<%BANNER%>

Bacteriophages of Xanthomonas Campestris Pv. Begoniae; Their Occurrence, Survival and Potential Use As a Biological Cont...

Permanent Link: http://ufdc.ufl.edu/UFE0041099/00001

Material Information

Title: Bacteriophages of Xanthomonas Campestris Pv. Begoniae; Their Occurrence, Survival and Potential Use As a Biological Control Agent.
Physical Description: 1 online resource (148 p.)
Language: english
Creator: Kaesberg, Jeffrey
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: Plant Pathology -- Dissertations, Academic -- UF
Genre: Plant Pathology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: 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. 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.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jeffrey Kaesberg.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Strandberg, James O.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041099:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041099/00001

Material Information

Title: Bacteriophages of Xanthomonas Campestris Pv. Begoniae; Their Occurrence, Survival and Potential Use As a Biological Control Agent.
Physical Description: 1 online resource (148 p.)
Language: english
Creator: Kaesberg, Jeffrey
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: Plant Pathology -- Dissertations, Academic -- UF
Genre: Plant Pathology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: 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. 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.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jeffrey Kaesberg.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Strandberg, James O.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041099:00001


This item has the following downloads:


Full Text

PAGE 1

BACTERIOPHAGES OF XANTHOMONAS CA MPESTRIS PV. BEGONIAE; THEIR OCCURRENCE, SURVIVAL AND POTENTIAL USE AS A BIOLOGICAL CONTROL AGENT By JEFFREY KAESBERG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009 1

PAGE 2

Jeffrey Kaesberg 2

PAGE 3

To Shakyamuni and Nichiren, thank you 3

PAGE 4

ACKNOWLEDGEMENTS First, I would like to thank my parents for their prolonged support over the years. None of this would have been possible without them. I would also like to thank the rest of my family and friends for advice and support. I owe a great deal of gratit ude to Dr. James Strandberg w ho has served as a colleague, mentor and friend to me over the years. His acad emic and financial support made this project possible. I would also like to thank my other committee memb ers, Dr. Jeff Jones, Dr. Dave Norman and Dr. Donna Duckworth who have provided academic support and advice to me over the years. I would like to thank the faculty and staff of the Mid-Florida Research and Education Center (MREC) who have been helpful, friendly and brought a smile to my face, especially Greg Alexander, Chris Fooshee and Ho lly McNaught. Special thanks are also given to Dr. Richard Beeson and Mr. Ed Tillman; they were extremely he lpful towards the completion of this project. I would like to express gratitude to Drew Bryne, Patricia Ramos and Marlene Saldivar, who were helpful to me while I worked in the Strandberg lab. 4

PAGE 5

TABLE OF CONTENTS page ACKNOWLEDGEMENTS .............................................................................................................4LIST OF TABLES ...........................................................................................................................8LIST OF FIGURES .......................................................................................................................10ABSTRACT ...................................................................................................................... .............15 CHAPTER 1 BACTERIAL LEAF SPOT OF BEGONIA ...........................................................................17The Foliage Industry .......................................................................................................... .....17Bacterial Spot ..........................................................................................................................19Disease Management of Begonia Leaf Spot ...........................................................................20Project Goal and Objectives ...................................................................................................212 THE OCCURRENCE OF BACTERIOPHAGES ..................................................................233 CHARACTERIZATION OF BACTERIOP HAGES ISOLATED FROM NURSERIES AND FOLIAGE GROWING ENVIROMENTS IN FLORIDA ............................................29Materials and Methods ...........................................................................................................29Bacterial Strains ...............................................................................................................29Phage Isolation from Soil, Irrigation Systems, Water Canals, and Plant Tissue .............30Plaque Assays ..................................................................................................................31Phage Isolation from Plaque Assays ...............................................................................31Preparation of Bacteriophage Cultures ............................................................................32Spot Testing .....................................................................................................................32Extraction of Bacteriophage DNA ..................................................................................33Electron Microscopy of Isol ated Bacteriophages of Xcb ................................................33Results .............................................................................................................................34Initial Characterization of Isolated Bacteriophages ........................................................34Analysis of Bacteriophage DNA .....................................................................................35Electron Microscopy of Select Bacteriophages ...............................................................35Discussion .................................................................................................................... ....364 PERFORMANCE OF BACTERIOPHAGES AS A BIOLOGICAL CONTROL OF BACTERIAL SPOT OF BEGONIA ......................................................................................50Introduction .................................................................................................................. ...........50Materials and Methods ...........................................................................................................52Bacteriophage Formulation fr Biological Control ...........................................................52Biological Control of Bact erial Spot of Begonia ............................................................53 5

PAGE 6

Results .............................................................................................................................57Discussion .................................................................................................................... ....595 ENVIRONMENTAL FACTORS THAT AFFECT THE SURVIVAL AND REPRODUCTION OF BA CTERIOPHAGES .......................................................................77Introduction .................................................................................................................. ...........77Materials and Methods ...........................................................................................................79Plants Used for Assaying Phage Survival .......................................................................79Phage Population Dynamics on Leaf Surfaces ................................................................80Comparison of Growing Environmen ts on Bacteriophage Survival ...............................81Phage Survival under Filtered Solar Radiation ...............................................................82Bacteriophage Survival on Inert Surfaces. ......................................................................83Light Spectra of Solar Radiation Reaching the Plants in Different Environments .........84Phage Survival in Controlled Environments ...................................................................84Role of Relative Humidity on Phage Survival ................................................................85Infection of Begonia Inoculum Plants with Xcb .............................................................86Bacteriophage Infecti on on Leaf Surfaces under Favorable Conditions .........................87Bacteriophage Infection on Begonia Leaf Surfaces under Greenhouse Conditions .......88Phage Population Dynamics on Begonia Leaf Surfaces of Plants Infected with Xcb .....90Identifying Possible Key Factors That Aff ected the Biological Control Experiments ...90Phage Populations in Irrigation Runoff ...........................................................................91Infection Rate of Bacteriophage in Nutrient Broth Culture ............................................92Infection Rate of Bacteriophage to Bacteria on Vigna Phylloplanes under Laboratory Conditions .................................................................................................93Results .....................................................................................................................................93Phage Population Dynamics on Leaf Surfaces ................................................................93Comparison of Growing Environmen ts on Bacteriophage Survival ...............................95Phage Survival under Filtered Solar Radiation ...............................................................96Bacteriophage Survival on Inert Surfaces .......................................................................98Light Spectra of Solar Radiation Reaching the Plants in Different Environments .........98Phage Survival in Controlled Environments ...................................................................99Role of Relative Humidity on Phage Survival ..............................................................102Infection of Begonia Inoculum Plants with Xcb ...........................................................102Bacteriophage Infecti on on Leaf Surfaces under Favorable Conditions .......................103Bacteriophage Infection on Begonia Leaf Surfaces under Greenhouse Conditions .....103Phage Population Dynamics on Begonia Leaf Surfaces of Plants Infected with Xcb ...106Identifying Possible Key Factors that Aff ected the Biological Control Experiments ...106Phage Populations in Irrigation Runoff Water ..............................................................108Infection Rate of Bacteriophage in Nutrient Broth Culture ..........................................109Infection Rate of Bacteriophage on Vigna Phylloplane under Laboratory Conditions ..................................................................................................................110Discussion .................................................................................................................... .........1106 OVERALL SUMMARY AND CONCLUSIONS ...............................................................139REFERENCE LIST .....................................................................................................................142 6

PAGE 7

BIOGRAPHICAL SKETCH .......................................................................................................148 7

PAGE 8

LIST OF TABLES Table page 3-1 Bacterial strains used in this study .....................................................................................3 93-2 Bacteriophages isolated from nursery or greenhouse production environments during this study. ...........................................................................................................................403-3 Bacteriophages isol ated during this study.........................................................................433-4 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 di fferent isolates of bacteriophage. A + means a completely clear and concise plaque. A +/- means an opaque plaque was observed. A means no plaque was observed ........................................................473-5 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 di fferent isolates of bacteriophage. A + means a completely clear and concise plaque. A +/- means an opaque plaque was observed. A means no plaque was observed. .......................................................483-6 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 di fferent isolates of bacteriophage. A + means a completely clear and concise plaque. A +/- means an opaque plaque was observed. A means no plaque was observed. .......................................................494-1 Average disease damage ratings from bacterial spot of begonia incited by Xanthomonas campestris pv. begoniae during the second bi ological control experiment..................................................................................................................... .....724-2 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. ...........................................................................................724-3 Student Newman Keuls Mean Separati on of the Area under the Disease Progress Curve (MAUDPC) for the Biologi cal Control Experiment 2. ...........................................734-4 Average disease damage ratings from bacterial spot of begonia incited by Xanthomonas campestris pv. begoniae during the fourth biological control experiment..................................................................................................................... .....744-5 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

PAGE 9

4-6 Average disease damage ratings from bacterial spot of begonia incited by Xanthomonas campestris pv. begoniae during the fourth biological control experiment..................................................................................................................... .....754-7 Average disease damage ratings from bacterial spot of begonia incited by Xanthomonas campestris pv. begoniae during the fifth biological control experiment ....765-1 Average Measured Light IntensitiesD in different environments examined in this study. ........................................................................................................................ ........1245-2 Changes in bacteriophage population sizes over time on begonia leaves in a greenhouse environment. .................................................................................................1295-3 Changes in bacteriophage population sizes over time on begonia leaves in a greenhouse envi ronment ..................................................................................................1325-4 Changes in bacteriophage population sizes over time on begonia leaves in a greenhouse environment. .................................................................................................134 9

PAGE 10

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 ch aracterized 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). ....................................................................................................................... .........443-2 Images of each of the bacteriophage used in the biological cont rol 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-J onsII 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. ................................................................................................................... .....453-3 Images of six other bacteriophages partia lly characterized in th is 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. ................................................................................464-1a Irrigation apparatus used to apply phage and other treatments in the fifth biological control experiment. ........................................................................................................... .654-1b Irrigation apparatus used to apply phage and other treatments in the fifth biological control experiment. ........................................................................................................... .664-1c Detail of the underside of the irrigation a pparatus used in the fifth biological control experiment, which shows the arrangement and type of application nozzles. ....................674-2 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. .......................................684-3a A healthy begonia leaf. Note the waxy leaf surface. ........................................................694-3b Typical leaf damage classes which corre spond to the pictorial leaf damage key (Fig. 4-2) used in the biologica l control experiments. ................................................................704-4 Average levels of bacterial spot damage on begonias at the end of the first biological control experiment (one month). 1. Untr eated 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. E rror bars indicate st andard deviation. .........71 10

PAGE 11

4-5 Disease progress curves of bacteria l spot damage on begonias for different treatments in the second experiment. 1. Un treated 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. E rror bars indicate st andard deviation. .........714-6 Disease progress curves from bacterial sp ot 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. ...........................................734-7 Disease progress curves from bacterial sp ot on begonias for different treatments in the fifth biological control experiment. 1. Irrigated w ith water alone. 2. Irrigated with water supplemented with spent NB. 3. Irrigated with water supplemented with a phage cocktail. Error bars i ndicate standard deviation. ....................................................755-1 Emission spectrum of GE F40BL UVA tubes and of Ushio G15T8E UVB tubes as provided by the manufacturer. .........................................................................................1165-2 Changes in bacteriophage populations on cowpea leaves over time in three different environments: (i) in an open field under fu ll sun, (ii) in a plastic-covered greenhouse, (iii) in a plastic-covered greenhouse under an 80% shade cloth. Error bars indicate standard deviation. ...........................................................................................................1165-3 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 th e greenhouse with overhead irrigation (I). ....1175-4 Changes in bacteriophage populations on cowpea leaves over time in four environments: (i) in a plastic-covered gr eenhouse, (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. .............................................................................................1175-5 Measured transmission spectrum of sola r radiation through gr eenhouse glass in the ultraviolet region. UVB light is 260 320 nm in wavelength. UVA is 320 400 nm in wavelength. ..................................................................................................................1185-6 Changes in bacteriophage population size over time on cowpea leaves (i) in an open field under full sun (ii) under regular greenhous e glass. Error bars indicate standard deviation. .................................................................................................................... ......1185-7 Measured transmission spectrum of solar radiation transmitted by blue-filter composed of greenhouse glass and blue cellophane in the ul traviolet region. ................1195-8 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

PAGE 12

5-9 Measured transmission spectrum of solar radiation transmitted by red-filter composed of greenhouse glass and blue cellophane in the ul traviolet region. ................1205-10 Changes in bacteriophage population size over time on cowpea leaves (i) in the open under full sun (ii) under a red-cellophane greenh ouse glass filter. Error bars indicate standard deviation. ...........................................................................................................1205-11a Transmission spectrum from ultraviolet thr ough infrared wavelengths of light of four types of filters used in th is study, (i) blue-cellophane and greenhouse glass, (ii) redcellophane and greenhouse gl ass, (iii) cold mirror, (iv) Museum Glass. ........................1215-11b Transmission spectra from four types of gl ass filters used in th is study in ultraviolet and partial visible wavelengths; (i) blue-c ellophane and greenhouse glass, (ii) redcellophane and greenhouse gl ass, (iii) cold mirror, (iv) Museum Glass. ........................1225-12 Measured transmission spectrum of so lar radiation through Museum Glass in the ultraviolet region. .............................................................................................................1235-13 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 mi rror. Error bars indicate standard deviation. .............................................................................................1235-14 Measured emission spectrum of unfilter ed solar radiation in the UV and partial visible wavelengths. .........................................................................................................1245-15 Light radiation provided by the UVA tubes (A), th e UVA tube light radiation through Museum Glass (B), changes in b acteriophage 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 standa rd deviation. Panes A and B show the types of light the phage were e xposed to in pane C. .................................................................1255-16 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 li ght and protected by the Museum Glass (lower). Error bars indicate standard de viation. Panes A and B show the types of light the phage were exposed to in pane C. .....................................................................1265-17 Changes in bacteriophage population size over time on cowpea leaf surfaces in the growth room at (i) 100% relative hu midity, (ii) 45 % relative humidity. .......................1275-18 Disease progress curves for bacteria l spot damage on begonia following different incubation times in a humid environment for 0, 2, 4, 8, 16, and 32 hr. ...........................1275-19 Relative phage population achieve d 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

PAGE 13

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 stan dard deviation. .................................1285-21 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. .............................................................................................1295-22 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. .............................................................................................1305-23 Changes in bacteriophage population size over time on begonia leaves in a greenhouse environment (i) J ph age alone, (ii) J phage with Xcb. Error bars indicate standard deviation. .............................................................................................1305-24 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. .............................................................................................1315-25 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. .............................................................................................1315-26 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. .............................................................................................1325-27 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. .............................................................................................1335-28 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. .............................................................................................1335-29 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. .............................................................................................1345-30 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. .............................................................................................1355-31 Changes in bacteriophage population over time on Xcbinfected begonias in a greenhouse environment. .................................................................................................135 13

PAGE 14

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. .............................................................................................1365-33 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 st andard deviation. ......................................................................1375-34 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 irriga tion. Error bars indicate standard deviation. .............................................................................................1375-35 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. ..........................................1385-36 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 st andard deviation. ............................................................................138 14

PAGE 15

Abstract of Dissertation Pres ented 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 CA MPESTRIS 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 cen tral 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. Th ere was not much diversity among the phages studied in regard to morphology and genome charact eristics. From these ten phages, four phages were used in biological control experiments in the greenhouse to contro l 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, th e other phage may be a Cystovirus or Levivirus. 15

PAGE 16

Five attempts at biological control usi ng these bacteriophages were not successful because of environmental conditions in the green house, which greatly favored the pathogen. In the first four biological contro l experiments, a mixture of th e 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 nurse ry industries was examined. A variety of different filters and light sources were used to examine the effects on b acteriophage populations on both leaf surfaces and on iner t surfaces. Two important negative factors found in this study were desiccation and inac tivation 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 labo ratory conditions at the same rate as in nutrient broth. Under gree nhouse conditions, these pha ges could not infect Xanthomonas campestris pv. begoniae on leaf surfaces. This st udy identified other negative factors, such as ultraviolet li ght and desiccation that need to be addressed for successful biological control of bacterial spot in nur sery and foliage production systems. 16

PAGE 17

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 Co mmerce 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 m illion 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 pl ants grown for aesthetic value is the Begoniaceae. The sale of begonias in Florida am ounted to nearly $3.5 million in 2004. Begonias grow naturally in trop ical 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 prev iously 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

PAGE 18

The cultivation of begonias has a long and distin guished 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 a nd 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 weat her until Nathaniel Ward invented a portable greenhouse in 1835 (66). These greenhouses could keep plants at warm temperatures during transport. New begonia species slowly trickl ed 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 to day, 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 ar e monoecious, the number of male and female flowers varies. Horticulturalists group begonias into three ma in groups: tuberous, rhizomatous, and fibrous (root). There are many popular tuberous begonias grown; these plants can feature large double flowers (56). The most popular sp ecies 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 m eans always flowering. There are a multitude 18

PAGE 19

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 favorab le environment and moderate levels of nutrients and moisture. However, when enviro nmental 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 advers ely 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 losse s 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. Ba cteria can enter the plant through wounds, stomata, or hydathodes. Once inside the apoplast, infection utilizes a 19

PAGE 20

type-III secretion system (31). The bacteria also secrete helper and accessory proteins into the intercellular spaces. These secreted proteins supp ort 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 inci te a hypersensitive response (H R), where there is a pronounced localized cell death. A hypersensitive response is thought to inhibit bacter ial cell growth because of a decrease in the apopla stic 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, irre gular necrotic areas with yellow borders. Older leaves and petioles turn yellow as the infection b ecomes systemic. When the vascular tissues are colonized, the plant can wilt (12). Systemically-inf ected leaves and flowers are easily abscised. Disease Management of Begonia Leaf Spot Lipp et al. (41) explains that the main cont rol measures are sanita tion and exclusion of plants with disease symptoms. An industry-wide standard chem ical 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

PAGE 21

When Xanthomonas is present in, or on a host plan t, the bacterial population could possibly support a population of bacteriophages (pha ges). Most bacterial populations in nature are capable of supporting large popu lations of phages (52, 71). Bacteriophages are viruses that prey on host bacteria; this is a very specific pred ator-prey relationship and is frequently pathovar, and even strain specific. This specificity doe s 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 th e phage may persist for long periods without a living host. The use of copper for disease control has lead to concerns in term s of the environment and in terms of the pathogens developing pesticide resistance (33). Therefore, alternate disease control strategies are being developed. Zaccar delli et al. (77) first proposed the use of bacteriophages preventatively as a biological contro l 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 re garding bacteriophages that infect Xcb. When bacteriophages are applied to le af surfaces to control plant pa thogenic 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

PAGE 22

investigate the occurrence of b acteriophages that can infect Xcb and establish a collection of strains that attack Xcb and which are common in Florida, (i i) partially characterize selected phages from this collection, (iii) use some of these se lected bacteriophages in an attempt at a biological control of bacterial spot of begonia, and (iv) study bacteriophage survival on leaf surfaces under a variety of artif icial and natural environments, to determine which negative stresses are most significant. 22

PAGE 23

CHAPTER 2 THE OCCURRENCE OF BACTERIOPHAGES Through diverse circumstances, phage popula tions can accumulate and survive in a wide variety of environments. The abundance of both bacteria and phage in the biosphere is quite large (5, 14, 71). A general estimate is that there are ten times as many phage as bacteria. Another enthusiastic estimate claims there are 1030 bacteriophages in th e biosphere (14). However, the abundance of phage is dependent on the abundance of host bacteria and the survival of phage; thus the levels of phage populations can vary greatly in different environments. There are abundant data concerning phage existen ce and survival in the soil (4, 5), but only a few studies regarding phage survival on plants in ag ricultural systems. However, there are no studies available regarding phage occurrences and survival in controlled environments, such as in nurseries, greenhouses, shade houses, or similar production systems. Bacteriophage occurrence in other environm ents, such as marine phages, has been extensively investigated (9, 10, 52, 53, 71). There is an abundance of studies on aquatic phage in fresh water also (71). Generally, there are two commonly-used methods for estimating the populations of bacteriophage (4, 5). One method relies on a tr ansmission electron microscope (TEM); where a direct count of virus-like partic les (VLPs) is performe d. The other method is a plaque assay where a sample suspected of contai ning phage is mixed with the bacterial host and plated with soft agar. Successful infections become evident as clear areas, or plaques (1) in the agar. For example, direct phage counts using a TEM showed an abundance of phage-like particles in the rhizosphere of sugar beet. This was followed by plaque assays using both Serratia sp. and Pseudomonas sp. (5). These results showed that a phage count by traditional plaque assay methods is likely to greatly underestimate the true population level. In other words, 23

PAGE 24

there are likely more phage presen t in a given sample than can be detected via a plaque assay. Furthermore, phages of Serratia were shown to vary seasonally (4). The above mentioned and similar studi es have suggested a plausible phenomenon known as the Great Plate Count Anomaly (GPCA) which implies that there are many phages present within a given ecosystem which might not have been detected by conventional plaque assays because all of the bacterial hosts that su pport them have not yet been identified or grown in culture (71). Moreover, if a fluorescent stain such as SYBR Gold is used to stain all VLPs in a sample, and then observed with epifluorescence microscopy, there are likely to be many more VLPs observed than are indica ted with a plaque assay. Most natural bacterial populat ions are frequently infected with phage. The phage population and the bacterial population co-exist (71). Bacteriophages have been found in nearly every environment that contains a supportive bacterial population. Breitbart et al. (10) studied bacteriophages occurring in hot springs located in California. They stained samples with SYBR Gold, examined them under epifluorescence microscopy, and observed numbers of VLPs ranging from 7.0 X 104 to 7.0 X 106 VLPs/ml. It should be noted that the temperature of these hot springs was sufficient to excl ude eukaryotic organisms, so it is likely all the VLPs observed were bacteriophages (11). In another study, hot springs around the world were examined and 115 different bacteriophages of Thermus spp. were found. Four phage families were represented: Myoviridae, Siphoviridae Inoviridae and Tectiviridae (76). This was the first documentation of the latter two families found in such environments. Starting in the 1950s, Bacteriophage Lambda was used as a model system for many molecular studies. These findings formed a f oundation for many of the molecular techniques 24

PAGE 25

used today. Lambda was also im portant in revealing the principl es of recombination and lytic versus lysogenic virus life cycles. Bacteriophages are also present in many f oods consumed by humans. Foods produced by fermentation processes with lactic acid bacteria such as cheeses and sauerkraut can have very high phage populations ( 49). Unfortunately, when phages contaminate these food-processing systems, they are usually detrimental to the pr ocess. For example, bacteriophage can be a nuisance in the dairy industry where they may att ack starter bacteria used in milk fermentation. These fermentation systems and associated phage have been well studied. Interestingly, there are several studies that show re sistance of lactic acid bacteria to bacteriophages (49). In marine environments, populations of bacterio phage can be very high (14). Bergh et al. (9) studied eutrophic estuarine water and found the count of bacteria to be 106 cells/ml and the bacteriophage count to be 107 particles/ml. Marine phage popul ations are strongly affected by two opposing forces: the synthesis of new virions by lytic infections a nd phage inactivation by environmental factors. A major cause of bacteriophage inactivati on in marine environments is solar radiation, particularly the ultr aviolet spectrum [(UV), (74)]. Protozoan grazing also reduces ma rine phage populations, but not as significantly as solar radiation (63). Wilhelm et al (72) found that in marine e nvironments, ultraviolet B (UVB) radiation could cause phage DNA da mage within the genome and t hus reduce infectivity. More about the effects of UV light on phage inactiv ation will be discussed in Chapter 3. Within soil, Davies et al. ( 17) found that the inactivation rate of bacteriophage PRD1 (used as a surrogate for human pathological viruses) was signif icantly affected by the biotic status of the soil. The inactiva tion rate was much greater when the phage was exposed to soil and sewage biota than when the phage was exposed to soil and sewage biota that had been gamma 25

