MSU LIBRARIES \r RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. PATHOLOGIC VARIATION IN MALAWIAN ISOLATES OF PSEUDOMONAS SYRINGAE PV. PHASEOLICOLA (BURK.) YOUNG, DYE, AND WILKIE, AND IMPLICATIONS FOR BREEDING DISEASE RESISTANT BEANS By Wilson A. B. Msuku 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 1984 ABSTRACT PATHOLOGIC VARIATION IN MALAWIAN ISOLATES OF PSEUDOMONAS SYRINGAE PV. PHASEOLICOLA (BURK.) YOUNG, DYE, AND WILKIE, AND IMPLICATIONS FOR BREEDING DISEASE RESISTANT BEANS By Wilson A. B. Msuku Halo blight of beans (Phaseolus vulgaris L.) incited by Pseudomonas syringae pv. phaseolicola (Burk.) Young, Dye, Wilkie is an important disease in Malawi. Thirty two isolates of P. syringae pv. phaseolicola obtained from various bean growing areas of Malawi were separated into four patho— types on the basis of their differential pathogenicity. Pathotype determinations were based on both foliage and pod reaction of the five bean cultivars, Red Mexican U13, Red Mexican UI34, Great Northern 123, Jubila and Namajengo. Cultivars Nasaka and Montcalm were included as susceptible and resistant controls respectively. Standard isolates of race 1 and race 2 were included for comparison. Thirteen Malawian isolates plus race 1 isolate were grouped as patho- type 1; sixteen Malawian isolates plus race 2 isolate were determined to be pathotype 2; two isolates made up pathotype 3; and one isolate represented pathotype 4. Wilson A. B. Msuku The isolates were also ranked for virulence on the basis of l) the diameter of water—soaked lesions caused on pods and 2) number of water—soaked lesions induced on spray inoculated leaves of the susceptible cultivar Nasaka. The most virulent isolates were those of pathotype 2 while isolates belonging to pathotype l were least virulent. Virulence was positively correlated to the rate of toxin production by P. syringae pv. phaseolicola isolates. How— ever, studies showed no relationship between virulence and motility. Antisera produced against seven Malawian P. syringae pv. phaeolicola isolates grouped the Malawian isolates into 5 distinct serotypes. However, all but one Malawian iso- lates serologically shared some antigenic properties. This isolate was the only isolate exhibiting rough colony form. The genetic basis of resistance to pathotype 2 of P. syringae pv. phaseolicola was investigated in crosses between the resistant cultivar Montcalm and eight Malawian cultivars/ lines, Nasaka, 600¥lD, 1212D, 2586D, 2589B, 26OOD, 2609D and 2610D. Analysis of F1 and F2 progenies showed that resistance in Montcalm was controlled by two dominant comple— mentary genes. In addition, an allelomorphic series of three alleles was assumed to control resistance in comple— mentation with either of two genes in 1212D. Studies also indicated linkage between resistance and flower color. In memory of my grandfather Isaac N. Luhanga ii ACKNOWLEDGMENTS The author wishes to express his sincere appreciation and gratitude to the following: Dr. A. W. Saettler for his interest, encouragement and guidance throughout his study. Dr. J. D. Kelly, who served as a member of the Guidance Committee, for his guidance in the genetic studies and his invaluable criticisms in the preparation of this manuscript. Drs. D. Fulbright and C. Stephens for their kind ser- vice in the Guidance Committee, understanding and critical review of this manuscript. Tom Harpstead, Bonnie Wright, Sally Evans, S. D. Manyela and K. K. Mthobwa for their technical assistance. His wife Doreen, and children Lindizga, Mwabi and Bongani for their help, love and understanding. F.A.O. of the United Nations for granting the scholar- ship to study in the United States of America, this fellow- ship is gratefully appreciated. iii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . LIST OF FIGURES . . . . . . . . INTRODUCTION AND LITERATURE REVIEW . . . . . . . GENERAL MATERIALS AND METHODS . . Sources and Isolation of Isolates . . Seed Source . . . . . . . . . . . . . . DETERMINATION OF RACES OR PATHOTYPES Materials and Methods . . . Pod Inoculation Techniques . Seedling Injection Technique . . . . . . . . Spray Inoculation Technique . . . . . Results . . ... . . . . . . . . . . . . . . . . Race Determination . . . . . . . . . . . Virulence Determination . TOXIN PRODUCTION STUDIES . . . . . . . . . . . Materials and Methods . . . . . . . . . . . . . Bacterial Strains . . . . . . . . . . Media Toxin Bioassay . . . . . Results . . . . . . . . . . . . . . . . . iv Page vii ix 11 12 12 12 14 15 16 16 22 26 27 27 28 28 29 MOTILITY STUDIES . . . . . . Materials and Methods . . . Halo blight strains . Media . . . . . . . . Procedure . . . Results . . . . . . . . . . SEROLOGICAL STUDIES . . . . . Materials and Methods . Antigen preparation and immunization of rabbits . Collection and fractionation of antisera . . . . . . . Serological Techniques Results . . . . . . Reaction of antisera against steamed cell antigens . . . . . Absorption test . . DISCUSSION . . . . . . . . . . INHERITANCE OF RESISTANCE TO HALO BLIGHT IN Phaseolus vulgaris L. Materials and Methods . Bacterial Isolates . . . Bean Plants . . . . . . Results . . . . . . . Discussion . . . . . . . . BREEDING IMPLICATIONS AND STRATEGIES Bean Production in Malawi Page 32 33 33 33 33 34 39 39 39 4O 41 43 43 45 51 58 59 59 59 65 72 75 75 Page Breeding implications . . . . . . . . . . . . . . 76 Pathogenic Variation . . . . . . . . . . . . . 76 Method Of inoculation, quality and quantity of inoculum . . . . . . . . . . . . . 78 Developmental stage and nutritional status of the host . . . . . . . . . . . . . . 80 Site for evaluating breeding materials . . . . 80 Method of evaluating disease reaction . . . . 81 Breeding Strategies . . . . . . . . . . . . . . . 82 Source of resistance . . . . . . . . . . . . . 82 Inoculation of plants with mixtures of Psp pathotypes . . . . . . . . . . . . . . 83 Use of diseased seed as disease Spreaders . . 84 LITERATURE CITED ... . . . . . . . . . . . . . . . . 85 vi LIST OF TABLES Table Page 1. Location, altitude, host and identifica— tion of the Pseudomonas syringae pv. phaseolicola isolates . . . . . . . . . . . 10 2. Foliage reaction of bean cultivars to inoculation with several isolates of Pseudomonas syringae pv. phaseolicola . . . l7 3. Pod reactions of bean cultivars inoculated with several isolates of Pseudomonas syringae pv. phaseolicola . . . . . . . . . 21 4. Average diameter and range (mm) of water— soaked spots induced by 34 isolates of Pseudomonas syringae pv. phaseolicola on pods of bean cultivar Nasaka . . . . . . 23 5. Average number and range of water-soaked lesions induced by 34 isolates of Pseudomonas syringae pv. phaseolicola on leaves of bean cultivar Nasaka . . . . . 23 6. Inhibition zones of Escherichia coli induced by several isolates of Pseudomonas syringae pv. phaseolicola . . . . . . . . . 31 7. A comparison of bacterial migration rates and numbers of lesions induced on leaves of bean cultivar Nasaka by six Pseudomonas syringae pv. phaseolicola isolates . . . . 38 8. Serotype grouping of several Pseudomonas syringae pv. phaseolicola isolates . . . . 46 9. Reactions of different halo blight isolates in tube agglutination tests against Pseudomonas syringae pv. phaseolicola antisera . . . . . . . . . . . . . . . . . 47 lO. Reactions of different halo blight iso— lates to Pseudomonas syringae pv. phaseolicola antisera in agar gel double—diffusion tests . . . . . . . . . . 48 vii 11. 12. 13. 14. 15. 16. 17. Reaction of homologous and heterologous Pseudomonas syringae pv. phaseolicola antigens to absorbed antisera . . . Precipitation (mm) and mean temperature (°C) during the growing season in major bean growing districts of Malawi . . Seed characteristics of Phaseolus vulgaris genotypes used as parents in inheritance study . . . . . . . . . . . Reaction of Phaseolus vulgaris genotypes to pathotype 2 of Pseudomonas syringae pv. phaseolicola . . . . . . . . . . . Parental, F1 and F2 reactions to pathotype 2 of Pseudomonas syringae pv. phaseolicola and expected ratios of resistant (R), and susceptible (S) plants . ... . . . . . . . . . . . . . Proposed genotypes of 9 Phaseolus vulgaris genotypes based on their reaction to infection by pathotype 2 of Pseudomonas syringae pv. phaseolicola . . . . . . Segregation of plants for halo blight (pathotypes 2) reactions in F2 of bean crosses . . . . . . . . . . . . . . . viii Page 49 61 63 64 68 69 71 LIST OF FIGURES Figure Page 1. Leaves of bean cultivars of Nasaka (left) and Red Mexican U134 (right) showing typical water-soaked lesions surrounded by chlorotic halos . . . . . . . 8 2. Symptom types on green pods of Jubila: 3='SC' type = susceptible reaction- con- centric patterns of green and brown lesions; l3='SN' type = susceptible reaction- dark brown necrotic lesions with macerated tissue; 12='RN' type = resistant reaction- dry needle hole with brown necrotic halo, tissue not macerated; l4='SW' type = susceptible reaction— light to dark green water-soaked lesions with or without bacterial ooze; and C='RD' type = resistant reaction- dark needle holes with no chlorosis . . . . . . . . . . l9 3. Histogram showing frequency distribution of 34 isolates of Pseudomonas syringae pv. phaseolicola in relation to diameter of water—soaked spots caused on pods of bean cultivar Nasaka. LSD.05=1.1 . . . 24 4. Linear relationship between virulence (expressed as number of lesions pro— duced on inoculated leaf) and toxin production of Pseudomonas syringae pv. phaseolicola isolates . . . . . . . . . . 3O 5. Test tubes showing bacterial migration bands in a basal semi-solid medium . . . . 35 6. Movement of bacterial migration bands of Pseudomonas syringae pv. phaseolicola isolates in semi—solid medium . . . . . . . 36 7. Smooth (HB15) and rough (HB35) colony forms of Pseudomonas syringae pv. phaseolicola . 