The Pennsylvania State University. The Graduate School. College of Agricultural Sciences ANTIBIOTIC RESISTANCE IN PENNSYLVANIA STONE FRUIT ORCHARDS

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1 The Pennsylvania State University The Graduate School College of Agricultural Sciences ANTIBIOTIC RESISTANCE IN PENNSYLVANIA STONE FRUIT ORCHARDS A Dissertation in Plant Pathology by Sarah J. Capasso 2016 Sarah J. Capasso Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2016

2 ii The dissertation of Sarah Capasso was reviewed and approved* by the following: María del Mar Jiménez Gasco Associate Professor of Plant Pathology Dissertation Advisor Chair of Committee Beth K. Gugino Associate Professor of Vegetable Pathology Gary W. Moorman Professor Emeritus of Plant Pathology Kari A. Peter Assistant Professor of Tree Fruit Pathology Mary Ann Victoria Bruns Associate Professor of Soil Science/Microbial Ecology Carolee T. Bull Professor of Plant Pathology and Systematic Bacteriology Head of the Department of Plant Pathology and Environmental Microbiology *Signatures are on file in the Graduate School

3 iii ABSTRACT Bacterial spot (caused by Xanthomonas arboricola pv. pruni) is the most important bacterial disease of peach and nectarine in the eastern United States. The antibiotic oxytetracycline is used to mitigate the yield limiting symptoms of this disease. Despite that, yield loss remains high in susceptible stone fruit cultivars, raising concern among growers over the development of antibiotic resistance in the causal pathogen. Previous surveys of the stone fruit orchard bacterial community indicated the presence of oxytetracycline resistant epiphytic bacteria. This was significant because epiphytic or nontarget bacteria are thought to harbor more resistance genes and often before coexisting pathogens. Under a strong selection pressure such as repeated antibiotic applications, transfer of resistance genes from epiphytic bacteria to pathogenic bacteria is favored. Therefore, when evaluating antibiotic resistance development in pathogenic bacteria, nontarget bacteria must also be considered. The overall goal of this research was to determine the consequences of repeated oxytetracycline applications in commercial Pennsylvania stone fruit orchards, including management factors related to the incidence of bacteria carrying tetracycline resistance genes and the sensitivity of X. arboricola pv. pruni isolates to oxytetracycline. Tetracycline resistance genes, tet(a), tet(b), and tet(c), were found in epiphytic bacteria recovered from commercial stone fruit orchards and research blocks at the PSU Fruit Research and Extension Center. The most common carriers of these resistance genes were bacteria that belonged to the genera Pantoea and Pseudomonas where tet(b) was most commonly associated with the former and tet(c), the latter. Bacteria carrying these tetracycline resistance genes could grow on media amended with greater than 450 ug/ml of oxytetracycline, three times that of the rate used in the field to manage bacterial spot. While

4 iv the incidence of tetracycline resistance genes in epiphytic bacteria significantly differed (P > ) among the sampled commercial orchards, this was not related to oxytetracycline use (P = ). When tested in the experimental stone fruit blocks, again, the distribution of bacteria positive for tetracycline resistance genes was not related to bactericide treatment (2013: P = 0.407; 2014: P = 0.520). Other management factors including tree age, cultivar, and sampling date were. Sensitivity to oxytetracycline among Xap isolates significantly differed (P > ) among those collected from commercial stone fruit orchards. While no tetracycline resistance genes were found in any of the sampled Xap isolates and overall sensitivity remained high (MIC < 25 µg/ml), oxytetracycline use was a significant factor associated with oxytetracycline sensitivity (P > ). Further research should be conducted to determine the molecular mechanism associated with the variability in sensitivity in the Xap isolates, including novel and untested resistance genes, adaptive resistance, or mutation.

5 v TABLE OF CONTENTS LIST OF TABLES... vii LIST OF FIGURES... viii ACKNOWLEDGEMENTS... x Chapter 1 Introduction...1 Bacterial Spot of Stone Fruit in the Eastern United States.1 Disease Significance...1 Symptoms and Disease Development...2 Causal Organism...6 Disease Management...8 Use of Antibiotics in Agriculture...10 Antibiotic Resistance...11 Mechanisms of Resistance...13 Tetracycline Resistance...13 Bacterial Diversity of the Phyllosphere...16 Research Goals and Objectives...19 Justification...20 Literature Cited...23 Chapter 2 Tetracycline resistance genes in epiphytic bacteria collected from Pennsylvania stone fruit orchards...34 Abstract...34 Introduction...35 Materials and Methods...37 Results...42 Discussion...45 Literature Cited...52 Chapter 3 The effects of bactericide use on bacterial epiphytes for the management of bacterial spot in Pennsylvania stone fruit orchards...65 Abstract...65 Introduction...66 Materials and Methods...70 Results...76 Discussion...80 Literature Cited...86

6 vi Chapter 4 Oxytetracycline sensitivity of Xanthomonas arboricola pv. pruni isolates collected from Pennsylvania stone fruit orchards Abstract Introduction Materials and Methods Results Discussion Literature Cited Chapter 5 Discussion Literature Cited Appendix...139