PAGE 26

irradiated. Furthermore, it was shown that humic acid increased the survival of bacteriophage PRD1. Dissolved organic matter of this type is believed to provide more sites for viral attachment and assist in l ong distance transport (27). The GPCA implies a large and significant underestimation of phage population as described above. This is very evident in marine systems. One study analyzed 200 L of seawater in which 5000 viral genotypes were found (11). Plaque assay counts compared to direct TEM counts underestimated the phage population by 100-1000 fold. Although this assumes that all the VLPs observed are alive. As mentioned above, a bacteriophage is usua lly very host-specific for the particular bacterial species it will infect. Host ranges of bacteriophages are frequently species, pathovar, and even strain specific. The occurrence of inf ection of one phage within different Genera of bacteria is not a common occurrence (52). Fo r this reason, a phage that can infect a plant pathogenic bacterium is not likely to infect an epiphytic bacterium. The notion of using bacteri ophage to control plant pat hogenic bacteria has been frequently presented, and bacteriophages have been used as a biological c ontrol of bacterial plant disease incited by several species and pathovars of Xanthomonas (6, 7, 8, 24, 25, 77). Bacteriophages have been successful in some cases and relatively ineffective in other cases for this use because they have been found to be relatively short-lived on leaf surfaces depending on the host plant and the outdoor environment (7, 24, 51). Enviro nments common to the nursery industry are very different than those of field-grown tomatoes or peaches. For example, the effects of ultraviolet light, desiccation, and adsorption may not be as profound in greenhouse environments, potentially making b acteriophage a feasible means of controlling bacterial spot in such production systems. 26

PAGE 27

The concept of treating bacterial infections w ith bacteriophages has ex isted nearly as long dHerelles discovery. DHerell e isolated phages of a Shiga bacillus, a common cause of dysentery. He found these phages to be obligat ory and parasitic on only the Shiga pathogens, and not on any other tested bacterium (57). He went on to find phages of other pathogenic bacteria. DHerelle realized this potential to tr eat these diseases and obtained favorable results treating typhoid, dysentery, urin ary tract infections and even bubonic plaque (18, 19). In the 1920s, some of the techniques devel oped by dHerelle were applied to plant systems. The concept of the term bacteriophage coined by dHerelle was not even used by the entire scientific community during this time. Many researchers referred to the effects of bacteriophage as the lytic princi ple. In 1923, Gerretsen et al. (29) isolated a lytic agent that was active against root nodule organisms from se rradella, lupine and clover (34). The following year, an inhibitory substance, that might have been a phage was isolated from cabbage that had been rotted by a fluorescent organism (42). Bacteriophages of Erwinia carotovora were isolated by Coons and Kotilia in 1925. A first t ype of biological control was attempted using carrot slices, which were then either inoculated with Erwinia alone or Erwinia plus the bacteriophage. The samples were then incubated at 25 C for three days and the slices with the phage showed fewer symptoms of soft rot ( 15, 34). Another study during the same period demonstrated lytic principles from juice from a tobacco plant with wildfire disease that were active against Phytomonas tabaci A high potency bacteriopha ge was isolated from soil beneath infected peach trees that could infect Phytomonas pruni (3, 34). There were other studies during this period that demonstrated the lytic principle ag ainst phytopathological bacteria, but the concept of us ing bacteriophage to treat or prevent bacterial diseases de27

PAGE 28

emphasized because antibiotics became the preferred antibacterial agent in the west after World War II. Another use for of bacteriophages that wa s first published shortly after WW II was phage typing (21). Klement (36, 37) used ba cteriophages to distinguish between different strains of Xanthomonas on bean. Besides identifying bacterial strains, phages were also used for diagnostic purposes. Sutton et al. (65) found a robust bacteriopha ge that could infect several pathovars of Xanthomonas and used macerate from infected plan ts in plaque assays to show the presence of particular Xanthomonas strains. This phage could infect multiple pathovars but not every strain within a particular pathovar. Another useful appl ication with bacteriophages was first proposed by Zaccardelli et al. (77) in 1992. This was to apply phages preventatively to control bacterial spot on certain plants of monetary value. Th is subject will be more thoroughly discussed in Chapter 4. 28

PAGE 29

CHAPTER 3 CHARACTERIZATION OF BACTERIOPHAGES ISOLATED FROM NURSERIES AND FOLIAGE GROWING ENVIROMENTS IN FLORIDA Bacterial populations in nature comm only support associated populations of bacteriophages (71). Populations of plant pathogenic Xanthomonas spp. from infected plants can also support bacteriophages; a lthough this phenomenon has not been reported for nursery and greenhouse environments. A primary objective of this study was to investigate the occurrence of bacteriophages of Xanthomona s in nursery and greenhouse producti on systems. If phages could exist there, they might be useful for biological control of diseases caused by Xanthomonas. Greenhouse micro-ecology as it affects bacterial plant pathogens and their phages has not yet been studied extensively. This study is novel in this regard. Samples were collected from the soil and water drainage areas in commercial greenhouses and from irrigation systems in commercial production systems. Additionally, sample s were taken from bacteria-infected plants submitted to the University of Florida Mid-Fl orida Research and Education Center (MREC) Plant Clinic. Materials and Methods Bacterial Strains To determine the occurrence and nature of Xcb and its associated bacteriophage, nurseries and greenhouses near Apopka, FL area were visited, and begonias showing symptoms of bacterial spot were collecte d. Samples were also collected from plants brought to the MREC Plant Clinic. Lesions were excised, macerated in sterile tap water then st reaked out on nutrient agar [(NA), (0.3% beef extract, 0.5% peptone, 1.5% agar, pH 7.3) ] and also streaked out on a tetrazolium chloride medium [(TZC), (1 % peptone, 0.5% glucose, 1.8% agar, 0.001% tetrazolium chloride)]. Colonies typical of Xanthomonas spp. (based on appearance) were restreaked on TZC two additional times and were confirmed to be Xanthomonas by PCR using 29

PAGE 30

primers to amplify a section of Xanthomonas hrpB gene following a method described by Obradovic et al. (51). Phage Isolation from Soil, Irrigation Systems, Water Canals, and Plant Tissue As phages of plant pathogenic bacteria ar e commonly found in soil to a depth of 0-4 cm, soil samples were collected from locations proximat e to the diseased plants or 0-4 cm from the surface in nearby runoff water drainage areas. Two popular methods were utilized to extract phage from soil. For the fi rst, the techniques were adapte d from Ashelford et al. (4). Approximately 10 g of soil was placed in a steril e 50 ml conical centrifuge tube that then was filled to the top with tap water, and allowed to stand 20 min with periodic inversions. The tubes were then centrifuged at 1,500 rpm for 20 min a nd the supernatants were passed through a 0.2 m syringe filter (Cat.# SLGP033RS, Millipore Corp., Bedford Md.) Soil filtrates were platetested immediately (protocol follows) or stored at 4C. A second method for extracting phage from soil or tissue was also used if phages were thought to be present at very low numbers (B. Balogh, personal communication, 2005). This is an enrichment method that allows the phage to reach higher populations. The enrichment broth (EB) contained nutrient broth [(NB), (0.3% beef extract, 0.5% peptone; pH 7.3)] and 2.5% CaCO3 To the EB, 5 g of soil or pl ant tissue was added along with 800 l of an active culture (0.7 OD) of Xcb. This suspension was incubated overnight at 28C with constant shaking at 120 rpm. Then, it was transferred to a sterile 50 ml conical centrifuge tube and centrifuged at 1,500 rpm for 20 min. The supernatant was passed through a 0.2 m syringe filter and used immediately in a plaque a ssay or stored at 4C. Water samples from irrigation systems were collected in 50 ml conical tubes then centrifuged at 8,000 rpm for 10 min. Approximately 45 ml was collected from each tube and passed through a 0.2 m syringe filte r and used immediately in a plaque assay or stored at 4C. 30

PAGE 31

Plaque Assays The following protocols were developed fo llowing personal communications with B. Balogh, University of Florida, Plant Pathol ogy Department. Plaque assays estimate both abundance and diversity of phages in soil and wa ter samples. In the developed procedure, Xcb or other pathovars of Xanthomonas spp. were grown in NB for 18 hr until the optical density (OD) was approximately 0.7. Cultures were then centr ifuged at 4,000 rpm for 10 min and the cells resuspended in 10 mM MgSO4. The bacterial suspension and phage samples were combined at a 1:1 ratio (the total volume was 1 ml), mixed gentl y, and the phage were allowed to adsorb to the host cells for 20 min with periodic inversions of the microcentrif uge tubes. After the allotted adsorption time, the phage and bacterial mixture was added to a sterile Petri dish. Warm (40C) nutrient agar with yeast extract [(YNA), (0.3% b eef extract, 0.5% pepton e, 0.2% yeast extract, 0.6% agar)] was then added, mixe d gently, allowed to solidify, then the plates were incubated overnight at 28C. Phage Isolation from Plaque Assays To isolate individual phage samples from a pla que assay plate, indi vidual plaques were picked from single plaques with a sterile t oothpick and streaked on a freshly prepared YNAXcb lawn plate. The plates were incubated overnight at 28 C. Approximately 3 ml of sterile tap water was added to each plate. The plates were allowed to gently shake at 56 rpm for 20 min, and then the water was collected and centrifuge d at 15,000 rpm for 10 min. The supernatant was passed through a 0.2 m syringe filte r into a sterile 2.0 ml screw-cap vial and stored at 4C. Phage titer was estimated by performing a serial dilution series and spotting each concentration on a freshly poured YNAXcb lawn plate (method de scribed below). 31

PAGE 32

Preparation of Bacteriophage Cultures Bacteriophage grown in a nutrient broth culture overnight can achieve very high population levels; typically 109 PFU/ml (Plaque Forming Unit). In brief, 50 500 ml of NB was inoculated with a loopful Xcb cells from a Petri dish, followed by inoculation with 10 20 l of a particular bacteriophage suspension (about 108 PFU/ml). The culture was grown at 28C oscillating at 150 rpm overnight. After inc ubation, the culture was centrifuged at 8,000 rpm for 10 min and passed through a 0.2 m filter, as descri bed previously. Serial dilutions were made and spotted on an Xcb lawn plate to estimate phage titer. For preparing larger volumes, a 500 ml filter holder and receiver apparatus (Cat# 300-4050, Nalgene N unc International, Rochester, NY) and a 47 mm diameter 0.2 m Nitrocellu lose filter (Cat.# SA 1J789H5, Millipore Corp., Bedford Md.) were used. Syringe filters were not used because viruses were observed to excessively adsorb to the filter surfaces (26). Spot Testing Plaque assays provide the best estimate of the viable titer of bacteriophage in a given sample. However, plaque assays are time cons uming. A faster method to estimate the number of phages present is through spot testing (B. Balogh, personal communication, 2005). In brief, a small aliquot of a bacteriophage s ource or culture is seri ally diluted 1:10; so the concentrations are 100, 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7, etc. A fresh lawn plate of of YNA and Xcb is prepared. When the agar has set, a 10 l aliquot of each dilution is pipetted on a different location on the plate. After an overnight incubation at 28 C, the titer of viable phage can be estimated: a spot will appear wherever an amount of viable phage was placed. Titer can be extrapolated to the nearest power of ten. This method can only estimate tite r to the power of ten and not more exactly, but it is useful for fast estimation. 32

PAGE 33

Extraction of Bacteriophage DNA A method adapted from Sambrook and Russell (58) was used to extract the DNA of the bacteriophages as follows. The phage were grown overnight in a NB culture as described above. The culture was centrifuged at 8,000 rpm for 10 min and the supernatant was passed through a 0.2 m filter. A stock solution of Pancreatic DNase I [1 g/ml (Cat.# D5025, Sigma Aldrich, St. Louis, MO); 50% glycerol, 20 mM Tris (pH 7.5), 1 mM MgCl2,] was diluted into a DNase I dilution buffer [10 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1 mM MgCl2; 3 l/ml] to form a working DNase solution which was added to the phage sample 1:10 and incubated at 37 C for 30 min. Following incubation, 40 l of 2.5 X SDS-EDTA solution [0.4% (w/v) SDS, 30 mM EDTA, 20% Sucrose, 0.5% DNA loading dye] was added and the sample was incubated at 65C for 5 min. The phage DNA was then gel elect rophoresed at 50 V for 1 hr on a 1.0% TAE [(40 mM Tris, 20 mM EDTA, 120 mM Sodium Acetat e, 0.017% Glacial Acetic Acid), pH 8.0] agarose gel containing 0.5 g/ml of ethidium br omide. Regions of the gel that contained DNA were excised and purified (P romega Wizard Purification system, Cat. # A9281, Promega Corp. Madison, WI.) following th e manufacturers protocol. Electron Microscopy of Isolated Bacteriophages of Xcb Bacteriophages were grown in NB culture and centrifuged and filtered as described above. Five microliters of a phage suspen sion was applied to 300 mesh Formvar Carbon B copper grids (Product # 1GC300, Ted Pella Inc. Re dding, CA.). The grids were allowed to dry in a desiccator. Droplets of approximately 50 l of 2% methylamine tungstate (Product # 18353, Ted Pella Inc. Redding, CA.) were applied to a small sheet (a pprox. 5 cm X 20 cm) of Parafilm (Product # PM-992, Pechiney Plastic Packaging. Me nasha, WI), one droplet per phage sample. The grids were placed on the droplets sample side down for 30 sec, removed, returned to proper orientation and then further dried under desi ccation. They were then examined with a 33

PAGE 34

transmission electron microscope (Zeiss EM-10, Carl Zeiss Inc. Jenna, Germany) or (Hitachi H7600, Hitachi High Technologies America, Inc. Pleasanton, CA). A magnification of 67,000 X showed the general field which was examined at 120,000 X, 150,000 X and 200,000 X to show the details of phage morphology. Use and assi stance with the TEMs were provided by The Interdisciplinary Center for Biot echnology Research (ICBR) at the University of Florida. Results Using the phage isolation tec hniques described above with Xanthomonas strains collected during this study or provided by D.J. Norman (Table 3-1), 150 isolates of phage that could infect Xcb were found, 91 of which were unique and diffe rent phages (Table 3-2, 3-3, 3-4, 3-5) based on infection profiles. The strains were differentia ted based on their ability to infect different strains of Xcb (Table 3-6, 3-7, 3-8). The majority of these phages were collected from soil proximate to diseased plants, from irrigation syst ems, recycled water, a nd drainage canals at various nurseries and greenhouses. However, many of these pha ges were found in nursery and greenhouse environments where begonias were not presently grown. It is the authors conclusion that phage commonly accumulate in recycl ed water, irrigation, and drainage systems. It is unlikely, however, that phage of Xanthomonas be found in nascent well water. Initial Characterization of Isolated Bacteriophages Once a group of phages was isolated from a source using the above procedures, spot testing on different strains of Xanthomonas was used to better characterize the strains (Table 33). Ten of these bacteriophages that infect Xcb were selected for further study. (Table 3-3) These ten were selected as representative speci es from various locations where phage were collected. From this group, four bacteriophages were selected for the biological control portion of this study and they are designated: Pxcp-A (P), Pxcb-AgSt III A (A), Pxwm-Jons II A (J) and Pxad-5sC (D). These four bacteriophages were chosen because they had broad in vitro host 34

PAGE 35

ranges (Table 3-3). Of all the phages isolated in this study, the J phage ha d the ability to infect the largest number of strains of Xcb that were tested (see table 3-3) and was chosen for the survival assays in this study. The other six phage s that were not used fo r a biological control were selected as representatives of phages isolat ed from other sources. These phages are named: Pxcb-AgSt IIa, Pxcb-AgSt IV A, Pxcb-Rus II B, Pxcb-P1, Pxcb-P3, and Pxcb-P7. Analysis of Bacteriophage DNA Bacteriophage DNA from each of the four phages selected for biological control experiments was extracted as described above a nd visualized on a 1.0% TAE agarose gel (Fig 31). This figure shows the four biocontr ol phage DNA samples alongside a 1 Kb ladder (Cat#3412A, Takara Bio Inc., Otsu, Shiga, Japan). The bands visualized i ndicated that all four phage had linear, non-segmented DNA genomes. Kodak 1D 3.6 image analysis software was used to estimate the size of each phages genom e to be approximately 12,500 base pairs (bps) in length. These results combined with TEM data s uggest that all four of these biocontrol phages are related. Restriction dige stion of each of the four phage genomes was done with Alu I, Bam H I, BstZ I, Eco R I, Hpa II, Sac I, and Xho I, but none of these endonucleases cut any of the genomes. Electron Microscopy of Select Bacteriophages Each of the ten bacteriophages selected were grown in NB, centrifuged, and filtered as described above. The filtered cultures were th en transported to the ICBR Electron Microscopy core facility at UF where they were applied to the grids; the negative stain was applied and were examined as mentioned above. All of these bact eriophages appeared to be of similar sizes and morphologies. Each image in Figure 3-2 and Figure 3-3 was acquired at 67,000 X, 120,000 X, 150,000 X or 200,000 X magnifications. Nine of these different virions were approximately 6070 nm in diameter. The phage II A had a diameter of less then 50 nm. For the four biological 35

PAGE 36

control bacteriophages, combini ng these results from the DNA analysis and TEM images and considering their size and mo rphology, all four of these phage s may belong to the family, Tectiviridae [National Center for Biotec hnology Information (Gen-bank), http://www.ncbi.nlm.nih.gov]. However at present, the described species of Tectiviridae feature genomes of about 14,900 bps. Ther e is one report of a hot-spri ng Tectivirus with a genome size of about 31,000 bps (79). Five of the other six phages viewed have mo rphologies that also resemble Tectiviruses. The phage II A possi bly could also be a Tectivirus, or perhaps a Cystovirus or Levivirus. Although to make determinant taxonomical judgments, further molecular characterization of all the phages needs to be done. Discussion It is likely that bacteriophage can be found anywhere in nature where a supportive bacterial population exists or has existed recentl y. When attempting to isolate phage of a plant pathogenic bacterium, it is important to use the exact isolate or strain of pathogen of interest as a host. This can be viewed as using the proper bait to detect the presence of phage of interest. However, some bacteriophages are often not able to infect different isolat es or strains of the same pathovar of bacteria. Therefore, using a different bacterial strain, even of the same pathovar, may not detect any phage in a plaque assay. This process is slightly analogous to phage typing (25), in that a stra in of a certain pathovar from an unrelated location may not be a suitable host to phage present in the samples bei ng tested. However, using other unrelated or closely related bacteria as baits, it is possible that other phage can be found that infect the host of interest. Also, in nursery and gr eenhouse environments, phage appear to play a large role in the micro-ecology. This field is stil l relatively new to microbiology and it is likely that in the future, there will be numerous additional reports of interesting and useful phage occurrences. Unexpectedly, many of the phage isolat es found in this study that infect Xcb were not found in 36

PAGE 37

close association with begonias infected with Xcb. Of the ten phages studied in detail, six were found with Xcb as the initial bait. Four of these ten phages were selected for use as a potential biological control. These four phage s were selected based on having broad in vitro host ranges as shown in Table 3-6, 3-7, 3-8. Phage can persist in soil that is not desiccated or soil that is not exposed to high amounts of solar radiation. The D phage was reco vered from soil below a greenhouse bench where Anthuriums were grown, however, bacterial spot caused by Xanthomonas axonopodis pv. dieffenbachiae was previously a problem in this greenhouse. At the time of collection, no active Xanthomonas infections were observed in this greenhouse. The A phage was found in recycled greenhouse irrigation water using Xcb as the bait. There was a wide variety of foliage plants grown in this greenhouse. This wa ter sample yielded ma ny different phages of Xanthomonas spp. (other pathovars were also used as bait, see Table 3-2). Since many of these phages have the ability to infect multiple pathovars of different Xanthomonas spp., they could possibly be useful as a biocontrol agent for ot her Xanthomonads. The phages II-A and IV-A were collected from greenhouse soil immediately below benches of begonias that showed signs and symptoms of Xanthomonas and bacterial spot. The phages Pxcb-P1, P3, and P7 were also collected from soil from pots of begonias with bacterial spot. The pha ge Pxcb-Rus 2-4 is interesting in that it was the onl y phage isolated in this study fr om foliar tissue; all the others were collected from irrigation runoff or soil adjacent to an infection. The Rus phage was isolated from a ruscus plant infected with Xanthomonas axonopodis pv. diffenbachiae. The phage was obtained following an enrichment of this leaf tissue, followed by plaque assays. The results of study suggests that bacteriophages that are original ly found associated with an unrelated Xanthomonas spp. may be used as a biological control for other xanthomonads and 37

PAGE 38

perhaps other related species. Perhaps some of the phages with large host ranges could be used to prevent bacterial spot on di fferent foliage plants. 38

PAGE 39

39 Table 3-1. Bacterial stra ins used in this study Strain Orig in Provided by X. axonopodis dieffenbachiae Cal 1b Cal 2c Cal 3d Anmare AnCrHf 1520 X. campestris pv. begoniae 873 1076 1143 1276 1314 1500 1501 1503 1567 1800 Escarg ICh Nurs.i X. campestris pv. campestris Xcc j X.campestris pv. passiflorae Xcp 2k X. off Wax Myrtle XWM Florida Florida Florida Florida Florida Florida Florida Florida Florida Florida Florida Florida Florida Florida Florida Florida Florida Florida Florida Florida Florida Florida this study this study this study this study this study D.J. Normana D.J. Norman D.J. Norman D.J. Norman D.J. Norman D.J. Norman D.J. Norman D.J. Norman D.J. Norman D.J. Norman D.J. Norman this study this study this study this study this study this study a University of Florida, Mid-Florid a Research and Education Center b Caladium with bacterial spot from the MREC plant clinic c Caladium with bacterial spot from the MREC plant clinic d Caladium with bacterial spot from the MREC plant clinic e Anthurium with bacterial spot from the MREC plant clinic f Anthurium with bacterial spot from the MREC plant clinic g Begonia escargot with bacteria l spot from a local nursery h Begonia iron cross with bacterial s pot from the MREC plant clinic i Begonia with bacterial spot from the MREC plant clinic j Crucifer with bacterial spot from the MREC plant clinic k Passion vine with bacterial spot from the MREC teaching garden l Wax myrtle with bacterial spot from the MREC plant clinic

PAGE 40

Table 3-2. Bacteriophages isolat ed from nursery or greenhous e production environments during this study. Bacteriophages that were studied further are indicated by initials in parentheses. Phage Original Hosta Source Pxcb-AgSt III A (A) Pxad-5s C (D) Pxwm-Jons II A (J) Pxcp-A (P) Pxcb-AgSt I A-H (II A) Pxcb-AgSt III B Pxcb-AgSt III C Pxcb-AgSt III D Pxcb-AgSt III F Pxcb-AgSt IV A-B (IVA) Pxcb-AgSt IV C Pxcb-AgSt IV D Pxcb-ICS A-E Pxcb-Rus 2-4 (Rus) Pxcb-MG I A Pxcb-P1 (P1) Pxcb-P2-P8 (P3, P7) Pxcb-Jons I A Pxcb-Jons II A Pxcb-Jons II B-E Xcbescar Xad-1520 Xan-wax myrtle Xcp 2 Xcbescar Xcbescar Xcbescar Xcbescar Xcbescar Xcbescar Xcbescar Xcbescar XcbIC Xcbescar Xcbescar Xcbescar Xcbescar Xcbescar Xcbescar Xcbescar Recycled irrigation water b Below an Anthurium benchcIrrigation drainage canald Below a passion vinee Below a begonia benchf Recycled irrigation waterb Recycled irrigation waterb Recycled irrigation waterb Recycled irrigation waterb Below a begonia benchf Below a begonia benchf Below a begonia benchf MREC plant clinicg MREC plant clinicg MREC plant clinicg MREC plant clinicg MREC plant clinicg Irrigation linei Irrigation drainage canald Irrigation drainage canald 40

