THESlS This is to certify that the thesis entitled _l_N_ VITRO AND _IN VIVO INHIBITION OF BACTERIAL AND FUNGAL PATHOG NS OF BEANS BY BACTERIAL ANTAGONISTS presented by LINDA TULLY PONTIUS has been accepted towards fulfillment of. the requirements for MS degree in BOTANY 8 PLANT PATHOLOGY Major professor 6- - Date 3 83 0-7 639 MSU LIBRARIES ” RETURNINQMNAIERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. “f at"? w am. a a “a”?! 'l .. n I . .. ~ «: ‘, o ‘- ‘fi w.» ’p; 1. . ll: ‘6'. .‘I: I‘. ‘.~ .‘ ‘1 a“ IN VITRO AND IN VIVO INHIBITION OF BACTERIA: AND FUNGAE PATHOGENS OF BEANS BY BACTERIAL ANTAGONISTS By Linda Tully Pontius A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1983 ABSTRACT IN VITRO AND IN VIVO INHIBITION OF BACTERIAI‘AND FUNGAE‘PATHOGENS OF BEANS BY BACTERIAL ANTAGONISTS By Linda Tully Pontius Internal bacterial contaminants from bean seed were screened in vitrg and in 2129 for the ability to inhibit growth of bacterial and fungal plant pathogens. I9 yiggg studies were conducted to test the effect of different culture ages on the inhibitory activity of promising antagonists. Bean plants (Phaseolus vulgaris L.) were inoculated with mixtures of bacterial pathogens and antagonists. Several antagonists prevented infection, but only for short periods of time. Longer periods of protection did occur, but not consistently. In greenhouse studies seed inoculation with several bacterial antagonists protected bean seedlings subse— quently inoculated with Xanthomonas campestris pv. phaseoli which causes common bacterial blight of beans. Spray inoculation of plants with antagonist 143a significantly decreased the Severity of bean anthracnose (Colletotri- chum lindemuthianum) and bean rust (Uromyces phaseoli). ACKNOWLEDGMENTS I would like to thank the members of my committee, Dr. E. J. K103 and Dr. C. L. Schneider, for their interest in this research as well as their guidance. I thank my husband, Greg, for his assitance in statistical analysis of the data and for his continuous encouragement and support. I am especially grateful to Dr. A. W. Saettler for giving me the opportunity to do this research. I thank him for his advice and suggestions. I also appreciate the many hours he spent editing this paper. ii INTRODUCTION AND LITERATURE REVIEW MATERIALS AND METHODS TABLE OF CONTENTS Isolation and In Vitro Screening of Antagonists ...... The Effect of Temperature on the Ability of Antagonists to Inhibit Bacterial Pathogens of Beans ........................................ The Effect of Culture Age on In Vitro In- hibition by Antagonists of Bacterial and Fungal Pathogens ................................ Physiological Tests .................................. Pathogenicity Tests .................................. Screening of Antagonists In Vivo ..................... Use of Mixed Inoculation Techniques to Screen Antagonists for Ability to Protect Bean Plants from Fuscous, Common, and Halo Blight of Beans ................................. The Effect of Application Time on the Ability of Antagonists to Protect Greenhouse Grown Bean Plants from Bacterial and Fungal Pathogens The Effect of Mode of Application on the Ability of Antagonists to Protect Greenhouse Grown Bean Plants from Bacterial and Fungal Pathogens The Effect of Media on the Ability of Antagonist 143a to Control Uromyces phaseoli Infections on Greenhouse Grown Bean Plants ................. Viability of Isolate 143a on Spray Inoculated Bean Leaves ..................................... Control of Damping-Off of Sugar Beet Seedlings with Antagonist 143a ............................ iii OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 22 23 28 28 29 29 3O 32 35 36 37 Page Field Experiments Conducted in 1981 .................. 3 Use of Antagonists to Control Anthracnose and White Mold of Beans Under Field Conditions ...................................... 38 Treatment of Bean Seeds with Antagonists for Protection of Plants from Bacterial and Fungal Pathogens Under Field Conditions ......... 40 Protection of Bean Plants from Common Blight and Halo Blight Using Spray Applications of Various Antagonists .......................... 42 RESULTS .............................................. 46 In Vitro Studies with Antagonistic Bacteria .......... 46 Physiological and Pathogenicity Tests ................ 60 In V129 Studies with Antagonistic Bacteria ........... 61 Screening of Antagonists In Vivo Using Mixed Inoculation Techniques fEr the Ability to Control Fuscous, Common and Halo Blight of Beans ........................................ 61 The Effect of Time of Application on the Ability of Antagonists to Control Bacterial and Fungal Bean Pathogens on Greenhouse Grown Bean Plants ............................... 64 The Effect of Mode of Application on the Ability of Antagonists to Control Bacterial and Fungal Pathogens of Beans ................... 68 The Effect of Media on the Ability of Antagonist 143a to Control Uromyces phaseoli Infections on Greenhouse Grown Bean Plants ................. 68 Viability of Isolate 143a on Spray Inoculated Bean Leaves ..................................... 71 Control of Damping-Off of Sugar Beet Seedlings with Antagonist 143a ............................ 72 Field Experiments .................................... 72 DISCUSSION ........................................... 75 APPENDIX A Sources of Bacterial Antagonists ......... 84 APPENDIX B Sources of Plant Pathogenic Fungi ........ 87 APPENDIX C Sources of Plant Pathogenic Bacteria ..... 89 iv Table 10. LIST OF TABLES Inhibition of plant pathogenic fungi in vitro by various antagonists ................. Inhibition of plant pathogenic fungi in vitro by antagonist 143a ..................... The effect of different culture media on inhibitory activity of antagonists against Xanthomonas phaseoli var. fuscans (X Pseudomonas syringae pv. phaseoIicoIE—(Pp), andiPseudomonas syringae pv. syringae (PS) Lg vitro ..................................... The effect of temperature on the ability of antagonists to inhibit bean bacterial pathogens in vitro ........................... Effect of culture age on Ln vitro inhibition by antagonists of X. campestrls pv. haseoli (X ), P. syringae pv. phaseolicola (P25, and P. syr1ngae pv. syringae (PS) ................ Screening of antagonists Ln vivo for ability to control X. campestris pv. phaseoli var. fuscans (pr) in beans ....................... Screening of antagonists Ln vivo for the ability to control X. campestris pv. haseoli (Xp) and P. syringae pv. phaseolicola (PBS in beans ..................................... The effect of application time on ability of antagonist 139b to control X. campestris pv. phaseoli (X) in beans ....................... Control of Xanthomonas campestris pv. phaseoli (X ) in beans by preinoculation at var1ous t1mes with antagonists ....................... The effect of preinoculation of Montcalm bean plants with antagonist 143a on severity of anthracnose (C. lindemuthianum) .............. V Page 48 49 50 51 52 62 63 65 66 67 11. 12. 13. 14. The effect of seed treatment with various antagonists on disease severity in Manitou variety plants subsequently inoculated with Xanthomonas campestris pv. phaseoli XE ......................................... The effect of culture medium on ability of antagonist 143a to control bean rust (Uromyces phaseoli) on greenhouse grown‘bean plants ............................ Control of damping—off of sugar beet seedlings with antagonist 143a ......................... Sources of Bacterial Antagonists ............... vi Page 69 7O 73 84 LIST OF FIGURES Figure 1. Inhibition of various plant pathogenic fungi by filtrates from different aged cultures of antagonist 143a ................. 2. Inhibition of S. sclerotiorum by filtrates from different aged cuItures of antagon- ist 143a .................................... 3. Inhibition of isolate a of Xanthomonas campestris pv. haseoli by culture filtrates from.