50 ix INTRODUCTION AND LITERATURE REVIEW Halo blight of beans (Phaseolus vulgaris L.) is a bacterial disease caused by Pseudomonas syringae pv. phase- olicola (Burk.) Young, Dye, and Wilkie, (Psp). The disease was first described and reported from the state of New York by Burkholder in 1926 (5). Since then it has been reported from many countries of Central and South America, Africa and Europe (12, 28, 34, 50, 60). In Malawi, halo blight is one of the most widespread, destructive and economically important plant diseases of dry beans (12) and is among the diseases that develop early in the growing season in most bean growing areas of the country. The disease affects all parts of the plant above ground (5, 64). Characteristic leaf symptoms (Figure l) initially appear as small, brown water—soaked spots on the undersides. Later, a halo—like zone of greenish—yellow tissue develops around the water—soaked area. In cases of systemic plant infection, a general foliage chlorosis, leaf malformation and stunting may result without the appearance of much leaf spot infection. On pods, lesions appear as green water- soaked spots which may enlarge and coalesce. Stem lesions appear as reddish necrotic longitudinal lesions. Very small spots may appear on the seed, or the entire seed may be destroyed. In pigmented seed it is difficult to distinguish the spots caused by halo blight bacteria from those caused by other bean pathogens such as Colletotrichum lindemuthianum (Sacc. and Magn.) Scrib. (causative organism of bean anthra- cnose) and Xanthomonas campestris pv. phaseoli (E.F.S.) Dows. (causative organism of bean common blight). The pathogen Psp is seed borne (48) and can survive in infected plant residues (50, 64). Wilson, cited by Zaumeyer and Thomas (64) reported that soil carryover was not an important source of Psp inoculun from one growing season to the next in Australia. The bacteria reportedly survived for nine months after ingestion and passage through sheep (52). Knowledge as to how a pathogen survives and disseminated is very important for developing successful control strategies. It is clear, also, that contaminated seed is important for both local and long distant transport of the disease. Burkholder indicated that successful infection by halo blight bacteria could only take place through wounds. How- ever, later studies (50, 60, 64) revealed that the pathogen also gained entrance through stomata during periods of free moisture or high relative humidity (95-98% for 24 hours after inoculation). Light also influences the plant and nature of its response to Psp (25). Incidence of halo blight in the plant is generally greatest under conditions of low temperatures and high rela— tive humidity or free moisture (38, 50, 64). Patel and Walker (38) reported that development of halos and pronounced systemic chlorosis developed more commonly at l6—20°C than 24—28°C. They also noted that age and nutritional status of the host greatly influenced disease development. Older leaves were more tolerant than younger leaves, while extreme— ly low and high levels of Nitrogen, Phosphorus and Potassium retarded the development of both halo blight and common blight symptoms. While seed is generally the main source of primary inoculum, spread of Psp between leaves and plants in the field is believed to be due mainly to splash and wind dispersal during periods of rainfall (60). Incidence of halo blight was lower in a bean/maize association than in bean monoculture (32, 50) suggesting that maize may have served as a physical barrier to disease spread between bean plants in the association. Insects, animals and man also aid dissemination of the pathogen within and between fields (64). The pathogen Psp exhibits very distinct morphological, physiological and nutritional characteristics. Cells of Psp appear as single straight rods (44, 50) and are motile with peritrichous flagella. The bacteria are gram negative, strictly aerobic and do not require growth factors. Poly— B—hydroxy butyrate is not accumulated as an intracellular carbon reserve. In artificial media, especially those defi— cient in iron, the bacteria produce a diffusable fluorescent pigment. Arginine dihydrolase activity is absent. Gluta- rate, mesotartrate, DL-glycerate, iso—ascorbate, erythriol, sorbitol, meso-inositol or N—caproate are not utilized but the bacteria do utilize D—gluconate, L (+) arabinose, suc- rose, succinate, DL-B—hydroxybutyrate, transaconitate, L-serine, L-alanine and p—hydroxy benzoate. The optimum temperature for growth is 20-23°C. White to cream colonies with a bluish hue are produced which may be either smooth or rough (I, 64). The great losses in worldwide and specifically Malawi bean production associated with the halo blight disease warrant the development of control measures. Measures such as the use of disease-free seed and eradicant chemicals are being utilized with some success (12). Disease—free seed is produced in Malawi by growing bean seed under furrow irrigation during the dry cool season (May—August). A bi— weekly spray program with copper oxychloride at 4.5g per litre has effectively controlled halo blight (12). Workers elsewhere (43, 50, 64) have also reported that halo blight can effectively be controlled chemically, using bordeaux mixture, copper oxide, streptomycin sulfate and dihydrostrep— tomycin sulfate. However, control with chemicals may not always be effective or practical (50, 62). Moreover, chemi— cals are relatively expensive for growers in developing countries such as Malawi and may be difficult to obtain. Use of resistant varieties as a means to control halo blight has been successful in several areas of the world (3, ll, 50, 63, 64). Unfortunately, Malawi has no program devoted to the development of halo blight resistant bean varieties. Such a breeding program must take into considera- tion both variability in the pathogen and presence of gene— tic resistance in the host. Pathogenic variation in Psp has been confirmed numerous times (6, 9, 24, 25, 26, 28, 37, 49) since Jensen and Living- ston (26) distinguished 3 pathogenicity groups in 13 halo blight isolates on the basis of symptoms produced in sus- ceptible Red Kidney bean. Group 1 isolates induced lesions with halos, group 2 isolates induced lesions without halos, and group 3 isolates induced lesions with and without halos. The authors also observed that isolates which were able to produce lesions with halos or those that produced mixed lesions also induced the greatest number of lesions which led to their observation that toxin-producing strains were more virulent than nontoxin-producers. Patel and Walker in 1965 (37) established the existence of races 1 and 2 in the United States. The distinction between these 2 races was based on disease reactions induced in cultivar Red Mexi— can UI3. Red Mexican UI3 is resistant to race 1 but sus- ceptible to race 2. Since then the presence of these two races have been reported from numerous other countries (6, l4, l7, 19, 25, 28, 61). However, numerous workers believe that the designation of only 2 races is not valid (47, 49, 54, 55). Schroth et a1 (49) suggested that there are num— erous strains of Psp with varying degrees of virulence. Schuster and his colleagues (47) reported the isolation of a halo blight isolate (possible race 3) in Nebraska which was more virulent than known race 2 isolates. Szarka and Velich (54, 55) also observed that Psp does not consist of two races only but of a series of strains or pathotypes which can be grouped on the basis of increasing pathogenicity. Variation in virulence of halo blight strains has been attri— buted to differences in the rate at which they produce toxin (25, 26, 41) and in motility (33, 35). Mulrean and Schroth (33) suggested that motility in Psp may be regulated by chemicals produced by potential infection sites on leaves. Other than virulence, Psp isolates can also be differentiated by use of serological techniques. Serology has become a very rapid and useful technique for detection and identification of various plant pathogenic bacteria (2, 15, 18, 51, 56, 58). Serological studies of halo blight by Taylor (56), Guthrie (l6) and Guthrie et a1 (18) indicate that Psp antiserum is pathovar specific but not race specific. Good knowledge of the genetic variability and accurate identification and/or recognition of a pathogen is very important in the control of plant diseases. It is also equally important to understand the genetic inheri- tance of resistance of a host plant to a particular disease, especially, where disease resistance is chosen as a means of controlling that disease. Plant resistance to halo blight is well known and is thought to involve both specific and general resistance mechanisms to strains differing in virulence (50). While resistant bean cultivars have been recognized for some time, the general pattern of inheritance of resistance to the disease is not well understood (SO, 63). Some workers report that disease reactions in segregating populations suggest a single major gene, with resistance due to the recessive condition (36, 46, 57). Others (7, 8, 20, 22, 36) report that resistance is due to one or several major dominant genes. Coyne and Schuster (8) indicated that although a single major dominant gene primarily determines resistance in great Northern Nebraska #1, numerous modifying genes also affect the degree of expression of resistance. Hong and Frazier (22) report made a similar interpretation. Quantitative inheritance has also been suggested in certain segregating populations (23). The present study addressed two objectives, namely 1) characterization of the range of pathogenic potential in isolates of Psp collected from different ecological zones of bean production in Malawi, and 2) investigation of the inheritance of resistance in crosses between several Malawian bean lines and Montcalm, a halo blight resistant dark red kidney bean cultivar developed at Michigan State