7 vii LIST OF TABLES Table 2-1: Oxytetracycline sensitivity of bacterial epiphytes with different tetracycline resistance genes...58 Table 2-2: Orchard management factors associated with recovery of epiphytic bacteria from Pennsylvania orchards positive for tetracycline resistance genes...59 Table 3-1: 2013 evaluation of bacterial spot incidence and severity on leaves and fruit on 'Beekman' and 'Snow King' in the FREC block...89 Table 3-2: 2014 evaluation of bacterial spot incidence and severity on leaves and fruit on 'Easterglo', 'Beekman', and 'Snow King' in the FREC block...90 Table 3-3: Factors associated with the number of colony forming units per gram of peach leaf tissue collected in Table 3-4: Factors associated with the number of colony forming units per gram of peach leaf tissue collected in Table 3-5: Oxytetracycline sensitivity of bacterial epiphytes with different tetracycline resistance genes...93 Table 4-1: Orchard management factors associated with the level of sensitivity of Xap isolates to oxytetracycline

8 viii LIST OF FIGURES Figure 1-1: Lesions on peach and nectarine leaves...3 Figure 1-2: Symptoms on peach fruit...4 Figure 1-3: Symptoms of bacterial spot on peach...5 Figure 2-1: Combined 2012 and 2013 bacterial communities from the sampled commercial orchards based on 16S identification of collected bacterial colonies...60 Figure 2-2: The percentage of colony forming units per gram of leaf material recovered from Kings B media amended with 0, 10, and 25 µg/ml oxytetracycline...61 Figure 2-3: Percent of isolates collected in 2012 and 2013 from commercial stone fruit orchards positive for teta, tetb, and tetc...62 Figure 2-4: Percent of isolates of each identified genus positive for tet R genes...63 Figure 2-5: The effect of the number of oxytetracycline applications made in the year of and year prior to sample collection, the tree age, the management method, and the oxytetracycline application method on the incidence of tetracycline resistance genes found in bacterial epiphytes collected from commercial orchards...64 Figure 3-1: Colony forming units per gram of leaf tissue collected in 2013 on different sampling dates...94 Figure 3-2: Colony forming units per gram of leaf tissue collected in 2013 and 2014 on 0, 10, and 25 µg/ml oxytetracycline...95 Figure 3-3: Colony forming units per gram of leaf tissue collected in 2013 from the cultivars Beekman, Red Haven, and Snow King and in 2014 from the cultivars Beekman, Easternglo, and Snow King...96 Figure 3-4: Colony forming units per gram of leaf tissue collected in 2014 on different sampling dates...97 Figure 3-5: The percentage of collected bacterial epiphytes positive for the presence of tet(a), tet(b), and tet(c) in 2013 and Figure 3-6: Percent of bacterial isolates positive and negative for tetracycline resistance genes (teta, tetb, or tetc) in 2013 and 2014 with respect to bactericide treatment..99 Figure 3-7: The incidence of epiphytic bacteria positive for teta, tetb, and tetc among species belonging to the genera Pseudomonas, Pantoea, Bacillus, Xanthomonas, and Curtobacterium

9 ix Figure 4-1: Percent of Xap isolates collected in 2011 and 2012 from commercial stone fruit orchards that are considered sensitive and less sensitive to 25 µg/ml oxytetracycline Figure 4-2: The effect of tree age, the oxytetracycline application method, the sample collection year, dormant copper use, and the number of oxytetracycline and copper applications made in the year of and year prior to sample collection on the percent of Xap isolates sensitive and less sensitive to 25 µg/ml oxytetracycline

10 x ACKNOWLEDGEMENTS This research was supported with funds from the State Horticultural Association of Pennsylvania (SHAP), the Pennsylvania Peach and Nectarine Marketing Board, the Penn State College of Agriculture, and the Penn State Department of Plant Pathology and Environmental Microbiology. I thank my major advisor, Dr. María del Mar Jiménez Gasco and the members of my committee, Drs. Kari A. Peter, Beth K. Gugino, Gary W. Moorman, and Mary Ann Bruns for their advice and time. I appreciate all of the support and help I have received from the members of the Penn State Department of Plant Pathology and Environmental Microbiology. I am also appreciative of the help I have received from Brian Lehman, Dr. Marcie Lehman and Kari Showers from Shippensburg University, Dr. Emily Pfuefer, Terry Salada, Dr. Henry Ngugi, Dr. Noemi Halbrendt, Teresa and Joanna Krawczyk, Kristina Gans, and the wonderful summer students at the Fruit Research and Extension Center. Finally, I am grateful for the love and encouragement I have received from my family and friends.

