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The Role of Tanos and Its Components in the Management of Bacterial Spot of Tomato and Pepper, and Bacterial Leaf Spot o...

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Title: The Role of Tanos and Its Components in the Management of Bacterial Spot of Tomato and Pepper, and Bacterial Leaf Spot of Lettuce
Physical Description: 1 online resource (72 p.)
Language: english
Creator: Fayette, Joubert
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: cymoxanil, famoxadone, lettuce, pepper, tanos, tomato, 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: Tank mixes of pesticides are often used in plant disease control. The effects of Tanos and its components, Famaxadone and Cymoxanil, in combination with copper and mancozeb in some instances, were evaluated on the in vitro growth of strains of Xanthomonas perforans, X. euvesicatoria, X. campestris pv vitians, on disease control of bacterial spot of pepper and tomato in the greenhouse, and on the population dynamics of Cur strains of X. perforans and X. euvesicatoria on tomato leaves and pepper leaflets. Both in vitro and greenhouse trials demonstrated that Tanos and its components do not have any bactericidal activity. In some instances, Tanos tended to promote bacterial growth. However, the addition of Tanos to the Copper/Mancozeb mixture tended to induce a modest synergistic increase in the toxicity of the combination. It appears that both components of Tanos, Cymoxanil and Famaxadone, are essential for synergistic interaction since the addition of Cymoxanil or Famaxadone to copper did not induce significant reduction of bacterial populations in comparison to copper alone. In the greenhouse trials, levels of disease control were similar for Kocide alone, and Kocide/Tanos in most the trials. In some instances, the mixture Kocide/Tanos led to similar level of disease control as the standard mixture of Kocide/Manzate, and Kocide + Tanos + Manzate. The use of Famaxadone + Cymoxanil could possibly reduce the application of copper, which might help reduce the selection of copper-resistant Xanthomonas strains. Tanos and its components did not have any bactericidal activity against the epiphytic populations of X. perforans, and X. euvesicatoria on tomato leaves and pepper leaflets. Different mixtures with copper, Famaxadone, Cymoxanil, and Tanos generally did not differ at statistical level in a given sampling period. In one trial with a strain of X. perforans, the standard mixture copper/mancozeb did consistently reduce the pyllosphere populations in each sampling period in comparison to the control.
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 Joubert Fayette.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Roberts, Pamela D.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042254:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042254/00001

Material Information

Title: The Role of Tanos and Its Components in the Management of Bacterial Spot of Tomato and Pepper, and Bacterial Leaf Spot of Lettuce
Physical Description: 1 online resource (72 p.)
Language: english
Creator: Fayette, Joubert
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: cymoxanil, famoxadone, lettuce, pepper, tanos, tomato, 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: Tank mixes of pesticides are often used in plant disease control. The effects of Tanos and its components, Famaxadone and Cymoxanil, in combination with copper and mancozeb in some instances, were evaluated on the in vitro growth of strains of Xanthomonas perforans, X. euvesicatoria, X. campestris pv vitians, on disease control of bacterial spot of pepper and tomato in the greenhouse, and on the population dynamics of Cur strains of X. perforans and X. euvesicatoria on tomato leaves and pepper leaflets. Both in vitro and greenhouse trials demonstrated that Tanos and its components do not have any bactericidal activity. In some instances, Tanos tended to promote bacterial growth. However, the addition of Tanos to the Copper/Mancozeb mixture tended to induce a modest synergistic increase in the toxicity of the combination. It appears that both components of Tanos, Cymoxanil and Famaxadone, are essential for synergistic interaction since the addition of Cymoxanil or Famaxadone to copper did not induce significant reduction of bacterial populations in comparison to copper alone. In the greenhouse trials, levels of disease control were similar for Kocide alone, and Kocide/Tanos in most the trials. In some instances, the mixture Kocide/Tanos led to similar level of disease control as the standard mixture of Kocide/Manzate, and Kocide + Tanos + Manzate. The use of Famaxadone + Cymoxanil could possibly reduce the application of copper, which might help reduce the selection of copper-resistant Xanthomonas strains. Tanos and its components did not have any bactericidal activity against the epiphytic populations of X. perforans, and X. euvesicatoria on tomato leaves and pepper leaflets. Different mixtures with copper, Famaxadone, Cymoxanil, and Tanos generally did not differ at statistical level in a given sampling period. In one trial with a strain of X. perforans, the standard mixture copper/mancozeb did consistently reduce the pyllosphere populations in each sampling period in comparison to the control.
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 Joubert Fayette.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Roberts, Pamela D.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042254:00001


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THE ROLE OF TANOS AND ITS COMPONENTS IN THE MANAGEMENT OF
BACTERIAL SPOT OF TOMATO AND PEPPER, AND BACTERIAL LEAF SPOT OF
LETTUCE

















By

JOUBERT FAYETTE


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

2010

































2010 Joubert Fayette
































To my parents, Nativida Codio and Gerard Fayette who have always provided the best
for my personal and professional growth









ACKNOWLEDGMENTS

I would like to thank my committee members Drs. Pamela D. Roberts, Kenneth L.

Pernezny and Jeffrey B. Jones for this opportunity to pursue a master and also their

support, constructive criticism and guidance during the research project and the

preparation of this manuscript.

I would like to thank all those who helped me in this project: Dr. Ryan Donahoo,

Dr. Robert E. Stall, Jerry Minsavage, Rod Sytsma and David Ballesteros. There were

people who supported me in different ways at different times, and I am grateful for their

assistance: Katia Vernord, Lemane Delva, Edrice Fleurimond, Esnan Ambeau, Patricia

Desir, Pierre-Paul Audate and Marc Evens Jean-Jacques.

I thank my father, Gerard Fayette, for his love. I also thank Sheila, Breton, Carl

Renand, Wolfert, Brillant and Patricia Fayette and Immacula Guerrier for their constant

support and enthusiasm.









TABLE OF CONTENTS

page

A C KNOW LEDG M ENTS ............... ....................... ................ ............... 4

L IS T O F T A B LE S ........................ ................. ........... .... ............................... 7

LIS T O F F IG U R E S .................................................................. 8

ABSTRACT .............. .. ........ .. ............ ..........................9

CHAPTER

1 BACTERIAL SPOT OF PEPPER AND TOMATO ............................. ............... 11

History and Strain Diversity............................................... .................... 11
E p ide m io lo gy .................................................................................................. 15
D disease C control .................................................................................................. 16
Copper-Resistant Strains................................ ............... 17
Evaluation of Copper-Resistant Strains .................................................. 19

2 BACTERIAL LEAF SPOT OF LETTUCE ........................................................ 22

E co no m ic Im pacts ............... ....................................................................... ... 24
Sym ptoms..................... ...................... ............... 25
E p ide m io lo gy .................................................................................................. 2 5
C o n tro l ................................................... ............................................... 2 6

3 CHEMICAL CONTROL AND INTERACTIONS BETWEEN FUNGICIDES ............. 28

Interactions Between Fungicides ................ ........... ... ............. .. .. .............. 29
Mode of Action of Copper, Tanos, Cymoxanil and Famaxadone ............................ 30
C opper ................. ................. ............ ... ... ............................ 30
Mancozeb................................ ......................30
Tanos, Cymoxanil and Famaxadone ............................................. 31

4 THE ROLE OF TANOS AND ITS COMPONENTS IN THE CONTROL OF
BACTERIAL SPOT OF TOMATO AND PEPPER, AND BACTERIAL LEAF
S PO T O F LETT U C E ........................... ...... ................................... 33

Materials and Methods................................ ............... 34
S tra in s ........................................................................ .... ........ 3 4
In vitro Assays ............... .............................. 34
Greenhouse Experiment ................... ........ ................. .............. 35
Inoculation and assay procedure ............ ... ........ ..... ......... ........ 35
R eating and statistical analysis ............ .. ........ .. .......... .. ............... 36
Results .............................................. 37









In vitro assays .................. ............ ......... .. ...................... 37
Disease Control on Tomato in the Greenhouse .......... ......... .. ........... .. 43
Disease Control on Pepper in the Greenhouse......... ............ ............ .. 43
D discussion .............. ......... ........ ... ................................... 43

5 INFLUENCE OF TANOS AND ITS COMPONENTS IN THE POPULATION
DYNAMICS ON THE LEAVES .................. ......................... ........ ......... 47

Materials and Methods....................................... .......... 50
Strains and Inoculum ............................................................ 50
Leaf Sampling and Assay Procedure .......... ........... ..... ...... ........... 50
R e s u lts ............ .......... ... ... .......... .................................................. ...... ............... 5 1
D discussion .............. ......... ........ ... ................................... 56

6 CO NCLUSIO NS ........................ ...... .............. ........... .......... 60

LIST O F R E FE R E N C ES ............ .......... ...................................... ............... 62

B IO G RA PH IC A L S K ETC H ............ .......... ...... .......... ........................... ............... 72









LIST OF TABLES


Table page

1-1 Resistance in tomato and pepper and avirulence genes that interact with
them ....... .......................................... .......... 14

2-1 Difference among Xanthomonas campestris pv vitians and Xanthomonas sp.
strains, based on carbon sources in Biolog GN microplate assay (Sahin et al.
2003) ............................................................... ..... ..... ......... 23

4-1 List of the treatments and their respective rate ............... .............................. 35

4-2 In vitro trials with the copper-resistant strain T4 (Xanthomonas perforans)
after incubation with various chemical compounds............... .. ........... 38

4-4 In vitro trials with the copper-sensitive strain L7 (Xanthomonas campestris
pv. vitians) after incubation with various chemical compounds ........... .......... 40

4-5 Numbers of lesions caused by Xanthomonas perforans strain T4 on tomato
plants treated with chemicals in greenhouse trials ................. .............. ....... 41

5-1 Populations dynamics of a strain ofX. euvesicatoria on pepper leaflets......... 52

5-2 Populations dynamics of a strain ofX. euvesicatoria on pepper leaflets......... 53









LIST OF FIGURES


Figure page

1-1 Symptoms of bacterial spot on pepper leaves and tomato fruits ................... 12

3-1 Structure of the fungicide Mancozeb ...... ...................... ............ 31

3-2 Structure of the active ingredients in the formulation of Tanos............. ........... 32

5-1 Population dynamics of strain of X. euvesicatoria on pepper leaflets,Trial 1 ...... 57

5-2 Population dynamics of a strain of X. euvesicatoria on pepper leaflets, Trial 2.. 58









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

THE ROLE OF TANOS AND ITS COMPONENTS IN THE MANAGEMENT OF
BACTERIAL SPOT OF TOMATO AND PEPPER, AND BACTERIAL LEAF SPOT OF
LETTUCE

By

Joubert Fayette

August 2010

Chair: Pamela D. Roberts
Major: Plant Pathology

Tank mixes of pesticides are often used in plant disease control. The effects of

Tanos and its components, Famaxadone and Cymoxanil, in combination with copper

and mancozeb in some instances, were evaluated on the in vitro growth of strains of

Xanthomonas perforans, X. euvesicatoria, X. campestris pv vitians, on disease control

of bacterial spot of pepper and tomato in the greenhouse, and on the population

dynamics of Cur strains of X. perforans and X. euvesicatoria on tomato leaves and

pepper leaflets.

Both in vitro and greenhouse trials demonstrated that Tanos and its components

do not have any bactericidal activity. In some instances, Tanos tends to promote

bacterial growth. However, the addition of Tanos to the copper/mancozeb mixture tends

to induce a modest synergistic increase in the toxicity of the combination. It appears that

both components of Tanos, Cymoxanil and Famaxadone, are essential for synergistic

interaction since the addition of Cymoxanil or Famaxadone to copper did not induce

significant reduction of bacterial populations in comparison to copper alone. In the

greenhouse trials, levels of disease control were similar for copper alone, and









copper/Tanos in most the trials. In some instances, the mixture copper/Tanos led to

similar level of disease control as the standard mixture of copper/mancozeb, and copper

+ Tanos + Mancozeb.

Tanos and its components do not have bactericidal activity against the epiphytic

populations of X. perforans, and X. euvesicatoria on tomato leaves and pepper leaflets.

Different mixtures with copper, Famaxadone, Cymoxanil, and Tanos generally did not

differ at stastical level. In one trial with a strain of X. perforans, the standard mixture

copper/mancozeb did consistently reduce the pyllosphere populations in each sampling

period in comparison to the control.









CHAPTER 1
BACTERIAL SPOT OF PEPPER AND TOMATO

Vegetable production is one of the most important agricultural activities in Florida.

This state ranks first in the United States in terms of fresh-market tomato and pepper

value. U.S consumption of fresh tomatoes increased 71%, from 7.0 kg per capital in

1991 to 9.4 kg per capital in 2006. In 2007, the Florida tomato industry was valued at $

464 million (USDA, ERS, 2008a). The country relies heavily on Florida for the supply of

fresh peppers from October through June. In 2007, Florida harvested 17 500 acres of

bell pepper, valued at over $ 183 million (USDA, ERS, 2008b).

Both crops are affected by many diseases. Bacterial spot is one of the major ones

and occurs wherever tomato and pepper are grown. It is particularly troublesome in

tropical and subtropical areas (Jones et al., 1998a). Production is affected by the

disease, which can result in great economic losses. In some fields, loss of foliage may

fluctuate between 50-70% (Pohronezny and Volin, 1983). Crop losses result from the

reduction in yield due to defoliation and severely spotted fruits, which are not suitable

for the market (Jones, 1991). Bacterial spot is a major concern to transplant growers

because many states that receive seedlings from Florida require these transplants to be

free of the pathogen. (Sun et al.,2002).

History and Strain Diversity

Bacterial spot disease of tomato (Solanum lycopersicum) was first observed in

1914 in South Africa and described as a tomato canker by Ethel Doidge (1920). The

causal agent was named Bacterium vesicatorium. Gardner and Kendrick (1921)

described a similar disease in the United States and referred to it as bacterial spot.

They named the causal bacterium B. exitiosum; Doidge's name took precedence A









similar disease on pepper (Capsicum annum) was described by Gardner and Kendrick

(1923). Numerous studies showed that the bacterial pathogens from pepper and

tomato used in the initial studies induce disease in both plants, and it was believed for

many years that cross infection could occur in field (Stall et al.,, 2009).






















Figure 1-1. Symptoms of bacterial spot on pepper leaves and tomato fruits

Symptoms induced by these different xanthomonads are similar. The

characteristic symptoms include the development of small, brown to black lesions of 1-3

mm diameter, with or without yellow halos, that affect all the aboveground organs

(Figure 1-1). Subsequent enlargement and coalescence of spots occur later, which

leads to the browning of the entire leaf and defoliation (Kucharek, 1994). However,

there are some small differences, such as a shot-hole symptom induced by X. perforans

on tomato (Stall et al.,, 2009). The pathogen can be readily isolated from infected

tissue. It is a gram-negative, rod-shaped bacterium. It is motile with a single polar

flagellum, strictly aerobic, and measures 0.7 to 1.0 pm by 2.0-2.4 pm. Other









characteristics include production of a yellow, water-insoluble pigment, xanthomonadin

and an extracellular polysaccharide (EPS) named xanthan.

Based on pathogenicity profiles, many races have been identified. A race is

characterized by its ability to grow on a cultivar with or without specific genes for

resistance (Pernezny et al. 2008a). The letters P, T and PT are respectively assigned to

races pathogenic to pepper only, tomato only or both pepper and tomato (Gore and

O'Garro, 1999). All Capsicum genotypes tested are resistant to strains of Xanthomonas

that are only pathogenic to tomato, and conversely, all Lycopersicon genotypes

evaluated show resistance to strains of the pepper group. Thus, specific genotypes of

pepper and tomato have been used to characterize races of xanthomonads pathogenic

to theses hosts (Table 1-1). Both genera (Capsicum and Lycopersicon) contain genes

for resistance to Xanthomonas, but the genes cannot be transferred between these

genera through natural hybrizidation (Jones et al., 1998). Several avirulence genes

have been identified in xanthomonads associated with tomato. Strains of X.

euvesicatoria, tomato race 1 (T1), carry the avirulence gene avrRxv which induces a

hypersensitive response (HR) on the genotype genotype H7998 with the corresponding

resistance gene Rxv (Whalen et al. 1993). Strains of X. perforans, tomato race 3 (T3)

contain the avrXv3 that induce an HR on H7981 (Minsavage et al., 1996). Another

avirulence gene, avrXv4, was found in X. perforans strains based on HR reactions with

the Xv4 resistance gene of the tomato genotype LA716 (Lycopersicum pinnellii). Thus,

strains of X. perforans, containing the avrXv4, but lacking a functional avrXv3, were

designated tomato race 4 (Astua-Monge et al., 2000a; 2000b).









Table 1-1. Resistance in tomato and pepper and avirulence genes that interact with
them
Resistance Bacterium
Source Species Effector Location
Pepper
Bsl PI 163192 C.a annuum AvrBsl Plasmid
Bs2 PI 260435 C. chacoense AvrBs2 Chromosome
Bs3 PI 271322 C. annuum AvrBs3 Plasmid
Bs4 PI 235047 C. pubescens AvrBs4 Plasmid
Bs5 PI 163192 or C. annuum NDc ND
PI 271322 C. annuum ND ND
Bs6 PI 163192 C. annuum ND ND
PI 271322 C. annuum ND ND
BsT Commercial C. annuum AvrBst Plasmid
pepper
Tomato
rxl,rx2,rx3 Hawai 7988 S.b lycopersicum AvrRxv Chromosome
Xv3 P1128216 & S. pimpinellifolium & S. AvrXv3 Chromosome
Hawai 7981 lycopersicum
Xv4 716 S. pennellii AvrXv4 Chromosome
Bs4 Commercial S. lycopersicum AvrBs4 Plasmid
tomato
Source : Stall et al., 2009
a Capsicum
b Solanum
C not determined
Several avirulence genes, with their corresponding resistance genes, have also

been characterized in pepper (Table 1-1). Ten races of X. euvesicatoria have been

identified based on hypersensitivity reactions with the resistance genes Bsl, Bs2, Bs3

and Bs4 (Stall et al.,,2009).

Tomato race 1 (T1) was commonly found in Florida before the appearance of T3 in

Florida in 1991. In vitro tests showed that T3 strains are antagonistic to T1 strains

(Jones et al., 1997). Tudor (1999) determined that T3 strains produce bacteriocin-like

substances that have biological activity against T1 strains. The antagonism of T3

against T1 may lead to a shift in race composition towards T3 strains in Florida, with a









bacteriocin production confering a competitive advantage for T3 strains (Jones et al.,

1997).

In 1982, pepper race 2 (P2) was the primary race in Florida (Cook and Stall,

1982), but by the late 1980s, pepper race 1 (P1) had become the prevalent race in

Florida. In 1988, 89% of the strains were represented by P2, but in 1989, P2 strains

accounted for only 15% (Pohronezny et al., 1992).

Shifts in race populations can occur several ways: introduction of a new race by

seeds or other plant propagative materials, introduction of resistant cultivars which may

select for new races, and competitive advantage (Jones et al., 1997). Based on the

widespread use of copper-based bactericides in Florida, it appears that copper

resistance gave P2 strains a competitive advantage (Jones et al., 1997), which could

explain why P2 strains were prevalent before 1989 in Florida (Pohronezny et al., 1992).

It has been proposed that specific races were associated with specific regions

(Cook and Stall, 1982). However, other reports mention the establishment of the same

races in a number of locations (Jones et al. 1995; O'Garro and Tudor, 1994). It has

been suggested that the introduction of pepper and tomato seeds may serve as sources

of races that eventually became endemic in production (Pohronezny et al.,1992). The

pathogen race structure may also be altered by mutation and selection. A report

referred to the emergence of the race P3 from P1 and P2 by plasmid loss (Kousik et al.,

1996).

Epidemiology

A number of different sources of primary inocula have been identified for the

bacterial spot pathogen. The spectrum of hosts of bacterial spot is not limited to

peppers and tomatoes, but also includes a few other solanaceous plants (Stall et al.,









2009). The pathogen may stay alive between seasons in lesions on volunteer plants

and it does not remain in the soil without crop debris for more than 6 weeks in the

Florida summer. Seeds may be an important source of inoculum (Jones, 1991).

Commercial seeds are usually treated with a bactericide in order to reduce seedborne

primary inoculum (Stall et al., 2009). A low incidence of contaminated seed can induce

a high incidence of disease in the field due to epiphytic multiplication and distribution of

the pathogen in the phyllosphere (Louws et al. 2001). Cotyledon leaves generally

become infected after the emergence from an infested seedcoat. Seedlings and

transplants become infected by rain splash or windblown particles from nearby infected

plants. Infected transplants carry the bacterial inocula to the field and there may induce

further contamination (Jones, 1991).

The bacteria can enter the plant when there are favorable conditions for disease

development, including a threshold epiphytic population or significant wounding wounds

engendered by wind-driven sand, insect punctures or mechanical means facilitate the

ingress of the pathogen (McGuire et al. 1991; Jones, 1991). Natural opening such as

stomates and hydathodes can also serve as an entry point (Ramos and Volin, 1987).

High humidity is also an important factor; high humidity has been shown to increase

infection of X. euvesicatoria by 10- to 100- fold on tomato leaves in comparison to low

humidity (Timmer et al.1987).

Disease Control

Cultural practices such as good field sanitation (Pohronezny et al. 1990), crop

rotation, the use of disease- and pathogen-free transplants and the elimination of

solanaceaous weeds such as ground cherry and nightshade reduce primary

inoculum(Kucharek, 1994). Cultural practices alone are not sufficient for good control









and need to be complemented with other approaches. Streptomycin was commonly

used in the 1950s for the control of bacterial spot, but was no longer recommended by

the 1960s due to resistance by the bacteria to this antibiotic (Stall and Thayer, 1962).

Other approaches to management include the application of bacteriophages mixes

(Flaherty et al. 2000; Balogh et al. 2003), activators of the plant immune response that

induce systemic- acquired resistance such as acibenzolar-S-methyl (Louws et al. 2001).

