ml suspension of R15-l at day 0. Values are average of three replications. . . . . 57 Figure Page Population trends of Xanthomonas phaseoli (R15-1 mutant) in and on pods of resistant (Tepary, Arizona-Buff), moderately—resistant (G.N.‘Valley) and susceptible (Tuscola) bean genotypes. Pods (flat-pod stage) were inoculated by gentle spryaing to run—off with a 1.0x107 cell/ml suspension of RlS—l at day 0. Values are average of three replications. . . . . . . . . . . . . . . . . . 61 PART III. SURVIVAL AND TRANSMISSION OF BEAN BLIGHT BACTERIA (XANTHOMONAS PHASEOLI AND E, PHASEOLI VAR. FUSCANS) IN TISSUES OF SUSCEPTIBLE AND RESISTANT PLANT SPECIES Recovery of RlS-l (Xp) from dry leaf tissue, maintained in field soil or stored under laboratory i conditions, on YCA supplemented with 15 ug/ml ‘ rifampin (R), 100 ug/ml cycloheximide (C), and 100 ug/ml PCNB (P). Photographs taken after six days incubation at room temperature . . . . . . . . . . . . . 95 Disease symptoms on mature pods of Tepary (Arizona— Buff), G.N. Valley, and Tuscola bean genotypes. Pods at the flat green stage of development were inoculated by scratching the dorsal suture with a syringe containing 1.0x107 cells/ml of R15- 1 (Xp mutant). . . . . . . . . . . . . . . . 100 Seed obtained from pods of Tepary (Arizona-Buff), G.N. Valley, and Tuscola bean genotypes, inoculated at the flat green pod stage of plant development by scratching the dorsal suture of the pods with a syringe containing 1.0xlO7 cells/m1 of R15-1 (Xp mutant). . . . . . . . . . . . . . . . . . . . . . . 102 APPENDIX Transmission electron micrographs of spongy mesophyll cells in resistant Tepary bean (Phaseolus acutifolius) leaves showing the interaction of host cell wall and common blight bacteria (Xanthomonas phaseoli) . . . . . 119 Cross section of bacteria at three hours after inoculation. Note group of bacteria aligned close to the host cell walls. (x 12 000) . . . . . . . . . . . 119 Cross section of bacterium in intercellular space three hours after inoculation. Note loose fibrillar material on the host cell wall in close proximity to the bacterium (arrow). (x 60 000) , . . . . . . . . . 119 Page 3,4,5. Cross sections of bacteria attached to host cell wall matrix 18 hours after inoculation. Note matrix attached to bacterial cell (arrows). ~ (Fig. 3 and 5 x 32 000; Fig. 4 x 25 000). . . . . . . . 119 Cross section of intercellular space 96 hours after inoculation. Structure (arrow) that resembles those typically reported to completely encapsulate bacteria (x 52 000) . . . . . . . . . . . . . 119 Transmission electron micrographs of spongy mesophyll cells in Xanthomonas blight susceptible cv. Tuscola (Phaseolus vulgaris) leaves . . . . . . . . . 120 Bacteria in susceptible host 18 hours after inoculation. Note large number of bacterial cells (x 16 000). . . . . . . . . . . . . . . . . . . . . 120 Bacteria in the lesion area of susceptible host genotype eight days after inoculation (x 3 200) . . . . . 120 xi GENERAL INTRODUCTION AND LITERATURE REVIEW Common and fuscous blight caused by Xanthomonas phaseoli (E.F. Smith) Dowson (Xp) and g, phaseoli var. fuscans (Burk.) Starr and Burk (pr) respectively, are among the most serious seed-borne bacterial diseases of dry edible and green beans throughout the world. These bacteria are distributed worldwide and continue to be serious production-limiting factors for dry beans in many areas of Latin America, as well as in the humid Great Lakes regions of the U.S. and Canada (34). Chemical control measures available at the present time are not entirely satisfactory. Although practical short-term control is possible through the use of disease-free seed grown under irrigation in dry areas, effective long-term control depends upon development of resistant cultivars. Considerable effort has been directed toward finding resistant germplasm useful to breeding (27, 14) and absence of immunity to common and fuscous blights further underscores the role of blight in bean production. Burkholder (4) was the first to conduct extensive screening of bean germplasm for resistance to common blight; he found that none of the cultivars tested were immune, although some differed in disease severity. In 1946 Burkholder and Bullard (5) reported varietal susceptibility trials to xpf; all cultivars tested were susceptible except two, which showed a low level of resistance. Coyne 33 51 (15) tested an extensive collection of plant introductions of Phaseolus species and varieties and breeding lines of Phaseolus vulgaris to natural field infections in Nebraska and subsequently rescreened apparently tolerant selections in the greenhouse. Twelve PI accessions were highly tolerant under both tests, although none were fully immune; those that were highly tolerant were also of late maturity. Major programs of breeding Xanthomonas resistance into dry edible bean types were not initiated until 10-15 years ago. Programs of this type are located at the University of Nebraska, Michigan State University-USDA, Puerto Rico-USDA, Canada, CIAT (Colombia) and Cornell University. It has long been recognized that certain accessions of Tepary bean (Phaseolus acutifolius) are resistant to bacterial blight (27), but the interspecific cross can only be made with.P, vulgaris as the female and by employing embryo culture. In early attempts four fertile F plants were obtained by Honma (25); F bulk populations 1 from the F2 plants tested by inoculation showed a normal distribution of reaction grades. Great Nbrthern Nebraska No. l originated from 3 one of these families. Great Northern Nebraska No. 1 has been recognized as possessing good resistance to blight bacteria and a selection made within it, sel. 27 (27) has been used in breeding improved Great Northern varieties Tara (8), Jules (9), and valley (11). The inheritance of the disease reaction to §. phaseoli has been reported by several workers. In crosses between Great Northern 1140 and Great Northern Nebraska No. 1 selection 27, Coyne 35 a1 (16), found that the latter contributed several genes for resistance to the hybrid. In advanced self-pollinated or back-cross generations, the pattern of segregation suggested polygenic inheritance for resistance. Pompeu and Crowder (33) reported that resistance was conditioned by several partially dominant genes in crosses between two bligth susceptible and two blight resistant bean cultivars. This character was quantitative and highly heritable. Coyne and Schuster (13) reported that PI 207262 (Colombia) possesses different genes for resistance to Xp Nebraska isolate than GN Nebraska 1, sel. 27. In crosses between PI 207292 x GN 1140, nearly complete dominance for a tolerant disease reaction and early-flowering (maturity) was observed in the F ; a bimodal 1 distribution of disease reaction ratings was noted in the F (10). 2 This is the first report of qualitative genetic control of reaction to Xp in Phaseolus vulgaris L. Two collections of P, coccineus reported as resistant to g. phaseoli according to Coyne §E_al_(15), might also be used as sources of resistance for incorporation in P. vulgaris since the two species are easily intercrossed. Vakili recently reported (46) that scarlet runner beans (Phaseolus coccineus L.) possess higher level of resistance than dry beans (P, vulgaris L.) to various diseases and pests under greenhouse and field conditions. For this reason a breeding scheme was set up to select plants with the widest and highest levels of resistance to a number of diseases, including bacterial blight. The presence of pathogenic variation in Xp was initially suggested in 1956 by Smale and Worley (42) who detected pathogenic differences in individual colonies of stock Xp cultures. Definitive evidence that variation is present in Xp isolates from various geographical areas was presented by Schuster gt §1_in Nebraska (39, 40, 41). Ekpo (20) and Saettler and Ekpo (35) confirmed the existence of pathogenic variation in Xp and extended the existence of such variation to pr. In a recent study at CIAT (6) greenhouse experiments were done to determine if pathogenic variation of common bacterial blight was due to distinct races or to variations in isolate virulence. Six cultivars with different degrees of resistance or susceptibility were inoculated with isolates from Latin America and the United States. Virulence between isolates and resistance of the cultivars varied, but there was no interaction at the P=0.05 level between isolates and cultivars to imply the presence of races. In a further study, they found that isolates from different locations in the Americas were as virulent as Xp 123 from Colombia. The authors suggested the use of the most highly virulent local isolate of the blight pathogen when evaluating germplasm for resistance. That bean leaves of different ages are not equally susceptible to Xp infection has been known for some time. Goes (22) reported that as leaf age increased, Xp susceptibility also increased. 0n the other hand, Patel and Walker (32), noted that the youngest rather than the oldest leaves were the most susceptible. Both of these studies were made with plants in the vegetative stage of development, however, and did not simulate a natural field situation of Xp disease development. In the field, Xanthomonas bacterial blights become most visible at or just following the blossom stage, generally, symptoms are observed initially on the lower, older leaves. Secondary spread of the pathogen occurs most rapidly after this time. The importance of evaluating breeding material in various stages of growth was emphasized by Coyne gtflal (13, l4, 17) who determined that plants of both susceptible and moderately resistant lines were more susceptible to Xp when in a reproductive stage of development. Increased susceptibility to Xp and pr when plants are in reproductive as compared to vegetative stage of development was recently reported to be a common phenomenon by Saettler and Ekpo (35). Even though developmental stage is important when evaluating breeding material for Xanthomonas resistance, previous work.on inheritance of Xp tolerance based on inoculation of vegetative plants is not negated (25, 33). In a recent study by Yoshii.gtfl§l (49), Phaseolus germplasm.was screened for field reaction to Colombian isolates of Xp. Two P, acutifolius lines, PI 169932 and Tepary Nebraska Accession No. 10, had the highest degree of resistance because there were no foliage or pod symptoms. None of four thousand P. vulgaris entries tested were free from blight symptoms and foliage reaction was not correlated with pod reaction or growth habit. Coyne and Schuster (12) suggested that bean blight reactions may be due to a recombination of different genes controlling the response of different plant parts to bacterial infection and emphasized the importance of obtaining resistance in both leaves and pods to this pathogen. Epiphytic survival and multiplication on surfaces of host and non-host plants has been described for several plant pathogenic bacteria (19, 21, 23, 26, 28, 29, 30, 36, 43). Several workers have shown that symptomless tissue of bean cultivars and lines possessing different levels of resistance (1, 14, 20) or susceptibility (20, 45, 48) can contain detectable levels of Xp and pr. The increase of the pathogen in the absence of symptoms may be of epidemiological impor- tance by serving to build up inoculum for secondary spread. It has been suggested that additional research studies be initiated on the epi- and endophytic phases of Xp and pr relative to resistant and susceptible tissues (34). Under natural conditions Xp and pr enter the leaves through natural openings such as stomata and hydatodes or through.wounds. The bacteria invade the intercellular spaces, causing a gradual dissolution of the middle lamella. Later the cells begin to dis- integrate with the formation of bacterial pockets (52). Systemic movement of Xp was first noted by Barlow (2) in 1904 and later studied by Burkholder (3). Histological studies by Zaumeyer (50, 51) indicated that bacteria may enter the stem through the stomata of the hypocotyl end epicotyl through the vascular elements leading from the leaf, and from infected cotyledons. According to Burkholder the behavior of Xp after infecting the vascular system of the host plant depends upon environmental conditions and on the variety of beans. In many instances, no external lesions or death of the plant parts occur until blossom time, or even after pod set. Burkholder also stated that one of the most important points in the behavior of the blight bacteria is their ability to enter the pods through the vascular system and infect the seeds without causing lesions on the pod surface. In a recent investigation, Weller (47) studied the multiplication of blight bacteria in field-grown Navy beans by monitoring populations from seedling till the early reproductive stage. All seeding parts of seedlings grown from infected seeds became colonized by blight bacteria immediately after germination. Multiplication of R10 and Ra (pr and Xp rifampin-resistant mutants, respectively) was described by a series of growth curves displaced over time, each leaf becoming colonized as it differentiated from the main stem. Maximal bacterial populations and symptoms were detected first in the older leaves and later in the younger leaves. The spread of R10 and Ra was facilitated by rain, bud colonization, and systemic movement. According to the author, the pattern of bacterial multiplication offers an explanation for the late appearance of field symptoms typical of bacterial blights. No study has been conducted on the movement and distribution of blight bacteria relative to tolerant or resistant bean germplasm. Transport of a bacterial pathogen in seed is an important means of survival and dissemination in time and space. Infection sites of seed—borne bacterial pathogens may be in contaminating trash, as surface infection (i.e., limited colonization of the seed coat or hilum area) or as a deep-seated infection (colonization of the embryo or other internal structures). It has been demonstrated that the bacterial blight organism is harbored.below the seed coat (52); bacteria apparently enter the sutures of the pods from the vascular system of the pedicel and then pass into the funiculus and through the raphe leading into the seed. The micropyle also serves as a point of entry into the seed. The bacteria either remain in the seed coat or pass into the region of the cotyledons during seed germination. Both, externally and internally infected seed have been mentioned as important sources of primary inoculum and dissemination of blight bacteria (34). For these reasons disease control is based on seed certification programs to maintain clean seed stocks. Cope- land 23 31 (7) have described the process of producing certified bean seed from breeder and foundation seed stocks. Certified seed raised from foundation seed is certified only after the crop has been inspected and the resulting seed tested for the presence of bacterial blight organisms. There is no doubt that such programmes have been successful in reducing seed infection by the bacterial pathogens; nevertheless, outbreak of common and fuscous blights persist and some fields are rejected annually for certification. In a recent study on the ecology of Xp and pr in Navy beans, Weller (47) reported that seeds externally infested with.blight bacteria were shown to be an important source of primary inocula and 14% of commercial Navy bean seed lots were so contaminated. Symptomless seed internally bearing Xp and pr was identified as potential primary inocula sources and seeds with symptoms were always associated with visibly-infected pods. Coyne 32 31 (18) reported that Xp infected seed (internal infection) was detected in susceptible varieties but was not detected in tolerant lines. Nevertheless, the authors reported that seed transmission of Pseudomonas phaseolicola has been found in some halo blight tolerant beans and has caused serious problems to seed producers. The possible transmission of common and fucous blight bacteria in seed of resistant bean germplasm is important relative to breeding and certification programs, and emphasizes the need for research in this area. How the blight organisms overwinter, especially in the northern states where the winters are long and temperatures low, has been variously argued. Zaumeyer (52) stated that there are several plants other than beans that are susceptible to Xp, and in the south, where a succession of crOps is grown throughout the year, the perpetuation of the organism by a series of different hosts is not improbable. Muncie (31) isolated the blight organism from diseased bean stubble that remained in the field over winter. Zaumeyer (50) related observations that lend support to the possible overwintering in the field. Schuster (37, 38) reported overwintering of Xp and pr in refuse of beans and in two weeds in Nebraska. On the other hand, there are also reports of non-overwintering of Xp and pr. Hedges(24), at Arlington-Virginia, placed lima bean leaves infected with Xp into pots in the fall and kept them buried during the winter. The next 10 spring the author was unable to obtain any infection on snap beans planted in these pots. Sutton and wallen (44), reported that Xp and pr were not isolated from fields known to be infected with either one or both of the pathogens. In a more recent study, Weller (47) reports that pr and Xp isolates were not detected in bean stem and leaf tissue which was buried in, or laid on, field soil during the winters following the 1975, 1976 and 1977 growing seasons. The overall purpose of this investigation was to study the effect of host genotype on multiplication, movement, distribution and survival of bean blight bacteria. It is hoped that the information obtained from the study will improve our understanding of and provide further insight into the ecology of Xanthomonas blight as related to the host-parasite relationship, and will contribute practical informa— tion for bean blight breeding programs. 10. LITERATURE CITED ALLINGTON, W.B., and D.W. CHAMBERLAIN. 1949. Trends in the population of pathogenic bacteria within leaf tissues of susceptible and immune plant species. Phytopathology 39: 656-6600. BARLOW, B. 1904. A bacterial disease of beans. Ontario Agr. Coll. Bul. 136:9-13. BURKHOLDER, W.H. 1921. The bacterial blight of the bean: a systemic disease. Phytopathology 11:61-69. BURKHOLDER, W.H. 1924. varietal susceptibility among beans to the bacterial blight. Phytopathology 14:1-7. BURKHOLDER, W.H., and E.T. BULLARD. 