University. It is intended that the results of these studies would greatly assist the bean breeding program being developed at Bunda College of Agriculture, Lilongwe, Malawi. Figure 1. Leaves of bean cultivars of Nasaka (left) and Red Mexican UI34 (right) showing typical water—soaked lesions surrounded by chlorotic halos. GENERAL MATERIALS AND METHODS Sources and Isolation of Isolates Thirty two isolates of Psp were Obtained from various bean growing regions of Malawi during the 1981/82 growing season. In addition, isolates race 1 and race 2 received from Dr. D. Hagedorn (University of Wisconsin), were in— cluded for comparison (Table 1). All Malawi strains were isolated from infected bean plant tissues, mainly leaves and pods. Pieces of diseased tissue including small areas of adjoining healthy tissue were excised from naturally infected plants. The pieces were surface sterilized with 10% sodium hypochlorite for 1 minute and rinsed twice with sterile glass distilled water. The tissue pieces were then transferred into sterile plastic petri dishes containing four to five drops of sterile glass distilled water and crushed to release the bacterial cells. With the aid of a sterile loop, the bacterial sus— pensions were streaked onto plates containing Kings medium B agar (KMB) (Peptone, 20g; K HPO .3H 2 4 2 4 2 gylcerol,15 ml; agar, 20g; and distilled water, lOOOml), 0, 2.5g; MgSO .7H 0, 6g; and incubated at 25°C. Bacterial colonies resembling Pseudomonas were purified by a series of single colony transfers and then verified 10 TABLE 1. Location, altitude, host and identification of the Pseudomonas syringae pv. phaseolicola isolates Location District Altitude(m) Identification Mhuju Rumphi 1200 HBl, HB3 Ehehleni Mzimba 1230 HB4, HB5, HB6, HB7, HB8, HB9, HB10, HBll Mzimba Hills Mzimba 1290 HB12 Dowa Hills Dowa 1350 HBl3, HB14, HB15, HB16, HB17, HBl8, HB19 Bunda Lilongwe 1020 HB20, HB23, HB24 Chitedze Lilongwe 1020 HB25, HB26, HB27 Kadembo Dedza 1467 HB29 Chilobwe Ntcheu 1440 HB34, HB35 Bvumbwe Thyolo 870 HB36, HB37 Phalombe Mulanje 1060 HB38, HB39, HB4O Dr. D. Hagedorn University 500 Race 1, Race 2 Of Wisconsin, U.S.A. 11 as Psp by biochemical and pathogenicity tests. These tests included fluorescent pigment production on KMB, gram nega— tive rod shaped cells, negative reaction in arginine dihydro- lase and oxidase tests, nonutilization of casein, hyper- sensitivity in tobacco leaves and production of typical water-soaked lesions on inoculated leaves Of the two sus- ceptible bean cultivars, Nasaka and Manitou. Isolates were maintained on KMB slants at 4°C and in infected plant tissue samples stored at 4°C. Seed Source Seed of Nasaka and Namajengo were Obtained from The Bean Research Programme of Bunda College of Agriculture, Malawi. Red Mexican U1 3 seed was obtained from Dr. D. Hagedorn.(University of Wisconsin) and increased in the greenhouse. Seed of the remaining bean cultivars Manitou, Great Northern 123, Red Mexican UT 34, Jubila and Montcalm were obtained from stocks maintained by Dr. A. W. Saettler at Michigan State University. DETERMINATION OF RACES OR PATHOTYPES Despite the importance of halo blight in Malawi, no information is available as to which races exist in the country. Therefore, the aim of this study was to determine the pathogenic variation and virulence of thirty two isolates of Psp collected from different bean growing districts of Malawi. The determination of race or pathotype (24) for each of the thirty two isolates was based on pod and foliage reactions of five bean cultivars; Red Mexican U1 3, Red Mexican UI 34, Great Northern 123, Jubila and Namajengo. Cultivar Nasaka was always used as a susceptible control. In later studies, Montcalm variety was added as a tolerant control. Materials and Methods Pod Inoculation Technique: Inoculum Preparation- Each Psp isolate was grown on KMB for 24-48 hours at 25°C. Suspensions were made by add— ing 20ml of sterile glass distilled water to each plate, and dislodging the bacteria. Suspensions were then trans- ferred to a 2x14.4cm test tube and diluted to an optical density of 0.3 (approx. 1x106cfu per ml) at a wave length 12 13 of 620 mu using a spectronic 20 colorimeter (Bausch and Lomb Co.). Inoculation of Pods— Bean pods were removed from either field grown or greenhouse grown plants. Debris was removed by washing in running tap water for ten minutes, and the pods were then surface sterilized by immersion in either 10% sodium hypochlorite or 90% ethyl alcohol for 1 minute, and then rinsing in sterile glass distilled water. Pods were examined for freedom from superficial injuries and selected for uniformity in size and maturity. Two lots were selected. One lot was composed of pods with well filled seeds and the other lot was composed of succulent immature pods, partially filled or no seeds in the pods. One pod from each lot was inoculated with one of the thirty four Psp isolates. Pods were pricked while immersed in the bac— terial suspension (13) contained in sterilized plastic petri dishes. Depending on pod size (variable with individual bean variety) 4—6 stabs were performed to a depth of 2mm using a flame sterilized dissecting needle. Inoculations were restricted to one side of the pod. The pods were then placed side by side in moist plastic boxes with the inocu— lated sides up and incubated at 20i2°C. Pods were examined and observations made of lesion types. Quantitative measure— ments of lesion size were also made. l4 Seedling Injection Technique: Growing of Bean Plants- Two series of plants of each cultivar were utilized in this study. One set of plants was grown in a growth chamber maintained at 25il°C and 16 hours of light prior to inoculation. After inoculation the temperature was reduced to 21il°C. In this experiment, plants were grown in 10cm diameter plastic pots (3 plants per pot) containing a pasteurized 3:1 mixture of soil and vermiculite. In the second set, plants were grown in a greenhouse with temperature fluctuating between 24-28°C. Clay pots of 12.5cm diameter were used with a 3:1 mixtflre of either soil/vermiculite or soil/peat. Plants were watered as needed with tap water. Plant Inoculation- Plants possessing fully expanded primary leaf and partially unfolded trifoliolate leaf were selected for injection (approximately 10-12 days after plant— ing). Bacterial inoculum was introduced into the plant by use of a 250 5/8 (15.9mm) needle attached to a 3m1 sterile disposable syringe. The needle was passed obliquely through the stem at the primary leaf node. Fluid was slowly dripped out as the needle was withdrawn (42). Plants were observed for disease reactions up to 3 weeks after injection. The most common susceptible symptoms consisted of wilting of the primary leaves, accompanied by systemic chlorosis in the top leaves, stunted growth and green water—soaked lesions at the point of inoculation. 15 Spray Inoculation Technique: Growing of Bean Plants— Five plants of each variety were grown in the greenhouse in 20cm diameter clay pots containing a pasteurized 3:1 mixture of either soil and peat or soil and vermiculite respectively. Plants were maintained at temperatures that ranged between 21 and 25°C and were watered twice a day throughout the period of the experiment. A soluble 15:30:15 (NPK) fertilizer was applied to plants once a week starting 10 days after the date Of planting. Plant Inoculation- Plants were spray inoculated 15 days after planting, when plants possessed two fully ex- panded trifoliolate leaves. In preliminary experiments with Manitou, two spray inoculation techniques namely, water- soaking and spray to run—off, were compared. In the water- soaking method, the lower surface (abaxial) of all leaves present were water—soaked by spraying bacterial suspensions at a pressure of 15psi. This method caused permanent injury to the sprayed leaves. In the other method both upper and lower surfaces of trifoliolate leaves were gently sprayed to liquid run-off with the bacterial suspension using an aerosol sprayer. Inoculations were always performed during the late afternoon hours of the day in order to avoid the high daytime temperatures. All inoculated plants were kept in the greenhouse where temperatures ranged between 21-25°C. The plants were periodically examined, and leaf reaction were recorded up to 10 days after inoculation. Water-soaked 16 lesions were observed on the abaxial surface of leaves ino- culated by both methods 6 days after inoculation. Results Race Determination Test Foliage Reactions— Large areas of water—soaking at the site of inoculation indicated a susceptible reaction for both inoculation techniques used. Notes were also taken relative to the amount of systemic chlorosis caused by toxin translocation. Plants were rated as resistant if they showed (a) brown necrotic lesions with only slight water- soaking at the site of inoculation (b) hypersensitive reac- tion alone and/or (c) absence of any visible symptom. Both inoculation techniques gave identical results. All isolates were pathogenic to cultivar Nasaka, in which water-soaked lesions appeared at the primary leaf node of injected plants and on inoculated leaves 3-6 days after spray inoculation, (Table 2). Some of the plants developed systemic chlorosis. Cultivar Montcalm was resistant to all isolates. Thirteen Malawian isolates plus isolate race 1 were unable to attack any of the differential cultivars. These isolates either induced small brown necrotic lesions on the inoculated leaves and stem, or induced no obvious symptoms. Isolates HB4 and HB6, both from the Ehehleni, Mzimba district, were not pathogenic to Red Mexican U1 3, Red Mexican UI 34, Great Northern 123 and Namajengo. However, they were pathogenic to cultivar Jubila, where severe drooping of primary leaves 17 TABLE 2. Foliage reaction of bean cultivars to inoculation with several isolates of Pseudomonas syringae pv. phaseolicola. 