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Etiology of Botryosphaeria Stem Blight on Southern Highbush Blueberries in Florida and Quantification of Stem Blight Res...

Permanent Link: http://ufdc.ufl.edu/UFE0022845/00001

Material Information

Title: Etiology of Botryosphaeria Stem Blight on Southern Highbush Blueberries in Florida and Quantification of Stem Blight Resistance in Breeding Stock
Physical Description: 1 online resource (63 p.)
Language: english
Creator: Wright, Amanda
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: blight, blueberry, botryosphaeria, breeding, heritable, parva, resistance, rhodina, ribis, screen, stem, vaccinium
Plant Pathology -- Dissertations, Academic -- UF
Genre: Plant Pathology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The southern highbush blueberry (SHB) industry in Florida is an early-season high-dollar niche market increasing in acreage and market value. Stem blight caused by Botryosphaeria dothidea is a serious disease of SHB in Florida. In recent years, growers have reported increased economic losses due to stem blight and have reported differences in cultivar susceptibility. In 2007, 360 samples of stems and crowns with stem blight symptoms were collected from SHB in Florida. Botryosphaeria spp. were isolated from 85% of samples collected. Phylogenic analysis of internal transcribed spacer region showed at least three spp. occur on SHB in Florida: B. dothidea, B. rhodina, and an unresolved clade consisting of B. parva and B. ribis species. Environmental factors and genetic make-up were investigated as potential contributors to perceived differences in cultivar susceptibility. Progeny differed significantly by which parents were used to make the cross. Parents that produce stem blight resistant progeny were identified. A technique was devised to screen for stem blight resistant progeny. There was no correlation between percent lesion length in either replicated 05 or 07 trials. Lack of repeatability was due in part to a limited number of replicates, and experimental modifications. Botryosphaeria was recovered from control plants, indicating plants were infected with the fungus prior to inoculation. Disease-free material and more replicates need to be used for further experimentation.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Amanda Wright.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Harmon, Phillip.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022845:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022845/00001

Material Information

Title: Etiology of Botryosphaeria Stem Blight on Southern Highbush Blueberries in Florida and Quantification of Stem Blight Resistance in Breeding Stock
Physical Description: 1 online resource (63 p.)
Language: english
Creator: Wright, Amanda
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: blight, blueberry, botryosphaeria, breeding, heritable, parva, resistance, rhodina, ribis, screen, stem, vaccinium
Plant Pathology -- Dissertations, Academic -- UF
Genre: Plant Pathology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The southern highbush blueberry (SHB) industry in Florida is an early-season high-dollar niche market increasing in acreage and market value. Stem blight caused by Botryosphaeria dothidea is a serious disease of SHB in Florida. In recent years, growers have reported increased economic losses due to stem blight and have reported differences in cultivar susceptibility. In 2007, 360 samples of stems and crowns with stem blight symptoms were collected from SHB in Florida. Botryosphaeria spp. were isolated from 85% of samples collected. Phylogenic analysis of internal transcribed spacer region showed at least three spp. occur on SHB in Florida: B. dothidea, B. rhodina, and an unresolved clade consisting of B. parva and B. ribis species. Environmental factors and genetic make-up were investigated as potential contributors to perceived differences in cultivar susceptibility. Progeny differed significantly by which parents were used to make the cross. Parents that produce stem blight resistant progeny were identified. A technique was devised to screen for stem blight resistant progeny. There was no correlation between percent lesion length in either replicated 05 or 07 trials. Lack of repeatability was due in part to a limited number of replicates, and experimental modifications. Botryosphaeria was recovered from control plants, indicating plants were infected with the fungus prior to inoculation. Disease-free material and more replicates need to be used for further experimentation.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Amanda Wright.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Harmon, Phillip.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022845:00001


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1 ETIOLOGY OF BOTRYOSPHAERIA STEM BLIGHT ON SOUTHERN HIGHBUSH BLUEBERRIES IN FLORIDA AND QUANTIFIC ATION OF STEM BLIGHT RESISTANCE IN BREEDING STOCK By AMANDA FAITH WATSON A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

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2 2008 Amanda Faith Watson

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3 To my family and friends for all their gifts of roots and wings

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4 ACKNOWLEDGMENTS I thank Jon W right for his patience, love, kindness, humor, and strength throughout this process. I thank my parents for their guidance an d support. I thank my sister for her humor and encouragement. I thank my major advisor Dr Harmon and my committee members, for their instruction and patience. I thank Ms. Patricia Hill and Ms. Ca rrie Yankee for their willingness to help. I thank the Florida Bluebe rry Growers association for thei r funding and project support.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4 LIST OF TABLES ...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 ABSTRACT ...................................................................................................................... ...............9 CHAP TER 1 LITERATURE REVIEW .......................................................................................................11 Breeding Southern Highbush Blueberries in Florida ..............................................................11 Breeding for Botryospha eria Resistance ................................................................................12 Stem Blight of Blueberries .................................................................................................... .13 Botryosphaeria dothidea ........................................................................................................13 Host Range ..............................................................................................................................14 Disease Cycle ..........................................................................................................................14 Plant Health and Disease Transmission .................................................................................. 16 Management Options for Stem Blight .................................................................................... 17 Taxonomy of Botryosphaeria .................................................................................................18 Botryosphaeria Anam orphs ....................................................................................................19 Higher Classification of Botryosphaeria ................................................................................20 2 QUANTIFICATION AND IDENTIFICATION OF BO TRYOSPHAERIA SPP. CAUSING STEM BLIGHT ON SOUTHERN HIGHBUSH BLUEBERRIES IN FLORIDA ....................................................................................................................... ........24 Introduction .................................................................................................................. ...........24 Materials and Methods ...........................................................................................................25 Plant Material Collection .................................................................................................25 DNA Extraction, Amplification and Phylogenic analysis ............................................... 26 Pathogenicity ...................................................................................................................27 Results .....................................................................................................................................28 Field Survey, Fungal Isolation, a nd Molecular C haracterization .................................... 28 Phylogenetic Characterization .........................................................................................29 Pathogenicity ...................................................................................................................30 Discussion .................................................................................................................... ...........30

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6 3 SCREENING FOR AND QUANTIFICATION OF STEM B LIGHT RESISTANCE IN SOUTHERN HIGHBUSH BLUE BERRY BREEDING STOCK ......................................... 42 Introduction .................................................................................................................. ...........42 Methods ..................................................................................................................................43 Field Evaluation .............................................................................................................. .43 Clone Replicates and Inoculation .................................................................................... 44 Results .....................................................................................................................................45 Heritability Study ............................................................................................................ 45 Trials 1&2 (07 Clones) ....................................................................................................45 Trial 3&4 (05 Clones) .....................................................................................................45 Discussion .................................................................................................................... ...........45 LIST OF REFERENCES ...............................................................................................................54 BIOGRAPHICAL SKETCH .........................................................................................................63

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7 LIST OF TABLES Table page 1-1 Comparison of morphologi cal characteristics of B. dothidea, B. ribis, and B. parva .......22 2-1 Incidence of coloni e s consistent with Botryosphaeria growth habit ................................. 32 2-2 Preliminary species identification of isolates with Botryospha eria growth habit ............. 33 2-3 Representative Isolates from sample collections used in phylogenic analysis ..................34 2-4 Botryosphaeria sequences from GenBank used in phylogenic analysis ........................... 36

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8 LIST OF FIGURES Figure page 2-1 Botryosphaeria sym tpoms ................................................................................................. 37 2-2 Conidial morphology of Botryosphaeria spp.. .................................................................. 38 2-3 Asci of either Botryosph aeria parva or B. ribis ................................................................39 2-4 Single-gene ITS phylogeny ................................................................................................ 40 2-5 Audpc values for isolates used in pathogenicity study. .....................................................41 3-1 Mean progeny disease score of pare nts of the 2005 clone evaluation ...............................47 3-2 Mean progeny disease score of pare nts of the 2004 clone evaluation. ..............................48 3-3 Mean progeny disease score of pare nts of the 2003 clone evaluation.. .............................49 3-4 Trial 1 average percent lesion length of 07 clones ............................................................ 50 3-5 Trial 2 average percent le sion length of 07 clones ........................................................... 51 3-6 Trial 3 average percent le sion lengths of 05 clones. .......................................................... 52 3-7 Trial 4 average percent lesion length of 05 clones ............................................................ 53

