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Angular Leaf Spot of Strawberry

Permanent Link: http://ufdc.ufl.edu/UFE0021457/00001

Material Information

Title: Angular Leaf Spot of Strawberry Disease Control Strategies and Association of Pseudomonas syringae with Lesions
Physical Description: 1 online resource (62 p.)
Language: english
Creator: Cooper, Gary T
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: angular, bacterial, leaf, spot, strawberry, xanthomonas
Plant Pathology -- Dissertations, Academic -- UF
Genre: Plant Pathology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Angular leaf spot (ALS) of strawberry is a bacterial disease caused by Xanthomonas fragariae. Cultivars of strawberry used in Florida vary in their susceptibility to ALS and there are no strawberry cultivars that are immune. Recommendations for management of ALS include using pathogen-free stock, limiting overhead irrigation, and applying chemical treatments. A trial to evaluate various materials for control of ALS was conducted at the Gulf Coast Research and Education Center during the 2005?06 and 2006?07 seasons. During the 2006?07 season, the susceptibility to ALS of various cultivars and breeding selections was evaluated. Spray materials were applied on 7- and 14-day schedules to ?Strawberry Festival? plants and disease severity was evaluated on a scale of 0 to 6. Disease incidence was evaluated at the end of the 2006?07 season. Temperature and rainfall/irrigation data were collected and analyzed for both seasons to assess the effect of environmental conditions on ALS. Treatments that included Actigard at the higher rate, Kocide, Copper Count N, Badge, and Kasumin had significantly lower disease severity than untreated controls. Results from these studies indicate that there are some good alternatives to copper for managing ALS. Cultivars Treasure and Ruby Gem were mildly susceptible to ALS. Cultivars Camino Real, Sweet Charlie, Albion, Festival, Sugar Baby, Carmine, and advanced selections FL 99-164, Festival #9, and FL 00-51 were moderately susceptible whereas cultivars Winter Dawn and Camarosa, and advanced selections FL 99-117, and FL 01-116 were highly susceptible. Lastly, we report an association between X. fragariae and Pseudomonas spp. based on laboratory inoculations in which lesions of angular leaf spot were different between plants inoculated with X. fragariae only and a mixture of X. fragariae and Pseudomonas spp.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Gary T Cooper.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Peres, Natalia.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021457:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021457/00001

Material Information

Title: Angular Leaf Spot of Strawberry Disease Control Strategies and Association of Pseudomonas syringae with Lesions
Physical Description: 1 online resource (62 p.)
Language: english
Creator: Cooper, Gary T
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: angular, bacterial, leaf, spot, strawberry, xanthomonas
Plant Pathology -- Dissertations, Academic -- UF
Genre: Plant Pathology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Angular leaf spot (ALS) of strawberry is a bacterial disease caused by Xanthomonas fragariae. Cultivars of strawberry used in Florida vary in their susceptibility to ALS and there are no strawberry cultivars that are immune. Recommendations for management of ALS include using pathogen-free stock, limiting overhead irrigation, and applying chemical treatments. A trial to evaluate various materials for control of ALS was conducted at the Gulf Coast Research and Education Center during the 2005?06 and 2006?07 seasons. During the 2006?07 season, the susceptibility to ALS of various cultivars and breeding selections was evaluated. Spray materials were applied on 7- and 14-day schedules to ?Strawberry Festival? plants and disease severity was evaluated on a scale of 0 to 6. Disease incidence was evaluated at the end of the 2006?07 season. Temperature and rainfall/irrigation data were collected and analyzed for both seasons to assess the effect of environmental conditions on ALS. Treatments that included Actigard at the higher rate, Kocide, Copper Count N, Badge, and Kasumin had significantly lower disease severity than untreated controls. Results from these studies indicate that there are some good alternatives to copper for managing ALS. Cultivars Treasure and Ruby Gem were mildly susceptible to ALS. Cultivars Camino Real, Sweet Charlie, Albion, Festival, Sugar Baby, Carmine, and advanced selections FL 99-164, Festival #9, and FL 00-51 were moderately susceptible whereas cultivars Winter Dawn and Camarosa, and advanced selections FL 99-117, and FL 01-116 were highly susceptible. Lastly, we report an association between X. fragariae and Pseudomonas spp. based on laboratory inoculations in which lesions of angular leaf spot were different between plants inoculated with X. fragariae only and a mixture of X. fragariae and Pseudomonas spp.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Gary T Cooper.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Peres, Natalia.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021457:00001


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ANGULAR LEAF SPOT OF STRAWBERRY: DISEASE CONTROL STRATEGIES AND
ASSOCIATION OF Pseudomona~s syringe WITH LESIONS

















By

GARY TODD COOPER


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2007

































O 2007 Gary Todd Cooper





























To my wife Aimee, for without her love, support, and sacrifice, it would not have been possible;
to my parents Gary and Ailene and my sisters Dedra and Leah for their love and support, and to
my friend Frank Smith, who also has been there for me from the very beginning; I thank you all.









ACKNOWLEDGMENTS

I thank the chair and members of my supervisory committee, Dr. Natalia Peres, Dr. Jeffrey

B. Jones and Dr. Craig Chandler, for their guidance and patience. I also thank Mrs. Teresa Seijo

for her tremendous contribution to my training and Dr. James Mertely for his time, vast

experience, and advice. Finally, I wish to thank Dr. Jane Polston for all of her guidance that

helped keep me motivated.












TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. ...............4.....


LI ST OF T ABLE S ............. ...... ...............7...


LI ST OF FIGURE S .............. ...............8.....


AB S TRAC T ......_ ................. ............_........9


CHAPTER


1 INTRODUCTION ................. ...............11.......... ......


2 EVALUATION OF SPRAY MATERIALS FOR CONTROL OF ALS, AND IMPACT
OF ENVIORNMENTAL CONDITIONS ................. ...............18........... ....


Introducti on ................. ...............18.................
Materials and Methods .............. ...............21....
R e sults............... . .. ........ ...............23.......
2005-06 Chemical Trial .............. ...............23....

Di sease severity ................. ...............23.......... ......

Phytotoxicity .............. ...............24....
Marketable weight ................. ...............24.................

Temperature and rainfall .............. ...............25....
2006-07 Chemical Trial .............. ...............25....

Di sease severity ................. ...............25.......... ......
Di sease incidence .............. ...............26....

Phytotoxicity .............. ...............26....
Marketable weight ................. ...............26.................

Temperature and rainfall .............. ...............27....
Discussion ................. ...............27.................


3 RESISTANCE OF FLORIDA STRAWBERRY CULTIVARS TO ANGULAR LEAF
SPOT............... ...............37..


Introducti on ............. ...... ._ ...............37...
Materials and Methods .............. ...............39....
R e sults.............. ......_ ...............40...
Cultivar Evaluation ............. ...... .__ ...............40...

Marketable Weight ............. ...... ...............41...
Discussion ............. ..............41._ __.......












4 ASSOCIATION OF ANGULAR LEAF SPOT LESIONS AND Pseudomonas spp.IN
SYMPTOMATIC TISSUE ISOLTIONS OF STRAWBERRY ................. .....................46


Introducti on ................. ...............46.................
Materials and Methods .............. ...............47....
Tissue Isolations ................ .. ...............47..

Hypersensitive Response Tests .............. ...............47....
Fatty Acid Analy si s .............. ...............47....
Oxidase Test ................. ...............48.................
Ice Nucleation............... ...............4

Pathogenicity Tests............... ...............48.
Results and Discussion .............. ...............49....
Tissue Isolations .............. ...............49....
Characterization of Strains .............. ...............49....

Pathogenicity Test ................. ...............49.......... .....


SUMMARY ................. ...............56.......... ......


LIST OF REFERENCES ................. ...............57........... ....


BIOGRAPHICAL SKETCH .............. ...............62....










LIST OF TABLES


Table page

2~1 Treatments and application schedules for control of angular leaf spot in annual
strawberry for the 2005-06 season in Florida. .....__.....___ .......... .. .........3

2~2 Treatments and application schedules for control of angular leaf spot in annual
strawberry for the 2006-07 season in Florida. .....__.....___ .......... .. .........3

2~3 Severity of angular leaf spot, phytotoxocity, and market weight of annual strawberry
for the 2005-06 season in response to the application of various spray materials. ...........33

2~4 Severity of angular leaf spot, phytotoxocity, and market weight of annual strawberry
for the 2006-07 season in response to the application of various spray materials. ...........34

3~1 Disease incidence of angular leaf spot in cultivars of annual strawberry in Florida for
2006-07 season. .............. ...............45....

4~1 Characterization of strains associated with lesions of angular leaf spot on strawberry
during 2005-06 and 2006-07 season. .............. ...............53....










LIST OF FIGURES


Figure page

2~1 Environmental conditions and disease severity for 2005-06. .......... .....................35

2~2 Environmental conditions and disease severity for 2006-07. .......... .....................36

4~1 Angular leaf spot symptoms on strawberry from inoculations ofX fragariae and
Pseudomona~s spp. ............ ...............55.....









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

ANGULAR LEAF SPOT OF STRAWBERRY: DISEASE CONTROL STRATEGIES AND
ASSOCIATION OF Pseudomona~s syringe WITH LESIONS

By

Gary Todd Cooper

August 2007

Chair: Natalia A. Peres
Major: Plant Pathology

Angular leaf spot (ALS) of strawberry is a bacterial disease caused by Xanthomona~s

fragariae. Cultivars of strawberry used in Florida vary in their susceptibility to ALS and there

are no strawberry cultivars that are immune. Recommendations for management of ALS include

using pathogen-free stock, limiting overhead irrigation, and applying chemical treatments. A

trial to evaluate various materials for control of ALS was conducted at the Gulf Coast Research

and Education Center during the 2005-06 and 2006-07 seasons. During the 2006-07 season, the

susceptibility to ALS of various cultivars and breeding selections was evaluated. Spray materials

were applied on 7- and 14-day schedules to Strawberry Festival' plants and disease severity was

evaluated on a scale of 0 to 6. Disease incidence was evaluated at the end of the 2006-07 season.

Temperature and rainfall/irrigation data were collected and analyzed for both seasons to assess

the effect of environmental conditions on ALS. Treatments that included Actigard at the higher

rate, Kocide, Copper Count N, Badge, and Kasumin had significantly lower disease severity than

untreated controls. Results from these studies indicate that there are some good alternatives to

copper for managing ALS. Cultivars Treasure and Ruby Gem were mildly susceptible to ALS.

Cultivars Camino Real, Sweet Charlie, Albion, Festival, Sugar Baby, Carmine, and advanced









selections FL 99-164, Festival #9, and FL 00-51 were moderately susceptible whereas cultivars

Winter Dawn and Camarosa, and advanced selections FL 99-117, and FL 01-116 were highly

susceptible. Lastly, we report an association between X fragariae and Pseudomona~s spp. based

on laboratory inoculations in which lesions of angular leaf spot were different between plants

inoculated with X. fragariae only and a mixture of X. fragariae and Pseudomona~s spp.









CHAPTER 1
INTTRODUCTION

Florida ranks second in the nation in strawberry production, accounting for about 12

percent of the total U. S. supply of fresh product and generating more than $200 million in sales

annually (9). There are several important diseases of strawberry that are costly to control. Most

of those diseases are caused by fungal pathogens and are managed through the use of a fungicide

program. Angular leaf spot (ALS) is the only economically important bacterial disease of

strawberry in the U.S.

Although ALS-like symptoms had been observed in Utah in 1927, ALS was not reported

as a bacterial disease caused by Xanthomona;s fragariae until 1962 (24, 37), when it was

observed in Minnesota. No additional reports of the disease occurred until 1966 when ALS was

found in wisconsin, producing severe losses reaching 75-80% of the crop (8). Hildebrand et al.

(14) reported that ALS had been observed in California for 10-15 years; however, it was not

considered an important disease since its occurrence in the field was sporadic. Hildebrand et al.

(14) proposed the name bacterial blight of strawberry rather than angular leaf spot. However,

since the symptoms described by Kennedy and King (24) were more frequent than those

described by Hildebrand et al. (14), the current name remains angular leaf spot. Finally, in 1971,

ALS was reported in Florida (16). ALS had been observed in fields starting in mid-January of

1968 and reports of yield loss and poor plant condition were attributed to ALS. Since evidence

of infection was not found in Florida nurseries except in very mild cases during 1969, ALS was

not considered to be an important threat. Recent observations by growers in Florida suggest that

plantings may now suffer more severe ALS infections and potentially greater losses to the

disease.










By 1993, ALS of strawberry was occurring in all parts of the world (49), and it became

apparent that the international shipment of infected transplants was responsible for the rapid

spread of this disease. At the time of a review by Maas et al. (37), ALS was occurring in North

America, as well as Europe, Africa, South America, and Australia; however, efforts to eradicate

the disease in Australia were successful (39). In 2001, Janse et al. (18) discovered a similar, but

new bacterial disease of strawberry in northern Italy. Although this disease is caused by a

bacterium in the genus Xanthomona~s, symptoms of the two diseases differ. This new disease,

bacterial blight, has not been reported in the U.S.

Kennedy and King (24) described the symptoms of ALS as light-green, angular, water-

soaked lesions on the under surface of the leaf. The term angular refers to the noncircular

appearance of the lesions. When primary infections occur in the interveinal tissue, the vascular

system in the leaf limits movement of the pathogen, which limits ALS lesions to the parenchyma

tissue. When held to a transmitted light source, the spots are translucent, angular and easily

distinguishable from other types of foliar damage during the early stages of infection. If

conditions are favorable, these lesions can increase in number and size and eventually coalesce

and become necrotic. The advanced symptoms may become more difficult to distinguish from

other leaf infections such as common leaf spot, caused by the fungal pathogen Mycosphaerella

Jfralgariae (24).

Although parenchymatous foliar tissue is targeted primarily, Hildebrand et al. (14) reported

that vascular tissue could also be infected resulting in tissue collapse. Symptoms associated with

a vascular infection are similar to those of foliar infection in their translucence; but lesions are

concentrated in tissue adj acent to veins. Foliar infections are usually randomly distributed on the

leaf in contrast to vascular infections. Although not reported as a vascular infection at the time,










examples of both vascular and foliar symptoms can be seen in the first report of ALS on

strawberry by Kennedy and King (24). Vascular infections that produce the symptoms

associated with lesion concentration in areas adj acent to veins are the result of infection of

strawberry crowns by X. fragariae (4, 14, 37).

Additionally, blighting of petioles and infection of flowers and runners can occur (1 1). I.

fragariae is not pathogenic to the strawberry fruit; however, severe infection of the calyx

produces obvious symptoms and the calyx will turn brown making fruit unmarketable (31, 43).

Xanthomona;s fragariaea is a rod-shaped, Gram-negative bacterium. Typically, the cells are

non-motile; however some may have a single polar flagellum (50, 7). I. fragariae is aerobic,

non-capsulate, and non-spore-forming. Typical colonies are circular, convex, and mucoid.

Although they produce xanthomonadin, the colonies are pale yellow and young colonies may

appear translucent. Growth on nutrient agar and in nutrient broth is poor. Colony formation may

require 3 to 5 days incubation at 20 to 240C on a suitable medium such as the Wilbrink' s

medium (26, 48).

fragariae is listed as a quarantine pest by the European Plant Protection Organization

(7). Often, plants infected with ALS are asymptomatic. Because strawberry is propagated

vegetatively and transplants are shipped worldwide, movement of infected transplants often goes

undetected and is thought to be the reason for distribution ofX. fragariae worldwide (37). Thus,

good detection techniques are important to avoid movement of the disease.

In the early to mid nineties, efforts to develop rapid detection of ALS with molecular and

serological assays resulted in more efficient techniques for confirmation ofX. fragariae. One

technique, Enzyme-Linked Immunosorbent Assay (ELISA), was effective in detecting X.

fragariae in symptomatic tissue, but, unfortunately, ELISA was ineffective for detection on









asymptomatic tissue or tissue infected with bacterial populations below 104 ofu/ml (37, 49). A

more effective technique for detection ofX fragariae and for detection of epiphytic bacteria is

based on the Polymerase Chain Reaction (PCR). With the advent of PCR and its adaptation to

plant disease diagnosis, studies began to develop primers for specific detection ofX. fragariae

(13, 45, 47). The molecular and serological detection techniques developed by Rowhani et al.

(49), Roberts et al. (47), and Pooler et al. (45) not only improved diagnosis of infected

symptomatic material, but hastened the ability to detect the pathogen in asymptomatic

transplants as well. The combination of tissue isolation and the new molecular and serological

techniques proved successful and diagnosis of ALS was considerably improved (45, 47, 49).

Strawberry production in Florida differs from northern regions in that new transplants are

obtained annually from northern or high elevation nurseries to establish strawberry fields.

Transplants can be infected and escape detection as leaves may remain asymptomatic until

favorable conditions occur. In the study by Roberts et al. (48), nearly 50% of the shipments

contained boxes of transplants with ALS symptoms.

To determine the host range of X. fragariae, Kennedy and King (24) inoculated 35

different types of plants by infiltration with cell suspensions. None of the thirty-five potential

hosts, except strawberry, became infected. Since their initial tests, the only other reported host

for X. fragariae has been a couple ofPotentilla species.

Conditions that favor ALS are high humidity, moderate or low temperatures in some cases

near zero Celsius, and good plant growth (8, 25). When humidity is high, the underside of the

leaf can have a slimy mucous layer of bacterial exudates covering the lesions (8, 36, personal

observation). In pathogenicity tests where inoculated plants were incubated under different









moisture conditions, few lesions were observed on plants left on open benches compared to those

placed in moisture chambers (8).

Bacterial pathogens are frequently spread by rain as is the case with Xanthomona~s

axonopodis py. citri, the causal agent of citrus canker of citrus (3). Another mechanism of

bacterial transmission occurs via mechanical transmission by working in the field when plants

are wet from rain, or more frequently from the early morning dew. Intuitively, limiting the

amount of overhead irrigation during the growing season would be helpful in controlling the

spread of ALS in strawberry fields. As early as 1966, Epstein questioned the use of overhead

irrigation and pointed out its potential impact on ALS (8). However, in Florida, strawberry is

farmed annually and the use of bare-root transplants each season makes overhead irrigation

essential for transplant establishment. Florida strawberry field temperatures are high during

October, and without adequate overhead irrigation until transplants are established, transplants

would dehydrate and die. Additionally, strawberry in Florida is a winter crop and air

temperatures can fall below freezing in the evening and early morning hours. Overhead

irrigation is the most cost effective method to protect the flowers of the strawberry plants from

freezes. Unfortunately, this freeze protection method spreads the pathogen in the field and new

symptoms are seen frequently following freeze events (37, 48, personal observation).

Reports of losses associated with ALS are variable. Epstein (8) reported losses from 75-

80%; yet in Florida, Roberts et al. (48) reported much lower losses of approximately 8%. No

other reports on the effect of ALS on yield have been published since 1997. Thus, more

information is needed on the effect of ALS on yield for the currently grown cultivars and on the

cost effectiveness of treatment programs. Since the pathogen does not cause fruit rot, and only

infects the calyx when disease pressure is high, ALS rarely causes a direct reduction in yield.









However, it may indirectly reduce yield by reducing photosynthetically active leaf area. In a

recent study by Mertely et al. (40), leaf and fruit sanitation were compared to the standard

fungicide program for management of Botrytis Fruit Rot and leaf sanitation actually reduced

yield. A possible explanation suggested by Mertely et al. (40) was that yield loss may be a result

of the loss of photosynthetically active tissue and nutrients that could be mobilized by plants

brought about by removal of tissue.

Management of plant diseases is usually achieved by employing several techniques. The

first method of control is avoidance. Pathogen-free seed or transplants should be used to avoid

introducing a pathogen to a field. Sanitation or eradication, the removal of infected tissue or

plants, may be useful if applied correctly. Resistance to bacterial pathogens is a useful

management tool if available. The use of soil sterilization, anti-bacterial sprays (including

inorganic compounds and antibiotics) are other methods for control of plant diseases (1). Some

of these techniques may be practical and effective in strawberry production in Florida, but others

are not. For example, sanitation has been studied in annual strawberry to manage a different

disease and had adverse affects, and antibiotics have not been successful in field application for

disease control. The most effective way of preventing ALS is the use of pathogen-free

transplants (37, 44). However, disease-free transplants may not be readily available. Copper-

based pesticides have been effective for control of bacterial diseases on strawberry and other

crops, but phytotoxicity on strawberry plants limit rates and frequencies of application (43).

Plant resistance is a useful tool for disease management; however, there are no resistant cultivars

of strawberry currently available.

There were four primary obj ectives of this research. The first obj ective was to evaluate

different spray materials currently available for use in an integrated management program for









control of ALS of strawberry. The second obj ective was to quantify the effect of ALS and the

effect of the treatments for ALS management on marketable yield. The third obj ective was to

determine whether there were differences in susceptibility among commercial cultivars currently

grown in Florida or advanced selections from a breeding program. The final obj ective was to

determine if an association of Pseudomona~s spp. with lesions of angular leaf spot exists.









CHAPTER 2
EVALUATION OF SPRAY MATERIALS FOR CONTROL OF ALS, AND IMPACT OF
ENVIORNMENTAL CONDITIONS

Introduction

Angular leaf spot (ALS), caused by Xanthomona;s fragariae, is a bacterial disease of

strawberry (24). The disease was first observed in Minnesota by Kennedy and King in 1959, but

was not reported until 1962 (24). ALS is primarily a foliar disease; however the calyx of the fruit

can be infected as well. If the calyx is severely infected, the tissue will turn necrotic and fruit is

not marketable as fresh fruit (43, 31). Symptoms of ALS in the early stages are easily diagnosed

and distinguishable from other foliar infections (24). With ALS, tissue can be infected and

asymptomatic making it very difficult to control the spread of infected plant material.

