.3". \{1~‘:.uu'v ' .3... ".6" “17.23": . “ vat: ‘} Lfix:f:‘tl VV‘QV'.V".‘ 8‘4. " 105, ' .n whey“ 1., Lil", 4x I1)?” . ." “viii: “Va 1 .' ‘-_.L v '7‘“ u (L; 3:3? $3341.“le .("i‘ . .4 v ’.‘",1:' 0,: ‘:‘.‘I' 1 I ‘7 Laswo%%5 MICHIGAN STATE UNI VERS L“ L LLLL LLLL LIBRARY Michigan State University ’f'h‘k‘iw This is to certify that the thesis entitled Copper Resistance in Pseudomonas sxringae pv. szringae and Population Dynamics of B. s. pv. szringae and g. g. pv. morsprunorum on Cherry presented by George Wilfred Sundin has been accepted towards fulfillment of the requirements for M. S . degree in Mammalogy Date 2“/2 0/ Y7 / / 0-7639 MS U is an Afl‘irmative Action/Equal Opportunity Institution MSU LIBRARIES 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. COPPER RESISTANCE IN ESEUDOMONAS SYBINGAE PV. §YRIN§A§ AND POPULATION DYNAMICS OF 2. S. PV. SYRINGAE AND E. S. PV. HOBSPRUNORQM ON CHERRY BY George Wilfred Sundin 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 1989 5973323 ABSTRACT COPPER RESISTANCE IN BSEQDQMQNAS SXBIESAE PV. SYRINGAE AND POPULATION DYNAMICS OF B. S. PV- §XBIE§AE AND 2- £- PV- MQB§£BHEQBQM 0N CHERRY BY George W. Sundin Both W firings: pv. misses and B- s. pv. mgggprgngrgm were recovered from apparently healthy blossoms, leaves, and dormant buds collected from sweet and sour cherry trees in Michigan. Copper-resistant 2. g. pv. gyringgg were recovered from bulked blossom samples from nine and 12 orchards in 1987 and 1988, respectively. Among 46 copper-resistant isolates screened, 43 contained a single plasmid of approximately 46, 55, 61, 67, or 73 kilobase pairs (kb) depending on the source of the isolates. Copper resistance, and the 61 kb plasmid, was donated in filter matings to three recipients from six donor strains, and from one donor to one recipient in matings conducted in bean leaves. Populations of copper-sensitive strains of 2. g. pv. syringgg were reduced significantly more than those of copper-resistant strains on bean leaves sprayed with cupric hydroxide. ACKNOWLEDGEMENTS I would like to thank Dr. Alan L. Jones for his patience in reviewing this manuscript and for his guidance throughout my two years at Michigan State University. I would also like to thank Dr. D. W. Fulbright and Dr. A. W. Saettler for serving on my graduate committee. I would also like to extend my sincere thanks and appreciation to Dr. Tyre J. Proffer for countless hours of beneficial interaction. I would also like to thank Drs. c. Schlichting, c. J. Hillson, C. Uhl, and W. Merrill for initially stimulating my interest in botany, microbiology, and plant pathology. In addition, I thank Dr. B. D. Olson for additional experimental data which are presented in Chapter 1. ii TABLE OF CONTENTS GENERAL INTRODUCTION AND . . . . . . . Literature Cited . . . . . . . . . CHAPTER 1 - Overwintering and Population Dynamics of W guinea: pV- mines: and 2- a. 1W- morgngngxgn on sweet and sour cherry trees . . . . “SM“ 0 O O O O I O O O O O O O O 0 MATERIALS AND METHODS . . . . . . . Epiphytic populations on blossoms Colonization of cherry leaves . Detection in naturally infested buds Inoculation and sampling of leaf scars RESULTS 0 O O O O O O O O O O O O O O Epiphytic populations on blossoms Colonization of cherry leaves . Overwintering in buds . . . . . Overwintering in leaf scars . . DIstSION O O O O O O O O O O O O 0 0 LITERATURE CITED . . . . . . . . . . . CHAPTER 2 - Ecology and Genetics of Copper Resistance inWWPV-m--.... ABSTRAfl O O O O O O O O O O O O O O 0 MATERIALS AND METHODS . . . . . . . . Source of copper-resistant bacteria Identification of copper-resistant bacteria . . . Plasmid characterization of copper-resistant strains . . . . . . . Bacterial conjugation . . . . . Tn5 mutagenesis . . . . . . . . . DNA hybridizations . . . . . . Survival of 2. s. pv. _yring§§ on c bean plants . . . . . . . . iii opper sprayed Page 10 16 16 18 18 19 20 22 22 23 23 24 31 35 37 37 39 39 41 41 42 44 44 45 RESULTS . . . . . Source of copper-resistant bacteria . Identification of copper-resistant bacteria . Plasmid characterization of copper-resistant strains . . Bacterial conjugation . Tns mutagenesis . Survival of 2. g. pv. syringgg on copper sprayed bean plants DISCUSSION . . . . LITERATURE CITED . iv 47 47 48 48 50 53 53 57 60 LIST OF TABLES Page CHAPTER 1 Table 1.1 Detection of epiphytic Pseudomonas syringag pv. syringag (Pss) and 2. g. pv. morsprunorum on blossoms in Michigan cherry orchards . . . . . . . . . . . . . . . . . . . . 25 Table 1.2 Recovery of Pseudomonas gyringag pv. syringae (Pss) and 2. g. pv. morsprunorgm (Psm) from dormant cherry buds in East Lansing, MI, following inoculation of the trees on 29 April 1986 with a suspension containing both bacteria . 28 Table 1.3 Recovery of Pseudomonas syringae pv. syringae (Pss) and E. g. pv. mogsprunorgm (Psm) from dormant cherry buds in six orchards in Oceana county . . . . . . . . . . . . . . . . . . 29 Table 1.4 Recovery of rifampicin-resistant Esgudomonas syginggg pv. gyginggg (PssR) and g. g. pv. morsprunorum (Pst) from buds of Montmorency sour cherry in spring after inoculation of adjacent leaf scars the previous autumn with four concentrations of inoculum . . . 30 CHAPTER 2 Table 2.1 Recovery of Pseudomonas syginggg pv. syringag from cherry blossom washes on media with and without copper. Blossom samples were collected from orchards that contained copper-resistant bacteria in 1987 or were sprayed with copper in 1988 . . . . . . . . . . . 49 Table 2. 2 Frequency of conjugal transfer of copper resistance inP Pseudomonas syringae pv. syringgg O O O O O O O O O O O O I O O O O O I 52 Table 2.3 Differences in effect of a single copper treatment with 4. 8 g/L Kocide 101 (cupric hydroxide) on the recovery of copper-resistant and copper-sensitive strains of Pseudomongss _y;i_ggg pv. gyrigggg isolated from cherry blossoms on bean leaves sampled at four intervals following copper spray . . . . . . 56 V LIST OF FIGURES Page CHAPTER 1 Figure 1.1 Seasonal populations in 1986 of fluorescent pseudomonads per gram (fresh weight) of leaves recovered from symptomless sweet and sour cherry trees collected in an orchard at East Lansing, MI. Each data point is the mean of four replicates for sweet cherry and three replicates for sour cherry. Vertical bars are standard errors . . . . . . . . . . . . . . . . . 27 CHAPTER 2 Figure 2.1 Agarose gel electrophoresis of cleared lysates of copper-resistant strains of W amines: pV- 5111111993 isolated from cherry orchards in Michigan in 1987. Lane 1, 318-4; lane 2, 43A—1: lane 3, 45A-3; lane 4, 228-4; lane 5, 478-4: lane 6, 39B-1; lane 7, 183-5 0 e e e e e e e e e e e e e e e e e e e e e 51 Figure 2.2 Agarose gel electrophoresis of cleared lysates of donor, recipient, and transconjugant strains of Egggggmgngg pv. . Lane 1, 31B-4: lane 2, 27A-4 ; lane 3, 27A-4a: lane 4, 70-7: lane 5, 31A-53tr' lane 6, 31A-5a; lane 7, 27A-4a; lane 8, 17A-5s r; lane 9, 27-17a . 54 vi GENERAL INTRODUCTION Bacterial canker is a recent and important problem of sweet and sour cherry (Brynn; gyiun L. and 2. ggzasus L. respectively) in Michigan (30,33). The disease is caused by each of two bacteria, Pseudomonas 3121333; pv. mgrgprgngrgm (wormald) Young et a1. and P. g. pv. syringes van Hall. The disease is interrelated with frost injury in that some strains of 2. g. pv. gyxinggg are ice nucleation-active (INA), and can catalyze ice formation at temperatures as high as -1 C (37). Frost injury to sensitive plants occurs only if ice is formed (36). Under field conditions, frost injury usually occurs within the temperature range of -2 to -5 c (37).' Strains of four different bacteria have been shown to be active ice nuclei, Erwinig hgzhigglg (Lohnis) Dye, We Wis pm We. 2mm: flggrgsggns Migula, and many pathovars of 2‘ syringe: (27,31,39,43). These bacteria are active ice nuclei at temperatures above -2 C, while other bacterial strains become INA only at temperatures around -10 C (37). Ice nucleation and frost injury are important epidemiologically because frost damage has been shown to predispose plants to disease by providing wounds through which INA bacteria may invade host cells causing infection which in many cases does not occur unless preceded by frost damage (6,32,52,60,65,66). 2. g. pv. gyzingag is the most common INA bacterium, and has been found as a resident epiphyte on symptomless leaves and blossoms on a number of plant hosts (40). Ice nucleation-active 2. g. pv. gyzinggg have been implicated in frost damage to a number of annual crop plants and may play a role in frost injury to fruit crops in many areas of the world (25,35,67). INA bacterial pathogens are able to maintain a resident or epiphytic stage in their life cycle in which they multiply on the surfaces of healthy plants providing inoculum without disease incidence (28,35). Populations of INA 2. a. pv. syringgg increase rapidly following bud break on fruit trees remaining consistently high throughout the bloom period (25,35). These data indicate that populations of INA 2. g. pv. syringe; are highest when fruit trees and annual plants are most susceptible to frost injury, i.e. during spring when mild frosts are most likely to occur. Active 2. a. pv. syringe; ice nuclei are sufficient to limit the supercooling ability of many frost sensitive plants at temperatures above -5 c (37) . Deciduous fruit trees are highly susceptible to frost injury from prebloom to postbloom (26,53). Proebsting, et al. showed that in the absence of INA bacteria, detached flowers from a variety of fruit trees including apricot, peach, sweet cherry, and pear could supercool to temperatures between 95 and -8 C without injury. However, when inoculated with INA bacteria, ice formed in these same flowers at temperatures between -3 and -4 C (53,54,55). The major source of ice nucleation in fruit trees, however, may not be bacterially related. An intrinsic source of ice nuclei associated with woody stem tissue has been characterized in sweet cherry, peach, pear, and plum (3,4,26). The intrinsic ice nucleus is active at temperatures between -1.5 and -5 C regardless of whether INA bacteria are present. In one study, INA bacteria were found to have no influence on mortality of flowers and fruitlets of five deciduous fruit species in the field over a 6 year period (56). The bacterial canker pathogens, 2. ;. pv. eyringe; and 2.