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Etiology and Management of Recent Outbreaks of Pepper Anthracnose in Florida

Permanent Link: http://ufdc.ufl.edu/UFE0021951/00001

Material Information

Title: Etiology and Management of Recent Outbreaks of Pepper Anthracnose in Florida
Physical Description: 1 online resource (88 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

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

Notes

Abstract: In the last 4 to 6 years, anthracnose has become an increasingly serious disease on immature, green pepper fruit in Florida. This contrasts with earlier reports of anthracnose as strictly a ripe-rot disease of mature, colored pepper fruit. The species of Colletotrichum associated with anthracnose on both immature and ripe pepper in Florida were identified. Based on reactions with PCR-specific primers, 28 of 50 isolates associated with anthracnose lesions from Florida were identified as C. acutatum, including 22 of 22 recovered from immature, green fruit. Six of the C. acutatum isolates were associated with typical lesions on ripe, colored fruit, but only in fields where lesions on green fruit were also observed. In contrast, all 17 isolates identified by PCR as C. gloeosporioides were recovered from lesions found only on ripe, colored fruit from fields where no lesions on green fruit were initially observed. No isolates were identified as C. capsici or C. coccodes. Isolates of C. gloeosporioides grew up to twice as fast in vitro as isolates of C. acutatum, suggesting a way to tentatively differentiate pepper isolates without PCR testing. In addition, C. gloeosporioides produced conidia that were slightly larger than those produced by C. acutatum. In field and laboratory pathogenicity tests, anthracnose isolate HB05 recovered from bell pepper was not pathogenic on tomato or strawberry when artificially inoculated on ripe and unripe fruit in the field. However, anthracnose lesions did form on detached, wounded fruit of all three crops in the laboratory. This result suggests that laboratory wound-inoculation studies might not be a reliable method to determine the natural host range of Colletotrichum spp. on various crops in the field. Three fungicide field trials were conducted on pepper (?Revolution?) artificially inoculated with an isolate of C. acutatum recovered from pepper (HB05) to evaluate azoxystrobin (Quadris 250SC), famoxadone plus cymoxanil (Tanos 50WG), copper hydroxide (Kocide 2000), mancozeb (Manzate 75WG), acibenzolar-S-methyl (Actigard 50WG), and fludioxanil plus cyprodinil (Switch 50WG) for control of pepper anthracnose. In one of the three trials, difenoconazole (Inspire 250EC) was included. All treatments provided significant control of anthracnose symptoms on fruit in comparison to the untreated control. Overall, azoxystrobin, fludioxanil plus cyprodinil, difenoconazole, and mancozeb provided the highest amount of uninfected, healthy fruit per plot, while famoxadone plus cymoxanil, copper hydroxide, and acibenzolar-S-methyl provided the least amount of healthy fruit per plot among all of the treatments. The name 'early anthracnose' is proposed for the disease on immature, green fruit caused by C. acutatum.
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.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Pernezny, Kenneth L.
Local: Co-adviser: Datnoff, Lawrence E.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021951:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021951/00001

Material Information

Title: Etiology and Management of Recent Outbreaks of Pepper Anthracnose in Florida
Physical Description: 1 online resource (88 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

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

Notes

Abstract: In the last 4 to 6 years, anthracnose has become an increasingly serious disease on immature, green pepper fruit in Florida. This contrasts with earlier reports of anthracnose as strictly a ripe-rot disease of mature, colored pepper fruit. The species of Colletotrichum associated with anthracnose on both immature and ripe pepper in Florida were identified. Based on reactions with PCR-specific primers, 28 of 50 isolates associated with anthracnose lesions from Florida were identified as C. acutatum, including 22 of 22 recovered from immature, green fruit. Six of the C. acutatum isolates were associated with typical lesions on ripe, colored fruit, but only in fields where lesions on green fruit were also observed. In contrast, all 17 isolates identified by PCR as C. gloeosporioides were recovered from lesions found only on ripe, colored fruit from fields where no lesions on green fruit were initially observed. No isolates were identified as C. capsici or C. coccodes. Isolates of C. gloeosporioides grew up to twice as fast in vitro as isolates of C. acutatum, suggesting a way to tentatively differentiate pepper isolates without PCR testing. In addition, C. gloeosporioides produced conidia that were slightly larger than those produced by C. acutatum. In field and laboratory pathogenicity tests, anthracnose isolate HB05 recovered from bell pepper was not pathogenic on tomato or strawberry when artificially inoculated on ripe and unripe fruit in the field. However, anthracnose lesions did form on detached, wounded fruit of all three crops in the laboratory. This result suggests that laboratory wound-inoculation studies might not be a reliable method to determine the natural host range of Colletotrichum spp. on various crops in the field. Three fungicide field trials were conducted on pepper (?Revolution?) artificially inoculated with an isolate of C. acutatum recovered from pepper (HB05) to evaluate azoxystrobin (Quadris 250SC), famoxadone plus cymoxanil (Tanos 50WG), copper hydroxide (Kocide 2000), mancozeb (Manzate 75WG), acibenzolar-S-methyl (Actigard 50WG), and fludioxanil plus cyprodinil (Switch 50WG) for control of pepper anthracnose. In one of the three trials, difenoconazole (Inspire 250EC) was included. All treatments provided significant control of anthracnose symptoms on fruit in comparison to the untreated control. Overall, azoxystrobin, fludioxanil plus cyprodinil, difenoconazole, and mancozeb provided the highest amount of uninfected, healthy fruit per plot, while famoxadone plus cymoxanil, copper hydroxide, and acibenzolar-S-methyl provided the least amount of healthy fruit per plot among all of the treatments. The name 'early anthracnose' is proposed for the disease on immature, green fruit caused by C. acutatum.
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.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Pernezny, Kenneth L.
Local: Co-adviser: Datnoff, Lawrence E.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021951:00001


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ETIOLOGY AND MANAGEMENT OF RECENT OUTBREAKS OF PEPPER ANTHRACNOSE IN FLORIDA By TYLER L. HARP A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1

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2008 Tyler L. Harp 2

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To my wife, Cheryl, who has stood by me throu ghout this experience with patience, admiration, consideration, and love, And to my children, daughter Jo rdan and sons Caleb and Canaan, who continue to provide for me the energy and entertainment needed to enjoy each day, And to my mother, who unexpectedly and trag ically passed on during the writing of this dissertation, but has always given me the inspir ation and determination to make things happen, and who continues to live on a nd be with me in spirit. 3

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ACKNOWLEDGMENTS I would like to thank Dr. Ken Pernezny and members of my supervisory committee for their guidance and support. Also, I give spec ial thanks to Dr. Paul Kuhn for all of his encouragement, wisdom, advice, and direction. W ithout his support for this project, it would not have been done. I thank Dr. Charlie Mellinger of Glades Crop Care, Jupiter, Florida, for providing anthracnose-infected peppers from Hendry Co. Thanks also go to Dr. David Langston, University of Georgia, Tifton, for providing is olate GA01 and useful discussions; and to Dr. Robert McGovern, University of Florida, for providing isolates MG01-MG03, all used in this study. I also thank Midway Farms in St. Lucie Co., Florida, for allowi ng the sampling of pepper fields for anthracnose-infected pepper fruit; and to Leslie Fuquay, Syngenta Crop Protection (Greensboro, NC), for statistical analysis and recommendations. I give special thanks to Dr. Sally Miller and Melanie Lewis-Ivey, of The Ohio State University, for hosting my visit to Wooster, Ohio and allowing me to use their laboratory. In addition to providing help and assistance, th eir work on pepper anthracnose provided the foundation for which my project was built. I am indebted to them for their graciousness and hospitality, and proud to have had the privilege to work in their laboratory. Lastly, I thank Syngenta Crop Protection, fo r allowing me the oppor tunity and providing the resource to support this endeavor. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 ABSTRACT .....................................................................................................................................9 CHAPTER 1 INTRODUCTION TO PE PPER ANTHRACNOSE..............................................................11 2 ETIOLOGY OF RECENT OUTBREAKS OF PEPPER ANTHRACNOSE IN FLORIDA...............................................................................................................................23 Introduction .............................................................................................................................23 Materials and Methods ...........................................................................................................25 Isolates .............................................................................................................................25 PCR Amplification ..........................................................................................................26 Growth Rate in vitro ........................................................................................................27 Conidial Measurements ...................................................................................................27 Results .....................................................................................................................................28 PCR Amplification with Species-Specific Primers .........................................................28 Colony Growth Rate ........................................................................................................28 Conidial Measurements ...................................................................................................29 Discussion ...............................................................................................................................29 3 HOST RANGE OF PEPPER ANTHRACNOSE ISOLATES RECOVERED FROM PEPPER IN FLORIDA...........................................................................................................36 Introduction .............................................................................................................................36 Materials and Methods ...........................................................................................................39 Host Range Field Trials ...................................................................................................39 Plants ...............................................................................................................................39 Inoculum for Field a nd Laboratory Evaluations .............................................................40 Field Treatment Plots ......................................................................................................40 Laboratory Detached Fruit ..............................................................................................41 Field Inoculations ............................................................................................................42 Laboratory Inoculation ....................................................................................................42 Disease Assessments .......................................................................................................43 Results .....................................................................................................................................43 Inoculation Field Trials ...................................................................................................43 Detached-Fruit Inoculation ..............................................................................................44 Discussion ...............................................................................................................................45 5

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4 CHEMICAL CONTROL OF PEPPER ANTHRACNOSE...................................................59 Introduction .............................................................................................................................59 Materials and Methods ...........................................................................................................61 Fungicide Field Trials ......................................................................................................61 Pepper Plants ...................................................................................................................61 Inoculum Production .......................................................................................................62 Fungicide Treatments ......................................................................................................62 Fungicide Applications ....................................................................................................62 Artificial Inoculation .......................................................................................................63 Disease Assessments .......................................................................................................63 Results .....................................................................................................................................64 Disease Assessments .......................................................................................................64 Fungicide Field Trial 1 ....................................................................................................65 Fungicide Field Trial 2 ....................................................................................................66 Fungicide Field Trial 3 ....................................................................................................66 Discussion ...............................................................................................................................67 5 SUMMARY AND DISCUSSION.........................................................................................77 REFERENCE LIST .......................................................................................................................81 BIOGRAPHICAL SKETCH .........................................................................................................88 6

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LIST OF TABLES Table page 2-1. Isolates of Colletotrichum spp. recovered from pepper fields throughout Florida or Georgia ...............................................................................................................................32 3-1. Plot size and planting conditions for each crop evaluated in Field Trial 1 and Field Trial 2 .................................................................................................................................51 3-2. Mean number of lesions per plot on pepper, strawberry and tomato fruit in inoculated, un-inoculated, a nd water-sprayed treatments for Field Trial 1 and Field Trial 2 at 10 days following an artificial inoculation with Colletotrichum acutatum .......52 4-1. Plot size and planting conditions fo r peppers in the fungicide field trials .........................72 4-2. Effect of fungicides on marketable yi eld of pepper artificial ly inoculated with Colletotrichum acutatum in three trials conducted in Florida in 2006 and 2007. .............73 7

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LIST OF FIGURES Figure page 1-1. Pepper anthracnose lesions on green, unr ipe bell pepper fruit recovered from Palm Beach Co., Florida in 2004.. ..............................................................................................22 2-1. Agarose PCR gel of isolates of Colletotrichum spp. collected from Florida and Georgia that have produced am plified DNA fragments with either CaInt2 (20 isolates from left) or CgInt (13 isolates from right) species-specific primer.. ................................33 2-2. Average radial growth per day of 45 isol ates representing the two species of pepper anthracnose isolates recovered from Florida as determined by PCR.. ..............................34 2-3. Isolates of Colletotrichum gloeosporioides and C. acutatum recovered in 2004 from pepper fruit in Florida growing on potato dextro se agar in continuous darkness at 30 C for 5 days ...................................................................................................................35 3-1. Ripened jalapeno and bell peppe r with anthracnose lesions caused by Colletotrichum gloeosporioides .................................................................................................................53 3-2. Unripe bell pepper with anthracnose symptoms caused by Colletotrichum acutatum ......54 3-3. Strawberry, tomato, and pepper plants fr om un-inoculated and inoculated plots in Field Trial 1.. ......................................................................................................................55 3-4. Detached, wound-inoculated fruit of strawberry and pepper fruit three days after inoculation.. ........................................................................................................................56 3-5. Detached, wound-injected fruit of to mato and pepper fruit five days after the inoculation with a c onidial suspension of Colletotrichum acutatum .................................57 3-6. Detached, wound-inoculated tomato and tomato and pepper 12 days after inoculation with a conidial suspension of Colletotrichum acutatum ....................................................58 4-1. Fully sized, harvestable fruit from the treated pepper plots inoculated with Colletotrichum acutatum ...................................................................................................74 4-2. Heavy infection of the flowers and newly-formed fruit as a result of the artificial inoculation by Colletotrichum acutatum ...........................................................................75 4-3. Harvested pepper from treated and untrea ted pepper plots in F ungicide Field Trial 3.. ...76 5-1. Pepper anthracnose isolate on unripe, green be ll pepper caused by Colletotrichum acutatum in Florida 8

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ETIOLOGY AND MANAGEMENT OF RECENT OUTBREAKS OF PEPPER ANTHRANCOSE IN FLORIDA By Tyler L. Harp May 2008 Chair: Ken Pernezny Cochair: Lawrence Datnoff Major: Plant Pathology In the last 4 to 6 years, anthracnose has become an increasingly serious disease on immature, green pepper fruit in Florida. This co ntrasts with earlier repor ts of anthracnose as strictly a ripe-rot disease of mature, co lored pepper fruit. The species of Colletotrichum associated with anthracnose on bot h immature and ripe pepper in Florida were identified. Based on reactions with PCR-specific primers, 28 of 50 isolates associated with anthracnose lesions from Florida were identified as C. acutatum including 22 of 22 recovered from immature, green fruit. Six of the C. acutatum isolates were associated with typical lesions on ripe, colored fruit, but only in fields where lesions on green fruit we re also observed. In contrast, all 17 isolates identified by PCR as C. gloeosporioides were recovered from lesions found only on ripe, colored fruit from fields where no lesions on green frui t were initially observe d. No isolates were identified as C. capsici or C. coccodes. Isolates of C. gloeosporioides grew up to twice as fast in vitro as isolates of C. acutatum suggesting a way to tentativel y differentiate pepper isolates without PCR testing. In addition, C. gloeosporioides produced conidia that were slightly larger than those produced by C. acutatum 9