ALS is favored by high humidity and low temperatures similar to those experienced in

Florida strawberry fields in the winter months. Temperatures are relatively high during the day,

frequently reaching 18 to 220C, and nights are cool, with air temperatures at ground level

dropping to between 0 and 50C at times. These conditions result in high surface moisture on

plants permitting colonization by the pathogen. Studies by Hildebrand et al. (15) and Epstein (8)

determined the importance of post-inoculation humidity, and as early as 1966, Epstein (8)

questioned the impact that overhead irrigation might have on ALS epidemics.

Losses associated with ALS are not clear. Reports of losses reaching 75 to 80% by Epstein

differ greatly from reports in Florida that indicated losses of approximately 8% (8, 48).

Currently, ALS is managed with a variety of techniques. The most effective and efficient

technique is the use of pathogen-free planting stock (37, 43). Cultural practices such as limiting

overhead irrigation are important in reducing the spread of inoculum in fields. Unfortunately,

most growers are dependent on the use of overhead irrigation to protect their crop during freeze

events. Currently, the only compounds recommended for control of ALS are copper-based










products. However, there is a danger in using copper-based products since copper is phytotoxic

to strawberry plants (48). Thus, new compounds need to be evaluated to determine if better

control alternatives exist. Systemic acquired resistance (SAR) inducers, and new antibiotic

formulations and biological control agents have shown some promise in managing bacterial

diseases and increasing yield in other crops (33, 41, 42). In this study, five types of spray

materials were tested including copper-based treatments, SAR inducers, kasugamycin antibiotic

treatments, biological control products, and surfactants.

Copper-based spray programs have been proved to be effective to control bacterial spot of

tomato, caused by Xanthomona~s campestris, and citrus canker, caused by Xanthomona~s

axonopodis py. citri (5, 20, 21, 52). In a previous study by Roberts et al. (48), copper hydroxide

has shown some suppression of ALS and served as the standard treatment for disease control in

this study. Additionally, other copper formulations are available such as basic copper sulfate,

copper ammonia complexes, and copper oxychloride. These formulations were evaluated to see

whether they were able to reduce disease without causing phytotoxicity.

Products that induce the systemic acquired resistance (SAR) were not available at the

time of the study by Roberts et al. (48). Recent studies indicated that the SAR compound

acibenzolar-S-methyl (ASM) was a good alternative to copper for reducing disease severity in

bacterial spot of tomato (41, 35). A group of SAR inducers including ASM were evaluated for

efficacy in control or suppression of ALS. Potassium phosphite is a well-known inducer of

resistance especially to diseases caused by Oomycetes and has been effective in studies of

bacterial spot of peaches, peppers, and tomato (19, 46, 22, 17). The mixture of famoxadone and

cymoxanil, although not described as an SAR, is a systemic pesticide that targets protein

complex III in the mitochondria thus limiting ATP production and ultimately energy production.










These products penetrate the plant and cannot be washed off by rain. Tanos, the mixture of these

chemicals, has been shown to significantly reduce numbers of lesions in bacterial speck of

tomato, caused by Pseudoomonas syringae py. tomato (28, 29).

Antibiotics have not been widely used in the field because of limited residual activity and

potential problems with the development of resistance in the pathogen. Recently, Kasumin, a

product not yet labeled for use in the U.S. has been studied for control of bacterial diseases of

pepper, tomato, citrus, and walnut; all of which are caused by different pathovars of

Xanthomonas campestris (30, 17). Kasumin significantly reduced disease in all four studies, and

is an effective bactericide/fungicide that has been registered and used in other countries for years

now. Kasugamycin, the active ingredient in Kasumin, is an aminoglycoside antibiotic from

streptomyces, and was included in this study to investigate whether it may also provide control

of ALS on strawberries.

Two biological control products were also evaluated--a spore suspension of

Streptomyces lydicus (Actinovate) and Bacillus subtilus (Serenade Max). In separate studies on

control of bacterial leaf spot of tomato and pepper, Actinovate and Serenade Max reduced

disease compared to the untreated control (17, 33). The bacteria in these products may colonize

leaf surfaces of strawberry plants making it more difficult for X. fragariae to obtain leaf surface

nutrients.

Efforts to improve coverage of pesticides used for control of other economically

important strawberry diseases may require a surfactant to be added to the formulation for

maximum efficacy. Surfactants alter the properties of water and reduce hydrophobicity to plant

surfaces. Unfortunately, the materials used for management of fungal diseases may possibly aid

bacterial pathogens in reaching areas with natural openings overcoming the natural physical









barriers of the plant. In a study by Gottwald et al. (10), surfactants were found to enhance

bacterial infections on citrus. A surfactant was included in this study to determine if the use of

surfactants in strawberry production increases ALS severity.

The goal of this study was to evaluate several new chemical and biological products to

determine if any available products minimize or control ALS during the growing season, and by

doing so, increase yield. The effects of temperature and rainfall/overhead irrigation on disease

severity during the growing season were also analyzed.

Materials and Methods

Field experiments evaluating the use of products for ALS control and the impact of ALS

on yield were conducted at the University of Florida Gulf Coast Research and Education Center

in Wimauma, Florida during the 2005-06 and 2006-07 growing seasons in fields managed using

current conventional strawberry farming practices. On October 6, 2005 and October 16, 2006,

bare-root transplants of the cultivar Strawberry Festival from Canada were planted in raised,

fumigated beds covered with black plastic mulch. Soil had been fumigated with methyl bromide

and chloropicrin at a ratio of 67:33 and at a rate of 397 kg/ha. Beds were 1.2 m apart, center to

center, and were 0.7 m wide. Two offset rows of plants were planted on each bed. Plants were

spaced 28 cm in the row with 38 cm between the rows. Overhead irrigation was applied for 10

days to establish transplants and then plants were irrigated and fertilized by drip tape.

Treatments were arranged in a randomized complete block design in four successive beds. Plots

for each treatment contained 12 plants and were 2.9 m long. In this study, 15 and 17 treatments

including control groups were compared for the 2005-06 and 2006-07 growing seasons,

respectively (Tables 2~1 and 2~2). Transplants were obtained from nurseries known to have

ALS to ensure a source of inoculum.









Five types of products were selected for study. These included copper-based products,

systemic acquired resistance (SAR) products that induce natural defense mechanisms of the

plant, the antibiotic kasugamycin, biological compounds, and a surfactant. In all cases,

applications were made with a CO2 backpack sprayer that delivered 950 L/ha at 275 kPa with a

two-nozzle wand. Treatments were applied on a seven-day schedule beginning December 2 and

November 22 and continuing through February 22 and February 28 for 2005-06 and 2006-07

seasons, respectively.

Evaluations of disease severity, phytotoxicity, and marketable yield were conducted for

both seasons. Disease severity was evaluated three times during each season on a scale from 0 to

6 where 0 = no lesions; 1 = a few lesions on the entire plant; 2 = a few lesions on more than one

leaflet with no general necrosis; 3 = several lesions on leaflets with or without concentration of

lesions near the veins and necrosis; 4 = numerous lesions present and some partial leaflet blight;

5 = some older leaves killed and others extensively blighted; 6 = all older leaves killed and

middle age leaves blighted. For each evaluation, the middle eight plants per plot were rated

individually on February 10 and 24, and March 14 for the 2005-06 season, and December 28,

January 25, and February 22 for the 2006-07 season. At the end of the 2006-07 growing season,

five leaves from six plants in each plot, or a total of 30 leaves or 90 leaflets, were collected

arbitrarily and evaluated for disease incidence.

Phytotoxicity evaluations were conducted in all treatments once per season on January 6

and February 28 for the 2005-06 and 2006-07 seasons, respectively, after damage was observed

in plots. Phytotoxicity was rated on a scale of: 0 = no phytotoxicity; 1 = light damage; 2 =

moderate damage; 3 = severe damage.









Marketable yield was determined by harvesting the fruit twice per week from 20

December, 2005 to 17 March, 2006 and 15 December, 2006 to 16 March, 2007 for a total of 25

and 27 harvests for each season respectively. Fruit was hand picked and graded on the day of

harvest. Fruit were considered marketable if there were no visible symptoms of ALS on the

calyx or other fruit rot diseases, and fruit weight for each berry was 10 g or greater.

Minimum, maximum, and average temperature and rainfall data were collected for 2005-

06 and 2006-07 seasons from the Florida Automated Weather Network (FAWN\) and total

rainfall and overhead irrigation data were collected from the GCREC weather station for

comparison to determine freeze events versus rain events. Increases in disease severity were

then compared to dates with heavy precipitation.

Data were subj ected to analysis of variance (ANOVA) and the means separated by Least

Significant Difference (LSD, P I 0.05). Statistical analyses of disease severity, disease

incidence, phytotoxicity, and yield were performed using software package Statistix 8 (Statistix

8, Tallahassee FL).

Results

2005-06 Chemical Trial

Disease severity

The severity of ALS disease was evaluated on February 10, 24, and March 14, 2006 for the

2005-06 season. An overall disease severity rating for the season was calculated by averaging

the disease severity for each treatment for the three evaluation dates. Differences in ALS disease

severity were significant for each evaluation period and for the overall season (P<0.05). Plants

treated with Actigard and Kocide 2000 at the higher rate had significantly lower disease severity

than the plants in the untreated control plots for the February 10 evaluation (P<0.05). There

were no differences between the remainder of the treatments and the untreated control. Plants









treated with Prophyt alternated with Kocide or Kocide + Tanos, Actigard, and Kocide 2000, had

significantly lower disease severity compared to plants in the untreated control plots for the

February 24 evaluation (P<0.05). There were no differences between the remainder of the

treatments and untreated control for the February 24 evaluation. Plants treated with Prophyt

alternated with Kocide or Kocide + Tanos, and Kocide 2000 had significant differences in

disease severity for the March 14 evaluation (P<0.05). There were no differences between the

remainder of the treatments and untreated control for the March 14 evaluation. Overall, plants

treated with Prophyt alternated with copper, Actigard, and Kocide 2000 had significantly less

disease than plants in the untreated control plots. Treatments with K-Phite, Prophyt alone or

alternated with Actigard or Kasumin, Actinovate, Kasumin, Serenade Max, and Kinetic were not

significantly different from the untreated control (Table 2~3).

Phytotoxicity

Phytotoxicity was evaluated on January 6 and there were significant differences between

treatments (P<0.05). All plots treated with products containing copper had some damage. Plots

treated with Actinovate plus Silicone 100, and the untreated control plots had some

phytotoxicity; however plots treated with these two products had significantly less phytotoxicity

than plots treated with copper-based products. The remaining treatments with no copper

component were not phytotoxic (Table 2~3).

Marketable weight

Differences in fruit yield were significant (P<0.05) among treatments. Plants treated with

Kinetic at 665.5 ml/ha on a 7-day schedule produced 27,516 kg/ha of fresh fruit and plants

treated with a mixture of Serenade Max, Biotune, and Kocide on a 7-day schedule produced

20,661 kg/ha fresh fruit for the highest and lowest yields per treatment, respectively. The









untreated control produced 24,291 kg/ha and was not significantly different in yield to any of the

other treatments. Therefore, control of ALS did not appear to influence yield (Table 2~3).

Temperature and rainfall

Temperature and rainfall/overhead irrigation data were analyzed from December 1 to

March 26 for the 2005-06 season. Two freeze events occurred during this season, on January 8,

2006 and February 14, 2006. Plants received 23.4 mm and 26.9 mm of overhead irrigation on

January 8 and February 14, respectively. One major rainfall event occurred during the 2005-06

season on February 3 and 4. Plants received 29.5 mm and 1 1.2 mm of precipitation, respectively

(Figure 2~1A). The freeze event and overhead irrigation coincided with an increase in disease

severity observed during the 2005-06 season (Figures 2~1A, IB).

2006-07 Chemical Trial

Disease severity

The severity of ALS was evaluated on December 28, January 25, and February 22 for the

2006-07 season for all treatments. Disease severity was also evaluated on January 12 and

February 9 for the untreated control and a few selected treatments. Differences in ALS severity

were significant (P<0.05) for each evaluation period and the overall season (Table 2~4). Plants

treated with Actigard at the higher rate, Kocide 2000, Copper-Count-N, Badge, and Kasumin +

Kocide had significantly lower disease severity than the plants in the untreated control plots for

the December 28, January 25, and February 22 evaluations, and for the overall season (P<0.05).

Plants treated with Kocide 3000 had significantly lower disease severity for the January 25

evaluation (P<0.05). Treatments with Cuprofix Ultra 40D, Kinetic, Serenade Max, and Actigard

at the lower rate, Kasumin with Captan, and Kasuran were not significantly different from

untreated controls in disease severity for the entire season (Table 2~4).









Disease incidence

Differences in disease incidence were significant among treatments (P<0.05). All treated

plants had significantly lower disease incidence than the plants in the untreated control plots

(P<0.05). Disease incidence in the untreated control plots was 77% whereas the range of disease

incidence in all other plots was between 32% and 58%. Plots treated with Badge, Kocide 3000,

Actigard at the lower rate alternated with Kocide, Kasuran, and Copper-Count-N had

significantly lower disease incidence with 32%, 37%, 39%, 41%, and 44% disease incidence,

respectively. However, those treatments were not significantly different than many of the other

treatments (Table 2~4).

Phytotoxicity

Phytotoxicity was evaluated on February 28 and there were significant differences among

treatments (P< 0.05). Plants treated with products containing copper had some damage. Plants

treated with Actigard only, Kinetic, and the untreated control had no phytotoxicity symptoms

and ratings were not significantly different from each other or from treatments with Cuprofix

Ultra, Actigard at the lower rate alternated with Kocide, Kasumin alternated with Kocide, or

Kasumin + Captan alternated with Kocide (Table 2~4).

Marketable weight

Differences in fruit yield between treatments were not significant (P=0.47). Plants treated

with Actigard applied on a 14-day schedule at 26.6 g/ha produced 28,711 kg/ha of fresh fruit and

plants treated with Kinetic at 665.5 ml/ha on a 7-day schedule produced 23,794 kg/ha fresh fruit

for the highest and lowest numerical yields per treatment, respectively. The untreated control

produced 25,375 kg/ha and was not significantly different in yield to any other treatment (Table

2~4).










Temperature and rainfall

Temperature and rainfall/overhead irrigation data were analyzed from December 1 to

March 26 for the 2006-07 season. One freeze event occurred during this season on February 17,

2007. Plants received 13.5 mm of overhead irrigation. Nine major rainfall events occurred

during the 2006-07 season on the days of December 23 and 25, January 3, 24, 25, and 28,

February 2 and 13, and March 16 when plants received 11.2, 30.5, 10.7, 18.0, 15.5, 19.6, 25.1,

16.5, and 12.4 mm of precipitation, respectively (Figure 2~2A). A major increase in disease for

the 2006-07 season coincided with the large amount of rainfall and low temperatures in late

December. These optimal conditions for ALS were associated with an increase in disease

severity from approximately 2.5 to approximately 4.5 in period of one month (Figures 2~2A,

2B).

Discussion

This study demonstrated that suppression of ALS is possible with the use of some

products. In the 2005-06 season, disease suppression was achieved mostly with products that

contained copper, which was the case in previous studies where copper was used to control

bacterial diseases (20, 21). However, Actigard, a non-copper based product that induces systemic

acquired resistance, also suppressed ALS, and has controlled bacterial spot of tomato (41).

Disease was suppressed most effectively with the higher rate of Kocide 2000, but the

disadvantage of this treatment was that it was also phytotoxic. Prophyt treatments when

alternated with copper had disease severity indexes lower than untreated controls, but treatments

with Prophyt alone did not achieve control. The reduction of disease in the Prophyt/copper

treatments was probably a result of the copper, and therefore these treatments were not included

in the 2006-07 trial. Actigard on the other hand, contained no copper and suppressed ALS as









effectively as the lower rate of Kocide 2000. The biocontrol agents had no effect on ALS, and

Kinetic, a surfactant which was thought might actually increase disease, had no effect.

The 2006-07 trial included some additional copper treatments such as basic copper sulfate

and copper oxychloride, but copper hydroxide (Kocide 2000) at the high rate was not evaluated

again because of its tendency to be phytotoxic. Additional Actigard treatments, including

different rates alternated with copper, were added, and additional antibiotic treatments with

higher rates of Kasumin alternated or mixed with copper were added. The additional treatments

did not result in any new outcomes. The effective treatments remained copper, Actigard, and

Kasumin with copper. Effective copper treatments included copper hydroxide and copper

oxychloride + copper hydroxide; however, treatments with copper sulfate were not effective.

Treatments with Kasumin were effective; however, these treatments included a copper

component and it is likely that efficacy may be due to the copper rather than the antibiotic.

Actigard alone again suppressed disease as effectively as the lower rate of copper. At the end of

the 2006-07 season, disease incidence was evaluated to determine if disease assessment could be

improved. This evaluation proved successful for differentiating all treatments from the untreated

control. However, disease incidence did not correlate to disease severity ratings and this

assessment would need to be refined for field use throughout the season.

The use of copper for treatment of ALS is of concern to growers because copper is

phytotoxic to strawberry plants. Every treatment containing copper for both seasons showed at

least some phytotoxicity. Two treatments, that did not even include copper, had trace amounts of

phytotoxicity probably from spray drift due to their proximity to treatments receiving copper.

Plants that were treated with Kocide at the lower rate had half as much damage as plants treated









with the higher rate. Actigard was the only treatment that provided disease suppression without

phytotoxic side effects.

The data on the combined effect of ALS and the management of ALS on yield were

inconclusive. In some cases, there were significant differences in marketable yield between

treatments, but there were no significant differences in yield between treated and untreated plants

for the 2005-06 season. Plants treated with copper at the higher rate had the most phytotoxicity

and also had yields similar to plants that were not treated with copper and had no phytotoxicity.

Thus, phytotoxicity did not seem to affect yield. The 2006-07 season was similar in that yield

differences occurred between a few treatments, but none of the treatments differed from the

untreated control. In the 2006-07 season, plants treated with Actigard alone at the lower rate had

a significantly lower incidence of disease than untreated plants, but there was no significant

difference in marketable yield between the two groups of plants. Contrary to results obtained in

tomato (41), it is possible that resistance to ALS in strawberry, whether inherent in the cultivar or

artificially induced, could come at a cost to yield. This study also demonstrated the impact that

temperature and precipitation or overhead irrigation had on the epidemiology of ALS as

previously described (25, 37). Two freeze events occurred during the 2005-06 season during

which overhead irrigation was used to protect the crop. The first freeze event occurred on

January 8. A month later the first evaluation was conducted, but by this time, ALS had become

epidemic. In 2006-07, however, the first evaluation was conducted much earlier in the season,

when disease severity was relatively low. Four days prior to the first evaluation there was a

maj or precipitation event which coincided with an extreme drop in temperature. This was not a

freeze event, but conditions for development of an ALS epidemic were optimal. About four









weeks after the temperature drop and rain, disease severity had almost doubled. Levels of

disease severity were similar to those in the 2005-06 season four weeks after a freeze event.

Despite an increasing number of products labeled for control of ALS, this disease will be

difficult to control if conditions are optimal for dissemination of the pathogen. Evaluations of

disease incidence rather than disease severity may help to more accurately assess the level of

control being achieved since disease incidence is an obj ective evaluation whereas disease

severity is a subj ective evaluation. Based on the information that was gathered in this study,

Actigard is a good alternative to copper for suppressing ALS in strawberry and providing a level

of control similar to copper. There is no phytotoxicity associated with Actigard so plants are not

suffering from efforts to manage the disease. Unfortunately, while Actigard appeared to have no

negative effect on yield, in this study there was also no positive effect on yield compared to

untreated control plants. One possibility for a lack of observable differences in yield may be that

plot sizes were too small. Another possibility may be that although differences in disease

severity were significant, differences were not enough to reflect differences in yield.