For many years, the standard recommendation has been the application of copper

compounds. In various studies, it has been shown that the addition of maneb or

mancozeb to copper bactericides increases their bactericidal activity. It is important to

note that the carbamates, mancozeb or maneb did not control the bacteria when used

alone (Marco and Stall, 1983). In some instances, copper-mancozeb mixtures resulted

in a reduction of bacterial populations on tomato leaves and improved disease control

(Jones et al., 1991). However, this combination was not effective when weather

conditions were optimal for disease development; positive yield responses were rarely

obtained in conditions where copper-resistant strains were present (Jones and Jones,

1985).

Copper-Resistant Strains

Foliar application of chemicals such as fixed copper compounds has been

routinely used to try to control the disease. Different copper compounds: cupric

hydroxide, tribasic Cu sulfate, Cu ammonium carbonate, Cu oxychloride sulfate, and Cu

salts of fatty and rosin acids; are used in the control of bacterial spot. The use of these

chemicals has led to the frequent occurrence of copper-resistant strains of vegetable

bacterial pathogens (Marco and Stall, 1983; Bender et al., 1990; Cooksey, 1990).









Two apparently independent lines of copper resistance genes in xanthomonads

associated with bacterial spot pepper and tomato are known. Copper-resistance genes

were found on 188- to 200-kb self-transmissible plasmids in strains from Florida and

Oklahoma (Bender et al., 1990; Stall et al.,, 1986) and on a 100-kb non-self

transmissible plasmid in a strain from California (Cooksey et al.,1990). Chromosome-

encoded copper resistance was found in a strain XvP26 from Taiwan which contains a

small plasmid (15 kb) (Basim et al. 1999; Canteros et al.,1995).

Bactericides have been used in plant disease control for decades. Nevertheless, in

the 1980s, resistance to the most commonly used bactericide, copper, was detected

(Marco and Stall, 1983). It may be that copper resistance occurred much earlier but was

overlooked by plant pathologists (Cooksey,1990). Moreover, the widespread

combination of an ethylene bisdithiocarbamate fungicide with copper, which enhances

the toxicity of copper sprays, might not lead to earlier detection of copper-resistant

pathogens in the field (Marco and Stall, 1983). It seems that resistance has been

present in the field for many years since strains isolated in 1968 were found to be

copper-resistant (Cooksey, 1990).

The increasing use of antimicrobial agents has led to a strong selective force,

favoring the survival of bacterial strains to such agents, either by mutation or by

acquisition of R-plasmids (Davies and Smith, 1978). In laboratory assays, chromosomal

mutations for bacterial resistance can be induced, but the role of such resistance in field

populations is not well understood (Cooksey, 1990). In natural isolates of most bacteria,

metal-and antibiotic-resistance genes are usually found on plasmids and transposons

(Cooksey, 1994; Silver and Misra, 1988). Canteros et al., (1995) determined the









plasmid profile of 522 strains of xanthomonads associated with bacterial spot of pepper

and tomato. They were from both culture collections from different geographic locations

and strains isolated from commercial fields in Florida. High diversity, in terms of number

of plasmids and plasmid size (3 to 300 kb), was observed. Such diversity could be the

result of frequent plasmid transfer between bacterial strains within the pyllosphere.

It is important to note that plasmid-borne resistance determinants are easier to

identify through conjugal plasmid transfer to bactericide-sensitive strains (Cooksey,

1990). Both mechanisms of copper-resistance gene evolution may be correlated in

some species. For example, chromosomal genes were found to be similar to plasmid-

borne copper-resistance genes in pseudomonads, and such genes are involved in

copper uptake and management in Escherichia coli (Cooksey, 1993).

The presence of these plasmids possibly provides a selective metabolic

advantage to the bacterial strains in comparison to their plasmid-free relatives and also

provides extra genetic material. This material may be involved in antibacterial resistance

that cannot be linked to the mutation of host chromosomal genes (Davies and Smith,

1978).

Evaluation of Copper-Resistant Strains

The existence of copper-resistant strains in the fields can be inferred from the poor

control of bacterial spot with applications of copper bactericides at recommended rates

(Martin et al., 2004). The continuous application of such pesticides results in selection of

resistant strains until a resistant population becomes an important component in

disease epidemics (Martin et al., 2004). However, in the absence of selection pressure,

copper-resistant strains can revert to copper sensitive ones (Gore and O'Garro, 1999).









The basis of the reversion might be due to the loss of plasmids encoding copper

resistance (Stall et al.,,1986).

Several methods have been reported in the literature to assess the resistance

among bacterial strains (Gore and O'Garro, 1999; Marco and Stall, 1983; Zevenhuizen

et al. 1979; Pernezny et al. 2008). Stall et al., (1986) found plates of nutrient agar

amended with 200 pg/ml CuSO4.5H20 were useful in the screening of resistance to

copper among pepper strains of Xanthomonas euvesicatoria. Strains from Barbados,

associated with bacterial spot of pepper and tomato, that produced confluent growth on

nutrient agar amended with 200 pg/ml CuSO4.5H20, were considered resistant to

copper and those that failed to grow were considered sensitive (Gore and O'Garro,

1999). This procedure (Stall et al., 1986) was used to assess the copper resistance of

tomato strains of Xanthomonas perforans (Jones et al. 1991) and strains of

Xanthomonas campestris pv. vitians (Pernezny et al. 1995). In Australia, Martin et al.

(2004) used a low-complexing casitone yeast-extract-glycerol broth medium to

determine copper tolerance. This medium is characterized by minimal tendency to bind

the copper to components of the medium. The rationale for this choice is the fact that

most of the copper remains in the ionic form, thus ensuring maximum toxicity to the

bacteria. Copper-resistant bacterial strains survived at 1.0 mM CuSO4 in this medium.

(Zevenhuizen et al. 1979). Marco and Stall (1983) reported sensitivity to copper based

on the viability of cells after exposure to copper solutions. Sensitive strains were killed in

suspensions in which the concentrations of soluble amount of copper were 1-2 mg/L. A

concentration of 13 mg/L was necessary to kill the copper-resistant strains.









Pernezny et al. (2008b) reported that the protocol and especially the culture

medium chosen to screen bacterial strains can affect the classification of strains based

on sensitivity to copper. In their study, these authors found that most of the strains were

classified as resistant when using glucose nutrient agar (GNA) amended with copper

(Stall et al., 1986) and sensitive when using casitone-yeast extract, CYE, (Andersen et

al. 1991) amended with copper. Their results support GNA + Cu as a more suitable

medium to screen Xanthomonas strains from Florida. However, another factor that may

influence the choice of the medium for screening for copper resistance is the

host/pathogen system (Pernezny et al. 2008b). CYE amended with copper was useful

to classify Pseudomonas cichorii strains into highly resistant, moderately resistant and

sensitive strains (Pohronezny et al. 1994).









CHAPTER 2
BACTERIAL LEAF SPOT OF LETTUCE

Bacterial leaf spot of lettuce, Lactuca sativa L, (BLSL) was first reported in 1918 in

the United States (Brown, 1918). She proposed that the causal agent be named

Bacterium vitians. Since the first report, the disease has been observed in many states

including Florida (Pernezny et al.,1995), Ohio (Salin and Miller, 1997), and California

(Barak et al., 2001). They also have been reports from India (Wallis and Joubert, 1972),

France (Allex and Rat, 1990), Canada (Toussaint, 1999) and Turkey (Sahin, 2000).

In 1951, Elliot reported that three xanthomonds are associated with BLSL:

Xanthomonas vitians, X. lactucae and X. lactucae-scariolae (Elliot, 1951). Further

characterization showed that these three described species were not distinct, but rather

synonyms for X. vitians (Burkholder, 1954). In 1995, a reclassification of xanthomonads

was proposed that include 20 Xanthomonas DNA homology groups (Vauterin et al.,

1995). Based on DNA-DNA hybrizations and Biolog profiles, the current taxon

Xanthomonas campestris pv vitians was divided in two groups, strains of A and B.

(Vauterin et al. 1995). Using tetrazolium violet as a redox indicator, the Biolog system

identifies bacteria based on metabolic activity in the presence of 95 different carbon

sources (Toussaint, 1999). Group B strains, including the pathovar reference strain

LMG 938, renamed X. hortorum pv vitians, show high relatedness with X. campestris

pv. pelargonii and X. campestris pv. hederae. The pathovar reference strain LMG 937

was the only strain that was included in the taxon X. axonopodis pv. vitians and fell into

Group 9 strains that include 34 X. campestris pathovars and X. axonopodis (Vauterin et

al. 1995). However, Stefani et al. (1994) and Barak et al. (2001) reported that the strain

LMG 937 was not pathogenic on lettuce. Sahin et al. (2003) also found that the strain









LMG 937 was nonpathogenic on lettuce, but was weakly pathogenic on tomato and

pepper, inducing small numbers of necrotic spots. This strain, isolated in 1917, might be

misidentified, mislabeled or might have lost pathogenicity on lettuce after many years in

storage (Sahin et al. 2003).

Table 2-1. Difference among Xanthomonas campestris pv vitians and Xanthomonas sp.
strains, based on carbon sources in Biolog GN microplate assay (Sahin et al.
2003)
X. campestris Xanthomonas sp.
pv. vitians
Carbon substrate Group A Group B LMG 937
a -D-Lactose +
D-Melibiose + +
D-Raffinose + +
Formic acid -- +
a -Hydroxybutyric acid + +
a -Ketobutyric acid + +
Glycyl-L-aspartic acid +
L-serine + +
L-Threonine +
Glycerol -- +


Despite the uncertain affinity of the reference strain LMG 937, a recent study

supports the separation of X. campestris pv. vitians strains into at least two groups

(Table 2-1) (Sahin et al. 2003). Group A strains cause both local and systemic

symptoms, whereas Group B strains, including the pathovar reference strain LMG 938,

induced only distinct necrotic spots. Due to their systemic spread in the plant, Group A

strains may represent a greater threat to lettuce production. It was also found that the X.

campestris pv. vitians type strain, LMG 937, and California strain B-53, both isolated

from lettuce, are different from Group A and B strains and were not pathogenic on

lettuce. Such separation was supported by monoclonal antibodies, fatty acid methyl

ester analysis (FAME), sodium dodecyl sulfate polyacrylamide gel electrophoresis









(SDS-PAGE), repetitive extragenic palindromic (Rep-PCR) fingerprinting studies.

However, there were no differences in the sequences of 16S-23S rDNA spacer regions

of four representative strains (Sahin et al. 2003). Barak et al. (2001) also found that the

spacer regions of strains from different geographical origins were identical, but no

genetic evidence of different groups pathogenic on lettuce. Analysis of ribosomal RNA

is a well established tool to study the relationship among bacteria (Woese, 1987). Due

to the high level of sequence conservation that can exist among rRNA genes (16s, 23S

and 5S) at the genus and species levels, the spacer regions-variable sequences

separating these genes can be a useful taxonomical tool (Jensen et al. 1993). Several

pathovars of Pseudomonas syringae could not be differentiated on the basis of RFLP

analysis of PCR-amplified rrs (16S) and rrl (23S) genes. However, P. syringae pv.

tomato strains were differentiated from other pathovars based on RFLP analysis of the

internal transcribed spacer region 1 (ITS1) (Manceau and Horvais, 1997).

The nomenclatural change proposed by Vauterin et al. (1995) has not be fully

accepted by the scientific community. Schaad et al. (2000) rejected the reclassification

of X. campestris pv. vitians type A as X. axonopodis pv. vitians and X. campestris pv.

vitians type B as X. hortorum pv. vitians until more phylogenetic information is available.

Recent literature continues to identify the pathogen as X. campestris pv. vitians

(Pernezny et al., 2002; Robinson et al., 2006). In this thesis, the causal agent of

Bacterial leaf spot of lettuce (BLSL) is referred as X. campestris pv. vitians (Xcvi).

Economic Impacts

Bacterial leaf spot reduces the quality and yield of lettuce and increase the risks of

postharvest losses (Carisse et al. 2000). Bacterial leaf spot have been reported in fields

of all major market types of lettuce including leaf, crisphead, butterhead and romaine









(Pernezny et al., 1995; Toussaint, 1999; Carisse et al., 2000). In Florida, BLSL continue

to be a major concern for farmers because of the favorable conditions for disease

development in the subtropical climate of southern Florida (Pernezny et al. 2002).

Under favorable conditions, the disease can damage head leaves of crisphead lettuce,

making the produce unmarketable (Toussaint, 1999).

Symptoms

The pathogen induces small, angular leaf lesions, about 1-2 mm in diameter,

along the margin of leaves, which are water-soaked, dark brown or olive colored. The

lesions become V-shaped, translucent and progress along the veins (Sahin and Miller,

1997). Coalescence of lesions results in large necrotic regions (Bull et al. 2007).

Another type of symptom consists of individual black spots dispersed on the leaf surface

(Sahin and Miller, 1997). Plants with BLSL symptoms are more susceptible to other

fungal diseases including Botrytis cinerea, Sclerotinia sclerotiorum and Rhizoctonia

solani (Carisse et al. 2000; Toussaint, 1999).

Epidemiology

Warm, humid, and rainy environmental conditions are conducive for the

development of BLSL (Barak et al. 2001; Toussaint, 1999). There is some inconsistency

related to the reports of optimum temperature for infection. Brown (1918) found the

optimum temperature for in vitro growth of Xcvi ranges from 26 to 28 C. Toussaint

(1999) reports that the optimal growth temperature in vitro is 28 C. Based on growth

chamber studies, Robinson et al. (2006) determined that the optimum temperature for

infection was 22.7 C. Robinson et al. (2006) suggested to collect strains from different

areas in order to compare temperature optima, since the differences observed in the

reports may be related to variation among strains from different locations.









X. campestris pv vitians (Xcvi) can have an epiphytic stage on leaf surfaces before

the induction of symptoms, based on scanning electron microscopy of asymptomatic

leaves infected by Xcvi strains or by dilution plating after the maceration of

asymptomatic leaves in phosphate buffer (Toussaint, 1999). Sahin and Miller (1997)

found relatively high populations of Xcvi, ranging from 109 to 1012 CFU/ g fresh weight

leaf tissue, on leaves inoculated with a concentration of 108 CFU/mL. They concluded

that some of the detected populations result from bacteria surviving epiphytically on leaf

surfaces.

The pathogen is also known to survive on diseased plant debris for short periods

of time and in association with weeds (Barak et al. 2001; Sahin and Miller, 1997). Barak

et al. (2001) found that populations of Xcvi can colonize and survive in association with

crop debris for at least 5 months in California. Contaminated debris can serve as an

inoculum source for subsequent crops. Contaminated seeds are also an important

source of inoculum and are also the major mean of long-distance dissemination of the

bacterium (Sahin and Miller, 1997). However, the bacteria have not been consistently

recovered from commercial seed lots. Contaminated debris may be more important for

BSLS development than seedborne inoculum (Barak et al. 2001). Jones et al. (1986)

reported a similar situation for bacterial spot of tomato, in which inoculum sources other

than seed, such as volunteer tomato plants and crop residue, appear to play a more

important function in the epidemiology of the tomato disease.

Control

For an effective management of BLSL, an integrated approach is needed.

Management tactics should include seed treatment, crop rotation, elimination of wild

host plants in or around lettuce fields, and avoidance of overhead irrigation. Other tools









are copper-based fungicides in association with mancozeb and the use of disease-

resistant cultivars (Sahin, 1997; Toussaint, 1999; Carisse et al. 2000 ).

Pernezny et al. (1995) reported that the romaine (cos) and butterhead types were

more susceptible to BLSL than crisphead lettuce. Sahin and Miller (1997) reported that

only the red leaf cv. Redline, among nine commercial cultivars grown in Ohio, was

resistant to the disease. Carisse et al (2000) also found that butterhead and cos types

were the most susceptible cultivars, with the green-leaf types less susceptible.

Since seeds are probably a major source of inoculum, seed treatment is an

essential component of an integrated management program. Several seed treatments

are suggested. Carisse et al. (2000) found that the most efficient seed treatment was a

1% of sodium hypochlorite soak for 5 to 20 min. Pernezny et al. (2002) reported that

seedborne inoculum was reduced below 10% with a 1% sodium hypochlorite treatment,

but they recommended a 15 minutes soaking time for better efficacy. They also also

found that a mixture of copper hydroxide and mancozeb and solutions of aqueous 3 to

5% hydrogen peroxide effectively eradicated X. campestris pv vitians associated with

lettuce seeds.









CHAPTER 3
CHEMICAL CONTROL AND INTERACTIONS BETWEEN FUNGICIDES

There are few bactericides among the increasing numbers of manufactured

pesticides (Mew and Natural, 1993). Chemical control of many bacterial diseases has

been in general a major challenge. Pathogen variability, high risk for development of

resistant strains, rapid population growth, and few available chemical-based options all

contribute to the difficulty in management of bacterial diseases (Jones et al. 2007).

The discovery of Bordeaux mixture in 1880's is considered to be an important step

in the history of chemical control. Bordeaux mixture is part of the first generation of

fungicides, which also includes other inorganic chemicals. The development of

dithiocarbamates constitutes the second generation of fungicides (De Waard et al.,

1993). These fungicides are surface protectants. They do not enter the plant tissues

and are only effective if applied in advance of infection (Sbragia, 1975). Third-

generation fungicides (e.g.: benzimidazoles, carboxamides, phenylamides) are mainly

systemic and help in the control of established infections (Waard et al., 1993; Baldwin

and Rathmell, 1988). The fourth generation of fungicides (e.g.: Trycycazole,

probenazole) includes compounds that are non toxic in in vitro trials, but control plant

disease by interfering with processes involved in pathogen penetration, or by enhancing

plant defense responses (Waard et al., 1993).

Antibiotics, such as streptomycin, have been used in agriculture to control

phytopathogenic bacteria (Mew and Natural, 1993). Extensive use of this antibiotic has

increased the prevalence of streptomycin-resistant strains in bacterial populations,

which reduces the efficacy of streptomycin-based control (Cooksey, 1990), including

bacterial spot of tomato and pepper (Thayer and Stall, 1961). However, the agricultural









use of antibiotics with medical applications is discouraged due to potential transfer of

resistance genes from phytopathogenic bacteria to those associated with animals and

humans (Mew and Natural, 1993).

Interactions Between Fungicides

Tank mixes of pesticides are often used in plant disease control. Pesticides are

combined in order to widen the spectrum of biological activity, to delay the selection of

resistant strains, and exploit synergistic interactions (Gisi et al., 1985). The efficacy of a

mixture may be equal to the additive effects of the single substances, or may be

sometimes superior or inferior to these additive effects. (Scardavi, 1965; Samoucha and

Cohen, 1986). Synergism is referred as a phenomenon in which the total response of

an organism to the mixture is higher than the sum of responses to the individual

components (Scardavi, 1965). When the efficacy of the mixture is below the arithmetical

sum of the effects of individual components, it is referred to antagonism (Samoucha and

Cohen, 1986).

Several synergistic interactions between fungicides have been reported in the

literature (Gisi et al. 1985; Marco and Stall, 1983; Roberts et al. 2008). Synergistic

interactions occurred when oxadixyl, mancozeb and Cymoxanil, were mixed in different

concentrations against sensitive strains of Phytophthora infestans in vivo (Gisi et al.

1985). Marco and Stall (1983) reported that the mixture of copper and mancozeb

induced a better control of bacterial spot of pepper than copper alone. The mechanism

by which this enhancement of copper toxicity occurs is unknown; however, one

suggestion is that the EBDC fungicide may induce an increase in the amount of soluble

copper (Cooksey, 1990). In vitro tests showed that mancozeb increased the soluble

copper in the suspension (Marco and Stall (1983). Roberts et al. (2008) reported









synergistic interactions between Tanos and copper, including mancozeb in some

instances, in the control of bacterial spot of tomato.

Mode of Action of Copper, Tanos, Cymoxanil and Famaxadone

Copper

Copper is an integral part of many enzymes involved in many vital processes.

Copper serves as a protein cofactor in fundamental redox reactions that involve

enzymes such as cytochrome oxidase and superoxide dismutase (SOD). It is vital in

cellular respiration and free radical defense mechanisms (Harris and Gitlin, 1996).

Normally bound to proteins, Cu may be released and become free and serves as a

catalyst in the formation of highly reactive hydroxyls radicals (Gaetke and Chow, 2003).

Thus, if levels of free ions increase, a number of toxic effects can occur in cells

(Cooksey, 1994). Copper is generally biocidal, affecting plants, fungi and bacteria.

Cupric ion, Cu2+, is the toxic form. It denatures proteins and competes with essential

metals for biding sites on coenzymes. Copper, at concentrations higher than 1 pM, is a

potent inhibitor of photosynthetic electron transport (Mohanty et al., 1989).

Mancozeb

The organic sulfur compounds comprise one of the most important and versatile

group of fungicides. They include thiram, ferbam, nabam, maneb, zineb and mancozeb.

These fungicides derive from dithiocarbamic acid. It is believed that the

dithiocarbamates are fungitoxic because they are metabolized to the isothiocyanate

radical, -N=C=S. This radical induces the inactivation of the sulfhydryl group (-SH) in

amino acids and in enzymes within pathogen cells and consequently inhibits the

production and function of these compounds (Agrios, 1997).









HS
-N iiS


N II Sm
^N-gS--S
HS

Sn
Figure 3-1. Structure of the fungicide Mancozeb
Mancozeb belongs to the ethylenebisdithiocarbamate group, which also include

maneb and zineb. It is a broad-spectrum fungicide that is useful in the control of many

foliage and fruit diseases of many vegetables. Mixture of maneb with zinc or zinc ion

results in the formulations known as maneb zinc (sold as Manzate D). Mancozeb (sold

as Manzate 200, Dithane M-45, Pencozeb) is a polymer of maneb and a zinc salt.

Zineb is sold as Dithane Z-79 (Agrios, 1997).

Tanos, Cymoxanil and Famaxadone
In the U.S, Famaxadone is used in combination with Cymoxanil in the formulation

of Tanos DF (water dispersible granules with 25% Famaxadone/25% Cymoxanil) for the

control of various fungal diseases on fruiting vegetables, potatoes, cucurbits, and head

lettuce. Famaxadone is in FRAC group 11 fungicide and belongs to the

oxazolidinedione class of chemicals. It is highly inhibitory to spore germination and

mycelial growth of sensitive isolates. It inhibits the fungal mitochondrial respiratory chain

at Complex III, which induces a reduction in production of ATP by the fungal cells

(Environmental Protection Agency, EPA, 2003).