PAGE 41

Table 3-2. Continued Phage Original Hosta Source Pxad-3e A-B Pxad-4s A Pxad-4s B Pxad-4s C Pxad-5s B Pxad-phage A Pxad-6e A-B Pxad-6e C Pxad-6e D Pxad-6e E Pxad-6e F-G Pxad-6e H Pxad-6e I Pxad-6e J Pxad-6e K Pxad-AgSt III A Pxad-AgSt III B Pxad-AgSt III C Pxad-AgSt III D Pxad-AgSt III E Pxad-AgSt IV A Pxad-AgSt IV B Pxad-AgSt IV C Pxad-AgSt IV D Xad-120 Xad-120 Xad-120 Xad-120 Xad-120 Xad-120 Xad-120 Xad-120 Xad-120 Xad-120 Xad-120 Xad-120 Xad-120 Xad-120 Xad-120 Xad-120 Xad-120 Xad-120 Xad-120 Xad-120 Xad-120 Xad-120 Xad-120 Xad-120 Below an Anthurium benchcBelow an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Recycled irrigation waterb Recycled irrigation waterb Recycled irrigation waterb Recycled irrigation waterb Recycled irrigation waterb Below a begonia benchf Below a begonia benchf Below a begonia benchf Below a begonia benchf 41

PAGE 42

Table 3-2. Continued Phage Original Hosta Source Pxad-AgSt IV E Pxad-P1, P3-P8 Pxad-P2 Pxcm-GSF B Pxcm-AgSt III A-B Pxcm-AgSt III C Pxcm-AgSt IV A, C Pxcm-AgSt IV B Pxcm-AgSt IV D Pxcp-P1 Pxcp-P2 Pxcp-PB Pxcp-AgSt IV A Pxcp-AgSt IV B Pxcp-AgSt IV C Pxcp-AgSt IV D Pxcp-AgSt IV E Pxcp-AgSt IV F Pxcp-AgSt IV G Pxcp-AgSt IV H Pxcp-AgSt IV I Pxcp-AgSt IV J Xad-120 Xad-120 Xad-120 Xan crape myrtle Xan crape myrtle Xan crape myrtle Xan crape myrtle Xan crape myrtle Xan crape myrtle Xcp 2 Xcp 2 Xcp 2 Xcp 2 Xcp 2 Xcp 2 Xcp 2 Xcp 2 Xcp 2 Xcp 2 Xcp 2 Xcp 2 Xcp 2 Below a begonia bench f Below an Anthurium benchc Below an Anthurium benchc Geranium soilj Recycled irrigation waterb Recycled irrigation waterb Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below a Passion Vinee Below a Passion Vinee Below a Passion Vinee Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc 42

PAGE 43

Table 3-2. Continued Phage Original Hosta Source Pxcc-AgSt IV A Pxcc-AgSt IV B Pxcc-AgSt IV C Pxcc-AgSt IV D Pxcc-AgSt IV D Pxcc-AgSt IV F Pxcc-AgSt IV G Pxcc-AgSt IV H Pxcc-AgSt IV I Pxcc-AgSt IV J Xcc Xcc Xcc Xcc Xcc Xcc Xcc Xcc Xcc Xcc Below an Anthurium benchcBelow an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc Below an Anthurium benchc _____________________________________________________________________________________________________________________ a Bacterial hosts described in Table 3-1 b Water from water holding tank in a nursery growing a variety of foliage plants c Soil from below a greenhouse bench with previous bacterial spot infections d From an irrigation drainage canal in an outdoor nursery e Soil from below a passion vine with bacterial spot f Soil from below a begonia bench wi th begonias with bacterial spot g Plants brought to the MREC plant di sease clinic with bacterial spot h From an enrichment of diseased tissue from a ruscus plant with bacterial spot i Water from a directly from an irri gation line in an outdoor nusery jSoil from the pot of a geranium with bacterial spot 43

PAGE 44

Figure 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 char acterized in this study. From left to right, they are Pxcb-AgSt III A (A), Pxad-5 sC (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

PAGE 45

D J P A Figure 3-2. Images of each of the bacteriophage used in the biological control portion of this study. The image labeled D is the bacteriophage Pxad-5sc. The image labeled J is the bacteriophage Pxwm-JonsII A. The image labeled P is the bacteriophage Pxcp-A. The image labeled A is the bact eriophage Pxcb-AgSt III A. The sizing scales are for approximation purposes. These phages were imaged with a Zeiss EM10 electron microscope. 45

PAGE 46

Rus II A P7 P3 P1 IV A Figure 3-3. Images of six othe r bacteriophages partially character ized in this study. The image labeled II A is the bacteriophage Pxcb-A gSt IIA. The image labeled Rus is the bacteriophage Pxcb-Rus II B, the image labeled P7 is Pxcb-P7, the image labeled P3 is the bacteriophage Pxcb-P3, the image labeled P1 is the bacteriophage Pxcb-P1, and the image labeled IV A is the bacteriophage 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

PAGE 47

Table 3-3. 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 pla que. A +/- means an opaque plaque was observed. A means no plaque was observed Phage Bacterial Strain 47

PAGE 48

Table 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 di fferent isolates of bacteriophage. A + means a completely clear and concise plaque. A +/- means an opaque plaque was observed. A means no plaque was observed. Phage Bacterial Strain 48

PAGE 49

Table 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 pla que. A +/- means an opaque plaque was observed. A means no plaque was observed. Phage Bacterial Strain 49

PAGE 50

CHAPTER 4 PERFORMANCE OF BACTERIOPHAGES AS A BIOLOGICAL CONTROL OF BACTERIAL SPOT OF BEGONIA Introduction Prevention of bacterial infections of plants has relied primarily on the use of copper bactericides for many years. Coppe r is toxic to microbia l life at low to moderate concentrations because it can generate free radical s, which damage lipid membranes and DNA (69). Teixeira et al. (67) observed that as little as 0.25 mM of copper was en ough to inhibit the growth of a copper-resistant mutant of Xanthomonas axonopodis pv. c itri. For agricultural crops, cupric hydroxide is a commonly applied form of copper. In a recent study, the fungicide Fluazinam [3Chloro-N-(5-chloro-2,6-dinitro-4-trifluoromethylphenyl)-5-trifluoromethy l-2-pyridinamine] and the bactericide Greenshield (a quaternary ammo nium compound) were compared with cupric hydroxide. Each one provided reasonable control for some bacterial pathogens but not others. The overall protection from Fluazi nam or Greenshield was inferior to cupric hydroxide (13). Numerous other studies have produced widely varying result s. Although copper is useful, prolonged use of copper has resulted in bacteria (both epiphytic and path ogenic) that express resistance genes for copper (69). These re sistance systems are varied and found in both chromosomal and plasmid DNA. These are not resi stance systems in a strict sense as copper is a required co-factor for some enzymatic activity (67) Rather, these operons encode proteins that are responsible for removing excess copper; so th is is tolerance and not resistance. A few copper-tolerant strains of Xanthomonas campestris pv. vesicatoria were found to possess a copABCD operon; which is a copper induced resistan ce system. The operon encodes a protein, CopR that has a conserved binding motif (cop box). When copper binds to CopR, the other genes in the operon are expressed and excessive atoms of copper are pu mped out (67). The 50

PAGE 51

frequent appearance of copper-res istant strains, in addition to environmental concerns for copper and other heavy metals, makes alternatives to controlling bacterial pathogens desirable. Antibiotics have also been used since the 1950s to control certain ba cterial diseases of horticulturally-important plants. The two most commonly used antibiotics today are oxytetracycline and streptomycin (45). In the United States, antibiotic use to control plant diseases now accounts for less than 0.5% of the total use of antibiotics for the country. Antibiotic use to control bacterial plant dis ease was very common in the 1950s because they were used at low doses and showed only minima l phytotoxicity. However, antibiotic resistant strains of Erwinia Pseudomonas and Xanthomonas began to appear quic kly as early as the 1950s and continue to appear today (16, 45). Accordingly, the use of antibiotics for plant disease control has declined greatly. A different and novel method for controlling bacterial plant disease has been the use of bacteriophage of Xanthomonas campestris pv. pruni to control bacterial spot on peach trees (77). Bacteriophages were sprayed on the trees as a preventative control. Since then, similar methods have been used to control bacterial spot of geranium caused by Xanthomonas campestris pv. pelargonii (24) and bacterial spot of tomato caused by Xanthomonas campestris pv. vesicatoria (24, for a review, see reference 33). Bacteriophages were also used successfully to control Xanthomonas leaf blight of onion (39). However, there have been severa l problems with the use of phage to control plant disease. As will be discussed in Chapter 5, UV light and other environmental factors greatly aff ect the survival of phage on leaf surfaces. To overcome some of these problems, Balogh et al. (7) tested different formulations fo r phage suspensions to control bacterial spot of tomato. These included pre-gelatinized corn flour, casecrete, and powdered skim milk. Skim milk was found to be the mo st effective and inexpensive for enhancing the 51

PAGE 52

survival of bacteriophage on leaf surfaces. A dditionally, the ideal time for application of bacteriophage mixtures to tomato was found to be at dusk because the leaves usually remained wet for several hours from dew, etc. The use of bacteriophage for biological control was further tested with less success than for tomato (6, 7, 24) by Balogh (8) to control citrus canker ( Xanthomonas axonopodis pv. c itri ). Using bacteriophage as a biocontrol was also tried (unsuccessfully) to contro l walnut blight caused by Xanthomonas campestris pv. juglandis (47). Generally, phage survive poorly on leaves, but if they were internal to the plant, perhaps survival could be improved. Hypothetically, a bacteriophages sma ll size should allow for passive uptake by plants and tran slocation in the transpiration stream. The point of ingress would likely be wounds or small openings caused by r oot growth, transplantation or cultivation. However, in experiments to achieve even a dete ctable presence of phage within the leaves of tomato and bean plants, the plan ts needed to be grown hydroponically, and required that roots be injured by cutting immediately prior to phage exposure (70). These root abrasions facilitated phage entry into the xylem tissue of the plants and into the transpiration stream. Generally, poor survival in field experiments has often been cite d as a primary reason some uses of a biological control with bacteriophages have been unsuccessf ul (5, 6, 47). Greenhouses and other controlled environments provide more favorable conditions for bacteriophage to persist on leaf surfaces, possibly making bacterial disease control more feas ible than in the field. An objective of the present study was to evaluate the use of bacteriophage as a biological control of bacterial spot of begonia, which is commonly pr oduced in greenhouses. Materials and Methods Bacteriophage Formulation fr Biological Control Four of the phages described in Chapter 3, Pxcb-AgSt III A (A), Pxad-5sC (D), PxwmJonsII A (J), and Pxcp-A (P) were used for this portion of the study. Instead of using only one 52

PAGE 53

bacteriophage, a mixture of four phages was used to prevent the ba cterium from becoming resistant to a single phage. Phages were produced using large NB cultures containing Xcb and the selected phages, as described in Chapter 3. The phages were grown indi vidually in flasks of NB, then centrifuged and filtered as described in Chapter 3. The phage suspensions were pooled and formulated based on the results of Balogh et al. (2003) with 0.75% (w/v) instant nonfat dry milk. Additionally, 0.1% (v/v) of the agricultural surfactant AG 98 (80% octylphenoxypolyethoxyethanol, Rohm & Haas Co., Philadelphia, PA) was added to the phage preparation before application to provide wettin g and better distribution on leaf surfaces, as begonia leaves have a thick waxy cuticle. Estimation of bacteriophage population. A sample of each bacteriophage NB culture prior to pooling was diluted with sterile tap water to 1X, 0.1 X, 0.01 X, 0.001 X, 1.0 X 10-4, 1.0 X 10-5, 1.0 X 10-6, and 1.0 X 10-7. Each dilution was then used in a spot test, as described in Chapter 3. Biological Control of Ba cterial Spot of Begonia Experimental design. All of the biologica l control experiments were carried out in a plastic-covered greenhouse. A greenhouse bench wa s shaded with an 80% shade cloth to make the environment more suitable for begonias. An ir rigation line was set up in the center of the bench, which provided water from overhead risers. The first th ree experiments used sprinkler nozzles that had an output of 49.5 L per hr and the fourth experime nt used nozzles with an output of 105.6 L per hr. The irrigation was applied so that each pot received approximately 20 ml of water total in each replication. Daily irrigation for the first three experiments was initiated at midnight and was intermittent: 1 min of irrigation per 10 min interval. This process continued for 5 hr. In the fourth experiment, the bench was irrigated once starting at 05:00 and lasted for 53

PAGE 54

15 min. This provided adequate water for the plan ts. Irrigation in the fi fth biological control experiment is detailed below. Infection of Begonia inoculum plants with Xcb Begonias growing in 15 cm pots, were watered well and had saucers placed under the pots immediately before inoc ulation. To infect begonias with Xcb, bacteria were grown in NB culture overn ight. The cells were centrifuged at 4,000 rpm for 10 min and the supernatant was discar ded. The cells were re-suspended in saline [0.8% (w/v) NaCl] and applied to begonia plan ts with a chromatography sprayer [(Cat.# Z529745-1EA) (Sigma-Aldrich, St. Louis, MO.)] at 17.2 kPa. Following inoculation, the plants were placed in plastic bags and the edges of the bags were tu cked between the bench and the saucer to provide a humid environment. The pl ants were left in this condition overnight, and then the bags were removed. Symptoms of b acterial spot developed in about seven days. Biological Control Experiments. For the first three biolog ical control experiments, begonias ( Begonia semperflorens cv. cultorum ) growing in 10 cm pots were arranged in the greenhouse similar to the method of Flaherty et al. (28). Each replicat ion consisted of nine plants. The center begonia (inoculum plant, see above) was infected with Xcb and showed symptoms of bacterial spot. Su rrounding the inoculed plant, eight healthy begonias were placed. There were thirty-two plots total, eight per tr eatment, arranged randomly across the bench. All of the applications of bacteriophage or copper we re made with a chromatography sprayer. Four bacteriophages were chosen based on host range (see Chapter 3) and prepared as already described. In each of the first four experiment s, four treatments were applied: 1. untreated begonias, no agent for controlling bacterial spot. 2. plots sprayed twice a week with the phage mixture (until they were well-covered, but not dripping). 3. Plots spra yed three times a week with the phage mixture in the same manner. 4. Plots treated with c opper sulfate pentahydrate 54

PAGE 55

once a week [(Phyton 27, Source Technologies Biol ogicals Inc., Edina, MN) (21.36% EC diluted to 0.2% for application)]. Treatment with P hyton 27 or other copper form ulations is standard procedure in the foliage industry to combat bacterial disease, a nd presumed to be the benchmark for acceptable control in these experiments. For the fourth biological cont rol experiment, begonia plugs ( Begonia semperflorens cv. cultorum ) were transplanted into 10-cm square pots. Each plot had a cente r plant in a 15 cm pot that was already infected with Xcb. Surrounding to center plant were thirty-one healthy begonias. There were thirty-two plots total; eight plots per trea tment, randomly arranged across the bench and the treatments and application of phage and copper were the same as detailed above. Treatment 2 was applied at approxima tely 08:00 on Monday and Friday. Treatment 3 was applied at approximately 08:00 on Monday, Wednesday a nd Friday. Treatment 4 was applied at approximately 14:30 on Monday. The fifth biological control experiment invol ved a different method of application. A square table (91 cm X 91 cm) was constructed. The height was sta ndard table height (81 cm). Above the table, a 4 L PVC vessel was mounted approximately 8 cm above the table. At the base of the vessel were nine brass-barbed hose connections. Mounted with in and under the table were nine sprinkler heads forming a 3 X 3 square. Tygon tubing connected the brass fittings to the sprinklers (Fig. 4-1a-c). Three different treatm ents were used for this experiment: 1. water, 2. water supplemented with spent NB [(6%), (method follows)] or 3. water supplemented with a phage mixture (the A, D, J, P mixture mentioned above, but not supplemented with milk). To prepare the spent NB (for treatment 2), one liter of NB was inoculated with Xcb and incubated overnight shaking at 130 rpm at 28C. After in cubation, the broth was centrifuged at 8,000 rpm for 10 min and the supernatant passed through a 0.2 m filter as described in Chapter 3 and then 55

PAGE 56

autoclaved for 20 min. To prepare the phage mi xture (for treatment 3), four 250 ml cultures of each of the four biocontrol phages, A, D, J, and P was grown overnight at 28 C shaking at 130 rpm, centrifuged and filtered as de scribed above. The four cultures were then mixed to create a 1 L suspension. For treatments 2 and 3, the resp ective additive was mixed with the water before being poured in the vessel. For treatment 1, each respectiv e begonia plot was irrigated with 3.5 L of water. For treatment 2, each respective begoni a plot was irrigated with 3.5 L of water supplemented with spent NB (6%). For treatment 3, each plot of respective begonias was irrigated with 3.5 L of water supplemented with the phage mixture (6%). To provide the air pressure necessary for irri gation, a compressor was attached to the top of the vessel with Tygon tubing; pressure was regulated at 413 kPa. A brass ball valve controlled pressure delivery. Begonias were arra nged in the same manner as for the first three biocontrol experiments. An Xcb -infected plant in the center was surrounded by eight healthy begonias forming a 3 X 3 grid. The begonias were aligned directly below the shower heads of the table (see Fig. 4-1c). Disease Assessment. Disease damage levels for all th e biological control experiments were rated using a leaf damage key (Fig 4-2). Images of a healthy begon ia leaf, in addition to leaves in each disease damage rating class are shown in Figs. 43a and b. In the first experiment, all the leaves greater than 5 cm in width were rated after one month. For the second and third experiments, individual leaves were ma rked at the beginning of the experiments with dots of acrylic paint and were rated weekly using the leaf da mage key; disease progress was estimated by plotting weekly rating values. The ma rked leaves were only rated and not sampled. The leaf damage key was used to estimate total da mage to each plant in each plot in experiment 56

PAGE 57

4 and disease damage ratings were plotted weekly The treated plants were only rated week to week and not destructively sampled. For the fift h experiment, the leaf damage key was used to estimate total damage to each plant; these data were then plotted to estimate disease progress rates. Statistical Analyses. The leaf damage ratings were compiled and disease progress curves were plotted. The means of the rated pl ots for each treatment were plotted each week to estimate disease progress. To determine if a significance difference existed between treatments in each experiment, disease data from individual plots in each treatment were plotted. The area under the disease progress curve (AUDPC) was cal culated using SigmaPlot (Systat Software, Chicago, IL) for each plot (Tables 4-2, 4-5, 4-7) All the AUDPCs for each treatment were then assessed by analysis of varian ce (ANOVA) using SAS 9.0 (SAS Software, Cary, NC). Results In the first experiment, the irrigation provided a mist-like environment for 5 hr each night starting at midnight. Disease damage was rated on all the leaves greater th an 5 cm in width after one month using a leaf damage key (Fig. 4-2) an d the data showed very little difference between the treatments (Fig. 4-4). The average leaf damage rating for treatment 1 was 3.1 and the average leaf damage rating for tr eatment 2 was 3.4. Average leaf damage ratings for treatments 3 and 4 were 3.4 and 3.1 respectively. None of th e treatment ratings were significantly different. Neither the phage mixtures nor th e application of copper signifi cantly controlled disease when compared to the untreated control because cond itions favored the pathogen too much. For the second experiment, irrigation was the same as for Experiment 1 with intermittent irrigation starting at midnight and ending at 05:00. Disease damage for each marked leaf was rated once per week. The disease progress curves for each treatment were similar (Fig. 4-4). Disease damage was rated at days: 3,7,14, 21, 28 and 35. The respective ratings for treatment 1 57

PAGE 58

were 0, 0, 0.5, 2.0, 2.7, 4.3, and 5.5. The respective disease damage ratings for treatment 2 were 0, 0, 0.4, 2.3, 3.9, 5.3, and 6.3. At days 3,7,14, 21, 28 and 35, the disease ratings for treatment 3 were 0, 0, 0.2, 2.1, 3.9, 4.7, and 6.3 and for treatment 4 the respective disease damage ratings were 0, 0, 0.2, 1.9, 2.9, 4.0, and 5.6. A su mmary of disease damage ratings is shown in Table 41 and the area under the disease progress curve (AUDPC) data and stat istical analysis for Experiment 2 are shown in Table 4-2. The biologi cal control plots, treatm ent 2 (sprayed with the phage mixture 2 X per week) and treatment 3 (spr ayed with the phage mixture 3 X a week) were significantly worse than treatmen ts 1 (untreated control) and 4 [(treated with copper sulfate pentahydrate), (F=23.515, P=0.05)]. Student Newman Keuls analysis post-hoc (Table 4-3) revealed that treatments 1 and 4 were not signifi cantly different from one another and treatments 2 and 3 were not significantly different from one another. However, treatments 1 and 4 were significantly better (less disease) than treatments 2 and 3. For the third experiment, conditions were identical to those of the s econd. The treatments were also identical to those in experiments 1 a nd 2. Individual leaves were marked at the beginning of the experiment and disease was ra ted weekly. Unfortunately, leaf wetness and possibly other environmental conditions or the so urce of the plants resu lted in a fungal disease epidemic caused by Myrothecium roridum ; no usable data could be collected. For the fourth experiment, the sprinkler nozzl es were changed so that a higher volume of water was delivered to each pot. This shorte ned the required irrigation time to 15 min (this delivered 20 ml of water to each pot). Irrigation started at 05:00. The leaves remained wet until approximately 08:00. Again, there were four treat ments, each prepared a nd applied as described for experiment one. However, instead of nine plan ts per replicate, there were 32 plants per plot. One infected begonia (inoculum plant) in a 15 cm pot was surrounded by 31 healthy begonias, 58

PAGE 59

each in 10 cm pots. The leaf damage key was used to estimate a disease damage rating for each plant. Disease was rated at days: 7, 14, 21, 28, 35, and 42. The ratings for treatment 1 at those respective days were 0,0, 0.3, 1.5, 2.1, 2.6, a nd 3.0 [(Fig. 4-5), (Table 4-4)]. The respective ratings for treatment 2 were 0, 0, 0.3, 1.9, 2.0, 2. 4, and 3.1 [(Fig. 4-5), (T able 4-4)]. The ratings for treatment 3 were 0, 0, 0.4, 1.9, 2.0, 2.3, a nd 2.9 respectively [(Fig. 4-5), (Table 4-4)]. The respective ratings for treatment 4 we re 0, 0, 0.3, 1.7, 2.0, 2.3, and 3.0 [(Fig 4-5), (Table 4-4)]. The AUDPC data and statistical analysis for the fourth biological contro l experiment are shown in Table 4-5. The was no significant difference between the treatment values [(F=0.57, P=0.05), (Table 4-5)]. The fifth experiment utilized a different means of applica tion of phage than what was used for the previous four experiments. The be gonias were irrigated three times per week using the irrigation apparatus detailed earlier. The total volume app lied to each plot was 3.5 L at approximately 15:00 each treatment day. Treatme nts were: 1. water alone, 2. water plus spent NB (described above), or 3. water plus bacterio phage cocktail (described above). Disease was rated using the leaf damage key (Fig. 4-2) once per week for five weeks, days 7, 14, 21, 28, and 35. For treatment 1, the respective disease damage ra tings were 0, 0, 0.1, 0.8, 0.9, and 2.1 [(Fig. 4-7), (Table 4-6)]. For treatment 2, the respective disease damage rati ngs were 0, 0, 0.2, 0.8, 0.9, and 2.0 [(Fig. 4-7), (Table 4-6)]. The respective disease damage rating for treatment 3 were 0, 0, 0.2, 0.7, 0.9, and 1.8 [(Fig. 4-7), (Table 4-6) ]. The AUDPC data was statistically analyzed (Table 4-7) and there is no si gnificant difference between the treatment values [(F=0.77, P=0.05), (Table 4-7)]. Disease damage progressed similarly in all the plots. Discussion Under daily overhead irrigation, neither a bacteriophage mixture nor cupric hydroxide provided adequate protection against bacterial spot of begonia. It can be speculated that lack of 59