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In field and laboratory pathogenicity tests, anth racnose isolate HB05 recovered from bell pepper was not pathogenic on tomato or strawberry when artificially inoculated on ripe and unripe fruit in the field. However, anthracnos e lesions did form on detached, wounded fruit of all three crops in the laboratory. This result s uggests that laboratory wound-inoculation studies might not be a reliable method to determine the natural host range of Colletotrichum spp. on various crops in the field. Three fungicide field trials we re conducted on pepper (Revolution) artificially inoculated with an isolate of C. acutatum recovered from pepper (HB05) to evaluate azoxystrobin (Quadris 250SC), famoxadone plus cymoxanil (Tanos 50WG), copper hydroxide (Kocide 2000), mancozeb (Manzate 75WG), acibenzolar-S-met hyl (Actigard 50WG), and fludioxanil plus cyprodinil (Switch 50WG) for control of pepper anthracnose. In one of the three trials, difenoconazole (Inspire 250EC) was included. A ll treatments provided significant control of anthracnose symptoms on fruit in comparison to the untreated control. Overall, azoxystrobin, fludioxanil plus cyprodinil, difenoconazole, a nd mancozeb provided the highest amount of uninfected, healthy fruit per pl ot, while famoxadone plus cymoxanil, copper hydroxide, and acibenzolar-S-methyl provided the least amount of healthy fruit per plot among all of the treatments. The name early anthracnose is proposed for the disease on immature, green fruit caused by C. acutatum 10

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CHAPTER 1 INTRODUCTION TO PEPPER ANTHRACNOSE Peppers ( Capsicum sp.) are herbaceous plants with fru it that are cultivated and consumed worldwide. The fruit are eaten as a fresh vegeta ble or dehydrated for use as one of the largest and most important spice commodities in the world. Pepper fruit or its extracts are widely used in foods, sauces, medicines, and cosmetics, and will continue to be an important vegetable and spice crop in many regions of the world. Capsicum species originated in the tropical Amer icas and are believed to have been consumed by humans since about 7500 BC (MacN iesh, 1964). The plants are thought to be among the oldest cultivated crops in the Ameri cas, with Native Americans reportedly growing and harvesting peppers between 5200 and 3400 BC (Heiser, 1976). Christopher Columbus is credited with bringing peppers to Europe on his return trip from the Ne w World, after reportedly naming the fruit red pepper, due to its similarity in texture and taste to the unrelated black pepper, Piper nigrum (Bosland, 1996). Capsicum spread rather quickly throughout Europe and into Asia, becoming an increasingly important an d prized spice for many civilizations. In the United States, pepper continues to be an important vegetable crop, not only for use as a spice but as an important component of the fresh vegeta ble market. During 2004, bell pepper was grown on over 23,000 hectares in the U.S. with a current market value of nearly $600 million. Florida, which ranks 2 rd to California among U.S. pepper-produci ng states, harvested over 7,400 hectares in 2004 with a current market value of over $218 million (USDA annual agricultural statistics www.usda.gov\nass). The genus Capsicum belongs to the Solanaceae family, along with eggplant, petunia, potato, tobacco, and tomato, and currently consists of about 25 species. The most economically important domesticated species, C. annum is the species to which most commercial cultivars 11

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belong. The center of genetic diversity of this sp ecies is in Mexico, with a secondary center in Mesoamerica (Bosland, 1996). Four other domesticated species exist: C. baccatum, C. pubescens, domesticated in the Peruvian Andes, and C. frutescens and C. chinense, domesticated in the Amazon Basin a nd Central America, respectively. Colletotrichum frutescens and C. chinense, are better known as tabasco and habane ro pepper, respectively. The remaining wild species are rarely utilized by man, but are likely to contain a va luable reservoir of genes that could be used to produce plants with enhanced genetic traits such as in creased yield or disease resistance. In Florida, peppers are an important co mmercial vegetable crop. They are grown throughout Florida, and are generally rotated with tomatoes or cucurbits. Like tomatoes, peppers are grown using the plastic mulch system with e ither drip or seep irrigation and in many cases are also staked and tied with st ring. Typically, pepper cultivation oc curs twice a year in Florida, in both fall and spring seasons. Actual planting and harvesti ng dates depend on the specific location within Florida. The fu rther south the location, the less likely for a frost to occur, and therefore the earlier they can be planted in the spring. Further north in the state, peppers are planted earlier in the fall and later in the spring. According to the USDA report -2003 Acreage, Yield, Production and Value of Florid a Vegetables (www.nass. usda.gov/fl/rtoc0v.htm), bell peppers accounted for 1,820 harvested hectares in the fall season and 5,170 hectares in the spring, with a total of 7,160 hectares harvested (o ut of 7,200 planted). The yields per hectare were 4,667 kg in the fall and 5,268 in the spring for an average of 4,968 kg per hectare. A total of 222 million kg were sold with a total annu al value of nearly $200 million. The 10-year statewide average yield for bell peppers as of 2003 is 5,400 kg per hectare (Maynard et al., 2003). This amount exceeds the reported value of cabbage, cucumbers, potatoes, snap beans, or 12

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squash during the 2002-2003 vegetable season in Fl orida. Tomato was the only vegetable crop with a higher monetary value ($546,699,000). This report does not include yields for jalapeno, habanero, or other specialty peppers, which are al so grown in Florida, particularly in South Floridas Miami-Dade and Palm Beach Counties. Cultivars of these specialty peppers include: Aruba, Key West, and Key Largo (C ubanelle-type peppers); and Habanero, Milta jalapeno, Xatapa jalapeno, Grande jalapeno Hungariane, Hot Wa x, Messilla, Long Thin Red Cayenne, and Large Red Thick Cayenne as specialty hot peppers (Li et al., 2000). In recent years, bell peppers have not been grown on a significant scale in Miami-Dade Co. As with most vegetable crops in Florida, peppe rs are susceptible to various diseases, pests, and disorders that can affect fruit quality and reduce yields. Some of the traditionally important diseases of pepper in Florida include bacteria l spot, frogeye leaf spot, Phytophthora blight, powdery mildew, and various viral diseases (Pernezny, et al., 2003). In some cases, cultivars of pepper have been developed with resistance to bacterial spot, Phytophthor a blight, as well as some poty virus diseases (Bosland, 1996). Many of these diseases are controlled with various fungicides, such as maneb and copper for bacterial spot, mefenoxam, metalaxyl, and dimethomorph for Phytophthora blight, or azoxystrobin for powdery mildew (Maynard et al., 2003). All of these diseases can have severe cons equences in terms of fr uit quality and yield if not controlled by chemical or other means, such as crop rotation or soil fumigation. Another increasingly important disease of peppe r in Florida is pepper anthracnose, caused by Colletotrichum spp. (Roberts et al., 1998). This dise ase has been reported in Florida on various cultivars of C. annum as well as an earlier report on C. chinense, or Jamaican Scotch bonnet pepper, in which incidence varied from 25 to 50% (McGovern and Polston, 1995). At least four different species of Colletotrichum have been reported in th e U.S. to cause anthracnose 13

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of pepper: C. acutatum C. capsici C. coccodes and C. gloeosporioides (Hadden, 1989; Marvel et al., 2003; Roy et al., 1997). Ho wever, only three species have been reported to cause this disease in Florida, C. capsici C. coccodes, and C. gloeosporioides (McGovern and Polston, 1995; Roberts et al., 1998). At the present time, C. acutatum has not been reported on pepper in Florida. Many of these species are likely pathogens or saprophytes of other important agricultural crops or weed species in Florida. For example, C. gloeosporioides has been reported to attack 470 host genera worldwide (Sutton, 1980). The earliest report of anthracnos e disease of pepper was by B. D. Halsted in 1891 (Halsted, 1891) where the causal agents were identified, perhaps mistakenly, as Gloeosporium piperatum and Colletotrichum nigrum Since that time, much debate has revolved around th e identification of species of Colletotrichum causing anthracnose on pepper and the potential host range of these species (Alexander and Pernezny, 2003; Hadden, 1989; Manandhar et al., 1995a; Roberts et al., 2001). Some of these species are likely to have wide host ranges among plants in the vicinity of pepper fields, and this information could be help ful in determining proper cultural techniques to reduce the incidence and severity of this disease in pepper. Although anthracnose of pepper is becoming s eemingly more prevalent and serious on pepper in the U.S., in parts of Asia it is considered the most important dis ease of pepper (Hong and Hwang, 1998; Jetiyanon et al., 2003; Manan dhar et al., 1995b; Ma nandhar et al., 1995c; Qing et al., 2002). Therefore, much of the recent research regarding pepper anthracnose has been conducted in Korea, Thailand, and other Asian countries. In the U.S., reports of anthracnose have occurred in Louisiana (Ha dden and Black, 1988), Miss issippi (Roy et al., 1997); Virginia (Marvel et al., 2003); Georgia (David Langston, personal communication), Ohio (Lewis-Ivey et al., 2004), and Florida (McGovern and Polston, 1995, Roberts et al., 2001). 14

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However, the disease is likely to occur in other southern states where warm temperatures and high humidity are prevalent, conditions that are reported to be conducive for disease development (Hong and Hwang, 1998; Kwon and Lee, 2002; Qing et al., 2002). Considering the disease is seed-borne (Grover and Bans al, 1970; Manandhar et al., 1995; Sangchote and Juangbhanich, 1984), over-winters in infected pl ant debris (Hadden, 1989; Kwon and Lee, 2002; Smith and Crossan, 1958), and the wide host-range of the fungal species in volved (Bailey et al., 1992; Freeman et al., 2001; Hadden, 1989; Horowitz et al., 2002; Prusky and Plumbley, 1992), it seems likely that pepper anthracnose will only continue to increase in importance in the southeastern United States, including Florida. In Asia, much effort has been conducted to ev aluate whether ripe or unripe fruit are more likely to be the site of initial infection. In Florida, the disease has traditionally been associated with ripened fruit (Roberts et al., 2001, Ken Pernezny, personal communication), but in Georgia and Ohio, lesions on ripe and unripe (green) fru it have been reported (L ewis-Ivey et al., 2004; David Langston, personal communication). More re cently, the disease on unripe fruit in Florida has been observed (Fig. 1-1). Interestingly, much work has been done that demonstrates incompatible reactions of C. gloeosporioides infection on un-wounded ripened fruit, and compatible reactions on un-wounded green or un ripe fruit (Kim et al., 1999; Kim et al., 2001, Manandhar et al., 1995a; Manandhar et al., 1995b; Manandhar et al ., 1995c; Oh et al., 1999a; Oh et al., 1999b). These compatible or incompatible in teractions are based on the ability or inability of the fungus to cause disease when inoculated on the surface of either ripe or unripe fruit. Furthermore, inoculation with conidia after wounding the fruit does not differentiate infection between ripe or unripe fruit, as both stages of fruit are equall y prone to infection using this method (Kim et al., 1999). 15

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More recently, Kim et al., (2001) reported that a pepper esterase gene, designated PepEST was highly expressed in ripe fru it, but not in unripe fruit. This gene has been cloned and demonstrated to prevent appressorium formati on when the PepEST protein was amended with conidia of C. gloeosporioides and inoculated on compatible unr ipe fruit. Although the PepEST protein is reported to have no fungicidal activity, it inhibits appressorium formation in a dosedependent manner (Kim et al., 2001) It has been proposed in th is study that the recombinant PepEST protein affects one or more signal tr ansduction pathways invol ved in appressorium formation, based on other reported experiment al results with the rice blast fungus, Magnaporthe grisea Another gene, designated PepTLP for pepper thaumatin-like protein, was isolated and found to be expressed in the ripe fruit but not un ripe fruit upon fungal inf ection, leading to higher levels of PepTLP mRNA and PepTLP protein in the infect ed ripe fruit (Kim et al., 2002). The authors suggested that ripe pe pper fruit are protected because of the presence of the PepTLP protein in the intercellula r spaces of ripe fruit and the subseq uent absence of fungal colonization. These studies add support to the fact that the fungus is more virulent on un-wounded unripe green fruit than on un-wounded ripe fruit. This con cept is particularly interesting, due to the fact that in Florida most lesions are found on ripened fruit, which are thought to be more susceptible (Alexander and Pernezny, 2003; Robert s et al., 2001). It is increasingly clear from these reports that infections from C. gloeosporioides are likely occurring on the fruit prior to ripening, although visible lesions and evidence of disease may only occur after ripening. Other work with Colletotrichum infection on other hosts, particularly on tropical fruits, has demonstrated the fungus can undergo a period of fa irly long latency after initial infection. In these reports, the growth of the fungus is cont ained only within the ep idermal layer until after fruit ripening, at which point the pathogen is th en able to invade host cells and cause disease 16

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(Bailey et al., 1992). Colletotrichum gloeosporioides on avocado fruit underwent appressorium formation and penetration into unripe avocado fru it, and the subsequent hypha appeared to rest beneath the cuticle until the fruit began to ripen (Prusky and Plumbley, 1992). At that point, the hyphae go on to invade the cell walls, causing ce ll death and forming the characteristic anthracnose lesions. In another study of C. capsici and Glomerella cingulata on pepper fruit, infection remained quiescent on im mature fruits and only developed after the fruits became ripe (Adikaram et al., 1983), even t hough appressoria did germinate to form penetration pegs and penetrate the host surfac e within 65 hours after inoculation. In other studies, some of the appressoria of C. musae on banana adhered to the fruit su rface, but remained quiescent and ungerminated, while other appressoria produced penetrating hyphae on th e unripe fruits. The initially quiescent appressoria eventually did germin ate, but resulted in lesions only after the fruit began to ripen (Muirhead and Deverall, 1981). Prusky and Plumbl ey (1992) provided many such examples of quiescence during Colletotrichum infection on various fruits such as avocado, banana, mango, and pepper. In this summary, the authors quote Verhoe ff (1974) and Swinburne (1983) in defining a quiescent infection as a qui escent or dormant parasitic relationship which, after a time, changes to an active one. Some de gree of quiescence or latency likely plays a role in C. gloeosporioides infection of pepper, and could be used to explain observations in Florida of lesion development only on ripened red peppe r fruit by this particular species. The ability of the pepper anth racnose fungus to infect unrip e green pepper fruit and not ripened red fruit could provide valuab le insight into strategies for ma nagement of this disease. In Florida, it is currently not unde rstood at what stage of deve lopment the fungus invades the pepper fruit to create the lesions that form on ripened fruit. Based on the work by Kim et al., (2001) and others, it is apparent that the fungus is initiating infection on green fruit. This 17

PAGE 18

knowledge, along with further research, could provi de insights that could ultimately lead to major changes to our approach to management of this disease on pepper in Florida. Another factor of potentially great importance for this diseas e in Florida is the source of inoculum. As previously mentioned, C. gloeosporioides has a very wide host range including weed species as well as many commercially cultivated crops in Florida, such as mango and strawberry. Anthracnose is curr ently considered to be the mo st important fungal disease of mango in Florida (Ken Pernezny, personal communicat ion), and in some areas of south Florida, mango is grown in close proximity to pepper fiel ds. Strawberry could also be a source or reservoir of inoculum. Colletotrichum gloeosporioides causes crown rot of strawberry and is believed to spread to commercial strawberry fiel ds in Florida from local weed species (Legard, 2000). Certainly, in addition to agricultural crops, the possibility of alternate weed hosts can not be ruled out as a source of inoculum for peppers The identification of such alternate hosts is complicated by the fact that the fungus may survive on weed or crop hosts without detectable disease symptoms. Colletotrichum acutatum recovered from strawberry was shown to survive on inoculated pepper, eggplant, a nd tomato as mycelia and appre ssoria that failed to germinate resulting in epiphytic growth without invasion of host cells (Horowitz et al., 2002). If penetration of the plant did o ccur, it was only after several da ys and was restricted to the intercellular areas of the first cell layer and did not necessarily cause any visible damage to the plant tissue. Freeman et al., (2001) recovered C. acutatum from healthy looking asymptomatic plants of the weed genera Vicia and Conyza and found they were highly pathogenic on strawberry. Both C. acutatum and C. gloeosporioides are important pathogens of pepper and strawberry. It is certainly quite possible that many poten tial external sources of C. gloeosporioides inoculum occur that might initia te the disease in peppers. 18