3, Reactions of Bean Cultivar Designated Halo Blight Isolate NAS UI3 U134 GN123 JUB NAM MONT Pathotype Race 1,HB1,HB3,HB5, HB7,HB8,HB9,HB10, HB11,HB12,HB13, HB23,HB29,HB4O Race 2,HB14,HB15, HB16,HB17,HB18, HB19,HB20,HB24, HB25,HB26,HB27, HB34,HB36,HB37, HB38,HB39 HB4,HB6 S R R R S R R 3 HB35 S R R R S s R V 4 aNAS=Nasaka, UI3=Red Mexican UI3, U134=Red Mexican UI34, GN123=Great Northern 123, JUB=Jubila, NAM=Namejengo, MONT=Montcalm. bS=Susceptible reaction - stunting of inoculated plants, wilt— ing and drooping of primary leaves usually followed by systemic chlorosis of top leaves. R=Resistant reactions - brown necrotic lesions and absence Of any visible symptom. 18 was followed by systemic chlorosis 4—6 days after inocula- tion. The isolates induced large amounts of water—soaked lesions on spray inoculated leaves 6 days after inoculation. Isolate HB35, from Chilobwe in Ntcheu district, was patho- genic to Jubila and Namajengo. Sixteen Malawian isolates plus isolate race 2 incited a susceptible water—soaking reaction with or without systemic chlorosis in all differen— tials. Various susceptible reactions were noted 3—6 days after inoculation, including stunting, drooping of primary leaves (mainly in injected plants) and water—soaked lesions, usually followed by systemic chlorosis. The Malawian halo blight isolates were grouped into four pathogenicity groups or pathotypes (Table 2). Pod Reaction- Disease reactions were evident in most isolate/pod combinations 2-3 days after inoculation. Final results were recorded at 4 days. The reactions were sepa- rated into five symptom classes (Figure 2) as follows. RD = Resistant reaction — Dry needle hole with no chlorosis. RN = Resistant reaction — Dry needle hole with brown necrotic halo, tissue not macerated. SW = Susceptible reaction — Light to dark green water-soaked lesion with or without bacterial ooze. SN = Susceptible reaction - Dark brown lesion with macerated tissue. SC = Susceptible reaction - Lesions with concentric patterns of green and brown. l9 Figure 2. Symptom types on green pods of Jubila: 3='SC' type = susceptible reaction— concentric patterns of green and brown lesions; 13='SN' type = susceptible reaction— dark brown necrotic lesions with macerated tissue; 12='RN' type = resistant reaction— dry needle hole with brown necrotic halo, tissue not macerated; l4='SW' type = susceptible reac- tion— light to dark green water-soaked lesions with or with- out bacterial ooze; and C='RD' type = resistant reaction- dark needle holes with no chlorosis. 20 Table 3 summarizes pod reactions of the seven culti- vars when inoculated with different halo blight isolates. Pods of bean cultivar Nasaka were uniformly susceptible to all isolates. Susceptible reactions induced by halo blight isolates were observed 2—3 days after inoculation. All isolates in the pathotype 1 category (foliage reaction) were not pathogenic on pods of Montcalm. However, these isolates did induce variable reactions in the other five differentials. Isolates HB8 and race 1 were not pathogenic to pods of Red Mexican U1 3, Red Mexican UI 34 and Great Northern 123, but were pathogenic to pods of Jubila and Namajengo. Isolates HB3, HBlO and HB13 were pathogenic to pods of all five differentials. The remaining nine iso— lates (Table 3) were non-pathogenic to pods of all five differentials. All isolates of pathotype 2 induced sus- ceptible reactions on pods of all bean cultivars including Montcalm. The reaction induced by isolates HB4 and HB6 on pods of Red Mexican U1 3, Red Mexican UI 34, Great Northern 123, Jubila and Namajengo were similar to HB8 and race 1 isolates. Both susceptible and resistant reactions were evident in pods of Montcalm inoculated with HB4 and HB6. However, the water-soaked spots induced by these isolates were not as extensive as those on pods of other cultivars. Isolate HB35 induced water-soaked lesion on pods of all bean cultivars except Montcalm. 21 TABLE 3. Pod reactions of bean cultivars inoculated with several isolates of Pseudomonas syringae pv. phaseolicola. Reaction of Bean Cultivara’ Foliage Halo Blight Isolate NAS UI3 U134 GN123 JUB NAM MONT Pathotype HB8,Race 1 SW RN RN RN SW SW RN 1 HB1,HB5,HB7,HB9, SW, RD, RD, RD, RD, RD, RN 1 HB11,HB12,HB23, SN RN RN RN RN RN HB29,HB40 HB3,HB10,HB13 SW,SN SN SN SN SN SN RN 1 SC Race 2,HB14,HB15, HB16,HB17,HB18, HB19,HB20,HB24, HB25,HB26,HB27, SW SW SW SW SW SW fig, 2 HB34,HB36,HB37, HB38,HB39 HB4,HB6 SW RN RN RN SW SW SW,SN 3 HB35 SW SW SW SW SW SW RN 4 aNAS=Nasaka, U13=Red Mexican UI3, U134=Red Mexican U134, GN123=Great Northern 123, JUB=Jubila, NAM=Namajengo, MONT: Montcalm. b RD=Resistant reaction - dry needle hole with no chlorosis. RN=Resistant reaction — dry needle hole with brown necrotic halo, tissue not macerated. SW=Susceptible reaction — light to dark green water—soaked lesion with or without bacterial ooze. SN=Susceptible reaction — dark brown necrotic lesion with macerated tissue. SC=Susceptible reaction - lesions with concentric patterns of green and brown lesions. 22 Virulence Determination Szarka and Velich (55) and Buruchara and Pastor-Corrales (6) observed that virulence of Psp can be quantified on the basis of diameter of water—soaked spots around infection points. In this study, pods of bean cultivar Nasaka were used to determine the variation in virulence among the iso— lates from each of the four established foliage pathotypes. In addition, numbers of water-soaked lesions induced by the same isolates on spray inoculated leaves of Nasaka were recorded. Plants were grown, inoculated and maintained in the greenhouse as previously described. Ten days after inoculation, first trifoliolate leaves were harvested and numbers of lesions on each leaf counted. Mean diameters and range of the water—soaked spots for each of the four pathotypes are shown in Table 4. The frequency distribution of isolates in relation to mean dia— meter of water-soaked spots on pods of Nasaka are shown in Figure 3. Significant differences (LSD 0.05=1.l) were observed between and within pathotypes, especially patho— types 1 and 2. In general, isolates belonging to pathotype 1 had lower mean diameter values than the other pathotypes. However, isolates in pathotype l as a group some isolates were more virulent statistically than others. Isolate HB8 showed the highest mean diameter value (4.9mm) followed by isolate race 1 with mean diameter value of 4.5mm. Isolates belonging to foliage pathotype 2 as a group also ranged from less virulent to more virulent. Generally, 23 TABLE 4. Average diameter and range (mm) of water-soaked spots induced by 34 isolates of Pseudomonas syringae pv. phaseolicola on pods of bean culti— var Nasaka. Pathotype 1 2 3 4 # of isolates l4 l7 2 1 average diameter 3.7 4.7 4.2 5.0 range 2.3—4.9 2.8—7.0 4.0-4.4 TABLE 5. Average number and range of water—soaked lesions induced by 34 isolates of Pseudomonas syringae pv. phaseolicola on leaves of bean cultivar Nasaka. Pathotype l 2 3 4 # of isolates l4 l7 2 1 average # of lesions 26 427 187 620 range 10-98 170-709 125-249 24 15 14 Pathotype 13 -1 12 1:2 [[1113 “ [XXXM 10 9 8 FREQUENCY ; 7/ .1 3.0 5.1 6.0.6.1 7.ol 7/ MEAN DIAMETER (mm) OF WATER—SOAKED SPOTS Figure 3. Histogram showing frequency distribution of 34 isolates of Pseudomonas syringae pv. phaseolicola in rela- tion to diameter of water-soaked spots caused on pods of bean cultivar Nasaka. LSD.05=1.1 25 most isolates were more virulent than those of pathotypes l and 3. Except for isolate race 2, no significant differ— ences were observed between HB35 (belonging to foliage patho— type 4) and the more virulent isolates of pathotype 2. A similar trend was observed when the isolates were evaluated based on the number of water-soaked lesions induced on inoculated leaves (Table 5). TOXIN PRODUCTION STUDIES Toxins are known to be significant causal factors in the development of several destructive plant diseases (29, 39). Symptoms associated with fungal and bacterial toxins in diseased plants have been observed and documented by numerous workers (29). Classical examples of pathogenic organisms that produce toxins related to the development of diseases in plants are Helminthosporium species, Alter— naria species, Agrobacterium species, Pseudomonas species and Rhizobium species (29, 39, 45). Bean halo blight caused by Psp is characterized by the formation of a chlorotic halo around the infection site an the subsequent development of systemic chlorosis. Studies on host—parasite relationships indicated that a bacterial toxin is involved in the production of these symptoms (4, 21, 30, 53). Accumulation of ornithine in infected or toxin treated tissue has been observed. Therefore, halo blight toxin is a potent and specific inhibitor of the enzyme L— ornithine—carbamyl transferase (OCT). This enzyme catalyzes the conversion of ornithine to citrulline. Halo blight toxin has been observed to inhibit the growth of Escherichia coli strain K12 (53) in minimal medium, which led to the development of a quick method for bioassaying halo toxin 26 27 production by different Psp isolates. Halo blight toxin and toxins produced by other phytobacteria have been termed as "virulence factors" (45). This denotes that their pre— sence in infected plants is associated with an increase in severity of the disease. Variation in virulence in Psp isolates have been well documented, and such virulence dif— ferences have been associated with differences in the rate of toxin production (25, 50). Hence, the Objective of this study was to compare the rate of toxin production of some halo blight isolates belonging to the four pathogenic groups (Table 2). In a preliminary study, all Malawian isolates plus race 1 and race 2 were tested for their effect on growth of E. £211. All the 34 isolates except HB3, HB7, HB9, HBll, HB12 and HB13 inhibited E. ggli_growth (Figure 4). A moder— ately high correlation coefficient (r=0.695) was