N
H H
/ON N N N-N
N NIW 0
0 0

a) Cymoxanil b) Famaxadone

Figure 3-2. Structure of the active ingredients in the formulation of Tanos
Cymoxanil is in FRAC group 27 fungicide and belongs to the cyanoacetamide-

oxime class of chemicals. The trade name of Cymoxanil is Curzate 60 DF (E.I. du Pont

de Nemours and Company, Wilmington, DE). Curzate 60 DF is to be tank-mixed with a

protectant fungicide such as mancozeb (EPA, 1998). It is primarily active against

oomycetes (e.g. Phytophthora, Pseudoperonospora). Cymoxanil induces local systemic

activity and post-infection activity for the first half of the incubation period (Sujkowski et

al., 1995).









CHAPTER 4
THE ROLE OF TANOS AND ITS COMPONENTS IN THE CONTROL OF BACTERIAL
SPOT OF TOMATO AND PEPPER, AND BACTERIAL LEAF SPOT OF LETTUCE.

The mixture of copper and mancozeb has been used for many years for bacterial

disease control in vegetables. However, there is some health concerns related to the

use of these fungicides. Mancozeb (Dithane 50 DF) is registered as a general

fungicide by the U.S Environmental Protection Agency (EPA). It is a complex of

manganese ethylene bisdithiocarbamate (EBDC) and zinc salt. In the presence of

moisture, oxygen and biological systems, the EBDC fungicides may break down (U.S.

Environmental Protection Agency, 1992). Under such conditions, they can be easily

degraded with the formation of products like ethylenethiourea (imidazolidine-2-thione,

ETU) (Lentza-Rizos 1990). ETU has induced cancer in animals and has been identified

as a group B2 probable human carcinogen by the EPA (U.S. Environmental Protection

Agency, 1992). Based on this fact, they might be some restrictions in the use of

mancozeb in the future. Therefore, it is important to investigate alternative compounds

for the control of bacterial spot.

A report on Tanos (Tanos 50 DF, 25 % a.i each of Famaxadone and Cymoxanil,

E.I du Pont de Nemours and Company, Wilmington, DE), mixed with copper and

mancozeb in somes instances, induced equal or better control of bacterial spot in field

trials than copper and mancozeb alone (Roberts et al., 2008). No significant reduction in

bacterial populations was observed in laboratory assays with Tanos (Pernezny et al.,

2008b). Suppression of bacterial spot is included on the label for Tanos when this

fungicide is tank-mixed with a full dose of copper-based fungicides.

The objectives of this study are to:









Evaluate the effect of the combination of Tanos and Kocide on resistant tomato

and pepper copper strains and a lettuce strain.

Evaluate bactericidal activity of Tanos, Cymoxanil and Famaxadone alone and in

combination with Kocide.

Compare control by the mixture of Tanos and Kocide with the mixture of

mancozeb and Kocide.

Materials and Methods

Strains

Strains of X. euvesicatoria (P6), X. perforans (T4) and of X. campestris pv. vitians

(L7) were studied in vitro and in the greenhouse using combinations of Tanos,

Cymoxanil, Famoxidone, Mancozeb with Kocide. The product Tanos and its

components were also tested alone. Copper-tolerant tomato race 4 of X. perforans and

copper-tolerant pepper race 6 of X. euvesicatoria were provided by J.B. Jones,

University of Florida, Gainesville. X. campestris pv. vitians strain L7, isolated in 1995

from Everglades Agricultural Area, was provided by K.L. Pernezny, University of Florida.

For long term storage, bacterial cultures were stored in sterile 15% aqueous glycerol

solution at -70 oC. Working cultures were maintained on glucose-nutrient agar slants

(GNA).

In vitro Assays

Inoculum and assay procedure: Bacterial suspensions were prepared using 24-

h cultures in a sterile phosphate-buffered saline (Leben et al. 1968). The suspensions

were adjusted to A600 = 0.3, which approximately equals 5x108 CFU/ml. Chemical

suspensions (Table 4-1) were prepared in the laboratory in sterile distilled water at

dosages equivalent to those recommended for field use. With a ratio of 1:1, the bacterial









suspension was mixed with the chemical suspensions in beakers (Table 4-1). Beakers

were maintained at room temperature. Every 15-20 minutes, the beakers were manually

shaken during a 4-hour period. Then, suspensions were filtered with sterile Whatman

no. 1 filter paper. A set of 10-fold dilutions were carried out. A volume of 50 uL from

dilutions was pipetted per GNA plate and a sterile bent glass rod was used to distribute

the inoculum over the plate. Plates were incubated at 280C for 72 h and only plates with

less than 400 hundred colonies were counted. There were three replicates of each

treatment for each experiment. Data were logarithmically transformed (base 10) and

then analyzed with the Statistical Analysis System (SAS version 9.1, SAS Institute,

Cary, NC). An analysis of variance and a mean separation using the Least Significant

difference (LSD) were carried out.

Table 4-1. List of the treatments and their respective rate
Treatments Chemical g/L
T1 Control (water)
T2 Kocide 3000 + Manzate 75DF 1.2 + 2.4
T3 Kocide 3000 + Tanos 50DF + Manzate 75DF 1.2 + 0.6 + 2.4
T4 Kocide 3000 1.2
T5 Kocide 3000 + Tanos 50DF 1.2 + 0.6
T6 Tanos 50 DF 0.6
T7 Kocide 3000 + Cymoxanil 1.2 + 0.21
T8 Kocide 3000 + Famaxadone 1.2 + 0.46
T9 Cymoxanil 0.21
T10 Famaxadone 0.46


Greenhouse Experiment

Inoculation and assay procedure

The greenhouse component included separate trials with pepper and tomato. All

the procedures were similar except the strains P6 and T4 were used in pepper and

tomato. Tomato and pepper plants seedlings were transplanted in 10 cm-diameter pots









in a commercial soil mix Fafard no. 2 (Conrad Fafard, Inc., Agawan, MA.). Each pot

received 5 to 10 g of a slow-release fertilizer (Osmocote, 15-9-12; Sierra Chemical Co.,

Milpitas, CA). The plants were maintained in an air-conditioned greenhouse. Tomato

plants at 3-4 weeks of age and pepper plants at 4-5 weeks were sprayed with the

chemicals suspensions listed in Table 4-1 using a hand-held pump sprayer. Six plants

were spayed per chemical suspension. Control consisted of six plants sprayed with

sterile tap water. Once the plants were dry, they were transported to a growth-room for

2 days in which environmental conditions included a temperature of 280C, a 12-hour

light and a 12-hour dark cycle. Using a hand-held pump sprayer, both adaxial and

abaxial leaf surfaces were inoculated by spraying until run-off with a bacterial

suspension (A600 = 0.3), consisting of a 24-hour culture suspended in tap water. All

plants were immediately encased in transparent, polyethylene, plastic bags for a 48-h

period. The bags were then removed and the plants were returned to the greenhouse

for the remainder of the experiment. The treatments were distributed in a randomized

complete block design. The experiment was done twice.

Rating and statistical analysis

Fourteen days after inoculation, the plants were rated as disease incidence by

counting the number of lesions on the fifth leaf (from bottom to top) on tomato or as

disease severity by estimating the percent of leaf area affected by bacterial spot on

pepper. Data were transformed either logarithmically in case of number of lesions or

using the Horsfall-Barrett scale with pepper data. Statistical analysis was carried using

SAS (SAS version 9.1, SAS Institute, Cary, NC). The analysis included variance and a

mean separation using the Least Significant difference (LSD).









Results


In vitro assays

Tanos alone and its components, as expected, did not have any direct bactericidal

effects on the strains T4, P6 and L7 (Tables 4-2, 4-3). In some trials (Table 4-4),

bacterial populations increased significantly in comparison to the control. With the

strains T4 and P6, Kocide 3000 + Tanos + Manzate 75DF appeared to be the most

bactericidal in four of the six trials, whereas Kocide 3000 + Manzate 75DF was the most

toxic in two of the six trials. There is also a trend for Tanos to induce a modest increase

in the toxicity of Kocide in comparison to Kocide alone. For the strain T4, in all three

trials, the combination of Kocide + Tanos was significantly better than Kocide alone;

however, no statistical difference was detected in two of three trials with the strain P6

(Table 4-3) and in only one trial, Kocide + Tanos resulted in a larger reduction of

bacterial populations than Kocide alone. The addition of Cymoxanil or Famaxadone to

Kocide do not appear to increase the toxicity of Kocide with no stastical differences in

populations in most of the trials.









Table 4-2. In vitro trials with the copper-resistant strain T4 (Xanthomonas perforans) after incubation with various
chemical compounds
Trial la Trial 2 Trial 3


Tanos
Famaxadone
Cymoxanil
Control
Kocide
Kocide + Famaxadone
Kocide + Cymoxanil
Kocide + Tanos +
Mancozeb


9.38 A
9.38 A
9.27 A
9.20 A
6.42 B
6.41 B
6.21 B
2.16 C


Cymoxanil
Famaxadone
Control
Tanos
Kocide + Famaxadone
Kocide + Cymoxanil
Kocide
Kocide + Mancozeb


9.46 A
9.44 A
9.34 A
9.33 A
6.95 B
6.75 B
6.51 B
5.53 C


Famaxadone
Tanos
Cymoxanil
Control


Kocide
Kocide +
Kocide +
Kocide +


Famaxadone
Cymoxanil
Tanos


Kocide + Tanos 1.50 C Kocide + Tanos 4.59 D Kocide + Mancozeb 0.29 F
Kocide + Mancozeb 0.00 D Kocide + Tanos + 1.98 E Kocide + Tanos + 0.00 G
Mancozeb Mancozeb
aData are expressed as Log CFU. Numbers in columns followed by the same letter are not significantly different at
P< 0.05 according to the LS Means test.


8.92 A
8.82 A
8.72 A
8.37 A
3.96 C
3.82 C
3.48 D
3.16 E









Table 4-3. In vitro trials with the copper-resistant strain P6 (Xanthomonas euvesicatoria) after incubation with various
chemical compounds


Famaxadone
Cymoxanil
Control
Tanos
Kocide + Tanos
Kocide
Kocide + Cymoxanil
Kocide + Famaxadone
Kocide + Tanos +
Mancozeb
Kocide + Mancozeb


Cymoxanil
Control
Famaxadone
Tanos
Kocide
Kocide + Famaxadone
Kocide + Tanos
Kocide + Cymoxanil
Kocide + Mancozeb

Kocide + Tanos +
Mancozeb


Trial 2
9.96 A
9.95 A
9.93 A
9.87 A
7.52 B
7.40 B
7.29 B
7.19B
2.22 C

0.56 D


Control
Cymoxanil
Famaxadone
Tanos
Kocide
Kocide + Cymoxanil
Kocide + Famaxadone
Kocide + Tanos
Kocide + Mancozeb

Kocide + Tanos +
Mancozeb


Trial 3
9.39 A
9.31 A
9.18A
8.97 A
5.77 B
5.54 B
4.76 C
3.18 D
0.44 E

0.00 E


Trial 1l
9.57 A
9.55 A
9.52 A
9.48 A
6.84 B
6.49 B
6.26 BC
5.69 CD
5.58 D

1.61 E


aData are expressed as Log CFU. Numbers in columns followed by the same letter are not significantly different at
P< 0.05 according to the LS Means test.









Table 4-4. In vitro trials with the copper-sensitive strain L7 (Xanthomonas campestris pv. vitians) after incubation with
various chemical compounds
Trial 1a Trial 2


Tanos
Cymoxanil
Control
Famaxadone
Kocide + Tanos
Kocide
Kocide + Cymoxanil
Kocide + Famaxadone
Kocide + Tanos +
Mancozeb


9.69 A
9.64 A
9.56 A
9.50 A
0.00 B
0.00 B
0.00 B
0.00 B
0.00 B


Tanos
Famoxadone
Control
Cymoxanil
Kocide
Kocide + Famaxadone
Kocide + Tanos
Kocide + Cymoxanil
Kocide + Mancozeb


9.54 A
9.49 A
9.45 A
9.44 A
0.00 B
0.00 B
0.00 B
0.00 B
0.00 B


Kocide + Mancozeb 0.00 B Kocide + Tanos + 0.00 B
Mancozeb
aData are expressed as Log CFU. Numbers in columns followed by the same letter are not significantly different at
P< 0.05 according to the LS Means test.









Table 4-5. Numbers of lesions caused by Xanthomonas perforans strain T4 on tomato plants treated with chemicals in
greenhouse trials


Famaxadone
Tanos
Cymoxanil
Control
Kocide + Famaxadone
Kocide + Cymoxanil
Kocide + Tanos
Kocide
Kocide + Mancozeb

Kocide + Tanos +


Trial 1l
2.47 A
2.45 A
2.35 A
2.31 A
1.42 B
1.41 B
1.41 B
1.32 BC
1.12C

1.10C


Famaxadone
Tanos
Control
Cymoxanil
Kocide
Kocide + Famaxadone
Kocide + Tanos
Kocide + Cymoxanil
Kocide + Tanos +
Mancozeb
Kocide + Mancozeb


Trial 2
2.64 A
2.42 A
2.37 B
2.34 B
2.09 C
1.94 C
1.82 D
1.66 E
1.61 E

1.53 F


Control
B Tanos
Famaxadone
Cymoxanil
Kocide
D Kocide + Tanos
E Kocide + Cymoxanil
F Kocide + Famaxadone
F Kocide + Tanos +
Mancozeb
Kocide + Mancozeb


Mancozeb
aData are expressed as Log of number of lesions on 5th leaf. Numbers in columns followed by the same letter are not
significantly different at P< 0.05 according to the LS Means test.


Trial 3
2.86 A
2.80 A
2.76 AB
2.56 ABC
2.52 ABC
2.41 BC
2.41 BC
2.32 C
2.27 C

2.23 C









Table 4-6. Effects of Xanthomonas euvesicatoria strain P6 on
Trial 1a


Cymoxanil
Control
Famaxadone
Tanos
Kocide
Kocide + Famaxadone
Kocide + Cymoxanil
Kocide + Tanos
Kocide + Mancozeb +
Tanos
Kocide + Mancozeb


10.00 A
9.83 A
9.83 A
9.17A
7.00 B
6.83 B
6.17 BC
5.83 BC
5.00 C


Cymoxanil
Famaxadone
Tanos
Control
Kocide + Famaxadone
Kocide
Kocide + Cymoxanil
Kocide + Tanos
Kocide + Mancozeb


4.67 C Kocide + Mancozeb +


Tanos
aData are expressed as Horsfall Barett rating (HB). Numbers
different at P< 0.05 according to the LS Means test.


pepper plants treated with chemicals in greenhouse trials
Trial 2 Trial 3
8.80 A Tanos 11.67 A
8.60 A Cymoxanil 11.00 AB
8.50 A Famaxadone 10.67 AB
8.40 A Control 10.33 B
6.80 B Kocide + Famaxadone 7.17 C


6.50 BC
6.40 BC
5.83 C
3.67 D


Kocide
Kocide + Cymoxanil
Kocide + Tanos
Kocide + Mancozeb


3.50 D Kocide + Mancozeb +
Tanos
in columns followed by the same letter are


6.50 CD
6.00 DE
5.50 DEF
5.17 EF

4.83 F


not significantly









Disease Control on Tomato in the Greenhouse

Plants treated with Tanos, Famaxadone or Cymoxanil did not induce any disease

control in all three trials. In the first trial, a similar level of control was obtained between

the Kocide alone, and Kocide + Mancozeb or Mancozeb + Tanos + Kocide. The

addition of Tanos, Cymoxanil or Famaxadone to Kocide resulted in a similar disease

control to Kocide alone. In the second trial, only the combination Kocide + Famaxadone

was similar to Kocide alone; the other mixtures that contained Kocide had significantly

reduced disease severity. There was a lot of variation in disease control in disease

control.

Disease Control on Pepper in the Greenhouse

In the first trial, the disease severity of the untreated plants had a mean rating of

9.83 (HB rating, Table 4-6), which was not significantly different from the means of

plants treated with Tanos, Cymoxanil or Famaxadone. A similar pattern was observed

for the two other trials. In trials 1 and 3, the mixture Tanos/Kocide resulted in a similar

disease control to the standard combination of Kocide/ Manzate. Tanos tends to

increase the efficacy of the mixture Kocide/Manzate in two of the trials. In all trials,

Kocide alone provided a similar level of control as the combination of Kocide with

Tanos, Cymoxanil or Famaxadone.

Discussion

In this study, we were interested in dissecting the components of Tanos-

Cymoxanil and Famaxadone- in the control of bacterial spot of tomato and pepper, and

bacterial leaf spot of lettuce. In Florida, the control of bacterial spot is primarily based on

copper-mancozeb sprays. The EBDC can break down in the environment and can

generate carcinogenic residue on fruits (US EPA, 1992). The use of EBDCs may be









further regulated on food crops. Therefore, it is desirable to investigate alternative

compounds as a potential substitute for mancozeb.

Tanos, Cymoxanil and Famaxadone did not have any inhibitory activity against the

in vitro growth of X. perforans, X. euvesicatoria and X. campestris pv. vitians. In some of

the trials, the chemicals appeared to promote bacterial growth as previously reported by

Roberts et al. (2008). In all trials, greenhouse data reflected the trends shown by

Cymoxanil, Famaxadone and Tanos. No disease suppression was observed with plants

treated with these chemicals. However, Roberts et al. (2008) reported disease

suppression with applications of Famaxadone in greenhouse experiments. Pernezny et

al. (2008b) found that mancozeb and Famaxadone/Cymoxanil did not have any

inhibitory effects on X. campestris pv. vitians, X. perforans and X. euvesicatoria in vitro.

They coucluded that the reports of disease suppression with Famaxadone/Cymoxanil

(Roberts et al., 2008) might not be due to bactericidal activity but rather due to another

mode of action, such as systemic-acquired resistance.

As previously reported by Pernezny et al. (2008b), there is a trend for Tanos to

induce a modest synergistic increase in the toxicity of Kocide and Manzate to X.

perforans and X. euvesicatoria. In four of the six in vitro trials with the strains X.

euvesicatoria and X perforans, this reduction was statistically significant (4.2). There is

a trend of synergism for the mixture of Tanos and Copper for all in vitro trials with the

strain X. perforans (Table 4-2). However, increased disease control was not obtained

with the greenhouseb data since there was no statistical difference between Kocide

alone, and Kocide/Tanos for disease control in five of the six trials (Tables 4-5, 4-6). In

some instances, the mixture Kocide/Tanos led to similar level of disease control as the









standard mixture of Kocide/Manzate, and Kocide + Tanos + Manzate. Roberts et al.

(2008) reported that various mixtures of Famaxadone, Famaxadone + Cymoxanil,

mancozeb, and copper, all supressed bacterial spot in comparison to the applications

of the copper-mancozeb standard. The use of Famaxadone plus Cymoxanil and

mancozeb could possibly reduce the application of copper, which might help reduce the

selection of copper-resistant Xanthomonas strains and the accumulation of copper in

the environment (Roberts et al., 2008).

In vitro and greenhouse trials pointed to copper/mancozeb, including Tanos in

some instances, as the best treatment. Addition of mancozeb to copper usually resulted

in increased mortality of the pathogen. This might be due to a higher concentration of

Cu2+ ions in solution (Marco and Stall, 1983). However, Jones et al. (1991)

hypothesized that soluble copper may contribute to toxicity against the Cur strains, but

does not appear to be the primary component involved in the toxicity of the

copper/mancozeb combination to Cur strains of X. c. vesicatoria. Other workers

demonstrated that zinc is involved in the observed control of bacterial spot (Adaskaveg

and Hine, 1985). Mancozeb may also chelate copper ions, therefore increasing their

availability to certain sites in bacterial cells (Medhekar and Boparai, 1981). If levels of

free ions increase, a number of toxic effects can occur in cells (Cooksey, 1994). Copper

is a potent inhibitor of photosynthetic electron transport at concentrations higher than 1

pM (Mohanty et al., 1989).

In one trial with a strain of X. perforans, T4, (Table 4-5), no difference was

obtained with Cymoxanil and all the treatments that contained copper, including the

standard copper/mancozeb mixture. No significant difference was obtained between the









untreated plants and those treated with Cymoxanil. Pernezny et al. (2008b) reported

that the addition of mancozeb to copper did not induce a sufficient reduction of bacterial

populations for a strain of X. euvesicatoria. Populations were still above 1x106 CFU/mL

after a 2-hour exposure in vitro. These observations support the suboptimal

performance of the standard copper/mancozeb mixture under optimal conditions for

disease development (Jones and Jones, 1985).









CHAPTER 5
INFLUENCE OF TANOS AND ITS COMPONENTS IN THE POPULATION DYNAMICS
ON THE LEAVES

Microbial population dynamics on leaves are related to four processes:

immigration, emigration, growth and death (Kinkel, 1997). Several patterns have been

generally reported for leaf surface populations: a) aggregation of phyllosphere microbial

populations within individual leaves, b) variability among leaves and among plants, c)

increase over the growing season, d) high variability over short period of time,

associated with specific environmental events (e.g., rainstorms), and e) existence of

seasonal patterns among phyllosphere populations (Kinkel, 1997).

The terms epiphytic, phylloplane, resident and leaf-surface bacteria are

interchangeably used in the literature (Beattie and Lindow, 1995; Hirano and Upper,

1983 ). It is generally admitted that epiphytic bacteria are able to live (multiply) on plant

surfaces (Hirano and Upper, 1983; Leben, 1965). Leben (1963, 1965) used the terms

"residents" and "casuals" to differentiate bacteria that can multiply on leaf surfaces from

those that may reach the leaves by chance but can not multiply. From a functional

perspective, epiphytic bacteria are considered those that can be removed from above

ground plants parts by washing (Hirano and Upper, 1983). In contrast, endophytic

bacteria are considered to be those that can live in the leaf intercellular spaces,

substomatal cavities, or vascular tissues and have been functionally defined as those

bacteria that remain after the removal of the epiphytic bacteria (Beatie and Lindow,

1995) Both the surfaces and internal regions of the leaves can be colonized by foliar

pathogens (Hirano and Upper, 1990; Leben, 1965) and active exchange occurs

between the internal and external population (Bashan et al., 1981). Foliar pathogens

can have access to internal leaf tissues from the surface (Beattie and Lindow, 1995).