PAGE 60

persistence and inactiva tion of bacteriophage due to enviro nmental stresses (e.g. UV radiation and desiccation) would explain th e failure to achieve significant reductions in disease damage. Changes to the application were made and despite slight modifications, none of the five trials achieved significant protection from leaf spot damage. In the first experiment, there were four types of treated plots: 1. untre ated control plots, 2. plots sprayed with a phage mixture twice a week, 3. plots spra yed with a phage mixture three times a week and 4. plots sprayed with copper sulfate pentahydr ate once a week, at the rate indicated by the manufacturer (P hyton 27). After one month, disease was rated using a leaf damage key and there was no difference in leaf damage when comparing the begonias sprayed with phage to the begonias spraye d copper sulfate pentahydrate and to the begonias that were not treated (Fig. 4-4). Modifications were made for subsequent biological cont rol experiments. For the second and third biological cont rol experiments, individual leaves were marked with dots of acrylic paint. Disease damage on the ma rked leaves was rated weekly. Disease progress was plotted and progressed at the same rate for all the plots (Fig. 4-5). In other words, none of the three treatments showed less disease than the untreated control plots. Analysis of the variance revealed that the plots of begonias treate d with bacteriophage (treatments 2 and 3) were significantly worse than the untreated plots (tre atment 1) or the plots treated with copper sulfate pentahydrate [( treatment 4), (F=23.52, P=0.05)]. In the third experiment, the same experiment al conditions were used. Disease was again rated weekly, however, an unwelcome fungus, Myrothecium roridum invaded and confounded disease to the point where no usab le data could be collected. In the fourth experiment, several changes we re made to decrease the period of leaf wetness; leaves only remained wet for approximately 3 hours. The same four treatments used in 60

PAGE 61

experiments 1-3 were used. Disease was rated weekly. However, once again, the disease progress curves did not show a difference between th e treated plots and the untreated plots. This was the most robust experiment in this study, there were over one thous and plants and the F value was only 0.57 (Tab. 4-5). These results im ply that environmental conditions in the first four of these experiments were unfavorable for di sease control and highly favored the pathogen. When environmental conditions are very favorable for the pathogen, treatments are less likely to provide adequate pr otection (41). A change in application method was utilized in the fifth biol ogical control experiment. As will be shown in Chapter 5, a reason why the first four biological control experiments were unsuccessful was that the phage we re rinsed off the leaf surfaces during irrigation and thus were not available to offer disease control. For the fifth biological control, the bacteriophage cocktail was mixed with the irrigation water directly, befo re the plants were irrigated. Hypothetically, this associates the phage w ith the Xanthomonas inoculum. The second treatment, water amended with spent NB served as an additional c ontrol to show that the nutrient broth did not affect the results. Each day the plants were irrigated with the pha ge cocktail, a small aliquot of the irrigation water from treatment 3 was reserved, serially dilu ted and spotted on an Xcb lawn plate to estimate the phage popul ation size of the suspension. This titer was consistently 107 PFU/ml. However, it has been recently suggest ed the for success with using bacteriophages to control bacterial diseases on plants the phage population size in the applicant needs to be at least 108 PFU/ml (S.T. Abedon, personal communicati on, 2008) and the phage population in this experiment was only 107 PFU/ml. Results that will be disc ussed in Chapter 5 will show that even if the phage population is 109 PFU/ml, successful infection of Xcb may not occur because phage need to be in a water suspension to adsorb and infect the host. On begonia leaves in 61

PAGE 62

greenhouse environments suitable for begonias, th e desiccation is very rapid and the water suspension the phage need evaporates before a successful infection can be initiated. Moderately successful prevention of Xanthomon as infections of diffe rent crop plants has been achieved with bacteriophages (6, 7, 8, 24, 25, 39). But the environmental conditions in all of these studies were very different than those used in the present study. For tomato, initial attempts showed the bacteriophage were inact ivated quickly on leaf surfaces under solar radiation. Two major changes were made to make phage application to tomato crops more effective. Research indicated th e best time to apply phage was at dusk. Furthermore, Balogh (7) tested different formulations of phage mixtures and found the addition of sk im milk gave the best protection. Jagger (32) discu ssed how UV light can damage molecules and cause radical oxygen to be released, which then may damage DNA or proteins. Protein in the milk may provide a substrate for these reactive species to bind before the active oxygen can damage the phage. In citrus, bacteriophage did provide some protection agai nst Xanthomonas infections, although protection was inferior to copper (8). With Geraniums, conditions were similar to conditions in the present study and bacteriophage did provide a significant reduction in disease damage (25). However, the bacteriophage mixt ure was applied daily, which may not be costeffective or practical for a grower. The use of bacteriophage to pr event disease caused by Xanthomonas in onions was successful and the phage were found to persist on leaf surfaces longer than in the present study. In the onion st udy, it was speculated th at leaf morphology of the onion plant could orient the leaves so the pha ge were better shielded from negative factors that cause inactivation, such as UV light (39) Leaf morphology may c ontribute to reasons why bacteriophage were able to reduce the severity of bacterial spot of tomato (6, 7, 24). In the present study, the phage mixture was formulated with skim milk (7), which does offer some 62

PAGE 63

protection against UV. However, another substan tial negative factor affecting phage in this study was that the phage were simply rinsed off during overhead irrigation (see Chapter 5). It may be possible to develop a formulation that enhances persistence, but still allows effectiveness; further research needs to be ca rried out. Lang (39) proposed controlling bacterial spot by alternating phage and copper sprayi ngs; although Balogh (8) f ound that alternating copper and phage did not provide superior protection against bact erial pathogens of citrus. Perhaps other biological controls can be alternated or combined with phage and copper to yield an effective control. Hypothetically, mixing bact eriophage with the irri gation water should work better because Xanthomonas spreads by splashing wate r; thus, the phage is quickly and directly associated with the pathogen. Nevertheless, this approach did not work either. Possible causes that contributed to these failures will be addressed in Chapter 5. A very significant negative factor affecting ph age viability and persistence is desiccation. Because begonias have a thick waxy cuticle th at sheds water well; the phage might dry out quickly. A reason bacteriophages can survive be tter on tomato leaves than begonia leaves may be due to the microscopic threedimensional structures of tomato leaves. Tomato leaves have abundant trichomes whereas begonia leaves do not. The trichomes may de lay water evaporation and reduce desiccation and other forms of damage This suggests another reason why biocontrol did not work in the present study. Another explanation is that simply the wr ong phages were selected. These four phages were selected based on their host range in the labo ratory and not based on their ability to survive and infect Xanthomonas on begonia leaf surfaces. It is inte resting to note that some of the instances of a successful biological c ontrol of bacterial spot incited by Xanthomonas used commercial bacteriophage preparations (e.g. Om nilytics, Salt Lake City, UT), whereas some 63

PAGE 64

unsuccessful biological controls used phages found in the local environment. Studies presented in Chapter 5 show that the biocontrol phages sele cted in the present study simply cannot infect Xcb on begonia leaf surfaces. Phage survival on leaf surfaces will also be addressed in Chapter 5. However, the species of plant is also importa nt because the characteristics of leaves from species to species vary greatly and might affect phage survival. More effective bacteriophages must be screened for better survival on begonia leaf surfaces and have th e ability to infect the host bacterium on begonia leaf surfaces in greenhouse environments. 64

PAGE 65

Figure 4-1a. Irrigation apparatus used to apply phag e and other treatments in the fifth biological control experiment. 65

PAGE 66

Figure 4-1b. Irrigation apparatus used to apply phage and other treatments in the fifth biological control experiment. 66

PAGE 67

Figure 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

PAGE 68

Figure 4-2. Leaf Damage Key. Class 1 repr esents 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

PAGE 69

Figure 4-3a. A healthy begonia leaf Note the waxy leaf surface. 69

PAGE 70

Figure 4-3b. Typical leaf damage classes whic h correspond to the pictorial leaf damage key (Fig. 4-2) used in the biologi cal control experiments. 70

PAGE 71

Treatment 1234 Average Disease Damage Rating 0 1 2 3 4 5 Figure 4-4. Average levels of b acterial spot damage on begonias at the end of the first biological control experiment (one month). 1. Untr eated 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. Time (days) 0510152025303540 Leaf Damage Rating 0 2 4 6 8 Untreated Control Sprayed with phage 2 X per week Sprayed with phage 3 X per week Sprayed with copper Figure 4-5. Disease progress curves of bact erial spot damage on begonias for different treatments in the second experiment. 1. Un treated 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

PAGE 72

Table 4-1. Average disease damage ratings from bacterial spot of begonia incited by Xanthomonas campestris pv. begoniae during the second bi ological control experiment. Days Average Disease Ratings Treatment 1a 2b 3c 4d 0 3 7 14 21 28 35 0 0 0.5 2.0 2.7 4.3 5.5 0 0 0.4 2.3 3.9 5.3 6.3 0 0 0.2 2.1 3.9 4.7 6.3 0 0 0.2 1.9 2.9 4.0 5.6 a Untreated control b Sprayed with phage mixture 2 X per week c Sprayed with phage mixture 3 X per week d Sprayed with copper sulfate pentahydrate Table 4-2. Area under the diseas e progress curves (AUDPC) of disease damage from each plot of begonia incited by Xanthomonas campestris pv. begoniae during the second biological control experiment. Plot AUDPC F crit Treatment 1a 2 b 3c 4 d 1 2 3 4 5 6 7 8 76.2 78.8 85.1 91.5 89.7 73.0 88.2 88.3 101.2 100.2 112.5 110.3 110.8 109.7 98.3 90.5 94.2 101.7 94.0 97.5 96.2 98.3 106.7 101.5 92.0 74.7 76.7 79.1 81.6 77.6 87.5 86.4 4.35 Fz 23.52 a Untreated control b Sprayed with phage mixture 2 X per week c Sprayed with phage mixture 3 X per week d Sprayed with copper sulfate pentahydrate Z The likelihood that there is a significant difference between the data sets as calculated by the analysis of the variance. P=0.05. 72

PAGE 73

Table 4.3. Student Newman Keuls Mean Se paration of the Area under the Disease Progress Curve (MAUDPC) for the Biologi cal Control Experiment 2. Treatment MAUDPC1, 2,3 1 Control 2 Phage 2X Week 3 Phage 3X Week 4 Copper 83.859a 104.195b 98.762b 81.939a 1 Mean separation by Student Newman Kuels method. 2 Means followed by the same letter are not significantly different at P=0.05 level 3 Based on Analysis of the variance, 8 replications. Time (days) 51015202530354045 Leaf Damage Rating 0 1 2 3 4 5 6 Untreated Control Sprayed w/Phage 2X Week Sprayed w/phage 3X Week Sprayed with Copper Figure 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 standa rd deviation. 73

PAGE 74

Table 4-4. Average disease damage ratings from bacterial spot of begonia incited by Xanthomonas campestris pv. begoniae during the fourth biological control experiment. Days Average Disease Ratings Treatment 1a 2b 3c 4d 0 7 14 21 28 35 42 0 0 0.3 1.5 2.1 2.6 3.0 0 0 0.3 1.9 2.0 2.4 3.1 0 0 0.4 1.9 2.0 2.3 2.9 0 0 0.3 1.7 2.0 2.3 3.0 a Untreated control b Sprayed with phage mixture 2 X per week c Sprayed with phage mixture 3 X per week d Sprayed with copper sulfate pentahydrate Table 4-5. Area under the disease progress curves (AUDPC) fr om disease damage from each plot of begonia incited by Xanthomonas campestris pv. begoniae during the fourth biological control experiment. Plot AUDPC F crit Treatment 1a 2 b 3c 4 d 1 2 3 4 5 6 7 8 55.7 70.9 58.2 75.1 68.6 60.1 49.5 55.1 64.0 54.0 58.1 58.0 55.0 53.1 67.1 56.1 51.8 49.4 58.6 61.4 61.4 54.4 58.2 62.7 60.8 58.3 68.2 53.0 55.7 52.0 59.1 49.5 4.35 Fe 0.57 a Untreated control b Sprayed with phage mixture 2 X per week c Sprayed with phage mixture 3 X per week d Sprayed with copper sulfate pentahydrate e The likelihood that there is a significant differen ce between the data sets as calculated by the analysis of the variance. P=0.05. 74

PAGE 75

Time (days) 510152025303540 Disease Damage rating 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Water only Water + SpNB Water + Phage Cocktail Figure 4-7. Disease progress curves from bacterial spot on begonias for different treatments in the fifth biological control e xperiment. 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. Table 4-6. Average disease damage ratings from bacterial spot of begonia incited by Xanthomonas campestris pv. begoniae during the fourth biological control experiment. Days Average disease damage rating Treatment 1a 2 b 3c 0 7 14 21 28 35 0 0 0.1 0.8 0.9 2.1 0 0 0.2 0.8 0.9 2.0 0 0 0.2 0.7 0.9 1.8 a irrigated with water b irrigated with water supplemented with spent NB c irrigated with water supplemented with phage mixture 75

PAGE 76

Table 4-7. Average disease damage ratings from bacterial spot of begonia incited by Xanthomonas campestris pv. begoniae during the fifth biologi cal control experiment Plot AUDPC Fcrit Treatment 1a 2 b 3c 1 2 3 4 5 23.6 22.3 22.3 40.3 24.6 33.3 29.3 19.7 24.5 33.5 20.6 19.3 25.4 23.2 22.8 6.94 F d 0.77 a irrigated with water b irrigated with water supplemented with spent NB c irrigated with water supplemented with phage mixture d The likelihood that there is a significant difference between the data sets as calculated by the analysis of the variance. P=0.05. 76

PAGE 77

CHAPTER 5 ENVIRONMENTAL FACTORS THAT AFFECT THE SURVIVAL AND REPRODUCTION OF BACTERIOPHAGES Introduction The above-ground parts of leaf y plants are known collectivel y as the phyllosphere. For biological control, the site where a bacteriophage is likely to infect its host is on the leaf surface, the phylloplane. Leaf surfaces present a very diffe rent environment for infection, than laboratory conditions. The phylloplane has a unique microe nvironment with varying humidity, temperature, pH, and osmotic potential and ther e can be varying levels of free moisture and radiation (40). Water is essential as it provides a medium for the phage to interact with its host. The conditions in a greenhouse may differ greatly from the outdoors and be unsatisfactory for a bacteriophage to infect its host. Attempts at biological control of Xcb, as reported in Chapter 4 were not effective. One explanation could be a reduced ab ility of the phages to infect Xcb on leaf surfaces as compared to infection under labo ratory conditions in liquid media. Another factor could be the efficiency of the infection process. This poses th e question; how does the infection rate of Xcb by phage on leaf surfaces compare to the inf ection rate in a flask of NB, wh ere conditions are obviously more optimum? In addition to the infection efficiency of the phage on the leaf surface, impediments to phage survival such as environmental stresses provided by solar radiati on and desiccation could also affect the infection rate as well as phage survival and persistence (40). On the leaf surface, phage frequently encounter le thal levels of ultraviolet (UV) radiation. Interestingly, some epiphytic bacteria have developed the ability to produce pigments that provide some UV protection. The most tolerant strains of epiphytes produce pink or orange pigments that are thought to provide some protection from radical oxygen produced by UV by supplying a substrate for these reactive atoms or molecules to bind to (63). Phage inactivation in 77

PAGE 78

aquatic environments due to UV from solar radia tion has been well documented (50). In marine environments, UVB light was shown to damage phage DNA and thus decrease infectivity (72). It has been speculated that UV radiation is also a large contributor to phage inactivation on leaf surfaces in many environments (6, 7, 24, 33). Ra diation levels are likely to differ in various environments commonly encountered in folia ge production as compared to outdoor crop production. Additional causes of bacteriophage inactivation may be desiccation, adsorption to inert surfaces and limited persistence they are simp ly washed off during irri gation or rainfall. Mechanisms of phage inactivation by UVC (100 nm to 280 nm) and UVB (280 nm to 320 nm) are similar, but not iden tical. UVC causes direct damage to DNA, such as single or double-stranded lesions. It also may cause certain bases to dimeri ze. Inactivation by UVB is far less damaging; although the presence of active oxygen, produced by ot her compounds that are excited by UVB, can contribute to extensive pha ge inactivation (32). UVB can react with a variety of substrates and result in free O2, or -OH being released. UVB also causes lesions and base dimers to form in DNA. This process is si milar to UVC, but occurs at a far reduced rate. The damaging effect of UV radiation on DNA is also demonstated by work with E. coli where photoreactive DNA repair enzymes were not activated when the organism was exposed to UVB light (32). Sint on et al. (61) found a very profound effect after exposing bacteria to different wavelengths of UV. There was a sharp logarithmic increase in inactivation of E. coli cells between 313 nm and 260 nm. This rate of inact ivation for bacteriophage on leaf surfaces by radiation of these wavelengths may be even mo re detrimental since bacteriophages outside the host have no known means to repair DNA damage. Because of the reported influence of the ma ny environmental factors on phage survival, the objectives of this porti on of the study were to invest igate survival of selected Xcb phages on 78

PAGE 79

leaf surfaces in environments co mmonly encountered in the nurs ery and foliage industry and to assess some selected Xcb phages abilities to infect their host on the leaf surface. The key factors, once identified, that cause phage inactivation may be reduced in environmentally controlled systems as in nursery or foliage production which can differ greatly from outdoor production systems. Some of these suspected factors were simulated in controlled were identified and quantified. Thus, three contro lled environments common to foliage production were compared to an open field, a shade hous e, a plastic-film greenhouse, and a glass green house with shade paint applied. More specifically, factors that were inves tigated were effects of sunlight and UVA and UVB exposur e, desiccation of phage, and atmospheric water vapor on phage survival. Materials and Methods The first series of experiments will focus on bacteriophage survival and persistence on various surfaces in the absence of the bacterial ho st. The second series of experiments will focus on Xanthomonas infection of begonias with or without phage, and then phage survival and persistence in the presence of the bacterial host. Plants Used for Assaying Phage Survival Because begonia leaves are sel dom uniform in regard to leaf size and orientation and are covered by a thick waxy layer, Vigna unguiculata California Blackeye, commonly called cowpeas were used as surrogate plants for so me of the phage survival experiments on leaf surfaces. Cowpea cotyledons are fast to emerge, cons istent in size and orientation, and the plants can tolerate different environments well as opposed to begonias, which prefer moderate temperatures and the shaded conditions. Cowpeas were used as a surrogate plant system to study phage survival in greenhouse and foliage producti on environments and to assess the effects of 79

PAGE 80

UV radiation. The survival studies only used Xcb plaque assay plates to detect viable phage. No Xcb bacteria were concurrently applied to leaf surfaces in these experiments. Phage Population Dynamics on Leaf Surfaces Each of the four biocontrol phages was grown in NB overnight at 28C and oscillated at 130 rpm as described in Chapter 3. The culture s were centrifuged at 8,000 rpm for 10 min and passed through a 47mm diameter 0.2 m nitrocel lulose filter (Cat#SA1J 789H5, Millipore Corp., Bedford, MA) using a Nalgene f ilter holder and rece iver apparatus (Cat# 300-4050, Nalge Nunc Inter., Rochester, NY). The phage broth was amended with 0.1% AG 98 [(v/v), (80% octylphenoxypolyethoxyethanol, Rohm & Haas Co., Phila delphia, PA)] before application. This additive is a wetting agent; it breaks the surf ace tension so the treatments are more evenly distributed on leaf surfaces. The phage prepara tions were applied to tr ue leaves and cotyledons of cowpeas with a chromatography sprayer [( Cat.# Z529745-1EA) (Sigma-Aldrich, St. Louis, MO.)] at 17.2 kPa. At this pressure, the spray volume was 0.5 ml per sec. Each selected leaf was sprayed from a distance of approximatel y 30 cm for 1 sec for uniformity. The phagesprayed cowpeas were placed in one of three di fferent locations: (i) in a growth room under regular fluorescent lighting[(eight 40 W cool white fluorescent tubes), (approximately 60 cm from the light source to leaf surface)], (ii) in the greenhouse unde r an 80% shade cloth and (iii) in the greenhouse under an 80% shad e cloth with overhead irrigation. The plants were kept in their respective environments for two days befo re sampling them to allow the environmental stresses to influence population levels. To determine the populations of bacteriophage on leaf surfaces, leaves from the sprayed plants were se lected at random and placed individually in a tared zip-lock 10 cm X 15 cm polyethylene bag (C at. # 01-816-1C, Fisher Scientific, Pittsburgh, PA), and weighed. Added to each bag was 50 ml of sterile tap water; with 0.1% Tween 80 80

PAGE 81

(Fisher cat.# T164-500). The bags were gen tly shaken at 50 rpm for 20 min at room temperature. From each sample, 2 ml of the liquid was collected and centrifuged at 15,000 rpm for 10 min. The supernatant was diluted with sterile tap water to 1X, 0.1X, 0.01X, 0.001X, 0.0001X, and plaque assayed as described in Chapter 3. The leaf weights were used to estimate the area of the leaf; the data are presented as plaque forming unit (PFU) per square centimeter (cm2). To better detect low phage populations, whol e plants from each environment described above were brought to the laboratory and selected leaves were placed in plastic Petri dishes with slots cut in the sides for the peti oles and lined with moist filter pa per. Four 100 l droplets of Xcb (in 10 mM MgSO4) were placed on each leaf with a pipe tte. The dish lids were secured and the leaves were incubated overnight at room temp. Follo wing overnight incubation, the cell/phage droplets were removed with a pipette serially diluted a nd plaque-assayed using methods already described above. Comparison of Growing Environments on Bacteriophage Survival Bacteriophage J was used for the leaf surviv al assays. It was chosen because of its broad host range as discussed in Chapter 3. A m odified protocol adapted from Balogh et al. (7) was utilized. Nutrient brot h was inoculated with both Xcb and phage was grown overnight at 28 C, oscillating at 130 rpm. Nutrient broth cultures of only Xcb were also started for subsequent plaque assays. After incubation, the phage su spension was centrifuged at 8,000 rpm for 10 min and passed through a 47 mm diameter 0.2 m filter, as described above and in Chapter 3. Serial dilutions of the phage suspension were made post-filtration and each dilution was spotted on an Xcb lawn plate to estimate the viable phage populat ion at the beginning of the experiment. The phage suspensions applied to leaves were am ended with 0.1% AG 98 to allow the applied suspension to spread more evenly on leaf surfaces. 81

