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 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
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 iv
the incidence of tetracycline resistance genes in epiphytic bacteria significantly differed (P
> 0.0001) among the sampled commercial orchards, this was not related to oxytetracycline use (P = 0.0855). 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 > 0.0001) 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 > 0.0001). 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.
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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 vi
Chapter 4 Oxytetracycline sensitivity of Xanthomonas arboricola pv. pruni isolates collected from Pennsylvania stone fruit orchards ...... 101
Abstract ...... 101 Introduction ...... 101 Materials and Methods ...... 104 Results...... 107 Discussion ...... 110 Literature Cited ...... 117
Chapter 5 Discussion ...... 126
Literature Cited ...... 134
Appendix ...... 139
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 2013 ...... 91
Table 3-4: Factors associated with the number of colony forming units per gram of peach leaf tissue collected in 2014 ...... 92
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 ...... 123
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 tetR 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 2014 ...... 98
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 ...... 100 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 ...... 124
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 ...... 125
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.
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. 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). 3
A B C D Fig. 1-1. 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. 4
A B C
Fig. 1-2. 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.
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. 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 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 (41 102 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 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 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 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 11 a pesticide in 1974, oxytetracycline is produced by the actinomycete Streptomyces rimosus
(Chropra and Roberts, 2001). This antibiotic prevents the association of the aminoacyl- tRNA 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 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. 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 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 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 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 (http://ardb.cbcb.umd.edu/cgi/search.cgi?db=R&and0
=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 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 bio- insecticide (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 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, 19
2000). 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 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 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 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.
23
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34
Chapter 2
Tetracycline resistance genes in epiphytic bacteria collected from Pennsylvania
stone fruit orchards
ABSTRACT
In this study, epiphytic bacteria collected from commercial orchards were identified to genus using 16S ribosomal sequences. The incidence of tetracycline resistance genes was determined and the management factors associated with the distribution of tetracycline resistance genes were evaluated. A total of eight bacterial genera were identified and included Pantoea (39.9%), Xanthomonas (31.9%), Pseudomonas (15.7%), Bacillus
(6.3%), Curtobacterium (2.9%), Staphylococcus (2.6%), Frondihabitans (0.6%), and
Rahnella (0.3%). Pantoea spp. were found in all eight orchards while Bacillus spp. were found in only the organic orchards. Tetracycline resistance genes, tetA, tetB, and tetC, were found in five of the eight sampled orchards. TetB was most commonly associated with
Pantoea spp. while tetC was most often found in Pseudomonas spp. The incidence of tetracycline resistance genes significantly differed (P > 0.0001) among the sampled orchards so multiple management factors were evaluated to determine the possible source of variability in incidence among the orchards. The number of oxytetracycline applications made in the year of and in the year prior to sample collection was not significantly associated with the incidence of resistance genes (P = 0.0855); however, tree age (P >
0.0001) and spray application method (i.e.: alternate row middle versus complete) (P =
0.0002) were significant factors influencing the distribution of tetracycline resistance 35 genes. A greater percentage of tetracycline resistance genes were recovered from old trees
(> 10 years) than young trees while trees that had been sprayed using the alternate row middle method supported a greater percentage of bacterial epiphytes positive for tetA, tetB, and tetC.
INTRODUCTION
Bacterial spot, caused by Xanthomonas arboricola pv. pruni (Xap), has become an increasingly difficult disease to manage in the north eastern United States. Warm and wet environmental conditions, conducive to bacterial spot development, regularly favor bacterial spot development on highly susceptible stone fruit cultivars (Zehr et al., 1996). In years when bacterial spot is particularly severe, even the most resistant cultivars exhibit symptoms of bacterial spot, including foliar yellowing and “shot hole” lesions, twig cankers, and yield limiting spots on fruit (Ritchie, 1995). Pathogenic Xap may also survive on the surface of the leaves as an epiphyte without any associated symptoms (Shepard and
Zehr, 1994; Battilani et al., 1999; Zehr et al., 1996). Due to the warm and humid environment in the north eastern U.S., symptoms of bacterial spot may be quite severe when the pathogen is left unmanaged.
Oxytetracycline, a tetracycline antibiotic, was first used as an agricultural pesticide in the 1970’s and is currently the only antibiotic registered for use on stone fruit for bacterial spot (Chropra and Roberts, 2001). Therefore, oxytetracycline has been one of the most popular antimicrobials used to manage bacterial spot in the Pennsylvania stone fruit industry despite its relatively high expense (McManus et al., 2002). Oxytetracycline is 36 formulated as an oxytetracycline calcium complex, dissolved in water, and applied as a foliar spray with a boom sprayer as a fine mist. The antibiotic prevents the association of the aminoacyl-tRNA with the ribosome, halting protein synthesis, and bacterial growth
(Chropra and Roberts, 2001). Due to the reversible association of the antibiotic with the ribosome, the lack of systemic activity within the leaves, and the rapid breakdown of the material by UV light and wash off by rainfall (Christiano, 2010), this antimicrobial must be regularly reapplied every 10 to 14 days throughout the growing season. Because of that, it may be sprayed up to 10 times per season depending on the cultivar and time of harvest.
Oxytetracycline does not specifically target Xap when applied to trees and will thus prevent the growth of any bacteria sensitive to it. It has been hypothesized that due to repeated applications of an antibiotic, the remaining bacterial community is enriched for those bacteria resistant to that antibiotic, either intrinsically, through random mutations, or through acquisition of antibiotic resistance genes (Levy and Marshall, 2004). Intrinsic resistance, or insensitivity, is the innate ability of bacteria to withstand the activity of an antimicrobial agent and is vertically inherited. While mutations that confer resistance to tetracycline antibiotics have been identified, tetracycline resistance primarily occurs through the acquisition of genes on mobile genetic elements, including conjugative, mobilizable, and nonconjugative plasmids as well as transposons (Chopra and Roberts,
2001). These nontarget bacteria, living in close association with the bacterial pathogen, are thought to harbor these resistance genes and then could potentially transfer them to the pathogen. In some cases, these nontarget bacteria carry more antibiotic resistance genes and acquire them before their pathogenic neighbors (Roberts, 1989; Roberts et al., 1999).
In fact, this scenario was hypothesized after streptomycin resistance in Erwinia amylovora 37
(the causal agent of fire blight in pome fruit) was found where resistant isolates of E. amylovora shared similar SmR genes as streptomycin resistant isolates of Pantoea agglomerans, a common orchard epiphyte (Chiou and Jones, 1993). Overall, the transfer of resistance genes from a donor to a host has been well documented (Roberts, 2012).
Because of the substantial antibiotic use and the persistent yield loss due to bacterial spot, many PA stone fruit growers have expressed concern over antibiotic resistance in
Xap. The risk of antibiotic resistance in Xap cannot be examined without evaluating the potential sources of resistance genes that may be acquired by Xap (McGhee and Sundin,
2011). Moreover, it is crucial to explore the effects of repeated antibiotic use on these closely associated epiphytic bacteria, including their diversity and the distribution of tetracycline resistance genes. Therefore, the objectives of this study were to monitor and identify populations of bacterial epiphytes, including those resistant to oxytetracycline, and to determine the incidence of tetracycline resistance genes among commercial stone fruit orchards in PA.
MATERIALS AND METHODS
Collection, isolation, and identification of epiphytic bacteria from commercial stone fruit orchards: Leaf samples were collected in 2012 and 2013 from eight commercial orchards located in Adams, Franklin, Lancaster, Chester, and Delaware Counties,
Pennsylvania. Two of the orchards had an organic certification while the remaining used conventional management practices. All leaf samples were taken within a period of two weeks and were collected from a total of 12 trees of a single cultivar in each orchard where 38 the leaves of four trees were bulked into a composite sample and processed together. Each orchard was sampled once within the two-week time period. Leaves were chosen randomly, regardless of symptoms, at a height of about 135 cm from all around the tree.
Trees were sampled in “V” formation, where 4 trees from each point were sampled.
Numerous cultivars of peach, nectarine, and apricot were included. At least 20 grams of leaves from each tree were collected into new paper bags and put on ice or chilled at 4C until taken to the lab and processed immediately.
Epiphytes were washed from the leaves. Twenty grams of each bulk sample were transferred into a sterile 500ml lidded jar containing 400ml of chilled 1M potassium phosphate buffer with a drop of Tween 20 (Polyoxyethylene(20)sorbitan monolaurate;
Agdia; Elkhart, IN). The leaves were shaken on a rotary arm shaker for 30 minutes before being sonicated for 5 minutes (McGhee and Sundin, 2011). A milliliter from each sample was taken to complete serial dilutions on fresh Kings B media (McGhee and Sundin, 2011) amended with 0, 10, and 25 µg/ml oxytetracycline (oxytetracycline dehydrate; Sigma-
Aldrich; St. Louis, MO). A total of 100ml of the rinsate was store at -80C for future evaluation. Plates were incubated at 22C for 96 hours in the dark. The bacterial colonies were counted and a subset of the colonies were randomly chosen from all of the concentrations of antibiotic amended media. Selected colonies were then transferred to nutrient broth (Schaad et al., 2001) and grown for 24 to 48 hours before being preserved at
-80° C in 25% sterile glycerol.
The selected bacterial isolates were identified to genus level through sequencing of the 16S rRNA. The frozen culture was used as the template and was introduced to each 25
µl reaction with a sterile toothpick. Choice Taq Mastermix (Denville Scientific; Holliston, 39
MA) was used. The forward primer 530F (5’-GTGCCAGCAGCCGCGG-3’) and the reverse primer 1494R (5’-TACGGCTACCTTGTTACGAC-3’) amplified a fragment sized
1kb (Neilan et al, 1985; Weisburg et al., 1991). The thermocycler parameters included a
112C heated lid and an initial denaturation of 96C for 10 minutes. After that, 34 cycles were completed of 96C denaturation for 30 seconds, 55C annealing for 1 minute, and 72C elongation for 30 seconds. The products were stored at 4C and run through 1.5% agarose gel in TRIS-Acetate-EDTA (TAE) buffer using EZ-Vision loading dye from AMRESCO
(Solon, OH). Gels were visualized under a KODAK gel imaging system. With each cycler run, a positive and negative control were included. PCR products were cleaned using
ExoSAP-IT (Affymetrix, Inc., Santa Clara, CA) and were prepared for sequencing at the
Genomics Core Facility of the Huck Institute of Life Sciences at Penn State University
(University Park, PA). Sequence analysis was completed using standard nucleotide blast
(BLASTN) from the National Center for Biotechnology Information
(http://blast.ncbi.nlm.nih.gov/ Blast.cgi). Only alignments with an E-value of 0.0 were accepted.
Oxytetracycline sensitivity of epiphytic bacteria: The minimum inhibitory concentration (MIC) of a selection of epiphytic bacteria to oxytetracycline was determined using the agar dilution method as previously described (Wiegand et al., 2008). For each isolate, 3 to 5 morphologically similar colonies from a fresh agar grown culture were selected using a sterile loop. The colonies were transferred to a sterile capped plastic tube containing 2ml sterile 0.1M potassium phosphate buffer. To ensure a similar number of bacterial cells was being used to determine the MIC, the turbidity of the vortexed suspension was assessed by measuring the absorbance. An absorbance between 0.08 and 40
0.13 (equal to 1x108 colony forming units per milliliter [cfu/ml]) at OD625 nm was achieved by adding more buffer or bacterial material. All bacterial suspensions were used within 30 minutes to ensure the accuracy of the measured turbidity. A final amount of 104 cfu’s was used to determine the MIC after 3 days of incubation at 26C. Sensitivity of each isolate was replicated 3 times. Growth comparable to the growth of bacteria on 0 µg/ml oxytetracycline was considered “growth” while light bacterial films and no visible growth were considered “no growth”. The MIC was considered the lowest concentration of oxytetracycline that inhibited the growth of the tested isolate. The MIC for several isolates of Pantoea, Pseudomonas, and Xanthomonas positive and negative for tet(A), tet(B), and tet(C) was determined on Kings B media amended with 10 concentrations of oxytetracycline (0, 50, 100, 150, 200, 250, 300, 350, 400, and 450 µg/ml).
Screening of tetracycline resistance genes of epiphytic bacteria: Bacterial epiphytes were screened for the presence of three previously described tetracycline resistance genes, tet(A), tet(B), and tet(C), using PCR and specific primers. These genes are among the most common genes found in Gram negative bacteria (Roberts, 2012). The frozen culture was used as the template and was introduced to each 25 µl reaction with a sterile toothpick. Choice Taq Mastermix (Denville Scientific; South Plainfield, NJ) was used. The tet(A)-F forward primer (5’-TTGGCATTCTGCATTCACTC-3’) and the reverse primer tet(A)-R (5’-GTATAGCTTGCCGGAAGTCG-3’) amplified a 494bp product (GenBank accession no.: X75761). Forward primer tet(B)-F (5’-
CAGTGCTGTTGTTGTCATTAA-3’) and reverse primer tet(B)-R (5’-
GCTTGAATACTGAGTGTTAA-3’) amplified a 571bp product (GenBank accession no.:
V00611). The tet(C)-F forward primer (5’-CTTGAGAGCCTTCAACCCAG-3’) and the 41 reverse primer tet(C)-R (5’-ATGGTCGTCATCTACCTGCC-3’) amplified a 418bp product (GenBank accession no.: J01749) (Ma et al., 2007). The thermocycler parameters included a 112C heated lid and an initial denaturation of 96C for 10 minutes. After that, 34 cycles were completed of 96C denaturation for 30 seconds, 55C annealing for 1 minute, and 72C elongation for 30 seconds. The PCR products were stored at 4C and run through
1.5% agarose gel in TAE buffer using EZ-Vision loading dye (AMRESCO; Solon, OH).
Gels were visualized under the KODAK gel imaging system. With each cycler run, a positive and negative control were included. Positive controls for tetB and tetC were isolates of previously screened Pantoea spp. and tetA was an isolate of Pseudomonas sp. recovered from the PSU Fruit Research and Extension Center. The negative control was a sensitive isolate of Xap. Bacteria that were considered “tetR positive” were positive for only one of the three tetracycline resistance genes assayed for in this study.
Statistical analysis for orchard management factors associated with the incidence of tetR positive bacteria: During sample collection, a survey on bacterial spot management practices as well as orchard characteristics was completed by the corresponding grower or farm manager. Data were collected on oxytetracycline use, application method, organic certification, and tree age. The number of oxytetracycline applications made in the year of and the year prior to sample collection were divided into 2 categories, 3 applications and less and greater than 3 applications. Tree age was divided into two age categories of less than 10 years and older than 10 years. There were two applications methods that included
“alternate row middle” where one side of the row of trees is sprayed every other application and “complete” where both sides of the tree are sprayed each application. When assessing the orchard management factors affecting the distribution of tetracycline resistance genes, 42 the data obtained for tetA, tetB, and tetC for 2012 and 2013 were combined. The data analyzed consisted of binary response variable where isolates negative for a resistance gene were marked “0” and those positive for a resistance genes were marked “1”. Initially, a
Pearson’s chi-square statistic was calculated using the FREQ procedure of SAS (version
9.2; SAS Institute Inc.). After that, the data were modeled with the maximum likelihood method using the GLIMMIX procedure of SAS and were evaluated based on the type III tests of fixed effects, similar to that used by Pfeufer and Ngugi (2012). This generalized linear mixed (GLM) model allowed for the evaluation of all the predictors of tetR incidence together. The GLIMMIX procedure in SAS may be used to model correlations where the distribution may not be normal and the response variable is binary. Model estimates and standard errors along with 95% confidence ratios were obtained with the lsmeans statement.
RESULTS
Bacterial identification: A total of 364 and 284 bacterial isolates were collected and saved from eight commercial orchards in 2012 and 2013, respectively. Of those, 352 isolates were identified to the genus level. A total of 8 genera were identified over the two years and included Pantoea (39.9%), Xanthomonas (31.9%), Pseudomonas (15.7%),
Bacillus (6.3%), Curtobacterium (2.9%), Staphylococcus (2.6%), Frondihabitans (0.6%), and Rahnella (0.3%). Species of Bacillus and Curtobacterium were more common among the organic orchards (orchards 7 and 8 in Fig. 2-1), whereas Pantoea spp., Xanthomonas 43 spp., and to a lesser extent, Pseudomonas spp. made up the largest percentage of bacteria classified from the conventional orchards (orchards 1 to 6 in Fig. 2-1).