§ifferent aged cultures of antagonist 143a .......................... vii Page 54 58 INTRODUCTION AND LITERATURE REVIEW Research on biological control of plant pathogenic microorganisms is aimed at developing effective, environ- mentally safe, economical, and technically sound methods of disease control. Such control may be achieved either by introduction of microorganisms antagonistic to plant pathogens, or by manipulation of the environment in such a way as to favor inhibitory activity of natural antagonist populations. In recent years, increased interest has been shown in the control of plant diseases by bacterial antag- onists. Much of this interest was directly due to the discovery of a particular bacterium (35) which effectively controlled crown gall disease caused by some strains of Agrobacterium radiobacter var. tumefaciens. A large number of plant species are attacked by A. radiobacter var. tumefaciens including fruit trees, roses, and grapes (19,32); the pathogen enters these plants through wounds. Only pathogen strains possessing a Specific plasmid, the Ti plasmid, cause disease (53). A segment of DNA from the virulence plasmid is passed to and becomes incorporated into host cell DNA (50). Unregu- lated division of host cells is induced, leading to gall formation (19). 2 In addition to pathogenic agrobacteria, strains were discovered which did not possess the Ti plasmid and did not cause disease. The ratio of pathogens to nonpathogens was high in soil around diseased plants but low in soil around healthy plants (35). This discovery suggested the possi— bility of biological control of pathogenic strains through the use of nonpathogenic agrobacteria, Further investiga- tions showed that one strain of nonpathogenic agrobacterium, A. radiobacter var. radiobacter strain 84, protected tomato seedlings from crown gall when the pathogen/nonpathogen ratio was less than one (19). Strain 84 was shown to produce a specific antibiotic (Agrocin 84) which inhibits most of the pathogenic agro- bacteria (16). Currently strain 84 is being used commer- cially to control crown gall of stone fruit and roses; inoculation of plants with this strain effected virtually 10 X control of crown gall under field conditions. This is greater control than that achieved by use of standard bactericides (32). The treatment is also inexpensive and simple, requiring no special equipment; cost of inocula- tion is only a few cents per plant (32). Bacterial antagonists may inhibit pathogens in several ways: through antibiosis, by direct parasitism, by altering the environment to make it unfavorable for growth of the pathogen, or by stimulating host plant defenses (3). Anti- biosis is the mechanism whereby strain 84 controls crown gall infections caused by Agrocin 84-sensitive strains of 3 A. radiobacter var. tumefaciens (l9). Antibiosis is also implicated as the mode of action in control of Pseudomonas syringae pv. phaseolicola by a strain of P. fluorescens (48). Preinoculation of bean plants by inserting tooth- picks coated with P. fluorescens in the stem between the cotyledons, protected leaves above this point from infec- tion by P. syringae pv. phaseolicola. P. fluorescens could not be recovered from the protected leaves, suggesting that an antibiotic substance produced by the bacterium was transported up within the plant (48). Bacteria may directly parasitize bacterial and fungal plant pathogens by causing lysis. In most cases lysis is believed accomplished through the production of enzymes which attack components of the pathogen cell wall, causing disintegration (3). Bdellovibrio bacteria are known to have the ability to lyse other bacteria (3,38). An Arthro- bacter Sp. was shown to cause lysis of Pythium debaryanum hyphae in vitro. When tomato seed was dipped in a suspen- sion of the antagonist and planted in soil infected with P. debaryanum, a 37% reduction in disease resulted. The bacterium.was thought to produce proteases or glucanases which were primarily responsible for lysis (31). A strain of Bacillus pgmilus was isolated which caused lysis of germ tubes from uredospores of Puccinia graminis, P. coronata, and P. graminis f. sp. tritici in vitro (33). When bact- erial suspensions were applied to wheat leaves prior to 4 inoculation with the pathogen, disease was significantly reduced (33). Several bacteria control plant pathogens by presumably altering the environment in such a way as to make it unfav- orable for pathogen growth. Antagonists may accomplish this by altering the pH of the substrate or by depleting the sub- strate of nutrients required for growth of the pathogen. Erwinia herbicola affects growth of several bacterial patho- gens by lowering the pH of the substrate (9,37). Hsieh and Buddenhagen (1974) found E. herbicola inhibited Xanthomonas oryzae 19 vitro (15). When pH of the media was adjusted to 7.0, no inhibition was observed. They also found that while live cells of P. herbicola provide some protection to rice plants, killed cells, which did not lower the pH, provided no protection (15). Blakeman and Fraser found that germina- tion of Botrytis cinerea conidia on Chrysanthemum leaves was inhibited by bacteria present on the leaves (2). Antifungal compounds were not detected in droplets containing the bacteria, and subsequent experiments revealed that antagon- istic bacteria removed amino acids from the environment at a rate faster than the fungus. Depletion of nutrients from the environment was proposed as the cause of inhibition in this case (4). How bacterial antagonists inhibit plant pathogens by stimulation of host plant defenses is not well understood. Several excellent articles dealing with this subject are found in the literature (12,27,36,52). Because antagonists 5 that inhibit only by this method are not identified using normal screening procedures for bacterial antagonists, this topic will not be discussed extensively. The subject is, however, mentioned later in the discussion of in 21539 screening procedures. General procedures to detect bacterial antagonists in- volve isolation, and in_zi££g screening, followed by in 2139 testing and/or screening (41). Most researchers have iso- lated antagonists from the same species of plant which they wish to protect from disease (5,7,10,21-25,31,33,45—48). In some cases, antagonists were isolated from soil (6,11,17, 29,40,49). Soil isolation is often used when the pathogen invades host seed or root tissues. An antagonistic isolate obtained from or near the plant part to be protected is preferable, since such isolates are theoretically better adapted to the particular plant part; therefore, these isolates are more effective in controlling disease, than an antagonist taken from a completely different site (1,6). Potential bacterial antagonists are screened LE giggg for ability to inhibit pathogens by several methods. One of the most common methods is to place potential antagonist and pathogen near each other in an agar medium. Inhibition of pathogen growth is evidenced by formation of an inhibi- tion zone. Antagonists found to inhibit pathogens 12.21EEE are then subjected to L3 vivo testing. In some cases only antagonists causing formation of especially large inhibition zones were retained (1,21). 6 Type of media used, incubation temperature, and test- ing technique all affect the results of Lg 21329 screening tests. Relative to effect of media, Weinhold and Bowman found that a Bacillus sp. produced greater antibiosis on soybean tissue extract agar than on barley extract agar (54). Vasudeva et. al. reported that culture filtrates of a B. subtilis isolate grown in dextrose asparagin phosphate media reduced Fusarium udum colony growth by 61%; whereas culture filtrates from potato dextrose broth cultures of the antagonist reduced colony growth by only 13% (51). Dunleavy found inhibitory compounds produced by B. subtilis more active at pH 4.0 (8). Teliz-Ortiz and Burkholder found that P. fluorescens inhibited P. syringae pv. phase - licola actively on potato glucose agar but not on nutrient agar (48). Spiers found that although A. radiobacter var. radiobacter strain 84 produced Agrocin 84 on all media test- ed, inhibition was not. Agar plugs were removed from such plates and placed in holes in plates containing media nor- mally allowing inhibition, sensitive A. radiobacter var. tumefaciens isolates were inhibited (42). The temperature at which test plates are incubated influences Lg vitro inhibition of a particular pathogen by an antagonist. In one study, five isolates of Bacillus sp. were tested for inhibition of Rhizoctonia solani at different temperatures (26). Each antagonist grew at temp- eratures below 12°C but none inhibited the pathogen at temperatures below 12°C (26). Vasudeva et. al. found 7 maximum growth inhibition of Fusarium udum colonies by B. subtilis at 24-340C while size of inhibition zones de- creased at higher or lower temperatures (51). Techniques of testing may also affect results. When potential bacterial antagonists were placed on one side of the plate and Typhula idahoensis mycelia on the other, fungal colony growth was inhibited by several bacteria. When