11 1 Chapter 1 Introduction BACTERIAL SPOT OF STONE FRUIT IN THE EASTERN UNITED STATES Disease Significance: Peach (Prunus persica (L.) Batsch) is the second most important fruit crop in the eastern United States after apple. In 2011, the eastern US peach crop was valued at over $220 million with significant production from the states of Alabama, Georgia, Maryland, Michigan, New Jersey, New York, North Carolina, Ohio, Pennsylvania, South Carolina, Virginia, and West Virginia, (National Agricultural Statistics Service). In the same year, over 17,000 ha of farm land were dedicated to the production of peaches in the eastern US. Peach production, however, is severely limited by bacterial spot of stone fruit, caused by Xanthomonas arboricola pv. pruni (Smith) Vauterin et al. (Xap). Bacterial spot was first described by F.E. Smith in 1903 on Japanese plum in a Michigan fruit orchard (Smith, 1903) and in Europe in 1920 by Italian scientists (Battilani et al., 1999). Considered the most important bacterial disease of peach and nectarine in the eastern US, bacterial spot epidemics are especially severe in the southeastern US and the mid-atlantic regions where the weather is warm, wet, and conducive to rapid disease development. For example, in 2005 bacterial spot reduced Georgia s peach crop value by 15%; a loss of almost $5 million (Buttner et al., 2002). Such economic losses are not uncommon in New Jersey and Pennsylvania where 100% fruit loss has been observed on highly susceptible cultivars in years where weather conditions favored bacterial spot development.

12 2 Symptoms and Disease Development: Symptoms of bacterial spot occur on leaves, twigs, and fruit. On leaves, angular, vein delimited lesions at the leaf tip, mid-rib, and/or along the leaf margin occur from early spring to fall (Fig. 1-1A) (Zehr et al., 1996). Initially, foliar lesions appear water-soaked (Fig. 1-1B) but eventually these darken in color and the centers of lesions abscise from the leaf, developing a shot hole appearance. Young leaves that are partially expanded are most susceptible to the development of symptoms as bacteria infested water may become trapped in the leaf, prolonging the leaf wetness period. In contrast, young leaves that have not expanded are not susceptible to bacterial spot because the leaf cells are too tightly packed together to allow the establishment of bacteria inside of the leaf. Premature leaf drop and leaf yellowing is common, effectively defoliating the tree and reducing the overall photosynthetic competence of the remaining leaves when disease severity is high (Fig. 1-3A). Severe premature defoliation reduces fruit quality and size due to an increase risk of fruit sun burn and poor nutritional uptake (Fig. 1-1C). Crop loss also results from severely damaged fruit associated with bacterial spot symptoms. On fruit, the earliest lesions occur about three weeks after petal fall (Ritchie, 1995). Initially, lesions appear water-soaked and eventually become dark-brown as they enlarge and age. Early season fruit infections that occur before pit hardening develop lesions that extend deep into the fruit (Fig. 1-2A). Late season lesions that develop after pit hardening are shallow and may cause the skin to crack if the lesions coalesce (Fig. 1-2B) (Ritchie, 1995). Bacterial spot lesions often favor secondary infection by the brown rot fungus, Monilinia fructicola, as well as other pre- and post-harvest fungal rots (Fig. 1-2C). Twig symptoms consist of cankers that initially appear water soaked and eventually enlarge, cracking the surface of the bark (Fig. 1-3B).

13 3 A B C D Fig Lesions on peach and nectarine leaves. A) Characteristic symptoms of bacterial spot including yellowing and angular lesions; B) New bacterial spot lesions that appear water soaked; C) Characteristic symptoms of nitrogen deficiency; D) Injury caused by copper.

14 4 A B C Fig Symptoms on peach fruit. A) Early season infection on peach occurs before pit hardening. Lesions extend deep into the fruit; B) Late season lesions develop after pit hardening, are shallow, and may cause the peach skin to crack when coalesced; C) Bacterial spot lesions favor secondary infection by the brown rot fungus, Monilinia fructicola.

15 5 A B Fig. 3. Symptoms of bacterial spot of peach. A) Severe premature defoliation of a peach tree puts fruit in risk of sunburn and premature fruit drop; B) Bacterial spot canker lacking vegetative growth.

16 6 Bacterial spot of stone fruit is a polycyclic disease with many secondary disease cycles that occur throughout the growing season that are highly dependent upon temperature and leaf wetness (Zehr et al., 1996). Bacteria overwinter in cankers on twigs that are first visible during bloom (Ritchie, 1995). Although cankers are the primary source of inoculum in the spring, bacteria may also overwinter in infected buds and leaf scars on the surface of the tree and in infected leaves on the soil surface below the tree (Ritchie, 1995; Zaccardelli et al., 1998). Under certain conditions, Xap can survive as an epiphyte on hosts and non-hosts for several weeks without causing disease since the bacteria have been isolated from symptomless plant tissue (Shepard and Zehr, 1994; Battilani et al., 1999; Zehr et al., 1996). Bacteria are spread from cankers by wind-driven rain to leaves and fruit (Battilani et al., 1999; Ritchie, 1995). Disease incidence and severity is highest on leaves and fruit located around cankers. Foliar infections provide additional inoculum for secondary infections of leaves and fruit. New infections are favored by wet weather between 14 C and 30 C, but become less frequent as temperatures rise above 30 C and the environment dries (Battilani et al., 1999; Zehr et al., 1996). Causal Organism: Xanthomonas arboricola pv. pruni (Xap) is the causal agent of bacterial spot of Prunus species. Susceptible hosts include peach, nectarine, Japanese plum, apricot, the plum-apricot hybrids (e.g., aprium, pluot, plumcot, and apriplum), and almond (Ritchie, 1995). Xap is a Gammaproteobacterium belonging to the genus Xanthomonas and is a Gram-negative, rod shaped, aerobic, flagellated bacterium with a single polar flagellum (Buttner et al., 2002). It forms yellow colonies in culture due to the xanthomonadin pigment (Starr et al., 1977; Starr, 1981) that are also glossy in appearance

