TOWARDS ORCHID IPM: TOOLS AND MOLECULAR TECHNIQUES FOR THE DIAGNOSIS AND MANAGEMENT OF SELECTED ORCHID PESTS AND DISEASES IN FLORIDA

By

ROBERT ALLEN CATING

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2010

1

© 2010 Robert Allen Cating

2

To my Grandmother, who always believed in me

3 ACKNOWLEDGEMENTS

There have been many people who have had an extraordinary influence on my research, the writing of this dissertation, and my development as a scientist, most notably my committee members Dr. Aaron Palmateer, Dr. Marjorie Hoy, Dr. Randy Ploetz, Dr. Robert McMillan, and

Dr. Wagner Vendrame. I found that my committee 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 members, 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, answered 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 growers who donated time and , and I am sincerely grateful for the time and interest each of them put into assisting me with my work:

Kerry Herndon, Bill Peters, Dr. Martin Motes, Duane and Donna Goodwin, Suzanne Farnsworth,

Robert Fuchs, Michael Coronado, Jose Esposito, and Jack Batchelor. I discovered early in my research that it was going to be difficult to obtain funding to perform orchid related research. I express my sincere gratitude to the Redland Orchid Festival, Inc. and the Redland Professional

Orchid Growers for coming forward and providing the funding for my work.

4 Also, there were people who supported me in different 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.

5 TABLE OF CONTENTS

page

LIST OF TABLES ...... 10

LIST OF FIGURES ...... 11

ABSTRACT ...... 13

CHAPTER

1 LITERATURE REVIEW ...... 15

Introduction ...... 15 Integrated Pest Management ...... 18 What is Integrated Pest (Disease) Management? ...... 18 Management Tactics of an Integrated Pest Management Program ...... 20 Mechanical and physical control ...... 21 Cultural control ...... 22 Chemical control ...... 22 Biological control ...... 23 Research Objective ...... 24 Chapter 2: Identification and Characterization of a Soft-Rot Isolated from Vanda, , , and Orchids in Florida...... 25 Objectives ...... 27 Chapter 3: Culture of Pseudocercospora that Affect Orchids ...... 28 and identification ...... 28 Molecular systematics ...... 29 Growth and sporulation of Cercosporoid fungi in culture ...... 30 Objective ...... 31 Chapter 4: A Comparison of the Standard and High-Fidelity Polymerase Chain Reactions (PCR) in the Detection of Pseudocercospora odontoglossi from Orchids and its Use in the Diagnosis of Orchid Diseases ...... 31 High-fidelity PCR ...... 32 Objectives ...... 33 Chapter 5: The use of Silwet L-77 as an Integrated Pest Management Tool ...... 34 Objectives ...... 35 Appendices ...... 35

2 IDENTIFICATION AND CHARACTERIZATION OF A SOFT-ROT PATHOGEN ISOLATED FROM Vanda, Phalaenopsis, Oncidium, AND Tolumnia ORCHIDS IN FLORIDA ...... 39

Introduction ...... 39 Materials and Methods ...... 41 Isolation and Initial Tests ...... 41 Fatty Acid Analysis ...... 42

6 Carbohydrate Utilization Tests ...... 42 Molecular and Phylogenetic Analysis ...... 42 Pathogenicity Tests ...... 45 Results ...... 46 Isolation and Identification of Strains ...... 46 Fatty Acid Analysis ...... 46 Carbohydrate Utilization Tests ...... 46 Molecular and Phylogenetic Analysis ...... 47 Pathogenicity Tests ...... 48 Discussion ...... 48

3 CULTURE OF Pseudocercospora SPECIES THAT AFFECT ORCHIDS ...... 80

Introduction ...... 80 Materials and Methods ...... 81 Growth and Sporulation of P. dendrobii on Different Media ...... 81 Effects of Temperature on Growth and Sporulation of Pseudocercospora spp. on V-8 Agar ...... 83 Effects of Nutrient Reduction on Sporulation of Pseudocercospora sp. from a Tolumnia Orchid ...... 83 Results ...... 84 Growth and Sporulation of P. dendrobii on Different Media ...... 84 Effects of Temperature on Growth and Sporulation of three Pseudocercospora spp. on V-8 Agar ...... 85 Effects of Nutrient Reduction on Sporulation of Pseudocercospora sp. from a Tolumnia Orchid ...... 85 Discussion ...... 85

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 ...... 91

Introduction ...... 91 Materials and Methods ...... 92 Evaluation of High-Fidelity PCR by Use of Plasmids ...... 92 Deoxyribonucleic acid (DNA) extraction ...... 92 High-fidelity PCR protocol ...... 93 Molecular Cloning ...... 94 Comparison of Standard and High-Fidelity PCR ...... 95 Comparison of the High-Fidelity and Standard PCR in Detecting Sclerotium rolfsii and Dickeya sp. ( chrysanthemi) from Phalaenopsis Orchids ...... 95 Results ...... 97 Evaluation of High-Fidelity PCR by Use of Plasmids ...... 97 Comparison of the High-Fidelity and Standard PCR in Detecting Sclerotium rolfsii and Dickeya sp. (Erwinia chrysanthemi) from Phalaenopsis Orchids ...... 97 Discussion ...... 98

7 5 SILWET L-77 IMPROVES THE EFFICACY OF HORTICULTRAL OILS FOR CONTROL OF BOISDUVAL SCALE Diaspis boisduvalii (: DIASPIDIDAE) AND THE FLAT MITE Tenuipalpus pacificus (ARACHNIDA: ACARI: TENUIPALPIDAE) ON ORCHIDS ...... 103

Introduction ...... 103 Materials and Methods ...... 105 Phytotoxicity Study ...... 105 Silwet + Oil Efficacy Trial (Boisduval Scale) ...... 106 Silwet + Oil Efficacy Trial (Flat Mite) ...... 107 Results ...... 108 Phytotoxicity Study ...... 108 Silwet + Oil Efficacy Trial (Boisduval Scale) ...... 108 Silwet + Oil Efficacy Trial (Flat Mite) ...... 108 Discussion ...... 109

6 SUMMARY AND CONCLUSIONS ...... 119

7 REFLECTIONS ...... 123

APPENDIX

A PHYSIOLOGICAL DISORDERS OF ORCHIDS: OEDEMA ...... 125

Symptoms ...... 125 Diagnosis ...... 125 Management ...... 126

B PHYSIOLOGICAL DISORDERS OF ORCHIDS: MESOPHYLL CELL COLLAPSE ....131

Symptoms ...... 131 Cause, Diagnosis, and Control ...... 131

C BLACK ROT OF ORCHIDS CAUSED BY Phytophthora palmivora AND Phytophthora cactorum: SYMPTOMS, DIAGNOSIS, AND MANAGEMENT ...... 135

Host Range ...... 135 Symptoms ...... 135 Diagnosis ...... 136 Management ...... 136 Nursery Recommendations for Phytophthora ...... 136 Fungicide Options ...... 137 Growing Media and Storage ...... 137 Containers ...... 137 Bench Sanitation ...... 138 Water Supply and Hand Watering ...... 138 New Plants Brought into the Nursery, Greenhouse, or Growing Area ...... 138

8 D FIRST REPORT OF Sclerotium rolfsii ON Ascocentrum AND Ascocenda ORCHIDS IN FLORIDA ...... 141

Identification ...... 141 Pathogenicity Tests ...... 141

E A DIAGNOSTIC GUIDE FOR BLACK ROT OF ORCHIDS CAUSED BY Phytophthora palmivora AND Phytophthora cactorum FOR DIAGNOSTIC CLINICS AND DIAGNOSTICIANS ...... 144

Introduction ...... 144 Disease ...... 145 Pathogen ...... 145 Taxonomy ...... 145 Symptoms and Signs ...... 146 Range within the ...... 146 Geographic Distribution ...... 146 Pathogen Isolation ...... 146 Pathogen Identification ...... 147 Molecular Diagnostics ...... 148 Pathogen Storage ...... 148 Pathogenicity Tests ...... 149

F FIRST REPORT OF BACTERIAL SOFT ROT ON Tolumnia ORCHIDS CAUSED BY A Dickeya sp...... 152

Identification ...... 152 Pathogenicity Tests ...... 153

REFERENCES ...... 155

BIOGRAPHICAL SKETCH ...... 175

9 LIST OF TABLES

Table page

1-1 Pseudocercospora and species reported to occur on orchids and their common hosts...... 38

2-1 Orchid isolate collection information and initial tests for Dickeya (Erwinia) 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 utilization 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

10 LIST OF FIGURES

Figure page

1-1 Examples of economically important orchids grown as ornamental plants or for cut flowers...... 36

1-2 Difficulties in plant disease diagnosis...... 37

2-1 ClustalX 16S rDNA alignment of 18 Dickeya isolates obtained from orchids using primers pair 27f/1495r ...... 52

2-2 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...... 68

2-3 Maximum likelihood phylogenetic analysis of Dickeya spp. orchid strains based on 16S rDNA gene sequences...... 71

2-4 Bayesian phylogenetic analysis of Dickeya spp. based on 16S rDNA gene sequences. ...72

2-5 Maximum Likelihood phylogenetic analysis of Dickeya spp. strains based on a portion of the pelADE pectolytic enzyme gene cluster...... 73

2-6 Bayesian analysis of Dickeya spp. based on a portion of the pelADE pectolytic enzyme gene cluster...... 74

3-1 Growth of Pseudocercospora dendrobii on 10 different media...... 89

3-2 Measurements of culture diameter at 3, 7, 11, and 13 d at 15, 20, 25, and 30 ± 1ºC on V-8 agar...... 90

4-1 A comparison of high-fidelity 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...... 101

4-2 A comparison of high-fidelity and standard PCR in the detection of two important orchid from five inoculated Phalaenopsis plants...... 102

5-1 Examples of orchids used in the phytotoxicity study after post-spray quality rating. None of the orchids developed symptoms of phytotoxicity on leaves, stems, flowers, buds or roots...... 116

5-2 Cattleya mericlone orchids before treatment with water, petroleum oil, or Silwet L- 77 + petroleum oil ...... 117

5-3 Cattleya mericlone orchids infested with Boisduval scale 1 wk after second treatment .118

A-1 Examples of oedema on various orchids...... 127

11 A-2 Examples of oedema on various orchids...... 128

A-3 Diagnosing oedema in orchids...... 129

A-4 Differentiating scale from oedema blisters ...... 130

B-1 Examples of mesophyll cell collapse in orchids ...... 134

C-1 Black rot of orchids, symptoms and signs...... 140

D-1 Ascocenda orchids with Southern blight caused by S. rolfsii ...... 143

E-1 Black rot of orchids, symptoms and signs ...... 150

E-2 A comparison of sexual reproductive structures of P. palmivora and P. cactorum...... 151

F-1 Tolumnia orchids with symptoms of a soft rot bacterial infection caused by Dickeya sp...... 154

12

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

TOWARDS ORCHID IPM: TOOLS AND 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:

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 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 and disease problems were frequently encountered and which were considered the most severe. They

13 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.