the test was repeated placing the bacteria in the middle of the fungal colonies no inhibition of the path- ogen resulted (18). ln_vLE£g_screening has several other limitations, in addition to problems in interpreting the results, due to the effects of cultural environment and testing technique. Because the testing environment differs greatly from actual ig_yizg conditions, antagonists successful at inhibiting pathogens ig_yiggg_may not necessarily be successful 12 vivo. These inconsistencies may arise because: (1) media employed may provide a nutrient Spectrum different from that found in the host environment, and (2) effects of normal host environment and microflora on the antagonist/ pathogen interaction are not encountered in_vitro. Frequently, bacteria which inhibit pathogens ig_vitro through antibiosis fail to do so in_vivo, possibly because nutrients available for antibiotic production in_vitro may not be available in/on the host plant (21,24,28,30,45). Even if antibiotics are produced LE vivo they may be 8 inactivated, degraded or adsorbed onto soil particles or plant tissues (3,39). Failure of successful Lg ELLLQ antagonists to control pathogens Lg ngg may also be due to loss of viability of the antagonist Lg yLyg. In one study, a bacterial antag- onist inhibitory to Colletotrichum lagenarium Lg vitro gave no control when applied to cucumber plants in the field prior to pathogen inoculation. Further study reveal- ed that 99% of the antagonist cells were no longer viable 24 hours after application. Viability was not significant- ly improved by adding nutrients to the inoculum (25). Loss of viability may have been due to environmental conditions, to antagonism from other naturally occurring microorganisms, or to competitive weaknesses of the antagonist as compared to normal microflora. Even when disease control is obtained Lg ngg, it can not be assumed that the mode of inhibition is the same as that Lg YiE£9~ Siminoff and Gottlieb found that Strepto- myces griseus inhibited B. subtilis Lg vitro by the pro- duction of antibiotics. S. griseus also inhibited growth of S. subtilis in soil. When a mutant S. griseus, unable to produce antibiotics, was added to soil, growth of S. subtilis was similarly inhibited. Therefore, control orig- inally obtained was not due to antibiosis (39). It is difficult to predict Lg ngg screening perform- ance of antagonists which do not exhibit activity Lg ELLLQ (5). Despite the limitations of Lg vitro screening, the 9 procedure remains an inexpensive, rapid way to screen large numbers of potential antagonists; thus it is almost always used as the initial Step in the screening proce— dure. The many successful attempts at biological control using bacterial antagonists originally selected 13.21EEQ indicate its utility (6,8,11,22,31,33,40,46,48,49). After Lg_yLE£g screening of potential antagonists, Lg_vivo tests are performed to determine whether such isolates will control specific diseases on greenhouse or field grown plants. Many factors affect the outcome of these tests, including mode of antagonist application, nutrients available in the inoculum, application time and populations of the antagonist relative to the pathogen. In most cases, potential antagonists are applied to the target by only one mode so it is difficult to deter- mine to what extent mode of application affects Lg vivo screening results. In the few cases where several modes of application were attempted, results suggest that the mode is an important factor. A. radiobacter var. radio- bacter strain 84 decreased crown gall on peach trees by 95% when roots of trees were dipped in the inoculant prior to transplanting. When both seeds and roots were so treated, 99% control was achieved. When just seeds were inoculated, only 7 % control was obtained (16). Strain 84 was more effective in controlling pathogenic strains insensitive to Agrocin 84 when applied to the seed and roots rather than when applied to aerial plant 10 parts (32). Swineburne controlled Nectria galligena infection of apple trees when S. subtilis was applied to the leaf scar tissue. Bacteria applied to internodal areas did not persist and did not control disease (46). In contrast, P. fluorescens controlled P. syringae pv. phaseolicola infections of bean equally well whether applied as a spray, by a toothpick method, or as a seed treatment (48). Since antibiotic production can be enhanced Lg ELEEQ by nutrient modifications, attempts have been made to in- crease inhibitory activity of antagonists Lg ngg by including nutrients in the inoculum. S. subtilis con- trolled P. solani more effectively in greenhouse tests when a nutrient solution high in nitrogen was added to soil containing the antagonist (8). A bacterial antag— onist used to control cucumber anthracnose was more ef- fective when applied in nutrient broth, soybean meal broth or glucose (23,24). S. subtilis significantly reduced rust infections (Uromyces phaseoli) on greenhouse grown beans only when Eugonbroth was applied with the bacterium; Eugonbroth alone provided no protection from infection (44). The time at which the antagonist is applied in re- lationship to the time of inoculation or infection by the pathogen also affects disease control. P. fluorescens protected bean plants from infection by P. syringae pv. phaseolicola only when applied prior to the pathogen (48). P. subtilis controlled Uromyces phaseoli infections on ll greenhouse grown bean plants only when applied 0-3 days prior to the pathogen. Weekly applications to plants in the field, however, did not significantly control bean rust (44). Strain 84 of A. radiobacter var. radiobacter was more effective in reducing crown gall on Mazzard cherry trees when applied after the pathogen than when applied prior to the pathogen (32). P. cepacia sprayed weekly on field grown peanuts reduced Cercospora leaf spot by 38%; weekly applications were necessary because antagonist populations declined rapidly after applica- tion (43). Populations of antagonists relative to those of the pathogen may also affect efficiency of the antagonist. Strain 84 was only found effective in reducing crown gall on tomato when the pathogen/nonpathogen population ratio was less than 1 (19). S. herbicola was more effective in controlling X. ggyggg infections of rice when the pathogen/ nonpathogen population ratio was less than 1 (15). In summary, a large number of factors may influence the results of Lg ngg testing. Antagonists successful in controlling disease in the protected environment of a mist chamber or greenhouse are often ineffective in the field (7,20,25,26,43). In one study, B. subtilis pro- tected corn from corn root infections in the field during a wet season but did not give protection during two dry growing seasons. A fungal antagonist showed the ability to protect corn during the two dry seasons but was less 12 effective during the wet season. It was suggested that a mixture of antagonists might have protected the seedlings under both extremes of field conditions (20). The fact that mixtures of bacterial antagonists have not been tested to control disease is probably a shortcoming of the screening procedures used to identify potential antagon- ists (1). Successful control of bean rust and halo bacterial blight with bacterial antagonists suggests that biological control methods may be useful in controlling some other bean diseases (44,48). Bean halo blight and common bacteri— al blights are generally managed through preventive meas- ures such as use of clean seed and tolerant varieties (14). Other bean diseases, such as white mold can be partially controlled by cultural practices and chemical applica- tions (13,34). In each case however, the diseases can be difficult and expensive to control once pathogen inoculum is present in the field. Development of biological control agents for control of bean diseases could provide safe and inexpensive methods to supplement current control methods. 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Phytopathology 50: 119-123. Thirumalachar, M. J., and M. J. O'Brien. 1977. Suppression of charcoal rot in potato with a bacter- ial antagonist. Plant Disease Reporter 61: 543- 546. Thomashow, M. F., R. Nutter, K. Postle, M. D. Chilton, F. R. Blattner, A. Powell, M. P. Gordon, and E. W. Nester. 