‘;. pv. nereprnngrnn are most active in spring and autumn when cool, wet weather favors growth and dissemination (33). Following the budburst stage of bud development and weekly during bloom, sprays of tribasic copper sulfate are recommended for control of bacteria in Michigan sour cherry orchards (46,50). Only a dormant spray of Bordeaux mixture or of fixed copper are used on sweet cherries. Copper sprays continuing into the summer were found to have a phytotoxic effect on leaves (50). Spray applications of copper compounds have also been recommended for sweet cherry in Oregon (51). Two sprays of Bordeaux mixture, at the white bud stage and at petal fall reduced the leaf spot phase of the disease in England (47), although the petal fall spray was highly toxic to leaves. Three sprays of the antibiotic streptomycin, at full bloom, 75% petal fall, and 100% petal fall reduced leaf spot symptoms by 93 to 96%, however Bordeaux mixture was more effective at reducing autumn populations than streptomycin (16). Other attempts to control bacterial canker include breeding programs for production of resistant cherry varieties. Breeding and screening for resistance, however, is a long and rigorous process wrought with problems (23). More recent efforts aimed at gaining frost control involve attempts to control INA bacterial populations, either through use of copper sprays (41), or by biological control consisting of suppression of INA bacteria by ice nucleation- negative (INN), antagonistic bacteria utilizing miscellaneous bacterial strains selected in the field (14), or genetic INN mutants of 2. e. pv. eyringe; produced in the laboratory (34,38). Copper compounds are desirable as control agents in Michigan because of their low cost, low toxicity to mammals, and because they also have activity against some important fungal pathogens of cherry (50). Copper ions have many sites of action in bacterial cells and denature proteins and may also act as respiratory poisons (22). Most bacteria and fungi are killed by free copper ion concentrations higher than 0.5 ppm (11,22). Extensive usage of copper compounds to control plant diseases, however, has led to the development of resistance in the bacterial species 3. gr pv. We (Doidqe) Dye (45). B- s. pv. senate (Okabe) Young (7), and g. eyringe; (2). Copper resistance in bacteria has been reported from diverse sources and, where found, has been associated with prior exposures to copper ions (1,2,7,11,13,17,18,45,62). In a number of instances, including those of plant pathogens, copper resistance genes have been associated with conjugative plasmids conferring resistance through unexplored mechanisms (7,18,29,59,62). Bacterial genes encoding resistance to antibiotics and/or heavy metals are associated with conjugative plasmids in a majority of cases (21,61). In some instances, a single conjugative plasmid may encode resistance genes to a number of different antibiotics and heavy metals (21,22,48,63). The process of conjugation enables resistant bacteria to transfer resistance genes to a variety of related bacteria (67). Ecologically, the process of conjugation enables bacteria to genetically adapt to the presence of elevated levels of toxic metal ions at a much faster rate than by spontaneous mutation or natural selection. Although many heavy metal ions are essential for bacterial growth, elevated levels of those same metal ions become toxic to bacterial cells mainly because of their ability to denature protein molecules (22). However, a wide variety of microorganisms have been found to possess a range of resistance or tolerance mechanisms against heavy metal poisoning, most of which feature some method of detoxification (21,22,61). Heavy metal tolerance is defined as bacterial survival and resistance as bacterial growth in the presence of toxic levels of metal ions (24). Heavy metal resistance, in many cases, is also genetically stable, in that organisms retain resistance even after extended periods of growth in the absence of metal ions (24). A problem inherent in metal toxicity research is that there are no standard concentrations of specific metal ions defining boundaries between resistance and sensitivity (63). Also, much in vitro work on metal resistance is conducted using solid agar media containing ingredients which may modify toxic ions (10). For example, copper ions are easily complexed and are only toxic to bacteria in the form of free ions or in complexes in which the copper atom retains its Cu2+ valence (5). The use of high nutrient agar media also amended by adjusting the pH requires greater concentrations of metal ions which elevates levels of resistance in yitrg. Copper resistance is quantitative or incomplete in that resistance levels may vary among different strains of the same bacterial species (2,7,45,64). Levels of copper resistance in plant pathogen populations are sufficient to preclude control of these bacteria in some areas by standard copper sprays. Alternate control methods for 2. ;. pv. genes; combining copper sprays with inoculation of a copper- resistant nonpathogenic strain of 3. ;. pv. renerg are under investigation in California (15). The mechanisms of copper resistance in bacteria associated with conjugative plasmids have yet to be elucidated. Possible mechanisms of copper resistance include non- specific exclusion of toxic ions from entering the cell, selective uptake and detoxification of ions through copper binding proteins, effluxing copper ions once they have entered the cell, and production and excretion of compounds which bind or precipitate copper ions outside the cell. Bacterial cell walls are highly anionic and are capable of binding heavy metal ions (9). While Gram positve cell walls were found to bind more metal ions than Gram negative walls, in both cases quantities of bound metal ions were found to exceed the number of available binding sites within the walls. It is conjectured that initially bound metal ions act as nucleation sites for growth of metal aggregates which continues until limited by available space within the wall (9). Extracellular capsular polysaccharides of the Gram negative bacterium Eieneieiie e;r;g;n;; were found to effectively bind copper ions. Encapsulated E. e;r;g;n;e cells displayed a significantly higher survival rate than a noncapsulated mutant strain in distilled water containing 10 ppm Cu2+ (11). E. ggii mutants lacking a specific outer membrane protein (protein b) were resistant to copper ions (43). Protein b is believed to act as a pore involved in nonspecific uptake of metabolites. Protein b mutants could not utilize low concentrations of available metabolites due to nonspecific exclusion from the cell (43). Activated sludge, which consists of both living and dead bacterial cells and their extracellular polysaccharides has been shown to absorb both zinc and copper ions readily (24). Copper resistance determinants from a 35 kilobase (kb) conjugative plasmid in 2. ;. pv. LQEESQ were localized to a. 4.5 kb DNA fragment (8). The fragment was subsequently sequenced and found to encode for four proteins (46). One of the proteins was found to share similarities in a region of 50 amino acids with bacterial azurin proteins (58) which are copper-containing proteins. It was suggested that these regions could contain repeated copper-binding domains and play a role in copper resistance (46). In yeast, copper resistance is mediated by synthesis of a low molecular weight metal-binding protein, metallothionein (MT) (19). MTs are a class of low molecular weight cysteine-rich proteins which bind heavy metals (12). MT production is inducible in response to treatment with copper ions (12). Levels of copper resistance are mediated by the copy number of the gene coding for MT production (20). There has been one report of an MT-like protein produced by a prokaryotic organism (49). Plasmid-borne copper resistance in E. g;ii_is inducible, and thought to be mediated through an intracellular binding protein coupled with an efflux mechanism which ejects excess copper ions (57). Cells induced to copper resistance accumulate less copper than uninduced cells. The induction process protects the cell against toxic copper concentrations while also maintaining internal concentrations adequate for normal copper-dependent cell functions (57). Copper-resistant strains of Myggpeggerinn egrgfniegenm were able to remove copper from culture media by a sulfate- dependent precipitation as copper sulfide, a trait mediated by a plasmid (18). Production of hydrogen sulfide and citric acid by copper-resistant yeast cells has also been implicated in the complexing of free copper ions (22). The objectives of this study were to 1) determine if 2. ;. pv. eyringe; and R. ;. pv. unreprnngrnn could overwinter in sweet and sour cherry buds in Michigan, ii) determine if copper-sensitive and copper-resistant strains of 2. ;. pv. eyringe; and 2. ;. pv. mgreprnngrnn are present in Michigan cherry orchards, iii) determine if copper resistant bacterial strains can survive epiphytically on plants sprayed with copper bactericides, iv) determine if the copper resistance phenotype can be transferred from a resistant to a sensitive 2. ;. pv. eyringe; strain via conjugation, and v) determine if the copper resistance gene(s) are plasmid borne or in the bacterial chromosome. 10 Literaturs.§ited 1. Adaskaveg, J. E., and Hine, R. B. 1985. Copper tolerance and zinc sensitivity of mexican strains of Xanthemenes ssmneefzie PV- xesisaterie. causal agent of bacterial spot of pepper. Plant Dis. 69:993-996. 2. Andersen, G. L., and Lindow, S. E. 1986. Occurrence and control of copper tolerant strains of Peendgngne; eyringe; on almond and citrus in California. Phytopathology 76:1118. 3. Ashworth, E. N., and Davis, G. A. 1984. Ice nucleation within peach trees. J. Amer. Soc. Hort. Sci. 109(2):198-201. 4. Ashworth, E. N., Anderson, J. A., and Davis, G. A. 1985. Properties of ice nuclei associated with peach trees. 5. Avakyan, z. A. 1971. Comparative toxicity of free ions and complexes of copper and amino acids to nriiie. Microbiology 40:363-368. 6. Azad, E., and Schaad, N. W. 1988. The relationship of Xenthgmgnss SEEPQSLIifl PV- trenslussne t0 frost and the effect of frost on black chaff development in wheat. Phytopathology 78395-100. 7. Bender, C. L., and Cooksey, D. A. 1986. Indigenous plasmids in Pseudemgnes syringes pV- tenets: conjugative transfer and role in copper resistance. J. Bacteriol. 165:534-541. 8. Bender, C. L., and Cooksey, D. A. 1987. Molecular cloning of copper resistance genes from Beenggngne; eyringe; pv. renetg. J. Bacteriol. 169:470-474. 9. Beveridge, T. J. 1984. Mechanisms of the binding of metallic ions to bacterial walls and the possible impact on microbial ecology. In: Current Perspectives in Microbial Ecology, M. J. Klug and C. A. Raddy eds, pp. 601-607. Washington, D. C.: American Society for Microbiology. 10. Bird, N. P., Chambers, J. G., Leech, R. W., and Cummins, D. 1985. A note on the use of metal species in microbiological tests involving growth media. J. Appl. Bacteriol. 59:353-355. 11. Bitton, G., and Friehofer, V. 1978. Influence of extracellular polysaccharides on the toxicity of copper and cadmium toward fiiepeieiie e;rgg;n;_. Microb. Ecol. 4:119- 125. 11 12. Butt, T. R., and Ecker, D. J. 1987. Yeast metallothionein and applications in biotechnology. Microbiol. Rev. 51:351-364. 13. Calomiris, J. J., Armstrong, J. L., and Seidler, R. J. 1984. Association of metal tolerance with multiple antibiotic resistance of bacteria isolated from drinking water. Appl. Environ. Microbiol. 47:1238-1242. 14. Cody, Y. 8., Gross, D. C., Proebsting, E. L. Jr., and Spotts, R. A. 1987. Suppression of ice nucleation-active eyringe; by antagonistic bacteria in fruit tree orchards and evaluations of frost control. Phytopathology 77:1036-1044. 15. Cooksey, D. A. 1988. Reduction of infection by assessmenas syringes pv. senate using a nonpathogenic. copper resistant strain combined with a copper bactericide. Phytopathology 78:601-603. 16. Crosse, J. E. 1957. Streptomycin in the control of bacterial canker of cherry. Ann. Appl. Biol. 45:226-228. 17. Dutta, G. N., and Devriese, L. A. 1981. Sensitivity and resistance to growth promoting agents in animal lactobacilli. J. Appl. Bacteriol. 51:283-288. 18. Erardi, F. x., Failla, M. L., and Falkinham.III, J. O. 1987. Plasmid-encoded copper resistance and precipitation by Mysebssferium esrefnlasenn- Appl. Environ- Hicrobicl- 53:1951-1954. l9. Fogel, S., and Welch, J. W. 1982. Tandem gene amplification mediates copper resistance in yeast. Proc. Natl. Acad. Sci. USA 79:5342-5346. 20. Fogel, 8., Welch, J. W., and Karin, M. 1983. Gene amplification in yeast: CUPl copy number regulates copper resistance. Curr. Genet. 