PAGE 19

In addition to the identity of an external i noculum source, such as weed species or other crops, little is known regarding internal inoculum sources of the disease on pepper plants. Although it is already known the dise ase is seed-borne in pepper a nd can spread to healthy plants via infected crop debris, it is not currently well understood at what growth stage in peppers that Colletotrichum becomes established. In lychee ( Litchi chinensis ), a fruit tree crop grown in south Florida, anthracnose caused by C. gloeosporioides is considered the most important and destructive disease. It was recently shown by Da vis (2003) that greater numbers of conidia were consistently detected on inflores cence tissues than on leaves. Later in the season, mature fruits that were picked and placed in a moist chamber developed lesions from which C. gloeosporioides was isolated. This confirms that the presence and accumulation of inoculum in the flowers likely contributes to the disease on fruit later in the season. This clearly could have implications on spray timing and other control measures. In peppers, it is not known whether inoculum in the flowers could contribute to disease development in the subsequent fruits, but such knowledge may be critical for implementation of successful and efficient control measures. Lastly, knowledge of the specific species of Colletotrichum responsible for the disease is also of critical im portance. Although C. gloeosporioides has been documented as a pathogen of pepper in Florida, C. acutatum has not been reported and may differ in host range, survival, and other epidemiological characteristics. In Ohio, C. acutatum is considered a more aggressive pathogen of pepper than other Colletotrichum species and is capable of causing yield losses up to 90% (Lewis-Ivey et al., 2004). In addition, it ha s been proposed that this species does not differentiate from ripe or unr ipe fruit and can cause lesions on both (Sally M iller, personal communication). If C. actuatum can be isolated from unripe pe pper in Florida, this certainly would have significant implicati ons on the understanding and management of this disease. 19

PAGE 20

Therefore, determining the etiology of this disease is crucial to providing knowledge and implementing control measures for pepper anthracnose. In this study, over fifty isolates of Colletotrichum sp were recovered from infected fruit collected from various pepper-growi ng regions throughout southern Flor ida. In two of the fields that were sampled, anthracnose lesions were only observed on ripened fruit. In these locations, the peppers were not harvested a nd therefore allowed to ripen, a nd an abundance of lesions were detected on nearly every fruit th roughout the entire field. In the remaining locations, lesions were found on green, unripe fruit from younger fields that were intended to be harvested. In these fields, anthracnose lesions were found only in specific areas, or loci, and were not predominant throughout the field. Using P CR and species-specific primers for both C. gloeosporioides and C. acutatum the conserved ITS regions of DNA were identified in most isolates as either one or the other of these two species. The isolates recovered from the fields containing only ripened fruit were identified as C. gloeosporioides while those recovered from green fruit were identified as C. acutatum This was the first report of C. actuatum recovered from pepper in Florida, and this discovery high lights what could be a significant threat to the pepper industry in Florida and will most likely require enhanced management strategies that include the application of pesticides. In addition, increased knowle dge of the epidemiology of C. acutatum isolates recovered from pepper, such as their host range on other cr ops grown adjacent to peppers, would aid in the understanding and prevention of disease epidemics in pepper a nd other important crops in Florida, such as strawberry and tomato, both of which are host to more than one species of Colletotrichum (Freeman et al., 2001; Lewis-Ivey et al., 2004; Prusky and Plumbley, 1992). In this study, an isolate from pepper was used to in oculate field-grown stra wberries, tomatoes and 20

PAGE 21

peppers to determine if this species of Colletotrichum on pepper could spread to nearby strawberry or tomato fields and create further di sease epidemics. In addi tion, since no data have been generated to evaluate certain fungicides for control of C. acutatum on pepper in Florida, various fungicides were eval uated in field trials inocul ated with an isolate of C. acutatum recovered from pepper in Florida. Re sults show that the isolates of C. acutatum recovered from pepper are not pathogenic on either strawberry or tomato, and that several fungicides do provide good to outstanding control of this disease, even under heavy disease pressure. The results from this study are likely to aid in the understandi ng and management of pe pper anthracnose in Florida and provide further insight into this potentially devastat ing disease. Hopefully, this discovery of C. acutatum as a pathogen of pepper in Florida will spark further research that will undoubtedly continue to enhance our understanding of this pat hogen and provide management options needed to control this disease in the future. 21

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Figure 1-1. Pepper anthracnose lesions on green, unripe bell pepper fruit recovered from Palm Beach Co., Florida in 2004. Traditionall y, the disease was observed only on fullysized ripened, colored fruit (usually red) ; however, the occurrence of anthracnose symptoms on developing green, unripe fruit was becoming increasingly common since the late 1990s in Fl orida pepper fields. 22

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CHAPTER 2 ETIOLOGY OF RECENT OUTBREAKS OF PEPPER ANTHRACNOSE IN FLORIDA Introduction Florida is second only to Califor nia in production of peppers ( Capsicum annuum L.) in the United States. Most acreage is planted to sw eet bell pepper with the bulk of the production during the winter months (September to May) in the southeastern and southwestern areas of the state (Maynard et al., 2003). In th e 2003 2004 season, 223,605, 454 kg of pepper were harvested from more than 6,880 hectares with a total annual value of $175, 654, 000, second only to tomatoes in farm-gate value in the state. Anthracnose has emerged as an increasingly significant disease of pepper in Florida in recent years (Roberts et al., 2001). It has been observed on both sweet bell peppers and specialty peppers, such as cubanelle, jalapeno, and scotch bonnet (C. chinense ) (McGovern and Polston, 1995). The disease is characterized by sunken, necr otic lesions on the surface of pepper fruit and usually contain an abundance of tan or salmon-co lored conidia. Traditionally, the disease in Florida has been primarily associated with ripene d fruit that have already turned from green to the ripened color of the cultivar (usually red) Therefore, the causal agents were generally thought of as mostly ripe-rot pathogens (Ale xander and Pernezny, 2003; Roberts et al., 2001). On immature, unripe green fruit, typical anthracn ose symptoms were generally not observed, and therefore the disease was not considered a si gnificant problem on bell peppers harvested as mature green fruit (by far the bulk of the harv ested acreage in Florida). Within the last few years, however, the disease has been observed on immature green pepper fruit grown in Florida. A similar outbreak of anthracnose on immature, green fruit has occurred in Ohio (Lewis-Ivey et al., 2004). The causal agent of the Ohio epidemic was identified as Colletotrichum acutatum (Simmonds). Although at least four different species of Colletotrichum have been reported in 23

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the U.S. to cause anthracnose of pepper, C. gloeosporioides (Penz.), C. capsici, C. coccodes, and C. acutatum (Alexander and Pernezny, 2003; Haden, 1989; Haden and Black, 1998; Lewis-Ivey et al., 2004; Marvel et al., 2003; Roy, 1996), C. acutatum has never been identified as a pepper pathogen in Florida (McGovern and Po lston, 1995; Roberts et al., 2001). In Ohio, C. acutatum was reported as more aggressive than other Colletotrichum species known to infect pepper, capable of causing losses in marketable yield of up to 100% (Lewis-Ivey et al., 2004). Probabl y of more significance, C. acutatum will attack both ripe, colored fruit and immature, green fruit, unlike other species that are strictly ripe-rot pathogens. Since the epidemics of anthracnose on unripe, immature green fruit in Florida have occurred at roughly the same time as those in Ohio, it is possible that C. acutatum is responsible for the disease in Florida as well. Various methods previously have been described to determine the species of Colletotrichum causing pepper anthracnose, including both molecular (K im et al., 2002; LewisIvey et al., 2004) and morphol ogical (Haden, 1989; Kim et al., 1999; Lewis-Ivey et al., 2004) techniques. Species-specific primers based on the rDNA internal transcribed spacer (ITS) regions of different species have been used to differentiate C. gloeosporioides and C. acutatum (Brown et al., 1996; Freeman et al., 2000; Le wis-Ivey et al., 2004; Mills et al., 1992; Sreenivasaprasad et al., 1996). Molecular methods generally have been preferred over morphological methods (Lewis-Ivey et al., 2004; Marvel et al., 2003), because the morphology between species often are quite similar and a certain degree of mor phological variation is considered acceptable within a species of Colletotrichum (Sutton, 1992). Colony growth rate on artificial media in growth chambers ha s been used to differentiate between C. acutatum and C. gloeosporioides recovered from pepper (Haden, 1989). The other species reported to occur on 24

PAGE 25

pepper, C. coccodes and C. capsici are easily distinguished due to the production of abundant sclerotia in culture by C. coccodes, and by the distinct falcate or curved shape of conidia produced by C. capsici (Bailey et al., 1992; Haden, 1989; Roy, 1996; Sutton, 1992). The purpose of this study was (i) to isolate and identify the species of Colletotrichum causing pepper anthracnose in Flor ida from both ripened, colored fr uit and immature, green fruit, and (ii) identify morphologi cal characteristics useful for differe ntiation of isolates identified to species by PCR-DNA analysis. Materials and Methods Isolates Fifty isolates were recovered from both ripe, colored and immature, green symptomatic pepper fruit from various commercial farms throughout Flor ida. An additional isolate from a diseased green bell pepper in 2005 from southern Georgi a was provided by D. Langston (Table 2-1). Forty-seven of the Florida isol ates were recovered from inf ected pepper fruit during the 2004 2005 vegetable season, and three isolates from pe pper were originally recovered in the mid 1990s by R. McGovern. Two isolates, Ca Mil-1 ( C. acutatum ) and GD ( C. gloeosporioides ) (Lewis-Ivey et al., 2004), were us ed as reference isolates in the PCR studies. The 50 isolates from Florida were collected from three diffe rent pepper-growing areas: Indian River and St. Lucie Co. in east-central Florida, Palm Beach Co in southeast Florida, and Collier and Hendry Co. in southwest Florida (Table 2-1). Fungi were isolated by rinsing symptomatic fruit with deionized water and placing in a closed plastic co ntainer containing a moist paper towel (ca. 100% humidity) for 24 h. Conidia from lesions on the surface of the fruit were removed with a sterile loop, which was then streaked onto the surface of 10-cm-diameter Petri plates containing water agar (15 grams agar per 1000 mL distilled water), a nd allowed to grow for 12 to 18 h. Up to four germinating, single-spores per isolation were identified under a dissectio n microscope (40), 25

PAGE 26

removed with a sterile needle, and transferred to a Petri dish cont aining 25 mL of potato dextrose agar (PDA) and allowed to grow at 30C for 7 d. One isolate per lesion was selected for storage and further study. In some cases, two isolates were obtained from different lesions on the same fruit. Isolates were transferred to P DA plates containing sm all (approximately 5-mm ) pre-cut sterilized pieces of filter paper (Whatman #4) pl aced directly on the surface, and incubated at 20C for 14 d with continuous light. The individual pieces of colonized filter paper were then removed from the surface of the agar using sterile forceps, allowed to dry in empty Petri-dishes for 14 d, and placed in vials for long-term storage at -4C. Isolates were recovered as needed by transferring filter paper units to PDA and in cubating plates a minimum of 3 d at 20C 2 PCR Amplification Polymerase chain reaction (PCR) amplification was used to putatively identify isolates as species of C. acutatum or C. gloeosporioides using species-specific primers as previously described (Lewis-Ivey et al., 2004; Mills et al., 1992; Sreenivasaprasa d et al., 1996). The species-specific primers for C. gloeosporioides (CgInt; 5'-GGCCTCCCGCCTCCGGGCGG-3') (Mills et al., 1992) and for C. acutatum (CaInt2; 5'-GGGGA AGCCTCTCGCGG -3') (Sreenivasaprasad et al., 1996) from the ITS 1 region of the rDNA were used in combination with the conserved primer ITS 4. Before conducting PCR, the DNA of each isolate was extracted according to the protoc ol previously described (Lee a nd Taylor, 1990) and modified by Lewis-Ivey et al., (2004). Each 25 L reaction mixture contained: 2.5 L of extracted DNA (50 ng/ L), 0.125 of each 10 M primer, 0.08 l 10 mM dNTP, 0.5 L Taq Polymerase (5 U/ L), 1.5 L of 25 mM MgCl 2 2.5 L 10X polymerase buffer, and 16.9 L sterile de-ionized water. The PCR was performed with a MJR PTC-100 thermocycler (MJ Research Inc., Waltham, MA) using the following temperature-cycle program: 5 min at 94 C, 30 cycles of 1.5 min at 94 C, 2 min at 55 C, and 3 min at 72 C, followed by a 10 min final extension at 72 C. The PCR 26

PAGE 27

products (7 L) were mixed with 3 L of loading dye (5 mg bromphenol blue, 5 mL 5X TBE, 2g sucrose) and separated by horizontal gel electrop horesis in 1.5% agarose in 0.5X TBE buffer at 110 V for 150 min. Gels were then stained in dilute ethidium bromide (2 g/mL), visualized under UV light, and photographed using the Kodak Electrophoresis Documentation and Analysis System (EDAS) 290 (Eastman Kodak Company, New Haven, CT). The PCR procedure was conducted three times for each isolate. Growth Rate in vitro Radial growth rate (mm) was determined for each isolate. Isolates were grown on PDA for 3 to 5 d and were transferred to each of three replicate PDA Petri-dishes using plugs made with a sterile #3 cork-borer. Plates were placed into a growth chamber (Enviro ch amber, Detroit, MI) at 30 C in continuous darkness, and arranged within the growth chamber in a completely randomized design. At 5 d, the radius of each co lony was measured and recorded. Mean growth rates were calculated for all isolates and were compared statistically using ANOVA ( P <0.05). The experiment was repeated once. Conidial Measurements Isolates VB07, VB09, MF05, MF08, MG01, HB 01, HJ01, HC02, and GA01 were grown on PDA for 5 d under continuous fluorescent light at 25 C to promote sporulation. Conidia were suspended in sterile water using a sterile loop and mounted on a microscope slide. Length and width were measured for 25 conidia per isolate using an ocular scale at 700 magnification (10 ocular, 70 objective) using brig ht field microscopy (Leitz, Germ any). The length and width were compared statistically using a onesided t-test between the two species. 27