Oxytetracycline sensitivity of bacterial epiphytes: Bacteria positive and negative for tetracycline resistance genes (tetA, tetB, and tetC) for the three most common genera of bacterial epiphytes, Pantoea, Psuedomonas, and Xanthomonas, were evaluated (Table
2-1). In all cases, tetR positive bacteria (those found to harbor tetA, tetB, or tetC) had a minimum inhibitory concentration (MIC) of greater than 450 µg/ml oxytetracycline. TetR negative (those found to lack tetA, tetB, or tetC) Pseudomonas spp. had a similar MIC while species of Pantoea and Xanthomonas negative for tetA, tetB, or tetC had MICs of less than 50 µg/ml oxytetracycline.
Tetracycline resistance genes in bacterial epiphytes: Bacterial colonies growing on 25 µg/ml oxytetracycline were recovered from all commercial orchards (Fig. 2-2). The three tetracycline resistance genes (tetA, tetB, or tetC) assayed for, however, were not found in all orchards. Bacteria carrying tetracycline resistance genes were recovered from media amended with 0, 10, and 25 µg/ml oxytetracycline. Using PCR and primers for tetA, tetB, and tetC, tetracycline resistance genes were found in only five of the eight sampled orchards. Overall, tetR genes were found in 13.0% of 684 isolates collected and screened in 2012 and 2013 (Fig. 2-3). The incidence of tetA, tetB, and tetC among orchards differed significantly (χ2 =160.22; P = <.0001) with frequency of isolation ranging from 0% in three of the orchards to greater than 44% of the isolates screened. TetB was the most common of the three tetR genes screened (8.6% of the total screened isolates), followed by tetC
(3.9%), and tetA (0.5%). TetB was found wherever tetR genes were found except orchard 44
8, where only tetA and tetC were found. No bacterial isolates were positive for more than one tetR gene.
Of the eight genera identified in the recovered bacteria, the three tetracycline resistance genes were only found in Pantoea, Pseudomonas, Xanthomonas and Rahnella
(Fig. 2-4). Only one isolate of Rahnella was recovered and the tetR gene found in that isolate was tetA. In Xanthomonas, tetC was found. In Pantoea and Pseudomonas, tetA, tetB, and tetC were found. TetB was most commonly found in isolates of Pantoea (37.6% of the 141 Pantoea isolates) compared to those of Pseudomonas (1.8% of the 55
Pseudomonas isolates). In contrast, tetC was most commonly found in isolates of
Pseudomonas (30.9% of the 55 isolates) compared to those of Pantoea (5.0% of the 141 isolates) and Xanthomonas (0.9% of 112 isolates).
Orchard factors and management practices associated with the distribution of tetracycline resistance genes in collected bacterial epiphytes: The distribution of tetracycline resistance genes significantly varied among orchards based on the Pearson chi- square statistic (χ2 = 160.47; P <0.0001) (Fig. 2-3). The number of oxytetracycline applications was a significant factor affecting the incidence of tetracycline resistance genes whereby 27.1% of isolates from orchards with more than three applications of the antibiotic were tetR positive compared with only 4.7% of isolates for orchards sprayed less frequently
(Χ2 = 67.36; P <0.0001; Fig. 2-5). This was not supported by the GLM model (odds ratio
= 0.544; 95% CI = 0.271 to 1.089; P = 0.0865 (Table 2-2). Tree age was significantly associated with the incidence of tetR positive bacterial epiphytes (Χ2 = 77.51, P < 0.0001).
A larger percentage of tetR positive bacteria were collected from older trees (28.6%) than younger trees (4.3%) (Fig. 2-4). The GLM model indicated that the isolates from younger 45 trees had a significantly lower likelihood of being tetR positive (odds ratio = 0.132; 95%
CI = 0.068 to 0.257; P < 0.0001) (Table 2-2). Bacterial epiphytes collected from conventionally managed orchards had a greater percentage of tetR positive bacteria than those collected from certified organic orchards (21.0% versus 1.1%; χ2 = 54.84; P <
0.0001) (Fig. 2-4). Lastly, bactericide application method (i.e.: “complete” application where both sides of the tree are applied with antibiotic each time the tree is sprayed versus
“alternate row middle” where every other side of the tree is applied with antibiotic each time the tree is sprayed) was a significant factor influencing the distribution of tetracycline resistance genes (χ2 = 24.96; P < 0.0001) (Fig. 2-5). Trees sprayed in an “alternate row middle” fashion had an increased incidence (21.7%) of tetracycline resistant bacteria than trees sprayed in a “complete” manner (8.0%). Based on the GLM model, epiphytes from orchards using the “alternate row middle” spray method were more than three times as likely to be tetR positive than epiphytes from orchards using the “complete” spray method
(i.e.: on both sides of the tree each time) (odds ratio = 3.119; 95% CI = 1.719 to 5.657; P
= 0.0002) (Table 2-2).
DISCUSSION
The identification of the bacterial epiphytes recovered from the commercial orchards showed that Pantoea spp. are ubiquitous among culturable bacteria, found in all sampled orchards. In general, Pantoea spp. have been isolated from a wide variety of environments including plants, soil, water, humans, and animals. Pantoea spp. are often found as epiphytes but more recently as emerging pathogens of valuable crops as well as 46 opportunistic pathogens of humans (Weinthal et al., 2007; Grimont and Grimont, 2005;
Delétoile et al., 2009). Pantoea spp. have also been recovered in apple orchards in similar studies (Schnabel and Jones, 1999; McGhee and Sundin, 2011, Yashiro and McManus,
2012). Although not recovered from all orchards, species of Pseudomonas and
Xanthomonas were also commonly identified. Pseudomonas spp. thrive in diverse niches and have been found as human and plant pathogens as well as bacterial epiphytes (Schnabel and Jones, 1999; McGhee and Sundin, 2011). In fact, Pseudomonas spp., more specifically
P. fluorescens A506, have been investigated and marketed for use as a biocontrol agent of fire blight (Stockwell and Stack, 2007). As previously mentioned, Xap and other species of Xanthomonas can survive as epiphytes so it comes as no surprise that it was found in most of the orchards, even where bacterial spot was not found (orchard 7 – Fig. 2-1)
(McGhee and Sundin, 2011; Shepard and Zehr, 1994; Battilani et al., 1999; Zehr et al.,
1996). Bacillus spp. were only recovered from the organic orchards (orchards 7 and 8 –
Fig. 2-1). This suggests that both growers used a Bacillus containing biofungicide
(marketed under the product names Serenade, Serenade Max, Serenade Optimum, etc.) to manage bacterial spot or some other disease or natural populations of the bacteria were able to thrive under a reduced spray program. Neither organic grower reported the application of such a product prior to sampling.
Although bacteria able to grow on 25 µg/ml oxytetracycline were recovered from all orchards, bacteria harboring the three tetracycline resistance genes assayed for were not
(Fig. 2-2, Fig. 2-3). First, this could be because those bacteria growing on 25 µg/ml were carrying a resistance gene not evaluated in this study. While tetA, tetB, and tetC are the most common tetracycline resistance genes among Gram negative bacteria (Schnabel and 47
Jones, 1999; Roberts, 2012), there are currently 40 additional tetracycline resistance genes not screened for in this study. Second, those bacteria could have a novel form of tetracycline resistance caused by, an as yet, uncharacterized resistance gene or, although rare, a mutation resulting in resistance (Nonaka et al., 2005). Finally, those bacteria could be intrinsically insensitive to oxytetracycline (Levy and Marshall, 2004). Bacteria carrying tetracycline resistance genes, in this study, were able to grow on high levels of oxytetracycline – greater than 450 µg/ml oxytetracycline compared to the field rate of 150
µg/ml used by growers (Table 2-1). Pseudomonas spp. lacking tetA, tetB, and tetC were able to grow at those elevated antibiotic levels while Pantoea spp. and Xanthomonas spp. lacking tetA, tetB, and tetC could not. This indicates that the tested Pseudomonas spp. possibly carried a determinant of resistance, like a resistance gene not screened for in this study.
Epiphytic bacteria positive for tetA, tetB, and/or tetC were recovered from 5 of the
8 sampled orchards and were not limited to the conventionally managed orchards. Both tetA and tetC were found in orchard 8 (Fig. 2-3), an organic orchard where no oxytetracycline had ever been used. This orchard was previously woodland, never used for farming, and cleared specifically for this organic peach block. This finding reaffirms the results of many previous studies where antibiotic resistance genes have been identified in places where antibiotics were never used (Smith, 1967; Houndt and Ochman, 2000;
Hughes and Datta, 1983; Mindlin et al., 2001, 2005; Walson et al., 2001; Bartoloni et al.,
2004).
The most common tetracycline resistance gene, of those evaluated in this study, was tetB (Fig. 2-3). Recovered from four of the orchards, tetB confers the most common 48 form of tetracycline resistance among Gram negative bacteria – an energy dependent efflux pump (Pringle et al., 2007; Spaunaric et al., 2005). Although tetB is limited to Gram- negative bacteria (Roberts, 2012), this gene has the largest host range of tet genes associated with Gram-negative bacteria (Chopra and Roberts, 2001). TetB was found almost exclusively in Pantoea spp. with few exceptions in Pseudomonas spp. (Fig. 2-4).
TetC was recovered from only 2 of the sampled orchards (Fig.2-3) and was most commonly recovered from Pseudomonas spp. The association of Pseudomonas spp. with tetC and
Pantoea spp. with tetB is consistent with the results of the study conducted by Schnabel and Jones in Michigan apple orchards (1999). However, in that study, they also found that
Pseudomonas spp. were associated with tetA while in our study, the incidence of tetA was small and was associated with three genera (Fig 2-4).
It was hypothesized that the variability in tetR incidence was due to the different management strategies and orchard factors employed by growers. Management and individual orchard factors differed greatly among the surveyed orchards so it was no surprise that incidence of tetracycline resistance genes differed significantly (P < 0.0001) among the sampled orchards (Fig. 2-3). It was not expected; however, that oxytetracycline use was not a significant factor influencing the incidence of tetR genes (F = 2.97; P =
0.0855) (Table 2-2). Even though conventional orchards having a significantly higher percentage of tetracycline resistance genes than organic orchards (P < 0.0001), not all conventional orchards used oxytetracycline to manage bacterial spot. The number of oxytetracycline applications varied greatly among the sampled orchards where the organic orchards and a conventional orchard sprayed no oxytetracycline, one grower applied the antibiotic 13 times, and the others applied the bactericide somewhere in between during 49 the year of and the year prior to sample collection. Oxytetracycline has typically been applied on a calendar basis but growers may apply it more or less based on the susceptibility of the peach cultivars, environmental conditions, and perceived disease pressure. It is likely that the presumed selection pressure caused by frequent applications of oxytetracycline is not strong even to significantly increase the incidence of tetracycline resistance genes among epiphytic bacteria. Although unexpected, this result is frequently supported by the scientific literature. Yashiro and McManus (2012) were able to culture streptomycin resistant bacteria more frequently from orchards that had not been sprayed with streptomycin than those that had been sprayed with the antibiotic. In fact, in this study, the orchard with the greatest percentage of tetracycline resistance genes did not coincide with the greatest number of oxytetracycline applications and resistance genes were found where no antibiotics had ever been applied.
The age of trees also varied greatly among the sampled orchards where trees ranged in age from 3 to 16 years. This was similar to that found by Pfeufer and Ngugi (2012) where a larger percentage of Venturia inaequalis isolates resistant to fenbuconazole were collected from older trees (greater than 20 years). This was expected because older trees have been exposed to more antibiotics than younger trees. However, because the results already suggest that oxytetracycline was not related to the incidence of tetR genes, it cannot be assumed that the incidence was higher because the trees had been exposed to more oxytetracycline. This was the assumption made in the study by Pfeufer and Ngugi (2012) but in this instance, some factors other than the exposure to repeated applications of oxytetracycline must influence this phenomenon (Yashiro and McManus, 2012). For example, older trees have also had more time to acquire a larger phytobiome including a 50 greater number of tetracycline resistant bacteria, although this hypothesis has not been thoroughly evaluated among the scientific literature.
Finally, oxytetracycline application method was significantly associated with the incidence of tetA, tetB, and tetC in the epiphytic bacteria (Fig. 2-5, Table 2-2). The incidence of tetracycline resistance genes recovered from leaves was three times greater for those applied using the alternate row middle method compared to those applied in a complete manner. In general, less oxytetracycline is used each growing season with the alternative row middle method. Therefore, it was initially expected that this method would be associated with less tetR epiphytic bacteria. However, the reduced spray volume in this method results in incomplete coverage of the foliage, especially for large and densely foliated trees. There is evidence to suggest that repeated applications of sublethal levels of the antibiotic lead to a buildup of antibiotic resistance in bacterial populations due to increased mutation rates and selection for resistance as well as for the selection of acquired resistance genes in bacterial communities (Kohanski et al., 2010; Stokes and Gillings,
2011; Chopra and Roberts, 2001). In the case of oxytetracycline and bacterial spot, the antibiotic must come in contact with the bacteria in order to prevent growth and infection.
Complete application of the foliage also ensures that, not only is more of the tree coming into contact with the product, but that it is coming in contact with an effective dose of the product.
The culture based method used to recover and identify bacterial epiphytes as well as antibiotic resistance genes likely limited the number of bacterial genera identified and the incidence of resistance genes found. In fact, less than 1% of bacteria are thought to be culturable under standard laboratory conditions (Amann et al., 1995). Because only the 51 culturable bacteria were screened for tetracycline resistance genes, it is likely that a greater number of bacteria carry tetracycline resistance genes. Nevertheless, studies utilizing culture based methods are valuable because they enable one to readily assess antibiotic sensitivity and other physiological characteristics of the cultured bacteria (McGhee and
Sundin, 2010). This study would be improved if a culture independent method, such as one using Illumina sequencing technology, was employed to better examine the effects of bacterial spot management on microbial communities (Caporaso et al., 2012). In addition, the investigation on the incidence of tetracycline resistance genes could be enhanced using qPCR and DNA extracted from the entire microbial community. This is a technique used to quantify levels of targeted resistance genes that aids in the comparison of genes recovered over varying time scales and environments (Walsh et al., 2010). The addition of more tetracycline resistance genes also would have improved this research.
Nevertheless, this study has shown that determinants of resistance do exist in many of the sampled orchards and could potentially serve as a source of resistance genes that could be acquired by Xap. Although the repeated applications of oxytetracycline serve as a selection pressure to support antibiotic resistant bacteria, the results of this study indicate that oxytetracycline use over the two year period prior to this study and the incidence of tetracycline resistance genes are not related in the sampled Pennsylvania stone fruit orchards. Improved methods would ensure a more thorough understanding of the influence of oxytetracycline on tetracycline resistance and the stone fruit phyllosphere.
52
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58
Table 2-1. Oxytetracycline sensitivity of bacterial epiphytes with different tetracycline resistance genes Genusa tetR status nb MICc Pantoea tetR Negative 3 <50 µg/ml tetA 1 >450µg/ml tetB 9 >450µg/ml tetC 2 >450µg/ml Pseudomonas tetR Negative 2 >450µg/ml tetA 0 . . . . . tetB 1 >450µg/ml tetC 2 >450µg/ml Xanthomonas tetR Negative 2 <50 µg/ml tetA 0 . . . . . tetB 0 . . . . . tetC 1 >450µg/ml a Only bacteria belonging to the most common genera identified were evaluated. b n = sample size of evaluated bacterial isolates. c MIC= minimum inhibitory concentration.