Surfaces structures, such as stomata (Gitaitis et al., 1981), hydathodes (Bretschneider

et al., 1989) have been reported as entry sites for many foliar pathogens. Internal

population can egress onto the surface. The formation of lesions may increase the

amount of cells that egress and subsequently spread the pathogen on the leaf surface

(Beattie and Lindow, 1995).

Epiphytic phytopathogenic bacteria can provide inoculum for disease development

and for spread to the surface of other plants and plant parts (Hirano and Upper, 1983;

Beattie and Lindow, 1995). In a given pathosystem, each epiphytic phytopathogenic

bacterium has a finite and extremely low probability of inducing disease. Subsequently,

when resident populations are sufficiently high on individual leaves, the probability of

causing disease is greater under favorable conditions (Hirano and Upper, 1883). Large

epiphytic populations have been associated with time of disease onset and with

increased levels of disease (Beattie and Lindow, 1995). Under field conditions, detection

of disease was always observed at a population level of at least 5 x 106 cells per navy

bean leaflet of either X. campestris pv. phaseoli or X. campestris pv. phaseoli var.

fuscans (Weller and Saettler, 1980). For some pathosystems, including brown spot

disease of beans (Lindemann et al. 1984) and halo blight of oats (Hirano et al. 1981), a

quantitative relationship has been established between epiphytic population size and

probability of disease occurrence. Increases in resident populations of P. syringae pv.

tomato result in higher incidence of bacterial speck on tomato with a 10- to 12-day lag

required for infection and symptom expression (Smitley and McCarter, 1982). Studying

the role of hrp genes in the fitness of P. syringae on beans, Hirano et al. (1997; 1999)









found that an intact type III secretion system is required for the growth, and possibly the

survival of P. syringae in the phyllosphere.

It is generally accepted that bacteria must gain access to the internal tissues and

establish large endophytic populations for successful infection. Therefore, the internal

populations, not the epiphytic populations, are essential for disease development.

However, there is a strong correlation between high epiphytic population sizes and high

probability of disease occurrence in some foliar diseases. One reason for this is that

increased epiphytic populations potentially result in higher endophytic population sizes

(Beattie and Lindow, 1995). However, it was been shown that large shoot surface

populations of phytopathogens can exist in the absence of disease (Hirano and Upper,

1983, Leben, 1965). Thus, large resident populations of a phytopathogen may increase

the probability of endophytic populations, but their presence does not ensure the

development of large endophytic populations that results in disease. Based on infectivity

titration experiments, a bacterial concentration of 104 cfu/ml was enough to initiate

disease in compatible host/pathogen inoculations (Robinson et al., 2006). In the

presence of large epiphytic population size, the extent of ingress, which relies on the

number of entry sites available (Ramos and Volin, 1987) and environmental conditions

(Daub and Hagerdorn, 1979), is a major factor that influences disease induction. The

number of entry sites is influenced by host genotype, leaf age, position on leaf surfaces

and wounds (Ramos and Volin, 1987; Beattie and Lindow, 1995).

Jones et al. (1991) reported that copper and a mixture of copper and mancozeb

reduced the epiphytic population of X. campestris pv. vesicatoria in comparison to the

untreated plants. A positive correlation, between epiphytic populations and disease









severity, was also found. In one test, Pernezny and Collins (1997) found that copper

sprays reduced X. campestris pv. vesicatoria populations on pepper leaflets in 99% in

comparison to a reduction of 51 % in buds. This study was undertaken to determine the

effects of Tanos and its components alone in combination with copper and mancozeb,

in some instances, on the population dynamics of Cur strains of X. perforans and X.

euvesicatoria on tomato leaves and pepper leaflets.

Materials and Methods

Strains and Inoculum

Rifampicin-resistant P6 and T4 strains were selected as follow : 25 uL aliquots of

24-h-old bacteria culture, grown in Nutrient Broth (Laboratories Difco), were spread on

Nutrient Agar (NA, Laboratories Difco ) plates amended with rifampicin (25 pg/mL).

Plates were incubated for 3 days at oC. Rifampicin-resistant colonies (Rif T4; Rif P6)

were selected. RifT4 or RifP6 strains were grown for 24-h on NA amended with

rifampicin, and then flooded with a solution of 0.01 M MgS04. Suspensions were

adjusted to an optical density of 0.30 at 600 nm with a spectrophotometer. The

inoculation of the plants, the time in the growth chamber and the application of the

chemical suspensions were as previously described for the greenhouse component in

the previous chapter.

Leaf Sampling and Assay Procedure

Three inoculated leaflets per treatment were randomly sampled 0, 2, 4, 6, 8, 10

and 12 days after inoculation, and then at days. Each leaflet was placed in a 50 mL tube

weighed and then mixed with a volume of 10 mL of peptone buffer per gram of tissue.

The buffer contains (per liter) 5.3 g of KH2PO4, 8.6 g of Na2HPO4, and 1 g of bacto

peptone (McGuire et al. 1986). Tubes were shaken on a rotary shaker at 200 rpm for 45









min. Serial 10-fold dilutions were made in of 0.01 M MgS04. A 50-pl aliquot of different

serial dilutions was plated onto each of three plates of Nutrient Agar amended with

rifampicin. After incubation at 280C for 3 days, typical colonies of X. perforans or X.

euvesivatoria were counted. Statistical analysis was carried out with logo transformed

data. Data were expressed as logo CFU/g of tissue. The analysis included variance and

a mean separation using the Least Significant difference (LSD).

Results

Epiphytic populations of X. euvesicatoria show a variable pattern among the

treatments in both trials. In one trial with a strain of Xanthomonas euvesicatoria (Table

5-2) and one trial with Xanthomonas perforans (Table 5-3), some statistical differences

(P 0.05 ) were obtained among some treatments in some sampling days (Day 0, Day

2, Day 4, and Day 6) whereas no statistical difference was obtained for the remainder of

the experiment. Generally, the addition of Tanos, and its components to Kocide in some

instances, did not reduce epiphytic populations at significant level in comparison to

Kocide alone, and the control in some days. The general trend is that the populations in

different treatments are the same over the period during which the samplings were

carried out. After inoculation (Day 0), the epiphytic populations of X. euvesicatoria of all

plants treated with copper and Famaxadone were similar, whereas these populations

were significantly lower than those of untreated plants, or plants treated with Cymoxanil,

or Tanos. In samplings days 2 and 4, epiphytic populations on leaflets treated with

Tanos were higher than on untreated control plants.










Table 5-1. Populations dynamics of a strain of X. euvesicatoria on pepper leaflets
Day Oa Day 2 Day 4 Day 6 Day 8 Day 12
Famaxadone 9.73 Control 9.86 Kocide + 5.00 Cymoxanil 6,73 Kocide + 10.98 Kocide 9.36
A A Tanos A A Tanos + A A
Mancozeb
Cymoxanil 9.66 Cymoxanil 9.76 Cymoxanil 4.82 Famaxadone 6.39 Cymoxanil 10.94 Tanos 9.12
A A A AB AB A
Control 9.65 Tanos 9.75 Tanos 4.44 Kocide + 6.34 Kocide + 10.94 Famaxadone 9.01
A A AB Famaxadone AB Famaxadone AB A
Tanos 9.62 Kocide 8.71 Famaxadone 4.43 Control 6.26 Control 10.92 Kocide + 8.33
A B AB AB AB Cymoxanil A
Kocide + 8.06 Kocide + 8.40 Control 4.23 Kocide 5.99 Tanos 10.92 Kocide + 8.32
Famaxadone B Tanos BC AB ABC AB Famaxadone A
Kocide 7.87 Famaxadone 8.30 Kocide + 3.66 Kocide + 5.77 Kocide + 10.91 Control 8.16
B BC Famaxadone AB Cymoxanil ABC Cymoxanil AB A
Kocide + 7.63 Kocide + 8.25 Kocide + 3.55 Tanos 5.35 Kocide + 10.91 Kocide + 8.05
Tanos BC Famaxadone BC Mancozeb AB ABC Tanos AB Tanos + A
Mancozeb
Kocide + 7.17 Kocide + 8.06 Kocide 3.49 Kocide + 5.28 Famaxadone 10.89 Kocide + 7.82
Cymoxanil C Cymoxanil C AB Tanos ABC AB Tanos A
Kocide + 6.52 Kocide + 5.91 Kocide + 2.94 Kocide + 4.32 Kocide + 10.88 Kocide + 7.56
Tanos + D Mancozeb D Cymoxanil B Tanos + BC Mancozeb B Mancozeb A
Mancozeb Mancozeb
Kocide + 5.58 Kocide + 5.81 Kocide + 2.88 Kocide + 3.79 Kocide + 10.86 Cymoxanil 7.54
Mancozeb E Tanos + D Tanos + B Mancozeb C Tanos + B A
Mancozeb Mancozeb Mancozeb


significantly different at


aData are expressed as Log CFU/g. Numbers in columns followed by the same letter are not
P< 0.05 according to the LS Means test.











Table 5-2. Populations dynamics of a strain of X. euvesicatoria on pepper leaflets
Day 0 a Day 2 Day 4 Day 6 Day 8 Day 10 Day 12


Famaxadon 8.08 Tanos 7.94 Tanos 6.92 Tanos 7.50 Tanos 8.89 Tanos 11.9 Control 11.19A
e A A A A A 8A
Control 7.61 Cymoxanil 7.00 Famaxadon 6.18 Famaxadon 6.76 Control 8.15 Control 10.2 Cymoxanil 10.36
A AB e AB e AB AB 5 AB AB
Cymoxanil 6.81 Control 6.83 Cymoxanil 5.83 Cymoxanil 5.86 Famaxadon 7.72 Kocide + 9.50 Tanos 10.2 AB
AB AB AB ABC e ABC Cymoxanil AB
Tanos 6.79 Famaxadon 6.71 Control 5.53 Kocide + 5.81 Cymoxanil 7.69 Famaxadon 8.38 Famaxadon 10.02
AB e ABC ABC Famaxadon ABC ABC e BC e AB


e
Kocide 5.81 Kocide 5.96 Kocide 5.45 Kocide +
BC BD BC Tanos
C
Kocide + 5.61 Kocide + 5.53 Kocide + 4.74 Control
Famaxadon BC Tanos BD Famaxadon BD
e D C e C
Kocide + 5.09 Kocide + 5.47 Kocide + 4.14 Kocide +
Tanos + BC Tanos + BD Tanos CD Cymoxanil
Mancozeb D Mancozeb C E
Kocide + 4.46 Kocide + 5.23 Kocide + 3.58 Kocide
Cymoxanil CD Famaxadon DC Cymoxanil DE


e
Kocide + 4.83 Kocide + 4.72 Kocide +
Tanos CD Mancozeb D Tanos +
Mancozeb
Kocide + 4.00 Kocide + 4.71 Kocide +
Mancozeb D Cymoxanil D Mancozeb


3.33 Kocide +
ED Tanos +
Mancozeb
3.25 Kocide +
E Mancozeb


5.05 Kocide
BC
D
5.04 Kocide +
BD Cymoxanil


7.40 Cymoxanil 8.38 Kocide +
ABCD BC Cymoxanil

6.70 Kocide + 7.85 Kocide
ABCD Famaxadon BC


7.63 Kocide +
BC Tanos

6.43 Kocide +
CD Famaxadon
e
5.64 Kocide +
CD Mancozeb

4.49 Kocide +
D Tanos +


C e
4.94 Kocide + 6.57 Kocide
BD Tanos + ABCD
C Mancozeb
4.87 Kocide + 6.18 Tanos +
BD Famaxadon BCD Kocide


C e
4.28 Kocide +
CD Mancozeb

3.61 Kocide +
D Tanos


5.28 Kocide +
CD Tanos +
Mancozeb
4.96 Kocide +
D Mancozeb


Mancozeb
aData are expressed as Log CFU/g. Numbers in columns followed by the same letter are not significantly different at
P< 0.05 according to the LS Means test.


9.14
ABC

7.69
BCD

6.77 CD


6.18D


5.14 D


5.14 D











Table 5-3. Populations dynamics of a strain of X. perforans on tomato leaves
Day Day Day Day Day Day Day
Oa 2 4 6 8 10 12
Cymoxanil 8.77 Control 10.64 Control 9.07 Cymoxanil 9.34 Control 10.60 Control 10.40 Famaxadone 11.47
A A A A A A A
Tanos 8.49 Tanos 9.50 Famaxadone 8.93 Famaxadone 9.27 Tanos 10.37 Famaxadone 10.07 Tanos 10.43
A AB A A AB A AB
Control 8.47 Famaxadone 9.47 Tanos 8.88 Control 9.17 Cymoxanil 9.40 Cymoxanil 9.83 Control 9.97
A AB AB A ABC AB BC
Famaxadone 8.46 Cymoxanil 9.10 Cymoxanil 8.47 Tanos 9.13 Kocide 8.73 Tanos 9.00 Cymoxanil 9.23
A B AB A BCD ABC CD
Kocide 7.76 Kocide 7.53 Kocide + 7.50 Kocide 7.33 Famaxadone 8.70 Kocide 8.37 Kocide 8.13
AB C Cymoxanil BC B BCD BCD DE
Kocide + 7.21 Kocide + 7.07 Kocide + 7.03 Kocide + 7.30 Kocide + 8.43 Kocide + 8.33 Kocide + 7.93
Tanos BC Famaxadone CD Famaxadone DC Famaxadone B Cymoxanil CDE Tanos BCD Cymoxanil E
Kocide + 6.73 Kocide + 6.43 Kocide 6.80 Kocide + 7.13 Kocide + 8.03 Kocide + 8.27 Kocide + 7.90
Famaxadone BCD Tanos + CD CD Cymoxanil B Tanos CDE Cymoxanil BCD Famaxadone E
Mancozeb
Kocide + 6.19 Kocide + 6.33 Kocide + 6.37 Kocide + 6.73 Kocide + 7.53 Kocide + 8.20 Kocide + 7.60
Tanos + CD Tanos CD Tanos CDE Tanos + B Famaxadone DE Famaxadone CD Tanos EF
Mancozeb Mancozeb
Kocide + 6.17 Kocide + 6.27 Kocide + 5.77 Kocide + 6.37 Kocide + 7.00 Kocide + 7.40 Kocide + 6.60
Cymoxanil CD Cymoxanil CD Mancozeb DE Tanos BC Tanos + E Mancozeb CD Tanos + FG
Mancozeb Mancozeb
Kocide + 5.66 Kocide + 5.83 Kocide + 5.10 Kocide + 5.40 Kocide + 6.77 Kocide + 7.17 Kocide + 6.20
Mancozeb D Mancozeb D Tanos + E Mancozeb C Mancozeb E Tanos + D Mancozeb G
Mancozeb Mancozeb
aData are expressed as Log CFU/g. Numbers in columns followed by the same letter are not significantly different at
P< 0.05 according to the LS Means test.









As in trial 1 for X euvesicatoria, a similar trend of similar treatments was observed

over time in trial 2. Specific reductions of populations could be observed during a giving

sampling day, but such differences were not consistent over time, except the

combination of copper/mancozeb in relation to the untreated plants. The copper and

mancozeb combination consistently reduced epiphytic populations of X. euvesicatoria

compared to those of the control (Table 5.2) during all the sampling days (exception:

day 6). All plants treated with Kocide and another chemical generally presented similar

populations than plants treated with Kocide alone. The addition of Tanos to Copper

seems to promote modest bactericidal activity in the reduction of epiphytic populations

since plants treated with this mixture presented the lowest epiphytic populations -

although at levels no statistically different- in most of the sampling days. However, the

addition of Tanos did not appear to increase the bactericidal property of the standard

mixture of copper/mancozeb since plants treated with copper/mancozeb generally

presented lower epiphytic populations than plants treated with Tanos + copper +

mancozeb in most of the samplings days.

As observed in trials with X. euvesicatoria, the population dynamics of X.

perforans show a variable pattern among the treatments (Table 5.3). The treatments

that contain copper generally induced a similar level of control of epiphytic bacterial

populations although population sizes varied greatly among the different treatments with

copper. Moreover, the mixture Kocide / Mancozeb tends to be the best treatment in

terms of reduction of epiphytic populations in comparison to the untreated plants. The

addition of Tanos to Copper seems to have a modest effect since in most sample times,









plants treated with Kocide/Tanos generally have lower populations than those treated

with kocide alone.

Discussion

Epiphytic bacteria are able to multiply on plant surfaces (Hirano and Upper, 1983).

Large epiphytic populations have been associated with time of disease onset and with

disease progression (Beattie and Lindow, 1995). For foliar pathogens, like

xanthomonads associated with bacterial spot of pepper and tomato, foliar application of

chemicals can modify the populations dynamics on the leaves. The effects of copper-

based bactericides on phyllosphere populations have been reported in some studies.

(Scheck and Pscheidt,1998; Pernezny and Collins, 1997; Jones et al. 1991). The aim of

this study was to evaluate the effects of Tanos, Cymoxanil and Famaxadone alone, and

in combination with Kocide and in some instances, mancozeb, on the epiphytic

populations of a strain of X. euvesicatoria and a strain of X. perforans.

The general trend is that many treatments were similar in a given sampling period

and over time. In both trials with a strain of X. euvesicatoria, P6, all treatments that

contain Kocide generally did not differ significantly over time although population sizes

vary among them in a given sampling day. Specifically, the addition of Tanos, either to

copper or to copper/mancozeb, did not result in better bactericidal activity in terms of

reduction in phyllosphere populations.















Log CFU/g ---- '






0 2 4 6 8 10
Days

-Control Kocide+ Mancozeb Kocide+ Tanos+ Mancozeb
Kocide -Kocide-+ Tanos Kocide+i Curzate
Kocide+ Famoxadone -Curzate Famoxadone
-Tanos



Figure 5-1. Population dynamics of strain of X. euvesicatoria on pepper leaflets,Trial 1

In trial 1 with a strain of X. euvesicatoria, the general trend is that all treatments

were similar over time. Significant reductions of phyllosphere populations among some

treatments during the first sampling days were not consistent over time. In this trial, the

treatments that contain copper, including the standard copper/mancozeb, did not reduce

the phyllosphere populations although plants treated with the standard mixture

presented the lowest populations (no statistical difference) in most of the sampling days.















Log CFU/g ,. r,




3 i------i-----i------i-----i-----i
0 2 4 6 8 10 12
Days

-Control Kocide+ Mancozeb Kocide+ Tanos + Mancozeb
Kocide Kocide-+ Tanos Kocide+i Curzate
Kocide+ Famoxadone -Curzate Famoxadone
-Tanos


Figure 5-2. Population dynamics of a strain of X. euvesicatoria on pepper leaflets, Trial
2

In one trial with a strain of X. euvesicatoria (Table 5-2 and Figure 5-2), and

another one with a strain of X. perforans (Table 5-3), the copper/mancozeb mixture

appears to be the best treatment over time in terms of reductions in phyllosphere

populations. In contrast with the trial 1 with X. euvesicatoria (Table 5-1 and Figure 5-1),

the standard mixture consistently reduced the pyllosphere populations in comparison to

the untreated plants. In a greenhouse study where a copper-resistant strain of

X.c.vesicatoria was applied to tomato foliage, plants treated with copper or with copper

and mancozeb mixture resulted in lower bacterial populations than untreated plants

(Jones et al., 1991). Lower bacterial populations on the leaf may reduce initial inoculum,

and consequently, affect bacterial spot epidemics (Pernezny and Collins, 1997). A

quantitative relationship has been established between epiphytic population size and









probability of disease occurrence for some pathosystems (Lindemann et al. 1984;

Hirano et al. 1981). Lindemann et al. (1984) demonstrated that a threshold of 104 cfu

per gram of leaflet tissue was a good predictor of brown spot incidence on snap beans,

caused by Pseudomonas syringae pv. syringae. Very high populations of this pathogen

constituted a more reliable predictor of disease severity than for disease incidence.

Leaf surface chemistry may be an important factor in the interaction between

copper bactericides and copper-resistant strains. Copper sprays applied to leaves exist

primarily as insoluble deposits of copper salts (Menkissoglu, and Lindow, 1991b).

Components of leachates on the tomato leaf surface may modify bacteria sensitivity to

copper (Jones et al. 1991). Binding of copper to environmental constituents affects the

biological availability of copper and can reduce the toxicity of copper towards

microorganisms (Gadd and Griffith, 1978). In vitro studies showed that the toxicity of

Cu2 to copper-sensitive and copper-tolerant strains of Pseudomonas syringae was

reduced in the presence of organic compounds including glucose, fructose, sucrose,

succinate and citrate (Menkissoglu and Lindow 1991a). Field trials demonstrated that

the concentration of soluble and completed forms of copper was abundant on navel

orange and beans leaves following spray application, but had no significant toxicity

towards strains of P. syringae (Menkissoglu and Lindow, 1991b). The leachates on

pepper and tomato may interact with copper, and therefore could disminish the amount

of soluble copper and Cu2' which might result in less toxicity towards the epiphytic

populations of X. perforans and X. euvesicatoria on tomato and pepper leaflets

respectively.









CHAPTER 6
CONCLUSIONS

Tank mixes of pesticides are often used in plant disease control. In this study, we

evaluated Tanos ands its components- Cymoxanil and Famaxadone-, in association

with copper, in the control of bacterial spot of tomato and pepper, and bacterial leaf spot

of lettuce. Pesticides are combined in order to widen the spectrum of biological activity,

to delay the selection of resistant strains and to exploit synergistic interactions (Gisi et

al., 1985).