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9 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ETIOLOGY OF BOTRYOSPHAERIA STEM BLIGHT ON SOUTHERN HIGHBUSH BLUEBERRIES IN FLORIDA AND QUANTIFIC ATION OF STEM BLIGHT RESISTANCE IN BREEDING STOCK By Amanda Faith Watson December 2008 Chair: Philip Harmon Major: Plant Pathology The southern highbush blueberry (SHB) industry in Florida is an ea rly-season high-dollar niche market increasing in acreage and ma rket value. Stem blight caused by Botryosphaeria dothidea is a serious disease of SHB in Florida In recent years, growers have reported increased economic losses due to stem blight and have report ed differences in cultivar susceptibility. In 2007, 360 samples of stems and crowns with st em blight symptoms were collected from SHB in Florida. Botryosphaeria spp. were isolated from 85% of samples collected. Phylogenic analysis of internal transcribed spacer region showed at least thr ee spp. occur on SHB in Florida: B. dothidea, B. rhodina, and an unresolved clade consisting of B. parva and B. ribis species. Environmental factors and geneti c make-up were investigated as potential cont ributors to perceived differences in cultivar susceptibilit y. Progeny differed significantly by which parents were used to make the cross. Parents that produce stem blight resistant pr ogeny were identified. A technique was devised to screen for stem blight re sistant progeny. There was no correlation between percent lesi on length in either replicated 05 or 07 trials. Lack of repeatability was due in part to a limited number of replicat es, and experimental modifications. Botryosphaeria was recovered from control plants, indi cating plants were infected with the

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10 fungus prior to inoculation. Disease-free material and more replicates need to be used for further experimentation.

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11 CHAPTER 1 LITERATURE REVIEW Breeding Southern Highbush Blueberries in Florida The genus Vaccinium (E ricaceae) contains three major crops: blueberry, cranberry, and lingonberry. About 400 species are cl assified in the genus. Species are native to all continents except Australia and Antarctica. Vaccinium is divided into five groups: Cyanococcus (blueberry), Myrtillus Oxycoccus (cranberry), Vaccinium, and Vitis-idaea (lingonberry) Cyanococcus, Myrtillus, and Oxycoccus, have a polyploidy series (2n=2x, 4x, 6x= 24, 48, 72) (60,111). Tretraploid highbush (V. corymbosum ) and hexaploid rabbiteye ( V. ashei ) blueberries have been bred at the University of Florida. Breeding programs have not merged because the tetraploid x hexaploid crosses produce pentaploids, which have reduced male fertility and have dark fruit color (57). Professor Ralph Sharp began the breeding progr am at the University of Florida (57). Superior northern highbush blue berry (NHB) cultivars from Mich igan and New Jersey provided initial breeding stock. These cultivars were poor ly adapted to Florida s subtropical climate; therefore, Florida native species were used to produce cultivar s with better adaptation (57,58). Florida native species successfully incorporated into breeding stock have included diploid and tretraploid V. corymbosum spp. from north central Florida, V. darrowi, V. elliottii, and most recently V. arboreum (57). The complex crosses have produced varieties with low chill hour requirements that ripen a month ahead of the earliest rabbiteye blueberries (115). Recurrent selection has been implemented to simultaneously change traits controlled by hundreds of genes (59). Recurrent selection is based on two principles. The first is heterozygous parents yield variable progeny. Th e second is that if progeny that are extreme in the expression

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12 of certain characteristics are crossed; the second generation pr ogeny will be variable, and some will be more extreme in the selected character than their parents (58). The University of Florida breeding program has two main goals: selection of breeding stocks and cultivar selection. A total of 100 seed lings are grown from each individual cross for a total of 15,000 seedlings per generation. From the 15,000 seedlings, 200 are selected as parents for the next generation. The process is continued generation after generation. Cultivar selection has four stages. Stage I consists of 15,000 seedlings planted in high density plots. After one year, stage I plants ar e rated for desirable frui t size, firmness, flavor, ripening time, and bush defects. The best 500 plants are selected, and evaluated in stage II. The rest of the plants are discarded. Stage II plants are rated for th ree years; the best 150-200 plants are numbered, and approximately 40 softwood cuttings are rooted from each plant. These best clones are planted in 15-plant plots using commer cial spacing. The clones are rated over three years for survival, and bush and berry qualit y. The superior 12 to 15 stage III clones are vegetatively propagated and planted on multiple farm s. The stage IV plants are evaluated for three to six years by the breeder and growers. On average, between one and two clones are selected for cultivar release each year ( Lyrene personal communication ). Breeding for Botryosph aeria Resistance Stem blight on SHB is caused by Botryosphaeria dothidea Fungicide utilization for control of Botryosphaeria disease is inconsistent ( 11,15,24,47,50,83). Irrigation management and pruning practices have given little success combating the disease (71,73,81,87). Resistant cultivars produced though breeding e fforts offers growers cost effici ent control options with little or no added inputs. Various levels of susceptibility to Botryosphaeria diseases have been noted in Vaccinium spp. dogwood, mango, and peach (25,32,36,75,86,90). Differences in cultivar susceptibility have

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13 been attributed to cultivar genetics, plant stre ss, age of tissue used for inoculation, wound age, and inoculum virulence (30,32,75,86,113). Disease i ndexes, the use of fresh highly virulent isolates, and succulent stem tissue for inoculat ion have been reported to help standardize resistance screening methods (9,30,99). Buckley (1990) concluded narrow since heri tability was greater than broad since heritability for stem blight resistance (25). Both additive and non-additive genetic effects are involved in resistance which is conferred from the low bush blueberry ( V. angustifolium ) in populations from Michigan, New Jersey, and Nort h Carolina (25). However, Gupton and Smith (1989) concluded there was a larg e nonadditive genetic variance, and SCA and GCA were equal, suggesting that only moderate progress could be ma de in stem blight re sistance breeding (44). Stem Blight of Blueberries Blueberry stem blight is caused by Botryosphaeria dothidea. In the early stages of infection, leaves on affected branches appear yell ow or reddish. Leaves turn brown and remain attached on stems girdled by B. dothidea Pecan-brown discolored st em tissue typically occurs on one side of an affected bran ch. Discolored vascular tissue ex tends from a few inches to the length of the branch (116). Botryosphaeria dothidea Botryosphaeria dothidea is a f ilamentous ascostromatic ascomycete. Its pycnidial anamorph is Fussicocum asculi (3). The teleomorph is associated with stem blight; however it is infrequently encountered in nature. The an amorph frequently found on infected tissue is predominantly used for identification (97,105). Botryosphaeria dothidea can live as a saprophyte or endophyte, and is an opportunistic pathogen of wounded and stressed hosts (76,97,106)

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14 Colony characteristics are olive gr ay to violet black in color, with thick to wispy aerial mycelium that darkens with age. Margins are smoo th, becoming crenulate with age. Conidia can be produced on media, and are si milar to those produced in natu re. Optimum temperature for growth is 25-28C, and the growth range is 4 to 30C (31,96). Host Range Botryosphaeria spp. have a broad host range infecting m any woody fruits, trees, and herbaceous plants (3). Twenty plant families, 34 genera, and 50 plant species are known to be susceptible to B. dothidea Rosaceae, Juglandaceae, and Palmaceae are the most well known (100). Botryosphaeria dothidea also infects other economically important crops including apple, blueberries, eucalyptus, grapes, mangos, peach, and pistachios (22,69,80,90,98,110,116). Disease Cycle The diseas e cycle of B. dothidea is one of opportunism. Typically, B. dothidea persists as a soil saprophyte or as an endophyte (76,97,106). A latent infection period begins with host tissue colonization (117). For pistachios, latent infec tion periods are most frequent during the month with the most rainfall (76). Apple white ro t infection occurs after petal fall and symptoms do not appear until 6-8 w eeks before harvest (50) Drought stress and wounding predispose a plant to infection by Botryosphaeria spp. (23,34,65,87). Botryosphaeria dothidea can enter host tissue through lenticels, stomata, or small openings in the bark (23,69,72,88,91,113). Resistance to B. dothidea is related to fungal development after infection rather than establishm ent (72). In apples and blueberries infection develops through open stomata or lenticels, the host cell layer beneath the epidermis undergoes cell division. The thickened periderm layer restricts B. dothidea to the outer portion of the lesion. After six weeks, small reddish brown le sions appear. The fungus does not move through the vascular tissue (23,72).