59
Table 2-2. Orchard management factors associated with recovery of epiphytic bacteria from Pennsylvania orchards positive for tetracycline resistance genesa
Factorb Num. dfc Den. Dfc Odds Ratio 95% CId F value P value Tree Age 1 644 0.132 0.068-0.257 35.49 <0.0001 Application Method 1 644 3.119 1.719-5.657 14.07 0.0002 Oxytetracycline Applications 1 644 0.544 0.271-1.089 2.97 0.0855 a The tetracycline resistance genes included tetA, tetB, and tetC and were pooled together for the purpose of this analysis. b Factor responses were based on the grower surveys collected at sampling where management or orchard factors included tree age (<10 years or >10 years), application method (alternate row middle or complete spray application), and the number of oxytetracycline applications (≤3 applications or >3 applications). c Num. Df is the numerator degrees of freedom while Den. Df is the denominator degrees of freedom adjust for bias with the Kenward- Roger's method (Pfuefer and Ngugi, 2012; Kenward and Roger, 1997). d Confidence interval 60
70
60
50
40
30
20 Percent Percent ofIsolates 10
0
Orchard
Pantoea Xanthomonas Pseudomonas Bacillus Curtobacterium Staphylococcus Frondihabitans Rahnella
Fig. 2-1. Combined 2012 and 2013 bacterial communities from the sampled commercial orchards based on 16S identification of collected bacterial colonies. Bacteria were identified to the genus level using standard nucleotide blast (BLASTN) from the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/ Blast.cgi). Orchards 1 through 6 were conventionally managed orchards and orchards 7 and 8 were organic orchards. Species of Pantoea (black - recovered from all orchards), Xanthomonas (white), and Psedomonas (blue) were the most common bacteria recovered from the 8 orchards. Bacillus spp. (pink) were only recovered from the two organic orchards (7 and 8). In smaller percentages, Curtobacterium (red), Staphylococcus (green), Frondihabitans (yellow), and Rahnella (orange) spp. were also recovered.
61
100 2012 80
60
40
Percent Percent ofCFU/g 20
0 1 2 3 4 5 6 7 8 Total Orchard
100 2013 80
60
40
Percent Percent ofCFU/g 20
0 1 2 3 4 5 6 7 8 Total Orchard
Fig. 2-2. The percentage of colony forming units per gram of leaf material recovered from Kings B media amended with 0 (green), 10 (yellow), and 25 (red) µg/ml oxytetracycline in 2012 (top) and 2013 (bottom). No samples were collected from orchard 2 in 2012. Orchards 1 through 6 were conventionally managed orchards while orchards 7 and 8 were organic. Bacteria were recovered from 25 µg/ml oxytetracycline from all sampled orchards but the majority of bacteria were recovered from media amended with no oxytetracycline.
62
40
20 Percent Percent ofIsolates
0
Orchard
TetA Positve TetB Positive TetC Positive TetR Negative
Fig. 2-3. Percent of isolates collected in 2012 and 2013 from commercial stone fruit orchards positive for tetA (gray), tetB (black), and tetC (blue). Orchards 1 through 6 were conventionally managed while orchards 7 and 8 were organic. The distribution of tetracycline resistance genes significantly varied among the sampled orchards (Χ2 = 160.47, P <0.0001) based on the Pearson chi-square statistic. Tetracycline resistance genes were found in orchards 3, 4, 5, 6, and 8 and in total were found in 13.0% of all screened isolates. TetB was the most common resistance gene and was found wherever resistance genes were recovered except for those recovered from orchard 8 where only tetA and tetC were found.
63
100
80
60
40 Percent Percent ofIsolates
20
0
Bacterial Genus
TetA Positve TetB Positive TetC Positive TetR Negative
Fig. 2-4. Percent of isolates of each identified genus positive for tetR genes. Tetracycline resistance genes were found in species of Pantoea (43.3%), Pseudomonas (34.6%), Rahnella (100%), and Xanthomonas (0.9%). Overall, 23.3% of the 352 identified isolates were positive for one tetracycline resistance gene. No isolate was positive for more than one resistance gene. TetA (pink) was found in isolates of Pseudomonas and Rahnella. TetB (black) was most commonly associated with Pantoea spp. while tetC (green) was associated with Pseudomonas spp.
64
100
80 TetR Positive
60 TetR Negative
40 Percent Percent of Isolates (n 648) = 20
0
Orchard Management Factor
Fig. 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 (ARM = alternate row middle) on the incidence of tetracycline resistance genes found in bacterial epiphytes collected from commercial orchards. Based on the Pearson chi-square statistic, the incidence of tetR positive bacteria significantly differed (P < 0.0001) for all of these factors.
65
Chapter 3
The effects of bactericide use for the management of bacterial spot in Pennsylvania
stone fruit orchards
ABSTRACT
In this study, epiphytic bacteria were collected throughout the 2013 and 2014 growing seasons from May to September from the product evaluation site at the Penn State
Fruit Research and Extension Center to determine the effects of specific bactericide use on bacterial epiphytic communities and the incidence of tetracycline resistance genes.
Collected 3 to 5 days after bactericide application, the number of colony forming units per gram leaf tissue (cfu/g) was not significantly correlated with bactericide treatment.
However, the number of cfu/g did significantly differ among sampling dates with the last sampling date in September providing the greatest number of cfu/g in both years. The number of cfu/g also varied among cultivars with ‘Snow King’ having the largest number of cfu/g each year. Tetracycline resistance genes, tetA, tetB, and tetC, were also found in a small percentage of the bacterial epiphytes collected each year. These resistance genes were most commonly found in species of Pseudomonas and Pantoea. Although the number of bacteria positive for tetracycline resistance genes significantly differed among bactericide treatments, the largest percentage was collected from trees that had been treated with copper mixed with vegetable oil. In addition, all treatments, even the untreated control, supported populations of bacteria carrying tetracycline resistance genes, suggesting repeated exposure to oxytetracycline alone does not directly result in an
66 increase of tetracycline resistance among epiphytic bacterial communities. Nevertheless, the results of the product evaluation test support the use of copper products in rotation with other bactericides to reduce to use of a single product and best manage bacterial spot.
INTRODUCTION
Bacterial spot, caused by the Gram negative bacterium Xathomonas arboricola pv. pruni, regularly limits stone fruit production across the eastern United States. 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. In Pennsylvania,
100% fruit loss has been observed on highly susceptible cultivars in years where weather conditions favored bacterial spot development. Such was the case in the 2013 growing season, when bacterial spot was particularly bad on apricots and plums across much of southern Pennsylvania (PA). Severe bacterial spot epidemics are often associated with defoliation, poor fruit quality, and a greater susceptibility to brown rot caused by the fungus
Monilinia fructicola (Ritchie, 1995; Bardsley, 2010). Although symptoms on fruit are the primary focus of chemical management practices, foliar and twig symptoms are critical targets of chemical bactericides, as well. The primary bactericides used to manage bacterial spot are the antibiotic oxytetracycline and various formulations of copper. Starting at shuck split/petal fall when the petals have fallen off of the blossom and the fruit is small, applications of bactericides begin and continue throughout the season until harvest at an
67 interval of ten to 14 days. Depending on the cultivar and time of harvest, up to ten applications may be made per season.
The survey of Xap isolates collected from PA commercial stone fruit orchards in
2011 and 2012 in addition to a subsequent test for antibiotic sensitivity of those isolates indicated that levels of antibiotic sensitivity in Xap isolates remain high and oxytetracycline-based products remain effective (Chapter 4). However, antibiotic sensitivity varied greatly among orchards based on the number of antibiotic applications made per year. For example, in orchards where antibiotics had not been used or were used sparingly, Xap isolates were highly sensitive to oxytetracycline compared to isolates collected from orchards where the antibiotics had been regularly used. Although Xap isolates able to grow in media amended with the 150 µg/ml oxytetracycline field rate have not yet been found in PA, the results of this survey indicated that the selective force associated with repeated applications of oxytetracycline can result in a reduction in sensitivity of Xap isolates (Chapter 4).
Copper has been used as an alternative and has a multi-site mode of action since it targets nonspecific sites on multiple organisms. Commonly applied in the spring and fall when trees are dormant to reduce overwintering Xap inoculum, copper is now often applied during the season to effectively manage bacterial spot. The severe phytotoxicity often associated with copper compounds when applied in wet or slow drying conditions, however, limits its effectiveness as a bactericide (Lalancette and McFarland, 2007).
Vegetable oil has been investigated as a safener in the management of bacterial canker on cherries (Allen, 1988). A safener is a product used to reduce the injury associated with copper. Vegetable oil, however, has not been evaluated as a safener with respect to copper
68 used in bacterial spot management. This combination could be a useful option for growers during the season. In addition, the development of copper formulations that promise to be less phytotoxic are potentially useful alternatives as well (www.certisusa.com).
Bacterial spot is not easily managed on highly susceptible, consumer preferred, stone fruit cultivars, and has therefore, greatly hindered the development of organic peach and nectarine orchards in the eastern US. The only bacterial spot management strategies for organic production are the cultural practices of planting in well-draining soils, pruning branches to allow increased airflow, managing populations of ring nematodes in the soil that injure tree roots, as well as planting resistant stone fruit cultivars (Shepard et al., 1999;
Zehr et al., 1996). However, no peach or nectarine cultivar is completely resistant to the disease and crops not managed with conventional bactericides are at a particular risk of developing severe bacterial spot epidemics. Fortunately, products marketed for organic production are becoming available and show potential for bacterial spot management in organic and conventional orchards. Lime sulfur, although not a new product, is approved for use in organic stone fruit production and has been used to manage bacterial spot on less susceptible stone fruit cultivars, when disease pressure is low. However, lime sulfur often results in 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). Biofungicides containing species of Bacillus, including
Double Nickel (Bacillus amyloliquefaciens strain D747) (www.certisusa.com) and
Serenade Opti (Bacillus subtilis strain QST 713) (www.bayercropscience.us) possess multiple modes of action against fungi and bacteria, making the development of resistance to them less likely to occur. However, neither have been evaluated against the strong
69 disease pressure found in Pennsylvania stone fruit orchards or for their compatibility with conventional management practices.
Successful bacterial spot management must focus not only on reducing disease symptoms but on mitigating the risk of antibiotic resistance. The survey conducted to monitor the diversity of epiphytic bacteria in commercial orchards indicated that antibiotic resistant bacteria (i.e.: bacteria growing on 25 µg/ml oxytetracycline) were present in all of the orchards surveyed (Chapter 2). Previous studies have shown that unrelated bacterial species readily transfer antibiotic resistance genes among species and therefore pose a potential risk to antibiotic use because they are an ever present source of resistance genes
(Roberts, 1996). Moreover, bacterial diversity differed among the orchards in this survey, with greatest differences observed between conventional and organic orchards. Because there are so few products regularly used to manage bacterial spot (copper and oxytetracycline), resistance to one or both product types would severely threaten the production of stone fruit in the eastern U.S. Alternatives and rotation programs used to reduce the use of a single chemical should be evaluated. Rotating chemicals with different modes of action will help reduce the selective pressure posed by a particular chemistry thereby reducing the risk of resistance development. In addition, monitoring populations of epiphytic bacteria will help determine the effect of each bactericide program on non- target bacterial communities with the goal of increasing bacterial diversity and reducing the development of antibiotic resistance. Although alternative chemicals for bacterial spot management have been tested before, none have been tested in relation to their effects on antibiotic resistance. Therefore, the objectives of this study were to monitor the effects of specific bactericides on disease severity and incidence, to identify the distribution of
70 tetracycline resistance genes in epiphytic communities following bactericide treatments, and to determine the best bactericide program, including new potential chemicals and rotation programs, to reduce bacterial spot symptoms and tetracycline resistance among non-target bacteria.
MATERIALS AND METHODS
Bactericide application schedule: Collection of epiphytic bacteria and bactericide evaluation was conducted in the Plant Pathology Peach and Nectarine Block at the Penn
State Fruit Research and Extension Center, Biglerville, PA. This is a 4-cultivar
(‘Easternglo’ nectarine, ‘Beekman’ peach, ‘Snow King’ peach, and ‘Sweet Dream’ peach;
Guard trees: ‘Redhaven’ peach) peach and nectarine block that was planted in 2006 and will hereafter be referred to as ‘FREC block’. This experimental block received standard fungicide and insecticide applications according to the commercial practices in the northeastern United States. Treatments were applied with an airblast sprayer calibrated to apply 378.54 liters per hectare at 400 psi. In 2013, the treatments evaluated included: (1) untreated check without dormant copper application, (2) untreated check with dormant copper application, (3) Kocide 3000 0.61g/L (46.1% copper hydroxide; Dupont;
Wilmington, DE), (4) FireLine 1.80g/L (18.3% oxytetracycline; AgroSource; Cranford,
NJ), (5) Lime Sulfur 5.02ml/L (30% calcium polysulfide)/ Sulfur 11.89g/L/ Kocide 3000
0.61g/L rotation, (6) Regalia 10.04ml/L (5% extract of Reynoutria sachalinensis; Marrone
Bio Innovations, Inc. Davis, CA)/ Serenade Max 3.59g/L (14.6% QST 713 strain of dried
Bacillus subtilis; AgraQuest; Davis, CA) rotation. Kocide 3000 contains copper hydroxide
71 while FireLine contains the active ingredient of oxytetracycline hydrochloride. Regalia and
Serenade Max are both compliant with the regulations of the National Organic Program
(http://www.ams.usda.gov/AMSv1.0/nop) and contain extract of Reynoutria sachalinensis which activates the plant’s defense system and Bacillus subtilis (strain QST 713), respectively. Treatments were applied on April 25-26 (PF - petalfall/shucksplit), May 6
(C1), May 17 (C2), May 30 (C3), June 12 (C4), June 24 (C5), July 11 (C6), July 24 (C7), and August 2, 2013 (C8). In 2014, the treatments evaluated included: (1) untreated check without dormant copper application, (2) FireLine (1.80g/L) (PF-C6), (3) MasterCop
(1.24ml/L) (21.46% copper sulfur pentahydrate; Adama, Raleigh, NC) (PF-C6), (4)
MasterCop (1.24ml/L) (PF, C2, C4, C6) / Rampart (53% Mono- and dipotassium salts of phosphorous acid) (250ml/L) (C1, C3, C5) rotation (5) MasterCop (1.24ml/L) (PF, C2, C4,
C6) / Serenade Optimum (1.05g/L) (26.2% QST 713 strain of dried Bacillus subtilis) rotation (C1, C3, C5), and (6) MasterCop (1.24ml/L) + Vegetable Oil (750ml/L) (PF-C6).
MasterCop (http://www.adama.com/) contains copper sulfate pentahydrate while Rampart
(http://www.lovelandproducts.com/) contains the active ingredient potassium phosphite.
Serenade Optimum (https://www.bayercropscience.us/) also contains Bacillus subtilis
(strain QST 713) but is formulated to contain a larger percentage of the active ingredient than Serenade Max. Treatments were sprayed on May 12 (PF – petalfall/shucksplit), May
22-23 (C1), June 2 (C2), June 16 (C3), June 25 (C4), July 7 (C5), and July 22 (C6).
Bactericide evaluation: Bactericide evaluation took place in the FREC block in
2013 and 2014. In 2013, 30 random leaves per tree were assessed for percent bacterial spot severity. Leaves were rated from 0 through 100%. In 2014, 5 shoots were randomly tagged and 10 leaves per shoot, starting from the base of the shoot, were assessed for percent
72 bacterial spot severity (0-100%) twice during the season. At harvest 25 fruit per tree were assessed for percent bacterial spot severity in 2013 and 2014. Treatment was assessed statistically using analysis of variance (ANOVA) and Fisher’s Protected LSD test on
MINITAB (www.minitab.com).
Collection and isolation of bacterial epiphytes from product evaluation site: Leaf samples were collected in 2013 and 2014 from the FREC block 3 to 5 days after a bactericide application except for the last collection which was approximately a month after harvest. In 2013, leaves were collected from the cultivars ‘Redhaven’, ‘Beekman’, and ‘Snow King’ at the beginning, middle, and end of the season (May 20, June 28, and
August 31). For the remaining sample collections, ‘Beekman’ was used and was sampled on June 3, June 17, July 15, and July 29. In 2014, leaves were collected from the cultivars
‘Easternglo’, ‘Beekman’, and ‘Snow King’ three times during the season (June 5, June 27, and September 11). ‘Beekman’ was used for the remaining sample collections on June 20,
July 11, and July 25. Leaves were chosen randomly, regardless of symptom severity, at a height of about 135 cm from all around the tree. Leaves were collected into new paper bags and were immediately processed.