Table 2~1. Treatments and application schedules for control of angular leaf spot in annual strawberry for the 2005-06 season in
Florida

Treatment (active ingredient) (rate/ha) Schedule
Control
K-Phite (phosphorous acid) (5.8 L) 14-day
Prophyt (potassium phosphite) (5.8 L) 14-day
Prophyt (potassium phosphite) (5.8 L) alt. Actigard (acibenzolar-S-methyl) (53.2 g) 7-day
Prophyt (potassium phosphite) (5.8 L) alt. Kasumin (kasugamycin) (1.2 L) 7-day
Prophyt (potassium phosphite) (5.8 L) alt. Kocide 2000 (copper hydroxide) (1.7 kg) 7-day
Prophyt (potassium phosphite) (5.8 L) alt. [Kocide 2000 (copper hydroxide) (1.7 kg) + Tanos (famoxadone +
cymoxanil) (567 g)] 7-day
Actinovate (Streptomyces lydicus)(850.5 g) + Silicone 100 (295.8 ml) 7-day
Actigard 50WG (acibenzolar-S-methyl) (53.2 g) 14-day
Kasumin (kasugamycin (1.2 L) 14-day
SKocide 2000 (copper hydroxide) (0.9 kg) 7-day
Kocide 2000 (copper hydroxide) (1.7 kg) 7-day
Serenade Max (Bacilhts subtilis) (1.1 kg) + Biotune (sodium lauryl sulfate, sodium dodecylbenzene sulfonate, and
polyoxyethylene (20) sorbitan monooleate) (2.9 L) 7-day
Serenade Max (Bacilhts subtilis) (1.1 kg) + Biotune (sodium lauryl sulfate, sodium dodecylbenzene sulfonate, and
polyoxyethylene (20) sorbitan monooleate) (2.9 L) + Kocide 2000 (copper hydroxide) (0.9 kg) 7-day
Kinetic (organosilicone) (665.5 ml) 7-day









Table 2~2. Treatments and application schedules for control of angular leaf spot in annual strawberry for the 2006-07 season in
Florida

Treatment (active ingredient) (rate/ha) Schedule
Control
Kocide 2000 (copper hydroxide) (0.9 kg) 7-day
Kocide 3000 (copper hydroxide) (= GFJ52) (1.0 kg) 7-day
Cuprofix Ultra 40D (copper sulfate) (0.8 kg) 7-day
Copper-Count-N (copper ammonia complex) (2.4 L) 7-day
Badge (copper hydroxide + copper oxychloride) (1.1 L) 7-day
Badge (copper hydroxide + copper oxychloride) (2.1 L) 7-day
Kinetic (organosilicone) (665.5 ml) 7-day
Serenade Max (Bacillus subtilis) (1.1 kg) + Biotune (sodium lauryl sulfate, sodium dodecylbenzene sulfonate, and
polyoxyethylene (20) sorbitan monooleate) (1.2 L) + Kocide 2000 (copper hydroxide) (0.9 kg) 7-day
Actigard 50WG (acibenzolar-S-methyl) (26.6 g) 14-day
Actigard 50WG (acibenzolar-S-methyl) (26.6 g) alt Kocide 2000 (copper hydroxide) (0.9 kg) 7-day
w Actigard 50WG (acibenzolar-S-methyl) (53.2 g) 14-day
Actigard 50WG (acibenzolar-S-methyl) (53.2 g) alt Kocide 2000 (copper hydroxide) (0.9 kg) 7-day
Kasumin (kasugamycin) (295.8 ml = 100 ppm) alt. Kocide 2000 (copper hydroxide) (0.9 kg) 7-day
Kasumin (kasugamycin) (295.8 ml) + Kocide 2000 (copper hydroxide) (0.9 kg) alt Kocide 2000 (copper hydroxide)
(0.9 kg) 7-day
Kasumin (kasugamycin) (5.8 L) + Captan 80WDG captain ) (1.7 kg) alt Kocide 2000 (copper hydroxide) (0.9 kg) 7-day
Kasuran (kasugamycin + copper oxychloride) (2.0 kg = 100 ppm) 7-day










Table 2~3. Severity of angular leaf spot, phytotoxocity, and market weight of annual strawberry for the 2005-06 season in response to
the application of various spray materials.


Mkt Wt
(kg/ha)
24291abcd
25678abc
27054ab
27097ab
26474ab
22355cd
26528ab
26671ab
23603bcd
25516abc
24611abc
21928cd
21993cd
20661d
27516a


DSI z1
4.94abx
4.38abc
5.11ab
4.56ab
4.41abc
4.32bc
4.60abc
5.12ab
3.85cd
4.94ab
3.88ab
3.18d
5.04ab
4.88ab
5.19a


Phytotox
0.50de
0.00e
0.00e
0.00e
0.00e
1.75b
1.00cd
0.25e
0.00e
0.00e
1.25bc
2.50a
0.00e
1.00cd
0.00e


Treatment
Control
K-Phite (5.8 L)
Prophyt (5.8 L)
Prophyt (5.8 L) alt. Actigard (53.2 g)
Prophyt (5.8 L) alt. Kasumin (1.2 L)
Prophyt (5.8 L) alt. Kocide 2000 (1.7 kg)
Prophyt (5.8 L) alt. [Kocide 2000 (1.7 kg) + Tanos (567 g)]
Actinovate (850.5 g) + Silicone 100 (295.8 ml)
Actigard 50WG (53.2 g)
Kasumin (1.2 L)
SKocide 2000 (0.9 kg)
Kocide 2000 (1.7 kg)
Serenade Max (1.1 kg) + Biotune (2.9 L)
Serenade Max (1.1 kg) + Biotune (2.9 L) + Kocide 2000 (0.9 kg)
Kinetic (665.5 ml)


D SI2
5.44abc
5.32abcd
5.66ab
5.16bcde
5.47abc
4.85def
4.85def
5.78a
4.79ef
5.34abcd
4.38fg
4.03g
5.75a
5.12cde
5.69a


DSI3
6.03abc
5.69bcd
6.17ab
5.56cde
5.88abc
5.32de
5.07e
6.37a
5.50cde
5.94abc
5.29de
4.47f
6.25ab
5.89abc
6.19ab


D SIAvg
5.47abc
5.13bcde
5.64ab
5.09cde
5.25abcd
4.83def
4.83def
5.76a
4.71ef
5.41abc
4.51f
3.89g
5.68a
5.29abcd
5.69a


z DSI disease severity index on a scale of 0 = none to 6 = severe. Evaluations were conducted on 2/10, 2/24, and 3/14/2006;
Phytotoxicity rated on a scale of 0 = none to 3 = severe; x Treatment means within columns followed by the same letter are not
significantly different by Fisher's protected LSD (P<0.05)










Table 2~4. Severity of angular leaf spot, phytotoxocity, and market weight of annual strawberry for the 2006-07 season in
response to the application of various spray materials.


Dis
incidence"
77a
49bcde
37efg
45cdef
44cdefg
36fg
32g
48bcdef

56bc
53bcd


Mkt Wt
(kg/ha)
25375abc
28030ab
25547abc
26016abc
26038abc
27482ab
24860bc
23794c

27086abc
28711a

27456ab
26595abc


Treatment
Control
Kocide 2000 (0.9 kg)
Kocide 3000 (1.0 kg)
Cuprofix Ultra 40D (0.8 kg)
Copper-Count-N (2.4 L)
Badge (1.1 L)
Badge (2.1 L)
Kinetic (665.5 ml)
Serenade Max (1.1kg) + Biotune (1.2L) +
Kocide 2000 (0.9 kg)
Actigard 50WG (26.6 g)
Actigard 50WG (26.6 g) alt Kocide 2000 (0.9
w kg)
Actigard 50WG (53.2 g)
Actigard 50WG (53.2 g) alt Kocide 2000 (0.9
kg)
Kasumin (295.8 ml = 100 ppm) alt. Kocide
2000 (0.9 kg)
Kasumin (295.8 ml) + Kocide 2000 (0.9 kg) alt
Kocide 2000 (0.9 kg)
Kasumin (5.8 L) + Captan 80WDG (1.7 kg) alt
Kocide 2000 (0.9 kg)
Kasuran (2.0 kg = 100 ppm kasugamycin + Cu)


DSI z1
2.35ax
1.00ef
1.91abcd
1.88abcd
1.44cdef
1.47cdef
1.44cdef
2.22ab

2.22ab
1.97abc


D SI2
4.28a
3.16e
3.54bcde
3.78abcde
3.47bcde
3.44cde
3.22de
4.10ab

4.00abc
3.85abcd


DSI3
4.50a
3.79f
4.25abcde
4.40abc
4.03bcdef
4.00cdef
3.88ef
4.35abcd

4.54a
4.44ab


DSIAvg
3.71a
2.65f
3.23abcde
3.56abcd
2.98cdef
2.97cdef
2.85def
3.56ab

3.58ab
3.42abc


Phytotox
0.00d
1.00bc
1.00bc
0.50cd
1.25ab
1.00bc
1.75a
0.00d

1.25ab
0.00d

0.25d
0.00d


1.82abcd 3.85abcd 4.38abc 3.35abcd 39efg
0.88f 3.28de 3.81f 2.66f 53bcd


1.63abcde 3.41cde 4.00cdef 3.0125cdef

1.50bcdef 3.69abcde 4.17abcdef 3.12bcdef


45cdef

47bcdef

45cdef


1.00bc 25331abc


0.25d


25461abc


1.22def


3.28de


3.94def 2.82ef


1.00bc 25983abc

0.25d 25780abc
1.50ab 27661ab


1.63abcde 3.85abcd 4.38abc 3.28abcde 58b
1.88abcd 3.85abcd 4.28abcde 3.34abcde 41defg


" Percentage of disease incidence on a total of 120 randomly collected leaves for each treatment. Evaluation was conducted on
3/31/2007; x Treatment means within columns followed by the same letter are not significantly different by Fisher' s protected LSD
(P<0.05); = Phytotoxicity rated on a scale of 0 none to 3 severe; z DSI disease severity index on a scale of 0 = none to 6 =
severe. Evaluations were conducted on 12/28/2006, 1/25, and 2/22/2007.














35.0 25.0


30.0
-20.0

25.0-


20.0 -5.


15.0
o 10.0


10.0 -

-5.0




0 0 .














a04
















Figure 2~1. Environmental conditions and disease severity for 2005-06. A) Total
rainfall/overhead irrigation (mm, vertical bars) and average daily temperature (oC,
line) during the 2005-06 annual strawberry season for evaluation of products for
control of angular leaf spot. Arrows indicate freeze events when overhead irrigation
was used. B) Angular leaf spot disease severity index on leaves of control plots of
cultivar Strawberry Festival over time in 2005-06 annual strawberry season.














35.0 30.0


30.0 ----2 .
25.0 -

20.

20.00 -
=- 15.
15.00 -

-5. 100

10.0


5.0 -5.0




5.






4.5


3.5

~3-

~2.5

2-
a 1.5


0.5

0






Figure 2~2. Environmental conditions and disease severity for 2006-07. A) Total
rainfall/overhead irrigation (mm, vertical bars) and average daily temperature (oC,
line) during 2006-07 annual strawberry season for evaluation of products for control
of angular leaf spot. Arrow indicates freeze event. B) Angular leaf spot disease
severity rating for control plots of cultivar Strawberry Festival over time in 2006-07
annual strawberry season.









CHAPTER 3
RESISTANCE OF FLORIDA STRAWBERRY CULTIVARS TO ANGULAR LEAF SPOT

Introduction

Angular Leaf Spot (ALS) of strawberry, caused by Xanthomona;s fra~gariae, is a bacterial

disease of strawberry first observed in Minnesota in 1959 and subsequently reported in 1962 by

Kennedy and King (24). ALS now occurs worldwide and it is thought that the shipment of

infected asymptomatic tissue is responsible for its rapid distribution (37). Symptoms of ALS as

described by Kennedy and King (24) are lesions on the abaxial surface of the leaf that are green,

angular, and water-soaked. Lesions are translucent and easily distinguishable from healthy

tissue. Diagnosis of ALS in the early stage, before lesions become necrotic, is easily done by

observing translucent lesions with transmitted light. Once necrosis occurs, diagnosis becomes

more difficult as symptoms can be confused with other foliar diseases such as common leaf spot

caused by the fungal pathogen M\~ycosphaerella fragag~riae (24).

ALS is primarily a foliar pathogen of strawberry plants; however infection can occur in the

parenchymatous tissue of the calyx. Strawberry fruit itself is not parasitized; however, if

infection of the calyx is severe enough, the tissue can become necrotic making fruit

unmarketable as fresh fruit (31, 43). Less commonly, X. fragariae can infect crowns of

strawberry plants resulting in a vascular infection (4, 14, 37). In vascular infections, lesions are

concentrated around vascular tissue rather than distributed randomly over leaf surface. At the

time of Kennedy and King's first report of ALS in strawberry, X fCragariae was not thought to

infect vascular tissue; however, their report contains images of leaves with vascular infection as

well as parenchymatous tissue infection (24).

ALS is favored by high humidity and low temperatures; conditions common to Florida

strawberry fields in the winter months. Temperatures are relatively high in the daytime









frequently reaching 18 to 22 OC and nights are cool, with temperatures dropping to between 0

and 5 oC at times. These conditions result in high surface moisture on plants permitting

colonization by the pathogen. Optimal conditions make this disease very difficult to control.

Management of ALS requires an integrated approach. The easiest way to control ALS is

to use pathogen-free stock (37). Unfortunately, clean plants are not always available, and plants

infected with X fCragariae are commonly asymptomatic and escape detection until favorable

conditions occur. Limiting overhead irrigation and working in Hields after plants are dry is useful

for limiting the spread of inoculum in Hields, however certain times require overhead irrigation in

Florida Hields. Strawberry is planted in early to mid-October and mid-day temperatures are high.

Overhead irrigation is necessary to establish plants in the field before they receive drip irrigation.

Additionally, strawberry is a winter crop in Florida and air temperatures can drop below freezing

requiring overhead irrigation to protect the flowers of the plants from freezing.

Chemical application of copper compounds has previously been effective (48), however

the use of copper products is limited since copper is phytotoxic to strawberry plants (43, 48).

Other spray materials were evaluated in this thesis research and the SAR product acibenzolar-S-

methyl (ASM) was effective in reducing disease severity compared to untreated control without

being phytotoxic to the plants.

Resistant cultivars would be useful for management of ALS; however no resistant cultivars

have been developed. Kennedy and King (25) evaluated 64 cultivars with X fCragariae to

observe reactions to ALS and Hildebrand et al. (15) investigated the factors affecting infection

and cultivar reaction for 22 cultivars. All cultivars in those studies were susceptible to ALS, and

only two, Sweet Charlie and Chandler, are used for production in Florida. Compared to the other

cultivars, Sweet Charlie and Chandler were among the most susceptible in Hildebrand's study










(15). Maas et al. (3 8) identified two small-fruited genotypes, a native E. virginiana and a E

virginian2a x xanana~ssa hybrid, that are highly resistant to ALS, and a recent study by Lewers

et al. (32) has led to understanding the number of genes involved and the heritability of

resistance to ALS, but further research with larger populations is needed to develop cultivars

with resistance and other desirable traits.

There is speculation from growers that among cultivars grown in Florida the currently

most popular cultivar Strawberry Festival is more susceptible than others. However, there are no

data available to determine if differences in ALS resi stance/susceptibility among cultivars

currently grown in Florida exist. The objective of this study was to compare resistance to ALS

in cultivars of strawberry currently produced in Florida and four advanced selections from a

breeding program, and determine the effect ALS has on yield.

Materials and Methods

Field experiments to evaluate different strawberry cultivars for resistance to ALS were

conducted at the University of Florida Gulf Coast Research and Education Center in Wimauma,

Florida during the 2006-07 growing season in fields managed using current conventional

strawberry farming practices. Between October 12 and October 25, 2006, bare-root transplants

of the cultivars Albion, Camarosa, Camino Real, Carmine, Strawberry Festival, Ruby Gem,

Sugar Baby, Sweet Charlie, Treasure, Winter Dawn, and advanced selections Festival #9, FL 99-

117, FL 99-164, FL 00-51, and FL 01-116 (Table 3~1) were planted in raised, fumigated beds

covered with black plastic mulch. Soil had been fumigated with methyl bromide and

chloropicrin at a ratio of 67:33 at 397 kg/ha. Centers of the beds were 1.2 m apart and beds were

0.7 m wide. Two rows of staggered plants were planted in each bed. Plants were spaced 28 cm

in the row with 38 cm between the rows. Space between plots for each treatment was 1.1 m.

Overhead irrigation was applied for 10 days to establish transplants and then plants were










irrigated and fertilized by drip tape. Treatments were arranged in a randomized complete block

design with four replications in successive beds. Plots for each treatment contained 12 plants

and were 2.9 m long.

Disease incidence of plants naturally infected was evaluated on April 6, 2007. Based on

the results from the products evaluations, disease incidence provided a better separation among

treatments than disease severity. Thus, disease incidence was selected for this cultivar

evaluation. Five leaves were collected from six plants in each plot, for a total of 30 leaves or 90

leaflets. Marketable yield was determined by harvesting fruit twice per week for each plot. Fruit

was hand picked and graded twice per week from 12 December, 2006 to 30 March, 2007 for a

total of 32 harvests. Fruit were considered marketable if there were no visible symptoms of ALS

on the calyx or other fruit rot diseases, and fruit weight for each berry was 10 g or greater. Data

were subj ected to analysis of variance (ANOVA) and the means were separated using Fisher' s

Protected Least Signifieant Difference (LSD, P10.05). Statistical analyses of disease incidence

and yield were performed using software package Statistix 8 (Statistix 8, Tallahassee FL).

Results

Cultivar Evaluation

Differences in cultivar susceptibility were significant (P<0.05) with the range of percent

disease incidence from 21% to 75%. One cultivar, Treasure, had a significantly lower disease

incidence compared to all other cultivars with 21% disease incidence (Table 3~1). The

genotypes with the highest disease incidence were advanced selections FL 99-117 and FL 01-

116 with 75% and 70% disease incidence, respectively, and cultivars Winter Dawn and

Camarosa with 74% and 68% disease incidence, respectively (Table 3~1). Nine cultivars had

disease incidences that were not significant from each other, but were significantly different from

cultivars with the highest or lowest disease incidence. These included 'Camino Real', FL 99-










164, Festival #9, 'Sweet Charlie', 'Albion', 'Strawberry Festival', 'Sugar Baby', 'Carmine', and

FL 00-51 (Table 3~1).

Marketable Weight

Differences in fruit yield between cultivars were significant (P<0.05) with a range of

17,976 kg/ha to 41,112 kg/ha. Cultivars producing the most fruit were Strawberry Festival,

Camarosa, Camino Real, and Ruby Gem with 41,112, 39,811, 38,403, and 37,938 kg/ha,

respectively, and differences between these four were not significant. 'Sugar Baby' and Festival

#9 produced the least amount of fruit with 17,976 kg/ha and 19,350 kg/ha, respectively, and

differences between these two were not significant. Among treatments that produced slightly

more fruit than 'Sugar Baby' and Festival #9 were advanced selections FL 01-116, FL 99-117,

and FL 00-51 which produced 24,739, 24,790, and 24,988 kg/ha respectively. 'Albion', 'Winter

Dawn', 'Sweet Charlie', 'Treasure', FL 99-164, and 'Carmine' had significantly higher yields

than 'Sugar Baby', Festival #9, FL 01-116, FL 99-117, and FL 00-51 but also had significantly

lower yields than 'Strawberry Festival', 'Camarosa', 'Camino Real', and 'Ruby Gem' (P<0.05).

The incidence of ALS was not related to yield (P=0.08). 'Treasure', the most resistant cultivar,

produced 34,098 kg/ha of fruit which was only slightly higher than, and not significantly

different from the most susceptible cultivar, Winter Dawn, which produced 31,346 kg/ha. In

comparison, the moderately susceptible cultivar Strawberry Festival produced the highest yield

of 41,1 12 kg/ha. Differences in yield could not be attributed to ALS (Table 3~1).

Discussion

This study demonstrates that all cultivars of strawberry grown in Florida are susceptible

to ALS; however, the degree of susceptibility varies greatly. ALS susceptibility among Florida

cultivars can differ by as much as 50%, as was observed between 'Treasure' and 'Winter Dawn'.

No cultivars of strawberry are immune to ALS and for purposes of this discussion, cultivars are










placed in one of three groups: cultivars that are mildly, moderately, or highly susceptible to

ALS. Previous studies have evaluated susceptibility in a number of cultivars, however only two,

'Sweet Charlie' and 'Chandler' are currently used in Florida (25, 15). Hildebrand et al. (15)

compared the reactions of 22 cultivars of strawberry to ALS. 'Chandler' and 'Sweet Charlie'

were included in that study and compared to the other cultivars in that trial, they were highly

susceptible. However, compared to cultivars used in Florida, 'Sweet Charlie' is only moderately

susceptible. Although 'Chandler' is not used as much as other current cultivars, and although it

was not included in this study, it is likely that 'Chandler' would perform similarly to 'Sweet

Charlie' based on the similar susceptibilities reported by Hildebrand et al. (15).

Strawberry Festival' is currently the most popular cultivar of strawberry used in Florida.

One reason for the popularity of 'Strawberry Festival', apart from desirable consumer traits, is its

high potential yield. Although popular, growers have speculated that Strawberry Festival' is

highly susceptible to ALS and those questions have been addressed in this study. 'Strawberry

Festival' is a moderately susceptible cultivar compared to the other cultivars grown in Florida,

with a disease incidence rating of 58.3%. Despite a moderate disease incidence rating,

' Strawberry Festival' produced the highest total yield of all the cultivars tested, slightly over

7000 kg/ha more than 'Treasure', the cultivar most resistant to ALS in our study.

The results from this study are not conclusive to indicate if ALS significantly impacts yield

among cultivars with different levels of susceptibility. Differences in yield and disease incidence

were significant between cultivars, however there was no correlation observed between disease

incidence and yield. One explanation for the lack of differences in yield among cultivars with

different levels of susceptibility to ALS is that other important diseases of strawberry also

reduced yield in certain cultivars. 'Treasure' and 'Camarosa' are highly susceptible to









anthracnose fruit rot, caused by Colletotrichum acutatum, and 'Sweet Charlie' and 'Camino

Real' are highly susceptible to gray mold, caused by Botrytis cinerea. These two diseases

combined caused losses of 13.1, 8.6, 3.8, and 12.0% of the total number of berries harvested

from each of these cultivars, respectively (data not shown). In contrast, 'Winter Dawn' and

'Carmine' lost less than 2.0% of the fruit to anthracnose and gray mold. Thus, in addition to

natural differences in yield usually observed among cultivars, variable losses due to other

diseases were observed making it difficult to correlate differences in yield to ALS. In addition,

ALS is a foliar disease and losses are much more difficult to quantify than losses due to fruit

diseases because they are not a direct result of infection. In addition, certain cultivars reach peak

blooms at different points during the season. 'Winter Dawn' for example is typically planted

early because it produces very quickly and has its peak production before Strawberry Festival',

which has a more steady production throughout the season and usually peaks in late

February/March. ALS was evaluated at the end of the season. It may be of interest to see what

levels of disease exist at peak production times for different cultivars. Perhaps an experiment

comparing plants infected with ALS and plants free of ALS would accurately evaluate the effect

of ALS on yield; however, maintaining plots free of ALS in close proximity to plots infected

with ALS have been proven to be very difficult.