17 7 because of xanthan gum, an exopolysaccharide (Corey and Starr, 1957; Jansson et al., 1975). The species X. arboricola consists of the pathovars corylina (causing bacterial hazelnut blight), fragariae (causing bacterial leaf blight of strawberry), juglandis (causing walnut blight), poinsetticola type C strains, populi (causing bacterial canker of poplar), celebensis (causing wilt of banana), and pruni (causing bacterial spot of stone fruits) (Vauterin et al., 1995). Originally known as Xanthomonas pruni, the species has undergone numerous reclassifications. In the mid 1990 s, it was reclassified from Xanthomonas campestris pv. pruni to Xanthomonas arboricola pv. pruni (Vauterin et al., 1995). Found in all representative strains from around the world, including the U.S., Xap harbors a 41-kb ( bp) plasmid, pxap41. Unique to this pathovar, this plasmid is the only one found in Xap with an estimated four copies per cell which is considered a low copy number. This plasmid is stable within Xap since attempts at plasmid curing in the laboratory have failed (Pothier et al., 2011). Genes conferring resistance to copper and streptomycin have not been found on this plasmid despite previous reports of resistance determinants carried by plasmids in other xanthomonads (Stall et al., 1986; Minsavage et al., 1990). This plasmid has a mosaic structure with genes worth noting encoding proteins presumably associated with virulence, including the genes xope3, mltb, and xope2. The gene xope3 encodes a protein that is a member of the HopX/AvrPphE effector family found in a wide range of bacteria. The gene mltb, located nearby, encodes for a type III secretion helper protein (Moreira et al., 2010), orthologs of which can also be found in many plant pathogenic bacteria. Because these two genes are usually located on the chromosome of other xanthomonads (Noël et al., 2003), it has been hypothesized that this 7-kb region has undergone a recent chromosome-plasmid DNA exchange through

18 8 horizontal gene transfer (Moreira et al., 2010). The gene xope2, thought to encode a protein associated with localization in plant cells (Thieme et al., 2007), is conveniently located next to a transposase gene. This also suggests recent acquisition though horizontal gene transfer (Moreira et al., 2010). A conservation of these genes among xanthomonads suggests that they contribute to virulence and are necessary for pathogenicity. Overall, there is very little genetic diversity among strains of Xap from different geographic locations and different hosts, suggesting that Xap is a relatively new plant pathogen (Zaccardelli et al., 1999). A worldwide collection of 109 Xap strains were studied using amplified fragment length polymorphism (AFLP) markers. This study showed that Xap populations have the highest diversity in North America compared to the low diversity of European populations. Bacterial populations from Italy and France are nearly genetically identical but are different from populations in North America (Zaccardelli et al., 1999). The low diversity found in Europe suggests that the population has undergone a recent bottleneck as a result of the introduction of the pathogen aided by humans (Brannen, 2006). The geographical distribution of bacterial spot is limited to where stone fruit are grown and in places where the environment favors disease development. Disease Management: There are few effective management strategies for bacterial spot in the eastern United States. The first is to plant less susceptible stone fruit cultivars. Unfortunately, no cultivar is completely resistant to bacterial spot and even the least susceptible cultivars exhibit symptoms and potential yield loss when environmental conditions are conducive to rapid disease development. Moreover, consumer preference for highly susceptible stone fruit cultivars forces growers to abandon this management

19 9 strategy altogether. Cultural practices such as proper site selection for well-draining soil, weed and nutrient management, and pruning to increase air flow within the tree canopy as well as to remove overwintering cankers may reduce the incidence and severity of bacterial spot but are often labor intensive, expensive, and overlooked management strategies (Shepard et al., 1999; Zehr et al., 1996). Therefore, bacterial spot is generally managed with repeated applications of bactericides including copper compounds and the antibiotic oxytetracycline. Copper compounds have been used as dormant sprays (i.e., applications made while the tree is dormant) providing a prophylactic protection of trees against infection by Xap and reducing the inoculum that may remain on the surface of the tree before the bacteria overwinter in infected leaf scars in the fall as well as when bacteria become active in the spring (Ritchie, 1995; Ngugi et al., 2009). Copper compounds have also been used as cover sprays (i.e., applications made during the growing season) to reduce bacterial spot symptoms. However, copper at high rates is phytotoxic often resulting in leaf discoloration, curling, and premature defoliation (Fig. 1-9) (Lalancette and McFarland, 2007). To a lesser extent, lime sulfur has been used to manage bacterial spot on less susceptible stone fruit cultivars when disease pressure is low but often leaves a persistent powdery white (and often smelly) residue on the surface of the fruit and may also be phytotoxic to leaves if applied under certain environmental conditions (J. Travis, personal communication). The antibiotic oxytetracycline has been used with significant disease suppression but it is registered for use on peaches and nectarines only; however, it is likely used on other susceptible stone fruit in Pennsylvania as well (personal communication with multiple Pennsylvania growers). Applied at 7 to 14 day intervals, up to 10 applications