1946. varietal susceptibility of beans to Xanthomonas phaseoli var. fuscans. Plant Dis. Rep. 30:446-448. CENTRO INTERNACIONAL DE AGRICULTURA TROPICAL (CIAT). 1978. Bean Program. Ann. Rept. 1977, 84 pp. COPELAND, L.0., M.W. ADAMS, AND D.C. BELL. 1975. An improved seed programme for maintaining disease-free seed on field beans (Phaseolus vulgaris). Seed Sci. and Technol. 3:719- 724. COYNE, D.P., and M.L. SCHUSTER. 1969. 'Tara', a new Great Nerthern dry bean variety tolerant to common blight. Bull. Nebr. Agric. Exp. Sta. 506:1-10. COYNE, D.P., and M.L. SCHUSTER. 1970. 'Jules', a Great Northern dry bean tolerant to common blight bacterium (Xanthomonas phaseoli). Plant Dis. Rep. 54:557-558. COYNE, D.P., and M.L. SCHUSTER. 1973. First report of a qualitative genetic control of the reaction to Xanthomonas phaseoli in Phaseolus vulgaris L. Ann. Rept. Bean Impr. Coop. 16:17. 11 11. 12. 13. 14. 15. l6. 17. 18. 19.‘ 20. 21. 12 COYNE, D.P., and M.L. SCHUSTER. 1974. 'Great Northern Valley' dry bean. Hort. Science 9:482. COYNE, D.P., and M.L. SCHUSTER. 1974. Differential reaction of pods and foliage of beans (Phaseolus vulgaris) to Xanthomonas phaseoli. Plant Dis. Rep. 58:278-282. COYNE, D.P., and M.L. SCHUSTER. 1974. Inheritance and linkage relations of reaction to Xanthomonas phaseoli (E.F. Smith) Dowson (common blight), stage of plant development and plant habit in Phaseolus vulgaris L. Euphytica 23:195-204. COYNE, D.P., and M.L. SCHUSTER. 1974. Breeding and genetic studies of tolerance to several bean (Phaseolus vulgaris L.) bacterial pathogens. Euphytica 23:651-656. COYNE, D.P., M.L. SCHUSTER. and S. AL—YASIRI. 1963. Reaction studies of bean species and varieties to common blight and bacterial wilt. Plant Dis. Rep. 47:534-537. COYNE, D.P., M.L. SCHUSTER, and L. HARRIS. 1965. Inheritance, heritability, and response to selection for common blight (Xanthomonas phaseoli) tolerance in Phaseolus vulgaris field bean crosses. Proc. Amer. Soc. Hort. Sci. 86:373-379. COYNE, D.P., M.L. SCHUSTER, and K. HILL. 1973. Genetic control of reaction to common blight bacterium in beans (Phaseolus vulgaris) as influenced by plant age and bacteri- ial multiplication. J. Am” Soc. HOrt. Sci. 98:94-99. COYNE, D.P., M.L. SCHUSTER, and S. MAGNUSON. 1976. Effect of tolerant and susceptible dry bean germplasm on seed transmission of Xanthomonas phaseoli. Ann. Rept. Bean Imp. Coop. 19:20. CROSSE, J.E. 1963. Bacterial canker of stone-fruits.~4V. A comparison of leaf-surface populations of Pseudomonas mors-prunorum in autumn on two cherry varieties. Ann. appl. Biol. 52:97-104. EKPO, E.J.A. 1975. Pathogenic variation in common (Xanthomonas phaseoli) and fuscous (Xanthomonas phaseoli var. fuscans) bacterial blights of bean (Phaseolus vulgaris L.). Ph.D. Thesis, Michigan State Univ., East Lansing, 127 pp. ERCOLANI, G.L., D.J. HAGEDORN, A. KELMAN, and R.E. RAND. 1974. Epiphytic survival of Pseudomonas syringae on hairy vetch in relation to epidemiology of bacterial brown spot of bean in Wisconsin. Phytopathology 64:1330-1339. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 13 GOSS, Raw. 1940. The relation of temperature to common and halo blight of beans. Phytopathology 30:258-264. HASS, J.H., and J. ROTEM. 1976. PseudomonaS‘lachrymans absorption, survival and infectivity following precision inoculation of leaves. Phytopathology 66:992-997. HEDGES, F. 1946. Experiments on the overwintering in the soil of bacteria causing leaf and pod spots of snap and lima beans. Phytopathology 36:677-678. HONMA, S. 1956. A bean interspecific hybrid. J. Hered. 47: 217-220. KENNEDY, B.W., and G.L. ERCOLANI. 1978. Soybean primary leaves as a site for epiphytic multiplication of Pseudomonas glycinea. Phytopathology 68:1196-1201. LEAKEY, C.L.A. 1973. A note on Xanthomonas blight of beans (Phaseolus vulgaris (L.) Savi) and prospects for its control by breeding for tolerance. Euphytica: 132-140. LEBEN, c., G.C. DAFT, andiA.F. SCHMITTHENNER. 1968.. Bacterial blight of soybeans: population levels of Pseudomonas glycinea in relation to symptom development. Phytopathology 58:1143-1146. MEW, T.W., and B.WI KENNEDY. 1971. Growth of Pseudomonas glycinea on the surface of soybean leaves. Phytopathology 61:715—716. MILLER, T.D., and M.N. SCHROTH. 1972. Monitoring the epiphytic population of Erwinia amylovora on pear with a selective medium. Phytopathology 62:1175-1182. MUNCIE, J.H. 1914. Two Michigan bean diseases. Mich. Agr. Exp. Sta. Spec. Bul. 68, 12 pp. PATEL, P.N., and J.C. WALKER. 1963. Relation 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. POMPEU, A.S., and L.V. CROWDER. 1972. Inheritance of resist- ance of Phaseolus vulgaris (dry beans) to Xanthomonas phaseoli Dows. (common blight). Cien. Cult. (S) Paulo) 24: 1055-1063. SAETTLER, A.W. 1977. Breeding dry edible beans (Phaseolus vulgaris L.) for tolerance to Xanthomonas bacterial blights. Fitopatologia Brasileira 2: 179-186. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 14 SAETTLER, A.W., and EKPO, E.J.A. 1975. Pathogenic variation in Xanthomonas phaseoli and g. phaseoli var. fuscans. Ann. Rept. Bean Imp. Coop. 18:67-70. SCHNEIDER, RIW., and R.G. GROGAN. 1977. Bacterial speck of tomato: Sources of inoculum and establishment of a resident population. Phytopathology 67: 388-394. SCHUSTER, M.L. 1967. Survival of bean bacterial pathogens in the field and greenhouse under different environmental conditions. Phytopathology 57:830 (Abstr.). SCHUSTER, M.L. 1970. Survival of bacterial pathogens of beans. Ann. Rept. Bean Imp. Coop. 13:68-70. SCHUSTER, M.L. and D.P. COYNE. 1971. New virulent strains of Xanthomonas phaseoli. Plant Dis. Rep. 55:505-506. SCHUSTER, M.L., and D.P. COYNE. 1975. Genetic variation in bean bacterial pathogens. Euphytica 24:143-147. SCHUSTER, M.L., D.P. COYNE, and B. HOFF. 1973. Comparative virulence of Xanthomonas phaseoli strains from Uganda, Colombia, and Nebraska. Plant Dis. Rep. 57:74-75. SMALE, B.C., and J.F. WORLEY. 1956. Evaluation of 2, 3, 5- triphenyl tetrazolium chloride for obtaining pathogenic types from stock cultures of halo blight and common blight organisms. Plant Dis. Rep. 40:628. STALL, R.E., and .A.A. COOK. 1966. Multiplication of Xanthomonas vesicatoria and lesion development in resistant and susceptible paper. Phytopathology 56:1152- 1153. SUTTON, M.D., and V.R. WALLEN. 1967. Phage types of Xanthomonas phaseoli isolated from beans. Can. J. Bot. 45: 267-280. THOMAS Jr., W.D., and GRAHAM, R.W. 1952. Bacteria in apparently healthy Pinto beans. Phytopathology 42:214. VAKILI, N.G. 1978. Germplasm of multiple disease resistance scarlet runner beans. Internal Report M.I.T.A., 4 pp. (Unpublished). 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., East Lansing, 137 pp. 48. 49. 50. 51. 52. 15 WELLER, D.M., and A.W. SAETTLER. 1977. Population studies of Xanthomonas phaseoli var. fuscans in field grown navy pea beans. Ann. Rept. Bean Imp. Coop. 20:76-79. YOSHII, K., G.E. GALVEZ-E., and G. ALVEREZ-A. 1978. Screening bean germplasm for tolerance to common blight caused by Xanthomonas phaseoli and the importance of pathogenic variation to varietal improvement. Plant Dis. Rep. 62: 343—347. ZAUMEYER, W.J. 1930. The bacterial blight of beans caused by Bacterium phaseoli. U.S. Dept. Agr. Tech. Bul. 186, 36 pp. ZAUMEYER, W.J. 1932. Comparative pathological histology of three bacterial diseases of bean. Jour. Agr. Res. 44:605- 632. ZAUMEYER, W.J., and H.R. THOMAS. 1957. A monographic study of bean diseases and methods for their control. U.S. Dept. Agri. Tech. Bull. 808, 255 pp. PART I PRELIMINARY STUDIES Isolation of rifampin-resistant mutants of Xanthomonas phaseoli (Xp) and Xanthomonas phaseoli var. fuscans (pr). Epiphytic growth of Xp and pr on susceptible and resistant plant tissue. Population trends of Xanthomonas phaseoli in bean germplasm as related to disease reaction. 1.1. Isolation of rifampin-resistant mutants of Xanthomonas phaseoli (Xp) and Xanthomonas phaseoli var. fuscans (pr). The lack of efficient culture medium selective for bean blight bacteria and the presence of heterogenous populations of microorgan- isms (fungi and bacteria) growing saprophytically on the surface of field-grown beans, suggested the possible utility of antibiotic- resistance mutants for the study of plant pathogenic bacteria. Antibiotic-resistance has been used only relatively recently in the study of diseases caused by plant pathogenic bacteria. Lewis and Goodman (19) used streptomycin resistant Erwinia amylovora to study mode of penetration and movement of fire blight bacteria in apple leaf and stem tissue. Similar types of mutants were used by Gowda and Goodman (10) to permit selective isolation of E. amylovora from shoot, stem and root of apple. Stall and Cook (14) studied the multiplication of Xanthomonas vesicatoria in susceptible and resistant peppers with a streptomycin-resistant mutant of Xanthomonas oryzae for studying pathogen ecology and as a method for detecting the presence of the bacteria in rice seed. Gardner and Redo (9) studied systemic movement of Erwinia rubifaciens in walnuts by the use of a double mutant resistant to rifampicin and neomycin. More recently rifampin resistance has been used in ecological studies of Agrobacterium tumefaciens (l, 21), and Pseudomonas coronofaciens the causal agent of halo blight of rye (4). Wellwer and Saettler (28) reported the usefulness of rifampin mutants as tools for studying Xp and pr under field conditions. 16 17 The objective of this preliminary study was the development of rifampin-resistant Xp and pr to be used in an antibiotic selective system for qualitative and quantitative ecological studies. Naturally-occurring rifampin-resistant mutants were selected from wild type isolates Xp 15 and pr l7 (highly virulent Michigan isolates) by spreading 108 cells on plates of YCA (YCA, 10 gm yeast extract, 2.5 gm CaCO 15 gm agar of 1000 ml distilled water) 3, supplemented with 50 ug/ml rifampin. Mutants were first screened based on colony characteristics and growth in culture media (YCA) and standard physiological test for Xanthomonas (6, 28). Also, mutants and wild types were compared serologically using the Ouchterlony technique (14). Pathogenicity was tested under greenhouse conditions by: 1) injecting a bacterial suspension (5.0x107 cells/ml) into stems of lZ-day-old Seafarer and Manitou bean seedlings and 2) spraying the undersurfaces of the leaves to a watersoaked appearance with a bacterial suspension of 1.0x107 cells/m1. Two selected mutants one each of Xp and pr were also compared with their respective wild types for growth and pathogenicity, by gently spraying the undersurfaces of field-grown navy bean leaves (cultivar Seafarer) with a bacterial suspension of 1.0x107 cells/ml and recording symptom development. The isolates retained their rifampin-resistant phenotypes after repeated subculturing on YCA in the absence of rifampin and retained pathogenicity as tested by inoculations to beans. The isolates were resistant to greater than 250 ug/ml rifampin. 18 Isolates R15-l (Xp) and R17 (pr) were identical to the respective wild types in cultural, physiological, serological, and pathological tests and were selected for use in detailed epidemiol- ogical studies. 1.2. Epiphytic growth of Xp on susceptible and resistant plant tissue. A preliminary study was undertaken to determine whether susceptible and resistant leaf tissues are inherently capable of supporting epiphytic growth.of bean blight bacteria. Plants of bacterial blight resistant (Phaseolus acutifolius, P597 - CIAT and Arizona-Buff) and susceptible navy bean cultivars Seafarer and Tuscola (Phaseolus vulgaris); soybean cv. Hark (Glycine max), COWpea cv. Mississippi Silver Pea (Vigna unguiculata), corn WG 4A (Zea mays), sugar beet US-20 (Beta vulgaris), and two common weeds, lambsquarters (Chenopodium alba) and pigweed (Amaranthus retroflexus), were grown in the growth chamber with air temperature maintained at 25 C and 16 hours photoperiod. To prepare the inocula, R15-l (Xp mutant) was grown at room temperature (24 :_1C) for 48 hours, suspended in sterile-distilled water and adjusted turbidimetrically to 2.0x108 viable cells/m1. Bacterial suspensions were lightly sprayed by means of a‘DeVilbiss atomizer to run-off on the lower and upper surfaces of the leaves, without producing watersoaking. Bean plants possessed fully expanded second trifoliolate leaves and weed plants were at the vegetative stage of growth- 19 Growth of R15-l on leaves was determined at intervals after inoculation using leaf-impression cultures. Direct leaf-prints were made by gently pressing the upper and lower surface of leaves onto plates of YCA supplemented with 50 ug/ml rifampin and 25 ug/ml cycloheximide, for one minute. Bacterial growth was evaluated by estimating the percentage of the leaf-print area covered with bacteria after 72 hours of incubation at room temperature. High bacterial populations were detected on leaves of susceptible bean cultivars Seafarer and Tuscola, the resistant genotype P597 (Phaseolus acutifolius) and on the leaves of soybean (cultivar Hark), cowpea (cultivar Mississippi Silver pea), sugar beet (US-20) and pigweed, although in the five last species populations tended to decline about 12 days after inoculation (Table 1). On corn (WG-4A) and lambquarters the bacterial population tended to decline on the third day after inoculation, but remained at detectable levels 18 to 21 days after inoculation, respectively; at that time leaves of lambsquarters were almost senescent. Bacteria were detected both, on the upper and lower surfaces of the leaves, although at higher concentrations on the lower surface. Pathogenicity of R15-1 isolated 21 days after inoculation, from each of the different materials under study, was tested by inoculating a susceptible bean cultivar. No change in the virulence of the isolate was observed. The increase of a pathogen in the absence of symptoms in susceptible and resistant tissue may be of epidemiological importance by serving to build up inoculum prior to infection or as a source of 20 .meoumewm oncomflc camoomouome mo monommum mouMOHccfl mflmonucmumm a .mucoEflHomxo owns» mo cmmum>m on» who moumeflumo one .wmm n .Hlmam mo cowmcommsm HS\mHHoo oaxm m nuw3 nucleon o» coumanoocfi ouoz mo>moq + “womnmm u ++ “wmsuom “soHumnoocH mo mason Nb Houmc nuzonm Hmwuouomn nufl3 oouo>oo mono unflum mood on» no wmh +++ +++..ANV Adv + + + + + ++ ++++ ++++ ++++ msxoamouumu mscucmumem + + + + + + + ++ ++++ mane asa60doemso + + + + ++ ++ ++ +++ ++++ Amaummag mummy omnmo : + + + + + + ++ ++++ Amsme.mmmv «a 03 + + ++ ++ +++ +++ ++++ ++++ ++++ Amumaoowsmcs mcma>v .m.m.z + + + + ++ ++ ++++ ++++ ++++ Axes chUSHoV xnmm + ++ ++ ++ ++ +++ ++++ ++++ ++++ Amsaaomeuoom amv some I}; “+++.v A+++V +++ ++++ ++++ ++++ ++++ ++++ Ag .3 «Joanne A+++V 1+++C iii +++ ++++ ++++ ++++ ++++ ANV++++ Ag .3 umummmmm Hm ma ma NH m o m H “so: H seaumaooocH nouwm when mmflommm .AavmacOE :memwwulmow so mucHHmImme an cocweuouoo mo mowoomm unoclcoc can umoc HcHo>om so “Himamv “Hoomcnm moccaosucmx mo mao>oa cowumHsmom o>flucaom .H wands 21 inoculum for secondary spread and, also could provide pathogen cells which survived unfavorable times. Leben (16) suggested, on the basis of work with Xanthomonas vesicatoria, that pathogenic bacteria possessed a "resident phase" in their life cycle, and this was defined as the capacity for multiplication on the surface parts of healthy tissue. Since then, several studies have confirmed that a number of bacterial plant pathogens possess a resident phase, which may be associated with leaves, buds or flowers of host and non-host plants (5, 8, 11, 13, 15, 23). More recently, Leben (17) proposed to expand the term resident to include all types of associations of microflora with healthy plants, including the surface and interior plants, above and below ground. The results obtained in this preliminary study suggest that leaves of susceptible and resistant bean genotypes and non-host plants may support epiphytic multiplication of blight bacteria and that the bacteria may possess a resident phase in its life cycle. It remains to be determined to what extent the epiphytic capability of bean blight bacteria is epidemiologically important for the disease under field conditions. 1.3 Population trends of Xanthomonas phaseoli (Xp) in bean germplasm as related to disease reaction. Because Xanthomonas blight bacteria are capable of colonizing bean plants without the production of symptoms, we decided to examdne a wide range of Phaseolus germplasm relative to their reactions to 22 Xp and as to their ability to support the population build-up of Xp. Germplasm sources selected for study (Table 2) represent important sources of resistance to diseases, insects, and nematodes; such sources are being utilized in most of the major dry bean breeding programs throughout the world (12). Six experiments were conducted under controlled conditions at the Botany and Plant Pathology Greenhouse, Michigan State University, East Lansing, Michigan, during February through June 1978. Navy bean cultivar Seafarer was used as susceptible check in all the experiments. Plants were grown in a standard soil mixture in 16 cm diameter clay pots and watered alternately as needed with.Rapid-Gro (1 teaspoon per 2 liters of water) and tap water. Temperature was maintained at 27 1.2 C and daylight was supplemented with 14 hours of fluorescent lighting. A spontaneous mutant (RlS-l) of Xanthomonas phaseoli resistant to 50 ppm rifampin obtained by conventional selective plating methods and found to possess virulence equivalent to the parental wild type (Xp 15, high virulent Michigan isolate) was used in these experiments. Inoculum was prepared from two day-old YCA (YCA: 10 g yeast extract, 2.5 g calcium carbonate, and 15 g agar per 1000 ml distilled water) cultures incubated at room temperature (24 i 1 C). Bacterial cells were washed from plates and suspended in sterile distilled water. Plants were inoculated when the first trifoliolate leaves were fully expanded by gentle spraying from a DeVilbiss sprayer to run-off with a 1.0x107 cells/m1 bacterial suspension, on the lower and upper surfaces of the leaves with no visible water soaking. 