14 CHAPTER 1 LITERATURE REVIEW

Introduction

The Orchidaceae is believed to be the largest family 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 flower 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 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 US dollars in 2006 (Jerardo 2006). Orchids are the second most economically important flowering plant produced in the United States, and Florida and

California are the top producing states (Jerardo 2006). Although poinsettias earn the largest dollar amount in sales, orchids have shown the greatest 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 intriguing, 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 centuries as a flavoring agent, autumnalis Lindl. and Laelia grandiflora (La Llave & Lex.) Lind. are used to make candies and pastries in Mexico, and 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 (Arditti 1992; Kong et al. 2003). Orchids used in traditional medicine include Bletilla striata [Thunb.] Rchb.f. (used to treat tuberculosis,

15 hemoptysis, gastric and duodenal ulcers, and cracked skin), Gastrodia elata Blume (headaches,

dizziness, blackouts, numbness of the limbs, epilepsy, spasms, and migraines), spp.

(sores of the lips), and Oncidium spp. (lacerations) (Kong et al. 2003).

Recent investigations into the chemical compounds found in orchids have yielded surprising

results. Moscatilin, a bibenzyl 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 antiproliferative effect against certain cancer cells

(choriocarcinoma, lung cancers, and stomach cancers) (Ho & Chen 2003). Anti-fungal

compounds have also been isolated and characterized from several different orchids. For

example, the achlorophyllous orchid Gastrodia elata Blume forms a parasitic relationship with

the 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 digestion of the hyphae and nutrient

absorption occurs (Wang et al. 2001). Gastrodianin also has been shown to confer disease resistance to several root pathogens of tobacco (Nicotiana tabacum L. cv. Wisconsin) after

Agrobacterium-mediated 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 shown to be important during

different developmental stages of the plant (Shimura et al. 2007).

Other novel compounds have been described. Habenariol, isolated from the aquatic orchid

Habenaria repens Nutt., deters feeding by freshwater crayfish (Wilson et al. 1999), gymnopusin

from Maxillaria densa Lindl. is phytotoxic to duckweed (Valencia-Islas et al. 2002), and two

16 stilbenoids from livida (Lindl.) Rauschert have a vasorelaxation effect in rats

(Estrada-Soto et al. 2006). Several other phytochemicals have been isolated from orchids used in

traditional medicines and are currently being investigated (Kovács 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 popularity 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 continues to grow, so will the desire for pest and

disease information. For example, the question and answer section of Orchids magazine,

published by the American Orchid Society, frequently 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 known 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 has been done. In addition to viruses, other economically important diseases affecting orchids include rusts (Meléndez & 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.

17 Nelson, T. A. Tousson, & Marasas (syn. Fusarium moniliforme J. Sheld, var. subglutinans

Wollenweb & Reinking) and F. proliferatum (Matsushima) (Broadhurst & Hartill 1996;

Ichikawa & Aoki 2000)); southern blight caused by Sclerotium rolfsii Sacc. (Bag 2004; Cating et

al. 2009a, see Appendix D); bacterial soft 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 integrated pest management program is available.

Integrated Pest Management

What is Integrated Pest (Disease) Management?

Integrated pest management (IPM) and integrated disease management (IDM) are control

strategies that seek to reduce 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 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). As their names suggest, these strategies seek to “integrate” multiple tactics to reduce pest

18 populations or diseases to acceptable levels without relying solely on chemical control.

Although IDM has been used when specific reference is made to disease management, plant

pathogens also have been considered “pests” and IPM includes both and pathogens.

True IPM must consider information from multiple sources, such as from economists,

horticulturalists, and entomologists, and the role of plant pathology in IPM “must be that of a partner within the entire IPM team” (Jacobsen 1997).

Geier (1966) described traditional 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 them” and “integrated control is intended to imply some degree of discrimination 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 suitable techniques and methods in as a compatible manner as possible and maintains the pest population at levels below those causing economic injury” (Food and Agriculture Organization 1975).

The first steps in establishing an IPM program 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 the identification of nonchemical preventative actions (Flint & van den Bosch 1981; Dreistadt et al. 2001). Dreistadt 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;

19 3. accurate diagnoses of pest problems;

4. development of control action guidlines; and

5. the use of effective management tools.

A successful IPM program “integrates” these components into a workable, sustainable system to

prevent pest and disease outbreaks and keep damage at acceptable levels when they do occur.

Management Tactics of an Integrated Pest Management Program

No single pest management tactic will completely 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 pests and diseases on a given 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), which is the threshold at which yields are reduced

(Stern et al. 1959; Flint & van den Bosch 1981; Luckmann & Metcalf 1994; Peterson & Hunt

2003; Bethke & Cloyd 2009). However, EILs are less useful for ornamental crops since they are

sold based on aesthetic appearance and, therefore, 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, which in many cases is possible with only morphological criteria,

plant disease diagnoses usually require several steps, including isolation of the pathogen in pure

culture followed by pathogen identification with morphological characteristics (fungi), biological

tests () or other criteria (viruses) (Louws et al. 1999; Alvarez 2004; Daughtrey & Benson

2005; Finetti Sialer & Rosso 2007). These procedures are labor intensive and time consuming,

20 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 & Tisserat 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 control 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 plant pathology include avoidance, exclusion,

protection, eradication, and resistance. 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 and physical control tactics as those that mechanically destroy pests or introduce a barrier to prevent an infestation. Mechanical and physical control tactics take advantage of “weak links” in the pest’s life cycle or behavior patterns to reduce or prevent infestations (Flint & van den Bosch 1981). Common types of

21 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 implemented specifically for pest control, cultural control tactics are modifications of current practices that alter the environment, making it less favorable for pests or diseases (Flint & van den Bosch 1981).

Cultural control tactics include sanitation, water and fertilizer management, crop rotation, and harvesting practices (for example, using a clean razor blade or flame-sterilized tool to harvest cut flowers to prevent virus transmission) (Flint & van den Bosch 1981). For orchids, cultural practices are crucial for orchid health and include using the correct type of potting mix and pot size, and appropriate humidity and 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 and components of IPM programs (Flint & van den

Bosch 1981).

Chemical control

Although chemical pesticides have been the primary method of pest control since the 1940s, associated ecological and safety concerns continue to rise (Lewis et al. 1997). Chemicals temporarily reduce pest populations but do not contribute to permanent 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;

22 4. toxic residues on food and forage crops;

5. potential damage to human and health and the environment;

6. legal issues resulting from environmental contamination or pesticide drift.

The overall goal of an “integrated” program should be to choose a chemical that can be “fit” into an ecological system, rather than “imposing” 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 pesticides virtually useless against certain organisms (Brent 1995; Hoy 1995). Brent (1995) states that in order to reduce the development of pesticide resistance, one should rotate pesticides, limit treatments, use recommended label rates, and integrate with non-chemical control methods which should include physical, cultural, and biological control tactics.

Biological control

Stern et al. (1959) indicated that biological control and chemical control are not necessarily alternative objectives; in many 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 (Stern et al. 1959; Flint & van den Bosch 1981;

Huffaker 1985; Hajek 2004). Flint & van den Bosch (1981) described three aspects of biological control: (1) naturally occurring biological control, (2) classical biological control, and (3) preservation of native, natural enemies. Naturally occurring biological control 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 classical biological control, natural enemies are deliberately introduced into an area to reduce a pest population.

23 After the identification of the pest and its origin is determined, a search for natural enemies

in the pest’s native habitat is conducted and potential natural enemies are identified. After extensive laboratory, greenhouse, and field trials, the natural enemies may be released and re- establish the pest-natural enemy relationship (Flint & van den Bosch 1981; Dreistadt 2001). For reviews of biological control concepts and applications, see Stern 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 biological control of plant pathogens use such mechanisms as antibiosis (production of a chemical by one organism that directly inhibits another), parasitism (reduction of a plant pathogen by another pathogen), competition (biological control agent and pathogen compete for resources), and the stimulation of plant defense mechanisms by a hypovirulent strain of a pathogen. 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 pathogen 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 describes 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 disease problems they frequently encounter and consider the most

24 severe. Through these consultations, it was determined 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 diagnostic tool. The specific objectives of each chapter are discussed below:

Chapter 2: Identification and Characterization of a Soft-Rot Pathogen Isolated from Vanda, Phalaenopsis, Oncidium, and Tolumnia Orchids in Florida

Pectolytic enzyme-producing bacteria can be devastating plant pathogens. Pectolytic enzymes (pels) macerate plant tissue by degrading plant cell-wall components which leads to soft, water-soaked lesions that can enlarge rapidly (Pérombelon & Kelman, 1980; Kotoujansky

1987; Barras et al. 1994; Kazemi-Pour et al. 2004). Although many bacteria produce pectolytic enzymes, most that cause soft-rot diseases in plants belong to the genera Erwinia,

Pectobacterium or Dickeya (Dickey 1979; Kotoujansky 1987; Aysan 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 lady’s slipper orchids (Cyprepedium,

Paphiopedilum, and Phragmipedium), but has been reported to cause brown rots on other orchids

as well (Phalaenopsis, Aerides, ) (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-soaked 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

25 degrading enzymes macerate tissue (Daniels et al. 1988). Pectobacterium carotovora causes

soft-rots on many plants (Pérombelon and Kelman 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 indistinguishable from those caused by Dickeya

spp.

Recently, Erwinia chrysanthemi (Burkholder et al. 1953) and 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 sequences, and phenotypic characters. Dickeya spp. cause

diseases in many different plant species (Pérombelon & Hyman 1986; Hugouvieux-Cotte-Pattat

et al. 1992; Yap et al. 2005; Lee & yu 2006) and are serious orchid pathogens (Cating et al.

2009b).

Since the 16S rDNA gene of Escherichia coli (Migula 1895) Castellani and Chalmers 1919

was sequenced in 1972 (Ehresmann et al. 1972), it has been used extensively for bacterial

identification (Stackebrandt & Goebel 1994; Kolbert & Persing 1999; Clarridge III 2004; Janda

& Abbott 2007; Woo et al. 2008) and phylogenetic analyses (Weisburg 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 strains 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)

26 indicated that two closely related species that could be distinguished with 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 orchids. Although soft-rot pathogens typically have wide host ranges, recent isolates from orchids have not fallen in previously described taxa. For example, Cating et al. (2009b) described a Dickeya sp. that caused soft-rot symptoms in Tolumnia orchids; fatty acids, 16S rDNA sequences 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, additional 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 were 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 described 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 isolates phylogenetically with 16S rDNA and pel gene sequences.

