<%BANNER%>

Towards Orchid IPM

Permanent Link: http://ufdc.ufl.edu/UFE0041471/00001

Material Information

Title: Towards Orchid IPM Tools and Molecular Techniques for the Diagnosis and Management of Selected Orchid Arthropod Pests and Diseases in Florida
Physical Description: 1 online resource (175 p.)
Language: english
Creator: Cating, Robert
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

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: Towards Orchid IPM: Tools and Molecular Techniques for the Diagnosis and Management of Selected Orchid Arthropod Pests and Diseases in Florida The Orchidaceae is believed to be the largest family of flowering plants, with an estimated 19,000 species. Orchid sales in the United States have increased steadily since 1997, and exceeded 140 million US dollars in 2006. Orchids are the second most economically important flowering plant produced in the United States, and Florida is one of the top producers in the nation. Orchids are sensitive to chemical pesticides. Thus, it would be desirable to establish an integrated pest management (IPM) program in order to reduce arthropod pest and disease problems, while minimizing the impact of the control measures on non-target organisms (natural enemies), humans, and the environment. Currently, no comprehensive IPM programs exist for orchid production. The overall objective of this dissertation was to develop new diagnostic procedures and control strategies for several important orchid pests and pathogens that would be compatible with, and could be used to establish, an overall IPM program for this crop. Commercial orchid growers and hobbyists in Florida were asked what insect and disease problems were frequently encountered and which were considered the most severe. They indicated that bacterial soft-rot diseases caused by Dickeya spp., leaf spots caused by Pseudocercospora spp., and infestations of Boisduval scale, Diaspis boisduvalii Signoret, were the most problematic. Therefore, these problems were primary focuses of this dissertation. Eighteen bacterial isolates were collected from orchids with soft-rot disease symptoms and, through the use of biochemical tests, carbohydrate utilization tests, fatty-acid analysis and 16S rDNA and pelADE gene sequences, isolates were determined to be distinct from currently identified species of Dickeya; they may represent new species. Pseudocercospora spp. cause leaf spots in several orchid taxa that can be severe. A first step towards understanding these diseases is to culture the causal fungi and produce conidia for pathogenicity tests, which could be used later for studies on host range and epidemiology. It was determined that V-8 agar produced the best mycelial growth at 25?C, and that sporulation was induced after actively growing V-8 agar cultures were transferred to water agar. In order to select the most appropriate management options for plant diseases, pathogens must be identified correctly and quickly. High-fidelity PCR greatly increased the ability to detect P. odontoglossi (Prill & Delacr.) U. Braun, Dickeya spp., and Sclerotium rolfsii Sacc. directly from plant tissue and could be useful in managing diseases with long latent periods, difficult to recover pathogens, or ambiguous symptoms. Improved control options were developed for Boisduval scale and a flat mite, two important arthropod pests that can be difficult to control. Silwet L-77 (0.05%), an organosilicone adjuvant, increased the efficacy of 3 horticultural oils and significantly improved control of these pests. Information from this work was provided to orchid growers in six extension bulletins, diagnostic guides, and disease notes.
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 Robert Cating.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Palmateer, Aaron J.
Local: Co-adviser: Hoy, Marjorie A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041471:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041471/00001

Material Information

Title: Towards Orchid IPM Tools and Molecular Techniques for the Diagnosis and Management of Selected Orchid Arthropod Pests and Diseases in Florida
Physical Description: 1 online resource (175 p.)
Language: english
Creator: Cating, Robert
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

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: Towards Orchid IPM: Tools and Molecular Techniques for the Diagnosis and Management of Selected Orchid Arthropod Pests and Diseases in Florida The Orchidaceae is believed to be the largest family of flowering plants, with an estimated 19,000 species. Orchid sales in the United States have increased steadily since 1997, and exceeded 140 million US dollars in 2006. Orchids are the second most economically important flowering plant produced in the United States, and Florida is one of the top producers in the nation. Orchids are sensitive to chemical pesticides. Thus, it would be desirable to establish an integrated pest management (IPM) program in order to reduce arthropod pest and disease problems, while minimizing the impact of the control measures on non-target organisms (natural enemies), humans, and the environment. Currently, no comprehensive IPM programs exist for orchid production. The overall objective of this dissertation was to develop new diagnostic procedures and control strategies for several important orchid pests and pathogens that would be compatible with, and could be used to establish, an overall IPM program for this crop. Commercial orchid growers and hobbyists in Florida were asked what insect and disease problems were frequently encountered and which were considered the most severe. They indicated that bacterial soft-rot diseases caused by Dickeya spp., leaf spots caused by Pseudocercospora spp., and infestations of Boisduval scale, Diaspis boisduvalii Signoret, were the most problematic. Therefore, these problems were primary focuses of this dissertation. Eighteen bacterial isolates were collected from orchids with soft-rot disease symptoms and, through the use of biochemical tests, carbohydrate utilization tests, fatty-acid analysis and 16S rDNA and pelADE gene sequences, isolates were determined to be distinct from currently identified species of Dickeya; they may represent new species. Pseudocercospora spp. cause leaf spots in several orchid taxa that can be severe. A first step towards understanding these diseases is to culture the causal fungi and produce conidia for pathogenicity tests, which could be used later for studies on host range and epidemiology. It was determined that V-8 agar produced the best mycelial growth at 25?C, and that sporulation was induced after actively growing V-8 agar cultures were transferred to water agar. In order to select the most appropriate management options for plant diseases, pathogens must be identified correctly and quickly. High-fidelity PCR greatly increased the ability to detect P. odontoglossi (Prill & Delacr.) U. Braun, Dickeya spp., and Sclerotium rolfsii Sacc. directly from plant tissue and could be useful in managing diseases with long latent periods, difficult to recover pathogens, or ambiguous symptoms. Improved control options were developed for Boisduval scale and a flat mite, two important arthropod pests that can be difficult to control. Silwet L-77 (0.05%), an organosilicone adjuvant, increased the efficacy of 3 horticultural oils and significantly improved control of these pests. Information from this work was provided to orchid growers in six extension bulletins, diagnostic guides, and disease notes.
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 Robert Cating.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Palmateer, Aaron J.
Local: Co-adviser: Hoy, Marjorie A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041471:00001


This item has the following downloads:


Full Text

PAGE 1

1 TOWARDS ORCHID IPM: TOOLS AN D MOLECULAR TECHNIQUES FOR THE DIAGNOSIS AND MANAGEMENT OF SELECTED ORCHID ARTHROPOD PESTS AND DISEASES IN FLORIDA By ROBERT ALLEN CATING 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 2010

PAGE 2

2 2010 Robert Allen Cating

PAGE 3

3 To my Grandmother, who always believed in me

PAGE 4

4 ACKNOWLEDGEMENTS There have been many people who have had an extraordinary influence on my research, the writing of this dissertation, and my developmen t as a scientist, most notably my committee members Dr. Aaron Palmateer, Dr. Marjorie Ho y, Dr. Randy Ploetz, Dr. Robert McMillan, and Dr. Wagner Vendrame. I found that my comm ittee was superbly balanced between those members who fostered my desire to get as much diagnostic, extension, and field experience as possible and those who guided me through daily laboratory methods, procedures, and lab management. In addition to my committee memb ers, there were other faculty and staff who were crucial for the completion of this dissertation. Dr. Carol Stiles and Patti Rayside taught me laboratory procedures for working with fungi, Dr. Jeff Jones, Ellen Dickstein, and Jason Hong assisted me frequently with bacteriology, Dr Jane Polston answered my questions about virology, and Dr. Janice Uchida, at the University of Hawaii, an swered my questions directly related to orchid pathology. Dr. Ayyamperumal Jeyaprakash, who was always patient, assisted me in the lab on a daily basis teaching me molecular biology and phylogenetic analysis procedures. There were several commercial orchid gr owers who donated time and plants, and I am sincerely grateful for the time and interest each of them put into assist ing me with my work: Kerry Herndon, Bill Peters, Dr. Martin Motes, Duane and Donna Goodwin, Suzanne Farnsworth, Robert Fuchs, Michael Coronado, Jose Esposito, a nd Jack Batchelor. I discovered early in my research that it was going to be difficult to obtai n funding to perform orchid related research. I express my sincere gratitude to the Redland Orchid Festival, In c. and the Redland Professional Orchid Growers for coming forward a nd providing the funding for my work.

PAGE 5

5 Also, there were people who supported me in di fferent ways at different times, and I am grateful for their assistance: Roger, Jesse, and Cielo Shannon, Heidi Bowman, Amit Gupta, Mauro Tudares, Rio St. John, Tara Tarnowski, Asha Brunings, Linley Dixon, and Dalia and Robert Stubblefield.

PAGE 6

6 TABLE OF CONTENTS page LIST OF TABLES .........................................................................................................................10LIST OF FIGURES .......................................................................................................................11ABSTRACT ...................................................................................................................... .............13 CHAP TER 1 LITERATURE REVIEW .......................................................................................................15Introduction .................................................................................................................. ...........15Integrated Pest Management ...................................................................................................18What is Integrated Pest (Disease) Management? ............................................................ 18Management Tactics of an Integr ated Pest Management Program .................................20Mechanical and physical control ..............................................................................21Cultural control ........................................................................................................22Chemical control ......................................................................................................22Biological control .....................................................................................................23Research Objective .................................................................................................................24Chapter 2: Identification and Characterizati on of a Soft-Rot Pathogen Isolated from Vanda, Phalaenopsis Oncidium and Tolumnia Orchids in Florida...................................25Objectives .................................................................................................................... ...........27Chapter 3: Culture of Pseudocercospora Species that Affect Orchids .................................. 28Taxonomy and identification ................................................................................... 28Molecular systematics .............................................................................................. 29Growth and sporulation of Ce rcosporoid fungi in culture ....................................... 30Objective ..................................................................................................................... ............31Chapter 4: A Comparison of the Standa rd and High-Fidelity Polymerase Chain Reactions (PCR) in the Detection of Pseudocercospora odontoglossi from Cattleya Orchids and its Use in the Dia gnosis of Orchid Diseases ................................................... 31High-fidelity PCR ....................................................................................................32Objectives .................................................................................................................... ...........33Chapter 5: The use of Silwet L-77 as an Integrated Pest Management Tool ......................... 34Objectives .................................................................................................................... ...........35Appendices .................................................................................................................... .........352 IDENTIFICATION AND CHARACTERIZAT ION OF A S OFT-ROT PATHOGEN ISOLATED FROM Vanda, Phalaenopsis, Oncidium AND Tolumnia ORCHIDS IN FLORIDA ....................................................................................................................... ........39Introduction .................................................................................................................. ...........39Materials and Methods ...........................................................................................................41Isolation and Initial Tests ................................................................................................41Fatty Acid Analysis ......................................................................................................... 42

PAGE 7

7 Carbohydrate Utilization Tests ........................................................................................ 42Molecular and Phylogenetic Analysis ............................................................................. 42Pathogenicity Tests ..........................................................................................................45Results .....................................................................................................................................46Isolation and Identification of Dickeya Strains ............................................................... 46Fatty Acid Analysis ......................................................................................................... 46Carbohydrate Utilization Tests ........................................................................................ 46Molecular and Phylogenetic Analysis ............................................................................. 47Pathogenicity Tests ..........................................................................................................48Discussion .................................................................................................................... ...........483 CULTURE OF Pseudocercospora S PECIES THAT AFFECT ORCHIDS .......................... 80Introduction .................................................................................................................. ...........80Materials and Methods ...........................................................................................................81Growth and Sporulation of P. dendrobii on Different Media ......................................... 81Effects of Temperature on Gr owth and Sporulation of Pseudocercospora spp. on V-8 Agar ......................................................................................................................83Effects of Nutrient Reduction on Sporulation of Pseudocercospora sp. from a Tolumnia Orchid ..........................................................................................................83Results .....................................................................................................................................84Growth and Sporulation of P. dendrobii on Different Media ......................................... 84Effects of Temperature on Grow th and Sporulation of three Pseudocercospora spp. on V-8 Agar .................................................................................................................85Effects of Nutrient Reduction on Sporulation of Pseudocercospora sp. from a Tolumnia Orchid ..........................................................................................................85Discussion .................................................................................................................... ...........854 A COMPARISON OF THE STANDARD AND HIGH-FIDELITY POLYMERASE CHAIN REACTIONS (PCR) IN THE DETECTION OF Pseudocercospo ra odontoglossi FROM Cattleya ORCHIDS AND ITS USE IN THE DETECTION OF Sclerotium rolfsii AND A Dickeya sp. FROM Phalaenopsis ORCHIDS .............................. 91Introduction .................................................................................................................. ...........91Materials and Methods ...........................................................................................................92Evaluation of High-Fidelity PCR by Use of Plasmids .................................................... 92Deoxyribonucleic acid (DNA) extraction ................................................................ 92High-fidelity PCR protocol ...................................................................................... 93Molecular Cloning ...........................................................................................................94Comparison of Standard and High-Fidelity PCR ............................................................ 95Comparison of the High-Fidelity and Standard PCR in Detecting Sclerotium rolfsii and Dickeya sp. (Erwinia chrysanthemi) from Phalaenopsis Orchids ........................ 95Results .....................................................................................................................................97Evaluation of High-Fidelity PCR by Use of Plasmids .................................................... 97Comparison of the High-Fidelity and Standard PCR in Detecting Sclerotium rolfsii and Dickeya sp (Erwinia chrysanthemi) from Phalaenopsis Orchids ........................ 97Discussion .................................................................................................................... ...........98

PAGE 8

8 5 SILWET L-77 IMPROVES THE EFFICACY OF HORTI CULTRAL OILS FOR CONTROL OF BOISDUVAL SCALE Diaspis boisduvalii (HEMIPTERA: DIASPIDIDAE) AND THE FLAT MITE Tenuipalpus pacificus (ARACHNIDA: ACARI: TENUIPALPIDAE) ON ORCHIDS ...................................................................... 103Introduction .................................................................................................................. .........103Materials and Methods .........................................................................................................105Phytotoxicity Study .......................................................................................................105Silwet + Oil Efficacy Trial (Boisduval Scale) ...............................................................106Silwet + Oil Efficacy Trial (Flat Mite) .......................................................................... 107Results ...................................................................................................................................108Phytotoxicity Study .......................................................................................................108Silwet + Oil Efficacy Trial (Boisduval Scale) ...............................................................108Silwet + Oil Efficacy Trial (Flat Mite) .......................................................................... 108Discussion .................................................................................................................... .........1096 SUMMARY AND CONCLUSIONS ...................................................................................1197 REFLECTIONS ................................................................................................................... .123 APPENDIX A PHYSIOLOGICAL DISORDERS OF ORCHIDS: OEDEMA ........................................... 125Symptoms ...................................................................................................................... .......125Diagnosis ..............................................................................................................................125Management .................................................................................................................... .....126B PHYSIOLOGICAL DISORDERS OF ORCHID S: MESOPHYLL CELL COLLAPSE ....131Symptoms ...................................................................................................................... .......131Cause, Diagnosis, and Control ..............................................................................................131C BLACK ROT OF ORCHIDS CAUSED BY Phytophthora palmivora AND Phytophthora cactorum: SYMPTOM S, DIAGNOSIS, AND MANAGEMENT ................ 135Host Range ............................................................................................................................135Symptoms ...................................................................................................................... .......135Diagnosis ..............................................................................................................................136Management .................................................................................................................... .....136Nursery Sanitation Recommendations for Phytophthora ..............................................136Fungicide Options .........................................................................................................137Growing Media and Storage ..........................................................................................137Containers .................................................................................................................... ..137Bench Sanitation ............................................................................................................138Water Supply and Hand Watering .................................................................................138New Plants Brought into the Nurser y, Greenhouse, or Growing Area ......................... 138

PAGE 9

9 D FIRST REPORT OF Sclerotium rolfsii ON Ascocentrum AND Ascocenda ORC HIDS IN FLORIDA ........................................................................................................................141Identification ................................................................................................................ .........141Pathogenicity Tests ...............................................................................................................141E A DIAGNOSTIC GUIDE FOR BLACK RO T OF ORCHIDS CAUSED BY Phytophthora palmivora AND Phytophthora cactorum FOR PLANT DIAGNOSTIC CLINICS AND DIAGNOSTICIANS .................................................................................. 144Introduction .................................................................................................................. .........144Disease ..................................................................................................................................145Pathogen ...................................................................................................................... .........145Taxonomy ...................................................................................................................... .......145Symptoms and Signs ............................................................................................................ .146Host Range within the Orchidaceae ..................................................................................... 146Geographic Distribution ....................................................................................................... 146Pathogen Isolation ................................................................................................................146Pathogen Identification .........................................................................................................147Molecular Diagnostics ......................................................................................................... .148Pathogen Storage ..................................................................................................................148Pathogenicity Tests ...............................................................................................................149F FIRST REPORT OF BACTERIAL SOFT ROT ON Tolumnia ORCHIDS CAUSED BY A Dickeya sp. .................................................................................................................152Identification ................................................................................................................ .........152Pathogenicity Tests ...............................................................................................................153REFERENCES .................................................................................................................... ........155BIOGRAPHICAL SKETCH .......................................................................................................175

PAGE 10

10 LIST OF TABLES Table page 1-1 Pseudocercospora and Cercospora species repo rted to occur on orchids and their common hosts. ...................................................................................................................38 2-1 Orchid isolate collection information and initial tests for Dickeya ( Erwinia) genus determination. ................................................................................................................ ....75 2-2 16S rDNA GenBank BLAST search and MIDI results for orchid strains ..................... 77 2-3 Characteristics that differentiate Dickeya sp. as described by Samson et al., (2005) compared to the Dickeya spp. isolated from orchids used in this study and D. chrysanthemi reference strain ATCC #11663.. .................................................................. 78 2-4 Carbohydrate uti lization test results for Dickeya spp. isolated from orchids used in this study compared to reference strains Dickeya chrysanthemi Erwinia carotovora carotovora ( E. c. c.) and Pectobacterium cyprepedii ........................................................79 5-1 Pre-spray and post-spray quality ratings (range=1-10) of orchids used in the phytotoxicity study...........................................................................................................113 5-2 Evaluation of Silwet L-77 (0.05%) in combination with two oils for control of Boisduval scale infestations in Cattleya mericlone orchids. ........................................... 114 5-3 Evaluation of Silwet L-77 (0.05%) in combination with Prescription Treatment Ultra Pure Oil for control of flat mite ( Tenuipalpus pacificus ) infestations in Dendrobium or Grammatophyllum orchids. ....................................................................115

PAGE 11

11 LIST OF FIGURES Figure page 1-1 Examples of economically important orchid s grown as ornamental plants or for cut flowers................................................................................................................................361-2 Difficulties in plant disease diagnosis. ............................................................................... 372-1 ClustalX 16S rDNA alignment of 18 Dickeya isolates obtained from orchids using primers pair 27f/1495r ....................................................................................................... 522-2 Clustal X DNA alignment of a portion of the pectate lyase gene cluster of orchid Dickeya isolates using primer pair ADE1/ADE2 and a portion of the pectate lyase gene cluster from four GenBank Dickeya species accessions. ..........................................682-3 Maximum likelihood phylogenetic analysis of Dickeya spp. orchid strains based on 16S rDNA gene sequences.................................................................................................712-4 Bayesian phylogenetic analysis of Dickeya spp. based on 16S rDNA gene sequences. ... 722-5 Maximum Likelihood phylogenetic analysis of Dickeya spp. strains based on a portion of the pelADE pectolytic enzyme gene cluster. ....................................................732-6 Bayesian analysis of Dickeya spp. based on a portion of the pelADE pectolytic enzyme gene cluster. .......................................................................................................... 743-1 Growth of Pseudocercospora dendrobii on 10 different media.. ......................................893-2 Measurements of culture diameter at 3, 7, 11, and 13 d at 15, 20, 25, and 30 1C on V-8 agar. ............................................................................................................................904-1 A comparison of high-fidelity and sta ndard PCR using plasmid pRC17 containing the 571-bp ITS1, 5.8S, and ITS2 rDNA of Pseudocercospora odontoglossi as a template and primers ITS4/ITS5. ..................................................................................... 1014-2 A comparison of high-fidelity and standa rd PCR in the detection of two important orchid pathogens from five inoculated Phalaenopsis plants. .......................................... 1025-1 Examples of orchids used in the phytotoxi city study after post-spray quality rating. None of the orchids developed symptoms of phytotoxicity on leaves, stems, flowers, buds or roots. ....................................................................................................................1165-2 Cattleya mericlone orchids before treatment with water, petroleum oil, or Silwet L77 + petroleum oil ............................................................................................................ 1175-3 Cattleya mericlone orchids infested with Boisdu val scale 1 wk after second treatment 118A-1 Examples of oedema on various orchids. ........................................................................ 127

PAGE 12

12 A-2 Examples of oedema on various orchids. ........................................................................ 128A-3 Diagnosing oedema in orchids. ........................................................................................ 129A-4 Differentiating scale insect s from oedema blisters .......................................................... 130B-1 Examples of mesophyll cell collapse in orchids .............................................................. 134C-1 Black rot of orchids, symptoms and signs. ...................................................................... 140D-1 Ascocenda orchids with Southern blight caused by S. rolfsii ..........................................143E-1 Black rot of orchids, symptoms and signs ....................................................................... 150E-2 A comparison of sexual reproductive structures of P. palmivora and P. cactorum .......151F-1 Tolumnia orchids with symptoms of a soft rot bacterial infection caused by Dickeya sp. ........................................................................................................................... ..........154

PAGE 13

13 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 TOWARDS ORCHID IPM: TOOLS AN D MOLECULAR TECHNIQUES FOR THE DIAGNOSIS AND MANAGEMENT OF SELECTED ORCHID ARTHROPOD PESTS AND DISEASES IN FLORIDA By Robert Allen Cating May 2010 Chair: Aaron Palmateer Cochair: Marjorie A. Hoy Major: Plant Pathology The Orchidaceae is believed to be the largest fa mily of flowering plants, with an estimated 19,000 species. Orchid sales in the United St ates have increased steadily since 1997, and exceeded 140 million US dollars in 2006. Orchid s are the second most economically important flowering plant produced in the United States, an d Florida is one of th e top producers in the nation. Orchids are sensitive to chemi cal pesticides. Thus, it would be desirable to establish an integrated pest management (IPM) program in order to reduce arthropod pest and disease problems, while minimizing the impact of the co ntrol measures on non-target organisms (natural enemies), humans, and the environment. Currently, no comprehensive IPM programs exist for orchid production. The overall objective of this dissertation was to develop new diagnostic procedures and control strategies for several important orchid pe sts and pathogens that would be compatible with, and could be used to esta blish, an overall IPM pr ogram for this crop. Commercial orchid growers a nd hobbyists in Florida were as ked what insect and disease problems were frequently encountered and whic h were considered the most severe. They

PAGE 14

14 indicated that bacterial soft-rot diseases caused by Dickeya spp., leaf spots caused by Pseudocercospora spp., and infestations of Boisduval scale, Diaspis boisduvalii Signoret, were the most problematic. Therefore, these problem s were primary focuses of this dissertation. Eighteen bacterial isolates were collected from orchids with soft-rot disease symptoms and, through the use of biochemical te sts, carbohydrate utiliza tion tests, fatty-acid analysis and 16S rDNA and pelADE gene sequences, isolates were determ ined to be distinct from currently identified species of Dickeya ; they may represent new species. Pseudocercospora spp. cause leaf spots in several orchid taxa that can be severe. A first step towards understanding these diseases is to culture the causal fungi and produce conidia for pathogenicity tests, which could be used later for studies on host range and epidemiology. It was determined that V-8 agar produced the best mycelial growth at 25 C, and that sporulation was induced after actively growing V-8 agar cultures were transferred to water agar. In order to select the most appropriate management options for plant diseases, pathogens must be identified correctly and quickly. High-fidelity PCR gr eatly increased the ability to detect P. odontoglossi (Prill & Delacr.) U. Braun, Dickeya spp., and Sclerotium rolfsii Sacc. directly from plant tissue and c ould be useful in managing dis eases with long latent periods, difficult to recover pathogens, or ambiguous symptoms. Improved control options were developed for Boisduval scale and a flat mite, tw o important arthropod pests that can be difficult to control. Silwet L-77 (0.05%), an organosi licone adjuvant, increased the efficacy of 3 horticultural oils and significantly improved control of these pests. Information from this work was provided to orchid growers in six extensi on bulletins, diagnostic guides, and disease notes.

PAGE 15

15 CHAPTER 1 LITERATURE REVIEW Introduction The Orchidaceae is believed to be the largest fa mily of flowering plants (Bechtel et al. 1992; Dressler 1993; Tsavkelova et al. 2008) with estimations of around 19,000 species (Atwood 1986; Dressler 1993). Orchids are grown for the cut fl ower trade, as potted plants, as landscape and garden specimens, and they inspire the creation of art, jewelry, stamps and literature (Arditti 1992) (Figure 1-1). According to the USDA Ec onomics, Statistics, and Market Information System, orchid sales in the United States have increased steadily since 1997 and sales were estimated to exceed 140 million US dollars in 20 06 (Jerardo 2006). Orchids are the second most economically important flowering plant produ ced in the United States, and Florida and California are the top producing states (Jerardo 2006). Although poinsetti as earn the largest dollar amount in sales, orchids have shown the gr eatest rate of growth in sales (Jerardo 2006). Statistics from the Aalsmeer flower auction in the Netherlands show that Phalaenopsis orchids comprised 5% of the market in 1983, and 66% in 1994 (Griesbach 2000). It is clear that the popularity of orchids is growing as more people discover these in triguing, beautiful and, in many cases, long-lasting flowers. In addition to being grown as ornamental plants, other uses of orchid flowers and tubers do exist. Vanilla planifolia Andrews has been used for cen turies as a flavoring agent, Laelia autumnalis Lindl. and Laelia grandiflora (La Llave & Lex.) Lind. are used to make candies and pastries in Mexico, an d the dried tubers of Orchis tridentate Muhl. ex Willd. have been used to make salep a starch meal (Arditti 1992). Orchids also have been used in herbal and traditional medicines in many parts of the world (Ardit ti 1992; Kong et al. 2003). Orchids used in traditional medicine include Bletilla striata [Thunb.] Rchb.f. (used to treat tuberculosis,

PAGE 16

16 hemoptysis, gastric and duodena l ulcers, and cracked skin), Gastrodia elata Blume (headaches, dizziness, blackouts, numbness of the limbs, epilepsy, spasms, and migraines), Epidendrum spp. (sores of the lips), and Oncidium spp. (lacerations) (Kong et al. 2003). Recent investigations into the chemical com pounds found in orchids have yielded surprising results. Moscatilin, a bibenz yl derivative derived from Dendrobium loddigesii Rolfe, has been shown to be an antiplatlet agent (Chen et al 1994) and, more recently, mosactilin, derived from D. loddigessi was reported to have an antiproliferat ive effect against certain cancer cells (choriocarcinoma, lung cancers, and stomach cancers) (Ho & Chen 2003). Anti-fungal compounds have also been isolat ed and characterized from seve ral different orchids. For example, the achlorophyllous orchid Gastrodia elata Blume forms a parasitic relationship with the fungus Armillaria mellea (Vahl: Fr.) Kummer. Gastrodia elata produces an antifungal compound known as gastrodianin, which is used by the orchid to control Armillaria penetration and sequester the fungal hyphae to the cortical layer where dige stion of the hyphae and nutrient absorption occurs (Wang et al. 2001). Gastrodi anin also has been shown to confer disease resistance to several root pathogens of tobacco ( Nicotiana tabacum L. cv. Wisconsin) after Agrobacteriummediated transformation with GAFP-1 (gastrodianin), illustrating the potential for its use in the development of disease-tolerant plants (Cox et al. 2006). Two other antifungal compounds, lusianthrin and chrysin, have been isolated from the orchid Cypripedium macranthos Sw. var. rebunense (Kudo) Miyabe et Kudo and s hown to be important during different developmental stages of th e plant (Shimura et al. 2007). Other novel compounds have been described. Habe nariol, isolated from the aquatic orchid Habenaria repens Nutt., deters feeding by freshwater cr ayfish (Wilson et al. 1999), gymnopusin from Maxillaria densa Lindl. is phytotoxic to duckweed (Valen cia-Islas et al. 2002), and two

PAGE 17

17 stilbenoids from Scaphyglottis livida (Lindl.) Rauschert have a vasorelaxation effect in rats (Estrada-Soto et al. 2006). Several other phytochemi cals have been isolated from orchids used in traditional medicines and are currently being inve stigated (Kovcs et al. 2008). These include phenanthrenes, dihydrophenanthrenes bibenzyls, lectin, chalcone dihydrochalcone derivatives, thunalbene, phenanthropyran, and dimeric phenanthrenes (Majumder et al. 1994, 1995, 1998; Zenteno et al. 1995; Leong et al. 1997, 1999; Majumder & Majumder 1999, 2001; Manako et al. 2001). It appears that the popula rity of orchids as ornamentals and their potential as sources of novel chemical compounds will assure a place for the orchid industry in the United States and other parts of the world. As orchid popularity cont inues to grow, so will the desire for pest and disease information. For example, th e question and answer section of Orchids magazine, published by the American Orchid Society, freq uently contains questions submitted by hobbyists and commercial growers concerning the identification and control of orchid pests and diseases. When compared to many other horticultural crops, very little is know n about orchid pests and diseases. With the exception of orchid viruses (Hu et al. 1993, 1994, 1995; Fry et al. 2004; Jones 2005; Khentry et al. 2006; Zheng et al. 2008a, 2008b; Vaughan et al. 2008), the scientific literature contains limited information concerning pathogenicity tests, reports of new diseases, and studies involving host ranges of most orchid pathogens and methods of diagnosing orchid pest and disease problems; however, some work ha s been done. In addition to viruses, other economically important diseases affecting orchids include rusts (Melndez & Ackerman 1993, 1994; Pereira & Barreto 2004); vascular wilts caused by Fusarium spp. (Burnett 1965); leaf spots caused by Cercospora/ Pseudocercospora spp. (Crous & Braun 2003) and Phyllosticta spp. (Uchida & Aragaki 1980; Uchida 1994), Fusarium subglutinans (Wollenweb & Reinking) P. E.

PAGE 18

18 Nelson, T. A. Tousson, & Marasas (syn. Fusarium moniliforme J. Sheld, var. subglutinans Wollenweb & Reinking) and F. proliferatum (Matsushima) (Broadhurst & Hartill 1996; Ichikawa & Aoki 2000)); s outhern blight caused by Sclerotium rolfsii Sacc. (Bag 2004; Cating et al. 2009a, see Appendix D); bacterial so ft rots and leaf spots caused by Dickeya sp (Cating et al. 2008, 2009b, see Appendix F), Pectobacterium carotovora (Jones 1901) Waldee 1945 emend. Hauben et al. 1998, (Liau et al. 2003; Chan et al. 2005 ; Sjahril et al. 2006), Erwinia cyprepedii (Hori) Bergey et al. and Acidovorax spp.; and root, stem, leaf, and pseudobulb rots, commonly called black rot, caused by Phytophthora spp. (Uchida 1994; Ilieva et al. 1998; Cating et al. 2009, see Appendices C and E). In addition, there are several physiological problems, such as oedema and mesophyll cell collapse, which can perplex the hobbyist or commercial grower (Cating et al. 2007; Cating & Palmateer 2009; see Appendices A and B). Orchid disease information has been compiled in book chapters for commercial growers and hobbyists (Ark 1959; Hadley et al. 1987). Burnett (1965) described some of the most important diseases of orchids, while the American Orchid Society has published a small handbook on orchid diseases written for hobbyists (American Orchid Society 2002) However, alternatives to chemical control are rarely discussed, and no integr ated pest management program is available. Integrated Pest Management What is Integrated Pest (Disease) Management? Integrated pest management (IPM) and inte grated disease management (IDM) are control strategies that seek to redu ce or avoid plant damage by arthropod pests or diseases while minimizing the impact of the control measures on non-target organisms (e .g. beneficial insects), humans, and the environment by utilizing control tact ics that disrupt natural control factors (i.e. natural enemies) as little as possible (Stern et al. 1959; Flint & van den Bosch 1981; Dreistadt 2001). As their names suggest, these strategies seek to integrate multiple tactics to reduce pest

PAGE 19

19 populations or diseases to accepta ble levels without relying solely on chemical control. Although IDM has been used when specific refere nce is made to disease management, plant pathogens also have been considered pests and IPM includes both ar thropods and pathogens. True IPM must consider information from multiple sources, such as from economists, horticulturalists, and entomologist s, and the role of plant pathol ogy in IPM must be that of a partner within the entire IP M team (Jacobsen 1997). Geier (1966) described traditiona l pest control as hardly more than bulldozing nature without thought of consequences and pest management as acceptance of the continued existence of potentially harmful species, albeit at tolerable levels of abundance. Of integrated control, Geier stated that it differs in essence from previous nomenclature in that it refers not to the nature and technology of means, but to a way of using th em and integrated control is intended to imply some degree of discriminatio n in the conventional use of pesticides. At a meeting in Rome, Italy in 1975, the Food and Agriculture Organization defined IPM as a pest management system that, in the context of the associated environment and the population dynamics of the pest species, utilizes all suita ble techniques and methods in as a compatible manner as possible and maintains the pest popu lation at levels below those causing economic injury (Food and Agriculture Organization 1975). The first steps in establishing an IPM progr am should focus on understanding the biology of the crop and the identification of associated arthropods pests and pathogens, the influence of the surrounding ecosystem on the crop and pests, and th e identification of no nchemical preventative actions (Flint & van den Bosch 1981; Dreistadt et al. 2001). Drei stadt et al. (2001) described five key components of a successful IPM program: 1. preventing problems; 2. regular monitoring of crops and growing areas for pests;

PAGE 20

20 3. accurate diagnoses of pest problems; 4. development of control action guidlines; and 5. the use of effective management tools. A successful IPM program integra tes these components into a workable, sustainable system to prevent pest and disease outbreaks and keep dama ge at acceptable levels when they do occur. Management Tactics of an Integrated Pest Management Program No single pest management tactic will comple tely control every pest problem (Flint & van den Bosch 1981; Dreistadt et al. 2001; Bethke & Cloyd 2009). Therefore, multiple control tactics are required to adequately control all pe sts and diseases on a give n crop (Flint & van den Bosch 1981; Hoy 1994). An important decision-making tool in IPM programs for crops that can tolerate some pest damage is an economic injury level (EIL), whic h is the threshold at wh ich yields are reduced (Stern et al. 1959; Flint & van den Bosch 1981; Luckmann & Metcalf 1994; Peterson & Hunt 2003; Bethke & Cloyd 2009). However, EILs are le ss useful for ornamental crops since they are sold based on aesthetic appearance and, theref ore, have virtually no tolerance for pest and disease damage (Bethke & Cloyd 2009). Another crucial component of an IPM program is the accurate identification of the pest or pathogen (Flint & van den Bosch 1981; Dreistadt et al. 2001; Flint & Gouveia 2001). In contrast to arthropod identification, whic h in many cases is possible with only morphological criteria, plant disease diagnoses usually re quire several steps, including is olation of the pathogen in pure culture followed by pathogen identification with morphological characteris tics (fungi), biological tests (bacteria) or other crit eria (viruses) (Louws et al. 1999; Alvarez 2004; Daughtrey & Benson 2005; Finetti Sialer & Rosso 2007) These procedures are labor intensive and time consuming,

PAGE 21

21 and identifications require highly trained personnel. In some cases, species determinations can only be made by taxonomic specialists. In addition, some pathogens are obligate parasites (i.e. plant viruses, some bacteria and fungi) or difficult to grow on artificial media. Diagnosis can be further complicated by the fact that different diseases may cause similar symptoms. For example, bacterial soft-rots caused by Pectobacterium carotovora subsp. carotovora or Dickeya chrysanthemi (Burkholder et al. 1953) Samson et al. 2005 can sometimes be confused with early symptoms caused by the fungus Sclerotium roflsii (Figure 1-2). The identification of organisms that produce similar symptoms or those that cannot be cultured can be greatly aided by molecular diagnostics techniques, particular the polymerase chain reaction (PCR) (Vincelli & Ti sserat 2008). Only after an accurate diagnosis is obtained can a management plan be established (Alvarez 2004; Daughtrey & Benson 2005). Since integrated, multiple control tactics are most effective, management plans should include mechanical, physical, cultural, and chemical contro l strategies (Stern et al. 1959; Flint & van den Bosch 1981; Louws et al. 1999; Dreistadt et al. 2001; Daughtrey & Benson 2005). Traditional management terms used in pl ant pathology include a voidance, exclusion, protection, eradication, and resist ance. Plant disease management uses different terminology, but tactics for disease control can be classified into the same categories as those used for entomology. For example, exclusion, which is the most cost-effective management strategy (Ploetz 2007), could also be considered a type of mechanical, physical, or cultural control. Mechanical and physical control Flint & Gouveia (2001) define mechanical a nd physical control t actics as those that mechanically destroy pests or introduce a barrie r to prevent an infestation. Mechanical and physical control tactics take advantage of weak links in the pests life cycle or behavior patterns to reduce or prevent infestations (F lint & van den Bosch 1981). Common types of

PAGE 22

22 mechanical and physical control tactics include using light traps, and the creation of barriers, such as the addition of screens. For a review of physical control options see Vincent et al. (2003). Cultural control In contrast to mechanical and physical control tactics, which are implem ented specifically for pest control, cultural control tactics are modi fications of current pr actices that alter the environment, making it less favorable for pest s or diseases (Flint & van den Bosch 1981). Cultural control tactics include sanitation, wate r and fertilizer management, crop rotation, and harvesting practices (for example, using a clean razor blade or flamesterilized tool to harvest cut flowers to prevent virus transmission) (Flint & van den Bosch 1981). For orchids, cultural practices are crucial for orchid health and incl ude using the correct type of potting mix and pot size, and appropriate humidity a nd light duration/intensity. Due to the development of pesticide resistance or absence of effective chemicals, cultural control options have become more important as a pest management tactics a nd components of IPM programs (Flint & van den Bosch 1981). Chemical control Although chemical pesticides have been the pr imary method of pest control since the 1940s, associated ecological and safety concerns contin ue to rise (Lewis et al. 1997). Chemicals temporarily reduce pest populations but do not contribute to perman ent pest reduction (Stern et al. 1959). Stern et al. (1959) listed problems that were associated with traditional pesticides: 1. the development of resistance; 2. secondary outbreaks of non-target arthropods; 3. elimination of non-target organisms, such as natural enemies, which may allow the pest population to quickly return;

PAGE 23

23 4. toxic residues on food and forage crops; 5. potential damage to human and animal health and the environment; 6. legal issues resulting from environmen tal contamination or pesticide drift. The overall goal of an integrated program shoul d be to choose a chemical that can be fit into an ecological system, rather than imposi ng insecticides on the ecosystem (Stern et al. 1959). By understanding that the control of arthropod pests is a complex ecological problem, we may consider the effects of an insecticide on all the arthropods in a system, not just the target organism. The development of resistance can render pes ticides virtually useless against certain organisms (Brent 1995; Hoy 1995). Brent (1995) states that in or der to reduce the development of pesticide resistance, one should rotate pest icides, limit treatments, use recommended label rates, and integrate with non-chemical control methods which should include physical, cultural, and biological c ontrol tactics. Biological control Stern et al. (1959) indicated th at biological control and chemi cal control are not necessarily alternative objectives; in ma ny cases they may be complementary, and, with adequate understanding, can augment each other. Biological control can be summarized as the use of natural enemies to lower pest populations (S tern et al. 1959; Flin t & van den Bosch 1981; Huffaker 1985; Hajek 2004). Flin t & van den Bosch (1981) describe d three aspects of biological control: (1) naturally occurring bi ological control, (2) classical biological control, and (3) preservation of native, natural enemies. Natura lly occurring biological c ontrol is constantly at work to some degree, but may not be effective when a pest is introduced into a new area where natural suppressive factors are absent. In clas sical biological contro l, natural enemies are deliberately introduced into an area to reduce a pest population.