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Multiplication of R15-l was followed at intervals after inoculation during the vegetative and early reproductive stage of growth of plants, except for P, coccineus lines that did not flower under greenhouse conditions. The number of viable bacterial cells was determined from a sample of ten leaflets, randomly sampled from different inoculated leaves at each assay period, by grinding them with 50 ml of 0.01 M phosphate buffer, pH 7.2, with a mortar and pestle. After appropriate serial dilutions, suspensions were plated on YCA supplemented with 50 ug/ml rifampin and 25 ug/ml cycloheximide; colonies were counted after four days of incubation at room temperature. Populations of blight bacteria are expressed on the basis of number of colony forming units (CFU) per 100 cm2 leaf tissue (approximate average area of one leaf). In order to study systemic colonization within different bean genotypes, seedlings with primary leaves fully expanded of resistant Tepary, moderately resistant MSU-51319 and susceptible Seafarer, were inoculated by watersoaking an area of one cm diameter in the center of the lower surfaces with a 5.0x107 cells/m1 suspension of R15-l mutant. Successive leaves on the main axis were subsequently assayed for the presence of the mutant; samples consisted of 15 leaflets per replication. At the end of the assay period, sections of stems, previously surface sterilized (five minutes in 2.5% NaOCl and rinsed in sterile distilled water), were assayed following the 25 same procedure described above for leaf samples. Populations of bac- teria are expressed on the basis of CFU per gram fresh weight of tissue. The genotypes were evaluated for disease reaction on a total plant basis according to the following scale: 0.0 = no visible symptoms of the disease; 1.0 = a few blight lesions, 5% leaf infection; 2.0 = 5.-10% leaf infection; 3.0 = 10-20% leaf infection, lesions large and spreading; 4.0 = 25-50% leaf infection, many lesions coalescing; 5.0 = 50-100% leaf infection, numerous plants dead. Where statistical analysis was performed, the data were transformed to common logarithm, and analyzed as a split-plot design. Genotypes were considered as the whole plot factor and were arranged in a randomized complete block design with three replications. The sub-plot factor was time. Significant differences among treatments were estimated using least significant ranges (L.S.R.) obtained from Tukey's w-procedure (25). The data presented in Table 3 summarize the results of three experiments with diverse bean germplasm related to multiplication pattern of R15-1 and disease reaction. The results showed that in almost all genotypes, bean blight bacteria multiplied rapidly until eight days after inoculation reaching population levels of 107 to 108 cells per leaf; the only exceptions were G.N. Nebr. No. 1, sel. 27, iPI 165421, and PI 325596 in which relatively lower levels of Ioacteria were obtained. N0 disease symptoms were observed at this time in any of the genotypes, even though the leaves contained high 26 Houmm when ma can .m .H um conEcm ouoz AoumHOHHomwuu boa + mHmenmv mo>moa cmucasoocfl aaco HMOH womloa manwma> on u o canon ecu o» mcwouoooo mflncb panda Houou o so come ouoz Amov cowuomom oncomao .coflucasoocw noumm mamc om um coamecm ouo3 mm>moa coucasoocwncon aflco “GOAHMHDUOGA Ave .cmoc muccam umoe .cowuoomcw mood wooalom u m can «mcaomoamoo mcoamoa acme .coHoomMCH mama womamm u v “monoconmm can momma mcoamoa .cowuoomce u m «cowuoomcfi mama woalm u m «cofluoomcfl mood mm .m:0amoa unmeab 30m c u a «meoumewn .mcofluooaamon moan» mo mmmno>m who mosam> Amy Amy .AucnuoE mxv Humam mo cowmcommSm HE\mHaoo hoaxo.a m Suez unclean ou coucaooocw muoB ooocmmxo madam mood oumHOHHomaHu umuflm on» cuflz musoam Adv O.m hOHN¢.m OJN mcdfim.vt 0.0 oOHNNwH 0.0 :OHXm.v Hwhmmmwm o.H hSeam...“ m.o soaxo.a o.o hSxma o.o .oaxm.e mosmam Hm m.N BOHNv.m N.N oOme.H 0.0 mOHNm.N 0.0 :Ome.m Ht Odom MEMQMH4 m.o ROHNQ.M m.o oOHNm.m 0.0 ROHXm.h 0.0 :dem.m mmvaH Hm o.H hoaxe.a m.o soaxm.a 0.0 5018.6 6.0 moaxm.a assuaoH N.H NOon.h m.O mOme.H 0.0 hodxm.N 0.0 :OHXm.N wwmfiwmm OfiMCH H.o moaxm.~ o.o hoaxm.fl o.o moaxv.¢ o.o .ono.m daemon Hm m.o mOHNO.m 0.0 hOme.H 0.0 wOme.> 0.0 :OHX©.N hm .Hmm .H# .QOZ .Z.U o.m soaxm.o o.~ coaxe.~ o.o hoaxe.m o.o moaxo.a vasoma Hm O.H mOHNO.v 0.0 oOHxN.H 0.0 hOHX¢.® 0.0 :OHXm.m NHNHHM Hm v.0 ROHNm.H N.O hOHNm.m 0.0 ROHxH.® 0.0 :OHNN.m mmmMON Hm N.O boaxm.m 0.0 BOHNH.N 0.0 moaxm.m 0.0 :OHNm.H @mmmmm Hm m.o eonH.H m.o soaxm.m o.o coaxH.H o.o .oaxm.m mmooom Hm O.H wOHNm.H O.H oOHXH.m 0.0 N.O.nunm.~u 0.0 :Ome.v mam HOUMSOM o.m sonxo.a m.H soaxe.m o.o soaxo.a o.o moaxa.~ mam oonxms O.H N.OLUnm.N m.O oOHXN.¢ 0.0 N.O._U_n.m.m 0.0 :OHXm.m Nvmmv HHOGHOU MO Avvvm .MQ 0H .mQ m (MD H :cowuowsoosw.uoumo.mmdo_ EmmHmEHoO Amov coauommm oncomao + some mama So ooH\DmO Amy Amy N mccoeosumcx on EmchEnom comb ucmumwmownoooumEoc can .IuoomcH .Iommomwc macaum> mo maceuomom .Avvflaoommcm .m mam¢9 27 population levels of RlS-l. Although bacteria continued to multiply slowly, populations tended to reach a stationary phase 16 days after inoculation. R15-l was recovered from non-inoculated leaves of all of the germplasm sources, although a few or no visible blight symptoms were present at that time. Population trends of R15-l and disease reactions in inoculated trifoliolate leaves of two P, coccineus lines, as compared with susceptible cultivar Seafarer, are presented in Table 4. The analysis of variance of the data indicated significant differences at 1% level for genotype, time, and genotye/time interactions. Bacteri- al growth patterns were similar in resistant and susceptible leaves until five days after inoculation, although maximal bacterial populations were lower in the former. At this time populations in the resistant genotypes reached stationary phase and at day 15 started to decline. Populations in Seafarer continued to increase, although slowly until 15 days after inoculation, remaining stable at day 20. Statistically there were no significant differences between the two P, coccineus lines in levels of bacterial populations, at any assay period and both were significantly different from Seafarer after the fifth day. Even though relatively high bacterial concentrations were determined in leaves of the P, coccineus lines, no visible disease symptoms were observed throughout the experiment. Population trends and disease reaction in trifoliolate leaves of resistant Tepary bean and PI 207262, and susceptible Seafarer bean genotypes, are presented in Table 5. The analysis of variance 28 .onscoooumIB m.%oxpe an Hm>oa m.o n w.uc econommwc wHucmonacmfim uos mum Hopped 08mm on» nuw3 cEsHoo meow opp ca memo: « .m wanna mom soaumflnomoc How “.m.o .cofluomom oncomflo Amy .m:0epcoaamon cons» mo omnum>c mum mosam> Amy .o amp um Himam mo cowmcommsm HE\mHHoo ROHXH c cows unclean o» coumasoocfl mums mo>cwa cacaoHHomeuaHHV m.N .. O.H. . V.N.o . 0.0 0.0 .m.n n moaxv.a b coaxv.m n moaxm.a n Roaxo.m c moaxm.m Hoummoom o.o o.o 0.0 0.0 0.0 .m.o c moaxm.m c moaxm.m m ROHxH.H no Roaxm.a c moaxH.H Mmlmm 0.0 0.0 0.0 0.0 0.0 Hmv.m.o m coaxm.m c coaxm.m m Roaxn.m o GOHXm.m an moaxm.a Humv om .mHX... .. oH. m, H :oflumasoocw Hound mama omwuocow 538... mmmH «.8 ooHBmo I. .Havmomhuocom coon Hmwhoman> .m .Hmumwcomv poHumoomSm can Hmocfla mocceoooo msaoomccm .Mmlmm one Humvv ucmumwmou mo mo>moH mHMHOHHomeu cw Anemone Humamv HHommmnm mmcoaocuccx mo mucous coaumasmom .v mamas 29 .wuscooouma3 m.>mxse an Ho>oH m.o H «van ucouomwflo >Hucmoamwcmam uoc mum Manama 05mm on» cows cssHoo ween ecu :H mcmoz « .m manna mom cowumfluomoc mom "A.m.ov coHuocom oncomoflo Amy .chADmOHHmoH gonna mo ommuo>m on» one menace/AmV .0 mom on almam mo cowmcmmmsm HE\mHHoo hoard m nuwz unclcou ou poundsoocfl ouo3 mo>mma mumHoaHowaue HHV m.~ m.o o.o 0.6 0.0 .m.o .Ome.m QOHxh.m h3366 .Ome.H m mOmeH Hmummmmm ~.o o.o o.o 0.0 0.0 .m.a N.2."on hOmeH .Oon.m .onm.H n HOme.G momeom Hm 0.0 0.0 0.0 o.o o.o .m.a .Ome.H mOHxHH monmH HOer.H m .Ome.m AmuHezumeoanav m sundae o.o 0.0 0.0 0.0 o.o 1m1.m.o .onm.e mOHxNH HOme.H monmH «m .Ome.o AesoumumeoNHuav a sundae om mH 0H m H cofiumHnoocw Houmm mmmo omwuocow NORM m0 EU .m Ame m H N OOH\D o .Aavmmmmuocmm coon Huoummmomv oabwudoomsm can Hmomhom He can .m5HHOMHusom .m..ccob wummoev occumflmou mo mo>moH oumHoHHomwuu ca Huccusa Humamv “Hoommcm mmcoeocucmx mo monouu coaucasmon .m mqmds 30 of the data showed highly significant differences for genotype, time and genotype/time interactions. Differences in bacterial growth. patterns in the genotypes were evident early after inoculation. In both.of the Tepary lines, A and B, RlS-l populations increased slightly between one and five days, when maximum bacterial concentrations of 1.8x105 and 1.7x105 cells per leaf respectively, were recorded; populations then remained in stationary phase and began to decline 15 days after inoculation. 0n the contrary, multiplication patterns of RlS-l in leaves of PI 207292 and Seafarer showed a continuous increase throughout the assay periods, although the rate of increase was slower in the former, resulting in maximal bacterial populations at day 20 of 8.0x107 and 5.5x108, respectively. Statistically there were not significant differences between the two Tepary lines in levels of bacterial populations at any assay period; both were statistically different from 207262 and Seafarer throughout the experiment, except at day l; and PI 207262 from Seafarer since day 10. At 20 days after inoculation, no bacterial blight symptoms were observed on either of the Tepary beans, and only slight symptoms were observed on PI 207262. Population levels of R15—1 in leaves of resistant (Tepary), moderaltely-resistant (MSU-51319), and susceptible (Seafarer) bean genotypes, at different assay periods after inoculation of the primary leaves (seedling stage), are presented in Table 6. In Tepary, bacteria were detected only in inoculated primary leaves; in genotypes with intermediate levels of resistance, bacteria were .msoumewm omcwmflc canflmfi> mo oocmmoum oumoflccfl memocucoumm « .mcofluMOMHmou women no wmmuo>m mum moonSAmV .AHImHmV accuse ax mo coamcommSm HE\mHHoo Roaxo.m c ch3 wmoa on» mo Hoodoo ecu :H umuosnwo so 0:0 mo comm cm mcfixmomuouc3 >n commasooca mums mo>coa wumeflnm AHV moaxm.a 0.0 0.0 0.0 0.0 oucaoflaowflua cum Hwoaxm.av woaxo.m moaxm.m 0.0 0.0 mumHOHHowHHB com HmOHxO.cV AROHXN.NV Boaxoo :oaxm.m 0.0 oumHoHHowflua umH Amoaxn.mv HoOHxM.vV Aooaxa.ov QOHxH.N mOHXm.n mumeflum monommom 0.0 0.0 0.0 0.0 0.0 oumHoHHOMHHB cum 1 moaxm.a 0.0 0.0 0.0 0.0 oDMHoHHOMHHB can 3 HROHxv.Hv soaxh.a hoaxo.m :onH.H 0.0 oumHoHHOMHHB umH Asoaxm.hv «Amoaxm.av moaxm.m moaxh.H moaxo.o >HmEHHm mamamibmz moaxm.a moaxm.h soaxm.a Roaxw.a moaxm.m xumeflum Hmwsmnmcouwumv hummoe vN ma NH 0 H coaumasoocfi Houwm w>co omxuozoo HNVoSmmflu cmwum mo m\DmU .Havmomauocom coon Huoummmomv meHumoowsm can .Amamamuomzv ucnuwflmoulwaoucuoooe .Hhummoev unnumflwou mo mucmam ca auscuofi Humamv HHoommsm mocOEocucmx mo mHo>oH coHumasmom .0 mqmde 32 recovered from the primary, first and second trifoliolate leaves, as well as from internodes between primary and secondary leaves (Table 7L In the susceptible genotype bacteria were recovered from primary, first, second, and third trifoliolate leaves and from all internodes between the primary and third trifoliolate leaves. No systemic symptoms were recorded in Tepary, and only a brown necrotic reaction that sharply delimited the inoculated area from healthy tissue on the primary leaves was evident. Blight symptoms were present on inoculated primary leaves of MSU-513l9 and Seafarer, and systemic symptoms on first, and on first and second trifoliolate leaves respectively. These studies indicate that most of the germplasm.sources being utilized in bean breeding programs throughout the world may be potential "symptomless carriers" of the common blight pathogen Xanthomonas phaseoli. The data confirms previous claims that large populations of bacteria may develop in inoculated leaves of lines and cultivars with intermediate levels of resistance, although different disease reactions develop as compared with susceptible ones (3, 7, 22). Valladares 32 El (26) and Yoshii ggnal (29), reported that low populations of blight bacteria can exist in Tepary beans in the absence of visible symptoms, although no quantitative data were given to the observations. An important finding in these preliminary studies is the fact that Xp can systemically-colonize the uninoculated leaves of all germplasm sources, frequently with.the development of little or no visible disease symptoms, and that the systemic movement of the 33 .mcoHHMOHHmoHIucmHm madman o>fim scum coxmu mHoB muconv .HHImHmv banana ax mo concmmmSm HE\mHHoo oaxo.m c sues mm>moa mumsflnm on» no Hoodoo onu ca Houoenflc so one no comm so mcwxmomumum3 an ommum mcwacoom on» on coucasoocfl ouo3 magmas HHV + + + + + + + Houcmcmm I I + + + + + mHmHmIsz I I I I I I + comm humane mumHOHHOMHHB m oucHoflHOMHHB m oumHoHHOMHue H mo>moq - cum Scum com Baum umH Scum humeflum omxuocoo muucm pecan msowum> cw HImHm mo >Ho>ooom Hmv . av mom>uocom coon maneumoomsm can ucmumflmou ca mo>moa SHMEHHQ mo coflucaooocfl Houmm when «N accuse HImHmV HaoommnmImmcoeocucox mo auo>ooom .5 mamme 34 bacteria may be affected by the host genotype. Several studies on phytopathogenic bacteria have indicated that inoculum.may be available for dissemination early in the course of the disease even before symptoms are evident. This has been reported for Pseudomonas glyginea in soybeans (18), g} pruni in peach (20), and recently for Xp and pr in susceptible navy bean cultivars (27). The results presented here suggest that this may also be the case in blight resistant bean genotypes, that support an epiphytic growth of the bacteria. LITERATURE CI TED LITERATURE CITED ANDERSON, A.R., and L.W. MOORE. 1976. Survival of Agrobacterium in soil and on pea roots. Proc. Am. Phytopathol. Soc. 3: 258 (Abstr.). CENTRO INTERNACIONAL DE AGRICULTURA TROPICAL. (CIAT). 1975. Genetic improvement of dry beans, Phaseolus vulgaris and germplasm resources. Bean Production Systems Program. CIAT, Colombia, 65 pp. COYNE, D.P., M.L. SCHUSTER, and K. HILL. 1973. Genetic control of reaction to common blight bacterium in bean (Phaseolus vulgaris) as influenced by plant age and bacterial multiplication. J. Amer. Soc. Hort. Sci. 98:94-99. CUNFER, B.M., N.W. SHAAD, and D.D. MOREY. 1978. Halo blight of rye: multiplicity of symptoms under field conditions. Phytopathology 68:1545—1548. de LANCE, A., and C. LEBEN. 1970. Colonization of cucumber seeds by Pseudomonas lachrymans in relation to leaf symptoms. Phytopathology 60:1865-1866. DYE, D.W. 1962. The inadequacy of the usual determinative tests for the identification of Xanthomonas spp. N.Z. J. Sci. 5: 393-416. EKPO, E.J.A., and A.W. SAETTLER. 1976. Pathogenic variation in Xanthomonas phaseoli and g. phaseoli var. fuscans. Plant Dis. Rep. 60:80-83. FRYDA, S.J., and J.D. OTTA. 1978. Epiphytic movement of Pseudomonas syringae on spring wheat. Phytopathology 68: 1064—1067. GARDNER, J.M., and C.I. KADO. 1973. Evidence for the systemic movement of Erwinia rubrifaciens in Persian walnuts by the use of double-antibiotic markers. Phytopathology 63:1085- 1086. 35 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 36 GOWDA, 8.5., and R.N. GOODMAN. 1970. Movement and persistence of Erwinia amylovora in shoot, stem, and root of apple. Plant Dis. Rep. 54:576-580. HAGEDORN, D.J., R.E. RAND, and G.L. ERCOLANI. 1972. Survival of Pseudomonas syringae on hairy vetch in relation to epidemiology of bacterial brown spot of bean. Phytopathol- ology 92:762 (Abstr.). HSIEH, S.P.Y., I.W. BUDDENHAGEN, and H.E. KAUFFMAN.V 1974. An improved.method of detecting the presence of xanthomonas oryzae in rice seed. Phytopathology 64:273-274. KENNEDY, B.W”,and G.L. ERCOLANI. 1978. Soybean primary leaves as a site for epiphytic multiplication of Pseudomonas glycinea. Phytopathology 68:1196-1201. KLEMENT, Z. 1975. Method for the separation of closely related bacteria by gel diffusion test. Proceedings, First WOrkshop on Phytobacteriology. Univ. of Missouri, Columbia. 73 pp. LAUB, C.A., and R.E. STALL. 1967. An evaluation of Solanum nigrum and Physalis minima as suscepts of Xanthomonas vesicatoria. Plant Dis. Rep. 51:659-661. LEBEN, C. 1963. Multiplication of Xanthomonas vesicatoria on tomato seedlings. Phytopathology 53:778-781. LEBEN, C. 1974. Survival of plant pathogenic bacteria. Spec. Circ. 100. Ohio Agric. Res. Devel. Center, Wooster, OH. 21 p. LEBEN, c., G.C. DAFT, and A.F. SCHMITTHENNER. 1962. Bacterial blight of soybean: Population levels of Pseudomonas glycinea in relation to symptom development. Phytopathol- ogy 58:1143-1146. LEWIS, S.M., and R.N. GOODMAN. 1965. Mode of penetration and movement of five blight bacteria in apple leaf and stem tissue. Phytopathology 67:139-144. MILES, W.G., R.H. DAINES, and J.W. RUE. 1977. Presymptomatic egress of Xanthomonas pruni from infected peach leaves. Phytopathology 67:895—897. MOORE, L.W. 1977. Prevention of crown gall on prune roots by bacterial antagonists. Phytopathology 67:139-144. SCHAREN, A.L. 1959. Comparative population trends of Xanthomonas phaseoli in susceptibel, field tolerant and resistant hosts. Phytopathology 49:425-428. 23. 24. 25. 26. 27. 28. 29. 37 SCHNEIDER, R.W., and R.G. CROGAN. 1977. Bacterial speck of tomato: sources of inoculum and establishment of a resident population. Phytopathology 67:388—394. STALL, R.E., and A.A. COOK. 1966. Multiplication of Xanthomonas vesicatoria and lesion development in resistant and susceptible peppers. Phytopathology 56:1152-1154. STEEL, G.D., and J.H. TORRIE. 1960. Principles and Procedures of Statistics. New York: McGraw-Hill Book Company, Inc. 481 pp. VALLADARES, N., D.P. COYNE, M.L. SCHUSTER, and B. HOFF. 1977. Reaction of Phaseolus germplasm to different strains of Xanthomonas phaseoli and E. phaseoli var. fuscans. Ann. Rept. Bean Imp. Coop. 20:74-75. WELLER, D.M. 1978. Ecology of Xanthomonas phaseoli var. fuscans in navy (pea) beans (Phaseolus vulgaris L.). Ph.D. Thesis, Michigan State Univ., East Lansing, 137 pp. 