27 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 morphological 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 cause 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 (Chupp 1954). In addition, Fresenius described three other 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, characteristics of hila, and other

morphological characters), Fresenius and subsequent 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 absence 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).

28 Chupp (1954) listed 155 plant families that were infected by Cercospora species. He made

generic divisions based on conidial morphology, color and septation, 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 characteristic used for separating true Cercospora from closely allied genera was the hilum, or scar on the conidia and the scar on the conidiophores (conidiogenous loci). The presence and thickness of a hilum was used by Deighton to classify species without a thickened scar into Pseudocercospora, Pantrospora, Cercoseptoria, Pseudocercosporella,

Denticularia, and Mycocentrospora, removing them from Cercospora proper, which has a thickened, distinct hilum on the conidia 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 identify 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 phylogenetic 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;

Ávila 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

29 relatedness of two sympatrically separated populations of Cercospora zeae-maydis Tehon & E.

Y. Daniels. Goodwin et al. (2001) examined genetic variability and evolutionary 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, were 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 characteristics, and morphological characteristics to examine the synonomy of

C. apii and C. beticola. Although they caused disease symptoms 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 construction of phylogenetic trees. However, in order to do this, these fungi need to be grown 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 spores 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

30 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 notorious for slow growth and reduced conidium production on artificial media (Nagel 1934; Ekpo & Esuruoso 1978).

Stavely & Nimmo (1968) examined the relationship of pH and nutrition to growth and sporulation of C. nicotianae Ellis & Everh. They determined that growth and sporulation was best on media that contained DL-leucine, sucrose, and yeast extract. 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 associated 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 pathogenicity tests, host-range studies, pesticide trials, store and deposit isolates in culture collections, and increase our understandings of these fungi and the diseases they cause.

Objective

The objective of this research was to develop methods to grow and sporulate orchid- associated 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 since it was first used to sequence

31 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; Arneson et al. 2008), and to examine genetic 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 (=long PCR), which incorporates a second heat- stable DNA polymerase with 3’-exonuclease activity, 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 containing an n-terminal 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 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 target DNA in the presence of large amounts of genomic DNA from hosts or the target organism. Vickers and Graham (1996) were able to use a high-fidelity PCR protocol to amplify a single-copy gene (Bar), a marker for the selection of transgenic plants, in the presence of barley genomic DNA; it consistently amplified the target gene, whereas standard

PCR only occasionally produced results.

32 High-fidelity PCR has also detected bacterial infections and 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 characterize a new microsporidium species from the

predatory mite Metaseiulus occidentalis (Nesbitt) (Becnel et al. 2002), to identify and distinguish

two parasitoids of the brown citrus 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 endotoxin-producing bacteria in varying amounts of

dust mite DNA (Valerio et al. 2005). Because of the increased sensitivity of the high-fidelity

PCR and ability to detect without an intermediary culturing step, 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.

33 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 plant and can quickly move 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, Arachnida:

Acari: Tetranychidae) and several 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 primary tools used in pest 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, alternatives to these chemicals are needed.

Furthermore, pesticides are phytotoxic 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 effectiveness of limonene for the control of mealybugs

(Pseudococcus longispinus Targioni-Tozzetti) by reducing the surface tension around their wax-

covered bodies (Hollingsworth 2005). Tipping et al. (2003) demonstrated that Silwet L-77 alone

is toxic to Pacific spider mite (Tetranychus pacificus McGregor) eggs, grape mealybug

(Pseudococcus maritimus Ehrhorn) crawlers, western flower thrips (Frankliniella occidentalis

Pergande), and cotton aphid (Aphis gossypii Glover). In addition, Silwet L-77 was toxic to

nymphs of the Asian citrus psyllid (Diaphorina citri Kuwayama) and significantly increased the

34 effectiveness of insecticides; eggs of D. citri were killed with one-fourth the label rates of

imidacloprid or abamectin and adults were killed with one-fourth or one-half the label rate of

imidacloprid (Srinivasan et al. 2008). Silwet L-77 also can increase the efficacy of fungicides.

Silwet L-77 or another surfactant (Kinetic) improved the activity of the protectant fungicide

maneb against potato early blight and dry bean (Gent et al. 2003).

Objectives

The objectives of this chapter were to determine (1) if Silwet L-77 was phytotoxic to several

commonly cultivated orchid genera, and (2) if Silwet L-77 increased the effectiveness of three

horticultural oils against Boisduval scale and the flat mite (Tenuipalpus pacificus) on orchids.

Appendices

According to Agrios (2001), Plant Pathology is a science with a practical goal:

“..to develop information, materials, and techniques that will lead to the management or control of plant diseases 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 identification 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 diagnostic guides were produced and are located in the appendices.

35 A B

C D

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

36

A B

CC DD

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 these orchids by three different pathogens.

37 Table 1-1. Pseudocercospora and Cercospora species reported on orchids Species Reported Host Taxa References

Pseudocercospora dendrobii (H.C. Burnett) U. Braun & Dendrobium species and hybrids, including nobile and Burnett 1965; Hsieh & Goh Crous, in Crous & Braun lautoria types 1990; Crous & Braun 2003

Pseduoercospora peristeriae (H. C. Burnett) U. Braun & Burnett 1965; Crous & Braun Peristeria elata Crous, comb. nov. 2003 Epipactis latifolia, E. palustris, Phaius grandifolius, Bletia purpurea (Florida native orchid), Eulophia, Ansellia onidioph, Brassia, Calanthe, Catasetum, Chysis aurea, Coelogyne massangeana, Cycnoches Cercospora epipactidis C. Massalonga Burnett 1965; Alfieri et al. 1994 chlorochilon, Cyrtopodium, Gongora, Lycaste, Maxillaria, Mendoncella fimbriata, Monomeria, Pescatorea, X Phaiocalanthe, Xylobium squalens, and Zygopetalum Ascocenda, , Brassocattleya, , Broughtonia, Cattleya, Caularthron bicornutum, Dendrobium, Epicattleya, Chupp 1954; Burnett 1964; Ellis Pseudocercospora odontoglossi (Prill & Delacr.) U. Epidendrum, Laelia, Laeliocattleya, Pleurothallis 1976; Alfieri et al. 1994; Braun Braun talpinaria, Potinara, Rodricidium, & Hill 2002 secunda, Schombocattleya Schombodiacrium, and Sophrolaeliocattleya Pseudocercospora cymbidiicola U. Braun & C. F. Hill sp. Cymbidium orchids in New Zealand Braun & Hill 2002 Nov Cercospora epidendronis Bolick, nom. nud. Epidendrum Alfieri et al. 1994

Alfieri et al. 1994; Crous & Cercospora cyprepedii Ellis & Dearn Paphiopedilum Braun 2003 Angraecum, Cattleya Macrangraecum, Cercospora angraeci Feuillebois & Roum. Macroplectrum sesquipedale Laelia, Oncidium, and Crous & Braun 2003 Odontoglossum alexandrae

38 CHAPTER 2 IDENTIFICATION AND CHARACTERIZATION OF A SOFT-ROT PATHOGEN ISOLATED FROM Vanda, Phalaenopsis, Oncidium, AND Tolumnia ORCHIDS IN FLORIDA

Introduction

Pectolytic enzyme-producing bacteria can be devastating plant pathogens. Pectolytic enzymes (pels) macerate plant tissue by degrading plant cell-wall components which leads to soft, water-soaked lesions that can enlarge rapidly (Pérombelon & Kelman, 1980; Kotoujansky

1987; Barras et al. 1994; Kazemi-Pour et al. 2004). Although many bacteria produce pectolytic enzymes, most that cause soft-rot diseases in plants belong to the genera Erwinia,

Pectobacterium or Dickeya (Dickey 1979; Kotoujansky 1987; Aysan 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 lady’s 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-soaked 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 (Pérombelon & 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).

39

Symptoms can develop rapidly and are virtually indistinguishable 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 sequences, and phenotypic characters. Dickeya spp. cause diseases in many different plant species (Pérombelon and Hyman 1986; Hugouvieux-Cotte-Pattat et al. 1992; Yap et al. 2005; Lee & yu 2006) and are serious orchid pathogens (Cating et al.

2009b).

Since the 16S rDNA gene of Escherichia coli (Migula 1895) Castellani and Chalmers 1919 was sequenced in 1972 (Ehresmann et al. 1972), it has been used extensively for bacterial identification (Stackebrandt & Goebel 1994; Kolbert & Persing 1999; Clarridge III 2004; Janda

& Abbott 2007; Woo et al. 2008) and phylogenetic analyses (Weisburg 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 strains 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 with 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.

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Very little is known about soft-rot causing bacteria in orchids. Although soft-rot pathogens typically have wide host ranges, recent isolates from orchids have not fallen in previously described taxa. For example, Cating et al. (2009b) described a Dickeya sp. that caused soft-rot symptoms in Tolumnia orchids; fatty acids, 16S rDNA sequences 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, additional 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 different 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; Palacio-Bielsa et al. 2006); and (2) perform phylogenetic analyses 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 isolated according to Schaad et al. (2001), and grown on nutrient agar (NA) at 27˚C for 24 h. Single colonies were transferred to crystal-violet pectate medium (CVP) (Schaad et al. 2001) and incubated at 27˚C for 24 h to assess pectolytic activity.

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Colonies that pitted CVP, indicating pectate degradation, were transferred to nutrient agar- glycerol-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 sensitivity, oxidase, phosphatase, and acid production from (+)-D- arabitol and sorbitol and the KOH test were performed 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 other 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 Bacterial Identification and Fatty Acid Analysis

Laboratory at the University of Florida, Gainesville 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 (bioMérieux, Durham, NC) was used to determine utilization of 49 different carbohydrates by the 18 orchid and four reference isolates. Tests were performed according to the manufacturer’s 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 strain, the 16S rRNA gene was amplified via high-

42 fidelity PCR, in which 50-μL reaction volumes contained 50 mM TRIS (pH 9.2), 16 mM ammonium sulfate, 1.75 mM MgCl2 , 350 μM 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 μL of sterile mineral oil and were amplified using three linked temperature profiles: (i) 94˚C for 2 min; (ii) 10 cycles 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 cluster (Tamaki et al. 1988; Hugouvieux-

Cotte-Pattat et al. 1992; Nassar et al. 1996; Palacio-Bielsa et al. 2006) was also amplified with the above genomic DNA, using primers ADE1 (ADE1 (5’-

GATCAGAAAGCCCGCAGCCAGAT -3’) and ADE2 (5’-

CTGTGGCCGATCAGGATGGTTTTGTCGTGC -3’) (Nassar 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 Corp., Carlsbad, CA). Before ligation into the cloning vector,

PCR products were cleaned using the QIAquick PCR purification kit (Qiagen, Valencia, CA) following the manufacturer’s recommendations and eluted in 50 µL 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 purification by mixing the 50-µL DNA sample with 5.75 µL of 10X high-fidelity buffer (50 mM TRIS, pH 9.2, 16 mM ammonium sulfate, 1.75 mM MgCl2), 100

43 mM dATP, and 1 unit of Taq polymerase (Bioline), and 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 following the manufacturer’s recommendations

(Invitrogen Corp.). Transformed E. coli colonies were selected from 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 EcoRI restriction enzyme followed by gel electrophoresis 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 for 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 using the sequence analysis software MacDNASIS version 3.7 (Hitachi, San Bruno, CA, USA). Sequences were compared with similar sequences in the GenBank database (National Center for Biotechnology Information, Bethesda, MD) using the Basic Alignment Search Tool (BLAST) search 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

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(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 before performing phylogenetic analysis.