PAGE 82

Cotyledons and true leaves of cowpeas were sprayed using the same chromatography sprayer mentioned above with the same conditions also described above; each leaf was sprayed for approximately one second each from a distance of approximately 30 cm. The plants were placed in four groups which were then placed in one of the four designated environments (open field, shade house, plastic greenhouse, and gla ss greenhouse). A zero-time sample was taken, followed by sampling once every 3 hr for the first 9 hr and then once at every 24, 48, and 72 hr after application. To estimate the relative popu lations of viable bacter iophage, leaves were selected at random and placed individually in a tared zip-lock polyethylene bag [(10 cm X 15 cm), (Cat. # 01-816-1C, Fisher Scientific, Pittsbur gh, PA)], and weighed. Fifty milliliters of sterile tap water; with 0.1% Tween 80 (Fisher Ca t.# T164-500) was added to each bag. The bags were gently shaken at 50 rpm for 20 min at room temperature. From each sample, 2 ml of the liquid was collected and centrifuged at 15,000 rpm for 10 min. The supernatant was serially diluted by tenfold dilutions to 10-4 and plaque assayed as described above. The leaf weights were used to estimate the leaf area ; the data are presented as PFU/cm2 (plaque forming unit per cm2. Phage Survival under Filtered Solar Radiation A window-like wooden frame supported with legs like a table was constructed to suspend various types of window glass a nd other filters or colored ce llophane over test plants to determine the effects particular light wavelengths on bacteriophage survival. The panes of glass or filters were approximately 61 cm2, and the frame stood 122 cm tall. The transmission spectra of the different types of glass used with and without cellophane were measured using a spectrophotometer (Cary 5000 Spectrophotometer, Varian Inc. Palo Alto, CA) at the Center for Research and Education in Optics and Lasers (CREOL ) at the University of Central Florida. 82

PAGE 83

Poplawsky et al. (57) found that in Xanthomonas spp., the function of its xanthomonodin pigment is not for protection from UV, but rather, for protection from blue light. Thus, in this study, assays to measure the effects of particular wavelengths of visible light were performed. Colored cellophane was supported between two panes of greenhouse glass. The wooden frame mentioned above supported the panes. Blue and re d cellophane were chosen; blue because of the Poplawsky et al. (54) study and red because it transmits light with wavelengths of low energy. Cowpeas were sprayed with a phage suspensi on as already described and divided into two groups. One group was placed under the wooden frame holding the filters to select certain wavelengths of sunlight. The other was expos ed direct solar radiation. Under regular greenhouse glass (without cellophane), leaves were sampled at a zero time and then at 30, 60, 90, 120, 150 and 1200 min. Under greenhouse glass w ith blue cellophane, phage-sprayed cowpea leaves were sampled at 0, 45, 120, 200, and 300 min. Under greenhouse glass with red cellophane, phage-sprayed cowp ea leaves were sampled at 0, 60, 120, 180, 240, 300, and 360 min. In each experiment, leaves were collected, weighed, rinsed in sterile tap water (with 0.1% Tween 80), centrifuged, diluted and pla que assayed as described above. Bacteriophage Survival on Inert Surfaces. The effects of different wavelengths of light on the survival of bacteriophage on an inert surface were also investigated. Cellulose strips were cut from dialysis membrane (Cat.# 08667E, Fisher Scientific, Pittsbur gh, PA) and placed in a small cardboard frame with a cardboard back. Different types of glass were placed on th is frame and the whole apparatus was placed in the open sun. Phage survival on cellulose under diffe rent types of glass was tested. The types of glass that were used were a cold mirror, greenhou se glass, and Museum Glass. A cold mirror (model # NT47-305, Edmund Optics, Barrington, NJ) reflects visible light partially transmits UVA light and completely transmits infrared light (for a transmission spectrum, see below). The 83

PAGE 84

transmission spectra of greenhouse glass, greenhouse glass with bl ue cellophane, greenhouse glass with red cellophane, Museum glass and a cold mirror are presented in Fig. 5-2, and discussed below. A 10 l droplet of a phage-NB suspension (pre pared as described above) was placed with a pipette on a cellulose strip and allowed to dry. The strip wa s then placed in the cardboard frame and the specified piece of glass was secured above the strip. The assembly was placed outdoors in direct sunlight for 2 hr and samples were collected every 30 min. A control strip with a 10 l droplet of the same phage preparat ion was placed next to th e apparatus unprotected from solar radiation. After exposure, the test strips were placed individually in empty Petri dishes. Warm YNA mixed with Xcb was poured over the strip. Th e plate was swirled, the agar was allowed to set and the plates were incubated at 28 C overnight. Light Spectra of Solar Radiation Reaching the Plants in Different Environments The spectra of direct solar ra diation and of solar radiation filtered through different types of glass or filters were measured with a spectrometer (Model #. OSM-400, Newport Corporation, Irvine, CA). The same window-like frame mentioned above was used to support the different pieces of glass. The sensor of the spectrom eter was placed at plant height when the measurements were made. This spectrometer wa s also used to measure the emission spectra of artificial lighting described below. Additionally, a UV meter (Model# UVM-SS, Apogee Instruments, Logan, UT) and a photometer (LI-185B, Li-cor, Inc., Lincoln, NE) were used to estimate the intensities of light reaching th e cowpea leaves in these experiments. Phage Survival in Controlled Environments Effects of bacteriophage surviv al on leaf surfaces exposed to visible and UV light sources were investigated. In environment rooms, the wooden frame mentioned above was used to support a piece of Conservation Clear Museum Glass (TruVue, Inc., McCook, IL); this glass 84

PAGE 85

is opaque to UVA and UVB (Figs 5-11, 5-15, and 516). Fluorescent lights that emit exclusively UVA or UVB light (described below) were used in conjunction with the Museum glass. Light with a peak emission in the UVA range was pr ovided with General Electric fluorescent bulbs (product # F40BL, General Electric Corp., Fa irfield, CT) which have a peak emission wavelength of 352 nm (UVA). The manufacturers emission spectrum is shown in Fig. 5-1. To assay the effects of UVB light, fluorescent bulbs manufactured by Ushio (model # G15T8E, Ushio America, Inc., Cypress, CA) were us ed. The emission spectrum of the UVB tubes provided by the manufacturer has a peak emissi on of 306 nm (Fig. 5-1) The intensity of photosynthetic light (Table 5-1) from these UV tubes and from solar radiation in the four environments mentioned above was measured with a photometer and the intensity of UVA and UVB light from was measured with the UV meter mentioned above. Eight UVA or UVB bulbs were placed above the bench in a growth room. The approximate distance from the tops of the cowpea plants to the lights was 60 cm. A large piece of plywood divided the bench surf ace in half. Cowpeas sprayed with phage were placed on one half and received light without filtration. On the other half of the bench, the wooden frame (mentioned above) was used to support the Museum Glass placed just below the lights. Cowpeas sprayed with phage were placed below the glass. Leaves were sampled at zero time, again at 4 hr, 24 hr, 48 hr and 72 hr for the UVA lights. U nder the UVB lights, leaves were sampled at 15 min, 30 min, 45 min, 60 min, and at 90 min. Sa mpled leaves were weighed, rinsed, and plaque assayed as described above. Role of Relative Humidity on Phage Survival To measure the effects of relative humidity (RH) on phage survival, a plastic box (30cm X 25cm X 10cm) with slots melted in the side for the leaf petiole so that a tight chamber was formed. Leaves sprayed with phage were placed into this chamber. Water was placed in the 85

PAGE 86

chamber, the lid was secured, and the edges and petiole port were sealed with modeling clay. Pure water created an environment with 100% RH. Immediately next to the chamber was a second replicate of phage-sprayed cowpeas. The RH outside the box was measured to be 45%. The plants were exposed to Cool-White fluorescent lighting throughout the experiment. Leaves from both environments were sampled st arting with a zero time and then once at 144 hrs and again at 196 hrs. The viable phage population was estimated with leaf rinses, centrifugation, serial dilution and plaque a ssays as described above. Infection of Begonia Inoculum Plants with Xcb The following series of experiments investigat ed phage persistence in the presence of a susceptible bacterial host, Xcb. The method for inoculating begoni as that was utilized in the previous chapter may have provided conditions highly favorable to the bacterial pathogen. In other words, inoculating a begonia with Xcb followed by an overnight incubation in a humid environment may create an environment extrem ely favorable for the pathogen under such conditions; if the phages reduce th e bacterial population only slightly, it may not be detectable with the methods employed. To determine optimum conditions for infection of begonia with Xcb, the following experiment was performed. Be gonias growing in 15 cm pots were placed in saucers and watered well immediately before inoculation. To infect begonias with Xcb (to incite bacterial spot), Xcb was grown in NB culture overnight. The cells were centrifuged at 4,000 rpm for 10 min and the supernatant was discarded. The cells were re-suspended in 10 mM MgSO4. The cells were serially diluted and plated on NA and the concentration of the inoculum was estimated to be 2 X 108 CFU/ml. The inoculum was applied to the begonias with a chromatography sprayer as described above with an inoculum volume of approximately 1.5 ml per plant. To determine the optimal incubation time in a very humid environment, the plants 86

PAGE 87

were placed in plastic bags and the edges of the bags were tu cked between the bench and the saucer to provide water vapor sa turated conditions. Begonias were arranged in six plots. The first plot was incubated 0 hr in the humid environment. The second through sixth were incubated 2, 4, 8, 16, and 32 hr respectively. After each incubation period, the plastic bags were removed. Symptoms of bacteria l spot developed in about 7 days and disease damage was rated once per week for three weeks using the le af damage key described in Chapter 4. Bacteriophage Infection on Leaf Surfaces under Favorable Conditions It has been speculated that desiccation greatly limits the time that a phage can persist on leaf surfaces (33, 39). As mentioned earlier cowpeas were used instead of begonias to determine the efficiency of infection of Xcb by phage on leaf surfaces; in the laboratory instead of the greenhouse. To create favorable conditions for infection on leaf surfaces, slots were cut into the sides of plastic Petri dishes for the petioles. With moist filter paper in each dish and the lid secured, a moist chamber is created. On th e laboratory bench, cowpea plants and the Petri dishes were arranged as follows. The plants we re divided into four groups; one group for each of the four biocontrol phages. The Petri dishes were supported at l eaf height. Moist filter paper was placed in each dish. Leaves were secured in dividually inside the Petri dishes and the lids were added. Xanthomonas was grown overnight in a NB culture, centrifuged, the supernatant poured off and the cells re-suspended in 10 mM MgSO4 as described in Chapter 3. Four 50 l droplets of this Xcb suspension were placed on each leaf with a pipette. To assay the phages ability to infect Xcb on a leaf surface, each of the four bi ocontrol phages was grown overnight in a NB culture, centrifuged, and filtered as described in Chapter 3. All four phage suspensions were diluted individually to 103 PFU/ml. To each 50 l droplet of Xcb a 50 l aliquot of one of the phages was added. This yielded four, 100 l samples on each leaf. The lids were secured 87

PAGE 88

and the leaves were incubated overnight at room temperature. The next day, each droplet was removed with a pipette, serially diluted, a nd plaque assayed as described in Chapter 3. Bacteriophage Infection on Begonia Leaf Surfaces under Greenhouse Conditions Initial experiments. Desiccation was suspected as a major negative factor affecting bacteriophage infection of Xcb on begonia leaf surfaces. In the first attempt to measure this effect, host cell infection from pha ge applied to leaves that were wet when the host was applied to phage applications were compared with host ce ll infections on leaves that were dry when the host bacterium was applied. Cultures of each of the four biological control bacteriophages were grown in NB overnight, centrifuged and filtered as detailed previously. A NB culture of Xcb was also grown overnight, centrifuged, the supernatant discarded a nd the cells re-suspended in 10 mM MgSO4 as described previously. Each phage su spension was then diluted 1:1000 in 10 mM MgSO4. Aliquots of each phage culture and of the bacterial suspension were saved for serial dilutions and spot testing for th e phage and dilution plating of th e bacterium in order to estimate the respective populations. The viable ti ter for each phage pre-dilution was 109 PFU/ml; after dilution it was 105 PFU/ml. The population size of the bacterial suspension was 108 CFU/ml. Seventy-two begonia plants were divided even ly into four plots; one plot for each biological control bacteri ophage. During phage and Xcb applications, a cardboard barricade was placed around each plot to reduce spray drift. Each plant in each plot was sprayed with a given diluted phage suspension (described above) unt il sufficiently covered, but not dripping. Immediately, after the phage was applied, half of the plants were removed and the remaining plants were sprayed with the Xcb suspension. The phage suspension did not have time to dry. The plants that were removed after the phage su spension was applied were held until the phage application dried, then sprayed with the bacterial suspension. Leaves were sampled at 0hr, 24 hr, and 48 hr. The process was repeated for the rema ining three plots with the other three respective 88

PAGE 89

bacteriophages in the same manner. The same sample bags mentioned above were used. The sampled leaves were brought to the laboratory fo r processing and plaque assaying using the same methods described in the phage persis tence section of this chapter. Additional experiments to determine if the bacteriophages could infect Xcb on leaf surfaces were also undertaken. Each of the ten bacteriophages that were partially characterized (see Chapter 3) was grown overnight in NB culture, centrifuged, and filtered as described above, but the phage cultures were not diluted. A small aliquot was kept for seri al dilutions and spot testing, which indicated the viable titer of each phage culture was 109 PFU/ml. A separate culture of NB was inoculated with only Xcb at the same time the phage cultures were started. The bacterial culture was centrifuged at 4,500 rpm, the supernatant discarded, and the cells were re-suspended in 10 mM MgSO4. The cell population in the bacter ial culture was determined via dilution plating to be 108 CFU/ml. One hundred eighty begonia plants were placed in saucers, wa tered well, and divided into ten plots. Each plot was designated for one of the ten phage characterized in Chapter 3. A NB culture of each phage was grown overnight, centrif uged and filtered as detailed in Chapter 3. A NB culture of Xcb was also gown and prepared as menti oned above. A cardboard barricade was placed around the plot during spraying to reduce spray drift. Each plot was sprayed with the respective phage suspension until sufficiently co vered but not dripping. Then, half of the sprayed plants were removed and the remaining pl ants were sprayed the bacterial suspension. The begonias that were removed were then moved b ack into the plot. Leaves were sampled at 0, 24, 48, and 72 hr. The leaves were processed and plaque assayed as described in the phage persistence section of this chapter. 89

PAGE 90

Phage Population Dynamics on Begonia Le af Surfaces of Plants Infected with Xcb If a bacteriophage can infect and multip ly on its host on leaf surfaces, the phage population on a leaf surface should decline less rapidly and may even increase in good conditions and the presence of the host compared to on leaves with phage, but without the host. To measure the persiste nce and the infection pot ential of bacteriophages on begonias that were already infected with bacterial spot, begonia plants were inoculat ed as described earlier with Xcb and plants showing adequate symptoms were sel ected. Using the leaf damage key (Chapter 4), these plants were rated as class 3. To determine if bacteriophage sprayed on Xcb -infected begonia plants can infect Xcb from an infected site can occu r in greenhouse environments, the J phage was grown overnight in a NB culture, centrif uged and filtered as described in Chapter 3. A culture of Xcb was grown overnight, centrifuged at 4000 rpm for 10 min, the supernatant poured off and the culture was re-suspended in 10 mM MgSO4. Six infected begonias were arranged per plot. A cardboard barrier was secured around the plot while it was being treated to reduce spray drift. Each plant was sprayed with the appropriate phage until covered, but not dripping. Leaves were sampled at 0, 24, 48, and 72 hr and processed and plaque assayed as described in the phage persistence section in this chapter. Identifying Possible Key Fact ors That Affected the Biol ogical Control Experiments One reason that the biological control attemp ts in the discussed in Chapter 4 may have been unsuccessful could be that one or more of the additives (AG 98, skim milk, etc.) to the phage mixture may have reacted adversely. The following set of experiments examined each additive. Begonias were placed in eight groups; twelve plants pe r group. Effects of individual components of the biocontrol mixture which were: 1. sterile tap water; 2. spent nutrient broth; 3. spent NB + 0.75% skim milk; 4. spent NB + 0. 1% AG 98; 5. A phage culture; 6. D phage culture; 7. J phage culture; 8. P phage cult ure were tested as follows. The four phage 90

PAGE 91

cultures and spent nutrient broth were prepared as described in Chapter 4. A culture of Xcb was grown overnight, centrifuged, the supernatant poured off, and th e cells were re-suspended in 10 mM MgSO4. The viable titer in each phage culture was 109 PFU/ml. The population of the Xcb culture was estimated at 108 CFU/ml. All the begonia pots we re placed in saucers and wellwatered. Each of the eight plots was then spra yed with its respective treatment until sufficiently wet, but not dripping with the same chromatogr aphy sprayer at the same pressure described previously. After the begonias received their respec tive treatments, they were immediately sprayed with Xcb After inoculation, the plants were covered with plastic bags and the edges were tucked between the saucer and the bench, just as described above in the infection of inoculum plants section. The plants were incu bated in this humid environment for eight hours. Disease was rated after 7 days and again at 14 days using the leaf damage key described in Chapter 4. The wetting agent AG 98 was suspected to have a negative effect on biological control. To test this, each of the four biocontrol phage s was grown overnight, centrifuged and filtered as mentioned before. Each phage culture was amen ded with 0.1% AG 98. Each plot of healthy begonias, placed in saucers and watered well was sprayed with a phage culture then immediately sprayed with a suspension of Xcb (in 10 mM MgSO4). A cardboard barrier was placed around the plots during treatment to reduce spray drift. The plants were incubated in a humid environment just as in infection of inoculum plan ts section. The plants were incubated for 8 hr, and then the bags were removed. Disease wa s rated seven days and again after 14 days. Phage Populations in Irrigation Runoff Desiccation and damage from UV light were two possible negative factors that affected the results in the biological cont rol experiments described in Chap ter 4. Another negative factor could have been that the phages were rinsed off the leaves during ir rigation, making them 91

PAGE 92

unavailable to attack Xcb To confirm this notion, each of the four biocontrol phages was grown overnight in a NB culture and prepared as desc ribed previously. Indivi dual begonias in 10 cm pots were sprayed with one of these phages, and placed in the greenhouse under the irrigation system that was utilized for the fourth biocont rol experiment; the bench was irrigated for 15 min daily at 05:00. Polystyrene cups (30 ml) were em bedded in the soil next to the begonias. The lip of the cup was a few millimeters above the soil. Fresh cups were used each day. For the next four days, irrigation water that had splashed or dripped off the begonias and accumulated in the cups was collected and the viable phage populat ion estimated by the fo llowing method. Water samples were brought into the laboratory. From each sample, 2 ml was collected and centrifuged at 15,000 rpm for 10 min. The supernatants were then serially diluted and tested in plaque assays as described above. Infection Rate of Bacteriophage in Nutrient Broth Culture Another negative factor that could have aff ected a bacteriophages ability to control bacterial spot of begonia may be that the rate of infection may be signi ficantly reduced on leaf surfaces. The rate of infection of Xcb in a NB culture was determined for each of the four biocontrol phages as follows. Four 50 ml NB cultures of Xcb were grown overnight. Each phage culture was diluted to 103 PFU/ml and 100 l of phage inoculum was added to each flask. A zero-time aliquot of 0.5 ml wa s taken and plaque assayed. Ev ery hour, 0.5 ml aliquots were taken, diluted appropriately, then plaque assayed. The process was repeated for 6 hr. The burst rate of each phage was determined by observing th e time when an exponential increase in the phage population occurred. 92

PAGE 93

Infection Rate of Bacteriophage to Bacteri a on Vigna Phylloplanes under Laboratory Conditions As discussed above, the rate of infection of Xcb by the biological control bacteriophages could be one of the negative factors that cau sed the biological control attempts to be unsuccessful. To estimate th e rate of infection of Xcb on leaf surfaces, cowpeas were used instead of begonia, just as in the phage persiste nce section of this chap ter. Bacteriophage NB cultures of each of the four bi ocontrol phages, A, D, J, and P (see Chapter 4) were grown, centrifuged and filtered (see Chapter 3). The cultures were amended with 0.1% AG 98 (see Chapter 4), and sprayed on cowpea leaves just as in the phage persistence section of this chapter. The plants were kept overnight in a growth ro om. The cowpea plants (four plants per phage) were brought into the laboratory and four leaves treated with each phage were enclosed in Petri dishes containing moistened filter paper with slots cut in the sides for the petioles. A 50 ml NB culture of Xcb was grown overnight at 28C oscillating at 130 rpm, centrifuged at 4,000 rpm, the supernatant was discarded and the ce lls were re-suspended in 10 mM MgSO4. Four 100 l aliquots of Xcb were placed to each leaf. Beginning with a zero-time, four droplets from one entire leaf was collected with a pipette, serially diluted, a nd plaque assayed as described in the phage persistence sectio n of this chapter. Results Phage Population Dynamics on Leaf Surfaces Cowpea plants, sprayed with the J phage were placed in one of three locations: (i) in an open field under full sunlight, (ii) in a plastic-covered greenhouse, and (iii) in the same plasticcovered greenhouse, but also under 80% shade clot h. Leaves were sampled once an hour for 6 hr and phage populations were estimated by plaque assay as described above. The initial phage population of all three groups was 1.07 X 106 PFU/cm2 (Plaque Forming Units per square 93

PAGE 94

centimeter). After 1 hr, the phage population in the open (full sun) had decreased 8.8 fold (Fig. 5-2), in the plastic-covered greenhouse after 1 hr, the phage population on leaf surfaces decreased 3.7 fold and in th e plastic covered greenhouse under the shade cloth, the phage population on leaf sufaces decreased 2.7 fold. After 2 hr, the phage popul ation on leaf surfaces had decreased 286 fold in the open, 17.2 fold in the plastic-covered greenhouse and 4.4 fold under the shade cloth. After 3 hr, the phage population on leaf surfaces in the had decreased 3.64X 103 fold in the open, 87 fold in the plastic-covered greenhouse, and 16 fold under the shade in the plastic-covered gr eenhouse (Fig. 5-2). After 4 hr the phage population on leaf surfaces decreased 5.91 X 103 fold in the open, 507 fold in the plastic-covered greenhouse, and 52 fold under the shade in the plastic-covere d greenhouse. The phage population on leaf surfaces decreased 5.94 X 105 fold in the open after 5 hr. After 5 hr, the phage population size on leaf surfaces decreased 2.37 X 103 fold in the plastic-covered greenhouse and decreased 78 fold in the plastic-covered gree nhouse under the shade (Fig. 5-2). When bacteriophage persisten ce was poor, a different techni que to estimate the relative viable population size was used. Cowpeas were sprayed individually with each of the four biological control bacteriophages and the plants placed in, (i) an open field under full sun, (ii) a plastic-covered greenhouse under a shade cloth, and (iii) a plasticcovered greenhouse under a shade cloth with daily overhead irrigation. After two days, the plants were brought into the laboratory and the host ( Xcb ) was used to allow the phage pr esent to reproduce as described above. These data obtained were relative values compared to the initial number of viable phage on leaf surfaces in th e respective environments. The A phage persisted best in the shaded greenhouse environment; the re lative population was 90 PFU/cm2. In the growth room, the relative phage population for the A phage was 60 PFU/ cm2 (Fig. 5-3). The A phage persisted 94

PAGE 95

the poorest in the irrigated greenhouse envi ronment; the relative population was 5 PFU/ cm2. The D phage persisted best in the shaded greenhouse environment; achieving a relative population of 1600 PFU/ml. The relative D phage population in the growth room was 850 PFU/ cm2, and 50 PFU/ cm2 in the irrigated greenhouse envi ronment (Fig. 5-3). The relative population size J phage in the shaded greenhouse environment was 1860 PFU/ cm2, in the growth room environment was 1750 PFU/ cm2, and was 10 PFU/ cm2 in the irrigated greenhouse environment (Fig. 5-3). The P phage persisted best in the shaded environment (Fig. 5-3). The relative population of the P phage in the shaded greenhouse environment was 1860 PFU/ cm2, in the growth room environment was 960 PFU/ cm2 and in the irrigated greenhouse environment was 140 PFU/ cm2. Comparison of Growing Environments on Bacteriophage Survival The J bacteriophage was sprayed on cotyledon s and true leaves of cowpea plants. The plants were then divided into four groups and placed in one of f our different environments: (i) in a plastic-covered green house, (ii) in a glass gr eenhouse with shade paint, (iii) in an open field (with full sun exposure) and (iv) in a shade house. Leaves were sampled at random over time and the viable phage population size was determined as described above. In the plastic-covered greenhouse, the phage population decreased 18 fold in the first 3 hr (Fig. 5-4). After 27 hr, the phage population decreased 1660 fold. Af ter 50 hr, the measured phage population increased 15 fold, but reproducti on did not occur; no host was present. The apparent increase in phage population size was likel y due to variability in the orientation of sampled leaves. Leaf orientation affects the angle that the electromagnetic radiation contacts the phage. After 72 hr, the phage population decreased 5.44 X 105 fold, to zero. In a glass greenhouse with shade paint, th e phage population on cowpea leaves decreased 1.1 fold after 3 hr, 96 fold after 27 hr, and anot her 3 fold after 50 hr. After 72 hr, the phage 95

