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Bateriophages of Xanthomonas campestris pv. begoniae; Their occurrence, survival and potential use as a biological control agent

ProQuest Dissertations and Theses, 2009
Dissertation
Author: Jeffrey Kaesberg
Abstract:
Bacterial spot of begonia, caused by Xanthomonas campestris pv. begoniae (Xcb ), can be a very serious disease during begonia production. In nature, bacterial populations commonly support populations of bacteriophages, which have sometimes been used to suppress susceptible bacterial populations. In this study, bacteriophages of Xcb were collected and isolated from foliage production and greenhouse environments in central Florida. Many of the phages isolated could infect several strains and, in some cases, multiple species in the genus Xanthomonas. From this collection, ten bacteriophages were selected based on their reaction to isolates of bacterial plant pathogens in the genus Xanthomonas. They were also evaluated based on phage morphology and genome characteristics. There was not much diversity among the phages studied in regard to morphology and genome characteristics. From these ten phages, four phages were used in biological control experiments in the greenhouse to control bacterial spot of begonia. Each of the four biological control bacteriophages studied had a linear dsDNA genome of approximately 12.5 kilobases and nine of the these ten phages appear to belong to the family Tectiviridae , based on morphology. Based on morphology, the other phage may be a Cystovirus or Levivirus. Five attempts at biological control using these bacteriophages were not successful because of environmental conditions in the greenhouse, which greatly favored the pathogen. In the first four biological control experiments, a mixture of the four phages was applied to begonias. In the fifth biocontrol experiment, the same phage mixture was mixed and applied with the irrigation water. No significant level of control was achieved in any of the experiments. Several negative environmental factors, which affected phage survival and persistence, were identified; some were studied in detail. Bacteriophage survival on leaf surfaces in environments common to the foliage and nursery industries was examined. A variety of different filters and light sources were used to examine the effects on bacteriophage populations on both leaf surfaces and on inert surfaces. Two important negative factors found in this study were desiccation and inactivation from high energy light, such as UV. It was also shown that the four phages could not persist well on begonia leaves and were washed off during overhead irrigation during the first four biological control experiments. Bacteriophages also could not infect Xanthomonas campestris pv. begoniae on leaf surfaces under laboratory conditions at the same rate as in nutrient broth. Under greenhouse conditions, these phages could not infect Xanthomonas campestris pv. begoniae on leaf surfaces. This study identified other negative factors, such as ultraviolet light and desiccation that need to be addressed for successful biological control of bacterial spot in nursery and foliage production systems.

TABLE OF CONTENTS

page

ACKNOWLEDGEMENTS .............................................................................................................4   LIST OF TABLES ...........................................................................................................................8   LIST OF FIGURES .......................................................................................................................10   ABSTRACT ...................................................................................................................................15 CHAPTER 1 BACTERIAL LEAF SPOT OF BEGONIA ...........................................................................17   The Foliage Industry ...............................................................................................................17   Bacterial Spot ..........................................................................................................................19   Disease Management of Begonia Leaf Spot ...........................................................................20   Project Goal and Objectives ...................................................................................................21   2 THE OCCURRENCE OF BACTERIOPHAGES ..................................................................23   3 CHARACTERIZATION OF BACTERIOPHAGES ISOLATED FROM NURSERIES AND FOLIAGE GROWING ENVIROMENTS IN FLORIDA ............................................29   Materials and Methods ...........................................................................................................29   Bacterial Strains ...............................................................................................................29   Phage Isolation from Soil, Irrigation Systems, Water Canals, and Plant Tissue .............30   Plaque Assays ..................................................................................................................31   Phage Isolation from Plaque Assays ...............................................................................31   Preparation of Bacteriophage Cultures ............................................................................32   Spot Testing .....................................................................................................................32   Extraction of Bacteriophage DNA ..................................................................................33   Electron Microscopy of Isolated Bacteriophages of Xcb ................................................33   Results .............................................................................................................................34   Initial Characterization of Isolated Bacteriophages ........................................................34   Analysis of Bacteriophage DNA .....................................................................................35   Electron Microscopy of Select Bacteriophages ...............................................................35   Discussion ........................................................................................................................36   4 PERFORMANCE OF BACTERIOPHAGES AS A BIOLOGICAL CONTROL OF BACTERIAL SPOT OF BEGONIA ......................................................................................50   Introduction .............................................................................................................................50   Materials and Methods ...........................................................................................................52   Bacteriophage Formulation fr Biological Control ...........................................................52   Biological Control of Bacterial Spot of Begonia ............................................................53   5