Phylogenetic analyses were performed with the 16S rDNA and pelADE datasets using PAUP version 4.0b10 for maximum likelihood (ML) (Swofford 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 Tests (hLRTs) and Akaike Information Criterion

(AIC) parameters obtained for both data sets were used to preset 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. The 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 probabilities obtained from

1,000,000 generations with the first 2,500 trees discarded as burnin (MrBayes).

Pathogenicity Tests

Pathogenicity tests were performed 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 µL 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 µL of sterile water and four of each were not 45 inoculated. Plants were incubated in a greenhouse at 20.6-29.1˚C and 37-90% RH under a 16L:

8D photoperiod, and evaluated for symptom development after 24 h. To complete Koch’s postulates and confirm that the inoculated strains could be recovered from symptomatic tissue, bacteria were isolated and the following tests were performed as described above: pectate degradation on CVP, oxidase, phosphatase, indole, arabinose, erythromycin sensitivity, indigoidine production, growth at 39˚C, and PCR using ADE1/ADE2 primers.

Results

Isolation and Identification of Dickeya Strains

A large number of bacterial colonies were isolated from symptomatic tissue of each plant on

NA. Single colonies were transferred to CVP and produced pits, indicating pectolytic enzyme activity. All strains were Gram negative (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 not currently in the MIDI database.

Carbohydrate Utilization Tests

All 18 orchid isolates utilized glycerol, L-arabinose, D-ribose, D-xylose, D-galactose, D- glucose, D-fructose, D-manose, L-rhamnose, inositol, D-manitol, N-acetylglucosamine, arbutin, esculin ferric citrate, salicin, D-saccharose, and gentiobiose, but did not utilize erythritol, L- xylose, D-adonitol, methyl-ßD-xylopyranoside, L-sorbose, dulcitol, D-sorbitol, methyl-αD- mannopyranoside, methyl-αD-glucopyranoside, D-melezitose, amidon (starch), glycogen,

46 xylitol, D-turanose, D-lyxose, D-tagatose, D-fucose, 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-cellobiose, and none of the orchid isolates utilized D-maltose, D-lactose, and D-trehalose (Table 2-4).

Molecular and Phylogenetic Analysis

BLAST searchs with the 16S rDNA sequences indicated that the 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 (Cating 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 aligned 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 (bootstrap 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 contained isolates from Tolumnia, Oncidium, and Phalaenopsis with a posterior probablility of 75 (Fig. 2-4, clade 2) and the other which contained the two Vanda isolates with a posterior probability of 100 (Fig. 2-4, clade 1). 47

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 aligned with four similar sequences in GenBank (Fig.

2-2). Maximum likelihood phylogenetic analysis based on the 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 same 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-inoculated 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 utilization, and production of the expected 420-bp PCR product with primers ADE1/ADE2.

Discussion

Eighteen isolates of bacteria that caused soft-rot in four orchid genera were characterized biochemically and genetically. Initial 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, these isolates matched none of the six Dickeya taxa that were described by Samson et al. (2005); several differences were evident (Table 2-3). The Florida orchid strains utilized (-)-D-arabinose, but D. chrysanthemi bv. chrysanthemi and D.

48 chrysanthemi bv. parthenii do not. The Florida orchid strains did not utilize lactose, but D. dadantii and D. zeae do. Dickeya dieffenbachiae does not utilize (+)-D-melibiose and (+)-D- raffinose, 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 orchid 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; Hadley 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 strains in this study were compared to

P. carotovora , P. cyprepedii (ATCC# 29267), and the type strain of D. chrysanthemi (ATCC#

11663) to determine if there were biochemical characteristics 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 isolates 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 and 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 used frequently to construct phylogenies and identify bacteria, but may not be useful in analyses below the genus level, especially among closely related species in the Enterobacteriaceae (Naum et al. 2008; Zeigler 2003). Fox et al.

(1992) concluded that 16S rDNA sequences may not be useful in species identifications, but can

49 be an important tool for strain identifications when 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 (Hugouvieux-Cotte-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 support (94) in the Bayesian analysis.

The results of this study suggest that there are two distinct 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 addition, 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 the causes of these diseases, but indicate that additional research is needed. Whether these strains exist on other orchid taxa, their respective host ranges, ecology, and whether they have endemic or exotic origins are unknown. More isolates should be collected from members of different orchid genera to determine their distributions and whether they are new species. The latter studies could include identifying

50 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 chemicals (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 pathogen identification (Miller & Martin 1988;

Lévesque 1997; Toth et al. 2001). For example, if the orchid strains had specific environmental requirements for disease development, these might be manipulated to reduce disease incidence or severity after pathogen presence was revealed. Furthermore, pathogen identification is required for epidemiological studies and surveys of geographical distribution, which can be particularly important when searching for disease resistance (Ploetz 2007).

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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 alignment of 16S rDNA sequences of 18 Dickeya isolates from orchids in Florida 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 , D. dianthicola, D. paradisiaca, D. zeae, D. dieffenbachiae, D. chrysanthemi, and two isolates of Pectobacterium atrosepticum. A hyphen indicates a nucleotide deletion.

52

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.

53

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.

54

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.

55

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.

56

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.

57

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.

58

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.

59

730 740 750 760 770 780 790 800 810 ...... D. zeae (AF520711) AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- D. dieffenbachiae (AF520712) AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- D. chrysanthemi (U80200) AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- Dickeya sp. 0723-1 V AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- Dickeya sp. 0723-2 V AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- Dickeya sp. 1114-4 P AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTT Dickeya sp. 0827-3 O AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- Dickeya sp. 1114-3 P AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCCGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- Dickeya sp. 1015-2 T AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- Dickeya sp. 0911-2 T AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- Dickeya sp. 1015-4 T AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- Dickeya sp. 1114-16 P AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- Dickeya sp. 1015-5 T AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- Dickeya sp. 0911-3 T AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- Dickeya sp. 1015-1 T AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- Dickeya sp. 0911-1 T AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- Dickeya sp. 1015-3 T AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- Dickeya sp. 0827-2 O AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACTCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- Dickeya sp. 1114-1 P AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- Dickeya sp. 0827-4 O AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- Dickeya sp. 0827-1 O AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- D. dadantii (AF520707) AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- D. dianthicola (AF520708) AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- D. paradisiaca (AF520710) AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTT- P. atrosepticum (EF178668) AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGACTTGGAGGTT- P. atrosepticum (EF530555) AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGACTTGGAGGTT-

Figure 2-1. cont.

60

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.

61

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.

62

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.

63

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.

64

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.

65

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.

66

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.

67

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 partial 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 nucleotide deletion.

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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.

69

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.

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

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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 with high posterior probability value (100). T = Tolumnia, P = Phalaenopsis, O = Oncidium, V = Vanda.

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

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

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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 CVP Indole Indigoidine Erythromycin Oxidase Phosphatase (+)-D- Sorbitol collected production production sensitivity Arabitol 0723-1 Leesburg, FL Vanda MCa 23 July + + + + - + - - 2007

0723-2 Leesburg, FL Vanda MC 23 July + + + + - + - - 2007

0827-1 Homestead, Oncidium 27 Aug + + + + - + - - FL MC 2008

0827-2 Homestead, Oncidium 27 Aug + + + + - + - - FL MC 2008

0827-3 Homestead, Oncidium 27 Aug + + + + - + - - FL MC 2008

0827-4 Homestead, Oncidium 27Aug + + + + - + - - FL MC 2008

0911-1 Homestead, Tolumnia 11 Sept + + + + - + - - FL fantasy 2008 ‘Brandon’ 0911-2 Homestead, Tolumnia 11 Sept + + + + - + - - FL fantasy 2008 ‘Brandon’ 0911-3 Homestead, Tolumnia 11 Sept + + + + - + - - FL fantasy 2008 ‘Brandon’

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Table 2-1, cont. Isolate Origin Orchid host Date CVP Indole Indigoidine Erythromycin Oxidase Phosphatase (+)-D- Sorbitol genus collected production production sensitivity Arabitol 1015-1 Homestead, Tolumnia 15 Oct + + + + - + - - FL sp. 2008

1015-2 Homestead, Tolumnia sp. 15 Oct + + + + - + - - FL 2008

1015-3 Homestead, Tolumnia sp. 15 Oct + + + + - + - - FL 2008

1015-4 Homestead, Tolumnia sp. 15 Oct + + + + - + - - FL 2008

1015-5 Homestead, Tolumnia sp. 15 Oct + + + + - + - - FL 2008

1114-1 Homestead, Phalaenopsis 14 Nov + + + + - + - - FL MC 2008

1114-3 Homestead, Phalaenopsis 14 Nov + + + + - + - - FL MC 2008

1114-4 Homestead, Phalaenopsis 14 Nov + + + + - + - - FL MC 2008

1114-16 Homestead, Phalaenopsis 14 Nov + + + + - + - - FL MC 2008 a MC=mericlone

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Table 2-2. 16S rDNA GenBank BLAST search and MIDI results for orchid strains. Based on these results and results 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.

16S rDNA MIDI Strain 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

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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 D. dad. + D. ch. p. D. ch. ch. D. dif. D. dia. D. par. D. chry. Vanda Oncidium Tolumnia Phalaenopsis D. z. (control)b Isolates Isolates Isolates isolates Characteristic (33)a (4) (5) (5) (16) (6) (2) (4) (8) (4)

(-)-D - +c - - + - + - + + + + Arabinose

Inulin - - + - V(88) - + - - - -

Lactose + V(75) V(20) - - V(17) + - - - -

(+)-D- + + + - V(44) V(83)+ + + + + Melibiose

(+)-D- + + + - V(44) V(83)+ + + + + Raffinose

Growth at + + + + - V(83) + + + + + 39° C

Mannitol + + + + + - + + + + +

aNumber of strains bType specimen ATCC #11663 c+, 90-100% of strains positive; -, 90-100% of strains negative; V(n) percent of strains positive.