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Results PCR Amplification with Species-Specific Primers Twenty-eight of the 50 pepper isolates from Florida were identified as C. acutatum using the species-specific primer CaInt2 in conjunction with the ITS4 primer for C. acutatum Seventeen isolates were identified as C. gloeosporioides, using the CgInt and ITS4 primers specific for C. gloeosporioides. The C. gloeosporioides isolates were recovered exclusively from ripe, colored pepper fruit in St. Luci e and Indian River Co. in 2004, and in the mid 1990s in Collier Co. The C. acutatum isolates were recovered in 2004 from anthracnose lesions on green, immature fruit, or on ripe, colore d fruit in close proximity in the same fields. PCR reactions of several represen tative isolates are shown (Fi g. 2-1) and compared with the previously published refere nce isolates, Ca Mil-1 ( C. acutatum ) and GD ( C. gloeosporioides ) (Lewis-Ivey et al., 2004). Fi ve pepper isolates (VB01, VB 03, VB04, VB05, and VB06) did not produce a PCR product with either primer mixture. The isolate from immature, green fruit in southern Georgia (GA01), also was identified as C. acutatum (Fig. 2-1). Colony Growth Rate In two separate tests, is olates identified by PCR as C. gloeosporioides grew significantly faster ( P <0.0001) than those identified as C. acutatum (Figs. 2-2 and 2-3). The 17 isolates of C. gloeosporioides grew an average of 5.91 mm/day in Test 1 and 5.93 mm/day in Test 2, while the 28 isolates of C. acutatum grew an average of 2.96 mm/day and 3.54 mm/day in Test 1 and Test 2, respectively. The five isolates that did not produce an identif iable PCR product (VB01, VB03, VB04, VB05, and VB06), grew at a mean rate of 5.85 mm/day (data not shown), consistent with that of the growth rate for isolates of C. gloeosporoides. The isolate identified by PCR as C. acutatum from green pepper fruit in Georgia (GA01) grew at a mean rate of 3.37 mm/day (data not shown), consistent with the known growth rate of isolates of C. acutatum 28

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Conidial Measurements Conidial length and width were measured for five isolates previously identified by PCR as C. gloeosporioides, as well as four isolates of C. acutatum The isolates designated as C. gloeosporioides had an average conidial size of 17.96 6.37 m (standard error = 0.146 0.041), whereas isolates designated as C. acutatum had an average conidial size of 16.79 4.49 m (standard error = 0.172 0.049). The length and width were statistically analyzed using a one-way ANOVA t-test, and both we re significantly larger for C. gloeosporioides ( P <0.0004 and P <0.0001, respectively). Discussion Colletotrichum acutatum has been identified as the causal ag ent of the recent epidemics of anthracnose on immature, green pepp er fruit in Florida. This conclusion is based primarily on reaction of DNA from all isolat es from lesions on immature, green fruit with PCR-specific primers for C. acutatum This is the first report of an extensive collection of Colletotrichum isolates in the United States from immature peppe r anthracnose lesions that definitively identify the pathogen as C. acutatum All isolates identified by PCR as C. gloeosporioides were recovered from ripe, colored fruit, neve r from immature, green fruit. A few C. acutatum isolates were from mature, colored fruit, indicating that C. acutatum can attack pepper fruit during all stages of maturity. Colletotrichum gloeosporioides on the other hand, seems to be strictly a ripe-rot pathogen on pepper. These results parall el those reported recently based on two isolates in Ohio (Lewis-Ivey et al., 2004). Pepper can now be added to the list of hosts for C. acutatum in Florida (Brown et al., 1996; Lahey et al., 2004; Legard, 2000; Pere s et al., 2005; Timmer and Brown, 2000) and other locations (A daskaveg and Frster, 2000; Bailey et al., 1992; Correll et al., 2000; Freeman, 2000; Freeman et al., 1998; Fr eeman et al., 2001; Peres et al., 2002). Although only one isolate from Georgia was included in our study, it seems likely that 29

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anthracnose on immature, green pepp ers in Georgia is also caused by C. acutatum More isolates from Georgia need to be recovered and id entified to confirm this contention. Growth rates in culture we re clearly different for C. acutatum and C. gloeosporioides Isolates of C. gloeosporioides grew 50 to 200 % faster than those of C. acutatum These observations serve as the basis for a suggestion that colony growth rate under very specific conditions (30C in complete dar kness on PDA plates) can be used to tentatively separate these two species. Other researchers (Brown et al., 19 96; Haden, 1989; Kim et al ., 1986; Marvel et al., 2003; Sutton, 1992) have also suggested that colony growth rate of isolates can be of taxonomic significance. Tolerance to benomyl (Adaskav eg and Hartin, 1997; Be rnstein et al., 1995; Freeman et al., 1998; Peres et al ., 2004) also tends to vary be tween these two species. The difference in conidial size between the two species was less distinct than growth rate differences in our studies. However, conidia of C. acutatum were significantly smaller than conidia of C. gloeosporioides in both length and width. Differentiation between these two species on conidial size alone could prove difficult, due to size variation within an isol ate and the similarity of spore shape between the two species. However, fo r laboratories without access to many modern molecular techniques, colony growth rates, coni dial size, and other phe notypic characteristics may be very important for initial id entification of fungal isolates. On a particular host, Colletotrichum species may exist as a hemi-biotroph or necrotroph using the terminology of Bailey et al., (1992) and OConnell et al., (2000), and more recently Dieguez-Uribeondo et al., (2005). When C. gloeosporioides attacks pepper fruit, its lifestyle is probably that of a hemi-biotroph. Most likely, it initiall y colonizes the space directly below the cuticle. Only as the fruit ripens, does it produce enzymes that kill tissue and allow for development of the typical sunken lesions characteristic of anthr acnose. When immature, green 30

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bell pepper fruit were inoculated with C. gloeosporioides in field plots, lesions did not form until 45 d later when the pepper fruit ripe ned and turned red (Harp, unpublished). Colletotrichum acutatum in contrast, seems to establish as a necrotroph soon after colonization of immature, green fruit, producing symptoms in 7 to 10 d, long before fruit turn color (Lewis-Ivey et al 2004). However, one cannot easily predict how a particular species will react. For example, C. acutatum acts as a hemi-biotroph, not a n ecrotroph, on apple, blueberry, and peach (Bernstein et al., 1995; Jones et al., 1996; Milholland, 1995; Peres et al., 2005; Zaitlin et al., 2000), and probably other crops (Prus ky and Plumbley, 1992; Ti mmer et al., 1998). Because most bell pepper in Florida is harves ted at a mature green stage, until recently, anthracnose had been a problem only when crops we re extended to the co lored fruit stage. Indeed, in the past, some grower s and extension personnel referred to anthracnose as ripe-rot to reflect its impact on ripe, colored fru it only. However, the recent emergence of C. acutatum as a pathogen of immature, green fruit raises th e status of anthracnose to a potentially major disease problem throughout the indu stry. This likely means that anthracnose c ontrol measures must be initiated earlier and followed dilig ently throughout the crop cycle. This new anthracnose disease is sufficiently different from the traditional ripe-rot anthracnose to merit, in our opinion, a distinctive name. We propose the name early anthracnose for the disease of immature, green pepper fruit caused by C. acutatum The presence of early anthracnose on pepper in Florida could ha ve dire consequences for pepper growers throughout the st ate. Floridas humid and wet environment is most likely conducive to anthracnose diseases and could be cause for potent ially dramatic yield losses. More research is needed on this pepper diseas e, such as pathogen host range and efficacy of fungicides. 31

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Table 2-1. Isolates of Colletotrichum spp. recovered from pepper fields throughout Florida (sample no. 1 7) or Georgia (sample no. 8). Sample no. Isolate designation z Location (Co.) No. of Isolates Host Sample Description Species recovered 1 MF01-MF09 St. Lucie 9 Red bell Olympus C. gloeosporioides 2 VB01-VB10 Indian River 10 Red jalapeno Milta C. gloeosporioides 3 PB01-PB06 Palm Beach 6 Green bell Brigadier C. acutatum 4 HB01-HB08 Hendry 8 Green/Red bell Aristotle C. acutatum 5 HC01-HC09 Hendry 9 Green Cubanelle Aruba C. acutatum 6 HJ01-HJ05 Hendry 5 Green/Red jalapeno Tormenta C. acutatum 7 MG01-MG03 Collier 3 Red bell, Scotch bonnet, Thai C. gloeosporioides 8 GA01 Tift (GA) 1 Green bell C. acutatum z All isolates were recovered in 2004 and 2005 except for those collected from Collier Co. (MG01 MG03), collected in the mid 1990s by R. McGovern, University of Florida. 32

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1 kb plus Ca-Mil-1 HB01 HB03 HB04 HB05 HJ01 HJ04 HJ05 HC02 HC06 HC07 HC08 HC09 PB01 PB02 PB03 PB04 PB05 PB06 GA01 VB07 VB08 VB09 VB10 MF05 MF06 MF07 MF08 MF09 MG01 MG03 MG02 GD Figure 2-1. An agarose PCR gel of isolates of Colletotrichum spp. collected from Florida and Georgia that have produced am plified DNA fragments with either CaInt2 (20 isolates from left) or CgInt (13 isolates from right ) species-specific primer. Ca Mil-1 and GD are reference isolates of C. acutatum and C. gloeosporioides respectively. 33

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0 1 2 3 4 5 6 7 8 9 10Test 1Test 2Growth (mm/day) C. gloeosporioides C. acutatum a a b b Figure 2-2. Average radial growth per day of 45 isolat es representing the two species of pepper anthracnose isolates recovered from Florida as determined by PCR. Colony radius (mm) was measured for three colonies per isolate after mycelial plugs were allowed to grow on artificial media at 30C for 5 da ys in two separate te sts (Test 1 and 2). The species of each isolate was determined previously by PCR using species specific primers for Colletotrichum gloeosporioides (CgInt/ITS4) and C. acutatum (CaInt2/ITS4). Each of the17 isolates designated as C. gloeosporioides grew at a significantly faster rate ( P <0.0001) in both tests than did the 28 isolates designated as C. acutatum 34

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Figure 2-3. Isolates of Colletotrichum gloeosporioides (top) and C. acutatum (bottom) recovered in 2004 from pepper fruit in Florida growi ng on potato dextrose agar in continuous darkness at 30C for 5 days. Note that the isolates of C. acutatum grew slower than isolates of C. gloeosporioides 35

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CHAPTER 3 HOST RANGE OF PEPPER ANTHRACNOSE IS OLATES RECOVERED FROM PEPPER IN FLORIDA Introduction Colletotrichum acutatum has been recently identified as an anthracnose pathogen of pepper in Florida (chapter 2). In that study, isolates of the anthracnose fungus were recovered from heavily infected peppers throughout Florida and identified as either C. acutatum or C. gloeosporioides using PCR amplification with ITS species-specific primers. Interestingly, the C. gloeosporioides isolates were recovered only fr om ripened, red fruit, whereas C. acutatum isolates were recovered primarily from unripe, green fruit. In the fields containing C. gloeosporioides lesions were not observed until most or all of the fr uit had ripened, after which an abundance of lesions appeared over a short period of time on nearly every fruit throughout the field (Fig. 3-1). However, in the fields where C. acutatum was recovered from green, unripe fruit (Fig. 3-2), anthracnose symp toms were found only within is olated loci. Heavy disease was observed on a few plants that we re within close proximity to each other, but less disease was observed moving outward from that point. Within those loci, lesions were predominant on green fruit, ranging from newly-formed small fruit to fully-sized green fruit (Fig. 3-2). However, symptoms were also found on an occasiona l adjacent ripened, red fruit. Colletotrichum gloeosporioides has a reportedly wide host range including many weed species (Bailey et al., 1992; OConne ll et al., 2000), and thus it is very likely that this species of Colletotrichum exists naturally in Florida, either symptomatically or asymptomatically, on host plants that occur adjacent to pepper fields. Th is could help to explai n the source and abundance of inoculum that must be necessary to cause the anthracnose symptoms on nearly every fruit that is commonly observed in fields of ripened pepper infected with C. gloeosporioides (Fig. 3-1). In contrast, C. acutatum has a much more limited host range (Bailey et al., 1992; Legard, 2000; 36

PAGE 37

Peres et al., 2005), and is genera lly thought not to occur naturall y on weed hosts to the same extent as C. gloeosporioides (Freeman et al., 1998; Freeman, 2000). As a result, widespread infection of C. acutatum in pepper fields was not observed (Bernstein et al., 1995; Harp et al., 2006; Lewis-Ivey et al., 2004; Marv el et al., 2003). Instead, symp toms appeared initially in localized foci, perhaps originating from a singl e to few infected plants or fruits. On strawberry, where both C. acutatum and C. gloeosporioides cause anthracnose symptoms, C. acutatum has been isolated from lesions on nursery-grown transplants prior to being planted in the field (Legard, 2000; Peres et al., 2005). Most nurse ry-grown strawberry transplants that are planted in Florida are typi cally produced in other states (e.g. North Carolina or California), or in Canada. In these repor ts, the pathogen was appa rently introduced on the transplants prior to arriving in Florida with the tr ansplants serving as the primary inoculum in the field (Legard, 2000; Peres et al., 2005). In contrast, C. gloeosporioides has not been recovered from transplants and is believed to infect strawberry only after transplants are set in the field. The source of inoculum is probably weed hosts or infected debris (Freeman et al., 1998; Harp et al., 2003; Legard, 2000). In additi on, research has been conducted in Florida that demonstrated the inability of C. acutatum isolates recovered from strawberry to survive in crop debris or soil for any extended period of time, such as during the summer months when strawberries are not grown (Legard, 2000). Therefore, it is unlikely in Florida that C. actuatum recovered from strawberry survived off-season in soil debris or a local alternative hos t (Bailey et al., 1992; Kwon and Lee et al., 2002; Legard, 2000; OConnell et al., 2000). The same could be true with pepper, and this likely explains why the occurrence of C. acutatum on pepper is typically found in the field in randomly positioned loci, whereas the occurrence of C. gloeosporioides on pepper is found widespread throughout the field and dete cted at once on virtually every susceptible 37

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(ripened) fruit within that field. Perhaps C. acutatum on pepper, as with strawberry, is seedborne or somehow infects the nurse ry-grown transplants, whereas C. gloeosporioides on pepper originates from a local source, such as a w eed host, and arrives on pepper after planting (Freeman et al., 1998; Freeman et al., 2001; Harp et al., 2003; Legard, 2000). Since C. acutatum infects immature, unripe green fruit, we are calling this disease early anthracnose. The implications for pepper gr owers are more significant than for ripe-rot anthracnose, caused by C. gloeosporioides, which only appears to cause symptoms on ripened, red fruit. Most pepper fruit harvested in Florid a are harvested as fully-sized green fruit, and therefore anthracnose caused by C. gloeosporioides poses little or no threat under these circumstances. Colletotrichum gloeosporioides infection of pepper has been previously described in Florida (McGovern and Polston, 1995; Roberts et al., 2001) a nd overall accepted as a ripe rot disease on pepper and other crops (Alexander and Pernezny, 2003; Maynard et al., 2003; Milholland, 1995). The possibility of C. gloeosporioides cross-infection from pepper to other crops has not been investig ated, and likely would be of lit tle consequence considering the wide host range and abundant inoculum source of this species already present in nature. However, with the recent introduction of C. acutatum on pepper, it is possible that early anthracnose of pepper could have disease management implications for other crops grown in Florida, especially those that grow in proximity to pepper and are known hosts of C. acutatum such as strawberry (Legard, 2000) and tomato (C orrell et al., 2000; Guerber et al., 2003). In this study, an isolate of C. acutatum (HB05) was recovered from green bell pepper grown in Hendry, Co., Florida and used to artific ially inoculate field-gr own strawberries and tomatoes during the spring of 2006 and 2007. In addition, this isolate was tested for the ability to cause lesions on detached strawberry and tomato fruit harvested from the same fields used in 38