Obtained between virulence (based on the number of leaf lesions) and toxin production by the same isolates. Materials and Methods The method used for bioassaying halo blight toxin pro- duction by the isolates was the one described by Staskawicz and Panopoulos (53). Bacterial Strains Four Malawian isolates of halo blight (HB6, HB8, HB26, and HB35) were used in this study. In addi— tion, cultures of race 1 and race 2 and G50Tox_ (obtained from Dr. S. S. Patil, University of Hawaii at Manoa) halo blight 28 isolates were included. We Obtained E. coli strain K12 from Dr. N. J. Panopoulos, University of California, Berkeley for use as an indicator strain for toxin production. Media Halo blight isolates were grown and maintained in tube slants of solid KMB. For toxin bioassays, the organ- isms were grown in Minimal salt Agar. Composition of Minimal Salt Agar Flask l Agar 20g Glass distilled water 500ml Flask 2 Glycerol .3ml K2HPO4 10.5g KHZPO4 4.5g (NH4)2S04 1.0g Sodium Citrate 0.5g Glass distilled water 500ml The two flasks were autoclaved separately. To flask 2, 0.5ml of 1% Vitamin B and 1 m1 of MgSO .7H20 (20g/100ml 4 of glass distilled water stock) were added. Both chemicals were filter sterilized. The two flasks were then mixed and plates poured, 10ml per plate. Toxin Bioassay The cultures of Psp were grown on minimal salt agar. Each isolate was transferred from the stock culture into isolated spots on minimal agar plates by use 29 of sterile toothpicks (4 spots per plate). The cultures were then incubated for 72 hours at l8il°C, the optimum temperature for toxin production (53). After 72 hours of growth, the cultures were killed using anhydrous Ethyl ether [(CH3CH2)20]. The plates were then overlaid with a 24 hour old culture of E. 22;; strain K12 by mixing 2ml of E. 33;; suspension (Optical density 0.02—0.03 at wavelength of 620mu) with an equal volume of 2% molten water agar. The plates were sealed with parafilm and incubated at 30°C for 13 hours. Clear inhibition zones were observed after 12-13 hours following the overlaying of Ethyl ether treated Psp plates with E. coli suspension. Results As expected, a nontoxigenic isolate of Psp, G50tox- did not inhibit the growth of E, 33;; strain K12. The re- maining isolates produced diffusible toxins which inhibited the growth of E. ggll. Growth inhibition of E. 33E; is reported to result from inhibition of the enzyme L-ornithine- carbamyl transferase which resulted in a phenotypic require— ment for arginine (53). Different Psp isolates induced significantly different zones of inhibition at P 0.05 (Table 6). Isolate HB26 and race 2 both of foliage patho- type 2 produced inhibition zones statistically larger than isolates HB35, HB8, and race 1. (pathotypes l and 4). Isolate race 2 produced inhibition zones slightly larger than HB6 though the differences were not significant. 30 750 H338 O 700 r=O.695 = -73.334+l7.78lx HB 8 H826 x= 11.316+0.027y HB35 b ‘ 3334 SE 600 3325 x (Di-J Em 3314 HB39O H319 HP 0 o UJ<1 ‘38 ... 500 Qn—l mo MEI-a oz mozaemHa 20 25 2O 15 10 TIME (hr) 'I 38 TABLE 7. A comparison of bacterial migration rates and numbers of lesions induced on leaves of bean cul— tivar Nasaka by six Pseudomonas syringae pv. phaseolicola isolates. Distance (mm) moved over time (hr) Number of Foliage Isolate l 5 10 15 22 Lesions Pathotype HB8 14.0 6.8 4.0 + + 44 1 HB6 12.5 3.8 3.5 3.2 + 125 3 HB39 11.3 4.9 3.5 2.2 + 545 2 Race 2 9.8 2.4 2.0 2.4 3.0 169 2 HB35 8.0 3.2 1.8 2.3 2.5 620 4 Race 1 5.8 1.1 0.6 0.1 0.07 98 l + = Bacterial migration band had already reached the bottom of the tube (tube length 65mm) SEROLOGICAL STUDIES Serology is a rapid and useful technique for the detec- tion and/or identification of several plant pathogenic bac- teria (2, 15, 18, 51, 56, 58). Serological studies of Psp have been limited (16, 18, 56). These earlier studies sug- gest that Psp antisera are pathovar but not race specific. The present study was conducted to determine whether different foliage pathotypes (Table 2) in Malawi isolates also differed serologically. Materiaksand Methods Halo blight isolates— Eight of the Malawian halo blight isolates (HB6, HB15, HB17, HB26, HB27, HB35, HB36 and HB39) were used as antigens for production of antisera. The iso— lates were chosen as representative of the major bean growing regions of the country. Antigen preparation and immunization of rabbits- Cultures used for antisera production were prepared according to the method of Allan and Kelman (2). Bacterial cells were grown on KMB agar at 25°C for 48 hours. Cells were harvested by suspending them in buffered saline (PBS— 0.01M NazHPOA- KHZPO4 buffer at pH 7.2; containing 0.85% w/v NaCl) and washed by centrifugation for 15 minutes at 10,000 rpm in 39 40 a Sorvall SSI centrifuge with SS-34 rotor. The washing procedure was repeated three times. Cells were then fixed by dialyzing against 2% aqueous glutaraldehyde (25ml of cell suspension to 100ml of glutaraldehyde) at 4°C for 3—4 hours, followed by dialysis against PBS with frequent changes at 4°C for 20 hours. Cells in the suspension were adjusted to 2-4 x 109 cells/m1 prior to immunization of rabbits. Immunization of rabbits- One New Zealand white female rabbit (2.25-2.7 kg) was immunized for each bacterial isolate. Normal sera were obtained prior to immunization. The rab— bits were injected at l (0.3m1), 4 (0.5m1), 7 (1.0m1), ll (1.0ml), l8 (1.0m1), 25 (1.0ml), and 32 (1.0ml) days. Blood for sera was collected 14, 21, 28, and 35 days after the first immunization. The bacterial suspensions were injected into the rabbits intravenously using the marginal ear vein. The first injection of the series was always made as near the tip of the ear as possible. Succeeding injections were made closer to the animals head so that the scar tissue would not interfere with injections. All injections were done on one ear. The marginal ear vein in the other ear was used for bleeding. The first bleeding was made from the site near the middle of the ear and succeeding punctures were made distal to the head. Collection and fractionation of antisera- The procedure of Allan and Kelman (2) was used to fractionate the antisera. Blood was collected in a sterile plastic centrifuge tube and allowed to stand undisturbed for 3 hours at room temperature. 41 Tubes were placed in a 4°C refrigerator overnight. The fluid serum was carefully removed by decanting into fresh sterile centrifuge tubes, and the antiserum fractionated by the dropwise addition of saturated ammonium sulfate solu— tion. To each m1 of antiserum, 2/3 ml of saturated (NH4)2SO4 was added dropwise with stirring. The mixture was stirred for at least 2-4 hours to allow complete precipitation. The mixutre was then centrifuged for 20 minutes at 10,000 rpm in Sorvall SS1 centrifuge with 88-34 rotor. The precipi- tate was dissolved in PBS to the initial antiserum volume. The sample was reprecipitated and centrifuged two times more and then dialyzed against PBS for 40 hours to remove all (NH4)ZSO4. Dialysis was conducted in a coldroom set at 4"C. Dialyzed antisera were centrifuged one additional time to remove precipitate. Sodium azide was added to the antiserum at 500ppm as a preservative. Antisera were stored in small volumes (3ml per tube) at -20°C until required. Serological Techniques Agglutination tests— Both test tube and microagglutination tests were employed in this study. Bacterial cells of each 9cfu/ml and steamed for isolate were adjusted to 3—4 x 10 60 minutes. For test tube agglutination tests, 0.2m1 of a one tenth dilution of antibody was overlayed with an equal volume of steamed bacterial cells. The resultant reaction was recorded after one hour. In microagglutination test, drops of one tenth dilution of antibody were put in 15mm2 42 squares etched in 90mm2 plastic trays. Then a drop of each antigen was put over a drop of either PBS (control) or anti— body. Observations were recorded at 20 and 60 minutes. Ouchterlony gel double diffusion test (ODD)- The ODD method utilizes the precipitation of antigens and antibodies in a gel medium rather than a fluid medium. Lines of precipita- tion will form when specific antigen and antibody come to- gether in equivalent proportions by diffusing through the agar media. In this study the ODD agar contained: 8.5g Difco puri— fied agar, 10ml of a solution containing 1% w/v orange G, 30ml of a solution containing 10mg/ml NaN and 960ml of 39 buffered saline. Sodium azide was added to prevent bacterial and fungal contamination, while orange G was added to increase contrast during photography (58). Plastic petri dishes of 9cm diameter were filled with 15ml of medium. A serological test consisted of one central hole of 5mm diameter surrounded by six holes each of 5mm diameter also. Normally, the central hole contained the antisera and the surrounding holes contained the antigens. All plates were maintained in covered plastic boxes to avoid desiccation and readings were taken every 2 days. Preparation of the absorption-antisera- Agglutinin—absorp— tion was used to prepare "type serum" which contained the antibodies necessary to identify (by agglutination or preci— pitation) major determinants (58). 43 Procedure:— l. lml of the desired antiserum was diluted 1:10 with PBS and mixed with lml of packed steamed cells of the selected antigen in a 12 x 75 mm test tube. 2. Tubes were incubated for 4 to 5 hours in 45°C water bath and then refrigerated overnight. 