7:1-9. 21. Foster, T. J. 1983. Plasmid-determined resistance to antimicrobial drugs and toxic metal ions in bacteria. Microbiol. Rev. 47:361-409. 22. Gadd, G. M., and Griffiths, A. J. 1978. Microorganisms and heavy metal toxicity. Microb. Ecol. 34:303-317. 23. Garrett, C. M. E. 1981. Screening for resistance in Prune; to bacterial canker. Proc. 5th. Int. Conf. Plant Pathogenic Bacteria, California, pp. 525-530. 12 24. Griffiths, A. J., Hughes, D. E., and Thomas, D. 1975. Some aspects of microbial resistance to metal pollution. In Minerals and the Environment, ed. M. J. Jones, pp. 387-394. Institution of Mining and Metallurgy, Washington D. C. 25. Gross, D. C., Cody, Y. S., Proebsting, E. L. Jr., Radamaker, G. R., and Spotts, R. A. 1983. Distribution, population dynamics, and characteristics of ice nucleation- active bacteria in deciduous fruit tree orchards. Appl. 26. Gross, D. C., Proebsting, E. L. Jr., and Andrews, P. K. 1984. The effects of ice nucleation-active bacteria on temperatures of ice nucleation and freeze injury of Prune; flower buds at various stages of development. J. Amer. Soc. Hort. Sci. 109:375-380. 27. Hirano, S. S., Maher, E. A., Kelman, A., and Upper, C. D. 1978. Ice nucleation activity of fluorescent plant pathogenic pseudomonads. Proc. 4th. Int. Conf. Plant Pathogenic Bacteria, Angers, France. 2:717-724. 23. Hirano, s. s., and Upper, c. D.’ 1983. Ecology and epidemiology of foliar bacterial plant pathogens. Ann. Rev. Phytopathology 21: 243-269. 29. Ishihara, M., Kamio, Y., and Terawaki, Y. 1978. Cupric ion resistance as a new genetic marker of a temperature sensitive R plasmid, Rtsl in E;;h;ri;nie4;;ii. Bioch. Biophys. Res. Comm. 82:74-80. 30. Jones, A. L. 1971. Bacterial canker of sweet cherry in Michigan. Plant Dis. Rep. 55:961-965. 31. Kim, H. R., Orser, C., Lindow, S. E., and Sands, D. C. 1987. Xenthsmgnes semesstris.pvo trenelussns strains active in ice nucleation. Plant Dis. 71:994-997. 32. Element, 2., Rozsnyay, D. S., Balo, E., Panczel, M., and Prileszky, Gy. 1984. The effect of cold on development of bacterial canker in apricot trees infected with Pseudemgnas airings: pv. exrinsee- Physiolo P1. Path- 24:237-246. 33. Latorre, B. A., and Jones, A. L. 1979. Peenegngne; n;r_prnn;rnn, the cause of bacterial canker of sour cherry in Michigan, and its epiphytic association with P. _yringe;. Phytopathology 69: 335- 339. 34. Lindemann, J., and Suslow, T. V. 1987. Competition between ice nucleation-active wild type and ice nucleation- deficient deletion mutant strains of Pseudongneg gyringe; and E. {ingreegene biovar I and biological control of frost injury on strawberry blossoms. Phytopathology 77:882-886. 13 35. Lindow, S. E. 1982. Population dynamics of epiphytic ice nucleation-active bacteria on frost sensitive plants and frost control by means of antagonistic bacteria. In: Plant Cold Hardiness and Freezing Stress, ed. P. H. Li, pp. 395- 416. New York: Academic. 36. Lindow, S. E. 1983. Methods of preventing frost injury through control of epiphytic ice nucleation active bacteria. Plant Dis. 67:327-333. 37. Lindow, S. E. 1984. The role of bacterial ice nucleation in frost injury to plants. Ann. Rev. Phytopath. 21:363-384. 38. Lindow, S. E. 1987. Competitive exclusion of epiphytic bacteria by ice-mutants of 2;;nggngneeleyringe;. Appl. Environ. Microbiol. 53:2520-2527. 39. Lindow, S. E., Arny, D. C., and Upper, C. D. 1978. Erxinina 3 an active ice nucleus incites frost damage to Maize. Phytopathology 68:523-527. 40. Lindow, S. E., Arny, D. C., and Upper, C. D. 1978. Distribution of ice nucleation active bacteria on plants in nature. Appl. Env. Microbiol. 36:831-838. 41. Lindow, s. E., and Connell, J. H. 1984. Reduction of frost injury to almond by control of ice nucleation active bacteria. J. Amer. Soc. Hort. Sci. 109(1):48-53. 42. Lutkenhaus, J. F. 1977. Role of a major outer membrane protein in E;gh;ri;nie ggii. J. Bacteriol. 131:631-637. 43. Maki, L., Galyon, E. L., Chang-Chien, M., and Caldwell, D. R. 1974. Ice nucleation induced by Peendgngne; eyringe;. Appl. Microbiol. 28:456-460. 44. Marco, G. M., and Stall, R. E. 1983. Control of bacterial spot of pepper initiated by strains of pv. yeeigengrie that differ in sensitivity to copper. Plant Dis. 67:779-781. 45. Mellano, M. A., and Cooksey, D. A. 1988. Nucleotide sequence and organization of copper resistance genes from Peenggngne; eyringe; pv. LQEQLQ- J. Bacteriol. 170:2879- 46. Michigan State University Extension Service. 1988. Fruit Spraying Calendar. Michigan State Univ., East Lansing. 14 47. Montgomery, H. B. S., and Moore, M. H. 1945. The control of bacterial canker and leaf-spot in sweet cherry. J. Pomology 21:155-163. 48. Novick, R. P., and Roth, C. 1968. Plasmid-linked resistance to inorganic salts in Stepnyigegggn; enr;n;. J. Bacteriol. 95:1335-1342. 49. O1afson, R. W., Loya, S., and Sin, R. G. 1980. Physiological parameters of prokaryotic metallothionein production. Bioch. Biophys. Res. Comm. 95:1495-1503. 50. Olson, B. D., and Jones, A. L. 1983. Reduction of REQHQQEQEEE 52118939 PV- EQIREIBEQIEE on Montmorency sour cherry with copper and dynamics of the copper residues. Phytopathology 73:965-971. 51. Oregon State University Extension Service. 1976. Oregon Plant Disease Control Handbook. Oregon State Univ., Corvallis. 52. Panagopolous, C. G., and Crosse, J. E. 1964. Frost injury as a predisposing factor in blossom blight of pear caused by P. eyringe; van Hall. Nature 202:1352. 53. Proebsting, E. L. Jr., Andrews, P. R., and Gross, D. 1982. Supercooling young developing fruit and floral buds in deciduous orchards. HortScience 17(1):67-68. 54. Proebsting, E. L. Jr., and Andrews, P. K. 1982. Supercooling and Prnnn; flower bud hardiness. In Plant Cold Hardiness and Freezing Stress, ed. P. H. Li, pp. 529-539. New York:Academic. 55. Proebsting, E. L. Jr., and Mills, H. H. 1978. Low temperature resistance of developing flower buds of six deciduous fruit species. J. Amer. Soc. Hort. Sci. 103(2):192-198. 56. Proebsting, E. L. Jr., and Gross, D. C. 1988. Field evaluations of frost injury to deciduous fruit trees as influenced by ice nucleation-active Beenggngne; eyringe;. J. Amer. Soc. Hort. Sci. 113:498-506. 57. Rouch, D., Camakarkis, J., Lee, B. T. O., and Luke, R. K. J. 1985. Inducible plasmid-mediated copper resistance in Eeeh;ri;nie 291i. J. Gen. Microbiol. 131:939-943. 58. Ryden, L. 1985. Structure and evolution of the small blue proteins. In: Copper Proteins and Copper Enzymes, ed. R. Lontie, pp. 157-182. Boca Raton: CRC Press. 15 59. Stall, R. E., Loschke, D. C., and Jones, J. B. 1986. Linkage of copper resistance and avirulence loci on a self- transmissible plasmid in Xenrngngnee‘genpeerri; pv. yeeigetgrie. Phytopathology 76:240-243. 60. Sule, S., and Seemuller, E. 1987. The role of ice formation in the infection of sour cherry leaves by mines: pv. mines:- Phyt0pathology 77:173- 177. 61. Summers, A. O., and Silver, S. 1978. Microbial transformations of metals. Ann. Rev. Microbiol. 32:637- 672. 62. Tetaz, T. J., and Luke, R. K. J. 1983. Plasmid- controlled resistance to copper in E;;h;ri;hie,g;ii. J. Bacteriol. 154:1263-1268. 63. Trevors, J. T., Oddie, K. M., and Beliveau, B. H. 1985. Metal resistance in bacteria. FEMS Microbiol. Rev. 32:39-54. 64. Trevors, J. T. 1987. Copper resistance in bacteria. Microbiol. Sci. 4:29-31. 65. Weaver, D. J. 1978. Interaction of Peendgngne; and freezing in bacterial canker on excised peach twigs. Phytopathology 68:1460-1463. 66. Weaver, D. J. 1981. Ice nucleation by Peenggngne; eyringe; associated with canker production in peach. Phytopathology 71:109-110. 67. Willetts, N., and Wilkins, B. 1984. Processing of plasmid DNA during bacterial conjugation. Microbiol. Rev. 48:24-41. 68. Yankofsky, S. A., Levin, z., and Moshe, A. 1981. Association with citrus of ice nucleating bacteria and their possible role as causative agents of frost damage. Curr. Microbiol. 5:213-217. 69. Young, J. M. 1978. Survival of bacteria on Prune; leaves. Proc. 4th. Int. Conf. Plant Pathogenic Bacteria, Angers, France. 2:779-786. CHAPTER 1 Overwintering and population dynamics of Pseugomones mingle: pv. mines: and 124- pm W on sweet and sour cherry trees ABSTRACT Apparently healthy blossoms, leaves, and dormant buds collected from sweet and sour cherry trees in Michigan were examined for the presence of Eeenggngnee gyringe; pv. eyringe; and P. eyringe; pv. nereprnngrnn. Trees inoculated with B. ;. pv. eyringe; and 2. ;. pv. nereprnngrnn during the prebloom period carried populations of about 107 colony- forming units (cfu)/g on sweet cherry leaves and 106 cfu/g on sour cherry leaves throughout the summer, and the bacteria were recovered from dormant buds taken from these trees the following winter. 2. ;. pv. eyringe; and g. e. pv. unreprnngrnn were detected in bulked blossom samples from 26% and 55% of the commercial orchards sampled in 1986 and 1987, respectively. Populations of green-fluorescent bacteria on blossoms ranged from 103 to over 106 cfu/g (fresh weight). In orchards with high epiphytic populations during bloom, 2. e. pv. eyringe; and 2. e. pv. o s no were detected in 19% and 11% of the dormant buds collected from four sweet and two sour cherry orchards, respectively. 16 17 Rifampicin-resistant strains of both pathovars were recovered in late spring from dormant sour cherry buds adjacent to leaf scars inoculated the previous autumn. Recovery of marked strains increased as inoculum concentrations increased. These data indicate that 2. 1. PV- mines: and P. 1. 1"“ menu: overwinter in buds of sour cherry as well as sweet cherry. Bacterial canker is an important problem of sweet and sour cherry in Michigan (16), and of sweet cherry and plum in the neighboring province of Ontario, Canada (1,10). On sour cherry (mambo. WWW- m;r;nrnn;rnn,(Worma1d) Young et al. is the major causal organism of bacterial canker and is commonly associated with epiphytic populations of B. ;. pv. eyringe; van Hall (14). On sweet cherry (P. eyinn L.), both pathovars are found as residents on symptomless tissues and as incitants of bacterial canker (14,20). Buds are an overwintering site for E. e. pv. mgreprnngrnn on sweet cherry in the United Kingdom (7,11), and for both 2. ;. pv. eyringe; and 2. e. pv. nerenrnngrnn on sweet cherry in South Africa (19). In Michigan, both pathovars are associated with buds of sour cherry from early spring through late autumn (14), but whether these bacteria overwinter in buds of either sweet or sour cherry has not been investigated. We report the presence of g. e. pv. eyringe; and 2. ;. pv. 18 mgrenrnngrnn in apparently healthy buds from an experimental orchard following inoculation of leaf surfaces the previous spring, from commercial orchards with high populations of bacteria during bloom but lacking disease symptoms, and from buds adjacent to leaf-scars inoculated with marked strains. We also report on the incidence of ice nucleation-active 2. ;. pv. eyringe; associated with buds and blossoms. MATERIALS AND METHODS mm: minim: en blesaems. During 1986 end 1987. blossom samples were collected from a total of 26 sweet and 31 sour and 15 sweet and 38 sour cherry orchards respectively (Table 1.1). The orchards were located in western Michigan in the counties of Berrien, van Buren, Leelanau, Grand Traverse, Mason, Oceana, Kent, and Manistee. Two blossom clusters were collected from each of 40 trees per orchard. The blossoms were bulked by orchard in plastic bags and transported to the laboratory