PAGE 24

24 After the identification of the pest and its origin is determined, a search for natural enemies in the pests native habitat is conducted and potential natural enemies are identified. After extensive laboratory, greenhouse, and field trials, the natural enemies may be released and reestablish the pest-natural enemy relationship (F lint & van den Bosch 1981; Dreistadt 2001). For reviews of biological control con cepts and applications, see Ster n et al. (1959), Flint & van den Bosch(1981), Huffaker (1985), Hoy (1994), Price (2002), and Hajek (2004). Preservation of natural enemies typically involves using nontoxic pesticides or by modifying the environment in other ways for their maintanence. Biological control of plant disease also can be an important component of an integrated management plan. Organisms used in biol ogical control of plan t pathogens use such mechanisms as antibiosis (production of a chem ical by one organism that directly inhibits another), parasitism (reduction of a plant pat hogen by another pathogen), competition (biological control agent and pathogen compete for resour ces), and the stimula tion of plant defense mechanisms by a hypovirulent strain of a pathoge n. Although the goal of biological control in entomology is to reduce pest populations, it is disease reducuction in plant pathology. Thus, reducing populations of a given pa thogen may not be sufficient to achieve this goal (Bull 2001). Research Objective The overall objective of this research is to develop new diagnostic procedures and control strategies for several important orchid pests and pathogens that are compatible with an overall IPM program. Currently, no comprehensive IPM programs exist for orchid production; therefore, this dissertation de scribes tools that can be used in the establishment of an IPM program. Several commercial growers and hobbyists around the state of Florida were contacted and asked what orchid insect and di sease problems they frequently en counter and consider the most

PAGE 25

25 severe. Through these consultations, it was determ ined that soft-rot diseases, leaf spots caused by Pseudocercospora spp., and infestations of Boisduval scale, Diaspis boisduvalii Signoret, were considered to be the most problematic; they were the targets of the described research. In addition, high-fidelity PCR was examined as a plant-disease diagnosti c tool. The specific objectives of each chapter are discussed below: Chapter 2: Identification and Characteriza tion of a Soft-Rot Pathogen Isolated from Vanda Phalaenopsis Oncidium and Tolumnia Orchids in Florida Pectolytic enzyme-producing bacteria can be devastating plant pa thogens. Pectolytic enzymes (pels) macerate plant tissue by degradin g plant cell-wall components which leads to soft, water-soaked lesions that can enlarge rapidly (Prombe lon & Kelman, 1980; Kotoujansky 1987; Barras et al. 1994; Kazemi-Pour et al. 2004) Although many bacteria produce pectolytic enzymes, most that cause soft-rot dis eases in plants belong to the genera Erwinia Pectobacterium or Dickeya (Dickey 1979; Kotoujansky 1987; Ay san et al. 2003; Gardan et al. 2003; Samson et al. 2005; Pitman et al. 2008). Three species cause disease in orchids: Pectobacterium cyprepedii (formerly Erwinia cyprepedii ), Pectobacterium carotovora (formerly Erwinia carotovora), and Dickeya spp (formerly Erwinia chrysanthemi) (Ark 1959; Burnett 1965; Bradbury 1986; Hadley et al. 1987; Liau et al. 2003; Chan et al. 2005; Sjahril et al. 2006; Cating et al. 2008, 2009b). Pectobacterium cyprepedii is a serious pathogen of ladys slipper orchids ( Cyprepedium Paphiopedilum and Phragmipedium ), but has been reported to cause brown rots on other orchids as well (Phalaenopsis Aerides Catasetum ) (Ark 1959; Bradbury 1986; Alfieri et al. 1994). Although P. cyprepedii is closely related to the soft-rot causing P. carotovora it causes distinct symptoms. They start as small, round, water-soake d lesions and eventually turn dark brown and dry (Hadley et al. 1987). In contrast, P. carotovora causes a true soft-rot in which cell-wall

PAGE 26

26 degrading enzymes macerate tissue (Daniels et al. 1988). Pectobacterium carotovora causes soft-rots on many plants (Prombelon and Kelm an 1980; Schuerger and Batzer 1993; Chuang et al. 1999; Toth et al. 2003) and is a serious pathogen of many orchid species (Ark 1959; Burnett 1965; Bradbury 1986; Hadley et al. 1987; Liau et al. 2003; Chan et al. 2005; Sjahril et al. 2006). Symptoms can develop rapidly and are virtually indistinguis hable from those caused by Dickeya spp. Recently, Erwinia chrysanthemi (Burkholder et al. 1953) and Brenneria paradisiaca (Fernandez-Borrero and Lopez-Duque 1970) Hauben et al. 1999 were reexamined, reclassified, and divided into six taxa within the novel genus Dickeya (Samson et al 2005): D. dadantii Samson et al. 2005, D. chrysanthemi bv. parthenii Samson et al. 2005, D. chrysanthemi bv. chrysanthemi Samson et al. 2005, D. dieffenbachiae Samson et al. 2005, D. dianthicola Samson et al. 2005, and D. paradisiaca Samson et al. 2005 These divisions were based on DNA:DNA hybridization, serology, 16S rDNA seque nces, and phenotypic characters. Dickeya spp cause diseases in many different plant species (P rombelon & Hyman 1986; Hugouvieux-Cotte-Pattat et al. 1992; Yap et al. 2005; Lee & yu 2006) and are serious orchid pat hogens (Cating et al. 2009b). Since the 16S rDNA gene of Escherichia coli (Migula 1895) Castella ni and Chalmers 1919 was sequenced in 1972 (Ehresmann et al. 1972), it has been used exte nsively for bacterial identification (Stackebrandt & Goebel 1994; Kolbert & Persing 1999; Clarridge III 2004; Janda & Abbott 2007; Woo et al. 2008) an d phylogenetic analyses (Weisbur g et al. 1991; Ludwig et al. 1998; Kwon et al. 1997; Samson et al. 2005; Naum et al. 2008). Although 16S rRNA gene sequences are useful to identify st rains to the level of family or genus, they are often not useful in species identification (Naum et al. 2008; Staley 2009). For example, Fox et al. (1992)

PAGE 27

27 indicated that two closely related species that could be distinguished w ith phenotypic characters, Bacillus globisporus Larkin and Stokes 1967 and B. psychrophilus (ex Larkin & Stokes 1967) Nakamura 1984, nom. rev., had 99.8% similar 16S rDNA sequences. Very little is known about soft -rot causing bacteria in orch ids. Although soft-rot pathogens typically have wide host ranges, recent isolates from orchids have not fallen in previously described taxa. For example, Ca ting et al. (2009b) described a Dickeya sp. that caused soft-rot symptoms in Tolumnia orchids; fatty acids, 16S rDNA seque nces and biochemical tests did not place this pathogen in a described species. Similarly, a Dickeya sp. from Oncidium orchids in China was initially identified as D. chrysanthemi but 16S rDNA sequences demonstrated 98% similarity to D. dadantii and D. dianthicola (Li et al. 2009). Thus, a dditional tests or sequence analyses of more than the 16S rDNA gene may be needed to understand these new orchid pathogens (Kwon et al. 1997). Louws et al. (1999) described the three Ds of PCR based genomic analysis of phytobacteria as diversity, detection, and disease diagnosis. They indicated that an assessment of population genetic diversity was needed to establish a stable taxonomy; only then can new strains quickly be characterized and methods for diagnosis and management of new strains be developed. Objectives The objectives of this chapter we re to: (1) identify and characterize 18 isolates of a soft-rot bacterium isolated from four different orchid hosts, (Vanda, Phalaenopsis Oncidium and Tolumnia ) using standard biochemical tests as desc ribed by Schaad et al. (2001), fatty acid analysis, 16S rDNA sequences, and the pectate lyase coding gene cluster ( pel) (Nassar et al. 1996; Palacio-Bielsa et al. 2006); and (2) to analyze these isolat es phylogenetically with 16S rDNA and pel gene sequences.

PAGE 28

28 Chapter 3: Culture of Pseudocercospora Species that Affect Orchids Cercospora and related fungi ( Cercosporidium Pseudocercospora, Cercosporella etc.) cause diseases on leaves, stems, pedicels, fruit, and bracts on numerous plant species in many plant families (Chupp 1954; Deighton 1973, 1976, 1979; Ellis 1971, 1976). More than 3,000 cercosporoid species have been described (Solheim 1930; Chupp 1954; Deighton 1973, 1976, 1979; Ellis 1971, 1976) while 659 are currently recognized as valid species based on the reexamination of morphologica l characters (Crous & Braun 2003). In addition to causing diseases on important food, tree and ornamental crops (Crous 1998; Crous & Braun 2003), at least eight Cercospora or Pseudocercospora have been reported to caus e leaf spot diseases on at least 40 orchid genera. Pseudocercospora and Cercospora species that have been found on orchids and their common hosts are listed in Table 1-1. Taxonomy and identification Fresenius based the genus Cercospora on the species C. apii Fresen, Beitr., which is found on celery (Chupp 1954). In addition, Fresenius described three othe r species, but chose C. apii as the type species (Solheim 1930). Because the morphological characters used to identify these species are ambiguous (i.e. color of conidiophores and spores, char acteristics of hila, and other morphological characters), Fresenius and subseq uent taxonomists grouped species that did not truly have hyaline conidia like C. apii within the genus Cercospora (Solheim 1930). Taxonomists have sought to reclassify falsely identified Cercospora species in other genera based on conidial color (Ellis 1971). However, these reclassifications were based on other characteristics, such as presence or absence of external mycelium, simple or branched conidiophores, presence or absenc e of stromae and, if present, the type and shape of conidia (Solheim 1930). However, this system has not proven useful and has not been adopted by taxonomists (Deighton 1976; Crous & Braun 2003).

PAGE 29

29 Chupp (1954) listed 155 plant families that were infected by Cercospora species. He made generic divisions based on conidial morphology, color and septat ion, and used conidial width and base morphology for species determinations Deighton (1973, 1976) moved some previously described species of Cercospora to other genera such as Pseudocercospora Pantospora, Cercoseptoria, Cercosporella, Pseudocercospora, and Pseudocercosporidium For Deighton (1976), the most important character istic used for separating true Cercospora from closely allied genera was the hilum, or scar on the conidia and the scar on the coni diophores (conidiogenous loci). The presence and thickness of a hilum wa s used by Deighton to classify species without a thickened scar into Pseudocercospora, Pantrospora, Ce rcoseptoria, Pseudocercosporella Denticularia, and Mycocentrospora, removing them from Cercospora proper, which has a thickened, distinct hilum on the conidi a and conidiophores (Deighton 1973, 1976). Previously, many species of Cercospora were identified based on host associations. However, Groenewald et al. (2006) demonstrated that C. beticola Sacc. and C. apii were not completely host specific, as previously believed. Therefore, identifications of other cercosporoid fungi based on host may not be accurate. Molecular systematics As the use of molecular techniques to identi fy fungal pathogens becomes more common, it may be possible to use these techniques to identify Cercospora and Cercospora-like species and to develop new, more-accurate phylogenet ic trees. To correctly identify many Cercospora and Pseudocercospora species, ribosomal and protein-coding genes have been utilized (Wang et al 1998; Stewart et al 1999; Crous et al. 2001; Goodwin et al 2001; Beilharz & Cunnington 2003; vila et al. 2005). Dunkle and Levy (2000) used amplified fragment length polymorphism (AFLP) analysis, internal transcribed spacer (ITS) regions, and 5.8S ribosomal DNA (rDNA) to examine the

PAGE 30

30 relatedness of two sympatrical ly separated populations of Cercospora zeae-maydis Tehon & E. Y. Daniels. Goodwin et al. ( 2001) examined genetic variability and evolut ionary history of Cercospora species from cereal crops based on the ITS region. Based on these data, they concluded that the morphologically similar Cercospora zeae-maydis groups I and II were actually two distinct species. They also confirmed that two species, Cercospora sorghi Ellis & Everh. and Cercospora sorghi var. maydis Ellis & Everh ., which were separated by Chupp (1954) based on morphological features, we re different species. In addition, C. sorghi var. maydis isolates had identical ITS sequences to those of C. apii, C. asparagi Sacc., C. beticola Sacc., C. hayi Calp ., C. kikuchii T. matsumoto & Tomoy, and C. nicotianae Ellis & Everh. These results supported the hypothesis that some Cercospora species may have wider host ranges than previously believed. Groenewald et al. (2005) used host-range studies and multilocus sequence data, AFLP analysis, cultural characteristic s, and morphological characteristic s to examine the synonomy of C. apii and C. beticola Although they caused disease sympto ms on reciprocal hosts and were morphologically indistinguishable, they were distinct species based on molecular data. The use of molecular techniques can elucidate some of the difficulties with identifying Cercospora and related genera and assist in the constructi on of phylogenetic trees. However, in order to do this, these fungi need to be gr own from single conidia and produce sporulating cultures for disease studies, morphological identifications and deposit in culture collections. Growth and sporulation of Cercosporoid fungi in culture Some cercosporoid fungi can be cultured easily and readily produce spor es in culture (Nelson & Campbell 1990; Karaoglanidis & Bardas 2006). For example, C. cruenta Sacc. and C. canescens Ellis and Martin (Ekpo & Esuruoso 1978), and C. zeae-maydis Tehon & E. Y. Daniels (Asea et al. 2005) and C. zebrina Pass. (Nelson & Campbell 1990) were cultured on V-8

PAGE 31

31 juice agar, whereas other species were grown on other media (Balis & Payne 1971; Sah & Rush 1988; Hartman et al. 1991; Wang et al. 1998; Crous et al. 2001; Beilharz & Cunnington 2003). However, some cercosporoid species are notorio us for slow growth and reduced conidium production on artificial media (Nagel 1934; Ekpo & Esuruoso 1978). Stavely & Nimmo (1968) examined the relatio nship of pH and nutri tion to growth and sporulation of C. nicotianae Ellis & Everh. They determined that growth and sporulation was best on media that contained DL-l eucine, sucrose, and yeast extr act. Good results also were obtained on V-8 agar supplemented with tobacco extract. Sporulation was enhanced on V-8 medium when the pH was adjusted to 3.5-4.5 (Stavely & Nimmo 1968). Balis & Payne (1971) successfully cultured C. beticola Sacc. on a beet-leaf dextrose medium. Although cercosporoid species have been asso ciated with orchid hosts, very little information is available concerning their biology and pathogenicity, and no literature exists on appropriate media for their growth and sporulation. If these fungi were successfully cultured, it would be possible to conduct pat hogenicity tests, host-range studi es, pesticide trials, store and deposit isolates in culture coll ections, and increase our understa ndings of these fungi and the diseases they cause. Objective The objective of this research was to de velop methods to grow and sporulate orchidassociated Pseudocercospora species. Chapter 4: A Comparison of the Standard and High-Fidelity Polymerase Chain Reactions (PCR) in the Detection of Pseudocercospora odontoglossi from Cattleya Orchids and its Use in the Diagnosis of Orchid Diseases The polymerase chain reaction (PCR) has become widely used since the discovery (Chien et al. 1976) and subsequent use of heat-stable DNA polymerase for in vitro replication of DNA (Saiki et al. 1988) and has been used to diagnose plant disease si nce it was first used to sequence

PAGE 32

32 viroid RNA (Puchta & Sanger 1989; Trout et al. 1997; Yokomi et al. 2008). When compared to many other plant-disease diagnostic procedures (culturing, ELISA, etc.), the PCR is relatively fast and sensitive (Tsai et al. 2006). In addition to plant-disease diagnosis, the PCR is now used to amplify DNA for phylogenetic studies (Stewart et al. 1999; Crous et al 2001; Palmateer et al. 2003; Chaverri et al 2005; Dettman et al. 2006 ), in genomic analyses (Nadeau et al. 1992; Lashkari et al. 1997; Arne son et al. 2008), and to examine geneti c diversity within populations of plant pathogens (Urena-Padilla et al. 2002; Zhang et al. 2005; Winton et al. 2006). High-fidelity PCR Although the PCR is fast and sensitive, it is generally unable to produce sequences of more than 5 kb (Barnes 1994). High-fidelity PCR (=l ong PCR), which incorporates a second heatstable DNA polymerase with 3-exonuclease act ivity, has been shown to produce longer sequences than standard PCR, with products as large as 35 kb (Barnes 1994). The addition of the proofreading enzyme to the reaction cont aining an n-terminal deletion mutant of Taq polymerase was shown by Barnes (1994) to rem ove mismatched base pairs, allowing strand synthesis to proceed. The use of the proofreading enzyme alone did not amplify the target DNA, which may have occurred because of the degradation of the primers by the 3-exonuclease activity of the enzyme when used in excessive amounts (Barnes 1994). In addition to producing longer sequences than standard PCR, high-fidelity PCR has been shown to efficiently amplify ta rget DNA in the presence of la rge amounts of genomic DNA from hosts or the target organism. Vickers and Grah am (1996) were able to use a high-fidelity PCR protocol to amplify a single-copy gene ( Bar ), a marker for the selecti on of transgenic plants, in the presence of barley genomic DNA; it consistently amplified the target gene, whereas standard PCR only occasionally produced results.

PAGE 33

33 High-fidelity PCR has also detected bacterial infections a nd microbial associations in arthropods. Jeyaprakash and Hoy (2000) demonstrated that high-fidelity PCR was more sensitive than standard PCR in detecting Wolbachia infections in arthropods. When plasmids containing the wsp gene were amplified in the presence of arthropod DNA, high-fidelity PCR consistently amplified 1 fg of plasmid DNA containing the wsp gene but standard PCR only detected 1 ng of plasmid. Hoy et al. (2001) also showed that the high-fidelity PCR was more sensitive than standard PCR in detecting the citrus greening bacterium Candidatus Liberobacter asiaticus in the presence of genomic DNA from citrus psyllids, citrus trees, or citrus psyllid parasitoids. Furthermore, high-fidelity PCR was used to detect and char acterize a new microsporidium species from the predatory mite Metaseiulus occidentalis (Nesbitt) (Becnel et al. 2002), to identify and distinguish two parasitoids of the brown citr us aphid (Persad et al. 2004), to examine the microbial diversity of Metaseiulus occidentalis and its prey, Tetranychus urticae Koch (Hoy & Jeyaprakash 2005), and to amplify 16S ribosomal sequences of e ndotoxin-producing bacteria in varying amounts of dust mite DNA (Valerio et al. 2005). Because of the increased se nsitivity of the high-fidelity PCR and ability to detect without an intermediary culturing ste p, it should be useful in plant disease diagnosis. Objectives The objectives of this chapter were to determine (1) if the high-fidelity PCR was more sensitive than standard PCR in the detection of Pseudocercospora sp. from orchids and (2) if high-fidelity PCR was more sensitive than standard PCR in detecting fungal and bacterial pathogens directly from infected plant tissue.

PAGE 34

34 Chapter 5: The use of Silwet L-77 as an Integrated Pest Management Tool Boisduval scale, Diaspis boisduvalii is one the most important orchid pests. It can be difficult to control using traditional chemical methods because females possess a hard covering that impedes direct contact with pesticides (Hamon 2002; Johnson 2009). Boisduval scale is commonly introduced into an orchid collection on an infested pl ant and can quickly move to a variety of orchids (Johnson 2009). Mites also can be a major problem on cultivat ed orchids (Johnson 2008). Mite species that are known pests of orchids include the two-spotted spider mite ( Tetranychus urticae Arachnida: Acari: Tetranychidae) and seve ral flat mites (Tenuipalpidae), such as the orchid mite ( Tenuipalpus orchidarum Parfitt), the phalaenopsis mite ( Tenuipalpus pacificus Baker), and the oncidium mite (Brevipalpus oncidii Baker) (Johnson 2008). Synthetic organic pesticides have been the pr imary tools used in pe st control on orchids (Lewis et al. 1997). In order to preserve as many natural enemies as possible and have the least impact on the environment and human health, al ternatives to these chemicals are needed. Furthermore, pesticides are phytotox ic to many orchids (Johnson 2008). Surfactants are commonly used in agriculture to increase the spread and retention of herbicides and pesticides on plants (Tu et al. 2001). Silwet L-77, an organosilicone surfactant, has been shown to increase the effectivene ss of limonene for th e control of mealybugs ( Pseudococcus longispinus Targioni-Tozzetti) by reducing th e surface tension around their waxcovered bodies (Hollingsworth 2005). Tipping et al (2003) demonstrated th at Silwet L-77 alone is toxic to Pacific spider mite ( Tetranychus pacificus McGregor) eggs, grape mealybug ( Pseudococcus maritimus Ehrhorn) crawlers, we stern flower thrips ( Frankliniella occidentalis Pergande), and cotton aphid ( Aphis gossypii Glover). In addition, S ilwet L-77 was toxic to nymphs of the Asian citrus psyllid ( Diaphorina citri Kuwayama) and significantly increased the

PAGE 35

35 effectiveness of insecticides; eggs of D. citri were killed with one-f ourth the label rates of imidacloprid or abamectin and adults were killed with one-fourth or one-h alf the label rate of imidacloprid (Srinivasan et al. 2008). Silwet L-77 also can increas e the efficacy of fungicides. Silwet L-77 or another surfactan t (Kinetic) improved the activity of the protectant fungicide maneb against potato early blight and dry bean rust (Gent et al. 2003). Objectives The objectives of this chapter were to determ ine (1) if Silwet L-77 was phytotoxic to several commonly cultivated orchid genera, and (2) if S ilwet L-77 increased the effectiveness of three horticultural oils agai nst Boisduval scale and the flat mite ( Tenuipalpus pacificus) on orchids. Appendices According to Agrios (2001), Plant Pathol ogy is a science with a practical goal: ..to develop information, materials, a nd techniques that will lead to the management or control of plant dis eases and, thereby, increase yields and quality of plants and plant products. One of the objectives of my dissertation was to develop extension and educational materials in order to educate and assist all types of growers in the identific ation and management of orchid pests and diseases. To meet this objective, several Electron Data Information Source (EDIS) publications, first reports of new diseases of orchids, and diagnos tic guides were produced and are located in the appendices.

PAGE 36

36 Figure 1-1. Examples of economically important or chids grown as ornamental plants or for cut flowers. (A) Laelia anceps; (B) Cattleya orchid; (C) Cymbidium orchid; (D) Vanda orchids. A B C D

PAGE 37

37 Figure 1-2. Southern blight, caused by Sclerotium rolfsii on an (A) Ascocenda orchid and (B) Phalaenopsis orchid; and soft-rots caused by Dickeya chrysanthemi on a (C) Oncidium orchid and Pectobacterium carotovora on a (D) Phalaenopsis orchid. Note the similar symptoms that are caused on th ese orchids by three different pathogens. D C D B A D C

PAGE 38

38 Table 1-1. Pseudocercospora and Cercospora species reported on orchids Species Reported Host Taxa References Pseudocercospora dendrobii (H.C. Burnett) U. Braun & Crous, in Crous & Braun Dendrobium species and hybrids, including nobile and lautoria types Burnett 1965; Hsieh & Goh 1990; Crous & Braun 2003 Pseduoercospora peristeriae (H. C. Burnett) U. Braun & Crous, comb. nov. Peristeria elata Burnett 1965; Crous & Braun 2003 Cercospora epipactidis C. Massalonga Epipactis latifolia E. palustris Phaius grandifolius Bletia purpurea (Florida native orchid), Eulophia Ansellia onidioph Brassia, Calanthe, Catasetum, Chysis aurea Coelogyne massangeana Cycnoches chlorochilon Cyrtopodium, Gongora, Lycaste, Maxillaria, Mendoncella fimbriata, Monomeria, Pescatorea, X Phaiocalanthe Xylobium squalens and Zygopetalum Burnett 1965; Alfieri et al. 1994 Pseudocercospora odontoglossi (Prill & Delacr.) U. Braun Ascocenda, Brassavola Brassocattleya, Brassolaeliocattleya, Broughtonia, Cattleya, Caularthron bicornutum, Dendrobium, Epicattleya Epidendrum, Laelia, Laeliocattleya, Pleurothallis talpinaria, Potinara, Rodricidium, Rodriguezia secunda, Schombocattleya Schombodiacrium, and Sophrolaeliocattleya Chupp 1954; Burnett 1964; Ellis 1976; Alfieri et al. 1994; Braun & Hill 2002 Pseudocercospora cymbidiicola U. Braun & C. F. Hill sp. No v Cymbidium orchids in New Zealand Braun & Hill 2002 Cercospora epidendronis Bolick, nom. nud. Epidendrum Alfieri et al. 1994 Cercospora cyprepedii Ellis & Dearn Paphiopedilum Alfieri et al. 1994; Crous & Braun 2003 Cercospora angraeci Feuillebois & Roum. Angraecum Cattleya Macrangraecum, Macroplectrum sesquipedale Laelia, Oncidium, and Odontoglossum alexandrae Crous & Braun 2003

PAGE 39

39 CHAPTER 2 IDENTIFICATION AND CHARACTERIZAT ION OF A SOFT-ROT PATHOGEN ISOLATED FROM Vanda, Phalaenopsis, Oncidium AND Tolumnia ORCHIDS IN FLORIDA Introduction Pectolytic enzyme-producing bacteria can be devastating plant pa thogens. Pectolytic enzymes (pels) macerate plant tissue by degradin g plant cell-wall components which leads to soft, water-soaked lesions that can enlarge rapidly (Prombe lon & Kelman, 1980; Kotoujansky 1987; Barras et al. 1994; Kazemi-Pour et al. 2004) Although many bacteria produce pectolytic enzymes, most that cause soft-rot dis eases in plants belong to the genera Erwinia Pectobacterium or Dickeya (Dickey 1979; Kotoujansky 1987; Ay san et al. 2003; Gardan et al. 2003; Samson et al. 2005; Pitman et al. 2008). Three species cause disease in orchids, Pectobacterium cyprepedii (formerly Erwinia cyprepedii ), Pectobacterium carotovora (formerly Erwinia carotovora), and Dickeya spp (formerly Erwinia chrysanthemi) (Ark 1959; Burnett 1965; Bradbury 1986; Hadley et al. 1987; Liau et al. 2003; Chan et al. 2005; Sjahril et al. 2006; Cating et al. 2008, 2009b). Pectobacterium cyprepedii is a serious pathogen of ladys slipper orchids ( Cyprepedium Paphiopedilum and Phragmipedium ), but has been reported to cause brown rots on other orchids as well (Phalaenopsis Aerides Catasetum ) (Ark 1959; Bradbury 1986; Alfieri et al. 1994). Although P. cyprepedii is closely related to the soft-rot causing P. carotovora it causes distinct symptoms. They start as small, round, water-soake d lesions and eventually turn dark brown and dry (Hadley et al. 1987). In contrast, P. carotovora causes a true soft-rot in which cell-wall degrading enzymes macerate tissue (Daniels et al. 1988). Pectobacterium carotovora causes soft-rots on many plants (Prombelon & Kelman 1980; Schuerger & Batzer 1993; Chuang et al. 1999; Toth et al. 2003) and is a serious pathogen of many orchid species (Ark 1959; Burnett 1965; Bradbury 1986; Hadley et al. 1987; Liau et al. 2003; Chan et al. 2005; Sjahril et al. 2006).

PAGE 40

40 Symptoms can develop rapidly and are virtually indistinguis hable from those caused by Dickeya spp. Recently, Erwinia chrysanthemi (Burkholder et al. 1953) and Brenneria paradisiaca (Fernandez-Borrero & Lopez-Duque 1970) Hauben et al. 1999 were reexamined, reclassified, and divided into six taxa within the novel genus Dickeya (Samson et al 2005): D. dadantii Samson et al. 2005, D. chrysanthemi bv. parthenii Samson et al. 2005, D. chrysanthemi bv. chrysanthemi Samson et al. 2005, D. dieffenbachiae Samson et al. 2005, D. dianthicola Samson et al. 2005, and D. paradisiaca Samson et al. 2005 These divisions were based on DNA:DNA hybridization, serology, 16S rDNA seque nces, and phenotypic characters. Dickeya spp cause diseases in many different plant species (P rombelon and Hyman 1986; Hugouvieux-Cotte-Pattat et al. 1992; Yap et al. 2005; Lee & yu 2006) and are serious orchid pat hogens (Cating et al. 2009b). Since the 16S rDNA gene of Escherichia coli (Migula 1895) Castella ni and Chalmers 1919 was sequenced in 1972 (Ehresmann et al. 1972), it has been used exte nsively for bacterial identification (Stackebrandt & Goebel 1994; Kolbert & Persing 1999; Clarridge III 2004; Janda & Abbott 2007; Woo et al. 2008) an d phylogenetic analyses (Weisbur g et al. 1991; Ludwig et al. 1998; Kwon et al. 1997; Samson et al. 2005; Naum et al. 2008). Although 16S rRNA gene sequences are useful to identify st rains to the level of family or genus, they are often not useful in species identification (Naum et al. 2008; Staley 2009). For example, Fox et al. (1992) indicated that two closely related species that could be distinguished w ith phenotypic characters, Bacillus globisporus Larkin and Stokes 1967 and B. psychrophilus (ex Larkin & Stokes 1967) Nakamura 1984, nom. rev., had 99.8% similar 16S rDNA sequences.

PAGE 41

41 Very little is known about soft -rot causing bacteria in orch ids. Although soft-rot pathogens typically have wide host ranges, recent isolates from orchids have not fallen in previously described taxa. For example, Ca ting et al. (2009b) described a Dickeya sp. that caused soft-rot symptoms in Tolumnia orchids; fatty acids, 16S rDNA seque nces and biochemical tests did not place this pathogen in a described species. Similarly, a Dickeya sp. from Oncidium orchids in China was initially identified as D. chrysanthemi but 16S rDNA sequences demonstrated 98% similarity to D. dadantii and D. dianthicola (Li et al. 2009). Thus, a dditional tests or sequence analyses of more than the 16S rDNA gene may be needed to understand these new orchid pathogens (Kwon et al. 1997). Louws et al. (1999) described the three Ds of PCR based genomic analysis of phytobacteria as diversity, detection, and disease diagnosis. They indicated that an assessment of population genetic diversity was needed to establish a stable taxonomy; only then can new strains quickly be characterized and methods for diagnosis and management of new strains be developed. The objectives of this research were to: (1) characterize 18 soft-rot isolates that were recovered from four diffe rent orchid hosts ( Vanda Phalaenopsis Oncidium and Tolumnia ) with standard biochemical tests (Schaad et al. 2001) fatty acid analysis, 16S rDNA sequences, and the pectate lyase coding gene cluster ( pel) (Nassar et al. 1996; PalacioBielsa et al. 2006); and (2) perform phylogenetic anal yses with 16S rDNA and pel gene sequences. Materials and Methods Isolation and Initial Tests Plants with soft-rot symptoms were obtained from commercial orchid growers in central and south Florida (Table 2-1). Bacteria were isol ated according to Schaad et al. (2001), and grown on nutrient agar (NA) at 27 C for 24 h. Single colonies were tran sferred to crystal-violet pectate medium (CVP) (Schaad et al 2001) and incubated at 27 C for 24 h to assess pectolytic activity.

PAGE 42

42 Colonies that pitted CVP, i ndicating pectate degradation, were transferred to nutrient agarglycerol-manganese chloride medium (NGM) (Lee & Yu 2006) to assess indigoidine pigment production (Starr et al. 1966; Reverchon et al 2002; Lee & Yu 2006). Tests for indole production, erythromycin sensitiv ity, oxidase, phosphatase, a nd acid production from (+)-Darabitol and sorbitol and the KOH test were pe rformed according to Schaad et al. (2001). Growth at 39 C was determined by stab-inoculating NA plates with a 24-h-old culture as described by Schaad et al. (2001). In addition to the isolates from Florida orchids, four reference isolates were used in these and ot her tests that are described below: P. carotovora (obtained from J. Bartz, Department of Plant Pathology, University of Florida, Gainesville), E. chrysanthemi (ATCC No. 11663), Pectobacterium cypripedii (ATCC No. 29267), and Acidovorax avenae subsp. cattleyae (ATCC No. 10200). Fatty Acid Analysis The 18 orchid isolates were sent to the Bact erial Identification and Fatty Acid Analysis Laboratory at the University of Florida, Gainesvi lle for fatty acid analysis using the MIDI system (Sherlock version TSBA 4.10; Microbial Identification 16 System, Newark, DE). Carbohydrate Utilization Tests The API 50 CH test kit (bioMrieux, Durham, NC) was used to determ ine utilization of 49 different carbohydrates by the 18 orchid and four reference isolates. Tests were performed according to the manufacturers recommendations for members of the Enterobacteriaceae and evaluated after 24 and 48 h. Molecular and Phylogenetic Analysis The 18 orchid isolates were grown on NA for 24 h at 27 C. Two loops of cells were then removed from culture surfaces and genomic DNA was extracted with Puregene DNA isolation reagents (Qiagen, Valencia, CA). For each stra in, the 16S rRNA gene was amplified via high-

PAGE 43

43 fidelity PCR, in which 50L reaction volumes contained 50 mM TRIS (pH 9.2), 16 mM ammonium sulfate, 1.75 mM MgCl2 350 M each of dATP, dGTP, dCTP and dTTP, 800 pmol of primers 27f (5 -GAGAGTTTGATCCTG GCTCAG-3 ) and 1495r (5 TACGGCTACCTTGTTACGA-3 ) (Weisburg 1991), 1 unit of Accuzyme (Bioline, Taunton, MA), and 5 units of Taq DNA polymerase (Bioline) (Barnes, 1994). Samples in thin-walled microfuge tubes were covered with 100 L of sterile mineral oil and were amplified using three linked temperature profiles: (i) 94 C for 2 min; (ii) 10 cycl es of denaturing at 94 C for 10 s, annealing at 55 C for 30 s, and extension at 68 C for 1 min; (iii) 25 cycles of 94 C for 10 s, annealing at 55 C for 30 s, and extension at 68 C for 1 min plus an additional 20 s during each consecutive cycle (Jeyaprakash and Hoy 2000; Hoy et al. 2001). A portion of the pelADE pectolytic enzyme gene cluste r (Tamaki et al. 1988; HugouvieuxCotte-Pattat et al. 1992; Nassar et al. 1996; Palaci o-Bielsa et al. 2006) was also amplified with the above genomic DNA, using primers ADE1 (ADE1 (5GATCAGAAAGCCCGCAGCCAGAT -3) and ADE2 (5CTGTGGCCGATCAGGATGGTTTTGTCGTGC -3) (Na ssar et al, 1996). With the exception of a 63 C annealing temperature, the same PCR parameters were used. The above 16S rDNA (1508 bp) and pelADE gene (420 bp) sequences were cloned using the TOPO T/A cloning kit (Invitrogen Co rp., Carlsbad, CA). Before ligation into the cloning vector, PCR products were cleaned using the QIAquick PCR purification kit (Qiagen, Valencia, CA) following the manufacturers recommendations and eluted in 50 L of sterile glass-distilled, glass-collected water. To facilitate ligation into the cloning vector, a 3 A-overhang was added to the PCR product after purif ication by mixing the 50-L DNA sample with 5.75 L of 10X high-fidelity buffer (50 mM TRIS, pH 9.2, 16 mM ammonium sulfate, 1.75 mM MgCl2), 100

PAGE 44

44 mM dATP, and 1 unit of Taq polymerase (Bioline), a nd running the reaction in a thermal cycler (Perkin Elmer DNA Thermal Cycler 480) at 72 C for 45 min. The product was immediately cloned into the TOPO T/A cloning vector fo llowing the manufacturers recommendations (Invitrogen Corp.). Transformed E. coli colonies were selected fr om plates containing X-GAL, IPTG, and ampicillin and grown overnight in LB broth containing ampicillin at 37 C. Plasmids were extracted using the Qiagen Plasmid Mini Prep Kit (Qiagen, Valencia, CA) and digested with Eco RI restriction enzyme followed by gel elect rophoresis on a 2% agarose TAE gel stained with ethidium bromide to confirm the correct size of the insert. Purified plasmids were sent to the Interdisciplinary Center fo r Biotechnology Research Core Facility at the University of Florida, Gainesville for sequencing. The partial 16S gene sequence and a portion of the pelADE gene cluster were sequenced in both directions and contigs were created usi ng the sequence analysis software MacDNASIS version 3.7 (Hitachi, San Bruno, CA, USA). Sequen ces were compared with similar sequences in the GenBank database (National Center for Biotechnology Information, Bethesda, MD) using the Basic Alignment Search Tool (BLAST) sear ch program (Altschul et al. 1990). Similar sequences chosen from GenBank for use in the 16S phylogenetic analysis include D. zeae (AF520711), D. dieffenbachiae (AF520712), D. dadantii (AF520707), D. dianthicola (AF520708), D. paradisiaca (AF520710), D. chrysanthemi (U80200), and Pectobacterium atrosepticum (EF178668 and EF530555) as outgroups (Slawiak et al. 2009). Four pelADE gene sequences were available in GenBank and were used in the pelADE gene alignment (D. chrysanthemi M33584 and X17284), D. dadantii (CP001654), and D. zeae (CP001655). Sequences were aligned using the CLUSTAL X v. 1.83 (Thompson et al. 1997) after setting parameters for pairwise and multiple alignments to gap opening=15 and gap extension=6.66

PAGE 45

45 (Hall 2001). Sequences alignments were examined and adjusted by eye using MacDNASIS version 3.7. The pelADE gene sequences were translated and aligned first as amino acids, then translated back to DNA sequences befo re performing phylogenetic analysis. Phylogenetic analyses were pe rformed with the 16S rDNA and pelADE datasets using PAUP version 4.0b10 for maximum likelihood (ML) (S wofford 2002) and MrBayes version 3.1.2 for Markov chain Monte Carlo (MCMC) analysis (Huelsenbeck & Ronquist 2001). Modeltest version 3.7 (Posada & Crondall 1998) was used to select the best analysis method for all analyses. The hierarchial Likelihood Ratios Te sts (hLRTs) and Akaike Information Criterion (AIC) parameters obtained for both data sets were used to pres et MrBayes and ML in PAUP. The hLRTs and AIC parameters for the 16S best-fit model (TrN+I +G) were: Base=(0.2540, 0.2332, 0.3207, 0.1921); Rmat=(1.0000, 2.9593, 1.0000, 1.0000, 4.6971, 1.0000); Rates=gamma; Shape=0.9021; Pinvar=0.7126. Th e hLRTs and AIC parameters for the pelADE best-fit model (TrNef+G) were : Base=(0.1824, 0.2521, 0.2949, 0.2705); Rmat=(1.0000, 10.1198, 1.0000, 1.0000, 3.5779, 1.0000); Rates=gamma; Shape=0.4304; Pinvar=0. Clade stability was determined by 1,000 bootstrap replications (ML) or the posterior proba bilities obtained from 1,000,000 generations with the first 2,500 trees discarded as burnin (MrBayes). Pathogenicity Tests Pathogenicity tests were performe d with four isolates: 1015-1 from Tolumnia 1114-1 from Phalaenopsis 0827-2 from Oncidium and 0723-1 from Vanda. Isolates were grown on NA for 24 h at 27 C, and cells were scraped from the medium surface, suspended in sterile tap-water, and adjusted to a concentration of 108 cells ml-1 (Tsror (Lahkim) et al. 2009); 100 L was injected into four plants each of Tolumnia mericlones, Phalaenopsis hybrids, and Oncidium mericlones in 10.6-cm plastic pots, and Vanda hybrids in 10.6-cm plastic baskets. As controls, four plants of each were inoculated with 100 L of sterile water and four of each were not

PAGE 46

46 inoculated. Plants were inc ubated in a greenhouse at 20.6-29.1 C and 37-90% RH under a 16L: 8D photoperiod, and evaluated for symptom deve lopment after 24 h. To complete Kochs postulates and confirm that the i noculated strains could be rec overed from symptomatic tissue, bacteria were isolated and the following test s were performed as described above: pectate degradation on CVP, oxidase, phosphatase, indol e, arabinose, erythromycin sensitivity, indigoidine producti on, growth at 39 C, and PCR using ADE1/ADE2 primers. Results Isolation and Identification of Dickeya Strains A large number of bacterial colonies were isol ated from symptomatic tissue of each plant on NA. Single colonies were transferred to CVP and produced pits, indicat ing pectolytic enzyme activity. All strains were Gram nega tive (KOH test) and placed in the genus Dickeya based on the biochemical test results in Table 2-1 (Schaad et al. 2001; Samson et al. 2005; Lee & yu 2006; Tsror (Lahkim) et al. 2009). Fatty Acid Analysis Fatty acid analyses indicated that the 18 orchid isolates were most similar to Erwinia chrysanthemi with similary index ratings (SIM) from 0.723 to 0.963 (Table 2-2); however, it should be noted that the Dickeya species described by Samson et al. (2005) are no t currently in the MIDI database. Carbohydrate Utilization Tests All 18 orchid isolates utilized glycerol, L-arabinose, D-ribose, D-xyl ose, D-galactose, Dglucose, D-fructose, D-manose, L-rhamnose, inos itol, D-manitol, N-acetylglucosamine, arbutin, esculin ferric citrate, salicin, D-saccharose, a nd gentiobiose, but did not utilize erythritol, Lxylose, D-adonitol, methyl-D-xylopyranoside, L-sorbose, dulcitol, D-sorbitol, methylDmannopyranoside, methylD-glucopyranoside, D-melezito se, amidon (starch), glycogen,

PAGE 47

47 xylitol, D-turanose, D-lyxose, D-tagatose, D-fuco se, L-fucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-ketogluconate, and potassium 5-ketogluconate. Two of the Phalaenopsis isolates utilized amygdalin, whereas the Vanda (2) Oncidium (4) and Tolumnia (8) isolates did not. All Vanda, Oncidium and Phalaenopsis isolates utilized D-ce llobiose, and none of the orchid isolates utilized D-maltose, D-l actose, and D-trehalose (Table 2-4). Molecular and Phylogenetic Analysis BLAST searchs with the 16S r DNA sequences indicated that th e orchid isolates were 94-99 % similar to P. chrysanthemi or E. chrysanthemi (E values 0.0) (Table 2-2). The sequences of two isolates (0723-1, 0723-2) from Vanda shared the highest similarity with GenBank accession EU526397, which was previously isolated from a Vanda orchid in Florida (C ating et al. 2008). The sequences for isolates from Oncidium Tolumnia and Phalaenopsis shared the highest similarity with GenBank accession FM946179, isolated from an Oncidium orchid in China (Li et al. 2009). These sequences are alig ned in Fig. 2-1 with those of six Dickeya and two P. atrosepticum accessions from GenBank. Maximum likelihood phylogenetic analysis of the 16S rDNA data placed the orchid isolates in two distinct clades (Fig. 2-3). Clade 1 was well-supported (boot strap value = 98) and contained the two Vanda isolates. Clade 2 was less well supported (bootstrap = 73) and contained the remaining orchid isolates from Tolumnia Oncidium and Phalaenopsis as well as D. dadantii, D. dianthicola and D. paradisiaca from GenBank. Dickeya zeae and D. dieffenbachiae formed a clade distinct (bootstrap = 87), and D. chrysanthemi was distinct from the other taxa. Similarly, Bayesian analysis of the 16S rDNA data produced the same orchid clades, one of which c ontained isolates from Tolumnia Oncidium and Phalaenopsis with a posterior probablility of 75 (Fig. 2-4, clad e 2) and the other which contained the two Vanda isolates with a posterior probabi lity of 100 (Fig. 2-4, clade 1).