Results .............................................................................................................................57   Discussion ........................................................................................................................59   5 ENVIRONMENTAL FACTORS THAT AFFECT THE SURVIVAL AND REPRODUCTION OF BACTERIOPHAGES .......................................................................77   Introduction .............................................................................................................................77   Materials and Methods ...........................................................................................................79   Plants Used for Assaying Phage Survival .......................................................................79   Phage Population Dynamics on Leaf Surfaces ................................................................80   Comparison of Growing Environments on Bacteriophage Survival ...............................81   Phage Survival under Filtered Solar Radiation ...............................................................82   Bacteriophage Survival on Inert Surfaces. ......................................................................83   Light Spectra of Solar Radiation Reaching the Plants in Different Environments .........84   Phage Survival in Controlled Environments ...................................................................84   Role of Relative Humidity on Phage Survival ................................................................85   Infection of Begonia Inoculum Plants with Xcb. .............................................................86   Bacteriophage Infection on Leaf Surfaces under Favorable Conditions .........................87   Bacteriophage Infection on Begonia Leaf Surfaces under Greenhouse Conditions .......88   Phage Population Dynamics on Begonia Leaf Surfaces of Plants Infected with Xcb .....90   Identifying Possible Key Factors That Affected the Biological Control Experiments ...90   Phage Populations in Irrigation Runoff ...........................................................................91   Infection Rate of Bacteriophage in Nutrient Broth Culture ............................................92   Infection Rate of Bacteriophage to Bacteria on Vigna Phylloplanes under Laboratory Conditions .................................................................................................93   Results .....................................................................................................................................93   Phage Population Dynamics on Leaf Surfaces ................................................................93   Comparison of Growing Environments on Bacteriophage Survival ...............................95   Phage Survival under Filtered Solar Radiation ...............................................................96   Bacteriophage Survival on Inert Surfaces .......................................................................98   Light Spectra of Solar Radiation Reaching the Plants in Different Environments .........98   Phage Survival in Controlled Environments ...................................................................99   Role of Relative Humidity on Phage Survival ..............................................................102   Infection of Begonia Inoculum Plants with Xcb ...........................................................102   Bacteriophage Infection on Leaf Surfaces under Favorable Conditions .......................103   Bacteriophage Infection on Begonia Leaf Surfaces under Greenhouse Conditions .....103   Phage Population Dynamics on Begonia Leaf Surfaces of Plants Infected with Xcb ...106   Identifying Possible Key Factors that Affected the Biological Control Experiments ...106   Phage Populations in Irrigation Runoff Water ..............................................................108   Infection Rate of Bacteriophage in Nutrient Broth Culture ..........................................109   Infection Rate of Bacteriophage on Vigna Phylloplane under Laboratory Conditions ..................................................................................................................110   Discussion .............................................................................................................................110   6 OVERALL SUMMARY AND CONCLUSIONS ...............................................................139   REFERENCE LIST .....................................................................................................................142   6

BIOGRAPHICAL SKETCH .......................................................................................................148  

7

LIST OF TABLES Table page

3-1 Bacterial strains used in this study .....................................................................................39   3-2 Bacteriophages isolated from nursery or greenhouse production environments during this study. ...........................................................................................................................40   3-3 Bacteriophages isolated during this study.........................................................................43   3-4 Infection profiles of bacteriophage isolated in the study as determined by plaque assays. The Horizontal row across the top of each table shows different isolates of Xcb. The vertical row on the left shows the different isolates of bacteriophage. A “+” means a completely clear and concise plaque. A “+/-” means an opaque plaque was observed. A “-” means no plaque was observed ........................................................47   3-5 Infection profiles of bacteriophage isolated in the study as determined by plaque assays. The Horizontal row across the top of each table shows different isolates of Xcb. The vertical row on the left shows the different isolates of bacteriophage. A “+” means a completely clear and concise plaque. A “+/-” means an opaque plaque was observed. A “-” means no plaque was observed. .......................................................48   3-6 Infection profiles of bacteriophage isolated in the study as determined by plaque assays. The Horizontal row across the top of each table shows different isolates of Xcb. The vertical row on the left shows the different isolates of bacteriophage. A “+” means a completely clear and concise plaque. A “+/-” means an opaque plaque was observed. A “-” means no plaque was observed. .......................................................49   4-1 Average disease damage ratings from bacterial spot of begonia incited by Xanthomonas campestris pv. begoniae during the second biological control experiment..........................................................................................................................72   4-2 Area under the disease progress curves (AUDPC) of disease damage from each plot of begonia incited by Xanthomonas campestris pv. begoniae during the second biological control experiment. ...........................................................................................72   4-3 Student Newman Keuls Mean Separation of the Area under the Disease Progress Curve (MAUDPC) for the Biological Control Experiment 2. ...........................................73   4-4 Average disease damage ratings from bacterial spot of begonia incited by Xanthomonas campestris pv. begoniae during the fourth biological control experiment..........................................................................................................................74   4-5 Area under the disease progress curves (AUDPC) from disease damage from each plot of begonia incited by Xanthomonas campestris pv. begoniae during the fourth biological control experiment. ...........................................................................................74   8