on ice. Two sets of 20 blossoms, composed of one flower per cluster, were selected from each sample, weighed, and suspended in 20 mL sterile 0.01 M potassium phosphate buffer (pH 7.2). The samples were shaken vigorously for 30 min on a Burrell wrist action shaker. The wash solution was serially diluted three times in the same buffer and 0.1 mL aliquots plated onto King's medium B (13) amended with 50 ug/mL cycloheximide (KBc). Colonies of fluorescent pseudomonad bacteria were counted after 3 days incubation at 22°C. 19 Randomly selected representative bacterial colonies were suspended in 0.5 mL sterile phosphate buffer and tested for ice nucleation activity at -5 C with the droplet freezing procedure of Lindow et a1. (17). Ten 10-u1 drops per isolate were spotted onto paraffin covered aluminum foil boats which were floated on a refrigerated water bath (Lauda RM 20: Brinkmann Instruments). If all the droplets froze within 15 min, the isolate was considered ice nucleation active (INA). Up to ten colonies from each orchard were subjected to pathogenicity tests using immature green cherry fruit (12) and were identified as 2. ;. pv. eyringe; or 2. ;. pv. nereprnngrnn based on the GATTa tests (14). Celenizatien.:f gnerry i;ey;;. Napoleon sweet cherry and Montmorency sour cherry trees in an orchard in East Lansing, MI, were inoculated with a bacterial suspension. A group of 12 sweet cherry trees and another of 12 sour cherry trees were subdivided into three 4-tree replicates of sour cherry and four 3-tree replicates of sweet cherry. Two strains isolated in Michigan, 2. ;. pv. eyringe; strain HTl from sweet cherry and 2. ;. pv. mgrenrnngrnn strain TTl from Montmorency sour cherry, were mixed in an approximate 1:1 ratio in 102 L phosphate buffer to give a final concentration of 107 colony-forming units (cfu) per milliliter. The bacterial suspension was sprayed onto the trees on 29 April 1986 with a handgun sprayer at 300 psi. Populations of bacteria on the leaves of the inoculated trees were determined daily beginning 30 April 1986 and 20 three times per week beginning 1 July 1986. From each replication, a single leaf was collected from each of 15-20 fruiting spurs selected at random. During the period of active growth, the youngest fully expanded leaf was sampled: after growth stopped, the second and third leaves from the tip were sampled. All samples were collected between 8:00 and 8:30 a.m. Approximately 2 g of leaf tissue for each replicate were shaken on a wrist action shaker for 1 hr in an Erlenmeyer flask containing 20 mL of phosphate buffer. The wash solution was serially diluted and 0.1 mL aliquots were plated onto KBc. Colonies of fluorescent pseudomonad bacteria were counted after 3 days incubation at 22°C. W in naturalist infused mm. right fruiting spurs from each of five trees were collected on 15 December 1986, 13 January 1987, and 14 February 1987 from the inoculated sweet and sour cherry trees in East Lansing, MI, that had been used to study colonization of leaves. Two fruiting spurs from each of 15 trees were collected from four sweet and two sour cherry orchards in Oceana county on 9 March 1987, and two fruiting spurs from each of 25 trees were collected from one sweet and two sour cherry orchards on 15 December 1987 and 16 February 1988. Orchards selected for sampling had harbored high populations of bacteria on blossom tissue in the previous spring. Each fruiting spur was surface sterilized in a solution of 10% bleach for 2 min, rinsed in sterile water for 30 sec, and.allowed to air dry in a laminar flow hood for 10-15 min. Apparently 21 healthy buds were excised individually from each spur and ground in a sterile Pyrex tissue grinder containing 2 mL of sterile 0.01 M potassium phosphate buffer (pH 7.2). The solution was serially diluted and 0.1 mL aliquots plated in duplicate onto KBc. The number of colonies of fluorescent bacteria were counted after 3 days incubation at 22°C. At least four randomly selected colonies per bud were tested for INA activity at -5°C and subjected to the GATTa tests (14). Ineeeletien end Mine :1 leaf sears- Rifampicin- resistant strains of P. ;. pv. eyringe; (PssR) were selected by spreading 108 cfu of 2. ;. pv. eyringe; onto KBc medium containing 50 ug/mL rifampicin (KBrc) (Calbiochem-Behring Corp., La Jolla, CA). Eleven isolates recovered from leaves of sweet cherry were screened for rifampicin-resistant strains. Colonies that developed after 4 days incubation at 22°C were considered resistant to rifampicin. Pathogenicity of parental and PssR strains were tested by inoculating tobacco leaves and Montmorency sour cherry fruit and leaves with inoculum concentrations of 106 cfu/mL in 0.01 M phosphate buffer, pH 7.2. A strain of PssR similar in pathogenicity to the parental isolate was used in this study. The rifampicin resistant strain of 2. ;. pv. mgreprnngrgn (Pst) used in this study was selected by Latorre and Jones (15). Inoculum of Pst and PssR was prepared from 2-day-old cultures grown on King's medium B at 22°C. Suspensions in phosphate buffer were adjusted 22 turbidimetrically to contain 108 cells per milliliter and lower concentrations were obtained by serial dilution. Three Montmorency sour cherry trees were inoculated with each isolate on 21 September and 15 October 1979: and on 19 August, 11 September, and 8 October 1980. One week before inoculation, the leaf blade was cut off at the tip of the petiole to stimulate the formation of an abscission layer. On each date, the petiole was removed from each spur and a 0.05 mL drop of inoculum was placed on the exposed leaf scar. Inoculum concentrations were 10‘, 10°, 108 cfu/mL in 1979 and 102, 10‘, 106 cfu/mL in 1980. Each inoculum concentration was applied to 18 leaf scars per tree. Bacterial suspensions were kept in an ice bath during the inoculation period. Spurs with inoculated leaf scars were collected on 15 April 1980 and 14 April 1981, and individually frozen at -20°C. The buds were swollen but not open when collected. Isolations were made by dicing the buds of each spur with a sterile razor blade and placing the pieces in 10 mL phosphate buffer. After 1 hr, the suspension was shaken for about 1 min, and 0.1 mL aliquots were plated onto KBrc. The presence of rifampicin-resistant strains was recorded after S-days incubation at 22°C and the percentage of infected leaf scars was calculated for each replication. RESULTS Eeienxtie eeeeletien: en eleeeems- Green-fluorescent 23 bacteria were isolated from 44 of 110 blossom samples collected from sweet and sour cherry orchards in 1986 and 1987 (Table 1.1). In 1986, 2. ;. pv. eyringe; was detected in blossom samples taken from 11 of 31 sour and 2 of 26 sweet cherry orchards and 2. ;. pv. nereprnngrun was detected in 1 of 31 sour and 2 of 26 sweet cherry orchards. In 1987, 2. e. pv. eyringe; was detected in 5 of 15 sweet and 19 of 38 sour cherry orchards and P. ;. pv. mgreprnngrnn was detected in 4 of 15 sweet and 12 of 38 sour cherry orchards. Ice-nucleaction-active 2. ;. pv. eyringe; were detected in 77.3% of the 44 orchards with green-fluorescent bacteria. Celenizerign 91 en;rry 1;ey;;. Cherry strains HTl and TT1 colonized leaves of sweet and sour cherry following spray inoculation on 29 April 1986 (Fig. 1.1). Populations of green-fluorescent bacteria recovered from leaves increased during the first 2 wk of May to levels of 106-107 cfu/g fresh weight. Populations of bacteria on leaves of sweet cherry were usually 10 times greater than populations on leaves of sour cherry. Populations of bacteria on the leaves increased markedly from 9 to 13 June following several days of cool, wet weather. Leaf spots typical of infections of the bacterial canker pathogens were noted 5 days later. From mid-June through late July, populations of bacteria recovered from the leaves remained relatively constant. gyeryingering in DBQE- Fluorescent pseudomonad bacteria 24 were detected in 64.9% of the sweet cherry buds and 7.0% of the sour cherry buds collected from the East Lansing orchard during the winter of 1986-87 (Table 1.2). From the 48 infested sweet cherry buds, 2. ;. pv. eyringe; was recovered from 43 and 2. ;. pv. mgreprnngrnn from 5 buds. INA 2. ;. pv. eyringe; were detected in 12 buds. All six infested sour cherry buds contained 2. ;. pv. eyringe; and four of these buds contained INA bacteria. Fluorescent pseudomonads were recovered from buds collected on 9 March 1987 in three of four sweet cherry and one of two sour cherry orchards in Oceana county, and from two sweet and one sour cherry orchard sampled on 16 December 1987 and 17 February 1988 (Table 1.3). A total of 11% of both sweet cherry and sour cherry buds were infested. From the 31 infested sweet cherry buds, 2. ;. pv. eyringe; was recovered from 16 buds and B-.§- pv. nereprnngrnn and unidentified fluorescent pseudomonads were recovered from 15 buds. INA 2. ;. pv. eyringe; were detected in two buds. From the 15 infested sour cherry buds, 2. ;. pv. eyringe; was recovered from seven buds and 2. ;. pv. nereprnngrnn and unidentified fluorescent pseudomonads were recovered from eight buds. INA 2. eyringe; were recovered from five buds. gyeryin§;ring in 1;er egere. In the spring of 1980 and 1981, Pst and PssR were recovered from buds adjacent to leaf scars inoculated in the autumn of 1979 and 1980, respectively (Table 1.4). In both years, recovery of Pst and PssR from buds increased as inoculum concentrations were 25 Table 1.1. Detection of epiphytic {eggggggnle gyrinne; pv; 81218881 (Pss) and 2. e. pv. mm (Pam) on hlossaas in Michigan cherry orchards. Total Species populationb present Orchard Comty Year (lo: cfuls) nu" Pas p.- Swaat cherry orchards Gregory Laelanau 1888 5.21 0/4 - + Melchik Laelanau 1888 5.70 0/8 - + Aria Menistaa 1888 4.87 5/5 + - Daly Oceana 1888 4 . 72 4 / 4 + - Eackart Mason 1887 5.77 0/10 - + Harmon Meson 1887 5.88 5I8 + - Lister Mason 1887 8.00 4/14 + + Van lortwiok Mason 1887 8.30 5/5 + - DaRuitar Oceana 1887 8.08 2/10 + + Glover Oceana 1887 5.58 10/10 + - Ire-star Oceana 1887 8 . 28 0/ 10 - + Sour cherry orchards Baler Grand Traverse 1888 5.80 10/10 + - Lardla Grand Traverse 1888 5.80 SIS + - Viaahill Grand Traverse 1888 3.10 5/5 + - Underwood Grand Traverse 1888 3.00 2/3 + - Gregory Laelanau 1888 4.38 0/5 + - Korson Lealaneu 1888 4.81 10I10 + - Nueant Laelanau 1888 4.87 10/10 + - Paterson Mason 1888 8.18 5/10 + + Daly Oceana 1888 3.88 5/5 + - DaRuitar Oceana 1888 5.04 5/5 + - Beer Oceana 1888 5.32 8/7 + - Bayer Berrian 1887 3.81 1/4 + + troupe Grand Traverse 1887 5.80 9/10 + - Dietrich Kent 1887 5.70 5/7 + + Klank Kent 1987 4.71 0/5 + - Bardanhaaan Laelanau 1987 5.32 5/5 + - Nunant Laelanau 1887 3.61 5/8 + - Stanak Laelanau 1887 5.85 0/8 - + Brya Rd. Mason 1887 4.72 1/12 + + Christoffarson Mason 1887 5.30 5/10 + - Dittmer Mason 1887 4.98 2/8 + + Harmon Mason 1887 5.30 0/7 - + Paterson Mason 1887 5.04 5/8 + + Daly Oceana 1887 8.08 10/10 + - anlay Oceana 1887 4.11 0/4 - + Kokx Oceana 1987 4.20 8/11 + - 28 Table 1.1 (cont.) Total Species populationb prssat Orchard County Year (log cfulg) INAc Pss Pam Duskirk Va Duran 1887 8.88 8/11 + + Colgra Van Dara 1887 4.74 717 + - Dowd Va Duran 1887 8.78 10/ 10 + - Bessel Van Duran 1887 5.23 4/4 + - Msachun Va Duran 1887 5.54 5/8 + + Pugslsy Va Dura 1887 5.34 0/10 + + Winkle Va Bura 1887 8.28 7/ 14 + + .Dactaria were not detected in 2 sweet and 20 sour cherry orchards in 1888 and in 8 sweet ad 18 sour cherry orchards in 1887. These orchards were not listed in the table. The limit of detection for the dilutia plating assay was 1.8 log (colony-forming units per gra of blossas tissue). bHighest bacterial population recorded from two bulked 20 blossom saplas. climber of ice nucleatim-sctiva (INA) isolates over the umber of isolates tested. dPss ad Pam were identified based a: the GATTA determinative tests (14). Isolates idatifisd as Pss were positive for gelatin liquefactica: and aasculin hydrolysis but negative for tyrosinasa activity and tartrata utilization; those identified as Pas were negative for gelatin liquefaction and assculin hydrolysis but positive for tyrosinasa activity and tartrats utilization. b 1 LL Log(Bocterio per gram of leaf) .9 $.3-‘9- (I A 0‘ . i 27 I'r"r7"'I'""r"'I'VV']"VVIVV'TYITVVVIIIYIIVIUIIItvv‘v1rrTvvvaIvvv'vvvr'tvvtvair'vrvTIvv 1 fi ‘ , SweetChorry ' - + M 3. " | ’4 1 1‘ , \ Vii/{r " ' "4 \ + ”I ‘f’ Sour Cherry + ' I" ‘3' ’ h! Ivvvv'vvtrrfitv'vvvvlvvvtlvvV'V'VTVIIVIIYIItv"IIUV'IIVVIITVV'VYVV'T‘IU‘IVITI‘VVVI'I'IV'VT 3O 5 l0 I5 20 25 30 5 IO I5 20 25 30 5 IO I5 20 25 30l MAY JUNE JULY Figure 1.1. Seasonal population in 1986 of fluorescent pseudomonads per gram (fresh weight) of leaves recovered from symptomless sweet and sour cherry trees collected in an orchard located at East Lansing, MI. Each data point is the mean of four replicates for sweet cherry and three replicates for sour cherry. Vertical bars are standard errors. 