Based on these findings, it is clear that ALS affects cultivars differently, but, under the

conditions that the plants were exposed to during the 2006-07 season, the differences were not

dramatic enough to have a significant impact on yield. Strawberry Festival' is a good choice

based on marketability and it also produces high yields regardless of moderate levels of disease

incidence. Efforts to identify resistance while maintaining desirable consumer traits and high

yield are not complete, but recent work focused on obtaining a cultivar with all these traits has









been promising. Of the four advanced selections from a breeding program, one was only mildly

susceptible. Advanced selection 99-164, with a disease incidence rating of 48.3%, had a low to

moderate susceptibility and had numbers similar to 'Treasure' with respect to yield. Recently,

Lewers et al. (32) determined that three to four unlinked loci are involved in conferring ALS

resistance. They concluded resistant progeny could be selected; however, studies with large

populations would be necessary to produce cultivars that were not only resistant to ALS, but

were also good producers of quality fruit. The potential for a resistant cultivar is on the horizon

and given time, the combination of a resistant cultivar with high yield and quality fruit is a

reasonable possibility










Table 3~1. Disease incidence of angular leaf spot in cultivars of annual strawberry in Florida for
2006-07 season.

Disease Incidence Mkt Wt
Cultivar/Selection (%) (kg/ha)
Albion 55.8cdx 29011ef
Camarosa 68.3abc 39811ab
Camino Real 47.5de 38403abc
Carmine 61.8abcd 35289bcd
Strawberry Festival 58.3bcd 41112a
Festival #9 51.0d 19350g
Ruby Gem 32.5ef 37938abc
Sugar Baby 60.0abcd 17976g
Sweet Charlie 55.8cd 31564de
Treasure 20.8f 34098cd
Winter Dawn 74.3ab 31346de
99-117 75.0a 24790f
99-164 48.3de 34897cd
00-51 64.0abcd 24987f
01-116 70.0abc 24739f
x Means within columns followed by the same letter are not significantly different by Fisher' s
protected LSD (P<0.05)









CHAPTER 4
ASSOCIATION OF ANGULAR LEAF SPOT LESIONS AND Pseudomona~s spp. WITH
SYMPTOMATIC TISSUE OF STRAWBERRY.

Introduction

Angular Leaf Spot (ALS), caused by Xanthomona;s fragariae, has been described by

Kennedy and King as dark green angular water soaked lesions on the under side of the leaf (24).

Under optimal conditions these lesions can increase in size and number, and in early mornings

when surface moisture from dew or rain is abundant, a mass of bacterial exudate appears on the

under surface covering the water soaked lesions (24, 37, 8, 36, personal observation). The

function of this exudate, whether it may be for survival during unfavorable conditions or

plugging vascular tissues, is not known (37). Eventually lesions become necrotic and are more

difficult to differentiate from other diseases such as common leaf spot caused by M~ycosphaerella

fragariae.

Recently, detection of X. fragariae has improved by using serological and molecular

techniques (49, 47, 45), however; isolation of the pathogen is still difficult. Isolation ofX.

fragariae requires 3 to 5 days incubation at 20 to 240 C on a suitable medium such as Wilbrink' s

medium. The morphology and growth characteristics make direct isolation difficult as growth of

the bacterium is slow and faster growing saprophytes mask the growth ofX. fragariae in culture

plates (7, 49). Colonies of X. fragariae are minute and translucent rather than yellow at the early

stages of growth.

During the 2005-06 and 2006-07 strawberry seasons, numerous tissue isolations were

performed to obtain X. fragariae strains. Isolations were made under optimal conditions when

lesions were oozing and symptoms were easily identifiable as ALS; however, Pseudomona~s spp.

were consistently isolated from the lesions. We report on the association of Pseudomona~s spp.

with angular leaf spot lesions from symptomatic tissue on strawberry.












Tissue Isolations

During the 2005-06 and 2006-07 strawberry seasons, leaf samples from 'Strawberry

Festival', 'Winter Dawn', and 'Sweet Charlie' were collected from fields at the GCREC, and

leaf samples from breeding advanced selections 99-117, 01-116, 97-39, and 99-164 were

collected from a farm in Floral City, FL. Tissue isolations were performed by dissecting a small

piece of tissue containing one lesion (~2mm x 2mm) that exhibited visual oozing when viewed

under the stereoscope and placing the lesion in a 1.5 ml microcentrifuge tube with 200C1l to 1 ml

of sterile water or 0.01 M magnesium sulfate solution (MgSO4-7H20). Tissue was macerated in

tubes with a microcentrifuge pestle and the suspension was streaked on nutrient agar or

Wilbrink' s media or both. Plates were incubated at 24 to 280 C for 24 to 72 h. Individual

colonies were transferred to culture plates to isolate pure cultures.

Hypersensitive Response Tests

Selected strains ofPseudomona~s spp. isolated from field samples and a strain of

Xanthomona;s fragariaea (ATCC 33239) were tested to determine if they induced a hypersensitive

response in tomato and tobacco. The fluorescent pseudomonads and X. fragariae strains were

grown on nutrient agar for 24 hours and on Wilbrinks medium 48 to 72 hours, respectively. The

bacteria were suspended in sterile tap water. Mature tobacco leaves and four week old tomato

plants were infiltrated with a water control or bacterial suspensions adjusted to 0.10oD at 590 nm.

Plants were examined 16 to 48 hours later for a hypersensitive response.

Fatty Acid Analysis

A single colony of each strain was transferred from a nutrient agar culture to trypticase soy

broth agar, and after 24 h of growth at 280C approximately 40 mg of cells was collected for

analysis. The bacterial fatty acids were derivatized to their methyl esters, separated by gas


Materials and Methods










chromatography, and identified using the Microbial Identifieation System software (version 3.6;

MIDI, Newark, DE).

Oxidase Test

Cultures identified as fluorescent Pseudomona~s spp. by fatty acid analysis were tested for

indophenol oxidase production. Pseudomona~s spp. were incubated overnight and tested with

oxidase reagent droppers (Becton, Dickinson and Company; Sparks, MD.)

Ice Nucleation

Ice nucleation activity was tested for the fluorescent Pseudomona~s spp. isolated from Hield

samples and Pseudomona~s spp. received from tissue isolations from California, North Carolina,

and Vermont. Suspensions were prepared from 24 h cultures grown on NA and adjusted to

A590=0.1. Aluminum weigh dishes were placed on the surface of the alcohol/ice mixture with

the temperature at approximately -100C and 10 drops of suspension from each isolate were

placed in the aluminum dish. The number of drops that froze after 1 minute was recorded for

each strain.

Pathogenicity Tests

'Strawberry Festival' and 'Sweet Charlie' plants obtained from a region free of ALS in 6-

inch pots were inoculated with suspensions of X. fragariae, Pseudomona~s spp., a mixture of both

species, or water control (sterile tap water or MgSO4 7H20). Suspensions were prepared and

diluted to A590=0. 1 from 24 h Pseudomona~s spp. cultures, and 72 h X. fragariae cultures. Two

groups with two plants per treatment, one set left at room temperature and the other placed in a

growth chamber, were inoculated and placed in plastic bags immediately following inoculation.

The growth chamber was set to temperature fluctuation of 30C to 180C and a 12 hour

photoperiod. Plants were observed daily for symptoms after one week post-inoculation.









Results and Discussion


Tissue Isolations

Of the numerous leaf samples collected to isolate the causal agent of ALS of strawberry,

none resulted in plates that produced a pure culture of X. fragariae. Interestingly, many of the

plates contained almost pure cultures of white colonies, and on Wilbrink' s media, these same

colonies produced a green pigment. It appeared that these cultures consistently isolated from

leaves with symptoms of ALS were Pseudomona~s spp. There were six samples from the

GCREC and 11 samples from Floral City (Table 4~1). During this same time, we also received

1, 4, and 10 strains isolated from symptomatic ALS lesions from North Carolina, Vermont, and

California, respectively, that appeared to be Pseudomona~s spp. (Table 4~1).

Characterization of Strains

Isolates 13-15, 27-29, and 31-36 were all identified as Pseudomona~s spp. by fatty acid

analysis (Table 4~1). Those same isolates were evaluated to see if they induced hypersensitive

responses (HR) in tomato and the oxidase reaction. All but one of the strains, number 33, failed

to induce an HR or induced only a weak HR. Oxidase production was weak in most strains

tested. Initially these strains tested slightly oxidase positive but eventually turned negative. Two

strains, numbers 33 and 36, were oxidase negative. Ice nucleation activity was tested for several

strains. Strains 33 from Floral City, 52 and 54 from Vermont, and strains 70 and 71 from

California all exhibited strong ice nucleation activity.

Pathogenicity Test

Pathogenicity tests for all Pseudomona~s spp. strains were negative (Table 4~1). As a

result of isolating almost exclusively Pseudomona~s spp. from the tissue samples, we began to

suspect some type of interaction or association between lesions of ALS caused by X. fragariae

and these Pseudomona~s spp.









Bacteria employ a number of strategies to successfully colonize host plants and more

specifically, epiphytes are bacteria that can colonize and survive on plant surfaces (2).

Pseudonzona~s syringe is a well known pathogen of many crops, but it has also been reported as

an epiphyte and can exist as aggregates on surfaces of leaves where scarce nutrients are available

(6, 12, 27, 23). For example, some P. syringe strains produce indole-3-acetic acid (IAA), which

is capable of inducing nutrient leakage on the surface of plants thus making nutrients available

for survival (2). Many bacteria produce extracellular polysaccharides which can modify the

environment by helping cells adhere to leaf surfaces and preventing desiccation of cells (2).

Frequently, a mass of bacterial exudate is associated with ALS lesions during periods of high

humidity.

Another mechanism that can assist bacteria in colonization is ice nucleation. During freeze

events, ice nucleation active bacteria can injure leaf surfaces increasing frost damage to plants

which may provide more openings for pathogenic bacterial ingress (6, 34, 53). Given the fact

that some of the P. syringe strains associated with strawberry were ina+, it is possible that this

served some role in colonization of the leaves. However, no symptoms were observed in

pathogenicity tests with only P. syringe strains on strawberry.

An association of P. syringe and a Xanthonzona~s species has previously been reported in

citrus lesions by Stall et al. (51). They reported that P. syringe was often associated with citrus

canker lesions in Argentina. While P. syringe by itself produced symptoms typical of citrus

blast, an association of P. syringe and Xanthonzona~s canspestris py. citri produced symptoms

not typical of either citrus blast or citrus canker. Pathogenicity tests on strawberry with X.

fragariae, P. syringe, and a mixture of both organisms had different symptoms as well. Two

plants inoculated with X. fragariae left at room temperature developed typical ALS lesions









between 6 and 14 days that were described by Kennedy and King (24) in laboratory inoculations;

however, no bacterial oozing or tissue necrosis developed with these symptoms as was observed

in the field (Figure 4~1A). Additionally, the level of humidity had an effect on the expression of

these early symptoms. When removed from high humidity, expression of ALS symptoms in

plants inoculated with X. fragariae was reduced. Plants inoculated only with P. syringe did not

develop symptoms, but the combination of the two organisms produced symptoms in two plants

in the growth chamber that were identical to those seen in the field during the season (Figure

4~1B). After three weeks, lesions on strawberry plants inoculated with a mixture of X. fragariae

and Pseudomona~s spp. developed oozing lesions that became necrotic at the site of the lesion.

After lesions began oozing, both plants remained in bags but were transferred to room

temperature where development of symptoms progressed rapidly.

In order to determine if the Pseudomona~s strains were colonizing ALS lesions in

strawberry, isolations of lesions at various stages of infection were performed and populations of

Pseudomona~s strains were enumerated. Isolations were made from a leaf with no symptoms,

early lesions (no oozing), advanced lesions (oozing, no necrosis) and necrotic tissue.

Populations of Pseudomona~s were not different between asymptomatic tissue and early lesions;

however, populations increased by three orders of magnitude between early lesions and

advanced lesions. Populations on necrotic tissue were too numerous to count (data not shown).

This preliminary study indicates that an association between ALS lesions and

Pseudomona~s strains exists based on the observations that the Pseudomona~s spp. tested

exacerbated the symptoms caused by X fCragariae. More data is necessary to statistically

compare populations of Pseudomona~s spp. associated with ALS lesions. Because strawberry

was not an apparent host to the Pseudomona~s strains isolated, and if the Pseudomona~s spp. are










responsible for the exacerbation of the symptoms, there may be need for a new management

strategy for ALS in strawberry.









Table 4~1. Characterization ofPseudomona~s strains associated with lesions of angular leaf spot on strawberry during 2005-06
and 2006-07 season.


Strain
Designation
12
13
14
15
16
23
24
25
27
28
29
31
32
33
34
35
36
50
51
52
53
54
60
61
62
70


Colony
Color
White
White
White
White
White
White
White
White
White
White
White
White
White
White
White
White
White
White
White
White
White
White
White
White
White
White


Ice
Nucleation
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
10/10
1/10
ND
ND
2/10
10/10
5/10
10/10
ND
ND
ND
ND
10/10


Path
Test
Neg
Neg
Neg
Neg
Neg
ND
ND
ND
ND
ND
ND
ND
ND
Neg
Neg
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Neg


Location
GCREC, FL
GCREC, FL
Floral City, FL
Floral City, FL
Floral City, FL
GCREC, FL
GCREC, FL
GCREC, FL
GCREC, FL
Floral City, FL
Floral City, FL
Floral City, FL
Floral City, FL
Floral City, FL
Floral City, FL
Floral City, FL
Floral City, FL
North Carolina
Vermont
Vermont
Vermont
Vermont
California
California
California
California


Fatty Acid
ND
Pseudomona~s s) ringae py. tomato
Pseudomona~s s) ringae py. tomato
Pseudomona~s s) ringae py. tomato
ND
ND
ND
ND
Pseudomona~s putida biotype B


Oxidase
ND
Weak +
Weak +
Weak +
ND
ND
ND
ND
Pos
Weak +
Weak +
Weak +
Weak +
Neg
Weak +
Weak +
Neg
ND
ND
ND
ND
ND
ND
ND
ND
ND


HR
Neg
Weak
Neg
Weak
ND
ND
ND
ND
Neg
Neg
Neg
Neg
Neg
Pos
Weak
Neg
Neg
ND
ND
ND
ND
ND
ND
ND
ND
ND


Pseudomona~s s) ringae py.
Pseudomona~s s) ringae py.
Pseudomona~s syringae py.
Pseudomona~s s) ringae py.
Pseudomona~s s) ringae py.
Pseudomona~s s) ringae py.
Pseudomona~s syringe py.
Pseudomona~s syringe py.
ND
ND
ND
ND
ND
ND
ND
ND
ND


tomato
tomato
tomato
tomato
atrofaciens
tomato
tomato
tomato










71 Califomnia
72 Califomnia
73 Califomnia
74 Califomnia
75 Califomnia
79 Califomnia
ND = Not determined


White ND
White ND
White ND
White ND
White ND
White ND


ND 10/10
ND 0/10
ND 0/10
ND 2/10
ND 0/10
ND 1/10


Neg
ND
ND
ND


























































Figure 4~1. Angular leaf spot symptoms on strawberry. A) Angular leaf spot symptoms on
strawberry inoculated with X. fragariae only. B) Bacterial oozing and slight necrosis from ALS
lesions on strawberry inoculated with X. fragariae and Pseudomona~s spp.









CHAPTER 5
SUMMARY

Overall, the goal of this research was to develop more effective management practices for

angular leaf spot of strawberry. To accomplish that goal field studies comparing different

chemical and biological products were evaluated, cultivar resistance for all cultivars of plants

used in Florida was evaluated, differences in yield between treatments and untreated controls

were compared, the impact of environmental conditions on control of angular leaf spot was

analyzed, and finally a preliminary study on the association ofPseudomona~s spp. and lesions of

angular leaf spot was performed. From these studies we have learned that Actigard is a suitable

alternative to copper-based products for suppressing angular leaf spot without inducing the

negative side effect of phytotoxicity; however, we were not able to conclusively determine if

suppression of angular leaf spot or minimizing the damage from phytotoxicity could increase

yield. This study also produced the first data comparing resistance to angular leaf spot for all

cultivars grown in Florida. Similarly, we learned that differences occur in cultivar resistance and

this resistance may be useful as part of a management strategy; however, the effect of angular

leaf spot on yield remains a question. The impact of environmental conditions was analyzed and

we learned that if optimal conditions occur in the field; management of this disease will be

difficult regardless of methods. Finally, by processing a number of symptomatic tissue samples

and performing pathogenicity tests on the strains resulting from isolations, we observed that

there was an association between X. fragariae and Pseudomona~s spp. based on laboratory

inoculations in which symptoms differed between plants inoculated with X. fragariae and

Pseudomona~s spp. More work is required to better understand this interaction. Further studies to

understand this association may lead to new more effective management of angular leaf spot.










LIST OF REFERENCES


1. Agrios, G. 2005. Plant Pathology. Elsevier Academic Press, Burlington, MA. Pp. 625-626.

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strategies. Phytopathology 89: 353-359.

3. Bock, C.H., Parker, P.E., and Gottwald, T.R. 2005. Effect of simulated wind-driven rain on
duration and distance of dispersal ofXanthomona~s axonopodis py. citri from canker-infected
citrus trees. Plant Dis. 89:71-80.

4. Central Science Laboratory. 2003. Angular leaf spot on strawberry, EC listed diseases, Ref.
no. QIC/34. Crown Copyright, Sand Hutton, York. http ://www.defra.gov.uk/planth/qic.htm
October 18, 2006.

5. Conover, R.A., and Gerhold, N.R. 1981. Mixtures of copper and maneb or mancozeb for
control of bacterial spot of tomato and their compatibility for control of fungus diseases.
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BIOGRAPHICAL SKETCH

Gary Todd Cooper was born in Columbus, Ohio to Gary and Ailene Cooper. He and his

family moved to southwest Florida and he graduated from Port Charlotte High School in 1987.

After high school graduation, he worked in his family's business until 1991. He entered the

automotive industry in 1991 and worked as an automatic transmission repair technician. While

working as a repair technician, he began his undergraduate degree attending night classes and

eventually transitioned into a career in science by accepting a position studying bioaerosols at

Battelle Memorial Institute in Columbus, Ohio. He received his Bachelor of Science in life

science from Otterbein College after completing eight years of part-time school. Immediately

following graduation, he accepted an offer to pursue a Master of Science in plant pathology at

the University of Florida. He has been married since October 12, 2002 to Aimee, who is also a

graduate of Otterbein College. He is looking forward to pursuing his Ph.D. at the University of

Florida in the fall of 2007.





PAGE 1

1 ANGULAR LEAF SPOT OF STRAWBERRY: DISEASE CONTROL STRATEGIES AND ASSOCIATION OF Pseudomonas syringae WITH LESIONS By GARY TODD COOPER A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007

PAGE 2

2 2007 Gary Todd Cooper

PAGE 3

3 To my wife Aimee, for without her love, support, and sacrifice, it would not have been possible; to my parents Gary and Ailene and my sisters De dra and Leah for their l ove and support, and to my friend Frank Smith, who also has been there fo r me from the very beginning; I thank you all.