1980. Recombination between higher plant DNA and the Ti plasmid of Agrobacterium tumefaciens. Proc. Natl. Acad. Sci. 77: 6448-6452. Vasudeva, R. S., A. C. Jain, and K. G. Nema. 1952. Investigations of the inhibitory action of Bacillus subtilis on Fusarium udum Butl. , the fungus causing wilt of Pigeon-Pea4 (Ca anus ca an (L. ) Millsp. ). Ann. Appl. Biol. 229- 238. Wade, M., and P. Albersheima 1979. Race-specific molecules that protect soybeans from Phytophthora me as erma var. sojae. Proc. Natl. Acad. Sci. 76: 2433-4437. Watson, B., T. C. Currier, M. P. Gordon, M. D. Chilton, and E. W. Nester. 1975. Plasmid required for viru- lence of Agrobacterium tumefaciens. J. Bacteriology 123: 255-264. Weinhold, A. R., and T. Bowman. 1968. Selective in- hibition of the potato scab pathogen by antagonistic bacteria and substrate influence on antibiotic production. Plant and Soil 28: 12-24. MATERIALS AND METHODS Isolation and In Vitro Screening of Antagonists A total of 214 bacterial isolates were screened for ability to inhibit Xanthomonas campestris pv. phaseoli in_vitro. In addition, 131 of the isolates were screened for the ability to inhibit Pseudomonas syringae pv. phaseo- licola, and 58 were screened for the ability to inhibit Pseudomonas syringae pv. syringae in vitro. Most (140) of the isolates were obtained by means of the lacuna method (8), as follows. Bean seeds were surface sterilized for 2 minutes in 2.5% sodium hypochlorite (2.5% Cl'), and rinsed twice for 1 minute each in sterile distill- ed water (SDW). The seeds were allowed to air dry, and ground to a fine powder in a mill (screen size 40p). Sam- ples of ground seed (8.5 g) from each seed lot were then suspended in 50 ml of Kings Medium B Broth (20 g peptone, 2.5 g KZHPOA'BHZO, 6 g MgSO4'7H20, 15 m1 glycerol in 1000 ml distilled water) (4) or in 50 m1 BYE CHJg yeast extract in 1000 ml 0.01M P04 buffer pH 7.2) and the mixtures in- cubated on a rotary shaker at room temperature (22 : 20C). After 48 hours, serial dilutions of the cultures were 4 6 7 8 made and 0.1 m1 of the 10‘ , 10'5, 10’ , 10' , and 10' 19 20 dilutions were mixed with 0.1 m1 of a pathogen suspension (adjusted to 0. D. 0.3 at 600 nm) plus 4.0 ml of soft agar (10 g agar in 1000 ml distilled water). Absorbance was determined using a Bausch and Lomb Spectronic 20 Spectro- photometer. Suspensions were poured over a hard agar (15 g agar in 1000 ml distilled water) base layer. Soft BYE over a hard BYE base layer was used to test antagonists from cultures incubated in BYE, while soft KMB was used over a hard KMB base layer to test antagonists from cul- tures incubated in liquid KMB. Antagonistic bacteria selected for further study were those giving distinct areas of inhibition in the pathogen lawn. These colonies were picked out and maintained on modified YCA (10 g yeast extract, 2.5 g calcium carbonate, and 15 g agar in 1000 ml distilled water) or KMB plates for further testing. If more than one isolate with identi- cal colony characteristics was obtained from a given seed lot, only one was retained. Seed lots used, seed type and antagonistic isolates obtained are given in Appendix A. Numerous (58) isolates of internal bean seed contamin- ants were obtained from Mintarsih Adimihardja (presently at Kampus University, Lampung, Indonesia), and 6 isolates were obtained from Maureen Mulligan (graduate student, Department of Botany and Plant Pathology, Michigan State University, East Lansing MI). In addition 4 isolates were obtained from bean stem sections after grinding the stem sections in buffered saline and streaking the liquid onto 21 KMB and YCA plates. The source of each of these isolates is given in Appendix A. All isolates were further tested for inhibitory act- ivity by spotting or streaking the bacteria on t0p of a soft agar layer containing the pathogen of interest. Most frequently used was a 10 ml base layer of Nutrient Agar (Difco) with a NBGA soft layer consisting of 8 g Nutrient Broth (Difco), 5 g glucose, and 10 g agar in 1000 ml dis- tilled water. Occasionally NBGYE (consisting of 8 g Nutrient Broth, 5 g glucose, 5 g yeast extract and 10 g agar in 1000 ml distilled water) was used for the soft agar layer. Certain of the antagonists were also tested on Bean Pod Agar (Difco). Single colonies from 2-3 day old antag- onist cultures were streaked on t0p of a soft BPA layer (15 g Difco Bean Pod Agar in 1000 ml distilled water) con- taining the pathogen of interest; BPA was also used for the base layer in these experiments. This was done to test the effect of different cultural media on inhibitory activity. Some antagonists were tested for the ability to inhibit growth of plant pathogenic fungi in culture. In general, tests were performed on PDA (Difco Potato Dextrose Agar), while tests for inhibition of Colletotrichum lindemuthianum were performed on EPA, and tests for the inhibition of Phytophthora megasperma were conducted on LBA (Difco Lima Bean Agar). The source of all fungal isolates used through- out this study are given in Appendix B. 22 A 5 mm diameter plug of mycelium was removed from the advancing edge of a 4—8 day old colony of each fungal iso- late using a cork borer. Each mycelial plug was placed on a fresh plate of PDA, and a 5,41 droplet of a turbid suspension of the antagonist in SDW was spotted onto the plate 2 cm from the mycelial plug. Control plates were those in which only SDW was spotted beside the mycelial plug. Studies included 3 replications and 1 control for each antagonist/pathogen combination. Plates were observed after 4-7 days incubation in the dark at 25°C. The Effect of Temperature on the Ability of Antagonists to'lnhibit Bacterial’Pathogens of Beans Experiments were performed to determine possible temperature effects on the inhibitory activity of antagon- ists. In these studies, NBGA was used for both soft and base layers of media. Pathogens were grown on a shaker for 36 hours in liquid BYE, adjusted to an absorbance of 0.25 at 600 nm, and 0.1 ml of the pathogen suspension mixed into 4.0 ml of soft agar. The mixture was poured over the 10 ml base layer. Single colonies were then taken from 48 hour old YCA or KMB cultures of each antagonist using sterile Q-tips and streaked on the soft agar layer. Control plates for each pathogen were streaked with sterile Q-tips rubbed on the surface of uninoculated YCA or KMB plates. Up to 5 or 6 replications were made for each antagonist/pathogen combination at each temperature. Plates were incubated 23 in the dark at 15, 20, 30 and 36°C. After 48 hours, inhibition zones were measured and recorded. The Effect of Culture Age on In Vitro Inhibition by Antagonists of Bacterial—and Fungal Pathogens Turbid suspensions of selected antagonists were pre- pared in SDW and.5,yl droplets of the suspensions or of SDW were placed in the middle of plates containing 10 ml BPA. The isolates were allowed to grow for l to 9 days before being killed by 30 minutes exposure to chloroform vapor. Control plates were similarly exposed to chloroform vapor. A 4.0 ml soft layer of BPA containing the bacterial pathogen of interest was then poured over each plate. Pathogens included Xanthomonas campestris pv. phaseoli (KB), Pseudomonas syringae pv. phaseolicola (Pp), and Pseudomonas syringae pv. Syringae (Pg). The sources of all bacterial pathogens used throughout this study are given in Appendix C. In each case, 4 replications were made for each antag- onist (of a particular culture age)/pathogen combination. Plates were observed after 1, 2 and 3 days, and inhibition zones recorded. Isolate 143a, an endospore forming bacterium, could not be killed with chloroform, therefore a number of eXperi— ments were performed to detect activity of inhibitory com- pounds in the culture filtrates of different aged cultures. In the first experiment, inhibition of bacterial pathogens was tested by first mixing 0.1 ml of a suspension of EB 24 isolate 520 or pr R10 with 4.0 ml of soft NBYEA. This suspension was poured over a NA base layer and allowed to harden. Fungal inhibition was tested by first spreading 0.2 ml of a mycelial suspension of Sclerotinia sclero- tiorum or Phoma betae on plates containing 10 ml each of PDA. Isolate 143a was grown in NBGCC (8' gNB, 5 g glucose, and 5 g calcium carbonate in 1000 ml distilled water) for 24 hours. The culture was passed through a 0.2,upore size filter (Nalgene presterilized disposable filter unit) using a vacuum of approximately 1200 mm Hg. Sterile filter paper discs 1 cm in diameter were dipped in the culture filtrate and two such discs were placed in plates contain- ing the test bacterial or fungal pathogen. Control plates contained the pathogen and two filter discs dipped in filter sterilized NBGCC. Seven replications were prepared for each pathogen and plates were