28 Table 1-2- Recovery of Essuggmgnas airings: pV- syringes (Pss) and E. g. pv. mgrgpzungrgm (Psm) from dormant cherry buds in East Lansing, MI, following inoculation of the trees in 29 April 1986 with a suspension containing both bacter a Number of buds Buds infested containing Sampling date Fractiona % Pss Psm INA? Buds from sweet cherry 15 December 1986 17/28 60.7 14 3 0 13 January 1987 18/26 69.2 16 2 9 14 February 1987 13/20 65.0 13 0 3 Buds from sour cherry 15 December 1986 3/36 8.3 3 0 1 13 January 1987 2/26 7.7 2 0 2 14 February 1987 1/20 5.0 1 0 1 Mean populations (log colony-forming units (and variance) per infested bud) Cherry species Pss Psm INA Sweet 3.55 (1.34) 4.18 (1.02)' 4.05 (0.92) Sour 2.98 (0.69) 0.0 2.65 (0.32) aNumber of buds containing green fluorescent bacteria over the tetal number of buds in each sample at a limit of detection of 1.3 log colony forming units per bud. sts and Psm were identified based on the GATTa determin- ative tests (14) and ice nucleation-activity was determined using the procedure of Lindow et al. (17). 29 Table 1-3- Recovery of W 912113933 1W- 511111935 (Pss) and 2-.§- pv. mgzgngngrum (Psm) from dormant cherry buds in six orchards in Oceana county Number of b ds Buds infested containingB Cherry Orchard species Fractiona % Pss Psm INA Sampled on 9 March 1987 Trommater sweet 7/24 29.2 7 0 0 Beer sweet 0/18 0.0 0 0 0 Daly sweet 4/18 22.2 4 0 0 Silver Hills sweet 5/20 25.0 4 l 2 Tubbs sour 0/17 0.0 0 0 0 Daly sour 4/19 21.0 4 0 l Sampled on 16 December 1987 Trommater sweet 5/50 10.0 0 5 0 Daly sweet 4/50 8.0 1 3 0 Daly sour 6/50 12.0 1 5 1 Sampled on 17 February 1988 Trommater sweet 5/50 10.0 0 5 0 Daly sweet 1/50 2.0 0 l 0 Daly sour 5/50 10.0 2 3 3 Mean populations (log colony-forming units (and variance) per infested bud) Cherry species Pss Psm INA Sweet 3.42 (1.33) 2.73 (0.83) 3.33 (0.30) Sour 3.04 (0.84) 2.52 (1.06) 3.53 (0.70) aNumber of buds containing green fluorescent bacteria over the total number of buds in each sample at a limit of detection of 1.3 log (colony forming units per bud). sts and Psm were identified based on the GATTa determinative tests (14) and ice nucleation activity was determined by the procedure of Lindow et al. (17). 30 Table 1.4. Percent recovery of rifampicin-resistant zgggggmgggg gyginggg pv. IIILBISS (Peak) and E. ;. pv. ggggpggggggg (Pst) from buds of Montmorency sour cherry in spring after the inoculation of adjacent leaf scars the previous eutua with four concatrations of inoculun - Buds yielding Pst (x)‘ Buds yielding PssR (2) 16666166166 dateb 1o2 10‘ 106 108 Heanc 102 10‘ 106 108 Mean 21 September 1878 -d 25 56 61 47+18 - o 7 14 7+11 5 06666.: 1878 - 5 60 78 28+28 - 12 24 54 30+22 Means for 1878° 15+13 58+16 70+18 6+3 15+1o 34+26 18 August 1880 2 24 62 - 28+25 2 8 55 - 22+26 11 September 1880 2 34 64 - 34+28 2 25 65 - 31+28 6 06666.: 1880 2 61 80 - 48+35 2 16 71 - 30+32 Means for 1880 2+3 4o+18 69+11 2+3 16+15 64+15 ‘Bach value is the mea of three replications with 12 to 18 buds per replicate. Buds were collected on 15 April 1980 ad 14 April 1981. Infection was established by isolating on King's medias B aaded with 50 ug/ml. rifapicin ad 25 ug/ml. cycloheximide. hLeaf scars were inoculated with e 0.05 ml. droplet of phosphate buffer containing 102, 10‘, 106, or 108 cells per milliliter. cfleas with stadard errors for concentration and for date of inoculation. - not inoculated 31 increased. Recovery of PssR from buds near leaf scars inoculated 5 October 1979 was higher than from buds near leaf scars inoculated 21 September 1979, while Pst was recovered from a similar number of buds. In 1980, the recovery of Pst and PssR from buds was higher from inoculations on 8 October than on 19 August. DISCUSSION The recovery of 2. a. pv. mm and 2. s. pv. mgrsprnngrnn from dormant buds of sweet cherry during and following cold winters in Michigan is consistent with the results of similar studies in regions with milder winters (6,19), and it supports the conclusion that buds of sweet .cherry are an overwintering site for these bacteria. Our results extend this concept to sour cherry. Previously, 2. .s. pv. syringag was found to overwinter in buds of apple in New York (3,4) and 2. g. pv. papglang (Rose) Dhanvantari was found to overwinter in buds of Mutsu apple in Ontario and New York (2,3,4). Thus, three pathovars of 2. gyringgg have been shown capable of surviving in buds of fruit trees in the Great Lakes fruit growing region of North America. Surveys of Michigan cherry orchards during the bloom period indicated that a number of orchards contained detectable numbers of 2. g. pv. gyringgg, including INA strains, and of 2. g. pv. mgrgprgngrum on blossoms. We suspect bacteria were present also in the majority of the orchards that did not yield pseudomonad bacteria. We did 32 not detect them because of the small sample size and because samples were often taken ahead of wet weather favorable for dissemination. In 1986, we were able to repeat sample in a few orchards following rain and detected bacteria in orchards in which we did not detect them initially. As noted previously by Leben (16), it is likely that bacteria in buds colonize blossom and leaf tissue as they unfold from the bud. Detection of bacteria on blossoms would depend on selecting a colonized blossom from an infested bud or for rain to disseminate the bacteria from colonized blossoms to uncolonized blossoms. The overwintering of Pst and PssR in buds of Montmorency sour cherry following leaf scar inoculations in autumn agrees with reports of infection of cherry through leaf scars (7-9,ll). As in the United Kingdom (8), infection through leaf scars with Pst was greater in late autumn then in early autumn. In our study, the incidence of infection through leaf scars by E. g. pv. gyringgg and 2. g. pv. morgprgngrgm were similar, while Crosse and Garrett (9) found the incidence of infection with a strain of B. s. pv. syringe; from Oregon was less than with a strain of 2. g. pv. mgrgpxgngzgm from the United Kingdom. These differences in efficiency of leaf scar infection between 2. g. pv. syringes and 2. g. pv. mgzgprungrum may be related in the way infection was determined in the two studies. Crosse and Garrett (9) estimated infection by visual observation of symptoms; we based our data on actual isolations of 33 bacteria. Although our results with rifampicin-resistant strains suggest that the establishment of 2. a. pv. syringe; and 2. 5. pv. mgzfiprnngznn in buds collected from commercial orchards and from an experimental orchard with high populations of bacteria during summer occurred by invasion in autumn through leaf scars, such a conclusion may not be entirely correct. Recent studies on systemic movement of 2. g. pv. mgrgpznngxnn indicate entry of bacteria into the vascular system of sweet cherry in tissue away from the site of inoculation (20). Furthermore, a rifampicin-resistant 2. s. pv. mgrgngngrgn was detected in the vascular system of sour cherry shoots 5, 10, and 20 cm downward from the point of inoculation and subsequent canker formation (B. A. Latorre and A. L. Jones, unpublished). The mutant was not detected in symptomless tissue until 3 months after inoculation, and detection coincided with the onset of normal leaf senescence and defoliation. When trees enter dormancy in autumn, possibly there is movement of bacteria, similar to the annual movement of metabolites from leaves, from symptomless tissue, cankers, and wounds including leaf scars to other parts of the tree. This theory could explain the detection of 2. g. pv. syringgg in the wood of sweet cherry trees in Oregon (5). Also, if movement of bacteria occurs annually, it would explain our observation that a high proportion of buds in old sweet cherry trees contained pseudomonad bacteria. 34 As noted previously (4,5,18,19), the association of 2. g. pv. syringag, including INA strains, and of 2. g. pv. mgrgprnngznm with apparently healthy buds, along with the systemic nature of the bacteria, has important implications to the control of bacterial canker in tree fruit nurseries and orchards in the Great Lakes region. It explains why sprays of bactericides during the dormant period are unlikely to succeed. Also, budwood for propagation may carry the bacteria and increase the likelihood of dissemination through nursery stock. 35 LITERATURE CITED 1. Allen, W. R., and V. A. Dirks. 1978. Bacterial canker of sweet cherry in the Niagara Peninsula of Ontario: Essggsmsnss species involved and cultivar susceptibilities. Can. J. Plant Sci. 58:363-369. 2. Bedford, K. E., B. H. MacNeill, and W. G. Bonn. 1984. Survival of a genetically marked strain of the blister spot pathogen Pseudomonas.axringas PV- 8889188: in leaf scars and buds of apple. Can. J. Plant Pathol. 6:17-20. 3. Burr, T. J., and B. Katz. 1982. Evaluation of a selective medium for detecting Essngsmsnss syringss pv. 88831888 and 2. arrinsas PV- airingas in apple ereherde- Phytopathology 72:564-567. 4. Burr, T. J., and B. H. Ratz. 1984. Overwintering and distribution pattern of Pseudomonas syringes pV- 88881383 and pv. syringss in apple buds. Plant Dis. 68:383-385. 5. Cameron, 8. R. 1962. Mode of infection of sweet cherry by Pseudomonas arringas- Phytopathology 52:917-921- 6. Cameron, 8. R. 1970. Pseudomonas content of cherry trees. Phytopathology 60:1343-1346. 7. Crosse, J. E. 1956. Bacterial canker of stone-fruits. II. Leaf scar infection of cherry. J. Bortic. Sci. 31:212- 224. 8. Crosse, J. E. 1957. Bacterial canker of stone-fruits. III. Inoculum concentration and time of inoculations in relation to leaf-scar infection of cherry. Ann. Appl. Biol. 45:19-35. 9. Crosse, J. E., and C. M. E. Garrett. 1966. Bacterial canker of stone-fruits. VII. Infection experiments with morsnrunerun and B. airingae- Ann- Appl- 3101- 58:31-41. 10. Dhanvantari, B. N. 1969. Occurrence of bacterial canker of sweet cherry and plum in Ontario. Can. Plant Dis. 11. Hignett, R. C. 1974. Absorption of Essgdsmsnss msrsprgnssgm through leaf scars. J. Gen. Microbiol. 80:501- 506. 12. Jones, A. L. 1971. Bacterial canker of sweet cherry in Michigan. Plant Dis. Rep. 55:961-965. 36 13. King, B. O., M. K. Ward, and D. E. Raney. 