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Table 2-4. Carbohydrate utilization 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 D.ch. P.c. P.cy. Vanda Oncidium Tolumnia Phalaenopsis Carbohydrate Isolates Isolates Isolates isolates (2) (4) (8) (4) Amygdalin -b - - - - - V(50)

D-Cellobiose + + + + + - +

D-Maltose + - + - - - -

D-Lactose + + + - - - -

D-Trehalose + ------

aIn addition to the carbohydrate results reported in this table, other carbohydrates were tested ; however, those results were not different 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-acetylglucosamine, arbutin, esculin ferric citrate, salicin, D-saccharose, and gentiobiose. All orchid strains tested were negative for erythritol, L- xylose, D-adonitol, methyl-ßD-xylopyranoside, L-sorbose, dulcitol, D-sorbitol, methyl-αD- mannopyranoside, methyl-αD-glucopyranoside, D-melezitose, amidon (starch), glycogen, xylitol, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-ketogluconate, potassium 5-ketogluconate. b+, 90-100% of strains positive; -, 90-100% of strains negative; V(n) percent of strains positive.

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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 described (Solheim 1930; Chupp 1954; Deighton 1973,

1976, 1979; Ellis 1971, 1976), but only 659 are currently recognized as valid (Crous & Braun

2003). Eight Cercospora or Pseudocercospora species have been associated 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 (Chupp 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 spores 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 other media (Balis & Payne 1971; Sah & Rush 1988;

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Hartman et al. 1991; Wang et al. 1998; Crous et al. 2001; Beilharz & Cunnington 2003).

However, some cercosporoid species are notorious for slow growth and reduced conidium production on artificial media (Nagel 1934; Ekpo & Esuruoso 1978).

Stavely & Nimmo (1968) examined the relationship of pH and nutrition to growth and sporulation of C. nicotianae Ellis & Everh. They determined that growth and sporulation was best on media that contained DL-leucine, sucrose, and yeast extract. 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 associated 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 pathogenicity tests, host-range studies, pesticide trials, store and deposit isolates in culture collections, and increase our understandings of these fungi and the diseases they cause.

The objective of this chapter was to develop methods to grow and sporulate orchid- associated 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 transcribed spacers 1 and 2 (ITS1 and ITS2) were amplified using primers

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ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) and ITS5 (5’-

GGAAGTAAAAGTCGTAACAAGG-3’) (protocol described in chapter IV) as described by

White (1990) and compared to ITS sequence data in GenBank.

Single spores were isolated directly from sporulating lesions and suspended in 500 µL of sterile water, after which the suspension was briefly 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 germinated spores were cut out, placed on V-8 agar

(44 mL of V-8 juice, 12 g of Bacto-agar (Difco Laboratories, Detroit, MI) and 2.4 g of

CaCO3/L), and incubated for 2 weeks at 25ºC ± 1ºC 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. ¼-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 Bacto- agar/L);

4. ¼-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 contained 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 medium (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).

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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 hybrid, were incubated at 15, 20, 25 and 30°C ± 1°C under

12L:12D fluorescent light. The experiment was performed 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 after 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 cercosporoid 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). Single- spore 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 scalpel and spread on WA plates, and incubated in growth chambers at 15, 20, 25 and 30°C ± 1°C under constant fluorescent light (Ekpo &

Esuruoso 1978). Cultures were examined after 3, 4, and 5 days for sporulation by flooding

83 plates with 1 mL of sterile water followed by gentle rubbing with a sterile glass rod. Spore production was calculated by counting the number of spores present in three 10-μL 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 treatment means were separated using Fisher’s LSD

test. Levene’s 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) was comparable on V-8, ¼-V-8, PDA, ¼-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 ¼-V-8, ¼-PDA, DOLA+L, and WA+leaves media grew very close to the surface

of the media, which would inhibit the removal 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 mycelia 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 better than the other media (Fig. 3-1). Sparse,

inconsistent sporulation was observed on the ¼-V-8, WA+leaves, PDA, and ¼- 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;

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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 25°C (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 25°C 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 30°C than at 15°C (P<0.0001), while on day 7, 11, and 13, 25 and 30°C 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

Pseudocercospora sp. from a Tolumnia orchid and transferred to WA. Conidia developed at

25ºC (3 days, mean of 837/mL; 4 days, 860/mL; and 5 days, 831/mL), but did not develop at 15,

20, or 30ºC.

Discussion

Many media have been used to culture cercosporoid 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 fairly specific nutritional and environmental requirements for growth and sporulatation. For 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

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PDA. Sporulation was observed on V-8, PDA, and cowpea-stem decoction agar; however, abundant sporulation was observed only on V-8 agar between 24 and 25ºC 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) used V-8 agar to produce conidia of C. zeae-maydis for plant inoculations, as did Karaoglanidis & Bardas (2006) with C. beticola. Nelson & Campbell (1990) used V-8 agar to determine that C. zebrina Pass. grew best at 24ºC, whereas Wang et al. (1998) used V-8 to produce conidia of C. zeae-maydis isolates for molecular analysis. Conidia might also be needed 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, ¼ V-8, PDA, ¼ 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 molecular analyses. Due to its multiple uses, V-8 agar was chosen for further temperature and sporulation studies.

Pseudocercospora dendrobii and a Pseudocercospora sp. isolated from a Bulbophyllum hybrid both grew significantly better at 25ºC than at 15, 20, or 30ºC. 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 well at 25 and 30ºC; others cercosporoid species also grow at higher temperatures (Sah & Rush 1988; Hartman et al.

1991).

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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 occurred as the leaf senesced or abscised (R. Cating, personal observation). Thus, it was hypothesized that a sudden change in nutrition might stimulate sporulation, a phenomenon 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, consistent, albeit low (822 to 867 conidia mL-1), sporulation was

achieved at 25ºC 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 the 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 nonsporulating cercosporoid fungus could be used as substitutes for conidia in pathogenicity studies (Donzelli & Churchill 2007). Conidia of another species that were harvested directly from infected leaves have been used in disease studies (e.g. P. mori

(Hara) Deighton; Babu et al. 2002), but they would not be useful for pathogenicity tests and host- range 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 nonpathogenic parasite on this host. The

87 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 25ºC under 12L:12D photoperiod. In addition, a

Pseudocercospora sp. from Tolumnia produced conidia at 25ºC under 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 other orchid-associated species to sporulate.

Conidia from single–spore isolates would be preferred when fulfilling Koch’s postulates and evaluating the host-range of different taxa in the Orchidaceae. In addition, this procedure would assist those who need to identify isolates morphologically.

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A 40 a ab ab abc bc bc 30 c d de 20 e

Diameter (mm) 10

0 -8 es A -8 A A +L A L S V v D V L D A W +A A h a P O P L A + gt Le D th O L LA n n g D O O re e en D D St D tr 4 + S 1/ A /4 W 1

B

Figure 3-1. Growth of Pseudocercospora dendrobii on 10 different media. (A) Colony diameter after 3 weeks. Blue bars represent 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 are not significantly different. Variances were not significantly different as determined by a Levene’s test for homogeneity of variance. (B) Photo of fungal growth on media used in the experiment.

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Figure 3-2. Measurements of culture diameter at 3, 7, 11, and 13 d at 15, 20, 25, and 30 ± 1ºC on V-8 agar. P. dendrobii (red) from a hybrid Dendrobium, and Pseudocercospora sp. from a hybrid Bulbophyllum grew best at 25ºC on each day, while P. odontolgossi (green) from a Cattleya mericlone grew best at 20, 25, and 30ºC on day 3, but grew best at 25 and 30ºC on day 7, 11, and 13. Data 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 Levene’s test for homogeneity of variance.

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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 eliminated 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. The 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; Lévesque 2001; McCartney 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 standard PCR, high-fidelity PCR (=long PCR) can amplify sequences of up to 35 kb (Barnes 1994).

The addition of a small amount of the proofreading enzyme to the reaction containing a n- terminal 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’-exonuclease 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 large amounts of non-target DNA. Vickers & Graham

91

(1996) used high-fidelity PCR to consistently amplify a single-copy gene (Bar), a marker for the selection of transgenic plants, in the presence of barley genomic DNA; standard PCR only occasionally amplified the target gene. High-fidelity PCR also can detect bacterial infections and microbial associations in arthropods. Jeyaprakash & Hoy (2000) demonstrated that high- fidelity 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 the presence of genomic DNA from the Asian citrus psyllid, citrus trees, or citrus psyllid parasitoids (Hoy et al. 2001). Furthermore, high-fidelity PCR has been used to: detect and characterize a new microsporidium species from the predatory mite Metaseiulus occidentalis (Nesbitt) (Becnel et al. 2002); identify and distinguish two parasitoids of the 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 sequences of endotoxin-producing bacteria in varying amounts of dust mite DNA (Valerio et al. 2005).

The objectives of this study were to: 1) determine if high-fidelity PCR is more sensitive than standard PCR in detecting fungal DNA in the presence of plant genomic DNA; and 2) determine if high-fidelity PCR is more sensitive 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 liquid nitrogen and ground in 8 ml of CTAB buffer (2% cetyltrimethylammonium bromide, 100 mM Tris pH 7.5, 20 mM EDTA, 1%

92 polyvinyl pyrrolidone, and 1.4 M NaCl) for 10 min and aliquoted into eight tubes. The samples were then incubated at 60ºC for 16 h. After two chloroform extractions, the eight DNA aliquots were combined into four and precipitated in 2-propanol and resuspended in 100 μL of sterile water. All samples were pooled to make a 400-μL 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 characters (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 approximately 2 X 2 cm was scraped from the surface of the plate with a sterile wooden applicator stick and placed in a 0.5-mL tube. 100 μL of extraction solution (Extract-n-Amp, Sigma-Aldrich, St. Louis, MO) was added and the sample ground for 5 min with a sterile plastic pestle and heated to 95˚C for 10 min. 100 μL of dilution solution (Extract-n-Amp, Sigma-Aldrich) was added to the sample after which the sample was briefly vortexed. 30 μL of the extracted DNA was added to 270 μL 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 50-μL reaction volume contained 50 mM TRIS, pH