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2007 by wound-inoculation in the la boratory. If anthracnose symp toms were to be observed on inoculated strawberries or tomatoes, this c ould have significant implications for disease management on tomatoes, strawberries, or other crops susceptible to C. acutatum that grow in Florida adjacent to pepper fields. Additionally, it could lead to insight regarding the source of inoculum on peppers, and also provide cultural recommendations for managing this disease on all such crops. The purpose of this study was to evaluate if C. acutatum recovered from pepper is pathogenic to two important crops grown in Florida, tomato and strawberry, under both field and laboratory conditions. Materials and Methods Host Range Field Trials Two host-range field trials were conducted in field plots located at the Syngenta Vero Beach Research Center, Indian River Co., Flor ida, during consecutive growing seasons for strawberry, tomato, and pepper. The first field trial was initiated in the fall of 2006 (Field Trial 1), and the second trial in the fall of 2007 (Field Trial 2). In addi tion, fruits were harvested from the same fields used in Field Trial 2 (outside of the testing area) to conduct a wound-inoculation study in the laboratory (Laboratory Trial 1). Plants In Field Trial 1, strawberry plants (Cameros a and Chandler) were obtained as bareroot transplants produced in Cana da and provided courtesy of Carl Grooms, Inc., Plant City, FL, and hand-transplanted on 29 October, 2006. Th e transplants were planted in double rows on raised beds under plastic mulch a nd single center drip-tube irrigation. The cult ivars, Camerosa and Chandler, were planted together in the same plot (six plants each per plot with 12 plants total per plot), and the trial consisted of thr ee replications. Tomatoes (FL 47) and peppers (Revolution) were planted on 01 March, 2007, on raised beds with plastic mulch and drip 39

PAGE 40

irrigation. Pre-plant fungicides, mefenoxam (Ridomil Gold SL, 1.2 L / Ha) and PCNB (Terraclor Super X, 7.1 L / Ha), were broadcas t-incorporated into th e soil along with diazinon (Diazinon AG500, 2.4 L / Ha) for soil fungi and insect control, respectively. A rotational spray program of spinosad (Spintor, 0.44 L / Ha) emamectin benzoate (Proclaim, 0.29 L / Ha) and lambda-cyhalothrin (Warrior, 0.29 L / Ha) were applied on all crops on a 7 to 14-day interval for insect control. In Field Tria l 2, strawberry plants were obtai ned as plugs from Norton Creek Farms (Fischer, NC), and hand-planted on 22 October, 2007. Peppers (Revolution) and Tomatoes (FL 47) were planted on 10 Octobe r, 2007, and all crops in Field Trial 2 were planted and maintained under the same cultu ral conditions as in Field Trial 1. Inoculum for Field and Laboratory Evaluations Isolate HB05 was grown on PDA at 20 C under continuous lighting for 7 days as previously described (Chapter 2; Harp et al., 2006). Conidia were harvested by flooding cultures with de-ionized water and using an L-shaped gl ass rod to remove coni dia into solution. The conidial suspension was adjusted to a concentration of 2.5 x 10 4 conidia per mL using a hemacytometer. For the field inoculations, the conidial concentra tion was prepared in 5 L of deionized water, whereas for the laboratory inocul ations, approximately 50 mL of inoculum was prepared. Field Treatment Plots In each crop, three treatment plots were arra nged in a randomized, complete-block design and three replications. The treatments consiste d of un-inoculated, inocul ated, and water-sprayed control plots using the same de-ionized water used in the inoculated plots except without conidia. In Field Trial 1, the row spacing for each crop was 1.5 m, and the plant spacing was 45.7cm for tomatoes and peppers (8 plants per plot), and 30.5 cm for strawberries (12 plants per plot). The 40

PAGE 41

plot sizes were 3 x 1.5 m for tomatoes and pepper s, and 3.6 x 1.5 m for st rawberries. In Field Trial 2, the row spacing for each crop was 1.5 m, and the plant spacing was 60.9 cm for tomatoes and peppers (15 and 8 plants per plot, respectively), and 45.7 cm for strawberries (15 plants per plot). The plot sizes were 6 x 1.5 m for to matoes, 4.6 x 1.5 m for peppers, and 9.1 x 1.5 m for strawberries. The plot sizes and relevant plantin g details for each crop in Field Trial 1 and Field Trial 2 are summari zed in Table 3-1. Laboratory Detached Fruit Tomato, strawberry, and pepper fruit were obtained from th e border rows of the same fields used in Field Trial 2. These rows were sprayed with the same insecticide maintenance treatments as the plots within Field Trial 1 a nd Field Trial 2 but did not receive any fungicide treatments or inoculations. The fruit were collected on 19 January 2008, and washed with deionized water in the laboratory. A total of 40 strawberry fruit and 20 tomato fruit were collected that represented all stages of fruit development from small, unripe green fruit to fully-sized, ripened red fruit. Out of the 40 strawberry fruit, 20 were injected with the conidial suspension (see Laboratory inoculations) and 20 injected w ith water. Out of 20 tomato fruit, 10 were injected with the conidial suspension and 10 we re injected with water. Eighteen pepper fruit were also collected that ranged from medium-sized, unripe green fruit to fully-sized harvestable green fruit. Twelve pepper fru it were injected with the conidial suspension while the remaining six were injected with water. A few peppers had st arted to ripen and were pa rtially red in color. After the fruit were rinsed in de-ionized wate r for three minutes, they were dipped in a 10% bleach solution (300 mL of Clorox bleach in 3000 mL of de-ionized water) for 30 seconds and immediately placed in a tub of de-ionized (5000 mL) water for an additional 30 seconds. Fruit were then removed from the tub and allowed to air dry. Plastic c ontainers (Tupperware, 41

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Hartford, CT) were lined with moist paper towe ls and used as incubation chambers for the detached, inoculated and un-inoculated fruit. Field Inoculations Inoculations were conducted for Field Tr ial 1 and Field Trial 2 on 18 May, 2007 and 21 December, 2007, respectively. Each inoculat ion was conducted at approximately 2300 h EST, when the dew point was within 3 to 5 C of the ambient temperature, ensuring dew formation and leaf wetness for at least 8 hours. The nighttime temperatures during the inoculations for Field Trial 1 and Field Trial 2 were between 18 and 20 C, and the humidity was between 88 and 92%. The treatments were applied over the top of the plants using a 10 L backpack pump-sprayer (Solo 435, Detroit, WI) until run-off, ensuring good coverage of the fruit and foliage. For each inoculation, the same inoculum batch was used on all three crops within a span of one hour. The water-sprayed treatments were applied prior to the inoculated treatments using the same backpack pump sprayer containing de-ionized wa ter but with no conidia. For strawberry and tomato plants, both unripe green fruit, and ripened red fruit, occurred in the treated plots. For pepper plants, unripe green fruit ranging from very small to fully-sized fruit, but not red-ripened fruit, occurred in the plots at the time of the inoculations. Laboratory Inoculation Using a black Sharpie, circles (approximate ly 3 cm in diameter) were drawn on each pepper and tomato fruit to identify the wound-inoculation site. On the strawberries, which could not be easily marked, the inocula tion site was located on the side of the fruit facing directly upward after placing in the plastic containers. Fruits were inoculated using a 1 cc syringe (25G needle, Becton Dickinson and Co., Rutherford, NJ) containing a coni dial suspension of C. acutatum (conidial concentration of 10 3 per mL) in de-ionized water. Controls consisted of 42

PAGE 43

similarly treated fruit injected with de-ionized water without conidia. For the wound-treatments, the tip of the syringe was used to penetrate the skin of each fruit and a small amount (approximately 0.01 mL) of either the conidial su spension or de-ionized water without conidia was injected under the skin. In many cases, a small amount of liquid formed a droplet on the surface of the wound. The fruit were allowed to re main in the sealed pl astic containers with moist paper towels (100% hum idity) at approximately 20 C for 5 days. At 3 and 5 days after inoculation, the fruit were assessed for the deve lopment of lesions, and the presence or absence of lesions on each fruit was recorded. Disease Assessments For Field Trial 1 and Field Trial 2, fruit in each plot was assessed for anthracnose symptoms at 10 days after inoculation and agai n 7 days later. The number of lesions were counted in each plot for all th ree crops and for all three treatme nts within each crop, inoculated, uninoculated, and the water-treated control. For Laboratory Trial 1, the fruits were assessed for lesions and scored as infected or uninfected after 3 days fo r strawberry and pepper, and 5 days for tomato. Results Inoculation Field Trials In both Field Trial 1 and Field Trial 2, mode rate to heavy anthracnose symptoms were observed on inoculated pepper fruit within 7 to 10 days after inoc ulation in the inoculated plots (Fig. 3-2). However, no symptoms were observed in the water-treated or unt reated control. In Field Trial 1, there was a mean of 22.7 lesions among fruit in the inoculated pepper plots after 10 days. A mean of 39.3 lesions were recorded in Field Trial 2 (Table 32). In both trials, no lesions were found on pepper in either untreated or water-treated control plots. 43

PAGE 44

In the tomato and strawberry plots, no le sions or anthracnose symptoms were observed among fruit in any of the treatment plots at both 10 (Table 3-2) and 17 days after treatment (data not shown). The number of lesions counted in eac h of the plots at 10 days after inoculation for all treatments in both trials wa s identical to the numb er of lesions after 17 days. Although both unripe, green fruit and ripened, red fruit were inoc ulated in the strawberry and tomato plots, no lesions or anthracnose symptoms were observed on any fruit (Fig. 3-3). These results confirm that the C. acutatum isolate recovered from pepper is pathogenic on pepper. However, this isolate was not pathogenic on fi eld-grown strawberry or toma toes following an artificial inoculation during environmental conditions highly conducive to disease development. Detached-Fruit Inoculation In the detached fruit study (Laboratory Trial 1), 12 out of 12 (100%) of the woundinoculated peppers formed an anthracnose lesion at the site of inoculation within 3 days. No lesions occurred in the six fruit that were inject ed with de-ionized water. Interestingly, both strawberry and tomato fruit th at were wound-inoculated with C. acutatum conidia also formed lesions, whereas those fruit injected with de-ioni zed water did not (Figs. 3-4 and 3-5). The number of fruit that were wound-inoculated and formed lesions was 17 out of 20 (85%) for strawberry, and 10 out of 10 (100%) for tomato. Th e lesions formed within 3 days on strawberry and pepper (Fig. 3-4), and within 5 days on tomato (Fig. 3-5). The characteristic salmon-colored conidial matrix could be observed within the le sions on the strawberry and pepper fruit (Fig. 34), but were initially less obvious on tomato fruit. However, mi croscopic examination confirmed the presence of conidia on all th ree types of fruit after 5 days (data not shown) (Fig. 3-5), and large lesions were eventually observed with profuse sporulatio n after 15 days on tomato and pepper (Fig. 3-6). Although anthracnose symptoms on tomato and strawberry did not occur in 44

PAGE 45

the field inoculations, wound-inocul ations on detached fruit did pr oduce lesions characteristic of anthracnose disease (Figs. 3-4, 3-5 and 3-6). Discussion Colletotrichum acutatum is a devastating pathogen of ma ny crops and the focal point of a great deal of research in the United States and abroad (Bailey et al., 1992; Correll et al., 2000; Freeman et al., 2000; Freeman et al., 2001; Guerbe r et al., 2003; Kim et al., 1986; Kim et al., 2002; Kwon and Lee, 2002; Legard, 2000; Park and Yoon, 2003; Park, 2007; Peres et al., 2005; Prusky and Plumbley, 1992; Robe rts and Snow, 1990; Zong-Ming et al., 2007). The fact that this fungus is now confirmed as a pathogen of pe pper in Florida causing early anthracnose only adds to the significance of this pathogen as a continual and emerging threat to pepper crops throughout the world (Black and Wang, 2007; Hadden and Black, 1988). Although C. gloeosporioides was already recognized as a ripe-rot an thracnose pathogen of pepper in Florida (Alexander and Pernezny, 2003; McG overn and Polston, 1995; Robe rts et al., 2001), the addition of C. acutatum as a pathogen of pepper brings new impli cations to pepper growers attempting to manage anthracnose. In additi on, it adds new challenges to othe r potential host crops that grow adjacent to pepper, such as tomato or strawber ry, or those rotated as a plant-back crop into harvested pepper fields such as tomato, cucurbit s, or even another crop of peppers. Certainly, understanding and comprehending the epidemiology and host range of this pathogen on peppers would lead to more informed decisions concerni ng crop rotation and other cultural practices. Isolate HB05 from pepper pr oduced typical anthracnose symptoms in the field and laboratory. Only in the la boratory, using wounded and detached fruit, did it produce anthracnose-like lesions on strawberry and tomato. These observations cast doubt on host-range reports developed for this or other pathogens ba sed solely on detached fruit assays. One recent example is the result by Black and Wang (2007), wh ere little correlation was found between field 45

PAGE 46

inoculations and laboratory woundinoculations of anthracnose pa thogens on different varieties of pepper fruit. Although certain varieties did express a partial resistance to artificial inoculation of C. acutatum in the field, these same varieties provi ded for no such conclusion when detached fruit were wound-inoculated in the laboratory (Black and Wang, 2007). Perhaps of even more concern, some researchers have drawn conclusions regarding the host range of certain species of Colletotrichum by exclusive use of detached-fruit studies (Freeman et al., 1998; Hong and Hwang, 1998; Kim et al., 2001; Manandhar et al., 1995a). Our study challenges the validity of previous reports that used woundinoculations to determine pathoge nicity of different species of Colletotrichum on pepper fruit. Currently in Florida, C. acutatum is now well-known as a pathoge n of citrus (Brown et al., 1996; Lahey et al., 2004; Peres et al., 2004; Timmer et al., 1998) and strawberry (Harp et al., 2003; Legard, 2000; Peres et al., 2005). The fungus has also been reported as a pathogen on lychee (Davis, 2003), where infectio n was shown to initiate unnoti ced in the flowers and then become symptomatic in the subsequent fruits. Additionally, the fungus is a pathogen of certain ornamental crops, such as flowering dogwood in central and north Fl orida (Strandberg and Chellemi, 2002), where it causes do gwood anthracnose and is responsible for significant losses and constraints to flowering dogwood production. On pepper, C. acutatum infection has not been reported previously in Florida; however, there are recent repor ts from other states such as Ohio (Lewis-Ivey et al., 2004), Virginia (Marve l et al., 2003) and more recently in Georgia (Chapter 2), where the disease appears to be emer ging as a significant threat to peppers and is gaining considerable attention (Correll et al., 2007). This work demonstrates that C. acutatum is indeed a pathogen of pepper in Florida and is ba sed for the first time on a large collection of 46