1954. Two simple media for the demonstration of pycocyanin and fluorescin. J. Lab. Clin. Med. 44:301-307. 14. Latorre, B. A., and A. L. Jones. 1979. Essngsmsnss msrspznnsrgn, the cause of bacterial canker of sour cherry in Michigan, and its epiphytic association with 2. syringss. Phytopathology 69:335-339. 15. Latorre, B. A., and A. L. Jones. 1979. Evaluation of weeds and plant refuse as potential sources of inoculum of Rssgdsmsnss in bacterial canker of cherry. Phytopathology 69:1122-1125. 16. Leben, C. 1988. Relative humidity and the survival of epiphytic bacteria with buds and leaves of cucumber plants. Phytopathology 78:179-185. 17. Lindow, S. E., D. C. Arny, and C. D. Upper. 1978. Erxinis : an active ice nucleus incites frost damage to Maize. Phytopathology 68:523-527. 18. Mansvelt, E. L., and M. J. Mattingh. 1987. Pseudomonas syringss pv. syringss associated with apple and pear buds in South Africa. Plant Dis. 71:789-792. 19. Roos, I. M. M., and M. J. Hattingh. 1986. Pathogenic Essggsmsnss spp. in stone fruit buds. Phytophylactica 20. Roos, I. M. M., and M. J. Hattingh. 1987. Systemic invasion of cherry leaves and petioles by Essggsmsnss syringss pv. mszsprnnssgn. Phytopathology 77:1246-1252. CHAPTER 2 Ecology and Genetics of Copper Resistance in 25888882838 axringae pv. sxrinsae ABSTRACT Copper-resistant Essndomenas.sxrinsae pV- sxringae.were recovered from blossoms of sweet and sour cherry from nine and 12 orchards in Michigan in 1987 and 1988, respectively. Isolates which failed to grow on media amended with 0.16 to 0.48 mM cupric sulfate were considered sensitive. Isolates that grew on media amended with 0.64 to 0.96 mM cupric sulfate were classified as having low resistance and those that grew on media amended with 1.12 to 1.28 mM cupric sulfate were classified as having high resistance. Among 17 copper-resistant isolates collected in 1987, 14 contained a single plasmid of approximately 46, 55, 61, 67, or 73 kilobase pairs (kb) and three with low resistance lacked a detectable plasmid. A 61 kb plasmid was present in all 29 copper-resistant isolates selected on media containing 0.80 mM cupric sulfate in 1988. Among 15 copper-sensitive isolates, one isolate contained a plasmid of 58 kb. All 2. s. pv. msrsngnszgm from cherry were copper-sensitive. Copper resistance and a 61 kb plasmid were transferred in filter matings to three copper-sensitive recipient strains of 2. s. pv. sysingss from each of six copper-resistant 37 38 donor strains and to two recipient strains from a transconjugant used as a donor strain. Resistance was also transferred from one donor to one recipient when suspensions of bacteria were infiltrated into bean leaves. Copper resistance was not transferred in filter matings when E. s. pv. msxspgnnssgn was the recipient. Populations of copper- sensitive strains of 2. s. pv. syringss were reduced significantly more than those of copper-resistant strains in tests performed on bean leaves sprayed with cupric hydroxide. Bacterial canker is a recent and important problem of sweet and sour cherry (zzgngs syign L. and B. sssssgs L., respectively) in Michigan (7,11). The disease is caused by each of two bacteria, Pssngsmsnss syringss pv. sygingss van Hall and 2. s. pv. msrsngnszgn (Wormald) Young et al (7). Bacterial populations are often high in spring and in autumn when cool, wet weather favors growth and dissemination (11). In Michigan, sprays of tribasic copper sulfate are applied weekly for about 6 wk starting with the bud burst stage of bud development to prevent infection of leaves, blossoms, and fruit of sour cherry. On sweet cherry, a dormant treatment only of Bordeaux mixture or of fixed copper is applied in orchards where bacterial canker has been a problem. Prior to the use of copper for bacterial canker, copper fungicides were used in many sour cherry orchards to 39 control cherry leaf spot, caused by stssmys s hismslis Higgins. Resistance to copper has been reported in the bacterial plant pathogens 2338121121an musing pV- 11881263236 (Doidge) Dye and 2. s. pv. tsmsts (Okabe) Young et a1 (1,15). In both bacteria, resistance to copper was associated with conjugative plasmids (1,20). Copper resistance has also been associated with conjugative plasmids in the Gram negative bacterium Essnszishis 9911 (23) and is plasmid encoded in Myssssstsrign ssrsfulsssgm (5). Selection of resistant strains in all cases was associated with prior exposures to copper ions. Because of the long history of copper usage on cherries in Michigan, we undertook a study to determine if copper- resistant 2. s. pv. syringss and 2. s. pv. nsrspggnszgm were present in cherry orchards and if copper-resistant strains were able to survive on plants sprayed with a copper bactericide. We also investigated whether the gene(s) involved in copper resistance were plasmid-borne or located in the chromosome. MATERIALS AND METHODS mmwxwm. In 1987, epiphytic fluorescent pseudomonads were isolated from blossom samples collected from sweet and sour cherry orchards located in western Michigan as previously described (22). The sensitivity to copper of 154 isolates from that 40 study was determined on Casitone-yeast extract-glycerol medium (CYE) (24) amended with 0 to 1.28 mM cupric sulfate in increments of 0.16 mM. The cupric sulfate was added to the medium and the pH adjusted to 6.0 before autoclaving. Bacteria to be screened were grown for 48 hr on King's medium B (KB) (9) amended with 50 ug/ml cycloheximide (KBc) in petri plates at 23 C, and then streaked on CYE amended with 0.64 mM cupric sulfate. Bacteria with confluent growth on the medium were considered copper-resistant, and those that failed to grow were considered copper-sensitive. To determine the minimal inhibitory concentration (MIC) of copper necessary to prevent confluent growth, copper- resistant isolates were streaked on CYE amended with 0.80, 0.96, 1.12, and 1.28 mM cupric sulfate and copper-sensitive isolates were streaked on CYE amended with 0.16, 0.32, and 0.48 mM cupric sulfate. In 1988, blossom samples were collected from three sweet and 10 sour cherry orchards located in the western Michigan counties of Mason, Oceana, and Van Buren. Orchards that were sampled either contained copper-resistant bacteria in 1987 or received copper applications in 1988. Two spurs with blossom clusters were collected from each of 100 trees per orchard. Samples were placed in plastic bags and kept in a cooler until processed. A sample of 200 blossoms, one blossom from each spur, was placed in a 2 L flask and washed with 100 ml 0.01 M potassium phosphate buffer (pH 7.2) (K- buffer) by shaking the flask vigorously by hand for 2 min. 41 The wash solution was centrifuged for 10 min at 6,000 rpm in a Sorvall GSA rotor and the pellet resuspended in 10 ml K- buffer. Aliquots (0.1 ml) from the original and from 10'2 to 10’5 dilutions of the original sample were plated (two replications) on KBc and CYE amended with 0.80 mM cupric sulfate. Petri plates were incubated in an inverted position at 23 C for 3-4 days after which colonies of fluorescent pseudomonads were counted. Ten isolates arising on CYE from each of 12 orchards were randomly selected and tested for copper MIC as described above. Four copper- sensitive isolates from one orchard were tested on CYE amended with 0.16, 0.32, and 0.48 mM cupric sulfate. Identification cf ccpccrzrcsictant bacteria- Isolates of fluorescent pseudomonads from both 1987 and 1988 were identified based on ice nucleation-activity at -5 C as determined by the droplet freezing procedure of Lindow et a1 (13) and the GATTa determinative tests (11). Isolates that were ice nucleation-active and GATTa+ were considered 2. s. pv. sysingss and isolates that were ice nucleation negative and GATTa' were considered 2. s. pv. mssspznnszum. 21m chenctcricaticn cf ccppcnrccistcnt strains- A total of 46 copper-resistant strains of B. s. pv. sysisgss were screened for plasmids using a modification of the method of Kado and Liu (8). One ml of a 10 ml overnight culture grown in Luria broth (LB) (16) was centrifuged, and the pellet suspended in 0.15 ml E buffer (0.04 M Tris, pH 7.9: 0.04 M Na-acetate, 0.02 M EDTA). The suspension was 42 lysed with 0.30 ml SDS-lysis buffer (0.05 M Tris, pH 12.6: 3% SDS), incubated for 45 min at 65 C, and extracted twice with two volumes pheno1:chloroform:isoamyl alcohol (25:24:1), followed by a final extraction with two volumes chloroform:isoamyl alcohol (24:1). Plasmid DNA was separated by electrophoresis for 2.5 hr at 5 V/cm through a horizontal 0.7% agarose gel submersed in E buffer. Gels were stained for 30 min with ethidium bromide (0.5 ug/ml), destained for 5 min in 0.01 M MgClz, and photographed with Polaroid type 55 Land film using Wratten red filters under 302-nm UV light. Strain SW2 of Egginis stswsrtii (4) was used as a molecular size marker in plasmid characterization. Bsstsrisl ssniugstisn. Copper-resistant strains 3lB-4, 70-7, 71-7, 72-9, 77-8, and 79-2 (2. s. pv. syringss) were used as donors and streptomycin or rifampicin-resistant mutants of copper-sensitive strains 17A-5, 27A-4, and 31A-5 (B. s. pv. syringss) and 101A-3 and 115A-1 (B. s. pv. msrsprgnszgm) were used as recipients in conjugation experiments. All donors were highly resistant to copper and harbored a 61 kb plasmid. Recipient strains were inhibited by 0.16 mM cupric sulfate in CYE medium. Antibiotic resistant strains were selected by spreading approximately 108 cells on KB amended with 25 ug/ml streptomycin or 100 ug/ml rifampicin. Both antibiotics were filter-sterilized and added to autoclaved media at 45 C. After 3 days incubation at 23 C, colonies were selected that had growth rates similar to the parent strain, were ice nucleation- 43 active, and GATTa+ (11). Matings were conducted using 20 m1 overnight cultures grown in LB broth. The broth cultures were centrifuged, and the pellets resuspended in 1 ml K-buffer. Matings were conducted on a medium containing 1.5 g KZHPO4, 5 g yeast extract, 5 9 glucose, 10 g peptone, 2.5 g NaCl, and 15 g agar per liter by mixing approximately 108 donor cells with 5 x 108 recipient cells on 0.45 um Millipore filters. The plates were incubated for 18 hr at 23 C after which the cells were suspended in 2 ml K-buffer, serially diluted, and plated onto CYE and KB media amended with the appropriate antibiotics to determine transconjugant and recipient populations respectively. Colonies that grew on CYE amended with 0.80 mM cupric sulfate and 25 ug/ml streptomycin or 100 ug/ml rifampicin were considered punitive transconjugants. Controls consisting of donor and recipient cells alone were treated similarly to determine the frequency of spontaneous resistant mutants in the population. One to 10 transconjugant bacterial colonies were randomly selected per mating for plasmid characterization to establish transfer of the 61 kb plasmid. Matings between strains 31B-4 and 31A-5Str were also performed in bean leaves. Approximately 108 donor and 5 x 108 recipient cells in K- buffer were mixed and drawn into a sterile 10 ml plastic syringe. The end of the disposable tip of the syringe had previously been cut off and inverted with the large diameter opening facing outwards. The cell 44 suspension (0.2 ml) was infiltrated into the underside of bean leaves on intact plants, water-soaking the leaves. The plants were incubated in plastic bags for 1 and 3 days at 25 C after which the areas of infiltration were diced in 1 ml K-buffer and aliquots (0.1 ml) plated on CYE and KB amended with 25 ug/ml