incubated in the dark at 25°C. Plates containing bacterial pathogens were examined after 48 hours while those containing fungal pathogens were examined after 4 days. The presence of any inhibition zones around or beneath the filter discs was recorded. In the second experiment, antagonist 143a was grown in Eugonbroth (BBL) for 4 or 35 days on a shaker at room temperature. The 4 day old culture was filter sterilized using a 0.45,ppore size filter while the 35 day old culture was filter sterilized using a 0.2‘ppore size filter. Drop- lets (5 pl) of each culture filtrate were placed on plates 25 containing 10 m1 of BPA, allowed to dry for 1 hour, and then 4.0 ml of soft BPA containing 0.1 m1 of a suspension of ER 11 was poured over this base layer. Plates were incubated for 24 hours in the dark at 25°C then observed for the presence of inhibition zones. In a third experiment, 143a was grown in Eugonbroth for l, 2, 4, 6 or 9 days, and culture filtrates obtained by passing each through a filter of 0.45/upore size. A 10/41 droplet of one of the culture filtrates was spotted onto each of 4 plates containing 10 ml of PDA, and a myceli- al plug of S. sclerotiorum (5 mm in diameter) placed 1 cm from each droplet. Control plates were spotted with unin- oculated filter sterilized Eugonbroth. Radial growth of the fungal colonies in the direction of the culture filtrate was measured after 3 days. In the final experiment with 143a, flasks containing 75 ml of Eugonbroth were inoculated with 143a at various times (65-0 days) prior to filter sterilization. Cultures were grown at room temperature on a shaker. Control flasks containing Eugonbroth alone were also placed on the shaker for various time intervals prior to filter steriliza— tion. All cultures for a given experiment were filter sterilized the same day using Nalgene presterilized dis- posable filter units with apore size of 0.45 [4. Culture filtrates were tested immediately, after autoclaving 20 min- utes, after 30 days aging, and after 30 days aging plus autoclaving. 26 Pathogens used in these eXperiments included S. sclerotiorum, P. betae, Rhizoctonia solani, Fusarium solani f. sp. phaseoli, C. lindemuthianum, as well as Pp 11 and $2 a. Plates of bacterial pathogens were prepared by add- ing 3.0 m1 of a suspension (0. D. 0.3) of the bacteria in 0.01M P04 buffer pH 7.2 per 100 m1 warm (45°C), liquid NA. After mixing, 10 ml of the solution was added to each petri dish and allowed to harden. To test S. sclerotiorum, R. solani, and P. betae, mycelium of each was scraped off 4 day old PDA cultures and placed in SDW for 20 minutes and shaken frequently. Inoculum of P. sglagi f. sp. phaseoli was prepared by scraping spores from.7 day old PDA cultures and suspend- ing in SDW. A 0.2 ml aliquot of each mycelial or spore suspension was spread evenly over plates containing 10 m1 of PDA. Plates containing C. lindemuthianum were prepared by first scraping conidia from a 6-9 day old BPAB (22.5 g BPA and the extract from 20 g of navy bean seeds in a total of 1000 ml distilled water) culture of the pathogen. The conidia were suspended in SDW for 20 minutes with frequent shaking. A 0.2 m1 aliquot of the spore suspen- sion was spread evenly over plates containing 10 ml each of BPA. Four or 5 wells of 5 mm diameter were then cut with a cork borer in the agar of the seeded plates. wells were 27 spaced equidistant from one another and sides of the petri dishes. A 30;:1 aliquot of each test culture filtrate was placed in each well; one well in each plate contained fil- ter sterilized Eugonbroth as a control. At least 3 replications were prepared for each experiment for each pathogen/culture filtrate combination, and all plates were incubated in the dark at 25°C. Plates containing bacterial pathogens were observed after 48 hours, while those with fungal pathogens were observed after 4 days. In each case size of inhibition zones was recorded. To further study 143a inhibition of fungi in culture, 5’11 of 143a suspension was spotted onto Eugonagar plates. At 10 days after inoculation, 5 mm diameter plugs were re- moved from areas 2 mm from the bacterial colony. The plugs were transferred to fresh plates of PDA (20 ml/plate). Plugs of S. sclerotiorum mycelium were taken from 4 day old cultures of the fungus and placed on the PDA plates 20 mm from the Eugonagar plugs. After 6 days incubation at 25°C, S. sclerotiorum hyphae were removed from edges of inhibition zones, examined microscopically and trans- ferred to fresh PDA plates. Plates were retained for 20 days to determine whether hyphae were viable. To determine whether in vitro inhibition of S. Pings- muthianum by 143a was due to pH effects, 5 mm diameter plugs of pathogen strains alpha, beta and gamma were placed 5 cm from a colony of 143a. Medium used in these experiments was BPA prepared in 1000 ml 0.01M P0 buffer pH 7.2. 4 28 Plates were incubated in the dark at 25°C for 11 days at which time inhibition zone size was recorded. Two plates were used for each pathogen strain and the experi- ment repeated twice. Physiological Tests Attempts were made to identify several of the antagonists. Tests included: fluorescence on KMB (l), catalase (3), growth in Thioglycollate Broth (9), gram stain (l6), arginine dihydrolase (15), reduction of nitrate (ll), Voges-Proskauer Reaction (3), hydrolysis of starch (3), oxidase activity (6), and hypersensitivity in tobacco (5). PathogenicitygTests All antagonists used for in yigrg studies were first tested for pathogenicity to beans by the seedling injec— tion technique (10). Manitou kidney bean seedlings were injected at 10-12 days with turbid suspensions of each isolate; a minimum of 9 seedlings were used for each isolate. Isolates causing browning of stems, wilting, or any other abnormality in plant growth were discarded. 29 Screening of Antagonists In Vivo Use of Mixed Inoculation Techniques to Screen Antagonists f5r Ability to Protect Bean Plants from Fuscous, Common, andiHalo Blight of Beans Various antagonists were tested for ability to control halo, fuscous and common bacterial blight ig yiyg in two different experiments. In the first experiment, flasks containing 25 ml of BYE were inoculated with 0.1 ml sus- pension (0.3 0. D.) of the antagonist and 0.1 ml suspension (0.3 0. D.) of the pathogen. After 2 days incubation on a shaker at room temperature, the mixed cultures were injected into lO-day-old Manitou seedlings (10). Control seedlings were injected with BYE alone or the pathogen alone. After 17 days plants were observed for disease symptoms. As there was little or no variation in disease severity between plants in each pot, disease ratings were on a pot basis rather than individual plant basis. The experiment was repeated 3 times. In the second mixed inoculation experiment, equal suspension volumes of antagonist and pathogen were mixed and immediately injected into the primary leaf nodes of 10-12-day-old Manitou seedlings. Plants were observed 5, 10, 20, and 34 days after injection and rated for disease severity. In each mixed inoculation experiment, disease ratings were made using the following rating system: (-) healthy, (+) plants with stem browning, (++) plants with stem browning, slight wilting and, (+++) severely wilted plants. 30 The Effect of Application Time on the Ability of Antagonists to Protect'Gieefihouse Grown Bean Plants from Bacterial andl Fungal Pathogens Experiments were performed to determine whether time of antagonist application affected ability of the antag- onists to control common blight. Manitou bean plants were grown in the greenhouse (l plant/pot) at temperatures of 22-300C. Antagonists were grown in BYE at room temp- erature for 2 days on a shaker, and the cells sedimented by centrifugation for 15 minutes at 10,000g. Bacteria were suspended in 0.01M PO4 buffer, and adjusted to an 0. D. of 0.4. Plants were sprayed with the bacterial suspension to run off at various time intervals (9-1 days) prior to inoculation with the pathogen. Control plants were either unsprayed or sprayed with PO4 buffer alone. A minimum.of 8 pots of plants were sprayed for each antagonist at each time interval. The common blight pathogen, isolate $2.11: was pre- pared in the same manner as antagonists except that the inoculum suspension was adjusted to 0. D. 0.25. Plants were sprayed lightly to run off with the pathogen after d all plants possessed fully expanded 3r trifoliolate leaves. Plants were observed after 3 weeks and percentage of the 2nd trifoliolate covered with lesions recorded. Duncan's multiple range test was used in each experiment to compare disease severity of treated plants with that of the controls (13). 