PAGE 4

4 ACKNOWLEDGMENTS I thank the chair and members of my supervisory committee, Dr. Natalia Peres, Dr. Jeffrey B. Jones and Dr. Craig Chandler, for their guidance and patience. I also thank Mrs. Teresa Seijo for her tremendous contribution to my traini ng and Dr. James Mertely for his time, vast experience, and advice. Finally, I wish to tha nk Dr. Jane Polston for all of her guidance that helped keep me motivated.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ..............9 CHAPTER 1 INTRODUCTION..................................................................................................................11 2 EVALUATION OF SPRAY MATERIALS FOR CONTROL OF ALS, AND IMPACT OF ENVIORNMENTAL CONDITIONS..............................................................................18 Introduction................................................................................................................... ..........18 Materials and Methods.......................................................................................................... .21 Results........................................................................................................................ .............23 2005 Chemical Trial..................................................................................................23 Disease severity........................................................................................................23 Phytotoxicity............................................................................................................24 Marketable weight....................................................................................................24 Temperature and rainfall..........................................................................................25 2006 Chemical Trial..................................................................................................25 Disease severity........................................................................................................25 Disease incidence.....................................................................................................26 Phytotoxicity............................................................................................................26 Marketable weight....................................................................................................26 Temperature and rainfall..........................................................................................27 Discussion..................................................................................................................... ..........27 3 RESISTANCE OF FLORIDA STRAWBERRY CULTIVARS TO ANGULAR LEAF SPOT........................................................................................................................... ............37 Introduction................................................................................................................... ..........37 Materials and Methods.......................................................................................................... .39 Results........................................................................................................................ .............40 Cultivar Evaluation..........................................................................................................40 Marketable Weight..........................................................................................................41 Discussion..................................................................................................................... ..........41

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6 4 ASSOCIATION OF ANGULAR LEAF SPOT LESIONS AND Pseudomonas spp IN SYMPTOMATIC TISSUE ISO LTIONS OF STRAWBERRY.............................................46 Introduction................................................................................................................... ..........46 Materials and Methods.......................................................................................................... .47 Tissue Isolations..............................................................................................................47 Hypersensitive Response Tests.......................................................................................47 Fatty Acid Analysis.........................................................................................................47 Oxidase Test................................................................................................................... .48 Ice Nucleation................................................................................................................. .48 Pathogenicity Tests..........................................................................................................48 Results and Discussion......................................................................................................... ..49 Tissue Isolations..............................................................................................................49 Characterization of Strains..............................................................................................49 Pathogenicity Test...........................................................................................................49 SUMMARY........................................................................................................................ ...........56 LIST OF REFERENCES............................................................................................................. ..57 BIOGRAPHICAL SKETCH.........................................................................................................62

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7 LIST OF TABLES Table page 2~1 Treatments and application schedules for control of angular leaf spot in annual strawberry for the 2005 season in Florida....................................................................31 2~2 Treatments and application schedules for control of angular leaf spot in annual strawberry for the 2006 season in Florida....................................................................32 2~3 Severity of angular leaf spot, phytotoxocity, and market we ight of annual strawberry for the 2005 season in response to the application of various spray materials............33 2~4 Severity of angular leaf spot, phytotoxocity, and market we ight of annual strawberry for the 2006 season in response to the application of various spray materials............34 3~1 Disease incidence of angular leaf spot in cultivars of annual strawberry in Florida for 2006 season................................................................................................................. .45 4~1 Characterization of strains associated with lesions of a ngular leaf spot on strawberry during 2005-06 and 2006-07 season..................................................................................53

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8 LIST OF FIGURES Figure page 2~1 Environmental conditions a nd disease severity for 2005. .........................................35 2~2 Environmental conditions a nd disease severity for 2006. .........................................36 4~1 Angular leaf spot symptoms on strawberry from inoculations of X. fragariae and Pseudomonas spp. ............................................................................................................55

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9 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ANGULAR LEAF SPOT OF STRAWBERRY: DISEASE CONTROL STRATEGIES AND ASSOCIATION OF Pseudomonas syringae WITH LESIONS By Gary Todd Cooper August 2007 Chair: Natalia A. Peres Major: Plant Pathology Angular leaf spot (ALS) of strawberry is a bacterial disease caused by Xanthomonas fragariae Cultivars of strawberry used in Florida va ry in their susceptibility to ALS and there are no strawberry cultivars that are immune. Recommendations for management of ALS include using pathogen-free stock, limiting overhead irri gation, and applying chemical treatments. A trial to evaluate various materi als for control of ALS was conducted at the Gulf Coast Research and Education Center during the 2005 and 2006 seasons. During the 2006 season, the susceptibility to ALS of various cultivars and br eeding selections was evaluated. Spray materials were applied on 7and 14-day sche dules to Strawberry Festival pl ants and disease severity was evaluated on a scale of 0 to 6. Disease incide nce was evaluated at th e end of the 2006 season. Temperature and rainfall/irrigation data were co llected and analyzed for both seasons to assess the effect of environmental conditions on ALS. Treatments that include d Actigard at the higher rate, Kocide, Copper Count N, Badge, and Kasumin had significantly lower di sease severity than untreated controls. Results from these studies in dicate that there are so me good alternatives to copper for managing ALS. Cultivars Treasure and Ruby Gem were mildly susceptible to ALS. Cultivars Camino Real, Sweet Charlie, Albion, Fe stival, Sugar Baby, Carmine, and advanced

PAGE 10

10 selections FL 99-164, Festival #9, and FL 00-51 we re moderately susceptib le whereas cultivars Winter Dawn and Camarosa, and advanced se lections FL 99-117, and FL 01-116 were highly susceptible. Lastly, we re port an association between X. fragariae and Pseudomonas spp. based on laboratory inoculations in whic h lesions of angular leaf spot were different between plants inoculated with X. fragariae only and a mixture of X. fragariae and Pseudomonas spp.

PAGE 11

11 CHAPTER 1 INTRODUCTION Florida ranks second in the nation in st rawberry production, accounting for about 12 percent of the total U.S. supply of fresh produc t and generating more than $200 million in sales annually (9). There are several impor tant diseases of strawberry that are costly to control. Most of those diseases are caused by fungal pathogens and are managed through the use of a fungicide program. Angular leaf spot (A LS) is the only economically im portant bacterial disease of strawberry in the U.S. Although ALS-like symptoms had been observe d in Utah in 1927, ALS was not reported as a bacterial disease caused by Xanthomonas fragariae until 1962 (24, 37), when it was observed in Minnesota. No additional reports of the disease occurred until 1966 when ALS was found in Wisconsin, producing severe losses reachi ng 75% of the crop (8). Hildebrand et al. (14) reported that ALS had been observed in California for 10 year s; however, it was not considered an important disease since its occurren ce in the field was sporad ic. Hildebrand et al. (14) proposed the name bacterial blight of strawb erry rather than angular leaf spot. However, since the symptoms described by Kennedy and Ki ng (24) were more frequent than those described by Hildebrand et al. ( 14), the current name remains angul ar leaf spot. Finally, in 1971, ALS was reported in Florida (16). ALS had been observed in fields starting in mid-January of 1968 and reports of yield loss and poor plant conditi on were attributed to ALS. Since evidence of infection was not found in Fl orida nurseries except in very mild cases during 1969, ALS was not considered to be an important threat. Recen t observations by growers in Florida suggest that plantings may now suffer more severe ALS inf ections and potentially greater losses to the disease.

PAGE 12

12 By 1993, ALS of strawberry was occurring in all parts of the world (49), and it became apparent that the international shipment of in fected transplants was responsible for the rapid spread of this disease. At the time of a review by Maas et al. (37), AL S was occurring in North America, as well as Europe, Africa, South Americ a, and Australia; however, efforts to eradicate the disease in Australia were su ccessful (39). In 2001, Janse et al (18) discovered a similar, but new bacterial disease of strawberry in norther n Italy. Although this disease is caused by a bacterium in the genus Xanthomonas symptoms of the two diseases differ. This new disease, bacterial blight, has not been reported in the U.S. Kennedy and King (24) describe d the symptoms of ALS as light-green, angular, watersoaked lesions on the under surface of the leaf. The term angular refers to the noncircular appearance of the lesions. When primary infecti ons occur in the intervei nal tissue, the vascular system in the leaf limits movement of the pat hogen, which limits ALS lesions to the parenchyma tissue. When held to a transmitted light source, the spots are translucent, angular and easily distinguishable from other types of foliar damage during the early stages of infection. If conditions are favorable, these le sions can increase in number and size and eventually coalesce and become necrotic. The advanced symptoms ma y become more difficult to distinguish from other leaf infections such as common leaf spot, caused by the fungal pathogen Mycosphaerella fragariae (24). Although parenchymatous foliar tissue is targeted primarily, Hildebrand et al. (14) reported that vascular tissue could also be infected resulting in tissue colla pse. Symptoms associated with a vascular infection are similar to those of folia r infection in their translucence; but lesions are concentrated in tissue adjacent to veins. Foliar infections are usually randomly distributed on the leaf in contrast to vascular in fections. Although not re ported as a vascular infection at the time,

PAGE 13

13 examples of both vascular and foliar symptoms can be seen in the first report of ALS on strawberry by Kennedy and King (24). Vascul ar infections that produce the symptoms associated with lesion concentration in areas ad jacent to veins are the result of infection of strawberry crowns by X. fragariae (4, 14, 37). Additionally, blighting of petio les and infection of flowers and runners can occur (11). X. fragariae is not pathogenic to the st rawberry fruit; however, seve re infection of the calyx produces obvious symptoms and the calyx will tu rn brown making fruit unmarketable (31, 43). Xanthomonas fragariae is a rod-shaped, Gram-negative bacterium. Typically, the cells are non-motile; however some may have a single polar flagellum (50, 7). X. fragariae is aerobic, non-capsulate, and non-spore-forming. Typical co lonies are circular, convex, and mucoid. Although they produce xanthomonadi n, the colonies are pale yellow and young colonies may appear translucent. Growth on nutrient agar and in nutrient broth is poor. Colony formation may require 3 to 5 days incubation at 20 to 24C on a suitable medium such as the Wilbrinks medium (26, 48). X. fragariae is listed as a quarantine pest by the European Plant Protection Organization (7). Often, plants infected with ALS are as ymptomatic. Because strawberry is propagated vegetatively and transplants are sh ipped worldwide, movement of infected transplants often goes undetected and is thought to be the reason for distribution of X. fragariae worldwide (37). Thus, good detection techniques are important to avoid movement of the disease. In the early to mid nineties, efforts to deve lop rapid detection of ALS with molecular and serological assays resulted in more efficient techniques for confirmation of X. fragariae One technique, Enzyme-Linked Immunosorbent A ssay (ELISA), was effective in detecting X. fragariae in symptomatic tissue, but unfortunately, ELISA was ineffective for detection on

PAGE 14

14 asymptomatic tissue or tissue infected with bacterial populations below 104 cfu/ml (37, 49). A more effective technique for detection of X. fragariae and for detection of epiphytic bacteria is based on the Polymerase Chain Reaction (PCR). W ith the advent of PCR and its adaptation to plant disease diagnosis, studies began to develop primers for specific detection of X. fragariae (13, 45, 47). The molecular and se rological detection techniques developed by Rowhani et al. (49), Roberts et al. (47), and Pooler et al. (45) not only im proved diagnosis of infected symptomatic material, but hastened the ability to detect the pathogen in asymptomatic transplants as well. The combination of tissue isolation and the new molecular and serological techniques proved successful and diagnosis of ALS was considerably improved (45, 47, 49). Strawberry production in Florida differs from northern regions in that new transplants are obtained annually from northern or high elevation nurseries to es tablish strawberry fields. Transplants can be infected and escape detection as leaves may remain asymptomatic until favorable conditions occur. In the study by Robe rts et al. (48), nearly 50% of the shipments contained boxes of transp lants with ALS symptoms. To determine the host range of X. fragariae, Kennedy and King (24) inoculated 35 different types of plants by infiltration with cel l suspensions. None of the thirty-five potential hosts, except strawberry, became infected. Since th eir initial tests, the only other reported host for X. fragariae has been a couple of Potentilla species. Conditions that favor ALS are hi gh humidity, moderate or low temperatures in some cases near zero Celsius, and good plant growth (8, 25) When humidity is high, the underside of the leaf can have a slimy mucous layer of bacterial exudates cove ring the lesions (8, 36, personal observation). In pathogenicity tests where inoculated plants were incubated under different

PAGE 15

15 moisture conditions, few lesions were observed on plants left on open benches compared to those placed in moisture chambers (8). Bacterial pathogens are frequently sp read by rain as is the case with Xanthomonas axonopodis pv. citri the causal agent of citrus canker of citrus (3). Another mechanism of bacterial transmission occurs via mechanical tr ansmission by working in the field when plants are wet from rain, or more frequently from th e early morning dew. Intuitively, limiting the amount of overhead irrigation during the growin g season would be helpful in controlling the spread of ALS in strawberry fields. As ear ly as 1966, Epstein questioned the use of overhead irrigation and pointed out its pote ntial impact on ALS (8). Howeve r, in Florida, strawberry is farmed annually and the use of bare-root tran splants each season makes overhead irrigation essential for transplant establishment. Florid a strawberry field temp eratures are high during October, and without adequate overhead irrigati on until transplants are established, transplants would dehydrate and die. Additionally, strawb erry in Florida is a winter crop and air temperatures can fall below freezing in the ev ening and early morning hours. Overhead irrigation is the most cost effec tive method to protect the flowers of the strawberry plants from freezes. Unfortunately, this freeze protection met hod spreads the pathogen in the field and new symptoms are seen frequently following freeze events (37, 48, personal observation). Reports of losses associated with ALS are va riable. Epstein (8) reported losses from 75 80%; yet in Florida, Roberts et al. (48) reporte d much lower losses of approximately 8%. No other reports on the effect of ALS on yield have been published since 1997. Thus, more information is needed on the effect of ALS on yield for the currently gr own cultivars and on the cost effectiveness of treatment programs. Sin ce the pathogen does not cau se fruit rot, and only infects the calyx when disease pressure is high, ALS rarely causes a direct reduction in yield.

PAGE 16

16 However, it may indirectly reduc e yield by reducing photosynthetical ly active leaf area. In a recent study by Mertely et al. (40) leaf and fruit sanitation were compared to the standard fungicide program for management of Botrytis Fruit Rot and leaf san itation actually reduced yield. A possible explanation sugg ested by Mertely et al. (40) was that yield loss may be a result of the loss of photosynthetically active tissue and nutrients that could be mobilized by plants brought about by removal of tissue. Management of plant diseases is usually achieved by employing seve ral techniques. The first method of control is avoida nce. Pathogen-free seed or tran splants should be used to avoid introducing a pathogen to a field. Sanitation or eradication, the removal of infected tissue or plants, may be useful if applied correctly. Resistance to bacterial pathogens is a useful management tool if available. The use of so il sterilization, anti-bacte rial sprays (including inorganic compounds and antib iotics) are other methods for contro l of plant diseases (1). Some of these techniques may be practic al and effective in strawberry production in Florid a, but others are not. For example, sanitation has been studie d in annual strawberry to manage a different disease and had adverse affects, and antibiotics ha ve not been successful in field application for disease control. The most effective way of preventing ALS is the use of pathogen-free transplants (37, 44). However, di sease-free transplants may not be readily available. Copperbased pesticides have been effective for contro l of bacterial diseases on strawberry and other crops, but phytotoxicity on strawb erry plants limit rates and freque ncies of application (43). Plant resistance is a useful tool for disease management; however, there are no resistant cultivars of strawberry currently available. There were four primary objectives of this research. The first obj ective was to evaluate different spray materials currently available for use in an integrated management program for

PAGE 17

17 control of ALS of strawberry. The second objecti ve was to quantify the effect of ALS and the effect of the treatments for ALS management on marketable yield. Th e third objective was to determine whether there were differences in su sceptibility among commercia l cultivars currently grown in Florida or advanced selections from a breeding program. The final objective was to determine if an association of Pseudomonas spp. with lesions of a ngular leaf spot exists.

PAGE 18

18 CHAPTER 2 EVALUATION OF SPRAY MATERIALS FOR CONTROL OF ALS, AND IMPACT OF ENVIORNMENTAL CONDITIONS Introduction Angular leaf spot (ALS), caused by Xanthomonas fragariae is a bacterial disease of strawberry (24). The disease was first obser ved in Minnesota by Kennedy and King in 1959, but was not reported until 1962 (24). ALS is primarily a foliar disease; however the calyx of the fruit can be infected as well. If the calyx is severely infected, the tissue will turn necrotic and fruit is not marketable as fresh fruit (43, 31). Symptoms of ALS in the early stages are easily diagnosed and distinguishable from other fo liar infections (24). With AL S, tissue can be infected and asymptomatic making it very difficult to cont rol the spread of infected plant material. ALS is favored by high humidity and low temp eratures similar to those experienced in Florida strawberry fields in the winter months Temperatures are relatively high during the day, frequently reaching 18 to 22C, and nights are cool, with air temperatures at ground level dropping to between 0 and 5C at times. Thes e conditions result in high surface moisture on plants permitting colonization by the pathogen. Studi es by Hildebrand et al. (15) and Epstein (8) determined the importance of post-inoculation humidity, and as early as 1966, Epstein (8) questioned the impact that overhead ir rigation might have on ALS epidemics. Losses associated with ALS are not clear. Reports of losses reaching 75 to 80% by Epstein differ greatly from reports in Florida that indicated losses of approximately 8% (8, 48). Currently, ALS is managed with a variety of t echniques. The most effective and efficient technique is the use of pathoge n-free planting stock (37, 43). Cu ltural practices such as limiting overhead irrigation are important in reducing the spread of inocul um in fields. Unfortunately, most growers are dependent on the use of overhea d irrigation to protect their crop during freeze events. Currently, the only compounds recomm ended for control of ALS are copper-based

PAGE 19

19 products. However, there is a danger in usi ng copper-based products si nce copper is phytotoxic to strawberry plants (48). Thus, new compounds need to be evaluated to determine if better control alternatives exist. Systemic acquire d resistance (SAR) inducers, and new antibiotic formulations and biological control agents ha ve shown some promise in managing bacterial diseases and increasing yield in other crops (33, 41, 42). In this study, five types of spray materials were tested including copper-based tr eatments, SAR inducers, kasugamycin antibiotic treatments, biological contro l products, and surfactants. Copper-based spray programs have been proved to be effective to cont rol bacterial spot of tomato, caused by Xanthomonas campestris and citrus canker, caused by Xanthomonas axonopodis pv. citri (5, 20, 21, 52). In a previous study by Roberts et al. ( 48), copper hydroxide has shown some suppression of ALS and served as the standard treatment for disease control in this study. Additionally, other copper formulations are available such as basic copper sulfate, copper ammonia complexes, and copper oxychloride. These formulations were evaluated to see whether they were able to reduce disease without causing phytotoxicity. Products that induce the systemic acquired resistance (SAR) were not available at the time of the study by Roberts et al. (48). R ecent studies indicated that the SAR compound acibenzolarS -methyl (ASM) was a good alternative to copper for reducing di sease severity in bacterial spot of tomato (41, 35). A group of SAR inducers including ASM were evaluated for efficacy in control or suppression of ALS. Potassium phosphite is a well-known inducer of resistance especially to diseases caused by Oo mycetes and has been effective in studies of bacterial spot of peaches, peppers, and tomato (19, 46, 22, 17). The mixture of famoxadone and cymoxanil, although not described as an SAR, is a systemic pesticide that targets protein complex III in the mitochondria thus limiting AT P production and ultimately energy production.

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20 These products penetrate the plant and cannot be washed off by rain. Tanos, the mixture of these chemicals, has been shown to significantly reduc e numbers of lesions in bacterial speck of tomato, caused by Pseudomonas syringae pv. tomato (28, 29). Antibiotics have not been widely used in th e field because of limited residual activity and potential problems with the development of resistance in the pathogen. Recently, Kasumin, a product not yet labeled for use in the U.S. has been studied for control of bacterial diseases of pepper, tomato, citrus, and wa lnut; all of which are caused by different pathovars of Xanthomonas campestris (30, 17). Kasumin significantly reduced disease in all four studies, and is an effective bactericide/fungicide that has been registered and used in other countries for years now. Kasugamycin, the active ingredient in Ka sumin, is an aminoglycoside antibiotic from streptomyces, and was included in this study to investigate whethe r it may also provide control of ALS on strawberries. Two biological control products were al so evaluateda spore suspension of Streptomyces lydicus (Actinovate) and Bacillus subtilus (Serenade Max). In separate studies on control of bacterial l eaf spot of tomato and pepper, Ac tinovate and Serenade Max reduced disease compared to the untreated control (17, 33). The bacteria in these products may colonize leaf surfaces of strawberry plants making it more difficult for X. fragariae to obtain leaf surface nutrients. Efforts to improve coverage of pesticid es used for control of other economically important strawberry diseases may require a su rfactant to be added to the formulation for maximum efficacy. Surfactants al ter the properties of water a nd reduce hydrophobicity to plant surfaces. Unfortunately, the materials used for management of fungal diseases may possibly aid bacterial pathogens in reaching areas with na tural openings overcoming the natural physical

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21 barriers of the plant. In a study by Gottwald et al. (10), surfactants were found to enhance bacterial infections on citrus. A surfactant was in cluded in this study to determine if the use of surfactants in strawberry pr oduction increases ALS severity. The goal of this study was to evaluate severa l new chemical and bi ological products to determine if any available products minimize or control ALS during the growing season, and by doing so, increase yield. The effects of temper ature and rainfall/overhead irrigation on disease severity during the growing season were also analyzed. Materials and Methods Field experiments evaluating the use of produc ts for ALS control and the impact of ALS on yield were conducted at the University of Flor ida Gulf Coast Research and Education Center in Wimauma, Florida during the 2005 and 2006 growing seasons in fields managed using current conventional strawberry fa rming practices. On October 6, 2005 and October 16, 2006, bare-root transplants of the cultivar Strawberry Festival from Canada were planted in raised, fumigated beds covered with black plastic mulch. Soil had been fumigated with methyl bromide and chloropicrin at a ratio of 67:33 and at a rate of 397 kg/ha. Beds were 1.2 m apart, center to center, and were 0.7 m wide. Two offset rows of plants were planted on each bed. Plants were spaced 28 cm in the row with 38 cm between the rows. Overhead irrigation was applied for 10 days to establish transplants and then plants were irrigated and fer tilized by drip tape. Treatments were arranged in a randomized comple te block design in four successive beds. Plots for each treatment contained 12 plants and were 2.9 m long. In this study, 15 and 17 treatments including control groups were compared for the 2005 and 2006 growing seasons, respectively (Tables 2~1 and 2~2). Transplant s were obtained from nurseries known to have ALS to ensure a source of inoculum.

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22 Five types of products were selected for study. These included copper-based products, systemic acquired resistance (SAR) products that induce natural defense mechanisms of the plant, the antibiotic kasugamy cin, biological compounds, and a su rfactant. In all cases, applications were made with a CO2 backpack sprayer that deliver ed 950 L/ha at 275 kPa with a two-nozzle wand. Treatments were applied on a seven-day schedule beginning December 2 and November 22 and continuing through February 22 and February 28 for 2005 and 2006 seasons, respectively. Evaluations of disease severi ty, phytotoxicity, and marketab le yield were conducted for both seasons. Disease severity was evaluated three times during each season on a scale from 0 to 6 where 0 = no lesions; 1 = a few lesions on the en tire plant; 2 = a few lesions on more than one leaflet with no general necrosis; 3 = several lesions on leaflets w ith or without concentration of lesions near the veins and necrosis; 4 = numerous lesions present and some partial leaflet blight; 5 = some older leaves killed and others extens ively blighted; 6 = all older leaves killed and middle age leaves blighted. For each evaluation, the middle eight plants per plot were rated individually on February 10 and 24, and Ma rch 14 for the 2005 season, and December 28, January 25, and February 22 for the 2006 season. At the end of the 2006 growing season, five leaves from six plants in each plot, or a total of 30 leaves or 90 leaflets, were collected arbitrarily and evaluated for disease incidence. Phytotoxicity evaluations were conducted in all treatments once per season on January 6 and February 28 for the 2005 and 2006 seasons, respectively, after damage was observed in plots. Phytotoxicity was rated on a scal e of: 0 = no phytotoxicity; 1 = light damage; 2 = moderate damage; 3 = severe damage.