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. YOSHII, K., G.E. GALVEZ-E., and G. ALVARES-A. 1978. Screening bean germplasm for tolerance to common blight caused by Xanthomonas phaseoli and the importance of pathogenic variation to varietal improvement. Plant Dis. Rep. 62:343- 347. PART II EFFECT OF HOST GENOTYPE ON MULTIPLICATION AND DISTRIBUTION OF BEAN COMMON BLIGHT BACTERIA (XANTHOMONAS PHASEOLI) INT RODUCT ION Because certain phytopathogenic bacteria can multiply not only in their natural homologous hoast but also in quite unrelated heter- ologous plants, it is difficult to define host range and host specificity. Several workers have studied the population trends of plant pathogenic bacteria in resistant and susceptible tissues. Generally pathogens multiply rapidly to high levels in host plants in which they induce disease, (compatible or homologous relationships» however pathogens multiply at the same rate but attain lower population levels at the stationary phase in non-host plants (incompatible or heterologous relationships) (16). In an early study, Allington and Chamberlain (1) reported active multiplication of Pseudomonas glycinea and Xanthomonas phaseoli in leaves of resistant bean and soybean varieties. They suggested that ability of some pathogens to live and multiply in resistant hosts, under natural conditions, may influence the dynamics of disease resistance and adaptation of pathogens to new hosts. Stall and Cook (29) related hypersensitivity in pepper to bacterial concentration; the level of Xanthomonas vesicatoria cells associated with necrosis in hypersensitive pepper tissues was lower than that in susceptible tissue. Their observation suggested that 38 39 ability of bacteria to grow and multiply is a factor in pathogenicity Grosse (8) compared leaf-surface populations of Pseudomonas morsprunorum in two cherry varieties. Populations of bacteria were consistently higher on the leaf surface of the susceptible variety than on the resistant one. Mew and Kennedy (19) reported that race specificity of Pseudomonas glycinea correlates with the resident phase of the bacteria on leaf surfaces of soybean, and P. glycinea populations increased only on susceptible leaves. The relationship between ia_yiyg bacterial populations and plant pathogenicity was studied by Young (34), who inoculated leaves of Phaseolus vulgaris with pathogenic pseudomonads and non-patho- genic bacteria, and compared rates of multiplication at stationary phase. Multiplication of the homologous pathogen P: phaseolicola suggested that inhibitory factors did not regulate the behavior of the bacteria; heterologous pathogens multiplied at lower rates and to lower stationary phase population levels, and non-pathogens remained in stasis or declined slowly. Similar results were reported by Omer and Wood (21). P, phaseolicola introduced into leaves of bean cultivars multiplied logarithmically for three to five days reaching much higher populations in the susceptible cultivar than in the resistant cultivar, where the bacteria multiplied less rapidly. Bacteria introduced into the cotyledonary node, moved more rapidly upwards than downwards, and moved rapidly and further in susceptible than in resistant bean cultivars. Few studies have been conducted on the population dynamics of Xanthomonas blight bacteria in susceptible and resistant bean 40 genotypes. Coyne gE_al (6) and Ekpo and Saettler (12),.reported that different blight isolates”multiplied'to similar levels in both, susceptible and moderately resistant Great Northern bean cultivars, although different disease reactions developed. CoynegEHEIAC6) noted that plants inoculated in the vegetative stage exhibited higher levels of resistance and lower bacterial populations than plants inoculated at the reproductive stage. The importance of studying the reaction of the plants at different stages of development and at different intervals after inoculation, was emphasized. Almost all of the studies relative to population trends of phytopathogenic bacteria have looked only at multiplication of the organism in and/or on inoculated susceptible and resistant tissues. No comparative study has been conducted on bean blight bacteria relative to the movement and distribution of the pathogen in susceptible and resistant genotypes. This investigation compares multiplication and distribution patterns of Xanthomonas phaseoli in bean genetypes possesSing different levels of disease resistance. MATERIALS AND METHODS The experiments were conducted under field conditions at the Botany and Plant Pathology Research Farm, Michigan State University, East Lansing, Michigan, during the summers of 1977 and 1978. Host Genotypes Common blight susceptible navy bean cultivars Seafarer and Tuscola; moderately—resistant MSU-513l9 (MSU breeding line) and G.N. Valley; and, resistant Tepary beans (Phaseolus acutifolius), P597 (CIAT) and Arizona-Buff, were used throught the studies. Bacterial Isolate A spontaneous bacterial mutant resistant to rifampin was isolated from cells of Xanthomonas phaseoli isolate 15 (highly virulent Michigan isolate) growing on YCA (YCA: 10 g yeast extract, 15 g agar and 2.5 g calcium carbonate per 1000 ml glass distilled water), supplemented with 50 ug/ml rifampin. R15—1 mutant was morphologically, pathologically and serologically indistinguishable from wild-type rifampin-sensitive Xp 15. The mutant retained its rifampin-resistant phenotype after repeated subculturing on YCA in the absence of rifampin and retained pathogenicity as tested by host inoculations. 41 42 Bacteria were grown on YCA and stored in dry infested tissue at 4 C and in 40% v/v aqueous glycerol at -10 C. Experimental Plots Disease—free seeds of the different host genotypes were planted by hand in three-row plots of 3‘m length with 50 cm between rows. There were three replications of each treatment in all experiments. Inoculation Technique Suspensions of R15-1 were prepared from 48-hour-old cultures on YCA plates by rinsing the bacteria off the agar surface and suspending in sterile distilled water. Plants were inoculated by gentle spraying the lower and upper surfaces of primary leaves, trifoliolate leaves or pods to runoff with a Knapsack sprayer, using a bacterial suspension Containing l to 5.0x107 cells/m1. Leaves or pods were not watersoaked during inoculation. This procedure deposited about 0.021 ml inoculum/cm2 of leaf area as determined by weighing leaflets of known area before and after inoculation. Isolation Procedures The number of viable bacterial cells (CFU) was determined on samples of 21 inoculated trifoliolate leaves or 21 pods, randomly sampled from each replication at each assay period by lightly shaking the samples in 100 m1 of 0.01 M phosphate buffer, pH 7.2 (surface populations) and by homogenizing in the same amount of buffer in a Waring blender (internal populations). After appropriate serial dilutions, suspensions were plated on YCA supplemented with 43 50 ug/ml rifampin and 25 ug/ml cycloheximide; colonies were counted after four days of incubation at room temperature. Bacterial multiplication and movement within different bean genotypes, were studied in inoculated seedlings possessing fully expanded primary leaves. Successive leaves on the main axis were subsequently assayed for the presence of the mutant; samples consisted of 15 leaflets per replication. At reproductive stage (well filled-plump pods) five single plants from each replication were taken and assayed for the presence of the mutant in roots and stems, following the same procedure used for leaves and pods; tissues were previously surface sterilized (5 minutes in 2.5% NaOCl) and rinsed in sterile distilled water. Populations of blight bacteria are expressed on the basis of number of colony forming units (CFU) per 100 cm2 leaf tissue (approximate average area of one leaf) or per 10 cm2 pod tissue (approximate average area of one pod). Evaluation of Disease Reaction The genotypes were evaluated for disease reactions on both a foliage and on a pod basis. For foliage reactions: 0.0 = no disease; 1.0 = a few blight lesions, 5% leaf infection in the row; 2.0 = 5-10% infection in the row; 3.0 = 10-20% leaf infection, lesions large and spreading; 4.0 = 25-50% leaf infection, many lesions coalescing; 5.0 = 50-100% leaf infection, numerous plants dead. For pod reactions: 0.0 = no pod lesions observed; 1.0 <10 pod lesions in the row; 2.0 = >10 pod lesions in the row; 3.0 = >10 44 pod lesions in the row and visible infection of upper and lower suture. These rating scales have been used by Saettler and Adams (24) in evaluating breeding lines for Xp and pr resistance. Statistical Analysis The data were transformed to common logarithm, and analyzed as a split-plot design. Genotypes were considered as the whole plot factor and were arranged in a randomized complete block design with three replications. The sub-plot factor was time. Significant differences among treatments were estimated using least significant ranges (L.S.R.) obtained from Tukey's w-procedure (30). RESULTS Multiplication of Xp in and on LeaveS'and Pods of Resistant, Moderately-Resistant and Susceptible Bean Genotypes Leaf_populations, 1977 Population trends of Xp in and on leaves of moderately- resistant MSU-51319 and susceptible Seafarer cultivar, are shown in Figure 1 and Table 1. Population trends resembled a typical bacteri- al growth-curve with a three-day lag phase; six-day logarithmic or exponential growth phase with a mean generation (or doubling) time of 17.1 and 15.1 hours for MSU-513l9 and Seafarer respectively; and, a stationary phase where bacterial populations remained stable or declined slowly (Fig. 1). Surface populations of R15—1 followed a pattern similar to total populations and ranged from 7.6 to 26.1% and 2.6 to 18.7% of the total population in MSU-51319 and Seafarer respectively. Although maximal bacterial populations were lower in the moderately-resistant MSU-513l9, primarily during flower stage of development (Table 1), analysis of variance indicated no significant differences for genotype and genotype/time interactions. In both.genotypes, symptoms developed during the flower stage of development, about the time when maximum bacterial populations per leaf were obtained. Disease in MSU-51319 developed later and to a 45 FIGURE 1. 46 Population trends of Xanthomonas phaseoli (R15-l mutant) in and on trifoliolate leaves of moderately- resistant (MSU-51319) and susceptible (Seafarer) bean genotypes. Twenty-three day old plants (3rd and 4th trifoliolate leaves) were inoculated to run-eff with a 1.0x107 cells/m1 suspension of R15-1 at day 0. Values are average of three replications. 47 zo_._.<.._aooz_ much—4 m>ccE .:0HuoowcH wmoH womImN H o.v HOGHomonm can omHoH mconoH .coHuoowcH mmoH wONIOH n o.m Hzou or» :H COHuoomcH mmoH wOHIm n o. ocu :H GOHuoomcH mooH mm .mconoH uanHn 3ow m u o.H Homooch oz u 0.0 ":oHuomom omoomHo .nu3ouw wo ommum N “30H ads: I 0 m5 .0 Sop um HImHm mo concommsm HE\mHHoo HIOonH m :uH3 MNOIGSH on poucHsoocH onoz Hmo>noH oumHoHHOMHHu :uv cam puny mDGMHm oH0I>mc mm Amy .mcoHumoHHmou ooucu mo owmuozm one moHHHmSHHv o.m m.~ o.H 0.0 0.0 0.0 0.0 .m.o Hoson Hoonm Hoson com o>Humuomo> o>Humuomo> o>Humuomo> .o.m AOHXO.m hOmeH honm.m sOHxH.m hOmeH :Oon.m :OHXO.v Hence 8 mono.m oono.H GOHxn.v GOwa.m SOonH HOme.m mOme.m oommusm 4 monommom m.o o.o 0.0 0.0 0.0 0.0 0.0 va.m.o Hoson Hoson Hoson com o>Huouomo> o>Humuomo> o>Humuomo> Hmv.o.m GOon.n GOHNH.o oonm.m ROme.N oOHxv.m :OHxH.m :Ome.v Hmuoe nOme.m BOHxN.H GOHxv.H GOon.o mOHxN.m HOHXR.v m352m ooowusm mHmHmIsz mH mH NH 0 o m H AchofionDoocH Houmo mama om>uocoo coho ooH EU Dmo HHC m m 00H\ .mom>uo:om coon Huoumwcomv poHumoomow can HmHMHmIszV ucmbmHmouISHoumuoooe mo mo>moH ouMHoHHowHHu :0 can :H Abacuse HImHmV HHoooocm mmcoeocucmx mo mucouu cOHHMHsmom .H mamas 49 lesser degree than Seafarer. Population trends in and on leaves of P597 and Seafarer were similar until 16 to 20 days after inoculation (Fig. 2). ‘Maximum bacterial populations were recorded in the resistant genotype at 16 days after inoculation, populations then gradually declined; in susceptible Seafarer populations continued to increase. There were statistically significant differences in levels of total bacterial populations between genotypes at the end of the assay period, when plants were at the reproductive stage of development (Table 2). Xp populations on leaf surfaces of both genotypes varied throughout the experiment, ranging from 4.3 to 27% of the total population on P597 and from 1.0 to 16.7% on Seafarer. There were no visible disease symptoms on plants of P597. Pod populations, 1977 Population trends of.Xp in and on pods of MSU-51319 and Sea- farer are shown in Figure 3 and Table 3. Analysis of variance indicated significant differences for time, genotype and genotype/ time interactions. Bacterial growth patterns (Fig. 3) in both genotypes exhibited four-day lag phase, after which the patterns differed. In MSU-51319 Xp exhibited a four-day exponential growth. phase with a mean generation time of 23.7 hours; bacterial cell concentrations reached the maximum level at eight days after’ inoculation, after which.there was a stationary phase and a SlOW‘ decline in populations. In the susceptible genotype, bacteria remained in an exponential growth phase eight days; the bacteria entered a stationary phase at 12 days after inoculation and FIGURE 2. 50 Population trends of Xanthomonas phaseoli (R15-1 mutant) in and on trifoliolate leaves of resistant (P597, P. acutifolius) and susceptible (Seafarer) bean genotypes. Thirty-six day-old plants (3rd and 4th trifoliolate leaves) were inoculated to run-off with a 1.0x107 cells/ml suspension of R15-1 at day 0. Values are average of three replications. 51 29.2.5002. much; m>oxnfi >b Ho>oH mo. "mu no ucoHoMMHc no: ouo HouuoH oeom on» cuH3 cEsHoo oeom on» cH mcooza .H pooB oom coHumHHomoc mom "soHuooom omoomHo u .m.o Hey .U.m . uzono o omou s m m Hmv .o moo uo HImHm mo concommsm HE \mHHoo H.Oon..H o SHHB MMOIcoH ou oouoHsoocH oHoB Amo>ooH ouoHoHHOMHHu too too cwmv mucon GHOIhoo mmHNV .mGOHuooHHmou oouca mo omouo>o ouo moHHHo>HHV N.m N.m m.~ m.H 0.0 o.o 0.0 0.0 .m.o com com com uon uoson pom o>Huouomo> o>Huouomo> o>Huouooo> .w.m QROme.N o N.OHHGH o GObe.m o monh.m o GOme.H o mOon.H o :Oon.o b :OHxv.m Hobos GOme.m oOmeH mOHxhé mOHRHn mOmeH mOHRNK HOon.m m0on.m ooowusm Houomoom 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 va.m.o com com uon Hoonm com o>Huouomo> o>Huouomo> o>Huouomo> o>Huouomo> Hmv.u.m oGOHxv.m o GOon.h o mOerd o o353m o mOme.m o mOmeH o :Ome.m «o mOme.m Houoe mOmeH mOme.m SOHxv.m mOHXvn mOmeuH :Ome.H mOHxH.m mOme.m ooomnsm some coHuoHooocH Houmo whoa .momwuocom coon Huouomoomv poHumoomom coo AmsHHOMHusoo am .hmmmv ucoumHmoH mo mo>ooH omoHoHHOMHHu co coo :H Honouss HImHmv HHoonocm mocosocucox mo meson» cOHuoHsmom .N names n FIGURE 3. 53 Population trends of Xanthomonas phaseoli (R15-1 mutant) in and on pods of moderately-resistant (MSU-51319) and susceptible (Seafarer) bean geno- types. Pods (flat-pod stage) were inoculated by gentle spraying to run-off with a 1.0x107 cells/ml suspension of R15-l at day 0. Values are average of three replications. 54 zo_._.<._=ooz_ mu......._< m>oH mo. "yo no ucoHoMMHo no: oHo HouuoH oeom on» cqu cesHoo oeom onu cH mcooz« .ouousm com Momma mo :OHuoomcH oHnHmH> coo 30H ocu :H mconoH com OHA n o.m «Sou ocu :H mconoH com OHA u o.m “sou oau :H meHmoH com OHw u o.H Hoo>womno mSOHmoH moon on u 0.0 "GOHuooom omoomHo n .m.o Hey .nuzouo mo mmmum u .o.m1mv .0 Sop no HImHm mo cowmcommsm HEKmHHoo hOonH o nqu mmoIssu ou mcHonmm oHucom SQ touoHsoocH oHoB Homoum com uonv muomamv HHV mooHuoOHHmoH oouzu mo omouo>o ouo mosHo> 0.0 I 0.0 0.0 0.0 0.0 .m.o com mfisHm coHHHMIHHoS pom cooum meon com cooum meon com cooum uon com coon uon .o.m n monH.N a oOme.H o mOme.m o :onm.H o :Oon.m Hobos moHNm.m mOme.H mOme.N :onm.H :Oon.H ooomuom Houomoom m.o 0.0 0.0 0.0 0.0 va.m.o com meon coHHHMIHHoB pom sooum mEon com coowm meon com cooum uon com coonm uon Hm .o.m o :oHNH.m o :Ome.m o :Ome.m o HOHxH.m «o :onm.~ wouoe :onH.m :onv.m :Oon.o «Ome.m :onm.H ooomunm mHmHmIsz 9H NH , . w v H :OHuoHooocH noumo whoa omwuocoo mmufl 0% EU .ommuocom coon HHoHomoomv oHnHumoomsm oco HmHmHmIszv ucoumwmou I>Hououooofi mo moon :0 poo nH Hucouse HImHmv HHoomocm mocoeocucox mo mucouu coHuoHomom .m names 56 populations continued to increase slowly. There were statistically significant differences in population levels between the genotypes, at 12 and 16 days after inoculation (Table 2). Surface population of Xp on pods of MSU-513l9 were similar to the total populations, ranging from 60% to 70% throughout the assay period of the total populations. In susceptible Seafarer, surface populations ranged from 61.5% at day one to 86.2% at day eight and then decreased to 28% of the total population at day 16. Symptoms were first observed in Seafarer at 12 days after inoculation; disease symptoms in MSU-513l9 were observed only at the end of the assay period and to a much lower degree (Table 3). Leaf_p9pulations, 1978 Multiplication of Xp in and on leaves of resistant Tepary (Arizona-Buff), moderately-resistant G.N. Valley, and susceptible Tuscola bean genotypes, are shown in Figure 4 and Table 4. Analysis of variance of the data indicated highly significant differences for genotype, time and genotype interactions. Differences in bacterial growth patterns in the genotypes were evident shortly after inoculation (Fig. 4). In Tepary bean, Xp populations increased slightly during the first days after inoculation and then the populations remained in stationary phase throughout the rest of the experiment. In G.N. Valley bacterial populations showed a four-day lag phase, then increased exponentially for eight days with a mean generation time of 15.2 hours, reached maximum levels at day 12, after which the bacteria entered a stationary phase. No lag phase was evident in Tuscola; bacterial FIGURE 4. 57 Population trends of Xanthomonas phaseoli (R15-1 mutant) in and on trifoliolate leaves of resistant (Tepary, Arizona—Buff), moderately-resistant (G.N. Valley) and susceptible (Tuscola) bean genotypes. Twenty-five day-old plants (2nd and 3rd trifoliolate leaves) were inoculated to run-off with a 1.0xlO7 cells/ml suspension of R15-1 at day 0. Values are average of three replications 58 20:44:00.5. mmhud m>0H m.o H W.Ho ucouommwc #0: ohm HouuoH oEom onu SUH3 GESHOO oEom osu.cw mcooza .H oHuoB oom coHumHuomoc How "coHuooom omoomHo n .m.onC .cusouo mo omoum n .0. mg3 oo>ooH couoHsoocH SCAM ooHQfiom oommHu owo monEom Hocuo HHm .mo>ooH couoHooocHIcoc scum monEom osome monoOHccHHmv .o aoc uo HImHm mo mGOHmcommoo HS\mHHoo Oon. H o squ MMOIcou o» couoHooocH ouo3 Hmo>ooH ouoHOHHOMHHu cum coo ccmv mucon cHOIhoc mNHNv .mGOHuoOHHmoH oounu mo omouo>o oHo mofiHo>AHv m.m m.m o.m .,w. e.