9.2, 16 mM ammonium sulfate, 1.75 mM MgCl2 , 350 μM each of dATP, dGTP, dCTP and

dTTP, 800 pmol of primers (ITS4, 5’-TCCTCCGCTTATTGATATGC-3’ and ITS5, 5’-

GGAAGTAAAAGTCGTAACAAGG-3’) (White et al.1990), 1 unit of Accuzyme (Bioline,

Taunton, MA) and 5 units of Taq DNA polymerase (Bioline) (Barnes 1994). Samples were 93 covered with 100 μL of sterile mineral oil and were amplified 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 products were cleaned using the QIAquick PCR purification kit (Qiagen, Valencia, CA) following the manufacturer’s recommendations and eluted in 50 µL of sterile glass-distilled, glass-collected water. A 3’ A-overhang was added to the PCR product after purification to facilitate ligation into the cloning vector by mixing the 50-

µL DNA sample with 5.75 µL of 10X high-fidelity 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 (Perkin 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 manufacturer’s recommendations (Invitrogen Corp.). Transformed Escherichia coli colonies were selected from 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 EcoRI restriction enzyme followed by gel electrophoresis on a 2% agarose TAE gel stained with 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 University of Florida, Gainesville, Florida for DNA sequencing. 94

Comparison of Standard and High-Fidelity PCR

Escherichia coli cells containing plasmid pRC17 were 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) and quantified using a BioPhotometer G131 V1.35

(Eppendorf, Hamburg, Germany). To quantify the differences between the standard and the high- fidelity 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-µL reaction volume and contained 2.5 µL of 10X PCR

Buffer + Mg (Boehringer, Mannheim, Germany), 200 µM dATP, dGTP, dCTP and dTTP, 400 pM of primers ITS4 and ITS5 (White et al. 1990), and 0.2 units of Taq DNA polymerase

(Bioline). Samples were covered with 50 µL 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-fidelity 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,

95 and used to inoculate five Phalaenopsis plants, each with a 6-mm plug that was placed mycelium side down on the youngest leaf. Plants were placed in a greenhouse under 16L: 8D photoperiod at 25-30°C 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 section 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) protocol described above. Identification of the pathogen was confirmed by cloning and sequencing the PCR product using the procedures described above and comparing the sequence to that 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 Samson 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 (Eppendorf); into the youngest leaf of five

Phalaenopsis plants, 100 µL of the suspension was injected. 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. High- fidelity and standard PCR were performed with primer pair ADE1 (5’-

GATCAGAAAGCCCGCAGCCAGAT -3’) and ADE2 (5’-

CTGTGGCCGATCAGGATGGTTTTGTCGTGC -3’) (Nassar et al. 1996). With the exception of a 63˚C annealing temperature, the same PCR parameters were used. Identification of the pathogen was confirmed by cloning and sequencing the PCR product with the above procedures.

96

To confirm that reactions worked properly, two 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 previously described. All reactions were replicated twice on different days.

Results

Evaluation of High-Fidelity PCR by Use of Plasmids

To determine if the high-fidelity PCR is more sensitive than the standard PCR, initial experiments were conducted in which serially diluted 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-fidelity 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 samples were amplified using the standard PCR protocol, only the positive controls produced a band of the expected size (Figure 4-2, A).

Similarly, high-fidelity PCR detected Dickeya sp. directly from each of five inoculated plants,

97 but standard PCR only amplified the positive control (Figure 4-2, B). To confirm the identity of the pathogens, the positive reactions, including 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 sensitive than standard PCR in detecting a fungal and bacterial pathogen of Phalaenopsis orchids. High-fidelity PCR consistently detected as little as 1 fg, in the presence of host DNA, which is equivalent 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 ftsZ- and 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 detect plant pathogens, there 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 used. Diallo et al. (2009) found that competitor DNA 98 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 infection occurs or only when certain conditions for host susceptibility are met, can considerably reduce a plant inspector’s or diagnostician’s ability to detect a given pathogen. Environmental factors such as temperature and moisture are critical 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 latent 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 that the high-fidelity PCR protocol could become an important tool for diagnosticians and regulatory agencies, especially when pathogens are of quarantine significance, occur at low titers, 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 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 organisms, 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. Plant cellular contents (organic and inorganic compounds), including host genomic DNA, can interfere with the efficiency of standard PCR

(Vickers & Graham 1996; Wilson 1997; Vincelli & Tisserat 2008), making the diagnosis of plant

99 pathogens directly from plant tissue 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, can 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 bacterial 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 exist in a given sample.

Many techniques exist to increase the sensitivity 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 others (Vincelli & Tisserat 2008). However, high- fidelity PCR is relatively simple and requires only the addition of small amounts of a proof- reading enzyme to the Taq polymerase (Barnes 1994) and a slight increase in primer and dNTP concentrations due to the theoretical increase in product yield. Therefore, plant disease clinics and diagnosticians that already own thermocyclers can adopt high-fidelity PCR without the purchase of new equipment or additional training. By contrast, real-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.

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Figure 4-1. A comparison of high-fidelity 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 template 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 genomic DNA, the standard PCR protocol (C) amplified as little as 10 fg of template. M=molecular marker IV (Roche).

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Figure 4-2. A comparison of high-fidelity and standard PCR in the 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 high- fidelity 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).

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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 family 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 illustrated in the question and answer section of Orchids magazine published by the American Orchid Society, which frequently contains questions submitted by hobbyists and commercial growers concerning the identification and control of orchid pests and diseases. Common arthropod pests of orchids that can be difficult to control include the Boisduval scale Diaspis boisduvalii Signoret (Hemiptera:

Diaspididae) and several 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 scale 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) and several 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).

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Many orchids, some of which are highly valuable or rare, exhibit phytotoxicity when treated with pesticides (Johnson 2008). Therefore, alternatives to traditional pesticides are desirable, especially for hobbyists and smaller orchid producers. Synthetic organic 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 pesticides 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 the target pest or weed (Tu et al. 2001). Silwet

L-77 has been shown to increase the effectiveness of limonene for the control of mealybugs

(Pseudococcus longispinus Targioni-Tozzetti) by reducing the surface tension around their wax- covered bodies (Hollingsworth 2005). Tipping et al. (2003) demonstrated that Silwet L-77 alone is toxic to Pacific spider mite (Tetranychus pacificus McGregor) eggs, grape mealybug

(Pseudococcus maritimus Ehrhorn) crawlers, western flower thrips (Frankliniella occidentalis

Pergande), and cotton aphid (Aphis gossypii Glover). In addition, Silwet 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 applied using one-fourth or one-half the label rate of imidacloprid (Srinivasan et al. 2008). Silwet L-77 also can increase the efficacy of fungicides.

Silwet L-77 or another surfactant (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 chemical pesticides has been shown to increase their efficacy, even at lower-than-label rates.

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The objectives of this study were to (1) determine if Silwet L-77 can be used without causing phytotoxicity to eight commonly cultivated orchid genera, and (2) determine if Silwet L-

77 can increase the effectiveness of 3 horticultural oils against Boisduval scale (Diaspis boisduvalii) and the flat mite (Tenuipalpus pacificus) on orchids.

Materials and Methods

Phytotoxicity Study

Representatives of seven commonly cultivated orchid genera obtained 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 pre- and post-spray ratings based on overall vigor, color, and presence/absence of spots or necrotic areas on leaves, stems, flowers, or flower buds. Plants 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 fertilized 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 heptamethyltrisiloxane, 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 intervals with hand sprayers until completely covered and assessed for phytotoxicity damage one week after the second spray.

Plants (pre- and post-spray) were kept in a greenhouse at the Department of Entomology and

Nematology, University of Florida, Gainesville, FL at temperatures ranging from 21.0 to 35.0ºC and 45 to 95% RH and 16L:8D photoperiod from the time the plants were obtained until the end

105 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. Levene’s test for homogeneity of variance was performed (Hill &

Lewicki 2006).

Silwet + Oil Efficacy Trial (Boisduval Scale)

A commercial Cattleya orchid grower in central Florida 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 severity 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). Pre- and 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: 21-

40 scales; 3: 41-60 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: 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 hand 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 first 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 (Whitmire Micro-Gen Research Laboratories, 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 combination with petroleum oil. Plants were sprayed to run-off using a 3.8-L pump sprayer (H. D. Hudson Manufacturing 106

Company, Hastings, MN). Plants were treated within 1 hr of the 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 assessment 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 identification 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 was 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 consisted of 3 water controls, 3 oil treatments

(Prescription Treatment Ultra-pure Oil, Whitmire Micro-Gen Research Laboratories, Inc., St.

Louis, MO) and 3 oil (Prescription Treatment Ultra-pure) + 0.05% (v:v) Silwet treatments.

Plants were sprayed to run-off using a 3.8-L pump sprayer (H. D. Hudson Manufacturing

Company). Plants were treated within 1 hr of the pre-spray assessment.

Plants (pre- and post-spray) were kept in a greenhouse at the Tropical Research and

Education Center, University of Florida, Homestead, FL, under 50% shade with temperatures ranging from 18 to 30.5˚C and 45-80% RH. Post-spray assessment 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 Boisduval trial and the flat mite trial was performed by using the

PROC GLM procedure in SAS (SAS Institute 2002) and treatment means were separated using 107

Fisher’s LSD test. Levene’s test for homogeneity of variance was performed Hill and Lewicki

2006).

Results

Phytotoxicity Study

No evidence of phytotoxicity (burning, chlorosis, spots or other types of lesions) was observed on any of the 7 orchid genera tested in this study including leaves, stems, inflorescences or roots and there was no significant difference between the pre- and post-spray quality ratings (Table 5-1; Figure 5-1). Of the 117 orchids (all genera) 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 (ratings 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 Trial (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 Ultra-Fine 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 phytotoxicity on leaves, flowers, flower buds, or roots (data not shown) was observed in these experiments.

Mortality of female Boisduval scale was increased 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 water 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:

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P=0.0017, experiment 4b: P<0.0001) (Table 5-3). However, there 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 water 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 alone 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 & Schroeder 1996; Srinivasan et al. 2008; Cocco & Hoy

2008). This study demonstrates, for the first time, that Silwet L-77 could be an important management tool to increase the 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, allowing it to infiltrate plant leaf stomata (Neumann & Prinz 1974) and hydathodes (Zidack et al. 1992). It is plausible that this reduction in surface tension of water around the body of an arthropod may allow an infiltration of water into the spiracles, drowning the arthropod (Cowles et al. 2000; Shapiro et al. 2009).

In the present study, three petroleum oils were used. 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 using 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 addition of 0.05% (v:v) Silwet L-77 significantly increased the efficacy of all 3 oils tested against the Boisduval scale on Cattleya 109 orchids (Table 5-2) or the Ultra-Pure 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), perhaps 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 flat 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 Silwet 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 mites, 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 beneficial where tolerance for arthropod infestations is essentially zero and plants are sold and appreciated based on aesthetic value (Bethke & Cloyd 2009).

However, as suggested by Tipping et al. (2003) and Liu and Stansly (2000) it is essential that

100% coverage is achieved to obtain adequate control.