4-6 Average disease damage ratings from bacterial spot of begonia incited by Xanthomonas campestris pv. begoniae during the fourth biological control experiment..........................................................................................................................75   4-7 Average disease damage ratings from bacterial spot of begonia incited by Xanthomonas campestris pv. begoniae during the fifth biological control experiment ....76   5-1 Average Measured Light Intensities D in different environments examined in this study. ................................................................................................................................124   5-2 Changes in bacteriophage population sizes over time on begonia leaves in a greenhouse environment. .................................................................................................129   5-3 Changes in bacteriophage population sizes over time on begonia leaves in a greenhouse environment ..................................................................................................132   5-4 Changes in bacteriophage population sizes over time on begonia leaves in a greenhouse environment. .................................................................................................134  

9

LIST OF FIGURES Figure page

3-1 Extracted Bacteriophage DNA resolved on a 1% agarose TAE gel. The DNA ladder is a 1 kb ladder [Takara Bio Inc. (see above); the largest band of the ladder is 10 kb.] The four bands are from each of the phage characterized in this study. From left to right, they are Pxcb-AgSt III A (A), Pxad-5sC (D), Pxwm-JonsII A (J) and Pxcp-A (P). Genome size was determined to be 12,500 bp using Kodak 1D 3.6 software (see above). ................................................................................................................................44   3-2 Images of each of the bacteriophage used in the biological control portion of this study. In the top left, is an image of Pxad-5sc taken at 200,000 X magnification. The top right is the the phage Pxwm-JonsII A taken at 67,000 X magnification. Bottom right is Pxcp-A taken at 67,000 X magnification and the bottom right is Pxcb-AgSt III A taken at 67,000 X magnification. The sizing scales are for approximation purposes. These phages were imaged with a Zeiss EM-10 electron microscope. ........................................................................................................................45   3-3 Images of six other bacteriophages partially characterized in this study. Top left is the bacteriophage Pxcb-AgSt IIA, top right is Pxcb-Rus II B, center left is Pxcb-P7, center right is Pxcb-P3, lower left is Pxcb-P1, and lower right is Pxcb-AgSt IV A. The sizing bar in each image represents 100 nm. These phages were imaged with a Hitachi H-7600 electron microscope. ................................................................................46   4-1a Irrigation apparatus used to apply phage and other treatments in the fifth biological control experiment. ............................................................................................................65   4-1b Irrigation apparatus used to apply phage and other treatments in the fifth biological control experiment. ............................................................................................................66   4-1c Detail of the underside of the irrigation apparatus used in the fifth biological control experiment, which shows the arrangement and type of application nozzles. ....................67   4-2 Leaf Damage Key. Class 1 represents 0.3% leaf area damage (LAD), Class 2 represents 0.7% LAD, Class 3 represents 1.5% LAD, Class 4 represents 3% LAD, Class 5 represents 8% LAD, and Class 6 represents 14% LAD. .......................................68   4-3a A healthy begonia leaf. Note the waxy leaf surface. ........................................................69   4-3b Typical leaf damage classes which correspond to the pictorial leaf damage key (Fig. 4-2) used in the biological control experiments. ................................................................70   4-4 Average levels of bacterial spot damage on begonias at the end of the first biological control experiment (one month). 1. Untreated control. 2. Sprayed with a phage mixture twice a week. 3. Sprayed with a phage mixture three times a week. 4. Sprayed with copper sulfate pentahydrate. Error bars indicate standard deviation. .........71   10