~ , ~.H . 0.0. ‘ .o.o: _ ‘ o.o o.o , .m.o com com com HHoEm Hoonm com .mo> .mo> .mo> .mo> .0.m hOme.c oOHNm.v sonm.m caonm.N coonv.H OoOHx>.H OQOme.H QGOme.H b30Hxh.m Hobos o0me.v H.OHHHHH AOHNH.H honH..m mOme.m son~.N bOmeH m0me.H HOHx~.m ooomusm oHoomsB m.H N.H m.o m.o o.o o.o 0.0 0.0 0.0 .m.o com com com HHoEm Hoonm onm .mo> .mo> .mm>. .mm>_ .o.m m1QHSHH .m aQHSHH .c eOHHHH . m boonm . m boonc . m 3.0me . H £1.0me . m obem . H £9.0on . H Houoe mOon.H mOme.o mOme.m mono.m mOon.v uOHxN.m mOme.c mono.m HOme.H ooomwnm smHHm> .z .o o.o o.o o.o o.o 0.0 6.0 0.0 o.o o.o Hm1.m.n oomHHmam HosoHe umsoHn cam .mm> .mm> .mo> .mm> .mm> va.o.m o.o o.o HOon.Hv m.onm.m m.one.m m.onH.e m.Ome.m n.0me.H .mHOme.m Hence o.o o.o Hono.Hv .onv.~ Nonm.m Honm.m HOme.H Nonm.m HOon.H momousm HmmsmIocouHuev suomoe 14ml isms HAHNC om SH. NH m a H coHuoHsoocH Houmo mmoo ommuocow Ame ooum ooH So an 1H1 o m ooH\ o .momhuocom coon HoHoomsev oHnHumoomsm cco AhoHHo> .z.o. ucoumHmoquHououocoE .kuomoev acoumHmon chch Mo mo>ooH ouooHHomHHu so cco cH Hucouoe HImHmV HHoomocm mocoeocucox mo occonu cOHuoHsmom .o mamas 60 population increased exponentially until eight-days after inoculation, with a mean generation time of 16.4 hours, and then populations attained stationary phase. There were statistically significant differences in population levels between the genotypes throughout the assay periods, as shown in Table 4. Bacterial populations detected at the surface of the leaves showed growth patterns similar to total populations, ranging from about 1% to 7.5% on Tepary, 15% to 28% on G.N. Valley, and from 6.6% to 15.0% on Tuscola of the total population. Bacterial populations detected in and on uninoculated leaves of G.N. valley and Tuscola were similar to those previously found in and on inoculated tissue. Practically no bacteria were detected in non-inoculated leaves of Tepary bean. Also, Xp was found only on ‘pod samples of G.N. valley and Tuscola at 1.0x103 and 1.8x104 CFU per pod respectively. Disease symptoms were initially recorded in Tuscola at the early flower stage of plant development; symptoms in G.N.‘Valley developed later and to a lesser degree, and no symptoms were observed in Tepary (Table 4) . Pod populations, 1978 Population trends of Xp in and on pods of the three genotypes are presented in Figure 5 and Table 5. Analysis of variance indicated significant differences at 1% level for genotype, time, .and genotype/ tjnua.interactions. Bacterial growth patterns were similar in G.N. Valley and Tuscola, although levels of Xp populations were (generally- lower in the former (Fig. 5). After a four-day lag phase, bacteria FIGURE 5. 61 Population trends of Xanthomonas phaseoli (R15-l mutant) in and on pods of resistant (Tepary, Arizona-Buff), moderately-resistant (G.N. Valley) and susceptible (Tuscola) bean genotypes; Pods (flat-pod stage) were inoculated by gentle spraying to run—off with a 1.0xlO7 cells/ml suspension of R15—l at day 0. Values are average of three replications. 62 29.2.5002. cub“; «>40 R 0 modes»: lllllll IOI .I O I_<._.o._.u._3<>l A. D NOflum 3 ”I I 38.8.51... m r _ L L vaav 00d 3W9 0| 83d “:19 90') 63 I3 m.smxae an Hm>mH m.o .ruzono mo ommum n .o.m1mv oHuoom an couoHooooH ouoz Homoum com uonV mcom Hmv .ouocoooum ~o uo uooHoMMHc uoo ouo HouuoH oeom on» ouH3 oeoHoo oeom on» oH moooz« .m pooB oom oOHumHHomoc Mom "oOHuooom omoomHo .o >oc no HImHm mo oonoommSm HE\mHHoo ROon.H o o9H3 MMOIooH ou mloono .mooHuooHHmoH oouou mo omouo>o oHo mooHo> u .m.aivs HHV m.~ m.o 0.0 0.0 0.0 .o.o com mEon coHHHMIHHoB com oooum meon com oooum mEon com ooon uon com oooum uon .O.m o H.OHonm.~ o H.OmeH o mOmeH. n mOHxN.m o :Ome.b Houoe GOHXH.m GOon.v oOon.N nOHxNH :OHxH.c ooomnom oHoomoe «.0 0.0 0.0 0.0 0.0 .m.o com mafiHm coHHHMIHHoB com oooum maon com ooon mEon com oooum uon com ooon uon .O.m n GOme.N In BOme.H a GOHXM.H o :Ome.m o :Ome.m Houoa . mono.N SOHN~.H mOch.c :OHx~.v :Oon.h ooomuom smHHo> .z.o 0.0 0.0 0.0 0.0 0.0 va.m.o com mEon coHHHmIHHoB com oooum QESHm com oooum uon com oooum uon com oooum pon Amy .U.m o HOHNH.~ o HOme.m o :onh.m o :OHxH.m «o :Ome.N Houoe HOme.H MOHxn.v :Oon.m :Ome.H :Och.m ooomuom whomoe ngooHuoHooooH Houmo mxoo oouo om So 5 HHS c m 0H\ mo .momhuooom ooob HoHooooHC oHnHumoooom coo H>oHHo> .z.ov uooumHmothHouoHocoe..HmmomIooONH uooumHooH >HomHo morocco o0 coo oH Hboouoa HImHmV HHoomoom moooeoouoox mo mcoouu ooHuoHomom .m mqmoe no woomoev 64 multiplied exponentially for four days with mean generation times of 21.2 hours and 19.0 hours for G.N.‘Valley and Tuscola respectively. At day eight, Xp populations entered stationary phase in G.N. Valley, but continued to increase slowly in Tuscola. Bacterial population growth in Tepary bean remained in stationary phase, and started to gradually decline eight days after inoculation. There were statistically significant differences in bacterial populations between the genotypes, beginning four days after inoculation (Table 5) . In Tepary and G.N. Valley genotypes leaf surface populations of Xp represented a high percent of the total populations ranging from 71.4% to 81% and 50.8% to 84% respectively. In susceptible Tuscola genotype, leaf surface populations of Xp were high until day four but represent less than 30% of the total pOpulation for the remain- der of the experimental period. No Xanthomonas blight symptoms were observed on pods of Tepary bean; only few blight lesions were seen on pods of G.N.‘Valley at the end of the experiment. Multiplication, Movement and Distribution of Xp in Resistant, Moderately-Resistant and Susceptible Bean Genotypes Multiplication and movement of Xp (R15-l mutant) were studied in inoculated seedlings of the different bean genotypes, throughout the growing season of 1978. Population levels of Xp from primary leaves until early reproductive stage, representing bacterial multiplication on individual leaves as differentiated from the main axis, and symptom 65 expression, are presented in Table 6. In the resistant Tepary genotype, Xp was detected consistently only in inoculated primary leaves. In moderately-resistant G.N.‘Valley, bacteria were recovered from primary, first, second and 3rd and 4th trifoliolate leaves. In susceptible Tuscola, bacteria were isolated from.primary, first, second, 3rd & 4th, 5th.and 6th trifoliolate leaves. High.bacterial populations and symptoms were detected first in the older leaves and later in the younger ones, from the primary to the second trifoliolate leaves in G.N. Valley and from the primary to the fifth trifoliolate leaves in Tuscola. No symptoms and low levels of bacteria were detected in inoculated and first trifoliolate leaves in resistant Tepary. Bacteria systemically colonized stems of G.N. Valley and Tuscola plants but not of Tepary bean (Table 7). Xp populations recovered from mature plants of G.N. Valley and Tuscola were 1.2x102 and 6.5x104 CFU per g of stem tissue respectively; no visible evidences of infection were observed on stems of either genotypes. Bacteria were recovered only from the roots of TUScola, althOUgh not consistently and at low population levels. All attempts to isolate Xp from flowers of the different genotypes throughout the growing season were negative. Xp bacteria were recovered only from the pods and seeds with visible symptoms of the Tuscola and G.N. valley varieties. 66 .mEOumE>w omooch onoomOHooE mo oooomoum ouooHcoH mHmoruoouom« comHomno n no .HImHm mo oonoomwsm HE\oHHoo sono.H o nqu monoou on mo>ooH >HoEHHm on» moH>onm oHnoom >n couoHsoooH oHoB Homoum moHHcoomv ouoon cH0I>oc vHHNv .mooHuooHHooH oounu mo omouo>o ouo mooHo> HHS Amy .beh.H HOHx>.® 0.0 I I I I I I I .MHHH nuo Ccme.Hv:onm.H mono.s ~0HXh.v I I I I I I .wHuu cum fbeo.H:sonm.Hv monv.H non~.m HOHxH.N I I I I I .wHuu too a cum fibeo.H:n0Hxh.mvHpOon.vC BOme.H monm.v monHH I I I I .MHHu com no H 0Hx~.mCH ono.vCH OHXN.NV onm.m ono.H OHXm.m onh.o I I ouoHoHHowHuu A A s A A w m umuHm no no no A Owa.HVH Och.mv onm.H ono.m onm.v OHXm.m onm.H mo>ooH a A n o a m : whoEHHm oHoowoB tme.H 0.0 0.0 0.0 0.0 I I I I I .wHHu one o cum Hfime.mvmonm.v :Oon.m monmH :onv.m :Ome.s I I I I .wHuu com be H onm.Hv onm.e Oon.H Oer.m OHxN.H Ome.o OHxv.o I I mumHoHHooHuu n a m a u : m umHHm no no no A onw.HVL Oon.mv Ome.~ Ome.m OHxH.H Ome.m onH.m mo>ooH m m w m m : m 395”an soHHo> .z.o no 0.0 0.0 0.0 ono.Hv Oon.Hv 0on.H Oon.H I I ouoHoHHOMHuu H H H H UmHflm no no no ono.H onH.m onv.m onw.H Ome.m Ome.m Ome.m mo>ooH Amy 3 3 o o m m H whoEHHo xuomoe vm Om mm mm mH VH 0H 5 v H ooHuoHsoooH Houmo ozoa om>uoooo .mom>uooom coon HoHoowoBV oHnHumoomsm coo H>oHHo> .z.0v nooumHmouIzHououocoe .HmmomIooooHuo >Hodoev uooumHmoH oH Huoouoe HImHmC HHoomocm moooeocuoox mo mHo>oH ooHuoHsmom .0 moods 67 XIII!!! Ii ‘ .msoume>m uanHn mHnHmH> nufiz mommm cco mvom Eoum omnm>ooou mace oflumuoomavV .mfimum omNHHfiuwum mooMHSm scum umuoaomw ofiumuoomnmv .mcofluoofiammulucoam onqwm m>Hm Bony cmxou mums ouoanmv .HImHm mo cowmcmmmsm HE\mHHmo oaxo.H o nuns mmoIqsu ou mo>mmH anmsflum on“ mafiamnmm «Hanan an omumasoocfl mums Ammmum maflflomwmv muamam oHOImmo «A AC + + I + + + + + + + + 3083. + + I + I I + + + + I >o-o> .z.w I I I I I I I I + + I anomoe mommm moon“ muw3on mfimnm .mwuu .mwup .mwuu npo .manu .maun mm>owH Avv . HMV HOOK AHA nuo num w «in cam umufim angina o noan Amvoflooz :fimSoMflmImuw no nuzouu Hofiumuoom .va mommuocmm coon AoHoomsav oHnwumoomsm cco A>maao> .z.ov ucoumwmmHImHououwvoa .Aauo may ucoumflmwu mo mm>owH whoefium on» no :ofluoHnoocfi mcfl3oHHom munom ucoam msoHHo> Eoum Aucousa HImHmV HHoomonm,mo:OEonu:ox mo >Ho>oomm .5 mqmoe DISCUSSION Multiplication and distribution of Xanthomonas phaseoli (RlS-l rifampin—resistant isolate) in resistant, moderatelybresistant and susceptible bean genotypes during vegetative and reproductive stages of growth of the plants, were studied under field conditions during the growing seasons of 1977 and 1978. In general, growth curves in and on leaves of susceptible and resistant bean genotypes resembled a typical bacterial growth curve, with a three to four-day-lag phase, followed by a logarithmic or exponential phase that varied in the different experiments between four and 12 days, and a stationary phase. Mean generation times during exponential growth ranged from 15.1 to 21.2 hours in leaves and from 23.7 to 36.6 hours in pods. While bacterial growth patterns were similar in and on leaves and pods of moderately-resistant and susceptible genotypes, maximum bacterial populations were generally lower in the former, particularly during the reproductive stage of plant development. At this time, bacterial growth. in resistant genotypes showed an abrupt termination of the exponential phase, the Population then declined and remained stable. In susceptible genotypes, bacterial growth continued to increase but at a lower rate. 68 69 Growth patterns of Xp in tissues of resistant and susceptible genotypes, in general agree with those reported for this (6, ll, 25) and for other phytopathogenic bacteria (4, 8, 10, 16, 19, 29), where pathogen populations increase after inoculation irrespective of the host genotype, but the increases are less in the resistant than in the susceptible tissues. However bacterial growth.patterns in Tepary ArizonavBuff were different in that Xp was able to survive in inoculated tissues for relatively long periods of time and remained at stationary phase or declined only slowly after inoculation. While it has been previously suggested that Xp may grown in Tepary bean (31, 33), this is the first report that shows population trends of the bacteria under field conditions. Population trends of blight bacteria on the surface of resistant and susceptible bean leaves were similar to total populations and except for Tepary Arizona-Buff, large numbers of bacteria were available for dissemination early in the infection process; The behavior of blight bacterial populations at the surface of pod tissues was noticeable; in resistant genotypes, epiphytic bacteria averaged greater than 65% of the total population throughout the assay’period. In blight susceptible genotypes, most of the bacterial p0pu1ations were internal after 12 days. IBoth external and internal factors have been suggested to affect multiplication of bacteria in and on leaves and pod tissues of resistant cultivars L7, 9, 17, 19). On the other hand, independent genetic control of the differential reaction of foliage and pods of 7O bean to Pseudomonas phaseolicola (l4) and to Xanthomonas phaseoli L5, 7) has been reported. The importance of obtaining resistance in both. leaves and pods to these pathogens has been emphasized (6). Our results clearly indicate that even though.large populations of Xp grew in and on inoculated leaves and pods of moderately— resistant bean genotypes, the disease reactions on the resistant genotypes were less than those on the susceptible genotypes. The visible appearance of blight symptoms in the genotypes coincided closely with the transition from exponential to stationary phaSe of growth for the blight bacteria and after a minimal bacterial population was reached; however the incubation period was longer in resistant than in susceptible tissue. High Xp populations were also detected in non-inoculated symptomrfree leaves of both blight susceptible and moderately-resistant genotypes, which suggests that Xp may possess a 'resident phase' of growth in both. Such a growth- phase suggests that inoculum for secondary spread of Xp may occur in the absence of visible disease symptoms. No visible Xanthomonas blight symptoms were ever found on leaves of pods of Tepary beans during this study, results that agree with previous reports (31, 33). According to Lyon and Wood (18), after bacteria enter leaves through stomata, the populations may: 1) increase little if at all, and the leaf tissue is apparently not damaged, at least macro— scopically; 2) increase, but with no visible damage to the leaves; .3) increase over a short period and then decrease or remain more <>r less constant. This is associated with death of protoplast in an acute local reaction of tissue containing the bacteria, the 71 'hypersensitive' reaction; 4) number of bacteria increase over a period considerably longer than in 3) and reach higher levels, visible damage to leaves is delayed but damage is much more extensive, the typical susceptible reaction. In this study we simulated natural conditions of infection by gently spraying the inoculum.to runoff onto leaf surfaces, without wounding or internal soaking of the tissue. Under these conditions, the behavior of the bacteria in Tepary bean would fall into the alternative 2) described by Lyon and Wood. Nevertheless, we also observed in previous greenhouse experiments (Part 1, 3.3), that a characteristic hypersensitive reaction was produced when blight bacteria were infiltrated (watersoaked) into leaf tissue of Tepary bean. Under these circumstances, alternative 3) reflects the interaction. The behavior of Xp in genotypes with intermediate levels of resistance, suggests an alternative explanation. Even though bacteria increased exponentially over a period and reached high population values, the.maximal bacterial populations were lower than in blight susceptible genotypes and disease symptoms were clearly delayed. This interaction appears to be different from the typical susceptible reaction suggested in alternative 4) above. Little is known about the factor or factors that prevent loacterial multiplication in the absence of visible responses or how~ Ioacteria can multiply and not cause visible symptoms. Understandably, jpathologists have been more concerned with the susceptible reaction and with the visible damage of the hyper-sensitive reaction associated 72 with.resistance (18). According to Young (34), theories to account for the different behavior of bacteria have particular characteristics which allow them to multiply in plant tissue (23), and those in which preformed inhibitors in the plnat (15) or a post-infection reaction by the plant selectivity inhibits bacterial multiplication (20, 26). More recently, a phenomenon of attachment and envelopment of incompatible and/or saprophytic bacteria by plant cell walls has been reported in several systems (13, 22, 27, 28) and has been suggested as a major host defense mechanism against bacteria (27, 28). Than Xanthomonas phaseoli may move systemically in infected bean plants was previously reported by Barlow (2), Burkholder (3). and Zaumeyer (35, 36). Burkholder suggested that such systemiC‘movement may be affected by environmental conditions and by the host plant. Recently Weller (32) determined that all above and below~ground portions of seedlings grown from internally infected seeds were colonized by the blight bacteria immediately after germination. Spread of the bacteria in the expanded leaf canopy was facilitated by rain, bud colonization, and systemic movement. According to Weller, the overall rate at which bean plants are colonized is strongly affected by the growth rate of the bacterial population on each infected leaf. Our preliminary greenhouse experiments (Part 1, 3.3), indicated that systemic colonization of bean plants by Xp was affected by host genotype. The present results obtained in field studies further support these previous findings and are consistent with the general pattern of bean blight colonization of susceptible plants as reported 73 by Weller (32). Patterns of Xp colonization of moderately- resistant genotypes were similar to those in susceptible genotypes, although in the moderately-resistant genotypes the bacteria moved at a slower rate, levels of inoculum for systemic and rain-splashing spread were lower, and the bacteria were recovered closer to the primary site of infection. In the resistant Tepary genotype, bacteria were consistently detected only in the inoculated primary leaves and no colonization occurred beyond this point. Additional studies are necessary to determine the mechanisms involved in Tepary which limit the multiplication and spread of blight bacteria. The detection of high bacterial populations in moderately- resistant commercial cultivars may be important to bean breeders and seed producers. Hertofore, plant pathologists and breeders have selected for disease resistance on the basis of symptom development and severity. Bean breeding programs directed to the development of Xanthomonas blight resistance should include tests to monitor the leaf, pod, and seed population levels of the pathogen. Tepary bean (Phaseolus acutifolius) continues to be the best source of blight resistance presently available; certain accessions possessed the highest foliage and pod resistance of the germplasm tested to a range of Xanthomonas blight isolates, and also have shown resistance to systemic colonization by the bacteria. Breeding programs should emphasize the transfer of this resistance to Phaseolus vulgaris through interspecific hybridization. LITERATURE CITED ALLINGTON, W.R., and D.W. CHAMBERLAIN. 