31 The effect of application time on ability of 143a to protect bean seedlings from infection by S. lindemuthia- BEE;W33 also tested. Montcalm kidney bean seedlings were pretreated with the antagonist at various times prior to inoculation with the pathogen. Plants were pretreated by spray inoculation with a suspension of 143a (approximately 2.5 x 109 cfu/ml) in SDW or with SDW alone. Approximately 3.5 m1 of the bacterial inoculum or SDW was used per plant. The plants were then placed in a mist chamber with relative humidity above 95% until inoculation with strain beta of S. lindemuthianum. The seedlings were no more than 18 days old before inoculation with the pathogen. Inoculum.was prepared by flooding 6-10 day old BPAB cul- tures of S. lindemuthianum with SDW, scraping the agar surface and pouring the resulting spore suspension through two layers of cheesecloth into SDW. One plate of Q. lindemuthianum was used for each 150 ml of SDW (approxi- 7 mately 1 x 10 spores/ml); 0.1% v/v Tween 80 was added to the inoculum. Plants were removed from the mist chamber, spray inoculated with the pathogen and immediately returned to the mist chamber. Special attention was given to inocu- lating the undersides of leaves, the growing point and the stems. After an additional 8 days in the mist chamber, plants were removed to greenhouse benches for 2 days before disease ratings were made. 32 Plants were rated subjectively and quantitatively to determine disease severity. Subjective data was taken using the following rating system: healthy a few lesions on the stem or first trifoliolate leaf a moderate number of lesions on the stem and first trifoliolate leaf both first trifoliolate leaf and stem.with num— erous lesions = severe water-soaking and chlorosis of the first trifoliolate leaf = collapse of the first trifoliolate leaf, lesions on the leaf, stem, and petioles U1 P U) N l—‘O II Quantitative data was obtained by counting numbers of lesions on the stem from soil level to the point of attachment of the first trifoliolate leaf. Both sets of data were analyzed using Duncan's multiple range test. The Effect of Mode of Application on the Ability of Antagonists to Protect Greenhouse Grown Bean Plants from *Bacterial’and'Fungal'Pathogens Seed treatments with a variety of antagonists were studied to determine whether mode of inoculation affected degree of control of common blight of beans. Manitou bean seeds were surface sterilized by soaking in a solution of 2.5% sodium hypochlorite (2.5% Cl') for 2 minutes. The seeds were then rinsed twice for 1 minute each with SDW and allowed to dry. Cultures of each antagonist were grown for 48 hours in BYE on a shaker. Five Manitou seeds were then placed in each mixture of 10 ml soft BPA with 1.0 ml of an antagonist culture, or in 11 ml of soft BPA alone. Seeds were 33 incubated in the inoculum for 16 hours in the dark at 25°C then planted in the greenhouse in vermiculite with 1 seed per pot. One month after planting plants were sprayed lightly to run off with Kp ll. Pathogen inoculum.was prepared by centrifuging 48 hour old liquid BYE cultures of Sp 11 for 15 minutes at 10,000g. Sterile distilled water was added to adjust inocula to an 0. D. of 0.2. Twenty days after spray inoculation of plants with Sp 11 number of lesions on the first trifoliolate leaf of each plant was recorded. The experiment was designed with 5 blocks containing 1 replication (1 plant) for each treatment. Plants were randomized within each block and were separated by suffi- cient space to prevent physical contact between plants. After square root transformation of the data, to obtain homogeneous variance, seed treatments with different antag- onists were compared to control treatments using Duncan's multiple range test. Seed treatments were also evaluated in two experiments as a control for anthracnose. In the first experiment, seeds of the Michigan Improved Cranberry (MIC) variety naturally infected with S. lindemuthianum strain beta were obtained from field plants inoculated with the pathogen during the 1981 growing season. To determine whether seed treatment with 143a would reduce disease severity, seeds were dipped in SDW, soft BPA, or soft BPA containing 143a 9 (approximately 2.5 x 10 cfu/ml). BeCause of differences 34 in seed germination the number of plants used for each treatment varied from 14 in one experiment to 22 and 24 in the other experiments. Seeds were planted immediately after treatment in vermiculite or a mixture of vermiculite and sterile green- house soil. Upon germination, plants were transferred to a mist chamber for 7-10 days. Seven days after removal from the mist chamber, number of lesions on the portion of the stem from soil line to the primary leaf node was recorded. In the second experiment, uninfected seeds of the MIC variety were surface sterilized then dipped into SDW, soft BPA, or soft BPA containing 143a (approximately 2.5 x 109 cfu/ml), and planted. When all plants possessed fully lSt expanded trifoliolate leaves they were spray inoculated with S. lindemuthianum strain beta (approximately 1 x 107 spores/ml) and placed in a mist chamber for 8 days. Two days after removal from the mist chamber the number of lesions on the stem from soil line to the primary leaf node was recorded. The experiment was repeated twice: 27 plants were used for each treatment in each experiment. Log transformations were used to achieve homogeneous variance of the data from both experiments and Duncan's multiple range test was then used to compare lesion numbers found in the different treatments within each experiment. 35 The Effect of Media on the Ability of Antagonist 143a to Control’Uromyces phaseoli Infection on Greenhouse Grown Bean Plants The effect of different suspending solutions on the ability of 143a to control bean rust was tested. An isolate of B. subtilis (obtained from Dr. J. R. Stavely, USDA, Beltsville, MD) reported to protect bean seedlings from bean rust (12) was compared with 143a. Fourteen day old UI-ll4 pinto bean seedlings were spray inoculated with an average of 3.0 ml/plant of the following solutions: (1) SDW, (2) no spray, (3) sterile Eugonbroth, (4) 143a from a 2 day old Eugonagar culture suspended in SDW, (5) 143a in Eugonbroth (a two day old culture) or (6) B. ggp- pills in Eugonbroth (a two day old culture). All bacterial inocula were adjusted to an absorbance of 0.4 at 600 nm. Special care was exercised to completely cover both sides of each primary leaf with the appropriate inoculum. Plants were placed in a mist chamber at relative humidity greater than 95%. After two days plants were spray inoculated with an average of 2.0 ml/plant of a suspension of Uromyces phaseoli uredospores prepared by adding 5 mg of uredospores per 10 ml of SDW containing 0.1% v/v Tween 20. The experiment was designed to include 2 blocks with l replication of each treatment (consisting of 12 samples) randomly arranged.within each block. Each treatment was separated from others in the mist chamber by a row of pots containing UI—ll4 plants. 36 After 5 days in the mist chamber, plants were rated for number of lesions on each primary leaf. Data were transformed using a square root transformation to achieve homogeneous variance. Duncan's multiple range test was used to compare each treatment with the control treatment of interest. Results should be viewed with caution since the experiment was performed only once. Viability of Isolate 143a on Spray Inoculated Bean Leaves In order to determine whether antagonist 143a remains viable on spray inoculated bean leaves, leaf prints were obtained. Seven different varieties of beans were used including Manitou, Montcalm, Michigan Improved Cranberry, UI-ll4, GN Tara, Sanilac, and Black Turtle Soup beans. Plants possessing fully expanded 2nd trifoliolate leaves were spray inoculated with 143a (approximately 2.5 x 109 cfu/ml) in SDW. Control plants of each variety were spray inoculated with SDW alone. Inoculum.of 143a was prepared by centrifugation of 2 day old Eugonbroth cultures at 10,000g for 15 minutes. The pellet was resuspended in SDW and absorbance adjusted to 0.3 at 600 nm. One, three and eleven days after inocula- an trifoliolate was removed, tion, one leaflet of the and the upper surface pressed flat onto Eugonagar plates containing cycloheximide (50 ppm) for 1 minute (2). Plates were incubated at 25°C in the dark for 2 days. Percentage of leaflet area colonized