In vitro studies show a synergistic action between copper and Tanos against the in

vitro growth of a strain of Xanthomonas perforans. It was also found that Tanos tended

to induce a modest increase in the toxicity of Kocide and Mancozeb to X. perforans and

X. euvesicatoria. It appears that both components of Tanos, Cymoxanil and

Famaxadone, are required for synergistic action since the addition of Cymoxanil or

Famaxadone to copper did not induce significant reduction of bacterial populations in

comparison to copper alone. Tanos, and its components do not have any inhibitory

activity against the in vitro growth of the pathogens evaluated. In contrast, Tanos

appears to promote bacterial growth as previously reported by Robert et al. (2008).

Therefore, the mechanism by which this growth occurs could be desirable to elucidate.

There was a lot of variation in disease control in greenhouse studies. Plants

treated with the standard mixture copper/Mancozeb, including Tanos in some instances,

tended to have a lower disease incidence. But, no statistical difference was obtained in

most the trials between the copper/mancozeb and other treatments that contain copper.

Various mixtures of Famaxadone + Cymoxanil, mancozeb and copper were reported to

induce a similar disease suppression as copper/mancozeb. The use of Famaxadone +









Cymoxanil could possibly reduce the application of copper, which might help reduce the

selection of copper-resistant Xanthomonas strains and the accumulation of copper in

the environment (Roberts et al. 2008).

A variable pattern was also obtained in the study related to the populations

dynamics of X. perforans, and X. euvesicatoria on tomato and pepper leaflets. High

variability of epiphytic populations could be observed over a short period of time (Kinkel,

1997). All treatments that contain copper generally did not differ at statistical level in a

given sampling period. In one trial with a strain of X. perforans, the standard mixture

copper/mancozeb did consistently reduce the pyllosphere populations in each sampling

period in comparison to the control. The addition of Tanos to the copper/mancozeb

mixture did not appear to increase bacteridal activity of this mixture to epiphytic

populations.

The rates of chemicals that were used in this study are recommended dosis for

field applications. However, concentration of fungicides can be a major component in

synergistic interactions. Synergistic interactions occurred when oxadixyl, mancozeb and

Cymoxanil, were mixed in different concentrations against sensitive strains of

Phytophthora infestans in vivo (Gisi et al. 1985). Wadley's method of assessing

synergism uses an approach of different concentrations to construct the dose

responses of fungicides in mixture. Equally effective concentrations, EC-values, for

different levels of control, are computed (Gisi et al. 1985, Levy et al., 1986). Therefore,

the synergistic trend observed in some in vitro trials with strains of X. euvesicatoria and

X. perforans could be further supported by studying different concentrations of the

chemicals involved in the synergic interactions.









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BIOGRAPHICAL SKETCH

Joubert Fayette was born in Haiti. He graduated from the "College Les Normaliens

Reunis" High School in 2002. He attended "Faculte d'Agronomie et de Medecine

Veterinaire", State University of Haiti, from 2002 to 2003. Then, he moved to Costa Rica

where he earned his Bachelor of Science degree in agronomical engineering at

EARTH (Escuela de Agricultura de la Regi6n Tropical HOmeda) University in December

2007. In 2006, he did an undergraduate internship at Southwest Florida Research and

Education Center, University of Florida. During his undergraduate studies, he evaluated

several plants extracts against the in vitro growth of Xanthomonas perforans,

Phytophthora capsici and Colletotrichum gloesporioides. He attended the graduate

program at the University of Florida, College of Agricultural and Life Sciences,

Department of Plant Pathology, from August 2008 to August 2010. He conducted a

research project that was based on the evaluation of Tanos and its components, in

association with copper, and mancozeb in some instances, in the management of

bacterial spot of tomato and pepper, and bacterial leaf spot of lettuce.





PAGE 1

1 THE ROLE OF TANOS AND ITS COMPONENTS IN THE MANAGEMENT OF BACTERIAL SPOT OF TOMATO AND PEPPER, AND BACTERIAL LEAF SPOT OF LETTUCE By JOUBERT FAYETTE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA I N PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010

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2 2010 Joubert Fayette

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3 To my parents, Nativida Codio and Gerard Fayette who have always provided the best for my personal and professional growth

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4 ACKNOWLEDGMENTS I would like to thank my committee members Drs. Pamela D. Roberts, Kenneth L. Pernezny and Jeffrey B. Jones for this opportunity to pursue a master and also their support, constructive criticism and guidance during the research project and the preparation of this manuscript. I would like to thank all those who helped me in this project: Dr. Ryan Donahoo, Dr. Robert E. Stall, Jerry Minsavage, Ro d Sytsma and David Ballesteros. T here were people who supported me in different ways a t different times, and I am grateful for their assistance : Katia Vernord, Lemane Delva, Edrice Fleurimond, Esnan Ambeau Patricia Desir, Pierre Paul Audate and Marc Evens Jean Jacques I thank my father, Gerard Fayette, for his love. I also thank Sheila, B reton Carl Renand, Wolfert, Brillant and Patricia Fayette and Immacula Guerrier for their constant support and enthusiasm

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 BACTERIAL SPOT OF PEPPER AND TOMATO ................................ ................... 11 History and Strain Diversity ................................ ................................ ..................... 11 Epidemiology ................................ ................................ ................................ .......... 15 Disease Control ................................ ................................ ................................ ...... 16 Copper Resistant Strains ................................ ................................ ........................ 17 Evaluation of Copper Resistant Strains ................................ ................................ .. 19 2 BACTERIAL LEAF SPOT OF LETTUCE ................................ ................................ 22 Economic Impacts ................................ ................................ ................................ .. 24 Symptoms ................................ ................................ ................................ ............... 25 Epidemiology ................................ ................................ ................................ .......... 25 Control ................................ ................................ ................................ .................... 26 3 CHEMICAL CONTROL AND INTERACTIONS BETWEEN FU NGICIDES ............. 28 Interactions Between Fungicides ................................ ................................ ............ 29 Mode of Action of Copper, Tanos, Cymoxanil and Famaxadone ............................ 30 Copper ................................ ................................ ................................ .............. 30 Mancozeb ................................ ................................ ................................ ......... 30 Tanos, Cymoxanil and Famaxadone ................................ ................................ 31 4 THE ROLE OF TANOS AND ITS COMPONENTS IN THE CONTROL OF BACTERIAL SPOT OF TOMATO AND PEPPER, AND BACTERIAL LEAF SPOT OF LETTUCE. ................................ ................................ .............................. 33 Materials and Methods ................................ ................................ ............................ 34 Strains ................................ ................................ ................................ .............. 34 In vitro Assays ................................ ................................ ................................ .. 34 Greenhouse Experiment ................................ ................................ ................... 35 Inoculation and assay procedure ................................ ............................... 35 Rating and statistical analysis ................................ ................................ .... 36 Results ................................ ................................ ................................ .................... 37

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6 In vitro assays ................................ ................................ ................................ .. 37 Disease Control on Tomato in the Greenhouse ................................ ............... 43 Disease Control on Pepper in the Greenhouse ................................ ................ 43 Discussion ................................ ................................ ................................ .............. 43 5 INFLUENCE OF TANOS AND ITS COMPONENTS IN THE POPULAT ION DYNAMICS ON THE LEAVES ................................ ................................ ............... 47 Materials and Methods ................................ ................................ ............................ 50 Strains and Inoculum ................................ ................................ ........................ 50 Leaf Sampling and Assay Procedure ................................ ............................... 50 Results ................................ ................................ ................................ .................... 51 Discussion ................................ ................................ ................................ .............. 56 6 CONCLUSIONS ................................ ................................ ................................ ..... 60 LIST OF REFERENCES ................................ ................................ ............................... 62 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 72

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7 LIST OF TABLES Tab le page 1 1 Resistance in tomato and pepper and avirulence genes that interact with them ................................ ................................ ................................ ................... 14 2 1 Difference among Xantho monas campestris pv vitians and Xanthomonas sp. strains, based on carbon sources in Biolog GN microplate assay (Sahin et al. 2003) ................................ ................................ ................................ .................. 23 4 1 List of the treatments and their respective rate ................................ ................... 35 4 2 In vitro trials with the copper resistant strain T4 ( Xanthomonas perforans ) after incubation with various chemical compounds ................................ ............. 38 4 4 In vitro trials with the copper sensitive strain L7 ( Xanthomonas campestris pv. vitians ) after incubation with various chemical compounds .......................... 40 4 5 Numbers of lesions caused by Xanthomonas perforans strain T4 on tomato plants treated with chemicals in greenhouse trials ................................ ............. 41 5 1 Populations dynamics of a strain of X. euvesicatoria on pepper leaflets ........... 52 5 2 Populations dynamics of a strain of X. euvesicatoria on pepper leaflets ........... 53

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8 LIST OF FIGURES Figure page 1 1 Symptoms of bacterial spot on pepper leaves and tomato fruits ........................ 12 3 1 Structure of the fungicide Mancozeb ................................ ................................ .. 31 3 2 Structure of th e active ingredients in the formulation of Tanos ........................... 32 5 1 Population dynamics of strain of X. euvesicatoria on pepper leaflets,Trial 1 ...... 57 5 2 Population dynamics of a strain of X euvesicatoria on pepper leaflets, Trial 2 .. 58

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9 Abstract of T hesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science THE ROLE OF TANOS AND ITS COMPONENTS IN THE MANGEMENT OF BACTERIAL SPOT OF TOMATO AND PEPPER, AND BACTERIAL LEAF SPOT OF LETTUCE By Joubert Fayette August 2010 Chair: Pamela D. Roberts Major: Plant Patho logy Tank mixes of pesticides are often used in plant disease control The effects of Tanos and its components, Famaxadone and Cymoxanil in combination with copper and mancozeb in some instances were evaluated on the in vitro growth of strains of Xantho monas perforans, X. euvesicatoria, X. campestris pv vitians on di sease control of bacterial spot of pepper and tomato in the greenhouse, and on the population dynamics of Cu r strains of X. perforans and X. euvesicatoria on tomato leaves and pepper leaflet s. Both in vitro and greenhouse trials demonstrated tha t Tanos and its components do not have any bactericidal activity In some instances, Tanos tends to promote bacterial growth. However, the addition of Tanos to the c opper/ m ancozeb mixture tends to indu ce a modest synergistic increase in the toxicity of the combination It appears that both components of Tanos, Cymoxanil and Famaxadone are essential for synergistic inter action since the addition of Cymoxanil or Famaxadone to copper did not induce signi ficant reduction of bacterial populations in comparison to copper alone. In the greenhouse trials, levels of disease control were similar for c opper alone, and

PAGE 10

10 c opper /Tanos in most the trials In so me instances, the mixture copper /Tanos led to similar leve l of disease control as the st andard mixture of c opper / m an coze b and c opper + Tanos + M an cozeb Tanos and its com ponents do not have bacteri cid al activity against the epiphytic populations of X. perforans and X. euvesicatoria on tomato leaves a nd pepper leaflets. Different mixture s with c opper, Famaxadone Cymoxanil and Tanos general l y did not diffe r at stastical level In one trial with a strain of X. perforans, the standard mixture copper/mancozeb did consistently reduce the pyllosphere populations in each sampling p eriod in comparison to the control.

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11 CHAPTER 1 BACTERIAL SPOT OF PEPPER AND TOMATO Vegetable production is one of the most important agricultural activities in Florida. This state ranks first in the United States in terms of fresh marke t tomato and pepper value. U.S co nsumpt ion of fresh tomatoes increase d 71%, from 7.0 kg per capita in 1991 to 9.4 kg per capita in 2006. In 200 7 the Florida tomato industry was value d at $ 464 million ( USDA, ERS, 2008 a ). T he country relies heavily on Florida for the supply of fresh peppe rs fr om October through June. In 2007 Fl orida harvested 17 500 acres of bell pepper, valued at over $ 183 million (USDA, ERS, 2008 b ). Both crops are affected by many diseases. B acterial spot is one of the major one s and occurs wherever tomato and pepper a re grown. It is particularly troublesome in tropical and subtropical areas ( Jones et al. 1998 a ). Production is affected by the disease, which can result in great economic losses. In some fields, loss of foliage may fluctuate between 50 70% (Pohronezny and Volin, 1983). Crop losses result from the reduction in yield due to defoliation and severely spotted fruits, which are not suitable for the market (Jones, 1991). Bacterial spot is a major concern to transplant growers because many states that receive seed lings from Florida require these trans plants to be free of the pathogen (Sun et al., 2002). History and Strain D iversity B acterial spot disease of tomato ( Solanum lycopersicum ) was first observed in 1914 in South Africa and described as a tomato canker by Ethel Doidge (1920). The causal agent was named Bacterium vesicatorium Gardner and Kendrick (1921) described a similar disease in the United States and referred to it as bacterial spot. They named the causal bacterium B exitiosum ; Doidge cedence A

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12 similar disease on pepper ( Capsicum annum ) was described by Gardner and Kendrick (1923). Numerous studies showed that the bacterial pathogens from pepper and tomato used in the initial studies induce disease in both plants, and it was believed for many years that cross infection could occur in field ( Stall et al., 2009 ). Figure 1 1. Symptoms of bacterial spot on pepper leaves and tomato fruits Symptoms induced by these different xanthomonads are similar. The characteristic symptoms incl ude the development of small brown to black lesions of 1 3 mm diameter, with or wi thout yellow halos, that affect all the aboveground organs (Figure 1 1 ). Subsequent enlargement and coalescence of spots occur later, which leads to the browning of the enti re leaf and defoliation (Kucharek, 1994). However, there are some sm all differences, such as a shot hole symptom induced by X perforans on tomato ( Stall et al., 2009). T he pathogen can be readily isolated from infec ted tissue. It is a gram negative, rod shaped bacterium. It is motile with a single polar flagellum, strictly aerobic, and measures 0.7 to 1.0 m by 2.0 2.4 m Other

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13 characteristics include production of a yellow, water insoluble pigment, xanthomonadin and an extracellular pol ysaccharide (EPS) named xanthan. Based on pathogenicity profile s many races have been identified. A race is charact erized by its ability to grow on a cultivar with or without specific genes for resistance ( Pernezny et al. 2008a ). The letters P, T and PT are respectively assigned to races pathogenic to pepper only, tomato only or both pepper and tomato (Gore and Capsicum genotypes tested are resistant to strains of Xanthomonas that are only pathogenic to tomato, and conversely, all Lycopersicon genotype s evaluated show resistance to strains of the pepper group. Thus, specific genotypes of pepper and tomato have been used to characterize races of xanthomo nads pathogenic to theses hosts (Table 1 1 ). Both genera ( Capsicum and Lycopersicon ) contain genes for resistance to Xanthomonas but the genes cannot be transferred between these genera through natura l hybrizidation (Jones et al. 1998). Several avirulence genes have been identified in xanthomonads associated with tomato. Strains of X. euvesicatoria, to mato race 1 (T1), carry the avirulence gene avrRxv which induce s a hypersensitive response (HR) on the genotype genotype H7998 with the corresponding resistance gene Rxv (Whalen et al. 1993). Strains of X. perforans tomato race 3 (T3) contain the avrXv3 that induce an HR on H7981 (Minsavage et al. 1996). Another avirulence gene, avrXv4, was found in X. perforans strains based on HR reactions with the Xv4 resistance gene of the tomato genotype LA716 ( Lycopersicum pinnellii ). Thus, strains of X. perforans containing the avrXv4, but lacking a functional avrXv3, were designated tomato race 4 (Astua Monge et al., 2000a; 2000b).

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14 Table 1 1. Resistance in tomato and pepper and avirulence genes that interact with them Resistance Bacterium Source Speci es Effector Location Pepper Bs1 PI 163192 C.a annuum AvrBs1 Plasmid Bs2 PI 260435 C. chacoense AvrBs2 Chromosome Bs3 PI 271322 C. annuum AvrBs3 Plasmid Bs4 PI 235047 C. pubescens AvrBs4 Plasmid Bs5 PI 163192 or C. annuum NDc ND PI 271322 C. ann uum ND ND Bs6 PI 163192 C. annuum ND ND PI 271322 C. annuum ND ND BsT Commercial pepper C. annuum AvrBst Plasmid Tomato rx1,rx2,rx3 Hawai 7988 S.b lycopersicum AvrRxv Chromosome Xv3 PI128216 & Hawai 7981 S. pimpinellifolium & S. lycopersicum A vrXv3 Chromosome Xv4 716 S. pennellii AvrXv4 Chromosome Bs4 Commercial tomato S. lycopersicum AvrBs4 Plasmid Source : Stall et al., 2009 a Capsicum b Solanum c not determined S everal avirulence genes, with their corresponding resistance genes, have also been characterized in peppe r (Table 1 1 ). Ten races of X. euvesicatoria have been identified based on hypersensitivity reactions with the resistance genes Bs 1 Bs 2 Bs 3 and Bs 4 (Stall et al.,, 2009). Tomato race 1 (T1) was commonly found in Florida before the appearance of T3 in Florida in 1991. In vitro tests show ed that T3 strains are antagonistic to T1 strains (Jones et al. 1997). Tudor (1999) determined t hat T3 strains produce bacteriocin like substance s that have biological activity against T1 strain s. The antagonism of T3 against T1 may lead to a shift in race composition towards T3 strains in Florida, with a

PAGE 15

15 bacteriocin production confer ing a competitive advantage for T3 strains (Jones et al., 1997). In 1982, pepper race 2 (P2) was the primary race in Florida (Cook and Stall, 1982), but by the late 1980s, pepper race 1 (P1) had become the prevale nt race in Florida. In 1988, 89 % of the strains were represented by P2, but in 1989, P2 strains accounted for only 15% (Pohronezny et al., 1992). Shifts in race populations can occur several ways: introduction of a new race by seeds or other plant propagative materials, introduction of resistant cultivars which may select for new races, and competitive advantage (Jones et al. 1997). Based on the widespread use of copper based bactericides in Florida, it appears that copper resistance gave P2 strains a competitive advantage (Jones et al., 1997), which could explain why P2 strains were prevalent before 1989 in Florida (Pohronezny et al., 1992). It has been pro posed that specific races were associated with specific regions (Cook and Stall, 1982). However, other reports mention the establishment of the same races in a number of locations (Jones et al. 1 It has been suggested that th e introduction of pepper and tomato seeds may serve as sources of races that eventually became endemic in production (Pohronezny et al.,1992). The pathogen race structure may also be altered by mutation and selection. A report referred to the emergence of the race P3 from P1 and P2 by plasmid loss (Kousik et al., 1996). Epidemiology A number of differ ent sources of primary inocula have been identified for the bacterial spot pathogen. The spectrum of hosts of bacterial spot is not limited to peppers and toma toes, but also includes a few other solanaceous plants ( Stall et al.,

PAGE 16

16 2009). The pathogen may stay alive between seasons in lesions on volunteer plants and it do es no t remain in the soil without crop debris for more than 6 weeks in the Florida summer Seed s may b e an important source of inoculum (Jones, 1991). Commercial seeds are usually treated with a bactericide in order to reduce seedborne primary inoculum ( Stall et al., 2009). A low incidence of contaminated seed can induce a high incidence of disease in the field due to epiphytic multiplication and distribution of the pathogen in the phyllosphere (Louws et al. 2001). Cotyledon leaves generally be come infec ted after the emergence from an infested seedcoat. Seedlings and transplants become infected by r ain splash or windblown particles from nearby infected plants. Infected transplants carry the bacterial i nocula to the field and there may induce further contam ination (Jones, 1991 ). The bacteria can enter the plant when there are favorable conditions for disease develo pment, including a threshold epiphytic population or significant wounding w ounds engendered by wind driven sand, insect punctures or mechanical means facilitate the ingress of the pathogen ( McGuire et al. 1991; Jones, 1991 ). Natural opening s uch as stomates and hydathodes can also serve as an ent r y point (Ramos and Volin, 1987) High humidity is also an important factor; high humidity has been shown to increase infection of X. euvesicatoria by 10 to 100 fold on tomato leaves in comparison t o low humidity (Timmer et al.1987). Disease C ontrol Cultural practices such as good field sanitation (Pohronezny et al. 1990), crop rotation, the use of disease and pathogen free transplants and the elimina tion of solanaceaous weeds such as ground cherr y and nightshade reduce primary inoculum(Kucharek, 1994) Cultural practices alone are not sufficient for good control

PAGE 17

17 and need to be complemented with other approaches. St reptomycin was commonly used in the 1950s for the control of bacterial spot, but w as no longer recommended by the 1960s due to resistance by the bacteria to this ant ibiotic (Stall and Thayer, 1962). Other approaches to management include the application of bacteriophage s mixes (Flaherty et al. 2000; Balogh et al. 2003 ) activators of t he plant immune response that induce s ystemic acquired resistance such as acibenzolar S methyl (Louws et al. 2001). For many years, the standard recommendation has been the application of copper compounds. In various studies, it has been shown that the ad dition of maneb or mancozeb to copper bactericides increase s their bactericidal activity. It is important to note that the carbamates, mancozeb or maneb did not control the bacteria when used alone (M arco and Stall, 1983). In some instances, copper mancoz eb mixtures resulted in a reduction of bacterial population s on tomato leaves and improved disease control (Jones et al., 1991). However, this combination was not effective when weather conditions were opti mal for disease development; positive yield respon ses were rarely obtained in conditions where copper resistant strains were present (Jones and Jones, 1985). Copper Resistant S trains Foliar application of chemicals such as fixed copper compounds has been routinely used to try to control the disea se. Diff erent copper compounds: cupric hydroxide, tribasic Cu sulfate, Cu ammonium carbonate, Cu oxychloride sulfate, and Cu salts of fatty and rosin acids; are used in the control of bacter ial spot The use of these chemicals has led to the frequent occurrence o f copper resistant strains of vegetable bacterial pathogens (Marco and Stall, 1983; Bender et al., 1990; Cooksey, 1990).