Tipping et al. (2003) demonstrated that Silwet L-77 alone increased 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 Stansley (2003) also suggest that complete coverage is necessary for good control due to contact activity. For orchids, this includes the abaxial and adaxial leaf surfaces, stems, pseudobulbs, inflorescences, under sheaths

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(Cattleya orchids), and possibly the roots. This requirement may preclude 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 overhead booms, which may make it difficult obtain complete coverage. In addition, because there is no residual activity, re-applications may be necessary to obtain acceptable control.

Several caveats must be considered before using Silwet L-77 + petroleum oil to treat arthropod infestations in orchids. Although no evidence of phytotoxicity was observed in any of the orchids used in this study, representatives of only 7 genera of orchids were tested. It is quite possible that other genera, intergeneric hybrids, or certain cultivars may be susceptible to phytotoxicity. It is recommended that growers check a representative of the plants to be treated for signs of phytotoxicity before extending treatment to large groups of plants. In addition, environmental conditions may play a role in phytotoxicity, 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) suggest 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 the use of an adjuvant, such as Silwet L-77, may increase the incidence of bacterial diseases (Gottwald et al. 1997). The decrease in water tension could allow entry sites for plant pathogenic bacteria into leaf stomata (Melotto 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

111 could be due to the absence of bacteria or a conducive environment, so caution should be exercised if it is known that pathogens are present or there is a history of bacterial infections.

There are several advantages to using oil + Silwet L-77 to control orchid arthropod pests. No arthropod is known to have developed resistance to petroleum oil (M. A. Hoy, Dept.

Entomology, University of Florida, personal communication). Therefore, Silwet L-77 could be used as an adjuvant with petroleum oils to increase 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 expensive than some pesticides (Liu & Stansley

2000) and may assist in the conservation of natural enemies, one of the goals of an integrated pest management program. Wood et al. (1997) noted that Silwet L-77 had little or no effect on adults or larvae of lady beetles and green lacewings, suggesting that Silwet L-77 could be an important tool for an integrated pest management program.

We can conclude that Silwet L-77 increases the efficacy of oil against Boisduval scale and the flat mite on orchids. Because complete coverage is required, its use may be most relevant to hobbyists or small commercial growers who want to use a chemical control with reduced toxic effects on non-target organisms, the environment, and human health.

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Table 5-1. Pre-spray and post-spray quality ratings (range=1-10) of orchids used in the phytotoxicity study. Mean pre-spray Mean post- quality rating spray quality Orchid genera tested Treatment n ±SEa P rating ±SEa P Dendrobium hybrids Water 10 7.9 ±0.4 0.27 8.1 ±0.3 0.74 Silwet 10 7.3 ±0.4 7.9 ±0.2 Oncidium KBR 456 Water 10 8.1 ±0.2 0.55 8.3 ±0.2 0.66 Silwet 10 8.3 ±0.2 8.4 ±0.2 Doritaenopsis hybrids Water 10 7.2 ±0.1 0.78 8.0 ±0.0 1.00 Silwet 7 7.1 ±0.1 8.0 ±0.0 Paphiopedilum maudiae type Water 7 5.6 ±0.3 0.16 6.0 ±0.4 0.84 Silwet 8 6.1 ±0.2 6.0 ±0.3 Epidendrum MC Water 10 6.5 ±0.3 0.88 7.2 ±0.2 0.28 Silwet 7 6.5 ±0.2 6.7 ±0.2 Cymbidium Golden Elf Water 10 7.8 ±0.1 0.63 7.9 ±0.1 1.00 Silwet 10 7.7 ±0.2 7.9 ±0.1 Cattleya MC Water 3 6.3 ±0.3 0.37 6.7 ±0.8 0.12 Silwet 3 6.0 ±0.0 6.0 ±0.0

aMeans were analyzed using the PROC GLM procedure of the SAS software program. Levene’s test for homogeneity of variances showed equal variances within each orchid genus. Plant 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

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Table 5-2. Evaluation of Silwet L-77 (0.05%) in combination with two oils for control of Boisduval scale infestations in Cattleya mericlone orchids.

Mean post-spray rating of scale density ±SEac Scale mortality (%) ±SEbc

Date, environmental conditions at Mean pre-spray rating of time of treatment, and type of oil scale densitya used n Scale mortality (%) Water Oil Silwet + Oil P Experiment 1a, Sept. 22, 2007. 30 10.0 10.0 ±0.0a 8.4 ± 1.1a 3.7 ±0.7b <0.0001 25.5°C, 64% RH. Petroleum Oil 0.0 2.0 ±2.0a 44.0 ±10.8b 93.0 ±5.0c <0.0001 435 (2%)

Experiment 1b, Oct. 27, 2007. 30 10.0 10.0 ±0.0a 6.2 ±0.6b 1.8 ±0.4c <0.0001 24.4°C, 62% RH. Petroleum Oil 0.0 8.0 ±2.9a 47.0 ±3.7b 86.0 ±5.0c <0.0001 435 (2%)

Experiment 2a, Nov. 10, 2007. 30 10.0 9.8 ±0.1a 8.7 ±0.3b 2.2 ±0.4c <0.0001 25.5°C, 70% RH. Prescription 0.0 3.0 ±2.1a 39.0 ±4.8b 89.0 ±6.6c <0.0001 Treatment Ultra Fine Oil (1%)

Experiment 2b, Nov. 26, 2007. 30 10.0 9.7 ±0.2a 7.3 ±0.2b 1.4 ±0.2c <0.0001 25.0°C, 62% RH. Prescription 0.0 2.0 ±1.3a 46.0 ±3.1b 91.0 ±2.3c <0.0001 Treatment Ultra Fine Oil (1%) aAssessment rating of scale density based on number of female scales present. 0: 0 scales; 1: 1-20 scales; 2: 21-40 scales; 3: 41-60 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: 181-200+ scales. bMortality was calculated by randomly selecting 10 remaining scales on each plant and determining if they were alive or dead by using a metal probe and a hand lens.cTreatment means were analyzed using the PROC GLM procedure and means separated using Fishers’ LSD with P=0.05. Levene’s test for homogeneity of variance was also performed. Treatments with the same letter within a row are not significantly different from each other.

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Table 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.

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, 9 27.7 ±5.8a 94.8 ±2.7b 98.7 ± 1.3b <0.0001 49% RH

Experiment 4a, Dendrobium orchids. Jan.14, 2009. 20.8° C, 53% RH 9 25.5 ±2.2a 65.4 ±0.6b 98.8 ±1.2c 0.0017

Experiment 4b, Dendrobium orchids. Jan. 22, 2009. 24.5° C, 60% RH 9 19.6 ±1.6a 38.1 ±1.9b 98.4 ±1.6c <0.0001 aTreatment means were analyzed using the PROC GLM procedure and means separated using Fishers’ LSD with P=0.05. Levene’s test for homogeneity of variance was also performed. Treatments with the same letter within a row are not significantly different from each other.

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A B

C D

Figure 5-1. Examples of orchids used in the phytotoxicity 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.

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A B

C D

Figure 5-2. Cattleya mericlone orchids before treatment with water, petroleum oil, or Silwet L- 77 + 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 the sheath (lower left).

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A B

C D

Figure 5-3. Cattleya mericlone orchids infested with Boisduval scale 1 wk after second treatment with A) water control; B) Prescription Treatment Ultra-Fine oil alone; C and D) Prescription Treatment Ultra-Fine oil + Silwet L-77 showing that scales were removed from the orchids with the oil + Silwet L-77 combination.

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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 while minimizing the impact of the control measures

on non-target organisms (beneficial insects), humans, 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 control strategies for several important orchid pests and pathogens that will be compatible with an overall IPM program. This objective was met by demonstrating that the high-fidelity 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 two 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 important tool for diagnosticians and regulatory agencies, especially

when pathogens are of quarantine significance, occur at low titers, 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 valuable attributes that contribute to its wide range of uses and users.

It also was determined, through the use of the high-fidelity PCR, biochemical tests, and fatty acid analysis, that there are strains of soft-rot causing bacteria from orchids in Florida that have not been characterized previously. Because the phenotypic and genotypic characters of bacteria

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can be highly variable, in many cases multiple methods are required for identification (Alvarez

2004); therefore, multiple tests were used to identify and characterize the orchid 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 using portions of the 16S rDNA gene and pelADE pectolytic enzyme gene cluster, two distinct orchid 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 the 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 studies both within and without the Orchidaceae. This information could have an impact on the management of the disease. The selection of appropriate management strategies is dependent upon the correct identification of the pathogen (Miller & Martin 1988; Lévesque 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 manipulated to prevent or lessen the severity of the disease. Soft-rot diseases are extremely difficult to control with chemicals (Aysan et al.

2003). Therefore, the first line of defense is exclusion, i.e. starting with healthy plant material, which is dependent upon identification of the pathogen and determining its origin. Furthermore,

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pathogen identification is required for epidemiological studies and surveys of geographical

distribution, which can be particularly important when searching for disease resistance (Ploetz

2007).

In addition to the work with these novel Dickeya isolates from Florida 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 25ºC 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 25ºC 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 produce inoculum for pathogenicity

tests, for confirming Koch’s postulates, and for evaluating host-range within the Orchidaceae

without contaminating the inoculum with other pathogens that may be present on diseased

leaves. In addition, because spores are needed for morphological identification, these results

could aid diagnosticians and plant-health practitioners who encounter orchid diseases and wish to

grow isolates for morphological or molecular identification.

As mentioned above, one of the goals 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 that the use of Silwet L-77 + a light horticultural oil increased

control of Boisduval scale insects and a flat mite (Acari: Tenuipalpidae) on orchids while

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reducing the requirement for toxic pesticides. In addition to the 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 have developed resistance to petroleum oil.

Therefore, Silwet L-77 could be used as an adjuvant with petroleum oils to increase their effectiveness with the development of resistance a minimal concern (Butler et al. 1993). Also, the use of petroleum oil with the addition of Silwet 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 management program. Wood et al. (1997) noted that

Silwet L-77 had little or no effect on adults or larvae of lady beetles and green lacewings, suggesting that Silwet L-77 could be an important 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 commercial growers who want to use a chemical control that has reduced toxic effects on non-target organisms, the environment, and human health.

Currently, no IPM program specifically designed for orchids exists. The techniques and tools developed in this dissertation could serve 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 from 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 communication of research results to the potential end users, no IPM program can be adopted.

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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 fortunate 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 Pathogens. 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 responsibilities, I was an invited speaker at over 15

Florida orchid society meetings and orchid shows and I enjoyed speaking about orchid diseases

and management. Interacting with growers and hobbyists 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 spent most of my time in Marjorie Hoy’s 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. Hoy’s direction, 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, even if he/she doesn’t 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 connecting the dots, and filling in the blanks after that.