PAGE 47

isolates. Therefore, it has signi ficant implications for growers attempting to manage anthracnose on pepper. The fact that C. acutatum is responsible for the recent outb reaks of early anthracnose in Florida (Harp et al., 2006) has potential implications for other crops where C. acutatum is a pathogen, such as citrus and strawberry. Variou s management strategies have been implemented to facilitate the control of anthracnose diseases on these cr ops. Tomatoes, an economically important crop in Florida, have not been reported as a host of C. acutatum in the U.S. However, other species of Colletotrichum that infect strawberry and pepper, such as C. gloeosporioides, do infect tomato (Freeman et al., 1998; Hadden, 1989; Lewis-Ivey et al., 2004; Legard, 2000). The most important anthracnose pathogen of tomato, C. coccodes (Dillard, 1992; Fa rley, 1976; Hong and Hwang, 1998; Peres et al., 2002; Tsror and Johns on, 2000) also is reported as a pathogen of pepper (Hadden, 1989; Harp et al ., 2006; Lewis-Ivey et al., 2004; Legard, 2000; Roberts et al., 2001). Therefore, C. acutatum on pepper might be a threat to tomato in the U.S., particularly since this species has been recovered from tomato in Ne w Zealand (Guerber et al., 2003). In addition, C. acutatum is a well-known pathogen of strawber ry, and therefore one could easily speculate that this diseas e on strawberry could spread to pepper, or vise versa. In this study, a C. acutatum isolate recovered from pepper was found to be non-pathogenic in field inoculations on both tomato and strawberry. Since no cross-in fection occurred during ideal conditions in the field, infected pepper fields in close proximity to strawberry or tomato probably do not constitute a serious threat to the latter crop. The same conclusion would not be reached based strictly on a de tached-fruit bioassay conducted in the laboratory. In our laborat ory experiment, lesions were formed on woundinoculated fruit of tomato and strawberry from the same isolate of C. acutatum recovered from 47

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pepper used in the field tests. Theref ore, one must conclude that although C. acutatum did not cause anthracnose symptoms when applied to he althy, attached field-gr own strawberries or tomatoes, a detached, wounded fruit could beco me symptomatic under artificial conditions. Perhaps severe wounding and forcible injection of spores is necessary on these fruits for infection to occur, but this is unlikely under field conditions based on two seasons of study. One deficiency in these investigations was the use of only one isolate of C. acutatum from pepper for all of the pathogenicity tests. Isolates may va ry in ability to infect strawberry and tomato naturally in the field. Studies using more isolates would be a fruitful area for further research but may be limited by the logistics of conducti ng such large-scale field experiments. More recently, enhanced mol ecular techniques beyond the trad itional use of ITS sequence data (Chapter 2) have be en employed in studies of Colletotrichum and have further delineated species boundaries in this taxonomically complex genus (Correll et al., 2007; Correll et al., 2000; Guerber et al., 2003). Although a great deal of work has traditionally been used to identify and distinguish species of Colletotrichum based on the conserved ITS region (Adaskaveg and Forster, 2000; Brown et al., 1996; Freeman et al ., 2001; Harp et al., 2006; Horowitz et al., 2002; Lewis-Ivey et al., 2004; Mills et al., 1992; Peres et al., 2005; Sreenivasaprasad et al., 1996; Timmer and Brown, 2000), more contemporary appr oaches have more recently been employed to identify and examine subspecies populations within C. acutatum and other species of Colletotrichum (Correll et al., 2007; Du et al., 2005; Guerber et al., 2003; Peres et al., 2005). One method analyzes and compares the genetic sequence variation of a 900-bp intron of the glutamine synthetase gene be tween a diverse collection of C. acutatum isolates from different hosts (Guerber et al., 2003; Liu and Correll, 2000). Several sub-species populations, or clades, were identified and shown in some examples to correlate with differences in host range within C. 48

PAGE 49

acuatum (Correll et al., 2007; Correll et al., 2000; Pere s et al., 2005). If these and other genetic differences can be found to corres pond directly to differences in host range, then this would support the thought that isolates of C. acutatum recovered from pepper are potentially different than isolates recovered from strawberry or citrus, and therefore pose less of a threat for crossinfection. Indeed, isolate HB05 from pepper was re cently found to be different from strawberry and citrus isolates collected in Florida in Dr. James Corrells laboratory at the University of Arkansas (Harp and Correll, unpublished). Furt hermore, by RFLP analysis, HB05 was found to be most closely related to isolates recovere d from pepper in Taiwan (Correll, personal communication), which were grouped into the mt DNA haplotype D3 (Guerb er et al., 2003). This study has demonstrated that a highly virulent isolate of C. acutatum recovered from pepper could be used to re-infect pepper plan ts in the field, but did not infect adjacent strawberry, a known host of C. acutatum Although wound-inoculations did produce disease symptoms on detached fruit, molecular differe nces between the pathogen recovered from pepper and those recovered from strawberry could be the reason that a pepper isolate could not cause disease on field-grown strawberry fruit. It is likely that genetic differences between C. acutatum isolates from different hosts could affect the molecular sign aling and recognition needed by a specific pathogen to identify and de tect a potential host. If th e pathogen does not recognize a substrate as a host, it simply will not undergo the transformations needed to be pathogenic on that host, such as spore germin ation and / or appressorium formation and development. It appears that the isolates of C. acutatum recovered from pepper a nd used in this study are genetically different than isolates of C. acutatum recovered from strawberry or citrus, and therefore pose no threat to nearby strawberry fields or citrus groves. Th e reverse is also likely true, in that isolates recovered from strawberry or citrus woul d not cause disease in pepper. 49

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However, it is interesting that pe pper isolates will still form lesions on strawberry and tomato with wound-inoculation of detached fruit. Pe rhaps wounding the fruit allows the pathogen to bypass the molecular recognition needed for ingre ss, and once inside the cuticle the fungus can continue to germinate, grow, and produce coni dia. This is not unlike the fungus growing on artificial media, where appressoria and other pathogenic structures ar e typically not produced, but the fungus continues to grow and reproduce. Certainly, more research is needed to draw further conclusions on ability for isolates of th e same species to infect different hosts, both by molecular characterization and epidemio logy studies (Peres et al., 2005). For now, Florida growers can feel fairly confid ent that an epidemic of pepper anthracnose in a given season is not necessari ly a significant threat to nearby tomato or strawberry crops. Certain species of Colletotrichum such as C. gloeosporioides are known for their wide host range and potential devastation to many crops (OConnell et al., 2000; Sutton, 1992). Although the recent discovery of C. acutatum on pepper and the implicati ons of this disease on younger, underdeveloped fruit is a grave concern, it seems lik ely that this species is selective to bell and chili pepper, and does not lik ely threaten other valuable crops that are hosts of C. acutatum strains. Even so, the knowledge of the inoc ulum source on pepper and the ability of this pathogen to maintain a viable inoculum rese rvoir between pepper crops would be of great significance to assist in the management of this disease. This study hopes to provide a firm starting point to evaluate the epidemiology of pepper anthracnose caused by C. acutatum and should contribute to the understanding and management of this disease now and into the future. 50

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Table 3-1. Plot size and planting conditions for each crop evaluate d in Field Trial 1 and Field Trial 2. Trial / crop Plot size(m) w No. of plants x Row spacing(m) y Plant spacing(cm) z Field Trial 1 Tomatoes 3.0 x 1.5 8 1.5 45.7 Strawberries 3.7 x 1.5 12 1.5 30.5 Peppers 3.0 x 1.5 8 1.5 45.7 Field Trial 2 Tomatoes 6.1 x 1.5 15 1.5 61.0 Strawberries 9.1 x 1.5 15 1.5 45.7 Peppers 4.6 x 1.5 8 1.5 61.0 w Length x width (m). All crops were transplanted on raised beds covered in white plastic mulch with drip irrigation. x The number of plants within each pl ot. Tomatoes and peppers were planted on raised beds in a single row, while strawberries were planted in a double row. y Raised beds were 30 cm tall, 1.5 m wide, and 1.5 m apart. z Centimeters between plants within a row. For strawberries, the plants are 61.0 cm apart al ong the row and between the double row plants. 51

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Table 3-2. Mean number of lesions per plot on peppe r, strawberry and tomato fruit in inoculated, un-inoculated, and water-sprayed treatments for Field Trial 1 and Field Trial 2 at 10 days following an artificial inoculation with Colletotrichum acutatum The inoculation occurred only in the inoculated treatments No infection occurred on either strawberry or tomato fruit in any of the treatments. No. of lesions ____________________________________ Trial / crop Inoculated x Un-inoculated y Water-sprayed z treatment treatment treatment Field Trial 1 Peppers 22.7 a 0.0 0.0 Strawberries 0.0 b 0.0 0.0 Tomatoes 0.0 b 0.0 0.0 Field Trial 2 Peppers 39.3 a 0.0 0.0 Strawberries 0.0 b 0.0 0.0 Tomatoes 0.0 b 0.0 0.0 x Pepper, strawberry, and tomato plots were inoc ulated with a conidial suspension (2.5 x 10 5 ) of C. acutatum and the number of lesions per pl ot were counted for each crop. y Each crop in this treatment did not receive any inocul ation or application of water. z Each crop in this treatment was sprayed with de-ionized water but without the addition of conidia. 52

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B A Figure 3-1. Ripened jalapeno (A) and bell pepper (B) with anthracnose lesions caused by Colletotrichum gloeosporioides. Once ripened, the pepper fruit become susceptible to infection by C. gloeosporioides and symptoms occur at once throughout the entire field. 53

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Figure 3-2. Unripe bell pepper with anthracnose symptoms caused by Colletotrichum acutatum Unlike anthracnose symptoms which appear on strictly ripened, red fruit caused by C. gloeosporioides C. acutatum can cause symptoms on unripe, green pepper fruit, including very young fruit (right). 54

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Un-inoculated Inoculated Figure 3-3. Strawberry, tomato, and pepper plants from un-inoculated (left) and inoculated plots (right) in Field Trial 1. Only the pepper fruit in the inoc ulated plots developed lesions observed within 7 to 10 days after the artificial inoculation with Colletotrichum acutatum (pictured above at 14 days after inoculation). In the strawberry and tomato plots, both green unripe fruit, and red-ripe ned fruit were inoculated, but no lesions formed up to 21 days after the inoculation. 55

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Figure 3-4. Detached, wound-inoculated fruit of strawberry (left) and pepper fruit (right) three days after inoculation. At left, the top two strawberry fruit were injected with deionized water while the bottom two were wound-inoculated with a spore suspension of Colletotrichum acutatum isolate HB05 recovered from pepper. At right, detached pepper fruit was wound-inoculated as a positive control. Conidia were observed within the lesions on stra wberry and pepper after onl y 3 days following the woundinoculation. 56

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Figure 3-5. Detached, wound-injected fruit of to mato (left) and pepper fruit (right) five days after the inoculation with a conidial suspension of Colletotrichum acutatum In each photograph, the fruit on the left was injected with de-ionized wate r, and the fruit on the right wound-inoculated with a conidial suspension of C. acutatum isolate HB05 recovered from pepper. Five days after the inoculation, lesions on the inoculated tomato fruit became apparent and conidia were recovered from the lesion. 57

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Figure 3-6. Detached, wound-inoculated tomato (top) and tomato and pepper (bottom) 12 days after inoculation with a conidial suspension of Colletotrichum acutatum Profuse sporulation, observed as a salmon-colored conidial matrix exuding from the lesion, was apparent on both types of fruit. 58

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CHAPTER 4 CHEMICAL CONTROL OF PEPPER ANTHRACNOSE Introduction Pepper anthracnose is a potent ially devastating disease of pe pper in most regions where pepper is grown (Alexander and Pernezny, 2003; Hadden and Black, 1998; Kwon and Lee, 2002). Although traditionally thought of as a ripe-rot disease, anthracnose caused by Colletotrichum acutatum has been recently recovered from immature, green pepper fruit in Ohio (Ivey, et al., 2004), Louisiana (Haden, 1998), Virginia (Marvel et al., 2003), Georgia, and Florida (This dissertation, chapter 2). Th erefore, unlike the ripe rot phase of anthracnose typically caused by C. gloeosporioides or C. coccodes (Alexander and Pernezny, 2 003; Lewis-Ivey et al., 2004; Roberts et al., 2001) an thracnose disease caused by C. acutatum would warrant additional chemical control measures in order to harvest healthy, fully-sized gree n fruit. Although a few fungicides are labeled for pepper anthracnose co ntrol, no data evaluating efficacy of these products in Florida for control of early anthracnose caused by C. acutatum are currently available. At present, most pepper fi elds throughout Florida under go a pesticide spray program which often includes both insecticides and fungicides. Typicall y, these products are tank-mixed and used to target insect pest s, such as the pepper weevil, a nd diseases such as Phytophthora blight, powdery mildew, frogeye leaf spot, and ba cterial spot. Anthrac nose disease, caused by C. gloeosporioides or C. coccodes, is traditionally considered a ripe-rot disease occurring only on ripened, red pepper and is not targ eted with pesticides unless red pepper is the intended harvested product. For pepper harvested green, which in cludes the majority of the acreage in Florida (Maynard et al., 2003), anthracnose caused by C. gloeosporioides is typically not considered a disease needing special chemical treatment. However, now that C. acutatum has been confirmed 59

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to infect immature green pepper fruit in Florida, pepper growers need to in clude this disease as a target in their pest management program. Currently, there are five commonly used fungicides labeled for anthracnose of pepper in Florida. These include azoxystrobin (Quadr is), famoxadone plus cymoxanil (Tanos), pyraclostrobin (Cabrio), maneb, and copper hydroxide (Kocide or Champ). The first three fungicides contain an active ingred ient of the strobilu rlin class of chemistry, and are effective against a broad range of pathogens on a large number of crops. The remaining two pesticides are usually applied as a tank mix on pepper for control of bacterial spot. However, when applied separately, copper or maneb can be used for contro l of anthracnose or other fungal diseases, such as Phytophthora blight. In this study, six fungicides and one syste mic-acquired resistant (SAR) pesticide were evaluated for efficacy against artificially inoculated C. acutatum on pepper plants in Florida. The isolate used for the artificial inoculati on (HB05) was originally recovered from an anthracnose lesion on an unripe, green pepper fruit in 2004 from Hendry, Co., Florida, and was among those responsible for a severe epidemic in a growers field that caused nearly 50% reduction in yield (Harp, personal observation). HB05 was used to artificially inoculate pepper plants treated with various fungicides to evalua te the efficacy of these products against pepper anthracnose. The evaluation of pesticides agai nst this potentially devastating new disease in Florida will assist growers in de veloping successful management stra tegies to control this disease and minimize the potentially severe economic losse s that could result. The purpose of this study was to determine the efficacy of seven pesticides against pepper anthracnose in Florida and to provide insight into chemical management r ecommendations necessary to optimize control of this disease. 60