PAGE 48

48 The ADE1/ADE2 primer pair generated a 420 -bp band for the orchid strains that corresponded to the pelADE pectolytic enzyme gene cluster of Dickeya sp. After sequences were trimmed of 13 to 25 bases, they were aligne d with four similar sequences in GenBank (Fig. 2-2). Maximum likelihood phyloge netic analysis based on th e conserved portion of the pelADE pectolytic enzyme gene cluster revealed three clades (Fig. 2-5): Clade 3 contained the Phalaenopsis and Oncidium isolates with D. dadantii (bootstrap = 100), all but one Tolumnia isolate fell in clade 2 (bootstrap = 54), and the Vanda isolates formed clade 1 (bootstrap = 100). Bayesian analysis of these data produced the sa me clades, with posterior probabilities of 100 for clade 3, 94 for clade 2, and 100 for clade 3 (Fig. 2-6). Pathogenicity Tests Each of the four strains caused soft-rot symptoms on all inoculated plants 24 h after inoculation, but not on mock-inoc ulated or non-inoculated plants. From each of the 64 inoculated plants Dickeya sp. was recovered on NA after 24 h at 27 C, based on the growth of isolates at 39 C, pectate degradation on CVP, indole indigoidine and phosphatase production, erythromycin sensitivity, and arabinose utiliz ation, and production of the expected 420-bp PCR product with primers ADE1/ADE2. Discussion Eighteen isolates of bacteria that caused soft-ro t in four orchid genera were characterized biochemically and genetically. In itial results indicated that the isolates were members of the Dickeya genus (Table 2-1). Subsequent 16S rDNA and fatty acid analyses identified the isolated strains as either E. chrysanthemi or P. chrysanthemi each of which were transferred to Dickeya by Samson et al. (2005). However, thes e isolates matched none of the six Dickeya taxa that were described by Samson et al. (2005); several differe nces were evident (Table 2-3). The Florida orchid strains utilized (-)-D-arabinose, but D. chrysanthemi bv. chrysanthemi and D.

PAGE 49

49 chrysanthemi bv. parthenii do not. The Florida orchid strain s did not utilize lactose, but D. dadantii and D. zeae do. Dickeya dieffenbachiae does not utilize (+)-D-m elibiose and (+)-Draffinose, and D. paradisiaca does not utilize mannitol, whereas the orchids strains utilized all three carbohydrates. And the orchid strains grew at 39 C whereas D. dianthicola does not. Based on these results, the studied orch id strains may represent new species. In addition to Dickeya spp., another soft-rot pathogen, P. carotovora causes soft-rot disease in orchids (Ark 1959; Burnett 1965; Bradbury 1986; Ha dley et al. 1987; Liau et al. 2003; Chan et al. 2005; Sjahril et al. 2006) and P. cyprepedii causes leafspots (Ark 1959; Bradbury 1986; Hadley et al. 1987; Alfieri et al. 1994). Therefore, orchid stra ins in this study were compared to P. carotovora P. cyprepedii (ATCC# 29267), and th e type strain of D. chrysanthemi (ATCC# 11663) to determine if there were biochemical char acteristics that could be used to diagnose the Florida orchid soft-rot diseases. The orchid isolates differed from the above reference strains in the utilization of five carbohydrates (Table 2-4). The orchid is olates could be distinguished based on their inability to utilize D-lactose, and the Tolumnia isolates could be distinguished further by their inability to utilize D-cellobiose. In both the Bayesian and ML phylogenetic analyses of the 16S rDNA data, Vanda isolates formed a distinct clade with high bootstrap a nd posterior probablility values. Isolates from Tolumnia Oncidium and Phalaenopsis formed a distinct clade in the Bayesian analysis, but grouped with D. dadantii, D. dianthicola and D. paradisiaca in the ML analysis (Fig. 2-3). As mentioned above, 16S rDNA sequences are us ed frequently to construct phylogenies and identify bacteria, but may not be useful in an alyses below the genus level, especially among closely related species in the Enterobacteriac eae (Naum et al. 2008; Zeig ler 2003). Fox et al. (1992) concluded that 16S rDNA sequences may not be useful in species identifications, but can

PAGE 50

50 be an important tool for strain identifications wh en they are represented in sequence databases. Given the inconsistent value of 16S rDNA data, another gene, pelADE was used to further resolve the orchid isolates. Pectate lyases (pels) that are produced by Dickeya spp. (formerly Erwinia chrysanthemi ) are important pathogenicity determinants (HugouvieuxCotte-Pattat et al. 1996; Nassar et al. 1996). With restriction fragment length polymorphism (RFLP) analysis of a 420-bp fragment of the pelADE gene cluster, Nassar et al. (1996) identified six groups among 78 strains of E. chrysanthemi In the present study, pelADE sequence data revealed three clades in both ML and Bayesian analyses (Figs. 2-5 and 2-6). The Phalaenopsis and Oncidium isolates grouped with D. dadantii and the Vanda isolates formed a distinct clade, each with bootstrap and posterior probability values of 100. Tolumnia isolates formed another clade with a bootstrap value of 54 in the ML analysis, but higher suppor t (94) in the Bayesian analysis. The results of this study s uggest that there are two di stinct orchid clades in Dickeya in Florida, one isolated from Vanda orchids and another from Tolumnia orchids. Isolates from Phalaenopsis and Oncidium formed distinct clades in neither the 16S nor pelADE analyses, and were most similar to D. dadantii (Figs. 2-6 and 2-7). In addi tion, carbohydrate utilization tests indicated that the orchid isolates could be differentiated from the Dickeya taxa that were described by Samson et al (2005) (Table 2-3). The present results provide new information on th e causes of these diseases, but indicate that additional research is needed. Whether these st rains exist on other orchid taxa, their respective host ranges, ecology, and whether they have e ndemic or exotic origins are unknown. More isolates should be collected from members of different orchid gene ra to determine their distributions and whether they are new species. The latter studies co uld include identifying

PAGE 51

51 novel biological and pathological attributes and increased the phylogenetic resolution of these bacteria with, for example, sequence data from additional genes, such as those for indigoidine production (Lee & Yu) and recA (recombinase A) (Waleron et al. 2002; Young & Park 2007). Clearly, this information impacts the management of soft-rot diseases of orchids. Soft-rot diseases are extremely difficult to control with chem icals (Aysan et al. 2003), and the first line of defense, exclusion, depends on accurate pathogen identification. The selection of appropriate management strategies can also depend on path ogen identification (Miller & Martin 1988; Lvesque 1997; Toth et al. 2001). For example, if the orchid strains ha d specific environmental requirements for disease development, these might be manipulated to reduc e disease incidence or severity after pathogen presence was revealed. Furthermore, pathogen id entification is required for epidemiological studies and surveys of geogr aphical distribution, which can be particularly important when searching for disease resistance (Ploetz 2007).

PAGE 52

52 10 20 30 40 50 60 70 80 90 . . D. zeae (AF520711) ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGGGCGGTAGCACAAGGGAGCTTGCTCCCTGGGTGACGAGCGGCGGACGGGTGAG D. dieffenbachiae (AF520712) ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGGGCGGTAGCACAAGGGAGCTTGCTCCCTGGGTGACGAGCGGCGGACGGGTGAG D. chrysanthemi (U80200) ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGGGCGGTAGCACAAGGGAGCTTGCTCCC-GGGTGACGAGCGGCGGACGGGTGAG Dickeya sp. 0723-1 V ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGTAGCACAAGGGAGCTTGCTCCCTGGGTGACGAGCGGCGGACGGGTGAG Dickeya sp. 0723-2 V ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGTAGCACAAGGGAGCTTGCTCCCTGGGTGACGAGCGGCGGACGGGTGAG Dickeya sp. 1114-4 P ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGTAGCACAGAGGAGCTTGCTCCTTGGGTGACGAGCGGCGGACGGGTGAG Dickeya sp. 0827-3 O ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGTAGCACAAAGGAGCTTGCTCCTTGGGTGACGAGCGGCGGACGGGTGAG Dickeya sp. 1114-3 P ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCAGCGGGGGGAAGCTTGCTTCCCTGCCGGCGAGCGGCGGACGGGTGAG Dickeya sp. 1015-2 T ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCAGCGGGGGGAAGCTTGCTTCCCCGCCGGCGAGCGGCGGACGGGTGAG Dickeya sp. 0911-2 T ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCAGCGGGGGGAAGCTTGCTTCCCCGCCGGCGAGCGGCGGACGGGTGAG Dickeya sp. 1015-4 T ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCAGCGGGGGGAAGCTTGCTTCCCCGCCGGCGAGCGGCGGACGGGTGAG Dickeya sp. 1114-16 P ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCAGCGGGGGGAAGCTTGCTTCCCCGCCGGCGAGCGGCGGACGGGTGAG Dickeya sp. 1015-5 T ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCAGCGGGGGGAAGCTTGCTTCCCCGCCGGCGAGCGGCGGACGGGTGAG Dickeya sp. 0911-3 T ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCAGCGGGGGGAAGCTTGCTTCCCCGCCGGCGAGCGGCGGACGGGTGAG Dickeya sp. 1015-1 T ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCAGCGGGGGGAAGCTTGCTTCCCCGCCGGCGAGCGGCGGACGGGTGAG Dickeya sp. 0911-1 T ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCAGCGGGGGGAAGCTTGCTTCCCCGCCGGCGAGCGGCGGACGGGTGAG Dickeya sp. 1015-3 T ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCAGCGGGGGGAAGCTTGCTTCCCCGCCGGCGAGCGGCGGACGGGTGAG Dickeya sp. 0827-2 O ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCAGCGGGGGGAAGCTTGCTTCCCCGCCGGCGAGCGGCGGACGGGTGAG Dickeya sp. 1114-1 P ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCAGCGGGGGGAAGCTTGCTTCCCCGCCGGCGAGCGGCGGACGGGTGAG Dickeya sp. 0827-4 O ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCAGCGGGGGGAAGCTTGCTTCCCTGCCGGCGAGCGGCGGACGGGTGAG Dickeya sp. 0827-1 O ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCAGCGGGGGGAAGCTTGCTTCCCTGCCGGCGAGCGGCGGACGGGTGAG D. dadantii (AF520707) ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCAGCGGGGGGAAGCTTGCTTCCCCGCCGGCGAGCGGCGGACGGGTGAG D. dianthicola (AF520708) ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCAGCGGGGGGAAGCTTGCTTCCCCGCCGGCGAGCGGCGGACGGGTGAG D. paradisiaca (AF520710) ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCAGCGGGGGGAAGCTTGCTTCCCCGCCGGCGAGCGGCGGTCGGGTGAG P. atrosepticum (EF178668) -----------------------------------------------------------------------CGAGCGGCGGACGGGTGAG P. atrosepticum (EF530555) -----------------------------------------------------------------------CGAGCGGCGGACGGGTGAG Figure 2-1. ClustalX alignmen t of 16S rDNA sequences of 18 Dickeya isolates from orchids in Flor ida using primers pair 27f/1495r (Weisburg et al. 1991); they are from: V= Vanda, P= Phalaenopsis O= Oncidium and T= Tolumnia For comparison, GenBank sequences are included from Dickeya dadantii D. dianthicola D. paradisiaca, D. zeae D. dieffenbachiae, D. chrysanthemi and two isolates of Pectobacterium atrosepticum. A hyphen indicates a nucleotide deletion.

PAGE 53

53 100 110 120 130 140 150 160 170 180 . . D. zeae (AF520711) TAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCTTCGGACCAAAGAGGGGGACC D. dieffenbachiae (AF520712) TAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCTTCGGACCAAAGAGGGGGACC D. chrysanthemi (U80200) TAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCTTCGGACCAAAGAGGGGGACC Dickeya sp. 0723-1 V TAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCTTCGGACCAAAGTGGGGGCTC Dickeya sp. 0723-2 V TAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCTTCGGACCAAAGTGGGGGCTC Dickeya sp. 1114-4 P TAATGTCTGGGGATCTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACC Dickeya sp. 0827-3 O TAATGTCTGGGGATCTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACC Dickeya sp. 1114-3 P TAATGTCTGGGGATCTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACC Dickeya sp. 1015-2 T TAATGTCTGGGGATCTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACC Dickeya sp. 0911-2 T TAATGTCTGGGGATCTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACC Dickeya sp. 1015-4 T TAATGTCTGGGGATCTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACC Dickeya sp. 1114-16 P TAATGTCTGGGGATCTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACC Dickeya sp. 1015-5 T TAATGTCTGGGGATCTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACC Dickeya sp. 0911-3 T TAATGTCTGGGGATCTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACC Dickeya sp. 1015-1 T TAATGTCTGGGGATCTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACC Dickeya sp. 0911-1 T TAATGTCTGGGGATCTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACC Dickeya sp. 1015-3 T TAATGTCTGGGGATCTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACC Dickeya sp. 0827-2 O TAATGTCTGGGGATCTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACC Dickeya sp. 1114-1 P TAATGTCTGGGGATCTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACC Dickeya sp. 0827-4 O TAATGTCTGGGGATCTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACC Dickeya sp. 0827-1 O TAATGTCTGGGGATCTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACC D. dadantii (AF520707) TAATGTCTGGGGATCTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACC D. dianthicola (AF520708) TAATGTCTGGGGATCTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATGACGTCGCAAGACCAAAGTGGGGGACC D. paradisiaca (AF520710) TAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCTTCGGACCAAAGTGGGGGCTC P. atrosepticum (EF178668) TAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCTTCGGACCAAAGAGGGGGACC P. atrosepticum (EF530555) TAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCTTCGGACCAAAGAGGGGGACC Figure 2-1. cont.

PAGE 54

54 190 200 210 220 230 240 250 260 270 . . D. zeae (AF520711) TTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAAAGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG D. dieffenbachiae (AF520712) TTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAAAGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG D. chrysanthemi (U80200) TTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAAAGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG Dickeya sp. 0723-1 V TTCGGACCTCACGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAAAGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG Dickeya sp. 0723-2 V TTCGGACCTCACGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAAAGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG Dickeya sp. 1114-4 P TTCGGGCCTCACGCCATCGGATGAACCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG Dickeya sp. 0827-3 O TTCGGGCCTCACGCCATCGGATGAACCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG Dickeya sp. 1114-3 P TTCGGGCCTCACGCCATCGGATGAACCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG Dickeya sp. 1015-2 T TTCGGGCCTCACGCCATCGGATGAACCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG Dickeya sp. 0911-2 T TTCGGGCCTCACGCCATCGGATGAACCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG Dickeya sp. 1015-4 T TTCGGGCCTCACGCCATCGGATGAACCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG Dickeya sp. 1114-16 P TTCGGGCCTCACGCCATCGGATGAACCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG Dickeya sp. 1015-5 T TTCGGGCCTCACGCCATCGGATGAACCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG Dickeya sp. 0911-3 T TTCGGGCCTCACGCCATCGGATGAACCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG Dickeya sp. 1015-1 T TTCGGGCCTCACGCCATCGGATGAACCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG Dickeya sp. 0911-1 T TTCGGGCCTCACGCCATCGGATGAACCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG Dickeya sp. 1015-3 T TTCGGGCCTCACGCCATCGGATGAACCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG Dickeya sp. 0827-2 O TTCGGGCCTCACGCCATCGGATGAACCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG Dickeya sp. 1114-1 P TTCGGGCCTCACGCCATCGGATGAACCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG Dickeya sp. 0827-4 O TTCGGGCCTCACGCCATCGGATGAACCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG Dickeya sp. 0827-1 O TTCGGGCCTCACGCCATCGGATGAACCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG D. dadantii (AF520707) TTCGGGCCTCACGCCATCGGATGAACCCAGATGGGATTAGCTAGTAGGTGAGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG D. dianthicola (AF520708) TTCGGGCCTCACGCCATCGGATGTGCCCAGATGGGATTAGCTGGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG D. paradisiaca (AF520710) TTCGGACCTCATGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGCGGGGTAAAGGCCCACCTAGGCGACGATCCCTAGCTGGTCTG P. atrosepticum (EF178668) TTCGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGCGGGGTAATGGCCCACCTAGGCGACGATCCCTAGCTGGTCTG P. atrosepticum (EF530555) TTCGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGCGGGGTAATGGCCCACCTAGGCGACGATCCCTAGCTGGTCTG Figure 2-1. cont.

PAGE 55

55 280 290 300 310 320 330 340 350 360 . . D. zeae (AF520711) AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCT D. dieffenbachiae (AF520712) AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCT D. chrysanthemi (U80200) AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCT Dickeya sp. 0723-1 V AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCT Dickeya sp. 0723-2 V AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCT Dickeya sp. 1114-4 P AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAAGCCT Dickeya sp. 0827-3 O AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCT Dickeya sp. 1114-3 P AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCT Dickeya sp. 1015-2 T AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCT Dickeya sp. 0911-2 T AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCT Dickeya sp. 1015-4 T AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCT Dickeya sp. 1114-16 P AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCT Dickeya sp. 1015-5 T AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCT Dickeya sp. 0911-3 T AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCT Dickeya sp. 1015-1 T AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCT Dickeya sp. 0911-1 T AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCT Dickeya sp. 1015-3 T AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCT Dickeya sp. 0827-2 O AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCT Dickeya sp. 1114-1 P AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCT Dickeya sp. 0827-4 O AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCT Dickeya sp. 0827-1 O AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCT D. dadantii (AF520707) AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCT D. dianthicola (AF520708) AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCT D. paradisiaca (AF520710) AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCT P. atrosepticum (EF178668) AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCT P. atrosepticum (EF530555) AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCT Figure 2-1. cont.

PAGE 56

56 370 380 390 400 410 420 430 440 450 . . D. zeae (AF520711) GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGGGGCAGGCTTAATACGTCTGTTC D. dieffenbachiae (AF520712) GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGGGGCAGGCTTAATACGTCTGTTC D. chrysanthemi (U80200) GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGGAACAAGGTTAATACCTTTGTTC Dickeya sp. 0723-1 V GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGGGATAGAGTGAATACCTTTATCC Dickeya sp. 0723-2 V GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGGGATGAAGTTAATACCTTTATCC Dickeya sp. 1114-4 P GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGGGTGAGTTTAATAACCTTACCG Dickeya sp. 0827-3 O GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGACAAGGTTAATAACCTTGTCG Dickeya sp. 1114-3 P GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGGTAAGGTTAATAACCTTATCG Dickeya sp. 1015-2 T GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGGCAAGGTTAATAACCTTGTCG Dickeya sp. 0911-2 T GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGGCAAGGTTAATAACCTTGTCG Dickeya sp. 1015-4 T GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGGCAAGGTTAATAACCTTGTCG Dickeya sp. 1114-16 P GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGGCAAGGTTAATAACCTTGTCG Dickeya sp. 1015-5 T GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGGCAAGGTTAATAACCTTGTCG Dickeya sp. 0911-3 T GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGGCAAGGTTAATAACCTTGTCG Dickeya sp. 1015-1 T GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGGTAAGGTTAATAACCTTGTCG Dickeya sp. 0911-1 T GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGGTAAGGTTAATAACCTTGTCG Dickeya sp. 1015-3 T GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGGTAAGGTTAATAACCTTACCG Dickeya sp. 0827-2 O GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGGTAAGGTTAATAACCTTACCG Dickeya sp. 1114-1 P GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGGTAAGGTTAATAACCTTGCCG Dickeya sp. 0827-4 O GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGGCAAGGTTAATAACCTTGTCG Dickeya sp. 0827-1 O GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGGCAAGGTTAATAACCTTGTCG D. dadantii (AF520707) GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGGGACAAGGTTAATAACCTTGTTC D. dianthicola (AF520708) GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGGAGCAGGGTTAATAACCTTGTTC D. paradisiaca (AF520710) GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGGGACAGGCTTAATACGTGTGTTC P. atrosepticum (EF178668) GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCAGTAAGGTTAATAACCTTGCTG P atrosepticum (EF530555) GATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCAGTAAGGTTAATAACCTTGCTG Figure 2-1. cont.

PAGE 57

57 460 470 480 490 500 510 520 530 540 . . D. zeae (AF520711) ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG D. dieffenbachiae (AF520712) ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG D. chrysanthemi (U80200) ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG Dickeya sp. 0723-1 V ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG Dickeya sp. 0723-2 V ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG Dickeya sp. 1114-4 P ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG Dickeya sp. 0827-3 O ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAACACGGAGGGTGCAAGCGTTAATCGGAATGACTGG Dickeya sp. 1114-3 P ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG Dickeya sp. 1015-2 T ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG Dickeya sp. 0911-2 T ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG Dickeya sp. 1015-4 T ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAACCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG Dickeya sp. 1114-16 P ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG Dickeya sp. 1015-5 T ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG Dickeya sp. 0911-3 T ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG Dickeya sp. 1015-1 T ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG Dickeya sp. 0911-1 T ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG Dickeya sp. 1015-3 T ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG Dickeya sp. 0827-2 O ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG Dickeya sp. 1114-1 P ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG Dickeya sp. 0827-4 O ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG Dickeya sp. 0827-1 O ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG D. dadantii (AF520707) ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG D dianthicola (AF520708) ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG D paradisiaca (AF520710) ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG P. atrosepticum (EF178668) ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG P atrosepticum (EF530555) ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATGACTGG Figure 2-1. cont.

PAGE 58

58 550 560 570 580 590 600 610 620 630 . . D. zeae (AF520711) GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG D. dieffenbachiae (AF520712) GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG D. chrysanthemi (U80200) GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG Dickeya sp. 0723-1 V GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG Dickeya sp. 0723-2 V GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGGGCTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG Dickeya sp. 1114-4 P GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG Dickeya sp. 0827-3 O GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG Dickeya sp. 1114-3 P GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG Dickeya sp. 1015-2 T GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG Dickeya sp. 0911-2 T GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG Dickeya sp. 1015-4 T GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG Dickeya sp. 1114-16 P GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG Dickeya sp. 1015-5 T GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG Dickeya sp. 0911-3 T GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG Dickeya sp. 1015-1 T GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG Dickeya sp. 0911-1 T GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG Dickeya sp. 1015-3 T GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG Dickeya sp. 0827-2 O GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG Dickeya sp. 1114-1 P GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG Dickeya sp. 0827-4 O GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG Dickeya sp. 0827-1 O GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG D. dadantii (AF520707) GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG D dianthicola (AF520708) GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG D paradisiaca (AF520710) GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG P atrosepticum (EF178668) GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG P atrosepticum (EF530555) GCGTAAAGCGCACGCAGGCGGTCTGTTAAGTTGGATGTGAAATCCCCGGG-CTTAACCTGGGAACTGCATTCAAAACTGACAGGCTAGAG Figure 2-1. cont.

PAGE 59

59 640 650 660 670 680 690 700 710 720 . . D. zeae (AF520711) TCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA D. dieffenbachiae (AF520712) TCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA D chrysanthemi (U80200) TCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA Dickeya sp. 0723-1 V TCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA Dickeya sp. 0723-2 V TCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA Dickeya sp. 1114-4 P TCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA Dickeya sp. 0827-3 O TCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA Dickeya sp. 1114-3 P TCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA Dickeya sp. 1015-2 T TCTCGTAGAGGGGGGTGGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA Dickeya sp. 0911-2 T TCTCGTAGAGGGGGGTGGAATTCCAGGTGTAGCGGTGAAGTGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA Dickeya sp. 1015-4 T TCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGACGGCCCCCTGGACGA Dickeya sp. 1114-16 P TCTCGTAGAGGGGGGTAGAATTCCGGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA Dickeya sp. 1015-5 T TCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA Dickeya sp. 0911-3 T TCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA Dickeya sp. 1015-1 T TCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCAGTGGCGAAGGCGGCCCCCTGGACGA Dickeya sp. 0911-1 T TCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA Dickeya sp. 1015-3 T TCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA Dickeya sp. 0827-2 O TCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA Dickeya sp. 1114-1 P TCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA Dickeya sp. 0827-4 O TCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA Dickeya sp. 0827-1 O TCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGGAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA D. dadantii (AF520707) TCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA D. dianthicola (AF520708) TCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA D. paradisiaca (AF520710) TCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA P. atrosepticum (EF178668) TCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAA P. atrosepticum (EF530555) TCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAA Figure 2-1. cont.

PAGE 60

60 730 740 750 760 770 780 790 800 810 . . D. zeae (AF520711) AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTD. dieffenbachiae (AF520712) AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTD chrysanthemi (U80200) AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTDickeya sp. 0723-1 V AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTDickeya sp. 0723-2 V AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTDickeya sp. 1114-4 P AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTT Dickeya sp. 0827-3 O AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTDickeya sp. 1114-3 P AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCCGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTDickeya sp. 1015-2 T AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTDickeya sp. 0911-2 T AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTDickeya sp. 1015-4 T AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTDickeya sp. 1114-16 P AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTDickeya sp. 1015-5 T AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTDickeya sp. 0911-3 T AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTDickeya sp. 1015-1 T AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTDickeya sp. 0911-1 T AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTDickeya sp. 1015-3 T AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTDickeya sp. 0827-2 O AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACTCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTDickeya sp. 1114-1 P AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTDickeya sp. 0827-4 O AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTDickeya sp. 0827-1 O AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTD dadantii (AF520707) AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTD dianthicola (AF520708) AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTD paradisiaca (AF520710) AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTP atrosepticum (EF178668) AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGACTTGGAGGTTP atrosepticum (EF530555) AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGACTTGGAGGTTFigure 2-1. cont.

PAGE 61

61 820 830 840 850 860 870 880 890 900 . . D. zeae (AF520711) GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATGGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC D. dieffenbachiae (AF520712) GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATGGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC D. chrysanthemi (U80200) GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC Dickeya sp. 0723-1 V GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC Dickeya sp. 0723-2 V GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC Dickeya sp. 1114-4 P GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC Dickeya sp. 0827-3 O GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC Dickeya sp. 1114-3 P GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC Dickeya sp. 1015-2 T GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC Dickeya sp. 0911-2 T GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC Dickeya sp. 1015-4 T GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC Dickeya sp. 1114-16 P GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGGGCCGCAAGGTTAAAACTCAAATGAATTGAC Dickeya sp. 1015-5 T GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC Dickeya sp. 0911-3 T GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC Dickeya sp. 1015-1 T GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC Dickeya sp. 0911-1 T GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC Dickeya sp. 1015-3 T GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC Dickeya sp. 0827-2 O GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC Dickeya sp. 1114-1 P GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC Dickeya sp. 0827-4 O GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC Dickeya sp. 0827-1 O GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC D dadantii (AF520707) GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC D dianthicola (AF520708) GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC D paradisiaca (AF520710) GTGGTCTTGAACCGTGGCTTCCGGAGCTAACGCGTTAAATGGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC P atrosepticum (EF178668) GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC P atrosepticum (EF530555) GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGG-CCGCAAGGTTAAAACTCAAATGAATTGAC Figure 2-1. cont.

PAGE 62

62 910 920 930 940 950 960 970 980 990 . . D. zeae (AF520711) GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAAGCCTGCAGA D. dieffenbachiae (AF520712) GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAAGCCTGCAGA D. chrysanthemi (U80200) GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAAGCCTGCAGA Dickeya sp. 0723-1 V GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGCGAACCCTGTAGA Dickeya sp. 0723-2 V GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGCGAACCCTGTAGA Dickeya sp. 1114-4 P GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGCGAAGACTGCAGA Dickeya sp. 0827-3 O GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGCGAAGACTGCAGA Dickeya sp. 1114-3 P GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGCGAAGACTGCAGA Dickeya sp. 1015-2 T GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGCGAAGACTGCAGA Dickeya sp. 0911-2 T GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGCGAAGACTGCAGA Dickeya sp. 1015-4 T GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGCGAAGACTGCAGA Dickeya sp. 1114-16 P GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGCGAAGACTGCAGA Dickeya sp. 1015-5 T GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGCGAAGACTGCAGA Dickeya sp. 0911-3 T GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGCGAAGACTGCAGA Dickeya sp. 1015-1 T GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGCGAAGACTGCAGA Dickeya sp. 0911-1 T GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGCGAAGACTGCAGA Dickeya sp. 1015-3 T GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGCGAAGACTGCAGA Dickeya sp. 0827-2 O GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGCGAAGACTGCAGA Dickeya sp. 1114-1 P GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGCGAAGACTGCAGA Dickeya sp. 0827-4 O GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGCGAAGACTGCAGA Dickeya sp. 0827-1 O GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGCGAAGACTGCAGA D dadantii (AF520707) GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGCGAACCCTGTAGA D dianthicola (AF520708) GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTTAGCAGA D paradisiaca (AF520710) GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAAGACTGCAGA P atrosepticum (EF178668) GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAATTTGGCAGA P atrosepticum (EF530555) GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAATTTGCTAGA Figure 2-1. cont.

PAGE 63

63 1000 1010 1020 1030 1040 1050 1060 1070 1080 . . D. zeae (AF520711) GATGCGGGTGTGCCTTCGGGAGCTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG D. dieffenbachiae (AF520712) GATGCGGGCGTGCCTTCGGGAGCTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG D. chrysanthemi (U80200) GATGCGGGTGTGCCTTCGGGAGCTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG Dickeya sp. 0723-1 V GATACGGGGGTGCCTTCGGGAACGCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGCTGTGAAATGTTGGGTTAAGTCCCGCAACG Dickeya sp. 0723-2 V GATACGGGGGTGCCTTCGGGAACGCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG Dickeya sp. 1114-4 P GATGCGGTCGTGCCTTCGGGAACGCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG Dickeya sp. 0827-3 O GATGCGGTCGTGCCTTCGGGAACGCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG Dickeya sp. 1114-3 P GATGCGGTCGTGCCTTCGGGAACGCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG Dickeya sp. 1015-2 T GATGCGGTCGTGCCTTCGGGAACGCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG Dickeya sp. 0911-2 T GATGCGGTCGTGCCTTCGGGAACGCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG Dickeya sp. 1015-4 T GATGCGGTCGTGCCTTCGGGAACGCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG Dickeya sp. 1114-16 P GATGCGGTCGTGCCTTCGGGAACGCTGAGACAGGTGCTGCATGGCTGTCGCCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG Dickeya sp. 1015-5 T GATGCGGTCGTGCCTTCGGGAACGCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG Dickeya sp. 0911-3 T GATGCGGTCGTGCCTTCGGGAACGCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG Dickeya sp. 1015-1 T GATGCGGTCGTGCCTTCGGGAACGCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG Dickeya sp. 0911-1 T GATGCGGTCGTGCCTTCGGGAACGCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG Dickeya sp. 1015-3 T GATGCGGTCGTGCCTTCGGGAACGCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG Dickeya sp. 0827-2 O GATGCGGTCGTGCCTTCGGGAACGCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG Dickeya sp. 1114-1 P GATGCGGTCGTGCCTTCGGGAACGCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG Dickeya sp. 0827-4 O GATGCGGTCGTGCCTTCGGGAACGCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG Dickeya sp. 0827-1 O GATGCGGTCGTGCCTTCGGGAACGCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG D dadantii (AF520707) GATGCGGGGGTGCCTTCGGGAACGCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG D dianthicola (AF520708) GATGCGGTGGTGCCTTCGGGAGCTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG D paradisiaca (AF520710) GATGCGGTTGTGCCTTCGGGAGCTCTGAGACAGGTGCTGCATGGCTGTCGGCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG P atrosepticum (EF178668) GATGCCTTAGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG P atrosepticum (EF530555) GATAGCTTAGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACG Figure 2-1. cont.

PAGE 64

64 1090 1100 1110 1120 1130 1140 1150 1160 1170 . . D. zeae (AF520711) AGCGCAACCCTTATCCTTTGTTGCCAGCAC-TTCGGGTGGGAACTCAAGGGAGACTGCCGGTGATAAACCGGAGAAAGGTGGGGATGACG D. dieffenbachiae (AF520712) AGAACAACCCTTATCCTCTGTTGCCAGCAC-TTCGGGTGGGAACTCAGGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG D. chrysanthemi (U80200) AGCGCAACCCTTATCCTCTGTTGCCAGCACGTTATGGTGGGAACTCAGGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG Dickeya sp. 0723-1 V AGCGCAACCCTTATCCTTTGTTGCCAGCAC-TTCGGGTGGGAACTCAAGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG Dickeya sp. 0723-2 V AGCGCAACCCTTATCCTTAGTAGCCAGCAGGTAAAGCTGGGCACTCTAGGGAGACTGCCAGGGATAACCTGGAGGAAGGCGGGGATGACG Dickeya sp. 1114-4 P AGCGCAACCCTTATCCTCTGTTGCCAGCAC-TACGGGTGGGAACTCAGGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG Dickeya sp. 0827-3 O AGCGCAACCCTTATCCTCTGTTGCCAGCAC-TACGGGTGGGAACTCAGGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG Dickeya sp. 1114-3 P AGCGCAACCCTTATCCTCTGTTGCCAGCAC-TACGGGTGGGAACTCAGGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG Dickeya sp. 1015-2 T AGCGCAACCCTTATCCTCTGTTGCCAGCAC-TACGGGTGGGAACTCAGGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG Dickeya sp. 0911-2 T AGCGCAACCCTTATCCTCTGTTGCCAGCAC-TACGGGTGGGAACTCAGGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG Dickeya sp. 1015-4 T AGCGCAACCCTTATCCTCTGTTGCCAGCAC-TACGGGTGGGAACTCAGGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG Dickeya sp. 1114-16 P AGCGCAACCCTTATCCTCTGTTGCCAGCAC-TACGGGTGGGAACTCAGGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG Dickeya sp. 1015-5 T AGCGCAACCCTTATCCTCTGTTGCCAGCAC-TACGGGTGGGAACTCAGGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG Dickeya sp. 0911-3 T AGCGCAACCCTTATCCTCTGTTGCCAGCAC-TACGGGTGGGAACTCAGGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG Dickeya sp. 1015-1 T AGCGCAACCCTTATCCTCTGTTGCCAGCAC-TACGGGTGGGAACTCAGGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG Dickeya sp. 0911-1 T AGCGCAACCCTTATCCTCTGTTGCCAGCAC-TACGGGTGGGAACTCAGGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG Dickeya sp. 1015-3 T AGCGCAACCCTTATCCTCTGTTGCCAGCAC-TACGGGTGGGAACTCAGGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG Dickeya sp. 0827-2 O AGCGCAACCCTTATTCTCTGTTGCCAGCAC-TACGGGTGGGAACTCAAGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG Dickeya sp. 1114-1 P AGCGCAACCCTTATCCTCTGTTGCCAGCAC-TACGGGTGGGAACTCAGGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG Dickeya sp. 0827-4 O AGCGCAACCCTTATCCTCTGTTGCCAGCAC-TACGGGTGGGAACCCAGGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG Dickeya sp. 0827-1 O AGCGCAACCCTTATCCTCTGTTGCCAGCAC-TACGGGTGGGAACTCAGGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG D dadantii (AF520707) AGCGCAACCCTTATCCTCTGTTGCCAGCAC-TTCGGGTGGGAACTCAGGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG D dianthicola (AF520708) AGCGCAACCCTTATCCTCTGTTGCCAGCACGTTATGGTGGGAACTCAGGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG D paradisiaca (AF520710) AGCGCAACCCTTATCCTTTGTTGCCAGCACGTGATGGTGGGAACTCAAGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG P atrosepticum (EF178668) AGCGCAACCCTTATCCTTTGTTGCCAGCGCGTAATGGCGGGAACTCAAAGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG P atrosepticum (EF530555) AGCGCAACCCTTATCCTTTGTTGCCAGCGCGTAATGGCGGGAACTCAAAGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACG Figure 2-1. cont.