1949. Trends.in the population of pathogenic bacteria within leaf tissues of susceptible and immune plant sciences; Phytopathology 39:656-660. BARLOW, B. 1904. A bacterial disease of beans. Ontario Agr. Coll. Bul. 136:9—13. BURKHOLDER, WgH. 1921. The bacterial blight of the bean: a systemic disease. Phytopathology 11:61-69. CHAND, J.N., and J.C. WALKER. 1964. Relation of age of leaf and varietal resistance to bacterial multiplication in cucumber inoculated with Pseudomonas lachrymans. Phyto- pathology 54: 49—50. COYNE, D.P., and M.L. SCHUSTER. 1974. Differential reaction of pods and foliage on beans (Phaseolus vulgaris) to Xanthomonas phaseoli. Plant Dis. Rep. 58:278m282. COYNE, D.P., M.L. SCHUSTER, and K. HILL. 1973. Genetic control of reaction to common blight bacterium in bean (Phaseolus vulgaris) as influenced by plant age and bacterial multiplication. J. Amer. Soc. HOrt. Sci. 98:94-99. COYNE, D.P., M.L. SCHUSTER, and B. HOFF. 1977. Epiphytic populations of Xanthomonas phaseoli on tolerant and susceptible leaves and pods of Phaseolus vulgaris L. Ann. Rept. Bean Imp. Coop. 20:75-76. CROSSE, J.E. 1963. Bacterial canker of stone-fruits. V. A comparison of leaf-surface populations of Pseudomonas-mors- prunorum in autumn on two cherry varieties. Ann. appl. BioL 52:97—104. DAUB, M.E., and D.J. HAGEDORN. 1976. Studies on resistance of Phaseolus to bacterial brown spot of bean (Pseudomonas syringae). Proc. Amer. Phytopathol. Soc. 3:234 (Abstr.). 74 10. 11. 12. l3. 14. 15. l6. 17. 18. 19. 20. 21. 75 DIACHUM, S., and J. TROUTMAN. 1954. Multiplication of Pseudomonas tabaci in leaves of Burley tobacco, Nicotiana longiflora and hybrids. Phytopathology 44:186—187. EKPO, E.J.A. 1975. Pathogenic variation in common (Xanthomonas phaseoli) and fuscans (Xanthomonas phaseoli var. fuscans) bacterial blights of bean (Phaseolus vulgaris L.). Ph.D. Thesis, Michigan State Univ., East Lansing, 127 pp. EKPO, E.J.A. and A.WL SAETTLER. 1976. Pathogenic variation in Xanthomonas phaseoli and M. phaseoli var. fuscansx Plant Dis. Rep. 60:80-83. GOODMAN, R.N., P.Y. HUANG, and J.A. WHITE. 1976. Ultrastructural evidence for immobilization of an incompatible bacterium, Pseudomonas pisi, in tobacco leaf tissue. Phytopathology 66:754—764. HILL, K., D.P. COYNE, and M.L. SCHUSTER.v 1972. Leaf, pod, and systemic chlorosis reactions in PhaseoluS‘vulgaris to halo blight controlled by different genes. J. Am. Soc. Hortic. Sci. 97:494-498. KELMAN, A., and V. SEQUEIRA. 1972. Resistance in plants to bacteria. Proc. of the Royal Soc., London, E, 181:247-266. KLEMENT, Z., G.L. FARKAS, and L. LOVREKOVICH. 1964. Hyper- sensitive reaction induced by phytopathogenic bacteria in the tobacco leaf. Phytopathology 54:474-477. LYON, F., and R.K.S. WOOD. 1973. Antibacterial substances~in hypersensitive responses induced by bacteria. Nature 242: 532-533. LYON, F.L., and R.K.S. WOOD. 1976. The hypersensitive reaction and other responses of bean leaves to bacteria. Ann. Bot. 40:479—491. MEW, T.W., and B.W. KENNEDY. 1971. Growth of Pseudomonas glycinea on the surface of soybean leaves. Phytopathology 61:715-716. MOUSTAFA, F.A., and R. WHITTENBURY. 1970. Properties which appear to allow phytopathogenic pseudomonads to counteract plant defence mechanism. Phytopathologische Zeitschrift 67:214—224. 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. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 76 ROEBUCK, P., R. SEXTON, and J.W. MANFIELD. 1978. Ultrastructural observations on the development of the hypersensitive re- action in leaves of Phaseolus vulgaris-cv. Red Mexican inoculated with,Pseudomonas phaseolicola Grace 1). Physio— logical Plant Pathology 12:151-157. RUDOLPH, K. 1972. The halo—blight toxin of Pseudomonas phaseolicola: Influence of host-parasite relationship and counter effect of metabolites. In: 'Phytotoxins in Plant Diseases'. Edited by R.K.S. Whod, A. Ballio, and A. Graniti. Academic Press, London. SAETTLER, A.W., and M.Wp ADAMS. 1974. Breeding navy (pea) beans for bacterial blight tolerance. Research Rept., saginaw Valley Bean-Beet Research Pram, Mich. State Univ., Agr. Expt. Sta. 4:46-50. SCHAREN, A.L. 1959. Comparative population trends of Xanthomonas phaseoli in susceptible, field tolerant and resistant hosts. Phytopathology 49:425—428. SEQUEIRA, L. 1972. Prevention of the hypersensitive reaction. Proc. of the Third Intern. Conf. of Plant Path. Bacteria. Edited by H.P. Maas Geesteranus. Pudoc, Wageningen. 365 pp. SEQUEIRA, L., G. GAARD, and G.A. De ZOETEN. 1977. Interaction of bacteria and host cell walls: its relation to mechanism of induced resistance. Physiological Plant Pathology 10: 43—50. SING, V.O., and SCHROTH, M.N. 1977. Bacteria-plant cell surface interactions: active immobilization of saprophytic bacteria in plant leaves. Science 197:759-761. STALL, R.E., and A.A. COOK. 1966. Multiplication of Xanthomonas vesicatoria and lesion development in resistant and susceptible pepper. Phytopathology 56:1152-1153. STEEL, G.D., and J.H. TORRIE. 1960. Principles and Procedures of Statistics. New York: McGraw—Hill Book Company, Inc. 481 pp. VALLADARES, N., D.P. COYNE, M.L. SCHUSTER, and B. HOFF. 1977. Reaction of Phaseolus germplasm to different strains of Xanthomonas phaseoli and g. phaseoli fuscans. Ann. Rept. Bean Imp. Coop. 20:74-75. 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. East Lansing, 137 pp. 33. 34. 35. 36. 77 YOSHII, K., G.E. GALVEZ-E., and G. ALVAREZ-A. 1978. Screening bean germplasm for tolerance to common blight caused by Xanthomonas phaseoli and the importance of pathogenic variation to varietal improvement. Plant Dis. Rep. 62:343- 347. YOUNG, J.M. 1974. Development of bacterial populationS‘in_vivo in relation to plant pathogenicity. N.Z. Journal of Agricultural Research 17:105—113. ZAUMEYER, W.J. 1929. Seed infection by Bacterium phaseoli. Phytopathology 19:96 (Abstr.). ZAUMEYER, W.J., and H.R. THOMAS. 1957. A monographic study of bean diseases and methods for their control. U.Sk Dept. Agri. Tech. Bull. 868, 255 pp. PART III (SURVIVAL AND TRANSMISSION OF BEAN BLIGHT BACTERIA (XANTHOMONAS PHASEOLI AND 3(_. PHASEOLI VAR. FUSCANS) IN TISSUES OF SUSCEPTIBLE AND RESISTANT PLANT SPECIES INTRODUCTION Bacterial survival has been studied extensively in numerous groups, however little information is available relative to survival of plant pathogenic bacteria. A better understanding of the means of survival and mechanism of transmission of phytopathogenic bacteria are important elements in any effort to improve the control of these pathogens. Although plant pathogenic bacteria are non-sporeforming, many are tolerant to desiccation and survive in a state of reduced metabolism and decreased sensitivity to environmental variables (hypobiosis), for relatively long periods (17). It has been suggested that many bacterial pathogens survive well under some conditions if they are in aggregates and protected by bacterial exudate (ooze), substances commonly found in infected cankers, living or dead plant parts, and in seeds. Debris from diseased plants have been always considered a possible source for seasonal carryover of the pathogens. Recent studies have also shown that phytopathogenic bacteria can survive in protected positions on healthy leaves of host as well as non-host Plants (2, 4, 5, 6, 13, 20, 23). The ability of plant pathogenic bacteria to grow epiphytically on susceptible and resistant plant 78 79 tissue may be of epidemiological importance by serving to build up inoculum prior to infection; such growth may provide pathogen cells for dissemination and season—to—season survival (17). Ercolani gt El (6) recovered Pseudomonas syringae throughout the year from leaf surfaces of healthy yigia villosa (hairy vetch), and correlated natu— ral outbreaks of bean brown spot with the epiphytes on non-susceptible hairy vetch. Isaka (12) reported that Xanthomonas oryzae was able to overwinter in association with various weed plants growing in rice fields, and Laub and Stall (15) suggested that g. vesicatoria may be disseminated to weed plants, survive as a resident through the summer period and serve as source of inoculum to tomato and pepper plants. Recently, Latorre and Jones (14) reported Pseudomonas syringae isolated from weeds would infect sour cherry leaves; suggesting weed populations of P, syringae were a source of inoculum for bacterial canker. Little information is available on the possible role of weed plants in the survival and dissemination of Xp and pr. Gardner (7) and Sabet gt al (21) suggested that different isolates of Xp may infect a number of weeds under natural conditions, and Schuster (25, 26) reported that Xp overwintered in bean and weed refuses under Nebraska field conditions. Presence of plant pathogenic bacteria in seeds is an important means of survival and dissemination in time and space; most seed-borne bacteria survive as long as the seed remains viable (27). Seed transmission is the primary means for dissemination of the bean bacterial diseases. walker and Patel (29), Guthrie gt a} 80 (9), and Wallen and Sutton (30) have indicated that low infection levels of Pseudomonas phaseolicola or Xanthomonas phaSeoli in bean seeds are capable of initiating heavy field infections and crop losses under epiphytotic conditions. Grogan and Kimble (8) reported that P. phaseolicola was transmitted through bean seeds harvested from a field where the disease was not detected during the growing season. Internally-infected seed has been mentioned as the main source of primary inoculum in xanthomonas bean bacterial blights (22, 35). Zaumeyer (33, 35) indicated that X. phaseoli may cause a systemic invasion of the bean plant under certain conditions, and that the‘ bacteria may pass into the developing seed through the vascular system of the plant, without producing visible symptoms. The blight organism may also enter the pod cavity either via stomata of the pod or by breaking through.the vascular tissue of the pod suture, the bacteria then pass into the funiculus and the raphe or the micropyle leading into the seed. It has been mentioned that P, phaseolicola was found in some halo blight resistant beans and has caused serious problems to seed producers (3). Our field experiments (Part II) showed that Xp can multiply to high population levels in and on leaves and pods, and systemically colonize bean genotypes with intermediate levels of resistance. It was therefore desirable to further investigate the possible seed transmission of Xp in resistant bean genotypes. This study was primarily concerned with: (i) survival and field-overwintering of Xp and pr in leaf tissue of susceptible and 81 resistant bean genotypes and of non-host species; (ii) secondary spread of Xp between blight susceptible and resistant bean cultivars, and weeds; (iii) transmission of Xp in seeds of bean genotypes with. different levels of disease resistance. MATERIAL AND METHODS This study included experiments conducted under field and green— house conditions. Field experiments were done at the Botany and Plant Pathology Research Farm, Michigan State University, East Lansing, Michigan, during the 1977 and 1978 growing seasons. In greenhouse experiments plants were grown at 27 i 2 C, and in day light supplemented with 14 hours of fluorescent lighting, in a standard soil mixture in 16 cm diameter clay pots and watered alternately as needed with Rapid—Gro (1 teaspoon per 2 liters of water) and tap water. Bacterial Isolates Naturally—occurring rifampin—resistant mutants, RlS—l of Xanthomonas phaseoli and R17 of §. phaeoli var. fuscans, were obtained by concentional selective plating methods (18) and found to possess virulence equivalent to the parental wild types (Xp 15 and pr 17, Michigan isolates). Inoculation Techniques Bacterial cells were washed from plates of two-day-old YCA (YCA: 10 g yeast extract, 2.5 9 calcium carbonate, and 15 g agar per 1000 ml distilled water) cutures incubated at room temperature (24 :_l C) 82 83 and suspended in sterile-distilled water at concentrations of 1x107 to 5x107 cells/ml of RlS—l or R17. Inoculum was applied to plants in the vegetative stage of development by gentle spraying with a DeVilbiss sprayer (in the greenhouse) or with a Knapsack sprayer (in the field); inoculum was applied to run—off on the lower and upper leaf surfaces or by waterSoaking the leaves (24). Determination of Bacterial Populations Multiplication and spread of RlS—l was monitored at intervals after inoculation. Populations of viable bacterial cells were assayed from ten or 21 randomly sampled leaflets replicated three times, by homogenizing the tissue in 0.01 M phosphate buffer, pH 7.2. After appropriate serial dilutions, suspensions were plated on YCA medium supplemented with 50 ug/ml rifampin adn 25 ug/ml cycloheximide. Colonies were counted after four days incubation at room temperature. Populations of blight bacteria were expressed on the basis of number of colony forming units (CPU) per 100 cm2 leaf tissue (approximate average area of one leaf), or CPU per gram dry weight of tissue. Survival in Leaf Tissues Infected materials for overwinter studies were obtained from infected leaf tissues of bean genotypes grown in the greenhouse and the field. Leaves were harvested 22 days after inoculation and dried at room temperature. Uniform weighed samples of pulverized dried tissue were wrapped in fine mesh nylon bags tied with a nylon string. Initial numbers of viable bacterial cells of R15—l or R17 per g of dry tissue were determined at the same time. In November 84 1977, samples (three replications for each material) were placed outside at three localities: Saginaw, St. Louis and East Lansing (Michigan). Tissue samples were placed: (a) on the soil surface, and (b) 20 cm below soil surface. Tissue samples were retrieved during June 1978 and returned to the laboratory. To assay for viable blight bacteria, portions of each tissue sample were homogenized in phosphate buffer and after appropriate serial dilutions, suspensions were plated on YCA supplemented with 150 ug/ml rifampin, 100 pg/ml cycloheximide, and 100 ug/ml penfachloronitr«L (PCNB). Other portions of the sample were initially incubated in BYE (BYE: 5 g yeast extract in 1000 ml 0.01 M phosphate buffer pH 7.2) supplemented with the above chemicals and subsequently plated on solid media and also infiltrated into bean leaves. Comparison tissue samples were maintained at room temperature and assayed for viable bacteria at six month intervals over a two year period. Secondary Spread Alternating ten meter—long rows of MSU—513l9 (moderately- resistant) and Tuscola (susceptible) bean genotypes, were planted using commercial planting procedures with 50 cm between the rows. When plants possessed fully expanded second trifoliolate leaves, the rows of MSU-513l9 were inoculated by spraying with a suspension of R15-l. In a separate experiment, ten rows of cultivar Tuscola were planted following the above specifications. When bean plants were at the vegetative stage of growth (second trifoliolate leaf fully expanded), selected plots of the 85 Tuscola plants and of weed plants (Chenopodium alba and Amaranthus retroflexus), growing between and in the bean rows, were inoculated as above with RlS-l. Multiplication and spread of R15-l, from inoculated MSU-51319 to Tuscola in the first experiment, and from inoculated beans to weeds and from inoculated weeds to bean plants in the second, were monitored at intervals after inoculation. Bacterial populations were determined as described before. R15-l growth on the leaf surface of weeds was also detected using leaf-impression cultures ("leaf-print") (16) on the rifampin—selective medium. Seed Transmission I, Greenhouse study. Xanthomonas resistant Tepary bean Arizona-Buff (Phaseolus acutifolius): moderately—resistant W-ll7 (USDA, Puerto Rico), and susceptible Seafarer navy bean cultivar, were used in this experiment. When plants were at the flat green stage of development, approximately 50 pods of each genotype were inoculated by scratching along part of the dorsal suture with the needle of a sterile syringe containing 5.0xlO7 cells/ml of RlS-l. At normal maturity the pods were removed and the seeds separated into those without visible symptoms, and those showing some type of visible symptoms of blight infection. Internally—borne blight bacteria were isolated from individual surface-sterilized (3 minutes in 2.5% NaOCl and rinsed twice in sterile-distilled water) seeds, in both, solid (YCA-R: 10 g yeast extract, 2.5 g calcium carbonate, 50 mg rifampin, 25 mg cycloheximide, 15 g agar in 1000 ml distilled water), and liquid (BYE-R: 10 g yeast extract, 86 50 mg rifampin, 25 mg cycloheximide in 1000 ml .01 M phosphate buffet pH 7.2) selective media. Individual seeds were first placed hilum down directly on YCA-R and incubated 18 hours at room temperature, and then transferred to 7 ml test tubes containing 3 ml of BYE—R for 48 hours shaker incubation. Bacteria from tubes with turbidity were streaked on YCA-R to confirm the presence of RlS-l mutant. In this way, each individual seed was checked for internal blight infection by two methods. The total number of seeds with visible blight symptoms and symptomless seeds tested for each genotype are presented in Table 8. II. Field study. The following bean genotypes were selected for field studies on the basis of their reported reactions to bacterial blight: resistant Tepary bean Arizona-Buff, moderately- resistant Great Northern Nebraska #1 selection 27, G.N. Valley, G.N. Jules and MSU-Sl3l9, and susceptible Tuscola. At the flat green stage of plant development, approximately 200 pods of each genotype were inoculated with a bacterial suspension containing 1.0xlO7 cells/ml of RlS-l, following the same procedures for inoculum preparation and inoculation technique as in the greenhouse study. Pods were collected at normal maturity, and the seeds separated into those with visible symptoms and those without symptoms. Internally—borne bacteria were isolated from individual-surface sterilized seeds using the techniques described previously. 