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15 Invasion of wounded or succulent stems re sults in rapid breakdown of phloem and cortical tissues for almond, apple, blue berry, mango, melaleuca, and peach (17,23,43,72,90,91). After invasion, mycelium moves ra pidly down the vascular tissue. Lateral movement occurs slowly through pits and intercellular spaces (23,43,72,91). Hyphae advance by colonizing all cell types, including callus pare nchyma, cortical parenchyma, xylem ray parenchyma, trachieds, and vessels (17,91). Plant mortality results from partial or complete occlusions of the vascular tissue by tyloses and mycelium (23,72,90,91). Callus and lignified cells containing tannins do not restrict host colonization (17,91). Partially submerged pycnidial stro mata develop on stems colonized by B. dothidea, and are important sources of inocula for pistachios (69). Pycnidia mature after 12 days at temperatures ranging from 10-36C for apple an d pistachio (23, 69). Peak pyc nidial production occurs at 30C (23,69). The epidemiology of B. dothidea has been researched for th e following diseases: apple white rot, fungal gummosis of peach, as well as panicle and shoot blight of pistachio (8,17,19,23,31,61,69,70,73,76,81,88,113). Conidial production ar ises between 10-30C four to six weeks after inoculation. Peak sporulation develops at 24C (31,69). For apples, peaches, and pistachios, spore germination occurs four to six hours after inocul ation at temperatures ranging from 25-35C (23,70,113). In a pple, conidial germ tubes c onsistently grow toward the wounded area of the stem suggesting a chemotatic re sponse (23). Conidial germination declines with decreasing relative humidity for apple white rot infections. Germination is favored between 98-100% relative humidity. Less than 5% of coni dia germinate at 95% relative humidity (105). Twelve hours of moisture is necessary for penetra tion of lenticels, stomata, fruit, and wounds of pistachios (69,73). Interrupted we tness periods of one hour or mo re irreversibly stop infection

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16 for apple black rot and significantly reduce disease incidence for B. obtusa (8). For apple white rot infection air drying of twenty minutes significantly reduces conidial viability (105). Inoculum can be found throughout a growing se ason for blueberry, peach, and pistachio (33,69,73,76,88,113). Pycnidia produced on pistachios during current or prior growing seasons provide inoculum for new infections throughout the year (69). Coni dia have been detected from February to November in blueberries, peach, and pistachio orchards (33,69,113). The highest levels of conidial inoculum have been record ed between May to July for blueberry in North Carolina and from July to mid-August for peac h in Georgia (33,88,113). Rainfall is required for spore dispersal. Light rain is more conducive for spore deposition and infection than heavy rain for dissemination in blueberry and peach orch ards (33,113). The for pistachios number of continuous rainy days and increa sed summer temperatures are posit ively correlated with disease severity (73,76,88). Plant Health and Disease Transmission Plants are predisposed to disease when stre ssed. Drought lim its photosynthetic production and the accumulation of carbohydrat es aid the plant in disease defense (21,52,79). Pathogens responsible for stress-related dis eases are usually facultative sa prophytes, are latently present on host tissue, and attack when the hos t weakens (21,61,79). Susceptibility to B. dothidea increases as plant water potential ( ) decreases (34,61,87,95). Birch trees ha ve a threshold between -12 to -13Mpa predisposing them to infection. Disease resistance to B. dothidea can be restored within 3-5 days after turgor pressure restoration. Suscep tibility to disease is reversible between 14 to 18 MPa; greater than -18MPa birch trees are irreversibly predis posed to disease (34,95). Protein synthesis, enzyme synthesis, and carbohydrate production decrease in droughtstressed plants (21). Hyphal gr owth inside healthy birch stem s is irregular and contorted compared to large round hyphal growth inside stre ssed stems. Lytic activity on invading fungal

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17 hyphae is suppressed in stressed plants (68). When aspens ar e drought stressed, catechol and salicin, compounds inhibitory to Hypoxlon mammatum, are suppressed (52). Stored carbohydrates are utilized due to limited photos ynthetic production, and callus formation is limited during fungal invasion (79). Higher plants cannot grow at below 0 Mpa, whereas many fungal pathogens can grow at below zero (1,21,40,46,65,78). Spore germination, germ tube elongation, and mycelia growth of B. dothidea increases from 0 to -2.0 MPa (65). Mycelia growth dec lines after -2.0MPa (40,65). Mycelial growth increases as water pot entials decrease for other fungi including Botrytis squamosa, Monilinai fructicola, and Macrophomina phaseolina (1,46,78). Management Options for Stem Blight Cultural and m anagement options for control of Botryosphaeria disease are similar in many cropping systems including apple, blueberr ies, grape, and peach. Fungicides have provided growers with short term crop protectio n and have limited disease incidence (11,45). Benomyl and strobilurin and DMI fungici des reduced external symptoms of Botryosphaeria blight; however, the infection wa s not prevented in apple,blue berry, cut flower, grape, and pistachio cropping systems ( 15, 24,29, 37,45,81). Root dip treatments for container-grown blueberry nursery plants limits B. dothidea development but does not provide long term control (29). Treatments of captan and difolatan impr oved peach tree fruit yield, and trunk diameter; however, infection was not prevented (11). An al ternative control to trad itional fungicides could be paclobutrazol (PBZ), a gibbe rellin inhibitor. PBZ reduced mycelial growth and spore germination for a broad range of woody pathogens including: Armillaria gallica, Botryosphaeria dothidea, and Fusarium roseum (47). PBZ enhanced tolerance to environmental stresses and has reduced foliar dis eases including dollar spot (28, 47).

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18 Fungicides have been more effective controls for Botryosphaeria fruit rots. Partial pressure infiltration of prochloraz and pyrimethanil co ntrolled mango stem end rot (83). Fungicidal applications approximately 10wks after bud break have reduced fungal foliar and fruit diseases of cranberry (49). Late season applications of tebuconazole reduced latent apple white rot infections (50). Fungicidal resistance has occurred in Alternaria alternaria, Monilinia fruticola, Sclerotinia homeocarpa, and Venturia inaequalis (17,41,102,121). The sensitivity of B. dothidea to tebuconazole and iprodione was evaluated ( 64,66). Resistant isolates were produced in vitro and retained high levels of virulence on pistachios. Tebuconazole retained efficacy while iprodione could not control mycelia growth of resistant is olates (64,66). Integrated pest management (IPM) program s including orchard sani tation and irrigation management have effectively reduced disease in cidence. Altering the trajectory angle of sprinklers from 23 to 12 and drip irrigation have reduced spore rel ease, dispersal, and germination in pistachio orchards (70,71,73). Reducing irrigation time from 24 to 12 hours also reduced the incidence of pani cle and shoot blight, and 24 hour irrigation periods are not recommended for apple due to increased disease incidence (70,81). Removal of blighted shoots from pistachio orchards removed sources of i noculum for current and prior seasons (45,73). Stems infected with Botryosphaeria are pruned out during peach dormancy and chipped to increase decomposition (11,22). Taxonomy of Botryosp haeria The genus Botryosphaeria was described by Cesati and De Notaris in 1863. Botryosphaeria originally included twelve species lack ing a complete morphological description or a type species (39, 35, 96,101). Barr (1972) designated B. dothidea as the lectotype species for the genus because it was originally included in the initial description (96).

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19 Botryosphaeria has high morphological plasticity and de spite obvious similarities researchers described new species occurring on different hosts (82,96,97). Von Arx and Muller (1954) synonomized many Botryosphaeria species into either B. quercuum or B. dothidea species complexes. Differences in anamorph morphology prohibited re searchers from accepting the synonymization of B. ribis with B. dothidea (48,89,90,91,101,120). Others accepted the grouping according to the Inte rnational Rules of Nomenclature (116, 82). Smith and Stanoz indicated para phyly within the species complex B. dothidea ; B. ribis was phylogenticially separated from B. dothidea (101). Cluster analysis and conidial morphology reinforced the separation of B. ribis from B. dothidea and B. parva (48, 101). Based on multiallelic data sets, Slippers (97) va lidated previous studies (48,101,120). B. ribis was no longer considered a synonym for B. dothidea (96). The study allowed for accurate identification of Botryosphaeria spp. associated diseases on commercia l crops including grapes, mango, pome and stone fruits (97,98,109,110). Botryosphaeria Anamorphs Eighteen anam orph genera have been associated with Botryosphaeria including Diplodia, Dothiorella, Fusicoccum, Lasiodiplodia, Phylosticta, and Sphaeropsis (39,48,101). Denaman combined the anamorphs of Botryosphaeria into two main lineages: Diplodia, pigmented conidia, and Fusicoccum hyaline conidia (39). Zhou and St anoz supported Denmans findings and proposed the conidial groups Hyala and Brunnea (120). The conidial groupings of Diplodia and Fusicoccum were disputed. Zhou and Stanoz noted that B. dothidea and B. corticis were less closely related to other Fusicoccum spp compared with Diplodia taxa (119). Crous refute d the two anamorph lineages of Botryosphaeria and noted many intermediate conidial characters between Diplodia and Fusicoccum (35). Using single gene phylogeny, ten anamorph lineages were recognized within Botryosphaeriaceae