20 10 (personal communication with fruit tree growers) may be made per season depending on the cultivar susceptibility and date of harvest. USE OF ANTIBIOTICS IN AGRICULTURE Antibiotics, substances produced by one microorganism that inhibit the growth of or kill other microorganisms, have been used in plant agriculture since the 1950 s. Used to manage diseases of valuable crops and ornamental plants caused by bacteria and phytoplasmas, the antibiotics used in plant production make up a small percentage of the overall antibiotics used in agriculture and in general, the United States. Few antibiotics are registered for use on plants and include streptomycin, an aminoglycoside antibiotic; oxytetracycline, a tetracycline antibiotic; gentamicin, an aminoglycoside antibiotic; oxolinic acid, a synthetic quinolone antibiotic; and kasugamycin, an aminoglycoside antibiotic (McManus et al., 2002). Antibiotics tend to be more expensive than metal-based bactericides such as copper and sulfur; however, they are effective at low rates and are rarely phytotoxic. The greatest use of antibiotics in plant agriculture has been to manage fire blight (caused by Erwinia amylovora) of apple and pear (Psallidas and Tsiantos, 2000). Due to issues with antibiotic resistance, streptomycin, oxytetracycline, and most recently, kasugamycin, have all been used to manage this disease of pome fruit. The antibiotic oxytetracycline is the only registered antibiotic for the management of bacterial spot of stone fruit in the U.S. and is marketed as Mycoshield or Fireline. It is formulated as an oxytetracycline calcium complex, dissolved in water, and applied as a foliar spray with a boom sprayer as a fine mist. Discovered in 1948 and first registered as

21 11 a pesticide in 1974, oxytetracycline is produced by the actinomycete Streptomyces rimosus (Chropra and Roberts, 2001). This antibiotic prevents the association of the aminoacyltrna with the ribosome, effectively halting protein synthesis. Oxytetracycline is considered a bacteriostat because it only prevents bacterial growth, likely because its association with the ribosome is reversible. In addition to that, this antibiotic is a protectant with no systemic activity within the leaves which limits its effectiveness to those bacteria that remain epiphytic (i.e., bacteria that have not yet invaded the leaf) (Christiano, 2010). In contrast, the antibiotic streptomycin, used to manage fire blight of pome fruit, is locally systemic within the nectaries of open flowers. Streptomycin applied 24 hours before or after inoculation with E. amylovora will prevent blossom infection (Gouk et al., 1999). Like most foliar applied agrichemicals, oxytetracycline is susceptible to rapid breakdown after application due to UV light exposure and wash off due to rainfall. According to Christiano et al. (2010), oxytetracycline is degraded to ineffective levels in less than two days under sunny, dry conditions and in only two minutes in a heavy precipitation event. According to the Environmental Protection Agency (EPA), oxytetracycline has a low degree of toxicity associated with environmental exposure to humans, birds, fish, and honey bees and the risk associated from eating a piece of fruit treated with oxytetracycline is considered negligible (EPA R.E.D. FACTS). ANTIBIOTIC RESISTANCE Antibiotic resistance is not a new phenomenon and actually predates the regular use of antibiotics by humans. Soil dwelling fungi and bacteria that naturally produce antibiotics

22 12 often harbor genes conferring resistance to their own antibiotics (Martin and Liras, 1989; Hopwood, 2007; Tahlan et al., 2007). These natural antibiotics exert a negative selection pressure on neighboring organisms. Therefore, the genes conferring resistance to antibiotics enhance the fitness of bacteria living in the environment by providing tolerance to these poisonous compounds (Allen et al., 2010; Poole, 2005). Evidence of antibiotic resistance and conjugative plasmids exists in bacteria collected before the beginning of the antibiotic era in the mid-twentieth century (Smith, 1967; Hughes and Datta, 1983; Houndt and Ochman, 2000). In addition to that, plasmids carrying resistance genes extracted from pre-antibiotic era bacteria fall into the same incompatibility group as those more recently recovered (Hughes and Datta, 1983). Furthermore, transposons carrying mercury resistance genes recovered from bacteria found in ancient permafrost are related to those more recently found in bacterial pathogens (Mindlin et al., 2001, 2005). Therefore, it is not surprising to find antibiotic resistance genes in places where antibiotics are not regularly used or applied, including antibiotic resistant Salmonella and Shigella species isolated from humans living in remote regions of Nepal where antibiotics are rarely, if ever, used (Walson et al., 2001). The same was true of residents in an isolated area of Bolivia where high levels of antibiotic resistant E. coli were found (Bartoloni et al., 2004). In each case, these antibiotic resistance genes were similar to those found in environments regularly exposed to antibiotics, demonstrating that these genes persist in the environment without an apparent selection pressure (i.e., regular antibiotic use) (Allen et al., 2010). Even though antibiotic resistance genes are common in natural environments, humans and the use of antibiotics in medicine and agriculture have undoubtedly increased the frequency of antibiotic resistant bacteria in the environment.