87 The total number of seeds with visible blight symptoms and symptomless seeds tested for each genotype are presented in Table 9. In a separate field study plants of Tepary (Arizona—Buff), G.N. Valley, and Tuscola were inoculated by gentle spraying to run—off with a 1.0x107 cells/ml suspension of RlS—l at different stages of plant development, viz. seedling stage, third trifoliolate stage, blossom stage, and small flat pod stage. Pods were harvested at normal maturity and internal seed infection assayed by direct plating of seeds on the rifampin—selective medium or after 48 hours incubation in BYE-R. RESULTS Multiplication of §p (R15-l) in Leaves of Beans and Non-Host Species Growth patterns of Xp after gentle spray inoculation of susceptible (Tuscola), moderately—resistant (W-ll7) bean genotypes, to non-host species soybean (cv. Hark) and lambsquarters (Chenopgdium alba), are shown in Table 1. While bacterial growth patterns were similar in both bean genotypes, maximum bacterial populations were generally lower in the moderately-resistant W-ll7, particularly' during the reproductive stage of plant development; different disease reactions developed on the genotypes. Xp populations remained stable in leaves of soybean until eight days after inoculation; then declined at 12 days and again stabilized until the end of the experiment. Bacterial populations in leaves of lambsquarters remained more or less stable throughout the assay period, although at lower population levels as compared with soybean. No visible disease reaction was observed in leaves of soybean and lambsquarters throughout the experiment. Secondary Spread of Xp from Resistant to Susceptible Bean Genotypes Xp was initially detected in non—inoculated leaves of Tuscola plants ten days after the MSU-4l3l9 plants were inoculated (Table 2). Recovery of the bacteria in susceptible Tuscola followed several 88 89 .omoo moofimoH xcoE .coauomwcw wooH womlmm u o.v “38 we» :H :oHpowocH HmmH HOHIm u o.m Homooomno o: n 0.0 oHoom onu on mcflouoooo mucon msouoeos .GOHUUOMCH mooH wooalom n Hmcfloooumm too omHoH mcoflmoH .coHuquGH mood womIoH n n3ou onu on cofiuoowcfi mooH mm mfimon ncoam Houou o co woos oHo3 wooeuooom omoomao u o.m chwomoHooo o.m .mconmH urmHHn 3mm m u o.H .m.D Ame .mCOHuooHHmou oounu mo omouo>¢ Awe .0 wow uo Hlmam mo coflmcommom HE\mHHoo oaxo.H o nuns wmoIcou on pwuoHSUOGH mums mucoam czoumIomooncooum tHoIhoo om mo mo>omn a . AHV . . . . . . 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At that time, bacterial populations in the moderately-resistant MSU-51319 were about 106 cells per leaf, but no visible disease symptoms were visible. Population trends of Xp in both genotypes followed similar growth patterns, although different disease reactions developed. At the end of the assay period (32 days after inoculation of MSU-513l9), Tuscola exhibited typical blight symptoms (lo-20% leaf infection in the row, lesions large and spreading), while MSU-513l9 showed a few very scattered blight lesions on the lower leaves. Reciprocal Secondary Spread of Xp Between Susceptible Bean Cultivar to Weeds Xp multiplied in inoculated leaves of weed and bean plants, although at lower growth rates in the former (Table 3). A substantial proportion of the total Xp population on weeds was detected on leaf surfaces as determined by direct leaf prints on the rifampin-selective medium. Reciprocal secondary spread was first detected after several days of heavy rains, 12 days after inoculation, when Xp had reached exponential growth in both bean and weed leaves, with average population levels of 7.5xlO7 and 1.4x106 bacterial cells per leaf, respectively. Survival of gp (RlS-l) and pr (R17) in Dry Tissues of Susceptible and Resistant Bean Genotypes and Non-Host Species High population levels of Xp and pr were recovered 36 days after leaf inoculation on all of the species studied (Table 4). Xp and pr isolated from each of the dry tissue samples were 92 .mEoumE>m omoomHt onoomOHooE mo oocomoum muoOHccH mHmonucoHom« .HImHm mo :oncommom HE\mHHmo Oon.H o nuHB uwolcsu on mcHonmm oHucom >n o >oo no touoHooocH ouo3 mucon AHV HHOHxH.mV H.OonH honm.N 0.0 0.0 m AHOme.Ng mOon.H :Obe.H o.o 0.0 v HHOme.NV HHOHxH.HV :OHxH.m o.o 0.0 m HooHNm.hv AROHxN.HV :OHxN.m 0.0 0.0 N AcouoHooooHIcocv HBOHNN.>V HROme.HV :Oon.m 0.0 0.0 H oHoomoe :Ome.m :OHxh.N oonv.H :onm.m monNN m :OHXN.v :Ome.H oOon.H mOHxH.H NOwa.m v :Ome.o :Owa.m mOme.H :Ome.o NOme.o m :Ome.m :Ome.N oOme.H m0on.H HOHxv.m N HoouoHsooch :Ome.h :OHxh.N mOHxN.H :OHxh.h mOHXvH H momma HOme.N :OHxH.m NOHxH.v 0.0 0.0 m Honv.H :onH.H NOHxv.m 0.0 0.0 v HOHxh.m :OHxN.h NOHxN.v 0.0 0.0 m HQon.m :OHxH.m 0.0 0.0 0.0 N ApouoHooocHIcocv HOHXm.N HOHxH.N N013....” 0.0 0.0 H mtomz BOme.w mOHxH.H nOme.h mOHxH.H :Ome.H m Asono.hV AnoHXm.hv HHOme.mV mOme.H :OHxH.H v HROHx©.vV HQOHxN.HV HHOme.mv SOHxN.H :OHx@.H m HoOme.Hv HaOHxN.HV HHOHxv.mv mOHxN.m :onh.H N HtouoHSOOCHV HHOon.mV HQOHxH.HV *Hhonm.hv mOon.H :OHxh.N H oHoomoB vN mH NH o H coHuooHHmom N coHuoHooocH Houwo mmoo omnm mood SO OOH\DhO .mcothooo pHon Hops: H:Uoo3 mHm=V moonmoHuoH monocouoeo poo H:muouuo5wmnfioH:v onHo fioHcomoconU on HoHoomSBV omzuocom coon oHnHumoowom cvoBuon Huoouofi HImHmv HHommonm moGOEonucox wo pooumm >Hoocooom HoooumHoom .m mnmoe 93 .onsnonomfion Soon no omononm m>oo vH nonno moomen mooH >no :H tocHEnonoo mo3 hHm coo HImHm no HDMUV mHHoo HoHHonoon oHnoH> no Hones: onenmv .HU «NINNV onononomeon econ no ooan coo coHnoHooocH nonmo whom NN uonmo>non ono3 mo>ooH oonoHooocHHNV .hHm pco HlmHm mo GOHmcommom HE\mHHoo OHXm o nan oommHn mooH mconOmIHonoB >n omnoHooocH mums wncon oHoINoo mm on on n HHS :OHxh.o , :OHxH.N HHHHoomono ooHnoocHnomv mmonm pnoxcnom HOon.N :Oon.m HoHHonHHmHEonno ononneov cows mom moon: monNH €3ch 5:302 $23:ch xomHm HOon.H Sonm.N Hwoonwonnon monnconoeov coma mHm mOme.H o0on.N HonHo ESHtomoconov mnonnooomneoq HOme.m mOHxNH HmNoE ooNv «v 03 :Oon.H DOHxN.m AmHnomHo> onomv 0N .m.D HOme.H hOHxNN HonoHoOHomco oomH>v .m.m.z ronHS m.OonS :85 932:8 has honoS House $388.. We nmumoowm 5015.... .286. SHEER. .ms some ..onH.o QOHva 6338.. .MC mHmHmIsmz sOHxh.H aOHxvo HmoHHOMHnooo Ame ammo sOHxN.H HOHxv.m HmoHHOMHnooo .mv HwnomIocoanov >nomoe hHm HImHm moHoon HmvoommHB moon >no «0 m\Dmu ANV .HHancon ozonmIomsoncoonm no oomen mooH who cH Hmcoomow .Ho> HHoomonm .Mv hHm coo HHHoomonm moGOEonncoxv HImHm no mHo>oH coHnoHomom .v mHmoe 94 pathogenic as tested by host inoculations. Weighed samples of these dry leaf tissues and tissues obtained from field-grown plants (Table 5), were utilized to study between—season bacterial survival. Neither Xp (RlS-l) nor pr (R17) was detected in any of the dry leaf tissue samples maintained on or buried in field soil at three different localities from November 1977 to June 1978. Samples were colonized by a heterogenous population of soil microorganisms, predominantly bacteria and fungi, as determined by plating serial dilutions of the samples on YCA medium. These microorganisms were inhibited in YCA supplemented with rifampin, cycloheximide, and PCNB, but allowed the growth of RlS-l and R17 (rifampin-resistant mutants of Xp and pr, respectively) as determined by plating serial dilutions of similar tissue samples stored under laboratory conditions (Fig. 1). Both bacterial isolates have survived and retained pathogenicity after two years storage in the laboratory of dry infected tissue samples of susceptible and resistant bean genotypes. Population levels of R17 recovered from these samples tested at six month intervals over a two year period, are shown in Table 6 . XP was recovered from samples of infected plants left standing in the field following the 1978 growing season in October, November and December 1978, but no bacteria were recovered in January, February, March, and May 1979. 95 lAI-YCAOROCQP Ln-v u FIGURE 1. Recovery of R15-l (Xp) from dry leaf tissue, maintained in field soil or stored under laboratory conditions, on YCA supplemented with 15 ug/ml rifampin (R), 100 ug/ml cycloheximide (C), and 100 ug/ml PCNB (P). Photographs taken after Six days incubation at room temperature. 96 .oHanmoomom n m “nooanmon >HononocoE n m: “nooanmon u m "oOHnooom omommHoA3 .mooHnoOHHmoH monnn no omono>o who mooHo> .onononomson Econ no omononm mxoc mH nonwo moommHn mooH anc oH cooHEnonoc mo3 hHm coo HImHm no HDmOV mHHoo HoHHonoon oHnoH> no Monaco onBHmv .Ho VNINNV onononomoon soon no cmHHc coo mcom mfion coHHHMIHHoB commommom mnoon oonz conmo>non ono3 mo>ooHHNV .HnoonoE waxy hHm coo Anoonoo mxv HImHm no oonoomwom HE\mHHoO Oon.m o onH3 MMOIooH on conoHooooH onus Amm>ooH onoHOHHOMHnn one coo cnmv mnoon cHoINoc MN H. HHS a0me.m oOme.H m nonomoom mOHNmN :OHxh.m m2 onoa mOon.m HOHx®.m m2 mHMHmIDmE HOon.m HOme.m o ammo hHm HImHm AHlooHnooom oomeB moon >nc m\Dmo omoomHo ANvomwnooou Amy .Avaom>nooom ooon oBOHmIcHon no oommHn mooH wnc oH Hmooomow .no> HHoomonm .Mg hHm coo HHHoomonm mooooonnooxv HImHm no mHo>oH ooHnoHomom .m mHmoB 97 .oHanmoomnm u m «nooanmon HHonoHocoE u m: “nooanmoH u m "oOHnooom omoomHQ n .m.o Ave .cownom omononm oooo nonuo concocooo ono3 mnmon mnHoHoomonnom Hmv .mooHnooHHmon noon no omouo>o ono mooHo>. ANV .cooHENonoc oOHnoHomom HoHuonoon HoHnHoH onn coo no H H.mNV onononomfion Econ no coHHc .ooHnoHooooH Honmo mmoc NN conmo>non onus mo>ooH conoHooooH .nHm mo oOHmoommom HE\mHHoo QOHNOH o onH3 oommHn onn monoomInonoB Nn conoHooooH woos mnoon ozonmuomoooooonm mo mo>ooH onoHoHHOMHHB HHV houch. . H Noon . H Noon . N sono . N None . m m nonomomm mOon .m mods . m mods . a Node . m $on . a mo mHmHmIomz soda...“ hOoné honmAL. hOme.m hodouim m: R How .He .onmz .z.o :Ome.m JOond :Omed :onmo ”354 m HmmsmIosoNHué Hoodoo. em 3 NH o 88.333 HoHnonoom Hmvomononm Honwo monooz HoHnHoH om>noooo ANvoommHB mo n3 HMO m\DmU .HHvonononomEon Econ no cononm mom>nooom coon noonowch mo oommHn nooH conooon Nnc Econ HnoonoE nHmv max no >no>ooom .o mqmoe 98 Survival and Seed Transmission of Xp (RlS—l) in Seed of Resistant and Susceptible Bean Genotypes Xp was only recovered from seed samples (seeds with no symptoms) harvested from symptomless pods of field-grown plants inoculated by gentle spraying the bacterial suspension at the small-flat pod stage of plant development (Table 7). G.N. Valley and Tuscola exhibited similar levels of surface bacterial populations but susceptible Tuscola showed the highest incidence of internal seed infection. Relatively few bacteria were externally detected in seed samples from Tepary bean. when plants of resistant and susceptible bean genotypes were inoculated by scratching the dorsal suture of the pods at the flat green stage with a syringe containing the bacterial suspensions, different disease reactions were observed. At normal maturity, only pods of the susceptible Tuscola exhibited typical Xanthomonas blight symptoms extending beyond the inoculated areas; G.N. Nebr. No. l sel. 27, G.N. Jules, G.N. Valley, MSU-513l9, and W-ll7 all showed brown— necrotic reactions and sometimes few and small watersoaked zones around the scratches; only a light-brown necrotic reaction was observed on the inoculated pods of Tepary bean (Figure 2). Seeds with different degrees of Xanthomonas blight symptoms and seeds with no visible symptoms, were harvested from inoculated pods of all the bean genotypes (Figure 3). In both experiments, Xp (RlS-l) was recovered from about 40 to 50% of seeds exhibiting any type of visible symptoms of internal blight infection in resistant genotypes, and from about 70% in susceptible bean genotypes. Also, Xp was 99 oHanmoomom n m «nooanmoHINHononoccE u xx “nooanmoH u m "ocHnooom omoomHo H.m.oVHmv .moEHn oonon conoomon mos nooEHHomxo one .mocHnooHHmon oounn no omonc>o cno mcoHo> .ocHEonnchho HE\m: mN coo onEoMHn HE\m: om + mam oH monEom onn no ocHnonocoH mnoon we Honwo no oHcoE o>ancHomIonEoan won :0 Nanoan conon noonHo one; mcoom oonB .Hoooz am.N oH monooHE my mcoom coNHHHHonm coomnom oH conconcc mos nnzcnm HoHuoncon oz Hes .oHcmE o>HnooHomIonEoan onn oc moHnon coo HN.> rev Howmon ononmmcom z Ho.o oH HHoooz mm.N oH monooHE my mcoom couHHHnonm woomnom moHcoHHm >n cooHEnonoc mos ocHnoHomom HoononoHHmv .358: m2 moHstwsoHoso Ham HHE\m1 omv onEoan + o0» oc moHnon coo .onooHE coo How AN.h may nonwon mnoommcnm E Ho.o oH comm won mononm zn cooHEMoncc mo3 Hmcoom mmoHEcnmEHm OOHV monEom comm mo ocomnom won :0 HImHm no ocHnoHoannHHNV .nooEmcHo>oc noon no omonm o>anomon con no HImHm mo ochocmmom HE\wHHco FOon.H o onHB wmcloon on monoHQm oHnoom kn conoHoccoH oHoB mnoonHHV :OHXN.H mOon.H I I I m oHoomSB mOHNcH mOHxH.N I I I m: NoHHo> .z.o I HOon.Hv I I I o HwnomIoocNHnov Hey wnomoe HoononoH ooownom moosch onoHcHH mc>ooH hnoEHHm A .m.o E E -83 EH 3 mccm non HHoEm ommncoou nooemcHo>oo noon no omonm mcoom OOH\DmU . anooEmcHo>oc noon no momonm noonomec no conoHoocoH mom>nooom ooon oonnn no mnoon oo mcow mmoHEcnmENm Ecnw conmo>non mccom Econ HnoonoE HImHmv mx mo >Hm>ocom .h mqmoe FIGURE 2 . 100 Disease symptoms on mature pods of Tepary (Arizona— Buff), G.N. Valley, and Tuscola bean genotypes. Pods at the flat green stage of development were inoculated by scratching the dorsal suture with a syringe containing 1.0xlO7 cells/ml of R15—l (Xp mutant). FIGURE 3. Seed obtained from pods of Tepary (Arizona—Buff) , G.N. Valley, and Tuscola bean genotypes, inoculated at the flat green pod stage of plant development by scratching the dorsal suture of the pods with a syringe containing 1.0x107 cells/ml of RlS-l (Xp mutant) . 104 recovered from 5.4, 6.5 and 11.8% of symptomless seeds in the genotypes Tepary, W—ll7, and Seafarer, respectively, in the greenhouse experiment (Table 8); and from 1.3, 2.0, 1.9, 1.3, 2.0, and 10.4% in the genotypes Tepary, G.N. Nebr. No. l sel. 27, G.N. Valley, G.N. Jules, MSU-Sl3l9, and Tuscola, respectively, in the field experiment (Table 9). 105 .oHanmocmom u m «nooanmonINHononoccE n m: “nooanmcH u m ”ocanoom omoomHo n .m.o Hmv .oHccE onEoan cHooHH oH ooHnonocoH mnocn we >n co3oHHom coo mnocn mH Hon oHcoE.o>anoHomIonEoMHn cnoH NHnocHHc ozcc EDHHo cocon nwnHw woos ocomm .mcoom HHUooZ wm.N oH monooHE.mV coNHHHHonmIocomnom HoocH>HcoH Econ conoHcmH oHoB oHchoom HNV .HnoonoE axe HImHm no HE\mHHoc mOon.H moHoHonocc omoHnam o no oHcoco won onH3 muonow Homncc onn moannoHcm >n ccnoHsocoH oHoB omxnooom ncoo mo mnoon o3cnmlomoonoconm oc mccm om HHonoEchHmmo HHV w.HH :va\hH . . o.wo Om\vm m nononowm m.o mmH\HH 0.0m om\mN m2 nHHIz q.m wcH\m o.c¢ om\MN m AmmomIoocNHnov Nnomoa w I Honce\conconoH w _ .HoncH\concomoH mEcnmEHm mEonmENm oHnHmH> onHo mccom mcoom concomoH >HHoononoH omwncoow oz onHo mcoom HNV .HHV HHcomoom .M nnH3 conoHoccoH mnoon osonm owocoooonm Econ conmc>non comm oH oannoon nanHn no oooochoH .m momma .oHanmocwom u m “nooanmonINchonoccE n m: “nooanmon u m "ooHnooom omoomHD u H.m.ov Hmv .oHcoE onEoMHn cHoUHH oH ooHnonocoH mnoco we >n cosoHch coo mnoon mH new oHcoE o>HnooHomIoneoan cnoH NHnOcHHc ozoc eoHHn cooon nmnHw onoz mcoom .mcoom HHoooz mm.N oH monooHE my coNHHHHonmIocomnom HoocH>HcoH Ecnm conoHcmH oncz oHHoncom HNv .HnoonoE mxv HImHm no HE\mHHoc hOon.H moHoHonooo omoHMNm oHHnonu o no oHcooo won onHB muonow Hochc onn moannoncw an ccnoHocooH who: omwncoom ncoo mo mnoon ozcnmIcHon oc mccm OON hHonoEchnmmo HHS V.OH BQNH on 8H}: m 2085 m.H Home} ca 03):. m mnzmIoooNHnE Hoodoo. % o.m 0mm? we ooqma oz 32.“.-sz 1 m.H 03$ 2. oOHSH. m2 HmHHS, .z.o m.H 093 Hm o0H\Hm m2 mmHzH. .z.o o.m 8m} 9. 8:9. as R How He .Swz .z.o a Houoeomnooofi » Hmuoehvmnomofi .m . 9 inc mEcnmENM mEcnmENm oHnHwH> omxncooc oz and; $58 on? £68 mcoom concomoH NHHooncnoH HNV .HHVHHcomonm um onHB conoHoccoH mnoon ozonm cHon Econ conmc>noo coon oH oHnonoon nanHn no oooochoH .m mHmoB DISCUSSION Results of these studies further support our previous findings (Part 1, 1.2) that leaves of susceptible and resistant bean genotypes and non—host plants may support epiphytic multiplication of bean blight bacteria. Several workers have reported plant pathogenic bacteria surviving on healthy host and non-host tissues (2, 4, 5, 6, 13, 20, 23). The data on secondary spreas of Xp between blight resistant and susceptible beans, and weed plants indicated that inoculum was available for dissemination early after colonization of the plants, suggesting that secondary spread, primarily due to rain splashing, occurs in the field prior to symptom expression. Such an epidemiological pattern suggests that moderately-resistant bean cultivars could serve as 'symptomless carriers' of bean blight bacteria. While epiphytic growth of Xp on weed plants has been previously suggested (7, 21), our results indicate that the bacteria may be a resident on weed species. The inherent ability of leaves of host and non-host species to support epiphytic growth of blight bacteria may be of relative importance under Michigan bean growing conditions. In Latin America, particularly in the tropics, however, 107 108 environmental conditions allow more than one successive crop during the year; also, beans are frequently cultivated in association with other crops and heavy weed infestations are common problems in bean fields. Survival of phytopathogenic bacteria associated with.plant residues in soil is well recognized (17, 27) and debris from diseased plants have been always considered a possible source for seasonal carryover. Circumstantial evidence suggested that infected plant refuses may play a role in long-term survival of bean blight bacteria (10, 34) and Schuster (25, 26) reported overwintering of Xp and pr in bean and weed refuses in Nebraska. However, there are also reports of none overwintering of these pathogens (ll, 28, 31). The results of our study indicate that infected leaves of host and non—host plants, whether on the soil surface or buried below ground, do not allow between-season survival or Xp and pr in Michigan. Several factors may influence the survival of plant pathogenic bacteria in nature. It has been reported that maintenance under dry conditions commonly favors bacterial survival in plant residues (17, 26, 27, 32), mainly because in these tissues bacterial cells remain in a reduced state of metabolism (17). Bacterial exudates (ooze) have also been mentioned as playing an important role in the survival of the pathogens by preventing desiccation and affording protection. Wilson SE.