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20 including an unresolved clade: (Diplodia/Lasiodiplodia/Tiar osporella), Botryosphaeria (Fusicoccum anamorphs), Macrophomina, Neoscyt alidium, Dothidotthia (Dothiorella anamorphs), Neofusicoccum ( Botryosphaeria -like teleomorphs, Dichomera-like synanamorphs), Pseudofusicoccum ( Fusicoccum and Diplodia -like synanamorphs), B. quercuum ( Diplodia-like anamorph), and Guignardia ( Phyllosticta anamorphs) (35). Higher Classification of Botryosph aeria Luttrell granted formal taxonomic status to the subclass Locoascomycetes defined by a bitunicate ascus-wall and pseudothecia (3,54,55,94). All other filamentous ascomycetes were segregated to the Euascomycetes (13,56). Se paration from the unitunitcate ascomycetes was widely accepted. The placement and number of orders within the groups was disputed. Lutrell placed Botryosphaeria in the Pleosporales (56). vonArx and Mller did not support the placement of Guignardia and Botrosphaeria, two closely related ge nera into separate orders (Dothideales and Pleos porales). Instead one order, the Dothideales, was delimited containing two sub-orders and 24 families (3,35,39,51). Botrosphaeria remained in Botryosphaeriaceae and was relocated to the Doth ideales (35,39). Barr agreeing with Luttrel, disagreed with the consolidation, created ten or ders based on dicaryon and ascus type (10). By the end of the 1980s two systems of classification existed that of Barr & Luttrel, and vonArx & Mller. Berbee and Spatafora rejected the monophyly of Loculoascomycota, and questioned class validity (13,55,103). Studies retained sister group status of the Dothideiales and Pleosporales, while the Chaetothyriales formed a sister group with Eurotiomycetes. The Loculoascomycetes were split into two classes, the Chaetothyr iomycete (lichenized pyrenomycete) and the Dothideomycetes (51,54,55).

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21 Disagreement concerning subdivision within the Dothideomycetes was unresolved. The weight of taxonomic characters was heavily di sputed and included: centrum development, pseudothecia, and pseudoparaphyses characte ristics (10,56,94). Two Dothideomycete lineages predominate: the pseudoparaphyste Pleospor omycetidae (Pleosorales ) and aparaphysate Dothideomycetidae (Dothideales, Capnodi ales, and Myriangiales) (94). Botryosphaeriaceae did not group phylogentically within any of the previously described orders. Higher taxonomic classification has been enigmatic because of the intermediate morphology: pseudoparaphyses are present in imma ture and absent in mature fruiting bodies (3,10,51,54,94). A new order, Botryosphaeriale s was created to accommodate phylogenic separation (94).

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22Table 1-1. Comparison of mor phological characteristics of B. dothidea, B. ribis, and B. parva (96,82). B. dothidea B. ribis B. parva Ascostroma Position Erumpent through bark Erumpent through bark Erumpent through bark Size 200-500 m 100-400 m Unknown Ascomata Pseudothecia Ps eudothecia Pseuodtheica Color Brown to black Brown to black Brown to black Shape Botryose aggregate of up to 100, sometimes solitary or globose Botryose aggregate of 5-50, globose Caespitose aggregate 5-50 (100) per cluster Size n/a 175-250 m 150-250 m Opening Central ostiole, to emergent Central ostiole, papillate or not Non-papillate or short conical papilla Asci Description 8-spored, bitunicate, clavate 8-spored, bitunicate, clavate 8-spored, bitunicate Shape Filiform Filiform Ellipsoide to fusoid Size 63-125 x 16-20 m 80-120 x 17-20 m 75-143 (-210) x 17-21 m Paraphyses Peudoparaphyses 2-4 m wide Peudoparaphyses 2-4 m wide N/A Ascospores Description Unicellular, biseriate in ascusU nicellular, biserate in ascus Unicellular Color Hyaline, smooth with granular contents Hyaline, smooth with granular contents Hyaline, smooth Shape Fusoid to ovoid Fusoid to elli psoide Broadly ellipsoide to fusoid Size (17-) 19-24 (-32) x (6-) 7-8 (10) m (14-) 18-23 (-27) x 6-8 (-10) m (14-) 18-23 (-26) x (7-) 8-10 (11) m

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23Table 1-1 Continued. B. dothidea B. ribis B. parva Anamorph General Indistinguishable from pseudothecia Indistinguishable from pseudothecia Indistinguishable from pseudothecia Pycnidia N/A Solitary or imbedded Locule 100-150 m Condiogenous cells Color Hyaline Hyaline Hyaline Size 6-20 x 4-5 m 6-22 x 2-5 m N/A Shape Holoblastic, subcylindrical Holoblastic, subcylindrical N/A Conidia Color Hyaline, smooth with granular contents, rarely becoming septate with age Hyaline, smooth with granular contents, rarely becoming septate with age Hyaline, becoming light brown and 1-2 septate with age Size (17-) 18-20 (-22) x 4-5 m (16-) 19-23 (-24) x 5-6 (-7) m (11-) 14-18 (-23) x 5-7 (-10) m Shape Narrowly or irregularly fusiform Fusiform Ellipsoid

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24 CHAPTER 2 QUANTIFICATION AND IDENTIFICATION OF BOTRYOSPHAERIA SPP. C AUSING STEM BLIGHT ON SOUTHERN HIGHBUSH BLUEBERRIES IN FLORIDA Introduction The Florida southern highbush blueberry (S HB) industry is an ear ly-season high-dollar niche m arket increasing in acreag e and market value (114). Commercial production has more than doubled since the early 1980s ; currently Florida ranks 5th in the United States for commercial acreage (104,115). Fungal vascular di seases have become a growing problem for commercial blueberry growers. These pathogens will enter through flower buds, lenticels, stomata, and wounds and colonize the xyl em and phloem (23,69,72,88,91,114). Infected bushes are then weakened and exhibit dieback on stems a nd branches. Severe infection results in bush mortality by partial or complete occlusion of va scular tissue in the crow n. Symptoms include dead branches with attached leaves, and pecan brown discoloration extending the length of the affected branch (72,116). Vascular tissue is mottl ed in the crown of plants killed by dieback. Stem blight caused by Botryosphaeria dothidea (Moug.:Fr.) Ces & DeNot. is associated with these described symptoms (Fig 2-1) (116). Botryosphaeria spp. have a wide host range and geogr aphical distributio n (100). These fungi are largely considered drought-stress opportunistic pathogens living as saprophytes or endophytes most of the time (39,73,76,97,106). Since the genus was founded in 1863 (Moug.:Fr.) Ces & DeNot., different Botryosphaeria species have been identified causing cankers and blights on woody hosts (18,27,43,45,53,82,90,100,109,116). Species identification has been difficult because multiple species have been found parasitizing the same host (17,19,20,110,109). Virulence of and symptoms caused by Botryosphaeria spp. have been reported to be different depending on cultivars and location (97, 109).

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25 The teleomorph, Botryosphaeria is infrequently associated with disease symptoms (96,116). The anamorphs of Botryosphaeria occur frequently on infected tissue, and are primarily used for identification (48, 111,118). Anamorphic characteristics are continuous between species and have high phenotypic plas ticity (48, 96). Often the connection between sexual and asexual states has not been made (118). Eighteen anamorph genera have been associated with Botryosphaeria Currently, ten lineages are recognized within the Botryosphaeriaceae (35,96). Phylogenic studies using morphological characterization and genomic data have contributed to the clarification of Botryosphaeria taxonomy (35,48,96,101,120,121). Data have allowed for the positive and rapid identification of Botryosphaeria spp. parasitizing apples, grapes, mango, and pistachio (6,7,62,63,77,96,98,109,110). Witcher and Clayton described B. dothidea as the causal agent of stem blight of blueberries (116). They noted the morphology strongly resembled B. ribis, which was annotated for B. dothidea under von Arx and Muller (116). Based on multi-gene phylogeny, B. ribis is considered a separate species from B. dothidea (96). To date, B. dothidea has been most commonly associated with stem blight and dieb ack infections; however, other fungi such as Diplodia spp., Macrophoma spp, and Phomopsis spp. have been found causing similar symptoms (2). The objective of the study was to determine the incidence of B. dothidea causing stem blight and dieback infections on SHB in Florida. Materials and Methods Plant Material Collection Infected crowns and stem s were sampled fr om two farms in Florida: one located in Alachua Co. and the second located 225.3km south in Polk Co. A farm-wide survey of disease was taken at each location at four-month interv als (Jan-Feb, Jun-Jul, and Oct-Nov.) in 2007;