4-5 Disease progress curves of bacterial spot damage on begonias for different treatments in the second experiment. 1. Untreated control. 2. Sprayed with a phage mixture twice a week. 3. Sprayed with a phage mixture three times a week. 4. Sprayed with copper sulfate pentahydrate. Error bars indicate standard deviation. .........71   4-6 Disease progress curves from bacterial spot on begonias for different treatments in the fourth experiment. 1. Untreated control. 2. Sprayed with a phage mixture twice a week. 3. Sprayed with a phage mixture three times a week. 4. Sprayed with copper sulfate pentahydrate. Error bars indicate standard deviation. ...........................................73   4-7 Disease progress curves from bacterial spot on begonias for different treatments in the fifth biological control experiment. 1. Irrigated with water alone. 2. Irrigated with water supplemented with spent NB. 3. Irrigated with water supplemented with a phage cocktail. Error bars indicate standard deviation. ....................................................75   5-1 Emission spectrum of GE F40BL UVA tubes and of Ushio G15T8E UVB tubes as provided by the manufacturer. .........................................................................................116   5-2 Changes in bacteriophage populations on cowpea leaves over time in three different environments: (i) in an open field under full sun, (ii) in a plastic-covered greenhouse, (iii) in a plastic-covered greenhouse under an 80% shade cloth. Error bars indicate standard deviation. ...........................................................................................................116   5-3 Measured relative population size of each biocontrol phage (A, D, J, P) on cowpea leaves in three environments; (i) the growth room (R), (ii) under the shade in the greenhouse (S), (iii) under the shade in the greenhouse with overhead irrigation (I). ....117   5-4 Changes in bacteriophage populations on cowpea leaves over time in four environments: (i) in a plastic-covered greenhouse, (ii) in a glass greenhouse with shade paint, (iii) in an open field under full sun, (iv) in a shade house. Error bars indicate standard deviation. .............................................................................................117   5-5 Measured transmission spectrum of solar radiation through greenhouse glass in the ultraviolet region. UVB light is 260 – 320 nm in wavelength. UVA is 320 – 400 nm in wavelength. ..................................................................................................................118   5-6 Changes in bacteriophage population size over time on cowpea leaves (i) in an open field under full sun (ii) under regular greenhouse glass. Error bars indicate standard deviation. ..........................................................................................................................118   5-7 Measured transmission spectrum of solar radiation transmitted by blue-filter composed of greenhouse glass and blue cellophane in the ultraviolet region. ................119   5-8 Changes in bacteriophage population size over time on cowpea leaves (i) in the open under full sun (ii) under a blue-cellophane greenhouse glass filter. Error bars indicate standard deviation. .............................................................................................119   11

5-9 Measured transmission spectrum of solar radiation transmitted by red-filter composed of greenhouse glass and blue cellophane in the ultraviolet region. ................120   5-10 Changes in bacteriophage population size over time on cowpea leaves (i) in the open under full sun (ii) under a red-cellophane greenhouse glass filter. Error bars indicate standard deviation. ...........................................................................................................120   5-11a Transmission spectrum from ultraviolet through infrared wavelengths of light of four types of filters used in this study, (i) blue-cellophane and greenhouse glass, (ii) red- cellophane and greenhouse glass, (iii) cold mirror, (iv) Museum Glass. ........................121   5-11b Transmission spectra from four types of glass filters used in this study in ultraviolet and partial visible wavelengths; (i) blue-cellophane and greenhouse glass, (ii) red- cellophane and greenhouse glass, (iii) cold mirror, (iv) Museum Glass. ........................122   5-12 Measured transmission spectrum of solar radiation through Museum Glass in the ultraviolet region. .............................................................................................................123   5-13 Changes in bacteriophage population size over time on cellulose (i) in the open in the full sun or under (ii) under Museum Glass, (iii) under cold mirror. Error bars indicate standard deviation. .............................................................................................123   5-14 Measured emission spectrum of unfiltered solar radiation in the UV and partial visible wavelengths. .........................................................................................................124   5-15 Light radiation provided by the UVA tubes (A), the UVA tube light radiation through Museum Glass (B), changes in bacteriophage population size over time [(C), (i)] under the UVA light, (ii) under the UVA light and protected by the Museum Glass (lower). Error bars indicate standard deviation. Panes A and B show the types of light the phage were exposed to in pane C. .................................................................125   5-16 Light radiation provided by the UVB tubes (A), the UVB tube light radiation through Museum Glass (B), changes in bacteriophage population size over time [(C), (i)] under the UVB light, (ii) under the UVB light and protected by the Museum Glass (lower). Error bars indicate standard deviation. Panes A and B show the types of light the phage were exposed to in pane C. .....................................................................126   5-17 Changes in bacteriophage population size over time on cowpea leaf surfaces in the growth room at (i) 100% relative humidity, (ii) 45 % relative humidity. .......................127   5-18 Disease progress curves for bacterial spot damage on begonia following different incubation times in a humid environment for 0, 2, 4, 8, 16, and 32 hr. ...........................127   5-19 Relative phage population achieved by each bacteriophage on cowpea leaf surfaces. Aliquots of Xcb and the respective phage were mixed and incubated overnight on leaf surfaces in a humid environment. Error bars indicate standard deviation. ..........................................................................................................................128   12