23 13 Mechanisms of Resistance: There are multiple mechanisms by which bacteria overcome the toxic effects of antibiotics. The first is to reduce the buildup of the antibiotic within the cell. A bacterium may do this by altering the permeability of the membrane, preventing the antibiotic from entering in great quantities or by actively removing the antibiotic from the cell once inside with efflux pumps. Because the antibiotic works on a specific target within the bacterial cell, the bacterium may alter this target through a mutation rendering the antibiotic ineffective against the modified target (i.e., target alteration). An antibiotic may be broken down within the cell by intracellular enzymes through a process called enzymatic detoxification (Depardieu et al., 2007). Finally, the antibiotic may bypass its target through a shift in metabolism (Agrios, 2005). Multiple mechanisms may exist within the same bacterium resulting in multidrug resistance. Whatever the mechanism, antibiotic resistance is often costly for the bacterium and is associated with a loss of fitness including reduced growth rates in the absence of antibiotics (Schulz zur Wiesch et al., 2010). Nevertheless, these antibiotic resistant bacteria can persist in an environment where they have a competitive advantage over bacteria that lack antibiotic resistance, such as an orchard receiving repeated antibiotic applications. Bacteria may become resistant to an antibiotic after a genetic mutation or after acquiring a mobile genetic element such as a transposon or plasmid harboring an existing resistance gene (Depardieu et al., 2007). Tetracycline Resistance: Tetracycline (i.e., oxytetracycline) resistance was first found in the 1950 s, soon after tetracycline antibiotics were discovered (Roberts, 2012). There are currently 43 different tetracycline resistance genes that confer resistance to these antibiotics through four different mechanisms. The chief resistance mechanism, conferred

24 14 by 27 different genes [tet(a), tet(b), tet(c), tet(d), tet(e), tet(g), tet(h), tet(j), tet(v), tet(y), tet(z), tet(30), tet(31), tet(33), tet(39), tet(41), tet(k), tet(l), tet(38), teta(p), tet(40), otr(b), otr(c), tcr, tet(42), tet(35), and tet(43)], is an energy-dependent efflux pump. This protein is located in the membrane and exports tetracycline antibiotics from the cell. It reduces the concentration of the antibiotic within the cell so that most of the ribosomes maintain their function. This is the most common mechanism found in Gram-negative bacteria (Pringle et al., 2007; Spaunaric et al., 2005) and in fact, the tet(a), tet(b), tet(c), tet(d), tet(e), tet(g), and tet(h) genes are limited to Gram-negative bacteria (Roberts, 2012). Moreover, the tet(b) gene has the largest host range of tet genes associated with Gram-negative bacteria (Chopra and Roberts, 2001). A total of 11 genes [tet(m), tet(o), tet(s), tet(w), tet(32), tet(q), tet(t), tet(36), otr(a), tetb(p), and tet(44)] code for ribosomal protection proteins. These cytoplasmic proteins force bound tetracycline to release it from the primary binding site on the ribosome, restoring the function of the ribosome (Connell et al., 2003a; Connell et al., 2003b). Inactivating enzymes are encoded by 3 genes [tet(x), tet(37), and tet(34)]. These enzymes, such as NADP-dependent monooxygenase, break down tetracycline antibiotics within the bacterial cell (Di Francesco et al., 2008). In addition, these three genes are limited to Gram-negative bacteria and in most cases are accompanied by genes coding for efflux and/or ribosomal protection proteins. A single gene, tet(u), conferring a low level of resistance compared to other resistance genes, has been found with an, as yet, unknown resistance mechanism. Bacteria may carry many copies of the same gene, two or more different tet genes with the same resistance mechanism, or two or more different tet genes with varying mechanisms of resistance (Nonaka et al., 2005). In addition, mosaic tet genes exist among those genes conferring

25 15 resistance through ribosomal protection proteins with two or more regions of known tet genes, essentially forming a hybrid of tet genes (Roberts, 2012). Although mutations altering the 16S rrna and conferring resistance to tetracycline antibiotics have been identified in a few bacteria, tetracycline resistance primarily occurs through the acquisition of genes on conjugative plasmids, mobilizable plasmids, nonconjugative plasmids, transposons, and conjugative transposons. Conjugation is the process through which two bacterial cells pass genetic material from one to the other. Conjugative plasmids differ from mobilizable plasmids in that the latter lacks the DNA to form a mating pair during conjugation (i.e., tra genes) but still contains the genetic material to initiate the start site of replication (i.e., orit genes). Nonconjugative plasmids lack the DNA material for conjugation and require an additional mobile element in the same host for the horizontal transfer of the plasmid. Transposons, often referred to as jumping genes, are small mobile pieces of DNA that also carry genes for transposition (i.e., movement from one location in the genome to another). Conjugative transposons form circular intermediates during conjugation and carry all the necessary genes for conjugation. In addition, tet genes associated with conjugative transposons are more likely to move among unrelated bacteria because they lack incompatibility systems like many plasmids (Nonaka and Suzuki, 2002). Incompatibility refers to the instance when a plasmid (conjugative or mobilizable) cannot be transferred to a host cell that already harbors a plasmid with the same origin of replication (Fournier et al., 2006). This ultimately reduces the host range of the tet genes associated with these plasmids. In addition, mobile elements often carry additional genes conferring resistance to one or more antibiotics or heavy metals in many combinations that may be lost or added at any time and are the reason these