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26 thirty samples of each symptom were collected from both farms during a survey period. Other samples outside the survey included isolates from Vaccinium ashei, SHB samples from the Florida Extension Plant Disease Clinic, Ilex spp, and four isolates from a foliar ring spot symptom caused by Botryosphaeria on the SHB cultivars Millenni a, and Star (Table 3). Excess bark of blueberry samples was removed to expose discolored vasc ular tissue; margins were excised and cut into small pieces. Sa mple pieces were surface disinfected in 10% household bleach for one minute and washed with ta p water. Blueberry samples were dried with a paper towel and plated on 85-mm petri dishes containing V8 agar (BD, Sparks MD) amended with 0.01mg of rifampicin (rif) and 0.25g of am picillin sodium salt (amp). Cultures were incubated at 25C for five days. Botryosphaeria isolates were obtained by transferring mycelia fragments from the margins of growing colonies. Infected crowns and stems not plated were left in sample bags at room temperature for two weeks. After incubation all samples were checked for the presence of sporulating structures. The number of samples with spor ulating structures per farm and sample period was counted. Sexual fruiting bodies were single-spored using seri al dilutions plated onto potato dextrose agar (PDA) (BD, Sparks MD). Mycelia fragments from the margin of single colonies were excised. Isolates obtained are currently maintained in co llection in the Department of Plant Pathology at the University of Florida. DNA Extraction, Amplificatio n and Phylogenic analysis Genom ic DNA from select co lonies consistent with Botryosphaeria growth was extracted from pure cultures using Qiagen Dneasy Kit (Qiagen 69106 Gmbh, Germany). After extraction oligonucleotide primers ITS1 and ITS4 (Integrat ed DNA Technologies, Inc Coralville, IA) were used to amplify part of the in ternal transcribed spacer region including the 5.8S region of rDNA. Polymerase chain reaction (PCR) was completed by combining 10 L of REDExtract-N-Amp

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27 PCRmix (Sigma, Saint Louis MI), 2 L of each primer, 2 L PCR grade water, and 4 L of purified fungal DNA. The reaction was carried out in a thermal cycler (Brinkman Instruments Inc., Westbury NY) as follows: denaturization 3 min at 94oC followed by 35 cycles of denaturization at 94 oC for 60s, annealing at 55 oC for 60s, and extension at 72 oC for 2min. Five L of each PCR product were separated by ge l electrophoresis in 1.2% agarose gels (FisherScientific, Fair Lawn NJ) containing 1 L of ethidium bromide in a 1.0x tris-borate buffer (Sigma, St. Louis MI). Five L of PCR products were placed on half of a 96 well PCR plate and were sent to University of Fl oridas Interdisciplinary Center for Biotechnology Research (ICBR) for bidirectional sequencing. Sequences were edited using the softwa re program Sequencher 4.6 (Gene Codes Corp. Ann Arbor MI), locally aligne d using ClustalX 2.06-macosx and manually aligned using the computer software McClade 4.08 OSX. Phylogenetic analysis was completed using PAUP 4.0bl0 (PPC). Alignment gaps were treated as missing data. Representa tive isolates from the survey (Table 2-3) were compared using phyloge nic analysis to relate d sequences published in GenBank (Table 2-4). The Pestalotia isolate from the Ilex spp. was used as the outgroup for phylogenic analysis. Maximum parsimony analysis was performed using the heuristic search option (TBR branch swapping). Bootstrap values were evaluated using 1,000 replicates and 100 random sequence additions, saving no more than 10 trees greater than 264 to test branch strength. Tree length, consiste ncy index (CI), and re tention index (RI) were recorded for all analyses. Pathogenicity Eight clones of the cu ltivar Misty were a rranged in a random ized complete block (RCB) design in greenhouse inoculation trials. Pl ants were pruned be fore inoculation. Botryosphaeria isolates were grown for three days on V8 agar amended with rif and amp. Four isolates were

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28 chosen from each of the three clades from the ITS phylogeny: B. dothidea, possible B. parva or B. ribis species and B. rhodina. MixFC-6 ITS sequence was similar to B. rhodina isolates in Genbank. Likewise 07-30 was similar to B. dothidea ; while WsuF-29 and WWF-47 were similar to either B. parva or B. ribis The positive control, isolate 04-40 was similar to either B. parva or B. ribis. A sterile agar plug of V8 amended with rif and amp was the negative control. Eight millimeter plugs were excised from the colony margin of each species, and placed on a pruned stem. Lesion lengths were measured in centime ters once every week for three weeks. Audpc values were calculated from lesions lengths. Da ta was analyzed in SAS (SAS Institute, Cary, N.C.) using a general linear model. Waller Duncan k-ratio t -test (k =100) was used to separate mean lesion length differences between isolates. Experiment was repeated thrice. Results Field Survey, Fungal Isolation, and Molecular Characterization Colonies consistent with Botryosphaeria growth were isolated from, 99 out of 120 samples in the winter, 92 out of the 120 samples in th e summer, and 115 out of 120 samples in the fall (Table 2-1). Incidence of Botryosphaeria spp. did not vary significantly between sample periods and locations. Overall, colonies consistent with Botryosphaeria growth were isolated from 85% of the 360 samples. Other fungal genera isolated from blueberry samples were Alternaria spp. Pestalotia spp., and Phomopsis spp. Identification of isolates from colonies consistent with Botryosphaeria growth was based on ITS sequence data (Table 2-2). The total number of isolates sequenced from the winter, summer, and fall collection periods were 78, 63, a nd 78, respectively. Fungi isolated included: B. parva or B. ribis B. rhodina, and other fungi such as Alternaria and Phomopsis Botryosphaeria spp. were interspersed between crown and flag samples (Table 2-2, 2-3). However, B. rhodina was recovered at a larger pe rcentage from crown samples. B. dothidea was

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29 isolated twice from samples outside the survey area. Isol ation frequency of Botryosphaeria spp. remained consistent and did not va ry between sample periods. One sample was found to have pycnidial frui ting bodies during the wi nter and one during the summer collection periods. The greatest number s of fruiting structures were observed in the fall. Eleven samples had pycnidia, and two sa mples had perithecia on the farm in Alachua Co., Fl. Eighteen samples had pycnidia and two sample s had perithecia on the farm in Polk Co, Fl. All samples found having pe rithecia, were either B. parva or B. ribis species (Fig 2-3). Winter and summer pycnidial fruiting bodies were B. rhodina. Pycnidial of B. rhodina were recovered from ten samples from Alachua Co., and from seve nteen of the samples from Polk Co. Pycnidia of either B. parva or B. ribis were recovered once from each location in the fall. Phylogenetic Characterization ITS sequences of Floridian Botryosphaeria isolates were compared with homologous ITS sequences published in GenBank. Of the 533 nucleotides analyzed 101 characters were parsimony informative. Maximum parsimony anal ysis yielded one tree (length = 264, CI= .905 RI= .985). Botryosphaeria specie s having hyaline thin-walled conidia grouped within a clade; supported by a 94% bootstrap value (Fig 2-4). Species included B. dothidea, B. corticis, B. parva, and B. ribis and have Fusicoccum or Fusicoccumlike anamorphs. Intraspecific variation was present in the B. dothedia clade. B. parva and B. ribis isolates could not be resolved and grouped in a single clade with high intraspecific variation. B. rhodina isolates, Diplodia anamorphs, formed a sister clade to the Fusiccocium isolates, no intraspecific variation was present within the clade. The th ree clades were strongly supporte d with bootstrap values of 97, 100, and 100 percent.