PAGE 18

18 Two apparently independent lines of co pper resistance genes in x anthomonads associated with bacte rial spot pepper and tomato are known Copper resistance genes were found on 188 to 200 kb self transmissible plasmid s in strains from Florida and Oklahoma (Bender et al., 1990; Stall et al., 1986) and on a 100 kb non self transmissible plasmid in a strain from California (Cooksey et al.,19 90). C hromosome encoded co pper resistance was found in a strain XvP26 from Taiwan which co ntains a small plasmid (15 kb) (Basim et a l. 1999; Canteros et al.,1995). Bactericides have been used in plant disease control f or decades. Nevertheless, in the 1980s resistance to the most common ly used bactericide copper, was detected (Marco and Stall, 1983). It may be that copper resistance occurred much earlier but was overlooked by plant pathologist s (Cooksey,1990). Moreover, the widespread combination of an eth ylene bisdithiocarbamate fungic ide with copper, which enhance s the toxicity of copper sprays, might not lead to earlier detection of copper resistant pathogens in the field (Marco and Stall, 1983). It seems that resistance has been present in the field for many years since strains isolated in 1968 were found to be copper resistant (Cooksey, 1990). The increasing use of antimicrobial agents has led to a strong selective force, favoring the survival of bacterial strains to such agents, either by mutation or by acquisition of R plasmids (Davies and Smith, 1978). In laboratory assays, chromosomal mutations for bacterial resistance can be induced, but the role of such resistance in field populations is not well understood (Cooksey, 1990). In natural isolates of most bacteria, metal and antibiotic resistance genes are usually found on plasmids and transposons (Cooksey, 1994; Silver and Misra, 1988). Canteros et al., (1995) determined the

PAGE 19

19 plasmid profile of 522 strains of xanthomonads associated with bacterial spot of pepper and tom ato. They were from both culture collections from different geographic locations and strains isolated from commercial fields in Florida High diversity, in terms of number of plasmids and plasmid size (3 to 300 kb), was observed. Such div ersity could be the result of frequent p lasmid transfer between bacterial strains within the pyllosphere. It is important to note that plasmid borne resistance determin ants are easier to identify through conjugal plasmid transfer to bactericide sensitive strains (Cooksey, 1 990). Both mechanisms of copper resistance gene evolution may be correlated in some species. For example, chromosomal genes were found to be similar to plasmid borne copper resistance genes in pseudomonads and such genes are involved in copper uptake and management in Escherichia coli (Cooksey, 1993). The presence of these plasmids possibly provides a selective metabol ic advantage to the bacterial strains in comparison to their pl asmid free relatives and also provides extra genetic mate rial This material may be involved in antibacterial resistance that cannot be linked to the mutation of host chromosomal genes (Davies and Smith, 1978). Evaluation of C opper R esistant S trains The existence of copper resistant strains in the fields can b e inferred from the poor control of bacterial spot with applications of copper bactericides at recommended rates ( Martin et al. 2004). The continuous application of such pesticides results in selection of resistant strains until a resistant population bec omes an important component in disease epidemics ( Martin et al. 2004). However, in the absence of selection pressure, copper resistant strains can revert to co pper sensitive ones

PAGE 20

20 The basis of the reversion might be due to the l os s of plasmids encoding copper resistance ( Stall et al., ,1986). Several methods have been reported in the literature to assess the resistance among bacterial strains ( et al. 1979 ; Pernezny et al. 2008). Stall et al., (1986) found plates of nutrient agar amended with 200 g/ml CuSO 4 .5H 2 0 were useful in the screening of resistance to copper among pepper strains of Xanthomonas euvesicatoria Strains from Barbados, associated with bacterial spot of pep per and tomato, that produced confluent growth on nutrient agar amended with 200 g/ml CuSO 4 .5H 2 0, were considered resistant to copper and those that fail ed 1999). This procedure ( Stall et al., 1986) wa s used to assess the copper resistance of tomato strains of Xanthomonas perforans (Jones et al. 1991) and strains of Xanthomonas campestris pv. vitians (Pernezny et al. 1995). In Australia, Martin et al. (2004) used a low complexing casitone yeast extract glycerol broth medium to determine copper tolerance. This medium is characterized by minimal tendency to bind the copper to components of the medium. The rationale for this choice is the fact that most of the copper remains in the ionic form, thus ensurin g maximum toxicity to the bacteria. Copper resistant bacterial strains survived at 1.0 mM CuSO 4 in this medium. (Zevenhuizen et al. 1979). Marco and Stall (1983) reported sensitivity to copper based on the viability of cells after exposure to copper soluti ons. Sensitive strains were killed in suspensions in which the concentrations of soluble amount of copper were 1 2 mg/L. A concentration of 13 mg/L was necessary to kill the copper resistant strains.

PAGE 21

21 Pernezny et al. (2008 b ) reported that the protocol and e specially the culture medium chosen to screen bacterial strains can affect the classification of strains based on sensitivity to copper. In their study, these authors found that most of the strains were classified as resistant when using glucose nutrient a gar (GNA) amended with copper ( Stall et al., 1986) and sensitive when using casitone yeast extract, CYE, (Andersen et al. 1991) amended with copper. Their results support GNA + Cu as a more suitable medium to screen Xanthomonas strains from Florida. Howev er, another factor that may influence the choice of the medium for screening for copper resistance is the host/pathogen system (Pernezny et al. 2008 b ). CYE amended with copper was useful to classify Pseudomonas cichorii strains into highly resistant, moder ately resistant and sensitive strains (Pohronezny et al. 1994).

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22 CHAPTER 2 BACTERIAL LEAF SPOT OF LETTUCE Bacterial leaf spot of lettuce, Lactuca sativa L, (BLSL) was first reported in 1918 in the United States (Brown, 1918). She proposed that the causal a gent be named Bacterium vitians Since the first report, the disease has been observed in many states including Florida (Pernezny et al.,1995), Ohio (Salin and Miller, 1997), and C alifornia (Barak et al., 2001). They also have been reports from I ndia (Wall is and Joubert, 1972), France (Allex and Rat, 1990), Canada (Toussaint, 1999) and Turkey (Sahin, 2000). In 1951, Elliot reporte d that three xanthomonds are associated with BLSL : Xanthomonas vitians, X. lactucae and X. lactucae scariolae (Elliot, 1951). F urther characterization showed that these three described species were not distinct, but rather synonyms for X vitians (Burkholder, 1954). In 1995, a reclassification of xanthomonads was proposed that include 20 Xanthomonas DNA homology groups (Vauterin e t al. 1995). Based on DNA DNA hybrizations and Biolog profiles, the current taxon Xanthomonas campestris pv vitians was divided in two groups, strains of A and B. (Vauterin et al. 1995). Using tetrazoli um violet as a redox indicator, the Biolog system ide ntifies bacteria based on metabolic activity in the presence of 95 different carbon sources (Toussaint, 1999). Group B strains, including the pathovar reference strain LMG 938, renamed X. hortorum pv vitians, show high relatedness with X. campestris pv p elargonii and X. campestris pv hederae The pathovar reference strain LMG 937 was the only strain that was included in the taxon X axonopodis pv vitians and fell into Group 9 strains that include 34 X. campestris pathovars and X. axonopodis (Vauterin et al. 1995). However, Stefani et a l. (1994) and Barak et al. (2001 ) reported that the strain LMG 937 was not pathogenic on lettuce. Sahin et al. (2003) also found that the strain

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23 LMG 937 was nonpathogenic on lettuce, but was weakly pathogenic on tomato and pepper, inducing small number s of necrotic spots. This strain, isolated in 1917, might be misidentified, mislabeled or might have lost pathogenicity on lettuce after many years in storage (Sahin et al. 2003). Table 2 1. Difference among Xanthomonas campes tris pv vitians and Xanthomonas sp. strains, based on carbon sources in Biolog GN microplate assay (Sahin et al. 2003) X. campestr i s pv. vitians Xanthomonas sp. Carbon substrate Group A Group B LMG 937 D Lactose + D Melibiose + + D Raffinose + + Formic acid + Hydroxybutyric acid + + Ketobutyric acid + + Glycyl L aspartic acid + L serine + + L Threonine + Glycerol + Despite the uncertain affinity of the re ference strain LMG 937, a recent study supports the separation of X. campestris pv vitians strains into at least two groups (Table 2 1 ) (Sahin et al. 2003). G roup A strains cause b oth local and systemic symptoms, whereas Group B strains, including the pa t hovar reference strain LMG 938, induced only distinct necrotic spots. Due to their systemic spread in the plant, Group A strains may represent a greater threat to lettuce production. It was also found that the X campestris pv vitians type strain, LMG 93 7, and California strain B 53, both isolated from lettuce, are different from Group A and B strains and were not pathogenic on lettuce. Such separation was suppo rted by monoclonal antibodies, f atty acid methyl ester analysis (FAME), s odium dodecyl sulfate polyacrylamide gel electrophoresis

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24 (SDS PAGE), repetitive extragenic palindromic (Rep PCR) fingerprinting studies. However, there w ere no difference s in the sequences of 16S 23S rDNA spacer regions of four representative strains (Sahin et al. 2003). Barak et al. ( 2001 ) also found that the spacer regions of strains from different geographical origins were identical, but no genetic evidence of different groups pathogenic on lettuce. Analysis of ribosomal RNA is a well established tool to study the relationshi p among bacteria (Woese, 1987). Due to the high level of sequence conservation that can exist among rRNA genes (16s, 23S and 5S) at the genus and species levels, the spacer regions variable sequences separating these genes can be a useful taxonomical tool (Jensen et al. 1993). Several pathovars of Pseudomonas syringae could not be differentiated on the basis of RFLP analysis of PCR amplified rrs (16S) and rrl (23S) genes. However, P syringae pv tomato strains were differentiated from other pathovars base d on RFLP analysis of the internal transcribed spacer region 1 (ITS1) (Manceau and Horvais, 1997). The nomenclatural change proposed by Vauterin et al. (1995) has not be fully accepted by the scientific community. Schaad et al. (2000) rejected the reclassi fication of X campestris pv vitians type A as X axonopodis pv. vitians and X. campestris pv vitians type B as X. hortorum pv vitians until more phylogenetic information is available. Recent literature continues to identify the pathogen as X. campestri s pv vitians (Pernezny et al., 2002; Robinson et al., 2006). In this thesis, the causal agent of Bacterial leaf spot of lettuce (BLSL) is referred as X. campestris pv vitians (Xcvi). Economic I mpacts Bacterial leaf spot reduce s the quality and yield of lettuce and increase the risks of postharvest losses (Carisse et al. 2000). Bacterial leaf spot have been reported in fields of all major market types of lettuce including leaf, crisphead, butterhead and romaine

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25 (Pernezny et al., 1995; Toussaint, 1999; Car isse et al., 2000). In Florida, BLSL continue to be a major concern for farmers because of the favorable conditions for disease development in the subtropical climate of southern Florida (Pernezny et al. 2002). Under favorable conditions, the disease can damage head leaves of crisphead lettuce, making the produce unmarketable (Toussaint, 1999). Symptoms The pathogen induces small, angular leaf lesions, about 1 2 mm in diameter, along the margin of leaves, which are water soaked, dark brown or olive colore d The lesions become V shaped, translucent and progress alo ng the veins ( Sahin and Miller, 1997). Coalescence of lesions results in large necrotic regions (Bull et al. 2007). Another type of symptom consists of individual black spots dispersed on the leaf surface ( Sahin and Miller, 1997). Plants with BLSL symptoms are more susceptible to other fungal diseases including Botrytis cinerea, Sclerotinia sclerotiorum and Rhizoctonia solani (Carisse et al. 2000; Toussaint, 1999). Epidemiology Warm, humid, and rai ny environmental conditions are conducive for the development of BLSL (Barak et al. 2001; Toussaint, 1999). There is some inconsistency related to the reports of optimum temperature for infection. Brown (1918) found the optimum temperature for in vitro gro wth of Xcvi ranges from 26 to 28 C. Toussaint (1999) reports that the optimal growth temperature in vitro is 28 C. Based on growth chamber studies, Robinson et al. (2006) determined that the optimum temperature for infection was 22.7 C. Robinson et al. (2006) suggest ed to collect strains from different areas in order to compare temperature optima since the differences observed in the reports may be related to variation among strains from different locations.

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26 X. campestris pv vitians (Xcvi) can have an epiphytic stage on leaf surface s b efore the induction of symtoms, based on scanning electron microscopy of asymptomatic leaves infected by Xcvi strains or by dilution plating after the maceration of asymptomatic leaves in phosphate buffer (Toussaint, 1999) Sahin and Miller (1997) found relatively high populations of Xcvi, ranging from 10 9 to 10 12 CFU/ g fresh weight leaf tissue, on leaves inoculated with a concentration of 10 8 CFU/mL. They conclude d that some of the detected populations result from bacteri a surviving epiphytically on leaf surfaces. The pathogen is also known to survive on diseased plant debris for short period s of time and in association with wee ds (Barak et al. 2001; Sahin and Miller, 1997). Barak et al. (2001) found that populations of X cvi can colonize and survive in association with crop debris for at least 5 month s in California. C ontaminated debris can serve as an inoculum source for subsequent crops. Contaminated seeds are also an important source of inoculum and are also the major mean of long distan ce dissemination of the bacterium (Sahin and Miller, 1997). However, the bacteria have not been consistently reco vered from commercial seed lots. Contaminated debris may be more important for BSLS development than seedborne inoculum (Bar ak et al. 2001). Jones et al. (1986) reported a similar situation for bacterial spot of tomato, in which inoculum sources other than seed, such as volunteer tomato plants and crop residue, appear to play a more important function in the epidemiology of the tomato disease. Control For an effective management of BLSL, an integrated approach is needed. M anagement tactics should include seed treatment, crop rotation, elimination of wild host plant s in or around lettuce field s, and avo idance of overhead irrigat ion. Other tools

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27 are copper based fungicides in association with mancozeb and the use of disease resistant cultivars (Sahin, 1997; Toussaint, 1999; Carisse et al. 2000 ). Pernezny et al. (1995) reported that the romaine (cos) and butterhead types were mo re susceptible to BLSL than crisphead lettuce. Sahin and Miller (1997) reported that only the red leaf cv. Redline, among nine commer cial cultivars grown in Ohio, was resistant to the disease. Carisse et al (2000) also found that butt erhead and cos types were the most susceptible cultivars with the green leaf types less susceptible. Since seeds are probably a major source of inoculum, seed treatment is an essential component of an integrated management program. Several seed treatments are suggested. Caris se et al. (2000) found that the most efficient seed treatment was a 1% of sodium hypochlorite soak for 5 to 20 min. Pernezny et al. (2002) reported that seedborne inoculum was reduced below 10% with a 1% sodium hypochlorit e treatment, but they recommended a 15 minutes soaking time for better efficacy. They also also found that a mixture of copper hydroxide and mancozeb and solutions of aqueous 3 to 5 % hydrogen peroxide effective ly eradicated X campestris pv vitians associated with lettuce seeds.

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28 CHAPTER 3 CHEMICAL CONTROL AND INTERACTIONS BETWEEN FUNGICIDES There are few bactericides among the increasing numbers of manufactured pesticides (Mew and Natural, 1993). Chemical control of many bacterial diseases has been in general a major challenge. Pathogen va riability, high risk for development of resistant strains, rapid population growth, and few available chemical based options all contribute to the difficulty in management of bacterial diseases (Jones et al. 2007). The discovery of Bordeaux mixture in 188 in the history of chemical control. Bordeaux mixture is part of the first generation of fungicides, which also includes other inorganic chemicals. The development of dithiocarbamates constitutes the second generati on of fungicides ( De Waard et al., 1993). These fungicides are surface protectants. They do not enter the plant tissues and are only effective if applied in advance of infection (Sbragia, 1975). Third generation fungicides (e.g.: benzimidazoles, carboxamid es, phenylamides) are mainly systemic and help in the control of established infections (Waard et al., 1993; Baldwin and Rathmell, 1988). The fourth generation of fungicides (e.g.: Trycycazole, probenazole) includes compounds that are non toxic in in vitro trials, but control plant disease by interfering with processes involved in pathogen penetration, or by enhancing plant defense responses (Waard et al., 1993). Antibiotics, such as streptomycin, have been used in agriculture to control phytopathogenic ba cteria (Mew and Natural, 1993). Extensive use of this antibiotic has increased the prevalence of streptomycin resistant strains in bacterial populations, which reduces the efficacy of streptomycin based control (Cooksey, 1990), including bacterial spot of tomato and pepper (Thayer and Stall, 1961). However, the agricultural

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29 use of antibiotics with medical applications is discouraged due to potential transfer of resistance genes from phytopathogenic bacteria to those associated with animals and humans (Mew a nd Natural, 1993). Interactions Between F ungicides Tank mixes of pesticides are often used in plant disease control. Pesticides are combined in order to widen the spectrum of biological activity, to delay the selection of resistant strains, and exploit sy nergistic interactions (Gisi et al., 1985). The efficacy of a mixture may be equal to the additive effects of the single substances, or may be sometimes superior or inferior to these additive effects. (Scardavi, 1965; Samoucha and Cohen, 1986). Synergism is refe rred as a phenomenon in which the total response of an organism to the mixture is higher than the sum of responses to the individual components (Scardavi, 1965). When the efficacy of the mixture is below the arithmetical sum of the effects of indivi dual components, it is referred to antagonism (Samoucha and Cohen, 1986). Several synergistic interactions between fungicides have been reported in the literature (Gisi et al. 1985; Marco and Stall, 1983; Roberts et al. 2008). Synergi stic interactions occ ured when oxadixyl, mancozeb and Cymoxanil were mixed in different concentrations against sensitive strains of Phytophthora infestans in vivo (Gisi et al. 1985). Marco and Stall (1983) reported that the mixture of copper and mancozeb induced a better cont rol of bacterial spot of pepper than copper alone. The mechanism by which this enhancement of copper toxicity occurs is unknown; however, one suggestion is that the EBDC fungicide may induce an increase in the amount of soluble copper (Cooksey, 1990). In vitro tests showed that mancozeb increased the soluble copper in the suspension (Marco and Stall (1983). Roberts et al. (2008) reported

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30 synergistic interactions between Tanos and copper, including mancozeb in some instances, in the control of bacterial spo t of tomato. Mode of A ction of Copper, Tanos Cymoxanil and Famaxadone Copper Copper is an integral part of many enzymes involved in many vital processes. C opper serves as a protein cofactor in fundamental redox reactions that involve enzy mes such as cyto chrome oxidase and superoxide dismutase (SOD). It is vital in cellular respiration and free radical defense mechanisms (Harris and Gitlin, 1996). Normally bound to proteins, Cu may be released and become free and serves as a catalyst in the formation of h ighly reactive hydroxyls radicals (Gaetke and Chow, 2003). Thus, if levels of free ions increase a number of toxic effects can occur in cells (Cooksey, 1994). Copper is generally biocidal, affecting plants, fungi and bacteria. Cupric ion, Cu 2+ is the to xic form. It denatures proteins and competes with essential metals for biding sites on coenzymes. Copper, at concentrations higher than 1 M, is a potent inhibitor of photosynthetic electron transport ( Mohanty et al., 1989 ). Mancozeb The organic sulfur co mpounds comprise one of the most important and versatile group of fungicides. They include thiram, ferbam, nabam, maneb, zineb and mancozeb. These fungicides derive from dithiocarbamic acid. It is believed that the dithiocarbamates are fungitoxic because t hey are metabolized to the isothiocyanate radical, N = C = S This ra dical induces the inactivation of the sulfhydryl group ( SH) in amino acids and in enzymes within pathogen cells and consequently inhibits the production and function of these compounds (Agrios, 1997).

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31 Figure 3 1. Structure of the fungicide Mancoze b Mancozeb belongs to the ethylenebisdithiocarbamate group, which also include maneb and zineb. It is a broad spectrum fungicide that is useful in the control of many foliage and fruit diseases of many vegetables. Mixture of maneb with zinc or zinc ion res ults in the formulations known as maneb zinc (sold as Manzate D). Mancozeb (sold as Manzate 200, Dithane M 45, Pen cozeb) is a polymer of maneb and a zinc salt. Zineb is sold as Dithane Z 79 (Agrios, 1997). Tanos Cymoxanil and Famaxadone In the U.S, Famax adone is used in combination with Cymoxanil in the formulation of Tanos DF (water dispersible granules with 25% Famaxadone / 25% Cymoxanil ) for the control of various fungal d iseases on fruiting vegetables potatoes, cucurbits, and head lettuce. Famaxadone i s in FRAC group 11 fungicide and belongs to the oxazolidinedione class of chemicals. It is highly inhibitory to spore germination and mycelial growth of sensitive isolates. It inhibits the fungal mitochondrial respiratory chain at Complex III, which induce s a reduction in production of ATP by the fungal cells (Environmental Protection Agency, EPA, 2003).

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32 a) Cymoxanil b) Famaxadone Figur e 3 2. Structure of the active ingredients in the formulation of Tanos Cymoxanil is in FRAC group 27 fungicide and belongs to the cyanoacetamide oxime class of chemicals. The trade name of Cymoxanil is Curzate 60 DF (E.I. du Pont de Nemours and Company, Wilmington, D E). Curzate 60 DF is to be tank mixed with a protectant fungicide such as mancozeb (EP A, 1998) It is primarily active against oomycetes ( e.g. Phytophthora, Pseudoperonospora ). Cymoxanil induces local systemic activity and post infection activity for the first half of the incubation period ( Sujkowski et al. 1995).