PAGE 96

population size appeared to increase 14 fold, but th is was probably due to leaf variability and random sampling. Overall, the phage populatio n decreased 330 fold after 75 hr on cowpea plants in a glass greenhouse with shade paint (Fig. 5-4). In the open field with full exposure to sola r radiation, the phage population decreased 128 fold in the first 3 hr (Fig. 5-4). After 27 hr, the phage population si ze had decreased 6.9 X 105 fold to zero. In the shade house, the phage population decreas ed 1.1 fold in 3 hr, 9400 fold in 27 hr, and another 2.3 fold in 50 hr (Fig. 5-4). After 72 hr, the population appeared to increase 9 fold. However, no host was present; reproduction did not occur. The discrepancy is due natural variability in leaf orientation. Overall, the phage population size decreased 24,000 fold on cowpea leaves on plants in a shade house over 72 hr. Phage Survival under Filtered Solar Radiation Different glass filters, with or without color cellophane, were used to measure the effects of different wavelengths of light on bacteriophage survival on l eaf surfaces of cowpeas. The transmission spectrum of UV light with regular greenhouse glass showed that UVA light is welltransmitted (Fig. 5-5). Under regular greenhouse glass, at zero time, the phage population on cowpea leaves under the glass was 72 fold higher than the ph age population on cowpea leaves on plants that were in full sunli ght (Fig. 5-6). After 30 min the population had decreased by 37 fold under glass and only 1.4 fold in full sunlight After 60 min the phage population under greenhouse glass decreased an additional 4.5 fo ld from the previous time and the phage population in full sun decreased 3.8 fold from the previous time. At 60 min, the phage population under greenhouse glass was 2.4 fold highe r than the population in full sun. At 90 min the phage population under glass was 10 fold higher than the popul ation in the open. After 120 min the phage populations under glass and in the fu ll sun were measured to be nearly equal. 96

PAGE 97

After 150 min, the phage population size under gla ss was roughly 2 fold higher than the phage population in full sun. After 24 hours, the pha ge population on cowpea l eaves under glass was 23 fold higher than the phage population size on cowpeas that were in full sunlight (Fig 5-6). Overall, under regular greenhouse glass, bacter iophage populations on leaf surfaces of cowpea plants decreased 1370 fold in 24 hr. In full sun light, the phage population decreased 450 fold. It should be noted that the starting population under glass was 72 fold higher than the starting population in full sunlight. Blue cellophane was used in conjunction w ith greenhouse glass to study the effects of blue-colored light on phage surviv al. The blue cellophane did impede some of the UV light that reached the leaf surface (Fig. 5-7). Leaves were sample at the time intervals 0, 45, 120, 200, and 300 min. At zero time, the phage population was 7.5 fold higher on leaves sampled from plants from the open, under full solar radiation. U nder blue glass, phage population on cowpea leaves decreased 1, 1.8, 7.4, and 30.4 fold respectiv e to the sampling times (Fig. 5-8). The phage population in full sun did decrease at a slightly fa ster rate. With respect to the sampling times, the phage population on cowpea leav es in the open (full sun) decr eased 2.4, 8.8, 60.7, and 392.4 fold (Fig. 5-8). Red cellophane was also used with regular greenhouse glass to make a red filter. Red light is lower in energy than blue light. Th e transmission spectrum of light in the UV range through red-colored glass is shown in Fig. 5-9. At zero time, the phage population on cowpea leaves under red filter was 1.6 fold higher than the phage population on cowpea leaves in full sun (Fig. 5-10). This is due to e xperimental variation in phage cu ltures and variation in leaf orientation. Leaves were sampled at 0, 60, 120, 180, 240, 300, and 360 min. Under red-colored glass, the phage population on cowpea leaves decreased 1.3, 2.5, 34.7, 10.1, 95.5, and 6400 fold 97

PAGE 98

respectively (Fig. 5-10). In the open field, in full sunlight, the phage population on cowpea leaves decreased 1.4, 3.0, 68.9, 106.4, 552.1, and 1600 fold respectively. Bacteriophage Survival on Inert Surfaces Bacteriophage survival on an inert su rface (cellulose) under solar radiation was investigated using different filters : (i) a cold mirror, (ii) Museum Glass, and (iii) in the open, under full sun. The transmission spectra of the filte rs used are shown in Fig. 5-11a, 5-11b. In full sun, phage population size on cellulose decreased 87 fold in 1 hr (Fig. 5-13). After 2 hr, phage population size on cellulose in the open decreased 28 fold to zero. Overall, phage population decreased 2300 fold in full sunlight. Under Museum Glass (Fig. 5-11a, 5-11b, 5-12) the phage population decreased 4.3 fold in 1 hr (Fig 5-13) and decreased 11.5 fold in 2 hr. Overall, the phage population under Museum Glass decreased 49 fold. Under a cold mirror (Fig 5-11 a, 5-11b), th e phage population decrease d 1.6 fold in 1 hr (Fig. 5-13). After 2 hr, populations appear to increase 1.4 fold, but this was likely due to variability in leaf orientation and not reproduction. Overall, phage population on cellulose under a cold mirror decreased 1.6 fold. Light Spectra of Solar Radiation Reaching the Plants in Different Environments A Newport spectrometer was used to measure the emission spectrum of unfiltered solar radiation at plant height (Fig 5-14). The intensity of both tota l photosynthetic and UV light was measured as described above. In the open fiel d (full solar radiation), the total photosynthetic light measured 162.0 Wm-2sec-1 and the UV light measured was 116.3 mol (photons) m-2sec-1 (Table 5-1). In the plastic-covered greenhouse, th e light intensity was approximately half as in full sun measuring 79.5 W m-2sec-1 for total photosynthetic light and 79.5 mol m-2sec-1 for UV light. In the shade house, light was approximately 25% as intens e as full sun, measuring 31.0 W 98

PAGE 99

m-2sec-1 for total photosynthetic light and 30.1 mol m-2sec-1 for UV light. In the glass greenhouse, total photosynthetic li ght was about 10% than measur ed in the open field (16.5 Wm2sec-1), UV light was minimal, 0.65 mol m-2sec-1. Different types of glass filters were utili zed in this study. Regular greenhouse glass permitted 135 W m-2sec-1 of total photosynthetic light to pa ss (Table 5-1) and transmitted 90.0 mol m-2sec-1 of UV light. The blue-colored greenhous e glass (with a blue cellophane filter, described above) transmitted 57.0 Wm-2sec-1 of total photosyntheti c light and 36.0 mol m-2sec-1 of UV light. The red-colored greenhouse glass transmitted 57.0 W m-2sec-1 of total photosynthetic light and 43.0 mol m-2sec-1 of UV light. The Muse um Glass passed 135.0 W m2sec-1 of total photosynthetic light and 23.5 mol m-2sec-1 of UV light. These readings were taken at plant height, so th e UV light detected was likely reflected from the surrounding environment. Museum Glass does not tr ansmit UV light (Fig 5-11, 5-12). The total photosynthetic light and the UV light were meas ured from the UVA and the UVB fluorescent lights used in this study. The UVA tubes emitted 2.25 W m-2sec-1 of total photosynthetic light a nd 12.7 mol photons m-2sec-1 of UV light. The UVB lights emitted 0.1 W m-2sec-1 of total photosynthetic light and 11.5 mol photons m-2sec-1 of UV light. The emission data for both sets of UV tubes are presented below. Phage Survival in Controlled Environments The Newport spectrometer described above was used to measure a detailed emission spectrum of UVA fluorescent tubes (Fig. 5-15). An emission peak at 352 nm (manufacturers claim) was verified. Cowpea plants were spra yed with bacteriophage and placed under the UVA lights. One half of the group was protected by the Museum Glass. The spectra of light at plant height under this protection are shown in Fig. 5-15 ; no significant UV intensiti es were observed. 99

PAGE 100

When the Museum Glass protected the bact eriophage from some UV radiation, the population decreased 1.3 fold in the first 4 hr. After 24 hr, the populati on decreased 1.5 fold. After 48 hr, the population size appear s to increase 1.4 fold, but this is probably not due to phage reproduction, but to natural variability in leaf orientation. After 72 hr, the population had decreased 34 fold. Overall, the phage populatio n size decreased 47 fold when protected by the Museum Glass. Without the UV protection of the Museum Glass, bacteriophage population size on cowpea leaves decreased 1.4 fold in the first 4 hr. This population under the Museum Glass was 254 fold greater than the phage population not prot ected from some UV by the Museum Glass at 4 hr. Overall, the phage populati on decreased 27 fold in 24 hr and decreased 43 fold in 48 hr. At 24 hr, the phage population under the Museum Glass was 115 fold higher than the phage population on cowpea leaves that were not prot ected by the Museum Glass. After 72 hr, the population appeared to increase 3.6 fold, but this increase is no t due to reproduction, Xcb was not present, just natural variability in leaf orie ntation. Overall, th e phage population on cowpea leaves decreased by 443 fold in full sunlight. The Newport spectrometer detailed above was also used to measure the emission spectrum of the UVB fluorescent tubes (Fig. 5-16 ). The manufacturers claimed peak emission (306 nm) was confirmed. Cowpea plants were sprayed with bacteriophage and placed under the UVB fluorescent lights; half were fully exposed and the other half of the plants were protected by the Museum Glass (as in the UVA experiment reported above). When the Museum Glass was placed between the UVB lights and the phage-sprayed cowpea plants, the phage population size increased 4.3 fold in the first 15 min (Fig. 5-16). But this is not due to reproduction because no bacterial host was present; this is due to variation in 100

PAGE 101

leaf orientation. After 30 min, the phage population on cowpea leaves under the UVB light filtered by the Museum Glass decreased 15.6 fold At 45 min, the phage population on cowpea leaf surfaces under the UVB light plus the Museum Glass increased 5.2 fold, but this increase is not due to reproduction, Xcb was not present on leaf surfaces. The increase was due to variation in leaf orientation. The phage population on cowpea leaf surfaces under UVB light plus Museum Glass increased 1.4 fold in 60 min. Th is increase is not due to reproduction because no host was present. Rather, this increase is due to random sampling and natu ral variation in leaf orientation. At 90 min, the phage population on cowpea leaf surfaces decreased 1.6 fold (Fig. 516). Overall, phage populations on cowpea leaf surfaces under the UVB lights filtered through Museum Glass increased 1.2 fold. But this ap parent increase was clea rly not due to phage reproduction, Xcb was not present. Bacteriophage populations on co wpea leaf surfaces under the UVB lights increased 1.05 fold in 15 min (Fig. 5-16). The increase is due to natural variability in leaf orientation and random sampling and not phage reproduction. At 15 min, the phage population size was 4.1 fold less on leaf surfaces under the UVB lights then under the UVB lights filtered through Museum Glass. At 30 min, phage population under UVB light decreased 7.5 fold. The phage population on cowpea leaf surfaces under UVB lights was 2 fold less than the phage population on cowpea leaf surfaces under the UVB light filtered by Muse um Glass. After 45 min, the phage population on cowpea leaves under UVB light decreased 23 fold; this was 235 fold less than the phage population on cowpea leaves under the UVB lights filtered by Museum Glass. The phage population on cowpea leaf surfaces under UVB light at 60 min decreased 36 fold. The phage population on leaf surfaces under UVB light at 60 min was 1.2 X 104 fold less than the phage population on leaf surfaces unde r the UVB light filtered by Muse um Glass. At 90 min, the 101

PAGE 102

phage population on leaf surfaces under the UVB lights filtered by Museum Glass increased 220 fold. This increase was also not due to phage reproduction. Overall, the phage population on cowpea leaf surfaces under UVB light decreased 6.0 X 103 fold (Fig. 5-16). Role of Relative Humidity on Phage Survival The J phage was sprayed on cowpea leaves and the plants were divided into two plots; one plot exposed to 100% relative humidity (RH) and the other plot e xposed to 45% relative humidity. This experiment took place in the growth room, so other environmental stresses were minimal. After 44 hr, the phage population on cowpea leaves in 100% RH was 4.0 X 104 PFU/cm2 (Fig. 5-17) and at 45% RH the phage population on cowpea leaves was 3.2 X 104 PFU/cm2. At 144 hr, the phage population on cowpea leaves at 100% RH was 2.5 X 104 PFU/cm2 and for leaves at 45% RH, the phage population on cowpea leaves was 1.3 X 103 PFU/cm2. At 192 hr, the phage population at 10 0% RH and at 45% RH was 6.0 PFU/cm2 (Fig. 5-17). Infection of Begonia Inoculum Plants with Xcb Six plots of begonias were inoculated with Xcb and incubated in a humid environment, each plot for a different length of time. The incu bation times were: (i) 0 hr, (ii) 2 hr, (iii) 4 hr, (iv) 8 hr, (v) 16 hr, and (vi) 32 hr. Disease was rated at 7 days and at 14 days after inoculation by comparing leaves with the leaf damage key de scribed in Chapter 4. For the 0 hr incubationtime the average leaf damage rating was 0.2 after 7 days and 0.6 after 14 days (Fig. 5-18). For the 2 hr incubation-time the average leaf damage rating after 7 days was 0.2 and after 14 days, 0.6. For the 4 hr incubation-tim e average leaf damage rating after 7 days was 0.4 and after 14 days, 0.8. The average leaf damage ratings for begonias incubated 8 hr we re 0.4 at 7 days and 0.8 at 14 days. For the 16 hr incubation-time the leaf damage rating was 0.4 at 7 days and 1.0 at 102

PAGE 103

14 days. For the 32 hr incubation-time the leaf damage rating was 0.4 at 7 days and 1.2 at 14 days (Fig. 5-18). Bacteriophage Infection on Leaf Surfaces under Favorable Conditions Bacteriophage infection of Xcb under favorable conditions in the laboratory was tested for the four biological control bacteriophages (m ethod previously described). These conditions provided a humid environment at room temperat ure, as opposed to the greenhouse which can be much warmer and the leaves more prone to desi ccation. After an ove rnight incubation in a favorable environment, the A phage achieved a population of 8.1 X 109 PFU/ml (Fig. 5-19). The D phage population after an overnight incubati on in a favorable environment was 4.0 X 109 PFU/ml. After an overnight incubation in a favorable environment, the J phage population was 2.1 X 109 PFU/ml and the P phage population after an overnight incubation in a favorable environment was 7.3 X 109 PFU/ml (Fig. 5-19). Bacteriophage Infection on Begonia Le af Surfaces under Greenhouse Conditions To asses the potential of a bacteriophage as a biological control, the phage must be able to (i) multiply to a large populat ion and (ii) reduce disease se verity. Desiccation was also suspected to be a significant factor that reduced the bacteriophages ability to infect its host on leaf surfaces. To verify this, bacteriophage we re sprayed on begonias, then immediately sprayed with Xcb before the applications had time to dr y. For comparison purposes, a second group of plants was also sprayed with bacteriophage allowed to dry, and then sprayed with Xcb For both treatment regimes, the A phages population decreas ed to zero within 24 hr (Fig. 5-20). When the phage and Xcb were applied concurrently, population of the D phage decreased 157 fold within 24 hr and decreased to zero within 48 hr When the D phage was applied and allowed to dry, the viable population decrease d 3 fold in 24 hr, 30 fold in 48 hr, and 68 fold overall. When the J phage was applied concurrently with Xcb the viable population decreased to zero within 24 103

PAGE 104

hr. When the J phage was applied, allowed to dry, before the host was applied, the population decreased 34 fold in 24 hr and to zero in 48 hr. When the P phage was sprayed concurrently with Xcb the population decreased 2.5 fold within 24 hr, 3 fold in 48 hr, and over 7 fold overall. When the P phage was sprayed, allowed to dry, and then Xcb applied, the population size decreased 39 fold in 24 hr, and to zero in 48 hr (Fig. 5-20). The infection abilities of each of the ten phages partially char acterized in Chapter 3 were also examined. Each phage culture was not diluted; concentrated suspensions (109 PFU/ml) were applied. In each plot, half of the begonias were sprayed with phage alone and the rest of the begonias in the plot were sprayed with both the particular phage along with and Xcb. This was to determine if any of these phages could infect Xcb on begonia leaf surfaces under greenhouse conditions. For the bacter iophages A, D, J, and P, leaves sampled at 0, 4, 24, 48, and 72 hr and processed and plaque assayed as de scribed above. The A phage alone population on begonia leaf surfaces decreased by 3, 21, 86, a nd 1230 fold with respect to the sampling times (Fig 5-21, Table 5-2). The A phage population that was concurrently applied with Xcb decreased by a similar rate by 7, 110, 270, and 1220 fold with respect to the sampling time. When sprayed on begonia plants alone, without Xcb, the D phage population decreased by 2, 46, 204, and 1300 fold at the times 0, 4, 24, 48, and 72 hr (Fig. 5-22, Table 5-2). When the D phage and Xcb were applied concurrently, the populat ion decreased by 2, 20, 200, and 1460 fold with respect to the sampling times. Plots of begonias were sprayed with either the J alone or the J pha ge concurrently with Xcb and the population was monitored at 0, 4, 24, 48, and 72 hr. When applied alone to begonia leaves, the J phages populati on decreased by 2, 13, 46, and 306 fold with respect to the 104

PAGE 105

sampling times (Fig 5-23, Table 5-2). The J phage with Xcb population decreased by 5, 39, 150, and 550 fold at the respective sampling times. The P phage population alone on begonia leaves decreased by 2, 22, 24, and 800 fold at the sampling times 0, 4, 24, 48, and 72 hr (Fig. 5-24, Table 5-2). When the P phage was applied to begonia leaves concurrently with Xcb, the population decreased by 4, 35, 95, and 4700 fold with respect to the sampling times. Begonia leaves sprayed with the phage IIA were sampled at 0, 24, 48, and 72 hr. After 24 hr, the IIA population had decreased 37 fold (Fig. 5-25, Table 5-3). In 48 hr, the IIA population had decreased 2600 fold and after 72 hr the population had decreased 13,000 fold. When the phage IIA was applied concurrently with Xcb, the population decreas ed 133 fold in 24 hr. The population had decreased 12,000 fold in 48 hr and had decreased 29,000 fold in 72 hr (Fig 5-25, Table 5-3). The phage IVA population alone decreased 18 fo ld in 24 hr, 154 fold in 48 hr and 42,500 fold in 72 hr (Fig. 5-26, Table 5-3). When the phage IVA was applied concurrently with Xcb, the population decreased 55 fold in 24 hr, 134 fold in 48 hr and 65,000 fold in 72 hr. In 24 hr, the Rus phage population (without Xcb ) had decreased 28 fold. In 48 hr, the Rus phage population had decreased 1200 fold and in 72 hr, 183,000 fold (Fig. 5-27, Table 5-3). When the Rus phage was applied concurrently with Xcb, the population decrea sed 52 fold in 24 hr, 700 fold in 48 hr and 1100 fold in 72 hr. The P1 phage alone was sprayed on begonia l eaves and the population was estimated at the times 0, 24, 48, and 72 hr. After 24 hr, the P1 phage population had decreased 415 fold (Fig. 5-28, Table 5-4). After 48 hr, the P1 populat ion had decreased 13,100 fold and after 72 hr, 105

PAGE 106

13,600 fold. When the P1 phage was applied concurrently with Xcb, the population decreased 220 fold in 24 hr, 19,600 fold in 48 hr and 272,000 fold in 72 hr. Begonia leaves were sprayed with the phage P3 and leaves were sampled at 0, 24, 48, and 72 hr to estimate the population. In 24 hr, th e P3 population had decreased 51 fold (Fig. 5-29, Table 5-4). The P3 population ha d decreased 3230 fold in 48 hr and had decreased 5,500 fold in 72 hr. When the P3 phage was applied concurrently with Xcb, the population decreased 790 fold in 24 hr, 6,300 fold in 48 hr and 91,000 fold in 72 hr. When sprayed on begonia leaves alone, the phage P7 population decreased 7 fold in 24 hr, 290 fold in 48 hr and 190 fold in 72 hr (Fig. 5-30, Table 5-4). This increase is not due to phage reproduction, Xcb was not applied. This apparent increase is due to natural variation in leaf orientation. When the P7 pha ge was applied concurrently with Xcb, the population decreased 2 fold in 24 hr, 145 fold in 48 hr and 255 fold in 72 hr. Phage Population Dynamics on Begonia Le af Surfaces of Plants Infected with Xcb The J phage was sprayed on begonias that were previously infected with Xcb and were showing typical symptoms of bacterial spot The initial phage population was 9.9 X 105 PFU/cm2. After 24 hr, the phage population size decrea sed 5.8 fold (Fig. 5-31). After 48 hr, the phage population size increased by 1.2 fold. But this increase is likely due to natural variability in leaf orientation. Identifying Possible Key Fact ors that Affected the Biol ogical Control Experiments The effects of the individual components of the bacteriophage mixture that was used during the biological control experiments was studied in detail. Begonia plots were sprayed with each of the major components, and then inoculated with Xcb. Disease was rated weekly for three weeks. The individual components were: (i) Ster ile Tap Water (STW), (ii) Spent Nutrient Broth [(SpNB), (see Chapter 4)], (iii) nonf at skim milk (dissolved in SpNB), (iv) AG 98 (dissolved in 106

PAGE 107

SpNB, (v) the A phage, (vi) the D phage, (vii) the J phage, and (viii) the P phage alone. Methods are detailed above in section of this chapter. At 7 days, the average leaf damage rating for the STW plot was 1.4 (Fig. 5-32). For the SpNB plot, the average disease rating was 1.4. At 7 days, the plot treated with skim milk had an average disease rating of 1.5. The AG 98 plot had an average disease rating of 1.3 at 7 days. The averag e disease rating at 7 days were 1.3, 1.5, 0.8, and 0.4, for the A phage, D phage, J phage, and P phage respectiv ely (Fig. 5-32). At 14 days, the average disease rating for the STW plot was 1.8. The average disease rating for the SpNB plot was 1.3 (Fig. 5-32). Th e average disease rating at 14 days for the skim milk plot was 1.6. At 14 days, the average disease rating for the AG 98 plot was 1.2. For the A phage, D phage, J phage, and P phage, at 14 days, the average disease ratings were 0.9, 1.5, 1.0, and 1.1 respectively (Fig. 5-32). At 21 days, the average disease ratings for the STW, SpNB, skim milk, and AG 98 were 2.6, 1.9, 2.4, and 1.5 respectively (Fig. 5-32). The average disease ratings for the A phage plots, the D phage plots, the J phage plots, and the P phage plots were 1.8, 2.1, 1.9, and 2.3 respectively. The agricultural wetting agent, AG 98, was utilized during the first four biological control attempts and was suspected as to possibl y being a negative factor. Each of the four biocontrol phages was grown and prepared as de scribed above, fortified with 0.1% AG 98, and sprayed on individual plots of hea lthy begonias. A fifth plot was sprayed with STW. Then all the plots were inoculated with Xcb. Disease was rated at days 7, 14, 21, and 24 (methods described above). At day 7, the average diseas e damage rating for the STW plot was 1.4 (Fig. 533). The disease ratings at day 7 for the A phage plus AG 98 plot, the D phage plus AG 98 plot, 107