streptomycin as with filter matings. Controls consisted of donor and recipients cells infiltrated alone to determine the frequency of spontaneous mutation. In; mgtsgsnssis. Overnight cultures of strain SMlo of E. 9911 (19) containing the suicide plasmid pSUP1011 and transconjugant copper-resistant strain 27A-4a of P. s. pv. syringss grown in LB broth were centrifuged, the pellets washed in 10 ml sterile 0.85% NaCl, centrifuged again, and the pellets resuspended in 1 ml 0.85% NaCl. Filter matings were conducted at 28 C as described above. After incubation, the cells were resuspended in 1 ml 0.85% NaCl, serially diluted, and plated on M9 minimal medium (16) amended with 12.5 ug/ml kanamycin. To assay for kanamycin- resistant mutants lacking the copper-resistance phenotype, colonies arising on M9 were replica plated on KB amended with 12.5 ug/ml kanamycin (KBkan) and on CYE amended with 0.80 mM cupric sulfate. Copper-sensitive mutants were those that grew on KBkan but not on CYE. DNA Hybsigizstisss. Biotinylated DNA probes were constructed from plasmid pSUPlOll. The plasmid was isolated from bacteria grown in 250 ml cultures using the procedure of Birnboim and Daly (2). Precipitated plasmid DNA was 45 suspended in 4.6 ml E buffer, to which 5.5 g cesium chloride and 75 ul bis-benzimide (10 mg/ml) was added. The solution was then transferred to a 6 ml ultracentrifuge tube. The plasmid DNA was purified by centrifugation at 50,000 rpm in a Sorvall TV-865 rotor at 20 C for 17 hr and removed from the tubes through an 18-gauge needle. The bis-benzimide was removed from the solution by repeated extraction with isopropanol saturated with water and cesium chloride. The DNA was precipitated with 1.5 volumes E buffer and 2.2 volumes ice cold 100% ethanol followed by incubation for 2 hr at -20 C. Precipitated DNA was pelleted by centrifugation for 15 min at 12,500 rpm, rinsed once with 70% ethanol, once with 100% ethanol, and finally suspended in 20 ul E buffer. The plasmid DNA was labelled with biotin-7-dATP using a nick translation kit as described by the manufacturer (Bethesda Research Laboratories, Gaithersburg, MD). Unincorporated nucleotides were removed by spun column chromatography (14). Plasmid DNA of kanamycin-resistant transconjugants from agarose gels was transferred to GeneScreen Plus nylon membranes (New England Nuclear (Dupont) Boston, MA) by the method of Southern (21) as modified by the manufacturer. Posthybridization washes, filter blocking, and detection of homologous sequences were performed as specified in the Blu-GENE alkaline phosphatase DNA detection system (BRL). 51111111619112. amurinccccnccpccrcmycdbccn 46 plants. Populations of copper-resistant strains 18A-5, 22B-4, 318-4, and 64B-3, of copper-sensitive strains 18A-3, 19A-2, 478-1, and 52A-2, and of transconjugant copper- resistant strain 27-17a were compared on bean leaves sprayed with cupric hydroxide. Bean plants were used instead of cherry because of their rapid growth and their ability to harbor high epiphytic populations of bacteria. All strains were isolated from cherry blossoms in 1987 (22), except 27- 17a was produced in a mating between strains 27A-4a and 17A- 5. Inoculum was prepared by growing each strain overnight in 20 ml LB broth. The cultures were centrifuged for 10 min at 4,000 rpm in a clinical centrifuge, the pellets resuspended in 1 ml K-buffer, diluted in 150 ml buffer, and adjusted turbidimetrically using a Bausch and Lomb spectrophotometer at 620 nm. Navy bean (znsssslns,yn1gn:1s var. Seafarer) plants were grown in 473 ml plastic cups (three plants per cup) in the greenhouse to a height of approximately 15 cm. Groups of 15 plants were spray inoculated with an atomizer containing a suspension of approximately 107 bacterial cells per milliliter until the leaves were uniformly wet. The plants were then transferred to a dew chamber (Model I-60DL, Percival Manufacturing Co., Boone, IA) maintained at 19 C with a 12 hr light/dark schedule. Three days after inoculation, both adaxial and abaxial leaf surfaces were sprayed to runoff with Kocide 101 (cupric hydroxide) at 4.8 g/L. Control plants were not sprayed. 47 Samples of either leaf disks or excised tissue consisting of approximately 0.3 g were taken from one leaf on each of three plants. There were two replications of each sample. The plants were sampled 1, 2, 4, and 7 days after copper application, always when leaf surfaces were dry. Plants inoculated with strain 27-17a were also sampled immediately before copper application. Each sample was placed in a 250 ml flask and washed with 20 ml K-buffer for 30 min on a Burrell wrist action shaker. Aliquots (0.1 m1) from the original and from 10'1 to 10"3 dilutions of the original washings were plated (two replications) on KBc and distributed evenly with an L-shaped glass rod. Colonies of fluorescent bacteria were counted after 3 days incubation at 23 C. RESULTS sccrcc cf copper resistant tactcria- Testing of fluorescent bacteria on cupric sulfate-amended CYE resulted in the identification of three groups of isolates based on the concentration of cupric sulfate required to inhibit the growth of each isolate. Isolates were considered sensitive if they failed to grow on media amended with 0.16 to 0.48 mM copper sulfate, as having low resistance if they grew on media amended with 0.64 to 0.96 mM cupric sulfate, and as having high resistance if they grew on media amended with 1.12 to 1.28 mM cupric sulfate. In 1987, 137 of 154 isolates (89.0%) failed to grow on CYE 48 amended with 0.64 mM cupric sulfate and were classified as copper-sensitive. The remaining 17 isolates (11.0%) grew on CYE amended with 0.64 mM cupric sulfate and following furthur testing, nine isolates were classified as having low and eight as having high resistance. In 1988, populations of fluorescent bacteria on blossoms in 13 orchards averaged 6.87 log colony forming units per gram (cfu/g) fresh weight (Table 2.1). Recovery of bacteria on CYE amended with 0.80 mM cupric sulfate and on KBc was high in 12 of 13 orchards. Ten isolates from each orchard were selected and the MIC for each isolate was determined on copper-amended CYE. Forty isolates were classified as low and 80 as highly resistant to copper. Four isolates from orchard M were classified as copper-sensitive. Iccntificaticncfccppcrzrcsictanthactcria. A11137 copper-resistant isolates from both 1987 and 1988 were identified as 2. s. pv. syringss based on positive results for the GATTa tests and ice nucleation activity. In 1987, 98 and 39 copper-sensitive isolates were identified as 2. s. pv. syringss and 2. s. pv. nszsprnnsrnn, respectively. In 1988, the four copper-sensitive isolates from orchard M were identified as 2. s. pv. syningss. Blacmicchcractcritaticncfccppcrcrccictantctrainc- Fifteen of 102 randomly selected copper-sensitive isolates from 1987 and 1988 were screened for plasmids and only one contained an indigenous, cryptic plasmid of approximately 58 kb. However, among the copper-resistant isolates identified 49 Table 2.1. Recovery of Pseudmfi m pv. m from cherry blossua washes on media with ad without copper. Blossan saples were collected from orchards that contained copper-resistat bacteria in 1887 or were sprayed with copper in 1888 Bacterial populationsx Copper resistace (log cfu/g fresh weight) levely Orchard: County :86” are" 168 high .4“ Mesa 7.01 8.43 0 10 8“ Mesa 7.22 6.48 o 10 8“ Mason 7.26 6.63 3 7 D Oceaa 7.58 7.13 l 9 8“ Oceaa 7.06 6.86 2 8 P Oceaa 7.14 8.39 8 4 6 Va Bura 8.81 8.94 5 5 B Va Bura 8.89 8.82 5 5 1 Va 86:66 6.88 6.81 5 5 .1“ Va Bura 8.81 6.65 4 6 1:“ Va Duran 6.80 6.83 4 6 1.“ Va 66m 6.80 6.84 5 5 :6“ Va Bura 4.80 0.00 0 0 2Serial dilutions of washings of 200 blossom per orchard were plated in duplicate on each media ad the nunber of colonies counted after 3 days incubation at 23 C. Counts represent the average umber of colonies per gra of blossom tissue. yCopper resistace level detenained by confluat growth on CYE median aended with cupric sulfate. Low - inhibited by 0.84 - 0.88 of! cupric sulfate, high - inhibited by 1.12 - 1.28 m cupric sulfate. A total of 10 isolates were tested per orchard. xOrchards C, D, and 6 were sweet cherry, remaining orchards were sour cherry. wKBc is King’s median B (8) mended with 50 ug/ml cycloheximide. vCYEc is Casitone-yeast extract glycerol median (22) mended with 0.80 or: cupric sulfate. uCopper resistant bacteria were also detected in this orchard in 1987. 50 in 1987, three isolates with low resistance lacked detectable plasmids, six isolates with low resistance contained a single plasmid which was approximately 46, 55, 61, 67, or 73 kb in size, depending on the orchard the isolates were from, and eight isolates with high resistance all contained a plasmid of either 46 or 61 kb in size (Figure 2.1). The three isolates lacking plasmids were from the same orchard and grew on CYE with 0.64 mM cupric sulfate. Among 29 copper-resistant isolates screened in 1988, 11 isolates with low and 18 isolates with high resistance each contained a single plasmid of approximately 61 kb. Copper-resistant isolates maintained their plasmids and resistance phenotypes after as long as 1 year of growth encompassing 50 transfers on KB in the absence of copper ions. fisstszisl ssningstisn. Frequency of transfer of copper-resistance of 1.28 mM in CYE medium ranged from 3.43 x 10'3 to 3.37 x 10'8 transconjugants per recipient cell of B. s. pv. sysingss (Table 2.2). Repeated attempts to transfer copper resistance into two strains of 2. s. pv. nszsprnnsrnn were unsuccessful. Spontaneous mutants arose infrequently and the observed rates of spontaneous mutation to copper or to antibiotic resistance were always less than 10-10 per cell. The frequency of transfer was dependent on the recipient, as it differed as much as four orders of magnitude using the same donor but a different recipient. 51 1234567 61 kb —: Figure 2.1. Agarose gel electrophoresis of cleared lysates of copper-resistant strains of Pseudomonas syringae pv. syringae isolated from cherry orchards in Michigan in 1987. Lanes 1-7 are strains 3lB-4, 43A-1 45A-3, 228-4, 478-4, 393- 1, and 18A-5, respectively. Table 2. 2. 52 resistance in Bccuccmcnac cxrincac pv. Frequency of conjugal transfer of copper Recipient strainsb 8:32:58 17a-5Str 278-4rif 3111-5Btr 3lB-4 1.81 x 10'5 3.48 x 10“ 3.43 x 10‘3 70-7 5.78 x 10" 4.21 x 10"8 2.72 x 10'5 71-7 2.33 x 10'5 3.20 x 10“ 6.04 x 10"5 72-9 1.33 x 10" 4.21 x 10"8 4.82 x 10‘5 77-8 3.37 x 10’5 6.88 x 10"7 5.66 x 10"5 79-2 8.53 x 10" 6.18 x 10'8 3.16 x 10" 271-46c 1.96 x 10" --- 2.41 x 10'6 “Frequency of spontaneouso was always less than 10 mutation of donors or recipients per cell. bSpontaneous antibiotic resistant copper-sensitive mutants, str - streptomycin and rif - rifampicin. cCopper-resistant transconjugant constructed in mating of strains 318-4 and 27A-4. 