3. Contents of the tube were centrifuged at 2,000g for 20 minutes and the supernatant was carefully removed to a clear test tube. Efficiency of the absorption was deter— mined by a standard agglutination test against the homolo— gous antigen and procedures in l and 2 were repeated as necessary. Results Of the eight antisera produced to Malawian halo blight isolates, only seven were retained for this study. Anti- serum produced to antigen HB26 became contaminated either during production or in storage and was discarded. Reaction of antisera against steamed cell antigens- Antisera titers in both tube and microagglutination tests ranged from l:l60—l:2560. Antiserum HB35 had the lowest titer, while antisera HB6, HB17, HB27, and HB36 all had a titer of 1:2560. Reaction of halo blight antisera against all Malawian isolates were evaluated by means of agglutination (Table 9) and agar gel double diffusion (Ouchterlony) (Table 10) tests. Both tests were performed with antisera diluted 1:10 with 44 PBS. Tests gave comparable results when antisera were dilu— ted l:lO and antigen concentrations were 3-4 x 109cfu/ml. In general tube agglutination tests were more sensitive when compared to microagglutination and Ouchterlony tests. The Ouchterlony test was useful from the standpoint that it yielded clear and distinct results. However, it did require 2 or more days for completion. A total of 26 Malawian isolates plus races 1 and 2 Psp were tested against the seven antisera. Antisera HB6 and HB17 reacted with all antigens except HB35. Antiserum HB36 reacted with all antigens except HBll, HB12, and HB35. Antiserum HB27 did not react with antigens HBll, HB12, Hb23, and HB35. Antisera HB15 and HB39 reacted with all except HB9, HBll, HB12, HB23 and HB35. Antiserum HB35 only reacted very strongly with the homologous and race 2 antigens. The Malawian isolates were then grouped into 5 serotypes (Table 8). The five Psp serotypes do not correlate completely with the foliage pathotypes described earlier. Antigens of isolates in foliage pathotypes 2 and 3 react with all antisera except HB35. Antigen HB35 foliage pathotype reacts only with the homologous antiserum which suggests that this isOlate is quite distinct from the other isolates. The antigens of isolates in foliage pathotype 1 varied in their reactions with different antisera. Antigen HB9 failed to react with antisera HB15 and HB39, while antigens HBll and HB12 did not react with antisera HB15, HB27, HB36 and HB39 45 and antigen HB23 did not react with antisera HB15, HB27 and HB39. Absorption test— This test was conducted to assess serologi— cal identity among the Psp isolates. Three antisera HB6, HB27 and HB39 were used in cross absorption tests to detect related antigens. Each of the Psp isolates that positively reacted with each of these antisera was allowed to react with the antiserum and the absorbed antiserum was obtained as described in the previous section. For identity deter— mination, homologous antigen and the antigen that was used to absorb the antiserum were allowed to react with the ab- sorbed antiserum. If the homologous antigen did not react with the absorbed antiserum then the two antigens were re- lated. 0n the other hand, if the absorbed antiserum reacted with the homologous antigen and the heterologous antigen did not react with the absorbed antiserum, then there was no identity. The results (Table 11) reveal that isolate HB6 is sero- logically related to all isolates that reacted with its antiserum except for HB3, HB5, HB9 and HB23; isolate HB27 was serologically unrelated to HB3, HB5 and HB9; and isolate HB39 is serologically unrelated to HB3 and HB5. The four isolates HB3, HB5, HB9, and HB23 all belong to foliage patho- type 1 unlike isolate HB6 which is in foliage pathotype 3, and HB27 and HB39 which are in foliage pathotype 2. 46 TABLE 8. Serotype grouping of several Egggdomonas syringae pv. phaseolicola isolates. Antiserum Proposed Antigen HB6 HB17 HB36 HB27 HB15 HB39 HB35 Serotype HB3,HB4,HB6,HB8,HB14, HBlS,HB16,HBl7,HB18, + + + + + + _ 1 HB19,HB20,HB24,HB25, HB26,HB27,HB34,HB36, HB37,HB38,HB39,Race 1, Race 2 HB9 + + + + - - — 2 HB23 + + + — — — — 3 HBll,HB12 + + — - - - - 4 H335 - - - — — — + 5 47 TABLE 9. Reactions of different halo blight isolates in tube agglutination tests against Pseudomonas syringae pv. phaseolicola antisera. ANTISERA USEDa ANTIGENb HB6 HB15 HB17 HB27 HB35 HB36 HB39 HB3 +C + + + —d + + HB4 + + + + - + + HBS + + + + — + + HB6 + + + + - + + HB8 + + + + - + + HB9 + - + + — + — HB11 + - + — - — — HB12 + — + — — — - HB14 + + + + - + + HBlS + + + + - + + HB16 + + + + — + + HB17 + + + + - + + HB18 + + + + - + + HB19 + + + + — + + HBZO + + + + — + + HB23 + — + — — + — HB24 + + + + - + + H825 + + + + - + + HB26 + + + + - + + HB27 + + + + - + + HB34 + + + + - + + HB35 - — - - + — — HB36 + + + + - + + HB37 + + + + — + + HB38 + + + + — + + H839 + + + + — + + Race 1 + + + + — + + Race 2 + + + + + + + aAntisera prepared against 2% gluteraldehyde fixed cells. bAntigen gonsisted of steamed cells adjusted to about 3-4 x 10 cfu/ml. c . . . + = agglutination: d— = no reaction. Reactions recorded 2 hr. after setting the experiment. TABLE 10. ANTISERA USEDa Reactions of different halo blight isolates to Pseudomonas syringae pv. in agar gel double-diffusion tests. phaseolicola antisera ANTIGENb H36 H815 H817 H827 H835 H836 H839 c d HB3 + + +2 + — + + H84 +2 +2 +3 + - + + H85 + + +2 + - + + HB6 +2 +2 +3 + - + + H88 +2 +2 +3 + - + + HB9 + — +2 + - + - H811 +2 — +2 - - — - H812 + - +2 — - — - H814 +2 +2 +3 + - + + H815 +2 +2 +3 + - + + H816 +2 +2 +3 + - + + H817 +2 +2 +3 + - + + H818 +2 +2 +3 + — + + H819 +2 +2 +3 + — + + H820 +2 +2 +3 + - + + H823 + — +3 — — + - H824 +2 +2 +3 + - + + H825 +2 +2 +3 + - + + H826 +2 +2 +3 + - + + H827 +2 +2 +3 + - + + H834 +2 +2 +3 + — + + H835 — — - — +2 — — H836 +2 +2 +3 + - + + H837 +2 +2 +3 + - + + HB38 +2 +2 +3 + - + + Race 1 +2 + +3 + — + + Race 2 +2 +2 +3 + + + + aAntisera prepared against 2% gluteraldehyde fixed cells. bAntigen go 3—4 + II x 10 band formation no band formation nsisted of steamed cells adjusted to about cfu/ml. 49 TABLE 11. Reaction of homologous and heterdogmm Pseudomonas syringae pv. phaseolicola antigens to absorbed antisera. Absorbed Reaction Absorbed Reaction Absorbed Reaction Antiserum HET HOM Antiserum HET HOM Antiserum HET HOM HB6/HB3 + HBZ7/HB3 — + HB39/HB3 - + H36/H34 — H827/HB4 — — H839/H84 — — HB6/HB5 + HB27/HES — + HB39/HB5 - + HB6/HB6 — HB27/HB6 - — HB39/HB6 - — HB6/HB8 - HB27/HB8 — — HB39/HB8 - HB6/HB9 + HB27/HB9 - + HB6/HB11 — HB6/HB12 — HB6/H814 - 3327/3314 - — HB39/H814 — — H86/H815 — H827/H815 - - H839/H815 - - HB6/HB16 - HB27/HB16 - - HB39/HB16 - - HB6/HB17 - HB27/HB17 — — HB39/HB17 — — HB6/HB18 — HB27/HB18 — — HB39/HB18 - — HB6/HB19 - HB27/HB19 — - HB39/HB19 - - HB6/HB20 - HB27/HB20 — — HB39/HB20 - - HB6/H823 + HB6/HB24 — HB27/HB24 — — HB39/HB24 - - HB6/HB25 - HB27/HB25 — - HB39/HB25 — — HB6/HB26 - HB27/HB26 - — HB39/HB26 — — HB6/H827 - HB27/HB27 - - HB39/HB27 — - HB6/HB34 — HB27/HB34 - - HB39/HB34 — - HB6/HB36 — HB27/HB36 — — HB39/HB36 — — HB6/HB37 - HB27/HB37 - — HBB9/HB37 - - HB6/HB38 — HB27/HB38 - - HB39/HB38 — — HB6/HB39 - HB27/HB39 — — HB39/HB39 - - HB6/Race l — HB27/Race l - - HB39/Race l - - HB6/Race 2 — — HB27/Race 2 - - HB39/Race 2 — — HET = heterologous antigen used to absorb the corresponding antiserum HOM homologous antigen aNumerator = Antiserum; and Denominator = Absorbing antigen 50 Figure 7. Smooth (HB15) and rough (HB35) colony forms of Pseudomonas syringae pv. phaseolicola. DISCUSSION 0n the basis of selective pathogenicity, 32 Psp iso- lates from Malawi were placed into four distinct strains/ pathotype groups (Hubbeling 1974). Pathotype 1 consisted of 13 Malawian isolates plus isolate race 1. Pathotype 2 consisted of 16 Malawian isolates plus isolate race 2. Isolates HB4 and HB6 constituted pathotype 3, while only isolate HB35 was assigned to pathotype 4. The HB35 isolate also differed from the rest of the Malawian isolates in terms of colony morphology as it was the only isolate to produce rough colonies in artificial media (Figure 7). Both smooth and rough colony types have been reported for Psp (l, 36). The existence of pathogenic variation in Psp is of importance to breeding resistance to the halo blight disease. Earlier, Patel and Walker (37) described two races of Psp based essentially on infection of the Red Mexican UI3 culti- var. Later, Schroth, Vitanza and Hildebrand (49) suggested the existence of more than two races/pathotypes within Psp when virulence of a strain toward a given bean cultivar was considered as a criterion for establishing a race. They also observed that neither race 1 nor 2 Psp was homolo— gous with regard to virulence when tested in numerous bean 51 52 cultivars. This opinion is also supported by Szarka and Velich (54, 55) who suggested that Psp consists of a series of strains, which can be distinguished by their increasing pathogenicity. The present study utilized foliage reactions of seven bean differentials to distinguish Psp isolates. Our findings support the existence of more than two races or pathotypes in Psp. Had we only used the Red Mexican UI3 cultivar as a differential for pathogenic variation, the Malawian isolates would have comprised two groups instead of four. Extensive surveys to determine the relative pre- valence of these different pathotypes in the country is certainly needed in order for a breeding program to be suc- cessful. Virulence studies revealed that the Malawian isolates of Psp varied in virulence from weak to high. Isolates belonging to foliage pathotype l were generally the weakest in virulence. Ecologically, most of the group 1 isolates were obtained from warmer areas as opposed to the more viru— lent isolates in groups 2, 3, and 4 which were obtained from cooler areas of the Malawian bean growing regions. This observation could be one reason why halo blight is more prevalent in cooler than warmer areas of the country. Temperature is known to affect toxin production by Psp (26, 53). Jensen and Livingston (26) found that several isolates which induced halo-less lesions at 22°C did so also at 16°C and 28°C. In contrast, most other isolates produced halo-less lesions at 28°C, but typical halo lesions 53 at 16°C and 22°C. These results indicated that in nature, both toxigenic and nontoxigenic strains of Psp exist. The