5-20 Changes in bacteriophage population size over time on cowpea leaves (i) the A phage, leaves wet, (ii) A phage, leaves dry, (iii) D phage leaves wet, (iv) D phage, leaves dry, (v) J phage, leaves wet, (vi) J phage, leaves dry, (vii) P phage, leaves wet, (viii) P phage, leaves dry. Error bars indicate standard deviation. .................................128   5-21 Changes in bacteriophage population size over time on begonia leaves in a greenhouse environment (i) A phage alone, (ii) A phage with Xcb. Error bars indicate standard deviation. .............................................................................................129   5-22 Changes in bacteriophage population size over time on begonia leaves in a greenhouse environment (i) D phage alone, (ii) D phage with Xcb. Error bars indicate standard deviation. .............................................................................................130   5-23 Changes in bacteriophage population size over time on begonia leaves in a greenhouse environment (i) J phage alone, (ii) J phage with Xcb. Error bars indicate standard deviation. .............................................................................................130   5-24 Changes in bacteriophage population size over time on begonia leaves in a greenhouse environment (i) P phage alone, (ii) P phage with Xcb. Error bars indicate standard deviation. .............................................................................................131   5-25 Changes in bacteriophage population size over time on begonia leaves in a greenhouse environment (i) IIA phage alone, (ii) IIA phage with Xcb. Error bars indicate standard deviation. .............................................................................................131   5-26 Changes in bacteriophage population size over time on begonia leaves in a greenhouse environment (i) IVA phage alone, (ii) IVA phage with Xcb. Error bars indicate standard deviation. .............................................................................................132   5-27 Changes in bacteriophage population size over time on begonia leaves in a greenhouse environment (i) Rus phage alone, (ii) Rus phage with Xcb. Error bars indicate standard deviation. .............................................................................................133   5-28 Changes in bacteriophage population size over time on begonia leaves in a greenhouse environment (i) P1 phage alone, (ii) P1 phage with Xcb. Error bars indicate standard deviation. .............................................................................................133   5-29 Changes in bacteriophage population size over time on begonia leaves in a greenhouse environment (i) P3 phage alone, (ii) P3 phage with Xcb. Error bars indicate standard deviation. .............................................................................................134   5-30 Changes in bacteriophage population size over time on begonia leaves in a greenhouse environment (i) P7 phage alone, (ii) P7 phage with Xcb. Error bars indicate standard deviation. .............................................................................................135   5-31 Changes in bacteriophage population over time on Xcb-infected begonias in a greenhouse environment. .................................................................................................135   13

5-32 Disease progress of bacterial spot of begonia with different preventative treatments treated with (i) sterile tap water (STW), (ii) spent nutrient broth (SpNB), (iii) skim milk, (iv) AG98, (v) A phage, (vi) D phage, (vii) J phage, (viii) P phage. Error bars indicate standard deviation. .............................................................................................136   5-33 Disease progress of bacterial spot of begonia with different preventative treatments (i) treated with sterile tap water (STW), (ii) treated with the A phage plus AG 98, (iii) the D phage plus AG 98, (iv) the J phage plus AG98, (v) the P phage plus AG 98. Error bars indicate standard deviation. ......................................................................137   5-34 Bacteriophage populations measured in the irrigation runoff from begonia plants sprayed with (i) the A phage, (ii) the D phage, (iii) the J phage, (iv) the P phage. The plants were sprayed once and then subjected to daily overhead irrigation. Error bars indicate standard deviation. .............................................................................................137   5-35 Changes in bacteriophage population size over time in nutrient broth with Xcb culture (i) A phage, (ii) D phage, (iii) J phage, (iv) P phage. ..........................................138   5-36 Changes in bacteriophage population size over time on cowpea leaf surfaces in a humid environment with Xcb (i) A phage, (ii) D phage, (iii) J phage, (iv) P phage. Error bars indicate standard deviation. ............................................................................138  

14

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

BACTERIOPHAGES OF XANTHOMONAS CAMPESTRIS PV. BEGONIAE; THEIR OCCURRENCE, SURVIVAL AND POTENTIAL USE AS A BIOLOGICAL CONTROL AGENT.