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23 Marketable yield was determined by harv esting the fruit twice per week from 20 December, 2005 to 17 March, 2006 and 15 Decembe r, 2006 to 16 March, 2007 for a total of 25 and 27 harvests for each season respectively. Fruit wa s hand picked and graded on the day of harvest. Fruit were considered marketable if there were no visible symptoms of ALS on the calyx or other fruit rot diseases, and fruit we ight for each berry was 10 g or greater. Minimum, maximum, and average temperature and rainfall data we re collected for 2005 06 and 2006 seasons from the Florida Automated Weather Network (FAWN) and total rainfall and overhead irrigati on data were collected from the GCREC weather station for comparison to determine freeze events versus rain events. Increases in disease severity were then compared to dates w ith heavy precipitation. Data were subjected to analysis of vari ance (ANOVA) and the means separated by Least Significant Difference (LSD, P 0.05). Statistical analyses of disease severity, disease incidence, phytotoxicity, and yield were performe d using software package Statistix 8 (Statistix 8, Tallahassee FL). Results 2005 Chemical Trial Disease severity The severity of ALS disease was evaluate d on February 10, 24, and March 14, 2006 for the 2005 season. An overall disease severity ratin g for the season was calculated by averaging the disease severity for each treatment for the th ree evaluation dates. Differences in ALS disease severity were significant for each evaluation period and for the overall season ( P 0.05). Plants treated with Actigard and Kocide 2000 at the higher rate had signi ficantly lower disease severity than the plants in the untreated contro l plots for the February 10 evaluation ( P 0.05). There were no differences between the remainder of the treatments and the untreated control. Plants

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24 treated with Prophyt alternated with Kocide or Kocide + Tano s, Actigard, and Kocide 2000, had significantly lower disease severity compared to plants in the untreated control plots for the February 24 evaluation ( P 0.05). There were no differences between the remainder of the treatments and untreated control for the Februa ry 24 evaluation. Plants treated with Prophyt alternated with Kocide or Kocide + Tanos, and Kocide 2000 had significant differences in disease severity for the March 14 evaluation ( P 0.05). There were no differences between the remainder of the treatments and untreated contro l for the March 14 evaluation. Overall, plants treated with Prophyt alternated with copper, Actigard, and Ko cide 2000 had significantly less disease than plants in the untre ated control plots. Treatments with K-Phite, Prophyt alone or alternated with Actigard or Kasumin, Actinova te, Kasumin, Serenade Ma x, and Kinetic were not significantly different from the untreated control (Table 2~3). Phytotoxicity Phytotoxicity was evaluated on January 6 and there were significant differences between treatments ( P 0.05). All plots treated with products cont aining copper had some damage. Plots treated with Actinovate plus Silicone 100, and the untreated control plots had some phytotoxicity; however plots treate d with these two products had si gnificantly less phytotoxicity than plots treated with copper-based products. The remaining treatments with no copper component were not phytotoxic (Table 2~3). Marketable weight Differences in fruit yield were significant ( P 0.05) among treatments. Plants treated with Kinetic at 665.5 ml/ha on a 7-day schedule prod uced 27,516 kg/ha of fresh fruit and plants treated with a mixture of Serenade Max, Biot une, and Kocide on a 7day schedule produced 20,661 kg/ha fresh fruit for the highest and lowe st yields per treatme nt, respectively. The

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25 untreated control produced 24,291 kg/ha and was not si gnificantly different in yield to any of the other treatments. Therefore, control of ALS did not appear to influence yield (Table 2~3). Temperature and rainfall Temperature and rainfall/overhead irrigati on data were analyzed from December 1 to March 26 for the 20056 season. Two freeze events occurred during this season, on January 8, 2006 and February 14, 2006. Plants received 23. 4 mm and 26.9 mm of overhead irrigation on January 8 and February 14, respectively. On e major rainfall event occurred during the 2005 season on February 3 and 4. Plants received 29.5 mm and 11.2 mm of pr ecipitation, respectively (Figure 2~1A). The freeze event and overhead irri gation coincided with an increase in disease severity observed during the 2005 6 season (Figures 2~1A, 1B). 2006 Chemical Trial Disease severity The severity of ALS was evaluated on D ecember 28, January 25, and February 22 for the 2006 season for all treatments. Disease severi ty was also evaluated on January 12 and February 9 for the untreated control and a few sel ected treatments. Differences in ALS severity were significant ( P 0.05) for each evaluation period and the ove rall season (Table 2~4). Plants treated with Actigard at the higher rate, Ko cide 2000, Copper-Count-N, Badge, and Kasumin + Kocide had significantly lower dis ease severity than the plants in the untreated control plots for the December 28, January 25, and February 22 evaluations, and for the overall season ( P 0.05). Plants treated with Kocide 3000 had significantl y lower disease severi ty for the January 25 evaluation ( P 0.05). Treatments with Cuprofix Ultra 40D, Kinetic, Serenade Max, and Actigard at the lower rate, Kasumin with Captan, and Kasuran were not signifi cantly different from untreated controls in disease severity for the entire season (Table 2~4).

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26 Disease incidence Differences in disease incidence were significant among treatments ( P 0.05). All treated plants had significantly lower disease incidence th an the plants in the untreated control plots ( P 0.05). Disease incidence in the untreated contro l plots was 77% whereas the range of disease incidence in all other plots was between 32% and 58%. Plots treated with Badge, Kocide 3000, Actigard at the lower rate alternated w ith Kocide, Kasuran, and Copper-Count-N had significantly lower disease incidence with 32%, 37%, 39%, 41%, and 44% disease incidence, respectively. However, those treatments were not significantly different than many of the other treatments (Table 2~4). Phytotoxicity Phytotoxicity was evaluated on February 28 and there were significant differences among treatments ( P 0.05). Plants treated with products cont aining copper had some damage. Plants treated with Actigard only, Ki netic, and the untreated contro l had no phytotoxicity symptoms and ratings were not significantl y different from each other or from treatments with Cuprofix Ultra, Actigard at the lower rate alternated wi th Kocide, Kasumin altern ated with Kocide, or Kasumin + Captan alternated with Kocide (Table 2~4). Marketable weight Differences in fruit yield between treatments were not significant ( P =0.47). Plants treated with Actigard applied on a 14-day schedule at 26 .6 g/ha produced 28,711 kg/ha of fresh fruit and plants treated with Kinetic at 665.5 ml/ha on a 7-day schedule produced 23,794 kg/ha fresh fruit for the highest and lowest numer ical yields per treatment, resp ectively. The untreated control produced 25,375 kg/ha and was not si gnificantly different in yield to any other trea tment (Table 2~4).

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27 Temperature and rainfall Temperature and rainfall/overhead irrigation data were analyzed from December 1 to March 26 for the 20067 season. One freeze event occurred during this season on February 17, 2007. Plants received 13.5 mm of overhead irri gation. Nine major rainfall events occurred during the 2006 season on the days of December 23 and 25, January 3, 24, 25, and 28, February 2 and 13, and March 16 when plants received 11.2, 30.5, 10.7, 18.0, 15.5, 19.6, 25.1, 16.5, and 12.4 mm of precipitation, respectively (Figur e 2~2A). A major increase in disease for the 2006 season coincided with the large amount of rainfall a nd low temperatures in late December. These optimal conditions for ALS we re associated with an increase in disease severity from approximately 2.5 to approximately 4.5 in period of one month (Figures 2~2A, 2B). Discussion This study demonstrated that suppression of ALS is possible with the use of some products. In the 2005 season, di sease suppression was achieved mostly with products that contained copper, which was the case in previ ous studies where copper was used to control bacterial diseases (20, 21). However, Actigard, a non-copper based product that induces systemic acquired resistance, also suppressed ALS, and ha s controlled bacterial spot of tomato (41). Disease was suppressed most effectively w ith the higher rate of Kocide 2000, but the disadvantage of this treatment was that it was also phytotoxic. Prophyt treatments when alternated with copper had diseas e severity indexes lower than unt reated controls, but treatments with Prophyt alone did not achieve control. The reduction of diseas e in the Prophyt/copper treatments was probably a result of the copper, and therefore these treatments were not included in the 2006 trial. Actigard on the other ha nd, contained no copper and suppressed ALS as

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28 effectively as the lower rate of Kocide 2000. Th e biocontrol agents had no effect on ALS, and Kinetic, a surfactant which was thought might actually increase disease, had no effect. The 2006 trial included some additional copper treatments such as basic copper sulfate and copper oxychloride, but copper hydroxide (Kocid e 2000) at the high rate was not evaluated again because of its tendency to be phytot oxic. Additional Actigar d treatments, including different rates alternated with copper, were adde d, and additional antibiotic treatments with higher rates of Kasumin alternat ed or mixed with copper were added. The additional treatments did not result in any new outcomes. The effective treatments remained copper, Actigard, and Kasumin with copper. Effective copper tr eatments included copper hydroxide and copper oxychloride + copper hydroxi de; however, treatments with coppe r sulfate were not effective. Treatments with Kasumin were effective; however, these treatments included a copper component and it is likely that efficacy may be due to the copper rather than the antibiotic. Actigard alone again suppressed disease as effectivel y as the lower rate of copper. At the end of the 2006 season, disease incidence was evaluated to determine if disease assessment could be improved. This evaluation proved successful for di fferentiating all treatments from the untreated control. However, disease incidence did not co rrelate to disease seve rity ratings and this assessment would need to be refine d for field use throughout the season. The use of copper for treatment of ALS is of concern to growers because copper is phytotoxic to strawberry plants. Every treatmen t containing copper for both seasons showed at least some phytotoxicity. Two treatments, that did not even include copper, had trace amounts of phytotoxicity probably from spray drift due to th eir proximity to treatments receiving copper. Plants that were treated with Kocide at the lower rate had half as much damage as plants treated

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29 with the higher rate. Actigard was the only tr eatment that provided disease suppression without phytotoxic side effects. The data on the combined effect of ALS and the management of ALS on yield were inconclusive. In some cases, there were signif icant differences in marketable yield between treatments, but there were no sign ificant differences in yield betw een treated and untreated plants for the 2005 season. Plants treated with copper at the higher rate had the most phytotoxicity and also had yields similar to plants that were not treated w ith copper and had no phytotoxicity. Thus, phytotoxicity did not seem to affect yi eld. The 2006 season was similar in that yield differences occurred between a few treatments, but none of the treatments differed from the untreated control. In the 2006 s eason, plants treated with Actigar d alone at the lower rate had a significantly lower incidence of disease than untr eated plants, but there was no significant difference in marketable yield betw een the two groups of plants. C ontrary to results obtained in tomato (41), it is possible that resistance to ALS in strawberry, whether inhere nt in the cultivar or artificially induced, could come at a cost to yiel d. This study also demonstrated the impact that temperature and precipitation or overhead ir rigation had on the epid emiology of ALS as previously described (25, 37). Two freeze ev ents occurred during the 2005 season during which overhead irrigation was used to protec t the crop. The first freeze event occurred on January 8. A month later the first evaluation was conducted, but by this time, ALS had become epidemic. In 2006, however, the first evaluati on was conducted much earlier in the season, when disease severity was relatively low. Four days prior to the firs t evaluation there was a major precipitation event which coincided with an extreme drop in temperature. This was not a freeze event, but conditions for development of an ALS epidemic were optimal. About four

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30 weeks after the temperature drop and rain, dis ease severity had almost doubled. Levels of disease severity were similar to those in the 2005 season four weeks after a freeze event. Despite an increasing number of products labele d for control of ALS, this disease will be difficult to control if conditions are optimal for dissemination of the pathogen. Evaluations of disease incidence rather than disease severity may help to more accurately assess the level of control being achieved since di sease incidence is an objectiv e evaluation whereas disease severity is a subjective evaluation. Based on th e information that was gathered in this study, Actigard is a good alternative to copper for supp ressing ALS in strawberry and providing a level of control similar to copper. There is no phytotox icity associated with Actigard so plants are not suffering from efforts to manage the disease. Un fortunately, while Actigard appeared to have no negative effect on yield, in this study there was also no positive effect on yield compared to untreated control plants. One possibility for a lack of observable differences in yield may be that plot sizes were too small. Another possibili ty may be that although differences in disease severity were significant, differences were not enough to reflect differences in yield.

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31Table 2~1. Treatments and application sche dules for control of angular leaf spot in annual strawberry for the 2005 season i n Florida Treatment (active i ngredient) (rate/ha) Schedule Control K-Phite (phosphorous acid) (5.8 L) 14-day Prophyt (potassium phosphite) (5.8 L) 14-day Prophyt (potassium phosphite) (5.8 L) alt. Actigard (acibenzolar-S-methyl) (53.2 g) 7-day Prophyt (potassium phosphite) (5.8 L) alt. Kasumin (kasugamycin) (1.2 L) 7-day Prophyt (potassium phosphite) (5.8 L) alt. Kocide 2000 (copper hydroxide) (1.7 kg) 7-day Prophyt (potassium phosphite) (5.8 L) alt. [Kocide 2000 (copper hydroxide) (1.7 kg) + Tanos (famoxadone + cymoxanil) (567 g)] 7-day Actinovate ( Streptomyces lydicus )(850.5 g) + Silicone 100 (295.8 ml) 7-day Actigard 50WG (acibenzolar-S-methyl) (53.2 g) 14-day Kasumin (kasugamycin (1.2 L) 14-day Kocide 2000 (copper hydroxide) (0.9 kg) 7-day Kocide 2000 (copper hydroxide) (1.7 kg) 7-day Serenade Max ( Bacillus subtilis ) (1.1 kg) + Biotune (sodium lauryl sulfat e, sodium dodecylbenzene sulfonate, and polyoxyethylene (20) sorbitan monooleate) (2.9 L) 7-day Serenade Max ( Bacillus subtilis ) (1.1 kg) + Biotune (sodium lauryl sulfat e, sodium dodecylbenzene sulfonate, and polyoxyethylene (20) sorbitan monooleate) (2.9 L) + Kocide 2000 (copper hydroxide) (0.9 kg) 7-day Kinetic (organosilicone) (665.5 ml) 7-day

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32Table 2~2. Treatments and application sche dules for control of angular leaf spot in annual strawberry for the 2006 season i n Florida Treatment (active i ngredient) (rate/ha) Schedule Control Kocide 2000 (copper hydroxide) (0.9 kg) 7-day Kocide 3000 (copper hydroxide) (= GFJ52) (1.0 kg) 7-day Cuprofix Ultra 40D (coppe r sulfate) (0.8 kg) 7-day Copper-Count-N (copper ammonia complex) (2.4 L) 7-day Badge (copper hydroxide + copper oxychloride) (1.1 L) 7-day Badge (copper hydroxide + copper oxychloride) (2.1 L) 7-day Kinetic (organosilicone) (665.5 ml) 7-day Serenade Max ( Bacillus subtilis ) (1.1 kg) + Biotune (sodium lauryl sulfat e, sodium dodecylbenzene sulfonate, and polyoxyethylene (20) sorbitan monooleate) (1.2 L) + Kocide 2000 (copper hydroxide) (0.9 kg) 7-day Actigard 50WG (acibenzolar-S-methyl) (26.6 g) 14-day Actigard 50WG (acibenzolar-S-methyl) (26.6 g) alt Kocide 2000 (copper hydr oxide) (0.9 kg) 7-day Actigard 50WG (acibenzolar-S-methyl) (53.2 g) 14-day Actigard 50WG (acibenzolar-S-methyl) (53.2 g) alt Kocide 2000 (copper hydr oxide) (0.9 kg) 7-day Kasumin (kasugamycin) (295.8 ml = 100 ppm) alt. Kocide 2000 (copper h ydroxide) (0.9 kg) 7-day Kasumin (kasugamycin) (295.8 ml) + Kocide 2000 (copper hydroxide) (0.9 kg) alt Ko cide 2000 (copper hydroxide) (0.9 kg) 7-day Kasumin (kasugamycin) (5.8 L) + Captan 80WDG (captan) (1.7 kg) alt Kocide 2000 (copper hydroxide) (0.9 kg) 7-day Kasuran (kasugamycin + copper oxyc hloride) (2.0 kg = 100 ppm) 7-day

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33Table 2~3. Severity of angular leaf spot, phytotoxocity, and mark et weight of annual strawberry for the 2005 season in respo nse to the application of various spray materials. Treatment DSI z1 DSI2 DSI3 DSIAvg Phytotoxy Mkt Wt (kg/ha) Control 4.94abx 5.44abc 6.03abc 5.47abc 0.50de 24291abcd K-Phite (5.8 L) 4.38abc 5.32abcd5.69bcd 5.13bcde0.00e 25678abc Prophyt (5.8 L) 5.11ab 5.66ab 6.17ab 5.64ab 0.00e 27054ab Prophyt (5.8 L) alt. Actigard (53.2 g) 4.56ab 5.16bcde5.56cde 5.09cde 0.00e 27097ab Prophyt (5.8 L) alt. Kasumin (1.2 L) 4.41abc 5.47abc 5.88abc 5.25abcd0.00e 26474ab Prophyt (5.8 L) alt. Kocide 2000 (1.7 kg) 4.32bc 4.85def 5.32de 4.83def 1.75b 22355cd Prophyt (5.8 L) alt. [Kocide 2000 (1.7 kg) + Tanos (567 g)] 4.60abc 4.85def 5.07e 4.83def 1.00cd 26528ab Actinovate (850.5 g) + Silicone 100 (295.8 ml) 5.12ab 5.78a 6.37a 5.76a 0.25e 26671ab Actigard 50WG (53.2 g) 3.85cd 4.79ef 5.50cde 4.71ef 0.00e 23603bcd Kasumin (1.2 L) 4.94ab 5.34abcd5.94abc 5.41abc 0.00e 25516abc Kocide 2000 (0.9 kg) 3.88ab 4.38fg 5.29de 4.51f 1.25bc 24611abc Kocide 2000 (1.7 kg) 3.18d 4.03g 4.47f 3.89g 2.50a 21928cd Serenade Max (1.1 kg) + Biotune (2.9 L) 5.04ab 5.75a 6.25ab 5.68a 0.00e 21993cd Serenade Max (1.1 kg) + Biotune (2.9 L) + Kocide 2000 (0.9 kg) 4.88ab 5.12cde 5.89abc 5.29abcd1.00cd 20661d Kinetic (665.5 ml) 5.19a 5.69a 6.19ab 5.69a 0.00e 27516a z DSI = disease severity index on a scale of 0 = none to 6 = severe. Evaluations were conducted on 2/10, 2/24, and 3/14/2006; y Phytotoxicity rated on a scale of 0 = none to 3 = severe; x Treatment means within columns followed by the same letter are not significantly different by Fishers protected LSD ( P 0.05)

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34Table 2~4. Severity of angular leaf s pot, phytotoxocity, and market weight of annual strawberry for the 2006 season in response to the application of various spray materials. Treatment DSI z1 DSI2 DSI3 DSIAvg Dis incidencew Phytotoxy Mkt Wt (kg/ha) Control 2.35ax 4.28a 4.50a 3.71a 77a 0.00d 25375abc Kocide 2000 (0.9 kg) 1.00ef 3.16e 3.79f 2.65f 49bcde 1.00bc 28030ab Kocide 3000 (1.0 kg) 1.91abcd 3.54bcde 4.25abcde 3.23abcde 37efg 1.00bc 25547abc Cuprofix Ultra 40D (0.8 kg) 1.88abcd 3.78a bcde 4.40abc 3.56abcd 45cdef 0.50cd 26016abc Copper-Count-N (2.4 L) 1.44cdef 3.47bcde 4.03bcdef 2.98cdef 44cdefg 1.25ab 26038abc Badge (1.1 L) 1.47cdef 3.44cde 4.00cdef 2.97cdef 36fg 1.00bc 27482ab Badge (2.1 L) 1.44cdef 3.22de 3.88ef 2.85def 32g 1.75a 24860bc Kinetic (665.5 ml) 2.22ab 4.10ab 4.35abcd 3.56ab 48bcdef 0.00d 23794c Serenade Max (1.1kg) + Biotune (1.2L) + Kocide 2000 (0.9 kg) 2.22ab 4.00abc 4.54a 3.58ab 56bc 1.25ab 27086abc Actigard 50WG (26.6 g) 1.97abc 3.85abcd 4.44ab 3.42abc 53bcd 0.00d 28711a Actigard 50WG (26.6 g) alt Kocide 2000 (0.9 kg) 1.82abcd 3.85abcd 4.38abc 3.35abcd 39efg 0.25d 27456ab Actigard 50WG (53.2 g) 0.88f 3.28de 3.81f 2.66f 53bcd 0.00d 26595abc Actigard 50WG (53.2 g) alt Kocide 2000 (0.9 kg) 1.63abcde 3.41cde 4.00cdef 3.0125cdef 45cdef 1.00bc 25331abc Kasumin (295.8 ml = 100 ppm) alt. Kocide 2000 (0.9 kg) 1.50bcdef 3.69abcde 4.17a bcdef3.12bcdef 47bcdef 0.25d 25461abc Kasumin (295.8 ml) + Kocide 2000 (0.9 kg) alt Kocide 2000 (0.9 kg) 1.22def 3.28de 3.94def 2.82ef 45cdef 1.00bc 25983abc Kasumin (5.8 L) + Captan 80WDG (1.7 kg) alt Kocide 2000 (0.9 kg) 1.63abcde 3.85abcd 4.38abc 3.28abcde 58b 0.25d 25780abc Kasuran (2.0 kg = 100 ppm kasugamycin + Cu) 1.88abcd 3.85abcd 4.28abcde 3.34abcde 41defg 1.50ab 27661ab w = Percentage of disease incidence on a to tal of 120 randomly collected l eaves for each treatment. Evaluation was conducted on 3/31/2007; x = Treatment means within columns followed by the same letter are not significantly differe nt by Fishers protected LSD ( P 0.05); y = Phytotoxicity rated on a s cale of 0 = none to 3 = severe; z DSI = disease severity index on a scale of 0 = none to 6 = severe. Evaluations were conduc ted on 12/28/2006, 1/25, and 2/22/2007.