Epiphytes were washed from the leaves. A total of 20 grams of each bulk sample were transferred into 400ml of chilled 1M potassium phosphate buffer in a 500ml sterile glass jar with a drop of tween20 (Polyoxyethylene(20)sorbitan monolaurate; Agdia;
Elkhart, IN). The leaves were shaken on a rotary arm shaker for 30 minutes before being sonicated for 5 minutes (McGhee and Sundin, 2011). A milliliter from each sample was taken to complete serial dilutions and 100ml of the rinsate was store at -80C for future evaluation. Serial dilutions were carried out on fresh Kings B media amended with 0, 10,
73 and 25 mg/L oxytetracycline (oxytetracycline dehydrate; Sigma-Aldrich; St. Louis, MO).
Duplicates of three dilution series for each antibiotic concentration were completed and incubated at 22C for 96 hours in the dark. The bacterial colonies were counted and a subset of the colonies were randomly chosen from all of the concentrations of antibiotic amended media. Selected colonies were then transferred to nutrient broth (Schaad et al., 2001) and grown for 24 to 48 hours before being preserved at -80° C in 25% sterile glycerol.
Oxytetracycline sensitivity of epiphytic bacteria: The minimum inhibitory concentration (MIC) of a selection of bacterial epiphytes to oxytetracycline was determined using the agar dilution method as previously described (Wiegand et al., 2008). For each isolate, 3 to 5 morphologically similar colonies from a fresh agar grown culture were selected using a sterile loop. The colonies were transferred to a sterile capped plastic tube containing 2ml sterile 0.1M potassium phosphate buffer. To ensure a similar number of bacterial cells was being used to determine the MIC, the turbidity of the vortexed suspension was assessed by measuring the absorbance. An absorbance between 0.08 and
0.13 (equal to 1x108 cfu ml-1) at OD625 nm was achieved by adding more buffer or bacterial material. All bacterial suspensions were used within 30 minutes to ensure the accuracy of the measured turbidity. A final amount of 104 cfu was used in triplicate to determine the MIC after 3 days of incubation at 26C. Growth comparable to the growth of bacteria on 0 mg/L oxytetracycline was considered “growth” while light bacterial films and no visible growth were considered “no growth”. The MIC was considered the lowest concentration of oxytetracycline that inhibited the visible growth of the tested isolate. The
MIC for several isolates of Pantoea and Pseudomonas spp. positive and negative for tet(A), tet(B), and tet(C) was determined. Pantoea and Pseudomonas spp. were the most common
74 species of bacteria recovered from the sampled orchards. The MIC was determined on
Kings B media amended with 10 concentrations of oxytetracycline (0, 50, 100, 150, 200,
250, 300, 350, 400, and 450 µg/ml).
Tetracycline resistance genes, epiphytic bacteria, and bacterial identification. As in chapter 2, epiphytic bacteria were screened for the presence of three previously described tetracycline resistance genes, tet(A), tet(B), and tet(C), using PCR and specific primers. These genes are among the most common genes found in Gram negative bacteria
(Roberts, 2012). The frozen culture was used as the template and was introduced to each
25 µl reaction with a sterile toothpick. Choice Taq Mastermix from Denville Scientific
(Holliston, MA) was used. The tet(A)-F forward primer (5’-
TTGGCATTCTGCATTCACTC-3’) and the reverse primer tet(A)-R (5’-
GTATAGCTTGCCGGAAGTCG-3’) amplified a 494bp product (accession no.: X75761).
Forward primer tet(B)-F (5’-CAGTGCTGTTGTTGTCATTAA-3’) and reverse primer tet(B)-R (5’-GCTTGAATACTGAGTGTTAA-3’) amplified a 571bp product (accession no.: V00611). The tet(C)-F forward primer (5’-CTTGAGAGCCTTCAACCCAG-3’) and the reverse primer tet(C)-R (5’-ATGGTCGTCATCTACCTGCC-3’) amplified a 418bp product (accession no.: J01749) (Ma et al., 2007). The thermocycler parameters included a 112C heated lid and an initial denaturation of 96C for 10 minutes. After that, 34 cycles were completed of 96C denaturation for 30 seconds, 55C annealing for 1 minute, and 72C elongation for 30 seconds. The products were stored at 4C and run through 1.5% agarose gel in TAE buffer using EZ-Vision loading dye (Amresco; Solon, OH). Gels were visualized under the KODAK gel imaging system. With each cycler run, a positive and negative control were included.
75
Bacterial isolates positive for a tetracycline resistance gene were identified to genus level through sequencing of the 16S rDNA. The frozen culture was used as the template and was introduced to each 25 µl reaction with a sterile toothpick. Choice Taq Mastermix
(Denville Scientific; Holliston, MA) was used. The forward primer 530F (5’-
GTGCCAGCAGCCGCGG-3’) and the reverse primer 1494R (5’-
TACGGCTACCTTGTTACGAC-3’) amplified a fragment sized 1kb. The thermocycler parameters included a 112C heated lid and an initial denaturation of 96C for 10 minutes.
After that, 34 cycles were completed of 96C denaturation for 30 seconds, 55C annealing for 1 minute, and 72C elongation for 30 seconds. The products were stored at 4C and run through 1.5% agarose gel in TAE buffer using EZ-Vision loading dye (Amresco; Solon,
OH). Gels were visualized under the KODAK gel imaging system. With each cycler run, a positive and negative control were included. PCR products were cleaned using ExoSAP-
IT (Amresco; Solon, OH) and were prepared for sequencing at the Genomics Core Facility of the Huck Institute of Life Sciences at Penn State University (University Park, PA).
Sequence analysis was completed using standard nucleotide blast (BLASTN) from the
National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/ Blast.cgi).
Only alignments with an E-value of 0.0 were accepted.
Statistical analysis for bactericides associated with the incidence of tetR genes and the distribution of bacterial epiphytes: Bacterial plate counts were used to calculate colony forming units per gram (cfu/g). The cfu/g was log10 transformed to normalize the data distribution before statistical analysis. The general linearize model in MINITAB was used to determine significant differences among the number of colonies per gram of leaf tissue due to sample date, the oxytetracycline concentration of the media, bactericide treatment
76 of trees where samples were collected, and the cultivar of trees where samples were collected. Tukey’s test was used to determine significant differences among each individual factor. Plate counts were analyzed separately for each year.
For the incidence of tetracycline resistance genes, the proc FREQ procedure in SAS
(Chapter 2 & 4) was used to determine any significant association of resistance genes with year and bactericide treatment. Because the incidence of tetracycline resistance genes was so few, the data was combined across years and cultivar.
RESULTS
Efficacy of alternative bactericides: In 2013, disease severity of foliar and fruit symptoms was assessed for ‘Beekman’ and ‘Snow King’. Because data on the lime sulfur, the extract of R. sachalinensis (Regalia), and the B. subtilis (Serenade) treatments were collected at a later time (September versus August for the analysis shown), they were not included in the analysis. Also, data on the disease severity of fruit is missing and is therefore not included. For both cultivars, oxytetracycline (FireLine) provided the best reduction of foliar bacterial spot symptoms; however, this was not significantly different than both untreated treatments in ‘Beekman’ or the untreated treatment with dormant copper (Kocide 3000) application in ‘Snow King’ (Table 3-1). For fruit severity of
‘Beekman’, copper performed the best but not significantly better than oxytetracycline. In
‘Snow King’, copper and oxytetracycline performed equally and significantly better than the untreated controls for managing disease severity on fruit.
77
In 2014, foliar and fruit disease severity and incidence was assessed for
‘Easternglo’, Beekman’, and ‘Snow King’ in July for the analysis shown in Table 3-2. The severity and incidence of fruit symptoms was assessed at harvest (‘Easternglo’ on July 25,
‘Beekman’ on August 5, and ‘Snow King’ on August 11). For ‘Easternglo’, MasterCop
(the active ingredient being copper) preformed the best at reducing foliar disease severity but only significantly better than oxytetracycline (FireLine) and the untreated control.
There was no significant difference between the disease severity of oxytetracycline and the untreated control. For disease severity of ‘Easterglo’ fruit, copper (MasterCop) preformed the best but only significantly better than the untreated control and the rotation of copper with phosphorous acid (Rampart). For the foliar disease severity of ‘Beekman’, copper was the best treatment but it was not significantly better than copper rotated with phosphorous acid or copper mixed with vegetable oil. Copper alone as well as copper rotated with B. subtilis (Serenade Optimum) preformed equally the best but only significantly better than the untreated control in disease severity of ‘Beekman’ fruit. For ‘Snow King’ foliar disease severity, copper mixed with vegetable oil reduced severity the best but only significantly more than oxytetracycline and the untreated control which were not significantly different from each other. In ‘Snow King’ fruit, the rotation of copper and B. subtilis reduced disease severity the most but not significantly more than the rotation of copper and phosphorous acid.
Collection of epiphytic bacteria: In both years, bacterial epiphytes were recovered and counted across all sampling dates, cultivar, treatment, and oxytetracycline amended media. In 2013, significant factors influencing the number of bacterial colony forming units per gram of leaf tissue were the sample date (F = 25.86, P < 0.0001), the concentration of
78 oxytetracycline in the media (F = 50.96, P < 0.0001), and cultivar (F = 11.27, P < 0.0001).
Bactericide treatment, however, was not significant (F = 1.02, P = 0.407) (Table 3-3). The largest number of cfu/g leaf tissue was collected on the last sampling date of 2013, August
31 (Fig. 3-1). This sampling occurred 29 days after the last cover application and was used to show what happens to the number of bacteria on the leaves after bactericide applications have ceased. However, this number was not significantly different from the cfu/g collected on July 15. The smallest number of cfu/g was collected on June 17, the third sampling date.
The smallest number of cfu/g were collected from media amended with 25 µg/ml oxytetracycline in 2013 (Fig. 3-2). The largest number of cfu/g were collected from the cultivar 'Snow King', although it was only significantly higher than those collected from
'Red Haven' (Fig. 3-3).
In 2014, sample date (F = 20.08, P < 0.0001), oxytetracycline concentration of sample media (F = 71.49, P < 0.0001), and cultivar (F = 11.59, P < 0.0001), but not treatment (F = 0.84, P = 0.520), were again, significant factors influencing the number of colony forming units per gram of leaf tissue collected from the FREC block (Table 3-4).
Again, the last sampling date (51 days after the last bactericide application), September,
11, had the largest number of cfu/g, significantly more than the other sampling dates (Fig.
3-4). The number of cfu/g significantly differed among each concentration of antibiotic amended media where the most cfu/g were collected from media amended with 0 µg/ml oxytetracycline and the least were collected from media amended with 25 µg/ml (Fig. 3-
2). Again, the largest number of cfu/g were collected from the cultivar ‘Snow King’, significantly more than the cultivars ‘Beekman’ and ‘Easternglo’ (Fig. 3-3). The largest
79 number of cfu/g was collected from the copper + vegetable oil treatment, although bactericide treatment was not a significant factor.
Oxytetracycline sensitivity of epiphytic bacteria: Bacteria positive and negative for tetracycline resistance genes for the three most common genera of bacterial epiphytes,
Pantoea, Psuedomonas, and Xanthomonas, were evaluated (Table 3-5). In all cases, tetR positive bacteria had a minimum inhibitory concentration (MIC) of greater than 450 µg/ml oxytetracycline. TetR negative Pseudomonas spp. had a similar MIC while species of
Pantoea and Xanthomonas negative for a tetracycline resistance gene had MICs of less than 50 µg/ml oxytetracycline.
Tetracycline resistance genes and bacterial identification: A total of 418 and 309 bacterial epiphytes were collected and screened for the presence of tetA, tetB, and tetC, in
2013 and 2014, respectively. In 2013, 3.4%, 3.6%, and 1.2% of isolates were tetA, tetB, or tetC, respectively, with a total of 8.1% of isolates tetR positive. In 2014, 2.9%, 8.7%, and
5.8% were tetA, tetB, or tetC, respectively, with a total of 17.5% of isolates tetR positive.
This variation in incidence of tetracycline resistance genes in the collected bacterial epiphytes between 2013 and 2014 was significant (Χ2 = 124.26, P < 0.0001) (Fig. 3-5).
Combined data for 2013 and 2014 for all tetR positive bacteria indicate that the incidence of bacteria positive for tetracycline resistance genes (tetA, tetB, or tetC) significantly differed among sampled bacteria collected from different bactericide treatments (Χ2 =
32.98, P < 0.0001) (Fig. 3-6). Bacteria recovered from the copper mixed with vegetable oil treatment had the greatest incidence of tetracycline resistance; 32.7% of isolates were positive for tetA, tetB, or tetC. Bacteria collected from trees that had been sprayed with the antibiotic oxytetracycline had the second largest incidence of tetracycline resistance genes,
80
16.1% of isolates were tetR positive. The incidence of tetracycline resistance genes for bacteria collected from untreated trees (7.0%) was not the lowest, as expected, but bacteria collected from trees treated with the extract of R. sachalinensis (Regalia) rotated with B. subtilis (Serenade) was (5.4%).
Only bacteria positive for tetA, tetB, or tetC were identified to genus level. Of the tetR bacteria, 46.5% were Pantoea spp., 43.7% were Pseudomonas spp., 5.6% were
Xanthomonas spp., 2.8% were Curtobacterium spp., and 1.4% were Bacillus spp. (Fig. 3-
7). Bacteria positive for tetA were species of Pseudomonas (87.0%), Pantoea (8.7%), and
Bacillus (4.4 %). Bacteria positive for tetB were species of Pseudomonas (2.9%), Pantoea
(80.0%), Xanthomonas (11.4%), and Curtobacterium (5.7%). Bacteria positive for tetC were species of Pseudomonas (76.9%) and Pantoea (23.1%).
DISCUSSION
Bacterial spot is a difficult disease to manage. Variable environmental conditions and susceptibility of stone fruit only increase the difficulty of managing this often sporadic disease (Ritchie, 1995). Product efficacy evaluation is important to increase the number of products available to manage bacterial spot so that the antibiotic oxytetracycline and copper are not over used. Finding alternative products is also important to the organic tree fruit industry and to consumers who value organic production. Finding alternatives and evaluating bactericide efficacy is difficult because rating disease severity is time consuming (Bardsley and Ngugi, 2012) and statistically, the same products do not produce the same results on cultivars with varying levels of susceptibility to bacterial spot year after year. Such was the case in this study where, although the products tested differed between
81 years, standards such as oxytetracycline and copper containing products varied in efficacy
(Tables 3-1 and 3-2). This was similar to the results found previously (Bardsley, 2010) where oxytetracycline was not statistically more efficacious than the untreated control; however, disease rating method, confusion over copper injury and disease symptoms, and severe defoliation probably contributed to these results as they likely do in every evaluation
(Bardsley, 2010).
In this study, products were evaluated for their ability to mitigate symptoms and for the effects they had on bacterial communities and the incidence of tetracycline resistance genes. In 2013 and 2014, bactericide treatments did not significantly influence the number of colony forming units per gram of leaf tissue (Table 3-3 and 3-4). Bactericide treatment was also not a significant factor influencing the number of cfu/g recovered from media amended with 0, 10 or 25 µg/ml (data not shown). This result was unexpected; however, it is similar to those results found by McGhee and Sundin (2011) where epiphytic bacterial populations on apple leaves were not significantly different after treatment with streptomycin (Agri-Mycin), kasugamycin (Kasumin), or water. Although more cfu/g were recovered from untreated trees in 2013, the greatest number of cfu/g were collected from trees treated with copper (MasterCop) mixed with vegetable oil in 2014. In addition, that same treatment, copper mixed with vegetable oil, had the smallest foliar disease severity rating for ‘Snow King’ and ‘Easternglo’ in 2014. The lack of significant differences in cfu/g among treatments may be that the 3 to 5 day interval between bactericide application and sample collection was enough time for bacterial populations to recover from the presumed initial reduction in bacterial community due to the bactericide application. It may
82 also be because the structure of the community differed after treatment but the size of the culturable community did not.