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33 CHAPTER 4 THE ROLE OF TA NOS AND ITS COMPONENTS IN THE CONTROL OF BACTERIAL SPOT OF TOMATO AND PEPPER, AND BACTERIAL LEAF SPOT OF LETTUCE. The mixture of copper and mancozeb has been used for many years for bacterial disease control in vegetables However there is some health con cerns related to the use of these fungicide s Mancozeb (Dithane 50 DF ) is registered as a general fungicide by the U.S Environm ental Protection Agency (EPA). It is a complex of manganese ethylene bisdithiocarbamate (EBDC) and zinc salt. In the presence o f moisture, oxygen and biological systems, the EBDC fungicides may break down (U.S. Environmental Protection Agency, 1992). Under such conditions, they can be easily degraded with the formation of products like ethylenethiourea (imidazolidine 2 thione, ETU ) (Lentza Rizos 1990). ETU has induced cancer in animals and has been identified as a group B2 probable human carcinogen by the EPA (U.S. Environmental Protection Agency, 1992). Based on this fact, they might be some restrictions in th e use of mancozeb in the future. T herefore it is important to investigate alternative compounds for the control of bacterial spot. A report on Tanos ( Tanos 50 DF 25 % a.i each of Famaxadone and Cymoxanil E.I du Pont de Nemours and Company, Wilmington, DE), mixed with copp er and mancozeb in somes instances, induced equal or better control of bacterial spot in field trials than copper and mancozeb alone (Roberts et al., 2008). No significant reduction in bacterial population s was observed in laboratory assays with Tanos (Pe rnezny et al., 2008 b ). Suppression o f bacterial spot is included on the label for Tanos when this fungici de is tank mixed with a full dose of copper based fungicides. The objectives of this study are to:

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34 Evaluate the effect of the combination of Tanos and Kocide on resistant tomato a nd pepper copper strains and a lettuce strain Evaluate bactericid al activity of Tanos, Cymoxanil and Famaxadone alone and in combination with Kocide Compare control by the mixture of Tanos and Kocide with the mixture of m ancoz eb and K ocide. Materials and Methods Strains Strains of X. euvesicatoria (P6), X. perforans (T4) and of X. campestris pv. vitians (L7) were studied in vitro and in the greenhouse using combination s of Tanos Cymoxanil Famoxidone, Mancozeb with Kocide. The product Tanos and its components were also tested alone. Copper tolerant tomato race 4 of X. perforans and copper tolerant pepper race 6 of X. euvesicatoria were provided by J.B. J ones, University of Florida, Gainesville. X. campestris pv vitians strain L7, isolated in 1995 from Everglades Agricultural Area, was provided by K.L. Pernezny, University of Florida. For long term storage, bacterial culture s were stored in sterile 15% aque ous glycerol solution at 70 C. W or king cultures were maintained on glu cose nutrient agar slants (GNA). In v itro A ssays Inoculum and assay procedure : B acterial suspension s w ere prepared using 24 h culture s in a sterile phosphate buffered saline (Leben et al. 1968) The suspension s were adjusted to A 600 = 0.3 which approxim ately equals 5x10 8 CFU/ml. Chemical s uspensions ( Table 4 1 ) were prepared in the laboratory in sterile distilled water at dosages equivalent to those recommended for field use. With a ratio of 1:1, the bacterial

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35 suspension was mixed with the chemical su spensions in beakers (Table 4 1) Beakers were maintained at room temperature. Every 15 20 minutes, the beakers were manually shaken during a 4 hour period. Then, suspensions were filtered with s terile Whatman no. 1 filter paper A s et of 10 fold dilutions were carried out. A volume of 50 uL from dilutions was pipe tted per GNA plate and a sterile bent glass rod was used to distribute the inoculum over the plate. Plates were incubated at 28C for 72 h and only plates with less than 400 hundred colonies were counted. There were three replicates of each treatment for each experiment. Data were logarithmically transformed (base 10) and then analyzed with the Statistical Analysis S ystem (SAS version 9.1, SAS Institute, Cary, NC). An analysis of variance and a me an separation using the Least Significant difference (LSD) were carried out. Table 4 1. List of the treatments and their respective rate Treatments Chemical g/L T1 Control (water) T2 Kocide 3000 + Manzate 75 DF 1.2 + 2.4 T3 Kocide 3000 + Tanos 50D F + Manzate 75 DF 1.2 + 0.6 + 2.4 T4 Kocide 3000 1.2 T5 Kocide 3000 + Tanos 50 DF 1.2 + 0.6 T6 Tanos 50 DF 0.6 T7 Kocide 3000 + Cymoxanil 1.2 + 0.21 T8 Kocide 3000 + Famaxadone 1.2 + 0.46 T9 Cymoxanil 0.21 T10 Famaxadone 0.46 Greenhouse E xperiment I noculation and assay p rocedure The greenhouse component include d separate trials with pepper and tomato. All the procedures were si milar except the strains P6 and T4 were used in pepper and tomato Tomato and pepper plants seedlings were transplanted in 10 cm diameter pots

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36 in a commercial soil mix Fafard no. 2 (Con rad Fafard Inc., Agawan, MA.). Each pot received 5 to 10 g of a slow release fertilizer (Osmocote, 15 9 12; Sierra Chemical Co., Milpitas, CA). The plants were maintained in an air conditioned g reenhouse. Tomato plants at 3 4 weeks of age and pepper plants at 4 5 weeks were sprayed with the chemica ls suspensions listed in Table 4 1 using a hand held pump sprayer. Six plants were spayed per chemical suspension. Control consisted of six plants spra yed with sterile tap water. Once the plants were dry, they were transported to a growth room for 2 days in which environmental conditions include d a temperature of 28C, a 12 hour light and a 12 hour dark cycle. Using a hand held pump sprayer, both adaxial and abaxial leaf surfaces were inoculated by spraying until run off with a bacterial s uspension (A 600 = 0.3 ), consisting of a 24 hour culture suspended in tap water. All plants were immediately encased in transparent, poly ethylene, plastic bags for a 48 h period. The bags w ere then removed and the plants were returned to the greenhouse for the remainder of the experiment. The treatments were distributed in a r andomized complete block design. The experiment was done twice. Rating and s tatistical a nalysis F ourteen days after inoculation, t he plants were rated as disease incidence by counting the number of lesions on the fifth leaf (from bottom to top) on tomato or as disease severity by estimating the percent of leaf area affected by bacterial spot on peppe r Data were transformed either logarithmically in case of number of lesions or using the Horsfall Barrett scale with pepper data Statistical analysis was carried using SAS (SAS version 9.1, SAS Institute, Cary, NC ). The a nalysis included variance and a m ean separation using the Lea st Significant difference (LSD).

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37 Results In v itro assays Tanos alone and its components, as expected, did not have any direct bactericidal effec ts on the strains T4, P6 and L7 (Tables 4 2, 4 3). In some trials (Table 4 4 ), bact erial populations increased significantly in comparison to the control. With the strains T4 and P6, Kocide 3000 + Tanos + Manzate 75 DF appea red to be the most bactericidal in four of the six trials, whereas Kocide 3000 + Manzate 75 DF was the most toxic in two of the six trials. There is also a trend for Tanos to induce a modest increase in the toxicity of Kocide in comparison to Kocide alone. For the strain T4, in all three trials, the combination of Kocide + Tanos was signifi cantly better than Kocide alone ; however, no statistical difference was detected in two of three trials with the strain P6 (Table 4 3 ) and in only one trial, Kocide + Tanos resulted in a larger reduction of bacterial populations than Kocide alone. The addition of Cymoxanil or Famaxadone to Kocide do not appear to increase the toxicity of Kocide with no stastical differences in populations in most of the trials.

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38 Table 4 2 In vitro trials with the copper resistant strain T4 ( Xanthomonas perforans ) after incubation with various chemical compounds Trial 1 a Trial 2 Trial 3 Tanos 9.38 A Cymoxanil 9.46 A Famaxadone 8.92 A Famaxadone 9.38 A Famaxadone 9.44 A Tanos 8.82 A Cymoxanil 9.27 A Control 9.34 A Cymoxanil 8.72 A Control 9.20 A Tanos 9.33 A Control 8.37 A Kocide 6.42 B Kocide + Famaxadone 6.95 B Kocide 3.96 C Kocide + Famaxadone 6.41 B Kocide + Cymoxanil 6.75 B Kocide + Famaxadone 3.82 C Kocide + Cymoxanil 6.21 B Kocide 6.51 B Kocide + Cymoxanil 3.48 D Kocide + Tanos + Mancozeb 2.16 C Kocide + Mancozeb 5.53 C Kocide + Tano s 3.16 E Kocide + Tanos 1.50 C Kocide + Tanos 4.59 D Kocide + Mancozeb 0.29 F Kocide + Mancozeb 0.00 D Kocide + Tanos + Mancozeb 1.98 E Kocide + Tanos + Mancozeb 0.00 G a Data are expressed as Log CFU. Numbers in columns followed by the same letter are not significantly different at P< 0.05 according to the LS Means test.

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39 Table 4 3 In vitro trials with the copper resistant strain P6 ( Xanthomonas euvesicatoria ) after incubation with various chemical compounds Trial 1 a Trial 2 Trial 3 Fa maxadone 9.57 A Cymoxanil 9.96 A Control 9.39 A Cymoxanil 9.55 A Control 9.95 A Cymoxanil 9.31 A Control 9.52 A Famaxadone 9.93 A Famaxadone 9.18 A Tanos 9.48 A Tanos 9.87 A Tanos 8.97 A Kocide + Tanos 6.84 B Kocide 7.52 B Kocide 5.77 B Kocide 6.49 B Kocide + Famaxadone 7.40 B Kocide + Cymoxanil 5.54 B Kocide + Cymoxanil 6.26 BC Kocide + Tanos 7.29 B Kocide + Famaxadone 4.76 C Kocide + Famaxadone 5.69 CD Kocide + Cymoxanil 7.19 B Kocide + Tanos 3.18 D Kocide + Tanos + Mancozeb 5.58 D Kocide + Mancoz eb 2.22 C Kocide + Mancozeb 0.44 E Kocide + Mancozeb 1.61 E Kocide + Tanos + Mancozeb 0.56 D Kocide + Tanos + Mancozeb 0.00 E a Da ta are expressed as Log CFU Numbers in columns followed by the same letter are not significantly different at P< 0.0 5 according to the LS Means test.

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40 Table 4 4 In vitro trials with the copper sensitive strain L7 ( Xanthomonas campestris pv. vitians ) after incubation with various chemical compounds a Da ta are expressed as Log CFU Numbers in columns followed by the same letter are not sig nificantly different at P< 0.05 according to the LS Means test. Trial 1 a Trial 2 Tanos 9. 69 A Tanos 9.5 4 A Cymoxanil 9.64 A F amoxadone 9.49 A Control 9.56 A Control 9.4 5 A Famaxadone 9. 50 A Cymoxanil 9.4 4 A Kocide + Tanos 0.00 B Kocide 0.00 B Kocide 0.00 B Kocide + Famaxadone 0.00 B Kocide + Cymoxanil 0.00 B Kocide + Tanos 0.00 B Kocide + Famaxadone 0.00 B Kocide + Cymoxanil 0.00 B Kocide + Tanos + Mancozeb 0.00 B Kocide + Mancozeb 0.00 B Kocide + Mancozeb 0.00 B Kocide + Tanos + Mancozeb 0.00 B

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41 Table 4 5 Numbers of lesions caused by Xanthomonas perforans strain T4 on tomato pl ants treated with chemicals in g reenhouse trials Trial 1 a Trial 2 Trial 3 Famaxadone 2.47 A Famax adone 2.64 A Control 2.86 A Tanos 2.45 A Tanos 2.42 AB Tanos 2.80 A Cymoxanil 2.35 A Control 2.37 B Famaxadone 2.76 AB Control 2.31 A Cymoxanil 2.34 B Cymoxanil 2.56 ABC Kocide + Famaxadone 1.42 B Kocide 2.09 C Kocide 2.52 ABC Kocide + Cymoxanil 1.41 B Kocide + Famaxadone 1.94 CD Kocide + Tanos 2.41 BC Kocide + Tanos 1.41 B Kocide + Tanos 1.82 DE Kocide + Cymoxanil 2.41 BC Kocide 1.32 BC Kocide + Cymoxanil 1.66 EF Kocide + Famaxadone 2.32 C Kocide + Mancozeb 1.12 C Kocide + Tanos + Mancozeb 1.61 EF Kocide + Tanos + Mancozeb 2.27 C Kocide + Tanos + Mancozeb 1.10 C Kocide + Mancozeb 1.53 F Kocide + Mancozeb 2.23 C a Data are expressed as Log of number of lesions on 5 th leaf Numbers in columns followed by the same letter are not significantly diffe rent at P< 0.05 according to the LS Means test.

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42 Table 4 6 Effects of Xanthomonas euvesicatoria strain P6 on pepper pl ants treated with chemicals in g reenhouse trials Trial 1 a Trial 2 Trial 3 Cymoxanil 10.00 A Cymoxanil 8.80 A Tanos 11.67 A Contro l 9.83 A Famaxadone 8.60 A Cymoxanil 11.00 AB Famaxadone 9.83 A Tanos 8.50 A Famaxadone 10.67 AB Tanos 9.17 A Control 8.40 A Control 10.33 B Kocide 7.00 B Kocide + Famaxadone 6.80 B Kocide + Famaxadone 7.17 C Kocide + Famaxadone 6.83 B Kocide 6.50 BC K ocide 6.50 CD Kocide + Cymoxanil 6.17 BC Kocide + Cymoxanil 6.40 BC Kocide + Cymoxanil 6.00 DE Kocide + Tanos 5.83 BC Kocide + Tanos 5.83 C Kocide + Tanos 5.50 DEF Kocide + Mancozeb + Tanos 5.00 C Kocide + Mancozeb 3.67 D Kocide + Mancozeb 5.17 EF Kocide + Mancozeb 4.67 C Kocide + Mancozeb + Tanos 3.50 D Kocide + Mancozeb + Tanos 4.83 F a Data are exp ressed as Horsfall Barett rating (HB) Numbers in columns followed by the same letter are not significantly different at P< 0.05 according to the LS Means test.

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43 Disease Control on T omato in the G reenhouse Plants treated with Tanos Famaxadone or Cymoxanil did not induce any disease control in all three trials. In the first trial, a similar level of control was obtained between the Kocide alone, and K ocide + Mancozeb or Mancozeb + Tanos + Kocide. The addition of Tanos Cymoxanil or Famaxadone to Kocide result ed in a similar disease control to Kocide alone. In the second trial, only the combination Kocide + Famaxadone was similar to Kocide alone; the o ther mixtures that contained K ocide ha d signifi cantly reduced disease severity. There was a lot of variation in disease control in disease control. Disease C ontrol on P epper in the G reenhouse In the first trial, the disease severity of the untreated plant s had a mean ra ting of 9.83 (HB rating, Table 4 6 ) which was not significant ly different from the means of plants treated with Tanos Cymoxanil or Famaxadone A s imilar pattern was observed for the two other trials. In trials 1 and 3, the mixture Tanos /K ocide resulted in a similar disease control to the standard combination of Kocide/ Manzate Tanos tends to increase the efficacy of the mixture Kocide/Manzate in two of the trials. In all trials, Kocide alone provided a similar level of control as the comb inasion of Kocide with Tanos Cymoxanil or Famaxadone Discussion In this study, we were interested in dissecting the components of Tanos Cymoxanil and Famaxadone in the control of bacterial spot of tomato and pepper, and bacterial leaf spot of lettuce. In Florida, the control of bacterial spot is primarily based on copper mancozeb sprays. The EBDC can break down in the environment and can generate carcinogenic residue on fruits (US EPA, 1992). The use of EBDC s may be

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44 further regulated on food crops. The refore, it is desirable to investigate alternative compounds as a potential substitute for mancozeb. Tanos Cymoxanil and Famaxadone did not have any inhibitory activity against the in vitro growth of X. perforans, X. euvesicatoria and X. campestris pv vi tians In some of the trials, the chemicals appeared to promote bacterial growth as previously reported by Roberts et al. (2008). In all trials, greenhouse data reflected the trends shown by Cymoxanil Famaxadone and Tanos No disease suppression was obser ved with plants treated with these chemicals. However, Roberts et al. (2008) reported disease suppression with applications of Famaxadone in greenhouse experiments. Pernezny et al. (2008b) found that mancozeb and Famaxadone / Cymoxanil did not have any inhib itory effects on X. campestris pv. vitians, X. perforans and X. euvesicatoria in vitro They coucluded that the reports of disease suppression with Famaxadone / Cymoxanil (Roberts et al., 2008) might not be due to bactericidal activity but rather due to anot her m ode of action, such as systemic acquired resistance. As previously reported by Pernezny et al. (2008b), there is a trend for Tanos to induce a modest synergistic increase in the toxicity of Kocide and Manzate to X. perforans and X. euvesicatoria In f our of the six in vitro trials with the strains X. euvesicatoria and X perforans this reduction was statistically significant ( 4.2 ). There is a trend of synergism for the mixture of Tanos and Copper for all in vitro trials with the strain X. perforans (Ta ble 4 2 ). However, increased disease control was not obtained with the greenhouse b data since there was no statistical difference between Kocide alone, and Kocide/ Tanos for disease control in five of the six trials (Tables 4 5, 4 6) In some instances, the mixture Kocide/ Tanos led to simila r level of disease control as the

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45 standard mixture of Kocide/Manzate, and Kocide + Tanos + Manzate. Roberts et al. (2008) reported that various mixtures of Famaxadone Famaxadone + Cymoxanil mancozeb, and copper all su pressed bacterial spot in comparison to the applications of the copper mancozeb standard. The use of Famaxadone plus Cymoxanil and mancozeb could possibly reduce the application of copper, which might help reduce the selection of copper resistant Xanthomon as strains and the accumulation of copper in the environment (Roberts et al., 2008). In vitro and greenhouse trials pointed to copper/mancozeb, including Tanos in some instances, as the best treatment. Addition of mancozeb to copper usually resulted in in creased mortality of the pathogen. This might be due to a higher concentration of Cu 2+ ions in solution (Marco and Stall, 1983). However, Jones et al. (1991) hypothesized that soluble copper may contribute to toxicity against the Cu r strains, but does not appear to be the primary component involved in the toxicity of the copper/mancozeb combination to Cu r strains of X. c. vesicatoria Other workers demonstrated that zinc is involved in the observed control of bacterial spot (Adaskaveg and Hine, 1985). Manco zeb may also chelate copper ions, therefore increasing their availability to certain sites in bacterial cells (Medhekar and Boparai, 1981). I f levels of free ions increase a number of toxic effects can occur in cells (Cooksey, 1994). Copper is a potent in hibitor of photosynthetic electron transport at concentrations higher than 1 M ( Mohanty et al., 1989 ). In one trial with a strain of X. perforans T4, (Table 4 5 ), no difference was obtained with Cymoxanil and all the treatments that contain ed copper, in cluding the standard copper/mancozeb mixture. No significant difference was obtained between the

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46 untreated plants and those treated with Cymoxanil Pernezny et al. (2008b) reported that the addition of mancozeb to copper did not induce a sufficient reducti on of bacterial populations for a strain of X euvesicatoria Populations were still above 1x 10 6 CFU/ mL after a 2 hour exposure in vitro These observations support the suboptimal performance of the standard copper/mancozeb mixture under optimal conditions for disease development (Jones and Jones, 1985).

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47 CHAPTER 5 INFLUENCE OF TANOS AND ITS COMPONENTS IN THE POPULATION DYNAMICS ON THE LEAVES Microbial population dyn amics on leaves are related to four processes: immigration, emigration, growth and death (K inkel, 1997). Several patterns have been generally reported for leaf s urface populations: a) aggrega tion of phyllosphere microbial populations within individual leaves, b) var iability among leaves and among plants, c) increase over the growing season, d) high variability over short period of time, associated with specific environmental events (e.g., rainstorms) and e) existence of seasonal patterns among phyllosphere populations (Kinkel, 1997). The terms epiphytic, phylloplane, resident and leaf surface bacteria are interchangeably used in the literature (Beattie and Lindow, 1995; Hirano and Upper, 1983 ). It is generally admitted that epiphytic bacteria are able to live (multiply) on plant surfaces (Hirano and Upper, 1983; Leben, 1965). Leben (1963, 196 5) used the terms those that may reach the leaves by chance but can not multiply. From a functional perspective, epiphytic bacteria are considered those that can be removed from above ground plants parts by washing (Hirano and Upper, 1983 ) In contrast, endophytic bacteria are considered to be those that can live in the leaf intercellular spaces, substomatal cavities, or vascular tissues and have been functionally de fined as those bacteria that remain after the removal of the epiphytic bacteria ( Beatie and Lindow, 1995 ) Both the surfaces and internal regions of the leaves can be colonized by foliar pathogens (Hirano and Upper, 1990; Leben, 1965) and active exchange o ccurs between the internal and external population (Bashan et al., 1981). Foliar pathogens can have access to internal leaf tissues from the surface (Beattie and Lindow, 1995).