PAGE 65

65 1180 1190 1200 1210 1220 1230 1240 1250 1260 . . D. zeae (AF520711) TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGTATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT D. dieffenbachiae (AF520712) TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGTATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT D. chrysanthemi (U80200) TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT Dickeya sp. 0723-1 V TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGTATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT Dickeya sp. 0723-2 V TCAAATCATCATGCCCCTTATGATCTGGGCTACACACGTGCTACAATGGTGGCTACAAAGGGAAGCAACCCTGTGAAGGTGAGCAAATCC Dickeya sp. 1114-4 P TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGTATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT Dickeya sp. 0827-3 O TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGTATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT Dickeya sp. 1114-3 P TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGTATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT Dickeya sp. 1015-2 T TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGTATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT Dickeya sp. 0911-2 T TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACGCGTGCTACAATGGCGTATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT Dickeya sp. 1015-4 T TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGTATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT Dickeya sp. 1114-16 P TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGTATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT Dickeya sp. 1015-5 T TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGTATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT Dickeya sp. 0911-3 T TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGTATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT Dickeya sp. 1015-1 T TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGTATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT Dickeya sp. 0911-1 T TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGTATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT Dickeya sp. 1015-3 T TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGTATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT Dickeya sp. 0827-2 O TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGTATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT Dickeya sp. 1114-1 P TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGTATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT Dickeya sp. 0827-4 O TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACATGCTACAATGGCGTATACAAAGAGAAGCGACCTCGCGAGGGCAAGCGGACCT Dickeya sp. 0827-1 O TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGTATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT D dadantii (AF520707) TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT D dianthicola (AF520708) TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT D paradisiaca (AF520710) TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGATCT P atrosepticum (EF178668) TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGTATACAAAGAGAAGCGAACTCGCGAGAGCCAGCGGACCT P atrosepticum (EF530555) TCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGTATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCT Figure 2-1. cont.

PAGE 66

66 1270 1280 1290 1300 1310 1320 1330 1340 1350 . . D. zeae (AF520711) CATAAAGTACGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG D. dieffenbachiae (AF520712) CATAAAGTACGTCGTAGTCCGGATTGGAGTCTGCAACTAGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG D. chrysanthemi (U80200) CATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG Dickeya sp. 0723-1 V CATAAAGTACGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG Dickeya sp. 0723-2 V CAAAAAGGCCATCCCAGTTCGGATTGTAGTCTGCAACTCGACTACATGAAGCTGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTG Dickeya sp. 1114-4 P CATAAAGTACGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG Dickeya sp. 0827-3 O CATAAAGTACGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG Dickeya sp. 1114-3 P CATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG Dickeya sp. 1015-2 T CATAAAGTACGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTGGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG Dickeya sp. 0911-2 T CATAAAGTACGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG Dickeya sp. 1015-4 T CATAAAGTACGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG Dickeya sp. 1114-16 P CATAAAGTACGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG Dickeya sp. 1015-5 T CATAAAGTACGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG Dickeya sp. 0911-3 T CATAAAGTACGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG Dickeya sp. 1015-1 T CATAAAGTACGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG Dickeya sp. 0911-1 T CATAAAGTACGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG Dickeya sp. 1015-3 T CATAAAGTACGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG Dickeya sp. 0827-2 O CATAAAGTACGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG Dickeya sp. 1114-1 P CATAAAGTACGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG Dickeya sp. 0827-4 O CATAAAGTACGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG Dickeya sp. 0827-1 O CATAAAGTACGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG D dadantii (AF520707) CATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG D dianthicola (AF520708) CATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG D paradisiaca (AF520710) CATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGGATCAGAATGCCGCGGTG P atrosepticum (EF178668) CATAAAGTACGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG P atrosepticum (EF530555) CATAAAGTACGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTG Figure 2-1. cont.

PAGE 67

67 1360 1370 1380 1390 1400 1410 1420 . D. zeae (AF520711) AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCT D. dieffenbachiae (AF520712) AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCT D. chrysanthemi (U80200) AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCT Dickeya sp. 0723-1 V AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCT Dickeya sp. 0723-2 V AATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCGGATATGCCCGAAGTCAGTGACCCAACCG Dickeya sp. 1114-4 P AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCT Dickeya sp. 0827-3 O AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTGACCT Dickeya sp. 1114-3 P AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCT Dickeya sp. 1015-2 T AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCT Dickeya sp. 0911-2 T AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCT Dickeya sp. 1015-4 T AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCT Dickeya sp. 1114-16 P AGTACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCT Dickeya sp. 1015-5 T AATACGTTCCCGGGCCATGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCT Dickeya sp. 0911-3 T AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCT Dickeya sp. 1015-1 T AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCT Dickeya sp. 0911-1 T AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCT Dickeya sp. 1015-3 T AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCT Dickeya sp. 0827-2 O AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCT Dickeya sp. 1114-1 P AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCT Dickeya sp. 0827-4 O AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCT Dickeya sp. 0827-1 O AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCT D dadantii (AF520707) AATACGTTCCCGGGCCTTGTACCCACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCT D dianthicola (AF520708) AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCT D paradisiaca (AF520710) AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCT P atrosepticum (EF178668) AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAG-------P atrosepticum (EF530555) AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAG-------Figure 2-1. cont.

PAGE 68

68 10 20 30 40 50 60 70 80 90 . . D. chrysanthemi (M33584) CTGTGGCCGATCAGGATGGTTTTGTCGTGCAGCTCGAAGCGGCTATTAGAAACGGTAACGTAGTCGGAGCCGCGCTTGATATCCAGCGAG [90] D. chrysanthemi (X17284) CTGTGGCCGATCAGGATGGTTTTGTCGTGCAGCTCGAAGCGGCTATTAGAAACGGTAACGTAGTCGGAGCCGCGCTTGATATCCAGCGAG [90] D. dadantii (CP001655) CTATGACCGATGAGCATGGTTTTGTCATGCTGTTCGAAACGACTGTTGGAGACCGTGACGTAGTCGGCGCCGCGCTTGATATCCAGCGCA [90] D. zeae (CP001655) CTGTGGCCGATCAGGATGGTTTTGTCATGCAGCTCGAAGCGGCTGTTAGAGATGGTGACGTAATCTGAACCGCGCTTGATATCCAGTGCA [90] Dickeya sp. 1015-5 T CTGTGGCCGATCAGGATGGTTTTGTCGTGCAGTTCGAAGCGGCTGTAAGAAATGGTGACGAAGTCGGACCCTTTCTTGATATCCAGCGCG [90] Dickeya sp. 1015-4 T CTGTGGCCGATCAGGATGGTTTTGTCGTGCAGTTCGAAGCGGCTGTTGGAAACGGTCACATAGTCGGAGCCGCGTTTGATATCCAGTGCG [90] Dickeya sp. 1015-1 T CTGTGGCCGATCAGGATGGTTTTGTCGTGCAGTTCGAAGCGGCTGTAAGAAATGGTGACGAAGTCGGACCCTTTCTTGATATCCAGCGCG [90] Dickeya sp. 1015-3 T CTGTGGCCGATCAGGATGGTTTTGTCGTGCAGTTCGAAGCGGCTGTAAGAAATGGTGACGAAGTCGGACCCTTTCTTGATATCCAGCGCG [90] Dickeya sp. 0911-2 T CTGTGGCCGATCAGGATGGTTTTGTCGTGCAGTTCGAAGCGGCTGTAAGAAATGGTGACGAAGTCGGACCCTTTCTTGATATCCAGCGCG [90] Dickeya sp. 0911-1 T CTGTGGCCGATCAGGATGGTTTTGTCGTGCAGTTCGAAGCGGCTGTTGGAAACGGTCACATAGTCGGAGCCGCGTTTGATATCCAGTGCG [90] Dickeya sp. 0911-3 T CTGTGGCCGATCAGGATGGTTTTGTCGTGCAGTTCGAAGCGGCTGTAAGAAATGGTGACGAAGTCGGACCCTTTCTTGATATCCAGCGCG [90] Dickeya sp. 1015-2 T CTGTGGCCGATCAGGATGGTTTTGTCGTGCAGTTCGAAGCGGCTGTAAGAAATGGTGACGAAGTCGGACCCTTTCTTGATATCCAGCGCG [90] Dickeya sp. 1114-1 P CTGTGGCCGATCAGGATGGTTTTGTCGTGCTGATCGAACAGGCTGTTGGAGATGGTAACGTAGTCGGAACCGCGTTTGATATCCAGCGCG [90] Dickeya sp. 1114-3 P CTGTGGCCGATCAGGATGGTTTTGTCGTGCTGATCGAACAGGCTGTTGGAGATGGTAACGTAGTCGGAACCGCGTTTGATATCCAGCGCG [90] Dickeya sp. 0827-1 O CTGTGGCCGATCAGGATGGTTTTGTCGTGCTGATCGAACAGGCTGTTGGAGATGGTAACGTAGTCGGAACCGCGTTTGATATCCAGCGCG [90] Dickeya sp. 0827-4 O CTGTGGCCGATCAGGATGGTTTTGTCGTGCTGATCGAACAGGCTGTTGGAGATGGTAACGTAGTCGGAACCGCGTTTGATATCCAGCGCG [90] Dickeya sp. 0723-1 V CTGTGGCCGATCAGGATGGTTTTGTCGTGCAGTTCAAAGCGGCTGTTGGAAATGGTGACATAGTCAGACCCTTTCTTGATATCCAGTGCA [90] Dickeya sp. 0723-2 V CTGTGGCCGATCAGGATGGTTTTGTCGTGCAGTTCAAAGCGGCTGTTGGAAATGGTGACATAGTCAGACCCTTTCTTGATATCCAGTGCA [90] 100 110 120 130 140 150 160 170 180 . . D. chrysanthemi (M33584) CCGTCATGCTGCACATATTTTTCGCCGTTTTTGGTGGTGTATTTGTCGTCGGTGAGGCTGCCGTCGCTGATGGTGACATGGTCAACCCAG [180] D. chrysanthemi (X17284) CCGTCATGCTGCACATATTTTTCGCCGTTTTTGGTGGTGTATTTGTCGTCGGTGAAGCTGCCGTCGCTGATGGTGACATGGTCAACCCAG [180] D. dadantii (CP001655) CCGTCGTGTTGCACATAGGTTTCACCGTCTTTGGTGGTGTACATATCGTCGGTAAAACTGCCGTCGCTGATGGTGACGTGATCGACCCAT [180] D. zeae (CP001655) CCGTCATGCTGGACATATTTTTCGCCATTTTTGGTGGTGTATTTGTCATCCGTAAAGCTACCGTCGCTGATGGTCACGTGATCAACCCAT [180] Dickeya sp. 1015-5 T CCGTCGTGCTGGACGTATTTTTCACCGTTTTTGGTGGTGTATTTGTCATCGGTGAAGCTGCCGTCGCTGATGGTAACATGGTCAACCCAG [180] Dickeya sp. 1015-4 T CCATCGTGCTGGACATATTTTTCGCCGTTTTTGGTGGTGTATTTGTCATCGGTGAAGCTGCCGTCGCTGATGGTAACATGGTCAACCCAG [180] Dickeya sp. 1015-1 T CCGTCGTGCTGGACGTATTTTTCACCGTTTTTGGCGGTGTATTTGTCATCGGTGAAGCTGCCGTCGCTGATGGTGACATGGTCAACCCAG [180] Dickeya sp. 1015-3 T CCGTCGTGCTGGACGTATTTTTCACCGTTTTTGGTGGTGTATTTGTCATCGGTGAAGCTGCCGTCGCTGATGGTGACATGGTCAACCCAG [180] Dickeya sp. 0911-2 T CCGTCGTGCTGGACGTATTTTTCACCGTTTTTGGTGGTGTATTTGTCATCGGTGAAGCTGCCGTCGCTGATGGTGACATGGTCAACCCAG [180] Dickeya sp. 0911-1 T CCATCGTGCTGGACATATTTTTCGCCGTTTTTGGTGGTGTATTTGTCATCGGTGAAGCTGCCGTCGCTGATGGTAACATGGTCAACCCAG [180] Dickeya sp. 0911-3 T CCGTCGTGCTGGACGTATTTTTCACCGTTTTTGGTGGTGTATTTGTCATCGGTGAAGCTGCCGTCGCTGATGGTGACATGGTCAACCCAG [180] Dickeya sp. 1015-2 T CCGTCGTGCTGGACATATTTTTCGCCGTTTTTGGTGGTGTATTTGTCATCGGTGAAGCTGCCGTCGCTGATGGTGACATGGTCAACCCAG [180] Dickeya sp. 1114-1 P CCATCGTGCTGGACGTAAGTTTCGCCGTCTTTGGTGGTGTACATGTCATCAGTAAAGCTGCCGTCACTGATGGTGACGTGGTCGACCCAC [180] Dickeya sp. 1114-3 P CCATCGTGCTGGACGTAAGTTTCGCCGTCTTTGGTGGTGTACATGTCATCAGTAAAGCTGCCGTCACTGATGGTGACGTGGTCGACCCAC [180] Dickeya sp. 0827-1 O CCATCGTGCTGGACGTAAGTTTCGCCGTCTTTGGTGGTGTACATGTCATCAGTAAAGCTGCCGTCACTGATGGTGACGTGGTCGACCCAC [180] Dickeya sp. 0827-4 O CCATCGTGCTGGACGTAAGTTTCGCCGTCTTTGGTGGTGTACATGTCATCAGTAAAGCTGCCGTCACTGATGGTGACGTGGTCGACCCAC [180] Dickeya sp. 0723-1 V CCGTCGTGCTGGACGTATTTTTCGCCGTTTTTAGTGGTGTATTTGTCATCGGTAAAGCTGCCGTCGCTGATGGTGACATGGTCAACCCAG [180] Dickeya sp. 0723-2 V CCGTCGTGCTGGACGTATTTTTCGCCGTTTTTAGTGGTGTATTTGTCATCGGTAAAGCTGCCGTCGCTGATGGTGACATGGTCAACCCAG [180] Figure 2-2. Clustal X DNA alignment of part ial pectate lyase gene sequences of 18 Dickeya isolates from orchids in Florida using primer pair ADE1/ADE2 (Nassar et al. 1996); they are from: V= Vanda, P= Phalaenopsis O= Oncidium and T= Tolumnia For comparison, sequences are included from GenBank accessions M33584, X17284, CP001655, and CP001655. A hyphen indicates a nucle otide deletion.

PAGE 69

69 190 200 210 220 230 240 250 260 270 . . D. chrysanthemi (M33584) ACGTGGTCTGTGCTGTCAAT---CACTACTGCGTCCCACTCGGCGTTCCAACCATCTCCCTCTTCGTAATGCGGCGCCACGTCCACCGGC [267] D. chrysanthemi (X17284) ACGTGGTCTGTGCTGTCAAT---CACTACTGCGTCCCACTCGGCGTTCCAGCCATCTCCCTCTTCGTAATGCGGCGCCACGTCCACCGGC [267] D. dadantii (CP001655) ACGTGCTGGGCGTTGTTGAT---GTTCATGCCATCCCATTCGGCGTTCCAGCCGTCACCGTCTTCATAGTGAGGTGCAACATCCACCGGG [267] D. zeae (CP001655) ACATGATCGGTACTGTCGAT---CACCACGGCGTCCCATTCAGCATTCCAGCCGTCCCCATCTTCATAATGCGGCGCCACGTCTACCGGC [267] Dickeya sp. 1015-5 T ACGTGGTCTGTGCTATCAAT---CACCACTGCGTCCCACTCGGCGTTCCAGCCATCCCCTTCTTCGTAATGCGGCGCTACGTCTACCGGC [267] Dickeya sp. 1015-4 T ACGTGGTCTGAGTTATCAAT---CACCGCGGCGTCCCACTCGGCGTTCCAACCATCCCCTGTTTCGTAATGCGGCGCCACATCCACCGGC [267] Dickeya sp. 1015-1 T ACGTGGTCTGAGTTATCAAT---CACCGCGGCGTCCCACTCGGCGTTCCAACCATCCCCTGTTTCGTAATGCGGCGCCACATCCACCGGC [267] Dickeya sp. 1015-3 T ACGTGGTCTGAGTTATCAAT---CACCGCGGCGTCCCACTCGGCGTTCCAACCATCCCCTGTTTCGTAATGCGGCGCCACATCCACCGGC [267] Dickeya sp. 0911-2 T ACGTGGTCTGAGTTATCAAT---CACCACTGCGTCCCACTCGGCGTTCCAGCCATCCCCTTCTTCGTAATGCGGCGCTACGTCTACCGGC [267] Dickeya sp. 0911-1 T ACGTGGTCTGTGCTATCAAT---CACCACTGCGTCCCACTCGGCGTTCCAGCCATCCCCTTCTTCGTAATGCGGCGCTACGTCTACCGGC [267] Dickeya sp. 0911-3 T ACGTGGTCTGAGTTATCAAT---CACCGCGGCGTCCCACTCGGCGTTCCAACCATCCCCTGTTTCGTAATGCGGCGCCACATCCACCGGC [267] Dickeya sp. 1015-2 T ACGTGGTCTGAGTTATCAAT---CACCGCGGCGTCCCACTCGGCGTTCCAACCATCCCCTGTTTCGTAATGCGGCGCCACATCCACCGGC [267] Dickeya sp. 1114-1 P ACGTGGTGAGCGCCGTTGGTGATGTTCATGCCATCCCATTCGGCATTCCAACCGTCGCCTTTTTCGTAATGCGGTTCTACGTCAATTGGC [270] Dickeya sp. 1114-3 P ACGTGGTGAGCGCCGTTGGTGATGTTCATGCCATCCCATTCGGCATTCCAACCGTCGCCTTTTTCGTAATGCGGTTCTACGTCAATTGGC [270] Dickeya sp. 0827-1 O ACGTGGTGAGCGCCGTTGGTGATGTTCATGCCATCCCATTCGGCATTCCAACCGTCGCCTTTTTCGTAATGCGGTTCTACGTCAATTGGC [270] Dickeya sp. 0827-4 O ACGTGGTGAGCGCCGTTGGTGATGTTCATGCCATCCCATTCGGCATTCCAACCGTCGCCTTTTTCGTAATGCGGTTCTACGTCAATTGGC [270] Dickeya sp. 0723-1 V ACGTGGTCTGTACTGTCAA---CCACCACCGCATCCCATTCTGCGTTCCAGCCATCCCCATCTTCGTAATGCGGCGCTACGTCTACCGGT [267] Dickeya sp. 0723-2 V ACGTGGTCTGTACTGTCAA---CCACCACCGCATCCCATTCTGCGTTCCAGCCATCCCCATCTTCGTAATGCGGCGCTACGTCTACCGGT [267] 280 290 300 310 320 330 340 350 360 . . D. chrysanthemi (M33584) GTTTCGATGTACAGGTTACGCAGGATAACGTTGCTGACGCCTTT---------CACCACCAGCGAACCGTTGGTGAATTTGCCTTTGTTG [348] D. chrysanthemi (X17284) GTTTCGATGTACAGGTTACGCAGGATAACGTTGCTGACGCCTTT---------CACCACCAGCGAACCGTTGGTGAATTTGCCTTTGTTG [348] D. dadantii (CP001655) GTTTCGATATAGACGTTACGAACGATGACGTTGGTCACATCCTT---------GATGATCAACGAGCCGTTGGTGAGTTTCGCATCAGTA [348] D. zeae (CP001655) GTTTCGATGTACAGGTTACGCAGGATAACGTTGCTGACGCCTTT---------CACAACCAGCGAACCGTTGGTGAATTTTCCTTTGTTG [348] Dickeya sp. 1015-5 T GTTTCGATATACAGGTTACGCAGGATAACGTTGCTGACGCCCTT---------CACCACCAGCGAACCGTTGGTGAATTTGCCTTTGCTG [348] Dickeya sp. 1015-4 T GTTTCGAGATACAGGTTACGCAGAATAACGTTGCTGACACCTTT---------CACTACCAGCGAACCGTTGGTGAACTTGCCGTTGCTG [348] Dickeya sp. 1015-1 T GTTTCGAGATACAGGTTACGCAGAATAACGTTGCTGACACCTTT---------CACTACCAGCGAACCGTTGGTGAACTTGCCGTTGCTG [348] Dickeya sp. 1015-3 T GTTTCGAGATACAGGTTACGCAGAATAACGTTGCTGACACCTTT---------CACTACCAGCGAACCGTTGGTGAACTTGCCGTTGCTG [348] Dickeya sp. 0911-2 T GTTTCGATATACAGGTTACGCAGGATAACGTTGCTGACGCCTTT---------CACCACCAGCGAACCGTTGGTGAATTTGCCTTTGCTG [348] Dickeya sp. 0911-1 T GTTTCGATATACAGGTTACGCAGGATAACGTTGCTGACGCCTTT---------CACCACCAGCGAACCGTTGGTGAATTTGCCTTTGCTG [348] Dickeya sp. 0911-3 T GTTTCGAGATACAGGTTACGCAGAATAACGTTGCTGACACCTTT---------CACTACCAGCGAACCGTTGGTGAACTTGCCGTTGCTG [348] Dickeya sp. 1015-2 T GTTTCGAGATACAGGTTACGCAGAATAACGTTGCTGACACCTTT---------CACTACCAGCGAACCGTTGGTGAACTTGCCGTTGCTG [348] Dickeya sp. 1114-1 P GTCTGGATGTAGACGTTACGGATGATGACGTTATTGGTGCCGTCGGTACCGTCGATAATCAGAGAACCGTTGATGAATTTGGCGTCGGCG [360] Dickeya sp. 1114-3 P GTCTGGATGTAGACGTTACGGATGATGACGTTATTGGTGCCGTCGGTACCGTCGATAATCAGAGAACCGTTGATGAATTTGGCGTCGGCG [360] Dickeya sp. 0827-1 O GTCTGGATGTAGACGTTACGGATGATGACGTTATTGGTGCCGTCGGTACCGTCGATAATCAGAGAACCGTTGATGAATTTGGCGTCGGCG [360] Dickeya sp. 0827-4 O GTCTGGATGTAGACGTTACGGATGATGACGTTATTGGTGCCGTCGGTACCGTCGATAATCAGAGAACCGTTGATGAATTTGGCGTCGGCG [360] Dickeya sp. 0723-1 V GTTT---------CGATATACAGGTTGCGTAGAATAACGTTGCTGACGCCTTTCACCACCAAAGAGCCGTTGGTGAATTTGCCTTTGTTA [348] Dickeya sp. 0723-2 V GTTT---------CGATATACAGGTTGCGTAGAATAACGTTGCTGACGCCTTTCACCACCAAAGAGCCGTTGGTGAATTTGCCTTTGTTA [348] Figure 2-2, cont.

PAGE 70

70 370 380 390 400 . D. chrysanthemi (M33584) CCAATACCGATGATGGTGGTGTTGGACGGAATGCTGATCTGGCTGCG [395] D. chrysanthemi (X17284) CCAATACCGATGATGGTGGTGTTGGACGGAATGCTGATTTGGCTGCG [395] D. dadantii (CP001655) CCGATACCGAAGATGGTGGTATTAGACGGGATGCTTAACTGGCTGCG [395] D. zeae (CP001655) CCGATGCCGATGATGGTGGTATTGGCGGGAATACTGATCTGGCTGCG [395] Dickeya sp. 1015-5 T TCGATACCGATGATGGTGGTATTGGACGGAATGCTGATCTGGCTGCG [395] Dickeya sp. 1015-4 T CCGATACCGATGATGGTGGTATTGGACGGAATGCTGATCTGGCTGCG [395] Dickeya sp. 1015-1 T CCGATACCGATGATGGTGGTATTGGACGGAATGCTGATCTGGCTGCG [395] Dickeya sp. 1015-3 T CCGATACCGATGATGGTGGTATTGGACGGAATGCTGATCTGGCTGCG [395] Dickeya sp. 0911-2 T TCGATACCGATGATGGTGGTATTGGACGGAATGCTGATCTGGCTGCG [395] Dickeya sp. 0911-1 T TCGATACCGATGATGGTGGTATTGGACGGAATGCTGATCTGGCTGCG [395] Dickeya sp. 0911-3 T CCGATACCGATGATGGTGGTATTGGACGGAATGCTGATCTGGCTGCG [395] Dickeya sp. 1015-2 T CCGATACCGATGATGGTGGTATTGGACGGAATGCTGATCTGGCTGCG [395] Dickeya sp. 1114-1 P CCAATCCCGATCACGGTGGTGTTAGCCGGAATGTTGATCTGGCTGCG [407] Dickeya sp. 1114-3 P CCAATCCCGATCACGGTGGTGTTAGCCGGAATGTTGATCTGGCTGCG [407] Dickeya sp. 0827-1 O CCAATCCCGATCACGGTGGTGTTAGCCGGAATGTTGATCTGGCTGCG [407] Dickeya sp. 0827-4 O CCAATCCCGATCACGGTGGTGTTAGCCGGAATGTTGATCTGGCTGCG [407] Dickeya sp. 0723-1 V CCAATACCGATGATGGTGGTGTTGGAAGGAATACTGATCTGGCTGCG [395] Dickeya sp. 0723-2 V CCAATACCGATGATGGTGGTGTTGGAAGGAATACTGATCTGGCTGCG [395] Figure 2-2, cont.

PAGE 71

71 Figure 2-3. Maximum likelihood phylogenetic analysis of Dickeya strains isolated from Florida orchids based on 16S rDNA gene sequences indi cates that the strains studied separate into two clades: Clade 1 contains the two Dickeya strains from Vanda and clade 2 contains the remaining or chid strains grouped with D. dadantii, D. dianthicola and D. paradisiaca. T = Tolumnia P = Phalaenopsis O = Oncidium V = Vanda.

PAGE 72

72 Figure 2-4. Bayesian phylogenetic analysis of Dickeya strains isolated from Florida orchids based on 16S rDNA gene sequences results in two orchid clades well separated from the six previously described Dickeya species (Samson et al. 2005). Similar to the ML analysis, the Vanda isolates form a distinct clade w ith high posterior probability value (100). T = Tolumnia P = Phalaenopsis O = Oncidium V = Vanda.

PAGE 73

73 Figure 2-5 Maximum Likelihood phylogenetic analysis of Dickeya strains isolated from Florida orchids based on a portion of the pelADE pectolytic enzyme ge ne cluster results in three clades that are distinct from D. chrysanthemi T = Tolumnia P = Phalaenopsis O = Oncidium V = Vanda.

PAGE 74

74 Figure 2-6. Bayesian analysis of Dickeya strains isolated from Flor ida orchids based on a portion of the pelADE pectolytic enzyme gene cluster re sults in three well supported clades. Tree topology is the same as the ML anal ysis (Figure 2-5) with high posterior probability values. T = Tolumnia P = Phalaenopsis O = Oncidium V = Vanda.

PAGE 75

75 Table 2-1. Orchid isolate collection information and initial tests for Dickeya genus determination (Schaad et al. 2001; Samson et al. 2005; Lee et al. 2006; Kaneshiro et al. 2008). Isolate Origin Orchid host Date collected CVP Indole production Indigoidine production Erythromycin sensitivity Oxidase Phosphatase (+)-DArabitol Sorbitol 0723-1 Leesburg, FL Vanda MCa 23 July 2007 + + + + + 0723-2 Leesburg, FL Vanda MC 23 July 2007 + + + + + 0827-1 Homestead, FL Oncidium MC 27 Aug 2008 + + + + + 0827-2 Homestead, FL Oncidium MC 27 Aug 2008 + + + + + 0827-3 Homestead, FL Oncidium MC 27 Aug 2008 + + + + + 0827-4 Homestead, FL Oncidium MC 27Aug 2008 + + + + + 0911-1 Homestead, FL Tolumnia fantasy Brandon 11 Sept 2008 + + + + + 0911-2 Homestead, FL Tolumnia fantasy Brandon 11 Sept 2008 + + + + + 0911-3 Homestead, FL Tolumnia fantasy Brandon 11 Sept 2008 + + + + +

PAGE 76

76 Table 2-1, cont. Isolate Origin Orchid host genus Date collected CVP Indole production Indigoidine production Erythromycin sensitivity Oxidase Phosphatase (+)-DArabitol Sorbitol 1015-1 Homestead, FL Tolumnia sp. 15 Oct 2008 + + + + + 1015-2 Homestead, FL Tolumnia sp. 15 Oct 2008 + + + + + 1015-3 Homestead, FL Tolumnia sp. 15 Oct 2008 + + + + + 1015-4 Homestead, FL Tolumnia sp. 15 Oct 2008 + + + + + 1015-5 Homestead, FL Tolumnia sp. 15 Oct 2008 + + + + + 1114-1 Homestead, FL Phalaenopsis MC 14 Nov 2008 + + + + + 1114-3 Homestead, FL Phalaenopsis MC 14 Nov 2008 + + + + + 1114-4 Homestead, FL Phalaenopsis MC 14 Nov 2008 + + + + + 1114-16 Homestead, FL Phalaenopsis MC 14 Nov 2008 + + + + + a MC=mericlone

PAGE 77

77 Table 2-2. 16S rDNA GenBank BLAST search and MIDI results for orchid strains. Based on these results and re sults in Table 2-1, the preliminary identification of strains isolated from Vanda, Phalaenopsis Oncidium and Tolumnia orchids from Florida is that they are Dickeya sp. Strain 16S rDNA MIDI Similarity (%) Strain/Accession # SIM Identification 0723-1 V 99 Erwinia chrysanthemi EU526397 0.901 E. chrysanthemi 0723-2 V 94 Erwinia chrysanthemi EU526397 0.906 E. chrysanthemi 0827-1 O 99 Pectobacterium chrysanthemi FM946179 0.880 E. chrysanthemi 0827-2 O 99 Pectobacterium chrysanthemi FM946179 0.929 E. chrysanthemi 0827-3 O 98 Pectobacterium chrysanthemi FM946179 0.887 E. chrysanthemi 0827-4 O 99 Pectobacterium chrysanthemi FM946179 0.910 E. chrysanthemi 0911-1 T 99 Pectobacterium chrysanthemi FM946179 0.723 E. chrysanthemi 0911-2 T 99 Pectobacterium chrysanthemi FM946179 0.795 E. chrysanthemi 0911-3 T 99 Pectobacterium chrysanthemi FM946179 0.931 E. chrysanthemi 1015-1 T 99 Pectobacterium chrysanthemi FM946179 0.961 E. chrysanthemi 1015-2 T 99 Pectobacterium chrysanthemi FM946179 0.907 E. chrysanthemi 1015-3 T 99 Pectobacterium chrysanthemi FM946179 0.963 E. chrysanthemi 1015-4 T 99 Pectobacterium chrysanthemi FM946179 0.775 E. chrysanthemi 1015-5 T 99 Pectobacterium chrysanthemi FM946179 0.815 E. chrysanthemi 1114-1 P 99 Pectobacterium chrysanthemi FM946179 0.808 E. chrysanthemi 1114-3 P 99 Pectobacterium chrysanthemi FM946179 0.887 E. chrysanthemi 1114-4 P 98 Pectobacterium chrysanthemi FM946179 0.795 E. chrysanthemi 1114-16 P 99 Pectobacterium chrysanthemi FM946179 0.832 E. chrysanthemi

PAGE 78

78 Table 2-3. Characteristics that differentiate D. dadantii ( D. dad.), D. zeae ( D. z .), D. chrysanthemi bv. parthenii ( D. ch. p.), D. chrysanthemi bv. chrysanthemi ( D. ch. ch .), D. dieffenbachiae ( D. dif .), D. dianthicola ( D. dia.), and D. paradisiaca ( D. par.) as described by Samson et al., (2005) compared to the Dickeya strains isolated from orchids used in this study and D. chrysanthemi reference strain ATCC #11663. Dickeya taxa described by Samson et al. (2005) Orchid isolates used in this study Characteristic D. dad. + D. z. (33)a D. ch p (4) D. ch. ch. (5) D. dif (5) D. dia (16) D. par. (6) D. chry. (control)b Vanda Isolates (2) Oncidium Isolates (4) Tolumnia Isolates (8) Phalaenopsis isolates (4) (-)-D Arabinose +c + + + + + + Inulin + V(88) + Lactose + V(75) V(20) V(17)+ (+)-DMelibiose + + + V(44) V(83)+ + + + + (+)-DRaffinose + + + V(44) V(83)+ + + + + Growth at 39 C + + + + V(83)+ + + + + Mannitol + + + + + + + + + + aNumber of strains bType specimen ATCC #11663 c+, 90-100% of strains positive; -, 90-100% of st rains negative; V(n) perc ent of strains positive.

PAGE 79

79 Table 2-4. Carbohydrate utiliz ation test results for Dickeya strains isolated from Florida orchids used in this study compared to reference strains Dickeya chrysanthemi ( D.ch .) ATCC #11663, Pectobacterium carotovora ( P.c.) and Pectobacterium cyprepedii ( P.cy .) ATCC#29267. Reference strains Orchid isolates used in this studya Carbohydrate D.ch P.c. P.cy. Vanda Isolates (2) Oncidium Isolates (4) Tolumnia Isolates (8) Phalaenopsis isolates (4) Amygdalin b V(50) D-Cellobiose + + + + + + D-Maltose + + D-Lactose + + + D-Trehalose + aIn addition to the carbohydrate resu lts reported in this table, ot her carbohydrates were tested ; however, those results were not di fferent that reference strain D. ch Orchid strains and D. ch were positive for glycerol, L-arabinose, D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-manose, L-rhamnose, inositol, D-manitol, N-acet ylglucosamine, arbutin, esculin ferric citrate, salicin, D-saccharose, and gentiobiose. All orchid strains tested were negative for erythritol, Lxylose, D-adonitol, methyl-D-xylopyranoside, L-sorbose, dulcitol, D-sorbitol, methylDmannopyranoside, methylD-glucopyranoside, D-melezito se, amidon (starch), glycogen, xylitol, D-turanose, D-lyxose, D-tagatose, D-fuco se, L-fucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-ketogluconat e, potassium 5-ketogluconate. b+, 90-100% of strains positive; -, 90-100% of strains negative; V( n) percent of st rains positive.

PAGE 80

80 CHAPTER 3 CULTURE OF Pseudocercospora SPECIES THAT AFFECT ORCHIDS Introduction Cercospora and related fungi ( Cercosporidium Pseudocercospora, Cercosporella Paracercospora) cause diseases on leaves, stems, pedicels, fruit, and bracts on numerous species in many plant families (Chupp 1954; Deighton 1973, 1976, 1979; Ellis 1971, 1976). More than 3,000 cercosporoid species have been descri bed (Solheim 1930; Chupp 1954; Deighton 1973, 1976, 1979; Ellis 1971, 1976), but only 659 are current ly recognized as valid (Crous & Braun 2003). Eight Cercospora or Pseudocercospora species have been asso ciated with at least 40 orchid genera: 1. P. dendrobii (H.C. Burnett) U. Braun & Crous (Burnett 1965; Hsieh & Goh 1990; Crous & Braun 2003), 2. P. peristeriae (H. C. Burnett) U. Braun & Crous comb. nov. (Burnett 1965; Crous & Braun 2003), 3. P. odontoglossi (Prill & Delacr.) U. Braun (C hupp 1954; Burnett 1964; Alfieri et al. 1994; Braun & Hill 2002), 4. P. cymbidiicola U. Braun & C. F. Hill sp. nov. (Braun & Hill 2002), 5. C. epipactidis C. Massalonga (Burnett 1965; Alfieri et al. 1994), 6. C. epidendronis Bolick, nom. nud. (Alfieri et al. 1994), 7. C. cyprepedii Ellis & Dearn (Alfieri et al. 1994; Crous & Braun2003), and 8. C. angraeci Feuillebois & Roum. (Crous & Braun 2003). Some cercosporoid fungi can be cultured easily and readily produce spor es in culture (Nelson & Campbell1990; Karaoglanidis & Bardas 2006). For example, C. cruenta Sacc. and C. canescens Ellis and Martin (Ekpo & Esuruoso 1978), C. zeae-maydis Tehon & E. Y. Daniels (Asea et al. 2005), and C. zebrina Pass. (Nelson & Campbell 1990) were cultured on V-8 juice agar, whereas other species were grown on othe r media (Balis & Payne 1971; Sah & Rush 1988;

PAGE 81

81 Hartman et al. 1991; Wang et al. 1998; Crous et al. 2001; Beilharz & Cunnington 2003). However, some cercosporoid species are notorio us for slow growth and reduced conidium production on artificial media (Nagel 1934; Ekpo & Esuruoso 1978). Stavely & Nimmo (1968) examined the relatio nship of pH and nutri tion to growth and sporulation of C. nicotianae Ellis & Everh. They determined that growth and sporulation was best on media that contained DL-l eucine, sucrose, and yeast extr act. Good results also were obtained on V-8 agar supplemented with tobacco extract. Sporulation was enhanced on V-8 medium when the pH was adjusted to 3.5-4.5 (Stavely & Nimmo 1968). Balis & Payne (1971) successfully cultured C. beticola Sacc. on a beet-leaf dextrose medium. Although cercosporoid species have been asso ciated with orchid hosts, very little information is available concerning their biology and pathogenicity, and no literature exists on appropriate media for their growth and sporulation. If these fungi were successfully cultured, it would be possible to conduct pat hogenicity tests, host-range studi es, pesticide trials, store and deposit isolates in culture coll ections, and increase our understa ndings of these fungi and the diseases they cause. The objective of this chapter was to develop methods to grow and sporulate orchidassociated Pseudocercospora species that could be used to produce mycelia and spores for experimental work. Materials and Methods Growth and Sporulation of P. dendrobii on Different Media Pseudocercospora dendrobii conidia were isolated from sporulating lesions on leaves of a hybrid Dendrobium collected in Homestead, FL. Initial identification of the fungus was based on its morphology and host association (Hsieh & Goh 1990). In addition, the 5.8S rDNA gene and flanking internal transcribe d spacers 1 and 2 (ITS1 and ITS2 ) were amplified using primers

PAGE 82

82 ITS4 (5-TCCTCCGCTTATTGATATGC-3) and ITS5 (5GGAAGTAAAAGTCGTAACAAGG-3) (protocol desc ribed in chapter IV) as described by White (1990) and compared to ITS sequence data in GenBank. Single spores were isolated directly from s porulating lesions and suspended in 500 L of sterile water, after which the suspension was briefl y vortexed, and spread onto water-agar plates. Plates were incubated at 25 C under a 12L:12D photoperiod for 24 h. After 24 h, plates were examined under a dissecting microscope and germin ated spores were cut out, placed on V-8 agar (44 mL of V-8 juice, 12 g of Bacto-agar (D ifco Laboratories, Detroit, MI) and 2.4 g of CaCO3/L), and incubated for 2 weeks at 25C 1C under fluorescent, 12L:12D, light; 6-mm plugs were then removed and placed fungus-side down on the following media: 1. V-8 (described above); 2. -V-8 (same recipe as V-8, except 11.1 mL of V-8 juice/L was added); 3. potato-dextrose agar (PDA) (24 g of PDA broth (Difco Laboratories) and 12 g of Bactoagar/L); 4. -PDA (same recipe as PDA, except 6 g of PDA broth/L was added); 5. water agar (WA) (12 g of Bacto-agar/L); 6. WA+dried, autoclaved Dendrobium leaves broken into small pieces sprinkled on top of the medium; 7. Dendrobium orchid-leaf agar (DOLA), which containe d an orchid-leaf extract (80 g of Dendrobium leaves boiled in 1 L of water, followed by filtration through 2 layers of cheese cloth and adjustment to a volume of 1 L), 19 g of Bacto-agar, 4 g of CaCO3, 5 g of sucrose, and 3.6 g of yeast extract/L; 8. DOLA+L, which contained the base medi um (DOLA) described above + 1.64 g of leucine/L; 9. DOLA+AS, which contained the base medium (DOLA) + 1.64 g of asparagine/L; and 10. DOLA+A, which contained the base medium (DOLA) + 1.64 g of alanine/L (Stavely & Nimmo 1968).