toxigenic strains would then produce toxin depending on the prevailing temperatures. Optimim temperature for toxin production in Psp is 18°C (53), the temperature used in these studies. The data indicate that more virulent isolates produced more toxin and a moderately high correlation (r= 0.695) was observed between virulence (as measured by water- soaked lesions on inoculated leaf) and toxin production of all 34 isolates (Figure 4). Several other workers have reported similar observations (25, 26, 50, 53). The six Malawian isolates (H83, HB7, HB9, HB11, HB12 and HB13) which were least virulent consistently failed to inhibit the E. 22;; tester strain in minimal agar. This suggests that these isolates are either nontoxigenic or toxigenic with very little toxin production. The relationship in Malawian isolates between virulence and toxin production could be associated with ecological adaptation of the Psp isolates. For example, the more viru— lent, greater toxin—producing Psp isolates were mainly iso- lated in cooler bean production areas. In contrast, the less virulent and non or low toxin—producing isolates are mainly present in warmer production areas. The specific attraction of motile plant pathogenic organisms to chemicals (chemotaxis) is a well—documented phenomenon (27, 35, 40, 65). Bacteria chemotaxis can be demonstrated by various methods including the movement of 54 bacterial migration bands in a semi-solid medium containing different chemicals (27). The mechanism whereby bacteria are attracted and how the bacterial flagella are directed towards a source of stimulation is still not clear (56). In the present work Psp was shown to be motile. The data, however, showed no relationship between virulence and moti— lity. Panopoulos and Schroth (35) also carried out a similar study. They looked at the relationship between virulence and motility using motile and nonmotile strains of Psp. Using virulence index (the ratio of water—soaked lesions produced in primary leaves to the number of viable cells introduced into the leaf) as the basis for comparison of virulence, they obtained similar values for both motile and non—motile strains. They therefore, concluded that loss of motility had no effect on the inherent virulence of the strains. However, they further noted that when the inoculum was applied to the leaves externally, either by spraying or dipping, differences in infectivity between motile and nonmotile strains were substantial. They defined infectivity as the capacity of the pathogen to enter the plant and incite disease. The authors also observed that in general motile strains incited 2 to 12 times more lesions than the corresponding nonmotile mutants with paralized flagella. Kelman and Hruschka (27) examined relationship between motility and virulence of E. Solanacearum and noted that virulent strains were nonmotile while a virulent strains 55 were, in most cases, devoid of flagella as opposed to the avirulent ones which were usually flagellated and highly motile. Motility would seem to play a very important role in Psp and other bacteria. This would be so, especially, during ingress and egress of the pathogen from the host plant. Because after dissemination by rain, vectors or other agen— cies, swimming enables a greater percentage of cells to reach the nutritionally favorable sites. However, our re— sults show that motility is not a determining factor for virulence. In serological studies our primary objective was to determine whether the established foliage pathotype groups would also differ serologically. The results of this investi— gation indicate that except for HB35 Psp isolate of patho— type 4 group, all Malawian Psp isolates serologically share some antigenic characteristics. Psp antisera HB17 and HB6 each from foliage pathotype 2 and 3 respectively, reacted with all antigens except for HB35. Isolate HB35 was the only Psp isolate exhibiting rough colony form. The colonies of this isolate appeared a little larger than the smooth forms, had a wavy margin and a crinkled dry-looking surface (Figure 7). It is generally agreed that the change from smooth to rough form involves an alteration in the structure of the somatic antigen (1), hence rough form isolates should be serologically different from smooth forms. Adam and Pugsley (1) demonstrated that flagellar antigens were the 56 only antigenic qualities shared by both smooth and rough forms of Psp. They noted that flagellar antigen is heat- labile. Hence, to demonstrate the serological difference between smooth and rough forms they used heat-treated anti- gen. They heat-treated the bacterial fragments at 100°C for 1 hour. No serological cross reactions between isolates resulted when heat-treated antigens for all serological tests. In the present study; we utilized heat-treated antigens for all serological tests. This therefore, would explain why neither antiserum HB35 nor antigen HB35 reacted with either the rest of the antigens or the other antisera respectively. Earlier studies (l6, 18, 56) have also shown that Psp antisera are pathovar but not race specific. The data suggest the occurance of at least five sero— groups among Malawian Psp isolates. This nonhomogenuity among isolates could be the result of geographical separa— tion. Isolates HB15 (Dowa) and HB27 (Lilongwe) from central region; and isolates HB36 (Thyolo) and HB39 (Mulanje) from the southern region were serologically homogeneous with all isolates from both central and southern regions except HB23. But, they were not related serologically to most Psp isolates from the northern region. Similar observations have been reported for isolates of sugarbeet pathogen (Erwinia carotovora var atroseptica (51). Stanghellini et al reported that there were two distinct serogroups in the sugarbeet pathogen, and such serological biotypes represented widely separated sugarbeet production areas (51). 57 The results of the absorption tests suggest serological relationships among some of the Malawian isolates. Partial relationships here refers to a situation where Psp isolates share some antigenic properties but not all of them. Iso- lates HB3, HB5, HB9 and HB23 were observed to be partially related to isolates HB6, HB27 and HB39. The former isolates belong to foliage pathotype 1 while the latter belong to foliage pathotypes 2 (HB27 and HB39) and 3 (HB6). This partial relation shown by isolates of group 1 may be due to either slight structural differences in some of their somatic antigens or to the fact that the somatic antigens of these isolates are in some degree heat labile. INHERITANCE OF RESISTANCE TO HALO BLIGHT IN Phaseolus vulgaris L. Halo blight (Psp), a seed-borne bacterial disease of beans is a serious problem in Malawi. Weather conditions in Malawi favor infection and distribution of halo blight in field crops of beans due to the cool, rainy and windy conditions during the growing season (Table 12). Control of this disease in developed countries involves the growing of seed crops in semi-arid areas, notably Idaho in the USA, where the pathogen is unlikely to survive and multiply (10, 20). Crop rotation and chemical sprays are other control measures. In Malawi, almost 90% of bean production is con— ducted by subsistent farmers. Such farmers do not have the resources to purchase disease free seed or chemicals. As a result many of them plant seed saved from their previous crop. Therefore, the availability of resistant varieties. to these farmers would be a considerable value for control— ling the destructive halo blight disease. In a review article on disease resistance, Zaumeyer and Meiners (63) state that the general pattern of inheri- tance of resistance to halo blight is not well understood. Different patterns of qualitative genetic control have been 58 59 reported in the evidence, depending on the host cultivar and pathogen races used in the study (20). The present study was undertaken to provide background information for a Malawian project focused towards the develop— ment of halo blight resistant varieties for Malawian farmers. The mode of inheritance of resistance to one Malawian halo blight isolate (HB39) was investigated in crosses between several Malawian bean lines and Montcalm, a halo blight resistant Michigan bean cultivar. Materials and Methods Bacterial isolate- A Malawian foliage pathotype 2 isolate HB39 of Psp was used in this study. The isolate was grown and maintained on KMB agar as described previously. Bean Plants— Crosses were made in the greenhouse between Montcalm cultivar and eight Malawian bean lines/cultivars which were selected on the basis of their acceptability (Nasaka and 60041D) and previous history of exhibiting some field tolerance. Montcalm is a large dark red kidney bean cultivar (Table 13) with excellent resistance to halo blight. In previous experiments Montcalm proved resistant to halo blight isolates belonging to the Michigan isolates of patho- types 1 and 2. The Malawian lines 2586D, 2589D, 2600D, 2609 and 2610 have small yellow seed (Table 13); 1212D is a white navy bean; 600¥1D is a red kidney bean and Nasaka is a tan kidney bean. All lines evaluated under Malawi field conditions except Nasaka and 60041D exhibited some 60 field tolerance to halo blight. However, when they were subjected to HB39 halo blight strain in Michigan field and greenhouse conditions all except 1212D, appeared to be sus- ceptible (Table 14). Line 1212D was moderately resistant. In the summer of 1983, the parental, F1 and F2 genera— tions of crosses between Malawian lines and Montcalm were planted on June 14, in the field at the Botany Research Farm of Michigan State University, East Lansing. Nasaka, planted in alternate rows between the test lines was used as a source of inoculum for the disease. All plants were inoculated at 22 days after planting (July 6, 1983), with a bacterial suspension using an 18 litbr knap—sack sprayer. during this time, the first trifoliolate leaf of most plants was fully expanded. The suspension was prepared by washing the bacteria contained in ten KMB petri plates into 18 liters of water. Eight and sixteen days after the first inocula- tion (July 14 and July 22 respectively) plants were reinocu- lated to insure heavy infection of stems and leaves of sus- ceptible plants. Three separate readings for disease reac— tions were made. Plants were rated on a scale of 1-5 basis (1=no visible symptoms; 2=l—25% infection; 3=26-50% infection; 4=51-75% infection; and 5=severe stunting, 76—100% leaf infection and death of plant due to disease). The flower color of plants was also recorded since resistant Montcalm and 1212 lines were the only white flowered parents. Resis- tant plants with large kidney shaped seed and preferable seed color (mainly red and tan colors) were harvested. 