However, the percentage of tetR positive bacteria collected from trees in the FREC block were significantly different among bactericide treatments (Χ2 = 32.98, P ≤ 0.0001)
(Fig. 3-6). The treatment of MasterCop mixed with vegetable oil supported the largest percentage (32.7%) of tetR positive bacteria. Again, this treatment ranked the best for reducing foliar disease severity in ‘Snow King’ and ‘Easternglo’ and was associated with the largest cfu/g leaf tissue (though not significant) in 2014. Although the percentages of tetR positive bacteria significantly differed among treatments, the results suggest that tetracycline resistance genes are not associated with oxytetracycline use alone. In fact, all of the treatments, even the untreated control supported tetracycline resistance genes. This is in agreement with numerous studies that show the common recovery of antibiotic resistance genes from environments not exposed to antibiotics (Smith, 1967; Houndt and
Ochman, 2000; Hughes and Datta, 1983; Mindlin et al., 2001, 2005; Walson et al., 2001;
Bartoloni et al., 2004; Yashiro and McManus, 2012). This also fits with the results of chapter 2 where tetracycline resistance genes were found in an organic orchard and the number of oxytetracycline applications was not a significant factor affecting the distribution of tetracycline resistant bacteria recovered from commercial stone fruit orchards.
Again, bacteria carrying tetracycline resistance genes were able to grow on high levels of oxytetracycline – greater than 450 µg/ml oxytetracycline compared to the field rate of 150 µg/ml (Table 3-5). TetR negative Pseudomonas spp. were able to grow at those elevated antibiotic levels while tetR negative Pantoea spp. and Xanthomonas spp. could
83 not. The percentage of bacteria carrying tetracycline resistance genes varied significantly between 2013 and 2014 (Fig. 3-5). This is similar to what was found in chapter 2 where the distribution of resistance genes also differed between years. This study also focused only on tetA, tetB, and tetC, all of which were detected in the FREC block in 2013 and
2014. Bacteria carrying one of these three genes were identified to one of the genera including Pseudomonas, Pantoea, Bacillus, Xanthomonas, or Curtobacterium (Fig. 3-7).
Bacteria belonging to Pseudomonas and Pantoea were the most common where Pantoea spp. made up the largest percentage of tetB positive bacteria and Pseudomonas spp. made up the largest percentage of tetA and tetC positive bacteria. These were consistent with the results found in chapter 2 and the study conducted by Schnabel and Jones in Michigan
Apple Orchards (1999). TetB also had the largest host range of tetR genes tested in this study (4 compared to 3 for tetA and 2 for tetC) (Roberts , 2012; Chopra and Roberts, 2001).
Again, tetracycline resistance genes were found in species of Xanthomonas (chapter 2) and should be further examined to confirm this result and investigate a potential role they may play in the development of tetracycline resistance in Xap.
In addition to the examination of the incidence of tetracycline resistance genes and the association of bactericide treatment on the number of cfu/g, this study also focused on additional factors that influenced the magnitude of bacterial epiphytes found on the surface of peach leaves. The number of cfu/g leaf tissue was significantly influenced by sampling date, the oxytetracycline concentration of the sampling media, and cultivar of sample collection in 2013 and 2014 (Tables 3-3 and 3-4) (P < 0.0001). In both years, the last sampling date of the year that occurred 29 and 51 days after the last bactericide application in 2013 and 2014, respectively, had the largest number of cfu/g leaf tissue (Figs. 3-1 and
84
3-4). This, along with nonsignificant differences in the number of cfu/g collected on different sampling dates from only the untreated control (data not shown), suggest this increase in cfu/g is a large recovery or buildup of the number of bacteria in the community after a long (i.e.: longer than 3 to 5 days) period without bactericide exposure (Wiuff et al.,
2005; Levin and Rozen, 2006). A greater number of bacteria were recovered on media amended with 0 µg/ml oxytetracycline than 10 and 25 µg/ml of the antibiotic, significantly only more than the 25 µg/ml in 2013 and 10 and 25 µg/ml in 2014 (Fig. 3-2). This was similar to that found in chapter 2 where the number of cfu/g recovered from media amended with 25 µg/ml oxytetracycline was less than that of those collected from plates amended with 0 and 10 µg/ml of the antibiotic. This suggests that tetracycline sensitive bacteria dominate the culturable bacterial community. Finally, in both years, ‘Snow King’ had the largest number of cfu/g leaf tissue among the sampled cultivars – significantly only more than ‘Redhaven’ in 2013 and more than ‘Beekman’ and ‘Easternglo’ in 2014. While all of the sampled cultivars, with the exception of ‘Redhaven’, are considered highly susceptible to bacterial spot, ‘Snow King’ had the highest foliar disease severity in 2013 and 2014
(Tables 3-1 and 3-2). This is similar to a study on potato by Sessitsch et al. (2002) where they found that unique bacterial communities were associated with specific potato cultivars. They found that healthy plants had a significantly higher diversity of bacterial endophytes than unhealthy plants when exposed to environmental stressors.
Because this study focused only on culturable bacteria and not the whole bacterial community, it is impossible to know how specific bactericides, time, or cultivar effect the total community structure. This would have provided more useful data from which to draw conclusions. As in chapter 2, deep sequencing techniques that are culture independent
85 would certainly enhance this study. In addition, the variability in product performance makes making recommendations to fruit tree growers difficult. Importantly, resistance genes are not associated with the “failure” of oxytetracycline containing products, such as
FireLine, since that product performed the best for foliar disease severity in 2013 (Table
3-1) and often, although not always, better than the untreated control in 2014 (Table 3-2).
In addition, this study could not demonstrate an elevated level of tetracycline resistance genes due to oxytetracycline application. In fact, a copper treatment supported the highest level of tetracycline resistance genes in this study. Perhaps, longer term studies where the split plot designs are not rotated each year for treatment would help better emulate common practices in commercial orchards where the same treatments are applied year after year to the same trees. Here, products and rotations containing copper performed just as well, if not better, than oxytetracycline (Tables 3-1 and 3-2). Recommendations should be made to use copper in rotations, when possible, in order to mitigate the symptoms of bacterial spot and prevent the overuse of copper or oxytetracycline.
86
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Allen, W.R. 1988. Bacterial Canker of Sweet Cherry. Ministry of Agriculture and
Food, Ontario, Canada. NO. 88-0886.
Bardsley, S.J. 2010. Studies on the epidemiology and management of bacterial spot
of peach and nectarine in Pennsylvania. The Pennsylvania State University.
Master’s Thesis.
Bardsley, S.J., and Ngugi, H.K. 2013. Reliability and accuracy of visual methods
to quantify severity of foliar bacterial spot symptoms on peach and nectarine.
Plant Pathology 62:460-474.
Bartoloni, A., Bartalesi, F., Mantella, A., Dell’Amico, E., Roselli, M., Strohmeyer, M.,
Gamboa Barahona, H., Barro´ n V.P., Paradisi, F., and Rossolini, G.M. 2004. High
prevalence of acquired antimicrobial resistance unrelated to heavy antimicrobial
consumption. Journal of Infectious Disease 189: 1291-1294.
Chopra, I. and Roberts, M. 2001. Tetracycline antibiotics: Mode of action, applications,
molecular biology, and epidemiology of bacterial resistance. Microbiology and
Molecular Biology Reviews 65: 232-260.
Houdt, T. and Ochman, H. 2000. Long-term shifts in patterns of antibiotic resistance
in enteric bacteria. Applied and Environmental Microbiology 66: 5406-5409.
Hughes, V.M. and Datta, N. 1983. Conjugative plasmids in bacteria of the ‘pre-antibiotic’
era. Nature 302: 725-726.
Lalancette, N., and McFarland, K.A. 2007. Phytotoxicity of copper-based bactericides
to peach and nectarine. Plant Disease 91: 1122-1130.
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Levin, B.R., and Rozen, D.E. 2006. Non-inherited antibiotic resistance. Nature Reviews
Microbiology 4: 556-562.
Ma, M., Wang, H., Yu, Y., Zhang, D., and Liu, S. 2007. Detection of antimicrobial
resistance genes of pathogenic Salmonella from swine with DNA microarray.
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McGhee, G.C., and Sundin, G.W. 2011. Evaluation of kasugamycin for fire blight
management, effect on nontarget bacteria, and assessment of kasugamycin
resistance potential in Erwinia amylovora. Phytopathology 101: 192-204.
Mindlin, S., Minakhin, L., Petrova, M., Kholodii, G., Minakhina, S., Gorlenko, Z., and
Nikiforov, V. 2005. Present-day mercury resistance transposons are common
in bacteria preserved in permafrost grounds since the Upper Pleistocene. Research
in Microbiology 156: 994-1004.
Ritchie, D.F. 1995. Bacterial spot. Pages 50-52 in: Compendium of Stone Fruit
Diseases. J.M. Ogawa, E.I. Zehr, G.W. Bird, D.F. Ritchie, K. Uriu, and J.K.
Uyemoto, eds. The American Phytopathological Society, St. Paul, MN.
Roberts, M.C. 2012. Acquired tetracycline resistance genes. In: Antibiotic discovery
and development. Springer, New York, pp. 543–568.
Schaad, N.W., Jones, J.B., and Chun, W. 2001. Laboratory Guide for Identification
of Plant Pathogenic Bacteria. Third Edition. St Paul, MN: APS.
Schnabel, E.L. and Jones, A.L. 1999. Distribution of tetracycline resistance genes and
transposons among phylloplane bacteria in Michigan apple orchards. Applied
and Environmental Microbiology 65: 4898-907.
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Sessitsch, A., Reiter, B., Pfeifer, U., and Wilhelm, E. 2002. Cultivation-
independent population analysis of bacterial endophytes in three potato
varieties based on eubacterial and Actinomycetes-specific PCR of 16S
rRNA genes. FEM Microbiology Ecology 39: 23-32.
Shepard, D.P., Zehr, E.I., and Bridges, W.C. 1999. Increased susceptibility to bacterial
spot of peach trees growing in soil infested with Croconemella xenoplax. Plant
Disease 83: 961-963.
Smith, D.H. 1967. R. factor infection of Escherichia coli lyophilized in 1946. Journal
of Bacteriology 94: 2071-2072.
Walson, J.L., Marshall, B., Pokhrel, B.M., Kafle, K.K. and Levy, S.B. 2001. Carriage
of antibiotic-resistant fecal bacteria in Nepal reflects proximity to Kathmandu.
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Wiuff, C., Zappala, R.M., Regoes, R.R., Garner, K.N., Baquero, F., and Levin, B.R. 2005.
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populations. Antimicrobial Agents Chemotherapy 49: 1483-1494.
Yashiro, E., and McManus, P.S. 2012. Effect of streptomycin treatment on bacterial
community structure in the apple phyllosphere. PLoS ONE 7: e37131. doi:
10.1371/journal.pone.0037131.
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Plant Disease 80: 339-341.
89
Table 3-1. 2013 evaluation of bacterial spot incidence and severity on leaves and fruit on 'Beekman' and 'Snow King' in the FREC block. 'Beekman' 'Snow King'
Foliar Fruit Fruit Foliar Fruit Fruit a c Treatment Severityb Incidence Severity Severity Incidence Severity d 1 Untreated w/out dormant cu 2.1b 90.0a 49.0a 7.8a 100.0a 43.8a 2 Untreat w/dormant cu 1.1b 90.0a 52.0a 4.1b 98.0ab 43.8a 3 Kocide 3000 4.1a 73.0ab 7.0b 4.5b 54.0d 8.3b 4 FireLine 0.8b 78.0ab 17.8b 3.5b 77.0c 8.3b 5 Lime Sulfur 2.2 60.0 . . . . . 6.7 85.0 . . . . . Sulfur Kocide 3000 6 Regalia 5.7 87.0 . . . . . 11.6 92.0 . . . . . Serenade Max a Treatments were sprayed on April 25-26 (PF - petalfall/shucksplit), May 6 (C1), May 17 (C2), May 30 (C3), June 12 (C3), June 24 (C4), July 11 (C5), July 24 (C6), and August 2, 2013 (C7). b For foliar disease severity assessment, 30 random leaves per tree were assessed twice for percent bacterial spot severity. The assessment shown here was taken in August for treatments 1-4 and in September for treatments 5 and 6. c At harvest, 25 fruit per tree were assessed for percent bacterial spot severity by visually assessing the percent area of fruit covered with bacterial spot lesions. Disease incidence was also assessed. d Values within columns followed by the same letter(s) are not not significantly different (P ≤ 0.05) according to Fisher's Protected LSD test. Treatments 5 and 6 were not included in the analysis because leaves were assessed at different times. The values for fruit severity for treatments 5 and 6 are missing.
90
Table 3-2 2014 evaluation of bacterial spot incidence and severity on leaves and fruit on 'Easterglo' nectarine, 'Beekman' peach, and 'Snow King' peach in the FREC block. 'Easternglo' Nectarine 'Beekman' Peach 'Snow King' Peach
Foliar Foliar Fruit Fruit Foliar Foliar Fruit Fruit Foliar Foliar Fruit Fruit a c Treatment Incidenceb Severity Incidence Severity Incidence Severity Incidence Severity Incidence Severity Incidence Severity d 1 Untreated 69.0 b 2.4 a 92.0 a 5.1 a 80.0 a 2.4 a 71.0 a 4.3 a 95.3 a 5.4 a 97.3 ab 17.3 a 2 Oxytetracycline (FireLine) 85.0 a 2.3 a 89.0 a 3.8 a-c 82.0 a 1.8 ab 58.0 a-c 1.7 bc 95.5 a 5.8 a 95.0 ab 11.9 b 3 Copper (MasterCop) 39.0 c 1.0 b 73.0 bc 2.6 cd 47.0 bc 0.7 d 38.0 d 1.1 c 77.0 b 3.5 b 97.0 ab 8.5 c Copper (MasterCop) 4 47.36 c 1.0 b 84.0 ab 4.8 ab 49.5 bc 1.0 cd 65.0 ab 2.3 b 77.5 b 3.2 b 99.0 a 6.3 cd Phosphorous Acid (Rampart) Copper (MasterCop) 5 Bacillus subtilis QST 713 40.5 c 0.9 b 75.0 bc 3.1 b-d 46.5 bc 1.5 bc 39.0 d 1.1 c 80.5 b 3.3 b 95.0 ab 5.1 d (Serenade Optimum) Copper (MasterCop) + 6 vegetable oil 43.0 c 0.8 b 88.0 a 3.2 b-d 51.5 bc 1.1 cd 48.0 cd 1.3 bc 75.0 bc 2.8 bc 4.6 b 7.2 a a Treatments were sprayed on May 12 (PF – petalfall/shucksplit), May 22-23 (C1), June 2 (C2), June 16 (C3), June 25 (C4), July 7 (C5), and July 22 (C6). b For foliar disease incidence and severity assessment, 5 shoots were randomly tagged and 10 leaves per shoot, starting from the base of the shoot, were assessed for percent bacterial spot incidence and severity (0-100%) twice during the season. The assessment shown here was taken in July 2014. c At harvest ('Easternglo' nectarine on July 25, 'Beekman' peach on August 5, and 'Snow King' peach on August 11), 25 fruit per tree were assessed for percent bacterial spot severity by visually assessing the percent area of fruit covered with bacterial spot lesions. Disease incidence was also assessed. d Values within columns followed by the same letter(s) are not not significantly different (P ≤ 0.05) according to Fisher's Protected LSD test.
91
Table 3-3. Factors associated with the number of colony forming units per gram of peach leaf tissue collected in 2013
Factor DFa Adj SSb Adj MSc F P Sample Date 6 208.643 34.774 25.86 <0.0001 Oxytetracycline Concentration 2 137.057 68.529 50.96 <0.0001 Bactericide Treatment 5 6.853 1.371 1.02 0.407 Cultivar 2 30.318 15.159 11.27 <0.0001 Error 218 293.167 1.345 Total 233 687.532 R2 = 57.36% a Degrees of freedom. b Adjusted sum of squares. c Adjusted mean square.