PAGE 83

83 The pH of all media was adjusted to 6.5 with 1 M HCl (Stavely & Nimmo 1968), and 25 mL of each was poured into sterile plastic 100 X 15 mm petri plates and cooled. The experiment was performed twice and replicated six times in a completely randomized design (CRD). Colony diameters were measured and plates examined for sporulation weekly for 3 weeks. Effects of Temperature on Growth and Sporulation of Pseudocercospora spp. on V-8 Agar Three single-spore isolates of Pseudocercospora recovered as described above, were transferred to fresh V-8 agar plates to assess the impact of temperature. The P. dendrobii isolate described above, and two from Weirsdale, FL, P. odontoglossi from a Cattleya mericlone and an undescribed from a Bulbophyllum hybrid, were incubated at 15, 20, 25 and 30C 1C under 12L:12D fluorescent light. The experiment was pe rformed twice and replicated eight times in a CRD. Colony diameters were measured on days 3, 7, 11, and 13, and plates were examined for sporulation under a dissecting microscope afte r a week and were destructively sampled by washing the plates with 1 mL of water at the end of weeks 2 and 3. Effects of Nutrient Reduction on Sporulation of Pseudocercospora sp. from a Tolumnia Orchid To determine if a sudden reduction in media nutrition has an effect on sporulation, an unreported cercosporoi d isolate from a Tolumnia orchid collected in Homestead, FL was used. Based on morphology (Hsieh & Goh 1990; Crous et al. 2001; Crous & Braun 2003) and sequence of the 5.8S rDNA and flanking ITS1 and ITS2 regions using primers ITS4 and ITS5 (White 1990), this isolate was confirmed as Pseudocercospora sp. (Crous et al. 2001). Singlespore cultures were made as described above, grown on V-8 agar for 7 days, 6-mm plugs were chopped into small pieces with a sterile scalpe l and spread on WA pl ates, and incubated in growth chambers at 15, 20, 25 and 30C 1C under constant fluorescent light (Ekpo & Esuruoso 1978). Cultures were examined afte r 3, 4, and 5 days for sporulation by flooding

PAGE 84

84 plates with 1 mL of sterile water followed by ge ntle rubbing with a sterile glass rod. Spore production was calculated by counting the num ber of spores present in three 10L drops, and averaging the number to estimate the number of spores mL-1. The experiment was performed twice and replicated five times in a CRD. ANOVAs were performed with the PROC GLM procedure in SAS (SAS Institute 2002), and treat ment means were separated using Fishers LSD test. Levenes test for homogeneity of variance was performed (Hill & Lewicki 2006). Results Growth and Sporulation of P. dendrobii on Different Media After 3 weeks, growth (colony diameter) wa s comparable on V-8, -V-8, PDA, -PDA, WA+leaves, DOLA, and DOLA+L and significantly greater than that on WA, DOLA+AL, and DOLA+AS media ( P <0.0001) (Fig. 3-1). However, mycelium was closely appressed to the surface ofthe -V-8, -PDA, DOLA+L, and WA+leav es media grew very close to the surface of the media, which would inhibit the remova l of mycelia for use in molecular analyses. Sequences obtained from the isolates used in this study were deposited in GenBank (P. dendrobii accession # TBA, P. odontoglossi accession # TBA, Pseudocercospora sp. from Bulbophyllum accession # TBA, and Pseudocercospora sp. from Tolumnia accession # TBA). Full-strength V-8 and PDA produced aerial my celia that were easily removed. DOLA and DOLA+L produced better mycelial growth than DOLA+A and DOLA+AS, but no medium that contained orchid leaf extracts performed bette r than the other media (Fig. 3-1). Sparse, inconsistent sporulation was observed on the -V-8, WA+leaves, PDA, and PDAAll media on which conidia were not reliable. Therefore, conidium production was not quantified, and subsequent experiments focused on increasing spore production. V-8 was used in the remaining experiments due to good mycelium production on it and its frequent use to sporulate other cercosporoid fungi (Beckman & Payne 1983; Nelson & Campbell1990; Wang et al. 1998;

PAGE 85

85 Stewart et al. 1999; Dunkle & Levy 2000; Goodwin et al. 2001; Asea et al. 2005; Karaoglanidis & Bardas 2006). Effects of Temperature on Growth and Sporulation of three Pseudocercospora spp. on V-8 Agar On day 3, P. dendrobii grew significantly better at 25C ( P <0.0001), as well as on day 7 ( P <0.0001), day 11 ( P <0.0001) and 13 ( P <0.0001) (Figure 3-2). Similarly, the Bulbophyllum isolate grew best at 25C on day 3 (P <0.0001), day 7 ( P <0.0001), day 11 ( P <0.0001), and day 13 ( P <0.0001). In contrast, on day 3 the P. odontoglossi isolate grew significantly better at 20, 25, and 30C than at 15C ( P <0.0001), while on day 7, 11, and 13, 25 and 30C produced the best growth ( P <0.0003, P <0.0001, and P <0.0001, respectively). Conidia were not observed on any of the media. Effects of Nutrient Reduction on Sporulation of Pseudocercospora sp. from a Tolumnia Orchid In each of two experiments, 6-mm-dia plugs from 7-day-old V-8 agar cultures of a P seudocercospora sp. from a Tolumnia orchid and transferred to WA. Conidia developed at 25C (3 days, mean of 837/mL; 4 days, 860/mL; and 5 days, 831/mL), but did not develop at 15, 20, or 30C. Discussion Many media have been used to culture cerco sporoid fungi (Balis & Payne 1971; Beckman and Payne 1983; Sah & Rush 1988; Hartman et al 1991; Wang et al. 1998; Crous et al. 2001; Beilharz & Cunnington 2003). Some species have fa irly specific nutritional and environmental requirements for growth and sporulatation. Fo r example, Ekpo & Esuruoso (1978) cultured C. cruenta and C. canescens on ten different media. Although neither species grew well on most media, C. cruenta grew well on V-8 and nutrient agar and C. canescens grew best on V-8 and

PAGE 86

86 PDA. Sporulation was observed on V-8, PDA, and cowpea-stem decoction agar; however, abundant sporulation was observed only on V-8 ag ar between 24 and 25C under constant light. Other cercosporoid fungi also grow well on V-8 juice agar Beckman & Payne (1983) concluded that C. zeae-maydis could grow on several media, but spore production was best on V-8 agar under 12 hr of fluorescent light per day. Asea et al. (2005) us ed V-8 agar to produce conidia of C. zeae-maydis for plant inoculations, as did Ka raoglanidis & Bard as (2006) with C. beticola Nelson & Campbell (1990) used V-8 agar to determine that C. zebrina Pass. grew best at 24C, whereas Wang et al. (1998) used V-8 to produce conidia of C. zeae-maydis isolates for molecular analysis. Conidia might also be need ed for taxonomic purposes. To these ends, V-8 agar has been used for several different cercosporoid fungi. In this study, P. dendrobii grew well equally on V-8, V-8, PDA, PDA, WA+leaves, DOLA, and DOLA+L. Although leaf extracts enhanced the growth of other cercosporoid fungi (Balis & Payne 1971; Hartman et al. 1991), orchid -leaf extracts did not increase the growth P. dendrobii Pseudocercospora dendrobii produced easily removed mycelia on V-8 and PDA which would facilitate DNA extractions for mol ecular analyses. Due to its multiple uses, V-8 agar was chosen for further temp erature and sporulation studies. Pseudocercospora dendrobii and a Pseudocercospora sp. isolated from a Bulbophyllum hybrid both grew significantly better at 25C than at 15, 20, or 30C. These results are similar to those of Ekpo & Esuruoso (1978), Beckman & Payne (1983), and Nelson & Campbell (1990) with other cercosporoid species. However, P. odontoglossi grew equally we ll at 25 and 30C; others cercosporoid species also grow at higher temperatures (S ah & Rush 1988; Hartman et al. 1991).

PAGE 87

87 Initial attempts to promote sporulation of P. dendrobii, P. odontoglossi, and a Pseudocercospora sp. isolated from a Bulbophyllum orchid were not successful on different media or at different temperatures. On naturally infected orchids, Pseudocercospora infections were most common on older leaves and, in most cases, sporulation oc curred as the leaf senesced or abscised (R. Cating, personal observation). Thus, it was hypothesized that a sudden change in nutrition might stimulate sporulation, a phe nomenon that was reported previously for C. cruenta and C. canescens (Ekpo & Esuruoso 1978). When a Pseudocercospora sp. from Tolumnia was transferred from V-8 to WA, consis tent, albeit low (822 to 867 conidia mL-1), sporulation was achieved at 25C under constant light These results suggest that a change in nutrient level may induce sporulation in other orchid -associated members of this genus. Different taxa should be tested, as should different sizes of transfer plugs to increase th e numbers of conidia that are produced. Plugs or mycelial fragments of an isolate of P. dendrobii from actively growing cultures were used to artificially inoculate Dendrobium orchids. After 6 months, no disease developed (data not shown). Although conidia of this fungus were not tested, others have demonstrated that hyphal fragments of a nonsporula ting cercosporoid fungus could be used as substitutes for conidia in pathogenicity studies (Donzelli & Churchill 2007). C onidia of another species that were harvested directly from infected leav es have been used in disease studies (e.g. P. mori (Hara) Deighton; Babu et al. 2002), but they woul d not be useful for pathogenicity tests and hostrange studies, due to uncertainties over their single or multiple species composition. Given the inability to reproduce symptoms on Dendrobium with what should have been useful inoculum of P. dendrobii it is possible that this fungus is a no npathogenic parasite on this host. The

PAGE 88

88 pathogenicity, or lack thereof, of this and other cercoporoid fungi on orchids needs to be investigated more thoroughly. In this study, P. dendrobii P. odontoglossi, and a Pseudocercospora sp. from Bulbophyllum were successfully cultured on V-8 agar at 25C under 12L:12D photoperiod. In addition, a Pseudocercospora sp. from Tolumnia produced conidia at 25C unde r constant light when plugs were transferred from actively growing V-8 agar cultures to WA. Additional work is needed to determine if the same procedure would induce ot her orchid-associated species to sporulate. Conidia from singlespore isolates would be preferred when fulfilling Kochs postulates and evaluating the host-range of differe nt taxa in the Orchidaceae. In addition, this procedure would assist those who need to identify isolates morphologically.

PAGE 89

89 Figure 3-1. Growth of Pseudocercospora dendrobii on 10 different media. (A) Colony diameter after 3 weeks. Blue bars re present media that produced significantly better growth than WA, DOLA+AL ( Dendrobium orchid-leaf agar + alanine), and DOLA+AS ( Dendrobium orchid-leaf agar + asparagine). Data for diameter were analyzed using the ANOVA procedure (P<0.0001) in SAS and the means were compared using LSD at P 0.05. Treatments with the same letter ar e not significantly different. Variances were not significantly different as determin ed by a Levenes test for homogeneity of variance. (B) Photo of fungal growth on media used in the experiment. 1 / 4 S t r e n g t h V 8 W A + D e n L e a v e s P D A V 8 D O L A 1 / 4 S t r e n g t h P D A D O L A + L W A D O L A + A L D O L A + A S Diameter (mm) 0 10 20 30 40 abc a ab bc d ab bc de e c A B

PAGE 90

90 Figure 3-2. Measurements of culture diameter at 3, 7, 11, and 13 d at 15, 20, 25, and 30 1C on V-8 agar. P. dendrobii (red) from a hybrid Dendrobium and Pseudocercospora sp. from a hybrid Bulbophyllum grew best at 25C on each day, while P. odontolgossi (green) from a Cattleya mericlone grew best at 2 0, 25, and 30C on day 3, but grew best at 25 and 30C on day 7, 11, and 13. Da ta for diameter were analyzed using ANOVA ( P <0.0001) in SAS and the means were compared using LSD at P 0.05. Variances were not significantly different as determined by a Levenes test for homogeneity of variance.

PAGE 91

91 CHAPTER 4 A COMPARISON OF THE STANDARD AND HIGH-FIDELITY POLYMERASE CHAIN REACTIONS (PCR) IN THE DETECTION OF Pseudocercospora odontoglossi FROM Cattleya ORCHIDS AND ITS USE IN THE DETECTION OF Sclerotium rolfsii AND A Dickeya sp. FROM Phalaenopsis ORCHIDS Introduction The polymerase chain reaction (PCR) has become widely used since the discovery (Chien et al. 1976) and subsequent use of heat-stable DNA polymerase for in vitro replication of DNA (Saiki et al. 1988). This elimin ated the task of adding fresh DNA polymerase at the beginning of each PCR cycle (Mullis & Faloona 1987), and led to the development of programmable thermocyclers that automated the procedure. Th e PCR is now used routinely in phylogenetic studies (Stewart et al. 1999; Crous et al. 2001; Palmateer et al. 2003; Chaverri et al 2005; Dettman et al. 2006), genomic analyses (Nadeau et al. 1992; Lashkari et al. 1997; Arneson et al. 2008), plant-disease diagnoses (Trout et al. 1997; Lvesque 2001; Mc Cartney 2003; Alvarez 2004; Ward et al. 2004; Yokomi et al. 2008), and to examine genetic diversity in plant pathogens (Urena-Padilla et al. 2002; Zhang et al. 2005; Winton et al. 2006). However, standard PCR usually does not amplify sequences of more than 5 kb and those between 2 and 5 kb are amplified inefficiently (Barnes 1994). Unlike sta ndard PCR, high-fidelity PCR (=long PCR) can amplify sequences of up to 35 kb (Barnes 1994). The addition of a small amount of the proofr eading enzyme to the reaction containing a nterminal deletion mutant of Taq polymerase was shown by Barnes (1994) to remove mismatched base pairs, allowing strand synthesis to proceed. The use of the proofreading enzyme alone does not amplify target DNA and may, due to its 3-e xonuclease activity, degrade excess primers in the reaction mix (Barnes 1994). In addition to producing longer sequences than standard PCR, high-fidelity PCR efficiently amplifies target DNA in the presence of larg e amounts of non-target DNA. Vickers & Graham

PAGE 92

92 (1996) used high-fidelity PCR to cons istently amplify a single-copy gene ( Bar ), a marker for the selection of transgenic plants, in the presen ce of barley genomic DNA; standard PCR only occasionally amplified the target gene. High-fi delity PCR also can detect bacterial infections and microbial associations in arthropods. Jeyaprakash & Hoy (2000) de monstrated that highfidelity PCR was more sensitive than standard PCR in detecting Wolbachia infections: 1 fg of plasmid DNA containing the wsp gene was amplified versus only 1 ng of plasmid with standard PCR. High-fidelity PCR was also more sensitive than standard PCR in detecting the citrus greening pathogen, Candidatus Liberobacter asiaticum, in th e presence of genomic DNA from the Asian citrus psyllid, citrus tr ees, or citrus psyllid parasitoids (Hoy et al. 2001). Furthermore, high-fidelity PCR has been used to: detect a nd characterize a new microsporidium species from the predatory mite Metaseiulus occidentalis (Nesbitt) (Becnel et al. 2002); identify and distinguish two parasitoids of th e brown citrus aphid (Persad et al. 2004); examine the microbial diversity of Metaseiulus occidentalis and its prey, Tetranychus urticae Koch (Hoy & Jeyaprakash 2005); and amplify 16S ribosomal se quences of endotoxin-pr oducing bacteria in varying amounts of dust mite DNA (Valerio et al. 2005). The objectives of this study were to: 1) determin e if high-fidelity PCR is more sensitive than standard PCR in detecting fungal DNA in the pr esence of plant genomic DNA; and 2) determine if high-fidelity PCR is more sensitiv e than standard PCR in detecting Sclerotium rolfsii Sacc. and Dickeya sp. (=Erwinia chrysanthemi ) directly from orchid tissues. Materials and Methods Evaluation of High-Fidelity PCR by Use of Plasmids Deoxyribonucleic acid (DNA) extraction Cattleya leaf tissue (3.866 g) was frozen in li quid nitrogen and ground in 8 ml of CTAB buffer (2% cetyltrimethylammonium bromid e, 100 mM Tris pH 7.5, 20 mM EDTA, 1%

PAGE 93

93 polyvinyl pyrrolidone, and 1.4 M NaCl) for 10 min and aliquoted into eight tubes. The samples were then incubated at 60C for 16 h. After two chloroform extractions, the eight DNA aliquots were combined into four and precipitat ed in 2-propanol and resuspended in 100 L of sterile water. All samples were pooled to make a 400L sample of genomic orchid DNA. Plant DNA quantity and quality was estimated using an Eppendorf BioPhotometer G131 V1.35 (Eppendorf, Hamburg, Germany). The leaf spot fungus Pseudocercospora odontoglossi was isolated from a Cattleya hybrid and identified based on morphological character s (Ellis 1976; Crous & Braun2003) and host. Single spores were grown on V-8 juice agar for 2 weeks at 25 C under artificial light at 12L: 12D photoperiod. A section of mycelium approxima tely 2 X 2 cm was scraped from the surface of the plate with a sterile wooden applicat or stick and placed in a 0.5-mL tube. 100 L of extraction solution (Extract-n-Amp, Sigma-Aldric h, St. Louis, MO) was added and the sample ground for 5 min with a sterile pl astic pestle and heated to 95 C for 10 min. 100 L of dilution solution (Extract-n-Amp, Sigma-Al drich) was added to the sample after which the sample was briefly vortexed. 30 L of the extracted DNA was added to 270 L of sterile water (Harmon et al. 2003). High-fidelity PCR protocol To compare high-fidelity and standard PCR, plasmids that contained ITS1, 5.8S, and ITS2 rDNA sequences, were generated with DNA extracted from Pseudocercospora odontoglossi as described above with high-fidelity PCR. The 50L reaction volume contained 50 mM TRIS, pH 9.2, 16 mM ammonium su lfate, 1.75 mM MgCl2 350 M each of dATP, dGTP, dCTP and dTTP, 800 pmol of primers (ITS4, 5-T CCTCCGCTTATTGATATGC-3 and ITS5, 5GGAAGTAAAAGTCGTAACAAGG-3) (White et al.1990), 1 unit of Accuzyme (Bioline, Taunton, MA) and 5 units of Taq DNA polymerase (Bioline) (Barnes 1994). Samples were

PAGE 94

94 covered with 100 L of sterile mineral oil and were am plified using three linked temperature profiles: (i) 94 C for 2 min; (ii) 10 cycles consisting of denaturing at 94 C for 10 s, annealing at 53 C for 30 s, and extension at 68 C for 1 min; (iii) 25 cycles consisting of 94 C for 10 s, annealing at 53 C for 30 s, and extension at 68 C for 1 min plus an additional 20 s during each consecutive cycle (Jeyaprakash & Hoy 2000; Hoy et al. 2001). Molecular Cloning The ITS1, 5.8S, and ITS2 rDNA sequences amplified by high-fidelity PCR from P. odontoglossi were cloned using the TOPO T/A cloning kit (Invitrogen Corp., Carlsbad, CA). Before ligation into the cloning vector, PCR pr oducts were cleaned using the QIAquick PCR purification kit (Qiagen, Valencia, CA) follo wing the manufacturers recommendations and eluted in 50 L of sterile gla ss-distilled, glass-collected water. A 3 A-overhang was added to the PCR product after purification to facilitate ligation into th e cloning vector by mixing the 50L DNA sample with 5.75 L of 10X highfidelity buffer (50 mM TRIS, pH 9.2, 16 mM ammonium sulfate, 1.75 mM MgCl2), 100 mM dATP, and 1 unit Taq polymerase (Bioline). The reaction was placed in a thermal cycler (P erkin Elmer DNA Thermal Cycler 480) at 72 C for 45 min. The product was immediately cloned into the TOPO T/A cloning vector following the manufacturers recommendations (In vitrogen Corp.). Transformed Escherichia coli colonies were selected from plates c ontaining X-GAL, IPTG, and ampicil lin and grown overnight in LB broth containing ampicillin at 37 C. Plasmids were extracted using the Qiagen Plasmid Mini Prep Kit (Qiagen, Valencia, CA) and digested with Eco RI restriction enzyme followed by gel electrophoresis on a 2% agarose TAE gel stained w ith ethidium bromide to confirm the correct size of the insert. Purified plasmids (pRC17) were sent to the Interdisciplinary Center for Biotechnology Research Core Facility at the Univ ersity of Florida, Gain esville, Florida for DNA sequencing.

PAGE 95

95 Comparison of Standard and High-Fidelity PCR Escherichia coli cells containing plasmid pRC17 we re grown in LB broth containing ampicillin and grown overnight at 37 C. The plasmids were extracted and purified using the Qiafilter Plasmid Midi Prep Kit (Qiagen) a nd quantified using a BioPhotometer G131 V1.35 (Eppendorf, Hamburg, Germany). To quantify the di fferences between the standard and the highfidelity PCR, serially diluted (1000 ng to 1 fg) plasmid pRC17 DNA was spiked with 10 ng of Cattleya genomic DNA. The high-fidelity PCR was the same as described above, and the standard PCR was performed in a 25-L reac tion volume and contained 2.5 L of 10X PCR Buffer + Mg (Boehringer, Mannheim, Germa ny), 200 M dATP, dGTP, dCTP and dTTP, 400 pM of primers ITS4 and ITS5 (Wh ite et al. 1990), and 0.2 units of Taq DNA polymerase (Bioline). Samples were covered with 50 L of sterile mineral oil and were amplified using the following temperature profile: (i) 94 C for 5 min; (ii) 35 cycles consisting of denaturing at 94 C for 30 s, annealing at 53 C for 30 s, and extension at 72 C for 1 min. Two negative controls were used in each set of high-fide lity and standard PCR reactions, one containing plant genomic DNA alone, the other without DNA. As a comparison, a set of standard PCR reactions were performed using the same serial diluted plasmid pRC17 DNA, but without the addition of 10 ng of plant DNA. All reactions were replicated twice on different days. Comparison of the High-Fidelity and Standard PCR in Detecting Sclerotium rolfsii and Dickeya sp. (Erwinia chrysanthemi) from Phalaenopsis Orchids At three times on different days, Phalaenopsis orchids were artificially inoculated with two economically important orchid pathogens: S. rolfsii and a Dickeya sp. previously known as E. chrysanthemi (Cating et al. 2008; Samson et al. 2005). The Sclerotium rolfsii isolate was recovered from an Ascocenda orchid, identified based on morphological characteristics and ITS1, 5.8S, and ITS2 rDNA sequences (Cating et al. 2009a), grown for 7 days on V-8 medium,

PAGE 96

96 and used to inoculate five Phalaenopsis plants, each with a 6-mm pl ug that was placed mycelium side down on the youngest leaf. Plants were placed in a greenhouse under 16L: 8D photoperiod at 25-30C and 56-88% RH. After 2 days, inoculated leaves were removed and washed in a 10% bleach solution for 30 s followed by two 1-min rinses with sterile water. Leaf tissue was patted dry with sterile paper towels and a 5 X 5 mm sec tion was removed from the margin of the lesion from each of the inoculated plants. With primer pair ITS4/ITS5 (White et al. 1990), high-fidelity and standard PCR were performed with DNA that was extracted from each of the five samples using the Extract-n-Amp (Sigma-Aldrich) protoc ol described above. Identification of the pathogen was confirmed by cloning and sequenc ing the PCR product us ing the procedures described above and comparing the sequence to th at that was deposited previously in GenBank (GQ358518) (Cating et al. 2009a). The Dickeya sp. was isolated from a Phalaenopsis orchid, and identified based on characteristics described by Sams on et al. (2005), including 16S rDNA sequence. It was grown on nutrient agar for 24 h at 27 C, suspended in sterile tap water, and adjusted to a concentration of 1 X 108 with a BioPhotometer G131 V1.35 (Eppendor f); into the youngest leaf of five Phalaenopsis plants, 100 L of the suspension was in jected. Plants were incubated in a greenhouse under 16L: 8D photoperiod at 26-30 C and 56-90% RH. After 24 h, symptoms developed and DNA was extracted using the procedure described above for S. rolfsii Highfidelity and standard PCR were performed with primer pair ADE1 (5GATCAGAAAGCCCGCAGCCAGAT -3) and ADE2 (5CTGTGGCCGATCAGGATGGTTTTGTCGTGC -3) (Na ssar et al. 1996). With the exception of a 63 C annealing temperature, the same PCR para meters were used. Identification of the pathogen was confirmed by cloning and sequencing the PCR product with the above procedures.

PAGE 97

97 To confirm that reactions worked properly, tw o positive controls were used in each set of reactions that consisted of genomic DNA from the S. rolfsii or the Dickeya sp. isolates extracted using the Puregene protocol pr eviously described. All reac tions were replicated twice on different days. Results Evaluation of High-Fidelity PCR by Use of Plasmids To determine if the high-fidelity PCR is mo re sensitive than the standard PCR, initial experiments were conducted in which serially di luted plasmids containing the 571-bp region of the ITS1, 5.8S, and ITS2 rDNA from P. odontoglossi were quantified and amplified in the presence of 10 ng of host DNA (Figure 4-1). High-fidelity PCR was six orders of magnitude more sensitive than the standard PCR when detecting P. odontoglossi DNA in the presence of host DNA: bright bands of the correct size were generated with high-fidelity PCR to 1 fg of target (Figure 4-1, A), whereas standard PCR detected only 1 ng of plasmid DNA (Figure 4-1, B). However, in the absence of host DNA standard PCR detected plasmid DNA as low as 10 fg (Figure 4-1, C). Thus, high-fideli ty PCR is more sensitive than standard PCR in the presence of plant DNA, but the efficiency of standard PCR was improved dramatically in the absence of plant DNA. No amplification was observed in either the no-DNA or plant-alone reactions and the same results were obtained both times the experiment was repeated. Comparison of the High-Fidelity and Standard PCR in Detecting Sclerotium rolfsii and Dickeya sp (Erwinia chrysanthemi) from Phalaenopsis Orchids High-fidelity PCR detected S. rolfsii in all five inoculated plants and both positive controls (Figure 4-2, A). However, when the same samp les were amplified using the standard PCR protocol, only the positive cont rols produced a band of the e xpected size (Figure 4-2, A). Similarly, high-fidelity PCR detected Dickeya sp directly from each of five inoculated plants,

PAGE 98

98 but standard PCR only amplified the positive control (Figure 4-2, B). To confirm the identity of the pathogens, the positive reactions, includ ing the controls, were cloned and sequenced according to the above method and determined to be the correct pathogen (data not shown). Discussion High-fidelity PCR was shown to be more sensit ive than standard PCR in detecting a fungal and bacterial pathogen of Phalaenopsis orchids. High-fidelity PCR c onsistently detected as little as 1 fg, in the presence of host DNA, which is eq uivalent to 207 copies of the ITS1, 5.8S, and ITS2 rDNA. These results are similar to those obtained by Jeyaprakash & Hoy (2000) who detected with high-fidelity PCR as little as 1 fg (=100 copes) of the Wolbachia wsp sequence in the presence of genomic DNA of the parasitoid wasp, Tamarixia radiata Waterston. In addition, when total DNAs from six arthropods ( Toxoptera citricida Kirkaldy, Diaphorina citri Kuwayama, Anastrepha suspensa Low, Phyllocnistis citrella Stainton, Diaphorencyrtus aligarhensis Shafee, Alam and Agarwal, and Tamarixia radiata) were amplified with ftsZand wsp -specific primers for Wolbachia five of the arthropods were positive for Wolbachia with high-fidelity PCR, but none were with standard PCR (Jeyaprakash & Hoy 2000). Similarly, Hoy et al. (2001) demonstrated that the high-fidelity PCR could detect as little as 1 fg of citrus greening pathogen DNA while in the presence of citrus DNA or citrus psyllid ( D. citri ) DNA. Although standard PCR is frequently used to dete ct plant pathogens, ther e are finite limits to its sensitivity. For example, Nielsen et al. (2002) detected between 1 and 10 pg of pure fungal DNA of Botrytis spp. that cause neck rot of onion, and Morrica et al. (1998) detected between 1 and 50 ng of Fusarium oxysporum f. sp. vasinfectum DNA from infected cotton. Tsai et al. (2006) could not amplify less than 1 pg of Phytophthora palmivora DNA, but amplified as little as 10 fg when a nested PCR protocol was use d. Diallo et al. (2009) found that competitor DNA

PAGE 99

99 of Pectobacterium carotovorum subsp. carotovorum or P. atrosepticum reduced detection limits of Dickeya sp. from 0.1 ng to 1.0 ng when added to PCR reactions. Latent infection, wherein plant pathogens cause symptoms long after infe ction occurs or only when certain conditions for hos t susceptibility are met, can considerably reduce a plant inspectors or diagnosticians abi lity to detect a given pathogen. Environmental factors such as temperature and moisture are critic al in disease development, and pathogens can remain latent in the host after infection if disease-conducive conditions are not met (Kotoujansky 1987; Toth et al. 2003; Palacio-Bielsa et al. 2006 ; Swanson et al. 2007). Since late nt pathogens can be difficult to detect on artificial media or with standard PCR protocols, false negatives/escapes from detection are not uncommon. The present results indicate th at the high-fidelity PCR protoc ol could become an important tool for diagnosticians and regulatory agencies, especially when pathogens are of quarantine significance, occur at low titers, are latent, or ca nnot be cultured. The sensitivity of this method, its utility with impure DNA samples, and amplifi cation of target DNA directly from plant tissue are all valuable attributes that contribute to its wide range of uses and users. According to Vincelli & Tisserat (2008), the use of nucleic acid-based technology for the detection of plant pathogens should be used more frequently in order to provide data to support a diagnosis, particularly for non-culturable organi sms, for pathogen species determination, and to contribute sequence data to GenBank. However, when using the PCR in a diagnostic setting, particularly when detecting plant pathogens directly from plant tissue, inhibited DNA amplification can be a major problem. Plan t cellular contents (o rganic and inorganic compounds), including host genomic DNA, can inte rfere with the efficiency of standard PCR (Vickers & Graham 1996; Wilson 1997; Vincelli & Tisserat 2008), making the diagnosis of plant

PAGE 100

100 pathogens directly from plant ti ssue extremely difficult and one of the most common factors that limit obtaining accurate results. To increase the applicability and reliability of the PCR in microbe detection, Wilson (1997) indicated that methods for the removal of PCR inhibitors were needed. For example, large amounts of genomic DNA, whether from the host or target organism, ca n inhibit PCR reactions (Tebbe & Vahjen 1993; Wilson, 1997; Hoy et al. 2001). Our results show that by incorporating a second polymerase with proof-reading ability, it was possible to reduce false negatives when the standard PCR was used to detect fungal and b acterial plant pathogens in the presence of such inhibitors. Thus, high-fidelity PCR may enable a dramatic improvement in the detection of a wide range of plant pathogens, especially when low template concentration, contaminating DNA, or amplification inhibitors exis t in a given sample. Many techniques exist to increase the sensitivit y of PCR. These include nested PCR (Ward et al. 2004), immunocapture (Louws et al. 1999 ; Ward et al. 2004), multiple displacement amplification (Dean et al. 2002), as well as ot hers (Vincelli & Tisserat 2008). However, highfidelity PCR is relatively simple and require s only the addition of sm all amounts of a proofreading enzyme to the Taq polymerase (Barnes 1994) and a sli ght increase in primer and dNTP concentrations due to the theore tical increase in product yield. Th erefore, plant disease clinics and diagnosticians that already own thermocycl ers can adopt high-fide lity PCR without the purchase of new equipment or addi tional training. By contrast, re al-time PCR is more sensitive than standard PCR, but requires the purchase of expensive equipment and requires additional training, making it cost-prohibitive in many cases.

PAGE 101

101 Figure 4-1. A comparison of high-fi delity and standard PCR using plasmid pRC17 containing the 571-bp ITS1, 5.8S, and ITS2 rDNA of Pseudocercospora odontoglossi as a template and primers ITS4/ITS5 (White et al. 1990). (A) High-fidelity PCR amplified as few as 207 copies (1 fg) of templa te in the presence of 10 ng of Cattleya DNA, while the standard PCR (B) required at least 200 million copies (1 ng) of template for amplification. In the absence of host genom ic DNA, the standard PCR protocol (C) amplified as little as 10 fg of templa te. M=molecular marker IV (Roche).

PAGE 102

102 Figure 4-2. A comparison of high-fi delity and standard PCR in th e detection of two important orchid pathogens from five inoculated Phalaenopsis plants. (A) S. rolfsii could be detected in all five plants using fungal ITS4/ITS5 (White et al. 1990) and the highfidelity PCR protocol (lanes 5-9), while it could be detected in no plants with standard PCR (lanes 14-18). Similarly in (B) the high-fidelity PCR could detect Dickeya sp. ( Erwinia chrysanthemi ) in inoculated plants using primers ADE1/ADE2 (Nassar et al. 1996) (lanes 5-9) while it could be detected in no plants with standard PCR (lanes 14-18). M=molecular marker XIV (Roche).

PAGE 103

103 CHAPTER 5 SILWET L-77 IMPROVES THE EFFICACY OF HORTICULTRAL OILS FOR CONTROL OF BOISDUVAL SCALE Diaspis boisduvalii (HEMIPTERA: DIASPIDIDAE) AND THE FLAT MITE Tenuipalpus pacificus (ARACHNIDA: ACARI: TENUIPALPIDAE) ON ORCHIDS Introduction The Orchidaceae is believed to be the largest fa mily of flowering plants (Bechtel et al. 1992; Dressler 1993; Tsavkelova et al. 2008) with approximately 19,000 named species (Atwood 1986; Dressler 1993). Orchids are the second most economically important flowering plant produced in the United States and sales continue to grow (Jerardo 2006). As orchid popularity continues to grow, so will the desire for pest and disease information, as illust rated in the question and answer section of Orchids magazine published by the American Orchid Society, which frequently contains questions submitted by hobbyists and co mmercial growers concerning the identification and control of orchid pests and diseases. Co mmon arthropod pests of orchids that can be difficult to control include the Boisduval scale Diaspis boisduvalii Signoret (Hemiptera: Diaspididae) and severa l species of mites. Boisduval scale is one the most important orchid pests and can be difficult to control using traditional chemical methods because females possess a hard covering that makes coverage difficult (Hamon 2002; Johnson 2009). Boisduval scal e is commonly introduced into an orchid collection on an infected plant and can move quickly to a variety of orchids (Johnson 2009). Mites also can be a major problem on cultivated orchids (Johnson 2008). Mite species that are known pests of orchids include the two-spotted spider mite ( Tetranychus urticae Koch, Arachnida: Acari: Tetranychidae) a nd several flat mites (Tenuipalpid ae) such as the orchid mite ( Tenuipalpus orchidarum Parfitt), the phalaenopsis mite ( Tenuipalpus pacificus Baker), and the oncidium mite (Brevipalpus oncidii Baker) (Johnson 2008).

PAGE 104

104 Many orchids, some of which are highly valuable or rare, exhibit phyto toxicity when treated with pesticides (Johnson 2008). Therefore, alternatives to traditional pesticides are desirable, especially for hobbyists and smaller orchid producers. Synthetic or ganic pesticides historically have been the primary tactic used in pest control on orchids. In order to preserve as many natural enemies as possible and have the least impact on the environment and human health, more alternatives to traditional pesticid es are needed. One alternative to traditional pesticides is Silwet L-77, an organosilicone surfactant. Surfactants commonly are used in agriculture to increase the spread of herbicides or pesticides on plants, and thus improve control of th e target pest or weed (Tu et al. 2001). Silwet L-77 has been shown to increase the effectiv eness of limonene for the control of mealybugs ( Pseudococcus longispinus Targioni-Tozzetti) by reducing th e surface tension around their waxcovered bodies (Hollingsworth 2005). Tipping et al (2003) demonstrated th at Silwet L-77 alone is toxic to Pacific spider mite ( Tetranychus pacificus McGregor) eggs, grape mealybug ( Pseudococcus maritimus Ehrhorn) crawlers, we stern flower thrips ( Frankliniella occidentalis Pergande), and cotton aphid ( Aphis gossypii Glover). In addition, S ilwet L-77 alone has been shown to be toxic to nymphs of the Asian citrus psyllid ( Diphorina citri Kuwayama) and can significantly increase mortality of D. citri eggs when applied with one -fourth the label rates of imidacloprid or abamectin or of adults when appl ied using one-fourth or on e-half the label rate of imidacloprid (Srinivasan et al. 2008). Silwet L-77 also can increase the e fficacy of fungicides. Silwet L-77 or another surfactan t (Kinetic) improved the activity of the protectant fungicide maneb using potato early blight or dry bean rust as model systems (Gent et al. 2003). Thus, the addition of Silwet L-77 as an adjuvant to chemi cal pesticides has been shown to increase their efficacy, even at lower-than-label rates.