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30 Pathogenicity Audpc values were si gnificantly different ( p < 0.001) between Botryosphaeria spp (Figure 2-5). B. dothidea AUDPC values were significantly lower than the other Botryosphaeria spp. The positive control, isolate 0440, had a significantly higher AUDPC value than B. parva or B. ribis isolate WsuF-29, and B. rhodina isolate MixFC-6. Discussion This study constitu tes the first attempt to assess the presence and diversity of fungal species causing stem blight and dieback infections in Florida. Based on partial sequence analysis of the ITS region, at least three Botryosphaeria species were isolated from crowns and branches of SHB from Alachua and Polk Co., Fl. B. dothidea B. parva, and B. ribis were previously recognized as pathogens of SHB in Florida (2). The association of B. rhodina with SHB in Florida has not been reported. Stem blight and dieback of Flor ida SHB has been attributed to B. dothidea and occasionally to other fungi such as Diplodia spp Macrophoma spp, and Phomopsis spp (2). However, B. parva, B. ribis and B. rhodina were recovered from stem blight and canker infections more often than B. dothidea or any other fungal genus (T able 2-1, 2-2), indicating the former species may be a more important cause of SHB mortality than previously recognized. Difficulties distinguishing Botryosphaeria species are common because the group of fungal organisms has many taxonomic and nomenclatural ambiguities (39, 96). Teleomorphs of Botryosphaeria are infrequently encountered in nature, and are difficult to produce in vitro (8, 116) Species identification has been based on an amorph characteristics such as colony and conidial morphology (39,48,96,101,109). Differentiati on based on conidial characteristics is difficult because characters vary with age and type of media (48) (Fig 2-2). Botryosphaeria spp. have overlapping host ranges, and consequently multiple species can parasitize the same host

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31 (17,22,96,110,111). Results found herein support previous studies (39, 96,110,111), sexual states were infrequently recovere d from sample material, and multiple Botryosphaeria species were found on SHB in Florida. DNA sequence comparisons accurately identified Botryosphaeria spp. recovered from Florida SHB. Results of ITS phylogenetic an alysis supports previous work classifying Botryosphaeria anamorphs into two groups: Diplodia and Fusicoccum (48, 39,109,120) (Fig 22). Current phylogenetic research suppor ts multiple conidial lineages within Botryosphaeriaceae (35,98). However, species found on Florida SHB separated into two distinct groups. The phylogeny is not a complete sampling of the family; however, the di fferentiation between Diplodia and Fusiccocium conidia is important as a di agnostic tool a llowing species differentiation. No intraspecific variation was observed within the B. rhodina clade, indicating a uniform population, possibly due to limited sexual recombinat ion. Intraspecific vari ation was present in the B. dothidea and the unresolved B. parva/B. ribis clades. The presence of isolates from different hosts and geographic locations could expl ain the variation. However, variation between Florida B. parva/ B. ribis isolates could either be due to se xual recombination, or that species could not be distinguished based solely on the ITS sequence data. Previous studies using single gene phylogenies, RFLP and RAPD makers ha ve been unable to separate the species (6,7,98,101). The EF1region has been show to distinguish the two sp ecies (96,111). B. parva and B. ribis are difficult to differentiate molecularly and morphologically; pathogenicity is very similar (96,111). Currently, fu rther molecular, morphological, and pathogenicity studies designed to help elucidate the B. parva/B. ribis clade are now underway.

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32 Table 2-1. Incidence of co lonies consistent with Botryosphaeria growth habit Survey Period No. Percent Winter Alachua Co. Flag 25 83% Crown 27 90% Subtotal 52 87% Polk Co. Flag 18 60% Crown 29 97% Subtotal 47 78% Winter Total 99 83% Summer Alachua Co. Flag 24 80% Crown 23 77% Subtotal 47 78% Polk Co Flag 24 80% Crown 21 70% Subtotal 45 75% Summer Total 92 76% Fall Alachua Co. Flag 30 100% Crown 30 100% Subtotal 60 100% Polk Co. Flag 26 87% Crown 29 99% Subtotal 55 92% Fall Total 115 96%

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33 Table 2-2. Preliminary species identifi cation of isolates consistent with Botryosphaeria growth habit; no samples outside the survey area were included. Preliminary identification was determined by comparing ITS region with isolates published in GenBank. Survey B. parva-ribis B. rhodina Other No. PercentNo. PercentNo.Percent Winter Alachua, Co Flag 14 82% 3 18% 0 Crown 13 65% 7 35% 0 Subtotal 27 73% 10 27% 0 Polk Co Flag 17 85% 2 10% 1 5% Crown 12 57% 9 43% 0 Subtotal 29 71% 11 27% 1 2% Winter Total 56 72% 21 27% 1 1% Summer Alachua, Co Flag 18 95% 1 5% 0 Crown 9 60% 4 27% 2 13% Subtotal 27 79% 5 15% 2 6% Polk Co Flag 11 69% 3 19% 2 12% Crown 11 85% 2 25% 0 Subtotal 22 76% 5 17% 2 7% Summer Total 49 78% 10 16% 4 6% Fall Alachua, Co Flag 20 87% 3 13% 0 Crown 14 78% 4 22% 0 Subtotal 34 83% 7 27% 0 Polk Co Flag 14 70% 4 20% 2 10% Crown 11 65% 6 35% 0 Subtotal 25 68% 10 27% 2 5% Fall Total 59 76% 17 22% 2 2%

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34Table 2-3. Representative Isolates from sample collections used in phylogenic analysis Species Origin Date Collected Host Abbreviation. B. parva or B. ribis Archer Aug-04 SHB A0440 B. rhodina Apopka May-05 Illex cassine A05161 B. parva or B. ribis Alachua Jun-07 SHB 3010B B. rhodina Hawthorne Jun-06 SHB A0636 B. dothidea Wildwood Feb-07 SHB A0730 B. dothidea Archer Aug-07 SHB ArcherRingSpotM B. parva or B. ribis Archer Aug-07 SHB ArcherStarRingSpot B. parva or B. ribis Windsor Aug-07 SHB BBC2 B. rhodina Floral City May-07 SHB FerrisFarm B. parva or B. ribis Waycross, GA May-07 SHB GAC1 B. parva or B. ribis Waycross, GA May-07 SHB GAC3 Pestalotia Gainesville Jul-07 Ilex spp. Holley1 B. parva or B. ribis Hawthorne May-07 Vaccinium ashei rbe2 B. rhodina Windsor Dec-06 SHB WDSP2 B. parva or B. ribis Windsor Aug-07 SHB WindsorRingSpot-1 B. parva or B. ribis Windsor Aug-07 SHB WindsorRingSpot-2 B. rhodina Polk Co Oct-07 SHB MixFC151 B. parva or B. ribis Polk Co Oct-07 SHB MixFC221 B. parva or B. ribis Polk Co Oct-07 SHB MixFC42 B. parva or B. ribis Polk Co Oct-07 SHB MixFC7 B. rhodina Polk Co Oct-07 SHB MixFF1 B. parva or B. ribis Polk Co Oct-07 SHB MixFF15 B. rhodina Polk Co Oct-07 SHB MixFF19 B. parva or B. ribis Polk Co Oct-07 SHB MixFF8 B. rhodina Polk Co Jul-07 SHB MixSuC14 B. parva or B. ribis Polk Co Jul-07 SHB MixSuC282 B. parva or B. ribis Polk Co Jul-07 SHB MixSuC51 B. parva or B. ribis Polk Co Jul-07 SHB MixSuF13

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35Table 2-3 Continued. B. rhodina Polk Co Jul-07 SHB MixSuF7 B. rhodina Windsor Nov-07 SHB WFC21 B. parva or B. ribis Windsor Nov-07 SHB WFC25 B. parva or B. ribis Windsor Nov-07 SHB WFC6 B. parva or B. ribis Windsor Nov-07 SHB WFF10 B. parva or B. ribis Windsor Nov-07 SHB WFF29a3 B. parva or B. ribis Windsor Nov-07 SHB WFF9 B. rhodina Windsor Nov-07 SHB WFF92 B. parva or B. ribis Windsor Jun-07 SHB WsuC17 B. rhodina Windsor Jun-07 SHB WsuC21 B. parva or B. ribis Windsor Jun-07 SHB WSuC5 B. parva or B. ribis Windsor Jun-07 SHB WsuC61 B. parva or B. ribis Windsor Jun-07 SHB WSuC9 B. parva or B. ribis Windsor Jun-07 SHB WSuF16 B. rhodina Windsor Jun-07 SHB WsuF22 B. parva or B. ribis Windsor Jun-07 SHB WSuF29 B. parva or B. ribis Windsor Jan-07 SHB WWC38 B. rhodina Windsor Jan-07 SHB WWC47 B. parva or B. ribis Windsor Jan-07 SHB WWF37 B. rhodina Windsor Jan-07 SHB WWF46 B. parva or B. ribis Windsor Jan-07 SHB WWF47 B. rhodina Windsor Feb-07 SHB WMixC35 B. parva or B. ribis Polk Co Feb-07 SHB WmixC4 B. parva or B. ribis Polk Co Feb-07 SHB WmixF13 B. parva or B. ribis Polk Co Feb-07 SHB WmixF14 B. parva or B. ribis Polk Co Feb-07 SHB WmixF15 B. rhodina Polk Co Feb-07 SHB WmixF27