92
Table 3-4. Factors associated with the number of colony forming units per gram of peach leaf tissue collected in 2014
Factor DFa Adj SSb Adj MSc F P Sample Date 5 319.84 63.97 20.08 <0.0001 Oxytetracycline Concentration 2 455.50 227.75 71.49 <0.0001 Bactericide Treatment 5 13.45 2.69 0.84 0.520 Cultivar 2 73.85 36.93 11.59 <0.0001 Error 201 640.34 3.19 Total 215 1531.63 R2 = 58.19% a Degrees of freedom. b Adjusted sum of squares. c Adjusted mean square.
93
Table 3-5. Oxytetracycline sensitivity of bacterial epiphytes with different tetracycline resistance genes Genusa tetR status nb MICc Pantoea tetR Negative 3 <50 tetA 1 >450 tetB 9 >450 tetC 2 >450 Pseudomonas tetR Negative 2 >450 tetA ...... tetB 1 >450 tetC 2 >450 Xanthomonas tetR Negative 2 <50 tetA ...... tetB ...... tetC 1 >450 a Only bacteria belonging to the most common genera identified were evaluated. b n = sample size c MIC= minimum inhibitory concentration in µg/ml.
94
9
8
7
6
5
4
3
CFU per gram(log10) 2
1
0 140 (B) 154 (B) 168 (C) 179 (B) 196 (AB) 210 (B) 243 (A) Sample Date
Fig. 3-1. Colony forming units per gram (CFU per gram) of leaf tissue collected in 2013 on different sampling dates. The numbers below the bars indicate the Julian day for each sample date where 140 = May 20, 154 = June 3, 168 = June 17, 179 = June 28, 196 = July 15, 210 = July 29, and 243 = August 31. For each date, the number of cfu/g recovered on media amended with 0 µg/ml (green), 10 µg/ml (yellow), and 25 µg/ml (red) oxytetracycline is represented. Different letters following the Julian day indicate significantly different cfu/g according to Tukey’s test.
95
7
6
5
4
3
2 CFU per gram(log10) 1
0
Oxytetracycline Concentration of Media
Fig. 3-2. Colony forming units per gram (CFU per gram) of leaf tissue collected in 2013 (left bars) and 2014 (right bars) on 0, 10, and 25 µg/ml oxytetracycline. Different letters following the antibiotic concentration indicate significantly different cfu/g according to Tukey’s test. The data collected from 2013 and 2014 were analyzed separately.
96
9 8 7 6 5 4 3
2 CFU per gram(log10) 1 0 'Beekman' 'Red Haven' 'Snow King' 'Beekman' 'Easternglo' 'Snow King' (A) (B) (A) (B) (B) (A) Cultivar
Fig. 3-3. Colony forming units per gram (CFU per gram) of leaf tissue collected in 2013 (left bars) from the peach cultivars ‘Beekman’, ‘Red Haven’, and ‘Snow King’ and in 2014 (right bars) from the peach cultivars ‘Beekman’ and ‘Snow King’ as well as the nectarine ‘Easternglo’. For each cultivar, the number of cfu/g recovered on media amended with 0 µg/ml (green), 10 µg/ml (pink), and 25 µg/ml (red) oxytetracycline is represented. Different letters following the cultivar indicate significantly different cfu/g according to Tukey’s test.
97
9
8
7
6
5
4
3
CFU per gram(log10) 2
1
0 156 (B) 171 (B) 177 (B) 192 (B) 206 (B) 254 (A) Sample Date
Fig. 3-4. Colony forming units per gram (CFU per gram) of leaf tissue collected in 2014 on different sampling dates. The numbers below the bars indicate the Julian day for each sample date where 156 = June 5, 171 = June 20, 177 = June 26, 192 = July 11, 206 = July 25, 254 = September 11. For each date, the number of cfu/g recovered on media amended with 0 µg/ml (green), 10 µg/ml (yellow), and 25 µg/ml (red) oxytetracycline is represented. Different letters following the
Julian day indicate significantly different cfu/g according to Tukey’s test.
98
100
80
60
40
Percent Percent ofIsolates 20
0 2013 (n = 418) 2014 (n = 309) Tetracycline Resistance Genes Per Year
TetA TetB TetC TetR Negative
Fig. 3-5. The percentage of collected bacterial epiphytes positive for the presence of tetracycline resistance genes in 2013 and 2014. TetA (blue), tetB (gray), tetC (red), and tetR negative bacteria were represented in both years.
99
100 TetR Positive 80 TetR Negative 60
40
20 Percent Percent ofIsolates
0
Treatment
Fig. 3-6. Percent of epiphytic bacteria positive (black) and negative (gray) for tetracycline resistance genes (tetA, tetB, or tetC) in 2013 and 2014. Treatment 1 = copper (Kocide 3000: 46.1% copper hydroxide; Dupont; Wilmington, DE or MasterCop: 21.46% copper sulfur pentahydrate; Adama, Raleigh, NC), treatment 2 = copper rotated with Rampart (53% Mono- and dipotassium salts of phosphorous acid), treatment 3 = copper rotated with Serenade (14.6% QST 713 strain of dried Bacillus subtilis; AgraQuest; Davis, CA), treatment 4 = copper and vegetable oil, treatment 5 = lime sulfur rotated with copper, treatment 6 = oxytetracycline (18.3% oxytetracycline; AgroSource; Cranford, NJ), treatment 7 = Regalia (5% extract of Reynoutria sachalinensis; Marrone Bio Innovations, Inc. Davis, CA) rotated with Serenade, and treatment 8 = untreated. The incidence of tetR positive bacteria differed significantly (Χ2 = 32.98, P <0.0001) among bacterial epiphytes collected from differing bactericide treatments based off of the Pearson chi-square statistic.
100
100
80
60
40
Percent Percent ofIsolates 20
0 TetA (n = 24) TetB (n = 131) TetC (n = 27) Total (n = 182) Bacterial Epiphytes
Pseudomonas Pantoea Bacillus Xanthomonas Curtobacterium
Fig. 3-7. The incidence of epiphytic bacteria positive for tetA, tetB, and tetC among species belonging to the genera Pseudomonas (green), Pantoea (yellow), Bacillus (red), Xanthomonas (blue), and Curtobacterium (orange).
101
Chapter 4
Oxytetracycline sensitivity of Xanthomonas arboricola pv. pruni isolates
collected from Pennsylvania stone fruit orchards
ABSTRACT
Xanthomonas arboricola pv. pruni isolates (n = 830) were collected from commercial stone fruit orchards in Pennsylvania. The sensitivity of these isolates to oxytetracycline was evaluated on a culture medium amended with 25 µg/ml of the antibiotic and were screened for the presence of tetracycline resistances genes (tetB and tetC). The relationship between the incidence of isolates capable of growth on medium amended with 25 µg/ml of oxytetracycline and orchard management factors was determined. Neither tetB or tetC genes were found in the Xap isolates tested. However, there was variability in sensitivity of Xap to oxytetracycline among isolates collected from different orchards. The number of oxytetracycline and copper applications made in the year of and the year prior to sample collection (P > 0.0001), tree age (P = 0.0006), sample collection year (P > 0.0001), and oxytetracycline application method (i.e.: alternate row middle or complete application) (P > 0.0001) were significant factors influencing this variability in sensitivity.
INTRODUCTION
102
Bacterial spot, caused by Xanthomonas arboricola pv. pruni (Smith) Vauterin et al. (Xap), is considered the most important bacterial disease of stone fruit in the eastern
United States where peach production is second only to apple in yield per year and dedicated farm land. Stone fruit cultivation in the eastern United States is largely centered in 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). Pennsylvania is the fifth largest producer of peaches in the country behind California, South Carolina, Georgia, and New Jersey. Nearly 17,000 ha of orchards are concentrated in the south central part of the state.
Bacterial spot severely limits peach production in the eastern United States, including Pennsylvania, where the weather is often warm wet and conducive to rapid disease development. Bacterial spot is a polycyclic disease and is heavily dependent on temperature and leaf wetness. Symptoms are often yield limiting and include lesions on fruit and leaves as well as cankers on twigs and branches (Zehr et al., 1996). Lesions on leaves first appear as water soaked, angular spots that eventually darken to brown and often fall out of the leaves, leaving a shot-hole appearance. Leaf yellowing and premature defoliation is also common (Ritchie, 1995). Severe premature defoliation reduces the overall photosynthetic competence of the remaining leaves when disease severity is high.
Defoliation caused by bacterial spot is frequently associated with reduced fruit quality due to poor nutritional uptake as well as fruit sun burn owing to the over exposure of ripening fruit. Bacterial spot lesions also occur on fruit where early lesions, water soaked in appearance, first form about three weeks after petal fall and eventually darken and enlarge.
Coalescing lesions may cause the skin to crack which regularly favors secondary infection 103 by the brown rot fungus, Monilinia fructicola, as well as other pre- and post-harvest fungal rots (Ritchie, 1995). Twig symptoms consist of cankers that initially appear water soaked and eventually enlarge, cracking the surface of the bark. Bacteria overwinter in cankers that form on branches and twigs.
Bacterial spot is a difficult disease to manage, especially on highly susceptible stone fruit cultivars. Because of that, tree fruit growers rely heavily on the use of chemical bactericides, including copper compounds and the antibiotic oxytetracycline, to mitigate disease symptoms. 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 overwintering inoculum (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 but at high enough rates are phytotoxic and often result in leaf discoloration, curling, and premature defoliation (Lalancette and
McFarland, 2007). The antibiotic oxytetracycline has been used with significant disease suppression but is registered for use on peaches and nectarines only. Applied at a ten to 14 day interval, up to ten applications may be made per season depending on the cultivar susceptibility and date of harvest.
Despite repeated applications of these chemical bactericides, severe symptoms of bacterial spot often develop and yield loss is frequently high. Many Pennsylvania stone fruit growers have expressed concern over the potential development of antibiotic resistance in the causal pathogen, Xap to the antibiotic oxytetracycline. Resistance to tetracycline antibiotics, such as oxytetracycline, is well characterized with 43 different known tetracycline resistance genes and four mechanisms of resistance (Roberts, 2012; 104
Pringle et al., 2007; Spaunaric et al., 2005; Connell et al., 2003a; Connell et al., 2003b; Di
Francesco et al., 2008; Nonaka et al., 2005; Chopra and Roberts, 2001). Although mutations altering the 16S rRNA, 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 (Chopra and Roberts, 2001).
Nevertheless, oxytetracycline resistance has not been confirmed in Xanthomonas spp.
Therefore, the objectives of this research were to determine the current levels of oxytetracycline sensitivity in Xap, the prevalence of two tetracycline resistance genes (tetB and tetC) in Xap, and the orchard management practices and factors associated with oxytetracycline sensitivity and the incidence of resistance genes.
MATERIALS AND METHODS
Sample collection, bacterial isolation, and identification of Xap isolates:
Extensive sampling of commercial stone fruit orchards in Pennsylvania was conducted in
2011 and 2012. Xap isolates were collected in June and July from six and eight commercial stone fruit orchards, respectively, in 2011 and 2012. The orchards were located in Adams,
Franklin, Lancaster, Chester, and Delaware Counties, PA. Isolates were also collected from two research plots located at the Penn State Fruit Research and Extension Center (PSU
FREC) in Biglerville, PA. Attempts to sample the two organic orchards (see chapter 2) were made but because no symptoms of bacterial spot were found, Xap was not collected from either organic orchard. While most of the orchards sampled in the chapter 2 for 105 epiphytic bacteria were also sampled for bacterial spot, additional orchards were sampled for bacterial spot in this chapter and therefore the number assigned to orchards in figures showing the results in chapter 2 are not identical to those referred to in this chapter.
Symptomatic leaf tissue was collected from 20 trees throughout a single orchard block in a “W” sampling pattern due to the random distribution of this disease in the orchard. Two leaves per tree were used to isolate Xap by slicing a bacterial spot lesion under sterile water with a sterilized scalpel and allowing the bacteria to stream into the sterile water. A loop was used to transfer streaming bacteria to a plate of sucrose peptone agar (Schaad et al.,
2001). Two characteristic yellow colonies per leaf were then sub-cultured and verified as
Xap through PCR and specific primers Y17coF and Y17CoR (Pagani, 2004; Pothier et al.,
2011a). Isolates were then transferred to nutrient broth (Schaad et al., 2001) and grown for
24 to 48 hours before being preserved at -80° C in sterile 25% glycerol.
Oxytetracycline sensitivity of Xap isolates: Xap isolates were grown in Kings B broth amended with six concentrations (0, 5, 10, 15, 20, and/or 25 µg/ml) of the antibiotic oxytetracycline (oxytetracycline dehydrate; Sigma-Aldrich; St. Louis, MO) in triplicates in a 96-well plate. Each well was filled with 300 µl of sterile amended broth. A sterile toothpick was used to inoculate each well, except for the negative control wells, by dipping the end lightly into a 3 to 4 day culture grown on Kings B agar. A spectrophotometer was used to measure optical density at 600nm to determine cell growth at 0, 24, 48, and 72 hours. Sensitivity was evaluated based on the ratio of absorbance values measured at 0 and
72 hours for isolates grown in media amended with 25 µg/ml. Isolates were classified as
“sensitive” if the ratio of absorbance values measured at hour 0 and hour 72 was less than or equal to 1.4 (i.e.: Abs72/Abs0 ≤ 1.4). An isolate was classified as “less sensitive” if the 106 ratio of absorbance values measured at hour 0 and hour 72 was greater than or equal to
1.45 (i.e.: Abs72/Abs0 ≥ 1.45). The classification of “resistant” was reserved only for those isolates carrying tetracycline resistance genes.
Identification of tetracycline resistance genes in Xap: Xap isolates were screened for the presence of two previously described tetracycline resistance genes, tet(B) and tet(C), using PCR and specific primers tet(B)-F, tet(B)-R, tet(C)-F, and tet(C)-R (Ma et al.,
2007). These two genes were the most common genes found in epiphytic bacterial communities collected from some of the same orchards (Chapter 2). The frozen culture was used as the template and was introduced to each 25 µl reaction with a sterile toothpick.
Choice Taq Mastermix (Denville Scientific; Holliston, MA) was used. Forward primer tet(B)-F (5’-CAGTGCTGTTGTTGTCATTAA-3’) and reverse primer tet(B)-R (5’-
GCTTGGAATACTGAGTGTTAA-3’) amplified a 571bp product (GenBank accession no.: V00611). The tet(C)-F forward primer (5’-CTTGAGAGCCTTCAACCCAG-3’) and the reverse primer tet(C)-R (5’-ATGGTCGTCATCTACCTGCC-3’) amplified a 418bp product (GenBank accession no.: J01749) (Ma et al., 2007). The thermocycler parameters included a 112C heated lid and an initial denaturation of 96C for 10 minutes. After that, 34 cycles were completed of 96C denaturation for 30 seconds, 55C annealing for 1 minute, and 72C elongation for 30 seconds. The products were stored at 4C and run through 1.5% agarose gel in TAE buffer using EZ-Vision loading dye (Amresco; Solon, OH). Gels were visualized under a KODAK gel imaging system. With each cycler run, a positive and negative control were included. Positive controls for tetB and tetC were isolates of previously screened Pantoea spp. recovered from the PSU Fruit Research and Extension
Center in Chapter 3. The negative control was a sensitive isolate of Xap. 107
Grower survey on management factors and data analysis: During sample collection, a handwritten survey of bacterial spot management practices was completed by the corresponding grower or farm manager (Appendix). Data were obtained on the number of oxytetracycline and copper applications made in the year of and the year prior to sample collection, the use of dormant copper, and tree age. The spray coverage method was also surveyed to determine if growers applied oxytetracycline and copper in complete sprays
(i.e.: spray both sides of the tree) or alternate row middle (i.e.: spray only one side of the tree every other application).
The data analyzed consisted of a binary response variable with a score of “0” for isolates that were considered “sensitive” or negative for a resistance gene and “1” for those considered “less sensitive” or positive for a resistance gene. Initially, a Pearson’s chi- square statistic was calculated using the FREQ procedure of SAS (version 9.4; SAS
Institute Inc.) to assess the association between orchard management factors and the incidence of shifted sensitivity of Xap isolates as well as the incidence of tetR genes. The relationship between orchard management factors and the incidence of tolerant isolates and the incidence of tetR genes was further evaluated using generalized linear mixed models using the GLIMMIX procedure of SAS, similar to that used by Pfeufer and Ngugi (2012).