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35 A 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.012/1 12/15 12/29 1/12 1/26 2/9 2/23 3/9 3/23 Total rainfall (m m 0.0 5.0 10.0 15.0 20.0 25.0Avg Temperature (C ) B 0 1 2 3 4 5 6 712/1/05 12/15/05 12/29/05 1/12/06 1/26/06 2/9/06 2/23/06 3/9/06 3/23/06 Disease Severit y Figure 2~1. Environmental conditions and disease severity for 2005. A) Total rainfall/overhead irrigation (mm, vertical bars) and average daily temperature (C, line) during the 2005 annual strawberry season for evaluation of products for control of angular leaf spot Arrows indicate freeze events when overhead irrigation was used. B) Angular leaf spot disease se verity index on leaves of control plots of cultivar Strawberry Festival over time in 2005 annual strawberry season.

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36 A 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.012/1 12/15 12/29 1/12 1/26 2/9 2/23 3/9 3/23 Total rainfall (m m 0.0 5.0 10.0 15.0 20.0 25.0 30.0Avg temperature C B 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 512/1/2006 12/15/2006 12/29/2006 1/12/2007 1/26/2007 2/9/2007 2/23/2007 3/9/2007 3/23/2007 Disease Severit y Figure 2~2. Environmental conditions and disease severity for 2006. A) Total rainfall/overhead irrigation (mm, vertical bars) and average daily temperature (C, line) during 2006 annual strawberry season fo r evaluation of products for control of angular leaf spot. Arrow indicates fr eeze event. B) Angular leaf spot disease severity rating for control plots of cultiv ar Strawberry Festival over time in 20067 annual strawberry season.

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37 CHAPTER 3 RESISTANCE OF FLORIDA STRAWBERRY CULTIVARS TO ANGULAR LEAF SPOT Introduction Angular Leaf Spot (ALS) of strawberry, caused by Xanthomonas fragariae is a bacterial disease of strawberry first observed in Minne sota in 1959 and subsequently reported in 1962 by Kennedy and King (24). ALS now occurs worldw ide and it is thought that the shipment of infected asymptomatic tissue is responsible for its rapid distribution (37). Symptoms of ALS as described by Kennedy and King (24) are lesions on the abaxial surface of the leaf that are green, angular, and water-soaked. Lesi ons are translucent and easily distinguishable from healthy tissue. Diagnosis of ALS in the early stage, be fore lesions become necrotic, is easily done by observing translucent lesions with transmitted light. Once necrosis occurs, diagnosis becomes more difficult as symptoms can be confused with other foliar diseases such as common leaf spot caused by the fungal pathogen Mycosphaerella fragariae (24). ALS is primarily a foliar pathogen of strawberry plants; however infection can occur in the parenchymatous tissue of the calyx. Strawberry fruit itself is not para sitized; however, if infection of the calyx is se vere enough, the tissue can b ecome necrotic making fruit unmarketable as fresh fruit (31, 43). Less commonly, X. fragariae can infect crowns of strawberry plants resulting in a va scular infection (4, 14, 37). In vascular infections, lesions are concentrated around vascular tissue rather than distributed random ly over leaf surface. At the time of Kennedy and Kings first report of ALS in strawberry, X. fragariae was not thought to infect vascular tissue; however, their report contains images of l eaves with vascular infection as well as parenchymatous tissue infection (24). ALS is favored by high humidity and low te mperatures; conditions common to Florida strawberry fields in the winter months. Temperatures are re latively high in the daytime

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38 frequently reaching 18 to 22 C and nights are cool, with temperatures dropping to between 0 and 5 C at times. These conditions result in high surface moisture on plants permitting colonization by the pathogen. Optimal conditions make this disease very difficult to control. Management of ALS requires an integrated approach. The ea siest way to control ALS is to use pathogen-free stock (37). Unfortunately, cl ean plants are not always available, and plants infected with X. fragariae are commonly asymptomatic and escape detection until favorable conditions occur. Limiting overhead irrigation and wo rking in fields after pl ants are dry is useful for limiting the spread of inoculum in fields, how ever certain times require overhead irrigation in Florida fields. Strawberry is pl anted in early to mid-October a nd mid-day temperatures are high. Overhead irrigation is necessary to establish plants in the field befo re they receive drip irrigation. Additionally, strawberry is a winter crop in Fl orida and air temperatures can drop below freezing requiring overhead irrigation to protect th e flowers of the plants from freezing. Chemical application of copper compounds has previously been effective (48), however the use of copper products is limited since copper is phytotoxic to strawberry plants (43, 48). Other spray materials were evaluated in this thesis research and the SAR product acibenzolarS methyl (ASM) was effective in re ducing disease severity compared to untreated control without being phytotoxic to the plants. Resistant cultivars would be useful for manage ment of ALS; however no resistant cultivars have been developed. Kennedy and King (25) evaluated 64 cultivars with X. fragariae to observe reactions to ALS and Hildebrand et al. (1 5) investigated the factors affecting infection and cultivar reaction for 22 cultivar s. All cultivars in those studies were susceptible to ALS, and only two, Sweet Charlie and Chandler, are used for production in Florida. Compared to the other cultivars, Sweet Charlie and Chandler were am ong the most susceptible in Hildebrands study

PAGE 39

39 (15). Maas et al. (38) identified two small-fruited genotypes, a native F. virginiana and a F. virginiana x F. ananassa hybrid, that are highly resistant to ALS, and a recent study by Lewers et al. (32) has led to understanding the number of genes involved and the heritability of resistance to ALS, but further research with larger populations is needed to develop cultivars with resistance and ot her desirable traits. There is speculation from growers that among cultivars grown in Florida the currently most popular cultivar Strawberry Festival is more susceptible than others. However, there are no data available to determine if differences in ALS resistance/susceptibility among cultivars currently grown in Florida exist. The objective of this study was to compare resistance to ALS in cultivars of strawberry currently produced in Florida and four adva nced selections from a breeding program, and determine the effect ALS has on yield. Materials and Methods Field experiments to evaluate different strawb erry cultivars for resistance to ALS were conducted at the University of Florida Gulf Coas t Research and Education Center in Wimauma, Florida during the 2006 growing season in fi elds managed using current conventional strawberry farming practices. Between Octobe r 12 and October 25, 2006, bare-root transplants of the cultivars Albion, Camarosa, Camino Real, Carmine, Strawberry Festival, Ruby Gem, Sugar Baby, Sweet Charlie, Treasure, Winter Daw n, and advanced selections Festival #9, FL 99117, FL 99-164, FL 00-51, and FL 01-116 (Table 3~1) were planted in raised, fumigated beds covered with black plastic mulch. Soil had been fumigated with methyl bromide and chloropicrin at a ratio of 67:33 at 397 kg/ha. Centers of the beds were 1.2 m apart and beds were 0.7 m wide. Two rows of staggered plants were planted in each bed. Plants were spaced 28 cm in the row with 38 cm between the rows. Sp ace between plots for each treatment was 1.1 m. Overhead irrigation was applied for 10 days to establish transplants and then plants were

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40 irrigated and fertilized by drip tape. Treatments were arranged in a randomized complete block design with four replications in successive beds. Plots for each treatment contained 12 plants and were 2.9 m long. Disease incidence of plants naturally inf ected was evaluated on April 6, 2007. Based on the results from the products evaluations, dis ease incidence provided a better separation among treatments than disease severi ty. Thus, disease incidence wa s selected for this cultivar evaluation. Five leaves we re collected from six plants in each plot, for a total of 30 leaves or 90 leaflets. Marketable yield was determined by harv esting fruit twice per week for each plot. Fruit was hand picked and graded twice per week from 12 December, 2006 to 30 March, 2007 for a total of 32 harvests. Fruit were considered mark etable if there were no visible symptoms of ALS on the calyx or other fruit rot diseases, and fr uit weight for each berry was 10 g or greater. Data were subjected to analysis of variance (ANOVA) and the means were separated using Fishers Protected Least Significant Difference (LSD, P 0.05). Statistical analyses of disease incidence and yield were performed using software pack age Statistix 8 (Stati stix 8, Tallahassee FL). Results Cultivar Evaluation Differences in cultivar susceptibility were significant ( P 0.05) with the range of percent disease incidence from 21% to 75%. One cultivar, Treasure, had a significantly lower disease incidence compared to all other cultivars w ith 21% disease incidence (Table 3~1). The genotypes with the highest dise ase incidence were advanced selections FL 99-117 and FL 01116 with 75% and 70% disease incidence, re spectively, and cultivars Winter Dawn and Camarosa with 74% and 68% dise ase incidence, respectively (Tab le 3~1). Nine cultivars had disease incidences that were not significant from each other, but were significantly different from cultivars with the highest or lowest disease in cidence. These included Camino Real, FL 99-

PAGE 41

41 164, Festival #9, Sweet Charlie, Albion, Straw berry Festival, Sugar Baby, Carmine, and FL 00-51 (Table 3~1). Marketable Weight Differences in fruit yield between cultivars were significant ( P 0.05) with a range of 17,976 kg/ha to 41,112 kg/ha. Cultivars producing th e most fruit were Strawberry Festival, Camarosa, Camino Real, and Ruby Gem with 41,112, 39,811, 38,403, and 37,938 kg/ha, respectively, and differences between these four we re not significant. Sugar Baby and Festival #9 produced the least amount of fruit with 17,976 kg/ha and 19,350 kg/ha, respectively, and differences between these two we re not significant. Among treat ments that produced slightly more fruit than Sugar Baby and Festival #9 were advanced selections FL 01-116, FL 99-117, and FL 00-51 which produced 24,739, 24,790, and 24,988 kg/ ha respectively. Albion, Winter Dawn, Sweet Charlie, Treas ure, FL 99-164, and Carmine had significantly higher yields than Sugar Baby, Festival #9, FL 01-116, FL 99-117, and FL 00-51 but al so had significantly lower yields than Strawberry Festival, Camarosa, Camino Real, and Ruby Gem ( P 0.05). The incidence of ALS was not related to yield ( P =0.08). Treasure, the mo st resistant cultivar, produced 34,098 kg/ha of fruit which was only sl ightly higher than, and not significantly different from the most susceptible cultivar Winter Dawn, which produced 31,346 kg/ha. In comparison, the moderately susceptible cultivar St rawberry Festival produced the highest yield of 41,112 kg/ha. Differences in yield could not be attributed to ALS (Table 3~1). Discussion This study demonstrates that all cultivars of strawberry grown in Florida are susceptible to ALS; however, the degree of susceptibility va ries greatly. ALS susceptibility among Florida cultivars can differ by as much as 50%, as was ob served between Treasure and Winter Dawn. No cultivars of strawberry are immune to ALS and for purposes of this discussion, cultivars are

PAGE 42

42 placed in one of three groups: cultivars that ar e mildly, moderately, or highly susceptible to ALS. Previous studies have eval uated susceptibility in a number of cultivars, however only two, Sweet Charlie and Chandler ar e currently used in Florida ( 25, 15). Hildebrand et al. (15) compared the reactions of 22 cultivars of strawberry to ALS. Chandler and Sweet Charlie were included in that study and compared to the ot her cultivars in that tr ial, they were highly susceptible. However, compared to cultivars used in Florida, Sweet Char lie is only moderately susceptible. Although Chandler is not used as much as other current cultivars, and although it was not included in this study, it is likely that Chandler woul d perform similarly to Sweet Charlie based on the similar susceptibili ties reported by Hildebrand et al. (15). Strawberry Festival is current ly the most popular cultivar of strawberry used in Florida. One reason for the popularity of Strawberry Festival apart from desirable consumer traits, is its high potential yield. Although popul ar, growers have speculated th at Strawberry Festival is highly susceptible to ALS and those questions ha ve been addressed in this study. Strawberry Festival is a moderately susceptible cultivar co mpared to the other cult ivars grown in Florida, with a disease incidence rating of 58.3%. De spite a moderate disease incidence rating, Strawberry Festival produced the highest total yield of all the cultivar s tested, slightly over 7000 kg/ha more than Treasure, the cultivar most resistant to ALS in our study. The results from this study are not conclusive to indicate if ALS signi ficantly impacts yield among cultivars with different levels of susceptibility. Differences in yield and disease incidence were significant between cultiv ars, however there was no corre lation observed between disease incidence and yield. One explanation for the la ck of differences in yield among cultivars with different levels of susceptibility to ALS is that other important diseases of strawberry also reduced yield in certain cultiv ars. Treasure and Camaros a are highly susceptible to

PAGE 43

43 anthracnose fruit rot, caused by Colletotrichum acutatum and Sweet Charlie and Camino Real are highly susceptibl e to gray mold, caused by Botrytis cinerea These two diseases combined caused losses of 13.1, 8.6, 3.8, and 12.0% of the total number of berries harvested from each of these cultivars, respectively (dat a not shown). In contrast, Winter Dawn and Carmine lost less than 2.0% of the fruit to an thracnose and gray mold. Thus, in addition to natural differences in yield usually observed among cultivars, variable losses due to other diseases were observed making it difficult to correl ate differences in yield to ALS. In addition, ALS is a foliar disease and losses are much more difficult to quantify than losses due to fruit diseases because they are not a direct result of in fection. In addition, certa in cultivars reach peak blooms at different points during the season. W inter Dawn for example is typically planted early because it produces very quickly and has its peak production before Strawberry Festival, which has a more steady production throughout the season and usually peaks in late February/March. ALS was evaluated at the end of th e season. It may be of interest to see what levels of disease exist at peak production times for different cultivars. Perhaps an experiment comparing plants infected with ALS and plants free of ALS would accurately evaluate the effect of ALS on yield; however, maintaining plots free of ALS in close proximity to plots infected with ALS have been proven to be very difficult. Based on these findings, it is clear that ALS affects cultivars differently, but, under the conditions that the plants were exposed to dur ing the 2006-07 season, the differences were not dramatic enough to have a significant impact on yield. Strawberry Festival is a good choice based on marketability and it also produces high yields regardless of moderate levels of disease incidence. Efforts to identify resistance while maintaining desirable consumer traits and high yield are not complete, but recent work focused on obtaining a cultivar with all these traits has

PAGE 44

44 been promising. Of the four advanced selectio ns from a breeding program, one was only mildly susceptible. Advanced selection 99-164, with a disease incidence rating of 48.3%, had a low to moderate susceptibility and had numbers similar to Treasure with respect to yield. Recently, Lewers et al. (32) determined that three to four unlinked loci are i nvolved in conferring ALS resistance. They concluded re sistant progeny could be selected ; however, studies with large populations would be necessary to produce cultivars that were not only resistant to ALS, but were also good producers of quality fruit. The potential for a resistant cultivar is on the horizon and given time, the combination of a resistant cu ltivar with high yield and quality fruit is a reasonable possibility

PAGE 45

45 Table 3~1. Disease incidence of a ngular leaf spot in cultivars of annual strawberry in Florida for 2006 season. Cultivar/Selection Disease Incidence (%) Mkt Wt (kg/ha) Albion 55.8cdx 29011ef Camarosa 68.3abc 39811ab Camino Real 47.5de 38403abc Carmine 61.8abcd 35289bcd Strawberry Festival 58.3bcd 41112a Festival #9 51.0d 19350g Ruby Gem 32.5ef 37938abc Sugar Baby 60.0abcd 17976g Sweet Charlie 55.8cd 31564de Treasure 20.8f 34098cd Winter Dawn 74.3ab 31346de 99-117 75.0a 24790f 99-164 48.3de 34897cd 00-51 64.0abcd 24987f 01-116 70.0abc 24739f x Means within columns followed by the same lett er are not significantly different by Fishers protected LSD ( P 0 .05)

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46 CHAPTER 4 ASSOCIATION OF ANGULAR LEAF SPOT LESIONS AND Pseudomonas spp. WITH SYMPTOMATIC TISSUE OF STRAWBERRY. Introduction Angular Leaf Spot (ALS), caused by Xanthomonas fragariae has been described by Kennedy and King as dark green angular water soaked lesions on the under side of the leaf (24). Under optimal conditions these lesions can increa se in size and number, and in early mornings when surface moisture from dew or rain is abund ant, a mass of bacterial exudate appears on the under surface covering the water soaked lesi ons (24, 37, 8, 36, personal observation). The function of this exudate, whether it may be for survival during unfa vorable conditions or plugging vascular tissues, is not kno wn (37). Eventually lesions become necrotic and are more difficult to differentiate from other dis eases such as common leaf spot caused by Mycosphaerella fragariae. Recently, detection of X. fragariae has improved by using serological and molecular techniques (49, 47, 45), however; is olation of the pathogen is st ill difficult. Isolation of X. fragariae requires 3 to 5 days incuba tion at 20 to 24 C on a suitable medium such as Wilbrinks medium. The morphology and growth characteristics make direct isolation difficult as growth of the bacterium is slow and faster grow ing saprophytes mask the growth of X. fragariae in culture plates (7, 49). Colonies of X. fragariae are minute and translucent rather than yellow at the early stages of growth. During the 2005 and 2006 strawberry seasons, numerous tissue isolations were performed to obtain X. fragariae strains. Isolations were made under optimal conditions when lesions were oozing and symptoms were easily identifiable as ALS; however, Pseudomonas spp. were consistently isolated from the le sions. We report on the association of Pseudomonas spp. with angular leaf spot lesions fr om symptomatic tissue on strawberry.

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47 Materials and Methods Tissue Isolations During the 2005 and 2006 strawberry seas ons, leaf samples from Strawberry Festival, Winter Dawn, and Sweet Charlie were collected from fields at the GCREC, and leaf samples from breeding advanced se lections 99-117, 01-116, 97-39, and 99-164 were collected from a farm in Floral City, FL. Tissu e isolations were performed by dissecting a small piece of tissue containing one lesion (~2mm x 2 mm) that exhibited visual oozing when viewed under the stereoscope and placing the lesion in a 1.5 ml microcentrifuge tube with 200l to 1 ml of sterile water or 0.01 M magnesium sulfate solution (MgSO4H2O). Tissue was macerated in tubes with a microcentrifuge pestle and the suspension was streaked on nutrient agar or Wilbrinks media or both. Plates were incuba ted at 24 to 28 C for 24 to 72 h. Individual colonies were transferred to culture plates to isolate pure cultures. Hypersensitive Response Tests Selected strains of Pseudomonas spp. isolated from field samples and a strain of Xanthomonas fragariae (ATCC 33239) were tested to determ ine if they induced a hypersensitive response in tomato and tobacco. The fluorescent pseudomonads and X. fragariae strains were grown on nutrient agar for 24 hours and on Wilbri nks medium 48 to 72 hours, respectively. The bacteria were suspended in steril e tap water. Mature tobacco l eaves and four week old tomato plants were infiltrated with a water control or bacterial suspensions adjusted to 0.1OD at 590 nm. Plants were examined 16 to 48 hours later for a hypersensitive response. Fatty Acid Analysis A single colony of each strain was transferred from a nutrient agar culture to trypticase soy broth agar, and after 24 h of growth at 28C ap proximately 40 mg of cells was collected for analysis. The bacterial fatty acids were derivatiz ed to their methyl esters, separated by gas

PAGE 48

48 chromatography, and identified using the Microbial Identification System software (version 3.6; MIDI, Newark, DE). Oxidase Test Cultures identified as fluorescent Pseudomonas spp. by fatty acid analysis were tested for indophenol oxidase production. Pseudomonas spp. were incubated overn ight and tested with oxidase reagent droppers (Becton, Dickinson and Company; Sparks, MD.) Ice Nucleation Ice nucleation activity was tested for the fluorescent Pseudomonas spp. isolated from field samples and Pseudomonas spp. received from tissue isolations from California, North Carolina, and Vermont. Suspensions were prepared fr om 24 h cultures grown on NA and adjusted to A590=0.1. Aluminum weigh dishes were placed on the surface of the al cohol/ice mixture with the temperature at approximately -10C and 10 drops of suspension from each isolate were placed in the aluminum dish. The number of dr ops that froze after 1 minute was recorded for each strain. Pathogenicity Tests Strawberry Festival and Sweet Charlie pl ants obtained from a region free of ALS in 6inch pots were inoculated with suspensions of X. fragariae Pseudomonas spp., a mixture of both species, or water control (sterile tap water or MgSO4 7H2O). Suspensions were prepared and diluted to A590=0.1 from 24 h Pseudomonas spp. cultures, and 72 h X. fragariae cultures. Two groups with two plants per treatment, one set left at room temperature and the other placed in a growth chamber, were inoculated and placed in plastic bags immediat ely following inoculation. The growth chamber was set to temperatur e fluctuation of 3C to 18C and a 12 hour photoperiod. Plants were observed daily for symptoms after one week post-inoculation.