PAGE 105

105 The objectives of this study were to (1) de termine if Silwet L-77 can be used without causing phytotoxicity to eight comm only cultivated orchid genera, and (2) determine if Silwet L77 can increase the effectiveness of 3 hor ticultural oils agai nst Boisduval scale (Diaspis boisduvalii) and the flat mite ( Tenuipalpus pacificus) on orchids. Materials and Methods Phytotoxicity Study Representatives of seven commonly cultivated orchid genera obtai ned from commercial orchid growers in south Florida were used: Doritaenopsis Luchia Lip Sog. F714 X Han-Ben Girl RL in 10.2-cm pots, Cymbidium Golden Elf in 15.2-cm pots, Dendrobium cultivars Blue Sampran, Burana Stripe, Lady Pink, Lena Pink, Pegasus, and Salaya Fancy, all in 10.2-cm pots, Epidendrum mericlones in 10.2-cm pots, Oncidium KBR in 10.2-cm pots Paphiopedilum Maudiae types in 10.2-cm pots, and Cattleya mericlones in 10.2-cm pots (Figure 5-1). Plants received preand post-spray ratings based on over all vigor, color, and pr esence/absence of spots or necrotic areas on leaves, stems, flowers, or flower buds. Plan ts received a quality rating on a scale from 1 to 10, with 10 being undamaged and 1 being so severely damaged that the plant could not be sold. Plants were watered and fert ilized as appropriate for each type of orchid. Plants were treated within 1 hr of the pre-spray assessment. Treatments consisted of a water control or Silwet L-77 (99.5% polyalkyleneoxide modified he ptamethyltrisiloxane, Helena Chemical Co.) at 0.05% (v:v) and the number of replicates of each orchid type is shown in Table 1. Plants, including inflorescences, were sprayed twice at 7-d inte rvals with hand sprayers until completely covered and assessed for phytotoxicity damage one week after the second spray. Plants (preand post-spray) we re kept in a greenhouse at the Department of Entomology and Nematology, University of Florida, Gainesville, FL at temperatures ranging from 21.0 to 35.0C and 45 to 95% RH and 16L:8D photoperiod from th e time the plants were obtained until the end

PAGE 106

106 of the experiment. Statistical analysis was performed by using the PROC GLM procedure in SAS (SAS Institute 2002) to compare the pre-spray rating with the control, and the post-spray rating with the control. Levenes test fo r homogeneity of variance was performed (Hill & Lewicki 2006). Silwet + Oil Efficacy Tr ial (Boisduval Scale) A commercial Cattleya orchid grower in central Flor ida with a heavy Boisduval scale infestation participated in the efficacy trial. Thirty Cattleya orchid hybrids and mericlones in 15.2-cm pots were selected based on size and severi ty of scale infestation (200+ female scales per plant and nymphs and males were so abundant that much of the foliage appeared white) (Figure 5-2). Preand post-spray assessments were performed 2 ways: 1) The density of female scales present was estimated according to the following scale: 0: 0 scales ; 1: 1-20 scales; 2: 2140 scales; 3: 41-60 scales; 4: 61-80 scales; 5: 81100 scales; 6: 101-120 scales; 7: 121-140 scales; 8: 141-160 scales; 9: 161180 scales, 10: 181-200+ scales. 2) Mortality was calculated by examining 10 randomly chosen female scales remaining on each plant after the post-spray assessment. Each scale was examined with a ha nd lens to determine if it was alive or dead. Female scales were used in both assessments because males tend to cluster, making them difficult to count. Two separate trials were conducted. In the fi rst trial, 2% (v:v) petroleum oil 435 (Growers 435, Growers Fertilizer Co., Lake Alfred, FL) was used and in the second trial 1% (v:v) Prescription Treatment Ultra-Fine Oil (Whitmir e Micro-Gen Research La boratories, Inc., St. Louis, MO) was used. Thirty Cattleya orchids were selected as described above and used in each trial (10 water controls, 10 oil treatments and 10 oil + 0.05% (v:v) Silwet treatments). Because Silwet L-77 is not labeled for use as a pesticide, it was only used in co mbination with petroleum oil. Plants were sprayed to run-off using a 3.8-L pump sprayer (H. D. Hudson Manufacturing

PAGE 107

107 Company, Hastings, MN). Plants were treated within 1 hr of th e pre-spray assessment. Plants were kept in a greenhouse at 18.0 to 27.0 C and 60-80% RH. Plants in each trial were treated once a week for 2 weeks and the post-spray a ssessment was done 1 week after the second treatment. Each trial was performed twice. Silwet + Oil Efficacy Trial (Flat Mite) Two types of orchids (Grammatophyllum and Dendrobium) that were infested with flat mites were obtained from 2 commercial orchid growers in south Florida. Samples were sent to the Florida Division of Plant Industry, Gainesville, Florida for species id entification and were confirmed to be Tenuipalpus pacificus Pre-spray assessments of mite densities were performed by randomly selecting a single leaf of each plant and counting the number of adult mites present. The leaf was marked and the same leaf wa s used in the post-spray assessment. Nine Grammatophyllum orchids in 15.2-cm baskets or nine Dendrobium orchids in 10.2-cm pots were used in the efficacy trial and cons isted of 3 water controls, 3 oil treatments (Prescription Treatment Ultra-pu re Oil, Whitmire Micro-Gen Re search Laboratories, Inc., St. Louis, MO) and 3 oil (Prescription Treatment Ul tra-pure) + 0.05% (v:v) Silwet treatments. Plants were sprayed to run-off using a 3.8L pump sprayer (H. D. Hudson Manufacturing Company). Plants were treated within 1 hr of the pre-spray assessment. Plants (preand post-spray) were kept in a greenhouse at the Tr opical Research and Education Center, University of Florida, Ho mestead, FL, under 50% shade with temperatures ranging from 18 to 30.5 C and 45-80% RH. Post-spray as sessment was performed 24 h after spraying. The Dendrobium trial was repeated once but, due to a lack of infested plants, the Grammatophyllum trial was not repeated. Statistical analysis for the Bo isduval trial and the flat mite trial was performed by using the PROC GLM procedure in SAS (SAS Institute 20 02) and treatment means were separated using

PAGE 108

108 Fishers LSD test. Levenes test for homogene ity of variance was performed Hill and Lewicki 2006). Results Phytotoxicity Study No evidence of phytotoxicity (burning, chloro sis, spots or other types of lesions) was observed on any of the 7 orchid genera test ed in this study incl uding leaves, stems, inflorescences or roots and there was no significa nt difference between the preand post-spray quality ratings (Table 5-1; Figur e 5-1). Of the 117 orchids (all ge nera) used in the phytotoxicity study, 114 received the same rating or better after treatment with Silwet L-77, while 3 of the orchids received a reduced rating. Of these 3, 2 were water controls (rati ngs reduced from 9 to 8.5 and 6 to 5) and 1 was treated with Silwet L-77 (7 to 6.5). Silwet + Oil Efficacy Tr ial (Boisduval Scale) When applied at a rate of 0.05% (v:v), Silwet L-77 significantly increased the efficacy of the 435 light horticultural oil (experiment 1a: P <0.0001, experiment 1b: P <0.0001) and the Prescription Treatment UltraFine oil (experiment 2a: P <0.0001, experiment 2b: P <0.0001) in reducing Boisduval scale populations on the Cattleya orchids in each experiment under the described conditions (Table 5-2; Figures 5-2 and 5-3). No phyt otoxicity on leaves, flowers, flower buds, or roots (data not shown) was observed in these experiments. Mortality of female Boisduval scale was increase d with the addition of 0.05% Silwet L-77 to each oil in each experiment and ranged from 86 to 93%, which is significantly better than the oil alone (39 to 47%) or the wa ter control (2% to 8%) ( P <0.0001) (Table 5-2). Silwet + Oil Efficacy Trial (Flat Mite) Silwet L-77 significantly improved the efficacy of the Prescription Treatment Ultra-Pure oil in reducing the number of flat mites on Dendrobium orchids in both experiments (experiment 4a:

PAGE 109

109 P =0.0017, experiment 4b: P <0.0001) (Table 5-3). However, th ere was no significant difference between the oil or oil + Silwet L-77 treatments when used to treat Grammatophyllum orchids for flat mite infestations, although the oil alone and the oil + Silwet L-77 treatments were both significantly better than the wa ter control (experiment 3: P <0.0001). Discussion The results show that the addition of 0.05% Silwet L-77 to petroleum oil 435, Prescription Treatment Ultra-fine oil, or Prescription Treatment Ultra-pure oil significantly increased the efficacy of these oils against the Boisduval scale and the flat mite (Tables 5-2, 5-3). Previous research has demonstrated that Silwet L-77 alon e is toxic to several other important arthropod pests (Tipping et al. 2003; Cowles et al. 2000; Hollingsworth 2005; Wood et al. 1997; Liu & Stansly 2000; Imai et al. 1995; Purcell & Schr oeder 1996; Srinivasan et al. 2008; Cocco & Hoy 2008). This study demonstrates, for the first tim e, that Silwet L-77 could be an important management tool to increase th e efficacy of oil against Boisduval scale and the flat mite on orchids. The reason for the increased efficacy is not clear. Silwet L-77 greatly reduces the surface tension of water, allowi ng it to infiltrate plant leaf stomata (Neumann & Prinz 1974) and hydathodes (Zidack et al. 1992). It is plausible that th is reduction in surface tension of water around the body of an arthropod may allow an infiltr ation of water into th e spiracles, drowning the arthropod (Cowles et al. 2000; Shapiro et al. 2009). In the present study, three petroleum oils were us ed. After the initial work with the 435 light horticultural oil, the study was repeated using oil labeled for use on orchids, the Prescription Treatment Ultra-Fine oil. Upon completion of the experiments usi ng the Ultra-Fine oil, we were informed by the manufacturer that it would no longer be available, but a replacement, Prescription Treatment Ultra-Pure oil, was available. The a ddition of 0.05% (v:v) Silwet L-77 significantly increased the efficacy of all 3 oils tested against the Boisduval scale on Cattleya

PAGE 110

110 orchids (Table 5-2) or the UltraPure oil against the flat mite on Dendrobium orchids (Table 5-3) when compared to the water control. There was no significant difference between the oil alone and the oil + Silwet L-77 treatment when treating Grammatophyllum orchids for flat mite infestations (Table 5-3), pe rhaps due to the fact that Grammatophyllum orchids have large, thin leaves that show very little pitting as a result of feeding by the flat mite. In contrast, Dendrobium orchids have smaller, thicker leaves that produce deep pitting from flat mite feeding, and fl at mites are often located in these pits. Because the pitting the Grammatophyllum orchid foliage was very superficial, the mites were easily removed by both oil or oil + Silwet whereas the addition of the S ilwet L-77 to oil appeared to better penetrate into pits containing flat mites on the Dendrobium leaves, removing them. Due to the lack of available Grammatophyllum orchids infested with mite s, the experiment was only performed once and should be repeated. It was discovered after experiment 1a (Table 5-2) that the addition of oil to Silwet L-77 not only increased mortality of Boisduval scale, but also removed them from the plant (Figure 5-3 B,C ). This property could be be neficial where tolerance for arth ropod infestations is essentially zero and plants are sold and appreciated base d on aesthetic value (Bethke & Cloyd 2009). However, as suggested by Tipping et al. (2003) and Liu and Stansl y (2000) it is essential that 100% coverage is achieved to obtain adequate control. Tipping et al. (2003) demonstrat ed that Silwet L-77 alone in creased mortality of Western flower thrips, Pacific spider mite, and cotton aphid, ( 93.8%), but 100% mortality was not achieved, possibly due to lack of complete coverage. Liu and St ansley (2003) also suggest that complete coverage is necessary for good contro l due to contact activity. For orchids, this includes the abaxial and adaxial leaf surfaces, stems, pseudob ulbs, inflorescences, under sheaths

PAGE 111

111 ( Cattleya orchids), and possibly the root s. This requirement may precl ude the use of oil + Silwet L-77 by large commercial orchid growers where pesticides are generally delivered to large groups of plants by irrigation or by large ove rhead booms, which may make it difficult obtain complete coverage. In addition, because there is no residual activity, re -applications may be necessary to obtain a cceptable control. Several caveats must be considered before using Silwet L-77 + petroleum oil to treat arthropod infestations in orchids. Although no ev idence of phytotoxicity was observed in any of the orchids used in this study, repr esentatives of only 7 genera of or chids were tested. It is quite possible that other genera, intergeneric hybrids, or certain cultivars may be susceptible to phytotoxicity. It is recommended th at growers check a representative of the plants to be treated for signs of phytotoxicity before extending treatme nt to large groups of plants. In addition, environmental conditions may play a role in phyt otoxicity, so label recommendations for the oil should be followed. Humidity also may be important for increased efficacy and should be taken into consideration. Liu and Stansley (2003) s uggest that environmental conditions (drying) may affect efficacy, and Imai et al. ( 1995) demonstrated that the toxicity of Silwet L-77 to the green peach aphid Myzus persicae (Sulzer) increased from 23.8% 7.2 at 30% RH to 99.3% 0.46 at 90% RH. It has been suggested that th e use of an adjuvant, such as Silwet L-77, may increase the incidence of bacterial diseases (G ottwald et al. 1997). The decrea se in water tension could allow entry sites for plant pathogenic ba cteria into leaf stomata (Melo tto et al. 2008), which may allow bacteria to gain entry and cause infection (Gottwald et al. 1997; Zidack et al. 1992). However, no bacterial diseases were observed on any of the orchids tested throughout this study. This

PAGE 112

112 could be due to the absence of bacteria or a conducive environment, so caution should be exercised if it is known that pat hogens are present or th ere is a history of bacterial infections. There are several advantages to using oil + Silwet L77 to control orchid arthropod pests. No arthropod is known to have de veloped resistance to petr oleum oil (M. A. Hoy, Dept. Entomology, University of Florida, personal co mmunication). Therefore, Silwet L-77 could be used as an adjuvant with petroleum oils to in crease their effectiveness with the development of resistance a minimal concern (Butler et al. 1993 ). Also, the use of a petroleum oil with the addition of Silwet L-77 is safer and may be less e xpensive than some pesticides (Liu & Stansley 2000) and may assist in the conser vation of natural enemies, one of the goals of an integrated pest management program. Wood et al. (1997) note d that Silwet L-77 had little or no effect on adults or larvae of lady beetles and green lacew ings, suggesting that Silwet L-77 could be an important tool for an integrated pest management program We can conclude that Silwet L77 increases the efficacy of oil against Boisduval scale and the flat mite on orchids. Because complete covera ge is required, its use may be most relevant to hobbyists or small commercial growers who want to use a chemical contro l with reduced toxic effects on non-target organisms, the environment, and human health.

PAGE 113

113 Table 5-1. Pre-spray and post-spray qua lity ratings (range=1-10) of orchids used in the phytotoxicity study. Orchid genera tested Treatment n Mean pre-spray quality rating SEa P Mean postspray quality rating SEa P Dendrobium hybrids Water 10 7.9 .4 0.27 8.1 .3 0.74 Silwet 10 7.3 .4 7.9 .2 Oncidium KBR 456 Water 10 8.1 .2 0.55 8.3 .2 0.66 Silwet 10 8.3 .2 8.4 .2 Doritaenopsis hybrids Water 10 7.2 .1 0.78 8.0 .0 1.00 Silwet 7 7.1 .1 8.0 .0 Paphiopedilum maudiae type Water 7 5.6 .3 0.16 6.0 .4 0.84 Silwet 8 6.1 .2 6.0 .3 Epidendrum MC Water 10 6.5 .3 0.88 7.2 .2 0.28 Silwet 7 6.5 .2 6.7 .2 Cymbidium Golden Elf Water 10 7.8 .1 0.63 7.9 .1 1.00 Silwet 10 7.7 .2 7.9 .1 Cattleya MC Water 3 6.3 .3 0.37 6.7 .8 0.12 Silwet 3 6.0 .0 6.0 .0 aMeans were analyzed using the PROC GLM procedure of the SAS software program. Levenes test for homogeneity of variances showed equal variances within ea ch orchid genus. Plant receiv ed a quality rating on a scale from 1 to 10, with 10 being undamag ed and 1 being so severely damaged that the plant could not be sold

PAGE 114

114 Table 5-2. Evaluation of Silwet L-77 ( 0.05%) in combination with two oils for co ntrol of Boisduval scale infestations in Cattleya mericlone orchids. Mean post-spray rating of scale density SEac Scale mortality (%) SEbc Date, environmental conditions at time of treatment, and type of oil used n Mean pre-spray rating of scale densitya Scale mortality (%) Water Oil Silwet + Oil P Experiment 1a, Sept. 22, 2007. 25.5C, 64% RH. Petroleum Oil 435 (2%) 30 10.0 0.0 10.0 .0a 2.0 2.0a 8.4 1.1a 44.0 10.8b 3.7 .7b 93.0 5.0c <0.0001 <0.0001 Experiment 1b, Oct. 27, 2007. 24.4C, 62% RH. Petroleum Oil 435 (2%) 30 10.0 0.0 10.0 .0a 8.0 2.9a 6.2 .6b 47.0 3.7b 1.8 .4c 86.0 5.0c <0.0001 <0.0001 Experiment 2a, Nov. 10, 2007. 25.5C, 70% RH. Prescription Treatment Ultra Fine Oil (1%) 30 10.0 0.0 9.8 .1a 3.0 2.1a 8.7 .3b 39.0 4.8b 2.2 .4c 89.0 6.6c <0.0001 <0.0001 Experiment 2b, Nov. 26, 2007. 25.0C, 62% RH. Prescription Treatment Ultra Fine Oil (1%) 30 10.0 0.0 9.7 .2a 2.0 1.3a 7.3 .2b 46.0 3.1b 1.4 .2c 91.0 2.3c <0.0001 <0.0001 aAssessment rating of scale density based on number of female scales present. 0: 0 scales; 1: 1-20 scales; 2: 2140 scales; 3: 4 1-60 scales; 4: 61-80 scales; 5: 81-100 scales ; 6: 101-120 scales; 7: 121-140 scales; 8: 141-160 scales; 9: 161-180 scales, 10: 181200+ scales. bMortality was calculated by randomly selecting 10 remaining scales on each plant and dete rmining if they were alive or dead by using a metal probe and a hand lens.cTreatment means were analyzed us ing the PROC GLM pro cedure and means separated using Fishers LSD with P =0.05. Levenes test for homogeneity of vari ance was also performed. Treatments with the same letter within a row are not significantly different from each other.

PAGE 115

115 Table 5-3. Evaluation of Silwet L-77 (0.05%) in combination with Prescription Trea tment Ultra Pure Oil fo r control of flat mit e ( Tenuipalpus pacificus ) infestations in Dendrobium or Grammatophyllum orchids. Mean % mites removed SE after 24 ha Type of orchid used and environmental conditions at time of treatment n Water Oil Silwet + Oil P Experiment 3. Grammatophyllum orchids. Jan.11, 2009. 30.5 C, 49% RH 9 27.7 .8a 94.8 .7b 98.7 1.3b <0.0001 Experiment 4a, Dendrobium orchids. Jan.14, 2009. 20.8 C, 53% RH 9 25.5 .2a 65.4 .6b 98.8 .2c 0.0017 Experiment 4b, Dendrobium orchids. Jan. 22, 2009. 24.5 C, 60% RH 9 19.6 .6a 38.1 .9b 98.4 .6c <0.0001 aTreatment means were analyzed using the PROC GLM pr ocedure and means separated using Fishers LSD with P =0.05. Levenes test for homogeneity of variance was also performed. Treatments with the same letter within a row are not significantly different f rom each other.

PAGE 116

116 Figure 5-1. Examples of orchids used in the phyto toxicity study after post-spray quality rating. None of the orchids developed symptoms of phytotoxicity on leaves, stems, flowers, buds or roots. A) Flowering Dendrobium and Phalaenopsis orchids; B) Doritaenopsis Luchia Lip Sog. F714 X Han-Ben Girl RL in 10.2-cm pots; C) Paphiopedilum Maudiae type; D) Flower of a Cattleya mericlone. A B C D

PAGE 117

117 Figure 5-2. Cattleya mericlone orchids before treatment with water, petroleum oil, or Silwet L77 + petroleum oil (Table 5-2). A-C) Abaxial leaf surfaces with heavy Boisduval scale infestations; D) Pseudobulb of Cattleya mericlone orchid. Boisduval scales can be seen extending below th e sheath (lower left). A B D C

PAGE 118

118 Figure 5-3. Cattleya mericlone orchids infested with Bois duval scale 1 wk af ter second treatment with A) water control; B) Prescription Treatment Ultra-Fine oil alone; C and D) Prescription Treatment Ultra-Fine oil + S ilwet L-77 showing that scales were removed from the orchids with th e oil + Silwet L-77 combination. A B C D

PAGE 119

119 CHAPTER 6 SUMMARY AND CONCLUSIONS Integrated pest management (IPM) is a pest control strategy that seeks to reduce or avoid plant damage by arthropod pests or diseases whil e minimizing the impact of the control measures on non-target organisms (beneficial insects), hum ans, and the environment by utilizing control tactics that disrupt natural control factors (i.e. natural enemies) as little as possible (Stern et al. 1959; Flint & van den Bosch 1981; Dreistadt 2001). The overall objective of this dissertation was to develop new diagnostic procedures and co ntrol strategies for several important orchid pests and pathogens that will be compatible w ith an overall IPM program. This objective was met by demonstrating that the highfidelity PCR protocol is more efficient than the standard PCR protocol at detecting Pseudocercospora odontoglossi while in the presence of host genomic DNA. Detection of the 5.8S rDNA and the tw o flanking ITS regions was improved from 207 million copies of template DNA being required using the standard PCR, to needing only 207 copies with the high-fidelity PCR. In addition, the high-fidelity PCR protocol consistently detected two important orchid pathogens, Sclerotium rolfsii and Dickeya sp., directly from host tissue, while the standard PCR protocol could not. The results indicate that the high-fidelity PCR protocol could be an importa nt tool for diagnosticians and regulatory agencies, especially when pathogens are of quarantin e significance, occur at low titer s, are latent, or cannot be cultured. The sensitivity of this method, its utility with impure DNA samples, and amplification of target DNA directly from plant tissue are all va luable attributes that contribute to its wide range of uses and users. It also was determined, through the use of the hi gh-fidelity PCR, biochemical tests, and fatty acid analysis, that there are strain s of soft-rot causing bacteria from orchids in Florida that have not been characterized previously. Because the phenotypic and genotypic characters of bacteria

PAGE 120

120 can be highly variable, in many cases multiple methods are required for identification (Alvarez 2004); therefore, multiple tests were used to identi fy and characterize the or chid bacterial isolates used in this study. Results show that there are new strains of undescribed Dickeya spp. that cause soft-rot symptoms in orchids that can be distinguished from previously characterized Dickeya species. When performing phylogenetic analyses us ing portions of the 16S rDNA gene and pelADE pectolytic enzyme gene cluste r, two distinct orch id clades were found. Carbohydrate tests corroborate the conclusion that the bacterial isolates from Vanda and Tolumnia may be new Dickeya species: the carbohydrate profiles of the orchid strains do not match any currently known Dickeya species. This is the first time th e orchid bacterial strains have been characterized, and no information exists about pathogenicity, host-range, ecology, or where and how the strains entered Florida. More isolates should be collected from members of different orchid genera to determine the distribution of the orchid strains. In order to determine if the orchid strains are sufficiently different to be new species, other work should be done, such as DNA:DNA hybridization, immunological tests, and host-range studi es both within and without the Orchidaceae. This information could have an impact on the management of the disease. The selection of appropriate manageme nt strategies is dependent upon the correct identification of the pathogen (Miller & Martin 1988; Lvesque 1997; Toth et al. 2001). For example, it is possible that the orchid strains have certain environmental conditions that are conducive for disease development, and these requirements could be mani pulated to prevent or lessen the severity of the disease. Soft-rot diseases are extremely di fficult to control with chemicals (Aysan et al. 2003). Therefore, the first line of defense is exclusion, i.e. star ting with healthy plant material, which is dependent upon identification of the path ogen and determining its origin. Furthermore,

PAGE 121

121 pathogen identification is requi red for epidemiological studies and surveys of geographical distribution, which can be particul arly important when searching for disease resistance (Ploetz 2007). In addition to the wo rk with these novel Dickeya isolates from Flor ida orchid genera, culturing methods for Pseudocercospora spp. causing leaf spots in Dendrobium Cattleya Bulbophyllum and Tolumnia orchids were developed. In this study, P. dendrobii P. odontoglossi and an undescribed Pseudocercospora sp. from Bulbophyllum were successfully cultured on V-8 agar at 25 C under 12L:12D photoperiod. In addition, an undescribed Pseudocercospora sp. from Tolumnia produced spores in culture when transferring plugs from actively growing cultures on V-8 agar to water agar at 25C under constant light. Although transferring plugs from the actively growing isolate on V-8 to WA successfully induced sporulation with the Tolumnia isolate, further work should be done to determine if the same procedure induces other orchid-associated species to sporulate. By producing Pseudocercospora spores in culture from a single spore, it will be possible to pr oduce inoculum for pathogenicity tests, for confirming Kochs postulates, and fo r evaluating host-range within the Orchidaceae without contaminating the inoculum with othe r pathogens that may be present on diseased leaves. In addition, because spores are need ed for morphological identification, these results could aid diagnosticians and plant-health practitione rs who encounter orchid diseases and wish to grow isolates for morphological or molecular identification. As mentioned above, one of the goa ls of an IPM program is to select control measures that have the least impact on non-target organisms, humans, and the environment. In this dissertation, it was demonstrated th at the use of Silwet L-77 + a light horticultural oil increased control of Boisduval scale insect s and a flat mite (Acari: Te nuipalpidae) on orchids while

PAGE 122

122 reducing the requirement for toxic pesticides. In addition to th e reduced negative impact on the environment, there are several other advantages to using oil + Silwet L-77 to control orchid arthropod pests. No arthropod is known to ha ve developed resistance to petroleum oil. Therefore, Silwet L-77 could be used as an adjuvant with petr oleum oils to increase their effectiveness with the development of resistan ce a minimal concern (Butler et al. 1993). Also, the use of petroleum oil with the addition of Silw et L-77 is safer and may be less expensive than some pesticides (Liu & Stansley 2000) and may assist in the conservation of natural enemies, one of the goals of an integrated pest mana gement program. Wood et al. (1997) noted that Silwet L-77 had little or no eff ect on adults or larvae of la dy beetles and green lacewings, suggesting that Silwet L-77 could be an importa nt tool for an integrated pest management program We concluded that Silwet L-77 increases the efficacy of oil against Boisduval scale and the flat mite on orchids, if it is applied correctly. Because complete coverage is required, its use may be most relevant to hobbyists or small commerci al growers who want to use a chemical control that has reduced toxic effects on non-target orga nisms, the environment, and human health. Currently, no IPM program specifically designe d for orchids exists. The techniques and tools developed in this dissertation could serv e as the foundation for IPM programs for orchids and the tools discussed herein could be used to diagnose and control other important orchid arthropod pests and diseases. Finally, key information learned fr om this dissertation research has been incorporated into 6 publications aimed at orchid hobbyists and large-scale orchid growers, as well as cooperative extension agents. Without communi cation of research results to the potential end users, no IPM program can be adopted.

PAGE 123

123 CHAPTER 7 REFLECTIONS As I look back on my graduate student career in the Plant Pathology department at the University of Florida, I realize that I am fort unate to have received a multifaceted education. I took many courses in the department, my favorites being Fungal Plant Pathogens, Plant Disease Diagnosis, and Bacterial Plant Pat hogens. In addition to course work, I was a teaching assistant for Fungal Plant Pathogens and Plant Disease Diagnosis, both taught by Carol Stiles, and found the experience to be rewarding, challenging, and fun. To supplement my course work and TA responsib ilities, I was an invite d speaker at over 15 Florida orchid society meetings and orchid show s and I enjoyed speaking about orchid diseases and management. Interacting with growers and h obbyists turned out to be an extremely valuable experience, and these experiences helped me realized how important it is to produce extension materials. I am grateful to my advisor, Aaron Palmateer and to Carol Stiles for supporting me in my desire to gain extension as well as research and teaching experience. Toward the end of my academic program, I sp ent most of my time in Marjorie Hoys lab learning molecular techniques and applying these to my research. In addition to routine lab work, I learned some very valuable lessons while under Dr. Hoys directio n, some not as quickly as I should have: 1. It is necessary to work on multiple projects simultaneously. 2. It is to the students benefit, ev en if he/she doesnt think so, to set clear, realistic goals and deadlines and meet them. 3. Everything is easier to write with an outline! I found that when I had trouble writing, especially a discussion section, if I outlined it and wrote out the main points, suddenly things fell into place. It was a matter of c onnecting the dots, and filling in the blanks after that. 4. WRITE AS YOU GO. This is especially true of the materials and methods.

PAGE 124

124 5. Write down everything in your notebook, even if you think it is not important. For instance, what was the humidity in the gree nhouse during inoculations ? Not only does this make the experiment reproducible, but it may have an effect on the results! If your notebook is clear and complete, the materials and methods will be much easier to write. 6. Work a consistent schedule. 7. Ask for help when you need it! This include s seeking out the assist ance of a statistician, which can save hours later.

PAGE 125

125 APPENDIX A PHYSIOLOGICAL DISORDERS OF ORCHIDS: OEDEMA Oedema is a physiological disorder of orchid s caused by overwatering. The excess water is absorbed by the roots quicker than it is lost by the leaves, which can cause swelling of plant cells and produce a lesion resembling a blister. This condition frequently occurs during periods of cool weather when water quantity and/or freque ncy is not reduced. The blister-like symptoms can appear on upper or lower leaf surfaces, stems, petals, or sepals. Symptoms Symptoms of oedema can take on many forms. Generally, small, swollen blister-like structures develop that may become corky with age (Figures A-1 and A-2), and are usually round, but may have other shapes (Figures A-1and A-2). The plan t tissue beneath the blister will frequently remain green (Figure A-3, A). Occas ionally, blisters may be misidentified as scale insects because of shape and size. However, scale insects can be easily rubbed off, while oedema blisters can not (Figure A-4). If a cr oss section through symptomatic tissue is made, enlarged cells can be seen below the epidermis (o uter-most layer of cells on a leaf), which gives the lesion a raised or swollen appearance (Figure A-3, B). Diagnosis Orchid diseases and disorders can be caused by living pathogens (bacteria and fungi), or environmental conditions (heat, cold, light). Making the distinction between biotic (living organism) and abiotic (non-living) causes of the di sorder is crucial for effective orchid health management. For example, spraying a chemical fungicide would be a waste of time and money if the cause of leaf damage is sunburn or oedema. As a general guide, you may have oedema if the following occur:

PAGE 126

126 Blisters are slightly raised without a yellow halo or water-soaked margins, which may indicate the presence of a pathogen. Figure A-3, C shows an example of a lesion with a yellow halo, while water soaking can be seen in Figure A-3, D. Blisters are round and/or have a cork y layer over the top (Figure A-2, D). Green tissue can be seen underneath of top layer (Figure A-3, A). Oedema blisters cannot be rubbed off. Scal e insects can easily be removed (Figure A-4). Management Many consumers are unwilling to purchase plants with visible symptoms of an underlying disorder. Therefore, the prevention of oedema can be economically advantageous in certain orchid genera that are prone to oedema, such as Phalaenopsis species and hybrids. Once an orchid has oedema, the lesions are permanent. However, symptomatic plants can produce new, healthy growth when more favorable growing c onditions return. Because oedema is not caused by a pathogen, no disease contro l measures are required. In order to minimize oedema, growers should make sure that plants are watered correctly and not receiving excessive moisture in the form of rain if left outdoors, particularly during cool weather. The condition of the gr owing medium should also be examined to make sure it is not retaining too much water because of decompositi on. Reducing the amount of organic material in potting mix may also reduce occurrences of oedema Improve the flow of air over the leaves by spacing plants farther apart and increasing ventilations.

PAGE 127

127 Figure A-1. Examples of oedema on various orchids. A) Phalaenopsis with symptoms of oedema. Notice the numerous round spots sca ttered over the leaf surface; B) A closeup view of an odema blister on a Phalaenopsis ; C) Oedema on the lower surface of a Phalaenopsis leaf; D) Oedema on the lower surface of a Rhynchostylis leaf. A B D C

PAGE 128

128 Figure A-2. Examples of oedema on various orch ids. A) Oedema on the upper surface of a Cattleya leaf; B) Oedema on the lower surface of a Cattleya leaf; C) Oedema blisters on the flower of Encyclia cordigera ; D) Oedema on the lower surface of a Bulbophyllum leaf. B D C A

PAGE 129

129 Figure A-3. Diagnosing oedema in or chids. A) Oedema blister on a Phalaenopsis showing green tissue underneath; B) A cr oss section through an odema blister of the same Bulbophyllum in figure A-2 (D). Notice how the cells below the upper epidermis have enlarged, causing a swolle n or blister-like appearance; C) Bacterial leaf spots on a Vanda caused by Acidovorax sp. On the left, dark spot s can be seen with yellow margins or halos, while on the right the yellow has become more widespread; D) Bacterial disease on an Oncidium caused by Burkholderia sp. Water soaking can be seen as a watery, fluid-fi lled area surrounding the lesion. A B C D

PAGE 130

130 Figure A-4. Differentiating scale in sects from oedema blisters. A) A scale insect on the lower side of a Cattleya leaf; (B) In this figure, the same scale insect is being lifted with a metal probe. Scale insects can be easily removed, while oedema blisters cannot. Published as: CATING, R. A., PALMATEER, A. J., STILES, C. M., HARMON, P. F., AND DAVISON, D.A. 2007. Physiological disorders of orchids: Oedema, PP 224. Electronic Data Information Source, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida. B A

PAGE 131

131 APPENDIX B PHYSIOLOGICAL DISORDERS OF ORCHIDS: MESOPHYLL CELL COLLAPSE Mesophyll cell collapse is a condition that occurs in many orchid genera. This physiological disorder can be caused by exposure to low water temperatures or low air temperatures, which leads to damage to the mesophyll cells within the leaf (Sheehan 2002). For many growers, this is a difficult condition to diagnose b ecause the symptoms are frequently discovered 6-8 weeks after the exposure to damaging temperatures (Jones 2004; Sheehan 2002). Symptoms Initial symptoms include localized or wide-s pread sunken/yellow areas (Figure B-1) which later may turn dry and necrotic (Figure B-1, C). Once a leaf is damaged, it is permanent. Injured leaves may be colonized by saprophytic fungi, which can lead some growers to believe the disorder is caused by a fungus, prompting them to apply unnecessary chemicals. Cause, Diagnosis, and Control Very few scientific data have been reported concerning this condition. However, the data available do suggest that the seve rity of symptoms is related to several factors: temperature, length of exposure to low temperatures, and age of leaves (McConne ll & Sheehan 1978). Young leaves appear to be more susceptible to chilling injury, while mature leaves appear to be more resistant (McConnell & Sheehan 1987). Sheehan ( 1987) reported that temperatures of 1.6-7.2 C can cause mesophyll cell collapse in young Phalaenopsis leaves. Sheehan (2002) also states that young leaves will show symptoms after 2 hours at 7.2 C, with lower temperatures causing more rapid development; however, mature leaves can withstand a longer exposure at 1.6 C without detrimental effects. In contrast, Yamada et al. (2002) reported that no damage occu rred to orchid leaves when exposed to chilling at -2 C after an entire day of exposure, pr ompting them to conclude that the

PAGE 132

132 orchid was chilling-resistant. However, assessmen t of damage was made at the ultra-structural level, while no long-term assessment of changes in the tissue was made. In addition, the orchid chosen for the study, Paphiopedilum insigne (Wall. ex Lindl.) Pfitzer, can frequently withstand cool temperatures and even requires relatively cool temperatures to grow and flower properly (Bechtel et al. 1992). Furthermore, mesophyll cell collapse occurs more frequently in orchids that have thick, fleshy leaves such as Phalaenopsis while P. insigne has relatively thin leaves and may not be prone to this conditi on. Therefore, these data cannot be used to establish guidelines for other orchid genera, which may have much higher temperature requirements. Blanchard & Runkle (2006) found that me sophyll cell collapse occurred in Phalaenopsis Miva Smartissimo X Canberra at day/night temperatures of 20 /14 C, 26 /14 C, 26 /20 C, and 29 /17 C, but not when held at a constant temperat ure. This suggests that a decline in night temperature may play a role in the development of mes ophyll cell collapse. In order to properly diagnose mesophyll cell collapse, a thorough hi story of the growing conditions is crucial. High-low thermometers placed at various locations around the greenhouse or growing area can record temperature changes a nd may indicate areas that are prone to drafts or low temperatures. In Flor ida, daily temperature records can be obtained through the FAWN weather data system (fawn.ifas.ufl.edu), which may be helpful when trying to establish the cause of mesophyll cell collapse in or chids grown outdoors. In some cases, weather/environmental data may not be available and a more thorough exam ination of the plant may be required to rule out a pathogen as the cause of symptoms. Howeve r, if no fungal, bacterial, or viral pathogens are found, then adverse environm ental conditions may be the cause of the observed damage.

PAGE 133

133 After a diagnosis is made, the cau se of temperature variation s hould be identified in order to prevent recurrences of the condition. When one suspects mes ophyll cell collapse, it is important to: Examine temperature records for the previous 6 weeks and look for cool temperatures or drastic fluctuations in day/night temperatures. Determine if there are areas in the greenhouse or growing area that are prone to drafts. Make sure when watering that, the water is not too cold, which al so can cause mesophyll cell collapse. When in doubt, visit the Florida Plant Diagnostic Network ( http://fpdn.ifas.ufl.edu ) for contact inform ation and direct links to one of the Florida Extension Plant Diagnostic Clinics (FEPDC). However, keep in mind that thorough records are essential to diagnose this condition properly, and this information should be provided to the diagnostician.

PAGE 134

134 Figure B-1. Examples of mesophyll cell collapse in orchids. A) Laelia anceps ; B) A close-up view of the L. anceps in A; C) Phalaenopsis Published as: CATING, R. A., AND PALMATEER, A. J. 2009. Physiological disorders of orchids: Mesophyll cell collapse, PP 265. Elect ronic Data Information Source, Florida cooperative Extension Service, Ins titute of Food and Agricultural Sc iences, University of Florida A B C

PAGE 135

135 APPENDIX C BLACK ROT OF ORCHIDS CAUSED BY Phytophthora palmivora AND Phytophthora cactorum: SYMPTOMS, DIAGNOSIS, AND MANAGEMENT Black rot of orchids can be caused by several pathogens. Frequently, black rot is caused by Phytophthora palmivora or Phytophthora cactorum (Hine 1962; Uchida 1994; Orlikowski & Szkuta 2006). Although P. cactorum and P. palmivora are very similar, they can be distinguished by morphological ch aracters or by molecular diagnos tic techniques (Tsai et al. 2006). Another organism, Pythium ultimum can also cause black rot. Pythium ultimum can be morphologically differentiated from P. cactorum and P. palmivora ; however, P. ultimum is less commonly seen in orchids. Although Phytophthora and Pythium are different genera, their life cycles, epidemiology, and control are similar. Host Range Phytophthora palmivora and P. cactorum have been known to cause disease in several orchid genera including Aerides Ascocenda Brassavola Dendrobium Gongora, Maxillaria Miltonia Oncidium Paphiopedilum Phalaenopsis Rhynchostylis and Schomburgkia as well as some less commonly grown genera (Alfieri et al. 1994; Orlikowski & Szkut a 2006). However, the disease is frequently seen in Cattleya orchids and their hybrids, such as Brassocattleya and Laeliocattleya Symptoms Small, black lesions can be observed on the root s or basal portion of the pseudobulbs. As the lesions age, they enlarge and may engulf the en tire pseudobulb and leaf (figure C-1, A-C). The pathogen can spread through the rhizome to othe r portions of the plant. Under favorable environmental conditions, mycelia may be obser ved growing on parts of the plant (C-1, C). Eventually, the entire plant will be killed.

PAGE 136

136 Diagnosis Diagnosis of black rot caused by P. palmivora and P. cactorum is based on morphology of the pathogen or through the use of molecular techniques. Mor phological identific ation is based on characteristics of the mycelium, shape of z oosporangia (asexual reproductive structures) and the presence and shape of oospores (sexual reproductiv e structures). If zoosporangia are present, a nd are roughly lemon-shaped with a short pedicel (stalk at the base of the spore) after the zoosporangia have detached and have papilla (small swelling on the tip of the spore, see figure C-1, D), one can be fairly certain it is a Phytophthora species. In most cases, identifying the pathogen to genus will pr ovide enough information to identify proper management strategies (for identification to the species level, see Appendix E). Management Phytophthora palmivora and P. cactorum are considered water molds and require water to spread the spores and to germinate on new hosts. The spores can easily spread in irrigation water and can splash from one plant to another duri ng watering (Uchida 1994). In addition, zoospores, which are motile, are normally considered the infective spore and can move readily when free water is available. Therefore, it is crucial to remove infected plants immediately to prevent further spread, reduce periods of prolonged leaf wetness, and provide ad equate ventilation (to facilitate drying). Elevating the plants above the ground or keeping them on a solid surface can also help prevent infections. To prevent black rot, growers sh ould also consider the following: Nursery Sanitation Recommendations for Phytophthora Fungicides should be consider ed as a tool for managing Phytophthora but if not used properly (according to the manufacturers label) they will not be effective or may cause damage, such as phytotoxicity. However, they are the pr imary defense in an existing crop and provide at least some level of control when used properly.