Orchard management factors with a significant effect on Xap sensitivity to oxytetracycline were determined with type III tests of fixed effects. Parameter estimates and standard errors of significant orchard management factors along with odds ratios and their 95% confidence intervals were obtained with the lsmeans statement.
RESULTS 108
Oxytetracycline sensitivity of Xap isolates: A total of 351 and 479 Xap isolates, collected in 2011 and 2012, respectively, were tested for oxytetracycline sensitivity. In general, sensitivity to 25 µg/ml oxytetracycline was high among Xap isolates. Only 16.0% of the 830 Xap isolates tested grew adequately on 25 µg/ml oxytetracycline and were considered less sensitive while 84.0% were sensitive to 25 µg/ml. All Xap isolates were completely inhibited by 50 µg/ml oxytetracycline. The incidence of less sensitive isolates varied significantly among the sampled orchards (Χ2 = 198.18, P <0.0001) (Fig. 4-1). Xap isolates collected from orchards 4, 9, and 12 were all considered sensitive and the largest percentage of less sensitive isolates came from orchards 8 (37.7%), 10 (54.4%), and 11
(35.2%) (Fig. 4-1).
Management factors and orchard practices associated with the sensitivity of Xap isolates to oxytetracycline: Based on the Pearson chi-square statistic (χ2 = 83.73; P
<0.0001), the number of oxytetracycline applications was a significant factor affecting the variability of Xap sensitivity among sampled orchards whereby 27.1% of isolates collected from orchards sprayed with more than three applications of oxytetracycline were considered less sensitive while only 3.8% of isolates from orchards with three or fewer applications were less sensitive to oxytetracycline (Fig. 4-2). This conclusion was supported by the results of the mixed effects model which indicated that the odds of an isolate from orchards receiving greater than three oxytetracycline applications being shifted in sensitivity (i.e. less sensitive) were more than 11 times those of an isolate from orchards with three or fewer applications (odds ratio = 11.459; 95% CI = 6.403 to 20.511;
P <0.0001) (Table 4-1). Tree age was significantly associated with Xap sensitivity to 25 109
µg/ml oxytetracycline (χ2 = 11.81, P = 0.0006). A larger percentage (19.5%) of less sensitive Xap isolates were collected from older trees than from younger trees (10.4%)
(Fig. 4-2). This was also supported by the mixed effects model (odds ratio = 3.280; 95%
CI = 2.020 to 5.327; P < 0.0001) (Table 4-1). Application method (i.e.: “complete” application versus “alternate row middle”) was a significant factor influencing the sensitivity of Xap isolates to 25 µg/ml oxytetracycline (χ2 = 15.36, P < 0.0001) (Fig. 4-2).
Trees sprayed in an “alternate row middle” fashion had a smaller percentage (11.3%) of less sensitive isolates than those collected from trees that had been sprayed in a “complete” manner (21.3%). This conclusion, however, was not supported by the fixed effects model
(odds ratio = 0.942; 95% CI = 0.489 to 1.816; P = 0.8584) (Table 4-1). Year of sampling was also a significant factor influencing the variability in sensitivity of Xap isolates based on Pearson’s chi-square statistic (χ 2 = 19.0; P < 0.0001) (Fig. 4-2). Nearly twice as many less sensitive Xap isolates were collected in 2011 (22.5%) as were collected in 2012
(11.3%). Finally, based on the mixed effects model, the number of copper applications made in the year of and the year prior to sample collection was a significant factor influencing Xap sensitivity to 25 µg/ml oxytetracycline (odds ratio = 0.207; 95% CI =
0.108 to 0.397; P <0.0001) (Table 4-1). Broken into two groups, a larger percentage
(27.6%) of Xap isolates collected from trees sprayed with three or fewer applications of copper were considered less sensitive compared to the 10.3% of Xap isolates collected from trees sprayed with greater than three applications of copper (Fig. 4-2). In addition, over four times as many less sensitive Xap isolates were collected from orchards that did not use dormant copper (44.6% versus 10.3%) (χ 2 = 101.35; P < 0.0001). 110
Tetracycline resistance genes in Xap isolates: A total of 449 and 679 isolates, collected from 2011 and 2012, respectively, were screened for the presence of tet(B) and tet(C), the most common tetracycline resistance genes found in bacterial epiphytes from some of the same orchards (Chapter 2). None of the Xap isolates, however, were positive for these two tetracycline resistance genes. Positive controls, previously characterized bacteria positive for either tet(B) or tet(C), produced PCR bands at their respective sizes.
DISCUSSION
The antibiotic oxytetracycline is applied to trees at a concentration of 150 µg/ml.
None of the Xap isolates in this assay grew at 50 µg/ml of oxytetracycline. The results of this research indicate that total resistance (i.e.: resistance to 150 µg/ml and above) to oxytetracycline in Xap has not occurred in the sampled orchards. This observation is in agreement with other investigations on tetracycline resistance in Xap (McManus et al.,
2002). The resistance genes investigated in this study, tetB and tetC, were not found in any of the Xap isolates collected from commercial or research stone fruit orchards. In another study on mostly European isolates of Xap, Pothier et al. (2011b) also found no resistance genes. This was not surprising given that tetracycline resistance or any other resistance to antibiotics has not been found in Xap (McManus et al., 2002). No tetracycline resistance has been reported in the Antibiotic Resistance Data Base (ARDB) in any other
Xanthomonas spp. (http://ardb.cbcb.umd.edu/cgi/search.cgi?db=R&and0=O&term
=xanthomonas&field=a&). Streptomycin resistance, however, has been found in pathovars of X. campestris (Sundin, 2000; Minsavage et al., 1990). Resistance to copper, a common 111 antimicrobial compound used to manage bacterial spot of stone fruit, has also been found in species of Xanthomoas but has not yet been reported in Xap (Stall et al., 1986; Basim et al., 2005; Behlau et al., 2011).
The results of this study suggest a high variability in sensitivity to oxytetracycline to populations of Xap from different orchards (Fig. 4-1). As in chapter 2, it was hypothesized that this variability was due to the different management strategies and orchard factors employed by growers and farm managers. Based on the responses collected through the survey completed by each orchard manager, these factors were evaluated and included tree age, sample collection year, spray application method, oxytetracycline applications, and copper use.
The number of oxytetracycline applications varied greatly among the sampled orchards where oxytetracycline was applied between 0 and 13 times in the year of and the year prior to sample collection. The number of oxytetracycline applications was related to the variability in sensitivity of Xap isolates to 25 µg/ml oxytetracycline (Table 4-1).
Moreover, orchards 8, 10, and 11 (Fig. 4-1) with the highest percentages of isolates shifted in sensitivity (i.e. less sensitive) also applied the greatest number of oxytetracycline applications (between eight and 13 applications). The data obtained in this study indicate that frequent applications of oxytetracycline may reduce the sensitivity of Xap populations to the antibiotic. This was expected. These results are in agreement with research conducted on Escherichia coli isolates pre-exposed to various classes of antibiotics. Those exposed to antibiotics were able to grow on media with higher levels of antibiotic than those not previously exposed to the antibiotics (Wiuff et al., 2005). Similar observations have been made in plant pathogenic fungi such as Venturia inaequalis where isolates exposed to more 112 fungicide applications also had a higher relative growth and relative growth rate on media amended with the same fungicide than isolates obtained from orchards that had been treated with less fungicide (Pfeufer and Ngugi, 2012).
Copper applications also differed among orchards in varying amounts during the growing season to manage bacterial spot, either alone or in rotation with oxytetracycline.
The number of applications ranged from a single half application (as applied with the alternate row middle method) to 15 applications of copper in the year of and the year prior to sample collection. Copper was a significant factor influencing the variability of sensitivity of Xap isolates to 25 µg/ml (Table 4-1). Unlike oxytetracycline applications, however, Xap collected from trees sprayed with greater than three applications of copper had a smaller percentage of less sensitive isolates than those collected from trees that were sprayed with less than three applications of copper. Dormant copper also greatly reduced the number of Xap isolates capable of growth at 25 µg/ml oxytetracycline. This supports the theory behind using copper in that it reduces the overall number of oxytetracycline applications and prevents oxytetracycline resistant bacteria as well as those that have become less sensitive to the antibiotic from dominating the population by lowering the initial population of Xap surviving from the previous season prior to the start of antibiotic use in the spring. In other studies, increased exposure to one antimicrobial has been associated with reduced sensitivity to other antimicrobial compounds (Wiuff et al., 2005;
Kurenbach, 2015). The effect of copper use on copper sensitivity in Xap was not addressed in this study. Copper resistance has been reported in other species of Xanthomonas so caution should be used when using copper as a sole bactericide to manage bacterial spot
(Stall et al., 1986; Basim et al., 2005; Behlau et al., 2011) (Fig. 4-2). 113
The age of trees also varied greatly among the sampled orchards with a range in age from 2 to 16 years and was a significant factor influencing the variability in sensitivity of Xap isolates to 25 µg/ml oxytetracycline. Xap isolates collected from older trees were three times more likely to be less sensitive than isolates collected from younger trees (Table
4-1; Fig. 4-2). A possible interpretation of this is that older trees presumably have received a greater exposure to oxytetracycline over the years than younger trees, thereby increasing the likelihood of becoming less sensitive. This observation, again, was similar to that found by Pfeufer and Ngugi (2012) where a larger percentage of V. inaequalis isolates resistant to fenbuconazole were collected from older trees (greater than 20 years).
The method of oxytetracycline application was significantly associated with the variability in Xap sensitivity (Table 4-1). A greater percentage of Xap isolates collected from trees that received complete coverage with oxytetracycline were considered shifted in sensitivity (i.e. less sensitive) than those collected from trees that were sprayed with the alternate row middle application method. Growers that used this method used less oxytetracycline overall which may relax the selection pressure imposed on bacteria to become less sensitive. However, in order for oxytetracycline to be effective, it must come in contact with the bacteria to prevent growth and infection. Complete coverage of the foliage, while it may use more oxytetracycline, also ensures that more of the tree comes into contact with an effective dose of the product. Other studies have shown that repeated applications of less effective levels of the antibiotic lead to antibiotic resistance due to adaptive resistance (Kohanski et al., 2010; Stokes and Gillings, 2011; Chopra and Roberts,
2001; Wiuff et al., 2005; Kurenbach, 2015). 114
The percentage of tolerant isolates also varied significantly between years (Fig. 4-
2). A larger percentage of shifted isolates were collected in 2011 than 2012. On average, nearly the same number of oxytetracycline applications were made in both years (5.5 applications in 2011 and 5.4 applications in 2012). Bacterial spot is highly dependent upon the weather and is often worse in some years than others (Ritchie, 1995). Primary inoculum levels and weather conditions often influence disease severity (Zehr et al., 1996; Ritchie,
1995). The 2011 isolates were collected in June rather than July like the 2012 isolates.
Application of oxytetracycline is often more concentrated in the beginning of the season due to more frequent rain and dew events than in the dryer July weather in Pennsylvania.
While it should be expected that the 2012 Xap isolates have a higher percentage of tolerant isolates, this observation indicates that sensitivity to oxytetracycline is fluid within a single growing season with more less sensitive isolates in the beginning of the season than at the end of the growing season. Further investigation should be done to determine if disease severity, weather, and time of the growing season are also related to the variability in sensitivity of Xap to oxytetracycline. Nevertheless, this also indicates that the percentage of less sensitive isolates is not gradually increasing overtime; however, a longer term study is needed to evaluate this trend.
While potential management factors associated with the variability in sensitivity in
Xap isolates to 25 µg/ml oxytetracycline have been identified, the mechanisms causing this variability in a population of bacteria, otherwise lacking genetic diversity, have not. It was expected that this variability was caused by the acquisition of tetR genes by Xap isolates.
However, tet(B) and tet(C), the most common tetracycline resistance genes found in epiphytic bacteria (Chapters 2 and 3) were not found in the screened Xap isolates. The 115 reduction of sensitivity recorded in the Xap isolates was not comparable to that associated with the acquisition of tetA, tetB, and tetC which conferred high levels (> 450 µg/ml oxytetracycline) of resistance in bacteria like Pantoea spp. and Pseudomonas spp. in chapter 2. There are tetracycline resistance genes that confer low levels of resistance to tetracycline antibiotics (Nonaka et al., 2005; Roberts, 2012). Certain mutations conferring low level resistance to oxytetracycline are possible although rare (Chopra and Roberts,
2001). Moreover, a novel tetracycline resistance gene could be the cause as new and even chimeric resistance genes (genes made up of two or more resistance genes) are continuously adding to the 43 currently documented tetracycline resistance genes (Roberts,
2012). Finally, resistance gene acquisition and gene mutation are not the only mechanism of antibiotic resistance. Non-inherited resistance, in the form of adaptive resistance or phenotypic tolerance, often results in low level, reversible resistance (Wiuff et al., 2005;
Kurenbach, 2015). Adaptive resistance occurs in bacteria after they are exposed to gradually increasing levels of antibiotic. After the exposure to the antibiotic has ceased, the bacteria return to the non-resistant phenotype, making this resistance reversible. For example, Wiuff et al. (2005) showed that genetically sensitive Escherichia coli CAB1 was phenotypically tolerant to five different classes of antibiotics (ampicillin, ciprofloxacin, rifampin, streptomycin, and tetracycline) after gradual exposure to them. This tolerance was reversible after antibiotic exposure ended. Sandoval Motta et al. (2015) found that this phenomenon is related to the variability in gene expression regulating efflux pumps and is costly to cell growth. While adaptive resistance studies are gaining popularity, most have focused on bacterial growth and antibiotic sensitivity in vitro. It is unclear what role adaptive resistance may play in the sensitivity of Xap to oxytetracycline in the field. 116
Nevertheless, a mechanism for this variability in sensitivity of Xap to oxytetracycline should be further examined to better understand potential mechanisms involved in the development of resistance to oxytetracycline in Xap.
Overall, the results of this study indicate that the reason oxytetracycline has repeatedly failed to provide effective management of bacterial spot is not due to antibiotic resistance in the pathogen populations – Xap remains sensitive to the field rate of oxytetracycline (150 µg/ml). Rather, oxytetracycline rapidly degrades when exposed to
UV light, effective for two days under optimal conditions, and is easily washed from the leaves after a heavy rain, only two minutes in a heavy downfall (Christiano et al., 2010).
Because oxytetracycline lacks systemic activity, it is not able to prevent the growth of bacteria that have already invaded and colonized the internal leaf tissues. Carefully timed applications of the antibiotic are critical to minimize degradation from environmental conditions and to target the largest population of Xap on the surface of the leaves before ingress into the leaves. Further research on the best timing of oxytetracycline rather than application on a calendar basis would likely improve the perceived effectiveness of this antibiotic in the field. The use of the alternate row middle method of oxytetracycline application could also contribute to the antibiotic’s lack of effectiveness. Even though a greater percentage of sensitive isolates were recovered from orchards that used the alternate row middle method, this method often provides incomplete coverage of the leaves and therefore a greater incidence of disease. Complete coverage may be necessary to reduce disease incidence to acceptable levels, despite its association with a greater percentage of less sensitive Xap isolates. Special care should be used for the best possible application to ensure the most effective use of oxytetracycline. 117
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123
Table 4-1. Orchard management factors associated with the level of sensitivity of Xap isolates to oxytetracycline Factora Num. Dfb Den. Dfb Odds Ratio 95% CIc F valued P value Tree Age 1 824 3.280 2.020-5.327 23.12 <0.0001 Year 1 824 0.387 0.243-0.614 16.25 <0.0001 Application Method 1 824 0.942 0.489-1.816 0.03 0.8584 Oxytetracycline Applications 1 824 11.459 6.403-20.511 67.62 <0.0001 Copper Applications 1 824 0.207 0.108-0.397 22.64 <0.0001 a Factor responses were based on the grower surveys collected at sampling. Management or orchard factors included tree age (<9 years or >9 years), year of sampling (2011 or 2012), oxytetracycline application method (alternate row middle or complete spray application), the number of oxytetracycline applications (≤3 applications or >3 applications), and number of copper applications (<3 applications or ≥3 applications. b Num. Df is the numerator degrees of freedom while Den. Df is the denominator degrees of freedom adjust for bias with the Kenward- Roger's method (Pfuefer and Ngugi, 2012; Kenward and Roger, 1997). c confidence interval. d F-value is ratio of mean squares.