26 16 genes are readily able to move within and among bacterial communities and environments (Chopra and Roberts, 2001; Pasquali et al., 2004). For example, the tet genes coding efflux proteins are most often found on plasmids while those that code for ribosomal protection proteins are most commonly associated with conjugative transposons (Tauch et al., 2002; Recchia and Hall, 2005). In addition, transposons carrying tetracycline resistance genes are similar, if not identical, in tetracycline resistant bacteria recovered from the environment, animals, or humans (Roberts, 1996). Overall, the transfer of resistance genes from a donor to a host has been well documented (Roberts, 2012). Tetracycline resistance or any other antibiotic resistance, however, has not been found or investigated in Xap (McManus et al., 2002) and a survey of the literature, as well as a search of an online database of resistance genes ( =O&term=xanthomonas&field=af&) supports this as well as that tetracycline resistance has not been reported in any other xanthomonad. BACTERIAL DIVERSITY OF THE PHYLLOSPHERE A single species of bacteria rarely inhabits a host or environment alone. Most often, many species of bacteria form aggregates and comprise a diverse community of microorganisms (Lindow, 2002; Morris et al., 1997; Morris et al., 1998). Although many fungi, yeasts, algae, protozoa, and nematodes reside in the phyllosphere, bacteria, especially pigmented bacteria, make up the largest part of this community (Lindow and Brandl, 2003). Historically, pathogens get most of the attention among scientists and researchers, but more recently epiphytic bacteria have been receiving more attention for

27 17 the potential roles they play in their respective environments. These bacteria may benefit the host by providing nutrients or protecting it from harmful pathogens (Antonio et al., 1999). For example, research on probiotics in humans and livestock has shown that these beneficial bacteria in the gut microbiome may improve overall health and even prevent disease in their hosts (Kalliomäki et al., 2001). Epiphytic bacteria (i.e., bacteria living on the surface of leaves) have also been used to manage diseases in plants. For example, Pseudomonas fluorescens A506 has been used to manage fire blight of apple and pear by colonizing the pistil of the flower and therefore limiting the resources (e.g., nutrients and space) available to E. amylovora (Wilson and Lindow, 1993). This biocontrol agent has also been used to prevent frost damage on apple and pear flowers by preventing ice nucleation (Lindow, 1993). Many Bacillus spp. have been used with great success as biocontrol agents in agricultural settings including Bacillus thuringiensis as a bioinsecticide (Powell and Jutsum, 1993), Bacillus subtilis, Kodiak, that stimulates plant growth and reduces the incidence of diseases caused by Fusarium and Rhizoctonia (Turner and Backman, 1991; Backman et al., 1994), and Bacillus cereus UW85 that produces antibiotics to prevent damping off of alfalfa (Handelsman et al., 1990). In addition to these beneficial roles as disease reducers and plant growth promoters, these epiphytic bacteria also provided their pathogenic neighbors with an extra source of genetic material, including antibiotic resistance genes. Often epiphytic and pathogenic bacteria in the same environment have the same resistance genes and mobile elements (e.g., plasmids and transposons) (Chopra and Roberts, 2001). In some cases, these nonpathogenic bacteria carry more antibiotic resistance genes and acquire them before their pathogenic neighbors (Roberts, 1989; Roberts et al., 1999). A survey of Michigan apple

28 18 orchards for tetracycline resistance genes, many with streptomycin resistant E. amylovora causing fire blight, showed that tetracycline resistant bacterial epiphytes were recovered from apple blossoms and leaves in sites that had never received oxytetracycline applications (Schnabel and Jones, 1999). In addition to that, a greater number of tetracycline resistant bacteria were recovered from orchards where oxytetracycline had been used for a longer time, suggesting a selection for tetracycline resistant bacteria (Schnabel and Jones, 1999). The majority of bacterial epiphytes were Pantoea spp. carrying the tet(b) gene and Pseudomonas spp. carrying tet(a), tet(c), and tet(g) genes. These genes were most often found associated with transposable elements; however, attempts to transfer tetracycline resistance genes from epiphytic bacteria to E.coli through conjugation in the lab failed (Schnabel and Jones, 1999). Kasugamycin resistant epiphytic bacteria were also recovered from apple blossoms and leaves in another survey of Michigan apple orchards (McGhee and Sundin, 2010). These bacteria included Pantoea agglomerans, Pseudomonas graminis, Pseudomonas syringae, and Stenotrophomonas species. It is unknown if these kasugamycin resistance genes reside on a transposable element; however, the majority of these epiphytic bacteria were also resistant to streptomycin. Finally, the most probable source of streptomycin resistance genes in resistant isolates of E. amylovora recovered from Michigan apple orchards is P. agglomerans, a common epiphytic bacterium found in apple orchards, since it carried the same transposable elements, plasmid pea34 with transposon Tn5393, that was found in streptomycin resistant isolates of E. amylovora (Schnabel and Jones, 1999; Sobiczewski et al., 1991). Laboratory studies have also demonstrated a high incidence of transmission of pea34 from P. agglomerans to E. amylovora (Chiou and Jones, 1993; Jones and Schnable,