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49 Results and Discussion Tissue Isolations Of the numerous leaf samples collected to is olate the causal agent of ALS of strawberry, none resulted in plates that produced a pure culture of X. fragariae Interestingly, many of the plates contained almost pure cult ures of white colonies, and on Wilbrinks media, these same colonies produced a green pigment. It appeared that these cultures consistently isolated from leaves with symptoms of ALS were Pseudomonas spp. There were six samples from the GCREC and 11 samples from Floral City (Table 4~1). During this same time, we also received 1, 4, and 10 strains isolated from symptomatic ALS lesions from North Carolina, Vermont, and California, respectively, that appeared to be Pseudomonas spp. (Table 4~1). Characterization of Strains Isolates 13, 27, and 31 were all identified as Pseudomonas spp. by fatty acid analysis (Table 4~1). Those same isolates were evaluated to see if th ey induced hypersensitive responses (HR) in tomato and the oxidase reacti on. All but one of the strains, number 33, failed to induce an HR or induced only a weak HR. Oxidase production was weak in most strains tested. Initially these strains tested slightly ox idase positive but eventually turned negative. Two strains, numbers 33 and 36, were oxi dase negative. Ice nucleation activity was tested for several strains. Strains 33 from Floral City, 52 a nd 54 from Vermont, and strains 70 and 71 from California all exhibited str ong ice nucleation activity. Pathogenicity Test Pathogenicity tests for all Pseudomonas spp. strains were negative (Table 4~1). As a result of isolating almost exclusively Pseudomonas spp. from the tissue samples, we began to suspect some type of interaction or asso ciation between lesions of ALS caused by X. fragariae and these Pseudomonas spp.

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50 Bacteria employ a number of st rategies to successfully co lonize host plants and more specifically, epiphytes are bacteria that can colonize and survive on plant surfaces (2). Pseudomonas syringae is a well known pathogen of many crops but it has also been reported as an epiphyte and can exist as aggreg ates on surfaces of leaves wher e scarce nutrients are available (6, 12, 27, 23). For example, some P. syringae strains produce indole-3acetic acid (IAA), which is capable of inducing nutrient leakage on the su rface of plants thus making nutrients available for survival (2). Many bacteria produce extr acellular polysaccharides which can modify the environment by helping cells adhere to leaf surf aces and preventing desic cation of cells (2). Frequently, a mass of bacterial exudate is associated with ALS lesions during periods of high humidity. Another mechanism that can assi st bacteria in colonization is ice nucleation. During freeze events, ice nucleation active bact eria can injure leaf surfaces in creasing frost damage to plants which may provide more openings for pathogenic b acterial ingress (6, 34, 53). Given the fact that some of the P. syringae strains associated with strawberry were ina+, it is possible that this served some role in colonization of the leav es. However, no symptoms were observed in pathogenicity tests with only P. syringae strains on strawberry. An association of P. syringae and a Xanthomonas species has previously been reported in citrus lesions by Stall et al (51). They reported that P. syringae was often associated with citrus canker lesions in Argentina. While P. syringae by itself produced sympto ms typical of citrus blast, an association of P. syringae and Xanthomonas campestris pv. citri produced symptoms not typical of either citrus bl ast or citrus canker. Pathogeni city tests on strawberry with X. fragariae P. syringae and a mixture of both organisms had different symptoms as well. Two plants inoculated with X. fragariae left at room temperature developed typical ALS lesions

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51 between 6 and 14 days that were described by Ke nnedy and King (24) in la boratory inoculations; however, no bacterial oozing or ti ssue necrosis developed with these symptoms as was observed in the field (Figure 4~1A). Additionally, the leve l of humidity had an effect on the expression of these early symptoms. When removed from hi gh humidity, expression of ALS symptoms in plants inoculated with X. fragariae was reduced. Plants inoculated only with P. syringae did not develop symptoms, but the combination of the two organisms produced symptoms in two plants in the growth chamber that were identical to th ose seen in the field during the season (Figure 4~1B). After three weeks, le sions on strawberry plants i noculated with a mixture of X. fragariae and Pseudomonas spp. developed oozing lesions that became necrotic at the site of the lesion. After lesions began oozing, both plants remained in bags but were transfered to room temperature where development of symptoms progressed rapidly. In order to determine if the Pseudomonas strains were colonizing ALS lesions in strawberry, isolations of lesions at various stages of infection we re performed and populations of Pseudomonas strains were enumerated. Isolations we re made from a leaf with no symptoms, early lesions (no oozing), advanced lesions (oozing, no necrosis) and necrotic tissue. Populations of Pseudomonas were not different between asym ptomatic tissue and early lesions; however, populations increased by three orders of magnitude between early lesions and advanced lesions. Populations on necrotic tissue were too numerous to count (data not shown). This preliminary study indicates that an association between ALS lesions and Pseudomonas strains exists based on the observations that the Pseudomonas spp. tested exacerbated the symptoms caused by X. fragariae More data is necessary to statistically compare populations of Pseudomonas spp. associated with ALS le sions. Because strawberry was not an apparent host to the Pseudomonas strains isolated, and if the Pseudomonas spp. are

PAGE 52

52 responsible for the exacerbation of the sympto ms, there may be need for a new management strategy for ALS in strawberry.

PAGE 53

53Table 4~1. Characterization of Pseudomonas strains associated with lesions of angular leaf spot on strawberry during 2005-06 and 2006-07 season. Strain Designation Location Colony Color Fatty Acid HR Oxidase Ice Nucleation Path Test 12 GCREC, FL White ND Neg ND ND Neg 13 GCREC, FL White Pseudomonas syringae pv. tomato Weak Weak + ND Neg 14 Floral City, FL White Pseudomonas syringae pv. tomato Neg Weak + ND Neg 15 Floral City, FL White Pseudomonas syringae pv. tomato Weak Weak + ND Neg 16 Floral City, FL White ND ND ND ND Neg 23 GCREC, FL White ND ND ND ND ND 24 GCREC, FL White ND ND ND ND ND 25 GCREC, FL White ND ND ND ND ND 27 GCREC, FL White Pseudomonas putida biotype B Neg Pos ND ND 28 Floral City, FL White Pseudomonas syringae pv. tomato Neg Weak + ND ND 29 Floral City, FL White Pseudomonas syringae pv. tomato Neg Weak + ND ND 31 Floral City, FL White Pseudomonas syringae pv. tomato Neg Weak + ND ND 32 Floral City, FL White Pseudomonas syringae pv. tomato Neg Weak + ND ND 33 Floral City, FL White Pseudomonas syringae pv. atrofaciens Pos Neg 10/10 Neg 34 Floral City, FL White Pseudomonas syringae pv. tomato Weak Weak + 1/10 Neg 35 Floral City, FL White Pseudomonas syringae pv. tomato Neg Weak + ND ND 36 Floral City, FL White Pseudomonas syringae pv. tomato Neg Neg ND ND 50 North Carolina White ND ND ND 2/10 ND 51 Vermont White ND ND ND 10/10 ND 52 Vermont White ND ND ND 5/10 ND 53 Vermont White ND ND ND 10/10 ND 54 Vermont White ND ND ND ND ND 60 California White ND ND ND ND ND 61 California White ND ND ND ND ND 62 California White ND ND ND ND ND 70 California White ND ND ND 10/10 Neg

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5471 California White ND ND ND 10/10 Neg 72 California White ND ND ND 0/10 ND 73 California White ND ND ND 0/10 ND 74 California White ND ND ND 2/10 ND 75 California White ND ND ND 0/10 ND 79 California White ND ND ND 1/10 ND ND = Not determined

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55 A B Figure 4~1. Angular leaf spot symptoms on stra wberry. A) Angular leaf spot symptoms on strawberry inoculated with X. fragariae only. B) Bacterial oozing a nd slight necrosis from ALS lesions on strawberry inoculated with X. fragariae and Pseudomonas spp.

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56 CHAPTER 5 SUMMARY Overall, the goal of this research was to de velop more effective management practices for angular leaf spot of strawberry. To accomplis h that goal field studies comparing different chemical and biological products we re evaluated, cultivar resistan ce for all cultivars of plants used in Florida was evaluated, differences in yield between treatments and untreated controls were compared, the impact of environmental co nditions on control of angular leaf spot was analyzed, and finally a prelimin ary study on the association of Pseudomonas spp. and lesions of angular leaf spot was pe rformed. From these studies we have learned that Actig ard is a suitable alternative to copper-based pr oducts for suppressing angular l eaf spot without inducing the negative side effect of phytotoxic ity; however, we were not able to conclusively determine if suppression of angular leaf spot or minimizi ng the damage from phytotoxicity could increase yield. This study also produced the first data co mparing resistance to angul ar leaf spot for all cultivars grown in Florida. Similarly, we learne d that differences occur in cultivar resistance and this resistance may be useful as part of a mana gement strategy; however, the effect of angular leaf spot on yield remains a quest ion. The impact of environmental conditions was analyzed and we learned that if optimal conditions occur in th e field; management of this disease will be difficult regardless of methods. Finally, by processing a number of symptomatic tissue samples and performing pathogenicity tests on the strain s resulting from isolat ions, we observed that there was an association between X. fragariae and Pseudomonas spp. based on laboratory inoculations in which symptoms differ ed between plants inoculated with X. fragariae and Pseudomonas spp. More work is required to better unders tand this interaction. Further studies to understand this association may lead to new more effective management of angular leaf spot.

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57 LIST OF REFERENCES 1. Agrios, G. 2005. Plant Pathol ogy. Elsevier Academic Press, Burlington, MA. Pp. 625-626. 2. Beattie, G.A. and Lindow, S.E. 1999. Bact erial colonization of leaves: a spectrum of stratgies. Phytopathology 89: 353. 3. Bock, C.H., Parker, P.E., and Gottwald, T.R. 2005. Effect of simulated wind-driven rain on duration and distance of dispersal of Xanthomonas axonopodis pv. citri from canker-infected citrus trees. Plant Dis. 89:71. 4. Central Science Laboratory. 2003. Angular leaf spot on strawbe rry, EC listed diseases, Ref. no. QIC/34. Crown Copyright, Sand Hutton, York. http://www.defra.gov.uk/planth/qic.htm October 18, 2006. 5. Conover, R.A., and Gerhold, N.R. 1981. Mixtures of copper and maneb or mancozeb for control of bacterial spot of tomato and their compatibility for contro l of fungus diseases. Proc. Fla. State Hort. Soc. 94:154. 6. Dulla, G., Marco, M., Quinones, B., a nd Lindow, S. 2005. A closer look at Pseudomonas syringae as a leaf colonist. ASM News. 71: 469. 7. EPPO/CABI. 1997. Quarantine pets for Europe. 2nd Edition. Edited by Smith, I.M., McNamara, D.G., Scott, P.R., and Holderness, M. CABI International, Wallingford, UK. EPPO A2 List: No. 135. 8. Epstein, A.H. 1966. Angular leaf spot of strawberry. Plant Dis. Rep. 50:167. 9. Florida Agricultural Statistical Directory. 2006. Division of Marketing and Development. http://www.nass.usda.gov/fl/ Pp 10, 13. 10. Gottwald, T.R., Graham, J.H., and Riley, T.D. 1997. The influence if spray adjuvants on exacerbation of citrus bacterial spot. Plant Dis. 81:1305-1310. 11. Gubler, W.D., Feliciano, A.J., and Bordas, A.C. 1999. First report of blossom blight of strawberry caused by Xanthomonas fragariae and Cladosporium cladosporiodies in California. Plant Dis. 83: 400. 12. Hansen, M.A. 2000. Angular leaf spot of cu cumber. Virginia Cooperative Extension Plant Disease Fact Sheets. Publication 450-700W. 13. Henson, J.M., and French, R. 1993. The pol ymerase chain reaction and plant disease diagnosis. Annu. Rev. Phytopathol. 31:81. 14. Hildebrand, D.C., Schroth, M.N., and Wilhelm, S. 1967. Systemic invasion of strawberry by Xanthomonas fragariae causing vascular collapse Phytopathol. Notes 57:1260.

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58 15. Hildebrand, P.D., Braun, P.G., Renderos, W.E ., Jamieson, A.R., McRae, K.B., and Binns, M.R. 2005. A quantitative method for inoculating strawberry leaves with Xanthomonas fragariae factors affecting infection, and cultivar reactions. Can. J. Plant Pathol. 27:16. 16. Howard, C.M. 1971. Occurrence of strawberry angular leaf spot, Xanthomonas fragariae in Florida. Plant Dis. Rep. 55:142. 17. Ivors, K.L., Milks, D.C., and Holmberg, C. 2006. Evaluation of spray programs for control of bacterial leaf spot of tomato, 2006. Plant Disease Management Reports 1:V089 1. 18. Janse, J.D., Rossi, M.P., Gorkink, R.F.J., Derks, J.H.J., Swings, J., Janssens, D., and Scortichini, M. 2001. Bacterial leaf blight of strawberry ( Fragaria (x) ananassa ) caused by pathovar of Xanthomonas arboricola not similar to Xanthomonas fragariae Kennedy and King. Description of the causal organism as Xanthomonas arboricola pv fragariae (pv. nov., comb. Nov. Plant Pathol. 50:653. 19. Johnson, D.A., Inglis, D.A., and Miller, J.S. 2004. Control of potato tuber rots caused by Oomycetes with foliar applications of phosphorous acid. Plant Dis. 88: 1153. 20. Jones, J.B., and Jones, J.P. 1983. Bacterial leaf spot diseases on tomatoes in Florida and the control of two such diseases with bacteriocides. Proc. Fla. State Hort. Soc. 96:101. 21. Jones, J.B., Woltz, S.S., Kelly, R.O., and Harris, G. 1991. The role of ionic copper, total copper, and select bacteriocides on control of bacterial spot of tomato. Proc. Fla. State Hort. Soc. 104:257. 22. Katawczik, M.L., and Ritchie, D.F. 2005. Evalua tion of sprays for the control of bacterial spot of peppers, 2005. Fungicide and Nematicide Tests 61: V060. 23. Keinath, A.P., Wechter, W.P., and Smith, J.P. 2006. First report of b acterial leaf spot on leafy brassica greens caused by Pseudomonas syringae pv. maculicola in South Carolina. Plant Dis. 90: 683. 24. Kennedy, B.W. and King, T.H. 1962. Angular leaf spot of strawberry caused by Xanthomonas fragariae sp. nov. Phytopathology 52:873. 25. Kennedy, B.W., and King, T.H. 1962. Studies on epidemiology of bacterial angular leaf spot on strawberry. Plant Dis. Rep. 46:360. 26. Koike, H. 1964. The aluminum-cap method for te sting sugarcane varietie s against leaf scald disease. Phytopathology 55: 317. 27. Koike, S.T., Cintas, N.A., and Bull, C.T. 2000. Bacterial blight, a ne w disease of broccoli caused by Pseudomonas syringae in California. Plan t Health Progress 10:1094.

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59 28. Lange, H.W., Borsick Herman, M.A., and Smart, C.D. 2005. Comparing efficacy of foliar and soil treatments for bacterial speck of to mato, 2005. Fungicide and Nematicide Tests 61: V055. 29. Lange, H.W., Borsick Herman, M.A., and Smart, C.D. 2006. Comparing efficacy of foliar and soil treatments for bacterial speck of to mato, 2006. Plant Disease Management Reports 1: V009. 30. Langston Jr., D.B. 2005. Evaluation of bact eriocides and biological control materials for suppressing bacterial spot of bell pepper tr ansplants in Georgia, 2005. Fungicide and Nematicide Tests 61: V146. 31. Legard, D.E., Ellis, M., Chandler, C.K., and Pr ice, J.F. 2003. Integrated management of strawberry diseases in wint er fruit production areas. In the strawberry: a book for growers, Pp 111. N. Childers (ed.). Institute of F ood and Agricultural Sciences, Horticultural Sciences Department, University of Florida, Gainesville. Norm Ch ilders Publications. 246pp 32. Lewers, K.S., Maas, J.L., Hokanson, S.C., Gouin, C., and Hartung, J.S. 2003. Inheritance of resistance in strawberry to bacteria l angular leafspot disease caused by Xanthomonas fragariae J. Amer. Soc. Hort. Sci. 128: 2009. 33. Lewis Ivey, M.L., Mera, J.R., and Miller, S. A. 2004. Evaluation of fungicides for the control of bacterial leaf spot of bell peppers, 2004. F&N Tests Vol 60: V096. 34. Lindow, S.E., Arny, D.C., Upper, C.D. 1982. B acterial ice nucleation: a factor in frost injury to plants. Plant Physiol. 70: 1084. 35. Louws, F.J., Wilson, M., Campbell, H.L., Cupples, D.A., Jones, J.B., Shoemaker, P.B., Sahin, F., and Miller, S.A. 2001. Field contro l of bacterial spot and bacterial speck of tomato using plant activator. Plant Dis. 85: 481. 36. Louws, F. 2007. Bacterial angular leaf spot. The Strawberry Grower. April:2 37. Maas, J.L., Pooler, M.R., and Galletta, G.J. 1995. Bacterial angular leafspot disease of strawberry: present status and prospects for c ontrol. Advances in Strawberry Research 14:18. 38. Maas, J.L., Gouin, C., Hokanson, S.C., Hartung, J.S. 2002. Strawberry parent clones US 4808 and US 4809 resistant to bacterial angular leafspot disease caused by Xanthomonas fragariae HortScience 35: 128-131. 39. McGechan, J.K., and Fahy, P.C. 1976. Angular leaf spot of stra wberry. Xanthomonas fragariae: First record of its occurrence in Australia and attempts to eradicate the disease. Aust. Plant. Pathol. Soc. Nwsl. 5: 57-59.

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60 40. Mertely, J.C., Chanler, C.K., Xiao, C.L., and Legard, D.E. 2000. Comparison of sanitation and fungicides for management of Botrytis fr uit rot of strawberry. Plant Dis. 84:1197. 41. Obradovic, A., Jones, J.B., Momol, M.T., Ba logh, B., and Olsen, S.M. 2004. Management of tomato bacterial spot in the field by foliar applications of bacteriophages and SAR inducers. Plant Dis. 88:736. 42. Obradovic, A., Jones, J.B., Momol, M.T., Ol sen, S.M., Jackson, L.E., Balogh, B., Guven, K., and Iriarte, F.B. 2005. Integr ation of biological control ag ents and systemic acquired resistance inducers against bacterial spot on tomato. Plant Dis. 89: 712. 43. Peres, N.A., Rondon, S.I., Price, J.F., and Cantli ffe, D.J. 2004. Angular leaf spot: a bacterial disease in strawberries in Florida. Flor ida Cooperative Extension Service. PP120:1. 44. Peres, N.A., and Roberts, P. 2005. 2005 Fl orida Plant Disease Management Guide: strawberry. Florida Cooperative Extension Service. PDMGV3-50:1. 45. Pooler, M.A., Ritchie, D.F., and Hartung, J.S. 1996. Genetic relationships among strains of Xanthomonas fragariae based on random amplified polymorphic DNA PCR, Repetitive extrageninc palindromic PCR, and Enterobacter ial repetitive intergenic consensus PCR data and generation of multiplexed PCR primers useful for the identification of this phytopathogen. Appl. and E nviron. Microbiol. 62:3121. 46. Ritchie, D.F. 2005. Copper materials, FlameO ut, ProPhyt, and Seranade ASO for bacterial spot management on peaches, 2005. Fungi cide and Nematicide Tests 61: STF007. 47. Roberts, P.D., Jones, J.B., Chandler, C.K., Sta ll, R.E., and Berger, R.D. 1996. Survival of Xanthomonas fragariae in summer nurseries in Florida detected by specific primers and nested polymerase chain r eaction. Plant Dis. 80:1283. 48. Roberts, P.D., Berger, R.D., Jones, J.B., Ch andler, C.K., and Stall, R.E. 1997. Disease progress, yield loss, and control of Xanthomonas fragariae on strawberry plants. Plant Dis. 81:917. 49. Rowhani, A., Feliciano, A.J., Lips, T., and G ubler, W.D. 1994. Rapid identification of Xanthomonas fragariae in infected strawberry leaves by Enzyme-Linked Immunosorbent Assay. Plant Dis. 78:248. 50. Schaad, N.W., Jones, J.B., and Chun, W. 2001. Laboratory guide for identification of plant pathogenic bacteria, 3rd edition. APS Press. St. Paul, MN. Pp. 176. 51. Stall, R.E., Marco, G.M., and Canteros, B.I. 1984. Association of Pseudomonas syringae pv. syringae with citrus canker in Argentina. Proc. Int. Soc. Citriculture 1: 389. 52. Timmer, L. W.; Garnsey, S. M., and Graham, J. H. eds. Compendium of Citrus Diseases, 2nd ed. St. Paul, MN: APS Press, St Paul, MN

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61 53. Wowk, B. and Fahy, G.M. 2002. Inhibition of bacterial ice nucleation by polyglycerol polymers. Cryobiology 44: 14.

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62 BIOGRAPHICAL SKETCH Gary Todd Cooper was born in Columbus, Ohio to Gary and Ailene Cooper. He and his family moved to southwest Florida and he gra duated from Port Charlotte High School in 1987. After high school graduation, he worked in his familys business until 1991. He entered the automotive industry in 1991 and worked as an au tomatic transmission repair technician. While working as a repair technician, he began his undergraduate degree attending night classes and eventually transitioned into a career in scie nce by accepting a position studying bioaerosols at Battelle Memorial Institute in Columbus, Ohio. He received his Bachelor of Science in life science from Otterbein College after completing eight years of part-time school. Immediately following graduation, he accepted an offer to pursue a Master of Science in plant pathology at the University of Florida. He has been marri ed since October 12, 2002 to Aimee, who is also a graduate of Otterbein College. He is looking forward to pursuing his Ph.D. at the University of Florida in the fall of 2007.


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