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36Table 2-4. Botryosphaeria sequences from Genbank used in phylogenic analysis Isolate Species Host Collector Origin Accession # CBS119047 B. corticis V. corymbosum Oudemans PV New Jersey DQ299245 CAP234 B. dothidea Olea europaea Lazzizera C Italy EF638749 Bd.SC.PH-34.04 B. dothidea P. persica Schnabel G South Carolina DQ177876 CBS 116741 B. dothidea Populus nigra Phillips AJL Portugal AY640254 UCD1125NA B. parva V. vinifera Urbez-Torres California DQ233612 CMW1130 B. parva Sequoia gigantean Swart S South Africa AY236945 CBS110301 B. parva V. vinifera Phillips AJL Portugal AY259098 EU249466* B. parva E. lacrimans Dreaden TJ Florida EU249466 STE-U 4438 B. parva V. vinifera Hallen F R.S.A AY343467 CMW7799 B. parva Persica americana Pegg KG Australia AY615184 CM55 B. rhodina Theobroma cacao Rubini MR Brazil AY754002 WAC9853 B. rhodina V. vinifera Wood P Australia AY727849 UCD921SN B. rhodina V. vinifera Urbez-Torres Mexico EU012370 CMW13496 B. rhodina Acacia mangium Mohali S Venezuela DQ103529 STE-U 4379 B. ribis P. cynaroides Saywood C Zimbabwe AF452525 CMW_14025 B. ribis Syzygium cordatum Pavlic D South Africa DQ316080 CMW7773 B. ribis Ribis sp. Slippers B New York AY236936 CMW7230* Botryosphaeria sp. Eucalyptus Nakabonge G Uganda AY228098 CBS447.62 L. pseudotheobromae Citrus aurantium Smudlers C Suriname EF622081 CBS304.79 L. pseudotheobromae Rosa sp. Unknown Netherlands EF622079 CBS190.73 L. theobromae Persea Americana Bos WS Tanzania EF622068

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37 Figure 2-1. Symptoms of stem blight. A) Flagging symptom of stem blight. B) Pecan brown discoloration on one side of th e vascular tissue associated with stem blight symptoms. C) Severe die-back infecti on. D) Discolored vascular ti ssue associated with stem blight infection of the crown.

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38 Figure 2-2. Conidial morphology of Botryosphaeria associated anamorphs., photographs were taken at 40x magnification. Conidia were pr oduced directly on PDA. A) Immature Lasiodiplodia theobromae (teleomorph is B. rhodina) conidia (WmixC35). B) Mature L. theobromae (teleomorph is B. rhodina) conidia with dark brown longitudinal. striations (WmixC35) C) Conidia of a Neofusicocum anamorph of either B. parva or B. ribis (WWC38). D) Mature conidia of Fusicoccum aesculi (teleomorph is B. dothidea (ArcherRingSpotM).

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39 Figure 2-3. Light micrograph was taken with a dissecting microscope at 1.5x magnification. Micrographs of spores were taken with a compound microscope 40x magnification. A) Perithecia protruding from a plant stem. B) Immature acsi of B. parva or B. ribis from Polk county Florida (MixFC7) C) Asci of B. parva or B. ribis from Alachua county Florida (WFF29).

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40 Figure 2-4. Single-gene ITS phylogeny using representative isolates from Alachua Co., GenBank, and Polk Co., FL.

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41 0 20 40 60 80 100 120 140 160 180B. dothedia (07-30)B. rhodina (MixFC-6)B. parva-ribis (WsuF29) B. parva-ribis (WFF-47)B. parva-ribis (04-40)Audpc Values MSD= 37.59BAC A A B B Figure 2-5. The audpc values for isolates used in pathogenicity study. Uninoculated control plants developed no symptoms and were not included. Columns topped with the same letter are not significantly different according to Waller Duncan k-ratio t -test ( k =100) the minimum significant difference (MSD) = 37.59.

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42 CHAPTER 3 SCREENING FOR AND QUANTIFICATION OF STEM B LIGHT RESISTANCE IN SOUTHERN HIGHBUSH BLUEBERRY BREEDING STOCK Introduction Stem blight on southern highbush blueberries (SHB) is caused by Botryosphaeria spp. in Florida. Stem blight costs growers time and money by causing mortality and by reducing yield. Growers have noticed that some cultivars have higher mortality rates than others suggesting a potential difference in cultivar susceptibilit y. Varying levels of susceptibility to Botryosphaeria pathogens have been noted in blueberry, dogwood, mango, and peach (25,32,36,75,86,90). Susceptibility has been attributed to cultivar, age of tissue used for inoculation, wound age, and inoculum virulence (30,32,86,114). The use of dis ease indexes, highly virulent isolates, and non-woody stem tissue have been reported to standardize resistance screening methods (9,30,100). Buckley (1990) concluded that narrow sense heritability was greater than broad sense heritability for stem blight resistance (25,32). Both additive and non-additive genetic effects are involved in resistance which is deri ved from the low bush blueberry ( V. angustifolium ) in populations from Michigan, New Jersey and No rth Carolina. Buckley recommended that progeny could be screened for the identif ication of superior parents (25). The UF breeding program uses recurrent selec tion which is based on two principles. The first is heterozygous parents yield variable proge ny. The second is that progeny that are extreme in the expression of certain characteristics, and when crossed to produce a second generation progeny, will be variable, and some seedlings will be more extreme in character expression than their parents (58). Cultivar selection at UF ha s four stages. In stage I, 15,000 seedlings are planted in high density plots. After one year, stage I plants are rated for desirable bush defects, firmness, flavor, fruit size, and ripening time. Th e best 500 plants are selected and advanced to

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43 stage II. The rest of the plants are discarded. Stage II plants are rated for three years; the best 150-200 plants are numbered and marked for as exual propagation, approximately 40 softwood cuttings are rooted from each plant. The be st clones are planted in 15-plant plots using commercial spacing. The clones are rated over three years for survival, and for other bush and berry qualities. The superior 12 to 15 stage I II plants are asexually propagated and planted on multiple farms. The stage IV plants are evaluated for three to six years by the breeder and growers. On average one or two plants are selected for cultivar release each year ( Lyrene personal communication ). Progeny are diverse and have varying levels of stem blight mortality in stage III evaluation plots. Variation could be due to different levels of inoculum, va riations in field conditions, or varying levels of resistance. If differences ar e due to resistance, then progeny with the same parents should be more similar in levels of re sistance than progeny of different parents. If resistance has a strong genetic component, select ion for resistance should be possible given an effective screening tool. Therefore, resistance to Botryosphaeria was quantified using blueberry clones being evaluated for cultivar potential, and a screening protocol was devised to select the most resistant seedlings. Methods Field Evaluation Stage III 2003, 2004, and 2005 evaluation plots lo cated in Windsor, FL were rated for disease. E ach clone had 15 replicate plants per plot The clones were rated on a 0 to 2 scale with healthy plants receiving a zero, symptomatic plan ts receiving a one, and dead plants receiving a two. The average disease score of each clone was determined by dividing plant ratings by fifteen. The disease score of each clone rated was assigned to both parents in the cross as a progeny disease score (PDS). PDS data for parents with three or less offspring replicates were

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44 discarded. A general linear model in SAS (SAS Institute, Cary, N.C.) was used to analyze variance in data, and mean PDS of pa rents were separate d by Waller-Duncan k-ratio t -test ( k =100). Clone Replicates and Inoculation Genetically unique plants desi gnated by pedigree num ber were clonally propagated by soft wood cuttings. Clones were separated by pedi gree, randomized between and within pots by randomly selecting four unique clones which were planted per gallon pot in Canadian peat. Plants were cut with scissors below the top 3 or 4 leaves. Scissors we re surface-sterilized with 95% ethanol between pots. Plant height was measured. Eight 3-day-ol d culture plates of B. parva or B. ribis isolate 04-40 were ground w ith 400ml of sterile wate r in a blender. The suspensions were sprayed onto ster ile plates of media, and fungal growth was assessed. Plants were sprayed with one of the suspensions until runoff. Control plants were sprayed with a similar suspension of sterile media and water. A paper towel was moistened with sterile water and placed on top of the plants. Pots were bagged and placed in a 25C incubator with 12h of light per day for two weeks. Lesion lengths were measured in centimeters weekly for one month after the plants were removed from the incubator. Percent lesion length (PLL) was calcu lated by dividing lesion length by plant height. Average PLL was calculate d for each clone evaluated. Variation in data was analyzed using a generalized linear model in SAS (SAS Institute, Cary, N.C.) with class variables of pot and clone, Waller-Duncan k-ratio t -test (k =100) assessed mean separations between clones. The experiment was repeated twice with unique clonal accessions from 2005 and 2007 selected by the breeder.