PAGE 137

137 Getting Phytophthora under control requires a long-term strategy that focuses on changing and improving procedures and materials to reduce the opportunity for spread or reintroduction of the pathogen. Successful management of Phytophthora in the nursery has been accomplished when dramatic alterations of management practices were undertaken. Some growers keep a clean laboratory or surgical room in mind as they think about their nursery sanitation procedures. Fungicide Options Preventative applications of foestyl-AL (A liette, Flanker, Prokoz Avalon), potassium phosphate (Alude, Fungi-phite, Topaz), propamo carb hydrochloride (Banol), trifloxystrobin (Compass), Bacillus subtilis QST 713 (Rhapsody), dimethomorph (Stature), Mefenoxam (Subdue), or etridiazole (Terrazole 35%) may aid in reducing disease spread, but complete control can only be achieved if the inf ected planting material is destroyed. Growing Media and Storage Use only unopened, bagged growing media stored on a covered, paved surface that can be periodically washed down with a 1:3 ratio of bleach (sodium hypochlorite) to water (bleach works by oxidizing or destroying th e molecular bonds in microorgani sms). It is likely that the pathogen will move into your growing media if it is not bagged or completely covered. Use only disinfected tools and hands (disposable latex gloves that can be purchased at a grocery store or professional cooking equipment store) when potting or repotting plants. Avoid mixing bleach with acids which may cause the produc tion of toxic chlorine gas. Always use in good ventilation. Containers Store new pots in sanitized areas. The best op tion is to use new potting containers, but if this is not feasible, submerge pots in a 1:3 ratio of bleach (sodium hypochl orite) to water with agitation for a minimum of 10 minutes.

PAGE 138

138 Bench Sanitation Make sure that bench surfaces are high enough a bove the soil surface to avoid splashing from the ground below. Sanitize all bench surfaces and tools used to groom or work with plants before each use. It is highly recommended that di sposable razor blades be used to work with plants. These can be purchased at most home or garden supply stores. Use one new razor blade per plant and dispose of it after use. This can greatly reduce the spread of several types of orchid diseases, including fungi, bacteria, and viruses. If tools are used, there are several methods av ailable for sanitation. These include: 1) 25% chlorine bleach (3 parts water to 1 part bleach); 2) 25% pine oil cleaner (3 parts water to 1 part pine oil); 3) 50% rubbing alc ohol (70% isopropyl, equal part s alcohol and water); 4) 50% denatured alcohol (95%, equal pa rts alcohol and water); 5) 5% quaternary ammonium salts (DO NOT mix quaternary ammonia with bleach). Soak tools for 10 minutes and rinse repeatedly in clean water. The wooden portions of benches may be very difficult to sanitize because they are porous. Scrubbing to remove algae, scum, m ildew, and dirt before treatment may help. Water Supply and Hand Watering Surface water (ponds or reservoirs) should not be used unless disinfected. If hand watering, be sure to periodically saniti ze the hose, nozzles, and wands with a bleach solution and rinse well. Hang hoses and wands in an area where the ends will not contact soil or other potentially contaminated surfaces. New Plants Brought into the Nursery, Greenhouse, or Growing Area Any new plants brought into the gr owing area should be kept isol ated from other plants for a minimum of 6 weeks to observe the plants and wa tch for the development of any disease or pest symptoms (quarantine). This can greatly decreas e the possibility of introducing new pathogens or pests into the growing area. Make sure that different tools and bench spaces is used for plants

PAGE 139

139 under quarantine. Make sure to consider other potential contamination surfaces, such as plant transport trailers or cart surfaces.

PAGE 140

140 Figure C-1. Black rot of orchid s, symptoms and signs. A) Cattleya hybrid with black rot symptoms caused by P. cactorum ; B) A close-up view black rot symptoms in figure A; C) Cattleya orchid with visible mycelium on the apical portion of pseudobulbs; D) A sporangium of P. palmivora Sporangium is papillate (small swelling on left) with a short pedicel (right). Published as: CATING, R. A., PALMATEER, A. J., STILES, C. M., RAYSIDE, P. A., AND DAVISON, D. A. 2009. Black rot in orchids caused by Phytophthora palmivora and Phytophthora cactorum, PP 260. Electronic Data Information Source, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida. C A B D

PAGE 141

141 APPENDIX D FIRST REPORT OF Sclerotium rolfsii ON Ascocentrum AND Ascocenda ORCHIDS IN FLORIDA Southern blight caused by Sclerotium rolfsii is known to occur on several economically important orchid hosts, including Vanda species and hybrids (Alfieri Jr. et al. 1984; Farr et al. 1989; Alfieri Jr. et al. 1994; Bag 2004). In the summer and fall of 2008, an outbreak of southern blight on Vanda orchids was seen in several commerci al nurseries and landscapes throughout South Florida. More than a dozen orchids were af fected at one of the lo cations, and symptoms of S. rolfsii were observed on Ascocentrum and Ascocenda orchids, which also are common in the trade and demand a resale value ranging fr om $20 to $150 for specimens in bloom. Identification Affected Ascocentrum and Ascocenda orchids were found severely wilted at the apex, while around the base of the plants, tan, soft, water-soaked lesions were present (F igure D-1, A). As the lesions progressed, leaves around the base of th e plants began to fall off, leaving the stems bare (Figure D-1, B). After 2 days, white, flabel late mycelia were seen progressing up the stem and numerous, tan-to-brown sclerotia were present (Figures D-1, C and D). Leaves and portions of the stems were plated on aci dified potato dextrose agar (A PDA) and grown at 25C. White, flabellate mycelium and tan sclerotia approximately 2 mm in diameter were produced in culture and microscopic examination revealed the pr esence of clamp connections. The fungus was identified as S. rolfsii and a voucher specimen was deposited with the ATCC. A PCR was performed on the ITS1, 5.8S rDNA, and ITS2 us ing primers ITS4 and ITS5 as described by White et al. (2001) and the sequence was de posited in GenBank (Accession No. GQ358518). Pathogenicity Tests Pathogenicity of an isolate was tested by placing 6-mm plugs taken from APDA plates directly against the stem of five different Ascocentrum and Ascocenda orchids and five were

PAGE 142

142 untreated controls. Plants were housed under 50% shade, 60 to 95% relative humidity, and temperatures ranging from 23.8 to 31.2C. Within 7 days, all inoculated plants developed symptoms that were identical to those observed on the original plants and S. rolfsii was consistently reisolated from symptomatic tissue. Ascocentrum and Ascocenda were previously reported under miscellaneous orchid species and hybrids as hosts for S. rolfsii (Alfieri Jr. et al. 1984). However, this report was ambiguous and the most current edition does not report the host fungus combination (Alfieri et al. 1994). To our knowledge, this is the first report of S. rolfsii affecting Ascocentrum and Ascocenda orchids.

PAGE 143

143 Figure D-1. Ascocenda orchids with Southern blight caused by S. rolfsii. A) Ascocenda orchid with early symptoms of Southern blight. Early symptoms freque ntly resemble soft rot bacterial infections; B) Ascocenda orchid that has lost lower leaves due to Southern blight; C-D) Numerous sclerotia on the stems of two Ascocenda orchids. Published as: CATING, R. A., PALMATEER, A. J., AND MCMILLAN, R. T. JR. 2009. First report of southern blight caused by Sclerotium rolfsii in Ascocenda and Ascocentrum orchids in Florida. Plant Dis. 93: 963. C D B A

PAGE 144

144 APPENDIX E A DIAGNOSTIC GUIDE FOR BLACK ROT OF ORCHIDS CAUSED BY Phytophthora palmivora AND Phytophthora cactorum FOR PLANT DIAGNOSTIC CLINICS AND DIAGNOSTICIANS Introduction According to the USDA Economics, Statistics, and Market Information System, orchid sales in the United States have increased steadily since 1997 and sales were estimated to exceed 140 million dollars in 2006 (Jerardo 2006). Orchids are the second most economically important flowering plant produced in the United States, an d Florida and California are the top producers in the nation (Jerardo 2006). Although poinsettias have the largest dollar amount in sales, orchids have shown the greatest rate of growth in sales (Jerardo 2006). It is clear that the popularity of orchids is growing as more people discover thes e intriguing, beautiful and, in many cases, longlasting flowers. Orchids are susceptible to nu merous disease-causing agents such as fungi, bacteria, and viruses. Many common and economically important diseases affecting or chids include viruses (Jensen 1959; Farr et al. 1989; Hu et al. 1993; Hu et al. 1995),vascular-wilt pathogens (i.e. Fusarium spp., Foster 1955), leaf spots (i.e. Cercospora/ Pseudocercospora spp. (Chupp 1953; Hsieh & Goh 1990; Crous & Braun 2003), Phyllosticta spp. (Uchida and Aragaki 1980; Uchida 1994), bacterial soft rots such as Dickeya chrysanthemi (Cating et al. 2008) and Erwinia carotovora (Chan et al. 2005), and root, st em, leaf and pseudobulb rots (i.e. Phytophthora spp.) commonly called black rot. Several species of Phytophthora have been reported to occu r on orchids and cause economic damage worldwide (Uchida 1994; Erwin & Ribeiro 1996; Ilieva et al. 1998). Of these, P. cactorum and P. palmivora have the widest host range across orchid genera and they are the most common species affecting commercial orchid production in Florida. Therefore, our

PAGE 145

145 objective is to provide a resource that covers important diagnostic information including symptoms and signs associated with the diseas e, morphological, molecular, and pathological characteristics, taxonomy of the causal agents host range and geogr aphic distribution, and isolation and storage techniques. Disease Disease in orchids caused by Phytophthora cactorum and P. palmivora can be referred to by several names: black rot, crown rot, heart rot, or black rot of leaves (Hadley et al. 1987). Pathogen There are reports of other Phytophthora spp. causing disease on orch ids, however, black rot, in commercial orchid production thro ughout Florida is most commonly caused by Phytophthora cactorum and P. palmivora (Hine 1962; Uchida 1994; Erwi n & Ribeiro 1996; Orlikowski & Szkuta 2006). Although P. cactorum and P. palmivora are similar, they can be differentiated by morphological characteristics or by th e use of molecular diagnostic techniques (Tsai et al. 2006). Pythium ultimum, can also cause black rot. Pythium ultimum can be differentiated from Phytophthora based on morphology; however, P. ultimum is less commonly seen in orchids. Refer to Erwin & Ribeiro (1996) for more details on comparing the morphology of Pythium and Phytophthora Taxonomy Phytophthora cactorum and P. palmivora are members of the family Pythiaceae, in the kingdom Stramenopila. Recently, Blair et al. (2 008) produced a comprehensive phylogeny for the genus Phytophthora based on multiple loci. The reader is referred to that publication for current phylogenetic information. Additional taxonomic information is available in Erwin & Ribeiro (1996) and online taxonomic databases include www.phytophthoradb.org (Park et al. 2008).

PAGE 146

146 Symptoms and Signs Small black lesions can be observed on the roots or basal portion of the pseudobulbs. As the lesions age, they enlarge and may engulf the en tire pseudobulb and leaf (figures E-1, A and D) and white mycelium and sporangia may be seen growing directly on the plant material. The pathogen can spread through the rhizome to other portions of the plant. Eventually, the entire plant may die. Host Range within the Orchidaceae Phytophthora cactorum and P. palmivora have been known to cause disease on many different orchid genera, including Aerides, Ascocenda, Brassavola, Dendrobium, Gongora, Maxillaria, Miltonia, Oncidium, Paphiopedilum, Phalaenopsis, Rhynchostylis, Schomburgkia as well as some less commonly grown genera (Far r et al. 1989; Simone & Burnett 1995; Erwin & Ribeiro 1996; Orlikowski & Szkuta 2006). The disease is frequently seen on Cattleya orchids and their hybrids, such as Brassocattleya and Laeliocattleya Geographic Distribution According to Cline et al. (2008), there are 87 accepted species in the genus Phytophthora and about half of the accepted species occur in the United States. Phytophthora cactorum and P. palmivora have wide host ranges and occur on fruit, vegetable, tree, a nd ornamental crops throughout the world (Erwin & Ribeiro 1996). Pathogen Isolation Both species of Phytophthora can be readily isolated from diseased tissue. Wash the symptomatic plant tissue under a gen tle stream of tap water. Use a clean blade to slice multiple sections of tissue from the plant. To increase isolation frequency, one sh ould avoid selecting tissue that has dried and take tissue from margins with actively progressing lesions. Tissue sections should then be plated directly onto P5ARPH medium (Jeffers & Martin 1986). Seal the

PAGE 147

147 plates with parafilm and incubate in the dark at 27-32C. Examine for Phytophthora growth after 3-5 days. Pathogen Identification Diagnosis of black rot caused by P. cactorum and P. palmivora is based on morphology of the pathogen or through the use of molecular techniques. Mor phological identific ation is based on characteristics of the mycelium, shape of zoosporangia and the presence and shape of oospores and characteristics of the antheridia. Phytophthora zoosporangia are roughly lemonshaped with a short pedicel (stalk at the base of the spore) after the zoosporangium has been detached and contains a papilla (small swelling on the tip of the spore, figure E-1, C). In most cases, identifying the pathogen to genus will provide enough information to develop proper prevention and control strate gies. If it is nece ssary to identify the pathogen to species, oospores can be helpful in identifying the pathog en at the species level, although they are not always produced. Phytophthora cactorum is homothallic and produces sex bodies on several common media used in the plant diagnostic labo ratory (Erwin & Ribeiro 1996; Gallegly & Hong 2008). Phytophthora palmivora is heterothallic and sex bodies are formed when A1 and A2 mating types are paired in culture. A suitabl e medium for the formation of sex bodies (both Phytophthora species) is lima bean ag ar (Gallegly & Hong 2008). The location of attachment of the antheridiu m is an important diagnostic characteristic (Erwin & Ribeiro 1996). Antheridia which are the male reproductiv e structures, attach to the oogonia of P. cactorum and P. palmivora in different locations. In Figure E-2 (A), the oogonium stalk of P. palmivora has grown through the anth eridium and in Figure E2 (B), the antheridium of P. cactorum can be seen attached to th e side of the oogonium stalk.Both Phytophthora species produce abundant, papillate sporangi a that are caducous with short pedicels directly on orchid host tissue. For maximum sporangia production, maintain infected orchids at 25-28C for P.

PAGE 148

148 cactorum whereas slightly warmer temperat ures 30-33C are more favorable for P. palmivora (personal observation). Note th at the morphological characteristic s of sporangia are very similar for isolates of P. cactorum and P. palmivora so characteristics of the mycelium, and the presence and shape of oospores and characteristic s of the antheridia are necessary for species delineation. Molecular Diagnostics Some laboratories and plant disease clinics may be able to isolate DNA for molecular diagnostics involving a PCR and DNA sequencing. The internal transcribed spacer region (ITS) can be used for species identification (Cooke & Duncan 1997; Brasier et al. 1999); however, the ITS region cannot always distinguish different species (Martin & Tooley 2004), so multiple criteria should be used to make species id entifications (Cooke et al. 2000b). Recently, we sequenced the ITS1, 5.8S rRNA gene, and ITS2 regions from a P. palmivora isolate affecting Cattleya orchids in Florida and the sequence is available in GenBank (accession # GQ131800). Species specific primers for the ITS region are available for both Phytophthora species including P. palmivora isolates from orchids (Lacourt et al. 1997; Tsai et al. 2006). In addition to DNA sequencing, other molecular techniques have been used to identify Phytophthora species. These include single-strand -conformation polymorphism (SSCP) of ribosomal DNA (Gallegly & Hong 2008; Kong et al. 2003), restriction fragment analysis (Cooke et al. 2000a), and isozyme analysis (Mchau & Coffey 1994). Pathogen Storage Due to the economic importance of Phytophthora infestans and other Phytophthora species, extensive work has been perfor med investigating methods for st orage of certain species (Goth 1981; Peters et al. 1998; Erwin & Ribeiro 1996). Storage of Phytophthora isolates has proven to

PAGE 149

149 be difficult and frequent transfers may be necessary to maintain viability; however, transfers can cause mutations or other changes (Erwin & Ribeiro 1996). Ko (2003) demonstrated that isolates of P. palmivora can be stored in st erile water at room temperature for as long as 23 years. To store isolates in this ma nner, remove plugs from isolates actively growing on V-8 juice agar and place 4 plugs in 7 ml of sterile distilled water in a sterile test tube and tighten the cap. Store the tubes in a test tube rack on a laboratory shelf. Another method for storage of isolates involve s the use of liquid nitrogen (Tooley 1988; Erwin & Ribeiro 1996). In this method, plugs of agar containing the isol ate are removed and stored in polypropylene vials containing 10% glycerol or DMSO and placed in liquid nitrogen (Tooley 1988). However, a pretreatment of -80 C may be required to successf ully recover the isolate (Tooley 1988). For detailed information on cryo preservation, see Dahmen et al. (1983) and Tooley (1988). Although cryopreser vation is simple and can preserve the isolate in the original genetic state, it requires special e quipment (liquid nitrogen tanks, -80 C freezer for pretreatment) and can be expensive to maintain (Tool ey 1988; Erwin & Ribeiro 1996; Ko 2003) Pathogenicity Tests Hine (1962) tested th e pathogenicity of P. palmivora on members of several different orchid genera by using zoospore suspensions (no concentr ations given) and V-8 agar blocks. More recently, Orlikowski and Szkuta (2006) performed pathogenicity tests of P. palmivora on Phalaenopsis Dendrobium Cymbidium and Epidendrum orchids. In the described method, 3mm plugs were removed from P. palmivora cultures grown on potato dextrose agar (PDA) and placed on orchid leaf blades and roots. Plant material was placed on sterile, moist blotting paper in polystyrene boxes and incubated at 22 to 25C in the dark. Small black lesions were initially observed at the point of inoculation and after 3 days the disease progressed causing affected tissue to appear water soaked and black in color.

PAGE 150

150 Figure E-1. Black rot of orchids, symptoms and signs. A-B) Cattleya orchids with visible mycelia of Phytophthora palmivora ; C) A single sporangium of P. palmivora ; D) Oncidium orchid with symptoms of black rot caused by P. palmivora C B A C D

PAGE 151

151 Figure E-2. A comparison of sexua l reproductive structures of P. palmivora and P. cactorum A) Amphigynous antheridium and oogonium of P. palmivora ; B) Paragynous antheridium and oogonium of P. cactorum A B

PAGE 152

152 APPENDIX F FIRST REPORT OF BACTERIAL SOFT ROT ON Tolumnia ORCHIDS CAUSED BY A Dickeya sp. Tolumnia orchids are small epi phytic orchids grown for their attr active flowers. In the fall of 2008, approximately 100 Tolumnia orchids with soft, brown, macer ated leaves were brought to the University of Florida Extension Plant Dia gnostic Clinic in Home stead (Figure F-1). Ten plants were randomly selected and bacteria we re isolated from the margins of symptomatic tissues of each of the 10 plants on nutrient agar according to the method described by Schaad et al. (2001). Four reference strains were used in all tests, including the molecular tests: Erwinia carotovora subsp. carotovora (obtained from J. Bartz, Department of Plant Pathology, University of Florida, Gainesville), E. chrysanthemi (ATCC No. 11662), Pectobacterium cypripedii (ATCC No. 29267), and Acidovorax avenae subsp. cattleyae (ATCC No. 10200). Identification All 10 of the isolated bacteria were gram nega tive, grew at 37C, degraded pectate in CVP (crystal violet pectat e) medium, grew anaerobically, produc ed brown pigment on NGM (nutrient agar-glycerol-manganese chloride) medium (Lee & Yu 2006), were sensitive to erythromycin, and produced phosphatase. Three of the strains were submitted for MIDI analysis (Sherlock version TSBA 4.10; Microbial Identifica tion, Newark DE) (SIM 0.732 to 0.963), which identified them as E. chrysanthemi A PCR assay was performed on the 16S rRNA gene with primers 27f and 1495r described by Weisburg et al. ( 1991) from two of the isolates and a subsequent GenBank search showed 99% id entity of the 1,508-bp se quence to that of Dickeya chrysanthemi (Accession No. FM946179) (formerly E. chrysanthemi ). The sequences were deposited in GenBank (Accessi on Nos. GQ293897 and GQ293898).

PAGE 153

153 Pathogenicity Tests Pathogenicity was confirmed by injecting approximately 100 l of a bacterial suspension at 1 108 CFU/ml into leaves of 10 Tolumnia orchid mericlones. Ten plan ts were also inoculated with water as controls. Plants were placed in a greenhouse at 29C with 60 to 80% relative humidity. Within 24 h, soft rot symptoms appeared on all inoculated leaves. The water controls appeared normal. A Dickeya sp. was reisolated and identi fied using the above methods (biochemical tests and MIDI), fulfilling Koch's postulates. To our knowledge, this is the firs t report of a soft rot caused by a Dickeya sp. on Tolumnia orchids. Although 16S similarity and MIDI re sults suggest the isol ated bacteria are D. chrysanthemi because of its close similarity with other Dickeya spp., these results are not conclusive. Further work should be conducted to confirm the iden tity of these isolates. Through correspondence with South Florida Tolumnia growers, it appears this disease has been a recurring problem, sometimes affecting international orchid shipments where plant losses have been in excess of 70%.

PAGE 154

154 Figure F-1. Tolumnia orchids with symptoms of a soft rot bacterial infection caused by Dickeya sp. Published as: CATING, R. A., PALMATEER A. J., MCMILLAN, R. T. JR., AND DICKSTEIN, E. R. 2009. First report of a soft rot in Tolumnia orchids caused by a Dickeya sp. Plant Dis. 93:1354.

PAGE 155

155 REFERENCES AGRIOS, G. N. 2001. Plant pathology, pp. 779-782 In O. C. Maloy and T. D. Murray [eds.], Encyclopedia of plant pathology, volume 2. John Wiley & Sons, Inc., New York, NY. ALFIERI JR., S. A., LANGDON, K. R., WEHLBURG, C., AND KIMBROUGH, J. W. 1984. Index of plant diseases in Fl orida (revised). Florida Departme nt of Agriculture & Consumer Services, Division of Plan t Industry, Bulletin 11. 389 pp. ALFIERI JR., S. A., LANGDON, K. R., KIMBROUGH, J. W., ELGHOLL, N. E., AND WEHLBURG, C. 1994. Diseases and disorders of pl ants in Florida. Florida Department of Agriculture & Consumer Services, Divi sion of Plant Industry, Bulletin 14. 1114 pp. ALTSCHUL, S. F., MADDEN, T. L., SCHFFER A. A., ZHANG, J ., ZHANG, Z., MILLER, W., AND LIPMAN, D. J. 1997. Gapped BLAST a nd PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402. ALVAREZ, A. M. 2004. Integrated approaches fo r detection of plant pathogenic bacteria and diagnosis of bacterial diseases. Annu. Rev. Phytopathol. 42: 339-366. AMERICAN ORCHID SOCIETY, 2002. Orchid pests and diseases revised edition. American Orchid Society, Delray Beach, FL. 118 pp. ARDITTI, J. 1992. Fundamentals of orchid biol ogy. John Wiley & Sons, Inc, New York, NY. 700 pp. ARK, P. A.1959. Fungal and bacteria l diseases of orchids pp. 378-420 In : Withner, C. L. [ed.], The orchids: a scientific survey. Th e Ronald Press Comp any, New York, NY. ARNESON, N., HUGHES, S., HOULSON, R., AND DONE, S. 2008. Whole-genome amplification by degenerate oligonucleotide primed PCR (DOP-PCR). CSH Protocols doi:10.1101/pdb.prot4919. ASEA, G., LIPPS, P. E., PRATT, R. C ., GORDON, S. G., AND ADIPALA, E. 2005. Development of greenhouse inoculation proce dues for evaluation of partial resistance to Cercospora zeae-maydis in maize inbreds. J. Phytopathol. 153: 647-653. ATWOOD, J. T. JR. 1986. The size of the Orch idaceae and the systematic distribution of epiphytic orchids. Selbyana 9: 171-186. VILA, A., GROENEWALD, J. Z., TR APERO, A., AND CROUS, P. W. 2005. Characterisation and epitypification of Pseudocercospora cladosporioides the causal organism of Cercospora leaf spot of olives. Mycol. Res. 109: 881-888. AYSAN, Y., KARATAS, A., AND CINAR, O. 2003. Biological control of bacterial stem rot caused by Erwinia chrysanthemi on tomato. Crop Prot. 22: 807-811.

PAGE 156

156 BABU, A. M., KUMAR, V., A ND GOVINDAIAH. 2002. Surface ultras tructural studies on the infection process of Pseudocercospora mori causing grey leaf spot disease in mulberry. Mycol. Res. 106: 938-945. BAG, T. K. 2004. Two new orchid hosts of Sclerotium rolfsii in India. Plant Pathol. 53: 255. BALIS, C., AND PAYNE, M. G. 1971. Tr iglycerides and cercosporin from Cercospora beticola : fungal growth and cercosporin production. Phytopathology 61: 1477-1484. BARNES, W. 1994. PCR amplification of up to 35-kb DNA with high fi delity and high yield from bacteriophage templates. Proc. Natl. Acad. Sci. USA 91: 2216-2220. BARRAS, F., VAN GIJSEGEM, F., AND CHATTER JEE, A. K. 1994. Extracellular enzymes and pathogenesis of soft-rot Erwinia Annu. Rev. Phytopathol. 32: 201-234. BECHTEL, H., CRIBB, P.,AND LAUNERT, E. 1992. The manual of cultivated orchid species, third edition. The MIT Press, Cambridge, MA. 586 pp. BECKMAN, P. M., AND PAYNE, G. A. 1983. Cultu ral techniques and conditions influencing growth and sporulation of Cercospora zeae-maydis and lesion development in corn. Phytopathology 73: 286-289. BECNEL, J. J., JEYAPRAKASH, A., HOY, M. A., AND SHAPIRO, A. 2002. Morphological and molecular characterization of a new microsporidium species from the predatory mite Metaseiulus occidentals (Nesbitt) (Acari, Phytoseiidae). J. Invertebr. Pathol. 79: 163-172. BEILHARZ, V., AND CUNNINGTON, J. 2003. Two new closely related species of Pseudocercospora on unrelated host families from southeastern Australia. Mycol. Res. 107: 445-451. BETHKE, J. A., AND CLOYD, R. A. 2009. Pesticid e use in ornamental production: what are the benefits? Pest Manage. Sci. 65: 345-350. BLAIR, J. E., COFFEY, M. D., PARK, S. K., GEISER, D. M., AND KANG, S. 2008. A multilocus phylogeny for Phytophthora utilizing markers derived fr om complete genome sequences. Fungal Genet. Biol. 45: 266-277. BLANCHARD, M. G., AND RUNKLE, E. S. 2006. Temperature during the day, but not during the night, controls flowering in Phalaenopsis orchids. J. Exp. Bot. 57: 4043-4049. BRADBURY, J. F. 1986. Guide to plant pathogen ic bacteria. CAB International Mycological Institute, Surrey, UK. 332 pp. BRASIER, C. M., COOKE, D. E. L., AND DUNCAN, J. M. 1999. Origin of a new Phytophthora pathogen through interspecific hybridization. Proc. Natl. Acad. Sci. USA 96: 5878-5883.

PAGE 157

157 BRAUN, U., AND HILL, C. F. 2002. Some new micromycetes from New Zealand. Mycol. Prog. 1: 19-30. BRENT, K. J. 1995. Fungicide resistance in crop pathogens: how can it be managed? Fungicide Resistance Action Committee Monograph numbe r 1. Global Crop Protection Federation, Brussels, Belgium. 49 pp. BROADHURST, P. G., AND HARTILL, W. F. T. 1996. Occurrence of Fusarium subglutinans on Cymbidium orchids in New Zealand. Plant Dis. 80: 711. BULL, C. T. 2001. Biological control, pp. 130-135 in O. C. Maloy and T. D. Murray, Encyclopedia of plant pathology, vol. I. John Wiley & Sons, Inc., New York, NY. BURKHOLDER, W. H., MACFADDEN, L. H., AND DIMOCK, A. H. 1953. A bacterial blight of chrysanthemums. Phytopathology 43: 522-525. BURNETT, H. C. 1964. Leafspot, Cercospora odontoglossi Prillieux and Delacrois. Plant Pathology Circular No. 28, State of Florida Department of Agriculture, Division of Plant Industry. BURNETT, H. C. 1965. Orchid diseases. State of Fl orida Department of Agriculture, Division of Plant Industry, Gainesville, FL. 57 pp. BUTLER, G. D. JR., HENNEBERRY, T. J., STANSLEY, P. A., AN D SCHUSTER, D. J. 1993. Insecticidal effects of sel ected soaps, oils, and deterg ents on the sweetpotato whitefly (Homoptera: Aleyrodidae). Fla. Entomol. 76: 161-167. CATING, R. A., PALMATEER, A. J., STILES C. M., HARMON, P. F., AND DAVISON, D.A. 2007. Physiological disorders of orchids: oedema, PP 224. Electronic Data Information Source, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida. CATING, R. A., HONG, J. C., PALMATEER, A. J., STILES, C. M., AND DICKSTEIN, E. R. 2008. First report of Dickeya chrysanthemi ( Erwinia chrysanthemi ) on Vanda orchids in Florida. Plant Dis. 92: 977. CATING, R. A., AND PALMATEER, A. J. 2009. Physiological di sorders of orch ids: mesophyll cell collapse, PP 265. Electronic Data Informa tion Source, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida. CATING, R. A., PALMATEER, A. J., AND MCMI LLAN, R. T. JR. 2009a. First report of southern blight caused by Sclerotium rolfsii in Ascocenda and Ascocentrum orchids in Florida. Plant Dis. 93: 963. CATING, R. A., PALMATEER, A. J., MCMILL AN, R. T. JR., AND DICKSTEIN, E. R. 2009b. First report of a soft rot in Tolumnia orchids caused by a Dickeya sp. Plant Dis. 93:1354.

PAGE 158

158 CATING, R. A., PALMATEER, A. J., STILES, C. M., RAYSIDE, P. A., AND DAVISON, D. A. 2009c. Black rot in orchids caused by Phytophthora palmivora and Phytophthora cactorum PP 260. Electronic Data Information Source, Florid a Cooperative Extension Se rvice, Institute of Food and Agricultural Sciences, University of Florida. CHAN, Y. L., LIN, K. H., SANJAYA, L .L. J., CHEN, W. H., AND CHAN, M.T. 2005. Gene stacking in Phalaenopsis orchid enhances dual tolerance to pathogen attack. Transgenic Res. 14: 279-288. CHAVERRI, P., SAMUELS, G. J., AND HODGE, K. T. 2005. The genus Podocrella and its nematode-killing anamorph Harposporium. Mycologia 97: 433-443. CHEN, C. C., WU, L. G., KO, F. M., AND TE NG, C. M. 1994. Antiplatelet aggregation principles of Dendrobium loddigessi J. Nat. Prod. 57: 1271-1274. CHIEN, A., EDGAR, D. B., AND TRELA, J. M. 1976. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus J. Bacteriol. 127: 1550-1557. CHUANG, D. W., KYEREMEH, A. G., GUNJ I, Y., TAKAHARA, Y., EHARA, Y., AND KIKUMOTO, T. 1999. Identificat ion and cloning of an Erwinia carotovora subsp. carotovora bacteriocin regulator gene by insertional mutagenesis. J. Bacteriol. 181: 1953-1957. CHUPP, C. 1954. A monograph of the fungus genus Cercospora Published by the author, Ithaca, NY. CLARRIDGE III, J. E. 2004. Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infecti ons diseases. Clin. Microbiol. Rev. 17: 840-862. CLINE, E. T., FARR, D. F., AND ROSSMAN, A. Y. 2008. A synopsis of Phytophthora with accurate scientific names, host range, and geographic distribution. Online. Plant Health Progress doi:10.1094/PHP-2008-0318-01-RS. COCCO, A., AND HOY, M. A. 2008. Toxicity of organosilicone adjuvants and selected pesticides to the Asian citrus psyllid (H emiptera: Psyllidae) and its parasitoid ( Tamarixia radiata ) (Hymenoptera: Eulophidae). Fla. Entomol. 91: 610-620. COOKE, D. E. L. AND DUNCAN, J. M. 1997. Phylogenetic analysis of Phytophthora species based on ITS1 and ITS2 sequences of the ribosomal RNA gene repeat. Mycol. Res. 101: 667677. COOKE, D. E. L., DUNCAN, J. M., WILLI AMS, N. A., HAGENAAR-DE WEERDT, M., AND BONANTS, P. J. M. 2000a. Identification of Phytophthora species on the basis of restriction enzyme fragment analysis of the in ternal transcribed space r regions of ribosomal RNA. EPPO Bulletin 30: 519-523.

PAGE 159

159 COOKE, D. E. L., DRENTH, A., DUNCAN, J. M., WAGELS, G., AND BRASIER, C. M. 2000b. A molecular phylogeny of Phytophthora and related oomycetes. Fungal Genet. Biol. 30: 17-32. COWLES, R. S., COWLES, E. A., MCDE RMOTT, A. M., AND RAMOUTAR, D. 2000. Inert formulation ingredients with activity: t oxicity of trisiloxane surfactant solutions to twospotted spider mites (Acari: Tetra nychidae). J. Econ. Entomol. 93: 180-188. COX, K. D., LAYNE, D. R., SCORZA, R., AND SCHNABEL, G. 2006. Gastrodia anti-fungal protein from the orchid Gastrodia elata confers disease resistance to r oot pathogens in transgenic tobacco. Planta 224: 1373-1383. CROUS, P. W. 1998. Mycosphaerella spp. and their anamorphs a ssociated with leaf spot diseases of Eucalyptus. Mycol. Mem. 21: 1-170. CROUS, P. W., AND BRAUN, U. 2003. Mycosphaerella and its anamorphs: 1. Names published in Cercospora and Passalora Centraalbureau voor Schimm elcultures, Urtrecht, The Netherlands. 571 pp. CROUS, P. W., KANG, J. C., AND BRAUN, U. 2001. A phylogeneti c redefinition of anamorph genera in Mycosphaerella based on ITS rDNA sequence a nd morphology. Mycologia 93: 10811101. DAHMEN, H., STAUB, T., AND SCHWINN, F. J. 1983. Technique for long-term preservation of phytopathogenic fungi in liquid nitrogen. Phyopathology 73: 241-246. DANIELS, M. J., DOW, J. M., AND OSBOURN, A. E. 1988. Molecular genetics of pathogenicity in phytopathogenic bacter ia. Ann. Rev. Phytopathol. 26; 285-312. DAUGHTREY, M. L., AND BENSON, D. M. 2005. Principles of plant health management for ornamental plants. Annu. Rev. Phytopathol. 43: 141-169. DEAN, F. B., HOSONO, S., FA NG, L., WU, X., FARUQI, A ., BRAY-WARD, P., SUN, Z., ZONG, Q., DU, Y., DU, J., DRISCOLL, M., SONG, W., KINGSMORE, S. F., EGHOLM, M., AND LASKEN, R. S. 2002. Comprehensive human genome amplification using multiple displacement amplification. Proc Natl. Acad. Sci. 99: 5261-5266. DEIGHTON, F. C. 1973. Studies on Cercospora and allied genera. IV. Cercosporella Sacc.; Pseudocercosporella gen. nov. and Pseudocercosporidium gen. nov. Mycol. Pap. 133, C.M.I. Kew, Surrey, UK. 62 pp. DEIGHTON, F. C. 1976. Studies on Cercospora and allied genera. VI. Pseudocercospora Speg., Pantospora Cif. And Cercoseptoria Petr. Mycol. Pap. 140, C.M.I. Kew, Surrey, UK. 168 pp. DEIGHTON, F. C. 1979. Studies on Cercospora and allied genera. VII. New species and redispositions. Mycol. Pap. 144, C.M.I. Kew, Surrey. 156 pp.

PAGE 160

160 DETTMAN, J. R., JACOBSON, D. J., AND TAYL OR, J. W. 2006. Multilocus sequence data reveal extensive phylogenetic species diversity within the Neurospora discreta complex. Mycologia 98: 436-446. DIALLO, S., LATOUR, X., GROBOILLOT, A ., SMADJA, B., COPIN, P., ORANGE, N., FEUILLOLEY, M. G. J., AND CHAVALIER, S. 2009. Simultaneous and selective detection of two major soft rot pathogens in potato: Pectobacterium atrosepticum ( Erwinia carotovora subsp. atrosepticum ) and Dickeya spp. ( Erwinia chrysanthemi ). Eur. J. Plant Pathol. 125: 349-354. DICKEY, R. S. 1979. Erwinia chrysanthemi : a comparative study of phenotypic properties of strains from several hosts and other Erwinia species. Phytopathology 69: 324-329. DONZELLI, B. G. G, AND CHURCHILL, A. C. L. 2007. A qua ntitative assay using mycelial fragments to assess virulence of Mycosphaerella fijiensis Phytopathology 97:916-929. DREISTADT, S. H. 2001. Integrated pest manageme nt for floriculture and nurseries. University of California Statewide Inte grated Pest Management Project. DANR Pub. 3402, Oakland, CA. 422 pp. DRESSLER, R. L. 1993. Phylogeny a nd classification of the orchid family. Dioscorides Press, Portland, OR. 314 pp. DUNKLE, L. D., AND LEVY, M. 2000. Genetic relatedness of African and United States populations of Cercospora zeae-maydis Phytopathology 90: 486-490. EHRESMANN, C., STIEGLER, P., MACKIE, G. A., ZIMMERMANN, R. A., EBEL, J. P., AND FELLNER, P. 1978. Primary sequence of the 16S ribosomal RNA of Escherichia coli Nucleic Acids Res. 2: 265-278. EKPO, E. J. A., AND ESURUOSO, O. F. 1978. Growth and sporulation of Cercospora cruenta and Cercospora canescens Can. J. Bot. 56: 229-233. ELLIS, M. B. 1971. Dematiaceous hyphomycetes. CA B International, New York, NY. 608 pp. ELLIS, M. B. 1976. More dematiaceous hyphomycetes. CAB International, New York, NY. 512 pp. ERWIN, D. C., AND RIBEIRO, O. K. 1996. Phytophthora diseases worldwide. APS Press, St. Paul, MN. 562 pp. ESTRADA-SOTO, S., LPEZ-GUERRERO, J. J., VILLALOBOS-MOLINA, R., AND MATA, R. 2006. Endothelium indepe ndent relaxation of aorta rings by two stilbenoids from the orchid Scaphyglottis livida Fitoterapia 77: 236-239.

PAGE 161

161 FARR, D. F., BILLS, G. F., CHAMURIS, G. P., AND ROSSMAN, A. Y. 1989. Fungi on plants and plant products in the United States. The Amer ican Phytopathological Society, St. Paul, MN. 1,252 pp. FINETTI SIALER, M. M., AND ROSSO, L. 2007. Mol ecular detection in integrated pest and disease management, pp. 305-328 In A. Ciancio and K. G. Mukerj i [eds.], General concepts in integrated pest and disease management Springer, Dordrecht, The Netherlands. FLINT, M. L., AND GOUVEIA, P. 2001. IPM in practic e: principles and me thods of integrated pest management. University of Ca lifornia, DANR Pub. 348, Oakland, CA. 296 pp. FLINT, M. L., AND VAN DEN BOSCH, R. 1981. In troduction to integrated pest management. Plenum Press, New York, NY. 240 pp. FOOD AND AGRICULTURE ORGANIZATION. 1975. Report of th e FAO panel of experts on integrated pest control, Oct. 15-25, 1974, FAO-UN Meeting Report. Rome, Italy. 19 pp. FOSTER, V. 1955. Fusarium wilt of Cattleyas. Phytopathology 45: 99-602. FOX, G. E., WISOTZKEY, J. D., AND JURT SHUK, JR., P. 1992. How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. Int. J. Syst. Bacteriol. 42: 166-170. FRY, C. R., ZIMMERMAN, M. T., AND SCOTT, S. W. 2004. Occurrence of Colombian datura virus in the terrestrial orchid, Spiranthes cernua J. Phytopathol. 152: 200-203. GALLEGLY, M. E. AND HONG, C. 2008. Phytophthora: Identifying species by morphology and DNA fingerprints. APS Pr ess, St. Paul, MN. 168 pp. GARDAN, L., GOUY, C., CHRI STEN, R., AND SAMSON, R. 2003. Elevation of three subspecies of Pectobacterium carotovorum to species level: Pectobacterium atrosepticum sp. nov., Pectobacterium betavasculorum sp. nov. and Pectobacterium wasabiae sp. nov. Int. J. Syst. Evol. Micro. 37: 559-568. GEIER, P.W. 1966. Management of insect pests. Annu. Rev. Entomol. 11: 471-490. GENT, D. H., SCHWARTZ, H. F ., AND NISSEN, S. J. 2003. Effe cts of commercial adjuvants on vegetable crop fungicide coverage, abso rption, and efficacy. Plant Dis. 87: 591-597. GOODWIN, S. B., DUNKLE, L. D., AND ZISMAN, V. L. 2001. Phylogenetic analysis of Cercospora and Mycosphaerella based on the internal transc ribed region of ribosomal DNA. Phytopathology 91: 648-658. GOTH, R. W. 1981. An efficient te chnique for prolonged storage of Phytophthora infestans Am. J. Potato Res. 58: 257-260.