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Materials and Methods Fungicide Field Trials Fungicide field trials were conducted duri ng three different peppe r-growing seasons in Florida, fall 2006 (Fungicide Field Trial 1), sp ring 2007 (Fungicide Field Trial 2), and fall 2007 (Fungicide Field Trial 3). All th ree trials were conducted at th e Syngenta Vero Beach Research Center, Indian River Co., Florida. Pepper Plants Seven-week-old pepper (Re volution) transplants were purchased from Speedling nursery in Sun City, Florida, and transplanted in single rows on raised beds under white plastic mulch (1.5 m centers) with drip irrigation. Pre-plant pesticid es, mefenoxam (Ridomil Gold SL, 1.2 L / ha), PCNB (Terraclor Super X, 7.1 L / ha), and diazinon (Diazinon AG500, 2.4 L / ha) were broadcast-incorporated into the soil for so il-borne fungi and insect control, respectively. Approximately 168 kg / ha of fertilizer (10-10-10) was also applied to the soil prior to planting with two drip-line injections of fertilizer conduc ted approximately six weeks apart after planting. A rotational spray program of spinosad (Spintor 0.44 L / ha), emamectin benzoate (Proclaim, 0.29 L / ha), and lambda-cyhalothrin (Warrior, 0.29 L / ha) were applied on a 7 to 14-day interval for insect control. The planting da tes for Fungicide Field Trial 1, 2, and 3 were 06 October, 2006, 06 March, 2007, and 15 October, 2007, respectively. In Fungicide Field Trial 1 and 2, the row spacing was 1.5 m, and the plant sp acing was 45.7 cm with 10 plants per plot (plot size 4.6 x 1.5 m). In Fungicide Field Trial 3, th e row spacing was 1.5 m, and the plant spacing was 60.9 cm with 6 plants per plot (plot size 3.7 x 1.5 m). Plot sizes and relevant planting details for each fungicide field tria l are shown in Table 4-1. 61

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Inoculum Production Isolate HB05 was recovered from pepper as pr eviously described (Chapter 2) and grown for seven days on PDA under contin uous lighting at 20C. Conidi a were harvested by rinsing cultures with de-ionized water and filtering through three laye rs of cheesecloth to remove mycelia. Conidial concentra tion was adjusted to 2.5 x 105 conidia per mL in 5 L of de-ionized water using a hemacytometer. Fungicide Treatments In all three trials, treatments consisted of an untreated check, azoxystrobin (Quadris 250SC, 1.02 L / ha), famoxadone plus cymoxanil (Tanos 50WG, 0.56 kg / ha), mancozeb (Manzate 75WG, 1.68 kg / ha), acibenzolar-S-methyl (Actig ard 50WG, 0.05 kg / ha), copper hydroxide (Kocide 2000 53.8DF, 2.24 kg / ha), and fludioxanil plus cypr odinil (Switch 62.5WG, 0.84 kg / ha). In Fungicide Field Trial 3, difenoconazole (Inspire 250 EC, 0.51 L / ha) was included. Each trial consisted of either three (Fungicide Field Trial 1 and 3) or four (Fungicide Field Trial 2) weekly applications beginning at late flowering to ear ly fruit set (fruit size 25 to 50% of harvestable size). Fungicide Applications Applications were conducted using a back-pack CO sprayer with a hand-made sprayboom containing three nozzles (Tee-Jet hollow-c one size 8) at 40.6 cm spacing. The two end nozzles dropped 10.1 cm below center and pointed in ward at a 45 angle. The spray pressure was adjusted to 2.1 x 105 pascals, and all applications we re conducted weekly at 7 to 10-day intervals using a spray volume of 325 L / ha. In Fungicide Field Trial 1 and 2, seven treatments were included, while Fungicide Field Trial 3 contained eight treat ments (see Fungicide Treatments). For Fungicide Field Trial 1, the application dates were 29 November, 2006, 06 December, 2006, and 13 December, 2006. For Fungi cide Field Trial 2, the application dates 2 62

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were 04 May, 2007, 11 May, 2007, 18 May, 2007, and 25 May, 2007, and for Fungicide Field Trial 3, the application dates were 14 D ecember, 2007, 21 December, 2007, and 30 December, 2007. No rainfall occurred within 4 hours after any of the applica tions, except for in Field Trial 3, where rainfall did occur within 2 hours afte r the second applicati on (21 December, 2007). Within the treatment plots, the primary pepper (first pepper formed on primary inflorescence) was picked within one day prior to the first appl ication in all three trials to allow for improved development and growth of th e remaining secondary peppers. Artificial Inoculation An artificial inoculation was conducted for each trial within one day following the second (Fungicide Field Trial 1 and 3) or third (Fungicide Field Trial 2) application. For Fungicide Field Trial 1, 2, and 3 the inoculations took pl ace during the evening (a pproximately 2300 hr) on 07 December, 2006, 18 May, 2007, and 21 December, 2007, respectively. Night-time was chosen to ensure adequate conditions for infectio n, either during or just prior to dew formation under the same conditions as described in Chapter 3. Inoculum was applied to the plants using a back-pack pump sprayer (Solo 425 pump spra yer, Detroit, MI) until run-off, ensuring good coverage of fruit and foliage. Inoculum was a pplied to each pepper plant in all plots, and in some cases, plants in between the marked plots. A total of approximately 5 L of inoculum was used for each fungicide field trial. For all three inoculations, at least 8 hours of leaf wetness was obtained on the night of the inoculation. Disease Assessments Assessments were made by harvesting fully-si zed, green pepper fru it and evaluating each fruit for lesions. The number of fruit with le sions was recorded for each plot, along with the number of healthy fruit. However, due to the nature of this disease upon artif icial inoculation, 63

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many of the younger fruit and flowers became severely infected and either fell off or aborted. Therefore, a large number of fruit that would have been counted as infected were never developed and so were not counted in the assess ments. For this reason, the amount of healthy fruit recovered per plot represented a better indi cation of treatment performance and was used as the primary assessment for all trials. For Fungicide Field Trial 1, only one harvest was conducted (28 December, 2006), while two harvests were conducted for Fungicide Field Trial 3 (06 January, 2008 and 17 January, 2008) and three ha rvests for Fungicide Field Trial 2 (25 May, 2007, 31 May, 2007, and 07 June, 2007). Results Disease Assessments Within 7 days after the inoculation, lesions be gan to appear on pepper fruit in the untreated check plots for all three fungicide field trials. In most of the treated plots, both healthy and infected fruit were observed (Fig. 4-1). Harves ts were made when the majority of fruit was fully-sized and comparable in size and shape to green bell peppers harvested commercially. Fruit were not allowed to ripen or change color prior to picking. For eac h plot, fully-sized green fruit were harvested and the number of fruit with lesions was counted along with fruit that were free of symptoms. In most cases, the number of infected fruit and overall disease incidence could not be properly assessed sin ce many of the infected flowers and smaller fru it aborted prior to harvest (Fig. 4-2), especially in the untreated check plots. Nearly all of the undersized, developing fruit in the untreated plots containe d lesions and never grew to harvestable size before rotting and falling off of the plant (Fig. 4-2). Therefore, many of these smaller fruit never developed and could not, of course, be scored as infected. As a result, the number of infected fruit per plot did not provide an adequate indica tion of disease severity for these plots, and for many of the treated plots. For th at reason, the number of healthy fr uit per plot was chosen as the 64

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primary indicator of treatment performance, and is what is reported in the results. In the untreated check plots, nearly ev ery fully-sized, harvestable fru it showed lesions. All fungicide treatments significantly reduced the amount of inf ected fruit, both harvestable and developing, in all three fungicide field trials in comparison to the untreated check plots. In each trial, the number of healthy fruit harveste d per plot reflected the degree of efficacy for each of the fungicide treatments. Fungicide Field Trial 1 In this trial, pepper plants were of unusua lly low vigor prior to conducting the fungicide treatments and inoculation. The reason for the low vigor in this trial was not determined, and the amount of harvestable peppers per plot infected or uninfected was low. Regardless, applications were conducted and bot h healthy and infected fruit we re harvested 15 days after the last application. In the untreated check plots, a mean of 0.0 healthy fru it were harvested (Table 4-2), with a mean of 12 blemished fruit per plot Many fruit became infected prior to developing into a fully-sized harvestable pe pper fruit, and dropped prematurely (Fig. 4-2). Three treatments, azoxystrobin (Quadris), mancozeb (Manzate), and fludioxanil plus cyprodinil (Switch) provided the highest amount of healthy, harvestable fruit with mean numbers of 7.0, 8.0, and 8.3, respectively. The treatments with the least am ount of harvestable, he althy fruit were copper hydroxide (Kocide 2000), acibenzol ar-S-methyl (Actigard), and famoxadone plus cymoxanil (Tanos) with 3.0, 3.3, and 5.7 healthy fruit, respectively (Table 4-2). Copper hydroxide and acibenzolar-S-methyl provided si gnificantly less control than azoxystrobin, mancozeb, or fludioxanil plus cyprodinil ( P < 0.05). Further harvests from this trial were not possible, due to the reduced vigor of these plants, and the low amount of fruit that developed. 65

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Fungicide Field Trial 2 In this trial, plants were much more vigor ous than in Fungicide Field Trial 1, and three harvests were collected at 1, 6, and 13 days af ter the last applicati on. The total number of healthy fruit collected from all thr ee harvests was analyzed by ANOVA ( P < 0.05) and means were separated by Fishers Protected LSD (Table 42). In this trial, the treatments with the highest amount of healthy fr uit were mancozeb (Manzate), azoxystrobin (Quadris), copper hydroxide (Kocide 2000), and fludioxanil plus cyprodinil (Switch) with 31.3, 30.3, 29.0, and 26.0, healthy fruit, respectively. No significan t difference in healthy fruit was found among these three treatments. The treatments associat ed with the least amount of healthy, uninfected fruit were the untreated check plot, acibenzolar-S-methyl (Actigard), and famoxadone plus cymoxanil (Tanos) with 3.8, 16.0, and 17.0 healthy fruit, respectively (Table 4-2). There was a clear significant difference between the three best treatments, the three least effective treatments, and the untreated check plots (Table 4-2). Fungicide Field Trial 3 Moderate to good vigor occurred in this pepper trial, and two harvests were obtained at 7 and 17 days after the last applic ation. The total number of h ealthy fruit collected from both harvests was determined and si gnificantly analyzed by ANOVA ( P < 0.05) and means were separated by Fishers Protected LSD (Table 4-2). In this trial, the treatments with the highest amount of healthy fruit were azoxystrobi n (Quadris), difenoconazole (Inspire), famoxadone plus cymoxanil (Tanos), fludioxanil plus cyprodinil (Switch), acibenzolar-S-methyl (Actigard) and mancozeb (Manzate) with 33.3, 29.0, 24.3, 23.5, 23.0, and 20.8 healthy fruit, respectively. Azoxystrobin provided the best cont rol and was significantly superior to all other treatments except for difenoconazole (Table 4-2; Fig. 4-3). The treatments that pr ovided the least amount of healthy, uninfected fruit were the untreated check plot and copper hydroxide (Kocide 2000), 66

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with 6.3, and 15.8 healthy fruit, respectively (T able 4-2). Copper hydroxide, which provided notably better control in the second trial, was among the least eff ective treatment in this trial. In addition, mancozeb, the other non-systemic fungici de evaluated in the trial, also provided slightly less control in this trial in comparison to the other fungicides in the other two trials. Discussion Fungicide field trials were conducted to evaluate various commercial fungicides for efficacy against pepper anthracnose in Florida. For this study, the isolat e used to artificially inoculate the fungicide pl ots was an isolate of Colletotrichum acutatum recovered from Florida and therefore offered an opportunity to evaluate an anthracnose epidemic that might occur in Florida pepper fields. The fact that all fungicides evaluated in th ese trials provided moderate to good control in the presence of a severe epidemic l eads us to conclude that fungicidal sprays can be an important tool in the integrated management of this disease in Florida. In some reports, this disease is purportedly difficult to contro l chemically (Hadden and Black, 1988; Kwon and Lee, 2002; Lewis-Ivey et al., 2004). The results reported herein clearly identify fungicides that can be used effectively under signif icant pepper anthracnose epidemics. The fungicides evaluated in this study represent a fairly broa d range of chemistries, mode of actions, and costs. Two of the nine fungici des tested are strobilu rlins, azoxystrobin and famoxadone, and these compounds are known to be highly active against certain species of Colletotrichum including C. acutatum (Peres et al., 2005). Tanos the fungicide brand that contains famoxadone, also contains cymoxan il, an oomycete fungicide with known activity against late blight and downy m ildews. This component of Tanos has no activity on anthracnose but is added to widen the activity of Tanos to include the oomycetes, a significantly large and important group of plant pathogens. According to th e label, Tanos is required to be applied in mixture with another fungicide containing an alternate mode of action, such as mancozeb, 67

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chlorothalonil, or a copp er-containing fungicide. According to the results obtained from these trials, where famoxadone and cymoxanil provide d moderate to good control, the addition of mancozeb to this mixture might have significantly improved efficacy. With applications of straight mancozeb, good to excelle nt control was obtained in all three trials. Considering the price of application and spectrum of disease cont rol, mancozeb clearly provides a cost-effective option that provides reasonable e fficacy. Unfortunately, it is not currently labeled for use on pepper in Florida. Copper hydroxide, which also provided fair to good co ntrol in two of the three trials, might also increas e the efficacy of famoxadone and cymoxanil (Tanos) and therefore serve as a possible tank-mix co mbination. In one of the two trials where copper hydroxide was less effective (Trial 3), rainfa ll occurred within two hours following the second application, and may have contributed to chemical wash-off and reduced efficacy. In this trial, the artificial inoculation was conducted on the ev ening of this application, so the need for a systemic or rainfast product at this applic ation was probably crucial. Th e other strobilurlin fungicide, azoxystrobin (Quadris), provided ou tstanding control in all three tr ials and overall provided the highest amount of harvestable fruit of any treatment (Table 4-2). Certainly, the use of azoxystrobin should be considered by any grower faced with heavy anthracnose disease pressure, and prudence dictates that azoxystrobin be mixed or alternat ed with other fungicides with an alternate mode of action to re duce resistance development, as directed by the label. Other fungicides currently not labeled for peppe r anthracnose in Florida that provided good to outstanding control in our tests were fludioxanil plus cyprodinil (Switch) and difenoconazole (Inspire). Switch, which is a premix of fludioxan il and cyprodinil, is commonly used in Florida for control of Botrytis blight and anthracnose on strawberry; it is not su rprising that it provided good control for anthracnose on pepper. Both in gredients in this fungi cide have activity on C. 68

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acutatum so this might help explain the increased efficacy of this product. In addition, this product and would have the extra benefit on pepper of controlling Botrytis blight. On strawberry, fludioxanil plus cyprodinil (Switc h) is among the most effective fungicide combinations for anthracnose caused by C. acutatum (Harp, unpublished) as well as Botrytis blight (Dr. Jim Merteley, University of Florida, personal communication). Difenoconazole (Inspire) is a new fungicide from Syngenta Crop Protection that will be labeled for many leaf spot diseases, including early blig ht of tomato. Based on the resu lts from one trial, this product certainly looks promising for control of pe pper anthracnose should it ever achieve EPA registration for use on pepper. One other product evaluated in this trial wa s acibenzolar-S-methyl (Actigard). This product is not a typical fungicide, but work s by activating the SAR (systemic acquired resistance) pathway in plants. In short, acib enzolar-S-methyl activates various resistance genes ( R genes) in plant cells that work to generate resistance proteins (R proteins) that help fight off attack by intruding pathogens. The mechanisms involved can be quite complex; however, it represents a fairly well-studied system in molecular plant pat hology and is the focus of a great deal of academic research. In this study, artificial inoculations were conducted at either seven (Fungicide Trial 1 and Fungicide Tr ial 3) or 14 (Fungicide Trial 2) days after the fi rst application in order to allow time for the SAR pathway to be come activated and resistance expressed. If the artificial inoculation had been on the evening of the first ap plication, acibenzolar-S-methyl would probably not have had sufficient time to activate the plant-resistance pathway and therefore probably would have failed to provide acceptable control. Although this treatment was not among the most effective of those tested, it did provide significantly improved control over the untreated check plots, and therefore could be quite useful in a program for integrated 69