53 Among the 20 matings, the highest frequency of transfer was between two strains (313-4 and 31A-5) isolated from the same sour cherry orchard. The copper-resistant transconjugant 27A-4a was able to donate copper resistance to the two remaining recipients. Transconjugant colonies, randomly chosen from selection plates, always contained the 61 kb plasmid (Figure 2.2). Frequency of transfer of copper-resistance between strains 31B-4 and 31A-5, infiltrated into bean leaves, averaged 3.57 x 10'6 per recipient cell over five matings. This rate was approximately 103 lower than the filter mating involving the two strains but was higher than that of five other filter matings involving recipients 17A-5 and 27A-4. No spontaneous mutants arose in the infiltration experiments. In: nntsgsnss1s. Matings between E. 9911 (pSUP1011) and transconjugant copper-resistant strain 27A-4a produced kanamycin resistant transconjugants at an average rate of 4.6 x 10’7 per recipient cell. A total of 1,544 transconjugant colonies from a series of independent matings were screened for loss of copper resistance and two mutants (0.13%) were found that had lost resistance to copper. The plasmid DNA of the copper-sensitive mutants was larger than 61 kb and Southern blot hybridizations demonstrated an insertion of pSUP1011 DNA into the plasmid of strain 27A-4a. swabs-primincsccnccppcrcpmcdpccn 199299. Populations of copper-sensitive strains 18A-3, 19A- 2, 47B-1, and 52A-2 were reduced more than populations of 54 Figure 2.2. Agarose gel electrophoresis of cleared lysates of donor, recipient, and transconjugant strains of Pseudomonas syringae pv. syringae. Lang 1, donor strain 3lB-4: lane 2, recipient strain 27A-4r1 7 lane 3, transconjugant 27A-4a; éane 4, donor strain 70-7; lane 5, recipient strain 31A-5s r; lane 6, transconjugant 31A-5a; lane 7, transconjugant onor strain 27A-4a; lane 8, recipient strain 17A-5 r; lane 9, transconjugant 27-17a. 55 copper-resistant strains 18A-5, 228-4, 318-4, and 648-3 at all sampling intervals except for day 2 after a spray application of Kocide 101 (Table 2.3). Throughout the four sampling intervals of the experiment, populations of copper- sensitive strains 18A-3, 19A-2, and 52A-2, averaged 3.28 log cfu/g and strain 478-1 averaged 4.48 log cfu/g on copper sprayed bean leaves. Strains 18A-3, 19A-2, and 52A-2 were inhibited on CYE amended with 0.16 mM cupric sulfate while strain 478-1 was inhibited with 0.48 mM cupric sulfate. Populations of copper-resistant strains averaged 5.64 log cfu/g on copper sprayed bean leaves and in some cases exceeded populations on unsprayed controls. Populations of transconjugant strain 27-17a remained high on bean leaves sprayed with copper through the 7-day sampling period. Populations of strain 27-17a and copper- resistance donor strain 318-4 were consistently reduced less than the populations of the other copper-resistant strains. 56 Table 2.3. Differences in effect of a single copper treatment with 4.8 g/L Kocide 101 (cupric hydroxide) on the recovery of copper-resistant and copper-sensitive strains of W 51411189.! pv. m isolated from cherry blossom a bea leaves sapled at four intervals following copper spray"x Bacteria recovered Reduction in population frua unsprayed leaves on copper sprayed leaves (log cfulg leaf tissue) (log cfulg leaf tissue) Bacterial strainy Dayl Day2 Day4 Day7 Dayl Dayz Day4 Day7 18AF3. 6.87 5.66 6.67 6.50 3.25 b 1.73 b 2.88 6 3.26 6 IQAFZ. 6.50 5.86 6.36 6.43 6.50 6 3.31 6 2.70 6 3.20 6 478-1' 6.81 8.41 6.61 6.81 2.64 6 1.38 be 2.52 6 2.38 b 524-2' 6.40 6.46 6.26 5.86 2.16 d 3.28 6 2.47 6 1.88 b 18A-5: 5.76 5.82 6.48 6.67 0.76 6 1.23 be 1.18 b 0.76 c 228-4‘ 6.70 5.83 5.48 6.42 0.86 6 0.51 cd 0.00 c 0.64 c 318-4r 7.12 5.88 6.70 6.55 0.08 2 0.00 d 0.16 c 0.28 cd 648-3r 6.61 6.80 6.16 6.78 1.08 6 2.02 b 1.33 b 0.76 c 27-l7arz 5.59 5.65 5.58 5.21 0.00 f 0.00 d 0.15 c 0.04 d "Fleas within a column followed by the suns letter do not differ by Dunca's mltiple rage test (P - 0.05). “Readings of 0.00 indicate populations on copper sprayed leaves were higher than those on unsprayed controls. ys - copper-sensitive, r - copper-resistant. zCopper-resistat transconjugant constructed in mating between strains 274-46 and 17a-5“”. 57 DISCUSSION Copper-resistant 2. s. pv. syringss were recovered from blossoms from nine and 12 cherry orchards in Michigan in 1987 and 1988, respectively. Orchards harboring the resistant strains were located in four counties and were previously sprayed with copper. In 1988, populations of resistant bacteria on blossoms from seven orchards exceeded 107 cfu/g blossom tissue and at this level the populations were near the carrying capacity of the trees for 2. s. pv. syringss (18). The wide distribution of copper-resistant pseudomonads on cherry and their ability to survive and multiply on the surfaces of been plants sprayed with copper suggests that copper bactericides were no longer effective for reducing populations of 3. s. pv. syningss in these Michigan orchards. The detection of copper-resistant strains for two consecutive years in eight orchards suggests that these strains survive from season-to-season in the presence of copper applications. In the laboratory, copper-resistant 2. s. pv. syn1ngss remained resistant to copper and retained a plasmid after 1 yr of growth on media without copper ions. Similar findings on the stability of metal resistance have been reported by Griffiths et al (6). It is possible that copper-resistant B. s. pv. sy91ngss may persist in orchards for long periods even in the absence of copper applications, although the proportion of resistant strains in the 58 population may decline. Once a population of resistant strains has developed in an orchard, it is probably may not practical to use copper again in these orchards as populations of resistant strains will quickly redevelop. The spread of copper resistance within field populations of 2. s. pv. syxingss is likely enhanced by conjugation. Copper-sensitive strains of 2. s. pv. syr1ngss isolated from three cherry growing areas of Michigan were able to acquire the 61 kb plasmid from all six donors used in mating experiments, however, the frequency of transfer of copper resistance was highest in filter matings between bacteria isolated from the same orchard. The demonstration of conjugation in bean leaves indicates that transfer of the 61 kb plasmid and associated genes for copper resistance is not limited by the host. Plasmid transfer of antibiotic resistance in plants was previously demonstrated for B. s. pv. 91ys1nss (10). Recent reports of conjugative transfer of copper-resistance in B. s. pv. s9nss9 (1) and of streptomycin resistance in 2. s. pv.99991nns (3) suggests that Rssng9n9n9s has a high potential for developing resistance to antibiotics under selection pressure in the field. Copper-resistant strains of 2. s. pv. syringss harboring a 61 kb plasmid were detected in orchards separated from one another by up to 160 kilometers. An explanation for copper- resistant 2. s. pv. sys1n999 in a number of Michigan cherry orchards may be that the copper-resistant bacteria are 59 disseminated on nursery trees. In 1988, high populations of copper-resistant 2. s. pv. syzingss were recovered as epiphytes on blossoms of sweet cherry from trees used as a source for budwood. The retention of resistance once it is acquired and the ability of 2. s. pv. syn1ngns to grow epiphytically on a wide variety of plant hosts (12) would contribute to the buildup and survival of disseminated strains harboring. It is also likely, because the size of the plasmid in copper-resistant strains varied between orchards, that resistant strains were selected independently in some orchards from frequent use of copper sprays. The failure to recover any copper-resistant strains of 2. s. pv. n9nsnnnnsnnn in 1987 or 1988 suggests that populations of 2. s. pv. nsrsnxnn9rnn can still be effectively reduced with copper sprays. Repeated attempts at conjugation were unsuccessful, and it is possible that the 61 kb plasmid of 2. s. pv. syningss is incompatible with cells of B- c- pV- mcrcmncrcn. 6O LITERATURE CITATIONS l. Bender, C. L., and Cooksey, D. A. 1986. Indigenous plasmids in Ecccccmcnac syringes pV- tcnatc: conjugative transfer and role in copper resistance. J. Bacteriol. 165:534-541. 2. Birnboim, H. C., and Doly, J. 1979. A rapid extraction procedure for screening recombinant plasmid DNA. Nicleic Acids Res. 7:1513-1525. 3. Burr, T. J., Norelli, J. L., Katz, 8., Wilcox, W. F., and Hoying, S. A. 1988. Streptomycin resistance of syz1ngss pv. nsnn19ns in apple orchards and its association with a conjugative plasmid. Phytopathology 78: 410-413. 4. Coplin, D. L., Rowan, R. G., Chisholm, D. A., and Whitmoyer, R. E. 1981. Characterization of plasmids in Erg1n1s stsgsr§11. Appl. Environ. Microbiol. 42:599-604. 5. Erardi, F. M., Failla, M. L., and Falkinham III, J. O. 1987. Plasmid-encoded copper resistance and precipitation by Eycchactcricm ccrcfclaccun- Appl- Environ- Microbiol- 53:1951-1954. 6. Griffiths, A. J., Highes, D. E., and Thomas, D. 1975. Some aspects of microbial resistance to metal pollution. In: Minerals and the Environment, ed. M. J. Jones, pp. 387- 394. Institution of Mining and Metallurgy, Washington, D. c. 603 pp. 7. Jones, A. L. 1971. Bacterial canker of sweet cherry in Michigan. Plant Dis. Rep. 55:961-965. 8. Kado, C. I., and Liu, S. T. 1981. Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol. 145:1365-1373. 9. King, E. 0., Ward, M. K., and Raney, D. E. 1954. Two simple media for the demonstration of pyocyanin and fluorescen. J. Lab. Clin. Med. 44:301-307. 10. Lacey, G. H., and Leary, T. V. 1975. Transfer of antibiotic resistance plasmid RPl into gseugomonas 91y91n19 and Ese999nonss 999999119919 in vitro and in planta. J. Gen. Microbiol. 88:49-57. 11. Latorre, 8. A., and Jones, A. L. 1979. Eseudononas mozpsrunognn, the cause of bacterial canker of sour cherry in Michigan, and its epiphytic association with 2. syn1n999. Phytopathology 69:335-339. 61 12. Lindow, S. E., Arny, D. C., and Upper, C. D. 1978. Distribution of ice nucleation-active bacteria on plants in nature. Appl. Environ. Microbiol. 36:831-838. 13. Lindow, S. E., Arny, D. C., and Upper, C. D. 1978. h9r919919: a bacterial ice nucleus active in increasing frost damage to corn. Phytopathology 68:523- 527. 14. Maniatis, T., Fritsch, E. F., and Sambrook, J. 1982. Melecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. 545 pp. 15. Marco, G. M., and Stall, R. E. 1983. Control of bacterial spot of pepper initiated by strains of 39n§n9n9n9s 99n99s§n1s pv. yss199§9119 that differ in sensitivity to copper. Plant Dis. 67:779-781. 16. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. 466 pp. 17. Olson, B. D., and Jones, A. L. 1983. Reduction of Eccuccmcnac syringes pV- mcrccruncrum on Montmorency sour cherry with copper and dynamics of the copper residues. Phytopathology 73:965-971. 18. Proebsting, E. L. Jr., and Gross, D. C. 1988. Field evaluations of frost injury to deciduous fruit trees as influenced by ice nucleation-active 2s9999n9nss syz1n999. J. Am. Soc. Hortic. Sci. 113:498-506. 19. Simon, R., Priefer, U., and Puhler, A. 1983. A broad host range mobilization system for in vivo genetic engineering: Transposon mutagenesis in Gram negative bacteria. Biotechnology 1:784-791. 20. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. 21. Stall, R. E., Loschke, D. C., and Jones, J. B. 1986. Linkage of copper resistance and avirulence loci on a self- transmissible plasmid in zsn§n9n9nss campss§g1s pv. y9s19999z1s. Phytopathology 76:240-243. 22. Sundin, G. W., Jones, A. L., and Olson, 8. D. 1988. Overwintering and population dynamics of Escudononss syringes pV- syringes and B. c. pV- mcrcprcncrum on sweet and sour cherry trees. Can. J. Plant Pathol. 10:000-000. 62 23. Tetaz, T. J., and Luke, R. K. J. 1983. Plasmid- controlled resistance to copper in Essn9919n19 9911. J. Bacteriol. 154:1263-1268. 24. Zevenhuizen, L. P. T. M., Dolfing, J., Eshuis, E. J., and Scholtern-Korselman, J. 1979. Inhibitory effects of copper on bacteria related to the free ion concentration. Microb. Ecol. 5:139-164. STQTE UNIV. "‘Wjflgfiifim mmmmm wuumflifliflf 9300563