124
60
40
20 Percent Percent ofIsolates
0
Orchard
Fig. 4-1. Percent of Xap isolates collected in 2011 and 2012 from commercial stone fruit orchards that are considered “sensitive” (black bars) and “less sensitive” (gray bars) to 25 µg/ml oxytetracycline in vitro. The sensitivity of Xap isolates varied significantly among the sampled orchard (Χ2 = 198.18, P <0.0001) based on the Pearson chi-square statistic. All isolates collected from orchards 4, 9, and 12 were considered sensitive while Xap isolates collected from orchards 8, 10, and 11 had the highest percent of less sensitive isolates.
125
100
75 Percent Percent of Isolates (n 830) =
50
Orchard Management Factor
Fig. 4-2. The effect of tree age, the oxytetracycline application method (ARM = alternate row middle), 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” (black bars) and “less sensitive” (blue bars) to 25 µg/ml oxytetracycline in vitro. Based on the Pearson chi- square statistic, the percentage of “less sensitive” Xap isolates was significant for tree age (P = 0.0006), application method (P <0.0001), sample collection year (P <0.0001), dormant copper application (P < 0.0001), and oxytetracycline (P < 0.0001). Copper cover application was significant based on the fixed effects model (P < 0.0001).
126
Chapter 5
Discussion
The overall goal of this research was to determine the consequences associated with repeated applications of the antibiotic oxytetracycline in Pennsylvania stone fruit orchards with respect to bacterial spot caused by Xanthomonas arboricola pv. pruni (Xap). The first objective was to determine the incidence of tetracycline resistance among epiphytic bacteria in commercial orchards and the management strategies associated with tetracycline resistant bacteria (Chapter 2). Epiphytic bacteria were recovered from leaves collected from orchards. The epiphytes were identified to genus level and screened for the presence of tet(A), tet(B), and tet(C) using PCR and specific primers. Generalized linear mixed models were used to determine if management practices and orchard factors, including oxytetracycline and copper use, were associated with the incidence of tetracycline resistance genes (Chapter 2). The second objective was to evaluate the effects of bactericide use on the incidence of tetracycline resistance genes over the course of a growing season, while evaluating the efficacy of different bactericides and product rotation programs (Chapter 3). Epiphytes were collected three to five days after treatment application in the product evaluation site at the PSU FREC. They were counted and a total of 727 epiphytic isolates were collected, stored, and screened for tet(A), tet(B), and tet(C).
Bacteria positive for one of the three tetracycline resistance genes were identified to genus level. Foliar and fruit disease severity and incidence data on bactericide efficacy was also collected (Chapter 3). The final objective was to determine the sensitivity of Xap isolates, collected from commercial orchards, to the antibiotic oxytetracycline (Chapter 4). Xap was 127 isolated from symptomatic leaves, screened for oxytetracycline sensitivity on media amended with six concentrations (0, 5, 10, 15, 20, and/or 25 µg/ml) of the antibiotic oxytetracycline. Xap isolates were also screened for the presence of tet(B) and tet(C) genes.
Finally, generalized linear mixed models were used to determine the association of antibiotic sensitivity in Xap to bacterial spot management practices and orchard factors
(Chapter 4).
Studies similar to this were conducted to examine the prevalence of genes conferring resistance to tetracycline and kasugamycin among epiphytic bacteria in
Michigan apple orchards. These studies were done before the two antibiotics were used to replace streptomycin in orchards where streptomycin resistance in Erwinia amylovora (the causal agent of fire blight of pome fruit) was widespread (Schnabel and Jones, 1999;
McGhee and Sundin, 2011). Previous studies showed that nontarget (i.e.: epiphytic) bacteria often harbor antibiotic resistance genes earlier and more frequently than co- existing pathogenic populations (Levy and Marshall, 2004). For example, it has been hypothesized that the development of streptomycin resistance in E. amylovora occurred due to the acquisition of a plasmid carrying streptomycin resistance genes from the orchard epiphyte Pantoea agglomerans (Chiou and Jones, 1993). Because oxytetracycline is commonly used to manage bacterial spot, it was important to evaluate the prevalence of bacteria carrying known tetracycline resistance genes in Pennsylvania stone fruit orchards.
Previous collection of tetracycline resistant bacteria, including species of Panotea, from peach trees located at the PSU FREC in addition to raised concerns among stone fruit growers about the reduction in efficacy of oxytetracycline in the management of bacterial spot, prompted the evaluation of antibiotic sensitivity among Xap isolates and of epiphytic 128 bacteria carrying potentially transferable tetracycline resistance genes. It was hypothesized that the incidence of tetracycline resistance in epiphytic bacteria was positively correlated with oxytetracycline use (Chapters 2 and 3). It was also hypothesized that the sensitivity of Xap to oxytetracycline was related to oxytetracycline used in the management of bacterial spot and that the variability in sensitivity in Xap was due to the acquisition of tetracycline resistance genes from resistant epiphytic bacteria (Chapter 4).
The incidence of tetracycline resistant epiphytic bacteria was not related to oxytetracycline use (Chapter 2). Tetracycline resistance genes were found where no oxytetracycline had ever been applied, such as organic orchard 8 in Chapter 2. In epiphytic bacteria collected in the product evaluation site at the PSU FREC, treatment was not related to the incidence of tetracycline resistance genes (Chapter 3). The results of this research indicate that the use of oxytetracycline in the management of bacterial spot has not influenced the incidence of tetracycline resistance genes in epiphytic bacteria found in
Pennsylvania stone fruit orchards. Although not originally hypothesized, this was not entirely unexpected because numerous other studies have shown no relation between antibiotic use and resistant epiphytic bacteria (Smith, 1967; Houndt and Ochman, 2000;
Hughes and Datta, 1983; Mindlin et al., 2001, 2005; Walson et al., 2001; Bartoloni et al.,
2004; McManus, 2014). However, it is unclear why the incidence of tetracycline resistance genes in bacteria was not correlated with use of oxytetracycline. The results of this study as well as other studies seem to contradict what is expected. Selection of antibiotic resistance as a result of repeated antibiotic use has been previously identified in plant agricultural settings, specifically in E. amylovora (McManus, 2014). It is possible that the selection pressure is not as strong as previously thought. In fact, oxytetracycline readily 129 breaks down under environmental conditions (Christiano, 2010), and the product application techniques may not result in an even coverage of the leaf surface, depending on the foliage density and the structure of the canopy. This may leave some leaves and their epiphytic communities not exposed to oxytetracycline and thus, not subjected to selection (Shade et al., 2013). Based on results of Chapters 2 and 3, it is likely that oxytetracycline use alone is not a strong enough selection pressure but likely acts in concordance with other selectors including environmental conditions and disease management practices (including cultivar selection).
The most common culturable bacteria recovered from commercial orchards belonged to the genera Pseudomonas, Pantoea, and Xanthomonas. This was expected since both Pseudomonas and Pantoea spp. are common among epiphytic bacteria found in orchards (Schnabel and Jones, 1999; McGhee and Sundin, 2011, Yashiro and McManus,
2012). Xanthomonas spp. were also expected because Xap is often found as an epiphyte on the surface of the leaves (McGhee and Sundin, 2011; Shepard and Zehr, 1994; Battilani et al., 1999; Zehr et al., 1996). Xanthomonas spp. were present in orchards where bacterial spot was found. The two organic orchards, free of bacterial spot in 2012 and 2013, had a small percentage or no Xanthomonas spp. comprising their bacterial communities (Chapter
2). Bacillus spp. however, were only found in the two organic orchards. No orchard, including either of the organic orchards, reported using a Bacillus-based product. While variation in epiphytic bacteria was expected, it was not anticipated that bacterial genera would be excluded from the communities. Many studies have shown no difference between bacterial communities before and after bactericide application or among agricultural settings exposed to varying levels of antibiotics (Vanbroekhoven et al., 2004; Rodríquez et 130 al., 2006; Rodríguez-Sánchez et al., 2008; Yashiro and McManus, 2012; Yashiro et al.,
2011; Shade et al., 2013; Walsh et al., 2014). The results of this research suggest that the culturable bacterial communities associated with conventional and organic orchards were indeed different. While oxytetracycline use is the obvious explanation for these observed differences, the results of this research indicate that some other factor(s), such as cultivar or sample collection date (Chapter 3) may be responsible. Deep sequencing techniques, allowing the examination of a greater portion of the bacterial communities associated with stone fruit orchards could improve this research and enable a more thorough assessment of the bacterial diversity under antibiotic selection pressure.
The results of this research indicate that oxytetracycline use did seem to statistically influence the variability in sensitivity of Xap isolates collected from commercial orchards
(Chapter 4). This was originally hypothesized. In fact, copper use, either dormant or cover applications, greatly reduced the likelihood of Xap isolates growing on media amended with 25 µg/ml oxytetracycline (Chapter 4). This was expected because copper often replaced oxytetracycline applications and reduced the overall exposure of trees to the antibiotic. However, no Xap isolates positive for tet(B) and tet(C) were found. This was unexpected because it was originally predicted that the molecular mechanism causing the variability in sensitivity in Xap was a tetracycline resistance gene. Tet(B) and tet(C) were the most common resistance genes among the epiphytic bacteria (Chapters 2 and 3).
However, the development of antibiotic resistance is not limited to chromosomal mutation or the acquisition of a resistance gene. Non-inherited resistance such adaptive resistance or phenotypic tolerance results in a gradual loss of sensitivity to an antibiotic. This form of resistance is often reversible after exposure to an antibiotic ends (Kohanski et al., 2010; 131
Stokes and Gillings, 2011; Chopra and Roberts, 2001; Wiuff et al., 2005; Kurenbach,
2015). This phenomenon should be further examined in populations of Xap to understand how it may influence the development of resistance. It is unclear if or how high baseline sensitivity may become under increased exposure to oxytetracycline. Although effective alternatives to oxytetracycline such as copper and Bacillus-based products exist and could easily replace oxytetracycline (Chapter 4), the loss of any tool in the disease management toolbox is a limitation, not necessarily a direct threat, to production. Efforts to reduce the potential selection pressure associated with any one specific bactericide or management strategy should be taken.
Although tetracycline resistance has not previously been reported in any
Xathomonas spp., tetR positive isolates of Xathomonas spp. were found in Chapters 2 and
3. It was expected that isolates of Xanthomonas spp. carrying tetR genes would be found; however, it was also expected that these isolates would be recovered among epiphytic and pathogenic bacteria. These isolates, recovered only as epiphytic bacteria, will be further identified to species level and investigated to determine what role, if any, they play in the development of tetracycline resistance in Xap. It is possible that these Xanthomonas spp. are Xap and pathogenic to stone fruit trees.
This research was severely limited by the number of tetracycline resistance genes evaluated. There are over 40 currently known tetracycline resistance genes and while the three genes studied in this research are among the most common resistance genes in Gram- negative bacteria (Chopra and Roberts, 2001), the research would have been more comprehensive through the inclusion of more resistance genes in the screening. Indeed, thousands of epiphytic bacteria and Xap isolates were screened for the presence of tetA, 132 tetB, and tetC; however, an alternative strategy could have included screening for more resistance genes in fewer bacterial isolates. Another possible method could have used real- time PCR and DNA extracted from the bacterial leaf rinsate (Walsh et al., 2010; Popowska et al., 2012; Duffy et al., 2014; Snedecor and Cochran, 1989). This method could have allowed for the quantitative comparison of resistance genes among commercial orchards and bactericide programs. It could have also included a larger portion of the actual bacterial community that is not surveyed when using culture-based methods. Including more tetracycline resistance genes and a greater portion of the bacterial community could change the results and conclusions drawn from them. Moreover, those conclusions could potentially provide a better picture of the bacterial community, the distribution of tetracycline resistance genes, and the effects of oxytetracycline exposure on these bacteria.
Despite this, the results of studies utilizing culture-independent methods provide similar conclusions and do not show a clear association between the incidence of resistance genes and antibiotic use (Yashiro and McManus, 2011; Walsh et al., 2014; Shade et al., 2013).
This was the first time such a study on tetracycline resistance and oxytetracycline sensitivity in Xap and in epiphytic bacteria recovered from stone fruit orchards has been conducted with respect to bacterial spot management. Oxytetracycline used in the management of bacterial spot affects the incidence of tetracycline resistance genes in epiphytic bacteria and the sensitivity of Xap to 25 µg/ml oxytetracycline differently.
Oxytetracycline use was not directly related to the incidence of tetR positive bacteria; however, it was associated with a greater percentage of tolerant Xap isolates. Because this research did not show an association of oxytetracycline use and the incidence of tetracycline resistance genes or resistance in Xap, it is likely oxytetracycline will continued 133 to be used in bacterial spot management. Over time, studies revisiting tetracycline resistance and oxytetracycline sensitivity in Xap and epiphytic bacteria should be conducted as variation in environment and changes in management strategies may need such research. Certainly, culture-independent methods should be used to confirm the results of this research and enhance the overall study of oxytetracycline use in Pennsylvania stone fruit orchards.
134
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139
APPENDIX: Penn State Fruit Research and Extension Center Bacterial Spot of Peach and Nectarine – Grower Survey
Name:______Date:______
Size of Orchard:______Are multiple varieties grown in a single block? ______
Cultivar Age Bacterial Spot Pressure
1 2 3 4
1 2 3 4
1 2 3 4
1 2 3 4
Do you use Mycoshield or Flameout to manage bacterial spot? If yes, which one? ______
How many applications have been made this year so far? ______
Approximately how many applications were made last year? ______
Do you apply copper cover sprays to manage bacterial spot? ______
If yes, how many copper applications have been made this year so far?______
Approximately how many copper applications were made last year?______
What is the approximate time interval between bactericide sprays?______
Do you apply bactericides in complete sprays or alternate row middle? ______
Do you apply dormant copper?______If yes, do you apply it in the spring, fall, or at both times?______
Thank you for you participation!
Sarah Bardsley - [email protected] - 717-677-6116 ext 220 (office) - 610-716-7529 (cell) VITA
SARAH J. CAPASSO
EDUCATION Doctor of Philosophy in Plant Pathology expected May 2016 Master of Science in Plant Pathology August 2010 Bachelor of Science in Biology May 2008
AWARDS & GRANTS Roger C. Pearson Student Travel Award 2015 State Horticultural Association of Pennsylvania Research Grant 2015 PDA Peach and Nectarine Marketing Board Grant 2014 BS2 Antibiotic Resistance in Agroecosystems Workshop Travel Award 2014 Larry J. Jordan Endowment 2014 PDA Peach and Nectarine Marketing Board Research Grant 2013 State Horticultural Association of Pennsylvania Research Grant 2013 Lester P. Nichols Memorial Award 2013 13th I.E. Melhus Graduate Student Symposium and Travel Award 2013 Henry W. Popp Graduate Assistantship 2013 College of Agricultural Sciences Graduate Student Competitive Grant Program 2012 State Horticultural Association of Pennsylvania Research Grant 2012 Henry W. Popp Graduate Assistantship 2012 Annual Graduate and Undergraduate Research Expo Poster Competition 2012 Sunday Endowment 2010 Henry W. Popp Graduate Assistantship 2010
PROFESSIONAL ACTIVITIES AND AFFILIATIONS Plant Pathology Association 2008-present American Phytopathological Society 2009-present Gamma Sigma Delta 2010-present
PEER REVIEWED PUBLICATIONS Bardsley, S.J., and Ngugi, H.K. 2013. Reliability and accuracy of visual methods to quantify severity of foliar bacterial spot symptoms on peach and nectarine. Plant Pathology 62: 460-474. Bardsley, S.J. 2010. Studies on the epidemiology and management of bacterial spot of peach and nectarine in Pennsylvania. The Pennsylvania State University. Master’s Thesis.