61 TABLE 12. Precipitation (mm) and mean temperature (°C) during the growing season in major bean growing districts of Malawi. Growing Season* District October November December Prec. Temp. Prec. Temp. Prec. Temp. Rumphi 10.0 - 95.5 - 229.6 — Mzimba 9.2 22.2 40.8 22.5 220.8 21.0 Dowa 6.3 — 49.6 - 211.7 - Lilongwe 62.2 24.0 102.5 24.8 119.0 23.2 Dedza 20.8 19.1 75.1 20.0 256.7 — Ntcheu 16.7 21.6 72.7 21.2 160.9 20.4 Thyolo 26.7 22.7 146.1 23.2 147.5 22.6 Mulanje 79.5 20.9 137.3 21.9 288.2 20.9 *Data representing the means of four growing seasons (1979/80, 1980/81, 1981/82 and 1982/83). Prec. = Precipitation (mm), Temp. = Temperature (°C). 62 TABLE 12. (Cont'd) Growing Season January February March April District Prec. Temp. Prec. Temp. Prec. Temp. Prec. Temp. Rumphi 254.7 — ' 150.6 — 340.9 — 227.1 - Mzimba 149.8 19.8 170.9 19.9 178.4 19.7 57.7 19.3 Dowa 304.4 - 150.4 — 170.5 - 48.3 — Lilongwe 166.9 21.7 240.1 21.5 87.7 21.3 41.5 22.1 Dedza 242.9 18.0 185.8 18.3 199.8 17.7 35.4 16.9 Ntcheu 182.8 19.9 209.9 19.9 221.6 19.7 67.7 19.0 Thyolo 96.7 22.5 181.1 22.2 146.7 21.5 103.2 20.0 Mulanje 260.0 20.4 242.5 20.4 303.8 19.7 81.0 18.0 63 TABLE 13. Seed characteristics of Phaseolus vulgaris genotypes used as parents in inheritance study. Parent Seed Color 100 Seed Weight Shape Nasaka tan 44.9 Kidney 600%1D red 49.1 Kidney 1212D white 15.7 Navy bean 2586D yellow 44.2 Pea shaped 2589D yellow 43.2 Kidney 2600D yellow 35.8 Pea Shaped 2609D yellow 39.6 Kidney 2610D yellow 32.5 Kidney Montcalm dark red 50.5 Kidney 64 TABLE 14. Reaction of Phaseolus vulgaris genotypes to pathotype 2 of Pseudomonas syringae pv. phaseolicola. . a Disease Cultivar/line Rating Nasaka 5.0 2589D 3.8 2609D 3.1 2586D 2.8 60091D 2.5 2610D 2.3 ZOOOD 2.1 1212D 1.3 Montcalm 1.0 3A scale of 1-5 was adopted in recording the amount of disease present in each plant. (1 = no symptoms; 2 = 1-25% leaf infection; 3 = 26-50% leaf infection; 4 = 51-75% leaf infection; and 5 = severe stunting, 76% leaf infection and death of plant due to disease). The rating for each cultivar is a mean of 6-10 plants of each. 65 Results Parental, F1 and F2 plants were rated for disease reac- tion on a 1—5 scale. Plants possessing a rating between 1 and 2 were classified as resistant and plants with ratings from 2 and above were rated as susceptible. Flower color of the plants was also recorded. Reaction of Parental Plants. Plants of each parental variety were uniform and identical in their reactions, as reported in Table 14. All plants of Montcalm variety were resistant to halo blight isolate HB39; plants of line 1212D were moder— ately resistant and plants of each of the remaining varie— ties/lines were susceptible (Table 14). All plants of Mont- calm and 1212D possessed white blossoms while individual plants of each of the remaining Malawian varieties/lines possessed pink or purple flowers. Reaction of F1 Plants. All F1 plants from each cross were resistant to halo blight isolate HB39 (Table 15), suggesting that resistance in these crosses was dominant. All plants also had pink or purple blossoms which is dominant over white color. Reaction of F5 Plants. Table 15 shows the segregation patterns for halo blight reactions in F2 plants. A satis- factory fit to a 3:1 ratio of resistant to susceptible plants was observed in the F2 progeny of crosses Montcalm x 2586D and Montcalm x 2589D. This ratio indicates that resistance to halo blight was controlled by a single dominant gene 66 in these crosses. In the F2 populations involving Montcalm with Nasaka, 600/1D and 2610 a good fit to a 9:7 ratio of resistant to susceptible plants suggested the presence of two complementary dominant genes. Satisfactory fits to 54:10, 42:22 and 13:3 ratios of resistance to susceptible reactions were observed in F2 generations of Montcalm x 2600D, Montcalm x 2609D and Montcalm 1212D respectively. These data indicated that resistance reaction in these crosses was controlled by two complementary genes. Different segregation patterns in F2, plants (Table 15) could be explained by assuming that Montcalm carries 2 dominant genes, A and B for resistance against pathotype 2 strain of halo blight. These genes behave as complementary factors. In addition, there is a third gene at C locus with multiple allelic series (C1, C2 and C3). At this locus, alleles Cl and C3 confer resistance (R) and C2 confer sus— ceptibility (S). Again, the higher numbered alleles are dominant over the smaller numbered alleles. The order of dominance is C3 (R) over C2 over Cl (R). The 3R: 1S ratio in crosses of Montcalm with 2586D and 2589D indicated a single factor segregation. These results suggested that there was a single gene difference between Montcalm and 2586D and 2589D (Table 16), indicating that they carry either A or B dominant genes which alone are unable to confer resistance. In crosses of Montcalm with Nasaka, 600/1D and 2610D segregation ratios of 9R:7S were obtained, supporting a complementary factor hypothesis 67 where dominant genes A and B segregate and simultaneously interact to produce a resistant reaction. The observed ratios of 54R:lOS and 42R:228 for crosses involving Mont- calm with 2600D and 2609 respectively were expected if Mont- calm carried C2, 2600D carried C3 and 2609 carried C1 alleles. Thus, in F2 segregating population C3 in 2600D, and C1 in 2609D in complementary with A or B conferred resistance. The 13R:3S segregation ratio in the cross Montcalm x 1212D indicated the presence of recessive resistance in 1212D in addition to resistance by two complementary genes A or B and C1' The proposed genotypes of the 9 parents are pre- sented in Table 16. A satisfactory fit to a 3:1 ratio of plants with pink flowers and those with white flowers was obtained in F2 progency from all crosses. The results show that a single gene pair governed flower color in these crosses. The data also indicated that the gene for white flower color was linked to a gene for resistance. All F2 plants with white flowers in all crosses, were observed to be resis- tent (Table 17). The lack of independent assortment of genes into four expected classes as shown by high chi square value, for color and disease reactions indicate linkage between white flower color and resistance. 68 .Eamoucoz u use: .wmuomaxm u .me .cm>pomno u .mno .owumu nouooaxm mnu ou ufim woow 6 ma o=Hm> x umnu mmumowvca no.0um m>onm m=Hm> m* N .mflmuoano OHEoumxm Hmumcmw u0\ccm mcofimma mmma umxmomlumum3 nuw3 mucmaalm=0fiuummu mfinfiuamumsm n m .Eouaaxm mHnHmH> sum mo mucmmnm cam mCOfimmH owuonom: :30pn1mcowuommu ucmumflmmm H mm no.10o. ocH.o mama ca we sea moH m mam QNHNH\neoz mo.uma. o~o.o mmuma an mm HHH 033 m mxm moocm\neoz ma.-oa. m~.o cauem am ca 353 .omH m mam nooc~\necz we. Noo.o euo em em we me x mam aoHcN\eaoz n.1m. mme.a Ana oa am mm mm m mxm neoz\aHuooc ma.-ma. Nms.o eno 8e we mm 003 m mam mHuooc\naoz m.-m. mea.a nun cc me am we m mam nxannz\eeoz mo.umo. emo.o Hum ea me 083 N83 m mam nommm\eeoz n.1m. mc~.H Hum mm mm 83H oNH m mxm acmmw\naez cmosuom x mum .axm .mno .mxm .mno Hm ucoumm mmouo * N cane“ auflafinmnoum vmuomaxm manflummomsm ucmumfimmm mm meowuomou unwwan oamm .mucmam Amv manflummomsm cam .Amv acmumfimmu we moflumu cmuuoaxm cam maoowaommmnq .>a mmmcfiufim mmcoeocnmmm mo N masuonuma ou m:0fiuommu Nm cam Hm .Hmucoumm .ma m4m<9 69 TABLE 16. Proposed genotypes of 9 Phaseolus vulgaris geno- types based on their reaction to infection by pathotype 2 of Pseudomonas syringae pv. phaseolicola. Cultivar or line Proposed Genotype Montcalm AABBCZC2 2586D AAbbCzCz/aaBBCZC2 2589D AAbbCZCz/aaBBCZC2 Nasaka aabbCZC2 600-1D aabbC202 2600D aabbC3C3 2609D aabbClC1 1212D AAbbClCl/aabbClCl Complementary loci: A—B—; A-Cl; B—Cl; A-C3; and B—C3- R A,B,Cl and C 3 S a,b and C 2 7O 00.0v 00.0 I 300 A 0 00.0 n 300.0 1 00.3 I 03.03 u 03 00030333 000.0v 000.03 0 0300 00.03 H 0.0 n 00.3 1 30.03 u 03 00030330 3000.0v 030.00 0 0000 30.00 n 00.0 I 00.0 1 00.00 H 03 00030330 3000.0V 000.00 m 0000 00.00 u 003 I 00.0 1 00.00 u 00 00030330 00.0v 000.0 m 0000 00.0 n 000.0 1 00.0 I 00.33 u 03 00030330 3000.0v 000.00 N 0300 00.00 u 000.3 1 00.0 I 00.00 u 03 00030330 .00303 00300000 053 03 30M 000w 0 m0 0:00> Nx 0:3 03000000 no.0 n m 0>030 0000> m* .000N 0:0 Q0\ooo .030002 3303 50003002 wc0>00>c0 00003030000 mm :0 003000x0 00303 03030N30N .Qommm 0:0 mowmm L303 80003002 wc0>00>c0 0000303000w mm :0 003000x0 00303 Humumuom .003000xm n mxm .00>3mmno u mmo .5000300: u 300: 0.0.30600 .03 03000 71 0 00 00 03 003 00.0v 03.03 0 00 00 03 000 3030003062 03 00 00 03 030 0000.0v 30.03 0 00 00 00 000 030620031000 03 00 00 00 030 3000.0v 00.00 0 00 00 00 000 003100000002 03 30 00 00 030 3000.0v 00.00 0 30 00 00 000 003000203002 33 303 00 00 030 30.0V 00.33 0 003 00 00 000 00000003002 0 00 00 00 000 3000.0v 00.00 0 00 0 00 000 00000003002 *3 z x 0303000 0303000 0303000 0303000 00030 0 03303 3030 3030 03303 . 0030300000m 3003m0m0m 0030300000m 3003w0m0m 0003000m 000300030000m 0003000m 0030030m .mmmmouo 0003 00 mm 00 000030003 AN 0000300300v 30w00n 0000 300 030000 00 00030m03w0m .00 m0m