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anthracnose control. Acibenzolar-S-methyl is not currently labeled on pepper, but is labeled on tomato in Florida for control of bacterial spot, caused by Xanthomonas sp. The results presented in this study certainly show promise for this product on pepper and further research would be needed to evaluate rates, app lication timings, and the spectrum of disease control on pepper. Based on the results from all three trials, the most effective fungicides tested were azoxystrobin (Quadris), fludioxanil plus cyprodinil (Switch) and mancozeb (Manzate). Copper hydroxide (Kocide 2000), provided very good control in the second trial, but provided less control in the first and third trial. As with mancozeb, it is likely that copper hydroxide is susceptible to wash-off and adverse environmen tal conditions common fo r strictly protectant fungicides. Difenoconazole (Inspire) also provided outstanding c ontrol equivalent to azoxystrobin, but was only tested in one of three trials. In all trials, famoxadone plus cymoxanil (Tanos) and acibenzolar-S-methyl (Actigard) treatments appeared to have more disease than some of the other products and th erefore a lower amount of harvesta ble, healthy fruit. This was especially clear in Fungicide Field Trial 2, wher e both treatments provided significantly less uninfected, healthy fruit than all other chemical treatments. However, considering the heavy disease pressure that occurred in this trial as a result of the ar tificial inoculation, it could be argued that all treatments provided acceptable cont rol of this disease and could be successfully used in a chemical management program ai med at controlling pepper anthracnose. Although this study looked strictly at efficacy of these products applied alone, in reality a good fungicide program would consist of mixtures and alternations using products that contain different modes of action with perhaps a wider spectrum of target pathogens. Such a program would address resistance management concerns while providing optimal protection from an array of diseases at reasonable cost. A program such as azoxyst robin (Quadris) al ternated with 70

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mancozeb and / or fludioxanil plus cyprodinil (Switch) (if Botrytis blight is also present) would be ideal. If famoxadone plus cymoxanil (Tanos) is used, it should be mixed with copper hydroxide or maneb, as recommended on the label, and alternated with either azoxystrobin (Quadris) or fludioxanil plus cyprodinil (Switch). The active component in Tanos ( famoxadone) is cross-resistant to azoxystrobin, so proper rotation and tank-mixing should be practiced for resistant-management purposes. Acibenzolar-S-m ethyl (Actigard) should not be used alone under heavy disease pressure, but it is likely that Actigard would contribute to efficacy in a fungicide program and perhaps would be e ffective under light disease pressure. The purpose of this work was to evaluate vari ous labeled fungicides, such as azoxystrobin, copper hydroxide, and famoxadone plus cymoxanil for efficacy against a pepper anthracnose epidemic that might occur in pepper fields in Florida. In addition, some unlabeled fungicides were evaluated, since some of these could become labeled in the future. The overall intended aim of this study was to determine if chemical c ontrol of this potentially devastating disease was possible, and it appears from these results that acceptable control could be obtained from the use of fungicides. Certainly, further work is n ecessary to evaluate fungicide programs, i.e., alternations, timings, cost, etc., that could be implemented if pe pper anthracnose continues as a significant problem in the future. This work s hould provide a starting point to determine the optimal chemistries and spray programs necessary for this disease and assist pepper growers in their effort to manage early anthracnose of pepper. 71

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Table 4-1. Plot size and planting conditions for peppe rs in the fungicide field trials. All crops were transplanted on raised beds covered in white plastic mulch with drip irrigation. Fungicide Field Trial 1 Fungi cide Field Trial 2 Fungicide Field Trial 3 Plot size (m)w 4.6 x 1.5 4.6 x 1.5 3.7 x 1.5 No. plants/plot x 10 10 6 Row spacing (m)y 1.5 1.5 1.5 Plant spacing (cm)z 45.7 45.7 61.0 w Length x width (m). x The number of plants within each pl ot. Peppers were planted on raised beds in a single row. y Raised beds were 30 cm tall, 1.5 m wide, and 1.5 m apart. z Centimeters between plants within a row. 72

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Table 4-2. Effect of fungicides on marketable yield of pepper artifici ally inoculated with Colletotrichum acutatum in three trials conducted in Florida in 2006 and 2007. Number of Marketable Fruitv Treatmentu Field Trial 1w Field Trial 2x Field Trial 3y Untreated 0.0 dz 3.8 c 6.3 d Azoxystrobin 7.0 ab 30.3 a 33.3 a Famoxadone plus cymoxanil 5.7 abc 17.0 b 24.3 bc Mancozeb 8.0 a 31.3 a 20.8 bc Acibenzolar-S-methyl 3.3 bcd 16.0 b 23.0 bc Copper hydroxide 3.0 cd 29.0 a 15.8 c Fludioxanil plus cyprodinil 8.3 a 26.0 a 23.5 bc Difenoconazole NT NT 29.0 ab LSD 3.76 8.47 8.61 u Treatments were applied three (Fungicide Trial 1 and Fungicide Trial 3) or four (Fungicide Trial 2) times on a 7 to 10-day interval. v Number of total marketable fruit from all harvests per trial that were fully-sized, green fruit with no anthracnose lesions. w Field Trial 1 was conducted during the fall growing season, 2006, and the data represent the tota l number of marketable, healthy fruit from one harvest. x Field Trial 2 was conducted dur ing the spring growing season, 2007, and the data represent the total number of marketable, healt hy fruit from three harvests. y Field Trial 3 was conducted during the fall grow ing season, 2007, and the data represent the total number of marketable fruit from two harvests. z In each column, numbers followed by a different letter are significantly different ( P < 0.05) by Fishers Protected LSD. 73

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Figure 4-1. Fully sized, harvesta ble fruit from the treated pepper plots inoculated with Colletotrichum acutatum In each fungicide field trial, marketable peppers were found in treated plots that contained anthracnos e lesions (left) or we re healthy (right). Each fully-sized, marketable pepper, with or without lesions, was harvested and counted in each plot. 74

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Figure 4-2. Heavy infection of the flowers and ne wly-formed fruit as a result of the artificial inoculation by Colletotrichum acutatum In the untreated plots and many of the treated plots, the flowers (top left) and the newly-formed fruit became severely infected and usually aborted. Therefore, these fruit never obtai ned harvestable size and were not counted as i nfected fruit during the assessm ents. As a result, the number of infected, harvestable fruit pe r plot was not a good measure of disease levels in a particular treatment plot. Inst ead, the number of hea lthy fruit obtained was the best representation of treatment efficacy for a particular treatment plot. 75

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Figure 4-3. Harvested pepper from treated (left) and untreated (right) pepp er plots in Fungicide Field Trial 3. The peppers on the left were pick ed from plot 102 (azoxystrobin, 1.02 L / ha) and the peppers on the right were harv ested from plot 101 (Untreated). In this trial, azoxystrobin provided the least amount of infected peppers and the highest amount of marketable healthy peppers. 76

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CHAPTER 5 SUMMARY AND DISCUSSION Colletotrichum acutatum caused anthracnose lesions on pepp er fruit recovered from four pepper fields in Florida. The lesions formed on unripe, green fruit (Fig. 5-1), which was different than the well-described ripe-rot an thracnose that reportedly occurred on ripened, red fruit (Alexander and Pernezny, 2003; Roberts et al., 2001). In this study, a collection of 50 isolates was recovered from pepper anthracnose lesions on pepper fruit from younger fields that contained unripe, green fruit, as well as mature fields that cont ained ripened, red fruit. Using ITS species-specific primers (Lewis-Ivey et al., 2004; Sreenivasaprasad et al., 1996), the isolates recovered from unripe, green pe pper fruit were identified as C. acutatum and the isolates recovered from the ripened, red fruit were C. gloeosporioides This is the first report of C. acutatum as an anthracnose pathogen of pepper in Florida. The implications of a new anthracnose disease on pepper caused by C. acutatum could be quite significant. The disease is an aggressive pathogen of pepper in Asia, and has recently been reported in the U.S. (Lewis-Ivey et al., 2004; Marvel et al., 2003). It causes destructive, sunken lesions on developing and fully-sized pepper fruit that essentially destroy the fruit and significantly decrease marketable yields. Cl early, new management strategies would be necessary to control this disease in fields where C. acutatum becomes well-established. Considering the impact of this disease on pe pper in comparison to the ripe-rot form of anthracnose, we propose the na me early anthracnose for anthracnose of pepper caused by C. acutatum In Florida, C. acutatum is an important pathogen of strawberry, citrus, dogwood, and lychee. In strawberry, anthracnose outbreaks caused by C. acutatum are extremely destructive and were responsible for heavy losses. Chemical control is currently the best means to control 77

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strawberry anthracnose. Now that the pat hogen has been found on pepper, further research leading to new management strategies is needed for successful control of this potentially destructive disease. Field pathogenicity studies dem onstrated that an isolate of C. acutatum recovered from pepper was not directly pathogeni c on field-grown strawberries or tomatoes, both reported hosts of C. acutatum This provides some sense of assurance that anthracnose epidemics in pepper fields pose little threat to nearby strawberry or tomato fields. However, in the laboratory using wounded, detached fruit, anthracnose lesions coul d be induced on both strawberry and tomato by injecting conidia into the surface of the fruit. This demonstrates that using detached, wounded fruit is probably not a good i ndication of pathogenicity for Colletotrichum pathogens on fruit, and challenges the validity of reports that have used detached fruit studies to evaluate host range of Colletotrichum spp. Fortunately, chemical control measures do hold promise for managing pepper early anthracnose in Florida. Fungi cide field trials conducted over three seasons in east-central Florida have demonstrated that good efficacy can be obtained from labeled fungicides, even under heavy disease pressure. In addition, various unlabeled fungicides provided acceptable control and could be management options in th e future pending registration by the EPA. All fungicides tested provided significantly improved control over the untreated check plots and could be used successfully in a chemical c ontrol program. Results provide hope to pepper growers that preventive chemical control opti ons exist and could be implemented to control early anthracnose. Anthracnose disease on pepper in Florida caused by C. acutatum or early anthracnose, adds a new challenge for pepper growers throughout the state. The potentially destructive 78

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disease can spread quickly and cause heavy lo sses if not managed and controlled. Further research on early anthracnose is needed in Florid a to continue efforts to understand this disease and apply successful management strategies. 79

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Figure 5-1. Pepper anthracnose isolat e on unripe, green bell pepper caused by Colletotrichum acutatum in Florida. 80

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LIST OF REFERENCES Adaskaveg, J.E., Frster, H., 2000. Occurrence and management of anthracnose epidemics caused by Colletotrichum species on tree fruit crops in California. In: Prusky, D., Freeman, S., Dickman, M.B. (Eds.), Colletotrichum : Host Specificity, Pathology, and Host-Pathogen Interactions. American Phytopathological Society Press, St. Paul, MN, pp. 317. Adaskaveg, J.E., Hartin, R. J., 1997. Characterization of Colletotrichum acutatum isolates causing anthracnose of almond and peach in California. Phytopathology 87, 9797. Adikaram, N.K.B., Brown, A.E., Swinburne, T.R., 1983. Observations on infection of Capsicum annuum fruit by Glomerella cingulata and Colletotrichum capsici. Trans. Brit. Mycol. Soc. 80, 395-401. Alexander, S.A., Pernezny, K., 2003. Anthracnose. In: Pernezny, K., Roberts, P.D., Murphy, J.F., Goldberg, N.P., eds. Compendium of Pepper Diseases. American Phytopathological Society Press, St. Paul, MN, pp 9. Bailey, J.A., OConnell, R.J., Pring, R.J., Nash, C., 1992. Infection strategies of Colletotrichum species. In: Bailey, J.A., Jeger, M.J. (Eds.), Colletotrichum : Biology, Pathology and Control. CAB International, Wallingford, UK, pp 88. Bernstein, B., Zehr, E.E., Dean, R.A. Shabi, E., 1995. Characteristics of Colletotrichum from peach, apple, pecan, and othe r hosts. Plant Dis. 79, 478. Black, L.L, and Wang, T.C., 2007. Chili anthrac nose research at AVRDC 1993-2002. In: Oh, Dae-Geun and Ki-Taek Kim (Eds.), Abstracts of the First Intern ational Symposium on Chili Anthracnose. National Horticultural Research Institute, Rural Development of Administration, Republic of Korea. Bosland, P.W., 1996. Capsicums: Innovative uses of an ancient crop. In: Janick, J. (Ed.), Progress in New Crops. ASHS Press, Arlington, VA, pp. 479-487. Brown, A.E., Sreenivasaprasad, S., Timmer, L.W., 1996. Molecular characterization of slowgrowing orange and key lim e anthracnose strains of Colletotrichum from citrus as C. acutatum Phytopathology 86, 523. Correll, J.C., Cornelius, K., Feng, C., Ware, S. B., Gabor, B., and Harp, T.L., 2007. Overview of the phylogenetics spec ies concept in Colletotrichum as it relates to chili anthracnose. In: Oh, Dae-Geun and Ki-Taek Kim (Eds.), Abstra cts of the First International Symposium on Chili Anthracnose. National Horticultural Research Institute, Rural Development of Administration, Republic of Korea. 81

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BIOGRAPHICAL SKETCH Tyler L. Harp, son of Jack D. Harp of Vero Beach, Florida, and Janice L. Pingel of Edgemont, Arkansas, was born in 1970 in Saint L ouis, Missouri. At 12 years old, Tyler moved from Saint Louis to Mountain Home, Arkansas, where he attended Jr. High and High School. He graduated from the University of Arkansas, Fayetteville, where he worked on staff as a Research Specialist from 1994 to 1998, with a bachelors degree in biology and a Master of Science degree in plant pathology. During his time in Fayetteville, Tyler met and married his wife, Cheryl L., and had three children, Caleb Machin, Jordan Paisley, and Canaan Cole. Shortly after obtaining his M.S. degree in 1998, Tyler became employed with Zeneca Agricultural Products in the E xperimental Biology Department as a Research Scientist in Richmond, California. In 2001, Tyler accepte d a relocation to work for Syngenta Crop Protection in the Biological Research and Deve lopment Department as R&D Scientist at the Vero Beach Research Center in Vero Beach, Flor ida, where he remained for over seven years. In 2004, Tyler enrolled as a Ph.D. graduate student at the University of Florida, Gainesville, in the Plant Pathology Department. He graduated with a Ph.D. in plant pathology in 2008 and continues his employment with Syngenta Crop Pr otection. Shortly after finishing his degree, Tyler accepted an Intern ational Assignment to work at the Syngenta headquarters in Basel, Switzerland. Tyler has maintained an active membership in the American Phytopathological Society (APS) since 1994, and continues to dedicate his time and interests to the study and application of plant pathology. 88