4. WRITE AS YOU GO. This is especially true of the materials and methods.

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5. Write down everything in your notebook, even if you think it is not important. For instance, what was the humidity in the greenhouse 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 includes seeking out the assistance of a statistician, which can save hours later.

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APPENDIX A PHYSIOLOGICAL DISORDERS OF ORCHIDS: OEDEMA

Oedema is a physiological disorder of orchids 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 frequency 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 plant tissue beneath the blister will frequently remain green (Figure A-3, A). Occasionally, 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 cross section through symptomatic tissue is made, enlarged cells can be seen below the epidermis (outer-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 disorder 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:

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• 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 corky 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. Scale 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 conditions return. Because oedema is not caused by a pathogen, no disease control 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 growing medium should also be examined to make sure it is not retaining too much water because of decomposition. 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.

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A B

C D

Figure A-1. Examples of oedema on various orchids. A) Phalaenopsis with symptoms of oedema. Notice the numerous round spots scattered over the leaf surface; B) A close- up 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.

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A B

C D

Figure A-2. Examples of oedema on various orchids. 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.

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A B

C D

Figure A-3. Diagnosing oedema in orchids. A) Oedema blister on a Phalaenopsis showing green tissue underneath; B) A cross 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 swollen or blister-like appearance; C) Bacterial leaf spots on a Vanda caused by Acidovorax sp. On the left, dark spots 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-filled area surrounding the lesion.

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A B

Figure A-4. Differentiating scale insects 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.

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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 because 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-spread 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 severity of symptoms is related to several factors: temperature,

length of exposure to low temperatures, and age of leaves (McConnell & 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 occurred to “orchid” leaves when exposed to chilling at -2˚C after an entire day of exposure, prompting them to conclude that the

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orchid was chilling-resistant. However, assessment 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 condition. 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 mesophyll cell collapse occurred in Phalaenopsis

Miva Smartissimo X Canberra “450” 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 temperature. This suggests that a decline in night temperature may play a role in the development of mesophyll cell collapse.

In order to properly diagnose mesophyll cell collapse, a thorough history of the growing conditions is crucial. High-low thermometers placed at various locations around the greenhouse or growing area can record temperature changes and may indicate areas that are prone to drafts or low temperatures. In Florida, 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 orchids grown outdoors. In some cases, weather/environmental data may not be available and a more thorough examination of the plant may be required to rule out a pathogen as the cause of symptoms. However, if no fungal, bacterial, or viral pathogens are found, then adverse environmental conditions may be the cause of the observed damage.

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After a diagnosis is made, the cause of temperature variation should be identified in order to

prevent recurrences of the condition. When one suspects mesophyll 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 also can cause mesophyll cell collapse.

When in doubt, visit the Florida Plant Diagnostic Network (http://fpdn.ifas.ufl.edu) for contact information 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.

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A B

C

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. Electronic Data Information Source, Florida cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida

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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 characters or by molecular diagnostic 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 & Szkuta 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 roots or basal portion of the pseudobulbs. As the lesions age, they enlarge and may engulf the entire pseudobulb and leaf (figure C-1, A-C). The pathogen can spread through the rhizome to other portions of the plant. Under favorable environmental conditions, mycelia may be observed growing on parts of the plant (C-1, C).

Eventually, the entire plant will be killed.

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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. Morphological identification is based

on characteristics of the mycelium, shape of zoosporangia (asexual reproductive structures) and

the presence and shape of oospores (sexual reproductive structures).

If zoosporangia are present, and 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 provide 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 during 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 adequate 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 should also consider the following:

Nursery Sanitation Recommendations for Phytophthora

Fungicides should be considered as a tool for managing Phytophthora but if not used

properly (according to the manufacturer’s label) they will not be effective or may cause damage,

such as phytotoxicity. However, they are the primary defense in an existing crop and provide at

least some level of control when used properly.

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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 (Aliette, Flanker, Prokoz Avalon), potassium

phosphate (Alude, Fungi-phite, Topaz), propamocarb 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 infected 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 the molecular bonds in microorganisms). 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 production of toxic chlorine gas. Always use in

good ventilation.

Containers

Store new pots in sanitized areas. The best option is to use new potting containers, but if this

is not feasible, submerge pots in a 1:3 ratio of bleach (sodium hypochlorite) to water with

agitation for a minimum of 10 minutes.

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Bench Sanitation

Make sure that bench surfaces are high enough above 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 disposable 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 available 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 alcohol (70% isopropyl, equal parts alcohol and water); 4) 50%

denatured alcohol (95%, equal parts 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, mildew, 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 sanitize 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 growing area should be kept isolated from other plants for a minimum of 6 weeks to observe the plants and watch for the development of any disease or pest symptoms (quarantine). This can greatly decrease the possibility of introducing new pathogens or pests into the growing area. Make sure that different tools and bench spaces is used for plants

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

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A B

C

D

Figure C-1. Black rot of orchids, 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.

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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 commercial nurseries and landscapes throughout

South Florida. More than a dozen orchids were affected at one of the locations, 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 from $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 (Figure D-1, A). As the lesions progressed, leaves around the base of the plants began to fall off, leaving the stems bare (Figure D-1, B). After 2 days, white, flabellate 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 acidified potato dextrose agar (APDA) and grown at 25°C. White, flabellate mycelium and tan sclerotia approximately 2 mm in diameter were produced in culture and microscopic examination revealed the presence 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 using primers ITS4 and ITS5 as described by

White et al. (2001) and the sequence was deposited 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

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untreated controls. Plants were housed under 50% shade, 60 to 95% relative humidity, and temperatures ranging from 23.8 to 31.2°C. 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.

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A B

C D

Figure D-1. Ascocenda orchids with Southern blight caused by S. rolfsii. A) Ascocenda orchid with early symptoms of Southern blight. Early symptoms frequently 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.

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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, and 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 these intriguing, beautiful and, in many cases, long-

lasting flowers.

Orchids are susceptible to numerous disease-causing agents such as fungi, bacteria, and

viruses. Many common and economically important diseases affecting orchids 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, stem, leaf and pseudobulb rots (i.e. Phytophthora spp.) commonly called black rot.

Several species of Phytophthora have been reported to occur 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

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objective is to provide a resource that covers important diagnostic information including symptoms and signs associated with the disease, morphological, molecular, and pathological characteristics, taxonomy of the causal agents, host range and geographic 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 orchids, however, black rot, in commercial orchid production throughout Florida is most commonly caused by Phytophthora cactorum and P. palmivora (Hine 1962; Uchida 1994; Erwin & Ribeiro 1996; Orlikowski &

Szkuta 2006). Although P. cactorum and P. palmivora are similar, they can be differentiated by morphological characteristics or by the 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. (2008) 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).

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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 entire 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 (Farr 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, and 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 gentle stream of tap water. Use a clean blade to slice multiple sections of tissue from the plant. To increase isolation frequency, one should 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

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plates with parafilm and incubate in the dark at 27-32ºC. 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. Morphological identification 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 lemon-

shaped 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 strategies. If it is necessary to identify the pathogen to species, oospores can be helpful in identifying the pathogen 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 laboratory (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 suitable medium for the formation of sex bodies (both

Phytophthora species) is lima bean agar (Gallegly & Hong 2008).

The location of attachment of the antheridium is an important diagnostic characteristic

(Erwin & Ribeiro 1996). Antheridia, which are the male reproductive 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 antheridium and in Figure E-2 (B), the antheridium

of P. cactorum can be seen attached to the side of the oogonium stalk.Both Phytophthora species produce abundant, papillate sporangia that are caducous with short pedicels directly on orchid host tissue. For maximum sporangia production, maintain infected orchids at 25-28°C for P.

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cactorum, whereas slightly warmer temperatures 30-33°C are more favorable for P. palmivora

(personal observation). Note that the morphological characteristics 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 characteristics 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 identifications (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 performed investigating methods for storage of certain species (Goth

1981; Peters et al. 1998; Erwin & Ribeiro 1996). Storage of Phytophthora isolates has proven to

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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 sterile water at room temperature for as long as 23 years. To store isolates in this manner, 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 involves the use of liquid nitrogen (Tooley 1988; Erwin

& Ribeiro 1996). In this method, plugs of agar containing the isolate 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 successfully recover the isolate

(Tooley 1988). For detailed information on cryopreservation, see Dahmen et al. (1983) and

Tooley (1988). Although cryopreservation is simple and can preserve the isolate in the original

genetic state, it requires special equipment (liquid nitrogen tanks, -80˚C freezer for pretreatment)

and can be expensive to maintain (Tooley 1988; Erwin & Ribeiro 1996; Ko 2003)

Pathogenicity Tests

Hine (1962) tested the pathogenicity of P. palmivora on members of several different orchid

genera by using zoospore suspensions (no concentrations 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, 3- mm 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 25°C 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.

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A D

B

C

C

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.

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A B

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

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APPENDIX F FIRST REPORT OF BACTERIAL SOFT ROT ON Tolumnia ORCHIDS CAUSED BY A Dickeya sp.

Tolumnia orchids are small epiphytic orchids grown for their attractive flowers. In the fall of

2008, approximately 100 Tolumnia orchids with soft, brown, macerated leaves were brought to the University of Florida Extension Plant Diagnostic Clinic in Homestead (Figure F-1). Ten plants were randomly selected and bacteria were 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 negative, grew at 37°C, degraded pectate in CVP

(crystal violet pectate) medium, grew anaerobically, produced 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 Identification, 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% identity of the 1,508-bp sequence to that of Dickeya chrysanthemi (Accession No. FM946179) (formerly E. chrysanthemi). The sequences were deposited in GenBank (Accession Nos. GQ293897 and GQ293898).

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Pathogenicity Tests

Pathogenicity was confirmed by injecting approximately 100 μl of a bacterial suspension at 1

× 108 CFU/ml into leaves of 10 Tolumnia orchid mericlones. Ten plants were also inoculated

with water as controls. Plants were placed in a greenhouse at 29°C 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 identified using the above methods

(biochemical tests and MIDI), fulfilling Koch's postulates.

To our knowledge, this is the first report of a soft rot caused by a Dickeya sp. on Tolumnia

orchids. Although 16S similarity and MIDI results suggest the isolated 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 identity 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%.

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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.

154

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BIOGRAPHICAL SKETCH

Born in Louisville, Kentucky and raised in a small town in southern Indiana, Robert Cating

attended Crawford County High School in Marengo, Indiana followed by undergraduate study at

Indiana University-Bloomington and Indiana University-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 and 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. Robert 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 International in Apopka,

Florida as Director of Biotechnology Research.

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