§£ (32) reported that the longevity of Xp was considerably enhanced when bacteria were stored in exudate under different conditions of temperature, but failed to survive at high relative humidities. According to the authors, at high 109 relative humidity, the hygroscopic properties of the exudates would permit the retention of sufficient moisture to allow metabolic activity sufficient to exhaust the available reserve nutrients. On the other hand, Patrick (19) found a great abundance of microorganisms among the soil flora, capable of antagonizing most of the bacterial pathogens, and reported that Xanthomonas species were the most sensitive group. Obligately parasitic bdellovibrios, predations protozoa, and free living nematodes, have also been mentioned as influencing bacterial survival in nature (27). Moist conditions in the soil after placing the tissue samples during the fall, probably allowed an increased activity of micro- organisms; and it seems likely that blight bacteria were unable to stand the competition and gradually lost viability. The bacteria survived in dry tissues stored under laboratory conditions, where essentially no competing microorganisms could grow. The possibility that variation in survivability may be present in Xp and pr should not be overlooked in interpreting these results. In an early study, Burkholder (1) reported that bean seeds infected with common blight bacteria were obtained from symptomless pods. Recently, Weller (31) reported symptomless navy bean seed containing low population levels of blight bacteria, and that such seed was produced not only in visibly infected pods but also in symptomless pods. Results of our studies indicated that Xp was only recovered from seeds harvested from symptomless pods when plants were inoculated at the small—flat pod stage of development. This suggests that seed infection was primarily influenced by pod 110 colonization. Pods of Tepary bean exhibited the highest level of resistance and only few bacteria were recovered from the seed surfaces. Seed of moderately-resistant G.N. Valley and susceptible Tuscola exhibited similar levels of external contamination but internal seed infection was higher in the susceptible genotype. Pods of resistant and susceptible bean genotypes inoculated by scratching the dorsal suture with the needle of a syringe containing the bacterial suspension developed different disease reactions; however, seeds with and without disease symptoms of both susceptible and resistant genotypes, carried internal blight infection although at highest levels in the former. Transmission of bean blight bacteria in symptomless seed of both resistant and susceptible genotypes, suggests that tests to detect seed—borne bacterial blight should be a component in certified, blight-free bean seed production programs of all dry bean cultivars. 10. LITERATURE CITED BURKHOLDER, W.N. 1921. The bacterial blightof the bean: a systemic disease. Phytopathology 11:61-69. COYNE, D.P., M.L. SCHUSTER, and K. HILL. 1973. Genetic control of reaction to common blight bacterium in bean (Phaseolus vulgaris) as influenced by plant age and bacterial multiplication. J. Am. Hort. Sci. 98:94—99. COYNE, D.P., M.L. SCHUSTER, and S. MAGNUSON. 1976. Effect of tolerant and susceptible dry bean germplasm on seed transmission. Ann. Rept. Bean Imp. Coop. 19:20. CROSSE, J.E., and W.N. SHAFFER, Jr. 1969. Epidemiology of shoot blight caused by Erwinia amylovora. Phytopathology 59:1022-1023 (Abstr.). ERCOLANI, G.L. 1969. Epiphytic survival of Pseudomonas mors- prunorum Wormald from cherry and P, syringae van Hall from pear on the host and on the nonhost plant. Phytopathology Mediterr. 8:197—206. ERCOLANI, G.L., D.J. HAGEDORN, A. KELMAN, and R.E. RAND. 1974. Epiphytic survival of Pseudomonas syringae on hairy vetch in relation to epidemiology of bacterial brown spot of bean in Wisconsin. Phytopathology 64:1330-1339. GARDNER, M.W. 1924. A native weed host for bacterial blight of bean. Phytopathology 14:340. GROGAN, R.G., and K.A. KIMBLE. 1967. The role of seed contamination in the transmission of Pseudomonas phaseoli— cola in Phaseolus vulgaris. Phytopathology 57:28-31. GUTHRIE, J.W., D.M. HUBER, and H.S. FENWICK. 1965. Serological detection of halo blight. Plant Dis. Reptr. 49:297—299. HARRISON, F.C., and B. BARLOW. 1904. Some bacterial diseases of plants prevalent in Ontario. Ontario Agr. C01. and Exp. Farm Bul. 136, 20 pp. 111 11. 12. l3. 14. 15. l6. 17. 18. 19. 20. 21. 22. 23. HEDGES, F. 1946. Experiments on the overwintering in the soil of bacteria causing leaf and pod spots of snap and lima beans. Phytopathology 36:677-678. ISAKA, M. 1969. Studies on bacterial leaf blight of rice plant. On some grasses and weeds as carriers of the pathogen. Proc. Assoc. Pl. Prot. Kyushu 17:14-19. KENNEDY, B.W., and G.L. ERCOLANI. 1978. Soybean primary leaves as a site for epiphytic multiplication of Pseudomonas glycinea. Phytopathology 68:1196—1201. LATORRE, B.A., and A.L. JONES. 1978. Survival and patho— genicity to sour cherry of Pseudomonas syringae recovered from weeds and plant refuse. Phytopathology News 12:137 (Abstr.). LAUB, C.A., and R.E. STALL. 1967. An evaluation of Sclanum nigrum and Physalis minima as suspects of Xanthomonas ‘ vesicatoria. Plant Dis. Reptr. 51:659-661. LEBEN, C. 1961. Microorganism on cucumber seedlings. Phytopathology 51:553-557. LEBEN, C. 1974. Survival of plant pathogenic bacteria. Spec. Circ. l-O. Ohio Agric. Res. Devel. Center, Wooster, OH. 21 pp. LEDENBERG, J., and E.M. LEDENBERG. 1952. Replica plating and indirect selection of bacterial mutants. J. Bacteriol. 63:399-406. PATRICK, Z.A. 1954. The antibiotic activity of soil micro— organisms as related to bacterial plant pathogens. Can. J. Bot. 32:705—735. RINGGLE, J.H., and E.K. KLOS. 1972. Relationship of Erwinia. herbicola to Erwinia amylovora. Can. J. Bot. 50:1077— 1083. SABET, K.A., F. ISHAG, and O. KHALIL. 1969. Studies in the bacterial diseases on Sudan crops. VII. New records. Ann. appl. Biol. 63:357. SAETTLER, A.WQ, and S.K. PERRY. 1972. Seed-transmitted bacterial diseases in Michigan navy (pea) beans, Phaseolus vulgaris. Plant Dis. Reptr. 56:378-381. SCHNEIDER, R.W., and R.G. GROGAN. 1977. Bacterial speck of tomato: sources of inoculum and establishment of a resident population. Phytopathology 67:388—394. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 113 SCHUSTER, M.L. 1955. A method for testing resitance of beans to bacterial blights. Phytopathology 45:519-520. SCHUSTER, M.L. 1967. Survival of bean bacterial pathogens in the field and greenhouse under different environmental conditions. Phytopathology 57:830 (Abstr.). SCHUSTER, M.L. 1970. Survival of bacterial pathogens of beans. Bean Impr. Coop. 13:68-70. SCHUSTER, M.L., and D.P. COYNE. 1974. Survival mechanisms of phytopathogenic bacteria. Ann. Rev. Phytopathol. 12:199— SUTTON, M.D., and V.R. WALLEN. 1967. Phage types of Xanthomonas phaseoli isolated from beans. Can. J. Bot. 45:267-280. WALKER, J.C., and PATEL, P.N. 1964. Splash dispersal and wind as factors in epidemiology of halo blight bean. Phytopathology 54:140-141. WALLEN, V.R., and M.D. SUTTON. 1965. Xanthomonas phaseoli var. fuscans (Burk.) Starr and Burkh. on field bean in Ontario. Can. J. Bot. 43:437-446. 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., East Lansing, 137 pp. WILSON, H.A., V.G. LILLY, and J.G. LEACH. 1965. Bacterial polysaccharides. IV. Longevity of Xanthomonas phaseoli and Serratia marcescens in bacterial exudates. Phytopathology 55:1135-1138. ZAUMEYER, W.J. 1929. Seed infection by Bacterium phaseoli. Phytopathology 19:96 (Abstr.). ZAUMEYER, W.J. 1930. The bacterial blight of beans caused by bacterium phaseoli. U.S.D.A., Tech. Bul. 186, 36 pp. ZAUMEYER, W.J., and H.R. THOMAS. 1957. A monographic study of bean diseases and methods for their control. U.S.D.A. Tech. Bul. 868, 255 p. APPENDIX Preliminary Ultrastructural Evidence for Immobilization of Xanthomonas phaseoli in Tepary Bean (Phaseolus acutifolius) Leaves Common blight, caused by Xanthomonas phaseoli (Xp) is considered one of the most serious seed-borne diseases of dry edible and green beans in many production areas throughout the world (2, 7). Although practical short-term control is possible through the use of disease-free seed and crop rotation, long-term control depends on the development of disease resistant cultivars. Considerable effort has been directed toward finding resistant germplasm useful to breeding (2, 5, 7) and until now only certain accessions of Tepary bean (Phaseolus acutifolius) exhibit the highest levels of resistance to a range of Xanthomonas blight isolates. Our greenhouse and field studies compared multiplication and distribution patterns of Xp (RlS-l rifampin—resistant isolate) in bean genotypes possessing different levels of disease resistance, and the results confirmed previous findings. Although.Xp was able to survive in inoculated leaf tissues of Tepary bean (Arizona-Buff) for relatively long periods of time, bacterial populations remained at stationary phase and then declined. Also, Tepary exhibited resistance to systemic colonization, and Xanthomonas blight symptoms were never observed throughout the experiments. Elucidation of the physiological-biochemical bases for disease resistance in plant has remained as elusive a goal for researchers concerned with bacterial diseases as it has for those concerned with other pathogens. Attachment and envelopment (8) or immobilization (3) of incompatible and/or saprophytic bacteria by plant cell walls 114 115 appears to be a general phenomenon and has been reported in several host-bacteria systems (1, 3, 6, 8, 9). Such phenomena have been proposed as a major host defense mechanism against bacteria (3, 9). Recently Sequeira (8) suggested that this phenomenon may also occur in resistant host-parasite combinations. Xanthomonas-resistant Tepary bean (Arizona—Buff) and susceptible cv. Tuscola plants were greenhouse-grown in a standard soil mix and inoculated when the first trifoliolate leaf was fully expanded, 22 days after planting. Leaves were inoculated witth8 cells/ml suspension of Xp (R15—1 rifampin-resistant isolate), on the lower surfaces using a sprayer attached to a compressed air line (17 p.s.i.L As a result, the inoculation sites (approx. 0.5-1.0 mm diameter) of both resistant and susceptible leaves retained a watersoaked appearance for approximately three hours. At one, three, six and 18 hours, and four and eight days after inoculation, leaf samples were taken and prepared for transmission electron microscope (TEM). Sections one mm square were cut from inoculated areas of the leaf and placed in 5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4 C. The tissues were then washed twice with the same buffer during a one-hour period and post—fixed over- night in 1% osmium tetroxide in 0.1 M phosphate buffer at 4 C. The material was then washed, dehydrated in an ethanol series, and left overnight in 100% ethanol. After a transition period through acetone, sections were finally embedded in a mixture of Epon—Araldite/ ERL Epoxy Resine (4). Ultrathin sections were stained in uranyl acetate and then in lead citrate. Sections were examined with a 116 Philips 300 TEM, operated at 60 kV. Tepary bean (resistant) Three hours after inoculation, most bacteria in leaves of resistant Tepary bean were found in the intercellular spaces in close proximity to host cell walls (Fig. 1). Some loose fibrillar material, possibly released from the bean cell walls, were observed at that time between cell wall and the bacterium (Fig. 2). Attachment of Xp bacteria to host cell walls was detected 18 hours after inoculation (Fig. 3, 4, and 5). Macroscopically (48 hours after inoculation) the infiltrated sites possessed a clearly defined necrotic border between the watersoaked area and the surrounding green healthy tissue; by 72 to 96 hours all of the inoculated area was brown and necrotic; at the ultrastructural level, complete disruption of cellular organelles was observed. There was no evidence of multiplication by the immobilized bacteria, and complete encapsulation as described in tobacco leaves (3, 8) was never observed. However, "blister-like" structures were frequently found after 24 hours only in the cell walls of inoculated Tepary bean leaves (Fig. 6). Attachment occurred before observing hypersensitivity, which agrees with other bacteria/host cell wall interactions (3, 8). Tuscola bean (susceptible) In leaves of susceptible cultivar Tuscola, watersoaking reappeared at the infiltrated sites 24 hours after inoculation; by 96 hours the watersoaking had expanded beyond the inoculated sites and a faint chlorosis was observed at the upper leaf surfaces. Eight days 117 after inoculation typical Xanthomonas blight symptoms developed. At the ultrastructural level, no attachment of the bacteria to host cell walls was observed and Xp was seen to divide soon after in— oculation. At 18 hours, large numbers of bacteria were observed in the intercellular spaces (Fig. 7), and eight days after inoculation complete disorganization of the cytoplasms and host cells collapse was evident (Fig. 8). The interaction between bacteria and tobacco mesophyll cell walls and their relation to the various types of resistant reactions which occur in tobacco leaves, have been described in detail by Sequeira et_él (8). Attachment of incompatible strains of R. solanacearum to tobacco mesophyll cell walls appeared to be an essential step in the process that lead to HR. According to the authors, the attachment process may essentially be a "recognition" phenomenon that may involve interactions between specific constituents of the bacterial cell walls and binding sites on the host cell walls. Sing and Schroth (9) reported active immobilization of saprophytic bacteria in the intercellular spaces of bean leaves; immobilization was not observed when a pathogenic bacteria, Pseudomonas phaseolicola, was infiltrated into leaves of susceptible bean plants. The authors suggested that bean lectins may be involved in the attachment and encapsulation processes. Recently, Roebuck et 31 (6) reported that most P, phaseolicola cells "appeared to be attached" to host cell walls after infiltration of the bacteria in the intercellular spaces of leaves of a halo blight resistant bean cultivar. They suggested that bacterial attachment to host cell walls may allow the transfer of some 118 factor between bacteria and the host protoplast which may trigger an HR. According to Goodman gt a1 (3) "it is under conditions of low inoculum doses, which are more likely to occur in nature, that the active process of bacterial immobilization assumes real importance as a resistance mechanism". This study indicates that attachment of Xp, a bean pathogen, occurs in the intercellular spaces of leaves of blight-resistant Tepary bean (Arizona-Buff). Further investigation is required to determine whether the phenomenon is directly or indirectly involved in the resistance response of if other defense mechanisms are also operating in this host—pathogen interaction. Abbreviations used in Figures: B, bacterium; C1, chloroplast; Figures 1 to 5. Figure 1. Figure 2. Figures 3, 4, and 5. Figure 6. W, cell wall Transmission electron micrographs of spongy meso- phyll cells in resistant Tepary bean (Phaseolus acutifolius) leaves showing the interaction of host cell wall and common blight bacteria (Xanthomonas phaseoli). Cross section of bacteria at three hours after inoculation. Note group of bacteria aligned close to the host cell walls. (x 12 500). Cross section of bacterium in intercellular space three hours after inoculation. Note loose fibrillar material on the host cell wall in close proximity to the bacterium (arrow). (x 60 000). Cross sections of bacteria attached to host cell wall matrix 18 hours after inoculation. Note matrix attached to bacterial cell (arrows). (Fig. 3 and 5 x 32 000; Fig. 4 x 25 000). Cross section of intercellular space 96 hours after inoculation. Structure (arrow) that resembles those typically reported to completely encapsulate bacteria (x 52 000). 119 Figure 7 and 8. Figure 7. Figure 8. 120 Transmission electron micrographs of spongy mesophyll cells in Xanthomonas blight susceptible cv. Tuscola (Phaseolus vulgaris) leaves. Bacteria in susceptible host 18 hours after inoculation. Note large number of bacterial cells (x 16 000). Bacteria in the lesion area of susceptible host genotype eight days after inoculation (x 3 200). 121 122 123 o .0. .‘O- ‘.‘s ........l: ‘ buff ‘3 A ' ....:‘q'9o‘.1$:‘:’: , ‘ g. i“! \ ”3‘8: :0. .4: V U Literature Cited BOGERS, R.J. 1972. On the interaction of Agrobacterium tumefaciens with cells of Kalanchoe daigremontiana. In: Proc. 3rd Int. Conf. Plant Pathogenic Bacteria, ed., H.P. Maas Geesteranus, p.p. 239-50. Wageningen, the Netherlands, Cent. Agri. Publ. Proc. 365 pp. COYNE, D.P., and M.L. SCHUSTER. 1974. Breeding and genetic studies of tolerance to several bean (Phaseolus vulgaris L.) bacterial pathogens. Euphytica 23:651—656. GOODMAN, R.N., P.Y. HUANG, and J.A. WHITE. 1976. Ultra— structural evidence for immobilization of an incompatible bacterium, Pseudomonas pisi, in tobacco leaf tissue. Phytopathology 66:754—757. HOPPER, R.G., K.K. BAKER, and S.L. FLEGER. 1979. Exercises in Electron Microscopy: A Laboratory Manual for Biology and Medicine. Appendix 6. Published by MSU. pp. LEAKEY, C.L.A. 1973. A note of Xanthomonas blight of beans (Phaseolus vulgaris L.) and prospects for its control by breeding for tolerance. Euphytica: 132-140. ROEBUCK, P., R. SEXTON, and J.W. MANSFIELD. 1978. Ultra- structural observations on the development of the hyper- sensitive reaction in leaves of Phaseolus vulgaris cv. Red Mexican inoculated with Pseudomonas phaseolicola (race 1). Physiological Plant Pathology 12:151-157. SAETTLER, A.W. 1977. Breeding dry edible beans (Phaseolus vulgaris L.) for tolerance to Xanthomonas bacterial blights. Fitopatolcgia Brasileira 2:179-186. SEQUEIRA, L., G. GAARD, and G.A. DeZoeten. 1977. Interaction of bacteria and host cell walls: its relation to mechanism of induced resistance. Physiological Plant Pathology 10:43-50. SING, Y.O., and M.N. SCHROTH. 1977. Bacteria-plant cell surface interactions: active immobilization of saprophytic bacteria in plant leaves. Science 197:759-761. 124 IIIIIIIWIWIIHIIHIIIIllllllllll"llWIIWIIIIIIIIWHII