PAGE 108

the J phage plus AG 98 plot and the P phage plus AG 98 plot were 0.7, 0.8, 0.5, and 0.3 respectively. At day 14, the average disease damage rating for the STW plot was 1.8 (Fig 5-33). The disease ratings at day 14 for the A phage plus AG 98 plot, the D phage plus AG 98 plot, the J phage plus AG 98 plot and the P phag e plus AG 98 plot were 0.9, 0.5, 0.5, and 0.4 respectively. The average disease rating for the STW plot at day 21 was 2.6 (Fig. 5-33). For the four biocontrol bacteriophages, the A phage plus AG 98 plot, the D phage plus AG 98 plot, the J phage plus AG 98 plot and the P phage plus AG 98, the average disease ratings at day 21 were 0.9, 0.7, 1.0, and 0.9 respectively. At day 24, the average disease rating for the STW plot was 2.6 (Fig 5-33). The average disease ratings for the A phage plus AG 98 plot, the D phage plus AG 98 plot, the J phage plus AG 98 plot and the P phage plus AG 98 were 0.9, 0.8, 1.0, and 0.9 respectively. Analysis of the Variance indicates there is a significant differe nce between the phage-treated plots and the STW plots. (Fcrit = 3.05, F = 13.47) Phage Populations in Irrigation Runoff Water Individual begonia plants were sprayed with one of the four biocontrol bacteriophages and placed under daily overhead irrigation. Polyst yrene cups were used to collect a sample of the irrigation water that splashed off phage-treated begonias. This was to estimate the number of phage that was lost during irri gation over 5 days. The collected water was processed and plaque assayed as described above. Samples were taken at 1, 2, 3, 4, and 5 days after phage application. The numbers of A phage detected in the runoff water were 1.6 X 105 PFU (Fig. 5-34), 980 PFU, 210 PFU, 22 PFU, and 3.8 PFU, respectively. With respect to the sampling times, the numbers of the D phage detected in the runoff were 3.3 X 105 PFU, 3.7 X 104 PFU, 470 PFU, 1.6 X 103 PFU, and 100 PFU. And for the J phage were 2.0 X 105 PFU, 160 PFU, 2 PFU, 8 PFU, and 0 108

PAGE 109

PFU. At 1, 2, 3, 4, and 5 days after phage a pplication, the numbers of P phage detected in irrigation runoff were 2.9 X 105 PFU, 4.1 X 104 PFU, 490 PFU, 230 PFU, 1.2 PFU, respectively (5-34). Infection Rate of Bacteriophage in Nutrient Broth Culture Another negative factor that might have c ontributed to unsuccessful biological control experiments may have been that the infection ti me, or burst rate, may be increased on leaf surfaces as compared with liquid cultures. The reproduction rate in NB culture was determined. The initial inoculum was subtract ed from the plaque count for th ese growth rate data. Using methods described above, indi vidual log-phage cultures of Xcb were inoculated each with one of the biological control bacteriophages, A, D, J or P. Samples were taken from each flask at 0, 1, 2, 3, 4, 5, and 6 hr and processed and plaque assayed as described above. With respect to the sampling times, the A phage population was 0 PFU/ml, 0 PFU/ml, 630 PFU/ml, 7.9 104 PFU/ml, 2.40 X 107 PFU/ml, 6.10 X 109 PFU/ml, and 3.16 X 1010 PFU/ml (Fig. 5-35). The D phage population was 0 PFU/ml, 0 PFU/ml, 5.0 X 103 PFU/ml, 1.55 X 106 PFU/ml, 8.50 X 108 PFU/ml, 1.50 X 109 PFU/ml, and 5.00 X 109 PFU/ml respectively. The J phage population was 0 PFU/ml, 0 PFU/ml, N/A, N/A, 2.32 X 104 PFU/ml, 1.99 X 1010 PFU/ml, and 6.70 X 107 PFU/ml, respectively. Unfortunately, data could not be collected for the J phage at 2 and 3 hr because of a contaminant. The decrease in population for the J phage is due to natural variability. At the sampling times 0, 1, 2, 3, 4, 5, and 6 hr, the P phage population was 0 PFU/ml, 0 PFU/ml, 1.9 X 103 PFU/ml, 2.12 106 PFU/ml, 7.20 X 106 PFU/ml, 3.20 X 109 PFU/ml, and 1.20 X 1010 PFU/ml (Fig. 5-35). These data show that each of these four phages can complete an infection cycle in NB culture in approximately 2 hr. 109

PAGE 110

Infection Rate of Bacteriophage on Vigna Phylloplane under Laboratory Conditions Each of the four bacteriophages was prepared as already described and applied to cowpea leaves. Using methods described in the phage pers istence section, which u tilized Petri dishes to provide a humid environment, the phages and Xcb were allowed to interact and the phage populations was monitored at 0, 1, 2, 3, and 4 hr. A sharp increase in population size would indicate a burst; a complete re productive cycle. The average A phage population respective to the sampling times was: 53 PFU/cm2, 77 PFU/cm2, 82 PFU/cm2, 140 PFU/cm2, and 152 PFU/cm2 (Fig. 5-36). The average D phage population size was initially 17 PFU/cm2. Respective to the sampling times (1, 2, 3, and 4 hr) the average D population was 202 PFU/cm2, 194 PFU/cm2, 23 PFU/cm2, and 1634 PFU/cm2. This dramatic increase indicates a complete reproductive cycle had occurred; a burst (Fig. 5-36) The J phage had in initial population size of 85 PFU/cm2 and at 1, 2, 3, and 4 hr the population size was 60 PFU/cm2, 53 PFU/cm2, 31 PFU/cm2, and 1154 PFU/cm2, respectively. This sharp increase indicates a complete reproductive cycle (Fig. 5-36). At the sampling times 0, 1, 2, 3, and 4 hr, the average P phage population was 73 PFU/cm2, 134 PFU/cm2, 99 PFU/cm2, 64 PFU/cm2, and 443 PFU/cm2, respectively (Fig. 5-36). The roughly ten-fold increase in phage popul ation may indicate a complete reproduction cycle. Discussion Bacteriophages have been used to cont rol bacterial disease caused by other Xanthomonas species; some successful, some unsuccessful. For successful control, environmental stresses need to be identified and avoided. In this study investigated factors influencing biological control of bacterial s pot of begonia and some of the nega tive environmental stresses were elucidated. 110

PAGE 111

Phage persistence on leaf surfaces was poor and appears to be a limiti ng factor, especially under irrigation (Fig. 5-3). Alt hough without irrigation, conditions suitable for begonias should also allow phage to persist on leaf surfaces better than in some other environments (Fig. 5-2, 54). This study was important in that it was one of the first to extensively examine bacteriophage survival on leaf surfaces in nursery and greenhouse environments. The rates of phage inactivation on leaf surfa ces found in this study were similar to the rates reported in previous studies (6, 7, 8, 39, 47), which we re also in the open field or in the greenhouse. As expected, the time required for phage inactivation was slightly different in different environments. In the present study, the effects of different wavelengths of light on phage inactivation demonstrated that, in general, most wavelengths of light will cause some inactivation, although the energy of the wavelength is an important consid eration. The first appr oach in this study used different types of filters to f ilter solar radiation. Regular gr eenhouse glass appeared to offer some protection from UVB light (Fig. 5-6), but later results (see below) suggests the difference in phage population size on leaf surfaces under re gular greenhouse glass was only an anomaly. Poplawsky et al. (54) found that the function of the xanthomonad in pigment was for protection of Xanthomonas from blue light, and not UV. Results of the present study indicated that bacteriophage of Xcb were not greatly affected by blue li ght (Fig. 5-8) compared with unfiltered sunlight. However, because filtered solar ra diation was used as a light source for this experiment, some UV radiation was transmitted by the greenhouse glass to leaf surfaces, so the results of blue light causing inactivation of phage of Xcb were inconclusive. Ultraviolet light can be reflected from objects within the environment, just as visi ble light. Red light was also employed in the same manner as blue light with solar radiation providi ng the radiation source (Fig. 5-10). The results from that experiment we re also inconclusive for the same reasons as 111

PAGE 112

with the blue light; some UV radiation was also present from reflected solar radiation and transmission of UVA light. When UV light was completely excluded by the Museum Glass, which blocks UV light (Fig. 5-11, 12), the phage population on inert surf aces (cellulose) still decreased moderately over time (Fig. 5-13). This result indi cates that, in addition to desi ccation (discussed below), visible wavelengths and to a lesser degree, infrared radiation may cause phage inactivation on inert surfaces. On cellulose, under a cold mirror [whi ch reflects visible light and only transmits infrared light (Fig. 5-11)], bacteriophage population size on cellulose decreased by several factors of ten within 2 hr (Fig 5-13). These results demonstrate several wavelengths of light can have a detrimental effect on bacteriophage surv ival. The cold mirror protected phage from radiation energy better than Mu seum Glass or full sunlight and which suggests that longer wavelengths of light (lower ener gy) inactivate phages at a slower rate than higher energy light (like UV). This is as expected. The highest energy light tested in this study, UVB, inact ivated bacteriophage very quickly on leaf surfaces. UVA also inactivated bacteriophage on leaf su rfaces, although not as rapidly as UVB. The effects of these two type s of light on bacteriophage of plant pathogenic bacteria have not been studied exclusively befo re. The measured increase in phage population size in the UVB/Museum Glass experiment (Fig. 5-16) indicates that the apparent increase in phage population might be due to sampling problems caused by the variable orientation of leaf surfaces in relation to the light source because so me of the sampled leaf surfaces did not received enough UVB to inactivate the phage in the treatment period; it s hould be noted that the leaves selected at random. 112

PAGE 113

It is likely that plant habitat, plant and leaf orientation as well as leaf morphology have a role in protecting bacteriophage from inactivat ion by radiation. Phage do not survive well on broad, waxy leaves, such as begonias, whereas tomatoes and citrus have more leaves within their canopy that are shaded from sunlight. Wavele ngths of light other than UVA or UVB had a negative effect on bacteriophage, but they were not as profound as UV wavelengths. Assays to measure phage survival at different relative humidities (Fig 5-17) took place in a growth room, so other environmental stresses were minimal. Even so, phage populations on cowpea leaf surfaces decreased af ter 196 hr. Partial desiccation does not necessarily inactivate bacteriophage, but it does impact the infection pr ocess because bacteriophages need to be in an aqueous environment to infect the host. When the bacteriophage used in this study were incubated with Xcb on cowpea leaf surfaces in a humid e nvironment, the phage could replicate and achieved a high population size after an overnight incuba tion (Fig. 5-19). However, in the greenhouse, bacteriophage did not extensively infect Xcb on leaf surfaces of begonia (Fig. 5-20). This is despite the phage being in an aqueous suspension at the time of application. To simulate biologi cal control conditions, each of the ten phages characterized in Chapter 3 was sprayed on separate plots of begonias, then half of the plants in each plot was sprayed with Xcb (Fig. 5-21, 5-30). None of th ese phages recovered from leaf surfaces showed an increase in population size; th e phage population size with the bacterial host decreased at rates similar to pha ge populations without the host. This result generally agrees with work by Balogh (9), who found some phages c ould infect their host on leaf surfaces, while others could not. Some phage morphologies may be more suscepti ble to radiation damage than others. In this study, survival of bacteriophage Lambda was examined on cowpea leaves under different 113

PAGE 114

types of artificial light (data not shown ). Survival was evaluated under Cool-White fluorescent lights and under the UVA lights used in this study. Under both light sources, the starting population decreased 5 fold in less than 5 hrs. This result shows that visible light as well as UV light can have a negative effect and demons trates that different ph ages are inactivated at different rates. Bacteriophages us ed in this study were able to persist longer on cowpea leaves than bacteriophage Lambda. Perhaps phages familie s that are more resistant to UV can also be found and selected for better persistence, result ing in better disease control. In this study, the agricultural wetting agent, AG 98, was also suspected to have an adverse effect on bacteriophages. Begonias were sprayed with individual biocontrol phage suspensions amended with AG 98 or sprayed with sterile tap water. Disease damage was worse for the plants treated with sterile tap water tap water (Fig. 5-33). However, other experiments (Figs.5-32) do not show any additional protection from AG 98. Furthermore, this additive was used during the first four biological control experiments, which faile d to prevent bacterial spot of begonia from spreading. The wetting agent wa s used to more evenly distribute phage suspensions on leaf surfaces. Nutrient broth is water-based, and will bead without a wetting agent. Begonias have a thick waxy cuticle that may also suppress phage infection of Xcb. This creates an environment more subj ect to desiccation. Phage populati ons on leaf surfaces declined rapidly under overhead irrigation; ba cteriophages were detected in irrigation runoff (Fig. 5-34). Besides major negative stresses, such as UV light and desiccation, another reason the first four biocontrol experiments were unsuccessful was that the phage were simply rinsed off leaf surfaces during irrigation. 114

PAGE 115

Results of the present study i ndicate that all of the bacter iophages isolated from nursery environments could survive for as long as one week on begonia leaves in greenhouse environments, but these phages did not extensively infect Xcb on leaf surfaces. Experiments in this study indicate that an additional important f actor that affects bacteriophage survival is desiccation. As mentioned above, to infect the host, phages need to be in a water suspension and under greenhouse environments, water evaporates quickly; before the phage have a chance to adsorb to bacterial cells and in fect them. If methods to reduc e these negative factors, mainly desiccation and inactivation from high energy light, were found, the use of bacteriophages for a biological control of bacterial spot coul d work for many different plants. In this study, adequate water (i.e. leaf wetne ss period) appears to be a key factor. When cowpea leaves were maintained in a humid envi ronment overnight, abundant infection occurred. In fact, after an overnight incubation, the phage population size achieved was nearly the same size attained in a flask of NB -approximately 109 PFU/ml. The rate of infection in NB culture takes at least two hours for a burst [(reproduction), (Fig. 5-35)]; whereas infection on cowpea leaf surfaces requires at least 4 hr (Fig. 5-36) for a similar response under laboratory conditions. In the greenhouse, data from this study indica tes that the water susp ension evaporates too quickly from leaf surfaces for the phage to successfully infect Xcb The retarded burst rate does not provide the phage with adequate time to successfully infect. This study shows that major ne gative factors that affect b acteriophage in a biological control environment are a lack of persisten ce on plant surfaces accomp anied by inactivation by to high-energy light, and desiccati on. If these factors can be reduced or eliminated, control of bacterial spot on begonia in controlled environments could be achieved, just as it has for tomato and onion (5, 6, 37). 115

PAGE 116

Figure 5-1. Emission spectrum of GE F40BL U VA tubes as provided by the manufacturer and emission spectrum of Ushio G15T8E UVB t ubes as provided by the manufacturer. Time (hours) 01234567 Lof PFU/cm 2 0 1 2 3 4 5 6 7 Open Field Under Plastic Under Plastic + Shade Figure 5-2. Changes in bacter iophage populations on cowpea leaves over time in three different environments: (i) in an open field under fu ll sun, (ii) in a plas tic-covered greenhouse, (iii) in a plastic-covered greenhouse under an 80% shade cloth. Error bars indicate standard deviation. 116

PAGE 117

Location/Phage RARDRJRPSASDSJSPIAIDIJIP Log PFU/cm 2 0 1 2 3 4 5 Figure 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). Time (hours) 02 04 06 08 0 Log PFU/cm 2 0 2 4 6 8 Plastic Greenhouse Glass Greenhouse Open Field Shade House Figure 5-4. Changes in b acteriophage populations on cowp ea leaves over time in four environments: (i) in a plastic-covered gr eenhouse, (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

PAGE 118

Wavelength (nm) 260280300320340360380400 Abs (Relative Intensity) 0 1 2 3 4 5 6 Figure 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. Time (hours) 0 5 10152025 PFU/cm 2 0 2 4 6 Under Glass Open Field Figure 5-6. Changes in bacter iophage 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

PAGE 119

Wavelength (nm) 260280300320340360380400 Abs (Relative Intensity) 0 1 2 3 Figure 5-7. Measured transmission spectrum of solar radiation transmitted by blue-filter composed of greenhouse glass and blue cellophane in the ul traviolet region. Time (hours) 0123456 Log PFU/cm 2 3 4 5 6 7 Under Blue Filter Open Field Figure 5-8. Changes in bacter iophage 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

PAGE 120

Wavelength (nm) 260280300320340360380400 Abs (Relative Intensity) 0.5 1.0 1.5 2.0 2.5 3.0 Figure 5-9. Measured transmission spectrum of solar radiation transmitted by red-filter composed of greenhouse glass and blue cellophane in the ul traviolet region. Time (hours) 01234567 Log PFU/cm 2 0 1 2 3 4 5 6 7 Under Red Filter Open Field Figure 5-10. Changes in bacteriophage populatio n size over time on cowpea leaves (i) in the open under full sun (ii) under a red-cellopha ne greenhouse glass filter. Error bars indicate standard deviation. 120

PAGE 121

Figure 5-11a. Transmission spectrum from ultravio let 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, (i ii) cold mirror, (iv) Museum Glass. 121

PAGE 122

Figure 5-11b. Transmission spectra from four types of glass filters used in this study in ultraviolet and partial visible wavele ngths; (i) blue-cellophane and greenhouse glass, (ii) red-cellophane and greenhouse glass, (iii) cold mirror, (iv) Museum Glass. 122

PAGE 123

Wavelength (nm) 260280300320340360380400420 Abs (Relative Intensity) 0 1 2 3 4 5 6 Figure 5-12. Measured transmission spectrum of solar radiation through Museum Glass in the ultraviolet region. Time (hours) 0.0 0.5 1.0 1.5 2.0 Log Plaque Count 0 1 2 3 4 Open Field Museum Glass Cold Mirror Figure 5-13. Changes in bacter iophage 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

PAGE 124

Wavelenght (nm) 240260280300320340360380400420 Abs (Relative Intensity) 1 2 3 4 5 6 7 Figure 5-14. Measured emission spectrum of un filtered solar radiation in the UV and partial visible wavelengths. Table 5-1. Average Measured Light IntensitiesD in different environments examined in this study. Environment Total Photosynthetic RadiationA UV RadiationB Plastic-covered Greenhouse 79.5 56.0 Glass Greenhouse 16.5 0.65 Open Field 162.0 116.3 Shade HouseC 31.0 30.1 UVA Lights 2.25 12.7 UVB Lights 0.1 11.5 Greenhouse Glass 135.0 90.0 Blue Greenhouse Glass 57.0 36.0 Red Greenhouse Glass 57.0 43.0 Museum Glass 135.0 23.5 A W m-2s-1 B (mol photons m-2sec-1) C 70% Shade D average of three readings 124

PAGE 125

Wavelength (nm) 260280300320340360380400420 Abs (Relative Intensity) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 A Wavelength (nm) 260280300320340360380400 Abs (Relative Intensity) -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 B Time (hours) 02 04 06 08 0 Log PFU/cm 2 0 1 2 3 4 5 6 7 UVA UVA + Museum Glass C Figure 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) unde r the UVA light and protected by the Museum Glass (lower). Error bars indi cate standard deviation. Panes A and B show the types of light the phage were exposed to in pane C. 125

PAGE 126

Wavelength (nm) 260280300320340360380400420 Abs (Relative Intensity) -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 A Wavelength (nm) 260280300320340360380400420 Abs 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 B Time (minutes) 02 04 06 08 0 Log PFU/cm2 0 1 2 3 4 5 6 7 UVB UVB + MG C Figure 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) un der the UVB light and protected by the Museum Glass (lower). Error bars indi cate standard deviation. Panes A and B show the types of light the phage were exposed to in pane C. 126

PAGE 127

Time (hours) 20406080100120140160180200 Log PFU/cm 2 0 1 2 3 4 5 100% RH 45% RH Figure 5-17. Changes in bacteriophage populatio n size over time on cowpea leaf surfaces in the growth room at (i) 100% relative humi dity, (ii) 45% relative humidity. Time (days) 6 8 10 12 14 16 Leaf Damage Rating 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 0 hr. 2 hr. 4 hr. 8 hr. 16 hr. 32 hr. Figure 5-18. Disease progress curves for bact erial spot damage on be gonia following different incubation times in a humid environment for 0, 2, 4, 8, 16, and 32 hr. 127

PAGE 128

Phage ADJP Log PFU/ml 0 2 4 6 8 10 12 Figure 5-19. Relative phage population achie ved 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 envi ronment. Error bars indicate standard deviation. Time (hours) 010203040506 0 PFU/cm 2 0 100 200 300 A phage Leaves Wet A phage Leaves Dry D Phage Leaves Wet D Phage Leaves Dry J Phage Leaves Wet J Phage Leaves Dry P Phage Leaves Wet P Phage Leaves Dry Figure 5-20. Changes in bacteriophage populat ion size over time on cowpea leaves (i) the A phage, leaves wet, (ii) A pha ge, 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

PAGE 129

Time (hours) 02 04 06 0 8 0 Log PFU/cm 2 1 2 3 4 5 6 7 A Phage only A Phage + Xcb Figure 5-21. Changes in bacteriophage populat ion size over time on begonia leaves in a greenhouse environment (i) A phage alone, (ii) A phage with Xcb. Error bars indicate standard deviation. Table 5-2. Changes in b acteriophage population sizes ove r time on begonia leaves in a greenhouse environment. Bacteriophage and Xcb applied Measure populationsz Time A alone A + Xcb D alone D + Xcb J alone J + Xcb P alone P + Xcb 0 4 24 48 72 4.6X106 1.5X106 2.2X105 5.4X103 380 1.1X106 1.6X105 9.9X103 4.0X103 880 2.1X1061.3X1064.7X1041.1X1041.7X1032.0X1061.2X1069.8X1049.9X1031.4X1037.6X1053.1X1055.8X1041.6X1042.5X1031.1X1062.2X1052.8X1047.2X1032.0X1031.0X106 5.5X105 4.6X104 4.3X104 1.3X103 5.6X1061.4X1061.6X1055.9X1041.2X103 z PFU/cm2 129

PAGE 130

Time (hrs) 02 04 06 08 0 Log PFU/cm 2 2 3 4 5 6 7 D Phage only D Phage + Xcb Figure 5-22. Changes in bacteriophage populat ion size over time on begonia leaves in a greenhouse environment (i) D phage alone, (ii) D phage with Xcb. Error bars indicate standard deviation. Time (hrs) 02040608 0 Log PFU/cm 2 2 3 4 5 6 7 J Phage only J Phage + Xcb Figure 5-23. Changes in bacteriophage populat ion size over time on begonia leaves in a greenhouse environment (i) J ph age alone, (ii) J phage with Xcb. Error bars indicate standard deviation. 130

PAGE 131

Time (hours) 02 04 06 08 0 Log PFU/cm2 2 3 4 5 6 7 8 P Phage only P Phage + Xcb Figure 5-24. Changes in bacteriophage populat ion size over time on begonia leaves in a greenhouse environment (i) P phage alone, (ii) P phage with Xcb. Error bars indicate standard deviation. Time (hours) 02 04 06 08 0 Log PFU/cm2 1 2 3 4 5 6 7 8 Phage II A only Phage II A + Xcb Figure 5-25. Changes in bacteriophage populat ion 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

PAGE 132

Table 5-3. Changes in b acteriophage population sizes ove r time on begonia leaves in a greenhouse environment Bacteriophage and Xcb applied Measure populationsz Time IIA alone IIA + Xcb IVA alone IVA + Xcb Rus alone Rus + Xcb 0 24 48 72 3.7X106 1.0X104 1.4X103 280 1.1X107 8.0X105 880 370 5.1X105 2.9X104 3.3X103 12 7.5X105 1.4X104 5.6X103 12 3.1X106 1.1X105 2.6X103 17 2.2X106 4.3X106 3.2X103 2.0X103 z PFU/cm2 Time (hours) 01020304050607080 Log PFU/cm 2 0 1 2 3 4 5 6 7 Phage IV A only Phage IV A + Xcb Figure 5-26. Changes in bacteriophage populat ion 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