PAGE 162

162 GOTTWALD, T. R., GRAHAM, J. H., AND RI LEY, T. D. 1997. The influence of spray adjuvants on exacerbation of citrus bacterial spot. Plant Dis. 81: 1305-1310. GRIESBACH, R. J. 2000. Potted Phalaenopsis orchid production: histor y, present status, and challenges for the future. HortTechnology 10: 429. GROENEWALD, M., GROENEWALD, J. Z., BR AUN, U., AND CROUS, P. W. 2006. Host range of Cercospora apii and C. beticola and description of C. apiicola a novel species from celery. Mycologia 98: 275-285. HADLEY, G., ARDITTI, M., AND AR DITTI, J. 1987. Orchid diseases-a compendium, pp. 261328 In : Arditti, J. [ed.], Orchid biology reviews and perspectives, IV. Cornell University Press, Ithaca, NY. HAJEK, A. E. 2004. Natural enem ies: an introduction to biol ogical control. Cambridge University Press, Cambridge, UK. 396 pp. HALL, B. G. 2001. Phylogenetic trees made eas y. A how-to manual for molecular biologists. Sinauer Assoc., Sunderland, MA. 179 pp. HAMON, A. B. 2002. Orchid pests, pp. 35-49 In J. B. Watson [ed.], Orch id pests and diseases. American Orchid Society, Delray Beach, FL. HARMON, P. F., DUNKLE, L. D., AND LATIN, R. 2003. A rapid PCR-based method for the detection of Magnaporthe oryzae from infected perennial ry egrass. Plant Dis. 87: 1072-1076. HARTMAN, G. L., CHEN, S. C., AND WANG, T. C. 1991. Cultural studies and pathogenicity of Pseudocercospora fuligena the causal agent of black leaf mold of tomato. Plant dis. 75: 10601063. HILL, T., AND LEWICKI, P. 2006. Statistics: met hods and applications, first edition. StatSoft, Inc., Tulsa, OK. 800 pp. HINE, R. B. 1962. Pathogenicity of Phytophthora palmivora in the Orchidaceae. Plant Dis. Rep. 46: 643-645. HO, C. K., AND CHEN, C. C. 2003. Moscatilin from the orchid Dendrobium loddigesii is a potential anticancer agent. Cancer Invest. 21: 729-736. HOLLINGSWORTH, R. G. 2005. Limone ne, a citrus extract, for control of mealybugs and scale insects. J. Econ. Entomol. 98: 772-779. HOY, M. A. 1994. Parasitoids and predators in management of arthropod pests, pp. 129-198 in R. L. Metcalf and W. H. Luckmann, [eds]., Introd uction to insect pest management, third edition. John Wiley & Sons, New York, NY.

PAGE 163

163 HOY, M. A. 1995. Multitactic resistance managemen t: an approach that is long overdue? Florida Entomol. 78: 443-451. HOY, M. A., JEYAPRAKASH, A., AND NGUYEN, R. 2001. Long PCR is a sensitive method for detecting Liberobacter asiaticum in parasitoids undergoing risk assessment in quarantine. Biol. Control 22: 278-287. HOY M. A, AND JEYAPRAKASH, A. 2005. Microbial diversity in the predatory mite Metaseiulus occidentalis (Acari: Phytoseiid ae) and its prey, Tetranychus urticae (Acari: Tetranychidae). Biol Control 32: 427. HSIEH, W. H., AND GOH, T. K. 1990. Cercospora and similar fungi from Taiwan. Maw Chang Book Co., Taipei, Taiwan. 376 pp. HU, J. S., FERRERIA, S., WANG, M., BORT H, W. B., MINK, G., AND JORDAN, R. 1995. Purification, host range, serol ogy, and partial sequencing of Dendrobium mosaic potyvirus, a new member of the bean common mosaic virus subgroup. Phytopathology 85: 542-546. HU, J. S., FERREIRA, S., WANG, M., AND XU, M. Q. 1993. Detection of Cymbidium mosaic virus Odontoglossum ringspot virus Tomato spotted wilt virus and Potyviruses infecting orchids in Hawaii. Plant Dis. 77: 464-468. HU, J. S., FERREIRA, S., XU, M. Q., LU, M., IHA, M., PFLUM, E., AND WANG, M. 1994. Transmission, movement, and inactivation of Cymbidium mosaic and Odontoglossum ringspot viruses. Plant Dis. 78: 633-636. HUELSENBECK, J. P., RONQUIST, F. 2001. M RBAYES: Bayesian inference of phylogeny. Bioinformatics 17: 754-755. HUFFAKER, C. B. 1985. Biological control in integrated pest management: an entomological perspective, pp. 13-23 in M. A. Hoy and D. C. Herzog, [eds.] Biological control in agricultural IPM systems. Academic Press, Orlando, FL. HUGOUVIEUX-COTTE-PATTAT, N., CO NDEMINE, G., NASSER, W., AND REVERCHON, S. 1996. Regulatio n of pectinolysis in Erwinia chrysanthemi Annu. Rev. Microbiol. 50: 213-257. HUGOUVIEUX-COTTE-PATTAT, N., DOMIN GUEZ, H., AND ROBERT-BAUDOUY, J. 1992. Environmental conditions affect tran scription of the pectinase genes of Erwinia chrysanthemi 3937. J. Bacteriol. 174: 7807-7818. ICHIKAWA, K., AND AOKI, T. 2000. New leaf spot disease of Cymbidium species caused by Fusarium subglutinans and Fusarium proliferatum J. Gen. Plant Pathol. 66: 213-218. ILIEVA, E., MAN INT VELD, W. A., VEENBAAS-RIJKS, W., AND PIETERS, R. 1998. Phytophthora multivesiculata a new species causing rot on Cymbidium Eur. J. Plant Pathol. 104: 677-684.

PAGE 164

164 IMAI, T., TSUCHIYA, S., AND FUJIMORI, T. 1995. Aphicidal effects of Silwet L-77, organosilicone nonionic surfactant. Appl. Entomol. Zool. 30: 380-382. JACOBSEN, B. J. 1997. Role of plant pathology in integrated pest management. Ann. Rev. Phytopathol. 35: 373-391. JANDA, J. M. AND ABBOTT, S. L. 2007. 16S rRNA gene sequencing for bacterial identification in the diagnostic la boratory: pluses, perils, and p itfalls. J. Clin. Microbiol. 45: 2761-2764. JEFFERS, S. N. AND MARTIN, S. B. 1986. Comparison of two media selective for Phytophthora and Pythium species. Plant Dis. 70: 1038-1043. JENSEN, D. D. 1959. Virus diseasesin orchids, pp 431-458 in The Orchids: A Scientific Survey, C. L. Withner, [ed], The Rona ld Press Company, New York, NY. JERARDO, A. 2006. Floriculture and Nursery Crops Outlook, FLO-05. Economic Research Service, United States Department of Agriculture. http://www.ers.usda.gov/Publica tions/Flo/2006/09Sep/FLO05.pdf. Accessed August 21, 2009. JEYAPRAKASH, A., AND HOY, M. A. 2000. Long PCR i mproves Wolbachia DNA amplification: wsp sequences found in 76% of sixty-three ar thropod species. Insect Mol. Biol. 9: 393-405. JOHNSON, P. J. 2008. Mites on cultivated orchids. http://nathist.sdstate.edu/orchids/pests/mites.htm Accessed S eptember 9, 2009. JOHNSON, P. J. 2009. Scale insects on orchids. http://nathist.sdstate.edu/orchids/pests/scales.htm Accessed August 19, 2009. JONES, D. R. 2005. Plant virus es transmitted by thrips. Eur. J. Plant Pathol. 113: 119-157. JONES, S, 2004. Mesophyll col collapse. Orchids 73: 738-740. KANESHIRO, W. S., BURGER, M., VINE, B. G ., DE SILVA, A. S., AND ALVAREZ, A. M. 2008. Characterization of Erwinia chrysanthemi from a bacterial heart ro t of pineapple outbreak in Hawaii. Plant Dis. 92: 1444-1450. KARAOGLANIDIS, G. S ., AND BARDAS, G. 2006. Contro l of benzimidazoleand DMIresistant strains of Cercospora beticola with strobilurin fungicide s. Plant Dis. 90: 419-424. KAZEMI-POUR, N., CONDEMINE, G., AND HUGOUVIEWX-COTTE-PATTAT, N. 2004. The secretome of the plant pathogenic bacterium Erwinia chrysanthemi Proteomics 4: 31773186.

PAGE 165

165 KHENTRY, Y., PARADORNUWAT, A., TA NTIWIWAT, S., PHANSIRI, S., AND THAVEECHAI, N. 2006. Incidence of Cymbidium Mosaic Virus and Odontoglossum ringspot virus in Dendrobium spp. in Thailand. Crop Prot. 25: 926-932. KO, W. H. 2003. Long-term storage and su rvival structure of three species of Phytophthora in water. J. Gen. Plant Pathol. 69: 186-188. KOLBERT, C. P., AND PERSING, D. H. 1999. Ribosomal DNA sequencing as a tool for identification of bacterial pathogens. Curr. Opin. Microbiol. 2: 299-305. KONG, J. M., GOH, N. K., CHIA L. S., AND CHIA, T. F. 2003. Recent advances in traditional plant drugs and orchids. Acta Pharmacol. Sin. 24: 7-21. KONG, P., HONG, C., RICHAR DSON, P. A., AND GALLEGLY, M. E. 2003. Single-strandconformation polymorphism of ribosomal DNA for rapid species differentiation in genus Phytopthora Fungal Genet. Biol. 39: 238-249. KOTOUJANSKY, A. 1987. Molecular genetics of pathogenesis by soft-rot erwinias. Ann. Rev. Phytopathol. 25: 405-430. KOVCS, A., VASAS, A., AN D HOHMANN, J. 2008. Natural phenanthrenes and their biological activity. Phytochemistry 69: 1084-1110. KWON, S. W., GO, S. J., KANG, H. W., RYU, J. C., AND JO, J. K. 1997. Phylogenetic analysis of Erwinia species based on 16S rRNA gene sequen ces. Int. J. Syst. Bacteriol. 47: 10611067. LACOURT, I., BONANTS, P. J. M., VAN GENT-PELZER, COOKE, D. E. L., HAGENAAR DE WEERDT, SURPLUS, L., AND DUNCAN, J. M. 1997. The use of nested primers in the polymerase chain reaction for the detection of Phytophthora fragariae and P. cactorum in strawberry. Acta Hort. 439 Vol. 2. LASHKARI, D. A., MCCUSKER, J. H., AND DAVIS, R. W. 1997. Whole genome analysis: experimental access to all genome sequenced segments through larger-scale efficient oligonucleotide synthesis and PCR. Pr oc. Natl. Acad. Sci. USA 94: 8945-8947. LEE, Y. A., AND YU, C. P. 2006. A di fferential media for the isola tion and rapid detection of a plant soft rot pathogen, Erwinia chrysanthemi J. Microbiol. Meth. 64: 200-206. LEONG, Y. W., HARRISON, L. J., AND POW ELL, A. D. 1999. Phenanthrene and other aromatic constituents of Bulbophyllum vaginatum Phytochemistry 50: 1237-1241. LEONG, Y. W., KANG, C. C., HARRISON, L. J., AND POWELL, A. D. 1997. Phenanthrenes, dihydrophenanthrenes, and bibenzyls from the orchid Bulbophyllum vaginatum Phytochemistry 44: 157-165.

PAGE 166

166 LEWIS, W. J., VAN LENTEREN, J. C., PHATAK S. C., AND TUMLINSON, III., J. H. 1997. A total system approach to sustainable pest management. Proc. Natl. Acad. Sci. USA 94: 1224312248. LVESQUE, C. A. 1997. Molecular detection tools in integrated disease management: overcoming current limitations. Phytoparasitica 25: 3-7. LVESQUE, C. A. 2001. Molecula r methods for detection of pl ant pathogens-what is the future? Can. J. Plant Pathol. 24: 333-336. LI, B., QIU, W., FANG, Y., AND XIE, G. L. 2009. Bacterial stem rot of Oncidium orchid caused by a Dickeya sp. (ex. P. chrysanthemi ) in mainland China. Plant Dis. 93: 552. LIAU, C. H., LU, J. C., PRASAD, V., HSIAO, H. H., YOU, S. J., LEE, J. T., YANG, N. S., HUANG, H. E., FENG, T. Y ., CHEN, W. H., AND CHAN, M. T. 2003. The sweet pepper ferredoxin-like protein ( pflp ) conferred resistance against soft rot disease in Oncidium orchid. Transgenic Res. 12: 329-336. LIU, T. X., AND STANSLY, P. A. 2000. Insecticidal activity of surfactan ts and oils against silverleaf whitefly ( Bemisia argentifolii ) nymphs (Homoptera: Aleyrodidae) on collards and tomato. Pest Manage. Sci. 56: 861-866. LOUWS, F. J., RADEMAKER, J. L. W., AND DE BRUIJN, F. J. 1999. The three Ds of PCRbased genomic analysis of phytobacteria: divers ity, detection, and disease diagnosis. Annu. Rev. Phytopathol. 37: 81-125. LUCKMANN, W. H., AND METCALF, R. L. 1994. The pest-management concept, pp. 1-34 in R. L. Metcalf and W. H. Luckmann, [eds]., Introd uction to insect pest management, third edition. John Wiley & Sons, New York, NY. LUDWIG, W., AND SCHLEIFER, K. H. 1994. Bacterial phylogeny based on 16S and 23S rRNA sequence analysis. FEMS Microbiol. Rev. 15: 155-173. LUDWIG, W., STRUNK, O., KL UGBAUER, S., KLUGBAUER, N., WEIZENEGGER, M., NEUMAIER, J., BACHLEITNER, M., AND SCHLEI FER, K. H. 1998. Bacterial phylogeny based on comparative sequence analysis (review). Electrophoresis 19: 554-568. MAJUMDER, P. L., AND GHOSAL, S. 1994. Two stilbenoids from the orchid Arundina bambusifolia Phytochemistry 35: 205-208. MAJUMDER, P. L., LAHIRI, S., AND MUKHOT I, N. 1995. Chalcone and dihdrochalcone derivatives from the orchid Lusia volucris Phytochemistry 40: 271-274. MAJUMDER, P. L., PAL., S., AND MAJUMDER, S. 1999. Dimeri c phenanthrenes from the orchid Bulbophyllum reptans Phytochemistry 50: 891-897.

PAGE 167

167 MAJUMDER P. L., SEN, S., AND MAJUMDER, S. 2001. Phenanthrene derivatives from the orchid Ceologyne cristata Phytochemistry 58: 581-586. MAJUMDER, P. L., ROYCHOWDHURY, M., AND CHAKRABORTY, S. 1998. Thunalbene, a stilbene derivative from the orchid Thunia alba Phytochemistry 49: 2375-2378. MANAKO, Y., WAKE, H., TANAKA, T., SHIMOMURA, K., AND ISHIMARU, K. 2001. Phenanthropyran derivatives from Phalaenopsis equestris Phytochemistry 58: 603-605. MARTIN, F. N. AND TOOLEY, P. W. 2004. Identification of Phytophthora isolates to species level using restriction fragment length polymorphism analysis of a polymerase chain reactionamplified region of mitochondr ial DNA. Phytopathology 94: 983-991. MCCARTNEY, H. A., FOSTER, S. J., FRAAI JE, B. A., AND WARD, E. 2003. Molecular diagnostics for fungal plant pathoge ns. Pest Manag. Sci. 59: 129-142. MCCONNELL, D. B., AND SHEEHAN, T. J. 1978. Anatomical aspects of chilling injury to leaves of Phalaenopsis Bl. HortScience 13: 705-706. MCHAU, G. R. A. AND COFFEY, M. D. 1994. Isozyme diversity in Phytophthora palmivora : Evidence for a southeast Asia center of origin. Mycol. Res. 98: 1035-1043. MELNDEZ, E. J., AND ACKERMAN, J. D. 1993. The effects of a rust infection on fitness components in a natural population of Tolumnia variegata (Orchidaceae). Oecologia 94: 361367. MELNDEZ, E. J., AND ACKERMAN, J. D. 1994. F actors associated with a rust infection ( Sphenosphora saphena ) in an epiphytic orchid ( Tolumnia variegata ). Am. J. Bot. 81: 287-293. MELOTTO, M., UNDERWOOD, W., AND HE, S.Y. 200 8. Role of stomata in plant innate immunity and foliar bacterial dis eases. Annu. Rev. Phytopathol. 46: 101-122. MILLER, S. A., AND MARTIN, R. R. 1988. Molecu lar diagnosis of plan t disease. Ann. Rev. Phytopathol. 26: 409-432. MORICCA, S., RAGAZZI, A., KASUGA, T., AND MITCHELSON, K. R. 1998. Detection of Fusarium oxysporum f. sp. vasinfectum in cotton tissue by polymerase chain reaction. Plant Pathol. 47: 486-494. MULLIS, K. B., AND FALOONA, F. A. 1987. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reacti on. Methods Enzym. 155: 335-350. NADEAU, J. H., BEDIGIAN, H. G., BOUC HARD, G., DENIAL, T., KOSOWSKY, M., NORBERG, R., PUGH, S., SARGEANT, E., TU RNER, R., AND PAIGEN, B. 1992. Multilocus markers for mouse genome analysis: PCR amplif ication based on single primers of arbitrary nucleotide sequence. Mamm. Genome 3: 55-64.

PAGE 168

168 NAGEL, C. M. 1934. Conidial production in species of Cercospora in pure culture. Phytopathology 24: 1101-1110. NASSAR, A., PARRASSE, A., LEMATTRE, M., KOTOUJANSKY, A., DERVIN, C., VEDEL, R., AND BERTHEAU, Y. 1996. Characterization of Erwinia chrysanthemi by pectinolytic isozyme polymorphism and restriction fragment le ngth polymorphism analysis of PCR-amplified fragments of pel genes. Appl. Environ. Microbiol. 62: 2228-2235. NAUM, M., BROWN, E. W., MASON-GAMER, R. J. 2008. Is 16S a reliable phylogenetic marker to characterize relationships below the family level in the Enterobacteriaceae? J. Mol. Evol. 66: 630-642. NELSON, S. C., AND CAMPBELL, C. L. 1990. Ho st range and cultural characteristics of Cercospora zebrina from white clover in North Carolina. Plant Dis. 74: 874-878. NEUMANN, P. M., AND PRINZ, R. 1974. Evaluation of surfactants fo r use in the spray treatment of iron chlorosi s in citrus trees. J. Sci. Food Agric. 25: 221-226. NIELSEN, K., YOHALEM, D. S., AND JENSEN, D. F. 2002. PCR detection and RFLP differentiation of Botrytis species associated with neck rot of onion. Plant Dis. 86: 682-686. ORLIKOWSKI, L. B., AND SZKUTA, G. 2006. Phytophthora rot of some orchids-new disease in Poland. Phytopathol. Pol. 40: 57. PALACIO-BIELSA, A., CAMBRA, M. A., AND LPEZ, M. M. 2006. Characterisation of potato isolates of Dickeya chrysanthemi in Spain by a microtitre system for biovar determination. Ann. Appl. Biol. 148: 157-164. PALMATEER, A. J., MCLEAN, K. S., AND MORGAN-JONES, G. 2003. Concerning Phomopsis gossypii the causal organism of boll rot of cotton. Mycotaxon 87: 157-172. PARK, J., PARK B., VEERARA GHAVAN ,N., BLAIR, J. E., GE ISER D. M., ISARD, S., MANSFIELD, M. A., NIKOLAEVA, E., PARK, S. Y., RUSSO, J., KIM, S. H., GREENE, M., IVORS, K. L., BALCI, Y., PEIMAN, M., ERWI N D. C., COFFEY, M. D., JUNG, K., LEE Y. H., ROSSMAN, A., FARR, D., CLINE, E., GR N. J., LUSTER, D. G., SCHRANDT, J., MARTIN, F., RIBEIRO, O. K., MAKAL OWSKA, I., AND KANG, S. 2008. Phytophthora Database: A cyberinfrastru cture supporting the identification and monitoring of Phytophthora Plant Dis. 92: 966-972. PEREIRA, O. L., AND BARRETO, R. W. 2004. First report of Sphenospora kevorkianii (Raveneliaceae) on the orchid Catasetum fimbriatum in Brazil. Plant Pathol. 53: 256. PROMBELON, M. C. M., AND HYMAN, L. J. 1986. A rapid method for identifying and quantifying soft rot erwinias dire ctly from plant material based on their temperature tolerances and sensitivity to erythromycin. J. Appl. Bacteriol. 60: 61-66.

PAGE 169

169 PROMBELON, M. C. M., AND KELMAN, A. 1980. Ecology of the soft-rot Erwinia. Annu. Rev. Phytopathol. 12: 361-387. PERSAD, A. B., JEYAPRAKASH, A., AND HOY, M. A. 2004. High-fidelity PCR assay discriminates between immature Lipolexis oregmae and Lysiphlebus testaceipes (Hymenoptera: Aphidiidae) within th eir aphid hosts. Florid a Entomol. 87: 18-24. PETERSON, R. K. D., AND HUNT, T. E. 2003. The probalistic economic injury level: incorporating uncertainty into pest manage ment decision-making. J. Econ. Entomol. 96: 536542. PETERS, R. D., PLATT, H. W., AND HALL, R. 1998. Long-term survival of Phytophthora infestans in liquid media prepared from autoclaved seeds. Can. J. of Plant Pathol. 20: 165-170. PITMAN, A. R., WRIGHT, P. J., GALBRA ITH, M. D., AND HARROW, S. A. 2008. Biochemical and genetic diversity of pectolytic enterobacteria causing soft rot disease of potatoes in New Zealand. Australa sian Plant Pathol. 37: 559-569. PLOETZ, R. C. 2007. Diseases of tropical pere nnial crops: challenging problems in diverse environments. Plant Dis. 91: 644-663. POSADA, D., AND CRONDALL, K. A. 1998. Modeltest: Te sting the model of DNA substitution. Bioinformatics 14: 817-818. PRICE, J. F. 2002. Biological contro l of insects and mites, pp. 22-25 In Orchid pests and diseases, revised edition. American Orchid Society, Delray Beach, FL. PUCHTA, H., AND SANGER, H. L. 1989. Sequence an alysis of minute amounts of viroid RNA using the polymerase chain react ion (PCR). Arch. Virol. 106: 335-340. PURCELL, M. F., AND SCHROEDER, W. J. 1996. E ffect of Silwet L-77 and Diazinon on three tephritid fruit flies (Diptera: Tephritidae) and a ssociated endoparasitoids. J. Econ. Entomol. 89: 566-1570. REVERCHON, S., ROUANET, C., EXPERT, D., AN D NASSER, W. 2002. Ch aracterization of indigoidine biosynthetic genes in Erwinia chrysanthemi and role of this blue pigment in pathogenicity. J. Bacteriol. 2002. 184: 654-665. SAH, D. N., AND RUSH, M. C. 1988. Physiological races of Cercospora oryzae in the southern United States. Plant Dis. 72: 262-264. SAIKI, R. K., GELFAND, D. H., STOFFEL, S., SCHARF, S. J ., AND HIGUCHI, R. 1988. Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487.

PAGE 170

170 SAMSON, R., LEGENDRE, J. B., CHRISTEN, R., FISCHERLE SAUX, M., ACHOUAK, W., AND GARDAN, L. 2005. Transfer of Pectobacterium chrysanthemi (Burkholder et al. 1953) Brenner et al. 1973 and Brenneria paradisiaca to the genus Dickeya gen. nov. as Dickeya chrysanthemi comb. nov. and Dickeya paradisiaca comb. nov. and delineation of four novel species, Dickeya dadantii sp. nov., Dickeya dianthicola sp. nov, Dickeya dieffenbachiae sp. nov. and Dickeya zeae sp.nov. Inter. J. System. Evol. Microbiol. 55: 1415-1427. SAS INSTITUTE. 2002. SAS procedures guide version 9. SAS Institute, Cary, NY. 1040 pp. SCHAAD, N. W., JONES, J. B., AND CHUN, W., [eds], 2001. Laboratory guide for the identification of plant pathogenic bacteria, third edition. The American Phytopathological Society Press, St. Paul, MN. 373 pp. SCHUERGER, A. C., AND BATZER, J. C. 19 93. Identification and host range of an Erwinia pathogen causing stem rots on hydroponicall y grown plants. Plant Dis. 77: 472-477. SHAPIRO, J.P., SCHROEDER, W.J., AND STANS LY, P.A. 1998. Bioassay and efficacy of Bacillus thuringiensis and an organosilicone surfactant agai nst the citrus leafminer (Lepidoptera: Phylloenistidae). Fla. Entomol. 81: 201-210. SHEEHAN, T., 1987. Question Box. Am. Orchid Soc. Bull. 56: 733. SHEEHAN, T., 2002. Physiological disorders of orchids, pp. 4-21 In Orchid pests and diseases, revised edition. American Orch id Society, Delray Beach, FL. SHIMURA, H., MATSUURA, M., TAKADA, N., AND KODA, Y. 2007. An antifungal compound involved in symbiotic germination of Cypripedium macranthos var. rebunense (Orchidaceae). Phytochemistry 68: 1442-1447. SIMONE, G. W., AND BURNETT, H. C. 1995. Di seases caused by bacteria and fungi, pp. 3467 In Orchid pests and diseases. American Orchid Society, Delray Beach, FL. SJAHRIL, R., CHIN, D. P., KHAN, R. S., YAMAMURA, S., NAKAMURA, I., AMEMIYA, Y., AND MII, M. 2006. Transgenic Phalaenopsis plants with resistance to Erwinia carotovora produced by introducing wasa bi defensin gene using Agrobacterium method. Plant Biotechnol. 23: 191-194. SLAWIAK, M., VAN BECKHOVEN, J. R. C. M., SPEKSNIJDER, G. C. L., CZAJKOWSKI, R., GRABE, G., AND V AN DER WOLF, J. M. 2009. Biochemical and genetical analysis reveal a new clade of biovar 3 Dickeya spp. Strains isolated from potato in Europe. Eur. J. Plant Pathol. 125: 245-261. SOLHEIM, W. G. 1930. Morphol ogical studies of the genus Cercospora. Ill. Biol. Monogr. 12. 85 pp.

PAGE 171

171 SRINIVASAN, R., HOY, M. A ., SING, R., AND ROGERS, M. E. 2008. Laboratory and field evaluations of Silwet L-77 and Kinetic alone and in combination with imidacloprid and abamectin for the management of the Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae). Fla. Entomol. 91: 87-100. STACKEBRANDT, E., AND GOEBEL, B. M. 1994. Taxanomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int. J. Syst. Bacteriol. 44: 846-849. STALEY, J. T. 2006. The bacterial species dilemma and the genomic-phylogenetic species concept. Phil. Trans. R. Soc. B. 361: 1899-1909. STARR, M. P., COSENS, G., A ND KNACKMUSS, H. J. 1966. Formation Of the blue pigment indigoidine by phytopathogenic Erwinia Appl. Microbiol. 14: 870-872. STAVELY, J. R., AND NIMMO, J. A. 1968. Relati on of pH and nutrition to growth and sporulation of Cercospora nicotianae Phytopathology 58: 1372-1376. STERN, V. M., SMITH, R., VAN DEN BOSCH, R., AND HAGEN, K. S. 1959. The integration of chemical and biological contro l of the spotted alfalfa aphid. I. The integrated control concept. Hilgardia 29: 81-101. STEWART, E. L., LIU, Z., CROUS, P. W., AND SZABO, L. J. 1999. Phylogenetic relationships among some cercosporoid anamorphs of Mycosphaerella based on rDNA sequence analysis. Mycol. Res. 103: 1491-1499. SWANSON, J. K., MONTES, L. MEJIA, L ., AND ALLEN, C. 2007. Detection of latent infections of Ralstonia solanacearum race 3 biovar 2 in geranium. Plant Dis. 91: 828-834. SWOFFORD, D. L. 2002. PAUP*. P hylogenetic analysis using parsimony (*and other methods), version 4. Sinauer Associates, Sunderland, MA. TAMAKI, S. J., GOLD, S., ROBESON, M., MANULIS, S., AND KEEN, T. 1988. Structure and organization of the pel genes from Erwinia chrysanthemi EC16. J. Bacteriol. 170: 3468-3478. TEBBE, C. C., AND VAHJEN, W. 1993. Interferece of humic acids and DNA extracted directly from soil in detection and tran sformation of recombinant DNA from bacteria and a yeast. Appl. Environ. Microbiol. 59: 2657-2665. THOMPSON, J. D., GIBSON, T. J., PLEWNI AK, F., JEANMOUGIN, F., AND HIGGINS, D. G. 1997. The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided. Nucleic Acids Res. 24: 4876-482. TIPPING, C., BIKOBA, V., CHANDER, G. J ., AND MITCHAM, E. J., 2003. Efficacy of Silwet L-77 against several arthropod pests of ta ble grape. J. Econom. Entomol. 96: 246-250.

PAGE 172

172 TOOLEY, P. W. 1988. Use of uncontrolled freezing for liquid nitrogen storage of Phytophthora species. Plant Dis. 72: 680-682. TOTH, I. K., AVROVA, A. O., AND HYMAN L. J. 2001. Rapid identification and differentiation of the soft rot Erwinias by 16S-23S intergenic transcribed spacer-PCR and restriction fragment length polymorphism anal yses. Appl. Enviro. Microbiol. 67: 4070-4076. TOTH, I. K., BELL, K. S, HOLEVA, M. C., AND BIRCH, P. R. J. 2003. Soft-rot erwiniae: from genes to genomes. Mol. Plant Pathol. 4: 17-30. TROUT, C. L., RISTAINO, J. B., MADR ITCH, M., AND WANGSOMBOONDEE, T. 1997. Rapid detection of Phytophthora infestan s in late-blight infected pot ato and tomato using PCR. Plant Dis. 81: 1042-1048. TSAI, H. L., HUANG, L. C., ANN, P. J., AND LIOU, R. F. 2006. De tection of orchid Phytophthora disease by nested PCR. Botanical Studies 47: 379-387. TSAVKELOVA, E. A., BMKE, C., NETRUSOV, A. I., WEINER, J., AND TUDZYNSKI, B. 2008. Production of gibberellic aci ds by an orchid-associated Fusarium proliferatum strain. Fungal Genet. Biol. 45: 1393-1403. TSROR (LAHKIM), L., ERLICH, O., LEBIU SH, S., HAZANOVSKY, M., ZIG, U., SLAWIAK, M., GRABE, G., VAN DER WOLF, J. M., AND VAN DE HAAR, J. J. 2009. Assessment of recent outbreaks of Dickeya sp. (syn. Erwinia chrysanthemi ) slow wilt in potato crops in Israel. Eur. J. Plant Pathol. 123: 311-320. TU, M., HURD, C., AND RANDALL, J. M. 2001. Weed control methods handbook. The Nature Conservancy, http:// tncweeds.ucdavis.edu version: April, 2 001. Accessed August 19, 2009. UCHIDA, J. Y. 1994. Diseases of orchids in Hawaii. Plant Dis. 78: 220-224. UCHIDA, J. Y., AND ARAGAKI M. 1980. Nomenclature, pat hogenicity, and conidial germination of Phyllostictina pyriformis Plant Dis. 64: 786-788. URENA-PADILLA, A. R., MACKENZIE, S. J ., BOWEN, B. W., A ND LEGARD, D. E. 2002. Etiology and population genetics of Colletotrichum spp. causing crown and fruit rot of strawberry. Phytopathology 92: 1245-1252. VALENCIA-ISLAS, N. A., PAUL, R. N., SHIER, W. T., MATA, R., AND ABBAS, H. K. 2002. Phytoxicity and ultras tructural effects of gym nopusin from the orchid Maxillaria densa on duckweed ( Lemna pausicostata) frond and root tissues. Phytochemistry 61: 141-148. VALERIO, C. R., MURRAY, P., ARLIAN, L. G., AND SLATER, J. E. 2005. Bacterial 16S ribosomal DNA in house dust mite cultures. J. Allergy Clin. Immunol. 118: 1296-1300

PAGE 173

173 VAUGHAN, S. P., GRISONI, M., KUMAGAI M. H., AND KUEHNLE, A. R. 2008. Characterization of Ha waiian isolates of Cymbidium mosaic virus (CymMV) co-infecting Dendrobium orchid. Arch. Virol. 153: 1185-1189. VICKERS, J. E., AND GRAHAM, G. C. 1996. A protocol for th e efficient screening of transformed plants for bar, the selectable mark er gene, using the polymerase chain reaction. Plant Mol. Biol. Rep. 14: 363-368. VINCELLI, P., AND TISSERAT, N. 2008. Nucleic acid-based pat hogen detection in applied plant pathology. Plant Dis. 92: 660-669. VINCENT, C., HALLMAN, G., PANNETON, B., AND FLEURAT-LESSARD, F. 2003. Management of agricultural insects with physical control methods. Annu. Rev. Entomol. 48: 261-281. WALERON, M., WALERON, K., PODHAJSKA, A. J., AND OJKOWSKA, E. 2002. Genotyping of bacteria be longing to the former Erwinia genus by PCR-RFLP analysis of a recA gene fragment. Microbiology 148: 583-595. WANG, J., LEVY, M., AND DUNKLE, L. D. 1998. Sibling species of Cercospora associated with gray leaf spot of maize. Phytopathology 88: 1269-1275. WANG, X., BAUW, G., VAN DAMME, J. M., PE UMANS, W. J., CHEN, Z. L., MONTAGU, M. V., ANGENON, G., AND DILLEN, W. 2001. Gast rodianin-like mannose-binding proteins: a novel class of plant proteins with anti fungal properties. Plant J. 25: 651-661. WARD, E., FOSTER, S. J., FRAAIJE, B. A., AND MCCARTNEY, H. A. 2004. Plant pathogen diagnostics: immunological and nuc leic acid-based approaches Ann. Appl. Biol. 145: 1-16. WEISBURG, W. G., BARNS, S. M., P ELLETIER, D. A., AND LANE, D. J. 1991. 16S ribosomal DNA amplification for phyloge netic study. J. Bacteriol. 173: 697-703. WHITE, T. J., BRUNS, T., LEE, S., AND TAYL OR, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, pp. 315-322 In M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White [eds .], PCR protocols: a guide to methods and applications. Academic Press, San Diego, CA. WILSON, I. G. 1997. Inhibition and facilitation of nucleic acid amplification. Appl. Environ. Microbiol. 63: 3741-3751. WILSON, D. M., FENICAL., W., HAY, M., LINDQUIST, N., AND BOLSER, R. 1999. Habenariol, a freshwater feeding de terrent from the aquatic orchid Habenaria repens (Orchidaceae). Phytochemistry 50: 1333-1336. WINTON, L. M., HANSEN, E. M., AND STONE, J. K. 2006. Population structure suggests reproductively isolated lineages of Phaeocryptopus gaeumannii Mycologia 98: 781-791.

PAGE 174

174 WOO, P. C. Y., LAU, S. K. P., TENG, J. L. L., TSE, H., AND YUEN, K. Y. 2008. Then and now: use of 16S rDNA gene sequencing for bact erial identification and discovery of novel bacteria in clinical microbiology labora tories. Clin. Microbiol. Infec.14: 908-934. WOOD, B. W., TEDDERS, W. L ., AND TAYLOR, J. 1997. Control of pecan aphids with an organosilicone surfactant. HortScience 32: 1074-1076. YAMADA, T., KURODA, K., JITSUYAMA, Y., TAKEZAWA, D., ARAKAWA, K., AND FUJIKAWA, S. 2002. Roles of the plasma membrane and the cell wall in th e responses of plant cells to freezing. Planta 215: 770-778. YAP, M. N., YANG, C. H., BARAK, J. D., JAHN, C. E., AND C HARKOWSKI, A. O. 2005. The Erwinia chrysanthemi type III secretion system is re quired for multicellular behavior. J. Bacteriol. 187: 639-648. YOKOMI, R. K., MELLO, A. F. S., SAPONA RI, M., AND FLETCHER, J. 2008. Polymerase chain reaction based detection of Spiroplasma citri associated with citrus stubborn disease. Plant Dis. 92: 253-260. YOUNG, J. M., AND PARK, D. C. 2007. Relationships of plant pat hogenic enterobacteria based on partial atpD carA and recA as individual and concatenated nucleotide and peptide sequences. Syst. Appl. Microbiol. 30: 343-354. ZEIGLER, D. R. 2003. Gene sequences useful for predicting relatedness of whole genomes in bacteria. Int. J. Syst. Evol. Microbiol. 53: 1893-1900. ZENTENO, R., CHVEZ, R., PORTUGAL, D ., PEZ, A., LASCURAIN, R., AND ZENTENO, E. 1995. Purification of a N -acetyl-D-galactosamine specific lectin from the orchid Laelia autumnalis Phytochemistry 40: 651-655. ZHANG, J. X., FERNANDO, W. G. D., AND REMPHREY, W. R. 2005. Genetic diversity and structure of the Apiosporina morbosa populations on Prunus spp. Phytopathology 95: 859-866. ZHENG, Y. X., CHEN, C. C ., CHEN, Y. K., AND JAN, F. J. 2008a. Identification and characterization of a potyvirus causing chlorotic spots on Phalaenopsis orchids. Eur. J. Plant Pathol. 121: 87-95. ZHENG, Y. X., CHEN, C. C., CH EN, Y. K., YEH, S. D., AND JAN, F. J. 2008b. Identification and characterization of a tospoviru s causing chlorotic ringspots on Phalaenopsis orchids. Eur. J. Plant Pathol. 120: 199-209. ZIDACK, N. K., BACKMAN, P. A., AND SHAW, J. J. 1992. Promotion of bacterial infection of leaves by an organosilicone surfactant: implica tions for biological weed control. Biol. Control 2: 111-117.

PAGE 175

175 BIOGRAPHICAL SKETCH Born in Louisville, Kentucky and raised in a sm all town in southern Indiana, Robert Cating attended Crawford County High School in Mare ngo, Indiana followed by undergraduate study at Indiana University-Bloomington and Indiana Univer sity-Southeast. As an undergraduate, Robert studied piano, biology, near eastern languages and cultures, and jewish studies. An avid orchid grower, Robert encountered many orchid pest an d disease problems. The lack of information about these problems was the impetus for his study of orchid pests and diseases at the University of Florida, earning a Master of Science in entomology and a Doctor of Philosophy in plant pathology. Part of his time as a graduate student was spent at the Tropical Research and Education Center in Homestead, Florida. Robe rt enjoyed interacting with the numerous and exceptional orchid growers in the Redland and Homestead area, which is quickly becoming the center of the orchid universe. Robert accepted a position with Twyford In ternational in Apopka, Florida as Director of Biotechnology Research.