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45 Results Heritability Study Progeny sus ceptibility assessed in 15-plant clonal field plots di ffered significantly depending on which parents were used to make the cross. P-values for 2005 and 2004 evaluation plots were < 0.05. In 2003, evalua tion plots PDS were not significant ( p > 0.12). Parents were ranked by PDS (Figs 3-1, 3-2, 33) from least to most susceptible. Trials 1&2 (07 Clones) Between-pot variation was significant ( p < 0.1) in trials 1 and 2 (Fig 3-4 & 3-5). Clonal variation was not significant ( p > 0.1). There was no correlati on between average PL L and the clones used in trials one and two. Trial 3&4 (05 Clones) Average PLL was significant ( p < 0.05) for trial 3 (Fig 3-6). For trial 4, pot and clonal variation was not significant ( p > 0.1 ) at the time after plants were removed from the incubator (Fig 3-7). Clonal variation was significant ( p < 0.05) one week after th e end of the incubation period. Two weeks after the incubation period both pot and clone variables were significant ( p < 0.05). Minimum significant difference (MSD) decreased when both variables became statistically significant. There was no correlation between average PLL and the clones replicated in both trials. Discussion Parents were identified from progeny lineages having varying degrees of resistance (Fig 31, and Fig 3-2). The 2003 plot was not significan t because environmental factors were greater contributors to plant mortality in older plots (Fig 3-3). Resi stance was a continuous gradient from the least to most susceptible parents. Resu lts support Buckleys findi ngs that pedigree will influence progeny stem blight resistance. Therefor e, a reliable screening tool could be developed

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46 to select progeny with superior stem blight resistance. Clonal susceptibility to stem blight was not replicated using either the or clones. Lack of repeatability was due in part to low numbers of clonal replicates used throughout the trials. A larger number of replicates per clone would help to reduce the standard errors used in comparing the clone means. Differences between trials 1 & 2 contributed to variable results. Clones used in trial 1 were left in the incubator for three weeks instead of two unlike previous experiments. The three week incubation period left more time for infect ion many of the plants were dead by the end of the third week. Pots were not randomized in the incubator; some pots received less light because there were no lights on the bottom rack. Plan ts on the bottom rack were water soaked and greater disease incidence was observed. In trails three and four the incubator used had lights on the bottom shelf; all pots received equal amounts of light which helped to standardized the experiment. Stem blight symptoms were evident on the cont rol plants of trial four. No fungus grew on the petri plate sprayed with the control suspension. Botryosphaeria was re-isolated from the control plants having stem blight symptoms. These data suggest that cuttings used for trial 4 were already infected with Botryosphaeria before the trial began. Clones did not display visible symptoms of stem blight prior to inoculation. This indicated a possible fungal latent infection pe riod. Latent infection periods of Botryosphaeria have been reported for Proteaceae flowers, pistachio, and apple ( 39,50,73,76). Changes to propagation methods that could provide disease free material; and the addition of individual clonal replicates would help standardize th e screening procedure.

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47 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3a0052 a8619 a0231 a0213 Jew el a0128 a0019 a97140 a98295 a00198 a0212 a0219 a0106 a96138 a0235 a 9626 a98368 a0208 a9091 a98325 a9284 a0023 a93221 a98407 a9554 a0069 a0045 a0218 a0058 a732 a0207 a0202 SantaFe a0148 sbell a 9624 a0248 a0060ParentsMean Disease Score Pr < 0.001 MSD = 0.54 Figure 3-1. Mean progeny disease score of parents of the 2005 clone evaluation. Clones were dispersed randomly in the plot. The mean for each parent was based on 4 or more progeny clones. Pr is the ANOVA p-value and MSD is the minimum significant difference according to the Waller Duncan kratio ttest ( k =100).

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48 0 0.1 0.2 0.3 0.4 0.5 0.6a 0 054 a98409 a 0155 a 01 35 a0108 a 0 154 a 00 43 a 010 5 a9 71 30 a 016 9 a0039 a 01 06 a98 2 95 a 0 058 a 001 4 a 982 0 a 9 937 Jew e l a 963 2 Su r a 01 70ParentsMean Disease Score Pr < 0.01 MSD = 0.35 Figure 3-2. Mean progeny disease score of parents of the 2004 clone evaluation. Clones were dispersed randomly in the plot. The mean for each parent was based on 4 or more progeny clones. Pr is the ANOVA p-value and MSD is the minimum significant difference according to the Waller Duncan kratio ttest ( k =100).

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49 0 0.2 0.4 0.6 0.8 1 1.2a0056 a9 84 0 9 a94177 a96 9 6 a 971 4 7 a95 6 7 S.bell e a98297 M i ll en ni a 007 5 a00 1 9 a 932 2 1 Bluecris a9 41 1 5 Emerald a99 3 0 a0 04 3 a95 1 73 a0 03 4ParentProgeny Disease Score Pr > 0.12 MSD = 0.88 Figure 3-3. Mean progeny disease score of parents of the 2003 clone evaluation. Clones were dispersed randomly in the plot. The mean for each parent was based on 4 or more progeny clones. Pr is the ANOVA p-value and MSD is the minimum significant difference according to the Waller Duncan k-ratio t -test (k =100).

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50 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 3343054386688103195382221423853121668796435315315646891002825519684225104CloneAverage Percent Lesion Length 10/11 Clone Pr>0.73 10/18 Clone Pr>0.223 10/25 Clone Pr>0.312 Figure 3-4. Trial 1 average percent lesion length of 07 clones inoculated with Botryosphaeria isolate 04-40. Lesion lengths were measur ed at three dates. Pr is the ANOVA pvalue and MSD is the minimum significant difference according to the Waller Duncan k-ratio t -test ( k =100).

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51 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.101663121031958788382143348984225232243853281538543461043052551006696156196CloneAverage Percent Lesion Length 11/9 Clone Pr>0.127 11/15 Clonal Pr>0.185 11/21 Clone Pr>0.344 Figure 3-5. Trial 2 average percent lesion length of 07 clones inoculated with Botryosphaeria isolate 04-40. Lesion lengths were meas ured at three dates. Pr is the ANOVA p-value and MSD is the minimum significant difference according to the Waller Duncan kratio t -test ( k =100).

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52 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.1032932221553951715512544255925585606543552435189540052805451586562612975412535554502196563052295238CloneAverage Percent Lesion Length 1/28 Clone Pr>0.0032 MSD = 0.442 2/5 Clone Pr>0.0019 MSD = .48 2/11 Clone Pr>0.0092 MSD = .593 2/18 Clone Pr> 0.043 MSD = .70 Figure 3-6. Trial 3 average percent lesion lengths of 05 clones inoculated with Botryosphaeria isolate 04-40. Lesion lengths were meas ured at four dates. Pr is the ANOVA p-value and MSD is the minimum significant difference according to the Waller Duncan kratio t -test ( k =100).

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53 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.1055925606524355125189540056305171329321965442553955812975229586222154505412562652805355543552385451CloneAverage Percent Lesion Length in CM 2/25 Clone Pr>0.197 3/3 Clone Pr>0.048 MSD = 0.66 3/10 Clone Pr >0.0037 MSD = 0.44 Figure 3-7. Trial 4 average percent lesi on lengths of 05 clones inoculated with Botryosphaeria isolate 04-40. Lesion lengths were measur ed at three dates. Pr is the ANOVA pvalue and MSD is the minimum significant difference according to the Waller Duncan k-ratio t -test ( k =100).

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63 BIOGRAPHICAL SKETCH Am anda Faith Watson received a Bachelor of Science in biological science from Clemson University, May 2006. While attending Clemson, Ama nda participated in organizations such as the Clemson Wesley Foundation, Sigma Alpha, a nd Tiger Band. She worked for two summers at the Outdoor Lab in Clemson. Her undergradu ate research project was under Dr. Steven Jeffers. There she fulfilled Kochs postul ates on foliage blight of hostas caused by Phytophthora nicotianae While working in lab, she also helped with Phytophthora ramorum screening, and the maintenance of Clemsons Phytophthora collection. Upon graduation Amanda went to the Plant Pathology Department at the University of Florida to complete her Master of Science degree. There she worked on the etiology of stem blight of southern highbush blueberries (SHB) caused by Botryosphaeria, and the quantification of resistance in SHB breeding stock under Dr. Phil Harmon. While completing her masters Am anda presented her work at the Florida Phytopathological Society meeting, and at the Florida Blueberry Growers Association annual meetings Fall 2007 and Spring 2008. She was also an editor of the Plant Pathology news letter, and vice president of the Plant Pathology gradua te student association. Currently, Amanda plans to continue working on stem blight of blueberries under Dr. Phil Harmon.