PAGE 133

Time (hours) 01020304050607080 Log PFU/cm2 0 1 2 3 4 5 6 7 8 Phage Rus only Phage Rus + Xcb Figure 5-27. Changes in bacteriophage populat ion size over time on begonia leaves in a greenhouse environment (i) Rus phage alone, (ii) Rus phage with Xcb. Error bars indicate standard deviation. Time (hours) 01020304050607080 Log PFU/cm2 0 1 2 3 4 5 6 7 8 Phage P1 only Phage P1 + Xcb Figure 5-28. Changes in bacteriophage populat ion 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

PAGE 134

Table 5-4. Changes in b acteriophage population sizes ove r time on begonia leaves in a greenhouse environment. Bacteriophage and Xcb applied Measure populationsz Time P1 alone P1 + Xcb P3 alone P3 + Xcb P7 alone P7 + Xcb 0 24 48 72 9.4X106 2.3X104 720 700 1.3X107 6.1X104 680 50 2.3X106 4.6X104 720 420 1.4X107 1.7X104 2.2X103 150 1.0X105 1.5X104 350 540 1.4X105 5.7X104 950 540 z PFU/cm2 Time (hours) 01020304050607080 Log PFU/cm2 1 2 3 4 5 6 7 8 Phage P3 only Phage P3 + Xcb Figure 5-29. Changes in bacteriophage populat ion 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

PAGE 135

Time (hours) 01020304050607080 Log PFU/cm2 0 1 2 3 4 5 6 Phage P7 only Phage P7 + Xcb Figure 5-30. Changes in bacteriophage populat ion size over time on begonia leaves in a greenhouse environment (i) P7 phage alone, (ii) P7 phage with Xcb. Error bars indicate standard deviation. Time (hours) 010203040506 0 Phage Count (PFU/cm 2 ) 0.0 2.0e+5 4.0e+5 6.0e+5 8.0e+5 1.0e+6 1.2e+6 J Phage Count Figure 5-31. Changes in bact eriophage population over time on Xcbinfected begonias in a greenhouse environment. 135

PAGE 136

Time (days) 6810121416182022 Disease Damage Rating 0 1 2 3 4 5 6 7 STW SpNB Milk AG 98 A Phage D Phage J Phage P Phage Figure 5-32. Disease progress of bacterial spot of begonia with di fferent 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

PAGE 137

Time (days) 681012141618202224 Disease Damage Rating 0 1 2 3 4 STW A + AG 98 D + AG 98 J + AG 98 P + AG 98 Figure 5-33. Disease progress of bacterial spot of begonia with di fferent 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 st andard deviation. Time (hours) 0204060801 00 Log PFU/ml 0 1 2 3 4 5 6 A Phage D Phage J Phage P Phage Figure 5-34. Bacteriophage populations measured in the irrigation runo ff from begonia plants sprayed with (i) the A phage, (ii) the D pha ge, (iii) the J phage, (iv) the P phage. The plants were sprayed once and then subj ected to daily overh ead irrigation. Error bars indicate stan dard deviation. 137

PAGE 138

Time (hours) 01234567 Log PFU/ml 0 2 4 6 8 10 12 A Phage D Phage J Phage P Phage Figure 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. Time (hours) 01234 Log PFU/ml 1 2 3 4 A Phage D Phage J Phage P Phage Figure 5-36. Changes in bacteriophage populatio n 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 stan dard deviation. 138

PAGE 139

CHAPTER 6 OVERALL SUMMARY AND CONCLUSIONS Bacterial spot of begonia, caused by Xanthomonas campestris pv. begoniae can be very costly to growers. A conventiona l preventative treatment of bacterial spot has been the frequent application of cupric hydroxide or other copper compounds, but the possibility of resistance and environmental concerns about heavy metals make alternative approaches to copper preferable. Biological control of bacterial sp ot using bacteriophages has worked well in some cases but not in others (6, 7, 8, 24, 25, 33, 39, 47, 77), with noteworthy successes with tomato and onion. It was anticipated that it would also work well for Xcb. Accordingly, the first objective of this study was to investigate the occurrence of bacteriophages of Xcb in nursery and foliage production environments; this study is novel in this regard because the occurrence of phage in these environments has not been extensively investigated. This study is importa nt in that it shows that with the right host bacterium (the right bait), bacteriophages of plant pathogenic ba cteria can be found in these environments. Furthermore because of host specif icity to particular strains or pathovars, the bait should be the particular isolate of pathogen that incites the disease of interest. Many of the phages isolated that could infect Xcb were found with different pathovars of Xa nthomonas as the initial bait. Four of the phages found in this study were selected fo r biocontrol experiments based on having broader host ranges; six other phages we re selected as representative s from different environmental sources. The second objective was to partially characterize some of these phages. Ten phages from this collection were examined by TEM, and nine had similar morphology. Four phages were selected based on host range; all four were possibly related as they have, besides similar morphologies, similar DNA genome sizes. The f our bacteriophages were tested as a possible biological control of bacterial spot of begonia (the third objective). It was initially thought that 139

PAGE 140

because many environmental stresses are reduced in the greenhouse than in the field that these environments would be favorable for biological control experiments. Unfortunately, five experiments to evaluate biocont rol potential produced negative results because the conditions likely favored the pathogen while there were seve ral other negative factors that affected phage survival and persistence. Many of the negative factors affecting bacteriophage persistence, survival, and ability to infect its host on leaf surfaces were indentified and documented in Chapter 5 (fourth objective). It was shown that the phage could infect the host on cowpea leaf surfaces in laboratory conditions, but none of the ten phages characterized further (Chapter 3) could extensively infect Xcb on begonia leaf surfaces under greenhouse conditions; despite an eight hour incubation in a humid environment. The most significant factors identified were inactivation by to UV light, desiccation, and lack of persistence under irrigation. Excessive desiccation was very important. The thick waxy cuticles of begonia leaves did not retain water well, so the water containing the phage and Xcb suspensions quickly eva porated during daylight hours. Another factor that impacted pe rsistence was wash-off. It is likely that during the first four biological control experiments, where the leaves remained wet for approximately three hours, that most of the phage we re washed off the leaves during irrigation. This was documented in Fig. 5-34. There were two other negative f actors that affected the fifth biological control experiment, the first was that the initial concentration of phage was lower (approx. 107 PFU/ml) than used in previous published studies (6, 7, 8, 24, 25, 33, 39, 47, 77) and second was that since these plants were irrigated dur ing daylight hours, the leaves did not remain wet long enough because of excessive evaporation. 140

PAGE 141

Experiments presented in Chapter 5 showed that a major reason that none of the biological control experiments worked well is that none of the phages tested could infect Xanthomonas on begonia leaf surfaces under greenhouse conditions. The phage burst rate on cowpea leaves (in a humid environment) was approximately four hours. These studies establ ished key factors that should be considered for future research concerning conditions th at need to be optimized for successful biological control of ba cterial spot in greenhouse in gene ral. Bacteriophages need to be in a water suspension to infect the host. If the infection rate is too slow, the water present to provide an infection medium eva porates before a successful infec tion can occur. Prolonged leaf wetness may, in fact, provide an environment suitable for phageXcb infection; however a caveat to prolonged leaf wetness is that this may encourage other pathogens to infect (2). Future work should include: (i) screening more phages from production environmen ts and select ones that can infect Xcb on begonia leaf surfaces, (ii) with better phages, it is likely that larger volumes of phage cultures are needed so the viable titer in the application is at least 108 PFU/cm2 (S.T. Abedon, personal communication, 2008). Phages with faster rates of infection would be preferable, so an infection could be initiated befo re the water evaporates. (iii) mix or inject the phage into the irrigation and irriga te the plot 2-3 hr before dawn. These criteria can be useful when screening other phages fo r potential control of other bacterial diseases of plants in greenhouse environments. If thes e negative factors are alleviated the use of b acteriophage as prevention for bacterial spot on plants in the greenhouse may be an environmentally feasible option. 141

PAGE 142

REFERENCE LIST 1. Adams, M. 1959. Bacteriophages. Inters cience Publishers. New York, New York. 2. Agrios, G. 2005. Plant Pathology, 5th Edition. Elsevier Academic Press. Burlington, MA. 3. Anderson, H.W. 1928. Bacteriophage of Bacterium pruni Phytopathology. 18: 144. 4. Ashelford, K.E., Norris, S.J., Fry, J.C., Baily, M.J., and Day, M.J. 2000. Seasonal population dynamics and inte ractions of competing bacteriophages and their host in the rhizosphere. Applied and E nvironmental Microbiology. 62: 41934199. 5. Ashelford, K.E., Norris, S.J., Day, M.J. 2003. Elevated abundance of bacteriophage infecting bacteria in so il. Applied and Environm ental Microbiology. 69: 285289. 6. Balogh, B. 2002. Strategies for improving the efficacy of bacteriophages for controlling bacterial spot of tomato. M. S. thesis. University of Florida, Gainesville. 7. Balogh, B., Jones, J.B., Momol, M.T., Olson, S.M., Obradovic, A., King, P., and Jackson, L.E. 2003. Improved Efficacy of newly formulated bacteriophages for management of bacterial spot of tomato. Plant Disease. 87: 949-954. 8. Balogh, B. 2006. Characterization and use of Bacteriophages Asso ciated with Citrus Bacterial Pathogens for Disease Contro l. Ph.D. Dissertation. University of Florida, Gainesville. 9. Bergh, O., Borsheim, K.Y., Bratbak, G. and Heldal, M. 1989. High abundances of viruses found in aquatic e nvironments. Nature. 340:467-468. 10. Breitbart, M., Wegley, L., Leeds, S., Schoenfeld, T. and Rohwer, F. 2004. Phage community dynamics in hot springs. A pplied and Environmental Microbiology. 70:1633-1640. 11. Breitbart, M. and Rohwer, F. 2005. Here a virus, there a viru s, everywhere the same virus? Trends in Microbiology. 13(6):278-284. 12. Byrne, J. 2006. Bacterial leaf spot of begonia, another Xanthomonas. Michigan State University IPM newsletter. No 8, April 14, 2006. 13. Chase, A.R. 1992. Bacterial disease cont rol on ornamentals using aliette, kocide greenshield and ACS-66825. MREC -Apopka Research Report. http://mrec.ifas.ufl.edu/ Foliage/Resrpts/rh_92_10.htm 142

PAGE 143

14. Chibani-Chennoufi, S. Bruttin, A. Dillmann, M-L. and Brussow, H. 2004. PhageHost interaction: an ecological persp ective. Journal of Bacteriology. 186: 3677-3686. 15. Coons, G. H. and Kotilia, J.E. 1925. Tran smissible lytic princi ple (bacteriophage) in relation to plant pathogens Phytopathology. 15: 357-370. 16. Coyier, D.L. and Covey, R.P. 1975. Tolerance of Erwina amylovora to streptomycin sulfate in Oregon and Washington. Plant Dis. Rep. 59:849-852. 17. Davies, C.M., Logan, M.R., Rothwell, V.J ., Krogh, M., Ferfuson, C.M., Charles, K., Deer, D.A. and Ashbolt, N.J. 2006. Soil inactiva tion of DNA virues in septic seepage. Journal of Applied Microbiology. 100:2 365-374. 18. DHerelle, F. 1926. The bacteriophage and its behavior. Tr. by G.H. Smith. Williams and Wilkins Co., Baltimore. 19. DHerelle, F. 1930. The bacteriophage and its clinical application. Tr. by G.H. Smith. Md. C.C. Thomas Co., Baltimore. 20. Delannoy, E., Lyon, B.R., Marmey, P., Jalloul, A., Daniel, J.F. Montillet, J.L. Essenberg, M. and Nicole, M. 2005. Resitance of cotton toward Xanthomonas campestris pv. malvacearum Annual review of Phytopathology. 43:63-82. 21. Dunbar, J.M. 1948. Bacteriophage typing of untypable Salmonella typhi organisms. Nature. 162(4126):851 22. European and Mediterranean Plant Prot ection Organization. 2004. Data Sheets on Quarantine Pests, European Plant Protection Organization (EPPO). 23. Farrah, S.R. 1982. Chemical factors in fluencing adsorption of bacteriophage MS2 to membrane filters. Applied and Environmental Microbiology. 43(3): 659663. 24. Flaherty, J.E., Jones, J.B., Harbaugh, B.K., Somodi, G.C., and Jackson, L.E. 2000. Control of bacterial spot on tomato in the greenhouse and field with Hmutant bacteriophages. HortScience 35: 882-884. 25. Flaherty, J.E., Harbaugh, B.K., Jones, J.B., Somodi, G.C. and Jackson, L.E. 2001. H-mutant bacteriophages as a potential biocontrol of bact erial blight of geranium. HortScience. 36(1):98-100. 26. Florida Agricultural Statistics Service. 2005. Foliage, floriculture and cut greens. Florida Agriculture. http://www.nass.usda.gov/fl 143

PAGE 144

27. Foppen, J.W.A., Okletey, S., and Schijven, J.F. 2006. Effect of goethite coating and humic acid on the transport of b acteriophage PRD1 in columns of saturated sand. Journal of C ontaminant Hydrology. 85 287-301. 28. Hugouvieux, V. Barber, C.E. and Daniels, M.J. 1998. Entry of Xanthomonas campestris pv. c ampestris into Hydathodes of Arabidopsis thaliana Leaves: A System of Studying Early Infection Events in Bacterial Pathog enesis. Molecular Plant-Microbe Interactions. 11:6 537-543. 29. Gerretson, F.C., Grijns, A. and Sach, J. 1923. Das vorkommen eines bakteriophagen in der wurzelknollchen der leguminosen. Centralb l. Bakterio. Abt. 2, 60: 311-316. 30. Gill JJ, and Abedon ST. 2003. Bacteriophage Ecology and Plants. APSnet November. http://www.apsnet.org/online/feature/phages/ 31. Gurlebeck, D. Thieme, F. and Bonas, U. 2006. Type III effector proteins from the plant pathogen Xanthomonas and their role in the interaction with the host plant. Journal of Plant Physiology. 163:233-255. 32. Jagger, J. 1985. Solar-UV actions on liv ing cells. Praeger Press. New York. 33. Jones, J.B., Jackson, L.E., Balogh, B., Obradovic, A., Iriarte, F.B. and Momol, M.T. 2007. Bacteriophages for plant di sease control. Annual Review of Phytopathology. 45:245-262. 34. Katznelson, H. 1937. Bacteriophage in relati on to plant disease. The Botanical Review. 3(10): 499-521. 35. Kelland, L.R., Moss, S.H. and Davies, D.J.G. 1983. Damage to Bacterial Cell Membranes by UV Radiation in S unlight. Bioscience. 33:5 334-335. 36. Klement, Z. 1957. Two new bacteriophages for bacterial pathogens of the bean. Nature. 180: 41-42. 37. Klement, Z. 1959. Some new specifi c bacteriophages for plant pathogenic Xanthomonas spp. Nature. 184: 1248-1249. 38. Kucharek, T. 1994. Plant pathology fact sh eet: bacterial spot of tomato and pepper. University of Florida, Institute of Food and Agricultural Sciences, Plant Pathology Department. Cooperative Extens ion Service. Online publication. 39. Lang, J.M., Gent, D.H., and Schwartz H.F. 2007. Management of Xanthomonas leaf blight of onion with Bacteriophages and a plant activator. Plant Disease. 91:7 871-878. 144

PAGE 145

40. Lindow, S.E. and Brandl, M.T. 2003. Mi crobiology of the Phyllosphere. Applied and Environmental Microbiology. 69:4 1875-1883. 41. Lipp, R.L.; Alvarez, A.M.; Benedict A.A.; Berestecky, J. 1992. Use of monoclonal antibodies and pathogenicity tests to characterize strains of Xanthomonas campestris pv. dieffenbachiae. Phytopathology 82: 677-682. 42. Mallman, W.L. and Hemstreet, C. 1924. Isolati on of an inhibitory substance from plants. Jour. Agric. Res. 28: 599-602. 43. Martinez, A. 2005. Georgia plant diseas e loss estimates. University of Georgia Cooperative Extension. 1-24. 44. McCulloch, L. 1937. Bacterial leaf spot of Begonia. Journal of Agricultural Research. 54(8) 583-590. 45. McManus, P.S. and Stockwell, V.O. 2001. Antibiotic Use for Plant Disease Management in the United States. Plant Health Prog. http://www.Planthealthprogress.org/Current/reviews/antibiotic/top.htm 46. McManus, P.S., Stockwell, V.O., Sundin, G.W. and Jones, A.L. 2002. Antibiotic Use in Plant Agriculture. Annual Re view of Phytopathology. 40:443-465. 47. McNeil, D.L., Romero, S. Kandula, J., Stark, C., Stewart, A. and Larsen, S. 2001. Bacteriophages: A Potential Bioc ontrol Agent Against Walnut Blight ( Xanthomonas campestris pv. juglandis New Zealand Plant Protection. 54:220224. 48. Morris, W. ed. 1973. The American He ritage Dictionary. American Heritage Publishing. Boston, MA. 49. Neve, H., Kemper, U, Geis, A and Heller, K.J. 1994. Monitoring and characterization of lactococcal bacteriophage in a dairy plant. Kiel. Milchwirtsch. Forschungsber. 46:167-178. 50. Noble, R.T. and Fuhrman, J.A. 1997. Vi rus Decay and its causes in coastal waters. Applied and Environmental Microbiology. 63:77-83. 51. Obradovic, A., Mavridis, A., Rudolph, K., Janse, J.D., Arsenijevic, M., Jones, J.B.,Minsavage, G.W., Wang, J-F. 2004. Ch aracterization and PCR-based typing of Xanthomonas campestris pv. vesicatoria from peppers and tomatoes in Serbia. European Journal of Plant Pathology. 110: 285-292. 52. Okabe, N. and Goto, M. 1963. Bacteriophages of plant pathogens. Annual Review of Phtopathology. 1: 397-418. 145

PAGE 146

53. Paul, J.H., Sullivan, M.B., Segall, A.M., and Rohwer, F. 2002. Marine phage genomics. Comparative Biochemistry and Physiology. Part B 133 463. 54. Poplawsky, A.R., Urban, S.C. and Chun, W. 2000. Biological role of xanthomonadin pigments in Xanthomonas campestris pv. campestris Applied and Environmental Microbiology. 66 (12): 5123-5127. 55. Proctor, L.M. 1997. Advances in the st udy of marine viruses. Microscopy Research and Technique. 37:136-161. 56. Rochford, T. and Gorer, R. 1966.The Rochford book of flowering pot plants. Trinity Press. London, England. 57. Roux, R. 2007. On an invisible microbe anta gonistic toward dysenteri c bacilli: brief note by Mr. F. DHerelle, presented by Mr. Roux. Research in Mircro biology. 158: 553-554. 58. Sambrook, J. and Russel, D.W. 2001. Pr otocol 7: Assaying the DNA content of bacteriophage stocks and lysates by gel elect rophoresis. Molecular Cloning: A Laboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory and Press. Cold Spring Harbor, NY. 2.45-46. 59. Sathyanarayana, N; Reddy, OR; Latha, S; Rajak, RL. 1998. Interception of Xanthomonas campestris pv. dieffenbach iae on anthurium plants from the Netherlands. Plant Disease 82: 262. 60. Shields, P.A. and Farrah, S.R. 1983. Influe nce of salts on electros tatic interactions between poliovirus and membrane f ilters. Applied and Environmental Microbiology. 45 (2): 526-531. 61. Sinton, L.W. Davies-Colley, R.J. and Bell, R.G. 1994. Inactiv ation of enterococci and fecal coliforms from sewage and meat works effluents in seawater chambers. Applied and Environmental Microbiology. 60(6):2040-2048. 62. Stanley-Allen, K.L., Empey, N.R., Meade, D.S., Rzeznik, S.J., Streitwieser, M.L. and Strople, M.S. 2005. Preview of the benchmark input-output accounts for 2002. Survey of current business. Bu reau of Economic Analysis. 66-77. 63. Sundin, G.W. and Jacobs, J.L. 1999. U ltraviolet Radiation (UVR) sensitivity analysis and UVR survival strategies of a bacterial community from the phyllosphere of field-grown peanut ( Arachis hypogeae L.). Mircrob. Ecol. 38:2738. 64. Suttle, C.A. and Chen, F. 1992. Mechanisms and rates of decay of marine viruses in seawater. Applied and Envir onmental Microbiology. 58:3721-3729. 146

PAGE 147

65. Sutton, M.D., Katznelson, H. and Quadling, C. 1958. A bacteriophage that attacks numerous phytopathogenic Xanthomonas species. Ca nadian Journal of Microbiology. 4: 493-497. 66. Tebbitt, M.C. 2005. Begonias; Cultivation, Identification and History. Timber Press. Portland, Ore. 67. Teixeira, E.C., Franco de Oliveira, J.C., Marques Novo, M.T. and Bertolini, M.C. 2008. The copper resistance operon copAB from Xanthomonas axonopodis pathovar citri: gene inactivation results in copper sensitivity. Microbiology. 154(2):402-412. 68. University of California. 2002. Integrated Pest Management Online. Floriculture and Ornamental Nurser ies: Begonia. 69. Voloudakis, A.E., Reignier, T.M. a nd Cooksey, D.A. 2005. Regulation and Resistance to copper in Xanthomonas axonopodis pv. vesicatoria. Applied and Environmental Microbiology. 71:2 782-789. 70. Ward, R.L. and Mahler, R.J. 1982. Up take of Bacteriophage f2 Through Plant Roots. Applied and Environmen tal Microbiology. 43:5 1098-1103. 71. Weinbaur, M.G. 2004. Ecology of pr okaryotic viruses. Federation of Microbiological Societies Microb iology Reviews. 28: 127-181. 72. Wihelm, S.W., Jeffrey, W.H., Suttle, C. A.,and Mitchell, D.L. 2002. Estimation of biologically damaging UV levels in ma rine surface waters and DNA and viral dosimetersPhotochemistry and Photobiology. 76(3) 268-273. 73. Wright, C.A. and Beattie, G.A. 2004. Pseudomonas syrgingae pv. tomato cells encounter inhibitory levels of water stress during the hypersensitive response of Arabidopsis thaliana Proceedings of the National Academy of Science. 101:3269-3274. 74. Wommack, K.E., Hill, R.T., Muller, T.A. a nd Colwell, R.R. 1996. Effects of sunlight on bacteriophage viability and structur e. Appl. Environ. Microbiol. 62:241-250. 75. Wommack, K.E. and Colwell, R.R. 2000. Viroplankton: Viruses I aquatic ecosystems. Microbiology and Molecu lar Biology Reviews. 64(1):69-114. 76. Yu, M.X., Slater, M.R. and Ackermann, H.-W. 2006. Isolation and characterization of Thermus bacteriophages. Arch Virol. 151:663-679. 77. Zaccardelli, M., Saccardi, A., Bambin, E., and Mazzucchi, U. 1992. Xanthomonas campestris pv. pruni bacteriophages on peaches trees potential use for biological control. Phytopathologia Mediterra nea. 31: 133-140. 147

PAGE 148

148 BIOGRAPHICAL SKETCH Jeff Kaesberg was born in the Chicago area and moved to Florida when he was young. He graduated from Lake Mary High School in 1993 and earned his baccalaureate degree from the University of Central Florida in Biology in 1996. He earned a Master of Science from the University of Florida in Medical Science w ith a focus on human virology in 2001 and earned a second Masters of Science in Plant Pathology in 2005. He received his Doctor of Philosophy in Plant Pathology in December 2009. Currently, he is employed at the University of Floridas Mid-Florida Education and Research Center (MREC) in Apopka. He will continue this employment while he searches for a job in academia, industry, or with the USDA.