by bacteria with 37 colony characteristics identical to isolate 143a was recorded for both treated and control plants. Control of Damping-Off of Sugar Beet Seedlings with 143a Sugar beet seeds suspected of being internally in- fected with P. pggag were surface sterilized by soaking for 5 minutes in 0.5% sodium hypochlorite (0.5% Cl'). Seeds were allowed to dry and placed in petri dishes con- taining water agar (18 g agar in 1000 ml distilled water). After 3, 5 and 10 days, seeds were observed microscopically in order to determine number from.which mycelium charac- terisitic of P. 93525 grew out; P. 23523 hyphae are septate with vesicles. A total of 360 randomly selected seeds from the seed lot were observed, and percentage of seed infected was determined. To determine whether isolate 143a could control damping-off of sugar beet seedlings, seeds were treated for one and a half hours with 143a suspended in 0.5% w/v cellulose methyl ether (CME) (approximately 1 x 108 cfu/ seed) or CME (0.5% w/v) alone (14). Seeds were allowed to dry for 24 hours at 25°C. Treated seeds were then planted in sand (with a water content of 4.0%), 180 seeds per wooden flat and 2 flats per treatment. Each flat was covered with a plastic tOp and left at room temperature (23 : 3°C). After 8 and 16 days the number of seeds which did not germinate and the number of infected seedlings were recorded. Germination 38 and infection data for 143a treated seeds and for control seeds were compared using Duncan's multiple range test after an arcsin square root transformation. Field Experiments Conducted in 1981 During the summer of 1981, various experiments were performed in the field to test the ability of several potential antagonists to control bean pathogens. All experiments were conducted at the Michigan State University Botany Farm located in East Lansing, Michigan. Use of Antagonists to Control Anthracnose and White Mold of Beans Under Field Conditions An experiment was performed to determine whether spray inoculation with 143a would protect plants from infection with S. lindemuthianum strain beta. Seeds of the MIC variety were planted (5 seeds/ft) in 4 blocks with each block containing one 20 foot row of beans for each treat- ment. Treatments included: 1) Spray inoculation with approximately 1 x 108 cfu/ ml SDW of 143a, 7 and no days prior to inoculation with the pathogen 2) Inoculation with 143a, at the time of inoculation with the pathogen and 7 days later 3) Inoculation with 143a, 7 and 19 days after inocula- tion with the pathogen 4) Control (not spray inoculated) Pathogen inoculum was prepared as previously described for experiments in the greenhouse involving 9. lindemuthia- ppm. The pathogen was applied 39 days after planting when 2nd plants possessed fully expanded trifoliolate leaves. 39 At maturity, 70 pods were selected at random from each row and the number of sporulating lesions on each was recorded. A log transformation was used to obtain homo- geneous variance of the data. Duncan's multiple range test was used to compare treatments 1, 2 and 3 to control treatment #4. Experiments were also conducted to determine whether sprays of l43a to plants of Tuscola and MIC varieties at various times during the growing season would protect them from infection by S. sclerotiorum. Bacterial inoculum was prepared by suspending 2 day old PDA cultures in SDW. Approximately 1 x 108 cfu/ml were applied at a rate of 10 ml per foot of row at each inoculation time. The experiment with Tuscola plants was designed with 6 blocks, each block containing one 22 ft row per treatment. Treatments included: 1) Plants spray inoculated with 143a,24 and 32 days after planting 2) Plants spray inoculated with l43a,32, 38 and 47 days after planting 3) Plants spray inoculated with l43a,38 and 47 days after planting 4) Unsprayed control plants The same basic experiment was performed using MIC plants. In this case the experimental plot contained 4 blocks with one 18 ft row for each treatment in each block. Treatments included: 1) Plants spray inoculated with l43a,23 and 47 days after planting 2) Plants spray inoculated with l43a,47 and 59 days after planting 40 3) Plants spray inoculated with l43a, 59 days after planting 4) Unsprayed control plants Plants in both experimental plots became naturally infected with S. sclerotiorum. At harvest 40 plants were chosen at random from each row in both experiments. The percentage of plants out of 40 showing characteristic white mold symptoms (bleached appearance and/or sclerotia) was recorded. Duncan's multiple range test was used in each case to compare treatments 1, 2, and 3 to the control treatment . Treatment of Bean Seeds with Antagonists for Protection onglants from Bactefial and Fungal Pathogens Under Field Conditions Seeds of the MIC, Ouray pinto, and Seafarer navy bean varieties were treated with isolate 143a prior to planting to see whether this antagonist would provide protection from.naturally occurring disease pathogens. Seeds were surface sterilized in 2.5% sodium hypo- chlorite (2.5% Cl-) for 2 minutes, rinsed twice for 1 minute each in SDW, and allowed to dry for 24 hours at room temperature. Seeds were then placed in a side arm flask containing BYE alone or 48 hour old cultures of l43a 9 cfu/ml). Seeds in BYE (containing approximately 2.5 x 10 were vacuum infiltrated with the liquids for 3 minutes then rinsed 2 times for 1 minute each with SDW. Seeds were allowed to dry for 3 days at room temperature before planting. 41 The experiment contained 6 blocks with l replication (consisting of 50 seeds planted 5/ft) of each treatment per block. Since no natural infections developed in the beans in the experiment, treatments were only compared for their effect on emergence and final stand. Duncan's multi- ple range test was used to make these comparisons. In a similar experiment, seeds of MIC, Olathe pinto bean and Sacramento light red kidney bean varieties were surface sterilized as before, then vacuum infiltrated with BYE or 48 hour old BYE cultures of l43a. The seeds were planted using the same experimental design just described. However these plants were inoculated 26 days after planting with S. lindemuthianum strain beta. Inoculum of the patho- gen was prepared as previously described for greenhouse experiments. Plants were sprayed with the inoculum.on a cool humid evening at the rate of 10 ml inoculum/ft of row. To determine whether seed treatment with l43a provided protection from anthracnose, 70 mature pods were harvested at random from each row and number of sporulating lesions per pod recorded. Duncan's multiple range test was used after a square root transformation of the data, to compare number of sporulating lesions on pods from seeds treated with l43a and with BYE for each bean variety tested. Bean seeds were vacuum infiltrated with different antagonists to determine whether antagonists would provide effective protection of the plants from halo blight. Seeds 42 of Seafarer, Charlevoix and Manitou bean varieties were surface sterilized, rinsed and allowed to dry as described previously. Seeds of each variety were then vacuum infil- trated with BYE alone, 2 day old BYE cultures of antagonist 169, or 2 day old BYE cultures of 139b; absorbance of bacterial cultures used for inoculum.was adjusted to 0.3. Three blocks were used with 2 replications (each consist- ing of one 10 ft row) of each treatment in each block. Seedlings were spray inoculated with a 0.3 absorbance suspension of Pp R13 36 days after planting. Unfortunately, no disease symptoms were observed on any of the plants during the summer. Data is therefore only analyzed for any possible effects of isolate 169 or 139b on emergence or final stand count. Dunnett's two tailed test was used to make these comparisons (13). Protection of Bean Plants from Common Blight and Halo BlightlUsing Spray Applications of Various Antagonists Experiments were conducted to determine whether spray inoculation of Charlevoix bean plants with antagonists would protect them from common and halo bacterial blights under field conditions. The experimental design was that of a completely randomized block with 3 replications (each consisting of one 18 ft row) for each treatment. Inoculum of each antagonist was prepared by suspending the bacteria from 2 day old YCA cultures in SDW and adjusting absorbance to 0.3. Plants were sprayed lightly to run off with the suspension at a rate of 10 ml/ft using a knapsack sprayer. 