29 ). The phyllosphere is thought to be a place of prolific genetic exchange and diversity due to the close association of many species of bacteria and the high rates of plasmid transfer (Lindow and Leveau, 2002; Normander et al., 1998). RESEARCH GOALS AND OBJECTIVES The overall goal of this research is to gain a better understanding of the effects of antibiotic use on bacterial communities, including identifying sources of antibiotic resistance genes, establishing oxytetracycline sensitivity in Xap isolates, and characterizing management strategies that diminish oxytetracycline sensitivity and increase the incidence of antibiotic resistance genes in bacteria inhabiting stone fruit orchards. The objectives of Chapter 2 were to identify populations of epiphytic bacteria, including those resistant to oxytetracycline, and to determine management factors influencing the distribution of tetracycline resistance genes among commercial stone fruit orchards in PA. Bacterial epiphytes were recovered from leaves collected from orchards. They were identified and screened for the presence of teta, tetb, and tetc. Management factors were compared to the incidence of tetracycline resistance genes among bacterial epiphytes. It was hypothesized that oxytetracycline use is an important factor influencing the incidence of tetracycline resistance genes among the sampled orchards. The objectives of Chapter 3 were to monitor the effects of specific bactericides on not only foliar and fruit disease severity, but on the incidence of bacteria carrying tetracycline resistance genes as well. Bactericide programs were evaluated for efficacy by visually rating disease severity on leaves and fruit. To determine the incidence of resistance

30 20 genes among bactericide programs, epiphytic bacteria were isolated from leaves collected from the product evaluation site and were screened for the presence of teta, tetb, and tetc. It was hypothesized that trees treated with oxytetracycline would have a larger percentage of the bacterial community positive for the presence of tetracycline resistance genes. Because tetracycline resistance genes were found among epiphytic bacteria recovered from commercial orchards in Chapter 2, the objectives of Chapter 4 were to evaluate the incidence of tetracycline resistance genes in Xap and determine the current levels of oxytetracycline sensitivity. Symptomatic leaves were collected from commercial orchards. Xap was isolated from the leaves and screened for oxytetracycline sensitivity and the presence of tetb and tetc, the most common tetracycline resistance genes found in Chapter 2. Orchard management practices were evaluated to determine what factors, if any, were related to oxytetracycline sensitivity in Xap. It was hypothesized that Xap isolates collected from orchards where oxytetracycline had been used less or not at all would be more sensitive to oxytetracycline than isolates exposed to greater amounts of the antibiotic. JUSTIFICATION Bacterial spot is the most important bacterial disease of peach in the eastern United States where yield loss due to this disease is common. Because of that, stone fruit growers rely heavily upon the antibiotic oxytetracycline to mitigate disease symptoms and reduce yield loss. Repeated antibiotic use, such as the frequent and repeated applications made in Pennsylvania stone fruit orchards, exerts a strong selective pressure on bacterial populations to support the growth of antibiotic resistant bacteria. Despite the substantial

31 21 use of the antibiotic oxytetracycline, bacterial spot symptoms may remain severe and yield loss extreme on highly susceptible cultivars. Therefore, concern among Pennsylvania stone fruit growers over the development of antibiotic resistance in the causal agent of bacterial spot has been expressed. Repeated antibiotic applications not only influence pathogenic bacteria such as Xap, but epiphytic bacteria as well. Xap is a single resident in a complex phyllosphere. Many other microorganisms, including epiphytic bacteria (the primary focus of this research) reside on the surface of leaves as well. It is likely, but unknown what important ecological role they fill and that repeated antibiotic applications not only reduce diversity of these epiphytic bacteria but apply a strong selective pressure on these bacteria to acquire and harbor antibiotic resistance genes that can be potentially horizontally transferred to Xap. Tetracycline resistant Xap populations would have a devastating effect on stone fruit production in the eastern United States since it is not only the sole antibiotic registered for use on stone fruit, it is the most effective chemical bactericide for bacterial spot management. Therefore, it is necessary to identify these nonpathogenic bacteria and monitor the effect of repeated antibiotic use on the diversity of these bacterial populations, including the frequency of potentially transferrable tetracycline resistance genes. In addition, recent consumer demand for antibiotic free produce has put pressure on farmers, including fruit tree growers, to reduce antibiotic use in agriculture. This growing fear of antibiotics has already resulted in the phasing-out of the antibiotic oxytetracycline in organic stone fruit production, the development which has been greatly hindered due to the severity of bacterial spot in the eastern United States. This mounting demand also threatens the use of antibiotics in conventional tree fruit production since the

32 22 use of antibiotics in agriculture is often seen as needless and a perpetual threat to the effectiveness of clinical antibiotic use even though the administration of tetracycline antibiotics has been declining and the antibiotic oxytetracycline is currently not used in human medicine in the United States. Nevertheless, there is a genuine need to evaluate alternative bactericides and management methods not only on their ability to reduce the incidence and severity of bacterial spot but on the impact of the epiphytic bacterial community as well.

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