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48 Surfaces structures, such as stomata (Gitaitis et al., 1981), hydathodes (Brets chneider et al., 1989) have been reported as entry sites for many foliar pathogens. Internal population can egress onto the surface. The formation of lesions may increase the amount of cells that egress and subsequently spread the pathogen on the leaf surf ace (Beattie and Lindow, 1995). Epiphytic phytopathogenic bacteria can provide inoculum for disease development and for spread to the surface of other plants and plant parts (Hirano and Upper, 1983; Beattie and Lindow, 1995). In a given pathosystem, each ep iphytic phytopathogenic bacterium has a finite and extremely low probability of inducing disease. Subsequently, when resident populations are sufficiently high on individual leaves, the probability of causing disease is greater under favorable condition s (Hirano and Upper, 1883). Large epiphytic populations have been associated with time of disease onset and with increase d level s of disease (Beattie and Lindow,1995). Under field conditions, detection of disease was always observed at a population level o f at least 5 x 10 6 cells per navy bean leaflet of either X campestris pv phaseoli or X campestris pv phaseoli var. fuscans (Weller and Saettler 1980). For some pathosystems, including brown spot disease o f beans (Lindemann et al.1984) and halo blight of oats (Hirano et al. 1981), a quantitative relationship has been established between epiphytic population size and probability of disease occurrence. Increases in resident populations of P. syringae pv tomato result in higher incidence of bacterial spec k on tomato with a 10 to 12 day lag required for infection and sym ptom expression (Smitley and McCarter 1982). Studying the role of hrp genes in the fitness of P. syringae on beans, Hirano et al. (1997; 1999)

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49 found that an intact type III secretion syst em is required for the growth, and possibly the survival of P. syringae in the phyllosphere. It is generally accepted that bacteria must gain access to the internal tissues and establish large endophytic populations for successful infe ction. Therefore, th e internal populations, not the epiphytic p opulations, are essential for disease development However, there is a strong correlation between high epiphytic population sizes and high probability of disease occurrence in some foliar diseases. One reason for this is that increased epiphytic populations potentially result in higher endophytic population sizes (Beattie and Lindow, 1995). However, it was been shown that large shoot surface populations of phytopathogens can exist in the absence of disease (Hirano and Upper, 1983, Leben, 1965). Thus, large resident popu lations of a phytopathogen may increase the probability of endophytic populations, but their presence does not ensure the development of large endophytic populations that results in disease Based o n i nfecti vity titration experiments, a bacterial concentration of 10 4 cfu/ml was enough to initiate disease in compatible host/pathogen inoculations (Robinson et al., 2006). In the presence of large epiph ytic population size, the extent of ingress, which relies on the number of entry sites available (Ramos and Volin, 1987) and environmental conditions (Daub and Hagerdorn, 1979), is a major factor that influences disease induction. The number of entry sites is influenced by host genotype, leaf age, posit ion on leaf surfaces and wounds (Ramos and Volin, 1987; Beattie and Lindow, 1995). Jones et al. (1991) reported that copper and a mixture of copper and mancozeb reduced the epiphytic population of X campestris pv. vesicatoria in comparison to the untreat ed plants. A positive correlation, between epiphytic populations and disease

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50 severity, was also found. In one test, Pernezny and Collins (1997) found that copper sprays reduced X. campestris pv vesicatoria population s on pepper l eaflets in 99% in compari son to a reduction of 51 % in buds. This study was undertaken to determine the effects of Tanos and its components alone in combi nation with copper and mancozeb, in some instances, on the population dynamics of Cu r strains of X. perforans and X. euvesicato ria on tomato leaves and pepper leaflets. Materials and Methods Strains and I noculum R ifampicin resistant P6 and T4 strains were selected as follow : 25 uL aliquots of 24 h old bacteria culture grown in Nutrient B roth ( Laboratories Difco ), were spread on Nutrient Agar (NA, Laboratories Difco ) plates amende d with rifampicin ( 25 g /mL). Plates were incubated for 3 days at 0 C. Rifampicin resistant colonies (Rif T4; Rif P6) were selected. RifT4 or RifP6 strains were grown for 24 h on NA amended with rifampici n, and then flooded with a solution of 0.01 M MgS0 4 Suspensions were adjusted to an optical density of 0.30 at 600 nm with a spectrophotometer. The inoculation of the plants, the time in the growth chamber and the application of the chemical suspensions were as previously describe d for the greenhouse component in the previous chapter. Leaf Sampling and Assay P rocedure Three inoculated leaflets per treatment were randomly sampled 0, 2, 4, 6, 8, 10 and 12 days after inoculation, and then at days. Each lea flet was placed in a 50 mL tube weighed and then mixed with a volume of 10 mL of peptone buffer per gram of tissue. The buffer contains (per liter) 5.3 g of KH 2 PO 4 8.6 g of Na 2 HPO 4 and 1 g of bacto peptone ( McGuire et al. 1986). Tubes were shaken on a rot ary shaker at 200 rpm for 45

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51 min. Serial 10 fold dilutions were made in of 0.01 M MgS0 4 A 50 l aliquot of different serial dilutions was plated onto each of three plates of Nutrient Agar amended with rifampicin. After incubation at 28 0 C for 3 days, typic al colonies of X perforans or X euvesivatoria were counted. Sta tistical analysis was carried out with log 10 transformed data. Data were expressed as log 10 CFU/g of tissue. The a nalysis included variance and a mean separation using the Lea st Significant d ifference (LSD). Results Epiphytic populations of X. euvesicatoria show a variable pattern among the treat ments in both trials. In one trial with a strain of Xanthomonas euvesicatoria (Table 5 2) and one trial with Xanthomonas perforans (Table 5 3 ), some statistical differences were obtained among some treatments in some sampling days (Day 0, Day 2, Day 4, and Day 6) whereas no statistical difference was obtained for the remainder of the experiment. Generally, the addition of Tanos and its com ponents to Kocide in some instances, did not reduce epiphytic populations at significant level in comparison to Kocide alone, and the control in some days. The general trend is that the populations in different treatments are the same over the period durin g which the samplings were carried out. After inoculation (Day 0) the epiphytic populations of X. euvesicatoria of all plants treated with copper and Famaxadone we re similar, whereas these populations were significantly lower than those of untreated plant s, or plants treated with Cymoxanil or Tanos In samplings days 2 and 4, epiphytic populations on leaflets treated with Tanos were higher than on untreated control plants.

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52 Table 5 1 Populations dynamics of a strain of X. euvesicatoria on pepper leafl ets Day 0 a Day 2 Day 4 Day 6 Day 8 Day 12 Famaxadone 9.73 A Control 9.86 A Kocide + Tanos 5.00 A Cymoxanil 6,73 A Kocide + Tanos + Mancozeb 10.98 A Kocide 9.36 A Cymoxanil 9.66 A Cymoxanil 9.76 A Cymoxanil 4.82 A Famaxadone 6.39 AB Cymoxanil 10.94 AB Tanos 9.12 A Control 9.65 A Tanos 9.75 A Tanos 4.44 AB Kocide + Famaxadone 6.34 AB Kocide + Famaxadone 10.94 AB Famaxadone 9.01 A Tanos 9.62 A Kocide 8.71 B Famaxadone 4.43 AB Control 6.26 AB Control 10.92 AB Kocide + Cymoxanil 8.33 A Kocide + Famaxa done 8.06 B Kocide + Tanos 8.40 BC Control 4.23 AB Kocide 5.99 ABC Tanos 10.92 AB Kocide + Famaxadone 8.32 A Kocide 7.87 B Famaxadone 8.30 BC Kocide + Famaxadone 3.66 AB Kocide + Cymoxanil 5.77 ABC Kocide + Cymoxanil 10.91 AB Control 8.16 A Kocide + Ta nos 7.63 BC Kocide + Famaxadone 8.25 BC Kocide + Mancozeb 3.55 AB Tanos 5.35 ABC Kocide + Tanos 10.91 AB Kocide + Tanos + Mancozeb 8.05 A Kocide + Cymoxanil 7.17 C Kocide + Cymoxanil 8.06 C Kocide 3.49 AB Kocide + Tanos 5.28 ABC Famaxadone 10.89 AB Koci de + Tanos 7.82 A Kocide + Tanos + Mancozeb 6.52 D Kocide + Mancozeb 5.91 D Kocide + Cymoxanil 2.94 B Kocide + Tanos + Mancozeb 4.32 BC Kocide + Mancozeb 10.88 B Kocide + Mancozeb 7.56 A Kocide + Mancozeb 5.58 E Kocide + Tanos + Mancozeb 5.81 D Kocide + Tanos + Mancozeb 2.88 B Kocide + Mancozeb 3.79 C Kocide + Tanos + Mancozeb 10.86 B Cymoxanil 7.54 A a Data are expressed as Log CFU/g. Numbers in columns followed by the same letter are not significantly different at P< 0.05 according to the LS Means test.

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53 Table 5 2 Populations dynamics of a strain of X. euvesicatoria on pepper leaflets Day 0 a Day 2 Day 4 Day 6 Day 8 Day 10 Day 12 Famaxadon e 8.08 A Tanos 7.94 A Tanos 6.92 A Tanos 7.50 A Tanos 8.89 A Tanos 11.9 8 A Control 11.19 A Contro l 7.61 A Cymoxanil 7.00 AB Famaxadon e 6.18 AB Famaxadon e 6.76 AB Control 8.15 AB Control 10.2 5 AB Cymoxanil 10.36 AB Cymoxanil 6.81 AB Control 6.83 AB Cymoxanil 5.83 AB Cymoxanil 5.86 ABC Famaxadon e 7.72 ABC Kocide + Cymoxanil 9.50 AB Tanos 10.2 AB Tanos 6.79 AB Famaxadon e 6.71 ABC Control 5.53 ABC Kocide + Famaxadon e 5.81 ABC Cymoxanil 7.69 ABC Famaxadon e 8.38 BC Famaxadon e 10.02 AB Kocide 5.81 BC Kocide 5.96 BD C Kocide 5.45 BC Kocide + Tanos 5.05 BC D Kocide 7.40 ABCD Cymoxanil 8.38 BC Kocide + Cymoxani l 9.14 ABC Kocide + Famaxadon e 5.61 BC D Kocide + Tanos 5.53 BD C Kocide + Famaxadon e 4.74 BD C Control 5.04 BD C Kocide + Cymoxanil 6.70 ABCD Kocide + Famaxadon e 7.85 BC Kocide 7.69 BCD Kocide + Tanos + Mancozeb 5.09 BC D Kocide + Tanos + Mancozeb 5.47 BD C K ocide + Tanos 4.14 CD E Kocide + Cymoxanil 4.94 BD C Kocide + Tanos + Mancozeb 6.57 ABCD Kocide 7.63 BC Kocide + Tanos 6.77 CD Kocide + Cymoxanil 4.46 CD Kocide + Famaxadon e 5.23 DC Kocide + Cymoxanil 3.58 DE Kocide 4.87 BD C Kocide + Famaxadon e 6.18 BCD Tan os + Kocide 6.43 CD Kocide + Famaxadon e 6.18 D Kocide + Tanos 4.83 CD Kocide + Mancozeb 4.72 D Kocide + Tanos + Mancozeb 3.33 ED Kocide + Tanos + Mancozeb 4.28 CD Kocide + Mancozeb 5.28 CD Kocide + Tanos + Mancozeb 5.64 CD Kocide + Mancozeb 5.14 D Kocide + Mancozeb 4.00 D Kocide + Cymoxanil 4.71 D Kocide + Mancozeb 3.25 E Kocide + Mancozeb 3.61 D Kocide + Tanos 4.96 D Kocide + Mancozeb 4.49 D Kocide + Tanos + Mancozeb 5.14 D a Data are expressed as Log CFU/g. Numbers in columns followed by the same lette r are not significantly different at P< 0.05 according to the LS Means test.

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54 Table 5 3 Populati ons dynamics of a strain of X. perforans on tomato lea ves Day 0 a Day 2 Day 4 Day 6 Day 8 Day 10 Day 12 Cymoxanil 8.77 A Control 10.64 A Control 9.07 A Cymoxanil 9.34 A Control 10.60 A Control 10.40 A Famaxadone 11.47 A Tanos 8.49 A Tanos 9.50 AB Famaxadone 8.93 A Famaxadone 9.27 A Tanos 10.37 AB Famaxadone 10.07 A Tanos 10.43 AB Control 8.47 A Famaxadone 9.47 AB Tanos 8.88 AB Control 9.17 A Cymo xanil 9.40 ABC Cymoxanil 9.83 AB Control 9.97 BC Famaxadone 8.46 A Cymoxanil 9.10 B Cymoxanil 8.47 AB Tanos 9.13 A Kocide 8.73 BCD Tanos 9.00 ABC Cymoxanil 9.23 CD Kocide 7.76 AB Kocide 7.53 C Kocide + Cymoxanil 7.50 BC Kocide 7.33 B Famaxadone 8.70 BC D Kocide 8.37 BCD Kocide 8.13 DE Kocide + Tanos 7.21 BC Kocide + Famaxadone 7.07 CD Kocide + Famaxadone 7.03 DC Kocide + Famaxadone 7.30 B Kocide + Cymoxanil 8.43 CDE Kocide + Tanos 8.33 BCD Kocide + Cymoxanil 7.93 E Kocide + Famaxadone 6.73 BCD Kocid e + Tanos + Mancozeb 6.43 CD Kocide 6.80 CD Kocide + Cymoxanil 7.13 B Kocide + Tanos 8.03 CDE Kocide + Cymoxanil 8.27 BCD Kocide + Famaxadone 7.90 E Kocide + Tanos + Mancozeb 6.19 CD Kocide + Tanos 6.33 CD Kocide + Tanos 6.37 CDE Kocide + Tanos + Mancoz eb 6.73 B Kocide + Famaxadone 7.53 DE Kocide + Famaxadone 8.20 CD Kocide + Tanos 7.60 EF Kocide + Cymoxanil 6.17 CD Kocide + Cymoxanil 6.27 CD Kocide + Mancozeb 5.77 DE Kocide + Tanos 6.37 BC Kocide + Tanos + Mancozeb 7.00 E Kocide + Mancozeb 7.40 CD Ko cide + Tanos + Mancozeb 6.60 FG Kocide + Mancozeb 5.66 D Kocide + Mancozeb 5.83 D Kocide + Tanos + Mancozeb 5.10 E Kocide + Mancozeb 5.40 C Kocide + Mancozeb 6.77 E Kocide + Tanos + Mancozeb 7.17 D Kocide + Mancozeb 6.20 G a Data are expressed as Log CFU/ g. Numbers in columns followed by the same letter are not significantly different at P< 0.05 according to the LS Means test.

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55 As in trial 1 for X. euvesicatoria a similar trend of similar treatmen ts was observed over time in trial 2. Specific redu ctions of populations could be observed during a giving sampling day, but such difference s were not consistent over time, except the combination of copper/mancozeb in relation to the untreated plants. The copper and mancozeb combination consistently reduce d epiphytic populations of X. euvesicatoria compared to those of the control (Table 5.2 ) during all the sampling days (exception: day 6). All plants treated with Kocide and another chemical generally presented similar populations than plants treated with K ocide alone. The addition of Tanos to Copper seems to promot e modest bactericidal activity in the reduction of epiphytic populations since plants treated with this mixture presented the lowest epiphytic populations although at levels no statistically dif ferent in most of the sampling days. However, the addition of Tanos did not appear to increase the bactericidal property of the standard mixture of copper/mancozeb since plants treated with copper/mancozeb generally presented lower epiphytic populations t han plants treated with Tanos + copper + mancozeb in most of the samplings days. As observed in trials with X. euvesicatoria the population dynamics of X. perforans show a variable pattern among the treatments (Table 5.3 ). The treatments that contain copp er generally induced a similar level of control of epiphytic bacterial populatio ns although population size s varied greatly among the different treatments with copper. Moreover, the mixture Kocide / Mancozeb tends to be the best treatment in terms of reduc tion of epiphytic populations in comparison to the untreated plants The addition of Tanos to C opper seems to have a modest effect since in most sample t imes,

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56 plants treated with Kocide /Tanos generally have lower populations than those treated with kocide alone. Discussion Epiphytic bacteria are able to multiply on plant surfaces (Hirano and Upper, 1983). Large epiphytic populations have been associated with time of disease onset and with disease progression (Beattie and Lindow,1995). For foliar pathogens, like xanthomonads associated with bacterial spot of pepper and tomato, foliar application of chemicals can modify the populations dynamics on the leaves. The effects of copper based bactericides on phyllosphere populations have been reported in some studie s. (Scheck and Pscheidt,1998; Pernezny and Collins, 1997; Jones et al. 1991). The aim of this s tudy was to evaluate the effects of Tanos Cymoxanil and Famaxadone alone, and in combination with Kocide and in some instances, mancozeb, on the epiphytic popul ations of a strain of X. euvesicatoria and a strain of X. perforans The general trend is that many treatments were similar in a given sampling period and over time. In both trials with a strain of X. euvesicatoria P6, all treatments that contain Kocide generally did not differ significantly over time although population sizes vary among them in a given sampling day. Specifically, the addition of Tanos either to copper or to copper/mancozeb, did not result in better bactericidal a ctivity in terms of redu ction in phyllosphere populations.

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57 Figure 5 1 P opulation dynamics of strain of X. euvesicatoria on pepper leaflets Trial 1 In trial 1 with a strain of X. euvesicatoria the general trend is that all treatments were similar over time. Significant redu ctions of phyllosphere populations among some treatments during the first sampling days were not consistent over time. In this trial, the treatments that contain copper, including the standard copper/mancozeb, did not reduce the phyllosphere populations al though plants treated with the standard mixture presented the lowest populations (no statistical difference) in most of the sampling days.

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58 Figure 5 2 P o pulation dynamics of a strain of X euvesicatoria on pepper leaflets Trial 2 In one trial with a strain of X. euvesicatoria ( Ta ble 5 2 and Figure 5 2 ), and another one with a strain of X. perforans (Table 5 3 ), the copper/mancozeb mixture appears to be the best treatment ove r time in terms of reductions in phyllosphere populations. I n contrast with t he trial 1 with X. euvesicatoria ( Table 5 1 and Figu re 5 1 ), the standard mixture consistently reduce d the pyllosphere populations in comparison to the untreated plants. In a greenhouse study where a copper resistant strain of X.c.vesicatoria was applied t o tomato foliage, plants treated with copper or with copper and mancozeb mixture resulted in lower bacterial populations than untreated plants (Jones et al., 1991). Lower bacterial populations on the leaf may reduce initial ino culum, and consequently, affe ct bacterial spot epidemics (Pernezny and Collins, 1997). A quantitative relationship has been established between epiphytic population size and

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59 probability of disease occurrence for some pathosystems (Lindemann et al. 1984; Hirano et al. 1981). Lindemann et al. (1984) demonstrated that a threshold of 10 4 cfu per gram of leaflet tissue was a good predictor of brown spot incidence on snap beans, caused by Pseudomonas syringae pv. syringae Very high populations of this pathogen constituted a more reliable pr edictor of disease severity than for disease incidence. Leaf surface chemistry may be an important factor in the interaction between copper bactericides and copper resistant strains. Copper sprays applied to leaves exist primarily as insoluble deposits of copper salts (Menkissoglu, and Lindow, 1991b). Components of leachates on the tomato leaf surface may modify bacteria sensitivity to copper (Jones et al. 1991). Binding of copper to environment al constituents affects the biological availability of copper a nd can reduce the toxicity of copper towards microorganisms (Gadd and Griffith, 1978). In vitro studies showed that the toxicity of Cu 2+ to copper sensitive and copper tolerant strains of Pseudomonas syringae was reduced in the presence of organic compound s including glucose, fructose, sucrose, succinate and ci trate (Menkissoglu and Lindow 1991a). Field trials demonstrated that the concentration of soluble and complexed forms of copper was abundant on navel orange and beans leaves following spray applicati on, but had no significant toxicity towards strains of P. syringae (Menkissoglu and Lindow, 1991b). The leachates on pepper and tomato may interact with copper, and therefore could disminish the amou n t of soluble copper and Cu 2+ which might result in less toxicity towards the epiphytic populations of X. perforans and X. euvesicatoria on tomato and pepper leaflets respectively.

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60 CHAPTER 6 CONCLUSIONS Tank mixes of pesticides are often used in plant disease control In this study, we evaluated Tanos ands its components Cymoxanil and Famaxadone in association with copper in the control of bacterial spot of tomato and pepper, and bacterial leaf spot of lettuce. Pesticides are combined in order to widen the spectrum of biological activity, to d elay the s election of resistant strains and to exploit synergistic interactions (Gisi et al., 1985). In vitro studies show a synergistic action between copper and Tanos against the in vitro growth of a strain of Xanthomonas perforans It was also found that Tanos tended to induce a modest increase in the toxicity of Kocide and Mancozeb to X. perforans and X. euvesicatoria It appears that both components of Tanos, Cymoxanil and Famaxadone are required for synergistic action since the addition of Cymoxanil or Famax adone to copper did not induce significant reduction of bacterial populations in comparison to copper alone. Tanos, and its components do not have any inhibitory activity against the in vitro growth of the pathogens evaluated. In contrast, Tanos appears to promote bacterial growth as previously reported by Robert et al. (2008). Therefore, the mechanism by which this growth occurs could be desirable to elucidate. There was a lot of variation in disease control in greenhouse studies. Plants treated with the st andard mixture copper/Mancozeb, including Tanos in some instances, tended to have a lower disease incidence. But, no statistical difference was obtained in most the trials between the copper/mancozeb and other treatments that contain copper. Various mixtur es of Famaxadone + Cymoxanil m ancozeb and c opper were reported to induce a similar disease suppression as copper/mancozeb. The use of Famaxadone +

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61 Cymoxanil could possibly reduce the application of copper, which might help reduce the selection of copper r esistant Xanthomonas strains and the accumulation of copper in the environment (Roberts et al. 2008). A variable pattern was also obtained in the study related to the populations dynamics of X. perforans and X. euvesicatoria on tomato and pepper leaflets High variability of epiphytic populations could be observed over a short period of time (Kinkel, 1997). All treatments that contain c opper generally did not differ at stastistica l level in a given sampling period. In one trial with a strain of X. perforans, the standard mixture copper/mancozeb did consistently reduce the pyllosphere populations in each sampling p eriod in comparison to the control. The addition of Tanos to the copper/mancozeb mixture did not appear to increas e bacteridal activity of this mixture to epiphytic populations. The rates of chemicals that were used in this study are recommended dosis for field applications. However, concentration of fungicides can be a major component in synergistic interactions. S ynergi stic interactions occured when oxadixyl, mancozeb and Cymoxanil were mixed in different concentrations against sensitive strains of Phytophthora infestans in vivo (Gisi et al. 1985). ssing synergism uses an approach of differe nt concentrations to construct the d ose responses of fungicides in mixture Equally effective concentrations, EC values, for different levels of control, are computed (Gisi e t al. 1985, Levy et al., 1986). Therefore, the synergistic trend observed in some in vitro trials with strains of X. euvesicatoria and X. perforans could be further supported by studying different concentrations of the chemicals involved in the synergic interactions.

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72 BIOGRAPHICAL SKETCH Joubert Fayette was born in Haiti He graduated from t Runi where he earned his Bachelor of Sc ience degree in agronomical engeneering at EARTH (Escuela de Agricultura de la Regin Tropical Hmeda) University in December 2007 In 2006, he did an undergraduate internship at Southwest Florida Research and Education Center, University of Florida. During his undergraduate studies, he evaluated several plants extracts against the in vitro growth o f Xanthomonas perforans Phytophthora capsici and Colletotrichum gloesporioides He attended the graduate program at the University of Florida, College of Agricultural and Life Sciences, Department of Plant Pathology, from August 2008 to August 2010. He conducted a research project that was based on the evaluation of Tanos and its comp onents, in association with copper, and mancozeb in some instances, in the management of bacterial spot of tomato and pepper, and bacterial leaf spot of lettuce.