By

Jeffrey Kaesberg December 2009 Chair: James O. Strandberg Major: Plant Pathology

Bacterial spot of begonia, caused by Xanthomonas campestris pv. begoniae (Xcb), can be a very serious disease during begonia production. In nature, bacterial populations commonly support populations of bacteriophages, which have sometimes been used to suppress susceptible bacterial populations. In this study, bacteriophages of Xcb were collected and isolated from foliage production and greenhouse environments in central Florida. Many of the phages isolated could infect several strains and, in some cases, multiple species in the genus Xanthomonas. From this collection, ten bacteriophages were selected based on their reaction to isolates of bacterial plant pathogens in the genus Xanthomonas. They were also evaluated based on phage morphology and genome characteristics. There was not much diversity among the phages studied in regard to morphology and genome characteristics. From these ten phages, four phages were used in biological control experiments in the greenhouse to control bacterial spot of begonia.. Each of the four biological control bacteriophages studied had a linear dsDNA genome of approximately 12.5 kilobases and nine of the these ten phages appear to belong to the family Tectiviridae, based on morphology. Based on morphology, the other phage may be a Cystovirus or Levivirus. 15

Five attempts at biological control using these bacteriophages were not successful because of environmental conditions in the greenhouse, which greatly favored the pathogen. In the first four biological control experiments, a mixture of the four phages was applied to begonias. In the fifth biocontrol experiment, the same phage mixture was mixed and applied with the irrigation water. No significant level of control was achieved in any of the experiments. Several negative environmental factors, which affected phage survival and persistence, were identified; some were studied in detail. Bacteriophage survival on leaf surfaces in environments common to the foliage and nursery industries was examined. A variety of different filters and light sources were used to examine the effects on bacteriophage populations on both leaf surfaces and on inert surfaces. Two important negative factors found in this study were desiccation and inactivation from high energy light, such as UV. It was also shown that the four phages could not persist well on begonia leaves and were washed off during overhead irrigation during the first four biological control experiments. Bacteriophages also could not infect Xanthomonas campestris pv. begoniae on leaf surfaces under laboratory conditions at the same rate as in nutrient broth. Under greenhouse conditions, these phages could not infect Xanthomonas campestris pv. begoniae on leaf surfaces. This study identified other negative factors, such as ultraviolet light and desiccation that need to be addressed for successful biological control of bacterial spot in nursery and foliage production systems.

16

CHAPTER 1 BACTERIAL LEAF SPOT OF BEGONIA The Foliage Industry Humans have used plants for millions of years. At first, agriculture was just a means of providing food for themselves and for livestock. As civilization has become more advanced, plants have also been appreciated for their aesthetic value (2). The Bureau of Economic Analysis (BEA) within the US Department of Commerce classifies nearly all plants grown for food, fiber or aesthetic reasons into the same class – agriculture. In 2002, agriculture production in the US had a combined value of $18810.8 billion (62). Within agriculture, the cultivation of flowers is known as floriculture (48). Florida leads the nation in the production and sale of indoor foliage plants and in cut foliage. The national wholesale value of foliage plant production was $721 million in 2005 with Florida contributing 69.3% of that value (J. Chen, personal communication, 2009). Sales of potted indoor foliage and foliage hanging baskets were nearly $416 million in 2004. In 2005, central Florida had 910 nurseries that together, utilized over 24,000 acres of total space (J. Chen, personal communication, 2005, 26). A popular family of plants grown for aesthetic value is the Begoniaceae. The sale of begonias in Florida amounted to nearly $3.5 million in 2004. Begonias grow naturally in tropical regions of the world; in Central and South America, Africa and tropical regions of Asia. The name “Begonia” was termed by a French botanist Charles Plumier, to honor Michel Begon, a former governor of the French colony of Haiti (66). There were three other names previously given to this genus: Totoncaxoxo coyollin (Begonia gracilis) from Mexico, Tsjeria-narinampuli (B.malabarica) from India, and Aceris fructu herba anomala, flore tertrapetalo for a Caribbean species now called B. acutifolia. But the name Begonia was adapted by Linnaeus in his Species Plantarum of 1753 (66). 17