43 The following treatments were used: 1) Control plants (unsprayed) 2) Plants spray inoculated with isolate 139b, 23 and 36 days after planting 3) Plants spray inoculated with isolate 139b, 36 days after planting 4) Plants spray inoculated with isolate 169, 23 and 36 days after planting 5) Plants spray inoculated with isolate 169, 36 days after planting At 36 days after planting all plants were spray inoculated with a 0.3 absorbance suspension of isolate a of Sp in SDW. A similar experiment was conducted to determine if antagonist 195 would protect Charlevoix plants from halo blight. Inoculum for both antagonist and pathogen were prepared as described above, except that 195 was taken from KMB plates. Plants were spray inoculated with one of the following solutions: 1) Control plants (unsprayed) 2) Plants spray inoculated with isolate 195, 23 days after planting 3) Plants spray inoculated with isolate 195, 23 and 36 days after planting Plants were inoculated with Pp Bruder 53, 36 days after planting. In both experiments plants were observed throughout the summer for common blight or halo blight symptoms; unfortunately no disease developed. 10. LITERATURE CITED Buchanan, R. E., and N. E. Gibbons, eds. 1974. th Bergey's manual of determinative bacteriology. 8 ed. Williams and Wilkins, Baltimore. 1246 pp. Cafati, C. R. 1979. Effect of host genotype on multiplication, distribution and survival of bean common blight bacteria (Xanthomonas phaseoli). Ph. D. Thesis, Michigan State University, East Lansing. 124 pp. Gordon, R. E., W. C. Haynes, and C. H. Pang. 1973. The genus Bacillus. USDA Handbook. No. 427, 283 pp. King, E. 0., M. K. Ward and D. E. Raney. 1954. Two simple media for the demonstration of pyocyanin and fluorescein. .J.Lab. Clin. Med. 44: 301-307. Klement, Z., and R. Goodman. 1967. The hypersensi- tive reaction to infection by bacterial plant pathogens. Ann. Rev. Phytopath. 5: 17-44. Moore, L. W., A. Andersen, and C. I. Kado. 1980. Agrobacterium. pp. 17-30. N. W. Schaad ed. Laboratory guide for identification of plant patho- genic bacteria. The American Phytopathological Society, St. Paul. Neter, J., and W. Wasserman. 1974. Applied linear statistical models. Richard D. Irwin, Inc. Home- weed, Illinois. 842 pp. Ozeki, H., B. A. D. Stocker, and H. De Margerie. 1959. Production of colicine by single bacteria. Nature 184: 337-339. Ritchie, D., D. weller, and J. White. Date unknown. Isolation and identification of plant pathogenic bacteria. Unpublished manuscript, Michigan State University, East Lansing. 37 pp. Saettler, A. W. 1971. Seedling injection as an aid in identifying bean blight bacteria. Plant Disease Reporter 55: 703-706. 44 11. 12. l3. 14. 15. 16. 45 Sands, D. C., M. N. Scroth, and D. C. Hildebrand. 1980. Pseudomonas. pp. 36-43. N. W. Schaad ed. Laboratory guide for identification of plant pathogenic bacteria. The American Phytopathologi- cal Society, St. Paul. . Stavely, J. R., C. A. Thomas, C. J. Baker, and J. S. MacFall. 1981. Greenhouse control of bean rust with Bacillus subtilis. Phytopathology 71: 771. Steel, R. G. D., and J. H. Torrie. 1960. Principles and procedures of statistics. McGraw-Hill Book Company, Inc., New York. 481 pp. Suslow, T. V., and M. N. Scroth. 1982. Rhizo- bacteria of sugar beets: Effects of seed applica- tion and root colonization on yield. Phytopathology 72: 199-206. Thornley, M. J. 1960. The differentiation of Pseudomonas from other gram negative bacteria on the Basis of arginine metabolism. J. Appl. Bacteriol. 1: 37-52. Vidaver, A. 1980. Corynebacterium. pp. 12-16. N. W. Schaad ed. Laboratory guide for identifica- tion of plant pathogenic bacteria. The American Phytopathological Society, St. Paul. RESULTS In Vitro Studies with AntagoniStic Bacteria Initial screening of bacterial contaminants from bean seeds yielded over two hundred isolates which showed the ability to inhibit Sp, Pp, and P§_ip_yippg. Several of these isolates also inhibited one or more fungal plant pathogens ip_yiE£p_(Table 1). Further testing of antagonist 143a showed it to be inhibitory to a wide range of fungal plant pathogens (Table 2). Type of culture medium used was found to affect the ability of some antagonists to inhibit bacterial pathogens ip_yi££p (Table 3). Isolate 107 did not inhibit KR: R10 or Pp B53 when tested using a NBGA soft agar layer over 3 NA base layer, but inhibited both pathogens when tests were conducted using BPA soft and base layers. None of the antagonists inhibited Pg on NA based media while several (107, 169 and 195) successfully inhibited the pathogen on BPA. Incubation temperature also affected the inhibitory activity of some antagonists 1p.yippp (Table 4). Antagon- ist l43a inhibited Kpf, Pp and Pg isolates only over narrow temperature ranges with each genera of bacterial 46 47 pathogen inhibited over a different temperature range. In contrast antagonist 169 showed the same inhibitory act- ivity against Pp BB2 at all temperatures tested. Antagonists differed in inhibitory activity ip_yippp depending on culture age (Table 5). Isolate 139b showed activity against Sp 11 after 1 day of growth in culture, while isolate 161 showed activity only after 6 days of growth in culture. The culture age at which inhibitory activity was detected also differed for specific antagonists depending on the test pathogen. For example, isolate 139b inhibited Sp 11 at all culture ages, but inhibited Pg only after 6 days growth in culture. Preliminary experiments with antagonist 143a designed to detect activity of inhibitory compounds in culture filtrates of different ages showed no inhibition of pr 520, pr R10, S. sclerotiorum or P. betae by 24 hour culture filtrates filter sterilized with a 0.2‘fl pore size filter. However, inhibitory activity against Sp 11 was detected in filtrate from 4 and 35 day old Eugonbroth cultures of 143a. Culture filtrates from 6 and 9 day old cultures passed through 0.451ppore size filters significantly inhibited growth of S. sclerotiorum.when spotted onto plates 2 cm from mycelial plugs of the fungus. Filtrates from 6 day old cultures of 143a inhibited radial growth of the fungal colony to an average 18 mm in the direction of the filtrate while in control plates growth averaged 36 mm. 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SH msmwOSumm Hmwhouomn smon anHSGfi ou mummcowmucw mo muwaflnm man no mhnumuoaamu mo uommmm onH .q m4m ELOwoLo_:o op ocsmoqxo couwm o_nm_> ___um ocoz mum_commucm Axvm .AHV ocoz moum_a _0cucou __< .moum_a oumo__aoc __m uo: use oco ammo. um c_ pCJOw otoz mocoN co_u_n_;cm A+V .comOLHma ogy mo co_u_n_;:_ o: Anv .ucomoca moco~ co_u_n_;:_ A+v~ .m>mp m pcm N .— Loumm po>comno ocoz moum_m .co_u :mcmneoo comOLHma\Aomm oc:u_:o cm_:o_uuma m mov um_commucm sumo to» pocmaoca ouoz moum_a oumo__aoc c30u .Loam> EcOmoLo_;o ;u_3 po___x >_m:o_>oca mum_commucm mc_c_mucoo oum_a sumo co>o poc30a pcm «mm umOm mo .5 0.: Lu_3 pox_E ocoz comozumq sumo mo mot:u_:u v.0 >mp N sot» _E —.o mo mo_asmm .moE_u mc_pcoamoccou um coam> Ec0o0c0_;o cu pomoaxo ocoz Amum_commucm mc_xom_v moum_a _0cu:0u .coam> Ecow0co_;u cu oczmoaxo ou3c_e on m >n po__wx mc_on oLOmon m>mp m.— to» 30cm 0“ ooze—_m ocoz moum_0m_ .m .>a ommc_c>m am new .«mmv m_oo__oommca .>a ommc_cxm um .Ammv fig .3 3.5338 .w. to 32:83:... E 83325 ob? cl_ :0 man. 9.3.3 to Soto m 39:. 53 inhibitory activity was detected in plates containing culture filtrate from 1, 2 or 4 day old cultures of 143a. Further testing revealed a wide range of plant pathogenic fungi to be inhibited by culture filtrates of l43a of different ages (Figure 1). Of those tested P. pgpgg_appeared to be most sensitive to inhibitory substances in the filtrates while P. solani f. sp. phaseoli was found least sensitive. In general inhibitory activity reached a peak in 8-12 day old culture filtrates. Inhibitory activity remained stable or gradually decreased in filtrates from older cultures. Culture filtrates of 143a were found to inhibit S. sclerotiorum even after autoclaving, after aging for 30 days, or aging for 30 days then autoclaving prior to use (Figure 2). Both aging and autoclaving decreased inhibitory activity. Sp a was also inhibited by 143a culture filtrates (Figure 3). Autoclaving reduced inhibitory activity of culture filtrates. In general inhibitory activity against bacterial pathogens Sp 11 and Sp a, was highest in filtrates from 7-12 day old cultures. Culture filtrates from older cultures (50-65 days) showed no activity against either pathogen. In an experiment to determine how l43a inhibits S. sclerotiorum, plugs were taken 2 mm from a 10 day old colony of 143a. 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