The cultivation of begonias has a long and distinguished history that dates back to at least the 1400s when Begonia grandis was cultivated in its Chinese homeland (66). Begonia grandis is a frost-hardy species that was first grown for its medicinal properties and not for its aesthetic appeal. In Asia, many other begonia species are believed to have been used for human consumption as vegetables or greens, made into teas, or used for medicinal purposes to clean wounds and reduce swelling (66). Begonias were introduced to Europe in 1777, when a Jamaican species, Begonia minor, was sent to Kew Gardens in England. The transport of tropical plants via ship to Europe was difficult because of encounters with colder weather until Nathaniel Ward invented a portable greenhouse in 1835 (66). These greenhouses could keep plants at warm temperatures during transport. New begonia species slowly trickled into Europe and into the U.S. during the 1800s. As cultivation became less expensive and more species were introduced, many hybrids were created. A popular hybrid, resulting from crossing B. cucullata with B. schmidtiana, was B. semperflorens; a species that is widely popular today, commonly called wax begonias (66). The flowers of begonias are monoecious; with both genders of flower appearing on the same plant. Begoniaciae contains five genera with the genus Begonia containing the most species (56). While begonias are monoecious, the number of male and female flowers varies. Horticulturalists group begonias into three main groups: tuberous, rhizomatous, and fibrous (root). There are many popular tuberous begonias grown; these plants can feature large double flowers (56). The most popular species of rhizomatous begonia is Begonia rex (L.). Rex begonias are often grown for their foliage. Begonia semperflorens makes up the fibrous root group of begonia. The term “semperflorens” means “always flowering.” There are a multitude 18

of varieties within this species (54). Fibrous-root begonias are often used as bedding plants or as pot plants. For optimal growth, plants require a favorable environment and moderate levels of nutrients and moisture. However, when environmental conditions are favorable for pathogens, parasitic microorganisms can cause disease on plants (2). There are a variety of bacteria, fungi, viruses and other microorganisms that can adversely affect plants. Common diseases of begonia are bacterial spot (Xanthomonas campestris pv. begoniae), gray mold (Botrytis cinerea ) , Rhizoctonia aerial blight (Rhizoctonia solani), Myrothecium leaf spot (Myrothecium roridum), Southern blight (Sclerotium rolfsii), powdery mildew (Erysiphe cichoracearum, Oidium begoniae) and root and stem rot (Pythium splendens.) (13,68). Bacterial Spot Bacterial spot of begonia (Begonia sp.) was first described in 1937 by McCulloch (44). It can be a costly problem for growers; Xanthomonas campestris pv. begoniae (Xcb) is the causal agent. The genus Xanthomonas also contains several other notorious members, which are known to incite plant disease (2). Examples include: Xanthomonas campestris pv. campestris which causes black rot of many cruciferous plants; Xanthomonas axonopodis pv. citri incites citrus canker and Xanthomonas campestris pv. malvacearum incites bacterial spot on cotton (20). In 2005, the state of Georgia reported crop losses for tomato and bell pepper caused by Xanthomonas campestris pv. vesicatoria to be $6.0 million for tomato and $7.1 million for bell pepper. Additionally, Xanthomonas campestris pv. pruni caused a loss of $4.8 million on peach in Georgia in 2005 (32). Infection of begonia by Xcb commonly occurs under warm and humid conditions with splashing water being a common means of dispersal for the pathogen. Bacteria can enter the plant through wounds, stomata, or hydathodes. Once inside the apoplast, infection utilizes a 19

type-III secretion system (31). The bacteria also secrete helper and accessory proteins into the intercellular spaces. These secreted proteins support the injection of virulence effector proteins into the host cell (31). If the plant being invaded is not a host to the particular xanthomonad, these type-III proteins will incite a hypersensitive response (HR), where there is a pronounced localized cell death. A hypersensitive response is thought to inhibit bacterial cell growth because of a decrease in the apoplastic water potential (73). The type-III secretion system is composed of a group of transcriptional units in what is termed the hrp (hypersensitive reaction and pathogenicity) region. Comparing gene organization and regulation of the hrp regions among phytopathogenic bacteria indicates that there are two lineage groups. Noteworthy genera of Group I include Erwinia, Pantoea, and Pseudomonas syringae. Members of note in Group II include Ralstonia solanacearum and Xanthomonas spp. (31). When a begonia is first infected with Xcb, symptoms usually appear close to the leaf margin on the underside of the leaf and begin as small water-soaked lesions (3). These lesions are especially noticeable on the underside of the leaf (12). Later, the leaf spots become necrotic and brown and may coalesce, yielding large, irregular necrotic areas with yellow borders. Older leaves and petioles turn yellow as the infection becomes systemic. When the vascular tissues are colonized, the plant can wilt (12). Systemically-infected leaves and flowers are easily abscised. Disease Management of Begonia Leaf Spot Lipp et al. (41) explains that the main control measures are sanitation and exclusion of plants with disease symptoms. An industry-wide standard chemical control is to spray with copper. There are varieties of copper-based products available such as Phyton 27 ™or Kocide ™. Overhead irrigation and overcrowding should be avoided if possible (41). 20

When Xanthomonas is present in, or on a host plant, the bacterial population could possibly support a population of bacteriophages (phages). Most bacterial populations in nature are capable of supporting large populations of phages (52, 71). Bacteriophages are viruses that prey on host bacteria; this is a very specific predator-prey relationship and is frequently pathovar, and even strain specific. This specificity does not allow phages to infect other bacteria at random, but only phage-specific hosts. During an infection by a plant pathogenic bacterium, tissues from the infected plants may drop off into foliage growing environments, commonly in areas where irrigation drains. If phage are present and multiplying in the host bacteria, they may accumulate in the proximate soil, the irrigation systems and drainage canals. Xanthomonads do not survive for long periods in the soil, but the phage may persist for long periods without a living host. The use of copper for disease control has lead to concerns in terms of the environment and in terms of the pathogens developing pesticide resistance (33). Therefore, alternate disease control strategies are being developed. Zaccardelli et al. (77) first proposed the use of bacteriophages preventatively as a biological control of bacterial spot of peach which is incited by Xanthomonas campestris pv. pruni. There have been bacteriophages isolated that infect other species and pathovars of Xanthomonas (3, 6, 7, 8, 12, 24, 25, 30, 32, 33, 36, 37, 39, 40, 51, 52, 65, 77), but nothing has been published regarding bacteriophages that infect Xcb. When bacteriophages are applied to leaf surfaces to control plant pathogenic bacteria, environmental stresses, such as UV light and desiccation may inactivate the phages and reduce the efficacy of the biocontrol (7). Project Goal and Objectives The negative stresses may be reduced in greenhouse environments suitable for begonias, making biological control more feasible. The main objectives of this study were to (i) 21

Full document contains 149 pages
Abstract: Bacterial spot of begonia, caused by Xanthomonas campestris pv. begoniae (Xcb ), can be a very serious disease during begonia production. In nature, bacterial populations commonly support populations of bacteriophages, which have sometimes been used to suppress susceptible bacterial populations. In this study, bacteriophages of Xcb were collected and isolated from foliage production and greenhouse environments in central Florida. Many of the phages isolated could infect several strains and, in some cases, multiple species in the genus Xanthomonas. From this collection, ten bacteriophages were selected based on their reaction to isolates of bacterial plant pathogens in the genus Xanthomonas. They were also evaluated based on phage morphology and genome characteristics. There was not much diversity among the phages studied in regard to morphology and genome characteristics. From these ten phages, four phages were used in biological control experiments in the greenhouse to control bacterial spot of begonia. Each of the four biological control bacteriophages studied had a linear dsDNA genome of approximately 12.5 kilobases and nine of the these ten phages appear to belong to the family Tectiviridae , based on morphology. Based on morphology, the other phage may be a Cystovirus or Levivirus. Five attempts at biological control using these bacteriophages were not successful because of environmental conditions in the greenhouse, which greatly favored the pathogen. In the first four biological control experiments, a mixture of the four phages was applied to begonias. In the fifth biocontrol experiment, the same phage mixture was mixed and applied with the irrigation water. No significant level of control was achieved in any of the experiments. Several negative environmental factors, which affected phage survival and persistence, were identified; some were studied in detail. Bacteriophage survival on leaf surfaces in environments common to the foliage and nursery industries was examined. A variety of different filters and light sources were used to examine the effects on bacteriophage populations on both leaf surfaces and on inert surfaces. Two important negative factors found in this study were desiccation and inactivation from high energy light, such as UV. It was also shown that the four phages could not persist well on begonia leaves and were washed off during overhead irrigation during the first four biological control experiments. Bacteriophages also could not infect Xanthomonas campestris pv. begoniae on leaf surfaces under laboratory conditions at the same rate as in nutrient broth. Under greenhouse conditions, these phages could not infect Xanthomonas campestris pv. begoniae on leaf surfaces. This study identified other negative factors, such as ultraviolet light and desiccation that need to be addressed for successful biological control of bacterial spot in nursery and foliage production systems.